Characterization of human immunodeficiency virus type 1 vif gene in long-term asymptomatic individuals

We have determined the sequence of the human immunodeficiency virus type 1 (HIV-1) vif genes from a cohort of 42 long-term nonprogressors (LTNP) and compared these sequences to those of 8 late progressors. The coding potential of the vif open reading frame directly derived by nested PCR from uncultured peripheral blood mononuclear cell DNA was conserved in all 50 individuals. The nucleotide distances between vif sequences were not significantly different between LTNP and late progressors, indicating similar selections of viruses within both types of long-term HIV-1-infected subjects. However, a statistically significant correlation between an amino acid signature at position 132 of Vif and the viral load was found within LTNP. Namely, amino acid Ser was associated with low viral load and amino acid Arg with high viral load. This signature was also observed when LTNP with low viral load were compared to progressors. The Ser132 signature was introduced in place of Arg132 present in the HIV-1 YU-2 Vif prototype into chimeric viruses to assess the impact of Vif signature on the virus. While the replication properties in the SupT1 cell line were unmodified, the mutagenized virus revealed a fivefold decreased replication in activated PBMC, suggesting a possible role of this Vif signature for viral production in vivo.     

Defective nef alleles in a cohort of hemophiliacs with progressing and nonprogressing HIV-1 infection.

Deletion of the nef gene results in viral attenuation and confers protection against challenge with wild-type simian immunodeficiency virus in macaques. Regarding HIV-1 infection, a few long-term nonprogressors (LTNP) with nef deletions have been described. In this study, the nef genes of a group of seven LTNP and eight progressors, all belonging to the same cohort of infected hemophiliacs, were analyzed by cloning and sequencing from both virion RNA and peripheral blood mononuclear cell-associated proviral DNA. Defective nef sequences coexisted with full-length nef open reading frames in five of seven LTNP and two of eight progressors. The proportion of disrupted nef sequences within each individual was significantly higher in LTNP (ranging from 10 to 63%) than in progressors (ranging from 9 to 21%) (P = 0.013). Moreover, in-frame small deletions predicting to encode Nef were found in all RNA- and DNA-derived clones from one LTNP and four progressors. A chimeric virus in which the nef gene of NL4.3 was substituted with the nef allele containing the deletion of two alanines at position 49-50 found in two progressors showed a defective replicative capacity compared to NL4.3 virus. In summary, hemophiliacs with either progressing or nonprogressing HIV-1 infection are characterized by the presence of defective nef variants.     

Characterization of host genetic expression patterns in HIV-infected individuals with divergent disease progression

The course of HIV-1 infection shows a variety of clinical phenotypes with an important involvement of host factors. We compare host gene expression patterns in CD3+ T cells from two of these phenotypes: long-term non-progressor patients (LTNP) and matched control patients with standard HIV disease progression. Array analysis revealed over-expression of 322 genes in progressors and 136 in LTNP. Up-regulated genes in progressors were mainly implicated in the regulation of DNA replication, cell cycle and DNA damage stimulus and mostly localized into cellular organelles. In contrast, most up-regulated genes in LTNP were located at the plasmatic membrane and involved in cytokine-cytokine receptor interaction, negative control of apoptosis or regulation of actin cytoskeleton. Regarding gene interactions, a higher number of viral genes interacting with cellular factors were seen in progressors. Our study offers new comparative insights related to disease status and can distinguish differentiated patterns of gene expression among clinical phenotypes.     

Differential prevalence of the HLA-C -- 35 CC genotype among viremic long term non-progressor and elite controller HIV+ individuals

Susceptibility to HIV infection and disease progression are complex traits modulated by environmental and genetic factors, affecting innate and adaptive immune responses, among other cellular processes. A single nucleotide polymorphism (SNP) 35 kb upstream of the HLA-C gene locus (-35C/T) was previously shown to correlate with increased HLA-C expression and improved control of HIV-1. Here, we genotyped the -35C/T SNP in 639 subjects (180 uninfected patients, 304 HIV progressors and 155 LTNP) and confirmed the association of the -35C/T variant with the LTNP phenotype. The genotype frequencies in the general population subjects did not differ significantly from those seen in HIV progressors (p-value=0.472). However, a significant higher frequency of the protective CC genotype was identified when LTNP were compared either with HIV progressors alone (p-value<0.0001) or progressors and uninfected subjects together (p-value<0.0001). When considering aviremic LTNP alone (elite controllers; viral load below 50 copies/ml), the -35 CC genotype was not overrepresented compared to HIV progressors. Conversely, a significant association was found with the viremic LTNP groups (viral loads below 10,000 copies/ml). These results suggest that other factors alone or in combination with the -35 CC genotype may play an important role in differentiating the elite controller status from LTNP. Combination of different genetic variants may have additive or epistatic effects determining the HIV course of infection.     

CD4+T淋巴细胞的活化在HIV-1感染长期非进展者及典型进展者之间差异的研究

目的了解CD4 T淋巴细胞的活化在长期感染非进展者(LTNP)及典型进展者(TP)之间的差异,探讨HIV感染的免疫基础。方法收集24例HIV-1感染患者(7名LTNP患者,17名TP患者)及15例健康对照的抗凝血标本,流式细胞术检测CD4 T淋巴细胞的活化标志物CD38并检测其CD4计数等指标;bDNA方法检测患者体内的病毒载量。结果与正常人相比,LTNP患者的CD4 T淋巴细胞的活化的差异无统计学意义(P>0.05)而TP患者的活化程度明显增高(P<0.01);HIV感染者CD4 T淋巴细胞CD38的表达水平与CD4计数呈正相关,与病毒载量呈负相关。结论随着疾病的进展,CD4 T淋巴细胞的活化程度逐渐增高;LTNP患者CD4 T淋巴细胞的活化程度没有明显增高可能是其疾病长期不进展的原因之一。     

Immunogenic and tolerogenic signatures in human immunodeficiency virus (HIV)-infected controllers compared with progressors and a conversion strategy of virus control

Epidemiological studies have identified a small cohort of controllers of human immunodeficiency virus (HIV)-1 infection, who without treatment have no detectable virus, and others who progress at a variable rate. The objective of this study was to distinguish immune signatures in HIV controllers and progressors, by evaluating tolerogenic and immunogenic factors in untreated HIV-1 infected individuals. The recruited population was divided into putative elite controllers (PEC), long-term non-progressors (LTNP), normal progressors (NP) and fast progressors (FP). The proportion of regulatory T cells [T(regs) , CD4+ CD25+ forkhead box P3 (FoxP3+)], programmed death (PD)-1 and cytotoxic T lymphocyte antigen (CTLA)-inhibitory molecules and CD40L, CD69 and Ki67 activation markers were evaluated in peripheral blood mononuclear cells (PBMC) by flow cytometry. Significant differences were found between HIV controllers and HIV progressors, with up-regulation of T(regs) , PD-1 and CTLA-4 and decrease of CD40L expression in progressors compared with controllers. Expression of CD40L and concentrations of interleukin (IL)-6, CCL-3, and CCL-4 were significantly higher in PEC and LTNP than in NP and FP. In an attempt to convert immune signatures of progressors to those of controllers, seven agents were used to stimulate PBMC from the four cohorts. Treatment with CD40L and IL-4 or PD-1 antibodies in vitro were most effective in converting the immune signatures of progressors to those observed in controllers by down-regulating T(regs) and up-regulating CD40L expression in CD4+ T cells. The conversion concept merits translation to in vivo immune control of HIV infection.     

Genetic polymorphism of CCR5 gene and HIV disease: The heterozygous (CCR5/Δccr5) genotype is neither essential nor sufficient for protection against disease progression

Homozygous (Δccr5/Δccr5) and heterozygous (CCR5/Δccr5) deletions in the β-chemokine receptor 5 (CCR5) gene, which encodes for the major co-receptor for macrophage-tropic HIV-1 entry, have been implicated in resistance to HIV infection and in protection against disease progression, respectively. The CCR5/Δccr5 genotype was found more frequently in long-term nonprogressors (LTNP) (31.0%) than in progressors (10.6%, p < 0.0001), in agreement with previous studies. Kaplan-Meier survival analyses showed that a slower progression of disease, i.e. higher proportion of subjects with CD4⁺ T cell counts >500/μl (p = 0.0006) and a trend toward a slower progression to AIDS (p = 0.077), was associated with the CCR5/Δccr5 genotype. However, when LTNP were analyzed separetely, no significant differences in CD4⁺ T cell counts (p = 0.12) and viremia levels (p = 0.65) were observed between the wild-type (69 % of LTNP) and the heterozygous (31.0 %) genotypes. Therefore, there are other factors which play a major role in determining the status of nonprogression in the majority of LTNP. Furthermore, there was no evidence that the CCR5/Δccr5 genotype was associated with different rates of disease progression in the group of progressors. Taken together, these results indicate that the CCR5/Δccr5 genotype is neither essential nor sufficient for protection against the progression of HIV disease.     

Analysis of the HIV-1 nef gene in five intravenous drug users with long-term nonprogressive HIV-1 infection in Italy

Great variability in the course of human immunodeficiency virus type 1 (HIV-1) infection results from a complex interplay between host and virus factors. Some of the patients with prolonged nonprogressive infection have been reported to harbor virus variants with gross deletions in the accessory nef gene that has been implicated in in vivo pathogenicity in simian and mouse models. To investigate the role of nef-deleted HIV-1 in long-term nonprogressor (LTNP) drug addicts in Italy the nef sequence from proviral DNA was analyzed from five LTNPs and five rapid progressor controls. Only small (2-12 amino acids) in-frame deletions and insertions were detected in the N-terminal polymorphic and variable regions obtained from three LTNPs and one rapid progressor. There was no evidence of premature termination of the Nef protein and all of the identified functional motifs were well conserved in both groups. Phylogenetic analysis showed interdigitation of nef sequences obtained from LTNPs and rapid progressors. The nef sequence of one LTNP, however, diverged significantly from those of the other patients. Availability of two additional blood DNA samples obtained previously from this subject allowed to detect evolution of nef at 14-17 years of HIV-1 infection, including progressive deletions. Although alterations of nef may be relatively frequent and continue to evolve in LTNPs, this study of a small number of patients does not indicate that gross deletions or loss of functional motifs play a major role in delaying or halting disease progression in infected drug abusers in Italy.     

Gene expression profile in long-term non progressor HIV infected patients: In search of potential resistance factors

Long-term non-progressors (LTNP) represent a minority (1–5%) of HIV-infected individuals characterized by documented infection for more than 7–10 years, a stable CD4+ T cell count over 500/mm³ and low viremia in the absence of antiretroviral treatment. Protective factors described so far such as the CCR5delta32 deletion, protective HLA alleles, or defective viruses fail to fully explain the partial protection phenotype. The existence of additional host resistance mechanisms in LTNP patients was investigated here using a whole human genome microarray study comparing gene expression profiles of unstimulated peripheral blood mononuclear cells from LTNP patients, HIV-1 infected patients under antiretroviral therapy with CD4+ T cell levels above 500/mm³ (ST), as well as healthy individuals. Genes that were up- or downregulated exclusively in LTNP, ST or in both groups in comparison to controls were identified and classified in functional categories using Ingenuity Pathway Analysis. ST and LTNP patient groups revealed distinct genetic profiles, regarding gene number in each category and up- or downregulation of specific genes, which could have a bearing on the outcome of each group. We selected some relevant genes to validate the differential expression using quantitative real-time qRT-PCR. Among others, we found several genes related to the canonical Wnt/beta-catenin signaling pathway. Our results identify new possible host genes and molecules that could be involved in the mechanisms leading to the slower progression to AIDS and sustained CD4+ T cell counts that is peculiar to LTNP patients.     

IL6 and FAS/FASL gene polymorphisms may be associated with disease progression in HIV-1-positive ethnically mixed patients

The progression of AIDS depends on the complex host and virus interactions. The most important disease progression hallmarks are immune activation and apoptosis. In this study, we address the prevalence of polymorphisms related to proinflammatory and apoptotic genes, such as IFNG (+874T/A), TNF (308G/A), IL6 (−174G/C), IL8 (−251A/T), FAS (−670A/G), and FASL (−124A/G) in 160 ethnically mixed HIV-1-infected patients from multicentre cohorts with different clinical outcomes (13 elite controllers [EC], 66 slow long-term non-progressors [LTNPs], and 81 progressors [P]). The genotyping was accomplished by TaqMan-qPCR. Among all the polymorphisms analyzed in the cytokines, the IL6 −174G/C polymorphism showed a higher frequency of GG genotype in the LTNP and LTNP+EC groups as compared to the P group. Moreover, there was a significantly higher frequency of the G allele in the LTNP and LTNP+EC groups as compared to the P group. On the other hand, the levels of CD4⁺ T lymphocytes were higher among individuals showing the AA and AG genotypes for the FASL −124A/G polymorphism as compared to the GG genotype. Furthermore, the AG and AA genotypes were more frequent, as compared to the GG genotype, in individuals showing a lower viral load. In contrast, for the FAS −670A/G polymorphism, a significantly higher viral load was observed in individuals with the AG genotype as compared to the GG genotype. In conclusion, we found three genetic allelic variants of the IL6 −174G/C, FASL −124A/G, and FAS −670A/G polymorphisms that were related to disease progression and immunological and virological markers in cohorts of HIV-1-positive ethnically mixed patients.     

Mapping the genetic determinants of human immunodeficiency virus type 2 for cell tropism and replication efficiency

Summary.  Two distinct infectious molecular clones of human immunodeficiency type 2 (HIV-2) were analyzed for their biological properties in six cell lines. Fourteen chimeric and ten mutant viruses were constructed from these two viral genomes to localize the genetic determinants responsible for the phenotypes. Growth property of the viruses in the cell lines, together with the biochemical data, showed that a major determinant for the viral tropism resides in the env gene. In addition, in some cell lines, the accessory genes vif and nef affected the efficiency of virus replication. Thus, like HIV-1, mutations in the auxiliary and env genes of HIV-2 contributed much to the differences in virological characteristics. Accepted October 15, 1997 Received August 21, 1997     

Lack of association of SDF-1 3'A variant allele with long-term nonprogressive HIV-1 infection is extended beyond 16 years

We studied the frequency of the SDF-1 3'A allelic variant (801G-->A) in a cohort of white Spaniards made up of (1) HIV-1-infected long-term nonprogressors (LTNPs) older than 16 years of age (n = 57), (2) HIV-1-infected usual progressors (UPs; n = 107), and (3) a group of healthy controls (n = 100). The mutant SDF-1 3'A allele was observed in 28% of LTNPs, 19% of UPs, and 26% of healthy controls (P = not significant). Homozygosity for the 3'A mutation was detected in 7%, 4%, and 3% of LTNPs, UPs, and healthy controls, respectively (P = not significant). Polymorphism at the SDF-1 locus is not associated with LTNP disease of longer than 16 years in Spanish HIV-1-infected patients. This effect is independent of the CCR5Delta32 allele.     

中国HIV感染长期不进展者CD4+CD25+Foxp3+调节性T细胞变化研究

目的 对中国HIV感染长期不进展者(LTNP)CD4+CD25+Foxp3+调节性T细胞水平及其与疾病进展相关性进行研究,探讨CD4+CD25+Foxp3+ 调节性T细胞在LTNP保护机制中发挥的作用.方法 选取74名HIV-1感染者(LTNP、典型进展HIV组、AIDS组)及16名健康对照,应用流式细胞仪胞内染色技术在单细胞水平检测CD4+CD25+Foxp3+调节性T细胞表达水平,分析其与CD4+ T细胞数量、病毒载量、淋巴细胞活化、凋亡水平的相关性.结果 中国HIV感染LTNP CD4+CD25+Foxp3+ T细胞百分率明显低于典型进展HIV、AIDS组及健康对照组(P<0.05).HIV/AIDS患者CD4+CD25+Foxp3+ T细胞百分率与CD4+ T细胞显著负相关(r=-0.509,P<0.001),与病毒载量明显正相关(r=0.414,P<0.01),与CD4、CD8+ T细胞表面CD38、CD95表达水平明显正相关(P<0.05),与CD4、CD8+ T细胞表面HLA-DR表达无显著相关性.结论 中国HIV感染LTNP CD4+CD25+Foxp3+ 调节性T细胞百分率明显低于典型进展者,提示调节性T细胞与LTNP保护机制相关.     

Unusual substitutions in HIV‐1 Vif from children infected perinatally without progression to AIDS for more than 8 years without therapy

The HIV‐1 vif gene encodes for an accessory protein that is central for virus replication due mainly to its capacity to counteract the antiviral action of host APOBEC3 restriction factors. In order to evaluate whether HIV‐1 vif alterations account for a delayed progression to AIDS in children infected perinatally, the vif genes from a group of 11 patients who exhibited an extremely slow disease progression (slow progressors) were studied by direct sequencing. In addition, the vif genes from a group of 93 children with typical disease progression (typical progressors) were analyzed for comparison. Phylogenetic analysis indicated that sequences from slow progressors did not have a common origin, discarding a shared ancestor of reduced virulence. There were no differences in the diversity between the vif genes from slow and typical progressors. No gross defects showing a clear distinction among sequences from both groups of children were found. However, in the deduced Vif proteins, changes V13I, V55T, and L81M were observed only in sequences from slow progressors. By analyzing sequences stored in databases, these mutations were determined as unusual substitutions occurring at highly conserved Vif sites across different HIV‐1 clades, but were observed with an increased frequency in sequences from elite controllers. These mutations were in the Vif regions reported as relevant for protein activity. These findings suggest that the Vif sequences from slow progressors carry unusual substitutions, which may alter the protein function and may contribute to viral attenuation. J. Med. Virol. 84:1844–1852, 2012. © 2012 Wiley Periodicals, Inc.     

Transient marker stabilisation: a general procedure to construct marker-free recombinant vaccinia virus

Summary.  Recombinant vaccinia viruses based on the highly attenuated Modified Vaccinia Ankara (MVA) strain expressing HIV-1 antigen genes were constructed by a novel procedure involving the transient use of two marker genes. The selectable markers used, the Escherichia coli guanine phosphoribosyltransferase (gpt) and the ?-galactosidase (lacZ) genes, are not retained within the final recombinant virus. The transient marker stabilisation (TMS) procedure allows the generation of marker-free recombinant viruses in a series of simple plaque purification steps. HIV-l gag pol genes were inserted into two loci of vaccinia MVA demonstrating the efficiency of the procedure. Accepted November 10, 1997 Received September 14, 1997     

Transition from long-term nonprogression to HIV-1 disease associated with escape from cellular immune control

Transition from long-term nonprogressive infection to progressive HIV-1 disease presents an opportunity to investigate pathogenesis in a defined immunogenetic background. We studied a male long-term nonprogressor (LTNP) who remained asymptomatic and viremic and had normal CD4 T-cell counts without antiretroviral therapy for >18 years and then experienced a transition to disease progression. We analyzed the complete HIV-1 genomic RNA sequence from plasma and cellular immune responses to predefined human leukocyte antigen-matched autologous viral peptides spanning the viral genome, before and after progression. Serial viral sequences did not seem attenuated and consistently utilized coreceptor CCR5. LTNP status was associated with elongated V2 domains and broad low-level T-cell immune responses targeting several regions of the viral genome. The transition to progressive disease was associated with the acquisition of viral mutations conferring escape from CD8 T-cell responses. Multiple changes in HIV-1 sequence and loss of immune response over time most likely contributed to the transition from LTNP status to progressive disease. These data are relevant to vaccine design and identification of the correlates of protection from disease progression.     

Emergence of protease inhibitor resistance-associated mutations in plasma HIV-1 precedes that in proviruses of peripheral blood mononuclear cells by more than a year

HIV-1 genotype assay using plasma viruses has been widely applied for detection of resistant viruses in infected individuals, whereas there are only a few reports about proviral genotype in peripheral blood mononuclear cells (PBMCs). To determine which sample, plasma or PBMC, should be used for early detection of drug-resistant viruses during antiretroviral treatment, we analyzed 275 plasma-derived and 211 PBMC-derived HIV-1 protease sequences obtained from HIV-1-infected patients during protease inhibitor (PI) therapy. In 70 of 107 pairs (65.4%) of plasma and PBMC samples taken from the same blood draws, the numbers of PI resistance-associated mutations in the plasma-derived genotype were different from those in the PBMC-derived genotype. Plasma viruses had more PI resistance-associated mutations than PBMC proviruses (P = 0.0004). Analysis of serial samples showed that plasma-derived genotype assay could detect primary mutations about 425 days earlier than PBMC-derived genotype when the plasma viral load was less than 10(4 )copies/mL. Our data suggest that genetic turnover of PBMC proviruses is slower than that of plasma viruses and that time lag between emergence of mutations in plasma-derived and PBMC-derived genotypes correlates inversely with viral load. Plasma viruses should be the material of choice for early detection of drug resistance during antiretroviral treatment.     

Polymorphism of RANTES chemokine gene promoter is not associated with long-term nonprogressive HIV-1 infection of more than 16 years

To examine whether polymorphisms of the RANTES chemokine gene promoter are associated with long-term nonprogressive HIV-1 infection in white Spanish subjects, we performed a cross-sectional genetic association case-control study. Two-hundred sixty-seven white Spaniards were studied: 58 were HIV-1-infected long-term nonprogressors (LTNPs) of more than 16 years, 109 were HIV-1-infected usual progressors (UPs), and 100 were control subjects. Three RANTES single nucleotide polymorphisms (SNPs) at positions -28C>G, -109T>C, and -403G>A were assessed. The prevalence of the CCR5Delta 32 allele was also examined. Genotyping was performed using polymerase chain reaction and automatic sequencing analysis methods. Genotype and allele frequencies between the 3 groups were compared by the chi2 test and the Fisher exact test. The distribution of allelic variants of RANTES in controls, UPs, and LTNPs, respectively, was 3%, 2%, and 5% for -28G; 4%, 2%, and 2% for -109C; and 18%, 18%, and 18% for -403A (P = not significant). The differences were still nonsignificant when we exclusively analyzed individuals not carrying the CCR5Delta32 allele. We conclude that LTNP of more than 16 years is not associated with SNPs in the RANTES gene promoter in white Spanish HIV-1-infected subjects.     

Compatibility of Tat and Rev transactivators in the primate lentiviruses

Summary Primate immunodeficiency viruses carry a unique set of transacting regulator genes, which are essential for viral replication. The exchangeability of these Tat and Rev transactivators derived from viruses of the four major subgroups identified to date was assessed in transient transfection and infection assay systems. The human immunodeficiency virus type 1 (HIV-1), a major causative virus of human AIDS, efficiently activated the other viruses. In contrast, thetat andrev gene products of HIV-2, SIV_AGM (virus of the African green monkey), and SIV_MND (virus of the mandrill) did not fully transactivate the HIV-1. In particular, therev of HIV-1 was not substantially replaced by those of the other viruses. The result that HIV-1 is distinct from the other immunodeficiency viruses with respect to the compatibility of two transactivators gives a firm functional basis for the unique phylogenetic position of HIV-1.