Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-01-22
2002-09-03
Graser, Jennifer E. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S071100, C435S071200, C435S173300, C435S320100, C435S243000, C536S023100, C536S023700, C536S024320, C536S024100, C424S252100
Reexamination Certificate
active
06444445
ABSTRACT:
Brucella infects a significant number of people and livestock in developing countries and infects wild as well as domestic animals in the United States. In addition, Brucella is a potential biowarfare agent; strains of Brucella have been constructed with resistance to multiple antibiotics used to treat the disease. These strains pose a significant morbidity and mortality threat to exposed personnel. Brucellosis symptoms include recurring fever, chills and anxiety. Even though the disease is rarely fatal, once well established, the disease is difficult to treat since the bacteria reside in the bone marrow.
Two live attenuated Brucella strains currently approved for use as animal vaccines,
B. abortus
Strain 19 [Cheville, et al. (1993)
Am. J. Vet. Res.
54:1591-1597;
Brucellosis research: an evaluation
. Report of the subcommittee on Brucellosis Research, National Academy of Sciences. Washington, D.C.: National Academy Press, 1977:61-77] and
B. melitensis
strain Rev 1 [Jimenez de Bagues, M. P. et al. (1989)
Ann. Rech. Vet.
20:205-213; Pardon, P. et al. (1990)
Ann. Rech. Vet.
21:153-160], are not ideal vaccine strains. Both strains cause vaccinated animals to seroconvert and thus make subsequent serological diagnosis of brucellosis difficult [Jimenez de Bagues, M. P. et al. (1992)
Vet. Microbiol.
30:233-241]. Both strains can induce abortion (Jimenez de Bagues, 1989, supra; Corner, L. A. and Alton G. G. (1981)
Res. Vet. Sci.
31:342-344] and both can cause disease in humans [Blasco, J. M. and R. Diaz (1993)
Lancet
342:805; Young, E. J. (1983)
Rev. Inf. Dis.
5:821-842]. A more recent attenuated strain of
B. abortus
, RB51 [Schurig, G. G. et al. (1991)
Vet. Microbiol.
28:171-188], shows more promise as a live vaccine strain. RB51 is a rough strain that confers protection against infection by Brucella, yet does not cause seroconversion [Cheville, N. F. 1993, supra; Jimenez de Bagues, M. P. et al. (1994)
Infect. Immun.
62:4990-4996]. However, neither the genetic basis of the RB51 rough mutation nor the basis of attenuation is known. Also, RB51 carries resistance to rifampin, an antibiotic currently used to treat brucellosis.
Therefore, there is a need for a live attenuated Brucella vaccine strain, with a defined nonreverting genetic mutation, which does not cause seroconversion in the vaccinee, and which does not retain resistance to antibiotics used in the treatment of brucellosis.
SUMMARY OF THE INVENTION
The present invention fulfills the need described above. In this application is described attenuated rough strains of Brucella, containing genetically defined mutations, which will not cause seroconversion. The mutations in these attenuated Brucella strains were created by DNA deletion, the type of mutation least susceptible to genetic reversion and are therefore advantageous as vaccine strains. These vaccines strains do not retain resistance to an antibiotic useful for treatment of brucellosis.
More specifically, this invention relates to two genetically defined rough mutants of
Brucella melitensis
, WRR51 and WRRP1, as candidate strains for a live vaccine against brucellosis. These strains differ from Brucella live vaccines currently used in livestock because they have genetically defined mutations that were created by deleting DNA from the Brucella chromosome. Both strains have a lipopolysaccharide (LPS) defect and thus do not cause the seroconversion that complicates disease screening. Smooth strains currently approved for use in animals are not good candidates for human vaccines because though attenuated, they can still cause disease in humans. One of the vaccine strains of the present invention, WRRP1, is a double deletion mutant that is highly attenuated and is unlikely to cause disease in humans.
Briefly, the genetically defined rough mutants of Brucella were constructed by using a
Brucella abortus
VTRA1 chromosome containing a Tn5 insertion which conferred a rough phenotype [Winter, A. J. et al. (1996)
Amer. J. Vet. Res.
57:677-683]. The
B. abortus
gene containing the Tn5 insertion was cloned from the VTRA1 chromosome and the nucleotide sequence of the 2693 bp (SEQ ID NO:1) region containing the transposon insertion was determined. The Tn5 insertion was found to be located within an open reading frame of 1233 bp spanning nucleotides 883 through 2115 of SEQ ID NO:1 which coded for a gene that was distantly related (40% amino acid similarity) to the sequence of the
Salmonella enterica
LT2 rfbU, a gene encoding a mannosyltransferase [Liu, D. et al. (1993)
J. Bacteriol.
175:3408-2414]. A deletion of 607 bp was made in the putative rfbU gene and a cassette containing a chloramphenicol acetyl transferase gene (cat) was ligated into the deletion site to create rfbU/cat. The plasmid containing rfbU/cat, pRFBU1, was electroporated into
B. melitensis
strain 16M and electroporants with pRFBU1 integrated were selected on Brucella agar containing chloramphenicol. Southern DNA hybridization confirmed that the chloramphenicol resistant and ampicillin sensitive electroporants had the deletion mutation carrying the chloramphenicol resistance cassette in place of the wild type chromosomal locus resulting from a directed allelic exchange by a double crossover recombinational event. The deletion strain, designated WRR51, was confirmed to be rough by staining with crystal violet, and by lack of agglutination with an anti-LPS serum.
A purE deletion was then introduced into
B. melitensis
strain WRR51 by a similar allelic exchange procedure. PurE is an essential enzyme in the purine biosynthetic pathway. The resultant double deletion strain (&Dgr;rfbU &Dgr;purE) was designated WRRP1. The DNA flanking the transposon insertion was sequenced to determine the open reading frame that had been interrupted to cause the rough phenotype and was found to be rfbU. The complete sequence of Brucella rfbU is described for the first time in this application in SEQ ID NO:1.
Unlike the rough mutants of the present invention, none of the rough mutants described previously including
B. abortus
strain 2308 rfbU mutant, VTRA1, and the VTRA1 transposon mutation integrated into the chromosomes of
B. melitensis
and
Brucella suis
by allelic exchange to create VTRM1 and VTRS1, respectively [McQuiston, J. R. et al. (1995) Abstract, CRWAD, November 1995; Winter, A. J. et al. (1996)
Am. J. Vet. Res.
57:677-683] contained a defined mutation. In other words, the previously described mutant strains were produced by a transposon insertion which is a random event and can occur at any chromosomal location wherein the mutants of the present invention were produced by a directed allelic exchange to produce a unrevertable, defined deletion in the gene. A plasmid construct containing a synthetic copy of the putative rfbU gene that restored the smooth phenotype to the WRR51 deletion mutant of the present invention, did not restore the smooth phenotype to the VTRA1 transposon mutant. The inability to complement the transposon mutant indicates either that the transposon insertion confers a more general genetic defect in LPS biosynthesis (via a polar effect), or that the VTRA1 strain has additional mutations that affect LPS biosynthesis. The rough mutants of the present invention have a defined, nonreverting, deletion in the putative rfbU gene that was integrated into the chromosome by allelic exchange.
In order to construct the deletion in a rough strain, several factors had to be considered. The sequence of the flanking DNA (the rfbU gene) extending far enough in either direction of the deletion had to be known to allow for PCR or direct cloning of a large enough region of the Brucella chromosome. In addition, it was important to allow for a deletion of a significant portion of the rfbU gene to inactivate the gene in the first attempt; the actual crossover (allelic exchange) of the &Dgr;rfbU for the wild type was very difficult because it occurred at a very low frequency, and after several trials,
Boyle Stephen M.
Hadfield Ted L.
Hoover David L.
Lindler Luther E.
McOulston John R.
Arwine Elizabeth
Graser Jennifer E.
Harris Charles H.
The United States of America as represented by the Secretary of
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