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Symposia & Plenary Reviews: 13th CROI
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The 13th CROI: researchers and clinicians mark the 10th anniversary of HAART by measuring current deficiencies and celebrating new advances
Written for NATAP by David Margolis, MD
University of North Carolina
13th CROI, Feb 2006, Denver
Symposia and Plenary sessions:
In vitro cultivation of hepatitis C virus
The origin of AIDS
A genome that prevent HIV-infection
Can circumcision be used for HIV prevention in developing world
Topical microbicides development to prevent HIV transmission
Lessons Learned from In Vitro Cultivation of Hepatitis C:
Takaji Wakita of the Tokyo Institute for Neuroscience discussed the work of his groups and his collaborators in the development of a robust cell culture model for hepatitis C virus (HCV) infection. Heretofore, the lack of such a system has hampered the study of the HCV life cycle and the testing of effective HCV antivirals. As is well known HCV, a plus-strand RNA viruses, is a major cause of hepatitis worldwide. 170 million people are chronically infected, leading to chronic liver diseases, cirrhosis and hepatocellular carcinoma. Previous work had established partial expression of some HCV gene after they were transfected (articifically introduced) into the Huh-7 liver cell line in continuous culture. This was not sufficient for full study and testing of HCV.
Wakita's group cloned a HCV strain, called JFH-1,from a patient with fulminant hepatitis. This was a genotype 2a virus. After this viral genome was introduced into selected, permissive cell lines, viral RNA efficiently replicated. Importantly, for the first time viral particles were secreted into culture medium. Particles have a density of about 1.15-1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. These viral particles were infectious for both other cultured cells and for a chimpanzee, the only animal model in which HCV replicates.
However, cirrhosis and liver cancer are typically associated with genotype 1 HCV, which is more prevalent and relatively resistant to IFN therapy. More recently (PNAS Feb. 2004) the group reported production of infectious genotype 1a HCV in cells transfected with synthetic RNA derived from a prototype virus (H77-S). Viral proteins accumulated more slowly in H77-S transfected cells than in cells transfected with genotype 2a (JFH-1) RNA, but substantially more H77-S RNA was secreted into supernatant fluids. Most of the genotype 1a RNA was noninfectious, but some infectious particles can be recovered. Again infection is blocked by CD81 antibody.
This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies, and will benefit vaccine discovery programs.
How did AIDS get here?:
Paul Sharp from the Univ. of Nottingham began the plenary session on the conference's second day with a review of the primate origins of HIV. Evidence suggests that HIV appeared in Africa about 100 years ago. There are four chimpanzees subspecies in Africa that have evolved from a common ancestor, and as SIV exists in only two of these species, it can be shown that the primate SIV progenitor of HIV entered chimpanzees prior to the divergence of these two, the pan troglodytes and pan schweinfurthii.
Non-invasive sampling of urine and feces from wild chimps has enabled the identification of when and how SIV spread to man and became HIV. A chimpanzee virus, SIVcpz, spread into man during at least 3 (and perhaps as many as 7) different animal-to-human transmission events. These temporally separate events resulted in the genetically distinct classes of HIV known as clades: clade A and B in the earliest transmission event, M and N in the next, and O most last. Studies are now underway to understand the adaptation of SIV to replicating as HIV in humans, in the hope of better understanding AIDS pathogenesis.
A genome armed against HIV
Steve O'Brien of the NCI reviewed the understanding gained by his group and others over the past decade on human genes which either reduce or increase the transmission of HIV and the progression of HIV infection to AIDS. The best known protective human genetic trait is the "delta 32" deletion in the chemokine receptor CCR5. Along with the CD4 receptor, HIV requires a chemokine co-receptor, most often CCR5 to enter cells. About 1% of white Northern Europeans carry a 32-base pair deletion (termed delta 32) of CCR5 on both copies of their CCR5 genes that does not impair immune function but prevents infection by CCR5-using strains of HIV. Phylogenenetic have shown that this mutation occurred after migration of humans from Africa to Northern Europe. Linkage disequilibrium analysis suggests that the delta 32 event happened about 700 yrs ago, around the time of the Black Death, an epidemic of plague in Europe. Studies in transgenic mice in whom the CCR5 gene has been disrupted and expression of CCR5 is "knocked out" have shown that yersinia pestis, the plague bacteria, replicates with 30-fold lower efficiency in the macrophages of these mice. So it is argued that the Black Death exerted a selective pressure on Northern Europeans, resulting in some who carry a gene that generations later provides protection against a second, viral, plague.
Since the discovery of the delta 32 CCR5 mutation, at least 4 other mutations have been described that affect CCR5 receptor, and several other human leukocyte antigen (HLA) markers have been found that regulate T cell function and either increase or decrease risk of acquisition or progression of HIV. Six variations in genes related to the innate immune system (primitive immune responses not enacted by T or B cells) have also been described that alter the risk of HIV infection or disease.
O'Brien and others have searched for relationships between known human genotypes or haplotypes for relationships to the risk of HAART failure, post-HAART survival, and HAART-related toxicities. Many geneic markers have been found that have a statistical relationship to increased or decreased risk of progression to AIDS after HAART. However, thus far, these relationships are very complex, and it is not yet possible to guide clinical practice by genetic screening. However, this is certainly the goal of this work in the future.
O'Brien's group is now studying 332 single nucleotide polymorphisms (single site DNA variations in the human genome) within 8 known ARGs in a sample of over 2600 HIV-infected patients. The group hopes to construct a genetic map to predict risk of HIV disease and complications. The group is working out methods and controls for this endeavor, but O'Brien is optimistic that a genome-wide scan for useful ARGs can be performed.
Overall, various genetic markers, or AIDS restriction genes (ARGs) can be shown to increase the relative risk (>2.0) of morbidity and mortality in HIV infection. That is to say that those with such genes have a 2-fold increased risk of morbidity or mortality despite HAART. However, only about 10% of the total risk is explained by ARGs. This is similar to studies attempting to predict the risk of lung cancer, in which factors such as smoking increase the risk of lung cancer greatly, despite the fact that most smokers do not get lung cancer.
Surprising benefit from an unkind cut
Tom Quinn of Hopkins and the NIAID reviewed the maturing data that show that male circumcision confers protection against HIV infection. The biological basis for the protective effect of circumcision is not clear, but the foreskin is not keratinized and heavily enriched in dendritic cells, making it a potentially advantageous entry site for the virus. Quinn reported a number of epidemiological facts that suggested circumcision protects against HIV infection:
- the relative risk of HIV infection was 0.56 in circumcised men.
- the relative protective effect is even greater in men with high-risk demographics, in whom the relative risk in circumcised high-risk men for acquisition of HIV was only 0.29.
- Countries in which >80% of the men are circumcised have lower HIV prevalence than countries in which <20% of the men are circumcised.
- In a cohort of HIV sero-discordant partners in Rakai, none of 50 circumcised men acquired HIV, irregardless of their partner's viral load.
- Kenyan truck drivers are 2.5-fold more likely to be HIV infected if uncircumcised
- In circumcised Indian men presenting to STD clinics, 88% did not acquire HIV despite the fact that their acquisition rate of HSV, syphilis and GC rates were the same as uncircumcised men
This hypothesis has been preliminarily tested in a South African study in which sterile circumcision was provided to a cohort of at-risk men. A 75% reduction in HIV acquisition risk was reported, with 58 adverse events (3.8%) related to the procedure. The study's conclusions are limited, as follow-up is so far short-term, protection was incomplete, and it is unclear if these results will be generalizable outside of southern Africa. Two other studies are expected to be reported in the summer of 2006.
Quinn suggested that as a public health measure safe circumcision should be made available now, and further data developed. In a model of the effect of circumcision, a 50% reduction in relative HIV acquisition risk would cut HIV incidence from 0.8 per 100 person yrs to 0.4 in women, in addition to the 50% protective effect in men. But if 30% of men believed that sex was now safe and stopped using condoms, the beneficial effect would be abrogated.
The procedure was estimated to cost $69 in Rakai, and therefore would cost 1000 to 3000$ per HIV case prevented. This cost is similar to that of nevirapine prophylaxis for pregnant mothers, in which the cost of an averted infection is estimated to be $2500. Quinn felt that overall circumcision could reduce HIV and STD transmission, reduce cervical cancer, balanitis, and penile cancer. He suggested that preparations be made to offer safe surgery and educate to maintain low-risk behavior.
Topical Microbicides: the real front line of HIV prevention
Topical microbicides, preparations able to kill HIV on contact and prevent infection, are a critical complement to vaccines and other prevention strategies. They can protect women, for example, who cannot protect themselves by other means. Gels are in development and testing, but a plan is needed for future approaches if the results of trials expected in 2006-07 are not encouraging.
John Moore of Cornell provided an excellent overview of what is needed in the field. A microbicide must be safe, effective, affordable, and acceptable. To accomplish this, a preparation must not damage the epithelium, not alter bacterial flora, and must not cause inflammation (as has led to increased HIV transmission following the use of past microbicides).
Several HIV reverse transcriptase inhibitors, AZT-like drugs, are in development. Among them are TMC 120, UC781, and tenofovir. Reagents that block chemokine receptors such as cyanovirin, psc-rantes, and others are under study. In the SIV model, PSC-Rantes formulated in a gel protected monkeys from vaginal and rectal challenge, and did not induce inflammation. The "triple-therapy" cocktail of compound 167 (a Merck CCR5 inhibitor), combined with BMS 387806 (a Bristol CD4 blocking molecule), and c52L (a T-20 like fusion inhibitor molecule produced in engineered bacterial) were tested by Ron Veazey in macaques treated with progesterone to enhance their susceptibility to infection. 8 of 10, 6 of 8, and 3 of 5 monkeys were protected by each agent. 16 of 20 given two agents , all 3 of 3 given all 3 agents were protected from high-dose HIV mucosal challenge. In a delayed challenge SHIV (a SIV-HIV hybrid) experiment, microbicides were 80% effective 30 minutes after application, but only 33% protective at 12 hrs. 2 of 5 animals were protected against 5 daily high-dose challenges by daily triple application.
In addition to being effective, microbicides must be affordable. Moore estimated that a product could only cost $0.25-0.50/application. This is a challenge as the amount of compound needed to be effective varied from as little as 3 mg/application to 50 mg. Compounds must reach the millimolar range to protect in the monkey model, which is several-fold higher than in vitro models. In general, receptor blockers need to achieve higher concentrations than antivirals.
In the real world, the challenges will be great. Viral load (innoculum) is likely to be high in many settings as transmission is associated with other active STDs. A product requiring daily application is unlikely to achieve perfect adherence. The cheapest candidates as polyanion detergent-like antivirals at <0.01$ dose, antiviral drugs come in at $0.01-0.10/dose, and small peptides or molecules are the Rolls Royce at $1-20/dose. An interesting idea that Moore mentioned was Dean Hamer's proposal to engineer live bacterial flora to secrete antivirals, a clever and interesting idea that would need proof of concept in a model system, and acceptance by the public.
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