DNA-vaccines based on constructs derived from the genomes of human and animal pathogens

Abstract

Methods of eliciting an immune response in a subject by administering one or more large genomic DNA fragments are provided. Also provided are methods of identifying sequences encoding antigenic polypeptides. Also provided are vaccine compositions comprising one or more large genomic DNA fragments.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is related to U.S. provisional application Ser. No. 60 / 163,297, filed 3 Nov. 1999, from which priority is claimed pursuant to 35 U.S.C. §119(e)(1) and which application is incorporated herein by reference in its entirety.TECHNICAL FIELD [0002] The present invention relates generally to vaccine compositions and methods of use thereof. More particularly, the invention pertains to eliciting an immune response in a subject by administering one or more large genomic DNA fragments to the subject. BACKGROUND [0003] Vaccines which induce a cell-mediated immune response are emerging as important strategies in combating parasites, autoimmune disorders, allergic diseases and cancers. Conventional vaccination strategies generally involve administration of either “live” or “dead” vaccines. Ertl et al. (1996) J. Immunol. 156:3579-3582. The so-called live vaccines include attenuated microbes and recombinant molecules based on a living ...

AI Technical Summary

Benefits of technology

[0069] As described above, one advantage of the present invention is that the large genomic fragments include endogenous transcription and translation regulatory elements. Such regulatory control sequences include, for example, promoters (a sequence associated with initiating transcription), enhancers (a cis-acting sequence that enhances transcription) and other elements including those which cause the expression of a coding sequence to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Thus, in a preferred embodiment, the number of molecular modifications to the vectors and / or inserted polynucleotides is minimal.

[0070] It is also possible that selected nucleotide sequences within the vector constructs can be placed under the control of heterologous regulatory sequences such as a heterologous promoter, for example when the genomic fragments are derived from bacteria or other prokaryotes, and expression is desired in a eukaryotic subject. Modification of the sequences encoding the particular protein of interest may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it is attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame. The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site. Administration of Polynucleotides

[0071] The genomic fragments and ancillary substances described herein may be administered by any suitable method. In a preferred embodiment, described below, the polynucleotide fragments are administered by coating a suitable construct (e.g., cosmids or plasmids) containing the fragments onto core carrier particles and then administering the coated particles to the subject or cells. However, the genomic fragments may also be delivered using a viral vector or using non-viral systems, e.g., naked nucleic acid delivery.

[0073] A number of viral based systems have been used for gene delivery. For example, retroviral systems are known and generally employ packaging lines which have an integrated defective provirus (the “helper”) that expresses all of the genes of the virus but cannot package its own genome due to a deletion of the packaging signal, known as the psi sequence. Thus, the cell line produces empty viral shells. Producer lines can be derived from the packaging lines which, in addition to the helper, contain a viral vector which includes sequences required in cis for replication and packaging of the virus, known as the long terminal repeats (LTRs). The genomic fragment(s) of interest can be inserted into the vector and packaged in the viral shells synthesized by the retroviral helper. The recombinant virus can then be isolated and delivered to a subject. (See, e.g., U.S. Pat. No. 5,219,740.) Representative retroviral vectors include but are not limited to vectors such as the LHL, N2, LNSAL, LSHL and LHL2 vectors described in e.g., U.S. Pat. No. 5,219,740, incorporated herein by reference in its entirety, as well as derivatives of these vectors, such as the modified N2 vector described herein. Retroviral vectors can be constructed using techniques well known in the art. See, e.g., U.S. Pat. No. 5,219,740; Mann et al. (1983) Cell 33:153-159.

[0074] Adenovirus based systems have been developed for gene delivery and are suitable for delivering the genomic fragments described herein. Human adenoviruses are double-stranded DNA viruses which enter cells by receptor-mediated endocytosis. These viruses are particularly well suited for genetic transfer because they are easy to grow and manipulate and they exhibit a broad host range in vivo and in vitro. For example, adenoviruses can infect human cells of hematopoietic, lymphoid and myeloid origin. Furthermore, adenoviruses infect quiescent as well as replicating target cells. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis. The virus is easily produced at high titers and is stable so that it can be purified and stored. Even in the replication-competent form, adenoviruses cause only low level morbidity and are not associated with human malignancies. Accordingly, adenovirus vectors have been developed which make use of these advantages. For a description of adenovirus vectors and their uses see, e.g., Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921; Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al. (1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-629; Rich et al. (1993) Human Gene Therapy 4:461-476.

[0075] Adeno-associated viral vectors (AAV) can also be used to administer certain of the smaller genomic fragments (e.g., 5 kb) described herein. AAV vectors can be derived from any AAV serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or in part, preferably the rep and / or cap genes, but retain one or more functional flanking inverted terminal repeat (ITR) sequences. A functional ITR sequence is generally deemed necessary for the rescue, replication and packaging of the AAV virion. Thus, an AAV vector includes at least those sequences required in cis for replication and packaging (e.g., a functional ITR) of the virus. The ITR need not be the wild-type nucleotide sequence, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the sequence provides for functional rescue, replication and packaging.

Problems solved by technology

In contrast, the use of heterologous promoters in previously described expression library immunization would result in non-selective expression of the pathogen's genes.

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