This review examines some of the new technologies used to produce veterinary vaccines. In order to understand the intimate mechanism of these processes, it is necessary to briefly review the functioning of the immune system.
Immune recognition of foreign bodies
Immunity operates through clonal selection, whereby the B lymphocytes that produce antibodies and display them on the cell surface, and the T lymphocytes (helper and cytotoxic) which recognize a concrete antibody are activated, followed by multiplication and the development of immune defensive actions. In addition, immune memory is established since a part of the lymphocytes that responded to the primary infection are preserved and may posteriorly activate again in response to repeat invasion by the same type of microorganism. In this context, vaccination acts in a way similar to primary infection.
The response to pathogens found in the internal organic fluids or in the lumen of body cavities (respiratory and digestive tracts, etc.) involves the production of immunoglobulins — fundamentally IgG and IgA. However, viruses and some bacteria and parasites penetrate the cells before antibodies can be produced. In this sense, antibodies are water-soluble proteins that can diffuse into the extracellular fluid spaces and blood, but not across the lipid membranes of cells. As a result, they are unable to act upon intracytoplasmic pathogens. In order to afford protection against the latter, a cellular immune response must be induced.
Antigen-presenting cells (APCs), such as macrophages, patrol the body, ingesting (phagocytosing) antigens and fragmenting them into antigenic peptides. Pieces of these peptides bind to molecules of the major histocompatibility complex (MHC I and II), and are presented on the surface of the macrophages to B and T lymphocytes.
Once the antigen has been phagocytosed and digested, it passes through the reticuloendothelial system, and the peptidic antigen binds to MHC II molecules. This process in turn stimulates the B and T helper lymphocytes, resulting in the production of antibodies. Essentially, any inactivated vaccine administered via the parenteral route employs this same mechanism, and its function is limited to the induction of antibody production (fundamentally IgG, i.e., humoral immune response).
When the vaccine (or pathogen, in the case of infection) is live, however, it may pass into the cellular cytoplasm or cytosol (intracellular pathogens), and in this case the infested cell transports peptides that may be formed to its surface by using the MHC I system and thus activates cytotoxic T lymphocytes (cellular immune response). All body cells express MHC I molecules, as a result of which all transport the peptides produced during cytosol hydrolysis to the cell surface. This mechanism allows the body to continuously monitor the status of the different body cells and detect the production of anomalous proteins, as in tumor processes and infections, and to eliminate the affected cells.
Characterization of protective antigens and protective immune response
When a dead or attenuated vaccine is injected into an animal, an immune response occurs. However, this response may not afford protection, because the vaccine may not contain the adequate antigens, or because the induced immune response does not present the characteristics required for protection.
As we have already mentioned, a protective immune response to vaccination may be due to the production of antibodies (humoral immunity), the action of sensitized T lymphocytes (cellular immunity), or a combination of both. In general, systemic humoral immunity is thought to be particularly important in protecting against the extracellular phases of viral infections and systemic bacteremia, as well as in toxin-mediated diseases. In preparing these types of vaccines we must use antigens that are accessible to the immune system when the culprit bacteria or viruses are alive, i.e., flagella, pili, capsules, membrane proteins, extracellular toxins or endotoxins that are released with the death of the bacteria and which in many cases are responsible for the induction of tissue lesions. The antibodies against these external antigens act by agglutinating and inactivating the pathogen, opsonizing the bacteria to facilitate their phagocytosis, activating the complement system to destroy or opsonize bacteria, or by inhibiting bacterial adhesion to cell surfaces and thus complicating the bacterial colonization of host tissues.
The internal antigens, cytoplasmic antigens and some antigens immersed in the bacterial wall are of course also able to induce a humoral response, though in the live pathogen they are obviously not accessible to the antibodies.
Cell-mediated immunity is particularly important in protecting against intracellular bacteria, intracellular viral infectious phases, fungal infections and processes caused by protozoa. In this sense, cytotoxic T lymphocytes eliminate the infected host cells, thereby disrupting the replication process of the pathogen. External antigen expression is useful in this response, though intracellular antigens can also be used to prepare a vaccine, since any host cell exhibiting intracellular antigens of the pathogen on its surface will be eliminated by the host cellular immune defenses.
Secretory antibodies (IgA) are the most important immunoglobulins against those bacteria and viruses that must adhere to the epithelial surfaces in order to cause disease, as well as in diseases caused by toxins at mucosal surface level. It is very important to remember that the processes of greatest interest in veterinary practice (respiratory and digestive infections), or at least some of their phases, can be included here.
IgA are secreted in all mucosas. The importance of these secretory antibodies can be appreciated by taking into account that at least 80% of all antibody-producing plasma cells produce IgA, and that the IgA secreted into the intestinal lumen each day exceeds the amount of IgG released systemically.
In fact, the immune system can be divided into two functionally independent compartments: a systemic compartment comprising the bone marrow, spleen and lymph nodes, and another comprising the mucosas — including the mucosal lymphoid tissue and external secretory glands. The mucosal system is in turn intimately connected, and the immune response produced in one mucosa is transmitted to the rest, including the mammary glands (common mucosal immune system; see Fig. 1).
Fig.1: Common mucosal immune system
This characteristic of the mucosal immune system is very important, since an antigen introduced via the oral route in the correct manner will induce an immune response in the respiratory tract and vice versa. This type of immunity is effectively compartmentalized, and for example vaccines are being studied against human immunodeficiency virus infection (AIDS) administered via the rectal route in order to obtain responses in the genital tract.
It should be taken into account that IgA act on the external surfaces of the mucosal membranes, as a result of which they do not activate inflammatory responses. In contrast, IgG are not released to the exterior, and their action takes place directly upon the epithelial layers or beneath them — thus inducing inflammatory responses and therefore also lesions that can in turn constitute gateways for the penetration of other infections.
Good results are being obtained with human influenza or porcine Aujesky vaccines with a first does via the nasal route, producing a mucosal response destined to limit viral access, while the reminder dose is administered intramuscularly to neutralize the virus in its extracellular phases. As these are live vaccines, they also elicit a cellular immune response.
Characterization of the immune response
Having examined the important antigens, the characterization of a protective immune response requires the definition of which aspects of the immune reaction are responsible for the protection afforded (systemic humoral, mucosal, or cellular). All this information is necessary to ensure that a given in vitro potency test is also relevant for assessing in vivo efficacy, and for making sure that the relevant immune response is being measured against the adequate antigens.
It is very difficult to establish which elements of the immune response are responsible for protection, particularly in those diseases where the protection afforded is only partial, and where various components are implicated in protection. In fact, with the exception of very concrete cases, immune response (antibody titers or cell responses) have not been correlated to host protection. In human vaccines against Haemophilus influenzae, a correlation has indeed been established between an antibody titer against a well defined capsular polysaccharide and protection against meningitis. In this way it is possible to study (for example) whether maternal immunization also generates protective antibody titers in the offspring, and the duration of the protection afforded.
Many techniques have been developed for measuring antibody titers, including ELISA, hemagglutination, precipitation, virus neutralization, cytotoxicity tests, bactericidal activity in the presence of complement, toxin neutralization, bacterial opsonization, etc. These tests are normally simple to perform with blood serum, where IgG are therefore assayed. However, as has already been seen, IgA are normally important for immunity, preventing colonization and penetration of the mucosal barriers of the host. However, the determination of an IgA titer requires the collection of mucosal secretions, which considerably complicates the tests. In fact, even experimental vaccinal trials focus very little attention on IgA.
The cell response after vaccination can be detected by the presence of activated lymphocytes that recognize certain antigens and which actively multiply in the presence of these antigens — for example the delayed sensitivity test (as in tuberculin testing). In the same way, it is possible to study this proliferation or release of interleukins in vitro. However, the problem here is again the correlation between a given cell response and the protection against infection.
Because of our lack of knowledge of the way in which bacteria or viruses truly cause disease, it is very difficult to identify the antigens responsible for protection, and consequently it is impossible to conduct in vitro tests to measure the protection afforded by a given vaccine against the disease. In our opinion, the best approach is simply to continue using those vaccines that have demonstrated their efficacy in the field.
For this reason in vivo trials are conducted, in which a vaccinated animal is experimentally inoculated with a virulent strain. For example, in chicken vaccines against Pasteurella multocida, the evaluation is made in two phases. In a first phase the chickens that have been vaccinated twice are intramuscularly inoculated with a reference virulent strain, 14 days after the last vaccination. If 8 or more vaccinated animals survive, then the test is considered valid, and the results are evaluated according to the mortality of the vaccinated batch of animals.
In the same way, other tests are conducted in mice. In this context, it is necessary to extrapolate the results obtained, for example, in mice inoculated with Pasteurella via the intraperitoneal route, or in chickens via the intramuscular route (where exclusively mortality is assessed) to the protection obtained against a chronic respiratory process. The validness of these techniques can obviously be the subject of much debate.
An adjuvant contributes to achieve a potent immune response in various ways: 1) It acts as a deposit or reservoir, whereby the antigen is released progressively (mineral or oil compounds). In this way the macrophages phagocytose antigen for a longer period of time. 2) The adjuvant is able to present the antigen directly to the competent cells (macrophages, dendritic cells of the lymph nodes, etc.). For example, liposomes and microspheres avoid metabolization and elimination of the antigen contained within them — thus ensuring that the contained antigen is fundamentally presented to macrophages, which in turn transport the antigen to the lymph nodes where the immune response takes place. 3) Some adjuvants act as chemical immune stimulators of lymphoid cells (lipopolysaccharides, cytokines, lipid A, Freund, etc.). Of course, some vaccine formulations can combine several of these mechanisms. For example, an immune enhancer such as lipid A can be incorporated to the liposomes, followed by inoculation with aluminum hydroxide to thus create a reservoir.
However, it must be taken into account that the newest technological advances will not induce protection of the animal if the selected antigens are not adequate for the purpose.
Liposomes are lipid vesicles (Fig. 2). The external liposome membrane is composed of the same lipids as the cell membranes (green). This is very important, since the fact that the molecules used are not foreign to the host prevents the induction of immune rejection, and the same liposome formulation can be used for repeated vaccinations.
Any liposoluble antigen can be incorporated to the lipid membrane (red), while any hydrosoluble antigen can be included in the internal cavity of the liposome. This is very important, since other adjuvants are only useful for ensuring immune enhancement of proteins — and pathogens obviously also produce some other important antigens. On the other hand, it is also possible to incorporate some other liposoluble adjuvants to the liposome membrane (lipid A, lipopolysaccharide, etc.), or to the interior (MDP, cytokines, etc.). In addition, the liposome membranes can be made to fuse with the macrophage cell membrane, thereby delivering part of the antigen load directly to the cytosol fraction, with the resulting simultaneous induction of cellular and humoral responses.
The lipid composition of liposomes can also be modified, thereby changing the particle size; thus, a first population of small liposomes can deliver the antigen rapidly, while a second population of larger liposomes delivers antigen posteriorly. In fact, this is what happens with lyophilized liposomes, where following resuspension two liposome populations of different sizes are formed.
In order to protect against caries in humans, certain liposomes, that contain a capsular antigen of Streptococcus, are being investigated. However, in order to elicit a humoral response at mucosal level, the liposomes are administered included within a gelatin capsule via the oral route.
On our part, we are successfully testing the use of liposomes as an adjuvant to capsular antigens of Actinobacillus, Pasteurella, Streptococcus and Staphylococcus, and are initiating protocols to produce vaccines that can be used at the mucosal level (oral, nasal, rectal, lacrimal, vaginal, etc.).
Iscom are solid particles generated by combining an antigen with a biocompatible detergent and the adjuvant Quil-A, thus giving rise to minute structures (35 nm, Fig. 3). These particles can only be used with antigens that can be mixed with lipids and with Quil-A (normally proteins).
The same materials used in surgical sutures can be used as adjuvant. In vivo, these materials undergo more-or-less rapid non-enzymatic hydrolysis, depending on their composition, with release of the antigen in a gradual manner. However, their preparation implies the use of organic solvents that limit the type of candidate antigen to those that are both soluble and resistant to solvent action (i.e., only some proteins). As in the case of liposomes, the size of the microspheres can be modified, and it is even possible to include several of them within a larger microsphere.
The inoculation of small amounts of DNA (plasmids) in the host is being tested on an experimental basis, with good results. The underlying rationale is to allow the host cell to produce the protein (or fragment) encoded for by the DNA vaccine, without incorporating the vaccinal gene to its own chromosomes. The antigen thus produced is released and expressed on the cell surface, thereby inducing cellular and humoral responses.
The difficulty in this case is in obtaining a protein capable of affording protection and ensuring its correct expression within the host. For example, large proteins, toxins composed of various proteins, or glycosylated proteins are poorly expressed.
Some antigens, such as for example the capsular polysaccharides, are T cell independent. This essentially implies that they do not adopt the pathway described above (i.e., mediated by the MHC) for presentation by the macrophages to the T lymphocyte population. These antigens are recognized by the circulating B lymphocytes, which are partially activated as a result.
In order to overcome this obstacle, the capsular polysaccharide can be coupled to a transporter protein capable of binding to the MHC. This technology is widely used in human medicine in application to meningitis (Haemophilus influenzae and Streptococcus pneumoniae), but is rather expensive for application to veterinary medicine.
Alternatively, successful tests have been made to introduce these T-cell independent antigens in liposomes along with small T-cell dependent antigens, to in this way "trick" the immune system.
When a macrophage presents an antigen to a lymphocyte, or when a T helper lymphocyte recognizes a certain antigen, it releases a series of cytokines that enhance the ultimate immune response (i.e., antibody production and intervention of T cytotoxic cells). By administering these cytokines along with the antigen it is possible to achieve very similar effects. In addition, the released cytokines differ according to the responses induced (IgA, IgG, cytotoxicity); consequently, depending on the composition used, it is possible to direct the resulting immune response.
This technology is still in its infancy, however, since the truth is that we still know little of the mechanisms of action of the different cytokines. Moreover, cytokines are expensive, and some have a toxic potential. For this reason they may be useful in seriously ill patients where the result is important, e.g., in AIDS patients — but the approach is not advisable in children or healthy calves, for example.
Other substances act in a similar way (Quil-A, lipid A, MDP, etc.). These substances exert chemical action, favoring cytokine release by the inflammatory cells. If these substances are included in the vaccine formula, the immune response can be boosted in an unguided manner.
Empirical experience shows that much larger antigen doses are required to achieve immune responses when immunizing mucosal membranes. The reason for this is that antigens are eliminated due to the presence of mechanical barriers (epithelial cells) and chemical impediments (mucus), as well as by degradation and denaturalization as a consequence of enzyme and acid action. Moreover, the presence of pre-existing antibodies also contributes to eliminate the administered antigen. In this way, only small amounts of antigen actually reach the mucosal lymphoid tissues. A number of strategies have been designed in an attempt to overcome this problem:
- The antigens can be encapsulated in gelatin capsules in a way similar to the administration of certain drugs via the oral route. These capsules are effectively degraded by the alkaline pH found within the small bowel.
- The use of liposomes, Iscom and microspheres. This technology has been used adopting the intranasal, oral and intravaginal routes, etc.
- Conjugates with cholera toxin. The capacity of the toxin produced by Vibrio cholerae to bind to certain receptors of the intestinal epithelium is used for the immune enhancement of certain antigens that are administered together with the toxin or coupled to it.
- Live bacteria. The use of recombinant bacteria capable of colonizing the intestine (Salmonella, E. coli, Yersinia, etc.), or certain viruses (vaccinia, polio, rhinoadeno, mengo, etc.) expressing the antigen of choice is being widely tested as a vaccine. The system has clear advantages, for the use of these live vectors ensures immunity over a very long term — inducing humoral and cellular responses both at mucosal level and systemically. However, the responses obtained take place not only against the desired antigen but also against the vector, and the latter obviously dominates the associated immune response. This in turn prevents responses to other antigens with the same vector. Moreover, the antigens incorporated to the vector are limited to proteins, which pose problems for full expression, since their glycosylation is normally defective. Finally, the use of a vaccine with a certain vector precludes the use of another vaccine with the same vector, since as we have seen it would induce an intense response against the latter.
- Based on a similar idea, transgenic plants that express the desired antigen are also being considered as vectors