Clinical and genetic markers associated with tuberculosis, HIV-1 infection, and TB/HIV-immune reconstitution inflammatory syndrome outcomes.

BACKGROUND: Tuberculosis (TB) and AIDS are the leading causes of infectious disease death worldwide. In some TB-HIV co-infected individuals treated for both diseases simultaneously, a pathological inflammatory reaction termed immune reconstitution inflammatory syndrome (IRIS) may occur. The risk factors for IRIS are not fully defined. We investigated the association of HLA-B, HLA-C, and KIR genotypes with TB, HIV-1 infection, and IRIS onset. METHODS: Patients were divided into four groups: Group 1- TB+/HIV+ (n = 88; 11 of them with IRIS), Group 2- HIV+ (n = 24), Group 3- TB+ (n = 24) and Group 4- healthy volunteers (n = 26). Patients were followed up at INI/FIOCRUZ and HGNI (Rio de Janeiro/Brazil) from 2006 to 2016. The HLA-B and HLA-C loci were typed using SBT, NGS, and KIR genes by PCR-SSP. Unconditional logistic regression models were performed for Protection/risk estimation. RESULTS: Among the individuals with TB as the outcome, KIR2DS2 was associated with increased risk for TB onset (aOR = 2.39, P = 0.04), whereas HLA-B*08 and female gender were associated with protection against TB onset (aOR = 0.23, P = 0.03, and aOR = 0.33, P = 0.01, respectively). Not carrying KIR2DL3 (aOR = 0.18, P = 0.03) and carrying HLA-C*07 (aOR = 0.32, P = 0.04) were associated with protection against TB onset among HIV-infected patients. An increased risk for IRIS onset was associated with having a CD8 count ≤500 cells/mm3 (aOR = 18.23, P = 0.016); carrying the KIR2DS2 gene (aOR = 27.22, P = 0.032), the HLA-B*41 allele (aOR = 68.84, P = 0.033), the KIR2DS1 + HLA-C2 pair (aOR = 28.58, P = 0.024); and not carrying the KIR2DL3 + HLA-C1/C2 pair (aOR = 43.04, P = 0.034), and the KIR2DL1 + HLA-C1/C2 pair (aOR = 43.04, P = 0.034), CONCLUSIONS: These results suggest the participation of these genes in the immunopathogenic mechanisms related to the conditions studied. This is the first study demonstrating an association of HLA-B*41, KIR2DS2, and KIR + HLA-C pairs with IRIS onset among TB-HIV co-infected individuals.

Next-generation sequencing analyses of the emergence and maintenance of mutations in CTL epitopes in HIV controllers with differential viremia control.

BACKGROUND: Despite the low level of viral replication in HIV controllers (HICs), studies have reported viral mutations related to escape from cytotoxic T-lymphocyte (CTL) response in HIV-1 plasma sequences. Thus, evaluating the dynamics of the emergence of CTL-escape mutants in HICs reservoirs is important for understanding viremia control. To analyze the HIV-1 mutational profile and dynamics of CTL-escape mutants in HICs, we selected 11 long-term non-progressor individuals and divided them into the following groups: (1) viremic controllers (VCs; n = 5) and (2) elite controllers (ECs; n = 6). For each individual, we used HIV-1 proviral DNA from PBMCs related to earliest (VE) and latest (VL) visits to obtain gag and nef sequences using the Illumina HiSeq system. The consensus of each mapped gene was used to assess viral divergence, and next-generation sequencing data were employed to identify SNPs and variations within and flanking CTL epitopes. RESULTS: Divergence analysis showed higher values for nef compared to gag among the HICs. EC and VC groups showed similar divergence rates for both genes. Analysis of the number of SNPs showed that VCs present more variability in both genes. Synonymous/non-synonymous mutation ratios were < 1 for gag among ECs and for nef among ECs and VCs, exhibiting a predominance of non-synonymous mutations. Such mutations were observed in regions encoding CTL-restricted epitopes in all individuals. All ECs presented non-synonymous mutations in CTL epitopes but generally at low frequency (< 1%); all VCs showed a high number of mutations, with significant frequency changes between VE and VL visits. A higher frequency of internal mutations was observed for gag epitopes, with significant changes across visits compared to Nef epitopes, indicating a pattern associated with differential genetic pressure. CONCLUSIONS: The high genetic conservation of HIV-1 gag and nef among ECs indicates that the higher level of viremia control restricts the evolution of both genes. Although viral replication levels in HICs are low or undetectable, all individuals exhibited CTL epitope mutations in proviral gag and nef variants, indicating that potential CTL escape mutants are present in HIC reservoirs and that situations leading to a disequilibrium of the host-virus relationship can result in the spread of CTL-escape variants.

Reduction of inflammation and T cell activation after 6 months of cART initiation during acute, but not in early chronic HIV-1 infection.

OBJECTIVES: To investigate the impact of early combined antiretroviral therapy (cART) on inflammation biomarkers and immune activation during acute and early chronic HIV-1 infection. METHODS: We included 12 acute (AHI), 11 early chronic (EcHI), and 18 late chronic HIV-1-infected (LcHI) individuals who were treated with cART and 18 HIV-1-uninfected (HIV-neg) individuals. Plasmatic levels of inflammation biomarkers, CD8+CD38+HLA-DR+ T cell frequencies, CD4 T cell counts, CD4/CD8 ratio, total HIV-1 DNA and plasmatic viral load were evaluated. Mann-Whitney test, Pearson and Spearman correlation, and linear regression models were used for statistical analyses. RESULTS: IP-10, IL-18, and sCD163 were significantly elevated at pre-ART in the AHI and EcHI groups, showing a significant reduction after 6 months of cART in the AHI group, achieving similar levels to the HIV-neg group. For the EcHI group, the IP-10 and sCD163 levels were also significantly reduced on M6-ART; however, IP-10 levels remained higher than in the HIV-neg group, and no significant reduction of IL-18 levels was observed. The CD8+ T cell activation levels were elevated in the AHI and EcHI groups at pre-ART and showed a significant reduction on M6-ART, but they were similar to levels seen for HIV-neg only after 12 months of cART. At pre-ART, IP-10 levels but not IL-18 levels were positively correlated with HIV-1 viral load in the AHI group. CONCLUSIONS: Early initiation of cART in HIV infection can reduce systemic inflammation, but the earlier normalization of the inflammation markers was only observed when cART was initiated in the acute phase of infection. A slower dynamic of reduction was observed for CD8+ T cell activation.

Inflammasome genetic variants are associated with tuberculosis, HIV-1 infection, and TB/HIV-immune reconstitution inflammatory syndrome outcomes.

Background: Tuberculosis (TB) and AIDS are the leading causes of infectious diseases death worldwide. Here, we investigated the relationship between from single nucleotide polymorphisms (SNPs) of the NLRP3, CARD8, AIM2, CASP-1, IFI16, and IL-1ß inflammasome genes, as well as the profiles of secreted proinflammatory cytokines (e.g., IL-1ß, IL-18, IL-33, and IL-6) with the TB clinical profiles, TB-HIV coinfection, and IRIS onset. Methods: The individuals were divided into four groups: TB-HIV group (n=88; 11 of them with IRIS), HIV-1 group (n=20), TB group (n=24) and healthy volunteers (HC) group (n=10), and were followed up at INI/FIOCRUZ and HGNI (Rio de Janeiro/Brazil) from 2006 to 2016. Real-time PCR was used to determine the genotypes of the Single Nucleotide Polymorphism (SNPs), and ELISA was used to measure the plasma cytokine levels. Unconditional logistic regression models were used to perform risk estimations. Results: A higher risk for extrapulmonary TB was associated with the TT genotype (aOR=6.76; P=0.026) in the NLRP3 rs4612666 Single Nucleotide Polymorphism (SNP) and the C-C-T-G-C haplotype (aOR=4.99; P= 0.017) in the NLRP3 variants. This same Single Nucleotide Polymorphism (SNP) was associated with lower risk against extrapulmonary TB when the carrier allele C (aOR=0.15; P=0.021) was present. Among those with HIV-1 infections, a higher risk for TB onset was associated with the GA genotype (aOR=5.5; P=0.044) in the IL1-ß rs1143634 Single Nucleotide Polymorphism (SNP). In contrast, lower risk against TB onset was associated with the A-G haplotype (aOR=0.17; P= 0.026) in the CARD8 variants. Higher IL-6 and IL-33 levels were observed in individuals with TB. A higher risk for IRIS onset was associated with CD8 counts ≤ 500 cells/mm3 (aOR=12.32; P=0.010), the presence of extrapulmonary TB (aOR=6.6; P=0.038), and the CT genotype (aOR=61.06; P=0.026) or carrier allele T (aOR=61.06; P=0.026) in the AIM2 rs2276405 Single Nucleotide Polymorphism (SNP), whereas lower risk against IRIS onset was associated with the AT genotype (aOR=0.02; P=0.033) or carrier allele T (aOR=0.02; P=0.029) in the CARD8 rs2043211 Single Nucleotide Polymorphism (SNP) and the T-G haplotype (aOR=0.07; P= 0.033) in the CARD8 variants. No other significant associations were observed. Conclusions: Our results depict the involvement of genetic polymorphisms of crucial innate immunity genes and proinflammatory cytokines in the clinical outcomes related to TB-HIV coinfection.

Impact of HIV-1 Subtypes on AIDS Progression in a Brazilian Cohort.

Viral and host factors are known to play a role in the different patterns of AIDS progression. The cocirculation of HIV-1 subtypes B, F1, BBR, and BF1; the occasional detection of HIV-1 subtype D; and an increasing prevalence of subtype C and other recombinant forms have been described in Rio de Janeiro, Brazil. The aim of this study was to evaluate the potential association of HIV-1 subtypes circulating among HIV-1+ individuals in Rio de Janeiro with AIDS disease progression. For this purpose, 246 HIV-1 individuals under clinical and laboratory follow-up from 1986 to 2011 were classified according to their progression to AIDS in typical progressors (n = 133), rapid progressors (n = 95), and long-term nonprogressors (n = 18). The env-gp120 region was amplified and sequenced. Neighbor-joining phylogenetic inferences were performed in Mega 6 and bootscan analysis was performed in Simplot 3.5.1. The Kaplan-Meier method and Cox modeling were performed to determine the time until an AIDS-defining event based on the HIV-1 subtypes/variants. Similar AIDS progression rates were observed among individuals infected with HIV-1 subtype B and variant BBR. However, a direct association between more rapid AIDS progression and HIV-1 subtypes, D and BF1, was confirmed in the multivariate analysis, corroborating previous results. Our findings contribute to the investigation of the possible influence of HIV-1 subtypes in AIDS outcome.

Assessing the HIV-1 Epidemic in Brazilian Drug Users: A Molecular Epidemiology Approach.

Person who inject illicit substances have an important role in HIV-1 blood and sexual transmission and together with person who uses heavy non-injecting drugs may have less than optimal adherence to anti-retroviral treatment and eventually could transmit resistant HIV variants. Unfortunately, molecular biology data on such key population remain fragmentary in most low and middle-income countries. The aim of the present study was to assess HIV infection rates, evaluate HIV-1 genetic diversity, drug resistance, and to identify HIV transmission clusters in heavy drug users (DUs). For this purpose, DUs were recruited in the context of a Respondent-Driven Sampling (RDS) study in different Brazilian cities during 2009. Overall, 2,812 individuals were tested for HIV, and 168 (6%) of them were positive, of which 19 (11.3%) were classified as recent seroconverters, corresponding to an estimated incidence rate of 1.58%/year (95% CI 0.92-2.43%). Neighbor joining phylogenetic trees from env and pol regions and bootscan analyses were employed to subtype the virus from132 HIV-1-infected individuals. HIV-1 subtype B was prevalent in most of the cities under analysis, followed by BF recombinants (9%-35%). HIV-1 subtype C was the most prevalent in Curitiba (46%) and Itajaí (86%) and was also detected in Brasília (9%) and Campo Grande (20%). Pure HIV-1F infections were detected in Rio de Janeiro (9%), Recife (6%), Salvador (6%) and Brasília (9%). Clusters of HIV transmission were assessed by Maximum likelihood analyses and were cross-compared with the RDS network structure. Drug resistance mutations were verified in 12.2% of DUs. Our findings reinforce the importance of the permanent HIV-1 surveillance in distinct Brazilian cities due to viral resistance and increasing subtype heterogeneity all over Brazil, with relevant implications in terms of treatment monitoring, prophylaxis and vaccine development.

HIV-1 infection among injection and ex-injection drug users from Rio de Janeiro, Brazil: prevalence, estimated incidence and genetic diversity.

BACKGROUND AND OBJECTIVES: Due to their behavioral conditions and vulnerability, injection drug users (IDUs) are prone to multiple simultaneous or sequential infections with distinct HIV-1 subtypes and variants, making them a key population for molecular epidemiology surveillance. In the present study, we evaluated HIV-1 infection seroprevalence, genetic diversity and estimated incidence among IDUs and ex-injection drug users (ex-IDUs) from Rio de Janeiro, Brazil. STUDY DESIGN: Six hundred and eight IDUs and ex-IDUs, recruited between 1999 and 2001, were interviewed and agreed to donate 30 ml of blood. The serologic status for HIV infection was determined by two ELISAs and confirmed by IFA. CD4+ T-cell percentages were assessed by flow cytometry. HIV-1 positive samples were submitted to viral load quantification. DNA samples were PCR amplified and HIV-1 subtypes were determined using env and gag HMA. RESULTS AND CONCLUSIONS: Forty-eight (7.89%) individuals were seropositive for HIV-1 infection. The seroincidence of HIV-1 infection was estimated as 0.76%. HIV-1 env and gag subtyping identified 29 (69%) samples as belonging to subtype B, 7 (16.7%) to subtype F, and 6 (14.3%) discordant env/gag genomes infections, indicating the circulation of recombinant viruses in this population.

Association of single nucleotide polymorphisms in the lens epithelium-derived growth factor (LEDGF/p75) with HIV-1 infection outcomes in Brazilian HIV-1+ individuals.

The lens epithelium-derived growth factor p75 (LEDGF/p75), coded by the PSIP1 gene, is an important host co-factor that interacts with HIV-1 integrase to target integration of viral cDNA into active genes. The aim of this study was to investigate the association of SNPs in the PSIP1 gene with disease outcome in HIV-1 infected patients. We performed a genetic association study in a cohort of 171 HIV-1 seropositive Brazilian individuals classified as rapid progressors (RP, n = 69), typical progressors (TP, n = 79) and long-term nonprogressors (LTNP, n = 23). The exonic SNP rs61744944 and 9 tag SNPs were genotyped. A group of 192 healthy subjects was analyzed to determine the frequency of SNPs and haplotypes in the general population. Linkage disequilibrium (LD) analyses indicated that the SNPs analyzed were not in high LD (r2<0.8). Logistic regression models suggested that patients carrying the T allele rs61744944 (472L) were more likely to develop a LTNP phenotype (OR = 4.98; p = 0.05) as compared to TP group. The same trend was observed when LTNPs were compared to the RP group (OR = 3.26). Results of haplotype analyses reinforced this association, since the OR values obtained for the haplotype carrying allele T at rs61744944 also reflected an association with LTNP status (OR = 6.05; p = 0.08 and OR = 3.44; p = 0.12 for comparisons to TP and RP, respectively). The rare missense variations Ile436Ser and Thr473Ile were not identified in the patients enrolled in this study. Gene expression analyses showed lower LEDGF/p75 mRNA levels in peripheral blood mononuclear cells obtained from HIV-1 infected individuals. However, these levels were not influenced by any of the SNPs investigated. In spite of the limited number of LTNPs, these data suggest that the PSIP1 gene could be associated with the outcome of HIV-1 infection. Further analyses of this gene may guide the identification of causative variants to help predict disease course.

Analysis of HIV-1 BF pr/rt recombinant strains from Rio de Janeiro/Brazil reveals multiple unrelated mosaic structures.

Due to the co-circulation of HIV-1 subtypes B and F1 in Brazil, a large variety of unique BF1 recombinant forms (URFs_BF1) and four circulating BF1 recombinant forms (CRF28, CRF29, CRF39 and CRF40) have been described. The aim of this study was to investigate mosaic structure and phylogenetic relationship among several BF1 (protease-reverse transcriptase, pr/rt) recombinant sequences obtained from a group of patients from Rio de Janeiro, Brazil, from 1994 to 2005. Phylogenetic relationships were estimated by Bayesian and SplitsTree methods. Recombination breakpoints were analyzed by Bootscan. Those samples presenting the same recombinant pattern in the pr/rt region were investigated in the integrase (int) and envelope (env) regions as a screening method for the detection of potential new CRFs candidates. Third out of 61 pr/rt HIV-1 BF1 recombinant sequences analyzed depicted unique recombinant structures and were classified URFs_BF1. The other 31 samples segregated in eight well-supported phylogenetic clusters composed of at least three samples sharing the same recombination pattern. Analyses of the int and env regions from these 31 samples revealed that 11 samples were URFs_BF1. Three and four sequences corresponded, respectively, to the previously described CRF39 and CRF40. Three samples displayed a CRF28-like mosaic structure, and one sample a CRF29-like mosaic pattern. The other nine BF1 samples segregate in three distinct clusters with the same recombination profile and could represent good candidates for new CRFs_BF profiles. The HIV-1 BF1 epidemic in Rio de Janeiro is characterized by a high prevalence (67%) of URFs_BF1 and a low prevalence (4.9-6.6%) of each CRFs_BF previously identified in Brazil.

Distribution of CCR5 genotypes and HLA Class I B alleles in HIV-1 infected and uninfected injecting drug users from Rio de Janeiro, Brazil.

Host genetic factors play an important role in the HIV epidemic dynamics, and have been considered in studies assessing susceptibility/resistance to HIV-1 infection as well as clinical evolution. Class I and Class II HLA alleles have been associated with the heterogeneity of HIV-1 infection susceptibility, as protective or risk factors for HIV-1 transmission. Moreover, a 32-base pair deletion in the HIV-1 CCR5 gene-coding region confers resistance to HIV-1 infection in homozygous individuals for the deleted allele. In this study, DNA samples from HIV-1 infected and uninfected injecting drug users (IDUs) from Rio de Janeiro were PCR amplified to determine CCR5 genotypes based on the presence of the CCR5Delta32 mutation and typed for the HLA-B locus, in an attempt to assess possible associations between these genetic factors and susceptibility/resistance to HIV-1 infection. The distribution of CCR5 genotypes between the two IDU groups did not differ. The homozygous mutant genotype Delta32/Delta32 was not found in this study. Except for HLA-B*45 (4.0% vs. 3.0%; p=0.04) and for B*51 (12.1% vs. 4.4%; p=0.002), no statistically significant differences were made evident when analyzing the frequencies of each HLA-B allele between Caucasian and non-Caucasian IDUs. The most frequent HLA-B alleles were B*15; B*35; B*44 and B*51. Although some differences in the allele frequencies could be observed between the two IDU groups, none of these was statistically significant. Therefore, no putative association between these genetic markers and susceptibility/resistance to HIV-1 infection could be made evident in the present study. So far, the assessment of genetic markers among the IDU population has been restricted to North American, European, and Asian studies and this report represents a pioneer descriptive study of the distribution of CCR5 genotypes and HLA-B alleles in Rio de Janeiro, Brazil.

Lack of primary mutations associated with integrase inhibitors among HIV-1 subtypes B, C, and F circulating in Brazil.

BACKGROUND: Antiretroviral drugs targeting integrase (IN) have recently been approved for use in combined and salvage therapeutic interventions. OBJECTIVE: To evaluate the presence of natural polymorphisms and resistance mutations associated with IN inhibitors among HIV-1 subtypes B, C, and F samples obtained from drug-naive individuals and patients failing highly active antiretroviral therapy in Brazil. METHODS: Proviral DNA was obtained from blood samples of 105 HIV-1-positive drug-naive patients infected by B, C, or F subtypes and plasma viral RNA from 30 subtype B-infected individuals failing highly active antiretroviral therapy. The IN region was amplified by nested polymerase chain reaction and automatically sequenced for subtype determination. Translated amino acid sequences were inspected for IN mutations associated with antiretroviral resistance. RESULTS: Eleven mutations described as conferring in vitro resistance to IN strand transfer inhibitors were detected among the HIV-1 Brazilian samples. V72I and V201I were considered as polymorphisms. Major mutations associated with elvitegravir or raltegravir in vivo resistance (Q148K/H/R, N155H) were not detected. CONCLUSIONS: Although some naturally occurring polymorphisms were observed, the absence of major resistance mutations for the current IN inhibitors provides a good rationale for the introduction of these drugs in Brazil. These results highlight the importance of the continuous surveillance of IN genetic diversity.