Induction of an anti-hepatitis B surface antigen response in mice by noninternal image (Ab2 alpha) anti-idiotypic antibodies

Anti-idiotype antibodies to a mouse monoclonal antibody A-12 directed against HBsAg were produced in rabbits. The anti-Id consisted of an Ab-2 alpha preparation that did not display any detectable internal image activity. Immunization of BALB/c mice with the anti-Id (Ab-2 alpha) coupled to KLH induced an anti-HBs response without subsequent HBsAg exposure. No anti-HBs was detected in control groups of mice immunized with other rabbit anti-Id-KLH preparations. The anti-HBs containing sera from mice immunized with the Ab-2 alpha were able to inhibit the Id-anti-Id reaction, indicating that an Id-positive, anti-HBs response was induced. This idiotype is not normally expressed during the murine immune response to HBsAg and suggests that noninternal image anti-Id activates silent clones. This study, along with our previous results obtained with the use of internal image anti-Id, suggests that there is more than one Id network operational during the BALB/c murine immune response to HBsAg.     

Two major groups of neutralizing anti-gp120 antibodies exist in HIV-infected individuals. Evidence for epitope diversity around the CD4 attachment site.

The aim of this study was to dissect neutralizing anti-gp120 antibody populations in seropositive asymptomatic individuals. Murine anti-Id mAb were raised against polyclonal affinity-purified human anti-gp120 antibodies. These anti-Id mAb were used to fractionate anti-gp120 antibodies from a pool of HIV-positive sera into idiotypically distinct anti-gp120 antibody (Id+Ab) preparations. Immunochemical and neutralization studies indicated that all Id+Ab that neutralized HIV-1 in vitro interacted with either the V3 loop or the CD4 attachment site of gp120. The V3-specific Id+Ab neutralized HIV-1 in a strain-restricted manner. Id+Ab specific for the CD4 attachment site exhibited different spectra of neutralizing activities against multiple strains of HIV-1. This finding indicates that multiple, antigenically diverse epitopes reside around the CD4 attachment site of gp120. Significantly, depletion of the Id+Ab from affinity-purified total anti-gp120 antibodies abrogated most of the neutralizing activities of these antibodies, suggesting that neutralizing anti-gp120 antibodies consist of two major specificities, either to the V3 region or to the CD4 attachment site. The understanding of specificities and neutralizing activities of different anti-gp120 antibodies in seropositive healthy individuals will be helpful for designing effective vaccines and immunotherapeutic strategies for AIDS.     

A "network antigen" for human CD4. A murine monoclonal anti-idiotype to Leu-3a induces an anti-CD4 response in naive mice.

Previous studies have evaluated anti-CD4 mAb as idiotypic models of the HIV gp120-binding site for CD4. The success of this strategy depends upon the concept of internal image, whereby the binding paratope of the anti-CD4 structurally mimics the equivalent binding surface on HIV gp120. To test this concept of internal image, anti-idiotypic antibodies were raised against the anti-CD4, Leu-3a. If any of these anti-Id detect the paratopic idiotope on the anti-CD4 antibody, their own respective paratopes should structurally model the corresponding binding epitope on CD4 bound by Leu-3a. Consequently, the immunization of naive mice with the selected anti-Id should induce an anti-CD4 response which reflects the binding specificities of Leu-3a. Four anti-Id to Leu-3a were characterized and tested for their ability to induce anti-CD4 responses in naive animals. Although one anti-Id induced an anti-CD4 response in mice, no such response could be detected in other species. Thus the failure to raise anti-Id with internal image characteristics may provide an explanation for the lack of anti-gp120 activity reported in anti-Id antisera raised to multiple anti-CD4 antibodies.     

Monoclonal anti-idiotypic antibodies induce humoral immune responses specific for simian virus 40 large tumor antigen in mice.

Mouse monoclonal anti-Id antibodies were generated against a mouse mAb (Ab-1) preparation specific for SV40 large tumor Ag (T-Ag). Four monoclonal anti-Id preparations each inhibited the binding of the monoclonal anti-SV40 T-Ag Ab-1 preparation to SV40 T-Ag. These anti-Id preparations appeared to recognize similar idiotopes on the monoclonal anti-SV40 T-Ag Ab-1 based on competitive cross-inhibition studies. One of these anti-Id preparations, designated 57B, was examined further for its in vivo modulatory capacity in mice. This anti-Id induced an Ab-3 response in BALB/c mice that recognized SV40 T-Ag (Ag+) and expressed an Id that was shared by the monoclonal anti-SV40 T-Ag Ab-1 preparation (Id+). The Id expressed on the Ab-3 differed from the Id induced in BALB/c mice immunized with the nominal SV40 T-Ag. Furthermore, characterization of the humoral immune response induced by anti-Id immunization indicated that the Ab-3 also recognized different epitopes on SV40 T-Ag when compared to the anti-SV40 T-Ag Ab-1 preparation used to generate the anti-Id. These studies indicate that monoclonal anti-Id can be used to induce humoral immune responses to a viral encoded tumor-associated Ag in vivo with 1) and Id specificity that differs from that expressed on antibodies produced by immunization with the nominal Ag and 2) an epitope specificity distinct from the Ab-1 preparation used for the production of the anti-Id.     

Anti-idiotypic antibody response to monoclonal anti-CD4 preparations in nonhuman primate species.

A series of mouse monoclonal anti-CD4 preparations was characterized for the ability to recognize overlapping epitopes on CD4 and to inhibit HIV/simian immunodeficiency virus (SIV) syncytium formation. Based on this characterization, mAb able to recognize CD4 epitopes overlapping the HIV binding site were selected and used to immunize nonhuman primates to elicit the production of specific anti-Id antibodies. Five baboons and five rhesus monkeys were immunized with either individual or a cocktail consisting of several monoclonal anti-CD4 preparations. All the nonhuman primates produced specific anti-Id that recognized either private or cross-reactive Id depending on the monoclonal anti-CD4 used to generate the anti-Id response. Inhibition assays were performed to ascertain the ability of: 1) soluble CD4 to inhibit the Id-anti-Id reaction and 2) the various anti-Id to inhibit the CD4-monoclonal anti-CD4 reaction. These studies demonstrated that some of the anti-Id recognized a cross-reactive Id that was associated with the Ag-combining site. In addition, some of the anti-Id weakly recognized SIV gp120 by Western blot analysis. These studies may be useful in designing experiments that may lead to a better understanding of the CD4-HIV gp120 interaction and to the production of Id and/or anti-Id reagents that might be used to manipulate this virus-receptor interaction.     

Memory B cell antibodies to HIV-1 gp140 cloned from individuals infected with clade A and B viruses

Understanding the antibody response to HIV-1 in humans that show broad neutralizing serologic activity is a crucial step in trying to reproduce such responses by vaccination. Investigating antibodies with cross clade reactivity is particularly important as these antibodies may target conserved epitopes on the HIV envelope gp160 protein. To this end we have used a clade B YU-2 gp140 trimeric antigen and single-cell antibody cloning methods to obtain 189 new anti-gp140 antibodies representing 51 independent B cell clones from the IgG memory B cells of 3 patients infected with HIV-1 clade A or B viruses and exhibiting broad neutralizing serologic activity. Our results support previous findings showing a diverse antibody response to HIV gp140 envelope protein, characterized by differentially expanded B-cell clones producing highly hypermutated antibodies with heterogenous gp140-specificity and neutralizing activity. In addition to their high-affinity binding to the HIV spike, the vast majority of the new anti-gp140 antibodies are also polyreactive. Although none of the new antibodies are as broad or potent as VRC01 or PG9, two clonally-related antibodies isolated from a clade A HIV-1 infected donor, directed against the gp120 variable loop 3, rank in the top 5% of the neutralizers identified in our large collection of 185 unique gp140-specific antibodies in terms of breadth and potency.     

Analysis of the cross-reactive anti-gp120 antibody population in human immunodeficiency virus-infected asymptomatic individuals.

This study was undertaken to analyze the specificity and neutralizing properties of cross-reactive anti-gp120 antibodies (Abs) in the sera of two human immunodeficiency virus (HIV)-infected asymptomatic individuals. Two panels of murine monoclonal anti-idiotype Abs (anti-id MAbs) were established against cross-reactive polyclonal anti-gp120 Abs purified from HIV+ sera by sequential affinity chromatography using gp120SF2- and gp120IIIB-Sepharose columns. These panels of anti-id MAbs were then used to affinity purify idiotype-positive (Id+) anti-gp120 Abs from HIV+ sera. The recovery of each of these Id+ Abs by purification indicated that several idiotypically distinct cross-reactive anti-gp120 Abs are present in sera over a wide range of concentrations. Immunological and biological studies showed that although all of the Id+ Abs were reactive against gp120SF2 and gp120IIIB, they exhibited unique epitope specificities and distinct neutralizing activities. Most of the Id+ Abs were directed against epitopes in the CD4 attachment site (CD4 site epitopes) of gp120 and exhibited a spectrum of broadly neutralizing activities. On the other hand, a minor population of Id+ Abs showed specificity for the V3 region of gp120 and exhibited limited cross-neutralizing activities. Together, these studies indicate that the CD4 site epitope-specific Abs are heterogeneous with respect to their clonality, neutralizing activity, and concentration in sera. This heterogeneity suggests that anti-gp120 Abs to the CD4 attachment site are developed in response to multiple overlapping epitopes present on the original virus isolate and/or epitopes on mutated variants which emerged over time.     

Neutralization of HIV-1 by anti-idiotypes to monoclonal anti-CD4. Potential for idiotype immunization against HIV.

Anti-idiotypic antibodies were raised in rabbits against a panel of 11 murine mAb directed to the human CD4 receptor. Selection of mAb for vaccination was based on inhibition studies demonstrating that these mAb recognized CD4/V1 epitopes implicated in HIV-1-gp120 binding. Purified antisera showed high titer anti-Id activity and reacted specifically with Ag-combining site-related Id of the mAb used for their generation. Anti-Id either detected a private Id of the immunizing mAb or displayed a partial cross-reactivity with Id of other mAb to CD4. Eight anti-Id to six different mAb were shown to recognize determinants of recombinant HIV-1-gp120 or of HIV-1-gp160 as shown by ELISA and radioimmunoprecipitation assay. These anti-Id were capable of inhibiting HIV infection up to 100% in a MT-4 cell assay in vitro. In addition to neutralizing infectivity of cell-free virus, anti-Id to two mAb--the mAb IOT4a and 7.3F11--were also shown to inhibit HIV-induced syncytia formation up to 100%. Anti-Id to the mAb IOT4a, 7.3F11, and to the mAb anti-Leu3a interfered with rgp120 binding to cellular CD4 as assessed by flow cytometry. These results demonstrated that mAb specific for both CDR2- and CDR3-like regions of CD4 were capable of inducing HIV-1-gp120 cross-reacting anti-Id neutralizing HIV-1 in vitro. These studies may have implications for the development of a gp120 internal image based vaccine against HIV.     

Idiotype network components are involved in the murine immune response to simian virus 40 large tumor antigen

Summary Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (SV40 T-Ag), a monoclonal antibody specific for SV40 T-Ag (Ab-1 preparation), and a monoclonal anti-idiotypic antibody (anti-Id), designated 58D, were used to analyze the humoral immune response of Balb/c mice either immunized with recombinant SV40 T-Ag or challenged with SV40-transformed cells. Inhibition assays indicated that antibodies from mice immunized with SV40 T-Ag and from those bearing SV40 tumor inhibited the SV40 T-Ag/Ab-1 reaction. These data suggested that the antibody response in immunized or tumorchallenged mice recognized similar epitope(s) on SV40 T-Ag to that detected by the monoclonal Ab-1. These anti-(SV40 T-Ag) response antibodies also inhibited the Ab-1/anti-Id reaction and recognized the anti-Id in direct binding assays. Together, these data indicate that murine anti-(SV40 T-Ag) responses shared an idiotope with a monoclonal anti-(SV40 T-Ag) Ab-1 preparation. This idiotope, which is recognized by the monoclonal anti-Id preparation, 58D, appears to be involved in the humoral immune response to SV40 T-Ag in both SV40-T-Ag-immunized and tumor-bearing mice. The monoclonal anti-Id preparation may represent a focal point for manipulating the humoral immune response to tumors induced by SV40-transformed cells.     

Generation and specificity of monoclonal anti-idiotypic antibodies against human HIV-specific antibodies. I. Cross-reacting idiotopes are expressed in subpopulations of HIV-infected individuals

In this study we have generated monoclonal anti-idiotypic antibodies against human monoclonal and polyclonal anti-HIV antibodies in seropositive sera. A human anti-gp41 mAb (H2, IgM kappa) was used to immunize BALB/c mice and to prepare hybridoma anti-antibodies that react with H2 and not with normal human IgM. Similar monoclonal anti-antibodies were made in BALB/c mice immunized with Ig fraction prepared from a pool of HIV-seropositive sera. Both kinds of anti-idiotypic antibodies reacted with antibodies in pools of seropositive sera and with individual seropositive sera but not with normal human Ig or seronegative sera. The Id-positive Ig from single donors were isolated on two different anti-Id immunoabsorbents and shown to bind to p24 and gp120, respectively. The detection and isolation of idiotypically cross-reactive human anti-HIV antibodies from seropositive donors demonstrated, for the first time, the existence of shared Id expressed by antibodies against HIV Ag. The utility of cross-reacting anti-idiotypic antibodies as tools to dissect the network regulation of the anti-viral immunity in AIDS is discussed.     

Comparison of the murine humoral immune response to recombinant simian virus 40 large tumor antigen: Epitope specificity and idiotype expression

Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (SV40 T-Ag) was used to immunize inbred strains of mice to compare the humoral immune responses. Specifically we examined the epitope specificities and idiotype (Id) expression on anti-(SV40 T-Ag) responses induced in BALB/c and C57BL/6 inbred strains of mice. The predominant SV40 T-Ag epitopes recognized by the anti-(SV40 T-Ag) responses appeared to differ between these two inbred strains, this being based on the ability of sera to inhibit the binding of several murine monoclonal antibodies specific for SV40 T-Ag. In addition, anti-(SV40 T-Ag) responses produced in C57BL/6 mice failed to express a previously described cross-reactive Id expressed in the anti-(SV40 T-Ag) response in BALB/c mice. This cross-reactive Id is detected by a mouse monoclonal anti-Id, designated 58D, which has been shown to represent a potential focal point for manipulating the humoral immune response to SV40-induced tumors in BALB/c mice. Together, these data indicate that the functional duality of the humoral immune response, as assessed by epitope recognition and Id expression, differs between these two inbred strains of mice when immunized with a recombinant SV40 T-Ag.     

In vitro efficacy of anti-HIV immunotoxins targeted by various antibodies to the envelope protein.

Six different anti-HIV envelope antibodies and one irrelevant control antibody were coupled to ricin A chain and tested for their efficacy in inhibiting HIV tissue culture infections. The anti-HIV antibodies consisted of five monoclonals, three of murine and two of human origin, and one polyclonal preparation prepared by affinity purifying pooled serum antibodies from HIV-infected humans on rgp160. The binding specificity of the antibodies was defined by ELISA by using recombinant envelope proteins and synthetic peptides, and by flow cytometry on HIV-infected cells. The in vitro efficacy of the antibodies was tested by the abilities of the immunotoxins to inhibit protein synthesis in persistently infected cell lines and by their abilities to inhibit HIV production during both acute and persistent infection as measured with an HIV-specific focal immunoassay. The immunotoxins were tested against a panel of distinctly different HIV isolates. The results indicate the following: 1) A mAb to the immunodominant neutralizing loop was highly effective against homologous strains of HIV, but had no activity against heterologous HIV. 2) The efficacy of anti-gp41 mAb varied depending upon the epitope recognized and possibly the affinity of binding to gp41. 3) The polyclonal human anti-gp160 antibodies produced the immunotoxin with the broadest specificity for different HIV strains and the greatest specific activity. This is related to the polyclonal nature of the preparation rather than an increase in relative avidity of the antibody. 4) Activity of an immunotoxin is not a direct function of the binding of the antibody to the surface of infected cells. 5) The ability of an immunotoxin to halt the spread of infection through a tissue culture cell population is dependent upon the ability of the antibody to neutralize the virus as well as the activity of the toxin. Our data suggest that efficacious immunotoxins for the treatment of AIDS may be made with polyclonal anti-envelope antibodies derived from the serum of patients who have been infected with HIV or with appropriately chosen anti-gp41 antibodies.     

Human immune response to group A streptococcal carbohydrate (A-CHO). II. Antigen-independent stimulation of IgM anti-A-CHO production in purified B cells by a monoclonal anti-idiotopic antibody

The paper describes the induction by a monoclonal anti-idiotopic antibody (anti-Id mAb) of specific antibody production to group A streptococcal carbohydrate (A-CHO) in purified human B cells of several unrelated individuals. The anti-Id mAb, designated 16F498 (anti-Id498), recognizes a recurrent idiotope (Id 498) associated with the combining site of human antibodies to N-acetyl-D-glucosamine (GlcNAc), the immunodominant group of A-CHO. Id498 is expressed on IgM anti-GlcNAc antibodies but does not occur on IgG antibodies with the same specificity. It occurs also on a minor population of IgM antibodies without specificity for A-CHO. Id498 was found in 19 of 27 sera from unselected healthy donors and thus seems to be frequently expressed within the adult B cell repertoire. The in vitro induction of anti-A-CHO antibodies was analyzed in human B cells extensively depleted for T cells. Specific antibody secretion required cross-linked anti-Id which was achieved by coupling the mAb to agarose beads. No antibody secretion could be induced by soluble anti-Id (1 and 10 micrograms/ml). An optimal response required soluble T cell-derived factors which were added as a mixture of recombinant interleukin 2 with a T cell hybridoma supernatant that augments B cell growth and differentiation. Under these conditions an antigen-independent specific increase of IgM anti-A-CHO production (2.6- to 10-fold, or up to 2000 ng/ml respectively) could be induced in blood B cell populations of four of six normal individuals expressing the Id498 at serum level.     

Antiidiotypic vaccination against murine coronavirus infection.

Rabbit polyclonal antiidiotypic antibodies were generated against a neutralizing mAb specific for a conformational epitope on the S glycoprotein of murine hepatitis virus, strain A59 (MHV-A59). These anti-Id were directed predominantly against an Id that was undetectable in rabbit and rat anti-MHV-A59 sera and weakly represented in syngeneic and allogeneic antiviral sera. However, some partial idiotypic sharing was observed between the Id-bearing antibody and a mAb with a similar antigenic site specificity. The anti-Id inhibited the virus-binding and neutralizing activities of the immunizing antibody, demonstrating that they recognize paratope-associated idiotopes. Mice immunized with affinity-purified anti-Id developed MHV-A59-specific antibodies that neutralized viral infectivity to high titers. Moreover, these animals survived an otherwise lethal challenge with viral murine hepatitis virus, unlike control mice immunized with normal rabbit Ig. These results indicate that at least a subpopulation of the polyclonal anti-Id could induce a protective immune response directed toward a biologically important MHV-A59 epitope, and demonstrate the feasibility of antiidiotypic vaccination against a coronavirus infection.     

Anti-immunoglobulin antibodies. II. Expression of individual and cross-reactive idiotypes on syngeneic and homologous anti-idiotype antibodies

Syngeneic anti-(anti-Id) antibodies were prepared against BALB/c anti-A48Id antibodies, BALB/c anti-460Id monoclonal antibodies, and A/J anti-J558 IdI monoclonal antibodies. With these anti-(anti-Id) antibodies we identified cross-reactive idiotypes on syngeneic and homologous anti-A48Id and anti-460Id antibodies. By contrast, tbe idiotypic determinants of A/J anti-J558 IdI monoclonal antibodies were not shared by other syngeneic, homologous, or xenogenic anti-J558 IdI or IdX antibodies. These results suggest that idiotype-antiidiotype reactions that serve as regulatory controls within the immune system are characteristic for each particular antigen system, strain, or species and that such interactions make the system self-limited with respect to the whole antild repertoire.     

Synthetic peptides define the fine specificity of the human immunodeficiency virus (HIV) gp160 humoral immune response in HIV type 1-infected chimpanzees

The fine specificities of antibodies produced against human immunodeficiency virus type 1 (HIV-1) gp160 were examined in sera from 23 HIV-1-infected chimpanzees. These animals had been infected with one of six isolates of HIV-1. Sera were screened by enzyme-linked immunosorbent assay for reactivity against seven synthetic peptides corresponding to regions of gp160. Chimpanzees appear to remain healthy after infection with HIV-1, suggesting that these animals may prevent extensive spread of the virus in vivo through immunologic mechanisms. Antibody specificity to gp160 epitopes may play a key role in the defense against HIV-1-related disease. Approximately one-half of all chimpanzee sera contained antibodies reactive with peptide 846-860, which corresponds to the carboxyl terminus of gp41. Less than 10% of sera from HIV-1-infected humans that were examined contained antibodies reactive with peptide 846-860, suggesting that this region is not highly immunogenic in humans. Of the human sera containing antibodies reactive with this peptide, all were from individuals classified as Walter Reed stages 1 to 3. No sera from humans with advanced stages of the disease contained antibodies reactive with peptide 846-860. Peptide 600-611, which reportedly reacts with nearly all sera from HIV-infected humans, was reactive with less than one-half of sera from HIV-1-infected chimpanzees. The observed differences in antibody reactivity to gp160 peptides in sera from HIV-1-infected chimpanzees and humans suggest that each may generate antibodies against differing sets of HIV-1 epitopes. These differences may contribute to the lack of disease progression in chimpanzees after infection with HIV-1.     

应用ELISPOT方法筛选确定HIV-1B'/C亚型疫苗六种抗原的H-2d限制的T细胞表位

为了筛选和确定用于检测表达HIV-1 B’/C亚型病毒6种抗原(gp160、gag、polr、evt、at和nef)的艾滋病疫苗免疫小鼠后H-2d限制的特异性T细胞表位,本研究使用表达上述6种抗原的复制型DNA疫苗和非复制型重组痘苗病毒疫苗联合免疫BALB/c小鼠,通过矩阵设计将HIV-1 B(C)亚型6种相应抗原全序列肽库分别混合成肽池,使用肽池对免疫小鼠进行IFN-γELISPOT检测,根据检测结果确定肽库中特异反应的优势表位肽。结果显示:筛选到七条针对Gag的特异表位肽,其中有5条与文献报道相同,另2条为新表位肽;筛选到3条针对Pol蛋白特异表位肽,其中一条为新表位肽;筛选到2条针对gp160特异表位肽,其中一条为新表位肽;在Nef肽库中筛选到一条新的表位肽;从Tat肽库中筛选到3条表位肽,这三条肽在肽库中是连续的序列,都包含(或部分包含)网上公布的表位序列;在Rev肽库中没有筛选到能够产生阳性反应的特异性表位肽。本研究使用IFN-γELISPOT方法筛选和确定了可用于检测表达HIV-1 B’/C亚型病毒6种抗原(gp160、gag、pol、revt、at和nef)的艾滋病疫苗免疫小鼠后H-2d限制的特异性T细胞表位。     

Suppression of murine lupus nephritis by administration of an anti-idiotypic antibody to anti-DNA

The suppression of pathogenic antibodies to DNA in NZB/NZW f1 female mice was achieved by repeated inoculation of the mice with a monoclonal anti-idiotypic antibody (anti-Id). The anti-Id, an IgG1, kappa, was directed against a major cross-reactive idiotype (Id) on NZB/NZW IgG antibodies to DNA. One hundred micrograms of the anti-Id were inoculated i.p. every 2 wk, beginning at 6 wk of age (nondiseased mice--no circulating anti-DNA or proteinuria) or 20 wk of age (diseased mice--all with circulating anti-DNA, one-third with proteinuria). As controls, littermates received an IgG, kappa non-DNA-binding myeloma or no treatment. In the young mice, nephritis and anti-DNA antibodies appeared at the same time in all groups, and their circulating antibodies to DNA did not bear the target Id. In the older (20-wk-old) mice, survival was significantly prolonged because of delay in the onset of nephritis; the total quantities of antibodies to DNA were diminished, and the target Id, initially present on circulating IgG, was deleted. These benefits were transient; the suppression of antibodies was followed by the appearance of large quantities of anti-DNA that did not bear the major Id. Therefore, although administration of anti-Id was effective in reducing an undesirable antibody response after the target Id was present on circulating antibodies, the benefits were limited, probably by Id "switch" or by increased synthesis of pathogenic antibodies bearing a minor Id.     

Induction of neutralizing antibodies against human immunodeficiency virus type 1 primary isolates by Gag-Env pseudovirion immunization

A major challenge for the development of an effective HIV vaccine is to elicit neutralizing antibodies against a broad array of primary isolates. Monomeric gp120-based vaccine approaches have not been successful in inducing this type of response, prompting a number of approaches designed to recreate the native glycoprotein complex that exists on the viral membrane. Gag-Env pseudovirions are noninfectious viruslike particles that recreate the native envelope glycoprotein structure and have the potential to generate neutralizing antibody responses against primary isolates. In this study, an inducible cell line was created in order to generate Gag-Env pseudovirions for examination of neutralizing antibody responses in guinea pigs. Unadjuvanted pseudovirions generated relatively weak anti-gp120 responses, while the use of a block copolymer water-in-oil emulsion or aluminum hydroxide combined with CpG oligodeoxynucleotides resulted in high levels of antibodies that bind to gp120. Sera from immunized animals neutralized a panel of human immunodeficiency virus (HIV) type 1 primary isolate viruses at titers that were significantly higher than that of the corresponding monomeric gp120 protein. Interpretation of these results was complicated by the occurrence of neutralizing antibodies directed against cellular (non-envelope protein) components of the pseudovirion. However, a major component of the pseudovirion-elicited antibody response was directed specifically against the HIV envelope. These results provide support for the role of pseudovirion-based vaccines in generating neutralizing antibodies against primary isolates of HIV and highlight the potential confounding role of antibodies directed at non-envelope cell surface components.     

Comparison of recombinant human immunodeficiency virus gag precursor and gag/env fusion proteins and a synthetic env peptide as diagnostic reagents

Diagnostic reagents for detection of human immunodeficiency virus (HIV) exposure with improved reliability may be provided by viral encoded proteins produced by recombinant DNA techniques or by synthetic peptides corresponding to appropriate viral epitopes. We have expressed at high levels in E. coli a gag gene segment corresponding to approximately 97% of the p55 gag precursor protein, as well as a novel gag/env fusion protein that contains antigenic determinants in common with gag p24, env gp41, and env gp120. The gag and gag/env proteins were purified from insoluble inclusion bodies by sequential extraction with increasing concentrations of urea. These components were tested for reactivity with antisera to HIV proteins and peptides. We have also chemically synthesized a peptide corresponding to env residues 578-608, representing a portion of env gp41. The final preparation of gag and gag/env proteins in 8 M urea reacted with sheep anti-HTLV-III p24 gag antibodies and acquired immune deficiency syndrome (AIDS) patient sera. The gag/env fusion protein also reacted with rabbit anti-HIV env 500-511 peptide antibody. Both recombinant proteins and the env peptide were suitable as reagents for evaluation of serum samples by enzyme-linked immunosorbent assay (ELISA). Results of ELISA assays utilizing the recombinant viral proteins and synthetic peptide were in good agreement with results obtained using disrupted virus as antigen in ELISA assays and immunoblotting.