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HIV-1 escapes by acquiring mutations that differentially influence the course of infection. Unlike HIV-1 structural and enzymatic proteins, it remains elusive what extent the host immune-mediated selection pressure influences the variability of the accessory (Vif, Vpu, Vpr, and Nef) and regulatory (Tat and Rev) proteins. To address this, we analyzed the viral sequences encoding accessory and regulatory proteins from 446 human leukocyte antigen (HLA)-typed, chronically HIV-1 subtype B-infected, and treatment-naive individuals in Japan. We observed that Vpu and Vpr were the most and least polymorphic proteins with the average Shannon entropy scores of 0.63 and 0.38, respectively. Phylogenetically corrected methods identified a total of 161 HLA-associated polymorphisms; whereby Nef and Vpu had the highest (26.6%) and lowest (1.2%) proportion of amino acid sites associated with HLA-class I alleles, respectively. These results add further insight on the role of HLA-mediated selection pressure on HIV-1 sequence polymorphisms of HIV-1 accessory and regulatory proteins.

There have been no studies related to groups at the highest risk for HIV-1 infection in Korea before 1993. In this study, for the first time, we report the distribution of HIV subtypes in overseas sailors (OSs) and prostitutes who worked in brothels near U.S. military bases in Korea. We retrospectively determined the sequences of nef in 131 patients using reverse transcription polymerase chain reaction (RT-PCR). These patients composed of 102 OSs, 14 OS spouses, and 15 prostitutes. Phylogenetic analysis was performed using 128 Korean OSs, OS spouses, and prostitutes. The distribution of non-B subtypes (n = 105) was as follows: 39, CRF02_AG; 15, CRF01_AE; 7, A1; 7, A2; 6, D; 2, CRF06_cpx; 3, C; 6, G; 11, untypable; and 1 each for CRF09_cpx, CRF12_BF, CRF50_A1D, A3, AFG, H, F1, F2, and A. Of the 116 OSs and OS spouses, 101 (87%), 11 (9%), and 4 (3%) subjects had non-B, Western B, and Korean subclade B (KSB) HIV-1s, respectively. Among the 15 prostitutes, 10 had Western B (67%), 4 non-B (27%), and 1 KSB (7%) HIV-1s. All 14 couples, each comprising of an OS and his spouse, had the same subtype. KSB (5%) was detected in OSs and prostitutes in 1990 and 1994, respectively. Of the 131 patients analyzed in this study, 105 (80%), 21 (16%), and 5 (4%) were infected with the non-B, Western B, and KSB subtypes of HIV, respectively. In future, these data may provide an important foundation for analysis of HIV-1 subtypes in Korea.

HIV-infected individuals are at high risk of developing atherosclerosis and cardiovascular disease, in part, due to HIV-induced impairment of cholesterol metabolism. In vitro studies demonstrated that HIV-1 protein Nef inhibits activity of ABCA1, the main cellular cholesterol transporter, leading to cholesterol accumulation in macrophages and conversion of these cells into foam cells, characteristic for atherosclerosis. However, the mechanisms of Nef-mediated effects on cholesterol metabolism in vivo are not well characterized. In this study, we generated Nef-transgenic mice and evaluated the accumulation of neutral lipids in liver and aorta of these animals. Nef expression was low in all transgenic mice, with some mice carrying the Nef transgene, but not expressing the Nef RNA. Using Oil Red O staining, we demonstrated increased levels of neutral lipids in liver and aorta of mice expressing Nef relative to transgenic animals, with no detectable Nef expression or control wild-type mice. These results provide direct evidence that Nef promotes cholesterol deposition in tissues.

In this study, a novel CRF01_AE/07_BC HIV-1 recombinant form, with five breakpoints in the pol, vpr, env, and nef gene regions, was identified from a voluntary blood donor in North China. Our study showed that this HIV-1 recombinant form might be a second-generation recombinant form from two predominant strains in the men who have sex with men (MSM) population, with the CRF07_BC fragments belonging to a CRF07_BC lineage specific to Chinese MSM and the CRF01_AE fragments belonging to the CRF01_AE lineage 5 common among MSM in northern China. Our findings supported the high-risk blood donation behaviors of MSM, implied the increasing complexity of the HIV-1 epidemic among the Chinese MSM population, and called more attention to the effectiveness of the screening test for the growing number of new HIV-1 recombinant forms.

Using the early protein HIV Nef, new HLA class I binding epitopes of importance for immune responses to HIV were predicted for common African alleles. In total we identified 45 epitopes previously not described for the HLA alleles A*30:01, A*30:02, B*58:01, and C*07:01 and compared them to reported epitopes, primarily from HLA-A*02:01, from the Los Alamos database and our own vaccine studies. Related to its small size, the Nef gene/protein appears to be able to contribute effectively to confer both stronger and broader cellular immunogenicity to an HIV-1 vaccine. We also propose feasible mutations of such an additional vaccine antigen to preserve its immunogenicity, modified not to confer HLA or CD4+ down-regulating activities. This article includes data on a valuable HIV immunogenic component for a vaccine in Africa.

Approximately 20% of all HIV-1 mother-to-child transmission (MTCT) occurs in utero (IU). In a chronic HIV infection, HIV-1 exists as a complex swarm of genetic variants, and following IU MTCT, viral genomic diversity is restricted through a mechanism that remains to be described. The 5' U3R region of the HIV-1 long terminal repeat (LTR) contains multiple transcription factor (TF) binding sites and regulates viral transcription. In this study, we tested the hypothesis that sequence polymorphisms in the U3R region of LTR are associated with IU MTCT. To this end, we used single template amplification to isolate 517 U3R sequences from maternal, placental, and infant plasma derived from 17 HIV-infected Malawian women: eight whose infants remained HIV uninfected (NT) and nine whose infants became HIV infected IU. U3R sequences show pairwise diversities ranging from 0.2% to 2.3%. U3R sequences from one participant contained two, three, or four putative NF-κB binding sites. Phylogenetic reconstructions indicated that U3R sequences from eight of nine IU participants were consistent with placental compartmentalization of HIV-1 while only one of eight NT cases was consistent with such compartmentalization. Specific TF sequence polymorphisms were not significantly associated with IU MTCT. To determine if replication efficiency of the U3R sequences was associated with IU MTCT, we cloned 90 U3R sequences and assayed promoter activity in multiple cell lines. Although we observed significant, yet highly variable promoter activity and TAT induction of promoter activity in the cell lines tested, there was no association between measured promoter activity and MTCT status. Thus, we were unable to detect a promoter genotype or phenotype associated with IU MTCT.

HIV-1 Nef is an accessory protein necessary for HIV-1 virulence and rapid AIDS development. Nef promotes viral replication and infection by connecting CD4 and several other cell surface receptors to the clathrin adaptor protein AP2, resulting in the internalization and degradation of the receptors interacting with Nef. We investigated how Nef can mediate constitutive receptor endocytosis through the interaction of the dileucine motif in its C-terminal flexible loop (C-loop) with AP2, whereas AP2 binding of the transmembrane receptors usually results in an equilibrated (recycled) endocytosis. Our results indicated that in addition to the dileucine motif, there is a second motif in the Nef C-loop involved in the Nef–AP2 interaction. Nef-mediated CD4 downregulation was impaired when the residue in the hydrophobic region in the Nef C-loop (LL165HPMSLHGM173) was mutated to a basic residue K/R or an acidic residue E/D or to the rigid residue P, or when M168L170, L170H171, or G172M173 was mutated to AA. A pull-down assay indicated that AP2 was not coprecipitated with Nef mutants that did not downregulate CD4. Molecular modeling of the Nef C-terminal flexible loop in complex with AP2 suggests that M168L170 occupies a pocket in the AP2 σ2 subunit. Our data suggest a new model in the Nef–AP2 interaction in which the hydrophobic region in the Nef C-loop with the dileucine (L164L165) motif and M168L170 motif binds to AP2(σ2), while the acidic motif E174 and D175 binds to AP2(α), which explains how Nef through the flexible loop connects CD4 to AP2 for constitutive CD4 downregulation.

Induction of HIV-1-specific cytotoxic T lymphocyte (CTL) responses largely depends upon the presentation of CTL epitopes to the CD8(+) T cells aided by a large number of different HLA class I alleles. Although several studies showed the clustering pattern of HIV-1 CTL epitopes, the underlying reason for this tendency remains unresolved. Moreover, the hypothesis that the CTL epitope clusters tend to coincide with the conserved and hydrophobic regions of HIV-1 proteins has been challenged in recent times. The present study aims to characterize and compare the HIV-1 CTL epitope clusters in terms of restricting HLA alleles, hydrophobicity, and sequence conservation in a proteome-wide manner by including a large number of experimentally validated CTL epitopes from the HIV Molecular Immunology Database. CTL epitope cluster distribution analysis in a proteome-wide manner revealed that only two HIV-1 proteins, namely Nef and Gag, have significant cluster-forming capacity where their epitope localization coincides with the hydrophobic and conserved regions. Furthermore, analyses of proteasomal cleavage sites and HLA anchoring motif frequencies in the epitope-dense regions highlighted the role of specific HLA supertypes such as HLA B*07, HLA B*58, HLA A*02, and HLA A*03 in selecting the hydrophobic and conserved amino acid positions within Nef and Gag proteins to be presented as epitopes. Based on our results, we hypothesize that the cluster-forming tendency of HIV-1 CTL epitopes is not a proteome-wide feature confined to Nef and Gag proteins. Their cluster-forming tendency largely depends on the host HLA alleles that contribute significantly in selecting functionally constrained hydrophobic regions within the HIV-1 proteome.

Severe pulmonary hypertension (PH) associated with vascular remodeling is a long-term complication of HIV infection (HIV-PH) affecting 1/200 infected individuals vs. 1/200,000 frequency in the uninfected population. Factors accounting for increased PH susceptibility in HIV-infected individuals are unknown. Rhesus macaques infected with chimeric SHIVnef virions but not with SIV display PH-like pulmonary vascular remodeling suggesting that HIV-Nef is associated with PH; these monkeys showed changes in nef sequences that correlated with pathogenesis after passage in vivo. We further examined whether HIV-nef alleles in HIV-PH subjects have signature sequences associated with the disease phenotype. We evaluated specimens from participants with and without HIV-PH from European Registries and validated results with samples collected as part of the Lung-HIV Studies in San Francisco. We found that 10 polymorphisms in nef were overrepresented in blood cells or lung tissue specimens from European HIV-PH individuals but significantly less frequent in HIV-infected individuals without PH. These polymorphisms mapped to known functional domains in Nef. In the validation cohort, 7/10 polymorphisms in the HIV-nef gene were confirmed; these polymorphisms arose independently from viral load, CD4(+) T cell counts, length of infection, and antiretroviral therapy status. Two out of 10 polymorphisms were previously reported in macaques with PH-like pulmonary vascular remodeling. Cloned recombinant Nef proteins from clinical samples down-regulated CD4, suggesting that these primary isolates are functional. This study offers new insights into the association between Nef polymorphisms in functional domains and the HIV-PH phenotype. The utility of these polymorphisms as predictors of PH should be examined in a larger population.

Human immunodeficiency virus type 1 (HIV) infection of the central nervous system frequently causes HIV-associated neurocognitive disorders (HAND). The role of HIV Nef and other accessory proteins in HAND pathogenesis is unclear. To determine whether HIV nef undergoes adaptive selection in brain, we cloned 100 nef sequences (n = 30 brain and n = 70 lymphoid) from four patients with AIDS and HIV-associated dementia (HAD). Normalized nonsynonymous substitutions were more frequent at the divergence of lymphoid and brain sequences, indicating stronger adaptive selection in brain compared to lymphoid tissue. Brain-specific nonsynonymous substitutions were found within an NH(3)-terminal CTL epitope, the PACS1 binding motif, or positions predicted to be important for activation of the myeloid-restricted Src family tyrosine kinase Hck. These results suggest that adaptive selection of HIV nef in brain may reflect altered requirements for efficient replication in macrophages and brain-specific immune selection pressures.

The HIV-1 Nef protein is known to be secreted, and our group has shown that Nef is secreted from nef-transfected and HIV-1-infected cells in small exosome-like vesicles (d. 40-100 nm). The role of secreted Nef remains to be fully characterized. Thus, it is important to characterize the nature of and the mechanisms regulating Nef secretion. We hypothesized that specific structural domains on the Nef protein interact with components of the endosomal trafficking machinery, sorting Nef into multivesicular bodies (MVB) and packaging it in exosome-like vesicles. To identify those domains, a series of mutants spanning the entire nef sequence were made and cloned into the expression vector pQB1, which expresses the mutants as Nef-GFP fusion proteins. These constructs were used in transient transfection assays to identify sequences necessary for secretion of the Nef-GFP fusion protein. N-terminal domains were identified as critical for Nef-induced vesicle secretion: (1) a basic cluster of four arginine residues (aa 17, 19, 21, 22), (2) the phosphofurin acidic cluster sequence (PACS; Glu62-65), and (3) a previously uncharacterized domain spanning amino acid residues 66-70 (VGFPV), which we named the secretion modification region (SMR). Additional amino acids P25, 29GVG31, and T44 were identified in HIV-1 Nef as regulating its secretion. These residues have not been associated with other reported Nef functions. The myristoylation domain, ubiquitination lysine residues, and the C-terminal portion of Nef (aa 71-206) had no effect on secretion. A minimal HIV-1 Nef sequence, comprising the identified motifs, was sufficient for Nef-induced vesicle secretion.

Molecular interaction between the peptide/MHC class I complexes (pMHCs) and T cell receptor (TCR) is fundamental to the effector function of cytotoxic T lymphocytes (CTLs). Monoclonal antibody against pMHC with TCR-like specificity is a possible research tool for the antigen presentation. However, it is notoriously difficult to isolate monoclonal antibodies against pMHCs by the conventional hybridoma technique. To isolate monoclonal antibodies against an immunodominant HIV-1-derived CTL epitope in the nef gene, we panned phage clones from a human scFv phage display library. Eight Nef138-10/HLA-A*24(A24)-specific scFv clones were isolated and two of them (scFv#3 and scFv#27) were selected for further analysis. The clones stained A24-positive cells pulsed with Nef138-10 peptides specifically. We reconstituted humanized immunoglobulin Gs (IgGs) using a baculovirus expression system. Reconstituted IgGs kept the original specificities of the parental scFvs. The dissociation constants were 23 microM and 20 microM by Biacore, respectively. This is the first report of a successful generation of monoclonal antibodies against an HIV-1 CTL epitope loaded on an MHC class I molecule.

HIV-1 Nef protein plays a major role in viral immunopathogenesis, modulating surface expression of several immune receptors, altering signal transduction pathways, and enhancing viral infectivity, among other activities. Nef also exhibits great intersubtype diversity, but most studies have been focused only on Nef proteins from subtype B. Thus, little is known about the functional capacities of nonsubtype B Nef proteins in host cells. Here, we investigated cell surface regulation of MHC-I, MHC-II, the MHC-II-associated chaperone invariant chain (Ii), CD4, CD3, and CD28 in cells transfected or infected with five different Nef alleles including one HIV-1 subtype C and F allele. No significant difference among the Nef proteins regarding CD3, CD28, and MHC-II downregulation was observed. The NefC showed a slightly, yet significant, diminished capacity to downregulate MHC-I in all cells, as well as to downregulate CD4 in Jurkat cells and PBMCs. Strikingly, the two alleles from NefC and NefF were unable to upregulate the Ii chain both in transfected and infected cells. Moreover, the internalization rate of the surface Ii chain was only slightly affected by NefC and NefF, whereas it was drastically reduced by NefB. Nef domains known to be involved in Ii chain upregulation were conserved among the five alleles analyzed here. In summary, we identified two primary HIV-1 NefC and NefF alleles that are selectively impaired for Ii upregulation and that may help to elucidate the mechanism of this Nef function in the future. It will be important to determine whether the observed differences are HIV-1 subtype dependent and influence viral immunopathogenesis.

In this study, blood samples from newly HIV-1 infected men who have sex with men (MSM) were collected, and HIV-1 genotypes were identified based on gag p17-p24 and nef gene regions. We found that participants aged from 20 to 40 years old were the major infection group in Harbin. CRF01_AE was the predominant genotype, contributing to 84.7% of HIV-1 infections, followed by subtype B (4.7%), CRF07_BC (3.5%), and subtype B' (Thai B, 1.2%). Moreover, five unique recombinant forms (5.9%) were also identified, including genotypes 01B, 01C, and 01/02. The recombinant CRF01_AE/CRF02_AG was first reported in China. These results suggested that current HIV-1 genotype epidemic among MSM in Harbin is more complicated and that intersubtype recombinants have emerged. Therefore, timely regional epidemiological surveillance of HIV-1 genotype and development of prevention measures for new HIV-1 infections among MSM are quite important.

Multiple factors are known to contribute to nonprogressive disease in long-term nonprogressors (LTNP). We previously selected LTNPs, in which broadly neutralizing antibodies against HIV-1 very likely contribute to disease prevention. Here, we characterize those LTNPs further. We analyzed sequences of the viral genes env, nef, vpr, tat, and rev as well as the cellular ccr5, HLA-B*5701, and HLA-B*27 genes derived from eight LTNPs, as mutations in these genes have been associated with the LTNP status in some studies. Furthermore, we compared the replication rates of recombinant reporter viruses carrying envelope proteins from LTNPs to control viruses from patients with similar CD4 count and viral load. Concerning the cellular factors, none of the eight LTNPs showed the 32-base pair deletion in the ccr5 gene, and HLA-B*5701 and HLA-B*27 alleles were detected in only one LTNP, respectively. The reading frames for the regulatory genes nef, vpr, tat, and rev were all open. Although Env sequences from LTNPs differed from those of control patients with respect to the length of variable domains and the number of N-glycosylation sites, these differences were not statistically significant and did not lead to differences in infectivity of recombinant reporter viruses.

International audience; We screened the Neflaiprotein to identify new HLA-DR-restricted epitopes, because this small protein is expressed early during infection, and specific CD4(+) T cells are critical for effective immunity in HIV-1 infection. We synthesized a set of peptides that covers the sequence of the Nef protein, and performed binding assays using 10 common HLA-DR molecules. We defined four large regions in this protein able to bind very efficiently to eight HLADR molecules. We took advantage of healthy volunteers immunized with an HIV-1 lipopeptide vaccine that contains three of the four HLA DR-restricted regions to investigate their capacities to stimulate T cells. In 11 vaccinated volunteers, typed for their class II molecules, we were able to correlate sequences of the vaccine displaying binding activities to specific HLA-DR molecules and the induction of CD4(+) T cell proliferation. To identify potential HLA-DR epitopes, we synthesized 31 15-mer peptides and showed that 26 bound to one or more HLA-DR molecules. Interestingly, 12 of the 26 15-mer peptides identified are included in the sequence of lipopeptides. We used IFN-gamma ELISPOT and flow cytometer assays to investigate the capacity of these potential CD4(+) T cell epitopes to induce specific T cell responses. We showed that seven of these peptides were able to stimulate HIV-specific T cell responses in five of six tested volunteers. These cells are Nef-specific CD4(+) and CD4(+) CD8(+) T cells secreting IL-2/INF-gamma or IL-2 alone. To conclude, these 26 Nef HLA-DR-restricted peptides could be helpful to better evaluate CD4(+) deficiencies in HIV infection and, for new vaccine designs.

The importance of host cellular immune responses, particularly CD8(+) cytotoxic T-lymphocyte (CTL) responses, in control of human immunodeficiency virus type 1 (HIV-1) infection has been demonstrated in many clinical studies. These studies, along with vaccination challenge studies in rhesus macaques, indicate the importance of cellular immune responses against HIV-1. Toward this end, we evaluated anti-HIV-1 cellular immune responses in a cohort of 54 subjects who were chronically infected with HIV-1. By validation of IFN-gamma ELISpot assay, we established a dual cut-off criterion for scoring a positive response. The magnitude and frequency of cellular immune responses were measured against HIV-1 antigens (Gag, Pol, Nef, Rev, and Tat), using synthetic peptides as antigens in ELISpot assay. Here we showed that HIV-1 Gag, Pol, and Nef were frequent targets of T cell responses in these subjects, whereas Tat and Rev were less frequently recognized. We further evaluated the possible association between host cellular immune responses and corresponding plasma viral loads in this cohort. By performing ranking correlation analysis, we demonstrated a positive correlation between host viral loads and ELISpot responses of HIV Gag and Pol in untreated subjects. For the subjects under antiviral regimens, however, we did not find any significant association. Our findings suggest that the high levels of ELISpot responses in chronically infected subjects were reflective of their persistent viral infection.

The Nef protein of human immunodeficiency virus type 1 (HIV-1) has multiple functional domains, is immunogenic, and contains several cytotoxic T lymphocyte (CTL)-targeted epitopes. Several defined subfunctions of Nef are important for the pathogenesis of HIV-1 infection. In this study, we present the genetic diversity of the nef gene of 55 newly derived HIV-1 sequences obtained from Cameroonian patients. Four genetic subtypes and three circulating recombinant forms (CRFs) were identified: subtypes A (11%), G (7.3%), D (5.4%), F1 (1.8%), F2 (5.4%), CRF01_AE (5.4%), CRF02_AG (58.2%), and CRF11_cpx (1.8%). Two isolates clustered distinctly from the known HIV-1 genetic subtypes in nef and were designated as unclassified. Interestingly, the majority of all functional domains including the myristoylation signal, CD4 binding motif, beta turn motif, and the phosphorylation sites were well conserved in our cohort. Putative CTL-epitopic domains of the central portion of Nef were also well conserved, whereas those at the C-term were not. Our study demonstrated that despite high genetic diversity observed in the nef gene, most described functional domains and CTL epitopes were well conserved among Cameroonian HIV-1 subtypes. These findings could be used for the development of antiretroviral-acting therapeutics and anti-HIV-1 vaccines.

We showed that an anti-HIV lipopeptide vaccine injected to HIV-uninfected volunteers was well tolerated and able to induce a specific CD4(+) and CD8(+) T cell responses. The same vaccine was injected in HIV-1 chronically infected patients controlled by HAART to evaluate its immunogenicity. In this trial, 24 patients were immunized three times with a mixture of six lipopeptides (Nef 66-97, Nef 117-147, Nef 182-205, Gag 183-214, Gag 253-284, and Env 303-335) at 0, 3, and 6 weeks. We studied the HIV-1-specific CD4(+) T cell proliferative responses. The IFN-gamma secretion by activated CD8(+) T cells was evaluated, using an ex vivo ELISpot assay and 60 CD8(+) T cell epitopes derived from the vaccine. Before immunization (W0), anti-HIV CD4(+) T cell responses to Gag, Nef, and Env large peptides were detected in 7/23 (30%) analyzable patients. After three injections, 17/23 (74%) patients had a proliferative response and 16 of them induced new specific CD4(+) T cell responses. At W0, CD8(+) T cell responses to HIV-1 epitopes were detected in 6/23 (26%) patients. After vaccination, 16/23 (70%) patients showed CD8(+) T cell responses and 13 of these patients induced new T cell responses to 25 different HIV-1 epitopes. These HIV-1 epitopes were detected in patients with various HLA class I molecules (HLA-A2, -A3/A11, -A24, -B7 superfamily, -B8), as found in the majority of the white population. Lipopeptides induce new anti-HIV T cell responses in vaccinated infected patients and could be used as a new immunotherapy strategy. The majority of these responders induced specific new CD4(+) and CD8(+) T cell responses.

A multiHIV fusion gene expressing an antigenic fusion protein composed of regulatory HIV-1 proteins Rev, Nef, and Tat, as well as Gag p17/p24 and a stretch of 11 cytotoxic T lymphocyte (CTL) epitope clusters from Pol and Env, was cloned into a novel DNA vector named the Gene Transport Unit (GTU). A mouse H-2(d)-restricted HIV-1 gp120 epitope (RGPGRAFVTI) was cloned into the fusion gene as well. In addition to the HIV- 1 genes the GTU codes for a nuclear anchoring protein (bovine papilloma virus E2), ensuring the long maintenance of the vector and a high expression level of the selected immunogens. BALB/c mice were immunized with the GTU-MultiHIV DNA construct by different routes and regimens of immunization to assess the immunogenicity of the DNA vaccine in vivo. Mice developed strong CD8(+) CTL responses to HIV-1 Env and Gag measured by an ELISPOT-IFN-gamma assay and chromium release assay. In addition, T cell responses to regulatory proteins Rev, Nef, and Tat were induced. Antibody responses were detected to each of the HIV antigens encoded by the DNA construct. Minimal doses of the GTU-MultiHIV DNA delivered by gene gun were potent in inducing significant HIV-specific CTL responses. The equivalent doses of the conventional plasmid expressing MultiHIV DNA delivered by gene gun failed to do so. An ideal DNA vaccine should yield high expression of the viral antigens for a prolonged period of time, and expression of the multiple viral antigens is probably required for the induction of a broad and protective immune response. The GTU-MultiHIV DNA vaccine described is a good vaccine candidate that meets the above criteria.

We studied covariability of selected amino acid positions in globally dominant HIV-1 subtype C viruses. The analyzed sequences spanned the V3 loop, Gag p17, Gag p24, and five CTL epitope-rich regions in Gag, Nef, and Tat. The corresponding regions in HIV-1 subtype B were also evaluated. The analyses identified a great number of covarying pairs and triples of sites in the HIV-1B V3 loop (173 site pairs, 242 site triples). Several of these interactions were found in the earlier studies [e.g., the V3 loop covariability analyses by Korber et al. (Proc Natl Acad Sci USA 1993;90:7176-7180) and Bickel et al. (AIDS Res Hum Retroviruses 1996;12:1401-1411)] and have known biological significance. However, generally these key covarying sites did not covary in the HIV-1C V3 loop (total 17 covarying site pairs), suggesting that the V3 loop may have subtype differences in functional or structural operating characteristics. Covariability of positions 309 and 312 was observed in the immunodominant region HIV-1C Gag 291-320 but no covariability was found in the corresponding region of HIV-1B, and vice versa for Nef 122-141; these findings may reflect subtype-specific covariability within immunologically relevant regions. Gag p17 exhibited greater covariability and less diversity for HIV-1B than HIV-1C, raising the hypothesis that Gag p17 is highly immunodominant in HIV-1B and is especially important for HIV-1B vaccines. Information on covariability should be better exploited in assessments of HIV-1 diversity and how to surmount it with vaccine design.

HIV-1 Nef (Nef) is a myristoylated protein that contributes to HIV disease pathogenesis. Nef has a modulatory effect on T cell receptor (TCR) signaling, resulting in up-regulation of interleukin-2 (IL-2) production in stimulated T cells. Recent studies have shown that efficient TCR signaling requires enhanced association of TCR-signaling molecules with plasma membrane microdomains (lipid rafts) and fusion of rafts into larger structures. We utilized Jurkat T cell lines expressing wild-type Nef (Nef(wt)) and a myristoylation-deficient form of Nef (Nef(G)2(A)), from an inducible promoter, to determine the effects of Nef on the association of TCR-signaling molecules with rafts in nonstimulated T cells. In addition, the effect of Nef on raft size, before and after TCR activation by CD3 cross-linking, was also examined. Following induction, Nef(wt) was associated with both rafts and nonrafts, while Nef(G)2(A) was almost exclusively cytosolic. Induction of Nef(wt), but not Nef(G)2(A), coincided with an increased association of the src family tyrosine kinase, Lck, and TCRzeta with rafts, but not with nonrafts. Further, rafts were found to be significantly larger in CD3-activated T cells in the presence of Nef(wt) when compared to nonexpressing cells. We propose that myristoylated, raft-localized Nef primes resting T cells for activation by increasing the levels of signaling molecules within rafts, and that TCR activation is enhanced by the capacity of Nef to promote raft fusion.

Cytotoxic T lymphocytes (CTL) play a critical role in controlling human immunodeficiency virus-1 (HIV-1) and simian immunodeficiency virus (SIV) infections. However, in spite of developing a strong CTL response most HIV-1-infected patients eventually progress to AIDS. Amino acid changes in CTL epitope have been previously described and may permit HIV to escape from CTL immune responses. The importance of CTL selection pressure in controlling the course of viral evolution in HIV-infected patient remains debatable. For over a 10-year period, we longitudinally followed a patient for bulk unstimulated effector (eCTL) and stimulated memory CTL responses (mCTL) against the viral proteins Gag, Pol, and Nef. The patient showed a strong CTL response against Nef in unstimulated peripheral blood mononuclear cells with a peak during Month 40 of the follow-up. The mCTL response was also higher against Nef than Gag and Pol. PCR amplification and nucleotide sequencing of the plasma viral variants showed a viral variant with the epitope deletion that was detected early during the follow-up and essentially replaced the wild-type virus during the peak eCTL response. These studies support the importance of Nef epitope deletion as a mechanism for HIV-1 escape from CTL immune pressure.

Sequences from the nef/LTR overlap region of the human immunodeficiency virus type 2 (HIV-2) genome were amplified from uncultured peripheral blood mononuclear cells (PBMCs) from 40 HIV-2-infected individuals in The Gambia, West Africa. Additional sequences from the plasma of three blood donors were also derived. Analysis of HIV-2 U3 LTR transcription factor elements (PuB-1, p-ets, PuB-2, peri-kappa B, and NF-kappa B sites) indicated a relatively high level of conservation in vivo. The region immediately 3' of the nef termination codon, which exhibits clade-dependent specificity, was targeted by PCR to differentiate HIV-2 subtype A from subtype B infections, the two principal clinical HIV-2 subtypes. All clinical samples analyzed (n = 43) from The Gambia were identified as HIV-2 subtype A by a combination of LTR sequence analysis and subtype-specific amplification of subtypes A and B. Differential PCR amplification of the HIV-2 U3 LTR region represents a rapid means of differentiating subtype A from subtype B infections, the two dominant HIV-2 subtypes that are important in human disease.

DNA sequences and three distinct in vitro functions of Nef were evaluated in a group of seven perinatally infected children. nef gene sequences obtained before and after virus culture showed that one of the five non-/slow progressors harbored a virus with large deletions. nef genes from the remaining four children were full length but contained discrete changes at a higher frequency than the rapid progressors. In functional studies, 40 of 44 Nef proteins derived from the whole study group were capable of binding the cellular serine kinase p62, indicating that this function is well conserved among naturally occurring viruses. In contrast, representative Nef proteins derived from the long-term non-/slow progressors were found to be defective or far less capable of enhancing viral replication and/or viral infectivity in herpesvirus saimiri-transformed human T cells and peripheral blood mononuclear cells. On reversion of highly prevalent point mutations in the defective proteins, viral replication could be restored to wild-type levels. Our results suggest that nef genes derived from pediatric long-term nonprogressors have gross deletions in isolated cases but a higher prevalence of discrete changes that may impair Nef function in primary T cell assays, but not all functions reported for Nef.

Perturbation of astrocyte functions by HIV-1 infection may contribute to the pathogenesis of AIDS dementia complex (ADC). The present study investigated the possibility that astroglial transport of glutamate and aspartate, the major excitatory amino acids (EAAs) in the mammalian central nervous system (CNS), is altered by HIV-1 infection. Human U251 glioma cells were infected with the brain isolate SF162 of HIV-1. HIV-1 persisted in glial cells over several months. This nonproductive infection of glial cells was characterized by persistent expression of Nef over the time of the infection, and the transient presence of structural viral proteins, including the viral transmembrane glycoprotein gp41, which was detected during the initial 2 weeks following HIV-1 infection. The presence of gp41 in acutely HIV-1-infected glial cells coincided with a 36% decrease in D-[3H]aspartate uptake, owing to a reduction in the maximal transport capacity (vmax) for D-aspartate. The expression of typical astrocytic glutamate transporters EAAT1 and EAAT2 in U251 glioma cells was not altered by HIV-1 infection. To determine whether viral protein gp120, gp41, or Nef was involved in the impairment of EAA transport in acutely HIV-1-infected glial cells, effects of lentiviral lytic peptide type 1 (LLP-1) (corresponding to the carboxy terminus of gp41), recombinant SF2 gp120, and recombinant LAI Nef on D-[3H]aspartate uptake and the release of glutamate in glial cells were investigated. Only LLP-1 reduced D-[3H]aspartate uptake and facilitated the release of glutamate from glial cells in a concentration-dependent manner. These results suggest that the carboxy terminus of gp41 impairs EAA transport in glial cells, which may contribute to excitotoxic damage to neurons in HIV-1 infection of the CNS.

A major objective of current HIV-1 vaccination strategies is the induction of HIV-1-specific CD8+ MHC class I-restricted CTL responses, which are suggested to play a pivotal role in viral clearance and protection against HIV-1 disease progression. However, the marked genetic diversity of HIV-1 and existence of distinct viral subtypes or clades could potentially hinder the development of a universally efficacious HIV-1 vaccine. In this study we examined HIV-1 intraclade (B(LAI) versus B(MN)) Env gp160-specific CTL reactivity in recently HIV-1 clade B-infected individuals. We further evaluated the extent of interclade CTL cross-recognition of the divergent A and C Env gp160 subtypes, that are highly prevalent in the global pandemic. Freshly isolated PBMCs were stimulated in vitro with autologous PBMCs infected with recombinant vaccinia vectors expressing HIV-1 env, gag, pol, and nef genes derived from HIV-1 clade B. All 13 of the 19 HIV-1-seropositive subjects who elicited significant clade B Env gp160LAI CD8+ CTL responses also demonstrated comparable levels of CTL cross-reactivity against clade C92BR025 Env gp160. Nine of these individuals also showed extensive interclade CTL cross-recognition of clade A92UG037 Env gp160. Two HLA class I B7 donors had nondetectable intraclade CTL response against B Env gp160MN, while generating significant intraclade B(LAI) and interclade (A and C) Env gp160 CTL cross-reactivity. These observations serve to underscore the central importance of the HLA background of individuals in determining the pattern of immune reactivity to natural HIV-1 infection and presumably vaccines. Five donors studied also demonstrated broad CTL cross-reactivity against clade A92UG037 Gag p55, Pol, and/or Nef antigens. In conclusion, this present study indicates that there is a considerable degree of CD8+ CTL cross-recognition of the highly divergent HIV-1 Env gp160 subtypes during early phases of HIV-1 infection. Such findings suggest that HIV-1 vaccines based on a single clade that can induce extensive cross-clade immunity may demonstrate utility in diverse geographical regions.

HIV-1 Tat protein, which trans-activates HIV-1 expression, exerts many effects on host immune function. Meanwhile, PBMCs and pulmonary macrophages from HIV-1-infected patients produce only a small amount of IL-12, which plays an essential role in the development of helper T type 1 (Th1) cells, and in the generation of cytotoxic T lymphocytes. We examined the possibility that Tat suppresses IL-12 production by PBMCs from healthy donors. Tat significantly inhibited IL-12 production by human PBMCs stimulated with Staphylococcus aureus Cowan 1 strain (SAC) at concentrations between 5 and 40 ng/ml. Immunoabsorption by using polyclonal antibody to Tat abolished the suppression of the IL-12 production by Tat. Tat at the same concentrations did not affect IL-10, IL-6, or TNF-alpha production. Other HIV-1 proteins (Nef and gp120) did not influence IL-12 production. Tat also suppressed the expression of mRNA encoding the p40 chain of IL-12, whereas it did not affect the expression of mRNA encoding IL-10 and beta-actin. IL-12 production by monocytes, separated from PBMCs by the adhesion method, was also inhibited by Tat. These results suggest that Tat protein is one of the main causes of decreased IL-12 production by PBMCs (mostly by monocytes) from HIV-1-infected individuals.

Relationships were sought between specific anti-HIV cytotoxic T lymphocyte (CTL) responses (against structural and regulatory proteins of the HIV-1 LAI isolate) and plasma and cellular viral loads (VLs) in 17 recently HIV-1-infected patients including 3 displaying asymptomatic primary infection (PI) followed up for 12 months. Plasma VL was correlated directly with CD8 counts and inversely with CD4 counts. Cytotoxic reactions were observed in all patients and directed mainly against structural proteins. The earliest CTL responses were against Gag and Env proteins detected in 87 and 75% of the subjects, respectively, within the first month following PI. Anti-Env and Gag cytotoxic responses were inversely correlated with the plasma VL. Reactions against the pol gene products were thought to be either less involved in or less efficient for the initial decrease of viremia. Responses against regulatory gene products were weak and variable, apart from Nef, which was recognized by half of the subjects. Neutralizing antibodies were not detected before month 3, and were found only in six patients at subsequent times. Two of three patients with asymptomatic PI had a low viral burden and either a delayed response or one limited to a few protein CTL responses, suggesting that the magnitude of the CTL response depends on the initial plasma VL. The third patient displayed viral and CTL parameters identical to those of the patients with symptomatic PI. However, two subjects with symptomatic PI exhibited similarly low plasma VL and moderate CTL responses. Overall, the results suggest that the CTL response may not be the sole factor controlling viremia.

Myristoylated 21- and 25-residue N-terminal peptides of the Nef protein of HIV-1 lysed human erythrocytes and were cytotoxic toward a human CD4+ T cell line, CEM, and primary human peripheral blood mononuclear cells (PBMCs). The corresponding nonmyristoylated N-terminal peptides were only very weakly hemolytic and cytotoxic. A myristoylated peptide consisting of residues 31-50 of Nef was neither hemolytic nor cytotoxic. Alteration of the tryptophan residue at position 13 to a serine did not change the hemolytic and cytotoxic activity. Studies of the ultraviolet fluorescence of the tryptophan at position 5 in the peptide, using an artificial membrane system and fluorescence-quenching agents that inserted into the bilayer at different levels, suggested that myristoylation results in this residue being brought into contact with the upper hydrocarbon region of the lipid bilayer of the cell membrane. This tryptophan is flanked by a number of polar residues that would maintain it in this position, resulting in a considerable increase in disorder in the upper regions of the lipid bilayer, leading to its destabilization and to lysis. The cytotoxic activity of the myristoylated Nef fragments may, in part, explain the killing and deletion of cells, especially in lymphoid tissues, during HIV infection.

The partially CD4-expressing T cell clone, Vpr-1, which carries a latent vpr-defective HIV-1 genome and expresses HIV-1 Nef protein only, was permissive to superinfection by HIV-1. Superinfection of Vpr-1 with vif- or vpu-defective mutants, which were noncytopathic, reactivated the vpr-defective virus and led to homologous recombination and cytopathogenesis. The data provide an experimental model for homologous recombination being an important mechanism whereby HIV-1 acquires genetic heterogeneity, and when occurring among defective virus in vivo bestows novel biological activities and virulence.

We have studied two isoforms of Nef, Nef-27 and Nef-25, which were produced in E. coli. Nef-25 lacked the first 18 N-terminal residues of Nef-27 and both were nonmyristylated. Nef-27 fuses small unilamellar dipalmitoyl phosphatidylcholine vesicles (SUVs), as indicated by enhanced light scattering of SUVs and lipid mixing using concentration-dependent fluorescence dequenching. Nef-27 also causes the appearance of a shifted isotropic peak in the 31P NMR spectra of these vesicles, suggesting that protein interactions induce nonlamellar lipid structures. Recombinant Nef-25, which lacks only the 18 N-terminal residues of Nef-27, does not fuse vesicles and has little effect on the 31P NMR spectra. On the other hand, synthetic peptides consisting of 18 or 21 of the N-terminal residues of Nef-27 are strongly membrane perturbing, causing vesicle fusion and inducing isotropic peaks in the 31P NMR spectrum. Endogenous fluorescence spectra of the N-terminal peptide (21 residues) with SUVs show that the N-terminal sequence of Nef may achieve these perturbing effects by inserting its hydrophobic side into the lipid bilayer. Theoretical calculations using hydrophobic moment plot analysis indicate that short-length stretches (i.e., six amino acid residues) of the N-terminal sequence may insert into the lipid bilayer as multimeric alpha helices or beta sheets. The above-described membrane activities of Nef-27, which principally reside in its N-terminal domain, may play critical role(s) in certain functional properties of the full-length protein. For example, the fusogenic activity of the N-terminal sequence may be involved in the extracellular release of Nef-27, much of which appears to be associated with small membrane vesicles. The fusion activity may also be relevant to the ability of Nef-27 to downregulate CD4 and IL-2 receptors when this protein is electroporated into cultured lymphocytes, an activity not possessed by Nef-25.

Cellular distribution of HIV-1 Nef protein was studied by expressing the protein in mammalian cells. Cell extracts were fractionated by low- and high-speed centrifugation and by nonionic detergents. Two Nef-related proteins were expressed in COS cells, Nef-27kD and Nef-25kD. Nef-27kD, an N-myristoylated form of Nef, was found in the cytosol and in association with a particulate fraction of the cytoplasm. Treatment of the particulate cytoplasmic fraction with nonionic detergents, using three different protocols designed to isolate the cytoskeleton matrix, indicated that part of Nef was sensitive and part was resistant to detergent solubilization. These two cellular fractions represent membrane- and cytoskeleton-associated Nef. Nef-25kD, initiated from an in-frame AUG codon, was not modified with myristic acid at the amino terminus. Consequently, this protein was present in a soluble form in the cytosol. Furthermore, a mutant of Nef-27kD, in which the myristoylation signal is deleted, appears as a cytoplasmic soluble protein. To determine domains in Nef that are responsible for its subcellular distribution, successive internal deletions of 14-20 amino acids were introduced at the N-terminal portion of the protein. Five mutants were evaluated with respect to their cellular localization. One mutant (pSVLA-5), from which amino acids 73-88 were deleted, did not copurify with the detergent-insoluble fraction. The protein was, however, present in the particulate cytoplasmic fraction, presumably in association with membranes. Taken together, these results suggest that N-myristoylation of Nef affects its association with both membranes and cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

Reagents for evaluating non-clade B HIV-specific T cell responses are uncommon. Peptides based on highly conserved HIV-1 consensus group M sequences that are phylogenetically closer to most circulating strains may provide potential alternative reagents in populations with diverse infections, and may be relevant for vaccine design. Recognition of such reagents in clade A1-and D-infected populations has not been previously evaluated. Interferon (IFN)-γ ELISpot assay was used to evaluate T cell recognition of Gag and Nef peptides based on consensus group M sequences in 50 treatment-naive adults predominantly infected with HIV-1 clades A1 and D. Gag-induced T cell responses were correlated with gag sequence diversity. Infecting clades were determined from gag sequences for 45 of the 50 subjects as 40% clade A1 (18/45), 45% clade D (20/45), 2% clade C (1/45), 2% A1/C recombinant (1/45), 2% A1/D (1/45), 7% CRF10_CD (3/45), and 2% U (unclassifiable) (1/45). The mean genetic divergence and diversity of clade A and D gag region compared to group M consensus sequences at synonymous and nonsynonymous nucleotide and amino acid levels were not always significant. Gag peptides were targeted at significantly higher frequency [88% (44/50)] than Nef [64% (32/50)]; p=0.014, although their mean IFN-γ magnitudes were comparable ([3703 (95% CI 2567–4839)] vs. [2120 (95% CI 478–3762)]), respectively. Measurable virus-induced IFN-γ responses were detected in 96% (48/50) individuals, primarily targeting the more conserved Gag p24 and Nef central core regions. Use of these reagents to screen for HIV-specific IFN-γ responses may mitigate the challenge of viral diversity; although this targeting is apparently biased toward a few highly conserved epitopes.

The entire nef gene sequence of HIV-2, NIH-Z strain, has been cloned into the pJL6 expression vector and used for the synthesis of a 23-kDa protein in E. coli. The expressed protein is a fusion between the N-terminal 13 amino acids of the cII gene, 8 amino acids resulting from the ligation procedure, and the 180 amino acids that comprise the HIV-2 Nef sequence from the NIH-Z strain. The bacterially expressed Nef protein has been purified to apparent homogeneity on analytical scale (10-20 micrograms) by a combination of sequential detergent extraction, gel filtration, and reversed-phase high-performance chromatography. The expressed Nef protein is highly susceptible to proteolysis (chymotryptic-like activity) and this property accounts for the low yield obtained by gel filtration and RP-HPLC. Larger amounts (100 micrograms) of the purified Nef protein have been produced by a purification procedure that employs sequential detergent extraction, chromatography on Q-Sepharose in the presence of 7 M urea, and chromatography on hydroxylapatite, also in 7 M urea. The purified HIV-2 Nef protein has been used for the production of polyclonal and monoclonal antibodies. The milder method of purification should facilitate structure-function studies of the Nef protein and its role in the life cycle of HIV.

HIV-1 transcripts are generated through a complex alternative splicing mechanism, resulting in the production of multiple RNAs coding for each viral protein. HIV-1 RNA splicing has been analyzed mostly in in vitro assays, and in vivo data are scarce. Here we analyze HIV-1 transcripts generated in peripheral blood mononuclear cells of HIV-1-infected individuals by RT-PCR amplification and sequencing of RNA extracted from unstimulated cells. We identify several unusual or unreported transcripts, most of them splicing within the Nef coding sequence. The majority are predicted to code for a Nef C-terminal 34 amino acid peptide, but others code for Vpr, a truncated Tat, and a 41 amino acid peptide encoded in an antisense exon. We also identify nef and env transcripts splicing four nucleotides downstream of SA5. These results represent the first report on the in vivo generation of diverse novel HIV-1-spliced transcripts, frequently encoding a Nef C-terminal peptide.

Cellular immunogenicity of env gp160, nef p27, and gag p55 proteins of human immunodeficiency virus type 1 (HIV-1) was studied in mice immunized with vaccinia virus recombinants. Proliferative responses of spleen cells were comparable against env gp160, nef p27, and gag p25 recombinant proteins. No specific activity was observed against gag p18 protein. Env, nef, and gag-specific T-cell lines were generated by repeated stimulation of immune spleen cells with recombinant HIV-1 proteins. They were CD4 positive, proliferative, and also cytotoxic against HIV-transfected target cells. Specificity of the T-cell response against nef and gag protein was analyzed with synthetic peptides. Peptides nef 15, nef 16, and gag AM-30 were, respectively, reactive in nef- and gag-specific proliferative and cytolytic assays. The three peptides described have a relatively conserved amino acid sequence among HIV isolates and appear broadly immunoreactive among species.

Many aspects of HIV-1 pathogenesis are affected by Nef protein activity, and efforts have been made to study variation in the nef gene and how that variation relates to disease outcome. We studied the genetic diversity of the nef gene in distinct clones obtained from the same patient (intrahost) and in sequences obtained from different hosts (interhost). The set of sequences analyzed was obtained from HIV-1-infected Brazilian children and contained 112 clones from 25 children (intrahost samples), as well as 55 sequences from epidemiologically unlinked children (interhost samples). We found extensive site polymorphisms and amino acid length variations, mainly in the amino terminal region of the nef gene, between the myristoylation motif (MGxxxS) and the MHC-1 downregulation motif (Rxx). Analysis of the sequences deposited in the Los Alamos HIV sequences database ( www.hiv.lanl.gov ) indicated that the most frequent motif at the MHC-1 downregulation site in the subtype B strain is R(86%)A(64%)E(82%) (n = 1040) and R(78%)T(74%)E(56%) in the subtype C strain (n = 549). Conversely, the Brazilian subtype B isolates presented the motif R(81%)T(62%)E(67%) at this site (n = 64). A detailed analysis of selective pressures identified a concentration of codons under strong positive selection in the amino terminal region of the nef gene. We also determined that different sites are under positive selection in the subtype B and subtype C viruses. The amino acid composition in the MHC-1 downregulation motif of the nef gene in our sequences may indicate a distinct adaptive pattern of HIV-1 subtype B to the Brazilian host population.

HIV-1 mother-to-child transmission (MTCT) was evaluated in terms of the molecular characterization of the env and nef genomic regions and quantification of maternal RNA viral loads. Assignment of viral subtype was achieved by direct sequencing of PCR 1172 products amplified from proviral DNA in 45 HIV-1-nontransmitting mothers (NTM), along with 13 pairs of HIV-1-transmitting mothers (TM) and their infected children (C). Analysis of the env C2V3C3 and nef sequences revealed that subtypes G and B, and their genetic combinations (AG, BG), accounted for over 84.5% of all viruses identified. The genetic structure form envA-nefG was the most commonly observed, with a lower frequency in the NTM (13.3%) compared to the TM (23.1%) group. A greater number of genetic forms was observed among NTM, namely the presence of sequences assigned to subtypes D and F, as well as the intergenetic A/J, and C/U, recombinant forms, along with a mosaic provirus with a complex putative envA-nefEGE genetic structure. No significant differences were found when RNA viral loads were evaluated as a function of the viral subtypes. Nevertheless, a relatively high quantification of HIV-1 RNA was obtained in the NTM group, emphasizing the importance of the compliance and effectiveness of therapeutic schemes to control viral replication and reduce the risk of HIV vertical transmission. V3 sequences displaying features associated with the R5 phenotype dominated in both groups. Both C2V3C3 and Nef's functional domains were conserved during HIV-1 vertical transmission.

To provide the hard-won information on HIV-1 polymorphism from inmates in prison and to study the genetic variation of HIV-1 strains, we analyzed the characterization of the nucleotide and corresponding protein sequences of naturally occurring nef sequences derived from 16 HIV-1-positive inmates from prisons in China. Nested PCR and DNA sequencing were used to determine nef sequences in peripheral blood mononuclear cells. Phylogenetic analysis revealed a distribution of the viral sequences among subtype B, CRF_BC, and CRF_AE, with subtype B accounting for more than half of the genotypes. When compared with the consensus sequence, a certain amino acid sequence variability was observed. However, most of the described nef functional motifs were relatively well conserved in the majority of the sequences analyzed. Our results indicate that HIV-1 strains found in our study subjects may have a common origin and the description of motifs will produce important information for further studies of nef function.

We have shown that Korean red ginseng (KRG) intake is associated with gross deletions in the 5' LTR/gag (gDeltaLTR/gag) and nef genes (gDeltanef) of patients infected with subtype B of HIV-1. Here, we investigated these effects in three long-term survivors (LTSs) of subtype CRF02_AG of HIV-1. The three LTSs were diagnosed with HIV in 1987, 1988, and 1989, and have been treated with KRG for 7-15 years. Thirty-two samples of peripheral blood mononuclear cells were obtained from the subjects and used to amplify the 5' LTR/gag and nef genes via nested PCR. We obtained 88 amplicons in 5' LTR/gag and 128 amplicons in nef. The frequency of gDeltaLTR/gag was significantly higher (37.5%) in three LTSs than in control patients (8.6%, p0.01). Eight amplicons (9.5%) contained premature stop codon(s) in the gDeltaLTR/gag in three LTSs. Fourteen of the 128 nef amplicons (10.9%) contained the gDeltanef, which was present in only two (7.7%) of the 26 amplicons from control subjects. Interestingly, gDeltanef was detected 7 years after the reinitiation of KRG intake in an LTS and, coincidently, CD4 T cell counts and CD4/CD8 ratios rapidly increased. These data indicate that long-term intake of KRG has the therapeutic potential to induce gross deletions in HIV-1.

Twenty hemophiliacs were infected with Korean subclade B (KSB) of HIV-1 from two cash-paid plasma donors in Korea in 1990. Our previous studies revealed that Korean red ginseng (KRG) intake increases the frequency of gross deletion in the nef gene (gDeltanef). We investigated whether KRG and highly active antiretroviral therapy (HAART) affected the frequency of gDeltanef in the 20 hemophiliacs who share common characteristics of the HIV-1 source, mode of transmission, and infection time. Over a 10-year period, we obtained 522 nef amplicons by nested PCR using 172 samples of peripheral blood mononuclear cells. Of the 522 nef amplicons, 69 (13.2%) were gDeltanef. Despite a 2-fold higher monthly dose of KRG, the frequency of gDeltanef detection (3.2%) was significantly reduced during HAART compared with that prior to HAART (20.6%) (p < 0.001). gDeltanef was detected significantly more in patients treated with a monthly KRG intake of more than 60 g (26.8%) than in patients treated with a monthly KRG intake of less than 60 g (10.5%) (p < 0.05). These finding suggest that the frequency of gDeltanef is dependent on the amount of KRG intake, although further study is needed. These data might provide a new perspective on the pathogenesis of HIV-1.

Murine monoclonal antibodies (MAbs) raised against a recombinant nef protein fragment of human immunodeficiency virus type 1 (HIV-1) strain BH10 were characterized by an epitope mapping system using overlapping decapeptides. Four different immunogenic regions were identified. Ten human HIV-1-positive sera were tested in the same epitope mapping system, seven of these were reactive with four immunogenic regions. Two of the nef-specific epitopes recognized by human sera overlapped with the epitopes defined by the murine monoclonal antibodies. The reactivity of the monoclonal antibodies with the recombinant nef protein and with infected and uninfected cells were investigated in a variety of test systems. The results are discussed with respect to homologous regions of nef and cellular proteins.

A higher functionality of CD8(+) T cells might contribute to low-level HIV replication in long-term nonprogressors (LTNPs). However, the contrary could also be true, being the function of CD8(+) T cells modulated by HIV replication. We tested whether enhanced HIV replication following antiretroviral therapy interruption could modify the functional profile of HIV-specific CD8(+) responses. Production of MIP-1beta, IL-2, TNF-alpha, and CD107 expression by CD8(+) T cells in response to Gag and Nef optimal peptide pools was analyzed using polychromatic flow cytometry in nine HIV-infected individuals followed for 12 months after discontinuation of antiretroviral therapy. At baseline, CD8(+) T cell subsets with the greatest contribution to response were MIP-beta(+)TNF-alpha(-)IL-2(-)CD107(+) and MIP-beta(+)TNF-alpha(-)IL-2(-)CD107. Most responses were mediated by subsets expressing only one or two molecules. After 12 months of discontinuing antiretroviral therapy, no significant differences were observed in the functional profile of Gag- and Nef-specific CD8(+) responses. However, viral rebound induced a significant increase in the heterogeneity of Gag-specific CD8(+) responses. In summary, viral replication following discontinuation of antiretroviral therapy has no significant impact on qualitative aspects of HIV-specific CD8(+) responses. Thus, a higher functionality of CD8(+) responses does not seem to be the consequence of low-level virus replication.

The mechanism explaining the failure of HIV-specific CD8(+) T cell responses to successfully control HIV replication remains elusive. A total of 83 drug-naive HIV-infected individuals, 27 of whom were long-term nonprogressors (LTNP), was examined. The ability of CD8(+) T lymphocytes to produce three different cytokines (MIP-1beta, TNF-alpha, IL-2) in response to HIV Gag and Nef peptides and to polyclonal stimuli and the ability of HIV-specific CD8(+) T cells to expand in vitro were evaluated by multiparameter flow cytometry. In response to polyclonal stimulation, LTNP presented significantly higher levels of several CD8(+) T cell subsets than progressors. While most patients presented detectable Gag and Nef-specific CD8(+) responses, no significant differences in any of the CD8(+) functional T cell subsets were recognized when comparing LTNP and progressors. HIV responses were dominated by cells producing only MIP-1beta or TNF-alpha, being similar in LTNP and progressors. However, expansion of HIV-specific CD8(+) T cells was more frequent in LTNP than progressors, especially for cells producing MIP-1beta. LTNP show higher levels of CD8(+) responses against polyclonal stimuli than progressors. However, HIV-specific CD8(+) responses do not differ between them except for a more preserved ability of cells from LTNP to expand in vitro.

Both hydroxyurea (HU) and structured treatment interruptions (STI) have been investigated as therapeutic approaches to enhance immune responses in chronically HIV-infected individuals. HIV-specific T cell responses as well as T cell activation were analyzed longitudinally in 31 HIV-infected individuals who had been treated for the prior 12 months with didanosine (ddI) plus HU and thereafter completed three STI cycles consisting of 2 months off and 2 months on ddI-HU. Similar increases in plasma HIV-RNA were seen in each of the three cycles off therapy, whereas CD4 counts remained fairly stable along the study period. T cell activation paralleled the evolution of plasma HIV-RNA during the first STI cycle and waned afterward. At baseline most patients presented a high level of CD8+ responses to different HIV peptide pools and 23% of them had CD4+ responses to Gag and/or Env. The level of CD8+ responses against each pool was stable and did not increase during STI cycles, while CD4 responses tended to decline. However, the contribution of Nef-specific response to the total CD8 response tended to increase. In a multivariate model, both a higher baseline plasma HIV-RNA and a higher level of Nef-specific response contribution to the total CD8+ response were independently associated with lower plasma HIV-RNA increases during each of the three STI cycles. Nef-specific CD8+ responses might contribute to a better virological control of HIV replication following treatment interruptions in HIV-infected individuals and might be boosted by the immunomodulatory effect of HU.

Most HIV vaccine trials in the world are conducted with clade B while most circulating viral strains in Africa are non-B subtypes. We determined whether CD8+ T cells from HIV-1 intersubtype CRF02_AG-infected Ivorian individuals were able to recognize clade B epitopes. CD8+ T cell responses of nine HIV-1 intersubtype CRF02_AG-infected Ivorian patients and nine HIV-1 subtype B-infected French patients were studied using pools of HIV-1 clade B peptides (110 well-defined HIV CD8+ T cell epitopes) in an ELISPOT IFN-gamma assay. There was no difference in the number of recognized peptide pools between Ivorian and French cohorts (mean of four pools in both cases). Ivorian individuals had generated CD8+ T cell responses cross-reactive against HIV-1 subtype B and some individual peptides had been identified. Furthermore, sequence analysis of nef HIV genes of the Ivorian patients and nef cloning in two patients revealed very few variations between HIV- 1 intersubtype CRF02_AG and subtype B in nef immunodominant regions included in HIV clade B lipopeptide vaccines, currently tested in France.

The aim of this study was to define pluriepitopic regions in Gag and Nef possibly relevant in the perspective of a vaccine design in a vietnamese population. The protein sequences derived from gag and nef genes and phenotyping of the class I human leukocyte antigens (HLA) A and B alleles were established for 28 human immunodeficiency virus type 1 (HIV-1)-infected patients from Ho Chi Minh City, Vietnam. The protein sequences display polymorphism mutations as compared with a B reference strain (HXB2). The most frequently represented HLA-A and -B alleles were HLA-A11, A02, and A33 expressed by 35.7, 23.2, and 21.4% of the patients, respectively, and HLA-B75, B46, and B62 expressed by 35.7, 25, and 17.9% of the patients, respectively. This study allows us to determine four pluriepitopic regions in Gag and Nef that should be chosen for a vaccine design in a Vietnamese population.

Epidemiologically-linked HIV-1 transmission cohorts serve as excellent models to study HIV disease progression. The actual relationship between viral variability and HIV disease outcome can be extrapolated only through such rare epidemiologically linked HIV-1-infected cohorts. We present here a cohort of three patients with the source termed donor A (a nonprogressor) and two recipients B and C. Both recipients acquired HIV through blood transfusion from donor A and have progressed to AIDS. By analyzing 15 near full-length HIV- 1 genomes (8.7 kb each genome) from longitudinally collected peripheral blood cell samples (four time points for patient A, four for patient B, and seven from patient C), we were able to demonstrate transmission of HIV from donor A and epidemiologic linkage among members A, B, and C after 10 years of HIV infection. These analyses are novel in demonstrating that HIV-1-infected nonprogressing individuals bear the potential to transmit HIV-1 variants and that HIV variants, which led to a benign disease in a nonprogressor donor, were able to cause disease in other individuals. Overall, these studies highlight the utility of full genome sequencing in establishing epidemiologic linkage in a chronically infected HIV cohort after 10 years of initial infection.