The technical field of the invention is molecular biology and, in particular, vectors producing non-infectious particles that can be used to induce viral specific immune system responses.
Human immunodeficiency virus (HIV), the virus which causes acquired immune deficiency syndrome (AIDS), is a member of the retrovirus family. In particular, the HIV virus belongs to the lentivirus subfamily of retroviruses. The HIV virus contains two strands of single-stranded genomic ribonucleic acid (RNA) associated with two molecules of reverse transcriptase, an enzyme that catalyzes the process of “reverse transcription” to transcribe genomic RNA into double-stranded DNA. HIV also contains other nucleoid proteins, such as a protease enzyme and an integrase enzyme. The HIV genome, including the nucleoid proteins, is surrounded by a viral coat, known as the nucleocapsid, which consists of two layers of proteins. The HIV genome is further surrounded by an outer envelope coat, which is derived from the membrane of the host-cell.
The single-stranded RNA of the HIV genome encodes three different categories of proteins: the structural proteins encoded by the gag, pol and env genes; the regulatory proteins encoded by the tat and rev genes; and the accessory proteins encoded by the vpu, vpr, vif and nef genes. The HIV genome also has a repeated sequence, known as the long terminal repeat (LTR), at both the 5′ and the 3′ end of the genome. The 5′ LTR contains enhancer and promoter sequences that are necessary for viral transcription, while the 3′ LTR sequence is required for polyadenylating the transcripts that are created from the RNA genome. (Kuby, J., Immunology, 3rd ed. (W. H Freeman and Company (1997)).
Numerous studies have investigated the role of these different proteins, in particular, the role of the structural proteins encoded by the gag and pol genes. To date, attempts have focused on separately mutating proteins such as the reverse transcriptase (RT), integrase (In) and nucleocapsid (NC) in order to characterize and modify their function. (See e.g. Kim, et al., J. Biol. Chem., 271:4872–4878 (1996); Winters et al., J. Virol. 74:10707–10713 (2000); Lins et al., Biophys. J. 76:2999–3011 (1999); Ellison et al., J. Biol. Chem. 270:3320–3326 (1995); Zheng et al., Proc. Natl. Acad. Sci. 93:13659–13664 (1996); Leavitt et al., J. Virol. 70:721–728 (1996); Tanchou et al., J. Virol. 72:4442–4447 (1998); Druillennec et al., J. Biol. Chem. 274:11283–11288 (1999); and Schwartz et al., J. Virol. 71:9295–9305 (1997)).
In addition, attempts have been made to produce non-infectious virus particles, in particular by targeting the nucleocapsid protein. (See Aldovini et al., J. Virol. 64:1920–1926 (1990) and Poon et al., J. Virol. 70:6607–6616 (1996)). In U.S. Pat. No. 5,919,458, Aldovini et al., describes one approach to the construction of non-infectious HIV particles. This approach involves generating nucleotide alterations in the cis-acting RNA packaging site, also known as the Ψ site, and in the cysteine rich carboxy-terminal region of the gag gene, to create HIV mutants that were defective for RNA packaging. Another approach to constructing non-infectious HIV particles is described by Poon et al., J. Virol. 70:6607–6616, (1996). In this study, non-infectious particles were created by altering the nucleocapsid structure to preclude viral RNA incorporation. However, these studies relied on generating deletion mutants of the NC domain in which entire structural domains of the NC were removed in order to prevent recombination to wild-type HIV. As a consequence, the alteration in the protein structure of the HIV virus prevents the virion from acting as a suitable antigen to elicit an immune response. Furthermore, there still remained the concern for reversion to wild-type virus and the generation of infectious particles.
There is also an interest in the development of vaccines to HIV to protect against, or at least retard the progression of, AIDS. The potential efficacy of such vaccines has been suggested by studies in the simian AIDS model systems and in limited human trials. Challenging the patient with either an attenuated or inactivated whole virion confers some immunity. Studies have shown that vaccines composed of whole, inactivated virions of simian immunodeficiency virus (SIV) confer at least partial protection against challenge with live virus. (See eg., Langlois et al., Secience 255:292–293 (1992); Le Grand et al., Nature 355:684 (1992); Osterhaus et al., ibid., pp. 684–685; Cranage et al., ibid., pp. 685–686).
Production of inactivated HIV vaccines involves physical and chemical inactivation treatments necessary to render a non-infectious particle. However, such treatments can result in loss of immunogenicity due to partial destruction of the virions, thereby limiting the effectiveness of the immune response. A method that leaves virion structures intact, yet renders the virions non-infectious, would be a significant improvement in vaccine development.
One approach is the use of a DNA vaccine where the subject is inoculated with DNA molecules carrying a gene that encodes for a defective virion. DNA vaccines have a number of potential advantages over more conventional vaccine formulations. For example, multiple antigens can be expressed from a single DNA construct. In addition, because DNA vaccines express antigens in their native form, both humoral and cellular responses from the immune system are expected to be observed. Of particular importance is the generation of mucosal immunity because the major entry route for these retroviruses is typically through an orifice, which is lined with mucosa.
Accordingly, a need exists for producing a non-infectious virion which retains structural integrity in order to elicit an immune response, while preventing reversion to the wild-type virus. A need also exists for a DNA vaccine comprising a non-infectious virions capable of eliciting appropriate immune response in a subject without the risk of causing infection. A need also exists for DNA vaccines that can be specifically targeted to cells lining the passageways of the body.