Abstract:
The vaccines are prepared by isolating the Brucella genes complementing mutations in the purEK genes of Escherichia coli, physically mapping, determining the DNA sequence, constructing a defined deletion mutation by polynucleotide chain reaction (PCR), introducing a selectable marker into the deletion, and then selecting a purE mutant in Brucella arising by allelic exchange. The resulting Brucella require purines for growth because they lack the purE gene product that is required for the carboxylation of 5&#39;-phosphoribosyl-5-aminoimidazole.

Description:
This application is a continuation-in-part of U. S. Pat. application No. 08/334,129, filed Nov. 4, 1994, which is now abandoned. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to mutants of Brucella melitensis for use as a vaccine. 
     BACKGROUND OF THE INVENTION 
     Brucella infects a significant number of people and domestic herds in developing nations and domestic and wild animals of the United States. Brucellosis in man may be caused by a number of strains of Brucella including B. abortus (cattle), B. suis (hogs), B. rangiferi (caribou), B. melitensis (sheep and goats) and B. canis (dogs). The disease is acquired by exposure to secretions and excretions of infected animals and by ingestion of milk or milk products from infected animals. The current live-attenuated vaccine strains for animals cause disease in humans, abortions in adult animals and complicates immunological screening for infected animals, since they stimulate production of antibodies to lippopolysaccharide (LPS) which is detected in the milk and serum of vaccinated animals. These antibodies are detected in serological tests used to screen milk and meat for Brucella. 
     Two approved live-attenuated Brucella vaccines are currently used to vaccinate animals. Strain 19 is currently used to vaccinate animals against B. abortus infections. This live-attenuated strain has several characteristics that make it undesirable as a human vaccine. The attenuating mutation in this strain is unknown; it sero-converts vaccinated individuals making differentiation of infected and vaccinated individuals difficult and causes significant disease in humans. Another live-attenuated veterinary vaccine B. melitensis strain, REV1 has the same limitations as Strain 19. Recently, a rough mutation of B. abortus, RB51, has been tested and found to be safe and protective in a number of different animal models. This rough live-attenuated strain was derived from a rifampin resistant strain and the mutation causing the rough phenotype and attenuation is not identified. Although RB51 does not stimulate the production of antibodies to LPS, it stimulates portective immunity in animals and does not cause abortions in vaccinated animals. Since the nature of the genetic defect causing the rough phenotype is not known, it is not possible to predictably make a vaccine under appropriate manufacturing proceedures for human use. Brucella strains have not been used as live-attenuated vaccine carriers for recombinant proteins. 
     Brucella also presents a potential biowarfare threat for military personnel. Multiple antibiotic resistant strains of Brucella have been constructed by others and pose a significant threat to the morbidity and mortality of infected personnel. 
     O&#39;Callaghan (Infection and Immunity, Vol. 56, No. 2, pp 419-423 (1988)) describes deletion mutants of Salmonella having purE and purA deletions. Neither was effective as an oral vaccine. Hence, O&#39;Callaghan would direct interest of those of ordinary skill from the purE deletion mutants for use as vaccines. 
     Houng, et al, (Abstracts of the 94th General Meeting of ASM, page 133, Abstract D-208) teaches that the writers identified purEK genes and identified two polypeptides that are products of these genes. However, they do not teach any particular mutant, nor do they teach how to make any mutant. Furthermore, they do not suggest any particular use for any mutant. (Taken with O&#39;Callaghan, one of ordinary skill would not be directed to use such a mutant for a vaccine.) 
     Smith and Heffron (Infection and Immunity, Vol. 55, No. 11, pp 2774-2776 (1987)) teaches transposon Tn5 mutagenesis of Brucella abortus involving transposon Tn5. His construct is not made by the method of the invention. The instantly claimed invention does not involve introduction of Tn5 or Tn5 lac into a DNA sequence. Adams, in Advances in Brucellosis Research, Chapter 17, pages 250 to 276 (1990) suggests that Tn10 mutagenesis may be used to identify genes encoding virulence factors for adherence, invasion and intracellular survival, and that this method might be applicable to study of Brucella. He suggests no particular mutation. The instantly claimed invention was not suggested by Adams and was not made by the methods of Adams. Stocker in U.S. Pat. No. 4,735,801, suggests similar means of making mutants of Salmonella strains. Stocker introduced one or more modifications in biosynthetic pathways for products unlikely to be found in the disease susceptible host. Auxotrophic mutations are produced in one or more pathways by deletion or inversion of the target gene. These mutations can not be repaired by a single step and are referred to as non-reverting. In this patent he identifies aro, dap, pab and pur target genes for the construction of live vaccine strains. The pur gene targets claimed were those biosynthetic genes involved in the conversion of inosine monophosphate to adenine. He does not suggest the deletion mutants of the invention. 
     SUMMARY OF THE INVENTION 
     It is the purpose of this invention to provide a safe, effective vaccine to protect against brucellosis in man and animals. Live vaccines are provided and methods for preparing live vaccine for protection of a host from infection with Brucella species. The vaccines are prepared by isolating the Brucella genes complementing mutations in the purEK genes of Escherichia coli, physically mapping, determining the DNA sequence, constructing a defined deletion mutation by polynucleotide chain reaction (PCR), introducing a selectable marker into the deletion, and then selecting a purE mutant in Brucella arising by allelic exchange. The resulting Brucella require purines for growth because they lack the purE gene product that is required for the carboxylation of 5&#39;-phosphoribosyl-5-aminoimidazole. Since the purE mutation occurs before the synthesis of inosine monophosphate (IMP), these mutants can grow when supplied with exogenous adenine, hypoxanthine or guanine. In contrast, purA or purB mutants which block bio-synthetic steps after IMP can grow only when exogenous adenine is supplied. Unlike purE mutants of Salmonella, purE mutants of Brucella are substantially reduced in virulence and stimulate protective immunity from infection with wild-type Brucella. In contrast to purA or purB mutants of Salmonella, purE mutants of Brucella are capable of systemic spread from mucosal tissues. Therefore, purE mutants of Brucella are capable of stimulating mucosal, systemic and cellular immune responses to itself as well as to heterologous antigens expressed by Brucella. 
     The genetically engineered organisms of the invention grow at a significantly reduced rate when compared with the wild-type and are unable to escape the phagolysosome in human monocytes in cell culture. Hence, the organisms are useful for providing a safe, effective live vaccine carrier strain for production of antigen-specific cytotoxic T-cells and memory T-helper cells with specific reactivity for recombinant Brucella or non-brucella peptides. 
     The purE deletion mutant of B. melitensis is designed to be a live-attenuated vaccine for prevention of brucellosis. Since this strain remains as infectious as the wild-type 16M B. melitensis, and persists with reduced growth in human monocytes, it will deliver a high antigenic load at target mucosal surfaces and induce both cellular and humoral responses to the bacteria and any recombinant gene expressed by the bacteria. In addition, escape from the phagolysosome increases the processing and presentation of antigens by HLA class 1 proteins. These factors indicate methods of the invention would favor the production of antigen-specific cytotoxic T cells. The genetically engineered bacteria may be administered by method usual in the art, including injection. Methods that would avoid necessity of injection should be considered. The attenuated organisms may, for example, be administered orally in syrup, capsules, in carbonated buffers or nasally via a mist. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG. 1 shows the purE coding region of Brucella melitensis. 
     FIG. 2 shows the pURE3H: 7.9 kb HindIII insert of Brucella melitensis in pHC79. 
     FIG, 3 identifies purEK as was produced in accord with the deletions. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     This invention provides attenuated B. melitensis. The attenuated, genetically engineered organisms provide active immunity against Brucellosis species. The reduced growth rate of Brucella in the phagolysosome of human monocytes grown in cell culture renders the purE mutant a useful agent for use as a live, attenuated vaccine. 
     The purEK operon and purE gene have not previously been described for Brucella. The organism wherein the purE gene has been deleted from Brucella purE mutant is the first genetically characterized mutant of Brucella that fails to replicate in human monocytes. Previously, no genetically defined mutants of Brucella have been successfully developed for use as live, attenuated Brucella vaccines or as vaccine carriers. The purE mutant may also be a safe and effective live-vaccine carrier strain for the production of non-Brucella antigen-specific cytotoxic T-cells, and memory T-helper cells with specific reactivity for recombinant Brucella or non-Brucella peptides. A purE deletion mutant of B. melitensis should be a safe and effective live-attenuated vaccine for the prevention of brucellosis in humans and in animals when used alone or in combination with other deletion mutations in brucella genes such as aro, rfb, htr, rec or global regulatory genes. The purE deletion mutant can be used as a live-attenuated vaccine carrier for the stimulation of immune responses to bacterial, viral and fungal pathogens or any other antigens of interest that can be expressed by the carrier organism. It would be particularly appropriate to vaccinate dairy workers and other animal handlers and packing house workers. 
     The attenuated vaccine would also be useful for administration to lower animals that are likely to be infected with Brucella species. The administration of vaccine to military personnel that might be subjected to the organisms during biological warfare would also be appropriate. Finally, vaccination against brucellosis of travelers who will be likely to ingest unpasteurized milk should be considered. 
     The purEK operon of Brucella melitensis strain 16M was isolated and sequenced by complementing a purE::Tn10 mutation in Escherichia coli. Based upon the DNA sequence of this operon, deletions in the open reading frames (ORFs) that correspond to the purK and purE genes were defined. Deletion of the internal EcoRI fragment resulted in E. coli carrying the purE::Tn10 mutation growing on minimal media only in the presence of 5% CO 2 , confirming the identification of the purk ORF. A deletion mutation of the purE ORF was made by PCR amplification of pURE194 with oligonuceotides designed with an unique BglII restriction endonuclease site on the 5&#39; ends. These primers produced a plasmid pURE197 that lacked bp 435-713 of the purE ORF. E. coli purE::Tn10 carrying this plasmid grew only on minimal plates supplemented with purines. A Bam HI fragment carrying a kanamycin resistance cassette was cloned into the BglII site of pURE197 to make pURE198. Gene exchange in B. melitensis was accomplished by electroporation of pURE198 into B. melitensis strain 16M. The plasmid vector does not replicate in B. melitensis and kanamycin resistant colonies arise following single or double cross-over events. Double cross-over events between the wild-type chromosomal copy of the purEK operon and the purEKmK gene on the plasmid results in a B. melitensis that is KMr, AMPs and PurE-. 
     METHODS AND MATERIALS 
     A HindIII genomic library of B. melitensis was screened for transformants that complemented purE::Tn10 mutation in Escherichia coli strain RR1. A plasmid carrying 19 kilobases of B. melitensis DNA was isolated from E. coli growing on minimal media. A subcloned fragment of 3.9 kb (pURE193) that complemented the purE::Tn10 mutation in E. coli was isolated from the original DNA and sequenced (GenBank accession number U10241). Two open reading frames encoding for two polypeptides of 17 kd and 40 kd were found. A search of the DNA and protein databases showed 64.5% homology of the 17 kd peptide with purE and 41.5% homology of the 40 kd peptide with purK of E. coli. Deletion mutations of the putative purE and purK genes were made in B. Melitensis strain 16M by gene replacement. Deletion of the purK locus produced a strain of Brucella that grew faster in 5% CO 2  than in air, consistent with a PurK-phenotype. Deletion of the purE locus produced a Brucella strain that requires purines for growth on M9 minimal media, consistent with a purE mutation. A deletion mutation within purE was made by PCR amplification. These primers produced a fragment that deleted bp 435-713 of the pURE193. Next, a BamHI fragment carrying a kanamycin resistance cassette was cloned into an internal BglII site of the deletion mutant. The resulting plasmid was electro-porated into B. Melitensis strain 16M. DNA hybridization analysis of DNA isolated from two purE strains confirmed that the wild-type gene was replaced by a single copy of the purE deletion mutation carrying the kanamycin resistance cassette. Isolated human monocytes with wild-type 16 M and with purE- B. Melitensis strains BM198-7 and BM201-1 were infected. While the parent 16M strain increased over two logs in 72 hours, the purE mutants decreased by 2 logs. The purE mutation did not reduce the number of bacteria recovered one hour after infection of the monocytes when compared with wild-type 16M. These results show that the purE mutation reduces the capacity of B. Melitensis to grow within human monocytes without altering its capacity to infect the host&#39;s cells. 
     FIG. 1 shows the purE coding region of Brucella melitensis. Only the inserted portions of these recombinant plasmids are shown. Plasmid phenotypes were determined by complementation tests using E. coli RR1 purE::Tn10 mutant as the cloning host. Plasmid pURE19 was constructed by ligating the 4.0 kb HindIII-BgIII fragment of pURE3H into pUC19, which was previously digested with HindIII and BamHI. Plasmid pURE191 and pURE192 were constructed by subcloning the 3.8 kb HindII-CIaI and the 3.3 kb HindIII-Xbal fragments of pUREl9 into the corresponding sites of pACYC184 vector. Plasmid pURE193 was constructed by subcloning the 2.2 kb HindIII-SphI fragment of pUREl9 into its corresponding sites of pBR328. 
     FIG. 2 shows pURE3H wherein the 7.0 kb HindIII of B. melitensis has been inserted into pHC79. to provide the 13.4 kb pURE3H. 
     The DNA containing the purE mutation contains the following sequence: 
     
         __________________________________________________________________________GTGAGCGGAT AACAATTTCA CACAGGAAAC AGCTATGACC ATGATTACGC CAAGCTTCAGGAAATGGAAG ATCAGGTCAT TCCTGACATC ATTGCCTGAA CATTCAAGAA CACTATAGGGAAGAGCCGGG GTTCGCTGCT TGTCTTTTCA AGACCTTCCT CATGCCAACC GAAAAGCCAAGCAGGAAAGA CCGATGAGCG TTGATGTCGC CATTATCATG GGAAGCCAGT CCGATTGGGAAACCATGCAC CATGCAGCCG ACACATTGGA GGCGCTCGGC ATCTCCTTCG ACGCACGGATCGTTTCCGCC CATCGCACCC CTGACAGGCT GGTCGCCTTC GCCAAGGGGG CGAAACGGGAAGGCTTCAAG GTCATCATCG CAGGCGCCGG CCGCGCCCAC CTGCCCGGCA TGGCCGCTGCCATGACACCG CTTCCCGTCT TTGGCGTTCC AGTTCAATCC AAGGCGCTTT CGGGCCAGGATTCGCTACTC TCCATCGTAC AGATGCCAGC GGGTATTCCC GTCGGCACTC TCGCCATCGGCCGTGCAGGC GCGGTCAATG CCGCCCTTCT CGCCGCCGCC GTTCTGGCGC TATATGATGAAGCACTTGCC GCCCGTCTTG ATGAATGGCG CAAGGCGCAG ACCGAAAGCG TTGCAGAACGCCCCTCGAAC GAAGCCTGAT CCGGCGAGGT AGAAAATGGA CAAGACATCT CTCAAGCCCGGCTCCACCAT CGGCATTATC GGCGGGGGCC AGCTTGGCGT ATGCTCGCCA TGGCAGCGGCGCGCTTCGGT TATGAAACCA TAATCCTTGA GCCGCACGCG GTTGCCCGGC AGCACAGGTTGCCAATCGCC AGATTGTCGC CGCCTATGAT GACCCGAAGC GCTGGCCGAA ACTTGCCGCCGCTTCCGACG TCATCACCTA TGAATTTGAA AATGTGCCAG TCAGCGCCGC CGACAAGCTGGCTGAAACGG CGCTTGTTCT GCCCCCGCCC GCCGCACTGG AAATCTCTCA GGACCGCTTCACAGAAAAGC AGTTTCTCAA CGAAAGCGGC ATTGAAACCG CGCCCTGGCG GCTCGTGGATGACGAGGAAA CGCTCATCGC CGCGCTCGGC ACTTGGGCGG GCGTGGCATC CTCAAGATCGGCGTCTGGGT TATGACGGCA AGGGGCAGGT GCGCCTTGCC TCCCTCGATG AAACCCAGGCCTGCAACGCT TTTGCAGCCA TCAACAAGGC GCCTGCGATT CTCGAAGGCT TCGTGGAATTCGAGCGCGAA GTCTCCGTCA TCGCCGCGCG CGATCGCAGC GGCAATGTCG CCATCTTCGATCTTGCGGAA AACGTCCACA AGGATGGCAT TCTCGCCACG TCCACAGTGC CTGCCGCGATCAGCGTACAG ACGGCAGAAG CCGCGCGCAC AGCCGCCGAA AAACTGTTGC ACGCGCTGGACTATGTCGGT GTGCTGGGGC TTGAATTCTT CGTGCTGAAG GACGGCACGC TGCTCGCCAATGAATTTGCC CCGCGTGTGC ATAATTCGGG CCACTGGACG GAAGCAGCCT GCGCCATTTCCCAATTTGAG CAGCATATCC GCGCTGTGGC GGGACTGCCG CTCGGCAATA CGGATCGCCATAGCGACTGT GTGATGGAAA ACCTGATTGG CGACGATATC GAAAAGGTTC CGGCGATTCTCTGCGAGAAG AACGCCGTGC TGCATCTTTA CGGTAAAAAG GAAGCTCGCG CGGGCCGGAAAATAGGCCAT GTGACCCGCA TAAAGCCCCG CACAATTTAA GCTGCGCCGG GAATCTGCACGATTCCCGGC CCTTCCTGGC CGCCCGCCAA GAAATTCGGG GCCTCGGACC CTGAATCTGCGGCGCGGGAG TTGACATTTG CCTGAAACCT TGTGTATTTC GGCCAACTTC GGCACCTGAC__________________________________________________________________________ 
    
     which is designated as Sequence #1 herein and has been deposited in the GenBank wherein the accession number U10241 has been assigned. 
     The 17 kD and 40 kD polypeptides encoded by Sequence #1 are: 
     
         Met Ser Val Asp Val Ala Ile Ile Met Gly Ser Gln Ser Asp Trp Glu                                  (Sequence #2) 1               5                   10                  15Thr Met His His Ala Ala Asp Thr Leu Glu Ala Leu Gly Ile Ser Phe        20                 25                  30Asp Ala Arg Ile Val Ser Ala His Arg Thr Pro Asp Arg Leu Val Ala    35                  40                  45Phe Ala Lys Gly Ala Lys Arg Glu Gly Phe Lys Val Ile Ile Ala Gly50                  55                  60Ala Gly Arg Ala His Leu Pro Gly Met Ala Ala Ala Met The Pro Leu 65                  70                  75                  80Pro Val Phe Gly Val Pro Val Gln Ser Lys Ala Leu Ser Gly Gln Asp            85                  90                  95Ser Leu Leu Ser Ile Val Gln Met Pro Ala Gly Ile Pro Val Gly Thr       100                 105                 110Leu Ala Ile Gly Arg Ala Gly Ala Val Asn Ala Ala Leu Leu Ala Ala   115                 120                 125Ala Val Leu Ala Leu Tyr Asp Glu Ala Leu Ala Ala Arg Leu Asp Glu    130                 135                 140Trp Arg Lys Ala Gln Thr Glu Ser Val Ala Glu Arg Pro Ser Asn Glu145                 150                 155                 160AlaandMet Leu Ala Met Ala Ala Ala Arg Phe Gly Tyr Glu Thr Ile Ile Leu                                  (Sequence #3 1               5                   10                  15Glu Pro His Ala Val Ala Arg Gln His Arg Leu Pro Ile Ala Arg Leu       20                  25                  30Ser Pro Pro Met Met Thr Arg Ser Ala Gly Arg Asn Leu Pro Pro Leu    35                  40                  45Pro Thr Ser Ser Pro Met Asn Leu Lys Met Cys Gln Ser Ala Pro Pro50                  55                  60Thr Ser Trp Leu Lys Arg Arg Leu Phe Cys Pro Arg Pro Pro His Trp 65                  70                  75                  80Lys Ser Leu Arg Thr Ala Ser Gln Lys Ser Ser Phe Ser Thr Lys Ala            85                  90                  95Ala Leu Lys Pro Arg Pro Gly Gly Ser Trp Met Thr Arg Lys Arg Ser       100                 105                 110Ser Pro Arg Ser Ala Leu Gly Arg Ala Trp His Pro Gln Asp Arg Arg   115                 120                 125Leu Gly Tyr Asp Gly Lys Gly Gln Val Arg Leu Ala Ser Leu Asp Glu    130                 135                 140Thr Gln Ala Cys Asn Ala Phe Ala Ala Ile Asn Lys Ala Pro Ala Ile145                 150                 155                 160Leu Glu Gly Phe Val Glu Phe Glu Arg Glu Val Ser Val Ile Ala Ala           165                 170                 175Arg Asp Arg Ser Gly Asn Val Ala Ile Phe Asp Leu Ala Glu Asn Val       180                 185                 190His Lys Asp Gly Ile Leu Ala Thr Ser Thr Val Pro Ala Ala Ile Ser   195                 200                 205Val Gln Thr Ala Glu Ala Ala Arg Thr Ala Ala Glu Lys Leu Leu His    210                 215                 220Ala Leu Asp Tyr Val Gly Val Leu Gly Leu Glu Phe Phe Val Leu Lys225                 230                 235                 240Asp Gly Thr Leu Leu Ala Asn Glu Phe Ala Pro Arg Val His Asn Ser           245                 250                 255Gly His Trp Thr Glu Ala Ala Cys Ala Ile Ser Gln Phe Glu Gln His       260                 265                 270Ile Arg Ala Val Ala Gly Leu Pro Leu Gly Asn Thr Asp Arg His Ser   275                 280                 285Asp Cys Val Met Glu Asn Leu Ile Gly Asp Asp Ile Glu Lys Val Pro    290                 295                 300Ala Ile Leu Cys Glu Lys Asn Ala Val Leu His Leu Tyr Gly Lys Lys305                 310                 315                 320Glu Ala Arg Ala Gly Arg Lys Ile Gly His Val Thr Arg Ile Lys Pro           325                 330                 335Arg Thr Ile 
    
     Adams (cited supra) outlines a systematic approach to the valuation of the safety and efficacy of candiate Brucella vaccines. Appropriate vaccines are shown to have alter growth in vitro, reduced growth in macrophages, peeripheral blood monocytes or in tissue culture systems should be assayed in mice. Although the mouse model is not ideal, it is useful for deciding which candidate vaccines should be tested in the natural host. Under testing as described, B melitensus ΔpurE201 is attenuated for growth in both human and mouse macrophages. Further, pSD5, a plasmid carrying an expressed copy of the purE gene, restores the capacity of B. melitensis ΔpurE201 to grow on minimal media without exogenous purines and within human macrophages, confirming that the attnuated growth is due to the purE deletion and not due to some unknown secondary mutation. The purE mutation described was attenuated in human monocytes, in mice and in goats. 
     B melitensis ΔpurE201 as attenuated in the mouse was cleared from the spleens of mice by eight weeks following intraperitoneal injection. Wild-type 16M is not cleared from the spleens of mice. Table 1 shows that the ΔpurE201 is attenuated when given orally to mice. These results show that ΔpurE201 is unable to scavenge sufficient purines for persistent growth in tissues of the infected host. 
     
                       TABLE I______________________________________Oral attenuation of B meitensus ΔpurE201Infection of BALB/c mice after oral challenge with B. melitensisstrains  Week 1        Week 2  Week 4______________________________________16M      2/5           4/5     3/5purE201  0/5           0/5     0/5______________________________________ 
    
     To test the immunizing ability of ΔpurE201, BALB/c mice were injected with 10 5  colony forming units. Rising antibody titers against Brucella LPS was shown following vaccination. The antibody induced by ΔpurE201 cross reacts with LPS prepared from B. abortus 2308 LPS following vaccination. It was shown that the ΔpurE201 induces protective immunity from intranasal challenge with wildtype 16M. 
     TEST 
     PROTECTION AGAINST INTRANASAL CHALLENGE 
     Mice were immuinized with 10 5  B melitensis ΔpurE201 ip. then challenged 9 weeks later with 10 5  16M in harvested at 4 weeks to determine recovery of B melitensis. Among the nonimmunized mice, 8 of 10 were infected. Among those immunized with the mutant, only 2/10 were infected. 
     The study was extended with spleens being harvested at varying intervals. The mice were immunized with 10 5  B. melitensus ΔpurE201 i.p, then challenged 9 weeks later with 10 4  16M intranasally. (See Table 2 below.) 
     
                       TABLE 2______________________________________Protection against intranasal challenge.The number showing infected spleens at various intervals isindicated.     4 weeks   8 weeks 13 weeks______________________________________noniminunized       8/10        6/10    8/10purE-immunized       2/10        4/10    4/10______________________________________ 
    
     In view of the above, it is seen that vaccines produced by the defined mutations in E coli with plasmids carrying the mutated Brucella genes of the invention having the defined deletions provides non-reverting deletions mutants which stimulate protective immunity. 
     Improved safety may be attained by introducing a second defined attenuating mutation. In addition to mutations which impair ability to synthesize purine, other mutations may be effective. Examples of other attenuating mutations used alone or in combination with the purE included deletions in the biosynthetic pathway for LPS biosynthesis producing a rough phenotype. These mutations attenuate Brucella and prevent the vaccinated animal from seroconverting to LPS the predominant antibody detected in immune surveillance of animals. 
     Many deletions in genes required for homologous recombination have been shown to attenuate live-bacterial vaccines. A combination purE recA mutation of Brucella can be made by making a deletion of the recA gene based upon the published DNA sequence of this gene. 
     Living organisms respond to stress by producing a number of proteins referred to as heat-shock proteins. Deletion of one of these genes htrA has been shown to attenuate bacteria. A combination purE htrA vaccine strain can be made based upon the published sequence of the Brucella htrA gene. In addition to the double defined deletion mutations, combinations of three or more genes may be constructed. In all cases, those strains carrying a recA mutation will have the recA deletion added last, since this mutation will prevents gene replacement by recombination. Finally, bacterial RNA polymerase uses sigma factors to initiate gene transcription. Deletion mutants in sigma factor genes can be combined with purE to produce live-attenuated genes. 
     Live-attenuated Brucella can be used as a vaccine vector to deliver foreign antigens. Since Brucella is a mucosal pathogen and survives within host cells it should have the capacity to stimulate mucosal, systemic and cellular immune responses to the delivered antigen. Processing and presentation of antigens via Class 1 HLA surface molecules of antigen-presenting cells may stimulate strong cytotoxic T-cell responses by both CD4 and CD8 T-cells to the target antigen.