PrEP Works

The annual Conference on Retroviruses and Opportunistic Infections offered a broad survey of the state of HIV research, with new oral prevention results as the highlight

By Richard Jefferys

The 2015 Conference on Retroviruses and Opportunistic Infections (CROI) is likely to be remembered as a watershed moment for pre-exposure prophylaxis (PrEP)—the use of antiretrovirals to prevent HIV infection. Just as the 1996 International AIDS Conference launched highly active antiretroviral therapy into the mainstream, some attendees viewed this year’s CROI, held February 23-26 in Seattle, as the one that proved once and for all that PrEP is an effective HIV prevention strategy. PROUD and IPERGAY, two studies that were designed to evaluate PrEP’s effectiveness in men who have sex with men (MSM) in relatively real-world settings, reported reductions in HIV incidence that surprised even the investigators.

News from the vaccine research realm was not as dramatic, but an array of presentations illustrated recent progress, including advances in analyzing antibody functions and the development of a potential antibody simulacrum with unprecedented breadth and potency. Meanwhile, the pursuit of a cure remains at an early stage but is increasingly a focus at CROI, with several sessions addressing the state of the field, including areas of overlap with vaccine research such as the study of broadly neutralizing antibodies (bNAbs).

Oral PrEP results are easy to swallow

It isn’t news that PrEP works. Back in 2010 investigators first showed that oral Truvada (a pill combining the antiretrovirals tenofovir and emtricitabine) was 44% effective at preventing HIV infection in MSM and transgender women. At CROI, Sheena McCormack, professor of clinical epidemiology at the Medical Research Council Clinical Trials Unit at University College London, debuted results from the PROUD study, conducted in the UK, which was designed to assess how PrEP would perform in routine practice. To that end, investigators recruited high-risk MSM over 18 years old at sexual health clinics. High risk in this study was defined as having anal sex within the last 90 days without a condom and anticipating it would occur again in the next 90 days. Participants were randomized to receive Truvada immediately or after one year, in addition to standard-of-care prevention services. Follow up occurred every three months, in accordance with normal clinic practice.

The initial phase of the trial was intended as a pilot to explore whether recruitment and retention would be sufficient to embark on a larger efficacy trial, but in April 2014 the Data Safety Monitoring Board (DSMB) recommended evaluating efficacy due to the high HIV infection risk observed in the cohort. The DSMB then intervened again in October 2014 because the divergence between the number of HIV infections in the immediate and deferred PrEP groups was so significant that it would have been unethical to continue. Truvada was then offered to all participants.

At that point, 545 individuals were enrolled. The effectiveness analysis was based on 267 volunteers in the group that received Truvada from the start and 256 volunteers in the deferred group. The difference was striking: only three HIV infections occurred in the group receiving Truvada from the start, while there were 19 in the deferred group. Expressed in terms of incidence per 100 person-years, the comparison was 1.3 versus 8.9, indicating an efficacy of 86%. McCormack noted that this equated to one HIV infection averted for every 13 individuals on PrEP (referred to as the “number needed to treat”).

Of the three infections in the MSM randomized to receive Truvada immediately, two may have occurred just prior to enrollment (at which time the individuals were still HIV antibody negative and therefore not kept from enrolling in the study) and the other was in a participant who defaulted from follow up after receiving an initial Truvada prescription. When he was later diagnosed with HIV infection, he informed medical staff that he’d stopped taking PrEP. Six of the infections in the deferred arm could also have represented infections prior to study entry that were undetectable by antibody-based tests, but if all such cases were excluded from the analysis the reduction in incidence was still highly significant.

Adverse events were uncommon: of 13 cases where Truvada was interrupted due to a side effect (mostly gastrointestinal related), 11 volunteers successfully restarted PrEP. A drug resistance mutation associated with emtricitabine use was observed in three of six individuals who appeared to have been seroconverting at the time of starting PrEP, but there was no evidence of resistance to tenofovir. Rates of other sexually transmitted infections (STIs) were not significantly different between the arms, and rates of reported condom use did not change over the course of the study. Reported adherence to Truvada in this study was high, and this was confirmed by measuring drug levels in a 57-person sub-study.

McCormack pointed out that the HIV incidence in the deferred PrEP arm of PROUD was three times higher than anticipated based on analysis of the overall MSM population attending the clinics, suggesting that those at the very highest risk had chosen to volunteer for the study. “People who needed it really came forward,” McCormack said, and this emerged as a theme in PrEP presentations at CROI, raising the hope that if PrEP is made widely available, it will be used by populations that stand to benefit the most.

The resounding success of PROUD was duplicated—to the percentage point—in the results of the IPERGAY trial, which were presented by Jean-Michel Molina, chief of infectious diseases at the University of Paris Diderot. The purpose was to test whether “on demand” PrEP might be both efficacious and convenient in high-risk MSM from France and Canada. Participants were instructed to take two Truvada (or placebo) pills two to 24 hours prior to sex followed by two additional doses 24 and 48 hours afterward, in addition to receiving standard prevention counseling and condoms. The regimen was based on a study in macaques, which showed that intermittent Truvada could protect against an intra-rectal challenge with a hybrid simian immunodeficiency virus (SIV)/HIV (SHIV) challenge (Sci. Transl. Med. 2, 14, 2010).

As was the case for PROUD, the IPERGAY DSMB ended the randomized comparison in October 2014 due to the observed difference in HIV infections between the arms, and at that time Truvada was offered to all volunteers. The efficacy analysis was based on 176 participants from the Truvada-treated group and 177 placebo recipients.

Molina revealed that two HIV infections occurred in participants taking Truvada while 14 occurred among placebo recipients, a comparative incidence of 0.94 versus 6.6 per 100 person-years. The estimated efficacy was therefore 86% and the number needed to treat to prevent one HIV infection was calculated to be 18. Incidence was higher than anticipated in the placebo group, echoing PROUD’s suggestion that the MSM who enrolled were cognizant of their elevated risk of acquiring HIV. As with the primary results, the analyses of adherence, adverse events, STI rates, and sexual behavior over time all closely mirrored those of the PROUD study. The average number of Truvada pills consumed during the study was four per week, meaning monthly usage was close to half that of daily dosing. Molina concluded that “on demand” PrEP represents an attractive alternative to daily use for high-risk MSM.

Jared Baeten, professor of allergy and infectious diseases, epidemiology and global health at the University of Washington, addressed the use of both PrEP and antiretroviral therapy (ART) in a different population: heterosexual serodiscordant couples in Kenya and Uganda. Baeten’s goal was to investigate whether a combined strategy of PrEP use by the HIV-uninfected partner and ART initiation in the HIV-infected partner was feasible and reduced HIV transmission. In this setting, PrEP was administered on a time-limited basis, as a “bridge” until the HIV-infected partner had been on ART for six months (unless there were adherence issues or the individual’s viral load remained detectable despite treatment).

A total of 1,013 couples were enrolled, deemed to be at high risk of transmitting HIV based on a scoring system developed by Baeten and colleagues that takes into account risk factors such as younger age, lack of circumcision, and condomless sex in the prior month (J. Acquir. Immune Defic. Syndr. 62, 339, 2013). Of the HIV-uninfected partners, 33% were female and 67% male, and over 95% initiated PrEP with good adherence. ART was started by 80% of the HIV-infected partners, with more than 90% achieving viral load suppression. ART was prescribed in accordance with new guidelines recommending treatment for all HIV-infected individuals in serodiscordant relationships. Rather than employing a control group, which was considered unethical, Baeten constructed a counterfactual simulation model to predict likely HIV incidence in serodiscordant couples not using PrEP and in whom ART was prescribed based on previous guidelines (CD4+ T-cell count less than 350). The model was based on data from previous studies involving over 5,000 people.

In the current study, only two HIV infections were observed, an incidence of 0.2 per 100-person years. By contrast the model predicted 40 HIV infections, an incidence of 5.2 per 100-person years, therefore the estimated reduction compared to the model was 96%. The two individuals who seroconverted during the study reported breaks in PrEP use and had no Truvada detectable in plasma at the time their HIV infection was detected. In one case, the relationship with the HIV-infected partner had ended, suggesting a different transmission source, and in the other the partner had not started ART due to a relatively high CD4+ T-cell count. In response to a question from Glenda Gray, executive director of the Perinatal HIV Research Unit at the University of the Witwatersrand and current president of the South African Medical Research Council, Baeten stated that both the HIV infections were in women. Gray’s point was that there may still be some uncertainty about oral PrEP efficacy in women compared to men (one concern is possible differential tenofovir penetration into the vaginal versus rectal mucosa, see Sci. Transl. Med. 3, 112re4, 2011), but Baeten said that there appeared to be other explanations for the two seroconversions in this study.

Microbicide news disappoints

The encouraging news about oral PrEP that emerged at CROI was, unfortunately, accompanied by another blow to hopes that the antiretroviral tenofovir administered as a vaginal microbicide might be similarly efficacious. In 2010, the CAPRISA 004 trial reported a statistically significant 39% reduction in HIV incidence associated with use of 1% tenofovir gel by South African women at high risk of infection (Science 329, 1168, 2010). The microbicide was used pericoitally, meaning study participants were instructed to apply the gel within 12 hours before and 12 hours after sex. However, a subsequent trial of daily tenofovir gel administration, the VOICE study, was unable to demonstrate efficacy (N. Engl. J. Med. 372, 509, 2015). A confirmatory study known as FACTS 001, designed to evaluate the original CAPRISA 004 pericoital approach in a larger number of women in South Africa, also found no efficacy, as reported at CROI by Helen Rees, executive director of the Wits Reproductive Health and HIV Institute in Johannesburg.

The FACTS 001 trial was conducted at nine sites and enrolled 2,059 women aged 18-30 years. Because the results from VOICE indicated poor adherence contributed to the lack of efficacy, intensive adherence support was incorporated into the design. The efficacy analysis included 1,015 women in the tenofovir gel group and 1,014 who were randomized to receive a placebo gel. There was no significant difference in the number of HIV infections in the two groups, with 61 and 62 occurring in each group, respectively. Analyses of adherence bolstered previous findings that suggest the gel is not user-friendly—a paltry 13% of the participants applied it during more than 80% of sexual activity. There was some indication that a small proportion of women who had detectable tenofovir levels in cervical fluids and who reported having sex during the 10 days prior to sampling were protected. In this sub-group, HIV acquisition risk was reduced by 52%.

Rees stated that the results, while disappointing, represent important science that highlights the urgent need for prevention options that are easier to integrate into women’s lives. 

Striking an upbeat note for vaccines

In the self-described role of motivational speaker, Galit Alter, professor of medicine at the Ragon Institute of the Massachusetts Institute of Technology, Massachusetts General Hospital, and Harvard, delivered an upbeat overview of recent advances in HIV vaccine science at a workshop on the opening day of the conference. Alter delineated two approaches to successful vaccination: completely blocking HIV entry, or rapidly killing virus-infected cells at the site of exposure before systemic infection ensues. Citing recent data from Dan Barouch, associate professor of medicine and chief, Division of Vaccine Research in the Department of Medicine at Beth Israel Deaconess Medical Center and Harvard Medical School, Alter noted that the window of opportunity for extinguishing infection at the site of entry before a persistent HIV reservoir is established is very narrow, certainly less than three days in the SIV/macaque model (Nature 512, 74, 2014).

Hopes for blocking HIV entry with bNAbs have been bolstered by the discovery that a substantial proportion of HIV-infected individuals develop antibodies with neutralizing capacity over a period of two to three years after seroconversion. The appearance of bNAbs is associated with high initial viral load and greater viral diversity. Alter emphasized that this demonstrates the human immune system is capable of generating bNAbs, and that it therefore should be possible to recapitulate the process with immunization.

Progress is now being made toward designing immunogens that engage and activate specific naïve germline B cells that have the capacity to differentiate into bNAb-producing memory B cells. One strategy for coaxing the B cells along the correct differentiation pathway is to use sequential immunizations with different antigens designed to focus the resulting antibody response on conserved neutralization targets on the HIV envelope. To help achieve this goal, researchers are also investigating how to best exploit mechanisms that provide crucial help to developing B-cell responses, particularly follicular dendritic cells and T-follicular helper cells (Tfh).

Switching to approaches designed to rapidly eliminate virus-infected cells, Alter cited the example of Louis Picker’s replicating cytomegalovirus (CMV) vector-based SIV vaccine, which induces and maintains effector CD8+ T-cell responses at mucosal sites where infection occurs. By contrast, most vaccines induce central memory T cells. These require activation by viral antigens to generate a swarm of effector T cells and this process takes days, too long to prevent systemic HIV infection. A possible alternative method for sustaining effector T cells in mucosal tissues was recently described in a mouse model of herpes simplex infection. This strategy, dubbed “prime and pull,” employs vaccination to prime virus-specific T-cell responses, followed by local application of chemokines to pull the T cells to the mucosa where they establish a resident effector population (Nature 491, 463, 2012).

Beyond T cells, non-neutralizing antibody responses can also mediate killing of infected cells by recruiting innate effector cells such as natural killer (NK) cells and monocytes. Immune correlates studies have implicated an antibody-mediated cellular cytotoxicity (ADCC) mechanism in the protection observed in the RV144 trial, the first vaccine trial to show any efficacy in blocking HIV infection, spurring efforts to gain a deeper understanding of ADCC and develop means to fine-tune the response with vaccination. The relatively new sciences of systems biology and transcriptomics (the study of the RNA transcripts produced by the genome in response to a particular circumstance or stimulus) are also uncovering previously unknown immunological pathways that appear to contribute to the efficacy of licensed vaccines (Nat. Immunol. 15, 195, 2014), and these findings may also be applicable to HIV vaccine design.
 
Given all this, Alter concluded that the stage is set for significant progress toward a successful vaccine in the coming years, and several subsequent presentations at the conference expanded on areas touched upon in her talk.

Margaret Ackerman, assistant professor of engineering at Dartmouth College, took up the theme of enhancing protective antibody activities with an approach described as “systems serology.” Ackerman explained that antibodies possess domains that interact with Fc receptors on a variety of immune cell types including NK cells, monocytes, and dendritic cells. These interactions are critical for the induction of effector activities such as ADCC, and studies in both macaques and humanized mice have shown that even bNAbs depend on Fc receptor interactions to mediate optimal protection (Nature 449, 101, 2007; Cell 158, 1243, 2014).

The aim of systems serology is to capture data on all the key contributors to beneficial antibody activity to inform the design of vaccine strategies. One factor that contributes to better antibody activity is antibody subclasses. As an example of how vaccine regimens can modulate these responses, Ackerman compared the results of the RV144 trial—which tested the canarypox vector-based vaccine ALVAC in combination with an AIDSVAX B/E protein boost—and a previous trial known as VAX003 that tested AIDSVAX B/E alone. The RV144 vaccine regimen induced significantly greater amounts of immunoglobulin (IgG)3 antibodies, which are associated with potentiated effector function, while vaccination with only AIDSVAX in VAX003 skewed the response toward the IgG4 subclass of antibodies, which are linked to diminished Fc-mediated effector activity (Sci. Transl. Med. 6, 228ra38, 2014; Sci. Transl. Med. 6, 228ra39, 2014).

The glycosylation profiles of antibodies are also important. Ackerman found that in HIV-infected individuals, control of viral load in the absence of treatment is associated with the ability of virus-specific antibodies to recruit both NK cells and monocytes as effectors, which in turn is associated with more extensive antibody glycosylation (J. Clin. Invest. 23, 2183, 2013).

Ackerman’s lab has now developed a system that integrates over 1,000 salient measurements from a single sample to identify predictors of desired antibody activities. The approach has the potential to be combined with other techniques, including transcriptional profiling and assessments of cytokine responses, to better understand and modulate the response to vaccine candidates, with the ultimate goal of maximizing protective efficacy. Another possible application is to optimize the design of antibodies intended for delivery via passive immunization or gene transfer by improving their activity and bioavailability.

More shots, more protection?

Following the surprising results of RV144, researchers began preparations for a slew of studies designed to improve upon the modest 31% protection the vaccine regimen afforded. The RV305 trial, involving participants from RV144, was designed to investigate whether two additional booster shots with ALVAC, AIDSVAX B/E, or both (at baseline and 24 weeks later) would improve IgG3 antibody responses targeting the V1-V2 region of the HIV envelope and/or drive antibody gene maturation toward characteristics associated with bNAbs. Georgia Tomaras, associate director of research at the Duke University Human Vaccine Institute, presented results from this study at CROI.

An analysis of a blinded subset of participants in RV305 demonstrated significant enhancement of the IgG4 but not IgG3 antibody response compared to results obtained during RV144, in contrast to the desired outcome. There was evidence of a transient increase in ADCC activity and neutralization of tier-1 viruses (those that are considered the easiest to neutralize), but not broad neutralization, after the first but not second booster immunization. Detailed characterization of antibodies isolated from vaccinated volunteers showed an increase in somatic mutation frequency from a mean of 2.9% with the original regimen, to 6.7% after the additional boosters, and a greater prevalence of long HCDR3 sequences, which are a characteristic of bNAbs (2% versus 21% of antibodies with HCDR3 longer than 22 amino acids). Notably, there were a proportion of IgG3 antibodies identified among those with long HCDR3s. Ongoing studies are now exploring the specificities and functional attributes of the B-cell lineages responsible for producing these more matured antibodies. Tomaras, like Ackerman, highlighted the need to better understand the dynamics of antibody responses to vaccination and the implications for functionality.

Other researchers are also mining the original RV144 results for clues. In a late-breaker talk by Shelly Krebs, scientist and principal investigator at the US Military HIV Research Program (MHRP), data was presented on how vaccination influenced antibody evolution in RV144 participants from a different perspective. The question Krebs sought to answer was whether receipt of the vaccines affected the development of antibodies in those subjects who became HIV-infected during the trial despite vaccination. The hypothesis behind the study was that these individuals might generate neutralizing antibodies more rapidly than is normally seen in natural infection as a result of the priming of memory B-cell responses by immunization. But, as frequently happens in science, the results turned this hypothesis on its head.

There was no overall difference in the breadth or potency of antibody responses during post-infection follow-up but, strikingly, only placebo recipients developed antibodies with greater than 70% neutralization breadth. The single exception was an individual in the vaccine arm who only received a single injection of the ALVAC prime. There was some evidence for increasing neutralization capabilities over time, but it was significantly less in the vaccine recipients compared to placebo, or even compared to historical data from unvaccinated HIV-infected individuals. Krebs and colleagues are now attempting to explain this observation and are considering two possibilities: either that vaccine-induced antibodies restricted viral diversity and thereby limited neutralization breadth, or that those RV144 participants capable of making good antibody responses did not become infected, leaving only those with poor antibody responses in the group of vaccinees that acquired HIV. A new round of studies evaluating viral diversity and host genetics will be conducted to assess which of these two explanations appears more plausible, but for now, Krebs favors the latter.

A little help from your friends

The topic of Tfh help for B cells that was alluded to by Alter was discussed further by Hendrik Streeck, chief of the Cellular Immunology Section at MHRP. The vast majority of this CD4+ T-cell subset resides in lymph nodes, making routine analysis of Tfh responses challenging. Researchers have therefore been searching for markers that might reliably identify the small population of Tfh in the periphery. Streeck unveiled new evidence that production of the cytokine interleukin (IL)-21 is an ideal candidate for this marker.

In studies of HIV-infected individuals, HIV Gag-specific and Env-specific Tfh could be identified in blood based on IL-21 production, with both populations equally capable of providing help to CD8+ T-cell responses. However, there was a bifurcation when it came to B-cell responses—Gag-specific Tfh-supported B-cell proliferation and maturation, whereas Env-specific Tfh were better at driving antibody class switching (the shift from IgM to IgG antibody production). Interestingly, a preliminary analysis of samples from past vaccine trials revealed that the RV144 vaccine regimen, which provided slight protection, induced significantly higher levels of IL-21-producing Tfh in the periphery than HVTN 505, which evaluated the efficacy of a prime-boost regimen comprising a DNA prime and Ad5 vector boost encoding three different Env proteins from clades A, B, and C, along with clade B Gag, Pol, and Nef and failed to provide any protection against HIV infection (N. Engl. J. Med. 369, 2083, 2013). Streeck’s research suggests that more detailed investigations of Tfh will complement the work being conducted in the antibody arena.

Getting away without antibodies

Shortly before the start of CROI, a buzz-worthy paper in Nature captured headlines in publications from The New York Times to Science. The paper was on a potential alternative to traditional vaccination spearheaded by Michael Farzan, professor and vice chairman of the Department of Immunology and Microbial Science at The Scripps Research Institute in Florida (Nature 519, 87, 2015). Farzan’s approach involves a protein designed to bind to the sites on the HIV envelope that interact with the CD4 and CCR5 co-receptors, thereby blocking viral entry into target cells. Named eCD4-Ig, it has unprecedented breadth and potency, neutralizing 100% of a large panel of HIV isolates including tier-2 and 3 viruses, CXCR4-tropic viruses (those that gain entry using the alternative CXCR4 co-receptor), and those resistant to bNAbs targeting the CD4 binding site. Additionally, eCD4-Ig inhibits SIVmac239 and HIV-2 in vitro.

To deliver eCD4-Ig, Farzan adopted the adeno-associated virus (AAV) vector gene transfer approach developed by Phil Johnson, director of The Children’s Hospital of Philadelphia Research Institute. At CROI, Farzan noted that the potency of neutralization achieved with eCD4-Ig should translate into activity against HIV at the levels achievable in vivo with AAV. Furthermore, studies in macaques suggest eCD4-Ig is less immunogenic than bNAbs; “it looks more like self,” Farzan stated, meaning there is a reduced likelihood that it will provoke an anti- eCD4-Ig immune response that could block its antiviral effect. In an experiment conducted in macaques, an AAV encoding eCD4-Ig was mixed with a separate AAV encoding the gene for tyrosylprotein sulfotransferase 2 (TPST2). The TPST2 is needed to render the eCD4-Ig protein active via a process called sulfation. Four animals received the AAV mixture and four served as controls. Increasing doses of the challenge virus SHIV-AD8 were administered at weeks 8, 11, 16, 20, 26, and 34, with all controls eventually becoming infected. All eCD4-Ig immunized macaques remained uninfected and Farzan added that, since the publication of his paper, these animals have resisted two additional higher dose challenges of 1,600 and 3,200 picograms.

As was evidenced by quotes from other scientists in the media coverage of Farzan’s paper, the results have generated considerable excitement. Farzan believes that AAV-delivered HIV inhibitors, whether eCD4-Ig or bNAbs, are “closer to realization than any conventional vaccine approach.” Future plans include the development of an “off switch” that could curtail AAV production of encoded proteins in the event of safety issues, and therapeutic studies in SIV-infected macaques.

Antibodies and a cure

Interest in bNAbs and antibody effector functions is not only dominating the vaccine field these days. It has recently extended into cure research. A key rationale is that bNAbs may be able to promote destruction of HIV-infected cells, a task that cannot be accomplished by ART alone. Barouch reviewed the possible role of bNAbs in cure strategies. His laboratory bears credit for spurring interest in the topic with the publication of a paper in 2013 demonstrating that a cocktail of bNAbs could suppress viral replication and reduce proviral DNA in macaques infected with the hybrid SIV/HIV strain SHIV-SF162P3 (Nature 503, 224, 2013).

Barouch has since embarked on follow-up studies combining bNAbs and ART. He described results of an experiment intended to model chronic HIV infection in which a total of nine macaques were infected with SHIV-SF162P3 for seven months, then given either ART (comprising tenofovir, emtricitabine, and the integrase inhibitor dolutegravir) plus four monthly infusions of the bNAb PGT121, or ART alone. Five animals received the combination ART/antibody, while four received only ART. In both groups ART was maintained for 20 weeks. There was rapid and complete suppression of viral load in all macaques but significant declines in the viral reservoir in peripheral blood and gut—as measured by proviral DNA—were only seen in the bNAb recipients. The difference in proviral DNA levels between the groups was driven by three of the five macaques given bNAbs, with little change evident in the other two.

When ART was stopped, the two animals in the bNAb group showing no reduction in the reservoir experienced a typical viral load rebound, as did those on ART alone. But in the three cases where proviral DNA was diminished, there was an extended period of virological control, with one rebounding after nearly a year of sustained virus control. Barouch concluded that while bNAbs were not completely curative, they did appear to contribute to reservoir clearance. He suggested combinations of antibodies and agents capable of activating the latent reservoir may be needed to achieve greater effects, and larger macaque studies exploring this concept are now in progress.

Barouch also offered a glimpse at preliminary data from a macaque study modeling very early infection. Thirty-two animals were infected intra-rectally with SHIV-SF162P3 and divided into four groups of eight. The groups received no treatment, ART alone, ART plus the bNAb PGT121, or PGT121 alone. The interventions were initiated three days after challenge, with ART lasting for four weeks and PGT121 administered as a single infusion. The groups receiving ART or PGT121 briefly displayed detectable viral load prior to suppression, while recipients of the combination showed no viremia during treatment. After ART suspension, viral load rebounded in the majority of animals in all groups, but there was a significant delay in those given ART plus PGT121. Detailed virological and immunological analyses are ongoing.

Next steps include the development of bNAb cocktails, with Barouch noting that the combination of PGT121 with a recently discovered potent bNAb PGDM1400 (Proc. Natl. Acad. Sci. USA 111, 17624, 2014) was able to neutralize 98%-99% of a panel of circulating HIV isolates in vitro, suggesting a dual antibody approach could be highly effective. Human trials of PGT121 infusions are planned, first evaluating safety and then looking at its activity in different populations of HIV-infected individuals. The studies will include evaluations of antiviral activity in participants with detectable viral loads and effects on the reservoir in those on ART. Questioned about the mechanism by which bNAbs might impact the HIV reservoir, Barouch explained that some infected cells likely express the viral envelope on their surface, making them susceptible to ADCC or other antibody-mediated effector activities.

Passive immunization with bNAbs was also addressed by Diane Bolton, chief of Animal Models and Pathogenesis at MHRP, who outlined plans to conduct a clinical trial of the bNAb VRC01 in acutely HIV-infected individuals in Thailand. The trial, RV 398, is due to start later this year. As part of the planning process, Bolton performed a macaque study comparing ART (tenofovir, emtricitabine, and the integrase inhibitor raltegravir), ART plus VRCO1, and ART plus two other bNAbs selected due to their greater potency against the SHIV-SF162P3 challenge virus used in the experiment: VRC07-523 and PGT121. The design of Bolton’s monkey study differed from Barouch’s early infection experiment: animals were infected with SHIV-SF162P3 at baseline and on day 10, daily ART was begun in the first group, and a single infusion of the bNAbs was administered to the latter two groups. The bNAb recipients began receiving ART at day 21 following infection. This allowed Bolton to document that ART and the dual bNAb combination were equally potent at reducing acute viremia. VRC01 lowered viral load by about a log prior to ART, which Bolton pointed out was consistent with its lower in vitro potency against the challenge virus. All animals ultimately achieved undetectable viral loads on ART. Analysis of proviral DNA levels in lymph nodes on day 17 showed comparable declines across the groups, with one exception: those given dual bNAbs showed significantly less proviral DNA in naïve CD4+ T cells. There is a caveat, however. Bolton pointed out that the bNAbs used in the study were human so their Fc effector functions may not have been fully intact in macaques, which could lead to an underestimation of their impact. The RV 398 trial should reveal if superior activity is achievable in HIV-infected individuals.

James Whitney, assistant professor of medicine at Beth Israel Deaconess Medical Center and Harvard Medical School, delivered the cure-related presentation that garnered the most attention at the conference. Whitney debuted results from a macaque study designed to evaluate whether a toll-like receptor (TLR)7 agonist could reverse viral latency. TLRs are innate immune receptors involved in the recognition of pathogens and stimulation of several TLRs has been shown to activate latent HIV in vitro.

Ten macaques were infected intra-rectally with SIVmac251 and, 65 days later, started on a daily ART regimen of tenofovir, emtricitabine, and dolutegravir. At day 320 post-infection, four animals were administered escalating doses of the TLR7 agonist every other week (0.1mg/kg, 0.2mg/kg, and 0.3mg/kg) with the highest dose repeated four additional times. The other six animals received placebo. Receipt of the TLR7 agonist was associated with transient activation of both NK and CD8+ T cells, and there was some evidence of low-level CD4+ T-cell activation. Viral loads remained undetectable until after the second 0.3mg/kg dose, at which time two of the four macaques displayed transient viral load blips. The three subsequent TLR7 agonist doses prompted consistent blips in the two-log to three-log range in all four animals. Measurements of proviral DNA showed modest but significant reductions, including in the lymph nodes and colons in three out of the four macaques. No significant changes in any of the measurements occurred in the placebo group. When ART was interrupted, there was no difference in the kinetics of viral rebound between the groups, but set point viral loads were significantly lower in TLR7 agonist recipients.

Whitney explained that the TLR7 agonist used in the study is an analog GS-9620, a Gilead Sciences compound already in clinical development for hepatitis B and C infection. Early phase human trials have found GS-9620 to be safe and a study in people with HIV on ART has now been launched. GS-9620 also successfully activated latent HIV in CD4+ T cells isolated from HIV-infected individuals (abstract #417). Results from the clinical trial are eagerly anticipated.

The increasing enthusiasm for cure research remained evident at CROI, but John Mellors, director of the HIV/AIDS Program at the University of Pittsburgh, sought to quell any misconceptions about immediate prospects. “The reality is that progress will be slow, and will grind out over years to decades until we have a functional cure for a significant fraction of HIV-infected individuals—there are many pieces to the puzzle that need be put together to solve the problem.”

Richard Jefferys is Coordinator, Michael Palm Basic Science, Vaccines & Prevention Project at the Treatment Action Group.