HIV-1 superinfection occurs at varying frequencies in different at risk populations. Though seroincidence is decreased, in the negative partner of HIV-discordant couples after joint testing and counseling in the Zambia Emory HIV Research Project (ZEHRP) cohort, the annual infection rate remains relatively high at 7-8%. Based on sequencing within the gp41 region of each partner's virus, 24% of new infections between 2004 and 2008 were the result of transmission from a non-spousal partner. Since these seroconvertors and their spouses have disparate epidemiologically-unlinked viruses, there is a risk of superinfection within the marriage. We have, therefore, investigated the incidence and viral origin of superinfection in these couples.
Superinfection was detected by heteroduplex mobility assay (HMA), degenerate base counting of the gp41 sequence, or by phylogenetic analysis of the longitudinal sequences. It was confirmed by full-length env single genome amplification and phylogenetic analysis. In 22 couples (44 individuals), followed for up to five years, three of the newly infected (initially HIV uninfected) partners became superinfected. In each case superinfection occurred during the first 12 months following initial infection of the negative partner, and in each case the superinfecting virus was derived from a non-spousal partner. In addition, one probable case of intra-couple HIV-1 superinfection was observed in a chronically infected partner at the time of his seroconverting spouse's initial viremia. Extensive recombination within the env gene was observed following superinfection.
In this subtype-C discordant couple cohort, superinfection, during the first year after HIV-1 infection of the previously negative partner, occurred at a rate similar to primary infection (13.6% [95% CI 5.2-34.8] vs 7.8% [7.1-8.6]). While limited intra-couple superinfection may in part reflect continued condom usage within couples, this and our lack of detecting newly superinfected individuals after one year of primary infection raise the possibility that immunological resistance to intra-subtype superinfection may develop over time in subtype C infected individuals.
Reports of HIV-1 superinfection (re-infection) have demonstrated that the immune response generated against one strain of HIV-1 does not always protect against other strains. However, studies to determine the incidence of HIV-1 superinfection have yielded conflicting results. Furthermore, few studies have attempted to identify superinfection cases occurring more than a year after initial infection, a time when HIV-1-specific immune responses would be most likely to have developed. We screened a cohort of high-risk Kenyan women for HIV-1 superinfection by comparing partial gag and envelope sequences over a 5-y period beginning at primary infection. Among 36 individuals, we detected seven cases of superinfection, including cases in which both viruses belonged to the same HIV-1 subtype, subtype A. In five of these cases, the superinfecting strain was detected in only one of the two genome regions examined, suggesting that recombination frequently occurs following HIV-1 superinfection. In addition, we found that superinfection occurred throughout the course of the first infection: during acute infection in two cases, between 1–2 y after infection in three cases, and as late as 5 y after infection in two cases. Our results indicate that superinfection commonly occurs after the immune response against the initial infection has had time to develop and mature. Implications from HIV-1 superinfection cases, in which natural re-exposure leads to re-infection, will need to be considered in developing strategies for eliciting protective immunity to HIV-1.
Superinfection with HIV-1 occurs when an individual infected with one strain of HIV-1 acquires a second strain, from a different partner. There are more than 20 published cases of HIV-1 superinfection. These cases have raised concerns for HIV-1 vaccine design because they indicate that the immune response generated against natural infection is not always sufficient to protect against later exposures to the virus. However, it remains unclear how often HIV-1 superinfection occurs, especially at times in infection after an immune response would be expected. We investigated the incidence of HIV-1 superinfection in a cohort of 36 high-risk women followed for approximately five years after their first HIV-1 infections. We found seven cases of HIV-1 superinfection. Five cases occurred more than a year after the initial infection, a time when the immune response would have had time to develop and broaden. In three cases, the initial and superinfecting viruses were classified as the same HIV-1 genetic subtype, indicating a lack of protection against closely related viruses. Our results suggest that natural HIV-1 infection does not always elicit a protective immune response, an important consideration in developing strategies for HIV-1 vaccine design and testing.
Investigating the incidence and prevalence of HIV-1 superinfection is challenging due to the complex dynamics of two infecting strains. The superinfecting strain can replace the initial strain, be transiently expressed, or persist along with the initial strain in distinct or in recombined forms. Various selective pressures influence these alternative scenarios in different HIV-1 coding regions. We hypothesized that the potency of the neutralizing antibody (NAb) response to autologous viruses would modulate viral dynamics in env following superinfection in a limited set of superinfection cases. HIV-1 env pyrosequencing data were generated from blood plasma collected from 7 individuals with evidence of superinfection. Viral variants within each patient were screened for recombination, and viral dynamics were evaluated using nucleotide diversity. NAb responses to autologous viruses were evaluated before and after superinfection. In 4 individuals, the superinfecting strain replaced the original strain. In 2 individuals, both initial and superinfecting strains continued to cocirculate. In the final individual, the surviving lineage was the product of interstrain recombination. NAb responses to autologous viruses that were detected within the first 2 years of HIV-1 infection were weak or absent for 6 of the 7 recently infected individuals at the time of and shortly following superinfection. These 6 individuals had detectable on-going viral replication of distinct superinfecting virus in the env coding region. In the remaining case, there was an early and strong autologous NAb response, which was associated with extensive recombination in env between initial and superinfecting strains. This extensive recombination made superinfection more difficult to identify and may explain why the detection of superinfection has typically been associated with low autologous NAb titers.
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.
Identifying naturally-occurring neutralizing antibodies (NAb) that are cross-reactive against all global subtypes of HIV-1 is an important step toward the development of a vaccine. Establishing the host and viral determinants for eliciting such broadly NAbs is also critical for immunogen design. NAb breadth has previously been shown to be positively associated with viral diversity. Therefore, we hypothesized that superinfected individuals develop a broad NAb response as a result of increased antigenic stimulation by two distinct viruses. To test this hypothesis, plasma samples from 12 superinfected women each assigned to three singly infected women were tested against a panel of eight viruses representing four different HIV-1 subtypes at matched time points post-superinfection (∼5 years post-initial infection). Here we show superinfected individuals develop significantly broader NAb responses post-superinfection when compared to singly infected individuals (RR = 1.68, CI: 1.23–2.30, p = 0.001). This was true even after controlling for NAb breadth developed prior to superinfection, contemporaneous CD4+ T cell count and viral load. Similarly, both unadjusted and adjusted analyses showed significantly greater potency in superinfected cases compared to controls. Notably, two superinfected individuals were able to neutralize variants from four different subtypes at plasma dilutions >1∶300, suggesting that their NAbs exhibit elite activity. Cross-subtype breadth was detected within a year of superinfection in both of these individuals, which was within 1.5 years of their initial infection. These data suggest that sequential infections lead to augmentation of the NAb response, a process that may provide insight into potential mechanisms that contribute to the development of antibody breadth. Therefore, a successful vaccination strategy that mimics superinfection may lead to the development of broad NAbs in immunized individuals.
A broad and potent antibody response is considered essential for an effective HIV-1 vaccine that will protect against diverse circulating strains. Consequently, there is great interest in both the host and viral factors that impact the development of the neutralizing antibody (NAb) response in natural HIV-1 infections. HIV-infected individuals who become superinfected with a second virus from a different source partner represent unique cases for studying the antibody response, as superinfection reflects exposure to different HIV-1 antigenic variants, and hence may provide insight into the development of broadly NAbs. In support of this model, we show here that superinfected individuals develop broader and more potent NAb responses than singly infected individuals, a result that is likely due to the increased antigenic stimulation from two viruses compared to one. Our findings remained unchanged after controlling for other factors that have been shown to influence the NAbs response, such as CD4+ T cell count and viral load. This study demonstrates that superinfection yields antibodies that have the capacity to recognize diverse circulating HIV-1 variants. Therefore, further characterization of these superinfected individuals' NAb responses could lead to novel insights into pathways that elicit broadly NAbs.
Human Immunodeficiency virus type-1 (HIV) entry into target cells involves binding of the viral envelope (Env) to CD4 and a coreceptor, mainly CCR5 or CXCR4. The only currently licensed HIV entry inhibitor, maraviroc, targets CCR5, and the presence of CXCX4-using strains must be excluded prior to treatment. Co-receptor usage can be assessed by phenotypic assays or through genotypic prediction. Here we compared the performance of a phenotypic Env-Recombinant Viral Assay (RVA) to the two most widely used genotypic prediction algorithms, Geno2Pheno[coreceptor] and webPSSM.
Co-receptor tropism of samples from 73 subtype B and 219 non-B infections was measured phenotypically using a luciferase-tagged, NL4-3-based, RVA targeting Env. In parallel, tropism was inferred genotypically from the corresponding V3-loop sequences using Geno2Pheno[coreceptor] (5–20% FPR) and webPSSM-R5X4. For discordant samples, phenotypic outcome was retested using co-receptor antagonists or the validated Trofile® Enhanced-Sensitivity-Tropism-Assay.
The lower detection limit of the RVA was 2.5% and 5% for X4 and R5 minority variants respectively. A phenotype/genotype result was obtained for 210 samples. Overall, concordance of phenotypic results with Geno2Pheno[coreceptor] was 85.2% and concordance with webPSSM was 79.5%. For subtype B, concordance with Geno2pheno[coreceptor] was 94.4% and concordance with webPSSM was 79.6%. High concordance of genotypic tools with phenotypic outcome was seen for subtype C (90% for both tools). Main discordances involved CRF01_AE and CRF02_AG for both algorithms (CRF01_AE: 35.9% discordances with Geno2Pheno[coreceptor] and 28.2% with webPSSM; CRF02_AG: 20.7% for both algorithms). Genotypic prediction overestimated CXCR4-usage for both CRFs. For webPSSM, 40% discordance was observed for subtype A.
Phenotypic assays remain the most accurate for most non-B subtypes and new subtype-specific rules should be developed for non-B subtypes, as research studies more and more draw conclusions from genotypically-inferred tropism, and to avoid unnecessarily precluding patients with limited treatment options from receiving maraviroc or other entry inhibitors.
Evidence for human immunodeficiency virus type 1 (HIV-1) superinfection was sought among 37 HIV-1-positive street-recruited active injection drug users (IDUs) from the San Francisco Bay area. HIV-1 sequences from pairs of samples collected 1 to 12 years apart, spanning a total of 215 years of exposure, were generated at p17 gag, the V3-V5 region of env, and/or the first exon of tat and phylogenetically analyzed. No evidence of HIV-1 superinfection was detected in which a highly divergent HIV-1 variant emerged at a frequency >20% of the serum viral quasispecies. Based on the reported risk behavior of the IDUs and the HIV-1 incidence in uninfected subjects in the same cohort, a total of 3.4 new infections would have been expected if existing infection conferred no protection from superinfection. Adjusted for risk behaviors, the estimated relative risk of superinfection compared with initial infection was therefore 0.0 (95% confidence interval, 0.00, 0.79; P = 0.02), indicating that existing infection conferred a statistically significant level of protection against superinfection with an HIV-1 strain of the same subtype, which was between 21 and 100%.
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.
Because various HIV vaccination studies are in progress, it is important to understand how often inter- and intra-subtype co/superinfection occurs in different HIV-infected high-risk groups. This knowledge would aid in the development of future prevention programs. In this cross-sectional study, we report the frequency of subtype B and F1 co-infection in a clinical group of 41 recently HIV-1 infected men who have sex with men (MSM) in São Paulo, Brazil.
Proviral HIV-1 DNA was isolated from subject's peripheral blood polymorphonuclear leukocytes that were obtained at the time of enrollment. Each subject was known to be infected with a subtype B virus as determined in a previous study. A small fragment of the integrase gene (nucleotide 4255–4478 of HXB2) was amplified by nested polymerase chain reaction (PCR) using subclade F1 specific primers. The PCR results were further confirmed by phylogenetic analysis. Viral load (VL) data were extrapolated from the medical records of each patient.
For the 41 samples from MSM who were recently infected with subtype B virus, it was possible to detect subclade F1 proviral DNA in five patients, which represents a co-infection rate of 12.2%. In subjects with dual infection, the median VL was 5.3 × 104 copies/ML, whereas in MSM that were infected with only subtype B virus the median VL was 3.8 × 104 copies/ML (p > 0.8).
This study indicated that subtype B and F1 co-infection occurs frequently within the HIV-positive MSM population as suggested by large number of BF1 recombinant viruses reported in Brazil. This finding will help us track the epidemic and provide support for the development of immunization strategies against the HIV.
HIV-1 superinfection (SI) occurs when an infected individual acquires a distinct new viral strain. The rate of superinfection may be reflective of the underlying HIV risk in a population. The Centre for the AIDS Programme of Research in South Africa (CAPRISA) 004 clinical trial demonstrated that women who used a tenofovir-containing microbicide gel had lower rates of HIV infection than women using a placebo gel. Women who contracted HIV-1 during the trial were screened for the occurrence of superinfection by next-generation sequencing of the viral gag and env genes. There were two cases (one in each trial arm) of subtype C superinfection identified from the 76 women with primary infection screened at two time points (rate of superinfection, 1.5/100 person-years). Both women experienced a >0.5-log increase in viral load during the window when superinfection occurred. The rate of superinfection was significantly lower than the overall primary HIV incidence in the microbicide trial (incidence rate ratio [IRR], 0.20; P = 0.003). The women who seroconverted during the trial reported a significant increase in sexual contact with their stable partner 4 months after seroconversion (P < 0.001), which may have lowered the risk of superinfection in this population. The lower frequency of SI compared to the primary incidence is in contrast to a report from a general heterosexual African population but agrees with a study of high-risk women in Kenya. A better understanding of the rate of HIV superinfection could have important implications for ongoing HIV vaccine research.
Human immunodeficiency virus type 1 (HIV-1) superinfection refers to the acquisition of another strain by an already infected individual. Here we report a comprehensive genetic analysis of an HIV-1 superinfection acquired heterosexually. The infected individual was in a high-risk cohort in Tanzania, was exposed to multiple subtypes, and was systematically evaluated every 3 months with a fluorescent multiregion genotyping assay. The subject was identified in the window period and was first infected with a complex ACD recombinant strain, became superinfected 6 to 9 months later with an AC recombinant, and was monitored for >2.5 years. The plasma viral load exceeded 400,000 copies/ml during the first 9 months of infection but resolved to the set point of 67,000 copies/ml by 3 months after superinfection; the CD4 cell count was 377 cells/μl at 30 months. Viral diversity was evaluated with techniques designed to fully sample the quasispecies, permitting direct observation of the evolution, temporal fluctuation, and intercompartment dynamics of the initial and superinfecting strains and recombinants derived from them. Within 3 months of superinfection, seven different molecular forms were detected in gag and six were detected in env. The proportions of forms fluctuated widely over time in plasma and peripheral blood mononuclear cells, illustrating how challenging the detection of dually infected individuals can be. Strain-specific nested PCR confirmed that the superinfecting strain was not present until the 9 month follow-up. This study further defines the parameters and dynamics of superinfection and will foster appropriate studies and approaches to gain a more complete understanding of risk factors for superinfection and its impact on clinical progression, epidemiology, and vaccine design.
To date, the majority of HIV-1 phenotypic resistance testing has been performed with subtype B virus backbones (e.g. HXB2). However, the relevance of using this backbone to determine resistance in non-subtype B HIV-1 viruses still needs to be assessed. From 114 HIV-1 subtype C clinical samples (36 ARV-naïve, 78 ARV-exposed), pol amplicons were produced and analyzed for phenotypic resistance using both a subtype B- and C-backbone in which the pol fragment was deleted. Phenotypic resistance was assessed in resulting recombinant virus stocks (RVS) for a series of antiretroviral drugs (ARV's) and expressed as fold change (FC), yielding 1660 FC comparisons. These Antivirogram® derived FC values were categorized as having resistant or sensitive susceptibility based on biological cut-off values (BCOs). The concordance between resistance calls obtained for the same clinical sample but derived from two different backbones (i.e. B and C) accounted for 86.1% (1429/1660) of the FC comparisons. However, when taking the assay variability into account, 95.8% (1590/1660) of the phenotypic data could be considered as being concordant with respect to their resistance call. No difference in the capacity to detect resistance associated with M184V, K103N and V106M mutations was noted between the two backbones. The following was concluded: (i) A high level of concordance was shown between the two backbone phenotypic resistance profiles; (ii) Assay variability is largely responsible for discordant results (i.e. for FC values close to BCO); (iii) Confidence intervals should be given around the BCO's, when assessing resistance in HIV-1 subtype C; (iv) No systematic resistance under- or overcalling of subtype C amplicons in the B-backbone was observed; (v) Virus backbone subtype sequence variability outside the pol region does not contribute to phenotypic FC values. In conclusion the HXB2 virus backbone remains an acceptable vector for phenotyping HIV-1 subtype C pol amplicons.
Acquisition of more than one strain of human immunodeficiency virus type 1 (HIV-1) has been reported to occur both during and after primary infection, but the risks and repercussions of dual and superinfection are incompletely understood. In this study, we evaluated a longitudinal cohort of chronically HIV-infected men who were sexual partners to determine if individuals acquired their partners' viral strains.
Our cohort of HIV-positive men consisted of 8 couples that identified themselves as long-term sexual partners. Viral sequences were isolated from each subject and analyzed using phylogenetic methods. In addition, strain-specific PCR allowed us to search for partners' viruses present at low levels. Finally, we used computational algorithms to evaluate for recombination between partners' viral strains.
All couples had at least one factor associated with increased risk for acquisition of new HIV strains during the study, including detectable plasma viral load, sexually transmitted infections, and unprotected sex. One subject was dually HIV-1 infected, but neither strain corresponded to that of his partner. Three couples' sequences formed monophyletic clusters at the entry visit, with phylogenetic analysis suggesting that one member of the couple had acquired an HIV strain from his identified partner or that both had acquired it from the same source outside their partnership. The 5 remaining couples initially displayed no evidence of dual infection, using phylogenetic analysis and strain-specific PCR. However, in 1 of these couples, further analysis revealed recombinant viral strains with segments of viral genomes in one subject that may have derived from the enrolled partner. Thus, chronically HIV-1 infected individuals may become superinfected with additional HIV strains from their seroconcordant sexual partners. In some cases, HIV-1 superinfection may become apparent when recombinant viral strains are detected.
HIV superinfection, which occurs when a previously infected individual acquires a new distinct HIV strain, has been described in a number of populations. Previous methods to detect superinfection have involved a combination of labor-intensive assays with various rates of success. We designed and tested a next-generation sequencing (NGS) protocol to identify HIV superinfection by targeting two regions of the HIV viral genome, p24 and gp41. The method was validated by mixing control samples infected with HIV subtype A or D at different ratios to determine the inter- and intrasubtype sensitivity by NGS. This amplicon-based NGS protocol was able to consistently identify distinct intersubtype strains at ratios of 1% and intrasubtype variants at ratios of 5%. By using stored samples from the Rakai Community Cohort Study (RCCS) in Uganda, 11 individuals who were HIV seroconcordant but virally unlinked from their spouses were then tested by this method to detect superinfection between 2002 and 2005. Two female cases of HIV intersubtype superinfection (18.2%) were identified. These results are consistent with other African studies and support the hypothesis that HIV superinfection occurs at a relatively high rate. Our results indicate that NGS can be used for detection of HIV superinfection within large cohorts, which could assist in determining the incidence and the epidemiologic, virologic, and immunological correlates of this phenomenon.
The retroviral phenomenon of superinfection resistance (SIR) defines an interference mechanism that is established after primary infection, preventing the infected cell from being superinfected by a similar type of virus. This review describes our present understanding of the underlying mechanisms of SIR established by three characteristic retroviruses: Murine Leukaemia Virus (MuLV), Foamy Virus (FV), and Human Immunodeficiency Virus (HIV). In addition, SIR is discussed with respect to HIV superinfection of humans.
MuLV resistant mice exhibit two genetic resistance traits related to SIR. The cellular Fv4 gene expresses an Env related protein that establishes resistance against MuLV infection. Another mouse gene (Fv1) mediates MuLV resistance by expression of a sequence that is distantly related to Gag and that blocks the viral infection after the reverse transcription step. FVs induce two distinct mechanisms of superinfection resistance. First, expression of the Env protein results in SIR, probably by occupancy of the cellular receptors for FV entry. Second, an increase in the concentration of the viral Bet (Between-env-and-LTR-1-and-2) protein reduces proviral FV gene expression by inhibition of the transcriptional activator protein Tas (Transactivator of spumaviruses). In contrast to SIR in FV and MuLV infection, the underlying mechanism of SIR in HIV-infected cells is poorly understood. CD4 receptor down-modulation, a major characteristic of HIV-infected cells, has been proposed to be the main mechanism of SIR against HIV, but data have been contradictory. Several recent studies report the occurrence of HIV superinfection in humans; an event associated with the generation of recombinant HIV strains and possibly with increased disease progression. The role of SIR in protecting patients from HIV superinfection has not been studied so far.
The phenomenon of SIR may also be important in the protection of primates that are vaccinated with live attenuated simian immunodeficiency virus (SIV) against pathogenic SIV variants. As primate models of SIV infection closely resemble HIV infection, a better knowledge of SIR-induced mechanisms could contribute to the development of an HIV vaccine or other antiviral strategies.
To study the basis of cellular latency of human immunodeficiency virus (HIV), we have used a recombinant luciferase-encoding HIV (HXB-Luc) to superinfect nonproductively HIV-1-infected human leukemic cell lines. HXB-Luc contains the Photinus pyralis luciferase gene in place of the nef gene and provides a highly sensitive, simple assay for HIV infection and expression. To circumvent any superinfection block in latently infected cells, we also generated viruses pseudotyped with murine leukemia virus amphotropic envelope (HXB-Luc:ampho). The parental uninfected lines, U937 and A3.01, from which the latently infected cell lines U1 and ACH-2, respectively, were derived could be readily infected with pseudotyped or nonpseudotyped reporter viruses. However, superinfection of U1 cells with either HXB-Luc or HXB-Luc:ampho resulted in only low levels of luciferase activity. Like the endogenous provirus, HXB-Luc provirus could be efficiently activated by phorbol ester treatment of HXB-Luc:ampho-superinfected U1 cells. In contrast, superinfection of ACH-2 cells resulted in active expression of the secondarily introduced virus even in unstimulated cells and luciferase production higher than in the parental cell line A3.01. Thus, the proviral latency in U1 cells appears to result from a defect in the cellular environment (a trans effect), whereas the latency in ACH-2 is specific to the integrated provirus and is probably a cis effect due to the site of integration. These results demonstrate distinct modes of proviral latency in these two cell line models and may have implications in our understanding of the regulation and significance of cellular latency in HIV infection.
Since 1989, human immunodeficiency virus type 1 (HIV-1) has spread explosively through the heterosexual population in Thailand. This epidemic is caused primarily by viruses classified as "subtype E", which, on the basis of limited sequence comparisons, appear to represent hybrids of subtypes A (gag) and E (env). However, the true evolutionary origins of "subtype E" viruses are still obscure since no complete genomes have been analyzed, and only one full-length subtype A sequence has been available for phylogenetic comparison. In this study, we determined full-length proviral sequences for "subtype E" viruses from Thailand (93TH253) and the Central African Republic (90CR402) and for a subtype A virus from Uganda (92UG037). We also sequenced the long terminal repeat (LTR) regions from 16 virus strains representing clades A, C, E, F, and G. Detailed phylogenetic analyses of these sequences indicated that "subtype E" viruses do indeed represent A/E recombinants with multiple points of crossover along their genomes. The extracellular portion of env, parts of vif and vpr, as well as most of the LTR are of subtype E origin, whereas the remainder of the genome is of subtype A origin. The possibility that the discordant phylogenetic positions of "subtype E" viruses in gag- and env-derived trees are the result of unusual rates or patterns of evolution was also considered but was ruled out on the basis of two lines of evidence: (i) phylogenetic trees constructed for synonymous and nonsynonymous substitutions yielded the same discordant branching orders for "subtype E" gag and env gene sequences, thus excluding selection-driven evolution, and (ii) multiple crossovers in the viral genome are most consistent with the copy choice model of recombination and have been observed in other documented examples of HIV-1 intersubtype recombination. Thai and CAR "subtype E" viruses exhibited the same pattern of A/E mosaicism, indicating that the recombination event occurred in Africa prior to the spread of virus to Asia. Finally, all "subtype E" viruses were found to contain a distinctive two-nucleotide bulge in their transactivation response (TAR) elements. This feature was present only in viruses which also contained a subtype A 5' pol region (i.e., subtype A viruses or A/D and A/E recombinants), raising the possibility of a functional linkage between the TAR region and the polymerase. The implications of epidemic spread of a recombinant HIV-1 strain to viral natural history and vaccine development are discussed.
CCR5 antagonists are a powerful new class of antiretroviral drugs that require a companion assay to evaluate the presence of CXCR4-tropic (non-R5) viruses prior to use in human immunodeficiency virus (HIV)-infected individuals. In this study, we have developed, characterized, verified, and prevalidated a novel phenotypic test to determine HIV-1 coreceptor tropism (VERITROP) based on a sensitive cell-to-cell fusion assay. A proprietary vector was constructed containing a near-full-length HIV-1 genome with the yeast uracil biosynthesis (URA3) gene replacing the HIV-1 env coding sequence. Patient-derived HIV-1 PCR products were introduced by homologous recombination using an innovative yeast-based cloning strategy. The env-expressing vectors were then used in a cell-to-cell fusion assay to determine the presence of R5 and/or non-R5 HIV-1 variants within the viral population. Results were compared with (i) the original version of Trofile (Monogram Biosciences, San Francisco, CA), (ii) population sequencing, and (iii) 454 pyrosequencing, with the genotypic data analyzed using several bioinformatics tools, i.e., the 11/24/25 rule, Geno2Pheno (2% to 5.75%, 3.5%, or 10% false-positive rate [FPR]), and webPSSM. VERITROP consistently detected minority non-R5 variants from clinical specimens, with an analytical sensitivity of 0.3%, with viral loads of ≥1,000 copies/ml, and from B and non-B subtypes. In a pilot study, a 73.7% (56/76) concordance was observed with the original Trofile assay, with 19 of the 20 discordant results corresponding to non-R5 variants detected using VERITROP and not by the original Trofile assay. The degree of concordance of VERITROP and Trofile with population and deep sequencing results depended on the algorithm used to determine HIV-1 coreceptor tropism. Overall, VERITROP showed better concordance with deep sequencing/Geno2Pheno at a 0.3% detection threshold (67%), whereas Trofile matched better with population sequencing (79%). However, 454 sequencing using Geno2Pheno at a 10% FPR and 0.3% threshold and VERITROP more accurately predicted the success of a maraviroc-based regimen. In conclusion, VERITROP may promote the development of new HIV coreceptor antagonists and aid in the treatment and management of HIV-infected individuals prior to and/or during treatment with this class of drugs.
HIV-1 group M is classified into 9 subtypes, as well as recombinants favored by coinfection and superinfection events with different variants. Although HIV-1 subtype B is predominant in Europe, intersubtype recombinants are increasing in prevalence and complexity. In this study, phylogenetic analyses of pol sequences were performed to detect the HIV-1 circulating and unique recombinant forms (CRFs and URFs, respectively) in a Spanish cohort of antiretroviral treatment-naïve HIV-infected patients included in the Research Network on HIV/AIDS (CoRIS). Bootscanning and other methods were used to define complex recombinants not assigned to any subtype or CRF. A total of 670 available HIV-1 pol sequences from different patients were collected, of which 588 (87.8%) were assigned to HIV-1 subtype B and 82 (12.2%) to HIV-1 non-B variants. Recombinants caused the majority (71.9%) of HIV-1 non-B infections and were found in 8.8% of CoRIS patients. Eleven URFs (accounting for 13.4% of HIV-1 non-B infections), presenting complex mosaic patterns, were detected. Among them, 10 harbored subtype B fragments. Four of the 11 URFs were found in Spanish natives. A cluster of three B/CRF02_AG recombinants was detected. We conclude that complex variants, including unique recombinant forms, are being introduced into Spain through both immigrants and natives. An increase in the frequency of mosaic viruses, reflecting the increasing heterogeneity of the HIV epidemic in our country, is expected.
Transmission of human immunodeficiency virus (HIV) is no exception to the phenomenon that a second, productive infection with another strain of the same virus is feasible. Experiments with RNA viruses have suggested that both coinfections (simultaneous infection with two strains of a virus) and superinfections (second infection after a specific immune response to the first infecting strain has developed) can result in increased fitness of the viral population. Concerns about dual infections with HIV are increasing. First, the frequent detection of superinfections seems to indicate that it will be difficult to develop a prophylactic vaccine. Second, HIV-1 superinfections have been associated with accelerated disease progression, although this is not true for all persons. In fact, superinfections have even been detected in persons controlling their HIV infections without antiretroviral therapy. Third, dual infections can give rise to recombinant viruses, which are increasingly found in the HIV-1 epidemic. Recombinants could have increased fitness over the parental strains, as in vitro models suggest, and could exhibit increased pathogenicity. Multiple drug resistant (MDR) strains could recombine to produce a pan-resistant, transmittable virus.
We will describe in this review what is presently known about super- and re-infection among ambient viral infections, as well as the first cases of HIV-1 superinfection, including HIV-1 triple infections. The clinical implications, the impact of the immune system, and the effect of anti-retroviral therapy will be covered, as will as the timing of HIV superinfection. The methods used to detect HIV-1 dual infections will be discussed in detail. To increase the likelihood of detecting a dual HIV-1 infection, pre-selection of patients can be done by serotyping, heteroduplex mobility assays (HMA), counting the degenerate base codes in the HIV-1 genotyping sequence, or surveying unexpected increases in the viral load during follow-up. The actual demonstration of dual infections involves a great deal of additional research to completely characterize the patient's viral quasispecies. The identification of a source partner would of course confirm the authenticity of the second infection.
With millions of people infected worldwide, the evolution of HIV-1 in vivo has been the subject of much research. Although recombinant viruses were detected early in the epidemic, evidence that HIV-1 dual infections really occurred came much later. Dual infected patients, consisting of coinfected (second infection before seroconversion) and superinfected (second infection after seroconversion) individuals, opened up a new area of HIV-1 evolution studies. Here, we describe the in-depth analysis of HIV-1 over time in a patient twice superinfected with HIV-1, first with a subtype B (B2) strain and then with CRF01_AE after initial infection with a subtype B (B1) strain.
The nucleotide evolution of gag and env-V3 of the three strains followed a similar pattern: a very low substitution rate in the first 2–3 years of infection, with an increase in synonymous substitutions thereafter. Convergent evolution at the protein level was rare: only a single amino acid in a gag p24 epitope showed convergence in the subtype B strains. Reversal of CTL-epitope mutations were also rare, and did not converge. Recombinant viruses were observed between the two subtype B strains. Luciferase-assays suggested that the CRF01_AE long terminal repeat (LTR) constituted the strongest promoter, but this was not reflected in the plasma viral load. Specific real-time PCR assays based upon the env gene showed that strain B2 and CRF01_AE RNA was present in equal amounts, while levels of strain B1 were 100-fold lower.
All three strains were detected in seminal plasma, suggesting that simultaneous transmission is possible.
HIV superinfection (reinfection) has been reported in several settings, but no study has been designed and powered to rigorously compare its incidence to that of initial infection. Determining whether HIV infection reduces the risk of superinfection is critical to understanding whether an immune response to natural HIV infection is protective. This study compares the incidence of initial infection and superinfection in a prospective seroincident cohort of high-risk women in Mombasa, Kenya. A next-generation sequencing-based pipeline was developed to screen 129 women for superinfection. Longitudinal plasma samples at <6 months, >2 years and one intervening time after initial HIV infection were analyzed. Amplicons in three genome regions were sequenced and a median of 901 sequences obtained per gene per timepoint. Phylogenetic evidence of polyphyly, confirmed by pairwise distance analysis, defined superinfection. Superinfection timing was determined by sequencing virus from intervening timepoints. These data were combined with published data from 17 additional women in the same cohort, totaling 146 women screened. Twenty-one cases of superinfection were identified for an estimated incidence rate of 2.61 per 100 person-years (pys). The incidence rate of initial infection among 1910 women in the same cohort was 5.75 per 100pys. Andersen-Gill proportional hazards models were used to compare incidences, adjusting for covariates known to influence HIV susceptibility in this cohort. Superinfection incidence was significantly lower than initial infection incidence, with a hazard ratio of 0.47 (CI 0.29–0.75, p = 0.0019). This lower incidence of superinfection was only observed >6 months after initial infection. This is the first adequately powered study to report that HIV infection reduces the risk of reinfection, raising the possibility that immune responses to natural infection are partially protective. The observation that superinfection risk changes with time implies a window of protection that coincides with the maturation of HIV-specific immunity.
HIV-infected individuals with continued exposure are at risk of acquiring a second infection, a process known as superinfection. Superinfection has been reported in various at-risk populations, but how frequently it occurs remains unclear. Determining the frequency of superinfection compared with initial infection can help clarify whether the immune response developed against HIV can protect from reinfection – critical information for understanding whether such responses should guide HIV vaccine design. In this study, we developed a sensitive high-throughput method to identify superinfection and used this to conduct a screen for superinfection in 146 women in a high-risk cohort. This enabled us to determine if first HIV infection affects the risk of second infection by comparing the incidence of superinfection in this group to the incidence of initial infection in 1910 women in the larger cohort. We found that the incidence of superinfection was approximately half that of initial infection after controlling for behavioral and clinical differences that might affect infection risk. These results suggest that the immune response elicited in natural HIV infection may provide partial protection against subsequent infection and indicate the setting of superinfection may shed light on the features of a protective immune response and inform vaccine design.
Superinfection with drug resistant HIV strains could potentially contribute to compromised therapy in patients initially infected with drug-sensitive virus and receiving antiretroviral therapy. To investigate the importance of this potential route to drug resistance, we developed a bioinformatics pipeline to detect superinfection from routinely collected genotyping data, and assessed whether superinfection contributed to increased drug resistance in a large European cohort of viremic, drug treated patients.
We used sequence data from routine genotypic tests spanning the protease and partial reverse transcriptase regions in the Virolab and EuResist databases that collated data from five European countries. Superinfection was indicated when sequences of a patient failed to cluster together in phylogenetic trees constructed with selected sets of control sequences. A subset of the indicated cases was validated by re-sequencing pol and env regions from the original samples.
4425 patients had at least two sequences in the database, with a total of 13816 distinct sequence entries (of which 86% belonged to subtype B). We identified 107 patients with phylogenetic evidence for superinfection. In 14 of these cases, we analyzed newly amplified sequences from the original samples for validation purposes: only 2 cases were verified as superinfections in the repeated analyses, the other 12 cases turned out to involve sample or sequence misidentification. Resistance to drugs used at the time of strain replacement did not change in these two patients. A third case could not be validated by re-sequencing, but was supported as superinfection by an intermediate sequence with high degenerate base pair count within the time frame of strain switching. Drug resistance increased in this single patient.
Routine genotyping data are informative for the detection of HIV superinfection; however, most cases of non-monophyletic clustering in patient phylogenies arise from sample or sequence mix-up rather than from superinfection, which emphasizes the importance of validation. Non-transient superinfection was rare in our mainly treatment experienced cohort, and we found a single case of possible transmitted drug resistance by this route. We therefore conclude that in our large cohort, superinfection with drug resistant HIV did not compromise the efficiency of antiretroviral treatment.
HIV; Superinfection; Transmitted drug resistance; Sequence analysis
Infection with HIV-1 is characterized by genetic diversity such that specific viral subtypes are predominant in specific geographical areas. The genetic variation in HIV-1 pol and env genes is responsible for rapid development of resistance to current drugs. This variation has influenced disease progression among the infected and necessitated the search for alternative drugs with novel targets. Though successfully used in developed countries, these novel drugs are still limited in resource-poor countries. The aim of this study was to determine HIV-1 subtypes, recombination, dual infections and viral tropism of HIV-1 among Kenyan patients prior to widespread use of antiretroviral drugs.
Remnant blood samples from consenting sexually transmitted infection (STI) patients in Nairobi were collected between February and May 2001 and stored. Polymerase chain reaction and cloning of portions of HIV-1 gag, pol and env genes was carried out followed by automated DNA sequencing.
Twenty HIV-1 positive samples (from 11 females and 9 males) were analyzed. The average age of males (32.5 years) and females (26.5 years) was significantly different (p value < 0.0001). Phylogenetic analysis revealed that 90% (18/20) were concordant HIV-1 subtypes: 12 were subtype A1; 2, A2; 3, D and 1, C. Two samples (10%) were discordant showing different subtypes in the three regions. Of 19 samples checked for co-receptor usage, 14 (73.7%) were chemokine co-receptor 5 (CCR5) variants while three (15.8%) were CXCR4 variants. Two had dual/mixed co-receptor use with X4 variants being minor population.
HIV-1 subtype A accounted for majority of the infections. Though perceived to be a high risk population, the prevalence of recombination in this sample was low with no dual infections detected. Genotypic co-receptor analysis showed that most patients harbored viruses that are predicted to use CCR5.
Identifying microbial pathogens with zoonotic potential in wild-living primates can be important to human health, as evidenced by human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) and Ebola virus. Simian foamy viruses (SFVs) are ancient retroviruses that infect Old and New World monkeys and apes. Although not known to cause disease, these viruses are of public health interest because they have the potential to infect humans and thus provide a more general indication of zoonotic exposure risks. Surprisingly, no information exists concerning the prevalence, geographic distribution, and genetic diversity of SFVs in wild-living monkeys and apes. Here, we report the first comprehensive survey of SFVcpz infection in free-ranging chimpanzees (Pan troglodytes) using newly developed, fecal-based assays. Chimpanzee fecal samples (n = 724) were collected at 25 field sites throughout equatorial Africa and tested for SFVcpz-specific antibodies (n = 706) or viral nucleic acids (n = 392). SFVcpz infection was documented at all field sites, with prevalence rates ranging from 44% to 100%. In two habituated communities, adult chimpanzees had significantly higher SFVcpz infection rates than infants and juveniles, indicating predominantly horizontal rather than vertical transmission routes. Some chimpanzees were co-infected with simian immunodeficiency virus (SIVcpz); however, there was no evidence that SFVcpz and SIVcpz were epidemiologically linked. SFVcpz nucleic acids were recovered from 177 fecal samples, all of which contained SFVcpz RNA and not DNA. Phylogenetic analysis of partial gag (616 bp), pol-RT (717 bp), and pol-IN (425 bp) sequences identified a diverse group of viruses, which could be subdivided into four distinct SFVcpz lineages according to their chimpanzee subspecies of origin. Within these lineages, there was evidence of frequent superinfection and viral recombination. One chimpanzee was infected by a foamy virus from a Cercopithecus monkey species, indicating cross-species transmission of SFVs in the wild. These data indicate that SFVcpz (i) is widely distributed among all chimpanzee subspecies; (ii) is shed in fecal samples as viral RNA; (iii) is transmitted predominantly by horizontal routes; (iv) is prone to superinfection and recombination; (v) has co-evolved with its natural host; and (vi) represents a sensitive marker of population structure that may be useful for chimpanzee taxonomy and conservation strategies.
Cross-species transmissions of infectious agents from primates to humans have led to major disease outbreaks, with AIDS representing a particularly serious example. It has recently been shown that humans who hunt primates frequently acquire simian foamy virus (SFV) infections. Thus, these viruses have been proposed as an “early warning system” of human exposure to wild primates. In this study, we have tested this concept by developing non-invasive methods to determine the extent to which wild chimpanzees are infected with SFV. We analyzed more than 700 fecal samples from 25 chimpanzee communities across sub-Saharan Africa and obtained viral sequences from a large number of these. SFV was widespread among all chimpanzee subspecies, with infection rates ranging from 44% to 100%. The new viruses formed subspecies-specific lineages consistent with virus/host co-evolution. We also found mosaic sequences due to recombination, indicating that chimpanzees can be infected with multiple viral strains. One chimpanzee harbored an SFV from a monkey species, indicating cross-species transmission in the wild. These data indicate that chimpanzees represent a substantial natural reservoir of SFV. Thus, monitoring humans for these viruses should identify locations where human/chimpanzee encounters are most frequent, and where additional transmissions of chimpanzee pathogens should be anticipated.