Synthetic approaches to vaccine development against cholera-like heat-labile proteins
The role of synthetic adjuvants for enhancement of vaccines
Chairperson: I.F. Zavalla
A wide range of procedures has been developed to convert synthetic peptides into efficient immunogens. Several synthetic peptides have been produced corresponding to pieces of the B subunit of cholera toxin. The information necessary for peptide synthesis was derived from the amino-acid sequence of the B subunit and the choice of which peptides to synthesize was based on the known involvement of pieces of the B subunit in the biological activities of the intact toxin. The peptides were conjugated to tetanus toxoid (TT) with a number of synthetic carriers, with palmitic acid, or were polymerized with gluteraldehyde and injected into rabbits. Conjugates with TT, TGAL, palmitic acid and polymerized peptides were all good immunogens and elicited efficient anti-peptide antibodies.
The anti-peptide antibodies were screened for activity against the native molecule. Reactions were observed but no clear rules were found to account for the relationship between the titre of antibodies against either the peptide or the native molecule. The antibodies were also screened for their ability to block the biological activities of cholera toxin. The systems used were inhibition of cholera toxin adenylate cyclase and inhibition of toxin-induced fluid accumulation in isolated loops of rat intestine. Two of the peptides induced inhibiting antibodies and the most efficient was chosen for vaccine development.
The chosen peptide was also examined for its ability to elicit antibody responses which inhibit the biological effects of heat-labile toxins related to cholera toxin, e.g. Escherichia coli toxin, porcine-labile toxin, and heat-labile toxins from vibrios such as Vibrio minicus toxin. Once again the test used was inhibition of toxin-induced fluid accumulation in isolated gut loops. A peptide was chosen which induced broad protection.
Having identified the appropriate peptide, methods are being developed for its large-scale preparation. The chosen method generates synthetic DNA which encodes the peptide. The DNA has been inserted into the expressing vectors pAM1 and pAM2 which have many favourable properties, i.e. they support the production of the peptide fusion protein, transport that peptide to the surface and express, it without degradation. Both vectors expressed a fusion protein which was recognized by antibodies against the original synthetic peptide.
The fusion protein was injected into rabbits and was found to induce good antibody responses. The antibodies reacted with the synthetic peptide and neutralized biological activity of cholera toxin in the gut loop system. It appears that first steps in the production of a synthetic vaccine have now been made.
Points arising from the discussion
Discussion revolved around several general themes. How can a vaccine be developed which will induce antibody responses of a selected isotype? What carriers should be used with synthetic or genetically engineered peptides in order to avoid epitope-specific suppressions or rapid carrier clearance brought about by previous exposure of the host to the carrier? What adjutants should be used to promote efficient and safe responses? Many specific eases were discussed for each of these questions but no general rules were identified.
In immunization against infectious agents adjuvants are called upon to potentiate immune responses to protective antigens. This may take the form of increasing the magnitude of the response or changing the nature of the response, e.g. by biasing towards a particular immunoglobulin isotype or towards cell-mediated rather than humoral responses or vice versa. The availability of effective and safe adjuvants is particularly important when considering vaccines based on peptide subunits, since such small molecules on their own are invariably poorly immunogenic.
It was pointed out that the criteria for an adjuvant to be acceptable for use in animals are somewhat different from those that apply in humans. The primary considerations in using adjuvants in humans are safety and absence of side effects, whereas cost is a major factor in animals and minor side effects may be acceptable.
Overall, the most effective adjuvant for both antibody and cell-mediated immune responses in experimental situations is complete Freund's adjuvant (CFA). However, because of the undesirable reactions which this adjuvant provokes at the site of inoculation, it is not acceptable for use in vaccines. In addition it sensitizes to tuberculin which mitigates against its use in cattle. Studies on adjuvants were based mainly on the use of the peptidoglycan, muramyl dipeptide (MDP), which is the minimal chemical structure from the mycobacterium in CFA which will provide adjuvant activity. When MDP was incorporated into incomplete Freund's adjuvant it was found to replace completely the adjuvant activity of the mycobacterium.
MDP was also found to have adjuvant activity when used alone without oil. Furthermore, while inoculation of the adjuvant along with antigen was found to give optimal results, some adjuvant effect was observed when the adjuvant was administered at sites distant from the antigen (including the oral route). One disadvantage of MDP is that it has pyrogenic activity. However, by a simple chemical substitution, a derivative known as murabutide has been produced which is devoid of pyrogenic activity but has retained adjuvanticity. Several examples were presented which illustrate the potent adjuvant activity of this compound. These include one study which used 1 microgram of hepatitis B virus surface antigen. This dose of antigen on its own was poorly immunogenic, whereas a strong response was elicited when given with 100 micrograms of murabutide. The simultaneous use of murabutide plus alum resulted in complementation of the activity of the two adjuvants and, interestingly, resulted in a very low specific IgE response which was induced by antigen plus alum alone.
The use of peptides in immunization was considered, focusing particularly on the requirements for adjuvants and for carrier molecules when dealing with small peptides. Examples of peptides used included a subunit of the diptheria toxin and a peptide from the malaria circumsporozoite antigen. With the latter, murabutide was shown to be as effective as CFA, although the antibody elicited by this particular peptide reacted very weakly with the parent molecule. Nevertheless these antibodies were capable of inducing the circumsporozite protein precipitation which correlates with protective activity.
Small peptides (haptens) for use in immunization have to be linked to a carrier molecule to induce a significant antibody response. The potential problems accompanying the use of carriers were outlined. These include the possibility of hypersensitivity to the carrier molecule, rapid clearance of the hapten-carrier complex by a predominance of anti-carrier antibody after repeated boosts and induction of suppression to specific hapten epitopes. One approach to overcome some of the problems is to use polymerized haptens as carriers. Data were presented on the use of a foot-and-mouth disease virus peptide with the peptide itself used in polymerized form. This was administered either as a mixture with MDP or covalently linked to MDP-lysine. Both procedures resulted in strong biologically active antibody responses, although the MDP-conjugated antigen was superior. A further experiment involved testing a polymerized polypeptide prepared from four different peptide units. When given with either CFA or murabutide, this resulted in good immune responses to all four peptide components. Protective activity was demonstrated in the ease of diphtheria toxin and CS protein. In addition mice were challenged by a lethal dose of streptococci and were shown to be protected. The antibody response to the peptide could not be checked for protective activity. It was pointed out, however, that some peptides in polymer form do not induce easily detectable antibody responses to the native peptide. Finally, it was demonstrated that immunological castration of male mice could be achieved by administering LH-releasing hormone coupled to MDP-lysine (three doses).
Points arising from the discussion
These studies provided extremely valuable information on the efficacy of chemically defined adjuvants when used with a range of well-defined antigens presented in a number of different ways. Further studies are required to determine whether these compounds can be used effectively in domestic animals in quantities which are not financially prohibitive. One pilot study has been conducted using murabutide in humans with encouraging results. It was clear from the discussion that more information is required on the isotypes of antibody induced using these adjuvants and the duration of serum antibody and immunological memory. Persistence of significant levels of serum antibody is an important factor when considering immunity to Theileria sporozoites and would require the presence of antibody at the time of challenge.
Despite the many studies carried out on adjuvants, very little is known about their mode of action. There are indications that MDP and murabutide exert their effect on macrophages, possibly by increasing the release of immunological mediators. Further information on the mode of action of existing adjutants might provide a more rational basis on which to develop new compounds.