The United States of America, As Represented by the Secretary, Department of Health and Human Services

The United States of America As Represented by the Department of Health

The United States of America As Represented by the Secretary of

Other

The United States of America As Represented by the Secretary of Health

Stephen M Feinstone

Robert H Purcell

Marian Elaine Major

Jay A Berzofsky

John R Ticehurst

Ian D Gust

Richard J Daemer

Pablo Sarobe

Suzanne U Emerson

Jeffrey I Cohen

Jonathan E Silver

Pei Zhang

David Baltimore

Mutsunori Shirai

C David Pendleton

Gerardo Kaplan

Czeslaw Wychowski

Vincent R Racaniello

Bahige M Baroudy

Toshitaka Akatsuka

Hongying Duan

A specific epitope on the surface of the hepatitis C virus that induces a neutralizing antibody response in vivo and neutralizing monoclonal antibodies that bind specifically to the epitope are disclosed. The antibodies block hepatitis C virus from infecting cells.

Hepatitis C virus neutralizing antibody

Aspects of the present invention concern compositions that induce and/or improve an immune response to hepatitis C virus (HCV). Methods of making and using compositions that include epitopes of the HCV E2 structural protein involved in promoting or inhibiting neutralization of HCV are provided.

Neutralization of HCV

Provided are an isolated peptide having the amino acid sequence DLMGYIPAV (SEQ ID NO: 1), an isolated HCV core polypeptide comprising an L&#x2192;A substitution at amino acid position 139, an isolated HCV core polypeptide having the amino acid sequence of SEQ ID NO: 2, and a fragment of an HCV core polypeptide having fewer amino acids than the entire HCV core polypeptide and comprising the amino acid sequence of SEQ ID NO:1. Also provided are nucleic acids which encode the peptides and polypeptides of this invention, vectors comprising the nucleic acids of this invention and cells comprising the vectors and nucleic acids of this invention. Further provided are methods of producing an immune response in a subject and/or treating or preventing HCV infection in a subject, comprising administering to the subject, or to a cell of the subject, any of the compositions of this invention.

Modified HCV peptide immunogens

Modified HCV peptide vaccines

The present invention provides 1) an isolated peptide having the amino acid sequence DLMGYIPAV, (SEQ ID NO: 1); 2) an isolated HCV core polypeptide comprising an L&rarr;A substitution at amino acid position 139; 3) an isolated HCV core polypeptide having the amino acid sequence of SEQ ID NO: 2; and 4) a fragment of an HCV core polypeptide having fewer amino acids than the entire HCV core polypeptide and comprising the amino acid sequence SEQ ID NO: 1. Also provided are nucleic acids which encode the peptides and polypeptides of this invention, vectors comprising the nucleic acids of this invention and cells comprising the vectors and nucleic acids of this invention. The present invention further provides methods of producing an immune response in a subject and/or treating or preventing HCV infection in a subject, comprising administering to the subject, or to a cell of the subject, any of the compositions of this invention.

The present invention provides an attenuated hepatitis A virus useful as a vaccine.

Vaccine against hepatitis A virus

The cytotoxic T cell response to the protein encoded by the NS5 region of hepatitis C virus was determined using 28 peptides from NS5 which were selected by an amphipathicity algorithm as candidates for T cell epitopes. In BALB/c mice, a single relatively conserved epitope represented by a 16-residue synthetic peptide was presented by D.sup.d class I major histocompatibility complex (MHC) molecules to conventional CD4.sup.- CD8.sup.+ CTL. An exemplary peptide, which represents amino acid residues 2422-2437 of the polyprotein of the Chiron HCV1 isolate, had the amino acid sequence MSYSWTGALVTPCAAE [SEQ ID NO: 1]. A CTL line specific for this peptide recognized the two known natural variants of this NS5 sequence, each with conservative substitutions. Thus, CTL can recognize the product of the HCV NS5 gene, the probable RNA polymerase, in association with class I MHC molecules on model target cells and may recognize the same epitope on hepatocytes or any other cells infected with the virus.

Identification of peptides that stimulate hepatitis C virus specific

A full-length cDNA copy of an attenuated, cell culture-adapted hepatitis-A virus genome has been constructed. The HAV cDNA when inserted, without the oligo (dG) oligo (dC) tails, into an RNA transcription vector yielded a plasmid designated pHAV/7. Transfection of monkey kidney cells with pHAV/7 DNA yielded HAV. Transfection with RNA transcripts produced in vitro from pHAV/7 yielded about 10-fold more HAV than transfection with pHAV/7 DNA. HAV thus produced are useful as a vaccine.

Chimeric hepatitis A vaccine

Provided is the discovery and isolation of a cellular receptor for hepatitis A virus. Also provided is an isolated nucleic acid molecule comprising a nucleic acid encoding a polypeptide having the biological activity of a hepatitis A virus receptor. Also provided are vectors comprising the isolated nucleic acids encoding a hepatitis A virus receptor in host suitable for expression of the nucleic acids encoding the hepatitis receptor, fragments of the hepatitis A virus receptor, or homologs of the hepatitis A virus receptor. Further provided is a nonhuman transgenic animal which expresses a hepatitis A virus receptor, but does not express an endogenous, active hepatitis A virus receptor. Also provided is a method for screening drugs or vaccines utilizing the transgenic animal expressing a hepatitis A virus receptor. Also provided are methods for use of the purified hepatitis A virus receptor, such as separating hepatitis A virus from blood or other samples, detecting the presence of hepatitis A virus in a sample, determining the anti-hepatitis A virus binding of a compound, preventing hepatitis A virus infection in a subject, or treating a subject infected with hepatitis A virus. Also provided are nucleic acids which selectively hybridize with the nucleic encoding the hepatitis A virus receptor of the present invention.

Recombinant DNA encoding hepatitis A virus receptor

Methods for producing HAV cDNA, products thereof, and uses thereof, are described. HAV cDNA is produced, for example, by reverse transcribing HAV RNA and subsequently inserting the HAV cDNA into bacterial plasmids by genetic-engineering techniques. Transformed bacteria are then cloned and cultured to produce replicated chimetic plasmids containing the HAV cDNA. Such HAV cDNA is useful in assaying for the presence of HAV and in the production of HAV antigen and in the production of antibodies against HAV.

Methods of detecting hepatitis A virus

A method is described whereby new cDNA or RNA sequences can be introduced into or substituted for cDNA in any chosen position without specific sequence requirements using the polymerase chain reaction (PCR). The method entails the use of primers which are complementary to the 3' and 5' ends of the desired sequence to be inserted as well as to the 3' and 5' ends of the chosen site of insertion in the acceptor molecule. The desired sequence is amplified by PCR such that single stranded fragments are produced. The single stranded fragment of the desired sequence is then annealed to a single stranded acceptor molecule at the site of insertion and extended to produce a double stranded molecule. The double stranded molecule is then separated into two strands which are identical except that one of the strands contains the desired sequence inserted at the chosen site. A second double stranded molecule is then generated.

Techniques for producing site-directed mutagenesis of cloned DNA

A modification of the PCR technique is described which allows fragments of RNA or DNA to be amplified without prior knowledge of their sequence. The technique can be used to amplify viral nucleic acids present in small amounts in clinical material allowing, for example, the diagnosis of a particular virus infection or the discovery of new viruses.

Method for amplifying unknown nucleic acid sequences

Human hepatitis A virus (HAV), taken directly from human clinical specimens, can be isolated and serially passaged in primary African green monkey kidney (AGMK) cell cultures. This strain induced antibody to HAV in inoculated chimpanzees and is useful for vaccine.

Isolation of hepatitis A virus strain HM-175

Human hepatitis A virus (HAV) can be purified by a process in which master seed lots of HM-175 strain of hepatitis A virus are prepared from triply cloned virus by terminal dilution at passage level about 20 or preferably at least 20-30. The clones tested induced minimal or no hepatitis although significant antibody response was provoked in inoculated primates. A method of preparing the triply cloned inoculum is also described.

Hepatitis A virus purified and triply cloned

A method of inactivating a lipid virus contained in a dry protein carrier by contacting said virus-containing protein carrier for an abbreviated period of time at from 4.degree.-40.degree. C. with a composition including a halohydrocarbon treating agent and water dissolved in said treating agent. Preferred lipid viruses are Hepatitis B virus (HBV) and non-A, non-B Hepatitis (NANBH) virus.

Growing community of inventors

Washington, DC, United States of America

Stephen M Feinstone