Lymphocyte proliferative responses following immunization with human immunodeficiency virus recombinant GPI60 Geoffrey J. Gorse *z, Robert B. Belshe*, Frances K. Newman*, Sharon E. Frey* and the NIAID AIDS Vaccine Clinical Trials Network ~ Following immunization of healthy adult volunteers with baculovirus-derived HIV-1 gpl60 vaccine (rgpl60), we measured lymphocyte proliferation to rgpl60, the V3 loop peptide of gpl60, control proteins, and phytohaemagglutinin. Four persons received injections of 40 #g or 80 #9 doses of rgpl60 vaccine at times O, and 1, 6 and 18 months; one additional volunteer received only three injections of vaccine. Vaccination with rgpl60 induced lymphocyte proliferative responses to rgp l60, but not to V3 loop peptide. Repeated injection of rgp l60 even at low doses induced a cellular immune response which was not attributable to T-cell recognition of the V3 loop peptide. Keywords: Lymphocyte proliferation; human immunodeficiency virus; recombinant gp160; cell-mediated immunity

INTRODUCTION

MATERIALS AND METHODS

The immunological mechanisms which are protective against human immunodeficiency virus type 1 (HIV-1) infection are not yet known, although some combination of cellular and humoral immunity may be necessary. As part of a larger phase I trial of the immunogenicity and safety of a candidate HIV-1 vaccine 1, measurements were made of in vitro lymphocyte responsiveness to baculovirus-derived recombinant gpl60 (rgpl60), and of the V3 loop peptide (principal neutralizing determinant) of HIV-lmB gpl60 in recipients of baculovirus-derived rgpl60 at this institution. The rationale for testing a vaccine candidate that contains gpl60 includes the fact that HIV-I envelope glycoprotein (gpl60) contains the epitopes (including the V3 loop region) against which anti-HIV-1 neutralizing antibodies have been detected 2, the site of attachment of HIV-1 to the CD4 receptor 3 and T-cell epitopes 4. The V3 loop is a region of gpl60 which shows inter-strain variability in amino acid sequence and has been studied extensively as a target of neutralizing antibody. Thus, we were interested in exploring the ability of rgpl60 vaccine to induce helper T-cell memory to this immunologically important region.

Recombinant gpl60 vaccine The HIV-1 vaccine candidate (rgpl60, VaxSyn, MicroGeneSys Inc., Meriden, CT) is the envelope glycoprotein of HIV-1 (HIV-lnm strain). The rgpl60 gene was derived from an infectious molecular clone of HIV-15'6. The rgpl60 was produced in a continuous insect cell line by infection with a recombinant baculovirus expression vector which contained the rgpl60 gene 7. The vaccine was formulated with purified rgpl60 and aluminium phosphate gel (alum adjuvant). Insect cell and baculovirus proteins (up to 30% by weight) were present in the final product.

*Division of Infectious Diseases, Department of Internal Medicine, St Louis University School of Medicine and St Louis Veterans Affairs Medical Center, St Louis, MO, USA. t National Institutes of Health, Bethesda, MD, USA. tTo whom correspondence should be addressed at: Division of Infectious Diseases, Department of Internal Medicine, St Louis University School of Medicine, 1402 South Grand Blvd, St Louis, MO 63104, USA. (Received 2 July 1991 ; revised 19 November 1991; accepted 19 November 1991)

0264-410X/92/060383-O6 ~') 1992 Butterworth-HeinemannLtd

Subjects and immunization schedule Subjects in the study were healthy adult volunteers aged 18-55 years, without evidence of serious medical illnesses, as determined by history and physical examination. Subjects were seronegative for HIV-1 as determined by ELISA and Western blot assays (see below) prior to immunization. The Institutional Review Board approved the study and all participants gave written informed consent. At this institution two volunteers were vaccinated with 40 #g and two with 80/~g of rgpl60 intramuscularly at times 0, and 1,6 and 18 months. An additional volunteer received only the first three 80/~g doses of rgpl60. Preparation of peripheral blood mononuclear cells (PBL) PBL were separated from heparinized peripheral venous blood by Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density gradient centrifugation8 and suspended in

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RPMI 1640 medium supplemented with 10% heatinactivated pooled human AB (HAB) serum, penicillin G (250 U m l - 1 ) and gentamicin sulphate (50/~g ml- 1). PBL from normal, healthy unvaccinated adults were also obtained to serve as negative controls in the lymphocyte proliferation assays. PBL were cryopreserved at a concentration of 1 × 10 7 cells m1-1 in freezing medium (20% pooled human AB serum and 7.5% dimethylsulphoxide in RPMI 1640 medium) using a controlledrate freezing device (Cryomed, New Baltimore, MI), and were stored in liquid nitrogen for later use.

Lymphocyte proliferation assay On the day of the assay, PBL were thawed by immersion of cryules in a 37°C water bath. Cells were diluted in RPMI 1640 with 20% human AB serum and antibiotics, washed and suspended in RPMI 1640 with 10% human AB serum and antibiotics. Viability was greater than 70% as determined by trypan blue exclusion. Using a standard assay 9-11, PBL (105 viable cells per well) were added to 96-well microtitre plates (Flow Laboratories, McLean, VA). To these cell cultures, rgpl60 (MicroGeneSys), baculovirus-derived recombinant p24 (lk;licroGeneSys), V3 loop peptide (Multiple Peptide Systems, San Diego, CA), and baculovirus/insect cell protein preparation (MicroGeneSys) were each added in triplicate as the stimulating antigens. The V3 loop peptide was synthesized by solid phase procedures in conjunction with the method of simultaneous multiple peptide synthesis and consisted of 24 amino acids (HIV-I,, B strain, one letter amino acid code; N N T R K S I R I Q R G P G R A F V T I G K I G - a m i d e ) 4 ' t 2. The peptide preparation was 82.4% pure by analytical H P L C and the contaminating shorter peptides were deletion analogues. Phytohaemagglutinin-P ( P H A ; Difco Laboratories, Detroit, MI) at 5 p g m 1 - 1 was used as a non-specific mitogen to test lymphocyte responsiveness and was added to triplicate cell cultures 3 days after initially distributing the PBL into the culture plates. Unstimulated, medium control cultures included cells incubated in medium supplemented with 10%o human AB serum and antibiotics alone. Culture plates were incubated at 37°C for 6 days, 1.0 ~tCi of [3H]thymidine was then added to each well, the cells were harvested 20 h later, and counts per minute in each sample were determined in a liquid scintillation counter. Mean incorporation of [3H]thymidine was determined for triplicate determinations, and a stimulation index (SI) was derived by dividing the mean counts per minute incorporated in the presence of stimulating antigen or PHA by the mean counts per minute incorporated in the medium control cultures (cells with medium supplemented with only 10% human AB serum and antibiotics). The p24 antigen and the baculovirus/insect cell protein preparations were employed to document the degree of antigenic stimulation due to the contaminating insect cell and baculovirus proteins present in the rgpl60 preparation. Whole virus ELISA and Western blot antibody tests Serum from volunteers was tested for antibody to whole HIV-1 using commercially available kits by ELISA (Abbott Laboratories, North Chicago, IL) and by Western blot (DuPont, Wilmington, DE) according to manufacturers' guidelines.

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rgpl60 ELISA antibody test The initial step was to coat wells in 96-well Immulon 2 microtitre plates (Dynatech Laboratories, Chantilly, VA ) with rgpl60 (MicroGeneSys Inc. ) at 2 #g ml- 1. The plates were incubated at 4°C overnight and then washed the following day using water with 0.05% Tween-20. Serial 1 : 4 dilutions of test sera (lowest dilution was 1 : 25 ) were added, the plates were incubated at 37°C for 1 h and then washed again with the water plus 0.05% Tween-20. Background was determined by addition of the same serum dilutions to wells which had not been coated with rgpl60. The next step was the addition of rabbit anti-human IgG (gamma-chain) antibody conjugated with horseradish peroxidase (Dako Immunoglobulins, Denmark). After 30 min incubation at 37°C the plates were washed again with 0.05% Tween-20 in water and then orthophenylene diamine reagent (Abbott Laboratories, North Chicago, IL ) was added. After room temperature incubation for 30min, the reaction was stopped by the addition of 2.5 M H 2 S O 4. The absorbance was read at 490 nm. The antibody titre was that dilution of serum with an absorbance > 0.3 in rgp 160 coated wells which was at least twice background absorbance. Statistical methods Means of counts min -x values of l-3H]thymidine uptake and SI are reported with the standard error of the mean (SEM). Biologically significant lymphocyte proliferation was defined as an SI ~>3.0. A lack of statistical association between levels of I-3H] thymidine uptake by rgpl60 stimulated cells and that by PHA stimulated cells was confirmed using Kendall's rank correlation test.

RESULTS Four of the five vaccinees exhibited increased lymphocyte proliferation to rgpl60 following the third vaccine dose (Tables 1, 2 and 3). The remaining vaccinee (number 1, 40 #g dose recipient) did not have anti-HIV antibody detectable by whole virus ELISA or Western blot until after the fourth vaccine injection. All four 18-monthvaccine recipients exhibited increased lymphocyte proliferation to rgpl60 30 days following this vaccination. A higher level of proliferation measured at 30 days after the fourth dose was most obvious by SI, since in general the medium control [3H]thymidine uptake in counts min- 1 was lower at that time than at the two time points after the third vaccine dose although, in vaccinees 1 and 4, the rgpl60-stimulated counts min-1 were also higher than at 180 days following the third vaccine dose. Lymphocyte responsiveness to rgpl60 was demonstrable in only one of these four volunteers when measured 3 - 6 months after the fourth vaccine injection. The proliferative response to rgpl60 appeared less durable after the fourth vaccine injection than after the third. Of the volunteers' lymphocytes tested prior to the first vaccine injection, and control PBL from uninfected, nonvaccinated individuals run in parallel, none had a lymphocyte proliferative response to rgpl60 (SI 3 months postvaccination was better after three doses than after four, except in vaccinee number one who had essentially no response to vaccination until after the fourth vaccine dose. However, the proliferative response was definitely boosted by the fourth vaccine dose above levels measured 6 months after the third vaccination. Antibody to HIV-1 was less frequently detectable by whole virus ELISA than by Western blot and rgpl60 ELISA. Lymphocyte proliferation to the V3 loop peptide was not induced by baculovirus-derived rgpl60 vaccination in our study, although this peptide (amino acids 301 -324) overlaps with a cytotoxic T-cell epitope (amino acids 308-322 ) and a helper T-cell epitope cluster (amino

acids 317-348) 4. The cytotoxic T-cell epitope peptide (amino acids 308-322) has a sequence capable of folding in an amphipathic helical secondary structure, like that which has been found to be a good predictor of T-cell sites recognized by class II-restricted helper or proliferating T-cells is. One explanation for the lack of proliferation may be that the degree of overlap between the V3 loop and the helper T-cell cluster may not be sufficient, a n d / o r that the secondary structure of this peptide, while usually predictive of a helper T-cell epitope, was not in this case. However, Clerici et al. 16 reported that a peptide in the same region (amino acids 315-329) stimulated interleukin-2 production and lymphocyte proliferation by PBL from six of 29 and one of 22 asymptomatic HIV-seropositive donors, respectively. The induction of lymphocyte proliferation to V3 loop following priming with live recombinant vaccinia virus expressing gpl60 in the case of our positive control suggests that this peptide does not include a helper T-cell

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epitope and that the mode of antigen presentation to the immune system is an important factor in eliciting T-cell memory, as well. The clinical significance of lymphocyte proliferation to V3 loop peptide as a protective mechanism is currently unknown. Although deserving investigation, we did not explore anti-V3 loop cytotoxic T-cell activity which could occur since the V3 loop contains a cytotoxic T-cell epitope. While not investigated separately in our study, neutralizing antibody against HIV-1 tUBwas detectable in serum collected from vaccinee number 2 after the fourth rgpl60 dose at a 1:64 titre, but not in the other three recipients of a fourth dose at this institution 1. Thus, antibody against the V3 loop region or another neutralizing epitope may have been present in the serum of that vaccinee. Tacket et al. x7 and Keefer e t al. 18 reported similar levels of lymphocyte proliferation to rgpl60 following three injections of 40/~g or 80/~g of rgpl60 vaccine in volunteers at their institutions. Proliferative responses to rgpl60 were demonstrable in at least some vaccinees 28 days after the first rgpl60 vaccine dose 17'18. They did not report responses following a fourth vaccine dose, nor did they use peptide segments of HIV-1 envelope glycoprotein as stimulating antigens in vitro. This study indicates that a fourth injection boosted the lymphocyte proliferative response to rgpl60 even when given 12 months after the third injection, the boosted response post-fourth dose did not appear to be as durable as that following the third vaccine dose in three of our vaccinees, and no response to V3 loop peptide was detectable. Helper T-cell responsiveness was measured in a different way by Orentas et al. ~9 who reported isolation of CD4 + cytotoxic T-cell clones which lysed autologous target cells expressing gpl60 a n d / o r gpl20. The clones were derived from bulk PBL cultures obtained from three of eight recipients of a fourth injection of rgpl60 vaccine (40 #g or 80 big doses). One clone recognized a determinant contained within amino acid residues 546 645 of gp41. The results from the current study indicate that a baculovirus-derived HIV-1 recombinant envelope glycoprotein preparation can induce humoral and helper T-cell responses in humans. However, rgpl60 vaccination at these doses induced T-cell memory for portions of rgp 160 other than the V3 loop peptide. Whether this lack of sensitization was due to inter-subject variables such as restriction by major histocompatibility complex HLA type and might have been elicitable in other vaccinees if a larger sample had been tested is unclear. The presentation of gpl60 in the form of a non-replicating antigen to the host immune system may have limited any possible helper T-cell responses to the V3 loop peptide.

ACKNOWLEDGEMENTS The authors are grateful to Jean Reed, Mahendra Mandava, Gira Patel, Tina McNamee, Azamet Parsian and Joan Miller for technical assistance, and to June Bricker for secretarial assistance. This work was supported by the National Institute of Allergy and Infectious

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Diseases, contract no. NO1-AI-05064. We thank MicroGeneSys Inc., for provision of rgpl60, p24 and baculovirus/insect cell control protein for in vitro use in the lymphocyte proliferation assay.

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