Neutralization of divergent HIV-1 isolates by conformation-dependent human antibodies to Gp120.

The spectrum of human immunodeficiency virus type 1 (HIV-1) isolates neutralized by antibodies from HIV-1-infected humans is broader than the spectrum of isolates neutralized by sera from animals immunized with purified gp120 subunits. This broader neutralization was due, in part, to the presence of antibodies to conserved gp120 conformational epitopes. Purified conformation-dependent gp120-specific human antibodies neutralized a wider range of virus isolates than human antibodies directed to linear determinants in gp120 and were also responsible for the majority of the gp120-specific CD4-blocking activity of HIV-1-infected human sera. A gp120 subunit vaccine that effectively presents these conformation-dependent neutralization epitopes should protect against a broader range of HIV-1 variants than a vaccine that presents exclusively linear determinants.

Distinction of human immunodeficiency virus type 1 neutralization and infection enhancement by human monoclonal antibodies to glycoprotein 120.

There is increasing evidence that sera from HIV-1-infected individuals contain antibodies that enhance infection by HIV-1 in vitro. Previous work has demonstrated that complement receptors on T lymphoid cells and Fc receptors for IgG (Fc gamma R) on monocytic cells are required for enhanced infection by antibody-complexed HIV-1. Characterization of such infection-enhancing antibodies is essential because immunogenic epitopes which induce enhancing antibodies should be excluded from HIV-1 vaccines. This study was conducted to identify enhancing antibodies involved in Fc R-mediated enhancement of HIV-1 infection employing IgG human monoclonal antibodies (HMAbs) reactive against gp120 of HIV-1, which were produced by B cell lines derived from an HIV-1-infected individual. A potent neutralizing HMAb N70-1.5e did not enhance infection by HIV-1 (IIIB and MN strains), whereas HMAb N70-2.3a mediated enhancement of HIV-1 infection, but had little neutralizing activity. A competition radio immunoassay demonstrated that the two antibodies bind to distinct epitopes. These results indicated that enhancing and neutralizing antibodies can be induced by different epitopes on gp120, suggesting the potential for development of safe vaccines against HIV-1 by exclusion of immunogenic epitopes for enhancing antibodies. We made attempts to identify the epitope on gp120 that is recognized by the enhancing antibody N70-2.3a by using recombinant HIV-1 proteins and found that the antibody binds to a conformational site of nonvariable sequences in the carboxyl half (aa 272-509) of gp120.

Resistance of chimpanzees immunized with recombinant gp120SF2 to challenge by HIV-1SF2.

OBJECTIVE: To determine whether vaccination with recombinant HIV-1SF2 gp120 in a novel oil-in-water adjuvant emulsion, MF59, protects chimpanzees against challenge with HIV-1SF2, the homologous virus isolate. METHODS: Two vaccinated chimpanzees and two control animals were challenged with 25-50 animal infectious doses of a stock of HIV-1SF2 that had been grown in mitogen-activated human peripheral blood mononuclear cells (PBMC). The animals were monitored by a series of serologic [enzyme-linked immunosorbent assay (ELISA), Western blot, and neutralization assays] and virologic [virus culture, RNA and DNA polymerase chain reaction (PCR)] assays for infection. RESULTS: Both control animals showed evidence of seroconversion in ELISA and Western blot assays. In addition, virus was detected in the early, acute phase of infection of both control animals by (1) plasma RNA PCR, (2) virus culture, and (3) PBMC DNA PCR assays. One vaccinated animal showed no serologic or virologic evidence of infection. The other vaccinated animal has not seroconverted, and there was no evidence of plasma viremia. However, virus was detected at early timepoints in this animal's PBMC, and transient lymphoproliferation to HIV-1 proteins not in the vaccine was observed. These observations suggest that the former animal was protected from challenge while the latter may have experienced a transient or curtailed infection. CONCLUSION: Two types of vaccine-induced protective immune responses were observed when chimpanzees immunized with rgp120SF2 were challenged with the homologous virus isolate: a response consistent with the 'sterilizing immune response' documented in the chimpanzee model in previous studies, as well as one that did not completely protect from infection, showing curtailment of the acute phase and a failure of the animal to seroconvert.

The recognition site of type II restriction enzyme BglI is interrupted.

The Type II restriction endonuclease BglI recognizes the interrupted DNA sequence 5'-G-C-C-N-N-N-N-N-G-G-C-. This sequence occurs at all locations in over 33 000 base pairs of DNA sequence where the enzyme was found to cut DNA and nowhere else. All six of the specified bases are essential parts of the site since all groups of five of the six bases occur in the DNA sequences tested and none of them are cut by BglI. The length of the block of intervening unspecified positions must be exactly five since all other sizes between zero and 15 occur in the DNA sequences searched and none are cut by BglI. The 5'-terminal nucleotides of BglI cleaved phage G4 replicative form DNA and plasmid pER18 DNA were compared with the DNA sequences near the BglI sites on these DNAs. These results indicated that BglI cuts within the intervening unspecified region and produces single-stranded 3' termini that are three bases long. The BglI recognition site and cleavage points can thus be represented as follows: (Formula: see text). This study of the BglI recognition site was facilitated by the use of inexpensive microcomputers. A system of programs was developed that allowed analysis of over 33 kb of DNA sequences stored on flexible magnetic disks or audio cassettes. While these programs were generally written in the higher level language BASIC, some assembly language subroutines were utilized to reduce execution time.

Passive immune globulin therapy in the SIV/macaque model: early intervention can alter disease profile.

One of the major questions in AIDS is the role that the host immune system and the virus play in the dynamics of infection and the development of AIDS in an infected individual. In order to test the role of antibody in controlling viral infection, high-dose SIV-immune globulin was passively transferred to infected macaques early in infection. Immune globulin purified from the plasma of an SIV-infected long-term non-progressor macaque (SIVIG) or a pool of normal immune globulin (normal Ig) was infused into SIVsmE660-infected macaques (170 mg/kg) at one and fourteen days post infection. Animals were monitored for SIV-specific antibodies, viremia, plasma antigenemia, and clinical course. All animals were infected by SIV. At 16 months post infection, five macaques in the combined control groups have been euthanized, one as a rapid progressor with debilitating disease at 20 weeks post infection. Four macaques from the comparison groups have signs of AIDS, accompanied by high and increasing levels of virus and p27 antigenemia. One of the ten control animals had a very low virus load in plasma and peripheral blood and lymph node mononuclear cells at all times tested and has remained disease-free. In the SIVIG treatment group, two macaques were euthanized at 18-20 weeks due to AIDS, rapid progressors to disease. Three macaques in the SIVIG group had an initial high level of virus in plasma, peripheral blood mononuclear cells (PBMC), and lymph node mononuclear cells (LNMC), which dropped to baseline at 6 weeks post infection and has remained very low or negative for 16 months, a disease profile which has not been observed in untreated animals in this model to date. These macaques have remained clinically healthy. The sixth treated animal is also healthy, with very low virus burden that is detectable only by nested set polymerase chain reaction (PCR). All SIVIG-treated macaques had no detectable p27 plasma antigenemia for the first 10 weeks of infection, demonstrating that the IgG effectively complexed with the virus. The immunological correlates in the treated animals include development of de novo virus-specific antibodies and/or cytotoxic T cell (CTL), both of which are hallmarks of long term non-progressors. The two SIVIG-treated macaques that progress to disease rapidly had no detectable de novo humoral immune responses, as is often seen in rapid HIV disease in humans. Envelope-specific and virus neutralizing antibodies alone were not sufficient to prevent disease progression, as the plasma of both non-progressors as well as progressors had high titers of envelope-specific and neutralizing antibodies against SIVsmE660. Poor clinical prognosis was associated with moderate to high and increasing virus loads in plasma, PBMC, and lymph nodes. Good clinical prognosis correlated with low or undetectable post acute viremia in the peripheral blood and lymph nodes. We hypothesize that SIVIG reduced the spread of virus by eliminating or reducing plasma virus through immune complexes during the first four to 8 weeks of infection and then maintaining this low level of viremia until the host immune response was capable of virus control. Reduction of virus burden early in infection by passive IgG can alter disease outcome in SIV infection of macaques. Modifications of this strategy may lead to effective early treatment of HIV-1 infection in humans.

Protection from pathogenic SIV challenge using multigenic DNA vaccines.

To assess DNA immunization as a strategy for protecting against HIV infection in humans, we utilized SIVmne infection of Macaca fascicularis as a vaccine challenge model with moderate pathogenic potential. We compared the efficacy of DNA immunization alone and in combination with subunit protein boosts. All of the structural and regulatory genes of SIVmne clone 8 were cloned into mammalian expression vectors under the control of the CMV IE-1 promoter. Eight M. fascicularis were immunized twice with 3 mg of plasmid DNA divided between two sites; intramuscular and intradermal. Four primed macaques received a further two DNA immunizations at weeks 16-36, while the second group of four were boosted with 250 microg recombinant gp160 plus 250 microg recombinant Gag-Pol particles formulated in MF-59 adjuvant. Half of the controls received four immunizations of vector DNA; half received two vector DNA and two adjuvant immunizations. As expected, humoral immune responses were stronger in the macaques receiving subunit boosts, but responses were sustained in both groups. Significant neutralizing antibody titers to SIVmne were detected in one of the subunit-boosted animals and in none of the DNA-only animals prior to challenge. T-cell proliferative responses to gp160 and to Gag were detected in all immunized animals after three immunizations, and these responses increased after four immunizations. Cytokine profiles in PHA-stimulated PBMC taken on the day of challenge showed trends toward Thl responses in 2/4 macaques in the DNA vaccinated group and in 1/4 of the DNA plus subunit vaccinated macaques; Th2 responses in 3/4 DNA plus subunit-immunized macaques; and Th0 responses in 4/4 controls. In bulk CTL culture, SIV specific lysis was low or undetectable, even after four immunizations. However, stable SIV Gag-Pol- and env-specific T-cell clones (CD3+ CD8+) were isolated after only two DNA immunizations, and Gag-Pol- and Nef-specific CTL lines were isolated on the day of challenge. All animals were challenged at week 38 with SIVmne uncloned stock by the intrarectal route. Based on antibody anamnestic responses (western, ELISA, and neutralizing antibodies) and virus detection methods (co-culture of PBMC and LNMC, nested set PCR- of DNA from PBMC and LNMC, and plasma QC-PCR), there were major differences between the groups in the challenge outcome. Surprisingly, sustained low virus loads were observed only in the DNA group, suggesting that four immunizations with DNA only elicited more effective immune responses than two DNA primes combined with two protein boosts. Multigenic DNA vaccines such as these, bearing all structural and regulatory genes, show significant promise and may be a safe alternative to live-attenuated vaccines.

Fine analysis of humoral antibody response to envelope glycoprotein of SIV in infected and vaccinated macaques.

To characterize the serological response to SIV envelope, induced by vaccination with different envelope immunogens or by SIV infection, plasma samples from 11 cynomolgus macaques infected with simian immunodeficiency virus (SIV) and from 16 macaques vaccinated with three different recombinant envelope proteins were analyzed by (1) ELISA, using a variety of antigens including overlapping peptides encompassing the entire sequence of the envelope protein of SIV, and (2) competition assays, using neutralizing monoclonal antibodies to SIV gp120. Seven regions of SIV envelope were predicted to be antigenic. Peptides representing four of these, in the second and third variable regions (V2 and V3) and the fourth constant (C4) region of gp120 and the Gnann region of gp41, were recognized by the majority of sera from infected and vaccinated animals. Additional antigenic regions were identified in the first and fourth variable domains (V1 and V4) and the carboxy terminus (C5) of gp120 and in three additional regions of gp41. Most infected and vaccinated animals made antibodies that competed with the binding of the three conformational MAbs. Among the vaccinated groups, antibodies induced by vaccination with precursor glycoproteins (gp140 or gp160) recognized several additional gp120 epitopes when compared with antibodies induced by external glycoprotein gp130. Sera from infected animals showed a more restricted gp120 response (17 of 46 peptides recognized) compared to animals vaccinated with precursor glycoproteins (31 peptides recognized). The converse was true for antibodies to gp41. Sera from animals vaccinated with recombinant gp140, produced in insect cells, were the only group that failed to compete with the binding of conformational MAbs. Finally, the development of antibodies to specific epitopes of gp120 and gp41 revealed differences between long-term survivors and nonsurvivors, implying that responses to specific epitopes may be important in conferring resistance to disease progression.

Early postinfection antiviral treatment reduces viral load and prevents CD4+ cell decline in HIV type 2-infected macaques.

Reports of significant reductions in plasma viral load by anti-HIV drugs have raised the possibility that antiviral therapy, if initiated sufficiently early, may result in sustained control of infection and prolonged clinical benefits. We evaluated the effects of intervention coincident with infection using an antiviral nucleoside, d4T, in Macaca nemestrina infected with a highly pathogenic isolate of HIV-2 (HIV-2[287]). Infection with this virus reproducibly results in high viremia and rapid CD4+ cell depletion, allowing a sensitive measurement of the treatment effect on viral load and clinical outcome. Compared to the control group, d4T-treated macaques showed significantly lower (2-3 log10) plasma- and cell-associated viral load. No CD4+ cell decline was observed in the treatment group while on therapy with d4T whereas CD4+ cells of control macaques declined from a preinfection mean of 32% of PBMCs to below 10%. Notably, when d4T treatment was withdrawn after 16 weeks, five of the six macaques continued to control viral load and have maintained normal CD4+ cell level for more than a year. These results demonstrate that early antiviral intervention, even of a limited duration, may constitute an important strategy against lentiviral-induced disease.

Reduced virus load in rhesus macaques immunized with recombinant gp160 and challenged with simian immunodeficiency virus.

As a safe alternative to inactivated and live-attenuated whole-virus SIV vaccines, we have evaluated the potential of SIVmac239 gp160 expressed by recombinant vaccinia virus (vSIVgp160) and baculovirus (bSIVgp160) to protectively immunize rhesus macaques against intravenous (i.v.) infection with pathogenic SIVmac isolates. Macaques were immunized with live vSIVgp160 and/or bSIVgp160 protein partially purified from insect cells. The challenge viruses, propagated in rhesus peripheral blood mononuclear cells, consisted of the molecular clone SIVmac239 and another genetically similar, uncloned isolate, SIVmac251. Although antibodies that bind gp130 were induced in all animals following immunization with SIVgp160, neutralizing antibodies were undetectable 1 week prior to virus challenge. These results differ from those for macaques vaccinated with inactivated, whole SIV. All animals became infected after i.v. inoculation with 1-10 AID50 of either challenge virus. For animals challenged with SIVmac251, but not those challenged with SIVmac239, the cell-free infectious virus load in plasma of vSIVgp160-primed, bSIVgp160-boosted macaques was significantly lower than in unimmunized controls at 2 weeks postchallenge. Virus virulence, immunization regimen, and challenge with homologous or heterologous virus are factors critical to the outcome of the study. Immunization with surface glycoprotein may not necessarily provide protective immunity against infection but may reduce virus load. The relationship between reduction in virus load by vaccination and delay in onset of disease remains to be determined.

Functional and immunological characterization of SIV envelope glycoprotein produced in genetically engineered mammalian cells.

Retroviral envelope glycoproteins interact with cell receptors and are targets for antiviral immune responses in infected hosts. Macaque simian immunodeficiency virus (SIVmac) is a T-lymphocytopathic lentivirus which causes an AIDS-like disease in rhesus macaques. The envelope gene of SIVmac encodes a precursor glycoprotein (gp160) which is cleaved into an external domain (gp130) and a transmembrane domain (gp32). To investigate the functional and immunological properties of the SIV external envelope glycoprotein, we have used genetically engineered mammalian cells to produce recombinant gp130 (rgp130). The rgp130 has the appropriate molecular weight, is glycosylated, and has native conformation as determined by binding to the cell receptor for SIV, the CD4 antigen. Rhesus macaques immunized with purified rgp130 formulated in muramyl dipeptide adjuvant generated high titers of antienvelope antibodies. Antibodies from these macaques were tested for in vitro virus neutralization; very low or undetectable levels of neutralization were observed. In contrast, neutralizing antibodies were readily detected in sera from goats immunized with rgp130. With respect to cell-mediated immunity, proliferative responses to rgp130 were demonstrated in peripheral blood monocyte cells (PBMC) from macaques immunized with the recombinant glycoprotein as well as in PBMC from SIV-infected animals. These results show that rgp130 is functional and immunogenic; the potential of rgp130 for protective immunization remains to be determined.

Importance of hypervariable regions of HIV-1 gp120 in the generation of virus neutralizing antibodies.

Variants of the envelope gene of the HIV-SF2 isolate of HIV-1 with deletions of one or more of the hypervariable domains of gp120 were produced in genetically engineered yeast as nonglycosylated denatured polypeptide analogs of gp120. Purified antigens were used to immunize experimental animals to determine whether the removal of hypervariable regions from this type of gp120 immunogen had any effect on (1) the ability of the antigen to elicit virus neutralizing antibodies; and (2) the isolate specificity of the neutralizing antibodies that were elicited. The results of these studies demonstrate that, in addition to the previously identified V3 domain, at least two other hypervariable regions in gp120 are capable of eliciting neutralizing antibodies in experimental animals. However, when all five of the hypervariable regions were deleted, the resulting antigen was no longer capable of eliciting neutralizing antibodies. Finally, the neutralizing antibodies elicited by all of these nonglycosylated antigens were effective against HIV-SF2, the isolate from which the antigens were derived, but were not able to neutralize two divergent isolates, HIV-BRU or HIV-Zr6.

Titration of a vaccine stock preparation of human immunodeficiency virus type 1SF2 in cultured lymphocytes and in chimpanzees.

A large stock preparation of the HIV-1SF2 isolate has been derived after serial passage in human peripheral blood mononuclear cells (PBMCs). This viral stock has a titer of 10(4.9) TCID50 in human PBMCs and 10(4.2) TCID50 in chimpanzee PBMCs. By inoculation into animals the 50% chimpanzee infectious dose titer was found to be about 10(2.3). Virus isolation from animals was achieved on most occasions within 1-4 weeks after inoculation and then became transient. Viral RNA and DNA PCR analyses confirmed the virus infection of the chimpanzees. Anti-HIV antibody levels in the inoculated animals ranged from 1:400 to 1:6400 as measured by ELISA. About 680 vials of this stock preparation, frozen at -190 degrees C, are available for future studies of vaccines and antiviral therapies.

Consistent patterns of change during the divergence of human immunodeficiency virus type 1 envelope from that of the inoculated virus in simian/human immunodeficiency virus-infected macaques.

We have analyzed changes to proviral Env gp120 sequences and the development of neutralizing antibodies (NAbs) during 1 year of simian/human immunodeficiency virus SHIV-89.6P infection in 11 Macaca nemestrina macaques. Seven macaques had significant env divergence from that of the inoculum, and macaques with greater divergence had higher titers of homologous NAbs. Substitutions in sequons encoding potential N-linked glycosylation sites (PNGs) were among the first to be established, although overall the total number of sequons did not increase significantly. The majority (19 of 23) of PNGs present in the inoculum were conserved in the sequences from all macaques. Statistically significant variations in PNGs occurred in multiple macaques within constrained regions we term "hot spots," resulting in the selection of sequences more similar to the B consensus. These included additions on V1, the N-terminal side of V4, and the outer region of C2. Complex mutational patterns resulted in convergent PNG shifts in V2 and V5. Charge changes in Env V1V2, resulting in a net acidic charge, and a proline addition in V5 occurred in several macaques. Molecular modeling of the 89.6P sequence showed that the conserved glycans lie on the silent face of Env and that many are proximal to disulfide bonds, while PNG additions and shifts are proximal to the CD4 binding site. Nonsynonymous-to-synonymous substitution ratios suggest that these changes result from selective pressure. This longitudinal and cross-sectional study of mutations in human immunodeficiency virus (HIV) env in the SHIV background provides evidence that there are more constraints on the configuration of the glycan shield than were previously appreciated.

Infection with a molecularly cloned SIVsm virus elicits high titer homologous neutralizing antibodies with heterologous neutralizing activity.

We have evaluated the homologous and heterologous neutralizing antibody response in a cohort of six Macaca nemestrina infected with the cloned virus SIVsm62d that showed different levels of envelope diversification. Two progressor macaques developed AIDS by 1.5 years post-inoculation and four non-progressors were asymptomatic for 3 years of follow-up. All macaques developed high titers of neutralizing antibodies against homologous SIVsm viruses and intermediate titers against SIVsmB670. Heterologous virus neutralization of SIVmac, SIVmne, and HIV-2 was detected at much lower levels in both progressor macaques; only one of four non-progressors had evidence for broader neutralizing antibody activity. We noted changes in potential N-linked glycosylation (PNG) sites in V1/V2, C2, and V4 that were common to multiple macaques. These results support a model for viral neutralization where heterologous neutralization is, in part, driven by a strong homologous response and may be coupled to changes in PNG sites in envelope.

Human immunodeficiency virus type 1 subtype B ancestral envelope protein is functional and elicits neutralizing antibodies in rabbits similar to those elicited by a circulating subtype B envelope.

Human immunodeficiency virus type 1 (HIV-1) is a difficult target for vaccine development, in part because of its ever-expanding genetic diversity and attendant capacity to escape immunologic recognition. Vaccine efficacy might be improved by maximizing immunogen antigenic similarity to viruses likely to be encountered by vaccinees. To this end, we designed a prototype HIV-1 envelope vaccine using a deduced ancestral state for the env gene. The ancestral state reconstruction method was shown to be >95% accurate by computer simulation and 99.8% accurate when estimating the known inoculum used in an experimental infection study in rhesus macaques. Furthermore, the deduced ancestor gene differed from the set of sequences used to derive the ancestor by an average of 12.3%, while these latter sequences were an average of 17.3% different from each other. A full-length ancestral subtype B HIV-1 env gene was constructed and shown to produce a glycoprotein of 160 kDa that bound and fused with cells expressing the HIV-1 coreceptor CCR5. This Env was also functional in a virus pseudotype assay. When either gp160- or gp140-expressing plasmids and recombinant gp120 were used to immunize rabbits in a DNA prime-protein boost regimen, the artificial gene induced immunoglobulin G antibodies capable of weakly neutralizing heterologous primary HIV-1 strains. The results were similar for rabbits immunized in parallel with a natural isolate, HIV-1 SF162. Further design efforts to better present conserved neutralization determinants are warranted.

Progress and challenges in therapies for AIDS in nonhuman primate models.

Efforts to develop animal models for human immunodeficiency virus type-1 (HIV-1) vaccine testing have focused on lentivirus infection of nonhuman primates. A long-term goal of this primate research is to utilize the models to understand the mechanisms of pathogenesis leading to AIDS. Because the time to disease is compressed relative to HIV infection in humans, therapeutic strategies and compounds can be tested in nonhuman primate models in a shorter time frame and under more controlled conditions than are possible in many clinical studies. Recent interventive studies in primates using antiviral drugs or passive immune globulin (IgG) have demonstrated that multiple log reductions in plasma virus can be achieved and sustained, with accompanying health benefits. Information gained about timing and dosage may be of utility in designing clinical studies. The development of reliable and predictable animal models for effective therapies and vaccines against AIDS remains a critical priority for primate research.

Importance of conformation on the neutralizing antibody response to HIV-1 gp120.

We have investigated the role of conformation of HIV-1 gp120 on its potential efficacy as a subunit vaccine. The questions that we set out to answer were: 1) Are there neutralizing antibodies directed to conformational epitopes in gp120? 2) If so, what is the spectrum of virus isolates neutralized by these antibodies? 3) Is a conformationally correct gp120 subunit more effective in the induction of neutralizing antibodies than a denatured subunit? 4) Does native gp120 subunit vaccination induce a broader neutralizing response than a gp120 antigen that cannot display conformational epitopes? To address these questions, we characterized the gp120-specific antibody response of HIV-1-infected humans and of experimental animals immunized with recombinant native and nonnative gp120 subunits. Two versions of recombinant gp120 produced from the HIV-SF2 isolate of HIV-1 were employed in these studies: 1) a nonglycosylated, denatured version produced in genetically engineered yeast, which we presume is capable of presenting only linear determinants, and 2) a fully glycosylated, native version, produced in genetically engineered mammalian cells, that is capable of displaying linear as well as conformational epitopes. Antibodies directed exclusively to conformational epitopes in gp120 were purified from pooled HIV antibody-positive human sera using these two versions of HIV-SF2 gp120. These antibodies exhibited neutralizing activity, and this activity was effective in the neutralization of a different, broader spectrum of HIV-1 isolates than that of antibodies to linear determinants in gp120 purified from the same serum pool. When these two versions of HIV-SF2 gp120 were used as subunit immunogens in baboons, clear differences in their abilities to elicit neutralizing antibodies were observed. The native version was more effective in the induction of neutralizing antibodies effective against HIV-SF2, the homologous virus isolate. The isolate specificity of the neutralizing response to these two versions of HIV-SF2 gp120 also differed. The nonglycosylated version induced neutralizing antibodies that were effective against only the isolate, or closely related isolates, from which the antigen was derived. In contrast, the native version induced a neutralizing response that was effective against a broad panel of HIV-1 isolates, including at least one isolate that one would not expect to be neutralized by antibodies to the PND of HIV-SF2 gp120.