Use of laboratory-developed assays in global HIV-1 treatment-monitoring and research.

There has been a surge in the emergence of HIV-1 drug resistance in Low and Middle-Income Countries (LMICs) due to poor drug-adherence and limited access to viral load testing, the current standard for treatment-monitoring. It is estimated that only 75% of people living with HIV (PLWH) worldwide have access to viral load testing. In LMICs, this figure is below 50%. In a recent WHO survey in mostly LMICs, 21 out of 30 countries surveyed found HIV-1 first-line pre-treatment drug resistance in over 10% of study participants. In the worst-affected regions, up to 68% of infants born to HIV-1 positive mothers were found to harbour first-line HIV-1 treatment resistance. This is a huge public health concern. Greater access to treatment-monitoring is required in LMICs if the UNAIDS "third 95" targets are to be achieved by 2030. Here, we review the current challenges of viral load testing and present the case for greater utilization of Laboratory-based assays that quantify intracellular HIV-1 RNA and/or DNA to provide broader worldwide access to HIV-1 surveillance, drug-resistance monitoring, and cure-research.

A Novel Sample Selection Approach to Aid the Identification of Factors That Correlate With the Control of HIV-1 Infection.

Individuals infected with HIV display varying rates of viral control and disease progression, with a small percentage of individuals being able to spontaneously control infection in the absence of treatment. In attempting to define the correlates associated with natural protection against HIV, extreme heterogeneity in the datasets generated from systems methodologies can be further complicated by the inherent variability encountered at the population, individual, cellular and molecular levels. Furthermore, such studies have been limited by the paucity of well-characterised samples and linked epidemiological data, including duration of infection and clinical outcomes. To address this, we selected 10 volunteers who rapidly and persistently controlled HIV, and 10 volunteers each, from two control groups who failed to control (based on set point viral loads) from an acute and early HIV prospective cohort from East and Southern Africa. A propensity score matching approach was applied to control for the influence of five factors (age, risk group, virus subtype, gender, and country) known to influence disease progression on causal observations. Fifty-two plasma proteins were assessed at two timepoints in the 1st year of infection. We independently confirmed factors known to influence disease progression such as the B*57 HLA Class I allele, and infecting virus Subtype. We demonstrated associations between circulating levels of MIP-1α and IL-17C, and the ability to control infection. IL-17C has not been described previously within the context of HIV control, making it an interesting target for future studies to understand HIV infection and transmission. An in-depth systems analysis is now underway to fully characterise host, viral and immunological factors contributing to control.

Late-emerging strains of HIV induce T-cell homeostasis failure by promoting bystander cell death and immune exhaustion in naïve CD4 and all CD8 T-cells.

The mechanisms involved in the decline of CD4 and CD8 T-cells that lead to HIV-induced immune dysregulation are not clearly understood. We hypothesize that late-emerging strains of HIV, such as CXCR4-tropic (X4) virions, induce T-cell homeostasis failure by promoting significantly more bystander cell death, and immune exhaustion in naïve CD4 and all CD8 T-cells, when compared to strain of HIV, such as CCR5-tropic (R5) virions, found early during the course of infection. In the reported study, inactivated X4 virions induced greater bystander cell death in sort-purified naïve CD4 T-cells compared to R5 virions, which was significant (p=0.013), and in memory CD8 T-cells, though the latter was not significant. A clearer understanding of the mechanisms involved in HIV-induced depletion of T-cell numbers and function could lead to therapies that prevent T-cell death and restore immune function. These therapies could improve current anti-retroviral and cure-related treatments by boosting the immune system's own ability to combat the virus.

Discrepant amplification results during the development of an assay leads to reclassification of two AIDS reagent repository HIV-2 isolates as HIV-1.

The development and verification of HIV-2 assays depends in part on the availability of well-characterized samples, including those from reagent repositories. During the development of an HIV-2 RNA quantification assay, two HIV-2 viral isolates (CDC 301340 and CDC 301342) obtained from the NIAID AIDS Reagent and Reference Repository were not detected leading to an investigation. Two HIV-2 primers/probe sets of known performance in real-time viral RNA quantification assays, targeting different regions of the virus, also failed to generate RT-PCR products for these two isolates. These isolates were tested in the HIV-1 specific COBAS AmpliPrep/COBAS TaqMan HIV-1 Test v2.0 (Roche Molecular Diagnostics) and were quantified at high copy number. Other HIV-2 isolates tested were not amplified in the COBAS HIV-1 TaqMan assay. Furthermore, the discrepant isolates were highly reactive in an HIV-1 p24 antigen test while the other HIV-2 isolates showed very weak, if any, cross-reactivity with the HIV-1 p24 assay. Phylogenetic tree analysis of sequences from the protease-reverse transcriptase regions of the discrepant HIV-2 isolates mapped with HIV-1 Group M, Subtype CRF02_AG confirming these isolates were of HIV-1 origin and had been misclassified as HIV-2. The use of misclassified isolates in the verification of molecular and immunological assays can lead to misinterpretation of test results, misdirection of efforts into assay redesign and increased development costs. The results of this study were shared with the NIAID AIDS Reagent Program, leading to the reclassification of the two discrepant isolates as HIV-1.

The Use of Ultra-Sensitive Molecular Assays in HIV Cure-Related Research.

Ultra-sensitive laboratory assays based on the Polymerase Chain Reaction (PCR) are playing an increasingly important role in HIV cure-related research. This article reviews the different assays available and how they have evolved. There is a great need for their standardization and for the establishment of reference reagents and testing algorithms to evaluate potential HIV cure-related treatments.