The Safety and Immunogenicity of a Human Immunodeficiency Virus Type 1 (HIV-1) Recombinant gpl60 Candidate Vaccine in Humans Raphael Dolin, MD; Barney S. Graham, MD; Stephen B. Greenberg, MD; Carol O. Tacket, MD; Robert B. Belshe, MD; Karen Midthun, MD; Mary Lou Clements, MD; Geoffrey J. Gorse, MD; Brian W. Horgan, PA; Robert L. Atmar, MD; David T. Karzon, MD; William Bonnez, MD; Bruce F. Fernie, ScD; David C. Montefiori, PhD; Donald M. Stablein, PhD; Gale E. Smith, PhD; Wayne C. Koflf, PhD; and the NIAID AIDS Vaccine Clinical Trials Network
Objective: To evaluate the safety and immunogenicity of a human immunodeficiency virus type 1 (HIV-1) recombinant envelope glycoprotein (rgpl60) candidate vaccine in humans. Subjects: Healthy adults (72) who were seronegative for HIV-1 were randomly assigned to one of four groups. Interventions: The subjects were randomly assigned to receive 40 or 80 /xg of rgpl60,10 /ig of hepatitis B vaccine, or placebo in three doses (on days 0, 30, and 180), with an elective, nonblinded administration of a fourth dose on day 540. Measurements and Main Results: Neither clinical nor laboratory toxicity was encountered during a follow-up period exceeding 21 months. No effect of immunization was noted on lymphocyte counts, mitogenic responses, or delayed-type hypersensitivity. Serum antibody responses to HIV envelope proteins detected by Western blot were seen in 30 of 33 subjects (91%; 95% CI, 71% to 97%) receiving either 40- or 80-/*g doses of rgpl60 and were most commonly of weakly reactive intensity. Responses were first noted by Western blot after the second dose. They markedly increased in frequency after the third dose and declined over the next 12 to 18 months. The administration of a fourth dose resulted in homologous neutralizing activity in sera from 5 of 24 subjects (21%; CI, 7% to 37%) as well as in complement-mediated antibody-dependent enhancement in sera from 6 of 24 subjects (25%; CI, 10% to 42%). Antibody responses were detected by enzyme-linked immunosorbent assay (ELISA) less frequently than by Western blot, and these responses persisted for a shorter time. Conclusions: The administration of rgpl60 was well tolerated and safe, resulted in a high rate of antibody response by Western blot after the administration of the third and fourth doses, and generated serum neutralizing activity and complement-mediated antibody-dependent enhancement in some subjects after the fourth dose.
Annals of Internal Medicine. 1991;114:119-127. From the University of Rochester School of Medicine and Dentistry, Rochester, New York; Vanderbilt University, Nashville, Tennessee; Baylor College of Medicine, Houston, Texas; University of Maryland at Baltimore, Baltimore, Maryland; St. Louis University School of Medicine, St. Louis, Missouri; Johns Hopkins University School of Hygiene and Public Health and School of Medicine, Baltimore, Maryland; Georgetown University, Rockville, Maryland; The Emmes Corporation, Potomac, Maryland; MicroGeneSys, Inc., West Haven, Connecticut; and the NIAID AIDS, Rockville, Maryland. For current author addresses, see end of text.
1 he development of a safe, effective vaccine against human immunodeficiency virus (HIV) infection or HIVassociated disease (or both) is the object of intense research efforts throughout the world. The formulation and evaluation of candidate vaccines confront formidable conceptual and practical obstacles (1-3). Despite the fact that considerable information has emerged about the molecular virology and antigenic characteristics of HIV as well as about host responses to HIV infections, the critical measures of immunity and protection from HIV infection and HIV-associated disease are not yet fully understood. In addition, it has been suggested that some types of immune responses to HIV antigens might themselves contribute to immunosuppression (4-6) or might even enhance subsequent infection with HIV (7, 8). The recent establishment of practical animal models of infection with HIV or HIV-like agents and the evaluation of vaccine candidates in these models may generate information that will be important in the development of HIV vaccines (9, 10). Nonetheless, information about the behavior of candidate HIV vaccines in humans, particularly about the safety and immunogenicity of these vaccines (that is, "phase I data"), is clearly needed at early stages of development as well. The National Institute of Allergy and Infectious Diseases (NIAID) Acquired Immunodeficiency Syndrome (AIDS) Vaccine Clinical Trials Network therefore conducted a phase I trial of a candidate HIV subunit vaccine, rgpl60. The Network selected rgpl60, an HIV-1 envelope glycoprotein generated through recombinant DNA technology, as the first HIV vaccine candidate to be studied in humans for several reasons. First, because the HIV-1 envelope glycoprotein, gpl60, contains the epitope or epitopes against which anti-HIV neutralizing antibodies have been detected (11) as well as the site of attachment of HIV to the CD4 receptor (12), it is highly relevant for inclusion in vaccine preparations. Second, the use of a highly purified "recombinant" protein bypasses concerns of possible contamination of vaccine preparations with HIV nucleic acids or other HIV proteins. Finally, vaccines comprised of surface proteins have been successfully generated against some viral infections, such as those caused by hepatitis B viruses (13). This initial trial was carried out in healthy volunteers who were seronegative for HIV and who appeared to be at low risk for acquisition of HIV infection. Because ©1991 American College of Physicians
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this trial was one of the first in which an HIV vaccine candidate was used in humans, assessment of safety and immunogenicity was necessarily complex. Subjects were observed for acute reactions to the vaccines as well as for potential clinical and laboratory evidence of long-term adverse effects. These studies also included in-vitro and in-vivo assessments of possible immunosuppression. The immunogenicity of this candidate HIV vaccine was assessed by sequential measurements of serum antibodies to HIV by Western blot, enzymelinked immunosorbent assay (ELISA), and neutralization assays. The studies also included two control groups: One group received a placebo preparation, and the other received hepatitis B vaccine, a licensed vaccine whose reactogenicity and immunogenicity have been well characterized. Materials and Methods Recombinant gpl60 Vaccine The HIV vaccine candidate rgpl60, a glycoprotein of the envelope of HIV-1, was developed by MicroGeneSys, Inc., West Haven, Connecticut. The rgpl60 gene was derived from an infectious molecular clone of HIV-1, pNL4-3 (provided by Dr. Malcolm Martin, Laboratory of Molecular Microbiology, NIAID, Bethesda, Maryland) (14). The nucleotide sequence of this gene was previously reported by Wain-Hobson and colleagues (15). A baculovirus expression vector that contained the rgpl60 gene under the transcriptional control of the Autographa catifornica nuclear polyhedrosis virus polyhedrin promoter was constructed (16). The rgpl60 was produced in a continuous insect cell line derived from Spodoptera frugiperda by infection with the recombinant virus. The infected cells produced a membrane-bound rgpl60 that was extracted with sodium deoxycholate. The antigen was purified by gel filtration followed by lentil lectin affinity chromatography. Purified rgpl60 was dialyzed against 10-mM tris buffer, pH 8.0, and then formulated with an aluminum phosphate gel (alum adjuvant). Subjects Study subjects were healthy adult volunteers between 18 and 55 years of age and without evidence of serious medical illness, as determined by a history and a physical examination. In addition, the study subjects' laboratory values, including the hematocrit result, leukocyte count and differential, platelet count, total lymphocyte count, total T4-cell count (> 400 cells/ mm3), serum alanine aminotransferase level, creatinine level, and urinalysis result, were within the normal ranges. Study subjects did not have a history of immunodeficiency or of treatment with immunosuppressive medications. They had a reaction of at least 2 mm of induration to one or more of the antigens (other than tuberculin) in the Mlrieux Multitest battery of skin tests (see below). All candidate subjects with a reaction to tuberculin were subsequently skin-tested by intermediate-strength purified protein derivative, and, if induration of greater than 5 mm was noted, they were ineligible for participation. Women of childbearing potential who participated in the study had a negative pregnancy test at entry and agreed to use adequate birth control measures throughout the study and follow-up period. Study subjects were seronegative for HIV-1, as determined by ELISA and Western blot assays (see below) before immunization. Cultures of peripheral blood mononuclear cells for HIV-1 and polymerase chain reactions to detect HIV-1 genome in mononuclear cells obtained before entry from these subjects were also negative. In an attempt to reduce the likelihood of confounding the results of the study by an intercurrent HIV infection, persons whose behavior was identified by a screening questionnaire and interview as a factor putting them at high risk for HIV infection were excluded. In addition, persons with 120
a history of blood transfusions or who had received cryoprecipitates within 6 months of entry were also ineligible for participation. Because hepatitis B vaccine was to be administered to a "control" group in the study, study subjects also had to be seronegative for hepatitis B surface antibody, hepatitis B core antibody, and hepatitis B surface antigen. Subjects were recruited at each of the participating sites using procedures that previously had been used for recruiting normal volunteers for studies of other (non-HIV) vaccines by the NIAID vaccine evaluation centers. Recruitment activities also included widespread information campaigns describing the studies in local media and in meetings of community organizations concerned with the control of AIDS. Recruitment was followed by extensive discussions with persons who expressed an interest in participation. All study subjects signed informed consent forms for screening procedures as well as for participation in the study before entry. The informed-consent form contained a description of the possible risks of participation, including potential problems that might be caused by the development of positive HIV serology as determined by conventional ELISA tests. To address this latter problem, study subjects were provided with an identification card that stated that they were participants in an NIAID-sponsored AIDS vaccine trial and which contained a toll-free telephone number that could be called to verify such participation through the NIAID AIDS Program. The Program's commitment to maintaining the confidentiality of the results of HIV laboratory tests obtained in the course of the study was also stated in this form. Experimental Design Subjects were randomly assigned, in a double-blind fashion, to receive one of four preparations: 40 /xg of rgpl60, 80 p% of rgpl60, 10 p% of hepatitis B vaccine (Recombivax, Merck, Sharp and Dohme, Rah way, New Jersey), or placebo (alum adjuvant). Each of the study preparations was administered intramuscularly in the deltoid muscle in a volume of 1.0 mL according to the following schedule: initial immunization (day 0), a first booster dose at 1 month (day 30), and a second booster dose at 6 months (day 180). An independent data and safety monitoring board reviewed the findings of the study after the initial and booster doses were administered and approved administration of subsequent doses. After the data from the three-dose regimen of immunization were analyzed, a fourth dose was offered in a nonblinded manner to those subjects who wished to participate further in the study approximately 1 year after the administration of the third dose (day 540). Clinical Evaluation After intramuscular administration of the first dose, subjects were observed for 1 hour and were instructed to take their temperatures at home, 6 and 12 hours later. Any untoward effects that developed in the first 24 hours were reported to the study team and evaluated at that time. Symptoms, if present, were graded as mild, moderate, or severe. Subjects returned to the study site on days 1, 2, 3, and 4 for examination and clinical evaluation. For the next 10 days, subjects reported any symptoms that developed. Subjects then returned to the study site on days 14 and 28 for clinical evaluation. After administration of each booster dose (on days 30, 180, and 540), subjects were evaluated according to the follow-up schedule that was used after the initial immunization. In addition, subjects were clinically evaluated on days 60, 120, 270, 365, 455, and 635 after vaccination. Delayed-Type Hypersensitivity Tests The M6rieux Multitest battery of skin tests for delayed-type hypersensitivity was applied before initial immunization and on days 60, 210, and 365 (17). The battery contains a glycerin control and the following antigens: tetanus toxoid, diphtheria toxoid, Streptococcus group C, old tuberculin, Candida, Trichophyton, and Proteus. Skin tests results were noted at 48 hours, and the size of induration was recorded. A positive
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reaction to an antigen was defined as induration of 2 mm or greater induration than was associated with the glycerin control. Laboratory Studies Complete blood counts, differentials, platelet counts, total lymphocyte counts, T4 and T8 subset counts, serum alanine aminotransferase levels, and creatinine levels were obtained before immunization and on days 14, 28, 44, 60, 120, 178, 194, 210, 270, 365, 455, 540, 559, 575, and 635. These laboratory values were determined by the clinical hematology, chemistry, and immunology laboratories at each of the participating institutions. Serum specimens and peripheral blood mononuclear cells, a portion of which were immediately cryopreserved, were obtained before immunization and on days 14, 28, 44, 60, 120, 178, 194, 210, 270, 365, 455, 540, 559, 575, and 635. Cryopreserved mononuclear cells were intended for use in future studies of cell-mediated immunity, which were not a formal part of the multicenter trial. Serum specimens were assayed for antibody to HIV using several assays. Western blot assays were done using the commercially available Biotech-DuPont kit (DuPont Co., Wilmington, Delaware) (18). Reactions at each virus-associated band were graded as either absent (-); present, but less intense than the p24 band on the weakly reactive control strip (±); present and at least as intense as the p24 band on the weakly reactive control strip, but less intense than p24 on the strongly reactive control strip (+); or present and greater than or equal to the intensity of p24 on the strongly reactive control strip (+). Western blot assays were done in laboratories at the individual participating sites as well as at the central immunology laboratory (see below). For purposes of analysis, a serum specimen was considered to be positive if it was reactive by Western blot at both the participating site and the central unit. If the intensity of responses differed between the participating and central sites, the less intense grade was used for analysis. Enzyme-linked immunosorbent assays were done using the commercially available Abbott kit (Abbott Laboratories, North Chicago, Illinois) (19). The optical density was read at 492 nm, and serum specimens were interpreted as positive if the optical density value was greater than a calculated cutoff value (for example, a ratio of optical density to cutoff of more than one). These assays also were done at the individual study sites and the central unit, and arithmetic means of the values were calculated. A serum specimen was considered to be positive if positive values were obtained at both the individual site and the central unit. Neutralization assays were done at two sites: the central immunology laboratory at Georgetown University (Fernie and colleagues) where a previously described microtiter syncytiaformation inhibition assay in Molt-3 cells was used (20); and Vanderbilt University (Montefiori and colleagues) where a microneutralization assay in MT-2 cells, which used neutral red dye uptake as a measure of cytopathic effect, was used (21). The homologous HIV-1 HIB virus was used as the challenge strain in all of the above assays. In addition, serum responses were examined for complement-mediated antibody-dependent enhancement by Montefiori and colleagues (7) as previously described. Fresh human serum 1:40 was used as a source of complement.
shall University, Johns Hopkins University, University of Maryland, University of Rochester, and Vanderbilt University. At each institution, a detailed study protocol was approved by the respective institutional review boards before the initiation of the study, and the study was conducted according to the human experimentation guidelines of the U.S. Department of Health and Human Services. A central immunology laboratory at Georgetown University coordinated the immunologic studies and also did neutralization, Western blot, and ELISA assays. Data entry was carried out at each of the six AVEUs, and data were transmitted electronically to Emmes Corporation, Potomac, Maryland, which served as the central data management and statistical analysis unit. Results Subjects A total of 72 subjects participated, 12 at each of the six AVEUs. The first 36 volunteers were immunized between 28 February and 10 March 1988, and the second 36 volunteers were immunized between 9 April and 6 May 1988. The subjects ranged in age from 19 to 53 years (Table 1). Fifty-four percent of the subjects were women; 86% described themselves as heterosexual; and 14% described themselves as homosexual. Of the 72 subjects who entered the study, all but 5 completed the three-dose regimen. Of the 5 who did not, 2 received two doses of hepatitis vaccine; 1 received one dose of rgpl60, 40 /Ltg; 1 received one dose of rgpl60, 80 /ig; and 1 received two doses rgp!60, 80 /ig. The reasons for failure to complete the immunization regimen were unrelated to the development of adverse effects. Two subjects changed their minds about participating during the study, 1 moved away, and 2 acquired HIV infection while enrolled in the study and thus were ineligible to continue to participate. Neither of the latter 2 subjects was in the group that received the rgpl60 preparations, and both subjects have been referred for appropriate medical follow-up care. Infection in these 2 subjects was identified by the development of Western blot bands to HIV proteins other than the gpl60, gpl20, or gp41 proteins contained in the vaccine, which resulted in a Western blot pattern typical of HIV infection. The remaining 67 subjects have complied with 99.5% of subsequent visits related to the first three doses and have been followed for as many as 21 months. Of the subjects who completed the three-dose regimen, 52 elected to receive a fourth dose: 16 in the placebo group, 12 in the hepatitis B vaccine group, 14 in the rgpl60 group receiving the 40-/ug doses and 10 in the rgpl60 group receiving the 80-/ug doses.
Statistical Methods
Reactogenicity
Repeated measures analysis of variance was used to assess relative changes in individual hematologic and chemical safety measures. Other standard nonparametric tests for assessing the frequency and intensity of response were used. The sample size provides pairwise 80% power to detect a standard deviation change of one or more in mean paired differences for any univariate outcome. All P values are two-tailed and are not adjusted for multiple testing. Confidence intervals (CIs) of 95% were used.
All of the study preparations, including rgpl60, were well tolerated. Febrile reactions, as defined by an oral temperature of more than 37.4°C within 48 hours of immunization, were seen in 4 of 18 recipients of 40-jig doses of rgpl60, 4 of 18 recipients of 80-/ug doses of rgpl60, 3 of 18 recipients of hepatitis B vaccine, and 4 of 18 recipients of placebo. No subject manifested an oral temperature greater than 38.0°C within 48 hours after vaccination. Only 5 subjects reported severe systemic symptoms (headache, malaise, myalgias, or feverish feeling) after immunization. One of these subjects
Participating Sites The study was conducted at the six NIAID AIDS Vaccine Evaluation Units (AVEUs): Baylor College of Medicine, Mar-
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was in the placebo group; 2 were in the rgpl60 group receiving the 40-/ug doses; and 2 were in the rgpl60 group receiving the 80-/ug doses. Mild pain and tenderness, generally lasting less than 24 hours, were frequently present at the injection site in the recipients of all four study preparations. One placebo recipient and 2 recipients of the 80-/ug doses of rgpl60 reported transient severe pain after one of the injections. Small amounts of induration at the injection site (mean, 13 ± 3 mm) were noted in some recipients of all the study preparations. The frequency of induration was somewhat higher after injection of 40-/ug doses of rgpl60 (5 of 18 subjects) and 80-jtg doses of rgpl60 (8 of 18 subjects) than after injection of hepatitis B vaccine (2 of 18 subjects) or placebo (1 of 18 subjects) (P = 0.02). No other untoward event was noted during the study.
Immunogenicity Western Blot Antibodies Responses to HIV envelope antigens were detected by Western blot only in subjects who received rgpl60. The majority of antibody responses were to gpl60, gpl20, or both, whereas responses to gp41 were seen less frequently and occurred in subjects who had particularly strong responses to gpl60 and gpl20. The frequency and intensity of the serum antibody responses to gpl60 and gpl20 that were detected by Western blot are presented in Figure 2. Among subjects who completed the three-dose schedule, 16 of 17 who received the 40-/xg doses and 14 of 16 who received the 80-/ug doses manifested serum antibodies that were detected by Western blot at least once during the study (total, 30 of 33 subjects [91%; CI, 71% to 97%]). Antibody responses were initially detected by Western blot after the administration of the second dose (23% to 44%). The frequency of response markedly increased after the administration of the third dose (76% to 81%) and gradually declined to 12% to 13% after 15 to 18 months (days 455 to 540). After the administration of the fourth dose (day 540), the frequency of response rose again (79% to 90%) and appeared to be declining at the most recent follow-up evaluation, on day 635 (Figure 2). Western blot bands confirmed at two laboratories were exclusively ± or + in intensity (see the Methods), and the distribution of the intensity of reactions was similar after the administration of the third and fourth doses. Antibodies to gp41 were detected by Western blot less frequently, developed later in the study, and were of shorter duration than were anti-gpl60 and anti-gpl20 antibodies in recipients of either 40-/ig or 80-/xg doses (Figure 3). Anti-gp41 antibodies were first noted after the administration of the third dose, became undetectable from day 270 to day 365, and were seen again after the administration of the fourth dose on day 540 (Figure 3). There was no statistically significant difference in the frequency of antibody responses detected by Western blot between recipients of 40-/Kg doses and recipients of 80-/xg doses when it was examined during the study (P > 0.1); however, a somewhat higher rate of antibody response was detected by Western blot on day 120, after the administration of the second dose in the group
Laboratory Toxicities Using repeated measures analysis of variance, there were no important differences in hemoglobin level, total leukocyte count, lymphocyte count, platelet count, serum alanine aminotransferase level, or creatinine level among the study groups during the study (data not shown). Nor were there important differences among the study groups in CD4 or CD8 cell counts or percentages or in CD4:CD8 ratios during the study (Figure 1). Fresh or cryopreserved mononuclear cells were examined for mitogenic responses to phytohemagglutinin or concanavalin A at four of the study sites (Johns Hopkins, University of Maryland, University of Rochester, and Vanderbilt University). There were no important differences in mitogenic response among the study groups when they were examined during the study (data not shown).
Skin Test Reactivity As presented in Table 2, there were no important differences in skin test reactivity, either in the number of antigens to which subjects responded or in the cumulative size of the response to antigens, among the four study groups.
Table 1. Characteristics of Study Subjects Characteristic
Total
Treatment Group Placebo
Hepatitis B Vaccine
n
Sex Women
Men Sexual preference Homosexual Heterosexual Age, y Median Range
10 8
10 8
10 8
9 9
39 33
1 17
4 14
3 15
2 16
10 62 35
39
32
35
35
27-46
22-52
19-46
20-53
* Human immunodeficiency virus type 1 recombinant glycoprotein. 122
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specimens that were defined as positive by Western blot, 45 (27%) were defined as positive by ELISA.
Figure 1. Mean circulating CD4+ and CD8+ counts in subjects who received placebo (•), hepatitis B vaccine (O), 40-/ug doses of rgpl60 (A), or 80-/ig doses of rgpl60 (A). Arrows indicate when immunizations were administered.
Antibodies Detected by Neutralization Assays Assays for neutralizing antibodies were done on all serum specimens that were defined as positive by Western blot as well as on selected serum specimens that were defined as negative by Western blot by laboratories at Vanderbilt University and Georgetown University. Neutralization activity was not detected in any serum specimen until day 559 (14 days after the administration of the fourth dose) when Montefiori and colleagues at Vanderbilt University detected neutralizing activity in 5 of 24 serum specimens (21%; CI, 7% to 37%): Such activity was noted in the serum specimens of 3 of 14 subjects who received the 40-/ug doses and in the serum specimens of 2 of 10 subjects who received the 80-/ig doses (Table 3). It was detected using the microtiter assay in MT-2 cells, as noted in the Materials and Methods (21). Neutralizing activity was not detected in the same serum specimens in assays carried out in Molt-3 (20) cells by Fernie and colleagues at the central immunology laboratory at Georgetown University. Complement-mediated antibody-dependent enhancement, also first seen on day 559, was noted in 6 of 24 subjects (25%; CI, 10% to 42%): in 1 of 14 who
receiving the 80-/ig doses compared with the group receiving the 40-//g doses (P = 0.057; the Fisher exact test) (Figure 2). Antibodies Detected by Enzyme-Linked Immunosorbent Assay Seventeen percent of subjects who received the 40-/Ltg doses and 39% of those who received the 80-/ig doses manifested antibodies detected by ELISA at least once during the study. In general, antibodies were detected less frequently and for a shorter duration of time by ELISA than by Western blot. Antibodies were not detected by ELISA until after the administration of the third dose (day 194), when they were detected in 2 of 17 recipients (12%; CI, 1% to 29%) of the 40-/*g doses and in 7 of 16 recipients (44%; CI, 20% to 65%) of the 80-/ug doses (P = 0.039). The number of antibodies detected by ELISA waned rapidly; they were not detected in recipients of the 40-/*g or 80-/ug doses by days 270 and 365, respectively. After the administration of the fourth dose (day 540), antibodies were detected by ELISA in 14% of subjects who received the 40-/xg doses and in 70% of subjects who received the 80-/ug doses (P = .005). The mean ratios of observed optical density to cutoff values for ELISA in specimens obtained at various times during the study are presented in Figure 4. Two weeks after the administration of the third dose (day 194), there was a trend toward higher mean ratios in subjects who received the 80-/xg doses (1.42) than in subjects who received the 40-/ig doses (0.74) (P < 0.1). Two weeks after the administration of the fourth dose (day 559), the ratios were significantly higher in recipients of the 80-/Ltg doses (2.32) than in recipients of the 40-jig doses (0.67) (P = 0.04; Wilcoxon rank sum test). Of the 46 serum specimens that were defined as positive by ELISA, all but 1 (45 of 46) were also positive by Western blot, 41 with + intensity and 4 with ± intensity (see the Methods). Conversely, of the 168 serum
Figure 2..The percentage of subjects whose serum specimens were positive for antibody to gpl60, gpl20, or both by Western blot, according to the study day. The shading of bars indicates the intensity of the Western blot reactions, as graded on a semiquantitative scale described in the Methods (D = ±; • = +). Subjects received either 40-/ig doses of rgpl60 (top) or 80-/ug doses of rgpl60 (bottom). Arrows indicate when immunizations were administered.
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Table 2. The Number and Size of Reactions to Antigens among Study Groups Study Preparation Before Immunization (n = 72) Hepatitis B vaccine Placebo rgpl60t 40-/ug doses SO-fig doses
Application of M6rieux Skin Test* Day 210 Day 60 (n = 69) (n = 55)
Day 365 (n = 66)
2.61 (10.51) 2.67 (12.99)
2.18(8.81) 2.61 (12.03)
2.92 (12.08) 3.40 (13.99)
2.63(9.13) 2.67(10.15)
3.33 (12.26) 3.28(13.31)
2.88(11.56) 3.24 (12.58)
2.75 (10.78) 4.23 (17.82)
2.67 (9.44) 2.71 (9.88)
* The data are given as the mean number of antigens with an induration reaction of 2 mm or more compared with that of the glycerin control at 48 hours. The numbers in parentheses are the mean of the sum of induration (in millimeters) of the seven antigens minus induration of the glycerin control. t Human immunodeficiency virus type 1 recombinant glycoprotein.
received the 4 0 - ^ doses and in 5 of 10 who received the 80-/ug doses (Table 3). Three subjects had both complement-mediated antibody-dependent enhancement and neutralizing activity, 2 had neutralizing activity alone, and 3 had complement-mediated antibody-dependent enhancement alone. Neither neutralization nor complement-mediated antibody-dependent enhancement was seen in subjects who received hepatitis B or placebo preparations. Assays of serum specimens obtained after day 559 as well as assays with heterologous HIV strains will be done in the future.
Additional Observations A 37-year-old volunteer who received 40-/ig doses of rgpl60 became pregnant, despite her agreement to use adequate birth control measures during the study and admonitions about the importance of adhering to this requirement. The estimated date of conception was approximately day 210 of the study or 30 days after the administration of the third dose. This subject developed serum antibody responses of -I- intensity (see the Methods) by Western blot to gpl20 on days 1% and 210. Responses detected by Western blot eventually decreased to ± on day 288 and, thereafter, have been absent when examined through day 540. This subject never manifested serum antibodies detected by ELISA. On day 497, the subject delivered a normal, full-term infant without complications, except for a transient postpartum fever, and both mother and child are currently well. At the time of delivery and 1 month later, both mother and child tested negative for serum antibody to HIV by Western blot and ELISA. Discussion
Figure 3. The percentage of subjects whose serum specimens were positive for antibody to gp41 by Western blot, according to the study day. The shading of bars indicates the intensity of Western blot reactions, as graded on a semiquantitative scale described in the Methods ( • = ±; • = +). Subjects received either 40-/ug doses of rgpl60 (top) or 80-/ug doses of rgpl60 (bottom). Arrows indicate when immunizations were administered. 124
We conducted a phase I study of a candidate HIV vaccine, rgpl60, in healthy adult volunteers who were seronegative for HIV-1. In this population, administration of 40-/Ag or 80-/ug doses of rgpl60 in a three- or four-dose regimen appeared to be safe and well tolerated. Systemic reactions were uncommon and occurred no more frequently than in the groups that received either a placebo preparation or a licensed hepatitis B vaccine. Local reactions to rgpl60 administration were also mild and similar to those seen in recipients of hepatitis B vaccine or placebo preparations, although induration was noted somewhat more frequently in recipients of rgpl60. No evidence of long-term clinical or laboratory toxicities has been noted in a follow-up period that has now exceeded 21 months. To examine whether potential suppression of immune function might be associated with the administration of rgpl60, we also assayed several measures of immune function in our subjects. In these assessments, no effect of immunization was noted on total counts or percentages of circulating lymphocytes or on CD4 or CD8 lymphocyte subset counts. Nor did the administration of rgpl60 affect delayed-type hypersensitivity, as assayed by the application of seven antigens on days 0, 60, 210, and 365 of
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the study. Although assessment of in-vitro cell-mediated immune activity was not a formal part of the multicenter trial, mononuclear ceils from subjects at four of the participating sites were examined for mitogenic responses to phytohemagglutinin and concanavalin A, and suppression of proliferative responses was not noted. No evidence of immunosuppression, at least as assessed by the above in-vitro and in-vivo measures of immune function, was seen in our study. The immunogenicity of rgpl60 was assessed by measuring serum antibodies to HIV-1 using Western blot, ELISA, and neutralization assays. Serum antibody responses, directed primarily at gpl60 or gpl20 antigens, were detected by Western blot in 30 of 33 subjects who completed the three-dose regimen, and there was no statistically significant difference in responses detected by Western blot between the group that received the 40-pg doses and the group that received the 80-/ug doses P > 0.1. Despite this high frequency of responses, the intensity of antibody reactions detected by Western blot was generally low, with reactions confirmed at two laboratory sites falling into the ± or + categories (see the Methods). In general, these reactions detected by Western blot were of considerably lower intensity than those seen after naturally occurring HIV infection. Antibody responses were not detected by Western blot until after the administration of the second dose, and the frequency of responses clearly increased after the administration of the third dose on day 180. The seroprevalence rates of antibodies detected by Western blot gradually declined after the administration of the third dose, so that only 12% to 13% of subjects had antibodies detected by Western blot 15 to 18 months after the administration of the first dose. The administration of a fourth dose of 40 p% or 80 fjug of rgpl60 approximately 1 year after the administration of the third dose (day
Figure 4. The mean optical density (OD) to cutoff ratios in enzyme-linked immunosorbent assays for anti-HIV antibodies done on serum specimens obtained on various study days from all subjects who received placebo (•), hepatitis B vaccine (O), 40-/ug doses of rgpl60 (A), or 80-/xg doses of rgpl60 (A). Arrows indicate when immunizations were administered.
Table 3. Neutralizing Activity and Complement-Mediated Antibody-Dependent Enhancement (C'-ADE) at Day 559 in Serum Specimens from Recipients of rgp!60 Dose of rgpl60 vaccine, pg*
40
80
Neutralizing Titert
C'-ADE
128 64 32 < 16 < 16 < 16 < 16 < 16 < 16 < 16 < 16 < 16 < 16 128 64 < 16 < 16 < 16 < 16 < 16 < 16 < 16 < 16
Titert
16 < 16
< 16 < 16 < 16 < 16 < 16 < 16 < 16 < 16 < 16 < 16 < 16 64 32 32 32 16 < 16 < 16 < 16 < 16 < 16
* Human immunodeficiency virus type 1 recombinant glycoprotein. t As determined in the Methods (21). t As determined in the Methods (7).
540) resulted in a high rate of responses as detected by Western blot (79% to 90% of recipients). These rates appear to decrease gradually over the following 3 months, according to the latest available results (day 635). Serum antibodies were detected by ELISA (using a lysate of HIV-1 as a source of antigen) less frequently and for a shorter time than by Western blot. Recipients of the SO-fig doses had statistically significant, more frequent, and greater antibody responses detected by ELISA after the administration of the fourth dose of vaccine compared with those who received the 40-/ig doses, and a similar trend was present after the administration of the third dose. A recent observation indicates that antibody responses can be detected more frequently by ELISA when the immunogen rgpl60, rather than a whole virus lysate, is used as the antigen in the assay (22). The functional activity of serum antibodies detected by Western blot or ELISA techniques is uncertain with respect to protection against HIV infection or HIVassociated disease. We were therefore particularly interested in determining whether serum neutralizing antibodies were generated in our studies. Using an assay in MT-2 cells (21), modest levels of neutralizing activity against homologous virus were detected in sera from 5 of 24 subjects 2 weeks after the administration of a fourth dose of rgpl60, but not in samples that were obtained earlier. However, neutralizing activity was not detected in an assay in which Molt-3 cells were used (20). Whether the differences in these results reflect differences in the sensitivity of these assays in detecting relatively low levels of neutralizing antibodies generated
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by rgpl60 or whether these differences relate to other properties of the two assays is unclear and is under investigation. At present, the neutralizing activity of these serum specimens against heterologous HIV-1 strains has not been studied; such studies are planned for the future. The results of such studies are of considerable interest, because the ability to stimulate protection against several HIV-1 strains will be a critical goal in the development of effective HIV-1 vaccines. We also detected low levels of complement-mediated antibody-dependent enhancement in six serum specimens. These levels are considerably lower than those seen after naturally occurring HIV-1 infection, in which mean titers of 5000 have been seen (unpublished data). Although the role of complement-mediated antibodydependent enhancement in potentiating HIV infection in vivo is currently unknown, candidate HIV vaccines' ability to induce antibody enhancing activity should continue to be carefully monitored. As noted earlier, the assessment of the immunogenicity of rgpl60 in our collaborative trial focused on the development of humoral immunity, because of uncertainty about which in-vitro assays were optimal for assessing cell-mediated responses and because of the difficulty in standardizing such assays in a multicenter study. In-vitro studies of lymphocyte proliferative responses to HIV antigens in subjects in this study have been carried out by investigators at the University of Rochester and the University of Maryland and are reported in detail elsewhere (23, 24). The studies of Keefer and colleagues (23) showed that lymphocyte proliferative responses to purified rgpl60 developed in six of six recipients of rgplfSO, that responses in some instances developed before Western blot reactivity was noted, and that lymphocyte proliferative responses persisted for at least 18 months, even in subjects who no longer had detectable anti-HIV antibody by Western blot. In addition, circulating cytolytic T-cells directed at gpl60 recently have been detected in three of eight of the above subjects who received rgpl60 (25). In summary, the studies that we have conducted have defined some measures of the safety and immunogenicity of a candidate HIV-1 subunit vaccine. At the concentrations of immunogen that we examined and using a three-dose regimen, serum antibody responses occurred frequently, but were of low levels and of relatively short duration. The administration of a fourth dose of immunogen 1 year after the administration of the third dose resulted in a high incidence responses according to Western blot. At that time, the development of antibodies with certain functional properties was first noted in some subjects; in particular, neutralizing activity against the homologous virus strain was detected, albeit at low levels and in only one of two in-vitro assay systems that were used. No statistically significant difference P > 0.1 was noted in responses detected by Western blot or neutralization assays between recipients of the 40-/ug doses and recipients of the 80-/ug doses, although the frequency of responses detected by ELISA was higher in the group receiving the 80-/ig doses, particularly after the administration of the fourth dose. Our findings suggest that additional studies that examine the effects of using higher doses of rgpl60 are 126
warranted. Escalating-dose studies of rgpl60 whose experimental designs are somewhat different from that used in our study are currently being conducted by Kovacs and co-workers (26) at the NIH and will also be conducted by the NIAID AIDS Vaccine Clinical Trials Network. Different regimens of immunization with rgplfSO clearly need to be investigated as well. Our results suggest that at least a three-dose regimen will likely be required and that the administration of a fourth dose may be advantageous. Our results show that phase I investigations of candidate HIV vaccines can be conducted efficiently and successfully in healthy, HIV-seronegative subjects. Highly motivated and cooperative subjects can be recruited, and clinical and laboratory measures of the safety and immunogenicity of candidate vaccines can be assessed over extended periods. The results of our studies should be useful for the planning and conduct of phase I studies of other HIV candidate vaccines in humans as well as for further studies of rgpl60. Grant Support: By contracts (N01-AI-52577, N01-AI-72624, N01-AI62528, N01-AI-52575, N01-AI-52576, N01-AI-72623, N01-AI-82500, N01-AI-62515) from the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland. Requests for Reprints: Raphael Dolin, MD, Infectious Disease Unit, Department of Medicine, University of Rochester Medical Center, 601 Elmwood Avenue, Box 689, Rochester, NY 14642. Current Author Addresses: Drs. Dolin and Bonnez: University of Rochester Medical Center, 601 Elmwood Avenue, Box 689, Rochester, NY 14642. Drs. Graham, Karzon, and Montefiori: Vanderbilt University, Medical Center North, Nashville, TN 37232. Drs. Greenberg and Atmar: Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Dr. Tacket and Mr. Horgan: University of Maryland, Center for Vaccine Development, 10 South Pine Street, MSTF Building, Baltimore, MD 21201. Drs. Belshe and Gorse: St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104. Drs. Midthun and Clements: Johns Hopkins University, Hampton House, 624 North Broadway, Baltimore, MD 21205. Dr. Fernie: Division of Molecular Virology & Immunology, Georgetown University, 5640 Fishers Lane, Rockville, MD 20852. Dr. Stablein: The Emmes Corporation, 11325 Seven Locks Road, Suite 214, Potomac, MD 20892. Dr. Koff: AIDS Program, NIAID, Vaccine Research and Development Branch, 6003 Executive Boulevard, Rockville, MD 20892.
References 1. Fauci AS, Gallo RC, Koenig S, Salk J, Purcell RH. Development and evaluation of a vaccine for human immunodeficiency virus (HIV) infection. Ann Intern Med. 1989;110:373-85. 2. Koff WC, Hoth DF. Development and testing of AIDS vaccines. Science. 1988;24:426-32. 3. Barnes DM. Obstacles to an AIDS vaccine. Science. 1988;240:71921. 4. del Guercio P, Zanetti M. The CD4 molecule, the human immunodeficiency virus and anti-idiotypic antibodies. Immunology Today. 1987;8:204-5. 5. Ellrodt A, Le Bras P. The hidden dangers of AIDS vaccination. Nature. 1987;325:765. 6. Martinez ZC, Marcos MA, de la Hara A, et al. Immunological consequences of HIV infection: advantage of being low responder casts doubts on vaccine development. Lancet. 1988;1:454-7. 7. Robinson WE Jr, Montefiori DC, Mitchell WM. Antibody-dependent enhancement of human immunodeficiency virus type 1 infection. Lancet. 1988;1:790-4. 8. Takeda A, Tuazon CU, Ennis FA. Antibody-enhanced infection by HIV-1 via Fc receptor-mediated entry. Science. 1988;242:580-3. 9. Purcell RH. Animal models for the development of a vaccine for the acquired immunodeficiency syndrome. Ann Intern Med. 1989;110: 381-5. 10. Murphy-Corb M, Martin LN, Davison-Fairburn B, et al. A formalin-
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11.
12.
13. 14.
15. 16.
17.
18.
inactivated whole SIV vaccine confers protection in macaques. Science. 1989;246:1293-7. Looney D, Fisher AG, Putney SD, et al. Type-restricted neutralization of molecular clones of human immunodeficiency virus. Science. 1988;241:357-9. Matthews TJ, Weinhold KJ, Lyerly HK, et al. Interaction between the human T-cell lymphotropic virus type IIIB envelope glycoprotein gpl20 and the surface antigen CD4. Proc Natl Acad Sci USA. 1987;84:5424-8. Jacobson IM, Dienstag JL. Viral hepatitis vaccine. Annu Rev Med. 1985;36:241-61. Adachi A, Gendelman HE, Koenig S, et al. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986;59:284-91. Wain-Hobson S, Sonigo P, Danos O, et al. Nucleotide sequence of the AIDS virus, LAV. Cell. 1985;40:9-17. Cochran MA, Ericson BL, Knell JD, et al. Use of baculovirus recombinants as a general method for the production of subunit vaccines. In: Vaccines 87. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory; 1987:384-8. Kniker WT, Anderson CT, Roumeantzeff M. The MULTITEST system: a standardized approach to evaluation of delayed hypersensitivity and cell mediated immunity. Ann Allergy. 1979;43:73-9. DuPont Diagnostics. Human Immunodeficiency Virus (HIV): BiotechlDuPont HIV Western Blot Kit for Detection of Antibodies to HIV. Wilmington, Delaware: DuPont Diagnostics; 1987.
19. Abbott Laboratories, Diagnostics Division. Abbott HIV EI A. North Chicago, Illinois: Abbott Laboratories; 1987. 20. Vujcic LB, Shepp DH, Klutch M, et al. Use of a sensitive neutralization assay to measure the prevalence of antibodies to the human immunodeficiency virus. J Infect Dis. 1988;157:1047-50. 21. Montefiori DC, Robinson WE, Schuffman SS, Mitchell WM. Evaluation of antiviral drugs and neutralizing antibodies to human immunodeficiency virus by a rapid and sensitive microtiter infection assay. J Clin Microbiol. 1988;26:231-5. 22. Viscidi R, Ellerbeck E, Garrison L, et al. Characterization of serum antibody responses to recombinant HIV-1 gpl60 vaccine by enzyme immunoassay. AIDS Res Hum Retroviruses. 1990;6:1251-6. 23. Keefer MC, Bonnez W, Roberts NJ Jr, et al. HIV-1 recombinant gpl60 vaccine recipients demonstrate gpl60-specific lymphocyte proliferation prior to Western Blot reactivity. J Infect Dis. 1990 [In press]. 24. Tacket CO, Bagar SB, Munoz C, Murphy JR. Lymphoproliferative responses to mitogens and HIV-1 envelope glycoprotein among volunteers vaccinated with recombinant gpl60. AIDS Research and Human Retroviruses. 1990;6:535-42. 25. Orentas RJ, HUdreth JK, Obah E, et al. Induction of CD4+ human cytolytic T cells specific for HIV-infected cells by a gp 160 subunit vaccine. Science. 1990;248:1234-7. 26. Kovacs H, Easter ME, Davey R, et al. Safety and Immunogenicity of recombinant gpl60, a candidate AIDS vaccine. [Abstract 493]. 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA, 1990.
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Objective: To evaluate the safety and immunogenicity of a human immunodeficiency virus type 1 (HIV-1) recombinant envelope glycoprotein (rgpl60) candidate vaccine in humans. Subjects: Healthy adults (72) who were seronegative for HIV-1 were randomly assigned to one of four groups. Interventions: The subjects were randomly assigned to receive 40 or 80 /xg of rgpl60,10 /ig of hepatitis B vaccine, or placebo in three doses (on days 0, 30, and 180), with an elective, nonblinded administration of a fourth dose on day 540. Measurements and Main Results: Neither clinical nor laboratory toxicity was encountered during a follow-up period exceeding 21 months. No effect of immunization was noted on lymphocyte counts, mitogenic responses, or delayed-type hypersensitivity. Serum antibody responses to HIV envelope proteins detected by Western blot were seen in 30 of 33 subjects (91%; 95% CI, 71% to 97%) receiving either 40- or 80-/*g doses of rgpl60 and were most commonly of weakly reactive intensity. Responses were first noted by Western blot after the second dose. They markedly increased in frequency after the third dose and declined over the next 12 to 18 months. The administration of a fourth dose resulted in homologous neutralizing activity in sera from 5 of 24 subjects (21%; CI, 7% to 37%) as well as in complement-mediated antibody-dependent enhancement in sera from 6 of 24 subjects (25%; CI, 10% to 42%). Antibody responses were detected by enzyme-linked immunosorbent assay (ELISA) less frequently than by Western blot, and these responses persisted for a shorter time. Conclusions: The administration of rgpl60 was well tolerated and safe, resulted in a high rate of antibody response by Western blot after the administration of the third and fourth doses, and generated serum neutralizing activity and complement-mediated antibody-dependent enhancement in some subjects after the fourth dose.
Annals of Internal Medicine. 1991;114:119-127. From the University of Rochester School of Medicine and Dentistry, Rochester, New York; Vanderbilt University, Nashville, Tennessee; Baylor College of Medicine, Houston, Texas; University of Maryland at Baltimore, Baltimore, Maryland; St. Louis University School of Medicine, St. Louis, Missouri; Johns Hopkins University School of Hygiene and Public Health and School of Medicine, Baltimore, Maryland; Georgetown University, Rockville, Maryland; The Emmes Corporation, Potomac, Maryland; MicroGeneSys, Inc., West Haven, Connecticut; and the NIAID AIDS, Rockville, Maryland. For current author addresses, see end of text.
1 he development of a safe, effective vaccine against human immunodeficiency virus (HIV) infection or HIVassociated disease (or both) is the object of intense research efforts throughout the world. The formulation and evaluation of candidate vaccines confront formidable conceptual and practical obstacles (1-3). Despite the fact that considerable information has emerged about the molecular virology and antigenic characteristics of HIV as well as about host responses to HIV infections, the critical measures of immunity and protection from HIV infection and HIV-associated disease are not yet fully understood. In addition, it has been suggested that some types of immune responses to HIV antigens might themselves contribute to immunosuppression (4-6) or might even enhance subsequent infection with HIV (7, 8). The recent establishment of practical animal models of infection with HIV or HIV-like agents and the evaluation of vaccine candidates in these models may generate information that will be important in the development of HIV vaccines (9, 10). Nonetheless, information about the behavior of candidate HIV vaccines in humans, particularly about the safety and immunogenicity of these vaccines (that is, "phase I data"), is clearly needed at early stages of development as well. The National Institute of Allergy and Infectious Diseases (NIAID) Acquired Immunodeficiency Syndrome (AIDS) Vaccine Clinical Trials Network therefore conducted a phase I trial of a candidate HIV subunit vaccine, rgpl60. The Network selected rgpl60, an HIV-1 envelope glycoprotein generated through recombinant DNA technology, as the first HIV vaccine candidate to be studied in humans for several reasons. First, because the HIV-1 envelope glycoprotein, gpl60, contains the epitope or epitopes against which anti-HIV neutralizing antibodies have been detected (11) as well as the site of attachment of HIV to the CD4 receptor (12), it is highly relevant for inclusion in vaccine preparations. Second, the use of a highly purified "recombinant" protein bypasses concerns of possible contamination of vaccine preparations with HIV nucleic acids or other HIV proteins. Finally, vaccines comprised of surface proteins have been successfully generated against some viral infections, such as those caused by hepatitis B viruses (13). This initial trial was carried out in healthy volunteers who were seronegative for HIV and who appeared to be at low risk for acquisition of HIV infection. Because ©1991 American College of Physicians
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this trial was one of the first in which an HIV vaccine candidate was used in humans, assessment of safety and immunogenicity was necessarily complex. Subjects were observed for acute reactions to the vaccines as well as for potential clinical and laboratory evidence of long-term adverse effects. These studies also included in-vitro and in-vivo assessments of possible immunosuppression. The immunogenicity of this candidate HIV vaccine was assessed by sequential measurements of serum antibodies to HIV by Western blot, enzymelinked immunosorbent assay (ELISA), and neutralization assays. The studies also included two control groups: One group received a placebo preparation, and the other received hepatitis B vaccine, a licensed vaccine whose reactogenicity and immunogenicity have been well characterized. Materials and Methods Recombinant gpl60 Vaccine The HIV vaccine candidate rgpl60, a glycoprotein of the envelope of HIV-1, was developed by MicroGeneSys, Inc., West Haven, Connecticut. The rgpl60 gene was derived from an infectious molecular clone of HIV-1, pNL4-3 (provided by Dr. Malcolm Martin, Laboratory of Molecular Microbiology, NIAID, Bethesda, Maryland) (14). The nucleotide sequence of this gene was previously reported by Wain-Hobson and colleagues (15). A baculovirus expression vector that contained the rgpl60 gene under the transcriptional control of the Autographa catifornica nuclear polyhedrosis virus polyhedrin promoter was constructed (16). The rgpl60 was produced in a continuous insect cell line derived from Spodoptera frugiperda by infection with the recombinant virus. The infected cells produced a membrane-bound rgpl60 that was extracted with sodium deoxycholate. The antigen was purified by gel filtration followed by lentil lectin affinity chromatography. Purified rgpl60 was dialyzed against 10-mM tris buffer, pH 8.0, and then formulated with an aluminum phosphate gel (alum adjuvant). Subjects Study subjects were healthy adult volunteers between 18 and 55 years of age and without evidence of serious medical illness, as determined by a history and a physical examination. In addition, the study subjects' laboratory values, including the hematocrit result, leukocyte count and differential, platelet count, total lymphocyte count, total T4-cell count (> 400 cells/ mm3), serum alanine aminotransferase level, creatinine level, and urinalysis result, were within the normal ranges. Study subjects did not have a history of immunodeficiency or of treatment with immunosuppressive medications. They had a reaction of at least 2 mm of induration to one or more of the antigens (other than tuberculin) in the Mlrieux Multitest battery of skin tests (see below). All candidate subjects with a reaction to tuberculin were subsequently skin-tested by intermediate-strength purified protein derivative, and, if induration of greater than 5 mm was noted, they were ineligible for participation. Women of childbearing potential who participated in the study had a negative pregnancy test at entry and agreed to use adequate birth control measures throughout the study and follow-up period. Study subjects were seronegative for HIV-1, as determined by ELISA and Western blot assays (see below) before immunization. Cultures of peripheral blood mononuclear cells for HIV-1 and polymerase chain reactions to detect HIV-1 genome in mononuclear cells obtained before entry from these subjects were also negative. In an attempt to reduce the likelihood of confounding the results of the study by an intercurrent HIV infection, persons whose behavior was identified by a screening questionnaire and interview as a factor putting them at high risk for HIV infection were excluded. In addition, persons with 120
a history of blood transfusions or who had received cryoprecipitates within 6 months of entry were also ineligible for participation. Because hepatitis B vaccine was to be administered to a "control" group in the study, study subjects also had to be seronegative for hepatitis B surface antibody, hepatitis B core antibody, and hepatitis B surface antigen. Subjects were recruited at each of the participating sites using procedures that previously had been used for recruiting normal volunteers for studies of other (non-HIV) vaccines by the NIAID vaccine evaluation centers. Recruitment activities also included widespread information campaigns describing the studies in local media and in meetings of community organizations concerned with the control of AIDS. Recruitment was followed by extensive discussions with persons who expressed an interest in participation. All study subjects signed informed consent forms for screening procedures as well as for participation in the study before entry. The informed-consent form contained a description of the possible risks of participation, including potential problems that might be caused by the development of positive HIV serology as determined by conventional ELISA tests. To address this latter problem, study subjects were provided with an identification card that stated that they were participants in an NIAID-sponsored AIDS vaccine trial and which contained a toll-free telephone number that could be called to verify such participation through the NIAID AIDS Program. The Program's commitment to maintaining the confidentiality of the results of HIV laboratory tests obtained in the course of the study was also stated in this form. Experimental Design Subjects were randomly assigned, in a double-blind fashion, to receive one of four preparations: 40 /xg of rgpl60, 80 p% of rgpl60, 10 p% of hepatitis B vaccine (Recombivax, Merck, Sharp and Dohme, Rah way, New Jersey), or placebo (alum adjuvant). Each of the study preparations was administered intramuscularly in the deltoid muscle in a volume of 1.0 mL according to the following schedule: initial immunization (day 0), a first booster dose at 1 month (day 30), and a second booster dose at 6 months (day 180). An independent data and safety monitoring board reviewed the findings of the study after the initial and booster doses were administered and approved administration of subsequent doses. After the data from the three-dose regimen of immunization were analyzed, a fourth dose was offered in a nonblinded manner to those subjects who wished to participate further in the study approximately 1 year after the administration of the third dose (day 540). Clinical Evaluation After intramuscular administration of the first dose, subjects were observed for 1 hour and were instructed to take their temperatures at home, 6 and 12 hours later. Any untoward effects that developed in the first 24 hours were reported to the study team and evaluated at that time. Symptoms, if present, were graded as mild, moderate, or severe. Subjects returned to the study site on days 1, 2, 3, and 4 for examination and clinical evaluation. For the next 10 days, subjects reported any symptoms that developed. Subjects then returned to the study site on days 14 and 28 for clinical evaluation. After administration of each booster dose (on days 30, 180, and 540), subjects were evaluated according to the follow-up schedule that was used after the initial immunization. In addition, subjects were clinically evaluated on days 60, 120, 270, 365, 455, and 635 after vaccination. Delayed-Type Hypersensitivity Tests The M6rieux Multitest battery of skin tests for delayed-type hypersensitivity was applied before initial immunization and on days 60, 210, and 365 (17). The battery contains a glycerin control and the following antigens: tetanus toxoid, diphtheria toxoid, Streptococcus group C, old tuberculin, Candida, Trichophyton, and Proteus. Skin tests results were noted at 48 hours, and the size of induration was recorded. A positive
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reaction to an antigen was defined as induration of 2 mm or greater induration than was associated with the glycerin control. Laboratory Studies Complete blood counts, differentials, platelet counts, total lymphocyte counts, T4 and T8 subset counts, serum alanine aminotransferase levels, and creatinine levels were obtained before immunization and on days 14, 28, 44, 60, 120, 178, 194, 210, 270, 365, 455, 540, 559, 575, and 635. These laboratory values were determined by the clinical hematology, chemistry, and immunology laboratories at each of the participating institutions. Serum specimens and peripheral blood mononuclear cells, a portion of which were immediately cryopreserved, were obtained before immunization and on days 14, 28, 44, 60, 120, 178, 194, 210, 270, 365, 455, 540, 559, 575, and 635. Cryopreserved mononuclear cells were intended for use in future studies of cell-mediated immunity, which were not a formal part of the multicenter trial. Serum specimens were assayed for antibody to HIV using several assays. Western blot assays were done using the commercially available Biotech-DuPont kit (DuPont Co., Wilmington, Delaware) (18). Reactions at each virus-associated band were graded as either absent (-); present, but less intense than the p24 band on the weakly reactive control strip (±); present and at least as intense as the p24 band on the weakly reactive control strip, but less intense than p24 on the strongly reactive control strip (+); or present and greater than or equal to the intensity of p24 on the strongly reactive control strip (+). Western blot assays were done in laboratories at the individual participating sites as well as at the central immunology laboratory (see below). For purposes of analysis, a serum specimen was considered to be positive if it was reactive by Western blot at both the participating site and the central unit. If the intensity of responses differed between the participating and central sites, the less intense grade was used for analysis. Enzyme-linked immunosorbent assays were done using the commercially available Abbott kit (Abbott Laboratories, North Chicago, Illinois) (19). The optical density was read at 492 nm, and serum specimens were interpreted as positive if the optical density value was greater than a calculated cutoff value (for example, a ratio of optical density to cutoff of more than one). These assays also were done at the individual study sites and the central unit, and arithmetic means of the values were calculated. A serum specimen was considered to be positive if positive values were obtained at both the individual site and the central unit. Neutralization assays were done at two sites: the central immunology laboratory at Georgetown University (Fernie and colleagues) where a previously described microtiter syncytiaformation inhibition assay in Molt-3 cells was used (20); and Vanderbilt University (Montefiori and colleagues) where a microneutralization assay in MT-2 cells, which used neutral red dye uptake as a measure of cytopathic effect, was used (21). The homologous HIV-1 HIB virus was used as the challenge strain in all of the above assays. In addition, serum responses were examined for complement-mediated antibody-dependent enhancement by Montefiori and colleagues (7) as previously described. Fresh human serum 1:40 was used as a source of complement.
shall University, Johns Hopkins University, University of Maryland, University of Rochester, and Vanderbilt University. At each institution, a detailed study protocol was approved by the respective institutional review boards before the initiation of the study, and the study was conducted according to the human experimentation guidelines of the U.S. Department of Health and Human Services. A central immunology laboratory at Georgetown University coordinated the immunologic studies and also did neutralization, Western blot, and ELISA assays. Data entry was carried out at each of the six AVEUs, and data were transmitted electronically to Emmes Corporation, Potomac, Maryland, which served as the central data management and statistical analysis unit. Results Subjects A total of 72 subjects participated, 12 at each of the six AVEUs. The first 36 volunteers were immunized between 28 February and 10 March 1988, and the second 36 volunteers were immunized between 9 April and 6 May 1988. The subjects ranged in age from 19 to 53 years (Table 1). Fifty-four percent of the subjects were women; 86% described themselves as heterosexual; and 14% described themselves as homosexual. Of the 72 subjects who entered the study, all but 5 completed the three-dose regimen. Of the 5 who did not, 2 received two doses of hepatitis vaccine; 1 received one dose of rgpl60, 40 /Ltg; 1 received one dose of rgpl60, 80 /ig; and 1 received two doses rgp!60, 80 /ig. The reasons for failure to complete the immunization regimen were unrelated to the development of adverse effects. Two subjects changed their minds about participating during the study, 1 moved away, and 2 acquired HIV infection while enrolled in the study and thus were ineligible to continue to participate. Neither of the latter 2 subjects was in the group that received the rgpl60 preparations, and both subjects have been referred for appropriate medical follow-up care. Infection in these 2 subjects was identified by the development of Western blot bands to HIV proteins other than the gpl60, gpl20, or gp41 proteins contained in the vaccine, which resulted in a Western blot pattern typical of HIV infection. The remaining 67 subjects have complied with 99.5% of subsequent visits related to the first three doses and have been followed for as many as 21 months. Of the subjects who completed the three-dose regimen, 52 elected to receive a fourth dose: 16 in the placebo group, 12 in the hepatitis B vaccine group, 14 in the rgpl60 group receiving the 40-/ug doses and 10 in the rgpl60 group receiving the 80-/ug doses.
Statistical Methods
Reactogenicity
Repeated measures analysis of variance was used to assess relative changes in individual hematologic and chemical safety measures. Other standard nonparametric tests for assessing the frequency and intensity of response were used. The sample size provides pairwise 80% power to detect a standard deviation change of one or more in mean paired differences for any univariate outcome. All P values are two-tailed and are not adjusted for multiple testing. Confidence intervals (CIs) of 95% were used.
All of the study preparations, including rgpl60, were well tolerated. Febrile reactions, as defined by an oral temperature of more than 37.4°C within 48 hours of immunization, were seen in 4 of 18 recipients of 40-jig doses of rgpl60, 4 of 18 recipients of 80-/ug doses of rgpl60, 3 of 18 recipients of hepatitis B vaccine, and 4 of 18 recipients of placebo. No subject manifested an oral temperature greater than 38.0°C within 48 hours after vaccination. Only 5 subjects reported severe systemic symptoms (headache, malaise, myalgias, or feverish feeling) after immunization. One of these subjects
Participating Sites The study was conducted at the six NIAID AIDS Vaccine Evaluation Units (AVEUs): Baylor College of Medicine, Mar-
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was in the placebo group; 2 were in the rgpl60 group receiving the 40-/ug doses; and 2 were in the rgpl60 group receiving the 80-/ug doses. Mild pain and tenderness, generally lasting less than 24 hours, were frequently present at the injection site in the recipients of all four study preparations. One placebo recipient and 2 recipients of the 80-/ug doses of rgpl60 reported transient severe pain after one of the injections. Small amounts of induration at the injection site (mean, 13 ± 3 mm) were noted in some recipients of all the study preparations. The frequency of induration was somewhat higher after injection of 40-/ug doses of rgpl60 (5 of 18 subjects) and 80-jtg doses of rgpl60 (8 of 18 subjects) than after injection of hepatitis B vaccine (2 of 18 subjects) or placebo (1 of 18 subjects) (P = 0.02). No other untoward event was noted during the study.
Immunogenicity Western Blot Antibodies Responses to HIV envelope antigens were detected by Western blot only in subjects who received rgpl60. The majority of antibody responses were to gpl60, gpl20, or both, whereas responses to gp41 were seen less frequently and occurred in subjects who had particularly strong responses to gpl60 and gpl20. The frequency and intensity of the serum antibody responses to gpl60 and gpl20 that were detected by Western blot are presented in Figure 2. Among subjects who completed the three-dose schedule, 16 of 17 who received the 40-/xg doses and 14 of 16 who received the 80-/ug doses manifested serum antibodies that were detected by Western blot at least once during the study (total, 30 of 33 subjects [91%; CI, 71% to 97%]). Antibody responses were initially detected by Western blot after the administration of the second dose (23% to 44%). The frequency of response markedly increased after the administration of the third dose (76% to 81%) and gradually declined to 12% to 13% after 15 to 18 months (days 455 to 540). After the administration of the fourth dose (day 540), the frequency of response rose again (79% to 90%) and appeared to be declining at the most recent follow-up evaluation, on day 635 (Figure 2). Western blot bands confirmed at two laboratories were exclusively ± or + in intensity (see the Methods), and the distribution of the intensity of reactions was similar after the administration of the third and fourth doses. Antibodies to gp41 were detected by Western blot less frequently, developed later in the study, and were of shorter duration than were anti-gpl60 and anti-gpl20 antibodies in recipients of either 40-/ig or 80-/xg doses (Figure 3). Anti-gp41 antibodies were first noted after the administration of the third dose, became undetectable from day 270 to day 365, and were seen again after the administration of the fourth dose on day 540 (Figure 3). There was no statistically significant difference in the frequency of antibody responses detected by Western blot between recipients of 40-/Kg doses and recipients of 80-/xg doses when it was examined during the study (P > 0.1); however, a somewhat higher rate of antibody response was detected by Western blot on day 120, after the administration of the second dose in the group
Laboratory Toxicities Using repeated measures analysis of variance, there were no important differences in hemoglobin level, total leukocyte count, lymphocyte count, platelet count, serum alanine aminotransferase level, or creatinine level among the study groups during the study (data not shown). Nor were there important differences among the study groups in CD4 or CD8 cell counts or percentages or in CD4:CD8 ratios during the study (Figure 1). Fresh or cryopreserved mononuclear cells were examined for mitogenic responses to phytohemagglutinin or concanavalin A at four of the study sites (Johns Hopkins, University of Maryland, University of Rochester, and Vanderbilt University). There were no important differences in mitogenic response among the study groups when they were examined during the study (data not shown).
Skin Test Reactivity As presented in Table 2, there were no important differences in skin test reactivity, either in the number of antigens to which subjects responded or in the cumulative size of the response to antigens, among the four study groups.
Table 1. Characteristics of Study Subjects Characteristic
Total
Treatment Group Placebo
Hepatitis B Vaccine
n
Sex Women
Men Sexual preference Homosexual Heterosexual Age, y Median Range
10 8
10 8
10 8
9 9
39 33
1 17
4 14
3 15
2 16
10 62 35
39
32
35
35
27-46
22-52
19-46
20-53
* Human immunodeficiency virus type 1 recombinant glycoprotein. 122
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specimens that were defined as positive by Western blot, 45 (27%) were defined as positive by ELISA.
Figure 1. Mean circulating CD4+ and CD8+ counts in subjects who received placebo (•), hepatitis B vaccine (O), 40-/ug doses of rgpl60 (A), or 80-/ig doses of rgpl60 (A). Arrows indicate when immunizations were administered.
Antibodies Detected by Neutralization Assays Assays for neutralizing antibodies were done on all serum specimens that were defined as positive by Western blot as well as on selected serum specimens that were defined as negative by Western blot by laboratories at Vanderbilt University and Georgetown University. Neutralization activity was not detected in any serum specimen until day 559 (14 days after the administration of the fourth dose) when Montefiori and colleagues at Vanderbilt University detected neutralizing activity in 5 of 24 serum specimens (21%; CI, 7% to 37%): Such activity was noted in the serum specimens of 3 of 14 subjects who received the 40-/ug doses and in the serum specimens of 2 of 10 subjects who received the 80-/ig doses (Table 3). It was detected using the microtiter assay in MT-2 cells, as noted in the Materials and Methods (21). Neutralizing activity was not detected in the same serum specimens in assays carried out in Molt-3 (20) cells by Fernie and colleagues at the central immunology laboratory at Georgetown University. Complement-mediated antibody-dependent enhancement, also first seen on day 559, was noted in 6 of 24 subjects (25%; CI, 10% to 42%): in 1 of 14 who
receiving the 80-/ig doses compared with the group receiving the 40-//g doses (P = 0.057; the Fisher exact test) (Figure 2). Antibodies Detected by Enzyme-Linked Immunosorbent Assay Seventeen percent of subjects who received the 40-/Ltg doses and 39% of those who received the 80-/ig doses manifested antibodies detected by ELISA at least once during the study. In general, antibodies were detected less frequently and for a shorter duration of time by ELISA than by Western blot. Antibodies were not detected by ELISA until after the administration of the third dose (day 194), when they were detected in 2 of 17 recipients (12%; CI, 1% to 29%) of the 40-/*g doses and in 7 of 16 recipients (44%; CI, 20% to 65%) of the 80-/ug doses (P = 0.039). The number of antibodies detected by ELISA waned rapidly; they were not detected in recipients of the 40-/*g or 80-/ug doses by days 270 and 365, respectively. After the administration of the fourth dose (day 540), antibodies were detected by ELISA in 14% of subjects who received the 40-/xg doses and in 70% of subjects who received the 80-/ug doses (P = .005). The mean ratios of observed optical density to cutoff values for ELISA in specimens obtained at various times during the study are presented in Figure 4. Two weeks after the administration of the third dose (day 194), there was a trend toward higher mean ratios in subjects who received the 80-/xg doses (1.42) than in subjects who received the 40-/ig doses (0.74) (P < 0.1). Two weeks after the administration of the fourth dose (day 559), the ratios were significantly higher in recipients of the 80-/Ltg doses (2.32) than in recipients of the 40-jig doses (0.67) (P = 0.04; Wilcoxon rank sum test). Of the 46 serum specimens that were defined as positive by ELISA, all but 1 (45 of 46) were also positive by Western blot, 41 with + intensity and 4 with ± intensity (see the Methods). Conversely, of the 168 serum
Figure 2..The percentage of subjects whose serum specimens were positive for antibody to gpl60, gpl20, or both by Western blot, according to the study day. The shading of bars indicates the intensity of the Western blot reactions, as graded on a semiquantitative scale described in the Methods (D = ±; • = +). Subjects received either 40-/ig doses of rgpl60 (top) or 80-/ug doses of rgpl60 (bottom). Arrows indicate when immunizations were administered.
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Table 2. The Number and Size of Reactions to Antigens among Study Groups Study Preparation Before Immunization (n = 72) Hepatitis B vaccine Placebo rgpl60t 40-/ug doses SO-fig doses
Application of M6rieux Skin Test* Day 210 Day 60 (n = 69) (n = 55)
Day 365 (n = 66)
2.61 (10.51) 2.67 (12.99)
2.18(8.81) 2.61 (12.03)
2.92 (12.08) 3.40 (13.99)
2.63(9.13) 2.67(10.15)
3.33 (12.26) 3.28(13.31)
2.88(11.56) 3.24 (12.58)
2.75 (10.78) 4.23 (17.82)
2.67 (9.44) 2.71 (9.88)
* The data are given as the mean number of antigens with an induration reaction of 2 mm or more compared with that of the glycerin control at 48 hours. The numbers in parentheses are the mean of the sum of induration (in millimeters) of the seven antigens minus induration of the glycerin control. t Human immunodeficiency virus type 1 recombinant glycoprotein.
received the 4 0 - ^ doses and in 5 of 10 who received the 80-/ug doses (Table 3). Three subjects had both complement-mediated antibody-dependent enhancement and neutralizing activity, 2 had neutralizing activity alone, and 3 had complement-mediated antibody-dependent enhancement alone. Neither neutralization nor complement-mediated antibody-dependent enhancement was seen in subjects who received hepatitis B or placebo preparations. Assays of serum specimens obtained after day 559 as well as assays with heterologous HIV strains will be done in the future.
Additional Observations A 37-year-old volunteer who received 40-/ig doses of rgpl60 became pregnant, despite her agreement to use adequate birth control measures during the study and admonitions about the importance of adhering to this requirement. The estimated date of conception was approximately day 210 of the study or 30 days after the administration of the third dose. This subject developed serum antibody responses of -I- intensity (see the Methods) by Western blot to gpl20 on days 1% and 210. Responses detected by Western blot eventually decreased to ± on day 288 and, thereafter, have been absent when examined through day 540. This subject never manifested serum antibodies detected by ELISA. On day 497, the subject delivered a normal, full-term infant without complications, except for a transient postpartum fever, and both mother and child are currently well. At the time of delivery and 1 month later, both mother and child tested negative for serum antibody to HIV by Western blot and ELISA. Discussion
Figure 3. The percentage of subjects whose serum specimens were positive for antibody to gp41 by Western blot, according to the study day. The shading of bars indicates the intensity of Western blot reactions, as graded on a semiquantitative scale described in the Methods ( • = ±; • = +). Subjects received either 40-/ug doses of rgpl60 (top) or 80-/ug doses of rgpl60 (bottom). Arrows indicate when immunizations were administered. 124
We conducted a phase I study of a candidate HIV vaccine, rgpl60, in healthy adult volunteers who were seronegative for HIV-1. In this population, administration of 40-/Ag or 80-/ug doses of rgpl60 in a three- or four-dose regimen appeared to be safe and well tolerated. Systemic reactions were uncommon and occurred no more frequently than in the groups that received either a placebo preparation or a licensed hepatitis B vaccine. Local reactions to rgpl60 administration were also mild and similar to those seen in recipients of hepatitis B vaccine or placebo preparations, although induration was noted somewhat more frequently in recipients of rgpl60. No evidence of long-term clinical or laboratory toxicities has been noted in a follow-up period that has now exceeded 21 months. To examine whether potential suppression of immune function might be associated with the administration of rgpl60, we also assayed several measures of immune function in our subjects. In these assessments, no effect of immunization was noted on total counts or percentages of circulating lymphocytes or on CD4 or CD8 lymphocyte subset counts. Nor did the administration of rgpl60 affect delayed-type hypersensitivity, as assayed by the application of seven antigens on days 0, 60, 210, and 365 of
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the study. Although assessment of in-vitro cell-mediated immune activity was not a formal part of the multicenter trial, mononuclear ceils from subjects at four of the participating sites were examined for mitogenic responses to phytohemagglutinin and concanavalin A, and suppression of proliferative responses was not noted. No evidence of immunosuppression, at least as assessed by the above in-vitro and in-vivo measures of immune function, was seen in our study. The immunogenicity of rgpl60 was assessed by measuring serum antibodies to HIV-1 using Western blot, ELISA, and neutralization assays. Serum antibody responses, directed primarily at gpl60 or gpl20 antigens, were detected by Western blot in 30 of 33 subjects who completed the three-dose regimen, and there was no statistically significant difference in responses detected by Western blot between the group that received the 40-pg doses and the group that received the 80-/ug doses P > 0.1. Despite this high frequency of responses, the intensity of antibody reactions detected by Western blot was generally low, with reactions confirmed at two laboratory sites falling into the ± or + categories (see the Methods). In general, these reactions detected by Western blot were of considerably lower intensity than those seen after naturally occurring HIV infection. Antibody responses were not detected by Western blot until after the administration of the second dose, and the frequency of responses clearly increased after the administration of the third dose on day 180. The seroprevalence rates of antibodies detected by Western blot gradually declined after the administration of the third dose, so that only 12% to 13% of subjects had antibodies detected by Western blot 15 to 18 months after the administration of the first dose. The administration of a fourth dose of 40 p% or 80 fjug of rgpl60 approximately 1 year after the administration of the third dose (day
Figure 4. The mean optical density (OD) to cutoff ratios in enzyme-linked immunosorbent assays for anti-HIV antibodies done on serum specimens obtained on various study days from all subjects who received placebo (•), hepatitis B vaccine (O), 40-/ug doses of rgpl60 (A), or 80-/xg doses of rgpl60 (A). Arrows indicate when immunizations were administered.
Table 3. Neutralizing Activity and Complement-Mediated Antibody-Dependent Enhancement (C'-ADE) at Day 559 in Serum Specimens from Recipients of rgp!60 Dose of rgpl60 vaccine, pg*
40
80
Neutralizing Titert
C'-ADE
128 64 32 < 16 < 16 < 16 < 16 < 16 < 16 < 16 < 16 < 16 < 16 128 64 < 16 < 16 < 16 < 16 < 16 < 16 < 16 < 16
Titert
16 < 16
< 16 < 16 < 16 < 16 < 16 < 16 < 16 < 16 < 16 < 16 < 16 64 32 32 32 16 < 16 < 16 < 16 < 16 < 16
* Human immunodeficiency virus type 1 recombinant glycoprotein. t As determined in the Methods (21). t As determined in the Methods (7).
540) resulted in a high rate of responses as detected by Western blot (79% to 90% of recipients). These rates appear to decrease gradually over the following 3 months, according to the latest available results (day 635). Serum antibodies were detected by ELISA (using a lysate of HIV-1 as a source of antigen) less frequently and for a shorter time than by Western blot. Recipients of the SO-fig doses had statistically significant, more frequent, and greater antibody responses detected by ELISA after the administration of the fourth dose of vaccine compared with those who received the 40-/ig doses, and a similar trend was present after the administration of the third dose. A recent observation indicates that antibody responses can be detected more frequently by ELISA when the immunogen rgpl60, rather than a whole virus lysate, is used as the antigen in the assay (22). The functional activity of serum antibodies detected by Western blot or ELISA techniques is uncertain with respect to protection against HIV infection or HIVassociated disease. We were therefore particularly interested in determining whether serum neutralizing antibodies were generated in our studies. Using an assay in MT-2 cells (21), modest levels of neutralizing activity against homologous virus were detected in sera from 5 of 24 subjects 2 weeks after the administration of a fourth dose of rgpl60, but not in samples that were obtained earlier. However, neutralizing activity was not detected in an assay in which Molt-3 cells were used (20). Whether the differences in these results reflect differences in the sensitivity of these assays in detecting relatively low levels of neutralizing antibodies generated
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by rgpl60 or whether these differences relate to other properties of the two assays is unclear and is under investigation. At present, the neutralizing activity of these serum specimens against heterologous HIV-1 strains has not been studied; such studies are planned for the future. The results of such studies are of considerable interest, because the ability to stimulate protection against several HIV-1 strains will be a critical goal in the development of effective HIV-1 vaccines. We also detected low levels of complement-mediated antibody-dependent enhancement in six serum specimens. These levels are considerably lower than those seen after naturally occurring HIV-1 infection, in which mean titers of 5000 have been seen (unpublished data). Although the role of complement-mediated antibodydependent enhancement in potentiating HIV infection in vivo is currently unknown, candidate HIV vaccines' ability to induce antibody enhancing activity should continue to be carefully monitored. As noted earlier, the assessment of the immunogenicity of rgpl60 in our collaborative trial focused on the development of humoral immunity, because of uncertainty about which in-vitro assays were optimal for assessing cell-mediated responses and because of the difficulty in standardizing such assays in a multicenter study. In-vitro studies of lymphocyte proliferative responses to HIV antigens in subjects in this study have been carried out by investigators at the University of Rochester and the University of Maryland and are reported in detail elsewhere (23, 24). The studies of Keefer and colleagues (23) showed that lymphocyte proliferative responses to purified rgpl60 developed in six of six recipients of rgplfSO, that responses in some instances developed before Western blot reactivity was noted, and that lymphocyte proliferative responses persisted for at least 18 months, even in subjects who no longer had detectable anti-HIV antibody by Western blot. In addition, circulating cytolytic T-cells directed at gpl60 recently have been detected in three of eight of the above subjects who received rgpl60 (25). In summary, the studies that we have conducted have defined some measures of the safety and immunogenicity of a candidate HIV-1 subunit vaccine. At the concentrations of immunogen that we examined and using a three-dose regimen, serum antibody responses occurred frequently, but were of low levels and of relatively short duration. The administration of a fourth dose of immunogen 1 year after the administration of the third dose resulted in a high incidence responses according to Western blot. At that time, the development of antibodies with certain functional properties was first noted in some subjects; in particular, neutralizing activity against the homologous virus strain was detected, albeit at low levels and in only one of two in-vitro assay systems that were used. No statistically significant difference P > 0.1 was noted in responses detected by Western blot or neutralization assays between recipients of the 40-/ug doses and recipients of the 80-/ug doses, although the frequency of responses detected by ELISA was higher in the group receiving the 80-/ig doses, particularly after the administration of the fourth dose. Our findings suggest that additional studies that examine the effects of using higher doses of rgpl60 are 126
warranted. Escalating-dose studies of rgpl60 whose experimental designs are somewhat different from that used in our study are currently being conducted by Kovacs and co-workers (26) at the NIH and will also be conducted by the NIAID AIDS Vaccine Clinical Trials Network. Different regimens of immunization with rgplfSO clearly need to be investigated as well. Our results suggest that at least a three-dose regimen will likely be required and that the administration of a fourth dose may be advantageous. Our results show that phase I investigations of candidate HIV vaccines can be conducted efficiently and successfully in healthy, HIV-seronegative subjects. Highly motivated and cooperative subjects can be recruited, and clinical and laboratory measures of the safety and immunogenicity of candidate vaccines can be assessed over extended periods. The results of our studies should be useful for the planning and conduct of phase I studies of other HIV candidate vaccines in humans as well as for further studies of rgpl60. Grant Support: By contracts (N01-AI-52577, N01-AI-72624, N01-AI62528, N01-AI-52575, N01-AI-52576, N01-AI-72623, N01-AI-82500, N01-AI-62515) from the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland. Requests for Reprints: Raphael Dolin, MD, Infectious Disease Unit, Department of Medicine, University of Rochester Medical Center, 601 Elmwood Avenue, Box 689, Rochester, NY 14642. Current Author Addresses: Drs. Dolin and Bonnez: University of Rochester Medical Center, 601 Elmwood Avenue, Box 689, Rochester, NY 14642. Drs. Graham, Karzon, and Montefiori: Vanderbilt University, Medical Center North, Nashville, TN 37232. Drs. Greenberg and Atmar: Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Dr. Tacket and Mr. Horgan: University of Maryland, Center for Vaccine Development, 10 South Pine Street, MSTF Building, Baltimore, MD 21201. Drs. Belshe and Gorse: St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104. Drs. Midthun and Clements: Johns Hopkins University, Hampton House, 624 North Broadway, Baltimore, MD 21205. Dr. Fernie: Division of Molecular Virology & Immunology, Georgetown University, 5640 Fishers Lane, Rockville, MD 20852. Dr. Stablein: The Emmes Corporation, 11325 Seven Locks Road, Suite 214, Potomac, MD 20892. Dr. Koff: AIDS Program, NIAID, Vaccine Research and Development Branch, 6003 Executive Boulevard, Rockville, MD 20892.
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19. Abbott Laboratories, Diagnostics Division. Abbott HIV EI A. North Chicago, Illinois: Abbott Laboratories; 1987. 20. Vujcic LB, Shepp DH, Klutch M, et al. Use of a sensitive neutralization assay to measure the prevalence of antibodies to the human immunodeficiency virus. J Infect Dis. 1988;157:1047-50. 21. Montefiori DC, Robinson WE, Schuffman SS, Mitchell WM. Evaluation of antiviral drugs and neutralizing antibodies to human immunodeficiency virus by a rapid and sensitive microtiter infection assay. J Clin Microbiol. 1988;26:231-5. 22. Viscidi R, Ellerbeck E, Garrison L, et al. Characterization of serum antibody responses to recombinant HIV-1 gpl60 vaccine by enzyme immunoassay. AIDS Res Hum Retroviruses. 1990;6:1251-6. 23. Keefer MC, Bonnez W, Roberts NJ Jr, et al. HIV-1 recombinant gpl60 vaccine recipients demonstrate gpl60-specific lymphocyte proliferation prior to Western Blot reactivity. J Infect Dis. 1990 [In press]. 24. Tacket CO, Bagar SB, Munoz C, Murphy JR. Lymphoproliferative responses to mitogens and HIV-1 envelope glycoprotein among volunteers vaccinated with recombinant gpl60. AIDS Research and Human Retroviruses. 1990;6:535-42. 25. Orentas RJ, HUdreth JK, Obah E, et al. Induction of CD4+ human cytolytic T cells specific for HIV-infected cells by a gp 160 subunit vaccine. Science. 1990;248:1234-7. 26. Kovacs H, Easter ME, Davey R, et al. Safety and Immunogenicity of recombinant gpl60, a candidate AIDS vaccine. [Abstract 493]. 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA, 1990.
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