Incompletely closed HIV-1CH040 envelope glycoproteins resist broadly neutralizing antibodies while mediating efficient HIV-1 entry

Abstract

HIV-1 envelope glycoproteins (Envs) mediate viral entry and are sole target of neutralizing antibodies. Thus, HIV-1 Envs must maintain a delicate balance between evading neutralizing antibodies while still preserving viral compatibility to mediate entry into target cells. Here, we studied the viral entry effeciency, fitness, and replication of an incompletely closed, transmitted/founder HIV-1 Envs (CH040), which are highly resistant to most bnAbs. CH040 Envs mediated HIV-1 entry to target cells as efficient as other primary Envs, suggesting that antibody resistance and efficient viral entry can develop independently. Expression of CH040 Envs was comparable to other Envs and most CH040 variants that were rationally engineered to increase bnAb resistance showed no significant decrease in their ability to mediate HIV-1 entry. We detected robust in vitro spread of SHIV CH040 in pig-tailed macaque lymphocytes that was comparable to efficient spread of other SHIVs. Our study provides insights into the relationship between bnAb resistance and efficient HIV-1 entry.

Fig. 1. Pre-existing resistance of transmitted/founder CH040 Envs to bnAbs.

We measured the sensitivity of viruses pseudotyped with HIV-1_CH040 or, as control, HIV-1_AD8 Envs to bnAbs targeting Env vulnerability sites. a Sites of HIV-1 Env vulnerability mapped on available cryo-EM structure (adapted from Jeffy et al. mBio 2024). Residues labeled in pink are part of the gp120-gp41 interface that are not targeted by bnAbs. b Sensitivity of different T/F Envs to bnAbs. c, d Sensitivity of CH040 Env-mediated entry to antibodies that recognize internal Env epitopes (c) and to sCD4 (d). Panels b–d were adapted from Parthasarathy et al. Nat Commun 2024. e Effects of increasing concentrations of different bnAbs on viral entry mediated by HIV-1_CH040 or the control HIV-1_AD8 Envs (some or the raw data used to make the plots were taken from experiments reported by Parthasarathy et al. Nat Commun 2024). f Comparison of the sensitivity of HIV-1_CH040 Envs and the average sensitivity of between 26 and 1589 different Envs to different bnAbs. We calculated average sensitivity of different Envs using available data in HIV-1 databases [(HIResist; https://hiresist.umn.edu/) and (Los Alamos National Laboratory Pathogen Database; www.hiv.lanl.gov)]. Numbers in gray are the number of Envs used for each analysis (detailed data analysis is provided in Supplementary Data 1).

Fig. 2. Efficiency of HIV-1 entry mediated by different Envs.

a Relationship between number of pseudovirus particles (assessed by amount of p24) and viral entry into Cf2-Th/CD4 + CCR5+ target cells mediated by viruses pseudotyped with HIV-1_CH040 or indicated HIV-1 Envs. b Similar to (a) but for 13 T/F Envs, including CH040 Envs (adapted from Parthasarathy et al. Nat Commun 2024). c Western blot of viruses pseudotyoed with different HIV-1 Envs. ad8m env gene contains a codon optimized HIV-1_AD8 gp120 sequence and a native gp41sequence; jrfl env gene was codon-optimized; e7-ad8 and e7-yu2 env genes contain a native sequence of ad8 / yu2 env with a C-terminal sequence of NL-43 gp41. All other Envs are expressed from native T/F env sequences. Blots of three independent experiments are shown. d–e Flow cytometric analysis of Env expression on the surface of 293T cells for the specified Envs. f Cell–cell fusion kinetics of different Envs during 3–9 h. g Cell–cell transmission mediated by viruses pseudotyped with HIV-1_CH040 or indicated HIV-1 Envs. Results are representative (a–e) or average (f) of 2 independent experiments, each performed in at least two replicates. Results in panel (g) are the average of four technical replicates.

Fig. 3. Rational design of T/F CH040 Env variants to increase Env resistance to bnAbs.

a We mapped the amino acid changes introduced in gp120 CD4bs on an available cryo-EM structure of HIV-1_AD8 Envs (protein data base (pdb) ID: 8FAD). b Sensitivity of the specified CH040 Env variants to bnAbs. The specific Env changes and their related domains are indicated on the right. c Top—summary matrix of IC₅₀ calculated from the dose–response curves in panel (b); bottom - fold change in IC₅₀ of CH040 Env variants compared to the IC₅₀ of WT CH040 Envs. Color code is identical for the top and bottom matrixes of (c) and is shown on the right.

Fig. 4. Efficiency of HIV-1 entry mediated by engineered variants of CH040 Envs.

a Relationship between number of pseudovirus particles (assessed by amount of p24) and viral entry into Cf2-Th/CD4 + CCR5+ target cells mediated by viruses pseudotyped with WT HIV-1_CH040 or indicated engineered Env variants. b Western blot analysis of viruses pseudotyped with WT CH040 Envs or with CH040 Env variants. c–e Phenotypic characterization of CH040 Env variants. Cell–cell fusion (c), cell–cell transmission (d), and cold sensitivity (e) of viruses pseudotyped with WT HIV-1_CH040 Envs or indicated engineered Env variants.

Fig. 5. Viral replication kinetics and virus-cell fusion activity of SHIVs in pig-tailed macaque lymphocytes.

a Left, replication kinetics of indicated SHIVs in pig-tailed macaque (PtM) lymphocytes over a 12-day time course. Reverse transcriptase (RT) activity in viral supernatants is plotted against days post-infection. Right, area under the curve (AUC) for SHIV variants (indicated on the x-axis) calculated from the replication curves on the left. b Western blot analysis of virions (left) and whole cell extracts (right) harvested at twelve days post-infection from PtM lymphocytes infected with indicated SHIVs. Immunoblotting performed using anti-Env, anti-SIV p27, and anti-actin antibodies. c AUC for indicated SHIV variants normalized to per virion Env level (Env levels normalized to p27; Env:p27). Results (a, c) represent average ± sd of three independent experiments. d Representative flow cytometry plots indicating viral fusion (% of cells) detected by SHIV BlaM-Vpr assay. Fluorescence of uncleaved CCF2-AM uptake and cleaved CCF2-AM substrate was measured by Attune NxT flow cytometer using the VL1 and VL2 channels, respectively. Fusion activity of indicated SHIVs with PtM lymphocytes was measured as percentage of cells containing cleaved CCF-AM substrate. e Viral fusion (% of cells) for indicated SHIV variants (x-axis). Results represent average ± sd of five independent experiments. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test was used to assess statistically significant difference between the groups. ****p < 0.0001; ***p = 0.0002; **p = 0.0028.

Fig. 6. Graphical summary of CH040 Env phenotype.

Many HIV-1 strains exhibit efficient entry and low bnAb-resistance (i); but some strains, such as CH040, can very efficiently enter target cells and still highly resist multiple bnAbs (ii).