- Vaccine Immunology: Basics and BeyondYash Paul, Vipin M Vashishtha
- Elementary Epidemiology in VaccinationVipin M Vashishtha, Yash Paul
- Vaccination SchedulesYash Paul, Satish V Pandya
- Practice of VaccinationYash Paul, Satish V Pandya, Atul K Agarwal
- Vaccine-preventable Diseases SurveillanceMeeta Dhaval Vashi
- Vaccination in Special SituationsAbhaya K Shah
- Adverse Events following Immunization, Vaccine Safety and Misinformation against VaccinationMeeta Dhaval Vashi, Vivek R Pardeshi
- Cold Chain and Vaccine StorageDigant D Shastri
- Control and Eradication of Infectious DiseasesYash Paul, Priya Marwah
- Development and Licensing of VaccineGautam Rambhad, Canna Jagdish Ghia
- National Immunization Technical Advisory GroupMadhu Gupta, Adarsh Bansal
- Medicolegal and Ethical Issues in ImmunizationSatish Kamtaprasad Tiwari, Yash Paul
- Vaccine Schedules including National Immunization ProgramMeeta Dhaval Vashi, Vivek R Pardeshi
- Vaccine Hesitancy
1. 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 into the body. The vaccine may or may not seroconvert to vaccine whereas the process of inducing immune response, which can be “humoral” or “cell mediated” in the vaccine 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.
2. 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 (CMI)”. Antibodies are of several different types [immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA), immunoglobulin D (IgD), and immunoglobulin E (IgE)] and they differ in their structure, half-life, site of action, and mechanism of action. Humoral immunity is the principal defense mechanism against extracellular microbes and their toxins. B lymphocytes secrete antibodies that act by neutralization, complement activation, or by promoting opsonophagocytosis. CMI is the principal defense 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 natural killer (NK) cells. The cytotoxic T cells act by lysing infected cells.
3. What are innate and adaptive immunity?
Innate immunity comprises of the skin and mucosal barriers, phagocytes (neutrophils, monocytes, and macrophages), and the NK cells. It comes4 into play immediately on entry of the pathogen and is nonspecific. Adaptive immunity is provided by the B lymphocytes (humoral/antibody-mediated immunity) and T lymphocytes (cellular/CMI). 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 (Fig. 1 and Table 1).
4. What are “B” and “T” cells? What role do they play in regard to immunology of vaccines?
Immune system is almost nonexistent 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.
Fig. 1: Innate and adaptive immunity.Source: Abbas AK, Lichtman AH, Pillai S. Introduction to the immune system. In: Basic Immunology. 6th edition, Elsevier, 2019.
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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 humans. 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, and become activated in the spleen/nodes if (1) their T-cell receptor (TCR) binds to an antigen presented by a major histocompatibility complex (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 generates IgM. T helper 2 (Th2) cell leads to switch in the production from IgM to IgG and 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).
5. What are antigen-presenting cells (APCs) and dendritic cells? What functions do they perform?
Antigen-presenting cells 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 costimulation signals that act synergistically to activate antigen-specific T cells. APCs include B cells, macrophages, and dendritic cells (DCs), although only DCs are capable of activating naïve T cells (Fig. 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 (TLRs).
Vaccine antigens are taken up by immature 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 TLRs 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 [MHC-human leukocyte antigen (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
Fig. 2: Schematic presentation of a dendritic cell and its activation by pathogens.Source: Siegrist CA. Vaccine immunology. Presentation delivered at 20th Advanced Course of Vaccinology, Annecy, France 2019. Available from: https://www.advac.org/images/files/members/files_presentations/2019/0900_SIEGRIST_ADVAC_2019_Immunology_slides_-_9h-11h30.pdf(IL-1: interleukin-1; MHC: major histocompatibility complex)
Antigen-specific TCRs 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.
6. 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 costimulation signals (immunomodulators). Aluminum 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 analogs of TLRs, for example, CpG-ODN is used in new generation of Japanese encephalitis vaccines.
Most nonlive 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: (1) delivery systems that prolong the antigen deposit at site of injection, recruiting more DCs into the reaction and (2) 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 exceeds that of nonlive vaccines.
7. What are “germinal centers” and “marginal zone”?
Germinal centers (GCs) are dynamic structures that develop in spleen/nodes in response to an antigenic stimulation and dissolve after a few weeks.7 GCs contain a monoclonal population of antigen-specific B cells that proliferate and differentiate through the support provided by follicular DCs 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.
8. What do the terms “epitope” and “paratope” mean?
An “epitope” refers to the specific target against which an individual antibody binds. When an antibody binds to a protein, it is not binding to the entire full-length protein. Instead, it is binding to a segment of that protein known as an “epitope”. Hence, an epitope is a specific part of the antigen that elicits an immune response.
Binding between the antibody and the epitope occurs at the antigen-binding site, which is called a “paratope” and is located at the tip of the variable region on the antibody. This paratope is only capable of binding with one unique epitope.
9. 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.
10. What do the terms “CD4+ T cells” and “CD8+ T cells” stand for? What are the functions of these lymphocytes?
CD4+ T cells are those T lymphocytes that express the cluster of differentiation 4 (CD4) glycoprotein at their surface. CD4 is a glycoprotein expressed on the surface of T-helper cells, regulatory T cells, monocytes, macrophages, and DCs. It was discovered in the late 1970s and was originally known as Leu-3 and T4 (after the OKT4 monoclonal antibody that reacted with it) before being named CD4 in 1984. In humans, the CD4 protein is encoded by the CD4 gene. CD4 is a coreceptor that assists the TCR to activate its T cell following an interaction with an APC. 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 signaling cascade of an activated T cell. CD4 interacts directly with MHC class II molecules on the surface of the APC using its extracellular domain.
T cells expressing CD4 molecules (and not CD8) on their surface, therefore, are specific for antigens presented by MHC class 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.8
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 lymphocytes (CTLs) and destroy virally infected cells and tumor cells and are also implicated in transplant rejection (Fig. 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 interferon-γ (IFN-γ), tumor necrosis factor-α/-β (TNF-α/-β), interleukin-2 (IL-2) and IL-3, and supporting activation and differentiation of B cells, CD8+ T cells, and macrophages [T helper 1 (Th1) cells].
Producing IL-4, IL-5, IL-13, IL-6 and IL-10, and supporting B-cell activation and differentiation (Th2 cells).
11. What are toll-like receptors (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. TLRs are a key driver of innate immune response. They can recognize an invading pathogen through structurally conserved molecules of that particular pathogen collectively referred to as pathogen-associated molecular patterns (PAMPs) that are different from the host molecules. They later activate a cascade of immune responses that clear the infection. Many TLRs are now employed as adjuvants in novel vaccine products.
12. 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 body after administration and stimulate the immune system. The older concept that single dose of live vaccines induces lifelong immunity perhaps does not hold true, we need many doses of oral polio vaccine (OPV) and booster doses are required for live vaccines such as measles vaccine, varicella, rubella, and live oral typhoid vaccines. Inactivated vaccines may consist of whole-inactivated organisms such as whole-cell pertussis, typhoid, rabies; inactivated polio vaccine, modified exotoxins called “toxoids” such as diphtheria toxoid or tetanus toxoid; subunits such as polysaccharide antigens of Salmonella typhi, Haemophilus influenzae type b (Hib), and surface proteins of hepatitis B virus.9 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 ribonucleic acid (RNA)] allowing their recognition by pattern recognition receptors (PRRs)—TLRs. Following injection, viral particles rapidly disseminate throughout the vascular network and reach their10 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, DCs are activated at multiple sites, migrate toward 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 nonlive vaccines.
The strongest antibody responses are generally elicited by live vaccines that better activate innate reactions and thus better support the induction of adaptive immune effectors. Nonlive vaccines frequently require formulation in adjuvants, of which aluminum 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 deabsorbed and released, extending the duration of B- and T-cell activation, as well as the preferential induction of IL-4 by aluminum-exposed macrophages.
Very few nonlive 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 nonlive vaccines eventually wane.
13. 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 weeks of age onward 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 and repeated doses of vaccine do 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 help in providing mucosal protection and eradication of the carrier state. Few examples of T cell-independent vaccines include bacterial polysaccharide (PS) vaccines such as Streptococcus pneumoniae (S. pneumoniae), Neisseria meningitidis, Haemophilus influenzae (H. influenzae), and Salmonella typhi.
14. 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, in11 T cell-independent immune response, being B-cell mediated, younger children do not respond to such vaccines. A T cell-independent antigen like PS can be made into T cell dependent by the technique of conjugation. Such conjugated vaccines can be administered to children <2 years of age also. This technique is used to produce conjugated Vi typhoid, Hib, and pneumococcal and meningococcal vaccines.
15. 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 2). Capsular PSs 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 antigen-specific CD4+ T helper 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., deoxyribonucleic acid (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 Bacillus Calmette-Guérin (BCG)-induced T cells produce cytokines that contribute to macrophage activation and control of Mycobacterium 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 the 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). 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.12
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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.13
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 human immunodeficiency virus-1 (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).
16. What are the clinical scenarios where evidences of T-cells protection are available?
Bacillus Calmette-Guérin 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.
17. What happens once a vaccine is administered to a vaccinee?
Following injection, the vaccine antigens attract local and systemic DCs, 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 DCs/monocytes to the injection site. Secondly, the site of administration of killed vaccines is of importance; the intramuscular route which is well-vascularized and has a large number of patrolling DCs is preferred over the subcutaneous route. The site of administration is usually14 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.
18. 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 nonprotein, PSs in nature, reach the marginal zone of the spleen/nodes, and bind to the specific immunoglobulin surface receptors of B cells. In the absence of antigen-specific T cell help, B cells are activated, proliferated, and differentiated in plasma cells without undergoing affinity maturation in GCs. 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 <2 years due to immaturity of the marginal zones. As PS antigens do not induce GCs, bonafide memory B cells are not elicited. Consequently, subsequent reexposure to the same PS results in a repeat primary response that follows the same kinetics in previously vaccinated as in naïve individuals.
19. What is hyporesponsiveness of repeated doses of a vaccine referring to?
Revaccination with certain bacterial PSs, 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.
20. 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 [hepatitis B, hepatitis A, human papillomavirus (HPV), and toxoids] or conjugated protein carrier with PS antigens (Hib, meningococcal, and pneumococcal). 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 DCs trigger some antigen-specific B cells to migrate toward follicular dendritic cells (FDCs), initiating the GC reaction. In GCs, B cells receive additional signals from follicular T cells and undergo massive clonal proliferation, switch from IgM toward 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 GC 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, where15 they survive through signals provided by supporting stromal cells and this results in prolonged persistence of antibodies in the serum.
21. 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 reexposed 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.
22. 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 uptake of live vaccines is not 100% with the first dose. Hence, more than one 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 overtime).
23. What determines the 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 PS 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. Technically, 0, 1, and 6 months are the best immunization schedule; the first two doses are for induction and the long gap between the second and third doses 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.
24. What are the limitations of young age immunization?
Young age limits antibody responses to most vaccine antigens since maternal antibodies inhibit antibodies responses, but not T-cell response and due to limitation of B-cell response.
Immunoglobulin G 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,16 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 memory B 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., 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 postnatal age, as maternal antibodies wane.
25. Maternal antibodies interfere with neonatal immune responses, why hepatitis B, Bacillus Calmette-Guérin (BCG), and oral polio vaccine (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 of 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 OPV are significantly lower in infants than in adults.
26. How maternal antibodies can sometimes enhance T-cell responses of Bacillus Calmette-Guérin (BCG) vaccine administered at birth?
After administration of BCG, the maternal antibodies form immune complexes with the vaccine antigens. These immune complexes are taken17 up by more and more number of macrophages and DCs, which, in turn, are dissociated into their acidic phagolysosome compartment and are processed into small peptides. These peptides are displayed at the surface of APCs, thus available for binding by more number of CD4+ and CD8+ T cells.
27. 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 the World Health Organization (WHO) estimates, 2.5–3 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.
28. How limitations of young age immunization can be taken care of?
They can be countered by increasing the number of 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.
29. Which is the best vaccination schedule for nonlive vaccines acting on the principal of “prime-boost” mechanism?
Traditionally, 0–1–6 months schedule is considered as a most immunogenic schedule than 6–10–14 weeks or 2–3–5 months schedule for nonlive 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 (Fig. 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 weeks. However, for operational reasons and for early completion of immunization and attainment of protection, the 6, 10, 14 and weeks schedule is chosen in developing countries.18
Fig. 4: Schematic presentation of various components of 0, 1, and 6 months immunization schedule at cellular level.(IgA: immunoglobulin A; IgG: immunoglobulin G; IgM: immunoglobulin M)Source: Siegrist CA. Vaccine immunology. Presentation delivered at 20th Advanced Course of Vaccinology, Annecy, France 2019. Available from: https://www.advac.org/images/files/members/files_presentations/2019/0900_SIEGRIST_ADVAC_2019_Immunology_slides_-_9h-11h30.pdf
Accelerated infant vaccine schedules in which three vaccine doses are given at a 1-month interval (2, 3, 4 months or 3, 4, 5 months) result into lower responses than schedules in which more time elapses between doses (2, 4, and 6 months) or between the priming and boosting dose (3, 5, and 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.
30. 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 responses—primary 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.19
Fig. 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 immunoglobulin G (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 lifespan 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 antibody (6) during a few weeks—after which serum antibody titers decline initially with the same rapid kinetics as following primary (7) 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 (8) kinetics.Note: This generic pattern may not apply to live vaccines triggering long-term IgG antibodies for extended periods of time.Source: With permission from Siegrist CA. Vaccine immunology. In: Plotkin SA, Orenstein W, Offit P (Eds). Vaccines, 5th edition. Philadelphia: Saunders Elsevier; 2008. pp. 17-36.
Figure 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 lymph node. 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, nonconjugate, polysaccharide vaccines mainly induce 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.
31. What are the hallmarks of “memory B cell” responses?
These cells are only generated during T cell-dependent responses inducing GCs 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 reexposure to antigen. Memory B cells20 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 postprimary antibody titers 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, toward memory B cells. This phenomenon can be exploited by using small amount of expensive conjugate vaccines such as pneumococcal conjugate vaccine (PCV) followed by use of less expensive pneumococcal polysaccharide vaccine (PPV) as booster. Exposure to exogenous antigens may reactivate or favor the persistence of memory B cells.
32. 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 (travelers). Certain immunization schedules may not need boosters, if exposure provides regular natural boosters. Activation of immune memory and generation of protective antibodies usually take 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 and hepatitis B do 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 reexposed to antigen which drives their differentiation into antibody-producing plasma cells.
33. 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 reactions such as 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. Diphtheria, pertussis, and tetanus (DPT) is divided in three doses while rabies vaccine is divided in four or five doses.
34. 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.21 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, subclinical 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 nonimmune individuals in coming in contact with organisms (which is a beneficial for nonimmune people), but those immune following vaccination may be deprived of the benefit of repeated subclinical 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.
35. 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 is very large, so multiple doses are required.
It has been observed that natural infection with viral diseases provides very long or lifelong 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.
36. Do all inactivated vaccines need booster doses? How long after the primary doses are boosters advised? Why?
- No, certain vaccines such as hepatitis A, hepatitis B, HPV, etc., do not require frequent boosters.
- To believe that a single dose or a primary course of vaccination provides a lifelong immunity is a utopian thought. It does not exist because it could not exist. Not only inactivated, but even most live vaccines do need extra doses! Even for some of the highly efficacious live vaccines like measles, we need extra doses.
- The need and timing of boosters further depend on three factors: (1) related to the agent (microbe), (2) host, and (3) vaccine characteristics.
- Agent (microbe): If the incubation period of a disease is short than the time required for reactivation of memory B cells, plasma cells secrete antibodies. Boosters needed as incubation period is short in diseases such as diphtheria, tetanus, Hib, and pneumococcus, but not needed as incubation period is long such as in hepatitis B, hepatitis A, HPV, etc. It is generally considered that protection by22 toxoid-based vaccines requires the presence of antitoxin antibodies at time of toxin exposure. This is supported by the observation that despite the occurrence of many adult cases of diphtheria during a large outbreak in the former Soviet Union, a single vaccine dose raised strong antibody responses to this relatively poor immunogen. This confirmed that most patients had been immunized in childhood and had lost vaccine antibodies over time, but had persistent immune memory. This immune memory was however not sufficient to protect against diphtheria, a disease characterized by a short incubation period (1–5 days).
- Vaccine characteristics: It depends on the quality of immunogen and the vaccine-elicited immune responses. Take the case of another vaccine like flu vaccines. The immunity persists only for few months. Pertussis vaccines: you need frequent boosters. Japanese encephalitis (JE) vaccines: Old mouse brain: frequent boosters but new generation: only infrequently needed. We are now developing more new technology to have more durable immunity like use of nanoparticles, better adjuvants such as TLRs agonists, new delivery techniques, etc.
- Host: For example, in immunocompromised host with congenital/acquired immunodeficiency states (such as HIV, asplenia, complement deficiency, chronic illnesses, immunosuppressive drugs, malignancy, etc.), one needs frequent boosters to maintain a baseline antibodies level to accord protections.
37. Why cannot 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 cannot be administered at this age; (ii) different routes of administration, e.g., some are administered orally, others are administered parentally, some are administered intradermally (BCG), some are administered subcutaneously [measles, measles, mumps, and rubella (MMR), and 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 vaccines need two doses (such as measles, MMR, hepatitis A, and rotavirus vaccine), some vaccines need three doses (hepatitis B and HPV 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.
38. Few vaccines such as diphtheria, tetanus, and pertussis (DTP) vaccine causes higher incidence of fever, pain, tenderness, and other local reactions. Should antipyretics such as paracetamol, ibuprofen, etc., be prescribed prophylactically routinely?23
Prophylactic antipyretic administration decreases the postvaccination adverse reactions. Recent study finds that they may also decrease the antibody responses to several vaccine antigens.
Though prophylactic antipyretic administration leads to relief of the local and systemic symptoms after primary vaccinations, there is a reduction in antibody responses to some vaccine antigens without any effect on the nasopharyngeal carriage rates of S. pneumoniae and H. influenzae serotypes. Immunologically, this can be explained by reductions in “danger signals” due to anti-inflammatory effects of these compounds that “blunt” the antibodies production (Fig. 6). The development of fever or increase in the temperature postvaccination is due to the release of endogenous cytokines (IL-1 and TNF-α).
However, since the antibody response [geometric mean concentration (GMC)] was not reduced below seroprotection level, it is unlikely that prophylactic paracetamol (PCM) would have any detrimental effect for individual child concerned. Future trials and surveillance programs should also aim at assessing the effectiveness of programs where prophylactic administration of PCM is given. The timing of administration of antipyretics should be discussed with the parents after explaining the benefits and risks.
39. Immune memory is the function of adaptive immune system. Do innate immune systems also display memory responses?
Unlike the “adaptive” immunity, the “innate” immune system is supposed to have no memory responses. Recently, the phenomenon of vertebrate innate memory has experienced a renewed interest. This phenomenon is best exemplified by in vivo vaccination with the BCG that could induce a more effective host immune response to subsequent challenges, with a concomitant increase in resistance to unrelated infections. BCG, which can remain alive in the human skin for up to several months, triggers not only Mycobacterium-specific memory B and T cells but also stimulates the cells of the innate system (monocytes, neutrophils, macrophages, natural killer, DCs, etc.) for a prolonged period. The process by which BCG imparts immune memory to the innate system is known as “trained innate immunity”, which, in turn, is elicited by a phenomenon known as “epigenetic effect”.
“Epigenetic effect” is produced by the modification of gene expression rather than alteration of the genetic code or nucleotide sequencing. This effect is brought about by two main mechanisms, DNA methylation and histone modifications that alter innate immunity. BCG does epigenetic reprogramming in the training of innate cells, particularly monocytes. Upon pathogen X recognition by a receptor, “naïve” monocytes undergo epigenetic reprogramming and a metabolic shift and convert into “trained” monocytes, primed to respond more vigorously to nonspecific (pathogens X, Y, and Z) secondary stimulation. Unlike antigen-specific memory of the adaptive immune system, the second stimulation does not have to be with the same pathogen or antigen. Later on, these “trained” monocytes have a significantly higher production of several proinflammatory cytokines [such as IFN-γ, TNF-α, interleukins (IL-1β, IL-6, etc.] upon heterologous challenges, particularly T helper cell type 1 polarizing and typically monocyte-derived proinflammatory cytokines that help in rapid clearance of infection (Fig. 7).24
Fig. 6: “Danger signals”/adjuvants activate dendritic cells (DCs) directly or indirectly.(iNKT: invariant natural killer T; iTCR: invariant T-cell receptor; MPL: monophosphoryl lipid A; MSU: monosodium urate; PLG: poly-α-L-glutamine; TLRs: Toll-like receptors)Source: De Gregorio E, D’Oro U, Wack A. Immunology of TLR-independent vaccine adjuvants. Curr Opin Immunol. 2009;21(3):339-45.
These modified, activated, and “trained” cells can be stimulated by various nonrelated infectious (viruses, bacteria, fungi and their components, parasites) or noninfectious agents such as nanoparticles which lead to potent immune memory responses. This response explains the BCGs nonspecific protection against sepsis, pneumonia, and other pathogens. Both epigenetic changes and increased nonspecific immune responses could be detected up to 1 year after BCG vaccination.
40. What are the differentiating features of adaptive and innate memory responses?
Innate immune memory differs from adaptive memory for many aspects, including the lack of gene rearrangements, the involvement of epigenetic reprogramming, the type of cells involved (innate cells vs. T and B lymphocytes), and the receptors engaged in pathogen/antigen recognition (selective PRRs vs. antigen-specific T-cell and B-cell receptors). In general, innate memory is considered as a nonspecific short-lived phenomenon, as opposed to adaptive memory that is long-lived and highly specific.25
Fig. 7: “Trained innate immunity”—epigenetic reprogramming of monocytes. Upon pathogen X recognition by a receptor, naïve monocytes undergo epigenetic reprogramming and a metabolic shift and become primed to respond more robustly to nonspecific (pathogens X, Y, and Z) secondary stimulation.(DNA: deoxyribonucleic acid; IFN-γ: interferon-γ; IL-6: interleukin-6; TNF-α: tumor necrosis factor-α)Source: Vashishtha VM. Are BCG-induced non-specific effects adequate to provide protection against COVID-19? Hum Vaccin Immunother. 2020;7:1-4.
41. Can trained immunity may offer some protection against ongoing coronavirus disease 2019 (COVID-19) pandemic or other future viral pandemics?
Theoretically, the nonspecific, innate immunity shall provide some temporary protection against an unrelated viral pathogen through trained immunity. BCG is the vaccine to be tried for this purpose. It may have some utility owing to induction of strong, nonspecific, and innate immune responses in the vaccinated subjects. One should not expect significant inhibitory responses against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, but even “stop gap” protection and some attenuation of the disease may be expected against coronavirus disease 2019 (COVID-19). An extra dose of BCG to the healthcare workers and elderly people with comorbid conditions would be worth trying till a specific vaccine is developed (Figs. 8A to C).
Bacillus Calmette-Guérin is generally safe and well-tolerated. However, it is contraindicated in immunocompromised individuals, so one needs to be extra careful while administering BCG to these individuals. Whether the heterologous immunity would be adequate to neutralize the virus at its first portal of entry? Would the quantum of innate immune responses elicited in adults and elderly be at par with those produced in young children? Would BCG revaccination be safe in Mycobacterium tuberculosis-infected and uninfected populations? Would it be safe to administer a live vaccine to elderly with comorbidities? Which BCG strain would elicit the greatest nonspecific immunity? These are some of the queries that need urgent resolution.26
Figs. 8A to C: Trained immunity antiviral host defense and its role in a new viral pandemic. (A) BCG vaccination has been shown to protect against multiple viral pathogens; (B) Trained immunity leading to enhanced innate immune responses to different pathogens after a vaccination is mediated by metabolic and epigenetic rewiring in innate immune cells, which lead to increased gene transcription and improved host defense; and (C). Trained immunity as a tool for enhancing population immunity during a pandemic ahead of the availability of a specific vaccine.(BCG: Bacillus Calmette-Guérin; HSV: herpes simplex virus; IL-6: interleukin-6; RSV: respiratory syncytial virus; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TNF: tumor necrosis factor)Source: O’Neill LAJ, Netea MG. BCG-induced trained immunity: can it offer protection against COVID-19? Nat Rev Immunol. 2020;20(6):335-7.
As discussed above, the main argument against the ecological studies linking universal BCG use with protection against COVID-19 is the waning of BCG-induced immunity. But even if heterologous immune responses persist for a few weeks to few months, they should be able to provide some protection to the frontline health workers by immunomodulation.
SUGGESTED READING
- Advanced Course of Vaccinology (ADVAC). Presentation delivered at ADVAC. France: Springer; 2019.
- Das RR, Panigrahi I, Naik SS. The effect of prophylactic antipyretic administration on post-vaccination adverse reactions and antibody response in children: a systematic review. PLoS One. 2014;9(9):e106629.
- Netea MG, Joosten LAB, Latz E, Mills KHG, Natoli G, Stunnenberg HG, et al. Trained immunity: a program of innate immune memory in health and disease. Science. 2016;352(6284):aaf1098.
- O'Neill LAJ, Netea MG. BCG-induced trained immunity: can it offer protection against COVID-19? Nat Rev Immunol. 2020;20(6):335–7.
- Siegrist CA. Vaccine immunology. In: Plotkin SA, Orenstein W, Offit P (Eds). Vaccines, 5th edition. Philadelphia: Saunders Elsevier; 2008. pp. 17–36.
- Singhal T, Amdekar YK, Agarwal RK. IAP Guidebook on Immunization, 4th edition. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2009.
- Thacker N, Shendurkar N. Childhood Immunization: Issues and Options, 1st edition. New Delhi: Incal Communications; 2005.
- Vashishtha M. Manual of Advancing Science of Vaccinology. Mumbai: Indian Academy of Pediatrics; 2009.
- Vashishtha VM. Are BCG-induced non-specific effects adequate to provide protection against COVID-19? Hum Vaccin Immunother. 2020;7:1–4.