Consensus statement of the European guidelines on clinical management of HIV-1 tropism testing

Tenth International Congress on Drug Therapy in HIV Infection 7-11 November 2010 Glasgow, UKIntroduction: Testing for HIV tropism is recommended before prescribing a chemokine receptor blocker. To date, in most European countries HIV tropism is determined using a phenotypic test. Recently, new data have emerged supporting the use of a genotypic HIV V3-loop sequence analysis as the basis for tropism determination. The European guidelines group on clinical management of HIV-1 tropism testing was established to make recommendations to clinicians and virologists. Methods: We searched online databases for articles from Jan 2006 until March 2010 with the terms: tropism or CCR5-antagonist or CCR5 antagonist or maraviroc or vicriviroc. Additional articles and/or conference abstracts were identified by hand searching. This strategy identified 712 potential articles and 1240 abstracts. All were reviewed and finally 57 papers and 42 abstracts were included and used by the panel to reach a consensus statement. Results: The panel recommends HIV-tropism testing for the following indications: i) drug-naïve patients in whom toxicity or limited therapeutic options are foreseen; ii) patients experiencing therapy failure whenever a treatment change is considered. Both the phenotypic Enhanced Trofile assay (ESTA) and genotypic population sequencing of the V3-loop are recommended for use in clinical practice. Although the panel does not recommend one methodology over another it is anticipated that genotypic testing will be used more frequently because of its greater accessibility, lower cost and shorter turnaround time. The panel also provides guidance on technical aspects and interpretation issues. If using genotypic methods, triplicate PCR amplification and sequencing testing is advised using the G2P interpretation tool (clonal model) with an FPR of 10%. If the viral load is below the level of reliable amplification, proviral DNA can be used, and the panel recommends performing triplicate testing and use of an FPR of 10%. If genotypic DNA testing is not performed in triplicate the FPR should be increased to 20%. Conclusions: The European guidelines on clinical management of HIV-1 tropism testing provide an overview of current literature, evidence-based recommendations for the clinical use of tropism testing and expert guidance on unresolved issues and current developments. Current data support both the use of genotypic population sequencing and ESTA for co-receptor tropism determination. For practical reasons genotypic population sequencing is the preferred method in Europe.Ye

A21 HIV-1 sub-subtype F1 outbreak among MSM in Belgium

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Generation of representative primary virus isolates from blood plasma after isolation of HIV-1 with CD44 MicroBeads

Infection of cell cultures with cell-free virus isolated from HIV-infected patients is notoriously difficult and results in a loss of viral variation. Here, we describe viral sequences from PBMC, U87.CD4.CCR5 and U87.CD4.CXCR4 cell cultures and compare them to those from blood plasma from 12 patients from whom virus particles were isolated using CD44 MicroBeads. In both PBMC and U87.CD4.CCR5 cultures, 66% of the plasma viral strains were retrieved after culturing. In addition, coreceptor use was predicted based on the env-V3 sequence and tested in U87.CD4 cells expressing either CCR5 or CXCR4. Recovery was lower for the CXCR4-using viruses. Only 50% of the virus clusters predicted to use CXCR4 could be retrieved from cell cultures, while 71% of CCR5-using strains were found in U87.CCR5 cultures. Therefore, isolation of primary viruses with CD44 MicroBeads results in a good representation in cell culture of the in vivo divergence

Evaluation of immunoglobulin purification methods and their impact on quality and yield of antigen-specific antibodies

<p>Abstract</p> <p>Background</p> <p>Antibodies are the main effectors against malaria blood-stage parasites. Evaluation of functional activities in immune sera from Phase 2a/b vaccine trials may provide invaluable information in the search for immune correlates of protection. However, the presence of anti-malarial-drugs, improper collection/storage conditions or concomitant immune responses against other pathogens can contribute to non-specific anti-parasite activities when the sera/plasma are tested <it>in vitro</it>. Purification of immunoglobulin is a standard approach for reducing such non-specific background activities, but the purification method itself can alter the quality and yield of recovered Ag-specific antibodies.</p> <p>Methods</p> <p>To address this concern, various immunoglobulin (Ig) purification methods (protein G Sepharose, protein A/G Sepharose, polyethylene glycol and caprylic acid-ammonium sulphate precipitation) were evaluated for their impact on the quality, quantity and functional activity of purified rabbit and human Igs. The recovered Igs were analysed for yield and purity by SDS-PAGE, for quality by Ag-specific ELISAs (determining changes in titer, avidity and isotype distribution) and for functional activity by <it>in vitro </it>parasite growth inhibition assay (GIA).</p> <p>Results</p> <p>This comparison demonstrated that overall polyethylene glycol purification of human serum/plasma samples and protein G Sepharose purification of rabbit sera are optimal for recovering functional Ag-specific antibodies.</p> <p>Conclusion</p> <p>Consequently, critical consideration of the purification method is required to avoid selecting non-representative populations of recovered Ig, which could influence interpretations of vaccine efficacy, or affect the search for immune correlates of protection.</p

Comparison of phenotypic and genotypic tropism determination in triple-class-experienced HIV patients eligible for maraviroc treatment

BACKGROUND: Determination of HIV-1 tropism is a pre-requisite to the use of CCR5 antagonists. This study evaluated the potential of population genotypic tropism tests (GTTs) in clinical practice, and the correlation with phenotypic tropism tests (PTTs) in patients accessing routine HIV care. METHODS: Forty-nine consecutive plasma samples for which an original Trofile(TM) assay was performed were obtained from triple-class-experienced patients in need of a therapy change. Viral tropism was defined as the consensus of three or more tropism calls obtained from the combination of two independent population PTT assays (Trofile Biosciences, San Francisco, CA, USA, and Virco, Beerse, Belgium), population GTTs and GTTs based on ultra-deep sequencing. If no consensus was reached, a clonal PTT was performed in order to finalize the tropism call. This two-step approach allowed the definition of a reference tropism call. RESULTS: According to the reference tropism result, 35/49 samples were CCR5 tropic (R5) (patients eligible for maraviroc treatment) and 14/49 were assigned as non-R5 tropic. The non-R5 samples [patients not eligible for maraviroc treatment according to the FDA/European Medicines Agency (EMEA) label] group included both the CXCR4 (X4) samples and the dual and mixed CCR5/CXCR4 (R5/X4) samples. Compared with Trofile(TM) population PTTs, population GTTs showed a higher sensitivity (97%) and a higher negative predictive value (91%), but almost equal specificity and an equal positive predictive value. CONCLUSIONS: In line with recent reports from clinical trial data, our data support the use of population genotypic tropism testing as a tool for tropism determination before the start of maraviroc

Modeling dynamic interactions between pre-exposure prophylaxis interventions & treatment programs: predicting HIV transmission & resistance

Clinical trials have recently demonstrated the effectiveness of Pre-Exposure Prophylaxis (PrEP) in preventing HIV infection. Consequently, PrEP may soon be used for epidemic control. We model the dynamic interactions that will occur between treatment programs and potential PrEP interventions in resource-constrained countries. We determine the consequences for HIV transmission and drug resistance. We use response hypersurface modeling to predict the effect of PrEP on decreasing transmission as a function of effectiveness, adherence and coverage. We predict PrEP will increase need for second-line therapies (SLT) for treatment-naïve individuals, but could significantly decrease need for SLT for treatment-experienced individuals. If the rollout of PrEP is carefully planned it could increase the sustainability of treatment programs. If not, need for SLT could increase and the sustainability of treatment programs could be compromised. Our results show the optimal strategy for rolling out PrEP in resource-constrained countries is to begin around the “worst” treatment programs

Restricted Genetic Diversity of HIV-1 Subtype C Envelope Glycoprotein from Perinatally Infected Zambian Infants

Background: Mother-to-child transmission of HIV-1 remains a significant problem in the resource-constrained settings where anti-retroviral therapy is still not widely available. Understanding the earliest events during HIV-1 transmission and characterizing the newly transmitted or founder virus is central to intervention efforts. In this study, we analyzed the viral env quasispecies of six mother-infant transmission pairs (MIPs) and characterized the genetic features of envelope glycoprotein that could influence HIV-1 subtype C perinatal transmission. Methodology and Findings: The V1-V5 region of env was amplified from 6 MIPs baseline samples and 334 DNA sequences in total were analyzed. A comparison of the viral population derived from the mother and infant revealed a severe genetic bottleneck occurring during perinatal transmission, which was characterized by low sequence diversity in the infant. Phylogenetic analysis indicates that most likely in all our infant subjects a single founder virus was responsible for establishing infection. Furthermore, the newly transmitted viruses from the infant had significantly fewer potential N-linked glycosylation sites in Env V1-V5 region and showed a propensity to encode shorter variable loops compared to the nontransmitted viruses. In addition, a similar intensity of selection was seen between mothers and infants with a higher rate of synonymous (dS) compared to nonsynonymous (dN) substitutions evident (dN/dS\u3c1). Conclusions: Our results indicate that a strong genetic bottleneck occurs during perinatal transmission of HIV-1 subtype C. This is evident through population diversity and phylogenetic patterns where a single viral variant appears to be responsible for infection in the infants. As a result the newly transmitted viruses are less diverse and harbored significantly less glycosylated envelope. This suggests that viruses with the restricted glycosylation in envelope glycoprotein appeared to be preferentially transmitted during HIV-1 subtype C perinatal transmission. In addition, our findings also indicated that purifying selection appears to predominate in shaping the early intrahost evolution of HIV-1 subtype C envelope sequences