A caprine herpesvirus 1 vaccine adjuvanted with MF59™ protects against vaginal infection and interferes with the establishment of latency in goats

Abstract

The immunogenicity and the efficacy of a beta-propiolactone-inactivated caprine herpesvirus 1 (CpHV-1) vaccine adjuvanted with MF59™ were tested in goats. Following two subcutaneous immunizations, goats developed high titers of CpHV-1-specific serum and vaginal IgG and high serum virus neutralization (VN) titers. Peripheral blood mononuclear cells (PBMC) stimulated in vitro with inactivated CpHV-1 produced high levels of soluble IFN-gamma and exhibited high frequencies of IFN-gamma producing cells while soluble IL-4 was undetectable. On the other hand, control goats receiving the inactivated CpHV-1 vaccine without adjuvant produced only low serum antibody responses. A vaginal challenge with virulent CpHV-1 was performed in all vaccinated goats and in naïve goats to assess the efficacy of the two vaccines. Vaginal disease was not detected in goats vaccinated with inactivated CpHV-1 plus MF59™ and these animals had undetectable levels of infectious challenge virus in their vaginal washes. Goats vaccinated with inactivated CpHV-1 in the absence of adjuvant exhibited a less severe disease when compared to naïve goats but shed titers of challenge virus that were similar to those of naïve goats. Detection and quantitation of latent CpHV-1 DNA in sacral ganglia in challenged goats revealed that the inactivated CpHV-1 plus MF59™ vaccine was able to significantly reduce the latent viral load when compared either to the naïve goats or to the goats vaccinated with inactivated CpHV-1 in the absence of adjuvant. Thus, a vaccine composed of inactivated CpHV-1 plus MF59™ as adjuvant was strongly immunogenic and induced effective immunity against vaginal CpHV-1 infection in goats.

Figure 1. Serum and vaginal antibody responses in goats subcutaneously vaccinated with inactivated CpHV-1 plus MF59™.

Vaccines were given on day 0 and day 10. Sera and vaginal washes were collected on day 10 (white histograms) and day 20 (black or dashed histograms) to determine serum VN titers (A) or ELISA serum IgG titers (B) or ELISA vaginal IgG titers (C). Data are expressed as arithmetic mean ± SD. Data reported are cumulative from 4 independent experiments performed as described in Materials and Methods with a total of 8 goats in the naïve group, a total of 8 goats in the inactivated CpHV-1 only vaccinated group and a total of 12 goats in the inactivated CpHV-1 plus MF59™ vaccinated group. A double asterisk (**) denotes probability, p, <0.001 that the VN titers (A) or the serum IgG titers (B) or the vaginal IgG titers (C) in goats vaccinated with inactivated CpHV-1 plus MF59™ were equivalent to those of naïve goats on day 20 post immunization. The same probability, p, <0.001 was observed when VN titers (A), serum IgG titers (B) and vaginal IgG titers (C) in goats vaccinated with inactivated CpHV-1 plus MF59™ were compared to those of goats vaccinated with inactivated CpHV-1 only on day 20 post immunization. Statistical differences were calculated by the one-way ANOVA test followed by the Tukey's post-hoc test.

Figure 2. Serum IgG subclass responses in goats subcutaneously vaccinated with inactivated CpHV-1 plus MF59™.

Vaccines were given on day 0 and day 10. Sera were collected on day 20 to determine CpHV-1-specific IgG1 titers (white histograms) and CpHV-1-specific IgG2 titers (black histograms) by ELISA. Data are expressed as arithmetic mean ± SD. Data reported are cumulative from 4 independent experiments performed as described in Materials and Methods with a total of 8 goats in the naïve group, a total of 8 goats in the inactivated CpHV-1 only vaccinated group and a total of 12 goats in the inactivated CpHV-1 plus MF59™ vaccinated group. A single asterisk (*) denotes probability, p, <0.05 that the serum IgG1 titers are equivalent to the serum IgG2 titers in goats vaccinated with inactivated CpHV-1 plus MF59™. The Students' t-test was employed to study statistical differences.

Figure 3. IFN-gamma production by PBMC isolated from goats subcutaneously vaccinated with inactivated CpHV-1 and MF59™.

Vaccines were given on day 0 and day 10. Blood was drawn on day 20 and PBMC were isolated and stimulated in vitro as described in Materials and Methods. Soluble IFN-gamma in culture supernatants was measured by ELISA (A) while frequencies of IFN-gamma Spot Forming Cells (SFC) were measured by ELISPOT (B). Data (arithmetic mean ± SD) are cumulative from 4 independent experiments performed as described in Materials and Methods with a total of 8 goats in the naïve group, a total of 8 goats in the inactivated CpHV-1 only vaccinated group and a total of 12 goats in the inactivated CpHV-1 plus MF59™ vaccinated group. A double asterisk (**) denotes probability, p, <0.001 that the soluble IFN-gamma levels (A) or the IFN-gamma SFC (B) in goats vaccinated with inactivated CpHV-1 plus MF59™ were equivalent to those of naïve goats. The same probability, p, <0.001 was observed when soluble IFN-gamma levels (A) or IFN-gamma SFC (B) in goats vaccinated with inactivated CpHV-1 plus MF59™ were compared to those of goats vaccinated with inactivated CpHV-1 only. Statistical differences were calculated by the one-way ANOVA test followed by the Tukey's post-hoc test.

Figure 4. Vaginal viral shedding and clinical scores in goats vaccinated with inactivated CpHV-1 and challenged vaginally with virulent CpHV-1.

Vaccines were given on day 0 and day 10. All goats were challenged vaginally two weeks following the second vaccination as described in Materials and Methods with 4 ml of virulent CpHV-1 suspension (10⁵ TCID₅₀/50 µl). Naïve goats were included as unvaccinated controls. Goats were monitored daily for 14 days post-challenge and vaginal swabs were collected daily and employed to quantitate the viral shedding. Titration of the infectious virus in vaginal swabs was achieved by measuring the cytopathic effect of serial dilutions of the sample in MDBK cultures (A) while the number of CpHV-1 genomes in vaginal swabs was measured by real-time PCR (B). Clinical scores were also recorded daily following the challenge (C). Data (arithmetic mean ± SD) are cumulative from 4 independent experiments performed as described in Materials and Methods with a total of 8 goats in the naïve group, a total of 8 goats in the inactivated CpHV-1 only vaccinated group and a total of 12 goats in the inactivated CpHV-1 plus MF59™ vaccinated group. In (A), (B) and (C) the double asterisk (**) denotes probability, p, <0.001 that the CpHV-1 shedding titers (A) or the number of CpHV-1 genomes (B) or the clinical scores (C) in goats vaccinated with inactivated CpHV-1 plus MF59™ were equivalent to those of naïve goats. The same probability, p, <0.001 was observed when the CpHV-1 shedding titers (A) or the number of CpHV-1 genomes (B) or the clinical scores (C) in goats vaccinated with inactivated CpHV-1 plus MF59™ were compared to those of goats vaccinated with inactivated CpHV-1 only. In (C) the single asterisk denotes probability, p, <0.05 that the clinical scores in goats vaccinated with inactivated CpHV-1 only were equivalent to those of naïve goats. Data depicted in (A), (B) and (C), were used to calculate the AUC in order to perform statistical analyses. Statistical differences were calculated by the one-way ANOVA test followed by the Tukey's post-hoc test.

Figure 5. IFN-gamma SFC in PBMC isolated from goats subcutaneously vaccinated with inactivated CpHV-1 and challenged vaginally with virulent CpHV-1.

Vaccines were given on day 0 and day 10. All goats were challenged vaginally two weeks following the second vaccination with 4 ml of virulent CpHV-1 suspension (10⁵ TCID₅₀/50 µl) as described in Materials and Methods. Blood was drawn three weeks post-challenge and PBMC were isolated and stimulated in vitro as described in Materials and Methods. Frequencies of IFN-gamma Spot Forming Cells (SFC) were measured by ELISPOT. Data (arithmetic mean ± SD) are cumulative from 4 independent experiments performed as described in Materials and Methods with a total of 8 goats in the naïve group, a total of 8 goats in the inactivated CpHV-1 only vaccinated group and a total of 12 goats in the inactivated CpHV-1 plus MF59™ vaccinated group. A single asterisk (*) denotes probability, p, <0.05 that the IFN-gamma SFC in goats vaccinated with inactivated CpHV-1 plus MF59™ were equivalent to those of naïve goats. The same probability, p, <0.05 was observed when IFN-gamma SFC in goats vaccinated with inactivated CpHV-1 plus MF59™ were compared to those of goats vaccinated with inactivated CpHV-1 only. Statistical differences were calculated by the one-way ANOVA test followed by the Tukey's post-hoc test.

Figure 6. Distribution and quantitation of CpHV-1 genomic DNA in sacral ganglia from goats vaccinated with inactivated CpHV-1 and challenged with virulent CpHV-1.

Vaccines were given on day 0 and day 10. All goats were challenged vaginally two weeks following the second vaccination with 4 ml of virulent CpHV-1 suspension (10⁵ TCID₅₀/50 µl) as described in Materials and Methods. The five pairs of sacral ganglia were excised one month after challenge, the DNA was extracted from individual ganglion to detect and quantitate the number of CpHV-1 genomes by real-time PCR. Data (arithmetic mean ± SEM) are cumulative from 2 independent experiments i.e., experiment n.3 and experiment n.4, performed as described in Materials and Methods with a total of 6 goats in the naïve group, a total of 6 goats in the inactivated CpHV-1 only vaccinated group and a total of 6 goats in the inactivated CpHV-1 plus MF59™ vaccinated group. White circles (right ganglia), black circles (left ganglia). A single asterisk (*) denotes probability, p, <0.05 that the number of CpHV-1 genomes in the second (or in the fifth) pair of ganglia of goats vaccinated with inactivated CpHV-1 plus MF59™ was equivalent to that of naïve goats. A double asterisk (**) denotes probability, p, <0.001 that the number of CpHV-1 genomes in the third (or in fourth) pair of ganglia of goats vaccinated with inactivated CpHV-1 plus MF59™ was equivalent to that of naïve goats. Statistical differences were calculated by the one-way ANOVA test followed by the Tukey's post-hoc test.