Mucosal vaccination overcomes the barrier to recombinant vaccinia immunization caused by preexisting poxvirus immunity

Abstract

Overcoming preexisting immunity to vaccinia virus in the adult population is a key requirement for development of otherwise potent recombinant vaccinia vaccines. Based on our observation that s.c. immunization with vaccinia induces cellular and antibody immunity to vaccinia only in systemic lymphoid tissue and not in mucosal sites, we hypothesized that the mucosal immune system remains naive to vaccinia and therefore amenable to immunization with recombinant vaccinia vectors despite earlier vaccinia exposure. We show that mucosal immunization of vaccinia-immune BALB/c mice with recombinant vaccinia expressing HIV gp160 induced specific serum antibody and strong HIV-specific cytotoxic T lymphocyte responses. These responses occurred not only in mucosal but also in systemic lymphoid tissue, whereas systemic immunization was ineffective under these circumstances. In this context, intrarectal immunization was more effective than intranasal immunization. Boosting with a second dose of recombinant vaccinia was also more effective via the mucosal route. The systemic HIV-specific cytotoxic T lymphocyte response was enhanced by coadministration of IL-12 at the mucosal site. These results also demonstrate the independent compartmentalization of the mucosal versus systemic immune systems and the asymmetric trafficking of lymphocytes between them. This approach to circumvent previous vaccinia immunity may be useful for induction of protective immunity against infectious diseases and cancer in the sizable populations with preexisting immunity to vaccinia from smallpox vaccination.

Figure 1

Absence of P18–89.6R10- and P18-I10-specific CTL responses in the SP and PP in mice with preexisting immunity to vaccinia after s.c. reimmunization with recombinant viruses (a and b) and induction of P18–89.6R10- and P18-I10-specific CTL responses in the SP and PP in mice with preexisting immunity to vaccinia after IR reimmunization with recombinant viruses (c and d). BALB/c mice were immunized s.c. with vSC8 vaccinia virus at 5 × 10⁷ pfu (a–d). One month later these mice and control naive mice were reimmunized either s.c. (a and b) or IR (c and d) with either recombinant vaccinia MVA89.6 or vPE16 at 1 × 10⁸ pfu. Three weeks later the induction of P18–89.6R10- and P18-I10-specific CTL responses were studied in the SP (a and c) and PP (b and d). Results are means of 10 mice per group, with SP tested individually and PP pooled to obtain sufficient cell numbers. The percent specific ⁵¹Cr release was calculated as described in Materials and Methods. E:T, effector-to-target ratio.

Figure 2

Comparison of the induction of P18–89.6R10- and P18-I10-specific CTL responses in the SP of mice with preexisting immunity to vaccinia after either IR or IN reimmunization with recombinant viruses. (a) BALB/c mice were immunized s.c. with vSC8 vaccinia virus at 5 × 10⁷ pfu. One month later the mice were reimmunized IN with either recombinant vaccinia vPE16 or MVA89.6 at 1 × 10⁸ pfu. Three weeks later the induction of P18–89.6R10- and P18-I10-specific CTL responses were studied in the SP. The percent specific ⁵¹Cr release was calculated as described in Materials and Methods. E:T, effector-to-target ratio. (b and c) Induction of the systemic HIV-specific CTL responses by different routes of reimmunization of mice with 5 × 10⁷ pfu MVA89.6 (b) or vPE16 (c). Results are means of five mice per group tested individually. Killing of peptide-pulsed targets is compared with killing of unpulsed targets at an E/T of 50:1. Similar results were obtained at ratios of 25 and 12.5:1. In a–c, SEM of triplicate cultures were all <5% of the mean.

Figure 3

Enhancement of the splenic HIV-specific CTL response by mucosal (not systemic) treatment with rmIL-12 in mice with preexisting immunity to vaccinia. BALB/c mice (five mice per group) with preexisting immunity to vaccinia were immunized by the s.c. or IR route with recombinant vaccinia virus with or without 1 μg of rmIL-12 and also on day 5,10 and 15 (a). For s.c. immunization, the IL-12 was given i.p., whereas for IR immunization, the IL-12 was given IR in DOTAP with the virus. Boosting of P18–89.6R10-specific CTL responses in the SP of BALB/c mice (b and c). BALB/c mice (5/group) were immunized either s.c. (b) or IR (c) with MVA89.6 vaccinia virus at 5 × 10⁷ pfu. One month later the mice were reimmunized either s.c. or IR with the same recombinant vaccinia MVA89.6 at 1 × 10⁸ pfu. Three weeks later the induction of P18–89.6R10-specific CTL responses were studied in the SP. The percent specific ⁵¹Cr release was calculated as described in Materials and Methods. E:T, effector-to-target ratio.

Figure 4

Serum anti-gp120 IgG titer in BALB/c mice (5/group) with preexisting immunity to vaccinia after mucosal reimmunization with recombinant replication-deficient vaccinia virus expressing gp160 HIV-1 89.6 (MVA89.6) by different routes. Serum was assayed for the presence of anti-gp120 IgG by ELISA. Results are reported as the reciprocal of last dilution giving a positive ELISA response.