Using longitudinally sampled viral nucleotide sequences to characterize the drivers of HIV-1 transmission.

OBJECTIVES: Understanding the drivers of HIV-1 transmission is of importance for curbing the ongoing epidemic. Phylogenetic methods based on single viral sequences allow us to assess whether two individuals are part of the same viral outbreak, but cannot on their own assess who potentially transmitted the virus. We developed and assessed a molecular epidemiology method with the main aim to screen cohort studies for and to characterize individuals who are 'potential HIV-1 transmitters', in order to understand the drivers of HIV-1 transmission. METHODS: We developed and validated a molecular epidemiology approach using longitudinally sampled viral Sanger sequences to characterize potential HIV-1 transmitters in the Swiss HIV Cohort Study. RESULTS: Our method was able to identify 279 potential HIV-1 transmitters and allowed us to determine the main epidemiological and virological drivers of transmission. We found that the directionality of transmission was consistent with infection times for 72.9% of 85 potential HIV-1 transmissions with accurate infection date estimates. Being a potential HIV-1 transmitter was associated with risk factors including viral load [adjusted odds ratiomultivariable (95% confidence interval): 1.86 (1.49-2.32)], syphilis coinfection [1.52 (1.06-2.19)], and recreational drug use [1.45 (1.06-1.98)]. By contrast for the potential HIV-1 recipients, this association was weaker or even absent [1.18 (0.82-1.72), 0.89 (0.52-1.55) and 1.53 (0.98-2.39), respectively], indicating that inferred directionality of transmission is useful at the population level. CONCLUSIONS: Our results indicate that longitudinally sampled Sanger sequences do not provide sufficient information to identify transmitters with high certainty at the individual level, but that they allow the drivers of transmission at the population level to be characterized.

Stable HIV-1 reservoirs on dolutegravir maintenance monotherapy: the MONODO study.

OBJECTIVES: Dolutegravir (DTG) is a highly effective integrase inhibitor with a strong genetic resistance barrier and a potential role in simplified HIV maintenance treatment. We assessed the feasibility of DTG maintenance monotherapy and measured HIV reservoirs on DTG monotherapy. METHODS: An interventional, open-label, single-arm study including eight virologically suppressed HIV-1-infected patients switched to DTG 50 mg once daily for 24 weeks was performed. HIV-1 RNA levels in plasma and cerebrospinal and seminal fluids were measured at baseline and week 24, as well as HIV-1 DNA in peripheral cells and DTG concentrations in these compartments. RESULTS: HIV-1 RNA remained undetectable in all samples of blood, cerebrospinal fluid and sperm throughout the 24 weeks, except for one cerebrospinal fluid sample with a value of 28 HIV-1 RNA copies/mL at week 24. One patient discontinued the study because of a neurological side effect. There was no change in the mean HIV-1 DNA level between baseline and week 24. Plasma and cerebrospinal fluid DTG concentrations reached therapeutic levels in all patients in these two compartments. CONCLUSIONS: In this small sample of carefully selected patients, HIV-1 reservoirs were well controlled on DTG monotherapy over a period of 24 weeks. Viral suppression was also maintained throughout follow-up.

Mining for pairs: shared clinic visit dates identify steady HIV-positive partnerships.

OBJECTIVES: Here we examined the hypothesis that some stable HIV-infected partnerships can be found in cohort studies, as the patients frequently attend the clinic visits together. METHODS: Using mathematical approximations and shuffling to derive the probabilities of sharing a given number of visits by chance, we identified and validated couples that may represent either transmission pairs or serosorting couples in a stable relationship. RESULTS: We analysed 434 432 visits for 16 139 Swiss HIV Cohort Study patients from 1990 to 2014. For 89 pairs, the number of shared visits exceeded the number expected. Of these, 33 transmission pairs were confirmed on the basis of three criteria: an extensive phylogenetic tree, a self-reported steady HIV-positive partnership, and risk group affiliation. Notably, 12 of the validated transmission pairs (36%; 12 of 33) were of a mixed ethnicity with a large median age gap [17.5 years; interquartile range (IQR) 11.8-22 years] and these patients harboured HIV-1 of predominantly non-B subtypes, suggesting imported infections. CONCLUSIONS: In the context of the surge in research interest in HIV transmission pairs, this simple method widens the horizons of research on within-pair quasi-species exchange, transmitted drug resistance and viral recombination at the biological level and targeted prevention at the public health level.

Contribution of APOBEC3G/F activity to the development of low-abundance drug-resistant human immunodeficiency virus type 1 variants.

Plasma drug-resistant minority human immunodeficiency virus type 1 variants (DRMVs) increase the risk of virological failure to first-line non-nucleoside reverse transcriptase inhibitor antiretroviral therapy (ART). The origin of DRMVs in ART-naive patients, however, remains unclear. In a large pan-European case-control study investigating the clinical relevance of pre-existing DRMVs using 454 pyrosequencing, the six most prevalent plasma DRMVs detected corresponded to G-to-A nucleotide mutations (V90I, V106I, V108I, E138K, M184I and M230I). Here, we evaluated if such DRMVs could have emerged from apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3G/F (APOBEC3G/F) activity. Out of 236 ART-naive subjects evaluated, APOBEC3G/F hypermutation signatures were detected in plasma viruses of 14 (5.9%) individuals. Samples with minority E138K, M184I, and M230I mutations, but not those with V90I, V106I or V108I, were significantly associated with APOBEC3G/F activity (Fisher's P < 0.005), defined as the presence of > 0.5% of sample sequences with an APOBEC3G/F signature. Mutations E138K, M184I and M230I co-occurred in the same sequence as APOBEC3G/F signatures in 3/9 (33%), 5/11 (45%) and 4/8 (50%) of samples, respectively; such linkage was not found for V90I, V106I or V108I. In-frame STOP codons were observed in 1.5% of all clonal sequences; 14.8% of them co-occurred with APOBEC3G/F signatures. APOBEC3G/F-associated E138K, M184I and M230I appeared within clonal sequences containing in-frame STOP codons in 2/3 (66%), 5/5 (100%) and 4/4 (100%) of the samples. In a re-analysis of the parent case control study, the presence of APOBEC3G/F signatures was not associated with virological failure. In conclusion, the contribution of APOBEC3G/F editing to the development of DRMVs is very limited and does not affect the efficacy of non-nucleoside reverse transcriptase inhibitor ART.

Protease inhibitors to treat hepatitis C in the Swiss HIV Cohort Study: high efficacy but low treatment uptake.

OBJECTIVES: Direct-acting antiviral agents (DAAs) have become the standard of care for the treatment of chronic hepatitis C virus (HCV) infection. We aimed to assess treatment uptake and efficacy in routine clinical settings among HIV/HCV coinfected patients after the introduction of the first generation DAAs. METHODS: Data on all Swiss HIV Cohort Study (SHCS) participants starting HCV protease inhibitor (PI) treatment between September 2011 and August 2013 were collected prospectively. The uptake and efficacy of HCV therapy were compared with those in the time period before the availability of PIs. RESULTS: Upon approval of PI treatment in Switzerland in September 2011, 516 SHCS participants had chronic HCV genotype 1 infection. Of these, 57 (11%) started HCV treatment during the following 2 years with either telaprevir, faldaprevir or boceprevir. Twenty-seven (47%) patients were treatment-naïve, nine (16%) were patients with relapse and 21 (37%) were partial or null responders. Twenty-nine (57%) had advanced fibrosis and 15 (29%) had cirrhosis. End-of-treatment virological response was 84% in treatment-naïve patients, 88% in patients with relapse and 62% in previous nonresponders. Sustained virological response was 78%, 86% and 40% in treatment-naïve patients, patients with relapse and nonresponders, respectively. Treatment uptake was similar before (3.8 per 100 patient-years) and after (6.1 per 100 patient-years) the introduction of PIs, while treatment efficacy increased considerably after the introduction of PIs. CONCLUSIONS: The introduction of PI-based HCV treatment in HIV/HCV-coinfected patients improved virological response rates, while treatment uptake remained low. Therefore, the introduction of PIs into the clinical routine was beneficial at the individual level, but had only a modest effect on the burden of HCV infection at the population level.

Virological failure after 1 year of first-line ART is not associated with HIV minority drug resistance in rural Cameroon .

OBJECTIVES: The aim of this study was to describe clinical and virological outcomes in therapy-naive HIV-1-positive patients treated in a routine ART programme in rural Cameroon. METHODS: In a prospective cohort, 300 consecutive patients starting first-line ART were enrolled and followed for 12 months. Among 238 patients with available viral load data at Month 12, logistic regression was used to analyse risk factors for virological failure (≥1000 HIV RNA copies/mL) including clinical, immunological and virological parameters, as well as data on drug adherence. Population sequencing was performed to detect the presence of drug-resistance mutations in patients with virological failure at Month 12; minority drug-resistance mutations at baseline were analysed using next-generation sequencing in these patients and matched controls. RESULTS: At Month 12, 38/238 (16%) patients experienced virological failure (≥1000 HIV RNA copies/mL). Patients with virological failure were younger, had lower CD4 cell counts and were more often WHO stage 3 or 4 at baseline. Sixty-three percent of patients with virological failure developed at least one drug-resistance mutation. The M184V (n = 18) and K103N (n = 10) mutations were most common. At baseline, 6/30 patients (20%) experiencing virological failure and 6/35 (17%) matched controls had evidence of minority drug-resistance mutations using next-generation sequencing (P = 0.77). Lower CD4 count at baseline (OR per 100 cells/mm(3) lower 1.41, 95% CI 1.02-1.96, P = 0.04) and poorer adherence (OR per 1% lower 1.05, 95% CI 1.02-1.08, P < 0.001) were associated with a higher risk of virological failure. Unavailability of ART at the treatment centre was the single most common cause for incomplete adherence. CONCLUSIONS: Virological failure after 1 year of ART was not associated with minority drug resistance at baseline but with incomplete adherence. Strategies to assure adherence and uninterrupted drug supplies are pivotal factors for therapy success.

Efficacy of lead-in silibinin and subsequent triple therapy in difficult-to-treat HIV/hepatitis C virus-coinfected patients.

OBJECTIVES: The efficacy of current hepatitis C virus (HCV) triple therapy, including a protease inhibitor, is limited in HIV/HCV-coinfected patients with advanced liver fibrosis and nonresponse to previous peginterferon-ribavirin. These patients have a low chance (only 30%) of achieving a sustained virological response (SVR) during triple therapy and cannot wait for next-generation anti-HCV drugs. In a pilot study, we investigated the efficacy of a lead-in therapy with silibinin before triple therapy in difficult-to-treat patients. METHODS: Inclusion criteria were HIV/HCV coinfection with advanced liver fibrosis and documented failure of previous peginterferon-ribavirin treatment. Intervention was lead-in therapy with intravenous silibinin 20 mg/kg/day for 14 days. Subsequently, peginterferon-ribavirin combined with telaprevir was initiated for 12 weeks, followed by peginterferon-ribavirin dual therapy until week 48 after initiation of triple therapy. The outcome measurements were HCV RNA after silibinin lead-in, at weeks 2, 4 and 12 of triple therapy, and SVR at week 24 after the end of treatment. RESULTS: We examined six HIV/HCV-coinfected patients (four infected with genotype 1a). All had fibrosis grade METAVIR ≥F3 and were on fully suppressive antiretroviral therapy. Mean HCV RNA decline after silibinin therapy was 2.6 log10 IU/mL (range 2-3 log10 IU/mL). Five of the six patients were virologically suppressed at weeks 2 and 4, and all six at week 12 of triple therapy. One experienced a viral breakthrough thereafter. Four of five patients (80%) showed an SVR 24. One patient had an SVR 12 but has not yet reached week 24. CONCLUSIONS: A lead-in with silibinin before triple therapy is highly effective and increases the probability of HCV treatment success in difficult-to-treat HIV/HCV-coinfected patients with advanced liver fibrosis and previous failure of peginterferon-ribavirin.

[Evolution and Diversity of HIV-1]. / Evolution und Diversität von HIV-1.

The human immunodeficiency virus type 1 (HIV-1) is the cause of a devastating pandemic. HIV-1 infection leads to the disease AIDS (acquired immunodeficiency syndrome) after several years of clinical latency defined by the occurrence of opportunistic infections. The immune system of the patient is not capable of controlling the virus, not to mention to eliminate the virus. Nowadays, the antiretroviral therapy is very efficient; however, eradication of HIV-1 is currently not feasible. Countless efforts to develop an effective vaccine failed so far. One common cause for development of drug resistance during antiretroviral therapy and these fallibilities of the immune system and vaccine candidates is the rapid adaptability of HIV-1 and the associated high diversity of circulating virus variants world wide and within each patient.

Effects of in vivo CD8(+) T cell depletion on virus replication in rhesus macaques immunized with a live, attenuated simian immunodeficiency virus vaccine.

The role of CD8(+) T lymphocytes in controlling replication of live, attenuated simian immunodeficiency virus (SIV) was investigated as part of a vaccine study to examine the correlates of protection in the SIV/rhesus macaque model. Rhesus macaques immunized for >2 yr with nef-deleted SIV (SIVmac239Deltanef) and protected from challenge with pathogenic SIVmac251 were treated with anti-CD8 antibody (OKT8F) to deplete CD8(+) T cells in vivo. The effects of CD8 depletion on viral load were measured using a novel quantitative assay based on real-time polymerase chain reaction using molecular beacons. This assay allows simultaneous detection of both the vaccine strain and the challenge virus in the same sample, enabling direct quantification of changes in each viral population. Our results show that CD8(+) T cells were depleted within 1 h after administration of OKT8F, and were reduced by as much as 99% in the peripheral blood. CD8(+) T cell depletion was associated with a 1-2 log increase in SIVmac239Deltanef plasma viremia. Control of SIVmac239Deltanef replication was temporally associated with the recovery of CD8(+) T cells between days 8 and 10. The challenge virus, SIVmac251, was not detectable in either the plasma or lymph nodes after depletion of CD8(+) T cells. Overall, our results indicate that CD8(+) T cells play an important role in controlling replication of live, attenuated SIV in vivo.

Direct measurement of CD8+ T cell responses in macaques infected with simian immunodeficiency virus.

The simian immunodeficiency virus (SIV) macaque model system has been used extensively to study AIDS pathogenesis and to test candidate vaccines for their ability to protect against homologous or heterologous challenge with pathogenic SIV or SHIV. Recent studies suggest that stimulation of HIV-1-specific CTL responses is important for effective vaccination against HIV-1. While quantitative measurements of SIV-specific cytotoxic T lymphocyte (CTL) responses have been facilitated by the use of tetrameric peptide complexes, this technique is currently limited to the study of Mamu-A*01-positive rhesus macaques. Furthermore, very few SIV-specific CTL epitopes have been identified, and there is limited identification of other MHC alleles in macaques. In this study, cytokine flow cytometry (CFC) was used to quantify SIV-specific CD8+ antigen-reactive T cells in macaques infected with SIV. We found a strong correlation (r = 0.96, P < 0.001) between CD8+ antigen-reactive T cells stained with the Mamu-A*01 p11C, C-M tetramer and production of intracellular TNF-alpha in the CFC assay. Furthermore, the CFC assay was used to identify a novel SIV-specific CTL epitope in Envelope (SIV Env, a.a. 486-494, sequence AEVAELYRL). The use of the CFC assay facilitates the study of antigen-reactive T cell responses in SIV infection and vaccination.

Simian immunodeficiency virus-specific cytotoxic T lymphocytes and protection against challenge in rhesus macaques immunized with a live attenuated simian immunodeficiency virus vaccine.

In this study, we examined the role of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocytes (CTLs) in macaques immunized with an attenuated strain of simian immunodeficiency virus (SIVmac239Deltanef) in protection against pathogenic challenge with SIVmac251. Our results indicate that attenuated SIVmac239Deltanef can elicit specific CTL precursor cells (CTLp), but no correlation was observed between breadth or strength of CTLp response to structural proteins SIV-Env, -Gamg or -Pol (as measured by limiting dilution assay) and protection against infection. In one animal, we longitudinally followed the SIV-Gag-specific response to an MHC class I Mamu-A*01-restricted epitope p11C, C-M using a tetrameric MHC/peptide complex reagent. A low frequency of SIV p11C, C-M peptide-specific tetramer-reactive cells was present at the time of challenge but could be expanded in vitro. Surprisingly, the low level of Mamu-A*01/p11C, C-M-specific CTLs induced through attenuated SIVmac239Deltanef vaccination increased in the absence of detectable SIVmac251 or SIVmac239Deltanef proviral DNA. Overall, our results suggest that protection against infection in this model can be achieved through more than one mechanism, with SIV-specific CTLs being important in controlling SIVmac239Deltanef viral replication postchallenge.