Geoffrey J. Letchworth

Induction of a mucosal barrier to bovine herpesvirus 1 replication in cattle

Current vaccines for human and animal herpesviruses engender an immunity that may ameliorate disease but generally fails to prevent infection, latency, reactivation from latency, or spread through a population. By administering intranasally to cattle bovine herpesvirus type 1 virion envelope proteins combined with the potent mucosal immune system adjuvant, cholera toxin B subunit, we engendered a local antibody response that acted as a barrier to infection of mucosal epithelial cells and thereby prevented viral replication, consequently precluding disease, latency, and spread.

Monoclonal antibody analysis of bovine herpesvirus-1 glycoprotein antigenic areas relevant to natural infection

Neutralizing antigenic areas on the glycoproteins of bovine herpesvirus-1 (BHV-1) were identified by reciprocal competition radioimmunoassays using monoclonal antibodies. Three interrelated and two independent antigenic areas were identified on the 77-kDa (K) gIV envelope glycoprotein. Antigenic analysis of this protein has not been previously described. Four interrelated and one independent antigenic areas were found on the 97K gIII envelope glycoprotein. A third group of monoclonal antibodies reacting in Western blot with the 74K subunit of gI, a 130K disulfide-linked 74K/55K heterodimer, revealed four interrelated antigenic areas. All of the antigenic areas on all three glycoproteins were reactive with neutralizing monoclonal antibodies and all were targets for antibody-complement lysis. However, antibodies against gIV were the most efficient at neutralizing the virus and rendering infected cells susceptible to antibody-complement lysis. Convalescent sera from experimentally infected calves were used in a competitive radioimmunoassay to confirm that each antigenic area on the gI, gIII, or gIV glycoproteins was a target for bovine antibodies during primary infection with BHV-1.

Mucosal and systemic immunity to bovine herpesvirus-1 glycoprotein D confer resistance to viral replication and latency in cattle.

Mucosal immunity in the respiratory tract directed against bovine herpesvirus-1 (BHV-1) glycoprotein B forms an effective barrier against BHV-1 replication in cattle. Here we investigated the ability of a second BHV-1 glycoprotein, gD, to engender specific antibodies in nasal secretion and serum and protect against infection. We expected gD to give greater protection than gB because anti-gD antibodies prevent viral penetration into cells at much lower concentrations than anti-gB antibodies. Calves vaccinated once subcutaneously and thrice intranasally with affinity-purified BHV-1 gD had mucosal antibodies and three of five were protected against intranasal challenge by 10(7) p.f.u. of BHV-1. Four of the five vaccinated calves were proven free of BHV-1 latency by the lack of viral shedding following immunosuppression. The putative mucosal adjuvant, cholera toxin B subunit (CTB), did not significantly enhance mucosal immunity or protection against challenge or latency (P0.5) since only 4 of 6 gD plus CTB immunized calves were completely protected. Taken together, these data suggest that BHV-1 gD may be useful in a mucosal vaccine against BHV-1 infection in cattle but is less than totally effective when used alone.

Effects of formalin inactivation on bovine herpes virus-1 glycoproteins and antibody response elicited by formalin-inactivated vaccines in rabbits

The possibility of using a radioimmune assay (RIA) to measure the retention of antigenic determinants on gI, gIII, and gIV, the three major bovine herpes virus 1 (BHV-1) glycoproteins, during the formalin inactivation and aluminium hydroxide adsorption commonly used in the manufacture of killed virus vaccines was investigated. Monoclonal antibodies were used to measure thirteen previously identified glycoprotein epitopes in experimental vaccines containing live BHV-1 or formalin-killed BHV-1 and a commercial formalin-inactivated BHV-1 vaccine. All four epitopes on gI lost 80-97% of their ability to bind monoclonal antibodies. Two of five epitopes on gIII were preserved but the other three epitopes on gIII and five on gIV were reduced as much as 88%. To show that the measured loss of epitopes was biologically relevant, the vaccines were administered to rabbits and the resulting antibody responses against the same epitopes were evaluated by competition RIA. Little correlation was found between the measured antigenic site destruction and the rabbit response against those antigenic sites, suggesting that monoclonal antibody reactivity of a formalin-inactivated vaccine could not provide a meaningful measure of antigenic activity.

Epitope specificity and protective efficacy of the bovine immune response to bovine herpesvirus-1 glycoprotein vaccines

Bovine herpesvirus-1 (BHV-1) envelope glycoproteins gI, gIII and gIV were individually purified on monoclonal antibody affinity columns and injected intradermally into BHV-1 seronegative calves. The calves developed serum neutralizing antibodies that monospecifically precipitated the immunizing glycoprotein from a preparation of 125I-labelled BHV-1 envelope proteins. A competitive radioimmunoassay using the bovine antisera demonstrated that known functional epitopes had been retained in the glycoprotein vaccines. Calves immunized with the gI, gIII or gIV glycoproteins were not protected from intranasal challenge with BHV-1 and had levels and duration of viral shedding in their nasal secretions similar to those of non-immunized control calves.

Heterogeneity of neutralization-related epitopes within a bluetongue virus serotype

Twenty-four monoclonal antibodies raised against a 1962 Wyoming isolate of blue-tongue virus serotype 17 (BTV 17) were tested against 20 other field isolates of this serotype in a solid-phase radioimmunoassay (RIA), neutralization, and mouse passive protection tests. Of the 21 antibodies that bound in RIA to acetone-fixed BTV-infected cells, 18 bound to cells infected with any of the BTV 17 isolates and 3 detected minor antigenic differences in two isolates. The remaining 3 antibodies, that bound in RIA only to unfixed virus-infected cells detected additional differences. Of the 3 antibodies binding to unfixed virus-infected cells one bound to all but 2 isolates. A second antibody, 6C3A.2, bound only to the Wyoming isolate and passively protected mice against this isolate. The third antibody, 6C2A.4.2, bound to the Wyoming isolate and to 8 isolates from the mid-South U. S., but not to 12 isolates from California. Antibody 6C2A.4.2 neutralized the Wyoming and mid-South isolates to which it bound and passively protected mice against the Wyoming isolate but provided little or no protection against 4 California isolates tested. Polyclonal serum from mice immunized against Wyoming BTV 17 bound in RIA to all BTV 17 isolates and neutralized all isolates. Thus, three neutralization-related antigenic determinants were disclosed, one (perhaps a set) recognized by immune sera on all BTV 17 isolates, a second recognized by antibody 6C2A.4.2 on the Wyoming and 8 mid-South isolates, and a third recognized by antibody 6C3A.2 only on the Wyoming isolate. These differences may be important in selection of virus strains for vaccine development.

Yeast-secreted bovine herpesvirus type 1 glycoprotein D has authentic conformational structure and immunogenicity

Bovine herpesvirus-1 (BHV-1) glycoprotein D (gD), an envelope glycoprotein, engenders mucosal and systemic immunity protecting cattle from viral infection. Production of gD with authentic immunogenicity is required for a subunit vaccine. We placed the truncated BHV-1 gD gene, lacking its putative transmembrane and cytoplasmic domains, under the control of the methanol-inducible AOX1 promoter in the yeast Pichia pastoris. Truncated BHV-1 gD (tgD) was efficiently secreted into the culture medium as a 68 kDa protein using either the yeast alpha prepro or native BHV-1 gD signal sequences. The yeast-secreted tgD had N-linked glycosylation and appears to have authentic conformational structure and immunogenicity based on the following observations A panel of monoclonal antibodies recognizing five neutralizing epitopes reacted with yeast tgD. Sera from yeast tgD-immunized mice immunoprecipitated native BHV-1 gD and neutralized BHV-1 infection in vitro. Yeast tgD competitively blocked all reaction between native gD and monospecific gD polyclonal sera from cattle. Based on these data, yeast-derived BHV-1 tgD is an excellent candidate for a subunit vaccine.

Passively administered neutralizing monoclonal antibodies do not protect calves against bovine herpesvirus 1 infection

Monoclonal antibodies specific for defined epitopes on the gI, gIII and gIV envelope glycoproteins of BHV-1 were used individually or in glycoprotein-monospecific pools for passive immunization of young calves. Although serum antibody titres comparable to those found in naturally infected and recovered calves were achieved, passive immunization failed to prevent the growth of BHV-1 in nasal and ocular mucosa and did not decrease the duration of viral shedding.

Field Evaluation of a Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection of Vesicular Stomatitis Virus

Sporadic outbreaks of vesicular stomatitis (VS) in the United States result in significant economic losses for the U.S. livestock industries because VS is a reportable disease that clinically mimics foot-and-mouth disease. Rapid and accurate differentiation of these 2 diseases is critical because their consequences and control strategies differ radically. The objective of the current study was to field validate a 1-tube multiplexed real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay for the rapid detection of Vesicular stomatitis New Jersey virus and Vesicular stomatitis Indiana virus strains occurring in Mexico and North and Central America. A comprehensive collection of 622 vesicular lesion samples obtained from cattle, horses, and swine from throughout Mexico and Central America was tested by the real-time RT-PCR assay and virus isolation. Overall, clinical sensitivity and specificity of the real-time RT-PCR were 83% and 99%, respectively. Interestingly, VS virus isolates originating from a specific region of Costa Rica were not detected by real-time RT-PCR. Sequence comparisons of these viruses with the real-time RT-PCR probe and primers showed mismatches in the probe and forward and reverse primer regions. Additional lineage-specific primers and a probe corrected the lack of detection of the missing genetic lineage. Thus, this assay reliably identified existing Mexican and Central American VS viruses and proved readily adaptable as new VS viruses were encountered. An important secondary result of this research was the collection of hundreds of new VS virus isolates that provide a foundation from which many additional studies can arise.

Monoclonal antibodies against African swine fever viral antigens.

Monoclonal antibodies specific for African swine fever (ASF) viral proteins of 14, 32, 73, 174, and 240 kDa were produced and characterized. Immunoelectron microscopy detected the 73 kDa but not the 14-, 32-, or 240-kDa proteins at the surface of the virion. The 32-kDa protein was detected by radioimmunoassay 2 hr after infection of porcine monocytes and Vero cells, was detected in the seven widely divergent ASFV isolates tested, and stained brilliantly virus-infected cells in indirect immunofluorescence suggesting that monoclonal antibodies directed against this protein may be useful in ASFV diagnosis. Two monoclonal antibodies detected heterogeneity between ASF viruses.