Patent Grant
8084043
The present invention relates to the use of Pasteurella lipoprotein E (PlpE) in a subunit vaccine, and the use of vaccines containing PlpE to protect animals from diseases caused by P. multocida.
Pasterurella multocida is classified into family Pasteurellaceae, genus Pasteurella, and multocida means “causing many kinds of animal disease” in Latin. In the past, the taxonomic situation of Pasteurellaceae was ambiguous and arguable. However, according to the sequence analysis of 16s-RNA, Pasteurellaceae is classified into a gamma subgroup of purple bacteria, which is Gram-negative facultative anaerobe, and homologous to Enterobacteriaaceae. Pasteurella multocida is an important pathogen of domestic animals and an opportunistic pathogen of humans. Human infections with P. multocida largely arise from the bite of an infected carnivore, but other types of infections are occasionally reported (Hubbert W T, et al., Am J Public Health 1970; 60:1109-17). P. multocida has wide host range, and is the causative agent of fowl cholera in domestic birds, haemorrhagic septicaemia in cattle or sheep, and atrophic rhinitis in pigs (Hunt M L, et al., Vet Microbiol 2000, 72:3-25). Most P. multocida strains are highly pathogenic to murine and rabbits, which can result in acute septic symptoms at an infection dose of 1-10 CFU.
In domestic birds, P. multocida causes acute septic disorders in turkey, chicken, duck, and goose, which may lead to a great loss in economy. Of the five capsular serotypes (A, B, D, E and F) and 16 LPS serotypes, fowl cholera is mainly caused by serotypes A:1, A:3 and A:4 (Glisson J R. In: Saif Y M, editor. Diseases of poultry. Iowa State University Press, Ames, Iowa, 2003:657-90). Although both live-attenuated vaccines and bacterins are available, outbreaks of fowl cholera continue to occur. Live-attenuated vaccines have the disadvantage of reversion to virulence, while bacterins do not protect hosts against heterologous challenge (Bierer B W, Derieux W T, Poult Sci 1972; 51:408-16; and Rebers P A, Heddleston K L, Avian Dis 1977; 21:50-56). These disadvantages call for the development of a new type of vaccine for P. multocida.
In a previous report, a lipoprotein, designated Pasteurella lipoprotein E (PlpE), from Mannheimia haemolytica (formerly known as Pasteurella haemolytica), was found to be highly immunogenic in cattle (Confer A W, et al., Vaccine 2003; 21:2821-9). PlpE is a lipid-modified, surface-exposed outer membrane protein that is important in complement-mediated killing of M. haemolytica (Pandher K, et al., Infect Immun 1998; 66:5613-9). Addition of recombinant PlpE to the commercial M. haemolytica vaccine markedly enhanced the vaccine-induced resistance against experimental challenge with serotypes 1 and 6. A bioinformatics-based sequence search showed that a gene annotated PlpE is present in the published genome sequence of P. multocida strain pm-70 (serotype A:3) (May B J, et al., Proc Natl Acad Sci USA 2001; 98:3460-5). This gene has the potential to encode a lipoprotein of 335 amino acids that has 24.3% sequence identity with PlpE of M. haemolytica and 19.1% identity with OmlA of A. pleuropneumoniae. It has not yet been determined whether the PlpE of P. multocida could serve as a vaccine antigen by using mice and/or chicken as animal models.
Therefore, the present invention first finds out a new use of Pasteurella lipoprotein E (PlpE) in controlling infective diseases caused by P. multocida, or related disorders thereof, and further develops a subunit vaccine for protecting animal from diseases caused by P. multocida. The subunit vaccine of present invention is characterized by using recombinant PlpE protein as active antigen, which has no adverse side effect of forming fibrosarcoma. Additionally, the present subunit vaccine of P. multocida can provide a predominant protective effect against the challenge of homologous and/or heterologous serotypes in immunized animals over traditional inactivated or live-attenuated vaccines.
In one aspect, the present invention provides a subunit vaccine for protecting animal from diseases caused by P. multocida, which is characterized by comprising Pasteurella lipoprotein E (PlpE) as antigen, and a veterinary acceptable adjuvant. In one embodiment, the Pasteurella lipoprotein E is a protein having the amino acid sequence as listed in SEQ ID EF219452-EF219457 (SEQ ID NO:2, 4, 6, 8, 10 and 12), or an amino acid sequence with similarity of more than 90% to the amino acid sequence as listed in SEQ ID EF219452-EF219457 (SEQ ID NO:2, 4, 6, 8, 10 and 12). In another embodiment, the disease caused by P. multocida is fowl cholera in domestic birds, haemorrhagic septicaemia in cattle, or atrophic rhinitis in pigs.
In another aspect, the present invention provides a use of Pasteurella lipoprotein E in controlling infective diseases caused by P. multocida, or related disorders thereof. The disease caused by P. multocida infection may be fowl cholera in domestic birds, haemorrhagic septicaemia in cattle, or atrophic rhinitis in pigs. In other animals, such as mice, rabbits, cats, or dogs, P. multocida infection may cause haemorrhagic septicaemia.
The present invention will be further defined by reference to the following examples, which are set forth to assist in understanding the invention and should not be construed as specifically limiting the invention. Therefore, any modification or derivative made without departing from the spirit of this invention will be considered to fall within the scope of the invention.
A. Bacterial Strains and Genomic DNA Extraction of P. multocida
P. multocida standard strains X-73 (A:1), P-1059 (ATCC 15742) (A:3), and P-1662 (A:4) were grown at 37° C. in BRAIN-HEART INFUSION (BHI) broth (Difco Laboratories, MI, USA) for 18-24 hours. Bacterial genomic DNA was isolated using the DNEASY® TISSUE KIT (QIAGEN, Hilden, Germany).
B. Genetic Cloning and Expression Vector Construction for Recombinant Lipoprotein E (r-PlpE)
One set of primers, P1 and P2, was used to amplify the PlpE gene from P. multocida strain X-73. The amplified genes were then used for expressing recombinant PlpE (r-PlpE) in E. coli. These primers contained restriction enzyme (NcoI or XhoI) cutting sites at their 5′-ends (underlined sequences), followed by sequences specific to PlpE. The PCR product was cloned into the expression vector pET28a according to the manufacturer's instructions (Novagen, Inc. Madison, Wis.) to obtain a plasmid designated as pX73-PlpE. The identity of the insert in pET28a was verified by DNA sequence analysis. The amino sequence of r-PlpE protein is described in SEQ ID: 2.
C. Expression and Purification of Recombinant Lipoprotein E (r-PlpE)
Recombinant plasmid pX73-PlpE obtained in section B was transformed into E. coli strain BL21 (DE3) and recombinant protein was purified by nickel chromatography as previously described (Chang P C, et al., 2002, Avian Dis 46:570-80). In brief, E. coli strain BL21 (DE3) harboring the recombinant plasmid was cultured in LB medium at 37° C. until absorbance at 600 nm reached 0.6. Isopropylthio-β-D-thiogalactose (IPTG) was added to a final concentration of 0.4 mM, and the culture was grown for another 3 hrs. Cells were pelleted by centrifugation at 3000×g for 20 min, and resuspended in 2 ml of binding buffer (20 mM pH 7.9 Tris, 5 mM imidazole, 500 mM NaCl). The suspension was sonicated and centrifuged at 12,000×g for 40 min. The supernatant was collected and loaded into a column containing 2.5 ml of “His-bind” resin (Novagen). The column was washed with 25 ml of binding buffer and 15 ml of washing buffer (20 mM pH 7.9 Tris, 50 mM imidazole, 500 mM NaCl) to remove the unbound proteins. The bound protein was eluted with 15 ml of eluting buffer (20 mM pH 7.9 Tris, 250 mM imidazole, 500 mM NaCl), only the first 3 ml of the elute was collected. Protein concentration was determined using a “Protein Assay” kit (BIO-RAD, Hercules, Calif., USA).
The expression product and purity of the recombinant protein was observed by SDS-PAGE and Western blotting analysis, respectively. The results were showed in
As showed in
Western blot analyses using anti-hexa-histidine monoclonal antibody showed that this monoclonal antibody reacted with r-PlpB and r-PlpE (
Three experiments were conducted in BALB/c mice. In experiments 1 and 2, groups of 6-week-old mice were immunized subcutaneously with 10 micrograms of purified r-PlpB or r-PlpE in aluminum hydroxide adjuvant (Sigma-Aldrich Co., MO, USA), either alone or together with a bacterin composed of 1.25×107 or 2.5×107 CFU of formalin-inactivated P. multocida X-73 (A:1). Two weeks after immunization, mice were challenged with subcutaneous injection of 10-20 LD50 of strain X-73. In experiment 3, mice were immunized as described for experiments 1 and 2. Two weeks after immunization, mice were challenged with subcutaneous injection of 10 LD50 of strains P-1059 (A:3) or P-1662 (A:4), or strain T2A5 (which is a designated challenge strain used in drug inspection in Taiwan). All mice challenged were observed for 10 days and their survival rates were recorded. The results are summarized in Table 1. For statistical analysis, the survival rates were compared by Chi-squared tests using SAS® software (SAS Institute, Inc., Cary, N.C., USA). The mean times to death were compared using the GLM procedure in the same software. Differences were considered significant when p<0.05.
1Mice in the control group were not immunized.
2The LD50 of strains X-73 and P1662 in mice was <3 CFU, and that of P-1059 was 3.5 CFU.
3Different alphabetical characters indicate significant difference (p < 0.05) between groups.
In experiment 1, mice immunized with 10 microgram of purified r-PlpE were completely protected (100% survival) (Table 1, experiment 1). In contrast, mice immunized with 10 microgram of purified r-PlpB were not protected (0% survival) against challenge infection with 30 CFU (>10 LD50) of X-73 (serotype A:1). Mice immunized with a bacterin composed of 2×108 CFU of formalin-inactivated X-73 were completely protected (100% survival), whereas those immunized with a bacterin composed of a lower dose (1.25×107 CFU) of X-73 were not protected (17% survival) (Table 1, experiment 1). To investigate whether r-PlpB or r-PlpE could enhance the protective efficacy of the bacterin, mice were immunized with a bacterin composed of 1.25×107 CFU of X-73 supplemented with 10 microgram r-PlpB or r-PlpE. The results showed that r-PlpB did not significantly enhance the protective efficacy of the bacterin (30% survival, p>0.05) whereas r-PlpE did (100% survival, p<0.05) (Table 1, experiment 1).
In experiment 2, the challenge dose of X-73 was increased to 60 CFU (>20 LD50) and a bacterin composed of 2.5×107 CFU of X-73 was used. The results showed that mice immunized with 10 microgram of r-PlpB were not protected (10% survival) whereas those with 10 microgram of r-PlpE were significantly protected (80% survival, p<0.05) (Table 1, experiment 2). Mice immunized with a bacterin composed of 2.5×107 CFU of X-73 were moderately protected (50% survival). Mice immunized with the same bacterin supplemented with r-PlpB showed a survival rate of 40%, which was similar to that with the bacterin alone. In contrast, mice immunized with the bacterin supplemented with r-PlpE showed a survival rate of 90%, which was significantly higher than that with the bacterin alone (p<0.05) (Table 1, experiment 2).
In experiment 3, strains P-1059 (serotype A:3) and P-1662 (serotype A:4) were used as the challenge strains. The results showed that mice immunized with 10 microgram of r-PlpE were completely protected against challenge infection with 10 LD50 of P-1059 or >10 LD50 of P-1662 (Table 1, experiment 3). This result showed that r-PlpE, which was derived from X-73 (serotype A:1), conferred cross protection on mice against challenge with strains of serotypes A:3 and A:4. Additionally, mice immunized with 10 microgram of r-PlpE showed a survival rate of 90% when challenge with strain T2A5 (A:1), which was up to the proof inspection standard (survival rate of 60%) (p<0.05) (Table 1, experiment 3).
Three experiments in SPF chickens were conducted. In experiment 1, groups of 3-week-old SPF chickens were immunized subcutaneously with 100 micrograms of purified r-PlpB or r-PlpE in complete Freund's adjuvant (Sigma-Aldrich). Three weeks after the primary immunization, a booster immunization was conducted, and three weeks after booster immunization, chickens were challenged with intramuscular injection of 3.6×103 CFU of strain X-73 or 5.5×108 CFU of strain P-1662. In experiments 2 and 3, chickens were immunized subcutaneously twice with 125 micrograms of a crude extract of r-PlpE in a double emulsion adjuvant with a 3-week interval between immunizations. The crude extract was prepared by sonicating the pellet of E. coli that expressed r-PlpE. The double emulsion adjuvant contained Marcol 52 oil (63%), Arlacel A (7%), and Tween 80 (1.5%). Three weeks after booster immunization, chickens were challenged by intramuscular injection of 3.6×103−3.6×106 CFU of strain X-73 or 5.5×107−5.5×109 CFU of strain P-1662. All chickens challenged were monitored for 10 days and the survival rates were recorded. The results are summarized in Table 2. For statistical analysis, the survival rates were compared by Chi-squared tests using SAS® software (SAS Institute Inc., Cary, N.C., USA). The mean times to death were compared using the GLM procedure in the same software. Differences were considered significant when p<0.05.
1 Immunization was conducted twice with a 3-week interval. Chickens in the control group were not immunized.
2, 3 Different alphabetical characters indicate significant difference (p < 0.05) between immunization and control groups challenged with the same dose of X-73 or P-1662.
In experiment 1, chickens immunized twice with 100 microgram of purified r-PlpB showed a survival rate of 50% against challenge with X-73, but this survival rate was not significantly higher than that of the control group (30% survival, p>0.05). In contrast, chickens immunized twice with 100 microgram of purified r-PlpE showed a survival rate of 100%, which was significantly higher than that of the control group (p<0.05) (Table 2. experiment 1). This result suggests that r-PlpE but not r-PlpB conferred protection on chickens. A similar conclusion was reached when strain P-1662 was used as the challenge strain (Table 2, experiment 1).
In experiments 2 and 3, a crude extract of r-PlpB and r-PlpE (
In experiment 3, P-1662 was used as the challenge strain. The results showed that chickens immunized with 125 microgram of crude extract of r-PlpE had a survival rate of 50% against challenge with 5.5×107−5.5×109 CFU of strain P-1662. These rates were not significantly higher than those of the control groups (p>0.05), except when the challenge dose of P-1662 was 5.5×109 (p<0.05) (Table 2, experiment 3). The mean times to death of immunized chickens were not significantly longer than those of the control groups (p>0.05) (Table 2, experiment 3).
Two primers, P3 and P4, were used to amplify the PlpE genes from different reference strains of P. multocida, X-73 (A:1), pm-70 (A:3), P-470 (A:3), P-61 (D:3), P-1059 (A:3), P-1662 (A:4), and ATCC 12948 (D:11). The two primers were designed on the basis of the published genome sequence of P. multocida strain pm-70. P3 and P4 amplified the 1.0 kb DNA fragment containing the PlpE gene. The sequences of primers P3 and P4 were as follows. P3: 5′-ATG AAA CAA ATC GTT TTA AA-3′ (SEQ ID NO:13), and P4: 5′-TTA TTG TGC TTG GTG ACT TT-3′ (SEQ ID NO:14). The PCR products were purified with a QIAQUICK® GEL EXTRACTION KIT (QIAGEN) and sequenced from both directions using a BIGDYE® TERMINATOR CYCLE SEQUENCING KIT (Applied Biosystems, Foster City, Calif.) in an automatic sequencer (ABI-3730XL DNA ANALYZER®, Applied Biosystems). Sequences were compiled using the SEQMAN® program in the LASERGENE® package (DNASTAR Inc. Madison, Wis., USA). Open reading frames prediction and antigenic index assay were performed using the GENEQUEST and PROTEAN programs from the same package. Nucleotide and protein sequences were searched for homology in GenBank using the BLAST program provided by NCBI, USA.
The nucleotide sequences of the PlpE gene determined in this study are available in GenBank under the accession numbers EF219452-EF219457 (corresponding to the SEQ ID NO:1, 3, 5, 7, 9, and 11) in the appending sequence listing). All these PlpE genes were found to contain an open reading frame of 1008-1019 nt, encoding a PlpE protein of 37.4-37.7 kDa. Pair-wise sequence comparison showed that these PlpE proteins had 90.8-100% sequence identity with each other, suggesting that PlpE might serve as a cross-protective antigen. This is the first report of a recombinant P. multocida antigen that confers cross protection on animals. Therefore, a protein having the amino acid sequence as listed in SEQ ID NO:2, 4, 6, 8, 10, and 12, or an amino acid sequence with similarity of more than 90% to the amino acid sequence as listed in SEQ ID NO:2, 4, 6, 8, 10, and 12, is considered to exhibit highly similar protective effects, and contemplates to be included in the subunit vaccine of the present invention.
The above examples are given by way of illustration only, and should not be construed as specifically limiting the scope of present invention. Any variation of the invention described and claimed herein, including the substitution of all equivalents, which would be within the purview of those skilled in the art, is to be considered to fall within the scope of the invention incorporated herein.
The strain E. coli BL21 (DE3) containing the recombinant vector X73-plpE of the invention was deposited with the Agricultural Research Service Culture Collection (NARRL), on Feb. 29, 2008, as Deposit No. NARRL B-50117.
| Number | Date | Country | Kind |
|---|---|---|---|
| 96108321 A | Mar 2007 | TW | national |
| Number | Date | Country |
|---|---|---|
| 1 350796 | Oct 2003 | EP |
| Number | Date | Country | |
|---|---|---|---|
| 20090246229 A1 | Oct 2009 | US |
