Chapters
- • Vaccine Immunology: Basics and Beyond
- • Elementary Epidemiology in Vaccination
- • Vaccination Schedules
- • Practice of Vaccination
- • Vaccination in Special Situations
- • Adverse Events Following Immunization (AEFI), Vaccine Safety, and Misinformation Against Vaccination
- • Cold Chain and Vaccine Storage
- Are vaccination and immunization same?
Broadly speaking both terms appear to be same and frequently used interchangeably. However, there is minor technical difference. ‘Vaccination’ is a process of inoculating the vaccine/antigen in to the body. The vaccinee may or may not seroconvert to vaccine whereas the process of inducing immune response, which can be ‘humoral’ or ‘cell-mediated’ in the vaccinee is called ‘immunization’.
Vaccines can be administered through different routes e.g. nasal mucosa, gut mucosa or by injection which may be given intradermal, subcutaneous or intramuscular. This process is called ‘vaccination’ or ‘active immunization’. In case immunoglobulins or antisera are administered it is called ‘passive immunization’. Thus administration of immunoglobulins or antisera is not vaccination, although it provides immunity or protection for a short period.
- What are ‘humoral’ and ‘cell-mediated immunity’?
Vaccines confer protection against diseases by inducing both antibodies and T-cells. The former is called ‘humoral’ response and the latter, ‘cellular’ response or ‘cell-mediated immunity’. Antibodies are of several different types (IgG, IgM, IgA, IgD and IgE) and they differ in their structure, half life, site of action and mechanism of action. Humoral immunity is the principal defence mechanism against extracellular microbes and their toxins. B lymphocytes secrete antibodies that act by neutralization, complement activation or by promoting opsonophagocytosis. Cell mediated immunity (CMI) is the principal defence mechanism against intracellular microbes. The effectors of CMI, the T cells are of two types. The helper T cells secrete proteins called cytokines that stimulate the proliferation and differentiation of T cells as well as other cells including B lymphocytes, macrophages and NK cells. The cytotoxic T cells act by lysing infected cells.
- What are innate and adaptive immunity?
Innate immunity comprises of the skin and mucosal barriers, phagocytes (neutrophils, monocytes and macrophages) and the natural killer (NK) cells. 4It comes into play immediately on entry of the pathogen and is non specific. Adaptive immunity is provided by the B lymphocytes (humoral/antibody mediated immunity) and T lymphocytes (cellular/cell mediated immunity). The innate immune system triggers the development of adaptive immunity by presenting antigens to the B lymphocytes and T lymphocytes. Adaptive immunity takes time to evolve and is pathogen specific (Figure 1.1 and Table 1.1).
- What are ‘B’ and ‘T’ cells? What role do they play in regard to immunology of vaccines?
Immune system is almost not existent at birth; maternal antibodies transferred transplacentally provide some protection during early childhood. After birth baby comes in contact with microbes which gradually activate immune system. B cells form the most important component of immune system in the body. These are produced in liver in fetal life and mature in bone marrow in 5humans. In other species these cells mature in an organ called "bursa of Fabricius", thus these lymphocytes are called B cells. On activation by an antigen contained in microorganisms and vaccines, the B cells proliferate and get converted to plasma cells, which in turn produce antibodies. For effective production of antibodies, B cells need help from T helper cells. T lymphocytes are the 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.
B cells have immunoglobulin surface receptor, which binds with the appropriate antigen present on the infective pathogen. The processed antigen stimulates the B cell to mature into antibody secreting plasma cell and generate IgM. T helper2 (Th2) cell leads to switch in the production from IgM to IgG, IgA or IgD. The B cells can directly respond to the antigen and process the antigen, but the T cells do not react with the antigen directly unless processed and presented by special cells called antigen presenting cells (APCs).
- What are antigen-presenting cells (APCs) and dendritic cells? What functions do they perform?
Antigen presenting cells (APCs) are the 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 (Figure 1.2).
Dendritic cells are major APC in the body in addition to the B cells and the macrophages. The major role of these cells is to identify dangers, which is done by the special receptors on the APC named toll-like receptors (TLR).
Vaccine antigens are taken up by immature dendritic cells (DCs) activated by the local inflammation, which provides the signals required for their migration to draining lymph nodes. During this migration, DCs mature and their surface expression of molecules changes. DCs sense "danger signals" through their Toll-like receptors and respond by a modulation of their surface or secreted molecules. Simultaneously, antigens are processed into small fragments and displayed at the cell surface in the grooves of Major Histocompatibility Complex (MHC-HLA in humans) molecules. As a rule, MHC class I molecules present peptides from antigens that are produced within infected cells, whereas phagocytosed antigens are displayed on MHC class II molecules. Thus, mature DCs reaching the T cell zone of lymph nodes display MHC-peptide complexes and high levels of costimulation molecules at their surface. CD4+ T cells recognize antigenic peptides displayed by class II MHC molecules, whereas CD8+ T cells bind to class I MHC peptide complexes.6
Antigen-specific T cell receptors may only bind to specific MHC molecules (e.g. HLA A2), which differ among individuals and populations. Consequently, T cell responses are highly variable within a population.
- What are adjuvants? How do they affect performance of a vaccine?
Adjuvants are 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. Hence, the adjuvants improve the immunogenicity of vaccines. Many new generations of adjuvants are in fact analogues of TLRs, for example CpG-ODN used in new generation of Japanese encephalitis vaccines.
Most non-live vaccines require their formulation with specific adjuvants to include danger signals and trigger a sufficient activation of the innate system. These adjuvants may be divided into two categories: delivery systems that prolong the antigen deposit at site of injection, recruiting more dendritic cells (DCs) into the reaction, and immune modulators that provide additional differentiation and activation signals to monocytes and DCs. Although progress is being made, none of the adjuvants currently in use trigger the degree of innate immune activation that is elicited by live vaccines, whose immune potency far exceed that of non-live vaccines.
- What are ‘germinal centers’ and ‘marginal zone’?
Germinal centers (GCs) are 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 cells7 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.
‘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.
- What do the terms ‘epitope’ and ‘paratope’ mean?
An ‘epitope’, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that recognizes the epitope is called a ‘paratope’. Although epitopes are usually thought to be derived from non-self proteins, sequences derived from the host that can be recognized are also classified as epitopes.
T cell epitopes are presented on the surface of an antigen-presenting cell, where they are bound to MHC molecules. T cell epitopes presented by MHC class I molecules are typically peptides between 8 and 11 amino acids in length, whereas MHC class II molecules present longer peptides, and non-classical MHC molecules also present non-peptidic epitopes such as glycolipids.
Epitopes are sometimes cross-reactive. This property is exploited by the immune system in regulation by anti-idiotypic antibodies. If an antibody binds to an antigen's epitope, the paratope could become the epitope for another antibody that will then bind to it. If this second antibody is of IgM class, its binding can upregulate the immune response; if the second antibody is of IgG class, its binding can downregulate the immune response.
- What is the difference between ‘antibody affinity’ and ‘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 its function.
- What do the terms ‘CD4+T cells’ and CD8+ T cells’ stand for? What are the functions of these lymphocytes?
T lymphocytes are the 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.
CD 4+ T cells are those T-lymphocytes that express the CD4 glycoprotein at their surface. CD4 (cluster of differentiation 4) is a glycoprotein expressed on the surface of T helper cells, regulatory T cells, monocytes, macrophages, and dendritic cells. It was discovered in the late 1970s and was originally known as leu-3 and T4 (after the OKT4 monoclonal antibody that reacted 8with it) before being named CD4 in 1984. In humans, the CD4 protein is encoded by the CD4 gene. CD4 is a co-receptor that assists the T cell receptor (TCR) to activate its T cell following an interaction with an antigen presenting cell. Using its portion that resides inside the T cell, CD4 amplifies the signal generated by the TCR by recruiting an enzyme, known as the tyrosine kinase lck, which is essential for activating many molecules involved in the signalling cascade of an activated T cell. CD4 interacts directly with MHC class II molecules on the surface of the antigen presenting cell using its extracellular domain.
T cells expressing CD4 molecules (and not CD8) on their surface, therefore, are specific for antigens presented by MHC II and not by MHC class I (they are MHC class II-restricted). The short cytoplasmic/intracellular tail (C) of CD4 contains a special sequence of amino acids that allow it to interact with the lck molecule described above.
CD8 cells are those T-lymphocytes that express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis. CD8 cells are cytotoxic T cells (CTLs) and destroy virally infected cells and tumor cells, and are also implicated in transplant rejection (Figure 1.3).
CD8+ T cells do not prevent but reduce, control and clear intracellular pathogens by:
- directly killing infected cells (release of perforin, granzyme, etc.)
- indirectly killing infected cells through antimicrobial cytokine release
CD4+ T cells do not prevent but participate to the reduction, control and clearance of extra and intracellular pathogens by:
- producing IFN-γ, TNF-α/-β, IL-2 and IL-3 and supporting activation and differentiation of B cells, CD8+T cells and macrophages (Th1 cells).
- producing IL-4, IL-5, IL-13, IL-6 and IL-10 and supporting B cell activation and differentiation (Th2 cells).
- What are TLRs and their role in vaccine immunogenicity?
Toll-like receptors are a 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.
Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. They are single membrane-spanning non-catalytic receptors that recognize structurally conserved molecules derived from microbes. Once these microbes have breached physical barriers such as the skin or intestinal tract mucosa, they are recognized by TLRs which activates immune cell responses.
TLRs are a type of pattern recognition receptor (PRR) and recognize molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs).9
Figure 1.3: The T lymphocyte activation pathway(Source: Adapted from NIH Publication No. 03–5423, September 2003).
T-cell activation is triggered when a T cell encounters its cognate antigen, coupled to a MHC molecule, on the surface of an infected cell or a phagocyte. T cells contribute to immune defences in two major ways: some direct and regulate immune responses; others directly attack infected or cancerous cells.
TLRs together with the interleukin-1 receptors form a receptor superfamily, known as the "interleukin-1 receptor/toll-like receptor superfamily"; all members of this family have in common a so-called TIR (Toll-IL-1 receptor) domain.
Three subgroups of TIR domains exist. Proteins with subgroup 1 TIR domains are receptors for interleukins that are produced by macrophages, monocytes and dendritic cells and all have extracellular immunoglobulin (Ig) domains. Proteins with subgroup 2 TIR domains are classical TLRs, and bind directly or indirectly to molecules of microbial origin. A third subgroup of proteins containing TIR domains consists of adaptor proteins that are exclusively cytosolic and mediate signaling from proteins of subgroups 1 and 2.
TLRs are present in vertebrates, as well as in invertebrates. Molecular building blocks of the TLRs are represented in bacteria and in plants, and in the latter kingdom, are well known to be required for host defence against infection. The TLRs thus appear to be one of the most ancient, conserved components of the immune system.
Toll-like receptors bind and become activated by different ligands, which, in turn are located on different types of organisms or structures. They also have different adapters to respond to activation and are located sometimes at the cell surface and sometimes to internal cell compartments. Furthermore, they are expressed by different types of leucocytes or other cell types.
Following activation by ligands of microbial origin, several reactions are possible. Immune cells can produce signaling factors called cytokines which trigger inflammation. In the case of a bacterial factor, the pathogen might be phagocytosed and digested, and its antigens presented to CD4+ T cells. In the case of a viral factor, the infected cell may shut off its protein synthesis and may undergo programmed cell death (apoptosis). Immune cells that have detected a virus may also release anti-viral factors such as interferons.
The discovery of the Toll-like receptors finally identified the innate immune receptors that were responsible for many of the innate immune functions that had been studied for many years. Interestingly, TLRs seem only to be involved in the cytokine production and cellular activation in response to microbes, and do not play a significant role in the adhesion and phagocytosis of microorganisms.
- What are the differences between live attenuated and inactivated vaccines?
Live vaccines are attenuated (modified) live organisms, which have immunogenicity i.e. can generate antibodies, but have lost pathogenicity i.e. capability to cause disease. Live vaccines can be viral as well as bacterial. The live vaccine particles (viruses or bacteria) replicate or multiply in the 11body after administration and stimulate the immune system. The older concept that single dose of live vaccines induces life long immunity perhaps does not hold true, we need many doses of OPV, and booster doses are required for live vaccines like measles vaccine, varicella, rubella and live oral typhoid vaccines. Inactivated vaccines may consist of whole inactivated organisms like whole cell pertussis, typhoid, rabies; inactivated polio vaccine, modified exotoxins called ‘toxoids’ like diphtheria toxoid or tetanus toxoid; subunits like polysaccharide antigents of salmonella typhi, Hemophilus influenzae type-B (Hib), and surface proteins of hepatitis B virus. Conjugation of the polysaccharide with a protein carrier significantly improves the immune response.
Live viral vaccines do efficiently trigger the activation of the innate immune system, presumably through pathogen-associated signals (such as viral RNA) allowing their recognition by pattern recognition receptors-Toll-like Receptos. Following injection, viral particles rapidly disseminate throughout the vascular network and reach their target tissues. This pattern is very similar to that occurring after a natural infection, including the initial mucosal replication stage for vaccines administered through the nasal/oral routes. Following the administration of a live viral vaccine and its dissemination, dendritic cells are activated at multiple sites, migrate towards the corresponding draining lymph nodes and launch multiple foci of T and B cell activation. This provides a first explanation to the generally higher immunogenicity of live versus non-live vaccines.
The strongest antibody responses are generally elicited by live vaccines that better activate innate reactions and thus better support the induction of adaptative immune effectors. Non-live vaccines frequently require formulation in adjuvants, of which aluminium salts are particularly potent enhancers of antibody responses, and thus included in a majority of currently available vaccines. This is likely to reflect their formation of a deposit from which antigen is slowly de-absorbed and released, extending the duration of B and T cell activation, as well as the preferential induction of IL-4 by aluminium-exposed macrophages.
Very few non-live vaccines induce high and sustained antibody responses after a single vaccine dose, even in healthy young adults. Primary immunization schedules therefore usually include at least two vaccine doses, optimally repeated at a minimal interval of 3-4 weeks to generate successive waves of B cell and GC responses. These priming doses may occasionally be combined into a single "double" dose, such as for hepatitis A or B immunization. In any case, however, vaccine antibodies elicited by primary immunization with non-live vaccines eventually wane.
- What is the difference between T cell dependent and T cell independent immune response?
Certain antigens, primarily proteins, induce both B cell and T cell stimulation leading to what is called T cell-dependent immune response. Infants of 6 12weeks of age onwards are capable of T cell dependent response. This type of response usually results in higher titers of IgG type and long lasting. It also shows booster effects with repeated exposures.
On the other hand T cell independent response being only B-cell mediated is not possible below 2 years of age. It is predominantly IgM type with low titers. The response is short lasting, repeated doses of vaccine does not lead to boosting effect. IgA is not produced and hence there is no local mucosal protection with this type of antigens, while in case of T cell dependent response IgA antibodies are also produced which helps in providing mucosal protection and eradication of the carrier state. Few examples of T cell independent vaccines include bacterial polysaccharide (PS) vaccines such as S. pneumoniae, N. meningitidis, H. influenzae, S. Typhi.
- What are conjugate vaccines?
As already mentioned in answer to above question, regarding the difference between T cell dependent and T cell independent immune response that T cell-independent response being B cell mediated younger children do not respond to such vaccines. A T-cell independent antigen like polysaccharide can be made into T cell dependent by the technique of conjugation. Such conjugated vaccines can be administered to children less than 2 years of age also. This technique is used to produce conjugated Vi typhoid, Hib, pneumococcal and meningococcal vaccines.
- How do vaccines elicit their responses? Which are the main effectors of vaccine responses?
The nature of the vaccine exerts a direct influence on the type of immune effectors that are predominantly elicited and mediate protective efficacy (Table 1.2). Capsular polysaccharides (PS) elicit B cell responses in what is classically reported as a T-independent manner, although increasing evidence supports a role for CD4+ T cells in such responses. The conjugation of bacterial PS to a protein carrier (e.g. glycoconjugate vaccines) provides foreign peptide antigens that are presented to the immune system and thus recruits antigenspecific CD4+ Th cells in what is referred to as T-dependent antibody responses. A hallmark of T-dependent responses, which are also elicited by toxoid, protein, inactivated or live attenuated viral vaccines is to induce both higher-affinity antibodies and immune memory. In addition, live attenuated vaccines usually generate CD8+ cytotoxic T cells. The use of live vaccines/vectors or of specific novel delivery systems (e.g. DNA vaccines) appears necessary for the induction of strong CD8+ T cell responses. Most current vaccines mediate their protective efficacy through the induction of vaccine antibodies, whereas BCG-induced T cells produce cytokines that contribute to macrophage activation and control of M. tuberculosis. The induction of antigen-specific immune effectors (and/or of immune memory cells) by an immunization process does imply that these antibodies, cells or cytokines represent surrogates – or even correlates - of vaccine efficacy. This requires 13the formal demonstration that vaccine-mediated protection is dependent–in a vaccinated individual-upon the presence of a given marker such as an antibody titer or a number of antigen-specific cells above a given threshold. Antigen-specific antibodies have been formally demonstrated as conferring vaccine-induced protection against many diseases.
Passive protection may result from the physiological transfer of maternal antibodies (e.g. tetanus) or the passive administration of immunoglobulins or vaccine-induced hyperimmune serum (e.g. measles, hepatitis, varicella, etc.). Such antibodies may neutralize toxins in the periphery, at their site of production in an infected wound (tetanus) or throat (diphtheria).
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They may reduce binding or adhesion to susceptible cells/receptors and thus prevent viral replication (e.g. polio) or bacterial colonization (glycoconjugate vaccines against encapsulated bacteria) if present at sufficiently high titers on mucosal surfaces. The neutralization of pathogens at mucosal surfaces is mainly achieved by the transudation of vaccine-induced serum IgG antibodies. It requires serum IgG antibody concentrations to be of sufficient affinity and abundance to result into "protective" antibody titers in saliva or mucosal secretions. As a rule, such responses are not elicited by PS bacterial vaccines but achieved by glycoconjugate vaccines, which therefore prevent nasopharyngeal colonization in addition to invasive diseases.
Under most circumstances, immunization does not elicit sufficiently high and sustained antibody titers on mucosal surfaces to prevent local infection. It is only after having infected mucosal surfaces that pathogens encounter vaccine-induced IgG serum antibodies that neutralize viruses, opsonize bacteria, activate the complement cascade and limit their multiplication and spread, preventing tissue damage and thus clinical disease. That vaccines fail to induce sterilizing immunity is thus not an obstacle to successful disease control, although it represents a significant challenge for the development of specific vaccines such as against HIV-1. Current vaccines mostly mediate protection through the induction of highly specific IgG serum antibodies. Under certain circumstances, however, passive antibody-mediated immunity is inefficient (tuberculosis).
- What are the clinical scenarios where evidences of T-cells protection available?
BCG is the only currently used human vaccine for which there is conclusive evidence that T cells are the main effectors. However, there is indirect evidence that vaccine-induced T cells contribute to the protection conferred by other vaccines. CD4+ T cells seem to support the persistence of protection against clinical pertussis in children primed in infancy, after vaccine-induced antibodies have waned. Another example is that of measles immunization in 6-month-old infants. These infants fail to raise antibody responses because of immune immaturity and/or the residual presence of inhibitory maternal antibodies, but generate significant IFN-γ producing CD4+ T cells. These children remain susceptible to measles infection, but are protected against severe disease and death, presumably because of the viral clearance capacity of their vaccine-induced T cell effectors. Thus, prevention of infection may only be achieved by vaccine-induced antibodies, whereas disease attenuation and protection against complications may be supported by T cells even in the absence of specific antibodies. The understanding of vaccine immunology thus requires appraising how B and T cell responses are elicited, supported, maintained and/or reactivated by vaccine antigens.
15- What happens once a vaccine is administered to a vaccine? What are the first steps after immunization?
Following injection, the vaccine antigens attract local and systemic dendritic cells, monocytes and neutrophils. These activated cells change their surface receptors and migrate along lymphatic vessels, to the draining lymph nodes where the activation of T and B lymphocytes takes place. In case of killed vaccines there is only local and unilateral lymph node activation. Conversely for live vaccines there is multifocal lymph node vaccination due to microbial replication and dissemination. Consequently the immunogenicity of killed vaccines is lower than the live vaccines; killed vaccines require adjuvants which improve the immune response by producing local inflammation and recruiting dendritic cells/monocytes to the injection site. Secondly, the site of administration of killed vaccines is of importance; the intramuscular route which is well vascularised and has a large number of patrolling dendritic cells is preferred over the subcutaneous route. The site of administration is usually of little significance for live vaccines. Finally due to focal lymph node activation, multiple killed vaccines may be administered at different sites with little immunologic interference. Immunologic interference may occur with multiple live vaccines unless they are given on the same day/at least 4 weeks apart or at different sites.
- What are the immune responses of T cell independent antigens (i.e. polysaccharide vaccines) at the cellular level?
On being released from the injection site these antigens usually non-protein, polysaccharides in nature, reach the marginal zone of the spleen/nodes and bind to the specific Ig surface receptors of B cells. In the absence of antigen-specific T cell help, B cells are activated, proliferate and differentiate in plasma cells without undergoing affinity maturation in germinal centers. The antibody response sets in 2-4 weeks following immunization, is predominantly IgM with low titers of low affinity IgG. The half life of the plasma cells is short and antibody titers decline rapidly. Additionally the PS antigens are unable to evoke an immune response in those aged less than 2 years due to immaturity of the marginal zones. As PS antigens do not induce germinal centres, bona fide memory B cells are not elicited. Consequently, subsequent re-exposure to the same PS results in a repeat primary response that follows the same kinetics in previously vaccinated as in naïve individuals.
- What is hyporesponsiveness of repeated doses of a vaccine referring to?
Revaccination with certain bacterial polysaccharides (PS) - of which group C meningococcus is a prototype—may even induce lower antibody responses than the first immunization, a phenomenon referred to as hyporesponsiveness whose molecular and cellular bases are not yet fully understood.16
- What are the immune responses of T cell dependent antigens at the cellular level?
T-cell dependent antigens include protein antigens which may consist of either pure proteins (Hep B, Hep A, HPV, Toxoids) or conjugated protein carrier with PS antigens (Hib, meningo, pneumo). The initial response to these antigens is similar to PS antigens. However the antigen-specific helper T cells that have been activated by antigen-bearing dendritic cells trigger some antigen-specific B cells to migrate towards follicular dendritic cells (FDC's), initiating the germinal center (GC) reaction. In GC's, B cells receive additional signals from follicular T cells and undergo massive clonal proliferation, switch from IgM towards IgG/IgA, undergo affinity maturation and differentiate into plasma cells secreting large amounts of antigen-specific antibodies. Most of the plasma cells die at the end of germinal centre reaction and thus decline in antibody levels is noted 4-8 weeks after vaccination. However a few plasma cells exit nodes/spleen and migrate to survival niches mostly located in the bone marrow, where they survive through signals provided by supporting stromal cells and this results in prolonged persistence of antibodies in the serum.
- What are ‘memory B-cells’?
Memory B cells are those B-lymphocytes that generate in response to T-dependent antigens, during the GC reaction, in parallel to plasma cells. They persist there as resting cells until re exposed to their specific antigens when they readily proliferate and differentiate into plasma cells secreting large amounts of high-affinity antibodies that may be detected in the serum within a few days after boosting.
- What are the characteristics of immune response to live vaccines?
The live vaccines induce an immune response similar to that seen with protein vaccines. However, the take of live vaccines is not 100% with the first dose. Hence more than 1 dose is recommended with most live vaccines. Once the vaccine has been taken up, immunity is robust and lifelong or at least for several decades. This is because of continuous replication of the organism that is a constant source of the antigen. The second dose of the vaccine is therefore mostly for primary vaccine failures (no uptake of vaccine) and not for secondary vaccine failures (decline in antibodies over time).
- What determine intensity and duration of immune responses?
The nature of antigen is the primary determinant; broadly speaking live vaccines are superior (exception BCG, OPV) to protein antigens which in turn are superior to polysaccharide vaccines). Adjuvants improve immune responses to inactivated vaccines. Immune response is usually better with higher antigen dose (e.g. Hepatitis B). The immune response improves with increasing number of doses and increased spacing between doses. 17 Technically, 0, 1 and 6 months is the best immunization schedule; The first two doses are for induction and the long gap between the 2nd and 3rd dose allows for affinity maturation of B cells and clonal selection of the fittest B cells for booster and memory response. Extremes of age and disease conditions lower immune response.
- What are the limitations of young age immunization?
Young age limits antibody responses to most vaccine antigens since maternal antibodies inhibits antibodies responses but not T cell response, and due to limitation of B cell responses.
IgG antibodies are actively transferred through the placenta, via the FcRn receptor, from the maternal to the fetal circulation. Upon immunization, maternal antibodies bind to their specific epitopes at the antigen surface, competing with infant B cells and thus limiting B cell activation, proliferation and differentiation. The inhibitory influence of maternal antibodies on infant B cell responses affects all vaccine types, although its influence is more marked for live attenuated viral vaccines that may be neutralized by even minute amounts of passive antibodies. Hence, Antibody responses elicited in early life are short lasting. However, even during early life, induction of B memory cells is not limited.
Early life immune responses are characterized by age-dependent limitations of the magnitude of responses to all vaccines. Antibody responses to most PS antigens are not elicited during the first 2 years of life, which is likely to reflect numerous factors including: the slow maturation of the spleen marginal zone; limited expression of CD21 on B cells; and limited availability of the complement factors. Although this may be circumvented in part by the use of glycol-conjugate vaccines, even the most potent glycoconjugate vaccines elicit markedly lower primary IgG responses in young infants.
Although maternal antibodies interfere with the induction of infant antibody responses, they may allow a certain degree of priming, i.e. of induction of memory B cells. This likely reflects the fact that limited amounts of unmasked vaccine antigens may be sufficient for priming of memory B cells but not for full-blown GC activation, although direct evidence is lacking. Importantly, however, antibodies of maternal origin do not exert their inhibitory influence on infant T cell responses, which remain largely unaffected or even enhanced.
The extent and duration of the inhibitory influence of maternal antibodies increase with gestational age, e.g. with the amount of transferred immunoglobulins, and declines with post-natal age, as maternal antibodies wane.
- Maternal antibodies interfere with neonatal immune responses, why Hepatitis-B, BCG, and OPV are recommended at birth?
The first dose of Hepatitis-B which is administered at birth acts as ‘priming dose’ while subsequent doses provide an immune response even in presence 18of maternal antibodies. As mentioned above, maternal antibodies do not interfere with induction of memory B cells, certain degree of priming is allowed. However, hepatitis B vaccine induces lower primary IFN-γ responses and higher secondary Th2 responses in early life than adults. Similarly, antibodies of maternal origin do not exert their inhibitory influence on infant T cell responses. Since, BCG mainly works by inducing T cell immune response hence it can be given in the presence of maternal antibodies which may even enhance T cell responses. OPV is given at birth since there are no maternal IgA in the gut to neutralize the virus. However, IFN-γ responses to oral polio vaccine are significantly lower in infants than in adults.
- How maternal antibodies can sometimes enhance T-cell responses of BCG vaccine administered at birth?
After administration of BCG, the maternal antibodies form immune complexes with the vaccine antigens. These immune complexes are taken up by more and more number of macrophages and dendritic cells, which in turn are dissociated into their acidic phagolysosome compartment and are processed into small peptides. These peptides are displayed at the surface of antigen-presenting cells, thus available for binding by more number of CD4+ and CD8+ T cells.
- Considering the numerous limitations of young age immunization, why still vaccines are administered at much younger age in developing countries than in developed world?
This can be explained on the basis of disease epidemiology of vaccine-preventable diseases (VPDs). Since, majority of childhood infectious diseases cause early morbidity and mortality in poor, developing countries, hence the need to protect the children before wild organisms infect them. This is the reason why early, accelerated schedules are practiced in developing countries. According to W.H.O estimates, 2.5 to 3 million infants are born healthy but succumb to acute infections between the age of 1 and 12 months. These early deaths are caused by a limited number of pathogens, such that the availability of a few additional vaccines that would be immunogenic soon after birth would make a huge difference on this disease burden.
- How limitations of young age immunization can be taken care of?
They can be countered by increasing the number vaccine doses for better induction, use of adjuvants to improve immunogenicity of vaccines, and by use of boosters at later age when immune system has shown more maturity than at the time of induction. Increasing the dose of vaccine antigen may also be sufficient to circumvent the inhibitory influence of maternal antibodies, as illustrated for hepatitis A or measles vaccines.
19- Which is the best vaccination schedule for non-live vaccines acting on the principal of ‘prime-boost’ mechanism?
Traditionally, 0-1-6 month schedule is considered as a most immunogenic schedule than 6-10-14 weeks or 2-3-5 months schedules for non-live T cell dependent vaccines like Hepatitis-B vaccine. This is mainly due to proper spacing of the vaccine doses and adequate time interval between first few doses which act by inducing immune responses and last dose that works as boosters. Since, affinity maturation of B cells in GCs and formation of memory-B cells take at least 4-6 months, this schedule quite well fulfills these requirements. More than one dose is needed for better induction and recruitment of more number of GCs in young age considering young age limitations of immune system (Figure 1.4).
Immunization schedules commencing at 2 months and having 2 months spacing between the doses are technically superior to that at 6, 10 and 14 wks. However for operational reasons and for early completion of immunization and attainment of protection the 6, 10, 14 week's schedule is chosen in developing countries.
Figure 1.4: Schematic presentation of various components of 0-1-6 month immunization schedule at cellular level. Ag=Vaccine antigen, B=B lymphocyte, T=T lymphocyte, DC=Dendritic cell, M=Memory B lymphocyte, FDC=Follicular dendritic cell, SC=Stromal cells (in bone marrow)
Accelerated infant vaccine schedules in which 3 vaccine doses are given at a 1 month interval (2, 3, 4 or 3, 4, 5 months) result into lower responses than schedules in which more time elapses between doses (2, 4, 6 months), or between the priming and boosting dose (3, 5, 12 months). However, the magnitude of infant antibody responses to multiple dose schedules reflects both the time interval between doses, with longer intervals eliciting stronger responses, and the age at which the last vaccine dose is administered.
- What is a primary and secondary immune response?
When an antigen is introduced for the first time, the immune system responds primarily after a lag phase of up to 10 days. This is called the primary response. Subsequently, upon reintroduction of the same antigen, there is no lag phase and the immune system responds by producing antibodies immediately and this is called the secondary response. However, there are some differences in both these responsesprimary response is short-lived, has a lag phase, predominantly IgM type, and antibodies titers are low, whereas secondary response is almost immediate without a lag phase, titers persist for a long time, predominantly of IgG type, and antibodies titers are very high. Figure 1.5 describes the background developments at the cellular level and interactions of B cells, memory B cells and T cells at the follicular level in a lymphnode. The secondary response is mainly due to booster response and is seen with vaccines that work on a ‘prime-boost’ mechanism inducing T cells such as conjugate vaccines. On the other hand, non-conjugate, polysaccharide vaccines mainly induces primary response and the repeat dose produces another wave of primary response and not acts as a booster since they do not induce T cells.
- What are the hallmarks of ‘memory B cell’ responses?
These cells are only generated during T-dependent responses inducing germinal centers (GC) responses. These cells are resting cells that do not produce antibodies. Memory B cells undergo affinity maturation during several (4-6) months. A minimal interval of 4-6 months is required for optimal affinity maturation of memory B cells. Memory B cells rapidly (days) differentiate into antibody-secreting plasma cells upon re-exposure to antigen. Memory B cells differentiate into PCs that produce high(er)
affinity antibodies than primary plasma cells. As plasma cells and memory responses are generated in parallel in GCs, higher post-primary Ab titres reflect stronger GC reactions and generally predict higher secondary responses. During induction, a lower antigen dose at priming results in inducing B cells differentiation away from PCs, towards memory B cells. This phenomenon can be exploited by using small amount of expensive conjugate vaccines such as PCV (pneumococcal conjugate vaccine) followed by use of less expensive PPV(pneumococcal polysaccharide vaccine) as booster. Exposure to exogenous antigens may reactivate or favour the persistence of memory B cells.21
Figure 1.5: Correlation of antibody titers to various phases of the vaccine response
The initial antigen exposure elicits an extrafollicular response (1) that results in the rapid appearance of low IgG antibody titers. As B cells proliferate in germinal centers and differentiate into plasma cells, IgG antibody titers increase up to a peak value (2) usually reached 4 weeks after immunization. The short life span of these plasma cells results in a rapid decline of antibody titers (3), which eventually return to baseline levels (4). In secondary immune responses, booster exposure to antigen reactivates immune memory and results in a rapid (<7 days) increase (5) of IgG antibody titer. Short-lived plasma cells maintain peak Ab levels (6) during a few weeksafter which serum antibody titers decline initially with the same rapid kinetics as following primary immunization. Long-lived plasma cells that have reached survival niches in the bone marrow continue to produce antigen-specific antibodies, which then decline with slower kinetics.
Note: this generic pattern may not apply to live vaccines triggering long-term IgG antibodies for extended periods of time. (With permission from Siegrist CA. Vaccine Immunology. In Vaccines Ed. Plotkin SA, Orenstein W, Offit P. Saunders Elsevier, 5th Edition, 2008, pp 17-36)
- What are the implications of ‘immune memory’ for immunization programs?
Immune memory is seen with live vaccines/protein antigens due to generation of memory B cells which are activated on repeat vaccination/natural exposure. Immune memory allows one to complete an interrupted vaccine schedule without restarting the schedule. Hence, immunization schedule should never be started all over again regardless of duration of interruption. Regular boosters are not required to maintain immune memory during low risk periods (travellers). Certain immunization schedules may not need boosters if exposure provides regular natural boosters. Activation of immune memory and generation of protective antibodies usually takes 4-7 days. Diseases which have incubation periods shorter than this period such as Hib, tetanus, diphtheria and pertussis require regular boosters to maintain protective antibody levels. However diseases such as hepatitis A, hepatitis B 22do not need regular boosters as the long incubation period of the disease allows for activation of immune memory cells. This is to be noted that memory B cells do not produce antibodies unless re-exposed to antigen which drives their differentiation in to antibody producing plasma cells.
- Why is number of doses for each vaccine different ?
Live attenuated vaccines replicate (in case of viruses) or multiply (in case of bacteria) in the body thus the number of vaccine particles increases many folds which are capable of generating antibodies in large quantity to reach seroprotective levels. Due to some reasons not fully understood multiple doses of OPV are needed. On the other hand inactivated vaccines do not multiply in the body, and quantity of vaccine (antigens) required to provide full protection is large, fever and local reaction like swelling, tenderness and pain may be very severe if the required quantity of vaccine is administered at a time, so the quantity of vaccine is generally divided in two or more doses. DPT is divided in three doses while rabies vaccine is divided in four or five doses.
- Why do we need booster (booster doses) ?
The body starts antibody generation after administration of vaccines, which reach a peak after a period of time which is different for different vaccines. As already stated multiple doses of some vaccines have to be administered to attain optimal level of immunity. Over a period of time, which also varies for different vaccines, antibody level declines and revaccination or booster dose(s) is/are required to raise the antibody levels above the required protective levels.
In most cases sub clinical infection acts as a booster dose. As the percentage of vaccinated and immune population increases, circulation of the causative organisms declines in the community. This decline in circulation of organisms lessens the chances of non-immune individuals in coming in contact with organisms (which is a beneficial for non immune people), but those immune following vaccination may be deprived of the benefit of repeated sub clinical exposure leading to boosting effect. This is the reason that booster dose for varicella vaccine has been introduced in those countries where vaccine coverage is very high.
- Why we need to give only one dose of a particular vaccine while multiple doses are needed for another vaccine?
In general live vaccines generate antibodies to protective levels after administration while antigens need multiple doses, because the quantity required to generate antibodies to protective levels in very large, so multiple doses are required.
It has been observed that natural infection with viral diseases provides very long or life long protection while infections by bacteria do not provide any long lasting protection. Typhoid disease, skin infections and other infections caused by bacteria can recur again and again while second attack of measles or chickenpox occurs rarely if at all. Similarly antibodies produced by antiviral vaccines persist for much longer period as compared to antibodies produced by antibacterial vaccines.
- Why can't we have an ‘all-in-one vaccine’?
There are very strong scientific and logistic reasons against ‘all-in-one vaccine’. Scientific reasons are: (i) different ideal ages for different vaccines e.g. OPV, BCG and hepatitis B vaccines can be administered soon after birth, other vaccines can not be administered at this age, (ii) different routes of administration eg. some are administered orally, others are administered parentally, some intradermally (BCG), some subcutaneously (measles, MMR, varicella vaccines) and other vaccines are administered intramuscularly. Logistic reasons are: (i) some vaccines need to be administered as a single dose (BCG and varicella vaccines) some need two doses (like measles, MMR, hepatitis A, rotavirus vaccine), some vaccines need three doses (hepatitis B, and human papilloma virus vaccines) while other vaccines have to be administered at different intervals, and (ii) quantity of such an ‘all-in-one vaccine’ would be too large. Certainly the idea appears to be very attractive, but does not appear feasible in foreseeable future.
Suggested Reading
- Siegrist CA. Vaccine Immunology. In Vaccines Ed. Plotkin SA, Orenstein W, Offit P. Saunders Elsevier, 5th Edition, 2008, pp 17–36.
- Singhal Tanu, Amdekar YK, Agarval RK (eds) IAP Guide Book on Immunization. 4th Ed. IAP Committee on Immunization. New Delhi, Jaypee, 2009.
- Presentations delivered at 11th ADVAC at Annecy, France 2010 on May 10, 2010.
- Manual of Advancing Science of Vaccinology, Indian Academy of Pediatrics, 2009.
- Thacker N, Shendurkar N. Childhood Immunization-Issues and options. Incal Communications, New Delhi, First Ed, 2005.
- Wikipedia: the free encyclopedia. Available at: http://en.wikipedia.org/wiki/Main_Page.