WO2001083535A2 - Polypeptides for use as a vaccine and/or treatment for hiv infection - Google Patents

Abstract

Novel synthetic polypeptides are provided that represent a neutralizing epitope of the HIV envelope glycoprotein gp 120 that is purportedly involved in the binding of this molecule to its cognate cellular receptor CD4. The polypeptides may be used in vaccine formulations as well as in diagnostic immunoassays, immunotherapy and pharmaceutical compositions.

Description

PEPTIDES FOR USE AS A VACCINE AND/OR TREATMENT FOR HIV INFECTION

Government Interests This invention was made in part with the support of U.S. government grant 1R21 AI44395 from the National Institute of Health. Therefore, the United States government may have certain rights in the invention. Field ofthe Invention

The present invention relates to the field of treatment of HIV-l infection and particularly to methods of producing a vaccine for treatment or prophylaxis of HIV-l infection. Background of the Invention

Acquired Immune Deficiency Syndrome (AIDS) is the clinical manifestation ofthe infection of CD4 helper T-cells and other cell targets by the type-1 human immunodeficiency virus (HIV-l). AIDS is characterized by opportunistic infections and certain malignancies. Previous work has demonstrated that HIV infects T lymphocytes of the immune system by attaching its external envelope glycoprotein (gpl20) to the CD4 (T4) molecule on the surface of T lymphocytes and macrophages, thus using the CD4 (T4) molecule as a receptor to enter and infect these cells. After infecting the cell, the virus subverts the ability ofthe cell, and the immune system in general to fend off the virus. Retroviral envelope glycoproteins have been shown to be important in evoking a virus-specific neutralizing antibody response, as determined by the ability of sera containing anti-envelope antibodies to inhibit HIV-l infection in vitro and to protect animals from challenge with the virus in vivo. Specifically, the HIV external envelope glycoprotein gpl20 has been shown to be capable of inducing neutralizing antibodies (Nt-Abs). However, even though it has been well documented that in humans, a strong anti- HIV-l humoral response is mounted within days of initial infection (Pratt et al., J. Infect. Dis., 172:851 (1995)), it is clear that most individuals produce little, if any, broadly-Nt Abs. Both the humoral and cellular arms ofthe immune response are quantitatively and/or qualitatively inadequate in controlling sustained viral replication during the clinical asymptomatic phase of infection, and thus, in preventing the eventual onset of AIDS. Multiple factors are associated with the poor Nt activity seen in HIV-l infected people. First, the co-existence of Nt and infectivity-enhancing Abs may modulate the overall Nt activity; infectivity- enhancing Abs have been observed in HIV-l and other viral infections (Morens, Clin. Infect. Dis., 19:500 (1994)). Although their in vivo relevance to the pathogenesis of AIDS has not been established, infectivity-enhancing Abs can be identified in vitro in a significant proportion of sera (Homsy et al., J. Virol, 64:1437 (1990); Kostrikis et al., J. Virol, 70:445 (1996); Schutten et al, J. Gen. Virol, 78:999 (1997)). Moreover, several of these MAbs have been isolated from HIV-l infected people (Cavacini et al., AIDS Res. Hum. Retrov., 14:1271 (1998)). A second factor affecting the production of broadly-Nt Abs involves the envelope glycoproteins (Envs) in their different structural forms. On infective HIV-l particles, these exist as oligomeric complexes, composed of three interacting, gp41/gpl20 heterodimers (Weiss et al., J. Virol, 64:5674 (1990)). The accessibility of Nt epitopes on this native oligomeric structure is a key determinant for both the immunogenicity and antigenicity ofthe Env subunits. Conserved regions of gpl20 engaged in interactions with gp41 and the formation ofthe oligomer, are buried in the interior ofthe glycoprotein spike, maldng these domains unavailable for Ab binding (Kwong et al., Nature, 393:648 (1998); Wyatt et al., Nature, 393:705 (1998)); thus, this region is referred to as the "non-Nt face" of gpl20. Abs against this region are most likely elicited by non-virally-associated Env molecules (e.g., Envs that have sloughed off virus or infected cells, or cells producing recombinant Envs), and they are able to bind monomeric gpl20 but are not effective neutralizers (Moore and Sodroski, J. Virol, 70:1863 (1996) and references therein). Another identifiable region on the gpl20 molecule is the outer domain, also called the "silent face" (Wyatt et al. Nature, 393:705 (1998)); this region has a particularly dense concentration of N- linked high-mannose sugar residues. The heavy glycosylation of gpl20 seems to play a role in preventing the exposure of otherwise immunogenic or antigenic epitopes, as suggested by the studies of Reitter et al. (Nature Med., 4:679 (1998)) and Cheng-Mayer et al. (J. Virol, 73:5294 (1999)).

Third, transcient structures involved in infection, like the co-receptor binding site on gpl20, which is induced by CD4 binding, may be sites that are excellent for neutralization (LaCasse et al. Science, 283:357 (1998)), but are normally cryptic. Fourth, conserved epitopes on the viral envelope that are involved with broad neutralization, may have evolved to be difficult for the humoral response to recognize, either because only rarely-used Ab genes are involved, or because these conserved sites are highly restricted to a limited array of structural elements. Thus, antigenic sites on the virus are extremely limited, and the few conserved epitopes are very likely weakly-or non-immunogenic or cryptic. A fifth, probably major, element affecting the production of Nt Abs involves immune deviation.

Pan'en and Burton {Nature Med., 3:366 (1997)) recently proposed that most ofthe envelope protein presented to the immune system is not in the viral, oligomeric, membrane-associated form; rather, more prevalent are the non-native forms (e.g., monomeric gpl20). This idea has been challenged by others (Haigwood and Zolla-Pazner, AIDS 12 (Suppl A): S121 (1998)), but if true, it could explain much about the poor Nt activity seen in patients. Even in the presence of strong reactivity against the envelope, most ofthe Ab response is directed against the "virally-irrelevanfenvelope structures (the V3 loop, the silent face) outlined above. The oligomeric, virus-associated, envelope proteins and the free monomeric forms are structurally distinct, and have been shown to possess different immunogenic and antigenic properties (Richardson et al, J. Virol, 70:753 (1996); Beddows et al, J. Virol, 73: 1740 (1999); Gorny et al, Virology, 267:220 (2000)). Supporting this view are two studies that showed that primary-isolate neutralization correlates better with Ab binding to the oligomeric form of gpl20 than it does to the monomeric form (Fouts et al, J. Virol, 71 :2779 (1997); Parren et al, J. Virol, 72:3512 (1998)), and Cavacini et al. (J. Virol, 73:9638 (1999)) showed that the anti-gpl20 response of most patients binds very inefficiently to primary isolates. Thus, the non-viral forms ofthe envelope proteins function as decoy immunogens, deviating the immune response away from an anti-viral Ab production.

Various pharmaceutical treatments for HIV have been developed, however, these treatments have various associated drawbacks when compared to an immunological therapy. The elicitation of HIV-l specific cytotoxic T cells, and possibly Nt-Abs is regarded as a key feature in developing an HIV immunological therapy or prophylaxis.

Attempts at developing an immunological therapy or prophylactic are described for example, in US Patents 5,993,819 (Haynes et al.) 5,763,574 (Conley et al.) and in PCT application number PCT US90/05393 (Kieber-Emmons et al.) the contents of which are incorporated herein by reference. US Patent 5,9763,574 to Conley et al. describes novel synthetic peptides, which can be used as part of an anti-AIDS vaccine. These peptides mimic an epitope on the HIV protein gpl20, which is a binding site of a Nt-Ab. The peptides described therein were selected out of a large random or semi- random array or library, based on their binding affinity to a known neutralizing monoclonal antibody, 19b, which is also known as monoclonal antibody N70 19b. US Patent 5,993,819 to Haynes et al. describes peptides having amino acid sequences corresponding to antigenic determinants ofthe envelope protein of HIV for use in immunogenic preparations and vaccines. Such peptides include those corresponding to hydrophilic, charged regions of the HIV-l gpl20 Env. The peptides disclosed therein can have, for example, the sequence corresponding to amino acids 303-321 of HTLV-JJIB Env g l20. Peptides ofthe invention can also have sequences corresponding to the analogous SP-10 regions of HIV isolates other than HTLV-IH_B.

PCT application number PCT/US90/05393 relates to the identification of peptides and analogs thereof within the CD4 binding region of gpl20 that contain one or more neutralizing epitope(s) involved in the binding of gpl20 to its cellular receptor, CD4. More specifically, the invention is directed to peptides and immunologically equivalent analogs thereof from the gpl20 region from about amino acid 335 to about 517. However, these neutralizing sites on gpl20 are sites for Ab binding and not for T cell recognition.

It is now thought that even though some people who have long-term HIV-l infections, without the emergence of successful therapy, they will convert to frank AIDS. Thus, it is very important to develop a vaccine that produces sterile protection (i.e., prevents the initial infection by HIV-l). To date, three monoclonal (M) Abs have been isolated that neutralize a broad spectrum of HIV-l strains. They are defined by the human monoclonal (M) Abs bl2, 2G12 and 2F5. MAb bl2 binds to a discontinuous epitope that overlaps with the CD4-binding site on gpl20. MAb 2G12 recognizes a complex discontinuous epitope involving the C3-V4 region of gpl20 and carbohydrate (Trolda et al, J. Virol., 70:1100 (1996)). MAb 2F5 binds to a linear epitope on the ectodomain of gp41 (Conley et al, Proc. Natl. Acad. Sci USA, 91:3348 (1994); Muster et al, J. Virol, 69:6678 (1995); Trokla et al, AIDS, 10:587 (1996)); however, the simplicity ofthe this epitope is deceptive, since immunizations with recombinant influenza virus (Muster et al, J. Virol, 69:6678 (1995)) or fusion proteins bearing this epitope (Ecldiart et al, J. General Virol., 77:2001 (1996); Liang et al. Vaccine, 17:2862 (1999)) have failed to produce significant 2F5-like neutralizing Ab responses, indicating that the native epitope on gp41 is more complex than the 6-residue linear sequence. MAbs bl2, 2G12 and 2F5 have shown in vitro neutralizing activity against a wide variety of primary isolates (Burton et al. Science, 266:1024 (1994); Conley et al, Proc. Natl. Acad. Sci USA, 91:3348 (1994); D'Souza et al. J. Infect Dis., 197:1056 (1997); Parren et al, AIDS., 13:S137 (1999); Trolda et al, J. Virol, 69:6609 (1995)). Moreover, passive transfer of bl2, 2F5 and 2G12 can provide sterile protection if adequate concentrations are achieved before HIV-l exposure. Studies with 2F5, 2G12 and HIVIG showed that macaques were protected from intravenous (Mascola et al, J. Virol, 73:4009 (1999)) and vaginal (Mascola et al. Nature Med., 6:207 (2000)) challenge with the pathogenic SHIV ₈₉.₆p_D (Reimann et al, J. Virol. 70:6922 (1996)). Passive immunization with IgGl bl2 protects hu-PBL-SCID mice from HIV-l primary isolate challenge before and shortly after intravenous viral challenge; (Gauduin et al. Nature Med., 3:1389 (1997)), and macaques from vaginal challenge with pathogenic R5 SHIV ι₅₂5p.

The success of these passive immunization studies indicates an obvious goal in the development of prophylactic vaccine: to elicit Abs having neutralizing activities similar to those ofthe currently- known, broadly-neutralizing MAbs (bl2, 2G12 and 2F5). Yet, all recombinant envelope-based vaccine candidates tested so far in clinical trials have been unable to elicit significant, neutralizing responses against HIV-l primary isolates (Mascola et al, J. Infect. Dis. 173:340 (1996); Connor et al, J. Virol, 72: 1552 (1998); McCormack et al. Vaccine, 18: 1166 (2000)), even in cases in which bl2, 2F5 and 2G12 bound well to the immunizing subunit antigen, indicating that their respective epitopes are antigenic on these forms ofthe envelope proteins. Furthermore, these neutralizing epitopes are not recognized to any significant degree during natural infection; instead, as mentioned above, serum Abs having only weak cross-neutralizing titers are typically produced. Out ofthe large number of MAbs cloned from infected donors, bl2, 2G12 and 2F5 are the only ones reported so far that neutralize a broad spectrum of primary HIV-l isolates. Thus, although the epitopes known to mediate broad neutralization are present on recombinant envelope proteins, and on envelope proteins produced during natural infection, they do not elicit significant Nt-Ab responses against primary isolates.

The low apparent immunogenicity of these neutralizing epitopes on the envelope proteins may be circumvented if suitable small molecules mimicking them could be generated (i.e. molecules that bind tightly to the combining sites ofthe neutralizing MAbs), and then presented in such a form that they elicit the cognate Abs.

It is important to appreciate that the Nt epitope of gp 120 is a complex peptide conformation with primary, secondary and tertiary structure where amino acids interact both with each other to maintain the epitope's conformation and with the CD4 receptor to facilitate binding. Elucidation ofthe molecular basis for these structural and functional relationships in the native gpl20 amino acid sequences and their immunological significance remains a considerable scientific challenge. Duplication of these structural and functional relationships of a neutralizing epitope for use as a vaccine is the focus of the present invention. Brief Description of the Invention

The present invention is based on the identification of novel peptides that are specific for the anti- HrV-1 MAb bl2. More specifically, the present invention is based on the identification and characterization of peptides that binds specifically to MAb b 12.

In its broadest embodiment, the present invention is directed to polypeptides capable of eliciting HIV-l Nt-Abs. The peptides include polypeptide monomers having an amino acid consensus sequence:

(X)C(X)₂SDL(X)₃CI (SEQ ID No. 2) or (X)₇SDL(X)₃CI (SEQ TD No. 13) wherein X is any random amino acid residue.

In another embodiment, the consensus sequence of amino acids:

(X)C(X)₂SDL(X)₃CI (SEQ ID No. 2) further comprises a consensus sequence of amino acids: (X)C(X)(X")SDL(X)₃CI (SEQ ID No. 3) wherein X' is usually the hydrophobic amino acid leucine (L), although it may also be methionine (M); and X" is either an aromatic amino acid or serine (S).

In another embodiment, the consensus sequence of amino acids:

(X)₇SDL(X)₃CI (SEQ ID No. 13) further comprises a consensus sequence of amino acids:

(X)₅(X"')(X"")SDL(X)₃CI (SEQ ID No. 14) wherein X'" is usually the hydrophobic amino acid leucine (L), although it may also be methonine (M), threonine (T), valine (V), serine (S) or isoleucine (I), and X"" is an aromatic acid, usually tryptophan (W), although it may also be tyrosine (Y) or phenylalanine (F). One preferred amino acid sequence meeting the consensus sequence of amino acids SEQ ED No. 13 is:

REKRWIFSDLTHTCI (SEQ ID No. 12).

Another preferred amino acid sequence meeting the consensus sequence of amino acids SEQ ID No. 13 is:

HERS YMFSDLENRCI (SEQ ED No. 15) . The present invention is also directed to polynucleotide sequences SEQ ED Nos. 25, 26, 27, 28,

29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, and 43, each capable of expressing the polypeptide sequences ofthe present invention.

The present invention is also directed to a polypeptide di er that comprises first and second polypeptide monomers, each polypeptide monomer having an amino acid consensus sequence SEQ ED No. 13 wherein the amino acid residue X is any random amino acid residue. The preferred first and second polypeptide monomer sequence of one such polypeptide dimer is SEQ ED No. 12. Another preferred first and second polypeptide monomer sequence ofthe polypeptide dimer is SEQ ED No. 15. The amino acid consensus sequences of the polypeptides ofthe present invention comprise: (a) a sequence that overlaps with part of a CD4 binding region of a gp 120 envelope protein of at least one HEV-1 isolate, and preferably multiple HEV-1 isolates; (b) is a region that coincides with the binding site on gpl20 that contacts, and is responsible for the binding energy of MAb bl2; or (c) is an immunological, structural, or functional equivalent of said region or portion thereof. Analogous or homologous am o acid sequences are also within the scope of the present invention.

In other embodiments, the first and second polypeptide monomers ofthe polypeptide dimer are covalently linked together by a disulfide bond and as such the polypeptide dimer is conformationally restricted.

As the polypeptides ofthe present invention represent an immunologically specific neutralizing epitope on the native gpl20, it is preferable that the polypeptides, analogs, homologs and conjugates thereof be "immunologically, structurally, or functionally equivalent". As used herein, "immunologically equivalent" means that the polypeptides ofthe present invention and analogs, homologs, and conjugates thereof elicit the same or similar immune response as the native epιtope(s) on gpl20. "Functionally equivalent" as used herein means that the binding energy of MAb bl2 with the polypeptides ofthe present invention and analogs, homologs, and conjugates thereof is same or similar to the binding energy of MAb bl2 with the native epιtope(s) on gpl20. "Structurally equivalent" as used herein means the polypeptides, analogs, homologs and conjugates thereof should retain or be able to adopt the same three dimensional conformation that contributes to the binding of MAb bl2. Any other derivatives ofthe polypeptides ofthe invention that meet this immunological, structural, or functional criteria are within the scope ofthe present invention.

In one embodiment, the invention contemplates a polypeptide or any combination of polypeptides capable of eliciting HIV-l Nt-Abs, the polypeptide or combination of polypeptides selected from the group consisting of SEQ ED Nos. 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24. The present invention contemplates vaccines for generating HEV-1 Nt-Abs compπsing a polypeptide or any combination of polypeptides selected from the group consisting of SEQ ED Nos. 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24.

The present invention further contemplates vaccines for generating HEV-1 Nt-Abs comprising a segment of a polypeptide dimer, its derivative, or the entirety ofthe polypeptide dimer, said polypeptide dimer comprising the entirety of first and second polypeptide monomers or a segment of said first and second polypeptide monomers, said first and second polypeptide monomers each having a sequence of amino acid sequences SEQ ED No. 12 or SEQ ED No. 15.

Pharmaceutical and immunogenic compositions containing a polypeptide or combinations of polypeptides selected from the group consisting of SEQ ED Nos. 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 are also described. Pharmaceutical and immunogenic compositions containing a polypeptide dimer, said polypeptide dimer comprising first and second polypeptide monomers, said polypeptide monomers each having a sequence of ammo acid sequences SEQ ED No. 12 or 15 are also described. The present invention further includes a method for generating peptide-based vaccines for use as prophylaxis or immunotherapy. One such method requires coating a solid support with an effective amount of a composition including the peptides ofthe invention. Second, a serum sample containing polyclonal anti-gpl20 or anti-peptide antibodies contemplated by this invention is applied to the support, wherein a first plurality ofthe antibodies in the sample complex with the compound. The serum sample may be, for example, HEV positive human sera or sera from a mammalian host immunized with gpl20 or peptides ofthe present mvention. The solid support is then separated from the serum sample, and the first plurality of antibodies are eluted from the solid support. An immunogenic composition including the first plurality of antibodies is then formulated and used to immunize a mammalian host. A second plurality of polyclonal antibodies, which are lmmunogenically reactive with the first plurality of antibodies and immunogenically competitive with gpl20, is selected and purified from the sera ofthe host. Alternatively, a second plurality of monoclonal antibodies is selected by sacrificing the host and generating hybridomas using an antibody organ ofthe host. The second plurality of monoclonal antibodies are characterized as immunogenically reactive with the first plurality of antibodies and immunogenically competitive with gpl20. This second plurality of antibodies are gpl20 surrogates that may be used, for example, in the treatment or prevention of AEDS.

Also contemplated are kits incorporating the peptides of this invention. One such kit is for the preparation of a vaccination comprising a predetermined amount of a vaccine formula having the peptides of this invention. Another such kit is for the detection ofthe presence of HEV neutralizing antibodies directed to the peptides of this invention in a sample suspected of having such antibodies. Brief description of the Drawings

Figure 1: Binding of biotinylated IgGl bl2 to B2.1 phage (B2.1φ) and B2.1 synthetic peptide (B2.1 pep) by ELISA. Competition for IgGl bl2 binding to plate-adsorbed B2.1 phage by "in solution" competitors: 2 x 10¹⁰ B2.1 phage, 300 μM B2.1 synthetic peptide, 100 nM gp 120_Ba-_L (gpl20), f88-4 phage (f88 φ) and G45B unrelated peptide (G45B, sequence is VERSKAFSNCYPYDVPDYASLRS). BSA is bovine serum albumin and n. c. indicates no "in solution" competitor.

Figure 2: SDS-PAGE analysis ofthe f88-4 wild type phage (f88) and recombinant B2.1 phage (all others). Phage were untreated or treated with dithiothereitol (DTT), N-ethylmaleamide (NEM) or NEM followed by DTT, then analyzed by SDS-PAGE. Monomeric (M) and dimeric (D) recombinant pVIII are shown. Similar gels were either silver stained or the proteins were transferred to a membrane and subjected to western blotting with anti-phage Ab or IgGl bl2. Silver-stamed gel (A); western blot using IgGl bl2 to show the reactive dimer (B); western blot using rabbit anti-phage Ab to show the wild type and recombinant pVIII proteins (C).

Figure 3: Titration ofthe Fab bl2 (A), IgGl bl2 (B) and murine anti-B2.1 peptide serum (C) on different lmmoblized antigens. Two-fold dilutions of Fab and IgGl bl2 and four-fold mouse serum dilutions were reacted with biotinylated B2.1 directly adsorbed to ELISA wells (bio-B2.1), biotinylated B2.1 bound to immobilized streptavidin (SA + bio-B2.1), gpl20_Ba-_L,B2.1 recombinant phage, f88-4 phage, bovine serum albumin (BSA), ovalbumin and streptavidin (SA). Figure 4: Kinetics of binding of IgGl bl2 to B2.1 peptide -solution. Percent free Ab versus molar concentration of peptide; data (diamonds) and best-fit theoretical curve (A). Percent error from fit ofthe data 3A to the best-fit curves calculated for a range of Kds (B). The 95% confidence interval calculated for this experiment is 1.3-3.7 μM. Detailed Description of the Invention

A. Definitions

Amino Acid: The ammo acids described herein are preferably m the "L" isomeric form. However, ammo acids in the "D" isomeric form can be substituted for any L-ammo acid, as long as the desired functional property is retained by the polypeptide. NH₂ refers to the free ammo acid group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature , abbreviations for amino acids are shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE

SYMBOL

1 -Letter 3 -Letter AMINO ACID

Y Tyr tyrosme

G Gly glycine

F Phe phenylalanine

M Met methomne

A Ala alan e

S Ser serinine

I He isoleucine

L Leu leucine

T Thr threonine

V Val valine

P Pro proline

K Lys lysine

H His histidine

Q Gin glutamme

E Glu glutamic acid

Z Glx Glu and/or Gin w Trp tryptophan R Arg argmme

D Asp aspartic acid

N Asn asparagine

B Asx Asn and/or Asp

C Cys cysteine

X Xaa Unknown or other

It should be noted that all amino acid sequences represented herein by formulae have a left-to- right orientation in the conventional direction of amino terminus to carboxy terminus. In addition, the phrase "amino acid" is broadly defined to include modified and unusual amino acids. Furthermore, it should be noted that a dash at the beginning or end of an amino acid sequence indicates a peptide bond to a further sequence of one or more amino acids or a covalent bond to an ammo-terminal group such as NH₂ to a carboxy-terminal group such as COOH or CONH₂.

Polynucleotide: Multiple nucleotides linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g. cytosine (C), thymidine (T) or Uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G)). The term "polynucleotide" may refer to both polyribonucleotides and polydeoxyribonucleotides. Polynucleotides can be obtained from existing nucleic acid sources (e.g. genomic or cDNA), but can also be synthetic (e.g. produced by oligonucleotide synthesis) DNA or RNA.

Antibody: The term antibody in its various grammatical forms is used herein to refer to immunoglobulin molecules produced by animals in response to antigen that have the particular property of combining specifically with the immunogen that induced their formation. The term "antibody" also refers to immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the art as Fab, Fab', F(ab')₂ and F(v).

Antibody Combining Site: An antibody combining site is that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable regions that specifically binds (immunoreacts with) an antigen. The term immunoreact in its various forms means specific binding between an antigenic determinant-containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof.

Monoclonal Antibody: A monoclonal antibody in its various grammatical forms refers to a population of antibody molecules that contain only one species of antibody combining site capable of immunoreacting with a particular epitope. A monoclonal antibody thus typically displays a single binding affinity for any epitope with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combimng sites, each immunospecific for a different epitope, e g, a bispecific monoclonal antibody.

Immunogen: A molecule that provokes an immune response.

Antigen: A molecule, or portion thereof, that is recognized by an antibody or T-cell receptor. Conjugate: A reagent that is formed by covalently coupling a protein immunogen and a protein, polypeptide, or non-protemaceous molecule together such as keyhole-limpet hemocyanm and a peptide

Epitope or Antigenic Determinant: Specific region of an antigenic molecule that binds to an antibody or a T cell receptor.

Discontinuous epitope- A discontinuous epitope consists of a group of ammo acids that are not contiguous in the sequence, but are brought together by the folding ofthe polypeptide chain, or by the juxtaposition of two separate peptide chains.

Continuous epitope: A continuous epitope consists of a group of ammo acids that are contiguous in the sequence.

B. Vaccines and Therapeutic Compositions Comprising Peptides ofthe Invention The present mvention relates to peptides corresponding to immunogenic epitopes of HEV-1 for use as a vaccine and/or treatment in a mammal for HEV-1 infection. Such mammals include mice, Xenomouse™, rabbits, non-human primates, and humans. Vaccines can be either active or passive. An active vaccine causes the body to produce Abs and effector T cells (l e, launch an immune response) against an attacking organism, for example, the AEDS virus (HEV-1). Such a vaccine will comprise novel polypeptides of this invention. A passive vaccine will generally consist of pre-produced Abs, such as from mouse, rabbit or monoclonal antibody-producing line (or effector T cells made m a different animal), which can recognize and attack the virus, without the patient's body having to do anything, l e , it does not have to launch an immune response Such a vaccine will comprise pre-produced Abs generated to the novel polypeptides of this invention. Vaccines contemplated in this invention include the novel polypeptides of this invention capable of eliciting HIV-l Nt-Abs mixed in with the vaccine formulation, which can include an adjuvant. The major adjuvants that have been used with subumt vaccines include alum, Ribi incomplete Freund's adjuvant, and MF59 (an oil emulsion). The novel peptides ofthe invention may be conjugated to earner proteins that may enhance the peptide 's lmmunogemcity Such earner proteins may include, but are not limited to tetanus toxoid, keyhole limpet hemocyanm (KLH), bovme serum albumin (BSA), and hepatitis B virus core antigen, and filamentous phage The polypeptides ofthe present invention may also be conjugated to viral fusion proteins or expressed by viral constructs e g , polio virus/polypeptide, canary pox virus/polypeptide or a DNA vaccine Polypeptides comprising specific T-cell epitopes (such as those associated eith HEV-1), designed to elicit a cellular immune response may also be conjugated to the vaccine comprising the carrier protein and the peptides ofthe present invention, or expressed by a virus or DNA that encodes a recombinant vaccine that expresses the peptides ofthe present invention as a fusion protein Anti-idiotype vaccines are an alternative approach to generating a vaccine against particular pathological viruses and are contemplated by this mvention. To generate anti-idiotype vaccines agamst HIV-l, a polypeptide or combination of polypeptides ofthe present invention is injected into an animal in an immunizing amount so as to generate enough Abs to use as a vaccine. The Abs isolated from the animal are capable of recognizing the virus, since the polypeptides ofthe present invention overlap with part ofthe CD4 binding region ofthe gpl20 envelope protein of multiple HIV-l isolates, and are "first generation" Abs (Ab-1). These Ab-1 Abs can then be injected into another animal to produce a second generation of Abs (Ab-2) which recognizes the first generation, and which also mimics a part ofthe virus surface. The effect of this is to produce something which "looks" like the surface ofthe virus, and agamst which an immune response can be raised, but which does not carry any ofthe hazards implicit m using killed or attenuated virus. Thus, the Ab-2 Abs can be used as a vaccine. When injected into a patient, a third generation of Abs (Ab-3) will be generated agamst the Ab-2 Abs. Since the Ab-2 Abs are mimics of the virus antigen, the Ab-3 Abs can neutralize the virus.

The present invention contemplates therapeutic compositions useful for practicing the therapeutic methods described herein. Therapeutic compositions ofthe present mvention contain a physiologically tolerable carrier together with the peptides of this invention, dissolved or dispersed therein as an active ingredient and perhaps a physiologically-tolerable adjuvant.

As used herein, the terms "pharmaceutically acceptable", and grammatical vanations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.

The preparation of a pharmacological composition that contains active ingredients, such as the polypeptides of this invention, dissolved or dispersed therein is well understood in the art. Typically such compositions are prepared as sterile injectables either as liquid solutions or suspensions, aqueous or non- aqueous, however, solid forms suitable for solution, or suspensions, m liquid prior to use can also be prepared. The preparation can also be emulsified.

The polypeptides ofthe present invention can be mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods descnbed herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. En addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness ofthe active ingredient.

The therapeutic composition ofthe present invention can include pharmaceutically acceptable salts ofthe components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups ofthe polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, tnmethylamme, 2-ethylammo ethanol, histidine, proca e and the like.

Physiologically tolerable earners are well known m the art. Exemplary of liquid earners are sterile aqueous solutions that contain no materials addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contam more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, propylene glycol, polyethylene glycol and other solutes

Liquid compositions can also contain liquid phases in addition to and to the exclusion of water Such additional liquid phases are glycenn, vegetable oils such as cottonseed oil, organic esters such as ethyl oleate, and water-oil emulsions.

A therapeutically-effective amount of a peptide of this invention is a predetermined amount calculated to achieve the desired effect, l e , to elicit HEV-1 Nt-Abs m the sample or in the patient, and thereby decrease the amount of detectable HEV-1 in the sample or patient. En the case of in vivo therapies, an effective amount can be measured by improvements m one or more symptoms associated with HEV-mduced disease occurnng in the patient, or by serological decreases in HEV-1 antigens.

Thus, the dosage ranges for the administration of the monoclonal Abs ofthe invention are those large enough to produce the desired effect m which the symptoms ofthe HIV-l disease are ameliorated or the likelihood of infection decreased. The dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, the dosage will vary with the age, condition, sex and extent ofthe disease in the patient and can be determined by one of skill m the art

The polypeptides capable of eliciting HEV Nt-Abs of this invention can be administered parenterally by injection or by gradual infusion over time mucosally l e , as rectal or vaginal suppositories Although the HEV-1 infection is typically systemic and therefore most often treated by intravenous administration of therapeutic compositions, other tissues and delivery means are contemplated where there is a likelihood that the tissue targeted contains infectious HEV-1 Moreover, prophylactic compositions, meant to prevent the individual from contarctmg the infection after exposure to the virus, might best be administered as mucosal preparations, since the virus most often is transmitted by sexual contact Thus, the polypeptides capable of eliciting HEV Nt-Abs of the invention can be administered intravenously, mtrapeπtoneally, intramuscularly, mucosally, subcutaneously, mtracavity, transdermally, and can be delivered by peristaltic means

The therapeutic or prophylactic compositions containing novel polypeptides of this invention are conventionally administered subcutaneously, as by injection of a unit dose, for example The term "unit dose" when used in reference to a therapeutic composition ofthe present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle. The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically-effective amount. The quantity to be administered depends on the subject to be treated, capacity ofthe subject's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgement ofthe practitioner and are peculiar to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.

The invention also relates to a method for preparing a medicament or pharmaceutical composition comprising the polypeptides ofthe present invention, the medicament being used for immunotherapy of HEV-l disease.

C. Diagnostic Assay Methods The present invention contemplates various assay methods for determining the presence, and preferably amount, of an anti-HEV-1 Ab present in a sample, such as a biological fluid or tissue sample from a HEV-l -infected individual, using the polypeptides of this invention as an immunochemical reagent to form an immunoreaction product whose amount relates, either directly or indirectly, to the amount of anti-HEV-1 Ab in the sample. Those skilled in the art will understand that there are numerous well-known, clinical diagnostic chemistry procedures in which an immunochemical reagent of this invention can be used to form an immunoreaction product whose amount relates to the amount of HEV-l Ab present in a body sample. The polypeptides ofthe present invention can be used to detect, in bodily samples (e.g., serum and tissues) the presence of Abs that are functionally, structurally, or immunologically equivalent to the broadly neutralizing human MAb b 12.

The polypeptides ofthe present invention can be bound to many different carriers and used to detect the presence of bl2-like Abs. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature ofthe carrier can be either soluble or insoluble for purposes ofthe invention. Those skilled in the art will know of other suitable carriers for binding polypeptides, or will be able to ascertain such, using routine experimentation.

There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples ofthe types of labels, which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bio- luminescent compounds. Those of ordinary skill in the art will know of other suitable labels for binding to the Abs that bind to the polypeptides ofthe present invention, or will be able to ascertain such, using routine experimentation. Furthermore, the binding of these labels to the polypeptide-binding Abs can be accomplished using standard techniques common to those of ordinary skill in the art. For purposes ofthe invention, HEV-l may be detected by the polyclonal or monoclonal antibodies generated by the novel peptides ofthe invention, when present in samples of biological fluids and tissues. Any sample containing a detectable amount of HEV can be used. A sample can be a liquid such as urine, saliva, cerebrospinal fluid, blood, serum and the like, or a solid or semi-solid such as tissues, feces, and the like, or, alternatively, a solid tissue such as those commonly used in histological diagnosis.

A labeling technique, which may result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific anti-hapten antibodies. In some instances it may be desirable to couple the polypeptides of this invention to a hapten.

En one diagnostic embodiment, the invention contemplates screening HEV-1-infected patients for the presence of circulating anti-HEV-1 Abs immunoreactive with gpl20 that have a similar epitope immunospecificity when compared to a neutralizing Ab generated by the novel polypeptides of this invention. Such a screening method indicates that the HEV-l -infected patient is exhibiting a significant immune response to the virus, and provides useful information regarding disease status and prognosis.

The presence of anti-HEV-1 Abs cross-reactive with a novel polypeptide of this mvention indicates that a patient may have some degree of HEV-l neutralizing activity.

The diagnostic assay involves determining whether a patient has produced human anti-HEV-1 Abs immunoreactive with the same, similar or overlapping epitopes as the novel polypeptides ofthe invention, such that there is a likelihood that there is a useful neutralizing immune response in the patient. There are a variety of immunological assay formats that can be utilized to determine cross-reactivity of test and control polypeptides, and the invention need not be so limiting. Particularly preferred are competition assays for a common antigen, preferably in the solid phase.

Conditions for conducting the competition immunoreaction are well known in the art and can be varied according to recognized parameters in the contacting, the reaction admixtures, the maintenance step, the immunoreaction conditions and the detecting step. For example, the detection step can be conducted by use of a labeled polypeptide of this invention. E. Diagnostic Systems

The present invention also describes a diagnostic system, preferably in kit form, for assaying for the presence of bl2 or bl2-like Abs in a sample according to the diagnostic methods described herein. A diagnostic system includes, in an amount sufficient to perform at least one assay, the novel polypeptides ofthe present invention, as a separately packaged reagent.

In another embodiment, a diagnostic system is contemplated for assaying for the presence of an anti-HEV-1 Ab in a body fluid sample such as for monitoring the fate of therapeutically administered Ab, especially if the Ab is MAb bl2 or is bl2-like by utilizing the novel polypeptides ofthe present invention. The system includes, in an amount sufficient for at least one assay, a subject Ab as a control reagent, and preferably a pre-selected amount of HEV-l antigen comprising the novel polypeptides ofthe present invention, each as separately packaged immunochemical reagents. Instructions, for use ofthe packaged reagent are also typically included. "Instructions for use" typically include a tangible expression descnbmg the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time penods for reagent/sample admixtures, temperature, buffer conditions and the like. In embodiments for detecting potentially-neutralizing (bl2-lιke) antι-HEV-1 Ab a body fluid, a diagnostic system ofthe present invention can include a label or indicating means capable of signaling the formation of an lmmunocomplex containing a novel polypeptide ofthe present invention The word "complex" as used herein refers to the product of a specific binding reaction such as an antibody-antigen reaction Exemplary complexes are immunoreaction products As used herein, the terms "label" and "indicating means" their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any label or indicating means can be linked to or incorporated in an expressed protein, polypeptide, or antibody molecule that is part of an antibody or monoclonal antibody composition ofthe present invention, or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents Such labels are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems

The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fiuorochrome that is a useful lmmunofluorescent tracer. Suitable fluorescent labeling agents are fluorochromes such as fluorescent isocyanate (FIC), fluorescent isothiocyanate (FEEC), 5-dιmethylamme-l-naphthalenesulfonyl chloride (DANSC), tetramethylrhodamme isothiocyanate (TRITC), hssamme, rhodamme 8200 sulphonyl chlonde (RB 200 SC) and the like. Various mmunofluorescence analysis techniques are well known m the art

En preferred embodiments, the indicating group is an enzyme, such as horseradish peroxidase glucose oxidase, or the like In such cases where the principal indicating group is an enzyme such as HRP or glucose oxidase, additional reagents are required to visualize the fact that a receptor-hgand complex (immunoreactant) has formed. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diam obenzidine An additional reagent useful with glucose oxidase is 2,2'-amιno-dι-(3-ethyl-benzthιazolιne-G-sulfonιc acid) (ABTS) Radioactive elements are also useful labeling agents and are used illustratively herein An exemplary radiolabelmg agent is a radioactive element that produces gamma ray emissions Elements which themselves emit gamma rays, such as ^{1 4}1, ¹²⁵1, ^12SI, ¹³²I and ⁵¹Cr represent one class of gamma ray emission-producing radioactive element indicating groups Particularly preferred is ¹²⁵I. Another group of useful labeling means are those elements such as ⁿC, ¹⁸F, ¹⁵0 and ¹³N which themselves emit positrons The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body Also useful is a beta emitter, such ^ιπIndιum of ³H.

The linking of labels, I e , labeling of, polypeptides and proteins is well known in the art. For instance, antibody molecules pioduced by a hybndoma can be labeled by metabolic incorporation of radioisotope-contaimng ammo acids provided as a component in the culture medium. The techniques of protein conjugation or coupling through activated functional groups are particularly applicable. Alternatively, if a labeled polypeptide ofthe present invention, such as SEQ ED No. 15, was desired, it is possible to incorporate such label dunng synthesis ofthe polypeptide. Methods of synthesizing polypeptides are well known the art.

The diagnostic systems can also include, preferably as a separate package, a specific binding agent. A "specific binding agent" is a molecular entity capable of selectively binding a reagent species of the present invention or a complex containing such a species, but is not itself a polypeptide or antibody molecule composition ofthe present invention. Exemplary specific binding agents are second antibody molecules, complement proteins or fragments thereof, S. aureus protein A, and the like. Preferably the specific binding agent binds the reagent species when that species is present as part of a complex.

En preferred embodiments, the specific binding agent is labeled. However, when the diagnostic system includes a specific binding agent that is not labeled, the agent is typically used as an amplifying means or reagent. En these embodiments, the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species-contammg complex.

The diagnostic ldts ofthe present invention can be used in an "ELISA" format to detect the quantity of an antigen or antibody of this invention in a vascular fluid sample such as blood, serum, or plasma. "ELISA" refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen present in a sample. Thus, in some embodiments, a monoclonal or polyclonal antibody generated by the novel polypeptides ofthe present invention or the novel polypeptides themselves can be affixed to a solid matrix to form a solid support that comprises a package m the subject diagnostic systems. A reagent is typically affixed to a solid matrix by adsorption from an aqueous medium although other modes of affixation applicable to proteins and polypeptides well known to those skilled in the art, can be used.

Useful solid matrices are also well known in the art. Such materials are water insoluble and include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N.J.); agarose; beads of polystyrene beads about 1 micron to about 5 millimeters in diameter available from Abbott Laboratories of North Chicago, 111.; polyvmyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microtiter plate such as those made from polystyrene or polyvmylchloride. The reagent species, labeled specific binding agent or amplifying reagent of any diagnostic system descnbed herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form. Where the indicating means is an enzyme, the enzyme's substrate can also be provided m a separate package of a system. A solid support such as the before-descnbed microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.

The packaging materials discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems. The term "package" refers to a solid matrix or material such as glass, plastic (e.g., polyethylene, polypropylene and polycarbonate), paper, foil and the like capable of holding within fixed limits a diagnostic reagent such as a polypeptide ofthe present invention. Thus, for example, a package can be a bottle, vial, plastic and plastic-foil laminated envelope or the like container used to contain a contemplated diagnostic reagent or it can be a microtiter plate well to which microgram quantities of a contemplated diagnostic reagent have been operatively affixed, i.e., linked so as to be capable of being immunologically bound by an antibody or polypeptide to be detected.

The materials for use in the assay ofthe invention are ideally suited for the preparation of a kit. Such a kit may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each ofthe container means comprising one of the separate elements to be used in the method. For example, one ofthe container means may comprise a novel polypeptide ofthe invention, which is, or can be, detectably labelled. The kit may also have containers containing any ofthe other above-recited immunochemical reagents used to practice the diagnostic methods. EXAMPLES 1. Identifying the B2.1 peptide

Biotinylated IgGl bl2 (6, 7) was used to screen a panel of 11 peptide libraries displayed on the major coat protein of filamentous bacteriophage (pVIII), as described in (Bonnycastle et al. J. mol Biol, 258:747 (1996) the entire contents of which is hereby incorporated by reference). Two clones, Edl and Ed2, were identified that bound bl2; DNA sequencing revealed the amino-acid sequences of their displayed peptides, as shown in Table 1. The peptides displayed by these clones share the motif:

SDLX₃CI; however the Edl sequence bears two Cys residues, whereas Ed2 bears only a single Cys whose position is shared in both clones. Thus, a set of two phage sublibraries displaying the shared residues, and reflecting the Cys content ofthe two Ed clones, was constructed following (Bonnycastle et al. J. mol Biol, 258:747 (1996) the entire contents of which is hereby incorporated by reference). The resulting sublibraries bear the random-peptide sequences: XCX₂SDLX₃CI (Bl sublibrary, two fixed Cys) and X₇SDLX₃CI (B2 sublibrary, one fixed Cys), respectively. These sublibraries were screened with biotinylated IgGl bl2, yielding phage bearing the Bl and B2 peptide sequence families shown in Table 1. All but one ofthe selected phage clones bear two Cys residues, and all clones bound IgGl bl2 by direct phage ELISA, performed as described in (Bonnycastle et al. J. mol. Biol, 258:747 (1996) the entire contents of which is hereby incorporated by reference) .

The deduced amino-acid sequences ofthe peptides displayed by the phage clones isolated from the sublibraries revealed a more detailed consensus for both the Bl peptides alone and the B 1 and B2 peptides. Almost all clones selected from the Bl library contain Leu followed by an aromatic amino acid (usually Tyr) N-terminal to the fixed Ser.Asp.Leu sequence. Similarly, clones from the B2 library most often bear a hydrophobic residue (usually Leu) followed by an aromatic one (usually Trp) at this site. Most clones from the B2 sublibrary screening have a second Cys (selected for in the screening). The peptide displayed by only one clone, B2.1, contains a single Cys residue; this peptide sequence shares similarities with those of other clones from the B2 sublibrary, and even more similarity to the Ed2 sequence, in the region N-terminal to the fixed Ser.Asp.Leu sequence. The B2.1 phage was significant in binding more tightly to bl2 than the other clones. ELISA signals for almost all the clones were strong in assays performed with IgGl bl2; whereas binding was much reduced in assays using Fab bl2 when reacted with phage at 4 °C, and still lower when reacted at 37 °C (Table 1).

Fab bl2 bound only the B2.1 and Ed2 peptides with signals above background, and in a side-by-side titration experiment it was further demonstrated that binding of bl2 to B2. 1 significantly stronger than to Ed2 (data not shown), so the Ed2 peptide was not further characterized.

TABLE 1. Sequences and ELISA signals of peptide phage clones affinity-selected by biotynylated IgGl bl2.

OD '405-490

Clone Peptide Sequence* SEQ ED No. IgG bl2 Fab b 12

37°C 4°C 37°C

Edl T C LW SDL RAQ Cl nd nd nd

Bl library X C XX SDL XXX Cl

B1.2 G C LY SDL LAT Cl 1.321 0.013 0.019

Bl.ll N C LY SDL TES Cl 1.236 0.016 0.018

B1.9 N C LY SDL YAR Cl 1.223 0.013 0.015

B1.20 K C MY SDL LGI Cl 1.153 0.012 0.021

B1.10 D C LY SDL ESR Cl 0.818 0.015 0.021

B1.4 S C LY SDL LEL Cl 0.750 nd nd

B1.3 E C MS SDL ELR Cl 10 0.571 nd nd

B1.12 N C LW SDL EQF Cl 11 0.343 nd nd Ed2 REKRWfF SDL THT Cl 12 nd nd nd

B2 library XXXXXXX SDL XXX Cl 13

B2.1 HERSYMF SDL ENR Cl 15 1.236 1.071 0.219

B2.l l CSRNQLW SDL HGS Cl 16 1.207 0.012 0.016

B2.12 NNQGCLW SDL TAS Cl 17 1.189 0.013 0.017

B2.18 STTRCTW SDL YDS Cl 18 1.141 0.011 0.016

B2.8 QSSSCMW SDL FQQ Cl 19 0.992 0.016 0.018

B2.6 AQKQCTW SDL LSR Cl 20 0.903 0.019 0.018

B2.7 RPCRGVY SDL LDK Cl 21 0.886 0.016 0.020

B2.10 SSDHCLW SDL TMT Cl 22 0.644 nd nd

B2.3 LPSSCSW SDL LNR Cl 23 0.276 nd nd

B2.15 HTCAGTW SDL LST Cl 24 0.252 nd nd

gp!20 positive control 1.121 0.863 1.166

f88-4 negative control 0.075 0.014 0.019

# Bold residues indicate the fixed residues in the sublibraries nd indicates the experiment was not done

The cDNA expressing the polypeptides (shown in Table 1) displayed by the phage clones expressing the were subsequently sequenced by methods well known in the art. The nucleic acid sequences for each ofthe peptides shown in Table 1 are listed in Table 2.

Table 2. Nucleic acid sequences of the peptide phage clones affinity-selected by biotynylated

IgGl bl2.

Clone Nucleic Acid Sequence SEQ ED No.

B1.2 GGG TGT TTG TAT TCT GAT CTT CTG GCG ACG TGT ATA 25 Bl.ll AAT TGT CTG TAT TCT GAT CTT ACG CAG TCG TGT ATA 26 B1.9 AAT TGT CTT TAT TCT GAT CTT TAT GCG CGG TGT ATA 27 B1.20 AAG TGT ATG TAT TCT GAT CTT CTG GGG ATT TGT ATA 28 B1.10 GAT TGT CTT TAT TCT GAT CTT GAG TCG CGG TGT ATA 29

B1.4 AGT TGT CTG TAT TCT GAT CTT CTT GAG CTG TGT ATA 30

B1.3 GAG TGT ATG TGG TCT GAT CTT GAG CTT CGG TGT ATA 31

B1.12 AAT TGT CTG TGG TCT GAT CTT GAG CAG TTT TGT ATA 32

B2.1 CAT GAG CGG AGT TAT ATG TTT TCT GAT CTT GAG AAT AGG TGT ATA 33

B2.ll TGT AGT CGG 7AAT CAG CTT TGG TCT GAT CTT CAT GGG AGT TGT ATA 34

B2.12 AAT AAT CAG GGT TGT CTT TGG TCT GAT CTT ACT GCG AGT TGT ATA 35

B2.18 TCG ACT ACT CGG TGT ACT TGG TCT GAT CTT TAT GAT TCT TGT ATA 36

B2.8 CAG TCT TCG TCT TGT ATG TGG TCT GAT CTT TTT CAG CAG TGT ATA 37

B2.6 GCT CAG AAG CAG TGT ACG TGG TCT GAT CTT CTT TCT AGG TGT ATA 38

B2.7 CGT CCT TGT AGG GGT GTG TAT TCT GAT CTT CTG GAT AAG TGT ATA 39

B2.10 AGT TCT GAT CAT TGT CTG TGG TCT GAT CTT ACT ATG ACT TGT ATA 40

B2.3 TTG CCG TCG TCT TGT TCT TGG TCT GAT CTT CTG AAT CGT TGT ATA 41

NB21^* CAT GAA CGT TCT TAT ATG TTT TCT GAC CTG GAA AAC CGT TGC A C 42^~

* NB2.1 was not affinity-selected by biotynylated IgGl bl2 m the initial screening the peptide libraries. Subsequent to the sequencing ofthe cDNA of the B2.1 phage clone, the polynucleotide sequence of B2.1 was optimized for codon usage resulting m the sequence for NB2.1

2. Specificity of the B2.1 peptide for Mab bl2

The ability ofthe B2.1 peptide to bind to the antigen-b d g site of bl2 was assessed in a competition ELISA Biotinylated IgGl bl2 (1 nM) was pre-mcubated either with gpl20_{Ba L} (100 nM) and reacted with plate-adsorbed B2.1 phage. Results in Fig.l show that gpl20 blocked the binding of IgG 1 bl2 to immobilized B2. 1 phage, indicating that the peptide binds to the gpl20 antigen-bmdmg site of IgGl bl2. The binding to B2 1 phage was also blocked by B2 1 synthetic peptide (300 μM), non-biot ylated IgGl (100 nM) and the recombinant B2. 1 phage, but not by f88-4 phage or the unrelated synthetic peptide, G45B

The specificity ofthe B2.1 peptide for bl2 was also assessed. MAb bl2 was originally isolated from a phage-displayed Fab library constructed from the bone marrow of the HEV- 1 -infected donor, M, and subsequently screened with recombinant gpl20 (Burton et al, Proc. Natl Acad. Sci. USA, 88:10134 (1991) the entire contents of which is hereby incorporated by reference). To study the specificity ofthe B2.1 peptide for bl2, we tested whether B2.1 would select phage bearing bl2 out ofthe repertoire of expressed Fabs from donor M. Table 3 shows that yields of 10^"1 % were obtained after four rounds of panning the M phage library on B2.1 phage. Moreover, the Fabs from all twelve independent phage clones that were sequenced from this phage pool were identical to bl2 Thus, even though the M library contains a large number of other Fabs that recognize the CD4-bmdιng site of gpl20 (Barbas et al , /. Mol Biol, 230 812 (1993)), B2.1 selected only phage beanng the Fab bl2 TABLE 3. Percent yields for four successive rounds of affinity selection ofthe phage-displayed Fab library M with B2.1 phage.

Round Immobilized on plate Enput (TU x 10^s) Output (TU x 10⁴) % Yield

B2.1 phage 62 4.8 9.2 x 10^"5 f88-4 phage 62 4.0 7.8 x 10^"5 gp!20 62 9.6 1.8 x lO^"4

B2.1 phage 6.6 2.4 3.6 x lO^'4 f88-4 phage 6.6 1.6 2.4 x lO^"4 gpl20 6.6 140 2.1 x lO^"2

B2.1 phage 2.1 14 6.8 x lo-³ f88-4 phage 2.1 14 6.8 x lo-³ gpl20 2.1 3200 1.5

B2.1 phage 2.7 400 1.5 x lO^'1 f88 — 4 phage 2.7 19 8.8 x lO^'3 gp!20 2.7 110 4.1 x 10^"1

For the panning, 400 ng of pl20sF₂ and 5 x 10¹⁰ recombinant B2.1 or f88-4 phage were immobilized to the plate. Input and output phage are given in ampicillin-resistant transfecting units (TU).

3. Phage Bearing the B2.1 Peptide is a Homodimer

To produce synthetic peptides bearing the B2.1 sequence, we investigated the condition ofthe thiol group ofthe single Cys residue that is present in the B2.1 sequence. As multiple copies ofthe peptide-pVIII fusion protein are incorporated into the phage coat, the single Cys residue of B2.1-pVIII may potentially be in a reduced form (as a reduced thiol group) or disulfide-bridged to a second copy of the B2.1-p VIII fusion protein. If the B2.1 pep tide-p VIII fusion protein existed as a homodimer on the phage surface, it would have roughly twice the molecular weight ofthe pVEtl monomer. Thus, B2.1 phage were analyzed by SDS-PAGE using Tris-Tricine buffer as described (Zwick et al, J. Mol Biol, 300:307 (2000) the entire contents of which is hereby incorporated by reference). Phage samples were initially treated with the thiol-reactive reagent, N-ethylmaleimide (NEM) (Fig. 2 A) which blocks free thiols that might be present on the phage coat, and would prevent the formation of pVHI dimers after solubilization ofthe phage coat proteins with heat and SDS. Hence, if B2.1-p VIII fusions bear free thiols and are monomeric, reaction with NEM should prevent them from dimerizing after dissociation ofthe phage. Alternatively, if the B2.1-ρVIEI fusions exist on the phage coat as dimers (produced by disulfide bridging between displayed B2.1 peptides), treatment ofthe phage with NEM, followed by boiling in the presence of SDS, should not affect their migration as dimers. The results shown in Figure 2A reveal that the recombinant pVIII from B2.I phage migrates as a dimer that is not affected by NEM treatment; whereas it migrated as a monomer in samples treated with the reducing agent dithiothreitol (DTT). Samples sequentially treated with NEM and DTT also behaved as monomers. This proves that most or all ofthe B2.1 peptide displayed on the phage surface is homodimeric.

In contrast to the B2.1 dimers, the clones that display peptides containing two Cys residues produced monomer or, less often, a mixture of monomer and dimer, with monomers predominating (data not shown). This result suggests that, as opposed to B2.1, these clones bear mostly intra-chain disulfide bridges, consistent with the results of Zwick et al (J. Mol. Biol, 300:307 (2000)). Their survey of phage displayed peptides bearing one and two Cys residues showed that almost all containing two Cys residues are cyclic, whereas all of those bearing a single Cys residue form homodimers. 4. The Antigenicity of B2.1 homodimer requires an intact disulfide bridge

The requirement for an intact disulfide bridge for the antigenicity of B2.1, and of clones bearing cyclic peptides, was assessed by western blot experiments (Harlow et al. Antibodies. A laboratory manual. Cold Spring Harbor Laboratory, Newyork (1988) the entire contents of which is hereby incorporated by reference), using IgGl bl2 or rabbit, polyclonal anti-phage Ab for detection. Figure 2B shows that IgG 1 bl2 binds only to the B2.1-pVIII fusion in its dimeric form. Staining with IgGl bl2 was present at the site ofthe dimer, but not at the monomer; whereas both forms were detected by anti- phage Ab (Fig. 2C). A clone selected from the Bl sublibrary, bearing a peptide-pVIEI fusion containing two Cys residues, was also tested by western blot with IgGl bl2. It produced a much weaker band than that ofthe B2.1 phage, whereas blotting with anti-phage Ab produced a recombinant band with similar intensity to the B2.1 clone (data not shown). This supports the conclusions drawn from the ELISA data (Table 1), indicating that IgGl bl2 does not bind as tightly to peptides containing two Cys residues as it does to the B2.1 homodimer. Moreover, as with B2.1 homodimer, reduction by DTT of the intra-chain disulfide bridge of clones containing peptides bearing two Cys residues ablated bl2 binding in ELISA; thus, disulfide-bridging is also required for their antigenicity (data not shown). The location ofthe Cys residue (and hence the disulfide bridge) in the B2.1 sequence is crucial to its reactivity with b 12. Phage bearing mutations in the B2.1 peptide sequence were prepared, and assayed for their ability to bind IgGl bl2 and to produce homodimer and/or monomer bands on analysis by SDS- PAGE. As shown in Table 4, substitution of Cys with Ser ablated dimer formation and Ab binding. Interestingly, substitution of Ser₄ with Cys ablated binding, regardless of whether the a 14 was Cys: even the dimeric form of this mutant peptide did not bind bl2 significantly. Thus, the antigenicity of B2.1 is strongly affected by the presence and location ofthe disulfide bridge that produces homodimers.

Table 4. Binding of bl2 IgG to mutants ofthe B2.1 phage

IgGl bl2* SDS-PAGE f Western Blot #

Phage Clone Peptide sequence 3 nM 30 nM Dimer Monomer bl2 binding

B2.1 HERSYMFSDLENRCI L00 L04 (?) (?j (+) B2.1- Cys HERSYMFSDLENRSI 0.02 0.04 (-) (+) Θ

B2.1-5 'Cys HERCYMFSDLENRSI 0.02 0.05 (+) (+) (-)

B2.1-CC HERCYMFSDLENRCI 0.03 0.13 (-) (+) (-) f88-4 — 0.02 0.03 (-) Θ (-)

No phage — 0.02 0.03 n.a. n.a. n.a.

*Values are OD ₄₀5-₄9o) from direct phage ELISA.

t Wild type and mutant B2.1 phage were subjected to SDS-PAGE in the presence and absence of dithiothreitol (DTT); the "Dimer and "Monomer" columns show the results for non-treated and DTT- treated phage, respectively. The plus sign (+) indicates the detection of recombinant B2.1 -PVIII fusion band on silver-stained gels, the minus sign (-) indicates no band observed.

# A plus sign (+) indicates reactivity with IgGl bl2 in western blot, the minus sign (-) indicates no reactivity. n.a. is not applicable. . Affinity of Fab bl2, Mab bl2, and Murine anti-B2.1 peptide serum on synthetic B2.1 peptide, and gpl20_βa- and B2.1 recombinant phage.

To study the affinity ofthe B2.1 homodimer out ofthe context ofthe phage coat, a synthetic version ofthe B2.1 peptide was prepared as a disulfide-bridged homodimer, with the sequence: NH - HERSYMFSDLENRCEAAEGK-NH₂ (Multiple Peptide Systems, San Diego; monomer MW=2354.6; >95% pure and >95% dimer). This synthetic B2.1 peptide was used as a target to isolate phage bearing bl2 Fab from the M library; but no phage were selected (data not shown), indicating that the synthetic peptide does not bind bl2 as tightly as the phage-borne one. To verify the relatively weak interaction of the synthetic peptide with bl2 compared to phage-borne B2.1, a panning reconstruction experiment was perfonned in which phage bearing Fab bl2 were mixed with various amounts of phage bearing the unrelated Fab AD27/A47 (as background control phage). The Fab phage were panned side-by-side in wells coated with gp 120, B2.1 phage or B2.1 peptide. The results in Table 5 show that gpl20 and B2.1 phage enriched bl2 phage 50-100 fold better than that ofthe synthetic B2.1 peptide. Thus, the affinity of the phage-borne B2.1 for the bl2 Fab appears stronger than that ofthe synthetic peptide.

TABLE 5. Reconstruction panning of Fab bl2 phage (bl2 φ) vs. B2.1 peptide, B2.1 phage (B2.1 φ) and gp 120 Ba-L. Decreasing amounts of Fab bl2 phage were mixed with DP47/AD27 phage (DP47/AD27 φ), to a total of 10¹⁰ particles and screened one single round with the three antigens. Results are expressed as percent yields of ampicillin-resistant transfecting units.

bl2 φ ΪO¹⁵ ΪO⁵ ΪO ΪO⁷ Ϊ0* ΪO³ Ϊ0* —

DP47/AD27 φ - 9X10⁹ 10¹⁰ 10¹⁰ 10¹⁰ 10¹⁰ 10¹⁰ 10¹⁰ B2.1 peptide 2.7 e-3 3 e-3 1.5 e-4 1.6 e-4 4.5 e-4 1.2 e-4 3.4 e-4 1.6 e-4

B2.1 φ 2.1 e-1 1.4 e-1 7.7 e-2 3.1 e-4 5.8 e-5 1.3 e-4 2.7 e-4 2 e-4 gpl20 Ba-L 3.2 e-1 2 e-1 2.8 e-2 1 e-3 2.7 e-4 1.2 e-4 3.1 e-4 4.9 e-4

A biotinylated, synthetic version ofthe B2.1 peptide was also prepared, having the sequence: NH3-.HERSYMFSDLENRCIAAE-Orn(biotιn)-KK-NH₂ (Multiple Peptide Systems, monomer MW = 2767.6, >95% pure and 80% dimer). This peptide (bio-B2.I) was biotinylated so that it could be bound to immobilized streptavidin in ELISA wells, and directly detected during the production of conjugates for immunization, regardless of its IgG 1 bl2 antigenicity. The relative affinity of Mab bl2 for the B2.1 sequence presented in different forms was assessed by direct titrations using Fab and IgGl bl2. The titrations were performed on streptavidin-captured and plate-immobilized bio-B2.1 peptide, as well as with recombinant B2.1 phage (and gpl20 as positive control). Figure 3A shows that the binding of Fab bl2 to both plate-immobilized and streptavidin-captured synthetic B2.1 peptide was almost undetectable over background. In contrast, Fab binding to recombinant B2.1 phage was strong and followed a tifration curve similar to that of gpl20 (Fig. 3A), suggesting that the affinities of bl2 for gpl20 and phage- displayed peptide are similar. (Kds of 3 nM (gp 120_MN) and 9.1 nM (gp 120 _AI) have been reported by Roben et al. (J. Virol, 68:4821 (1994)) and Parren et al. (J. Virol. 72:3512 (1998)), respectively. Although the results were somewhat different when IgGl bl2 was used instead of Fab for the titration ELISA (which was most likely due to the avidity inherent to the IgG), a similar trend was observed (Fig. 3B). The IgGl bl2 reacted with both phage-displayed and synthetic B2.1 peptide; however, it bound more tightly to recombinant B2.1 phage than to either form ofthe synthetic peptide. Moreover, the Ab showed better binding to the plate-adsorbed peptide than to the streptavidin-captured one, thus it was able to discriminate between these two means of presenting the peptide. In contrast to IgGl bl2, the IgG from a mouse who had been immunized with a B2.1 conjugate vaccine showed no discrimination between the streptavidin-bound and plate-adsorbed forms of bιo-B2.1 (Fig. 3C), and binding to gpl20 was undetectable. These results indicate that ability of bl2 to discnminate between plate-immobilized and streptavidin-captured peptide is linked with its capacity to bind gpl20, again, suggesting that a specific B2.1 structure (or set of structures) is responsible for its antigenicity for b 12. It is apparent from these titration experiments that the most antigenic structure of the B2.1 sequence is best represented by the recombinant peptide in the context ofthe phage coat.

To assess the range of affinities ofthe different peptides for bl2, the m-solution binding affinity of IgG 1 bl2 for the B2.1 peptide was determined using a K ExA 3000 (Kinetic Exclusion Assay) instrument (Sapidyne Instruments, Enc, Boise, ED) (Blake et al, Immunochem. Technol. Environ. Applic, 258:747 (1996)), as described m (Craig et al, J. Mol. Biol. 281:183 (1998) the entire contents of which is hereby incorporated by reference). KmExA measurements involving in-solution monovalent antigen yields affinity constants that are independent ofthe Ab valency. The data in Figure 4 show that the interaction between IgGl bl2 and the free peptide closely follow a 2.5-μM Kd best-fit, theoretical curve derived from a simple, second-order kinetic model (Fig. 4A). Comparison ofthe % root-mean square deviation errors (Fig. 4B), produced from the fit of these data to the best-fit curves calculated for a range of Kds, reveal the accuracy ofthe Kd found for bl2 and B2.1 in solution. This Kd is ~200-fold higher than the 9.1-nM Kd measured for the interaction between Fab bl2 and recombinant gpl20 from HEV- I_LAI' as determined by surface plasmon resonance (Roben et al, J. Virol. 68:4821 (1994)). However, the Kd is lower than the —100 μM one found for a synthetic, cyclic peptide made from one ofthe clones isolated from the Bl library, by competition ELISA of that peptide with Fab bl2.

The Fab and IgGl titration data and the in-solution affinities of bl2 for B2.1 and gpl20 may be used to provide very rough reference values from which the affinity ofthe plate-bound and phage- displayed peptides could be interpolated. Given the range of 9 nM for gpl20_LAι and ~3 μM for the free peptide (and assuming that the affinity of free B2.1 is similar to that of bio-B2.1 captured on streptavidin), we speculate that the plate-adsorbed peptide binds with a Kd ranging between 20 nM and 500 nM. The phage-displayed, recombinant peptide shows the highest affinity for binding to Fab bl2, with the data suggesting a Kd value close to that of bl2 for gpl20.

Taken together, our results support the idea that the affinity of B2.1-bl2 interaction is dependent on the environment in which the peptide is presented to bl2. The data suggest the existence of different structures ofthe B2.1 homodimer, and indicate that the predominant structure of B2.1 in solution (and tethered, via biotin, to streptavidin) is either "unfolded" or "unstable", and different from the one(s) that it assumes in the context ofthe phage coat. Our results with synthetic and recombinant B2.1 peptides indicate that the structure ofthe homodimer could be further optimized to maximize its antigenicity. B2.1 binds bl2 preferentially when fused to the pVEH coat protein and displayed on the phage surface, perhaps, because the highly structured phage coat provides a more rigid and or stable environment to the peptide.

The polypeptides ofthe present invention may also be transferred from the filamentous phage coat to the N-terminus of maltose binding protein (MBP) for further characterization as described by Zwick et al (Anal. Biochem., 264:87 (1998), the entire contents of which is incorporated herein by reference). A variety of polypeptide sequences and lengths can be displayed, and the peptide:MBP fusions are amenable to analysis by various methods well known in the art. n this way, phage-derived peptides can be characterized in monomeric form and independent of phage-specifϊc effects.

The foregoing invention has been described in some detail by way of examples for purposes of clarity and understanding. It will be obvious to those skilled in the art form a reading ofthe disclosure that the synthetic peptides ofthe instant invention may differ slightly in amino acid sequence from the sequences ofthe polypeptides described herein without departing from the scope ofthe invention. Indeed, various modifications ofthe invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope ofthe appended claims.

Claims

Claims:

1. A polypeptide dimer capable of eliciting HEV-l neutralizing antibodies, said polypeptide dimer comprising first and second polypeptide monomers, said polypeptide monomers each having a consensus sequence of amino acids:

(X)₇SDL(X)₃CI SEQ ED No. 13 wherein X is any random amino acid residue.

2. The polypeptide dimer according to claim 1 wherein said consensus sequence of amino acids overlaps with part of a CD4 binding region of a gpl20 envelope protein of at least one HEV-l isolate.

3. The polypeptide dimer according to claim 1 wherein said consensus sequence of amino acids is a region that coincides with the binding site on gpl20 that contacts, and is responsible for the binding energy of MAb b 12.

4. The polypeptide dimer according to claim 1 wherein said consensus sequence of amino acids is an immunological, structural, and functional equivalent of a binding site on gpl20 that contacts, and is responsible for the binding energy of MAb bl2.

5. The polypeptide dimer according to claim 1 wherein each of said polypeptide monomers further comprises a consensus sequence of amino acids:

(X)₅(X'")(X"")SDL(X)₃CI SEQ ED No. 14 wherein X'" is M, L, T, V or S and X"" is W, Y, or F.

6. The polypeptide dimer according to claim 5 wherein X'" is L and X"" is W.

7. The polypeptide dimer according to claim 1 wherein each of said polypeptide monomers comprises a segment or the entirety of an amino acid sequence SEQ TD Nos. 12 or 15.

8. The polypeptide dimer according to any one of preceding claims 1-7 wherein said polypeptide dimer is conformationally restricted.

9. The polypeptide dimer according to claim 8 wherein each of said polypeptide monomers is covalently linked by a disulfide bond.

10. A polypeptide monomer capable of eliciting HEV-l neutralizing antibodies, said polypeptide monomer having a consensus sequence of amino acids: (X)₇SDL(X)₃CI SEQ TD No. 13 wherein X is any random amino acid residue.

11. The polypeptide monomer according to claim 10 wherein said consensus sequence of amino acids overlaps with part of a CD4 binding region of a gpl20 envelope protein of at least one HEV-l isolate.

12. The polypeptide monomer according to claim 10 wherein said consensus sequence of amino acids is a region that coincides with the binding site on gpl20 that contacts, and is responsible for the binding energy of MAb bl2.

13. The polypeptide monomer according to claim 10 wherein said consensus sequence of amino acids is an immunological, structural, and functional equivalent of a binding site on gpl20 that contacts, and is responsible for the binding energy of MAb bl2.

14. The polypeptide monomer according to claim 10 wherein each of said polypeptide monomers further comprises a consensus sequence of amino acids:

(X)₅(X'")(X"")SDL(X)₃CI SEQ ED No. 14 wherein X'" is M, L, T, V or S and X"" is W, Y, or F.

15. The polypeptide monomer according to claim 14 wherein X'" is L and X"" is W.

16. The polypeptide monomer according to claim 10 wherein said monomer comprises a segment of a polypeptide chain or the entirety of a polypeptide chain having the amino acid sequence selected from the group consisting of SEQ ED Nos. 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24.

17. The polypeptide monomer according to 16 wherein said polypeptide comprises a segment or the entirety of an amino acid sequence SEQ ED Nos. 12 or 15.

18. The polypeptide monomer according to claim 17 wherein said dimerized polypeptide is conformationally restricted.

19. The polypeptide monomer according to claim 18 wherein said dimerized polypeptide is covalently linked by a disulfide bond.

20. A polypeptide monomer capable of eliciting HEV-l neutralizing antibodies, said polypeptide monomer having a consensus sequence of amino acids:

(X)C(X)₂SDL(X)₃CI SEQ TD No. 2 wherein X is any random amino acid residue.

21. The polypeptide monomer according to claim 20 wherem said consensus sequence of ammo acids overlaps with part of a CD4 binding region of a gpl20 envelope protein of at least one HEV-l isolate.

22. The polypeptide monomer according to claim 20 wherem said consensus sequence of ammo acids is a region that coincides with the bmdmg site on gpl20 that contacts, and is responsible for the binding energy of MAb b 12

23. The polypeptide monomer according to claim 20 wherein said consensus sequence of ammo acids is an immunological, structural, and functional equivalent of a binding site on gpl20 that contacts, and is responsible for the binding energy of MAb bl2.

24. The polypeptide monomer according to claim 20 where each of said polypeptide monomers further comprises a consensus sequence of ammo acids: (X)C(X')(X")SDL(X)₃CI SEQ ED No. 3 wherem X' is L or M and X" is W, Y, or S.

25. The polypeptide monomer according to claim 24 wherein X' is L and X" is W

26. The polypeptide monomer according to claim 20 wherein said polypeptide monomer comprises a segment of a polypeptide chain or the entirety of a polypeptide chain having the ammo acid sequence selected from the group consisting of SEQ ED Nos 1, 4, 5, 6, 7, 8, 9, 10, and 11

27. The polypeptide monomer according to claim 26 wherem said polypeptide monomer is conformationally restricted

28. A vaccine for generating HEV-l neutralizing antibodies comprising a segment of a polypeptide dimer or the entirety of a polypeptide dimer, said polypeptide dimei comprising first and second polypeptide monomers, wherein each of said polypeptide monomers having an am o acid sequence SEQ ED Nos 12 or 15

29. A vaccine for generating HEV-l neutralizing antibodies comprising a segment of a polypeptide monomer or the entirety of a polypeptide monomer having an am o acid sequence selected from the group consisting of SEQ ED Nos. 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24.

30. An immunogenic composition comprising of. a) a polypeptide dimer comprising first and second polypeptide monomers, each of said polypeptide monomers having an ammo acid sequence SEQ ED Nos. 12 or 15; and b) an acceptable adjuvant or carrier protein.

31. The immunogenic composition according to claim 30, wherein said polypeptide monomers are covalently linked by a disulfide bond.

32. The immunogenic composition according to claim 30, wherein said peptide dimer is conformationally restricted.

33. An immunogenic composition comprising of: a) a polypeptide monomer having an amino acid sequence selected from the group consisting of SEQ ED Nos. 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24; and b) an acceptable adjuvant or carrier protein.

34. A pharmaceutical composition in dosage unit form suitable for administration to a mammal for eliciting HEV-l neutralizing antibodies, which comprises an immunologically effective amount of a polypeptide dimer, said polypeptide dimer comprising first and second polypeptide monomers, said polypeptide monomers each having a sequence of amino acids SEQ ED Nos. 12 or 15 in admixture with a suitable pharmaceutically acceptable diluent or carrier.

35. The pharmaceutical composition according to claim 34, wherein said polypeptide monomers are covalently linked by a disulfide bond.

36. The pharmaceutical composition according to claim 34, wherein said polypeptide dimer is conformationally restricted.

37. A pharmaceutical composition in dosage unit form suitable for administration to a mammal for eliciting HEV-l neutralizing antibodies, which comprises an immunogenically effective amount of a polypeptide monomer having the amino acid sequence selected from the group consisting of SEQ ED Nos. 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 in an admixture with a suitable pharmaceutically acceptable diluent or carrier.

38. The use ofthe polypeptide dimer according to any one of claims 1-9 for the manufacture of a medicament for eliciting HEV-l neutralizing antibodies in a mammal.

39. The use of the polypeptide monomer according to claim 16 for the manufacture of a medicament for eliciting HEV-l neutralizing antibodies in a mammal.

40. A kit for the preparation of a vaccination comprising a predetermined amount of a vaccine formula having the polypeptide dimer according to any one of claims 1-9.

41. A kit for the preparation of a vaccination comprising a predetermined amount of a vaccine formula having the polypeptide monomer according to claim 16.

42. A method of manufacturing a vaccine, which comprises incorporating the polypeptide dimer according to any one of claims 1-9 into a vaccine formula.

43. A method of manufacturing a vaccine, which comprises incorporating the polypeptide monomer according to claim 16 into a vaccine formula.

44. The method according to claim 42, further comprising the step of conjugating the polypeptide dimer according to any one of claims 1-9 with a suitable carrier protein.

45. The method according to claim 43, further comprising the step of conjugating the polypeptide monomer according to claim 16 with a suitable carrier protein.

46. A method of vaccinating a mammal comprising administering a vaccine for eliciting HEV-l neutralizing antibodies wherem said vaccine comprises the polypeptide dimer according to any one of claims 1-9.

47. The method according to claim 46 wherein the vaccine is administered in a single step.

48. The method according to claim 47 wherein the vaccine is administered in separate steps.

49. A method of vaccinating a mammal comprising administering a vaccine for eliciting HEV neutralizing antibodies, wherein said vaccine comprises the polypeptide monomer according to claim 16.

50. The method according to claim 49 wherein the vaccine is administered a single step.

51. The method according to claim 49 wherein the vaccine is administered in separate steps.

52. The method according to claim 46 or 49 wherein the mammal is a primate.

53 The method according to claim 52 wherein the primate is a human.

54. The method according to claim 46 or 49 wherein the mammal is a Xenomouse™.

55. A method for generating polyclonal antibodies for use as vaccines or in immunotherapy, comprising the steps of : a) coating a solid support with an effective amount of composition comprising a peptide according to claim 9 or claim 16; b) applying a serum sample containing anti-gpl20 antibodies, wherein a first plurality of said antibodies in said sample complex with said peptide, separating said solid support from said serum sample, and eluting said first plurality of antibodies complexed to said peptide from said solid support; c) immunizing a first mammalian host with an immunogenic formulation comprising said first plurality of antibodies; and d) selecting and purifying a second plurality of polyclonal antibodies from the sera of said first host, wherem said second plurality of polyclonal antibodies are characterized by their immunological reactivity with said first plurality of antibodies and immunological competition with gpl20.

56. A method for generating a monoclonal antibody for use as vaccines or in immunotherapy, comprising the steps of: a) coating a solid support with an effective amount of composition comprising a peptide according to claim 9 or claim 16; b) applying a serum sample containing anti-gpl20 antibodies, wherein a first plurality of said antibodies in said sample complex with said peptide, separating said solid support from said serum sample, and eluting said first plurality of antibodies complexed to said peptide from said solid support; c) immunizing a first mammalian host with an immunogenic formulation comprising said first plurality of antibodies; d) generating a hybridoma from an antibody producing organ of said first host, wherein said hybridoma is capable of producing a monoclonal antibody characterized by its immunological reactivity with said first plurality of antibodies and immunological competition with gpl20; and e) selecting and purifying said monoclonal antibody produced by said hybridoma.

57. A method for generating polyclonal antibodies for use as vaccines or in immunotherapy, comprising the steps of : a) immunizing a first mammalian host with an immunogenic formulation comprising a compound according to claim 9 or claim 16; b) applying a serum sample from said host to a solid support coated with gp 120, wherein a first plurality of said antibodies in said sample complex with said gpl20, separating said solid support from said serum sample, and eluting said first plurality of antibodies complexed to said peptide from said solid support; c) immunizing a second mammalian host with an immunogenic formulation comprising said first plurality of antibodies, and d) selecting and purifying a second plurality of polyclonal antibodies from the sera of said second host, wherein said second plurality of polyclonal antibodies are characterized by their immunological reactivity with said first plurality of antibodies and immunological competition with gpl20.

58. A method for generating a monoclonal antibody for use as vaccines or in immunotherapy, comprising the steps of: a) immunizing a first mammalian host with an immunogenic formulation comprising a peptide according to claim 9 or claim 16; b) applying a serum sample from said host to a solid support coated with gpl20, wherein a first plurality of said antibodies m said sample complex with said g l20, separating said solid support from said serum sample, and eluting said first plurality of antibodies from said solid support; c) immunizing a first mammalian host with an immunogenic formulation comprising said first plurality of antibodies; d) generating a hybridoma from an antibody producing organ of said second host, wherem said hybridoma is capable of producing a monoclonal antibody characterized by its immunological reactivity with said first plurality of antibodies and immunological competition with gpl20; and e) selecting and purifying said monoclonal antibody produced by said hybridoma.

59. Polyclonal antibodies generated according to the method of claim 55 or 57.

60. A monoclonal antibody generated according to the method of claim 56 or 58.

61. A monoclonal antibody that is immunologically reactive with the peptide of claim 9 or claim 16.

62. An immunoassay method for the detection of anti-HEV-1 antibodies comprising: a) coating a solid support with an effective amount of a peptide of claim 9 or claim 16 as an antigen; b) adding a test sera diluted with a buffer, wherein anti-HEV antibodies in the test sera form a complex with said peptide; c) incubating the mixture at room temperature; and d) detecting the presence of said complex.

63. An immunoassay method according to claim 62 wherein step (d) comprises introducing a second known antibody capable of complexing with said anti-HEV-1 antibody, wherein said second antibody is labeled with an enzyme, and adding a substrate which reacts with the enzyme to form a colored product.

64. An immunoassay method according to claim 62 wherein step (d) comprises introducing a second known antibody capable of complexing with said anti-HEV-1 antibody, wherem said second antibody is labeled with a radioactive isotope.

65. An immunoassay method according to claim 62 wherein the complex is detectable by chemiluminescence.

66. An immunoassay method according to claim 62 wherein the complex is detectable by agglutination.

67. An immunoassay test ldt for the detection of anti-HEV- 1 antibodies comprising: a) a solid support; b) an immunoadsorbant comprising a peptide according to claim 9 or 16, coated onto a solid support; and c) reagents for detecting a peptide/anti-HEV-1 antibody complex.

68. A polypeptide dimer capabale of forming a polypeptide/antibody complex with the monoclonal antibody of claim 61 in solution, said polypeptide dimer comprising first and second polypeptide monomers, said polypeptide monomers each having a sequence of amino acids: (X)₇SDL(X)₃CI SEQ ID No. 13 wherein X is any random amino acid residue.

69. A polypeptide dimer capabale of forming a polypeptide/antibody complex with the monoclonal antibody of claim 61 when said polypeptide dimer is attached on a solid support, said polypeptide dimer comprising first and second polypeptide monomers, said polypeptide monomers each having a sequence of amino acids:

(X)₇SDL(X)₃CI SEQ ED No. 13

70. A polypeptide dimer capabale of forming a polypeptide/antibody complex with the monoclonal antibody of claim 61 when said monoclonal antibody is attached on a solid support, said polypeptide dimer comprising first and second polypeptide monomers, said polypeptide monomers each having a sequence of amino acids:

(X)₇SDL(X)₃CI SEQ ED No. 13

71. A polypeptide dimer capabale of forming a polypeptide/antibody complex with a MAb bl2 or with the monoclonal antibody of claim 61 when said polypeptide dimer is expressed on the extracellular surface of a cell, said polypeptide dimer comprising first and second polypeptide monomers, said polypeptide monomers each having a sequence of amino acids: (X)₇SDL(X)₃CI SEQ TD No. 13

72. The polypeptide dimer according to claim 71 wherein said cell is a bacteriophage cell.

73. The polypeptide dimer according to any of claims 68-72 wherein each of said polypeptide monomers further comprises a sequence of amino acids:

(X)₅(X'")(X"")SDL(X₃)CI SEQ TD No. 14 wherein X'" is M, L, T, V, or S and X"" is W, Y, or F.

74. The peptide dimer according to any of claims 68-72 wherein each of said polypeptide monomers comprises a segment or the entirety of an amino acid sequence SEQ ED Nos. 12 or 15.

75. The polypeptide dimer according to claim 73 or claim 74 wherein said dimer is conformationally restricted.

76. The polypeptide dimer acording to claim 73 or claim 74 wherein each of said polypeptide monomers are covalently linked by a disulfide bond.

77. A polypeptide monomer capabale of foroiing a polypeptide/antibody complex with the monoclonal antibody of claim 61 in solution, said polypeptide monomer having a sequence of amino acids:

(X)C(X)₂SDL(X)₃CI SEQ ED No. 2 wherein X is any random amino acid residue.

78. A polypeptide monomer capabale of forming a polypeptide/antibody complex with the monoclonal antibody of claim 61 when said polypeptide monomer is attached on a solid support, said polypeptide monomers each having a sequence of amino acids:

(X)C(X)₂SDL(X)₃CI SEQ ED No. 2

79. A polypeptide monomer capabale of fonning a polypeptide/antibody complex with the monoclonal antibody of claim 61 when said monoclonal antibody is attached on a solid support, said polypeptide monomer each having a sequence of amino acids:

(X)C(X)₂SDL(X)₃CI SEQ TD No. 2

80. A polypeptide monomer capabale of forming a polypeptide/antibody complex with a MAb bl2 or with the monoclonal antibody of claim 61 when said polypeptide monomer is expressed on the extracellular surface of a cell, said polypeptide monomer having a sequence of amino acids:

(X)C(X)₂SDL(X)₃CI SEQ TD No. 2

81. The polypeptide monomer according to claim 71 wherein said cell is a bacteriophage cell.

82. The polypeptide monomer according to any of claims 77-81 wherein said polypeptide monomers further comprises a sequence of amino acids:

(X)C(X')(X")SDL(X)₃CI SEQ TD No. 2 wherein X' is L or M and X" is W, Y, or S.

83. The polypeptide monomer according to any of claims 77-81 wherein said polypeptide monomers comprises a segment or the entirety of an amino acid sequence selected from the group consisting of SEQ

ED Nos. 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24.

84. A polynucleotide sequence capable of expressing a polypeptide monomer, said polypeptide monomer capable of eliciting HEV-l neutralizing antibodies, said polynucleotide sequence selected from the group consisting of SEQ ED Nos. 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, and 43.

85. A polynucleotide sequence capable of expressing a polypeptide monomer, said polypeptide monomer capable of eliciting HEV-l neufralizing antibodies, said polypeptide monomer having an amino acid sequence selected form the group consisting of SEQ ED Nos. 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24.