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Title:  Major neutralization site of hepatitis E virus and use of this neutralization site in methods of vaccination and in methods of screening for neutralizing antibodies to hepatitis E virus

United States Patent:  6,930,176

Issued:  August 16, 2005

Inventors:  Schofield; Darren (Wantage, GB); Emerson; Suzanne U. (Kensington, MD); Purcell; Robert H. (Boyds, MD)

Assignee:  The United States of America as represented by the Secretary of the Department (Washington, DC)

Appl. No.:  148737

Filed:  November 30, 2000

PCT Filed:  November 30, 2000

PCT NO:  PCT/US00/32614

371 Date:  January 27, 2003

102(e) Date:  January 27, 2003

PCT PUB.NO.:  WO01/40270

PCT PUB. Date:  June 7, 2001

The invention describes the identification of major neutralization site of hepatitis E virus (HEV) and the use of this neutralization site in methods of vaccination and in methods of screening for neutralizing antibodies to HEV. The invention also describes the isolation and characterization of neutralizing chimpanzee monoclonal antibodies reactive to the neutralization site and the use of these antibodies in the diagnosis, treatment and prevention of HEV.

SUMMARY OF THE INVENTION

The present invention relates to the identification of a neutralization site of hepatitis E virus (HEV) which consists of one or more neutralization epitopes of HEV. The neutralization site is a polypeptide about 30 amino acids in length spanning from amino acids 578 to 607 of the ORF2 gene (capsid gene) of HEV. The neutralization site is conserved among genetically divergent HEV strains.

The invention also relates to the use of the neutralization site or the epitope(s) contained within the neutralization site as an immunogen to elicit the production in mammals of antibodies that can effectively neutralize one or more strains of HEV.

The invention also relates to the use of the neutralization site or the epitope(s) contained within the neutralization site as vaccine to effectively prevent, and/or reduce the incidence of HEV infection. An epitope or antigenic determinant is typically about six amino acid residues.

The invention also relates to pharmaceutical compositions comprising the neutralization site or the epitope(s) contained within the neutralization site.

The invention further relates to methods of producing neutralizing antibodies to HEV comprising administering the pharmaceutical compositions of the invention to a mammal in an amount effective to stimulate the production of neutralizing antibodies to HEV.

The present invention also relates to the isolation and characterization of two neutralizing chimpanzee monoclonal antibodies which are reactive with the neutralization site or the epitope(s) contained within the neutralization site of the invention. These monoclonal antibodies react with genetically divergent HEV strains.

The invention also relates to the heavy and light chain immunoglobulin variable region amino acid sequences of these neutralizing monoclonal antibodies to HEV, and to the nucleic acid molecules encoding the amino acid sequences.

The present invention also relates to the use of the neutralizing monoclonal antibodies of the invention in the detection of HEV infection in animals, especially mammals, and most especially humans.

The neutralizing monoclonal antibodies of the present invention are particularly advantageous for use in the development of prophylactic, therapeutic and diagnostic agents for the prevention and treatment of hepatitis E and detection of human HEV.

The invention therefore also relates to pharmaceutical compositions which comprise the neutralizing antibodies of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a peptide of at least 30 amino acids in length, spanning amino acids 578 to 607 of the open-reading frame 2 gene (capsid gene) of hepatitis E virus (HEV), which has been identified as a neutralization site of the virus.

The results described herein show that the peptide comprising amino acid 112 to amino acid 578 of the open-reading frame 2 gene reacted weakly with the neutralizing antibodies to HEV compared to the peptide comprising amino acid 112 to amino acid 607 which reacted strongly with the neutralizing antibodies to HEV. As the negative result obtained with the peptide comprising amino acid 112 to amino acid 578 may be due to a disruption of a neutralization epitope at the amino terminus of the 578-607 sequence, it is understood that the polypeptide of the invention may extend 5-10 amino acids amino-terminal to amino acid 578 such that it encompasses from about amino acid 572, or 573 to about amino acid 607, more preferably, from about amino acid 568 to about amino acid 607 of the open-reading frame 2 gene.

It is further understood that the neutralization site consists of one or more neutralization epitopes of HEV. The nature and the location of the neutralization epitope(s) within the neutralization site can be determined by deletional or mutational analyses described herein. A neutralization epitope is understood to be composed of at least 6 amino acids, preferably 6 to 8 amino acids.

As the neutralization site is conserved among genetically divergent HEV strains, it is understood that although the neutralization site of the invention was identified as a polypeptide about 30 amino acids in length spanning from amino acids 578 to 607 of the ORF2 gene of HEV strain SAR-55, the invention also encompasses a neutralization site and epitope(s) from corresponding regions of the ORF2 gene of other HEV strains.

It is further understood that substitution of amino acid residue(s) within the neutralization site or neutralization epitope(s) of the invention may result in polypeptides which have similar neutralization properties as the neutralization site or the neutralization epitope(s) set forth above, and which are capable of directing the production of antibodies that are reactive with the neutralization site or epitope(s) of the invention described above. It should be noted that the neutralization site set forth above represents a preferred embodiment of the present invention.

Deletional or mutational studies of the neutralization site of the invention will allow the engineering of broadly reactive neutralization epitopes of HEV. Such studies will also allow the engineering of genotype-specific epitopes of HEV which are useful as diagnostic agents for various genotypes of HEV.

The invention also relates to the use of the neutralization site or the neutralization epitope(s) of the invention as an immunogen to elicit the production in mammals of antibodies that can effectively neutralize one or more strains of HEV.

The term "antibodies" is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. 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, F_d and F(v) as well as chimeric antibody molecules.

In one embodiment, the neutralizing antibodies are produced by immunizing a mammal with a peptide or peptide fragments encoding the neutralization site or the neutralization epitope(s) of the invention. In another embodiment, the neutralizing antibodies are produced by immunizing a mammal with nucleic acids encoding the neutralization site or the neutralization epitope(s) of the invention. In yet another embodiment, the neutralizing antibodies are produced by immunizing a mammal with peptides bridging the ORF2 region of HEV. The antibody molecules may then be collected from the mammal if they are to be used in immunoassays or for providing passive immunity.

The antibody molecules of the present invention may be polyclonal or monoclonal. Monoclonal antibodies may be produced by methods known in the art. Portions of immunoglobulin molecules may also be produced by methods known in the art.

The antibody of the present invention may be contained in various carriers or media, including blood, plasma, serum (e.g., fractionated or unfractionated serum), hybridoma supernatants and the like. Alternatively, the antibody of the present invention is isolated to the extent desired by well known techniques such as, for example, by using DEAE SEPHADEX, or affinity chromatography. The antibodies may be purified so as to obtain specific classes or subclasses of antibody such as IgM, IgG, IgA, IgG₁, IgG₂, IgG₃, IgG₄ and the like. Antibodies of the IgG class are preferred for purposes of passive protection.

In addition to its use in generating neutralizing antibodies to HEV, the neutralization site or the neutralization epitope(s) of the invention can be used as an immunogen to stimulate the production of a protective humoral and/or cellular immune response to HEV.

In one embodiment, the immunogen may be a partially or substantially purified peptide or peptide fragments encoding the neutralization site or the neutralization epitope(s) of the invention. In another embodiment, the immunogen may be a construct in which the peptide or peptide fragments of the invention is incorporated into a live virus vector, for example, a vaccinia virus or adenovirus vector, which may contain neutralization epitopes of other pathogens. In yet another embodiment, the immunogen may be a construct in which the peptide or peptide fragments of the invention is incorporated into proteins such as the hepatitis B surface antigen or the hepatitis B core antigen. In another embodiment, the immunogen may be a construct in which the peptide or peptide fragments of the invention is incorporated into a mosaic protein which contains antibody binding site(s) of other antigens. In another embodiment, the immunogen may be a cell, cell lysate from cells transfected with a recombinant expression vector, or a culture supernatant containing the peptide encoding the neutralization site or the neutralization epitope(s) of the invention. In another embodiment, the immunogen may be a DNA construct encoding the neutralization site or the neutralization epitope(s) of the invention.

While it is possible for the immunogen to be administered in a pure or substantially pure form, it is preferable to present it as a pharmaceutical composition, formulation or preparation. For example, the immunogen can be used in a suitable diluent such as saline or water, or complete or incomplete adjuvants. In a preferred embodiment, the immunogen is coupled to a carrier to make the peptide more immunogenic. Examples of such carrier molecules include but are not limited to bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid, and the like. The immunogen can be administered by any route appropriate for antibody production such as intravenous, intraperitoneal, intramuscular, subcutaneous, and the like.

The effective amount of peptide per unit dose sufficient to induce an immune response depends, among other things, on the species of mammal inoculated, the body weight of the mammal and the chosen inoculation regimen, as well as the presence or absence of an adjuvant, as is well known in the art. Inocula typically contain peptide concentrations of about 1 microgram to about 50 milligrams per inoculation (dose), preferably about 10 micrograms to about 10 milligrams per dose, most preferably about 100 micrograms to about 5 milligrams per dose.

The term "unit dose" as it pertains to the inocula refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of active material (polypeptide) calculated to produce the desired immunogenic effect in association with the required diluent.

The immunogen may be administered once or at periodic intervals until a significant titer of anti-HEV antibody is produced. The antibody may be detected in the serum using an immunoassay.

To monitor the antibody response of individuals administered the compositions of the invention, antibody titers may be determined. In most instances it will be sufficient to assess the antibody titer in serum or plasma obtained from such an individual. Decisions as to whether to administer booster inoculations or to change the amount of the composition administered to the individual may be at least partially based on the titer.

The titer may be based on an immunobinding assay which measures the concentration of antibodies in the serum which bind to the neutralization site or the neutralization epitope(s) contained within the neutralization site of the invention. The ability to neutralize in vitro and in vivo biological effects of the viruses of this invention may also be assessed to determine the effectiveness of the immunization.

Where immunoassays are involved, such kits may contain a solid support, such as a membrane (e.g., nitrocellulose), a bead, sphere, test tube, microtiter well, rod, and so forth, to which a receptor such as an antibody specific for the target molecule will bind. Such kits can also include a second receptor, such as a labeled antibody. Such kits can be used for sandwich assays. Kits for competitive assays are also envisioned.

The invention also relates to the use of the immunogens of the present invention as vaccines for either a prophylactic or therapeutic purpose. When provided prophylactically, a vaccine(s) of the invention is provided in advance of any exposure to any one or more of the HEV strains or in advance of any symptoms due to infection of the viruses. The prophylactic administration of a vaccine(s) of the invention serves to prevent or attenuate any subsequent infection of these viruses in a mammal. When provided therapeutically, a vaccine(s) of the invention is provided at (or shortly after) the onset of infection or at the onset of any symptom of infection or any disease or deleterious effects caused by these viruses. The therapeutic administration of the vaccine(s) serves to attenuate the infection or disease. The vaccine(s) of the present invention may, thus, be provided either prior to the anticipated exposure to the viruses of this invention or after the initiation of infection.

The immunogens of the invention may be supplied in the form of a kit, alone, or in the form of a pharmaceutical composition.

The present invention also relates to neutralizing chimpanzee monoclonal antibodies to HEV, where the antibodies are isolated as Fab fragments from a phage display library prepared from RNA isolated from bone marrow lymphocytes of a chimpanzee experimentally infected with the HEV strain SAR-55, the hepatitis A virus (HAV), the hepatitis B virus (HBV), the hepatitis C virus (HCV), and the hepatitis D virus (HDV).

The present invention thus relates to neutralizing chimpanzee monoclonal antibodies having specified heavy (H) and light (L) chain immunoglobulin variable region amino acid sequences in pairs (H:L) which confer the ability to bind to the neutralization epitope of the invention.

The present invention therefore relates to the heavy chain immunoglobulin variable region amino acid sequences and the light chain immunoglobulin variable region amino acid sequences shown in FIG. 7.

The present invention also relates to nucleic acid molecules encoding the heavy and light chain immunoglobulin variable region amino acid sequences of this invention where these sequences are shown in FIG. 7.

Of course, due to the degeneracy of the genetic code, variations are contemplated in the sequences shown in FIG. 7 which will result in nucleic acid sequences that are capable of directing production of antibodies that are identical to the antibodies of the invention. It should be noted that the DNA sequences set forth above represent a preferred embodiment of the present invention.

The invention further relates to methods of making neutralizing chimpanzee monoclonal antibodies from the phage display library described herein. In a preferred embodiment, the method for isolating a neutralizing monoclonal antibody from the phage display library involves (1) using immunoaffinity techniques such as panning to select phage particles that immunoreact with the neutralizationepitope of the invention; (2) infecting bacteria with the selected phage particles; (3) preparing and analyzing the phagemid DNA from the colonies recovered; and (4) expressing and purifying soluble Fab fragments from clones of interest for further analysis.

The invention also relates to the use of the neutralizing monoclonal antibodies as diagnostic agents.

The antibodies can be used as an in vitro diagnostic agent to test for the presence of HEV in biological samples. In one embodiment, a sample such as biological fluid or tissue obtained from an individual is contacted with a diagnostically effective amount of one or more of the human monoclonal antibodies of this invention under conditions which will allow the formation of an immunological complex between the antibody and the HEV antigen that may be present in the sample. The formation of an immunological complex, which indicates the presence of HEV in the sample, is then detected by immunoassays. Such assays include, but are not limited to, radioimmunoassays, Western blot assay, immunofluorescent assay, enzyme immunoassay, chemiluminescent assay, immunohistochemical assay and the like.

The invention also relates to the use of the monoclonal antibodies of the invention in passive immunoprophylaxis and passive immunotherapy of HEV infection.

When used in passive immunotherapy, the patient is administered a therapeutically effective amount of one or more neutralizing human monoclonal antibodies. The passive immunotherapy of this invention may be practiced on individuals infected with HEV; passive immunoprophylaxis may be practiced on individuals at risk of HEV infection.

A prophylactically or therapeutically effective amount of a monoclonal antibody for individual patients may be determined by titrating the amount of antibody given to the individual to arrive at the therapeutic or prophylactic effect while minimizing side effects. The effective amount can be measured by serological decreases in the amount of HEV antigens in the individual. The plasma concentration for individuals receiving the treatment is typically between 0.1 ug/ml to 100 ug/ml.

The monoclonal antibodies of this invention may be administered via one of several routes including, but not limited to intravenous, intraperitoneal, intramuscular, subcutaneous, transdermal and the like.

The present invention therefore relates to pharmaceutical compositions comprising at least one antibody of the invention and a pharmaceutically acceptable carrier where such carriers may include physiologically acceptable buffers, for example, saline or phosphate buffered saline.

The present invention further relates to anti-idiotypic antibodies to the monoclonal antibodies of this invention. In one embodiment, an anti-idiotypic antibody can be prepared by immunizing a host animal with a monoclonal antibody of this invention by methods known to those of skill in the art. To eliminate an immunogenic response to the Fc region, antibodies produced by the same species as the host animal can be used or the Fc region of the administered antibodies can be removed. The anti-idiotypic antibodies produced can be used to prepare pharmaceutical compositions rather than using the monoclonal antibodies of this invention.

The present invention includes compositions of the antibodies described above, suitable for parenteral administration including, but not limited to, pharmaceutically acceptable sterile isotonic solutions. Such solutions include, but are not limited to, saline and phosphate buffered saline for intravenous, intramuscular, intraperitoneal, or subcutaneous injection, or direct injection into a joint or other area.

In providing the antibodies of the present invention to a recipient mammal, preferably a human, the dosage of administered antibodies will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history and the like.

In general, it is desirable to provide the recipient with a dosage of antibodies which is in the range of from about 5 mg/kg to about 20 mg/kg body weight of the mammal, although a lower or higher dose may be administered. In general, the antibodies will be administered intravenously (IV) or intramuscularly (IM).
 

Claim 1 of 6 Claims

1. An isolated neutralizing monoclonal antibody that is immunoreactive with hepatitis E virus (HEV), said antibody having heavy (H) chain immunoglobulin variable region amino acid sequence of SEQ ID NO: 1 and light (L) chain immunoglobulin variable region amino acid sequence of SEQ ID NO: 2.

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