Guoxin Wu

The HIV-1 envelope glycoprotein gp120 is prominently exposed on the surface of both HIV-1 virions and infected host cells, serving as a key marker of infection. gp120 plays a pivotal role in viral entry by interacting with the primary receptor, CD4, on host cells. Therapeutic strategies targeting the HIV-1 reservoir, such as anti-gp120 antibodies that trigger antibody-dependent cellular cytotoxicity (ADCC) and chimeric antigen receptor T (CAR-T) cells, rely on the presence of gp120 on the surface of infected cells to exert their effects. Consequently, accurate monitoring of gp120 expression on infected cells is essential for evaluating the pharmacological efficacy of these interventions. In this study, a sensitive, specific, and inexpensive enzyme-linked immunosorbent assay (ELISA) for quantifying HIV-1 gp120 glycoprotein was developed using a selected pair of anti-gp120 antibodies. The assay achieved a lower limit of quantitation (LLOQ) of 0.16 pM, demonstrating sensitivity comparable to that of the digital single molecule array (Simoa) platform, which exhibited a LLOQ of 0.23 pM and requires specialized instrumentation. The binding specificity of the antibodies used in the novel assay was confirmed using liquid chromatography–mass spectrometry (LC-MS), and the assay was pharmacologically validated with lysates obtained from 2D10 and MOLT IIIB cell lines. Furthermore, treatment of HIV-infected human primary CD4⁺ T cells with a targeted activator of cell kill (TACK) compound significantly reduced gp120 concentration in CD4⁺ T cell lysate compared to controls. The gp120 marker from infected cell lysates correlated with the number of gp120-positive cells detected by immunocytochemistry, as well as with HIV-1 p24 levels and cell-associated viral RNA measurements. In summary, a novel, simple, and sensitive HIV-1 gp120 ELISA has been developed and validated. This assay holds potential for investigating HIV-1 persistence and evaluating the efficacy of therapeutic agents targeting infected cells.

Despite the success of combination antiretroviral therapy (cART) to suppress HIV replication, HIV persists in a long-lived reservoir that can give rise to rebounding viremia upon cART cessation. The translationally active reservoir consists of HIV-infected cells that continue to produce viral proteins even in the presence of cART. These active reservoir cells are implicated in the resultant viremia upon cART cessation and likely contribute to chronic immune activation in people living with HIV (PLWH) on cART. Methodologies to quantify the active reservoir are needed. Here, an automated immunocytochemistry (ICC) assay coupled with computational image analysis to detect and quantify intracellular Gag capsid protein (CA) is described (CA-ICC). For this purpose, fixed cells were deposited on microscopy slides by the cytospin technique and stained with antibodies against CA by an automated stainer, followed by slide digitization. Nuclear staining was used to count the number of cells in the specimen, and the chromogenic signal was quantified to determine the percentage of CA-positive cells. In comparative analyses, digital ELISA, qPCR, and flow cytometry were used to validate CA-ICC. The specificity and sensitivity of CA-ICC were assessed by staining a cell line that expresses CA (MOLT IIIB) alongside a control cell line (Jurkat) devoid of this marker, as well as peripheral blood mononuclear cells (PBMCs) from HIV seronegative donors before or after ex vivo infection with an HIV laboratory strain. The sensitivity of CA-ICC was further assayed by spiking MOLT IIIB cells into uninfected Jurkat cells in limiting dilutions. In those analyses, CA-ICC could detect down to 10 CA-positive cells per million with a sensitivity superior to flow cytometry. To demonstrate the application of CA-ICC in pre-clinical research, bulk PBMCs obtained from mouse and non-human primate animal models were stained to detect HIV CA and SIV p27, respectively. The level of intracellular CA quantified by CA-ICC in PBMCs obtained from animal models was associated with plasma viral loads and cell-associated CA measured by qPCR and ELISA, respectively. The application of CA-ICC to evaluate the activity of small-molecule targeted activator of cell-kill (TACK) in clinical specimens is presented. Overall, CA-ICC offers a simple imaging method for specific and sensitive detection of CA-positive cells in bulk cell preparations.

Background: Latency remains a major obstacle to finding a cure for HIV despite the availability of antiretroviral therapy. Due to virus dormancy, limited biomarkers are available to identify latent HIV-infected cells. Profiling of individual HIV-infected cells is needed to explore potential latency biomarkers and to study the mechanisms of persistence that maintain the HIV reservoir. Methods: Single cell spatial transcriptomic characterization using the CosMx Spatial Molecular Imager platform was conducted to analyze HIV-infected cells in formalin-fixed paraffin-embedded sections of splenic tissue surgically obtained from an HIV-infected humanized mouse model. Regulation of over a thousand human genes was quantified in both viremic and aviremic specimens. In addition, in situ hybridization and immunohistochemistry were performed in parallel to identify HIV viral RNA- and p24-containing cells, respectively. Finally, initial findings from CosMx gene profiling were confirmed by isolating RNA from CD4 + T cells obtained from a person living with HIV on antiretroviral therapy following either PMA/Ionomycin or DMSO treatment. RNA was quantified using qPCR for a panel of targeted human host genes. Results: Supervised cell typing revealed that most of the HIV-infected cells in the mouse spleen sections were differentiated CD4 + T cells. A significantly higher number of infected cells, 2781 (1.61%) in comparison to 112 (0.06%), and total HIV transcripts per infected cell were observed in viremic samples compared to aviremic samples, respectively, which was consistent with the data obtained from ISH and IHC. Notably, the expression of 55 genes was different in infected cells within tissue from aviremic animals compared to viremic. In particular, both spleen tyrosine kinase (SYK) and CXCL17, were expressed approximately 100-fold higher. This data was further evaluated against bulk RNA isolated from HIV-infected human primary CD4 + T cells. A nearly 6-fold higher expression of SYK mRNA was observed in DMSO-treated CD4 + T cells compared to those stimulated with PMA/Ionomycin. Conclusion: This study found that the CosMx SMI platform is valuable for assessing HIV infection and providing insights into host biomarkers associated with HIV reservoirs. Higher relative expression of the SYK gene in aviremic-infected cells from the humanized mouse HIV model was consistent with levels found in CD4 + T cells of aviremic donors.

Promising therapeutic approaches for eradicating HIV include transcriptional activation of provirus from latently infected cells using latency-reversing agents (LRAs) and immune-mediated clearance to purge reservoirs. Accurate detection of cells capable of producing viral antigens and virions, and the measurement of clearance of infected cells, is essential to assessing therapeutic efficacy. Here, we apply enhanced methodology extending the sensitivity limits for the rapid detection of subfemtomolar HIV gag p24 capsid protein in CD4 ⁺ T cells from ART-suppressed HIV ⁺ individuals, and we show viral protein induction following treatment with LRAs. Importantly, we demonstrate that clinical administration of histone deacetylase inhibitors (HDACis; vorinostat and panobinostat) induced HIV gag p24, and ex vivo stimulation produced sufficient viral antigen to elicit immune-mediated cell killing using anti-gp120/CD3 bispecific antibody. These findings extend beyond classical nucleic acid endpoints, which are confounded by the predominance of mutated, defective proviruses and, of paramount importance, enable assessment of cells making HIV protein that can now be targeted by immunological approaches.

The latent reservoir in resting CD4+ T cells presents a major barrier to HIV cure. Latency-reversing agents are therefore being developed with the ultimate goal of disrupting the latent state, resulting in induction of HIV expression and clearance of infected cells. Histone deacetylase inhibitors (HDACi) have received a significant amount of attention for their potential as latency-reversing agents. Here, we have investigated the in vitro and systemic in vivo effect of panobinostat, a clinically relevant HDACi, on HIV latency. We showed that panobinostat induces histone acetylation in human PBMCs. Further, we showed that panobinostat induced HIV RNA expression and allowed the outgrowth of replication-competent virus ex vivo from resting CD4+ T cells of HIV-infected patients on suppressive antiretroviral therapy (ART). Next, we demonstrated that panobinostat induced systemic histone acetylation in vivo in the tissues of BLT humanized mice. Finally, in HIV-infected, ART-suppressed BLT mice, we evaluated the effect of panobinostat on systemic cell-associated HIV RNA and DNA levels and the total frequency of latently infected resting CD4+ T cells. Our data indicate that panobinostat treatment resulted in systemic increases in cellular levels of histone acetylation, a key biomarker for in vivo activity. However, panobinostat did not affect the levels of cell-associated HIV RNA, HIV DNA, or latently infected resting CD4+ T cells. We have demonstrated robust levels of systemic histone acetylation after panobinostat treatment of BLT humanized mice; and we did not observe a detectable change in the levels of cell-associated HIV RNA, HIV DNA, or latently infected resting CD4+ T cells in HIV-infected, ART-suppressed BLT mice. These results are consistent with the modest effects noted in vitro and suggest that combination therapies may be necessary to reverse latency and enable clearance. Animal models will contribute to the progress towards an HIV cure.

Reduction in cerebrospinal fluid (CSF) amyloid β42 (Aβ42) and elevation in total tau and phospho‐thr181 tau consistently differentiate between Alzheimer's disease (AD) and age‐matched control subjects. In contrast, CSF β‐site APP‐cleaving enzyme activity (BACE1) and soluble amyloid precursor proteins α and β (sAPPα and sAPPβ) are without consistent patterns in AD subjects. Plasma sampling is much easier, with fewer side effects, and is readily applied in primary care centers, so we have developed and validated novel plasma BACE activity, sAPPβ, and sAPPα assays and investigated their ability to distinguish AD from age‐matched controls. Plasma BACE activity assay was sensitive and specific, with signal being immunodepleted with a specific BACE1 antibody and inhibited with a BACE1‐specific inhibitor. Plasma sAPPβ and sAPPα assays were specific, with signal diluting linearly, immunodepleted with specific antibodies, and at background levels in APP knockout mice. In rhesus monkeys, BACE1 but not γ‐secretase inhibitor led to significant lowering of plasma sAPPβ with concurrent elevation of plasma sAPPα. AD subjects showed a significant increase in plasma BACE1 activity, sAPPβ, sAPPα, and Aβ42 (P < 0.001) compared with age‐matched controls. In conclusion, plasma BACE activity and sAPP endpoints provide novel investigative biomarkers for AD diagnosis and potential pharmacodynamic biomarkers for secretase inhibitor studies.

We have previously described a novel artificial NFEV β-secretase (BACE1) cleavage site, which when introduced into the amyloid-β precursor protein (APP), significantly enhances APP cleavage by BACE1 in in vitro and cellular assays. In this study, we describe the identification and characterization of a single chain fragment of variable region (scFv), specific to the EV neo-epitope derived from BACE1 cleavage of the NFEV-containing peptide, and its conversion to IgG1. Both the scFv displayed on phage and EV-IgG1 show exquisite specificity for binding to the EV neoepitope without cross-reactivity to other NFEV containing peptides or WT-APP KMDA cleavage products. EV-IgG1 can detect as little as 0.3 nmol/L of the EV peptide. EV-IgG1 antibody was purified, conjugated with alkaline phosphatase and utilized in various biological assays. In the BACE1 enzymatic assay using NFEV substrate, a BACE1 inhibitor MRK-3 inhibited cleavage with an IC₅₀ of 2.4 nmol/L with excellent reproducibility. In an APP_NFEV stable SH-SY5Y cellular assay, the EC₅₀ for inhibition of EV-Aβ peptide secretion with MRK-3 was 236 nmol/L, consistent with values derived using an EV polyclonal antibody. In an APP_NFEV knock-in mouse model, both Aβ_EV40 and Aβ_EV42 peptides in brain homogenate showed excellent gene dosage dependence. In conclusion, the EV neoepitope specific monoclonal antibody is a novel reagent for BACE1 inhibitor discovery for both in vitro, cellular screening assays and in vivo biochemical studies. The methods described herein are generally applicable to novel synthetic substrates and enzyme targets to enable robust screening platforms for enzyme inhibitors.

Amyloid-β peptide (Aβ) is generated by sequential cleavage of the amyloid precursor protein (APP) by β-site amyloid precursor protein cleaving enzyme 1 (β-secretase, or BACE1) and γ-secretase. Several reports demonstrate increased BACE1 enzymatic activity in brain and cerebrospinal fluid (CSF) from Alzheimer's disease (AD) subjects, suggesting that an increase in BACE1-mediated cleavage of APP drives amyloid pathophysiology in AD. BACE1 cleavage of APP leads to the generation of a secreted N-terminal fragment of APP (sAPPβ). To relate BACE1 activity better to endogenous APP processing in AD and control brains, we have directly measured brain sAPPβ levels using a novel APP β-site specific enzyme-linked immunosorbent assay. We demonstrate a significant reduction in brain cortical sAPPβ levels in AD compared with control subjects. In the same brain samples, BACE1 activity was unchanged, full-length APP and sAPPα levels were significantly reduced, and Aβ peptides were significantly elevated. In conclusion, a reduction in cortical brain sAPPβ together with unchanged BACE1 activity suggests that this is due to reduced full-length APP substrate in late-stage AD subjects. These results highlight the need for multiparameter analysis of the amyloidogenic process to understand better AD pathophysiology in early vs. late-stage AD.