Antonella Lagioia

Co-receptor switch during HAART is independent of virological success.

The influence of antiretroviral therapy on co‐receptor tropism remains controversial. To verify if co‐receptor tropism shift was affected by HAART, the evolution of proviral DNA V3 genotype after 12 months of a new antiretroviral regimen was compared between responder and non‐responder patients. Baseline blood samples were collected from 36 patients infected with HIV‐1 subtype‐B (18 naïve and 18 experienced) for virus isolation and env V3 genotyping from plasma HIV‐1 RNA and PBMC DNA. DNA V3 genotyping was repeated after 12 months from initiating HAART. WebPSSM was used for categorizing V3 sequences into X4 or R5; for analysis purposes, dual/mixed viruses were considered as X4. From the 10 (28%) patients changing their proviral DNA V3 genotype during therapy, six shifted from R5‐to‐X4 and four from X4‐to‐R5. The lack of reaching virological suppression was not associated with an X4‐to‐R5 (P = 0.25) or R5‐to‐X4 (P = 0.14) shift; time‐to‐viral suppression and CD4 increase were similar in both groups. No association was found between tropism shift and patient baseline characteristics including age, sex, CDC stage, CD4 count, viral load, exposure and length of previous HAART, enfuvirtide use in the new regimen, number of reverse transcriptase and protease resistance‐associated mutations. Conversely, CD4 nadir was correlated to emergence of X4 virus in proviral DNA (mean 27.2 ± 30.6 in R5‐to‐X4 shifting patients vs. 161.6 ± 150.6 in non‐shifting patients, P = 0.02). The occurrence of a tropism shift in both directions was independent of HAART use, irrespective of its efficacy. The CD4 count nadir was the only baseline characteristic able to predict an R5‐to‐X4 viral shift. J. Med. Virol. 81:2036–2044, 2009.

Impact of Mutations Outside the V3 Region on Coreceptor Tropism Phenotypically Assessed in Patients Infected with HIV-1 Subtype B

ABSTRACT HIV coreceptor tropism (CTR) testing is a prerequisite for prescribing a coreceptor antagonist. CTR is increasingly deduced by analyzing the V3 loop sequence of gp120. We investigated the impact of mutations outside V3 on CTR as determined by the enhanced-sensitivity Trofile assay (ESTA). Paired ESTA and gp120 sequencing (population sequencing; from codon 32 of the conserved C1 to the variable V5 domains) were obtained from 60 antiretroviral treatment (ART)-naïve patients (15 with AIDS) infected with subtype B HIV-1. For gp120 sequence analysis, nucleotide mixtures were considered when the second highest electropherogram peak was >25%; sequences were translated into all possible permutations and classified as X4, dual/mixed (DM), and R5 based on coincident ESTA results. ESTA identified R5 and DM viruses in 72 and 28% of patients, respectively; no pure X4 was labeled. Forty percent of AIDS patients had R5 strains. Thirty-two positions, mostly outside V3, were significantly (P < 0.05) different between R5 and DM sequences. According to multivariate analysis, amino acid changes at 9 and 7 positions within the C1 to C4 and V1 to V5 regions, respectively, maintained a statistical significance, as did the net charge of V3 and C4. When analyzing only R5 sequences, 6 positions in the variable regions were found which, along with the V4 net charge, were significantly different for sequences from early- and end-stage disease patients. This study identifies specific amino acid changes outside V3 which contribute to CTR. Extending the analysis to include pure X4 and increasing the sample size would be desirable to define gp120 variables/changes which should be included in predictive algorithms.

Genotypic analysis of the protease and reverse transcriptase of non-B HIV type 1 clinical isolates from naïve and treated subjects ☆

One hundred and ninety-two pol sequences of drug-naïve and drug-experienced subjects infected with non-B HIV-1 subtypes were analyzed to identify treatment-related amino acid changes which might be relevant for drug-resistance and possibly not included in the accepted mutation list for the B subtype. The correspondence analysis identified non-B-specific and subtype-specific polymorphisms which should not be mistaken for mutations. Multiple chi(2) were performed to detect the differences between naïve vs treated subjects and between different subtypes. To verify the contribution of each single mutation to the resistance levels as predicted by the Virtual Phenotype-LM, simple univariate linear regression was used with fold resistance as a dependent variable and individual mutations as predictors. Commonly accepted protease (PR) and reverse transcriptase (RT) positions along with mutants at RT positions 118 and 90 were significantly associated with treatment. Two unusual PR (K14R and I66F) and five RT positions (E28K, S68G, H221Y, L228R/H and P294A) were also associated with treatment (p<0.01). Only minimal variations were observed with respect to commonly accepted amino acid changes. All amino acid changes correlated with treatment influenced the resistance levels to each single drug. Our findings demonstrate that there are no substantial differences regarding known resistance-associated mutations and the newly emergent substitutions between non-B and B subtype strains.

Does HIV-1 co-receptor tropism correlate with fibrosis progression in HIV/HCV co-infected patients?

BACKGROUND In HIV/HCV co-infected patients, HIV-1 gp120 activates human hepatic stellate cells (HSCs) which play a key role in fibrosis pathogenesis. It is still unclear whether pro-fibrogenic effects are more attributable to X4 or R5 strains in vivo. OBJECTIVE To assess if HIV-1 X4 or R5 variants are associated with a different progression of fibrosis. STUDY DESIGN A total of 105 HIV/HCV co-infected patients were submitted to gp120 sequencing on proviral DNA and classified as X4 or R5 based on g2p (20% false positive rate). The fibrosis evolution was retrospectively determined by means of APRI and FIB-4 scores at 3-month intervals from the first anti-HCV-positive test. The association of co-receptor tropism with increased fibrosis scores was evaluated by linear mixed models. RESULTS X4 variants were found in 41 patients (39%). The median observation period was similar in X4 and R5 patients (17 years). No difference was observed between the two groups of patients, except for nadir CD4 which was lower in X4 compared to R5 (percentage, p=0.005, and absolute number, p=0.005). X4 and R5 patients did not significantly differ for FIB-4 and APRI score over time (p=0.5, and p=0.1, respectively). No association between HCV-RNA levels over time and co-receptor tropism was noted (p=0.9). Conversely, a significant correlation of fibrosis scores with gamma-glutamyl transferase levels, lower current CD4 count, HIV viremia and use of antiretrovirals was observed. CONCLUSIONS This retrospective analysis of fibrosis evolution did not support the evidence of a differing pro-fibrogenic activity for X4 and R5 HIV-1 variants in HIV/HCV co-infected patients.

V3 Sequences and Paired HIV Isolates from 52 Non-Subtype B HIV Type 1-Infected Patients

Viral isolation and V3 sequencing were performed for 52 patients with non-subtype B viruses. The HIV-1 isolation rate was 93%, and 98% of isolates were characterized as NSI. V3 sequences corresponding to NSI isolates were compared to non-subtype B sequences with corresponding SI isolates from the Los Alamos database. The two sequence groups significantly differed in number of sequences with 35 amino acids, net charge, Briggs coefficient, loss of NGS at positions 6-8, and 11/25 genotype (p < 0.0001). Substantial discrepancies in V3 variability were also observed. Basic amino acids at positions 8, 21, 23, and 24 were more frequent in SI sequences as were uncharged amino acids at positions 5, 6, 7, 8, 12, 13, 25, and 34. When characterizing paired viral isolates and V3 sequences from patients with non-subtype B HIV-1, current V3 sequence-based criteria from subtype B appeared to discriminate well between NSI and SI sequences from non-subtype B patients.

Improved Virological Outcome in Non-B Patients: A Possible Role for Baseline Coreceptor Tropism

TO THE EDITOR—We greatly appreciated the article of Scherrer et al [1] evaluating viral response to combination antiretroviral therapy (cART) in patients with subtype B (34.3% previously exposed to suboptimal therapies, P < .001) and nonsubtype B human immunodeficiency virus type 1 (HIV-1). The study brings to mind a similar article by Geretti et al [2], but we found particularly reasonable the choice of Scherrer et al to restrict the analysis to a single ethnic group (whites), thus avoiding potential biases caused by ethnicity. The authors demonstrated that virological failure was higher in patients with subtype B; in particular, subtype A and CRF02_AG infections were associated with lower virological failure rates. Because certain non-B subtypes do not seem more susceptible to some drugs, why should these patients have a better response to cART? A lower propensity to select for nucleoside reverse-transcriptase inhibitor– and protease inhibitor–related mutations has been suggested for some non-B subtypes by Soares et al [3]. It is our hypothesis that pretherapy differences might explain the improved

Comment on: Integrase strand-transfer inhibitor polymorphic and accessory resistance substitutions in patients with acute/recent HIV infection

Sir, We read with interest the paper of Ambrosioni et al. in naive patients with recent HIV infection, the authors reported a 13.9% prevalence of integrase strand-transfer inhibitor (INSTI) polymorphisms or substitutions (E157Q and Q95K), conferring low-level resistance to raltegravir and elvitegravir, by using highthroughput sequencing. Pre-treatment E157Q has also been reported as the only INSTI mutation responsible for non-virological response in a patient receiving a dolutegravir-based regimen, thereby reinforcing the need for genotypic analysis encompassing the integrase (IN) gene in all naive patients. According to current guidelines, plasma samples coincident with the first HIV-positive testing of patients referring to our clinic are regularly subjected to population sequencing for evaluation of primary resistance to NRTIs, NNRTIs and PIs. Since 2009, sequences including IN, gp41 and gp120 have also been obtained. At present, IN sequences from 370 consecutive patients with new HIV diagnosis are available. Among these patients, we retrospectively selected those with acute/recent HIV-1 infection (detectable HIV-1-RNA in plasma in the setting of a negative/indeterminate HIV-antibody test; 0.2% nucleotide ambiguity of reverse transcriptase and protease regions, respectively) diagnosed between January 2015 and June 2016, to mimic the same conditions as those of Ambrosioni et al., although by means of slightly different selection criteria. The WHO 2009 SDRM and the IAS lists (2015) were used to identify transmitted drug resistance-associated mutations for NRTIs/NNRTIs/PIs and relevant INSTI and fusion inhibitor mutations, respectively. IN substitutions/polymorphisms with 10 points according to the HIV Drug Resistance Database of Stanford University (https://hivdb.stanford.edu/) were also recorded. HIV-1 tropism was inferred with the geno2pheno algorithm (false-positive rate 10%). Epidemiological and clinical laboratory data were retrieved from our database. According to local regulations, approval of the Ethics Committee was not required due to the retrospective nature of the study. Thirty naive patients (86.7% males; 96.7% Italians; 56.7% MSM; median age 32 years) were included, eight (26.7%) with an acute HIV infection. At diagnosis, the median HIV-RNA was 40 500 copies/mL; the median absolute CD4! count and percentage were 501 cells/mm and 26%, respectively. A subtype B was attributed to 83.3% of clinical strains; most patients (73.3%) were infected with R5-tropic HIV-1. Overall, four patients (13.3%) had the E157Q (three patients) and T97A (one patient) IN mutations; the latter patient also had the unusual N155Y mutation (at this position N155H reduces raltegravir susceptibility .10-fold and elvitegravir susceptibility .30-fold; while N155S/T have somewhat less effect on raltegravir/ elvitegravir susceptibility). Three additional patients had L74I that, combined with primary INSTI-resistance mutations, appears to contribute to reducing susceptibility to INSTIs, including dolutegravir. Substitutions/polymorphisms in regions different from the integrase were observed in an additional seven patients; in particular, two epidemiologically unrelated subjects had a similar pattern of multiple NRTI mutations and a third person had the revertant T215S. Interestingly, two further patients with acute HIV infection had, respectively, the Q40H and V38E mutations for enfuvirtide. Finally, E138A and V108I for NNRTI resistance were detected in two additional subjects (Table 1). No clinical or virological variable (risk factor for HIV-1 transmission, acute infection, subtype, tropism, median CD4 count and viral load) appeared to correlate with the occurrence of INSTI mutations. Our data confirm the observations of Ambrosioni et al. describing a higher than previously reported frequency of INSTI substitutions/polymorphisms in a recent and relatively short period; in fact, we obtained a similar percentage despite using Sanger sequencing, with a detection threshold corresponding to a variant population frequency of 20%. Moreover, one seroconverter with a CRF02_AG HIV strain had the N155Y mutation, which, although not associated per se with reduced INSTI susceptibility, could evolve into the major N155H through a single nucleotide transition rather than a transversion. When analysing 215 IN sequences downloaded from the Los Alamos database (query: CRF02_AG subtype, sample data before 2007), an Asn155 was always found (https://www.hiv. lanl.gov/; data not shown). Overall, these data must warn about the possibility of an incoming wave of virological failure with first-line INSTIs. The detection in four patients of transmitted drug resistanceassociated mutations for NRTIs and signature mutations for enfuvirtide confirms the high stability of certain mutations (even in the absence of drug pressure and fitness advantages). In fact, it seems highly improbable that enfuvirtide mutations were transmitted

Reduced community viral load does not coincide with a reduction in the rate of new HIV diagnoses and recent infections: data from a region of southern Italy

We assessed whether changes in community viral load (CVL) over time were associated with the rate of new HIV diagnoses (NDs).