Title:  DNA vaccines against rotavirus infections

United States Patent:  6,165,993

Assignee:  University of Massachusetts Medical Center (Worcester, MA)

Filed:  March 2, 1998

This invention relates to methods of eliciting an immune response and/or protective immunity in a vertebrate by introducing into the vertebrate a DNA vaccine which consists essentially of DNA encoding an antigen or antigens, e.g., capsid proteins or polypeptides, of rotavirus. The uptake of the DNA vaccine by a host vertebrate results in the expression of the capsid protein, thereby eliciting humoral or cell-mediated immune responses, or both, which can provide protection against infection and/or prevent clinically significant rotavirus-caused disease. In addition, the invention demonstrates that an internal viral antigen provides protective immunity in a host. The host can be any vertebrate, including birds, piglets, and humans.

SUMMARY OF THE INVENTION

The invention relates to specific DNA vaccines and methods of providing protective immunity to vertebrates, particularly humans and pigs, against a rotavirus infection. "Protective immunity" conferred by the method of the invention can elicit humoral and/or cell-mediated immune responses to rotavirus infection, but more importantly interferes with the activity, spread, or growth of a rotavirus following a subsequent challenge after vaccination. The DNA vaccines of the invention are transcription units containing DNA encoding a rotavirus polypeptide or protein. In the method of the present invention, a DNA vaccine is administered to an individual in whom protective immunization is desired.

An object of the invention is to provide an immune response and protective immunity to an animal using a DNA vaccine encoding a rotavirus protein as it has the potential of achieving high levels of protection in the virtual absence of side effects. Such DNA vaccines are also stable, easy to administer, and sufficiently cost-effective for widespread distribution.

An object of the invention is provide protective immunity to an inoculated host. If the inoculated host is a female animal, an object of the invention is to provide protection in the offspring of that female.

The invention features a DNA vaccine containing a rotavirus DNA transcription unit (i.e., an isolated nucleotide sequence encoding a rotavirus protein or polypeptide). The nucleotide sequence is operably linked to transcriptional and translational regulatory sequences for expression of the rotavirus polypeptide in a cell of a vertebrate. Preferably the rotavirus polypeptide encoded by the DNA vaccine of the invention is VP4, VP6, and/or VP7. Preferably, the nucleotide sequence encoding the rotavirus polypeptide is contained in a plasmid vector.

The DNA vaccines can be administered to mammals such as pigs or humans susceptible to rotavirus infection and rotavirus-caused disease.

The DNA vaccines of the invention are preferably contained in a physiologically acceptable carrier for in vivo administration to a cell of a vertebrate. Administration of the DNA vaccines of the invention provide an immune response or protective immunity in the vertebrate to disease caused by rotavirus infection. Protective immunity is homologous, homotypic, heterotypic, or heterotypic.

As used herein, the term "homotypic," referring to viral protection or viral challenge, means that the inoculating antigen and the challenge antigen are derived from the same viral serotype.

As used herein, the term "heterotypic," referring to viral protection or viral challenge, means that the inoculating antigen and the challenge antigen are derived from different viral serotypes.

As used herein, the term "homologous," referring to viral protection or viral challenge, means that the inoculating antigen and the challenge antigen are derived from rotaviruses having the same species specificity.

As used herein, the term "heterologous," referring to viral protection or viral challenge, means that the inoculating antigen and the challenge antigen are derived from rotaviruses having different species specificity.

The invention also features a method of providing an immune response and protective immunity to a vertebrate against an infectious rotavirus. The method includes administering to a cell of a vertebrate a DNA transcription unit encoding a desired rotavirus antigen operably linked to a promoter sequence. Expression of the DNA transcription unit in the cell elicits a humoral immune response, a cell-mediated immune response, or both against the infectious rotavirus.

The promoter operably linked to the DNA transcription unit is of nonretroviral or retroviral origin. Preferably the promoter is the cytomegalovirus immediate-early enhancer promoter. The desired rotavirus antigen encoded by the DNA transcription unit is VP4, VP6, and/or VP7.

Protective immunity provided by administration of the DNA transcription unit of the invention is homologous, homotypic, heterotypic, or heterologous. The infectious rotavirus can be of the same strain or the same serotype as the rotavirus from which the DNA encoding a desired antigen is obtained. Alternatively, the infectious rotavirus can be of a different strain, a different serotype, or different species specificity as the rotavirus from which the DNA encoding a desired antigen is obtained.

The method of providing an immune response and protective immunity is practiced on a vertebrate, preferably a mammal such as a pig or other animal. The vertebrate can also be a human susceptible to infection by rotavirus and susceptible to disease caused by rotavirus. The human may be an infant less than 3 years of age, human caring for an infected infant, or an immunocompromised human of any age.

The DNA transcription unit of the method of the invention is preferably contained in a physiologically acceptable carrier and is administered to the vertebrate by routes including, but not limited to, inhalation, intravenous, intramuscular, intraperitoneal, intradermal, and subcutaneous administration. The DNA transcription unit in a physiologically acceptable carrier can also be administered by being contacted with a mucosal surface of the vertebrate.

Preferably, administration is performed by particle bombardment using gold beads coated with the DNA transcription units of the invention. Preferably, the gold beads are 1 .mu.m to 2 .mu.m in diameter. The coated beads are preferably administered intradermally, intramuscularly, by organ transfection, or by other routes useful in particle bombardment and known to those of ordinary skill in the art.

The term "immune response" refers herein to a cytotoxic T cells response or increased serum levels of antibodies to an antigen, or to the presence of neutralizing antibodies to an antigen, such as a rotavirus protein. The term "protection" or "protective immunity" refers herein to the ability of the serum antibodies and cytotoxic T cell response induced during immunization to protect (partially or totally) against disease caused by an infectious agent, such as a rotavirus. That is, a vertebrate immunized by the DNA vaccines of the invention will experience limited growth and spread of an infectious rotavirus.

The term "promoter sequence" herein refers to a minimal sequence sufficient to direct transcription. Also included in the invention is an enhancer sequence which may or may not be contiguous with the promoter sequence. Enhancer sequences influence promoter-dependent gene expression and may be located in the 5' or 3' regions of the native gene. Expression is constitutive or inducible by external signals or agents. optionally, expression is cell-type specific, tissue-specific, or species specific.

By the term "transcriptional and translational regulatory sequences" is meant nucleotide sequences positioned adjacent to a DNA coding sequence which direct transcription or translation of a coding sequence (i.e. facilitate the production of, e.g., VP4, VP6, or VP7 protein). The regulatory nucleotide sequences include any sequences which promote sufficient expression of a desired coding sequence (such as VP4, VP6, or VP7) and presentation of the protein product to the inoculated animal's immune system such that protective immunity is provided.

By the term "operably linked to transcriptional and translational regulatory sequences" is meant that a polypeptide coding sequence and minimal transcriptional and translational controlling sequences are connected in such a way as to permit polypeptide expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). In the present invention, polypeptide expression in a target vertebrate cell is particularly preferred.

The term "isolated DNA" means DNA that is free of the genes and other nucleotide sequences that flank the gene in the naturally-occurring genome of the organism from which the isolated DNA of the invention is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequences.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Claim 1 of 17 Claims

What is claimed is:

1. A method of eliciting an immune response against a rotavirus in a vertebrate, said method comprising administrating to the vertebrate a plasmid vector comprising one or more isolated nucleotide sequences each encoding a rotavirus polypeptide selected from the group consisting of VP4, VP6, and VP7, and transcriptional and translational regulatory sequences operably linked to the isolated nucleotide sequences, whereby expression of said nucleotide sequences in one or more cells in the vertebrate elicits a humoral immune response, a cell-mediated immune response, or both, against the rotavirus.

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