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. 2006 Mar 20;203(3):607-17.
doi: 10.1084/jem.20052005. Epub 2006 Feb 27.

Intensified and protective CD4+ T cell immunity in mice with anti-dendritic cell HIV gag fusion antibody vaccine

Christine Trumpfheller  1 , Jennifer S Finke, Carolina B López, Thomas M Moran, Bruno Moltedo, Helena Soares, Yaoxing Huang, Sarah J Schlesinger, Chae Gyu Park, Michel C Nussenzweig, Angela Granelli-Piperno, Ralph M Steinman
Affiliations

Intensified and protective CD4+ T cell immunity in mice with anti-dendritic cell HIV gag fusion antibody vaccine

Christine Trumpfheller et al. J Exp Med. .

Abstract

Current human immunodeficiency virus (HIV) vaccine approaches emphasize prime boost strategies comprising multiple doses of DNA vaccine and recombinant viral vectors. We are developing a protein-based approach that directly harnesses principles for generating T cell immunity. Vaccine is delivered to maturing dendritic cells in lymphoid tissue by engineering protein antigen into an antibody to DEC-205, a receptor for antigen presentation. Here we characterize the CD4+ T cell immune response to HIV gag and compare efficacy with other vaccine strategies in a single dose. DEC-205-targeted HIV gag p24 or p41 induces stronger CD4+ T cell immunity relative to high doses of gag protein, HIV gag plasmid DNA, or recombinant adenovirus-gag. High frequencies of interferon (IFN)-gamma- and interleukin 2-producing CD4+ T cells are elicited, including double cytokine-producing cells. In addition, the response is broad because the primed mice respond to an array of peptides in different major histocompatibility complex haplotypes. Long-lived T cell memory is observed. After subcutaneous vaccination, CD4+ and IFN-gamma-dependent protection develops to a challenge with recombinant vaccinia-gag virus at a mucosal surface, the airway. We suggest that a DEC-targeted vaccine, in part because of an unusually strong and protective CD4+ T cell response, will improve vaccine efficacy as a stand-alone approach or with other modalities.

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Figures

Figure 1.
Figure 1.
Immunization of T cells with one dose of anti–DEC-p24 fusion mAb vaccine. (A) BALB/c mice were injected i.p. with PBS, maturation stimulus alone (25 μg αCD40 mAb and 50 μg poly IC), 5 μg control Ig-p24 or anti–DEC-p24 mAbs and maturation stimulus, and 5 μg anti–DEC-p24 without maturation stimulus. After 17 d, splenic CD8+ T cells (top) or CD4+ T cells (bottom) were restimulated with CD11c+ DCs and peptide (AMQMLKETI, p24 197–205, 2 μg/ml), HIV gag p24 peptide pools (2 μg/ml), or medium alone for 2 d. IFN-γ secretion was evaluated by ELISPOT. (B) As in A, but immunization of BALB/c mice with graded doses of anti–DEC-p24 and a maturation stimulus. Data are representative of two to four similar experiments with two mice pooled in each experiment.
Figure 2.
Figure 2.
Strong CD4+ T cell responses to a single dose of anti–DEC-p24 fusion mAb vaccine. (A) BALB/c mice were immunized s.c. with graded doses of anti–DEC-p24 or control Ig-p24 mAbs and maturation stimulus. 19 d later, we assessed the percentage of IFN-γ+ CD4+ cells in gated CD3+ splenic T cells using gag p24 peptide pools. One of three similar experiments with two mice pooled in each experiment is shown. (B) As in A, but several experiments showing responses to 5 μg anti–DEC-p24 or control Ig-p24 mAbs with maturation stimulus given either s.c. or i.p. to BALB/c mice. Background activity (<0.1%) of nonvaccinated mice boosted with the HIV gag p24 peptide pools was subtracted from the percentage of IFN-γ+ CD4+ cells. Each point represents two mice pooled in each experiment. (C) BALB/c or C57BL/6 mice were treated s.c. with PBS or 2 and 5 μg anti–DEC-p24 or control Ig-p24 mAbs, respectively, each with maturation stimulus. IFN-γ and IL-2 production was monitored at 19 d. Numbers are the percentage of CD4+ cells producing cytokines to HIV gag p24 peptide pool 3 in BALB/c or pool 1 in C57BL/6 mice. These experiments were repeated two to four times with similar results. (D) Comparison of immune responses of wild-type and DEC-205−/− mice to 3 μg anti–DEC-p24 vaccine and maturation stimulus. IFN-γ secretion in response to HIV gag p24 peptide pools by CD4+ splenocytes as in A. Background frequencies (vaccinated mice boosted in medium only) were <0.01% in the top right quadrants. Data are representative of two similar experiments.
Figure 3.
Figure 3.
Enhanced efficacy of anti–DEC-p24 plus anti-CD40/poly IC relative to DNA and nontargeted protein vaccines. (A) C57BL/6 mice were injected s.c. with maturation stimulus and the proteins indicated above each panel. IFN-γ secretion was evaluated after 18 d in the spleen by intracellular cytokine staining as the percentage of IFN-γ CD4+ cells among CD3+ lymphocytes in response to HIV gag p24 peptide pool 4. Data are one of two to four similar experiments. (B) BALB/c mice were injected with 2 μg anti–DEC-p24 s.c. plus maturation stimulus, one or two doses of 50 μg DNA encoding HIV gag p24 protein i.m., or PBS. IFN-γ secretion in response to HIV gag p24 peptide pool 3 was evaluated as in A. Data shown are one of two to four similar experiments. (C) BALB/c mice were immunized i.p. with 5 μg anti–DEC-p24 or control Ig-p24 antibodies plus maturation stimulus, two doses of 50 μg DNA encoding HIV gag p24 protein i.m., or PBS. IFN-γ secretion was evaluated as in A, but the response to HIV gag p24 peptide pool 2 (containing a dominant peptide presented on MHC class I) and p24 pool 3 (containing a peptide presented on MHC class II) are shown for T cells directly stained for CD8 expression. Data shown are from one of two similar experiments.
Figure 4.
Figure 4.
Breadth and strength of responses to DC-targeted HIV gag vaccine. (A) 5 μg anti–DEC-p24 mAb in combination with maturation stimulus was administered either i.p. to BALB/c mice (top) or s.c. to C57BL/6 (middle) and C3H (bottom) mice. 18 d later, IFN-γ secretion in spleen CD3+ T cells was measured to medium or different HIV gag p24 peptide pools. Data are representative of two to four experiments with two mice pooled in each experiment. (B) C57BL/6 mice were immunized s.c. with 5 μg anti–DEC-p41 or control Ig-p41 mAbs and maturation stimulus. IFN-γ secretion in spleen CD3+ T cells was measured at 15 d in response to medium or different HIV gag p24 and p17 peptide pools. Data shown are from one of three experiments. (C) In three experiments C57BL/6 mice were vaccinated s.c. with 2 μg anti–DEC-p24 with maturation stimulus, and 19 d later, splenocytes were restimulated with graded doses of HIV gag p24 peptide number 6 from pool 1 (QAISPRTLNAWVKVV, p24 aa 145–159) or peptide number 8 from pool 4 (VDRFYKTLRAEQASQ, p24 aa 297–311).
Figure 5.
Figure 5.
Long-lived gag p24-memory to DC-targeted vaccine. BALB/c mice were immunized i.m. with two doses of 50 μg HIV gag p24 DNA or i.p. with 5 μg anti–DEC-p24 and control Ig-p24 mAb with maturation stimulus. C57BL/6 mice were similarly immunized, but the fusion antibodies and maturation stimulus were given s.c. CD4+ T cells were enriched by depleting MHC II+ and CD8+ cells from the lymph node (30 wk after BALB/c immunization) or spleen (at 19 wk for C57BL/6), CFSE labeled, and restimulated with CD11c+ DCs plus the peptide pools as indicated, anti-CD3 and anti-CD28, or medium. Proliferation was evaluated by CFSE dilution at 4 d. Numbers are the percentage of CD4+ proliferating T cells. Control PBS-immunized mice showed similar proliferative responses to control Ig-p24 mAb or two doses of DNA gag p24 vaccine. Data are representative of two to three experiments on the same batch of immunized mice.
Figure 6.
Figure 6.
A single dose of anti–DEC-p24 vaccine elicits CD4- dependent protection at a mucosal surface. (A and B) Groups of four to six BALB/c or C57BL/6 mice were immunized as in Fig. 1 and challenged at 2–3 wk with 5 × 104 PFU of vaccinia-gag intranasally. Weight was measured for 6–7 d, and then lung virus titers were evaluated by plaque assay. Data are representative of two experiments in BALB/c and one in C57BL/6. (C and D) C57BL/6 or IFN-γR knockout mice were immunized s.c. with 2 μg anti–DEC-p24 with maturation stimulus or with PBS and challenged with 2.5 × 104 PFU of vaccinia-gag 20 d later. (E and F) CD4+ T cells were depleted from C57BL/6 immunized mice with three doses of anti-CD4 mAb or control rat Ig 1–3 d before challenge. Data shown are one of three similar experiments. Weights are expressed as average body weight, whereas vaccinia plaque-forming titers (in CV-1 cells) are shown as a mean ± SD.
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