Targeting HIV Reservoirs with Engineered T Cells
Latent viral reservoirs in tissues remain a considerable obstacle to achieve HIV cure. We are exploring how engineered immune cells affect viral reservoir dynamics and can help eliminate persistent HIV reservoirs. We have developed chimeric antigen receptor (CAR) T cells designed to recognize and kill PD-1⁺ T follicular helper cells that harbor the virus deep within germinal centers in lymphoid tissues. Our preclinical work in nonhuman primates shows promise, with these CAR T cells successfully reaching germinal centers, killing PD-1+ T follicular helper cells and reducing viral RNA. However, the approach also revealed challenges, including unintended effects like depletion of memory CD8⁺ T cells, and, related to the loss of antiviral cellular immunity, disease progression.
To address these hurdles, ongoing projects are refining the technology using synthetic biology tools —such as drug-inducible ON switches—to create safer, more precise CAR T cells. The ultimate goal is to develop a controllable, targeted therapy that can clear HIV reservoirs and move closer to a functional cure.
Tissue-restricted antibody production as a mechanism controlling reactivation of herpes simplex virus: implications for vaccine design and therapy 
One of the enigmas of the HSV field is the absence of correlation between systemic titers of neutralizing antibodies (nAbs) and infection control. Current understanding of humoral immunity to HSV-2 is based on bulk metrics of circulating antibodies, however genital tissue represents the main battleground between HSV-2 shedding from neurons and host immune system, hence representing a unique immunological niche. Our premise is that studying local humoral responses will reveal critical, currently unidentified modalities and mechanisms for immune control of HSV-2. Although clearance and subsequent control of reactivating HSV-2 in tissue are believed to depend on T cells, clinical observations as well as experimental and mathematical models continue to present contradictory evidence, consistent with functional roles for local humoral responses in efficient virus control. We recently discovered that antibody-secreting cells (ASCs) are present in the dermis of human genital skin biopsies collected from active HSV-2 lesions but much more frequent in biopsies collected weeks after lesion healing– during virus containment phase. Increase in ASC frequency coincides with local but not systemic increase in HSV-2 antibodies. Moreover, skin ASCs are clonally expanded. Our current research pursues these observations to define a mechanistic link between locally increased levels of HSV-2 Abs, skin-resident ASCs and control of HSV-2 reactivation; observations of direct relevance for developing an HSV-2 vaccine.
We have two multi-faceted, complimentary projects in this arena:
Project 1: Detailed characterization of skin migratory B cells and resident ASCs to define transcriptional markers, clonal structure, local microenvironment and mechanisms of their recruitment and retention in skin. We use single-cell RNA sequencing (scRNASeq) to identify gene expression markers and specific cellular pathways activated in tissue B cells and ASCs. scBCRSeq of skin B cells and ASCs in combination with bulk sequencing of circulating B cells is used to reconstruct clonal evolution of local B cells and ASCs and identify “public” response. Observations made with these two approaches converge into experiments using spatial transcriptomics methods based on predesigned probes and/or direct sequencing of tissue RNA. We also use different microscopy techniques including immunofluorescence, in situ hybridization and laser-capture microdissection to validate results obtained via computational analysis of scRNAseq and spatial transcriptomics.
Project 2: HSV-specific antibodies isolated from skin-resident ASCs as new therapeutic modalities and guideline for vaccine design. Despite decades of research, human antibody response to HSV is poorly characterized. Epitopes of antibodies with portent anti-HSV activity are not mapped; prototypic/public antibody response to natural infection and/or vaccines is not characterized. Therefore, we don’t know what sites on HSV should be targeted by antibodies to achieve maximal anti-viral activity, whether these sites are immunodominant or natural response is skewed toward “decoy” epitopes. Because skin-resident ASCs are present at the very battlefront with HSV, the antibodies they secrete have high physiological relevance and therefore their epitopes and functional activity is of a great interest as it can inform both vaccine design and development of immunotherapeutics. We use variety of structural methods such as crystallography and cryo-electron microscopy (cryoEM) to map epitopes of antibodies isolated form skin-resident ASCs on HSV glycoproteins and then combine this knowledge with functional analysis of their anti-viral activity including neutralization, ability to inhibit cell-to-cell HSV spread and different antibody effector functions. The overarching goal is to decipher a mechanistic link between epitopes of highly potent anti-HSV antibodies with mechanism of their anti-viral activity.
Single Cell Sorting
We have established a technology to combine emulsion-based microfluidics with DNA, RNA and/or protein barcoding that allows recovery of natively paired TCRs (and BCRs) at the repertoire scale. This process is high-throughput, allowing up to 1 million cells per hour to be evaluated in individual 65 picoliter emulsion drops. Cells within droplets are lysed and target mRNA is reverse transcribed with target specific primers in a 2-step process that attaches both molecule specific and droplet specific barcodes to the cDNA. After subsequent recovery, the DNA is sequenced using the Illumina platform. Dual barcoding allows clustering of sequence reads to both molecules and cells of origin. We can also evaluate if direct in vitro co-culture with HSV-2 before single cell sorting allows selection of HSV-2 specific T-cell clones directly from tissue. We can recover thousands of T-cell clones from PBMC and biopsy tissues. The yield, throughput and consistency of the microfluidic single cell sorting approach, especially for identifying CD8+ T cells, are marked improvements over previously used technologies.
TCR reporter genes
Using laser capture microdissection (LCM), we can isolate individual cells from sequential genital biopsies during times of HSV-2 reactivation as well as clinical quiescence. Sequencing the TCR a and b chains from CD8+ T cells found in lesion and healed biopsies allows us to construct reporter TCRs in vitro to evaluate the antigen specificity of TRM CD8+ T cells. We have identified HSV-2 antigens that patient CD8 T cells recognize locally at the site of infection using an HSV-2 ORF library. These TCR reporter genes will help identify which antigens are biologically relevant targets for future HSV vaccines.
Spatiotemporal kinetics of virus in localized microenvironments using RNAscope
HSV-2 DNA is present in several genital tract regions during viral shedding. To specify spatiotemporal HSV dispersion, we have developed a grid system in which daily swabs are taken from 23 separate sites of a patient’s vulva for up to 30 days. During brief asymptomatic episodes, low HSV-2 quantities are confined to a few regions in which both viral load and location fluctuate over time. During longer, lesional episodes, variable viral quantities are detected throughout the genital tract. Even during times of clinical quiescence, the virus is widely dispersed, with heterogeneous viral loads.