Trispecific antibody targeting HIV-1 and T cells activates and eliminates latently-infected cells in HIV/SHIV infections.

Agents that can simultaneously activate latent HIV, increase immune activation and enhance the killing of latently-infected cells represent promising approaches for HIV cure. Here, we develop and evaluate a trispecific antibody (Ab), N6/αCD3-αCD28, that targets three independent proteins: (1) the HIV envelope via the broadly reactive CD4-binding site Ab, N6; (2) the T cell antigen CD3; and (3) the co-stimulatory molecule CD28. We find that the trispecific significantly increases antigen-specific T-cell activation and cytokine release in both CD4+ and CD8+ T cells. Co-culturing CD4+ with autologous CD8+ T cells from ART-suppressed HIV+ donors with N6/αCD3-αCD28, results in activation of latently-infected cells and their elimination by activated CD8+ T cells. This trispecific antibody mediates CD4+ and CD8+ T-cell activation in non-human primates and is well tolerated in vivo. This HIV-directed antibody therefore merits further development as a potential intervention for the eradication of latent HIV infection.

Potent anti-viral activity of a trispecific HIV neutralizing antibody in SHIV-infected monkeys.

Broadly neutralizing antibodies (bNAbs) represent an alternative to drug therapy for the treatment of HIV-1 infection. Immunotherapy with single bNAbs often leads to emergence of escape variants, suggesting a potential benefit of combination bNAb therapy. Here, a trispecific bNAb reduces viremia 100- to 1000-fold in viremic SHIV-infected macaques. After treatment discontinuation, viremia rebounds transiently and returns to low levels, through CD8-mediated immune control. These viruses remain sensitive to the trispecific antibody, despite loss of sensitivity to one of the parental bNAbs. Similarly, the trispecific bNAb suppresses the emergence of resistance in viruses derived from HIV-1-infected subjects, in contrast to parental bNAbs. Trispecific HIV-1 neutralizing antibodies, therefore, mediate potent antiviral activity in vivo and may minimize the potential for immune escape.

Principles Governing Establishment versus Collapse of HIV-1 Cellular Spread.

A population at low census might go extinct or instead transition into exponential growth to become firmly established. Whether this pivotal event occurs for a within-host pathogen can be the difference between health and illness. Here, we define the principles governing whether HIV-1 spread among cells fails or becomes established by coupling stochastic modeling with laboratory experiments. Following ex vivo activation of latently infected CD4 T cells without de novo infection, stochastic cell division and death contributes to high variability in the magnitude of initial virus release. Transition to exponential HIV-1 spread often fails due to release of an insufficient amount of replication-competent virus. Establishment of exponential growth occurs when virus produced from multiple infected cells exceeds a critical population size. We quantitatively define the crucial transition to exponential viral spread. Thwarting this process would prevent HIV transmission or rebound from the latent reservoir.

Clinical Trial of the Anti-PD-L1 Antibody BMS-936559 in HIV-1 Infected Participants on Suppressive Antiretroviral Therapy.

Background: Reversing immune exhaustion with an anti-PD-L1 antibody may improve human immunodeficiency virus type 1 (HIV-1)-specific immunity and increase clearance of HIV-1-expressing cells. Methods: We conducted a phase I, randomized, double-blind, placebo-controlled, dose-escalating study of BMS-936559, including HIV-1-infected adults aged >18 to <70 years on suppressive antiretroviral therapy with CD4+ counts >350 cells/µL and detectable plasma HIV-1 RNA by single-copy assay. Data on single infusions of BMS-936559 (0.3 mg/kg) versus placebo are described. The primary outcomes were safety defined as any grade 3 or greater or immune-related adverse event (AE) and the change in HIV-1 Gag-specific CD8+ T cell responses from baseline to day 28 after infusion. Results: Eight men enrolled: 6 received 0.3 mg/kg of BMS-936559, and 2 received placebo infusions. There were no BMS-936559-related grade 3 or greater AEs. In 1 participant, asymptomatic hypophysitis (a protocol-defined immune-related AE) was identified 266 days after BMS-936559 infusion; it resolved over time. The mean percentage of HIV-1 Gag-specific CD8+ T cells expressing interferon γ increased from baseline (0.09%) through day 28 (0.20%; P = .14), driven by substantial increases in 2 participants who received BMS-936559. Conclusions: In this first evaluation of an immunologic checkpoint inhibitor in healthy HIV-1-infected persons, single low-dose BMS-936559 infusions appeared to enhance HIV-1-specific immunity in a subset of participants. Clinical Trials Registration: NCT02028403.

Follicular CD8 T cells accumulate in HIV infection and can kill infected cells in vitro via bispecific antibodies.

Cytolytic CD8 T cells play a crucial role in the control and elimination of virus-infected cells and are a major focus of HIV cure efforts. However, it has been shown that HIV-specific CD8 T cells are infrequently found within germinal centers (GCs), a predominant site of active and latent HIV infection. We demonstrate that HIV infection induces marked changes in the phenotype, frequency, and localization of CD8 T cells within the lymph node (LN). Significantly increased frequencies of CD8 T cells in the B cell follicles and GCs were found in LNs from treated and untreated HIV-infected individuals. This profile was associated with persistent local immune activation but did not appear to be directly related to local viral replication. Follicular CD8 (fCD8) T cells, despite compromised cytokine polyfunctionality, showed good cytolytic potential characterized by high ex vivo expression of granzyme B and perforin. We used an anti-HIV/anti-CD3 bispecific antibody in a redirected killing assay and found that fCD8 T cells had better killing activity than did non-fCD8 T cells. Our results indicate that CD8 T cells with potent cytolytic activity are recruited to GCs during HIV infection and, if appropriately redirected to kill HIV-infected cells, could be an effective component of an HIV cure strategy.

Activation and lysis of human CD4 cells latently infected with HIV-1.

The treatment of AIDS with combination antiretroviral therapy (cART) remains lifelong largely because the virus persists in latent reservoirs. Elimination of latently infected cells could therefore reduce treatment duration and facilitate immune reconstitution. Here we report an approach to reduce the viral reservoir by activating dormant viral gene expression and directing T lymphocytes to lyse previously latent, HIV-1-infected cells. An immunomodulatory protein was created that combines the specificity of a HIV-1 broadly neutralizing antibody with that of an antibody to the CD3 component of the T-cell receptor. CD3 engagement by the protein can stimulate T-cell activation that induces proviral gene expression in latently infected T cells. It further stimulates CD8 T-cell effector function and redirects T cells to lyse these previously latent-infected cells through recognition of newly expressed Env. This immunomodulatory protein could potentially help to eliminate latently infected cells and deplete the viral reservoir in HIV-1-infected individuals.

Tracking epitope-specific T cells.

The tracking of antigen-specific T cells in vivo is a useful approach for the study of the adaptive immune response. This protocol describes how populations of T cells specific for a given peptide-major histocompatibility complex (pMHC) epitope can be tracked based solely on T-cell receptor (TCR) specificity as opposed to other indirect methods based on function. The methodology involves the adoptive transfer of TCR transgenic T cells with defined epitope specificity into histocompatible mice and the subsequent detection of these cells through the use of congenic or clonotypic markers. Alternatively, endogenous epitope-specific T cells can be tracked directly through the use of pMHC tetramers. Using magnetic bead-based enrichment and advanced multiparameter flow cytometry, populations as small as five epitope-specific T cells can be detected from the peripheral lymphoid organs of a mouse. The adoptive transfer procedure can be completed within 3 h, whereas analysis of epitope-specific cells from mice can be completed within 6 h.

CD4+ T cells that enter the draining lymph nodes after antigen injection participate in the primary response and become central-memory cells.

We explored the relationship between the time of naive CD4+ T cell exposure to antigen in the primary immune response and the quality of the memory cells produced. Naive CD4+ T cells that migrated into the skin-draining lymph nodes after subcutaneous antigen injection accounted for about half of the antigen-specific population present at the peak of clonal expansion. These late-arriving T cells divided less and more retained the central-memory marker CD62L than the T cells that resided in the draining lymph nodes at the time of antigen injection. The fewer cell divisions were related to competition with resident T cells that expanded earlier in the response and a reduction in the number of dendritic cells displaying peptide-major histocompatibility complex (MHC) II complexes at later times after antigen injection. The progeny of late-arriving T cells possessed the phenotype of central-memory cells, and proliferated more extensively during the secondary response than the progeny of the resident T cells. The results suggest that late arrival into lymph nodes and exposure to antigen-presenting cells displaying lower numbers of peptide-MHC II complexes in the presence of competing T cells ensures that some antigen-specific CD4+ T cells divide less in the primary response and become central-memory cells.

Naive and memory CD4+ T cell survival controlled by clonal abundance.

Immunity to a plethora of microbes depends on a diverse repertoire of naïve lymphocytes and the production of long-lived memory cells. We present evidence here that low clonal abundance in a polyclonal repertoire favors the survival and activation of naïve CD4(+) T cells as well as the survival of their memory cell progeny. The inverse relation between clonal frequency and survival suggests that intraclonal competition could help maintain an optimally diverse repertoire of T cells and an optimal environment for the generation of long-lived memory cells.

Nuclear export of Vpr is required for efficient replication of human immunodeficiency virus type 1 in tissue macrophages.

Retroviruses must gain access to the host cell nucleus for subsequent replication and viral propagation. Human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses are distinguished from the gammaretroviruses by their ability to infect nondividing cells such as macrophages, an important viral reservoir in vivo. Rather than requiring nuclear membrane breakdown during cell division, the HIV-1 preintegration complex (PIC) enters the nucleus by traversing the central aqueous channel of the limiting nuclear pore complex. The HIV-1 PIC contains three nucleophilic proteins, matrix, integrase, and Vpr, all of which have been implicated in nuclear targeting. The mechanism by which Vpr can display such nucleophilic properties and yet also be available for incorporation into virions assembling at the plasma membrane is unresolved. We recently characterized Vpr as a nucleocytoplasmic shuttling protein that contains two novel nuclear import signals and an exportin-1-dependent nuclear export signal (NES). We now demonstrate that mutation of this NES impairs the incorporation of Vpr into newly formed virions. Furthermore, we find that the Vpr NES is required for efficient HIV replication in tissue macrophages present in human spleens and tonsils. These findings underscore how the nucleocytoplasmic shuttling of Vpr not only contributes to nuclear import of the HIV-1 PIC but also enables Vpr to be present in the cytoplasm for incorporation into virions, leading to enhancement of viral spread within nondividing tissue macrophages.

Highly potent non-peptidic inhibitors of the HCV NS3/NS4A serine protease.

Screening of a diverse set of bisbenzimidazoles for inhibition of the hepatitis C virus (HCV) serine protease NS3/NS4A led to the identification of a potent Zn(2+)-dependent inhibitor (1). Optimization of this screening hit afforded a 10-fold more potent inhibitor (46) under Zn(2+) conditions (K(i)=27nM). This compound (46) binds also to NS3/NS4A in a Zn(2+) independent fashion (K(i)=1microM). The SAR of this class of compounds under Zn(2+) conditions is highly divergent compared to the SAR in the absence of Zn(2+), suggesting two distinct binding modes.