IAVI Report THE NEWSLETTER ON INTERNATIONAL AIDS RESEARCH VOL 7/NUM 2 MAY–AUGUST 2003 Do Clades Matter for HIV ? As thousands of people prepare to gather in Nairobi and New York for September’s AIDS meetings, a key question for vaccine developers is how to contend with the huge diversity of HIV strains circulating worldwide

BY PATRICIA KAHN

is famously the most genetically diverse than anything the public health field has ever attempt- HIVviral known—nowhere more so ed. Doing it with many different formulations, or with than in Africa—as well as one of the most rapidly repeated updates, would be even more challenging. mutating. That, plus the uneven global distribution of Nailing down the impact of HIV diversity on vac- its nine genetic subtypes, or clades, poses one of the cine responses is difficult, for several reasons. One is biggest scientific unknowns facing AIDS vaccine devel- that the current system for classifying HIV diversity is opers: is a single, “universal” vaccine against all strains based on genetic sequence, not immune properties, possible? Or will it be necessary to make a slew of dif- and hasn’t been translated into distinct “immuno- ferent vaccine formulations, each tailored to the most types”—which is what really matters for vaccines. common strains in a given region? Even worse, could it While that task is slowly being tackled for tar- Inside: mean that new formulations might be needed regular- geting cellular immunity, it may be impossible for neu- ly, as with flu vaccines? tralizing (NAbs), where clades don’t seem to In Memoriam: The answer will be key to how quickly, and at correlate with immune recognition. Balla Musa Silla 2 what cost, an AIDS vaccine can be widely distributed Another complication is that the clade issue has around the globe once a successful candidate is identi- become highly politicized. Until recently, vaccine Setting a Scientific fied. Manufacturing even a single formulation and get- development has had a lopsided focus on clade B Agenda for a House on ting it out quickly to adults and adolescents soon after strains, which dominate the epidemic in industrialized Fire: AIDS Prevention it is licensed will be far more complicated and costly countries but cause only about 12% of glob- Research Moves Ahead in Russia 3

continued on 2 ▼ Keystone Symposium: Updates on Trials, New NIH DETAILS PLANS FOR ANALYZING Candidates and Immune Basis of VAXGEN PHASE III DATA Protection 7

BY EMILY BASS AIDS Vaccines, from Monkeys to People: t the 24 June meeting of the In the plan’s ongoing first analysis of data with Thai investi- An Interview ANational Institutes of Health phase, an independent team will gators and VaxGen. with John Shiver 10 (NIH) AIDS Vaccine Research re-analyze existing data, looking at Johnston said that NIH devel- Working Group, Peggy Johnston, factors such as race, risk category oped this strategy after VaxGen Studying HIV Diversity head of the NIH AIDS vaccine and the viral strains that infected expressed reluctance to invest in Multi-Clade Regions: research effort, outlined a three- volunteers. A second team is dis- additional funds in the Thai trial. pronged plan for involvement of cussing potential additional analy- At the same meeting, VaxGen rep- Tanzania: At the Cross- NIH and the Centers for Disease ses of blood samples, such as resentative Marc Gurwith said that roads of Africa’s Control (CDC) in VaxGen’s com- broader characterization of anti- the company—which already has Major Clades 17 pleted and ongoing Phase III trials. body responses, and HLA typing. many samples from the nearly- The plan emerged after VaxGen The goal of the third component is completed Thai study—was col- Cameroon: “Final released data from its US- “to ensure completion” of the laborating fully with the independ- Common Pathway” European Phase III study and ongoing Phase III trial in Thailand, ent data analysis. At press time, for a Global Vaccine18 made the controversial claim that Johnston said. To achieve this, precise details of the NIH-initiated the vaccine showed efficacy in NIH, CDC and an as-yet unnamed consortia, and of VaxGen’s role, Vaccine Briefs 20 non-white minority groups. donor will work on statistical had not been specified. ◆ In Memoriam: Balla Musa Silla (1955-2003) Balla Musa Silla, found- Tall, elegant and well-spoken, Mr. Silla had a stately, captivating ing vice-President for presence. A colleague at Partners recalls a plenary speech he once Vaccine Preparedness gave. “Unlike the others on the podium, he had no slides; he simply at the International spoke and told the life story of a girl growing up in poverty. The tale AIDS Vaccine Initiative flowed naturally and illustrated the realities of injustice and the chal- (IAVI), and a visionary lenges to development, but on a deeply personal level. He put the leader in the fields of sentiment back into that auditorium in Geneva—he brought poverty population and interna- into the room. Colleagues from different parts of the world thought tional development, of their own people. This was his gift.” died on 27 June , 2003 Mr. Silla joined IAVI in September 2001, where he founded a at his home in Ossin- department tasked with building international support and aware- ing, New York. He was ness for AIDS vaccine development and helping to build developing 48. The cause of death country capacity for clinical testing. Once again, he proved to be was T-cell lymphoma. equally at home power-brokering with world leaders and listening Mr. Silla was born to the needs and concerns of underserved communities desperate- in The Gambia but ly in need of an AIDS vaccine. Most of all, he understood the cen- was truly a citizen of trality of AIDS in undermining development, as well as the potential the world, having lived and worked in over 27 countries. He was of to reverse the epidemic’s devastation in the world’s a tireless advocate for the needs and potential of the developing poor countries. world, a theme that runs through his professional and personal Mr. Silla left an indelible impression on those who worked life. His career began in The Gambia, first as a volunteer working with him, and will be remembered for his kindness, generosity of alongside the international community, and eventually as the gov- spirit and dynamic personality. “My memories are of a big, ernment’s Director for Population Affairs, where he was a direct strong, proud man, with a wide, mischievous grin, and a gracious advisor to the President. manner beyond any I have encountered,” says Craig McClure, In 1996, he was approached to lead an ambitious and untested who worked with him at IAVI on community mobilization. To his venture, Partners in Population and Development, an intergovern- former colleagues, he leaves a legacy of commitment to a vision mental alliance of Southern countries addressing issues of family of a better future. planning and reproductive health through South-to-South collabora- Mr. Silla is survived by his wife, Joan Millsap, and their two sons, tion. Partners was a unique organization of Southern governments Christopher and Andrew. working closely with each other and with a variety of NGOs and com- BONNIE BENDER AND FAWZIA RASHEED munity-based groups toward a shared goal. The pace at which the program grew bore witness to Mr. Silla’s deft diplomacy and his Bonnie Bender, IAVI’s Program Manager for Vaccine Preparedness, unstinting commitment to South-South cooperation. His vision was worked with Mr. Silla during his tenure with the organization. Dr. grounded in the belief that the solutions to development lay in devel- Fawzia Rasheed was his colleague at Partners, where she served as oping countries themselves. Senior Advisor on HIV/AIDS & STD and Policy Advisor. ▼ DO CLADES MATTER continued from 1 ally. That disconnect helped mobilize developing coun- cially in Africa.” tries to get involved with HIV vaccine testing, and But despite these challenges, there are promising spurred development of non-clade B candidates— developments, along with some sobering ones. A which now greatly outnumber new clade B-based growing body of data shows that immune responses to ones. But it also helped create a political logjam: Fears T-cell-based HIV vaccines, and to natural , that testing vaccines based on “unmatched” strains often recognize HIV proteins from different clades— exploits trial volunteers in developing countries some- fueling optimism that, at least for vaccines targeting cel- times engendered resistance even to early-stage trials of lular immunity, some cross-clade protection can be non-local clades, and raised pressures to tailor vaccine achieved. And studies in infected people are revealing candidates to ever-finer, single-country levels. that a few antibodies which neutralize primary HIV Yet only by comparing vaccine efficacy in strains also work against other clades, findings that are matched settings to partially or completely unmatched renewing hopes of designing immunogens able to elicit ones can the impact of HIV diversity ultimately be NAbs, a task that has long seemed intractable. resolved. Moreover, “a country-by-country approach to Things are also moving on the political front. “I vaccine development would be crippling,” says see a major shift,” says Jose Esparza, coordinator of the Francine McCutchan (Henry M. Jackson Foundation, WHO-UNAIDS HIV Vaccine Initiative, pointing as an Rockville), who leads the US military’s HIV global sur- example to a consensus document on clades released veillance program. “It would make it very, very slow to by the African AIDS Vaccine Programme at its June get vaccines suitable for some hard-hit regions, espe- meeting (see p. 20). “There’s now widespread recogni-

continued on 14 ▼ 2 IAVI REPORT Setting a Scientific Agenda for a House on Fire AIDS PREVENTION RESEARCH MOVES AHEAD IN RUSSIA

BY EMILY BASS

here’s nothing eye-catching highest prevalence of any political support for rapid, inno- Tabout the 8 Plus Clinic. No European country), Belarus and, vative interventions in high risk shingle outside gives away the more recently, Kazakhstan. groups like IDUs and sex work- building’s identity; the sole identi- Today, there are 230,000 docu- ers. President Vladimir Putin has fier is a small, colorful sign mented cases of HIV in Russia— yet to make a public statement tucked behind the bars that cover and the actual number is estimat- calling for a rapid response to the all the windows. A muddy yard ed at 800,000-1.5 million. The epidemic. Complicating matters, separates the one-story building face of the epidemic is young: AIDS must compete for attention from Gazovaya street in St 60% of all infections are in 21-40 with other diseases, including Petersburg, and an improvised year olds. Recent data show that hepatitis, multi-drug resistant stepping stone pathway offers increasing numbers of women , alcoholism and heart minimal protection from the slip- are being infected, and that rates disease, which have run rampant pery surface, which is particularly of heterosexual transmission are since the disintegration of Soviet- treacherous in late April when on the rise (see box, page 6). era medical services. the long winter begins to thaw. These trends are indices of an The lack of political will has But as modest as it looks on ominous trajectory: an epidemic financial and policy implications: the outside, the clinic is a land- spreading from high-risk groups the country’s annual AIDS budget mark in the Russian AIDS land- to the general population. of US$5.5 million is primarily scape. The clinic—a project of “Recently we attended parlia- used for mandatory HIV testing. the Biomedical Center, a non- mentary hearings where the Active IDUs are ineligible for profit research institute led by Russian authorities all started their treatment for HIV or common co- molecular biologist Andrei speeches by saying, “Attention, infections such as Kozlov—is conducting Russia’s attention, we are on the edge of a hepatitis or TB, and first prospective incidence study terrible disaster. Our house is on drug-replacement of HIV in intravenous drug users fire,” says Eduard Karamov, an programs which (IDUs). For over a year, the clin- outspoken virologist at Moscow’s swap methadone or ic, which is named for the num- Ivanovski Institute for . “I buprinorphen for ber of required study visits, has said, ‘Calm down, sit back, the injected drugs are tracked the spread of the fire started many years ago.’” illegal. The HIV care which infects more than 30% of Russia is not without that does exist is St Petersburg’s IDUs. It has also resources to combat this new handled by central- established itself as a trusted plague. Despite the Perestroika- ized Federal AIDS source of voluntary counseling era brain-drain which emptied Centers. In the major and testing, harm reduction and entire labs, the country has urban centers of basic medical care to IDUs, who retained a cadre of highly-trained Moscow and St generally risk being reported to research and medical profession- Petersburg, these the authorities, or arrested, each als. It also possesses a well-estab- centers each provide time they seek treatment. They lished regulatory system for antiretroviral therapy also face stigma in the general approving trials and products, (ART) to fewer than population: St Petersburg resi- and internal capacity to manufac- 2,000 people; in the dents were so unwilling to have a ture pharmaceuticals and vac- more remote areas, clinic serving IDUs in their back- cines (see sidebar, page 5). state-of-the-art thera- yards that “it took us a year just Marshalling these resources into py is simply to find a building,” says project projects like the 8 Plus clinic unheard of. coordinator Alla Shaboltas. could benefit Russia—and the Against this Inside the small building, world. IDU cohorts are a rarity in bleak backdrop Kozlov, Shaboltas and the clinic most countries with injection-driv- there are scattered team are on the frontlines of an en epidemics, as are models for points of light, most- exploding epidemic. Since 1998, IDU-friendly services. ly in the form of the number of reported HIV Unfortunately, the clinic is research sites and The entryway to 8 Plus Clinic. infections has doubled each the exception rather than the NGO-initiated projects. year—a frightening, exponential rule. By all accounts, Russia’s International funding supports growth curve. Similar crises are steps to combat the epidemic most, if not all, of the targeted building in neighboring countries, have been ineffective, hampered efforts to respond to the Russian including Ukraine (which has the by underfunding and lack of epidemic—from needle exchange

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clinics to prison-based prevention als,” he says. ble. Second: It’s unnecessary, programs. The 8 Plus Clinic is no Russia has also landed on because there is no AIDS prob- exception. The project is support- the international radar screen as a lem in Russia. Third: Who are ed by the the Biomedical Center potential site for efficacy trials of you going to give it to? Do you (which receives both international AIDS vaccines. One compelling want to defend the gays and and domestic funding) and the reason: with the exception of prostitutes?” recalls Sidorovich, a HIV Prevention Trials Network Thailand, almost no countries key member of the team which (HPTN), partnering with the with IDU epidemics have taken developed the Russian AIDS University of North Carolina. Too steps to develop cohorts or con- ELISA diagnostic in the late small to make a dent, too neces- duct research with these groups. 1980s. (The diagnostic had to be sary to be abandoned, these proj- With its mix of sexual and IDU field-tested in Uganda, because of ects beg the question of what transmission, Russia’s epidemic the low HIV incidence in Russia internationally-funded AIDS would also make it possible to at the time.) research can accomplish in the evaluate whether and how candi- Ultimately, the scientists won absence of political will to create date vaccines protect against a half-victory. Since 1997 a programs for the most affected intravenous compared to sexual Ministry of Science and groups—and what the cost will exposure (see IAVI Report, May- Technologies program to develop be if the world fails to find out. Jun 2002, p.6). Another key new vaccines and diagnostics has point: for reasons that are still included a funding stream for Growing research activity poorly understood, the clade A AIDS vaccine development. The question of whether or strain that has spread like wildfire Originally, the AIDS vaccine not research can play a catalytic in Russia shows astonishingly lit- development effort were centrally role in an AIDS response is not tle genetic diversity, with an aver- coordinated by the Biomedical unique to Russia. But unlike age of just 3% difference among Center. Since 2001 it has consist- many other regions of the world viral isolates from far-flung ed of three independent teams— where AIDS is spreading regions of the country. It’s a situ- each of which received roughly unchecked, Russia’s pre-existing ation that “eliminates one of the $200,000 in 2003. One, based at a infrastructure provides a solid variables [viral diversity] from a former bioweapons facility in foundation for initiating interven- ,” says molecular epi- Novosibirsk, focuses on vector- tions. Several groups are already demiologist Francine McCutchan based strategies, including salmo- building on this foundation, (Henry M. Jackson Foundation, nella constructs. The Biomedical including London’s Imperial Rockville, Maryland). Center is developing a DNA vac- College, Johns Hopkins In fact, Russian scientists cine, and a group at Moscow’s University, Yale University and were advocating for AIDS vac- Russian Institute of Immunology the University of North Carolina. cine research before the coun- (led by Igor Sidorovich and Russia has also received roughly try’s own epidemic exploded. including Eduard Karamov) is $12 million from the US Biomedical Center head Andrei developing a recombinant vac- Biotechnology Engagement Kozlov, virologist Eduard cine using the team’s novel adju- Program (BTEP), which aims to Karamov, and Igor Sidorovich vant, polyoxidonium (PO), which convert bioweapons capacity into from Moscow’s Institute of is widely used in a licensed public health-related research. A Immunology were among those Russian flu vaccine. small portion of this—less than scientists who began lobbying for The decision to split the pro- $1 million—is earmarked for state-funded AIDS vaccine gram into three separate efforts AIDS vaccine research and devel- research in the late 1980s, when may make it harder for each opment, which is underway at a the first handful of AIDS cases groups to assemble all of the ele- handful of sites, including the appeared in Russia. Their call for ments needed to bring a candi- Biomedical Center. action drew on the country’s date to trial. Missing pieces at Jonathan Weber (Imperial deep roots in vaccine production: one or more of the sites include College, London) is a co-investi- in its heyday the Soviet Union access to non-human primate gator on a Moscow-based study was a colossus in the vaccine facilities and GLP-compliant vivar- of the long-term impact of short- field, manufacturing 1/3 of the iums for small animal studies. course ART treatment during global supply of vac- Where improvements are being acute infection; he hopes to cine used in the eradication cam- made, it is with foreign dollars-- launch a microbicide trial in the paign, and producing a wide for example, BTEP funding is Urals. Weber says that his experi- array of human and veterinary helping to renovate the current ence with protocol approval, site vaccines for its sprawling empire. vivarium at the Biomedical development and staffing have In spite of this history, Center. In spite of these obsta- been uniformly positive. Based Russian officials did not warm to cles, the teams project a symbolic on this infrastructure, “Russia is the call. “The logic was like this. readiness—a best effort under dif- an ideal place to do clinical tri- First: An AIDS vaccine is impossi- ficult circumstances, designed to

4 IAVI REPORT Russia’s Current Vaccine Manufacturing Capacity show their government, and the The demise of the Soviet Union dealt a blow to vaccine and invest some of their returns into research and world, that there is AIDS vaccine producers, who had to adjust to the loss of state subsi- development. International groups including the research capacity in Russia. dies and purchase guarantees, and to the introduction of WHO and the International Science and Technology competition from foreign vaccine manufacturers. Today, Center (Moscow) are providing oversight in quality Stumbling blocks and Russian facilities—many of which were built between control training and funds for equipment upgrades. successes for trials the 1950s and the 1980s—are seeking to catch up and to Furthermore, Russian producers are showing The combination of passion- begin competing in the global market. A recent govern- increased concern that there is a domestic vaccine ate local scientists and foreign ment law set a 2005 deadline for bringing all plants up available to the population for each illness. dollars is promising—but there to international GMP (Good Manufacturing Practice) Strengthening domestic production also has pub- are still major hurdles to building standards for production practices, although Russian and lic health implications. While coverage with locally-pro- a comprehensive AIDS research international experts say that many many plants will not duced measles and mumps vaccines exceeds 90%, agenda. On a scientific level, one make this deadline. Currently the GMP status of Russian rubella coverage “is low because there is no domestic of the biggest stumbling blocks is production facilities varies considerably even within the vaccine,” says health ministry official Galina Lazikova. the lack of sound epidemiological same organization, ranging from WHO-accredited, to These developments are being closely watched data about the crisis. “There is no nearly-compliant, to facilities which are at least a decade by some AIDS vaccine researchers. “Russia may well history of cohort research in away from GMP status. Biomed (Moscow Region) and emerge as an important player in AIDS vaccine R&D. Russia,” says Shaboltas. Immunopreparat have at least one production line When an AIDS vaccine is finally licensed, Russia could In keeping with Soviet-era accredited by WHO. play a key role in manufacturing for many parts of the approaches, Russia has favored There are signs that the industry is adjusting to world,” says Don Burke, head of the Center for massive, mandatory-testing cam- the new conditions. Russian vaccine manufacturers Research at Johns Hopkins University. paigns over targeted HIV surveil- are beginning to apply new production technologies EMILY LOCKE lance. Since 1987 Russia has con- ducted over 200 million HIV tests—an average of 24 million a MAJOR VACCINE PRODUCTION FACILITIES IN RUSSIA year—in a relatively random selection of the population, Company (location); Vaccines produced Approximate # doses Upcoming products including job applicants, preg- State or private annually nant women, and hospital admit- Biomed (Moscow region) HBV, DIP, TET, DT, DTP 10-15 million tances. Given the many disincen- Private tives for IDUs to seek testing, this sampling method may not Biomed (Perm) DIP, TET, HBV, DT, TYP Unknown State grasp prevalence in this group, nor can it provide accurate inci- Combiotech (Moscow) HBV, DIP-TET-HBV, 5 million HAV-HBV, DPT-HBV-HIB Private DIP-PER-TET-HBV, HBV dence data. (preservative free) The ongoing HPTN trial at the 8 Plus Clinic will nail down Immunopreparat (Ufa) DIP, TET, DIP-TT, DPT, Unknown State INF, RAB some of this information. After screening nearly 1,000 IDUs— Institute of Poliomyelitis and OPV, TBE, RAB, YEL 100-150 million Viral Encephalitis (Moscow region) 30% of whom proved to be State already infected—the clinic is following 520 HIV-negative IDUs Moscow Enterprise of Bacterial MEA, MUM Previously produced RUB, MMR Preparations/Institute for Viral 100 million, expect- for 12 months, with biannual Preparations (Moscow) ed to produce this HIV tests. After six months the State amount after produc- trial has achieved a retention tion unit is certified. rate of over 80%. While this is St. Petersburg Institute of Vaccines INF, DIP, TYP, 5-6 million too low for vaccine efficacy tri- and Serums, Krasnoe Selo als, which last two to three (St Petersburg) years, it is a strong start with a State population often perceived as Vector (Koltsovo) HAV, MEA 100,000 HAV-HBV (collaborating difficult to engage in trials. The State with Combiotech) study’s community advisory Virion (Tomsk) HBV, TBE Unknown board even includes former State IDUs—a radical step for the DIP: diphtheria; DTP: diphtheria-tetanus-pertussis; HAV: hepatitis A virus; HBV: hepatitis B virus; INF: influenza; Russian research world. MEA: measles; MUM: mumps; OPV: oral ; PER: pertussis; RAB: rabies; RUB: rubella; So far, the study’s findings TBE: tick borne encephalitis; TYP: typhoid; TT: tetanus ; YEL: yellow fever. confirm the catastrophic state of Sources: http://www.privivka.ru/vaccines/complete.htm, data as of July 3 2003; the epidemic. Unofficial interim and personal communication with institute representatives. data analyses show an HIV inci- Emily Locke PhD, MPH (Johns Hopkins University) is based in St Petersburg, Russia, dence rate of about 3%. Rough where she is researching Russian vaccine development and production capacity. figures also show that the median

continued on 6 ▼ MAY–AUGUST 2003 5 ▼ RUSSIAN AIDS PREVENTION RESEARCH continued from 5

age is 24 years; over 60% have names—and so are unable to UNAIDS program in Russia, secondary education; 40% are report individuals’ HIV status or adding that “It looks like the employed full time; 62% live with drug-use to the authorities. country is being divided between their parents; 11% own a home. These regulations also curtail international donors, without Another cohort initiative is the treatment that can be provid- government coordination.” underway in Moscow, where epi- ed by research sites. Russia main- As small as it is, the domes- demiologists Chris Beyrer, Julie tains a centralized surveillance tic AIDS vaccine effort has Stachowiak (Johns Hopkins and treatment system for diseases already suffered from the historic University) and the NGO such as hepatitis B and C, which lack of coordination. The pro- AIDSInfoshare (which Stachowiak are endemic among drug users. gram has no provision for media co-founded) are building a sex As with HIV treatment, active outreach or public education, or worker cohort. After a series of drug users are ineligible for sub- for site preparedness activities. thwarted attempts to engage the sidized care, which is often mini- For now scientists shoulder these Moscow health system, Beyrer mal. 8 Plus project physician roles—with mixed results. In and Stachowski decided to train Natalya Khaldeeva would like to 2002, when the Moscow team sex workers and AIDSInfoshare treat her patients—nearly all of from the state-funded AIDS vac- staff (many of whom have med- whom have hepatitis B or C— cine development program sub- ical training) as lay epidemiolo- and the site’s funds might be able mitted a trial application (which gists. They have completed a to pay for it, but this offering is is still pending) for its candidate, qualitative phase and plan to not feasible under current laws. team scientist Irina Nikolaeva launch a 500-woman cohort in The laws constraining treat- wrote a press release. Neverthe- late 2003. ment of active IDUs would also less, the media carried erroneous Once cohorts are formed complicate large-scale vaccine tri- reports that Russia had discov- there are other hurdles, such as als, given the growing interna- ered an AIDS vaccine. laws that constrain provision of tional consensus that volunteers To address this, the WHO- treatment (including ART and who become infected should UNAIDS HIV/AIDS Vaccine have access to ART. But, Beyrer Initiative has proposed consen- says, research could help lever- sus-building activities to the RUSSIA’S HEALTH CRISIS age change. “These trials are a major Russian stakeholders. A long way off. We should proceed plan has yet to emerge—a delay ■ Estimated 2-4 million intravenous drug users (IDUs)—up with all due speed. If it turned which may reflect the fact that to 2.7% of the population. In some regions, more than out that the law was the primary the different teams work, by all 60% of IDUs are HIV-positive obstacle, then Russia might look accounts, more or less independ- ■ 22-fold increase in HIV prevalence among pregnant at ways to deal with it,” he says. ently of one another. women from 1998 to 2002 On the positive side, public A lack of central coordination statements by Vadim Pokrovksi, ■ More than half of registered HIV-positive individuals are Perhaps the greatest the head of Russia’s Federal co-infected with hepatitis C virus unknown is whether research AIDS Centers, led to a recent ■ Roughly 40% of HIV-positive people are co-infected with findings will affect government flurry of media attention; Russian TB policies. Without state implemen- scientists also report growing tation of key findings, research support from individual politi- ■ 1 in 10 prisoners have TB, 1/3 of them multi-drug resist- projects will only impact a frac- cians. Regionally, there is a ant TB tion of the population. Until growing AIDS activist movement, ■ Since 1965, the average Russian life expectancy has recently there was no govern- which recently held a summit on dropped from more than 67 years to 65 years ment monitoring to learn about treatment access in Minsk. successful interventions or to Whether these elements will identify the optimal use of inter- coalesce into a comprehensive hepatitis medications) to active national dollars flowing into response remains to be seen. “If drug users and other marginal- research and programs. In June we look at the democracy which ized groups. The 8 Plus Clinic the government formed an AIDS has emerged in Russia, it’s good has had to walk a fine line Advisory Council to provide this for people and even better for between complying with state guidance, but it remains to be ,” says Igor Sidorovich. regulations and addressing vol- seen whether this council will “Perestroika led to the destruc- unteers’ well-founded fears make policy recommendations tion of state systems, which are about the legal repercussions of which counter the existing gov- only now being rebuilt. Let’s seeking care. In the ongoing ernment positions. hope Russia is open not only to trial, for example, volunteers Observers are cautiously infection but to valuable technol- are known solely by their optimistic. The advisory council ogy and ideas. This is why our numerical identifiers. Doctors is “a very positive sign,” says team has remained here. We never learn their patients’ Pedro Chequer, head of the hope we survive.” ◆

6 IAVI REPORT Keystone Symposium: Updates on Trials, New Candidates and Immune Basis of Protection BY MARK BOAZ

ontinuing our report of April’s Keystone response than a single protein. Graham said CSymposium on HIV Vaccine Development, one of that macaque data showed no reduction in Gag the field’s premiere research conferences, we cover an responses when Env antigens are incorporated, and array of talks ranging from animal models to ongoing that data on the breadth of Env responses may be clinical trials (see also IAVI Report Apr-May 2003, p.1). available by the September AIDS Vaccine 2003 meet- Although there were no major surprises or new find- ing in New York. ings announced, the meeting gave a good overview of the broad efforts underway on many different fronts. High-dose study Juliana McElrath (University of Washington, Update on human clinical trials Seattle) presented data from HVTN 039, which tested Updates from ongoing vaccine trials and from a well-studied canarypox-based vaccine (ALVAC monkey studies of candidates due to enter Phase I vCP1452) given at the most commonly-used dose and 7.26 8 studies soon, featured heavily on the conference at one higher dose (10 and 10 TCID50). McElrath agenda. Since last year’s Keystone conference, four said that the immune responses to both doses, completely new vaccines have entered Phase I tri- assessed using the Elispot assay to detect IFN-gamma- als and several other Phase I and I/II studies were producing cells, were indistinguishable but low launched. (See database at www.iavi.org/trialsdb). (between 40 and 70 spot-forming cells per million white blood cells), leading McElrath to conclude that DNA candidates from the Vaccine use of the higher dose was not warranted. Some cross Research Center clade responses were seen to Gag, with three of the Barney Graham, who directs clinical studies at seven people who responded to subtype B Gag also the NIH Vaccine Research Center (VRC; Bethesda), responding to subtype C. discussed early results from Phase I trials of the Center’s new DNA vaccines. These constructs are the Preclinical studies in monkeys first step in a multi-clade, prime-boost strategy that Bob Johnston from the University of North will use adenovirus-based vaccines as a boost and Carolina gave an update on the development of HIV incorporate env from three different subtypes. vaccines using vectors of Venezuelan Equine The first VRC trial, started in late 2001, tested Encephalitis (VEE) virus. One potential advantage of DNA containing HIV gag and pol from subtype B at this approach is that VEE targets immature dendritic three doses (0.5 mg, 1.5 mg or 4 mg). Immune cells, which play a key role in presenting antigens to responses were determined by measuring interferon- the immune system. Together with the biotech com- (IFN)-gamma-producing cells using intracytoplasmic pany AlphaVax, Johnston’s team has engineered a cytokine staining (ICS) assays. Each group had seven replication-incompetent vaccine vector encoding HIV volunteers, two of whom received placebo. Graham clade C gag, which recently received approval from presented unblinded data on responses after the sec- ’s regulatory authorities for a Phase I ond of three . study. The trial will take place in Johannesburg and CD4 T-cell responses were present in 4/7 indi- four US sites of the US HIV Vaccine Trials Network viduals in the lowest-dose group, mainly to Gag but (see Vaccine Briefs, p. 20). also to Pol, and Gag-specific CD8 cells were found in Johnston described results showing partial pro- two of these volunteers. The 1.5 mg dose led to CD4 tection against disease in two studies of vaccinated and CD8 responses in 2/7 volunteers. No data is avail- macaques. In one, 2/6 macaques vaccinated with able yet for the 4 mg group. VEE plus env (gp160 and gp140) completely con- Graham also presented early results from the trolled an intrarectal challenge with SIVE660, one of more recently launched trial of multi-clade DNA con- the pathogenic SIV strains; the remaining four ani- taining gag, pol, and nef from subtype B and env mals showed partial control. In contrast, only 1/6 from subtypes A, B and C. Groups again consisted of unvaccinated monkeys partially controlled the chal- seven volunteers with two receiving placebo. After lenge, while the rest showed no control. Both CD8 two of three scheduled vaccinations, 3/7 volunteers and neutralizing responses were associat- in the 2 mg dose group had HIV-specific CD8 cells, ed with protection. and two of these three volunteers also had CD4 In the second study, eight macaques were vacci- T-cell responses. nated with VEE carrying env (gp160), gag and pol, and An important question for vaccines with multi- then challenged intravenously with SHIV89.6P. ple antigens is whether responses are reduced com- Vaccinated monkeys showed some protection from pared to single antigen vaccines. Another is whether disease compared to controls, with peak viral load using 3 different Env antigens induces a broader lowered about 10-fold and a 100-fold reduction in set-

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point. The correlates of protection were less clear in positive animals generally failed to control replica- this study, since pre-challenge CD8 T-cell responses tion of the escaped strain and showed no CD8 were only detectable in 2/8 monkeys and no neutral- responses to the mutated epitopes. But surprisingly, izing antibodies were found—although Johnston said in both of the monkeys negative for MamuA*01 and this may reflect differences between the different chal- B17—where there is no immune pressure against lenge viruses used in these studies. these epitopes—the Gag and Nef epitopes reverted back towards their original sequence, suggesting that Adeno-Associated Virus (AAV) the escape mutations reduce viral fitness. Watkins Phil Johnson (Children’s Research Institute, called this surprising finding a “saving grace” for Cincinnati) reviewed some new data on an HIV vac- CTL-based HIV vaccines, since it suggests that even cine using vectors of adeno-associated virus (AAV), when escape occurs, the escaped strains are unlike- focusing on recent safety studies. A version of this vac- ly to sweep through human populations, which are cine containing gag from clade C is slated for human genetically diverse; rather, important CD8 epitopes trials starting in late 2003. will probably be regenerated, thanks to the higher Johnson presented data from animal toxicology fitness they confer. and biodistribution studies, which are required for Viral escape in regulatory approval of clinical trials. No local or sys- Correlates of protection temic reactions to the vaccine were seen in a 150- Identifying the specific immune responses which monkeys is found rabbit toxicology study, and analysis showed only control HIV infection has long been a central issue for trace amounts of vaccine present in about 15% of the HIV vaccines, since this knowledge would greatly “to lower fitness— tissues sampled at 180 days—mostly in the muscle at simplify the tasks of designing and testing candidates. the injection site or in other highly perfused tissues. In Banff, several speakers reported on studies that a “saving grace” Importantly, no trace of vaccine was found in the tackled this problem from different angles. gonads, indicating that vector is not transmitted into Jeff Lifson (National Cancer Institute) gave an for CTL-based the genome of offspring. Overall, this safety profile is interesting talk describing examples of control of SIV similar to that seen with DNA vaccines. Two immu- infection, whether achieved through early antiretro- nized macaques sacrificed at 5 months also showed viral treatment (ART), vaccination, or spontaneous- vaccines? very low persistence of vector. ly, and the associated immune responses. One SIV- These studies also confirmed earlier findings that naïve macaque inoculated with the virulent the AAV vector does not integrate into cellular DNA SIVmac239 controlled viral replication following the (J.Virol 2003:77;3495). Integration of the unmodified initial peak to less than 20 copies/ml of plasma. CD8 AAV has been seen in cultured cells and initially raised responses seemed important in this control as these ”safety concerns about this approach. increased progressively over time and the viral load rebounded upon depletion of CD8+ cells. Of note, Viral escape: Can it generate Elispot responses in this animal during control of “immune-resistant” strains? SIVmac239 infection were lower than levels seen in David Watkins described a recent monkey study other animals that were unable to control this same which looked at some possible consequences of viral virus in other vaccine studies. Neutralizing antibod- escape in a vaccinated population. ies were low through the period of initial control, but In infected macaques and in some vaccination were present at high levels as the animal re-estab- studies, SIV and SHIV undergo mutations which lished viral control after the depletion of CD8+ lym- enable them to evade the CD8 responses that nor- phocyes. Conversely, a vaccinated animal controlling mally control viremia. Escape also occurs during HIV virus to <20 copies had detectable low-level Elispot infection, raising the possibility that T-cell-based vac- responses, yet upon CD8 depletion, no rebound of cines—which allow infection but prevent replica- virus was seen. A greater role may have been played tion—may lead to the selection of mutated viral in this animal by antibodies, which were detectable strains lacking crucial CD8 epitopes, and which all the way through. might then spread through populations unchecked In a different setting, a macaque which con- by immune control trolled SIVmacE660 infection following limited early To test this scenario, graduate student Tom ART also had modest levels of Elispot responses, but Friedrich created a strain of SIVmac239 with muta- was able to control a heterologous challenge with tions in each of the three most important epitopes SIVmac239. Lifson concluded that qualitative charac- (in the Gag, Tat and Nef proteins) targeted by CD8 teristics rather than the level of the immune response T-cells in monkeys of a specific genetic background were likely important in these animals, since low-level (MamuA*01 and B17). Four rhesus macaque mon- responses were associated with control of viremia. keys with this background, and 2 without (so they Early control probably also limited virus diversifica- target different epitopes) were then infected with tion, thereby facilitating sustained immune control. the mutant escape strain, which the researchers In a talk on CTL-based vaccines, Andrew called 3xSIV. McMichael (Oxford University) emphasized the Watkins said that, as expected, the MamuA*01/B17- importance of looking at memory cells in vaccinated

8 IAVI REPORT individuals, rather than at only the more short-term that this method may underestimate response levels, post-vaccination responses. He pointed out that CD8 especially to the more variable HIV proteins. memory responses in EBV-infected people do not The researchers analyzed CD8 responses in 6 strictly reflect the responses seen shortly after infection acutely infected patients using sequences based on due to a change in immunodominance: whilst direct- consensus virus or patients own (autologous) virus to ed at a similar range of epitopes, cells that showed the p24, Tat and Vpr proteins. The breadth and sum smaller responses after the initial infection come to of CD8 responses to Tat and Vpr were underestimat- dominate the memory response, whereas those that ed when consensus sequence was used rather than were initially dominant decrease proportionally (J. autologous sequence, with 8/24 peptides not being Immunol 2002;168:3309). Since vaccinees are most recognized with consensus sequence and an average likely to be exposed to virus when their immune magnitude of 179 SFC versus 514 SFC. In contrast, a responses are in the memory phase, he suggested it slight underestimation of CD8 responses to p24 were would be important to study these responses. seen when using consensus sequence compared to McMichael described several different assays that can autologous sequences, with 4/18 peptide responses be used to measure memory responses, including missed using consensus and a magnitude of 328 SFC Elispot and ICS to detect cytokines and cell markers, versus 464 SFC. The responses to consensus and and killing assays using cultured cells. He said the ICS autologous sequences differed more to Tat and Vpr tetramer assay and ICS cell proliferation assay (the than p24 because the virus was more different in these CFSE assay) would probably be the best ways to regions—approximately 10% in Tat and Vpr com- quantitate long-term memory. pared to only 1.8% in p24. Altfeld said that underestimation of responses to Measurement of immune responses the more variable HIV proteins may be a problem that Current measures used by researchers to deter- plagues research if autologous sequence is not used mine the level of HIV specific CD4 and CD8 T-cell for stimulation. (This was recently published in J.Virol responses focus mainly on production of IFN-gamma, 2003:77;7330.) a cytokine with antiviral activity that is relatively easy to detect by Elispot or ICS assays. However, some HIV transmission and envelope protein selection studies presented at Keystone add to the growing evi- From the swarm of different virus strains present dence indicating that this measurement is not suffi- in infected individuals, only a small subset is transmit- cient to identify all responding cells. ted sexually. This partly reflects the selection of virus- For example, data presented by Michael Betts es that use CCR5 as a co-receptor to enter cells, but (VRC, Bethesda, Maryland) showed that not all cells other viral properties also appear to play a role. Since which kill virally-infected cells produce IFN-gamma. it is these newly-transmitted strains which vaccines To do this, he used flow cytometry to examine CD8 must initially combat, a fuller characterization could T-cells which degranulate (by measuring the CD8 cell be enormously useful for vaccine design. surface degranulation markers CD107a and b), the Cynthia Derdeyn (University of Alabama) main mechanism by which they kill virally infected gave a thought-provoking presentation on an cells, in addition to IFN-gamma. These results suggest analysis of Env proteins in eight transmission that both the quality and quantity of HIV-specific CD8 pairs, drawn from a couples cohort T-cell responses determined by IFN-gamma measure- in Zambia. In addition to sequencing env genes ment may be underestimated. from 8 newly infected people and their partners, The magnitude of CD4 T-cell immune respons- the researchers also measured the sensitivity of es also appears to be underestimated by the meas- transmitted virus to neutralization by antibodies urement of IFN-gamma alone. Stephen De la Rosa present in the donor’s plasma. (VRC) illustrated this point with data on 9 individu- Derdeyn reported that the transmitted env als who received booster shots of either tetanus tox- sequences were all homogenous in the V1-V4 region, oid (TT) or . For the HepB-spe- regardless of the complexity of viral strains present in cific CD4 T-cells, and to a lesser extent the TT-spe- the donor. Interestingly, they also showed a compact cific cells, IL-2 and MIP-1 beta production were the variable loop structure, suggesting that variants with predominant cytokine responses, with IFN-gamma- larger V1-V2 and V4 loops were selected against dur- producing cells accounting for as little as 25% of the ing transmission, as were heavily glycosylated strains. responding CD4 cells. Recipient strains also proved to be roughly seven Other data suggested that the methods used to times more sensitive to neutralization with donor plas- stimulate HIV-specific cells so they can be measured ma than the donor’s viral population at large, indicat- are also part of the problem. Normally, researchers ing that strains which escaped neutralization are not stimulate cells with consensus or reference virus the source of transmitted virus. strains (or peptides) that are representative of circulat- Further understanding of exactly what is trans- ing viruses. However, a poster by Marylyn Addo and mitted should come from analysis of newly-infected Marcus Altfeld from Bruce Walker’s group individuals in the VaxGen trial, where over 300 full (Massachusetts General Hospital, Boston) suggests envelope genes have been sequenced. ◆

MAY–AUGUST 2003 9 AIDS Vaccines, From Monkeys to People

For many years, John Shiver, Merck’s Execu- Merck since 1991. He now oversees not only tive Director of Viral Vaccine Research, has the company’s HIV vaccine research activities been a regular speaker at major conferences but also its work on vaccines against other on HIV vaccines. With 11 clinical trials in viral . progress and an extensive pre-clinical program The previous IAVI Report [Feb-Apr 2003] AN for testing candidate vaccines in monkeys, reviewed Merck’s clinical program on HIV-1 Merck’s program ranks among the major HIV vaccines. Here, Shiver speaks with IAVI Report INTERVIEW vaccine development efforts. editor Patricia Kahn about the company’s newly- Shiver, who earned a Ph.D. in chemistry launched international trial and on lessons and then trained in immunology at the learned along the way about using the rhesus WITH National Institutes of Health, has been at macaque model for AIDS vaccine development.

Can you tell us about the international trial just tools for inducing cellular immunity. The next step— John launched with one of your vaccines? finding the best combinations—is unpredictable: you The trial will enroll 435 volunteers at sites in the still have to rely basically on intuition and the empiri- Shiver US, Puerto Rico, Brazil, Peru, Haiti, Thailand, South cism of seeing what happens. Africa and Malawi, which are part of the HIV Vaccine So it will take a while to figure out what our best Trials Network. We’re testing a “proof-of- options are. But we’ll make these decisions based on a concept” vaccine containing the HIV-1 gag substantial body of clinical data. gene in an adenovirus vector. The vaccine is already being studied in several hundred You mentioned genetic diversity of human popula- North American volunteers, so we know it tions as one rationale for the international trial. At looks safe and can induce good immune Keystone you presented some monkey data rele- responses. This trial will gather more safety vant to genetic differences and vaccine protection. and immunogenicity data in people of Can you describe these findings? diverse genetic backgrounds. We’re trying to develop a broad perspective about our challenge studies, by looking at choices like which What new information do you expect to monkeys or challenge viruses to use, and then seeing get by scaling up the numbers and what differences result from this choice. diversity of volunteers? Our earlier studies on adeno-based vaccines The key question is whether people used animals harboring the MamuA*01 MHC class I of diverse genetic backgrounds respond allele [a genetic marker associated with greater natural equally well to the vaccine—even though ability to control SIV and SHIV replication] and chal- they may target different epitopes. We’ll lenged with SHIV89.6P. These animals have controlled also analyze how well the vaccine-induced viremia to undetectable levels, with detectability being responses in these populations recognize about 50 virus copies per ml of plasma, and stayed HIV-1 antigens based on different clades, as there so far for 2-3 years. When we induced stronger we’re doing in the North American volunteers. [For fur- immune responses with a really good DNA prime ther discussion of vaccines and HIV clades, see article, plus adjuvant, and an adenovirus boost, it didn’t p.1.] And we’ll continue exploring the effect of pre- improve on this—we couldn’t crimp the primary existing immunity to the adenovirus vector. viremia further, and chronic viral load can’t go below undetectable. All animals showed a strongly dominant Your newer studies all involve adeno-based vac- response to the Gag CM9 , although they also cines. Are these now your main focus? responded to other epitopes. We already know a lot about our adeno-based The new experiments tested different adeno-based candidates, and see many advantages: immunological, vaccines against the same challenge strain, in monkeys convenience of construction, and so on. But we’re tak- with genetic backgrounds that don’t give immunodomi- ing a broad approach to viral vectors, exemplified by nant responses to Gag [MamuA*01-negative]. We found two studies we presented at the recent Keystone meet- that vaccination lowered viral set point 100-fold or ing. One is the testing of three different poxviruses in more compared with unvaccinated animals. That’s a prime-boost combinations with adeno. The other is our significant drop. But the load was still a few thousand work on different adenovirus serotypes, which we’ll copies, which is a lot more than 50. also test in various prime-boost combinations. This tells us that strong immunodominance for Our goal is to collect a set of vaccine tools and one Gag epitope confers a big advantage against SHIV analyze each one alone and in combination. Right challenge. But even in animals without this advantage, now, pre-clinical data suggest that prime-boost combi- two or three orders of magnitude is still a lot of con- nations of adenoviruses and poxviruses, or of different trol—actually, an outcome many people would proba- adenovirus serotypes, are our most promising vaccine bly be satisfied to see in a human study. Think about it:

10 IAVI REPORT if we start with a setpoint of 30,000, which is what’s One consequence is that, so far, it takes two dif- found in untreated people in the MACS cohort [a long- ferent types of vaccines to control SIV viremia in mon- term, prospective US-based cohort of HIV-positive men] keys. But we are seeing successes in monkeys, so and drop it by two logs, we’re in the hundreds. That there’s a strong rationale for moving these approaches should mean a much better clinical prognosis. into people. These results also tell us that SHIV is a more strin- To me the value of these challenge systems— gent challenge in MamuA*01-negative monkeys than in even if the disease they cause is exaggerated compared positive animals—another indication that genetic back- to humans—is that they help us discriminate among ground is important. candidates. If no vaccines impacted viremia or disease at all, we couldn’t learn anything about what to test in What else have you gleaned about genetic back- humans. But we do have vaccines that control SHIV or ground and control of viremia? SIV. So our foot is in the door. The task is to use ani- We’ve seen that even without a vaccine, mals for defining which combinations of vectors and MamuA*01-negative animals tend to have a worse clini- antigens open the door further, and then to translate cal outcome after challenge than the A*01-positives. this experience into human trials. They advance to AIDS much more rapidly. But I wouldn’t say that a particular vaccine Another observation is that unvaccinated animals approach is the one thing to move into people occasionally control virus spontaneously. I’ve seen this because it’s so great in monkeys. Instead, you use the with SIV, and even more with SHIV—we see about 1 animals to make a short list of what to take forward in 5 or 6 animals which do that, regardless of their into people. MHC background. I’m not sure why. But it shows that there’s a lot going on in individuals that determines dis- Given all these variables, what challenge model will ease course, whether they’re monkeys or people. you use from now on? That’s why human studies need to look at a lot of I’ll use both SIV and SHIV, and probably stick to diversity in volunteers, in terms of their genetic back- MamuA*01-negative animals for SHIV. I don’t think grounds, to really get a sense of how effective a vac- there’s any further room to mine for control of SHIV cine will be. And it’s why international studies are so with these vaccines in MamuA*01-positives. crucial—to start getting a handle on this. You mentioned that your successfully vaccinated You’ve also looked at different challenge viruses. monkeys are still protected 2-3 years after chal- What have you learned? lenge, with no sign of viral escape. That’s especially At Keystone, we showed data on SIVmac239, surprising since your vaccines carry only one HIV which is more pathogenic than SHIV, in both gene. Why does virus escape in some studies but MamuA*01-positive and -negative animals challenged not others? intrarectally. The question was whether vaccines that This is an important point. Our approach, which controlled SHIV89.6P would give at least partial control is based on our experience with anti-retroviral drugs, is against this challenge. that you need strong immune pressure against the Only one group did—the one given a DNA- virus. You need it as quickly possible, and it needs to prime, adeno boost, which also gave the best T-cell be fairly diverse. Even though our adenovirus carries responses. The relative control of viremia isn’t necessar- only gag, it’s very potent in monkeys—the magnitude ily due to these specific vaccines, but might happen of T-cell responses is strong prior to challenge, and it with any vaccines that get T-cell responses to this high- tends to recognize multiple Gag epitopes. er level. In the MamuA*01-positive animals, I collaborated on the DNA vaccine studies pre- DNA/adeno reduced SIV loads 10-30-fold below the sented at Keystone by Dan Barouch and Norman control levels, at least out to 260 days. We still need to Letvin [reporting viral escape for several animals in two follow this longer—it’s a young study. challenge studies ]. Those are early vaccines developed in my group, but that elicit relatively weak immune That’s more than what many studies find with SIV, responses. And where responses are weak, they also although still far from full protection. But is a chal- tend to be narrow. Less immune pressure on the virus lenge virus like SIVmac239 perhaps too stringent? means greater potential for escape. I don’t know. Well, yes, I know one thing. Both That doesn’t mean escape can’t happen where SHIV and SIV give extremely high viremia in responses are stronger and broader. But it stands to macaques—between 100,000 and over one million viral reason—and our data bear it out—that better contain- copies of virus per cc of plasma within a few weeks of ment of HIV is much more likely when there’s a challenge. They maintain this level for months, until stronger immune response from the start. AIDS-associated pathogenesis sets in. Clearly, that’s much more viremia, and faster pathogenesis, than HIV- By now Merck has tested certain approaches in 1 causes in people, where median viral setpoint is both monkeys and humans. How well did monkeys about 30,000. predict the human results?

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With adenovirus, many of our findings are very clinical safety and create the right type of information similar. You get the same magnitude of responses in for FDA review? How well can we make the vaccine? people and in monkeys, the same proportions of CD8 and CD4 contributions, the same durability of respons- In evaluating T-cell responses, most groups rely on es and diversity of epitopes. In terms of dose-response, assays that detect interferon-gamma. Are you satis- responses to adenoviruses in people exceed what we fied that this detects the right cells? saw in monkeys, at least in people without pre-existing I would not accept any single immunological antibodies to Ad5. parameter as the gatekeeper for clinical trials. We assess With DNA, immunogenicity decreased as we immune function based on many types of data. Besides moved from mice into bigger rodents like rats or interferon-gamma, we measure cytokines like IL-2 and guinea pigs, and then into monkeys. It took more TNF-alpha. We look at CD4 and CD8 contributions. We and more DNA, and the responses have a highly validated cytotoxicity assay, which corre- became lower. But we still came up with a dose and lates strongly with interferon-gamma responses for CD8 regimen in monkeys that consistently primed T-cell cells. Tetramer assays are great because they provide a responses. Even with DNA in saline, nearly every mon- reference point, an upper limit on the frequencies of key makes a sustained T-cell response, and the antigen-specific cells. And we look at cellular pheno- CRL1005 adjuvant increased responses by about 5-fold. types: Do the responding cells look more like activated But in people, that trend falls off: our best DNA or resting cells? Are they en route to a lymph node, or approach gives responses in only 40% of the volun- emerging from one? teers; they’re not very durable, and don’t synergize well It’s not clear what all these data mean. But with . The synergy between vac- they help you stay broad-minded about decisions, cines was there, though, from the animal work. and to remember that you don’t really know what These results also suggest another conclusion: you’re doing. cytokines and adjuvants generally don’t translate well That said, when I look for a common thread in from monkeys into humans. our challenge studies, I have to say that it’s the magni- tude and diversity of CD8 T-cell responses. So would you say that non-human primate models We can’t define a cutoff where you know that can help identify the best approaches, but not the above this level, every animal will control virus. The best dosage or immunization regimes? numbers are too limited. But at this point, it looks like In general, yes, but may depend on the animal species the stronger the response and the more epitopes it tar- and the specific vaccine. For example, we’ve done gets, the more likely an animal will control virus, at some testing of DNA vaccines in baboons, and found least for some time. Maybe we can still improve our that their dose response, and the impact of adjuvants, predictions by looking at subsets of these determinants. were close to what we saw in people. But I think we’re looking in the right direction.

So you could optimize your immunization regimens Why is it so hard to find definitive correlates of pro- in baboons after macaque studies identify the best tection in monkeys? overall candidates? I think there are indications of correlates, in terms The thought frequently occurs to me that, yes, I’d like of trends. But it’s hard to nail down more precisely to see responses in baboons with a particular regimen. without lots of data, which in turn means lots of mon- But these are large animals—30 or 40 kilos, not 3 or 4 keys. And you’ll never have enough monkeys with the kilos like macaques, so they’re much more difficult and types of determinants we’re looking at. The correlate expensive to work with. And our list of candidates is will probably be something like a probability of con- short enough to be manageable for clinical testing. trolling viremia as a composite of immune response Besides, you can only do so much, and clinical studies strength, diversity, CD4-CD8 contributions and ability to are our top priority right now. establish memory. Given the complexities of multi- component correlates, you won’t resolve this without a What are your criteria for deciding which approach- Phase III study in people. It’s not like hepatitis B vac- es to move into Phase I testing? cine, where if you get to a specific antibody titer, you’re There are lots of things. We’re working to gener- considered protected. ate complete datasets, characterizing and comparing vaccines in terms of T-cell responses—overall magni- Is Merck working on neutralizing antibodies? tude, CD4 versus CD8 contributions, how many epi- We’ve had our share of negative data. But we topes they recognize, and of course their ability to con- kept an active effort in this area, and showed some trol viremia after challenge. When you do the science results in posters at Keystone and in publications right, the answers are actually pretty clear as to which over the last year. These efforts are mostly directed vaccines look better. at and gp120-CD4 complexes. So far we Beyond how well the vaccine performs, a particu- haven’t been able to define an interesting immuno- lar vaccine may be more attractive for development gen based on these proteins, but there are still based on pragmatic things. Can we demonstrate pre- some approaches to try.

12 IAVI REPORT MACAQUE STUDIES TO OPTIMIZE VACCINE STRATEGIES information on key aspects of the structures involved in neutraliza- tion. This is done through crystallization of HIV envelope proteins arly this year IAVI launched a pre-clinical program aimed at opti- and monoclonal antibodies, which is now being executed by a robot- Emizing vaccine design, dosage and immunization regimen and ic system from Syrrx (San Diego, California), at the Scripps Research prime-boost combinations with its current crop of candidates. So far Institute (La Jolla, California) in collaboration with the Joint Center for it has launched the first vaccination studies in macaques and estab- Structural Genomics (a California- lished a core lab at the pharmaceutical company Becton Dickinson based consortium). The automat- (San Diego), where the analyses of immune responses in these stud- ed high-throughput platform can ies will be carried out. produce crystals in as little as two Ongoing studies are working out the best dosing regimes with weeks, in contrast to the usual DNA/MVA and the adeno-associated virus (AAV), and testing whether manual procedures, which gener- IAVI responses to AAV can be boosted with either AAV (using a different ally take months and are far more subtype than the prime) or with MVA. Future experiments will ana- costly. This system has now been news lyze which HIV genes are necessary for protective immunity, compare used successfully to determine different viral vectors head-to-head, and examine the ability of adju- the structure of several broadly neutralizing monoclonal antibodies, vants to enhance the immunogenicity of DNA vaccines. The findings including IgG1 b12, which recognizes the CD4 binding site, and of these studies will be published, and should help guide the design 2G12, a highly unusual antibody that recognizes a cluster of carbohy- of future vaccines. drates on gp120, among others directed at gp41 or the V3 loop. These efforts are being led by Alan Schultz at IAVI and Shiu-Lok The next step is the modification or enhancement of envelope Hu from the University of Washington. Collaborating non-human pri- proteins to expose important regions for neutralization. One design mate centers include the Oregon Health and Science University, approach is to mimic the native trimeric form of envelope spikes that Children’s Research Institute, and the University of Pittsburgh—with occur on the virus surface. One such candidate immunogen (gp140 possible future collaborations to include the Tulane National Primate GCN4) previously developed by the group of Joseph Sodroski (Harvard Research Center and the Air Force Research Institute for Medical University, Cambridge), an NAC member, has been shown to induce Science in Bangkok. neutralizing antibodies in rabbits and is being modified further to increase its immunogenicity. Sera from rabbits immunized with these novel immunogens will be tested against a broad range of viruses, CONSORTIUM UPDATE using ViroLogic’s high-throughput pseudovirion assay. Another ongoing study is comparing immune responses induced by DNA vaccines n 2002, IAVI, the National Institutes of Health Vaccine Research encoding env with responses to the envelope protein itself, since vac- ICenter (VRC), and a number of leading academic and industry labo- cines using DNA are potentially cheaper and easier to manufacture. ratories joined forces to create the Neutralizing Antibody Consortium The consortium has also established a repository to store and (NAC) (see IAVI Report, May-Jun 2002). In the year since its inception, distribute key reagents, such as the various envelope proteins and NAC members have made steady progress towards the design of monoclonal antibodies used in the crystallization studies. Looking immunogens that elicit broadly neutralizing antibodies to HIV. ahead, NAC members plan continued structural studies and produc- Members of the consortium are taking a systematic approach to tion of new immunogens, along with head-to-head immunogenicity developing effective immunogens. The first stage involves gathering studies in animals.

How important are neutralizing antibodies for mak- candidates to move into efficacy trials? ing a highly effective HIV vaccine? Our goal now is to finish defining our basic vac- There’s no doubt in my mind that a good anti- cines, through the one we consider most promising— body-inducing component will ultimately yield a better the trivalent vaccine with gag, pol and nef, which is vaccine. But I also think we can have a useful vaccine now going into people. We’re also expanding into pop- if a good antibody component isn’t found. ulations similar to those where efficacy studies will be By ‘good antibodies,’ I mean antibodies that neu- done. You’ll see more and more sites opening up that tralize most wild-type primary isolates of HIV. Adding are relevant to efficacy questions, whether it’s for stud- recombinant gp120 to a in one arm of a ies with the HVTN or new sites we’re opening up with trial, or putting an envelope gene into a viral vector, is the trivalent vaccine. not a solution, because the responses they induce We’re planning very aggressively. I can’t give you aren’t functional, in the sense that hey don’t meet the a time frame because it’s not defined yet. But we’re goal of what antibodies need to do. planning for diverse efficacy studies with a clade B vac- cine. While those efficacy studies are underway, we’ll Do you think sterilizing immunity is achievable? continue developing our other potential vaccine com- I think neutralizing antibodies might increase the ponents, so that we’ll have a short list of next best probability of getting sterilizing immunity, of not having choices by the time the first efficacy data come in. productive infections, and shifting average viremias Even a vaccine that works will probably have lower. How much depends on how good the T-cell shortcomings. We’ll see what these are, and with an part of the vaccine is. understanding of our next best tools, we’ll marry the two together and test efficacy again. That’s our Do you have a timetable for deciding which basic strategy. ◆

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tion across Africa that the question of diversity needs to epidemic, especially in regions where several clades co- be rigorously tested through well-designed clinical tri- circulate. Some of them, like the B/F recombinant als—including some in unmatched settings.” While the found in parts of South America, were previously clade issue is most relevant for efficacy trials, at this thought to be pure subtypes. (A few were already point there’s progress at the Phase I level: South known to be important circulating strains, especially the Africa—with an overwhelmingly clade C epidemic— A/G recombinant called CRF02,_AG, for Circulating has just approved a Phase I trial of a vaccine based on Recombinant Form, common in west and central Africa, clade A, shortly after giving the green light to its first and CRF01_AE, originally called clade E, in Southeast HIV vaccine study, which uses a clade C-based candi- Asia.) Even more surprising, says McCutchan, is the date (see p. 20). (Africa’s first HIV vaccine trial, a Phase high number of unique recombinants (those found so I study in Uganda in 1998-2000, also used a vaccine far only in a single person). For example, in Tanzania, from an unmatched clade—in this case, clade B, which where McCutchan and collaborator Michael Hoelscher is all that was available at the time.) (University of Munich and director of Tanzania’s Mbeya But at the same time, the picture of global diversi- Medical Research Program, MMRP) have dozens of full- ty—and the task of tracking it—are getting more com- length sequences from low-risk populations, at least plicated. That’s partly because new HIV variants and 40% are turning out to be unique recombinants (see recombinants are continuously generated, and estab- figure 3, p.17). Similarly, full-length sequencing of HIV lished ones move into new geographic areas. But it from low-risk adults in Uganda and Thailand, countries also reflects the growing use of more sophisticated that each have two important co-circulating strains, technologies to characterize HIV strains, which are found about 30% and 13% recombinants, respectively uncovering some unexpected layers of complexity. (see Figs. 1 and 2). Since recombination can only occur if two viral Describing global diversity strains have co-infected a single cell, these findings hint- At first glance, it might seem that classifying and ed at yet another unexpected layer of diversity: that tracking HIV variation around the world is a straightfor- double infections may be far more common than previ- ward, albeit mammoth, undertaking. But that hasn’t ously recognized. Consistent with this notion, a turned out to be so. prospective study of 600 female bar workers in south- The original definition of clades was based on western Tanzania suggests so far that, astonishingly, a short sequences, mostly within the HIV envelope, and substantial proportion of these high-risk women show has expanded to include nine clades (designated A preliminary evidence of double infection with HIV from through K, with no E or I). As the amount of sequence two different clades (see article, p.17). data grew over time—all captured in the NIH-support- But characterizing these dual infections and ed database now run by at the Los pinpointing when each one occurred is proving to Alamos National Laboratory (LANL) in be technically daunting, even with the project’s large New Mexico (www.-web.lanl.gov)—it collection of blood samples from individuals at dif- UGANDA became clear that each HIV gene shows a ferent time points. The difficulty is that the relative different, and characteristic, degree of vari- proportions of the two strains fluctuate widely over ation. Env heads the list, with up to 35% time, according to Hoelscher—one strain may sequence diversity between clades, 20% remain barely detectable for months, then suddenly A within one clade and even 10% in a sin- emerge to dominate the viral population in the AC 15% gle infected person. At the other end of blood. With superinfection now a hot issue in the CD the spectrum, the Gag and Pol proteins HIV field (see IAVI Report, Jul-Sep 2002), resolving show only 10-15% sequence divergence whether the infections happened more or less simul- D across clades. taneously, or if one occurred after the other was AD 55% But over the past few years, as more well-established (true superinfection), is a high pri- 23% HIV labs have established high-through- ority for these researchers. put sequencing technologies, the field has The issue has enormous implications for mapping accumulated hundreds of full-length HIV diversity, in terms of identifying the proportions of dif- sequences from around the world—allow- ferent strains circulating in a population, and for geno- ing large-scale comparison of whole typing HIV in samples used for studies of cross-clade genomes rather than just selected regions. immunity. And even though dual infections may prove Fig. 1: Proportion of different HIV The result: “a very rapid change in the less common outside highly-exposed groups like the subtypes and recombinants in circu- epidemiological picture,” says McCutchan, Tanzanian cohort, HIV varies up to 10% even in singly- lation, based on 47 full-length HIV whose updated map of global HIV diver- infected people. “I think we’ll find that sampling from a sequences. Samples represent inci- sity (included in this issue as a special single time point, and sequencing only one full clone, dent infections from a community- poster and available at has pretty poor power to reveal what’s going on,” says based cohort study in the Rakai dis- trict, 1999-present. www.iavi.org/iavireport) illustrates the 10 McCutchan. “We’ve uncovered a gigantic can of worms. major epidemiological patterns. It will take new technologies to sort this out.” Source: Francine McCutchan One big change is a growing aware- Yet there’s a bright spot in the data on HIV varia- ness of the role recombinants play in the tion, she adds. Looking at the global numbers, it

14 IAVI REPORT THAILAND emerges that four clades (A through D) plus two CRFs across clade are another potential factor— EC (01 and 02, both of which are about 70% clade A) although new data showing that a candi- account for over 90% of all infections worldwide. From date vaccine protects monkeys even this perspective, diversity can be boiled down to 4 key after one dose, which induces a weaker BE clades, plus small contributions from the non-A seg- response than a full prime-boost regimen ments of these two CRFs—a more manageable focus (Nature 424; 681: 2003), indicate that less- B for vaccine developers. than-optimal responses can suffice for a But the missing link remains to connect this successful vaccine. And 90% sequence increasingly detailed understanding of genetic diversity conservation between, say, the vaccine with a picture of immune properties. The usual way and an infecting Gag protein still corre- CRF01_AE of classifying viruses immunologically is based on sponds to an average of one amino acid serotype—a group of antigenically-related strains rec- difference per epitope, write Tomas ognized by a specific reference antibody, usually a Hanke and Andrew McMichael—which, neutralizing antibody. By this criterion, “there’s despite some “wobble” (tolerance for mis- absolutely no sense of serotypes for HIV,” says anti- match), could abrogate a significant num- Fig. 2: Proportion of different HIV body expert David Montefiori (Duke University, ber of responses across clades (Vaccine subtypes and recombinants in circu- Durham). “Although antibody binding tracks some- 20:1918;2001). Some loss was seen in the lation. Data based on 29 full-length what based on clade, we don’t know if neutralizing Ugandan canarypox vaccine study, where HIV sequences, most from incident serotypes even exist and can be defined.” He also cross-clade responses were sometimes as infections in the Rayong and Chon- points out that it’s extremely difficult to pinpoint NAb strong as those to vaccine strain (as meas- buri provinces (sites of an upcoming epitopes, which are often based on 3D shape and ured by peptide titrations in Elispot Phase III trial) and 41 samples from map to discontinuous sequences. assays), and sometimes weaker. incident infections in Northern Thai- On a more promising note, mapping T-cell epi- In any case, studies of cross-clade land. Both had 13% recombinants, but different proportions of the two topes (which are based strictly on sequence) and defin- recognition—including some with new types shown. For the recombinants, ing their patterns of cross-clade recognition is easier, non-clade B candidates—are yielding a E is shorthand for CRF01_AE. and work in this area is ongoing growing body of valuable information. And as Peggy Johnston (Assistant Source: Francine McCutchan Vaccines and cross-clade responses Director for AIDS Vaccines, National If there’s been one set of findings to cheer in the Institutes of Allergy and Infectious recent past, it’s the emerging clinical data suggesting Diseases) points out, “this will allow the field to that immune responses to T-cell-based vaccines fre- make decisions based on data, not conjecture,” she quently recognize at least some HIV strains of other says—decisions such as what vaccine strains are clades—albeit possibly fewer epitopes and/or at a most promising for testing in regions with particular lower magnitude. non-matching clades in circulation. For example, trials of Merck’s adenovirus-based Merck’s John Shiver, who heads the company’s candidate, which carries a clade B-derived gag gene, vaccine research program (see article, p.10), points to found that 10 out of 13 volunteers who responded to another potentially important (and under-recognized) vaccine also recognized peptides from clades A and C component of cross-clade protection: immune Gag. Consistent with these findings, an international responses to HIV antigens not present in the vaccine. study of people infected with HIV of different clades This notion emerged from Merck’s studies of its gag- detected similar frequencies of cross-clade T-cell only candidates in monkeys, which show “terrific” responses to Gag and to the relatively conserved pro- responses to Nef after challenge with either SIV or teins Pol and Nef, but far fewer responders to the more SHIV—responses rarely seen post-challenge in unvac- diverse Rev and Tat proteins. Cross-clade CTL respons- cinated animals. “This suggests that vaccines may only es are also seen with canarypox-based vaccines, as first need to generate enough of an immune response so shown in studies of Ugandan volunteers given clade B- that after infection, you can make lots of natural based vaccines and who often responded to some A responses to the infecting virus, whatever clade it is,” and D peptides (J Inf Dis 182:1350;2000). Overall, says he says. “The vaccine may not have to generate all Larry Corey, who directs the NIH-sponsored HIV the heterologous [cross-clade] coverage.” Vaccine Trials Network, “clades aren’t making the kind For vaccines that target the antibody-producing B- of difference people thought they would.” He also pre- cells, the picture remains much bleaker. So far, no vac- dicts that “cross-clade responses will be more the norm cine tested in humans has generated neutralizing anti- than the exception.” bodies (NAb) to anything beyond the vaccine strain But the case is far from proven. It’s unknown and a few closely related isolates, and there are few whether cross-reactivity will translate into cross-protec- ongoing clinical trials involving candidates that even tion, says Jaap Goudsmit, chief scientific officer at target this arm of the immune system. Crucell, a Dutch biotechnology company; without more Yet data on HIV-infected people show that cross- detailed data on which epitopes are and aren’t recog- neutralizing antibodies do exist—findings that have nized across clades, and which ones matter for protec- re-kindled efforts to find strategies for generating them tion, he’s reserving judgment. Lower levels of responses via vaccination.

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Designing Vaccines for Breadth Chiron’s gp140 immunogen, which should enter clin- All this leaves vaccine developers still operating ical trials this year); generating immunogens that largely in the dark in terms of how to design for max- bind to well-characterized, neutralizing broad Nabs, imal breadth. or which expose normally-hidden neutralizing epi- For T-cell-based vaccines, a common starting topes (fusion intermediates). point is to use the most conserved regions of HIV—first And for both B- and T-cell-based vaccines, sever- and foremost the gag gene (or protein), followed by al groups are combining immunogens from different pol and sometimes nef. These are usually derived from clades to create “cocktail” vaccines (for example, the a primary HIV isolate, sometimes selected for a particu- A/B/C candidate described in the article on p.7). In lar biological property such as use of the CCR5 recep- the future, cocktails could also contain mixtures of pri- tor, and/or origin in the geographic region where the mary isolates (or shape-based immunogens) plus con- vaccine will be used. In a variation on this theme, the sensus sequences. San Diego-based company Epimmune developed a candidate containing highly conserved epitopes (rather Vaccines and cross-clade responses than whole genes) from across the genome, selecting Last but far from least of the hurdles in developing further for those recognized by the most common HLA a broad HIV vaccine will be the challenge of determin- genotypes. This candidate recently entered Phase I clin- ing just how broadly protective a vaccine actually is. ical trials in the US and Botswana. Intended to help mobilize support for the steps this will More recently, researchers have begun looking at require, the new African AIDS Vaccine Programme doc- artificial sequences derived by computer analysis, rather ument emphasizes that clade-mismatched trials are a than actual circulating viruses, as sources of vaccine crucial part of the solution, and that politics must recog- strains. These are often consensus sequences, made by nize this reality. analyzing a set of sequences (say, primary isolates of The document also proposes some guidelines for clade C) and choosing the nucleotide found most com- decision-makers. For Phase I/II testing, it advocates monly at each position. Or they may be ancestral moving ahead with good candidates, regardless of the sequences, which represent the most likely common subtypes involved, for the sake of other benefits—such ancestor to a group of isolates. The rationale for these as building capacity for running trials, and for conduct- approaches—articulated in depth by Bette Korber ing scientific and ethics reviews; establishing dialogs (Science 296: 2354; 2002)—is that they minimize the among scientists, policy makers and communities; and genetic distance between the vaccine and the pool of gathering data on a vaccine’s ability to induce cross- circulating strains; in contrast, a primary isolate might clade responses in diverse populations. It also recom- be an “outlier,” genetically speaking, relative to many mends that decisions about efficacy trials should be other isolates of the same subtype. based on evidence of cross-reactivity between a candi- These sequences have not yet been incorporated date vaccine and the unmatched clades and/or CRFs into vaccines, although similarity to a clade consensus circulating in the trial population, along with a good sequence is sometimes used to help select primary iso- safety record from Phase I/II studies. lates for vaccine strains. But several groups are devel- From a scientific perspective, this testing is likely oping Env immunogens from consensus or ancestral to involve several different trial scenarios, says veteran sequences, and two teams reported at the 2003 Retro- vaccine developer Don Burke, who directs the Center virus conference that they seem to fold and function for Immunization Research at Johns Hopkins Univer- like real Env proteins, and to show potential as cross- sity. One is to compare the efficacy of vaccines based clade immunogens. Nancy Haigwood and Jim Mullins on a single clade in matched versus unmatched set- (University of Washington, Seattle) made two ‘proof- of- tings—the strategy Merck is likely to pursue with its concept’ ancestral clade B Env proteins, which so far clade B-based candidates. Another is to ask whether (as a DNA vaccine in rabbits) induce “reproducible but clade-matched vaccines work better than unmatched fairly low titers" of NAbs that cross-neutralize a clade C ones—studies that could be done with two different isolate, says Haigwood (poster 409). And researchers vaccines in one setting, or by testing one vaccine in a from Duke University, LANL and the University of Ala- region with multiple circulating clades, powering the bama reported on an immunogen made from a con- trial to detect efficacy in at least one of them. In all sensus Env sequence of all major clades, and which cases, getting an answer will require careful analysis of was recognized by several clade B and C antisera (from breakthrough infections. infected patients)—unlike Env from either clade, which Trial design, along with logistics, gets more com- reacted best with same-clade sera (poster 410). Both plicated for multi-clade candidates and/or sites with posters are available at www.retroconference.org/2003. multiple clades in circulation (see articles, pp. 17, 18), Other strategies for generating broad NAbs are since both these variables raise the number of volun- based on modifying the shape of the Env antigen, teers needed to identify statistically significant trends in rather than focusing on sequence (IAVI Report, Dec. vaccine cross-protection. But with the politics gradually 2002-Jan. 2003, p.1). These include development of aligning more closely with the science, and a growing native, trimeric structures; modification of Env to roster of potential trial sites in the picture, there should remove the more variable regions (exemplified by at least be a few less obstacles in the way. ◆

16 IAVI REPORT Studying HIV Diversity in Multi-Clade Regions

In the southernmost countries of Africa, where the AIDS epidemic is due almost exclusively to clade C HIV strains, the highly politicized issue of clades and vaccines has usually boiled down to decisions on whether to conduct trials only of candidates that closely match strains circulating locally, or to consider studies involving unmatched clades as well. But in other parts of Africa, HIV diversity is far higher—shifting the focus of the debate from the issue of getting a close match onto understanding how to induce the broadest possible protection. Here, we profile two clinical research sites—one in the highlands of Tanzania, the other in Cameroon’s capital city. Both are in countries with an especially broad diversity of circulationg HIV strains. And both have made tackling this diversity a cornerstone of their vaccine program.

ince the early days of the AIDS epidemic, the and the US military program, with support from the SMbeya region in southwestern Tanzania has been European Commission, to launch a systematic study Tanzania: At among the country’s hardest-hit areas. One glance at of double infection—basing their search on high- the map explains why: it’s located at two crossroads throughput methods they developed for detecting the Crossroads of the Trans-African highway—making Mbeya a hub HIV from two different clades in a single individual. for HIV spread along this major trucking route, which of Africa’s connects the capital city of Dar es Salaam with Dual infections in highly-exposed women Malawi and Zambia, and then further into southern- Looking for a population with very high expo- Major Clades most Africa. The region is also a bridge between the sure to HIV, the researchers focused on women eastern and southern African epidemics, with all three working in bars, local brew shops and guesthouses of the most globally important clades (A, C and D) in along the trucking route, many of whom live partly BY PATRICIA KAHN circulation—plus at least 40% unique recombinants from commercial sex work or casual partnerships. (see figure 3), the highest reported proportion any- Before launching a formal study, Oliver Hoffman—a where in the world. University of Munich physician who leads the team’s Today Mbeya is also home to a thriving cohort development—got to know the community HIV/AIDS clinical research effort that’s deeply through a baseline survey of 1,500 women, probing involved in studying this diversity, and is embedded their level of general knowledge about HIV/AIDS, in a regional program of HIV surveillance, prevention their risk behaviors, and in particular their move- and care. Under the umbrella of the Mbeya Medical ments within the region—key information for a Research Program (MMRP), a team of Tanzanians study involving 4 years of frequent follow-up in a working with University of Munich scientists, plus highly mobile group. The study got underway in collaborators and funders from the US, Europe and September 2000, recruiting a total of 600 Africa, is conducting two longitudinal studies with a women irrespective of HIV status—and total of nearly 4,000 participants. As the first of these resulting in a cohort with the staggering TANZANIA studies yields data showing that double HIV infec- prevalence of 68%, says MMRP coordi- tions are surprisingly common in the region, together nator Leonard Maboko. CD they are building capacity and laying groundwork for Now nearing the end of its third vaccine trials, which are likely to involve multi-clade year and with one more to go, the study AD “cocktail” candidates. (dubbed HISIS, for HIV SuperInfection A The MMRP has its roots in an intervention pro- Study), has a well-practiced rhythm. 17% gram launched 15 years ago at the Mbeya Referral Once every three months a mobile unit Hospital, through Tanzania’s Ministry of Health, the pulls into each of the 20 villages serving German Technical Cooperation and the University of as study nodes. The day before, volun- AC Munich. It gradually expanded to include broader teers and study staff meet for an infor- 23% surveillance and prevention activities, with research mal session on an AIDS-related topic, C entering the picture in 1995 when Michael Hoelscher such as living with HIV, or nutrition. By 43% (University of Munich), now the site’s scientific direc- 8 a.m. the next day, the mobile unit has tor, began subtyping HIV in blood samples collected set up a clinic with everything from D at antenatal clinics. By sequencing short regions of examination beds and tables to blood- the envelope gene, he found substantial proportions drawing equipment, and each woman of clades A, C and D. But when he did full genome goes through a full round of visits— Fig. 3: Proportion of different HIV sequencing in Francine McCutchan’s lab at the US interviews on HIV exposure and risk subtypes and recombinants in Military HIV Research Program (Maryland), it quickly behaviors, counseling sessions on pre- circulation, based on 30 full-length became clear that about half these samples were vention strategies, medical exams, blood HIV sequences. Samples were actually unique recombinants—suggesting that dou- draws and vaginal swabs and lavage. drawn from low-risk adults and ble infection, which had been documented in only a Acute diseases, including STIs and blood banks in the Mbeya region. few cases worldwide, might actually be relatively fre- opportunistic infections, are treated on Source: Francine McCutchan quent in the region. And that, in turn, led Hoelscher the spot, and trial staff provide medi-

continued on 18 ▼ MAY–AUGUST 2003 17 ▼ TANZANIA continued from 17

cines and condoms. A village contact person, who the CODE (COhort DEvelopment) study, a longitudi- keeps monthly tabs on volunteers between clinic vis- nal study of 3,100 uninfected and HIV-infected peo- its, searches out those who don’t appear. By noon, ple from a rural and an urban population, to analyze the field work is done. a variety of parameters important for trials—such as So far it’s clear that many of the women are HIV incidence, major risk factors, knowledge and atti- doubly infected, says Hoelscher, although immense tudes about vaccines, the course of HIV infection technical difficulties in tracking the two strains over (including CD4 and viral load setpoints) and overall time mean that it’s not yet known whether most of disease profile of the population. It will also compare them are true superinfections (i.e., where a second strategies for enrollment and follow-up. Like HISIS, infection happened after the first one was estab- the project offers free basic medical care, including lished) or occurred simultaneously (see article, p. 1). treatment for opportunistic infections, malaria and TB. But there are a few cases of “incident superinfec- Enrollment was complete within six months, using tions”—those which happened during the course of three different methods, and resulted in cohorts with the study, where the researchers are more certain of an HIV prevalence at uptake of 19.6% in the urban the timing. group and 14.4% in the rural one. Once they can definitively characterize enough A second milestone was the opening of a new infections, there are several key issues to tackle, such 12-room laboratory building next to the Mbeya as comparing cross-clade immune responses among Referral Hospital, which processes up to 60 samples doubly-infected women, women with single infec- a day (from HISIS and CODE) and where immuno- tions, and the few who remain uninfected despite logical methods like Elispot and flow cytometry are intense HIV exposure. The goal: insight into corre- now up and running. lates of protection against infection and—for those As they work towards vaccine trials, MMRP also already infected—against HIV of a second clade. At aims to establish anti-retroviral therapy (ART) treat- the same time, the Mbeya site is establishing the ment, not just for trial participants but for the broader immunological methods and laboratory capacity it community. Plans call for an ART feasibilility study will need for vaccine trials. next year to examine different regimens, along with supply and monitoring systems. The hope is to move Towards vaccine trials: cohorts, and a new lab quickly from Phase I studies in 2004 or 2005 (with With HISIS in full gear, last year brought two several European and multi-clade candidates in dis- other big steps for the MMRP. First was the launch of cussion) into larger trials. ◆

ith its richly diverse population of some 200 the site alongside CRESAR director Col. Eitel Cameroon: Wethnic groups speaking 80 different lan- Mpoudi-Ngole. Cameroon, like the rest of west and guages, Cameroon is often seen as “Africa in minia- central Africa, has a less severe HIV epidemic than “Final Common ture.” Sadly, that description also applies to its AIDS other sub-Saharan countries (although solid data on epidemic: the country is home to virtually every HIV prevalence and incidence is scarce). So the Pathway” for a known HIV subtype, plus the CRF02_AG recombi- feasibility of vaccine efficacy studies will become nant—second only to clade C in numbers of infec- clear once the Yaoundé team collects the needed Global Vaccine tions worldwide, according to UNAIDS—and a vast data on incidence (the rate of new infections)—a array of unique recombinants. key determinant of how many volunteers are need- That makes Cameroon among the most com- ed to measure efficacy. BY PATRICIA KAHN plex places in the world for an HIV vaccine—as well as one of the few where vaccine efficacy can The site’s beginnings be tested against a global range of subtypes and Cameroon’s vaccine program stems from a recombinants. And that’s just what the country is collaboration started in 1997 between Don Burke, gearing up to do, in partnership with the Johns Birx’ predecessor (now director of the Center for Hopkins School of Public Health and the US Immunization Research at Hopkins) and Mpoudi, Military HIV Research Program, as it builds the then the well-known head of the national AIDS small Center for Military Health Research (CRESAR) control program, whose openness and leadership in the capital city of Yaoundé into the hub of a on AIDS earned him the nickname Col. SIDA (the national HIV vaccine program. “Cameroon can be a French acronym for AIDS). With evidence emerg- final common pathway for testing a global vaccine,” ing that equatorial Africa has the highest HIV says Debbie Birx, who leads the US Military HIV diversity worldwide, the project’s initial focus was program. “If a vaccine shows efficacy here, it’s very on understanding how this diversity is generated, likely to work around the world.” and on expanding surveillance and prevention Whether the country can play that role will efforts, especially in rural villages and in hinge largely on success in identifying populations Yaoundé’s military garrisons. with high enough incidence to support vaccine tri- As work progressed on establishing VCT, train- als, says Nathan Wolfe (Johns Hopkins), who heads ing staff and building laboratory capacity, it became

18 IAVI REPORT CAMEROON IAVI Report clear that Cameroon offered G agricultural plantations with CRF11px F2 many advantages for vac- 25,000 workers and family CRF11_cpx EDITOR cine work. Chief among members. Both collabora- Patricia Kahn A2 them were strong political tions offer the advantages SENIOR WRITER support from one of the D of stable, mixed-gender Emily Bass few stable governments in a populations and established WRITER troubled region, and rela- health care infrastructures. Mark Boaz CRFO2_AG tively good infrastructure. A third potential group ASSISTANT MANAGING EDITOR Michael Hariton Another was the finding 47% is high-risk women, where Novel Strains DESIGN AND LAYOUT that about half of all infec- 33% Cameroon can build on its DESIGNdeFrancesco.com tions in the country experience from two Phase PRINTER involved the CRF02_AG III microbicide trials in the West End Graphics recombinant—an important mid- to late 1990’s—the FOUNDING EDITOR strain in west/central Africa source of some of the little David Gold (but rare elsewhere), and prevalence and incidence one that a vaccine for the Fig. 4: Proportion of different HIV data in Cameroon, says The IAVI Report is published bi-monthly by the International region must protect against. subtypes and recombinants in circu- Tamoufe, who served as lation, based on 30 full-length HIV AIDS Vaccine Initiative. To obtain a At the same time, new part- study coordinator of both subscription to the IAVI Report, or sequences. Samples were drawn trials. In the first one, which for reprint permission, send name, ners added support to the from HIV-infected people in rural growing effort: besides the enrolled 1,292 sex workers, address and request by e-mail to: villages, blood banks, hospitalized [email protected]; by fax to: US Military Program, these adults and STD clinics. HIV prevalence at uptake 1-212-847-1112; by mail: IAVI, included the US NIH screening was 16.8%, and 110 William Street, 27th floor, Source: Francine McCutchan Fogarty International Center, the trial found an incidence New York, NY 10038, USA. Centers for Disease Control of 6.6 infections per 100 All rights reserved. and Naval Health Research person-years. The second ©2003 Center, and Cameroon’s Ministry of Health. And in study, involving 1,251 high-risk women (excluding 2003, a collaboration with Merck on cross-clade sex workers), found 11% prevalence at uptake and immunity in HIV-infected people provided a first an incidence of 1.3 infections per 100 person-years. foray into vaccine-related work. Those trials also offered some valuable lessons on retaining volunteers, Tamoufe adds, which paid off Cohort pre-development in far less loss to follow-up in the second one (1.5% As the team now moves into finding potential compared with 20% over two years). Plans are now groups for vaccine studies, the military remains a underway to recruit 1,000 HIV-negative sex workers major focus. Its 60,000 members and their families from around Yaoundé for a two-year study similar to offer a stable population and an established system those in the military and plantation populations. The of free health care for active military personnel. Yaoundé team will also expand laboratory capacity IAVI is a scientific organization found- Surveillance work so far, which has involved about for evaluating immune responses to vaccines. ed in 1996 whose mission is to ensure 2,900 enlisted active-duty forces in Yaoundé, found While no specific candidate is “on deck” for the the development of safe, effective, accessible, preventive HIV vaccines about 10% HIV prevalence. Over the coming two site, the researchers envision a few possible scenar- for use throughout the world. years a new longitudinal study of 1,000 uninfected ios. One is to test vaccines against the CRF02_AG IAVI focuses on four key areas: people will gather incidence data, along with infor- recombinant. Another is to evaluate candidates that Accelerating scientific progress; edu- mation on knowledge and attitudes about vaccines. have already shown efficacy in clade-matched set- cation and advocacy; ensuring vac- And in a sign of the growing priority of HIV for tings, pitting them against the full onslaught of cine access and creating a more sup- portive environment for industrial African militaries, the Yaoundé team is collaborating Cameroon’s HIV’s diversity. involvement in HIV vaccine de- with three neighboring countries—Chad, Congo- velopment. Brazzaville and Gabon—to establish VCT, surveil- Studies on emerging diseases lance and staff training. Alongside its HIV work, the Yaoundé group IAVI is a UNAIDS Collaborating Centre. Its major financial support- Similar studies are also beginning at several uses its field experience in Cameroon’s remote ers include the Bill & Melinda Gates agricultural plantations. HIV risk in these popula- regions for another line of research: exploring the Foundation; the Rockefeller Sloan tions, says Ubald Tamoufe (program officer and origins of HIV and other viral diseases that enter and Starr foundations; the World coordinator of the cohort work) comes from “having human populations from non-human primates Bank; BD (Becton, Dickinson & Co.); thousands of low-income workers in isolated areas, (NHP). Central Africa has a huge diversity of wild and the governments of Canada, The Netherlands, United Kingdom, usually far from their home villages,” and from the NHP, along with forest-dwelling populations who United States, Ireland, Denmark, commercial sex trade that flourishes around the bi- hunt, butcher and eat them. But jump of a retrovirus Norway and Sweden. IAVI also has monthly paydays. One study will take place at a rub- into humans in a natural setting has never been doc- received support from the Vincent P. ber plantation of 6,000 workers and their 21,000 fam- umented—until the Yaoundé group’s recent demon- Belotsky Jr. Foundation, Cruisaid, the Elton John AIDS Foundation, James ily members in the Southern Province, where data stration of crossover by the simian foamy virus B. Pendleton Charitable Trust, Until from antenatal clinics suggest about 10% HIV preva- (SFV), based on both antibodies to viral proteins (in There’s a Cure Foundation, and other lence in adults. Another project involves the nearly 1% of 1,100 people sampled) and the pres- generous corporate, foundation and Cameroon Development Corporation, a group of ence of SFV DNA sequences. ◆ individual donors worldwide.

MAY–AUGUST 2003 19 vaccine

AUSTRALIAN CONSORTIUM LAUNCHES DNA-FOWLPOX TRIAL FIRST HIV VACCINE On 17 June the first volunteer was vaccinated in an Australian Phase I/IIa trial of a prime-boost TRIALS GET GREEN strategy that combines DNA- and fowlpox-based HIV vaccines. The trial, which will enroll 24 LIGHT IN SOUTH AFRICA volunteers, is sponsored by Australia’s National Center in HIV and Clinical On 6 June 2003, the South African Research and will be run at St Vincent’s Hospital (Sydney.) Medicines Control Council approved Fowlpox belongs to the same bird virus family as modified vaccinia Ankara virus (MVA) the country’s first HIV vaccine trial, and canarypox, which have also been developed as HIV vaccine platforms. Volunteers will and the first of a vaccine receive two 1 mg injections (0 and 4 weeks) of a DNA vaccine containing gag, rev, tat, vpu and based on clade C. The Phase I study truncated env genes from HIV-1 clade B, followed by an HIV-fowlbox boost with similar genes (HVTN 040) will test the safety and at week 8. It is the first study to test a DNA-fowlpox vaccine regimen in HIV-negative volun- immunogenicity of a candidate based teers. A therapeutic trial of fowlpox vaccine also containing IFN-gamma, an immune-modulating on a Venezuelan equine encephalitis cytokine, was conducted in HIV-positive individuals in 2002. Because participants in this study (VEE) vector containing HIV-gag from were on antiretroviral therapy, which reduces viral load, it was difficult to determine the bene- a South African clade C isolate. Clade fits of the vaccine-cytokine combination, although a follow-up study is now underway to assess C strains cause nearly all infections in control of viral load off therapy. Another preventive trial of similar vaccines containing HIV South Africa, and about 47% world- genes from the CFR_01AE recombinant, Thailand’s main circulating strain, is planned for wide. Thailand later this year. The vaccine was developed by Bob Johnston (University of North Carolina, Chapel Hill) and AlphaVax, NEW PROPOSAL FOR A GLOBAL AIDS VACCINE ENTERPRISE a biotechnology company (Research Triangle Park, North Carolina). The In an article titled, “The Need for a Global Vaccine Enterprise” (Science 300:2039;2003), 24 of the VEE vector is derived from a vaccine world’s leading vaccine researchers and advocates called for a major effort to expand and restructure designed to protect humans against a the search for an AIDS vaccine. Richard Klausner, Executive Director of the Bill & Melinda Gates South American virus that often caus- Foundation global health program, was lead author on the paper. es disease in horses. It targets mainly The proposal calls for a coordinated effort similar to the Human Genome Project, which dendritic cells, which play a key role divided and assigned roles to a diverse group of scientists to meet its goal. Similarly, the new vac- in inducing immune responses. cine enterprise would map out the entire “grid” of potential vaccine approaches. It would then In August, the MCC approved a assign tasks, allocate funds and ensure that participating teams of researchers collectively covered second Phase I trial (IAVI 011). This the entire grid. To accomplish this, the enterprise would establish of new Vaccine Development IAVI-sponsored study will test the Centers (VDCs), which could be actual institutes or virtual collaborations. The paper pointed out safety and immunogenicity of that VDCs could include efforts sponsored by existing funders, such as the National Institutes of HIVA.MVA, a candidate based on Health, IAVI and the European Union—all of whom were signatories to the article—could partici- modified vaccinia Ankara virus (MVA) pate in the enterprise. and containing a clade A gag consen- The VDCs would be part of an interconnected network that also includes manufacturing facili- sus sequence plus a string of CTL epi- ties, central laboratories, and clinical trial sites capable of enrolling a projected figure of 35,000 vol- topes from the gag, pol, nef and env unteers into clinical studies each year. The paper did not specify funding requirements or sources genes. It was designed by scientists at for this massive endeavor. In August, major vaccine stakeholders gathered at a Washington, D.C. the University of Nairobi and meeting hosted by the Bill & Melinda Gates Foundation and formed six working groups (product University of Oxford. discovery, product development, standardization of assays, manufacturing, clinical trials capacity, and The two trials will be conducted international regulatory and licensing issues) which will contribute to strategic framework. separately, but at the same South African sites: one in Soweto, the other AFRICAN AIDS VACCINE PROGRAMME MEETS IN ETHIOPIA in Durban. HVTN 040 will evaluate three dif- On 13-16 June nearly 200 scientists, trial investigators, national authorities, and community represen- ferent vaccine doses in a total of 96 tatives from Africa and around the world gathered for the second meeting of the African AIDS volunteers. Testing will begin in the Vaccine Programme, “Strategies for the Development of HIV Vaccine Trial Sites in Africa: Challenges US and proceed in South Africa once and Opportunities.” The meeting’s focus reflected “anticipation of a new wave of candidate HIV safety is established. vaccines based on strains prevalent in Africa,” said Jose Esparza, head of the WHO-UNAIDS vaccine IAVI 011 will take place at two program. AAVP’s primary funder. The meeting included intensive discussion of ethical issues, such European sites along with Durban as standard of care for trial participants and communities, and enrollment of women and adoles- and Soweto, and enroll 111 volun- cents, there were also updates on AAVP activities. In 2002, AAVP completed an assessment of the teers. HIVA.MVA is being studied needs and capacities of African ethical review committees, developed a consensus document on alone and as part of a prime-boost clade (see article, p.1) and established a working group on community issues. Looking ahead, AAVP combination with a DNA vaccine in plans to develop a template for national vaccine plans. Kenya, Uganda and the UK.

20 IAVI REPORT