Immunization of dogs and cats with a DNA vaccine against rabies virus.

The applicability of DNA immunization technology for vaccine development in companion animals was investigated by immunizing dogs and cats by the intramuscular (i.m.) and intradermal (i.d.) routes with a plasmid DNA vector encoding the rabies virus glycoprotein G. In dogs, administration of 100 microg DNA doses by the i.m. route resulted in stronger and more durable rabies virus neutralizing antibody (RVNA) titers than those obtained by i.d. inoculation. In contrast, i.m. vaccination of cats with a similar dose was less effective in terms of mean titer and seroconversion frequency. However, efficacy was improved by increasing the dosage to 300 microg of DNA per immunization. Interestingly, i.d. inoculation of cats appeared to be a superior route of delivery in this species, resulting in higher seroconversion frequency than i.m. administration. In addition, geometric mean RVNA titers in i.d. inoculated cats increased over four-fold during a seven month period following a second and final immunization. These results demonstrate that non-facilitated, naked DNA vaccines can elicit strong, antigen-specific immune responses in dogs and cats, and DNA immunization may be a useful tool for future development of novel vaccines for these species.

Genomic organization of the canine herpesvirus US region.

Canine herpesvirus (CHV) is an alpha-herpesvirus of limited pathogenicity in healthy adult dogs and infectivity of the virus appears to be largely limited to cells of canine origin. CHV's low virulence and species specificity make it an attractive candidate for a recombinant vaccine vector to protect dogs against a variety of pathogens. As part of the analysis of the CHV genome, the authors determined the complete nucleotide sequence of the CHV US region as well as portions of the flanking inverted repeats. Seven full open reading frames (ORFs) encoding proteins larger than 100 amino acids were identified within, or partially within the CHV US: cUS2, cUS3, cUS4, cUS6, cUS7, cUS8 and cUS9; which are homologs of the herpes simplex virus type-1 US2; protein kinase; gG, gD, gI, gE; and US9 genes, respectively. An eighth ORF was identified in the inverted repeat region, cIR6, a homolog of the equine herpesvirus type-1 IR6 gene. The authors identified and mapped most of the major transcripts for the predicted CHV US ORFs by Northern analysis.

Nucleotide sequence and transcriptional analysis of a portion of the bovine herpesvirus genome encoding genes homologous to HSV-1, UL25, UL26 and UL26.5.

We determined the nucleotide sequence of a 3,811 base pair (bp) region of the bovine herpesvirus type-1 (BHV-1) HindIII-A fragment covering map units 0.429-0.456, which contained the HSV-1 homologues of the UL25, UL26 and UL26.5 genes. Sequence overlap was verified with previously published BHV-1 sequences, resulting in contiguous sequence spanning from UL24 to UL27 (encoding glycoprotein B). We found that the UL25 open reading frame (ORF) overlapped that of UL24 by 16 nucleotides, an unexpected feature based on previous herpesvirus sequences. We previously showed that the BHV-1 UL26 and UL26.5 genes encoded proteins that functioned in proteinase and scaffolding activity during capsid assembly, similar to the homologues in herpes simplex virus type-1. Northern blotting was used to map the four major transcripts from this region. As expected from the ORF analysis, the BHV-1 UL25 transcript initiated within the UL24 ORF. The BHV-1 UL24, UL25, UL26 and UL26.5 transcripts all terminated at a common 3'-polyadenylation site and varied significantly in their relative abundance.

The bovine parainfluenza virus type-3 (BPIV-3) hemagglutinin/neuraminidase glycoprotein expressed in baculovirus protects calves against experimental BPIV-3 challenge.

Despite the availability of numerous vaccine schedules, "shipping fever", an acute bronchopneumonia brought on in part by a complex of bovine respiratory viruses, remains a major source of economic loss in the beef and dairy industries. We are exploring new strategies of bovine vaccine design which we hope may provide more effective and more cost-efficient control of these pathogens. In this report, we examined the possible use of subunit vaccines, using as an example the hemagglutinin/neuraminidase (HN) protein of bovine parainfluenza virus type-3 (BPIV-3) expressed in the baculovirus expression system. We showed that the protein was expressed at high levels, and was modified to a similar, but not identical size as the native HN protein expressed from BPIV-3 infected bovine cells. We further demonstrated antigenicity and biological activity of the expressed HN protein. Finally, we vaccinated colostrum deprived sera-negative calves with the baculo HN recombinant protein and challenged with BPIV-3. Vaccination induced excellent serum neutralizing antibody responses, and surprisingly, good mucosal antibody responses, even though the vaccine was administered parenterally. The vaccinated animals were well protected against challenge.

The bovine herpesvirus 1 maturational proteinase and scaffold proteins can substitute for the homologous herpes simplex virus type 1 proteins in the formation of hybrid type B capsids.

We determined the nucleotide sequence of a 3.5-kb region of the bovine herpesvirus 1 (BHV-1) genome which contained the complete BHV-1 homologs of the herpes simplex virus type 1 (HSV-1) UL26 and UL26.5 genes. In HSV-1, the UL26 and UL26.5 open reading frames encode scaffold proteins upon which viral capsids are assembled. The UL26-encoded protein is also a proteinase and specifically cleaves both itself and the UL26.5-encoded protein. The overall BHV-1-encoded amino acid sequence showed only 41% identity to the HSV-1 sequences and was most divergent in the regions defined to be involved in the scaffolding function. We substituted the proteins encoded by the BHV-1 homologs of the UL26 and UL26.5 open reading frames, expressed in baculovirus, for the corresponding HSV-1 proteins in an in vitro HSV-1 capsid assembly system. The proteins expressed from the BHV-1 UL26 and UL26.5 homologs facilitated the formation of hybrid type B capsids indistinguishable from those formed entirely with HSV-1-encoded proteins.

The UL45 gene product is required for herpes simplex virus type 1 glycoprotein B-induced fusion.

Herpes simplex virus type 1 (HSV-1) syncytial (syn) mutants cause formation of giant polykaryocytes and have been utilized to identify genes promoting or suppressing cell fusion. We previously described an HSV-1 recombinant, F1 (J.L. Goodman, M. L. Cook, F. Sederati, K. Izumi, and J. G. Stevens, J. Virol. 63:1153-1161, 1989), which has unique virulence properties and a syn mutation in the carboxy terminus of glycoprotein B (gB). We attempted to replace this single-base-pair syn mutation through cotransfection with a 379-bp PCR-generated fragment of wild-type gB. The nonsyncytial viruses isolated were shown by DNA sequencing not to have acquired the expected wild-type gB sequence. Instead, they had lost their cell-cell fusion properties because of alterations mapping to the UL45 gene. The mutant UL45 gene is one nonsyncytial derivative of F1, A4B, was found to have a deletion of a C at UL45 nucleotide 230, resulting in a predicted frame shift and termination at 92 rather than 172 amino acids. Northern (RNA) analysis showed that the mutant UL45 gene was normally transcribed. However, Western immunoblotting showed no detectable UL45 gene product from A4B or from another similarly isolated nonsyncytial F1 derivative, A61B, while another such virus, 1ACSS, expressed reduced amounts of UL45. When A4B was cotransfected with the wild-type UL45 gene, restoration of UL45 expression correlated with restoration of syncytium formation. Conversely, cloned DNA fragments containing the mutant A4B UL45 gene transferred the loss of cell-cell fusion to other gB syn mutants, rendering them UL45 negative and nonsyncytial. We conclude that normal UL45 expression is required to allow cell fusion induced by gB syn mutants and that the nonessential UL45 protein may play an important role as a mediator of fusion events during HSV-1 infection.

Architecture of the vir regulons of group A streptococci parallels opacity factor phenotype and M protein class.

Group A streptococci have traditionally been categorized into two broad groups based on the presence or absence of serum opacity factor (OF). Recent studies show that these two groups vary in a number of properties in addition to the OF phenotype, including sequence variations in the constant region of the antiphagocytic M protein genes, the presence or absence of immunoglobulin G Fc receptor proteins, and the presence or absence of multiple M protein-like genes situated in a tandem array. The M protein genes (emm) in OF- streptococcal strains are known to be part of a regulon of virulence-related genes controlled by the trans-acting positive regulatory gene, virR, situated just upstream of emm. In OF+ strains, however, the region adjacent to virR is occupied by an M protein-related, type IIa immunoglobulin G Fc receptor gene (fcrA), and the relative position of emm has not been determined. To further define the vir regulon in OF+ streptococci, we used the polymerase chain reaction to show that fcrA49 is situated immediately upstream of emm49 in the OF+ type 49 strain CS101. This result shows for the first time the separate identity and genetic linkage of these two genes in the vir regulon of an OF+ group A streptococcal strain and confirms our previous hypothesis that emm49 exists as the central gene in a trio of emm-like genes. Additionally, using DNA hybridizations, we found considerable sequence divergence between OF- and OF+ group A streptococci in virR and in the noncoding sequences between virR and the emm or fcrA expression site. We found, however, a high degree of sequence conservation in this region within each of the two groups of strains.

Identification of a divergent M protein gene and an M protein-related gene family in Streptococcus pyogenes serotype 49.

The antigenically variant M protein of Streptococcus pyogenes enhances virulence by promoting resistance to phagocytosis. The serum opacity factor (OF), produced by a subset of M serotypes, is also antigenically variant, and its antigenic variability exactly parallels that of M protein. OF-positive and OF-negative streptococci are also phenotypically distinguishable by a number of other criteria. In order to study the differences between OF-positive and OF-negative streptococci, we cloned and sequenced the type 49 M protein gene (emm49), the first to be cloned from an OF-positive strain. This gene showed evolutionary divergence from the OF-negative M protein genes studied previously. Furthermore, emm49 was part of a gene family, in contrast to the single-copy nature of previously characterized M protein genes.

Immunoprotection of rats against Haemophilus influenzae type B disease mediated by monoclonal antibody against a haemophilus outer-membrane protein.

Infant rats passively immunised with a murine monoclonal antibody against a cell-surface-exposed outer-membrane protein of Haemophilus influenzae by b (Hib) were protected against systemic Hib disease induced by intraperitoneal injection of virulent Hib cells. The immunoprotection mediated by this monoclonal antibody was specific for Hib strains carrying the protein antigenic determinant that it recognised; the antibody gave no protection against disease when passively immunised animals were challenged with a heterologous Hib strain. The immunoprotection given by an antibody against a Hib outer-membrane protein suggests that Hib outer-membrane proteins have potential for development as Hib vaccines.