Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
1999-10-06
2001-03-27
Scheiner, Laurie (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C424S229100, C424S204100, C424S199100, C424S205100, C435S320100, C435S235100, C536S023720
Reexamination Certificate
active
06207168
ABSTRACT:
BACKGROUND OF THE INVENTION
Herpes Simplex Virus (HSV) is a well-studied virus. Both distinguishable serotypes of Herpes Simplex Virus (HSV-1 and HSV-2) cause infection and disease ranging from relatively minor fever blisters on lips to severe genital infections, and generalized infections of newborns. HSV-1 and HSV-2 are 50% homologous at the DNA level, and polyclonal antibodies and MAbs to shared epitopes for one are cross-reactive to the other.
HSV-1 and HSV-2 have RR1 proteins (respectively designated ICP6 and ICP10) that contain a unique amino terminal domain. The HSV-2 unique domain codes for a ser/thr-specific PK which has auto- and transphosphorylating activity and has a transmembrane (TM) domain. Sequences which code for the PK domain cause neoplastic transformation and are associated with cervical cancer (HSV-2 oncogene). The unique terminal domain of the HSV-l RR1 protein (ICP6) also has PK activity but it is different from that of the HSV-2 oncoprotein, both structurally and functionally.
Original studies, using enzymatic assay conditions similar to those employed for ICP10 PK, concluded that ICP6 does not have PK activity, although the unique domain is retained (Chung et al., J. Virol. 63:3389-3398, 1989). This was not unexpected since the sequence of the unique PK domains showed only 38% homology (Nikas et al., Proteins: Structure, function and genetics 1:376-384, 1986). Further studies indicated that ICP6 has PK activity but only under different conditions. There are conflicting results about its ability to transphosphorylate other proteins (see Peng et al., Virology 216:184-196, 1996 for a review of the problem; particularly Table 1). The reason for the different PK activities of the ICP6 and ICP10 proteins is likely to be that the ICP6 PK ATP binding sites are located distantly from the rest of the catalytic motifs (Cooper et al., J. Virol 69:4979-4985, 1995). ICP6 also does not have a functional TM domain and it does not localize to the cell surface (Conner et al., Virology 213:615, 1995). The PK activity of the native ICP6 is very weak even under ideal conditions, such that its K
m
is 10-fold higher than that of ICP10 PK (Peng et al., Virology 216:184, 1996; Lee and Aurelian, in preparation).
The transforming activity of ICP6 is located within a genome fragment that is distant from that at which the HSV2 oncogene is located. Transformation in this system is morphologic (focus forming ability).
It has previously been shown that DNA sequences which encode for the amino-terminal one-third of ICP10 (amino acids 1-417) have oncogenic potential. Cells transfected with these DNA sequences evidence anchorage independent growth and cause tumors in animals. Transformation is seen in both rodent and human cells (Jariwalla et al., PNAS 77:2279-2283, 1980; Hayashi et al., PNAS 82:8493-8497, 1985; Smith et al., Virology 200:598-612, 1994; Hunter et al., Virology 210:345-360, 1995).
There are three functional domains within ICP10 amino acids 1-41 1: (i) an intracellular domain, at amino acids 106-411, which encompasses the PK catalytic domain with eight conserved catalytic motifs (amino acids 106-411), (ii) a TM, at amino acids 88-105, and (iii) an extracellular domain at amino acids 1-88 (Chung, et al., J. Virol. 63:3389-3395, 1989; Virology 179:168-178, 1990). The minimal size required for PK activity is amino acids 1-283 (pp29
Ia1
) (Luo et al., J. Biol. Chem. 266: 20976-20983, 1991). However, the PK activity of pp29
Ia1
has some properties different from the authentic ICP10 PK, presumably because it lacks part of the PK catalytic domain VI (Luo et al., J. Biol. Chem. 266: 20976-20983, 1991). The TM domain is also required (but insufficient) for PK activity (Luo and Aurelian, J. Biol. Chem. 267:9645-9653, 1992). Therefore, it can be concluded that the PK activity is localized within amino acids 88411 with an essential core at amino acids 88-283.
The unique HpaI site within the ICP10 coding region represents the 3′ end of the transforming region (Jariwalla et al., Proc. Nat. Acad. Sci. 77:2279-2283, 1980) and cuts the gene after the codon for amino acid residue 417. It is not known whether pp29
Ia1
has transforming activity. However, PK activity is required for neoplastic potential. PK negative mutants do not transform cells. This includes a mutant deleted in the TM domain and site directed mutants in the ATP binding sites (Lys
176
and/or Lys
259
) or the ion-binding site (Glu
209
) (Smith et al., Virology 200:598-612, 1994; Aurelian, L. Frontiers in Biology, in press). Because a PK
−
mutant deleted only in the TM domain does not have transforming activity (Smith et al., Virology 200:598-612, 1994), DNA sequences that code for ICP10 amino acids 106-411, but lack PK activity, are not intrinsically neoplastic. This demonstrates that: (i) the HSV-2 oncoprotein is located within ICP10 amino acids 1-411, and (ii) neoplastic potential requires a functional PK activity.
The function of ICP10 PK in virus growth/pathogenesis is unknown.
The HSV-2 ICP10 protein has intrinsic PK activity. This was shown by demonstrating that ICP10 PK activity is lost through site-directed mutagenesis. The oncogene also has SH3 -binding sites at positions 140, 149 and 396, which are required for interaction with signaling proteins. This interaction is required for transforming activity. Site directed mutagenesis was used to identify amino acids required for kinase activity and interaction with signaling proteins. Mutation of Lys
176
or Lys
259
reduced PK activity (5-8 fold) and binding of the
14
C-labeled ATP analog p-fluorosulfonylbenzoyl 5′-adenosine (FSBA), but did not abrogate them. Enzymatic activity and FSBA binding were abrogated by mutation of both Lys residues, suggesting that either one can bind ATP. Mutation of Glu
209
(PK catalytic motif III) virtually abrogated kinase activity in the presence of Mg
2+
or Mn
2+
ions, suggesting that Glu
209
functions in ion-dependent PK activity.
ICP10PK functions as a growth factor receptor involved in signaling and it binds the adaptor protein Grb
2
in vitro. The SH3-binding sites within the ICP10 PK domain (at positions 140, 149 and 396) are required for interaction with signaling proteins and, thereby transformation (Nelson et al., J. Biol. Chem. 271:17021-17027, 1996). Mutation of the ICP10 proline-rich motifs at position 396 and 149 reduced Grb
2
binding 20- and 2-fold respectively. Binding was abrogated by mutation of both motifs. Grb
2
binding to wild type ICP10 was competed by a peptide for the Grb2 C-terminal SH3 motif indicating that it involves the Grb
2
C-terminal SH3 (Nelson et al., J. Biol. Chem. 271:17021-17027, 1996).
The ICP10 PK catalytic domain also contains amino acids at position 106-178 that are responsible for binding a down-regulator of PK activity (ras-GAP). Deletion of amino acids 106-178 reduces, but does not abrogate, PK activity (Luo and Aurelian, J. Biol. Chem. 267:9645-9653, 1992). However, it abrogates ras-GAP binding, thereby increasing transforming potential (Nelson et al., manuscript in preparation).
The construction of the ICP10 PK virus is described by Peng et al. (Virology 216, 184-196, 1996). Briefly, the wild type sequences in a plasmid (TP101) that contains the HSV-2 BamHI E and T fragments were replaced with the 1.8kb SalI/BglII fragment from pJHL9. pJHL9 is a plasmid containing the ICP10 mutant deleted in the PK catalytic domain (Luo and Aurelian, J. Biol. Chem. 267:9645-9653, 1992). The resulting plasmid, TP9, contains sequences which code for ICP10 deleted in the PK catalytic domain flanked by 4 and 2.8 kb of HSV-2 DNA sequences at the 5′ and 3′ ends, respectively. The 10 kb HindIII/EcoRI fragment from TP9 was introduced by marker transfer into a virus (ICP10&Dgr;RR) in which the RR domain of ICP10 had been replaced with the LacZ gene. The resulting recombinant virus, designated ICP10&Dgr;PK, was obtained by selecting white plaques on a background of blue plaques after staining with X-gal. A few white plaques were picked and purified. Two were grown in
Pepper Hamilton LLP
Pouliquen Corinne M.
Scheiner Laurie
University of Maryland at Baltimore
Villacorta Gilberto M.
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