Impact of HIV-1 Backbone on Neutralization Sensitivity: Neutralization Profiles of Heterologous Envelope Glycoproteins Expressed in Native Subtype C and CRF01_AE Backbone

<div><p>Standardized assays to assess vaccine and antiviral drug efficacy are critical for the development of protective HIV-1 vaccines and drugs. These immune assays will be advanced by the development of standardized viral stocks, such as HIV-1 infectious molecular clones (IMC), that i) express a reporter gene, ii) are representative of globally diverse subtypes and iii) are engineered to easily exchange envelope (env) genes for expression of sequences of interest. Thus far, a subtype B IMC backbone expressing <i>Renilla</i> luciferase (LucR), and into which the ectodomain of heterologous <i>env</i> coding sequences can be expressed has been successfully developed but as execution of HIV-1 vaccine efficacy trials shifts increasingly to non-subtype B epidemics (Southern African and Southeast Asia), non-subtype B HIV-1 reagents are needed to support vaccine development. Here we describe two IMCs derived from subtypes C and CRF01_AE HIV-1 primary isolates expressing LucR (IMC.LucR) that were engineered to express heterologous gp160 Envs. 18 constructs expressing various subtypes C and CRF01_AE Envs, mostly acute, in subtype-matched and –unmatched HIV backbones were tested for functionality and neutralization sensitivity. Our results suggest a possible effect of non-<i>env</i> HIV-1 genes on the interaction of Env and neutralizing antibodies and highlight the need to generate a library of IMCs representative of the HIV-1 subtype spectrum to be used as standardized neutralization assay reagents for assessing HIV-1 vaccine efficacy.</p> </div

Titrations of CM235.LucR and ETH2220.LucR in different target cells.

<p>We also assessed infectivity of CM235.LucR (A) and ETH2220.LucR (B), in the cell lines TZMbl (two independent experiments) and A3R5 (two independent experiments), and in PBMC (three donors). RLU LucR was measured 72 hours after infection (dashed lines). Horizontal lines represent RLU cut off for LucR (dashed) activity.</p

Titrations in TZMbl cells of chimeric IMC.LucR before and after concentration.

<p>We measured CM235.LucR-620345 (A) and CM235.LucR-644039 (B) concentrated (open symbols) and original (plain symbols) virus stocks. Titrations were performed in duplicate, in a 4-fold dilution format, in TZM-bl cells. Firefly luciferase acitivity (RLU FF, bold lines) and Renilla luciferase activity (RLU LucR, dashed lines) were measured simultaneously in the same wells, 48 hours later. Horizontal solid and dashed lines indicate the cut-off values for FF and LucR, respectively.</p

Viral titrations in TZM-bl cells.

<p>Titrations of IMC.LucR only, CM235.LucR and ETH2220.LucR (A), of CM235.2 Env-based viruses (B) and of ETH2220.11B Env-based viruses (C). All titrations were performed in duplicate, in a 4-fold dilution format. Firefly luciferase acitivity (RLU FF, bold lines) was measured 48 hours later. For IMC.LucR only (A), Renilla luciferase activity (RLU LucR, dashed lines) was also measured simultaneously in the same wells. The different viral forms expressing Env of CM235.2 (B) and ETH2220.11B (C) were compared: virus stocks of pseudovirus (diamond), biological isolate (inverted triangle), parental IMC (triangle) and IMC.LucR (circle). Horizontal solid and dashed lines indicate the cut-off values for FF and LucR, respectively. </p

Neutralization profiles CM235.2 and ETH2220.11B Env-based viruses.

<p>Inhibition of infection using HIV-1+ sera and multiple viral forms (pseudovirus, biological isolate, original IMC and IMC.LucR) of CM235.2 (A) and ETH2220.11B (B) in the TZM-bl assay, using the cell-line encoded firefly luciferase reporter endpoint. Inhibition of infection using HIV-1+ sera and CM235.LucR (C) and ETH2220.LucR (D) in the TZM-bl and PBMC assay with three different donor PBMC as assay targets, using the IMC-encoded Renilla luciferase reporter endpoint. Values are the reciprocal sera dilution at which RLU was reduced by 50% compared to the level in virus control wells. Horizontal lines represent the threshold of detection (1:40 sera dilution); values at or under the line indicate 50% inhibition was not reached.</p

Neutralization sensitivity of Env expressed in subtype-matched vs mismatched backbones.

<p>Envs from three subtype C and six CRF01_AE isolates were exchanged into both the subtype C ETH2220.LucR and the CRF01_AE CM235.LucR backbones. Most of the Envs we tested were acute, except for 6838v7 and R2184. Neutralization titers for each stock were generated in TZM-bl cells using (A) 4E10 and (B) sCD4 and polyclonal plasma (C) 1026 and (D) 1028. The Y-axis represents the IC50 (A & B) or ID50 (C & D) values for each Env in both backbones. For statistical analysis, we compare, within Env subtype, neutralization sensitivity by reagent between different backbone constructs (Wilcoxon non-parametric test); p-values for C-envs are written in grey and for AE-envs in black. Horizontal lines represent the cut-off: for (A) and (B), values under the cut-off lines (25 ug/ml) represent neutralization sensitivity, when for (C) and (D) values under the cut-off (dilution 1:40) show resistance to neutralization. </p

IMC.LucR construction.

<p>Schematic representation of the construction of (A) primary isolate-derived IMC.LucR and (B) chimeric IMC.LucR-Env.</p