MRKAd5 HIV-1 Gag/Pol/Nef Vaccine-Induced T-Cell Responses Inadequately Predict Distance of Breakthrough HIV-1 Sequences to the Vaccine or Viral Load

Background: The sieve analysis for the Step trial found evidence that breakthrough HIV-1 sequences for MRKAd5/HIV-1 Gag/Pol/Nef vaccine recipients were more divergent from the vaccine insert than placebo sequences in regions with predicted epitopes. We linked the viral sequence data with immune response and acute viral load data to explore mechanisms for and consequences of the observed sieve effect. Methods: Ninety-one male participants (37 placebo and 54 vaccine recipients) were included; viral sequences were obtained at the time of HIV-1 diagnosis. T-cell responses were measured 4 weeks post-second vaccination and at the first or second week post-diagnosis. Acute viral load was obtained at RNA-positive and antibody-negative visits. Findings: Vaccine recipients had a greater magnitude of post-infection CD8+ T cell response than placebo recipients (median 1.68% vs 1.18%; p = 0.04) and greater breadth of post-infection response (median 4.5 vs 2; p = 0.06). Viral sequences for vaccine recipients were marginally more divergent from the insert than placebo sequences in regions of Nef targeted by pre-infection immune responses (p = 0.04; Pol p = 0.13; Gag p = 0.89). Magnitude and breadth of pre-infection responses did not correlate with distance of the viral sequence to the insert (p. 0.50). Acute log viral load trended lower in vaccine versus placebo recipients (estimated mean 4.7 vs 5.1) but the difference was not significant (p = 0.27). Neither was acute viral load associated with distance of the viral sequence to the insert (p>0.30). Interpretation: Despite evidence of anamnestic responses, the sieve effect was not well explained by available measures of T-cell immunogenicity. Sequence divergence from the vaccine was not significantly associated with acute viral load. While point estimates suggested weak vaccine suppression of viral load, the result was not significant and more viral load data would be needed to detect suppression.

Evaluation of Viral Load Thresholds for Predicting New World Health Organization Stage 3 and 4 Events in HIV-Infected Children Receiving Highly Active Antiretroviral Therapy

Background: This study evaluated a wide range of viral load (VL) thresholds to identify a cut-point that best predicts new clinical events in children on stable highly active antiretroviral therapy (HAART). Methods: Cox proportional hazards modeling was used to assess the adjusted risk for World Health Organization stage 3 or 4 clinical events (WHO events) as a function of time-varying CD4, VL, and hemoglobin values in a cohort study of Latin American children on HAART >= 6 months. Models were fit using different VL cut-points between 400 and 50,000 copies per milliliter, with model fit evaluated on the basis of the minimum Akaike information criterion value, a standard model fit statistic. Results: Models were based on 67 subjects with WHO events out of 550 subjects on study. The VL cut-points of >2600 and >32,000 copies per milliliter corresponded to the lowest Akaike information criterion values and were associated with the highest hazard ratios (2.0, P = 0.015; and 2.1, P = 0.0058, respectively) for WHO events. Conclusions: In HIV-infected Latin American children on stable HAART, 2 distinct VL thresholds (>2600 and >32,000 copies/mL) were identified for predicting children at significantly increased risk for HIV-related clinical illness, after accounting for CD4 level, hemoglobin level, and other significant factors.

HIV-1 tropism and CD4 T lymphocyte recovery in a prospective cohort of patients initiating HAART in Ribeirao Preto, Brazil

While human immunodeficiency virus (HIV)-1 chemokine co-receptors 5 tropism and the GWGR motif in the envelope third variable region (V3 loop) have been associated with a slower disease progression, their influence on antiretroviral response remains unclear. The impact of baseline V3 characteristics on treatment response was evaluated in a randomised, double blind, prospective cohort study with patients initiating highly active antiretroviral therapy with lopinavir or efavirenz plus azithothymidine/3TC (1:1) over 48 weeks. Similar virological and immunological responses were observed for both treatment regimens. The 43 individuals had a mean baseline CD4 T cell count of 119 cells/mm(3) [standard deviation (SD) = 99] and a mean viral load of 5.09 log(10) copies/mL (SD = 0.49). The GWGR motif was not associated with a CD4 T cell response, but predicted R5 tropism by the geno2pheno([clinical20%]) algorithm correlated with higher CD4 T cell levels at all monitoring points (p < 0.05). Moreover, higher false-positive rates (FPR) values from this analysis revealed a strong correlation with CD4 T cell recovery (p < 0.0001). Transmitted drug resistance mutations, documented in 3/41 (7.3%) cases, were unrelated to the assigned antiretroviral regimen and had no impact on patient outcomes. In conclusion, naive HIV-1 R5 infected patients exhibited higher CD4 T cell counts at baseline; this difference was sustained throughout therapy. The geno2pheno[clinical] option FPR positively correlated with CD4 T cell gain and may be useful in predicting CD4 T cell recovery.

Evaluation of HIV-1 resistance to antiretroviral drugs among 150 patients after six months of therapeutic interruption

Most of the antiretroviral (ARV) studies in Brazil have been reported in treatment-experienced and naive patients rather than in the setting of treatment interruption (TI). In this study, we analysed reasons given for TI and resistance mutations occurring in 150 HIV-1-infected patients who underwent TI. Of the patients analysed, 110 (73.3%) experienced TI following medical advice, while the remaining patients stopped antiretroviral therapy (ART) of their own accord. The main justifications for TI were: ARV-related toxicities (38.7%), good laboratory parameters (30%) and poor adherence (20%). DNA sequencing of the partial pol gene was successful in 137 (91.3%) patients, of whom 38 (27.7%) presented mutations conferring ARV resistance. A higher viral load prior to TI correlated with drug resistance (P < 0.05). Our results demonstrate that there are diverse rationales for TI and that detection of resistant strains during TI most likely indicates a fitter virus than the wild type. High viral loads coupled with unprotected sex in this group could increase the likelihood of transmission of drug-resistant virus. Thus, treating physicians should be alerted to this problem when the use of ARVs is interrupted.

Influence of HAART on Alternative Reading Frame Immune Responses over the Course of HIV-1 Infection

Background: Translational errors can result in bypassing of the main viral protein reading frames and the production of alternate reading frame (ARF) or cryptic peptides. Within HIV, there are many such ARFs in both sense and the antisense directions of transcription. These ARFs have the potential to generate immunogenic peptides called cryptic epitopes (CE). Both antiretroviral drug therapy and the immune system exert a mutational pressure on HIV-1. Immune pressure exerted by ARF CD8(+) T cells on the virus has already been observed in vitro. HAART has also been described to select HIV-1 variants for drug escape mutations. Since the mutational pressure exerted on one location of the HIV-1 genome can potentially affect the 3 reading frames, we hypothesized that ARF responses would be affected by this drug pressure in vivo. Methodology/Principal findings: In this study we identified new ARFs derived from sense and antisense transcription of HIV-1. Many of these ARFs are detectable in circulating viral proteins. They are predominantly found in the HIV-1 env nucleotide region. We measured T cell responses to 199 HIV-1 CE encoded within 13 sense and 34 antisense HIV-1 ARFs. We were able to observe that these ARF responses are more frequent and of greater magnitude in chronically infected individuals compared to acutely infected patients, and in patients on HAART, the breadth of ARF responses increased. Conclusions/Significance: These results have implications for vaccine design and unveil the existence of potential new epitopes that could be included as vaccine targets.

Missed opportunities for prevention of mother-to-child transmission of HIV-1 in the NISDI Perinatal and LILAC cohorts

Objective: To evaluate cases of mother-to-child transmission of HIV-1 at multiple sites in Latin America and the Caribbean in terms of missed opportunities for prevention. Methods: Pregnant women infected with HIV-1 were eligible for inclusion if they were enrolled in either the NISDI Perinatal or LILAC protocols by October 20, 2009, and had delivered a live infant with known HIV-1 infection status after March 1, 2006. Results: Of 711 eligible mothers, 10 delivered infants infected with HIV-1. The transmission rate was 1.4% (95% CI, 0.7-2.6). Timing of transmission was in utero or intrapartum (n = 5), intrapartum (n = 2), intrapartum or early postnatal (n = 1), and unknown (n = 2). Possible missed opportunities for prevention included poor control of maternal viral load during pregnancy; late initiation of antiretrovirals during pregnancy; lack of cesarean delivery before labor and before rupture of membranes; late diagnosis of HIV-1 infection; lack of intrapartum antiretrovirals; and incomplete avoidance of breastfeeding. Conclusion: Early knowledge of HIV-1 infection status (ideally before or in early pregnancy) would aid timely initiation of antiretroviral treatment and strategies designed to prevent mother-to-child transmission. Use of antiretrovirals must be appropriately monitored in terms of adherence and drug resistance. If feasible, breastfeeding should be completely avoided. Presented in part at the XIX International AIDS Conference (Washington, DC; July 22-27, 2012); abstract WEPE163. (c) 2012 Published by Elsevier Ireland Ltd. on behalf of International Federation of Gynecology and Obstetrics.

HIV-1 tropism and CD4 T lymphocyte recovery in a prospective cohort of patients initiating HAART in Ribeirão Preto, Brazil

While human immunodeficiency virus (HIV)-1 chemokine co-receptors 5 tropism and the GWGR motif in the envelope third variable region (V3 loop) have been associated with a slower disease progression, their influence on antiretroviral response remains unclear. The impact of baseline V3 characteristics on treatment response was evaluated in a randomised, double blind, prospective cohort study with patients initiating highly active antiretroviral therapy with lopinavir or efavirenz plus azithothymidine/3TC (1:1) over 48 weeks. Similar virological and immunological responses were observed for both treatment regimens. The 43 individuals had a mean baseline CD4 T cell count of 119 cells/mm³ [standard deviation (SD) = 99] and a mean viral load of 5.09 log10 copies/mL (SD = 0.49). The GWGR motif was not associated with a CD4 T cell response, but predicted R5 tropism by the geno2pheno[clinical20%] algorithm correlated with higher CD4 T cell levels at all monitoring points (p < 0.05). Moreover, higher false-positive rates (FPR) values from this analysis revealed a strong correlation with CD4 T cell recovery (p < 0.0001). Transmitted drug resistance mutations, documented in 3/41 (7.3%) cases, were unrelated to the assigned antiretroviral regimen and had no impact on patient outcomes. In conclusion, naÏve HIV-1 R5 infected patients exhibited higher CD4 T cell counts at baseline; this difference was sustained throughout therapy. The geno2pheno[clinical] option FPR positively correlated with CD4 T cell gain and may be useful in predicting CD4 T cell recovery.

Phylogeographic Analysis of HIV-1 Subtype C Dissemination in Southern Brazil

The HIV-1 subtype C has spread efficiently in the southern states of Brazil (Rio Grande do Sul, Santa Catarina and Parana). Phylogeographic studies indicate that the subtype C epidemic in southern Brazil was initiated by the introduction of a single founder virus population at some time point between 1960 and 1980, but little is known about the spatial dynamics of viral spread. A total of 135 Brazilian HIV-1 subtype C pol sequences collected from 1992 to 2009 at the three southern state capitals (Porto Alegre, Florianopolis and Curitiba) were analyzed. Maximum-likelihood and Bayesian methods were used to explore the degree of phylogenetic mixing of subtype C sequences from different cities and to reconstruct the geographical pattern of viral spread in this country region. Phylogeographic analyses supported the monophyletic origin of the HIV-1 subtype C clade circulating in southern Brazil and placed the root of that clade in Curitiba (Parana state). This analysis further suggested that Florianopolis (Santa Catarina state) is an important staging post in the subtype C dissemination displaying high viral migration rates from and to the other cities, while viral flux between Curitiba and Porto Alegre (Rio Grande do Sul state) is very low. We found a positive correlation (r(2) = 0.64) between routine travel and viral migration rates among localities. Despite the intense viral movement, phylogenetic intermixing of subtype C sequences from different Brazilian cities is lower than expected by chance. Notably, a high proportion (67%) of subtype C sequences from Porto Alegre branched within a single local monophyletic sub-cluster. These results suggest that the HIV-1 subtype C epidemic in southern Brazil has been shaped by both frequent viral migration among states and in situ dissemination of local clades.

Low-Cost Ultra-Wide Genotyping Using Roche/454 Pyrosequencing for Surveillance of HIV Drug Resistance

Background: Great efforts have been made to increase accessibility of HIV antiretroviral therapy (ART) in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance. Methods/Results: We have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in Sao Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we identified drug resistance mutations in 20% of treatment naive individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples. Conclusion: The developed ultra-wide sequencing approach described here allows multiplexing of at least 48 patient samples per sequencing run, 4 times more than the current genotyping method. This method is also 4-fold more sensitive (5% minimal detection frequency vs. 20%) at a cost 3-5 x less than the traditional Sanger-based genotyping method. Lastly, by using a benchtop next-generation sequencer (Roche/454 GS Junior), this approach can be more easily implemented in low-resource settings. This data provides proof-of-concept that next-generation HIV drug resistance genotyping is a feasible and low-cost alternative to current genotyping methods and may be particularly beneficial for in-country surveillance of transmitted drug resistance.

Characterization of Primary Isolates of HIV Type 1 CRF28_BF, CRF29_BF, and Unique BF Recombinants Circulating in Sao Paulo, Brazil

We report for the first time the genetic and biological characterization of 10 HIV-1 primary isolates representing CRF28_BF and CRF29_BF together with additional unique BF recombinant forms (URFs) obtained by PBMC cocultivation. Recombination is an important factor promoting the increase in the genetic diversity of HIV-1. Notably, more than 20% of HIV-1 sequences worldwide were recombinants. Several recombinant viruses were reported in Brazil, and six circulating recombinant forms (CRFs) have been identified (CRF28_BF, CRF29_BF, CRF31_BC, CRF39_BF, CRF40_BF, and CRF46_BF). CRF28_BF and CRF29_BF were found to infect almost 30% of the patients in Sao Paulo State. The near full-length genomes of these 10 primary isolates were amplified by nested PCR in three overlapping segments, purified, and sequenced. Three samples were related to CRF28_BF, three to CRF29_BF, and four were unique recombinant forms (URFs), as determined by their breakpoint profile determined with the jpHMM program. Additionally, the coreceptor usage of these isolates was investigated in vitro using GHOST assays, which revealed three dual-tropic (X4/R5) viruses, four lymphotropic (X4) viruses, and three macrophage-tropic (R5) viruses with different V3-loop motifs, which challenges the notion that GWGR-carrying viruses are macrophage-tropic only. In sum, we report a much-anticipated well-characterized panel of viruses representing CRF28_BF, CRF29_BF, and URFs from Sao Paulo State, Brazil.

Modeling the emergence of HIV-1 drug resistance resulting from antiretroviral therapy: Insights from theoretical and numerical studies

The use of antiretroviral therapy has proven to be remarkably effective in controlling the progression of human immunodeficiency virus (HIV) infection and prolonging patient's survival. Therapy however may fail and therefore these benefits can be compromised by the emergence of HIV strains that are resistant to the therapy. In view of these facts, the question of finding the reason for which drug-resistant strains emerge during therapy has become a worldwide problem of great interest. This paper presents a deterministic HIV-1 model to examine the mechanisms underlying the emergence of drug-resistance during therapy. The aim of this study is to determine whether, and how fast, antiretroviral therapy may determine the emergence of drug resistance by calculating the basic reproductive numbers. The existence, feasibility and local stability of the equilibriums are also analyzed. By performing numerical simulations we show that Hopf bifurcation may occur. The model suggests that the individuals with drug-resistant infection may play an important role in the epidemic of HIV. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

BF Integrase Genes of HIV-1 Circulating in Sao Paulo, Brazil, with a Recurrent Recombination Region

Although some studies have shown diversity in HIV integrase (IN) genes, none has focused particularly on the gene evolving in epidemics in the context of recombination. The IN gene in 157 HIV-1 integrase inhibitor-naive patients from the Sao Paulo State, Brazil, were sequenced tallying 128 of subtype B (23 of which were found in non-B genomes), 17 of subtype F (8 of which were found in recombinant genomes), 11 integrases were BF recombinants, and 1 from subtype C. Crucially, we found that 4 BF recombinant viruses shared a recurrent recombination breakpoint region between positions 4900 and 4924 (relative to the HXB2) that includes 2 gRNA loops, where the RT may stutter. Since these recombinants had independent phylogenetic origin, we argue that these results suggest a possible recombination hotspot not observed so far in BF CRF in particular, or in any other HIV-1 CRF in general. Additionally, 40% of the drug-naive and 45% of the drug-treated patients had at least 1 raltegravir (RAL) or elvitegravir (EVG) resistance-associated amino acid change, but no major resistance mutations were found, in line with other studies. Importantly, V151I was the most common minor resistance mutation among B, F and BF IN genes. Most codon sites of the IN genes had higher rates of synonymous substitutions (dS) indicative of a strong negative selection. Nevertheless, several codon sites mainly in the subtype B were found under positive selection. Consequently, we observed a higher genetic diversity in the B portions of the mosaics, possibly due to the more recent introduction of subtype F on top of an ongoing subtype B epidemics and a fast spread of subtype F alleles among the B population.

Calcium nitrate addition to control the internal load of phosphorus from sediments of a tropical eutrophic reservoir: Microcosm experiments

The main objective of this study was to perform laboratory experiments on calcium nitrate addition to sediments of a tropical eutrophic urban reservoir (Ibirite reservoir, SE Brazil) to immobilize the reactive soluble phosphorus (RSP) and to evaluate possible geochemical changes and toxic effects caused by this treatment. Reductions of 75 and 89% in the concentration of RSP were observed in the water column and interstitial water, respectively, after 145 days of nitrate addition. The nitrate application increased the rate of autotrophic denitrification, causing a consumption of 98% of the added nitrate and oxidation of 99% of the acid volatile sulfide. As a consequence, there were increases in the sulfate and iron (II) concentrations in the sediment interstitial water and water column, as well as changes in the copper speciation in the sediments. Toxicity tests initially indicated that the high concentrations of nitrate and nitrite in the sediment interstitial water (up to 2300 mg L-1 and 260 mg L-1, respectively) were the major cause of mortality of Ceriodaphnia silvestrii and Chironomus xanthus. However, at the end of the experiment, the sediment toxicity was completely removed and a reduction in the 48 h-EC50 of the water was also observed. Based on these results we can say that calcium nitrate treatment proved to be a valuable tool in remediation of eutrophic aquatic ecosystems leading to conditions that can support a great diversity of organisms after a restoration period. (C) 2012 Elsevier Ltd. All rights reserved.

High prevalence of the simultaneous excretion of polyomaviruses JC and BK in the urine of HIV-infected patients without neurological symptoms in São Paulo, Brazil

OBJECTIVE: To evaluate the prevalence of the urinary excretion of BKV and JCV in HIV-infected patients without neurological symptoms. METHODS: Urine samples from HIV-infected patients without neurological symptoms were tested for JC virus and BK virus by PCR. Samples were screened for the presence of polyomavirus with sets of primers complementary to the early region of JCV and BKV genome (AgT). The presence of JC virus or BK virus were confirmed by two other PCR assays using sets of primers complementary to the VP1 gene of each virus. Analysis of the data was performed by the Kruskal-Wallis test for numerical data and Pearson or Yates for categorical variables. RESULTS: A total of 75 patients were included in the study. The overall prevalence of polyomavirus DNA urinary shedding was 67/75 (89.3%). Only BKV DNA was detected in 14/75 (18.7%) urine samples, and only JCV DNA was detected in 11/75 (14.7%) samples. Both BKV and JCV DNA were present in 42/75 (56.0%) samples. CONCLUSION: In this study we found high rates of excretion of JCV, BKV, and simultaneous excretion in HIV+ patients. Also these results differ from the others available on the literature.