Simon Collins, HIV i-Base

Four of the six oral presentations in the ‘Treatment is prevention: the proof is here’ session reported on the results from HPTN 052. [1] This study had been unblinded four years earlier than planned due to a review by the data and safety monitoring board (DSMB), with all patients now being offered active treatment.

In summary, HIV-positive people on treatment had a 96% reduction in sexual transmission to their HIV-negative partners if they started ARVs with a CD4 count of 350-550 cells/mm3 compared to waiting until it dropped below 250 cells/mm3. As with all prevention studies, condoms, testing and intensive counselling was included throughout the study.

The main study results were presented by Myron Cohen from University of North Carolina. [2]

HPTN 052 screened over 10,000 couples in order to randomise 1763 HIV-positive people with CD4 counts 350-550 to either immediate or delayed HIV treatment (CD4 confirmed <250 or an AIDS-defining illness). Screening failure was mostly due to CD4 or other criteria in the positive partner, but 300 couples were already both HIV-positive. This was an international study predominantly recruiting in Africa (Botswana, Kenya, South Africa and Zimbabwe, n=954), Asia (India and Thailand, n=531) and Latin America (Brazil, n=276). Men and women were equally distributed as the positive partners. Median baseline CD4 count was 436 cells/mm3 (IQR 365-522) and viral load was approximately 25,000 copies/mL (IQR 6,000-80,000) respectively.

This was generally a low risk population with only 6-8% reporting recent unprotected sex and only 16% aged 18-25 years (~ 60% were 25-40 years and ~20% > 40 years).

The primary transmission endpoint was the prevention of virologically linked transmissions with a primary clinical endpoint of WHO Stage 4 events (including pulmonary TB, severe bacterial infections and death).

Transmission events (n=39) occurred significantly less frequently in the immediate (n=4) compared to the deferred (n=35) treatment arms, p<0.0001. Of these, only 28/39 were linked transmissions (within the couple) with 1 case in the immediate arm vs 27 cases in the deferred arm, p=0.001 (see below for details). Eleven transmissions were either unlinked or undetermined. This translated to incidence rates of linked transmission of 0.1 (95%CI 0.00-0.04) vs 1.7 (95%CI 1.1-2.5) per 100 person years respectively over a median follow-up of 1.7 years.

The single transmission in the immediate treatment arm was detected at the first follow-up visit. However viral diversification analysis estimated that transmission occurred prior to the positive partner initiating treatment (baseline 87,000 viral load) or certainly prior to viral suppression to <400 copies/mL which was recorded at day 28.

Other transmission risk factors were similar between arms, including rates of STIs (low at <5% in both index and partner at baseline and during the study), sexual activity (approximately 70%) and condom use (>90% by all throughout).

Viral suppression (<400 copies/mL) was maintained by >90% of participants in the immediate arm. There was a slow increase in this percentage over time in the deferred arm as people started treatment (from <10% over the first year, 20% by month 24 and increasing to 50% at month 45, though with much fewer patients). The median viral load closest to the time of transmission in the deferred arm was considerable at 80,000 copies/mL but had a wide range from 600 to 630,000 copies/mL.

In multivariate analysis, treatment was the strongest protective effect [HR=0.04, 95%CI 0.01-0.28] compared to condom use [HR=0.33; 95%CI 0.12-0.91]. Factors associated with increased transmission included baseline viral load [per log increment: HR 2.84, 95%CI 1.51-5.41] and baseline CD4 count [per 100 count increment: HR 1.24 95%CI 1.00-1.54].

The second presentation by Susan Eshelman from Johns Hopkins University School of Medicine focused on the analysis of linked transmission. [3] This included a helpful introduction to the three types of phylogenetic analyses used: phylogenetic analysis of HIV pol sequences using population sequencing, and statistical analysis of genetic distances from pol sequence pairs for the clearest cases (n=26), and phylogenetic analysis of env sequences obtained by deep sequencing for more complex cases (n=12). Together these provided a high level of reliability for indentifying whether the source of new infections was the HIV-positive partner or whether this was from another partner.

Transmissions in previous serodifferent couple studies have been from outside the main relationship in 25-50% cases.

The deep sequencing (‘ultradeep pyrosequencing’) supported linked two further cases and confirmed non-linkage for seven others (4 in the immediate and 3 in the deferred arm). Three cases remained unidentified (all in the deferred arm). Transmission linkage was not associated with index partner gender or CD4 count, geographical regions or time on study but this was strongly associated with study group and number of sexual partners in the three months prior to new seroconversions.

Results on the clinical outcomes for the HIV-positive participants in HPTN 052 were presented by Mina Hosseinipour from the UNC Project, Malawi. [4]

Results comparing the two groups were presented as ITT analyses and included the approximate 20% (184/877) people randomised to the deferred arm who started treatment during follow-up.

Over two years, median CD4 counts increased from 442 to 662 cells/mm3 in the immediate group compared to reducing from 428 to 390 cells/mm3 in the deferred arm. These differences are blunted as the deferred arm includes the response for the 20% people who started treatment. Viral suppression was achieved and maintained <400 copies/mL by >90% of the immediate arm. Less than 5% of patients on immediate treatment experienced virological failure during follow-up with most (60%) of these switching to a second-line combination.

The decision to start treatment in the deferred arm was driven by CD4 declines in 75% of cases. This occurred at a median count of 225 cells/mm3 (IQR 199–247), with 25% over people not starting until their CD4 count was less than 200. Treatment in both arms was predominantly AZT/3TC/efavirenz (70%) with ~10% using AZT/3TC/atazanavir, and ~10% using tenofovir/3TC/efavirenz. CD4 responses in the deferred arm were similar to absolute increase in the immediate treatment arm but remained significantly lower at all timepoints, reflecting the lower counts when starting treatment. Although there are fewer patients with longer duration of follow-up in the deferred arm, other studies have reported that baseline CD4 correlates with CD4 response after treatment.

The analysis by geographical region reported that about 80% of both the linked and unlinked transmission events occurred in African sites, likely a reflection of the higher background population prevalence rates in those countries, although the researchers highlighted higher rates of unprotected sex in the last week (by 9% vs 4% of African vs non-African) and higher sexual activity (>3 acts). However, baseline CD4 count, viral load and adjusted time to initiation, median adherence (99%) and treatment responses were similar between African and Asian sites.

Further details on clinical outcomes were presented by Beatriz Grinsztejn from the Oswaldo Cruz Foundation, Rio de Janeiro. [5]

Primary clinical events occurred at least once in 105 participants over 3304 person-years (PY) of follow-up; 40 in the immediate arm (2.4/100PY) and 65 in the delayed arm (4.0/100PY), hazard ratio (HR) 0.59, 95% CI: (0.40, 0.88), p=0.01. Seventeen people experienced more than one event. Time to event was significantly shorter in the deferred arm (HR 0.6, 95%CI 0.4, 0.9, p=0.01)

CD4 counts were significantly higher in the immediate arm vs deferred arms for all clinical endpoints (TB 518 vs 316; bacterial infection (mainly pneumonia) 551 vs 337 and death 476 vs 372 cells/mm3 respectively).

The between-arm difference was driven by extrapulmonary tuberculosis with 3 cases in the immediate versus 17 cases in deferred arms (p< 0.002). These were peripheral lymph nodes (2 vs 4), abdominal (0 vs 8), pleural (1 vs 3), skeletal (0 vs 1) and meningeal (0 vs 1). Isoniazid prophylaxis was only being used by 4% of patients in each arm at baseline.

Of the 23 deaths observed, there was no difference between arms: 10 in the immediate arm and 13 in the delayed arm [HR 0.77, 95% CI: (0.34, 1.76), NS p>0.5]. Causes of death were similar, but with 3 vs 3 suicides; 0 vs 2 accidents; and 3 vs 6 unknown).

Adverse events potentially related to ART were reported in 24% of subjects in the immediate arm and 5% in the delayed arm, but severe or life-threatening events occurred equally in 14% of each group and grade 4 lab events were also similar (in <1-2% of participants).

Since the DSMB recommendation in April 2011, all participants in the deferred arm have been offered HAART based on the strength of the study findings. This study continues to monitor all participants and results will add to clinical data from use of earlier vs later treatment in people with CD4 counts >350 cells/mm3.

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These results add to research that not only correlates viral load with risk of sexual transmission but specifically demonstrates a protective impact with treatment. The two cases of transmission in the early treatment arm (a second was discussed during the presentation) were both detected at the beginning of the study prior to the positive person reaching suppressed viraemia <400 copies/mL.

The fewer clinical endpoints from earlier treatment for the HIV-positive partners in this study are important but were driven by extrapulmonary TB. This clinical difference has significance for people in geographical regions where this study was run, but this aspect of the results was unexpected and has yet to be explained. A more generalisable benefit to people in Western countries is probably the reduced CD4 response in the deferred arm and this needs to be supported by longer follow-up. The ongoing START study will report on whether clinical benefits result from earlier treatment in Western countries.

It would be interesting to model the potential number of transmissions that have already been prevented over the last ten years from the seven million people globally on HAART. Given the financial constraints of access to treatment the additional impact on prevention should be included in future cost: benefit analysis.

The results from HPTN 052 clearly support offering an option for treatment to HIV-positive people who have HIV negative partners. This has been included in UK (BHIVA) guidelines for many years.

When access to treatment is limited with a waiting list using CD4 upper cut-offs to access treatment, those with the most severe medical should clearly be prioritised. However, the majority of the nine million people currently identified by UNAIDS and WHO analyses as requiring but not yet able to access treatment are likely to be undiagnosed. Broadening the CD4 criteria for access to treatment as prevention at higher CD4 counts is unlikely to directly deny access to treatment for more advanced patients.

It was unfortunate that a WHO guideline due to be launched at the IAS meeting, that included the recommendation for treatment people with CD4 counts higher than 350 and who have HIV-negative partners, based on the HPTN 052 study was withdrawn at the last minute. [6]

Although printed for a launch at the conference there is concern that while the scientific evidence is clear – and this should be the focus for clinical guidelines – practical issues on implementation have stalled their release perhaps under pressure from prominent WHO funders. It is difficult to understand how such a useful document that included broad community consultation and approval to the stage of print would have been retracted at such a late stage. WHO say this is due to a need to make “small modifications” and “to review their modeling data they used to inform investment structures”. The timeline for these changes are 2-3 months.

This plausibility for intervention from outside the extensive WHO guidelines writing and advisory panels is supported by an article in Science magazine that names the Gates Foundation specifically related to their interest in the latest PrEP results also being included. [7]

References

Unless stated otherwise, all references are the Programme and Abstracts of the 16th IAS Conference on HIV Pathogenesis, Treatment and Prevention, 17–20 July 2011, Rome.

1. Treatment is prevention: the proof is here. Oral abstract session: Monday 4.30-6.00pm.
2. Cohen M et al. Antiretroviral treatment to prevent the sexual transmission of HIV-1: results from the HPTN 052 multinational randomized controlled trial. Oral abstract MOAX0102. Webcast
3. Eshleman S et al. Analysis of genetic linkage of HIV from couples enrolled in the HIV Prevention Trials Network (HPTN) 052 trial. Oral abstract MOAX0103.
4. Hosseinipour M et al. Immunologic and virologic disease progression and responses to ART across geographic regions: outcomes from HPTN 052 study. Oral abstract MOAX0104. Webcast
5. Grinsztejn B et al. Effects of early versus delayed initiation of antiretroviral therapy (ART) on HIV clinical outcomes: results from the HPTN 052 randomized clinical trial. Oral abstract MOAX0105. Webcast.
6. WHO. Couples HIV testing and counselling and antiretroviral therapy for treatment and prevention in serodiscordant couples: Recommendations for a public health approach. 2011. Final version approved but not yet released. (PDF file)
7. Cohen J. New prevention data leads WHO to delay guidelines for couples. Science Insider (25 July 2011).

Simon Collins, HIV i-Base

Results from the double-blind placebo-controlled TDF2 study presented in an oral presentation in Rome provided additional supportive data for the benefit of daily oral tenofovir/FTC (Truvada) to reduce sexual heterosexual transmission. [1]

While the iPrEX study first reported a strongly protective impact in high risk MSM, the lack of protection in the FemPrEP study earlier this year has still to be explained. [2, 3]

TDF2 randomised 1200 sexually active HIV-negative adults (age 18-49: approximately 90% were between 21-29 years) and followed them for a year. Over 90% of participants were single with only 3% having low educations (primary or less) with >70% having secondary and >20% having post-secondary education. HIV testing was monthly and as with all prevention studies, intensive counselling on safer sex and free condom distribution was provided throughout the study. An indication of the background risk in this population is that 16% of people failing screening (197/2533) were excluded due to already being HIV-positive and 20% due to not being sexually active.

A slightly higher percentage of people in the active vs placebo arm (34% vs 31%) did not complete the study due to loss to follow-up, withdrawal of consent, relocations or other reason. The study had a good gender balance with 45% women.

With 33 seroconversions, primary efficacy results reported a 63% reduced risk of transmission with Truvada based on 9 new infections in the active arm compared to 24 in placebo group (difference 62%: 95%CI 21.5 to 83.4, p=0.0133).

When restricting the analysis (post hoc?) to infections within 4 weeks of a study visit (ie where the monthly visit schedule was being followed and the participant was under a prescription period) the association became stronger. Out of 23 seroconversions, 4 occurred in the active arm and 19 in the placebo group with 78% protection efficacy (95% CI 41.2 to 93.6, p=0.0053).

Although it was emphasised that the study was underpowered to draw any conclusion by gender, in an ITT analysis (33 cases) the intervention appeared protective in men (p=0.026) but not women (p=0.107) and in the observed results (23 cases) the protection was seen on women (p=0.021) but not men (p=0.065). Whilst interesting to see if a gender effect can shed light on the results from FemPREP, this will need to come from larger study numbers.

Resistance developed in one person enrolled in the active arm whose acute HIV infection was undiagnosed with K65R, M184V and A62V conferring nucleoside cross resistance. The person has achieved viral suppression after starting treatment with AZT/3TC/lopinavir/ritonavir. One person in the placebo group had low-level K65R suggesting an infection with drug resistant HIV.

Side effects were commonly reported in both arms, usually mild, with nausea (19% vs 7%, p=0.0001) and vomiting (11% vs 7%, p=0.005) occurring more significantly in the active arm compared to the placebo arm, but resolving within the first month. There were no differences in laboratory abnormalities with one case of elevated creatinine in the active group that resolved when treatment was stopped.

References

1. Thigpen MC et al. Daily oral antiretroviral use for the prevention of HIV infection in heterosexually active young adults in Botswana: results from the TDF2 study. Oral abstract WELBC01. Webcast.
2. Grant RM et al. Preexposure chemoprophylaxis for HIV prevention in men who have sex with men. NEJM. 23 November 2010 (10.1056/NEJMoa1011205). Free full access.
3. FHI statement on the FEM-PrEP HIV prevention study: FHI to initiate orderly closure of FEM-PrEP. (18 April 2011).

Simon Collins, HIV i-Base

The final presentation in the Treatment is Prevention session was a summary of the first results from the Partners PrEP study that randomised the HIV-negative partner in 4758 HIV serodifferent heterosexual couples to daily tenofovir (TDF) vs tenofovir/FTC (TVD) vs placebo in a 1:1:1 ratio. [1]

The results presented were based on a DSMB analysis a week before the conference that recommended unblinding the placebo arm and switching those participants to active drugs. This was based on significantly reduced risks of transmission in both the active arms and was 18 months earlier than the planned study endpoint.

This study was run in nine sites in Kenya and Uganda with all participants receiving intensive healthcare and adherence counselling including free condoms. The negative partners were seen monthly for counselling with HIV and pregnancy testing and the positive partners were monitored for their HIV care every three months.

Baseline demographics were similar across the three arms and included age (of the negative partner) 33 years (IQR 28-40), with the positive partner diagnosed a median of 0.4 years (IQR 0.1-2.0 years), CD4 count 490 cells/mm3 (IQR 370-660) and viral load 3.9 log copies/mL (3.2-4.5 logs).

Nearly all couples were married (98%) with duration of relationship a median of 7 years (IQR 3-14). The positive partner was a woman in 40% of couples. Approximately 20% of positive partners started treatment during the study for their own health.

Study retention was high with fewer than 5% discontinuations over 7337 person years of follow up (median 12 months). Adherence was also estimated high at 97% based on pill count using returned bottles (98% of bottles were returned).

Up to May 2011, there were 90 new infections, 12 of which were HIV-positive at randomisation (3 TDF, 3 TVD, 6 placebo). Of the 78 transmissions that occurred as events for the primary endpoint, 18, 14 and 47 occurred in the TDF, TVD and placebo arms respectively. The was an incidence of 0.74, 0.53 and 1.92 per 100 patient years that produced protection rates of 62% (95%CI 34-78%, p=0.0003) in the tenofovir and 73% (95%CI 49-85%, p<0.0001) in the tenofovir/FTC arms compared to placebo.

The study reported of a similar response between the two active arms (p=0.18 for comparison, NS). However, protection was numerically greater with the dual therapy and the gender analysis reported wider confidence intervals for tenofovir monotherapy with lower levels that were lower.

For women, protection rates were 68% (29-85%) and 62% (19%-82%) in the TDF vs TVD arms; for men these were 55% (4-79%) vs 83% (49-94%). The plausibility for greater protection from dual therapy would be extended with either lower adherence or less-than-daily dosing, both of which might be key public health factors for considering use of PrEP outside of clinical trials.

Safety results were very similar between all three arms for serious events and lab abnormalities including creatinine increases (1%, mainly grade 1) and reduced phosphorus (9%, equal across arms). As with other PrEP studies, nausea and diarrhoea were significantly more common in the active arms, but generally only for the first month of treatment.

As with other PrEP research, results from the pharmacokinetic will be important to correlate drug levels with level of protection and partner viral load with risk of infection. Resistance results will also be analysed.

Reference

Baeten J et al. Antiretroviral Pre-Exposure Prophylaxis for HIV-1 prevention among heterosexual African men and women: the Partners PrEP Study. Oral abstract MOAX0106. Webcast.

CROI was important this year because of the profile given to further studies supporting the role of treatment as prevention. Together they support the argument that universal treatment is perhaps the most powerful prevention tool we are likely to have for many years, perhaps with the potential to even eradicate the virus on a population level.

In a lecture prior to the main conference, Brian Williams from the South African Centre for Epidemiological Modelling and Analysis, Stellensbosch, detailed the modeling data for the direct and indirect impact of ARVs on prevention, [1] elaborating on the research paper published last year in the Lancet. [2]

At its most optimistic, this includes the potential for universal treatment to eliminate new infections in South Africa within 5-10 years on a cost neutral budget, at the same time saving millions of lives (and preventing millions of new infections). The science on which the model is based shows an impact on dramatically reducing infections that few can ignore.

The epidemiology for the model included low HIV infectivity (~0.001 per heterosexual encounter), 10-fold individual variability in infectivity, a slow epidemic doubling time (~1-3 years), a long period of potential infectiousness (5-15 years) and an average case reproduction number (~7 additional people infected per case): leading to a calculation showing that virtual eradication of HIV could be achieved if transmission could be reduced by 7-fold.

Viral load is commonly reduced by 10,000 times on treatment, and although infectivity reduces in smaller proportions (roughly in relation to the cubic root of viral load), the net impact of treatment on infectivity was estimated to be a 96% reduction.

The impact on reducing TB and for continuous treatment after pregnancy were also included, and for interventions based only on PrEP alone or in combination with ARVs. For South Africa, the model was based on a conservative treatment programme, treating at a CD4 count of 200 cells/mm3, but similar costs and benefits were shown when starting universal treatment at 350, 500 or even at diagnosis. The initial outlay (an adjusted US $60 billion) was compensated by lower cost of hospitalisations and reduced new infections, and saved an additional 3 million lives over 40 years, at stable costs.

The discussion after the presentation stressed the need for pilot operational research on each aspect of a universal treatment model, including willingness to test, virological response rates with earlier treatment, the actual impact on transmission – and the need to develop new heath structures to allow such scale-up.

A first step in confirming treatment reduces HIV transmission in real world settings was shown in results from the Partners in Prevention HSV/HIV Transmission (PARTNERS) Study in over 3400 serodifferent heterosexual couples in seven southern African countries (Botswana, Kenya, Rwanda, South Africa, Tanzania, Uganda, and Zambia).  The HIV-positive partner was a man in 32% and a woman in 68% of couples. [3]

This study previously reported that HSV therapy with daily acyclovir failed to protect against HIV infections, explained by a massive increase in localised CD4 target cells, and persistence for up to two months after the healing of HSV lesions.

All HIV-positive partners entered the study with CD4 counts >250 cells/mm3 and were not on treatment. Over two years, approximately 10% of study participants required HIV treatment for their own care, and this allowed for the HIV transmission rates to be compared by use of ARV treatment. Intensive risk reduction support was supplied throughout the study, to minimise HIV risk for the HIV-negative partners.

People with more advanced HIV at baseline were more likely to start treatment; with higher baseline viral loads (mean 4.4 vs 3.9 log copies/mL, p<0.001), and lower CD4 counts (375 vs 540 cells/mm3, p<0.001). A higher proportion of men than women (12% vs 9%, p=0.01) strated treatment, at slightly lower median CD4 counts (192 vs 204 cells/mm3, p=0.05). People starting treatment were also older (mean 35.2 vs 32.7 years, p<0.001).

ART was initiated at CD4 counts <200 cells/mm3 in 52% patients, between 200 and 349 cells/mm3 in 33%, and >350 cells/mm3 in 15% (30% of this group were for prevention of mother to child transmission).

New HIV infections were detected in 151 of the HIV-negative partners, over 24 months of follow-up, with testing and prevention support provided every 3 months. Phylogenetic analysis suggested that slightly less than one third (43/151) of the infections were not from the relationship partner. Five cases were excluded from the transmission analysis due to uncertain use of ARVs.

This left an overall transmission rate in 103 remaining transmissions of 2.1%.

Of these, 102/103 were in the non-ARV group (102/4558 person years; rate 2.24 95%CI 1.82-2.72) compared to 1/103 from partners using ARV treatment (1/233 person years; rate 0.37 95%CI 0.09-2.04). This produced an unadjusted relative risk of 0.17 (p=0.037), which became even more significant when adjusting for time on study and CD4 count, showing a 92% reduction in risk: RR=0.08 (95%CI 0.002, 0.57, p=0.004).

The single transmission case occurred in someone whose partner started treatment 18 days before the 9 month assessment, when they were still HIV-negative (details on whether this was by HIV-antigen or PCR testing were not provided), but who seroconverted by the month 12 evaluations. Viral load was undetectable at month 12 in the HIV-positive partner.

Details on CD4 count in the HIV-positive partner showed transmissions at all CD4 levels, with a considerably higher risk when the partner had a count <200 cells/mm3 (rate = 8.79 vs 2.79 at 200-350 and 1.7 at 350-500).

This is likely to be an indirect marker of higher viral load relating to more advanced infections, but surprisingly, the presentation provided no further information on viral load levels of the source partner, other than showing that after a median of 7 months treatment (IQR 3-12months) the median viral load dropped to undetectable, indicating excellent responses.

Importantly, and perhaps showing the positive results from the behavioural interventions, the percentage of visits at which people reported unprotected sex dropped from 6.2% to 3.7% at the pre- and post-treatment visits, respectively, with no change in frequency of sex.

Two other studies at CROI, in a largely MSM population in San Francisco, supported the impact of ARVs to reduce transmission.

Moupali Das-Douglas and colleagues from the San Francisco Department of Public Health and the University of California presented results from a model that estimated values for average and total community viral load (CVL) from 2004-2008 and then compared these with the expected and actual number of new diagnoses over the same period. [4]

Average CVL was defined as the mean of the most recent viral load of all reported HIV-positive individuals in a particular population, divided by the number of reported HIV-positive individuals in the population. Total CVL was the sum of the most recent viral loads of all HIV-positive individuals in a particular population.

The context for this study was an effective ‘test and treat’ programme that from 2004 to 2008 increased the percentage of MSM testing within 12 months from 65% to 72% and within 6 months from 41% to 53%. The percentage of HIV-positive MSM unaware of their status dropped from 24% to 14.5% (comparable UK figures vary from 30-50%). By 2008, 90% of patients in care were on HAART, with 72% virologically suppressed (<75 copies/mL).

The decreases in mean CVL and reductions in actual diagnoses (from 798 in 2004 to 434 in 2008) were both statistically significant (p=0.005), as were the decreases in total CVL (p=0.019) and percentage of virologically suppressed patients (p=0.002). The presentation acknowledged that a limitation in these results is that cases may be diagnosed chronic rather than new infections, which was addressed in methodology for expected and actual incidence rates.

However, using a more conservative meta-regression analysis (different to the reported abstract), the 30% reduction in CVL and almost 40% reduction in incidence (rather than cases) was not significant (p=0.3) due to the degree of imprecision in the estimates.

While this makes it too early to link CVL with incidence, the reductions in newly diagnosed and reported cases, at the same time as increased testing, greater ARV coverage and greater virological suppression strongly support close following of subsequent data from this model.

In a related poster, Edwin Charlebois and colleagues modeled the impact of earlier treatment and broader test and treat programmes in San Francisco, suggesting that HIV prevalence could fall from the current 25% to around 10% by 2030 if the programme shifted to universal test and treat. [5]

As this issue of HTB went to press, a policy shift in San Francisco to offer HIV treatment to all newly diagnosed patients, regardless of CD4 count or viral load, was announced by public health officials. [6]

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The positive correlation between viral load and risk of transmission for every route, whether sexual, from shared injection equipment, during pregnancy, at birth and from breast milk, and from needle stick exposure to health workers, is now convincingly demonstrated.  For some of these transmission routes, antiretroviral treatment to reduce viral load is already widely used to reduce transmission (principally for mother to child transmission, PEP and PEPSE).

Treatment dramatically extends life, reduces morbidity and should now be additionally valued for reducing transmission. An estimated 70% of HIV-positive people globally in need of immediate treatment for their own care are still unable to access it.

References:

Unless stated otherwise, all references are to the Programme and Abstracts of the 17th Conference on Retroviruses and Opportunistic Infections. 16-10 February 2010, San Francisco. All oral abstracts are available as webcasts.

http://www.retroconference.org

1. Williams B and Dye C. Put your money where your model is: ART for the prevention and treatment of HIV/AIDS.  Webcast: Guiding the global response. Tuesday 2.30pm.

http://www.retroconference.org/2010/data/files/webcast_2010.htm

2. Granich RM et al. Universal voluntary HIV testing with immediate antiretroviral therapy as a strategy for elimination of HIV transmission: a mathematical model. The Lancet, Volume 373, Issue 9657, p48-57, 3 January 2009. doi:10.1016/S0140-6736(08)61697-9.

http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(08)61697-9/abstract

3. Donnell D et al. ART and risk of heterosexual HIV-1 transmission in HIV-1 serodiscordant African couples: a multinational prospective study. 17th CROI 2010. Oral abstract 136.

http://www.retroconference.org/2010/Abstracts/39222.htm

Webcast: New Insights into Transmission and Acute Infection. Friday 9.30am.

http://www.retroconference.org/2010/data/files/webcast_2010.htm

4. Das-Douglas M et al. Decreases in community viral load are associated with a reduction in new HIV diagnoses in San Francisco. 17th CROI 2010. Oral abstract 33.

http://www.retroconference.org/2010/Abstracts/38232.htm

Webcast: Testing and Transmission. Wednesday 9.30am.

http://www.retroconference.org/2010/data/files/webcast_2010.htm

5. Charlebois E et al. Effect of Expanded ART Strategies on the MSM HIV Epidemic in San Francisco. 17th CROI 2010. Poster abstract 996.

http://www.retroconference.org/2010/Abstracts/39042.htm

6. San Francisco endorses starting HIV treatment immediately after diagnosis. (5 April 2010)

http://www.aidsmeds.com/articles/treatment_HIV_diagnosis_1667_18253.shtml

The protective mechanism for reducing heterosexual HIV transmission to circumcised men has been attributed to two factors relating to the properties of the inner foreskin: a thinner keratin layer reducing the physical barrier and a higher concentration of CD4 and Langerhans cells that are primary targets for infection. A third factor may be that the foreskin prolongs the time that fluid that contains HIV remains in contact with genital tissue. In theory, the size of the foreskin should also positively correlate to the risk from these mechanisms, and this is supported by results from a study published in the 23 October edition of the journal AIDS. [1]

HIV infection rates were collected from 965 men in Rakai, Uganda, who were recruited for two randomised circumcision studies. These men were initially HIV-negative and followed for a total of 3920 person years, prior to circumcision as part of the trial protocol. The results from these trials have already been reported. [2, 3]

After circumcision, the foreskin surface area was calculated (length x width; cm2) and infection rates prior to circumcision were calculated by quartile. Men who became infected compared to those who remained HIV-negative were found to have a significantly greater foreskin surface area (mean 43.3 (+2.1) vs 36.8 (+0.5) cm2 (p-0.01).

HIV incidence/100 person years (PY) was 0.80, 0.92, 0.90 and 2.48 for men with foreskin surface areas in the lower (7.0-26.3 cm2), second (26.4-35.0 cm2), third (35.2-45.5 cm2) and upper quartiles (45.6-99.8 cm2) respectively.

The incidence rate ratio (IRR) of HIV acquisition, after adjusting for age, education, religion, number of sex partners and condom use, was significantly higher for men in the highest compared to the lowest quartiles of foreskin surface area (IRR 2.37; 95%CI 1.05-5.31).

There was, however, no significant difference in HIV incidence between the lower three quartiles. In the adjusted analysis, older age (IRR 4.16; 95%CI 1.55, 11.19, and IRR 4.00; 95%CI 1.46, 10.74; for ages 25-30 and >30 respectively, each compared to 15-24 years), lower education level (0.40; 0.18, 0.91; secondary/tertiary vs primary/none) and catholic religion (IRR 0.37; 0.16, 0.82; Catholic vs non-Catholic) were also significantly associated with risk of HIV acquisition.

The authors concluded that their findings, in addition to the observational studies and randomised trials, add plausibility to the hypothesis that the foreskin is a tissue vulnerable to HIV acquisition.

They suggested that minimising retention of residual foreskin tissue after male circumcision using dorsal slit and sleeve procedures rather than the forceps-guided procedure (which leaves 0.5-1.0 cm of mucosal skin proximal to the corona) is a theoretical concern. However, they also reported that they did not observe any increased risk of HIV acquisition among men with smaller foreskin surface areas that were substantially larger than residual tissue retained after circumcision surgery.

comment

While the study states that these findings need to be replicated in other studies, it is difficult to see how this could be supported.

Firstly, although circumcision studies have shown protection against HPV, HSV and syphilis, men primarily want to be circumcised in order to reduce their risk of HIV infection, and should be told if they are HIV-positive at the time of surgery. It is unclear whether the men in this study would have undertaken circumcision, had they been made aware that they had already caught HIV prior to the intervention.

Secondly, now that circumcision had been proven to reduce heterosexual transmission in high prevalence settings, it is difficult to see why participants would be followed for any significant period prior to surgery.

References:

1. Kigozi G et al. Foreskin surface area and HIV acquisition in Rakai, Uganda (size matters). AIDS. 23(16):2209-2213, October 23, 2009. doi: 10.1097/QAD.0b013e328330eda8.
2. Gray RH et al. Male circumcision for HIV prevention in men in Rakai, Uganda: a randomised trial. Lancet 2007; 369:657-666.
3. Wawer MJ et al. Circumcision in HIV-infected men and its effect on HIV transmission to female partners in Rakai Uganda: a randomised controlled trial. Lancet 2009; 374:229-237.

Important limitations to the protective benefits from circumcision, prompted by a 2008 WHO review by Robert Bailey and colleagues, of complications during male circumcision in Kenya [1], were discussed in a recent editorial article in the 2 January 2010 journal AIDS. [2]

The original study, available online without subscription, deserves reading in full by anyone rushing to roll-out circumcision programmes on a community level.

The WHO study prospectively followed approximately 1000 men (IQR ~13-15, range 5-21 years), who were circumcised in July-August 2004, who were interviewed about complications 30-89 days after surgery. Twenty-four men were directly observed during circumcision and after 3, 8, 30 and 90 days.

The participants had either a traditional circumcision performed in a village or within a household compound, or a medical circumcision performed by someone the participant considered to be a clinician in a hospital, health centre, dispensary or private office. The researchers also interviewed 21 traditional and 20 clinical people who carried out the circumcisions.

After interviewing approximately two-thirds of participants and directly following the 24 cases, the researchers found very high rates of complications and decided to directly examine and interview the remaining 298 men, (range 45 – 89 days after circumcision).

One or more complications were reported by 35% men circumcised traditionally and by 17% men circumcised medically (OR 2.53; 1.89–3.38; p <0.001). These rates were significantly higher than the approximate 1-3% observed in clinical trials, or in infants circumcised in developed countries.

Although rates for each complication were not given, the most common self-reported complications were excessive bleeding, infections and excessive pain, with bleeding the most common. Pain upon urination, incomplete circumcision requiring repeat surgery, and lacerations of the glans, the scrotum and the thighs were also reported. Many traditional circumcisions continued to bleed and needed medical support.

Infections were equally common among subjects circumcised medically and traditionally. Those circumcised traditionally were more likely to report receiving antibiotics from local practitioners, often from “travelling nurses” with few or no qualifications. These informal practitioners often sold injections to address infections and bandaged the wound after applying gravacine (a talcum powder with penicillin). Whether it prevented infections we cannot be sure, but it tended to cake in the wound, delay healing and result in thick scarring and, in a few cases, permanent discolouration.

In 24% of the traditional cases and 19% of the medical cases, the wound had still not healed after 60 days (p=0.056) in contrast to 96% healed by 30 days in the randomised male circumcision in Kisumu, Kenya.

In the interviews with 298 men, traditionally circumcision was much more likely not to have healed (21% vs 10%, AOR 0.43; 0.22–0.84, p=0.014), to have significant swelling (14% vs 5%, AOR 3.20; 1.27–8.07, p=0.014), to have a culturally unacceptable amount of foreskin remaining (12% vs 3%, AOR 5.32; 1.54–18.31, p=0.008); and to higher trend to have lacerations (17% vs 10%, AOR 1.91; 0.93–3.91, p=0.077), and keloid scarring (17% vs 10%, AOR 1.99; 0.98–4,06, p=0.059.

Compared to developed country settings, delayed healing, swellings and lacerations were also prevalent among those circumcised medically.

The researchers concluded that “extensive training and resources will be necessary to build the capacity of health facilities in sub-Saharan Africa before safe circumcision services can be aggressively promoted for HIV prevention” and that “the rate of serious complications from traditional circumcisions should also serve as an alarm to ministries of health and the international health community that focus cannot only be on areas where circumcision prevalence is low”.

References:

1. Bailey RC, Egesah O, Rosenberg S. Male circumcision for HIV prevention: a prospective study of complications in clinical and traditional settings in Bungoma, Kenya. Bull World Health Organ 2008;86 (9):669-677.
   http://www.who.int/bulletin/volumes/86/9/08-051482/en/index.html
2. Crabb C. Male circumcision to prevent heterosexual HIV transmission gets (another) green light, but traditional circumcision in Africa has ‘shocking’ number of complications. AIDS. 24(1):N1-N2, January 2, 2010. doi: 10.1097/QAD.0b013e32832faec0

Earlier this year at the Conference on Retroviruses & Opportunistic Infections, Salim Abdool Karim presented data suggesting that a vaginally applied microbicide gel called PRO 2000 might offer some protection against HIV infection in high-risk women. [1]

The results were not statistically significant but represented a trend, suggesting a 30% reduction in risk of acquisition of the virus. Some commentators at the meeting noted that because this study had two separate control arms (a placebo gel and no gel), comparing the total number of control participants from both of these arms with the group that received PRO 2000 would render the result statistically significant. To his credit, Karim emphasised that such an analysis was not a pre-specified part of the protocol and was therefore inappropriate. He also pointed out that there was a larger, ongoing phase III study of PRO 2000 involving over 9,000 women that would provide a definitive answer as to the product’s efficacy.

The results from this trial, called MDP-301 and run by the UK Medical Research Council in close collaboration with Imperial College in London and investigators in four African countries, were announced on 14 December.  Disappointingly, the hint of efficacy seen in the smaller phase IIb was not duplicated: there were 130 HIV infections among the 3,156 women that received PRO 2000 gel, and 123 infections in the group of 3,112 women that received placebo gel.

The first news story reporting the result appeared in the Times newspaper (UK edition) the day before and broke the embargo on the MRC releases by several hours; it was subsequently taken offline before being reinstated. The article dramatically – but erroneously – characterises the PRO 2000 result as “a significant setback.” The whole purpose of large phase III efficacy trials is to definitively answer the question of whether an intervention works and, quite often, they don’t. In the case of PRO 2000, the microbicide is one of the last in a pipeline of products with relatively limited direct antiretroviral activity and, over the past several years, there has been increasing recognition in the field that more specific products are needed. Several such antiretroviral microbicides, such as the gel form of the drug tenofovir (Viread) are now in trials.

Source: TAG weblog. PRO 2000 microbicide gel does not pan out. (14 Dec 2009)

Further information on the MDP-301 trial:

Background materials from the trial’s sponsor, the Microbicide Development Programme of the UK Medical Research Council:

http://www.mdp.mrc.ac.uk

AVAC’s PRO 2000 resource page:

http://www.avac.org/ht/d/sp/i/3426/pid/3426

Global Campaign for Microbicides: http://www.global-campaign.org/MDP301.htm

Reference:

1. Karim SA et al. Safety and Effectiveness of Vaginal Microbicides BufferGel and 0.5% PRO 2000/5 Gel for the Prevention of HIV Infection in Women: Results of the HPTN 035 Trial. 16th CROI, 2009. Late breaker abstract 44LB.
   http://www.retroconference.org/2009/Abstracts/36659.htm

Acyclovir studies at IAS 2009

Connie Celum, the principal investigator on the HPTN 039 trial, together with Jai Lingappa, the medical director, and other members of the Partners in Prevention HSV-HIV Transmission Team presented findings at IAS2009 on a counterpart trial to HPTN 039. This study, in HIV serodiscordant couples, looked at whether suppressing HSV-2 in people dually infected with HSV-2 and HIV infections could reduce HIV transmissions to their HIV-negative partners. [1]

HPTN 039 was a double-blind placebo controlled trial of standard doses of acyclovir for HSV-2 suppression (400 mg twice daily) to prevent HIV acquisition among over 3200 African women and MSM in Peru and the United States who were HIV-negative and HSV-2 positive. HTPN 039 found that acyclovir suppression did not reduce HIV incidence compared to placebo. Results were reported at CROI2008 and subsequently in the Lancet. [2]

Celum presented the main results of this new double-blind placebo controlled trial, conducted at 14 sites in sub-Saharan Africa, including South Africa, Kenya, Zambia, Botswana, Tanzania, Rwanda and Uganda.

Of 6544 heterosexual HIV discordant couples screened, 3408 were enrolled. The inclusion criteria for the coinfected partner included a CD4 cell count >250/mm³. The other partner could be either HSV-2 positive or negative, but had to be HIV-negative. HIV-positive participants could not be eligible for ART at trial entry according to their country guidelines.

The HIV-positive partners were randomised to receive either 400mg acyclovir twice-daily or placebo twice-daily. Couples were followed for a maximum of 24 months. Participants were provided with ART if they became eligible for it according to country guidelines.

The primary endpoint was HIV infection in the HIV-negative partners. The secondary endpoints included plasma and genital HIV viral load in the HIV-positive partners, and HIV disease progression. The trial was set up so that if 88 ‘linked’ HIV transmissions (i.e. the virus transmitted from the enrolled partner to the seroconverting partner was determined by molecular sequencing to be linked) were observed, the trial would have high statistical power (90%) to see a 50% reduction in HIV transmissions in the acyclovir arm.

Baseline characteristics of the group included:

• 67% of HIV-positive partners were female;
• 65% of volunteers were <35 years old;
• average partnership duration was five years;
• 90% were cohabiting;
• a median of five sex acts were reported in the month prior to baseline measurements:
• 29% reported unprotected sex;
• 22% of the HIV-positive partners reported genital ulcer disease (GUD) in the prior three months;
• 4% of HIV-positive and 7% of HIV-negative partners reported outside partners respectively;
• median CD4 count was 460 cells/mm³ and median plasma HIV viral load was 4.2 log.

Monthly follow-up visits included medication provision, pill count and adherence support and individual and couple HIV risk reduction counselling. Every three months, HIV-positive partners were examined for GUD and plasma viral load and HIV-negative partners were tested for HIV and given risk reduction counselling. CD4 cell counts were taken every six months.

Retention was high. At 24 months, 92% of HIV-positive and 84% of HIV-negative participants were still in follow-up. Adherence measured by pill count was also high: 88% of all bottles were dispensed and 97% of dispensed bottle doses were taken.

No significant differences in incidence

There were 136 seroconversions at a rate of 2.8/100py (95% CI: 2.3-3.3), one after an incorrect drug kit was dispensed. Of the remaining 135, 68 occurred on the acyclovir arm and 67 on the placebo arm (HR: 0.92; 95%CI 0.60-1.41; p=0.70).

In a modified intention to treat analysis, 43 transmissions were linked by viral sequencing technology to partners on the acyclovir arm and 47 were linked to partners on the placebo arm. However, two in the acyclovir arm and four in the placebo arm were excluded from analysis because the study drug was withheld during pregnancy. Here too, there were no significant differences between the two arms. The sequencing methodology for this study was explained in a late breaker poster from Mary Campbell. [3]

Benefits of acyclovir

There were fewer GUD events in the acyclovir arm (217 vs 550; RR: 0.39; 95% CI: 0.32-0.48; p<0.001). HSV-2-positive GUD as determined by DNA PCR was also lower in the acyclovir arm (92 vs 336; RR: 0.27; 95%CI 0.2-0.36; p<0.001).

The acyclovir arm also had a 0.25 log reduction in plasma viral load (95%CI: 0.22-0.29).

A novel component of this study was evaluation of herpes suppression on HIV disease progression, an important secondary endpoint of the Partners in Prevention trial. In a separate analysis presented by Jairam Lingappa, 3,381 of the HIV-positive participants were followed up until a composite endpoint of first of CD4 cell count <200 cells/mm³, ART initiation, or death from non-trauma causes. [4]

In the acyclovir arm, 284 participants reached this endpoint versus 325 in the placebo arm (HR 0.83; 95%CI: 0.71-0.90; p=0.03). Similar reductions were found for each component of the composite endpoint analysed separately. However, Lingappa’s team further calculated that for every 43 people treated with the trial dose of acyclovir for a year, only one person would be prevented from attaining the composite endpoint. (We have previously reported findings demonstrating acyclovir and its pro-drug, valacyclovir’s effect on HIV plasma RNA levels, in the October 2006 and July/August 2008 issues of HIV Treatment Bulletin, but this is the first report documenting impact of herpes suppression on HIV disease progression.)

Among participants with CD4 counts >350 cells/mm³ at enrollment, acyclovir delayed the time to CD4 < 350 cells/mm³ (HR 0.81; 95%CI 0.71-0.93; p=0.002). Here, 20 people would need to be treated to prevent one person from progressing to a CD4 count < 350 cells/mm³.

Acyclovir effect on genital viral load

A late breaker poster by Jared Baeten et al presented the results of a substudy that examined genital HIV RNA concentrations as a surrogate marker for HIV infectivity. [5]

Endocervical and semen samples were collected from 2,521 (1,805 women and 716 men) of 3,408 HIV-positive participants. For 1,797 of these, plasma was concurrently taken. For the remainder a plasma viral load within six months was available. Since the genital samples were taken only once during the study, the genital viral load was analysed as a time-independent variable.

HIV was detected in 60% of endocervical swab samples and 57% of semen samples. The median endocervical HIV concentration was 3.2 log (IQR 2.08-3.87) overall. Genital HIV-1 concentrations were significantly lower among those randomised to acyclovir (median 2.98 vs 3.29 for endocervical swabs; p<0.001 and 2.38 vs 2.76 for semen; p=0.008). The key finding of the study was that genital HIV concentrations were higher among HIV transmitting couples, where transmission was genetically linked to the partner (3.44 vs 2.49 log copies/mL for semen, p<0.001 and 3.91 vs 3.18 log copies/swab for endocervical swabs, p<0.001). Each log increase in genital HIV-1 RNA concentration was associated with 1.85-fold increased odds of HIV transmission for semen (p<0.001) and 2.03-fold increased odds of transmission for endocervical swabs (p<0.001). The study found no significant difference in genital HIV concentration for participants whose partners acquired HIV from outside sexual partners versus those who did not transmit HIV.

However, despite a 73% reduction in GUD and 0.25 log decline in plasma HIV levels and an approximately 0.3 log decline in genital HIV levels, acyclovir conferred no reduction in
HIV transmission. The authors interpret the overall results of the trial to indicate that the plasma and genital tract HIV viral load reduction from herpes suppression with standard doses of acyclovir is too small to confer a protective effect against HIV transmission.

Future acyclovir trials

Nevertheless, given the promising effect of acyclovir on HIV viral load, Steve Reynolds described an ongoing double-blind placebo controlled trial in Rakai, Uganda. [6]

The purpose of the trial is to evaluate the effect of suppressive HSV-2 therapy among HIV-1/HSV-2 co-infected individuals on progression to AIDS, defined as CD4 count < 250 cells/mm³ or WHO stage IV disease. Volunteers with CD4 counts between 300 and 400 cells/mm³, not on ART, without WHO III/IV symptoms and no history of opportunistic infections, other than mucocutaneous Kaposi Sarcoma, candida or treated TB were eligible for inclusion. Enrollment was completed in November 2008. The trial assumes that 40% of individuals in the placebo arm will progress to CD4 counts <250 cells/mm³ or AIDS over 24 months and is powered to detect at least a 20% reduction in HIV disease progression in the intervention arm.

COMMENT

These studies show that a standard dose of acyclovir for HSV-2 suppression does not reduce HIV transmission. These are disappointing findings for an HIV prevention strategy that is already available.

A mechanism for the lack of protection has been suggested by Laurence Corey and colleagues in a recent paper in Nature Medicine. [7]

By analysing regular skin biopsies taken during acute lesions and over 20 weeks follow-up, they indentified a ‘massive localised infiltration’ of CD4 and CD8 cells, thereby increasing the targets for HIV infection. Eight weeks after lesions healed, these levels were still 8-fold higher (655 and 618 cells/mm² of skin, respectively, compared to 68 and 55 cells/mm² in unaffected skin samples).

This paper is reported in detail in the Basic Science section of this issue of HTB. [8]

It has been conventional wisdom that wider availability of acyclovir for patients with genital herpes outbreaks would reduce HIV transmissions. We now know this is incorrect, at least with the doses of acyclovir (400 mg twice daily) used in these trials. However, efforts to make acyclovir widely accessible should continue because herpes is a debilitating, unpleasant disease which acyclovir effectively treats and because HSV-2 in widely prevalent in both HIV-negative and HIV-positive people. One of the barriers to its accessibility remains its high price in many developing countries.

Despite the negative findings, this trial and its substudies have set a high standard for the testing of future HIV prevention interventions. Furthermore, modeling studies using the data from this trial provide a potential threshold of HIV plasma viral load reduction in HIV-infected persons that will be needed to impact HIV transmission.

We now need to know whether a therapeutic dose of acyclovir could delay the time until initiation of HIV treatment, and whether this would be cost effective. The trial in Rakai described by Steve Reynolds using 400 mg twice-daily dose will provide complimentary information to the Partners in Prevention trial.

Studies with higher doses of valacyclovir will evaluate whether greater reduction in plasma HIV levels is feasible compared to acyclovir 400 mg twice daily. However, this research could be overtaken by new developments in ART management, if guidelines recommend earlier treatment.

References
Unless othewise stated, all references are to the Programme and Abstracts of 5th IAS Conference on HIV Pathogenesis, Treatment and Prevention, 19-22 July 2009, Cape Town.

1. Celum C et al. Twice-daily acyclovir to reduce HIV-1 transmission from HIV-1 / HSV-2 co-infected persons within HIV-1 serodiscordant couples: a randomized, double-blind, placebo-controlled trial. 5th IAS 2009, Cape Town. Oral abstract WELBC101.
http://www.ias2009.org/pag/Abstracts.aspx?AID=3699
2. Celum C et al. Effect of aciclovir on HIV-1 acquisition in herpes simplex virus 2 seropositive women and men who have sex with men: a randomised, double-blind, placebo-controlled trial. Lancet, 21 June 2008. 371(9630):2109-19.
http://www.ncbi.nlm.nih.gov/sites/entrez/18572080
3. Campbell M et al. Determination of transmission linkage in the Partners in Prevention Study. 5th IAS 2009, Cape Town. Poster abstract LBPEC07.
http://www.ias2009.org/pag/Abstracts.aspx?AID=3756
4. Lingappa JR et al. Daily acyclovir delays HIV-1 disease progression among HIV-1/HSV-2 dually-infected persons: a randomised trial. 5th IAS 2009, Cape Town. Oral abstract WELBC102.
http://www.ias2009.org/pag/Abstracts.aspx?AID=3714
5. Baeten J et al. Genital HIV-1 RNA concentrations and heterosexual HIV-1 transmission risk. 5th IAS 2009, Cape Town. Poster abstract LBPEA07.
http://www.ias2009.org/pag/Abstracts.aspx?AID=3770
6. Reynolds SJ. HSV-2 Suppression Trial, Rakai, Uganda. Partners in Prevention presentation reported with permission.
7. Zhu J et al. Persistence of HIV-1 receptor–positive cells after HSV-2 reactivation is a potential mechanism for increased HIV-1 acquisition. Nature Medicine. Published online: 2 August 2009 | doi:10.1038/nm.2006.
http://www.nature.com/nm/journal/vaop/ncurrent/abs/nm.2006.html
8. Jefferys R. Immune surveillance below the radar: study offers explanation for acyclovir’s failure to reduce HIV risk. HTB September/November 2009. Vol 10, No 4. (Vol 2, No 3 of HTB South).

Two-dose intermittent pre-exposure prophylaxis (PrEP) with Truvada (tenofovir plus emtricitabine) protected male macaques from rectal exposure to a simian-HIV hybrid virus (SHIV) as well as daily Truvada did in an earlier study by scientists from the US Centers for Disease Control (CDC). [1]

But two-dose PReP did not protect all animals in any of the four 6-monkey groups who got the drugs at different times relative to SHIV exposure.

Trials of daily Truvada PrEP are now under way in different human populations at high risk of HIV infection. An earlier monkey trial of emtricitabine alone and tenofovir/emtricitabine found better protection from SHIV with the two drugs. [2]

In the new study Gerardo Garcia-Lerma and CDC colleagues rectally exposed 24 male Rhesus macaques to SHIV, a simian immunodeficiency virus with an HIV coat, once weekly over 14 weeks. They split the monkeys into four groups of 6, giving human-equivalent doses of Truvada to each group through a mouth-to-stomach tube at different times:

• Group 1: 2 hours before and 22 hours after SHIV exposure
• Group 2: 22 hours before and 2 hours after SHIV exposure
• Group 3: 3 days before and 2 hours after SHIV exposure
• Group 4: 2 hours after and 26 hours after (postexposure prophylaxis, or PEP)

In a comparison group of 24 untreated monkeys, 23 became infected with SHIV (detected in plasma and blood cells) after a median of 2 rectal exposures (range 1 to 12). In contrast 3 of 6 group-1 animals did not pick up SHIV after 14 exposures, 5 of 6 group-2 animals remained free of SHIV after 14 exposures, 5 of 6 group-3 animals remained uninfected after 14 exposures, and (in an ongoing study) 3 of 6 group-4 animals are free of virus after 12 exposures.

Compared with the untreated macaques, group 1 had a 3.7-fold lower risk of SHIV infection (P = 0.04), group 2 had a 15.5-fold lower risk (P = 0.008), and group 3 had a 14.0-fold lower risk (P = 0.01). Risk of SHIV infection in these three groups did not differ significantly from infection risk in monkeys who received daily Truvada PrEP in the earlier trial. In animals that did become infected, viremia was about 10-fold lower than in control animals. Resistant virus did not emerge in animals whose PrEP failed.

Garcia-Lerma and coworkers speculated “the long intracellular drug half-life of tenofovir and emtricitabine may explain the extended window for protection by the pre-exposure dosing and provides opportunities for different intermittent PrEP regimens.”

In a separate study also reported by NATAP, tenofovir/FTC formulated in a stable vaginally applied gel protected 6 of 6 female pigtail macaques from 20 SHIV exposures. [3]

Source
http://www.natap.org

References

1. Garcia-Lerma J, Cong M, Masciotra S, et al. Intermittent pre-exposure prophylaxis (PrEP) with oral Truvada protects macaques against repeated rectal SHIV exposures. 3rd International Workshop on HIV Transmission: Principles of Intervention. 31 July -2 August 2008, Mexico City. Abstract 40.
2. Garcia-Lerma JG, Otten RA, Qari SH, et al. Prevention of rectal SHIV transmission in macaques by daily or intermittent prophylaxis with emtricitabine and tenofovir. PLoS Med. 2008;5(2):e28
http://medicine.plosjournals.org/perlserv/?request=getdocument&doi=10.1371/journal.pmed.0050028.
3. Parikh UM, Sharma S, Cong M, et al. Complete protection against repeated vaginal SHIV exposures in macaques by a combination emtricitabine and tenofovir topical gel. 3rd International Workshop on HIV Transmission: Principles of Intervention. 31 July -2 August 2008, Mexico City. Abstract 41.

Vaginal microbicide research in humans took a recent setback with reports that two products did not protect women from HIV infection. But neither of those microbicides (Carraguard and cellulose sulfate) used antiretroviral agents to ward off HIV.

Urvi Parikh and colleagues at the US Centers for Disease Control (CDC) and Emory University randomized 14 female pigtail macaques to three study groups: 2 received no gel, 6 received a placebo gel (hydroxyethyl cellulose only), and 6 got the tenofovir/FTC gel (5% FTC plus 1% tenofovir, which is equivalent to one Truvada dose, in 2% hydroxyethyl cellulose). [1]

The tenofovir/FTC gel was clear, odorless, viscous, and stable at 37 degrees Celsius for 6 months. Technicians applied the gels vaginally 30 minutes before challenging the monkeys with SHIV, a simian immunodeficiency virus with an HIV coat, at a dose of about 1,160,000 RNA copies. After exposure the investigators used blood tests and polymerase chain reaction to search for SHIV in plasma. The challenges occurred twice weekly for 10 weeks.

Both monkeys who got no gel and 5 of the 6 who got the placebo gel became infected after a median of 3.5 challenges (range 2 to 11). All 6 pigtails treated with tenofovir/FTC before SHIV exposure remained free of infection after 20 challenges (p < 0.005 versus control groups). The CDC team could detect no viral RNA, no proviral DNA, and no viral antibody in any of the 6 tenofovir/FTC-treated animals.

Parikh and coworkers detected low levels of FTC (median 67 ng/mL) and tenofovir (median 22 ng/mL) in plasma samples 30 minutes after vaginal application. Those findings, the investigators proposed, suggest “rapid drug absorption with relatively higher levels of drug remaining in vaginal tissue.”

Ongoing trials are testing oral tenofovir/FTC (Truvada) as pre-exposure prophylaxis in humans, and a separate study at this workshop showed that oral tenofovir/FTC partially protected male monkeys from rectal SHIV exposure. [2]

An earlier study of 1% tenofovir without FTC applied rectally in a single high dose found only a 60% protection rate. [3]

Double therapy clearly did better in this vaginal trial, but Charles Boucher (Erasmus University, Rotterdam) worried about the wisdom of developing drugs for both prevention and treatment because acquired resistance could compromise use of those and other drugs.

A vaginal tenofovir/FTC gel has yet to be tested in humans.

Source
http://www.natap.org

References

1. Parikh UM, Sharma S, Cong M, et al. Complete protection against repeated vaginal SHIV exposures in macaques by a combination emtricitabine and tenofovir topical gel. 3rd International Workshop on HIV Transmission: Principles of Intervention. 31 July -2 August 2008, Mexico City. Abstract 41.
2. Garcia-Lerma J, Cong M, Masciotra S, et al. Intermittent pre-exposure prophylaxis (PREP) with oral Truvada protects macaques against repeated rectal SHIV exposures. 3rd International Workshop on HIV Transmission: Principles of Intervention. 31 July -2 August 2008, Mexico City. Abstract 40.
3. Cranage M, Sharpe S, Cope A, et al. Pre-exposure prophylaxis in macaques against rectal SIV challenge by mucosally applied PMPA: potential for complementation of microbicide and vaccination strategies. 14th Conference on Retroviruses and Opportunistic Infections. February 25-28, 2007. Los Angeles. Abstract 29.

…In heterosexual couples with enough other risk factors, transmission risk can climb as high as 1-in-10 for penile-vaginal sex and 1-in-3 for penile-anal sex…..heterosexual sex can be a remarkably efficient way to transmit HIV….

One commonly cited estimate of heterosexual HIV-1 transmission risk – 1 infection per 1000 sexual acts – is probably inaccurate because it fails to account for other factors that raise or lower the risk of HIV transmission. [1]

That conclusion emerged from a multi-study analysis by Kimberly Powers (University of North Carolina at Chapel Hill), who pinpointed five variables that have a potent impact on heterosexual HIV transmission. With enough cofactors in play, Powers estimated that men and women risk transmitting the virus once every three times they have insertive sex.

The investigators suspected the 1-per-1000 ratio may be too low to explain raging heterosexual HIV epidemics in many countries, partly because it does not factor in sexually transmitted infections (STIs), HIV disease stage, circumcision, and other variables known to boost or blunt transmission risk. Yet that ratio gets cited time and again in government reports, peer-reviewed studies, and media offerings, leaving the impression that heterosexual coitus is a highly inefficient way of infecting a partner. To get a better handle on sexual transmission dynamics, the North Carolina team systematically searched published studies estimating heterosexual infectivity of HIV-1. Then they used statistical tools to sort out infectivity differences according to risk cofactors.

Powers found 27 studies involving 15 distinct populations. Transmission estimates varied strikingly from one study to the next, depending on these cofactors. Estimates ranged from 0 transmissions after more than 100 penile-vaginal contacts to 1 transmission for every 3.1 episodes of heterosexual anal intercourse. The multistudy statistical analysis weighing the impact of cofactors identified five variables that boosted risk of HIV transmission:

• Transmission 33.8 times more likely with penile-anal sex than penile-vaginal sex.
• Transmission 8 times more likely for uncircumcised versus circumcised men.
• Transmission 6 times more likely with than without a genital ulcer disease.
• Transmission 2.5 times more likely with early versus mid-stage HIV infection.
• Transmission 1.85 times more likely with late versus mid-stage HIV infection.

Powers and colleagues concluded that the 1-in-1000 estimate adequately represents transmission risk only in stable couples with low rates of other transmission risk factors. In other words, 1-in-1000 “represents a lower bound” of a capacious risk spectrum. In heterosexual couples with enough other risk factors, transmission risk can climb as high as 1-in-10 for penile-vaginal sex and 1-in-3 for penile-anal sex.

The investigators encouraged researchers to consider such cofactors in future infectivity estimates, and they advised public health officials and clinicians to emphasise that heterosexual sex can be a remarkably efficient way to transmit HIV. The study will be published next week in Lancet Infectious Diseases.

Source
http://www.natap.org

References

1. Powers K, Poole C, Pettifor A, Cohen M. Rethinking the heterosexual infectivity of HIV-1: A systematic review and meta-analysis. 3rd International Workshop on HIV Transmission: Principles of Intervention. July 31-August 2, 2008, Mexico City. Abstract 14.

An Australian research group, led by David Wilson from the University of New South Wales, Sydney, published a study in the Lancet modelling the potential impact of a low-level residual transmission risk. [1] This was in response to the Swiss Statement relating to low-to-zero risk of HIV transmission when an HIV-positive person had shown a durable response to treatment (> 6 months with viraemia suppressed to <50 copies/mL) and other conditions are met (good adherence, no other STIs etc). [2]

The model looked at the risk of unprotected sexual transmission per act and cumulatively over many exposures, within couples where one partner is HIV-positive and the other is HIV-negative.

They assumed that each couple had 100 sexual encounters per year, and calculated a cumulative probability of transmission to the HIV-negative partner each year. Transmission risk assumptions were based on the Rakai data (a heterosexual Ugandan study from 1991 looking at transmission risks in 415 sero-defferent couples). The Rakai group reported that each ten-fold increment in viral load is associated with a 2.45-fold (95% CI 1.85-3.26) increase in the risk of HIV transmission per sexual contact. [3]

The model used per-transmission rates (from studies that hadn’t factored the impact of viral load) from 0.001 to 0.0005 per exposure and an assumption that on-treatment viral load was 10 copies/mL (see Table 1). Current transmission rates were estimated assuming 80% condom use, and 95% effect protection from condom use.

This modelling suggested per exposure risk rates from an HIV-positive partner on ARV treatment, fulfilling the above criteria,from 1 in 43,000 for anal sex to less than 1 in 220,000 for vaginal sex.

This modelling suggested an approximate four-fold increase in risk across all three groups if sero-different stopped using condoms.

While this is interesting, it didn’t closely relate to the motivation behind the Swiss Statement, nor the clinical situations in which they suggested it be applied. These were i) ability to safely conceive a baby without dependence on sperm-washing (a procedure that is
difficult to access or afford and which carries a reduced conception rate) and ii) to allay anxiety and worry over perceived risk of infection during regular sex (using condoms) and in the event of a condom break.

For many HIV-negative partners, single exposure risks of between 1 in 43,000 for anal sex to less than 1 in 220,000 for vaginal sex may often be acceptable in the context of general quality of life for family planning or a less anxious, healthy, sex life.

Importantly, a weakness in the model, from assuming a linear relationship between viral load and transmission was raised both in the original article and in an accompanying comment by Geoffrey Garnet from UCL and Brian Gazzard from the Chelsea and Westminster Hospital, published in the same issue of the Lancet. [4]

Assuming a log-linear relationship that supposes a risk at every level of viral load they argued “extrapolates the model beyond the available data, assuming that there is a continuous reduction in risk rather than a threshold below which no transmission is possible”.

The model doesn’t address the likelihood that most sero-different couples are likely to continue to use both ARVs and condoms, or the additional reduction in transmission risk from ARVs in this setting.

The comment concludes “In many ways, the Swiss statement provides the opportunity for positive public-health messages, by promoting adherence to treatment and concern over other sexually transmitted infections. The use of condoms, in addition to antiretrovirals, to further reduce risk and prevent other sexually transmitted infections can then also be promoted.”

COMMENT

More than ten years after HAART has been able to reduce viral load to very low levels, it is notable that we have no prospective trial results looking at its impact on transmission, on either a population or individual level. Lack of data on whether treatment brings most people below a minimum threshold for transmission is clearly key in any further discussion.

The lack of data on transmission risk for anal sex (heterosexual and MSM) is also worryingly sparse although some research groups are looking to address this, hopefully with funding for sufficiently powered conclusions, and with some urgency.

The focus on ARVs as a prevention strategy, also a key topic at the Mexico conference, is long overdue. Many research groups have highlighted that 25-50% of new diagnoses are likely to be driven by people who are undiagnosed, especially those recently infected when vireamia is highest by a magnitude of several logs. [5-9]

Many groups have also reported that risk behaviour generally falls after diagnosis, especially once patients are within care. [10]

Increased testing, reducing late diagnosis and seeing treatment as protective of both health and transmission risk – rather than as something to delay until as late as possible – are likely to develop as increasingly important themes for managing the HIV epidemic in the 21st century.

References
1. Wilson DP, Law MG, Cooper D et al. Relation between HIV viral load and infectiousness: a model-based analysis.The Lancet: 372, p314-320. ( 26 July 2008).
http://www.thelancet.com/journals/lancet/article/PIIS0140673608611150/fulltext
2. Vernazza P, Hirschel B, Bernasconi E, Flepp M. HIV seropositive persons without sexually transmitted diseases under fully suppressive
antiretroviral treatment do not sexually transmit HIV. Bulletin des médecins Suisses 2008; 89:165-169.
3. Quinn TC, Wawer MJ, Sewankambo N, et al. Viral load and heterosexual transmission of HIV type 1. Rakai Project Study Group. N Engl J Med 2000; 342: 921-929.

http://www.nejm.org/doi/full/10.1056/NEJM200003303421303

4. Garnett GP, Gazzard B. Comment: Risk of HIV transmission in discordant couples. The Lancet 2008; 372:270-271. (26 July 2008).
http://www.thelancet.com/journals/lancet/article/PIIS0140673608610892/fulltext
5. Brenner BG et al. High rates of forward transmission events after acute/early HIV-1 infection. J Infect Dis 195: 951-59, 2007.
6. Hughes G et al. Recent phylodynamics of the HIV epidemic among MSM in the UK 15th Conference on Retroviruses and Opportunistic Infections, Boston. Abstract 13, 2008.
http://www.retroconference.org/2008/Abstracts/31334.htm
7. Yerly S et al. The contribution of individuals with recent infection to the spread of HIV-1 in Switzerland: a 10-year survey. 15th Conference on Retroviruses and Opportunistic Infections, Boston. Abstract 512, 2008.
http://www.retroconference.org/2008/Abstracts/31699.htm
8. Pao D et al. Transmission of HIV-1 during primary infection: relationship to sexual risk and sexually transmitted infections. AIDS. 2005 Jan 3;19(1):85-90.
http://www.ncbi.nlm.nih.gov/pubmed/15627037
9. Pilcher CD et al. Amplified transmission of HIV-1: comparison of HIV-1 concentrations in semen and blood during acute and chronic infection. AIDS 2007 Aug 20;21(13):1723-30.
http://www.ncbi.nlm.nih.gov/pubmed/17690570
10 Metsch LR et al. HIV transmission risk behaviors among HIV-infected persons who are successfully linked to care. Clinical Infectious Diseases 15 August 2008, Vol. 47, No. 4: 577-584.
http://www.journals.uchicago.edu/doi/abs/10.1086/590153

In the context of quantifying levels of risk relating to natural conception in serodifferent couples in relation to the recent Swiss Statement relating to undetectable viral load and risk of transmission [1], Anne-Geneviève Marcelin from Hôpital Pitié Salpêtrière and colleagues reported rates of discordance between levels of HIV RNA in blood and semen in a cohort of 145 HIV-positive men enrolled in an assisted reproductive (sperm-washing) programme in Paris. [2]

The group, writing in a research letter to the August 2008 issue of AIDS, found that 5% of men in this group had detectable HIV RNA in semen.

264 paired blood and semen samples were collected between January 2002 and January 2008 with some patients providing up to six samples. Viral load was quantified using tests sensitive to 40 copies/mL and 200 copies/mL in blood and in seminal plasma respectively.

Thirty-two blood plasma samples were detectable (median 6,325 copies/ml (range = 222-28,300). Sixteen seminal plasma samples were detectable and the median level of HIV-1 RNA in semen was 1770 copies/ml (range = 255-25,100).

Overall, 234 paired samples were concordant, with 225 samples with undetectable HIV-1 RNA both in blood and semen (85.3%) and nine with detectable HIV-1 RNA in blood and semen (3.4%). However, 23 blood samples had detectable HIV-1 RNA although the seminal viral load was undetectable and seven seminal samples had detectable HIV-1 RNA although the blood viral load was undetectable (range 257-1230 copies/mL).

These seven discordant paired samples corresponded to seven distinct patients who had undetectable viral load in blood for greater than six months and no current STI’s. Interestingly, 6/7 had undetectable concordant results in blood and semen on at least one occasion during follow-up indicating variability over time. Antiretroviral drug levels in semen showed no relationship between choice of drug and viraemia: 3TC, FTC, tenofovir and indinavir showed higher penetration but were also included in many of the regimens that these seven patients were using.

The researchers concluded that their findings justify measuring HIV-1 RNA in semen when sero-different couples are planning a pregnancy. They also cautioned that a residual risk of transmission relating to these discordant results should be included in the information available to couples who would like to have unprotected sexual intercourse in the context of conceiving a baby.

COMMENT

It is interesting that level of discordance was intermittent in the seven patients with detectable viral load in semen, and that levels of viral load were generally low (maximum 1200 copies/mL).

As with the previous article, establishing whether a minimum threshold exists for transmission remains a crucial research question.

RNA testing of semen in the context of minimizing risks in the context of conceiving a baby without sperm-washing is clearly an additional safety measure that should be used whenever possible – along with limiting conception attempts to the most fertile days of the woman’s cycle and possibly use of single-doses of tenofovir/FTC PrEP and PEP.

Reference

1. Vernazza P, Hirschel B, Bernasconi E, Flepp M. HIV seropositive persons without sexually transmitted diseases under fully suppressive antiretroviral treatment do not sexually transmit HIV. Bulletin des médecins Suisses 2008; 89:165-169.
2. Marcelin A-G et al. Detection of HIV-1 RNA in seminal plasma samples from treated patients with undetectable HIV-1 RNA in blood plasma. Research Letter. AIDS:Volume 22(13)20 August 2008p 1677-1679.

The new PLoS Medicine features a study conducted by Martin Cranage and colleagues evaluating tenofovir gel as a potential rectal microbicide in the SIV challenge model. The researchers report that application of the gel two hours prior to exposure to the SIVmac251/32H challenge virus protected six out of nine macaques. Of the remaining three, two showed lowered viral loads post-infection compared to controls. Interestingly, most of the protected animals also displayed detectable SIV-specific T cell responses even though sensitive assays could find no trace of virus.

The PLoS editor’s summary raises the concern that these SIV-specific T cells may be associated with enhanced susceptibility to infection upon re-exposure; however, Cranage et al note in their discussion that transient tenofovir treatment immediately post SIV infection has been shown to lead to induction of SIV-specific T cell responses, and macaques <http://jvi.asm.org/cgi/content/full/75/21/10187> in this study subsequently resisted both homologous and heterologous SIV challenges. The question of whether the SIV-specific T cell responses observed in Cranage’s study have the potential to be protective can only be definitively addressed by another experiment in which the macaques are re-challenged with SIV.

An additional implication of these data is that human trials of microbicides and pre-exposure prophylaxis (PrEP) should include monitoring for HIV-specific T cell responses.

Source:
TAG Basic Science Blog 06 Aug 2008
http://tagbasicscienceproject.typepad.com/tags_basic_science_vaccin/2008/08/tenofovir-gel-a.html

Ref:
Cranage M et al. Prevention of SIV Rectal Transmission and Priming of T Cell Responses in Macaques after Local Pre-exposure Application of Tenofovir Gel. PLoS Medicine Vol. 5, No. 8, e157 doi:10.1371/journal.pmed.0050157
http://medicine.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pmed.0050157

A new and complex study has identified a possible genetic influence on susceptibility to HIV acquisition that preferentially impacts Africans and people of recent African descent. [1] The paper is available free on the website of the journal Cell Host & Microbe (link below).

The major points are as follows:

• The Duffy Antigen Receptor for Chemokines (DARC) [2] on red blood cells can bind a slew of different chemokines and also HIV itself (X4-using HIV isolates much more than R5-using isolates).
• A genetic change (called a single nucleotide polymorphism or SNP) can lead to a lack of DARC receptors on red blood cells. This SNP is very common in Africans and people of recent African descent, because it once protected against a form of malaria caused by the pathogen Plasmodium vivax.
• In a large cohort of African American individuals from the US military, having the DARC-negative SNP was associated with a significantly increased risk of having HIV infection, even in multivariate analyses controlling for various confounding variables. However, the confidence intervals on the multivariate analyses ppear to approach a relative risk of 1.0 at the low end (in other words, based on the numbers in this study, it is within the bounds of possibility that the SNP has little impact on susceptibility). The result appears to rest on the finding that perhaps ~60% of 814 HIV-negative African Americans lacked the DARC receptor compared to ~70% of 470 HIV-infected African Americans. [3]
• If the result holds up, the fact the SNP is nearly ubiquitous among African populations could contribute to the higher incidence of HIV infection on that continent (the researchers estimate it might explain ~11%).
• The absence of the DARC receptor is associated with lower levels of CCL5 (formerly called RANTES), a chemokine with strong anti-HIV activity, providing a possible mechanistic explanation of the finding. A prior study has reported that persistently exposed, uninfected sex workers have ten-fold higher levels of CCL5 in the genital tract compared to both infected individuals and uninfected study participants who had recently started sex work and thus had little prior exposure to HIV. [4] Higher CCL5 production has also been reported in highly exposed but uninfected gay men. [5]
• Although at first blush it might sound paradoxical, the absence of DARC was also associated with a slight but significant slowing of disease progression in the HIV-infected members of the study cohort. The researchers suggest that this is likely explained by the association between the presence of DARC and higher levels of pro-inflammatory chemokines such as CCL2. Once infection occurs, the authors propose, the presence of DARC may exacerbate immune activation due to this association with elevated levels of pro-inflammatory chemokines (immune activation is the single strongest predictor of the pace of disease progression in people with HIV). While this hypothesis remains speculative, the authors argue it is supported by an association they have reported previously; in that case, a SNP that increases CCL2 levels in European Americans was shown to be linked to reduced susceptibility to acquisition of HIV infection and also faster disease progression in infected individuals. [6] Another non-exclusive possibility raised by the authors is that the absence of DARC slows progression by preventing transfer of DARC-bound HIV particles to CD4 T cells.

In discussing their results, the study authors acknowledge that there is a possibility that the associations they have observed are connected to an unknown factor or factors that are linked to the DARC SNP, and stress that confirmation of these results in other cohorts will be necessary to ensure they are valid. If it does turn out that there is a protective effect mediated by elevated levels of CCL5, this information could potentially assist vaccine development, as it would suggest that using adjuvants that elevate CCL5 levels and/or inducing HIV-specific T cell responses that produce CCL5 could be useful strategies for HIV vaccines.

Source
TAG Basic Science Blog. Expression of the Duffy antigen receptor for chemokines may impact susceptibility to HIV infection. (16 Jul 2008)
http://tagbasicscienceproject.typepad.com/tags_basic_science_vaccin/2008/07/expression-of-t.html

References

1. He W, Neil S, Ahuja SK et al. Duffy Antigen Receptor for Chemokines Mediates trans-Infection of HIV-1 from Red Blood Cells to Target Cells and Affects HIV-AIDS Susceptibility. Cell Host and Microbe, Vol 4, 52-62, 17 July 2008.
2. http://en.wikipedia.org/wiki/Duffy_antigen_system
3. http://www.cellhostandmicrobe.com/content/article/image?uid=PIIS193131280800190X&imageid=fig2
4. http://www.ncbi.nlm.nih.gov/pubmed/16088822
5. http://www.ncbi.nlm.nih.gov/pubmed/15166527
6. http://www.ncbi.nlm.nih.gov/pubmed/12374865

Free online access
http://www.cellhostandmicrobe.com/content/article/fulltext?uid=PIIS193131280800190X