Human immunodeficiency virus type 1 gp120 envelope characteristics associated with disease progression differ in family members infected with genetically similar viruses.

The human immunodeficiency virus type 1 (HIV-1) envelope protein provides the primary contact between the virus and host, and is the main target of the adaptive humoral immune response. The length of gp120 variable loops and the number of N-linked glycosylation events are key determinants for virus infectivity and immune escape, while the V3 loop overall positive charge is known to affect co-receptor tropism. We selected two families in which both parents and two children had been infected with HIV-1 for nearly 10 years, but who demonstrated variable parameters of disease progression. We analysed the gp120 envelope sequence and compared individuals that progressed to those that did not in order to decipher evolutionary alterations that are associated with disease progression when individuals are infected with genetically related virus strains. The analysis of the V3-positive charge demonstrated an association between higher V3-positive charges with disease progression. The ratio between the amino acid length and the number of potential N-linked glycosylation sites was also shown to be associated with disease progression with the healthier family members having a lower ratio. In conclusion in individuals initially infected with genetically linked virus strains the V3-positive charges and N-linked glycosylation are associated with HIV-1 disease progression and follow varied evolutionary paths for individuals with varied disease progression.

Varied sensitivity to therapy of HIV-1 strains in CD4+ lymphocyte sub-populations upon ART initiation.

BACKGROUND: Although antiretroviral therapy (ART) has proven its success against HIV-1, the long lifespan of infected cells and viral latency prevent eradication. In this study we analyzed the sensitivity to ART of HIV-1 strains in naïve, central memory and effector memory CD4+ lymphocyte subsets. METHODS: From five patients cellular HIV-1 infection levels were quantified before and after initiation of therapy (2-5 weeks). Through sequencing the C2V3 region of the HIV-1 gp120 envelope, we studied the effect of short-term therapy on virus variants derived from naïve, central memory and effector memory CD4+ lymphocyte subsets. RESULTS: During short-term ART, HIV-1 infection levels declined in all lymphocyte subsets but not as much as RNA levels in serum. Virus diversity in the naïve and central memory lymphocyte populations remained unchanged, whilst diversity decreased in serum and the effector memory lymphocytes. ART differentially affected the virus populations co-circulating in one individual harboring a dual HIV-1 infection. Changes in V3 charge were found in all individuals after ART initiation with increases within the effector memory subset and decreases found in the naïve cell population. CONCLUSIONS: During early ART virus diversity is affected mainly in the serum and effector memory cell compartments. Differential alterations in V3 charge were observed between effector memory and naïve populations. While certain cell populations can be targeted preferentially during early ART, some virus strains demonstrate varied sensitivity to therapy, as shown from studying two strains within a dual HIV-1 infected individual.

Effect of chloroquine on reducing HIV-1 replication in vitro and the DC-SIGN mediated transfer of virus to CD4+ T-lymphocytes.

BACKGROUND: Chloroquine (CQ) has been shown to inhibit HIV-1 replication in vitro as well as in vivo and has been proposed to alter the glycosylation pattern of the gp120 envelope. These activities indicate that the compound can be used not only as an effective HIV-1 therapeutic agent but also as a modulator of the gp120 envelope protein structure enabling for the production of broader neutralizing Ab responses. RESULTS: We confirm here that HIV-1 replication on CD4+ T-lymphocytes can be reduced in the presence of CQ and show that the reduced replication is producer cell mediated, with viruses generated in the presence of CQ not being inhibited for subsequent infectivity and replication. By analysing the gp120 envelope protein sequences from viruses cultured long-term in the absence or presence of CQ we demonstrate variant evolution patterns. One noticeable change is the reduction in the number of potential N-linked glycosylation sites in the V3 region as well as within the 2G12 Ab binding and neutralization epitope. We also demonstrate that HIV-1 produced in the presence of CQ has a reduced capacity for transfer by Raji-DC-SIGN cells to CD4+ T-lymphocytes, indicating another means whereby virus transmission or replication may be reduced in vivo. CONCLUSION: These results indicate that CQ should be considered as an HIV-1 therapeutic agent with its influence exerted through a number of mechanisms in vivo, including modulation of the gp120 structure.

The HIV type 1 epidemic in Belarus: predominance of Eastern European subtype A strains and circulation of subtype B viruses.

To study the molecular epidemiology of HIV-1 in Belarus, where the rapid spread of HIV-1 has been registered since 1996, we obtained HIV-1 sequences from 30 individuals living in five cities in both the main geographic areas of the epidemic (Gomel and Minsk regions) and territories where spreading of the epidemic remains limited (Grodno region). Analysis of env V3 and gag p17/p24 sequences demonstrated that infections in all 12 injecting drug users and 14 of 18 individuals infected through sexual contacts were caused by subtype A viruses that are specific for the epidemic in the former Soviet Union (IDU-A viruses), while the remaining four infections were caused by phylogenetically unrelated to each other subtype B viruses. Extrapolation of these results to the total population of HIV-1-infected individuals in Belarus allowed us to estimate that IDU-A viruses account for nearly 95% of HIV-1 infections in Belarus.

Association between gp120 envelope V1V2 and V4V5 variable loop profiles in a defined HIV-1 transmission cluster.

OBJECTIVE: Variations in the HIV-1 gp120 Env variable loop sequences correlate with virus phenotypes associated with transmission and/or disease progression. We aimed to identify whether signature sequences could be identified in the gp120 Env between acute infection and chronic infection viruses obtained from a group of individuals infected with closely related viruses. METHODS: To analyse acute infection versus chronic infection viruses, we studied a transmission cluster of 11 individuals, in which six presented during acute infection and five during chronic infection. Multiple HIV-1 gp120 Env clones were sequenced from each patient with predicted amino acid sequences compared between the groups. RESULTS: Cluster analysis of V1V5 Env sequences (n = 215) identified that acute infection viruses had lower potential N-linked glycosylation site (PNGS) densities than viruses from chronic infection, with a higher amino acid length/PNGS ratio. We found a negative correlation between the V1V2 and V4V5 regions for both amino acid length (Pearson P < 0.01) and PNGS numbers (Pearson P < 0.01) during HIV-1 transmission. This association was lost following seroconversion. These findings were confirmed by analysing sequences from the Los Alamos database that were selected and grouped according to timing of transmission. This included acute infection sequences collected 0-10 days (n = 400) and chronic infection sequences 0.5-3 years postseroconversion (n = 394). CONCLUSION: Our observations are consistent with a structural association between the V1V2 and V4V5 gp120 regions that is lost following viral transmission. These structural considerations should be taken into consideration when devising HIV-1 immunogens aimed at inducing protective antibody responses targeting transmitted viruses.

HIV-1 autologous antibody neutralization associates with mother to child transmission.

The HIV-1 characteristics associated with mother to child transmission (MTCT) are still poorly understood and if known would indicate where intervention strategies should be targeted. In contrast to horizontally infected individuals, exposed infants possess inherited antibodies (Abs) from their mother with the potential to protect against infection. We investigated the HIV-1 gp160 envelope proteins from seven transmitting mothers (TM) whose children were infected either during gestation or soon after delivery and from four non-transmitting mothers (NTM) with similar viral loads and CD4 counts. Using pseudo-typed viruses we tested gp160 envelope glycoproteins for TZM-bl infectivity, CD4 and CCR5 interactions, DC-SIGN capture and transfer and neutralization with an array of common neutralizing Abs (NAbs) (2F5, 2G12, 4E10 and b12) as well as mother and infant plasma. We found no viral correlates associated with HIV-1 MTCT nor did we find differences in neutralization with the panel of NAbs. We did, however, find that TM possessed significantly higher plasma neutralization capacities than NTM (P = 0.002). Furthermore, we found that in utero (IU) TM had a higher neutralization capacity than mothers transmitting either peri - partum (PP) or via breastfeeding (BF) (P = 0.002). Plasma from children infected IU neutralized viruses carrying autologous gp160 viral envelopes as well as those from their corresponding mothers whilst plasma from children infected PP and/or BF demonstrated poor neutralizing capacity. Our results demonstrate heightened autologous NAb responses against gp120/gp41 can associate with a greater risk of HIV-1 MTCT and more specifically in those infants infected IU. Although the number of HIV-1 transmitting pairs is low our results indicate that autologous NAb responses in mothers and infants do not protect against MTCT and may in fact be detrimental when considering IU HIV-1 transmissions.

Transmission of two distinct HIV type 1 strains to an individual that were harbored for many years by another.

The concept of transmission bottlenecks in HIV-1 infection is well established. Coinfections and superinfections have been increasingly documented and provide a founding cause for the expansion of viral diversity through recombination. It is still relatively unclear how HIV-1 will propagate and evolve in individuals infected with more than one viral strain. Here we report on the parallel transmission of genetically distant viral strains cocirculating in one individual over many years to a single recipient.

HIV type 1 mother-to-child transmission facilitated by distinctive glycosylation sites in the gp120 envelope glycoprotein.

The human immunodeficiency virus type 1 (HIV-1) characteristics associated with mother-to-child transmission (MTCT) are still poorly understood. We studied a cohort of 30 mothers from Rwanda infected with HIV-1 subtype A or C viruses of whom seven infected their children either during gestation or soon after birth. CD4 counts and viral load did not significantly differ between nontransmitting mother (NTM) versus transmitting mother (TM) groups. In contrast to earlier studies we not only analyzed and compared the genotypic characteristics of the V1-V5 region of the gp120 envelope of viruses found in TM and their infected children, but also included data from the NTM. No differences were found with respect to length and number of potential N-glycosylation sites (PNGS) in the V1-V2 and the V1-V5 region. We identified that viruses with a PNGS on positions AA234 and AA339 were preferably transmitted and that viruses with PNGS-N295 showed a disadvantage in transmission. We also showed that the frequency of PNGS-N339 in the viruses of TM and infected children was significantly higher than the frequency in NTM in our cohort and in viruses undergoing sexual transmission while the frequency of PNGS-N295 in children was significantly lower than the frequency in TM and acute horizontal infections. Collectively, our results provide evidence that the presence of the PNGS-N339 site and absence of the PNGS-N295 site in the gp120 envelope confers an advantage to HIV-1 when considering MTCT.

Lack of in vivo compartmentalization among HIV-1 infected naïve and memory CD4+ T cell subsets.

Viral compartmentalization between naïve and memory CD4(+) T cell subsets has been described, but only for individuals who were receiving antiretroviral therapy (ART). We present here an extensive analysis of the viral quasispecies residing in the naïve, central and effector memory CD4(+) T cell subsets in a number of therapy naïve individuals and representing an array of HIV-1 subtypes. We longitudinally analyzed subset-specific infection and evolution in a subtype B infected individual who switches from CCR5 to dual CCR5/CXCR4 coreceptor usage. We show that the central memory subset, the predominantly infected subset, harbors a more diverse viral population compared to the others. Through sequence analysis of the env C2V3 region we demonstrate a lack of viral compartmentalization among all subsets. Upon coreceptor switch we observe a pronounced increase in the infection level of the naïve population. Our findings emphasize the importance of all CD4(+) T cell subsets to viral evolution.

CTL escape and increased viremia irrespective of HIV-specific CD4+ T-helper responses in two HIV-infected individuals.

We investigated whether development of mutations leads to loss of CD8 T-cell recognition in HIV-1 infection and is possibly linked to alterations in HIV-1-specific CD4(+) T-cell responses in 2 HIV-infected individuals. In patient, H434 full genome sequencing of HIV-1 biological clones at early and late time points during disease progression showed development of fixed mutations in 16 predicted HIV-specific CTL epitopes. Loss of T-cell recognition and reactivity against wild-type and mutant epitopes was observed primarily for the HLA-B27-restricted KK10 epitope and HLA-A2-restricted SL9 epitope. Similarly, in patient H671, decreasing numbers of HLA-A3-restricted CD8(+) T cells specific for the wild-type RK9 epitope was observed after CTL escape. Only in patient H434 loss of CTL responses was paralleled by a decrease in HIV-specific IL-2(+) CD4(+) T-helper responses. This suggests that loss of T-cell reactivity may not be directly linked to HIV-specific CD4(+) T-cell responses but that increased viremia after CTL escape may influence CD4(+) T-helper responses.

Broad cross-clade T-cell responses to gag in individuals infected with human immunodeficiency virus type 1 non-B clades (A to G): importance of HLA anchor residue conservation.

We aimed to identify cross-clade human immunodeficiency virus type 1 (HIV-1) specific T-cell responses among 10 HLA-typed individuals who were infected with non-B HIV-1 strains (A, AG, C, D, G, or F) and to correlate these responses with genetic variation in documented T-cell epitopes. T-cell reactivity was tested against peptide pools spanning clade B Gag, Pol, Nef, Rev, and Tat consensus, with Gag and Nef providing the highest responses. Nine individuals who responded to clade B Gag demonstrated cross-reactive T-cell responses against clade A and C Gag pools, while six of seven responders to Nef-B reacted to clade A and C Nef pools. An inverse correlation between the height of the T-cell responses and the sequence divergence of the HLA class I-restricted epitopes was identified when we compared autologous Gag and Nef sequences with the reactive consensus pools. This could be explained for the Gag sequences through observed variations in the HLA anchor residues. Through mapping of 30 amino acid cross-clade-reactive regions using Gag-B pools, we were able to link 58% (14/24) of the T-cell responses to regions containing previously described HLA class I-restricted epitopes. Forty-two percent (10/24) of the responses were directed to regions containing new epitopes, for which predicted HLA class I motifs could be recognized in 70% (7/10) of individuals. We demonstrate here that cross-clade T-cell responses are frequently induced in individuals infected with distinct HIV-1 clades, suggesting that interclade variation outside of HLA anchor residues may have less impact on vaccine-induced T-cell reactivity than previously thought.

Identification of sequential viral escape mutants associated with altered T-cell responses in a human immunodeficiency virus type 1-infected individual.

Control of viremia in natural human immunodeficiency virus type 1 (HIV-1) infection in humans is associated with a virus-specific T-cell response. However, still much is unknown with regard to the extent of CD8(+) cytotoxic T-lymphocyte (CTL) responses required to successfully control HIV-1 infection and to what extent CTL epitope escape can account for rises in viral load and ultimate progression to disease. In this study, we chose to monitor through full-length genome sequence of replication-competent biological clones the modifications that occurred within predicted CTL epitopes and to identify whether the alterations resulted in epitope escape from CTL recognition. From an extensive analysis of 59 biological HIV-1 clones generated over a period of 4 years from a single individual in whom the viral load was observed to rise, we identified the locations in the genome of five CD8(+) CTL epitopes. Fixed mutations were identified within the p17, gp120, gp41, Nef, and reverse transcriptase genes. Using a gamma interferon ELIspot assay, we identified for four of the five epitopes with fixed mutations a complete loss of T-cell reactivity against the wild-type epitope and a partial loss of reactivity against the mutant epitope. These results demonstrate the sequential accumulation of CTL escape in a patient during disease progression, indicating that multiple combinations of T-cell epitopes are required to control viremia.