Ultrasensitive quantification of HIV-1 cell-to-cell transmission in primary human CD4 ⁺ T cells measures viral sensitivity to broadly neutralizing antibodies

Abstract

HIV-1 can efficiently replicate by direct transmission from infected to uninfected CD4 + T cells at confined local sites designated virological synapses (VSs). VSs are formed by cell junctions between HIV-1 envelope glycoproteins (Envs) on an infected cell and CD4 on an uninfected cell. These sites facilitate highly efficient viral transmission and contribute to HIV-1 evasion from neutralizing antibodies, but accurate quantification of the efficiency of cell-cell transmission is still challenging. Here, we developed an ultrasensitive HIV-1 cell-to-cell transmission assay that triggers the expression of the nanoluciferase (nluc ) gene in target cells upon transmission and after reverse transcription of the HIV-1 RNA genome. The assay is based on the insertion of the nluc gene in an antisense orientation into the HIV-1 provirus. Nluc expression is blocked in virus-producing cells because the nluc gene contains an intron that can be efficiently spliced out only when mRNA is transcribed from the opposite (sense) strand. Thus, only sense mRNA that is spliced and subsequently reverse transcribed during transmission to target cells will support Nluc expression. Assay optimization resulted in a very low background, >99% splicing efficiency, high sensitivity, and wide dynamic range for the detection of cell-cell transmission in T cell lines and primary CD4 + T cells. The optimized reporter vector detects cell-cell transmission using single-round viral vectors and HIV-1 molecular clones, which provide viral proteins of different HIV-1 strains, and reproducibly measures the sensitivity of HIV-1 transmission to antibody neutralization in vitro . This assay will contribute to understanding the fundamental mechanisms of HIV-1 cell-to-cell transmission, allow evaluation of pre-existing or acquired HIV-1 resistance in clinical trials, and can be adapted to study other retroviruses. IMPORTANCE HIV-1 can efficiently transmit from one cell to another but accurate quantification of this mode of transmission is still challenging. Here, we developed an ultrasensitive assay to measure HIV-1 transmission between cells and to evaluate HIV-1 escape from broadly neutralizing antibodies in primary human T cells. This assay will contribute to understanding the fundamental mechanisms of HIV-1 cell-to-cell transmission, allow evaluation of pre-existing or acquired HIV-1 resistance in clinical trials, and can be adapted to study the biology of other retroviruses.

HIResist: a database of HIV-1 resistance to broadly neutralizing antibodies

“…These studies provide important information and guidance for HIV-1 vaccine design and development as well as for understanding the potential side effects, mode of administration, and the mechanisms of bnAb action during immunotherapy treatment. Notably, these studies highlight potential direct and indirect mechanisms of HIV-1 resistance to bnAbs, some of which have been already documented in multiple in vitro experiments ( Herschhorn et al 2011 , Yen et al 2014 , Lynch et al 2015 , Bar et al 2016 , Cale et al 2020 , Cervera et al 2021 , Herschhorn 2023 , Mazurov and Herschhorn 2023 ). Thus, combining Env sequence analysis and experimental approaches to understand the different pathways of HIV-1 resistance to bnAbs is beneficial for future robust application of bnAbs for medical interventions.…”

Alternative substitutions of N332 in HIV-1 AD8 gp120 differentially affect envelope glycoprotein function and viral sensitivity to broadly neutralizing antibodies targeting the V3-glycan

“…We have recently developed an ultrasensitive method to detect the transmission of HIV-1 between T cells (CEM to SupT1 cells). The assay uses a reporter lentivector that detects viral transmission in the co-culture of viral-producing cells and target cells ( 35 ). Reporter protein expression is blocked until HIV-1 completes reverse transcription in target cells, allowing to distinguish between viral-producing cells and target cells to which HIV-1 was transmitted.…”

“…Cell–cell transmission was measured as previously described ( 35 ). Briefly, 10 6 CEM CD4 + T cells were washed once with PBS (in 1.5-mL tubes) and electroporated in buffer R (Neon Transfection Reagent; Invitrogen, USA) with 3 µg of the reporter plasmid pUCHR-EF1a-inNluc, 2 µg of the packaging plasmid pNL4-3ΔEnv, and 1 µg of AD8 Env expression plasmids (WT or N332x variants).…”