Invited Review: Understanding the Basic Principles and Practical Aspects of Vaccinology
The role of vaccines in preventing disease in domestic animals and humans is reasonably well understood and documented. In zoological species immunity as a result of vaccination is largely assumed, but rarely certain, due to a variety of reasons including unknown species susceptibility and inability to prove vaccine efficacy. Current vaccination protocols in zoological collections are based on limited reports in the literature, anecdotal information, trepidation, animal value and irreplaceability, and the unknown consequences of administering vaccines on a routine basis. Zoo vaccinologists assume that a particular disease in a nondomestic species is caused by the identical etiologic agent and will develop an effective immune response to a vaccine produced for a known human or domestic animal pathogen. Vaccination strategies in zoological institutions should be based on principles of immunology, epidemiology of the disease in a particular species and the safety and efficacy of the specific vaccine. Since little is known about vaccinology in the wide variety of nondomestic species found in zoos, immunologic principles of domestic animal vaccines may serve as a model for vaccination protocols in exotic animals. However, to adequately design safe and effective vaccination programs for our animals there is a need to obtain information on true disease prevalence, species susceptibility and response to vaccination.
Vaccinology has changed significantly since many of the vaccines available today for domestic species were originally developed. Our understanding of the immune system has improved and expanded so now many aspects of immunology can be explained at the cellular and molecular level. Additionally, major discoveries have been made in the areas of cell and molecular biology, bacteriology, virology and parasitology which provide greater insight into the nature of the organisms against which the vaccines are designed. Veterinary vaccinologists have begun to take advantage of many of the advances made in the basic sciences. Transferring and applying the new scientific knowledge from modem biology to vaccinology has been slow and difficult because vaccinology, like most of medicine, remains as much an art as a science. Furthermore, vaccinology tends to be driven more by tradition and current wisdom than by scientific principles. It is a discipline that since its beginnings more than 200 years ago, has prospered through empiric approaches both for human and veterinary vaccines. The early success in development of vaccines to control many diseases that once caused significant morbidity and mortality clearly demonstrated that an understanding of immunology or any other basic science was not a prerequisite for a vaccine to be successful. Those successes were for diseases that are easily and effectively controlled by the immune system. Unfortunately, the diseases that more recent vaccines have been developed to prevent, present significant challenges to vaccinologists, since the empiric methods of the past will not work as effectively. The diseases that remain are often complex and not as easily controlled by the immune system as diseases for which many of the earlier vaccines were designed to prevent. In fact, some of the new vaccines are for diseases caused by the immune system or the immune system may facilitate survival of the pathogen. To produce the vaccines for the future, modem vaccinology will surely require not only a better understanding of basic immunology, but also a better understanding and utilization of certain advances made in molecular and cell biology, genetics, biochemistry, microbiology and parasitology. Perhaps equally important for modem vaccinologists to understand and consider will be subjects like economics, epidemiology, public health, management practices, animal husbandry and animal (including human) behavior, if new and improved vaccines are to be developed for the diseases which continue to cause significant problems.1-8
Thus the challenges for vaccinologists, regardless of the species to be vaccinated, will be to develop vaccines that are truly needed; are safe when administered to avoid immediate or future health problems for the vaccinees and nonvaccinated contacts; are effective in preventing disease in the vaccinees and hopefully effective in reducing the threat of disease for nonvaccinated individuals; are cost effective and; are stable and easily administered so they readily reach the target population that will benefit by receiving them. Vaccines developed for domestic species or humans and used in zoo species will present unique challenges. It will be critical when possible, and if required, that domestic animals vaccines be modified as needed to provide protective immunity in a safe and effective way for zoo species especially those that are rare and endangered. One cannot assume that a bovine vaccine will perform similarly in a Holstein (Bos taurus) cow and a gaur (Bos frontalis) cow.1
The Immune System
An understanding of the immune system provides a basis for understanding the nature of vaccine immunity. Two major types of immunity prevent or limit infectious diseases. The two types are nonspecific or innate immunity and specific or adaptive immunity. Adaptive immunity is characterized by specificity and memory and it is this adaptive immunity which is primarily or exclusively stimulated when an animal receives a vaccine. Ironically, in nature, it is innate or nonspecific immunity that prevents a majority of the pathogens from infecting and/or causing disease in animals. Innate immunity is important because it is the first line of defense and is immediately activated in response to inherent or elaborated chemical substances of the infectious agent. Examples of the innate immune system include skin defense mechanisms such as normal bacterial flora, hair, fatty acids, desquamation; intestinal tract normal flora, anaerobic conditions, pH, fluid flow, presence of lysozyme, mucus, and enzymes; and respiratory tract cilia, mucus, coughing and alveolar macrophages.
Although current vaccines often do not have a significant beneficial effect on innate immunity, immunomodulators (nonspecific immune stimulants) and some types of vaccines and drugs are targeted toward enhanced innate immunity as a nonspecific method for disease prevention. Certain of the innate factors play a role in specific immune responses. Notable among the soluble factors playing an important role in both innate and adaptive immunity are the interferons (IFNs), certain of the other cytokines (e.g., IL-1, TNF) and complement components. Cells involved in both innate and adaptive/specific immunity are, to a limited extent, neutrophils and natural killer cells, and more critically, monocytes and macrophages.1,2,4,8,9
Ontogeny of the Specific Immune System
The development of the specific immune system has been defined for several mammalian species. In most, if not all species, the genetic information for the specific immune system is present in the fertilized egg and within days the developing embryo has hematopoietic stem cells in its yolk sac. By the end of the first month of gestation in most species, hematopoiesis occurs in the fetal liver. The pluripotent stem cells in the liver are capable of differentiating into mast cells, basophils, megakaryocytes, erythrocytes, eosinophils, neutrophils, and monocytes and, especially critical for the development of the adaptive immune system, they eventually differentiate into the various types of lymphocytes. These include the T helper cell (TH), T cytotoxic cell (Tc) and B cells, particularly the plasma cells. Early in the second month of gestation lymphoid stem cells migrate from the liver to the developing bone marrow and certain of the cells migrate to the thymus for further maturation into T cells. Shortly after the bone marrow becomes populated with the lymphoid stem cells, the bone marrow becomes the main source of future lymphoid cells that migrate to the thymus for months to years, depending on the species. The B lymphocytes mature in the bone marrow under the influence of the stromal reticular cells and the T cells mature and are educated in the thymus. “Education” refers to the immune system’s cell-based discrimination between self and non-self. In the thymus, most T cells with anti-self specificity are eliminated or suppressed so an immune response does not develop to self-antigen. An immune response directed toward self-antigen can lead to autoimmune disease. Those T cells responsive to foreign antigens that provide cell mediated immunity (Tc and TH1 cells), those T cells that regulate (r) or suppress (s) the immune response (Tr or Ts) and those that provide help for B cells (T helper cells) leave the thymus to populate the peripheral lymphoid tissues.
In addition to B cell maturation in the bone marrow, the Peyer’s Patches are the site of B cell development for mucosal humoral immunity. The B cells that migrate to mucosal sites from the Peyer’s Patches produce primarily IgA that is secreted onto mucosal surfaces. Bone marrow, thymus and special areas in the Peyer’s Patches are referred to as central, or primary lymphoid tissues. However, these are not the sites where the lymphocytes encounter antigens. Instead, the primary lymphoid tissues are connected through blood and lymphatic channels to lymph nodes, spleen and lymphoid tissue found in the skin and at mucosal surfaces primarily of the gut, lung, and genital tract. These peripheral, or secondary lymphoid organs, are the sites where lymphocytes meet antigen. The mature, immunocompetent, but antigen naive cells are continually seeded from the primary lymphoid organs to secondary organs where they encounter antigens and perform their specific effector functions. By at least 1–2 weeks prior to birth in most, but not all, species the adaptive systemic immune system is well developed. However, the mucosal system often lags behind, not maturing until after birth.
There is a unique phenomenon that occurs at birth and persists for up to 2 weeks in certain species such as the domestic canine and feline. During this period, thermoregulatory control is not fully functional and therefore body temperatures rarely go above 35°C (95°F). The specific immune system, particularly monocyte/macrophage and lymphocyte function, is significantly suppressed by low body temperatures. Thus, the newborn puppy and kitten are heavily dependent during this period on their innate immune system, which is also partially affected by low temperatures. They are especially dependent on passively acquired immunity (antibody) which is obtained by absorption of the colostrum from the dam through the intestinal tract for up to 72 hours after birth since only a small amount (5%) of the passively acquired immunoglobulin passes via the placenta in these species. By 1–2 weeks of age, thermoregulation is functional and body temperatures become optimal (>37°C) for adaptive immunity. Thus, the temperature related immunosuppression disappears.2,4,9-15
However, at approximately 4–5 weeks of age, or at the time the puppy or kitten is weaned, another period of transient immunosuppression may occur. This immunosuppression usually lasts for about 1–2 weeks and is often due to nutritional deficiencies of vitamin E and selenium that is caused by low levels of these nutrients in the dam’s milk prior to weaning. Puppies and kittens fed commercial food supplemented with appropriate vitamins 1–2 weeks prior to weaning are more likely to avoid and/or shorten the duration of this immunosuppression. Deficiency in vitamin E and selenium affects cell types important for cell mediated and humoral immunity. It has been demonstrated in animals with severe vitamin E and selenium (Se) deficiency that certain vaccines fail to stimulate protective immunity, or they stimulate an immune response which is short lived. In the vitamin E/Se nutritionally deficient dog, vaccines that have a tendency to stimulate a hypersensitivity response are more likely to induce this adverse response and fail to provide protective immunity.16,17
In the bovine and other ruminant species the immune system is very well developed prior to birth and thermoregulation is well developed after birth. However, immunosuppression is still known to occur in these species just prior to and shortly after birth due to high levels of cortisol. Endogenous cortisol, sometimes produced at immunosuppressive levels, is required to initiate parturition. Environmental stress (poor husbandry, inclement weather, etc.) may also cause elevated levels of cortisol.14 This marked increase in cortisol can be highly immunosuppressive and interfere with effective vaccinal immunity in young animals.
By 6–8 weeks of age in most species the adaptive immune system should have recovered from any transient periods of immunosuppression, and if passively acquired maternal antibody (PAMA) is not present to interfere with active vaccine immunity, the young animals should be able to develop optimal immune responses to many of the vaccine antigens. However, most animals will have PAMA to many of the vaccine components, thus vaccines will often not immunize. To ensure vaccines are given at an age when PAMA no longer interferes, it is necessary to vaccinate until 12 weeks of age or older. To help ensure an optimal immune response to those components of a multi component vaccine that are of greatest concern for the young animal, vaccines with a limited number of components are recommended. For example, dogs at 6 weeks, should receive a vaccine with canine distemper and canine parvovirus in preference to one with many additional components that are probably not needed at that age. Then at 9 weeks and/or 12 weeks, vaccines with additional components could be given, if desired. Between 3–6 months of age, the canine and feline immune system is fully functional, interference by maternal antibody has disappeared, and the immune system should remain at optimal levels until an age associated immune senescence occurs in very old animals (dogs and cats >9 years of age). Similarly, the immune system of most species is fully functional by 3–6 months and there is usually little or no passively acquired antibody after 6 months of age to interfere with vaccine induced immunity in any of the domestic species. Since the persistence of passively acquired maternal antibody in zoo species has not been well studied it cannot be assumed that vaccines will effectively induce immunity at these same ages. Furthermore, if the maternal antibody titer results from natural infection rather than vaccinal immunity, the higher level of antibody from natural infection will persist for a much longer time than the lower amount of antibody from vaccination. Thus, the age a young zoo species can be effectively immunized may be different than for the domestic species depending on the source of antibody (i.e., infection or vaccination). For example, almost all domestic dog pups can be effectively immunized with canine parvovirus-2 (CPV-2) vaccines by 12–14 weeks, whereas the wolf (Canis lupus) pup from a dam that has never been vaccinated or infected could be immunized at 6 weeks of age. However, if the dam’s titer was caused by natural sub-clinical or clinical infection, the wolf pup may not respond until 16 to 18 weeks of age. Furthermore, the half-life of PAMA may be different among the various species.1,2,4,8,18,19
Immune senescence is a measurable decrease in immune function, primarily of T cells. However, B cell function, because of its dependence on T helper cells, can also be affected by age. Immune senescence is genetically controlled, thus the extent to which immunity is decreased, and the time that the decrease occurs, varies among individuals, breeds and species. It could be assumed that certain large dog breeds will experience a decline in immunity as early as 7 years of age, however, in most dog breeds a decline would probably not be seen until 10–15 years of age or older. Although immune senescence in cats has not been well studied, we would estimate immune suppression in most cats would occur several years later than in the dog, with a decline seen as early as 10 years for some cats but in most cats it would not occur until 13–18 years of age or older. In general, immune senescence would occur earlier in those breeds and species with the shortest life span, since it is one important factor contributing to decreased longevity. Because T cells and T cell functions decline more during age-related immune senescence than B cell function, vaccinal immunity dependent on B cells and antibody does not seem to be significantly affected in older animals immunized at an early age. This is likely to be the case because memory cells and memory-effector cells present from prior vaccination or infection are less dependent on T cell help than naive B cells or T cells (see discussion later). Unless significantly compromised by various environmental factors that can cause immunosuppression it is expected that during a majority of the life of a healthy individual, the adaptive immune system from vaccination is fully functional and the memory cells stimulated by vaccination should persist for certain, but not all vaccines for months or years. However, differences exist among species and a wild canid or bovid differ significantly from the domestic counterpart that received the same vaccine.7,8,18
The Cellular Immune Response
The antigen specific defense mechanisms, such as those triggered by vaccines or pathogens are dependent on the T cells and B cells found in the peripheral lymphoid tissues. Initiation of an adaptive immune response requires presentation of antigen by special cells, called antigen presenting cells (APCs), to the T and B cells that have receptors specific for the antigen epitope.
The professional antigen presenting cells include the macrophages, B cells, CD4+ T cells and dendritic cells. Dendritic cells include Langerhans cells and follicular dendritic cells (FDC) with the FDCs being unique in that they present antigen to B cells in the form of antigen-antibody complexes for months after immunization. A critical feature of an APC is the co-expression of class I and II MHC molecules on the cell membrane. Class II MHC molecules are found only on the professional APCs mentioned above, whereas class I MHC molecules are present on all nucleated cells. APCs elaborate a variety of cytokines and have other membrane molecules which are critical for activation of immune responses. Therefore, APCs are critical both for direct antigen presentation and for optimal activation of antigen specific lymphocytes.4,7,8
Antigen processing and presenting cells are critical to the development of an adaptive immune response. Vaccine antigens, like all other antigens, are processed in one of two ways depending on the type of vaccine. Noninfectious (killed) vaccines and modified live vaccines have antigens which are presented primarily in the context of class II major histocompatibility complex (MHC II) molecules to the CD4+T helper cells. This method of presentation is called the exogenous antigen pathway. In addition to this pathway, a second pathway exists for modified live vaccines with intracellular pathogens in which an infectious agent can infect any of a number of cells, including the APCs. After infection of the cell, new antigens produced in the cytosol moves to the endoplasmic reticulum to combine with the Class I MHC (MHC I) molecules. The MHC-Ag complex then moves to the surface of the cell to be presented to a CD8+ T cytotoxic cell (CTL) which is one of the primary lymphocyte classes important in cell-mediated immunity. This method of presentation is called the endogenous pathway. Although expression of antigen in the context of class I MHC molecules requires a live vaccine, a number of methods are under development to get noninfectious antigens to associate with class I molecules for the induction of CTL responses. Methods that have been partially successful under some conditions have included antigens mixed with liposomes or with certain adjuvants and placing antigens on biodegradable beads. Additional studies are required to find better methods to routinely activate CTLs when a noninfectious vaccine is used for diseases in which protective immunity is partially or completely dependent on CTLs.7,8,20,21
The T and B cells specific for an antigenic epitope will be activated to undergo clonal expansion which results in significant numbers of new antigenic specific lymphocytes that differentiate into effector cells and into memory cells. The effector T cells include T helper cells, T regulatory cells and T cytotoxic cells. The effector B cells are the antibody producing plasma cells that secrete specific antibody of the IgM, IgG, IgA and IgE classes.2,8,22,23
The antigen recognition receptor on a T cell is referred to as the T cell receptor (TCR) and on the B cell as the B cell receptor (BCR). Many additional receptors and molecules that serve as cofactors or stimulatory factors are also present on the surface of T and B cells to participate in activation, differentiation and expansion of these cells during antigen presentation. The BCRs are monomeric IgM molecules anchored in the B cell membrane. These immunoglobulins have the typical structure with four chains, two heavy chains composed of one variable and three to four constant regions and two light chains composed of one variable region and one constant region. The chains are connected by disulfide bonds. Antigen specificity of the membrane bound immunoglobulin receptor resides in the highly polymorphic amino terminal hypervariable regions of the heavy (VH) and light (VL) chains to form the antigen binding site. Memory B cells have receptors similar to the membrane IgM but include other Ig isotypes (e.g., IgG, IgA, IgE). The memory cells have the advantage of not first having to produce an IgM antibody when encountering antigen before a switch to the IgG, IgA or IgE classes take place since the switch has already occurred through previous encounters with antigen and T helper cells. Furthermore, the memory cells seem to be more easily activated during a secondary response than the naive cell stimulated during a primary response. This has the advantage of rapid and often significant increased amounts of the class of antibody providing the most appropriate protective response, notably IgG and IgA. Memory-effector B cells are B cells that survive and secrete antibody for months to years after initial antigenic stimulation.7,8,24-26
The antigen receptors on T and B lymphocytes differ structurally but all are members of the immunoglobulin superfamily, as are the MHC class I and class II molecules. An important functional similarity between TCRs and BCRs is they both, in combination with appropriate costimulatory molecules, activate the T cell or B cell respectively to undergo differentiation and clonal expansion. These receptors bind distinct epitopes of the antigen. The B cell receptor recognizes the shape of the antigenic epitope which is often composed of five to six amino acids or sugars, and the B cell receptor may bind structures as large as a virus particle. The TCR complex on the other hand binds peptide antigen in association with class I or II MHC molecules. T cell epitopes are processed antigen (peptide fragments) bound in MHC molecules and B cell epitopes are portions of unprocessed antigen in their native conformation. Thus, an antigenic epitope recognized by the B cell receptor would always be different from the epitope recognized by the T cells. Furthermore, since it is the T helper cell (CD4+) that recognizes antigen in the context of the MHC class II molecule and the T cytotoxic cell (CD8+) that recognizes the antigen in the context of MHC class I molecule, these two types of T cells would also recognize antigenic epitopes which differ. Additionally, only peptide determinants are presented by APCs to T cells, whereas B cells recognize not only peptides, but glycoprotein, lipoprotein, carbohydrate and lipid antigens as conformational determinants. Antigens as complex as those found in vaccines, and those in nature, have many epitopes. Some are B cell epitopes and others are T cell epitopes, but together they constitute the type and complexity of stimulus required to generate an immune response after vaccination or infection respectively. Virtually all vaccine antigens required for protective immunity are T dependent, thus T cell and B cells must cooperate to provide a protective immune response, whether that response is cell mediated and/or humoral.7,8,21,27
The immune response which develops to an infectious viral vaccine (e.g., modified live) will often be far more complete and complex than the response to a noninfectious vaccine with a purified protein (e.g., subunit vaccine) or a limited or large number of antigens (killed vaccine) for the same pathogen. The response to the more complete and complex array of antigenic epitopes expressed during in vivo replication of the attenuated pathogen in the MLV vaccine leads to a greater number of B and T cells being activated by the numerous epitopes presented to the immune system. This is important for induction of a protective immune response to the specific pathogen as well as pathogens that are antigenic variants of the original pathogen or possibly different serotypes of the pathogen. In general, there is more cross-reactivity for epitopes recognized by the TCR than BCR. Therefore, if a T helper cell or a CTL provides some level of protective immunity, a vaccine with appropriate T cell epitopes is likely to provide immunity not only to the specific pathogen, but also to closely related variants or serotypes of the pathogen. However, if antibody is the main protective factor, as is the case for many viral and bacterial pathogens, it is unlikely that immunity will be cross protective, especially when a noninfectious vaccine is used. This would be the case for various serovars (serotypes) of Leptospira sp. Vaccinal immunity to Leptospira, if present, is serovar specific, thus immunity for Leptospira serovars not present in the vaccine would not be expected.2,3,5,6,28,29,31
In contrast, there is excellent cross protective immunity between canine adenovirus type 1 (CAV-1) and canine adenovirus type 2 (CAV-2).30,31 Similarly, there is immunity to most, but not all, of the serotypes of feline caliciviruses due to cross-reactivity among serotypes. However, cross-reactivity may be improved by using additional or different serotypes of virus than are currently present in commercial vaccines. When disease is caused by a single or limited number of serotypes, development of an effective vaccine is much more easily achieved. When there are many antigenic variants, as with lentiviruses (e.g., HIV, FIV), it is very difficult if not impossible to make an effective vaccine.3,5,28 Thus, if a zoo species is infected by an uncommon serotype (or strain) of a pathogen, current vaccines may be poorly protective, if at all. Whereas, if there are no strain or serotypes of the pathogen, vaccinal immunity could be expected.
It has been known for many years that CD4+ T cells had two different functions: 1) to help B cells produce antibody (the classic helper T cell activity) and 2) to mediate delayed-type hypersensitivity or cell mediated immune functions ascribed to T effector cells other than CD8+ CTLs.7,8 Until recently, it was not clear whether these separate functions were accomplished by a single population of helper T cells each at different stage of maturation, or if two different populations of cells existed. The evidence now available would strongly suggest that there are two distinct CD4+ T helper cells in most, if not all, mammalian species. The two subsets of T helper cells have been imaginatively given a Thl and Th2 designation.4,7,8 In referring to the cells as Thl and Th2, it is suggested that the cells can be readily identified and easily separated, which is not the case. The difference between the two subsets of T cells is not based on unique cell determinants but by the cytokines the cells produce and by certain other biologic activities. In general, the Th1 cells facilitate a cell-mediated response with the production of certain cytokines important for that function, and the Th2 cells provide the classic helper response for antibody production by B cells. Vaccination strategies to selectively stimulate one type of response or T cell subset over another could provide an advantage for development of a protective immune response. Selection can be achieved by administering certain cytokines with antigen (e.g., IL-12), varying the dose of antigen, changing the route of administration of antigen and/or using a specific adjuvant. The potential importance of the role played by these two subsets of T cells is demonstrated by immunity to Leishmania infection in two different genetic strains of mice.7,8 The resistant strain C57BL/6 elicits a strong Th1 response and subsequent recovery, whereas BALB/C mice have a Th2 type response and develop severe disease. The production of IFN-γ by the Th1 cells was shown to be the important factor in protection since administration of anti-IFNγ monoclonal antibodies (MAb) which prevented IFNγs from being active, made the resistant mice susceptible. Susceptible mice administered anti-IL-4 MAb which inactivated IL-4, did not develop disease because the IL-4 was not able to block the Th1 cells from forming. It is important to recognize that studies in inbred genetic strains of mice are very artificial and/or exaggerated. After natural infection or vaccination the immune responses are likely to include both Thl and Th2, but the kinetics and the degree of these responses are likely to be very important for development of protective immunity. Therefore, if a vaccine can produce memory Th1 and Th2 cells at the time of challenge, the response should be protective. However, if a vaccine fails to induce or only poorly induces Thl, there would be little or no memory in this subset. If IFNγ is critical for protection from disease, the vaccine may not be effective. IL-12 and IL-4 are two cytokines that significantly influence the development of Th subset immunity, with IL-4 favoring a humoral response (Th2) and IL-12 a cell-mediated response (Th1). Thus, methods that influence the amount of IL-12 and IL-4 as well as other cytokines (IL-18, IL-6), are being used with some success to enhance protection associated with vaccines to several pathogens. In certain systems, efforts to direct the immune system look very promising. Hopefully, future vaccines designed to favor one T cell subset over another can provide better protective immunity than many current vaccines. This manipulation of T cell subsets might also be used to reduce the possibility of adverse reactions such as a type I hypersensitivity response.
The initial immune response that develops in the young animal after infection with a specific pathogen, or more importantly after vaccination, is of critical importance. This first encounter seems to determine the efficacy of the immune response when and if the pathogen is encountered at a later time. Development of a protective cellular and humoral immune response, and the development of T and B memory cells for multiple antigenic epitopes, is often best achieved by vaccines that most nearly simulate what occurs during natural infection. Vaccinal immunity is most often dependent on immunologic memory and not on the effector cells generated shortly after vaccination since the T and B effector cells stimulated after vaccination are usually short lived (living only for a few days to a few weeks). Memory is one of the two major characteristics of an adaptive immune response, specificity being the other. The success of vaccine induced immunity thus depends almost entirely on the development of memory T and B cells that can be activated to produce a rapid and effective protective response long after the original effector cells have disappeared. When a vaccine is developed it is then tested for its ability to induce cell mediated and/or humoral immunity, but more importantly the vaccine must provide a protective immune response shortly after vaccination. If the effector cells required for protection are present then it is highly likely that memory cells have also been stimulated by the vaccine. The memory cells, not the effector cells, survive to be activated by the next encounter with the same agent (e.g., virus, bacteria) as was present in the vaccine. Thus, protection shortly after vaccination is critical if the vaccine has any chance of inducing the appropriate memory cells which survive for long periods of time, providing protection for years or a lifetime with certain vaccines and up to 1 year with other vaccines. A vaccine that fails to protect the animal a few months after it is given will not provide protection a year or more later because the appropriate memory cells were not stimulated.30,32 Therefore, some vaccines cannot produce very effective protective immunity. Even worse is a partial, but nonprotective immune response, that can interfere with revaccination with the development of a response at a later time. Thus, no matter how many times the animal is vaccinated, protective immunity fails to develop.
Most of the experimental studies demonstrating that memory cells persist for the lifetime of the animal have been performed in mice, a species that rarely lives more than 3 years, therefore, studies in longer-lived species were required to determine the true life of memory cells. Memory cells and memory effector cells in humans have been demonstrated many years after vaccination in the absence of known antigenic stimulation. Memory cells in most species are not easily identified and only the demonstration of protection after a challenge infection can absolutely show that there are memory cells capable of protecting the animal from disease. There is, however, a general consensus among most immunologists based on experiments of nature, that memory B and T cells for certain antigens persist for very long periods of time in most species that have an adaptive immune system.
Another cell type that is very important is the memory effector B cell that, in the absence of overt antigen stimulation, produces antibody for months or years after infection or vaccination. These cells are more easily demonstrated by following antibody titers. In humans, it has been concluded, based on persistence of antibody and the demonstration of specific CTLs to certain viruses, that B cell and T cell memory can persist for at least 30–50 years. Studies in dogs have demonstrated that memory B cells, antibody producing memory effector B cells, and presumably the requisite T helper cells for canine distemper virus (CDV) persist for more than least 10 years.3,4,30,34 It was also demonstrated that protective immunity for CDV persisted for at least 7 years when vaccinated puppies were later challenged with virulent virus. Cats have memory-effector B cells for feline parvovirus (panleukopenia virus) that persist for more than 7 years. Similarly, animals vaccinated once as pups with CPV-2 were shown to have memory cells that protected them from infection and disease a minimum of 7 years later.2,31 These are a few examples in which modified live or killed viral vaccines were given at an early age then never again. Viral vaccines are generally believed to provide the longest and most effective immunity. Studies with killed vaccines also show long term memory but modified live products show even longer and stronger immunity. Vaccine licensing studies with noninfectious, killed rabies virus vaccines given once at 12–16 weeks of age show that dogs and cats have memory cells that persist for a minimum of 3 years and provide protective immunity from challenge with virulent rabies virus that is equivalent to that seen after 1 year. It should be noted that rabies vaccine is the only vaccine in which minimum duration of immunity studies are required by the United States Department of Agriculture (USDA).5,6 Only a few studies on maximum duration of immunity exist for domesticated species and no studies exist for maximum duration of immunity with vaccines used in zoo species.
One study where cats were vaccinated as kittens with killed combination vaccine containing feline panleukopenia (FPLV), calicivirus and herpes virus showed that memory and memory effector B cells, and presumably their respective T helper cells, were present at least 7 years after vaccination.34 An important observation from this feline study is that in spite of there being no revaccination, and the viruses of interest were not present where the cats were housed, the immune system remained stimulated since antibody was continually being produced by the memory-effector B cells.34 These studies show the immune response after vaccination with certain viral antigens can persist for many years. In contrast to studies with many modified live and killed viral vaccines where memory cells can be shown to provide a protective response for many years, it is difficult, if not impossible, to show that the bacterins (killed vaccines) for Leptospira sp. provide significant immunity for more than 4–6 months, if at all. Thus, protective immunity and immunologic memory varies significantly among components of a multi-component vaccine and for different types of vaccines (e.g., bacterial vs. viral) as well as among species.3,31 One animal in which there is a relatively short duration of immunity for most vaccine antigens is the horse. It is rare in this species to find vaccines that provide more than 1 year of immunity. Due to this fact, it is common practice to vaccinate nondomestic equine species on a year-to-year basis as well. Based on the aforementioned published studies, other unpublished studies, field observations, and general knowledge about natural disease and specific vaccines it is apparent that memory cells are capable of providing protective immunity in most animal species for many years or even a lifetime in species as long lived as human beings.29,31,35
Understanding the mechanisms involved in immunologic memory and developing methods to readily measure immunologic memory would facilitate the development and testing of new and improved vaccines. Thus, a number of laboratories are conducting studies to answer some of the important questions on this subject. Experience and observation with vaccines for dogs and cats suggest the following:
1. Modified live and killed viral vaccines that prevent diseases caused by systemic infections (e.g., CDV, CPV-2, FPLV) provide memory that persists for many years, probably the lifetime of the animal.
2. Modified live vaccines for viruses that cause local mucosal infections (e.g., canine parainfluenza virus, feline herpes) may have shorter lived memory than is seen for systemic infections but memory appears to last for several years although the effectiveness of these vaccines is limited.
3. Killed viral vaccines provide poor immunity against mucosal infections, thus there is no or only short-term memory.
4. Memory cells for some bacterial antigens seem to be reasonably long lived but memory for protection to the infections caused by other bacteria seem to be shorter lived than for most viruses probably due to the complexity of the immune response required for protection against bacteria.
5. Bacterins (killed bacterial vaccines like Leptospira or Bordetella) are not very effective in providing long-term immunity/memory.
6. Protection from systemic bacterial infection is often longer lived than protection from local mucosal infections with bacteria.
Improvements in bacterial vaccines and the development of parasite vaccines will be an important challenge for the future since the complexity of the organisms and the difficulty of preventing disease will likely lead to problems in developing vaccines that are effective and/or provide a long duration of immunity (e.g., greater than 1 year).30-32,34
An important factor in long-lasting protection from many pathogens of young animals is age related resistance that involves innate and adaptive immunity. This is especially true for those pathogens that primarily cause severe infections in younger animals due to a weakened (poorly developed) immune system. However, most age-related resistance against pathogens is totally or partially independent of specific immunity and independent of immunologic memory cells. For example, canine herpes virus infection in susceptible pups less than 3 weeks of age causes severe disease and high mortality, whereas infection of susceptible pups older than 6 weeks of age causes no significant clinical disease.1,2,35 Obviously this difference is not due to specific immunity or to memory cells. Instead, it is due to thermoregulatory control and to a maturation of a functional immune system. The decreased immunity and low body temperatures at less than 3 weeks of age makes this very young puppy highly susceptible to disease by this viral pathogen, as well as many other pathogens. When passively acquired maternal antibody is not present the animal is at severe risk of disease. Passively acquired maternal antibody is very important in protecting the young animal because active immunity is less than optimal; and the immune system is often unable to adequately protect against viral, bacterial pathogens and certain parasites. These same pathogens are unable to cause disease in an older animal unless the animal is immunosuppressed. In the young kitten, infection with feline leukemia virus (FeLV) will lead to persistent viremia and subsequent disease in a very high percentage of experimentally infected animals. Whereas, in older cats (1 year of age or older), the infection will generally result in protective immunity with no clinically apparent disease or persistent viremia, regardless of the vaccination status of the cat. Again, this example is not one of specific immunity or memory cells, but instead age maturation of innate and adaptive immunity in the cat. Therefore, when vaccinating a young animal to protect it from diseases which most often occur in young animals, specific immunologic memory is of importance—but this memory is not the only mechanism for lasting protection because age associated resistance and memory together will play a critical role in protection. In contrast, for pathogens that cause severe disease irrespective of age, memory cells are of critical and principle importance and age plays little or no role.1,3,31,36
When considering duration of immunity several principles apply:
1. Duration of immunity from vaccination will equal but will not be longer than duration of immunity after recovery from natural infection with a given pathogen.
2. Duration of immunity is shown to be greater for most viral vaccines than for bacterial vaccines.
3. Duration of immunity can be due to specific factors like memory cells as well as innate factors like age.
4. Duration of immunity can be extended for vaccines with short duration of immunity when the pathogen is present in the animal’s environment (natural vaccination).
Mucosal immunity requires special consideration when discussing vaccinal immunity. Although the immune responses that occur systemically can also occur locally, the mucosal immune system is partially independent of systemic immunity. The migration of specific B and T cells from mucosal sites such as the gut associated lymphoid tissue (GALT) and bronchial associated lymphoid tissues (BALT) to various other mucosal sites provides the host with a local immune response which is critically important for protection against certain pathogens. Local T cells, as well as T cells migrating from the systemic immune system to mucosal sites, provide both cytotoxic and helper functions. Also, large numbers of B cells, produced mainly in mucosal-associated lymphoid tissue (MALT) are responsible for IgA antibodies that are secreted onto mucosal surfaces. This IgA immunity is independent of systemic B cell responses to the same pathogen. The source of antigen naive B cells for local mucosal immunity in most mammals is mainly gait associated lymphoid tissue (e.g., Peyer’s Patches). Antigen-specific T cells and B cells, some of which may be activated by antigen in special regions of the Peyer’s Patches, and also antigen naive cells migrate through the efferent lymphatics, thoracic duct and systemic circulation to various effector sites to act locally and become part of mucosal immune system. Effector sites include the lamina propria of the gut, the respiratory tract, the reproductive tract and glandular tissues. MALT thus serves as a source of antigen reactive cells that can protect the mucosal surface from infection and invasion of pathogens that cause diseases at that site. MALT may also provide some limited protection from pathogens that enter the body at that site to cause systemic disease. Thus, local immunity at mucosal sites would serve as a critical first line of specific immunity. The induction of mucosal IgA responses depends on help provided by Th2 cells and the co-stimulation molecules and cytokines found in the systemic immune system. MALT contains both Th1 and Th2 cells, however, the Th2 cells required to help the large number of local B cells provide IgA antibody probably predominate as the T cell population. Although both CTLs and Th1 cells are present at mucosal sites to provide a cell mediated immune response, it is likely that the IgA antibody plays a more critical role in mucosal protection than do the T effector cells.4,7,8,37
Since a majority of pathogenic viruses and bacteria cause disease after entering the body at mucosal sites or by direct infection of mucosal tissue, methods to enhance mucosal immunity by vaccination could provide improved protective immunity at least to certain pathogens. To date, however, the development of noninfectious vaccines which provide effective mucosal immunity has been a challenge. A significant amount of research is directed toward improved vaccines that induce long-term protective immunity in various mucosal tissues. Local immunity is best stimulated by replicating antigens (e.g., modified live vaccine) at mucosal sites, whereas systemic immune responses can be stimulated parenterally by noninfectious vaccines (killed vaccines) as well as replicating antigens. Both routes of administration (parenteral and local) require adjuvants for noninfectious vaccines to avoid multiple injections and to achieve protective immunity. Special adjuvants are required for killed vaccines to be effective when administered locally (oral and/or intranasal) since many of the adjuvants used for parenteral immunization do not work. When one considers immunity to organisms that have mucosal tissue as a primary site of infection (e.g., canine parainfluenza virus, feline viral rhinotracheitis virus) the primary vaccinal immunity would often be local rather than systemic immunity. Therefore, if a modified live vaccine is used parenterally, the animal will develop a systemic immune response but may or may not develop local immunity. However, if the modified live vaccine is given locally the animal will often develop local and systemic immunity. In contrast, if a noninfectious (killed) vaccine is given parenterally the animal will not develop an effective local immune response but should develop a systemic immune response. The non-infectious vaccines presently available for dogs and cats when given locally (e.g., orally; intranasally) provide neither local nor systemic immunity.3,5,6,30,31,37 One major difficulty for immunization strategies with noninfectious oral vaccines, beyond the problems of eliciting a local antibody and cell-mediated immune response, is orally administered noninfectious vaccines may induce a state of systemic immunologic tolerance or unresponsiveness (anergy). “Oral tolerance,” as this process is called, may represent a natural avoidance of immune responses to ingested antigens that could lead to hypersensitivity reactions. Regardless of the reason for this tolerance, it does pose a significant problem and local immunization remains a major challenge to vaccinologists.4,7,8,37,38
For zoo species, oral vaccination may be desired since feeding vaccines would not require capture and restraint. However, special vaccines would need to be developed in order to immunize orally with certain modified live vaccines and with all noninfectious vaccines. Some of those methods are currently available (e.g., baited vaccines with MLV or recombinant vectored vaccines) and methods are being developed for oral administration of noninfectious vaccines (e.g., special immunomodulators like Cholera toxin B).
Types of Vaccines
There are two major types of vaccines: infectious and noninfectious. The infectious vaccines include virulent organisms, attenuated organisms, and live vectored organisms of viral, bacterial or parasite origin. Noninfectious vaccines include killed organisms, sub-units of the organisms, synthetic antigen, and anti-idiotypic antibodies.
In general, the most effective vaccines are the live infectious vaccines because they induce an immune response similar to that seen after recovery from natural infection. However, modified live vaccines are only safe in the host species they have been designed to protect. Attenuation is dependent on an effective immune system in the target species for the vaccine and vaccination of another species may result in severe disease and death. A good example is canine distemper virus vaccine. A very safe and highly effective product in the domestic dog, but capable of causing disease and death in the grey fox (Urocyon cinereoargenteus), the black footed ferret (Mustela nigripes) and certain other species. When vaccinating zoo species, noninfectious vaccines even though not always effective as infectious, would be the vaccine type of choice. This would be the case for several reasons including the possibility that the infectious vaccine may cause disease in the zoo species; an infectious vaccine in an unnatural host may be under significant mutational pressures, thus the vaccine organism may adapt to the new species to cause chronic disease; and the vaccine may be shed by the zoo species to cause the attenuated organism to become virulent even in the target domestic species. The obvious disadvantage of noninfectious vaccines in zoo species is reduced effectiveness; multiple doses (e.g., two to three) required for protective immunity; shorter duration of immunity and increased chance of an adverse reaction (hypersensitivity).3-6,39
Developing effective and safe vaccination programs for zoo species provide challenges and unique opportunities. First and foremost, it will be critical to show the vaccine will cause no significant adverse reactions in the animal. In general, the greatest concern in zoo species would occur with a modified live vaccine that causes disease and possibly death of the vaccinated animals. The example of canine distemper vaccines causing disease and death in certain wildlife/zoo species was discussed above. Because this problem is acute, severe and referenced in the literature, zoo vaccinologists recognize its potential danger and thus it is easily avoided. However, for other vaccines the adverse event may be more subtle. For example, a modified live herpes vaccine like bovine herpes type-I (BHV-1) is used in adult animals without an apparent affect. BHV-1 becomes latent, as all herpes viruses do, and when reactivated by stress (e.g., parturition) the virus is spread to young animals causing significant disease or spread to nonvaccinated pregnant adult animals to cause abortions. In domesticated cattle where most of the animals are immune as a result of natural infection or from vaccination, there are few or no problems when BHV-1 is reactivated. But in a highly susceptible species or nonvaccinated population severe adverse consequences may occur. When certain MLV vaccines are administered to animals,’ vaccinal virus is known to be shed in secretions and excretions. For example, intranasal BHV-1 and PI-3 viruses are readily shed in high amounts for days after vaccination. Whether or not the shed virus would cause disease in unknown, but infection of other susceptible animals could pose a direct risk to a highly sensitive species or it could cause the vaccine viruses to become more virulent through multiple animal passages.5,6
In contrast to attenuated live virus vaccines, discussed above, recombinant live viral vaccines may serve as a safe and effective alternative if available. For example, the canary pox-vectored canine distemper vaccine (Recombitek®, Merial, Inc., Duluth, Georgia USA) should be a safe vaccine for prevention of distemper. However, without studies in the specific species of interest it is difficult to know if the product is effective.
Noninfectious killed vaccines are not without safety problems. In general, killed vaccines are less effective than modified live vaccines in their ability to induce protective and lasting immunity. If immunity persists for only a few months, revaccination becomes an issue as it would not be possible to repeatedly revaccinate animals that need to be captured or darted due to the risk of injury and death. Killed vaccines are especially problematic with regard to causing hypersensitivity reactions. Risk-benefit analysis should always be an important aspect of vaccination, but for zoo species it is likely to be the first issue of concern, not a secondary issue as it is for domesticated species.
In spite of the vast amount of new knowledge in the basic sciences during the past 40 years, vaccinologists are left with many challenges for developing new and improved vaccines. Recombinant DNA biotechnology provides new and exciting methods that are essential to designing vaccines for the 21st century. This technology has already begun to provide opportunities to meet the challenges of making safe and effective vaccines. We can look forward to a bright future for veterinary vaccines as our understanding of the immune system improves and biotechnology is used to engineer products that stimulate immunity in ways not currently possible.3,5,6,31,40
These comparative immunologic principles and developing biotechnology will prove helpful in establishing rational zoo vaccination protocols only when several basic, unanswered questions are addressed. These include which species are truly susceptible to a particular etiologic agent; what is a protective response to vaccines in these species; which assay(s) should be used for detection of antibody response and/or presence of pathogen; how often should we vaccinate; how much vaccine should we provide; which is the best route of administration; how do we interpret serologic results; how do we prove efficacy without pathogen challenge studies; who has used what vaccine manufactured by who in what species; where do we find certain vaccines? These are just a few of many questions the zoo veterinarian is confronted with when deciding how to vaccinate a valuable, unique and often endangered species.
Therefore, the goal for veterinary vaccinologists must be to design an effective strategy to answer these vaccine questions for zoo species. A special zoo species vaccine interest group is proposed to organize and implement the effort to answer the aforementioned questions. A centralized information database on vaccine safety, adverse reactions, vaccine efficacy, known disease outbreaks, species serologic response to vaccination and species immunologic idiosyncrasies needs to be established. This basic, yet essential information is necessary if we are to truly provide protection from disease through vaccination for our ecologically, economically and esthetically important zoological specimens.
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