Combined effect of antiretroviral therapy and blockade of IDO in SIV-infected rhesus macaques.

Increased activity of IDO, which catalyzes the degradation of Trp into kynurenine (Kyn), is observed during HIV/SIV infection, and it may contribute to the persistence of HIV/SIV by suppressing antiviral T cell responses. We administered the IDO inhibitor 1-methyl-d-tryptophan (d-1mT) for 13 days to SIV-infected rhesus macaques receiving antiretroviral therapy (ART). d-1mT treatment increased the plasma levels of Trp, without reducing the levels of Kyn, suggesting only a partial effect on IDO enzymatic activity. Surprisingly, d-1mT significantly reduced the virus levels in plasma and lymph nodes of ART-treated animals with incomplete responsiveness to ART. In SIV-infected animals that were not receiving ART, d-1mT was ineffective in reducing the plasma viral load and had only a marginal effect on the plasma Kyn/Trp ratio. Increased IDO and TGF-beta mRNA expression in lymph nodes of ART-treated macaques after d-1mT treatment suggested that compensatory counterregulatory mechanisms were activated by d-1mT, which may account for the lack of effect on plasma Kyn. Finally, d-1mT did not interfere with the ART-induced T cell dynamics in lymph nodes (increased frequency of total CD4 T cells, increase of CD8 T cells expressing the antiapoptotic molecule Bcl2, and reduction of regulatory T cells). Thus, d-1mT appeared to synergize with ART in inhibiting viral replication and did not interfere with the beneficial immunologic effects of ART. Further studies are required to elucidate the immunologic or virologic mechanism by which d-1mT inhibited SIV replication in vivo.

Reduced protection from simian immunodeficiency virus SIVmac251 infection afforded by memory CD8+ T cells induced by vaccination during CD4+ T-cell deficiency.

Adaptive CD4(+) and CD8(+) T-cell responses have been associated with control of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) replication. Here, we have designed a study with Indian rhesus macaques to more directly assess the role of CD8 SIV-specific responses in control of viral replication. Macaques were immunized with a DNA prime-modified vaccinia virus Ankara (MVA)-SIV boost regimen under normal conditions or under conditions of antibody-induced CD4(+) T-cell deficiency. Depletion of CD4(+) cells was performed in the immunized macaques at the peak of SIV-specific CD4(+) T-cell responses following the DNA prime dose. A group of naïve macaques was also treated with the anti-CD4 depleting antibody as a control, and an additional group of macaques immunized under normal conditions was depleted of CD8(+) T cells prior to challenge exposure to SIV(mac251). Analysis of the quality and quantity of vaccine-induced CD8(+) T cells demonstrated that SIV-specific CD8(+) T cells generated under conditions of CD4(+) T-cell deficiency expressed low levels of Bcl-2 and interleukin-2 (IL-2), and plasma virus levels increased over time. Depletion of CD8(+) T cells prior to challenge exposure abrogated vaccine-induced protection as previously shown. These data support the notion that adaptive CD4(+) T cells are critical for the generation of effective CD8(+) T-cell responses to SIV that, in turn, contribute to protection from AIDS. Importantly, they also suggest that long-term protection from disease will be afforded only by T-cell vaccines for HIV that provide a balanced induction of CD4(+) and CD8(+) T-cell responses and protect against early depletion of CD4(+) T cells postinfection.

Immune activation driven by CTLA-4 blockade augments viral replication at mucosal sites in simian immunodeficiency virus infection.

The importance of chronic immune activation in progression to AIDS has been inferred by correlative studies in HIV-infected individuals and in nonhuman primate models of SIV infection. Using the SIV(mac251) macaque model, we directly address the impact of immune activation by inhibiting CTLA-4, an immunoregulatory molecule expressed on activated T cells and a subset of regulatory T cells. We found that CTLA-4 blockade significantly increased T cell activation and viral replication in primary SIV(mac251) infection, particularly at mucosal sites, and increased IDO expression and activity. Accordingly, protracted treatment with anti-CTLA-4 Ab of macaques chronically infected with SIV(mac251) decreased responsiveness to antiretroviral therapy and abrogated the ability of therapeutic T cell vaccines to decrease viral set point. These data provide the first direct evidence that immune activation drives viral replication, and suggest caution in the use of therapeutic approaches for HIV infection in vivo that increase CD4(+) T cell proliferation.

Interleukin-15 but not interleukin-7 abrogates vaccine-induced decrease in virus level in simian immunodeficiency virus mac251-infected macaques.

The loss of CD4(+) T cells and the impairment of CD8(+) T cell function in HIV infection suggest that pharmacological treatment with IL-7 and IL-15, cytokines that increase the homeostatic proliferation of T cells and improve effector function, may be beneficial. However, these cytokines could also have a detrimental effect in HIV-1-infected individuals, because both cytokines increase HIV replication in vitro. We assessed the impact of IL-7 and IL-15 treatment on viral replication and the immunogenicity of live poxvirus vaccines in SIV(mac251)-infected macaques (Macaca mulatta). Neither cytokine augmented the frequency of vaccine-expanded CD4(+) or CD8(+) memory T cells, clonal recruitment to the SIV-specific CD8(+) T cell pool, or CD8(+) T cell function. Vaccination alone transiently decreased the viral set point following antiretroviral therapy suspension. IL-15 induced massive proliferation of CD4(+) effector T cells and abrogated the ability of vaccination to decrease set point viremia. In contrast, IL-7 neither augmented nor decreased the vaccine effect and was associated with a decrease in TGF-beta expression. These results underscore the importance of testing immunomodulatory approaches in vivo to assess potential risks and benefits for HIV-1-infected individuals.

Decreased number of CD4+ and CD8+ T cells that express the interleukin-7 receptor in blood and tissues of SIV-infected macaques.

Acute HIV/SIV (human/simian immunodeficiency virus) infection results in severe CD4(+) T cell depletion in lymphoid compartments. During the chronic phase of infection, CD4(+) T cell numbers rebound in blood but remain low in the gut-associated lymphoid tissue (GALT), even when viral replication is suppressed by antiretroviral therapy (ART). Thus, strategies to repopulate lymphoid compartments may ameliorate the clinical outcome of HIV/SIV infection. Interleukin (IL)-7 is a key cytokine for the maintenance of homeostatic proliferation of T cells. In HIV/SIV infection, IL-7 expression is increased, likely to compensate for T cell loss, suggesting that supraphysiological administration of IL-7 could provide additional benefit. However, the ability of T cells to respond to IL-7 is dependent on the level of expression of the IL-7 receptor (IL-7R) in T cells in various body compartments. In here, we investigated the proportion of IL-7R(+) T cells in blood, spleen, gut, and genitourinary tract of healthy and SIV-infected macaques with various degrees of CD4(+) T cell depletion. We found that the percentage of T cells expressing IL-7R was significantly lower in both CD4(+) and CD8(+) T cell subsets in SIV-infected macaques than in healthy animals and this decrease directly correlated with the CD4(+) T cell number. Importantly, the proportion of CD4(+) and CD8(+) T cells expressing IL-7R in blood paralleled that found in tissues. IL-7R(+) T cells within the SIV-specific CD8(+) T cells varied and were lowest in most tissues of viremic macaques, likely reflecting continuous antigen stimulation of effector cells.

Improved vaccine protection from simian AIDS by the addition of nonstructural simian immunodeficiency virus genes.

An HIV-1 vaccine able to induce broad CD4+ and CD8+ T cell responses may provide long-term control of viral replication. In this study we directly assess the relative benefit of immunization with vaccines expressing three structural Ags (Gag, Pol, and Env), three early regulatory proteins (Rev, Tat, and Nef), or a complex vaccine expressing all six Ags. The simultaneous administration of all six Ags during vaccination resulted in Ag competition manifested by a relative reduction of CD8+ T cell and lymphoproliferative responses to individual Ags. Despite the Ag competition, vaccination with all six Ags resulted in a delay in the onset and a decrease in the extent of acute viremia after mucosal challenge exposure to highly pathogenic SIV(mac251). Reduced levels of acute viremia correlated with lower post-set point viremia and long-term control of infection. In immunized animals, virus-specific CD4+ T cell and lymphoproliferative responses were preserved during acute viremia, and the maintenance of these responses predicted the long-term virological outcome. Taken together, these results suggest that the breadth of the immune response is probably more important than high frequency responses to a limited number of epitopes. These data provide the first clear evidence of the importance of nonstructural HIV Ags as components of an HIV-1 vaccine.

CTLA-4 blockade decreases TGF-beta, IDO, and viral RNA expression in tissues of SIVmac251-infected macaques.

Regulatory T (T(reg)) cells are a subset of CD25(+)CD4(+) T cells that constitutively express high levels of cytotoxic T lymphocyte antigen-4 (CTLA-4) and suppress T-cell activation and effector functions. T(reg) cells are increased in tissues of individuals infected with HIV-1 and macaques infected with simian immunodeficiency virus (SIV(mac251)). In HIV-1 infection, T(reg) cells could exert contrasting effects: they may limit viral replication by decreasing immune activation, or they may increase viral replication by suppressing virusspecific immune response. Thus, the outcome of blocking T(reg) function in HIV/SIV should be empirically tested. Here, we demonstrate that CD25(+) T cells inhibit virus-specific T-cell responses in cultured T cells from blood and lymph nodes of SIV-infected macaques. We investigated the impact of CTLA-4 blockade using the anti-CTLA-4 human antibody MDX-010 in SIV-infected macaques treated with antiretroviral therapy (ART). CTLA-4 blockade decreased expression of the tryptophan-depleting enzyme IDO and the level of the suppressive cytokine transforming growth factor-beta (TGF-beta) in tissues. CTLA-4 blockade was associated with decreased viral RNA levels in lymph nodes and an increase in the effector function of both SIV-specific CD4(+) and CD8(+) T cells. Therefore, blunting T(reg) function in macaques infected with SIV did not have detrimental virologic effects and may provide a valuable approach to complement ART and therapeutic vaccination in the treatment of HIV-1 infection.

Subunit recombinant vaccine protects against monkeypox.

The smallpox vaccine Dryvax, a live vaccinia virus (VACV), protects against smallpox and monkeypox, but is contraindicated in immunocompromised individuals. Because Abs to VACV mediate protection, a live virus vaccine could be substituted by a safe subunit protein-based vaccine able to induce a protective Ab response. We immunized rhesus macaques with plasmid DNA encoding the monkeypox orthologs of the VACV L1R, A27L, A33R, and B5R proteins by the intradermal and i.m. routes, either alone or in combination with the equivalent recombinant proteins produced in Escherichia coli. Animals that received only DNA failed to produce high titer Abs, developed innumerable skin lesions after challenge, and died in a manner similar to placebo controls. By contrast, the animals vaccinated with proteins developed moderate to severe disease (20-155 skin lesions) but survived. Importantly, those immunized with DNA and boosted with proteins had mild disease with 15 or fewer lesions that resolved within days. DNA/protein immunization elicited Th responses and binding Ab titers to all four proteins that correlated negatively with the total lesion number. The sera of the immunized macaques recognized a limited number of linear B cell epitopes that are highly conserved among orthopoxviruses. Their identification may guide future efforts to develop simpler, safer, and more effective vaccines for monkeypox and smallpox.

Contrasting effects of low-dose IL-2 on vaccine-boosted simian immunodeficiency virus (SIV)-specific CD4+ and CD8+ T cells in macaques chronically infected with SIVmac251.

IL-2, the first cytokine discovered with T cell growth factor activity, is now known to have pleiotropic effects on T cells. For example, it can promote growth, survival, and differentiation of Ag-selected cells, or facilitate Ag-induced cell death of T cells when Ag persists, and in vivo, it is thought to contribute to the regulation of the size of adaptive T cell response. IL-2 is deficient in HIV-1 infection and has been used in the management of HIV-1-infected individuals undergoing antiretroviral therapy. In this study, we investigated how continuous low-dose IL-2 affected the CD4+ and CD8+ T cell response induced by two inoculations of a canarypox recombinant SIV-based vaccine candidate in healthy macaques chronically infected with SIVmac251. These macaques had normal levels of CD4+ T cells at the beginning of antiretroviral therapy treatment. Vaccination in the presence of IL-2 significantly augmented Gag-specific CD8+ T cell responses, but actually reduced Gag-specific CD4+ T cell responses. Although IL-2 at low doses did not change the overall concentration of circulating CD4+ or CD8+ T cells, it expanded the frequency of CD4+CD25+ T cells. Depletion of the CD4+CD25+ T cells in vitro, however, did not result in a reconstitution of Gag-specific CD4+ responses or augmentation of SIV-specific CD8+ T cell responses. Thus, we conclude that the decrease in virus-specific CD4+ T cell response may be due to IL-2-promoted redistribution of cells from the circulation, or due to Ag-induced cell death, rather than suppression by a T regulatory population.

Equivalent immunogenicity of the highly attenuated poxvirus-based ALVAC-SIV and NYVAC-SIV vaccine candidates in SIVmac251-infected macaques.

Therapeutic immunization of HIV-1-infected individuals may induce and/or enhance HIV-1-specific immune responses and decrease the dependency on antiretroviral drug treatment. However, repeated immunizations with live-recombinant vectors may induce vector-specific immune responses that interfere with the elicitation of vigorous immune responses to the desired antigen. Therefore, the use of mixed-modality vaccinations may be necessary to induce sustained virus-specific immune responses in HIV-1-infected individuals treated with antiretroviral therapy (ART). Thus, the relative immunogenicity of various vaccine modalities needs to be assessed. Here we compared the immunogenicity of two vaccine candidates, the canarypox-based ALVAC-SIV-gag-pol-env (ALVAC-SIV-gpe) and the vaccinia-based NYVAC-SIV-gag-pol-env (NYVAC-SIV-gpe), in rhesus macaques infected with SIVmac251 and treated with ART by 2 weeks postinfection. Both ALVAC-SIV-gpe and NYVAC-SIV-gpe vaccine candidates induced and/or enhanced a virus-specific CD8+ T cell response to a similar extent, as demonstrated by tetramer staining of Gag-specific CD8+ T cells. Similarly, both vaccines elicited comparable lymphoproliferative responses (LPRs) to the SIV p27 Gag and gp120 Env proteins. Thus, both these vaccine modalities alone or in combination may be suitable candidate vaccines for immune therapy of HIV-1-infected individuals.

A novel chimeric Rev, Tat, and Nef (Retanef) antigen as a component of an SIV/HIV vaccine.

The human immunodeficiency virus type 1 (HIV-1) regulatory proteins Rev, Tat, and Nef are expressed at early time post-infection and represent attractive targets to be included in a vaccine candidate for AIDS. However, the putative immunosuppressive activities of some of these proteins may limit their immunogenicity. To circumvent these issues, a novel chimeric polyprotein vaccine candidate (Retanef), comprising genetically modified and re-assorted rev, tat, and nef open reading frames of simian immunodeficiency virus (SIV), was constructed and optimized for its expression in mammalian cells. Retanef encodes a protein of approximately 55 kDa localized primarily in the cytoplasm of transfected cells. The Retanef gene expressed in context of an eucaryotic expression vector (DNA-SIV-Retanef) or cloned into a highly attenuated poxvirus-based NYVAC vector (NYVAC-SIV-Retanef) was used to immunize either naive rhesus macaques or macaques chronically infected with SIVmac251 undergoing anti-retroviral therapy (ART). Three immunizations of naive macaques with DNA-SIV-Retanef followed by a single NYVAC-SIV-Retanef boost induced a response to the Mamu-A(*)01-restricted Tat epitope (Tat_SL8, TTPESANL) demonstrated by staining with a specific tetramer and by direct cytolytic activity assays, as well as responses to Rev, Tat and Nef proteins demonstrated by ELISPOT assays using overlapping peptide pools encompassing the entire proteins. Immunization of infected macaques with either DNA-SIV-Retanef or NYVAC-SIV-Retanef expanded the frequency of Tat-specific tetramer-staining cells by two- to seven-fold. No adverse effects were observed in either naive or SIV-infected rhesus macaques. Thus, an analogous HIV-1-based chimeric vaccine may represent useful component of an HIV-1 vaccine.

High frequency of virus-specific CD8+ T cells in the central nervous system of macaques chronically infected with simian immunodeficiency virus SIVmac251.

Infection with human immunodeficiency virus or simian immunodeficiency virus (SIV) induces virus-specific CD8(+) T cells that traffic to lymphoid and nonlymphoid tissues. In this study, we used Gag-specific tetramer staining to investigate the frequency of CD8(+) T cells in peripheral blood and the central nervous system of Mamu-A*01-positive SIV-infected rhesus macaques. Most of these infected macaques were vaccinated prior to SIVmac251 exposure. The frequency of Gag(181-189) CM9 tetramer-positive cells was consistently higher in the cerebrospinal fluid and the brain than in the blood of all animals studied and did not correlate with either plasma viremia or CD4(+)-T-cell level. Little or no infection in the brain was documented for most animals by nucleic acid sequence-based amplification or in situ hybridization. These data suggest that this Gag-specific response may contribute to the containment of viral replication in this locale.

Emergence of cytotoxic T lymphocyte escape mutants following antiretroviral treatment suspension in rhesus macaques infected with SIVmac251.

Structured treatment interruption (STI) of antiretroviral drugs has been proposed as an alternative approach for managing patients infected with HIV-1. While STI is thought to spare drug-related side effects and enhance the HIV-1-specific immune response, the long-lasting clinical benefit of this approach remains uncertain, particularly in patients with long-standing HIV-1 infection. Here, we investigated the basis of unabated virological replication following different STI regimens in rhesus macaques that expressed the MHC class I Mamu-A*01 molecule treated during acute and long-standing infection with SIVmac251. An amino acid change at the anchor residue within the immunodominant Mamu-A*01-restricted Gag(181-189) CM9 epitope (T --> A) in one of six macaques with acute SIVmac251 infection and in three of four macaques with long-standing SIVmac251 infection (T --> A; T --> S; S --> C) was found in the majority of plasma virus. These amino acid changes have been shown to severely decrease binding of the corresponding peptides to the Mamu-A*01 molecule and, in the case of the T --> A change, escape from CTL. In one macaque with long-standing SIVmac251 infection, a mutation emerged that conferred resistance to one of the antiretroviral drugs (PMPA) as well. These results provide insights into the mechanism underlying the limited capacity of repeated interruption of antiretroviral therapy as an approach to restrain viral replication. In addition, these data also suggest that interruption of therapy may be less effective in chronic infection because of preexisting immune escape and that immune escape is a risk of interruption of therapy.

Containment of simian immunodeficiency virus infection in vaccinated macaques: correlation with the magnitude of virus-specific pre- and postchallenge CD4+ and CD8+ T cell responses.

Macaques infected with the SIV strain SIVmac251 develop a disease closely resembling human AIDS characterized by high viremia, progressive loss of CD4(+) T cells, occurrence of opportunistic infection, cachexia, and lymphomas. We report in this study that vaccination with the genetically attenuated poxvirus vector expressing the structural Ags of SIVmac (NYVAC-SIV-gag, pol, env) in combination with priming with DNA-SIV-gag, env resulted in significant suppression of viremia within 2 mo after mucosal exposure to the highly pathogenic SIVmac251 in the majority of vaccinated macaques. The control of viremia in these macaques was long lasting and inversely correlated to the level of both pre- and postchallenge Gag-specific lymphoproliferative responses, as well as to the level of total SIV-specific CD4(+) T lymphocyte responses at the peak of acute viremia as detected by intracellular cytokine-staining assay. Viremia containment also correlated with the frequency of the immunodominant Gag(181-189)CM9 epitope-specific CD8(+) T cells present before the challenge or expanded during acute infection. These data indicate, for the first time, the importance of vaccine-induced CD4(+) Th cell responses as an immune correlate of viremia containment. The results presented in this work also further demonstrate the potential of a DNA-prime/attenuated poxvirus-boost vaccine regimen in an animal model that well mirrors human AIDS.

Avipox-based simian immunodeficiency virus (SIV) vaccines elicit a high frequency of SIV-specific CD4+ and CD8+ T-cell responses in vaccinia-experienced SIVmac251-infected macaques.

The ability of ALVAC- or fowlpox-based simian immunodeficiency virus (SIV) vaccines to boost SIV-specific CD4+ and CD8+ T-cell responses was tested in 10 vaccinia-experienced macaques infected with SIVmac251. The CD8+ T-cell response to the dominant Gag(181-189) CM9 was quantitated in seven Mamu-A*01-positive macaques by tetramer staining, by ex vivo cytotoxic T-lymphocyte (CTL) activity, and by intracellular cytokine staining (ICS) with the specific Gag(181-189) CM9 peptide. The overall CD8+ T-cell response to Gag was assessed using a peptide pool encompassing the entire Gag protein followed by measurement of TNF-alpha production in ICS assay. Similarly, virus-specific CD4+ T-cell responses were measured by ICS for TNF-alpha following stimulation with the Gag-overlapping peptide and by proliferative response following stimulation with purified p27 Gag. The two vaccine modalities effectively boosted both CD4+ and CD8+ SIV-specific T-cell response despite prior exposure to the vaccinia-derivative NYVAC vector, suggesting that sequential boosting with either avipox-based vector vaccine candidate is a realistic approach in immune therapy of human immunodeficiency virus type 1 (HIV-1)-infected individuals.

Recombinant interleukin-7 induces proliferation of naive macaque CD4+ and CD8+ T cells in vivo.

Interleukin-7 (IL-7) regulates T-cell homeostasis, and its availability is augmented in lymphopenic hosts. Naive CD8+ T cells transferred to lymphopenic mice acquire a memory-like phenotype, raising the possibility that IL-7 is the biological mediator of this effect. Here, we provide direct evidence that IL-7 induces the acquisition of memory-cell markers not only in CD8+ T cells but also in CD4+ T-cell subsets in immune-competent Indian rhesus macaques. The increase of these memory-like populations was dependent on the dose of the cytokine, and these cells were found in the blood as well as secondary lymphoid organs. Memory-like CD4+ and CD8+ T cells acquired the ability to secrete tumor necrosis factor alpha and, to a lesser extent, gamma interferon following stimulation with a cognate antigen. The phenotypic change observed in naive T cells was promptly reversed after discontinuation of IL-7. Importantly, IL-7 induced cycling of both CD4+ and CD8+ central memory and effector memory T cells, demonstrating its contribution to the maintenance of the entire T-cell pool. Thus, IL-7 may be of benefit in the treatment of iatrogenic or virus-induced T-cell depletion.

Prior DNA immunization enhances immune response to dominant and subdominant viral epitopes induced by a fowlpox-based SIVmac vaccine in long-term slow-progressor macaques infected with SIVmac251.

A therapeutic vaccine for individuals infected with HIV-1 and treated with antiretroviral therapy (ART) should be able to replenish virus-specific CD4+ T-cells and broaden the virus-specific CD8+ T-cell response in order to maintain CD8+ T-cell function and minimize viral immune escape after ART cessation. Because a combination of DNA and recombinant poxvirus vaccine modalities induces high levels of virus-specific CD4+ T-cell response and broadens the cytolytic activity in naive macaques, we investigated whether the same results could be obtained in SIVmac251-infected macaques. The macaques studied here were long-term nonprogressors that naturally contained viremia but were nevertheless treated with a combination of antiviral drugs to assess more carefully the effect of vaccination in the context of ART. The combination of a DNA expressing the gag and pol genes (DNA-SIV-gp) of SIVmac239 followed by a recombinant fowlpox expressing the same SIVmac genes (FP-SIV-gp) was significantly more immunogenic than two immunizations of FP-SIV-gp in SIVmac251-infected macaques treated with ART. The DNA/FP combination significantly expanded and broadened Gag-specific T-cell responses measured by tetramer staining, ELISPOT, and intracellular cytokine staining and measurement of ex vivo cytolytic function. Importantly, the combination of these vaccine modalities also induced a sizeable expansion in most macaques of Gag-specific CD8-(CD4+) T-cells able to produce TNF-alpha. Hopefully, this modality of vaccine combination may be useful in the clinical management of HIV-1-infected individuals.

Modeling a safer smallpox vaccination regimen, for human immunodeficiency virus type 1-infected patients, in immunocompromised macaques.

We have modeled smallpox vaccination with Dryvax (Wyeth) in rhesus macaques that had depletion of CD4(+) T cells induced by infection with simian immunodeficiency virus or simian/human immunodeficiency virus. Smallpox vaccination induced significantly larger skin lesions in immunocompromised macaques than in healthy macaques. Unexpectedly, "progressive vaccinia" was infrequent. Vaccination of immunocompromised macaques with the genetically-engineered, replication-deficient poxvirus NYVAC, before or after retrovirus infection, was safe and lessened the severity of Dryvax-induced skin lesions. Neutralizing antibodies to vaccinia were induced by NYVAC, even in macaques with severe CD4(+) T cell depletion, and their titers inversely correlated with the time to complete resolution of the skin lesions. Together, these results provide the proof of concept, in macaque models that mirror human immunodeficiency virus type 1 infection, that a prime-boost approach with a highly attenuated poxvirus followed by Dryvax increases the safety of smallpox vaccination, and they highlight the importance of neutralizing antibodies in protection against virulent poxvirus.