Plasmids comprising a genetically altered feline...

Details Plasmids comprising a genetically altered feline... Plasmids comprising a genetically altered feline...

1995-06-07

2001-10-09

Myers, Carla J. (Department: 1655)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S320100, C435S235100, C435S236000, C435S440000, C435S445000, C435S471000, C435S475000, C435S476000, C536S023720

Reexamination Certificate

active

06300118

ABSTRACT:

FIELD OF THE INVENTION
The present invention pertains to the prophylaxis and treatment of disease caused by feline immunodeficiency virus (FIV), using genetically altered FIV virions. Specifically, a portion of the p10 gene, which encodes a protein responsible for packaging of the RNA into the virion, has been deleted. The resulting virions are produced in appropriate host cell lines and used to make vaccines comprising whole killed virions which do not comprise viral RNA.
BACKGROUND OF THE INVENTION
Feline immunodeficiency virus (FIV) infection is a significant health problem for domestic cats around the world. As in its human counterpart, infection with FIV causes a progressive disruption in immune function. In the acute phase of infection, the virus causes transient illness associated with symptoms such as lymphadenopathy, pyrexia, and neutropenia. Subsequently, an infected animal enters an asymptomatic phase of 1-2 years before clinical manifestations of immune deficiency become apparent, after which the mean survival time is usually less than one year.
FIV is a typical retrovirus that contains a single-stranded polyadenylated RNA genome, internal structural proteins derived from the gag gene product, and a lipid envelope containing membrane proteins derived from the env gene product (Bendinelli et al.,
Clin.Microbiol.Rev.
8:87, 1995). The gag gene is translated into a primary product of about 50 kDa that is subsequently cleaved by a viral protease into the matrix (p15), capsid (p25), and nucleocapsid (p10) proteins. The start and the end for each cleavage product of the GAG polyprotein are indicated in
FIGS. 2A-2E
underneath the open reading frame. The env gene yields a primary translation product of 75-80 kDa (unglycosylated molecular weight); in infected cells, the precursor has an apparent molecular weight of 145-150 kDa due to N-linked glycosylation. The env precursor is cleaved in the Golgi apparatus into the SU and TM proteins (also designated gp95 and gp40, respectively).
As discussed above, the gag gene of the feline immunodeficiency virus (FIV) is initially translated as a precursor polyprotein which is cleaved to yield the functionally mature matrix protein, capsid protein and nucleocapsid protein making up the core of virus (Elder et al., J. Virol. 67: 1869-76, 1993). The pot gene overlaps the gag gene by 112 nucleotides, and is in a −1 reading frame with respect to that of the gag gene. Thus, the gene is translated as a Gag-Pol fusion protein produced by ribosome frameshifting. The overlapping region contains frameshift signals, GGGAAAC and GGAGAAAC, located at the 3′ end of the gag gene (Morikawa et al., Virol. 186: 389-97, 1992).
The nucleocapsid protein, or p10, is a small basic protein, which is associated with the genomic RNA and may be required for viral RNA packaging (Egberink et al. J. Gen. Virol. 71: 739-743, 1990; Steinman et al., J. Gen. Virol. 71: 701-06, 1990). The p10 protein contains two cysteine arrays each consisting of 14 amino acid residues with the sequence C—X
2
—C—X
4
—H—X
4
—C (where X represents any amino acid and the subscript is the number of residues). Genetic studies with other retroviruses have shown that these two cysteine arrays are essential for viral RNA packaging (Rein et al., J. Virol. 68: 6124-29, 1994; Meric et al., J. Virol. 62: 3328-33; Gorelick et al., Proc. Natl. Acad. Sci. USA 85:8420-24, 1988). Therefore, deletion of these two cysteine arrays should, in theory, generate FIV virus particles which contains all viral proteins, but no viral genomic RNA. These FIV viral particles should be non-infectious and could be used to effect efficacious immune protection in vaccinated cats.
Most vaccines against FIV have failed to induce protective immunity. Ineffective vaccines have involved inactivated whole virus, fixed infected cells, recombinant CA and SU proteins, and a synthetic peptide corresponding to the V3 region of SU. In some cases, the vaccine actually enhanced infection after challenge. In one system, vaccination with paraformaldehyde-fixed virus or infected cells resulted in protective immunity (Yamamoto et al., J. Virol. 67:601, 1993), but application of this approach by others was unsuccessful (Hosie et al., in Abstracts of the International Symposium on Feline Retrovirus Research, 1993, page 50).
Thus, there is a need in the art for an effective whole killed virion vaccine against FIV.
SUMMARY OF THE INVENTION
The present invention pertains to the prevention or lessening of disease in cats caused by Feline Immunodeficiency Virus (FIV). Prevention or lessening of disease is understood to mean the amelioration of any symptoms, including immune system disruptions, that result from FIV infection.
The invention provides for a plasmid which encodes the FIV genome where said genome has had a portion of the gag gene, specifically the p10 (nucleocapsid) coding region, or a portion thereof, deleted. This deletion prevents the production of functional or whole p10 protein, which in turn, prevents the packaging of RNA into virions produced from transfection of this plasmid into an appropriate host cell, resulting in virions which do not contain RNA. Such virions will be described as “empty” virions. The invention also encompasses host cells transformed with the plasmid which produce the empty virions, and the empty virions themselves.
In another embodiment, the invention encompasses vaccines that comprise one or more empty virions described above, with a pharmaceutically acceptable carrier or diluent and a pharmaceutically acceptable adjuvant.
In yet another aspect, the invention provides methods for preventing or lessening disease caused by FIV, which is carried out by administering to a feline in need of such treatment the vaccines described above.


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