Regulatory Rapporteur
February 2025 | Vol. 22 | No. 2
Part 1: Elements of the immune response important to vaccine effectiveness
Abstract
This article explores the critical elements of the immune response that contribute to vaccine effectiveness, with reference to the European Medicine Agency’s (EMA) recent scientific guidance on clinical evaluation of new vaccines.
Vaccines exploit both the innate immune response, which provides a nonspecific first line of defence as well as the adaptive system that responds to infection through both antibody (humoral) and cellular (T-cell) responses. This article delves into the immune response to vaccination, highlighting how vaccines stimulate the immune system. It also explores the various antibody classes and their functions.
The article emphasises the importance of understanding both antibody and T-cell responses in vaccine development, noting that while antibody responses have been the primary focus, the roles of innate immunity and T-cell responses should not be underestimated. Developing a comprehensive understanding of the immune response during vaccine development allows for improved prediction of vaccine effectiveness and may help streamline the development process for novel vaccines.
Introduction
The European Medicines Agency (EMA) published a scientific guideline on ‘Clinical evaluation of new vaccines’, effective from 1 August 2023 (to be referred to henceforth as the ‘EMA scientific guideline’). This guideline specifies the need for characterisation of the immune response for each antigenic component of a vaccine. Such characterisation demands the inclusion of multiple exploratory immunological endpoints, which help to predict the potential therapeutic value of a vaccine early in its development.
Specifically, the EMA scientific guideline recommends evaluating secondary endpoints such as the induction of immune memory, cross-reactive antibodies, cross-priming (for example, induction of immune memory to a different antigen), cell-mediated immunity (CMI), the correlation between cytokine or gene expression profiles, and an immune correlate of protection. The successful development of a vaccine therefore requires diverse multi-disciplinary expertise such as bioanalytics, nonclinical, clinical, regulatory, biostatistics and, in particular, immunology.
The immune system comprises two distinct components, the innate and adaptive immune systems. The innate immune system provides a nonspecific first line of defence, whereas the adaptive system responds to infection by recognising non-self-antigens through both an antibody (humoral) and cellular (T-cell) response. Vaccination exploits both these immune system components to enhance protection against pathogens but regulatory focus has been on the adaptive immune response, the type and intensity of which can vary, driven by many factors, some related to the vaccine and others that are host- and environmentally-related.
The EMA scientific guideline expresses a preference that: ‘whenever possible, each immune parameter is assayed in a single central laboratory and that the same laboratories are used throughout the clinical development programme. If this is not possible, the potential impact of inter-laboratory variability on the results and conclusions of clinical trials (CT) should be addressed in the application dossier. Protocols should specify the assays to be used to evaluate immune parameters and key assays should be fully validated’.[1]
Antibody response to vaccination
Historically, vaccines have comprised attenuated live or killed viruses or bacteria or, in the case of subunit vaccines, isolated or recombinant antigens (for example, the hepatitis B vaccine), deactivated endotoxins (for example, diphtheria and tetanus) or polysaccharides from bacteria (for example, Haemophilus influenza type B and pneumococcal vaccines). The utility of vaccines is now being expanded with the application of innovative technologies; the most prominent of these is the messenger RNA (mRNA) vaccines but other approaches are also being adopted including the use of nanoparticle, DNA and viral vector vaccines, as well as enhancement of efficacy of the classical approaches by introducing novel adjuvants.[2]
The key objective of a vaccine is to prime and enhance the adaptive immune response against infectious organisms. The lymphocytes (B- and T-cells) are the key cells involved in adaptive immunity, and these are the cells that respond to vaccination and confer the immune response. Antibodies, secreted by B-cells, are the primary protective response arising from vaccination and are certainly the most studied response. T-cell response is, however, also important and its role following vaccination is likely under-rated. In fact, sometimes, the T-cell response provides the primary protection; for example, cellular immunity induced by Mycobacterium bovis (BCG) is the key protective function against tuberculosis. In the case of rotavirus, neutralising antibodies, non-neutralising antibodies, secretory antibodies, and cellular immune responses may all play a role, depending on the situation.[3] In the case of SARS CoV2, while the focus has been on neutralising antibody titre, in fact, immune memory and cellular immunity are also important.[4]
Harnessing the innate immune system
The adaptive immune response is facilitated and supported by the innate immune system, which provides a nonspecific but rapid first line of defence. Vaccines comprising attenuated live organisms, such as BCG or against smallpox or yellow fever, stimulate the innate immune system in the same way as an infectious organism would. To a lesser extent, this also applies to vaccines containing whole killed organisms. Where only the antigen subunit is included, an adjuvant is traditionally added to initiate an innate immune response. Historically, alum has been used as the adjuvant of choice, but newer adjuvants are now being deployed. The need and amount of adjuvant must be supported by safety and immunogenicity data, including the level of enhancement of the immune response in the clinical setting. [1]
Adjuvants may achieve their effect in many ways. These include the following:[5]
- Concentrating the antigen over a small area, which brings them into close proximity to each other and thereby makes them more easily recognised as foreign by the immune cells
- Retention of the antigen at the injection site, therefore prolonging the immune stimulus
- Inducing inflammation at the injection site, which attracts antigen presenting cells to that site
- Replicating pathogenic antigens that are recognised by the innate immune system, such as ligands for toll-like receptors (TLRs)
- Induction of cytokine release that attracts immune cells to the injection site
The cells involved in innate immunity are the leukocytes. These recognise and bind foreign molecular patterns typically associated with pathogens through TLRs and other pattern recognition receptors (PRRs), which include lipoproteins, foreign nucleic acid structures and proteins typically associated with viruses, bacteria or other infectious agents.[3]
TLR molecules can be used as adjuvants and provide an effective approach to stimulating an immune response. A TLR agonist absorbed on alum (AS04) has already been approved for use in a human papilloma virus vaccine as well as in a vaccine against hepatitis B. AS04 stimulates innate immunity by interaction via TLR-4, which recognises lipopolysaccharides.[6] AS01B used in a widely adopted shingles vaccine also interacts via TLR-4. CpG DNA is recognised by TLR-9 which binds bacterial DNA and is used as an adjuvant in a different hepatitis B vaccine. Other PRRs that can act as sensors for RNA, DNA and C-type lectins also represent excellent targets for adjuvant development.[7] Where novel adjuvants are used, this will need to be supported by nonclinical and extensive clinical safety data.[1]
There are eight defined subtypes of leukocytes (white blood cells) involved in innate immunity; these are basophils, neutrophils, eosinophils, mast-cells, macrophages, monocytes, dendritic cells and natural killer cells. Basophils and mast-cells are involved in release of cytokines such as histamine, while eosinophils, macrophages, monocytes and natural killer cells act by engulfing and digesting pathogens and diseased cells, a process known as phagocytosis.
When a TLR on the leukocyte surface binds to a target molecule, chemical messengers such as cytokines are released. Cytokines act in concert with each other, inducing or inhibiting inflammatory responses. The initial response to a pathogen associated antigen is the secretion of chemokines which attract cells of both the innate (leukocytes) and adaptive (lymphocytes) immune system to the site of infection or injury. Cytokines are then secreted; these can directly kill pathogens as well as facilitating wound healing and angiogenesis.[5] The EMA scientific guidelines recommend investigation of the correlation between cytokine profiles and antibody levels and events, such as immune-mediated adverse effects.
Another key mechanism by which the innate immune system facilitates the adaptive immune response is via antigen presentation to T-cells; in this respect it is the macrophages and dendritic cells that play a key role.
B-cell response
Of primary interest, following vaccination, is the increase in titer of circulating antibodies against the target infectious agent. The EMA guidelines require monitoring of the magnitude and kinetics of this response, for example, the time to reach peak antibody levels and the antibody decay curve.[1] Antibodies are secreted by the B-cells, which recognise foreign antigens via receptors on their surface. The B-cell receptors are extremely selective and bind only to a specific epitope (short amino acid sequence) of a foreign antigen. When a B-cell is activated by an antigen, it proliferates and differentiates into an antibody-secreting cell, known as a plasma cell. Most commonly B-cells reside in the follicles of lymph nodes, and these are responsible for generating the majority of high-affinity antibodies during an infection. Following infection or vaccination, the level of the circulating antibodies against the infectious agent gradually declines, but memory B-cells persist, enabling a rapid response to subsequent exposure. The EMA scientific guidelines suggest immune memory is studied as one of the objectives in vaccine trials.[1]
For infections with extended incubation times, the more rapid immune response, following vaccination, can completely prevent occurrence of illness and its transmission, whereas for infections with short incubation periods, a more rapid response serves to ameliorate rather than prevent infection. Long-lived plasma cells may also persist and continue to generate or synthesise antibodies and, if adequate levels are maintained, these provide extended protection against re-exposure and entirely prevent infection.[5][8] Interestingly, memory B-cells mutate over time, resulting in the development of a repertoire of antibodies which may possess increased binding affinity and/or the ability to neutralise emergent or different strains of an infectious organism. However, cross reaction with epitopes on healthy tissue can occur, leading to autoimmune conditions. Consequently, the potential for cross-reactivity should be considered as part of the CT programme.[1]
In addition to antigen-binding, antibodies display a second binding interaction through their Fc region, which interacts with cells of the adaptive and innate immune system to direct a multi-pronged immune assault against cells or organisms displaying the target epitope. Antibodies also bind a protein known as complement, activating the complement cascade which elicits a cytotoxic action against infected or foreign cells.
There are five classes of antibody produced by B-cells: IgM, IgG, IgA, IgD and IgE and sub classes of some of these, each playing different rolls. EMA guidelines recommend measuring total binding titers of the main immunoglobulin subclasses (IgM, IgG and IgA).[1] The first administration of a vaccine often produces a low immune response, initially generating low-affinity antibodies at low titers which are predominantly IgM. After about two weeks following first vaccination and generally more rapidly following subsequent inoculations (boosters), a stronger and more mature response occurs, typified by the production of higher titers of IgG antibodies. IgG is the most common type of antibody found in serums. This comprises about 75% of serum antibodies and exists as four subclasses (IgG-1, IgG-2, IgG-3, IgG-4), which react with components of the immune system in different ways. In addition to these subclasses, different subject specific allotypes and polymorphic forms exist (for example, of IgG-1) and these have been found to be associated with varying responses to vaccination and infection, for example against HIV.[9][10][11] The Fc region of antibodies bind to receptors expressed on the surface of cells of the innate immune system, such as NK cells, stimulating release of cytokines and/or inducing cytotoxicity; these Fc receptors also exist in several forms, e.g. Fc γR1, FcγR2a, FcγR2b, FcγR3a and FcγR3b and these too can display specific polymorphism which impacts strength of binding to the Fc region of the antibody impacting the strength of immune response.
IgA plays a key role in protecting mucosal surfaces and it may be advantageous to selectively induce an IgA response to protect against pathogens that target mucosal surfaces, a common port of entry. This can be achieved by administering vaccines intranasally and via other mucosal routes such as oral administration and use of mucosal adjuvants. There are significant hurdles to mucosal vaccine development, including incomplete knowledge of the nature of protective mucosal immune responses, and potential for poor local tolerance. Examples of mucosal adjuvants are cytosine-phosphonothioate-guanine (CpG) and adenosine diphosphate (ADP)-ribosylating enterotoxins, which stimulate an IgA antibody response in addition to a cellular immune response when included in vaccines administered via the mucosal route.[12] IgA deficiency is relatively common and estimated to affect one in 400 live births, although this varies among different ethnic groups.[13] While most people with selective IgA deficiency do not, as a rule, have recurrent infections, some may experience repeated infections, and IgA deficiency is reported to be associated with autoimmune disease and allergies.[13]
IgD is expressed in human nasal, lacrimal, salivary, mammary, bronchial, pancreatic and cerebrospinal fluids but its role is not well understood; it seems also to be involved in antigen recognition as it is primarily found on the surface of B-cells.[14] IgE is the least abundant antibody and is considered to be protective against parasites but is also very important in that it is responsible for allergy and anaphylaxis.
In addition to neutralisation of toxins and pathogens, antibodies can invoke a secondary attack on binding to cells expressing the target antigen such as viral infected cells, bacteria, parasites or cancer cells. The secondary attack is carried out by the cytotoxic T-cells, as well as cells of the innate immune system such as natural killer cells and macrophages.
The role of T-cells
T-cells directly attack cells expressing foreign antigens. There are a number of different types of T-cells that express different glycoproteins on their surface, most notably the cluster differentiation transmembrane glycoproteins CD4 and CD8, which can act as receptors or ligands and so facilitate interaction of T-cells with other immune cells. Cytotoxic T-cells express CD8, whereas helper T-cells express CD4. Helper T-cells do not kill cells directly but send signals in the form of cytokines that direct other immune cells such as cytotoxic T-cells, B-cells and macrophages to fight an infection. Flow cytometry and multiplex-immunoassays can help build useful insights into the T-cell response.
Genetically modified chimeric antigen receptors-T-cells (CAR-T-cells) have been developed as an individualised cell-based therapy against certain cancers. CAR-T-cells are produced by extracting T-cells from the patient, which are then modified to express receptors to antigens expressed on the patient’s cancer cells. This allows the T-cells to recognise antigens expressed on the cancer cells and activate the T-cells’ ability to kill these. CAR-T-cells have proved highly effective in controlling certain cancers such as acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL).
The general view is that antibodies supported by helper T-cells play a major role in preventing infection, whereas cytotoxic T-cells are required to control and clear established infection. As such cytotoxic T-cells do not prevent spread of infection but rather facilitate recovery, although the role of T-cells in preventing infection may be underrated; certainly T-cells protect against TB and there are reports that T-cells can immediately eradicate SARS-CoV-2 infection in some people.[4]
Like B-cells, cross-reactivity is also known to occur with T-cells. For example, cross-reactivity with antibodies against prior circulating coronaviruses has resulted in T-cell-driven immunity to SARS CoV2 in some people.[15][16] This observation has led to attempts to develop vaccines that specifically stimulate T-cells, but this has proved challenging.
Antigen processing
Following vaccination or infection, the associated antigens are processed in different ways depending, for example, on whether they were synthesised within the cell, as might occur following administration of an mRNA, DNA or live vaccine, or are located extra cellularly, as would be the case for a killed or subunit vaccine. Antigens synthesised within the cell generate peptides that are displayed on the cell surface, where they are recognised and then eliminated by CD8 T-cells. Most human cells can present antigens in this way, but some are specialised as antigen presenting cells (APC); these include B-cells, macrophages and dendritic cells.
Within the APC, protein antigens are broken down into small peptides which are displayed on the cell surface attached to a self-antigen known as a major histocompatibility complex (MHC). This step is important because T-cells are unable to recognise a foreign antigen unless it is attached to an MHC. The MHC varies in structure across the human population. There are two MHC classes, MHC-I and MHC-II. Each human cell expresses six MHC class I alleles (one HLA-A, -B, and -C allele from each parent) and six to eight MHC class II alleles (one HLA-DP and -DQ, and one or two HLA-DR from each parent, and combinations of these). The MHC variation in the human population is high, so that any two individuals who are not identical twins are almost certain to express differing combinations of MHC antigens.[5]
CD8 only binds to MHC-1, which is expressed on cytotoxic T-cells and most non-immune cells, resulting in activation of the cytotoxic T-cell. The activated CD8 T-lymphocytes directly attack cells expressing foreign antigens as might happen following viral infection or cancer; they are also able to secrete signaling proteins, known as cytokines, to recruit other immune cells.
CD4 is expressed on B-cells, macrophages and dendritic cells and binds to MHC-II, resulting in the activation of helper T-cells.[5] Unlike CD8 cytotoxic T-cells, the CD4 T-cells function by activating memory B-cells and cytotoxic T-cells to enhance the immune response. The specific adaptive immune response regulated by the helper T-cells depends on its subtype, such as T-helper1 (Th1) and T-helper2 (Th2) distinguished by the types of cytokines they secrete.[5] Th1-type cytokines tend to produce proinflammatory responses with interferon gamma as the major cytokine expressed; if unchecked they can lead to uncontrolled tissue damage. The Th2 response dampens the Th1 response through secretion of the anti-inflammatory cytokine IL-10. The Th2 response also includes secretion of interleukins 4, 5 and 13, which are associated with the promotion of IgE secretion and eosinophilic responses in allergy. The optimal response therefore requires balanced Th1 and Th2 response.[17]
An imbalance in the Th1 and Th2 response has been associated with the termination of the development of a formaldehyde-inactivated vaccine against respiratory syncytial virus. Infants vaccinated with the candidate FI vaccine developed more severe symptoms when exposed to natural infection and two babies died.[18] This phenomenon is known as enhanced respiratory disease exacerbation.
For completeness, it is also worth mentioning the more recently identified Th17 cells that are characterised by secretion of IL-17 and that may have evolved for microbial protection, for example extracellular bacteria and some fungi, where Th1 or Th2 immunity is not effective.[19]
Regulatory T-cells are yet another distinct population that provide the critical mechanism of tolerance, whereby immune cells can distinguish invading cells from ‘self’. Finally, tissue resident memory T-cells should be mentioned; these are thought to reside permanently within tissues and provide a first-line defence against pathogens.[20]
Conclusion
Enhanced understanding of the immune response has accelerated development of novel vaccines. The EMA and the Committee for Medicinal Products for Human Use have recently published a scientific guideline on the ‘Clinical evaluation of new vaccines,’ which emphasises the need to characterise the immune response for each antigenic component of a vaccine. Vaccination science exploits both the innate and adaptive immune systems to maximise protection in the ongoing battle against current and emergent infectious diseases. When the focus has been on the antibody responses, the role of innate immunity and T-cell response should not be underestimated. Part 2 of this article will address how understanding the immune response allows for improved prediction of vaccine effectiveness and may negate, or at least limit, the need for large, costly and time-consuming clinical effectiveness trials and meet the needs of diverse populations.
References
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