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.
HIV-1; Dual infection; Heteroduplex assay
Cameroon is a West African country where high genetic diversity of HIV-1 has been reported. The predominant CRF02_AG is involved in the emergence of more complex intersubtype recombinants. In this study, we sequenced the full-length genome of a novel unique recombinant form (URF) of HIV-1, 02CAMLT04 isolated in blood donors in urban Cameroon. Phylogenetic tree and bootscan analysis showed that 02CAMLT04 was complex and appeared to be a secondary recombinant derived from CRF02_AG and CRF22_01A1. The genomic composition of 02CAMLT04 strain showed that it is composed of three segments; twenty four percent of the genome is classified as CRF02_AG, spanning most of the envelope gene. The remaining seventy six percent of the genome is classified as CRF22_01A1. In addition, the sequence analysis of 13 full-length sequences from HIV-1 positive specimens received from Cameroon between 2002 and 2010 indicated that five specimens are pure CRF22_01A1 viruses, and six others have homology with CRF22_01A1 sequences in either gag, pol or env region where as 6% of strains contain portions of CRF22_01A1. Further study demonstrated that CRF22_01A1 is a primary prevalence strain co-circulating in Cameroon and is involved in complex intersubtype recombination events with subtypes (D or F), subsubtypes (A1 or F2) and CRFs (CRF01_AE or CRF02_AG). Our studies show that novel recombinants between CRF22_01A1 and other clades and recombinant forms may be emerging in Cameroon that could contribute to the future global diversity of HIV-1 in this region and world wide.
HIV-1; recombinant; genetic diversity; phylogenetic analysis; CRF22_01A1; CRF02_AG; Cameroon
For HIV recombination to occur, the RNAs from two infecting strains within a cell must dimerize at the dimerization initiation site (DIS). We examined the sequence identity at the DIS (697–731 bp, Hxb2 numbering engine) in patients superinfected with concordant HIV-1 strains and compared them to those with discordant strains. Viral RNA in sequential plasma from four subjects superinfected with subtype-discordant and two subjects superinfected with subtype-concordant HIV-1 strains was extracted, amplified (5′ LTR-early gag: 526–1200 bp, Hxb2 numbering engine), sequenced, and analyzed to determine their compatibility for dimerization in vivo. The concordant viruses infecting the two subjects exhibited identical sequences in the 35-bp-long DIS region while sequences from the discordant viruses revealed single nucleotide changes that were located in the DIS loop (715 bp), its flanking nucleotides (710 bp and 717 bp), and the DIS stem (719 bp). Evidence from in vitro experiments demonstrates that these in vivo changes identified can abolish dimerization and reduce recombination frequency. Therefore, these results revealing differences in the DIS of discordant strains versus the similarity noted for the concordant strains may contribute to the differences in the frequency of recombination in patients superinfected with such HIV-1 variants.
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.
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.
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.
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.
Little is known regarding the likelihood of recombination between any given pair of nonidentical HIV-1 viruses in vivo. The present study analyzes the HIV-1 quasispecies in the C1C2 region of env, the vif-vpr-vpu accessory gene region, and the reverse transcriptase region of pol. These sequences were amplified from samples obtained sequentially over a 12- to 33-month period from five dually HIV-1-infected subjects. Analysis of an average of 248 clones amplified from each subject revealed no recombinants within the three loci studied of the subtype-discordant infecting strains, whose genetic diversity was >11% in env. In contrast, two subjects who were initially coinfected by two subtype-concordant variants with genetic diversity of 7.4% in env were found to harbor 10 unique recombinants of these strains, as exhibited by analysis of the env gene. The frequent recombination observed among the subtype-concordant strains studied herein correlates with prior sequence analyses that have commonly found higher rates of recombination at loci bearing the most conserved sequences, demonstrating an important role for sequence identity in HIV-1 recombination. Viral load analysis revealed that the samples studied contained an average of 8125 virus copies/ml (range, 882–31,626 copies/ml), signifying that the amount of viral RNA in the samples was not limiting for studying virus diversity. These data reveal that recombination between genetically distant strains may not be an immediate or common outcome to dual infection in vivo and suggest critical roles for viral and host factors such as viral fitness, virus diversity, and host immune responses that may contribute to limiting the frequency of intersubtype recombination during in vivo dual infection.
High-risk cohorts in East Africa and the United States show rates of dual HIV-1 infection—the concomitant or sequential infection by two HIV-1 strains—of 50% to 100% of those of primary infection, and our normal-risk HIV-positive cohort in Cameroon exhibits a rate of dual infection of 11% per year, signifying that these infections are not exceptional. Little is known regarding the effect of dual infections on host immunity, despite the fact that they provide unique opportunities to investigate how the immune response is affected when challenged with diverse HIV-1 antigens. Using heterologous primary isolates, we have shown here that dual HIV-1 infection by genetically distant strains correlates with significantly increased potency and breadth of the anti-HIV-1 neutralizing antibody response. When the neutralization capacities of sequential plasma obtained before and after the dual infection of 4 subjects were compared to those of matched plasma obtained from 23 singly infected control subjects, a significant increase in the neutralization capacity of the sequential sample was found for 16/28 dually infected plasma/virus pairs, while only 4/159 such combinations for the control subjects exhibited a significant increase (P < 0.0001). Similarly, there was a significant increase in the plasma dilution capable of neutralizing 50% of virus (IC50) for 18/24 dually infected plasma/virus pairs, while 0/36 controls exhibited such an increase (P < 0.0001). These results demonstrate that dual HIV-1 infection broadens and strengthens the anti-HIV-1 immune response, suggesting that vaccination schemes that include polyvalent, genetically divergent immunogens may generate highly protective immunity against any HIV-1 challenge strain.
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.
drug-resistance mutations; HIV-1; NRTI; NNRTI; HAART; drug naïve
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.
Measurement of human immunodeficiency virus type 1 (HIV-1) plasma RNA levels using Roche AMPLICOR version 1.5 (HIV RNA) is an integral part of monitoring HIV-infected patients in industrialized countries. These assays are currently unaffordable in resource-limited settings. We investigated a reverse transcriptase (RT) assay as a less expensive alternative for measuring viral burden that quantifies RT enzyme activity in clinical plasma samples. A comparison of RT and HIV RNA assays was performed on 29 paired plasma samples from patients living in the United States and 21 paired plasma samples from patients living in Cameroon. RT levels correlated significantly with plasma HIV RNA viral loads in plasma from U.S. patients (r = 0.898; P < 0.001) and Cameroonian patients, a majority of whom were infected with HIV-1 clade type CRF02_AG (r = 0.669; P < 0.01). Among 32 samples with HIV viral load of >2,000 copies/ml, 97% had detectable RT activity. One Cameroon sample had undetectable RNA viral load but detectable RT activity of 3 fg/ml. The RT assay is a simple and less expensive alternative to the HIV RNA assay. Field studies comparing these assays in resource-limited settings are warranted to assess the practicality and usefulness of this assay for monitoring HIV-infected patients on antiretroviral therapy.
Sera from human immunodeficiency virus type 1 (HIV-1)-infected North American patients recognized a fusion protein expressing a V3 loop from a clade B primary isolate virus (JR-CSF) but not from a clade A primary isolate virus (92UG037.8), while most sera from Cameroonian patients recognized both fusion proteins. Competition studies of consensus V3 peptides demonstrated that the majority of the cross-reactive Cameroonian sera contained cross-reactive antibodies that reacted strongly with both V3 sequences. V3-specific antibodies purified from all six cross-reactive sera examined had potent neutralizing activity for virus pseudotyped with envelope proteins (Env) from SF162, a neutralization-sensitive clade B primary isolate. For four of these samples, neutralization of SF162 pseudotypes was blocked by both the clade A and clade B V3 fusion proteins, indicating that this activity was mediated by cross-reactive antibodies. In contrast, the V3-reactive antibodies from only one of these six sera had significant neutralizing activity against viruses pseudotyped with Envs from typically resistant clade B (JR-FL) or clade A (92UG037.8) primary isolates. However, the V3-reactive antibodies from these cross-reactive Cameroonian sera did neutralize virus pseudotyped with chimeric Envs containing the 92UG037.8 or JR-FL V3 sequence in Env backbones that did not express V1/V2 domain masking of V3 epitopes. These data indicated that Cameroonian sera frequently contain cross-clade reactive V3-directed antibodies and indicated that the typical inability of such antibodies to neutralize typical, resistant primary isolate Env pseudotypes was primarily due to indirect masking effects rather than to the absence of the target epitopes.
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 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.
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 cloned env genes. 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.
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.
We have examined the exposure and conservation of antigenic epitopes on the surface envelope glycoproteins (gp120 and gp41) of 26 intact, native, primary human immunodeficiency virus type 1 (HIV-1) group M virions of clades A to H. For this, 47 monoclonal antibodies (MAbs) derived from HIV-1-infected patients were used which were directed at epitopes of gp120 (specifically V2, C2, V3, the CD4-binding domain [CD4bd], and C5) and epitopes of gp41 (clusters I and II). Of the five regions within gp120 examined, MAbs bound best to epitopes in the V3 and C5 regions. Only moderate to weak binding was observed by most MAbs to epitopes in the V2, C2, and CD4bd regions. Two anti-gp41 cluster I MAbs targeted to a region near the tip of the hydrophilic immunodominant domain bound strongly to >90% of isolates tested. On the other hand, binding of anti-gp41 cluster II MAbs was poor to moderate at best. Binding was dependent on conformational as well as linear structures on the envelope proteins of the virions. Further studies of neutralization demonstrated that MAbs that bound to virions did not always neutralize but all MAbs that neutralized bound to the homologous virus. This study demonstrates that epitopes in the V3 and C5 regions of gp120 and in the cluster I region of gp41 are well exposed on the surface of intact, native, primary HIV-1 isolates and that cross-reactive epitopes in these regions are shared by many viruses from clades A to H. However, only a limited number of MAbs to these epitopes on the surface of HIV-1 isolates can neutralize primary isolates.
Because immunologic classification of human immunodeficiency virus type 1 (HIV) might be more relevant than genotypic classification for designing polyvalent vaccines, studies were undertaken to determine whether immunologically defined groups of HIV (“immunotypes”) could be identified. For these experiments, the V3 region of the 120-kDa envelope glycoprotein (gp120) was chosen for study. Although antibodies (Abs) to V3 may not play a major protective role in preventing HIV infection, identification of a limited number of immunologically defined structures in this extremely variable region would set a precedent supporting the hypothesis that, despite its diversity, the HIV family, like the V3 region, might be divisible into immunotypes. Consequently, the immunochemical reactivities of 1,176 combinations of human anti-V3 monoclonal Abs (MAbs) and V3 peptides, derived from viruses of several clades, were studied. Extensive cross-clade reactivity was observed. The patterns of reactivities of 21 MAbs with 50 peptides from clades A through H were then analyzed by a multivariate statistical technique. To test the validity of the mathematical approach, a cluster analysis of the 21 MAbs was performed. Five groups were identified, and these MAb clusters corresponded to classifications of these same MAbs based on the epitopes which they recognize. The concordance between the MAb clusters identified by mathematical analysis and by their specificities supports the validity of the mathematical approach. Therefore, the same mathematical technique was used to identify clusters within the 50 peptides. Seven groups of peptides, each containing peptides from more than one clade, were defined. Inspection of the amino acid sequences of the peptides in each of the mathematically defined peptide clusters revealed unique “signature sequences” that suggest structural motifs characteristic of each V3-based immunotype. The results suggest that cluster analysis of immunologic data can define immunotypes of HIV. These immunotypes are distinct from genotypic classifications. The methods described pave the way for identification of immunotypes defined by immunochemical and neutralization data generated with anti-HIV Env MAbs and intact, viable HIV virions.
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.