Immunological Basis of Vaccination

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D. Paul Lunn, BVSc, MS, PhD, MRCVS, Dip. ACVIM

Introduction rent vaccines is understanding what type of immune Equine veterinarians are faced with new commer- responses are required to protect against a specific cial vaccines, new efficacy claims, and potential side pathogen. effects, combined with an increased awareness of the questionable efficacy of established vaccines. Components of the Immune Response These complex issues require of equine veterinari- Our total immune defenses include both innate re- ans a more comprehensive understanding of vaccine sponses, such as neutrophils or complement, and immunology in order to be able to judge this infor- adaptive responses, mediated by lymphocytes, mation. The future promises further change and which result in immunological memory. Only controversy. The aim of this paper is to provide a adaptive responses can be induced by vaccination. review of the basic immunological processes that The specificity of adaptive responses, mediated by are critical to an understanding of the process of antibodies or by effector cells such as cytotoxic T- vaccination. lymphocytes (CTLs), is responsible for their capacity Currently a wide variety of vaccines are available for use in horses but the efficacy of these products to completely protect an animal against a particular varies widely despite the fact that many of these pathogen. The principal types of immune effectors, vaccines are similar in design, containing killed or- including antibodies and lymphocytes, relevant ac- ganisms or toxoids combined with simple adjuvants. cessory factors, and examples of infectious agents A significant reason for this is that this single type of against which they are most effective are listed in vaccination strategy will stimulate only one array of Table 1. There are some over-simplifications; for immune responses. In the case of infections like example, IgG is considered as a single type of im- tetanus, the inactivated toxoid vaccine generates munoglobulin, while in reality there are different neutralizing antibodies that are highly successful in sub-classes of IgG with different functions. To un- providing long-term complete protection. How- derstand how these different types of immune re- ever, in the case of viral infections, such as equine sponses are induced it is necessary to have a influenza virus, current inactivated virus vaccines rudimentary understanding of the biology of lym- fail to induce the complete spectrum of immune re- phocytes, as it is these cells that govern the adaptive sponses required for lasting and effective protection. immune responses that we need to generate with The first step to overcoming the limitations of cur- vaccines.

NOTES

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Proceedings of the Annual Convention of the AAEP 2000 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: VACCINATION

Table 1. Effector molecules and cells of the adaptive immune system, accessory factors, and examples of susceptible infectious agents. (From: Lunn DP, McCLure JT. Immunological principles of equine vaccination. In: Colahan PT, Merritt AM, Moore J, Mayhew IG, eds. Equine Medicine and Surgery 5 ed, St. Louis, MO: Mosby Inc., 1999;183–190)

Effector Accessory Factors Infectious Agents

IgG Complement, neutrophils Bacteria (Streptococcus sp. Clostridial exotoxins), viral neutralization IgA Alternative complement Respiratory, enteric and genital pathway infections, viral attachment IgE MAST cells Intestinal parasites IgM Complement, macrophages Encapsulated bacteria CTL (CD8ϩ) lymphocytes Perforin, lymphotoxin Viruses and intracellular bacteria TH1 (CD4ϩ) lymphocytes Macrophages, B lymphocytes Intracellular bacteria, viruses, fungi (IgG sub-isotypes) TH2 (CD4ϩ) lymphocytes B lymphocytes (IgA, IgE), Viruses, parasites MAST cells, Eosinophils

The Lymphocyte Family T cell Function Lymphocytes can be divided into different popula- Beyond this initial distinction of T and B lympho- tions with different specialized but coordinated cytes, the T cell family is divided into T-helper cells functions, and this family tree is illustrated in Fig- that express the CD4 surface molecule and cytotoxic ure 1. The major division within the lymphocyte T lymphocytes (CTLs) that express the CD8 mole- family is into T cells and B cells. The critical fea- cule. The exact reason why CTLs and T-helper ture separating these cell populations is that T cells cells express these molecules will become clear later, have on their surface an antigen receptor called the but let’s consider ﬁrst the basic features of what T-cell receptor (TCR) combined with a signaling these cells do and why they are important. The molecule called CD3, while B cells express immuno- role of CTLs is the easiest to explain. As their globulin molecules on their surface and use these name suggests, CTLs kill other cells within the directly as antigen receptors. There are two types body, and in the case of infectious disease they do of TCR, the ␣␤ and the ␥␦ TCR, but for our discus- this when cells are infected with a virus or an intra- sion of vaccine immunology we are only concerned cellular bacteria (Fig. 2). This specialization is ab- with the ␣␤-TCR–expressing T cells. There are solutely critical to ﬁghting these types of infections many other critical cell surface molecules, but un- and CTLs are an essential component of immune derstanding these is not essential to our discussion defenses. of vaccine immunology. Let’s examine the T cell and B cell families separately.

Fig. 2. Cytotoxic lymphocyte (CTL) killing. This sequence shows lysis of a virus infected target cell by a CTL. The target expresses antigens derived from the virus on its cell surface Fig. 1. Major divisions of the lymphocyte family. To the left of bound to MHC I molecules, which are recognized by the CTL, the diagram different populations of lymphocytes are distin- resulting in binding and release of effector proteins that trigger guished by expression of different cell surface molecules. To the cell death. The CTL survives the process and can go on to right of the diagram the distinctions are functional. another target.

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Proceedings of the Annual Convention of the AAEP 2000 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: VACCINATION induced by vaccination will determine whether the vaccine is either helpful or possibly even detrimen- tal, in protecting against disease. Therefore a key issue in vaccine development is the ability of different vaccination strategies to stimulate specific T- helper subsets.3 B cell Function The role of the B cell family is the production of a complex array of antibodies. In the horse all the major antibody classes have been identified: IgM, IgG, IgA, and IgE. Importantly, the IgG class can be divided into a number of subclasses, the best characterized and most important of which are IgGa, IgGb, and IgG(T). Each of these classes and Fig. 3. T-helper lymphocyte subsets. The TH1 and TH2 lymphocyte subsets provide help for macrophage activation and hu- subclasses are specialized for a specific role as indi- moral immunity, respectively. This is mediated by production of cated in Table 1, and discussed in detail below in the cytokines which have a regulatory effect on each other. context of IgA and mucosal immunity. The structure of an antibody molecule is shown for IgA in Figure 4, an antibody which is specialized for activity at mucosal surfaces. The other major immuno- If the CD8ϩ CTLs are responsible for destroying globulins involved in fighting microbial disease are infected target cells, what is the role of the other IgM, which is responsible for a rapid initial response major T lymphocyte subset, the CD4ϩ T-helper lym- to either infection or vaccination, and IgG, which phocytes? These lymphocytes, as their name im- increases after IgM but is produced in larger plies, “help” other effector cells to fight off amounts and with a higher affinity for its antigen. pathogens. It is currently believed that two differ- In addition, IgG responses can be very long-lasting ent subsets of T-helper cells, characterized by their and it is IgG that is responsible for recall responses cytokine production profile, may be responsible for on re-exposure to a pathogen or booster vaccination. determining the nature of the immune response to The maturation of B cells, illustrated in Figure 5, infectious agents.1 The subsets are the T-helper 1 provides an example of the process of lymphocyte subset (TH1) which stimulates cytotoxic and inflammatory functions, and the T-helper 2 subset (TH2) which stimulates strong antibody and allergic responses (Fig. 3). These two types of T-helper subsets and the cytokines they produce tend to suppress each other. As a result, in an immune response to a particular pathogen, either the TH1 or the TH2 will predominate and give rise to either an inflammatory/cytotoxic or a humoral immune response. Therefore, if an appropriate immune response to a pathogen is to be produced, vaccination must induce the appropriate T-helper response. An example of such a circumstance may be Rhodococcus equi infection in foals. Like other intracellular pathogens, such as Salmonella sp, this organism survives by parasitizing macrophages.2 In order to overcome such infections, the macrophage requires help from inflammatory TH1 cells that can activate the macro- Fig. 4. The IgA molecule. This schematic illustrates the major phage by secretion of cytokines such as IFN-␥ and features of immunoglobulin molecules. While the illustrated GM-CSF. In contrast, a TH2 response may be inef- IgA molecule is dimeric, with the two Ig units joined by the green fective in combating these intracellular organisms, “J-chain” and a series of red disulphide bonds, IgG molecules are and may actually be counterproductive by suppressing monomeric. Each Ig unit consists of two heavy chains and two TH1 activity. This may explain the lack of efficacy of light chains. The heavy chains have four subunits and the light killed vaccines against intracellular pathogens. chains two. One end of the Ig unit has a highly variable protein Several factors have been identified which may structure and is involved in antigen recognition, while the re- mainder of the Ig unit has a constant structure in each Ig class influence whether a TH1orTH2 type response will and subclass and determines the functional characteristics of the predominate and these include the type of antigen molecule, such as binding complement, or recognition by macro- presenting cell, dose of antigen, the type of adjuvant, phages or neutrophil FC receptors. This specialized dimeric IgA immunization route, and the cytokines present dur- molecule also has a blue secretory piece that increases its stabil- ing antigen presentation. The type of T-helper cell ity in the harsh mucosal environment.

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Proceedings of the Annual Convention of the AAEP 2000 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: VACCINATION We will focus on the antibody response to this pathogen, as this is probably a critical component of protective immunity to influenza. Until recently only killed vaccines were commercially available for equine influenza, but in young horses in particular they gave short-lasting protection at best.5 In contrast, equine influenza virus infection leaves horses protected for at least 6–12 months.6 To work out why vaccinal immunity compared so poorly with infection-acquired immunity, Dr. Kay Nelson per- formed an experiment to study local and circulating antibody responses and protection resulting from a conventional commercial vaccination or natural influenza virus infection.7 It was found that 3 months after administration of two doses of a con- Fig. 5. Maturation of B lymphocytes. Different stages of B lymphocyte development can be recognized by expression of im- ventional vaccine, ponies were left unprotected munoglobulin molecules. This maturation requires a series of when subjected to a challenge infection. In con- gene rearrangements in order to select the genes which will trast, 3 months after being given an initial influenza encode the antigen binding part of the immunoglobulin molecule virus infection, ponies were completely immune to a (variable region), and subsequently to select the genes that de- repeat challenge infection. The key local and se- termine the class or subclass of the antibody molecule. Initially, rum antibody responses to infection or vaccination immature B cells express IgM (the majority of peripheral blood B are shown in Figure 6. A critical difference be- cells), but after antigen exposure the B cell becomes activated and tween infection and vaccination was that infection may express any of the immunoglobulin classes or subclasses. induced high levels of IgA in nasal mucosal secre- This decision depends in large part on cytokine signals from tions whereas vaccination induced no IgA antibod- T-helper cells. Finally, activated B cells either mature into short-lived antibody secreting plasma cells, or become long-lived ies. In addition there were marked differences in memory B cells. the isotypes of IgG induced by infection compared to vaccination, with natural infection inducing IgGa and IgGb responses and conventional vaccines inducing IgG(T) responses. It is interesting to note development. There are two key processes that oc- that in the horse the IgGa and IgGb sub-isotypes are cur here. The first is a rearrangement of the germ capable of mediating important anti-viral activities line DNA of the B cell in order to determine the such as complement fixation and antibody-depen- exact structure of the immunoglobulin molecule an- dent cellular cytotoxicity, while IgG(T) responses tigen binding site it will later express on its surface can actually inhibit complement fixation and are or secrete. This process is mirrored in T cells when better adapted to neutralizing toxins such as those they determine the antigen binding specificity of the produced by the Clostridia sp.8,9 TCR they will subsequently express. This is a critical step in all lymphocyte development as it is responsible for the ability of the immune system to recognize a vast array of foreign antigens. Inter- estingly, a failure in this process, due to mutation in an enzyme gene, is responsible for the failure of B and T cell development that we see in Arabian severe combined immunodeficiency foals (SCID). A second key step in B cell development is the process of deciding what type of immunoglobulin class to produce. All B cells start out making IgM and IgD, but later make a final commitment to one specific class or subclass of immunoglobulin. This decision depends on a number of factors, but cytokines released by TH1orTH2 cells play a critical role in this decision.

Protective and Non-protective Immunity to Influenza Virus Fig. 6. Immune response to influenza virus. Equine immunoglobulin responses in two groups of four influenza naı¨ve ponies to At this stage we have explained the major compo- either influenza virus infection or conventional inactivated vac- nents of the adaptive immune response and it is a cine administration. The graphs show the mean nasal mucosal good time to put these in some context. The exam- IgA response to a viral infection (administered on Day 0), or the ple we will use is the immune response to equine IgG subclass responses to infection or a series of two vaccinations influenza virus, after either infection or vaccination. (second dose on Day 0).

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Proceedings of the Annual Convention of the AAEP 2000 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: VACCINATION that bind to MHC class I or class II molecules are, respectively, the CD8 or CD4 molecules (Fig. 9). The CD8 or CD4 molecules act as receptors for MHC molecules, and stabilize the interactions between T cells and antigens presenting or target cells. Therefore CTLs (CD8ϩ) can only respond to antigens presented by MHC I molecules, and T-helper lymphocytes (CD4ϩ) can only respond to antigens presented by MHC II molecules. This means that CTLs can recognize any cell in the body that is infected with an intracellular pathogen and is dis- playing components of that pathogen on its surface bound to MHC I molecules. In contrast, T-helper cells recognize antigens only on the MHC II expressing APCs, such as macrophages. Fig. 7. The role of professional antigen presenting cells (AP- C). In this ﬁgure pathogen invasion is followed by antigen up- At this point, an important practical implication take by a dendritic cell, the most potent of the APC family. The for vaccine development is apparent. CTLs have a dendritic cells become activated and migrate to a local lymph critical role in eliminating viral infection because node where they are extremely effective at stimulating naı¨ve T they are adapted to seek out and destroy infected cells including both T-helper cells and CTLs. cells. These cells can only respond to antigens presented by MHC I molecules. However, killed or inactivated antigens in vaccines are phagocytized by the antigen presenting cells and are therefore far Not all killed equine inﬂuenza vaccines perform as more likely to be presented by MHC II molecules. poorly as did the one used in this study, but this Therefore many conventional vaccines are unlikely example highlights that vaccines must induce the to be able to induce the CTL responses that are right kind of immune responses, and in the right essential for defense against some viral infections. places. Now that we understand the components of the immune response, there is one more basic immunological concept that must be grasped, and that is how the immune system sees an invading pathogen. This process is called antigen presentation.

Antigen Presentation T lymphocytes don’t respond directly to antigens present on the surface of a virus, for example. In- stead they recognize small processed antigen fragments that are present on the surface of either infected cells, or on cells that are specialized for capturing foreign antigens and presenting them on their surfaces. This latter type of cell is termed an antigen presenting cell (APC), and examples include macrophages and the highly efﬁcient dendritic cells (Fig. 7). After processing the antigen, short antigenic peptide fragments are presented on the cell surface bound to Major Histocompatibility mole- Fig. 8. Antigen presentation. This ﬁgure depicts MHC I antigen presentation to the left of the diagram, and MHC II antigen cules (MHC), which can be divided into the MHC presentation to the right. In MHC I antigen presentation (a) class I molecules which are present on all cells, and peptides generated by degradation of proteins in the cytoplasm the MHC class II molecules which are only present are transported into the endoplasmic reticulum (b). In this lo- on specialized antigen presenting cells. cation MHC I molecules bound by a membrane protein calnexin Antigens are processed for presentation by either (purple box) bind the peptides which allows release of the MHC I the endogenous pathway resulting in presentation molecules by the calnexin and transport through the Golgi com- by MHC I molecules, or by the exogenous pathway plex to the cell surface (c). In MHC II antigen presentation resulting in presentation by MHC II molecules as antigen is taken up by phagocytosis (1) into the endosome com- illustrated in Figure 8.10 This means that antigens partment and routed to lysosomes for degradation. Vesicles con- absorbed from outside the cell, by phagocytosis for taining MHC II molecules produced in the endoplasmic reticulum fuse with the endosomes (2) and the MHC II molecules bind with example, are presented by MHC II molecules. An- the degraded peptides for transport back to the cell surface tigens produced inside the cell, as is the case during (3). The MHC II molecules are prevented from binding the en- viral infection, are presented by MHC I molecules. dogenous peptides in the endoplasmic reticulum by the presence The implication of this for the T lymphocyte is ap- of invariant chain (brown box) which is only lost in the acidic parent when you understand that the molecules endosomal environment.

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Proceedings of the Annual Convention of the AAEP 2000 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: VACCINATION IgA-B cells to effector sites such as the lamina propria of the gut and respiratory tract, second signals from antigen presentation cells and from T-helper cells result in further differentiation into IgA pro- ducing plasma cells. Secretory IgA protects the body from bacteria and viruses principally by immune exclusion, i.e., by phys- ically preventing attachment to mucosal surfaces.11,15 After release of secretory IgA by plasma cells into the interstitium, it is transported across the epithelial cell and released at the luminal surface (Fig. 11). During its transit through the epithelial cell it is even possible that IgA can neutralize intracellular infections.16 Overall the mucosal immune system can function as an independent immunological or- Fig. 9. The role of T cell CD4 and CD8 molecules. T cells use their T-cell receptors to recognize processed antigen presented in gan, equipped with specialized tools to deal with the combination with either MHC I or MHC II molecules. T cells particular antigenic challenges faced by mucosal exclusively express either CD4 (T-helper cells) or CD8 (CTLs), surfaces. For many diseases, including influenza 17 18,19 and the CD4 molecule is required for interaction with MHC II virus and Streptococcus equi infection, a mu- molecules, while CD8 is required for MHC I interaction. As a cosal immune response may be the most effective result T-helper cells recognized antigens presented by MHC II type of immune protection. and CTLs only recognize antigens presented by MHC I molecules. Effective Vaccination We have reviewed many of the key functional ele- ments of the adaptive immune response, and some In addition, in the case of T-helper cells, several of the regulatory processes that control them. In factors involved in antigen presentation can influ- particular, we have identified three types of T lym- ence whether a TH1orTH2 response is induced. phocytes that mediate immunity: the CTL which It is apparent, therefore, that appropriate and effi- can destroy virus infected cells; the TH1 lymphocytes cient antigen presentation is an essential require- which can provide pro-inflammatory signals to acti- ment for any vaccine. vate cell mediated immunity (e.g., macrophage activation); and the TH2 lymphocyte which can drive Mucosal Immunity antibody production. Appropriately stimulating The components of the mucosal immune system are these regulatory and effector T lymphocyte re- no different from the immune system in the rest of sponses is an essential function of an effective vac- the body, but this “compartment” of the immune system is so important that it deserves special con- sideration. In the equine influenza study above there was a strong mucosal IgA response following infection, and there is excellent evidence that this type of immunity is critical for protection from many pathogens that invade mucosal surfaces. The mucosal surfaces of the gastrointestinal, respiratory, and genitourinary tracts are continuously exposed to foreign antigens, including potentially infectious bacteria and viruses. The adaptive mucosal immune responses that have evolved to protect the body against these challenges have distinct and specialized characteristics.11 The principal immunoglobulin produced by the mucosal immune system is secretory IgA, which is the most abundant immunoglobulin class in the body. Specialized antigen uptake cells in the Peyer’s Patches of the intestinal Fig. 10. Mucosal immunity. In this simplified overview of the tract or nasopharyngeal lymphoid tissues, termed mucosal immune system a virus (1) first encounters an M cell microfold or M cells, transport antigens to underly- (purple cell) overlying an aggregate of lymphocytes in mucosal ing mucosal associated lymphoid tissues or MALT associated lymphoid tissues (2). This results in production of 12,13 both IgG and IgA antibody responses and circulation of primed (Fig. 10). In the MALT antigen processing and antigen specific B lymphocytes in the blood system (3). These B presentation takes place, resulting in immunoglob- lymphocytes can travel to other mucosal surfaces where on re- ulin class-switching and activation of antigen-spe- exposure to antigen (4) they can secrete IgA which can be trans- 13 cific IgA-positive B cells. T-helper cells are ported across the mucosal epithelium to neutralize virus. The IgG, critical to this process.14 After homing of these in contrast, is restricted to the circulation and lamina propria.

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Proceedings of the Annual Convention of the AAEP 2000 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: VACCINATION nant hosts, with the risk of reversion to pathogenicity or contamination of the vaccine with other pathogens. Modified Live Vaccines A modified live vaccine (MLV) will produce proteins in the cytosol which will be presented by MHC I molecules and induce CTLs. MLV viral vaccines can be produced by attenuation in cell culture, by use of variants from other species (e.g., smallpox and vaccinia), or by development of temperature-sensi- tive mutants.20 The mutations in MLVs are often poorly defined and reversion to virulence is a constant threat. In future, MLVs may be available in which specific mutations are produced, using recom- Fig. 11. Effector mechanisms of IgA. Trans-epithelial IgA transport occurs when IgA molecules in the lamina propria (red binant DNA technology, which has predictable ef- dumbells) are bound by the polymeric Ig receptor (1). IgA is fects and cannot be reversed. Examples of such transported across the cell in transcytotic vesicles and released as approaches are the development of experimental secretory IgA at the lumenal side of the cell where virus neutral- equine herpesvirus (EHV-1) vaccines with deletions ization can occur (2). In addition, on viral infection of the cell, of specific glycoprotein genes.23 Current equine viral envelope proteins are synthesized in the rough endoplasmic MLVs include a highly efficacious Equine Viral Ar- reticulum (3) and then processed through the Golgi and exocytotic teritis Vaccine, and two relatively recent intranasal vesicles prior to insertion into the plasma membrane. Mature vaccines, one against S. equi and one against equine virions are then formed with new copies of the viral genome. influenza virus. During the transcytotic transport of IgA, fusion of IgA vesicles with exocytotic vesicles from the Golgi containing viral glycopro- Recombinant Vector Vaccines teins can occur (4), inhibiting further viral assembly. Both bacteria and viruses can be engineered, using recombinant DNA technology, to be carriers for defined antigenic polypeptides or peptide epitopes cine. What then are the types of vaccines that are from other pathogens. However, this is technically available to for this purpose? far more complicated for bacteria, given their much Current active vaccination strategies can be larger and more complex genomes.20 The advan- broadly divided into the administration of “live,” tage of such vectors is that they allow introduction of “dead,” and DNA vaccines, and these approaches genetic material encoding pathogen antigens into have been recently and extensively reviewed.3,20,21 host cells, with subsequent protein production and Live vaccines include attenuated microbes and re- antigen presentation by both MHC I and MHC II combinant vaccines that utilize a living vector, while pathways. A critical prerequisite to using this dead vaccines include killed whole pathogens, solu- technology is a knowledge of the protective antigens ble pathogen subunits, or protein subunits. Immu- of the specific pathogen of interest (see subunit vac- nization based on administration of plasmid DNA cines below). An example of suitable vector is ca- (variously termed “genetic,” “nucleic acid” or “DNA” narypox virus, which can infect mammalian cells immunization) is a radically different form of vacci- but is unable to produce viral progeny, and has been nation that enjoys many of the immunological and used as a vector for EHV-1 and equine influenza safety advantages of both live and dead vaccines. virus genes.24 This type of technology obviously Finally, passive vaccination remains a strategy with has great promise and is an area of active investi- unique advantages in specific circumstances. The gation. There are some safety concerns which are rest of this paper discusses these different vaccina- similar to those of classical MLVs, with the addition techniques and technologies. This subject has tional risk of contamination with adventitious recently been reviewed in detail,22 and therefore a agents and vector pathogenicity.25 brief overview is presented here. Dead Vaccines Live Vaccines Dead or killed vaccines remain attractive because of Live vaccines employ an organism that can continue their relative ease of preparation, lack of pathoge- to replicate in the horse but has attenuated patho- nicity, and inability to replicate and spread between genicity. They enjoy a number of advantages, such hosts. However, dead vaccines typically require as generating a broad range of immune responses multiple doses and regular boosters, and efficacy including a range of antibody-mediated responses frequently depends on use of potent adjuvants. and CTLs, having a generally long lasting duration of immunity, and typically requiring fewer doses.20 Inactivated Pathogen Vaccines While often successful in generating immunity they Inactivated whole pathogen vaccines are the most can be dangerous in immunocompromised or preg- common form of equine vaccine in current use.

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Proceedings of the Annual Convention of the AAEP 2000 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: VACCINATION Inactivation is achieved with agents such as thi- evidence of the efficacy of influenza virus ISCOM mersol or phenol in the case of bacteria, and formalin vaccines in horses.28,34,35 An important component or beta-proprionolactone for viruses. Historically of ISCOMs is Quil A, a component of a plant sapo- such vaccines have frequently proven highly immu- nin. Quil A is also found in several current equine nogenic, although their limitations in protecting vaccines, often in combination with alum, and while horses from respiratory pathogens represent an on- not as potent as an ISCOM, its inclusion does aug- going and serious problem.5,7,26 Quality control in ment adjuvanticity. the production of such vaccines can overcome some One particular type of adjuvant deserving special of these limitations,27 but development and use of mention are the mucosal adjuvants. Mucosal im- effective adjuvants represents the most important munity has a critical role in resistance to wide vari- tool for overcoming their limitations (see below).28,29 ety of pathogens such as equine influenza virus7 and S. equi.36 Generating mucosal IgA responses with Protein Vaccines killed vaccines is challenging, and the only effective Protein vaccines include naturally produced compo- mucosal adjuvants are the bacterial exdotoxins of nents of pathogens, such as the M-protein vaccine enteric bacterial pathogens such as cholera toxin for S. equi which is non-pathogenic and may pro- (CT) or the labile toxin of E. coli.37 This adjuvant mote fewer injection site reactions than whole bac- effect may depend on several known actions of CT, terial products. The most commonly used protein including enhancement of antigen presentation, pro- vaccine in horses is tetanus toxoid, which is pre- motion of B lymphocyte isotype differentiation, stim- pared by formalin inactivation of tetanus toxin and ulation of CD4ϩ T-helper lymphocytes, and incorporation with an alum adjuvant. induction of local and systemic memory responses. A disadvantage of using CT is that it can produce Recombinant Subunit Vaccines cholera diarrhea in humans,11 although it is well The explosion of knowledge in the field of recombi- tolerated in other species. Recently, encouraging nant DNA technology has led to the identification, results have been reported in horses vaccinated in- and in some instances, synthetic production of many tranasally with inactivated equine influenza virus of the specific antigens that are important for im- combined with cholera toxin B subunit.38 munity to pathogens. Such vaccines can include recombinant polypeptides, or peptide-based vac- DNA Vaccines cines containing a single antigenic epitope. Unfor- DNA vaccination results in the in vivo synthesis of tunately these purified proteins may be poorly antigenic proteins in a manner identical to that oc- immunogenic by themselves, and particularly so in curring in natural infection.25 This endogenous the case of peptide vaccines, and cannot overcome production results in presentation of antigens by the barriers that prevent MHC I presentation with- MHC I and presentation to CD8ϩ T cytotoxic lym- out the use of appropriate adjuvants. phocytes, and uptake and presentation of soluble proteins by MHC II to CD4ϩ T-helper lymphocytes. Adjuvants As a result DNA vaccination has been shown to Comprehensive explanations of the many types of induce both potent CTL and antibody responses. adjuvants is beyond the scope of this paper; how- Investigations of the use of DNA vaccines in horses ever, several excellent reviews have been pub- are at an early stage, but it has already been dem- lished,3,4,30,31 including one review of vaccine onstrated that they are effective at protecting horses adjuvants in use in veterinary products.32 The suc- from influenza virus infection and induce appropri- cess of killed vaccines frequently depends on the ate antibody isotype responses,39,40 and there is pro- adjuvant system used, as adjuvants can determine visional evidence of their potential for EHV-1 the form of immune response that will be stimulated vaccination.41,42 through stimulating either the TH1orTH2 regulatory lymphocyte subsets. Current adjuvants, such Passive Vaccination as alum, tend to stimulate TH2 responses while Passive vaccination is accomplished by administer- Freund’s Complete Adjuvant (FCA) is an example of ing preformed antibodies either as a plasma trans- an adjuvant that stimulates TH1 responses. How- fusion or in a concentrated form, as in commercially ever, although FCA can induce TH1 responses, it available tetanus antitoxin. This strategy can be cannot be used in commercial vaccines due to its side highly effective in diseases for which there is no effects. Another critical function of adjuvants is to available form of active vaccination (e.g., R. equi)or gain access to the cellular compartments that allow in high-risk situations when there is inadequate for MHC I presentation and CTL induction. One of time for protection to be generated by active vacci- the most promising adjuvants for this purpose is the nation. Generally passive vaccination should be Immune Stimulating Complex (ISCOM). ISCOM avoided when possible due to the risk of transmis- adjuvants have been associated with greatly in- sion of infection in serum-derived products. A creased antigen-specific antibody responses, and a prime example of this is the association with acute wide range of T cell responses including the induc- hepatic necrosis with a previous administration of tion of cytotoxic T-lymphocytes.33 There is already tetanus antitoxin.43

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AAEP PROCEEDINGS ր Vol. 46 ր 2000 9

Proceedings of the Annual Convention of the AAEP 2000