A new genetic vaccine platform based on an adeno-associated virus isolated from a rhesus macaque

Abstract

We created a hybrid adeno-associated virus (AAV) from two related rhesus macaque isolates, called AAVrh32.33, and evaluated it as a vaccine carrier for human immunodeficiency virus type 1 (HIV-1) and type A influenza virus antigens. The goal was to overcome the limitations of vaccines based on other AAVs, which generate dysfunctional T-cell responses and are inhibited by antibodies found in human sera. Injection of a Gag-expressing AAVrh32.33 vector into mice resulted in a high-quality CD8(+) T-cell response. The resulting Gag-specific T cells express multiple cytokines at high levels, including interleukin-2, with many having memory phenotypes; a subsequent boost with an adenovirus vector yielded a brisk expansion of Gag-specific T cells. A priming dose of AAVrh32.33 led to high levels of Gag antibodies, which exceed levels found after injection of adenovirus vectors. Importantly, passive transfer of pooled human immunoglobulin into mice does not interfere with the efficacy of AAVrh32.33 expressing nucleoproteins from influenza virus, as measured by protection to a lethal dose of influenza virus, which is consistent with the very low seroprevalence to this virus in humans. Studies of macaques with vectors expressing gp140 from HIV-1 (i.e., with AAVrh32.33 as the prime and simian adenovirus type 24 as the boost) demonstrated results similar to those for mice with high-level and high-quality CD8(+) T-cell responses to gp140 and high-titered neutralizing antibodies to homologous HIV-1. The biology of this novel AAV hybrid suggests that it should be a preferred genetic vaccine carrier, capable of generating robust T- and B-cell responses.

FIG. 1.

Impact of AAV capsids on the transgene-specific T-cell response. CB6F1 mice were injected i.m. with 26 AAV isolates from clades A, B, C, D, E, and F, as well as with isolates that do not cluster in a particular clade (X). (A) Antigen-specific CD8⁺ T-cell responses induced by different AAV vectors expressing HIV Gag were assayed by Gag tetramer staining at 3 weeks post-vector administration. (B) HIV Gag tetramer-specific CD8⁺ T-cell responses were further analyzed for memory phenotype, and data are shown as percentages of tetramer HIV Gag-positive TEM (CD127⁺/CD62L⁻) in total CD8⁺ T cells. Data are shown as mean results with standard deviations (n = 4).

FIG. 2.

Characterization of the immune response following AAV HIV Gag vaccination. Gag tetramer responses were monitored over time in CB6F1 mice that received i.m. administration of AAV8, AAVrh32.33, or SAdV24 encoding HIV Gag at two doses, 3 × 10⁹ (A) and 3 × 10¹⁰ (B) GC. (C) Phenotypic characterization. Three weeks following AAV vector administration, lymphocytes were isolated from whole blood and spleens of mice injected with AAV2/AAV8, SAdV24, and AAV2/AAVrh32.33 Gag-expressing vectors (3 × 10¹⁰ GC). Per lymphocyte source, on the left, a scatter plot of CD8⁺ T cells of a representative mouse (n = 5) illustrates the tetramer positivity (Tet⁺), as quantified by the percentage of Tet⁺ within the CD8⁺ population. On the right, CD62L marker expression versus that of CD127 is represented. Memory phenotype is represented with the Tet⁺/CD8⁺ population overlaid in red on the total lymphocyte population in black. The percentages of Tet⁺/CD8⁺ T cells and TEM⁺/Tet⁺ T cells are indicated within the individual panels. (D) Gag-specific antibody responses. Sera from mice immunized with AAV8, AAVrh32.33, or SAdV24 at the dose of 3 × 10¹⁰ or 1 × 10¹¹ GC were analyzed for anti-Gag IgG, and the results are presented as means with standard deviations (n = 4).

FIG. 3.

Dose response-effect of AAV prime on T- and B-cell responses. AAV8 or AAVrh32.33 expressing HIV Gag vectors were injected i.m. into CB6F1 mice (n = 4) at doses of 3 × 10⁹, 3 × 10¹⁰, and 1 × 10¹¹ GC; 8 weeks later, these mice received 1 × 10¹⁰ particles of SAdV24 Gag in parallel to a group of age-matched naïve mice. The production of CD8⁺ T-cell Gag tetramer responses from PBMCs (A and B) and anti-p24 Gag antibodies from sera (C and D) was monitored. The data are presented as the maximum response following the prime administration (peak prime), the measurement immediately prior to the boost administration (preboost), and peak response following the boost administration (peak boost). The value obtained when the same dose of SAdV24 was injected into naïve mice was called SAdV24 peak. Statistically significant differences (P < 0.05) between the peak boost and preboost data are marked with an asterisk. Data are shown as mean results with standard deviations.

FIG. 4.

Histopathology and Gag antigen expression levels around injection site. Mice were i.m. immunized with 3 × 10¹⁰ GC of AAV8 or AAVrh32.33 expressing HIV Gag. At day 7, 30, 60, 90, and 180 postimmunization, muscle tissue from the injected areas was obtained and analyzed for inflammation, infiltration, and Gag mRNA levels. (A) At 60 days post-vector administration, infiltrated cells were immunophenotyped with antibodies against CD8⁺ (dark blue), CD4⁺ (green), and FoxP3 (red). The bottom panels show additional staining with DAPI (light blue). (B) Table scoring the degree of infiltration at different time points and compared with SAdV24 as positive control (−, no infiltrates; ++++, strongest infiltration observed). Data for individual mice in each group are shown. (C) Gag mRNA levels from muscle injected with AAV8 or AAVrh32.33 by TaqMan. Data are shown as mean results with standard deviations.

FIG. 5.

Vaccine efficacy in the presence of pooled human Ig. Mice received either PBS or the indicated doses (mg) of pooled human Ig by intravenous administration prior to immunization. Recipient mice were immunized with 3 × 10¹⁰ GC of AAV2, -7, -8, or -rh32.33 expressing HIV Gag. The Gag-specific tetramer T-cell responses (A) and Gag-specific antibody responses (B) were measured at 3 weeks postimmunization in the presence or absence of pooled human Ig. The data from 8 to 10 mice in each group are shown as the mean results with standard deviations. Statistically significant differences (P < 0.05) between the IgG-treated vector group and PBS control group are marked with an asterisk. (C) Mice passively transferred with PBS or pooled human Ig (8 or 80 mg) were immunized with 1 × 10¹¹ GC of AAV8 or AAVrh32.33 expressing influenza virus type A NP or AAVrh32.33 expressing LacZ as the control. At day 35 postimmunization, all mice were challenged with 10 50% lethal doses of influenza virus strain PR8. The survival data analysis was plotted using GraphPad Prism (version 5.00 for Windows).

FIG. 6.

HIV gp140-specific IFN-γ response following AAV-Ad prime-boost vaccine regimen in cynomolgus macaques. Cynomolgus macaques were primed i.m. with 1 × 10¹² GC of AAV8 (n = 3) (A) or AAVrh32.33 (n = 4) (B) expressing HIV W61D gp140. Ten months after the prime administration, animals were boosted with 2 × 10¹¹ particles of SAdV24 HIV gp140. A control group of two naïve cynomolgus macaques received the same dose of SAdV24 HIV gp140. Freshly isolated PBMCs were assessed for IFN-γ ELISPOT responses after in vitro exposure to peptide pools A, B, and C spanning the HIV-1 gp140 proteins at different time points. The time points of boosting with SAdV24 HIV gp140 are indicated by arrows. Each bar represents the accumulative number of IFN-γ spot-forming cells (SFC) for each of the peptide pools. PBMCs without peptide stimulation showed <50 IFN-γ spot-forming cells. Animal C10363 died at week 59 of an intestinal volvulus.

FIG. 7.

Polyfunctional HIV gp140-specific T-cell responses following AAV-adenovirus prime-boost administration in cynomolgus macaques. Freshly isolated PBMCs were stimulated with the HIV gp140 peptide library. The secreted cytokines, including IFN-γ, IL-2, and TNF-α, as well as CD4⁺ and CD8⁺ surface markers, were stained by fluorescence-labeled antibodies and measured by flow cytometry as previously described (4). Representative plots are showing the percentages of total cytokine-secreting CD4⁺ (A) and CD8⁺ (B) T-cell responses 3 and 23 weeks post-AAV prime injection and 1 week post-SAdv24 boost injection (1B). (C and D) IFN-γ-, IL-2-, and TNF-α-cosecreting CD4⁺ and CD8⁺ T cells in response to the HIV-gp140 peptide library were plotted. The time points of boosting with SAdV24 HIV gp140 are indicated by arrows. Individual macaque data are shown. The bars represent the mean percentages of the total response.

FIG. 8.

HIV-specific CD8⁺ T-cell proliferative potential and NAb response following AAV-adenovirus prime-boost administration. (A) At 14 weeks postprime, the levels of CD8⁺ T-cell proliferation were assessed by a CFSE dye dilution assay. CFSE-labeled CD8⁺ T cells were stimulated ex vivo for 6 days with the HIV-1 gp140 peptide library. Proliferating cells were defined as the low-CFSE population among the total CFSE-labeled CD8⁺ population. Samples that had levels greater than four times the nonstimulated controls, and those higher than 1%, are indicated by an asterisk. (B) Anti-HIV NAb titers in sera were assayed at week 0, 4, 20, and 40 after AAV prime and at week 1 (1B), 5 (5B), and 15 (15B) after SAdV24 boost administrations. A control group primed with SAdV24 was also included. The bar represents the mean percentage of the total response.

FIG. 9.

HIV Env-specific IFN-γ responses from tissue-derived lymphocytes of nonhuman primates. At 36 weeks post-SAdV24 boost injection, all immunized macaques were necropsied, monocytes were isolated from the peripheral blood, spleen, mesenteric lymph nodes (LN), and large bowel, and HIV Env-specific IFN-γ responses were measured by IFN-γ ELISPOT assay. SFC, spot-forming cells.