Neutralization of a Clade B Primary Isolate by Sera from Human Immunodeficiency Virus Uninfected Recipients of Candidate AIDS Vaccines

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1 764 Neutralization of a Clade B Primary Isolate by Sera from Human Immunodeficiency Virus Uninfected Recipients of Candidate AIDS Vaccines Susan Zolla-Pazner, Carl Alving, Robert Belshe, VA Medical Center and New York University Medical Center, New Phillip Berman, Sherri Burda, Padmasree Chigurupati,* York; Walter Reed Army Institute of Research, Washington, DC; Saint Louis University School of Medicine, St. Louis, Missouri; Genentech, Mary Lou Clements, Anne-Marie Duliege, Inc., South San Francisco, San Francisco General Hospital, San Jean-Louis Excler, Catarina Hioe, James Kahn, Francisco, and Chiron Biocine, Emeryville, California; Johns Hopkins M. Juliana McElrath, Sandra Sharpe, Faruk Sinangil, University Center for Immunization Research, Baltimore, and National Kathelyn Steimer, Mary Clare Walker, Nabila Wassef, Institute of Allergy and Infectious Diseases, National Institutes of and Serena Xu Health, Bethesda, Maryland; Pasteur Mérieux/Connaught, Marnes-la- Coquette, France; University of Washington School of Medicine, Seattle The inability of antibodies induced by experimental human immunodeficiency virus type 1 (HIV- 1) vaccines to neutralize HIV-1 primary isolates may be due to a failure to elicit such antibodies, antigenic differences between the vaccine and the strains tested, insensitivity of the assays used, or to a combination of factors. New neutralization assays were used to determine the ability of candidate AIDS vaccines to generate neutralizing antibodies for clade B primary isolate BZ167, which is closely related in portions of its envelope to the immunizing strains. Sera from HIV-uninfected volunteers in vaccine trials were tested, and neutralizing activity was found in recipients of recombinant (r) gp120 MN or of rgp160 MN -containing canarypox boosted with rgp120 SF-2. Detection of antibodies that neutralize primary isolate BZ167 correlated with neutralizing activity for homologous vaccine strains. These data demonstrate that certain candidate AIDS vaccines can elicit antibodies that neutralize a primary isolate of HIV-1. Clinical use of many viral vaccines (such as those for rube- cell lines (laboratory-adapted HIV) has been shown to prevent ola, rubella, mumps, polio, influenza, hepatitis B, rabies, and infection of chimpanzees, hu-pbl-scid mice, and SCID-hu Japanese encephalitis virus) has been predicated on the demon- mice with laboratory-adapted viruses [2 9]. Similarly, in some stration that these vaccines elicit demonstrable neutralizing antibodies studies of active immunization of chimpanzees, protection has in the sera of recipients [1]. This practice is based, in been found to correlate with the presence of neutralizing anti- part, on the immunologic principle that the humoral arm of the bodies to laboratory-adapted strains of HIV [10 15]. More immune response plays a primary role in preventing many viral recently, protection of actively immunized chimpanzees from infections while the cellular arm of the immune response is a challenge with a high dose of cell-free HIV-1 was associated critical for control and eradication of virus-infected cells. with the presence of neutralizing antibodies to both laboratoryadapted To date, the clearest evidence for protection from human and primary strains of HIV [16], and the presence of immunodeficiency virus (HIV) infection also correlates with neutralizing antibodies to HIV primary isolates in the sera of the presence of neutralizing antibodies: Passive immunization pregnant women has been reported to correlate with protection with monoclonal or polyclonal antibodies or both with neu- of infants from maternal-fetal transmission of HIV [17]. tralizing activity for strains of HIV adapted for growth in T Consequently, there was initial enthusiasm when the first investigational AIDS vaccines given to human HIV-uninfected volunteers were shown to induce antibodies that could neutralize laboratory-adapted strains of HIV [18 23]. This enthusiasm Received 8 August 1996; revised 6 November waned, however, when it was reported that sera from vaccine Presented in part: Conference on Advances in AIDS Vaccine Development, recipients could not neutralize primary HIV isolates [24 26]. Bethesda, Maryland, February 1996 (abstract 36); XI International Conference Various hypotheses were suggested to explain this apparent on AIDS, Vancouver, Canada, July 1996 (abstract 0544). Informed consent was obtained from all subjects. Guidelines of the US discrepancy in neutralizing results. These include the follow- Department of Health and Human Services and from the institutional review ing: (1) primary isolates are intrinsically less sensitive to neuboards of the authors institutions were followed in the conduct of all clinical tralization than laboratory-adapted strains [24, 27 29], (2) antistudies. Financial support: NIH (AI-32424, AI-36085, AI-27742, AI-45207, AI- bodies induced by the vaccines are primarily directed to linear 45211, AI-45208, AI-45206, and AI-65305); Department of Veterans Affairs epitopes and not to conserved, discontinuous epitopes that (Research Center for AIDS and HIV Infection and Merit Review Funding). might be more biologically relevant [30], (3) the conformation Reprints or correspondence: Dr. Susan Zolla-Pazner, c/o VA Medical Center (Room 18124No), 423 E. 23rd St., New York, NY and exposure of relevant epitopes on envelope glycoproteins * Present affiliation: Paracelsian, Inc., Ithaca, New York. of primary isolates differ qualitatively from those of laboratory- Deceased. adapted strains, rendering the former more resistant to neutral- The Journal of Infectious Diseases 1997;175: ization [31], (4) the assays used have different levels of sensi by The University of Chicago. All rights reserved /97/ $01.00 tivity such that neutralizing antibodies for primary isolates that

2 JID 1997;175 (April) HIV-1 Vaccine Induced Neutralizing Antibody 765 might be present are below the threshold of detection in con- designated as FDA-2, is available from the AIDS Research and ventional neutralization assays [32], or (5) there is an antigenic Reference Reagent Program (catalog no. 1983) and was also used mismatch between the immunizing strains and the HIV strains in additional experiments described below. Sera from both placebo being tested for neutralization [24]. and candidate vaccine recipients, obtained before (preimmuniza- tion) and after immunization, were included in each AVEG panel. With the development of neutralization assays for primary Postimmunization serum specimens were selected to be representa- HIV isolates with increased sensitivity (described below and tive of HIV-specific binding and neutralizing antibody responses in [32]), the opportunity arose to address whether sera from induced in volunteers receiving the same treatment on the same recipients of various vaccine candidates contained neutralizing immunization schedule by the same route of administration, as antibodies that might previously have gone undetected. A clade determined by assays for neutralization of HIV MN and HIV SF-2 B HIV-1 primary isolate, BZ167, was selected for use because, performed at the AVEG Central Immunology Laboratory at Duke in reports of genetic analyses of a 630-bp portion of its envelope University (Durham, NC). AVEG panel 1 included 16 sera from a dose-escalation study [33], this isolate was found to be closely related to HIV SF-2 and HIV MN, strains from which several candidate vaccines of the Genentech rgp120 MN candidate vaccine, which included an were derived. Studies were undertaken with 84 specimens of arm in which the Genentech rgp120 IIIB product was given simulta- serum or plasma from HIV-positive and -negative control subthe prefix G in table 1. Results of this trial are published [19]. neously with the rgp120 MN product. These sera are designated by jects and from HIV-uninfected recipients of several candidate Sera in the second AVEG panel, designated by the prefix Np HIV vaccines, 74 of which were coded to ensure objective in table 1, were from two different AVEG trials. One group of testing. Experiments were designed to reveal whether primary sera was from recipients of the Biocine rgp120 SF-2 formulated in isolate neutralizing antibodies were induced and could be de- an adjuvant preparation consisting of alum-adsorbed liposomes tected in the sera of recipients of several candidate vaccines and made of phospholipids and cholesterol and containing monoto determine whether the different vaccines and immunizing phosphoryl lipid A [35]. The second group of sera was from recipients regimens induced different levels of these antibodies. Addipurified of the Genentech rgp120 MN subunit product with QS21, a tional experiments were designed to determine if detection of saponin adjuvant. An interim report describing the design neutralizing antibodies was dependent on the assay used and of these AVEG studies has been published [36]. whether, when detected, the level of primary isolate neutraliztable The third AVEG serum panel, designated by the prefix PB in 1, was derived from a dose-escalation and schedule of immu- ing antibodies correlated with the titers of antibodies that neunization variation trial of the Pasteur Mérieux/Connaught recombitralized the laboratory-adapted strains from which the vaccines nant canarypox-hiv gp160mn candidate vaccine (also designated as were derived. vcp125 or ALVAC gp160mn ) with and without boosting with Biocine rgp120 SF-2 in MF59 adjuvant. A recombinant canarypox expressing the rabies glycoprotein G (ALVAC RG ) was used as a Methods control vaccine preparation. A preliminary report of this trial was Subjects and specimens tested. Four panels of serum or plasma recently presented [37]. obtained from volunteers participating in phase I clinical trials of Known HIV-positive and -negative sera were used as controls candidate AIDS vaccines were tested. Trial volunteers were of and for studies of the relative neutralization sensitivity of various both sexes and between 18 and 60 years of age. All were HIV- HIV-1 primary isolates. Five HIV-positive serum and plasma preparations noninfected as determined by FDA-approved HIV-1 EIAs and were used. Sera F and L were from single blood samples Western blots. of 2 asymptomatic HIV-positive subjects infected for ú8 years. Table 1 summarizes the specimens tested and, where appropriate, Serum FDA-2, described above, was also used, as were a plasma the vaccine products used, the doses administered, the adju- pool from HIV-positive donors (provided by C. Hanson, California vants used or the formulations of the vaccine product, the immunization Department of Health Services, Berkeley) and a serum pool assem- schedules, the time at which blood samples were drawn, bled from 33 randomly picked HIV-positive subjects at the New and the trial designation for each of the 84 specimens tested. York VA Medical Center. HIV-negative sera were used as negative The Chiron Biocine company provided an unblinded panel of controls in all experiments. All sera were heat-inactivated at 56 C 10 plasma aliquots, designated by the prefix C in table 1, from for 30 min prior to use. subjects enrolled in a trial of their CHO cell expressed HIV-1 Neutralization assays. In all assays, heat-inactivated sera were rgp120 SF-2 subunit product formulated in MF59 adjuvant, a microfluidized used. Each plasma specimen supplied by Chiron Biocine was ad- oil-in-water emulsion [34], with or without escalating justed to 0.01 M with CaCl 2 to induce clotting before use. Four amounts of muramyl tripeptide phosphatidylethanolamine. The results different types of neutralization assays were performed. of the study have been published [23]. To determine the level of primary isolate neutralizing antibodies, The other three panels of sera were derived from volunteers clade B primary isolates BZ167 and US4 were obtained from participating in trials conducted by the National Institute of Allergy J. Mascola (Henry M. Jackson Foundation, Rockville, MD), and and Infectious Diseases (NIAID) sponsored AIDS Vaccine Evaluation isolates 91US056 and 92HT593 were obtained through J. Bradac Group (AVEG). The serum panels were coded and included and O. Sharma (NIAID AIDS Research and Reference Reagent HIV-positive and -negative control sera. One HIV-positive specimen, Program). Stocks of these isolates were produced in peripheral consisting of four separate blood samples from the same blood mononuclear cells (PBMC) as described previously [32]. HIV-infected subject, was provided as a blinded positive control Neutralization of these viruses was measured using three similar in two panels (specimens Np5 and PB5 in table 1). This serum, assays that differed primarily with respect to the target cell used.

3 766 Zolla-Pazner et al. JID 1997;175 (April) Table 1. Description of HIV-1 candidate vaccines and immunization regimens studied. Immunization Time blood was Vaccine Dose per schedule drawn in relation to developer Vaccine product injection Adjuvant or formulation (months) immunization Trial designation Specimen numbers Genentech rgp120 MN 100 mg Alum 0, 1, 6, 12 2 weeks after 4th AVEG 009 G1, G7, G11 rgp120mn 300 mg G3, rgp120mn 600 mg G4, G5, G8, G10 rgp120mn 300 / 300 mg G2, G6, G9, G12 / rgp120iiib Genentech rgp120mn 300 mg QS21 0, 1, 10 2 weeks after 3rd AVEG 016 Np6, Np8 Genentech rgp120mn 600 mg QS21 0, 1, 10 2 weeks after 3rd AVEG 016 Np10, Np11 Biocine rgp120sf mg Alum-absorbed liposomes 0, 2, 6 2 weeks after 2nd AVEG 015 Np16, Np17, Np19, Np21, Np23, with MPL or 3rd Np24, Np25, Np26 Biocine rgp120sf-2 50 mg MF59 { MTP-PE* 0, 1, 6, 12 2 weeks after 4th V6P1 C202, C205, C207, C224, C225, C227, C240, C241, C243, C260 Biocine rgp120sf-2 50 mg MF59 0, 1, 6, 12 2 weeks after 3rd AVEG 012B PB10, PB21, PB24 or 4th PMC/Biocine ALVACgp160MN 10 6 TCID50 MF59 0, 2, 9, 12 2 weeks after 4th AVEG 012A PB2, PB3, PB17, PB26 / rgp120sf-2 / 50 mg PMC/Biocine ALVACgp160MN 10 7 TCID50 MF59 0, 1 or 2, 9, 12 2 weeks after 4th AVEG 012B PB7, PB9, PB18, PB19, PB20 / rgp120sf-2 / 50 mg PMC ALVACgp160MN 10 6 or 10 7 NA 0, 1 or 2, 9, 12 2 weeks after 4th AVEG 012A or 012B PB1, PB11, PB15, PB22, PB25 TCID50 PMC/Biocine ALVACRG / TCID50 MF59 0, 1 or 2, 9, 12 2 weeks after 4th AVEG 012A or 012B PB12, PB14, PB16, PB23 rgp120sf-2 / 50 mg PMC ALVACRG TCID50 NA 0, 2, 9, 12 2 weeks after 4th AVEG 012A or 012B PB6, PB13 Genentech Placebo NA Alum 0, 1 or 2, 6 2 weeks after 2nd AVEG 009 Np2, Np3, Np28, Np30, Np32 or 3rd Genentech Placebo NA QS21 / alum or QS21 0, 1, 10 2 or 3 weeks after AVEG 016 Np4, Np12, Np13, Np14 alone 2nd or 3rd NA None NA NA NA Prior to 1st AVEG 015 or 016 Np7, Np9, Np18, Np20, Np29, Np31, Np33, Np34 NA None NA NA NA Prior to 1st AVEG 009 G13, G14 NA None NA NA NA Prior to 1st AVEG 012A or 012B PB4, PB8 NA HIV-negative NA NA NA NA NA Np22, Np27 serum pools NA HIV-positive NA NA NA NA NA G15, G16 serum pools NA HIV-positive NA NA NA NA NA Np5, PB5 serum NOTE. PMC, Pasteur Mérieux/Connaught; NA, not applicable; AVEG, AIDS Vaccine Evaluation Group; MPL, monophosphoryl lipid A. * Subjects C224, C225, and C227 received vaccine with MF59 and 1 mg of muramyl tripeptide phosphatidylethanolamine (MTP-PE); subjects C240, C241, and C243 received vaccine with MF59 and 10 mg of MTP-PE; subject C260 received vaccine with MF59 and 50 mg of MTP-PE. The first of these assays, the resting cell assay, was previously The third neutralization assay of primary isolate BZ167 used described in detail [32]. Briefly, frozen unstimulated PBMC were the T cell line CEM-SS as target cells. For this assay, thawed, and cells were used per culture. Serial serum dilutions 10 5 cells/well were used, and medium consisted of RPMI 1640 were incubated with BZ167 virus stocks for 1 h at 37 C and added supplemented with 10% fetal calf serum, 2% penicillin/streptomy- to cells in each well for 1.5 h, after which the virus-serum mixture cin, and 1% L-glutamine; all other parameters were identical to was aspirated and replaced with complete medium consisting of RPMI those described above supplemented with 10% heat-inactivated human AB serum, 2% All specimens were tested in two to four experiments, and in penicillin/streptomycin, and 1% L-glutamine. In some cases, the virusserum each experiment every specimen and control was performed with mixture was allowed to incubate with the cells overnight. For 4 or 6 replicate cultures. Thus, each data point presented is the assays using primary isolate BZ167, input virus ranged from 20 to mean of replicate cultures. 200 TCID 50 per well. For the other viruses, input was TCID 50 To determine the extent of neutralization, the mean nanograms per well. Twenty-four hours after infection, the cultures were supplemented of p24 per milliliter of each set of replicate cultures was determined with 0.8 mg/ml phytohemagglutinin (PHA) and 20 U/mL and the percentage of neutralization calculated as [1 0 (mean p24 human recombinant interleukin-2 (Boehringer Mannheim, Indianapolis). value in the presence of experimental sera/mean p24 value in the Six days after infection, the cells were harvested by adding 50 presence of control sera)] Control sera consisted of either ml/well 5% Triton-X in PBS, and a previously described noncommercial heat-inactivated individual HIV-negative human serum, heat-inac- p24 ELISA was used for the readout [38]. tivated pooled HIV-negative human AB sera, or, in the case of A related assay used PBMC/well that had been prestimulated specimens supplied by Chiron Biocine and from the AVEG 009 for 2 days with 0.8 mg/ml PHA. trial (designated with the prefix C or G [table 1 and figure 1]),

4 JID 1997;175 (April) HIV-1 Vaccine Induced Neutralizing Antibody 767 Figure 1. Neutralizing activity in 80 individual sera from controls (A D) and vaccinees (E K) vs. clade B primary isolate BZ167. Mean percentage of neutralization is shown for each specimen, which was derived from replicate cultures. Resting cell assay was used with sera diluted 1:20 or 1:25. Specimen numbers shown below each graph are cross-referenced to table 1. In F, results are shown for volunteers who received 4 different immunizing regimens: 100 mg/dose rgp120 MN (open bars), 300 mg/dose rgp120 MN (stippled bar), 600 mg/dose rgp120 MN (solid bars), and 300 mg/dose rgp120 MN plus 300 mg/dose rgp120 IIIB (hatched bars). heat-inactivated preimmunization blood samples from vaccine re- Assays of neutralization of the laboratory-adapted HIV SF-2 and cipients. In some instances, greater virus production was seen in HIV MN strains were performed at the AVEG Central Immunology the presence of test serum than in the presence of the known HIVpreviously Laboratory using the multiple virus dose neutralization assay as negative serum used as control. This increase in virus production described [21]. Briefly, each serum dilution was tested never met the definition of antibody-dependent enhancement [39]. in duplicate against six concentrations of virus; virus input ranged In all cases in which p24 production in the presence of antibody from 0.1 to 100 TCID 50 /well. The AA5 cell line was used as the exceeded that in the presence of the known HIV-negative control, target cells and a reverse transcriptase assay was performed at day the percentage of neutralization is shown as 0. 7 after infection. The TCID 50 for each control and test serum To define a cutoff value between positive and negative neutralizareduction was determined by the Reed and Muench formula; the infectivity tion, the data from 62 experiments with the HIV-negative specimens titers were defined as the reciprocal of the serum dilution shown in figures 1B D were analyzed. The amount of variation in that resulted in a reduction of TCID 50 by 1 order of magnitude. the resulting distribution reflects the variation from the assay technology and from the different specimens. The 95th percentile of this distribution, which corresponds to 31% neutralization, was used as Results the cutoff value. Consequently, cultures displaying 32% 50% neutralization were considered weakly positive and those displaying Four panels of specimens consisting of 84 samples of serum 51% 100% neutralization were considered strongly positive. or plasma were tested for neutralization of primary isolate

5 768 Zolla-Pazner et al. JID 1997;175 (April) BZ167 in the resting cell assay. The results from 4 of the 84 of recipients receiving various vaccines and placebos was tested specimens (Np11, C241, C260, and Np9) were excluded before against 2 laboratory-adapted HIV strains as well as against unblinding because of variability in replicate testing. The re- primary isolate BZ167. Infectivity reduction titers of 35 sera sults of neutralization assays with each of the remaining 80 were determined against HIV MN. The relationship of these titers specimens, tested at dilutions of 1:20 or 1:25, are shown in versus the percentage of neutralization achieved in the resting figure 1. All specimens, except those provided by Chiron Biocine cell assay against BZ167 was analyzed using the Spearman (and designated with the prefix C in table 1 and figure 1), rank correlation method. As shown in figure 2A, there is a were tested blind. Results shown represent the mean percentage direct association between the inverse of the HIV MN infectivity of neutralization for each specimen. reduction titer and the percentage of neutralization of BZ167 Neutralization of BZ167 by vaccinees sera in the resting cell achieved in the resting cell assay (Spearman rank r Å.6993; assay. All 4 HIV-positive specimens included in the panels dis- P õ.0001). Similarly, 42 sera from vaccine and placebo recipients played strong neutralizing activity, each giving ú97% neutralization were assayed for reactivity versus HIV SF-2. When all data of isolate BZ167 (figure 1A). All HIV-negative sera (figure were included in the analysis, a highly significant correlation 1B), sera from HIV-uninfected placebo recipients (figure 1C), and was observed (Spearman rank r Å.4321; P Å.004). However, preimmune sera from HIV-uninfected volunteers who would later inspection of the data, shown in figure 2B, indicates that there receive various candidate AIDS vaccines (figure 1D) lacked neutralizing was a group of 18 vaccinees who produced neutralizing anti- activity, as shown by õ31% neutralization. On the basis bodies with a titer 1:10 to SF-2 but little or no neutralizing of these blinded controls, the resting cell assay was shown to be antibodies to BZ167. Eight of these subjects received rgp 100% specific and 100% sensitive. 120 SF-2 in the liposome formulation. Ten recipients of other Sera from 3 of 3 volunteers who received 300 or 600 mg of candidate vaccines also produced neutralizing antibodies to SFrgp120 MN per dose with the QS21 adjuvant contained signifi- 2 but not to BZ167. These data suggest that even when using cant neutralizing activity (63% 77% neutralization at a serum immunogens that possess cross-neutralizing epitopes, some for- dilution of 1:20, figure 1E). When rgp120 MN, or a combination mulations will fail to induce or some recipients will fail to of rgp120 MN and gp120 IIIB, was administered at different doses produce (or both) cross-neutralizing antibodies. with alum rather than QS21 as adjuvant, 9 of 12 sera displayed Comparison of neutralizing activity determined using various significant neutralizing activity. Because of the small number assays for neutralization of primary isolates. Since of specimens tested at each dose, a dose-response relationship BZ167 is a syncytium-inducing virus and can grow both in could not be ascertained; however, even at the lowest dose PBMC and T cell lines, sera of several vaccinees were tested (100 mg), significant neutralizing activity was detected in the for their ability to inhibit the infectivity of BZ167 in different sera of 2 of the 3 recipients (figure 1F). neutralization assays in which the CEM-SS T cell line or un- As shown in figure 1G, sera from 5 of 11 volunteers who stimulated or PHA-stimulated PBMC were used as target cells. received four injections of 50 mg of rgp120 SF-2 in MF59 contained For these studies, sera were used from 4 recipients of the 300- significant neutralizing activity. In contrast, sera from mg dose of rgp120 MN plus 300-mg dose of rgp120 IIIB in alum recipients who received 37.5 mg of rgp120 SF-2 delivered in a (specimens G2, G6, G9, and G12; table 1, figure 1F). Results liposome formulation after two or three immunizations con- in figure 3A show that neutralizing antibodies for BZ167 could tained no detectable neutralizing activity (figure 1H). be detected in these 4 sera when CEM-SS cells were used as In studies of a prime/boost immunizing regimen, it was target cells. Neutralizing activity in the sera of these 4 vaccinees found that recipients of four doses of the priming construct diminished on dilution of the sera, with 50% neutralizing titers (ALVAC gp160mn, also designated as vcp125 [40]) had no serum ranging from Ç1:100 to 1:400 (equivalent to 0.25% 1.0% neutralizing activity (figure 1I). Recipients who were primed serum). Sera from specimen G6, which had displayed no sig- with the non-hiv ALVAC RG construct (expressing the rabies nificant neutralizing activity at 1:25 (4% serum) in the resting G glycoprotein) and subsequently boosted with rgp120 SF-2 in cell assay (figure 1F), was shown in the CEM-SS assay to MF59 responded sporadically, with only 1 of 4 recipients contain neutralizing antibodies for BZ167, although at a lower mounting a significant neutralizing response (figure 1J); this level than the sera of the other vaccinees tested. A preimmuni- was not dissimilar to the response of recipients of rgp120 SF-2 zation blood sample from an HIV-uninfected volunteer (G14) and MF59 administered without priming (figure 1G). However, contained no significant neutralizing activity. when volunteers were primed with ALVAC gp160mn and boosted Titration of the sera from the same recipients in the resting with rgp120 SF-2, 7 of 9 mounted significant neutralizing responses cell assay yielded the data shown in figure 3B. This experiment (figure 1K). recapitulates those summarized in figure 1F in which the 4 These results suggest that of the immunizing strategies studied, recipients of 300 mg of gp120 MN plus 300 mg of gp120 IIIB the most consistent neutralizing responses were mounted responded with different levels of neutralizing activity: Sub- by recipients of rgp120 MN in QS21 or alum and by recipients jects G9, G12, G2, and G6 had 63%, 59%, 49%, and 6% of the ALVAC gp160mn prime with the rgp120 SF-2 boost. neutralizing activity, respectively, at a serum dilution of 1:25. Correlation of neutralizing activity for laboratory-adapted Titration of sera from these 4 immunized subjects in the resting and primary strains of HIV. The neutralizing activity in sera cell assay displayed typical titration curves. The 50% neutraliz-

6 JID 1997;175 (April) HIV-1 Vaccine Induced Neutralizing Antibody 769 Figure 2. Correlation between neutralizing activity measured with sera (at 1:20 or 1:25 dilution) of vaccine and placebo recipients against primary isolate BZ167 in resting cell assay and infectivity reduction titers measured against laboratory-adapted strains HIV-1 MN (A) and HIV- 1 SF-2 (B).

7 770 Zolla-Pazner et al. JID 1997;175 (April) 1F), again showed little or no activity when titrated using this same neutralization format, although, as noted above, this serum did contain neutralizing activity for BZ167 when assayed using CEM-SS target cells (figure 3A). The HIV-negative preimmunization serum again displayed no demonstrable neutralizing activity. Titration of these same sera for neutralizing activity using PHA-stimulated PBMC as target cells failed to show any neutralizing activity in any of the sera tested (figure 3C). In this assay, the virus preparation, the amount of infectious virus ( TCID 50 per well), and the culture conditions used were identical to those used in the previous experiments (figure 3A and 3B); only the target cells were changed. The data from the experiments shown in figure 3 demonstrate that the target cell used contributes profoundly to the sensitivity of the assay and to the ability to detect neutralizing antibodies. Thus, the level of sensitivity of the assay using CEM-SS cells as target cells was greater than the sensitivity of the assay using unstimulated PBMC as target cells. Similarly, use of the unstimulated PBMC provided greater sensitivity than did PHA-stimulated PBMC. This conclusion was supported by neutralization studies with BZ167 and a human monoclonal antibody, D, specific for the V3 loop of gp120 of clade B HIV-1, which mediated 50% neutralization at 0.02, 0.16, and 0.83 mg of monoclonal antibody/ml in the assays using CEM-SS, unstimulated PBMC, and stimulated PBMC as target cells, respectively; neutralization was also more readily detected using unstimulated rather than stimulated PBMC as target cells when the p24 assays were performed at days 4 8 after infection (data not shown). Relative neutralization sensitivity of BZ167 among clade B primary isolates. While BZ167 was chosen for study because of its genetic relationship to the HIV vaccine strains and its growth characteristics in unstimulated PBMC, the neutralization by vaccinees sera could theoretically be explained if BZ167 were unusual in its sensitivity to antibody-mediated neutralization. Therefore, BZ167 and a panel of 3 other clade B primary isolates were tested for their ability to be neutralized by sera from 3 HIV-infected persons and by plasma and serum pools Figure 3. Titration of neutralizing activity for BZ167 in 3 assays prepared from HIV-infected subjects. Representative titration using sera from 1 HIV-uninfected control subject and 4 recipients of curves from these experiments are shown in figure 4. These data 300 mg/dose rgp120 MN plus 300 mg/dose rgp120 IIIB administered with show that each of the 4 viruses was neutralized by 1 of the 3 alum. Sera were tested in assays in which target cells consisted of HIV-positive sera and by 1 of the 2 polyclonal pools. BZ167 CEM-SS cells (A), unstimulated ( resting ) peripheral blood monowas the only virus that was neutralized effectively by all 5 nuclear cells (PBMC; B), or phytohemagglutinin-stimulated PBMC (C); all other conditions were held constant. Preimmunization blood specimens tested, whereas primary isolate 91US056 was neutralsamples (pre-bleeds) from HIV-uninfected volunteer G14, identified ized by 4 of 5 specimens and 92HT593 and US4 were neutralized in figure 1 and table 1, was used as control. Experimental sera included by 3 of 5. In considering data derived from two or three experithose from vaccine recipients G2, G6, G9, and G12, identified in ments with each serum-virus combination, the 50% geometric figure 1 and table 1. mean neutralizing titers of the polyclonal specimens that contained neutralizing activity were 1:100 1:158 for BZ167, 1:12 ing levels reflected the same relative activity that was detected 1:185 for 91US056, 1:32 1:82 for 92HT593, and 1: for when the sera were run at only one dilution (G9 ú G12 ú G2 US4. These data suggest that while neutralizing antibodies to ú G6), and 50% neutralizing titers for the reactive sera were BZ167 may be more prevalent, the antibody titers of positive between Ç1:70 and 1:200 (0.5% 1.5% serum). Specimen G6, sera suggest that BZ167 is not substantially more sensitive to which was previously negative in the resting cell assay (figure neutralization than the other viruses tested.

8 JID 1997;175 (April) HIV-1 Vaccine Induced Neutralizing Antibody 771 Figure 4. Neutralization of primary isolates BZ167, 91US056, 92HT593, and US4 by serum from HIV-positive subject F ( ), from HIV-positive subject L ( ), and by serum FDA-2, obtained from NIH AIDS Research and Reference Reagent Program ( ). Neutralization by pooled human plasma ( ) and by pooled human sera ( ) from HIVinfected persons is also shown. All specimens were titrated at doubling dilutions; virus input was within range of TCID 50 /well. for neutralization by vaccinees sera relative to the virus strains used as immunogens. Based on findings of antibodies induced with laboratory strains, one might predict that functional anti- bodies for primary isolates would also be restricted in their reactivity and that, therefore, primary isolates to be tested should ideally include strains closely related to the immunizing strain. Consequently, the use of randomly selected primary isolates may have resulted in the testing of viruses that did not share any neutralizing epitopes with the immunizing strains [25, 26, 29, 44], and thus, even if neutralizing antibodies reac- tive with primary isolates related to the immunizing laboratory- adapted strain had been induced, their presence could have gone undetected. Identification of primary isolates that share cross-neutraliz- ing epitopes with laboratory-adapted strains is not a trivial exercise. The degree and type of relatedness between viruses within a clade is dependent on the type of analysis that is done. Both monoclonal and polyclonal antibodies that neutralize sev- eral laboratory-adapted and primary isolates of HIV have been described (figure 4) [27, 45 50]. There is no known immuno- chemical test that correlates with neutralization, and neutraliza- tion serotypes do not appear to correlate with cladal groupings based on env or gag sequence analyses [51, 52]. Recent data Discussion Infection with HIV-1 elicits a vigorous antibody response that includes the production of antibodies that neutralize the virus. The antibody response to immunization with various investigational HIV-1 vaccine constructs is, however, much more limited than is the response to natural infection. This may be due in part to the much lower and shorter period of immunologic stimulation, to altered routes and mechanisms of antigen presentation, and to the restricted nature of the antigenic stimulus. Thus, while antibody responses to various candidate AIDS vaccines composed of envelope proteins derived from laboratory-adapted strains can be quite vigorous [26], the neutralizing activity detected is restricted in its breadth, reacting best with homologous and closely related laboratory strains and least well or not at all with more distantly related laboratory strains [19 21, 26]. With repeated boosts of the same immunogen, the breadth of reactivity usually increases but remains narrow [19, 41 43]. Given this relatively restricted breadth of neutralizing activity for laboratory-adapted strains of HIV in recipients of various candidate HIV vaccines, it is striking that little attention has been paid to the relationship of the primary isolates being tested

9 772 Zolla-Pazner et al. JID 1997;175 (April) based on neutralization assays suggest that neutralization serotypes may exist and that the groupings may be independent of clades, but the structural and antigenic basis of these groupings is still undefined [52]. Whether the epitopes that induce these cross-neutralizing antibodies participate in the binding of HIV to its receptor or coreceptors is also not known at this time. The findings reported above, that many sera of recipients of several different candidate AIDS vaccines neutralize a clade B primary isolate that is closely related to SF-2 and MN in the sequence of its gp120 in the C2, V3 V5 region, and that there is a highly significant correlation between serum neutralizing activity for SF-2 and BZ167, as well as for MN and BZ167, suggest that relatedness in this region may be important for inducing cross-neutralizing antibodies for primary isolates. The fact that the V3 loops of BZ167, SF-2, and MN are considerably disparate [32, 53] raises the possibility that anti-v3 antibodies may not be responsible for the neutralization observed in these studies. Genetic and antigenic analyses of other primary isolates that can be neutralized by these vaccinees sera may reveal critical neutralizing epitopes inducing cross-neutralizing antibodies. The positive results reported above with BZ167 and the previously published negative results with several randomly chosen primary isolates [25, 26, 29] support the hypothesis that the neutralizing activity induced by current HIV vaccines may be narrowly directed to viruses that are closely related to the immunizing strains and that primary isolates related to HIV SF-2 and HIV MN, defined by several different criteria, should be tested for their sensitivity to neutralization by vaccinees sera. The level of detection of neutralizing activity in sera also depends on the assay conditions used and the methods of reporting the results [54, 55]. Thus, neutralizing activity present in serum can be efficiently measured or may remain undetected, depending on such variables as the cell in which the virus was grown (the host cell effect) [56], the target cell used in the neutralization assay (the target cell effect) [32], the amount of virus used per culture, the quantity and quality of antibody used, the length of exposure of virus to antibody, the length of exposure of target cells to virus and antibody, and whether 50% or 90% neutralizing measurements or infectivity reduction are used as end points. None of these variables has been held constant in published HIV neutralization studies (reviewed in [54]), and variation in methodology explains, in part, the dis- crepant data in the published literature concerning the preva- lence and levels of neutralizing antibodies for primary HIV-1 isolates in the sera of HIV-infected and -immunized persons. That some of these variables may be relevant to success or failure in detecting neutralizing activity in the sera of HIV vaccine recipients is demonstrated by the data reported in figure 3. Thus, for example, neutralizing antibodies for BZ167, which were readily measured in an assay using CEM-SS as target cells, were detected at lower levels in a similar assay in which unstimulated PBMC were used as target cells. When PHAstimulated PBMC were used as target cells, no neutralizing activity was detected in these same sera, although when a lower input of virus was used and antibody was allowed to remain in the culture for 72 h rather than h, neutralization of BZ167 could be detected even with PHA-stimulated PBMC target cells (Wrin T, Berman P, unpublished data). These findings extend those in the literature with many other viruses (influenza virus, vesicular stomatitis virus, echovirus, feline immunodeficiency virus) showing that the nature of the target cell used in neutralization assays profoundly affects the level of sensitivity of the assay [32, 57 62]. Clearly, the most sensitive assays will allow the most effective detection when antibody levels are low. Sensitive detection in vitro, however, is meaningless if it does not correlate with protection in vivo. It is noteworthy, therefore, that in recent studies of chimpanzees immunized with a prime-boost regimen utilizing an adenovirusgp160 MN construct and an rgp120 SF-2 boost, protection from a high-dose, intravenous challenge with cell-free HIV SF-2 was correlated with neutralizing antibodies to BZ167 detected with the resting cell assay [16]. Even in protected chimpanzees, no neutralizing antibodies were detected in the less sensitive assay using PHA-stimulated PBMC. In conclusion, these studies demonstrate that recipients of certain investigational AIDS vaccines are capable of mounting an immune response that produces antibodies that neutralize a clade B primary isolate. The breadth of this response has yet to be ascertained. However, even if the response to current investigational AIDS vaccine products proves to be narrow in its specificity, the demonstration that some candidate HIV vaccines can induce antibodies that neutralize primary isolate BZ167 suggests that these vaccines possess sequences and req- uisite tertiary or quaternary structures (or both) that mimic relevant epitopes on at least a limited number of primary iso- lates. In addition, the detection of primary isolate neutralizing antibodies in the sera of recipients of some candidate HIV vaccines provides a benchmark for measuring relative levels of functional antibodies against primary isolates as new vac- cines are developed. Acknowledgment We wish to dedicate this article to the memory of Kathelyn Steimer, who was distinguished as a person of uncommon grace, intelligence, and integrity. 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