Evaluation of the VioOne HIV profile supplemental assay.

HIV is an ongoing global epidemic with estimates of more than a million new infections occurring annually. To combat viral spread, continuous innovations in areas including testing and treatment are necessary. In the United States, the Centers for Disease Control and Prevention recommend that laboratories follow an HIV testing algorithm that first uses a US Food and Drug Administration approved immunoassay to detect antibodies to HIV-1 or HIV-2 as well as HIV-1 p24 antigen in serum or plasma samples. An initially reactive specimen is tested by a supplemental assay for confirmation and to differentiate antibodies to HIV-1 or HIV-2. There are few Food and Drug Administration (FDA)-approved supplemental differentiation tests currently available. A multicenter investigation was conducted to determine the clinical performance for two independent versions of the Avioq VioOne HIV Profile Supplemental Assay (Avioq, Inc., Research Triangle Park, NC). The performance of both assay versions compared favorably with the performance parameters for the Geenius HIV 1/2 Supplemental Assay as published in that assay package insert (Bio-Rad Laboratories, Hercules, CA), the current gold standard for HIV supplemental testing. When comparing the two VioOne assays, version 2 (lacking HIV-2 p27 antibody detection) demonstrated improved reproducibility, specificity, and sensitivity as compared to its predecessor. IMPORTANCE We evaluated the reproducibility, sensitivity, and specificity data for two versions of the VioOne HIV Profile Supplemental Assay and compared these results back to similar results for the Geenius HIV 1/2 Supplemental Assay that are publicly available. Our study concluded that the VioOne HIV Profile Supplemental Assay compared favorably with the Geenius HIV 1/2 Supplemental Assay, thus providing an additional option for clinical laboratories to improve and expand their HIV testing capabilities.

Sensitivity and specificity of the new Bio-Rad HIV screening test, Access HIV combo V2.

Diagnosing of human immunodeficiency virus (HIV) types 1 and 2 requires a screening with a highly sensitive and specific enzyme immunoassay and a low detection limit for the HIV-1 p24 antigen to minimize the diagnostic window. The objective of the study was to determine the sensitivity, specificity, and p24 limit of detection of the Access HIV combo V2 assay. Retrospective part of sensitivity: 452 HIV-1 positive samples from 403 chronic (9 different HIV-1 group M subtypes, 22 different HIV-1 group M CRFs, and 3 HIV-1 group O), 49 primary HIV-1 infections, 103 HIV-2 positive samples assessed at Pitié-Salpêtrière Hospital, 600 untyped HIV-1, 10 subtype-D, and 159 untyped HIV-2 samples assessed in Bio-Rad Laboratories. Prospective part of clinical specificity: all consecutive samples in two blood donor facilities and Pitié-Salpêtrière (6,570 patients) tested with Access HIV combo V2 and respectively Prism HIV O Plus (Abbott) or Architect HIV Ag/Ab Combo (Abbott) for Ag/Ab screening, and Procleix Ultrio (Gen Probe) for HIV RNA screening. Limit of detection for p24 antigen was assessed on recombinant virus-like particles (10 HIV-1 group M subtypes/CRFs, HIV-1 group O). Sensitivity [95% confidence interval (CI)] of Access HIV combo V2 was 100% (99.63-100) for HIV-1 chronic infection, 100% (98.55-100) for HIV-2 chronic infection, and 100% (93.00-100) for HIV-1 primary infection. Specificity (95% CI) was 99.98 (99.91-100). Limit of detection for p24 antigen was around 0.43 IU/mL [interquartile range (0.38-0.56)], and consistent across the 11 analyzed subtypes/CRFs. Hence, with both high sensitivity and specificity, Access HIV combo V2 is a suitable screening assay for HIV-1/2 infection. IMPORTANCE: Bio-Rad is one of the leading human immunodeficiency virus (HIV) screening test manufacturers. This laboratory released in 2021 their new version of the Access combo HIV test. However, to date, there have been no studies regarding its performance, especially its limit of detection of the diverse p24 antigen. We present the sensitivity (chronic and primary HIV-1 infection and HIV-2 chronic infection), specificity (blood donors and hospitalized patients), and raw data for the p24/seroconversion panels the manufacturer gave to the European agencies.

Direct single-molecule imaging for diagnostic and blood screening assays.

Every year, over 100 million units of donated blood undergo mandatory screening for HIV, hepatitis B, hepatitis C, and syphilis worldwide. Often, donated blood is also screened for human T cell leukemia-lymphoma virus, Chagas, dengue, Babesia, cytomegalovirus, malaria, and other infections. Several billion diagnostic tests are performed annually around the world to measure more than 400 biomarkers for cardiac, cancer, infectious, and other diseases. Considering such volumes, every improvement in assay performance and/or throughput has a major impact. Here, we show that medically relevant assay sensitivities and specificities can be fundamentally improved by direct single-molecule imaging using regular epifluorescence microscopes. In current microparticle-based assays, an ensemble of bound signal-generating molecules is measured as a whole. By contrast, we acquire intensity profiles to identify and then count individual fluorescent complexes bound to targets on antibody-coated microparticles. This increases the signal-to-noise ratio and provides better discrimination over nonspecific effects. It brings the detection sensitivity down to the attomolar (10-18 M) for model assay systems and to the low femtomolar (10-16 M) for measuring analyte in human plasma. Transitioning from counting single-molecule peaks to averaging pixel intensities at higher analyte concentrations enables a continuous linear response from 10-18 to 10-5 M. Additionally, our assays are insensitive to microparticle number and volume variations during the binding reaction, eliminating the main source of uncertainties in standard assays. Altogether, these features allow for increased assay sensitivity, wide linear detection ranges, shorter incubation times, simpler assay protocols, and minimal reagent consumption.

Poly I:C and STING agonist-primed DC increase lymphoid tissue polyfunctional HIV-1-specific CD8+ T cells and limit CD4+ T-cell loss in BLT mice.

Effective function of CD8+ T cells and enhanced innate activation of DCs in response to HIV-1 is linked to protective antiviral immunity in controllers. Manipulation of DC targeting the master regulator TANK-binding Kinase 1 (TBK1) might be useful to acquire controller-like properties. Here, we evaluated the impact of the combination of 2´3´-c´diAM(PS)2 and Poly I:C as potential adjuvants capable of potentiating DC´s abilities to induce polyfunctional HIV-1 specific CD8+ T-cell responses in vitro and in vivo using a humanized BLT mouse model. Adjuvant combination enhanced TBK-1 phosphorylation and IL-12 and IFN-ß expression on DC and increased their ability to activate polyfunctional HIV-1-specific CD8+ T cells in vitro. Moreover, higher proportions of hBLT mice vaccinated with ADJ-DC exhibited less severe CD4+ T-cell depletion following HIV-1 infection compared to control groups. This was associated with infiltration of CD8+ T cells in the white pulp from the spleen, reduced spread of infected p24+ cells to LN, and with preserved abilities of CD8+ T cells from the spleen and blood of vaccinated animals to induce specific polyfunctional responses upon antigen stimulation. Therefore, priming of DC with PolyI:C and STING agonists might be useful for future HIV-1 vaccine studies.

Detecting Sources of Immune Activation and Viral Rebound in HIV Infection.

Anti-retroviral therapy (ART) generally suppresses HIV replication to undetectable levels in peripheral blood, but immune activation associated with increased morbidity and mortality is sustained during ART, and infection rebounds when treatment is interrupted. To identify drivers of immune activation and potential sources of viral rebound, we modified RNAscope in situ hybridization to visualize HIV-producing cells as a standard against which to compare the following assays of potential sources of immune activation and virus rebound following treatment interruption: (i) envelope detection by induced transcription-based sequencing (EDITS) assay; (ii) HIV-Flow; (iii) Flow-FISH assays that can scan tissues and cell suspensions to detect rare cells expressing env mRNA, gag mRNA/Gag protein and p24; and (iv) an ultrasensitive immunoassay that detects p24 in cell/tissue lysates at subfemtomolar levels. We show that the sensitivities of these assays are sufficient to detect one rare HIV-producing/env mRNA+/p24+ cell in one million uninfected cells. These high-throughput technologies provide contemporary tools to detect and characterize rare cells producing virus and viral antigens as potential sources of immune activation and viral rebound. IMPORTANCE Anti-retroviral therapy (ART) has greatly improved the quality and length of life for people living with HIV, but immune activation does not normalize during ART, and persistent immune activation has been linked to increased morbidity and mortality. We report a comparison of assays of two potential sources of immune activation during ART: rare cells producing HIV and the virus' major viral protein, p24, benchmarked on a cell model of active and latent infections and a method to visualize HIV-producing cells. We show that assays of HIV envelope mRNA (EDITS assay), gag mRNA, and p24 (Flow-FISH, HIV-Flow. and ultrasensitive p24 immunoassay) detect HIV-producing cells and p24 at sensitivities of one infected cell in a million uninfected cells, thereby providing validated tools to explore sources of immune activation during ART in the lymphoid and other tissue reservoirs.

Bryostatin-1 Decreases HIV-1 Infection and Viral Production in Human Primary Macrophages.

While combination antiretroviral therapy maintains undetectable viremia in people living with HIV (PLWH), a lifelong treatment is necessary to prevent viremic rebound after therapy cessation. This rebound seemed mainly caused by long-lived HIV-1 latently infected cells reverting to a viral productive status. Reversing latency and elimination of these cells by the so-called shock-and-kill strategy is one of the main investigated leads to achieve an HIV-1 cure. Small molecules referred to as latency reversal agents (LRAs) proved to efficiently reactivate latent CD4+ T cells. However, the LRA impact on de novo infection or HIV-1 production in productively infected macrophages remains elusive. Nontoxic doses of bryostatin-1, JQ1, and romidepsin were investigated in human monocyte-derived macrophages (MDMs). Treatment with bryostatin-1 or romidepsin resulted in a downregulation of CD4 and CCR5 receptors, respectively, accompanied by a reduction of R5 tropic virus infection. HIV-1 replication was mainly regulated by receptor modulation for bryostatin-1, while romidepsin effects rely on upregulation of SAMHD1 activity. LRA stimulation of chronically infected cells did not enhance HIV-1 production or gene expression. Surprisingly, bryostatin-1 caused a major decrease in viral production. This effect was not viral strain specific but appears to occur only in myeloid cells. Bryostatin-1 treatment of infected MDMs led to decreased amounts of capsid and matrix mature proteins with little to no modulation of precursors. Our observations revealed that bryostatin-1-treated myeloid and CD4+ T cells respond differently upon HIV-1 infection. Therefore, additional studies are warranted to more fully assess the efficiency of HIV-1 eradicating strategies. IMPORTANCE HIV-1 persists in a cellular latent form despite therapy that quickly propagates infection upon treatment interruption. Reversing latency would contribute to eradicate these cells, closing the gap to a cure. Macrophages are an acknowledged HIV-1 reservoir during therapy and are suspected to harbor latency establishment in vivo. However, the impact of latency reversal agents (LRAs) on HIV-1 infection and viral production in human macrophages is poorly known but nonetheless crucial to probe the safety of this strategy. In this in vitro study, we discovered encouraging antireplicative features of distinct LRAs in human macrophages. We also described a new viral production inhibition mechanism by protein kinase C agonists that is specific to myeloid cells. This study provides new insights into HIV-1 propagation restriction potentials by LRAs in human macrophages and underline the importance of assessing latency reversal strategy on all HIV-1-targeted cells.

Plasmacytoid dendritic cells have divergent effects on HIV infection of initial target cells and induce a pro-retention phenotype.

Although HIV infection inhibits interferon responses in its target cells in vitro, interferon signatures can be detected in vivo soon after sexual transmission, mainly attributed to plasmacytoid dendritic cells (pDCs). In this study, we examined the physiological contributions of pDCs to early HIV acquisition using coculture models of pDCs with myeloid DCs, macrophages and the resting central, transitional and effector memory CD4 T cell subsets. pDCs impacted infection in a cell-specific manner. In myeloid cells, HIV infection was decreased via antiviral effects, cell maturation and downregulation of CCR5 expression. In contrast, in resting memory CD4 T cells, pDCs induced a subset-specific increase in intracellular HIV p24 protein expression without any activation or increase in CCR5 expression, as measured by flow cytometry. This increase was due to reactivation rather than enhanced viral spread, as blocking HIV entry via CCR5 did not alter the increased intracellular p24 expression. Furthermore, the load and proportion of cells expressing HIV DNA were restricted in the presence of pDCs while reverse transcriptase and p24 ELISA assays showed no increase in particle associated reverse transcriptase or extracellular p24 production. In addition, pDCs also markedly induced the expression of CD69 on infected CD4 T cells and other markers of CD4 T cell tissue retention. These phenotypic changes showed marked parallels with resident memory CD4 T cells isolated from anogenital tissue using enzymatic digestion. Production of IFNα by pDCs was the main driving factor for all these results. Thus, pDCs may reduce HIV spread during initial mucosal acquisition by inhibiting replication in myeloid cells while reactivating latent virus in resting memory CD4 T cells and retaining them for immune clearance.

In-house ELISA protocols for capsid p24 detection of diverse HIV isolates.

BACKGROUND: The capsid p24 (CA-p24) antigen is a component of the viral capsid of human immunodeficiency virus (HIV) that has been commonly used for clinical diagnosis and monitoring of HIV infections in Enzyme-linked Immunosorbent Assays (ELISAs). Commercial CA-p24 ELISAs are widely used in research settings, but these kits are costly and have limited breadth for detecting diverse HIV isolates. METHODS: Commercial CA-p24 antibodies were used as capture and detection antibodies. Specific CA-p24 ELISAs were established with these antibodies and tested for the detection of HIV-1 isolates with the aim of developing in-house protocols to recognize HIV-1 infections in vitro for research purposes. RESULTS: Here we present four protocols for in-house ELISAs to detect HIV CA-p24 using commercial antibodies. The assays were able to detect the CA-p24 antigen of different HIV-1 isolates tested. Comparison between the protocols showed that these in-house ELISAs exhibit high specificity, sensitivity, and reproducibility for CA-p24 quantitation but their reactivity varied per HIV-1 isolate and subtype. CONCLUSIONS: These optimized ELISA protocols represent valuable tools to investigate HIV-1 infections in research facilities at a lower price than commercial CA-p24 kits.

Dysfunctional HIV-specific CD8+ T cell proliferation is associated with increased caspase-8 activity and mediated by necroptosis.

Decreased HIV-specific CD8(+) T cell proliferation is a hallmark of chronic infection, but the mechanisms of decline are unclear. We analyzed gene expression profiles from antigen-stimulated HIV-specific CD8(+) T cells from patients with controlled and uncontrolled infection and identified caspase-8 as a correlate of dysfunctional CD8(+) T cell proliferation. Caspase-8 activity was upregulated in HIV-specific CD8(+) T cells from progressors and correlated positively with disease progression and programmed cell death-1 (PD-1) expression, but negatively with proliferation. In addition, progressor cells displayed a decreased ability to upregulate membrane-associated caspase-8 activity and increased necrotic cell death following antigenic stimulation, implicating the programmed cell death pathway necroptosis. In vitro necroptosis blockade rescued HIV-specific CD8(+) T cell proliferation in progressors, as did silencing of necroptosis mediator RIPK3. Thus, chronic stimulation leading to upregulated caspase-8 activity contributes to dysfunctional HIV-specific CD8(+) T cell proliferation through activation of necroptosis and increased cell death.

Mucosal Priming with a Recombinant Influenza A Virus-Vectored Vaccine Elicits T-Cell and Antibody Responses to HIV-1 in Mice.

Recombinant influenza A viral (IAV) vectors are potential to stimulate systemic and mucosal immunity, but the packaging capacity is limited and only one or a few epitopes can be carried. Here, we report the generation of a replication-competent IAV vector that carries a full-length HIV-1 p24 gene linked to the 5'-terminal coding region of the neuraminidase segment via a protease cleavage sequence (IAV-p24). IAV-p24 was successfully rescued and stably propagated, and P24 protein was efficiently expressed in infected mammalian cells. In BALB/c mice, IAV-p24 showed attenuated pathogenicity compared to that of the parental A/PR/8/34 (H1N1) virus. An intranasal inoculation with IAV-p24 elicited moderate HIV-specific cell-mediated immune (CMI) responses in the airway and vaginal tracts and in the spleen, and an intranasal boost with a replication-incompetent adenovirus type 2 vector expressing the HIV-1 gag gene (Ad2-gag) greatly improved these responses. Importantly, compared to an Ad2-gag prime plus IAV-p24 boost regimen, the IAV-p24 prime plus Ad2-gag boost regimen had a greater efficacy in eliciting HIV-specific CMI responses. P24-specific CD8+ T cells and antibodies were robustly provoked both systemically and in mucosal sites and showed long-term durability, revealing that IAV-p24 may be used as a mucosa-targeted priming vaccine. Our results illustrate that IAV-p24 is able to prime systemic and mucosal immunity against HIV-1 and warrants further evaluation in nonhuman primates.IMPORTANCE An effective HIV-1 vaccine remains elusive despite nearly 40 years of research. CD8+ T cells and protective antibodies may both be desirable for preventing HIV-1 infection in susceptible mucosal sites. Recombinant influenza A virus (IAV) vector has the potential to stimulate these immune responses, but the packaging capacity is extremely limited. Here, we describe a replication-competent IAV vector expressing the HIV-1 p24 gene (IAV-p24). Unlike most other IAV vectors that carried one or several antigenic epitopes, IAV-p24 stably expressed the full-length P24 protein, which contains multiple epitopes and is highly conserved among all known HIV-1 sequences. Compared to the parental A/PR/8/34 (H1N1) virus, IAV-p24 showed an attenuated pathogenicity in BALB/c mice. When combined with an adenovirus vector expressing the HIV-1 gag gene, IAV-p24 was able to prime P24-specific systemic and mucosal immune responses. IAV-p24 as an alternative priming vaccine against HIV-1 warrants further evaluation in nonhuman primates.

Ultrasensitive Detection of p24 in Plasma Samples from People with Primary and Chronic HIV-1 Infection.

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 limit its application for clinical monitoring. The development of ultrasensitive protein detection technologies may help in overcoming these limitations. Here, we evaluated whether immune complex dissociation combined with ultrasensitive digital enzyme-linked immunosorbent assay (ELISA) single-molecule array (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-positive (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 interferon alpha (IFN-α) 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 most having undetectable viral loads. These people had different virological and immunological baseline characteristics compared with people who had undetectable p24 after cART. These results demonstrate that ultrasensitive p24 analysis provides an efficient and robust means 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. IMPORTANCE The introduction of combined antiretroviral treatment has transformed HIV-1 infection into 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-life 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 makes its quantification difficult 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 those 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.

A de novo approach to inferring within-host fitness effects during untreated HIV-1 infection.

In the absence of effective antiviral therapy, HIV-1 evolves in response to the within-host environment, of which the immune system is an important aspect. During the earliest stages of infection, this process of evolution is very rapid, driven by a small number of CTL escape mutations. As the infection progresses, immune escape variants evolve under reduced magnitudes of selection, while competition between an increasing number of polymorphic alleles (i.e., clonal interference) makes it difficult to quantify the magnitude of selection acting upon specific variant alleles. To tackle this complex problem, we developed a novel multi-locus inference method to evaluate the role of selection during the chronic stage of within-host infection. We applied this method to targeted sequence data from the p24 and gp41 regions of HIV-1 collected from 34 patients with long-term untreated HIV-1 infection. We identify a broad distribution of beneficial fitness effects during infection, with a small number of variants evolving under strong selection and very many variants evolving under weaker selection. The uniquely large number of infections analysed granted a previously unparalleled statistical power to identify loci at which selection could be inferred to act with statistical confidence. Our model makes no prior assumptions about the nature of alleles under selection, such that any synonymous or non-synonymous variant may be inferred to evolve under selection. However, the majority of variants inferred with confidence to be under selection were non-synonymous in nature, and in most cases were have previously been associated with either CTL escape in p24 or neutralising antibody escape in gp41. We also identified a putative new CTL escape site (residue 286 in gag), and a region of gp41 (including residues 644, 648, 655 in env) likely to be associated with immune escape. Sites inferred to be under selection in multiple hosts have high within-host and between-host diversity although not all sites with high between-host diversity were inferred to be under selection at the within-host level. Our identification of selection at sites associated with resistance to broadly neutralising antibodies (bNAbs) highlights the need to fully understand the role of selection in untreated individuals when designing bNAb based therapies.

A Δ42PD1 fusion-expressing DNA vaccine elicits enhanced adaptive immune response to HIV-1: the key role of TLR4.

Since its discovery in the 1990s, the DNA vaccine has been of great interest because of its ability to elicit both humoral and cellular immune responses while showing relative advantages regarding producibility, stability and storage. However, when applied to human subjects, inadequate immunogenicity remains as the greatest challenge for the practical use of DNA vaccines. In this study, we generated a DNA vaccine Δ42PD1-P24 encoding a fusion protein comprised of the HIV-1 Gag p24 antigen and the extracellular domain of murine Δ42PD1, a novel endogenous Toll-like receptor 4 (TLR4) agonist. Using a mouse model, we found that Δ42PD1-P24 DNA vaccine elicited a higher antibody response and an increased number of IFN-γ-producing CD4 and CD8 T cells. Moreover, mice with Δ42PD1-P24 DNA vaccination were protected from a subcutaneous challenge with murine mesothelioma cells expressing the HIV-1 p24 antigen. Importantly, the Δ42PD1-mediated enhancement of immune responses was not observed in TLR4 knockout mice. Collectively, these data demonstrate that the immunogenicity and efficacy of DNA vaccines could be improved by the fusion of the extracellular domain of Δ42PD1 to target the immunogen to dendritic cells.

A molecular basis for the control of preimmune escape variants by HIV-specific CD8+ T cells.

The capacity of the immune system to adapt to rapidly evolving viruses is a primary feature of effective immunity, yet its molecular basis is unclear. Here, we investigated protective HIV-1-specific CD8+ T cell responses directed against the immunodominant p24 Gag-derived epitope KK10 (KRWIILGLNK263-272) presented by human leukocyte antigen (HLA)-B∗2705. We found that cross-reactive CD8+ T cell clonotypes were mobilized to counter the rapid emergence of HIV-1 variants that can directly affect T cell receptor (TCR) recognition. These newly recruited clonotypes expressed TCRs that engaged wild-type and mutant KK10 antigens with similar affinities and almost identical docking modes, thereby accounting for their antiviral efficacy in HLA-B∗2705+ individuals. A protective CD8+ T cell repertoire therefore encompasses the capacity to control TCR-accessible mutations, ultimately driving the development of more complex viral escape variants that disrupt antigen presentation.

Universal amplification-free molecular diagnostics by billion-fold hierarchical nanofluidic concentration.

Rapid and reliable detection of ultralow-abundance nucleic acids and proteins in complex biological media may greatly advance clinical diagnostics and biotechnology development. Currently, nucleic acid tests rely on enzymatic processes for target amplification (e.g., PCR), which have many inherent issues restricting their implementation in diagnostics. On the other hand, there exist no protein amplification techniques, greatly limiting the development of protein-based diagnosis. We report a universal biomolecule enrichment technique termed hierarchical nanofluidic molecular enrichment system (HOLMES) for amplification-free molecular diagnostics using massively paralleled and hierarchically cascaded nanofluidic concentrators. HOLMES achieves billion-fold enrichment of both nucleic acids and proteins within 30 min, which not only overcomes many inherent issues of nucleic acid amplification but also provides unprecedented enrichment performance for protein analysis. HOLMES features the ability to selectively enrich target biomolecules and simultaneously deplete nontargets directly in complex crude samples, thereby enormously enhancing the signal-to-noise ratio of detection. We demonstrate the direct detection of attomolar nucleic acids in urine and serum within 35 min and HIV p24 protein in serum within 60 min. The performance of HOLMES is comparable to that of nucleic acid amplification tests and near million-fold improvement over standard enzyme-linked immunosorbent assay (ELISA) for protein detection, being much simpler and faster in both applications. We additionally measured human cardiac troponin I protein in 9 human plasma samples, and showed excellent agreement with ELISA and detection below the limit of ELISA. HOLMES is in an unparalleled position to unleash the potential of protein-based diagnosis.

Gag p24 Is a Marker of Human Immunodeficiency Virus Expression in Tissues and Correlates With Immune Response.

We demonstrate that human immunodeficiency virus (HIV) gag p24 protein is more readily detected in gut and lymph node tissues than in blood CD4+ T cells and correlates better with CD4 count during antiretroviral therapy (ART). Gut p24 levels also measurably decline with ART in natural controllers. During ART, gut p24 expression is more strongly associated both with HIV-specific CD8+ T-cell frequency and plasma soluble CD14 levels than gut HIV RNA expression. This study supports using gag p24 as a marker of HIV expression in HIV+ tissues to study effects of viral persistence and to monitor efficacy of treatment in HIV-based clearance studies.

Impact of HLA-B*52:01-Driven Escape Mutations on Viral Replicative Capacity.

HLA-B*52:01 is strongly associated with protection against HIV disease progression. However, the mechanisms of HLA-B*52:01-mediated immune control have not been well studied. We here describe a cohort with a majority of HIV C-clade-infected individuals from Delhi, India, where HLA-B*52:01 is highly prevalent (phenotypic frequency, 22.5%). Consistent with studies of other cohorts, expression of HLA-B*52:01 was associated with high absolute CD4 counts and therefore a lack of HIV disease progression. We here examined the impact of HLA-B*52:01-associated viral polymorphisms within the immunodominant C clade Gag epitope RMTSPVSI (here, RI8; Gag residues 275 to 282) on viral replicative capacity (VRC) since HLA-mediated reduction in VRC is a central mechanism implicated in HLA-associated control of HIV. We observed in HLA-B*52:01-positive individuals a higher frequency of V280T, V280S, and V280A variants within RI8 (P = 0.0001). Each of these variants reduced viral replicative capacity in C clade viruses, particularly the V280A variant (P < 0.0001 in both the C clade consensus and in the Indian study cohort consensus p24 Gag backbone), which was also associated with significantly higher absolute CD4 counts in the donors (median, 941.5 cells/mm3; P = 0.004). A second HLA-B*52:01-associated mutation, K286R, flanking HLA-B*52:01-RI8, was also analyzed. Although selected in HLA-B*52:01-positive subjects often in combination with the V280X variants, this mutation did not act as a compensatory mutant but, indeed, further reduced VRC. These data are therefore consistent with previous work showing that HLA-B molecules that are associated with immune control of HIV principally target conserved epitopes within the capsid protein, escape from which results in a significant reduction in VRC.IMPORTANCE Few studies have addressed the mechanisms of immune control in HIV-infected subjects in India, where an estimated 2.7 million people are living with HIV. We focus here on a study cohort in Delhi on one of the most prevalent HLA-B alleles, HLA-B*52:01, present in 22.5% of infected individuals. HLA-B*52:01 has consistently been shown in other cohorts to be associated with protection against HIV disease progression, but studies have been limited by the low prevalence of this allele in North America and Europe. Among the C-clade-infected individuals, we show that HLA-B*52:01 is the most protective of all the HLA-B alleles expressed in the Indian cohort and is associated with the highest absolute CD4 counts. Further, we show that the mechanism by which HLA-B*52:01 mediates immune protection is, at least in part, related to the inability of HIV to evade the HLA-B*52:01-restricted p24 Gag-specific CD8+ T-cell response without incurring a significant loss to viral replicative capacity.

Single-cell characterization and quantification of translation-competent viral reservoirs in treated and untreated HIV infection.

The phenotypic characterization of the cells in which HIV persists during antiretroviral therapy (ART) remains technically challenging. We developed a simple flow cytometry-based assay to quantify and characterize infected cells producing HIV proteins during untreated and treated HIV infection. By combining two antibodies targeting the HIV capsid in a standard intracellular staining protocol, we demonstrate that p24-producing cells can be detected with high specificity and sensitivity in the blood from people living with HIV. In untreated individuals, the frequency of productively infected cells strongly correlated with plasma viral load. Infected cells preferentially displayed a transitional memory phenotype and were enriched in Th17, peripheral Tfh and regulatory T cells subsets. These cells also preferentially expressed activation markers (CD25, HLA-DR, Ki67), immune checkpoint molecules (PD-1, LAG-3, TIGIT, Tim-3) as well as the integrins α4ß7 and α4ß1. In virally suppressed individuals on ART, p24-producing cells were only detected upon stimulation (median frequency of 4.3 p24+ cells/106 cells). These measures correlated with other assays assessing the size of the persistent reservoir including total and integrated HIV DNA, Tat/rev Induced Limiting Dilution Assay (TILDA) and quantitative viral outgrowth assay (QVOA). In ART-suppressed individuals, p24-producing cells preferentially displayed a transitional and effector memory phenotype, and expressed immune checkpoint molecules (PD-1, TIGIT) as well as the integrin α4ß1. Remarkably, α4ß1 was expressed by more than 70% of infected cells both in untreated and ART-suppressed individuals. Altogether, these results highlight a broad diversity in the phenotypes of HIV-infected cells in treated and untreated infection and suggest that strategies targeting multiple and phenotypically distinct cellular reservoirs will be needed to exert a significant impact on the size of the reservoir.

Antigen processing influences HIV-specific cytotoxic T lymphocyte immunodominance.

Although cytotoxic T lymphocytes (CTLs) in people infected with human immunodeficiency virus type 1 can potentially target multiple virus epitopes, the same few are recognized repeatedly. We show here that CTL immunodominance in regions of the human immunodeficiency virus type 1 group-associated antigen proteins p17 and p24 correlated with epitope abundance, which was strongly influenced by proteasomal digestion profiles, affinity for the transporter protein TAP, and trimming mediated by the endoplasmatic reticulum aminopeptidase ERAAP, and was moderately influenced by HLA affinity. Structural and functional analyses demonstrated that proteasomal cleavage 'preferences' modulated the number and length of epitope-containing peptides, thereby affecting the response avidity and clonality of T cells. Cleavage patterns were affected by both flanking and intraepitope CTL-escape mutations. Our analyses show that antigen processing shapes CTL response hierarchies and that viral evolution modifies cleavage patterns and suggest strategies for in vitro vaccine optimization.

HIV-1 evades virus-specific IgG2 and IgA responses by targeting systemic and intestinal B cells via long-range intercellular conduits.

Contact-dependent communication between immune cells generates protection but also facilitates viral spread. Here we found that macrophages formed long-range actin-propelled conduits in response to negative factor (Nef), a human immunodeficiency virus type 1 (HIV-1) protein with immunosuppressive functions. Conduits attenuated immunoglobulin G2 (IgG2) and IgA class switching in systemic and intestinal lymphoid follicles by shuttling Nef from infected macrophages to B cells through a guanine-exchange factor-dependent pathway involving the amino-terminal anchor, central core and carboxy-terminal flexible loop of Nef. By showing stronger virus-specific IgG2 and IgA responses in patients with Nef-deficient virions, our data suggest that HIV-1 exploits intercellular 'highways' as a 'Trojan horse' to deliver Nef to B cells and evade humoral immunity systemically and at mucosal sites of entry.