Basic Immunology

Abhay K Shah

SECTION 1

1Basic Vaccinology and General Considerations

Basic Immunology

Abhay K Shah
Basic Epidemiology in Vaccinations

Bhaskar Shenoy
Vaccination Schedules

Suhas V Prabhu
General Immunization Practices

Sanjay Srirampur
Documentation of Vaccination

Chetan Trivedi
Cold Chain Management

Sanjay Marathe, Srinivas G Kasi
Adverse Event following Immunization

Arun Wadhwa
Vaccination in Special Situations

Srinivas G Kasi
Vaccine Safety

Srinivas G Kasi
Setting Up a Vaccination Clinic

Vijay Kumar Guduru, Srinivas G Kasi
Combination Vaccines

P Sivaraman
Immunoglobulins for Passive Protection against Vaccine-Preventable Diseases

Raju C Shah, Pratima Shah2

Basic ImmunologyCHAPTER 1

Abhay K Shah

Q1. What is the function of immune system?

Ans. The immune system is an extremely important defense mechanism that can identify an invading organism, from outside the body (e.g., viruses, bacteria, parasites, allergens, etc.) or within the body, e.g., malignant cells, and destroy it.

Q2. What is immunity?

Ans. Immunity can be defined as a complex biological system endowed with the capacity to recognize and tolerate whatever belongs to the self, and to recognize and reject what is foreign (non-self).

Q3. What are antigens and antibodies?

Ans. A protein, toxin, or other substances of high molecular weight, to which the body reacts by producing antibodies and stimulates an immune response. Different organisms contain several different antigens.

Antibodies [Ab, immunoglobulins (Ig)] are protein molecules that bind specifically to a particular part of an antigen, called antigenic site or epitope. They are found in low levels in the blood and tissue fluids, including mucus secretions, saliva and breast milk. However, when an immune response is activated greater quantities are produced to specifically target the foreign material.

Q4. What is epitope and paratope?

Ans. 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” (Fig. 1).

Q5. What is the difference between immunization and vaccination?

Ans. Immunization is the immune response to any administered antigen whereas vaccination is the immune response elicited in the body with the help of the vaccine.4

Fig. 1: Epitope and paratope.

Fig. 2: Differentiation between innate and adaptive immunity.

Q6. What are innate and adaptive of immune response elicited in body in response to an antigen?

Ans. Immunity may be broadly classified as innate and adaptive immunity (Fig. 2).

Innate immunity comes into play within hours of the entry of an infective agent. The components of the innate immune system comprise of epithelial and mucosal barriers (mechanical), the antibacterial chemicals in these barriers, phagocytes (neutrophils, macrophages and NK cells) as well as complement. It is not very specific as it is triggered by structures shared by different microbes instead of specific microbial antigens. There is no immune memory. It plays a very important role as it is the first line of defense. It is also the effector pathway of adaptive 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 develop. The two arms of adaptive immunity are humoral immunity (B lymphocyte mediated) and cell mediated immunity (T lymphocyte mediated). It has intense diversity and is capable of responding to millions of antigens and possesses immune memory. Adaptive immunity takes time to evolve and is pathogen specific.5

Q7. What is trained immunity?

Ans. The ability of the innate immune system to develop adaptive features and provide long-term protection against unrelated pathogens is term trained immunity. Epigenetic modification of various transcriptional pathways, as well as metabolic reprogramming of innate immune cells by both endogenous and exogenous stimuli, is the main driving force for trained immunity. Recent studies have revealed that innate immune cells, especially monocytes and macrophages, can develop adaptive features after adequate priming. Akin to adaptive immune response, trained innate immunity is associated with a heightened immune response to reinfections. In general, trained immunity is known to provide relatively short-term protection ranging from about 3 months to 1 year. Monocytes are very short-lived cells; however, the heightened secondary response can be spotted even several months after the primary stimulation. This shows that the immune memory is created at the level of progenitor cells, in the bone marrow.

A wide range of stimuli, such as beta-glucan (fungal ligand) and BCG (Bacillus Calmette-Guérin), are known to induce trained immunity. In humans, BCG vaccination-mediated non-specific protection against secondary infections is believed to be caused by trained immunity. Induction of trained immunity is considered to be a potential therapeutic strategy to manage various health conditions associated with immune system malfunctioning, such as cancer. Moreover, triggering trained immunity via live vaccines, such as BCG, measles, and oral polio vaccines, can be an effective approach in treating patients with severe infectious diseases, such as coronavirus disease 2019 (COVID-19). Long-term boosting of innate immune responses, also termed “trained immunity,” by certain live vaccines (BCG, oral polio vaccine, measles) induces heterologous protection against infections through epigenetic, transcriptional, and functional reprogramming of innate immune cells.

Q8. What are B lymphocytes and T lymphocytes? What is their function?

Ans. B cells, also known as B lymphocytes, form the most important component of adaptive immunity (see Fig. 2). They provide humoral immunity by secreting antibodies. B-lymphocytes are produced in fetal liver and mature in the bone marrow in humans. In other species they mature in “Bursa of Fabricius” and hence named as B cells. B cells activated by the antigen present in the microbes and vaccines. These activated B cells are differentiated into antibody secreting plasma cells. For effective antibody B cells need help from T Helper cells.6

T cells or T lymphocytes are mediators of cell mediated immunity. They have a key importance in the immune system and are at the core of adaptive immunity. They originate in thymus and mature in periphery and get activated in spleen/nodes. T cells can be distinguished from other lymphocytes by the presence of a T-cell receptor on the cell surface. T cell does not interact directly with the vaccine antigen unless presented by antigen presenting cells.

Q9. Which are different types of T cells?

Ans. They are as under:

CD4+ helper cells: CD4+ helper cells help in the maturation of B cells into plasma cells and memory B cells. They also help activate cytotoxic T cells and macrophages. They become activated when they are presented with peptide antigens by major histocompatibility complex (MHC) class II molecules, which are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.
CD8+ cytotoxic cells: CD8+ cytotoxic cells cause lysis of virus-infected and tumor cells. They are also involved in transplant rejection. These cells recognize their targets by binding to antigen associated with MHC class I molecules which are present on the surface of all nucleated cells. Cytotoxic cells secrete hole forming proteins called perforins for its cytolytic action.
Natural killer T cells: They bridge the adaptive immune system with the innate immune system. While most T cells function based on recognition of MHC class molecules, natural killer T cells are able to recognize other antigen classes. Once activated, they are also able to perform the same functions as CD4+ and CD8+ cells.

Q10. Describe clinical implications of Th1, Th2, Th17 and Treg immune responses.

Ans. T cells play a central role in the adaptive immune response. T-helper (Th) cells can be classified into Th1, Th2, Th17 and Tregs cells. Th1 cells produce interleukin (IL)-2 and interferon (IFN) γ and are involved in cellular immunity, which help to eradicate infections by intracellular microbes which include certain viruses, protozoans, and intracellular bacteria, such as the mycobacteria.

Th2 cells, which produce IL-4, IL-5 and IL-13, are involved in humoral immunity, mainly against extracellular microorganisms.

Th17 cells play a role in host defense against extracellular pathogens, particularly at the mucosal and epithelial barriers, e.g., B pertussis, but aberrant activation has been linked to the pathogenesis of various autoimmune diseases. TH17 cells arise when the cytokines IL-6 and transforming growth factor (TGF)-β predominate during naive CD4 T-cell activation.

Regulatory T (TReg) cells are essential for maintaining peripheral tolerance, preventing autoimmunity and limiting chronic inflammatory diseases. However, they also limit beneficial responses by suppressing sterilizing immunity and limiting anti-tumour immunity. Suppression by inhibitory cytokines: interleukin-10 (IL-10), transforming growth factor-β (TGFβ) and the newly identified IL-35 are key mediators of TReg-cell function. Treg cells, which are CD4+/CD25+, regulate the functions of Th1, Th2 and Th17 cells.7

Clinical implications: In TB, T helper (Th)1 cytokines provide protection whereas Th2 and T regulatory (Treg) cytokines contribute to the pathogenesis and Th17 cytokines play a role in both protection and pathogenesis.

In respect to the type of cellular immunity, wP-containing vaccine induce a Th1 and Th17 skewed response whereas aP-containing vaccines mostly induce a Th2 skewed response.

Th1 responses have been traditionally elicited by live-attenuated, vector-based or Toll-like receptor ligand-adjuvanted formulations for optimal stimulation of the innate immune system and immunomodulation, while most of the present licensed alum-adjuvanted subunit vaccines fail to elicit Th1/Th17 immune responses.

Q11. What is humoral immunity?

Ans. Humoral immunity is mediated through B lymphocytes by secreting antibodies (immunoglobulins) that act by neutralization, complement activation or by promoting opsonophagocytosis, which results in early reduction of pathogen load and clearance of extracellular pathogens and their toxins. Some humoral antibodies prevent colonization, which is the first step in pathogenesis by encapsulated organisms such as Haemophilus influenzae type B (HIB), pneumococcal, meningococcal and non-capsulated organisms causing diphtheria and pertussis.

Q12. What are immunoglobulins? Which are different types of immunoglobulins? What is their role?

Ans. B cells have immunoglobulin surface receptors which bind with appropriate antigen that stimulates B cell to mature into antibody secreting plasma cells and generate immunoglobulins. Immunoglobulins are of different types (IgG, IgM, IgA, IgD and IgE) and they differ in their structure, half-life, site of action and mechanism of action. Scientists have identified nine chemically distinct classes of human immunoglobulins, four kinds of IgG and two kinds of IgA, plus IgM, IgE, and IgD. Immunoglobulins G, D, and E are similar in appearance.

The role of each type of immunoglobulin is as under (Fig. 3):

IgG, the major immunoglobulin in the blood, is also able to enter tissue spaces, able to cross placenta, it works efficiently to coat microorganisms, speeding their destruction by other cells in the immune system.8

Fig. 3: Functions of antibodies.
IgD is almost exclusively found inserted into the membrane of B cells, where it somehow regulates the cell's activation.
IgE is normally present in only trace amounts, but it is responsible for the symptoms of allergy.
IgA guards the entrance to the body. It concentrates in body fluids such as tears, saliva, and secretions of the respiratory and gastrointestinal tracts.
IgM usually combines in star-shaped clusters. It tends to remain in the bloodstream, where it is very effective in killing bacteria in early phase.

Q13. What is the main function of cell mediated or cellular immunity?

Ans. T cells are the effectors of cell mediated immunity (CMI). It is the principal defense mechanism against intracellular microbes. The T cell responses are more robust, long lasting and more cross protective than humoral responses hence modern vaccinology is being directed in this direction. The inherent T cell mediated immune regulatory mechanisms prevent any vaccines causing autoimmune diseases.

BCG is the only currently used human vaccine for which there is conclusive evidence that T cells are the main effectors.

Q14. What are antigen presenting cells (APC)?

Ans. Antigen-presenting cells (APCs) are a heterogeneous group of immune cells that mediate the cellular immune response by processing and presenting antigens for recognition by certain lymphocytes such as T cells. Classical APCs include dendritic cells, macrophages, Langerhans cells and B cells.9

Q15. What are the dendritic cells?

Ans. Dendritic cells (DCs) are antigen-presenting cells (also known as accessory cells) of the mammalian immune system. They act as messengers between the innate and the adaptive immune systems. Dendritic cells are present in those tissues that are in contact with the external environment, such as the skin (where there is a specialized dendritic cell type called the Langerhans cell) and the inner lining of the nose, lungs, stomach and intestines. They can also be found in an immature state in the blood. At certain development stages they grow branched projections, the dendrites that give the cell its name as dendritic cell. Once activated, they migrate to the lymph nodes where they interact with T cells and B cells to initiate and shape the adaptive immune response.

Q16. What role do dendritic cells play in immunity?

Ans. Dendritic cells are the only cells, capable of activating naïve T cells and play a crucial role in the induction of T cell response. They capture antigen, process then into small peptides, display them through MHC molecules and provide costimulation signals to activate antigen-specific T cells.

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 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 (Fig. 4).

Q17. What are toll-like receptors?

Ans. Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. They are single-pass membrane-spanning receptors usually expressed on sentinel cells such as macrophages and dendritic cells, that recognize structurally conserved molecules derived from microbes. Innate immunity is induced by exposure to evolutionarily conserved molecular structures termed pathogen-associated molecular patterns (PAMPs) that are expressed by a wide variety of infectious microorganisms. The recognition of PAMPs is mediated by pattern recognition receptors including TLRs, nod-like receptors and RIG-I-like receptors. Among them TLRs constitute one of the largest and most extensively studied classes of pattern recognition receptors. The innate immune response elicited by TLR activation is primarily characterized by the production of proinflammatory cytokines, chemokines, type I interferons (IFNs) and antimicrobial peptides. The ability of the immune system to recognize molecules that are broadly shared by pathogens is, in part, due to the presence of immune receptors called toll-like receptors that are expressed on the membranes of leukocytes including dendritic cells, macrophages, natural killer cells, cells of the adaptive immunity T cells, and B cells, and nonimmune cells. There are 13 TLRs, TLR 1 to TLR13, though the last three are not found in humans.10

Fig. 4: Role of dendritic cells in immunity.

Q18. How do vaccines mediate protection?

Ans. Vaccines play a crucial role in prevention, elimination and eradication of vaccine preventable diseases. This is best achieved by immunization programs capable of inducing long-term protection. This can be achieved by the maintenance of antigen-specific immune effectors and/or by the induction of immune memory cells.

Most of the currently available vaccines provide protection through induction of B cells and production of antigen-specific antibodies. Antibodies either neutralize the antigen or promote opsonophagocytosis which results in early reduction of pathogen load and clearance of extracellular pathogens.11

The role of cell mediated immunity in currently used vaccines (that have T cell dependent antigens) is mainly by supporting antibody protection. Other less common mechanisms by which cell mediated immunity works is by cytotoxic CD8+ T lymphocytes (CTL) that may limit the spread of infectious agents by recognizing and killing infected cells or secreting specific antiviral cytokines. Cellular immunity is essential for clearance of intracellular pathogens. The generation and maintenance of both B and CD8+ T cell responses is supported by growth factors and signals provided by CD4+ T helper (Th) lymphocytes, which are commonly subdivided into T helper 1 (Th1) and T helper 2 (Th2) subtypes.

BCG is the only currently used human vaccine for which there is conclusive evidence that T cells are the main effectors. Another instance is measles vaccination at 6 months during outbreak. 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. As a result these children may remain susceptible to measles infection, but are protected against severe disease 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.

Q19. Define active and passive immunity.

Ans. Active immunity is acquired through natural infection/immunization and is long lasting. Passive immunity is conferred by maternal antibodies or immunoglobulin/antitoxin sera preparations and is short lasting.

Q20. Which are main types of vaccines?

Ans. Vaccines may be broadly classified as follows:

Live attenuated vaccines: BCG, oral polio, measles, MMR, chicken pox, Rota virus yellow fever, live influenza vaccine, live hepatitis A.
Inactivated (Killed vaccines) may be:
1. Whole cell inactivated: Whole Cell Pertussis vaccines, Rabies, IPV, Hepatitis A.
2. Subunit vaccines: They differ from inactivated whole-cell vaccines, by containing only the antigenic parts which are necessary to elicit a protective immune response. They are as under—
  1. Protein vaccines:
    
    Inactivated toxins/toxoids (diphtheria/tetanus toxoids)
    Subunit vaccines: Acellular pertussis, HBV, some influenza.
  2. Polysaccharide vaccines:
    
    Pure polysaccharide: Comprising only of the polysaccharide—typhoid, PPPV, meningococcal
    Conjugated Hib, Typhoid, PCV Meningococcal—conjugation of the polysaccharide with a protein carrier (glycoconjugates) significantly improves the immune response.12
  3. Virus-like particle (VPL): HPV, Influenza
  4. DNA vaccines
  5. RNA vaccines.

Q21. What are live vaccines? How immunogenic are they?

Ans. Live attenuated vaccines (LAV) are derived from disease-causing pathogens (virus or bacteria) that have been attenuated under laboratory conditions. LAVs stimulate an excellent immune response as they mimic a natural infection. The vaccine virus/bacteria multiply and disseminate in multiple tissues and results in lymph node stimulation of dendritic cells at multiple sites. It also provides continual antigenic stimulation giving sufficient time for memory cell production. The activated DCs migrate towards the corresponding draining lymph nodes and launch multiple foci of T and B cell activation.

Q22. What are killed vaccines? How immunogenic are they?

Ans. Inactivated vaccines are made from microorganisms (viruses, bacteria, other organisms) that have been killed through physical or chemical processes. They can be whole or fractional subunit vaccines. Inactivated whole-cell vaccines are far less immunogenic as compared to live vaccines and the response may not be long lasting. Several doses of inactivated whole-cell vaccines may be required to evoke a sufficient immune response. In case of killed vaccines, there is only local and unilateral lymph node activation without associated dissemination and replication.

The immunogenicity of killed vaccine can be improved by various methods. Killed vaccines require adjuvants which improve the immune response by producing robust local inflammation and recruiting higher number of dendritic cells/monocytes to the injection site. Inactivated vaccines are more heat stable than live attenuated vaccines.

Q23. What are adjuvants?

Ans. Adjuvant is a substance that potentiates and/or modulates the immune responses to an antigen to improve their immunogenicity. They act by enhancing antigen presentation and/or by providing costimulation signals (immunomodulators). Aluminum salts are the most commonly used adjuvants in human vaccines. A toll-like receptors analog, named CpG ODNs, a new generation adjuvant, improves the function of professional antigen-presenting cells and boost the generation of humoral and cellular vaccine-specific immune responses:

Adjuvants help in the translocation of antigens to the lymph nodes where they can be recognized by T cells.
They provide physical protection to antigens which grants the antigen a prolonged delivery, providing additional time for upregulating the production of B and T cells needed for greater immunological memory in the adaptive immune response.13
They increase the capacity to cause local reactions at the injection site (during vaccination), inducing greater release of danger signals.
They induce the release of inflammatory cytokines which helps to not only recruit B and T cells at sites of infection but also increase transcriptional events leading to a net increase of immune cells as a whole.
They are believed to increase the innate immune response to antigen by interacting with pattern recognition receptors (PRRs) on or within accessory cells.

Q24. Does the route of administration of vaccines matter with the type of vaccine? How?

Ans. The site and route of administration of killed vaccines is of great importance. For killed vaccines intramuscular route is preferred over the subcutaneous route. As the muscles are well-vascularized and has a large number of patrolling dendritic cells. Hence, the vaccines which are supposed to be given intramuscularly should not be given subcutaneously and even if administered inadvertently that dose should be discounted, e.g., Rabies vaccine, Hepatitis B vaccine.

Intradermal route recruits the abundant dendritic cells in the skin and offers the advantage of antigen sparing, early and effective protection but the GMTs are lower than that achieved with IM and may wane faster. Dendritic cells are in highest number in the skin and hence marked reduction (e.g., 10-fold) of the antigen dose in intradermal immunization, e.g., ARV, IPV.

Finally due to focal lymph node activation, multiple killed vaccines may be administered at different sites with little immunologic interference.

The site of administration is usually of little significance for live vaccines. Immunologic interference may occur with multiple live vaccines unless they are given on the same day or at least 4 weeks apart or by different routes.

Q25. What are the characteristics of T cell independent immune response? Which vaccines do exhibit such response?

Ans. T cell independent immune response is elicited by B cells only and has following characteristics:

Only B cell response, T cell independent
Poorly immunogenic below 2 years due to immaturity of the marginal zones
Do not trigger GC activity
Weaker and shorter immune response
No induction of immune memory, hence no booster responses
There is no local immunity as IgA are not produced
Repeated doses lead to hypo responsiveness.

Bacterial (S. pneumoniae, N. meningitidis, H. influenzae, S. typhi) polysaccharide (PS) antigens exhibit T cell independent antigens.14

Q26. Describe the first steps after immunization.

Ans. Following vaccine injection, the vaccine antigens attract local and systemic dendritic cells, monocytes and neutrophils. Innate immune responses activate these cells by changing their surface receptors and migrate along lymphatic vessels, to the draining lymph nodes where the activation of T and B lymphocytes takes place. The type of response elicited will depend upon type of vaccine, its antigenic type and content and immune status of an individual. Vaccines that stimulate innate immunity effectively are better immunogens. This can be achieved by live vaccines, adjuvants, toll-like receptors (TLR) agonists, live vectors and DNA vaccines. Live vaccines are capable of activating innate immunity in a better way which is helpful for subsequent induction of adaptive immune effectors.

In the lymph nodes, the response to polysaccharide vaccines and protein/protein-conjugate vaccines are different.

Q27. What are the immune responses to polysaccharide vaccines?

Ans. On being released from the injection site they 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 activate, 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. As PS antigens do not induce germinal centers, bona fide memory B cells are not elicited (Fig. 5). 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.

Q28. What is hyporesponsiveness?

Ans. Revaccination with certain bacterial 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. Due to this phenomenon, only a single booster of either Pneumococcal or Meningococcal polysaccharide vaccine is recommended even in patients who require lifelong protection.

Fig. 5: Immune response to polysaccharide vaccines.

Q29. Which are characteristics of T cell dependent vaccines? Which vaccines do exhibit such response?

Ans.

Consistently immunogenic in infants beyond 6 months
Induces both T cell and B cell response
Immune response is robust long lasting and with higher titers of IgG response
High quality antibody
Booster response with repeated doses
No hyporesponsiveness.

Protein antigens which include pure proteins (Hepatitis B, Hepatitis A, HPV, Toxoids) or conjugation of PS antigens with a protein carrier (Hib, Pneumococcal, Meningococcal) are T cell dependent antigens.

Q30. What are the immune responses to protein and conjugated vaccines?

Ans. The immune enhancing effect of protein and of conjugate vaccines is assumed to result from an increase of carrier driven T-helper frequency and T-cell mediated costimulatory signals. Activation of germinal center (GC) is the key to such robust and long lasting immune response (Fig. 6).

Fig. 6: The germinal center (GC) response.Source: Stebegg M, Kumar SD, Silva-Cayetano A, Fonseca VR, Linterman MA, Graca L. Regulation of the germinal center response. Front Immunol. 2018;9:2469.

In response to a protein antigen reaching lymph nodes or spleen, B cells capable of binding to this antigen with their surface immunoglobulins undergo a brisk activation. In an extrafollicular reaction, B cells rapidly differentiate in plasma cells that produce low-affinity antibodies (of the IgM ± IgG/IgA isotypes) that appear at low levels in the serum within a few days after immunization (similar to PS antigens). Additionally, antigen-specific helper T cells that have been activated by antigen-bearing dendritic cells trigger some antigen-specific B cells to migrate toward follicular dendritic cells (FDCs) initiating the GC reaction. FDCs play an essential role in B cell responses: they attract antigen-specific B and T cells and capture/retain antigen for extended periods. B cells that are attracted by Ag-bearing FDCs become the founders of GCs. In GCs, B cells receive additional signals from follicular T cells and undergo massive clonal proliferation. This intense proliferation is associated to two major events: Ig class-switch from IgM toward IgG, IgA or IgE, and affinity maturation of the of B cells for their specific antigen which differentiate into plasma cells secreting large amounts of antigen-specific antibodies. At the end of the GC reaction, 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.

The development of this GC reaction requires a couple of weeks, such that hypermutated IgG antibodies to protein vaccine antigens first appear in the blood 10–14 days after priming. It is the magnitude of GC responses, i.e., the quality of DC, B cell, Tfh cell and FDC interactions, which controls the intensity of B cell differentiation into plasma cells, and thus the peak of IgG vaccine antibody reached within 4–6 weeks after primary immunization.

Q31. What is antibody affinity and avidity?

Ans. Antibody affinity refers to the strength with which the epitope binds to an individual paratope (antigen-binding site) on the antibody. High affinity antibodies bind quickly to the antigen, permit greater sensitivity in assays and maintain this bond more readily under difficult conditions.

Antibody avidity describes the sum of the epitope specific affinities with which an antibody binds to a complex antigen.

Q32. What are memory B cells?

Ans. Memory B cells are those B lymphocytes that are generated 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. Antigen-specific memory cells generated by primary immunization are much more numerous than naïve B cells initially capable of antigen recognition.17

Memory B cells do not produce antibodies, i.e., do not protect, unless re-exposure to antigen drives their differentiation into antibody producing plasma cells. This reactivation is a rapid process, such that booster responses are characterized by the rapid increase to higher titers of antibodies that have a higher affinity for antigen than antibodies generated during primary responses. The reactivation, proliferation and differentiation of memory B cells occur without requiring the induction and development of GC responses. This process is thus much more rapidly completed than that of primary responses.

Q33. Why do we need more than one dose, even for live vaccines?

Ans. The older concept that single dose of live vaccine induces life-long immunity is not true. The live vaccines induce an immune response similar to that seen with protein vaccines. However, live vaccines have limitations in the form of primary and secondary failures. Sometimes the take up of live vaccines is not 100% with the first dose (primary failure). Hence, more than 1 dose is recommended with these 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 such live vaccine will take care for primary vaccine failures (no uptake of vaccine). Secondary vaccine failures are associated with decline in antibody titers with passage of time and here also second dose of live vaccine becomes necessary. The examples are varicella and mumps vaccines.

Q34. What is primary and secondary (booster) immune response?

Ans. When an antigen is introduced for the first time, the immune response starts after a lag of 10 days or so. This is called primary response. Such response is short-lived, has a lag period, mainly IgM type with low titers of antibodies. In primary immune response, the antigen exposure elicits an extrafollicular response that results in the rapid appearance of low IgG antibody titers. As B cells proliferate in GCs and differentiate into plasma cells, IgG antibody titers increase up to a peak value usually reached 4 weeks after immunization. The short-life span of these plasma cells results in a rapid decline of antibody titers, which eventually return to baseline levels 3.

Secondary immune responses start on subsequent exposure (booster) to the same antigen. There is no lag phase, response starts in less than 7 days, persists for a long time, mainly IgG type with high antibody titers. Booster exposure to antigen reactivates immune memory B cells and results in a rapid (<7 days) increase of IgG antibody titer by a rapid proliferation of memory B cells and their evolution into abundant antibody secreting plasma cells. Short-lived plasma cells maintain peak Ab levels during a few weeks, after 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. This generic pattern may not apply to live vaccines triggering long-term IgG antibodies for extended periods of time (Fig. 7).18

Fig. 7: A schematic diagram showing a primary and secondary response.

Q35. Which are the determinants of intensity and duration of immune responses?

Ans. Both, primary and secondary immune responses after vaccination depend on various factors such as vaccine type, nature of antigen, vaccination schedule, genetic and environmental factors and age at immunization.

Vaccine Type

Broadly speaking live vaccines are superior (exception BCG, OPV) to protein antigens which in turn are superior to polysaccharide vaccines.

Live vs. inactivated: Higher intensity of innate responses, higher antigen content following replication and more prolonged antigen persistence generally result into higher antibodies (Ab) responses to live than inactivated vaccines.
Protein vs. polysaccharide: Recruitment of T cell help and induction of germinal centers (GCs) results into higher antibody responses to protein or glycoconjugate than to pure polysaccharide vaccines.
Adjuvants: Adjuvants improve immune responses to inactivated vaccines by either modulation of antigen delivery and persistence (depot or slow-release formulations) or enhancement of Th responses (immunomodulator) which may support or limit antibody responses.

Antigen Content

Polysaccharide antigens: Failure to induce GCs limit immunogenicity.
Protein antigens: Inclusion of epitopes readily recognized by B cells (B cell repertoire), inclusion of epitopes readily recognized by follicular helper T cells, elicitation of efficient follicular T cell help and the capacity of antigen to associate/persist in association to follicular dendritic cells (FDCs) result into higher antibody responses.19
Antigen dose: As a rule, higher antigen doses (e.g., Hepatitis B vaccine) increase the availability of antigen for B/T cell binding and activation, as well as for association with FDCs however there is a limiting dose for each.

Vaccination Schedule

The immune response improves with increasing number of doses and increased spaces between doses.

Other Factors

Age at immunization: Early life immune immaturity or age-associated immune senescence impairs immune responses to an administered vaccine.
Genetic factors: The capacity of antigen epitopes to associate to a large panel of MHC molecules increases the likelihood of responses in the population. MHC restriction may limit T cell responses. Gene polymorphisms in molecules critical for B and T cell activation/differentiation are likely to affect Ab responses. T cell responses differ markedly between individuals and populations because of genetic variability of MHC molecules (HLA A2).
Environmental factors: Mostly yet to be identified.

Q36. What is priming and boosting mechanism for killed vaccines?

Ans. The immune response improves with proper spacing of vaccine doses. Traditionally, “0-1-6” month schedule (prime and boost) is considered as a most immunogenic schedule than 6-10-14 week or 2,3,5 month or 2,4,6 month schedules for non-live T-cell dependent vaccines such as Hepatitis-B, vaccine. Here there is adequate time interval between first few doses for priming and inducing the 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. A 4 week minimal interval between primary doses avoids competition between successive waves of primary responses.

Q37. What should be the spacing between two or more vaccines?

Ans. The spacing between two or more live and/or killed vaccines is vaccines is given in Table 1.20

Table 1 Timing and minimal period of spacing between two doses of vaccines.

Antigen combination	Minimal interval between doses
2 or more inactivated antigens	None, can be administered simultaneously or at any interval between 2 doses
Inactivated and live antigens	None, can be administered simultaneously or at any interval between 2 doses
2 or more live	28 days minimum interval if not administered at the same visit

Q38. What is the importance of immune memory in immunization programs?

Ans. Immune memory allows one to complete an interrupted vaccine schedule without restarting the schedule. Immune memory is seen with live vaccines/protein antigens due to generation of memory B cells which are activated on repeat vaccination/natural exposure. 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 do not need regular boosters as the long incubation period of the disease allows for activation of immune memory cells.

Q39. What are limitations of immune responses during early life?

Ans. Transplacentally acquired maternal antibodies, and immaturity of immune system limit the immune responses during young age. 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. The extent and duration of the inhibitory influence of maternal antibodies increase with gestational age, e.g., with the amount of transferred immunoglobulins (Ig), and declines with postnatal age as maternal antibodies wane.

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 glycoconjugate vaccines, even the most potent glycoconjugate vaccines elicit markedly lower primary IgG responses in young infants.21

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.

Maternal antibodies inhibit only B cell induced antibodies responses but not T-cell response, which remain largely unaffected or even enhanced, e.g.,:

BCG may be given as the maternal antibodies actually enhance T cell responses.
OPV may be given as there are no maternal IgA in the gut to neutralize the virus.
Measles vaccine if given at the age of 6 months (in an outbreak situation) may work by inducing T cell immunity.

Q40. How are these limitations of young age immunization overcome?

Ans. This issue can be addressed favorably to a certain extent by increasing the number of a 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.

Q41. Why do we offer birth doses of BCG, OPV, HBV in spite of presence of maternal antibodies?

Ans.

BCG can be given as maternal antibodies actually enhance T cell responses. (BCG is T cell response and not affected by circulating maternal antibodies)
OPV may be given as there is no maternal IgA in the gut to neutralize the virus as maternal Ab are only IgG type. (Priming, so better seroconversion to subsequent doses)
Birth dose of HBV acts as priming dose so that subsequent doses are capable of eliciting immune response even in presence of maternal antibodies. This is because maternal antibodies do not interfere with induction of memory B cells allowing certain degree of much needed priming. (Important for prevention of both vertical and horizontal transmission)22

Q42. Why do we practice early accelerated schedule of 6-10-14 weeks despite young age limitations on immunization schedules?

Ans. Immunization schedules, practiced in developed world, commencing at 2 months and having 2 months spacing between the doses are considered technically appropriate. However, we do not follow it in our country. Disease epidemiology of vaccine-preventable diseases (VPDs) in a country often determines a particular vaccination schedule. Since, majority of childhood infectious diseases cause morbidity and mortality at an early age in developing countries, there is need to protect the children at the earliest opportunity through immunizations. This is the reason why early, accelerated schedules are practiced in developing countries despite the known limitations of young age immunization. So for both, operational reasons and for early completion of immunization, the 6, 10, 14 week's schedule is chosen in developing countries. Such a schedule has shown to give adequate protection in recipients.

Q43. Why do we have different number of doses for different age groups for inactivated vaccines?

Ans. For killed vaccines such as DPT, Hib, Pneumococcal and Hep B which are administered as early as birth/6 weeks, the first dose acts only as a priming dose while subsequent doses provide an immune response even in presence of maternal antibodies. However a booster at 15–18 months is required for durable immunity. As the age of commencement of vaccination advances the number of doses reduces (2 doses at 6–12 months followed by a booster dose and 1–2 doses between 12 and 23 months for Hib and Pneumococcal vaccines).

Q44. Do we need a vaccine after getting recovered from a particular disease?

Ans. In general natural infection with viral illness provides very long lasting or life-long immunity. Hence, viral vaccines such as MMR, Varicella, etc., are not advised after such diseases. On the other hand bacterial illnesses do not impart such protection, justifying the need for vaccination, e.g., diphtheria, tetanus, typhoid.

Chapter Notes

Basic ImmunologyCHAPTER 1