Binding moieties for human parvovirus B19

Details Binding moieties for human parvovirus B19 Binding moieties for human parvovirus B19

1998-11-05

2001-05-29

Scheiner, Laurie (Department: 1641)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S007920, C530S388300, C530S390100, C530S324000, C530S326000, C530S327000, C530S328000, C424S147100

Reexamination Certificate

active

06238860

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to methods and materials for detecting, clearing, or isolating parvovirus B19 and/or B19-like polypeptides from blood or other solutions containing it. The invention particularly provides polypeptides and recombinant bacteriophage expressing such polypeptides that are capable of binding to parvovirus B19 and/or B19-like polypeptides for the purpose of detecting, clearing, or isolating parvovirus B19 and/or B19-like viruses or polypeptides.
BACKGROUND OF THE INVENTION
Parvoviruses form the parvoviridea family which are common agents of animal diseases. Parvovirus B19 is thus far the only strain identified to infect humans. The first strong link between parvovirus B19 infection and human disease was reported by Cossart et al. in England during screening of healthy blood donors for hepatitis B surface antigen. See, Cossart et al.,
Lancet
, I: 72-73 (1975). “B19” refers to the designation of the sample from which this parvovirus was first isolated, and as the strain that is capable of infecting humans, it is often referred to as “human parvovirus B19”.
Parvovirus B19 is a non-enveloped, single-stranded DNA virus with a diameter of 22 nm, consisting of only the genome and a few structural and non-structural proteins. The capsid proteins are arranged with icosahedral symmetry and enclose the genome of approximately 5500 base pairs. Two large open reading frames are in the viral genome: The left open reading frame codes for non-structural proteins (NS1 and NS2) involved in viral replication and packaging; the right open reading frame codes for the structural proteins forming the viral capsid, VP1 (781 amino acids) and VP2 (554 amino acids). Both structural proteins are in the same reading frame and the entire sequence of VP2 is contained within VP1. VP2 is the major protein of the B19 capsid.
Parvovirus B19 is among the most resistant viruses known and has been identified as the causative agent of several diseases, including transient aplastic crisis (TAC) of hemolytic disease, the common childhood rash called “fifth disease”, a polyarthralgia syndrome in normal adults that may be chronic and that resembles rheumatoid arthritis in its clinical features, and some forms of chronic anemia and/or neutrpenia. Pregnant women infected with this virus frequently suffer serious disabilities including spontaneous abortion and hydrops fetalis.
As a blood-borne virus, parvovirus B19 has become a concern for organizations dealing with whole blood or blood products intended, e.g., for use in transfusions. Therefore, it is important to develop sensitive methods for detection of the virus in infected blood and methods for clearing the virus from blood drawn from an infected subject.
Techniques employing Polymerase Chain Reaction (PCR) have become prevalent in recent years for detecting the presence of parvovirus B19 in biological samples. For example, Schwarz et al. utilized a pair of oligonucleotide primers spanning the PstI-fragment of the B19 virus genome to detect the B19 viral DNA in sera of individuals in the incubation period and acute phase of parvovirus B19 infection. See, Schwarz et al.,
Scand. J. Infect. Dis.,
24:691-696 (1992). See, also, Musiani et al., who utilized nested PCR to detect B19 infection in immunocompromised patients (
J. Med. Virol.,
40:157-160 (1993)), and also Torok et al., who employed PCR as a tool to diagnose prenatal intrauterine infection with parvovirus B19 (
Clin. Infect. Dis.,
14:149-155 (1992)).
Another approach taken to detect the presence of viral products in the infected individual is the use of in situ hybridization with detectable probes. For example, Morey et al. reported intracellular localization of parvovirus B19 nucleic acid by in situ hybridization with digoxiginin-labeled probes (
Histochemical Journal,
25:421-429 (1993)). Later the same group employed a non-isotopic in situ hybridization technique in identifying parvovirus B19 infected cells using biotinylated probes (
J. Clin. Pathol.,
45: 673-678 (1992)). Although in situ hybridization is a rapid and specific means for localizing viral nucleic acid with a high degree of resolution, the sensitivity of this system is limited by the fact that hybridization occurs only at the surface of the section.
Further development of such assays has been hampered because parvovirus B19 cannot be isolated in conventional cell cultures and has only been propagated successfully in cultures of human bone marrow (Ozawa et al.,
Science,
233:883-886, (1986)), umbilical cord blood (Sosa et al.,
J. Med. Virol.,
36:125-130, (1992)), fetal liver (Yaegashi et al.,
J. Virol.,
63:6,2422-2426, (1989)), and cultures from peripheral blood stimulated by erythropoietin (Schwarz et al.,
J. Virol.,
66:1273-1276, (1992)). Another obstacle for development of such assays has been the possible existence of other parvoviruses and isotypes of parvovirus B19 that may also infect humans.
There is still a need, therefore, for sensitive and effective assays to detect the presence of B19 and/or B19-like viruses and subcomponents thereof, for ways to clear B19 and/or B19-like polypeptides from samples containing it (them), and for reagents that can bind B19 and/or B19-like polypeptides and which will be useful for detecting the presence of and/or clearing such viruses or polypeptides from samples, including blood.
In answer to the foregoing needs, a group of non-naturally occurring polypeptides has now been surprisingly discovered that bind specifically to parvovirus B19 and related polypeptides. Utilizing phage display technology, recombinant bacteriophage displaying polypeptides that recognize and bind to B19 capsid proteins have been identified and isolated. The phage products and isolated polypeptides have proved to be valuable reagents for effective detection and isolation of the B19 virus and B19-like polypeptides.
SUMMARY OF THE INVENTION
The present invention provides binding moieties for parvovirus B19 and/or B19-like viruses and polypeptide subcomponents of such viruses. Preferred binding moieties described herein are polypeptides and recombinant bacteriophage displaying such peptides which bind to parvovirus B19 and/or B19-like viruses, and most preferably to the virus's capsid proteins VP1 and/or VP2.
In specific embodiments, the invention provides binding moieties for B19 and/or B19-like polypeptides as well as methods for detection and removal of human parvovirus B19 and/or B19-like polypeptides from samples (particularly human whole blood or blood products) containing it. In particular, preferred embodiments disclosed herein provide polypeptides that bind to parvovirus B19 capsid proteins VP1 or VP2 or combinations of such proteins and provide methods for binding and/or removing such capsid proteins from solutions containing them. Preferred features include recombinant bacteriophage expressing exogenous DNA encoding parvovirus B19 binding polypeptides.
A preferred binding moiety for human parvovirus B19 and/or B19-like polypeptides according to this invention will be a polypeptide having an amino acid sequence including a sequence selected from the group consisting of:
X
1
-X
2
-Cys-X
3
-X
4
-X
5
-X
6
-X
7
-Cys-X
8
-X
9
(SEQ ID NO: 1),  I.
wherein X
1
is Phe or Leu or is not present; X
2
is Phe or Ser; X
3
is Arg, Gln, Ser, His, Ala, Leu, or Gly; X
4
is Phe, Tyr, Leu, or Trp; X
5
is Trp or Phe; X
6
is Tyr, Pro, or His; X
7
is Gly, Asn, Ser, Phe, or Asp; X
8
is His, Asp, Ser or Pro; X
9
is Pro, Ala, Phe, His, or Asp or is not present;
X
10
-Phe-Cys-X
11
-X
12
-Trp-X
13
-X
14
-X
15
-Cys-X
16
-X
17
(SEQ ID NO: 2),  II.
wherein X
10
is His, Ala, or Phe; X
11
is His, Trp, or Ser; X
12
is Phe or Leu; X
13
is Phe, Pro, or His; X
14
is Gly or His; X
15
is Gly or Asn; X
16
Pro, Leu, or Asp; X
17
is His or Asp; and
X
18
-Cys-X
19
-X
20
-X
21
-X
22
-X
23
-X
24
-X
25
-Cys-X
26
(SEQ ID NO: 3),  III.
wherein X
18
is Phe or Leu; X
19
is Trp, His, Gln or Pro; X
20
is Leu or Ala; X
21
is Trp or His; X
22
is Pro or Trp; X

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