Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Binds antigen or epitope whose amino acid sequence is...
Reexamination Certificate
1999-04-08
2002-08-20
Swartz, Rodney P (Department: 1645)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Binds antigen or epitope whose amino acid sequence is...
C424S164100, C424S184100, C424S185100, C424S190100, C435S243000, C530S300000, C530S350000, C536S023700
Reexamination Certificate
active
06436399
ABSTRACT:
BACKGROUND
This work was supported by grant RO1 AI33123 and grant RO1 A140934 from the National Institutes of Health. The Government has certain rights in this invention.
Human granulocytic ehrlichiosis (HGE), an emerging human infectious disease, is increasingly being recognized in the United States. Serological and PCR studies suggest that HGE infection also exists in Europe. HGE is caused by infection with an obligatory intracellular bacterium, HGE agent. Comparison of 16S rRNA gene sequences and ultrastructure indicates that the HGE agent is closely related to
Ehrlichia phagocytophila
, the agent of tick-borne fever, and
E. equi
, the agent of equine ehrlichiosis. The HGE agent is transmitted by the Ixodes sp. tick, and the white-footed mouse is considered to be the major reservoir of the HGE agent in the United States.
HGE infection is characterized by the presence of ehrlichial inclusions called morulae in human or animal peripheral blood granulocytes. The symptoms of HGE include chills, headache, myalgia, hematological abnormalities, including leukopenia and thrombocytopenia. HGE frequently requires prolonged hospitalization. When treatment is delayed due to misdiagnosis, HGE can be fatal.
Although several laboratories have used IFA testing, acute-phase blood smear, nested PCR, and culture isolation for diagnosis of HGE, each of these diagnostic tests has disadvantages. IFA testing using the HGE agent or
E. equi
-infected cells has been most widely used for serodiagnosis of ehrlichiosis. IFA requires a tissue culture system for preparation of HGE-infected cell antigen slides, fluorescent microscope, and trained persons especially for evaluation of the serum reactivity to the antigen on the slide. The nested PCR, while better suited to early diagnosis of HGE than IFA testing, requires thermocycler and trained personel, and the reagents are relatively expensive. The sensitivity of culture isolation in HGE diagnosis seems to be relatively lower than that of IFA test and the nested PCR. Moreover, diagnosis by culture isolation requires serologic evidence, PCR, or 16S rRNA gene sequence for confirmation of the identity of new isolates.
Accordingly, a convenient and sensitive method with high specificity for the diagnosis of HGE infection is still desirable.
SUMMARY OF THE INVENTION
The present invention relates to improved diagnostic tools for serodiagnosing HGE. diagnostic tools are structurally related proteins, collectively referred to hereinafter as the “Group 44 proteins”, and to antibodies to such proteins. As compared to each other, the Group 44 proteins comprise a single central hypervariable region of approximately 94 amino acid residues, a first conserved region and a second conserved region which flank the central hypervariable region. The first conserved region comprises 52 amino acids and is linked directly or through a linker of from 1 to 10 amino acids to the amino terminus, i.e., the N-terminus, of the hypervariable region. The second conserved region comprises about 56 amino acids and is linked either directly or through a linker of from 1 to 10 amino acids to the carboxy terminus, i.e., the C-terminus, of the hypervariable region. The hypervariable region of each Group 44 protein has a higher hydrophilicity, a higher antigenic index, and a higher surface probability than either the first conserved region or the second conserved region of the respective protein. The hypervariable region is basic and has an isoelectric point, i.e., a p1, of from about 7.1 to about 9.2 and a molecular mass, i.e., an Mr, of from about 8.5 kDa to about 11 kDa. The hypervariable region has a first semiconserved region of 8 amino acids near the amino terminus thereof and a second semiconserved region of 11 amino acids at the carboxy terminus thereof. The hypervariable region also contains a third semiconserved region of 6 amino acids. It is believed that the third semiconserved region, which is between and separated from the first semiconserved region and the second semiconserved region is perhaps involved in adhesion of the HGE agent to other cells.
The Group 44 proteins comprise the P44 protein, the P44-2 protein, the P44-12 protein, the P44-15 protein, the P44-18 protein, and the P44-19 protein and variants of such proteins. The P44 protein and variants thereof specifically bind to antibodies found in the sera from horses infected with the HGE agent and to antibodies in the sera from humans infected with the HGE agent and horse anti-
Ehrlichia equi
serum. The P44 protein and variants thereof do not bind to antibodies in sera from patients infected with
E.chaffeensis
or
Borrelia burgdorferi
. In one embodiment, the P44 protein comprises the amino acid sequence, SEQ ID NO: 2, shown in FIG.
1
.
The P44-2 protein has a molecular mass of about 45 kDa. In one embodiment the P44-2 protein comprises the amino acid sequence, SEQ ID NO: 4, shown in FIG.
2
.
The P44-12 protein has a molecular mass of about 41.2 kDa. In one embodiment the P44-12 protein comprises the amino acid sequence, SEQ ID NO: 6, shown in FIG.
3
. The P44-15 protein comprises a hypervariable region which encodes a polypeptide having a molecular mass of about 10.8 kDa. In one embodiment, the P44-15 protein comprises the amino acid sequence, SEQ ID NO: 8, shown in FIG.
4
. The P44-18 protein comprises a hypervariable region which encodes a polypeptide having a molecular mass of about 26.4 kDa. In one embodiment, the P44-18 protein comprises the amino acid sequence, SEQ ID NO:10 shown in FIG.
5
. The P44-19 protein comprises a hypervariable region which encodes a polypeptide having a molecular mass of about 8.6 kDa and an isoelectric point of about 8.8. The P44-19 protein comprises the amino acid sequence, SEQ ID NO: 12, shown in FIG.
6
.
The Group 44 proteins are immunogenic and, thus, are useful for preparing antibodies. The isolated proteins of the P44 family, either individually or as a panel of proteins, are useful for detecting antibodies to the HGE agent in the blood of patients with clinical signs of HGE. The isolated, individual Group 44 proteins are also useful in an immunogenic composition for ameliorating HGE. The isolated, individual Group 44 proteins are also useful in a vaccine for protecting against infection with the HGE agent.
The present invention also provides isolated polynucleotides or nucleic acids, referred to collectively hereinafter as the “Group 44 polynucleotides”, which encode the Group 44 proteins and fragments thereof. The P44 polynucleotides encode the P44 protein. One embodiment of the P44 polynucleotides comprises the nucleotide sequence, SEQ ID NO:1, shown in FIG.
1
. The P44-2 polynucleotides encode the P44-2 protein. One embodiment of the P44-2 polynucleotides comprises the nucleotide sequence, SEQ ID NO: 3, shown in FIG.
2
. The P44-12 polynucleotides encode the P44-12. One embodiment of the P44-12 polynucleotides comprises the nucleotide sequence, SEQ ID NO: 5, shown in FIG.
3
. The P44-15 polynucleotides encode the P44-15 protein. One embodiment of the P44-15 polynucleotides comprises the nucleotide sequence, SEQ ID NO: 7, shown in FIG.
4
. The P44-18 polynucleotides encode the P44-18 protein. One embodiment of the P44-18 polynucleotides comprises the nucleotide sequence, SEQ ID NO: 9, shown in FIG.
5
. The P44-19 polynucleotides encode the P 44-19 protein. One embodiment of the P44-19 polynucleotides comprises the nucleotide sequence, SEQ ID NO: 11, shown in FIG.
6
. The Group 44 polynucleotides are useful for preparing the Group 44 proteins and variants thereof. Group 44 polynucleotides which encode fragments of the Group 44 proteins are useful as primers and probes.
The present invention provides synthetic oligopeptides of 14-16 amino acids in length. Each of the oligopeptides comprise a sequence which is specific to one Group 44 protein. Such peptides are useful for preparing antibodies which are immunospecific for one or more Group 44 proteins.
The present invention also relates to antibodies which are immunospecific for and bind to members of the P44 famil
Ohashi Norio
Rikihisa Yasuko
Zhi Ning
Calfee Halter & Griswold LLP
Swartz Rodney P
The Ohio State University Research Foundation
LandOfFree
Nucleic acid encoding the major outer membrane protein of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Nucleic acid encoding the major outer membrane protein of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleic acid encoding the major outer membrane protein of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2955917