Duration of immunity in dogs vaccinated against leptospirosis with a bivalent inactivated vaccine.

Duration of immunity in dogs induced with current commercial inactivated leptospirosis vaccines and evaluated against experimental infection, to date, has hardly been documented. The purpose of the present work was to assess the duration of immunity in dogs that is attainable with a commercial inactivated bivalent leptospirosis vaccine. For this purpose, young dogs were vaccinated twice followed by challenge with either Leptospira interrogans serovar canicola or L. interrogans serovar icterohaemorrhagiae 5 weeks, 27 weeks or 56 weeks after the second vaccination. For assessment of the duration of immunity, titres of agglutinating serum antibodies were measured before and after challenge, and the effects of challenge on a variety of parameters were determined including reisolation of challenge organisms from blood, urine and kidney. Both challenge strains induced a generalised infection in control dogs, the canicola strain being most virulent. From the results with different parameters it appeared that the two vaccinations induced a high rate of protection from generalised infection with canicola and icterohaemorrhagiae at 5, 27 and 56 weeks after the second vaccination. In addition, after 56 weeks, still a high level of immunity against renal infection with sv. canicola and, as a consequence, urinary shedding of sv. canicola bacteria, was demonstrated. It was, therefore, concluded that with this vaccine, using this vaccination schedule, a duration of immunity of 1 year can be attained against infection with both serovars.

Epitopes on glycoprotein E and on the glycoprotein E/glycoprotein I complex of bovine herpesvirus 1 are expressed by all of 222 isolates and 11 vaccine strains.

Glycoprotein E (gE) of bovine herpesvirus 1 (BHV1) forms a complex with glycoprotein I (gI) and plays an important role in cell-to-cell spread mechanisms of the virus, but is not essential for propagation of the virus. To study the antigenic variability of BHV1 glycoprotein E, a set of six well characterised monoclonal antibodies (MAbs) was established using BHV1 gE and gI deletion mutants, eukaryotically expressed gE and gI and pepscan analysis. Two of these MAbs reacted with a linear gE epitope (MAbs 3 and 52), two reacted with a more conformation dependent gE epitope (MAbs 61 and 81) and two reacted with epitopes formed by a complex formed between gE and glycoprotein I (MAbs 67 and 75). With these six MAbs the gE expression of 222 BHV1 isolates and 11 BHV1 modified-live vaccine strains was studied in vitro, using an immunoperoxidase monolayer assay. All 222 BHV1 isolates and 11 vaccine strains were found to react with MAbs 61, 81 and 75. Three of the 222 isolates failed to react with MAb 67 and two of the vaccines reacted very weakly with MAbs 3 and 52. Analysis of the gE genes of these five aberrant isolates and the gE glycoproteins they expressed, did not show obvious size differences compared to wild-type BHV1. We conclude that the tested gE epitopes are highly conserved, including the epitopes formed by the gI/gE complex.

Epitopes on glycoprotein C of bovine herpesvirus-1 (BHV-1) that allow differentiation between BHV-1.1 and BHV-1.2 strains.

In cattle, bovine herpesvirus-1 (BHV-1) can cause a mild genital disease known as infectious pustular vulvovaginitis (IPV) and a more severe respiratory disease known as infectious bovine rhinotracheitis (IBR). On the basis of epidemiological data, it has been proposed that these diseases are caused by strains with different genotypes (IBR by BHV-1.1 and IPV by BHV-1.2 strains). By using a panel of 237 BHV-1 isolates, a monoclonal antibody (MAb 71) was found that failed to react with all 54 putative IPV strains in the panel, and another MAb (77) was found that did not react with 16 of these 54 IPV strains. Because MAbs 71 and 77 also failed to react with a BHV-1.1 glycoprotein C (gC)-deletion mutant, it was hypothesized that both MAbs recognize BHV-1.1 gC. By marker-rescue experiments and by expressing fragments of the BHV-1.1 gC gene in recombinant baculoviruses, it was shown that both MAbs indeed recognize BHV-1.1 gC. MAb 71 recognizes the N-terminal half and MAb 77 recognizes the C-terminal half of BHV-1.1 gC. In a PEPSCAN analysis with 12-mer oligopeptides, MAb 71 reacted with overlapping peptides containing gC amino acid residues 75-80 and MAb 77 did not react in this analysis. The differences in gC found in this study may contribute to the biological differences between BHV-1.1 and BHV-1.2.

Strains of bovine herpesvirus 1 that do not express an epitope on glycoprotein E in cell culture still induce antibodies that can be detected in a gE-blocking ELISA.

Two bovine herpesvirus 1 (BHV1) field strains that do not express an epitope on glycoprotein E (gE) in cell culture were inoculated into calves to examine whether their sera became positive in a gE-blocking ELISA that detects antibodies against gE. This gE-blocking ELISA uses one monoclonal antibody that is directed against the above mentioned epitope. All calves, except one, infected with these gE-epitope negative BHV1 strains, became positive in this gE-blocking ELISA, about two weeks later than in another gE-ELISA and a gB-ELISA. However, cattle infected with BHVI strains that do express this particular gE-epitope showed a similar type of antibody responses. These findings demonstrate that BHV1 strains that do not express a particular gE-epitope in cell culture, still can induce antibodies that are detected in a blocking ELISA that measures antibodies against that epitope.

Virulence, immunogenicity and reactivation of bovine herpesvirus 1 mutants with a deletion in the gC, gG, gI, gE, or in both the gI and gE gene.

Within the framework of developing a marker vaccine against bovine herpesvirus 1 (BHV1), several mutants with deletions in non-essential glycoprotein genes were constructed. Glycoprotein gC, gG, gI and gE single deletion mutants, a gI/gE double deletion mutant and a gE frame-shift mutant were made. The virulence and immunogenicity of these mutants were evaluated in specific-pathogen-free calves. Except for the gC deletion mutant, all mutants were significantly less virulent than the parental wild-type (wt) BHV1 strain Lam. The virulence of the gI and the gI-/gE- mutants was almost completely reduced. Upon challenge infection, the calves of the control group became severely ill, whereas all other calves remained healthy. The reduction of the virus shedding after challenge infection was related to the virulence of the strain of primary inoculation. Virus shedding was almost completely reduced in calves first inoculated with Lam-wt or with gC- and the least reduced in calves inoculated with gI- or gI-/gE-. Six weeks after challenge, all calves were treated with dexamethasone to study whether mutant or challenge virus or both could be reactivated. The gC- and the gG- mutants were reactivated, whereas none of the other mutants were reisolated. Reactivation of challenge virus was reduced in all calves inoculated with mutant viruses. The gC deletion mutant was too virulent and the gI and the gI/gE deletion mutants were the least immunogenic, but based on residual virulence and immunogenicity, both the gG and the gE deletion mutants are candidates for incorporation in live BHV1 vaccines. However, it also depends on the kinetics of the anti-gG and anti-gE antibody response after wild-type virus infection, whether these deletion mutants are really suitable to be incorporated in a marker vaccine.

An inactivated gE-negative marker vaccine and an experimental gD-subunit vaccine reduce the incidence of bovine herpesvirus 1 infections in the field.

An inactivated glycoprotein E-negative vaccine and an experimental glycoprotein D-subunit vaccine against bovine herpesvirus 1 (V1) were examined for their effectiveness in a randomized, double-bline, placebo-controlled field trial comprising 130 dairy farms. The use of these marker vaccines enabled us to monitor the incidence of infections in vaccinated populations. The aims of this trial were to evaluate whether these vaccines: (1) reduce the proportion of outbreaks in dairy herds; and (2) reduced virus transmission within dairy herds and to what extent. Vaccination with either of the two vaccines significantly reduced the proportion of herds wherein an outbreak occurred as well as the virus transmission within herds, as compared to placebo-treated herds. The estimated number of secondary cases caused by one infectious animal, expressed as the reproduction ratio R, was for both vaccines significantly > 1. This indicates that when BHV1 is introduced into vaccinated herds, major outbreaks may still occur.

Inactivated bovine herpesvirus 1 marker vaccines are more efficacious in reducing virus excretion after reactivation than a live marker vaccine.

A comparative study was carried out to evaluate the efficacy of three bovine herpesvirus 1 (BHV1) marker vaccines to reduce the reexcretion of virus after reactivation of latent BHV1. A live gE-negative vaccine, an inactivated gE-negative vaccine and an experimental gD-subunit vaccine were tested in three identical experiments in which cattle, latently infected with BHV1, were vaccinated twice before they were treated with high doses of dexamethasone. Virus excretion after dexamethasone treatment was compared with that in BHV1-infected, unvaccinated cattle which served as controls. All cattle, controls and vaccinees, excreted virus. However, the inactivated vaccines reduced virus excretion more efficiently than did the live vaccine.

An enzyme-linked immunosorbent assay to detect antibodies against glycoprotein gE of bovine herpesvirus 1 allows differentiation between infected and vaccinated cattle.

A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against glycoprotein gE (gE) of bovine herpesvirus 1 (BHV1). The assay is based on the use of two monoclonal antibodies directed against different antigenic domains on gE. Sera from uninfected cattle and cattle that had been repeatedly vaccinated with gE-negative marker vaccines scored negative, whereas sera from cattle naturally or experimentally infected with BHV1 field strains scored positive in the gE-ELISA. Antibodies against gE appeared in the serum around 11 days after infection. Cattle that were first vaccinated and then challenged, thus having less virus replication, also became gE-seropositive. The sensitivity and specificity of the gE-ELISA is high, and therefore the gE-ELISA is suitable for differentiating between infected cattle and vaccinated cattle with a gE-negative vaccine.

Advances in the development and evaluation of bovine herpesvirus 1 vaccines.

This review deals with conventional and modern bovine herpesvirus 1 (BHV1) vaccines. Conventional vaccines are widely used to prevent clinical signs of infectious bovine rhinotracheitis. The use of conventional vaccines, however, does not appear to have resulted in reduction of the prevalence of infection. Novel BHV1 marker vaccines comprise either mutants with a deletion in one of the non-essential genes, or subunit vaccines that contain one or more glycoproteins. These marker vaccines can be used in conjunction with companion diagnostic tests to differentiate between infected and vaccinated cattle. Their efficacy has been evaluated in vaccination-challenge experiments, transmission experiments and in field trials. The results demonstrate that the marker vaccines can contribute to the eventual eradication of BHV1. However, there remains room for improvement of BHV1 marker vaccines.

Persistence of antibodies against bovine herpesvirus 1 and virus reactivation two to three years after infection.

To study the long-term persistence of bovine herpesvirus 1 (BHV1) specific antibodies in cattle, serum samples were collected regularly over a period of two to three years after infection. The sera were titrated in a BHV1 neutralizing antibody test, a glycoprotein gB blocking ELISA and a glycoprotein gE blocking ELISA. Sera collected after infection were positive in all tests and the titres declined only minimally during the investigation period. Two to three years after infection, four animals were treated with dexamethasone to reactivate putatively latent virus. Virus was isolated from the nasal swabs from three animals, and PCR analysis proved the presence of BHV1-DNA in the trigeminal ganglia of the fourth animal. The results demonstrated that four animals still harboured latent BHV1, which was reactivated and excreted in three animals, two to three years after infection. Hence, the persistence of antibodies allowed the detection of latently infected cattle.

The role of glycoproteins gC, gE, gI, and gG in the induction of cell-mediated immune responses to bovine herpesvirus 1.

Mutant viruses with deletions in genes encoding non-essential glycoproteins are considered as promising bovine herpesvirus 1 (BHV1) vaccine candidates. The present study compared the influence of various gene deletions (gC, gE, gI, gG) on the induction of cell-mediated immune responses against the virus. The highest BHV1 specific lymphoproliferative response was observed in the group of calves inoculated with the gC- mutant. However, in all groups of inoculated calves, limiting dilution analysis showed marked individual variability in the number of BHV1 specific T lymphocytes that were stimulated. The same animals were then challenged with wild-type BHV1. In these animals, limiting dilution analysis did not reveal gE, gI nor gG as a major T lymphocyte antigen. However, further analysis suggested the T cell antigenicity of gE in a low number of BHV1 hyperimmunized calves. Stimulation of MHC unrestricted cytotoxicity was also evaluated after inoculation with the various deletion mutants. Cytotoxicity in gC- inoculated calves was as high as in BHV1 inoculated calves. In conclusion, among the BHV1 deletion mutants that were tested, the gC- mutant stimulated the best cell-mediated immune responses.

Early immunity induced by a live gE-negative bovine herpesvirus 1 marker vaccine.

We studied the early immunity induced by a live glycoprotein E (gE) negative bovine herpesvirus 1 (BHV1) marker vaccine. Three groups of specific-pathogen-free calves were either not vaccinated, or vaccinated two days or two hours before the introduction of a calf that was intranasally infected with wild-type BHV1 the day before. We quantified the shedding of gE-negative vaccine virus and of wild-type virus, using a double-staining immunoassay. In calves vaccinated two hours before the introduction of the infected calf, the shedding of wild-type virus was reduced, compared with that of the unvaccinated control calves. The shedding of wild-type virus was most significantly reduced in the calves that were vaccinated two days before: only very small amounts of wild-type virus were isolated. Wild-type virus was not detected at all in the samples from one of the five calves of that group. Furthermore, this calf was the only one in which we did not detect antibodies against gE. Hence, intranasal vaccination with a live gE-negative vaccine induced early immunity against a BHV1 contact infection. This suggests that this vaccine can be used efficaciously in the early stages of a BHV1 outbreak.

An attenuated bovine herpesvirus 1 marker vaccine induces a better protection than two inactivated marker vaccines.

A comparative study on the efficacy of 3 bovine herpesvirus 1 (BHV1) marker vaccines was carried out. An attenuated gE-negative vaccine, an inactivated gE-negative vaccine and an experimental gD-subunit vaccine were tested twice in a vaccination-challenge experiment in cattle. The attenuated vaccine induced the best clinical protection as evidenced by the total absence of clinical signs and fever in cattle. In addition, the attenuated vaccine reduced the shedding of challenge virus significantly more than the inactivated vaccines. Of the inactivated vaccines, the gE-negative vaccine induced a better clinical protection than the gD-subunit vaccine.

Virulence, immunogenicity and reactivation of seven bovine herpesvirus 1.1 strains: clinical and virological aspects.

Specific pathogen-free calves were inoculated intranasally with one of seven strains of bovine herpesvirus 1.1 (BHV 1.1) to identify a highly virulent strain for use in vaccination-challenge experiments. The calves were monitored clinically and virologically. Clear differences in virulence between the strains were observed. The Iowa strain was the most virulent; the four calves infected with the strain had the most severe clinical signs; two of them died and viraemia was detected in three of them. To evaluate the immunogenicity of the seven strains all the calves were challenged 16 weeks later with the Iowa strain. The calves of a control group showed the typical signs of a BHV1 infection, whereas all the other calves were protected against disease and shed little or no virus. Hence, the differences in virulence were not associated with differences in immunogenicity. After the calves had been treated with dexamethasone, differences were observed between the strains in the amount of virus that was excreted.

The use of marker vaccines in eradication of herpesviruses.

Marker vaccines are vaccines that allow serological differentiation between infected and vaccinated individuals. This differentiation is based on the absence of one or more microbial proteins in the vaccine that are present in the wild-type micro-organism. Consequently, after infection, but not after vaccination, an antibody response against that specific protein(s) can be detected. With a protein-specific antibody test infected individuals can thus be distinguished from vaccinated individuals. Marker vaccines against pseudorabies virus (PRV) and against bovine herpesvirus 1 (BHV1) infections have been developed, along conventional routes and by recombinant DNA technology. These vaccines have been shown to be efficacious in reducing (a) clinical signs after infection, (b) wild-type virus replication after infection, and (c) transmission of wild-type virus in the laboratory and in the field. At present, PRV vaccines that lack the gene for the glycoprotein gE are used worldwide in novel eradication programmes. The first phase of such a programme consists of systematic vaccination of pigs on a farm, in a region or an entire country. Experiences in the Netherlands show that it is feasible to eradicate PRV by the intensive use of marker vaccines. Whether, this also holds true for BHV1 is now under investigation.

Virulence and immunogenicity in calves of thymidine kinase- and glycoprotein E-negative bovine herpesvirus 1 mutants.

A deletion was introduced into the thymidine kinase (TK) gene of the BHV1 strain Lam and, or, the complete coding region of the glycoprotein E (gE) gene was deleted to reduce virulence and to make serological differentiation possible. The virulence and immunogenicity of these three BHV1 mutants (TK-, gE- and TK-/gE) were studied in specific-pathogen-free calves. Although inactivation of TK strongly reduced the virulence of the Lam strain, deletion of the gE gene alone sufficed to yield complete attenuation of the Lam strain for seven-week-old calves. The three mutants induced protective immunity against disease after challenge with a virulent BHV1 strain. The reduction of virus shedding after challenge was related to the virulence of the various strains. The immunogenicity of the mutants was also evidenced by the reduction of challenge virus shedding after dexamethasone treatment. None of the mutant viruses could be isolated after dexamethasone treatment. The results demonstrate that the gE- and TK-/gE- mutants are good candidates for incorporation in a BHV1 marker vaccine.

A glycoprotein E deletion mutant of bovine herpesvirus 1 infects the same limited number of tissues in calves as wild-type virus, but for a shorter period.

To gain insight into the role of glycoprotein E of bovine herpesvirus 1 (BHV-1), we compared the distribution of wild-type (wt) BHV-1 with that of a gE deletion mutant (gE-) in calves after intranasal inoculation. The wt-infected calves had severe clinical signs, but the gE(-)-infected calves were virtually free of clinical signs. At 3, 4, 7, 8, 44, 45, 50 and 51 days post-infection (p.i.), one calf from each group was killed and tissues were collected for virus isolation and PCR analysis. At 3, 4, 7 and 8 days p.i., infectious virus could be isolated only from the nasopharyngeal mucosa, parotid gland and nearby lymphoid tissues for both the wt- and gE(-)-infected calves. At 3 and 4 days p.i., virus titres in these tissues were comparable in both the wt- and gE(-)-infected calves. However, the virus titres were significantly reduced at 7 and 8 days p.i. in the gE(-)-infected calves, but not in the wt-infected calves. Semi-quantitative PCR analysis revealed that for the entire infection period (3 to 51 days p.i.) significantly more BHV-1 DNA was detected in the trigeminal ganglia (TG) of the wt-infected calves than in those of the gE(-)-infected calves. We conclude that the gE- mutant infects the same limited number of tissues as wt BHV-1, but for a shorter period.

An inactivated vaccine based on a glycoprotein E-negative strain of bovine herpesvirus 1 induces protective immunity and allows serological differentiation.

The bovine herpesvirus 1 (BHV1) strain Za is a conventionally attenuated strain with a 2.7 kb deletion that encompasses the complete coding region for glycoprotein E (gE). This gE-negative strain was used as whole-virus antigen in an inactivated virus vaccine. Three different antigen concentrations of this vaccine were evaluated for safety and efficacy in a vaccination-challenge experiment in calves. No adverse effects were observed in any of the calves vaccinated with the gE-negative vaccines. Calves given the vaccine with the highest antigen concentration were adequately protected against challenge; clinical symptoms were virtually absent and challenge virus shedding was significantly reduced as compared with unvaccinated calves. We developed a sensitive blocking enzyme-linked immunosorbent assay (ELISA) to detect antibodies against gE. After vaccination, calves did not produce antibodies against gE, but these antibodies were detectable within 2 weeks after challenge both in vaccinated and in unvaccinated calves. These results demonstrate the efficacy of a gE-negative inactivated BHV1 vaccine and the detectability of antibodies against gE after infection. The combined use of the marker vaccine and the gE-blocking ELISA makes it possible to differentiate between vaccinated animals and infected animals. This possibility may be very useful in BHV1 control programmes.

Quantitative assessment of the specific CD4+ T lymphocyte proliferative response in bovine herpesvirus 1 immune cattle.

We quantified the CD4+ T cell proliferation specific for bovine herpesvirus 1 (BHV-1) in peripheral blood mononuclear cells from cattle. The stimulation index as detected in proliferative assays performed in the presence of BHV-1 antigen is highly variable in immune cattle. By using proliferative assays performed after negative selection we showed that, as expected, CD4+ T cells were the limiting cell type for antigen-induced proliferation. Neither B, gamma delta T nor CD8+ cells seemed to be involved. The limiting dilution method was established to obtain quantitative estimations, namely frequencies of specific T cells. When limiting dilution cultures were supplemented with interleukin-2 (IL-2), an IL-2 induced unspecific cell proliferation masked the specific T cell proliferation. Natural killer cells were not the major cell type involved, but CD4+ lymphocytes themselves seemed to respond to IL-2 irrespective of the presence of antigen. When cultures were performed without addition of IL-2, the frequency of BHV-1 specific proliferative T cells could be obtained by the difference between the frequency of proliferating cells calculated in the presence and absence of antigen. The method provides a sensitive and quantitative means to measure the T cell immune response to BHV-1 vaccine candidates.

A glycoprotein E deletion mutant of bovine herpesvirus 1 is avirulent in calves.

A marker vaccine elicits an antibody response in the host that can be distinguished from the antibody response induced by a wild-type strain. To obtain a bovine herpesvirus 1 (BHV-1) marker vaccine, we constructed a glycoprotein E (gE) deletion mutant. This was obtained by removing the complete gE coding region from the BHV-1 genome. To attenuate the gE deletion mutant further, we also introduced a small deletion in the thymidine kinase (TK) gene. We selected three mutants: the gE deletion mutant, a TK deletion mutant and a gE/TK double deletion mutant, and examined their virulence and immunogenicity in calves. After intranasal inoculation, the TK deletion mutant showed some residual virulence, whereas the gE and gE/TK deletion mutants were avirulent. The calves inoculated with the deletion mutants were protected against disease after challenge exposure and shed significantly less virus than control calves. Deleting the gE gene, therefore, has little effect on the immunogenicity of BHV-1, but is sufficient to reduce the virulence of BHV-1 in calves. These findings led us to conclude that the gE deletion mutant is a good candidate for a modified live BHV-1 marker vaccine.