Introduction

COVID-19 COVID-19 COVID-19: Immunology, vaccines and epidemiology Introduction and guidance About this free course This version of the content may include video, images and interactive content that may not be optimised for your device. You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University – https://www.open.edu/openlearn/science-maths-technology/covid-19-immunology-vaccines-and-epidemiology/content-section-0 There you’ll also be able to track your progress via your activity record, which you can use to demonstrate your learning.

Unless otherwise stated, copyright © 2023 The Open University, all rights reserved. Intellectual property Unless otherwise stated, this resource is released under the terms of the Creative Commons Licence v4.0 http://creativecommons.org/licenses/by-nc-sa/4.0/deed.en_GB. Within that The Open University interprets this licence in the following way: www.open.edu/openlearn/about-openlearn/frequently-asked-questions-on-openlearn. Copyright and rights falling outside the terms of the Creative Commons Licence are retained or controlled by The Open University. Please read the full text before using any of the content. We believe the primary barrier to accessing high-quality educational experiences is cost, which is why we aim to publish as much free content as possible under an open licence. If it proves difficult to release content under our preferred Creative Commons licence (e.g. because we can’t afford or gain the clearances or find suitable alternatives), we will still release the materials for free under a personal end-user licence. This is because the learning experience will always be the same high quality offering and that should always be seen as positive – even if at times the licensing is different to Creative Commons. When using the content you must attribute us (The Open University) (the OU) and any identified author in accordance with the terms of the Creative Commons Licence. The Acknowledgements section is used to list, amongst other things, third party (Proprietary), licensed content which is not subject to Creative Commons licensing. Proprietary content must be used (retained) intact and in context to the content at all times. The Acknowledgements section is also used to bring to your attention any other Special Restrictions which may apply to the content. For example there may be times when the Creative Commons Non-Commercial Sharealike licence does not apply to any of the content even if owned by us (The Open University). In these instances, unless stated otherwise, the content may be used for personal and non-commercial use. We have also identified as Proprietary other material included in the content which is not subject to Creative Commons Licence. These are OU logos, trading names and may extend to certain photographic and video images and sound recordings and any other material as may be brought to your attention. Unauthorised use of any of the content may constitute a breach of the terms and conditions and/or intellectual property laws. We reserve the right to alter, amend or bring to an end any terms and conditions provided here without notice. All rights falling outside the terms of the Creative Commons licence are retained or controlled by The Open University. Head of Intellectual Property, The Open University Introduction and guidance Introduction Have you ever wondered why some people are very badly affected by a particular viral infection, while others are asymptomatic? Differences in the effectiveness of their immune systems play a part, but so do the type of virus, genetic variability in the human population, and whether a person has any immunity from previous infections or vaccination. This free badged course, COVID-19: Immunology, vaccines and epidemiology, will take you through some of the key science that underpinned the global efforts to control COVID-19. You will learn about virology, immunology and vaccinology. You will carry out practical online laboratory activities, in which you will learn how to detect antibodies against SARS-CoV2 using a standard antibody-detection technique called ELISA, an enzyme-linked immunosorbent assay. You will see how the detection of antibodies helped epidemiologists to track the course of the COVID-19 pandemic. You will also see how quantitation of antibodies against the spike protein was a critical measure, determining which vaccines would be most likely to protect against the disease. The course lasts 24 hours and is comprised of eight ‘weeks’. You can work through the course at your own pace, so if you have more time one week there is no problem with pushing on to complete a further study week. There will be weekly interactive quizzes, of which Weeks 4 and 8 will provide you with an opportunity to earn a badge to demonstrate your new skills. You can read more on how to study the course and about badges in the next sections. After completing this course, you will be able to: understand how the immune system protects against viral infection detect antibodies against COVID-19, using the ELISA technique distinguish individuals who have had a COVID-19 infection, from those who have had a COVID-19 vaccination understand how the detection of antibodies (serology) can be used to track an epidemic understand the different strategies developed for producing vaccines against COVID-19. Before you begin, you should have a basic knowledge of cell biology, and an understanding of how DNA and RNA hold genetic information. If you are unfamiliar with these areas, then you may find the following OpenLearn materials on molecular and cellular biology to be a good starting point: DNA, RNA and protein formation A tour of the cell. Moving around the course In the ‘Summary’ at the end of each week, you will find a link to the next week. If at any time you want to return to the start of the course, click on ‘Full course description’. From here you can navigate to any part of the course. It’s also good practice, if you access a link from within a course page (including links to the quizzes), to open it in a new window or tab. That way you can easily return to where you’ve come from without having to use the back button on your browser. The Open University would really appreciate a few minutes of your time to tell us about yourself and your expectations for the course before you begin, in our optional start-of-course survey. Participation will be completely confidential and we will not pass on your details to others.

What is a badged course? Digital badges are a new way of demonstrating online that you have gained a skill. Colleges and universities are working with employers and other organisations to develop open badges that help learners gain recognition for their skills, and support employers to identify the right candidate for a job. Badges demonstrate your work and achievement on the course. You can share your achievement with friends, family and employers, and on social media. Badges are a great motivation, helping you to reach the end of the course. Gaining a badge often boosts confidence in the skills and abilities that underpin successful study. So, completing this course could encourage you to think about taking other courses.

How to get a badge Getting a badge is straightforward! Here’s what you have to do: read each week of the course score 50% or more in the two badge quizzes in Week 4 and Week 8. For all the quizzes, you can have three attempts at most of the questions (for true or false type questions you usually only get one attempt). If you get the answer right first time you will get more marks than for a correct answer the second or third time. Therefore, please be aware that for the two badge quizzes it is possible to get all the questions right but not score 50% and be eligible for the badge on that attempt. If one of your answers is incorrect you will often receive helpful feedback and suggestions about how to work out the correct answer. For the badge quizzes, if you’re not successful in getting 50% the first time, after 24 hours you can attempt the whole quiz, and come back as many times as you like. We hope that as many people as possible will gain an Open University badge – so you should see getting a badge as an opportunity to reflect on what you have learned rather than as a test. If you need more guidance on getting a badge and what you can do with it, take a look at the OpenLearn FAQs. When you gain your badge you will receive an email to notify you and you will be able to view and manage all your badges in My OpenLearn within 24 hours of completing the criteria to gain a badge. Get started with Week 1.

Week 1: How the body recognises a viral infection Introduction This week, you will be introduced to a range of acute and chronic viral infections, focusing on acute viral infections. You will take a close look at influenza-A and SARS-CoV2, the causative agent of COVID-19, as key examples – not just because of the recent pandemic but also because they are very informative. The role of antibodies in protection against viral disease and reinfection is an important component of immunity. This area, and vaccine development, will be discussed later in the course, but for now you will start with an overview of viruses and then move on to see how the innate immune system recognises and responds to an acute viral infection. Video 1 Introduction to Week 1 DAVID MALE: The COVID-19 pandemic swept across the world in 2020–2022, with waves of infection in different countries, produced by successive variants of the SARS-CoV2 virus. The rapid spread was made possible by the high infectivity of the virus and the fact that a substantial proportion of infected individuals had no symptoms. But, why should the course of this novel infection be so variable in different individuals? It is partly due to the differences in the immune systems of each person, but it also relates to the dose and variant of virus encountered. The genome of the SARS-CoV2 virus was sequenced within days of the initial virus isolation and the importance of the viral spike protein which allows attachment to target cells, was immediately recognised. There followed an enormous research effort to produce vaccines which would induce antibodies against the spike protein. The production and distribution of millions of doses of vaccine followed on rapidly, and this required equally heroic efforts in biotechnology and logistics. Understanding how the immune system protects against viruses and how this knowledge can be applied to vaccine development and the control of a pandemic disease is one of the great stories of modern science.

By the end of this week, you should be able to: outline the range of different viral infections describe the structure and genomes of influenza-A and SARS-CoV2 viruses identify intracellular receptors of the innate system that recognise viruses understand how interferons delay the spread of an acute virus infection. The Open University would really appreciate a few minutes of your time to tell us about yourself and your expectations for the course before you begin, in our optional start-of-course survey. Participation will be completely confidential and we will not pass on your details to others. 1 Viral infections All viruses are small packages containing the genetic information needed to replicate themselves, but they can only do so by infecting cells of a host organism. However, beyond this basic description, viruses are enormously variable. Often they produce damage or disease in their host, including humans, and for this reason they are a very important group of pathogens. It would be impossible in a short course to cover the great range of viruses that cause disease in humans. However, we can look at the life-cycle of a typical virus infection in Video 2. Video 2 Life-cycle of a typical virus infection DAVID MALE: The diagram shows a generalised virus life-cycle. In step-1: A virus infects a cell by attaching to receptors on the cell surface and entering the cell. The virus is first taken into the cell in a vesicle called an endosome. The surface proteins of the virus fuses with the membrane of the endosome, releasing the virus’ genetic material into the cell. In step-2: The viral genome is replicated and viral proteins are synthesised by hijacking the cell’s machinery for replicating nucleic acids (DNA or RNA) and synthesising proteins. In step-3: New viruses are assembled within the infected cell, by packaging the viral genome inside new viral particles. Step-4 shows the new virus particles being released by budding from the surface of the infected cell, or in some cases directly killing the infected cell to allow escape. The virus can now spread to new cells within the host, or to a new host.

1.1 Acute infections The life-cycle shown earlier in Video 2 is typical of an acute virus infection, such as influenza or COVID-19. An acute infection is one where a person becomes infected, and the immune system then reacts to destroy the virus and virus-infected cells. In the end, the immune system prevails and there is no longer any of the virus left in that host – this is referred to as sterile immunity. Usually, an acute infection will last for a few weeks, at most. However, some virus infections can evade the immune system and lie low within cells of the host for months or years, producing no symptoms. Such infections are said to be latent. In addition, they may reactivate or stay continuously active over this period; in this case they are responsible for a chronic infection. Activity 1 Acute and chronic viral infections Allow 10 minutes Look at Table 1 below, which lists some viruses that produce disease in humans. From your previous knowledge or experience try to decide which of these viruses produce an acute infection and which produce a latent or chronic infection. Write your answers in the boxes provided in the last column. Then click on ‘Reveal feedback’ to see the answers. The first box has been filled in for you. Table 1 Viral infections

Virus	Disease	Acute or latent/chronic infection
SARS-CoV2	COVID-19	Acute
Rubella virus	German measles
Ebola virus	Ebola
Human immunodeficiency virus (HIV)	Acquired immune deficiency syndrome (AIDS)
Norovirus	Gastroenteritis (vomiting, diarrhoea)
Rhinovirus	Common cold
Epstein Barr virus	Glandular fever
Herpes simplex virus	Cold sores
Poliovirus	Poliomyelitis(Infantile paralysis)
Varicella zoster virus	Chicken pox, shingles
Mumps virus	Mumps

Here is the completed table: Table 1 Viral infections (completed)

Virus	Disease	Acute or latent/chronic infection
SARS-CoV2	COVID-19	Acute
Rubella virus	German measles	Acute
Ebola virus	Ebola	Acute
Human immunodeficiency virus (HIV)	Acquired immune deficiency syndrome (AIDS)	Chronic
Norovirus	Gastroenteritis (vomiting, diarrhoea)	Acute
Rhinovirus	Common cold	Acute
Epstein Barr virus	Glandular fever	Acute/Chronic
Herpes simplex virus	Cold sores	Latent
Poliovirus	Poliomyelitis(Infantile paralysis)	Acute
Varicella zoster virus	Chicken pox, shingles	Acute/Latent
Mumps virus	Mumps	Acute

Rubella virus, Ebola virus, Norovirus, Rhinovirus, Poliovirus and Mumps virus cause acute infections. Notice that acute infections can still cause very serious diseases and in some cases (eg polio) the damage lasts for a lifetime, even if the viral infection is relatively short. HIV causes a persistent chronic infection. Epstein Barr virus causes glandular fever, an acute infection, but usually persists as a symptomless chronic infection for years. Herpes simplex can remain latent for many years and sporadically reactivate to produce cold sores. Varicella zoster produces chicken pox as an acute illness, but becomes latent in some people and reactivates to produce shingles. As you may have deduced, chronic and latent infections are persistent infections that continue after the initial acute infection with that virus. Don’t worry if you did not get all of these. In reality, the outcome in any individual may be different from the usual course of infection. For example, in people who are immunosuppressed or immunodeficient, acute viral infections are often slower to clear and are more likely to become chronic infections.

1.2 Chronic and latent infections So what is the key difference between acute and chronic viral infections? Essentially, they have different reproductive strategies. After infection, viruses such as SARS-CoV2 and influenza replicate quickly and then spread by aerosol droplets, which are inhaled into the nose, throat and respiratory system – they complete their life cycle within days, before the immune system has fully geared up to eliminate them. Conversely, chronic infections such as Herpes simplex and HIV can evade the immune response for years. During this time, the virus is shed more slowly, sometimes sporadically and the method of transmission is often by direct contact between individuals. These persistent viruses have a variety of strategies for evading the immune response. Here are some examples: HIV mutates continuously within an individual to avoid being recognised by antibodies. HIV subverts the cellular machinery which promotes recognition of virus-infected cells. Herpes simplex remains latent in neurons, and deploys decoy proteins that fool the immune system into recognising the cell as not-infected. Epstein Barr virus (EBV) secretes a signalling molecule (cytokine), which deviates the immune response away from that which eliminates EBV-infected cells. Human papilloma virus (HPV) (Figure 1) produces very low levels of viral proteins, so the immune system has little foreign material to recognise.

Indeed, each of these viruses has numerous systems for evading immune responses. It is a fascinating area of research. However, to understand it requires a knowledge of how the immune system would normally eliminate a virus or a virus-infected cell. Before that you need to understand how the immune system recognises viruses and that is based on some knowledge of virus structure.

2 The anatomy of a virus Viruses come in a variety of different shapes and sizes. The simplest consist of a protein shell called the nucleocapsid, which contains the viral genetic material (nucleic acid). These are called non-enveloped viruses, to distinguish them from the more complex enveloped viruses, as shown in Figure 2.

Enveloped viruses also have a nucleocapsid containing the viral genome. The nucleocapsid is contained within a viral envelope, a composite structure which includes a phospholipid bilayer, derived from the plasma membrane of the host cells which produced the virus. Inside the membrane, viral matrix proteins connect the nucleocapsid to the envelope. The matrix is also important in organising the assembly of new virus particles. The envelope also contains viral proteins and some residual host proteins from the infected cell. The critical thing to notice is that some proteins are on the outside of the virus whereas others, such as the nucleocapsid and matrix proteins, are on the inside. This is important because it affects what parts of the virus can be recognised by different elements of the immune system. As you can see in Figure 3, enveloped viruses are released from infected cells by budding from the plasma membrane. Examples of enveloped viruses are influenza-A, HIV and SARS-CoV2. The transmission electron micrographs show different stages in the assembly (1, 2) budding (3) and release (4) of HIV from the surface of an infected cell. This process illustrates step 4 in Video 2.

In addition to the components described above, most viruses contain a number of enzymes and auxiliary proteins which are required to initiate infection of the cell and replication of the virus.

2.1 Types of virus You have now considered a great variety of viruses, which differ in size, shape and life-cycle, some of which are shown in Figure 4. In this course you will focus on Influenza-A, an orthomyxovirus, and SARS-CoV2, which is a coronavirus.

In addition to their obvious differences in size and shape, viruses are classified according to their genetic material and how it is replicated: DNA or RNA single-stranded (ss) or double stranded (ds) positive sense or negative sense; this relates to whether the genome directly encodes protein (positive) or whether it must be replicated before it can direct protein synthesis (negative). This is referred to as the Baltimore system of classification. The details of virus replication are beyond the scope of this course. It is sufficient to know that Influenza-A has a genome with eight segments of ssRNA, which is negative-sense, whereas SARS-CoV2 has a single segment of ssRNA, which is positive sense. One important point to note is that replication of the ssRNA genomes of these viruses involves an intermediate of dsRNA; double-stranded RNA is not a standard component of uninfected host cells.

2.2 Influenza-A and SARS-CoV2 You will now look in a bit more detail at influenza-A and SARS-CoV2, both of which cause acute respiratory infections. Activity 2 Comparing influenza-A and SARS-CoV2 Allow 5 minutes Take a moment to consider any other similarities. Try to identify three similarities. Write your answer in the box provided below. They are both enveloped viruses, with a ssRNA genome. They are spread by aerosol droplets and you might also have noted that they both cause pandemics, with waves of infection as new strains arise. Now try to identify three differences between influenza-A and SARS-CoV2. Write your answer in the box provided below. The genome of influenza-A is segmented (eight segments) and negative-sense, whereas that of SARS-CoV2 is a single, positive-sense segment of ssRNA. You might also have spotted that the shape of the viruses is different, as shown in Figure 4. Another difference between these viruses is in how they infect cells and exactly which cells can become infected. The first stage of infection is the binding of a viral surface protein to a protein receptor on the host cell. Since this interaction is highly specific it determines which cell types can be infected by each type of virus. As shown in Figure 5, the nucleocapsid contains viral protein and RNA (ribonucleoprotein) and the envelope of influenza-A has two external viral proteins – the haemagglutinin and neuraminidase.

The haemagglutinin (H) is responsible for attachment of the virus to the target cell. It binds to carbohydrate units (sialic acid) which are attached to a number of different proteins on the host cell surface. (Proteins with bound carbohydrate units are called glycoproteins). Because the target glycoproteins are quite widely distributed on different cell types, influenza-A can infect several different types of cell. The neuraminidase (N) is involved in virus release and spread. Strains of influenza-A are classified according to which haemagglutinin and neuraminidase they have, eg H3N2. An electron-micrograph of a corona virus is shown in Figure 6. It has prominent spikes on the outside, which give it a crown-like appearance, and this was the origin of the name for this group of viruses. You can see the structure of the virus shown diagrammatically in Figure 7 below.

The spike protein allows the virus to attach to target cells by binding to a protein called the ACE2 receptor (ACE2 = Angiotensin converting enzyme-2). The part of the spike protein which directly contacts the ACE2 receptor is the ‘receptor-binding domain (RBD)’. This detail is very relevant when we consider what antibodies are most effective in protecting against COVID-19 – antibodies which recognise the RBD are particularly important. Would you expect SARS-Cov2, to infect the same cells as influenza-A? If so, why? SARS-Cov2 binds to cells that have the ACE2 receptor, whereas influenza-A binds to a number of surface glycoproteins that are recognised by the haemagglutinin. So we might expect SARS-CoV2 and influenza-A to infect different sets of cells. Different strains of a virus can also selectively target distinct sets of cells and this is referred to as ‘viral tropism’. In humans the ACE2 receptor is found in many tissues, but at particularly high levels on the upper surface of epithelial cells facing into the alveoli of the lung and the lumen of the small intestine. It is also found on the endothelial cells which line the inside of blood vessels and in the smooth muscle of arteries. This observation partly explains why the lung is particularly targeted by COVID-19. But other tissues may be affected in more severe cases, when the virus spreads through the body.

2.3 Proteins encoded by SARS-CoV2 The genome of SARS-CoV2 is large for an RNA virus. It consists of a single piece of positive sense ssRNA with 29,903 nucleotides which encode 19 proteins, as shown in Figure 8. Two genes encode 16 non-structural proteins nsp1-nsp16. The genes for the four structural proteins (Spike, Envelope, Membrane glycoprotein, Nucleocapsid) and the auxiliary proteins (3a, 6,7,8,10) are indicated. There are untranslated regions (UTR) at the 5’ and 3’ ends of the genome. The non-structural and auxiliary proteins are required for virus replication, assembly and release.

New variants of SARS-CoV2 have shown mutations in many of these genes. However, it is variation in the spike protein that is of particular interest and importance, because antibodies against the spike protein are protective against infection, and the spike protein is the key component of all current vaccines against COVID-19 (Feb. 2023). Figure 9 shows the spike protein diagrammatically. It is a trimeric glycoprotein with two subunits, which is inactive until it comes into contact with a host cell. On contact, an enzyme on the host cell surface (TMPRSS2) cleaves and activates the spike protein so that it can now bind to the ACE2 receptor.

In the next section, you will now start to look at how the immune system can recognise a viral infection.

3 Recognition of viral infection To combat a virus infection, the immune system must first recognise the virus, and/or virus-infected cells. From your knowledge of the structure and life-cycle of an enveloped virus, identify three distinctive features of a virus or virus-infected cell, that could distinguish it from normal host cells. The virus has proteins encoded by the viral genome which are different from the host proteins, for example the spike protein, haemagglutinin, M-protein or nucleocapsid. If a cell is infected by an enveloped virus, then some viral proteins are inserted into the membrane of the cell, before the individual viruses assemble and bud off. The genome of influenza-A and SARS-Cov2 is RNA, whereas the genome of mammalian cells is DNA. The replication of these viruses involves dsRNA, which is not a regular component of host cells. As you will see, there are two major types of immune response – innate immune responses and adaptive immune responses. Adaptive immune responses improve with time, especially following repeated encounters with the same pathogen. In contrast, innate immune responses do not display immunological memory, and hence do not improve significantly over time. The adaptive immune system primarily recognises foreign proteins, such as virus-encoded proteins. Any biological molecule that can be recognised by the adaptive immune system is called an antigen. We will look at antigen-recognition and adaptive immune responses against viruses in Week 2. Adaptive immune responses take several days to become fully active. During this early period of infection, the innate immune system acts as a first line of defence. The innate immune system recognises ‘pathogen-associated molecular patterns (PAMPs)’ which are distinctive biological components of bacteria, viruses and fungi. For the rest of this week, you will consider some of these PAMPs and how they trigger innate immune responses to viruses.

3.1 Pathogen-associated molecular patterns (PAMPs) Cells of the body have internal receptors that allow them to recognise components of viruses. They belong to a family of ten receptors, called Toll-like receptors (TLRs), that recognise components of pathogens. Those most relevant for detection of viral infection are listed in Table 2. They are present in cells of the immune system and epithelial cells in mucous membranes – for example, at sites of potential virus infection. Table 2 Toll-like receptors that recognise viral infection

Receptor	Location	Recognises
TLR3	Endosome or cell surface	dsRNA
TLR7	Endosome	ssRNA
TLR8	Endosome	ssRNA

The importance of the TLRs is demonstrated by the rare individuals who lack them. For example, TLR3 deficiency is associated with susceptibility to herpes simplex infection. Notice that these receptors face into the endosome. When viruses such as SARS-CoV2 enter a cell they are first taken into an endosome, where the viral capsid is removed, releasing the viral RNA. The released RNA can be immediately recognised by the TLRs facing into the endosome. Recognition of the viral RNA triggers activation of the infected cell via a transcription factor, NFκB, which has been described as a ‘master-switch of inflammation’. One important action of NFκB is to induce the synthesis of Type-1 interferon (IFN), a signalling molecule that helps control viral infection. NFκB also induces synthesis and secretion of a variety of other signalling molecules, collectively called cytokines which control the development of inflammation; this is normally beneficial for controlling infection. However, excessive cytokines can damage host tissues. You may have heard the term ‘cytokine storm’, which refers to damage produced by excess cytokine production. In some patients, this was a particular problem with COVID-19 infection – where the virus infection was not well-controlled by the immune system, the collateral damage from the cytokines exacerbated the damage caused by the virus. You will now look at how interferon limits the spread of virus.

3.2 Interferons and anti-viral proteins When cells become infected, interferon (IFN) signals to neighbouring cells to induce synthesis of anti-viral proteins, which are normally inactive, but it puts the cell into a state of ‘alert’. Should the neighbouring cells later become infected, viral components activate the antiviral proteins, to shut down mRNA production and protein synthesis. This process is shown diagrammatically in Figure 10. An infected cell (1) releases IFN, which acts on receptors on neighbouring cells (2) to induce antiviral proteins (3). If that cell later becomes infected with a virus, the antiviral proteins are activated (4) to induce a virus-resistant state where protein synthesis is inhibited (5).

What advantage is there for a cell to shut down protein synthesis? What advantage is there for the infected host? There is no advantage to the individual cell, since all cells must synthesise protein to survive, however it also stops virus production in that cell and thus slows virus spread within the body. Interferons were originally identified for their role in limiting virus replication. However, they have numerous additional effects in the adaptive immune system. Specifically, they enhance the ability of all cells to present antigens to T lymphocytes (T cells), which are primarily responsible for elimination of virus-infected cells. You will hear a lot more about this in Week 2. But first we look at one more element of the innate immune system: how a replicating virus is detected.

3.3 Cytosolic receptors for viral infection After a virus has escaped from the endosome of an infected cell, virus components are located in the cytoplasm. Receptors are also present in the cytoplasm, that recognise and limit viral replication and spread. They belong to a family of receptors called the ‘Retinoic acid-inducible gene I (RIG1)-like helicases’ which are understandably abbreviated to RLHs. Two receptors are particularly important in this context: RIG-1 itself which recognises short dsRNA MDA5 which recognises long dsRNA Why would recognition of cytoplasmic dsRNA, enable a cell to identify an ongoing viral infection? dsRNA is an intermediate produced during replication of RNA genome viruses, which is not a regular component of host cells. After binding to dsRNA these receptors localise to mitochondria via an adapter protein IPS-1. They then activate transcription factors NFκB and IRF3 (Interferon regulatory protein-3) which translocate to the nucleus of the cell and activate transcription of a number of genes including the genes for type-1 interferon, as shown in Figure 11. You can also see that RIG-1 and MDA5 are cytoplasmic receptors for dsRNA. They lead to activation of transcription factors IRF3 and NFκB, which promote transcription of genes for type 1 interferons, and other cytokines

4 Week 1 quiz Check what you have learned this week by taking the end-of-week quiz. Week 1 practice quiz Open the quiz in a new window or tab, then return to this week when you’re done. 5 Summary This week, we introduced the wide range of viruses that produce disease in humans, before focusing on two respiratory viruses − influenza-A and SARS-CoV2. Both are enveloped viruses with an RNA genome that produce acute infections that are spread by aerosol droplets. Distinctive components of virus infection can be recognised by the immune system. The adaptive immune system recognises virus antigens, whereas the innate immune system recognises pathogen-associated molecular patterns. In the case of influenza-A and SARS-CoV2 the PAMPs are the ssRNA of the viral genome or the dsRNA, which is an intermediate of virus replication. TLRs recognise viral RNA in endosomes, while RLHs recognise dsRNA of replicating virus in the cytoplasm. After recognising the virus, infected cells secrete interferon, a cytokine which signals to neighbouring cells, to slow virus spread. Other cytokines induce inflammation, which normally helps control infection, but can contribute to host tissue damage. The actions of the innate immune system hold the line against the virus until the adaptive immune response gets into action, but that takes several days… And that is what you will learn about next week. You should now go to Week 2. Week 2: How the immune system combats viral infection Introduction During the COVID-19 pandemic, you will have heard a lot about antibodies and how vaccines induce neutralising antibodies that protect against infection. But antibodies are just one element of immune defence. Do you remember hearing about T cells and immunological memory? This week you will learn more about these and about the range of adaptive immune responses that combat viruses and deal with virus-infected cells. Audio 1 Introduction to Week 2 DAVID MALE: Adaptive immune responses are mediated by leukocytes, including lymphocytes, which come in various types – Cytotoxic T lymphocytes and Natural Killer cells, or NK cells, recognise and destroy virus-infected cells. B lymphocytes make antibodies which block virus binding to target cells and help macrophages, NK cells and the complement system to recognise a virus and virus antigens on infected cells. Antibodies come in different classes, which have different roles in protection. IgA antibodies are particularly important in protecting mucosal surfaces against respiratory viruses. The adaptive immune system displays immunological memory, which is the basis of vaccination – by immunising with a harmless antigen or attenuated pathogen, the immune system is primed to make a strong response if it subsequently encounters the real pathogen. Long term immunity resides in memory B and T cells, which persist, distributed throughout the body, even when antibody levels have declined. By the end of this week you should be able to: describe how T cells and NK cells recognise and destroy virus-infected cells outline how B cells recognise antigens and make antibodies list the major functions of different classes of antibody and their roles in combating a virus infection understand how innate and adaptive immune defences act in complementary ways understand the basis of immunological memory, which underpins vaccination. 1 Adaptive immune defences In Week 1, you learnt about innate immune responses, and some of the ways that the body can recognise a virus or virus-infected cell. This week, you will learn about the adaptive immune response to viruses. What is the key difference between the innate and adaptive immune responses? Innate immune responses do not improve following repeated encounters with the same pathogen. Adaptive immune responses are stronger and more effective each time the same antigen contacts a person’s immune system – in essence the immune system adapts. The principal cells of the adaptive immune system are white blood cells or leukocytes, which are distributed between various lymphoid organs such as lymph nodes and the spleen. Leukocytes traffic between organs via the blood and lymphatic system and interact with other cell types in the body in immune defence. Viruses are pathogens that live inside cells of the body but move between cells via the blood, tissue fluids and extracellular spaces. In each case, the immune system has to be able to recognise the pathogen and mount an appropriate response. The cells responsible for immune recognition are lymphocytes, a set of leukocytes that are found in blood and throughout the body, although they are particularly concentrated in lymphoid tissues and specialised areas of mucosal tissue. Lymphocytes fall into two basic categories: T cells, which develop in the thymus and B cells, which develop in the bone marrow. What term is used for a molecule that is recognised by a lymphocyte? Antigen – see Week 1, Section 2. A third group of lymphocytes which are important in antiviral defence is the Natural Killer or NK cells. However, unlike T cells and B cells, the defence provided by NK cells does not improve significantly following repeated encounters with the same virus. For this reason, NK cells are really part of the innate immune system, although it is easier to consider them alongside T cells, since they act in complementary ways. Video 1 below illustrates the principal cells of the immune system and explains how they are involved in immune defence against viruses. Video 1 Immune defence DAVID MALE: Two groups of lymphocytes can recognise and destroy virally-infected cells; these are the cytotoxic T-lymphocytes, or CTLs, and the Natural Killer, or NK, cells. B cells recognise antigens in extracellular spaces; if they become activated by contact with an antigen they recognise, they divide and differentiate into plasma cells which produce secreted antibodies. In order for B cells to become activated they normally need help from a group of helper T cells called TH2 cells, which also recognise the same antigen. Another group of helper T cells, the TH1 cells, activates macrophages. Macrophages internalise antigens and pathogens, including viruses, from the extracellular spaces and break them down internally. Other groups of T cells, such as the TH17 cells, are involved in the development of inflammation. All of these sets of T cells are subject to regulation, by another group of regulatory T cells or T regs.

If there is one key message you take away from this section, it is that T cells recognise antigens originating from inside the cells of the body, whereas B cells produce antibodies that recognise antigens in extracellular spaces and tissue fluids. For the rest of this week, you will look at how lymphocytes and antibodies protect against virus infection, starting with the cytotoxic T lymphocytes.

1.1 Cytotoxic T cells T cells recognise antigens through their T cell receptor (TCR). To be precise, T cells recognise antigen fragments presented to them by cell-surface molecules encoded by the major histocompatibility complex (MHC molecules), shown in Figure 1. There are in fact, two types of MHC molecule – class I and class 2. Here we will concentrate on MHC class I molecules. All cells of the body continuously sample their own internal proteins and present polypeptide fragments on the cell surface bound to MHC class I molecules. Internal molecules of the cell, which can be any intracellular molecule including a viral antigen (blue hexagon) are broken down into peptide fragments. These antigenic peptides are presented by MHC molecules on the cell surface, where they may be recognised by T cells that have an appropriate T cell receptor. (Note: in Figure 1 and later diagrams, the scale of the cells and the receptor molecules will differ.)

If the cell has become infected by a virus, then polypeptide fragments of viral proteins will also be presented by the MHC class I molecules. If a cytotoxic T cell recognises the antigen fragment+MHC molecule, then it can signal to the infected cell to induce apoptosis – programmed cell death. You will learn more about this later, but first you will look in a bit more detail at the MHC molecules.

1.2 MHC Molecules The MHC was originally identified because of its role in promoting rejection of foreign tissue grafts. However, this is not its true physiological function, which is presentation of a cell’s internal peptides for review by cytotoxic T cells. The MHC is a gene complex that encodes several different MHC class I and class 2 molecules. The genes are highly variable between different individuals – no two people have the same set of MHC genes (except identical twins). The antigenic peptides that are bound to MHC class I molecules are 8–10 amino-acid residues in length, and they are bound non-covalently in a cleft on the outer surface of the MHC molecule, as shown in Figure 2.

Exactly which peptides can bind to each MHC molecule depends on the amino-acid residues lining the antigen-binding cleft, which is different for each variant MHC molecule. Since everyone has different variants of the MHC molecules, the way that antigenic peptides are presented to T cells is different for each individual. Put simply, everyone’s immune system is genetically unique! In the next section, you will now look at where those antigenic peptides come from.

1.3 Antigen processing and presentation As previously noted, a cell samples its internal proteins and presents them on MHC class I molecules. The way it does this is called ‘antigen processing’ and is illustrated in Figure 3. Cells have an organelle called a proteasome, which breaks down cytosolic proteins into peptides that are transported into the endoplasmic reticulum by a transporter (TAP1/2), where they can associate with MHC class I molecules.

What determines whether a peptide will be able to bind to an MHC molecule? The amino acid residues lining the antigen-binding cleft on the MHC molecule interact with the amino acid residues in the peptide, so binding depends on both the variant of the MHC molecule and the sequence of the peptide. The antigenic peptides are trimmed to size by enzymes in the endoplasmic reticulum before the MHC molecule is transported to the plasma membrane in order to present the antigen.

1.4 Cytotoxicity When a cytotoxic T cell recognises a virus-infected cell, it can signal to it to induce apoptosis. Video 2 below shows a cytotoxic T cell engaging a target cell infected with the influenza virus. As you can see, a cytotoxic T cell (the smaller cell) binds to an influenza-infected target cell. The T cell has granules containing mediators that damage the membrane of the target cell and activate apoptosis. If the T cell recognises the target these granule associated mediators are released into the space between the cells. Apoptosis is seen as membrane blebbing (blobs) and condensation of the nucleus. Video 2 Apoptosis

The mechanisms by which the T cell induces apoptosis are beyond the scope of this course, but can be summarised as follows: Release of a molecule, perforin, which punches holes in the plasma membrane of the target cell. Release of granule-associated enzymes, which enter the target cell through the holes created, and activate the cell’s endogenous systems, for inducing apoptosis. Release of cytokines that bind to receptors on the target cell, that activate different endogenous pathways to apoptosis. As you can see, the T cell does not exactly kill the infected target cell, but causes it to die by suicide.

1.5 Natural Killer cells Many viruses have developed adaptations that help them to evade immune responses. A typical strategy is to prevent expression of MHC class I molecules so that viral antigens are not presented effectively to the cytotoxic T cells. However, the immune system has an alternative system for tackling this strategy – these are the Natural Killer cells, or NK cells. NK cells are a group of lymphocytes that normally recognise MHC molecules on cells of the body, via ‘killer-inhibitory receptors’ (KIR). If the NK cell recognises MHC molecules on a cell of the body, it leaves it alone. However, if a cell has lost its MHC molecules, perhaps because it has been infected by a virus trying to escape detection, then the NK cell is no longer inhibited, and it delivers a cytotoxic signal to the target cell, as illustrated in Figure 4. The mechanisms used to induce apoptosis are similar to those deployed by cytotoxic T cells.

NK cells are normally present and active in the body and are involved in immune defence from the earliest phases of infection. They do not have the high specificity for viruses that the cytotoxic T cells have. Why are cytotoxic T cells (CTLs) highly specific in their recognition of virus-infected cells? Each CTL has a T cell receptor, which specifically recognises an antigenic peptide presented on a specific MHC class I molecule. The activity of NK cells improves slightly with repeated encounters with the same antigen, but not in the way that the activity of CTLs does. As such, NK cells are normally considered to be part of the innate immune system.

1.6 B cells and antibodies B cells recognise antigens through their cell surface receptor for antigen, which is in fact a membrane-bound form of antibody. It is called the B cell receptor (BCR), and like T cells, each individual B cell has just one specificity – meaning that it can recognise a very limited range of antigens. However, taken as a whole, the entire population of B cells can recognise an enormous range of antigens. What happens to a B cell if it becomes activated following contact with the antigen it recognises? It divides and differentiates into plasma cells, which secrete antibody. The BCR and the secreted antibody are structurally very similar, and antibodies derived from a single clone of B cells will all have the same antigen-binding specificity. The BCR is a cell surface antigen receptor and is associated with molecules that signal cell activation (Igα, Igβ). The secreted antibody lacks the transmembrane segment that anchors the BCR in the plasma membrane of the B cell. Figure 5 compares the cell surface BCR and secreted form of antibody.

Secreted antibodies are also called immunoglobulins (Ig) and they come in a number of different classes. Three examples are IgG, IgM and IgA, which you will learn about later in the course. But first, let’s see how B cells become activated to divide and differentiate into plasma cells.

1.7 T cell help for antibody production In order for B cells to become active, they normally require help from helper T cells – TH2 cells. Figure 6 shows how B cells interact with TH2 cells by presenting antigen on MHC molecules. Notice, however, that it is slightly different from the antigen presentation shown in Figures 1 and 3; the B cell uses MHC class 2 molecules to present antigenic peptides to the TH2 cell, whereas the cytotoxic T cells are presented with antigenic peptides on MHC class I cells.

In effect, the antigen is recognised twice: once by the BCR and then by the TCR on the TH2 cell. Both cells must recognise the antigen before the B cell receives an activation signal. The activation signal consists of a combination of direct cell-cell signals and cytokines released by the T cell, which promote B cell division and differentiation. This process is described in Video 3 below. Video 1 Immune defence DAVID MALE: In this video, I will outline how T cells and B cells cooperate in antibody production. Here we have a population of mature B cells expressing surface antibody and T cells with T cell antigen receptors on their surface. At any one time in a population of lymphocytes, there will be millions of different clones of cells each carrying a different receptor, and therefore, only able to recognize a particular type of antigen. This means that in a normal immune system, there are always a small population of cells, which are able to recognize any specific antigen. If this were not the case, we would be completely unable to protect ourselves from pathogens. By circulating around the body, lymphocytes have the opportunity of encountering the antigen that they can actually recognise. One consequence of this is that most lymphocytes are never called upon to fight infection. They are simply there just in case they are needed to combat a new strain of pathogen that might enter the host. We’re going to look at how B cells and T cells differentiate and the mechanisms which bring this about. Here we have a virgin lymphocyte pool. Only one of these many different B cells is able to bind to that antigen successfully. This process known as clonal selection initiates a primary immune response, which causes the cell to undergo a series of irreversible changes. The antigen is taken up by binding to the cell’s surface immunoglobulin antigen receptor. It is internalised and processed. Antigen fragments are subsequently re expressed on the B cell surface associated with MHC class II molecules for presentation to Th2 cells. Receptors for cytokines including interleukin 4 are also induced on the B cell. If a Th2 cell recognises the antigenic peptide on the MHC molecule, it releases cytokines, which together with co stimulatory signals activate the B cell and trigger its division. Other cytokines released from the Th2 cell bind to the appropriate receptors on the B cell causing it to differentiate. Some of the B cells become plasma cells, which produce a secreted form of the original antibody. Consequently, they are also known as AFCs or antibody forming cells. Plasma cells are a different shape from B cells, and they have lost all their original cell surface antibody. They will go on dividing several times but within days or weeks, they will die. Other cells become memory cells. There are now more of them, and they have undergone a series of changes making them more efficient at reacting to the same antigen if they ever encounter it again. This means that there is always a supply of cells with the appropriate specificity to fight infection. Memory cells can live for many years, and their increased efficiency at fighting infection underlies the enhanced secondary immune response. Memory cells confer lasting immunity against a pathogen, which is the basic principle behind vaccination.

2 Immunological memory An important feature of the adaptive immune response is immunological memory – on subsequent encounters with the same antigen, the immune response is faster and more effective. This effect can be seen in the primary and secondary antibody responses against an antigen, as shown in Figure 7. Note: titre is a measure of how much antibody is present in the serum, and in this case it is shown on a logarithmic scale.

Notice four key differences between the primary and secondary antibody response. On the secondary response: The lag time before the appearance of antibodies is shorter. The peak titre is much higher. IgG antibodies predominate; these antibodies also bind more strongly to the antigen. The high levels of IgG antibodies are maintained for longer. There are three main explanations for the improved secondary immune response: The number of lymphocytes that can respond to the antigen increases by clonal division during the primary response. Some of the responding lymphocytes have undergone differentiation and maturation steps, which means they can react more swiftly and/or to lower levels of antigen stimulation; these cells act as ‘memory cells’. Memory cells are seeded into lymphoid organs and mucosal tissues around the body, so they are on-site to respond as soon as the antigen or pathogen is encountered again. These findings provide one of the rationales for vaccination. If a person has been vaccinated with a harmless variant of an antigen or pathogen and made a primary immune response against it, they are then able to mount a secondary immune response if they subsequently encounter the real pathogen.

2.1 Phases of the immune response So far, we have described several different types of immune responses that recognise free virus or virus-infected cells, but how do these responses relate to each other? Figure 8 shows when these reactions are most important during a response to an acute virus infection.

Interferon and NK cells are active during the earliest phases of infection. Adaptive immune responses mediated by lymphocytes develop a few days after infection and are involved in clearing infected cells. Antibody is released by plasma cells derived from differentiated B cells and persists for many months, although it gradually declines. Long-term adaptive immunity is primarily provided by the memory cells, which may be either B cells or T cells. One point you might have missed is that the number of lymphocytes initially available which can recognise a novel infection is relatively small. It takes several days for them to divide repeatedly in order to produce enough lymphocytes of the correct specificity to combat the novel infection. During this early phase of infection, interferons and NK cells are particularly important in slowing virus spread.

2.2 Overview of adaptive immune responses In this next section, you will watch a video that summarises how viruses infect cells and the variety of immune responses that combat them. Video 4 Virus infection and immune responses DAVID MALE: Viruses are unable to multiply on their own. They depend on the body’s own cells. To multiply, a virus must first enter the body. One of the ways it does this is through the mucous membranes of the gut or respiratory system. They are not able to infect cells indiscriminately, because they can only attach to cells expressing particular molecules on their surface. When a virus encounters such a cell, it binds using a complementary surface receptor protein. The virus is now able to penetrate the cell. The capsid, the part of the virus containing the foreign genetic material, enters the cytoplasm. There it breaks down, releasing its nucleic acid into the cell. Viruses have genomes which can be either RNA or DNA in single or double stranded forms. Many viruses replicate in the cytoplasm where their biomolecules are synthesised. These include more viral nucleic acid, nucleocapsid proteins and glycoproteins for the viral envelope if there is one. Some viruses incorporate their DNA into the nucleus. The components of the virus assemble in the cytoplasm and at the cell membrane. Viral envelope proteins protrude through the plasma membrane, and within the cytoplasm viral nucleic acids associate with capsid proteins to form a new nucleocapsid. The plasma membrane buds off to enclose this new nucleocapsid, causing a new virus to be released into the body. Many virus particles are produced simultaneously in each cell. Each released virus is potentially capable of infecting another cell. Neighbouring cells in the body are likely to be of the same type, and since the virus targets cells expressing specific host molecules on their surface, propagation of the infection is rapid. The immune system has several defence mechanisms against viral infection. Here, a virus binds to its target cell. As we saw, it fuses with the host cell, and releases its genetic material into the cytoplasm. When the foreign genetic material enters the cell and starts to synthesise viral products, the cell is stimulated to make type 1 interferons. These interferons can signal to neighbouring cells to directly inhibit viral synthesis. They do this by binding to interferon receptors which signal the production of antiviral proteins. The antiviral proteins can switch the cell into a virus resistant state if it should later become infected. If a virus now infects that cell, the anti-viral proteins become activated and block viral replication by breaking down the cell’s mRNA and stopping protein synthesis. The anti-viral proteins put the cell into stasis, which limits further replication of the virus and spread of infection. This holding action gives the immune system time to mobilise the T cell response. One consequence of interferon signalling is an increased production of major histocompatibility complex molecules, particularly class I molecules, which are synthesised within the endoplasmic reticulum, where they bind to peptide fragments of proteins synthesised in that cell. All of the peptides that have bound to MHC class I molecules are carried to the cell surface, including any viral peptides. Each T cell has an antigen receptor that can potentially recognise an antigenic peptide presented on an MHC class I molecule. Once attached there may be a direct inter-action between the molecules on the T cell and the infected target cell. An indirect signal may also be sent using, cytokines, such as tumour necrosis factor or lymphotoxin. In either case, this triggers the release of agents within the cell which disrupt nuclear DNA, and cause cell death by apoptosis. The cytotoxic T cell is now free to seek other similarly infected cells. Some viruses may attempt to evade immune responses by down-regulating the production of MHC class I molecules. But a cell which lacks MHC class I becomes susceptible to killing by natural killer cells. The body’s other defence mechanism is carried out by free immunoglobulin. Both infected cells and free virus present viral proteins on their surface. Antibodies can bind to viral proteins expressed on the surface of infected host cells. In this case they signal to components of the complement system, which attack the cell membrane and damage the cell by membrane attack complexes. Neutralising antibodies on encountering a virus, can also bind to its surface receptor for the host cells, making it unable to infect them.

3 Antibodies Clearly antibodies are important in protection against virus infection, but how exactly do they work? Before delving into that area, you will take a look at antibody classes because different antibody classes have different functions in immune defence. Recall the names of three different antibody classes and identify a distinguishing feature of one of these classes of antibody. IgG, IgM and IgA are the three main classes of antibody found in serum – the liquid component of blood. IgM is the first antibody produced in a primary immune response, whereas IgG is the major antibody produced in a secondary immune response (see Figure 7). An individual B cell initially produces an IgM antibody as its BCR. And if it is activated, it produces secreted IgM. As an immune response develops, and with help from TH2 cells, the B cell may switch to producing an IgG or IgA class of antibody. The antibody retains the same antigen-binding sites at the tips of the Y-shaped molecule, but the stem of the Y (Fc portion) is different for each class. How class-switching is effected at a molecular and cellular level is beyond the scope of this course. The key point is that IgM predominates in a primary response while IgG and IgA antibodies predominate in secondary immune responses.

3.1 Antibody classes Secreted IgG is produced as a monomer of the basic antibody structure (2 heavy chains, 2 light chains) but IgM is a pentamer and IgA is a dimer, as illustrated in Figure 9 and 10 below.

In Figure 9, the four polypeptide chains of an IgG molecule are folded into domains. The variable (V) domains of the heavy and light chains (VH, VL) form the antigen-binding sites. The remaining domains are relatively constant (C domains). The hinge confers segmental flexibility.

In Figure 10, each molecule is joined by an extra joining (J) chain. When IgA is secreted, it has an additional chain, the secretory component. (Note: carbohydrate units are shown in blue and inter-chain disulphide bonds in red.) How many antigen-binding sites does IgM have? Ten The multiple antigen-binding sites of IgM make it very efficient at cross-linking antigens and therefore useful as the first antibody produced in an immune response. However, the affinity of IgM for its target antigens is lower than the affinity of IgG and IgA antibodies produced later in an immune response. You will now look at how antibodies can protect against virus infection.

3.2 Protection of mucosal surfaces It was mentioned earlier that IgA antibodies can be produced as secreted molecules. In this case, the antibody is transferred across epithelial cells from the basal (tissue) side to the apical (exterior) side of the epithelium exposed at the mucosal surface. You can see the mechanism outlined in Figure 11.

Dimeric IgA produced by plasma cells binds to a poly-Ig receptor on the basal surface of an epithelial cell. It is transported across the cell in a vesicle and then released at the mucosal surface as secreted IgA. The secretory component of secreted IgA is derived from the poly-Ig receptor. IgA in mucosal secretions is particularly important in protecting the epithelium against infection with respiratory viruses, such as influenza, rhinoviruses and SARS-CoV2.

3.3 Anti-viral actions of antibodies Receptor blocking The simplest way that an antibody can interfere with virus replication is by blocking attachment to the host cell, as shown in Figure 12. Antibodies that do this are called ‘neutralising antibodies’.

What type of antibody would you expect to be most effective at blocking entry of SARS-CoV2 into a lung epithelial cell? Why? An IgA antibody that binds to the receptor binding domain (RBD) of the spike protein. An IgA antibody will be secreted onto the surfaces of the airways in the lung. An antibody that binds the RBD will be most effective at preventing attachment of the virus to the ACE2 receptor. Complement activation IgM and most IgG antibodies can activate the complement system. This is a group of proteins present in blood and tissue fluids that have many functions in controlling inflammation and damaging pathogens. Antibody bound to the virus surface or virus envelope activates the complement system causing deposition of complement molecules on the surface, which you saw earlier in Figure 12 above. This can have a number of anti-viral effects, including: It promotes uptake and breakdown of the virus by phagocytic cells, (eg macrophages). It directly damages the viral envelope. If an infected cell has virus molecules inserted in its plasma membrane, then antibodies can bind to these antigens and activate complement. Some complement components can punch holes in the target cell, thus killing it before replicating viruses can be assembled inside. In addition, complement system molecules can attract leukocytes to sites of infection, and enhance antigen presentation and antibody production. Promoting phagocytosis and NK cell activity Phagocytes, such as macrophages are important in destroying pathogens. They internalise them by a different pathway than a virus would normally take. Once inside the phagocyte, pathogens are broken down by a barrage of small toxic molecules and enzymes. Antibodies play an important role in this process by acting as adapters between the virus and the macrophage, as shown in Figure 13.

Antibody can also promote the activity of NK cells, allowing them to recognise viral antigens that have been inserted into the membrane of an infected cell, which you can see in Figure 14.

Activating intracellular defences Until recently, it was thought that antibodies could only recognise antigens in extracellular spaces, on the cell surface or in body fluids. It has now become apparent that antibodies can also combat many viruses inside cells. Moreover, this action can be effected by antibodies that are not conventional neutralising antibodies – for example, they may bind to internal proteins of the virus. Antibodies can enter the cell while bound to a virus, or may be taken up independently by pinocytosis, and they act in a variety of ways. Within an endosome antibodies can inhibit viral uncoating and the fusion mechanism which allows the viral genome to enter the cytosol of the infected cell. Within the cytosol, they can interfere with virus replication or assembly. In addition, some antiviral functions are mediated by a cytosolic receptor for antibody, TRIM21 (Tripartite Motif 21) which binds the Fc portion of IgG. TRIM21 binds to a virus that has any attached IgG antibody and tags the complex of antibody and viral protein to direct it to the proteasome, where the viral proteins are degraded and can then be presented on MHC class I molecules. Thus, TRIM21 enhances antigen presentation, as shown in Figure 15. This mechanism is most relevant for viruses that enter the cytoplasm intact – i.e., non-enveloped viruses. It is however possible for TRIM21 to target enveloped viruses that have entered the cytosol from the endosome and lost their envelope and external proteins. If antibodies against the internal viral proteins have independently entered the infected cell, then they can bind to the uncoated virus and engage TRIM21. The virus associated with an antibody in the cytoplasm as an immune complex is recognised by TRIM21 which ubiquitinates the complex. The addition of ubiquitin tags the components of the immune complex for rapid breakdown by the proteasome, so that viral peptides can be presented by MHC class I molecules.

4 Week 2 quiz Check what you have learned this week by taking the end-of-week quiz. Week 2 practice quiz Open the quiz in a new window or tab, then return to this week when you’re done. 5 Summary This week you learnt about the elements of the adaptive immune system that combat a virus infection. Cytotoxic T lymphocytes and NK cells act in a complementary fashion to detect virus-infected cells of the body. If they recognise an infected cell they signal to it to induce apoptosis, - programmed cell death. B cells can recognise viral antigens and, with help from TH2 cells, they divide and differentiate into plasma cells, which secrete antibody. Individual B cells switch from production of IgM to IgA or IgG. The different antibody classes have different functions. IgM is produced first in an immune response; IgG is the major antibody in the secondary immune response and IgA can be secreted across epithelial cells to protect mucous membranes. Antibodies protect against virus infection in a number of ways. Neutralising antibodies prevent virus from attaching to target cells. IgG and IgM antibodies can activate complement to damage enveloped virus or virus-infected cells. IgG can promote phagocytosis of virus by macrophages and allows NK cells to recognise infected cells. If they are internalised by an infected cell, IgG antibodies can interfere with virus replication and assembly, as well as promoting presentation of the viral antigens on MHC class 1 molecules, to cytotoxic T cells. Clearly, antibodies are very important in protection against virus infection. Indeed, the vaccines against COVID-19 were designed to induce high levels of neutralising antibodies against the SARS CoV2 spike protein. But how does one measure antibodies? That is what you will learn about next week, using a technique called ELISA, in a virtual laboratory. You should now go to Week 3. Week 3: ELISA – enzyme linked immunosorbent assay Introduction In the early days of the COVID-19 pandemic, a fair number of people thought that they had been infected with SARS-CoV2, but they had not had serious symptoms or typical symptoms of infection. But how could they know that it was COVID-19? Perhaps they had just had a regular cold. In the earliest months, qPCR and lateral flow tests for SARS-CoV2 were not widely available. By the time these tests were available, the virus would have long since gone from the body, and these tests would show a negative result. In fact, it was still possible to tell whether a person had come into contact with the virus because they would still have antibodies against it − antibodies last for many months. Audio 1 Introduction to Week 3 DAVID MALE: How can one tell if a person has been infected with a virus? An infection induces antibodies which persist for a lot longer than the virus itself. In effect, the infection leaves a signature of its presence – virus-specific antibodies. Anyone who has been infected, has antibodies against different components of the virus, and indeed they will be specific for the strain of virus that has infected them. Epidemiologists can use this information to track epidemics. A big problem with COVID-19 was that up to 40% of infected people were asymptomatic. But this only became apparent when random community testing for the virus or virus-specific antibodies was carried out. This week you will learn how to measure antibodies against SARS-CoV2 using a versatile assay called ELISA – an enzyme-linked immunosorbent assay. Much of your time will be spent in a virtual laboratory which will prepare you to do epidemiological and immunological studies in later weeks. By the end of this week you should be able to: outline the theoretical background and steps used in an ELISA carry out an ELISA in a virtual laboratory to detect antibodies to SARS-CoV2 spike protein report antibody titres derived from your assay interpret your results. 1 Measuring antibodies by ELISA ELISA is a very versatile assay used for measuring antibodies against pathogens in serum and body fluids. Variants of the assay are used to measure autoantibodies and cytokines. In this course, you will use a standard ELISA to measure antibodies against SARS-CoV2 in serum samples. ELISAs are usually carried out on 96-well plastic plates which have been ‘sensitised’ by binding an antigen to the surface of the wells. You can see the basic steps in an assay outlined in Figure 1.

The antigen bound to the plate determines the specificity of the ELISA test; e.g. SARS-CoV2 spike protein on the plate detects antibodies that are directed to spike protein. The test sera are applied to the plate in serial dilutions as explained in a video guide that you will watch later. A variety of ligands can be used to detect the bound antibodies. The two most often used are a second antibody which binds the first antibody or an antibody-binding protein (protein-G). A variety of enzymes may be coupled to the ligand; the two most often used are horse-radish peroxidase (HPO) or alkaline phosphatase. The chromogen must be matched appropriately to the enzyme. In these experiments, any antibodies that have bound will be detected with a second antibody coupled to horse-radish peroxidase (HPO), and the chromogen is a substance called tetra-methylbenzidine (TMB), which generates a blue end-product. The enzyme also requires hydrogen peroxide as a substrate. An example of a developed plate is shown in Figure 2. Do not be concerned if you do not understand the results shown in this figure − how to interpret the results will be explained later.

1.2 ELISA – experimental conditions As mentioned previously, you will be using one set of standard conditions in the ELISA laboratory to measure IgG antibodies against SARS-CoV2. To do this, you will watch the video guide again, but this time you will note down the conditions used. You will use these variable(s)/condition(s) when you use the laboratory later this week. Now go on to the next activity in which you will identify the assay conditions used in the protocol shown in Video 1 for the ELISA: Epidemiology laboratory. Activity 2 ELISA: Experimental conditions Allow 30 minutes Look at the table below For each action(s) in each step, note down the condition or variable in the blank cells in the table below. Table 1 ELISA protocol

Step	Action	Variable(s) /condition(s)
1. Serum sample	Select eight samples, one for each row of the plate
2. Dilution plate	Select transfer volume for serial dilution	One answer needed here
3. Primary antibody incubation	Select ELISA platePrimary incubation timeNumber of washesDuration of each wash	Four answers needed here
4. Secondary antibody preparation	Select secondary antibodyChoose volume of stock reagent	Two answers needed here
5. Second antibody incubation	Secondary antibody incubation timeNumber of washesDuration of each wash	Three answers needed here
6. Chromogen	Select the chromogenChromogen incubation time	Two answers needed here
7. ELISA plate reader	Wavelength of filter on plate reader	One answer needed here

When you have filled in the table, click on ‘Reveal answer’ to check your conditions are correct. These are the conditions you will use in your own assays. Note down these conditions ready for the next set of activities. The correct conditions are: Table 1 ELISA protocol (completed)

Step	Action	Variable(s) /condition(s)
1. Serum sample	Select eight samples, one for each row of the plate
2. Dilution plate	Select transfer volume for serial dilution	100 µl
3. Primary antibody incubation	Select ELISA platePrimary incubation timeNumber of washesDuration of each wash	Spike protein45 minutes35 minutes
4. Secondary antibody preparation	Select secondary antibodyChoose volume of stock reagent	Anti-human IgG, HPO conjugate4 µl
5. Second antibody incubation	Secondary antibody incubation timeNumber of washesDuration of each wash	45 minutes35 minutes
6. Chromogen	Select the chromogenChromogen incubation time	TMB20 minutes
7. ELISA plate reader	Wavelength of filter on plate reader	450 nm

If you had chosen ABTS as the chromogen, then the appropriate filter is 450nm. If the samples have very high levels of antibody, what adjustment would you make, so that the results will still be in range of the assay? Reduce the volume on the dilution plate transfer – eg 50µl transferred will give a 1:3 dilution series. If you use a higher concentration of secondary antibody than the recommended amount (1µg/ml), what effect will that have on the end result? The background values will be high. If you had used O-phenylene diamine (OPD) as the chromogen, what filter should you use on the plate reader? The 645nm filter.

1.3 Measuring antibodies to SARS-CoV2 spike protein Now it is your turn to try an assay in the ELISA: epidemiology laboratory. To be sure that you are carrying out your assays correctly, you have to directly reproduce the assay that is shown in Video 1 . Activity 3 Antibodies to SARS CoV2 spike protein Allow 30 minutes This time open the ‘ELISA experiment’. Then run an assay to detect IgG antibodies against SARS-CoV2 spike protein in the following eight samples, using the experimental conditions you noted down earlier in Activity 2: 1-Standard 2-Neg.Con N9921 C4443 C5050 H1151 F1949 Z8207 When you have completed the assay, you should have a plate that looks like Figure 3. (If you have arranged the samples in a different order on the plate, then the appearance will be different, but the results should be the same.) The results from the plate reader will be similar to Figure 4. However, note that the titres will depend on the dilution series chosen, and the identification of positive titres, (highlighted here) depend on the exact experimental conditions and the cut-off value selected.

2 Interpreting and reporting results The results from this type of ELISA are usually reported as the reciprocal of the highest dilution that shows a positive result. For example, if the highest dilution giving a positive result is 1:64, then the titre would be reported as 64. This has the advantage that a larger number indicates a greater amount of antibody in the serum. It should be emphasized that the type of ELISA that has been taught here is not highly accurate. If your results are out by one well, then you would report a value that is different by a factor of two from the stated value. The advantage of this assay is not in its accuracy, but in its speed and simplicity. It gives a rapid estimation of whether antibodies to the test antigen are present in significant amounts. As such it is very good for screening large numbers of samples in epidemiological studies. The results of the ELISA you carried out, show that four individuals (N9921, C5050, H1151, Z8207) had significant levels of antibodies to spike protein. (The low titre in C4443 is more likely due to a high background value in the assay rather than evidence of contact with SARS-CoV2 spike protein.) Finally, it is important to emphasize that the four positive samples do not necessarily imply that those four individuals have been infected with SARS-CoV2. Vaccination against COVID-19 also induces antibodies to spike protein, so we can say that these individuals have been vaccinated or infected, or possibly both. 3 Week 3 quiz Check what you have learned this week by taking the end-of-week quiz. Week 3 practice quiz. Open the quiz in a new window or tab, then return to this week when you’re done. 4 Summary This week you have learnt the ELISA technique for detecting antibodies, and shown how to use it to quantitate antibodies to SARS-CoV2 spike protein. You then had the opportunity to practice this assay in a virtual laboratory, and a simple method for recording antibody titres and interpreting the results was used. You may also have learnt something about technical aspects of the ELISA, and some basic laboratory science, such as making dilutions. Next week, you will use the technical knowledge gained here to carry out an epidemiological study and an immunological study using serum samples in the ELISA: epidemiology virtual laboratory. You should now go to Week 4. Week 4: Screening for SARS-CoV2 antibodies Introduction Knowing the proportion of people in a population that are immune to an infectious disease is really important – but why? One reason is that it can tell you something about how quickly an infection will spread or whether it will die out. Once a sufficient proportion of the population is immune to an infectious agent, then it will die out, or at least that strain of the pathogen will disappear, because there are insufficient susceptible individuals to maintain a chain of disease transmission. You are going to look into the relationship between transmission rates, herd immunity and vaccination rates later in the course. But before then you are going to investigate the incidence of antibodies to SARS-CoV2 in the sample set provided in the ELISA: Epidemiology laboratory. This will provide the basis for later investigations. Audio 1 Introduction to Week 4 DAVID MALE: Measuring antibodies against a pathogen, is one way of estimating the level of immunity in a population. It can also tell you how many people have been infected. This data is really important for epidemiological studies and designing vaccination programmes. Lateral flow tests for antibodies are convenient as they can be done at home but they only detect whether antibodies to a pathogen are present or not. In comparison, ELISA done in a laboratory provides quantitative data on antibodies of different classes. The aim of the investigation this week is to estimate the level of immunity to COVID-19 in different groups in August 2021. Do women make more antibodies than men? Do young people make stronger antibody responses than older people – here is your chance to find out. By the end of this week, you should be able to: outline the course of the COVID-19 epidemic and vaccination programme in the UK organise and carry out a laboratory-based investigation for antibodies against SARS-CoV2 record your data and carry out some simple data analysis. 1 Background to the investigation In Week 3, you were introduced to the sample set provided by the ELISA: epidemiology laboratory. The set of 60 serum samples were chosen to allow investigation of the level of immunity to SARS-CoV2 in the UK population in August 2021. This time point was chosen because it is particularly interesting and instructive. At this time a majority of the UK adult population had received at least one dose of the COVID-19 vaccine and a significant minority had been infected with SARS-CoV2. Both of these groups would be expected to have antibodies to the spike protein. The aim of the investigation is to determine what proportion of the population has antibodies to the spike protein. But first you will learn a little more about the COVID-19 epidemic in the UK to place your investigation in to context.

1.1 The COVID-19 epidemic in the UK By August 2021, the original strain of SARS-CoV2 first identified in Wuhan, China had been replaced in circulation by a number of ‘variants of concern’ (VOC), designated by the World Health Organisation (WHO) as variants with one or more of the following characteristics: increased transmissibility more severe disease reduced effectiveness of treatments or vaccines failure to be detected by current diagnostic tests. The new variants were designated by Greek letters (Figure 1), and as of August 2021, the delta variant accounted for nearly 99% of the cases in the UK.

At this time point the incidence of infection, which had been low during the spring and early summer was starting to rise in the community, as demonstrated in Figure 2. If you want to learn more about the course of the pandemic in the UK, all data during this period is available from Public Health England, and since December 2021, from the Health Security Agency.

1.2 COVID-19 vaccination in the UK You will be covering vaccines later in the course, but at this stage it will be helpful for you to see what was happening with the vaccination programme in the UK at the time of the investigation. A vaccination programme started in the UK in January 2021 with two doses of vaccine, typically spaced at an interval of 2−3 months. The programme started with medically vulnerable individuals and older people and then worked progressively down through the age groups to younger people. By the Summer of 2021, all of the adult population over the age of 18 had been offered vaccination. Similar programmes were taking place in many European countries during the early months of 2021, although the UK then delayed immunisation of younger age groups (<18 years) until later in the year. This profile of vaccination can be seen in the heat map (Figure 3).

Describe the incidence of vaccination in the UK population in August 2021, in relation to different age groups. Nearly all of the older population (age >70) had been vaccinated. The proportion falls successively with different age groups to ~50% in adults aged 18−-29. School age children and young adults (<18) had low levels of vaccination (<20%).

1.3 Screening programmes During the COVID-19 pandemic, two main tests were used to detect the presence of the virus: PCR test, which detects segments of the virus genome lateral flow test, which detects the presence of virus antigens. Both of these tests can detect the virus during a window of time when a person has recently been infected and is potentially infectious – typically this window lasts for about two weeks. In the UK the PCR tests became available early in 2020 and by May 2020 approximately 100,000 tests were being carried out daily. Lateral flow tests became available in late 2021. Antibody testing can also be carried out in different ways, including: ELISA lateral flow tests, configured to detect anti-viral antibodies. Antibodies can be detected from 1−2 weeks after infection, depending on the sensitivity of the test. This means that there is only a very short period of time when tests for virus and tests for antibodies will both give a positive result. An important difference between the tests is that the lateral flow tests could be carried out at home, and they give a positive/negative result; the PCR test (virus) and ELISA (antibodies) are carried out by trained staff, usually in a laboratory, and they provide quantitative results. It is important to note that testing for virus or anti-viral antibodies is carried out on different groups of people. The most reliable type of screening for assessing the prevalence of disease (virus) or immunity (antibodies) is carried out by random testing in the community. For notifiable diseases such as measles, data from general practice and hospitals may give a reliable measure of disease incidence, because virtually all affected individuals have symptoms and will be identified. COVID-19 was a notifiable disease. However there was initially a huge amount of uncertainty as to what proportion of individuals were asymptomatic, and therefore might not have come forward for testing. The advantage of random screening in the community is that it will identify people who are infected but who have no symptoms and do not know they are infected (virus +), or who had been infected in the past (antibody +). The samples available in the ELISA: epidemiology laboratory represent a random screen from across the UK taken in August 2021. For the remainder of this week, you will be analysing these samples for the incidence of antibodies to the SARS-CoV2 spike protein, which are referred to as S-antibodies.

2 Prevalence of antibodies to SARS-CoV2 In this section you will use the virtual ELISA: epidemiology laboratory to measure the prevalence and titre of antibodies to SARS-CoV2 spike protein. The laboratory includes serum samples from 60 individuals as well as the standard and negative control samples. You will also use the data you obtain to carry out some simple immunological investigations. For this purpose you are given some additional information on the age and sex of the individuals in Table 1, which can be downloaded here.

2.1 Laboratory investigation During this course, you will be carrying out a number of investigations on the samples in the ELISA: epidemiology laboratory, so you need to record your results as you proceed. You may wish to download and print a version of Table 2 to enter results or you can note your data in the blank boxes provided below. If you prefer you can also recreate the table in your notebook. In Table 2 below, there is space for 30 entries and the six results from the first ELISA (Week 3) have already been entered. You will analyse at least 24 more samples. Table 2 Data entry table for the investigation of antibodies against SARS-CoV2

Subject	Age	Sex	IgG S-antibody	IgG N-antibody
N9921	32	F	256
C4443	30	F	2
C5050	71	M	128
H1151	26	M	128
F1949	32	M	<2
Z8207	58	M	2048

Activity 1 Selection of serum samples Allow 20 minutes Begin by selecting 24 individuals from the list given in Table 1. You should choose subjects with a range of different ages – for example, at least ten subjects older than 50 years and at least ten subjects aged less than or equal to 50 years. You should also aim to select approximately equal numbers of female and male subjects. Now enter the identifier of the subjects chosen into column 1 of the data entry table and their age and sex into columns 2 and 3 respectively. Activity 2 Measurement of IgG S-antibodies by ELISA Allow 60 minutes Now that you have selected your samples, go to the ELISA: epidemiology laboratory and carry out assays to determine the titre of IgG antibodies against spike protein in each of your chosen samples. To do this you should use the assay protocol which you used and noted down in Week 3, Activity 2. If you are unsure how to carry out the investigation, you may wish to rewatch the relevant parts of the video guide provided again below. Video 1 ELISA laboratory video guide DAVID MALE Hello. I’m David Male from The Open University. In this video, I will show you how you can detect antibodies against the coronavirus SARS-CoV-2 using a technique called ELISA, an Enzyme-Linked Immunosorbent Assay. This technique is often used to quantitate antibodies in diagnostic laboratories or for research purposes. We have recreated this technique in a virtual laboratory so that you can carry out the assay for yourself and test a set of serum samples from August 2021 for the presence of antibodies against the virus. At that time, the majority of the UK adult population had received a vaccination against COVID-19 and a significant number had been infected and recovered from an infection with the virus. This diagram illustrates the key steps in an ELISA. We start with a 96-well polystyrene plate that has been sensitized with antigen. In this case, the antigen will be a component of the SARS-CoV-2 virus, either the external spike protein or an internal antigen of the virus called the nucleocapsid. In the first stage, diluted serum is applied to the wells on the plate. If there is specific antibody in the serum, it will bind to the antigen on the plate. The bound antibody is called the primary antibody. Any unbound proteins in the serum, including nonspecific antibodies, are then removed in a wash step. In the second stage, a ligand is applied to the plate, which binds specifically to the primary antibody. The ligand also has a coupled enzymatic portion, which is crucial for the following step. In some cases, the ligand itself may be an antibody that binds to the primary antibody. In this case, it would be called a secondary antibody. The plate is then washed again to remove any unbound ligand. Finally, a chromogen is put into the wells. The chromogen is a colorless chemical which generates a colored end product when acted upon by the enzyme of the ligand. The colored end product is detected on a plate reader. The more primary antibody is present, the more enzyme is bound to the plate and the more colored end product is produced. Hence, this technique can quantitate how much antibody is present in the initial serum sample. Now let’s take a look at how the virtual ELISA laboratory recreates the technique. The virtual laboratory allows many options in choosing different serum samples and the dilution series of these samples. You can also choose the time of the incubations with the antibodies and the number and duration of the wash steps. You can select from three different ligands, and there is also a choice of chromogens for the development step. Finally, the results are read on a plate reader. And it is important to select the correct wavelength filter. The results correspond with what you would see in a real laboratory as these experimental conditions are varied. Moreover, if you happen to choose conditions that aren’t quite right, then your results may not be optimal and perhaps even provide no usable results at all. So it’s important that you note exactly what you do at each of the steps. It is also important to use positive and negative controls in each assay. Once you have your experiments working well, I would encourage you to modify some of the conditions and see how this affects the results. This way, you will also learn a lot about technical aspects of the assay. The exact appearance and layout of the experiments within the onscreen experiment will depend on the web browser you are using and the screen size. In this introduction, I’m using a desktop computer and Chrome as the browser. Before we start, a quick word about timing. In the virtual laboratory, we have telescope time. So an incubation that would normally take 60 minutes can be done in one minute. The incubations are started and stopped on a timer like this. It means that an assay which would normally take two to three hours can be done in a few minutes. As mentioned, ELISA is normally done using 96-well polystyrene plates that have been sensitized with a specific antigen. They are relatively cheap, used once, and then discarded. If your ELISA experiment goes wrong, you can come back to the beginning and start again. Now let’s take a look at the laboratory. The first step is to choose serum samples. These are samples taken from individuals that may or may not contain the antibodies that you are going to test for. You can see eight tubes containing separated blood samples with serum at the top of the tube and red blood cells at the bottom. Each tube represents a sample from one patient. Up to eight different serum samples can be selected, one for each row of the ELISA plate. Select a box beneath one of the sample tubes and start to type in the identity of the sample you want to select. The program will present you with the options available, and you can then click to select them. Each sample you choose will eventually be assigned to one of the rows on the 96-well plate, which you will see in the next step. In this case, I’m going to use a standard as the first sample in row A. It is a positive control. And I choose a negative control as the second sample, which will go on row B of the plate. Samples from patients are normally coded so that laboratory staff cannot identify an individual by name and there can be no mistake about the exact identity of the sample. I have now selected six samples from the 60 available for the remaining rows of the plate. Step 2 shows a 96-well plate. You will see the identities of the samples chosen in step 1 are now listed beside each row on the plate. The task in this step is to perform a serial dilution of the serum samples. Put simply, a serial dilution is a successive dilution of a starting sample. You will see why this is important as you progress through the experiment. The serial dilution is performed by adding a known volume of serum sample to the leftmost well on the plate and mixing it with a known volume of diluent. A diluent is simply a liquid medium that is compatible with the sample, which we can use to dilute the sample. Every well on the plate contains 100 microliters of diluent. You need to take note of this volume so that you can work out what your serial dilution will be. To make a serial dilution, a volume of each of the serum samples chosen in step 1 is added to the left-most well on the plate, well 1. It is mixed with a diluent, and then a defined volume is transferred to well 2 and mixed. The same amount is transferred from well 2 to well 3, then from well 2 to well 4, and so on down the plate to well 12. This procedure is carried out with the multichannel pipette illustrated. By transferring a volume of the sample from well to well and mixing it with fresh diluent each time, each sample is successively diluted along the plate. In the virtual laboratory, you need to decide what volume you want to transfer from well to well by changing the setting on the pipette. In this case, I’m going to transfer 100 microliter volumes down the plate. Since there is 100 microliters of the diluent in each of the wells, transferring 100 microliters will produce a serial dilution where the concentration of the sample decreases by a half each time. In this case, well 1 is a one-in-two dilution of the serum sample. This is because 100 microliters of the serum sample was added to 100 microliters of the diluent. Well 2 would be a one-in-four dilution and so on. To do this, I will set 100 microliters on the pipette and press Transfer, which carries out the entire dilution series for me. You can now see the dilutions are given along the top of the plate. The pipette volume can be set at anything between 20 microliters and 100 microliters. By choosing other volumes on the multichannel pipette, you can make other serial dilutions, such as one where the concentration of the sample decreases by a third each time, giving one in three, one in nine, and one in 27 dilutions, and so on. It is up to you to choose what you think is an appropriate serial dilution for the ELISA. The doubling dilution series shown here is a good starting point. But if serum samples have very high titers of the antibody under investigation, then a higher dilution series may be required. In an ELISA, the antibody that binds to the antigen on the plate is called the primary antibody. In this step, I first have to choose the antigen-sensitized 96-well ELISA plate that I will use for the assay. The dropdown menu lists all the available options. Here, I have two options, and I want to detect antibodies to spike protein of the virus. When I choose the spike protein ELISA plate, I’m essentially getting a plate where an equal amount of the antigen is bound to every well in the plate. If there are relevant antibodies in the serum samples, then they will attach to the antigen on the base of the wells. Anyone who has been vaccinated or infected with SARS-CoV-2 is likely to have antibodies against the spike protein. But only individuals who have been infected are exposed to the internal proteins of the virus and will, therefore, usually also have antibodies against the nucleocapsid. At this stage, we have two 96-well plates. One is our antigen-sensitized plate, and the other contains the serial dilutions of our serum samples. We now transfer the contents of the serial dilution plate to the antigen-sensitized plate. This brings the antigen and any antibodies together so that they can interact. By pressing the Transfer button, diluted samples are transferred across from the serial dilution 96-well plate prepared in step 2. Importantly, the contents of each well in the serial dilution 96-well plate is moved into the corresponding position on the antigen-sensitized ELISA plate. You will notice that when you press Transfer, the wells all turn blue to indicate that liquid has been transferred. Now we incubate the samples on the antigen-sensitized plate by starting the clock. An incubation time of 45 minutes to one hour is recommended. I will stop the incubation now. If you incubate for a short time, the antibody has less time to bind to the antigen, and the signal at the end of the assay will be lower. If you incubate for a bit longer than one hour, it will not make much difference. The incubation allows the antigen and any antibodies in the samples to interact. This is such a strong interaction that we can wash the plate quite vigorously to remove any unbound antibodies and other serum proteins. In essence, washing the plates simply means removing all the liquid from each well and adding a volume of fresh washing buffer. You need to choose how many wash steps to do and for how long you leave the wash liquid to incubate during each wash. Click on the Wash Start button to begin a wash and press Wash Stop to end a wash. Three washes of five minutes each are the minimum recommended. If you do not wash the plate sufficiently, residual unbound antibodies in the serum samples will neutralize the detection antibody which will be added later. In step 3, the primary antibody in the serum sample, if indeed it is present, became bound to the antigen. The next step is to detect any bound antibody. This is done by using an enzyme-conjugated ligand, usually another antibody that we add to all of the wells in the 96-well plate. This detection antibody is called the secondary antibody because it binds to the primary antibody. The reason why we use a secondary antibody to detect the presence of the primary antibody will become clearer as you progress. In step 4, you are presented with three vials that contain secondary antibodies. There are three major classes of antibodies present in serum-- immunoglobulin M, immunoglobulin G, and immunoglobulin A, which are referred to as IgM, IgG, and IgA. Each of the secondary antibodies specifically detects one of the classes of serum antibodies. In this demonstration, I want to detect IgG antibodies. So I select the vial anti-human IgG, which is conjugated to the enzyme Horseradish Peroxidase, or HPO. You should also notice that the concentration of the secondary antibody is shown on the label. The stock of the anti-human IgG HPO conjugate has a concentration of 1.5 milligrams per mil. This is to concentrate it to use neat, so I need to prepare a dilution. The recommended concentration for the anti-IgG antibody in this assay is 0.6 micrograms per mil. And I need 10 mls of the solution in total to add to the 96-well plate. I, therefore, need to add 4 microliters of the anti-IgG stock solution to the 10 mls of diluent. The way to calculate the required volume of stock is shown in the panel. The volume of stock is the final concentration divided by the stock concentration multiplied by the final volume. That is 0.6 micrograms per ml divided by 1,500 micrograms per ml multiplied by 10,000 microliters. If you did not follow that, just take it on trust that you need four microliters of the stock anti-human IgG. The optimum concentration of secondary antibody depends on the reagent and batch. And in a real lab, each new batch of antibody would be tested. Typical optimum concentrations will be in the range 0.5 micrograms per ml to 5 micrograms per ml. The pipette volume can be set at anything between 2 microliters and 100 microliters. If you do not use enough of the secondary detection antibody, the signal at the end of the assay will be low. If you use too much, the background values will be high. And in any case, that would be wasteful of an expensive reagent. In step 5, the secondary detection antibody is added to every well on the plate. As before, this antibody is incubated on the plate for 45 to 60 minutes. This incubation allows the secondary antibody to bind to the primary antibody. If the incubation is too short, the signal at the end of the assay will be reduced. After the incubation, the unbound secondary antibody must be removed from the plate with washes. As before, a minimum of three five-minute washes is recommended. If you add an extra wash or make the washes slightly longer, it will make little difference. But you will find that three washes of five minutes are more efficient than, say, one wash of 15 minutes. In this step, we’re going to use the enzyme activity of the horseradish peroxidase linked to the secondary antibody to develop a colored product in each of the wells. To do this, I will add a chromogen solution to every well on the plate. In this case, there are three options. I will choose TMB, which is tetramethylbenzidine. It produces a blue-colored end product when acted on by horseradish peroxidase. Once the reaction has started, the color develops progressively with time. At an appropriate time of your choosing, the activity of the horseradish peroxidase enzyme should be stopped by adding sulfuric acid. You should allow enough time for the color to develop so that you can see visible differences between successive wells. If you run the reaction for too long, eventually, every well will develop color. Background values will increase. An incubation time of 5 to 30 minutes is typically used for these assays. In this case, I will stop the reaction after 20 minutes by the addition of sulfuric acid. You will notice that the sulfuric acid not only stops the activity of the enzyme but also turns the blue end product of the enzyme reaction bright yellow. Finally, we get to see the results of the assay by using a 96-well plate reader that can detect the concentration of the yellow-colored end product in each of the wells. Plate readers shine light at the 96-well plate and record how much light passes through each of the wells. Most plate readers can detect light of several different defined colors. The color is selected by placing an optical filter in the light path. To detect the yellow-colored end product, a filter of 450 nanometers is optimal. So I select that here. If I had chosen a different chromogen in the preceding step, then I would need a different filter at this point. For example, OPD, o-Phenylenediamine, produces a red end product, and the 645-nanometer filter is appropriate. Now we can read the absorbance values. The 96-well plate is taken into the plate reader and the absorbance read. The plate is then returned on the tray. In the final step, you can view the results which are presented in an 8 by 12 array corresponding to their position on the ELISA plate. The results can now be taken for analysis. Let’s look again at the appearance of the plate. You can see that the negative control on row B has essentially no color on any of the wells. The positive control on row A shows a progressive decrease in color across the plate at least up to well 8, which is a serum dilution of one in 256. The antibody titer is normally expressed as the reciprocal of the highest serum dilution that gives a detectable signal. So the positive control has a titer of 256. You can also see immediately that four of the samples in rows C to H are positive for IgG antibodies against spike protein and to a negative. A simple estimate by eye is very useful in healthcare settings where a plate reader is not available. But we can do better than this if we have the absorbance values from the plate reader. After you have read the plate using the appropriate filter, you should have a data set that looks like this. Notice that the absorbances are set out in an 8 by 12 grid which corresponds exactly to their position on the ELISA plate. The samples in wells 1 to 12 are in doubling dilutions, and the reciprocal of the dilution is now shown in the bar beneath the table. Look first at the positive control in row A. The most concentrated sample in well 1 has an absorbance of 1.305. And the absorbance values decrease progressively towards the most dilute sample in well 12. If I look at the negative control row, there is a background value, which is about 0.09. And the highest value in well B1 is 0.106. We can, therefore, say that an absorbance value above 0.12 is a good cutoff point for assessing whether there is a significant level of absorbance above the background. I’ve now highlighted all the wells above this threshold. From this, we can read off the titer in each sample. And this is now entered on the right-hand side. It’s really important to note that each assay will be slightly different depending on the exact conditions you use. You need to work this out for yourself by looking at your own data, and it will be different on each assay. Also, be aware that duplicate samples will usually give slightly different results reflecting the variation that is seen in this type of assay. The results will always be more accurate and reliable if the samples and standards are done in duplicate or triplicate and a mean value of the absorbance is used. I’ve now shown you how to determine the titer of IgG antibodies against SARS-CoV-2 spike protein using ELISA. You can do a lot of different analyses using this set of 60 serum samples. Here are some suggestions. You could test a larger group of samples for IgG antibodies against spike protein to estimate what proportion of the adult population in the UK had some immunity to SARS-CoV-2 in August 2021. You could test the sera for antibodies against nuclear capsid antigen to estimate what proportion of the population had had an infection with SARS-CoV-2. Both infected and vaccinated people will have antibodies to spike protein, but only people who have been infected are likely to also have antibody against nucleocapsid antigen. Given information on the age and sex of the individuals, which is available, you could determine whether women have higher levels of antibodies than men or whether younger people have more antibody than older people. You can also test for IgM antibodies. IgM antibodies arise early during an immune response and decline much more quickly than IgG. So comparing IgG and IgM antibodies in a single immune individual can give some indication on how long ago they were vaccinated or infected. If you are detecting IgM antibodies, you will find that a concentration of secondary antibody of 1 microgram per ml is optimal. You could test for IgA antibodies. IgA is important in protecting mucosal surfaces of the respiratory tract. You could then see whether the titers of IgG and IgA are correlated in different individuals. If you are testing for IgA antibodies, you will find that a secondary antibody detecting IgA is optimal at 4 micrograms per ml. As you can see, there are many investigations that could be done with this set of serum samples. I hope you have found this introduction to the ELISA technique useful and its application to investigation of COVID-19 antibodies interesting.

Each ELISA plate should have a standard control (positive control), a negative control, and space for six serum samples. Therefore, in order to have 24 samples, you will need to run four plates. If you have enough time, run some additional samples to give you more data to work with. Now enter the titre of each of the selected serum samples in column 4 of your data entry table. In the final section this week, you will start to analyse and interpret your data.

3 Data interpretation Screening surveys and epidemiological studies usually require thousands of data points. For example, the first study on home-screening of antibodies to SARS-CoV2 done in the UK using lateral flow tests included 10,000 subjects. Such large studies give robust data but are clearly beyond the scope of this course. Nevertheless, the aim is to give you a flavour of how such studies can be analysed using the data you have obtained in the ELISA laboratory. The aim of the next activity is to estimate the percentage of individuals who have IgG S-antibodies, from the samples in your data-set. Activity 3 Prevalence of antibodies to SARS-CoV2 Allow 10 minutes Take a cut-off point as a titre of 4 (dilution 1:4) and count the number of samples with a titre >4. Then estimate the percentage of individuals who are positive:

Percentage positive = \frac{number with titre > 4}{total number of samples} \times 100

You may recall that at the time the samples were taken (August 2021) 60-70% of the adult population in the UK had been vaccinated against COVID-19 (Figure 3) and a much smaller percentage had had a natural infection. Individuals with antibodies have a level of immunity against virus infection and considerable protection against severe disease. From your results, what percentage of individuals had some immunity to COVID-19 during this time?. Due to the way in which the vaccination programme was rolled out (older people first), you might also expect to see that a higher proportion of older people would have antibodies than younger people. However, you probably do not have enough samples to confirm whether this is true or not. Activity 4 Titres of antibodies to SARS-CoV2 Allow 20 minutes Part 1 Using your data for Table 2, you can now estimate how the titre of antibodies varies across different groups within the population. Statistical analysis is beyond the scope of this course and would require larger data-sets. Nevertheless, you can carry out some simple comparisons. To do this first identify those individuals who are positive for IgG S-antibodies (titre >4). Then carry out two comparisons. In each case compare the median titre in one group with the median titre in the other. The median is the individual who has the mid-point titre. For example, if the titres of a group of 11 individuals were: 8, 16, 64, 128, 128, 256, 512, 1024, 1024, 2048, 4096, then the median titre is 256, because this is the person in the mid-point of this group. Part 2 Now compare the median in female and male individuals. Are they different? Generally titres in males and females are quite similar. It would be surprising if the results in your samples were greatly different, but it might be so if your samples do not represent the overall population well. The larger the number of samples you have taken, the more likely they will accurately reflect the S-antibody titres in the whole population. Part 3 Next compare the median in older people (age >50) with those in younger people (age ≤50). Are they different? We expect that the titre in the older age group would be lower than the titre in the younger age group, but the interpretation of this is not quite so straightforward. Can you think of two possible explanations of why the S-antibody titres could be lower in older people? It may be that older people produce less antibodies. Alternatively, older people were mostly vaccinated early in 2021, and by this time their antibody titres will be declining. In comparison younger people were mostly vaccinated in Spring and early Summer, and antibody titres are usually highest 2−4 weeks after the second injection. Of course, it is possible that both explanations are true, but one cannot disentangle the two explanations from the data available so far. This investigation has shown you how data on antibodies can be used for monitoring immunity in the population, but results have to be interpreted with caution. Next week you will start to look at how this type of data informs vaccination programmes. 4 Week 4 quiz Now it’s time to complete the Week 4 badge quiz. It is similar to previous quizzes, but this time instead of answering five questions there will be fifteen. Week 4 compulsory badge quiz Remember, this quiz counts towards your badge. If you’re not successful the first time, you can attempt the quiz again in 24 hours. Open the quiz in a new tab or window then come back here when you’ve finished. 5 Summary This week you have been given an overview of the COVID-19 epidemic in the UK and the course of the vaccination programme during 2021. The programme prioritised vulnerable and older people, but by the end of the summer all adults had been offered vaccination. You then carried out an investigation to measure the prevalence and titres of IgG antibodies against SARS-CoV2 spike protein (S-antibodies) in a group of individuals selected from August 2021. Using your own data, you were able to estimate the percentage of the adult population who had some immunity to the virus at this time. You also carried out comparisons of the antibody titres in females vs males and older vs younger individuals. This type of data can be used to track epidemics and inform vaccination programmes. Next week, you will learn exactly how infection rates, herd immunity and vaccine effectiveness are related. You are now halfway through the course. The Open University would really appreciate your feedback and suggestions for future improvement in our optional end-of-course survey, which you will also have an opportunity to complete at the end of Week 8. Participation will be completely confidential and we will not pass on your details to others. Now go to Week 5. Week 5: Tracking infection Introduction How can one use antibodies to track infection? This week we start to look into the field of serology, the use of antibodies to identify infectious agents or diagnose disease. You will also start to learn some epidemiology, the study of disease in populations and how they spread. Audio 1 Introduction to Week 5 DAVID MALE: Epidemiology is the study of how diseases spread in populations. It is complicated because each disease is different. It will be simpler for us as we are just looking at COVID-19. But even looking at one disease, it varies between countries, and different groups of people, according to social, environmental, genetic and demographic differences. Serology, which is the study of antibodies, can help us understand what proportion of the population have been infected. Antibodies persist, long after an acute infection has passed, so they act as a marker of whether a person has previously contacted an infectious agent. You have already measured antibodies to SARS CoV2 in the virtual ELISA laboratory. This week you will extend your investigation of serum samples in the virtual ELISA laboratory, to distinguish people who have been vaccinated from those that have been infected. You will also see how serology can inform epidemiological studies. By the end of this week, you should be able to: understand the difference between disease incidence and prevalence calculate disease incidence from data provided outline how serology can inform epidemiological studies identify SARS-CoV2 infected individuals in the virtual laboratory by detection of N-antibodies and estimate the cumulative level of infection in the population. 1 Aspects of epidemiology Epidemiology is the study of diseases in populations and how they are transmitted. It is a broad and complex field of study, since each disease is unique in its incidence and the way it is transmitted. Because it deals with populations, epidemiology often deals with large data sets which require sophisticated statistical analysis. Furthermore, the findings of epidemiological studies must be considered in relation to environmental, geographic, demographic, cultural and genetic factors, all of which affect the incidence of disease and its transmission. In this course, the focus is just on the COVID-19 pandemic, and therefore can be very selective about which aspects of epidemiology are covered. The statistical analyses used in epidemiology are beyond the scope of the course. However, it is necessary to introduce you to some basic terminology.

1.1 Incidence and prevalence The incidence of an infectious disease is the rate at which new infections occur within a defined period of time. For a non-infectious disease the rate would be given as the number of newly diagnosed cases in a period of time. For many epidemiological studies the incidence is given as the number of new cases per year, per 100,000 population. For example, the incidence of lung cancer in the UK between 2016−2018 is given as 70 cases per 100,000 people per year. The incidence of this condition is highly dependent on the age of population group studied. For this reason, incidence rates are often ‘age standardised’ so that comparisons can be made between countries with different demographic profiles. Apart from age, what other factors might influence the incidence of lung cancer? You may have thought of gender, ethnic group, geographic location and social factors such as smoking. Many of these factors can be interrelated. As you can see, it is important to define exactly what population is being studied and when. If it is not stated, it often means the annual incidence in the entire population of a country or region. An alternative measure of the occurrence of disease is prevalence. This gives the total proportion of the population that are affected at any one point in time. It can be expressed as the number of affected individuals per 100,000 of the population. For more common diseases it may just be given as the percentage of the population affected at any one time point. Chronic conditions (e.g. diabetes) may last for many years, in which case the prevalence at any one time will be greater than the annual incidence. For acute infectious diseases such as COVID-19, which usually last for 7−10 days, the weekly incidence of the disease is a more useful measure, since it can show how the epidemic is changing from one week to the next. However, if you look again at the incidence of SARS-CoV2 cases in the UK (Figure 1) you will see that the figures actually show the daily incidence. It is therefore important to look at the period of time for which the incidence rate is quoted. For example, if the daily incidence of infection over a period of time is 10,000 cases, then the weekly incidence will be 10,000 x7 = 70,000.

Activity 1 Weekly incidence of disease Allow 10 minutes An interesting point can be seen in the data on this graph in relation to the weekly incidence of disease – can you see what it is? How could it be explained? Note your thoughts in the box below The bar chart shows the incidence of new infections detected each day, but it also shows a distinct weekly pattern (the regular notches on the overall profile). There are two possible explanations for this pattern: either infection rates vary on a weekly basis, depending on where people were located and what they were doing; or a more likely explanation is that the laboratories that were testing the specimens, handled or reported more cases during weekdays than at weekends. Sometimes, during the epidemic in the UK, the level of infection was reported as prevalence, for example, ‘At this time, 1 person in every 100 people in the UK has a COVID-19 infection.’ It is important to see the distinction between incidence and prevalence, since it can lead to confusion in understanding the absolute numbers affected. For example, suppose the incidence of new infections is 10,000 per day and the average duration of the infection is 10 days, then the prevalence during this period will be 10,000 x10 = 100,000. For an acute infectious disease, incidence and prevalence are both useful measures of how an epidemic is progressing. But, for many other conditions, incidence and prevalence give different types of information – beware and be aware of the distinction.

1.2 Calculating disease incidence In this section we will ask you to do some simple calculations of weekly disease incidence, which is made using the formula:

incidence = \frac{number of cases}{total (number of population x
                                            number of weeks)}

Here is a worked example: In an office with 140 employees, during February 2019 (4 weeks), 17 contracted influenza. What is the weekly incidence of influenza in this office? Incidence = 17/ (140 x 4) = 0.0304 cases per employee per week. Activity 2 Calculating disease incidence Allow 10 minutes In a secondary school, during the Winter term lasting from January – March 2021 (13 weeks) a number of students were recorded as absent due to COVID-19 infection. The data was broken down according to individual year groups (Table 1). Calculate the weekly incidence of infection in each year group. Don’t forget the units. Table 1 Individual year groups

Year group	Number of students	Number of cases
9	196	16
10	229	42
11	168	34
12	154	24

Which year group shows the highest incidence of COVID-19 infection? Did you see that year 11 has the highest incidence (0.0156 cases per student per week), even though the total number of cases was higher in year 10.

2 Detecting infection As we noted earlier, one of the best ways of measuring the rate of infection in a community is by regularly testing a panel of individuals who are representative of that community. In the earliest stages of the COVID-19 pandemic, it was clear that some individuals could be infected but have no symptoms, but it was not certain whether this was a large or small proportion of the population. This information was, however, very important, since asymptomatic people were more likely to go about their day-to-day activities as normal in the community and potentially infect those they interacted with. This information also informed public health measures such as lock-downs, isolation periods and the wearing of masks. The incidence of a disease such as COVID−19 is measured by detection of the pathogen, for example by PCR, lateral flow tests or laboratory culture of the infectious agent. These tests show positive for a limited period following infection – typically ~2 weeks for COVID-19. However, if infected people have no symptoms, they are unlikely to go forward for testing. Consequently, measuring the number of infections in people presenting to their doctor or a testing centre is likely to underestimate the true incidence of the disease in the community. An alternative to detection of the pathogen is to measure antibodies against the pathogen.

2.1 Serology Serology is the study of antibodies in blood serum and body fluids. It also relates to how antibodies can be used to distinguish between different pathogens and different strains of a pathogen. Serum, in this context, is the fluid component of blood containing soluble molecules such as antibodies and other proteins. Serum is formed after blood has clotted, removing the cellular elements of blood (red cells, white cells) and other components involved in formation of the clot (platelets and proteins of the blood clotting system). Serum should be distinguished from blood plasma which lacks cells, but which retains the components required for clotting. The pie chart in Figure 2 shows the relative amounts of the proteins in plasma.

As you can see, the three major classes of antibody (IgG, IgA, IgM) constitute about 25% of the total plasma protein.

2.2 Seroconversion Seroconversion refers to the point in time when an infected person has detectable antibodies against the infectious agent. For SARS-CoV2, seroconversion occurs at 7−14 days after the infection and antibody titres typically increase to a maximum at about one month after infection and then gradually decline, provided that the person does not become reinfected. Different classes of antibody last for different lengths of time in plasma in vivo, as shown in Table 2. The measure of persistence is the half-life of the antibody – the amount of time in which half of the original amount is lost. Notice also, that in humans there are four different subclasses of IgG (IgG1 – IgG4) and two of IgA (IgA1, IgA2), which have slightly different characteristics and functions. Table 2 Properties of human immunoglobulins. The concentration in serum of adults >18 years, is given as the normal range.

Antibody	Half-life (days)	Binding to macrophages	Complement activation	Mucosal transport	Serum conc. g/l
IgM	10	-	+++	+	0.5 -1.9
IgG1	21	+++	++	-	6.0 -16.0
IgG2	20	-	+	-
IgG3	7	+++	+++	-
IgG4	21	++	-	-
IgA1	6	-	-	++	0.8 -4.0
IgA2	6	-	-	++	0.8 -4.0

While antibodies may only last for a few months, antibody production from B cells and plasma cells lasts for many months after a secondary antigen challenge. and the memory of how to produce these antibodies, residing in memory cells, can last for many years. Consequently, when a person has undergone seroconversion, they usually have detectable antibodies against the pathogen for years. This is a generalisation, and it varies with the pathogen and the individual. One important question during the COVID-19 pandemic was how long the antibodies and immunity would last. The short answer is that antibodies and antibody production continued for many months following natural infection, and a level of immunity to severe disease lasted much longer than that. The main problem with SARS-CoV2 was not a decline in immunity to the original infection but that the virus mutated to evade the antibodies produced against earlier strains. You will return to this subject in Week 8 and delve further then.

2.3 Seroprevalence Seroprevalence is a measure of the prevalence of antibodies against an infectious agent. As you discovered in Weeks 3 and 4, this can provide useful information about levels of immunity in the population. In large studies it can also provide regional information. One of the first large-scale studies on COVID-19 antibodies took place in Spain in April – May 2020 and was reported in The Lancet. Figure 3 shows the seroprevalence of IgG antibodies against the SARS-CoV2 nucleocapsid protein, in different regions of the country. It is clear that the levels of infection in the central regions, including Madrid, were much higher than in the peripheral areas.

Seroprevalence can also be used to track changes in immunity across time. Figure 4 shows the percentage of samples having antibodies to SARS-CoV2 spike protein, from blood donors in different regions of the UK. The donations were screened by an ELISA (Euroimmun) which is similar to the one you carried out in Week 4. Notice that the time of this study covers the period from April to May 2020, when the UK vaccination programme was fully active. The progressive increase in antibodies against spike protein in blood donors, therefore, primarily reflects the increasing proportion who had been vaccinated.

Serology can also give information about the proportion of people who are infected but asymptomatic. In the study in Spain, noted above, and in other studies across Europe including Iceland it was found that 30−40% of people who had antibodies against SARS-CoV2 had reported no symptoms and mostly had not been tested for infection by PCR. A major difference between SARS-CoV2 and the original SARS is the high proportion of asymptomatic cases, which meant that it was much more difficult to identify and isolate infected people, to prevent transmission.

3 Laboratory investigation Last week, you measured the levels of IgG antibodies against SARS-CoV2 spike protein in a group of 30 individuals in order to assess the level of immunity in the population. This week, you are going to take that investigation one step further. Recall that the antibodies against spike protein could be due to vaccination or natural infection. But how could one specifically identify who had been infected? If a person has been infected they produce antibodies against all structural components of the virus, including the nucleocapsid. Antibodies against the nucleocapsid are called N-antibodies, to distinguish them from the S-antibodies that recognise spike protein. Activity 3 Measuring N-antibodies by ELISA Allow 50 minutes Go back to the data in Table 2 that you produced in Week 4. It should contain your own data on S-antibodies in the samples that you measured. You may have downloaded the table instead, which you can download again if you need to: Table 2. Now identify those individuals who have a titre of S-antibodies >8. You are going to retest these samples to see if they also have N-antibodies. Now go to the virtual ELISA: epidemiology laboratory, and measure the titre of IgG antibodies against nucleocapsid. You should carry out the assay in exactly the same way as previously except at step 3 of the assay, you should choose the nucleocapsid ELISA plate (Figure 5)

The standard has IgG N-antibodies (titre = 800) which should be used as a positive control, and the negative control serum has no significant N-antibodies (titre <4). You may need to do four ELISA plates in order to measure all of the samples that have S-antibodies. Put your results into the last column of Table 2.

3.1 Data interpretation You can now make an estimate of the prevalence of N-antibodies in the population, and by inference the number of people who have been infected with the virus. Take a titre of >4 as being a positive result.

incidence = \frac{number with N – antibodies}{Number tested} \times 100

At this time, about 10% of the UK population had been infected with SARS-CoV2. Because your sample is relatively small, it may not accurately reflect the whole population, but you should have found at least one individual with N-antibodies. When you are ready check your results. Do your results correspond? What percentage of the sample had N-antibodies? Of the 60 samples available, the following seven samples have IgG antibodies against nucleocapsid: C3111, C5930, F7812, H1151, H4439, M6723, N9921 From these figures we could estimate: Prevalence = 7/60 x100 ≈ 12%. If a person is seropositive for S-antibodies, but negative for N-antibodies, what can you infer? They have been vaccinated with SARS-CoV2 spike protein.

4 Week 5 quiz Check what you have learned this week by taking the end-of-week quiz. Week 5 practice quiz. Open the quiz in a new window or tab, then return to this week when you’re done. 5 Summary This week, we introduced you to some aspects of epidemiology, including the concepts of incidence and prevalence. Incidence is given as the rate of new disease cases over a defined period of time, whereas prevalence gives the total proportion of people affected at any one time. The value of antibody testing for SARS-CoV2 was outlined. For COVID-19, infected people usually undergo seroconversion 7-14 days after infection, and they then remain seropositive for many months, possibly years. Infection with the virus induces both S-antibodies and N-antibodies. The presence of N-antibodies distinguishes previously infected individuals from those that have been vaccinated against the spike protein. Seroprevalence, the proportion of people who are seropositive for antibodies, can be used to compare the cumulative level of infection in different regions or over a period of time. This information contributes to understanding disease spread and how it can be controlled. Particularly important is knowing what proportion of infected people are asymptomatic – in the case of COVID-19, up to 40% of cases were asymptomatic. Tests for infection (eg PCR) often underestimate the prevalence of the disease, when there are large numbers of asymptomatic cases, as they are less likely to come forward for testing. For this reason, random sampling in the community is the most reliable way of getting good estimates of disease prevalence. Finally you used the virtual laboratory to identify sera with N-antibodies as evidence of previous COVID-19 infection. You will take this investigation one step further next week. Now go to Week 6. Week 6: Epidemiology Introduction Do you remember the regular briefings given by the Chief Medical and Scientific Officers, at the height of the COVID-19 pandemic? There was a lot of concern about whether the number of infections were increasing or decreasing because that determined the public health measures needed to control the epidemic. Later, when vaccines became available, control of the epidemic shifted away from this as the vaccination programme did the heavy-lifting. Public health measures such as closure of public amenities, reduced travel on public transport and mask-wearing reduced effective contacts between individuals and delayed the progress of the COVID-19 pandemic in different countries. This week, you will be looking at aspects of epidemiology and modelling that underpinned the response to the pandemic. Video 1 Introduction to Week 6 DAVID MALE: This week, we’re going to take a look at some aspects of epidemiology and how it informs public health. A key concept in epidemiology is the reproduction number of an infection or its R-value. It describes the number of people who will become infected from a single infected individual. At any one time, this depends on how many people are susceptible to infection and how they are mixing in the community. The R-value is important because it can tell us whether an epidemic is growing or shrinking. With an R-value greater than 1, an epidemic is growing. And the larger the R-value, the faster the epidemic is growing. Understanding the rate of spread then helps determine what public health measures are needed to control an epidemic. In many countries, a major concern was that the health systems would become overwhelmed by a rapid influx of seriously ill patients. By reducing the R-value, public health measures slowed the initial wave of infection, preventing hospitals from being overwhelmed. In addition, it gave doctors time to identify the most effective treatments for serious disease. The closure of public amenities and restrictions on travel reduce the number of contacts between people. And other public health measures, such as social distancing, mask wearing, and good ventilation further reduced opportunities for the virus to transmit between individuals. These regulations all contributed to a reduction in the R-value and slowed the progress of the COVID-19 pandemic in different countries. A few countries, which had close control of their borders, introduced quarantine for visitors and comprehensive programs of COVID testing. And they were able to maintain very low levels of infection. But for most countries, waves of epidemic were checked only by implementation of widespread lockdowns. An important additional benefit of the public health measures was that it gave pharmaceutical companies time to develop vaccines and for countries to roll out their vaccination programs. Vaccination can reduce the rate at which an infection spreads, as there are fewer susceptible people. However, once vaccines were widely available, their main benefit was to protect against serious disease and allow removal of the restrictions on normal life.

//dog.open.ac.uk/PrintLive/nonCourse/OpenLearn/Courses/COV-19/assets/videos/intro_video_wk6/covid_19_wk_6_intro.jpg

By the end of this week, you should be able to: define the meaning of R0, RE and RT understand the implications of different values of these variables outline the assumptions underlying the determinations of these variables. calculate the critical immunisation threshold for different diseases. 1 Epidemiology and modelling From an epidemiological perspective, each disease is different but there are two main patterns – epidemic diseases and endemic diseases. The SARS-CoV2 pandemic was a classic example of an epidemic disease. It was effectively a new virus in humans and none of the human population had any immunity to it, consequently it produced epidemics in each country affected. A disease becomes endemic when it transmits continuously in a population, because there are always sufficient susceptible individuals to maintain the infectious agent. Some diseases, such as measles in an unvaccinated large population, are endemic because there are always enough susceptible children to maintain cycles of infection. In time, an epidemic would develop into a steady-state endemic disease – provided that the virus did not change and immunity lasted for a long-time. However for SARS-CoV2, the virus did mutate and continues to do so. Consequently we have been subjected to repeated waves of infection with new strains of the virus. It is important to distinguish successive waves of an epidemic disease (eg SARS-CoV2), from the regular repeated cycles of infection often seen with an endemic disease such as measles or chicken pox (Figure 1).

Do you think that influenza-A shows an endemic or epidemic pattern of disease? Influenza-A is another virus that mutates regularly and produces successive waves of epidemic infection, usually peaking in transmission during the winter months in the Northern and Southern hemispheres.

1.1 The basic reproduction number R0 An important concept in epidemiology and modelling is the basic reproduction number (R₀). When a person is infected with a disease, there is a period of time when they can transmit the infection to their contacts. The basic reproduction number of the infection is the average number of secondary infections that result from one infected person. This definition is represented in Figure 2.

The basic reproduction number is neither a risk nor a rate: it is just a number. It can take any positive value (or zero) and it specifically assumes that all contacts are potentially susceptible to infection. For most diseases this is clearly not true. While it was true at the start of the COVID-19 pandemic, resistance to reinfection gradually built up in the population as increasing numbers of people were infected, recovered and developed some immunity. Later on, the number of susceptible individuals was drastically reduced by the COVID-19 vaccination programmes. Despite its theoretical nature R₀ is a very useful measure, because it allows comparison of the infectivity of different pathogens or different strains of one pathogen. It is also a key parameter in determining what proportion of the population must be vaccinated in order to stop a disease spreading – this value is called the critical immunisation threshold (qc) and we will return to it later this week.

1.2 Understanding R0 The basic reproduction number (R₀) is important because it encapsulates the relationship between an infection and its physical and social environment. The number of secondary infections depends on the ability of the infectious organism to survive outside the host and to migrate from one host to the next, which in turn is contingent on biological and environmental factors. It depends on the infection–host interaction through, for instance, the duration of the infectious period. It is also affected by the frequency and type of contacts that take place within the population, which vary according to environmental, social and cultural factors. R₀ can also tell us about how quickly an infection is likely to grow in an unvaccinated, fully-susceptible population. Table 1 shows R₀ values for a number of virus diseases, Note that the values given are ranges, which will vary depending on the population and how the communities interact. Table 1 Range of R0 values for selected virus diseases.

Virus	R₀
Influenza	1 - 2
Hepatitis C	2 - 3
Ebola	1.5 – 2.5
Zika	1.5 – 4.1
HIV	2 - 5
SARS-CoV	2 - 3
SARS-CoV2	2.5 - 6
Mumps	7 - 10
Chickenpox	10 - 12
Measles	12 - 18

As you can see SARS-CoV2 falls around the middle of the range for virus diseases, but as it turned out different variants of SARS-CoV2 have different R₀ values.

1.3 The effective reproduction number RE Since most populations will have some level of immunity to an infectious disease, the rate of spread will be less than indicated by the R₀ value. The measure of how a disease spreads in real situations is given by another dimensionless variable R_E – the effective reproduction number (Figure 3), which is often just called the R-value.

Notice that R_E will never be greater than R₀ and will usually be less than R₀ because of the level of resistance or immunity in the population. Another point is that R_E can tell us something about whether an infection will develop into an epidemic or just die out. If R_E is >1 then the number of infections will gradually increase over time. Exactly how quickly an epidemic will develop depends partly on the value of R_E, and partly on how long it takes before each infected person becomes infectious themselves, ie the time-course of infection. Conversely if R_E <1 then the infection is self-limiting, because the number of infected people gradually decreases over time.

1.4 Understanding RE Because R_E depends on the particular circumstances of an infection, the value of R_E will be different dependent on the current local conditions. With reference to the COVID-19 pandemic: can you think of 5 different conditions that could affect the value of R_E. There is a lot to think about here, so take your time before you reveal the answer. The proportion of people in the population who were resistant to infection with SARS-CoV2 varied over time. It was dependent on whether a person had been infected with SARS-CoV2 or had been vaccinated against it. Immunity gradually declines over time, if a person does not become reinfected with the same virus. Also, as the pandemic developed, new variants arose which could partly evade the immunity produced against previous strains. Effectively the new strains resulted in a more rapid decline in resistance than normally occurs and increased susceptibility to reinfection. All of these factors relate to the intrinsic susceptibility of the population. Also R_E depends on how people interact with each other. Even before public health measures were introduced, many people started to reduce their social interactions so the level of effective contacts fell. Public Health measures, such as lock-downs, restricted access to public amenities and mask-wearing also all reduced the number of effective contacts between individuals. We should also consider the underlying assumption for R₀ and R_E – that the population is homogeneously mixing. This is obviously a simplification. Some people live in large families, others in Institutions, and some people live alone. The intrinsic level of social contact varies greatly for each of these groups. Another important factor is what type of work people were doing. During the pandemic, some people could and did work from home. Others, because of the nature of their occupation, travelled to work and may have been meeting other members of the public as part of their work. The number of effective contacts and the potential for transmission of an infectious agent depends on all of the factors noted here. Despite all of these variables affecting individuals, R_E is still a very useful concept, when applied to the population, as a whole, since it informs public health policies.

1.5 How RE changes over time We have already noted that R_E changes over time due to variable susceptibility, public health measures and vaccination. The value at any one time is important, since if R_E >1 an epidemic is growing, whereas when R_E <1 it is shrinking. Figure 4 shows the estimated value of R_E at different time points between July 2020 and October 2022. The values are given as ranges, and values vary above or below the critical value, R_E = 1.

It is important to understand that the values shown in Figure 4 are not the numbers of people affected by COVID-19, but they reflect whether numbers affected are increasing or decreasing. It is possible to have very few people affected but a high value of R_E, or many people affected and a low value of R_E. This type of data was fundamental for informing public health policies in the early phases of the pandemic, before vaccines were available. If R_E values were >1, then the numbers that would be infected over the following weeks or months could be projected. In order to prevent the huge numbers overwhelming medical services, public health measures were introduced to reduce contact rates and bring R_E <1. As one example, look at the R_E values in the month before January 2021 (Figure 4). The sudden rise in R_E at this time corresponds with the government decision to end lockdown restrictions on December 2nd 2020. Once the effect of this policy became evident, the lockdown had to be reinstated on 21st December in London and Southeast England and 26th December in other parts of the UK (Institute for Government analysis, 2021). With public health measures back in force, the R_E value fell below 1. Once vaccines became available the proportion of susceptible individuals was reduced and vaccination could be relied on to keep infection rates under control. Consequently, during 2021, there was a progressive shift from a reliance on public health measures (to reduce contacts) to the use of vaccines (to reduce susceptibles). [Note that when R_E values are stated in relation to particular time-points or over a period of time they are often referred to as R_Tvalues also called the net reproduction number. In many publications values of R_T or R_E are just given as R-values.]

1.6 Estimation of R0, RE and RT As R₀ is by definition a measure of disease spread in a totally susceptible population, and since such populations rarely exist, estimating R₀ for a disease often presents a challenge. Measurement of R_E is conceptually simpler, because it can be done empirically by measuring new infections. For uncommon infections such as Ebola, it is possible to directly identify how many people became infected from a single index case. While the numbers are usually small, it gives a direct measure of spread in a defined population where all individuals are susceptible. Also, in the case of Ebola, all infected contacts of the index case develop symptoms and can be identified. Consequently asymptomatic cases do not cause underestimation of R₀. The R₀ value for COVID-19 could also be estimated from the incidence of new infections. In the earliest stages of the COVID-19 pandemic, virtually everyone was susceptible, so R₀ was similar to R_E. However, at this time accurate data depended on the availability of reliable tests and screening programmes. In the early stages of the COVID-19 pandemic, testing for infection by PCR was patchy and selective. Moreover, as COVID-19 epidemics developed in different countries, people voluntarily reduced their level of social contacts and public health measures were introduced to limit spread, ie effective contacts were reduced. This means that the most reliable direct estimates of R₀ for SARS-CoV2 come from the period before these behavioural changes and public health policies came into effect. For endemic infections, which have reached a steady state in a population, it is possible to estimate R₀ indirectly, but this is beyond the scope of this course.

2 Herd immunity and immunisation This section introduces two important related concepts. Herd immunity describes the level of immunity that develops in a population, due to natural infection. Once a sufficiently large proportion of the population have become resistant, the disease can no longer spread because there are insufficient susceptible people and R_T <1. The level at which this occurs is the ‘Herd immunity threshold’ (HIT) and it is different for each disease. Critical immunisation threshold (qc) is the level of immunity that must be achieved in a population by vaccination, to stop a disease spreading. As you can see, it is conceptually very similar to the herd immunity threshold.

2.1 Herd immunity threshold In section 1, we discussed disease susceptibility and the proportion of the population who are susceptible. This value is designated by a variable ‘S’. In the illustrated example (Figure 5a) in a population of 12 individuals 9 are resistant and 3 are susceptible. S = 3/12 = 0.25 and the proportion resistant = 1 –S = 0.75

Recall that if R_T <1 then an infection will die out. The herd immunity threshold and the critical immunisation threshold occur when R_T =1. The value can be calculated from the basic reproduction number R₀. HIT = 1 – 1/R₀ Let us consider how this works for a disease with R₀= 4 HIT = 1 -1/4 = 0.75 If this is now expressed as the percentage of the population that must be resistant to stop a disease from spreading: HIT = 0.75 x100 = 75%. For a diagrammatic representation of this example, see Figure 5(b). In this case R₀=4 and there is only one secondary infection (R_E =1), because 3 of the 4 potential effective contacts are resistant. There is some simplification in the calculation of HIT, since it assumes that a person is either completely susceptible or totally resistant. In practice resistance develops over time following infection or vaccination, and the level of resistance can wane. Nevertheless, considering individuals to be susceptible or resistant is useful for modelling epidemics and planning vaccination programmes.

2.2 Critical immunisation threshold (qc) The critical immunisation threshold (qc) tells us what proportion of the population need to be vaccinated, in order to control an infection, and it is calculated in exactly the same way as the herd immunity threshold. qc = 1 – 1/R₀ x100 Activity 1 Calculation of qc Allow 15 minutes In this activity we ask you to calculate qc for 7 different virus diseases expressed as a percentage of the whole population. Use the equation to determine the values of qc and enter them into Table 2. Round your calculation to the nearest whole number. Table 2 Calculation of qc values – A range of R0 values is given for SARS-CoV2, but just a single typical value for the other viral infections.

Virus	R₀	qc
Influenza	2
Hepatitis C	3
Zika	4
SARS-CoV2	2.5 - 6
Mumps	8
Chickenpox	10
Measles	16

When you have completed your calculations, click to reveal the answers. What do you notice about the relationship between R₀ and qc? The higher the value of R₀, the higher is the value of qc. Table 2 Calculation of qc values – A range of R0 values is given for SARS-CoV2, but just a single typical value for the other viral infections.

Virus	R₀	qc
Influenza	2	50%
Hepatitis C	3	67%
Zika	4	75%
SARS-CoV2	2.5 - 6	60 - 83%
Mumps	8	87%
Chickenpox	10	90%
Measles	16	94%

For diseases such as mumps, chickenpox and measles, the level of vaccination coverage needed to prevent outbreaks is very high. When vaccine coverage of these childhood diseases falls below the required level in the population, outbreaks of the diseases occur. Also, when vaccination levels fall below the qc level, the age at which unvaccinated children contract the disease is older. The reason for this is that on average it will take longer before they encounter an infective with the disease, because the disease is less common in partially-vaccinated populations. Moreover, for some infections, disease is more serious if contracted later in life. Notice that if a population is protected because the level of vaccination exceeds the qc for a particular infection, then even people who are not immune have some protection, because the infectious agent no longer circulates in the community. However if everyone relies on other people being vaccinated, then the level of population immunity falls below qc and the infection returns. This has occurred in the UK recently for diseases such as measles, which require high levels of vaccine coverage to create herd immunity. Next week we will look at how vaccines are formulated and how effective they are, but it is worth remembering from you calculations here that the qc values for SARS-CoV2 range from 60 – 83%.

3 Laboratory investigation This week you will take your laboratory investigation one step further. The aim is to identify anyone who has been recently infected with COVID-19. Recall that last week you identified 7 serum samples that had antibodies against SARS-CoV2 spike protein and nucleocapsid, implying that they had been infected with the virus. Of the 60 samples available those that have both S-antibodies and N-antibodies are: C3111, C5930, F7812, H1151, H4439, M6723, N9921 Also recall that the first antibodies to be produced following infection are IgM, and it is only later that antibody production switches to IgG and IgA. Therefore, if you can identify sample(s) which have a relatively high IgM titre compared with IgG and IgA, it indicates that the person has been recently infected. Activity 2 Measurement of S-antibodies of different classes Allow 30 minutes In this activity, we ask you to measure the titres of IgM, IgG and IgA S-antibodies in the 7 samples from infected individuals. You should also include the standard in the 8th row of the plate. The assay will require three ELISA plates, one for IgM, one for IgG and one for IgA. ELISA; epidemiology on-line laboratory Carry out the assay according to the protocol you developed in week 3, with the following conditions: At step 1 of the assay you should select the samples; C3111, C5930, F7812, H1151, H4439, M6723, N9921 and the standard At step 3 of the assay you should choose plates sensitised with spike protein. At step 4, you will need to use 20µl anti-human IgM (HPO), 0.5mg/ml (final conc. 1µg/ml) Or 4µl anti-human IgG (HPO), 1.5mg/ml (final conc. 0.6µg/ml) Or 40µl anti-human IgA (HPO), 1.0mg/ml (final conc. 4µg/ml) [The S-antibody titres of the standard are IgM-120, IgG-1200 and IgA-140.] Record you results in a table like the one below (Table 3). Table 3 S-antibodies of different classes in previously infected individuals

Samples	S-antibodies
Samples	IgM	IgG	IgA
C3111
C5930
F7812
H1151
H4439
M6723
N9921
Standard

You should see that one of these samples has a relatively high IgM titre in comparison to IgG and IgA. This suggests that the person has had a relatively recent infection with SARS-CoV2. When you have examined your data and come to a conclusion, click to check your answer. Sample C5930 has IgM-160, IgG-120, IgA-80. The relatively high IgM titre suggests a recent infection. For the other samples the IgG titres are at least 8x higher than the IgM titre. 4 Week 6 quiz Well done for reaching the end of Week 6. Check what you’ve learned by taking the end-of-week quiz. Week 6 practice quiz Open the quiz in a new window or tab then come back here when you’ve finished. 5 Summary This week, we introduced some key concepts in epidemiology including: R₀, the number of people infected by a single infective in a totally susceptible population. R_E, the number of people infected by a single infective, in real circumstances. R_T, the number of people infected by a single infective at some defined time-point. The variables imply that when R >1 an epidemic is spreading, but if R<1 it will eventually die out. The measurement of these variables assumes homogeneous mixing in the population and that all individuals are either completely susceptible or completely resistant to infection – conditions which do not generally occur in the real world. Nevertheless the variables are useful for determining how an infection will spread in the population as a whole. The herd immunity threshold (HIT) and critical immunisation threshold (qc) define what proportion of the population must be resistant to infection, for it to stop it spreading. These variables can be calculated if R₀ is known. The value varies for each disease. The larger the value of R₀, the greater the proportion of people must be vaccinated to contain a disease. In your laboratory investigation, you identified one recently infected individual, using the ELISA to measure different classes of S-antibody. Now go to Week 7. Week 7: Vaccines 1 Active and passive immunisation The principle of vaccination is very simple – train the immune system to recognise and react against the infectious agent. This procedure is in fact ‘active immunisation’, so called because the person who receives the vaccine actively makes their own antibodies and T-cell responses against antigens in the vaccine. For most infections ‘active immunisation’ is the only form of immunisation available. However there is another form of treatment called ‘passive immunisation’.

1.1 Therapeutic antibodies In ‘passive immunisation’ a person is given antibodies that have been made in another person, an animal or in a laboratory. The technology for making antibodies in the laboratory was well established before the COVID-19 pandemic started. Consequently, at the start of the pandemic, when there was uncertainty about whether an effective vaccine could be produced in time, pharmaceutical companies put considerable effort into the production of human therapeutic antibodies against the SARS-CoV2 spike protein (Table 1). Table 1 Examples of therapeutic antibodies. FDA = Federal drug authority in the USA

Antibody	Class	Company	FDA or EU Authorised
Banlanivimab	IgG1	AbCellera Biologics / Eli LIlly	Nov. 2020 – April 2021
Bebtelovimab	IgG1	AbCellera Biologics / Eli LIlly	Feb. 2022 – Nov. 2022
Casirivimab/Imdevimab	IgG1	Regeneron Pharmaceuticals	Nov. 2021 -
Cilgavimab/Tixagevimab	IgG1	AstraZeneca	Dec. 2021 -
Banlanivimab/Etesivimab	IgG1	Junshi Biosciences / Eli LIlly	Feb. 2021 -

The antibodies produced were ‘monoclonal’, meaning that they came from a single clone of B cells, and unlike naturally-produced polyclonal antibodies, monoclonal antibodies bind to just one position on an antigen. In producing therapeutic antibodies, the aim was to have an antibody that bound the receptor-binding domain (RBD) on the spike protein which could prevent the virus binding to its target, the ACE2 receptor. These antibodies were initially given emergency authorisation, for use in preventing COVID-19 infection in vulnerable individuals and/or for treatment of hospitalised patients with serious disease. As the pandemic progressed it became clear that new variants of SARS-CoV2 sometimes evaded the protection produced by these antibodies. To reduce the risk of a virus variant evading immunity, formulations with two monoclonal antibodies (eg Casirivimab/ Imdevimab) were developed. Also, authorisation for use of these treatments was sometimes revoked or amended, if they became less effective. It must be emphasised that these therapeutic antibody treatments are no substitute for active immunisation. A single treatment with a therapeutic antibody can cost £1000 - £1500, which contrasts with the typical cost of a vaccine, £1.50 - £15. Also, for immunological reasons, infusion of therapeutic antibodies may inhibit endogenous antibody production. Hence the reasons that therapeutic antibodies are licensed only for vulnerable or seriously-ill patients. For the remainder of this week, we will look at active immunisation with vaccines containing virus or viral components.

1.2 Vaccine types There are several different ways to produce a vaccine against a virus (Figure 1). Traditional vaccines, used the virus itself but chemically inactivated in such a way that it could not produce an infection. Another route was to develop a variant of the virus that could replicate, but which did not produce any symptoms or pathology in the recipient. The two main types of polio vaccine were derived by these two strategies – inactivation or attenuation.

More recently, vaccines have been developed against individual components of a virus, for example against purified spike-protein of SARS-CoV2. One limitation here is knowing which component(s) of the virus are important for inducing immunity. Also, recall that the antigens which stimulate B cells and T cells are often different. Moreover an immune response to a single virus component is often less strong than the response to an inactivated or attenuated whole virus. For this reason, such antigens may be modified to make them more immunogenic, or to favour one type of immune response. The latest vaccines are produced by genetic engineering. The idea here is to use the genetic material of the virus, to induce production of viral components which then stimulate the immune response. It turns out that this approach, using mRNA for the SARS-CoV2 spike protein, was very successful in the race to develop effective COVID-19 vaccines.

1.3 COVID-19 vaccines By December of 2020, more than 200 vaccine candidates for COVID-19 were under development, with more than 50 being taken forward into trials on humans. A variety of approaches were made (Table 2). Table 2 Examples of COVID-19 vaccines taken through to clinical trials.

Vaccine type	Manufacturer (name)
mRNA for spike protein	Pfizer/ BioNTechModernaCureVac
Vector with gene for spike protein	Oxford/Astra Zeneca(Sputnik V)Johnson & Johnson /JanssenCanSinoBio (Convidecia)
Spike protein	Novavax/ GSK
Inactivated virus	ValnevaSinovac (CoronaVac)Sinopharm (BB1BP CorV)

Interestingly, it was the newest methods – vector vaccines, and mRNA vaccines, which came through first. One strategy for COVID-19 is to take the gene that encodes the spike protein and insert it into a harmless virus vector. The vector has very limited capacity to replicate, but it still produces the COVID-19 spike-protein which induces specific antibody production. This approach has been used by the Oxford/Astra-Zeneca and Russian Sputnik V vaccines. mRNA vaccines rely on the recipient’s cells taking up the gene and expressing it, so that virus antigens (but not virus) are produced by the cells of the body. This approach is relatively new and it was used by the Pfizer/Biontech and Moderna vaccines against COVID-19. This method has been so successful that it has revolutionised vaccine production, and is now being applied to other infectious diseases and cancer therapy.

1.4 Vaccine testing Vaccines undergo rigorous trials before they are released for general use. The one exception to this rule is where an infection is very dangerous or uncontrolled and it is necessary to put a vaccine into the field as quickly as possible. This was seen with an Ebola virus vector vaccine, in helping control outbreaks of Ebola in the Democratic Republic of Congo and Zaire. Where mortality from a virus infection is high, there is more tolerance of adverse reactions against the vaccine, and the normal extended testing programs can be abbreviated. A normal testing program is carried out in four phases. Phase-1 examines basic safety of the vaccine in healthy volunteers. Phase-2 expands the initial trial to a larger and more diverse group of individuals (older, younger, different ethnic groups, etc.). Phase-3 determines whether the vaccine is effective in a large cohort (thousands of people) in real-world conditions. Phase-4 trials look for any long-term effects of the treatment and may extend over many years. [Your laboratory investigation this week is to carry out antibody testing on volunteers in a phase-1 vaccine trial.] Due to the urgency to develop vaccines against COVID-19, volunteers were recruited as quickly as possible and where possible phase 1-3 trials were overlapped to reduce the time before results became available. Also, the vaccination schedules and doses were chosen in these trials as a best estimate of what would produce a good antibody response. Dosing and schedules were later refined pragmatically and as more data on effective schedules became available. For example, the initial schedule for the Pfizer/Biontech mRNA vaccine was 2 doses given 21 days apart. This schedule was approved in December 2020. However, when it came to roll out the vaccination programme, with limited supplies of vaccine it was thought that greater protection could be given to the population by immunising more people, but spacing out the first and second doses by 6-8 weeks. As it happened, the longer gap between doses actually produced a slightly better antibody response, and the aim of protecting more people sooner was epidemiologically sound.

1.5 Field tests To determine if a vaccine is truly effective it has to protect people from the naturally occurring infection in a phase-3 trial. The incidence of infection in vaccinated and non-vaccinated people is compared, to see what level of protection is given by the vaccine. The studies are designed to be double-blind, meaning that neither the clinician administering the vaccine nor the recipient, know whether they have received the real vaccine or a placebo. Data is then collected over several months or years to determine the incidence of infection in the two groups. Once sufficient data has accumulated (number of infections) the coding on the treatments (vaccine or placebo) is opened to see whether there is a difference between the groups and how large the difference is. If the prevalence of infection is low in the community, then it takes longer to see whether the vaccine is effective, because it requires sufficient infections, to obtain robust data. Ironically, when public health measures reduced the incidence of infection, it could then delay data accumulation on a phase-3 vaccine trial. All of the approved COVID-19 vaccines have been tested in real-world trials and found to be very effective. But notice that these trials are not exactly comparable, because different trials took place at different times and in different countries where the dominant strains of the SARS-CoV2 virus have been different. For this reason, a simple comparison of ‘vaccine effectiveness’ in these trials is not straightforward. As an example, you can see the design and outcomes of the phase-3 trial of the recombinant Oxford/AstraZeneca COVID-19 vaccine (AZD1222) at the US National Library of Medicine. Phase III Double-blind, Placebo-controlled Study of AZD1222 for the Prevention of COVID-19 in Adults

1.6 Vaccine effectiveness Vaccine effectiveness (VE) is defined as the percentage reduction of infection in a group of vaccinated people, compared with a similar unvaccinated group. The two key parameters are the attack rate in the vaccinated group (ARV), compared with the unvaccinated group (ARU). VE = (ARU – ARV)/ ARU x100% Activity 1 A phase III vaccine trial Allow 10 minutes The following activity is based on the data from a phase-3 trial of the Oxford/AstraZeneca AZD1222 vaccine for COVID-19. The trial was randomised, double-blind and all subjects were seronegative for SARS-CoV2 antibodies at the start of the trial. In this trial 17762 subjects were vaccinated and 8550 received placebo. 73 of the vaccinated group and 130 of the unvaccinated group became infected in the study period. We now ask you to calculate the vaccine effectiveness using the following steps: You should first calculate ARV and ARU. ARV = Number infected / Number vaccinated. ARU = Number infected / Number unvaccinated ARV = 73/ 17762 = 0.00411 ARU = 130/ 8550 = 0.0152 Then calculate VE, using the formula: VE = (ARU – ARV)/ ARU x100% Click to reveal the answer, once you have made your calculation. VE = (ARU – ARV)/ ARU x100% = (0.0152 – 0.0041)/ 0.0152 X100% = 73% Based on this data the vaccine appears to be effective in reducing the incidence of infection by 73%. Notice that vaccine effectiveness is normally defined by the attack rate – the reduction in the percentage of infected people. However it is possible to measure ‘effectiveness’ by other means, such as the number of people showing disease symptoms or the number requiring hospitalisation. In the COVID-19 pandemic, the major clinical concerns were the number of people going to hospital, the numbers in intensive care units and the number of deaths. It was very notable that the COVID-19 vaccines not only reduced incidence of infection, but also reduced hospitalisation and deaths. In effect the spectrum of disease severity, ranging from asymptomatic to hospitalisation was all shifted to the left (Figure 2).

2 Vaccination This week the laboratory investigation is to measure the development of antibodies in two subjects who have been enrolled in a phase-1 study to assess the effectiveness of a COVID-19 vaccine. You will be using the ELISA again, but with a different set of samples to the previous weeks. The first 10 minutes of the video describes the samples and data available. It is followed by a section which reminds you how to use the ELISA laboratory. Also note that the video refers to samples from 10 subjects, but in the version of the laboratory available to you, we have only included samples from the two subjects that you are investigating. In this demonstration a different chromogen, OPD was used. You can do this if you like (remember to use the 645nm filter on the plate reader), or you can use TMB as you have done in previous assays. As you watch the video, be sure to note the vaccination schedule and when the blood samples were taken (Figure 3).

Video 1 Detecting antibodies against SARS-CoV-2 DAVID MALE Hello. I'm David Male from the Open University. In this video, I will show you how you can detect antibodies against the coronavirus SARS-CoV-2 using a technique called ELISA, an enzyme-linked immunosorbent assay. This technique is often used to quantitate antibodies in diagnostic laboratories or for research purposes. We have recreated this technique in a virtual laboratory where you can test a set of serum samples from 10 individuals who have been immunized against SARS-CoV-2 and who have then been monitored for 200 days to test their levels of antibodies against the virus. This will show you how the immune response against the virus develops following vaccination. This diagram shows the time course of the study. A serum sample was taken on day zero immediately before the subjects were given their first vaccination. Samples were then taken at days 7, 14, and 21 to monitor the appearance of antibodies. Another sample was taken at day 56 before the second dose of vaccine, and the effect of the second vaccination was monitored at day 70. A final sample was taken about six months later to establish how quickly antibodies declined in the serum. Now, let's see how the antibodies can be measured using the ELISA technique. This diagram illustrates the key steps in an ELISA. The assay is done on 96-well polystyrene plates that have been sensitized with antigen. In this case, the antigen is the spike protein of the SARS-CoV-2 virus corresponding to the antigen used for the vaccination. In the first stage, diluted serum is applied to the wells on the plate. If there is specific antibody in the serum, it will bind to the antigen on the plate. The bound antibody is called the primary antibody. Any unbound proteins in the serum, including non-specific antibodies, are then removed in a wash step. In the second stage, a ligand is applied to the plate which binds specifically to the primary antibody. The ligand also has a coupled enzymatic portion, which is crucial for the following step. In some cases, the ligand itself may be an antibody that binds to the primary antibody, in which case, it would be called a secondary antibody. The plate is then washed again to remove any unbound ligand. Finally, a chromogen is put into the wells. The chromogen is a colorless chemical which generates a colored end product when acted on by the enzyme of the ligand. The colored end product is detected on a plate reader. The more primary antibody is present, the more enzyme is bound to the plate and the more colored end product is produced. Hence, this technique can quantitate how much antibody is present in the initial serum sample. Now let's look at how the virtual ELISA laboratory recreates the technique. The virtual laboratory allows many options in choosing different serum samples and the dilution series of these samples. You can also choose the time of the incubations with the antibodies and the number and duration of the wash steps. You can select from three different ligands which detect different classes of antibody. There is also a choice of three chromogens for the development step. Finally, the results are read on a plate reader. And it is important to select the correct wavelength filter. The results correspond with what you would see in a real laboratory as these experimental conditions are varied. Moreover, if you happen to choose conditions that aren't quite right, then your results may not be optimal and perhaps even provide no usable results at all. So it's important that you note exactly what you do at each of the steps. It's also important to use controls in each assay. Initially, you should follow the recommended experimental conditions. But once you have your experiments working well, I would encourage you to modify some of the conditions and see how this affects the results. This way, you will also learn a lot about technical aspects of the assay. The exact appearance and layout of the experiments within the on-screen experiment will depend on the web browser you are using and the screen size. In this introduction, I'm using a desktop computer and Microsoft Edge as the browser. Before we start, a quick word about timing. In the virtual laboratory, we have telescoped time, so an incubation that would normally take 60 minutes can be done in one minute. The incubations are started and stopped on a timer like this. It means that an assay which would normally take two to three hours can be done in a few minutes. As mentioned, ELISA is normally done using 96-well polystyrene plates that have been sensitized with a specific antigen. They are relatively cheap, used once, and then discarded. If your ELISA experiment goes wrong, you can come back to the beginning and start again by pressing the reset button. Now, let's take a look at the laboratory. The first step is to choose serum samples. These are samples taken from individuals that may or may not contain the antibodies you are going to test for. You can see eight tubes containing separated blood samples with serum at the top of the tube and red blood cells at the bottom. Up to eight different serum samples can be selected, one for each row of the ELISA plate. Select a sample by clicking the box beneath one of the sample tubes. You will see a menu with all the samples available. Each sample you choose will be assigned to one of the rows on the ELISA plate. In this case, I'm going to use a negative control as the first sample which will go on row A. I'm then going to choose a series of seven different samples from one subject, subject one or S1. The first sample is from day zero, and it goes on to row B. It is listed as S1 d0, meaning that it is from subject one on day zero. The second sample from subject one on day seven, called S1 d7, will go on row C. The third from day 14 will go on row D, and so on. I keep adding samples until I have one sample assigned to every row of the plate. I have now chosen a set of seven samples from subject one. But if you scroll through the list, you will find the samples from all the other subjects. Step two shows a 96-well plate. You will see the identities of the samples chosen in step one are now listed beside each row of the plate. The task in this step is to perform a serial dilution of the serum samples. Put simply, a serial dilution is a successive dilution of a starting sample. You will see why this is important as you progress through the experiment. The serial dilution is performed by adding a known volume of the serum sample to the left-most well on the plate and mixing it with a known volume of diluent. A diluent is simply a liquid medium that is compatible with the sample which we can use to dilute the sample. Every well on the plate contains 100 microliters of diluent. You need to take note of this volume so that you can work out what your serial dilution will be. To make a serial dilution, a volume of each of the serum samples chosen in step 1 is added to the leftmost well on the plate, well 1. It is mixed with a diluent, and then a defined volume is transferred to well 2 and mixed. The same amount is transferred from well 2 to well 3 then from well 3 to well 4, and so on down the plate to well 12. This procedure is carried out with the multichannel pipette illustrated. By transferring a volume of the sample from well to well and mixing it with fresh diluent each time, each sample is successively diluted along the plate. You need to decide what volume you want to transfer from well to well by changing the setting on the pipette. In this case, I'm going to transfer 100 microliter volumes down the plate. So I set 100 microliters on the pipette and press transfer. Since there is 100 microliters of the diluent in each of the wells, transferring 100 microliters will produce a serial dilution where the concentration of the sample decreases by a half each time. In this case, well 1 is a 1 in 2 dilution of the serum sample. Well 2 would be a 1 in 4 dilution, and so on. You can now see the dilutions are given along the top of the plate. The pipette volume can be set at anything between 20 microliters and 100 microliters. By choosing other volumes on the multichannel pipette, you can make other serial dilutions such as one where the concentration of the sample decreases by a third each time, giving 1 in 3, 1 in 9, 1 in 27, and so on. The doubling dilution series shown here is a good starting point. But if serum samples have very high titers of the antibody under investigation, then a smaller transfer volume giving a higher dilution series will be required. In an ELISA, the antibody that binds to the antigen on the plate is called the primary antibody. In step 3, I first have to choose the antigen-sensitized 96-well ELISA plate that I will use for the assay. The dropdown menu lists all the available options. Here I have only one option as I want to detect antibodies to spike protein of the virus. When I choose the spike protein ELISA plate, I'm using a plate where an equal amount of the antigen is bound to every well on the plate. If there are relevant antibodies in the serum samples, then they will attach to the antigen on the base of the wells. At this stage, we have two 96-well plates. One is our antigen-sensitized ELISA plate, and the other contains the serial dilutions of our serum samples. We now transfer the contents of the serial dilution plate to the antigen sensitized plate. This allows any spike-protein-specific antibodies to bind to the antigen on the plate. By pressing the Transfer button, diluted samples are transferred across from the serial dilution 96-well plate prepared in step 2. Importantly, the contents of each well in the serial dilution plate is moved into the corresponding position on the antigen-sensitized ELISA plate. You will notice that when you press Transfer, the wells all turn blue to indicate that liquid has been transferred. Now we incubate the samples on the antigen-sensitized plate by starting the clock. An incubation time of 45 minutes to 1 hour is recommended. [BEEPING] I will stop the incubation now. If you incubate for a short time, the antibody has less time to bind to the antigen, and the signal at the end of the assay will be lower. If you incubate for a bit longer than one hour, it will not make much difference. The incubation allows the antigen and any antibodies in the samples to interact. This is such a strong interaction that we can wash the plate quite vigorously to remove any unbound antibodies and other serum proteins. In essence, washing the plates simply means removing all the liquid from each well and adding a volume of fresh washing buffer. You need to choose how many wash steps to do and for how long you leave the wash liquid to incubate during each wash. Click on the Wash Start button to begin a wash, and press Wash Stop to end a wash. Three washes of five minutes each are the minimum recommended. [BEEPING] If you do not wash the plates sufficiently, residual unbound antibodies in the serum samples will neutralize the detection antibody which will be added later. Step 4 involves detection of any antibody that bound to the antigen in the preceding step. This is done by using an enzyme-conjugated ligand, usually another antibody that is added to all of the wells in the 96-well plate. This detection antibody is called the secondary antibody because it binds to the primary antibody that is bound to the antigen. In step 4, you are presented with three vials that contain secondary antibodies. There are three major classes of antibody present in serum-- immunoglobulin M, immunoglobulin G, and immunoglobulin A, which are referred to as IgM, IgG, and IgA. Each of the secondary antibodies specifically detects one of the classes of serum antibodies. In this demonstration, I want to detect IgG antibodies, so I select the vial anti-human IgG, which is conjugated to the enzyme horseradish peroxidase or HPO. You should also notice that the concentration of the secondary antibody is shown on the label. The stock of the anti-human IgG HPO conjugate has a concentration of 1.5mg per mil. This is too concentrated to use neat, so I need to prepare a dilution. The recommended concentration for the anti-IgG antibody in this assay is 0.6 micrograms per mil. And I need 10 mils of the solution in total to add to the 96-well plate. I therefore need to add 4 microliters of the anti-IgG stock solution to the 10 mils of diluent. The way to calculate the required volume of stock is shown in the panel. The volume of stock is the final concentration divided by the stock concentration multiplied by the final volume. That is 0.6 micrograms per mil divided by 1,500 micrograms per mil multiplied by 10,000 microliters. If you did not follow that, just take it on trust that you need 4 microliters of the stock anti-human IgG. The optimum concentration of secondary antibody depends on the reagent and batch. And in a real lab, each new batch of antibody would be tested to find its activity. Typical optimum concentrations will be in the range 0.5 micrograms per mil to 5 micrograms per mil. Recommended final concentrations of these three secondary antibodies are shown in the table. The pipette volume can be set at anything between 2 microliters and 100 microliters. If you do not use enough of the secondary detection antibody, the signal at the end of the assay will be low. If you use too much, the background values will be high. And in any case, that would be wasteful of an expensive reagent. In step 5, the secondary detection antibody is added to every well on the plate. As before, this antibody is incubated on the plate for 45 to 60 minutes. This incubation allows the secondary antibody to bind to the primary antibody. [BEEPING] If the incubation is too short, the signal at the end of the assay will be reduced. After the incubation, the unbound secondary antibody must be removed from the plate with washes. As before, a minimum of three five-minute washes is recommended. If you add an extra wash or make the washes slightly longer, it will make little difference. But you will find that three washes of five minutes are more efficient than, say, one wash of 15 minutes. In step 6, the enzyme activity of the horseradish peroxidase linked to the secondary antibody is used to produce a colored product in each of the wells. To do this, I will add a chromogen solution to every well on the plate. In this case, there are three options. I will choose OPD which is o-Phenylenediamine. It produces a red-colored end product when acted on by horseradish peroxidase. Once the reaction has started, the color develops progressively with time. At an appropriate time of your choosing, the activity of the horseradish peroxidase enzyme should be stopped by adding sulfuric acid. You should allow enough time for the color to develop so that you can see visible differences between successive wells. If you run the reaction for too long, eventually, every well will develop color and background values will increase. An incubation time of 5 to 30 minutes is typically used for these assays. In this case, I will stop the reaction after 20 minutes by the addition of sulfuric acid. [BEEPING] Finally, in step 7, we get to see the results of the assay by using a 96-well plate reader that can detect the concentration of the red end product in each of the wells. Plate readers shine light at the 96-well plate and record how much light passes through each of the wells. Most plate readers can detect light of several different defined colors. The color is selected by placing an optical filter in the light path. To detect the red-colored end product, a filter of 645 nanometers is appropriate, so I select that here. If I had chosen a different chromogen in the preceding step, then I would need a different filter at this point. For example, ABTS produces a green end product, and the 450 nanometer filter is appropriate. Now we can read the absorbance values. The 96-well plate is taken into the plate reader and the absorbance read. The plate is then returned on the tray. In the final step, you can view the results which are presented in an 8 by 12 array corresponding to their position on the ELISA plate. These results can now be taken for analysis. After you have read the plate using the appropriate filter, you should have a data set that looks like this. I've added a bar at the bottom showing the reciprocal of the serum dilution in each of the wells. The titer of antibody in a sample is usually read as the highest dilution that gives a detectable signal above the background of the assay or the negative control. If I look at the negative control in row A, there is a background value in wells 3 to 12 which is about 0.09. And the highest value in well A1 is 0.108. We can therefore say that an absorbance value above 0.11 is a good cutoff point for assessing whether there is a significant level of absorbance above the background. I have now highlighted all the wells above this threshold. From this, we can read off the titer in each sample. And this is now entered on the right-hand side. Titer is reported as the reciprocal of the highest serum dilution that gives a detectable signal in the assay. The result for subject number one shows a progressive increase in antibody titers up to day 21 following vaccination on day zero. The antibodies are then maintained at this level up to day 56 when the second vaccination was given, and this resulted in a further rise in the titer. The results would be more accurate and reliable if the samples were done in duplicate or triplicate. But it is really important to note that each assay will be slightly different depending on the exact conditions you use. You need to work this out for yourself by looking at your own data, and it will be different on each assay. Also, be aware that duplicate samples will usually give slightly different results reflecting the variation that is seen in this type of assay. I've now shown you how to determine the titer of IgG antibodies against SARS-CoV-2 spike protein using ELISA. You can do a lot of different analyses using this set of serum samples. Here are some suggestions. You could test all 10 subjects at day 21 to find the range of antibody titers induced by the first dose of vaccine. This will also show whether any of them failed to produce an antibody response. You could compare the titers of antibodies after the first and second vaccination to see what effect the second vaccination has on antibody titers. One important question is how long antibodies last for. This can be seen by comparing the titers in all 10 subjects at day 70 and day 200. You could measure IgA antibodies. IgA is important in protecting mucosal surfaces and is therefore very important in immunity to respiratory viruses. The intramuscular immunization with SARS-CoV-2 vaccine is expected to give a strong IgG response, but the IgA response may be less strong. Given information on the age and sex of the individuals, you could compare antibody titers in women and men or between younger and older individuals. The tables on this page give some extra information on the age and sex of the 10 subjects in the vaccine trial, which may be useful for your investigations. And there is a reminder underneath of when the samples were taken in relation to the vaccinations. As you can see, there are many investigations that could be done with this set of serum samples. I hope you have found this introduction to the ELISA technique useful and its application to investigation of COVID-19 antibodies interesting.

This protocol has a primary and secondary dose of vaccine separated by 8 weeks. Samples were taken in the first 21 days to detect appearance of antibodies. A sample was taken just before the second dose of vaccine and again 14 days after the second dose when maximum antibody titres were anticipated. The sample taken 6 months later was intended to detect how long antibodies remained in the serum.

2.1 Laboratory investigation The aim of this investigation is to detect how antibodies are induced following a primary and secondary injection of SARS-CoV2 spike protein. For a phase-1 vaccine trial, assays like this would be carried out on samples from 100s or 1000s of healthy volunteers. Activity 2 Quantitation of antibodies induced by vaccination Allow 30 minutes For this ELISA, you are only supplied with plates sensitised with spike protein; since the vaccination is against spike protein, there is no point in trying to detect antibodies against other viral components. IgG antibodies are most important for conferring immunity, so we ask you to quantitate IgG S-antibodies, using the ELISA protocol that you developed in week 4. ELISA website You will need to use at least two ELISA plates, one for each of the subjects. Set out each ELISA plate with samples from one of subjects, arranged in the order they were taken – day 0, 7, 14, 21, 56, 70, 200. You should also include the standard as a positive control. The day 0 sample will act as negative control in this assay, since the subjects were pre-selected to have no S-antibodies at the start of the trial. The results from each of your subjects should look something like Figure 4. Note that the subject shown in Figure 4 is different from the ones that you are measuring, and that the chromogen OPD has been used, which gives a red end-product. I don’t see this in OSL so haven’t looked at it, I assume it’s very similar to the other one but happy to look at it if someone sends me the link

Notice how IgG antibody titres rise in the 21 days after the first injection at day 0 and then rise sharply following the second injection at day 56. The levels only decline slightly over the following 6 months (d200). The two samples that you have investigated have a very similar time-course profile to this example although the peak titre at day 70 does vary between individuals. What was the titre of IgG antibodies to spike protein at day 70 in the two subjects you measured? The titres determined at day 70 were: S1 = 800 and S2 = 1600. The titres you measured should lie within one well of these values. For example, you might have estimated S1 titre as 512 or 1024, if you did a doubling dilution series. If time allows, you could also look at what happens to IgM or IgA titres in the two subjects. We expect IgM to appear slightly before IgG following the primary injection and decline quite quickly. The titre of IgA is likely to follow quite closely the appearance of IgG, but at a lower titre, because the vaccination was given by intramuscular injection, which tends to induce IgG, rather than IgA.

2.2 Comparison of vaccines As noted previously, it is difficult to directly compare vaccines which have undergone phase-3 trials in different countries or at different times. However it is possible to make some comparisons between vaccines in defined studies. Figure 5 shows antibody production produced by the Oxford/AstraZeneca (AZ) and Pfizer/Biontech vaccines in different age groups and in people with previous SARS-CoV2 infection. Antibodies were measured by an assay that is similar to the ELISA you have used (called RocheS) and the results on the y-axis are expressed as titres. The results are shown as ‘violin plots’, where the width of the ‘violin’ at any particular titre reflects the number of individuals having that level of antibody.

In the age 65-84 age group, which vaccine produced the stronger antibody response? What is the median titre, indicated by the white dot at the centre of the violins. In this age group, the AZ vaccine has a median titre of just over 1000. The Pfizer vaccine has a median titre just below 10,000. So the Pfizer vaccine produces a stronger antibody response in this study. What effect does vaccination have on antibody titres in people who have been previously infected? Both vaccines produce approximately a 100x increase in antibody titres in previously infected individuals, in comparison with previously infected, unvaccinated subjects. This result demonstrates the value of vaccination, even in individuals who have had a natural COVID-19 infection.

3 Vaccination programme The vaccination programme against COVID-19 in the UK started in January 2021, with priority given to older people and those with weak or suppressed immune systems. Over the following 6 months the programme worked down through the age groups and then paused in late Summer, as there was some debate about whether vaccination was necessary for school-age children. The programme was taken up again later in 2021 for the younger age groups, partly because a wave of COVID-19 infections had start to spread in the community from July 2021. Also by Autumn, the benefits of vaccination in children were becoming clearer – although COVID-19 is not usually a serious disease in children, preventing infection and disruption to their education was at least as important as protection against disease. In the UK the Pfizer/Biontech mRNA vaccine and the Oxford/AstraZeneca vector vaccines were both used in the initial vaccination programme. Later the mRNA vaccine from Moderna was also used. All of the initial vaccination schedules required two doses of vaccine spaced at an interval of 3-10 weeks. For booster doses, (3rd injection) which started in October 2021, only the mRNA vaccines were used. Hybrid vaccination – when a person is immunised with one type of vaccine and then boosted with another, was found to be just as effective as using the same vaccine throughout. The uptake of vaccination in the UK is shown in Figure 6.

It should be emphasized that the choice of vaccines to use, the prioritisation and scheduling of the programme was made pragmatically. In the early stages the supplies of vaccines were limited and the ability to deliver them in the community was dependent on the availability of suitably qualified staff who could carry out the immunisation procedure. In these conditions, decisions were taken to vaccinate those most vulnerable to severe disease, protect the health service from being overwhelmed and maximise benefit to the community, by reducing spread of infection.

3.1 Boosters and the duration of immunity As you may have noted from your laboratory investigation, antibody titres decline gradually after 6 months. This is partly due to the progressive loss of antibodies from the serum and partly because the antibody producing plasma cells stop production and die after 1 or 2 months. To maintain protection, it is necessary to give booster doses of vaccine. How often boosters are needed varies depending on the infectious agent. A major problem with SARS-CoV2 and influenza-A is that the viruses mutate regularly. Mutation particularly affects the external proteins involved in attachment to target cells, ie the spike protein of SARS-CoV2 and the haemagglutinin of influenza-A. Mutation of these proteins means that protective antibodies against them may no longer bind; the degree to which this occurs varies between individuals. At this point, it is worth repeating that protection against reinfection is primarily due to antibodies, but protection against disease involves T cells as well as antibodies. The antibodies mostly recognise epitopes exposed on proteins on the surface of the virus, but T cells recognise peptides (presented on MHC molecules) which may come from any part of the viral structural proteins, or even from non-structural proteins expressed in an infected cell. Consequently a variant that has evaded recognition by antibodies, may still be recognised by memory T cells. Next week will look at viral variants and how they evade immune responses.

4 Week 7 quiz Well done for reaching the end of Week 7. Check what you’ve learned by taking the end-of-week quiz. Week 7 practice quiz Open the quiz in a new window or tab then come back here when you’ve finished. 5 Summary This week we covered vaccination against SARS-CoV2 and the use of therapeutic antibodies for passive immunisation. Therapeutic antibodies are only used for vulnerable patients and for people with very serious illness. Antiviral vaccines can be made from inactivated virus or viral components. The most recent vaccines use genes encoding viral components incorporated into a vector, or as messenger RNA (mRNA). The vector vaccines and mRNA vaccines are a more recent technology, but they were the first ones to be approved for general use. More than 50 different COVID-19 vaccines went through trials in humans. Phase-1 trials assess vaccine safety in healthy individuals; phase-2 trials are on a larger scale and include different populations and demographics; Phase-3 trials assess vaccine effectiveness in real-world situations. Effectiveness is assessed as the percentage reduction in infection in the vaccinated group, compared with an unvaccinated group, although other measures of effectiveness may also be assessed, such as reduced number of disease cases or reduced severity of disease. The vaccination programme in the UK was rolled out in 2021, prioritising older people and medically vulnerable individuals. As immunity was gradually lost, booster doses of vaccine were needed to maintain protection. Viral variants can evade protection produced by antibodies against previous variants, but there is still considerable protection against serious illness. Now go to Week 8. Week 8: Variants and immunity 1 Variants of SARS-CoV2 At the start of the pandemic, there was much debate as to whether the original SARS-CoV2 virus, first identified in Wuhan, would mutate to produce new strains. Some viruses, (eg measles) are remarkably stable; the measles vaccine is 97% effective and has not required any substantial modification for many years. In contrast, other viruses such as influenza-A mutate often and it is necessary to produce new vaccines for the new strains each year. Viruses with an RNA genome tend to mutate more rapidly than those with a DNA genome, and those with a segmented genome such as influenza-A, can also change radically by a process called recombination. Since coronaviruses have an RNA genome, we might therefore have reasonably expected some new variants of SARS-CoV2 to arise. It turned out that SARS-CoV2 did mutate extensively, but only some of the variants produced were sufficiently advantaged compared with earlier strains, that they were able to spread in the community. Notice that here we are considering changes that could be advantageous for the virus. Viruses evolve in much the same way as other ‘life-forms’ and a strain with a genetic advantage will eventually replace earlier strains. Suggest three factors which could give a mutated virus an advantage over an earlier strain. The new strain might infect cells more effectively. It might go through its replication cycle more quickly. The assembly of the virus in infected cells or its release could be more efficient. The new strain might be able to evade the antibodies produced by an older strain. Notice that production of disease was not included in this list. A virus that produces an asymptomatic infection might actually have some advantage, because infected people continue to circulate unknowingly in the community, thereby promoting virus spread. However the production of some symptoms such as coughing or sneezing may be advantageous for a respiratory virus, because they promote production of aerosol droplets that transmit the virus. It is not simple. As you read through the following sections on new variants and virus evolution, think in terms of what is advantageous for the virus, rather than its human hosts.

1.1 Genomic sequencing Variants of SARS-CoV2 are detected by genomic sequencing – analysis of the full gene sequence of a virus isolate. Recall that the genome of SARS-CoV2 is 28,000 bases long and it encodes 4 structural proteins and a number of non-structural proteins (Figure 1).

Genomic sequencing takes considerably longer than detecting SARS-CoV2 infection by a lateral flow test or PCR. Once a positive sample has been identified, the virus genome is first extracted from the sample, isolated and then sequenced in segments. The sequences of overlapping segments are then assembled by computer, to produce the complete genome sequence of a variant. This process typically takes at least 2 days, but it is essential for tracking the appearance of new variants. At the time of writing (Jan. 2023) the Sanger Institute, which tracks variants in the UK, had sequenced 2.3 million SARS-CoV2 viral genomes.

1.2 Variation in the SARS-CoV2 genome Mutations in the SARS-CoV2 genome can occur in any of the structural or non-structural genes. For example, the alpha variant (1/Dec/2020) showed 23 mutations from the original strain. 17 of the mutations produced changes in the amino acid sequence of the encoded proteins and 6 did not. Table 8.1 shows the position and type of the 17 ‘non-synonymous’ mutations. Table 8.1 Non-synonymous mutations in the SARS-CoV2 alpha variant.

Gene(s)	Amino acid
Nsp1 – Nsp16	T1001I
	A1708D
	I2230T
Spike	SGF 3675-7 del.
	HV 69-70 del.
	Y144 del.
	N501Y
	A570D
	P681H
	T716I
	S982A
	D1118H
ORF 8	Q27stop
	R52I
	Y73C
Nucleocapsid	D3L
Nucleocapsid	S235F

Mutations in the gene sequence are described according to their position and the effect they have on the amino acid sequence. For example the mutation C3267T that occurred at position 3267 in the gene-sequence was a change from a cytosine base (C) to a thymidine (T). This mutation caused a consequent change in the amino acid sequence of the Nsp1-16 protein, T1001I, meaning that at position 1001 of the protein, a threonine residue (T) has been replaced with isoleucine (I). Notice also that there are 3 deletions in the gene encoding the spike protein that have produced deletions (del.) in the protein of 1-3 amino acids. The key point to take from this table is that the gene encoding the spike protein, which is 10% of the virus genome has more than 50% of the total mutations. In other words, mutation in new variants-of-concern tends to be clustered in the spike protein. Moreover the genome of the variants continued to mutate after it was first identified and sequenced. For example the mutation E484K affecting the spike protein occurred after the initial sequencing of the alpha variant. Some of the mutations that occurred in one variant appeared in other variants. For example N501Y present in the alpha variant also arose independently in the beta and gamma variants, which implies that this mutation confers some selective advantage in different strains.

1.3 Mutation in the spike protein As noted previously, mutations tend to cluster in the gene encoding the spike protein, but even there they are not evenly distributed. For example compared with the original Wuhan strain, Omicron BA.1 has a lot of mutations (15) in the receptor-binding domain (RBD), of which five have been shown to enhance binding to the ACE2 receptor. This strain also has a lot of mutation in the N-terminal domain (NTD) , 4 mutations, 3 deletions and one insertion (Figure 2).

Many of the neutralising antibodies bind to the RBD and NTD regions of the spike protein, so these mutations have allowed the omicron BA.1 variant to substantially evade the antibody response produced by previous variants. Since different people produce antibodies to different regions of the spike protein, the extent to which new variants evade antibodies produced by earlier strains varies between individuals.

2 Variants of concern Thousands of different genetic variants of SARS-CoV2 have been sequenced. A number of these are identified by the World Health Organisation (WHO) as variants of interest (VOI), perhaps because they have spread locally or have mutations that are characteristic of pandemic strains. If a strain spreads more widely it may be designated as a variant of concern (VOC). The four characteristics of a VOC were introduced in week 4 of this course. What are they? Increased transmissibility More severe disease Reduced effectiveness of treatments or vaccines Failure to be detected by current diagnostic tests A VOC strain does not necessarily have all of these characteristics. For example the omicron strain has greatly increased transmissibility over previous strains, but produces less severe disease than the delta strain. Also none of the VOCs to date have been able to completely evade the PCR detection tests or the lateral flow tests. One common feature of later VOCs was that they could partly evade antibodies produced by infection with earlier strains or the initial vaccine formulations which were based on the sequence of the Wuhan strain spike protein. In addition, mutations that allowed a strain to replicate more quickly or spread more effectively, provided a selective advantage and were seen in later VOCs. The effect of increased transmissibility is seen in table 8.2, which gives estimated R₀values for different VOCs. Notice that there is a range of estimated values due to the difficulties of estimating R₀ in different populations, discussed earlier. Table 8.1 Estimated values of R₀ for different SARS-CoV2 variants.

Variant	R₀
Wild-type (Wuhan)	1.4 – 2.5
Alpha	2.8 – 6.4
Beta	2.1 – 3.8
Gamma	2.5
Delta	5.2 – 6.7
Epsilon	1.2 – 1.4
Omicron BA.1	8.2

Remember that R₀ is the rate of spread in a totally susceptible population, so these values represent the basic transmissibility of the different strains. It is noticeable how the alpha delta and omicron strains which caused major waves of infection in the UK all have increasingly high R₀ values. In comparison the gamma and epsilon variants, which did not spread in the UK, have R₀ values similar to the original Wuhan strain.

2.1 Succession of variants Each of the VOCs has gradually replaced previous variants as the dominant strain in the pandemic although this has occurred at different times in different countries. This effect can be seen in the spread of the alpha variant, which was first identified in the UK in Kent in November 2020 from a sample taken in September. The variant spread rapidly in the UK and by January 2021 it accounted for more than half of the identified genomic sequences. By October 2021 it had disappeared from the UK, replaced mostly by the delta variant. By March 2022 the alpha variant was declared to be extinct worldwide (Figure 3).

2.2 Origin and spread of variants New VOCs are usually first identified in the country where they originated. However this depends on the availability of good genomic sequencing facilities and a programme of surveillance. For example, the gamma variant was first identified in Tokyo on Jan. 6th 2021, in 4 travellers arriving from Brazil. Presence of the variant was confirmed in Brazil on Jan. 12th 2021 and retrospective analysis of samples implied that it had been circulating widely in Manaus (Brazil) since December 2020. It is effectively impossible to identify exactly where and who was the first person to develop a new VOC. Variants are most likely to arise where large numbers of people are infected, but this is merely based on probability. Also it has been conjectured that variants are more likely to occur in people who have extended infections, possibly because they have a weak immune response. The delay in clearance of the virus in an immunosuppressed individual could allow the virus more time and opportunity for advantageous mutations to emerge. It is interesting to note that it was 1 year (2020) before the first VOC (alpha) emerged, and in this year the number of infections was relatively low, so there was less opportunity for variants to develop, and there was less selective pressure on the virus from immunity in the host population. Several VOCs emerged in 2021 and 2022 as more people became infected. The rate of spread of pandemic strains of disease has historically been dependent on the fastest form of transport available. For many previous centuries this has been ships and the original term quarantine refers to the isolation of ships carrying (or potentially carrying) infected individuals, for 40 days. More recently, air transport has been the means for the rapid spread of new strains of viruses, including influenza-A, SARS, MERS and SARS-CoV2. Data on the alpha variant shows how it initially spread in Western Europe and North America with later spread to Asia, South America and Africa (Figure 4). There are however some exceptions such as the early identification of the variant in India. This may reflect the travel links that these countries have with the UK, and it may also relate to the effectiveness of their genomic surveillance programmes.

2.3 Lineages of SARS-CoV2 Using genomic data, it is possible to reconstruct the lineages for SARS-CoV2, which are analogous to evolutionary family trees (Figure 5). This diagram shows each genome as a single point. The distance of each genome from the origin, reflects the number of mutations that have occurred since the first genome was published. The distance from other points, reflects the genetic distance between them. The lineage was constructed by estimating the fewest number of mutations needed to reach the given genome.

The diagram illustrates some interesting points about the evolution of the virus. The progressive accumulation of mutations seen in variants such as Gamma. The independent development and great diversity (branching) of the Delta variants. The VOCS which spread most rapidly (alpha, delta, omicron) also show the greatest diversity. A large number of tracked non-VOC lineages arising from the original Wuhan strain. It is likely that the virus will continue to evolve and produce new strains over the next few years.

2.4 Virus evolution What drives the evolution of a virus? Different strains of a virus are subject to two main types of selective pressure. In a susceptible host population, a strain that can spread more quickly and/or reproduce more quickly and efficiently has a selective advantage over other strains. The second pressure on a virus is created by immunity in the host population, which may come from infection or vaccination. With SARS-CoV2, we have seen both of these selective pressures acting on the virus. The increase in R₀ values in later variants indicates that the virus progressively adapted to the human population after it had initially jumped species from bats. The independent appearance of new variants that could evade the immune response against earlier variants, is evidence of the selective pressure produced by immunity in individuals and the population as a whole. It is worth noting here that a virus cannot mutate indiscriminately and still retain its ability to bind to target cells and replicate. Some viral structural components and enzymes must be retained. Immune responses (antibodies or T cells) recognising these less-variable viral components are likely to confer some immunity that acts across different strains. In the last part of the course, we will look in more detail as to how variants have partly evaded immunity produced by previous infections and how vaccines can be modified to provide continued protection against infection and disease.

3 Evasion of immunity As new variants of SARS-CoV2 emerged a major concern was that any new variant would completely evade the immune response produced by infection or by vaccination. The loss of immunity would lead to faster spreading infection, more serious disease, the necessity to reformulate vaccines and require new vaccination programmes. As suggested previously, the changes that have occurred in the spike protein have allowed new variants to avoid recognition by antibodies. This is seen most dramatically with the monoclonal antibodies that are used therapeutically.

3.1 Evasion of therapeutic antibodies Recall that monoclonal antibodies recognise just one position (epitope) on an antigen. If a mutation occurs in the epitope recognised by a monoclonal antibody then there is a high likelihood that the antibody will no longer recognise the epitope or bind to it much less well. Also recall that the therapeutic monoclonal antibodies were mostly selected to bind to the RBD of the spike protein, and this is the region that mutates most often. Figure 6 shows neutralisation curves for 4 therapeutic monoclonal antibodies for different SARS-CoV2 variants. The antibodies were raised against the original strain (D614G). In these graphs the concentration of serum is plotted against its ability to neutralise the virus in vitro. If a low concentration of serum causes good neutralisation (curve to the left of the x-axis) then it demonstrates that the antibody is effective against that variant. Notice how the original wild-type strain (D614G) and alpha strains are neutralised by Bamlanivimab, but the beta and delta strains are not. Etesivimab is effective against the D614G and delta strains, partly effective against alpha and ineffective against beta. Casirivimab is effective against D614G, alpha and delta, but has weak activity against beta. Imdevimab is effective against all four strains.

As some of the therapeutic monoclonal antibodies were seen to be ineffective against later virus variants, they were either withdrawn from use, or combined with other monoclonal antibodies, so that the new variant was less likely to be able to evade both of the therapeutic antibodies.

3.2 Evasion of natural antibody-mediated immunity The antibodies produced by a natural SARS-CoV2 infection or by a spike protein vaccine, are polyclonal; they are produced be many different clones of B cells and usually they will recognise a variety of different epitopes on the spike protein. It is therefore unlikely that a new variant of the spike protein could be so different from an earlier variant that it could completely evade the antibodies produced against an earlier variant or vaccine formulation. However each person produces a different spectrum of antibodies against epitopes on the spike protein, and virus variants will be better able to evade immunity in some people than others (Figure 7)

The three subjects had been naturally infected with an original (wild type (WT)) strain of the virus. In these plots a high dilution of plasma relates to a low concentration of plasma antibodies. The assays were carried out using two wild-type viruses (WT_1D7 and WT_2E1) which had been mutated in two positions (444 and 445) in the spike protein. The plasmas from COV-47 and COV-72 were almost as effective in neutralising mutated virus (orange lines) as the original virus (black lines). However the antibodies in COV-NY plasma were much less effective at neutralising the mutated variants- it requires 100x more antibody to produce a similar level of neutralisation. This result shows that a substantial amount of antibody in COV-NY plasma is binding to an epitope on the spike protein that involves amino acids 444 and 445. This variation between people in antibody production may explain why some people have been more susceptible to reinfection than others. Variation between individuals also applies to susceptibility to infection following vaccination.

3.3 Persistence of immunity Recall from week 2, that there are two major components of immune defence against viruses – T cells recognise and destroy virus-infected cells, whereas antibodies prevent virus spread within the body. Antibodies are also important for preventing reinfection, while T cells limit the damage produced by an infection and consequently reduce disease severity. It has been noticeable during the pandemic, that many people have become reinfected with new strains of the virus, but generally the new infections have produced less-serious disease symptoms. This suggests that T cell immunity persists well, even if the new strains can evade antibody-mediated immunity. The half-life of antibodies in plasma is a few weeks, depending on the class of the antibody. So there is a natural decline in immunity produced by antibodies. However the long-term ability to make antibodies and the ability of T cells to recognise virus-infected cells lies mostly in the memory populations of T and B cells. So some level of immunity lasts much longer than the antibodies. At this time (2023) we cannot know how long immunity to SARS-CoV2 would last if there were no reinfections. However it appears that new variants will continue to develop as the disease becomes endemic and less serious. Consequently previously infected or vaccinated individuals will be subjected to regular restimulation of their B cells and T cells and will always have some immunity. This can be enhanced by boosters with vaccines against new variants. In the final section of the course, we look at the prospects for adapting current vaccines to deal with new variants.

3.4 Adapting vaccines The majority of the COVID-19 vaccines are designed to induce an immune response against the spike protein. The mRNA vaccines have been particularly successful in this regard. Moreover, it is possible to modify an mRNA vaccine quite quickly; the gene sequence encoding the spike protein of any new variant can be rapidly identified and the new sequence is then used in the vaccine. This approach has been taken by both Pfizer/Biontech and Moderna. In January and February 2022 both companies produced a ‘bivalent vaccine’ which included both mRNA of the original spike protein and mRNA for the omicron BA.1 spike protein. These formulations were approved in the UK in August 2022 and used in the Autumn booster programme that year. Additional bivalent vaccines have since been developed (Figure 8).

New formulations are screened for safety and monitored in use to see whether they produce more adverse reactions than the original vaccine. The vector vaccines against spike protein can also be modified relatively quickly, by inserting a gene for the new sequence into the vector. Vector vaccines have some advantages in that they are cheaper to produce and require a simpler cold-chain than mRNA vaccines. However, there is the possibility that an immune response will be produced against components of the vector itself, which means that successive boosts with a vector vaccine become less effective. Production of a variant spike protein, to use in a component vaccine is certainly possible, but likely to be more complicated and time-consuming than modifying an mRNA or vector vaccine. Finally, one must not neglect the potential use of inactivated virus vaccines. Although these were the last to come through the development process during the COVID-19 pandemic, they do have one potential advantage. Since they include all the protein components of the virus (including the core structural proteins that mutate less) they may be more effective at producing cross-strain immune responses. An important final consideration is not just how quickly a variant-specific vaccine can be made in the laboratory, but also how long it takes to scale-up production. Clearly no-one wants to commit to the production of large quantities of vaccine unless it is genuinely necessary. For future years we can anticipate that new COVID-19 booster vaccines will be produced and given selectively to more vulnerable groups, much as influenza-A vaccines have in the past.

4 Week 8 quiz Well done for reaching the end of Week 8. Now it’s time to complete the Week 8 badged quiz. It’s similar to previous quizzes, but this time instead of answering five questions there will be fifteen, covering material from the last four weeks of the course. Week 8 compulsory badge quiz Remember, this quiz counts towards your badge. If you’re not successful the first time, you can attempt the quiz again in 24 hours. Open the quiz in a new window or tab then come back here when you’ve finished. 5 Summary Since it first appeared, the SARS-CoV2 virus has evolved and new pandemic strains have emerged. Many 1000s of variants have been identified by genome sequencing, but only a few of them are designated as variants of interest (VOI) , and fewer still variants of concern (VOC), that spread widely or produce particularly serious disease. New pandemic strains can displace earlier strains if they can replicate more efficiently, evade immune responses, or spread in the community more effectively. Genetic variation can occur throughout the genome, but it particularly affects the spike protein, because changes in the spike protein allow the virus to attach more effectively to target cells and/or evade antibodies. Using genomic sequences, it is possible to reconstruct lineages for the different VOCs. The lineages show how the alpha, delta and omicron variants, which have high R₀ values, have also diversified considerably. As new VOCs have emerged, they spread across the world from their point of origin, eventually replacing previous VOCs, which have gone extinct in the general population. New VOCs have partially evaded antibody-mediated immunity produced by previous strains or by the earlier vaccines. This means that reinfection with new strains does occur. However the residual antibody and T cell immunity still provides some protection, so that any disease from infection, is much less serious in a person who has been previously infected or vaccinated. Booster doses of vaccine can help maintain this residual immunity, and vaccines are being reformulated to include antigens from later variants. Audio 2 Valedictory audio DAVID MALE: We hope you have enjoyed this course on the Immunology and epidemiology of COVID-19. Much of what you have learnt applies more widely to other respiratory virus infections such as influenza. The scientific community has learnt an enormous amount from the COVID-19 pandemic, partly because it has been the first time that rapid genomic analysis was widely available to study viral variants and their global spread. In addition the technology for producing vaccines took a major step forward with the development of the mRNA and vector vaccines. We also hope that using the on-line ELISA laboratory has given you some insight into what occurs in laboratories that are developing vaccines or trying to estimate where a virus has spread in a community. The ELISA technique is widely used in immunology and related disciplines for measuring antibodies and other proteins in biological fluids; it is not just for detecting serum antibodies against SARS-CoV2. So the scientific and technical methods that you have learnt on this course are more generally applicable. If you want to take your studies further or achieve a qualification in this area, The Open University has a Health Science degree programme which includes modules that cover infection, immunity and public health and many other areas of the Health sciences. Now you’ve come to the end of the course, we would appreciate a few minutes of your time to complete this short end-of-course survey (you may have already completed this survey at the end of Week 4). References Prevalence of SARS-CoV-2 in Spain (ENE-COVID): a nationwide, population-based seroepidemiological study. Marina Pollán, Beatriz Pérez-Gómez, Roberto Pastor-Barriuso, Jesús Oteo, Miguel A Hernán, Mayte Pérez-Olmeda , Jose L Sanmartín, Aurora Fernández-García, Israel Cruz, Nerea Fernández de Larrea, Marta Molina, Francisco Rodríguez-Cabrera, Mariano Martín, Paloma Merino-Amador, Jose León Paniagua, Juan F Muñoz-Montalvo, Faustino Blanco, Raquel Yotti, on behalf of the ENE-COVID Study Group* The Lancet 2020, 366: 535-544. Humoral immune response to SARS-CoV-2 in Iceland. Daniel F. Gudbjartsson, Ph.D., Gudmundur L. Norddahl, Ph.D., Pall Melsted, Ph.D., Kristbjorg Gunnarsdottir, M.Sc., Hilma Holm, M.D. et al. N. Engl. J. Med 2020; 383: 1724-1734 DOI: 10.1056/NEJMoa2026116 Institute for Government analysis (2021) ‘Timeline of UK government coronavirus lockdowns and measures, March 2020 to December 2021’. Available at: https://www.instituteforgovernment.org.uk/sites/default/files/timeline-coronavirus-lockdown-december-2021.pdf Accessed: 11 February 2023 A Phase III Randomized, Double-blind, Placebo-controlled Multicenter Study in Adults, to Determine the Safety, Efficacy, and Immunogenicity of AZD1222, a Non-replicating ChAdOx1 Vector Vaccine, for the Prevention of COVID-19. https://clinicaltrials.gov/ct2/show/study/NCT04516746 Wei, J., Pouwels, K.B., Stoesser, N. et al. Antibody responses and correlates of protection in the general population after two doses of the ChAdOx1 or BNT162b2 vaccines. Nat Med (2022). https://doi.org/10.1038/s41591-022-01721-6 Viana, R., Moyo, S., Amoako, D.G. et al. Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa. Nature 603, 679–686 (2022). https://doi.org/10.1038/s41586-022-04411-y Zhanwei Du, Caifen Liu, Chunyu Wang et al. Reproduction numbers of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants: A systematic review and metaanalysis . Clinical Infectious Diseases, 75, e293–e295. https://doi.org/10.1093/cid/ciac137 Weisblum Y, Schmidt F, Zhang F, DaSilva J et al. Escape form neutralizing antibodies by SARS-CoV-2 spike protein variants. eLife 9:e61312. https://elifesciences.org/articles/61312 Acknowledgements This free course was written by Except for third party materials and otherwise stated (see terms and conditions), this content is made available under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 Licence. The material acknowledged below is Proprietary and used under licence (not subject to Creative Commons Licence). Grateful acknowledgement is made to the following sources for permission to reproduce material in this free course: Every effort has been made to contact copyright owners. If any have been inadvertently overlooked, the publishers will be pleased to make the necessary arrangements at the first opportunity. Figures Figure 1: https://dermnetnz.org/topics/viral-warts-images https://creativecommons.org/licenses/by-nc-nd/3.0/nz/ Figure 3: CNRI/Science Photo Library Figure 6: Public Domain. Photo Credit: Content Providers(s): CDC/Dr. Fred Murphy - This media comes from the Centers for Disease Control and Prevention’s Public Health Image Library (PHIL), with identification number #4814 Figure 9: © The Open University based on SARS-CoV-2 / COVID-19 live updates (lubio.ch) Audio visual Video 1: Introduction to the Course: © The Open University (2023) Images: Getty Images Every effort has been made to contact copyright owners. If any have been inadvertently overlooked, the publishers will be pleased to make the necessary arrangements at the first opportunity. Week 2 Figures Figure 1: adapted from: David Male, R. Stokes Peebles, Victoria Male (eds) (2020), (fig 1.15) in ‘Immunology 9th edition’, Elsevier Figure 2: adapted from: David Male, R. Stokes Peebles, Victoria Male (eds) (2020), (fig 6.9) in ‘Immunology 9th edition’, Elsevier Figure 5: adapted from David Male, R. Stokes Peebles, Victoria Male (eds) (2020) (fig10.1) in ‘Immunology 9th edition’, Elsevier Figure 7:adapted from Roitt, I. (1998) ‘Primary and secondary antibody responses’, Immunology, 5th edn. Mosby International Ltd Figure 10: adapted from Roitt, I. et al. (1998) Immunology, 5th edn. Mosby International Ltd Audio Visual Video 1: Immune defence: © The Open University. Diagram: adapted from Male. D, et al (eds) (2020), ‘Immunology 9th edition’, Elsevier Video 2: Apoptosis: ‘Immunology Interactive 3.0’ by Professor David Male, Professor Jonathan Brostoff and Professor Ivan Roitt. Copyright © David Male. Courtesy Professor David Male Video 3: Immune Defence: Courtesy Professor David Male Video 4: Virus infection and immune responses ‘Immunology Interactive 3.0’ by Professor David Male, Professor Jonathan Brostoff and Professor Ivan Roitt. Copyright © David Male. Courtesy David Male Week 3 Figures Figure 1: adapted from: David Male et.al (eds) (2020) in ‘Immunology 9th edition’ published by Elsevier Figure 2: adapted from: David Male et.al (eds) (2020) in ‘Immunology 9th edition’ published by Elsevier Audio Visual Video 1: ELISA laboratory video guide: courtesy: David Male © David Male Week 4 Figures Figure 1: SARS-CoV2 VOCs timeline: courtesy: David Male Figure 2: Daily confirmed COVID-19 cases in the UK: adapted from: https://coronavirus.data.gov.uk/details/healthcare?areaType=nation&areaName=England https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/ Figure 3: COVID-19 vaccination heat map.: adapted from: https://coronavirus.data.gov.uk/details/vaccinations?areaType=nation&areaName=England https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/ Audio Visual Video 1: ELISA laboratory video guide: courtesy David Male © David Male Week 5 Figures Figure 1: Daily incidence of SARS-CoV2 infections in the entire population of the UK adapted from: https://coronavirus.data.gov.uk/details/healthcare?areaType=nation&areaName=England https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/ Figure 2: The protein components of plasma. Courtesy: David Male Figure 3: Prevalence of SARS-CoV-2 in Spain (ENE-COVID): a nationwide, population-based seroepidemiological study Prof Marina Pollán, MD,(Figure 2) in: Prevalence of SARS-CoV-2 in Spain (ENE-COVID): a nationwide, population-based seroepidemiological study - The Lancet Figure 4: Seroprevalence of IgG antibodies to spike protein in UK blood donors: https://www.gov.uk/government/publications/national-covid-19-surveillance-reports/sero-surveillance-of-covid-19 https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/ Week 6 Figures Figure 4: Estimated values of RE in the UK: https://coronavirus.data.gov.uk/details/healthcare?areaType=nation&areaName=England https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/ Audio Visual Video 1: Introduction to week 6: © The Open University. Images: Getty images Week 7 Figures Figure 3: Schedule of a phase-1 vaccine trial. Courtesy David Male Figure 5: from Wei, J., Pouwels, K.B., Stoesser, N. et al. Antibody responses and correlates of protection in the general population after two doses of the ChAdOx1 or BNT162b2 vaccines. Nat Med (2022).Open Access Published: 14 February 2022 Figure 6: from https://coronavirus.data.gov.uk/details/vaccinations?areaType=nation&areaName=England https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/ Audio Visual Video 1: Detecting antibodies against SARS-CoV-2. Courtesy David Male © David Male Week 8 Figures Figure 2: (a)and (b) in Viana, R., Moyo, S., Amoako, D.G. et al. Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa. Nature 603, 679–686 (2022). https://doi.org/10.1038/s41586-022-04411-y open access Figure 3: from Figure 1 | Cumulative sequence count over time B.1.1.7 in https://cov-lineages.org/global_report_B.1.1.7 https://cov-lineages.org/ Figure 4 from Figure 2 | Date of earliest_B.1.1.7 detected in https://cov-lineages.org/global_report_B.1.1.7 https://cov-lineages.org/ Figure 5: https://www.npr.org/sections/goatsandsoda/2022/02/09/1047616658/take-a-look-at-sars-cov-2s-family-tree-its-full-of-surprises https://www.npr.org/https://creativecommons.org/licenses/by/4.0/ Figure 6: from Fig. 1: Neutralization of the SARS-CoV-2 variants D614G, Alpha, Beta and Delta by therapeutic monoclonal antibodies. Planas, D., Veyer, D., Baidaliuk, A. et al. Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization. Nature 596, 276–280 (2021). https://doi.org/10.1038/s41586-021-03777-9 Figure 7: in Weisblum Y, Schmidt F, Zhang F, DaSilva J et al. Escape form neutralizing antibodies by SARS-CoV-2 spike protein variants. eLife 9:e61312. https://elifesciences.org/ articles/ 61312 This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Figure 8: Photo by RINGO CHIU/AFP via Getty Images Don't miss out If reading this text has inspired you to learn more, you may be interested in joining the millions of people who discover our free learning resources and qualifications by visiting The Open University – www.open.edu/openlearn/free-courses. adaptive immune system The adaptive immune defence refers to the tailoring of an immune response to the particular foreign invader. It involves differentiating self from non self and involves B cells and T cells (lymphocytes). A key feature of the adaptive immune system is memory. Repeat infections by the same virus are met immediately with a strong and specific response. antigen Originally defined as any molecule which the body recognised as ‘non-self’, and against which an antibody was produced. This definition was extended to include any molecule that the body could recognise as foreign. This includes the fragments of molecules that are recognised by T lymphocytes. In the broadest sense, it has always been known that the immune system can recognise self molecules, even if it does not usually react against them. Consequently, the widest definition of an antigen is a molecule that can be recognised by the immune system, of which there are conventional non-self antigens and self molecules or autoantigens. anti-viral proteins A group of proteins that are induced by interferon, which when activated, inhibit protein synthesis and viral replication. chronic One that continues to produce disease symptoms and tissue damage over many months or years; some chronic infections (e.g. malaria) are characterised by periods of remission and relapse but the pathogen is never completely eliminated from the body. cytokines short-lived, short-range signalling molecules primarily synthesised and secreted by leukocytes that affect the activity of other cells participating in an immune response. glycoproteins A protein with one or more covalently attached carbohydrate groups (usually short sugar chains). Addition of such groups to proteins, termed glycosylation, is a form of post-translational modification. innate immune system The elements of the immune system that are continuously active and that do not depend on immune recognition of antigens by lymphocytes. Innate immune responses do not improve with repeated encounters with the same antigen or pathogen. interferons Cytokines that interfere with viral replication by the induction of anti-viral proteins. There are 3 main types of interferon IFNα, IFNβ and IFNγ. IFNγ, produced by active T lymphocytes and NK cells has many additional effects in controlling immune responses and inflammation. latent One in which the pathogens persist in or on the host’s body, but without producing symptoms; during the latent period, the host may or may not be infectious (i.e. capable of transmitting the pathogens to others). nucleocapsid The core of a virus containing its genetic material (DNA or RNA), within a protein coat (capsid). receptor-binding domain (RBD) For Sars CoV2, this is the region of the spike protein that binds to the ACE2 receptor on a cell, as the first step in virus infection of the cell. sterile immunity The complete elimination by the host’s immune response of the pathogens responsible for an infectious disease (e.g. the influenza virus is eliminated from the body as the illness resolves). toll-like receptors A group of receptors, located on the plasma membrane or on intracellular vesicles, that recognise components of pathogens (PAMPs) and transduce signals for inflammation. type-1 interferon (IFN) A cytokine produced by many cell types that signals to other cells to inhibit replication of viruses. viral envelope A phospholipid membrane that surrounds the nucleocapsid of some groups of virus. It is derived from the plasma membrane of the virus-infected cell. viral tropism The tendency of a particular virus to target specific cells which it can infect and then replicate within. antigen processing The process by which antigen is presented to lymphocytes in a form they can recognise. Most CD4+ T cells must be presented with antigen on MHC class II molecules, while CD8+ CTL cells only recognise antigen on MHC class I molecules. Antigen must be processed into peptide fragments before it can associate with MHC molecules apoptosis Type of cell death where particular cell populations die in a reproducible manner in every individual. Because of its predictable nature, this form of death was believed to occur as the result of a death ‘programme’, and so was named programmed cell death. Well-known examples are the loss of the cells between the digits (e.g. during the development of fingers). In adult tissues, cell death usually balances cell division, ensuring that tissues and organs retain the same size and structure as old cells are replaced. Apoptosis is a normal response in cells with DNA damage, serving to protect the body from cancer. B cells One of two main types of lymphocyte (cf. T cells) which, when activated, synthesises and releases huge quantities of soluble antibodies. IgA class of antibody that is prevalent in mucous secretions, and protects against infections in the gut, respiratory tree and genitourinary tract. IgG The main antibody in blood and tissue fluid. It has a large number of functions, including neutralising many toxic molecules, preventing viruses from attaching to cells, allowing phagocytes to recognise and internalise pathogens, and protecting the fetus and newborn babies. (It is the only antibody class that can cross the placenta.) IgM A class of antibody that is the first to be produced in an immune response. immunoglobulins An alternative name for soluble antibodies present in serum and tissue fluids. immunological memory the ability of the adaptive immune system to make an improved immune response on repeated encounters with the same antigen or pathogen. lymphatic system the connected system of lymphoid organs and lymphatic ducts present throughout the body. lymphocytes A major population of leukocytes including T cells, B cells and NK cells. lymphoid organs encapsulated organs such as thymus, lymph nodes and spleen and tonsils which contain collections of lymphocytes and cells involved in development of immune reactions. lymphoid tissues include both the encapsulated organs of the immune system (eg lymph nodes) and unencapsulated collections of lymphocytes found in mucosal tissues. MHC molecules A group of proteins involved in antigen presentation to T cells. NK cells A group of lymphocytes that have the intrinsic ability to recognise and destroy some virally infected cells and some tumour cells. proteasome an intracellular organelle that breaks down proteins into polypeptide fragments. T cells Lymphocytes that differentiate primarily in the thymus and are central to the control and development of immune responses. The principle subgroups are cytotoxic T cells (Tc) and T helper cells (Th). Assay A method for quantitating biological material – in this case antibodies. ELISA Enzyme linked immunosorbent assay, is a set of techniques used for detection and quantitation of antibodies or antigens. incidence of infection The number of new cases of an infection in a given number of people in a defined period of time. notifiable diseases Diseases that must by law be reported to health authorities. S-antibodies Antibodies against the SARS-CoV2 spike protein. variants of concern’ (VOC) Variants of the SARS CoV2 virus identified by the WHO as having any combination of these characteristics: increased rate of transmission; producing more serious disease; ability to evade treatments or immune responses produced by vaccines; not being identified by current test procedures. incidence The number of new cases of a disease arising in a given period, usually a year, expressed as a proportion of the population at risk (the incidence rate). N-antibodies antibodies against the SARS-CoV2 nucleocapsid. plasma The non-cellular fluid component of blood containing soluble molecules, including proteins. prevalence The proportion of the population with a particular infection or disease at a particular point in time, or during a given period. seroconversion The appearance of specific antibodies in the blood serum as a result of infection or vaccination. serology The study of antigens and antibodies in patients’ sera. seroprevalence The proportion of a population which have antibodies against a particular infection at a defined time point. serum (Plural, sera.) The part of the blood left behind after cells, platelets and fibrinogen have all been removed, usually by clotting. Basic reproduction number (R₀) The average number of individuals directly infected by a single typical infective if the population were totally susceptible.

450nm
Sample	1	2	3	4	5	6	7	8	9	10	11	12
A: 1-Standard	1.305	1.454	1.277	1.422	1.263	1.066	0.607	0.373	0.202	0.152	0.117	0.104
B: 2-Neg.Con	0.106	0.092	0.085	0.084	0.087	0.080	0.081	0.083	0.086	0.087	0.084	0.084
C: N9921	1.331	1.262	1.452	0.713	0.413	0.252	0.157	0.127	0.104	0.094	0.088	0.084
D: C4443	0.141	0.110	0.102	0.095	0.084	0.086	0.083	0.084	0.080	0.083	0.084	0.087
E: C5050	1.415	1.327	1.173	0.638	0.373	0.239	0.158	0.117	0.101	0.096	0.088	0.085
F: H1151	1.328	1.280	0.966	0.519	0.308	0.197	0.136	0.112	0.094	0.085	0.089	0.087
G: F1949	0.112	0.097	0.094	0.090	0.087	0.088	0.087	0.087	0.085	0.080	0.086	0.084
H: Z8207	1.314	1.454	1.334	1.418	4.458	1.427	0.921	0.485	0.272	0.188	0.125	0.103
	2	4	8	16	32	64	128	256	512	1024	2048	4096