Analysis of the human immunodeficiency virus type 1 gp41 membrane proximal external region arrayed on hepatitis B surface antigen particles.

Vaccine immunogens derived from the envelope glycoproteins of the human immunodeficiency virus type 1 (HIV-1) that elicit broad neutralizing antibodies remain an elusive goal. The highly conserved 30 amino-acid membrane proximal external region (MPER) of HIV gp41 contains the hydrophobic epitopes for two rare HIV-1 broad cross-reactive neutralizing antibodies, 2F5 and 4E10. Both these antibodies possess relatively hydrophobic HCDR3 loops and demonstrate enhanced binding to their epitopes in the context of the native gp160 precursor envelope glycoprotein by the intimate juxtaposition of a lipid membrane. The hepatitis B surface antigen (HBsAg) S1 protein forms nanoparticles that can be utilized both as an immunogenic array of the MPER and to provide the lipid environment needed for enhanced 2F5 and 4E10 binding. We show that recombinant HBsAg particles with MPER (HBsAg-MPER) appended at the C-terminus of the S1 protein are recognized by 2F5 and 4E10 with high affinity compared to positioning the MPER at the N-terminus or the extracellular loop (ECL) of S1. Addition of C-terminal hydrophobic residues derived from the HIV-1 Env transmembrane region further enhances recognition of the MPER by both 2F5 and 4E10. Delipidation of the HBsAg-MPER particles decreases 2F5 and 4E10 binding and subsequent reconstitution with synthetic lipids restores optimal binding. Inoculation of the particles into small animals raised cross-reactive antibodies that recognize both the MPER and HIV-1 gp160 envelope glycoproteins expressed on the cell surface; however, no neutralizing activity could be detected. Prime:Boost immunization of the HBsAg-MPER particles in sequence with HIV envelope glycoprotein proteoliposomes (Env-PLs) did not raise neutralizing antibodies that could be mapped to the MPER region. However, the Env-PLs did raise anti-Env antibodies that had the ability to neutralize selected HIV-1 isolates. The first generation HBsAg-MPER particles represent a unique means to present HIV-1 envelope glycoprotein neutralizing determinants to the immune system.

Formation of HIV-1 envelope-hepatitis B core antigen hybrids with high affinity for CD4.

We have identified an acceptor site on HIV gp120, where foreign protein sequences can be inserted while retaining the native conformation of gp120. The resulting hybrids showed dual antigenicity, normal glycosylation, and high affinity binding of the CD4 receptor. This site allows insertion of highly immunogenic proteins such as core antigen of hepatitis B virus. By combining the immunogenicity of the carrier protein with the antigenicity of gp120, these hybrids may lead to modified HIV-1 antigens with enhanced immunogenicity.

Genetic control of the immune response to HIV type 1 envelope glycoprotein 120 in mice: effects of MHC and transgenic human CD4.

HIV infection elicits a strong immune response to viral proteins, including broadly cross-reactive antibodies to envelope glycoprotein 120 (gp120). However, vaccination with recombinant gp120 generally produces lower titered antibodies with narrow specificity. We have examined host genes that may control the strength and breadth of the response to gp120 vaccines. Because of the complexity of the human MHC, we have focused on the response of MHC congenic mice, which share an identical genetic background, differing only in H-2 type. The antibody response to gp120 varied markedly with H-2 type. H-2a and H-2k mice gave consistently high antibody titers, while H-2s mice gave 100-fold lower titers, and H-2b mice gave low to intermediate responses. Nearly the same genetic control applied for antibodies to both unique and shared determinants and on a variety of different genetic backgrounds. Transgenic mice expressing human CD4 gave the same titers as normal H-2-matched controls. MHC-linked genetic control of the quantity and quality of antibodies indicate a requirement for T cell help in producing antibodies to unique and shared determinants of gp120.

The promise and pitfalls of monoclonal antibody therapeutics.

Recent experience has helped to clarify the best ways to use monoclonal antibodies to solve clinical problems. For example, imaging based on tumor antigens, rather than tumor size, will permit early detection of cancer and accurate staging. Blocking receptor-ligand interactions may permit therapeutic intervention in cell growth or function but activity may depend on the choice of an antiligand or antireceptor strategy. Humanized antibodies will achieve greater intensity and duration of therapy, while allowing repeat administration in chronic diseases.

Identification of a murine CD4+ T-lymphocyte response site in hepatitis C virus core protein.

The T cell response to a recombinant HCV truncated core protein (cp1-10) was measured in a proliferation assay. Based on a 10-fold greater response to this truncated core protein than to its shorter form (cp1-8), a predominant epitope was mapped to the carboxyl quarter of this sequence. This epitope was further mapped to a synthetic peptide corresponding to amino acids 121-140 of the core protein. The peptide was antigenic for T cells of all three H-2 types tested, H-2 r, b and d, and the proliferating T cells were CD4+. Besides inducing specific proliferation in vitro, peptide aa121-140 can prime helper T cells in vivo. When boosted with core protein, mice primed with peptide produced 64-fold higher antibody titer than without priming in 1 week. The identification of a broadly immunogenic T cell helper epitope on core protein may be important for vaccine design against HCV.

CD4-IgG binding threshold for inactivation of human immunodeficiency virus type 1.

The stoichiometry of human immunodeficiency virus type 1 (HIV-1) inactivation by soluble receptor CD4-IgG hybrid dimers (CD4-IgG) was examined. The extent of HIV-1 inactivation was measured in a sensitive plaque-forming assay, and the corresponding level of CD4-IgG binding was determined by immunofluorescence of infected cells. Ninety percent virus inactivation occurred at relatively low levels of CD4-IgG binding (10% of the saturating level). At even lower binding levels (1.4% of maximum binding), virus survival was 44%. Over a broad range of binding conditions, the survival curve followed a model in which viruses binding more than a threshold level of CD4-IgG were completely inactivated, while viruses binding less remained infectious. The data indicate that CD4-IgG binding to 1.4% of gp120 binding sites equals the threshold for inactivation. Thus, virus inactivation can begin when 3 CD4-IgG (of approximately 216 gp120 sites) bind per virion.

Genetic control of the murine humoral response to distinct epitopes of hepatitis C virus core protein.

Recombinant hepatitis C virus (HCV) core protein from aa1-164, designated cp1-10, was used to immunize mice. Antibodies to cp1-10 were produced in all seven strains of congenic mice; none of the strains could be considered low responders relative to the others. The mouse response against individual epitopes of HCV core protein varied from one strain to another: B10.RIII (H-2r) recognized all three peptides aa13-30, aa77-90, aa129-145; B10.D2 (H-2d), B10 (H-2b) and C3H.SW (H-2b) responded to aa13-30, aa77-90; B10.M (H-2f), B10.BR (H-2k) and C3H/Hej (H-2k) reacted with aa13-30 only. Competitive inhibition of binding demonstrated that antibody to the peptide was inhibited by cp1-10 protein and the corresponding peptide only. Recombinant HCV core protein is highly immunogenic and can elicit good antibody response in mice. The aa13-30 is a major epitope of HCV core protein in mice. The humoral response to the distinct epitopes was regulated by the H-2 genes. Further analysis indicated that the I-a locus of H-2 genes determined the antibody response to aa13-30 and 77-90. These results suggest that the variation of antibody responses to HCV in humans may partially contribute to different outcomes of HCV infection.

Enzymatic labeling of biologically active envelope glycoprotein gp120 of HIV-1.

We have found that the envelope glycoprotein gp120 of HIV-1 can be modified extensively by enzymatic oxidation of oligosaccharide chains without diminishing binding to its natural receptor, CD4. Using affinity purified galactose oxidase, over 20 sites per gp120 molecule were converted to chemically reactive aldehydes, as measured by 3H-BH4 reduction, while the conformation-dependent CD4 binding site remained intact. In contrast, periodate oxidation completely destroyed CD4 binding while producing fewer sites. Enzymatically labeled, biologically active gp120 should facilitate biochemical studies of receptor binding and viral inactivation by neutralizing antibodies.

Production of a soluble and secreted antigenic fragment of HBsAg in yeast.

We have produced a fragment of hepatitis B surface antigen (HBsAg) corresponding to amino acids 1-60 as a fusion protein with the alpha mating factor of yeast. The product is secreted from yeast as a soluble monomer that expresses HBsAg antigenicity. Unlike other heterologous fusion proteins, it is not processed by the Lys-Arg endoprotease, possibly due to a proline in the linker between the two coding sequences. The resulting soluble fragment will enable us to map the immunodominant sites of HBsAg recognized by T cells and to identify additional factors contributing to vaccine potency.

A predominant group-specific neutralizing epitope of human immunodeficiency virus type 1 maps to residues 342 to 511 of the envelope glycoprotein gp120.

Recombinant native human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins gp160 and gp120 (residues 1 to 511) expressed in insect cells quantitatively adsorbed the group-specific neutralizing antibodies found in human sera. However, these antibodies were not adsorbed by envelope fragment 1 to 471 or 472 to 857 or by both fragments sequentially, even though together they add up to the full-length gp160 sequence. A hybrid envelope glycoprotein was constructed with residues 342 to 511 of the HIV-1 sequence and residues 1 to 399 of the simian immunodeficiency virus type 1 sequence to vary the HIV-1 sequence while preserving its conformation. This hybrid glycoprotein quantitatively adsorbed human neutralizing antibodies, while native simian immunodeficiency virus type 1 envelope glycoprotein did not. These results identify a new neutralizing epitope that depends on conformation and maps to residues 342 to 511 of gp120. It overlaps the extended CD4-binding site but is distinct from the V3 loop described previously (K. Javaherian et al., Proc. Natl. Acad. Sci. USA 86:6768-6772, 1989; J. R. Rusche et al., Proc. Natl. Acad. Sci. USA 85:3198-3202). Since it is conserved among diverse HIV-1 isolates, this new epitope may be a suitable target for future vaccine development.

Anti-Leu3a induces combining site-related anti-idiotypic antibody without inducing anti-HIV activity.

Development of a vaccine for acquired immunodeficiency syndrome (AIDS) has proven difficult, and so alternative approaches such as idiotypic manipulation have been suggested. As applied to AIDS, this approach could involve immunizing with an anti-CD4 antibody resembling gp120, to induce anti-idiotypic antibodies which would bind to gp120. The CD4 binding site on gp120 is conserved, and so, such an immune response should protect against all variants. Induction of anti-human immunodeficiency virus (HIV) immunity has been reported using anti-Leu3a, and this result has led to testing in humans. Negative results obtained by others have been attributed to differences in immunization protocols. Because of the importance of this question, we reinvestigated the potential of anti-Leu3a to induce anti-HIV antibodies, compared with control immunizations with OKT4A (another anti-CD4 antibody) and the irrelevant Ig MOPC-21. Responses to anti-Leu3a showed induction of high-titer anti-idiotypic activity, and included combining-site-related activity. Yet sera showed no binding to gp160 above controls and no detectable neutralizing activity in a sensitive HIV plaque assay, so the anti-idiotypes induced were not internal images of CD4. We conclude that the pronounced anti-HIV responses reported with anti-Leu3a cannot be generalized, and thus that anti-Leu3a does not offer promise as an HIV vaccine. However, these results do not negate the promise of the idiotypic approach, and a vaccine for AIDS based on idiotype manipulation remains a possibility.

Human immunodeficiency virus 1. Predominance of a group-specific neutralizing epitope that persists despite genetic variation.

HIV-1 is known to show a high degree of genetic diversity, which may have major implications for disease pathogenesis and prevention. If every divergent isolate represented a distinct serotype, then effective vaccination might be impossible. However, using a sensitive new plaque-forming assay for HIV-1, we have found that most infected patients make neutralizing antibodies, predominantly to a group-specific epitope shared among three highly divergent isolates. This epitope persists among divergent isolates and rarely mutates, despite the rapid overall mutation rate of HIV-1, suggesting that it may participate in an essential viral function. These findings, plus the rarity of reinfections among these patients, suggest that HIV-1 may be more susceptible to a vaccine strategy based on a group-specific neutralizing epitope than was previously suspected.

Studies on peptides. CLXVII. Solid-phase syntheses and immunological properties of fragment peptides related to human malaria circumsporozoite protein.

A glycine-linked tetramer of Asn-Ala-Asn-Pro, a tandem repeated sequence of malaria circumsporozoite (CS) protein, was synthesized by the Boc-based solid phase method, followed by deprotection with 1 M trimethylsilyl trifluoromethanesulfonate-thioanisole in trifluoroacetic acid. In addition, three tetramer-related peptides were similarly synthesized, i.e., a 34-residue peptide [linked with TH, a proposed T-cell epitope of CS, at the C-terminus of the tetramer], a 46-residue peptide and a 59-residue peptide [linked with HA or HA', two proposed T-cell epitopes of influenza hemagglutinin protein, at the N-terminus of the above 34-residue peptide]. Their immunological properties were examined by enzyme-linked immunosorbent assay, for which three different congenic strains of mouse were used to raise the specific antibodies. Despite conjugation of T-cell epitopes to the tetramer, the mice of low-responder strains to the tetramer failed to produce any antibody specific to the tetramer. However, with the aid of recombinant interleukin 2 as an adjuvant, the low-responder mice produced antibody with relatively high titers.

Human T cell response to the surface antigen of hepatitis B virus (HBsAg). Endosomal and nonendosomal processing pathways are accessible to both endogenous and exogenous antigen.

We have studied the antigen specificity and processing requirements of three vaccine-induced cloned human T cell lines specific for HBsAg, the envelope protein of hepatitis B virus. Each T cell line recognized endogenously expressed antigen as well as exogenous antigen. Two clones required endosomal processing, both for exogenous and endogenous antigen; while the other T cell line depended on nonendosomal processing to generate antigenic peptides from both endogenous and exogenous antigen. Thus, the two processing pathways are accessible to exogenous and endogenous antigen. These results suggest that vaccine-induced T cells can participate actively in the immune response to live virus.

Protein antigenic structures recognized by T cells: potential applications to vaccine design.

In summary, our results using the model protein antigen myoglobin indicated, in concordance with others, that helper T lymphocytes recognize a limited number of immunodominant antigenic sites of any given protein. Such immunodominant sites are the focus of a polyclonal response of a number of different T cells specific for distinct but overlapping epitopes. Therefore, the immunodominance does not depend on the fine specificity of any given clone of T cells, but rather on other factors, either intrinsic or extrinsic to the structure of the antigen. A major extrinsic factor is the MHC of the responding individual, probably due to a requirement for the immunodominant peptides to bind to the MHC of presenting cells in that individual. In looking for intrinsic factors, we noted that both immunodominant sites of myoglobin were amphipathic helices, i.e., helices having hydrophilic and hydrophobic residues on opposite sides. Studies with synthetic peptides indicated that residues on the hydrophilic side were necessary for T-cell recognition. However, unfolding of the native protein was shown to be the apparent goal of processing of antigen, presumably to expose something not already exposed on the native molecule, such as the hydrophobic sides of these helices. We propose that such exposure is necessary to interact with something on the presenting cell, such as MHC or membrane, where we have demonstrated the presence of antigenic peptides by blocking of presentation of biotinylated peptide with avidin. The membrane may serve as a short-term memory of peptides from antigens encountered by the presenting cell, for dynamic sampling by MHC molecules to be available for presentation to T cells. These ideas, together with the knowledge that T-cell recognition required only short peptides and therefore had to be based only on primary or secondary structure, not tertiary folding of the native protein, led us to propose that T-cell immunodominant epitopes may tend to be amphipathic structures. An algorithm to search for potential amphipathic helices from sequence information identified 18 of 23 known immunodominant T-cell epitopes from 12 proteins (p less than 0.001). Another statistical approach confirmed the importance of amphipathicity and also supported the importance of helical structure that had been proposed by others. It suggested that peptides able to form a stable secondary structure, especially a helix, more commonly formed immunodominant epitopes. We used this approach to predict potential immunodominant epitopes for induction of T-cell immunity in proteins of clinical relevance, such as the malarial circumsporozoite protein and the AIDS viral envelope.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunologically relevant peptide antigen exists on the presenting cell in a manner accessible to macromolecules in solution.

Although studies of the association of antigen with APC have been complicated by antigen-processing requirements, recent studies have suggested that immunologically relevant antigen should be present on the APC surface. Nevertheless, blocking of antigen presentation with antibody to the antigen has not been demonstrable in most systems. To study this problem we developed a system using avidin to block presentation of amino-terminal biotinylated synthetic peptide 132-146 of sperm whale myoglobin (B132) to a murine T cell clone specific for this site in association with I-Ed. greater than 95% specific inhibition was observed with doses of B132 equipotent to unmodified peptide. Specific blocking could be observed: (a) after pulsing APC with antigen, washing, and incubating for a chase period of 8-16 h before addition of avidin and T cells to assure adequate time for intracellular trafficking and maximal display of antigen on the cell surface, or (b) when monensin is present during the antigen pulse to inhibit such traffic. Therefore, the inhibition appeared to be occurring at the cell surface unless dissociation and reassociation were constantly occurring. To distinguish these, B10.GD APC (I-Ed-negative) were pulsed with antigen and cocultured with B10.D2 APC (I-Ed-positive). No detectable antigen presentation resulted. Thus, minimal dissociation and reassociation between antigen and APC occurs and, consequently, blocking by extracellular solution-phase binding of avidin to antigen is unlikely. Taken together, these data suggest that the blocking is occurring at the cell surface. Thus, under physiologic conditions, immunologically relevant antigen necessary for T cell activation appears to be present on the APC surface and is freely accessible to macromolecules the size of avidin. These findings hold specific implications for models of antigen presentation for T cell recognition.