Rebecca L.R. Powell

High frequency of HIV-1 dual infections among HIV-positive individuals in Cameroon, West Central Africa.

Objectives:To determine the frequency of dual inter- and intra-subtype HIV-1 infection among a cohort of 64 longitudinally-studied, HIV-1-positive individuals in Yaoundé, Cameroon. Methods:Blood was collected every 3-6 months for up to 36 months and RNA was extracted from plasma. Gag fragment (HxB2 location 1577-2040) was amplified by nested RT-PCR, and mixed-time-point Heteroduplex Assays (HDAs) were performed. As heteroduplexes in this assay indicate ≥5% genetic discordance in the gag fragment, their presence reveals dual infection. Results were confirmed by phylogenetic analysis. Results:Heteroduplexes were generated by specimens of 10 subjects (15.6%). Kaplan-Meier nonparametric estimate of maintenance of single infection was calculated; the rate/year of a 2nd infection was found to be ~11%. Dual infection was identified in the final specimens of five subjects, after as much as 18 months follow-up, while for the remaining five subjects, dual infection was identified in interim specimens within an average of 10 months follow-up. Analysis of samples obtained after dual infection from each of these latter five subjects revealed two patterns: reversion to initial strain, or replacement of initial strain. Four subjects were dually-infected with HIV-1 strains of the same subtype, while 6 were infected with different subtypes. Conclusions:The high prevalence of recombinant HIV-1 strains in Cameroon may in part be explained by the high frequency of dual infection. In this genetically-diverse HIV-1 milieu, dual infections and the recombinant viruses they generate are strongly driving viral evolution, complicating vaccine strategies.

HIV-1 reverse transcriptase drug-resistance mutations in chronically infected individuals receiving or naïve to HAART in Cameroon

The most common first‐line, highly active anti‐retroviral therapy (HAART) received by individuals infected with HIV‐1 in Cameroon is the combination therapy Triomune, comprised of two nucleoside reverse transcriptase inhibitors (NRTI) and one non‐NRTI (NNRTI). To examine the efficacy of these drugs in Cameroon, where diverse non‐B HIV‐1 subtypes and recombinant viruses predominate, the reverse transcriptase (RT) viral sequences in patient plasma were analyzed for the presence of mutations that confer drug resistance. Forty‐nine HIV‐1‐positive individuals were randomly selected from those receiving care in HIV/AIDS outpatient clinics in the South‐West and North‐West Regions of Cameroon. Among the 28 patients receiving HAART, 39% (11/28) had resistance to NRTIs, and 46% (13/28) to NNRTIs after a median of 12 months from the start of therapy. Among those with drug‐resistance mutations, there was a median of 14 months from the start of HAART, versus 9 months for those without; no difference was observed in the average viral load (10,997 copies/ml vs. 8,056 copies/ml). In contrast, drug‐naïve individuals had a significantly higher average viral load (27,929 copies/ml) than those receiving HAART (9,527 copies/ml). Strikingly, among the 21 drug‐naïve individuals, 24% harbored viruses with drug‐resistance mutations, suggesting that HIV‐1 drug‐resistant variants are being transmitted in Cameroon. Given the high frequency of resistance mutations among those on first‐line HAART, coupled with the high prevalence of HIV‐1 variants with drug‐resistance mutations among drug‐naïve individuals, this study emphasizes the need for extensive monitoring of resistance mutations and the introduction of a second‐line HAART strategy in Cameroon. J. Med. Virol. 82:187–196, 2010.

Superinfection by Discordant Subtypes of HIV-1 Does Not Enhance the Neutralizing Antibody Response against Autologous Virus

Recent studies have demonstrated that both the potency and breadth of the humoral anti-HIV-1 immune response in generating neutralizing antibodies (nAbs) against heterologous viruses are significantly enhanced after superinfection by discordant HIV-1 subtypes, suggesting that repeated exposure of the immune system to highly diverse HIV-1 antigens can significantly improve anti-HIV-1 immunity. Thus, we investigated whether sequential plasma from these subjects superinfected with discordant HIV-1 subtypes, who exhibit broad nAbs against heterologous viruses, also neutralize their discordant early autologous viruses with increasing potency. Comparing the neutralization capacities of sequential plasma obtained before and after superinfection of 4 subjects to those of matched plasma obtained from 4 singly infected control subjects, no difference in the increase in neutralization capacity was observed between the two groups (p = 0.328). Overall, a higher increase in neutralization over time was detected in the singly infected patients (mean change in IC50 titer from first to last plasma sample: 183.4) compared to the superinfected study subjects (mean change in IC50 titer from first to last plasma sample: 66.5). Analysis of the Breadth-Potency Scores confirmed that there was no significant difference in the increase in superinfected and singly infected study subjects (p = 0.234). These studies suggest that while superinfection by discordant subtypes induces antibodies with enhanced neutralizing breadth and potency against heterologous viruses, the potency to neutralize their autologous viruses is not better than those seen in singly infected patients.

Utility of the Heteroduplex Assay (HDA) as a Simple and Cost-Effective Tool for the Identification of HIV Type 1 Dual Infections in Resource-Limited Settings

The predominance of unique recombinant forms (URFs) of HIV-1 in Cameroon suggests that dual infection, the concomitant or sequential infection with genetically distinct HIV-1 strains, occurs frequently in this region; yet, identifying dual infection among large HIV cohorts in local, resource-limited settings is uncommon, since this generally relies on labor-intensive and costly sequencing methods. Consequently, there is a need to develop an effective, cost-efficient method appropriate to the developing world to identify these infections. In the present study, the heteroduplex assay (HDA) was used to verify dual or single infection status, as shown by traditional sequence analysis, for 15 longitudinally sampled study subjects from Cameroon. Heteroduplex formation, indicative of a dual infection, was identified for all five study subjects shown by sequence analysis to be dually infected. Conversely, heteroduplex formation was not detectable for all 10 HDA reactions of the singly infected study subjects. These results suggest that the HDA is a simple yet powerful and inexpensive tool for the detection of both intersubtype and intrasubtype dual infections, and that the HDA harbors significant potential for reliable, high-throughput screening for dual infection. As these infections and the recombinants they generate facilitate leaps in HIV-1 evolution, and may present major challenges for treatment and vaccine design, this assay will be critical for monitoring the continuing pandemic in regions of the world where HIV-1 viral diversity is broad.

A heteroduplex assay for the rapid detection of dual Human Immunodeficiency Virus Type 1 infections

The predominance of circulating and unique recombinant forms (URFs) of Human Immunodeficiency Virus Type 1 (HIV-1) in Cameroon suggests that dual infection occurs frequently in this region. Despite the potential impact of these infections on the evolution of HIV diversity, relatively few have been detected. The failure to detect dual infections may be attributable to the laborious and costly sequence analysis involved in their identification. As such, there is a need for a cost-effective, more rapid method to efficiently distinguish this subset of HIV-positive individuals, particularly in regions where HIV diversity is broad. In the present study, the heteroduplex assay (HDA) was developed to detect dual HIV-1 infection. This assay was validated on sequential specimens obtained from 20 HIV+ study subjects, whose single or dual infection status was determined by standard sequence analysis. By mixing gag fragments amplified from the sequential specimens from each study subject in HDA reactions, it was shown that single and dual infection status correlated with the absence and presence, respectively, of heteroduplex bands upon gel electrophoresis. Therefore, this novel assay is capable of identifying dual infections with a sensitivity and specificity equivalent to that of sequence analysis. Given the impact of dual infection on viral recombination and diversity, this simple technique will be beneficial to understanding HIV-1 evolution within an individual, as well as at a population level, in West-Central Africa and globally.

P04-02. Increased breadth and potency of the neutralizing antibody response among dually-HIV-1-infected individuals

Methods Two sequential plasma samples from 4 dually-intersubtype-infected subjects, obtained ~6 months before and >12 months after the 2nd infection was identified, were tested against 7 heterologous viruses (5 primary isolates and 2 Tier1 viruses) representing subtypes A1, B, F2, G, and CRF02_AG in the GHOST cell neutralization assay. Additionally, 23 singly-infected control subjects matched for disease stage, CD4 counts, and time between samples were studied. Plasma was assayed at 1:80 dilution to compare each plasma pair; subsequently, plasma from duallyinfected subjects and 6 control subjects were assessed for magnitude and specificity of neutralization using plasma serial dilutions, 1:20–1:640.