Mucosal immunization with HIV-1 peptide vaccine induces mucosal and systemic cytotoxic T lymphocytes and protective immunity in mice against intrarectal recombinant HIV-vaccinia challenge

Abstract

Mucosal tissues are major sites of HIV entry and initial infection. Thus, the induction of a mucosal cytotoxic T lymphocyte (CTL) response is an important feature for an effective HIV vaccine. However, little is known about approaches to induce such a protective CTL response in the mucosa. Here for the first time we show that intrarectal immunization with a synthetic, multideterminant HIV peptide plus cholera toxin adjuvant induced long-lasting, antigen-specific CTL memory in both the inductive (Peyer's patch) and effector (lamina propria) mucosal sites, as well as in systemic sites (spleen), whereas systemic immunization induced specific CTL only in the spleen. Cholera toxin adjuvant, while enhancing the response, was not essential. The CTL recognized target cells either pulsed with HIV peptide or expressing endogenous whole envelope glycoprotein of Mr 160,000 (gp160). Exploring the requirements for CTL induction, we show that mucosal CTL responses are both interleukin 12 and interferon-gamma dependent by using antibody-treated and knock-out mice. Finally, to determine whether a mucosal response is actually protective against local mucosal challenge with virus, we show that intrarectal immunization with the synthetic HIV peptide vaccine protected mice against infection via mucosal challenge with a recombinant vaccinia virus expressing HIV-1IIIB gp160. These studies provide an approach to development of an HIV vaccine that induces CTL immunity in the mucosal and systemic immune systems and protects against mucosal infection with a virus expressing HIV-1 gp160.

Figure 1

(A) IR HIV peptide immunization induces both mucosal and systemic long-lasting immunity. In contrast, systemic immunization induces only systemic CTL. Closed squares show killing of P18IIIB-I10-pulsed targets and open diamonds show killing on unpulsed targets. (B) Induction of the mucosal and systemic CTL responses by different routes of immunization with synthetic peptide HIV vaccine. Killing of peptide-pulsed targets (▪) is compared with killing of unpulsed targets (□) at an effector-to-target ratio of 50:1. Similar results were obtained at ratios of 25 and 12.5:1. In both A and B, SEM of triplicate cultures were all<5% of the mean.

Figure 2

CT adjuvant is not essential for IR peptide vaccine induction of CTL. Mucosal peptide-induced CTL lyse targets endogenously expressing HIV gp160 envelope protein. Two different cell lines were used as a target cells: (i) 15–12 cells (BALB/c 3T3 fibroblasts transfected with HIV-1IIIB gp160 and endogenously expressing HIV gp160) (•, Left). These were compared with 18 Neo BALB/c 3T3 fibroblasts transfected with Neo^R alone as a control (▵, Left). (ii) P815 targets were tested in the presence (▪) or absence (◊) of I10 peptide (1 μM). SEM of triplicate cultures were all <5% of the mean.

Figure 3

(A) IR peptide induction of CTL is IL-12 dependent. One day before and one day after immunization with peptide mice were treated i.p. with anti-IL12 antibody (0.5 mg per injection; 4 mg per mouse total dose (Right) or were untreated (Left). For testing the peptide specificity of CTL, ⁵¹Cr-labeled P815 targets were pulsed with peptide at the beginning of the assay (▪) or without peptide (◊). (B) IR peptide induction of CTL is IFN-γ dependent. Wild-type BALB/c mice (Left) or IFN-γ^−/− (Right) were immunized as in A, and tested at 5 weeks. Killing of peptide-pulsed targets (▪) is compared with killing of unpulsed targets (░⃞) at an effector-to-target ratio of 50:1. Similar results were obtained at ratios of 25 and 12.5:1. (A and B) SEM of triplicate cultures were all <5% of the mean.

Figure 4

IR peptide immunization protects against IR challenge with HIV-gp160 expressing recombinant vaccinia virus. The left bar (▪) shows virus as the log of the number of pfus in ovaries of unimmunized mice and the right bar (□) shows virus titer in the ovaries of immunized mice. Bars = SEM of five mice per group. The difference is significant at P < 0.005 by Student’s t test.