The great advantage of virus-like particle (VLP) nano-vaccines is their structural identity to wild-type viruses, ensuring that antigen-specific B-cells encounter viral proteins in their natural conformation. “Wild-type” viral nanoparticles can be further genetically or biochemically functionalized with biomolecules (antigens and adjuvants). Flagellin is a potent inducer of innate immunity and it has demonstrated adjuvant effectiveness due to its affinity for toll-like receptor 5 (TLR5). In contrast to most TLR ligands, flagellin is a protein and can induce an immune response against itself. To avoid side-effects, we incorporated a less inflammatory and less immunogenic form of flagellin as an adjuvant into HIV-based nanoparticle B-cell-targeting vaccines that display either the HIV-1 envelope protein (Env) or a model antigen, hen egg lysozyme (HEL). While flagellin significantly enhanced HEL-specific IgG responses, anti-Env antibody responses were suppressed. We demonstrated that flagellin did not activate B-cells directly in vitro, but might compete for CD4+ T-cell help in vivo. Therefore, we hypothesize that in the context of VLP-based B-cell nano-vaccines, flagellin serves as an antigen itself and may outcompete a less immunogenic antigen with its antibody response. In contrast, in combination with a strong immunogen, the adjuvant activity of flagellin may dominate over its immunogenicity.

Defining how human host factors shape HIV-1 infection in vivo remains essential for the development of genetically engineered cell therapies. Here, we established a non-viral CRISPR/Cas9 ribonucleoprotein-based platform for efficient single and multiplex gene editing in primary human CD34 ⁺ hematopoietic stem and progenitor cells (HSPCs). Edited HSPCs retained viability, proliferative capacity, primitive immunophenotypes, and multilineage differentiation potential and supported targeted reporter knock-in independently of the cell source, cord blood (CB), bone marrow (BM), and mobilized from peripheral blood (MPB). Following differentiation, SAMHD1 knockout increased HIV-1 susceptibility of HSPC-derived macrophages, MX2 knockout also increased HIV infection in cells pre-stimulated with IFN-α2a, and CXCR4 knockout blocked X4-tropic HIV-1 infection of HSPC-derived megakaryocytes. We then transplanted CCR5-, SAMHD1-, or non-targeting control-edited HSPCs into immunodeficient mice and challenged reconstituted animals with R5-tropic HIV-1. CCR5 knockout prevented detectable viral spread, validating the model using a clinically relevant dependency factor. In contrast, SAMHD1 knockout accelerated viral dissemination, with earlier plasma viremia and 3.6-fold higher cumulative viremia, although endpoint viral burden was not significantly different from controls. These findings establish transplantation of CRISPR/Cas9-edited human HSPCs as a modular platform for dissecting HIV-1 host-factor function across hematopoietic lineages ex vivo and in vivo and for evaluating engineered cell-based strategies.

The unique ability of chimeric antigen receptor (CAR) T cells to infiltrate tissues is revolutionizing our perspectives for tackling severe, refractory and otherwise untreatable diseases. In HIV, CAR-T cells have been designed to target viral biomarkers, with limited success so far. Here, we investigated the possibility of redirecting CAR-T cells against a cellular biomarker of the HIV reservoir, the programmed cell death protein 1 (PD-1). We designed two second-generation 4-1BB-CARs using the scFv of either a blocking (bPD1-CAR) or a nonblocking (nbPD1-CAR) anti–PD-1 monoclonal antibody. The CAR avidity modulated T cell sensitivity, trogocytosis, and effector functions, independently of the PD-1 signaling domain. Both anti–PD-1 CAR-T cells could persist for 70 days in HIV-infected humanized mice, correlating with viral protection and a disruption of the lymphoid architecture in the white pulp of the spleen. Together, our results open strategic avenues for reducing the HIV reservoir by demonstrating the feasibility of depleting specific T cell subpopulations.