Immunization in Clinical Practice Nitin K Shah, Naveen C Thacker, Abhay K Shah, Alok Gupta
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1Basics in Immunization
  • Chapter 1 General Immunology and Principles of Vaccination
  • Chapter 2 General Guidelines for Vaccination
  • Chapter 3 Immunization Schedules
  • Chapter 4 Storage of Vaccines and Maintenance of Cold Chain
  • Chapter 5 Adverse Events Following Immunization
  • Chapter 6 Safe Injection Practices2

General Immunology and Principles of VaccinationCHAPTER 1

Suhas VPrabhu,
Rajiv KumarBansal
The Chinese and the Turks in the fifteenth century BC were the first to attempt to induce immunity to smallpox using dried crusts from smallpox lesions either by inhaling the crushed lesions or by inserting them into small cuts. These initial crude attempts at immunization led to further experimentation with immunization by Lady Mary Wortley Montagu in 1718 and Edward Jenner in 1798. Edward Jenner's experiments with cowpox to stimulate smallpox immunity are better known than the earlier attempts at immunization.
It is important to remember that the field of vaccinology evolved even when there was scanty knowledge available about the principles of immunology. Vaccines were devised and tested empirically after careful observation driven by the urgent need to prevent dreaded diseases like small pox and rabies. Over a period of time, with new research, we now know more about the principles of immunology as applicable to vaccination. It is important to know the basics of immune response to understand how the vaccines work. We also need to exploit this knowledge further to understand the needs of boosters for some of the vaccines and to develop more effective vaccines against diseases with intracellular pathogens such as human immunodeficiency virus (HIV), malaria, and kala azar etc.
The immune system is the most important organ of the body for its survival. It consists of major lymphoid organs such as spleen and lymph nodes as well as smaller lymphoid tissues lining the mucosal entry points of the body such as the airway (adenoids and tonsils) and the gut [(gut- associated lymphoid tissue (GALT). 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 the other in the body, which helps to disseminate immune messages throughout the body.
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 such as 4neutrophils, monocytes, macrophages as well as chemicals such as 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
Antigens have specific sites to which the antibody binds called epitope. There are multiple epitopes on the same antigen and there are multiple antigens on the same organism. Accordingly, the immune system produces multiple antibodies to the same organism. Only some of these antibodies are actually protective in nature and rest is not useful in this sense. In general, viruses contain lesser number of antigens than bacteria, fungi, or parasites.
Basic defense mechanisms are of two types: non-specific (innate) and specific (adaptive). 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’. Both the innate and adaptive systems are able to remember the past invaders.
Non-specific Immunity
It is also known as innate or non-adaptive immunity. It is present in every normal individual since birth and does not need 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 final effector pathway. The innate immune system's memory is robust and depends on pattern-recognition receptors that have evolved over millions of years to identify common invaders. All humans have the same innate memory.
The innate immunity 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 into action. It includes natural mechanical barriers such as skin, integument and mucosal linings; chemical barriers such as gastric acidity and gut enzymes; classical and alternate pathways of compliment systems; cytokines, chemokines and interferon α, β and γ; and the cells like macrophages, neutrophils, dendritic cells, natural killer cells.6 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. Complements help in initiating the inflammation and in sustaining the specific immune response and ultimate killing of the organism. Both systems of immune responses are interdependent, e.g. classical compliment 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 versus acquired, passive versus active and humoral versus 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 antibodies. Approximately 10% 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 the 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 signals for communication between many of these immune cells. Besides its role as helper cells to induce better antibody production by the B cells, the T cells are the most important cytotoxic cells, which are of great help in preventing invasion by and clearing of intracellular pathogens.
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 are 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 type may play a role against parasites and is also involved in allergies.
The 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 an antibody-secreting plasma cell. The T helper 2 (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 the absence of help from the Th2 cell, the antibody production is weak and predominantly IgM type as seen with many carbohydrate antigens.
During acute infection, IgM antibodies appear within a few days, peak at around 7–10 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 the IgM or after a few days, peaks it around 2–3 weeks and lasts for a very long time. It is usually seen best during the secondary response classically seen on re-exposure 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 from mucosa; whereas surface IgA is classically seen with localized mucosal infections such as in cholera and RSV infections.
While the B cells can directly respond to the antigen and process the antigen within, the T-cell receptor (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 cells 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 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 major histocompatibility complex (MHC) class I or II. Usually, a protein antigen leads to its expression with class II MHC and the intact organism leads to expression with class I MHC. The 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 co-stimulatory molecule on 6the 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.3
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.
The T cell lymphocyte is a very important cell in the immune response. It has T cell receptor (TCR) with α and 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 co-stimulatory 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. The CD4+ T cells react with MHC II on the APC, while CD8+ T cells react with the MHC I. The CD4+ T cells are called T helper cells and CD8+ T cells are also called cytotoxic T cells.
The CD4+ T cells are of two subtypes: Th1 and Th2 cells. The 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. The Th1 cells are responsible for the delayed hypersensitivity reaction and occur in the presence of interleukin-12 (IL-12) and IL-18. Besides this, the types of cytokines produced by Th1 and Th2 cells are also different. The 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 the presence of IL-4 produced by Th2 cell, it can convert to a form resembling Th2 cell and this is called type 2 cells.4 The 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 such as Mycobacteium tuberculosis, M. leprae, and some fungi. They are also important in surveillance against malignant cells. No wonder patients with T cell deficiency suffer from opportunistic infections, which are intracellular such as tuberculosis and fungal infection as well as peculiar cancers such as Kaposi's sarcoma in an HIV-infected person. The T cells communicate with one another and with other types of cells through production and release of substances called lymphokines. The B and T cells of the adaptive immune system have “updatable” memories which can remember the individual invaders one has encountered during his or her lifetimes, both common and rare. Adaptive memory is “personal” meaning that every person has a different adaptive memory.
Extracellular infections can be prevented, reduced and cleared by Th2 type response with strong production of antibodies. The 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. The Th1 and CD8+ cell responses will be needed to clear the infection, especially the intracellular viral infections.5
Primary vs. Secondary Immune Response
When the antigen is introduced for the first time, the immune system responds primarily after a 7lag phase of up to 10 days. On re-introduction of the same antigen, there is no lag phase and the immune system responds by producing antibodies immediately and this is called secondary response. There are some basic differences in both these responses. Primary response has lag phase, is of predominantly IgM type, is short lived and the titers are low. As compared to this, 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. 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, such as 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 a response which probably helps in providing mucosal protection and eradicating the carrier state. In contrast, in children below 1½ years of age, T-cell-independent responses, being only B cell mediated result in a predominantly IgM type antibody 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, the IgA is not produced and, hence, there is no local mucosal protection with this type of antigens. A T-cell- independent antigen such as polysaccharides can be made into a T-cell-dependent antigen 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 such as conjugated vaccines to Haemophilus influenzae type b (Hib), Pneumococcus, Vi antigen (Vi for virulence) of typhoid and Meningococcus.
Passive immunity is specific immunity, which is transferred passively to the recipient. It gives readymade immunoglobulins, which help 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 IgG type. This means that at birth, the child will have similar type of antibody pattern as the mother. This protects the child for the first few months till the time that he develops his own immunity after repeated exposure to various 8antigens after birth. The half-life and hence the protection offered will depend on the half-life of the specific antibody, e.g. the antibody against poliomyelitis does not protect for more than 4–6 weeks (the time of starting the polio vaccination in the baby), whereas the anti-measles antibody protects the child till 6–9 months (the reason for delaying the measles vaccine till 9 months). Not only does the passive immunity protect the newborn/infant against the specific diseases, it also interferes with the immune response to the concerned vaccine if given in the presence of maternal antibody such as 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, that may interfere with some live vaccines such as 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 humen hyperimmune globulin preparation
  3. Heterologous human hyperimmune globulin preparation.7
Pooled 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 intramuscular (IM) preparation as well as intravenous (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. It is also used 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 Human Hyperimmune Globulins
These were used in the past to prevent diseases such as 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 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 less morbidity and the person becomes immune without much suffering. Not all natural diseases lead to protective immunity; in naturally occurring tetanus or typhoid, repeated clinical courses are known unless vaccination is done. However, most of the time natural disease leads to strong protective immunity, which probably lasts lifelong, e.g. in measles 9or 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 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 kill the whole organism or a fraction of it. Fractional vaccine also includes toxoids (Diphtheria or tetanus toxoids) and subunit vaccine such as 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 their pathogenicity without altering their immunogenicity. Most live vaccines are viral vaccines such as measles, MMR [(measles, mumps, and rubella (German measles)], varicella, and oral polio vaccine (OPV). Some bacterial vaccines too are live vaccines such as Baccilus Calmette-Guerin (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 dose are required to maintain long-term protection. Live vaccines given orally such as OPV or Ty21a typhoid vaccines need multiple doses to induce lasting immunity. Another problem with live vaccines is side effects such as vaccine-induced disease, e.g. OPV-induced paralysis, which occurs due to reversal of the attenuated strain back to a virulent strains. Lastly, live vaccines are contraindicated in immune compromised individuals as the organism 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 kill the whole organisms such as whole cell typhoid vaccine or pertussis vaccine, or a fraction of it such as in acellular pertussis vaccine, toxoids such as tetanus toxoid, subunit vaccine, e.g. surface antigen of hepatitis B or polysaccharide such as 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.7 The immune response is not disturbed by the presence of previous antibodies; hence, these vaccines can be started early in life, e.g. diphtheria, pertusis, and tetanus (DPT) vaccination. 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.
Immune memory is very important for long-term protection against 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 such as proteins and conjugated 10polysaccharide antigens. This also explains protection even when the antibody levels may be undetectable in the blood, e.g. in hepatitis B after many years after immunization and this is the reason we do not need boosters for hepatitis B.
One can modify or augment the immune response by certain manipulations to achieve a high level of safety and efficacy; many newer vaccines rely on potent immune-stimulators: vaccine adjuvant. An adjuvant is any substance, compound or even strategy which results in the enhancement of adaptive immune responses when delivered together with an antigen.
The mechanism by which vaccine adjuvant enhance immune responses has historically been considered to be the creation of an antigen depot. From here, the antigen is slowly released and provided to immune cells over an extended period of time. This “depot” was formed by associating the antigen with substances able to persist at the injection site, such as aluminum salts or emulsions. The identification of pathogen-associated molecular patterns (PAMPs) has greatly advanced our understanding of how adjuvant work beyond the simple concept of extended antigen release and has accelerated the development of novel adjuvant.
Vaccine adjuvant are defined by the effect they have on innate and adaptive immune responses rather than their molecular structure or origin. Adjuvants come from a wide range of sources, are highly diverse, with no common structural or chemical features; exhibit their immune-enhancing/immune-skewing and activity through a broad variety of molecular and cellular mechanisms. Not only “immune potentiators” such as pathogen associated molecular patterns (PAMPs)—or their synthetic derivatives or the increasing number of small-molecule agonists which not only mimic their activity—but also particulate antigen-delivery systems are capable of initiating and/or enhancing immune responses and are classified as adjuvants.
Virtually, all vaccines require adjuvants in some form, endogenous or exogenous as very few antigens are inherently immunogenic. Without a component that engages either innate immune cells or additional receptors on lymphocytes such as complement receptors, most non-adjuvants, highly-purified antigens induce tolerance rather than immunity. Very few antigens, such as certain toxins, are capable of inducing antibody responses when administered without adjuvant.
An important criterion for the selection of modern adjuvant is their ability to promote the induction of strong CD8+ T cell responses. This subset of T cells is able to eliminate cells infected by viruses and other intracellular pathogens, which—once inside cells—are not accessible to antibodies.
The ability of a vaccine to: (a) induce a T cell response and (b) a specific type of T cell response (CD4+/CD8+; Th1/Th2/Th17) depends on a variety of factors such as the nature of the antigen, the vaccine platform (recombinant protein versus gene-based vaccines such as DNA or recombinant virus), the immunization regimen (homologous versus heterologous prime-boost), the route of administration, the immunization interval, and the frequently overlooked importance of formulation.
By inducing a pro-inflammatory environment, adjuvants, promote the immunogenicity of antigens in vaccines, enabling the recruitment and promotion of the infiltration of phagocytic cells, particularly antigen-presenting cells (APC), to the injection site. Adjuvants induce cytokine expression, activate APC, can enhance antigen presentation and modulate more downstream adaptive immune reactions (vaccine delivery systems, facilitating immune Signal 1). Adjuvants can act as immunopotentiators (facilitating Signals 2 and 3) exhibiting immune stimulatory effects during antigen presentation by inducing the expression of costimulatory molecules on APC. Together, these signals determine the strength of activation of specific T cells; thereby, also influencing the quality of the downstream T 11helper cytokine profiles and the differentiation of antigen-specific T helper populations.
New adjuvants should also target specific (innate) immune cells in order to facilitate proper activation of downstream adaptive immune responses and homing (Signal 4).
Killed organisms or purified antigens-derived vaccines usually require the use of adjuvants to maximize their effectiveness. Adjuvants may, however, cause local inflammation, and multiple doses or high doses of antigen increase the risk of producing hypersensitivity reactions. In addition, the latter are often more sensitive to proteases and nucleases that destroy them before uptake by APC.
Currently, there are only a few adjuvants that are included in licensed human vaccines, including the prominent one like alum, MF59® (oil emulsion), a squalene-based adjuvant system 03 (AS03®) and AS04® (monophosphoryl lipid A, MPL + alum).
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