Human Immunodeficiency Virus (HIV-l) gp160-Specific Lymphocyte Proliferative Responses of Mononuclear Leukocytes from HIV-l Recombinant gp160 Vaccine Recipients Michael C. Keefer, William Bonnez, Norbert J. Roberts, Jr., Raphael Dolin, and Richard C. Reichman

Infectious Disease Unit, Department of Medicine, University of Rochester School of Medicine and Dentistry, New furk

AIDS, caused by infection with the human immunodeficiency virus (HIV) ~ is a worldwide public health problem of enormous proportions. Development of effective methods to prevent HIV infection is paramount for control of AIDS and related disease, and some preventive measures such as education and serologic testing have already led to a decrease in HIV transmission in certain populations [1]. However, the current epidemic spread of sexually transmitted diseases and drug use [2,3] suggests that these measures cannot be relied upon as the sole means of prevention. The development of a safe and effective vaccine would greatly enhance our ability to control the AIDS epidemic. To address the need for HIV vaccines, the National Institute of Allergy and Infectious Diseases (NIAID), through its AIDS Vaccine Clinical Trials Network, is conducting phase I studies to evaluate the safety and immunogenicity of candidate vaccine preparations. The first NIAID-sponsored HIV type 1 (HIV-I)vaccine to enter phase I trials was a baculovirusexpressed recombinant envelope glycoprotein vaccine (rgpI60; MicroGeneSys, West Haven, CT). This vaccine has been

Received 18 June 1990; revised 14 September 1990. Presented in part: Vth International Conference on AIDS, Montreal, 1989. Informed consent was obtained from all subjects, and the studies were approved by the Research Subjects Review Board, University of Rochester School of Medicine and Dentistry. Financial support: National Institutes of Health (AI-52577, AI-25721); Wilmot Cancer Research Fellowship (to W.B.). Reprints or correspondence: Dr. Michael C. Keefer, Infectious Diseases Unit, Box 689, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642. The Journal of Infectious Diseases 1991;163:448-453 © 1991 by The University of Chicago. All rights reserved. 0022-1899/91/6303-0003$01.00

shown to be safe and to elicit a humoral immune response in a randomized, double-blind, placebo-controlled study of healthy individuals at low risk for HIV-I infection [3a]. Although induction of a humoral immune response is a wellrecognized feature of successful viral vaccines, induction of cellular immune responses will likely be an important additional characteristic of an effective vaccine for the prevention of HIV infection and disease [4]. To evaluate the cellular immune response to the baculovirus-expressed rgpI60 vaccine, we examined mononuclear leukocyte proliferative responses to gpI60 using cryopreserved peripheral blood mononuclear leukocytes (PBML) obtained from participants enrolled in the phase I trial at the University of Rochester.

Materials and Methods Study design. PBML were obtained from healthy, HIV-l-seronegative study volunteers at low risk for HIV infection. The rgp160 vaccine, produced using a baculovirus expression system and purified by gel filtration followed by lentil lectin affinity chromatography, was formulated with an alum adjuvant. The final product may have contained up to 30 % insect and baculovirus proteins by weight. Subjects were randomized to receive an intramuscular injection with either of two doses ofrgp160 (40 p,gor 80 p,g), another recombinant protein vaccine (Recombivax HB; Merck Sharp + Dohme, West Point, PA), or a placebo alum preparation (MicroGeneSys) at day 0, with boosters at 1 and 6 months. Serum was collected and frozen from each subject at study days 0, 14, 28, 42, 56, 120, 180, 194, 210,270,365,450, and 540. All sera were examined for anti-HIV-l antibodies using commercially available HIV-1 ELISA (Abbott Laboratories, North Chicago) and Western blot (Du Pont, Wilmington, DE) assays according to the manufacturers' instructions. PBML were obtained and cryopreserved from all subjects at days 0, 28, 56, 120,210, 270, 365, 450, and 540.

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The lymphocyte proliferative responses were studied of 12 volunteers enrolled in a phase I trial of a baculovirus-expressed recombinant human immunodeficiency virus type 1 (H1\'-1) envelope glycoprotein (rgpl60) vaccine. Six subjects received rgpl60 and three subjects each received recombinant hepatitis B vaccine or placebo at 0, 1, and 6 months. rgp160 and a control preparation, baculovirus-expressed recombinant Hl\'-1 p24~ were used as in vitro antigens. At day 56, all rgpl60 recipients had stimulation indexes (rgp160/rp24) >3.0, and five of six had differences in counts per minute (cpm) >1000. Stimulation indexes were 3.0. The six study participants who received hepatitis B vaccine or placebo had ~cpm values 95 %, was used as a control antigen (figure 1). Both antigens were received as 100 JLg/ml solutions in 5 roM Tris (pH 8), 150 mMNaCI, and 0.005% polysorbate 20 (rgpI60) or 0.005% Tween 20 (rp24). Separate PBML cultures containing purified phytohemagglutinin (PHA 16/17; Wellcome Laboratories, Beckenham, UK) at a final concentration ofO.5JLg/ml or RPMI 1640 alone were included in all assays. Lymphocyte proliferationassay. PBML cultures containing 1 x lOS cells in 0.2 ml RPMI 1640 supplemented with 0.02 M L-glutamine, 100 units of penicillin/ml, 50 JLg of gentamicin sulfate/ml, and 10% autologous plasma were established in U-shaped 96-well microtiter plates (Flow Laboratories, McLean, VA). rgp160 and rp24 antigens were added to quadruplicate wells in a concentration of 1 J,lg/ml, which was determined in preliminary experiments to produce optimal responses. Cultures were incubated at 37°C in 5 % C02 and 100% humidity for 6 days; 0.5 JLCi of tritiated thymidine (New England Nuclear, Boston) was then added to each well. Cultures stimulated with PHA were incubated for 2 days before addition of tritiated thymidine. After 16h of additional incubation, cultures were harvested using an automated cell harvester (Skatron, Sterling, VA)onto fiberglass filters. After drying, the filters were placed in liquid scintillation counting solution (Opti-fluor; Packard Instrument, Downers Grove, IL) and later counted in a liquid scintillation spectrometer (model LS9000; Beckman, Wakefield, MA), which yielded a counts per minute (cpm) value for each culture. The arithmetic mean cpm of quadruplicate cultures was determined and expressed both as Acpm and as a stimulation index (SI). Acpm was the difference in mean cpm/lff cells between cultures stimulated with rgp160 and cultures stimulated with rp24. SI equaled the mean cpm of cultures stimulated by rgp160 divided by the mean cpm of cultures stimulated by rp24. An SI >3.0 was considered positive. &pm values were used as an additional measurement to help confirm the result obtained by SI. A Acpm >1000 was considered suggestive of a positive result. All cpm data were interpreted blindly; the results of the serologic assays and the code defining vaccine recipients were not known before interpretation.


Keeferet al.


JID 1991;163 (March)

Table 1. Human immunodeficiency virus type 1 Western blot reactivity of Rochester vaccine study participants. Day of study 0*

Subject, vaccine




1-3, placebo 4-6, hepatitis B 7, rgpl60; 40 ug 8, rgpl60; 40 ug 9, rgpl60; 40 ug 10, rgpl60; 80 ug 11, rgpl60; 80f.tg 12, rgpl60; 80 ug











+ +

+ +




+ +

+ +

+ +




± ±





± ± ± ±










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. Q.


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Q. 0


Human immunodeficiency virus (HIV-1) gp160-specific lymphocyte proliferative responses of mononuclear leukocytes from HIV-1 recombinant gp160 vaccine recipients.

The lymphocyte proliferative responses were studied of 12 volunteers enrolled in a phase I trial of a baculovirus-expressed recombinant human immunode...
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