Drug resistance testing through remote genotyping and predicted treatment options in human immunodeficiency virus type 1 infected Tanzanian subjects failing first or second line antiretroviral therapy.

INTRODUCTION: Antiretroviral therapy (ART) has been successfully introduced in low-middle income countries. However an increasing rate of ART failure with resistant virus is reported. We therefore described the pattern of drug resistance mutations at antiretroviral treatment (ART) failure in a real-life Tanzanian setting using the remote genotyping procedure and thereafter predicted future treatment options using rule-based algorithm and the EuResist bioinformatics predictive engine. According to national guidelines, the default first-line regimen is tenofovir + lamivudine + efavirenz, but variations including nevirapine, stavudine or emtricitabine can be considered. If failure on first-line ART occurs, a combination of two nucleoside reverse transcriptase inhibitors (NRTIs) and boosted lopinavir or atazanavir is recommended. MATERIALS AND METHODS: Plasma was obtained from subjects with first (n = 174) or second-line (n = 99) treatment failure, as defined by clinical or immunological criteria, as well as from a control group of ART naïve subjects (n = 17) in Dar es Salaam, Tanzania. Amplification of the pol region was performed locally and the amplified DNA fragment was sent to Sweden for sequencing (split genotyping procedure). The therapeutic options after failure were assessed by the genotypic sensitivity score and the EuResist predictive engine. Viral load was quantified in a subset of subjects with second-line failure (n = 52). RESULTS: The HIV-1 pol region was successfully amplified from 55/174 (32%) and 28/99 (28%) subjects with first- or second-line failure, respectively, and 14/17 (82%) ART-naïve individuals. HIV-1 pol sequence was obtained in 82 of these 97 cases (84.5%). Undetectable or very low (<2.6 log10 copies/10-3 L) viral load explained 19 out of 25 (76%) amplification failures in subjects at second-line ART failure. At first and second line failure, extensive accumulation of NRTI (88% and 73%, respectively) and NNRTI (93% and 73%, respectively) DRMs but a limited number of PI DRMs (11% at second line failure) was observed. First line failure subjects displayed a high degree of cross-resistance to second-generation NNRTIs etravirine (ETR; 51% intermediate and 9% resistant) and rilpivirine (RPV; 12% intermediate and 58% resistant), and to abacavir (ABC; 49% resistant) which is reserved for second line therapy in Tanzania. The predicted probability of success with the best salvage regimen at second-line failure decreased from 93.9% to 78.7% when restricting access to the NRTIs, NNRTIs and PIs currently available in Tanzania compared to when including all approved drugs. DISCUSSION: The split genotyping procedure is potential tool to analyse drug resistance in Tanzania but the sensitivity should be evaluated further. The lack of viral load monitoring likely results in a high false positive rate of treatment failures, unnecessary therapy switches and massive accumulation of NRTI and NNRTI mutations. The introduction of regular virological monitoring should be prioritized and integrated with drug resistance studies in resource limited settings.

Role of translocated bacterial flagellin in monocyte activation among individuals with chronic HIV-1 infection.

Monocyte activation has been identified as a predictor of mortality and morbidity in HIV-1 infection. This study investigated translocated bacterial flagellin as a potential contributor to systemic monocyte activation via Toll-like receptor 5 (TLR5) stimulation.We demonstrated that circulating flagellin correlated to anti-flagellin, which was associated with soluble markers of microbial translocation (LPS, LBP) and monocyte activation (sCD14, sCD163). Flagellin exposure in vitro reduced monocyte TLR5 expression and the magnitude of reduction was correlated to anti-flagellin levels, indicative of previous flagellin exposure. Circulating anti-flagellin and basal TLR5 expression were both associated with basal and flagellin-stimulated monocyte cytokine production, where HIV + and HIV − differed in their cytokine patterns (IL-1ß, IL-6, IL-8).Our results suggest that translocated flagellin contributes to systemic immune activation in HIV-1 infection and reduces monocyte surface TLR5 expression resulting in a hyperactivated state with elevated basal cytokine production and reduced ability to respond to further TLR5 stimulation.

Gut microbiota diversity predicts immune status in HIV-1 infection.

OBJECTIVE: HIV-1 infection is characterized by altered intestinal barrier, gut microbiota dysbiosis, and systemic inflammation. We hypothesized that changes of the gut microbiota predict immune dysfunction and HIV-1 progression, and that antiretroviral therapy (ART) partially restores the microbiota composition. DESIGN: An observational study including 28 viremic patients, three elite controllers, and nine uninfected controls. Blood and stool samples were collected at baseline and for 19 individuals at follow-up (median 10 months) during ART. METHODS: Microbiota composition was determined by 16S rRNA sequencing (Illumina MiSeq). Soluble markers of microbial translocation and monocyte activation were analyzed by Limulus Amebocyte Lysate assay or ELISA. RESULTS: Several alpha-diversity measures, including number of observed bacterial species and Shannon index, were significantly lower in viremic patients compared to controls. The alpha diversity correlated with CD4 T-cell counts and inversely with markers of microbial translocation and monocyte activation. In multivariate linear regression, for every age and sex-adjusted increase in the number of bacterial species, the CD4 T-cell count increased with 0.88 (95% confidence interval 0.35-1.41) cells/µl (P = 0.002). After introduction of ART, microbiota alterations persisted with further reduction in alpha diversity. The microbiota composition at the genus level was profoundly altered in viremic patients, both at baseline and after ART, with Prevotella reduced during ART (P < 0.007). CONCLUSIONS: Gut microbiota alterations are closely associated with immune dysfunction in HIV-1 patients, and these changes persist during short-term ART. Our data implicate that re-shaping the microbiota may be an adjuvant therapy in patients commencing successful ART.

Arming of MAIT Cell Cytolytic Antimicrobial Activity Is Induced by IL-7 and Defective in HIV-1 Infection.

Mucosa-associated invariant T (MAIT) cells represent a large innate-like evolutionarily conserved antimicrobial T-cell subset in humans. MAIT cells recognize microbial riboflavin metabolites from a range of microbes presented by MR1 molecules. MAIT cells are impaired in several chronic diseases including HIV-1 infection, where they show signs of exhaustion and decline numerically. Here, we examined the broader effector functions of MAIT cells in this context and strategies to rescue their functions. Residual MAIT cells from HIV-infected patients displayed aberrant baseline levels of cytolytic proteins, and failed to mobilize cytolytic molecules in response to bacterial antigen. In particular, the induction of granzyme B (GrzB) expression was profoundly defective. The functionally impaired MAIT cell population exhibited abnormal T-bet and Eomes expression patterns that correlated with the deficiency in cytotoxic capacity and cytokine production. Effective antiretroviral therapy (ART) did not fully restore these aberrations. Interestingly, IL-7 was capable of arming resting MAIT cells from healthy donors into cytotoxic GrzB+ effector T cells capable of killing bacteria-infected cells and producing high levels of pro-inflammatory cytokines in an MR1-dependent fashion. Furthermore, IL-7 treatment enhanced the sensitivity of MAIT cells to detect low levels of bacteria. In HIV-infected patients, plasma IL-7 levels were positively correlated with MAIT cell numbers and function, and IL-7 treatment in vitro significantly restored MAIT cell effector functions even in the absence of ART. These results indicate that the cytolytic capacity in MAIT cells is severely defective in HIV-1 infected patients, and that the broad-based functional defect in these cells is associated with deficiency in critical transcription factors. Furthermore, IL-7 induces the arming of effector functions and enhances the sensitivity of MAIT cells, and may be considered in immunotherapeutic approaches to restore MAIT cells.

Multiparametric bioinformatics distinguish the CD4/CD8 ratio as a suitable laboratory predictor of combined T cell pathogenesis in HIV infection.

HIV disease progression is characterized by numerous pathological changes of the cellular immune system. Still, the CD4 cell count and viral load represent the laboratory parameters that are most commonly used in the clinic to determine the disease progression. In this study, we conducted an interdisciplinary investigation to determine which laboratory parameters (viral load, CD4 count, CD8 count, CD4 %, CD8 %, CD4/CD8) are most strongly associated with pathological changes of the immune system. Multiparametric flow cytometry was used to assess markers of CD4(+) and CD8(+) T cell activation (CD38, HLA-DR), exhaustion (PD-1, Tim-3), senescence (CD28, CD57), and memory differentiation (CD45RO, CD27) in a cohort of 47 untreated HIV-infected individuals. Using bioinformatical methods, we identified 139 unique populations, representing the "combined T cell pathogenesis," which significantly differed between the HIV-infected individuals and healthy control subjects. CD38, HLA-DR, and PD-1 were particularly expressed within these unique T cell populations. The CD4/CD8 ratio was correlated with more pathological T cell populations (n = 10) and had a significantly higher average correlation coefficient than any other laboratory parameters. We also reduced the dimensionalities of the 139-unique populations by Z-transformations and principal component analysis, which still identified the CD4/CD8 ratio as the preeminent surrogate of combined T cell pathogenesis. Importantly, the CD4/CD8 ratio at baseline was shown to be significantly associated with CD4 recovery 2 y after therapy initiation. These results indicate that the CD4/CD8 ratio would be a suitable laboratory predictor in future clinical and therapeutic settings to monitor pathological T cell events in HIV infection.

Superinfection with drug-resistant HIV is rare and does not contribute substantially to therapy failure in a large European cohort.

BACKGROUND: Superinfection with drug resistant HIV strains could potentially contribute to compromised therapy in patients initially infected with drug-sensitive virus and receiving antiretroviral therapy. To investigate the importance of this potential route to drug resistance, we developed a bioinformatics pipeline to detect superinfection from routinely collected genotyping data, and assessed whether superinfection contributed to increased drug resistance in a large European cohort of viremic, drug treated patients. METHODS: We used sequence data from routine genotypic tests spanning the protease and partial reverse transcriptase regions in the Virolab and EuResist databases that collated data from five European countries. Superinfection was indicated when sequences of a patient failed to cluster together in phylogenetic trees constructed with selected sets of control sequences. A subset of the indicated cases was validated by re-sequencing pol and env regions from the original samples. RESULTS: 4425 patients had at least two sequences in the database, with a total of 13816 distinct sequence entries (of which 86% belonged to subtype B). We identified 107 patients with phylogenetic evidence for superinfection. In 14 of these cases, we analyzed newly amplified sequences from the original samples for validation purposes: only 2 cases were verified as superinfections in the repeated analyses, the other 12 cases turned out to involve sample or sequence misidentification. Resistance to drugs used at the time of strain replacement did not change in these two patients. A third case could not be validated by re-sequencing, but was supported as superinfection by an intermediate sequence with high degenerate base pair count within the time frame of strain switching. Drug resistance increased in this single patient. CONCLUSIONS: Routine genotyping data are informative for the detection of HIV superinfection; however, most cases of non-monophyletic clustering in patient phylogenies arise from sample or sequence mix-up rather than from superinfection, which emphasizes the importance of validation. Non-transient superinfection was rare in our mainly treatment experienced cohort, and we found a single case of possible transmitted drug resistance by this route. We therefore conclude that in our large cohort, superinfection with drug resistant HIV did not compromise the efficiency of antiretroviral treatment.

Nuclear receptor-mediated induction of CYP450 by antiretrovirals: functional consequences of NR1I2 (PXR) polymorphisms and differential prevalence in whites and sub-Saharan Africans.

BACKGROUND: Antiretroviral therapy including HIV protease inhibitors and nonnucleoside reverse transcriptase inhibitors can both inhibit and induce expression of cytochrome P450s, potentially leading to drug interactions. However, information is lacking on the impact of genetic polymorphism on this interaction. METHODS: This study examines the prevalence of 33 polymorphisms in NR1I2 (pregnane X receptor [PXR]), CYP3A4, and CYP2B6 in 1013 white and sub-Saharan African patients with HIV; explores the inductive ability of 16 antiretrovirals on CYP3A4 and CYP2B6 promoter activity through nuclear receptors PXR and constitutive androstane receptor (CAR); and evaluates the influence of naturally occurring PXR genetic variants on antiretroviral activation. RESULTS: Seventeen polymorphisms were present at different frequencies between the two ethnicities. Darunavir, fosamprenavir, lopinavir, nelfinavir, tipranavir, efavirenz, and abacavir increased CYP3A4 and/or CYP2B6 promoter activity, some through constitutive androstane receptor but mainly through PXR. Addition of low-dose ritonavir enhanced levels of CYP promoter activity for several protease inhibitors. Some PXR variants displayed lower fosamprenavir- and lopinavir-induced CYP3A4 promoter activity than the PXR reference sequence, whereas efavirenz and nelfinavir induction was unchanged. CONCLUSIONS: The presence of NR1I2 polymorphisms can alter the induction of CYP3A4 and CYP2B6 promoter activity, potentially adding to the unpredictable nature of antiretroviral drug interactions. These polymorphisms differ in prevalence between whites and sub-Saharan Africans.

Selection of pfmdr1 mutations after amodiaquine monotherapy and amodiaquine plus artemisinin combination therapy in East Africa.

Despite the pharmacodynamic advantages with artemisinin-based combination therapy (ACT) and some potentially opposite molecular mechanisms of tolerance to amodiaquine (AQ)/desethylamodiaquine (DEAQ) and artesunate (ART), there is a risk for rapid decay in efficacy if the two drugs are unable to ensure mutual prevention against a selection and spread of drug-resistant parasites. We have studied if mutations in the pfcrt and pfmdr1 genes selected in recurrent infections after AQ monotherapy are also selected after AQ plus ART combination therapy. Samples for molecular analysis were derived from three clinical trials on children<5 years old with uncomplicated Plasmodium falciparum malaria; one AQ monotherapy study conducted in Kenya 2003 and two AQ plus ART combination therapy studies conducted in Zanzibar 2002-2003 and 2005, respectively. The PCR-adjusted treatment failure rates in the three studies were 19%, 8% and 9%, respectively. After monotherapy there was a significant selection of pfcrt 76T in re-infections (OR not calculable; p=0.048) and of pfmdr1 86Y in recrudescent infections (OR 8.0; p=0.048). No such selection was found after combination therapy. A selection of pfmdr1 1246Y and the pfmdr1 haplotype (a.a 86, 184, 1246) YYY was found in recrudescent infections both after monotherapy (OR 7.6; p=0.009 and OR 3.1; p=0.029) and combination therapy in 2005 (OR 3.6; p=0.017 and OR 5.4; p<0.001). Hence, pfmdr1 1246Y with synergistic or compensatory addition of pfmdr1 86Y and 184Y appears to be involved in AQ/DEAQ resistance and treatment failure. Our results suggest that ART may protect against a selection of these SNPs initially, but maybe not after continuous drug pressure in a population. However, treatment failure rate and spread of pfmdr1 SNPs may remain at a low level because of the suggested opposite selection by ART and the pharmacodynamic advantages with ACT.

Tumor-specific expression of the novel cytochrome P450 enzyme, CYP2W1.

A novel human cytochrome P450, CYP2W1, was cloned and expressed heterologously. No or very low CYP2W1 mRNA levels were detected in fetal and adult human tissues, expression was however seen in 54% of human tumor samples investigated (n=37), in particular colon and adrenal tumors. Western blotting also revealed high expression of CYP2W1 in some human colon tumors. In rat tissues, CYP2W1 mRNA was expressed preferentially in fetal but also in adult colon. The CYP2W1 gene was shown to encompass one functional CpG island in the exon 1-intron 1 region which was methylated in cell lines lacking CYP2W1 expression, but unmethylated in cells expressing CYP2W1. Re-expression of CYP2W1 was seen following demethylation by 5-Aza-2'-deoxycytidine. Transfection of HEK293 cells with CYP2W1 caused the formation of a properly folded enzyme, which was catalytically active with arachidonic acid as a substrate. It is concluded that CYP2W1 represents a tumor-specific P450 isoform with potential importance as a drug target in cancer therapy.