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HIV-1 Gag p24 has long been identified as an informative biomarker of HIV replication, disease progression and therapeutic efficacy, but the lower sensitivity of immunoassays in comparison to molecular tests and the interference with antibodies in chronic HIV infection limits its application for clinical monitoring. The development of ultrasensitive protein detection technologies may help overcoming these limitations. Here we evaluated whether immune-complex dissociation combined with ultrasensitive digital ELISA Simoa technology could be used to quantify p24 in plasma samples from people with HIV-1 infection. We found that, among different immune-complex dissociation methods, only acid-mediated dissociation was compatible with ultrasensitive p24 quantification by digital ELISA, strongly enhancing p24 detection at different stages of HIV-1 infection. We show that ultrasensitive p24 levels correlated positively with plasma HIV-RNA and HIV-DNA and negatively with CD4+ T cells in the samples from people with primary and chronic HIV-1 infection. In addition, p24 levels also correlated with plasma D-dimers and IFNα levels. P24 levels sharply decreased to undetectable levels after initiation of combined antiretroviral treatment (cART). However, we identified a group of people who, 48 weeks after cART initiation, had detectable p24 levels despite having undetectable viral loads. These people had different virologic and immunologic baseline characteristics when compared with people who had undetectable p24 after cART. These results demonstrate that ultrasensitive p24 analysis provides an efficient and robust mean to monitor p24 antigen in plasma samples from people with HIV-1 infection, including during antiretroviral treatment, and may provide complementary information to other commonly used biomarkers.ImportanceThe introduction of combined antiretroviral treatment has transformed HIV-1 infection in a manageable condition. In this context, there is a need for additional biomarkers to monitor HIV-1 residual disease or the outcome of new interventions, such as in the case of HIV cure strategies. The p24 antigen has a long half-live outside viral particles and it is therefore a very promising marker to monitor episodes of viral replication or transient activation of the viral reservoir. However, the formation of immune-complexes with anti-p24 antibodies difficult its quantification beyond acute HIV-1 infection. We show here that, upon immune-complex dissociation, new technologies allow the ultrasensitive p24 quantification in plasma samples throughout HIV-1 infection, at levels close to that of viral RNA and DNA determinations. Our results further indicate that ultrasensitive p24 quantification may have added value when used in combination with other classic clinical biomarkers.

For over a decade, multiple studies have disputed the notion of natural killer (NK) cells as purely innate lymphocytes by demonstrating that they are capable of putative antigen-specific immunological memory against multiple infectious agents including two critical global health priorities – HIV and influenza. However, the mechanisms underlying antigen specificity remain unknown. Herein, we demonstrate that antigen-specific human NK cell memory develops upon exposure to both HIV and influenza, unified by a conserved and epitope-specific targetable mechanism firmly dependent on the activating CD94/NKG2C receptor and its ligand HLA-E, and confirm these findings by three rigorous and novel assays. We validated the permanent acquisition of antigen-specificity by individual memory NK cells by single-cell cloning. We identified biomarkers of antigen-specific NK cell memory through RNA-Seq transcriptomic fingerprints and complex immunophenotyping by 30-parameter flow cytometry showing elevated expression of KLRG1, α4β7 and NKG2C. Finally, we show individual HLA-E-restricted peptides that may constitute the dominant response in HIV-1- and influenza-infected persons in vivo. Our findings clarify the mechanisms behind formation of antigen-specific memory NK cells, and suggest they could be targeted for future vaccines, cure strategies, or other therapeutic interventions.

The Infinium Human Methylation450 and Methylation EPIC BeadChips are useful tools for the study of the methylation state of hundreds of thousands of CpG across the human genome at affordable cost. However, in a wide range of experimental settings in particular for studies in infectious or brain-related diseases, human samples cannot be easily obtained. Hence, due to their close developmental, immunological and neurological proximity with humans, non-human primates are used in many research fields of human diseases and for preclinical research. Few studies have used DNA methylation microarrays in simian models. Microarrays designed for the analysis of DNA methylation patterns in the human genome could be useful given the genomic proximity between human and nonhuman primates. However, there is currently information lacking about the specificity and usability of each probe for many nonhuman primate species, including rhesus macaques (Macaca mulatta), originating from Asia, and African green monkeys originating from West-Africa (Chlorocebus sabaeus). Rhesus macaques and African green monkeys are among the major nonhuman primate models utilized in biomedical research. Here, we provide a precise evaluation and re-annotation of the probes of the two microarrays for the analysis of genome-wide DNA methylation patterns in these two Cercopithecidae species. We demonstrate that up to 162,000 of the 450K and 255,000 probes of the EPIC BeadChip can be reliably used in Macaca mulatta or Chlorocebus sabaeus. The annotation files are provided in a format compatible with a variety of preprocessing, normalization and analytical pipelines designed for data analysis from 450K/EPIC arrays, facilitating high-throughput DNA methylation analyses in Macaca mulatta and Chlorocebus sabaeus. They provide the opportunity to the research community to focus their analysis only on those probes identified as reliable. The described analytical workflow leaves the choice to the user to balance coverage versus specificity and can also be applied to other Cercopithecidae species.

HIV-1 successfully establishes long-term infection in its target cells despite viral cytotoxic effects. We have recently shown that cell metabolism is an important factor driving CD4+ T-cell susceptibility to HIV-1 and the survival of infected cells. We show here that expression of anti-apoptotic clone 11 (AAC-11), an anti-apoptotic factor upregulated in many cancers, increased with progressive CD4+ T cell memory differentiation in association with the expression of cell cycle, activation and metabolism genes and correlated with susceptibility to HIV-1 infection. Synthetic peptides based on the LZ domain sequence of AAC-11, responsible for its interaction with molecular partners, were previously shown to be cytotoxic to cancer cells. Here we observed that these peptides also blocked HIV-1 infection by inducing cell death of HIV-1 susceptible primary CD4+ T-cells across all T-cell subsets. The peptides targeted metabolically active cells and had the greatest effect on effector and transitional CD4+ T cell memory subsets. Our results suggest that AAC-11 survival pathway is potentially involved in the survival of HIV-1 infectable cells and provide a proof of principle that some cellular characteristics can be targeted to eliminate the cells offering the best conditions to sustain HIV-1 replication.IMPORTANCEAlthough antiretroviral treatment efficiently blocks HIV multiplication, it cannot eliminate the cells already carrying integrated proviruses. In the search for a HIV cure the identification of new potential targets to selectively eliminate infected cells is of the outmost importance. We show here that peptides derived from the anti-apoptotic clone 11 (AAC-11), which expression levels correlated with susceptibility to HIV-1 infection of CD4+ T-cells, induced cytotoxicity in CD4+ T-cells showing the highest levels of activation and metabolic activity, conditions known to favor HIV-1 infection. Accordingly, CD4+ T-cells that survived the cytotoxic action of the AAC-11 peptides were resistant to HIV-1 replication. Our results identify a new potential molecular pathway to target HIV-1 infection.