Recent advances on anti-HIV chimeric antigen receptor-T-cell treatment to provide sustained HIV remission.

PURPOSE OF REVIEW: Successful sustained remission of HIV infection has been achieved after CCR5Δ32/Δ32 allogeneic hematopoietic stem cell transplantation for treatment of leukemia in a small cohort of people living with HIV (PLWH). This breakthrough demonstrated that the goal of curing HIV was achievable. However, the high morbidity and mortality associated with bone marrow transplantation limits the routine application of this approach and provides a strong rationale for pursuing alternative strategies for sustained long-term antiretroviral therapy (ART)-free HIV remission. Notably, long-term immune-mediated control of HIV replication observed in elite controllers and posttreatment controllers suggests that potent HIV-specific immune responses could provide sustained ART-free remission in PLWH. The capacity of chimeric antigen receptor (CAR)-T cells engineered to target malignant cells to induce remission and cure in cancer patients made this an attractive approach to provide PLWH with a potent HIV-specific immune response. Here, we review the recent advances in the design and application of anti-HIV CAR-T-cell therapy to provide a functional HIV cure. RECENT FINDINGS: HIV reservoirs are established days after infection and persist through clonal expansion of infected cells. The continuous interaction between latently infected cells and the immune system shapes the landscape of HIV latency and likely contributes to ART-free viral control in elite controllers. CAR-T cells can exhibit superior antiviral activity as compared with native HIV-specific T cells, particularly because they can be engineered to have multiple HIV specificities, resistance to HIV infection, dual costimulatory signaling, immune checkpoint inhibitors, stem cell derivation, CMV TCR coexpression, and tissue homing ligands. These modifications can significantly improve the capacities of anti-HIV CAR-T cells to prevent viral escape, resist HIV infection, and enhance cytotoxicity, persistence, and tissue penetration. Collectively, these novel modifications of anti-HIV CAR-T cell design have increased their capacity to control HIV infection. SUMMARY: Anti-HIV CAR-T cells can be engineered to provide potent and sustained in-vitro and in-vivo antiviral function. The combination of anti-HIV CAR-T cells with other immunotherapeutics may contribute to long-term HIV remission in PLWH.

Generation of Anti-HIV CAR-T Cells for Preclinical Research.

The inability of people living with HIV (PLWH) to eradicate human immunodeficiency virus (HIV) infection is due in part to the inadequate HIV-specific cellular immune response. The antiviral function of cytotoxic CD8+ T cells, which are crucial for HIV control, is impaired during chronic viral infection because of viral escape mutations, immune exhaustion, HIV antigen downregulation, inflammation, and apoptosis. In addition, some HIV-infected cells either localize to tissue sanctuaries inaccessible to CD8+ T cells or are intrinsically resistant to CD8+ T cell killing. The novel design of synthetic chimeric antigen receptors (CARs) that enable T cells to target specific antigens has led to the development of potent and effective CAR-T cell therapies. While initial clinical trials using anti-HIV CAR-T cells performed over 20 years ago showed limited anti-HIV effects, the improved CAR-T cell design, which enabled its success in treating cancer, has reinstated CAR-T cell therapy as a strategy for HIV cure with notable progress being made in the recent decade.Effective CAR-T cell therapy against HIV infection requires the generation of anti-HIV CAR-T cells with potent in vivo activity against HIV-infected cells. Preclinical evaluation of anti-HIV efficacy of CAR-T cells and their safety is fundamental for supporting the initiation of subsequent clinical trials in PLWH. For these preclinical studies, we developed a novel humanized mouse model supporting in vivo HIV infection, the development of viremia, and the evaluation of novel HIV therapeutics. Preclinical assessment of anti-HIV CAR-T cells using this mouse model involves a multistep process including peripheral blood mononuclear cells (PBMCs) harvested from human donors, T cell purification, ex vivo T cell activation, transduction with lentiviral vectors encoding an anti-HIV CAR, CAR-T cell expansion and infusion in mice intrasplenically injected with autologous PBMCs followed by the determination of CAR-T cell capacity for HIV suppression. Each of the steps described in the following protocol were optimized in the lab to maximize the quantity and quality of the final anti-HIV CAR-T cell products.

The replication-competent HIV reservoir is a genetically restricted, younger subset of the overall pool of HIV proviruses persisting during therapy, which is highly genetically stable over time.

Within-host HIV populations continually diversify during untreated infection, and this diversity persists within infected cell reservoirs during antiretroviral therapy (ART). Achieving a better understanding of on-ART proviral evolutionary dynamics, and a better appreciation of how the overall persisting pool of (largely genetically defective) proviruses differs from the much smaller replication-competent HIV reservoir, is critical to HIV cure efforts. We reconstructed within-host HIV evolutionary histories in blood from seven participants of the Women's Interagency HIV Study who experienced HIV seroconversion, and used these data to characterize the diversity, lineage origins, and ages of proviral env-gp120 sequences sampled longitudinally up to 12 years on ART. We also studied HIV sequences emerging from the reservoir in two participants. We observed that proviral clonality generally increased over time on ART, with clones frequently persisting long term. While on-ART proviral integration dates generally spanned the duration of untreated infection, HIV emerging in plasma was exclusively younger (i.e., dated to the years immediately pre-ART). The genetic and age distributions of distinct proviral sequences remained stable during ART in all but one participant, in whom there was evidence that younger proviruses had been preferentially eliminated after 12 years on ART. Analysis of the gag region in three participants corroborated our env-gp120-based observations, indicating that our observations are not influenced by the HIV region studied. Our results underscore the remarkable genetic stability of the distinct proviral sequences that persist in blood during ART. Our results also suggest that the replication-competent HIV reservoir is a genetically restricted, younger subset of this overall proviral pool.IMPORTANCECharacterizing the genetically diverse HIV sequences that persist in the reservoir despite antiretroviral therapy (ART) is critical to cure efforts. Our observations confirm that proviruses persisting in blood on ART, which are largely genetically defective, broadly reflect the extent of within-host HIV evolution pre-ART. Moreover, on-ART clonal expansion is not appreciably accompanied by the loss of distinct proviral lineages. In fact, on-ART proviral genetic composition remained stable in all but one participant, in whom, after 12 years on ART, proviruses dating to around near ART initiation had been preferentially eliminated. We also identified recombinant proviruses between parental sequence fragments of different ages. Though rare, such sequences suggest that reservoir cells can be superinfected with HIV from another infection era. Overall, our finding that the replication-competent reservoir in blood is a genetically restricted, younger subset of all persisting proviruses suggests that HIV cure strategies will need to eliminate a reservoir that differs in key respects from the overall proviral pool.

The replication-competent HIV reservoir is a genetically restricted, younger subset of the overall pool of HIV proviruses persisting during therapy, which is highly genetically stable over time.

Within-host HIV populations continually diversify during untreated infection, and members of these diverse forms persist within infected cell reservoirs, even during antiretroviral therapy (ART). Characterizing the diverse viral sequences that persist during ART is critical to HIV cure efforts, but our knowledge of on-ART proviral evolutionary dynamics remains incomplete, as does our understanding of the differences between the overall pool of persisting proviral DNA (which is largely genetically defective) and the subset of intact HIV sequences capable of reactivating. Here, we reconstructed within-host HIV evolutionary histories in blood from seven participants of the Women's Interagency HIV Study (WIHS) who experienced HIV seroconversion. We measured diversity, lineage origins and ages of proviral sequences (env-gp120) sampled up to four times, up to 12 years on ART. We used the same techniques to study HIV sequences emerging from the reservoir in two participants. Proviral clonality generally increased over time on ART, with clones frequently persisting across multiple time points. The integration dates of proviruses persisting on ART generally spanned the duration of untreated infection (though were often skewed towards years immediately pre-ART), while in contrast, reservoir-origin viremia emerging in plasma was exclusively "younger" (i.e., dated to the years immediately pre-ART). The genetic and age distributions of distinct proviral sequences remained highly stable during ART in all but one participant in whom, after 12 years, there was evidence that "younger" proviruses had been preferentially eliminated. Analysis of within-host recombinant proviral sequences also suggested that HIV reservoirs can be superinfected with virus reactivated from an older era, yielding infectious viral progeny with mosaic genomes of sequences with different ages. Overall, results underscore the remarkable genetic stability of distinct proviral sequences that persist on ART, yet suggest that replication-competent HIV reservoir represents a genetically-restricted and overall "younger" subset of the overall persisting proviral pool in blood.

In vivo killing of primary HIV-infected cells by peripheral-injected early memory-enriched anti-HIV duoCAR T cells.

HIV-specific chimeric antigen receptor-T cell (CAR T cell) therapies are candidates to functionally cure HIV infection in people with HIV (PWH) by eliminating reactivated HIV-infected cells derived from latently infected cells within the HIV reservoir. Paramount to translating such therapeutic candidates successfully into the clinic will require anti-HIV CAR T cells to localize to lymphoid tissues in the body and eliminate reactivated HIV-infected cells such as CD4+ T cells and monocytes/macrophages. Here we show that i.v. injected anti-HIV duoCAR T cells, generated using a clinical-grade anti-HIV duoCAR lentiviral vector, localized to the site of active HIV infection in the spleen of humanized mice and eliminated HIV-infected PBMCs. CyTOF analysis of preinfusion duoCAR T cells revealed an early memory phenotype composed predominantly of CCR7+ stem cell-like/central memory T cells (TSCM/TCM) with expression of some effector-like molecules. In addition, we show that anti-HIV duoCAR T cells effectively sense and kill HIV-infected CD4+ T cells and monocytes/macrophages. Furthermore, we demonstrate efficient genetic modification of T cells from PWH on suppressive ART into anti-HIV duoCAR T cells that subsequently kill autologous PBMCs superinfected with HIV. These studies support the safety and efficacy of anti-HIV duoCAR T cell therapy in our presently open phase I/IIa clinical trial (NCT04648046).

Brief Report: Use of Remnant Specimens to Assess Use of HIV PrEP Among Populations With Risk of HIV Infection: A Novel Approach.

BACKGROUND: HIV-uninfected persons being evaluated for sexually transmitted infections (STIs) may be good HIV pre-exposure prophylaxis (PrEP) candidates. We measured PrEP use in a sentinel STI patient population. DESIGN: Cross-sectional study, New York City Sexual Health Clinics (January 2019-June 2019). METHODS: Remnant serum samples from 644 HIV-uninfected men who have sex with men (MSM) and 97 women diagnosed with chlamydia, gonorrhea, and/or early syphilis were assayed for tenofovir and emtricitabine levels using a validated liquid chromatography-mass spectrometry assay. Using paired test results and medical records, we assessed (1) prevalence and (2) correlates of PrEP use on the day of STI diagnosis (adjusted prevalence ratios [aPRs]). RESULTS: PrEP use among 741 patients was 32.7% [95% confidence interval (CI): 29.3 to 36.0]; 37.3% for MSM and 2.1% for women. PrEP use was high among White MSM (46.8%) and lowest among women. Among MSM with rectal chlamydia/gonorrhea or early syphilis, PrEP use was associated with age [aPR = 1.7 (95% CI: 1.2 to 2.4) for ages 25-34 years and aPR = 2.0 (1.4 to 2.9) for ages 35-44 years, vs. 15 to 24 years]; number of recent sex partners [aPR = 1.4 (1.0 to 2.0) for 3-5 partners, aPR = 2.1 (1.5 to 3.0) for 6-10 partners, aPR = 2.2 (1.6 to 3.1) for >10 partners, vs. ≤2 partners]; having sex/needle-sharing partners with HIV [aPR = 1.4 (1.1-1.7)]; and inconsistent condom use [aPR = 3.3 (1.8-6.1)]. Race/ethnicity, past-year STI diagnosis, and postexposure prophylaxis use were not associated. CONCLUSIONS: One in 3 people with newly diagnosed STIs had detectable serum PrEP, and PrEP use was exceedingly rare among women. Routinely collected remnant samples can be used to measure PrEP use in populations at high risk of HIV acquisition.

Self-Reported Use of HIV Preexposure Prophylaxis Is Highly Accurate Among Sexual Health Clinic Patients in New York City.

ABSTRACT: In New York City, 91% of sexually transmitted infection clinic patients reported preexposure prophylaxis (PrEP) use that matched the detection of PrEP in their serum. Self-report had 80% sensitivity and 96% specificity ( κ = 0.79) compared with measured PrEP. Our findings suggest that self-report may be a valid indicator of PrEP uptake.

T cell receptor-targeted immunotherapeutics drive selective in vivo HIV- and CMV-specific T cell expansion in humanized mice.

To delineate the in vivo role of different costimulatory signals in activating and expanding highly functional virus-specific cytotoxic CD8+ T cells, we designed synTacs, infusible biologics that recapitulate antigen-specific T cell activation signals delivered by antigen-presenting cells. We constructed synTacs consisting of dimeric Fc-domain scaffolds linking CD28- or 4-1BB-specific ligands to HLA-A2 MHC molecules covalently tethered to HIV- or CMV-derived peptides. Treatment of HIV-infected donor PBMCs with synTacs bearing HIV- or CMV-derived peptides induced vigorous and selective ex vivo expansion of highly functional HIV- and/or CMV-specific CD8+ T cells, respectively, with potent antiviral activities. Intravenous injection of HIV- or CMV-specific synTacs into immunodeficient mice intrasplenically engrafted with donor PBMCs markedly and selectively expanded HIV-specific (32-fold) or CMV-specific (46-fold) human CD8+ T cells populating their spleens. Notably, these expanded HIV- or CMV-specific CD8+ T cells directed potent in vivo suppression of HIV or CMV infections in the humanized mice, providing strong rationale for consideration of synTac-based approaches as a therapeutic strategy to cure HIV and treat CMV and other viral infections. The synTac platform flexibility supports facile delineation of in vivo effects of different costimulatory signals on patient-derived virus-specific CD8+ T cells, enabling optimization of individualized therapies, including HIV cure strategies.

HIV-1 diversity considerations in the application of the Intact Proviral DNA Assay (IPDA).

The Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a scalable method for intact HIV-1 reservoir quantification. This droplet digital PCR-based assay simultaneously targets two HIV-1 regions to distinguish genomically intact proviruses against a large background of defective ones, and its application has yielded insights into HIV-1 persistence. Reports of assay failures however, attributed to HIV-1 polymorphism, have recently emerged. Here, we describe a diverse North American cohort of people with HIV-1 subtype B, where the IPDA yielded a failure rate of 28% due to viral polymorphism. We further demonstrate that within-host HIV-1 diversity can lead the IPDA to underestimate intact reservoir size, and provide examples of how this phenomenon could lead to erroneous interpretation of clinical trial data. While the IPDA represents a major methodological advance, HIV-1 diversity should be addressed before its widespread adoption as a principal readout in HIV-1 remission trials.

Engineered Antigen-Specific T Cells Secreting Broadly Neutralizing Antibodies: Combining Innate and Adaptive Immune Response against HIV.

While antiretroviral therapy (ART) can completely suppress viremia, it is not a cure for HIV. HIV persists as a latent reservoir of infected cells, able to evade host immunity and re-seed infection following cessation of ART. Two promising immunotherapeutic strategies to eliminate both productively infected cells and reactivated cells of the reservoir are the adoptive transfer of potent HIV-specific T cells and the passive administration of HIV-specific broadly neutralizing antibodies also capable of mediating antibody-dependent cellular cytotoxicity (ADCC). The simultaneous use of both as the basis of a single therapeutic has never been explored. We therefore sought to modify HIV-specific T cells from HIV-naive donors (to allow their use in the context of allotransplant, a promising platform for sterilizing cures) so they are able to secrete a broadly neutralizing antibody (bNAb) directed against the HIV envelope to elicit ADCC. We designed an antibody construct comprising bNAb 10-1074 heavy and light chains, fused to IgG3 Fc to elicit ADCC, with truncated cluster of differentiation 19 (CD19) as a selectable marker. HIV-specific T cells were expanded from HIV-naive donors by priming with antigen-presenting cells expressing overlapping HIV antigens in the presence of cytokines. T cells retained specificity against Gag, Nef, and Pol peptides (218.55 ± 300.14 interferon γ [IFNγ] spot-forming cells [SFC]/1 × 105) following transduction (38.92 ± 25.30) with the 10-1074 antibody constructs. These cells secreted 10-1074 antibodies (139.04 ± 114.42 ng/mL). The HIV-specific T cells maintained T cell function following transduction, and the secreted 10-1074 antibody bound HIV envelope (28.13% ± 19.42%) and displayed ADCC activity (10.47% ± 4.11%). Most critically, the 10-1074 antibody-secreting HIV-specific T cells displayed superior in vitro suppression of HIV replication. In summary, HIV-specific T cells can be engineered to produce antibodies mediating ADCC against HIV envelope-expressing cells. This combined innate/adaptive approach allows for synergy between the two immune arms, broadens the target range of the immune therapy, and provides further insight into what defines an effective anti-HIV response.

Multispecific anti-HIV duoCAR-T cells display broad in vitro antiviral activity and potent in vivo elimination of HIV-infected cells in a humanized mouse model.

Adoptive immunotherapy using chimeric antigen receptor-modified T cells (CAR-T) has made substantial contributions to the treatment of certain B cell malignancies. Such treatment modalities could potentially obviate the need for long-term antiretroviral drug therapy in HIV/AIDS. Here, we report the development of HIV-1-based lentiviral vectors that encode CARs targeting multiple highly conserved sites on the HIV-1 envelope glycoprotein using a two-molecule CAR architecture, termed duoCAR. We show that transduction with lentiviral vectors encoding multispecific anti-HIV duoCARs confer primary T cells with the capacity to potently reduce cellular HIV infection by up to 99% in vitro and >97% in vivo. T cells are the targets of HIV infection, but the transduced T cells are protected from genetically diverse HIV-1 strains. The CAR-T cells also potently eliminated PBMCs infected with broadly neutralizing antibody-resistant HIV strains, including VRC01/3BNC117-resistant HIV-1. Furthermore, multispecific anti-HIV duoCAR-T cells demonstrated long-term control of HIV infection in vivo and prevented the loss of CD4+ T cells during HIV infection using a humanized NSG mouse model of intrasplenic HIV infection. These data suggest that multispecific anti-HIV duoCAR-T cells could be an effective approach for the treatment of patients with HIV-1 infection.

Rapid Elimination of Broadly Neutralizing Antibodies Correlates with Treatment Failure in the Acute Phase of Simian-Human Immunodeficiency Virus Infection.

Early human immunodeficiency virus type 1 (HIV-1) treatment during the acute period of infection can significantly limit the seeding of viral reservoirs and modify the course of disease. However, while a number of HIV-1 broadly neutralizing antibodies (bnAbs) have demonstrated remarkable efficacy as prophylaxis in macaques chronically infected with simian-human immunodeficiency virus (SHIV), intriguingly, their inhibitory effects were largely attenuated in the acute period of SHIV infection. To investigate the mechanism for the disparate performance of bnAbs in different periods of SHIV infection, we used LSEVh-LS-F, a bispecific bnAb targeting the CD4 binding site and CD4-induced epitopes, as a representative bnAb and assessed its potential therapeutic benefit in controlling virus replication in acutely or chronically SHIV-infected macaques. We found that a single infusion of LSEVh-LS-F resulted in rapid decline of plasma viral loads to undetectable levels without emergence of viral resistance in the chronically infected macaques. In contrast, the inhibitory effect was robust but transient in the acutely infected macaques, despite the fact that all macaques had comparable plasma viral loads initially. Infusing multiple doses of LSEVh-LS-F did not extend its inhibitory duration. Furthermore, the pharmacokinetics of the infused LSEVh-LS-F in the acutely SHIV-infected macaques significantly differed from that in the uninfected or chronically infected macaques. Host SHIV-specific immune responses may play a role in the viremia-dependent pharmacokinetics. Our results highlight the correlation between the fast clearance of infused bnAbs and the treatment failure in the acute period of SHIV infection and may have important implications for the therapeutic use of bnAbs to treat acute HIV infections.IMPORTANCE Currently, there is no bnAb-based monotherapy that has been reported to clear the virus in the acute SHIV infection period. Since early HIV treatment is considered critical to restricting the establishment of viral reservoirs, investigation into the mechanism for treatment failure in acutely infected macaques would be important for the therapeutic use of bnAbs and eventually towards the functional cure of HIV/AIDS. Here we report the comparative study of the therapeutic efficacy of a bnAb in acutely and chronically SHIV-infected macaques. This study revealed the correlation between the fast clearance of infused bnAbs and treatment failure during the acute period of infection.

Establishment of a Novel Humanized Mouse Model To Investigate In Vivo Activation and Depletion of Patient-Derived HIV Latent Reservoirs.

Curing HIV infection has been thwarted by the persistent reservoir of latently infected CD4+ T cells, which reinitiate systemic infection after antiretroviral therapy (ART) interruption. To evaluate reservoir depletion strategies, we developed a novel preclinical in vivo model consisting of immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells (PBMC) from long-term ART-suppressed HIV-infected donors. In the absence of ART, these mice developed rebound viremia which, 2 weeks after PBMC injection, was 1,000-fold higher (mean = 9,229,281 HIV copies/ml) in mice injected intrasplenically than in mice injected intraperitoneally (mean = 6,838 HIV copies/ml) or intravenously (mean = 591 HIV copies/ml). One week after intrasplenic PBMC injection, in situ hybridization of the spleen demonstrated extensive disseminated HIV infection, likely initiated from in vivo-reactivated primary latently infected cells. The time to viremia was delayed significantly by treatment with a broadly neutralizing antibody, 10-1074, compared to treatment with 10-1074-FcRnull, suggesting that 10-1074 mobilized Fc-mediated effector mechanisms to deplete the replication-competent reservoir. This was supported by phylogenetic analysis of Env sequences from viral-outgrowth cultures and untreated, 10-1074-treated, or 10-1074-FcRnull-treated mice. The predominant sequence cluster detected in viral-outgrowth cultures and untreated mouse plasma was significantly reduced in the plasma of 10-1074-treated mice, whereas two new clusters emerged that were not detected in viral-outgrowth cultures or plasma from untreated mice. These new clusters lacked mutations associated with 10-1074 resistance. Taken together, these data indicated that 10-1074 treatment depletes the reservoir of latently infected cells harboring replication competent HIV. Furthermore, this mouse model represents a new in vivo approach for the preclinical evaluation of new HIV cure strategies.IMPORTANCE Sustained remission of HIV infection is prevented by a persistent reservoir of latently infected cells capable of reinitiating systemic infection and viremia. To evaluate strategies to reactivate and deplete this reservoir, we developed and characterized a new humanized mouse model consisting of highly immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells from long-term ART-suppressed HIV-infected donors. Reactivation and dissemination of HIV infection was visualized in the mouse spleens in parallel with the onset of viremia. The applicability of this model for evaluating reservoir depletion treatments was demonstrated by establishing, through delayed time to viremia and phylogenetic analysis of plasma virus, that treatment of these humanized mice with a broadly neutralizing antibody, 10-1074, depleted the patient-derived population of latently infected cells. This mouse model represents a new in vivo approach for the preclinical evaluation of new HIV cure strategies.

Susceptibility to Neutralization by Broadly Neutralizing Antibodies Generally Correlates with Infected Cell Binding for a Panel of Clade B HIV Reactivated from Latent Reservoirs.

Efforts to cure human immunodeficiency virus (HIV) infection are obstructed by reservoirs of latently infected CD4+ T cells that can reestablish viremia. HIV-specific broadly neutralizing antibodies (bNAbs), defined by unusually wide neutralization breadths against globally diverse viruses, may contribute to the elimination of these reservoirs by binding to reactivated cells, thus targeting them for immune clearance. However, the relationship between neutralization of reservoir isolates and binding to corresponding infected primary CD4+ T cells has not been determined. Thus, the extent to which neutralization breadths and potencies can be used to infer the corresponding parameters of infected cell binding is currently unknown. We assessed the breadths and potencies of bNAbs against 36 viruses reactivated from peripheral blood CD4+ T cells from antiretroviral (ARV)-treated HIV-infected individuals by using paired neutralization and infected cell binding assays. Single-antibody breadths ranged from 0 to 64% for neutralization (80% inhibitory concentration [IC80] of ≤10 µg/ml) and from 0 to 89% for binding, with two-antibody combinations (results for antibody combinations are theoretical/predicted) reaching levels of 0 to 83% and 50 to 100%, respectively. Infected cell binding correlated with virus neutralization for 10 of 14 antibodies (e.g., for 3BNC117, r = 0.82 and P < 0.0001). Heterogeneity was observed, however, with a lack of significant correlation for 2G12, CAP256.VRC26.25, 2F5, and 4E10. Our results provide guidance on the selection of bNAbs for interventional cure studies, both by providing a direct assessment of intra- and interindividual variabilities in neutralization and infected cell binding in a novel cohort and by defining the relationships between these parameters for a panel of bNAbs.IMPORTANCE Although antiretroviral therapies have improved the lives of people who are living with HIV, they do not cure infection. Efforts are being directed towards harnessing the immune system to eliminate the virus that persists, potentially resulting in virus-free remission without medication. HIV-specific antibodies hold promise for such therapies owing to their ability to both prevent the infection of new cells (neutralization) and direct the killing of infected cells. We isolated 36 HIV strains from individuals whose virus was suppressed by medication and tested 14 different antibodies for neutralization of these viruses and for binding to cells infected with the same viruses (critical for engaging natural killer cells). For both neutralization and infected cell binding, we observed variation both between individuals and amongst different viruses within an individual. For most antibodies, neutralization activity correlated with infected cell binding. These data provide guidance on the selection of antibodies for clinical trials.

Potent In Vivo NK Cell-Mediated Elimination of HIV-1-Infected Cells Mobilized by a gp120-Bispecific and Hexavalent Broadly Neutralizing Fusion Protein.

Antibodies bound to human immunodeficiency virus type 1 (HIV-1) envelope protein expressed by infected cells mobilize antibody-dependent cellular cytotoxicity (ADCC) to eliminate the HIV-1-infected cells and thereby suppress HIV-1 infection and delay disease progression. Studies treating HIV-1-infected individuals with latency reactivation agents to reduce their latent HIV-1 reservoirs indicated that their HIV-1-specific immune responses were insufficient to effectively eliminate the reactivated latent HIV-1-infected T cells. Mobilization of ADCC may facilitate elimination of reactivated latent HIV-1-infected cells to deplete the HIV-1 reservoir and contribute to a functional HIV-1 cure. The most effective antibodies for controlling and eradicating HIV-1 infection would likely have the dual capacities of potently neutralizing a broad range of HIV-1 isolates and effectively mobilizing HIV-1-specific ADCC to eliminate HIV-1-infected cells. For this purpose, we constructed LSEVh-LS-F, a broadly neutralizing, defucosylated hexavalent fusion protein specific for both the CD4 and coreceptor gp120-binding sites. LSEVh-LS-F potently inhibited in vivo HIV-1 and simian-human immunodeficiency virus (SHIV) infection in humanized mouse and macaque models, respectively, including in vivo neutralization of HIV-1 strains resistant to the broadly neutralizing antibodies VRC01 and 3BNC117. We developed a novel humanized mouse model to evaluate in vivo human NK cell-mediated elimination of HIV-1-infected cells by ADCC and utilized it to demonstrate that LSEVh-LS-F rapidly mobilized NK cells to eliminate >80% of HIV-1-infected cells in vivo 1 day after its administration. The capacity of LSEVh-LS-F to eliminate HIV-1-infected cells via ADCC combined with its broad neutralization activity supports its potential use as an immunotherapeutic agent to eliminate reactivated latent cells and deplete the HIV-1 reservoir.IMPORTANCE Mobilization of antibody-dependent cellular cytotoxicity (ADCC) to eliminate reactivated latent HIV-1-infected cells is a strategy which may contribute to depleting the HIV-1 reservoir and achieving a functional HIV-1 cure. To more effectively mobilize ADCC, we designed and constructed LSEVh-LS-F, a broadly neutralizing, defucosylated hexavalent fusion protein specific for both the CD4 and coreceptor gp120-binding sites. LSEVh-LS-F potently inhibited in vivo HIV-1 and SHIV infection in humanized mouse and macaque models, respectively, including in vivo neutralization of an HIV-1 strain resistant to the broadly neutralizing antibodies VRC01 and 3BNC117. Using a novel humanized mouse model, we demonstrated that LSEVh-LS-F rapidly mobilized NK cells to eliminate >80% of HIV-1-infected cells in vivo 1 day after its administration. The capacity of LSEVh-LS-F to eliminate HIV-1-infected cells via ADCC combined with its broad neutralization activity supports its potential use as an immunotherapeutic agent to eliminate reactivated latent cells and deplete the HIV-1 reservoir.

T-Cell Receptor (TCR) Clonotype-Specific Differences in Inhibitory Activity of HIV-1 Cytotoxic T-Cell Clones Is Not Mediated by TCR Alone.

Functional analysis of T-cell responses in HIV-infected individuals has indicated that virus-specific CD8+ T cells with superior antiviral efficacy are well represented in HIV-1 controllers but are rare or absent in HIV-1 progressors. To define the role of individual T-cell receptor (TCR) clonotypes in differential antiviral CD8+ T-cell function, we performed detailed functional and mass cytometric cluster analysis of multiple CD8+ T-cell clones recognizing the identical HLA-B*2705-restricted HIV-1 epitope KK10 (KRWIILGLNK). Effective and ineffective CD8+ T-cell clones segregated based on responses to HIV-1-infected and peptide-loaded target cells. Following cognate peptide stimulation, effective HIV-specific clones displayed significantly more rapid TCR signal propagation, more efficient initial lytic granule release, and more sustained nonlytic cytokine and chemokine secretion than ineffective clones. To evaluate the TCR clonotype contribution to CD8+ T-cell function, we cloned the TCR α and ß chain genes from one effective and two ineffective CD8+ T-cell clones from an elite controller into TCR-expressing lentivectors. We show that Jurkat/MA cells and primary CD8+ T cells transduced with lentivirus expressing TCR from one of the ineffective clones exhibited a level of activation by cognate peptide and inhibition of in vitro HIV-1 infection, respectively, that were comparable to those of the effective clonotype. Taken together, these data suggest that the potent antiviral capacity of some HIV-specific CD8+ T cells is a consequence of factors in addition to TCR sequence that modulate functionality and contribute to the increased antiviral capacity of HIV-specific CD8+ T cells in elite controllers to inhibit HIV infection.IMPORTANCE The greater ex vivo antiviral inhibitory activity of CD8+ T cells from elite controllers than from HIV-1 progressors supports the crucial role of effective HIV-specific CD8+ T cells in controlling HIV-1 replication. The contribution of TCR clonotype to inhibitory potency was investigated by delineating the responsiveness of effective and ineffective CD8+ T-cell clones recognizing the identical HLA-B*2705-restricted HIV-1 Gag-derived peptide, KK10 (KRWIILGLNK). KK10-stimulated "effective" CD8+ T-cell clones displayed significantly more rapid TCR signal propagation, more efficient initial lytic granule release, and more sustained cytokine and chemokine secretion than "ineffective" CD8+ T-cell clones. However, TCRs cloned from an effective and one of two ineffective clones conferred upon primary CD8+ T cells the equivalent potent capacity to inhibit HIV-1 infection. Taken together, these data suggest that other factors aside from intrinsic TCR-peptide-major histocompatibility complex (TCR-peptide-MHC) reactivity can contribute to the potent antiviral capacity of some HIV-specific CD8+ T-cell clones.

Improving the CH1-CK heterodimerization and pharmacokinetics of 4Dm2m, a novel potent CD4-antibody fusion protein against HIV-1.

We previously described 4Dm2m, an exceptionally potent broadly neutralizing CD4-antibody fusion protein against HIV-1. It was generated by fusing the engineered single human CD4 domain mD1.22 to both the N and C termini of the human IgG1 heavy chain constant region and the engineered single human antibody domain m36.4, which targets the CD4-induced coreceptor binding site of the viral envelope glycoprotein, to the N terminus of the human antibody kappa light chain constant region via the (G4S)3 polypeptide linkers. However, therapeutic use of 4Dm2m was limited by its short in vivo half-life. Here, we show that a combination of three approaches have successfully increased the persistence of 4Dm2m in mice. First, to stabilize the scaffold, we enhanced heterodimerization between the heavy chain constant domain 1 (CH1) and kappa light chain constant domain (CK) by using structure-guided design and phage-display library technologies. Second, to address the possibility that long polypeptide linkers might render fusion proteins more susceptible to proteolysis, we shortened the (G4S)3 linkers or replaced them with the human IgG1 hinge sequence, which is naturally designed for both flexibility and stability. Third, we introduced two amino acid mutations into the crystallizable fragment (Fc) of the scaffold previously shown to increase antibody binding to the neonatal Fc receptor (FcRn) and prolong half-lives in vivo. Collectively, these approaches markedly increased the serum concentrations of 4Dm2m in mice while not affecting other properties of the fusion protein. The new 4Dm2m variants are promising candidates for clinical development to prevent or treat HIV-1 infection. To our knowledge, this is the first report on stabilized CH1-CK, which is potentially useful as a new heterodimerization scaffold for generation of bispecific and multispecific antibodies or proteins with a more favorable pharmacokinetic profile.

High-Throughput Humanized Mouse Models for Evaluation of HIV-1 Therapeutics and Pathogenesis.

Mice cannot be used as a model to evaluate HIV-1 therapeutics because they do not become infected by HIV-1 due to structural differences between several human and mouse proteins required for HIV-1 replication. This has limited their use for in vivo assessment of anti-HIV-1 therapeutics and the mechanism by which cofactors, such as illicit drug use accelerate HIV-1 replication and disease course in substance abusers. Here, we describe the development and application of two in vivo humanized mouse models that are highly sensitive and useful models for the in vivo evaluation of candidate anti-HIV therapeutics. The first model, hu-spl-PBMC-NSG mice, uses NOD-SCID IL2rγ(-/-) (NSG) mice intrasplenically injected with human peripheral blood mononuclear cells (PBMC) which develop productive splenic HIV-1 infection after intrasplenic inoculation with a replication-competent HIV-1 expressing Renilla reniformis luciferase (HIV-LucR) and enables investigators to use bioluminescence to visualize and quantitate the temporal effects of therapeutics on HIV-1 infection. The second model, hCD4/R5/cT1 mice, consists of transgenic mice carrying human CD4, CCR5 and cyclin T1 genes, which enables murine CD4-expressing cells to support HIV-1 entry, Tat-mediated LTR transcription and consequently develop productive infection. The hCD4/R5/cT1 mice develop disseminated infection of tissues including the spleen, small intestine, lymph nodes and lungs after intravenous injection with HIV-1-LucR. Because these mice can be infected with HIV-LucR expressing transmitted/founder and clade A/E and C Envs, these mouse models can also be used to evaluate the in vivo efficacy of broadly neutralizing antibodies and antibodies induced by candidate HIV-1 vaccines. Furthermore, because hCD4/R5/cT1 mice can be infected by vaginal inoculation with replication-competent HIV-1 expressing NanoLuc (HIV-nLucR)-, this mouse model can be used to evaluate the mechanisms by which substance abuse and other factors enhance mucosal transmission of HIV-1.

The Vaginal Acquisition and Dissemination of HIV-1 Infection in a Novel Transgenic Mouse Model Is Facilitated by Coinfection with Herpes Simplex Virus 2 and Is Inhibited by Microbicide Treatment.

UNLABELLED: Epidemiological studies have demonstrated that herpes simplex virus 2 (HSV-2) infection significantly increases the risk of HIV-1 acquisition, thereby contributing to the expanding HIV-1 epidemic. To investigate whether HSV-2 infection directly facilitates mucosal HIV-1 acquisition, we used our transgenic hCD4/R5/cT1 mouse model which circumvents major entry and transcription blocks preventing murine HIV-1 infection by targeting transgenic expression of human CD4, CCR5, and cyclin T1 genes to CD4(+) T cells and myeloid-committed cells. Productive infection of mucosal leukocytes, predominantly CD4(+) T cells, was detected in all hCD4/R5/cT1 mice intravaginally challenged with an HIV-1 infectious molecular clone, HIV-Du151.2env-NLuc, which expresses an env gene (C.Du151.2) cloned from an acute heterosexually infected woman and a NanoLuc luciferase reporter gene. Lower genital tract HIV-1 infection after HIV-Du151.2env-NLuc intravaginal challenge was increased ~4-fold in hCD4/R5/cT1 mice coinfected with HSV-2. Furthermore, HIV-1 dissemination to draining lymph nodes was detected only in HSV-2-coinfected mice. HSV-2 infection stimulated local infiltration and activation of CD4(+) T cells and dendritic cells, likely contributing to the enhanced HIV-1 infection and dissemination in HSV-2-coinfected mice. We then used this model to demonstrate that a novel gel containing tenofovir disoproxil fumarate (TDF), the more potent prodrug of tenofovir (TFV), but not the TFV microbicide gel utilized in the recent CAPRISA 004, VOICE (Vaginal and Oral Interventions to Control the Epidemic), and FACTS 001 clinical trials, was effective as preexposure prophylaxis (PrEP) to completely prevent vaginal HIV-1 infection in almost half of HSV-2-coinfected mice. These results also support utilization of hCD4/R5/cT1 mice as a highly reproducible immunocompetent preclinical model to evaluate HIV-1 acquisition across the female genital tract. IMPORTANCE: Multiple epidemiological studies have reported that genital herpes simplex virus 2 (HSV-2) infection increases the risk of HIV-1 sexual acquisition by severalfold. Understanding the underlying mechanisms by which HSV-2 facilitates HIV-1 infection and optimizing the efficacy of therapies to inhibit HIV-1 infection during HSV-2 coinfection should contribute to reducing HIV-1 transmission. Using our novel transgenic hCD4/R5/cT1 mouse model infectible with HIV-1, we demonstrated that HSV-2 infection enhances vaginal transmission and dissemination of HIV-1 infection while stimulating recruitment and activation of CD4(+) T cells and dendritic cells in the lower genital tract. HIV acquisition by hCD4/R5/cT1 mice vaginally coinfected with HSV-2 could be completely prevented in almost half the mice by preexposure prophylaxis (PrEP) with a novel gel containing tenofovir disoproxil fumarate (TDF), the tenofovir prodrug, but not with the tenofovir microbicide gel utilized in CAPRISA-004, VOICE, and FACTS-001 clinical trials. The hCD4/R5/cT1 mice represent a new preclinical mouse model to evaluate vaginal HIV-1 acquisition.