Journal of Clinical Immunology, Vol. 12, No. 6, 1992
Serum Antibodies to HIV-1 in Recombinant Vaccinia Virus Recipients Boosted with Purified Recombinant gp 160 DAVID C. M O N T E F I O R I , x'3 B A R N E Y S. G R A H A M , t S R I S A K U L K L I K S , 2 P E T E R F. W R I G H T , t and the N I A I D AIDS V A C C I N E C L I N I C A L T R I A L S N E T W O R K
were present in sera from two of these three vaccinees. None of the volunteers developed antisyncytial antibodies. These results indicate that inoculation with recombinant vaccinia followed by rgpl60 boosting is the most effective strategy to date for inducing serum antibodies to the envelope glycoproteins of HIV-1, but further study is needed to optimize the functionality and cross-reactivity of these responses.
Accepted: July 29, 1992
Serum antibody responses were studied in detail in four vaccinia-naive volunteers in a phase I trial evaluating primary vaccination with a recombinant vaccinia virus expressing the HIV-1 gpl60 envelope glycoprotein (HIVAC-le, Oncogen/Bristol-Myers Squibb), followed by booster immunization with baculovirus-derived rgpl60 (VaxSyn, MicroGeneSys). Prior to boosting, low-titer Fc receptor (FcR)-mediated, antibody-dependent enhancing (ADE) activity was detected in two of four volunteers but no IgM, IgG, IgA, neutralizing activity, or complementmediated ADE activity was detected. Two weeks after boosting, all four volunteers developed HIV-l-specific IgG with titers of 1:160 to 1:640 by immunofluorescence assay. IgG1 was present in sera from each individual, while lgG2 and IgG3 were present in sera from two individuals, and IgG4 was present in serum from one individual. IgM and IgA were undetectable in all sera. Only one volunteer had IgG to the heterologous HIV-1 isolates, RF, MN, and SF2, after boosting. Serum from this volunteer neutralized the vaccine strain, LAV/IIIB, but not the heterologous strains, RF, MN, and SF2. Antibodies from the remaining volunteers had no neutralizing activity. The neutralizing serum had a positive reaction in a peptide-based ELISA utilizing a peptide corresponding to the principal neutralizing domain of the third hypervariable region (i.e., V3 loop) of the envelope glycoprotein. Neutralizing activity was partially removed by adsorption to this peptide, suggesting that it contained a type-specific neutralizing vaccine epitope. A low titer (1:40 to 1:80) of complement-mediated ADE activity to HIV-1 IIIB was present in sera from three vaccinees after boosting. FcR-ADE activity for HIV-1 SF2 and SF-128A
KEY WORDS: Vaccine; AIDS; humoral immunity.
INTRODUCTION Several lines of evidence indicate that serum antibodies produced in response to HIV-1 infection can control the course of disease and, hence, will be important to vaccine development. Early infection with HIV-1 leads to the production o f virus-specific antibodies, whose initial appearance coincides with reductions in plasma concentrations of antigen and infectious virus (1-4). Studies of virus neutralization have demonstrated that initial isolates are neutralized by autologous sera in vitro but that genetic diversity eventually gives rise to neutralizationescape variants (5-7), which might contribute to the failure of the antibody response to provide prolonged control of infection. Still, clinical trials of passive immunization using h y p e r i m m u n e plasma from healthy, HIV-l-infected individuals have produced transient reductions in plasma antigenemia and viremia (8, 9). Further, passive immunization with a monoclonal antibody to the principal neutralizing domain of gpl20 (10) or with hyperimmune plasma from asymptomatic, HIV-l-infected individuals (11, 12) has protected chimpanzees from HIV-I infection. In addition, vaccine protection in chimpanzees has been achieved by active immunization with HIV-1 envelope subunit preparations (13, 14). Of concern is a set of naturally occurring antibodies
1Departments of Pathology and Medicine, Vanderbilt University Medical School, Nashville, Tennessee 37232. 2Cancer Research Institute, University of California, San Francisco, California 94143. 3To whom correspondence should be addressed at Department of Pathology, Vanderbilt University Medical School, Room C-3321, Medical Center North, Nashville, Tennessee 37232.
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which enhance HIV-1 infection in vitro (15-17). Increased titers of one type, Fc-receptor-mediated antibody-dependent enhancement (FcR-ADE), might signify disease progression (17), while the significance of another type, complement-mediated antibody-dependent enhancement (C'-ADE) remains in question (18, 19). Although nearly all infected individuals progress to AIDS despite the presence of HIV-l-specific antibodies, it remains to be seen whether the prior existence of humoral immunity induced by vaccination will prevent infection or alter the course of disease. A detailed analysis of serum antibody responses elicited during clinical trials of candidate vaccines is therefore critical. Two vaccine preparations recently completed phase I evaluation in uninfected volunteers enrolled in the AIDS Vaccine Clinical Trials Network sponsored by the National Institutes of Allergy and Infectious Diseases. The first of these was a recombinant HIV-1 envelope glycoprotein precursor, rgpl60 (VaxSyn, MicroGeneSys), synthesized in insect cells using a baculovirus expression system. Four inoculations with 40 or 80 p~g of this preparation over an 18-month period resulted in a high rate of seroconversion by Western blot after the third and fourth inoculations and generated weak serum neutralizing and C'-ADE antibodies after the fourth inoculation, in about 25% of volunteers (20). These antibody responses also included low levels of salivary antibodies detectable by Western blot in one of three vaccine recipients tested (21). Gpl60specific lymphoproliferative responses were detected soon after the first inoculation and persisted for at least 540 days (22, 23). In addition, class II major histocompatibility complex (MHC)-restricted, CD4 + cytotoxic T-lymphocyte (CTL) activity was identified in three of eight volunteers tested in this trial 2 weeks after the fourth inoculation (24). Three inoculations with a higher dose (640 Ixg) elicited antibody responses similar to those seen after four inoculations with 40- or 80-pog doses of this rgpl60 (20, Keefer M, Belshe R, Ctements M, Graham B, Corey L, Bolognesi D, Stablein D, Koff W, Fast P, Montefiori D, Smith G, Dolin R, and the NIAID AIDS Vaccine Clinical Trials Network. VIIIth International Conference on AIDS, Amsterdam, abstr. PoA2228). The second vaccine consisted of a recombinant vaccinia virus that expressed the HIV-1 gpl60 (HIVAC-le, Oncogen/Bristol-Myers Squibb). No HIV1-specific antibodies were detected in recipients of
MONTEFIORI ET AL.
this vaccine who had a history of smallpox vaccination (26). In vaccinia-naive individuals, however, HIVAC-le elicited weak antibody responses by Western blot in 16 of 22 volunteers. Neutralizing antibodies were detected in one of these individuals, while none of them developed detectable C'ADE antibodies (27). Interestingly, the duration of HIV-l-specific antibody in these recipients was considerably longer than in recipients of subunit vaccines. Recently, rgpl60 booster immunization of 12 previously vaccinia-naive HIVAC-le recipients induced higher antibody titers by enzyme-linked immunosorbent assay (ELISA) and virus neutralization than either vaccine alone (25). In this study, each vaccinee developed strong antibody responses by Western blot and ELISA, and eight had neutralizing antibodies. This represents a novel vaccine approach for which a more extensive evaluation of antibody response was warranted. Here we describe in greater detail the anti-HIV-I antibody response in four of four immunized volunteers enrolled at Vanderbilt University. MATERIALS AND METHODS
Vaccines. The recombinant vaccinia virus (venvNY5) used in this vaccine trial contained the entire coding sequence of gpl60 from the LAV strain of HIV-1 (28). This vaccine (HIVAC-le, Oncogen/Bristol-Myers Squibb, Seattle, WA) was constructed using vaccinia virus obtained from a commercial preparation of smallpox vaccine (Dryvax, Wyeth L a b o r a t o r i e s ) . Purified rgpl60 (VaxSyn), also derived from the LAV strain of HIV-1, was developed by MicroGeneSys, Inc., Meriden, CT. The gpl60 was purified from cell lysates after expression by a recombinant baculovirus in Spodoptera fugipoda cells as described (29). It should be noted that HIV-1 strain LAV, used in the construction of these vaccines, and HIV-1 strain IIIB, used throughout the studies described here, share greater than 98% sequence homology, especially in the V3 region, and are probably from the same isolate (30). Study Protocol. Volunteers were healthy adults, seronegative for vaccinia and HIV-1. HIVAC-le was administered using bifurcated needle punctures. Volunteers were inoculated 580-650 days later with 640 ~g of rgpl60 as described (25). Cells and Viruses. The CD4 + human lymphoblastoid cell lines MT-2 (31) and H9 (obtained from
Journal o f Clinical Immunology, Vol. 12, No. 6, 1992
SERUM ANTIBODIES IN HIV-IMMUNIZED HUMANS
R. C. Gallo, National Cancer Institute), were cultured at 37°C in RPMI-1640 containing 12% heatinactivated (56°C, 1 hr) fetal bovine serum and 50 p~g of gentamicin per ml. Stocks of the HIV-1 isolates IIIB, RF (obtained from R. C. Gallo), and MN (obtained from the National Institutes of Health, AIDS Research and Reference Reagent Program) were harvested from chronically infected cultures of H9 cells. Virus-containing culture fluids were clarified of cells by low-speed centrifugation and passed through 0.45-~m cellulose acetate filters (Millipore). HIV-1 SF2 was recovered from peripheral blood mononuclear cells (PBMCs) of a patient with oral candidiasis, while HIV-1 SF-128A was derived from spinal cord tissue of a patient with neurological AIDS (32). Both strains were obtained from Dr. Jay Levy and have been further propagated in normal human PBMCs maintained in fetal calf serum, glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 txg/ml), and 5-10% interleukin-2 (33). The culture medium containing freshly released virus was centrifuged at low speed to remove cells and cell debris. The fluid was then filtered through 0.45-p~m cellulose acetate filters (Millipore). The quantity of the virus was determined by reverse transcriptase activity (34) and the 50% tissue culture infectious dose (TCIDs0) in peripheral blood mononuclear cells (PBMCs) (35). Western Blot. Antibodies to HIV-l-strain IIIB antigens were detected by Western blot as described by the manufacturer (E. I. DuPont, Wilmington, DE). Immunofluorescence Assays. Antibodies to viral antigens were quantitated by indirect immunofluorescence assays using cells chronically infected with IIIB, RF, or MN strains of HIV-1. Slides of fixed, infected cells were prepared by air-drying and fixing in a 1:1 mixture of acetone:methanol for 30 rain. Sera were diluted in phosphate-buffered saline containing 0.1% globin-free bovine serum albumin (PBS-BSA) and incubated on slides for 30 rain at 36°C and 100% humidity. After two washes in carbonate buffer (0. I 1 M NazCO 3, 0.4 M NaHCO 3, 0.15 M NaC1, 0.05% Tween-80), the slides were incubated for 30 min at 36°C and 100% humidity with a 1:200 dilution of fluorescein-conjugated, IgG fraction of goat anti-human IgG (heavy and fight chain specific; Cappel) containing Evan's blue counterstain. Slides were again washed twice in carbonate buffer and then mounted using 50% glycerol and examined for fluorescence using a Nikon DIAPHOT-TMD-EF fluorescencemicroscope. Anti-
Journal of Clinical Immunology, Vol. 12, No. 6, 1992
431
body titers are defined as the last dilution yielding positive fluorescence. Additional experiments were done to determine the presence of anti-HIV-I immunoglobulin of various classes and subclasses using anti-human IgG1, IgG2, IgG3, IgG4, IgA, and IgM (Cappel, Organon Teknika Corp., Durham, NC). Cell lines expressing IgA (DAKIKI) and IgM (RPMI 1788) (both cell lines obtained from American Type Culture Collection, Rockville, MD) were used as positive controls. Peptide ELISA. Antibodies to rgpl60 or peptides corresponding to the principal neutralizing domain (PND) of gpl20 (36) were measured by ELISA. Peptides corresponding to amino acids 302-324, 308-324, and 312-327 of the HIV-1 IIIB gpl20 were obtained from American Bio-Technologies, Inc., Cambridge, MA. A negative control peptide corresponding to the Sindbis virus envelope glycoprotein was obtained from The Upjohn Company, Kalamazoo, MI. The rgpl60 was obtained from MicroGeneSys, Inc. Individual wells of 96-well Immulon-2 plates (Dynatech Laboratories, Inc., Chantilly, VA) were incubated overnight at 4°C with I00 ~1 of peptide in carbonate buffer (15 mM NazCO3, 35 mM NaHCO3, pH 9.8). The peptide solutions were then aspirated and the wells filled with 100 p~lof blocking buffer (Filter Paper Diluent, DuPont) containing 2.5% fetal bovine serum and incubated at 37°C for 4 hr. The plates were washed 4× with phosphate-buffered saline (PBS; 0.14 M NaC1, 2.7 mM KC1, 8 mM Na2HPO4, 1.7 mM NaHzPO4, pH 7.4) containing 0.05% Tween-20. Serial dilutions of volunteers' sera were made in wells containing borate buffer (0. I M boric acid, 47 mM sodium borate, 75 mM NaC1, 0.05%, v/v, Tween-20) + 2.5% fetal bovine serum and incubated at 37°C for 4 hr. Wells were then washed 4× with PBS/Tween-20 and received 100 ~1 of alkaline phosphatase-conjugated, goat anti-human IgG and IgM (Sigma Chemical Company, St. Louis, MO) and incubated at 37°C for 4 hr. The wells were again washed 4x with PBS/Tween-20 and incubated with 100 ~1 ofp-nitrophenylphosphate disodium hexahydrate (Sigma 104 phosphatase substrate) in diethanolamine buffer (0.9 M diethanolamine, 7 mM MgCI2, pH 9.8, with concentrated HC1). After color development, absorbance was read at 405 rim. The titer was reported as the highest logz reciprocal serum dilution that had an average absorbance reading greater than 0.1 and at least twice that of the negative control (Sindbis virus peptide).
432
Neutralizing Antibodies. HIV-1 IIIB neutralizing titers in sera from vaccinees were measured in an MT-2 cell cytopathic infection assay in microdilution plates as described previously (37). Briefly, twofold dilutions of heat-inactivated sera (56°C, 1 hr) were made in triplicate in a total of 100 ~1 of growth medium per well. Fifty microliters of virus (5 × l04 TCIDs0 of IIIB) was then added to all wells except for one row of eight noncytopathic control wells, which received growth medium in place of virus. After 1 hr of incubation, MT-2 cells (5 x 104) in 100 Ixl of growth medium were added to each well. Viable cells were quantitated colorimetrically by vital dye (neutral red) uptake 3 days later. Neutral red uptake is a linear function of cell viability, where light absorption at a 540-nm wavelength is linear from 0.025 to 0.85, corresponding to 2 x 104 to 25 × 104 viable cells/well (37). Percent protection was defined as the difference in absorption between test wells (cells + serum + virus) and virus control wells (cells + virus) divided by the difference in absorption between cell control wells (cells only) and virus control wells. Plates were harvested when cytopathic effect in virus control wells produced greater than a 50% reduction in viable cells. Neutralizing titers were defined as the reciprocal of the last dilution to provide at least 35% protection. The concentration of virus in the inoculum was determined by titration on MT-2 cells in this assay; the reciprocal dilution at which 50% of wells showed cytopathic effects after 2 weeks defined the titer (1 TCIDso). Antisyncytial Antibodies. Antisyncytial activity was measured in an assay analogous to the neutralizing antibody assay as described previously (38). Syncytium formation was induced by mixing MT-2 cells with H9/IIIB cells at a 10:1 ratio in the presence and absence of sera in 96-well microdilution plates. Serial dilutions of sera were made in triplicate. H9/IIIB cells (1.5 x 104) in 50 Ixl of growth medium were added to all wells except one row of eight noncytopathic control wells, which received uninfected H9 cells. MT-2 cells (1.5 × 105) in 100 ixl of growth medium were then added to each well. Syncytium formation leads to, and is directly proportional to, cytopathic effect in this assay. After incubation at 37°C for 20 hr, syncytium formation was observed microscopically, while viable cells were measured by vital dye uptake as described above. Antibody-Dependent Enhancement. C'-ADE was measured in MT-2-cell microtiter infection assays
MONTEFIORIET AL.
as described previously (15). The procedure is similar to the neutralizing antibody assay described above except that fresh human serum is added at a 1:40 dilution as a source of complement. Infection enhancement is evident by a reduction in viable cells relative to a set of virus control wells which contain cells, virus, and complement, but no antiHIV-1 serum. Therefore, the percentage viable cells was defined as the difference in A540 readings between the average of three test wells and the average of eight blank wells, divided by the difference in A54o readings of the average of eight virus control (no antibody) wells and eight blank wells. C'-ADE titers were defined as the reciprocal of the last dilution to produce at least a 20% reduction in viable cells. FcR-ADE was measured in human PBMCs as described by H o m s y et al. (17). Briefly, 100 I~1 of dilutions of heat-inactivated vaccinee's sera was mixed with an equal volume of virus-containing culture fluid which contained 100 TCIDso units of HIV-1. The mixtures were incubated at room temperature for 1 hr. Thereafter, 106 phytohemagglutinin (PHA)-stimulated PBMCs in 0.4 ml were added to each virus-antibody mixture and incubated at 37°C for 2 hr. The supernatant was removed and cells were resuspended in medium for several days. Culture fluids were harvested on days 3 and 5 for HIV-1 128A and on days 7 and 10 for HIV-1 SF2 and quantitated for amount of virus by reverse transcriptase activity. Enhancement of infection was measured by a greater than twofold increase in viral production from cultures receiving postvaccination serum as compared to those receiving prevaccination serum of the same individual.
RESULTS
HIV-1-Specific Ig Classes and Subclasses. HIV-1 IIIB-specific IgG was first detected by IFA in the sera of volunteers 2 weeks after they were boosted with rgpl60, where it was present in all four individuals at reciprocal titers of 160-640 (Table 1). Only one of the four volunteers (A20) had IgG that cross-reacted with antigens of HIV-1 strains RF and MN. Reciprocal titers of antibodies to HIV-I RF and MN were considerably lower (i.e., 8× lower for RF and 32× lower for MN). HIV-1 IIIB-specific IgG was detected in only one volunteer (A15) by 90 days after booster immunization (Table I). Journal of Clinical Immunology, Vol. 12, No. 6, 1992
SERUM ANTIBODIES IN HIV-IMMUNIZED HUMANS
433
Table I, Titers and Strain Specificity of Anti-HIV-1 IgG in Sera from Vaccinated Volunteers a Antibody titer (reciprocal dilution)
Volunteer
IIIB
RF
MN
IIIB
RF
MN
IIIB
RF
MN
90 days after rgpl60 IIIB
A10 At2 A15 A20
Serum Antibodies to HIV-1 in Recombinant Vaccinia Virus Recipients Boosted with Purified Recombinant gp 160 DAVID C. M O N T E F I O R I , x'3 B A R N E Y S. G R A H A M , t S R I S A K U L K L I K S , 2 P E T E R F. W R I G H T , t and the N I A I D AIDS V A C C I N E C L I N I C A L T R I A L S N E T W O R K
were present in sera from two of these three vaccinees. None of the volunteers developed antisyncytial antibodies. These results indicate that inoculation with recombinant vaccinia followed by rgpl60 boosting is the most effective strategy to date for inducing serum antibodies to the envelope glycoproteins of HIV-1, but further study is needed to optimize the functionality and cross-reactivity of these responses.
Accepted: July 29, 1992
Serum antibody responses were studied in detail in four vaccinia-naive volunteers in a phase I trial evaluating primary vaccination with a recombinant vaccinia virus expressing the HIV-1 gpl60 envelope glycoprotein (HIVAC-le, Oncogen/Bristol-Myers Squibb), followed by booster immunization with baculovirus-derived rgpl60 (VaxSyn, MicroGeneSys). Prior to boosting, low-titer Fc receptor (FcR)-mediated, antibody-dependent enhancing (ADE) activity was detected in two of four volunteers but no IgM, IgG, IgA, neutralizing activity, or complementmediated ADE activity was detected. Two weeks after boosting, all four volunteers developed HIV-l-specific IgG with titers of 1:160 to 1:640 by immunofluorescence assay. IgG1 was present in sera from each individual, while lgG2 and IgG3 were present in sera from two individuals, and IgG4 was present in serum from one individual. IgM and IgA were undetectable in all sera. Only one volunteer had IgG to the heterologous HIV-1 isolates, RF, MN, and SF2, after boosting. Serum from this volunteer neutralized the vaccine strain, LAV/IIIB, but not the heterologous strains, RF, MN, and SF2. Antibodies from the remaining volunteers had no neutralizing activity. The neutralizing serum had a positive reaction in a peptide-based ELISA utilizing a peptide corresponding to the principal neutralizing domain of the third hypervariable region (i.e., V3 loop) of the envelope glycoprotein. Neutralizing activity was partially removed by adsorption to this peptide, suggesting that it contained a type-specific neutralizing vaccine epitope. A low titer (1:40 to 1:80) of complement-mediated ADE activity to HIV-1 IIIB was present in sera from three vaccinees after boosting. FcR-ADE activity for HIV-1 SF2 and SF-128A
KEY WORDS: Vaccine; AIDS; humoral immunity.
INTRODUCTION Several lines of evidence indicate that serum antibodies produced in response to HIV-1 infection can control the course of disease and, hence, will be important to vaccine development. Early infection with HIV-1 leads to the production o f virus-specific antibodies, whose initial appearance coincides with reductions in plasma concentrations of antigen and infectious virus (1-4). Studies of virus neutralization have demonstrated that initial isolates are neutralized by autologous sera in vitro but that genetic diversity eventually gives rise to neutralizationescape variants (5-7), which might contribute to the failure of the antibody response to provide prolonged control of infection. Still, clinical trials of passive immunization using h y p e r i m m u n e plasma from healthy, HIV-l-infected individuals have produced transient reductions in plasma antigenemia and viremia (8, 9). Further, passive immunization with a monoclonal antibody to the principal neutralizing domain of gpl20 (10) or with hyperimmune plasma from asymptomatic, HIV-l-infected individuals (11, 12) has protected chimpanzees from HIV-I infection. In addition, vaccine protection in chimpanzees has been achieved by active immunization with HIV-1 envelope subunit preparations (13, 14). Of concern is a set of naturally occurring antibodies
1Departments of Pathology and Medicine, Vanderbilt University Medical School, Nashville, Tennessee 37232. 2Cancer Research Institute, University of California, San Francisco, California 94143. 3To whom correspondence should be addressed at Department of Pathology, Vanderbilt University Medical School, Room C-3321, Medical Center North, Nashville, Tennessee 37232.
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which enhance HIV-1 infection in vitro (15-17). Increased titers of one type, Fc-receptor-mediated antibody-dependent enhancement (FcR-ADE), might signify disease progression (17), while the significance of another type, complement-mediated antibody-dependent enhancement (C'-ADE) remains in question (18, 19). Although nearly all infected individuals progress to AIDS despite the presence of HIV-l-specific antibodies, it remains to be seen whether the prior existence of humoral immunity induced by vaccination will prevent infection or alter the course of disease. A detailed analysis of serum antibody responses elicited during clinical trials of candidate vaccines is therefore critical. Two vaccine preparations recently completed phase I evaluation in uninfected volunteers enrolled in the AIDS Vaccine Clinical Trials Network sponsored by the National Institutes of Allergy and Infectious Diseases. The first of these was a recombinant HIV-1 envelope glycoprotein precursor, rgpl60 (VaxSyn, MicroGeneSys), synthesized in insect cells using a baculovirus expression system. Four inoculations with 40 or 80 p~g of this preparation over an 18-month period resulted in a high rate of seroconversion by Western blot after the third and fourth inoculations and generated weak serum neutralizing and C'-ADE antibodies after the fourth inoculation, in about 25% of volunteers (20). These antibody responses also included low levels of salivary antibodies detectable by Western blot in one of three vaccine recipients tested (21). Gpl60specific lymphoproliferative responses were detected soon after the first inoculation and persisted for at least 540 days (22, 23). In addition, class II major histocompatibility complex (MHC)-restricted, CD4 + cytotoxic T-lymphocyte (CTL) activity was identified in three of eight volunteers tested in this trial 2 weeks after the fourth inoculation (24). Three inoculations with a higher dose (640 Ixg) elicited antibody responses similar to those seen after four inoculations with 40- or 80-pog doses of this rgpl60 (20, Keefer M, Belshe R, Ctements M, Graham B, Corey L, Bolognesi D, Stablein D, Koff W, Fast P, Montefiori D, Smith G, Dolin R, and the NIAID AIDS Vaccine Clinical Trials Network. VIIIth International Conference on AIDS, Amsterdam, abstr. PoA2228). The second vaccine consisted of a recombinant vaccinia virus that expressed the HIV-1 gpl60 (HIVAC-le, Oncogen/Bristol-Myers Squibb). No HIV1-specific antibodies were detected in recipients of
MONTEFIORI ET AL.
this vaccine who had a history of smallpox vaccination (26). In vaccinia-naive individuals, however, HIVAC-le elicited weak antibody responses by Western blot in 16 of 22 volunteers. Neutralizing antibodies were detected in one of these individuals, while none of them developed detectable C'ADE antibodies (27). Interestingly, the duration of HIV-l-specific antibody in these recipients was considerably longer than in recipients of subunit vaccines. Recently, rgpl60 booster immunization of 12 previously vaccinia-naive HIVAC-le recipients induced higher antibody titers by enzyme-linked immunosorbent assay (ELISA) and virus neutralization than either vaccine alone (25). In this study, each vaccinee developed strong antibody responses by Western blot and ELISA, and eight had neutralizing antibodies. This represents a novel vaccine approach for which a more extensive evaluation of antibody response was warranted. Here we describe in greater detail the anti-HIV-I antibody response in four of four immunized volunteers enrolled at Vanderbilt University. MATERIALS AND METHODS
Vaccines. The recombinant vaccinia virus (venvNY5) used in this vaccine trial contained the entire coding sequence of gpl60 from the LAV strain of HIV-1 (28). This vaccine (HIVAC-le, Oncogen/Bristol-Myers Squibb, Seattle, WA) was constructed using vaccinia virus obtained from a commercial preparation of smallpox vaccine (Dryvax, Wyeth L a b o r a t o r i e s ) . Purified rgpl60 (VaxSyn), also derived from the LAV strain of HIV-1, was developed by MicroGeneSys, Inc., Meriden, CT. The gpl60 was purified from cell lysates after expression by a recombinant baculovirus in Spodoptera fugipoda cells as described (29). It should be noted that HIV-1 strain LAV, used in the construction of these vaccines, and HIV-1 strain IIIB, used throughout the studies described here, share greater than 98% sequence homology, especially in the V3 region, and are probably from the same isolate (30). Study Protocol. Volunteers were healthy adults, seronegative for vaccinia and HIV-1. HIVAC-le was administered using bifurcated needle punctures. Volunteers were inoculated 580-650 days later with 640 ~g of rgpl60 as described (25). Cells and Viruses. The CD4 + human lymphoblastoid cell lines MT-2 (31) and H9 (obtained from
Journal o f Clinical Immunology, Vol. 12, No. 6, 1992
SERUM ANTIBODIES IN HIV-IMMUNIZED HUMANS
R. C. Gallo, National Cancer Institute), were cultured at 37°C in RPMI-1640 containing 12% heatinactivated (56°C, 1 hr) fetal bovine serum and 50 p~g of gentamicin per ml. Stocks of the HIV-1 isolates IIIB, RF (obtained from R. C. Gallo), and MN (obtained from the National Institutes of Health, AIDS Research and Reference Reagent Program) were harvested from chronically infected cultures of H9 cells. Virus-containing culture fluids were clarified of cells by low-speed centrifugation and passed through 0.45-~m cellulose acetate filters (Millipore). HIV-1 SF2 was recovered from peripheral blood mononuclear cells (PBMCs) of a patient with oral candidiasis, while HIV-1 SF-128A was derived from spinal cord tissue of a patient with neurological AIDS (32). Both strains were obtained from Dr. Jay Levy and have been further propagated in normal human PBMCs maintained in fetal calf serum, glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 txg/ml), and 5-10% interleukin-2 (33). The culture medium containing freshly released virus was centrifuged at low speed to remove cells and cell debris. The fluid was then filtered through 0.45-p~m cellulose acetate filters (Millipore). The quantity of the virus was determined by reverse transcriptase activity (34) and the 50% tissue culture infectious dose (TCIDs0) in peripheral blood mononuclear cells (PBMCs) (35). Western Blot. Antibodies to HIV-l-strain IIIB antigens were detected by Western blot as described by the manufacturer (E. I. DuPont, Wilmington, DE). Immunofluorescence Assays. Antibodies to viral antigens were quantitated by indirect immunofluorescence assays using cells chronically infected with IIIB, RF, or MN strains of HIV-1. Slides of fixed, infected cells were prepared by air-drying and fixing in a 1:1 mixture of acetone:methanol for 30 rain. Sera were diluted in phosphate-buffered saline containing 0.1% globin-free bovine serum albumin (PBS-BSA) and incubated on slides for 30 rain at 36°C and 100% humidity. After two washes in carbonate buffer (0. I 1 M NazCO 3, 0.4 M NaHCO 3, 0.15 M NaC1, 0.05% Tween-80), the slides were incubated for 30 min at 36°C and 100% humidity with a 1:200 dilution of fluorescein-conjugated, IgG fraction of goat anti-human IgG (heavy and fight chain specific; Cappel) containing Evan's blue counterstain. Slides were again washed twice in carbonate buffer and then mounted using 50% glycerol and examined for fluorescence using a Nikon DIAPHOT-TMD-EF fluorescencemicroscope. Anti-
Journal of Clinical Immunology, Vol. 12, No. 6, 1992
431
body titers are defined as the last dilution yielding positive fluorescence. Additional experiments were done to determine the presence of anti-HIV-I immunoglobulin of various classes and subclasses using anti-human IgG1, IgG2, IgG3, IgG4, IgA, and IgM (Cappel, Organon Teknika Corp., Durham, NC). Cell lines expressing IgA (DAKIKI) and IgM (RPMI 1788) (both cell lines obtained from American Type Culture Collection, Rockville, MD) were used as positive controls. Peptide ELISA. Antibodies to rgpl60 or peptides corresponding to the principal neutralizing domain (PND) of gpl20 (36) were measured by ELISA. Peptides corresponding to amino acids 302-324, 308-324, and 312-327 of the HIV-1 IIIB gpl20 were obtained from American Bio-Technologies, Inc., Cambridge, MA. A negative control peptide corresponding to the Sindbis virus envelope glycoprotein was obtained from The Upjohn Company, Kalamazoo, MI. The rgpl60 was obtained from MicroGeneSys, Inc. Individual wells of 96-well Immulon-2 plates (Dynatech Laboratories, Inc., Chantilly, VA) were incubated overnight at 4°C with I00 ~1 of peptide in carbonate buffer (15 mM NazCO3, 35 mM NaHCO3, pH 9.8). The peptide solutions were then aspirated and the wells filled with 100 p~lof blocking buffer (Filter Paper Diluent, DuPont) containing 2.5% fetal bovine serum and incubated at 37°C for 4 hr. The plates were washed 4× with phosphate-buffered saline (PBS; 0.14 M NaC1, 2.7 mM KC1, 8 mM Na2HPO4, 1.7 mM NaHzPO4, pH 7.4) containing 0.05% Tween-20. Serial dilutions of volunteers' sera were made in wells containing borate buffer (0. I M boric acid, 47 mM sodium borate, 75 mM NaC1, 0.05%, v/v, Tween-20) + 2.5% fetal bovine serum and incubated at 37°C for 4 hr. Wells were then washed 4× with PBS/Tween-20 and received 100 ~1 of alkaline phosphatase-conjugated, goat anti-human IgG and IgM (Sigma Chemical Company, St. Louis, MO) and incubated at 37°C for 4 hr. The wells were again washed 4x with PBS/Tween-20 and incubated with 100 ~1 ofp-nitrophenylphosphate disodium hexahydrate (Sigma 104 phosphatase substrate) in diethanolamine buffer (0.9 M diethanolamine, 7 mM MgCI2, pH 9.8, with concentrated HC1). After color development, absorbance was read at 405 rim. The titer was reported as the highest logz reciprocal serum dilution that had an average absorbance reading greater than 0.1 and at least twice that of the negative control (Sindbis virus peptide).
432
Neutralizing Antibodies. HIV-1 IIIB neutralizing titers in sera from vaccinees were measured in an MT-2 cell cytopathic infection assay in microdilution plates as described previously (37). Briefly, twofold dilutions of heat-inactivated sera (56°C, 1 hr) were made in triplicate in a total of 100 ~1 of growth medium per well. Fifty microliters of virus (5 × l04 TCIDs0 of IIIB) was then added to all wells except for one row of eight noncytopathic control wells, which received growth medium in place of virus. After 1 hr of incubation, MT-2 cells (5 x 104) in 100 Ixl of growth medium were added to each well. Viable cells were quantitated colorimetrically by vital dye (neutral red) uptake 3 days later. Neutral red uptake is a linear function of cell viability, where light absorption at a 540-nm wavelength is linear from 0.025 to 0.85, corresponding to 2 x 104 to 25 × 104 viable cells/well (37). Percent protection was defined as the difference in absorption between test wells (cells + serum + virus) and virus control wells (cells + virus) divided by the difference in absorption between cell control wells (cells only) and virus control wells. Plates were harvested when cytopathic effect in virus control wells produced greater than a 50% reduction in viable cells. Neutralizing titers were defined as the reciprocal of the last dilution to provide at least 35% protection. The concentration of virus in the inoculum was determined by titration on MT-2 cells in this assay; the reciprocal dilution at which 50% of wells showed cytopathic effects after 2 weeks defined the titer (1 TCIDso). Antisyncytial Antibodies. Antisyncytial activity was measured in an assay analogous to the neutralizing antibody assay as described previously (38). Syncytium formation was induced by mixing MT-2 cells with H9/IIIB cells at a 10:1 ratio in the presence and absence of sera in 96-well microdilution plates. Serial dilutions of sera were made in triplicate. H9/IIIB cells (1.5 x 104) in 50 Ixl of growth medium were added to all wells except one row of eight noncytopathic control wells, which received uninfected H9 cells. MT-2 cells (1.5 × 105) in 100 ixl of growth medium were then added to each well. Syncytium formation leads to, and is directly proportional to, cytopathic effect in this assay. After incubation at 37°C for 20 hr, syncytium formation was observed microscopically, while viable cells were measured by vital dye uptake as described above. Antibody-Dependent Enhancement. C'-ADE was measured in MT-2-cell microtiter infection assays
MONTEFIORIET AL.
as described previously (15). The procedure is similar to the neutralizing antibody assay described above except that fresh human serum is added at a 1:40 dilution as a source of complement. Infection enhancement is evident by a reduction in viable cells relative to a set of virus control wells which contain cells, virus, and complement, but no antiHIV-1 serum. Therefore, the percentage viable cells was defined as the difference in A540 readings between the average of three test wells and the average of eight blank wells, divided by the difference in A54o readings of the average of eight virus control (no antibody) wells and eight blank wells. C'-ADE titers were defined as the reciprocal of the last dilution to produce at least a 20% reduction in viable cells. FcR-ADE was measured in human PBMCs as described by H o m s y et al. (17). Briefly, 100 I~1 of dilutions of heat-inactivated vaccinee's sera was mixed with an equal volume of virus-containing culture fluid which contained 100 TCIDso units of HIV-1. The mixtures were incubated at room temperature for 1 hr. Thereafter, 106 phytohemagglutinin (PHA)-stimulated PBMCs in 0.4 ml were added to each virus-antibody mixture and incubated at 37°C for 2 hr. The supernatant was removed and cells were resuspended in medium for several days. Culture fluids were harvested on days 3 and 5 for HIV-1 128A and on days 7 and 10 for HIV-1 SF2 and quantitated for amount of virus by reverse transcriptase activity. Enhancement of infection was measured by a greater than twofold increase in viral production from cultures receiving postvaccination serum as compared to those receiving prevaccination serum of the same individual.
RESULTS
HIV-1-Specific Ig Classes and Subclasses. HIV-1 IIIB-specific IgG was first detected by IFA in the sera of volunteers 2 weeks after they were boosted with rgpl60, where it was present in all four individuals at reciprocal titers of 160-640 (Table 1). Only one of the four volunteers (A20) had IgG that cross-reacted with antigens of HIV-1 strains RF and MN. Reciprocal titers of antibodies to HIV-I RF and MN were considerably lower (i.e., 8× lower for RF and 32× lower for MN). HIV-1 IIIB-specific IgG was detected in only one volunteer (A15) by 90 days after booster immunization (Table I). Journal of Clinical Immunology, Vol. 12, No. 6, 1992
SERUM ANTIBODIES IN HIV-IMMUNIZED HUMANS
433
Table I, Titers and Strain Specificity of Anti-HIV-1 IgG in Sera from Vaccinated Volunteers a Antibody titer (reciprocal dilution)
Volunteer
IIIB
RF
MN
IIIB
RF
MN
IIIB
RF
MN
90 days after rgpl60 IIIB
A10 At2 A15 A20
