Francoise Audibert

Influence of helical organization on immunogenicity and antigenicity of synthetic peptides

Using cooperative effects of different peptide structures synthesized in tandem, we have induced an alpha-helical structure in water solution on a peptide which, alone, is unorganized. This structure is particularly relevant in this case as the selected model protein (type 24 M protein of Streptococcus pyogenes) is an extended coiled-coil system. We were thus able to assess the importance of organization or unorganization of a unique amino acid sequence with regards to its immunogenicity and antigenicity. Although in a classical manner, antibodies cross-reacting with the protein can be obtained with the short, unorganized peptide, we demonstrate that conformation-specific antibodies are raised when longer, organized peptides are used as immunogens.

Epitope specific immunity elicited by a synthetic streptococcal antigen without carrier or adjuvant

A polypeptide fragment of type 24 streptococcal M protein (pep M24) has been shown to raise protective anti-streptococcal antibodies in rabbits and humans when administered with adjuvants. More recently, such protective antibodies were shown to be evoked by a synthesized 35-residue sub-peptide fragment (S-CB7 synthetic cyanogen bromide fragment 7) of pep M24. We now show that the weak pep M24 immunogen induces high titers of long lasting antibodies when associated with murabutide, a synthetic derivative of MDP (NAcMur-L-Ala-D-Gln-n-butyl-ester) which is currently undergoing clinical trials. We demonstrate also that the polymerized synthetic S-CB7 administered without adjuvant or carrier evokes a strong epitope specific, protective immune response in mice primed with the parent pep M24. A booster dose of polymerized S-CB7 induced antibodies directed specifically against the S-CB7 structure whereas a booster dose of pep M24 evoked antibodies recognizing additional determinants of the whole pep M24 molecule.

Polyvalent synthetic vaccines: relationship between T epitopes and immunogenicity.

Three different synthetic polyvalent vaccines have been constructed by conjugating four synthetic peptides without any carrier protein. The peptides were copy fragments of two bacterial antigens (Streptococcus pyogenes M protein and diphtheria toxin), two parasitic antigens (circumsporozoite protein of Plasmodium falciparum and Plasmodium knowlesi), and one viral antigen (hepatitis B surface antigen). Outbred guinea-pigs immunized with polyvalent vaccine containing streptococcal, diphtheric, P. knowlesi and hepatitis peptides raised high specific antibody response against the four specificities. Individual T cell analysis demonstrated that hepatitis peptide bears T dominant epitope. A similar immune response was obtained with a second polyvalent vaccine where the P. knowlesi peptide had been replaced by the P. falciparum peptide. In both experiments the malarial peptides behave like pure B epitopes. Prediction of immunodominant helper T-cell antigenic sites were performed with the five peptides using computer algorithm. Hepatitis and diphtheric peptides were selected whereas the streptococcal peptide was rejected although it can experimentally contain a T epitope. To confirm this result animals were immunized with a third polyvalent vaccine which does not contain the hepatitis peptide. No T cell proliferation or antipeptide antibodies were detected. These results demonstrate that the cooperative immune response requires a certain degree of antigenic complexity for the induction of antibody response.

Protection of ruminants by Pasteurella haemolytica A1 capsular polysaccharide vaccines containing muramyl dipeptide analogs

The capsular polysaccharide (CP) of Pasteurella haemolytica serotype A1 is a poor immunogen for the prevention of pneumonic pasteurellosis of ruminants. To improve CP immunogenicity, vaccines were prepared with 1.0 mg CP dose-1 with and without the synthetic adjuvant, muramyl dipeptide (MDP; range 0.2-1.0 mg) or a lipophilic derivative, MDP-sn-glyceryl-dipalmitoyl (MDP-GDP; range 0.1-1.0 mg). The optimum effective concentration of adjuvant was first determined in lambs and calves and then the efficacy of CP +0.5 mg MDP and CP +1.0 mg MDP-GDP was compared with that of two commercial vaccines in calves. After immunization with CP, antibody titers in lambs and calves were typical of that seen with polysaccharide immunogens and characterized by an early IgM response followed by later IgG1 and IgG2 responses. CP + MDP or MDP-GDP vaccines induced significantly higher IgM, IgG1, and IgG2 titers. After transtracheal challenge of immunity with P. haemolytica serotype A1, extensive pulmonary consolidation containing P. haemolytica (10(6)-10(8) c.f.u. g-1) was seen in all lambs and calves vaccinated with CP alone and was not significantly different (P < 0.05) from the consolidation and concentrations of organisms in nonvaccinated challenge controls. In lambs, vaccines containing 1.0 mg CP +0.05 mg MDP or MDP-GDP significantly reduced pulmonary consolidation and concentrations of P. haemolytica in lung lesions. In calves, vaccines containing 0.2 mg MDP, 0.5 mg MDP, or 1.0 mg MDP-GDP also significantly reduced pulmonary consolidation and concentrations of P. haemolytica in lung lesions. Vaccines containing CP +0.5 mg MDP and CP +1.0 mg MDP-GDP induced high titer bactericidal antibodies by 7 days and were more efficacious than two commercial vaccines. Potentiation of CP with MDP or MDP-GDP has great promise in furthering the potential of CP as a vaccine immunogen for the prevention of pneumonic pasteurellosis.

Production of antibodies recognizing a hepatitis B virus (HBV) surface antigen by administration of Murabutide associated to a synthetic pre-S HBV peptide conjugated to a toxoid carrier

The influence of Murabutide, a muramyl peptide analog, on the response of mice to two synthetic hepatitis B virus antigens was compared with the activity of Al (OH)3 and of FCA. The synthetic peptides represented fragments either of the major component of the hepatitis B surface antigen (HBsAg) or of the pre-S region. They were used either conjugated to toxoid carrier or as a totally synthetic preparation, i.e. copolymerized with a streptococcal peptide. Our results demonstrated that treatment with Murabutide increased the levels of antibodies, modulated their specificity and allowed a better recognition of the natural antigens.

Construction of immunogens for synthetic malaria vaccines

The immunogenicity of a peptide consisting of eight repeats of the tetrapeptide sequence NANP (Asn-Ala-Asn-Pro) contained in the circumsporozoite protein of Plasmodium falciparum was investigated in mice under different modes of presentation. This peptide was able to produce biologically active antibodies when administered with adjuvant and linked to a protein carrier. However, a (NANP) peptide polymerized by carbodiimide was found to be immunogenic in the absence of protein carrier in H-2b mice. In contrast, the (NANP)8 peptide polymerized by glutaraldehyde was not immunogenic in the same strain. Furthermore, the efficacy of murabutide in saline, as an immunological adjuvant, was compared to the efficacy of Freunds complete adjuvant.

Covalent linkage of the synthetic adjuvant MDP to the synthetic polypeptide (T,G)-A—L changes the specificity of the immune response at the T and B cell level☆

It is now well established that the synthetic molecule MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine) can be a good adjuvant of immunity when covalently linked to antigen. The question raised in this work is whether conjugation of antigen to the immunomodulatory molecule MDP can modify the specificity of the antibodies and T cells induced following immunization. Using the well characterized synthetic polypeptide antigen, poly(L-Tyr,L-Glu)-poly(DL-Ala)--poly(L-Lys) [(T,G)-A--L], we show that immunization of C57B1/6 (H-2b) mice with MDP-(T,G)-A--L conjugate elicits at least two types of antibody directed against the poly(DL-Ala)--poly(L-Lys) (A--L) part of the antigen, and against new determinant(s) formed by MDP and a portion of the (T,G)-A--L molecule. Interestingly, the poly-(L-Try L-Glu) side chains thought to constitute the major antigenic determinants of the (T,G)-A--L molecule were not recognized. Lymph node cells from (T,G)-A--L immunized mice can be equally well stimulated in vitro by (T,G)-A--L or by MDP-(T,G)-A--L, whereas lymph node cells from MDP-(T,G)-A--L primed animals can be stimulated only when challenged by the conjugate used for immunization, and not by the free synthetic polypeptide (T,G)-A--L. The data presented here show that the coupling of a low mol. wt molecule such as MDP (mol. wt approx. 500) to an antigen can greatly modify the immune response directed against this antigen. Furthermore, (1) different antibody specificities are elicited depending upon whether the priming is done with free MDP and antigen or with MDP covalently linked to the antigen; (2) although still accessible on the conjugate, an epitope which represents the major antigenic determinant on the free polypeptide appears to be silent when presented on the conjugate; and (3) new determinant(s) formed by the chemical linkage of the polypeptide to the synthetic adjuvant are involved in the priming of T cells.

Modulation of the Immunogenic Characters of Protective Epitopes in Synthetic Vaccines

Protective antigenic determinants can be mimicked by chemical synthesis. To become part of efficient vaccines B and T epitopes must be inserted in appropriate structures so as to elicit a strong, long lasting protective immune response. To achieve this goal synthetic peptides can be either linked to a carrier or, preferably associated within totally synthetic constructions. Adjuvants such as synthetic muramyl dipeptides can afford tools not only to enhance the level of the immune response but also to modulate its characters. These points will be illustrated using synthetic malarial vaccine models and a totally synthetic polyvalent vaccine containing B and T epitopes. Finally, a model of prevention against experimental allergic encephalomyelitis will be discussed to illustrate how the immunogenic characters of a synthetic antigen can be modulated by association with adjuvants or by suitable coupling.