Michel Trudel

Identification of a Synthetic Peptide as Part of a Major Neutralization Epitope of Respiratory Syncytial Virus

A 7000 Mr cleavage fragment of the F1 subunit that carries the major neutralization epitope has been identified by chemical and enzymatic cleavage of the fusion protein of respiratory syncytial (RS) virus (Long strain) with an efficient RS virus-neutralizing monoclonal antibody. Based on the published mRNA-deduced sequence of the A2 strain, coupled to the hydropathicity profile and prediction of protein conformation, the neutralization epitope has tentatively been localized on the first third of the F1 protein N-terminal, probably in the region of amino acids 215Ser to 236Glu. Analysis of three peptides covering different portions of the 212Cys to 236Glu region of the F1 fusion protein identified a peptide (Cys X 216Asn to 236Glu) that reacted strongly with the neutralizing monoclonal antibody and that was efficient in blocking neutralization and in plaque-reducing assays, confirming that the neutralization epitope was localized in that region. Further analysis with two other synthetic peptides (212Cys to 222Glu and Cys X 221Ile to 236Glu) indicated that the dodecapeptide Ile-Glu-Phe-Gln-Lys-Asn-Asn-Arg-Leu-Leu-Glu mimicked either the whole or a major part of the neutralization epitope. This opens a promising avenue for the simple design of a synthetic peptide vaccine to control RS virus infection.

Complete protection of mice from respiratory syncytial virus infection following mucosal delivery of synthetic peptide vaccines

We have previously shown that intraperitoneal immunization of BALB/c mice with the 14 amino-acid long synthetic peptides G/174-187 and BG/174-187, representing the region 174-187 of the G-glycoprotein from human (H) and bovine (B) respiratory syncytial virus (RSV), respectively, completely protects animals from infection with the corresponding virus. A current goal in vaccine development being the delivery of noninvasive protective antigens via mucosal surfaces, we have evaluated the immunogenicity and protective efficacy of the two peptides when administered to mice by the intranasal (i.n.) route in the presence or absence of the cholera toxin (CT) as a mucosal adjuvant. The two peptides given alone induced the production of RSV-specific circulating IgG, as revealed by ELISA titers of immune sera. When the peptides were administered intranasally with CT, the higher IgG antibody titer which was induced was within the same order of magnitude as that obtained following i.n. immunization with live RSV or intraperitoneal injection with the peptides, thus demonstrating the stimulatory effect of the CT adjuvant. Moreover, although the peptides fail to induce a detectable level of secretory IgA, all animals immunized i.n. with peptide BG/174-187 (plus or minus CT) and all those immunized with peptide G/174-187 mixed with CT were completely resistant to infection by the corresponding virus. To our knowledge, this is the first study reporting that complete protection against a natural pathogen can be elicited by mucosally delivered synthetic peptides. This supports the usefulness of synthetic peptides in prophylactic vaccination.

Subgroup specific protection of mice from respiratory syncytial virus infection with peptides encompassing the amino acid region 174–187 from the G glycoprotein: the role of cysteinyl residues in protection

We identified subgroup specific protective epitopes represented by the amino acid regions 174-187 and 171-187 of the G glycoproteins from respiratory syncytial virus (RSV), subgroups A and B. Mice immunized with coupled synthetic peptides corresponding to either the region 174-187 containing a Cys186-->Ser substitution or to the native region 171-187 were completely resistant to RSV infection but only to the respective virus. The protective activities of the peptides 174-187 were dependent on the Cys186-->Ser substitution. In addition, a recombinant protein representing the region 125-203 of the A subgroup G glycoprotein expressed in Escherichia coli was capable without further treatment to completely protect animals against RSV subgroup A infection. We show that the combinations of cysteinyl residues (positions 173, 176, 182, and 186) retained within either synthetic peptides or the recombinant protein G125-203 greatly influenced their protective activities. This indicates that the region 171-187 is essential for the protection conferred by the G125-203 protein. Furthermore, our results strongly suggest that the peptides and recombinant proteins potencies are a function of a loop-like structure which is stabilized by intramolecular disulfide linkages between Cys176-Cys182 and Cys173-Cys186. This is further supported by the observation that chemical blocking of the sulfidryl groups in synthetic peptides completely eliminated their protective activity.

Detection of antibodies to individual proteins of rubella virus

Individual rubella virus structural polypeptides were electroeluted from SDS-polyacrylamide gels. The eluted polypeptides were used, without further purification, as antigens in ELISA assays for the detection of rubella-specific antibodies in patients sera. This provided a more sensitive detection method than that involving classical serological assays such as HI or VN or that using immunoprecipitation. Antisera against individual viral polypeptides were raised in mice. No haemagglutination inhibition activity was observed in any of these sera and weak virus neutralizing activity was only detected with antiserum to the E1 protein. Antisera to either the E1 or E2(a,b) complex proteins cross-reacted with both the E1 and E2(a,b) complex proteins.

Characterization and transcript mapping of a bovine herpesvirus type 1 gene encoding a polypeptide homologous to the herpes simplex virus type 1 major tegument proteins VP13-14

Using in vitro translation of hybrid-selected mRNA, we have previously shown that bovine herpesvirus type 1 HindIII fragment M encodes an abundant 94K polypeptide. Using immunoprecipitation and sequencing analyses, it has now been shown that the polypeptide is related to the major tegument protein VP8 and is homologous to the herpes simplex virus type 1 major tegument proteins VP13/14. The sequence of the VP8 gene (field isolate 34) is reported and compared to published data. Several differences between the sequences were detected, resulting particularly from base insertions/deletions generating three major frameshifts affecting an area of 87 amino acid residues of the encoded protein. In addition, sequence comparison revealed 29 single base alterations, excluding frameshift regions, producing 17 amino acid substitutions. Overall, 14.1% of the deduced amino acid sequences were divergent. We have also established that the last 152 nucleotides of the previously reported sequence correspond to the sequence of the minus not the sense strand. Finally, we report that the 4.4 kb transcript of the VP8 gene is initiated 39 nucleotides upstream from the translation start codon.

Electron microscopic evidence for bridges between bovine respiratory syncytial virus particles.

Electron microscopic examination of ultrathin sections of a continuous cell line of ovine kidney (OK) origin, infected by bovine respiratory syncytial virus (BRSV), revealed the presence of well defined bridges between virus particles. This is the first report of this novel structure. Observation of ultrathin sections of human RSV Long strain also grown on OK cells did not show inter-particle bridges and therefore suggested that this structure could be specific to BRSV. The biological significance of these bridges is not clear at this time; a possibility is that the bridges are formed by the fusion protein of BRSV which is known to cause cell fusion. Besides the structural implications, the importance is in relation to purification strategies for this virus, which must now take into account that most of the viral particles occur in large aggregates.

Modified immunoprecipitation procedure for the identification of human respiratory syncytial virus polypeptides.

When analysed by polyacrylamide gel electrophoresis, human respiratory syncytial virus harvested after a one step growth cycle and purified through a continuous sucrose density gradient was shown to be composed of nine structural proteins of 90, 68, 49, 42, 34, 28, 25, 19 an 13 kd. The 90, 49 and 19 kd polypeptides were identified as glycopolypeptides by glucosamine incorporation. A modified immunoprecipitation procedure confirmed the viral specificity of the 49, 42, 28, 25 and 19 kd polypeptides.