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Autoimmunity mediated by cross-reactive antibodies, elicited by HIV-1 envelope glycoproteins gp120/gp160, has been postulated to contribute to the pathogenesis of AIDS. Partial amino acid sequence homology between gp120/gp160 and several human host proteins, including MHC antigens and immunoglobulins, has been perceived as the basis for immunological cross-reactivity. Binding of antibodies from sera of HIV-1-infected individuals to selected host proteins and/or to synthetic peptides derived from them and the inhibitory activity of such sera in assays measuring the functional activity of T cells provided apparent support for the autoimmunity hypothesis, which is also relevant to the issue of safety of anti-HIV-1 vaccines. Considering the possibility that the detected autoantibodies may arise for reasons other than antibody responses to gp120/gp160, the immunological cross-reactivity between gp120/gp160 and the relevant host proteins was investigated using hyperimmune rabbit anti-gp120/gp160 and monoclonal antibodies. As determined from dilution end-point comparisons for polyclonal anti-gp120, the cross-reactivity of anti-gp120 with CD4 was undetectable (< 10(-5)%). The cross-reactivity of anti-gp120/gp160 with HLA-I and HLA-II antigens was also undetectable (< 4 x 10(-4)%) and that with other human proteins reported to have partial sequence homology with gp120/gp41 was < or = 0.013%. Anti-gp120/gp160 did not have detectable inhibitory effects in functional assays measuring proliferative T cell responses. Therefore, immunization with gp120/gp160 is unlikely to elicit harmful autoimmune responses.

A synthetic peptide, corresponding to the sequence (637-666) of the HIV-1 glycoprotein gp41, inhibited the replication of an array of HIV-1 strains. The peptide (637-666) selectively binds to the fusion domain at the N-terminus of gp41, suggesting that inhibition of HIV-1 infection is caused by blocking fusion of HIV-1 with cells or of infected cells with uninfected cells. Since this peptide has antiviral activity against both homologous and heterologous HIV-1 isolates and has no detectable cytotoxicity, it offers a novel approach to chemotherapy and prophylaxis of AIDS.

Earlier studies revealed that human interleukin 6 (IL-6) contains recognition sites for the hepatitis B virus (HBV) envelope (env) protein, and that IL-6 and anti-IL-6 antibodies, respectively, inhibited the interaction of cells expressing a receptor for HBV with the preS(21-47) segment of the HBV env protein, encompassing the complementary attachment site for IL-6. This suggested that IL-6 mediates HBV-cell interactions. We report that: (a) Chinese hamster ovary cells transfected with human IL-6 cDNA and Spodoptera frugiperda ovarian insect cells infected with recombinant baculovirus carrying human IL-6 cDNA expressed receptors for the preS(21-47) region of the HBV env protein, indicating that expression of IL-6 on the surface of cells is sufficient to endow them with receptors for HBV. (b) Among peptides covering the entire sequence of human IL-6 and the corresponding antipeptide antibodies, the peptide IL-6[35-66] and anti-IL-6[35-66] most effectively inhibited the interaction between human hepatoma HepG2 cells and the preS(21-47) ligand, suggesting that this region of the human IL-6 sequence encompasses a binding site for the HBV env protein. (c) Studies with replacement set peptides from the preS(21-47) sequence indicated that residues 21-25, 28, 31, 33-35, 39, and 43-45 can be replaced by alanine (serine) residues, while all the other residues are essential for maintaining the cell receptor/IL-6 binding activity. Further delineation of complementary sites on IL-6 and on the HBV env protein may contribute to the design of compounds inhibiting HBV replication.

The envelope of the hepatitis B virus (HBV) consists of three related proteins, designated S-, M- and L-protein, all of which share a common 226-amino acid residue sequence, corresponding to the S-protein that is sufficient for eliciting protective immunity against HBV. HBV variants, resulting from point mutations leading to replacements of amino acids within the S(122-160) segment of S-protein, have been recently recognized. In order to assure the continued success of vaccination against HBV and the adequacy of diagnostic tests for HBV envelope antigens and antibodies, it is necessary to understand the impact of amino acid replacements on the immunological recognition of S-protein at both the B- and T-cell levels. Immunologically tolerated and forbidden amino acid replacements within the S(139-147) segment of S-protein have already been discerned. The impact of amino acid substitutions within the S(122-136) segment on the immunological recognition of S-protein is analyzed in this report. Such replacements do not appreciably affect the binding of rabbit and goat anti-S antibodies to replacement set peptides, while decreased murine antibody binding was observed with some peptides having substitutions at residues 122, 123 and 133. On the other hand, amino acid substitutions within the (126-136) region, except those distinguishing serological subtypes of HBV from each other, abrogated murine T-cell proliferative responses to the peptides, while substitutions at residues 122, 123 and 125 had a lesser effect. Some of the peptides with amino acid substitutions peculiar to the variants had diminished stimulatory activity for T-cells from individuals vaccinated against hepatitis B. Amino acid substitutions in both the S(139-147) and S(122-136) segments of S-protein may potentially result in variant viruses escaping immunological surveillance based on current hepatitis B vaccines.

Synthetic peptides and anti-peptide antibodies have been widely used as probes to map B- and T-cell epitopes on proteins. Such probes also have the potential to delineate contact sites involved generally in protein-protein interactions or in association of domains within a protein. We applied peptide/anti-peptide probes to define: (1) regions on the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins gp120 and gp41 involved in the association between these two glycoproteins; and (2) sites on gp120/gp41, essential for the association of HIV-1 with the CD4 cell receptor. Results of this examination suggested the following: (1) two segments on gp120, encompassing residues (102-126) and (425-452), contribute to the binding site for CD4 and are expected to be juxtaposed in the folded gp120 chain; (2) portions of immunodominant gp120 and gp41 epitopes, encompassing residues (303-338) and (579-611), respectively, appeared to be involved in the gp120-gp41 association, as suggested by direct binding studies and by the limited accessibility of these epitopes on HIV-1 virions: other portions of gp120 also appeared to contribute to the association between these two glycoproteins; (3) there is a partial overlap between gp41 and CD4 binding sites on gp120; (4) the fusion domain and a segment (637-666) of gp41 are not accessible to antibodies after oligomerization of gp41; and 5) the gp120-gp41 association was blocked by aurintricarboxylic acid, suggesting the possibility of developing antiviral compounds interfering with HIV-1 assembly.

The major target organ for hepatitis B virus (HBV) is the liver. However, cells other than hepatocytes, including peripheral blood lymphocytes and monocytes, may become infected with HBV. The cell receptor binding site was assigned to the preS(21-47) segment of the HBV envelope protein. HBV receptors were detected on human liver and hepatoma cells, on B lymphocytes, and, as shown here, on monocytes, and T cell lines, activated by Escherichia coli lipopolysaccharide and concanavalin A, respectively. The cell receptors for HBV have not been characterized until now. The detection of HBV receptors and their "activation antigen" characteristic on distinct cells suggested paths for identification of the receptors with already defined cell surface proteins. This search revealed that interleukin 6 contains recognition sites for the preS(21-47) sequence and mediates HBV-cell interactions. Thus, HBV belongs to a group of viruses utilizing cytokines or cytokine receptors for replication and interference with the host immune system.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (gp120 and gp41) elicit virus-neutralizing antibodies (VNAB) and also antibodies enhancing HIV-1 infection (EAB). Several epitopes eliciting VNAB have been defined, the principal virus-neutralizing determinant being assigned to the V3 loop of gp120. To provide a background for a rational design of anti-HIV vaccines, it also appears important to define domains eliciting EAB. This was accomplished by screening antisera against synthetic peptides covering almost the entire sequence of gp120/gp41 for their enhancing effects on HIV-1 infection of MT-2 cells, a continuous T cell line. Many (16/30) of the antisera significantly enhanced HIV-1 in the presence of human complement. Antibodies to complement receptor type 2 (CR2) abrogated the antibody-mediated enhancement of HIV-1 infection. Antisera to V3 hypervariable loops of 21 distinct HIV-1 isolates were also tested for their enhancing effects on HIV-1IIIB infection. 11 of these sera contained VNAB and 10 enhanced HIV-1IIIB infection. All antisera with virus-enhancing activity contained antibodies crossreactive with the V3 loop of HIV-1IIIB, and the virus-enhancing activity increased with increasing serological crossreactivity. These results suggest that immunization with antigens encompassing V3 loops may elicit EAB rather than protective antibodies if epitopes on the immunogen and the predominant HIV-1 isolate infecting a population are insufficiently matched, i.e., crossreactive serologically but not at the level of virus neutralization.

Immunization of primates or humans with human immunodeficiency virus type 1 (HIV-1) glycoproteins usually elicited moderate immune responses to the principal neutralizing determinant (PND) located within the V3 hypervariable loop of gp120. Since an antibody response to the PND appears to be protective, experiments were carried out to determine the responsiveness of chimpanzees to immunization with synthetic peptides corresponding to the full-length V3 loop. Seven chimpanzees (4 preimmunized with gp160, 2 preimmunized with HIV-1 antigens unrelated to gp160, and 1 unimmunized) were vaccinated with a mixture of full-length V3 loop peptides from 21 distinct HIV-1 isolates (clones) either in unconjugated form or linked to carrier proteins from HIV-1 nef and gag P18, respectively. Six chimpanzees developed high levels of antibodies to the peptides (dilution endpoints 1: greater than 25,000), and 5 had high levels of antibodies to gp120 from HIV-1IIIB (endpoint titers 1: greater than 500,000). Chimpanzees immunized with peptide-carrier conjugates (4) had antibodies to the carrier proteins nef and gag P18, respectively (endpoint titers 1: greater than or equal to 35,000). Virus-neutralizing (VN) antibodies were detected in sera of 5 of 7 chimpanzees, but were present at titers of 1: greater than or equal to 400 only in sera of 2 chimpanzees. One of these was challenged with HIV-1 and was protected against infection, as reported elsewhere. The antibodies were primarily specific for the HIV-1 isolate used for primary immunization before boosting with peptides. The relatively low dilution endpoints of VN antibodies as compared with endpoints determined by site-specific immunoassays probably can be ascribed to imperfect mimicry of conformational epitopes by synthetic peptides. Nevertheless, sequential or simultaneous immunization with recombinant envelope glycoproteins of HIV-1 and selected synthetic peptides offers an approach for eliciting protective immunity against HIV-1.

Combining of subtype specific peptides from the hypervariable loop of the envelope glycoprotein gp120 of divergent HIV-1 isolates may help in designing a broadly protective immunogen against HIV-1 infection. To enhance the immunogenicity of such a polyvalent antigen, in the absence of oil-containing adjuvants, it is necessary to link the peptides to a protein carrier. It is preferable to use as carriers those proteins from HIV-1 itself which may contribute to eliciting protective immunity. The structural and non-structural proteins, gag P18 and nef, respectively, which can be prepared in high yields by recombinant DNA techniques in Escherichia coli, were selected for this purpose. The corresponding peptide-protein conjugates, each containing 21 distinct peptides, were prepared using the cross-linking reagents N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) or m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS). Conjugates prepared by the second method elicited approximately 10-100 times higher levels of antibodies recognizing the homologous peptides and the HIV-1 envelope glycoproteins. The sulfo-MBS conjugation procedure preserved the antigenicity of both gag P18 and nef and the respective conjugates elicited an immune response to these proteins. Despite the low immunization dose of single peptides (10 micrograms) present in the mixture of peptides collectively linked to the carriers, antibody responses to most of the individual peptides were high (dilution endpoints 1: greater than 16,000, 1: greater than 80,000 for the nef and gag P18 conjugates, respectively). Conjugates consisting of a multitude of HIV-1 envelope-derived peptides in combination with gag P18 and nef carriers are expected to elicit broadly protective immunity against distinct HIV-1 subtypes.

The positions of all 9 intrachain disulfide bonds within the envelope glycoprotein gp120 of the human immunodeficiency virus (HIV-1) have been established recently. Peptides expected to mimic some of the disulfide-bonded domains [(120-133)-(203-221); (133-138)-(164-203); (224-254); (391-425) and (385-392)-(425-452)] were synthesized. All peptides, except (120-133)-(203-221), elicited in immunized rabbits relatively high levels of antibodies reacting with gp120 in enzyme-linked immunosorbent assay (ELISA) and/or Western immunoblot assays. However, these antibodies failed to neutralize the infectivity of HIV-1. Combined with earlier reports concerning other gp120 loop peptides, these results confirm the uniqueness of the V3 (303-338) loop in encompassing a principal determinant(s) involved in virus neutralization.

The capacity of a preS1-specific monoclonal antibody (McAb) F35.25 to block the attachment of preS1-specific ligands to human hepatoma HepG2 cells was studied. In order to define more precisely the fine epitope specificity of McAb F35.25, its reaction with synthetic peptides derived from the preS1 sequence (12-53) was investigated. McAb F35.25 was found to recognize better synthetic peptide preS(21-47) from the adw 2 and ayw sequences than the synthetic peptide preS(32-53) adw 2. The shortest sequence recognized by McAb F35.25 among the peptide sequence studied was preS(32-47). The corresponding amino acid sequence (for HBV subtype adw 2) is PAFGANSNNPDWDFNP. As expected, it was found that McAb F35.25 inhibited the attachment of HepG2 cells to HBsAg-cellulose, as well as to preS(21-47)-cellulose, corresponding to two HBV subtypes adw 2 and ayw. Finally, the inhibitory effect of different peptides on the interaction of McAb F35.25 with HBsAg particles containing the preS1 sequence was also studied. The peptide preS(12-47) appeared to be the most effective inhibitor. Therefore, the McAb F35.25 is specific for the sequence preS1(X to 47), where (12 less than or equal to X less than 32). These results indicate that McAb F35.25 is probably virus-neutralizing and represents a reagent of great value to study the interaction between HBV and hepatocytes independently of d/y subtype changes.

It is essential for the development of strategies for prevention and therapy of human immunodeficiency virus (HIV-1) infections to define host factors playing a dominant role in determining the clinical outcome of infection. Antibodies directed against restricted regions of the HIV-1 glycoproteins gp120 and gp41 are likely to represent important factors involved in host defense against HIV-1. Definition of qualitative and quantitative differences in the spectrum of anti-gp120 and anti-gp41 antibodies between two vastly different groups of HIV-1-infected individuals, long-term asymptomatic carriers, and individuals with acquired immunodeficiency syndrome (AIDS) who died, might reveal the epitope specificity of antibodies contributing to prevention of clinical disease. To accomplish this goal, sera from both groups were assayed for antibodies recognizing synthetic peptides from gp120/gp41 which were shown in earlier experiments to mimic epitopes on the two HIV-1 glycoproteins. None of the sera recognized all of the distinct 27 peptides from gp120 and gp41. The spectrum of antibodies was distinct for each of the sera from both groups of HIV-1-infected individuals. Nevertheless, antibody responses distinguishing the two groups from each other were discerned. In particular, it was possible to predict the unfavorable outcome of disease by comparative measurements of levels of antibodies to a peptide (303-338), corresponding to the entire V3 hypervariable loop of gp120 and/or by providing evidence for declining levels of these antibodies during the course of infection. Antibodies recognizing additional peptides [(219-245), (280-306), (425-452), (658-682), (729-758), (808-845), and (845-862)] were significantly less prevalent in AIDS patients than in asymptomatic carriers. It appears possible that maintenance of high levels of the respective antibodies would contribute to preventing AIDS in HIV-1-infected individuals. Active immunization with antigens containing epitopes defined by the respective peptides and/or administration of the corresponding antibodies may be considered as a modality for therapy of HIV-1 infections.

The preS(21-47) sequence of the hepatitis B virus (HBV) envelope protein is involved in binding of the virus to cell receptors. A protein similarity search revealed a partial homology between this sequence and a segment of the human immunoglobulin A (IgA) heavy chain constant region, suggesting that the cell attachment site for HBV might be located on secretory component representing a receptor for polymeric IgA. Data presented herein do not support this hypothesis but provide evidence for immunological cross-reactivity between IgA and the preS(21-47) region of the HBV env protein.

Antibody mediated and cell mediated immune responses to the envelope glycoproteins gp120 and gp41 of the human immunodeficiency virus (HIV-1) are considered important for protection against infection and for attenuation of disease symptoms after infection. Virus neutralizing antibodies are mostly subtype specific and primarily directed against epitopes on a hypervariable loop from the V3 region of HIV-1 gp120. Such epitopes are recognized by helper and cytotoxic T-cells suggesting that all protective immune responses to HIV-1 are predominantly subtype specific. The extraordinary primary sequence variability of gp120 indicates that a combination of subtype specific components will be required to design a broadly effective protective immunogen against HIV-1. Peptides from hypervariable loops of the V3 region of 21 distinct HIV-1 isolates (clones) were synthesized and used to raise rabbit antisera. The antisera contained high levels of antibodies recognizing the homologous peptides and the parent gp120 sequence. The serological cross-reactivity between the distinct peptides was evaluated and related to amino acid divergence. The corresponding relationship approximated a linear regression with a correlation coefficient r = 0.718. The 21 peptides were combined into a single immunogen which elicited broadly reactive antibodies recognizing all 21 peptides as well as gp120 from the only isolate tested, HIV-1 IIIB. The results suggest the possibility of developing broadly protective HIV-1 immunogens by combining judiciously selected subtype specific peptides derived from envelope glycoproteins of divergent virus isolates.

Receptors for hepatitis B virus (HBV; subtype adw) were identified on the surface of human hepatoma HepG2 cells in earlier studies. The cell receptor binding site on HBV was assigned to the preS(21-47) region of the preS1 sequence of the envelope protein. Studies presented here show that (1) amino acid residue replacements within the preS(21-47) sequence distinguishing HBV subtypes adw and ayw, preserve the binding capacity of the HBV env protein for HepG2 cell receptors; (2) the inhibition of binding between HepG2 cells and preS1-specific ligands by antibodies is effective only if the subtype specificity of anti-preS1-specific antibodies and of the preS1-specific ligands are matched; (3) receptors for HBV were present on the surface of human cells of nonhepatic origin, including peripheral blood B-lymphocytes, some hematopoietic cell lines of the B-cell lineage, neuroblastoma, amnion, and embryonic carcinoma cell lines. Receptors for HBV on these cells appeared similar to the receptor on HepG2 cells by the following criteria: (a) recognition by antibodies raised against the receptor on HepG2 cells; (b) inhibitory activity of lysates prepared from these cells on the interaction between HepG2 cells and preS1-specific ligands; and (c) the inhibitory effect of lysates from HepG2 cells on the reaction of these cells with HBsAg- and preS(21-47)-cellulose. The presence of receptors for HBV on some cells of extrahepatic origin is in accordance with earlier observations indicating that hepadnaviruses are not strictly hepatotropic.

B and T cell epitopes expressed on the surface of S-protein, a major constituent of the envelope of hepatitis B virus (HBV), are essential for eliciting protective immunity against HBV infection. A segment of the S-protein sequence encompassing residues S(139-147) is a portion of overlapping B and T cell epitopes. This sequence is conserved among distinct serological subtypes of HBV and has a 77.8% homology with an analogous sequence in S-proteins of nonhuman mammalian hepadnaviruses. Rare subtypes and variants of HBV having amino acid replacements within the S(139-147) sequence were discerned recently. The impact of amino acid replacements within this sequence on its immunological recognition at both the B and T cell levels was explored by peptide replacement set analysis. Results of the analysis permit discrimination between tolerated and forbidden amino acid replacements and provide a background for the development of reagents and immunogens specific for emerging HBV variants.

Envelope glycoproteins, gp 120 and gp41, of the human immunodeficiency virus type 1 (HIV-1) elicit immune responses, including virus-neutralizing antibodies, which are expected to play a role in the defence against HIV-1 infection. Subregions of the gp120/gp41 sequence have immunosuppressive effects or may be implicated in autoimmune responses. Some of the immunodominant epitopes of gp120/gp41 do not contribute to protective immunity and act as immunological decoys. These circumstances emphasize the need to select from gp120/gp41 regions inducing protective responses. Towards this goal, 30 peptides covering approximately 87% of the HIV-1 strain BH10 gp120/gp41 sequence were synthesized. Antibodies in rabbit and human anti-HIV-1 sera recognized 28 and nine of the peptides, respectively, indicating that most of the gp120/gp41 sequence is immunogenic and secondly, that the antibody response to HIV-1 is restricted in infected humans. Most of the peptides, without conjugation to carriers, elicited high levels of anti-peptide (endpoints 1: greater than 10(4] and anti-gp120/gp41 (endpoints 1: greater than or equal to 10(3] antibodies. The highest levels of virus-neutralizing antibodies were elicited by peptide 306 to 338 from a hypervariable loop of gp120. Additional peptides from the full-length hypervariable loop (303 to 338) of HIV-1 BH10 and from 20 additional HIV-1 isolates were recognized differentially by human anti-HIV, suggesting that success of passive immunization may depend on a match between administered antibodies and the challenging HIV-1 strain, and also that active immunization with selected peptides from a hypervariable region of distinct HIV-1 isolates should be explored further as a method for prophylaxis against infection.

The effect on hepatitis B surface antigen (HBsAg) of reagents considered specific for each of the amino acid residues, lysine, methionine, cysteine, arginine, tyrosine, tryptophan, and glutamic (aspartic) acid, was studied. Based on the observed alterations of HBsAg antigenicity and the known amino acid sequence of the HBsAg polypeptide, a major antigenic determinant was localized within the sequence: Pro-Ser-Cys-Cys-Cys-Thr-Lys-Pro-Thr(Ser)-Asp-Gly-Asn-Cys-Thr-Cys-Ile-Pro-Ile-Pr o-Ser-Ser, corresponding to residues 135-155. The asparagine-linked saccharide chains are not essential for HBsAg antigenicity.

Partially purified preparations of e-antigen, obtained from sera of hepatitis B antigen carriers, were chromatographed on columns of immobilized single- or double-stranded DNA or on pyran-Sepharose. e-Antigen did not adsorb to any of these columns under conditions appropriate for the retention of various nucleic acid polymerases. Therefore, e-antigen and the DNA polymerase associated with Dane particles can be regarded as distinct proteins.

The 55 codons upstream of the gene sequence encoding the hepatitis B surface antigen (HBsAg) are called the pre-S(2) region. It has been proposed that polypeptides of high molecular weight that contain the pre-S(2) region should be included in future hepatitis B virus (HBV) vaccines. The pre-S(2) region and the S gene product [25 kilodalton (kD)] together compose a polypeptide of high molecular weight (33 kD). As an initial attempt to determine the relevance of the 33-kD polypeptide to development of an HBV vaccine, the murine immune response to pre-S(2)-encoded determinants as compared to S-encoded determinants on the same polypeptide was examined. The results indicate (i) the pre-S(2) region is significantly more immunogenic than the S region of HBsAg, (ii) the 26 amino acid residues at the NH2-terminus of the 33-kD polypeptide represent a dominant antibody binding site on the pre-S(2) region, (iii) the immune response to the pre-S(2) region is regulated by H-2-linked genes distinct from those that regulate the response to the S region, and (iv) immunization of an S region nonresponder strain with HBV envelope particles that contain both the pre-S(2) and S regions can circumvent nonresponsiveness to the S region.

A series of omega-aminoalkyl-agarose differing in the number of carbon atoms in the alkyl chains was prepared and used for chromatography of hepatitis B surface antigen (HBSAg). HBSAg was separated from the major part of serum proteins by adsorption to columns of 1,9-diaminononane or 1,10-diaminodecane linked to agarose beads (Sepharose 4B or 2B) followed by elution with 4 M-NaSCN.

Antibodies to the preS2 region of the hepatitis B virus (HBV) envelope protein and to human serum albumin (HSA) were allegedly detected at about the same level in sera of humans with acute or chronic hepatitis B [Hellström et al., 1986]. It was claimed that anti-HSA arises as a result of an immune response to the preS2 sequence and that it was involved in hepatocellular damage. Over 100 sera from animals and humans immunized with HBsAg containing preS2 sequences, or with synthetic peptides from the preS1, preS2, and S regions of the HBV env protein were assayed for anti-HSA. The results revealed the following: 1) Immunization with the native preS2 sequence or with unconjugated synthetic peptides derived from that sequence does not result in elicitation of anti-HSA. Therefore the alleged appearance of anti-HSA during hepatitis B cannot be directly related to an anti-preS2-specific immune response. 2) Some synthetic peptides, whether or not they were derived from the preS2 sequence, when linked to certain carriers, but not to others, elicited in rabbits an anti-HSA response, which was markedly lower than the response to the homologous peptide. These anti-HSA antibodies could be separated from anti-preS2-specific antibodies by affinity chromatography and did not recognize the synthetic peptide used for immunization. The use in active immunoprophylaxis of hepatitis B of unconjugated peptides from the preS2 sequence with proven high immunogenicity will avoid carrier/linker-mediated induction of antibodies not relevant to protection against HBV.

Proteins of the HBV envelope (env) are coded for by two adjacent regions of the HBV env gene: the pre-S and S regions. Antigenic determinants corresponding to amino acid sequences of both regions are recognized by human antibodies and are important in virus-neutralizing responses. Protective immune responses to HBV appear to be linked to the major HLA histocompatibility complex. Inbred and congenic strains of mice represent a model system relevant for studies on the genetic control of immune responsiveness of humans to HBV envelope proteins. Such mouse strains were ranked according to their antibody response to the S protein and divided into high [d,q], intermediate [a,k,b], and low [s] responders (letters in brackets indicate H-2 haplotype.) Selected pre-S antigenic determinants can be mimicked with high fidelity by synthetic peptide analogues that are immunogenic without any carriers. Thus it is possible to study directly the genetic control of immune responsiveness to pre-S epitopes mimicked by these peptides without having to consider the influence of carriers or of S protein. The results presented here show that inbred mouse strains can be ranked according to their antibody responses to the synthetic peptide pre-S(120-145) as follows: A/J[a] approximately equal to SWR/J[q] greater than C57BL/6J[b] approximately equal to AKR/J[k] approximately equal to SJL/J[s] much greater than DBA/2J[d] greater than BALB/cJ[d]. Only SJL/J[s] mice responded well to another synthetic peptide pre-S (12-32). Thus, H-2-linked genes regulating the immune response to S protein and to epitopes on pre-S-coded sequences are distinct. Anti-pre-S(120-145) responses in S protein-nonresponders circumvent this nonresponsiveness. This should be considered in the design of hepatitis B vaccines.

Indirect immunoperoxidase stainings for hepatitis B core and surface antigens (HBc&s Ag) were applied to formalin-fixed paraffin sections in 113 liver specimens from 56 patients. Many cytomorphologic staining characteristics of HBc&s Ag were illustrated, and the percentages of the cellular population positive for HBc&s Ag were estimated for all specimens in order to provide the basis for general analyses. The quantitative expressions and the topographic distribution of HBc&s Ag were assessed with respect to their significance and implication in histopathologic reactions. A definitive relationship or relevance was neither established nor completely excluded due to the size of samples. However, cytomorphologically the membranous expression of HBc&s Ag was shown often in association with acute lobular hepatitis and chronic active hepatitis. This observation supports the concept that the membranous expression may be the prerequisite for immune mediated hepatitic injury in viral hepatitis. In this study we also carried out indirect immunoferritin and immunoperoxidase electron microscopy for HBc&s Ag on formalin-fixed liver specimens. The results assured the validity of the light microscopic immunohistologic procedures. The immunoelectron microscopy confirmed the presence of core antigen in the Dane particles formation that takes place in the cisternae of endoplasmic reticulum. The significance of the cytoplasmic core antigen and the possible role of immunoelectron microscopy in the elucidation of mechanisms of hepatitic injury were discussed.

Radioimmunoassays were developed for antibodies to denaturated forms of HBsAg (reduced and alkylated in an isotonic buffer (RAHBs) or in 8 M urea-HBs) or reduced in 8M urea but not alkylated). These tests revealed that intact HBsAg and RA-HBs shared common antigenic determinants, termed Re[Imai et al, 1974]. Anti-Re is elicited in the course of immune response to intact HBsAg in humans and experimental animals. Denaturation in 8 M urea and alkylation causes the appearance of additional antigenic determinants, the specificity of which is affected by the reagent used for alkylation. Reduction in 8 M urea followed by treatment with iodoacetate reveals antigenic sites common for HBsAg and some human serum proteins. Antibodies specific for the Le Bouvier determinants of HBsAg are probably not the appropriate probe for detection of primary translational products of the hepatitis B virus genome.

An immune response to epitopes localized on the preS region of the hepatitis B virus (HBV) envelope (env) is elicited during recovery from HBV infection and appears to play a role in virus clearance. Anti-preS antibodies (Ab) are expected to be protective against HBV infection as indicated by the virus-neutralizing capacity of Ab to a preS2-specific synthetic peptide preS(120-145). However, there is considerable amino acid variability between preS regions corresponding to distinct serological subtypes of HBV, raising the question whether the preS sequences are sufficiently related immunologically to have the potential of inducing cross-protective immunity. To answer this question concerning the preS2 region, antisera to synthetic peptides preS(120-153) and preS(128-153) corresponding to subtypes adw2 and ayw, as well as to the native env [ayw] protein were raised. Using the resulting polyclonal Ab, an immunological relatedness between preS2 sequences of subtypes adw2 and ayw was demonstrated. On the other hand, Ab selected by affinity chromatography or by cloning hybridoma cells may recognize with strong preference subtype-specific determinants within the preS2 region when the compared antigens are in solution rather than on the solid phase. These findings have implication for the design of: (1) preS2-specific immunogens and (2) immunoassays for quantitation of preS2 sequences in HBV env proteins.

Additional antigenic sites, distinct from those present on spherical 20 nm diam. particles of hepatitis B surface antigen (HBsAg), are exposed on the surface of Dane particles and tubular forms of HBsAg. The immunological relationship of these sites to e-antigen, an antigen detected earlier in HBsAg-positive sera from patients with chronic hepatitis, cirrhosis or acute hepatitis but not in healthy HBsAg-carriers, was established by immune electron microscopy and affinity chromatography. These findings suggest that e-antigen may be potentially useful in active immunization against hepatitis B.

A synthetic peptide corresponding to residues 135-155 (S135-155) of the major protein component of HBsAg was conjugated to beta-galactosidase. This conjugate reacted with monoclonal anti-HBs antibodies having anti-alpha group specificity. The reaction was inhibited by: HBsAg of either subtype ad or ay; by unconjugated S135-155 or a shorter peptide S140-155, but not by unrelated peptides. Modification of lysine residues of either HBsAg or S135-155 reduced this inhibitory effect. These results indicate that Lys 141 is essential for maintaining the antigenicity of one of the epitopes responsible for the common alpha specificity of HBsAg and that studies involving the use of synthetic peptides and modifications of distinct amino acid residues in the native protein or in the peptide may help in characterizing epitopes of viral antigens in general.

As a prelude to the development of nucleic acid probes specific for non-A, non-B hepatitis virus(es) (NANBV), plasmas with alanine aminotransferase levels greater than or equal to 110 IU were assayed for DNA by a radioimmunoassay. Approximately 50% of such plasmas are expected to contain NANBV. One-hundred and seventy-eight of 420 plasma samples tested (42.4%) contained sequestered DNA resistant to DNAse I. The DNA has a molecular weight of approximately equal to 0.8 to 1.4 X 10(6) daltons and hybridizes with a 32P-labeled human DNA probe. The DNA in plasma is mostly bound to IgM. The presence of host DNA will have to be taken into account in planning experiments aiming at the preparation of nucleic acid probes specific for NANBV using infected plasmas as source material. Such experiments will have to utilize recombinant DNA technology and will require the separation of bacterial colonies containing recombinant DNA with viral DNA sequences from colonies with human DNA inserts. The feasibility of this approach is demonstrated using plasma-containing hepatitis B virus.

Human T-cell lymphotropic viruses designated HTLV III or LAV are considered to represent the causative agent(s) of the acquired immunodeficiency syndrome (AIDS). Individuals who have been infected with these viruses may generally be identified on the basis of a positive serological test for antibodies against the protein components of these viruses. Purified viruses or viral proteins have been utilized for developing such tests. Since AIDS may be transmitted by blood transfusion and by blood products, screening of donors for antibodies to HTLV III/LAV has become a necessity. Such screening may be facilitated by the application of assays based on the use of crude virus-infected tissue culture media avoiding elaborate, expensive and potentially hazardous virus purification steps. Serum specimens were mixed with an appropriate dilution of an HTLV III-infected tissue culture-derived fraction, obtained by precipitation with polyethylene glycol 6000 and treatment with Tween 80 and tri-n-butylphosphate (to disrupt virus particles), and incubated with polystyrene beads coated with antibodies to HTLV III/LAV (anti-HTLV III). Subsequently, washed beads were incubated with either 125I- or beta-lactamase-labeled anti-HTLV III. The radioactivity or enzymatic activity associated with the beads was proportionate to the quantity of HTLV III antigen originally added to the beads. The presence of anti-HTLV III in serum specimens resulted in decreased antigen binding and thus in decreased radioactivity or diminished beta-lactamase activity associated with the beads. The test was specific for antibodies to the approximately equal to 24 kDa core protein of HTLV III. The prevalence of these antibodies (given in parentheses) in distinct populations was as follows: random blood donors (0.33%); hemophiliacs (36.4%); random homosexual males (25.1%); homosexual males preselected on the basis of positive markers for infection with hepatitis B virus (50%); and those with persistent lymphadenopathy (70%).

B- and T-cell epitopes from three distinct regions of the hepatitis B virus (HBV) envelope (env) protein (preS1, preS2 and S) are involved in eliciting protective immunity. Since preS1 sequences inhibit the secretion of HBV env proteins from eukaryotic cells, it is difficult to prepare immunogens rich in preS1 sequences. This problem can be overcome by linking synthetic peptides from the preS1 region to particles containing both S and preS2 sequences. We describe here a novel approach for binding of synthetic peptides to exposed hydrophobic domains on HBV env proteins. Long chain fatty acids or mercaptans are covalently linked to synthetic peptides. Peptides with the attached hydrophobic tails interact strongly with HBV env proteins (S + preS2), whereby hybrid immunogens are generated. Such immunogens can be used in combination with alum, the only adjuvant approved for human use. The combination of the preS1 peptide [preS(12-47)] with particles containing the S and preS2 regions resulted in an immunogen which: (1) elicits a broad spectrum of protective antibodies; (2) circumvents the nonresponsiveness to: (a) preS1 epitopes in preS1-nonresponder strains of mice; and (b) S-protein in S-protein-nonresponder strains of mice; and (3) augments the immune response to S-protein. The combination of HBV env proteins with a synthetic peptide from the envelope of the human immunodeficiency virus (HIV-1) resulted in an immunogen eliciting anti-HIV-1. Hybrid immunogens consisting of viral proteins and of synthetic peptides represent a feasible approach for the design of future vaccines.

One of the open reading frames on hepatitis B virus (HBV) DNA comprises the coding region (designated the env gene) for the virus envelope proteins. Studies on messenger RNA transcription suggest that this gene has the potential to code for three related proteins: (1) a protein of 226 amino acids identified as a major protein constituent of the HBV envelope, termed S-protein; (2) a protein with 55 additional amino acids at the N-terminal coded for by a portion of the env gene upstream of the S-gene (pre-S); (3) a protein corresponding to the entire env gene (pre-S + S). Synthetic peptides from the N-terminals of proteins (2) and (3), and antisera to them have been used to study the occurrence and properties of pre-S sequences. The results presented here provide unambiguous evidence that all three env encoded proteins are present in HBV particles; synthetic peptides corresponding to the gene encoding pre-S are highly immunogenic and can be used in diagnostic tests for detection in human sera of antibodies preferentially recognizing HBV; such antibodies, specific for pre-S determinants, are elicited during hepatitis B infection and by immunization with HBV proteins (2) and (3); the hepatitis B vaccine licensed in the United States does not contain pre-S proteins; and the pre-S proteins of the HBV envelope contain domains specifically recognized by liver cells. These findings suggest that pre-S determinants are important in virus-neutralizing responses and should be present in HBV vaccines.

The acquired immunodeficiency syndrome (AIDS) may be transmitted by blood transfusions and by blood products from donors who have been infected with the lymphadenopathy-associated virus (LAV). Such donors may generally be identified on the basis of a positive test for antibodies-against LAV proteins. We have already described an anti-LAV assay based on the use of crude virus-infected tissue culture medium, which avoids elaborate, expensive and potentially hazardous virus purification steps. This test was operationally specific for antibodies to the approximately 24 kD core protein of the virus (P24; Neurath et al. J. Virol. Methods 11, 75, 1985). Molecular exclusion chromatography of crude LAV antigen preparations allows separation of most of P24 from larger proteins of LAV (PL). PL and 125I- or beta-lactamase-labeled anti-LAV were used as reagents for radioimmunoassay (RIA)--or enzyme-linked immunoassay (ELISA)--inhibition tests to detect antibodies directed predominantly against PL (anti-PL). Among 257 individuals belonging to groups at high risk of developing AIDS, 117 (45.5%) were positive for anti-PL and 108 (42%) for anti-P24, respectively. The 2 individuals among 600 random blood donors found to be anti-P24-positive in the preceding study also had anti-PL in their serum. Sera from 500 additional blood donors were screened for anti-PL and 1 of these was positive. The implication of these findings for screening of blood donors is discussed.

In a search for additional antigens associated with virus-induced human liver disease a radioimmunoassay (RIA) was developed using IgG from sera of a multiply transfused person. Polystyrene beads coated with IgG F(ab)'2 fragments, dinitrophenylated F(ab)'2 fragments and 125I-labelled anti-2,4-dinitrophenyl antibodies (Neurath & Strick, 1979) were used in the RIA. An apparently new antigen or the corresponding antibodies were detected in 155 serum specimens from 35/37 (94%)individuals who developed non-A, non-B hepatitis. The antigen was also present in hepatitis B surface antigen-negative sera of blood donors with normal (13.2%) and elevated levels of alanine aminotransferase (34%). The antigen has an approximate mol. wt. of 45,000, a buoyant density of 1.23 g/ml and an isoelectric point of 7.

A solid phase radioimmunoassay (RIA) for human fibroblast-derived beta (beta) interferon is described using dinitrophenylated (DNP) IgG from rabbit anti-interferon serum and quantitation of the antigen-bound antibodies by subsequently measuring the attachment of 125I-labeled anti-DNP. The sensitivity limit of the assay is 50 u of interferon corresponding to 0.5 ng of interferon protein. Preparations of alpha leukocyte-derived interferon show only marginal cross-reactivity (less than 0.01%) while the mouse interferons (mixture of alpha and beta)are negative in this assay. The simplicity, reproducibility and the possibility of using 125I-labeled anti-DNP as a universal reagent for different anti-interferon sera may widen the development of RIA tests for interferons both in research and clinical areas.

The preS region of the hepatitis B virus (HBV) envelope protein has the following properties: (1) exposure on the surface of the virus; (2) high immunogenicity; (3) involvement in the reaction of the virus with cell receptors and (4) elicitation of antibodies protective against infection. Attempts to mimic B- and T-cell epitopes on the native protein by synthetic peptides were highly successful. This success depended on identification of those regions within the preS sequences which are the most important for biological function of the virus and for immunity, and on the synthesis of long peptides (20-40 residues) containing both B- and T-cell epitopes. Results presented here highlight those subregions of the preS sequence which are the most essential for the antigenicity and immunogenicity of HBV.

Amino acid sequences coded for by the preS region of the hepatitis B virus (HBV) envelope gene are present both in HBV and in subviral hepatitis B surface antigen (HBsAg) particles. Consequently, anti-preS-specific antibodies are elicited during the course of HBV infection. Such antibodies are virus-neutralizing. Therefore, it is important to determine whether or not vaccination with HBsAg also induces an anti-preS-specific immune response. We describe here an enzyme-linked immunosorbent assay applicable for the screening of sera from vaccinated individuals for anti-preS antibodies. IgG from serum specimens was adsorbed to staphylococcal Protein A on a superparamagnetic support and subsequently mixed with a synthetic peptide analogue [preS(120-145)] covalently linked to beta-lactamase. The presence of anti-preS in serum specimens resulted in binding of the conjugated beta-lactamase to the magnetic support. The adsorbed enzyme was quantified colorimetrically.

A radioimmunoassay for hepatitis B e-antigen (HBeAg) is described. Polystyrene beads coated with IgG prepared from a human serum containing antibodies to HBeAg (anti-HBe) and anti-HBe IgG labelled with 125I-p-hydroxyphenylpropionic acid N-hydroxysuccinimide ester were used in the test. The radioimmunoassay was approximately 1,000-fold more sensitive than immunodiffusion. At least a transient presence of HBeAg in serum appears to be a common feature of infections by hepatitis B virus. The radioimmunoassay was instrumental in establishing conditions for identification of apparently free monomeric HBeAg. The HBeAg has an approximate mol. wt.of 35,000 and was recovered after isoelectric focusing in fractions with a pH between 4.25 and 4.8. Polyacrylamide gel electrophoresis revealed the presence in HBeAg of a polypeptide with an apparent mol. wt. of 17,000.

Recent data suggest that genes involved in the control of (1) immune responses of humans to HBsAg and (2) the susceptibility to the development of chronic hepatitis B are linked to the major HLA histocompatibility complex. Studies on the genetic regulation of anti-HBs responses and on the possible abrogation of nonresponsiveness to HBsAg in humans are difficult. In an attempt to develop a relevant animal model system, the anti-HBs response of inbred and congenic strains of mice was investigated. A great variation in anti-HBs responses among individual mice belonging to the same strains was observed. Nevertheless, it was possible to rank the inbred mouse strains studied according to their decreasing anti-HBs responses as follows: BALB/c[d] congruent to SWR/J[q] greater than C57BL/6J[b] congruent to DBA/2J[a] greater than AKR/J[k] greater than A/J[a] greater than CBA/CaJ[k] greater than SJL/J[s]. (Letters in brackets indicate H-2 haplotype). Only a small proportion of SJL mice had an anti-HBs response. Therefore, this strain may serve as a model for human nonresponders. Studies with the congenic strains B10.D2[d] and B10.S[s] indicated that genes conferring responsiveness to HBsAg are linked to the H-2 histocompatibility complex. However, genes not linked to H-2 also probably play a role in regulating anti-Hbs responses.

Peptides corresponding to amino acid residues 48-65 and 69-79 of the major polypeptide component of hepatitis B surface antigen (HBsAg) were synthesized and conjugated to protein (bovine serum albumin and keyhole limpet hemocyanin) and fully synthetic (polyglutaraldehyde and cross-linked liposomes) carriers. The peptide-liposome conjugates appeared the most consistent in eliciting antibodies to HBsAg. Results of competition assays between each of the free synthetic peptides and HBsAg for antibodies suggested that the synthetic analogues and the corresponding segments on intact HBsAg are structurally closely related.

Peptides synthesized for potential application as antiviral vaccines have been mostly tested in the form of conjugates with carrier proteins. The possible use of several distinct synthetic vaccines in prophylaxis would be facilitated by the availability of fully synthetic immunogens. A synthetic peptide corresponding to residues 135 to 155 ( P135 -155) of hepatitis B surface antigen (HBsAg) failed to elicit in free form anti-peptide antibodies or anti-HBs. However, polymers of P135 -155 (prepared by linking to diaminoalkanes ) and synthetic conjugates prepared by binding P135 -155 to liposomes or polylysine were immunogenic. A poor correlation was observed between anti-peptide and anti-HBs responses elicited by these conjugates. Glutaraldehyde-fixed liposomes appeared to be the carriers of choice for inducing anti-HBs.

We predicted the localization of a major hepatitis B surface antigen (HBsAg) determinant within residues 135-155 [Neurath, A.R., Strick, N. & Oleszko, W.R. (1981) J. Virol. Methods 3, 115-125]. A peptide corresponding to this sequence (P135-155) was synthesized, linked to macromolecular carriers, and used to immunize rabbits. In accordance with published data on shorter peptides within the same amino acid sequence, antibodies to HBsAg were elicited. A detailed analysis of the immune response to P135-155 revealed the following: A heterogeneous population of IgG and IgM antibodies reacting with P135-155 was detected in the antisera by a variety of radioimmunoassays and enzyme-linked fluorescence immunoassays. Only a subpopulation of these antibodies reacted with HBsAg. The equilibrium constant (K) for the reaction of the antibodies with HBsAg (K = 4 X 10(5) M-1) was approximately two orders of magnitude lower than K for the reaction with P135-155 and was below K for the reaction between HBsAg and antibodies elicited by HBsAg (K greater than 10(7) M-1). Preimmunization with P135-155 did not result in an enhanced response to subsequent immunization with HBsAg. Peptides more accurately mimicking determinants on HBsAg may have to be synthesized for possible application in antiviral prophylaxis.

Pre-S gene coded domains of the hepatitis B virus (HBV) envelope protein are highly immunogenic in experimental animals and humans. Their presence in HBV and hepatitis B surface antigen (HBsAg) particles leads to production of anti-pre-S-specific antibodies during the course of HBV infection. Since antibodies specific for pre-S domains are capable of preventing the attachment of HBV to hepatocytes and are virus neutralizing, it would seem desirable to produce HBV vaccines with a standardized level of pre-S determinants to ensure their potential for eliciting the same repertoire of protective antibodies as found after recovery from natural infection. However, a test with appropriate sensitivity for detecting pre-S determinants to ensure their potential for eliciting the same repertoire of protective antibodies as found after (ELISA) for detecting pre-S determinants in vaccines containing less than or equal to 20 micrograms of HBsAg. The components of this assay are (1) antibodies to a synthetic peptide pre-S (120-145) adsorbed to polystyrene beads, and (2) beta-lactamase-labelled antibodies purified from anti-HBV serum on the basis of their affinity for a pre-S (120-174) beta-galactosidase fusion protein produced in Escherichia coli. Results of an evaluation of the pre-S content of HBV vaccines from two different commercial sources are discussed.

Spherical 20 nm diam. particles of hepatitis B surface antigen (HBsAg), purified from some sera positive for hepatitis B e-antigen (HBeAg), are associated with antigenic determinants reacting with antibodies frequently present in sera containing anti-HBe. Treatment of HBsAg with the detergent Sarcosyl increased the exposure of these determinants which could be differentiated from HBeAg on the basis of immunological specificity and sensitivity to reduction and alkylation. The presence of these determinants appeared to be restricted to a subpopulation of HBsAg associated with IgG and albumin.