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

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.