Introduction
Normal immune response
The immune system provides protection against infectious agents. Classically, the system is divided into the innate immune system and the specific or acquired immune system. The innate immune system consists of cells (monocytes, macrophages, dendritic cells, neutrophils, eosinophils and natural killer cells) and molecules (complement, cytokines, chemokines etc) while the specific immune system is composed of lymphocytes. These include B lymphocytes producing antibody, and subsets of T lymphocytes including CD4+ T lymphocytes and CD8+ cytotoxic T lymphocytes. The CD4+T lymphocytes are further divided into TH1 cells producing inflammatory cytokines such as interferon Ɣ (IFN Ɣ ) and TH2 cells, as well as regulatory T cells and TH17 cells1,2.
The innate immune system recognizes the pathogen and subsequently activates the specific immune system3. Then these two systems act in concert against the infection. Pathogens that enter the body through skin/ mucous membranes are taken up by resident antigen presenting cells in these tissues. The main antigen presenting cell (APC) is the dendritic cell, the macrophage being another APC. The antigen presenting cells and molecules of the innate immune system have receptors (pattern recognition receptors) that can recognize conserved foreign molecules found only on pathogens (pathogen associated molecular patterns). Recognition is followed by activation of these cells and molecules. Dendritic cells along with the macrophage, found in the skin and othersites are crucial in the subsequent activation of the specific immune system1. The dendritic cell senses potential ‘danger’ when recognizing pathogen associated molecular patterns. Recognition is followed by uptake of the pathogen and activation of the dendritic cell and other antigen presenting cells.
This leads to,
• production of cytokines and chemokines resulting in inflammation
• up-regulation of co-stimulators on the antigen presenting cells essential for successful antigen presentation to T cells
• localization of the pathogen containing antigen presenting cells to the draining lymph node.
Blood borne pathogens are directly taken up by dendritic cells in the white pulp of the spleen.
During this process, the dendritic cells internalize the pathogens and present peptides derived from the microorganisms, in conjunction with major histocompatibility complex (MHC) class II molecules on its surface. Viruses infecting dendritic cells produce virus coded peptides in the cytoplasm. These peptides are presented in conjunction with MHC Class I molecules.
T and B cells have receptors that recognize antigen. Most circulating lymphocytes recognize non self-antigen2. Lymphocytes circulate in the body between blood and peripheral lymphoid tissue (cell trafficking). Activated dendritic cells present peptides derived from pathogens, in conjunction with MHC Class II molecules to CD4+ T cells in the T cell areas of the lymph nodes and spleen. The CD4+ T cell will be activated only if second signals are provided by co-stimulatory molecules on the surface of dendritic cells. These co-stimulators are up regulated only if pathogen associated molecular patterns are recognized by the dendritic cells. As these patterns are only found on pathogens, the dendritic cell will activate non-self reacting CD4 +T cells. Depending on the pathogen and the cytokine milieu around the reaction, the CD4+ T cells become either armed effector TH1 or TH2 cells or memory cells (2).
Dendritic cells which are activated by microorganisms such as M. tuberculosis produce cytokines that switch a naïve CD4+ T cell to an activated TH1 cell, while helminths and some bacterial pathogens induce a TH2 response. TH1 cells produce cytokines (IL2, IFN Ɣ) that activate CD8+cytotoxic T lymphocytes, macrophages and B lymphocytes, while TH2 cells activate B cells by producing IL4, 6 and 13.
B cells that recognize protein antigens need help from CD4+ T cells (TH1 and TH2) to produce antibody. The initial B cell response takes place extra follicularly (outside the germinal centre)2 and produces low affinity IgM and a small amount of IgG. This occurs within a few days of the infection/immunization and is short lived.This is followed by a response in the germinal centre. B cells move into the germinal centre and encounter their cognate antigen found on the surface of follicular dendritic cells. The B cell proliferates, producing a clone of daughter cells whose antigen binding receptors (immunoglobulin molecules found on the surface of the B cell) have undergone point mutations (somatic hypermutation). These mutations are confined to the antigen binding site of the receptor. B cells with receptors with a greater fit (affinity) would bind to the cognate antigen and survive, while those with a weaker fit would undergo apoptosis. The surviving B cells would differentiate into plasma cells or memory B cells. With time, high affinity (affinity maturation) IgG, IgA and IgE antibodies are produced (isotype switching) by plasma cells, some being long lived. Memory B cells are capable of producing high affinity, class switched antibody with great rapidity, after re-exposure to the same microorganism. Affinity maturation, isotype switching and memory need T cell help and are hallmarks of antibody responses to protein antigens. T cell help is provided in germinal centers by follicular helper T cells (TfH cells). This response takes 10-14 days to appear and terminates in 3-6 weeks. Peak antibody concentrations occur 4-6 weeks after primary immunization.
Polysaccharide epitopes such as the capsules of S pneumoniae and H influenzae, do not activate CD4+ T cells (T independent responses) (2). A subset of B cells in the marginal zone of the spleen, assisted by marginal zone macrophages, produce low affinity mainly IgM antibodies and medium affinity IgG (T independent antibodies). Polysaccharides arepoorly immunogenic in children under 2 years, till maturation of the marginal zone. As T independent responses do not produce memory cells, subsequent re-exposure evokes a repeat primary response. In some instances, revaccination with certain bacterial polysaccharides may even induce lower antibody responses than the first immunization, aphenomenon referred to as hyporesponsiveness (4).
Antibodies provide protection against extra cellular organisms, such as capsulate bacteria or viruses during an extra cellular phase. IgA provides mucosal immunity, preventing infection by bacteria and viruses through the mucosa; IgM provides quick responses to blood borne pathogens while IgG protects blood and tissues.
Protection against intracellular microorganisms is through cell mediated immunity. Viruses infect cells and produce virus derived proteins in the cytoplasm. Peptides derived from these proteins are presented on MHC Class I molecules by all nucleated cells. These are recognized by previously activated cytotoxic T lymphocytes and the infected cell is destroyed. Microorganisms residing in intracellular vesicles of macrophages such as M tuberculosis, are dealt with by TH1 cells activating the macrophage, resulting in intracellular killing of the bacteria.
Vaccines
Different types of vaccines have been produced (5).
• Live attenuated
• Killed/inactivated
• Subunit
• Recombinant
• Conjugate
Immune response to vaccines
Vaccine induced immunity is mainly due to IgG antibodies. Antibodies are capable of binding toxins and extracellular pathogens.The quality ofthe antibody (avidity), the persistence of the response and generation of memory cells capable of a rapid response to reinfection are key determinants of vaccine effectiveness. For protection against bacterial diseases that result from the production of toxins (tetanus and diphtheria) the presence of long lasting antitoxin antibody and memory B cells are necessary, ensuring the presence of antitoxin antibody at the time of exposure to the toxin. With viruses such as hepatitis B, undetectable antibody titers are seen in many vaccine recipients but due to the long incubation period of the virus, memory B cells can be reactivated in time to combat the infection.
For infections which originate at mucosal sites, transudation of serum IgG will limit colonization and invasion. This is due to pathogens being prevented from binding to cells and receptors in the mucosa. Transudation of IgG is not seen with polysaccharide vaccines. If the pathogens breach the mucosa IgG in serum will neutralize the pathogen, activate complement and facilitate phagocytosis, thereby preventing spread. Some vaccines (eg. oral polio, rotavirus and nasal influenza) will stimulate production of IgA antibody at mucosal surfaces and thereby limit virus shedding. Live, inactivated and subunit vaccines evoke a T dependent response, producing high quality antibody and memory B cells. Polysaccharide vaccines (eg. pneumococcal 23 valent vaccine) evoke a T independent response4 where the IgG produced is of poor quality (affinity) and memory B cells are not produced. However, conjugation of the polysaccharide with a protein (conjugate vaccines) evokes a T dependent response. Inactivated, subunit and conjugate vaccines will only evoke antibodies. Live viral vaccines will in addition activate cytotoxic T lymphocytes. These cytotoxic T lymphocytes limit the spread of infections by killing infected cells and secreting antiviral cytokines.
Antibody responses are ineffective against intracellular organisms such as M. tuberculosis. There is evidence that a CD4+TH1 response, with production of IFN Ɣ leading to activation of infected macrophages is elicited following BCG vaccination(6).
The quality of the immune response depends on the type of vaccine. Live viral vaccines evoke a strong immune response.
This is due to(7)
• having sufficient pathogen associated molecular patterns to efficiently activate immature dendritic cells, a key requirement for the development of specific immunity.
• the vaccine virus multiplying at the site of inoculation and disseminating widely, and being taken up by dendritic cells at many sites. These dendritic cells are then activated and are carried to many peripheral lymphoid organs, where activation of antigen specific B and T lymphocytes occur. As the immune response occurs at multiple sites, live viral vaccines evoke a strong immuneresponse persisting for decades. Due to the early and efficient dissemination of the virus, the site or route of inoculation does not matter (eg. SC versus IM). BCG vaccine acts similarly, by multiplying at the site of inoculation and at distant sites as well. Non-live vaccines may have enough pathogen associated molecular patterns to activate dendritic cells but in the absence of microbial replication this activation is limited in time and is restricted to the site of inoculation. As the immune response is restricted to the local lymph nodes, it is weaker than with a live vaccine. Therefore, repeated booster doses are necessary. As only the regional nodes are involved, multiple non live vaccines can be given, provided the inoculations are performed at different sites. Booster doses are ineffective with polysaccharide vaccines as memory B cells are not produced.
In addition, the route of inoculation is important. The dermis has many dendritic cells, and for example, the rabies vaccine given intradermally at 1/10th the IM dose can evoke an equally good response. Where the vaccine is not very immunogenic (eg. hepatitis B vaccine), IM injections are preferred over SC7,8 as muscle tissue has many dendritic cells, unlike adipose tissue.
Determinants of primary vaccine response
• Intrinsic immunogenicity of the vaccine.
• Type of the vaccine – Live viral vaccines elicit better responses than non-live vaccines. Non-live vaccines rarely induce high and sustained antibody responses after a single dose. Therefore, primary immunization schedules usually include at least two doses, repeated at a minimum interval of 4 weeks to generate successive waves of B cell responses. Even so, the response usually wanes with time.
• Dose – As a rule, higher doses of non-live antigens, up to a certain threshold, elicit higher primary antibody responses. This may be particularly useful when immunocompetence is limited eg. for hepatitis B immunization of patients with end stage renal failure.
• Nature of the protein carrier.
• Genetic composition of the individual.
• Age – responses at the extremes of age are weaker and less persistent.
Determinants of duration of vaccine response(7)
Plasma cells which produce antibodies are usually short lasting, while a few plasma cells produced in the germinal centre may survive for long periods in the bone marrow. These cells are responsible for the maintenance of protective antibodies for long periods. This occurs most efficiently with live vaccines, less efficiently with non-live vaccines, but not with polysaccharide vaccines. Live viral vaccines are the most efficient at evoking long lasting immune responses that may persist lifelong due to the presence of viral antigens that may regularly activate the immune system.
Interval between doses may be important. Two doses given one week apart may evoke a rapid short lived response, whereas 2 doses 4 weeks apart may be longer lasting. Vaccination at extremes of age or in patients with chronic disease may evoke short lived responses.
Adjuvants (9)
For non-live vaccines, adjuvants are incorporated to provide the ‘danger’ signal to the antigen presenting cells. Adjuvants are also needed to prolong the antigen delivery at the site of inoculation, thereby recruiting more dendritic cells. They should also be non-toxic.
The known adjuvants used in human vaccines are,
• Alum – an aluminum salt-based adjuvant.
• AS04 – a combination adjuvant composed of monophosphoryl lipid A adsorbed to alum.
• Oil-in-water emulsions – such as MF59 and AS03
Summary
All vaccines produce antibodies which can neutralize extracellular pathogens. Conjugate vaccines, toxoids, inactivated vaccines and live attenuated vaccines produce high affinity antibody and memory cells unlike polysaccharide vaccines. Polysaccharide vaccines are made more immunogenic by conjugation with a protein carrier.
Live viral vaccines evoke cytotoxic T lymphocyte responses which act against intracellular pathogens. Similarly, the BCG vaccine activates TH1 cells, whose cytokines help macrophages control M. tuberculosis. Live viral vaccines produce long lasting, even lifelong immunity compared to non-live vaccines.
References
1. Turvey SE, Broide DH. Innate immunity. J Allergy Clin Immunol 2010; 125: S24-32.
2. Bonilla FA, Oettgen HC. Adaptive immunity. J Allergy Clin Immunol 2010; 125: S33-40.
3. Iwasaki A, Medzhitov R. Regulation of adaptive immunity by the innate immune system. Science 2010; 327: 291-95.
4. Pace D. Glycoconjugate vaccines. Biol. Ther 2013: 13(1): 11-33.
5. Pulendran B, Ahmed R. Immunological mechanisms of vaccination. Nat Immunol 2011; 12(6): 509-17.
6. Ritz N, Hanekom WA, Robins-Browne R, Britton WJ, Curtis N. Influence of BCG vaccine strain on the immune response and protection against tuberculosis. FEMS Microbiol Rev 2008; 32: 821-841.
7. Siegrist CA. Vaccine Immunology. In: Plotkin SA, Orenstein W, Offit PA Eds. Vaccine Expert Consult 6th Ed Sauders 2012, p 15-32. 8. de Lalla F, Rinaldi E, Santoro D, et al. Immune response to hepatitis B vaccine given at different injection sites and by different routes: a controlled randomized study. Eur J Epidemiol 1988; 4: 256-8.
9. Alving CR, Peachman KK, Rao M, Reed SG. Adjuvants for human vaccines. Curr Opin Immunol 2012; 24(3): 310-15. Dr Rajiva de Silva Dip. Med. Micro, MD(Micro.) Consultant Immunologist, Medical Research Institute, Colombo
