Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

Summary

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

Abstract

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Introduction

Human immunodeficiency virus type 1 (HIV-1) entry, mediated by the trimeric viral envelope glycoproteins (Env) is the first step of the infectious cycle. Being the only exposed viral antigen presented at the surface of virions, the Env trimer elicits neutralizing and nonneutralizing antibodies. As such, it represents an interesting candidate for vaccine immunogen design. However, vaccination trials with Env in soluble or recombinant forms elicited responses with only minimal effectiveness against most primary HIV-1 isolates^1-3. Nonetheless, partial efficacy observed in the RV144 vaccine trial⁴ renewed interest in HIV-1 Env as an immunogen candida....

Protocol

1. Day 1 – Cell Culture

1. Plate 2 x 10⁴ human osteosarcoma (HOS) cells per well in an opaque, 96-well cell-culture plate suitable for luminescence reading. Use Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin. Incubate until next day at 37 °C, 5% CO₂.

2. Day 2 – Polyethylenimine (PEI) Transfection

1. Prepare transfection mix according to subsequent steps. Adjust reagents and DNA quantities according to the number of wells that are to be transfected with the same Env.
2. Tube A: Ad....

Results

Using the general procedure described above, we adapted the protocol to assay the impact of soluble CD4 (sCD4) and coexpressed cellular CD4 on the exposure of CD4i epitopes on either wild-type (wt) or mutated Env, as described previously^18,24,25,28. Figure 1 schematically represents the general procedure and the exposure of CD4i epitopes following treatment with sCD4 or by coexpression of cellular CD4¹⁸. In Figure 2, we used sCD4 to induce Env conformational changes.......

Discussion

This assay is optimized to detect the interaction of specific mAbs with HIV-1 trimeric Env expressed at the cell surface. Once the protocol has been established, it can be used at medium to high throughputs with low overall material costs and little amounts of antibodies. Since this assay is transfection-based, it can easily be adapted for coexpression of cellular proteins such as CD4 in order to study their effects on Env conformation.

However, the transfection base of this protocol also impl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HOS cells	ATCC	CRL-1543
White Opaque Tissue Culture Plate, 96-well, Flat Bottom	BD	353296
Polyethylenimine, linear, 25,000 MW	Polysciences	23966	Prepared in 1 mg/ml solution
Dulbecco's Modified Eagle Medium (DMEM)	Invitrogen	11995
Goat Anti-human IgG, Peroxidase Conjugated	Pierce	31413
Enhanced Chemiluminescence Substrate	PerkinElmer	NEL105001EA
TriStar LB 941, Plate Reader	Berthold Technologies