Anti-PD-1 chimeric antigen receptor T cells efficiently target SIV-infected CD4+ T cells in germinal centers.

Programmed cell death protein 1 (PD-1) is an immune checkpoint marker commonly expressed on memory T cells and enriched in latently HIV-infected CD4+ T cells. We engineered an anti-PD-1 chimeric antigen receptor (CAR) to assess the impact of PD-1 depletion on viral reservoirs and rebound dynamics in SIVmac239-infected rhesus macaques (RMs). Adoptive transfer of anti-PD-1 CAR T cells was done in 2 SIV-naive and 4 SIV-infected RMs on antiretroviral therapy (ART). In 3 of 6 RMs, anti-PD-1 CAR T cells expanded and persisted for up to 100 days concomitant with the depletion of PD-1+ memory T cells in blood and tissues, including lymph node CD4+ follicular helper T (TFH) cells. Loss of TFH cells was associated with depletion of detectable SIV RNA from the germinal center (GC). However, following CAR T infusion and ART interruption, there was a marked increase in SIV replication in extrafollicular portions of lymph nodes, a 2-log higher plasma viremia relative to controls, and accelerated disease progression associated with the depletion of CD8+ memory T cells. These data indicate anti-PD-1 CAR T cells depleted PD-1+ T cells, including GC TFH cells, and eradicated SIV from this immunological sanctuary.

CD20 CAR T cells safely and reversibly ablate B cell follicles in a non-human primate model of HIV persistence.

Chimeric antigen receptor (CAR) T cell therapies have demonstrated immense clinical success for B cell and plasma cell malignancies. We tested their impact on the viral reservoir in a macaque model of HIV persistence, comparing the functions of CD20 CAR T cells between animals infected with simian/human immunodeficiency virus (SHIV) and uninfected controls. We focused on the potential of this approach to disrupt B cell follicles (BCFs), exposing infected cells for immune clearance. In SHIV-infected animals, CAR T cells were highly functional, with rapid expansion and trafficking to tissue-associated viral sanctuaries, including BCFs and gut-associated lymphoid tissue (GALT). CD20 CAR T cells potently ablated BCFs and depleted lymph-node-associated follicular helper T (TFH) cells, with complete restoration of BCF architecture and TFH cells following CAR T cell contraction. BCF ablation decreased the splenic SHIV reservoir but was insufficient for effective reductions in systemic viral reservoirs. Although associated with moderate hematologic toxicity, CD20 CAR T cells were well tolerated in SHIV-infected and control animals, supporting the feasibility of this therapy in people living with HIV with underlying B cell malignancies. Our findings highlight the unique ability of CD20 CAR T cells to safely and reversibly unmask TFH cells within BCF sanctuaries, informing future combinatorial HIV cure strategies designed to augment antiviral efficacy.

Induction of durable remission by dual immunotherapy in SHIV-infected ART-suppressed macaques.

The eradication of the viral reservoir represents the major obstacle to the development of a clinical cure for established HIV-1 infection. Here, we demonstrate that the administration of N-803 (brand name Anktiva) and broadly neutralizing antibodies (bNAbs) results in sustained viral control after discontinuation of antiretroviral therapy (ART) in simian-human AD8 (SHIV-AD8)-infected, ART-suppressed rhesus macaques. N-803+bNAbs treatment induced immune activation and transient viremia but only limited reductions in the SHIV reservoir. Upon ART discontinuation, viral rebound occurred in all animals, which was followed by durable control in approximately 70% of all N-803+bNAb-treated macaques. Viral control was correlated with the reprogramming of CD8+ T cells by N-803+bNAb synergy. Thus, complete eradication of the replication-competent viral reservoir is likely not a prerequisite for the induction of sustained remission after discontinuation of ART.

Stable HIV decoy receptor expression after in vivo HSC transduction in mice and NHPs: Safety and efficacy in protection from SHIV.

We aim to develop an in vivo hematopoietic stem cell (HSC) gene therapy approach for persistent control/protection of HIV-1 infection based on the stable expression of a secreted decoy protein for HIV receptors CD4 and CCR5 (eCD4-Ig) from blood cells. HSCs in mice and a rhesus macaque were mobilized from the bone marrow and transduced by an intravenous injection of HSC-tropic, integrating HDAd5/35++ vectors expressing rhesus eCD4-Ig. In vivo HSC transduction/selection resulted in stable serum eCD4-Ig levels of ∼100 µg/mL (mice) and >20 µg/mL (rhesus) with half maximal inhibitory concentrations (IC50s) of 1 µg/mL measured by an HIV neutralization assay. After simian-human-immunodeficiency virus D (SHIV.D) challenge of rhesus macaques injected with HDAd-eCD4-Ig or a control HDAd5/35++ vector, peak plasma viral load levels were ∼50-fold lower in the eCD4-Ig-expressing animal. Furthermore, the viral load was lower in tissues with the highest eCD4-Ig expression, specifically the spleen and lymph nodes. SHIV.D challenge triggered a selective expansion of transduced CD4+CCR5+ cells, thereby increasing serum eCD4-Ig levels. The latter, however, broke immune tolerance and triggered anti-eCD4-Ig antibody responses, which could have contributed to the inability to eliminate SHIV.D. Our data will guide us in the improvement of the in vivo approach. Clearly, our conclusions need to be validated in larger animal cohorts.

Development of an anti-CAR antibody response in SIV-infected rhesus macaques treated with CD4-MBL CAR/CXCR5 T cells.

T cells expressing a simian immunodeficiency (SIV)-specific chimeric antigen receptor (CAR) and the follicular homing molecule, CXCR5, were infused into antiretroviral therapy (ART) suppressed, SIV-infected rhesus macaques to assess their ability to localize to the lymphoid follicle and control the virus upon ART interruption. While the cells showed evidence of functionality, they failed to persist in the animals beyond 28 days. Development of anti-CAR antibodies could be responsible for the lack of persistence. Potential antigenic sites on the anti-SIV CAR used in these studies included domains 1 and 2 of CD4, the carbohydrate recognition domain (CRD) of mannose-binding lectin (MBL), and an extracellular domain of the costimulatory molecule, CD28, along with short linker sequences. Using a flow cytometry based assay and target cells expressing the CAR/CXCR5 construct, we examined the serum of the CD4-MBL CAR/CXCR5-T cell treated animals to determine that the animals had developed an anti-CAR antibody response after infusion. Binding sites for the anti-CAR antibodies were identified by using alternative CARs transduced into target cells and by preincubation of the target cells with a CD4 blocking antibody. All of the treated animals developed antibodies in their serum that bound to CD4-MBL CAR/CXCR5 T cells and the majority were capable of inducing an ADCC response. The CD4 antibody-blocking assay suggests that the dominant immunogenic components of this CAR are the CD4 domains with a possible additional site of the CD28 domain with its linker. This study shows that an anti-drug antibody (ADA) response can occur even when using self-proteins, likely due to novel epitopes created by abridged self-proteins and/or the self-domain of the CAR connection to a small non-self linker. While in our study, there was no statistically significant correlation between the ADA response and the persistence of the CD4-MBL CAR/CXCR5-T cells in rhesus macaques, these findings suggest that the development of an ADA response could impact the long-term persistence of self-based CAR immunotherapies.

CAR/CXCR5-T cell immunotherapy is safe and potentially efficacious in promoting sustained remission of SIV infection.

During chronic human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infection prior to AIDS progression, the vast majority of viral replication is concentrated within B cell follicles of secondary lymphoid tissues. We investigated whether infusion of T cells expressing an SIV-specific chimeric antigen receptor (CAR) and the follicular homing receptor, CXCR5, could successfully kill viral-RNA+ cells in targeted lymphoid follicles in SIV-infected rhesus macaques. In this study, CD4 and CD8 T cells from rhesus macaques were genetically modified to express antiviral CAR and CXCR5 moieties (generating CAR/CXCR5-T cells) and autologously infused into a chronically infected animal. At 2 days post-treatment, the CAR/CXCR5-T cells were located primarily in spleen and lymph nodes both inside and outside of lymphoid follicles. Few CAR/CXCR5-T cells were detected in the ileum, rectum, and lung, and no cells were detected in the bone marrow, liver, or brain. Within follicles, CAR/CXCR5-T cells were found in direct contact with SIV-viral RNA+ cells. We next infused CAR/CXCR5-T cells into ART-suppressed SIV-infected rhesus macaques, in which the animals were released from ART at the time of infusion. These CAR/CXCR5-T cells replicated in vivo within both the extrafollicular and follicular regions of lymph nodes and accumulated within lymphoid follicles. CAR/CXR5-T cell concentrations in follicles peaked during the first week post-infusion but declined to undetectable levels after 2 to 4 weeks. Overall, CAR/CXCR5-T cell-treated animals maintained lower viral loads and follicular viral RNA levels than untreated control animals, and no outstanding adverse reactions were noted. These findings indicate that CAR/CXCR5-T cell treatment is safe and holds promise as a future treatment for the durable remission of HIV.

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.

Elimination of SHIV Infected Cells by Combinations of Bispecific HIVxCD3 DART® Molecules.

Bispecific HIVxCD3 DART molecules that co-engage the viral envelope glycoprotein (Env) on HIV-1-infected cells and the CD3 receptor on CD3+ T cells are designed to mediate the cytolysis of HIV-1-infected, Env-expressing cells. Using a novel ex vivo system with cells from rhesus macaques (RMs) infected with a chimeric Simian-Human Immunodeficiency Virus (SHIV) CH505 and maintained on ART, we tested the ability of HIVxCD3 DART molecules to mediate elimination of in vitro-reactivated CD4+ T cells in the absence or presence of autologous CD8+ T cells. HIVxCD3 DART molecules with the anti-HIV-1 Env specificities of A32 or 7B2 (non-neutralizing antibodies) or PGT145 (broadly neutralizing antibody) were evaluated individually or combined. DART molecule-mediated antiviral activity increased significantly in the presence of autologous CD8+ T cells. In this ex vivo system, the PGT145 DART molecule was more active than the 7B2 DART molecule, which was more active than the A32 DART molecule. A triple combination of the DART molecules exceeded the activity of the individual PGT145 DART molecule. Modified quantitative virus outgrowth assays confirmed the ability of the DART molecules to redirect RM CD3+ T cells to eliminate SHIV-infected RM CD4+ T cells as demonstrated by the decreased propagation of in vitro infection by the infected cells pre-incubated with DART molecules in presence of effector CD8+ T cells. While mediating cytotoxic activity, DART molecules did not increase proinflammatory cytokine production. In summary, combination of HIVxCD3 DART molecules that have broadly-neutralizing and non-neutralizing anti-HIV-1 Env specificities can leverage the host immune system for treatment of HIV-1 infection but will require appropriate reactivation of the latent reservoir.

Therapeutic Potential of IL-15 and N-803 in HIV/SIV Infection.

IL-15, a proinflammatory cytokine critical for the generation, maintenance, and homeostasis of T cell responses, is produced naturally in response to HIV/SIV infection, but has also demonstrated therapeutic potential. IL-15 can boost CD4+ and CD8+ T cell and NK cell proliferation, activation, and function. However, IL-15 treatment may cause aberrant immune activation and accelerated disease progression in certain circumstances. Moreover, the relationship between the timing of IL-15 administration and disease progression remains unclear. The IL-15 superagonist N-803 was developed to expand the therapeutic potential of IL-15 by maximizing its tissue distribution and half-life. N-803 has garnered enthusiasm recently as a way to enhance the innate and cellular immune responses to HIV/SIV by improving CD8+ T cell recognition and killing of virus-infected cells and directing immune cells to mucosal sites and lymph nodes, the primary sites of virus replication. N-803 has also been evaluated in "shock and kill" strategies due to its potential to reverse latency (shock) and enhance antiviral immunity (kill). This review examines the current literature about the effects of IL-15 and N-803 on innate and cellular immunity, viral burden, and latency reversal in the context of HIV/SIV, and their therapeutic potential both alone and combined with additional interventions such as antiretroviral therapy (ART) and vaccination.

Mathematical modeling of N-803 treatment in SIV-infected non-human primates.

Immunomodulatory drugs could contribute to a functional cure for Human Immunodeficiency Virus (HIV). Interleukin-15 (IL-15) promotes expansion and activation of CD8+ T cell and natural killer (NK) cell populations. In one study, an IL-15 superagonist, N-803, suppressed Simian Immunodeficiency Virus (SIV) in non-human primates (NHPs) who had received prior SIV vaccination. However, viral suppression attenuated with continued N-803 treatment, partially returning after long treatment interruption. While there is evidence of concurrent drug tolerance, immune regulation, and viral escape, the relative contributions of these mechanisms to the observed viral dynamics have not been quantified. Here, we utilize mathematical models of N-803 treatment in SIV-infected macaques to estimate contributions of these three key mechanisms to treatment outcomes: 1) drug tolerance, 2) immune regulation, and 3) viral escape. We calibrated our model to viral and lymphocyte responses from the above-mentioned NHP study. Our models track CD8+ T cell and NK cell populations with N-803-dependent proliferation and activation, as well as viral dynamics in response to these immune cell populations. We compared mathematical models with different combinations of the three key mechanisms based on Akaike Information Criterion and important qualitative features of the NHP data. Two minimal models were capable of reproducing the observed SIV response to N-803. In both models, immune regulation strongly reduced cytotoxic cell activation to enable viral rebound. Either long-term drug tolerance or viral escape (or some combination thereof) could account for changes to viral dynamics across long breaks in N-803 treatment. Theoretical explorations with the models showed that less-frequent N-803 dosing and concurrent immune regulation blockade (e.g. PD-L1 inhibition) may improve N-803 efficacy. However, N-803 may need to be combined with other immune therapies to countermand viral escape from the CD8+ T cell response. Our mechanistic model will inform such therapy design and guide future studies.

Protection of Newborn Macaques by Plant-Derived HIV Broadly Neutralizing Antibodies: a Model for Passive Immunotherapy during Breastfeeding.

Preventing human immunodeficiency virus (HIV) infection in newborns by vertical transmission remains an important unmet medical need in resource-poor areas where antiretroviral therapy (ART) is not available and mothers and infants cannot be treated prepartum or during the breastfeeding period. In the present study, the protective efficacy of the potent HIV-neutralizing antibodies PGT121 and VRC07-523, both produced in plants, were assessed in a multiple-SHIV (simian-human immunodeficiency virus)-challenge breastfeeding macaque model. Newborn macaques received either six weekly subcutaneous injections with PGT121 alone or as a cocktail of PGT121-LS plus VRC07-523-LS injected three times every 2 weeks. Viral challenge with SHIVSF162P3 was twice weekly over 5.5 weeks using 11 exposures. Despite the transient presence of plasma viral RNA either immediately after the first challenge or as single-point blips, the antibodies prevented a productive infection in all babies with no sustained plasma viremia, compared to viral loads ranging from 103 to 5 × 108 virions/ml in four untreated controls. No virus was detected in peripheral blood mononuclear cells (PBMCs), and only 3 of 159 tissue samples were weakly positive in the treated babies. Newborn macaques proved to be immunocompetent, producing transient anti-Env antibodies and anti-drug antibody (ADA), which were maintained in the circulation after passive broadly neutralizing antibody clearance. ADA responses were directed to the IgG1 Fc CH2-CH3 domains, which has not been observed to date in adult monkeys passively treated with PGT121 or VRC01. In addition, high levels of VRC07-523 anti-idiotypic antibodies in the circulation of one newborn was concomitant with the rapid elimination of VRC07. Plant-expressed antibodies show promise as passive immunoprophylaxis in a breastfeeding model in newborns. IMPORTANCE Plant-produced human neutralizing antibody prophylaxis is highly effective in preventing infection in newborn monkeys during repeated oral exposure, modeling virus in breastmilk, and offers advantages in cost of production and safety. These findings raise the possibility that anti-Env antibodies may contribute to the control of viral replication in this newborn model and that the observed immune responsiveness may be driven by the long-lived presence of immune complexes.

Mechanistic basis of post-treatment control of SIV after anti-α4ß7 antibody therapy.

Treating macaques with an anti-α4ß7 antibody under the umbrella of combination antiretroviral therapy (cART) during early SIV infection can lead to viral remission, with viral loads maintained at < 50 SIV RNA copies/ml after removal of all treatment in a subset of animals. Depletion of CD8+ lymphocytes in controllers resulted in transient recrudescence of viremia, suggesting that the combination of cART and anti-α4ß7 antibody treatment led to a state where ongoing immune responses kept the virus undetectable in the absence of treatment. A previous mathematical model of HIV infection and cART incorporates immune effector cell responses and exhibits the property of two different viral load set-points. While the lower set-point could correspond to the attainment of long-term viral remission, attaining the higher set-point may be the result of viral rebound. Here we expand that model to include possible mechanisms of action of an anti-α4ß7 antibody operating in these treated animals. We show that the model can fit the longitudinal viral load data from both IgG control and anti-α4ß7 antibody treated macaques, suggesting explanations for the viral control associated with cART and an anti-α4ß7 antibody treatment. This effective perturbation to the virus-host interaction can also explain observations in other nonhuman primate experiments in which cART and immunotherapy have led to post-treatment control or resetting of the viral load set-point. Interestingly, because the viral kinetics in the various treated animals differed-some animals exhibited large fluctuations in viral load after cART cessation-the model suggests that anti-α4ß7 treatment could act by different primary mechanisms in different animals and still lead to post-treatment viral control. This outcome is nonetheless in accordance with a model with two stable viral load set-points, in which therapy can perturb the system from one set-point to a lower one through different biological mechanisms.

Mitigation of endemic GI-tract pathogen-mediated inflammation through development of multimodal treatment regimen and its impact on SIV acquisition in rhesus macaques.

Here, we assessed the efficacy of a short-course multimodal therapy (enrofloxacin, azithromycin, fenbendazole, and paromomycin) to eliminate common macaque endemic pathogens (EPs) and evaluated its impact on gastrointestinal (GI) microbiota, mucosal integrity, and local and systemic inflammation in sixteen clinically healthy macaques. Treatment combined with expanded practices resulted in successful maintenance of rhesus macaques (RM) free of common EPs, with no evidence of overt microbiota diversity loss or dysbiosis and instead resulted in a more defined luminal microbiota across study subjects. Creation of a GI pathogen free (GPF) status resulted in improved colonic mucosal barrier function (histologically, reduced colonic MPO+, and reduced pan-bacterial 16s rRNA in the MLN), reduced local and systemic innate and adaptive inflammation with reduction of colonic Mx1 and pSTAT1, decreased intermediate (CD14+CD16+) and non-classical monocytes (CD14-CD16+), reduced populations of peripheral dendritic cells, Ki-67+ and CD38+ CD4+ T cells, Ki-67+IgG+, and Ki-67+IgD+ B cells indicating lower levels of background inflammation in the distal descending colon, draining mesenteric lymph nodes, and systemically in peripheral blood, spleen, and axillary lymph nodes. A more controlled rate of viral acquisition resulted when untreated and treated macaques were challenged by low dose intrarectal SIVmac239x, with an ~100 fold increase in dose required to infect 50% (AID50) of the animals receiving treatment compared to untreated controls. Reduction in and increased consistency of number of transmitted founder variants resulting from challenge seen in the proof of concept study directly correlated with post-treatment GPF animal's improved barrier function and reduction of key target cell populations (Ki-67+ CD4+T cells) at the site of viral acquisition in the follow up study. These data demonstrate that a therapeutic and operational strategy can successfully eliminate varying background levels of EPs and their associated aberrant immunomodulatory effects within a captive macaque cohort, leading to a more consistent, better defined and reproducible research model.

Evaluation of chimeric antigen receptor T cell therapy in non-human primates infected with SHIV or SIV.

Achieving a functional cure is an important goal in the development of HIV therapy. Eliciting HIV-specific cellular immune responses has not been sufficient to achieve durable removal of HIV-infected cells due to the restriction on effective immune responses by mutation and establishment of latent reservoirs. Chimeric antigen receptor (CAR) T cells are an avenue to potentially develop more potent redirected cellular responses against infected T cells. We developed and tested a range of HIV- and SIV-specific chimeric antigen receptor (CAR) T cell reagents based on Env-binding proteins. In general, SHIV/SIV CAR T cells showed potent viral suppression in vitro, and adding additional CAR molecules in the same transduction resulted in more potent viral suppression than single CAR transduction. Importantly, the primary determinant of virus suppression potency by CAR was the accessibility to the Env epitope, and not the neutralization potency of the binding moiety. However, upon transduction of autologous T cells followed by infusion in vivo, none of these CAR T cells impacted either acquisition as a test of prevention, or viremia as a test of treatment. Our study illustrates limitations of the CAR T cells as possible antiviral therapeutics.

Stem cell-derived CAR T cells traffic to HIV reservoirs in macaques.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) with CCR5- donor cells is the only treatment known to cure HIV-1 in patients with underlying malignancy. This is likely due to a donor cell-mediated graft-versus-host effect targeting HIV reservoirs. Allo-HSCT would not be an acceptable therapy for most people living with HIV due to the transplant-related side effects. Chimeric antigen receptor (CAR) immunotherapies specifically traffic to malignant lymphoid tissues (lymphomas) and, in some settings, are able to replace allo-HSCT. Here, we quantified the engraftment of HSC-derived, virus-directed CAR T cells within HIV reservoirs in a macaque model of HIV infection, using potentially novel IHC assays. HSC-derived CAR cells trafficked to and displayed multilineage engraftment within tissue-associated viral reservoirs, persisting for nearly 2 years in lymphoid germinal centers, the brain, and the gastrointestinal tract. Our findings demonstrate that HSC-derived CAR+ cells reside long-term and proliferate in numerous tissues relevant for HIV infection and cancer.

Immunotherapy during the acute SHIV infection of macaques confers long-term suppression of viremia.

We report that combination bNAb immunotherapy initiated on day 3 post-infection (PI) maintained durable CD8+ T cell-mediated suppression of SHIVAD8 viremia and preinoculation levels of CD4+ T cells in 9 of 13 treated monkeys during nearly 6 yr of observation, as assessed by successive CD8+ T cell-depletion experiments. In an extension of that study, two treatment interventions (bNAbs alone or cART plus bNAbs) beginning on week 2 PI were conducted and conferred controller status to 7 of 12 monkeys that was also dependent on control mediated by CD8+ cells. However, the median time to suppression of plasma viremia following intervention on week 2 was markedly delayed (85 wk) compared with combination bNAb immunotherapy initiated on day 3 (39 wk). In both cases, the principal correlate of virus control was the induction of CD8+ T cellular immunity.

Therapeutic vaccine-mediated Gag-specific CD8+ T-cell induction under anti-retroviral therapy augments anti-virus efficacy of CD8+ cells in simian immunodeficiency virus-infected macaques.

Anti-retroviral therapy (ART) can inhibit HIV proliferation but not achieve virus eradication from HIV-infected individuals. Under ART-based HIV control, virus-specific CD8+ T-cell responses are often reduced. Here, we investigated the impact of therapeutic vaccination inducing virus-specific CD8+ T-cell responses under ART on viral control in a macaque AIDS model. Twelve rhesus macaques received ART from week 12 to 32 after simian immunodeficiency virus (SIV) infection. Six of them were vaccinated with Sendai virus vectors expressing SIV Gag and Vif at weeks 26 and 32, and Gag/Vif-specific CD8+ T-cell responses were enhanced and became predominant. All macaques controlled viremia during ART but showed viremia rebound after ART cessation. Analysis of in vitro CD8+ cell ability to suppress replication of autologous lymphocytes-derived SIVs found augmentation of anti-SIV efficacy of CD8+ cells after vaccination. In the vaccinated animals, the anti-SIV efficacy of CD8+ cells at week 34 was correlated positively with Gag-specific CD8+ T-cell frequencies and inversely with rebound viral loads at week 34. These results indicate that Gag-specific CD8+ T-cell induction by therapeutic vaccination can augment anti-virus efficacy of CD8+ cells, which may be insufficient for functional cure but contribute to more stable viral control under ART.

Long-Term Delivery of an Anti-SIV Monoclonal Antibody With AAV.

Long-term delivery of anti-HIV monoclonal antibodies using adeno-associated virus (AAV) holds promise for the prevention and treatment of HIV infection. We previously reported that after receiving a single administration of AAV vector coding for anti-SIV antibody 5L7, monkey 84-05 achieved high levels of AAV-delivered 5L7 IgG1 in vivo which conferred sterile protection against six successive, escalating dose, intravenous challenges with highly infectious, highly pathogenic SIVmac239, including a final challenge with 10 animal infectious doses (1). Here we report that monkey 84-05 has successfully maintained 240-350 µg/ml of anti-SIV antibody 5L7 for over 6 years. Approximately 2% of the circulating IgG in this monkey is this one monoclonal antibody. This monkey generated little or no anti-drug antibodies (ADA) to the AAV-delivered antibody for the duration of the study. Due to the nature of the high-dose challenge used and in order to rule out a potential low-level infection not detected by regular viral loads, we have used ultrasensitive techniques to detect cell-associated viral DNA and RNA in PBMCs from this animal. In addition, we have tested serum from 84-05 by ELISA against overlapping peptides spanning the whole envelope sequence for SIVmac239 (PepScan) and against recombinant p27 and gp41 proteins. No reactivity has been detected in the ELISAs indicating the absence of naturally arising anti-SIV antibodies; moreover, the ultrasensitive cell-associated viral tests yielded no positive reaction. We conclude that macaque 84-05 was effectively protected and remained uninfected. Our data show that durable, continuous antibody expression can be achieved after one single administration of AAV and support the potential for lifelong protection against HIV from a single vector administration.

Immunotherapy with DNA vaccine and live attenuated rubella/SIV gag vectors plus early ART can prevent SIVmac251 viral rebound in acutely infected rhesus macaques.

Anti-retroviral therapy (ART) has been highly successful in controlling HIV replication, reducing viral burden, and preventing both progression to AIDS and viral transmission. Yet, ART alone cannot cure the infection. Even after years of successful therapy, ART withdrawal leads inevitably to viral rebound within a few weeks or months. Our hypothesis: effective therapy must control both the replicating virus pool and the reactivatable latent viral reservoir. To do this, we have combined ART and immunotherapy to attack both viral pools simultaneously. The vaccine regimen consisted of DNA vaccine expressing SIV Gag, followed by a boost with live attenuated rubella/gag vectors. The vectors grow well in rhesus macaques, and they are potent immunogens when used in a prime and boost strategy. We infected rhesus macaques by high dose mucosal challenge with virulent SIVmac251 and waited three days to allow viral dissemination and establishment of a reactivatable viral reservoir before starting ART. While on ART, the control group received control DNA and empty rubella vaccine, while the immunotherapy group received DNA/gag prime, followed by boosts with rubella vectors expressing SIV gag over 27 weeks. Both groups had a vaccine "take" to rubella, and the vaccine group developed antibodies and T cells specific for Gag. Five weeks after the last immunization, we stopped ART and monitored virus rebound. All four control animals eventually had a viral rebound, and two were euthanized for AIDS. One control macaque did not rebound until 2 years after ART release. In contrast, there was only one viral rebound in the vaccine group. Three out of four vaccinees had no viral rebound, even after CD8 depletion, and they remain in drug-free viral remission more than 2.5 years later. The strategy of early ART combined with immunotherapy can produce a sustained SIV remission in macaques and may be relevant for immunotherapy of HIV in humans.

Seeing Is Believing: Nuclear Imaging of HIV Persistence.

A major obstacle to HIV eradication is the presence of infected cells that persist despite suppressive antiretroviral therapy (ART). HIV largely resides outside of the peripheral circulation, and thus, numerous anatomical and lymphoid compartments that have the capacity to harbor HIV are inaccessible to routine sampling. As a result, there is a limited understanding of the tissue burden of HIV infection or anatomical distribution of HIV transcriptional and translational activity. Novel, non-invasive, in vivo methods are urgently needed to address this fundamental gap in knowledge. In this review, we discuss past and current nuclear imaging approaches that have been applied to HIV infection with an emphasis on current strategies to implement positron emission tomography (PET)-based imaging to directly visualize and characterize whole-body HIV burden. These imaging approaches have various limitations, such as the potential for limited PET sensitivity and specificity in the setting of ART suppression or low viral burden. However, recent advances in high-sensitivity, total-body PET imaging platforms and development of new radiotracer technologies that may enhance anatomical penetration of target-specific tracer molecules are discussed. Potential strategies to image non-viral markers of HIV tissue burden or focal immune perturbation are also addressed. Overall, emerging nuclear imaging techniques and platforms may play an important role in the development of novel therapeutic and HIV reservoir eradication strategies.