Bacterin that induces anti-OspA and anti-OspC borreliacidal antibodies provides a high level of protection against canine Lyme disease.

Groups of 15 laboratory-bred beagles were vaccinated and boosted with either a placebo or adjuvanted bivalent bacterin comprised of a traditional Borrelia burgdorferi strain and a unique ospA- and ospB-negative B. burgdorferi strain that expressed high levels of OspC and then challenged with B. burgdorferi-infected Ixodes scapularis ticks. The vaccinated dogs produced high titers of anti-OspA and anti-OspC borreliacidal antibodies, including borreliacidal antibodies specific for an epitope within the last seven amino acids at the OspC carboxy terminus (termed OspC7) that was conserved among pathogenic Borrelia genospecies. In addition, spirochetes were eliminated from the infected ticks that fed on the bacterin recipients, B. burgdorferi was not isolated from the skin or joints, and antibody responses associated specifically with canine infection with B. burgdorferi were not produced. In contrast, B. burgdorferi was recovered from engorged ticks that fed on 13 (87%) placebo-vaccinated dogs (P<0.0001), skin biopsy specimens from 14 (93%) dogs (P<0.0001), and joint tissue specimens from 8 (53%) dogs (P=0.0022). In addition, 14 (93%) dogs developed specific antibody responses against B. burgdorferi proteins, including 11 (73%) with C6 peptide antibodies (P<0.0001). Moreover, 10 (67%) dogs developed Lyme disease-associated joint abnormalities (P<0.0001), including 4 (27%) dogs that developed joint stiffness or lameness and 6 (40%) that developed chronic joint inflammation (synovitis). The results therefore confirmed that the bacterin provided a high level of protection against Lyme disease shortly after immunization.

Hybridization analysis of the gene encoding a hemolysin (suilysin) of Streptococcus suis type 2: evidence for the absence of the gene in some isolates.

A hemolysin gene was cloned from a virulent strain of Streptococcus suis type 2 strain 1933. Analysis of the gene and its product revealed that it is identical to a previously reported hemolysin (suilysin) of S. suis type 2. Southern hybridization analysis of the digested total genomic DNA from S. suis with the cloned hemolysin DNA sequences as probe indicated that the hemolysin gene is present as a single copy on the genome. Genomic DNA of 63 isolates of S. suis encompassing all known serotypes were examined by DNA hybridization and polymerase chain reaction (PCR) studies for the presence of the hemolysin gene homolog. The results of both techniques were identical and demonstrated the absence of the hemolysin gene in some isolates. In DNA hybridization studies, three DNA probes derived from the hemolysin encoding gene were used. Results showed that sequences encoding the C-terminal 257 amino acid residues (Probe 1) were the most conserved and hybridized to a 1.2 kb fragment in 32 (51%) strains and a 4.0 kb fragment in 23 (36%) strains respectively. Thus, Probe 2 hybridized to the DNA of 55 (87%) of the isolates tested. The first probe (Probe 1) comprising almost the entire hemolysin gene and the third probe (Probe 3) which consisted of the N-terminal sequences hybridized only to a 4.0 kb fragment in 23 (36%) of the strains tested. Eight (13%) of the strains tested were hybridization and PCR negative. The hybridization of the C-terminal end sequences (Probe 2) to the 1.2 kb fragment in 32 (51%) of the strains and the lack of hybridization of the probes to eight (13%) strains may suggest the presence of different types of hemolysin molecule in S. suis strains.

Expression and cellular distribution of baculovirus-expressed bovine herpesvirus 1 (BHV-1) glycoprotein D (gD) sequences.

Glycoprotein D (gD) of bovine herpesvirus 1 (BHV-1), a homolog of herpes simplex virus gD, represents a major component of the viral envelope and is a dominant immunogen. To study the antigenic properties of the different regions of gD, we have expressed the full-length gD encoding gene and overlapping fragments spanning various regions of the gD open reading frame in a baculovirus (Autographa californica nuclear polyhedrosis virus)--insect cell (Spodoptera frugiperda, SF-9) system. Maximum levels of expression for all proteins were obtained 48 to 72 h post infection of SF-9 cells by recombinant viruses. Full-length and truncated recombinant gD proteins reacted specifically with anti-gD monospecific serum as determined by immunoprecipitation and immunoblotting, indicating that the proteins retained their antigenicity. However, based on the reactivity with a panel of gD-specific monoclonal antibodies (Mabs), the full-length recombinant gD lacked proper expression for two highly neutralizing linear epitopes identified by Mabs R54 and 9D6. The rest of the epitopes appeared to be preserved and antigenically unaltered. Immunofluorescence studies of recombinant baculovirus infected SF-9 cells using gD monospecific serum, revealed no direct correlation between cellular localization of the expressed proteins and their amino acid sequences.

Evaluation of baculovirus-expressed bovine herpesvirus-1 (BHV-1) glycoproteins for detection and analysis of BHV-1-specific antibody responses.

Baculovirus (Autographa californica nuclear polyhedrosis)-expressed bovine herpesvirus-1 (BHV-1) glycoproteins B (gB), gC, and gD were developed and characterized. The recombinant proteins retained their antigenic properties as determined by immunoblotting against monoclonal antibodies. The proteins were examined as antigens for detection of BHV-1 infection and for the analysis of antibody responses to the individual viral proteins. A total of 115 bovine serum samples were tested for their reactivity with individual recombinant proteins from baculovirus-infected Spodoptera frugiperda (SF-9) cell lysates by enzyme-linked immunosorbent assay (ELISA), western blotting, and dot blotting assays. These serum samples were previously tested for BHV-1-specific antibodies by virus neutralization (VN) at the veterinary diagnostic laboratory. All 90 serum samples tested positive with VN were positive by ELISA against gC and gD, separately. However, reactivities of sera against gB were generally low and inconsistent. On the other hand, out of 25 sera that were negative with VN, 22 sera were consistent and gave negative results against gC or gD by ELISA, whereas reactivities with gB were inconsistent. Similar results were obtained when the sera were tested by western blotting and dot blotting. The positive sera consistently reacted strongly against gC and to a lesser extent gD. These results suggest that baculovirus expressed gC from infected cell lysate can be used as a potential diagnostic antigen for detection of anti-gC-specific antibody responses in BHV-1 infected cattle.

Fine mapping of bovine herpesvirus-1 (BHV-1) glycoprotein D (gD) neutralizing epitopes by type-specific monoclonal antibodies and sequence comparison with BHV-5 gD.

Overlapping fragments of the bovine herpesvirus-1 (BHV-1) glycoprotein (gD) ORF were expressed as trpE-gD fusion proteins in Escherichia coli to map linear neutralizing epitopes defined by BHV-1-specific MAbs. The MAbs 3402 and R54 reacted with the expressed fragments on Western blots that located the epitopes between the amino acids 52-126 and 165-216, respectively, of gD. Bovine covalescent sera with high neutralizing antibody titers against BHV-1 reacted with these bacterially expressed proteins containing both of the epitopes. Alignment of these sequences from BHV-1 with the corresponding region of the BHV-5 gD ORF sequences (reported here) identified several amino acid mismatches. Since the MAbs 3402 and R54 neutralize the BHV-1 and not BHV-5, it was presumed that these were important amino acids in defining the epitope. To further localize the neutralizing epitopes, synthetic peptides corresponding to these regions in the BHV-1 gD ORF were tested for their capacity to block monoclonal antibody neutralization of BHV-1 infectivity. The peptides encompassing amino acids 92-106 (3402 epitope) and amino acids 202-213 (R54 epitope) of the BHV-1 gD competed with BHV-1 for the binding by MAbs 3402 and R54, respectively, in a dose-dependent manner. Antisera produced in rabbits to these peptides conjugated to a carrier reacted strongly with a 30-kDa protein by Western blotting and had neutralizing antibody titers against BHV-1.

Comparison of bovine immune responses to affinity-purified bovine herpesvirus-1 antiidiotypes and glycoproteins.

Bovine immune responses to rabbit antiidiotypic antibodies (anti-Id) against neutralizing monoclonal antibodies to bovine herpesvirus-1 (BHV-1) envelope glycoproteins and to BHV-1 glycoproteins were compared. Glycoprotein-immunized animals produced high titers of anti-BHV-1 antibodies and were protected against BHV-1 challenge. Recombinant bovine interleukin-2 (rIL-2)-treated, anti-Id-immunized animals showed a slight reduction in clinical disease, and one calf produced BHV-1-neutralizing antibodies. Treatment with rIL-2 augmented non-BHV-1-specific immune responses. However, even with rIL-2 as an adjuvant, the mixture of polyclonal anti-Id did not elicit a consistent, protective BHV-1-specific immune response in calves.

Anti-idiotypic antibodies to bovine herpesvirus-1 inhibit virus infection in cell cultures.

A panel of murine monoclonal antibodies (MAbs) to bovine herpesvirus-1 (BHV-1) was prepared. Three of them were neutralizing MAbs and reacted against 130/75/50 kDa, 77 kDa, or 97 kDa glycoproteins (gp). A fourth non-neutralizing MAb recognized the 97 kDa gp. Competition radioimmunoassay demonstrated that each of the four MAbs reacted against a different virus epitope. Anti-idiotypic antibodies (anti-id) to the four MAbs were produced in rabbits and purified by sequential immunoaffinity chromatography. Each anti-id inhibited the binding of its respective MAb to BHV-1 in competitive ELISA and blocked BHV-1 neutralizing activity of the MAb. This inhibition suggested that the anti-ids were specific for the antigen binding site of the MAbs. Treatment of MDBK cells with anti-ids inhibited BHV-1 infection, which suggested that the anti-ids block a cellular component essential for virus infection. Absence of significant cross-reactivity among the anti-ids for heterologous MAbs indicated that they recognized unique determinants on the antigen binding site of the homologous MAb.

Anti-idiotypic antibodies mimic bovine viral diarrhea virus antigen.

Polyclonal rabbit anti-idiotypic antibodies (anti-ids) against two neutralizing murine monoclonal antibodies (mAbs) specific to a bovine viral diarrhea virus (BVDV) glycoprotein, 53 kDa, were produced, purified, and characterized. Each anti-id inhibited the binding of its respective mAb to BVDV antigen in a competitive ELISA and blocked the immunoprecipitation of the 53 kDa protein by the mAb. The anti-ids also inhibited the virus-neutralizing activity of their homologous mAbs. These results suggest that the anti-ids bear an internal image of a BVDV antigen and mimic neutralizing epitopes on the 53 kDa protein. Treatment of MDBK cells with the anti-ids inhibited BVDV infection, indicating that they block a cellular component, such as a virus receptor, required for virus adsorption or entry. Inhibition of the homologous mAb and lack of inhibition of the heterologous mAb indicate that the anti-ids are specific for the unique antigen-binding sites on the mAbs.

Induction of immune response to bovine herpesvirus-1 with anti-idiotypic antibodies.

Previously, we prepared rabbit anti-idiotypic (anti-Id) antibodies against murine monoclonal antibodies (MAbs) specific for the major bovine herpesvirus-1 (BHV-1) envelope glycoproteins. Glycoprotein III (gIII) contains neutralization epitopes and may be the virus attachment protein. Anti-Id antibodies to a neutralizing MAb that reacts with gIII were purified by sequential immunoaffinity chromatography. Immune responses to the purified anti-Id reagent and BHV-1 were compared in mice. Both groups of mice produced BHV-1-specific neutralizing antibodies. However, lymphocyte proliferative responses and interferon and interleukin-2 production were specific for the respective immunizing antigens. These results suggest that the anti-Id reagent may bear an internal image of a B-cell-stimulating epitope of glycoprotein gIII; however, this epitope does not stimulate a virus-specific cellular immune response in mice.