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The global intensification of antiretroviral therapy (ART) can lead to increased rates of HIV drug resistance (HIVDR) mutations in treated and also in ART-naive patients. ART-naive HIV-1-infected patients from Cameroon were subjected to a multimethod HIVDR analysis using amplification-refractory mutation system (ARMS)-PCR, Sanger sequencing, and longitudinal next-generation sequencing (NGS) to determine their profiles for the mutations K103N, Y181C, K65R, M184V, and T215F/Y. We processed 66 ART-naive HIV-1-positive patients with highly diverse subtypes that underlined the predominance of CRF02_AG and the increasing rate of F2 and other recombinant forms in Cameroon. We compared three resistance testing methods for 5 major mutation sites. Using Sanger sequencing, the overall prevalence of HIVDR mutations was 7.6% (5/66) and included all studied mutations except K65R. Comparing ARMS-PCR with Sanger sequencing as a reference, we obtained a sensitivity of 100% (5/5) and a specificity of 95% (58/61), caused by three false-positive calls with ARMS-PCR. For 32/66 samples, we obtained NGS data and we observed two additional mismatches made up of minority variants (7% and 18%) that might not be clinically relevant. Longitudinal NGS analyses revealed changes in HIVDR mutations in all five positive subjects that could not be attributed to treatment. In one of these cases, superinfection led to the temporary masking of a resistant virus. HIVDR mutations can be sensitively detected by ARMS-PCR and sequencing methods with comparable performances. Longitudinal changes in HIVDR mutations have to be considered even in the absence of treatment.

Current serological assays that are used for cross-sectional HIV incidence estimation have been shown to misclassify individuals with chronic infection. Limited information exists on the performance of cross-sectional incidence assays in Central Africa. HIV-positive individuals from Cameroon who were infected for at least 1 or 2 years were evaluated to determine the false recent ratio (FRR) of a two-assay algorithm, which includes the Limiting Antigen Avidity (LAg-Avidity) assay (normalized optical density units, ODn 1000 copies/ml). The subject-level FRR was 5.3% (95% confidence interval [CI], 2.1–10.5) for individuals infected for ≥1 year and 3.9% (95% CI, 0.8–11.0) for individuals infected for ≥2 years. These data suggest that the LAg-Avidity plus viral load incidence algorithm may overestimate HIV incidence rates in Central Africa.

HIV superinfection describes the sequential infection of an individual with two or more unrelated HIV strains. Intersubtype superinfection has been shown to cause a broader and more potent heterologous neutralizing antibody response when compared to singly infected controls, yet the effects of intrasubtype superinfection remain controversial. Longitudinal samples were analyzed phylogenetically for pol and env regions using Next-Generation Sequencing and envelope cloning. The impact of CRF02_AG intrasubtype superinfection was assessed for heterologous neutralization and antibody binding responses. We compared two cases of CRF02_AG intrasubtype superinfection that revealed complete replacement of the initial virus by superinfecting CRF02_AG variants with signs of recombination. NYU6564, who became superinfected at an early time point, exhibited greater changes in antibody binding profiles and generated a more potent neutralizing antibody response post-superinfection compared to NYU6501. In contrast, superinfection occurred at a later time point in NYU6501 with strains harboring significantly longer V1V2 regions with no observable changes in neutralization patterns. Here we show that CRF02_AG intrasubtype superinfection can induce a cross-subtype neutralizing antibody response, and our data suggest timing and/or superinfecting viral envelope characteristics as contributing factors. These results highlight differential outcomes in intrasubtype superinfection and provide the first insight into cases with CRF02_AG, the fourth most prevalent HIV-1 strain worldwide.

BACKGROUND—Since the identification of xenotropic murine leukemia virus–related virus (XMRV) in prostate cancer patients in 2006 and in chronic fatigue syndrome patients in 2009, conflicting findings have been reported regarding its etiologic role in human diseases and prevalence in general populations. In this study, we screened both plasma and peripheral blood mononuclear cells (PBMNCs) collected in Africa from blood donors and human immunodeficiency virus Type 1 (HIV-1)-infected individuals to gain evidence of XMRV infection in this geographic region. STUDY DESIGN AND METHODS—A total of 199 plasma samples, 19 PBMNC samples, and 50 culture supernatants from PBMNCs of blood donors from Cameroon found to be infected with HIV-1 and HIV-1 patients from Uganda were screened for XMRV infection using a sensitive nested polymerase chain reaction (PCR) or reverse transcription (RT)-PCR assay. RESULTS—Using highly sensitive nested PCR or RT-PCR and real-time PCR assays capable of detecting at least 10 copies of XMRV plasmid DNA per reaction, none of the 268 samples tested were found to be XMRV DNA or RNA positive. CONCLUSIONS—Our results failed to demonstrate the presence of XMRV infection in African blood donors or individuals infected with HIV-1. More studies are needed to understand the prevalence, epidemiology, and geographic distribution of XMRV infection worldwide.

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.

Broad HIV-1 genetic diversity in Cameroon provides a unique opportunity to monitor HIV-1 evolution and allows the detection of novel strains. We have genetically characterized the HIV-1 subtypes found in 156 samples from 90 drug-naive subjects in Yaounde, Cameroon collected from 2011 to 2013, using phylogenetic analysis of regions in gag and pol. We identified subtypes CRF02_AG (64.9%), CRF22_01A1 (7.1%), D (4.5%), F2 (3.9%), G (3.2%), CRF18_cpx (3.2%), CRF37_cpx (3.2%), CRF11_cpx (2.6%), CRF13_cpx (1.9%), A1 (1.3%), CRF01_AE (1.3%), CRF09_cpx (1.3%), A2 (0.6%), and H (0.6%). Sequence data for both the gag and pol regions were obtained from 62 subjects; for 59 of these subjects the two regions were identified as the same viral subtype while three subjects were discordant, A1/CRF02_AG (subject MDC006), CRF02_AG/F2 (subject MDC179), and a dual infection with CRF02_AG/F2 (subject MDC131). Longitudinal sequence data were obtained for 28 of these 62 subjects and confirmed the cross-sectional results. These data update subtype information for this area and highlight the necessity of such studies due to the numerous circulating subtypes, the ongoing superinfection, and the risk of emerging novel recombinant viruses.

The HIV vaccine-induced neutralizing antibodies (Abs) display low rates of mutation in their variable regions. To determine the range of neutralization mediated by similar human monoclonal Abs (mAbs) but derived from unselected chronically HIV-1 infected subjects, we tested a panel of 66 mAbs specific to V3, CD4 binding site (CD4bs) and V2 regions. The mAbs were tested against 41 pseudoviruses, including 15 tier 1 and 26 tier 2, 3 viruses, showing that the neutralization potency and breadth of anti-V3 mAbs were significantly higher than those of the anti-CD4bs and anti-V2 mAbs, and only anti-V3 mAbs were able to neutralize some tier 2, 3 viruses. The percentage of mutations in the variable regions of the heavy (VH) and light (VL) chains varied broadly in a range from 2% to 18% and correlated moderately with the neutralization breadth of tier 2, 3 viruses. There was no correlation with neutralization of tier 1 viruses as some mAbs with low and high percentages of mutations neutralized the same number of viruses. The electrostatic interactions between anti-V3 mAbs and the charged V3 region may contribute to their neutralization because the isoelectric points of the VH CDR3 of 48 anti-V3 mAbs were inversely correlated with the neutralization breadth of tier 2, 3 viruses. The results demonstrate that infection-induced antibodies to CD4bs, V3 and V2 regions can mediate cross-clade neutralization despite low levels of mutations which can be achieved by HIV-1 vaccine-induced antibodies.

Access to genotyping assays to determine successful antiretroviral treatment (ART) is limited in resource-constrained settings by high cost, suggesting the need for a cost-effective and simplified method to identify HIV-1 drug resistance (HIVDR) mutations. In this study, an amplification refractory mutation system (ARMS)-PCR assay was developed and used to investigate the most frequent HIVDR mutations affecting first-line ART in settings where WHO ART guidelines are applied. Seventy-five HIV-positive (HIV+) samples from Cameroon were used to assess the performance of this assay. Sequencing of HIV-1 reverse transcriptase was simultaneously performed for comparison, and discordant samples were tested with a Trugene HIV-1 genotyping kit. The ARMS-PCR assay was able to detect M184V, T215Y/F, K103N, and Y181C mutations with sensitivities of 96.8%, 85.7%, 91.3%, and 70%, respectively, and specificities of 90.6%, 95%, 100%, 96.9%, respectively, compared with data on sequencing. The results indicated the highest positive predictive value for K103N (100%) and the highest negative predictive value for M184V (97.5%). ARMS-PCR's limits of detection for mutations M184V, T215Y/F, K103N, and Y181C were

As people infected with the human immunodeficiency virus (HIV) in Sub-Saharan Africa live longer due to availability of antiretroviral treatment (ART), so is the rise of associated infections with their burdens on patients. But reliable data on the prevalence of co-infection with hepatitis B (HBV) or C (HCV) still remains sparse and many individuals with HIV do not know their co-infection status. This study attempted to estimate the seroprevalence and identify risk factors associated with hepatitis B and/or C co-infections in HIV-infected individuals from five Regions of Cameroon by screening 531 HIV infected subjects for the presence of HBV surface antigen (HBsAg) and antibodies to HCV (HCV-Ab). A Screening and a confirmatory Enzyme linked immunosorbent assay were used to detect presence of markers of infection. CD4 count levels were also examined. The results indicate that of the 531 participants, 68% were females and 32% males. Mean CD4 count was ~400 cells/μl. Seroprevalence rates for HBsAg and HCV-Ab were 23.7%, and 7.2%, respectively. Associations assessed using logistic regression revealed that HBsAg but not HCV-Ab positivity was linked to age, lower CD4 count and residing in an urban rather than in a rural setting. This high prevalence of co-infection with HBV raises the urgent need to systematically screen all newly diagnosed HIV cases for co-infection in Cameroon and other regions of sub-Saharan Africa where HIV accounts for the majority of the global infection, so as to improve management strategies for HBV infection and ART implementation.

Here we studied the patterns of generation of neutralizing antibodies (NAbs) and virus escape during non-B subtype HIV-1 chronic infection among asymptomatic patients, and established whether a correlation exists between the generation of NAbs and the kinetics of CD4 T-cell decline. Therefore, sequential viruses and plasma obtained at 6 months to one year intervals over a three years period from ten HIV-1 group M subtype A, CRF02_AG, G, and H infected treatment-naive individuals were tested in neutralization assays. Overall, NAbs were present in all ten individuals, and had the capacity to neutralize autologous virus obtained six months earlier. Eight of the ten subjects showed an increasing capacity to neutralize early viruses and a low capacity to neutralize contemporaneous and later time-point viruses. The neutralizing activities within these individuals resulted in emergence of neutralization resistant viruses, and with the subsequent generation of more NAbs to the emerging resistant viruses. In the remaining two individuals, the capacity to neutralize early, contemporaneous, and later time-point viruses remained conserved. While the kinetics of CD4 T-cell decline varied among all ten individuals, there was no correlation with the capacity to generate NAbs in that, sequential plasmas from individuals with moderately or rapidly declining CD4 T-cells were capable of neutralizing early sequential viruses. We conclude from this study that in non-B subtype chronically infected asymptomatic patients with moderately and rapidly declining CD4 T-cells, potent NAbs are readily generated as the virus evolves to escape the effect of these antibodies.

Both polyclonal and monoclonal human antibodies (Abs) to the V3 domain of HIV-1 gp120 display cross-clade neutralizing activity against primary isolates and T cell-adapted virus strains. The most broadly neutralizing of the human anti-V3 monoclonal Abs (mAbs), 447-52D, recognizes 14 amino acids, including the GPxR core epitope at the tip of the V3 loop. Monoclonal Ab 447-52D neutralized 92% of 38 primary isolates carrying the GPGR V3 motif regardless of whether the viruses belonged to clades A, B, F, or H; in contrast, none of 19 viruses with the GPGQ and other non-GPGR/Q sequences at the tip of the V3 loop was sensitive to mAb 447-52D. These data are consistent with the crystallographic resolution of a complex of the Fab fragment of mAb 447-52D with a V3 peptide that shows that the binding specificity of the mAb is due to recognition of the GPGR motif at the tip of the loop. The critical role of the Arg residue in this motif was determined using viruses pseudotyped with the envelope of primary isolate CA1 containing the GPGR motif or with a mutated envelope with a Gln (Q) replacing the Arg (R) at the tip of the loop. While the wild-type pseudovirus was neutralized by mAb 447-52D, the pseudovirus carrying the point mutation was resistant to neutralization. These data illuminate the structural basis for both the breadth and specificity of a broadly neutralizing human mAb and contribute to our understanding of the epitopes recognized by Abs that protect against infection with HIV-1.

Objective: To document infection with HIV type 1 (HIV-1) group M non-B subtypes in individuals living in New York City. Design: From October 1999 through April 2003, HIV-1-seropositive individuals were selected from 3 clinics in New York City based on having risk factors for infection with HIV-1 non-B subtypes. Methods: HIV-1 RNA was extracted from plasma samples, and partial gag, pol, or env genes were amplified by PCR analysis. The infecting HIV- 1 group M subtype was determined based on results of either heteroduplex mobility assay or sequencing and phylogenetic analysis. Results: Ninety-seven subjects were enrolled in the study. Of the 97 subjects, 91 (94%) were selected based on having emigrated from a non-European country, while 6 (6%) were native United States citizens. Subtypes were successfully determined in 53 (55%) of the 97 plasma samples tested. The subtypes in 2 plasma samples were unclassifiable. HIV-1 infections were classified as those due to the following group M subtypes: A (n = 4; 7%), B (n = 12; 22%), C (n = 8; 15%), F (n = 2; 4%), CRF01―AE-like (n = 7; 13%), CRF02―AG-like (n = 19; 34%), an intersubtype recombinant form G gag /A env (n = 1; 2%), and unclassifiable viruses (n = 2; 4%). Conclusion: This study reveals infection with a broad variety of HIV-1 group M subtypes mostly in the immigrant population of New York City as well as how several non-B subtypes are being introduced into the United States.

Background: The use of CCR5 antagonists involves determination of HIV-1 tropism prior to initiation of treatment. HIV-1 tropism can be assessed either by phenotypic or genotypic methods. Genotypic methods are extensively used for tropism prediction. However, their validation in predicting tropism of viral isolates belonging to group M non-B subtypes remains challenging. In Cameroon, the genetic diversity of HIV-1 strains is the broadest reported worldwide. To facilitate the integration of CCR5 antagonists into clinical practice in this region, there is a need to evaluate the performance of genotypic methods for predicting tropism of highly diverse group M HIV-1 strains. Methods: Tropism of diverse HIV-1 strains isolated from PBMCs from Cameroon was determined using the GHOST cell assay. Prediction, based on V3 sequences from matched plasma samples, was determined using bioinformatics algorithms and rules based on position 11/25 and net charge applied independently or combined according to Delobel’s and Garrido’s rules. Performance of genotypic methods was evaluated by comparing prediction generated with tropism assigned by the phenotypic assay. Results: Specificity for predicting R5-tropic virus was high, ranging from 83.7% to 97.7% depending on the genotypic methods used. Sensitivity for X4-tropic viruses was fairly low, ranging from 33.3% to 50%. In our study, overall, genotypic methods were less able to accurately predict X4-tropic virus belonging to subtype CRF02_AG. In addition, it was found that of the methods we used the Garrido rule has the highest sensitivity rate of over 50% with a specificity of 93%. Conclusion: Our study demonstrated that overall, genotypic methods were less sensitive for accurate prediction of HIV-1 tropism in settings where diverse HIV-1 strains co-circulate. Our data suggest that further optimization of genotypic methods is needed and that larger studies to determine their utility for tropism prediction of diverse HIV-1 strains may be warranted.

In order to protect against organisms that exhibit significant genetic variation, polyvalent vaccines are needed. Given the extreme variability of human immunodeficiency virus type 1 (HIV-1), it is probable that a polyvalent vaccine will also be needed for protection from this virus. However, to understand how to construct a polyvalent vaccine, serotypes or immunotypes of HIV must be identified. In the present study, we have examined the immunologic relatedness of intact, native HIV-1 primary isolates of group M, clades A to H, with human monoclonal antibodies (MAbs) directed at epitopes in the V3, C5, and gp41 cluster I regions of the envelope glycoproteins, since these regions are well exposed on the virion surface. Multivariate analysis of the binding data revealed three immunotypes of HIV-1 and five MAb groups useful for immunotyping of the viruses. The analysis revealed that there are fewer immunotypes than genotypes of HIV and that clustering of the isolates did not correlate with either genotypes, coreceptor usage (CCR5 and CXCR4), or geographic origin of the isolates. Further analysis revealed distinct MAb groups that bound preferentially to HIV-1 isolates belonging to particular immunotypes or that bound to all three immunotypes; this demonstrates that viral immunotypes identified by mathematical analysis are indeed defined by their immunologic characteristics. In summary, these results indicate (i) that HIV-1 immunotypes can be defined, (ii) that constellations of epitopes that are conserved among isolates belonging to each individual HIV-1 immunotype exist and that these distinguish each of the immunotypes, and (iii) that there are also epitopes that are routinely shared by all immunotypes.

To be successful with strategies involving passive immunization or the generation of neutralizing antibodies against HIV, it is crucial that we improve our understanding of the process of antibody-mediated HIV neutralization. We have used a neutralization assay based on polymerase chain reaction (PCR) that is more rapid and sensitive than the conventional p24 neutralization assay based on enzyme-linked immunosorbent assay (ELISA). PCR assays permit measurement of the number of infectious events and can detect small amounts of HIV-1 only a few days postinfection. In these studies, the human anti-V3 monoclonal antibody 694/98-D was used to neutralize the infectivity of the laboratory isolate HIVIIIB for CEM-SS cells. 8E5/LAV cells, which contain a single integrated copy of proviral DNA per cell, served as a standard to determine the amount of HIV-1 copies in infected CEM-SS cells. Evaluation of antibody-mediated neutralization was possible at 2 to 3 days postinfection, at a time when p24 readouts were not conclusive. We achieved >95% neutralization of HIVIIIB, and of its molecular clone HXB2, using the monoclonal antibody 694/98-D. This degree of neutralization is probably highly significant in vivo. Nevertheless, a small amount of both HIVIIIB and HXB2 ( approximately 5%) escapes neutralization and can consistently be detected after a few days by this sensitive assay. Experiments with different anti-HIV monoclonal antibodies and viruses showed that the assay could be applied to anti-V3 as well as anti-CD4 binding domain antibodies as well as HIV laboratory strains or primary isolates.

A previous study on cross-clade neutralization activity, identified three key isolates, MNlab (envB/gagB; X4 coreceptor), VI525 (envG/gagH, envA/gagA; R5X4) and CA9 (Group O; R5), that allowed discrimination of sera, likely or unlikely to neutralize primary HIV-1 isolates belonging to Group M (env clades A–H) and Group O. The prognostic ability of these three isolates was verified by means of an external validation on a different and larger set of sera. A total of 79 different sera (66 HIV-1, 10 HIV-2, 1 HIV-1+2 and 2 SIVcpz) were examined first for their capacity to neutralize the three key isolates, next sera were challenged against 12 other primary HIV-1 isolates of Group M (env A–H) and 2 isolates of Group O. Sera that neutralized all three isolates with an ID50 titer of ≥1/40, also neutralized the 14 other primary isolates belonging to different genetic groups and clades. Sera that did not neutralize all three isolates did not exert broad cross-neutralizing activity. The neutralizing activity was antibody-mediated because it was absorbed and eluted from a Prot-G column. Competition-neutralization experiments using recombinant gp120 (HIV-1 MNlab) reduced the neutralizing capacity, suggesting that the neutralizing antibodies were directed against the Env protein. Remarkably, the broad cross-neutralization activity was found primarily in African female patients. In conclusion, this study confirms that three isolates are sufficient to allow identification of broad cross-neutralizing sera. J. Med. Virol. 61:14–24, 2000. © 2000 Wiley-Liss, Inc.

To study the antigenic conservation of epitopes of human immunodeficiency virus type 1 (HIV-1) isolates of different clades, the abilities of human anti-HIV-1 gp120 and gp41 monoclonal antibodies (MAbs) to bind to intact HIV-1 virions were determined by a newly developed virus-binding assay. Eighteen human anti-HIV MAbs, which were directed at the V2, V3 loop, CD4-binding domain (CD4bd), C5, or gp41 regions, were used. Nine HIV-1 isolates from clades A, B, D, F, G, and H were used. Microtiter wells were coated with the MAbs, after which virus was added. Bound virus was detected after lysis by testing for p24 antigen with a noncommercial p24 enzyme-linked immunosorbent assay. The anti-V3 MAbs strongly bound the four clade B viruses and viruses from the non-B clades, although binding was weaker and more sporadic with the latter. The degrees of binding by the anti-V3 MAbs to CXCR4- and CCR5-tropic viruses were similar, suggesting that the V3 loops of these two categories of viruses are similarly exposed. The anti-C5 MAbs bound isolates of clades A, B, and D. Only weak and sporadic binding of all the viruses tested with anti-CD4bd, anti-V2, and anti-gp41 MAbs was detected. These results suggest that V3 and C5 structures are shared and well exposed on intact virions of different clades compared to the CD4bd, V2, and gp41 regions.

The HIV epidemic in Cameroon is marked by a broad genetic diversity dominated by circulating recombinant forms (CRFs). Studies performed more than a decade ago in urban settings of Southern Cameroon revealed a dominance of the CRF02_AG and clade A variants in >90% of the infected subjects; however, little is known about the evolving viral variants circulating in this region. To document circulating HIV viral diversity, four regions of the viral genome (gag, PR, reverse transcriptase, env) in 116 HIV-1 positive individuals in Limbe, Southern Cameroon, were PCR-amplified. Sequences obtained at the RT and protease regions were analyzed for mutations that conferred drug resistance using the Stanford Drug Resistance Database. The present study reveals a broad genetic diversity characterized by several unique recombinant forms (URF) accounting for 36% of infections, 48.6% of patients infected with CRF02_AG, and the emergence of CRF22_01A1 in 7.2% of patients. Three out of 15 (20%) treated patients and 13 out of 93 (13.9%) drug naive patients harbor drug resistance mutations to RT inhibitors, while 3.2% of drug naive patients harbor drug resistance mutations associated with protease inhibitors. The high proportion (13.9%) of drug resistance mutations among the drug naive patients reveals the ongoing transmission of these viruses in this region of Cameroon and highlights the need for drug resistance testing before starting treatment for patients infected with HIV-1. J. Med. Virol. 86:385–393, 2014. © 2013 The Authors. Journal of Medical Virology Published by Wiley Periodicals, Inc.

The global scale-up of antiretroviral (ARV) therapy has dramatically reduced human immunodeficiency virus (HIV) morbidity and mortality. Stavudine (d4T) has been among the most commonly used ARVs in resource-limited settings because of its efficacy, short-term tolerability, low cost, and availability in coformulated form. However, many countries are transitioning away from d4T, mainly because of mitochondrial toxicities associated with long-term d4T use. In 2010, the World Health Organization (WHO) recommended phasing out d4T even in patients without documented virological failure [1]. There are two distinct mutational pathways of d4T resistance with different implications for zidovudine (AZT) and tenofovir (TDF) cross-resistance, the primary candidates for replacing d4T [2–4]. However, genotypic resistance testing is usually not available in the resource-limited settings where d4T-containing regimens are most commonly used. It is therefore important to determine the extent of nucleoside reverse-transcriptase inhibitor (NRTI) cross-resistance in viruses from individuals with virological failure on d4T-containing first-line therapy undergoing genotypic resistance testing. In this study, we combined genetic sequence data from 35 different studies to characterize the patterns of NRTI resistance mutations in viruses from individuals with virological failure on the 2 widely used d4T-containing ARV regimens: d4T/lamivudine(3TC)/nevirapine(NVP) and d4T/3TC/efavirenz(EFV). We also examine the effects of HIV-1 subtype, duration of therapy, and concomitant nonnucleoside reverse-transcriptase inhibitor (NNRTI) on the selection of specific mutations. We focused our analysis on NRTI resistance mutations with differential effects on the residual activity of AZT compared with TDF. The implications of these data for optimal replacement of d4T and subsequent NRTI options are discussed.

Background 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. We investigated whether sequential plasma from subjects superinfected with discordant HIV-1 subtypes, who exhibit broad nAbs against heterologous viruses, also neutralize either or both of their discordant early autologous viruses with increasing potency.

Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.

To further refine our current nanoparticle-based HIV-1 p24 antigen assay, we investigated immune responses to p24 to identify diagnostically significant immune dominant epitopes (IDEs) in HIV-infected human sera, to address cross-reactivity of anti-p24 antibodies to different subtypes, and to identify new biomarkers that distinguish acute from chronic HIV infection for more accurate incidence estimation. We identified two major linear epitope regions, located in the CypA binding loop and adjacent helices and at the end of the C-terminal domain. Most sera (86%) from acutely HIV-1-infected individuals reacted with multiple peptides, while 60% and 30% of AIDS patient samples reacted with multiple and single peptides, respectively. In contrast, 46% and 43% of chronically HIV-1-infected individuals reacted with one and none of the peptides, respectively, and only 11% reacted with multiple p24 peptides, indicating a progression of immune responses from polyclone-like during acute infection to monoclone-like or a nonresponse to linear epitopes during chronic infection. Anti-p24 antibodies (subtype B) show broad cross-reactivity to different HIV-1 subtypes, and the synergistic action of different combinations of anti-HIV antibodies improves capture and detection of divergent HIV-1 subtypes. Our results indicate that the modified peptide immunoassay is sensitive and specific for the rapid identification of HIV-1 p24 IDEs and for investigation of immune responses to p24 during natural HIV-1 infection. The data provide the foundation for development and refinement of new assays for improved p24 antigen testing as future tools for rapid and accurate diagnosis as part of early intervention strategies and estimations of incidence.

Background Antibodies induced by a vaccine that will protect against infection by HIV-1 will be heterologous, yet, little is known on how virus evolution affects neutralization sensitivity to heterologous antibodies. Therefore, experiments were performed to test the sensitivity of sequential HIV-1 primary isolates to anti-HIV-1 monoclonal antibodies (mAbs) and to analyze changes in the neutralization epitopes.

To design a vaccine that will remain potent against HIV-1, the immunogenic regions in the viral envelope that tend to change as well as those that remain constant over time must be identified. To determine the neutralization profiles of sequential viruses over time and study whether neutralization patterns correlate with sequence evolution, 12 broadly neutralizing plasmas from HIV-1 subtype B-infected individuals were tested for their ability to neutralize sequential primary HIV-1 subtype B viruses from four individuals. Three patterns of neutralization were observed, including a loss of neutralization sensitivity by viruses over time, an increase in neutralization sensitivity by sequential viruses, or a similarity in the sensitivity of sequential viruses to neutralization. Seven to 11 gp160 clones from each sequential virus sample were sequenced and analyzed to identify mutational patterns. Analysis of the envelope sequences of the sequential viruses revealed changes characteristic of the neutralization patterns. Viruses that evolved to become resistant to neutralizing antibodies also evolved with diverse sequences, with most of the changes being due to nonsynonymous mutations occurring in the V1/V2, as well as in the constant regions (C2, C3, C4), the most changes occurring in the C3. Viruses from the patient that evolved to become more sensitive to neutralization exhibited less sequence diversity with fewer nonsynonymous changes that occurred mainly in the V1/V2 region. The V3 region remained constant over time for all the viruses tested. This study demonstrates that as viruses evolve in their host, they either become sensitive or resistant to neutralization by antibodies in heterologous plasma and mutations in different envelope regions account for these changes in their neutralization profiles.

Human anti-V3 monoclonal antibodies (mAbs) generated from HIV-1 infected individuals display diversity in the range of their cross-neutralization that may be related to their immunogenetic background. The study of the immunoglobulin (Ig) variable region gene usage of heavy chains have shown a preferential usage of the VH5-51 gene segment which was detected in 35% of 51 human anti-V3 mAbs. In contrast, human mAbs against other envelope regions of HIV-1 (anti-Env), including the CD4-binding domain, the CD4-induced epitope, and gp41 preferentially used the VH1-69 gene segment, and none of them used the VH5-51 gene. Furthermore, the usage of the VH4 family by anti-V3 mAbs was restricted to only one gene segment, VH4-59, while the VH3 gene family was used at a significantly lower frequency by all of the analyzed anti-HIV-1 mAbs. Multivariate analysis showed that usage of VH gene segments was significantly different between anti-V3 and anti-Env mAbs, and compared to antibodies from healthy subjects. In addition, the anti-V3 mAbs preferentially used the JH3 and D2-15 gene segments. The preferential usage of selected Ig gene segments and the characteristic pattern of Ig gene usage by anti-V3 mAbs can be related to the conserved structure of the V3 region.

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.

The HIV-1 epidemic among men who have sex with men (MSM) has been spreading throughout China. Shanghai, a central gathering place for MSM, is facing a continuously increasing incidence of HIV-1 infection. In order to better understand the dynamics of HIV-1 diversity and its influence on patient's immune status at baseline on diagnosis, 1265 newly HIV-1-infected MSM collected from January 2009 to December 2013 in Shanghai were retrospectively analyzed for genetic subtyping, CD4+T cell counts, and viral loads. HIV-1 phylogenetic analysis revealed a broad viral diversity including CRF01_AE (62.13%), CRF07_BC (24.51%), subtype B (8.06%), CRF55_01B (3.24%), CER67_01B (0.95%), CRF68_01B (0.4%), CRF08_BC (0.08%) and CRF59_01B (0.08%). Twenty-four unique recombination forms (URFs) (1.98%) were identified as well. Bayesian inference analysis indicated that the introduction of CRF01_AE strain (1997) was earlier than CRF07_BC strain (2001) into MSM population in Shanghai based on the time of the most recent common ancestor (tMRCA). Three epidemic clusters and five sub-clusters were found in CRF01_AE. Significantly lower CD4+T cell count was found in individuals infected with CRF01_AE than in those infected with CRF07_BC infection (P45 years of age were found to have lower CD4+T cell counts and higher viral loads than the patients with

The majority of global human immunodeficiency virus infections are caused by viruses characterized by a GPGQ motif at the tip of the V3 loop. Characterization of anti-V3 monoclonal antibodies (MAbs) that neutralize isolates with the GPGQ V3 motif is an important step in designing vaccines that will induce such Abs. Consequently, seven human anti-V3 MAbs derived from the cells of individuals infected with non-B-subtype viruses (anti-V3non-BMAbs) were generated from the cells of individuals from Africa infected with circulating recombinant forms CRF02_AG, CRF09_cpx, and CRF13_cpx, each of which contains a subtype Aenvgene. Sequence analysis of plasma viruses revealed a GPGQ motif at the apex of the V3 loop from six of the seven subjects and a GPGR motif from one subject. The MAbs were selected with fusion proteins (FP) containing V392UG037.8or V3JR-CSFfrom subtype A or B, respectively. In virus binding assays, five of the seven (71%) anti-V3non-BMAbs bound to V3-FPs from both subtype A and subtype B, while only four of the nine (44%) anti-V3BMAbs recognized both V3-FPs. Using two neutralization assays, both the anti-V3non-Band the anti-V3BMAbs neutralized subtype B viruses with similar activities, while the anti-V3non-BMAbs exhibited a tendency toward both increased potency and breadth of neutralization against non-B viruses compared to anti-V3BMAbs. Statistical significance was not achieved, due in large measure to the sizes of the MAb panels, but the overall pattern of data strongly suggests that viruses with the GPGQ motif at the tip of the V3 loop induce anti-V3 Abs with broader cross-neutralizing activity than do viruses with the GPGR motif.

To describe the presence of protease inhibitor (PI) resistance-associated mutations and subtype distribution in drug-naive villagers of six provinces of Cameroon, we sequenced the protease (PR) gene (297 bp) of 128 viruses. Secondary PI resistance-associated mutations were identified at five sites: L10I/V (16%), K20R (8%), M36I (98%), L63P (13%), and V77I (6%). No primary mutation in the PR was identified. Of the 128 specimens analyzed, subtypes A (11%), C(2%), D (6%), F2 (3%), G (6%), H (0.8%), J (6%), and CRF02_AG (60%) were identified. The mutations identified were not characteristic to any particular subtype. The absence of primary mutations, in addition to the few secondary mutations, gives good perspectives for PI treatment interventions in these rural areas.

Antibodies (Abs) against the V3 loop of the human immunodeficiency virus type 1 gp120 envelope glycoprotein were initially considered to mediate only type-specific neutralization of T-cell-line-adapted viruses. However, recent data show that cross-neutralizing V3 Abs also exist, and primary isolates can be efficiently neutralized with anti-V3 monoclonal Abs (MAbs). The neutralizing activities of anti-V3 polyclonal Abs and MAbs may, however, be limited due to antigenic variations of the V3 region, a lack of V3 exposure on the surface of intact virions, or Ab specificity. For clarification of this issue, a panel of 32 human anti-V3 MAbs were screened for neutralization of an SF162-pseudotyped virus in a luciferase assay. MAbs selected with a V3 fusion protein whose V3 region mimics the conformation of the native virus were significantly more potent than MAbs selected with V3 peptides. Seven MAbs were further tested for neutralizing activity against 13 clade B viruses in a single-round peripheral blood mononuclear cell assay. While there was a spectrum of virus sensitivities to the anti-V3 MAbs observed, 12 of the 13 viruses were neutralized by one or more of the anti-V3 MAbs. MAb binding to intact virions correlated significantly with binding to solubilized gp120s and with the potency of neutralization. These results demonstrate that the V3 loop is accessible on the native virus envelope, that the strength of binding of anti-V3 Abs correlates with the potency of neutralization, that V3 epitopes may be shared rather than type specific, and that Abs against the V3 loop, particularly those targeting conformational epitopes, can mediate the neutralization of primary isolates.

We have developed and validated an oligonucleotide probe hybridization assay for human immunodeficiency virus type 1 (HIV-1) circulating recombinant form (CRF) CRF02_AG. In the p17 coding region of the gag gene, a CRF02_AG-specific signature pattern was observed. Five working probes were designed to discriminate CRF02_AG infections from infections by all other documented subtypes and CRFs in an enzyme-linked immunosorbent assay-based oligonucleotide probe hybridization assay. Nucleic acids were extracted from a panel of HIV-1-positive plasma samples from Cameroon, Bénin, Côte d'Ivoire, Kenya, Zambia, and Belgium and from blood spots from The Gambia. CRF02_AG ( n = 147) and non-CRF02 ( n = 100) samples were analyzed to evaluate and validate the oligonucleotide probe hybridization assay. The CRF02_AG-specific oligonucleotide probe hybridization assay has a high sensitivity and specificity, with good positive and negative predictive values in regions of high and low prevalence. A validation of the assay with West and West Central African samples indicated a sensitivity of 98.4% and a specificity of 96.7%. The oligonucleotide probe hybridization assay as a diagnostic tool will allow for rapid screening for CRF02_AG. This could be used to track the HIV epidemic in terms of documenting the real prevalence of CRF02_AG strains and will complement efforts in vaccine development. Moreover, this technology can easily be applied in laboratories in developing countries.

Objective: To evaluate HIV-1 antibody seroprevalence and risk factors for HIV seropositivity in rural areas of Cameroon. Method: The prevalences of HIV antibodies in 53 villages in rural Cameroon visited during May–October 2000 were determined with an HIV1/2 rapid assay standard ELISA and western blot. Demographic data and risk factors were elicited via face-to-face interviews with a structured questionnaire. Results: HIV seroprevalence was 5.8% (243/4156 95% confidence interval [CI]=5.1–6.6) overall 6.3% (151/2394 95% CI=5.4–7.4) among females and 5.2% (92/1762 95% CI=4.3–6.4) among males. HIV seroprevalence among persons aged 15–70 years did not differ significantly by province (5.6% in Center 4.5% in East 6.9% in South and 5.8% in South-West) (p=.10). Analysis of age- and genderstandardized prevalence by village across provinces indicated a near-significant difference (nonparametric Wilcoxon signed rank test p=.06) with highest prevalence in South-West followed by South Center and East. Multivariate analysis revealed that single women were significantly more likely to be HIV seropositive than were married or widowed women. Women with a history of sexual relations while traveling were at significantly increased risk of HIV seropositivity (OR adjusted for age and marital status= 2.4 95% CI=1.4–9.7). Among men those who reported ever having a sexually transmitted disease were at significantly increased risk of HIVseropositivity (OR adjusted for age=1.8 95% CI=1.1–2.8). Conclusion: We have documented a wide range of HIV prevalences among rural villages of Cameroon. Age marital status (in women) and sexual risk factors appear to be associated with HIV infection in this setting. (author’s)

P34.11 LB Background: Superinfected HIV patients provide the unique opportunity to investigate the immune response after challenge with diverse HIV antigens and remain the major source for recombinant strain production. The competition and/or coexistence of two or more viral strains drive viral evolution and diversity, eventually triggering the generation of more broad and potent neutralizing antibodies (Abs) as compared to singly infected patients. The objective of this study was to analyze the genetic evolution of the viruses found within a superinfected Cameroonian patient, and to determine the neutralization sensitivity of the recombinant viruses to both autologous and heterologous neutralizing Abs. Methods: Longitudinal plasma samples from an HIV-1 superinfected Cameroonian patient were analyzed for their Env diversity, evolution, and recombination events. Pseudoviruses were generated from timepoints before, during, and after superinfection, and were then tested for sensitivity to homologous plasma and to heterologous neutralizing mAbs. Results: Env sequence analysis indicated that the CRF02_AG infected subject became superinfected with an F2 strain resulting in a massive increase in viral diversity. Later, the patient's viral repertoire was narrowed to quasispecies closely clustering with the initial strain. The highest neutralization was observed with autologous plasma from timepoints after superinfection but before the intiation of ART. Of interest, the superinfecting F2 strain was resistant to neutralization with most known broadly neutralizing antibodies (bnAbs) including PG09 and VRC01 while the original infecting CRF02_AG strain was sensitive to these bnAbs. Conclusions: The resistance of the F2 strain to known bnAbs urges the need to generate nAbs from such patients and to monitor the emerging recombinant strains. HIV-1 superinfected patients may serve for the generation of new bnAbs covering multiple subtypes and deliver important knowledge for future vaccine design.

P16.03 Background: A hallmark of HIV is its variability, which within group M viruses includes 9 subtypes and over 50 circulating recombinant forms (CRF)s. Cameroon is an epicenter of the disease known for circulation of many subtypes and CRFs, which provides a unique setting to study the immune response to HIV. It is crucial for a successful vaccine to elicit an adaptive immune response against a broad range of viral subtypes. Methods: We have performed subtype analysis of viral RNA isolated from the plasma of 63 HIV-1 infected Cameroonian subjects to verify non-B subtype infection and to monitor for recombination. From the non-B, drug naive cohort, we have isolated IgG (N=264) to screen for neutralization using a tier 2, pseudovirus panel including subtypes A1, B, C, and 01_AE. Samples capable of neutralizing multiple pseudoviruses at high potency were selected for further study to determine neutralization capacity against an extended panel and their ability to bind gp160 envelope peptides. Results: Subtypes A, D, F, G, and K were identified as well as many recombinant forms including the predominant subtype CRF02_AG. The neutralization screen concluded that 9.5% of the IgG samples were able to neutralize at least 2 pseudoviruses at low IgG concentrations. Neutralization of the extended panel revealed that these IgG samples were also able to neutralize 02_AG,G, and AC subtypes. We found that all of the broadly neutralizing IgG samples bound envelope protein gp41, over 80% bound gp120 proteins, 79% bound a scaffolded V1V2 peptide, and 100% of the samples bound cyclic V3 constructs. Conclusions: We have taken the first look at broad neutralization in a cohort infected with non-B subtype viruses and numerous CRFs. IgG from these patients is capable of neutralizing pseudoviruses across clades with high breadth and potency. We have shown that these samples bind strongly to envelope peptides and hope to continue to elucidate how these antibodies are conferring neutralization in order to guide future vaccine design.

We have used a virus-binding assay to examine conformational changes that occur when soluble CD4 (sCD4) binds to the surface of intact, native, primary human immunodeficiency virus type 1 virions. The isolates examined belong to seven genetic clades (A to H) and are representative of syncytium-inducing and non-syncytium-inducing phenotypes. Conformational changes in epitopes in the C2, V2, V3, C5, and CD4 binding domain (CD4bd) of gp120 and the cluster I and II regions of gp41 of these viruses were examined using human monoclonal antibodies that are directed at these regions. The studies revealed that sCD4 binding causes a marked increase in exposure of epitopes in the V3 loop, irrespective of the clade or the phenotype of the virus. Sporadic increases in exposure were observed in some epitopes in the V2 region, while no changes were observed in the C2, C5, or CD4bd of gp120 or the cluster I and II regions of gp41.

This protocol describes a simple assay that can be used to study the nature of exposure of antigenic epitopes and antigenic relatedness of different intact, native HIV-1 strains. The assay is based on the principle that mAbs coated on microtiter wells bind to epitopes on the surface of intact, native virions. The bound virion is then lysed to release p24, which is then quantitated (pg/ml) to give a measure of the amount of virion bound to the mAb. High p24 levels released after lysis correlate with high level capture of virions by mAbs, and as such, reflect good exposure of the epitope on the virion. Likewise, binding patterns of a specific mAb with different virus strains reveal information on their antigenic relatedness. In establishing this assay, the nature of exposure of antigenic epitopes and the antigenic relatedness of six intact, native HIV-1 virions of clades A, B, C, D, F and G were examined using anti-HIV-1 mAbs directed at epitopes in the V2, V3, CD4bd and C5 of gp120, and in clusters I and II of the gp41 region. Analysis of the binding data shows that mAbs directed at epitopes in the V3, C5 and gp41 Cluster I region bound best to the viruses examined, suggesting that these are the regions most exposed and conserved on intact, native HIV-1 virions of different clades. Epitopes in the V2 and CD4bd of gp120, and in gp41 cluster II, are not exposed on intact, native virions.

The relative resistance of human immunodeficiency virus type 1 (HIV-1) primary isolates (PIs) to neutralization by a wide range of antibodies remains a theoretical and practical barrier to the development of an effective HIV vaccine. One model to account for the differential neutralization sensitivity between Pls and laboratory (or T-cell line-adapted [TCLA]) strains of HIV suggests that the envelope protein (Env) complex is made more accessible to antibody binding as a consequence of adaptation to growth in established cell lines. Here, we revisit this question using genetically related PI and TCLA viruses and molecularly clonedenvgenes. By using complementary techniques of flow cytometry and virion binding assays, we show that monoclonal antibodies targeting the V3 loop, CD4-binding site, CD4-induced determinant of gp120, or the ectodomain of gp41 bind equally well to PI and TCLA Env complexes, despite large differences in neutralization outcome. The data suggest that the differential neutralization sensitivity of PI and TCLA viruses may derive not from differences in the initial antibody binding event but rather from differences in the subsequent functioning of the PI and TCLA Envs during virus entry. An understanding of these as yet undefined differences may enhance our ability to generate broadly neutralizing HIV vaccine immunogens.

Most strains of human immunodeficiency virus type 1 (HIV-1) which have only been carried in vitro in peripheral blood mononuclear cells (primary isolates) can be neutralized by antibodies, but their sensitivity to neutralization varies considerably. To study the parameters that contribute to the differential neutralization sensitivity of primary HIV-1 isolates, we developed a neutralization assay with a panel of genetically engineered cell lines (GHOST cells) that express CD4, one of eight chemokine receptors which function as HIV-1 coreceptors, and a Tat-dependent green fluorescent protein reporter cassette which permits the evaluation and quantitation of HIV-1 infection by flow cytometry. All 21 primary isolates from several clades could grow in the various GHOST cell lines, and their use of one or more coreceptors could easily be defined by flow cytometric analysis. Ten of these primary isolates, three that were CXCR4 (X4)-tropic, three that were CCR5 (R5)-tropic, and four that were dual- or polytropic were chosen for study of their sensitivity to neutralization by human monoclonal and polyclonal antibodies. Viruses from the X4-tropic category of viruses were first tested since they have generally been considered to be particularly neutralization sensitive. It was found that the X4-tropic virus group contained both neutralization-sensitive and neutralization-resistant viruses. Similar results were obtained with R5-tropic viruses and with dual- or polytropic viruses. Within each category of viruses, neutralization sensitivity and resistance could be observed. Therefore, sensitivity to neutralization appears to be the consequence of factors that influence the antibody-virus interaction and its sequelae rather than coreceptor usage. Neutralization of various viruses by the V3-specific monoclonal antibody, 447-52D, was shown to be dependent not only on the presence of the relevant epitope but also on its presentation. An epitope within the envelope of a particular virus is not sufficient to render a virus sensitive to neutralization by an antibody that recognizes that epitope. Moreover, conformation-dependent factors may overcome the need for absolute fidelity in the match between an antibody and its core epitope, permitting sufficient affinity between the viral envelope protein and the antibody to neutralize the virus. The studies indicate that the neutralization sensitivity of HIV-1 primary isolates is a consequence of the complex interaction between virus, antibody, and target cell.

Information is lacking on the prevalence of hepatitis C virus (HCV) infection in most African countries. An algorithm based on a combination of enzyme immunoassays (EIAs) with different formats (a commercial test, an HCV antibody [Ab] III test, and an HCV core Ab EIA) was used to estimate the prevalence of HCV infection in different population groups from southern Cameroon. An overall high prevalence was observed, with a significant increasing trend for both sexes with respect to age. A high proportion (67.4%) of HCV-positive sera were viremic as demonstrated by the reverse transcription-polymerase chain reaction. We conclude that the prevalence of HCV is high in southern Cameroon and increases linearly with age.

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 IC(50) titer from first to last plasma sample: 183.4) compared to the superinfected study subjects (mean change in IC(50) 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.

Our observations add to previous results on molecular epidemiology in different provinces in Cameroon demonstrating a high prevalence of CRF02_AG and subtype A accounting for 90% of circulating HIV-1 strains. The country-wide high prevalence of HIV-1 CRF02_AG in Cameroon compared with the relatively low prevalence of CRF02_AG in the Democratic Republic of Congo suggest an early founder effect of this AG recombinant in West Central Africa initiating major CRF02 epidemics. (excerpt)

To study how virus evolution affects neutralization sensitivity and to determine changes that occur in and around epitopes, we tested the ability of 13 anti-HIV-1 gp120 (anti-V2, anti-V3, anti-CD4bd and anti-carbohydrate) human monoclonal antibodies (mAbs) to neutralize sequential viruses obtained from five HIV-1 chronically infected drug naïve individuals. Overall, primary viruses collected from patients at first visit were resistant to neutralization by all anti-HIV-1 mAbs with the exception of one virus sensitive to IgG1b12. Four of the five patients' viruses evolved increased sensitivity to neutralization by anti-V3 mAbs. Virus collected from a patient obtained 31 months later, evolved increased sensitivity to anti-V2, anti-V3, and anti-CD4bd mAbs. Furthermore, the anti-V2 and anti-CD4bd mAbs also exhibited increased neutralization capacities against virus collected from a patient 29 months later. Of the seven anti-V3 mAbs, five showed increased potency to neutralize the evolved virus from a patient collected after 11 months, and three exhibited increased potency against viruses from two patients collected 29 and 36 months later. Anti-V3 mAbs exhibited the most breadth and potency in neutralizing the evolving viruses. Sequence analysis of the envelope regions revealed amino acid conservation within the V3 loop, while most of the changes identified occurred outside the core epitopes and in particular within the C3 region; these may account for increased neutralization sensitivity. These studies demonstrate that in vivo, HIV-1 can evolve increased neutralization sensitivity to mAbs and that the spectrum of neutralization capacities by mAbs can be broader when studied in longitudinal analysis.

One approach to the development of an HIV vaccine is to design a protein template which can present gp120 epitopes inducing cross-neutralizing antibodies. To select a V3 sequence for immunogen design, we compared the neutralizing activities of 18 anti-V3 monoclonal antibodies (mAbs) derived from Cameroonian and Indian individuals infected with clade AG and C, respectively. It was found that V3 mAbs from the Cameroonian patients were significantly more cross-neutralizing than those from India. Interestingly, superior neutralizing activity of Cameroonian mAbs was also observed among the nine VH5-51/VL lambda genes encoding V3 mAbs which mediate a similar mode of recognition. This correlated with higher relative binding affinity to a variety of gp120s and increased mutation rates in V3 mAbs from Cameroon. These results suggest that clade C V3 is probably weakly immunogenic and that the V3 sequence of CRF02_AG viruses can serve as a plausible template for vaccine immunogen design.

Background With the advent of entry inhibitors, monitoring of viral tropism in the clinical setting is important. Conventional methods are cell-based and lengthy, therefore V3 sequence based prediction algorithms are becoming increasingly attractive as monitoring tools. Here we report a comparative analysis of viral tropism of strains circulating in Cameroon where diverse and emerging variant strains are prevalent. Methods Viruses were isolated from 17 HIV positive individuals from three cities in Cameroon. Ghost cell lines expressing either CCR5 or CXCR4 with CD4 or CD4 alone (NIH AIDS Reagent Program) were used to determine co-receptor usage. HIV replication was determined by measuring p24 antigen levels. Plasma viral load (VL) was determined using the Versant bDNA assay. Nucleotide sequencing was performed on the V3 region and sequences were edited, aligned and translated into amino acids as described in the algorithm. Bio-informatics tools based on the 11/25 and charge rule were used to predict co-receptor usage. Results The majority of patient isolates in our study were CRF02_AG or CRF02_AG containing recombinants. Tropism of these complex viruses based on the cell culture assay was determined to be R5 in 15/17 (88.2%) patients. However, two patient isolates were dual tropic R5X4 and had drug-specific mutations. Of these two patients, one was on antiretroviral treatment with a VL of 20,899 copies/ml and the other was drug-naïve with 141,198 copies/ml. Genotype based prediction was overall in good agreement with phenotype for R5 viruses, where 93% (14/15) of results were comparable, dual tropic viruses being reported as X4 viruses by prediction. Conclusion Our results indicate that most HIV strains in Cameroon were R5 tropic and some harbored drug-resistant mutations. V3 sequence based prediction compared well with cell based assays for R5 strains and may be useful even in settings where highly diverse strains are prevalent.