Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-03-27
2001-11-27
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069100, C435S235100, C435S455000, C435S456000, C435S366000, C536S023500, C536S023510, C536S023520, C536S024100, C424S093100, C424S093200, C424S093600
Reexamination Certificate
active
06323024
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the fields of molecular biology and virology. More specifically, the present invention relates to a biologically engineered, attenuated Coxsackievirus and its use as a delivery vehicle for nucleic acids encoding antigenic or biologically active proteins.
BACKGROUND OF THE INVENTION
The coxsackieviruses, members of the family Picornaviridae, are divided into two groups, based essentially on their pathogenicity and replication in newborn mice. The Group B coxsackieviruses (CVB) are composed of six serotypes (1-6). Similar to other members of the Picornaviridae, the CVB genome is a single-stranded, messenger sense, polyadenylated RNA molecule (for review see Romero, J. R. et al.,
Current Topics in Microbiology and Immunology
223: 97-152, 1997). Genome analysis of the CVB shows that they are organized into a 5′ nontranslating region, a protein coding region containing a single open reading frame, a 3′ nontranslated region and a terminal poly-A tail, similar to other Picornaviruses. The CVB protein coding region can be further divided into three regions, P1, P2 and P3. P1 encodes the four capsid proteins VP4 (1A), VP2 (1B), VP3 (1C) and VP 1 (1D); P2 and P3 encode the non-structural proteins required for the CVB lifecycle: 2A (protease), 2B, 2C, 3A 3B (Vpg), 3C (protease) and 3D (polymerase) (See Romero et al., 1997, supra).
The genomes of CVB that have been fully sequenced are very similar to one another in length, ranging from 7389 nucleotides (CVB1) to 7402 nucleotides (CVB5) (Romero et al., 1997 supra). Variations in length are due to differences within the coding region of VP1 and VP2 (capsid proteins) and in the 5′ and 3′ non-translated regions. The 5′ non-translated regions also show remarkable similarity in length. For a detailed review of the similarities among the CVB genomes, refer to Romero et al, supra, 1997.
One of the six serotypes of the group B coxsackieviruses, Coxsackievirus B3 (CVB3), has been particularly well studied, and serves as a prototype for the other coxsackieviruses. The CVB3 genome is single molecule of positive sense RNA which encodes a 2,185 amino acid polyprotein. The single long open reading frame is flanked by a 5′ non-translated region (5′ NTR), 742 nucleotides long, and a much shorter 3′ NTR which terminates in a polyadenylate tract. Like the polioviruses (PVs), CVB3 shuts off host cell protein translation in infected HeLa cells. The near atomic structure of the CVB3 virion has been solved, demonstrating that the CVB3 capsid shares a similar capsid structure with genetically-related entero-and rhinoviruses.
Coxsackie B viruses are established etiologic agents of acute human inflammatory heart disease (reviewed in Cherry, J. D.
Infectious Diseases of the Fetus and Newborn Infant
, 4
th
ed., pp.404-446, 1995) and cardiac CVB3 infections may lead to dilated cardiomyopathy. Systemic CVB3 infections are common in neonates: often severe or life-threatening, they usually involve inflammation and necrosis of the heart muscle. One study of neonates under three months of age suggested a CVB infection rate as high as 360/100,000 infants with an associated 8% mortality (Kaplan, M. H., et al.,
Rev. Infect. Dis
. 5:1019-1032, 1983). Acute and chronic inflammatory heart disease afflicts approximately 5-8 individuals per one hundred thousand population annually worldwide (Manolio, T. A., et al.
Am. J. Cardiol
. 69: 1458-1466, 1992). Based upon molecular evidence of enteroviral involvement, approximately 20-30% of cases of acute inflammatory heart muscle disease and dilated cardiomyopathy involve an enteroviral etiology (see, e.g., Kandolf, R.
Coxsackieviruses
-
A General Update
, p. 292-318, 1988; and Martino, T. A., et al.,
Circ. Res
. 74:182-188, 1994).
The inflammatory process which characterizes enterovirus-induced inflammatory heart disease has been extensively studied in murine models (reviewed in Gauntt, C., et al.,
Medical Virology
, 8th ed., p. 161-182, 1989; Leslie, K., et al.,
Clin. Microbiol. Rev
. 2:191-203, 1989; Sole, M., and P. Liu.,
J. Amer. Coll. Cardiol
. 22 (Suppl.A):99A-105A, 1994; and Woodruff, J. F.,
Am. J. Pathol
. 101:425-484, 1980), but it remains unclear precisely what specific roles are played by the various components of the cell-mediated immune response in the induction of acute disease and continuation of the chronic state. However, it is clear that in the presence of an intact murine immune system, CVB3-induced inflammatory heart disease develops only following inoculation of mice with a cardiovirulent CVB3 strain (Chapman, N. M., et al.,
Arch. Virol
. 135:115-130, 1994; Gauntt, C. J., et al.,
J. Med. Virol
. 3:207-220, 1979; Tracy, S., et al.,
Arch. Virol
. 122:399-409, 1992; and Woodruff, J. F., and E. D. Kilbourne,
J. Infect. Dis
. 121:137-163, 1970).
Both cardiovirulent (able to induce disease) and non-cardiovirulent strains of CVB3 replicate well in hearts of experimentally-infected mice. Only cardiovirulent CVB3 strains, however, cause the significant cardiomyocyte destruction with subsequent cardiac inflammation which is characteristic of acute myocarditis (Chapman, N. M., et al.,
Arch. Virol
. 135: 115-130 (1994); and Tracy, S., et al.,
Arch. Virol
. 122:399-409, 1992). Non-cardiovirulent CVB3 is cleared from the experimentally-infected murine heart within 7-10 days post-infection, while infectious cardiovirulent CVB3 can remain detectable in hearts for up to 2 weeks post-infection (Klingel, K., et al,
Proc. Natl. Acad. Sci. U.S.A
. 89:314-318, 1992; Lodge, P. A., et al.,
Am. J. Pathol
. 128:455-463, 1987; and Tracy, S., et al.,
Arch. Virol
. 122:399-409, 1992). The fall in murine cardiac infectious CVB3 titer is coincident with the rise in anti-CVB3 neutralizing antibody titers and the ability of T cells to recognize CVB3 antigens (Beck, M. A., and S. Tracy,
J. Virol
. 63:4148-4156, 1989; Gauntt, C., et al.,
Medical Virology
, 8th ed., p. 161-182, 1989; and Leslie, K., et al.,
Clin. Microbiol. Rev
. 2:191-203, 1989). In addition to direct in situ hybridization evidence for enteroviral replication in human heart myocytes and for cardiovirulent CVB3 replication in murine heart myocytes, CVB3 infects a variety of cultured cardiac cell types including murine and human cardiomyocytes, murine fetal heart fibroblasts and cardiac endothelial cells.
Of great interest is that heart transplantation and acute enteroviral heart disease evoke a similar immune response in a host. Acute rejection of a transplanted heart involves primarily a Th1 type T cell response, the same type of T cell response that is observed in CVB3 induction of acute myocarditis in well-studied murine models of CVB3-induced inflammatory heart disease. Switching of this response to the Th2 type response, with a concomitant ablation of disease, has been accomplished in mice through parenteral administration of the key modulatory cytokines IL-4 or IL-10. However, parenteral administration of cytokines to humans often results in undesired clinical side effects.
Thus, the prior art is deficient in the use of an attenuated coxsackievirus as a gene delivery vector, specifically to target immunomodulatory or other biologically active genes or antigenic epitopes to selected cells, tissues or organs, including the heart. Such a mode of administration or gene delivery circumvents the undesirable side effects of parenteral administration of immunomodulatory agents, antigens or other therapeutic molecules. Thus, the present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
One object of the present invention is to provide viral vectors for therapeutic or prophylactic use in human disease by delivering nucleic acids encoding antigenic epitopes or specific biologically active gene products, such as (but not limited to) immunomodulatory cytokines, to target cells, tissues or organs in an individual.
Thus, according to one aspect of the invention, a viral vector for delivering a heterologous nucleic a
Chapman Nora M.
Kolbeck Peter
Malone, III James M.
Tracy Steven M.
Board of Regents of the University of Nebraska
Guzo David
Woodcock & Washburn LLP
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