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.

High IL-7 plasma levels may induce and predict the emergence of HIV-1 virulent strains in pediatric infection.

BACKGROUND: HIV-1 infection results in severe immunodeficiency when T-cell loss cannot be compensated. IL-7 is one of the main cytokines involved in the maintenance of T-cell homeostasis. However, IL-7 can also enhance HIV-1 replication in vivo and lead to an accelerated progression of AIDS. OBJECTIVE: The aim of our study was to evaluate if the increase of IL-7 levels in response to CD4+ T cell depletion could favor the emergence of HIV-1 strains with more aggressive phenotypes in pediatric infection. STUDY DESIGN: Plasma IL-7 levels were measured in 42 HIV-1 vertically infected infants at different times of infection, and HIV-1 isolates were obtained from primary cell cultures to determine replication rate and syncytium-inducing (SI) capability on MT-2 cell line. RESULTS: IL-7 levels were significantly higher in infants harboring HIV-1 SI strains compared to those with non-syncytium-inducing (NSI) viruses (p<0.0001). Likewise, IL-7 levels were higher in infants with rapid replicating viral strains versus those with slow replicating viruses (p=0.0006). Despite the strong negative correlation between IL-7 levels and CD4+ T lymphocyte counts (r=-0.55, p=0.0001), covariance analysis demonstrated that the high levels of IL-7 were associated with more virulent phenotype features (SI and rapid replicating strains) independently of CD4+ T cell depletion. In 19 of the 42 infants, longitudinal follow-up studies showed that SI to NSI phenotype switch can occur after HAART administration, with a reduction in IL-7 levels and an increase in CD4+ T cell counts. CONCLUSIONS: IL-7 response to T-cell depletion may enhance T-cell production, but at the same time may foster HIV-1 disease progression favoring the emergence of more virulent HIV-1 strains characterized by SI capability and rapid replication rate.

Role of 3 lipoprotein lipase variants in triglycerides in children receiving highly active antiretroviral therapy.

BACKGROUND: Lipoprotein lipase is a key enzyme in lipid metabolism, especially for plasma triglycerides (TGs). Genetic variants have been associated with lipid levels in healthy individuals, cardiovascular disease, obesity and diabetes. Our aim was to evaluate the influence of 3 polymorphisms: Hind III, Pvu II and S447X in plasma TG levels in human immunodeficiency virus-1-infected children under highly active antiretroviral therapy (HAART). METHODS: Fifty-two children diagnosed with human immunodeficiency virus-1 between 2005 and 2009 were retrospectively selected with at least 1 plasma TG level assessment. TG levels were examined before and after 1 year of HAART. Hypertriglyceridemia was defined as TG > 150 mg/dL. Hind III (H+/H-), Pvu II (P+/P-) and S447X (S/X) were determined by polymerase chain reaction and restricted fragment length polymorphism. The Wilcoxon sum-rank test was used to compare median plasma TG among groups. Also, allelic frequencies were estimated for these variants in an Argentinean population. RESULTS: Allelic frequencies for human immunodeficiency virus-1-infected children were: H-, 0.21; P-, 0.53; and X, 0.05 with no significant differences to controls. After 1 year of HAART, median TG levels were significantly lower in P-/P- (98.5 mg/dL) when compared with P+/P+ (180 mg/dL) (P = 0.039). The presence of the P- allele was associated with an 11-fold lower risk of hypertriglyceridemia. Hind III and S447X were not associated with TG at the selected time points. CONCLUSIONS: Our findings suggest a protective effect of lipoprotein lipase polymorphisms against hypertriglyceridemia in children after 1 year of HAART. These results could endorse a prompt nutritional or pharmacological intervention in patients lacking the P- allele.

Sequence variability in p6 gag protein and gag/pol coevolution in human immunodeficiency type 1 subtype F genomes.

Polymorphisms occurring at the p6gag protein of HIV-1 have been previously found to have an impact on viral fitness and antiretroviral (ARV) resistance, mainly on subtype B genomes. We compared p6gag variability in a large group of 165 subtype F gag-pol sequences, with 36 subtype B sequences from the same study source, and identified sites of gag-pol coevolution under ARV selection pressure. Subtype-specific differences in the frequency of point mutations, insertions, and deletions previously associated with ARV resistance were found. Also, in our dataset of subtype F genomes a strong association between mutation P5L in the p1/p6 cleavage region of gag and the nelfinavir (NFV) resistance mutation N88D(PR) was found with no impact on the preference for any of the NFV resistance pathways.

Hypercholesterolemia is associated with the apolipoprotein C-III (APOC3) genotype in children receiving HAART: an eight-year retrospective study.

Polymorphisms in apolipoprotein genes have shown to be predictors of plasma lipid levels in adult cohorts receiving highly active antiretroviral therapy (HAART). Our objective was to confirm the association between the APOC3 genotype and plasma lipid levels in an HIV-1-infected pediatric cohort exposed to HAART. A total of 130 HIV-1-infected children/adolescents that attended a reference center in Argentina were selected for an 8-year longitudinal study with retrospective data collection. Longitudinal measurements of plasma triglycerides, total cholesterol, HDL-C and LDL-C were analyzed under linear or generalized linear mixed models. The contribution of the APOC3 genotype at sites -482, -455 and 3238 to plasma lipid levels prediction was tested after adjusting for potential confounders. Four major APOC3 haplotypes were observed for sites -482/-455/3238, with estimated frequencies of 0.60 (C/T/C), 0.14 (T/C/C), 0.11 (C/C/C), and 0.11 (T/C/G). The APOC3 genotype showed a significant effect only for the prediction of total cholesterol levels (p<0.0001). However, the magnitude of the differences observed was dependent on the drug combination (p = 0.0007) and the drug exposure duration at the time of the plasma lipid measurement (p = 0.0002). A lower risk of hypercholesterolemia was predicted for double and triple heterozygous individuals, mainly at the first few months after the initiation of Ritonavir-boosted protease inhibitor-based regimens. We report for the first time a significant contribution of the genotype to total cholesterol levels in a pediatric cohort under HAART. The genetic determination of APOC3 might have an impact on a large portion of HIV-1-infected children at the time of choosing the treatment regimens or on the counter-measures against the adverse effects of drugs.

Relevance of early detection of HIV type 1 SI/CXCR4-using viruses in vertically infected children.

The aim of the study was to investigate the prevalence and persistence of syncytium-inducing (SI) strains in HIV-1-infected children along time of infection and to evaluate the influence of antiretroviral therapy and host factors on viral tropism. This is a retrospective analysis carried out in 267 HIV-1 vertically infected children from an Argentinean cohort. The viral phenotype was screened in MT-2 cells and coreceptor usage confirmed by the GHOST cell assay. Also, CD4(+) T cell count, viral load, antiretroviral therapy, and human CCR5-Δ32 and CCR2-64I genotypes were analyzed. A high frequency of HIV-1 SI/CXCR4-using variants (22%) was found among children within the first trimester of life, reaching 46% after 10 years of infection. At acute infection, zidovudine prophylaxis did not significantly affect the proportions of SI HIV-1 strains, while their presence was favored by the CCR5(+)/Δ32 genotype. Interestingly, the majority of the early SI strains did not persist over time, probably due to a higher susceptibility to antiretroviral (ARV) treatment or immunologic pressure. At the chronic stage, SI variants emerged even in the presence of HAART reaching 36% at 120 months of infection. Also the HIV-1 SI phenotype was associated with lower CD4(+) T cell counts all along the course of infection. These findings highlight the need to evaluate the presence of SI/CXCR4 variants early at primary infection. This will make it possible to optimize the use of CCR5 inhibitors in children who are apparently carriers of the R5 virus preventing early therapeutic failure due to the reemergence of SI strains from reservoirs.

Genetic and functional analysis of HIV type 1 nef gene derived from long-term nonprogressor children: association of attenuated variants with slow progression to pediatric AIDS.

Among persons infected by HIV-1, the rate of progression to AIDS is multifactorial being affected by host and viral factors, including the HIV-encoded negative factor (Nef). Our aim was to define whether variations in the nef gene as well as its functions may be associated with slower HIV disease course in infected children. The proviral HIV-1 nef gene was cloned, sequenced, and compared in children with contrasting disease course: 10 long-term nonprogressors (LTNP) and six rapid progressor (RP). The CD4 and MHC-I down-modulation ability of nef alleles derived from LTNP and RP children was analyzed. We observed that only one of our 10 LTNP had a protective genetic background, and out of them, 40% had defective nef genes, carrying substitutions at the (AWLEAQ(56-61)) and the (Rxx(22-24)) domains, and that those alleles were unable of down-regulate CD4 and MHC-I. The emergence or presence of Nef L58V substitution was associated with viral attenuation, indicated by a reduction in HIV viral loads, a persistent preservation of CD4(+) T cell counts, and lack of AIDS-related symptoms. Our results demonstrate that HIV-1 perinatally infected children carrying functionally defective nef HIV-1 strains have prolonged asymptomatic phases without therapy, suggesting a relevant role of CD4 and MHC-I down-modulation Nef domains on in vivo HIV-1 pathogenesis and pediatric immunodeficiency outcome.

APOBEC3-mediated editing in HIV type 1 from pediatric patients and its association with APOBEC3G/CUL5 polymorphisms and Vif variability.

The APOBEC3 proteins are cytidine deaminases that can introduce G→A mutations in the HIV-1 plus DNA strand. This editing process may inhibit virus replication through lethal mutagenesis (hypermutation), but could also contribute to viral diversification leading to the emergence of escape forms. The HIV-1 Vif protein has the capacity to counteract APOBEC3 factors by recruiting a CUL5-based ubiquitin ligase complex that determines their proteasomal degradation. In this work, we analyzed the APOBEC3-mediated editing in proviral HIV-1 from perinatally infected children (n=93) in order to explore its association with polymorphisms of APOBEC3G and CUL5 genes (APOBEC3G H186R, APOBEC3G C40693T, and CUL5 SNP6), the Vif protein variability, and also the time to AIDS development. To calculate the level of editing, we have developed an index exploiting the properties of a region within the HIV-1 pol gene that includes the central polypurine tract (cPPT). We detected a reduced editing associated with the CUL5 SNP6 minor allele and also with certain Vif variants (mutations at sites 46, 122, and 160), although we found no evidence supporting an impact of APOBEC3 activity on disease progression. Thus, our findings suggest that APOBEC3-mediated editing of HIV-1 could be modulated by host and virus genetic characteristics in the context of pediatric infection.

Effect of HIV-1 Vif variability on progression to pediatric AIDS and its association with APOBEC3G and CUL5 polymorphisms.

The APOBEC3G protein is a restriction factor that can inhibit the replication of HIV-1. The virus has the capacity to counteract this antiviral activity through the expression of the Vif accessory protein, which recruits a CUL5-based ubiquitin ligase complex that determines APOBEC3G proteasomal degradation. In this work we evaluated in a large pediatric cohort (i) whether single nucleotide polymorphisms of APOBEC3G and CUL5 genes (APOBEC3G H186R, APOBEC3G C40693T and CUL5 SNP6) can alter the risk of HIV-1 vertical transmission and/or the rate of progression to AIDS, (ii) the effect of HIV-1 Vif variants on the clinical course of disease, and (iii) whether the patient genotype for the studied polymorphisms could have an impact on Vif characteristics. We found no effect of the studied APOBEC3G or CUL5 genetic variants on vertical transmission or progression to pediatric AIDS. However, we detected an association of certain Vif alterations (a one amino acid insertion at position 61 and the substitutions A62D/N/S and Q136P) with an accelerated AIDS outcome. Additionally, we observed that the APOBEC3G C40693T and CUL5 SNP6 minor alleles were correlated with substitutions in Vif motifs that are involved in the interaction with APOBEC3G and CUL5 proteins, respectively. Our results suggest that Vif alterations may contribute to a rapid AIDS onset and that Vif variability could be influenced by APOBEC3G and CUL5 polymorphisms in children.

MDR1 3435T and 1236T alleles delay disease progression to pediatric AIDS but have no effect on HIV-1 vertical transmission.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in the MDR1 gene, coding for the drug transporter P-glycoprotein, may modulate the response to antiretroviral therapy and susceptibility to HIV-1 infection. We investigated whether the MDR1 SNPs C1236T (exon 12) and C3435T (exon 26) affect HIV-1 vertical transmission and progression to pediatric AIDS. METHODS: The MDR1 genotypes were identified by PCR-restriction fragment length polymorphism (RFLP) assays in 219 HIV-infected, 128 exposed uninfected children and 231 HIV-seronegative blood donors. Genotype and haplotype frequencies were estimated in the different groups. The median follow-up time of the infected cohort was 108 months and AIDS-free time was evaluated for the different MDR1 genotypes in 171 HIV-infected children. RESULTS: We found that both C1236T and C3435T polymorphisms were highly frequent in the studied groups (approximately 0.44) and showed strong linkage disequilibrium. There was no association between MDR1 genotypes and HIV-1 vertical transmission. However, a protective effect against progression to AIDS was associated with MDR1 3435CT, 1236CT and 1236TT genotypes (P = 0.005, P = 0.024 and P = 0.026, respectively). Moreover, haplotype pairs' analysis showed that the 3435CT/1236CT and 3435CT/1236TT exerted a significant protection against progression to pediatric AIDS (P = 0.0025 and P = 0.006, respectively). CONCLUSION: We conclude that in Argentinean children, MDR1 genotypes are associated with progression to AIDS, but they do not affect HIV-1 susceptibility by vertical transmission. These results support the notion that P-glycoprotein plays a role in HIV-1 infection independently from its role in drug transport.

HIV type-1 genotypic resistance profiles in vertically infected patients from Argentina reveal an association between K103N+L100I and L74V mutations.

BACKGROUND: Patterns and pathways of HIV type-1 (HIV-1) antiretroviral (ARV) drug resistance-associated mutations in clinical isolates are conditioned by ARV history and factors such as viral subtype and fitness. Our aim was to analyse the frequency and association of ARV drug resistance mutations in a group of long-term vertically infected patients from Argentina. METHODS: Plasma samples from 71 patients (38 children and 33 adolescents) were collected for genotypic HIV-1 ARV resistance testing during the period between February 2006 and October 2008. Statistically significant pairwise associations between ARV resistance mutations in pol, as well as associations between mutations and drug exposure, were identified using Fisher's exact tests with Bonferroni and false discovery rate corrections. Phylogenetic analyses were performed for subtype assignment. RESULTS: In protease (PR), resistance-associated mutations M46I/L, I54M/L/V/A/S and V82A/F/T/S/M/I were associated with each other and with minor mutations at codons 10, 24 and 71. Mutations V82A/F/T/S/M/I were primarily selected by the administration of ritonavir (RTV) in an historical ARV regimen. In reverse transcriptase, thymidine analogue mutation (TAM)1 profile was more common than TAM2. The non-nucleoside K103N+L100I mutations were observed at high frequency (15.5%) and were significantly associated with the nucleoside mutation L74V in BF recombinants. CONCLUSIONS: Associations of mutations at PR sites reflect the frequent use of RTV at an early time in this group of patients and convergent resistance mechanisms driven by the high exposure to protease inhibitors, as well as local HIV-1 diversity. The results provide clinical evidence of a molecular interaction between K103N+L100I and L74V mutations at the reverse transcriptase gene in vivo, limiting the future use of second-generation non-nucleoside reverse transcriptase inhibitors such as etravirine.