Induction of a mucosal cytotoxic T-lymphocyte response by intrarectal immunization with a replication-deficient recombinant vaccinia virus expressing human immunodeficiency virus 89.6 envelope protein

Abstract

To improve the safety of recombinant vaccinia virus vaccines, modified vaccinia virus Ankara (MVA) has been employed, because it has a replication defect in most mammalian cells. Here we apply MVA to human immunodeficiency virus type 1 (HIV-1) vaccine development by incorporating the envelope protein gp160 of HIV-1 primary isolate strain 89.6 (MVA 89.6) and use it to induce mucosal cytotoxic-T-lymphocyte (CTL) immunity. In initial studies to define a dominant CTL epitope for HIV-1 89.6 gp160, we mapped the epitope to a sequence, IGPGRAFYAR (from the V3 loop), homologous to that recognized by HIV MN loop-specific CTL and showed that HIV-1 MN-specific CTLs cross-reactively recognize the corresponding epitope from strain 89.6 presented by H-2Dd. Having defined the CTL specificity, we immunized BALB/c mice intrarectally with recombinant MVA 89.6. A single mucosal immunization with MVA 89.6 was able to elicit long-lasting antigen-specific mucosal (Peyer's patch and lamina propria) and systemic (spleen) CTL responses as effective as or more effective than those of a replication-competent vaccinia virus expressing 89.6 gp160. Immunization with MVA 89.6 led to (i) the loading of antigen-presenting cells in vivo, as measured by the ex vivo active presentation of the P18-89.6 peptide to an antigen-specific CTL line, and (ii) the significant production of the proinflammatory cytokines (interleukin-6 and tumor necrosis factor alpha) in the mucosal sites. These results indicate that nonreplicating recombinant MVA may be at least as effective for mucosal immunization as replicating recombinant vaccinia virus.

FIG. 1

(A) Recognition by the HIV-1 MN-specific CTL cell line of P815 target cells pulsed with different concentrations of P18MN peptides with substitutions. P815 targets were tested in the presence or absence of various concentrations of peptides as shown. (B) Killing of target cells pulsed with different concentrations of the peptides is compared with killing of unpulsed targets at an effector-to-target ratio (E:T) of 20:1. For both panels, standard errors of the means of triplicate cultures were all <5% of the mean.

FIG. 2

Induction of the 89.6-specific splenic CTL responses 2 and 4 weeks after i.r. immunization with replication-deficient MVA 89.6 and WR 89.6 expressing the HIV-1 envelope protein of HIV-1 strain 89.6. Induction of SP CTL responses 2 weeks (A) and 4 weeks (B) after i.r. immunization with the indicated recombinant viral vectors at the doses indicated to the right of panel B. Two weeks after i.r. immunization, SP cells were cultivated in vitro with 1 μM concentrations of the indicated peptides. SP cells were stimulated in vitro for two 7-day culture periods before assay. Cytolytic activity of CTL lines was measured by a 4-h assay with ⁵¹Cr-labeled P815 DBA/2 mastocytoma cell targets as described in Materials and Methods. E:T, effector-to-target ratio.

FIG. 3

Induction of PP and LP P18-89.6R10-specific CTL responses 2 weeks, 4 weeks, and 6 months after i.r. immunization with replication-deficient MVA 89.6 and WR 89.6. Induction of PP 89.6R10 (A)- and LP 89.6R10 (B)-specific CTL responses at the indicated times after i.r. immunization with MVA 89.6 and WR 89.6. Mice were immunized at doses as indicated to the right. The percent specific ⁵¹Cr release was calculated as described in the legend to Fig. 2. E:T, effector-to-target ratio.

FIG. 4

Induction of systemic and mucosal CTL responses 4 weeks after i.p. immunization with replication-deficient MVA 89.6 and WR 89.6. Mice were immunized i.p. with 10⁸ PFU of either virus, and responses were measured 4 weeks later as described in the legends to Fig. 2 and 3. E:T, effector-to-target ratio.

FIG. 5

Ex vivo antigen presentation activity of cells from mucosal and systemic sites for mucosal and systemic CTL lines after i.r. and i.p. immunization with replication-deficient MVA 89.6 and WR 89.6. The same mice were immunized by both i.r. and i.p. routes with 2 × 10⁸ PFU, and 48 h later APC were separated by MACS sorting for CD11c-positive cells from SP and PP. The PP (A) and SP (B) APC were cultivated in vitro in 96-well U-bottom plates with P18-89.6R10-specific or P18IIIB-specific CTL lines from SP and PP. The activation of the T-cell lines was studied by measuring IFN-γ concentration in culture supernatants. Similar results were obtained in three replicate experiments. The error bars represent the standard errors of the means.

FIG. 6

Proinflammatory cytokine (IL-6 and TNF-α) production by mucosal and systemic MC 48 h after i.r. immunization with replication-deficient MVA 89.6 and WR 89.6, measured after 4 days of in vitro stimulation with LPS (10 ng/ml). Cells were cultured at 250,000 per well in 96-well plates. Cytokines in the culture supernatants were measured by ELISA as described in Materials and Methods. The error bars represent the standard errors of the means.

FIG. 7

IL-6 production by MC 3 weeks after immunization with replication-deficient MVA 89.6 and WR 89.6, measured after 4 days of in vitro stimulation with P18-89.6R10 Env peptide (1 μM). Cells were cultured at 250,000 per well in 96-well plates. Cytokines in the culture supernatants were measured by ELISA as described in Materials and Methods. The error bars represent the standard errors of the means.