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 candidate. This was corroborated by a recent study describing that vaccine-elicited anti-Env antibodies were sufficient to generate a certain degree of protection against SIV and HIV challenges⁵.

After being synthesized in the endoplasmic reticulum, the Env glycoprotein precursor, gp160, undergoes various post-translational modifications that are critical for its ability to fuel the viral fusion process. The Env precursor must fold properly and associate in trimers before being cleaved into its extra-cytoplasmic gp120 and transmembrane gp41 subunits^6-10, with noncovalent interactions maintaining the gp120-gp41 liaison. The infected cell machinery is also responsible for heavily glycosylating Env, comprising about 50% of its total mass^11,12. The resulting complex structure allows Env to be conformationally flexible^13,14, while providing a metastability that is thought to allow Env to adapt and hide certain highly immunogenic epitopes that would otherwise be exposed^15-19, highlighting the importance to better understand the different conformations sampled by the native Env trimer.

To date, several techniques have been developed and successfully used to study Env conformational changes. However, they vary in their limitations, being often restricted to specific Env contexts. For example, surface plasmon resonance or immunoprecipitation assays using conformation specific monoclonal antibodies (mAbs), rely either on monomeric soluble or solubilized Env molecules which are known to be immunogenetically different from their trimeric forms^20,21. Recent studies also suggest that cleavage affects Env conformations resulting in the exposure of epitopes mainly recognized by nonneutralizing antibodies^14,22,23.

Here we describe in detail a method that allows for fast and easy determination of the conformation of cellularly-expressed Env trimers^18,24-26. Following transient transfection of Env in a human adherent cell line the binding of Env-specific antibodies is detected using a simple chemiluminescence reaction. This technique can also be used to characterize the conformational preference of conformation-dependent antibodies. Thus, this assay provides a robust and highly flexible detection method.

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: Add 10 ng Tat-encoding plasmid (such as pTat-III²⁷) and 150 ng Env-encoding plasmid to 5 µl DMEM supplemented with 25 mM HEPES.
3. The Tat-encoding plasmid is only required when using Tat-dependent Env-encoding plasmids such as pSVIII.
4. Tube B: Add 450 ng PEI (from a 1 µg/µl solution) to 5 µl DMEM.
5. Add content of tube B to tube A. Mix thoroughly by vortexing for 10 sec and incubate transfection mix 10 min at room temperature (22 °C).
6. Add 10 µl of the transfection mix per well of the 96-well plate. Incubate for 48 hr at 37 °C, 5% CO₂.

3. Day 4 – ELISA

1. Perform all experiments at room temperature to minimize possible endocytosis of Env/antibodies complexes.
2. Prepare 250 ml of Washing Buffer per plate being used at the same time. Washing Buffer is 1x Tris-buffered saline (TBS) pH 7.5 (50 mM Tris-Cl, pH 7.5; 150 mM NaCl), supplemented with 1 mM MgCl₂ and 1.8 mM CaCl₂.
3. Prepare 125 ml Blocking Buffer per plate by adding 1% nonfat dry milk and 5 mM Tris pH 8.0 to Washing Buffer.
4. Remove cell culture media and transfection mix (supernatant) from 96-well plate.
5. Add 100 µl of Blocking Buffer per well and incubate 20 min at RT.
6. Remove supernatant and add 50 µl of antibody (or serum) per well, diluted to appropriate concentration in Blocking Buffer. Typically, use a concentration of 1 µg/ml. Incubate 1 hr at RT.
7. Wash 3x with 100 µl Blocking Buffer and then repeat washing process 3x with 100 µl Washing Buffer.
8. Remove supernatant, add 100 µl Blocking Buffer, and incubate 5 min at RT.
9. Remove supernatant and add 50 µl of secondary antibody, diluted 1/3,000 in Blocking Buffer. Vary optimal antibody dilution according to manufacturer differences. Incubate 40 min at RT.
10. Wash 3x with 100 µl Blocking Buffer and then repeat washing process 3x with 100 µl Washing Buffer.

4. Data Acquisition

1. Remove supernatant from the plate and add 30 µl 1x enhanced chemiluminescence (ECL) substrate per well.
2. Acquire chemiluminescence signal for 1 sec/well on a suitable plate-reader according to manufacturer instructions. Reading time may differ according to hardware differences.

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