HIV-1 Envelope Subregion Length Variation during Disease Progression

The V3 loop of the HIV-1 Env protein is the primary determinant of viral coreceptor usage, whereas the V1V2 loop region is thought to influence coreceptor binding and participate in shielding of neutralization-sensitive regions of the Env glycoprotein gp120 from antibody responses. The functional properties and antigenicity of V1V2 are influenced by changes in amino acid sequence, sequence length and patterns of N-linked glycosylation. However, how these polymorphisms relate to HIV pathogenesis is not fully understood. We examined 5185 HIV-1 gp120 nucleotide sequence fragments and clinical data from 154 individuals (152 were infected with HIV-1 Subtype B). Sequences were aligned, translated, manually edited and separated into V1V2, C2, V3, C3, V4, C4 and V5 subregions. V1-V5 and subregion lengths were calculated, and potential N-linked glycosylation sites (PNLGS) counted. Loop lengths and PNLGS were examined as a function of time since infection, CD4 count, viral load, and calendar year in cross-sectional and longitudinal analyses. V1V2 length and PNLGS increased significantly through chronic infection before declining in late-stage infection. In cross-sectional analyses, V1V2 length also increased by calendar year between 1984 and 2004 in subjects with early and mid-stage illness. Our observations suggest that there is little selection for loop length at the time of transmission; following infection, HIV-1 adapts to host immune responses through increased V1V2 length and/or addition of carbohydrate moieties at N-linked glycosylation sites. V1V2 shortening during early and late-stage infection may reflect ineffective host immunity. Transmission from donors with chronic illness may have caused the modest increase in V1V2 length observed during the course of the pandemic

Virus Population Homogenization following Acute Human Immunodeficiency Virus Type 1 Infection

Understanding the properties of human immunodeficiency virus type 1 (HIV-1) variants capable of establishing infection is critical to the development of a vaccine against AIDS. Previous studies of men have shown that the HIV-1 env gene is homogeneous early in infection, leading to the suggestion that infection is established by a single transmitted variant. However, we report here that all of eight homosexual men evaluated beginning 3.7 to 9 weeks following onset of symptoms of acute infection harbored diverse virus populations in their blood, with median genetic distances averaging 1.08% in the env C2V5 region and 0.81% in the gag p17 gene. Within another 4.7 to 11 weeks, the variant lineage in env became more homogeneous, while gag sequences continued to diversify. Thus, the homogenization that has been reported to characterize acute infection is actually preceded by the replication of multiple virus variants. This early selective process focuses on viral properties within Env but not Gag p17. Hence, the viral homogeneity observed early in HIV-1 infection results from a selective process that occurs during the establishment of infection

Blood monocytes harbor HIV type 1 strains with diversified phenotypes including macrophage-specific CCR5 virus

BACKGROUND: Recent studies have shown that blood monocytes harbor human immunodeficiency virus type 1 (HIV-1) variants that are genotypically distinguishable from those in CD4(+) T cells. However, the biological function of monocyte-derived HIV-1 remains unclear. METHODS: Using pseudovirus assay, we analyzed the phenotype conferred by monocyte-derived HIV-1 envelopes from 8 patients. RESULTS: All pseudoviruses carrying monocyte-derived HIV-1 envelopes used CCR5; however, their use of additional coreceptors delineated 4 phenotypes in which viruses used (1) CCR5 only, (2) CCR5 and CXCR4, (3) CCR3 and CCR5, or (4) multiple coreceptors, including CCR1, CCR3, GPR15, CCR5, and CXCR4. More importantly, we observed 2 distinct cell tropism phenotypes for pseudoviruses carrying monocyte-derived envelopes: (1) monocyte-derived, macrophage-specific R5 (MDMS-R5) virus that, using CCR5 only, could infect monocyte-derived macrophages (MDMs) but not CD4(+) T cells and (2) dual tropic virus that infected both MDMs and primary CD4(+) T cells. We found blood monocytes harboring viruses with multiple phenotypes as early as 25 days before seroconversion and as late as 9 years after seroconversion. CONCLUSIONS: These data suggest that HIV-1 circulating in blood monocytes represents diverse HIV-1 with multiple phenotypes and that MDMS-R5 viruses may play an important role in infection with and persistence of HIV-1 within the monocyte/macrophage lineag

Evidence for Human Immunodeficiency Virus Type 1 Replication In Vivo in CD14(+) Monocytes and Its Potential Role as a Source of Virus in Patients on Highly Active Antiretroviral Therapy

In vitro studies show that human immunodeficiency virus type 1 (HIV-1) does not replicate in freshly isolated monocytes unless monocytes differentiate to monocyte-derived macrophages. Similarly, HIV-1 may replicate in macrophages in vivo, whereas it is unclear whether blood monocytes are permissive to productive infection with HIV-1. We investigated HIV-1 replication in CD14(+) monocytes and resting and activated CD4(+) T cells by measuring the levels of cell-associated viral DNA and mRNA and the genetic evolution of HIV-1 in seven acutely infected patients whose plasma viremia had been <100 copies/ml for 803 to 1,544 days during highly active antiretroviral therapy (HAART). HIV-1 DNA was detected in CD14(+) monocytes as well as in activated and resting CD4(+) T cells throughout the course of study. While significant variation in the decay slopes of HIV-1 DNA was seen among individual patients, viral decay in CD14(+) monocytes was on average slower than that in activated and resting CD4(+) T cells. Measurements of HIV-1 sequence evolution and the concentrations of unspliced and multiply spliced mRNA provided evidence of ongoing HIV-1 replication, more pronounced in CD14(+) monocytes than in resting CD4(+) T cells. Phylogenetic analyses of HIV-1 sequences indicated that after prolonged HAART, viral populations related or identical to those found only in CD14(+) monocytes were seen in plasma from three of the seven patients. In the other four patients, HIV-1 sequences in plasma and the three cell populations were identical. CD14(+) monocytes appear to be one of the potential in vivo sources of HIV-1 in patients receiving HAART

Combined Effect of CCR5-Δ32 Heterozygosity and the CCR5 Promoter Polymorphism −2459 A/G on CCR5 Expression and Resistance to Human Immunodeficiency Virus Type 1 Transmission

Exposed seronegative individuals (ES) with persistent high-risk sexual behavior may be less susceptible to human immunodeficiency virus type 1 (HIV-1) infection because they carry the chemokine receptor (CR) gene alleles CCR5 open reading frame (ORF) Δ32, CCR5 promoter −2459G, or CCR2 ORF 64I (CCR2-64I), all of which have been found to diminish HIV-1 infectivity and/or disease progression. To investigate this, we determined the haplotypes for these three genetic loci in 93 ES and 247 low-risk control individuals. To test if protective haplotypes exert their effect by modulating CR expression, we measured the protein expression of CCR5 and CXCR4 on circulating CD4(+) T cells and CD14(+) monocytes in 71 ES and 92 controls. To avoid investigator bias, the analysis was performed without knowledge of each subject's risk and genotype. The CCR5 −2459G allele was significantly enriched in ES Caucasian men, who constituted the majority (84%) of the ES cohort, compared to the control Caucasian men (P = 0.02). This increase was mostly attributable to a higher frequency of the −2459 A/G versus the −2459 A/A genotype in individuals heterozygous for the Δ32 allele (P = 0.012). No protective influence of the CCR2-64I allele was observed. The haplotypes CCR5 ORF Δ32/CCR5 −2459A (in complete linkage disequilibrium) and CCR5 ORF wt/CCR5 −2459G had a cumulative negative effect on the expression of CCR5, since we measured significantly reduced CCR5 densities on both T-helper cells and monocytes only when both haplotypes were present. Densities of CCR5 on lymphocytes and monocytes were correlated (r = 0.59; P < 0.0001), indicating concordance of CCR5 expression patterns across different cell types. We conclude that the CCR5 ORF Δ32/wt-CCR5 −2459 A/G genotype combination offers an advantage in resisting sexual HIV-1 transmission and that this effect is mediated by a relative paucity of CCR5 on potential target cells of HIV-1

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners▿

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses

Early Induction and Maintenance of Env-Specific T-Helper Cells following Human Immunodeficiency Virus Type 1 Infection

Mounting evidence points to a role for CD4(+) T-helper (Th) cell activities in controlling human immunodeficiency virus type 1 (HIV-1) infection. To determine the induction and evolution of Th responses following acute infection, we prospectively analyzed Env- and Gag-specific Th responses longitudinally for 92 patients with acute (n = 28) or early (n = 64) HIV-1 infection (median, 55 days postinfection [DPI]). The probability of detecting HIV-1-specific lymphoproliferative responses was remarkably low, and when present, the responses were more likely to be Gag specific than Env specific (16 versus 5%). Env-specific responses were significantly more common in patients presenting at <30 DPI than in those presenting at 30 to 365 DPI (21 versus 0.5%, P = 0.001). By contrast, Gag-specific responses occurred with similar frequencies among subjects presenting at <30 DPI and 30 to 365 DPI (13 versus 17%, P = 0.6). After treatment, and regardless of the duration of infection before therapy, Gag-specific Th responses predominated. Furthermore, some acutely infected subjects lost detectable Env-specific Th proliferative responses, which failed to reemerge upon treatment. Detailed analysis for one such subject revealed Env-specific lymphoproliferation at 11 DPI but no detectable Env-specific lymphoproliferation or ex vivo gamma interferon (IFN-γ) secretion at multiple subsequent time points. Env-specific CD4(+) T-cell clones from 11 DPI recognized six epitopes in both conserved and variable regions within gp120 and gp41, exhibited major histocompatibility complex-restricted cytotoxicity, and secreted high levels of antiviral cytokines. T-cell receptor clonal transcript analyses and autologous virus sequencing revealed that Th cells induced during acute infection were maintained and there were no Th escape mutations. Subsequent analysis for this subject and six of seven others revealed detectable IFN-γ-secreting cells, but only following in vitro gp160 stimulation. In summary, we conclude that Env-specific Th responses are elicited very early in acute infection and may precede Gag-specific responses. The inability to detect Env-specific Th responses over time and despite antiretroviral therapy may reflect low frequencies and impaired proliferative capacity, and viral escape is not necessary for this to occur

Successful isolation of infectious and high titer human monocyte-derived HIV-1 from two subjects with discontinued therapy.

HIV-1 DNA in blood monocytes is considered a viral source of various HIV-1 infected tissue macrophages, which is also known as "Trojan horse" hypothesis. However, whether these DNA can produce virions has been an open question for years, due to the inability of isolating high titer and infectious HIV-1 directly from monocytes.In this study, we demonstrated successful isolation of two strains of M-HIV-1 (1690 M and 1175 M) from two out of four study subjects, together with their in vivo controls, HIV-1 isolated from CD4+ T-cells (T-HIV-1), 1690 T and 1175 T. All M- and T- HIV-1 isolates were detected CCR5-tropic. Both M- HIV-1 exhibited higher levels of replication in monocyte-derived macrophages (MDM) than the two T- HIV-1. Consistent with our previous reports on the subject 1175 with late infection, compartmentalized env C2-V3-C3 sequences were identified between 1175 M and 1175 T. In contrast, 1690 M and 1690 T, which were isolated from subject 1690 with relatively earlier infection, showed homogenous env C2-V3-C3 sequences. However, multiple reverse transcriptase (RT) inhibitor resistance-associated variations were detected in the Gag-Pol region of 1690 M, but not of 1690 T. By further measuring HIV DNA intracellular copy numbers post-MDM infection, 1690 M was found to have significantly higher DNA synthesis efficiency than 1690 T in macrophages, indicating a higher RT activity, which was confirmed by AZT inhibitory assays.These results suggested that the M- and T- HIV-1 are compartmentalized in the two study subjects, respectively. Therefore, we demonstrated that under in vitro conditions, HIV-1 infected human monocytes can productively release live viruses while differentiating into macrophages