Transmission of highly virulent CXCR4 tropic HIV-1 through the mucosal route in an individual with a wild-type CCR5 genotype

Abstract

Nearly all transmitted/founder (T/F) HIV-1 are CCR5 (R5)-tropic. While previous evidence suggested that CXCR4 (X4)-tropic HIV-1 are transmissible, detection was not at the earliest stages of acute infection. Here, we identified an X4-tropic T/F HIV-1 in a participant in acute infection cohort. Coreceptor assays demonstrated that this T/F virus is strictly CXCR4 tropic. The participant experienced significantly faster CD4 depletion compared with R5 virus infected participants in the same cohort. Naïve and central memory CD4 subsets declined faster than effector and transitional memory subsets. All CD4 subsets, including naïve, were productively infected. Increased CD4⁺ T cell activation was observed over time. This X4-tropic T/F virus is resistant to broadly neutralizing antibodies (bNAbs) targeting V1/V2 and V3 regions. These findings demonstrate that X4-tropic HIV-1 is transmissible through the mucosal route in people with the wild-type CCR5 genotype and have implications for understanding the transmissibility and immunopathogenesis of X4-tropic HIV-1.

Figure 1. Genetic and phenotypic characterization of the 40700 T/F virus.

a, Highlighter plot showing near-full length viral genomes obtained at day 15 from the 1^st positive test for HIV-1 RNA. The sequences were obtained by SGA. The consensus sequence (black line on the top) is used as the master sequence. Genetic substitutions compared to the consensus sequence are color coded. b, Frequency distribution of the pair-wise Hamming Distance of the day 15 viral sequences. The frequency distribution follows Poisson distribution as determined by the Poisson-Fitter tool in the Los Alamos HIV Sequence Database. c, Replication kinetics of the 40700 IMC in purified CD4⁺ T cells from three healthy donors. The X4 tropic strain NL4.3 and an R5 tropic T/F virus CH058 were used as controls. All infections were performed in triplicate. The error bar represents the standard deviation (SD). d, Determination of coreceptor usage of the 40700 IMC in a panel of NP-2 cell lines expressing different HIV-1 coreceptors. The p24 concentration in the culture supernatant was determined on day 5 post infection. The X4 tropic strain NL4.3 and an R5 tropic T/F virus CH058 were used as controls. The infections were performed in duplicate. The error bar shows the SD.

Figure 2. Rapid CD4⁺ T cell depletion in participant 40700 after HIV-1 transmission.

a, Dynamics of CD4⁺ T cells in participant 40700 compared to R5 HIV-1 infected participants in the RV217 Thailand cohort. The rate of CD4 decline was calculated using a linear mixed effect model (LME). The statistical significance was determined using a normal distribution test. b, Dynamics of CD4 count, CD8 count, and plasma VL in participant 40700. The time frame when participant 40700 was on ART is highlighted in red (day 266 to 282). c, Dynamics of each CD4 subset in participant 40700 before ART initiation. Four different CD4 subsets (naïve, CM, EM, and TM) are color coded. The rate of CD4 subset decline was calculated by linear regression.

Figure 3. Quantification of total and integrated HIV-1 DNA and cell-associated HIV-1 RNA in participant 40700.

a, Total (light blue) and integrated (dark blue) HIV-1 DNA was quantified in each sorted CD4 subset at day 78 (from the 1^st positive test for HIV-1 RNA). The results are shown as DNA copies/10⁶ cells. The experiments were performed in duplicate. The error bar shows the SD. b, Dynamics of cell-associated HIV-1 RNA in each CD4 subset. The cell-associated HIV-1 RNA in each CD4 subset was quantified by amplifying the pol region and the tat/rev transcript. Different CD4 subsets are color coded. The pol region in the EM CD4 subset was not amplifiable at the first time point (day 20). The cell-associated HIV-1 RNA viral load is shown as copies/10⁶ cells.

Figure 4. Determination of CD4⁺ T cell activation and proliferation in participant 40700.

The expression of activation and proliferation markers on total CD4⁺ T cells as well as on each CD4 subset was determined by flowcytometry. The percentage of cells positive for each marker is shown. The time frame when participant 40700 was on ART (from day 266 to day 282) is highlighted in red.

Figure 5. Neutralization susceptibility of the 40700 T/F virus to a panel of bNAbs and the autologous neutralization activity induced in 40700.

a, Susceptibility of the 40700 T/F virus to a panel of bNAbs targeting different regions of the HIV-1 envelope. The IC₅₀ (μg/mL) of each bNAb is shown. The murine leukemia virus (MuLV) was used as negative control. b, Autologous neutralization activity induced by the 40700 T/F virus. Two participants infected by R5 tropic T/F viruses (40257 and 40094) were used as controls.

Figure 6. Phylogenetic tree and highlighter plot showing longitudinal viral evolution in participant 40700.

Longitudinal env sequences were obtained by SGA. Evolution of the 40700 T/F virus is illustrated by phylogenetic tree (left) and highlighter plot (right). Sequences from different time points (days from the 1^st positive test for HIV-1 RNA) are color coded. The phylogenetic tree was constructed using the maximum likelihood method. In the highlighter plot, the black line on the top represents the T/F virus. The red and green tics indicate non-synonymous and synonymous substitutions compared to the T/F virus, respectively. The variable loops are shaded in blue.