Many Keys to Protection, But Many Locks Remain

October’s HIV Research for Prevention conference highlighted the abundance of pathways that investigators are pursuing in the quest for an effective vaccine.

By Michael Keller

In mid-October, researchers in South Africa were finalizing plans to launch HVTN 702, the first large-scale efficacy trial of an HIV vaccine candidate since the landmark RV144 trial in Thailand showed that two vaccine candidates in a prime-boost regimen provided 31 percent efficacy after three years. Investigators hope that the modified regimen now being tested in HVTN 702 will be able to protect at least 50 percent of the 5,400 South African participants from HIV over the same period.

As the last arrangements were being made for this trial, more than 1,400 leading scientists, advocates, and government officials gathered in Chicago for the biennial HIV Research for Prevention (HIVR4P) conference. While South Africa was half a world away, the trial’s start was one element in the universe of vaccine discovery that imparted excitement and optimism for the development of useful and long-acting HIV prevention strategies. Another was the multi-country Antibody Mediated Prevention (AMP) Study, a second major trial that is testing whether regular infusions of a potent broadly neutralizing antibody (bNAb)—one of many isolated that can neutralize a broad swath of HIV isolates in circulation— can provide protection against HIV. This trial is testing the strategy researchers refer to as passive administration: rather than relying on the immune system to make the antibodies, the antibody itself is directly injected into the susceptible individual. Presenters at HIVR4P also showcased efforts to design vaccine immunogens that could train the immune system to make its own HIV-destroying antibodies. Taken together, the conference’s speakers presented incremental work toward the goal of developing an effective vaccine and other prevention modalities.

“This is a good year,” said William Snow, the former director of the Global HIV Vaccine Enterprise who recently stepped down from this post (see sidebar, below). “Now, after long preparation, the field has embarked on a number of efficacy trials. They should, in a short, comfortable number of years, clarify what really works and why, which in turn will set the best direction for truly effective products, be they bNAbs, potent vectors, native trimers, or rational immunogen design. The questions have been posed and the answers are forthcoming.”

Enterprise Appoints Interim Director

Gerald Voss, former head of GlaxoSmithKline’s (GSK) HIV Vaccine program and vice president of the Enterprise’s Board of Directors, is now interim director of the Global HIV Vaccine Enterprise Secretariat, replacing longtime AIDS vaccine advocate William Snow, who has stepped down. Voss will oversee the Secretariat while the organization charts its future direction. After conducting a strategic review, the Enterprise will seek a permanent director. Snow will remain a senior advisor to the Secretariat and the Board of Directors. Snow said he decided to take a step back because he felt someone else was needed to take the Enterprise to a new level. “I’m thrilled to say that Gerald has plenty of experience to take the reins,” says Snow. “His base of experience is much broader than mine and his connections are long-standing.” Voss has spent much of his career working on AIDS vaccines, primarily at GSK, but his work also involved malaria and tuberculosis research. As interim director, Voss says he will be seeking ways for the Enterprise to further the work that Snow and his team began. This includes capitalizing on recent developments. “There has been a lot of scientific progress and so we want to make sure that the Enterprise evolves to meet these new and exciting developments,” says Voss. —Mary Rushton Mary Rushton is a freelance writer based in Cambridge, Massachusetts..

bNAbs in the spotlight

The Phase III AMP Study, which started earlier this year and is expected to run for about two years, is just one application for the bNAbs that have recently become a major focus of HIV treatment and prevention. In this trial, around 3,900 participants in the Americas and sub-Saharan Africa will receive infusions of the bNAb VRC01, a potent monoclonal antibody that targets the highly conserved CD4+ binding site where HIV docks to infect T cells. More than 700 volunteers had been enrolled in the AMP study as of mid-October. Larry Corey, president and director emeritus of the Fred Hutchinson Cancer Research Center and co-chair of the study, said the best-case outcome of the trial would be that a low dose will provide a sufficient concentration of antibody in vivo to protect against infection. That result would open the door to creating a marketable subcutaneous or intramuscular injection that could protect against HIV. If it turns out that protection is only imparted at higher in vivo concentrations, it bodes less well for developing this approach and also suggests it may complicate vaccine efforts, he warns, as this concentration may be challenging to achieve. In fact, with a growing playbook of bNAbs now in hand, Corey was asked whether there were any plans to test a cocktail of antibodies. He said the idea is to eventually use a combination of bNAbs, but AMP is specifically designed to employ only one as a test of concept and to tease out its immunological functioning.

In the meantime, several researchers are working on tweaking bNAbs to improve their chances at protecting. John Mascola, director of the Vaccine Research Center (VRC) at the National Institute of Allergy and Infectious Diseases (NIAID), spoke at HIVR4P about various modifications VRC researchers are making to antibodies to increase their potency and half life. “We have now discovered hundreds of antibodies that are extremely potent,” said Kevin Whaley, the CEO of Mapp Biopharmaceuticals, a company developing and marketing antibody-based therapies and vaccines. “VRC01 came out so far ahead of the other candidates, but we need to look at these others. I think we have a revolution going on in the world of antibody discovery.”

The recent boom in antibody discovery might make it seem like bNAbs are common, but they are in fact naturally produced by a small subset of all HIV-infected people, and even then, typically only after years of infection. Part of the reason that bNAbs are rare and take so long to develop in HIV-infected individuals is that they are extensively mutated. B cells are the components of the immune system that recognize pathogens and give rise to antibodies. B cells with defined specificities exist before the body is ever exposed to a particular pathogen. Once these cells come in contact with a specific pathogen such as HIV, a chain reaction process begins that results in the induction of so-called germline antibodies that target specific sites on HIV Envelope (Env) protein. These germline antibodies do not, however, typically bind to or neutralize HIV Env. It is only after the B cells undergo successive rounds of somatic hypermutation, which involves B cell stimulation and the process of affinity maturation, that these B cells are capable of producing the types of bNAbs vaccine scientists are interested in.

Such bNAbs can target and bind to specific sites on multiple regions of the virus and some have protected nonhuman primates (NHPs) against infection with a simian immunodeficiency virus (SIV)/HIV hybrid known as SHIV (PNAS 109, 18921, 2012; among others). Many researchers think that a vaccine that induces a long-lasting and potent bNAb response would protect against HIV. Therefore, understanding bNAb specificities and determining how best to elicit them via vaccination has become a major focus of HIV vaccine research in recent years.

Dennis Burton, scientific director of IAVI’s Neutralizing Antibody Center (NAC) and chairman of the immunology and microbial science department at The Scripps Research Institute (TSRI) in La Jolla, CA, spoke at HIVR4P about progress in developing neutralizing antibody-based vaccine candidates. He said bNAbs have been identified that individually target widely conserved areas on the virus’s trimeric Envelope (Env) protein, including the apex, high mannose patch, CD4+ binding site, gp120-gp41 interface, and membrane-proximal external region.

Researchers studying bNAbs are now using a reverse vaccinology approach to try to develop vaccine candidates that can induce bNAbs. They start by determining what the unmated or germline form of the antibody is and then design a series of vaccine immunogens to stimulate B cells to induce these germline antibodies and then shepherd B cells through the process of making more mature, mutated antibodies that will hopefully be able to broadly neutralize HIV. If that sounds complicated, that’s because it is. But researchers are making progress using this step-wise approach to inducing bNAbs in animal models.

“We’ve been seeing if we can steer germline antibody maturation,” said Burton. “This field has made incredible progress. We now have many different rationally designed immunogens and immunization strategies that are being evaluated. Some of these are going to make it into humans.”

Devin Sok, an IAVI research scientist, presented research at HIVR4P on one approach to design immunogens that elicit bNAbs. Since 2013, William Schief of TSRI and IAVI’s NAC, Burton, Sok, and others have been designing a self-assembling nanoparticle immunogen called eOD-GT8 60-mer. The plan, what the team refers to as a “multi-step reductionist vaccine strategy,” is to use an engineered germline-targeting immunogen like eOD-GT8 to prime VRC01-class precursor B cells, then use successive booster immunogens that are increasingly more native-like to induce further rounds of somatic hypermutation, eventually inducing bNAbs with the ability to recognize and neutralize native HIV Envs.

In previous studies, theOD-GT8 60-mer immunogen was shown to activate the presumed B-cell precursors of the VRC01 class of antibodies (Science 349, 156, 2015). Then in a paper published earlier this year, researchers showed that more native-like boosting immunogens designed to guide the genetic and functional maturation of VRC01-class precursors induced by eOD-GT8 were able to induce antibodies that were similar to a partially mature VRC01-class antibody in transgenic mice (Cell 166, 1, 2016). Although these antibodies could only weakly neutralize native HIV, the researchers concluded that this sequential immunization strategy was capable of guiding maturation of antibody responses and is therefore “a significant milestone in HIV vaccine development.”

At HIVR4P, Sok showed that they were also able to use eOD-GT8 to boost the number of VRC01-class precursors in transgenic mice that can produce human immunoglobulin (Ig; Science 353, 1557, 2016). These VRC01-class precursors naturally appear very rarely in the body, so getting B cells to produce more of them is a critical early step. In the study, the team immunized mice with a dose of eOD-GT8 or a control. Twenty-nine percent of those that received the immunogen started producing a VRC01-class precursor, with tell-tale characteristics of VRC01-type antibodies, compared to none of the control group. “It looks like we’re not only activating them [VRC01 precursors], but that we are selecting for productive mutations that would enable higher affinity,” Sok said.

Bart Haynes, director of the Duke University Human Vaccine Institute, is also testing the sequential immunization strategy. At HIVR4P, he noted recent data showing there is less overlap in sequences of Ig genes in NHPs and humans, suggesting that NHP data resulting from sequential immunization strategies may not translate directly into humans.

Trimer mimics

Another approach to potentially induce bNAbs is immunizing with proteins that mimic the natural trimeric structure of HIV gp140, so-called native-like trimers. For years researchers struggled to obtain a stable, trimeric form of this notoriously floppy protein, but now with the structure in hand, researchers are testing trimeric immunogens. Marit Van Gils, a postdoctoral researcher at the University of Amsterdam’s Academic Medical Center, presented work using one of these trimeric immunogens, BG505 SOSIP.664 (see Research Brief, IAVI Report, Vol. 19, No. 2, 2015). Recently, she and a multinational team found that rabbits vaccinated with the immunogen produced neutralizing antibodies that targeted a vulnerable hole in the glycan shield, the protective carbohydrate molecules that are densely packed around HIV Env and shield the virus from antibody-mediated neutralization. These antibodies were found to be able to neutralize moderately resistant autologous viruses, but did not possess broad activity because the location of the hole in the glycan shield was specific to the HIV isolate used to create the immunogen (Cell Reports 16, 2327, 2016).

During the conference, Van Gils presented data extending this line of inquiry, showing that two rhesus macaques immunized with BG505 SOSIP.664 produced narrowly directed neutralizing antibodies, confirming results seen in the earlier rabbit study. She said this is a first step in an iterative vaccine design process that includes vaccinating animals and humans with the immunogen, isolating the antibodies that develop from it, characterizing them, and then redesigning the immunogen to get closer to the broadly neutralizing response that a vaccine would ideally induce to protect against diverse strains of HIV. Understanding how to broaden the response to effectively target HIV isolates with differing glycan shields will be key to this process.

Exploiting holes in HIV’s glycan shield is an emerging theme, according to Penny Moore, senior scientist at South Africa’s National Institute of Communicable Disease’s Centre for HIV and Sexually Transmitted Infections. In one study, a team including John Moore and Rogier Sanders of the Weill Medical College of Cornell University engineered native-like recombinant SOSIP.664 trimers that were based on Env genes from clade A, B, and C HIV isolates (PLoS Pathog. 9, 1005864, 2016). The trimer immunogens were given either at the same time or in sequence and effectively induced neutralizing antibodies in rabbits, but with limited neutralization breadth. Researchers found that antibodies elicited from different trimers primarily recognized holes in the glycan shield, and that in some cases the location of the holes was shared between the different isolates. With this knowledge, the group hopes that multiple trimers could induce broader neutralizing antibody responses that can home in on shared weak points.

These are just a sample of the bNAb-eliciting vaccine development routes under active investigation. But even with these promising developments, much still remains to be learned about how the ever-mutating virus might evade even bNAb responses. Jinal Bhiman, a postdoctoral fellow in South Africa’s National Institute for Communicable Diseases, presented evidence that CAP256-VR26—a class of antibodies that bind to the V1 and V2 regions on Env first isolated by the Centre for the AIDS Programme of Research in South Africa—could still attach to Env even after the virus had mutated to be resistant to neutralization. Further, those antibodies with higher levels of somatic hypermutation actually bound better to Env, but did not effectively neutralize the virus. This means that HIV can escape the neutralizing activity of a bNAb, while still allowing the antibody to bind to the viral Env protein, which is a surprising result. “This goes against everything I’ve ever been taught about neutralization,” said James Bradac, the chief of preclinical research and development at the US National Institute of Health’s (NIH) Division of AIDS.

Vector-based approaches

While researchers plug away at vaccine strategies to induce bNAbs, there is also considerable progress in advancing viral vector-based vaccine strategies into human clinical trials. Georgia Tomaras, research director of the Duke University Human Vaccine Institute, presented results from HVTN 100, the Phase I/II trial that served as a preliminary safety and immunogenicity test of the HVTN 702 regimen. The prime-boost study tested the ALVAC canary-pox-based vectored vaccine used in the RV144 trial, which had been modified to be specific to southern Africa’s clade C HIV strains, along with a bivalent HIV subtype C gp120 protein boost. In still unpublished data she presented at the conference, her group found that the regimen used in HVTN 100 elicited a robust mix of immune responses, some of which were higher and some lower compared to those seen in RV144.

Results that appeared superior in HVTN 100 included titers of IgG and IgG3 antibodies capable of binding the Env protein included in the vaccine, and HIV-specific CD4+ T-cell polyfunctionality. The latter is significant because in a separate presentation at HIVR4P, Nicole Frahm from the Fred Hutchinson Cancer Research Center reported that CD4+ T-cell polyfunctionality correlated with protection in RV144. However, antibodies recognizing the V1/V2 region of Env had less breadth in HTVN 100 compared to RV144 when tested against multiple HIV clade C Envs. IgG3 antibody responses against V1/V2, which were reported to be potentially important in RV144 (Sci. Transl. Med. 6, 228ra39, 2014), were also lower in HVTN 100. In terms of antibody functionality, antibody-dependent cellular phagocytosis was generally similar between the two trials, and assays for antibody-dependent cellular cytotoxicity (ADCC; the process by which antibodies facilitate destruction of virus-infected cells) produced evidence of better recognition of HIV-infected cells among HTVN 100 participants. Most importantly, however, all of the pre-defined immunogenicity criteria for launching the HVTN 702 efficacy trial were met.

Several other vector-based vaccine regimens are also being advanced post-RV144. In one Phase I trial, designated HVTN 094, researchers are evaluating a DNA prime and a modified vaccinia Ankara (MVA) boost made by biotech company GeoVax that produce virus-like particles. This approach has been shown to reduce the risk of SIV and SHIV infection in macaque studies.

In 48 HIV-uninfected volunteers, the 094 research team found that a regimen consisting of two or three doses of MVA was well tolerated and produced cellular and humoral immune responses. Susan Buchbinder, a University of California, San Francisco epidemiologist who led the investigation, presented unpublished data at the conference showing that the regimen induced ADCC. This regimen’s immunogenicity will be tested in the HVTN 114 trial involving 100 participants starting in December. “What’s unique about these vaccines is that they produce these virus-like particles that we believe presents the envelope in a more conformationally intact structure and, therefore, may generate more functional antibodies than what we might see with some other vaccine regimens,” said Buchbinder.

The virus’s rapid mutation and the resulting variability of circulating virus remains a challenge for vaccine development. One strategy scientists are using to counter this involves employing mosaic immunogens, which are a bioinformatically derived set of HIV gene sequences that encode whole HIV proteins that are designed to match those made by the majority of circulating HIV strains worldwide. Mosaic immunogens are meant to trigger a broad cellular immune response, and previous studies indicate that the mosaic approach can partially protect rhesus monkeys from SHIV through the action of both neutralizing and non-neutralizing antibodies (Cell 155, 531, 2013).

Lindsey Baden, director of clinical research at Brigham and Women’s Hospital’s Division of Infectious Diseases, discussed unpublished data from a Phase I trial called IPCAVD-006, which was the first in-human clinical trial of the mosaic approach. Twenty-five participants were split into two groups: those who had never been given an HIV vaccine candidate and those who had received an adenovirus serotype 26 (Ad26) vectored vaccine candidate up to five years before that was meant to elicit HIV Env-targeted antibodies. Volunteers in both groups received an MVA vector containing the mosaic insert by intramuscular injection. The vaccine candidate was found to be safe and well tolerated, and it elicited Env-directed B- and T-cell responses. This response was still seen up to a year after the vaccine was given, though response to Gag and Pol remained limited throughout the study period. Those who had previously received the Ad26 candidate had higher levels of immune response than those who had not, suggesting the Ad26 candidate had effectively primed the immune response.

Tomaras and colleagues also tested another prime-boost vaccine regimen in a study involving 184 South African participants (PLoS ONE, doi:10.1371/journal. pone.0161753). This regimen, an MVA vector-based prime containing genes encoding Gag, Reverse Transcriptase, Tat, Nef, and clade C Env HIV proteins followed by a Novartis-engineered subtype C envelope gp140 boost with an MF59 adjuvant, proved to induce the strongest peak neutralizing and binding antibody responses out of four vaccine regimens tested. Inclusion of the MVA prime contributed to both humoral and cellular immune responses, while a DNA-based candidate alone did not.

A path to remission?

In an unexpected twist, the study that garnered the most attention at HIVR4P was not about prevention at all. Rather it was about a treatment approach that led to sustained remission in NHPs. Just prior to the start of the conference, a study by Emory University and NIAID scientists revealed that ART along with administration of a mouse monoclonal antibody engineered for NHPs called Act1, which blocks the cellular receptor called α4β7 integrin on CD4+ T cells, had diminished SIV levels in infected monkeys to undetectable for as long as 23 months, even after they stopped all treatment (Science 354, 197, 2016). Their experiment started 18 infected macaques on 90 days of ART five weeks after infection. Then, nine weeks after infection, researchers initiated infusions of the recombinant monoclonal antibody on 11 animals every three weeks until week 32. The animals that received Act1 maintained “robust” virologic control for the rest of the study period, while viremia in the control group animals rebounded within two weeks. In addition, the experimental group’s diminished CD4+ T-cell count replenished to normal levels after ART and antibody treatment, a phenomenon not seen in the ART-alone group.

“Infusions of anti-alpha 4 beta 7 in nonhuman primates together with a course of ART post-infection with SIVmac239 induced long-term virological suppression following discontinuation of all therapy,” Anthony Fauci, a study author and head of NIAID, told an audience of hundreds during the conference’s first plenary. “CD4-positive T cells were restored in gut, blood, and certain peripheral lymphoid tissue.”

The α4β7 integrin is known to be a homing receptor that lymphocytes use to migrate to the gut. For years, Fauci’s lab has been looking into whether HIV gp120 binds to α4β7 on CD4+ T cells. In previous studies, they found that CD4+ T cells expressing high amounts of α4β7 were preferential targets for the invading virus (PNAS 106, 20877, 2009). In 2014, Fauci’s group also found that targeting α4β7 integrin prevented or delayed SIV transmission when given before repeat low-dose vaginal challenges (Nat. Med. 20, 1397, 2014).

How the antibody helped the study animals clear the virus for nearly two years remains unclear, but since August Fauci said they have been running an experiment to see if a similar effect will be seen in humans. They are performing a small open-label study of 15 to 25 volunteers using vedolizumab, a humanized anti-α4β7 monoclonal antibody already approved by the US Food and Drug Administration to treat Crohn’s disease and ulcerative colitis. “These studies may provide insight into potential mechanisms that could be pursued in the prevention of HIV infection and/or the maintenance of sustained virological remission following interruption of ART,” said Fauci. “All of this in my mind as a human immunologist and HIV clinician will only be important if it works in humans.”

David Margolis, director of the University of North Carolina School of Medicine’s HIV Cure Center, who was not involved in the research, said he was as surprised as other experts in the field at the study’s findings. “The work is impressive, and unexpected,” Margolis said. “This might lead to a way to prevent infection, or allow durable control of infection without lifelong antiviral drug therapy.”

Treatment is potent prevention

Another thread discussed at HIVR4P was the use of treatment as prevention. Myron Cohen, the University of North Carolina School of Medicine’s Associate Vice Chancellor for Global Health, offered more data from the HIV Prevention Trials Network (HPTN) 052 study at HIVR4P (NEJM 375, 830, 2016).

Cohen and his team analyzed data from almost 1,800 serodiscordant couples (where one partner is HIV-infected and the other isn’t) in nine countries and found a 93 percent HIV transmission reduction between partners when ART was started early compared to the late-starting ART group. This protective effect was stable over five years. “What’s really important is that we saw no transmission, zero transmission, when HIV replication was suppressed,” he said. “The only linked transmissions we saw were when the drug failed for whatever reason, and that was a rare event.”

The prospective, observational PARTNER (Partners of People on ART—A New Evaluation of the Risks) study found similarly good news (JAMA 316, 171, 2016). In this one, researchers followed 1,166 serodiscordant couples in 14 European countries and found no phylogenetically linked transmissions.

Cohen views the results of these treatment as prevention studies as just one of many rays of hope in defeating HIV. “Prevention research is really rapidly on the move. That’s the best way to look at it,” he said. “The ongoing studies in treatment as prevention are going to inform how to maximize this approach, and that’s exciting. Long-acting antiviral agents are almost certain to serve as critical new tools for treatment and prevention of HIV. The bNAbs are going to provide novel treatment and, in addition, they’re going to inform vaccine development in a way that’s not heretofore been possible. And, ultimately, all these new tools are going to lead to the kind of combination prevention that’s required to get to an AIDS-free generation.”

Michael Keller reports from the frontiers of science, technology, and international affairs. His writing has appeared online and in newspapers, magazines, and books, including the graphic novel Charles Darwin’s On the Origin of Species.