Claire Simard

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.

Experimental polyvalent ISCOMs subunit vaccine induces antibodies that neutralize human and bovine respiratory syncytial virus

The purpose of the present study was to evaluate experimentally, in guinea-pigs, the immunogenicity of respiratory syncytial (RS) virus subunit vaccines. Immunostimulating complexes (ISCOMs), made from the surface proteins of both human (Long) and bovine (A-51908) RS strains adsorbed to the adjuvant Quil A, were assayed for their capacity to induce neutralizing antibodies, in comparison to experimental live virus vaccines. Serums from animals vaccinated with either the human or bovine RS subunit vaccines were equally efficient in neutralizing human or bovine RS virus. ISCOMs prepared with bovine RS virus proteins were significantly (p less than 0.05%) more efficient than their human counterpart, in inducing neutralizing antibodies, suggesting their greater potential as a subunit vaccine.

Evidence that the amino acid region 124–203 of glycoprotein G from the respiratory syncytial virus (RSV) constitutes a major part of the polypeptide domain that is involved in the protection against RSV infection

The first 230 residues of the 298-amino acid glycoprotein G of respiratory syncytial virus (RSV) are sufficient to confer complete resistance to challenge with live RSV, whereas the first 180 residues completely failed (Olmsted et al. (1989) J. Virol. 63, 411-420). The characterization of a protective epitope corresponding to the amino acid region 174-187 of the G protein (Trudel et al. (1991) Virology 185, 749-757) suggests that interruption of this region in the 180 residue truncated polypeptide may be responsible for its inability to confer protection and consequently that the 174-187 region may play a major role in the protection effected by the protein G. To support these hypotheses, we examined the ability of the amino acid region 124-203 of glycoprotein G to confer protection. The corresponding peptide was expressed as a non-fusion protein in a recombinant vaccinia virus designated VG27. Immunization of BALB/c mice with this recombinant efficiently induced the production of antibodies capable of recognizing both the parental glycoprotein G and peptide 174-187. Furthermore, upon challenge with RSV, a significant decrease of infectious particles was found in the lungs of mice immunized with VG27 as compared with non-immunized mice. Our results suggest that the 124-203 amino acid region of the RSV G protein constitutes a major part of the domain involved in protection.

Synthetic peptides corresponding to the F protein of RSV stimulate murine B and T cells but fail to confer protection

SummaryWe have previously located a major neutralization site of the fusion protein of respiratory syncytial virus (RSV) in the polypeptide region extending from amino acids Ile221 to Glu232. In this report, 8 peptides corresponding to the six major hydrophilic regions of the F1 subunit were selected to analyse their immunogenic and protective capacities as well as their ability to block the high neutralization activities of 4 monoclonal antibodies (MAbs). Only 5 of the 8 peptides tested induced specific antibodies while all induced an in vitro interleukin-2 response of splenocytes from immunized mice. Peptide 3 (Ile221-Phe237) was able to elicit neutralizing antibodies, confirming our previous hypothesis concerning the location of a neutralization site. However, immunization with the latter did not induce significant reduction of virus in lungs of BALB/c mice upon challenge, probably due to an inadequate level of circulating neutralizing antibodies. Interestingly, peptides 2 (Asn216-Glu232), 3 (Ile221-Phe237), and 5 (Ser275-Ile288) blocked in vitro neutralization by four different F1 specific MAbs. A hypothesis is proposed to explain these results.

Immunization with a peptide derived from the G glycoprotein of bovine respiratory syncytial virus (BRSV) reduces the incidence of BRSV-associated pneumonia in the natural host.

Previous reports demonstrate that synthetic peptides corresponding to the amino acid region 174-187 of G glycoprotein from subgroups A and B human respiratory syncytial virus (HRSV), containing a Cys-->Ser substitution at position 186, confer complete resistance to immunized BALB/c mice against infection with the respective virus. In this report, we show that a Cys186-->Ser substituted peptide (BG/174-187) representing the corresponding region of the bovine (B) RSV G glycoprotein conferred complete protection of mice against BRSV challenge, suggesting that the 174-187 region of RSV G glycoproteins constitutes a dominant protective epitope which has been maintained throughout evolution. Furthermore, immunization of calves with peptide BG/174-187 efficiently induced the production of antibodies capable of recognizing both the parental G glycoprotein and peptide BG/174-187. Following challenge with live BRSV, although none of the animals were protected from upper respiratory tract disease, there were little or no gross pneumonic lesions in the four peptide-immunized calves. In contrast, moderate to extensive pneumonic lesions were observed in 2 out of 3 calves in the control group. Our results thus suggest that peptide BG/174-187 efficiently prevented BRSV-associated pneumonia in the natural host. The use of this system as a model is quite promising with regard to the development of a human synthetic vaccine.

Comparison of caprine, human and bovine strains of respiratory syncytial virus

SummaryA new continuous ovine kidney cell line allowing the growth of caprine, human and bovine respiratory syncytial virus was used to minimize host cell related variations for the direct comparison of the viral ultrastructures, serological relationships and structural protein profiles. Results show that all three strains are closely related although a closer relationship was found between bovine and caprine RS.

Gene contents in a 31-kb segment at the left genome end of bovine herpesvirus-1

We report the nucleotide sequence of a 31-kb segment at the left genome end of bovine herpesvirus-1 (BHV-1) and show that it comprises 19 different open reading frames (ORFs), including seven which have been described previously (circ, dUTPase, UL49.5, alpha TIF, VP8, glycoprotein C, and ribonucleotide reductase small subunit). The new sequence resulted in a correction at the C-terminus of glycoprotein C. All 19 ORFs exhibited strong amino acid sequence homology to the gene products of other alphaherpesviruses. The BHV-1 ORFs were arranged colinearly with the prototype sequence of herpes simplex virus 1 (HSV-1) in the range of the UL54 to UL37 genes. No BHV-1 homologs of the HSV-1 UL56, UL55, and UL45 genes were identified. The BHV-1 circ gene was the only gene without a HSV-1 counterpart. The additional ORFs 1 and 2 found at the left genome end of equine herpesvirus-1 (EHV-1) were absent in BHV-1. Among the newly sequenced BHV-1 ORFs are homologs of ICP27 (UL54), glycoprotein K (UL53), helicase-primase (UL52), DNA polymerase accessory protein (UL42), ribonucleotide reductase large subunit (UL39), and several virion proteins (UL49, UL46, UL43, UL41, UL38, UL37), most of which are strongly conserved in all herpesviruses. The possible functions of the proteins encoded within the sequenced region are assessed and features found are discussed.

Gene mapping of infectious bovine rhinotracheitis viral DNA genome.

SummaryA bovine herpesvirus I (BHV-I)HindIII genomic bank spanning 89% of the entire genome was constructed and individual fragments analyzed for their capacity to select specific mRNAs which were then expressed by in vitro translation assays. This procedure allowed the mapping of more than 20 viral polypeptides to discrete regions of the DNA genome. Some polypeptides map in neighboringHindIII fragments while most seem encoded in single fragments. In particular, the coding sequences for an abundant 94 kDa polypeptide, which is the potential unglycosylated precursor of gII glycoprotein, have been assigned to the small 3.6 kbpHindIII genomic fragment M. The localization of structural and non-structural gene-coding sequences will help to characterize viral polypeptides and eventually, a better understanding of BHV-I infection will be gained.

Sequencing and 5′- and 3′-end transcript mapping of the gene encoding the small subunit of ribonucleotide reductase from bovine herpesvirus type-1

The complete nucleotide sequence of the gene encoding the small subunit of ribonucleotide reductase (RNR) from bovine herpesvirus type-1 (BHV-1) was determined. The genomic DNA fragment sequenced also represented regions corresponding to the carboxy termini of RNR large subunit and of a virion protein causing host shut-off. The small subunit polypeptide was constituted of 314 amino acid residues totalling 35.25 kDa. The major transcription initiation and termination sites of the small subunit mRNA were located 95 bases upstream and 88 nucleotides downstream from the coding region, respectively. These findings indicate that the mRNA was 1128 bases long which correlated well with the size of the polyadenylated transcript detected in Northern blot analysis (1.3 kb). Within the RNR large subunit coding region, a TATA box and two CAAT box motifs were found 26, 104, and 190 nucleotides, respectively, upstream from the transcription initiation site of the small subunit mRNA. In contrast to previous studies (Slabaugh et al., J. Virol. 1988, 62, 519-527; Boursnell et al., Virology 1991, 184, 411-416), our comparative analysis of five herpesviruses, one iridovirus, and one poxvirus small subunit protein sequences suggested that the seven viruses arose from a common lineage.

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.