Mucosal immunization induces a higher level of lasting neutralizing antibody response in mice by a replication-competent smallpox vaccine: vaccinia Tiantan strain

Abstract

The possible bioterrorism threat using the variola virus, the causative agent of smallpox, has promoted us to further investigate the immunogenicity profiles of existing vaccines. Here, we study for the first time the immunogenicity profile of a replication-competent smallpox vaccine (vaccinia Tiantan, VTT strain) for inducing neutralizing antibodies (Nabs) through mucosal vaccination, which is noninvasive and has a critical implication for massive vaccination programs. Four different routes of vaccination were tested in parallel including intramuscular (i.m.), intranasal (i.n.), oral (i.o.), and subcutaneous (s.c.) inoculations in mice. We found that one time vaccination with an optimal dose of VTT was able to induce anti-VTT Nabs via each of the four routes. Higher levels of antiviral Nabs, however, were induced via the i.n. and i.o. inoculations when compared with the i.m. and s.c. routes. Moreover, the i.n. and i.o. vaccinations also induced higher sustained levels of Nabs overtime, which conferred better protections against homologous or alternating mucosal routes of viral challenges six months post vaccination. The VTT-induced immunity via all four routes, however, was partially effective against the intramuscular viral challenge. Our data have implications for understanding the potential application of mucosal smallpox vaccination and for developing VTT-based vaccines to overcome preexisting antivaccinia immunity.

Figure 1

Anti-VTT neutralizing antibody responses after the VTT vaccination. (a) A FACS-based neutralization assay was newly developed to measure the anti-VTT neutralizing antibody responses. (b) Serum samples were tested three weeks after the VTT vaccination. (c) Serum samples were tested six months after the VTT vaccination. Due to a limited amount of serum collected from each mouse, pooled sera, which contain an equal amount of serum of each vaccinated mouse in each group, were subjected to each experiment. The X-axis represents the serum dilution factor whereas the Y-axis indicates the percentage of viral inhibition. An IC₅₀ value is a serum dilution factor which achieves 50% of viral inhibition as indicated by the dashed horizontal line. The experiment was repeated twice with similar results obtained. Placebo (no VTT) mice were tested as well.

Figure 2

Anti-S neutralizing antibody responses in VTT-vaccinated mice after two MVTT-S challenges. (a) Serum samples of i.m., i.n., and i.o. VTT-vaccinated as well as of placebo-inoculated (no VTT) mice were tested. (b) Serum samples of s.c. VTT-vaccinated mice were tested. Pooled sera, which contain an equal amount of serum of each vaccinated mouse in each group, were subjected to each experiment. The X-axis represents the serum dilution factor whereas the Y-axis indicated the percentage of viral inhibition. The dashed horizontal line indicates IC₅₀ values. The experiment was repeated twice with similar results obtained.

Figure 3

Anti-S neutralizing antibody responses in VTT-vaccinated mice after two i.m. MVTT-S challenges. Serum samples collected three weeks after the second MVTT-S challenge were tested in this experiment. Pooled sera, which contain an equal amount of serum of each vaccinated mouse in each group, were subjected to each experiment. The X-axis represents the serum dilution factor whereas the Y-axis indicated the percentage of viral inhibition. The dashed horizontal line indicates IC₅₀ values. The experiment was repeated twice with similar results obtained. As controls, no VTT represents mice, who did not receive VTT-vaccination.

Figure 4

Anti-VTT neutralizing antibody responses after the first (a) and the second (b) MVTT-S challenges. Serum samples collected three weeks after each of the two MVTT-S challenges were included in this experiment. Pooled sera, which contain an equal amount of serum of each vaccinated mouse in each group, were subjected to each experiment. The X-axis represents the serum dilution factor whereas the Y-axis indicates the percentage of viral inhibition. The dashed horizontal line indicates IC₅₀ values. The experiment was repeated twice with similar results obtained. As controls, no VTT represents mice, who did not receive VTT-vaccination.