Immunization in Clinical Practice Nitin K Shah, Naveen Thacker
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BASICS IN IMMUNIZATION

General Immunology and Principles of Vaccination1

Nitin K Shah,
Naveen Thacker
 
INTRODUCTION
It is important to realize that the field of vaccinology evolved even when there was scanty knowledge available about the principles of immunology. This was driven more by the urgent need to prevent dreaded diseases like smallpox and rabies. With time, we now know more about the principles of immunology as applicable to vaccination. Now we need to exploit this field further to understand the needs of boosters for some of the vaccines and to develop more effective vaccines against diseases with intracellular pathogens like HIV, malaria, kala azar, etc.
 
IMMUNE SYSTEM
Immune system is the most important organ of the body for its survival. It consists of major lymphoid organs like spleen and lymph nodes as well as smaller lymphoid tissues lining the mucosal entry points of the body like the airway (adenoids and tonsils), gut [gut- associated lymphoid tissue (GALT)]. The immune cells are found in the reticuloendothelial system of many organs and in circulation. There is constant traffic of these cells from one point to other in the body, which helps to disseminate immune message throughout the body. It is important to know the basics of immune response to understand how the vaccines work.
The main aim of the immune system is to recognize ‘self’ from ‘foreign’ and to eliminate the supposedly harmful ‘foreign’ from the body. This is done through many afferent and efferent pathways, which work in unison and help the body fight infective organisms as well as cancerous cells. This is done through recognition of antigens on the foreign substances or organisms. The response mounted by the body is called immune response and it consists of producing either proteins called antibodies as in humoral response or specific cells called cellular response. Both these with the help from other cells like neutrophils, monocytes, macrophages as well as chemicals like complements and other cytokines elaborated by immune cells lead to ultimate clearance of the invading organism. The immune response is both specific and highly effective, e.g. anti-measles antibodies do not react with varicella virus and vice versa.1
Antigen has specific site called epitope to which the antibody binds. There are multiple epitopes on the same antigen and there are multiple antigens on the same organism. Accordingly, the immune system mounts multiple antibodies to the same organism. Only some of these antibodies are actually protective in nature and rest are not useful in this sense. In general, viruses contain lesser number of antigens than bacteria, fungi or parasites.2
 
Types of Immunity
The basic defense mechanisms are of two types, nonspecific and specific. Both are equally important for the survival of the human being. Both are interdependent in the ultimate goal of getting rid of what is ‘foreign’.
 
Nonspecific Immunity
It is also known as innate or nonadaptive immunity. It is present in every normal individual since birth and does not needs prior exposure to the organism nor is it specific against an individual organism. This is the oldest type of immunity in evolution which helps the body control invading organism before specific immune response is mounted and also helps the specific immune response to augment its efficacy by acting as the effector final pathway. It comes in picture within minutes to hours of exposure to any type of infection or organism. This is important as the specific immune response sometimes may take days to even weeks to come in picture. It includes natural mechanical barriers like skin, integument and mucosal linings; chemical barriers like gastric acidity, gut enzymes, etc.; classical and alternate pathways of complement systems; cytokines, chemokines and interferon α, β and γ; and the cells like macrophages, neutrophils, dendritic cells, natural killer cells.1 The complement system is again divided into two pathways, the classical and the alternate pathway both acting through a cascade of more than 19 proteins. The complements help in initiating the inflammation and in sustaining the specific immune response and ultimate killing of the organism. Both the systems of immune responses are interdependent, e.g. classical complement pathway depends on recognition of antigen-antibody complex and similarly γ interferon is produced by T cell (specific immunity arm) and natural killer cells (innate immunity arm).
 
Specific Immunity
This is the most important arm of immune system as proved by the fact that defects in this pathway are often life threatening in nature. Except for the transplacental transfer of immunoglobulin which offers protection to the newborn for a temporary period of time, it is not fully active at birth and develops gradually after birth on repeated exposure to the microbes in the surrounding. It can be divided into natural vs. acquired, passive vs. active, and humoral vs. cellular. The most important cells of this arm include the B lymphocytes, T lymphocytes, and their various subsets. On activation by an antigen, the B cells proliferate and get converted to plasma cells, which in turn produce the antibodies. Approximately 10 percent of the lymphocytes in the blood consist of B cells and they reside mostly in the peripheral lymphoid organs. For effective production of antibodies, B cells need help from T helper cells. They are produced in liver in fetal life and mature in bone marrow in humans. They are called B cells as in other species they mature in an organ called bursa of Fabricius.
On the other hand, T cells lead to cellular response and mature in thymus. The cellular response involves the T cells, macrophages and lymphokines, which are secreted by the lymphocytes and act as signal for communication between many of these immune cells. Besides its role as helper cells to induce better antibody production by B cells, the T cells are the most important cytotoxic cells, which are of great help in preventing and clearing intracellular pathogens.
 
Humoral Immune Response
This arm is mediated by the B cells which produce antibodies against the specific antigens on the microbes. The antibodies consist of heavy chains and light chains. There 3are two types of light chains, lambda and kappa chains, whereas there are five different types of heavy chains which identify the five types of immunoglobulins IgG, IgM, IgA, IgD and IgE. Of this, IgG, IgM and IgA are protective against pathogens. The IgE may play a role against parasites and is also involved in allergies.
B cells have immunoglobulin surface receptor, which binds with the appropriate antigen present on the infective pathogen. The antigen and the receptor complex are internalized and the antigen is processed within the cell. The processed antigen stimulates the B cell to mature into antibody secreting plasma cell. T helper2 (Th2) cell leads to switch in the production from IgM to IgG, IgA or IgD. The immunoglobulins thus produced are similar to the surface receptors and react with the antigen to produce antigen-antibody complex. This stimulates the macrophages, neutrophils and other effector cells to clear the infected cell and thus clear the infection. In absence of help from Th2 cell the antibody production is weak and predominantly IgM type as seen with many carbohydrate antigen.
During acute infection, IgM antibodies appear within a few days, peak at around 7 to 10 days and disappear in next few months to undetected levels. Hence, presence of IgM indicates recent infection. Similarly, IgM being a large molecule is not transferred transplacentally in a newborn. Hence presence of IgM antibodies indicates congenital infection in the newborn. The IgM response is usually seen in primary response, is short lived and the titers of the antibodies are lower. The IgG response usually picks up along with IgM or after a few days, peaks it around 2 to 3 weeks and lasts for a very long time. It is usually seen best during the secondary response classically seen on re-exposure to the concerned antigen and the titers are very high. The IgA response depends upon the route and the type of infection. Serum IgA is seen in organisms that invade the mucosa, whereas surface IgA is classically seen with localized mucosal infections like in cholera, RSV infection, etc.
 
Antigen Presenting Cells
While the B cells can directly respond to the antigen and process the antigen within, the TCR on the T cells do not react with the antigen directly unless processed and presented to it by special cells called antigen presenting cells (APC). Besides the B cell and the macrophages, dendritic cells are the major APC in the body. These cells are present at various places in the body including the Langerhans’ cells in the skin. The major role of these cells is to identify dangers, which is done by the special receptors on the APC named toll-like receptors (TLR), which recognize bacterial toxins or lipopolysaccharide.2
The antigen binds with the surface receptor on the APC and this is transported inside the cell and processed by endosomal or lysosomal activity. After the antigen, usually a peptide, is processed, it is presented on the surface of the APC along with the MHC class I or II. Usually, a protein antigen leads to its expression with class II MHC and intact organism leads to expression with class I MHC. Use of adjuvants can lead to expression with class I MHC molecules. The appropriate T cell then recognizes this processed antigen expressed on the surface of the APC in association with the MHC. Besides this, the APC also expresses costimulatory molecule on the surface for which the appropriate receptor is present on the T cell surface. This leads to stimulation of naïve T cell population, but it is not required for stimulation of memory cells.34
 
Cell-Mediated Immunity
This type of immunity is transferable by the lymphocytes and not antibodies and is mediated via T cells. They are called T cells as they mature in thymus. Though called cell-mediated immunity, it often involves the role of soluble chemicals called cytokines, which are secreted by and react upon T cells themselves besides the B cells and macrophages.
T cell lymphocyte is a very important cell in the immune response. It has T cell receptor (TCR) with α, β chains which binds with the antigen processed and expressed on the antigen presenting cell along with the MHC class I or II antigens. It also has receptors for costimulatory factor and for the various cytokines and chemokines released in the surrounding. It has many subsets, which carry out different functions. These cells are in circulation and in the lymphatic vessels. There are two essential types of T cells depending on the CD molecules expressed on the surface of the T cell, CD4+ T cells and CD8+ T cells. CD4+ T cells react with MHC II on the APC, while CD8+ T cells react with the MHC I. CD4+ T cells are called T helper cells and CD8+ T cells are also called cytotoxic T cells.
CD4+ T cells are of two subtypes Th1 and Th2 cells. Th2 cell response is the major factor for the stimulation of B cells, and for the switch in the production from IgM to other immunoglobulins, which occurs in the presence of IL4. Th1 cells are responsible for the delayed hypersensitivity reaction and occur in the presence of IL-12 and IL-18. Besides this, the types of cytokines produced is also different by Th1 and Th2 cells. CD8+ T cells recognize and target the infected cells in the body and hence are called cytotoxic T lymphocytes. This was first demonstrated with virus infected cells and later on with cells infected with bacteria as well as parasites. As the panel of cytokines released by CD8+ cells is similar to that of Th1 cells, both are classified as type 1 cells and Th2 as type 2 cells. In presence of IL-4 produced by Th2 cell, it can convert to a form resembling Th2 cell and this is called type 2 cell.4 T cell response is very important for T cell-dependent humoral response as discussed before and for immunity against certain organisms which are essentially intercellular pathogens like M. tuberculosis, M. leprae, fungal infections, etc. They are also important in surveillance against malignant cells. No wonder the patients with T cell deficiency suffer from opportunistic infections, which are intracellular like tuberculosis and fungal infection as well as peculiar cancers like Kaposi's sarcoma, e.g. in an HIV-infected person. T cells communicate with one another and with other types of cells through production and release of substances called lymphokines.
 
Role of Various Types of Immune Responses in Infections
Extracellular infections can be prevented, reduced and cleared by Th2 type response with strong production of antibodies. Th1 type response also may play a role to some extent, but CD8+ cells hardly play a role in this type of infection. In case of intracellular infections, antibodies may be able to prevent and reduce the infection, but they will not be able to clear the infections. Th1 and CD8+ cell responses will be needed to clear the infection, especially the intracellular viral infections.5
 
Primary versus Secondary Immune Response
When the antigen is introduced for the first time, the immune system responds primarily after a lag phase of up to 10 days. On re-introduction of the same antigen, there is no lag phase and the immune system responds 5by producing antibodies immediately and this is called secondary response. There are some basic differences in both these response. Primary response has lag phase, is of predominantly IgM type, is short lived and the titers are low. As compared, the secondary response is almost immediate, is of IgG type, is long lasting and the titers are very high. These differences are more with the antigens stimulating both B cells and T cells. Sometimes there is a negative phase where there is transient drop in the antibody levels immediately after the infection.1 The significance of this negative phase is not well known. Repeated exposure of the same antigen leads to more maturation of the immune response with better affinity and avidity of the antibodies and a longer time till which anamnestic response occurs. Affinity is the force with which the antigen-binding site on the antibody bonds with the epitopes and the combined such forces lead to avidity. High affinity and avidity antibodies are very useful in controlling infections.
 
T Cell-Dependent Immune Response
Certain antigens, mainly proteins, induce both B cell and T cell stimulation leading to what is called T cell-dependent immune response; whereas large molecular antigens like polysaccharides induce only B cell response as they are incapable of inducing T cell response on their own. The T cell-dependent response is usually prompt with higher titers, IgG type, and longer lasting. It also shows booster effects with repeated exposure. Infants of 6 weeks of age onwards are capable of T cell-dependent responses. Lastly, IgA antibodies are also produced in such response which probably helps in providing mucosal protection and eradicating the carrier state. As compared T cell-independent response being only B cell mediated is not possible below 1½ years of age. It is predominantly IgM type with low titers. The response is short lived, does not lead to boosting and such vaccines are actually revaccination rather than boosters when given repeatedly which produces the same type of response every time the antigen is introduced. Lastly, IgA is not produced and hence there is no local mucosal protection with this type of antigens. A T cell-independent antigen like polysaccharide can be made into T cell-dependent by the technique of conjugation, where a carrier protein is conjugated with the polysaccharide. When this conjugated moiety is presented to the T cell, it recognizes the protein carrier as an antigen and leads to internalization of the whole complex, which leads to the T cell now responding even to the carbohydrate antigen of the complex producing T cell response to the polysaccharide. This technique is very useful in producing vaccines like conjugated Hib, pneumococcal, Vi typhoid and meningococcal vaccines.
 
Passive Immunity
Passive immunity is specific immunity, which is transferred passively to the recipient. It gives readymade immunoglobulins, which help to fight infection immediately. However, it is for a temporary period and it wanes after a few weeks to a few months depending upon the half-life of the transferred immunoglobulins. Besides the natural transplacental passive transfer of the immunoglobulins in the newborn, the other examples of the passive immunity are infusing immunoglobulins in the person to protect him for a specific disease.
 
Transplacental Passive Immunity
The most common form of passive immunity is that given to the newborn from the mother. Immunoglobulins are transferred predominantly in the last trimester and are mainly of 6IgG type. This means that at birth the child will have similar type of antibody pattern as the mother. This protects the child for first few months till the time that he develops his own immunity after repeated exposure to various antigens after birth. Protection offered will depend on the half-life of the specific antibody, e.g. the antibody against poliomyelitis does not protect for more than 4 to 6 weeks (the time of starting the polio vaccination in the baby), whereas the anti-measles antibody protects the child till 6 to 9 months (the reason for delaying the measles vaccine till 9 months). Not only does the passive immunity protect the newborn and child against the specific diseases, it also interferes with the immune response to the concerned vaccine if given in the presence of maternal antibody like for measles as discussed before.
 
Acquired Passive Immunity
Immunoglobulins can be passively transferred by giving immunoglobulin preparation intramuscularly or intravenously. It can also be done inadvertently by infusing blood and blood products which also will infuse immunoglobulins, which may interfere with some, live vaccines like measles vaccine. There are three types of preparations, which will lead to passive transfer of the immunoglobulins. They are:
  1. Pooled human immunoglobulin preparation.
  2. Homologous hyperimmune globulin and
  3. Heterologous hyperimmune immunoglobulin preparation.6
Human immunoglobulins: This is prepared by pooled plasma from more than 100 healthy donors and fractionation of this plasma to produce the final product, which is available as IM preparation as well as IV preparation. As it contains a variety of antibodies, it is ideally suitable for replacement therapy in congenital and acquired immune deficiency with antibody deficiency. It is also used in many autoimmune disorders and for passive prophylaxis for measles or hepatitis A infection.
Homologous human hyperimmune globulins: This is obtained by pooling plasma from specific donors who have high titers of a specific antibody either due to repeated past natural exposure or due to vaccination. This preparation serves to protect against a specific disease. Of course, it will also have other types of antibodies too, albeit to a lesser extent. They are used for prophylaxis of diseases like hepatitis B, tetanus, varicella or rabies.
Heterologous hyperimmune globulins: These were used in past to prevent diseases like rabies or tetanus. It is obtained from animals mainly horse or rabbit who are hyperimmunized by repeated vaccination against the concerned disease and then collecting plasma which is fractionated to obtain pure product. Being an animal product, it can lead to severe allergic reactions including anaphylaxis, anaphylactoid reactions or serum sickness.
 
Active Immunity
Active immunity is developed by stimulating the immune system by antigens, which can lead to specific humoral or cellular immune response or both. It can happen in two ways, either by exposure to the wild pathogen naturally where the immunity develops after the person suffers from the disease which has chances of morbidity and even mortality; or by exposure to the antigens given as vaccines where the person has least morbidity and the person becomes immune without much suffering. Not all the natural diseases lead to protective immunity like in natural tetanus or typhoid where repeated clinical courses are known unless vaccination is done. Most of the 7times natural disease especially viral infection leads to strong protective immunity, which probably lasts life long, e.g. in measles or varicella. Vaccination, on the other hand, is introduction of antigens with the purpose of inducing immune response without leading to clinical disease. Vaccines are of different types as discussed below.
 
Vaccines
Vaccines can be live or inactivated and both can be bacterial or viral. Live vaccines are attenuated live organisms, which have immunogenicity without pathogenicity. Inactivated vaccines can be killed whole organism or a fraction of it. Fractional vaccine also includes toxoids (diphtheria or tetanus toxoids) and subunit vaccine like hepatitis B vaccine. They also include proteins or polysaccharides, which again can be unconjugated (Vi typhoid vaccine) or conjugated (Hib vaccine).
 
Live Vaccines
These are pathogens, which are modified in such a way that they lose the pathogenicity without altering their immunogenicity. Most live vaccines are viral vaccines like measles, MMR, varicella, OPV, etc. Some bacterial vaccines too are live vaccines like BCG and oral Ty21a typhoid vaccine. The pathogen is attenuated by serial passage of the wild type in tissue cultures or animals. The live vaccine multiplies inside the body after administration and stimulates the immune system. Injectable live vaccines thus need only one dose for development of long-term immunity, e.g. measles or MMR vaccine. The immunity is maintained subsequently, probably, by subclinical infections. However, when such vaccine is used universally, it will reduce or abolish the natural transmission leading to less chance of repeated subclinical exposures. This may lead to waning immunity after many years and may need artificial boosting, e.g. MMR where we now know that 2 doses are required to maintain long-term protection. Live vaccines given orally like OPV or Ty21a typhoid vaccines need multiple doses to induce lasting immunity. Another problem with live vaccines is side effects like vaccine- induced disease, e.g. OPV-induced paralysis (VAP), reversal of the attenuated strain back to virulent strain again like in OPV where vaccine-derived strains have been known to develop. Lastly, live vaccines are contraindicated in immune compromised individuals as it can replicate in such cases leading to disease. Attenuated and genetically modified viruses are used as vectors to introduce other antigens, e.g. canary poxvirus.
 
Inactivated Vaccines
Inactivated vaccines are either killed whole organisms like whole cell typhoid vaccine or pertussis vaccine, or a fraction of it like in acellular pertussis vaccine, toxoids like tetanus toxoid, subunit vaccine, e.g. surface antigen of hepatitis B or polysaccharide like pneumococcal vaccine. As the vaccine does not replicate in the body, it does not lead to clinical disease and is safe even in immune compromised host.6 The immune response is not disturbed by the presence of previous antibodies, hence these vaccines can be started early in life, e.g. DTP vaccination at 6 weeks of age. The first dose usually does not lead to protection and only primes the immune system. Subsequent doses lead to primary immune response, which protects the individual for a short time. Subsequent repetition of doses leads to boosting effect and long-term immunity. Hence, these vaccines need multiple primary and booster doses.8
 
Immune Memory
Immune memory is very important for long- term protection against the pathogens. Once the immune system is stimulated by an antigen, some of the stimulated B cells and T cells remain dormant in the body for years. When challenged by the same organism again, they divide immediately and lead to humoral and cellular protection against the disease. This happens with antigens which stimulate both B cells and T cells like proteins and conjugated polysaccharide antigens. This also explains protection even when the antibody levels may be undetectable in the blood, e.g. in hepatitis B many years after immunization and this is the reason we do not need boosters for hepatitis B.
 
Immunomodulation
One can modify or augment the immune response by certain manipulations. Adjuvants have been used to improve the immune response to certain vaccines. The best and the only adjuvants used since 1940s are the alum or aluminum salts. Adjuvants act via various mechanisms like acting as a depot at the site of injection to act as a stimulator of T cells and APCs. Besides the various other adjuvants tried, the most promising is the unmethylated CpG motifs present in the bacterial DNA which is promptly recognized as danger by the APCs leading to stimulation of the T cell function.7
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