Infectious clone of human parvovirus B19 and methods

#Human parvovirus B19 is the only member of the Parvoviridae family known to cause diseases in humans. Parvovirus B19 infection causes fifth disease in children, polyarthropathy syndromes in adults, transient aplastic crisis in patients with underlying chronic hemolytic anemia, and chronic anemia due to persistent infection in immunocompromised patients. Hydrops fetalis and fetal death have been reported after maternal infection with parvovirus B19 during pregnancy (Brown et al., 1994, Crit. Rev.Oncol./Hematol. 16:1-13).

#Parvovirus B19 exhibits a selective tropism for erythroid progenitor cells. The virus can be cultured in erythroid progenitor cells from bone marrow, fetal liver cells, and cell lines such as UT7/Epo or KU812Ep6. (Ozawa et al., 1986, Science 233:883-886; Brown et al., 1991, J. Gen. Vir.72:741-745; Komatsu et al., 1993, Blood 82:456-464; Shimomura et al., 1992, Blood 79:18-24; Miyagawa et al., 1999, J. Virol. Methods 83:45-54). Although the virus can be cultured in these cells very little virus is produced. The selective tropism of the virus is mediated in part by neutral glycolipid globoside (blood group P antigen), which is present on cells of the erythroid lineage (Brown et al., 1993, Science, 262:114-117). The presence of globoside on the surface of a cell is a determinant of viral tropism. Parvovirus B19 has a cytotoxic effect on erythroid progenitor cells in bone barrow and causes interruption of erythrocyte production. Human bone marrow cells that lack globoside on the cell surface are resistant to parvovirus B19 infection (Brown et al., 1994, N. Engl. J. Med., 33:1192-1196).

#The ends of the parvovirus B19 genome have long inverted repeats (ITR), which are imperfect palindromes that form double-stranded hairpins. The role of the ITRs in the parvovirus B19 viral life cycle is unknown due to the inability to produce an infectious clone containing complete ITR sequences. In other parvoviruses, ITRs play an important role in the viral life cycle: they serve as primers for the synthesis of the complementary strand of viral DNA and are essential for the replication, transcription, and packaging of virus DNA (Bems, K (1990) in Virology, eds. Fields et al. Raven Press Ltd, NY, ppl743-1763). Previous attempts to produce an infectious clone of parvovirus B19 were unsuccessful due to deletions in the ITR sequences and the instability of the ITRs in bacterial cells (Deiss et al., 1990, Virology 175:247-254; Shade et al., 1986, J. Virol. 58:921-936). Methods of consistently producing infectious B19 parvovirus in cell culture are not known.

#FIG. 1 shows a map of recombinant plasmid pB19-N8, which contains an insert comprising 4844 nucleotides of the B19 genome. The arrows indicate genes. Shaded arrows indicate genes in the B19 genome. The shaded circles at the 5′ and 3′ ends of the B19 genome indicate the ITR sequences.

#FIG. 2A shows the structure of the B19 terminal inverted repeats in hairpin form. The “flip” (SEQ ID NO: 1) and “flop” (SEQ ID NO:2) orientations at the 5′ end (+strand) are shown.

#FIG. 2B shows a comparison of nucleic acid sequences encoding the flip and flop forms of ITRs in B19 isolate J35 (SEQ ID NOS: 1 and 2), the B19 isolate reported by Deiss et al., 1990, Virology, 175:247-254 (SEQ ID NOS:3 and 4), and the flop form in B19-Hv (SEQ ID NO:37). Aligned positions are boxed in black. The numbering indicates the positions of nucleotides in the genomes of the respective B19 isolates.

#I. Definitions

#The term “antibody” is used in the broadest sense and specifically includes, for example, single anti-parvovirus B19 monoclonal antibodies, anti-parvovirus B19 antibody compositions with polyepitopic specificity, single chain anti-parvovirus B19 antibodies, and fragments of anti-parvovirus B19 antibodies. The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.

#“Antibody fragments” comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies (Zapata et al., 1995, Protein Eng., 8:1057-1062); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.