CD40L-adjuvanted DNA/modified vaccinia virus Ankara simian immunodeficiency virus (SIV) vaccine enhances protection against neutralization-resistant mucosal SIV infection.

Here, we show that a CD40L-adjuvanted DNA/modified vaccinia virus Ankara (MVA) simian immunodeficiency virus (SIV) vaccine enhances protection against a pathogenic neutralization-resistant mucosal SIV infection, improves long-term viral control, and prevents AIDS. Analyses of serum IgG antibodies to linear peptides of SIV Env revealed a strong response to V2, with targeting of fewer epitopes in the immunodominant region of gp41 (gp41-ID) and the V1 region as a correlate for enhanced protection. Greater expansion of antiviral CD8 T cells in the gut correlated with long-term viral control.

Vaccine-Induced Linear Epitope-Specific Antibodies to Simian Immunodeficiency Virus SIVmac239 Envelope Are Distinct from Those Induced to the Human Immunodeficiency Virus Type 1 Envelope in Nonhuman Primates.

To evaluate antibody specificities induced by simian immunodeficiency virus (SIV) versus human immunodeficiency virus type 1 (HIV-1) envelope antigens in nonhuman primate (NHP), we profiled binding antibody responses to linear epitopes in NHP studies with HIV-1 or SIV immunogens. We found that, overall, HIV-1 Env IgG responses were dominated by V3, with the notable exception of the responses to the vaccine strain A244 Env that were dominated by V2, whereas the anti-SIVmac239 Env responses were dominated by V2 regardless of the vaccine regimen.

Diminished viral control during simian immunodeficiency virus infection is associated with aberrant PD-1hi CD4 T cell enrichment in the lymphoid follicles of the rectal mucosa.

The inhibitory receptor programmed death-1 (PD-1) has been shown to regulate CD8 T cell function during chronic SIV infection; however, its role on CD4 T cells, specifically in the gut-associated lymphoid tissue, is less well understood. In this study, we show that a subset of CD4 T cells expresses high levels of PD-1 (PD-1(hi)) in the rectal mucosa, a preferential site of virus replication. The majority of these PD-1(hi) CD4 T cells expressed Bcl-6 and CXCR5, markers characteristic of T follicular helper cells in the lymph nodes. Following a pathogenic SIV infection, the frequency of PD-1(hi) cells (as a percentage of CD4 T cells) dramatically increased in the rectal mucosa; however, a significant fraction of them did not express CXCR5. Furthermore, only a small fraction of PD-1(hi) cells expressed CCR5, and despite this low level of viral coreceptor expression, a significant fraction of these cells were productively infected. Interestingly, vaccinated SIV controllers did not present with this aberrant PD-1(hi) CD4 T cell enrichment, and this lack of enrichment was associated with the presence of higher frequencies of SIV-specific granzyme B(+) CD8 T cells within the lymphoid tissue, suggesting a role for antiviral CD8 T cells in limiting aberrant expansion of PD-1(hi) CD4 T cells. These results highlight the importance of developing vaccines that enhance antiviral CD8 T cells at sites of preferential viral replication and support the need for developing therapeutic interventions that limit expansion of SIV(+)PD-1(hi) CD4 T cells at mucosal sites as a means to enhance viral control.

Dominant clonotypes within HIV-specific T cell responses are programmed death-1high and CD127low and display reduced variant cross-reactivity.

HIV epitope-specific T cell responses are often comprised of clonotypic expansions with distinct functional properties. In HIV(+) individuals, we measured programmed death-1 (PD-1) and IL-7Rα expression, MHC class I tetramer binding, cytokine production, and proliferation profiles of dominant and subdominant TCR clonotypes to evaluate the relationship between the composition of the HIV-specific T cell repertoire and clonotypic phenotype and function. Dominant clonotypes are characterized by higher PD-1 expression and lower C127 expression compared with subdominant clonotypes, and TCR avidity positively correlates with PD-1 expression. At low peptide concentrations, dominant clonotypes fail to survive in culture. In response to stimulation with peptides representing variant epitopes, subdominant clonotypes produce higher relative levels of cytokines and display greater capacity for cross-recognition compared with dominant clonotypes. These data indicate that dominant clonotypes within HIV-specific T cell responses display a phenotype consistent with ongoing exposure to cognate viral epitopes and suggest that cross-reactive, subdominant clonotypes may retain greater capacity to suppress replication of viral variants as well as to survive in the absence of strong antigenic signaling.

Enhanced PD-1 expression by T cells in cerebrospinal fluid does not reflect functional exhaustion during chronic human immunodeficiency virus type 1 infection.

During chronic viral infections, T cells are exhausted due to constant antigen exposure and are associated with enhanced programmed death 1 (PD-1) expression. Deficiencies in the PD-1/programmed death-ligand 1 (PD-L1) pathway are associated with autoimmune diseases, including those of the central nervous system (CNS). To understand the role of PD-1 expression in regulating T-cell immunity in the CNS during chronic infection, we characterized PD-1 expression in cerebrospinal fluid (CSF) and blood of individuals with chronic human immunodeficiency virus type 1 (HIV-1) infection. PD-1 expression was higher on HIV-specific CD8(+) T cells than on total CD8(+) T cells in both CSF and blood. PD-1 expression on CSF T cells correlated positively with CSF HIV-1 RNA and inversely with blood CD4(+) T-cell counts, suggesting that HIV-1 infection drives higher PD-1 expression on CSF T cells. However, in every HIV-positive individual, PD-1 expression was higher on T cells in CSF than on those in blood, despite HIV-1 RNA levels being lower. Among healthy HIV-negative controls, PD-1 expression was higher in CSF than in blood. Furthermore, frequencies of the senescence marker CD57 were lower on CSF T cells than on blood T cells, consistent with our prior observation of enhanced ex vivo functional capacity of CSF T cells. The higher PD-1 expression level on CSF T cells therefore does not reflect cellular exhaustion but may be a mechanism to downregulate immune-mediated tissue damage in the CNS. As inhibition of the PD-1/PD-L1 pathway is pursued as a therapeutic option for viral infections, potential effects of such a blockade on development of autoimmune responses in the CNS should be considered.

Expansion and exhaustion of T-cell responses during mutational escape from long-term viral control in two DNA/modified vaccinia virus Ankara-vaccinated and simian-human immunodeficiency virus SHIV-89.6P-challenged macaques.

In this study, we monitored the temporal breadths, frequencies, and functions of antiviral CD4 and CD8 T cells in 2 of 22 DNA/modified vaccinia virus Ankara-vaccinated macaques that lost control of a simian-human immunodeficiency virus 89.6P challenge by 196 weeks postchallenge. Our results show that both mutation and exhaustion contributed to escape. With the reappearance of viremia, responding CD8 and CD4 T cells underwent an initial increase and then loss of breadth and frequency. Antiviral gamma interferon (IFN-gamma)- and interleukin 2-coproducing cells were lost before IFN-gamma-producing cells and CD4 cells before CD8 cells. At euthanasia, all CD8, but no CD4, Gag epitopes detected during long-term control contained mutations.

Enhancement of human immunodeficiency virus (HIV)-specific CD8+ T cells in cerebrospinal fluid compared to those in blood among antiretroviral therapy-naive HIV-positive subjects.

During untreated human immunodeficiency virus type 1 (HIV-1) infection, virus-specific CD8(+) T cells partially control HIV replication in peripheral lymphoid tissues, but host mechanisms of HIV control in the central nervous system (CNS) are incompletely understood. We characterized HIV-specific CD8(+) T cells in cerebrospinal fluid (CSF) and peripheral blood among seven HIV-positive antiretroviral therapy-naïve subjects. All had grossly normal brain magnetic resonance imaging and spectroscopy and normal neuropsychometric testing. Frequencies of epitope-specific CD8(+) T cells by direct tetramer staining were on average 2.4-fold higher in CSF than in blood (P = 0.0004), while HIV RNA concentrations were lower. Cells from CSF were readily expanded ex vivo and responded to a broader range of HIV-specific human leukocyte antigen class I restricted optimal peptides than did expanded cells from blood. HIV-specific CD8(+) T cells, in contrast to total CD8(+) T cells, in CSF and blood were at comparable maturation states, as assessed by CD45RO and CCR7 staining. The strong relationship between higher T-cell frequencies and lower levels of viral antigen in CSF could be the result of increased migration to and/or preferential expansion of HIV-specific T cells within the CNS. This suggests an important role for HIV-specific CD8(+) T cells in control of intrathecal viral replication.

B cell responses to HIV antigen are a potent correlate of viremia in HIV-1 infection and improve with PD-1 blockade.

Infection with Human Immunodeficiency Virus Type 1 (HIV-1) induces defects of both cellular and humoral immune responses. Impaired CD4+ T cell help and B cell dysfunction may partially explain the low frequency of broadly neutralizing antibodies in HIV-infected individuals. To understand the extent of B cell dysfunction during HIV infection, we assessed the level of B cell activation at baseline and after stimulation with a variety of antigens. Increased levels of viremia were associated with higher baseline expression of the activation marker CD86 on B cells and with decreased ability of B cells to increase expression of CD86 after in vitro stimulation with inactivated HIV-1. In a series of cell isolation experiments B cell responses to antigen were enhanced in the presence of autologous CD4+ T cells. HIV infected individuals had a higher frequency of PD-1 expression on B cells compared to HIV- subjects and PD-1 blockade improved B cell responsiveness to HIV antigen, suggesting that inhibitory molecule expression during HIV-1 infection may contribute to some of the observed B cell defects. Our findings demonstrate that during chronic HIV infection, B cells are activated and lose full capacity to respond to antigen, but suppression of inhibitory pressures as well as a robust CD4+ T cell response may help preserve B cell function.

Reducing the activity and secretion of microbial antioxidants enhances the immunogenicity of BCG.

BACKGROUND: In early clinical studies, the live tuberculosis vaccine Mycobacterium bovis BCG exhibited 80% protective efficacy against pulmonary tuberculosis (TB). Although BCG still exhibits reliable protection against TB meningitis and miliary TB in early childhood it has become less reliable in protecting against pulmonary TB. During decades of in vitro cultivation BCG not only lost some genes due to deletions of regions of the chromosome but also underwent gene duplication and other mutations resulting in increased antioxidant production. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether microbial antioxidants influence vaccine immunogenicity, we eliminated duplicated alleles encoding the oxidative stress sigma factor SigH in BCG Tice and reduced the activity and secretion of iron co-factored superoxide dismutase. We then used assays of gene expression and flow cytometry with intracellular cytokine staining to compare BCG-specific immune responses in mice after vaccination with BCG Tice or the modified BCG vaccine. Compared to BCG, the modified vaccine induced greater IL-12p40, RANTES, and IL-21 mRNA in the spleens of mice at three days post-immunization, more cytokine-producing CD8+ lymphocytes at the peak of the primary immune response, and more IL-2-producing CD4+ lymphocytes during the memory phase. The modified vaccine also induced stronger secondary CD4+ lymphocyte responses and greater clearance of challenge bacilli. CONCLUSIONS/SIGNIFICANCE: We conclude that antioxidants produced by BCG suppress host immune responses. These findings challenge the hypothesis that the failure of extensively cultivated BCG vaccines to prevent pulmonary tuberculosis is due to over-attenuation and suggest instead a new model in which BCG evolved to produce more immunity-suppressing antioxidants. By targeting these antioxidants it may be possible to restore BCG's ability to protect against pulmonary TB.

Signature for long-term vaccine-mediated control of a Simian and human immunodeficiency virus 89.6P challenge: stable low-breadth and low-frequency T-cell response capable of coproducing gamma interferon and interleukin-2.

In 2001, we reported 20 weeks of control of challenge with the virulent 89.6P chimera of simian and human immunodeficiency viruses (SHIV-89.6P) by a Gag-Pol-Env vaccine consisting of DNA priming and modified vaccinia virus Ankara boosting. Here we report that 22 out of 23 of these animals successfully controlled their viremia until their time of euthanasia at 200 weeks postchallenge. At euthanasia, all animals had low to undetectable viral loads and normal CD4 counts. During the long period of viral control, gamma interferon (IFN-gamma)-producing antiviral T cells were present at unexpectedly low breadths and frequencies. Most animals recognized two CD8 and one CD4 epitope and had frequencies of IFN-gamma-responding T cells from 0.01 to 0.3% of total CD8 or CD4 T cells. T-cell responses were remarkably stable over time and, unlike responses in most immunodeficiency virus infections, maintained good functional characteristics, as evidenced by coproduction of IFN-gamma and interleukin-2. Overall, high titers of binding and neutralizing antibody persisted throughout the postchallenge period. Encouragingly, long-term control was effective in macaques of diverse histocompatibility types.