Detection of Binding Antibodies to Native and Recombinant Human ...

JOURNAL OF CLNICAL MICROBIOLOGY, Oct. 1992, p. 2606-2612

Vol. 30, No. 10

0095-1137/92/102606-07$02.00/0 Copyright © 1992, American Society for Microbiology

Detection of Binding Antibodies to Native and Recombinant Human Immunodeficiency Virus Type 1 Envelope Glycoproteins Following Recombinant gpl60 Immunization Measured by Flow Cytometry and Enzyme Immunoassays GEOFFREY J. GORSE,12* SHARON E. FREY,' FRANCES K. AND THE AIDS

NEWMAN,1"2 ROBERT B. BELSHE, 12

VACCINE CLINICAL TRIALS NETWORKt

Division of Infectious Diseases and Immunology, St. Louis University School of Medicine, St. Louis, Missouri 63104,1 and St. Louis Veterans Affairs Medical Center, St. Louis, Missouni 631062 Received 20 March 1992/Accepted 9 July 1992

The ability of antibody induced by vaccination with recombinant gpl60 (rgpl60) to bind to native and recombinant human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins was measured. Thirtythree HIV-1-seronegative healthy adult volunteers were injected four times with 40 or 80 ,g of an HIV-ILAv envelope glycoprotein candidate vaccine per dose. The vaccine consisted of rgpl60 produced in insect tissue culture cells infected with a recombinant baculovirus which contains the gpl60 gene from the HIV-1LAv strain. By using a flow cytometric indirect immunofluorescence assay (FIFA) to detect vaccine-induced antibody to native envelope glycoprotein expressed by target cells infected with H1V-lmB, sera from 9 of the 33 vaccinees were positive. These included sera from eight vaccinees which stained H1V-iMB-infected cells and sera from two vaccinees which stained target cells infected with HIV-lMN, a heterologous virus strain. None of the sera stained cells infected with the HIV-12F strain. Envelope glycoprotein-binding antibody was more frequently detectable in an enzyme-linked immunosorbent assay (ELISA) by using rgpl60 compared with that which was detectable in the FIFA with uninfected target cells which were pulsed with rgpl60 antigen. Positive correlations were observed between the rgpl60 FIFA and a whole-virus-lysate enzyme immunoassay, between the rgpl60 FIFA and the rgpl60 ELISA, and between the rgpl60 ELISA and the whole-virus-lysate enzyme immunoassay. The ability of sera from some volunteers who received rgpl60 vaccine to bind to HIV-1-infected cells suggests that further studies with this vaccine should be done.

Analysis of the humoral responses to human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein may include assays that measure the antibody which binds to antigen presented in various ways. We were interested in measuring antibodies which recognize the native envelope glycoprotein expressed on infected cell surfaces, since these antibodies may be more likely to be functional in vivo than those which bind to denatured protein only. The antibody response to an HIV-1rAv-based recombinant gpl60 (rgpl6O) vaccine (VaxSyn; MicroGeneSys, Inc., Meriden, Conn.) was evaluated in normal healthy adult volunteers by three assays: a flow cytometric indirect immunofluorescence assay (FIFA), an rgpl60 enzyme-linked immunosorbent assay (ELISA), and a whole-virus-lysate enzyme immunoassay

(EIA). These assays compared the ability of a candidate rgpl60 vaccine to induce antibody which binds to the gpl60 present on cells infected with HIV-1, the vaccine rgpl60 antigen, and the gpl60 present in whole virus lysate. The FIFA method with HIV-1IIIB-infected H9 cells has been found to be highly sensitive and specific for the detection of anti-HIV-1 antibody in sera from HIV-1-infected individuals (18). The FIFA method was used in the current study to measure antibody in sera from uninfected volunteers injected with an investigational vaccine, rgpl60. H9 cells infected with additional strains of HIV-1 and uninfected H9 cells pulsed with rgpl60 were also used as targets in the current study.

MATERIALS AND METHODS *

rgp160 vaccine. The HIV-1 vaccine candidate (rgpl6O; VaxSyn; MicroGeneSys) is the envelope glycoprotein of HIV-1 (HIV-1EAv strain). The gp160 gene was derived from an infectious molecular clone of HIV-1 (1, 23). The gpl60 was produced in a continuous insect tissue culture cell line by infection with a recombinant baculovirus expression vector which contained the gpl60 gene (2). The vaccine was formulated with purified gpl60 and aluminum phosphate gel (alum adjuvant). Subjects. Subjects in the study were healthy adult volunteers between the ages of 18 and 55 years without evidence of serious medical illnesses as determined by history and

Corresponding author.

t Current members of the network include Mary Lou Clements, Johns Hopkins University, School of Hygiene and Public Health and School of Medicine, Baltimore, Md.; Raphael Dolin, University of Rochester School of Medicine and Dentistry, Rochester, N.Y.; Lawrence Corey, University of Washington, Seattle; Barney S. Graham, Vanderbilt University, Nashville, Tenn.; Dani Bolognesi, Duke University, Durham, N.C.; and Patricia Fast, National Institute of Allergy and Infectious Diseases, Bethesda, Md. Former members who participated in the vaccine trial include Carol 0. Tacket, University of Maryland at Baltimore, Baltimore, Md.; Stephen B. Greenberg, Baylor College of Medicine, Houston, Tex.; and Bruce F. Fernie, Georgetown University, Rockville, Md. 2606

VOL. 30, 1992

ANTI-gp160 ANTIBODY RESPONSES FOLLOWING IMMUNIZATION

physical examination. Subjects were seronegative for HIV-1 as determined by EIA and Western blot (immunoblot) assays (see below) prior to immunization. They participated in a phase I trial of the immunogenicity and safety of the rgpl60 vaccine. The details of the clinical design and results of the clinical study have been reported elsewhere (4). The institutional review boards of the participating institutions approved the study, and all participants gave written informed consent.

The participants whose sera were evaluated in this study received either 40 or 80 ,ug of rgpl6O per dose intramuscularly on days 0, 30, 180, and 545. Sera from 24 volunteers who received all four vaccine injections and from 9 volunteers who received only the first three vaccine injections were available for testing. FIFA. The following three types of target cells were used: (i) cultures of uninfected H9 cells and CEM-NKR cells were negative control targets; (ii) cultures of H9 cells chronically infected with HIV-1IIIB, HIV-1RF, or HIV-1MN expressed native envelope glycoprotein and other HIV-1 antigens; and (iii) cultures of H9 and CEM-NKR cells were pulsed with rgpl6O (baculovirus expressed recombinant, nonreplicating gp160, which was the immunogen). The H9 cell line was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, from Robert Gallo (10-12) and is a single cell clone derived from HUT 78 cells, which are a human cutaneous T-cell lymphoma cell line. CEM-NKR is a human T-lymphoblastoid cell line and was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, from Peter Cresswell (7). The HIV-111B_-, HIV1RF-, and HIV-1MN-infected H9 cell lines (human T-cell lymphotropic virus type III [HTLV-III] strain B/H9, H9/ HTLV-IIIRF NIH 1983, and H9/HTLV-IIIm NIH 1984, respectively) were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, from Robert Gallo (5, 11-13, 17, 20). Uninfected H9 cells were added to cultures of H9 cells chronically infected with HIV-1 7 to 10 days before the assays were done to ensure good viral antigen expression on target cells. The immunofluorescence assay was patterned after one developed by Sligh et al. (18) to detect antibody against HIV-1IIIB-infected cells present in sera from HIV-1-infected individuals. Target cells chronically infected with HIV-1 were simply washed and were then incubated with test serum without prior incubation with rgpl6O. Uninfected target cells were initially washed and were then labeled with envelope glycoprotein by incubation with 2.5 ,ug of rgpl6O per 10 cells at 37°C for 90 min. In preliminary experiments, target cells incubated with as little as 1 ,ug of baculovirusderived gpl20-fluorescein isothiocyanate (American Biotechnologies, Inc., Cambridge, Mass.) expressed >90% positive fluorescence. After the 90-min incubation with rgpl6O (the immunogen), target cells were washed and resuspended in 150 ,ul of RPMI 1640 medium; 25 p,l of a 1:10 dilution of the human test serum was then added. After a 20-min incubation with the serum at 4°C, the cells were washed, and fluorescein isothiocyanate-conjugated F(ab')2 goat anti-human immunoglobulin G (IgG; Kallestad Diagnostics, Austin, Tex.) was added with bovine serum albumin for 30 min at 4°C to reduce nonspecific binding. After washing, the cells were fixed overnight in 1 to 2% (vol/vol) formaldehyde. After a final wash in phosphate-buffered saline (PBS), the logarithm of the green fluorescence intensity was analyzed on a FACScan

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flow cytometer (Becton Dickinson Immunocytometry Systems, Braintree, Mass.). The data were collected first into a forward-scatter versus right-scatter cytogram which displayed cell size and degree of granularity. This was gated to eliminate any particles which were not of sufficient size to be an intact cell. Cells passing through this gate were then displayed on a histogram of the logarithm of the green fluorescence intensity versus relative cell number. For each run of HIV-1-infected or rgpl60-pulsed H9 cells, cells stained with control sera from known HIV-1-seropositive (infected, not vaccinated) and HIV-1-seronegative individuals were used to set an appropriate gate with which to determine the fraction of cells stained with test vaccinee sera that were shifted into the positive fluorescence intensity region as defined by the gate (see Fig. 1A and B). The fraction of cells with intensity shift above the gate (termed percent positive fluorescence) was then used as a measure of antibody content. Sera which stained envelope glycoproteinexpressing cells all resulted in a similar pattern of a shift in a single peak of cells depicted on the flow cytometric histograms (see Fig. 1A and B). This pattern of the logarithm of the fluorescence intensity shift indicated that the target cells were a homogeneous population with respect to envelope glycoprotein expression. While determination of the shift in mean channel fluorescence intensity would be an alternative method of analysis, there is precedent for our method (16, 18). In our study, the two methods of data analysis should provide similar results because of the shape of the cell histograms, because all of the vaccinee test sera stained envelope glycoprotein-expressing targets such that in no case was the fluorescence intensity of more than 90% of the cells above the gate level and because we did not observe shifts in the mean channel fluorescence intensity without a shift in the fraction of cells with fluorescence intensity above the set gate level. The location of the gate in terms of the logarithm of fluorescence intensity varied slightly from assay to assay because of variables such as the relative intensity of the fluorochrome and other instrument settings on a particular day. This did not affect the reliability of the assay, because the gate setting was based on the negative and positive control readings on any particular assay day. Raw data derived from the flow cytometric histograms were used to determine the net percent positive fluorescence for each serum specimen and HIV-1 envelope glycoproteinexpressing target cell population, which was defined by the following equation: net percent positive fluorescence = [(percent positive fluorescence by using the test serum against HIV-1 antigen-expressing target cells) - (percent positive fluorescence by using the test serum against control target cells)] - [(percent positive fluorescence by using the negative control serum against HIV-1 antigen-expressing target cells) - (percent positive fluorescence by using the negative control serum against control target cells)]. A specimen was considered positive if the net percent positive fluorescence was .4.0, which was approximately 10 standard deviations (SDs) above the mean net percent positive fluorescence of prevaccination sera tested. Positive control sera from HIV-1-infected patients and hyperimmune IgG (HIVIG; Abbott Laboratories, Inc., Abbott Park, Ill.) derived from pooled plasma of HIV-1-seropositive asymptomatic donors (3) had net percent positive fluorescence of >50. Testing of each serum sample with target cells that did not express HIV-1 antigen provided an internal control for each sample so that we could correct for antibody binding to target cells which was not specific for viral antigen. Whole-virus-lysate ETA and Western blot. Sera from vol-

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GORSE ET AL.

J. CLIN. MICROBIOL.

TABLE 1. Comparison of FIFA, whole-virus-lysate EIA, Western blot, and rgpl60 ELISAM

Anti-HIV-111IB reactivity by Western blot,

FIFA (net % positive fluorescence)

Whole-virus-lysate EIA (OD/cutoff ratio)

Day of study

0 28 30 44 60 178 180 194 365 540 545 559 635

rgp160 ELISA reciprocal

antibody against:

antibody titer gpc6Orgp)2tbgp4e

rgp160-pulsed H9 cell targets

rgp160-pulsed CEMNKR cell targets

0.0 0.3

1.3 0.0

0.29 0.26

1.1 1.6 1.0

0.0 3.9 3.7

13.9 0.0 0.0

35.0 3.0

gp160

gp120

gp41