JOURNAL OF VIROLOGY, Jan. 1992, p. 172-182 0022-538X/92/010172-11$02.00/0 Copyright © 1992, American Society for Microbiology

Vol. 66, No. 1

Native but Not Denatured Recombinant Human Immunodeficiency Virus Type 1 gpl20 Generates Broad-Spectrum Neutralizing Antibodies in Baboons NANCY L. HAIGWOOD,1* PETER L. NARA,2 ERIC BROOKS,' GARY A. VAN NEST,' GARY OTT,1 KEITH W. HIGGINS,' NANCY DUNLOP,2 CARL J. SCANDELLA,1 JORG W. EICHBERG,3 AND KATHELYN S. STEIMER' Chiron Corporation, 4560 Horton Street, Emeryville, California 94608-29161; Virus Biology Section, National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 217012; and Southwest Foundation for Biomedical Research, P.O. Box 28147, San Antonio, Texas 782843 Received 26 July 1991/Accepted 30 September 1991

The protection of individuals from human immunodeficiency virus type 1 (HIV-1) infection with an envelope subunit derived from a single isolate will require the presentation of conserved epitopes in gpl20. The objective of the studies presented here was to test whether a native recombinant gpl20 (rgpl20) immunogen would elicit responses to conserved neutralization epitopes that are not present in a denatured recombinant gpl20 antigen from the same virus isolate. In a large study of 51 baboons, we have generated heterologous neutralizing activity with native, glycosylated rgpl2OSF2 but not with denatured, nonglycosylated env 2-3sF2. After repeated exposure to rgpl20SF2 formulated with one of several adjuvants, virus isolates from the United States, the Caribbean, and Africa were neutralized. The timing of the immunization regimen and the choice of adjuvant affected the virus neutralization titers both quantitatively and qualitatively. These results suggest that vaccination with native, glycosylated rgpl20 from a single virus isolate, HIV-SF2, may elicit a protective immune response effective against geographically and sequentially distinct HIV-1 isolates.

Efforts to develop a subunit vaccine against AIDS have focused on the envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). The gpl60 precursor and the external domain gpl20 bind to the CD4 receptor found on human T cells (28), and both have been shown to elicit virus-neutralizing antibodies in experimental animals (26, 38, 40, 49). gpl20 recently has been shown to induce a protective immune response in vaccinated chimpanzees (10); this protection correlated with the presence of high-titer neutralizing antibodies. Neutralizing antibodies have also been shown to neutralize HIV-1 ex vivo and to prevent the subsequent infection of chimpanzees (13). However, the virus in these experiments was a laboratory isolate of HIV-1, HIV-HTLVIIIB. Generation of protective immunity against a laboratory isolate of HIV-1 may be a simpler task than the generation of immunity effective against uncharacterized field isolates because of the sequence heterogeneity of the envelope (2, 7, 12, 30). Neutralizing antibodies induced by gpl20 subunit vaccination are generally effective against only the specific isolate, or closely related isolates, from which the immunizing antigens originate (5, 33, 41). These neutralizing antibodies have been shown to be directed against linear epitopes found in the third hypervariable region (V3) (16, 22, 37) containing the principal neutralizing determinant (25). Neutralization escape mutants with invariant V3 sequences that are resistant to V3-specific monoclonal antibodies have defined this region as both a conformational and a sequential epitope (34). Antibodies directed against conformational epitopes in viral envelope glycoproteins can neutralize the infectivity of *

other enveloped viruses (6, 29). The antibody repertoire of HIV-infected humans contains both isolate-specific antibodies (15) and a subset that is broadly cross-reactive in virus neutralization (8, 23, 52). We have shown that a portion of the broadly reactive neutralizing antibodies are directed against conserved conformational epitopes of gpl20 (46, 47). The protection of individuals from HIV-1 infection with an envelope subunit derived from a single isolate may require the presentation of conserved conformational epitopes in gpl20. We have produced a native, glycosylated recombinant gpl20 (rgpl2O) with full CD4-binding capability from HIVSF2, a laboratory-adapted prevalent field isolate of HIV-1 (25). The glycoprotein is secreted from mammalian cells. It represents full-length gpl20 without additional amino acids and is not appreciably cleaved in the V3 region (43). In order to carry out comparative immunogenicity studies with this antigen in a variety of adjuvants, we chose to work with baboons. These animals are primates, which we considered important for the evaluation of adjuvants being developed for use in humans, and are relatively plentiful, allowing reasonable group sizes for these comparative studies. In the studies described here, we have generated broad-spectrum neutralizing activity in baboons with native, glycosylated rgpl2OSF2 that was not achieved with another recombinant gpl20 antigen, the denatured, nonglycosylated env 2-3SF2' which has no CD4-binding capacity. After repeated exposure to rgpl2OSF2, baboons developed neutralizing activity effective against sequentially homologous and heterologous virus isolates, including two of African origin. The timing of the immunization regimen and the choice of adjuvant have significant qualitative and quantitative effects on the neutralization titers generated.

Corresponding author. 172

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MATERIALS AND METHODS Recombinant antigens. env 2-3SF2 (amino acids Ile-26 to Ala-510) was expressed intracellularly in yeast cells, extracted, and purified to >90% homogeneity as previously described (19). The 55,000-Da protein is not glycosylated and is incapable of binding to CD4 (1). rgp120SF2 (amino acids Glu-31 to Arg-509) was expressed as a secreted glycoprotein in stably selected Chinese hamster ovary (CHO) cells (51). The 120,000-Da protein was purified by nonaffinity methods to >90% homogeneity (43). Briefly, conditioned medium from CHO cells was concentrated by ultrafiltration and chromatographed sequentially by ion-exchange DEAE A-50, phenyl hydrophobic interaction, ether hydrophobic interaction, and gel filtration. Apparent molecular weight, purity, and heterogeneity were assessed by gel filtration high-pressure liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie brilliant blue or silver staining. The extent of glycosylation was determined by PAGE before and after deglycosylation with peptide-N-glycosidase F. Amino acid composition was determined with the Waters PicoTag system. Amino-terminal sequences were derived by Edman degradation on the Applied Biosystems gas phase sequenator. CD4 binding was determined by radioimmunoprecipitation of 35S-labeled recombinant, soluble human CD4 mixed with various amounts of purified env 2-3SF2 or rgpl20SF2. Complexes were coprecipitated with human HIV-positive immunoglobulin and quantified by densitometric scans of autoradiographs of gels. Reactivity to human HIV-positive immunoglobulin was assessed by standard Western immunoblotting procedures. Adjuvants. Adjuvants which had been shown to have different immunostimulatory potential in guinea pigs and goats were tested. These included aluminum hydroxide (alum; Alhydrogel; Superfos, Vedbaek, Denmark), incomplete Freund's adjuvant (IFA), IFA mixed with MTP-PE (N-

acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-[l'-2'-dipalmitoyl-sn-glycero-3'-hydroxyphosphoryloxy]-ethylamine sodium salt; Ciba-Geigy Ltd., Basel, Switzerland) (IFA/ MTP-PE), and a series of emulsions with and without added MTP-PE or nor-MDP (N-acetyl-nor-muramyl-L-alanyl-Disoglutamine; Ciba-Geigy Ltd.). MF59 is an emulsion containing 5% squalene, 0.5% Tween 80, 0.5% Span 85, and 400 pug of MTP-PE per ml prepared in the Microfluidizer (model llOY; Microfluidics Corp., Newton, Mass. [36]). MF58 and MF59 are identical except that the MTP-PE is added before microfluidization in MF59 and after microfluidization in MF58. MF1 and MF1l1 are microfluidized emulsions that contain only squalene and MTP-PE. MF1 contains 4% squalene and 1,000 jig of MTP-PE per ml. MF101 contains 6.4% squalene and 1,600 ,ug of MTP-PE per ml. The Syntex adjuvant formulation (SAF) emulsions were made with the Microfluidizer according to published procedures (27) and were formulated with nor-MDP (SAF/MDP) or with MTP-PE (SAF/MTP). The MTP-PE/low oil formulation (L/O) consists of 4% squalene, 0.008% Tween 80, and MTP-PE (42). For the MF58, MF58, MF1, and SAF formulations, equal volumes of emulsion and antigen solution were mixed by hand just before injection. For MF11, emulsion and antigen solution were- mixed at a 1:8 ratio just prior to injection. For IFA, IFA/MTP-PE, and L/0, antigens were mixed and emulsified by repeated passage through a syringe and needle immediately before injection. Antigens were bound to alum in phosphate-buffered saline by Vortex mixing for 1 min.

173

Immunization protocols. In the first rgpl20SF2 and env 2-3SF2 baboon studies, animals (Papio sp., male and female, eight animals per group) were immunized intramuscularly with 25 ,ug of env 2-3SF2 or 55 ,ug (based on fully glycosylated molecular mass of 120 kDa) of rgpl20SF2 formulated with IFA/MTP-PE (three animals), MF1 (three animals), or alum (two animals). Baboons received immunizations on an interrupted schedule, which has been shown to enhance immune responses (3). In these first studies, injections were administered intramuscularly five times at monthly intervals (weeks 0, 4, 8, 12, and 20), followed by a 6-month rest period and reboosting four additional times (weeks 55, 59, 65, and 81). In the second rgpl20SF2 baboon study, 17 animals were immunized a total of five times at weeks 0, 4, 8, 22, and 36 with rgpl20SF2 mixed with IFA (four animals), SAF/MDP (four animals), SAF/MTP (four animals), and MF59 (five animals). In the second env 2-3 study, 18 baboons were immunized a total of six times at weeks 0, 4, 8, 14, 24, and 30 with env 2-3SF2 mixed with SAF/MDP (two animals), MF1l1 (three animals), MF58 (three animals), MF59 (three animals), L/O (three animals), IFA (two animals), and alum (two animals). Sera were collected 2 weeks following each immunization or monthly between immunizations. The timing of the immunizations is depicted in Fig. 1. Antigen ELISA. Purified env 2-3SF2 was coated onto microtiter plates, and titers in serum samples were determined as described previously (19). The enzyme-linked immunosorbent assay (ELISA) titer corresponds to the reciprocal of the dilution that resulted in an optical density (OD) of 50% of the maximal signal in the assay. Titers reported are the average of duplicate assays. A standard human serum or baboon env 2-3SF2-specific serum was included in all assays for normalization of assays performed on different days. ELISA titers obtained when purified rgpl20SSF2 was used to coat the plates were indistinguishable from ELISA titers obtained when env 2-3SF2 was used. env 2-3SF2 values are reported here. Virus neutralization assays and virus isolates. Two virus neutralization assays were employed, a p249ag inhibition assay (19, 48, 49) and a syncytium formation infectivity (SFI) assay (31). Neutralization titers reported for samples tested in the SFI assay correspond to the reciprocal of the dilution of serum that reduced the syncytium-forming units of virus by 90%. Titers reported for the inhibition assay are the reciprocal of the dilution of serum that reduced p249ag antigen production by 50%. Prebleed samples for each animal were assayed at least once for each virus isolate and were uniformly negative for virus neutralization activity. The assays used to generate the data are noted in the figure legends and table footnotes. All sera were heat-inactivated (56°C, 30 min) prior to assay. HIV-SF2 was provided by J. Levy; HIV-HTLVIIIB and HIV-MN were provided by R. Gallo and F. Wong-Staal; HIV-BRU was obtained from F. Barre-Sinoussi; HIV-ZR6 was from A. Srinivasan (45). HIV-NDK was obtained from J.-C. Chermann (44). Data are reported as geometric mean titers (plus or minus the standard error of the geometric mean) for the different adjuvant groups. RESULTS Both env 2-3SF2 and rgp120s11 generate high-titer envelopespecific responses that are adjuvant dependent. In an effort to compare the ability of two versions of recombinant HIV-SF2 gpl20 antigens to generate neutralizing antibodies in primates, a total of eight baboons were immunized with each

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recombinant polypeptide. For this comparison, the antigens were formulated with three adjuvants: IFAIMTP-PE, MF1, and alum. The env 2-3SF2 and rgpl2OSF2 were similar with respect to purity, heterogeneity, amino acid composition, N-terminal sequence, and reactivity to human HIV-positive immunoglobulin in Western blot assays. They differed in CD4 binding, apparent molecular weight, and extent of glycosylation. Baboons were immunized multiple times inrgpl20SF2 baboon study I

tramuscularly, and the sera were assayed following each immunization for antigen-specific reactivities by env 2-3SF2 ELISA. A schematic diagram of the immunization regimen is shown in Fig. 1 (shown as rgpl2OSF2 baboon study 1 and env 2-3SF2 baboon study 1). Envelope-specific antibody titers, as measured by ELISA against env 2-3SF2, rose following each boost (Fig. 2). The animals were rested following the fifth immunization to allow env 2-3

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NEUTRALIZING-ANTIBODY GENERATION BY HIV-1 gpl2O

optimal immune recall (3). Note that titers for the alum and MF1 mixtures differed from those for the IFA/MTP-PE mixtures by approximately 10-fold, demonstrating the adjuvant dependence of the response. Baseline titers were reached after 6 months of rest, and the animals were then reboosted at monthly intervals. No significant increases in env 2-3SF2-specific ELISA titers above previous peak responses were observed following this boost. By these criteria, both env 2-3SF2 and rgpl20SF2 are highly immunogenic and essentially indistinguishable. Native, glycosylated rgpl20SF2 induced broader-spectrum neutralizing antibody than denatured, nonglycosylated env 2-3SF2. To compare the neutralizing activity of antibodies to these two versions of recombinant gpl20, antisera collected at various points in the immunization regime were tested in in vitro neutralization assays against both the homologous virus HIV-SF2 and the heterologous virus isolates. Shown are the results with serum samples obtained 2 weeks after the third (week 10), fifth (week 23), and sixth (week 57) immunizations (Fig. 3). Because of differences in growth rates and syncytium formation of the various HIV-1 laboratory isolates, two assays were employed to obtain the most reproducible and quantitative data, optimal for each virus isolate. HIV-SF2 neutralization was performed primarily with the p249ae inhibition assay (19, 48, 49), while HIV-MN, HIV-BRU, and HIV-HTLVIIIB neutralization was performed by an SFI assay (31). Absolute titers of sera analyzed by the two different neutralization assays differ, but the data are qualitatively similar, as we have reported previously (18). No direct comparisons of titers derived in the two assays can be made because of differences in their ranges and values obtained with human HIV-positive sera. However, many samples were assayed by both procedures after five immunizations. For these points, titers versus HIV-MN and HIV-SF2 are closely matched (data not shown). Overall, both the homologous HIV-SF2 and the heterologous neutralizing titers were higher in the IFAIMTP-PE-immunized animals than in the MF1- or alum-immunized animals. All eight env 2-3SF2-immunized animals neutralized HIVSF2, three of eight neutralized HIV-MN at low levels, and only one baboon had neutralizing activity at one time point against HIV-HTLVIIIB or HIV-BRU. In contrast, all eight animals immunized with rgpl20SF2 had neutralizing activity against HIV-SF2 and HIV-MN, and after the sixth immunization, two of eight had activity against HIV-BRU (data not shown) and five of eight had activity against HIV-HTLVIIIB. env 2-3SF2-immunized animals had low-level titers against HIV-MN in the IFA/MTP-PE and MF1 adjuvant groups, while broadening of the response of the rgpl2OSF2immunized animals to include HIV-MN was seen in all three adjuvant groups and to include HIV-HTLVIIIB was seen in two of the three adjuvant groups (IFA/MTP-PE and alum). Mean neutralization titers for most animals in each group were sustained or increased with the sixth immunization following the rest; with env 2-3-immunized animals, the peak of HIV-SF2 neutralization was seen after three immunizations and decreased after one or two additional boosts. The geometric mean neutralization titers for HIV-SF2 and HIV-MN among the rgp120SF2-immunized baboons in the IFA/MTP-PE group are similar to the mean titers of neutralizing activity normally observed with sera from HIV-infected humans and chimpanzees by these two virus neutralization assays (14, 32, 39). Animals immunized with rgpl20SF2 in the other two adjuvant formulations had titers that were, on average, 3- to 10-fold lower than the titers observed in the IFA/MTP-PE group. Geometric mean titers

175

of the rgpl20SF2-immunized baboons (IFA/MTP-PE group) versus HIV-HTLVIIIB were lower than the HIV-MN titers but in the range of neutralization titers shown to be protective in chimpanzees (14). Longitudinal analysis shows delayed development of heterologous neutralization. Analysis of all serum samples from the two highest-responding baboons further delineated differences in env 2-3SF2- and rgpl20SF2-immunized animals (Fig. 4). Additional boosting with env 2-3SF2 in baboon 2958 did not raise antibody or neutralization titers beyond the levels measured at week 10, and there was no detectable neutralization of HIV-HTLVIIIB. In the rgpl20SF2-immunized animal (baboon 2964), HIV-SF2, HIV-MN, and HIVHTLVIIIB titers increased following each boost, with the greatest increase observed following the rest. Patterns of neutralizing activity were similar for all three viruses, although titer magnitudes differed, as seen in the different scales used to graph the results. HIV-HTLVIIIB neutralization emerged later than those for the other two isolates on this immunization regimen. Heterologous activity of the neutralization response in the highest-responding gpl2O-IFA/MTP-PE animal. Serum samples collected from the highest-responding rgpl20SF2-immunized baboon (no. 2964) were tested for the ability to neutralize additional virus isolates (Fig. 5). The V3 region sequences of the virus isolates tested are shown, with the central GPGR sequences common to most isolates in the table highlighted. The highest neutralization titers observed were against HIV-SF2 and HIV-MN (1,000 and 600, respectively). All sera were assayed by the SFI assay. This example of similar titers of neutralizing activity against HIV-SF2 and HIV-MN suggests that these two isolates may be closely related serologically. A series of isolates from the HIV-BRU family were tested, and titers against HIV-HTLVIIIB and HIV-BRU were approximately three- to sixfold lower than titers measured against HIV-SF2 or HIV-MN. HXB3, an isolate found in the HIV-HTLVIIIB stock, and V32, a neutralization escape mutant isolated from an HIVHTLVIIIB-infected chimpanzee (34), were also neutralized, but less effectively than the uncloned stocks. V32 contains an invariant V3 sequence, matching that of HIV-BRU, but has changes outside the V3 loop (30a) that render it resistant to neutralization by antisera from the chimpanzee from which it was isolated. This chimpanzee serum is able to effectively neutralize wild-type HIV-BRU. These alterations most likely account for its approximately fivefold-lower neutralization titer compared with wild-type HIV-BRU. Titers against HIV-RF and HIV-CC were lower still, in the range of those of the escape mutant. Isolates HIV-ZR6 and HIV-NDK from Africa were not detectably neutralized after six immunizations, but low-level antibody titers were measured following additional boosting (see below). Repeated boosting generates neutralizing activity effective against African isolates. Repeated immunization of the IFA/ MTP-PE group of rgpl20SF2-immunized baboons was carried out to determine whether additional repeated exposure to gpl20 might result in antibodies effective in neutralizing an even broader range of isolates. Titers of neutralizing antibodies against HIV-SF2, HIV-MN, HIV-RF, and HIV-CC were not dramatically altered. However, repeated immunization did result in the appearance of low-titer neutralizing antibodies against two African isolates, HIV-ZR6 (titer, 4) and HIV-NDK (titer, 5). Similarly, in infected humans, titers against African isolates are often one or two orders of magnitude lower than titers against U.S. or European isolates (23). The temporal development of HIV-ZR6

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neutralization was examined by graphing the virus neutralization data (Fig. 6). As is evident in the graph, repeated boosting shifted the slope of the neutralization curve, so that neutralization was detected in serum sample 32, following nine immunizations. These results suggest that repeated boosting has activated or selected antibody-producing clones of additional specificities and/or higher affinities, thus broadening the response. Altered kinetics of development of heterologous neutralizing antibodies observed in a second rgpl2OsF7 study. Based on the encouraging results obtained in the first baboon study, particularly in the IFA/MTP-PE group, we designed a second immunogenicity study to test a series of adjuvants

designed to perform better than alum which would have potential for testing and approval in human subjects. One set consisted of microfluidized emulsions developed at Chiron, containing the immunostimulator MTP-PE. The other set comprised SAF formulated with either MDP or MTP-PE. Since the resting period had an apparently positive effect on the generation of neutralizing antibodies, we also altered the immunization regimen in the new study to incorporate more than one rest period (Fig. 1). In this second study, 16 baboons were immunized with rgpl20sF2 formulated with four adjuvants: MF59; SAF/MTP; SAF/MDP; and IFA. For comparison, we also set up an additional 18-animal study with env 2-3sF2 in which animals were immunized with env

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