Immunology of Vaccination

By: P. C. L Beverley

Scientific Head

The Edward Jenner Institute for Vaccine Research, Compton, Berkshire, UK

British Medical Bulletin 2002

The review describes some of the most important stages of an immune response and raises some of the issues that need to be resolved if progress toward a new generation of vaccines is to be made.

Properties of an Ideal Vaccine

Should give life-long immunityShould be broadly protective against all variants

of an organismShould prevent disease transmission, e.g. by

preventing sheddingShould induce effective immunity rapidlyShould be effective in all vaccinated subjects,

including infants and the elderlyShould transmit maternal protection to the fetusRequires few (ideally one) immunizations to

induce protectionWould not need to be administered by injectionShould be cheap, stable (no requirement for cold

chain), and safe

Adjuvant: Agents which increase the stimulation of the immune system by enhancing

antigen presentation (depot formulation, delivery systems) and/or by providing co-stimulation signals (immunomodulators). Aluminium salts are most often used in today’s vaccines.

Affinity, avidity: The antibody affinity refers to the tendency of an antibody to bind to a specific

epitope at the surface of an antigen, i.e., to the strength of the interaction. The avidity is the sum of the epitope specific affinities for a given antigen. It directly relates to its function.

Affinity maturation: Processes through which antigen-specific B cells undergo somatic hyper mutation

and affinity-based selection, resulting into B cells that produce antibodies with increased affinity over germ line antibodies.

Antibodies: Proteins of the immunoglobulin family, present on the surface of B lymphocytes,

secreted in response to stimulation, that neutralize antigens by binding specifically to their surface.

Antigen presenting cells: Cells that capture antigens by endo- or phagocytosis, process them into small

peptides, display them at their surface through MHC molecules and provide co-stimulation signals that act synergistically to activate antigen-specific T cells. Antigen presenting cells include B cells, macrophages and dendritic cells, although only dendritic cells are capable of activating naïve T cells.

B lymphocytes: Cells that originate in the bone marrow, mature in secondary lymphoid tissues,

become activated in the spleen/nodes when their surface immunoglobulins bind to an antigen and differentiate either in antibody secreting cells (plasma cells) or in memory B cells.

Carrier protein: A protein that is used as a template to which polysaccharide moieties are

chemically conjugated to generate glycoconjugate vaccines. It is currently considered that carrier proteins provide antigenic epitopes for recognition by CD4+ helper T cells, in particular follicular helper T cells.

CD4+ T helper 1 lymphocytes: CD4+ T cells that upon activation differentiate into cells that mainly

secrete IL-2, IFN-γ and TNF-β, exerting direct antimicrobial functions (viruses) and essentially providing support to cytotoxic T cells and macrophages.

CD4+ T helper 2 lymphocytes: CD4+ T cells that upon activation differentiate into cells that mainly

secrete IL-4, IL-6, IL-6, IL-10, IL-13, exerting direct antimicrobial functions (parasites) and essentially providing support to B lymphocytes.

Central memory T cells: Memory T cells trafficking through the lymph nodes, ready to proliferate

and generate a high number of effector cells in response to specific microbial peptides.

Chemokines: Small secreted proteins that function as chemoattractants, recruiting cells

that express the corresponding chemokine receptors at their surface and thus migrate towards higher concentrations of chemokines.

Costimulatory molecules: Molecules that become expressed at the surface of antigen presenting cells

upon activation and deliver stimulatory signals to other cells, namely T and B cells.

Dendritic cells: Cells that constantly sample the surroundings for pathogens such as

viruses and bacteria, detect dangers and initiate immune responses. Immature patrolling DCs have a high endocytic activity and low T cell activation potential. Contact with a pathogen induces maturation and the expression of certain cell-surface molecules, greatly enhancing their ability to activate T cells.

Effector memory T cells: Memory T cells patrolling through the body to detect specific microbial

peptides and capable of an immediate cytotoxic function in case of recognition.

Extrafollicular reaction: B cell differentiation pathways that occur outside of germinal centers,

in response to protein or polysaccharide antigens. Rapid, it generates B cells that are short-lived (days) and produce low affinity antibodies, without inducing immune memory.

Follicular dendritic cells: Stromal cells in spleen and nodes that upon activation express

chemokines (notably CXCL13) attracting activated antigen specific B and T cells, and thus nucleate the germinal center reaction. FDCs provide anti-apoptotic signals to GC B cells and support their differentiation into plasma cells or memory B cells.

Follicular helper T lymphocytes: CD4+ T cells that upon activation migrate towards follicular dendritic

cells and provide a most critical help to germinal center B cells, influencing isotype switching, affinity maturation and differentiation.

Germinal centers: Dynamic structure that develop in spleen/nodes in response to an

antigenic stimulation and dissolves after a few weeks. GCs contain a monoclonal population of antigen-specific B cells that proliferate and differentiate through the support provided by follicular dendritic cells and helper T cells. Immunoglobulin class switch recombination, affinity maturation, B cell selection and differentiation into plasma cells or memory B cells essentially occur in GCs.

Isotype switching: Switch of immunoglobulin expression and production from IgM to IgG,

IgA or IgE, occurring during B cell differentiation through DNA recombination.

Marginal zone: The marginal zone is the area between the red pulp and the white pulp of the

spleen. Its major role is to trap particulate antigens from the circulation and present it to lymphocytes.

Regulatory T cells: T cells that upon activation differentiate into cells that express specific cytokines (IL-

10, TGF-β/surface markers) and act to suppress the activation of the immune system through various mechanisms, maintaining immune homeostasis and tolerance to self antigens.

Somatic hypermutation: Process that introduces random mutation in the variable region of the B cell receptor

(i.e., immunoglobulin) locus at an extremely high rate, during B cell proliferation. This mechanism occurs through the influence of the activation-induced cytidine deaminase (AID) enzyme and generates antibody diversification.

T lymphocytes: Cells that originate in the thymus, mature in the periphery, become activated in the

spleen/nodes if 1) their T cell receptor bind to an antigen presented by an MHC molecule and 2) they receive additional costimulation signals driving them to acquire killing (mainly CD8+ T cells) or supporting (mainly CD4+ T cells) functions.

T-independent B cell responses: Differentiation pathway of B cells, mainly elicited by polysaccharides, that takes

place in the marginal zone and extrafollicular areas of spleen/nodes. Its hallmarks are to be rapid (days) but to elicit the transient (months) production of antibodies of low affinity, without inducing immune memory.

T-dependent B cell responses: Differentiation pathway of B cells elicited by protein antigens that recruits T and B

cells into germinal centers of spleen/nodes. Its hallmarks are to be slow (weeks) but to elicit long-lasting (years) production of antibodies of high affinity, and immune memory.

Toll-like receptors: Family of 10 receptors (TLR1 to TLR10) present at the surface of many immune

cells, which recognize pathogens through conserved microbial patterns and activate innate immunity when detecting danger.

Initiation of Immune ResponsesDanger Signals

A key stage of any immune response is the phase of initiation. Antigens must be recognized as foreign for an immune response to occur.

Microorganisms are usually recognized because they carry ‘danger’ signals that signal the immune system through conserved pattern recognition receptors.

Tissue damage also leads to the expression of self molecules that can also activate cells of the innate immune system.

The receptors for external and internal ‘danger’ signals are diverse. They include low affinity IgM, serum mannan binding protein, pentraxins and cellular receptors such as complement receptors, mannose and other lectin-like receptors for carbohydrates, the phosphatidylserine receptor, heat shock proteins and the recently described family of IL-1R-Toll-like molecules.

The latter may function as homodimers, but they frequently form heterodimers with other Toll-like receptors or may work in concert with other cell surface or soluble molecules such as CD14.

Pattern Recognition Receptors (PRP)

Pathogen Associated Molecular Patterns (PAMP)

Pattern Recognition ReceptorsReceptor Ligands

TLR2 + (TLR6 or TLRx) Bacterial lipoproteins, peptidoglycan TLR3 dsRNA TLR4 LPS, Taxol, HSP60 (human and chlamydial)

Fibronectin, F protein (respiratory syncytial virus) TLR5 Flagellin TLR9 CpG DNA FcγRs Pentraxin-opsonised zymosan, serum

amyloid P, C-reactive protein CR1 (CD35) Complement opsonised bacteria

and fungi CR3 (CD11b-CD18) Complement opsonised bacteria

and fungi CR4 (CD11b-CD18) M. tuberculosis CD43 leukosialin M. tuberculosis CD48 Enterobacteria Mannose receptor Mannosyl/fucosyl residues, P.

carinii, Candida albicans Scavenger receptor Apoptotic cells, Gram +ve cocci,

leipoteichoic acid MARCO E. coli CD14 P. aeruginosa

They recognize molecules that are often abundant, contain repeating subunits and are not produced by vertebrates.

These include bacterial polysaccharides and lipopolysaccharides, complex fungal polysaccharides, flagellin and bacterial DNA or viral RNA.

Initial recognition of micro-organisms as foreign is likely to take place in non-lymphoid tissues and the most important cells in this process are tissue resident macrophages and dendritic cells (DCs).

Activation of dendritic cells is crucial as these cells have been shown to be uniquely capable of initiating a primary immune response.

DCs are also actively pinocytic and take up soluble antigens as well as those bound by their surface receptors.

Uptake of antigen and ligation of one or more DC receptors, initiates three key processes:

1. antigen processing, 2. migration to lymph nodes, and 3. maturation of the DCs.

The Major Histocompatibility Complex (MHC)

MHC is a collection of tightly-linked genes found in every mammalian species studied to date; found on chromosome 6 in humans and also referred to as HLA (human leukocyte antigen) complex

MHC so named because gene products are major transplantation antigens

Biological Function is to bind peptides from degraded proteins and present them at the cell surface for T-cell recognition.

MHC genes organized into regions encoding 3 classes of molecules.

Human MHC Complex

Structure of MHC Class I Molecules

Structure of MHC Class II Molecules

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Comparison of the Properties & Function of MHC Class I and Class II Molecules

Antigen ProcessingAntigens entering cells by endocytosis are broken

down in lysosomal vesicles and peptides from them encounter major histocompatiblity class II antigens (MHC II) in a specialised intracellular loading compartment where the peptides are loaded onto MHC II molecules for transport to the cell surface (exogenous antigen processing).

Antigens synthesised in the cell, as is the case for viruses and other intracellular pathogens, are broken down to peptides by the proteasomes and the resulting peptides are transported into the rough endoplasmic reticulum for loading onto MHC class I molecules (endogenous antigen processing).

Loaded MHC molecules are then transported to the cell surface.

Following activation by ‘danger’ signals, surface expression of MHC increases greatly and subsequently antigen processing decreases.

The figure shows the two modes of antigen processing.

In the exogenous modes, antigens are captured from

the extracellular space, degraded to peptides in

endosomes and the peptides displayed on MHC II

molecules. Endogenous processing of intracellular

antigens is carried out by the proteasomes and the

resulting peptides are loaded on to MHC class I molecules

in the endoplasmic reticulum.

MHC Class I - Antigen Processing Pathway

MHC Class II - Antigen Processing Pathway

Migration and Maturation The DCs migrate from the tissues to the draining lymph nodes,

a process controlled by chemokines and their receptors. Thus in the tissues, DCs express CCR1, CCR5 and CCR6 – the

receptors for chemokines produced by tissue cells. Down-regulation of these receptors and up-regulation of

CXCR4 and CCR7 are induced by ‘danger’ signals and signals from inflammatory cytokines such as tumour necrosis factor-  (TNF- ) and interleukin-1 (IL-1).

This allows the DCs to receive chemotactic signals from the lymph node chemokines, secondary lymphoid tissue chemokine (SLC) and EBV-induced-receptor ligand chemokine (ELC;).

During migration and entry of DCs into the T-cell areas of nodes, the DCs show considerable changes in phenotype (maturation) in addition to the up-regulation of MHC.

The most important is the up-regulation of surface molecules that are important for interaction of DCs with T-cells.

CD40, CD80 and CD86 deliver crucial co-stimulatory signals for T-cell activation, while several members of the TNF–TNFR (tumour necrosis factor receptor) family of molecules are up-regulated.

These include CD40, Ox40 and 4-1BB and they appear to play important roles in differentiation of different types of effector T-cells. 

Important considerations for vaccine development

Two aspects of this complex series of processes are particularly crucial from the point of view of vaccines.

The first is the need for ‘danger’ signals to initiate responses. While whole micro-organisms, even if killed, may well deliver appropriate signals, subunit vaccines may be poorly immunogenic so that adjuvants are needed. In humans, the most commonly used is alum. Alum has been shown to favor Th2 responses in mice, inducing strong antibody responses.

This observation leads to the second crucial point, that the nature of the ‘danger’ signal has an important bearing on the type of immune response generated.

Clearly for vaccines where a Th1 type of response is required, alum may not be an appropriate adjuvant and, further more, the danger signals carried by the vaccine itself or a live vector must also be taken into account.

So far, few alternative adjuvants are available for routine use in humans. However, better understanding of the mechanisms of action of adjuvants and the signals that control differentiation of DCs and, therefore, T-cells will eventually allow design of vaccine-adjuvant preparations tailored to induce appropriate protective responses for particular infections.

 Immunological Memory Irrespective of the type of immune response

required for protection, for almost all vaccines long-lasting protection (memory) is a desirable objective.

It is also clear that memory is a dynamic state.

In both man and experimental animals, phenotypically defined memory cells have been shown to divide more rapidly than naive cells . 

This appears to be an inherent property of memory cells since division continues in the absence of antigen .

Constraints on the Duration of Memory Human T lymphocyte clones can only

undergo a finite number of cell divisions and, as they approach senescence, no longer express the co-stimulatory molecule CD28, can no longer up-regulate telomerase on activation, and show progressive shortening of telomeres .

These mechanisms may limit the duration of memory in the absence of re-exposure to antigen, which would recruit new clones.

In addition to these constraints on survival of individual clones, there is also the constraint of space in the memory pool.

Thus every time a new antigen is encountered and a new set of clones undergoes expansion and enters the memory pool, other cells must die to provide space. 

What factors favor one cell or clone over another in this competition for survival are not known. However, experimental evidence suggests that memory persists longer if the initial clonal expansion is large .

Alternatively, persistence of antigen may favor clonal survival as occurs in chronic infections such as EBV or CMV .

It is now clear that there is considerable heterogeneity among immune responses and it is thought that some memory cells may revert to a more slowly dividing state.

This suggests two alternative strategies for ensuring persistence of memory. Either vaccines should be designed to ensure maximal clonal expansion by providing an optimal dose of antigen and appropriate adjuvant, or

Vectors should be chosen to ensure long persistence of antigen.

Appropriate Immune Responses

Heterogeneity of immune responsesOne of the major discoveries of the modern era of immunology is that not all immune responses are the same. In truth, this is not a new discovery since it has been long been known that there are many types of immune response and these may be both beneficial, for example the development of neutralizing antibody to viruses, or pathological, for example the production of IgE antibody leading to anaphylaxis. What is new is the greatly increased, though not complete, understanding of how different types of response are generated.

Immune responses are influenced by many factors, but key cells that control the functions of other immune cells are the T helper cells (Th cells).

Two major types of Th cells have been described. Th1 effectors produce IL-2, IFN-gamma, etc and mediate ‘cellular’ immunity and antibody is not a prominent feature of the response.

In contrast, in Th2 cell responses, the dominant cytokines produced are IL-4, IL-5, IL-10, and IL–13. CD8 cytotoxic cells are not prominent and high titers of antibody may be produced, with a bias toward IgG as well as IgA and IgE.

In general, Th1 cell responses are adapted to deal with intracellular parasites through direct and indirect mechanisms – cell killing or production of cytokines (particularly IFN-gamma) that activate cellular protective mechanisms.

Th2 cell responses are particularly effective at coping with extracellular parasites through antibody-dependent mechanisms.

It should be emphasized that, although some response are almost exclusively Th1 or Th2, in most immune responses both Th1 and Th2 components can be detected.

Furthermore, many cytokines including TNF-alpha, IL-3, IL-6 and GM-CSF are produced by both Th1 and Th2 cells .

Control of T-cell Responses

It is important to understand what controls the development of a Th1 or Th2 biased response.

In experimental animals, the genetic background of the host has been shown to be important. 

Thus Balb/c mice mount a Th2 response to the parasite Leishmania major, while other strains of mice make a Th1 response. The former strain is susceptible to infection while others are resistant, demonstrating the important of making the ‘right’ type of response .

Treatment of susceptible Balb/c mice with antibody to the Th2 cytokine IL-4 or administration of the Th1-inducing cytokine IL-12, makes them resistant .

Apart from the genetic background of the host, it has been shown that the route, dose and form of the antigen and whether an adjuvant is given can have profound effects on the type of immune response generated .

More recent experiments show that treatment of DCs with products of micro-organisms such as lipopolysaccharide (LPS) or the worm antigen ES62, can bias their ability to stimulate Th1 and Th2 responses to a protein antigen .

There are 2 subtypes of DCs: DC1 and DC2These DC subtypes show different patterns of

cytokine production, which in turn influence Th cell generation. IL-12 produced by DCs has been shown to be a key cytokine for induction of Th1 cells while IL-4 and IL-10 are important for generation of Th2 cells .

These observations indicate why particular adjuvants may induce Th-biased responses.

Although the exact mechanisms of action of alum are not well understood, it induces strong Th2 responses in mice and humoral responses in man, presumably by inducing DCs to produce Th2-inducing cytokines .

In contrast, the experimental adjuvant, Freund's complete adjuvant, which contains BCG in mineral oil generates a strongly Th1-biased response.

ConclusionA key issue in vaccine development today is what

type of immune response is needed to best protect against the ‘difficult’ organisms for which there are currently no effective vaccines.

Most of the present generation of successful vaccines depend principally on generating high titres of antibody and many are given with the Th2-biasing adjuvant alum.

However, natural protection against many organisms, particularly intracellular parasites, is mainly Th1 in nature.

Furthermore, for organisms that vary rapidly (e.g. HIV, malaria) even if neutralizing antibody could be induced, escape variants would rapidly make this ineffective, as is the case with influenza virus.

For these difficult organisms, it remains unclear whether a strong cellular response, induced by a vaccine, could either prevent infection from becoming established or suppress it to a subclinical level compatible with normal life, although it is clear that the cellular immune response does contribute to protection against HIV.

Nevertheless, in the absence of concrete evidence that cellular immunity can be protective, many new vaccines are being designed to induce strong Th1 and CD8 cytotoxic T lymphocyte (CTL) responses.

To induce CTLs, presentation of antigen via MHC I is required and, as yet, the most effective way of doing this is through the use of live vectors that infect cells and thereby introduce antigens into the cytosol.

DNA has the advantage that, like live vectors, it can generate antigens inside cells, and the additional advantage is that the DNA may also code for genes such as cytokines that have adjuvant effects and can bias responses in a desired direction.