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INTRODUCTIONS

2 Making good drugs even better Jackie Oberst, Ph.D. Science/AAAS

3 ViiV Healthcare: At the forefront of HIV science John C. Pottage, Jr., M.D. ChiefScientificandMedicalOfficer, ViiV Healthcare

ABOUT ViiV HEALTHCARE

4 ViiV Healthcare: Disrupting and innovating to leave no HIV patient behind

RESEARCH ARTICLES

10 Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques Ling Xu, Amarendra Pegu, Ercole Rao et al.

15 High coverage of ART associated with decline in risk of HIV acquisition in rural KwaZulu-Natal, South Africa FrankTanser,TillBärnighausen,ErofiliGrapsaet al.

21 Modeling the dynamic relationship between HIV and the risk of drug- resistant tuberculosis Rinat Sergeev, Caroline Colijn, Megan Murray et al.

About the cover: The object in green is a representation of the HIV-1 virus. On the surface of the viral cell membrane are several glycoproteins (gp); gp41 in red, and gp120 trimers in yellow. The gp120 trimer is crucial for the HIV virus to bind to the human CD4t-cell receptor. Conceptualization and design by Max Lataillade and Mark Krystal (ViiV Healthcare), and John Wong (Global Nucleus).

Original content (pages 4–9) has notbeen peer-reviewed or assessedby Science. These articles can be cited using the following format: [AUTHOR NAME(S)] in ViiV Healthcare: Innovators and disruptors in HIV research and treatment (Science/AAAS, Washington, DC, 2018), p. [xx-xx].

This booklet was produced by the Science/AAAS Custom Publishing Office and sponsored by ViiV Healthcare.

Editor: Jackie Oberst, Ph.D.Writer: Alaina G. LevineProofreader/Copyeditor: Bob FrenchDesigner: Amy Hardcastle

ROGERGONCALVES, ASSOCIATE SALES DIRECTORCustom Publishing Europe, Middle East, and [email protected]+41-43-243-1358

© 2018 by The American Association for the Advancement of Science. All rights reserved. 13 July 2018

TABLE OF CONTENTS

ViiV Healthcare Innovators and disruptors in HIV research and treatment

1SCIENCE sciencemag.org

http://sciencemag.org

sciencemag.org SCIENCE

ViiV HEALTHCARE: INNOVATORS AND DISRUPTORS IN HIV RESEARCH AND TREATMENT

2

Making good drugs even betterWe invite the reader to further explore various milestones in HIV/AIDS treatment and to get a glimpse of what the future may hold for it.

T reatment of HIV/AIDS has come a long way. Thirty years ago, the virus was viewed as an automatic death sentence—people often died within a few months of being diagnosed. Now, HIV/AIDS has moved worldwide from an acutely fatal disease to one more chronic in nature. Patients are now referred to as “living with HIV” rather than simply having the disease. Early diagnosis and treatment with daily antiretrovirals have enabled patients to live nearly as long as someone who does not have HIV.

While great progress has been made in treating HIV, there is still much work to do. More than 1 million Americans are currently infected with HIV, and each year 50,000 more become infected, according to statistics from the U.S. Centers for Disease Control and Prevention (CDC). Awareness remains an issue: About one in seven Americans who have HIV don’t know they’re infected, so they may be unwittingly spreading the virus to others. The problem is even worse in developing nations, especially in parts of Africa, the CDC reports.

Furthermore, patients face a lifetime sentence of taking daily medications that carry side effects of their own, ranging from mild to life-threatening. For this reason, as well as limited access or fear of stigma, medication adherence is still an obstacle. Fewer than one in three Americans with HIV take antiretroviral medicines regularly enough to reduce the virus to undetectable levels, according to the U.S. National Institutes of Health.

Given that HIV treatment only works if it is continuous, studies are being performed to create and evaluate medications that might be taken less often, such as once a month, or in smaller quantities, such as two antiretrovirals daily instead of the usual three-drug cocktail. Some researchers are looking for a “sterilizing cure” in which all HIV virus is removed from the body, even from hidden reservoirs. Only one case exists of this type of cure—Timothy Brown, or the “Berlin Patient”—who received a bone-marrow transplant from a donor with a natural genetic resistance to HIV. This procedure is risky and is not feasible as a cure, but it does provide some insights to scientists who are pursuing other treatment methods, such as vaccines and gene editing.

Many companies—including ViiV Healthcare, the sponsor of this booklet—have enabled patients to live fuller, healthier lives, largely because of the medicines and discoveries made with their support. We invite the reader to further explore various milestones in HIV/AIDS treatment and to get a glimpse of what the future may hold for it.

Jackie Oberst, Ph.D.Science/AAAS Custom Publishing Office


SCIENCE sciencemag.org

INTRODUCTIONS

It has been over 35 years since the world became aware of the HIV/AIDS epidemic. In the 1980s, when I was starting out as an infectious diseases physician, researchers and health care professionals were trying to identify and understand a new virus that was infecting and killing large numbers of people, predominantly members of the gay community. This illness later became known as human immunodeficiency virus, or HIV.

Since the approval of azidothymidine (AZT), the first antiretroviral (ART), in 1987, there have been major advancements in HIV medicine, and what was once a death sentence can now be managed as a chronic disease. In the early days of the virus, fewer than 50% of people living with HIV (PLHIV) were expected to survive the first year after diagnosis, whereas today PLHIV can expect to live a regular lifespan.

Thanks to these advancements, the scientific community has been able to focus on improving treatment regimens and care for PLHIV, helping to reduce pill size and the number of pills required to a single, once-daily, fixed-dose tablet combining multiple drugs that suppress the virus.

Nevertheless, while we have seen remarkable success in HIV treatment, several challenges remain. Even with effective treatment, PLHIV who are now living longer may have to deal with side effects and health conditions caused by long-term exposure to their HIV medicines. Furthermore, many of these people will have to manage other conditions commonly associated with aging (e.g., diabetes, cardiovascular disease, or kidney disease) in addition to coping with the effects of their medication. This often means even more medication, which can lead to further complications and may significantly impact their quality of life.

At ViiV Healthcare, the only pharma company dedicated completely to HIV, we are focused on finding innovative medicines to improve outcomes for HIV patients and on understanding how best to prevent and treat the disease. Our scientists are committed to finding new ways to limit the impact of HIV on the nearly 37 million people living with the virus. Many of our scientists, including me, were there at the beginning; and we have dedicated our careers to improving the lives of PLHIV.

Although HIV therapy has made significant strides in recent years, until we have a cure we must continue to invest in the development of new medicines that can help all PLHIV, so that no one is ignored. We must also remain vigilant on issues such as viral resistance to treatment and the long-term effects of medication. Using a patient-centered approach to innovation, we are dedicated to advancing treatment options that could lessen the lifetime burden of HIV therapy on PLHIV.

Because we now have potent drugs, we are shifting our focus to tolerability and convenience. Believing that no patient should take more medicines than they need, ViiV Healthcare is exploring ways to reduce the number of HIV drugs that must be taken. If PLHIV can receive fewer medicines, we may be able to reduce drug interactions and improve tolerability—and if reducing medications can improve adherence, we can possibly reduce the chance of drug resistance and transmission.

As well as the five investigational medicines we currently have in the clinic, our investment in R&D supports new therapeutic options, such as antiretroviral drug candidates with novel mechanisms of action and the treatment of HIV-related immune dysfunction. We are actively collaborating with other commercial and academic organizations to work toward a cure, and we are also seeking new business alliances to guarantee that we will continue to deliver groundbreaking HIV treatments.

We are at the forefront of treatment development and research, and we are working with our international partners on multiple fronts to address the major barriers affecting global uptake and access of HIV medicines.

Despite the great progress that has already been made, there is a long way to go before an end to the HIV epidemic is in sight. At ViiV Healthcare, we are confident that we have both the scientific expertise and the innovative research pipeline necessary to make this ambition a reality—all consistent with our mission of ensuring that no person living with HIV gets left behind.

John C. Pottage, Jr., M.D.Chief Scientific and Medical Officer, ViiV Healthcare

ViiV Healthcare: At the forefront of HIV scienceWe are focused on finding innovative medicines to improve outcomes for HIV patients and on understanding how best to prevent and treat the disease.

3


I t was just over 30 years ago when the first antiretroviral for human immunodeficiency virus (HIV), developed by then-Burroughs Wellcome, was introduced. Azidothymidine, better known as AZT, was hailed as a game-changer in the fight against HIV, with the goal of extending the life

of patients. Over the last two decades, discovery and innovations have led to many more new mechanisms for treatment, and HIV patients are living longer lives. Indeed, the more than 40 therapies that have been designed in recent years, which include formulating multiple medicines into one pill, provide people with something they were not receiving at the dawn of the HIV crisis in the 1980s: an opportunity to control the virus like any other manageable, chronic condition.

However, people living with HIV (PLHIV) are now facing a new and evolving set of challenges, mostly associated with the cumulative effects of aging combined with being on antiretroviral therapy (ART) treatment for life. These can include issues such as long-term exposure to medicine, drug resistance, dosing schedules, drug interactions, and convenience.

To meet these challenges, one enterprise has emerged as a leader and strategic disruptor in the holistic treatment of PLHIV. Enter ViiV Healthcare: the world’s only pharmaceutical company focused 100% on HIV, with the most innovative HIV R&D program in the industry. Established in 2009 through a unique partnership between GlaxoSmithKline (GSK) and Pfizer, ViiV Healthcare now combines the HIV expertise of GSK, Pfizer, and Shionogi Limited, which became a shareholder in 2012 following a long-term collaboration.

One-of-a-kind pharmaceutical companyViiV Healthcare is unique in many respects. First, it is

solely focused on HIV. Most of its medical leadership and senior scientific staff have dedicated their entire professional lives to fighting and treating HIV. They have been in the trenches with clinicians and patients and have been listening and responding to their concerns

for decades. Mark Shaefer, a global medical lead, has been at ViiV since its founding in 2009, and says the company takes a purposeful, all-inclusive view in treating and working with patients. “For ViiV, it’s about more than making medicines—it’s also about affecting positive change in social systems as they relate to HIV and ensuring that all patients, wherever and whoever they are, can benefit from treatments and take back their lives,” he says. There are

currently 36.9 million PLHIV worldwide. ViiV Healthcare’s mission is to make sure that none of them get left behind.

ViiV Healthcare’s singular focus on HIV means that all its scientists are working to treat, prevent, or cure the same disease, and the company makes it a priority to have constant, open communication between scientific and clinical teams that traditionally do not collaborate so closely, says Max Lataillade, vice president, clinical development. “It is unusual for discovery and early development teams to work in such close proximity


ViiV HealthcareDisrupting and innovating to leave no HIV patient behind

Mark Shaefer, global medical lead

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and together in a pharma company,” he notes, and yet at ViiV Healthcare, this is the case. “What we have in our Branford, Connecticut site is a building where discovery and early development scientists and physicians work side by side, and very closely together. This interplay is unique. We are able to talk each other’s language and develop innovative drugs in a faster, more efficient manner.”

ViiV Healthcare’s team comprises over 1,000 staff members working in 15 countries. The company extends this geographical reach still further via its relationship with GSK, to make



its medicines available in more than 65 countries worldwide. It is equipped to move quickly in response to the needs of the HIV community, and has launched industry-leading access initiatives to help deliver on World Health Organization (WHO)/Joint United Nations Program on HIV and AIDS (UNAIDS) goals to reach all those needing treatment.

The bottom line? “Ultimately, we will get to a cure and then the company will no longer be needed,” says John C. Pottage, Jr., chief scientific and medical officer, who has been at ViiV since its inception. “The idea is not to expand and work in other therapeutic areas. We remain committed to HIV. There is always a worry that the market is satisfied or that we have done enough. We don’t believe we’ve reached that point, and we still perceive an ongoing medical need. And as long as we see that medical need, we will stay here. We are here for the long run. But we would love to be in a situation where we are not needed.”

Max Lataillade, vice president, clinical development

Chronology of HIV R&D

2014 1959 19831981 2012 2018 20132009 2016 1984

2018: ViiV Healthcare reports positive results for landmark phase III studies for a two- drug regimen of dolutegravir and lamivudine in treatment- naïve patients

2018 and beyond: ViiV Healthcare continues to be patient-focused and a disruptor in the HIV therapeutic space, as it explores new treatment options for HIV patients, including the world’s first long-acting injectables

2012: Shionogi Limited joins the company

2013: ViiV Healthcare ranks first ahead of all other pharmaceutical companies in Patient View’s “Corporate Reputation of Pharma” survey—it has claimed the top spot every year since.

2013: FDA approves Tivicay (dolutegravir)

2014: FDA approves Triumeq, ViiV Healthcare’s first dolutegravir-based fixed-dose combination

2016: ViiV Healthcare is recognized for its efforts to increase access to HIV/AIDS treatment for children by the Global Paediatric Antiretroviral Commitment-to-Action

2016: ViiV Healthcare launches phase III programme to evaluate a long-acting, injectable HIV treatment regimen

2017: Two-drug combination Juluca (dolutegravir/rilpivirine) is approved by the FDA for treatment of HIV-1 in virologically suppressed adult patients

1959: First documented case of HIV

1981: First established case of AIDS

Early 1980s: HIV and AIDS become epidemics

1983: Burroughs Wellcome begins research on HIV treatment, led by Dr. Marty St. Clair

1984: Screening of potential drug candidates to fight HIV begins in June

1984: Azidothymidine (AZT), an antiretroviral medication, is identified as a possible therapy

1984: St. Clair looks at her first assay with AZT and sees that every concentration entirely prevented virus replication

1987: AZT is approved by the U.S. Food and Drug Administration (FDA)

2009: ViiV Healthcare, the only pharmaceutical company focused 100% on HIV, is formed via a joint venture between GlaxoSmithKline and Pfizer

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HIV treatment has changed: View an animation charting the last 30 years of progress in just 30 seconds.

http://sciencemag.orghttps://www.youtube.com/watch?v=y_HlK_FLJAo

Ever adapting to the clinical environment for HIV treatments

One critical aspect of treating HIV that has changed over the decades and that significantly impacts how HIV research is conducted and translated into medicines relates to clinical insight. “For the last 15–20 years, the standard of care has revolved around a combination of three ARTs,” Pottage says. But many patients get HIV early—some in their teens and twenties—which requires them to be on drugs for the rest of their lives. “Now that we have potent drugs, the focus is shifting to tolerability and convenience. ViiV Healthcare is exploring whether

we can reduce the number of drugs taken. If PLHIV can receive fewer medicines, we may be able to reduce drug interactions and improve tolerability. If this can improve adherence, we could reduce the chance of drug resistance and transmission,” he says.

ViiV Healthcare has established and cemented its reputation for taking on challenges related to treatment and prevention. And the company has been able to do this because it is seen as a trusted partner and valued contributor in the global HIV communities it serves, and as an organization that isn’t satisfied with the status quo.

Within the HIV community, ViiV Healthcare continues to break ground by collaborating with diverse HIV communities through its Positive Action program. Deborah Waterhouse,

chief executive officer of ViiV Healthcare, who has been working in the pharmaceutical industry for more than 20 years, most recently with GSK before joining ViiV in 2017, says Positive Action gives the company the opportunity to seek out and build new connections and collaborations. “These relationships help to strengthen education, support services, and local health care capacity and capabilities, and to reduce stigma and discrimination directed towards PLHIV all over the world, focusing on key affected populations most at risk,” says Waterhouse. “Globally, ViiV supports more than 300 programs addressing the needs of PLHIV, including education as well as care and treatment projects. Nearly 200 of these programs are in Latin America, Africa, Asia, the Middle East, and Russia, where the need is greatest.”

ViiV Healthcare has a selective collaboration with the PENTA-ID Network and PENTA Foundation, a European pediatric research organization that supports “conducting world-class clinical studies, training programs, and infrastructure development initiatives in pediatric infectious disease, all with the aim of improving the lives of women and children through generation and application of scientific knowledge,” according to its website. “Kids, immunologically, are very different than adults,” says Jim Demarest, who has been with ViiV Healthcare from the beginning, and currently serves as global head of clinical virology and immunology. “We must understand the dynamic of the virus in children to help find ways to evaluate treatment strategies and remission—which means the patient can control the disease on their own, without antiretrovirals. Ultimately we want to get to a cure.”

Shifting the drug regimen paradigmViiV Healthcare’s current portfolio consists of 13

products that generated annual global sales of £4.35 billion (USD 5.75 billion) in 2017. This financial stability ensures the firm can take a sustainable, long-term view when investing in its pipeline of new medicines.

Integrase inhibitors, such as dolutegravir (DTG), are at the core of its current strategy. DTG was approved by the U.S Food and Drug Administration (FDA) in August 2013, and a fixed-dose combination of DTG, abacavir, and lamivudine was approved in August 2014. “The breadth and depth of data for DTG is unmatched, and we are not finished,” says Martin Gartland, vice president and medicine development leader for DTG. Having examined the potency and tolerability of DTG as part of three-drug regimens across a range of patients in the SINGLE, SPRING-2, FLAMINGO, ARIA, SAILING, INSPIRING, DAWNING, and VIKING-3 studies, ViiV Healthcare has recently turned its attention to two-drug regimens (2DRs). “We believe that because of the potency of DTG, only two drugs are necessary to control the virus, and that fewer drugs may be better for patients in the long-term,” Gartland says. One of these 2DRs is called Juluca—a combination of DTG and rilpivirine—and has been approved to treat adults with HIV-1 infections (whose virus is currently suppressed) on a stable regimen for at least six months, with no history of treatment failure and no known substitutions associated with resistance to the individual components. It has been recognized as a game-changer; the FDA said its approval represented a paradigm shift in the treatment of HIV-infected patients. ViiV is preparing to announce data from the GEMINI studies, two studies evaluating a different two-drug regimen (DTG and lamivudine) for the treatment of HIV-1 infection in a different group of patients (treatment-naïve patients), in the summer of 2018.

“Our mission is to develop the next generation of medicines for HIV. We are focused on innovative



6

“We must understand the dynamic of the virus in children to help find ways to evaluate treatment strategies and remission.”— Jim Demarest,global head of clinical virology and immunology


approaches that deliver meaningful advances,” says Pottage. “We are committed to leaving no patient behind.” This sweeping perspective is demonstrated by the scope of clinical trials that ViiV Healthcare conducts and supports, and by the diversity of typically underrepresented populations featured in the trials. “When we brought forward DTG, we did five studies as opposed to two. And we also compared the drug against the gold standard and looked at a wide variety of patients, including those first coming into the therapy, those who are running out of options, and everyone in between. We looked at female patients, those with coexisting tuberculosis, and those in developing countries,” says Pottage.

“We are bold—we are not afraid to push the envelope and try something different,” says Kimberly Smith, ViiV’s head of global research and medical strategy. “We recognize that DTG gives us the opportunity to explore alternatives to three-drug regimens. We have a philosophy that no one should take more drugs than they need, and we have a robust clinical development program to generate data with these two-drug regimens. Can two drugs work as well as three? Can we reduce long-term toxicities by removing one drug? We are seeking to answer these questions and more.”

Babafemi O. Taiwo, chief of Infectious Diseases in the Department of Medicine and the Gene Stollerman Professor of Medicine at Northwestern University Feinberg School of Medicine, has been working as an HIV researcher for 20 years and has collaborated with ViiV Healthcare since its inception, serving as principal investigator and investigator on several of its trials and studies. He notes the history of prescribing three drugs for HIV, and why it presented the perfect opportunity for ViiV Healthcare to engage in strategic

disruption on behalf of the community. “People said that the 3DR was not broken, so don’t fix it—but we realized that for patients on 3DR, they can be on drugs for 50 years or more, so the question was, ‘How can we improve their lives?’” he explains. “To use another construction—2DR—is pretty bold, and testifies to the confidence the company had in the quality of its product to try to recall this domain. It is a particularly risky move for a company to undertake, but if successful, could provide the most profound change in how we approach HIV therapeutics. This is the major legacy of ViiV.”

Smith, like many of her teammates, has taken care of HIV patients for years and spent 20 years as a physician and the principal investigator of the Rush University Medical Center Clinical Research Site of the AIDS Clinical Trials

Group (ACTG) in Chicago, Illinois. These types of experiences give ViiV Healthcare’s staff unparalleled insight surrounding patients’ medical needs. “We have a sense of the burden of HIV treatment, knowing you’re going to take these medications every day for the rest of your life,” she explains. “Add to that the fact that some of the medicines are tough to tolerate, especially over long periods of time, and can cause adverse effects. This is a ‘ball and chain’ [effect] of the meds impacting your life that we are driven to change.”

There are skeptics. “We are not the first to try a two-drug regimen,” notes Smith. “Others have tried and weren’t successful. The key is that it’s not any two drugs in combination—it must be the right two drugs for the right patient. The way you change a treatment paradigm is by delivering new data, building on the foundation of studies demonstrating the effectiveness of dolutegravir as part of a three-drug regimen and showing that the two-drug regimens containing dolutegravir can be just

SCIENCE sciencemag.org 7

The first reports of HIV began to surface two weeks before John Pottage started his fellowship at Rush Medical Center in Chicago in 1981. He describes it as a time of great fear of the unknown, when he quickly began to compare his job as an infectious diseases physician to that of a

detective trying to solve a mystery. “Because the disease wasn’t well understood, there were no real diagnostic tests to perform, and that meant we had to treat patients by getting close to them and understanding their issues and their needs.”

Fast forward to 2018, and Pottage is still a believer in that type of approach to patients, which he is using to help find new medicines—and ultimately a cure—for PLHIV. He is the first and only chief scientific and medical officer at ViiV, and has been a guiding force behind ViiV’s current portfolio of 13 medicines. He says he still uses the image of courage and resilience he saw in the people he treated at his Chicago clinic as inspiration, and he is extremely proud of what ViiV has been able to accomplish on their behalf. “It all circles back to understanding the clinical care and needs of the patient,” he says.

John Pottage: There from the beginning

John Pottage, chief scientific and medical officer



as effective. As the data now emerges, we are confident that physicians and patients will appreciate a valuable new option.”

Patient-centered careEvery patient with HIV is one patient

too many, notes the ViiV Healthcare team. And for that reason, the team takes a holistic approach to serving all patients, whoever they are, wherever they are, and in whatever phase of the disease they are. Whereas other pharmaceutical companies might focus only on developing medicines (and consequently conducting clinical trials) for young, white males, ViiV Healthcare sees an expanded need and has incorporated underserved populations in their trials, ensuring that its clinical trials go further. It has also created outreaches and other programs to better understand and deliver innovative solutions to patients from all walks of life and geographies. Whether it is a pediatric program, or one that serves women, transgender people, or families in Africa where HIV continues to be so heartbreaking and pronounced, ViiV Healthcare is making inroads.

“When you are a smaller company like ViiV, you are inherently more nimble and able to change,” says Mark Cockett, head of discovery at ViiV. “For bigger companies with rigid structures and processes, it is harder to rapidly adapt and find new ways of doing things with speed and innovation. Our goal is always to speed up patient care, and we try to focus on this.”

And because many of ViiV Healthcare’s researchers have extensive experience and interactions with clinicians and patients, the company is able to design medicines and support social programs that deliver meaningful results. “I’m very lucky that I work with investigators and patients,” says Marty St. Clair, senior director of clinical virology and one of the inventors of AZT. “Every patient is very important to me. I still meet patients today who tell me they were on AZT and that it kept them alive. I get to see the impact on patients, and that is everything. If you can’t see your drug’s impact, you are missing the real value of their discovery and development.”



8

For nearly 40 years, Marty St. Clair has been laser-focused on investigating HIV. Her name is legend within the global HIV eradication community for two simple reasons: She is an inventor of AZT, and she has never stopped working to uncover even better treatments for HIV. As a virologist who

attended the doctoral program at Duke University, where she studied retroviruses, she was a member of the R&D staff at Burroughs Wellcome when HIV and AIDS were declared epidemics in the early 1980s. “The scientific community didn’t believe that it was possible to discover an agent that would have activity against HIV,” she recalls, but she and her company were up for the task. In 1983, “I raised my hand to be part of the team to discover and deliver the first drug to treat HIV.”

The Burroughs Wellcome scientists urgently began a search for a potential new medicine against HIV. Thousands of potential candidates were run through screening tests, including marketed antivirals and late-stage investigational medicines. Screening began in June 1984. In November 1984, a candidate had been identified. When St. Clair ran the test to check its efficacy, the results were so spectacular that she thought she had made an error. “Every concentration of the assay prohibited HIV replication,” she says. “I showed my plates to my manager and said, ‘This is interesting. Do you think I forgot to put the virus in these 16 plates?’” But of course, it wasn’t a mistake—and she replicated the test to be sure. The compound was effective in stopping HIV progression.

“And then the world exploded,” she recalls. “We immediately set forth to develop this drug and collect data as quickly as possible to get FDA approval.” The medicine, which became known as AZT, was approved by the FDA on March 19, 1987. “Everybody worked hard to get this through. The need was unbelievable.”

St. Clair notices the difference in how HIV is treated now compared to the 1980s when it first emerged. “In the beginning, it was absolute desperation. People were dying at an alarming rate. We had to get the drug out there. It was a life-saving event,” she says. Now, she says, “we have moved from that to using combinations of drugs that have some toxicity issues. And now we use potent drugs that are giving patients their lives back. It’s an amazing difference.”

Today, as senior director of clinical virology at ViiV Healthcare, St. Clair is still actively involved in the fight against HIV and, more importantly, in serving patients around the world. With a goal of not only stopping the disease’s progression but giving every patient a superior quality of life, this pioneer combines bench research with constant interactions with patients and clinicians, a hallmark of both her firm and her own personal mission.

“People shake my hand and tell me they have lives and loves and careers because of the work we did in drug discovery. It couldn’t be more personal.”

Marty St. Clair: AZT discoverer and HIV pioneer



Invested for the long termCurrently, ViiV is developing another exciting

alternative: an injectable medication that would need to be taken just once per month. So instead of patients having to remember to take their pill(s) every day and being constantly reminded that they have HIV, they could receive a shot from a doctor instead. This innovative idea for HIV treatment comes directly from patient feedback.

In addition, ViiV has many other options in the pipeline. Its 2016 acquisition of the Bristol-Myers Squibb HIV R&D portfolio and discovery program shows a commitment to discovery and early clinical development. This acquisition has bolstered the firm’s pipeline with several complementary investigational assets: fostemsavir, an HIV attachment inhibitor that is a breakthrough therapy candidate for heavily treatment-experienced patients and is currently being studied

in phase III clinical trials; three maturation inhibitors, which would provide another way to block viral replication; combinectin, a novel biologic treatment with three distinct, nonoverlapping mechanisms of action currently in phase I development, which could offer patients an alternative to daily oral regimens; and novel mechanisms such as allosteric integrase inhibitors and capsid inhibitors to complement DTG-based regimens.

And what about the future? “We are currently developing novel drugs to address future medical needs 10 to 15 years from now,” notes Lataillade. “We are working hard to ultimately get ourselves out of business by finding a cure for patients. The nature of the HIV virus is that it always escapes the immune system. We must look five moves ahead to keep it in check, but also need to keep in mind the patient’s perspective. At ViiV Healthcare, our culture and strategies always revolve around the patient.”

Kimberly Smith was still a medical student when she began treating HIV patients at the University of Michigan Hospital. “I was drawn to this disease that was impacting young people,” she says. “In the ‘90s—the bad old days—we had very few medicines and they were very hard on the system. I was taking care of people who were very young, and the idea that they were not going to have a long life to share was devastating.” As both a researcher and a clinician who had worked with John Pottage prior to joining ViiV, Smith was on the frontlines of the fight against HIV, and she was beyond eager to provide medications and treatment that were lasting. In this intimate environment, working side-by-side with other doctors and counseling individual patients, Smith found her calling.

One of the first patients Smith treated was a young woman with HIV and AIDS who presented with pneumonia. She told Smith that she had just adopted a little boy who was only three and was heartbroken

that she wouldn’t live to see him grow up. “I told her that new medicines are coming, and that we are going to make sure you see him graduate from high school,” describes Smith. “That came true. We got her on better medicines. She got to see him off to college.” This encounter has stuck with Smith for over 20 years. “There is nothing like real patients’ experiences to give you the passion and the practical knowledge of how to pick the right medicine and treatment for an individual. It is critical to being a good and innovative drug maker.” Smith would interact with many other patients over the years who would shape her perspective as a clinician and a scientist. But that young woman never strays far from Smith’s mind, and they are still in touch. “For me it was tough to leave patient care, but I have taken that [experience] beyond looking at the person in front of me to sharing it with the world, and it is an honor for me to do that.”

9

Kimberly Smith: On the frontlines with HIV patients

Kimberly Smith, head of global research and medical strategy



10 sciencemag.org SCIENCE


Originally published 6 October 2017 in SCIENCE

HIV THERAPY

Trispecific broadly neutralizing HIVantibodies mediate potent SHIVprotection in macaquesLing Xu,1* Amarendra Pegu,2* Ercole Rao,1 Nicole Doria-Rose,2 Jochen Beninga,1

Krisha McKee,2 Dana M. Lord,1 Ronnie R. Wei,1 Gejing Deng,1 Mark Louder,2

Stephen D. Schmidt,2 Zachary Mankoff,2 LanWu,1 Mangaiarkarasi Asokan,2

Christian Beil,1 Christian Lange,1 Wulf Dirk Leuschner,1 Jochen Kruip,1 Rebecca Sendak,1

Young Do Kwon,2 Tongqing Zhou,2 Xuejun Chen,2 Robert T. Bailer,2 KeyunWang,2

Misook Choe,2 Lawrence J. Tartaglia,3,4 Dan H. Barouch,3,4 Sijy O’Dell,2 John-Paul Todd,2

Dennis R. Burton,4,5 Mario Roederer,2 Mark Connors,6 Richard A. Koup,2 Peter D. Kwong,2

Zhi-yong Yang,1 John R. Mascola,2† Gary J. Nabel1†

The development of an effective AIDS vaccine has been challenging because of viralgenetic diversity and the difficulty of generating broadly neutralizing antibodies (bnAbs).We engineered trispecific antibodies (Abs) that allow a single molecule to interact withthree independent HIV-1 envelope determinants: the CD4 binding site, the membrane-proximal external region (MPER), and the V1V2 glycan site. Trispecific Abs exhibited higherpotency and breadth than any previously described single bnAb, showed pharmacokineticssimilar to those of human bnAbs, and conferred complete immunity against a mixture ofsimian-human immunodeficiency viruses (SHIVs) in nonhuman primates, in contrast tosingle bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutictargets through a single protein, and they may be applicable for treatment of diversediseases, including infections, cancer, and autoimmunity.

Avariety of broadly neutralizing antibodies(bnAbs) have been isolated from HIV-1–infected individuals (1–3), but their poten-tial to treat or prevent infection in humansmay be limited by the potency or breadth

of viruses neutralized (4, 5). The targets of theseantibodies have been defined according to anunderstanding of the HIV-1 envelope structure(6–9). Although bnAbs occur in selected HIV-1–infected individuals (usually after several yearsof infection), it remains a challenge to elicit themby vaccination because broad and potent HIV-1 neutralization often requires unusual antibodycharacteristics, such as long hypervariable loops,interaction with glycans, and a substantial levelof somaticmutation. Strategies have thus shiftedfrom active to passive immunization, both to pro-tect against infection and to target latent virus(10–14).We and others have begun to explore com-

binations of bnAbs that optimize potency and

breadth of protection, thus reducing the likeli-hood of resistance and viral escape (15–17). Anti-bodies directed to the CD4 binding site (CD4bs),membrane-proximal external region (MPER),and variable-region glycans are among the com-binations that so far provide optimal neutraliza-tion (18). In addition, alternative combinationshave also been investigated for the immunotherapyof AIDS, specifically by directing T lymphocytesto activate latent viral gene expression and en-hance lysis of virally infected cells (19, 20). Be-causemultiple antibodiesmay help to reduce theviral replication that sustains chronic HIV-1 in-fection, we report here the generation of multi-specific antibodies designed to increase thepotential efficacy of HIV-1 antibodies for preven-tion or therapy.

Design of bispecific antibodies andevaluation of neutralization breadth

Although individual anti–HIV-1 bnAbs can neu-tralize naturally occurring viral isolates with highpotency, the percentage of strains inhibited bythese monoclonal Abs (mAbs) varies (21, 22). Inaddition, resistant viruses can be found in thesame patients from whom bnAbs were isolated,which suggests that immune pressure against asingle epitope may not optimally treat HIV-1infection or protect against it. We hypothesizedthat the breadth and potency of HIV-1 neutral-ization by a single antibody could be increasedby combining the specificities against differentepitopes into a single molecule. This strategywould be expected not only to improve efficacybut also to simplify treatment regimens, as well

as the regulatory issues required for clinicaldevelopment.To test this concept, we initially incorporated

prototype bnAbs to the CD4bs and MPER sitesinto a modified bispecific Ab. When two var-iable regions are linked in tandem, the distalsite typically retains its ability to bind antigenwhile the proximal binding is markedly dimin-ished. We therefore used an alternative configu-ration, termed CODV-Ig, that introduced linkersand inverted the order of the antibody bind-ing site in light and heavy chains to alter theorientation of the variable regions, allowing eachregion to interact with its target (23). Severalknown bnAbs, including VRC01, 10E8, PGT121,and PGT128 [reviewed in (1)], were evaluated fortheir ability to neutralize a select panel of viruseswith known resistance or sensitivity to these anti-bodies (fig. S1). Initially, we determined whetherthe position of the variable regions from VRC01and 10E8 in the proximal or distal positions (Fig.1A) could affect neutralization breadth and po-tency. Inclusion of both variable regions in eitherorientation in the bispecific antibody reducedthe number of resistant strains relative to theparental antibodies alone (Fig. 1B). Better po-tency was observed when VRC01 was proximaland 10E8 distal, although neither bispecific anti-body was as potent as a mixture of the two anti-bodies alone.To explorewhether other bnAbs could perform

better in the bispecific format, we evaluated twodifferent combinations: VRC01 plus PGT121, orVRC01 plus PGT128. For PGT121, expressionwasobserved only with VRC01 in the distal position.When this antibodywas compared to the paren-tal antibodies alone, it providedmarginally betterneutralization (Fig. 2A). In contrast, VRC01 couldbe expressedwith PGT128 in both positions, withgreater breadth observedwhen VRC01 was distal(Fig. 2B). Together, these data indicate that im-provements in breadth could be achieved with abispecific format; however, in this case, the po-tency was not consistently improved relative toeach Ab alone. We therefore sought an alterna-tive format to improve the potency and breadthof neutralization.

Generation and comparison of broadand potent trispecific antibodies

To achieve our goal, we used a previously un-described trispecific Ab format. Three speci-ficities were combined by using knob-in-holeheterodimerization (24) to pair a single arm de-rived from a normal immunoglobulin (IgG) witha double arm generated in the CODV-Ig. A panelof bnAbs was evaluated, including those directedagainst the CD4bs that included VRC01 and N6,as well as PGT121, PGDM1400, and 10E8 (fig. S1).A modified version of the latter, termed 10E8v4,was used because of its greater solubility (25).We first determined which bispecific arms showedthe best potency, breadth, and yield. This screen-ing analysis revealed that combinations contain-ing PGDM1400, CD4bs, and 10E8v4 showed thehighest level of production and greatest potencyof neutralization (fig. S2).

RESEARCH

Xu et al., Science 358, 85–90 (2017) 6 October 2017 1 of 5

1Sanofi, 640 Memorial Drive, Cambridge, MA 02139, USA.2Vaccine Research Center, National Institute of Allergy andInfectious Diseases, Bethesda, MD 20892, USA. 3Center forVirology and Vaccine Research, Beth Israel DeaconessMedical Center, Harvard Medical School, Boston, MA 02215,USA. 4Ragon Institute of MGH, MIT, and Harvard, Cambridge,MA 02139, USA. 5Department of Immunology andMicrobiology, International AIDS Vaccine Initiative (IAVI)Neutralizing Antibody Center, Center for HIV/AIDS VaccineImmunology and Immunogen Discovery, The ScrippsResearch Institute, La Jolla, CA 92037, USA. 6NationalInstitute of Allergy and Infectious Diseases, Bethesda, MD20892, USA.*These authors contributed equally to this work.†Corresponding author. Email: [email protected] (G.J.N.);[email protected] (J.R.M.)

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Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques

HIV THERAPY

Trispecific broadly neutralizing HIVantibodies mediate potent SHIVprotection in macaquesLing Xu,1* Amarendra Pegu,2* Ercole Rao,1 Nicole Doria-Rose,2 Jochen Beninga,1

Krisha McKee,2 Dana M. Lord,1 Ronnie R. Wei,1 Gejing Deng,1 Mark Louder,2

Stephen D. Schmidt,2 Zachary Mankoff,2 LanWu,1 Mangaiarkarasi Asokan,2

Christian Beil,1 Christian Lange,1 Wulf Dirk Leuschner,1 Jochen Kruip,1 Rebecca Sendak,1

Young Do Kwon,2 Tongqing Zhou,2 Xuejun Chen,2 Robert T. Bailer,2 KeyunWang,2

Misook Choe,2 Lawrence J. Tartaglia,3,4 Dan H. Barouch,3,4 Sijy O’Dell,2 John-Paul Todd,2

Dennis R. Burton,4,5 Mario Roederer,2 Mark Connors,6 Richard A. Koup,2 Peter D. Kwong,2

Zhi-yong Yang,1 John R. Mascola,2† Gary J. Nabel1†

The development of an effective AIDS vaccine has been challenging because of viralgenetic diversity and the difficulty of generating broadly neutralizing antibodies (bnAbs).We engineered trispecific antibodies (Abs) that allow a single molecule to interact withthree independent HIV-1 envelope determinants: the CD4 binding site, the membrane-proximal external region (MPER), and the V1V2 glycan site. Trispecific Abs exhibited higherpotency and breadth than any previously described single bnAb, showed pharmacokineticssimilar to those of human bnAbs, and conferred complete immunity against a mixture ofsimian-human immunodeficiency viruses (SHIVs) in nonhuman primates, in contrast tosingle bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutictargets through a single protein, and they may be applicable for treatment of diversediseases, including infections, cancer, and autoimmunity.

Avariety of broadly neutralizing antibodies(bnAbs) have been isolated from HIV-1–infected individuals (1–3), but their poten-tial to treat or prevent infection in humansmay be limited by the potency or breadth

of viruses neutralized (4, 5). The targets of theseantibodies have been defined according to anunderstanding of the HIV-1 envelope structure(6–9). Although bnAbs occur in selected HIV-1–infected individuals (usually after several yearsof infection), it remains a challenge to elicit themby vaccination because broad and potent HIV-1 neutralization often requires unusual antibodycharacteristics, such as long hypervariable loops,interaction with glycans, and a substantial levelof somaticmutation. Strategies have thus shiftedfrom active to passive immunization, both to pro-tect against infection and to target latent virus(10–14).We and others have begun to explore com-

binations of bnAbs that optimize potency and

breadth of protection, thus reducing the likeli-hood of resistance and viral escape (15–17). Anti-bodies directed to the CD4 binding site (CD4bs),membrane-proximal external region (MPER),and variable-region glycans are among the com-binations that so far provide optimal neutraliza-tion (18). In addition, alternative combinationshave also been investigated for the immunotherapyof AIDS, specifically by directing T lymphocytesto activate latent viral gene expression and en-hance lysis of virally infected cells (19, 20). Be-causemultiple antibodiesmay help to reduce theviral replication that sustains chronic HIV-1 in-fection, we report here the generation of multi-specific antibodies designed to increase thepotential efficacy of HIV-1 antibodies for preven-tion or therapy.

Design of bispecific antibodies andevaluation of neutralization breadth

Although individual anti–HIV-1 bnAbs can neu-tralize naturally occurring viral isolates with highpotency, the percentage of strains inhibited bythese monoclonal Abs (mAbs) varies (21, 22). Inaddition, resistant viruses can be found in thesame patients from whom bnAbs were isolated,which suggests that immune pressure against asingle epitope may not optimally treat HIV-1infection or protect against it. We hypothesizedthat the breadth and potency of HIV-1 neutral-ization by a single antibody could be increasedby combining the specificities against differentepitopes into a single molecule. This strategywould be expected not only to improve efficacybut also to simplify treatment regimens, as well

as the regulatory issues required for clinicaldevelopment.To test this concept, we initially incorporated

prototype bnAbs to the CD4bs and MPER sitesinto a modified bispecific Ab. When two var-iable regions are linked in tandem, the distalsite typically retains its ability to bind antigenwhile the proximal binding is markedly dimin-ished. We therefore used an alternative configu-ration, termed CODV-Ig, that introduced linkersand inverted the order of the antibody bind-ing site in light and heavy chains to alter theorientation of the variable regions, allowing eachregion to interact with its target (23). Severalknown bnAbs, including VRC01, 10E8, PGT121,and PGT128 [reviewed in (1)], were evaluated fortheir ability to neutralize a select panel of viruseswith known resistance or sensitivity to these anti-bodies (fig. S1). Initially, we determined whetherthe position of the variable regions from VRC01and 10E8 in the proximal or distal positions (Fig.1A) could affect neutralization breadth and po-tency. Inclusion of both variable regions in eitherorientation in the bispecific antibody reducedthe number of resistant strains relative to theparental antibodies alone (Fig. 1B). Better po-tency was observed when VRC01 was proximaland 10E8 distal, although neither bispecific anti-body was as potent as a mixture of the two anti-bodies alone.To explorewhether other bnAbs could perform

better in the bispecific format, we evaluated twodifferent combinations: VRC01 plus PGT121, orVRC01 plus PGT128. For PGT121, expressionwasobserved only with VRC01 in the distal position.When this antibodywas compared to the paren-tal antibodies alone, it providedmarginally betterneutralization (Fig. 2A). In contrast, VRC01 couldbe expressedwith PGT128 in both positions, withgreater breadth observedwhen VRC01 was distal(Fig. 2B). Together, these data indicate that im-provements in breadth could be achieved with abispecific format; however, in this case, the po-tency was not consistently improved relative toeach Ab alone. We therefore sought an alterna-tive format to improve the potency and breadthof neutralization.

Generation and comparison of broadand potent trispecific antibodies

To achieve our goal, we used a previously un-described trispecific Ab format. Three speci-ficities were combined by using knob-in-holeheterodimerization (24) to pair a single arm de-rived from a normal immunoglobulin (IgG) witha double arm generated in the CODV-Ig. A panelof bnAbs was evaluated, including those directedagainst the CD4bs that included VRC01 and N6,as well as PGT121, PGDM1400, and 10E8 (fig. S1).A modified version of the latter, termed 10E8v4,was used because of its greater solubility (25).We first determined which bispecific arms showedthe best potency, breadth, and yield. This screen-ing analysis revealed that combinations contain-ing PGDM1400, CD4bs, and 10E8v4 showed thehighest level of production and greatest potencyof neutralization (fig. S2).

RESEARCH


1Sanofi, 640 Memorial Drive, Cambridge, MA 02139, USA.2Vaccine Research Center, National Institute of Allergy andInfectious Diseases, Bethesda, MD 20892, USA. 3Center forVirology and Vaccine Research, Beth Israel DeaconessMedical Center, Harvard Medical School, Boston, MA 02215,USA. 4Ragon Institute of MGH, MIT, and Harvard, Cambridge,MA 02139, USA. 5Department of Immunology andMicrobiology, International AIDS Vaccine Initiative (IAVI)Neutralizing Antibody Center, Center for HIV/AIDS VaccineImmunology and Immunogen Discovery, The ScrippsResearch Institute, La Jolla, CA 92037, USA. 6NationalInstitute of Allergy and Infectious Diseases, Bethesda, MD20892, USA.*These authors contributed equally to this work.†Corresponding author. Email: [email protected] (G.J.N.);[email protected] (J.R.M.)

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RESEARCH ARTICLES

We then evaluated different combinations ofsingle-arm and double-arm specificities fromPGDM1400, CD4bs, and 10E8v4 Abs for theirexpression levels and activity against a smallpanel of viruses (fig. S3), leading ultimately tothe identification of trispecific antibodies VRC01/PGDM1400-10E8v4 and N6/PGDM1400-10E8v4as lead candidates. When analyzed against apanel of 208 viruses (18) and compared to theparental antibodies alone, the highest neutrali-zation potency and breadth were observed withN6/PGDM1400-10E8v4, with only 1 of the 208viruses showing neutralization resistance and amedian inhibitory concentration (IC50) of less than0.02 mg/ml (Fig. 3A). VRC01/PGDM1400-10E8v4also displayed high potency and breadth, andonly four resistant viruses were found. Someparental mAbs displayed either high breadth(e.g., 10E8, N6) or high potency (PGDM1400),but none displayed a combination of breadthand potency as optimal as the trispecific Abs(Fig. 3B). For example, the most potent and broadparental mAb, N6, was around 20% as potent asthe N6/PGDM1400-10E8v4 trispecific Ab andtargeted only a single epitope, which could in-crease the chance of viral escape mutations. As asingle recombinant protein, the trispecific Abs dem-onstrated potency and breadth superior to anysingle antibody yet defined (Fig. 3 and fig. S4).We also determined the binding affinity of

each component of the trispecific Ab and com-pared each to its parental Fab. The equilibriumbinding constant Kd of each binding site in thetrispecific Ab, determined by surface plasmonresonance, was comparable to the affinity of theparental Fab, with PGDM1400 showing a slightdecrease in affinity (factor of ~3) and VRC01 and10E8v4 exhibiting approximately a logarithmicincrease (fig. S5). In addition, the trispecific Abwas able to bind sequentially to each of the threeantigens (Fig. 3C), indicating that there is inde-pendent binding of each epitope.The N6 trispecific Ab also showed greater

potency and breadth relative to three relatedbispecific Abs when tested against a panel of20 viruses that were selected for resistance tobnAbs (table S1). This finding is consistent withprevious studies comparing the efficacy of mix-tures of two versus three bnAbs (18) and pro-vides additional support for the multitargetingconcept. In addition to their greater efficacy,the trispecific Abs also yielded higher proteinlevels and greater solubility than the bispecificmodel (compare fig. S2A and fig. S3), whichwould facilitate large-scale production and clin-ical translation.

Fc modification to extend half-life andcrystal structure

To identify the optimal candidate for furtherdevelopment, we determined the half-life of thetrispecific Abs in nonhuman primates (NHPs).We previously showed that in the context of theVRC01 mAb, mutations that increased bindingto the neonatal Fc receptor (FcRn), which recyclesIgG in intestinal epithelial cells and increaseslevels in the serum, extended half-life, enhanced


Fig. 1. CODV-Ig bispecific antibody design and neutralization titers of the VRC01/10E8bispecific antibodies. (A) CODV-Ig bispecific antibody design with two different orientations of 10E8and VRC01. (B) Neutralization titers (IC50), in mg/ml, of VRC01/10E8 bispecific Abs and parental Absagainst a select panel of 19 circulating HIV-1 strains; values highlighted in red, orange, and yellowindicate highest, medium, and lowest potency, respectively.

A B

Fig. 2. Neutralization titers of VRC01/PGT121- and VRC01/PGT128-based bispecific antibodies.(A and B) Neutralization titers (IC50), in mg/ml, of the VRC01/PGT121 (A) and VRC01/PGT128 (B)bispecific Abs against a select panel of 20 circulating HIV-1 strains, with colors as in Fig. 1.

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mucosal localization, and conferred more efficientprotection against lentivirus infection relative tothe wild-type antibody (26). One such mutationwas incorporated into the trispecific Abs as wellas the parental VRC01 and N6 Abs. Abs werethen infused into rhesus macaques, and serumlevels were analyzed over a 14-day time frame.Ab VRC01 displayed a longer half-life than the

more broad and potent N6, which was also di-rected to the CD4bs (Fig. 4, VRC01 versus N6).Similarly, the trispecific Ab containing VRC01showed greater persistence and a longer half-life (7.43 days, based on day 1–day 14 serum con-centrations) than the N6 trispecific (4.79 days) invivo (Fig. 4, VRC01/PGDM1400-10E8v4 and N6/PGDM1400-10E8v4). For this reason, and because

the N6 trispecific Ab yielded less product withdecreased solubility, we studied the VRC01/PGDM1400-10E8v4 trispecific Ab further.Further characterization was performed by

solving the crystal structure of the bispecificarm of the trispecific Ab, PGDM1400-10E8v4CODV Fab, at 3.55 Å resolution (Fig. 5, A and B).The light chain was well resolved in the electrondensity (with the exception of the two mostC-terminal residues), whereas the heavy chainshowed some regions of dynamic disorder.The most notable region consisted of part ofPGDM1400 complementary-determining regionheavy chain 3 (CDRH3) and the linker betweenPGDM1400 Fv and the heavy chain constantdomain (residues 280 to 305). Similar to theanti–interleukin-4 (IL-4)/IL-13 CODV Fab crystalstructures (23), the PGDM1400 and 10E8v4 Fvsopposed one another, with the CDRs well ex-posed to the solvent. The distance between theCDRH3s of PGDM1400 and 10E8v4 exceeded100 Å. The PGDM1400 and 10E8v4 Fvs super-posed very well with their respective parentalFv structures [RMSD (Ca) ≈ 1 Å] (fig. S6) (25, 27),confirming that their antigen-binding propertieshave been well preserved in the CODV format.Most important, the orientations of the CDRsin two Fvs were 180° from each other, whichsuggests that each antibody-combining site canindependently engage its antigen without ob-structing the other Fv structure. A model for thetrispecific Ab was constructed by combining thePGDM1400-10E8v4 CODV Fab with VRC01 (6)and the intact b12 (28) IgG crystal structures(Fig. 5C). Similar to a natural IgG, the distancebetween the monovalent fragment of antigenbinding (Fab) and CODV Fab was about 150 Å.Two of three antigens (gp120 core and gp41MPER) were also included in the model, althoughwe do not have direct evidence that all three HIVepitopes can be engaged simultaneously by asingle trispecific Ab.

Enhanced cross-protection anddecreased viral escape in vivo

To evaluate the VRC01/PGDM1400-10E8v4 tri-specific Ab for its ability to protect against in-fection, we used a mixture of two SHIVs thateach differed in neutralization sensitivity tothe parental bnAbs. In vitro assessment of thereplication-competent SHIV challenge stocksshowed that SHIV BaLP4 was sensitive to VRC01and the trispecific antibody but was resistantto PGDM1400 (Fig. 6A). In contrast, SHIV 325Cvirus was sensitive to PGDM1400 and the tri-specific Ab but was resistant to VRC01 (Fig. 6A).In a neutralization assay with an equal mixtureof SHIV BaLP4 and SHIV 325c, we observedthat the trispecific Ab could achieve completeneutralization of the viral mixture, whereasVRC01 or PGDM1400 could not (fig. S7). Whennaïve rhesus macaques were infused with thehalf-life–extended VRC01, PGDM1400, or VRC01/PGDM1400-10E8v4 (5mg/kg), respectively, serumconcentrations were maintained at levels of≥1 mg/ml formore than 14days for all Abs (Fig. 6B).A decrease in serum levels at later time points for


Fig. 3. Neutralization titers of trispecific antibodies and broadly neutralizing antibodies, andsequential binding of alternative Env epitopes. (A) Neutralization titers (IC50) of different bnAbsand trispecific Abs against a genetically diversepanel of 208 circulatingHIV-1 strains.The solid line denotesthe median IC50 neutralization titer of sensitive viruses; the dashed line indicates median titers of all208 viral strains. Percentages of resistant viruses are shown in the top line. (B) Breadth and potency ofthe trispecific Abs relative to other bnAbs. Colors associated with each Ab designate first-generationAbs of lower breadth (orange); second-generation bnAbs or an engineered Ab-like molecule, eCD4-IgG(brown); structurally enhanced bnAbs (green); or trispecifics (red). (C) Sequential binding of threeantigens to the trispecific Ab,VRC01/PGDM1400-10E8v4, in the indicated order.The RSC3 (45) antigenrepresents monomeric gp120 optimized for the CD4 binding site Ab VRC01. MPER peptide interactswith 10E8 (7); gp140 trimer for PGDM1400 was derived from the gp140DN6 (BG505) protein.

Fig. 4. Serum antibody levels in rhe-sus macaques infused with parentaland trispecific antibodies. Concen-trations of VRC01 (green), N6 (black),and the two indicated trispecific Abs(brown, blue) containing a Fc mutationto extend half-life were measured inserum over the course of 14 days afterintravenous administration of a single10 mg/kg dose of each antibody. Eachdata point represents the mean ± SEMof the values from two to six animalsper group (VRC01, n = 6; N6, n = 4;each trispecific Ab, n = 2) anddetermined in replicates from twoindependent experiments.



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RESEARCH ARTICLES

the trispecific Ab correlatedwith the developmentof monkey anti-human Abs but arose almost2 weeks after the SHIV challenge.To ensure an adequate challenge dose, we

first challenged naïve animals with each virusindependently. For SHIV 325c, four naïve rhesusmacaques were inoculated one time intrarectallywith 1 ml of undiluted viral stock. All four ani-mals were infected and showed persistent vire-mia for up to 90 days (fig. S8). For SHIV BaLP4,the same stock and dose of virus were used asdescribed in several of our prior publications(26, 29, 30). In total, 30 control animals werepreviously challenged with a single 1-ml intra-rectal inoculation of SHIV BaLP4, and all becameinfected.To assess in vivo protection, we challenged

NHPs mucosally with a mixture of these dif-ferentially sensitive SHIVs, 5 days after Ab in-fusion in two separate experiments, with fouranimals in each group. In total, six of eightmacaques (75%) infused with VRC01 alone, andfive of eight animals (62%) treated solely withPGDM1400, became infected. In contrast, noneof the eight animals in the trispecific-treatedgroup were infected (Fig. 6C; P = 0.0058, two-tailed Fisher exact test). These data confirm thatthe improved breadth and potency of the tri-specific Ab conferred protection against virusesthat otherwise show resistance to single bnAbsalone.

DiscussionNext-generation HIV-1 bnAbs

A hallmark of HIV-1 infection is the remarkablegenetic diversity of the virus. Since 2010, sub-stantial progress has been made in the identi-fication of bnAbs that show exceptional breadthand potency [reviewed in (1)]. Several of theseantibodies have progressed into clinical trialsfor prevention or treatment, and there is renewedinterest in exploring their potential in the clinicalmanagement of HIV-1 infection (5, 12, 14). Here,we explored the potential of different bnAbs tocombine into a single protein that confers pro-tection against diverse HIV-1 strains. Among theclasses of bnAbs, we found that trispecific Absderived from bnAbs with CD4bs, MPER, andV1V2 glycan specificities had broad specificity,were potent, and could be produced in sufficientquantities to allow evaluation in NHPs and even-tually in humans. When tested in NHPs with vi-ruses resistant to individual parental bnAbs, thetrispecific Ab demonstrated complete protectionagainst both viruses, whereas infection was es-tablished in most animals treated with individ-ual parental antibodies VRC01 and PDGM1400.In addition, the ability of this trispecific Ab totarget three independent epitopes may improvetreatment efficacy in humans.In HIV-1–infected patients, reductions in viral

load have been observed after one infusion of asingle bnAb, thus demonstrating the biologicalactivity of HIV-1 bnAbs (31–34). A modest exten-sion of viral rebound was also observed whenindividual bnAbs were infused after antiretroviraldrugs were discontinued in previously suppressed

HIV-1–infected subjects (32, 33). NHP and humanpassive transfer studies have also suggested thatsuch bnAbs can enhance antiviral immunity thatmay contribute to improved viral control (35, 36).In addition, NHP studies demonstrate the im-portance of mAb potency and prolonged anti-body half-life in mediating protection againstinfection (26, 29). The generation of trispecificAbs with improved potency and breadth mayfurther enhance the efficacy of either passive im-munity or passive-active immunization strategies.Although bnAbs show exceptional breadth and

potency, resistant viral strains have been de-tected in patients who make these Abs (6, 37)and among natural viral isolates (38–40), raisingthe concern that resistance and escape mutationsmay arise. Such escape mutations are producedfrequently with antiviral drug therapy (41), andcountermeasures to reduce the likelihood ofescape would increase the likelihood of develop-ing a globally relevant therapy. Such breadth ofcoverage might alternatively be generated byadministering multiple bnAbs, and protectiveefficacy in a NHP model has recently been dem-onstrated against a mixture of SHIV viruses usingan antibody cocktail (42), providing further sup-port for the multitargeting concept. CombinationmAb therapy increases the complexity, develop-ment pathway, cost, and regulatory burdens oftheir use for treatment or prevention, in contrastto a single biologic therapy. The potency of the

trispecific Abs described here also exceeds thatof a broad and potent recombinant form of CD4(43), termed eCD4-Ig (fig. S4), and this lattermolecule is also directed to a single, albeit highlyconserved, HIV-1 Env epitope. The availability of asingle protein that targets multiple independentepitopes on virus also reduces the potentialgeneration of escape mutations. This advantagecould be related, in part, to the presence of threeindependent binding specificities at all times, incontrast to mixtures of antibodies where selec-tive pressure by individual mAbs with shorterhalf-lives may wane.

Clinical translation

The trispecific Abs have not yet been evaluatedfor safety and efficacy in humans. Initial char-acterization of their half-life in NHPs suggeststhat they behave similarly to conventional anti-bodies, but it remains unknown whether theycould be immunogenic in vivo. The administra-tion of a bispecific antibody to the human cyto-kines IL-4 and IL-13, which uses a related formatand linkers (44), may provide guidance in thisregard. This bispecific antibody has been evalu-ated in humans, where single subcutaneous dosesof SAR156597, ranging from 10 to 300 mg/kg,were well tolerated in healthy subjects, with lowtiters of anti-drug antibody (ADA) in only 4 of36 subjects (44). This trial showed amean half-lifeof about 2 weeks (44), similar to natural mAbs.


Fig. 5. Crystal structure of the CODV Fab and a structure model of the trispecific antibody.(A) Configuration of the trispecific antibody, color-coded by parental antibody. Dark shades (purple orgreen) denote heavy chain peptides; light shades denote light chain peptides. (B) Crystalstructure of the PGDM1400-10E8v4 CODV Fab in side and top views. CDRH3s from the two Fvsare labeled to highlight the antigen-binding region gp41 MPER, which was modeled in bysuperposing PDB 5IQ9 onto the 10E8v4 Fv. (C) VRC01/gp120 structure (PDB 4LST) and theCODV Fab were modeled onto the b12 structure (PDB 1HZH) by overlaying the CH1-CL domains.Color codes are matched in (A), (B), and (C).



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Originally published 22 February 2013 in SCIENCE

Although further human trials are neededto assess the full potential of the trispecific Abplatform, the data from the NHP challenge studydescribed here, as well as the previous experiencein humans with bispecific Abs (44), suggest thatthe approach merits further clinical investiga-tion. Studies in HIV-infected subjects, alone or incombination with other immune interventions,will address the potential of trispecific Abs toprovide durable protective immunity against in-fection or sustained viral control in HIV-infectedsubjects during drug holidays or in the absenceof antiretroviral therapy. The recognition of in-dependent target sites with multispecific anti-bodies can also be applied to other infectiousdiseases, cancer, and autoimmunity. These anti-bodies can promote recognition and binding tocritical antigenic determinants on target cellswhile simultaneously allowing engagement ofimmune cells that can stimulate relevant effec-tor function without the complications and ex-pense of delivering multiple recombinant proteins.

REFERENCES AND NOTES

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(2017).11. A. S. Fauci, J. Am. Med. Assoc. 316, 143–144 (2016).12. A. Pegu, A. J. Hessell, J. R. Mascola, N. L. Haigwood, Immunol.

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324–333 (2017).14. M. Caskey, F. Klein, M. C. Nussenzweig, N. Engl. J. Med. 375,

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(1998).25. Y. D. Kwon et al., J. Virol. 90, 5899–5914 (2016).26. S. Y. Ko et al., Nature 514, 642–645 (2014).27. D. Sok et al., Proc. Natl. Acad. Sci. U.S.A. 111, 17624–17629

(2014).28. E. O. Saphire et al., Science 293, 1155–1159 (2001).

29. A. Pegu et al., Sci. Transl. Med. 6, 243ra88 (2014).30. K. O. Saunders et al., J. Virol. 89, 5895–5903 (2015).31. R. M. Lynch et al., Sci. Transl. Med. 7, 319ra206 (2015).32. K. J. Bar et al., N. Engl. J. Med. 375, 2037–2050 (2016).33. J. F. Scheid et al., Nature 535, 556–560 (2016).34. M. Caskey et al., Nat. Med. 23, 185–191 (2017).35. T. Schoofs et al., Science 352, 997–1001 (2016).36. Y. Nishimura et al., Nature 543, 559–563 (2017).37. X. Wu et al., Cell 161, 470–485 (2015).38. H.-X. Liao et al., Nature 496, 469–476 (2013).39. P. L. Moore et al., J. Virol. 87, 4882–4894 (2013).40. J. N. Bhiman et al., Nat. Med. 21, 1332–1336 (2015).41. World Health Organization, WHO HIV Drug Resistance Report

2012 (2012); www.who.int/hiv/pub/drugresistance/report2012/en/.

42. B. Julg et al., Sci. Transl. Med. 9, eaao4235 (2017).43. M. R. Gardner et al., Nature 519, 87–91 (2015).44. C. Soubrane et al., paper presented at the 18th International

Colloquium on Lung and Airway Fibrosis, Mont Tremblant,Quebec; www.iclaf.com/Final_ICLAF_Program.pdf, 127(2014).

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ACKNOWLEDGMENTS

We thank C. Lawendowski for excellent program management;A. E. Schroeer and B. DelGiudice for graphic arts support;K. Radošević, C. J. Wei, M. Hollis, S. Rao, and B. Zhang fororganizational support; S.-Y. Ko for pharmacodynamics analysis;H. Qiu and B. Brondyk for technical advice; and L. Hou andA. Park from Sanofi for expressing and purifying the10E8v4-PGDM1400 CODV Fab used in the crystallization. Thedata are tabulated in the main text and supplementary materials.The coordinates and crystal structure factors were deposited inthe Protein Data Bank (PDB) under code 5WHZ. L.X., Z.-y.Y.,G.J.N., R.R.W., J.B., J.K., E.R., W.D.L., C.B., C.L., M. Connors, J.R.M.,R.A.K., N.D-R., T.Z., P.D.K., Y.D.K., and A.P. are inventors on patentapplication WO 2017/074878 submitted by Sanofi U.S. and theNational Institutes of Health that discloses the use of anti-HIVantibodies. Supported by Sanofi Global R&D (L.X., L.W., E.R., J.B.,J.K., D.M.L., R.R.W., G.D., C.B., C.L., W.D.L., R.S., Z.-y.Y., andG.J.N.); the National Institute of Allergy and Infectious Diseases(NIAID) Division of Intramural Research and Vaccine ResearchCenter (A.P., N.D.-R., K.M., M.L., M.A., Y.D.K., T.Z., S.D.S., R.T.B., K.W.,M. Choe, Z.M., S.O., J.-P.T., X.C., M.R., M. Connors, P.D.K., R.A.K.,and J.R.M.); NIH grants AI096040, AI124377, and AI126603 (D.H.B.and L.J.T.); and NIAID grant UM1AI100663 and IAVI (D.R.B.). Authorcontributions: Z.-y.Y., M.R., P.D.K., R.A.K., J.R.M., D.R.B., and G.J.N.designed the research; Z.-y.Y., A.P., N.D.-R., E.R., J.B., J.K., R.S.,and R.B.T. carried out the research; Z.-y.Y., L.X., A.P., L.W., M.A.,D.M.L., R.R.W., G.D., K.M., M.L., C.B., C.L., W.D.L., S.D.S., R.T.B.,K.W., M. Choe, Z.M., S.O., J.-P.T., and X.C. performed the experiments;Y.D.K., T.Z., D.H.B., M.R., and M. Connors contributed newreagents/viral strains; Z.-y.Y., L.X., A.P., N.D.-R., R.R.W., G.D., P.D.K.,J.R.M., and G.J.N. analyzed the data; and Z.-y.Y., A.P., N.D.-R.,R.R.W., J.R.M., and G.J.N. wrote the paper. All authors reviewedthe paper. VRC01, VRC07, VRC07-523, and CAP256-VRC26.25antibodies are available from J.R.M. and 10E8 and N6 antibodies areavailable from M. Connors under a material transfer agreementwith NIH. All other requests for data and further information shouldbe directed to the corresponding authors.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/358/6359/85/suppl/DC1Materials and MethodsFigs. S1 to S8Tables S1 and S2References (46–48)

30 May 2017; accepted 28 August 2017Published online 20 September 201710.1126/science.aan8630


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Fig. 6. Trispecific and broad neutralizing antibody sensitivity of SHIVs, plasma antibodylevels, and viremia in rhesus macaques. (A) IC50 neutralizing titers, in mg/ml, of VRC01, PGDM1400,and VRC01/PGDM1400-10E8v4 against replication-competent SHIV BaLP4 or SHIV 325c, with colorsas in Fig. 1. (B) Plasma levels of VRC01, PGDM1400, and VRC01/PGDM1400-10E8v4 in rhesusmacaques (n = 8 on each arm, done in two separate experiments with four animals each). All animalswere administered 5 mg/kg of the indicated antibody intravenously. Each data point represents themean ± SEM of the values from all eight animals per group. (C) Plasma viral loads in rhesus macaques(n = 8 per group) challenged with a mixture of SHIV BaLP4 and SHIV 325c, 5 days after intravenousadministration of VRC01, PGDM1400, or VRC01/PGDM1400-10E8v4.



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RESEARCH ARTICLES

Originally published 22 February 2013 in SCIENCE

High Coverage of ART Associated withDecline in Risk of HIV Acquisition inRural KwaZulu-Natal, South AfricaFrank Tanser,1* Till Bärnighausen,1,2 Erofili Grapsa,1 Jaffer Zaidi,1 Marie-Louise Newell1,3

The landmark HIV Prevention Trials Network (HPTN) 052 trial in HIV-discordant couples demonstratedunequivocally that treatment with antiretroviral therapy (ART) substantially lowers the probability ofHIV transmission to the HIV-uninfected partner. However, it has been vigorously debated whethersubstantial population-level reductions in the rate of new HIV infections could be achieved in “real-world”sub-Saharan African settings where stable, cohabiting couples are often not the norm and whereconsiderable operational challenges exist to the successful and sustainable delivery of treatment and careto large numbers of patients. We used data from one of Africa’s largest population-based prospectivecohort studies (in rural KwaZulu-Natal, South Africa) to follow up a total of 16,667 individuals who wereHIV-uninfected at baseline, observing individual HIV seroconversions over the period 2004 to 2011.Holding other key HIV risk factors constant, individual HIV acquisition risk declined significantly withincreasing ART coverage in the surrounding local community. For example, an HIV-uninfectedindividual living in a community with high ART coverage (30 to 40% of all HIV-infected individualson ART) was 38% less likely to acquire HIV than someone living in a community where ART coveragewas low (



population to calculate the number of HIV-infectedindividuals in the surrounding local communityfor each cell on the grid. In the same manner, eachpatient on ARTwas geolocated to their homesteadof residence, and the number of individuals in thesurrounding local community on ART was com-puted for each cell on the grid. These data are thenused to derive ARTcoverage (which we define asthe proportion of all HIV-infected individualsreceiving ART) at every location. Given thescattered distribution of the population (home-steads are not concentrated into villages or com-

pounds as in many parts of Africa), the methodis well suited to this setting because it does notimpose any static geographical boundaries onthe data but uses the precise location of each in-dividual (fig. S2) to derive a sensitive community-level measure that is both responsive to localvariations and robust to the effects of randomnoise. The resulting ART coverage measure is atrue population estimate because its denominatorincludes all HIV-infected individuals in this com-munity, independently of whether they have everaccessed an ART program or not.

The group of 16,667 repeat HIV testers in thisstudy are 75% of all individuals (≥15 years ofage) ever observed to be HIV-uninfected throughtheAfrica Centre’s population-basedHIV surveil-lance (2004 to 2011). Themedian duration betweentests in these individuals was 1.8 years. We usedsurvival analysis to examine the effect of ARTcoverage in the surrounding local community on anHIV-uninfected individual’s risk of HIVacquisitionwhile controlling for a range of well-establisheddemographic, behavioral, economic, and environ-mental determinants of acquisition of new HIVinfection (16, 17). These control variables rule outconfounding by the individual and communitycharacteristics they capture. In the analysis an in-dividual from this group of repeat HIV testers wasconsidered to be at risk of acquiring HIVafter thedate of their first HIV-seronegative test.

Treatment for the first patients in the surveil-lance area began in September 2004, and by July2011, an estimated 37% of all HIV-infected adultsin the area had been successfully started on ART.Overall, the speed with which ART has beenscaled up in this population has been rapid butheterogeneous in time and space (Fig. 1A). In2007, 6.4% of the population lived in commu-nities where coverage of all HIV-infected indi-viduals exceeded 30%, and by the middle of2011 this figure had increased to 90.8%. Thegeography of ART coverage did not follow aclear spatial pattern over the period of observa-tion (2004 to 2011). HIV prevalence was con-sistently highest in the peri-urban communitiesnext to the National Road (Fig. 1B), where >30%of the adult population were infected with HIV.The lowest HIV prevalence occurred among com-munities in the more remote rural areas (


RESEARCH ARTICLES

individual’s risk of HIVacquisition (community-level HIV prevalence, marital status, household-levelwealth, and the number of sexual partners inthe past 12 months). After ruling out confound-ing by these factors, there was a steep and highlysignificant decline in an individual’s adjusted HIVacquisition hazard with increasing ART cover-age (Fig. 3B). Holding other factors equal, anHIV-uninfected person who lived in a communi-ty with ART coverage of 30 to 40% was on av-erage 38% (P < 0.0001) less likely to acquire HIVinfection than anHIV-uninfected person living ina community with ART coverage of 25% relative to the base categoryof



period dummy variables to our regression equation (table S4). After inclusion of these dummy variables, the ART coverage effect size estimates remain essentially unchanged.

The ART coverage result proved to be robust to a wide range of changes in the multiple sensitivity analyses we performed, including different survival model specifications (table S8), different age-eligibility criteria (table S10), different methods for imputing the date of HIV seroconversion (tables S5 and S6), and a range of different size (radius = 2.5 to 3.8 km) and shape kernels used in constructing the community-level variables (table S7). In the base-case analysis, values that were missing for two of the covariates, marital status and number of sexual partners in the past year, were imputed using multiple imputation (see supplementary materials). As a sensitivity analysis, we repeated our regressions with the sample of complete cases only, that is, the sample of individuals without any missing values. The ART coverage effect size estimates in this analysis remained very similar to those in the base-case analysis (table S9). As described above, the ART coverage results also persisted after we controlled for the influence of other prevention activities that had changed over the study period (table S3), as well as after adjusting for the trends in calendar time (table S4). Consistent with the “treatment-as-prevention” hypothesis, the decline in the risk of acquisition of HIV infection with increasing ART coverage in the older age groups (>35 years of age), where ART coverage was highest, was more pronounced than for the population as a whole (fig. S6). We also formally investigated whether selection effects could affect the results, by imputing HIV status for every individual who was eligible for testing in a particular year (see supplementary materials). We then used

Fig. 3. Results of the multivariable analysis showing an HIV-uninfected individual’s HIV aHR (95% CI) and associated P values for different categories of ART coverage, that is, the proportion of the total HIV-infected population receiving ART (A and B), and HIV prevalence (C) in the surrounding local community (derived using a standard Gaussian kernel of radius 3 km, as shown in Fig. 1, A and B). (A) displays the aHRs adjusted for systematic differences in age and sex, and (B) and (C) display the aHRs adjusted for all other variables in the final model (see Table 1 and table S2 multivariable analysis).

individual’s risk of HIVacquisition (community-level HIV prevalence, marital status, household-levelwealth, and the number of sexual partners inthe past 12 months). After ruling out confound-ing by these factors, there was a steep and highlysignificant decline in an individual’s adjusted HIVacquisition hazard with increasing ART cover-age (Fig. 3B). Holding other factors equal, anHIV-uninfected person who lived in a communi-ty with ART coverage of 30 to 40% was on av-erage 38% (P < 0.0001) less likely to acquire HIVinfection than anHIV-uninfected person living ina community with ART coverage of 25% relative to the base categoryof


RESEARCH ARTICLES

that capture the behavioral (18) or biological out-comes of other HIV prevention interventions: (i)the proportion of people who had ever had sex,(ii) the average number of sexual partners in thepast year, (iii) the point prevalence of concurrentsexual partnerships, (iv) the mean age differencebetween partners, (v) proportion using condomsat last sexual activity, and

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