Abstract:
The present invention is related with the field of Biomedicine. It comprises the engineering of the Pertactin protein (Prn) and using it as part of bacterial vaccines, and more precisely, as part of acellular vaccines against  Bordetella pertusis . The engineered Prn molecules comprise on their structure polimorfisms from different  B. pertussis  strains, and induce immune responses with protective capacity and opsonophagocytic activity when assayed as vaccines, higher than that generated by other pre-existing vaccines. The engineered Prn variants of the present invention are applicable in human and veterinary medicine.

Description:
TECHNICAL FIELD 
       [0001]    The present invention is related with the field of Biomedicine. It comprises the engineering of the Pertactin protein (Prn) and using it as part of bacterial vaccines, and more precisely, as part of acellular vaccines against  Bordetella pertusis . The engineered Prn molecules comprise on their structure polimorfisms from different  B. pertussis  strains, and induce immune responses with increased protective capacity and opsonophagocytic activity when assayed as vaccines, higher than that generated by other pre-existing vaccines. 
       BACKGROUND OF THE INVENTION 
       [0002]    Whooping Cough or  Pertussis  is an acute, highly infectious respiratory disease caused by the  Bordetella pertussis  bacterium, a microorrganism formerly isolated by Bordet and Gengou in 1906 [Bordet, J. and O. Gengou. Ann Inst Pasteur (Paris), 1906. 20: p. 731-41]. Recently, the annual morbidity of infections throughout the world was estimated in 48.5 millions. The disease is particularly severe in children with less than six months of age, with 90% of the casualties being associated to this ethareal group (300,000-400,000) [Crowcroft, N. S., et al. Lancet Infect Dis, 2003. 3(7): p. 413-8]. 
         [0003]    Several vaccines are available against  B. pertussis , distributed in two main groups according to their type: cellular vaccines, and, more recently, acellular vaccines. Vaccination has dramatically decreased the incidence of the disease, moving it from children towards teenager and adult populations. Several studies have shown teenagers as the major reservoir for  B. pertussis  and the main source for spreading of this disease among partially protected children. Therefore, whooping cough remains as an unsolved health problem, demanding the development of new vaccines for a better control of the epidemics and re-emergent outbreaks, and possibly to eradicate this disease in endemic regions [Cherry, J. D. Pediatrics, 2005. 115(5): p. 1422-7; Singh, M. and K. Lingappan, Chest, 2006. 130(5): p. 1547-53]. The  Bordetella  genera includes nine species, four of them being associated to infections in mammals ( B. holmesii, B. bronchiseptica, B. parapertussis  and  B. pertussis ), the last two being responsible for infections in humans [Mattoo, S., et al. Front Biosci, 2001. 6: p. E168-86]. Most of their virulence factors are regulated at transcriptional level by a two-components system denominated BvgA/S ( Bordetella  virulence genes Activator/Sensor) [Stibitz, S., et al. Nature, 1989. 338(6212): p. 266-9]. Among them, the most relevant factors are the  Pertussis  toxins (PT), the tracheal colonization factor, adenylate cyclase; and adhesins filamentous Phytohemagglutinin (PHA), Fimbriae (Fim) and Pertactin (Prn), the latter being the focus of the present invention. 
         [0004]    Prn is an outer membrane protein belonging to the family of type V autotransporter proteins. It is characterized by catalyzing its own transportation through the bacterial outer membrane [Henderson, I.R. Trends Microbiol, 2000. 8(12): p. 534-5]. The mature Prn is a protein of 68 kDa in  B. bronchiseptica  [Henderson, I.R. Infect Immun, 2001. 69(3): p. 1231-43.], 69 kDa in  B. pertussis  [Charles, I. G., et al. Proc Natl Acad Sci USA, 1989. 86(10): p. 3554-8] and 70 kDa in  B. parapertussis  [Li, L. J., et al. Mol Microbiol, 1991. 5(2): p. 409-17], respectively. Its structure consists on 16 paralel strands forming a β helix and a transversal section in V form [Emsley, P., et al. Nature, 1996. 381(6577): p. 90-2.]. Numerous loops protrude from this helicoidal core. One of them is the Arg-Gly-Asp triplete (RGD), a motif associated to tissue adherence [Leininger, E., et al. Infect Immun, 1992. 60(6): p. 2380-5; Emsley, P., et al. Nature, 1996. 381(6577): p. 90-2]. The presence of this motif and numerous proline-rich regions are related to Prn functions during adhesion. Experiments have shown that the Prn can mediate adhesion to cells of the respiratory epithelium [Everest, P., et al. Microbiology, 1996. 142 (Pt 11): p. 3261-8]. Nevertheless, assays on the inhibition by human sera of  B. pertussis  adhesion to A549 cultured cells (alveolar human epithelium) did not evidence Prn as a crucial element during that process under the tested conditions [Rodriguez, M. E., et al. FEMS Immunol Med Microbiol, 2006. 46(1): p. 39-47]. 
         [0005]    The Prn protein is part of acellular vaccines composed of three or more components. Acellualr vaccines can be composed of: 1) one component of PT, 2) two components: PT and PHA, 3) three components: PT, PHA and Prn, and 4) five components, including the three components previously mentioned and also the Fimbriae 2 (Fim2) and Fimbriae 3 (Fim3) proteins. In humans, the levels of the anti-Prn, anti-Fim2 and anti-PT antibodies correlate with protection levels against the disease [Cherry, J. D., et al. Vaccine, 1998. 16(20): p. 1901-6; Storsaeter, J., et al. Vaccine, 2003. 21(25-26): p. 3542-9]. 
         [0006]    The active immunization with Prn of  B. pertussis  and  B. bronchiseptica  induces a specific antibody response against Prn, confering protection in different animal models [Charles, I. G., et al. Eur J Immunol, 1991. 21(5): p. 1147-53; Roberts, M., et al. Vaccine, 1992. 10(1): p. 43-8]. Similarly, the passive administration of anti-Prn monoclonal antibodies (MAbs) protected mice in the model of respiratory challenge [King, A. J., et al. Microbiology, 2001. 147(Pt 11): p. 2885-95]. Protection levels in mice subjected to the intranasal challenge assay (INCA) were increased by adding Prn to vaccines containing PT and PHA [Guiso, N., et al. Vaccine, 1999. 17(19): p. 2366-76]. It has been recently shown that Prn is the only component of acellular vaccines which generates an antibody response of such a level that correlates to the opsonophagocytic activity [Hellwig, S. M., et al. J Infect Dis, 2003. 188(5): p. 738-42]. In spite of efficacious vaccines and the well established vaccination programs available, whooping cough is still endemic in regions of America, Europe and Asia, being considered as a re-emergent disease [Raguckas, S. E., et al. Pharmacotherapy, 2007. 27(1): p. 41-52]. One of the hypotheses trying to explain this phenomenon is based on the loss of efficacy, due to appearance of resistant strains [Mooi, F. R et al. Emerg Infect Dis, 2001. 7(3 Suppl): p. 526-8]. Prn is one of the most polymorphic proteins in  B. pertussis . It contains two variable regions designated as region 1 (R1) and 2 (R2), respectively, with repetitive amino acid sequences rich in proline Gly-Gly-X-X-Pro (GGXXP) and Pro-Gln-Pro (PQP) motifs. The R1 region is located in the protruding loop proximal to the aminoterminal sequence (N-terminal) and near to the RGD motif, while the R2 region is located near to the carboxyl terminal end (C-terminal) [Hijnen, M., et al. Infect Immun, 2004. 72(7): p. 3716-23]. Up to 12 different variants of Prn (Prn1, Prn2, Prn3 . . . Prn12) have been identified in  B. pertussis , as shown in the database of the National Center for Biotechnology Information of the United Staes of America (NCBI). Strains bearing the Prn1, Prn2 and Prn3 are distributed worldwide. Numerous strain characterization studies, either retrospective or of strains currently circulating, were carried out in American, European, Asian and Australian regions and showed a tendency towards a progressive persistence of Prn2 strains over Prn1 strains, the Prn2 strains predominating in most of the countries studied [Mooi, F. R., et al. Infect Immun, 1998. 66(2): p. 670-5; Cassiday, P et al. J Infect Dis, 2000. 182(5): p. 1402-8; Weber, C. et al. J Clin Microbiol, 2001. 39(12): p. 4396-403; Hallander, H. O., et al. J Clin Microbiol, 2005. 43(6): p. 2856-65; van Amersfoorth, S. C., et al. J Clin Microbiol, 2005. 43(6): p. 2837-43; Byrne, S, et al. BMC Infect Dis, 2006. 6: p. 53]. 
         [0007]    Current differences in the amino acid sequence of Prn between cellular (DTPc) or acellular (DPTa) vaccines and circulating strains is one of the factors supporting the hypothesis of the efficacy loss of vaccines available, due to the appearance of new strains. Studies in populations vaccinated with DPTc or DTPa, and non-vaccinated populations, in Netherlands and Italy indicated that these types of vaccines protect better against circulating strains similar to the vaccine strain [Mooi, F. R., et al. Infect Immun, 1998. 66(2): p. 670-5; Mastrantonio, P., et al. Microbiology, 1999. 145 (Pt 8): p. 2069-75]. In agreement with these findings, it was shown in the mice model that vaccination with DPTc differentially protects against strains bearing Prn1 and Prn2, indicating that changes in the Prn R1 region can confer resistance levels [King, A. J., et al. Microbiology, 2001. 147(Pt 11): p. 2885-95]. However, massive studies stratifying  B. pertussis  strains according to country of origin, vaccination status, and type of vaccines (DPTc and DPTa), did not show significant differences in the frequencies of prn, ptxC, ptxA or tcfA2 alleles for circulating strains and vaccination programs [van Amersfoorth, S. C., et al. J Clin Microbiol, 2005. 43(6): p. 2837-43]. The high prevalence of Prn2 strains in many countries is indicative of the favored transmission of these strains by means still unraveled, although the findings mentioned above hardly link the origin of new variants to vaccination. Remarkably, in the above mentioned study [van Amersfoorth, S. C., et al. J Clin Microbiol, 2005. 43(6): p. 2837-43], the three clinically isolated strains bearing allelles similar to that of the vaccines used were found only in non-vaccinated children. Either casual or not, it suggests that Prn1 strains are favored in niches devoid of specific immunity. On the other hand, the recent identification of a phage infecting  Bordetella  (BPP-1) by using Prn as primary receptor, suggested that variations in this protein might be triggered by selective pressures other than those imposed by the immune system [Liu, M., et al. Science, 2002. 295(5562): p. 2091-4]. The possible influences of both phenomena, together with other unknown factors leading to harmonized variations in  B. pertussis , are not excluded. 
         [0008]    The evolution of  Pertussis  epidemiology has been simulated by a mathematical model, integrating the incidence of the disease and the pathogen&#39;s transmission independently [Aguas, R., et al. Lancet Infect Dis, 2006. 6(2): p. 112-7]. This model predicts that regular boosting doses would not be capable of eliminating the severity grades of the disease, observed in current epidemics. It is highly probable that this should be caused by the short lifespan of the protection conferred by the available acellular vaccines (4-12 years), and also the variability of the immune response and the different types of vaccines. This model predicts as the most optimistic scenario that where vaccines could generate an immunity superior to the natural one, a paradigm still unreached by the cellular and acellular vaccines available. 
         [0009]    The main purpose of the present invention resides on the contribution to develop more efficacious acellular vaccines against Whooping Cough. The main work preceding the present invention were based on administering immunogenic preparations obtained by mixing Prn proteins (Nicole Guiso et al., WO 01/90143 A2 y US 2006/0008474 A1) or synthetic peptides of the Prn R1 region (Frederik Mooi et al., WO 02/00695 A2). Therefore, the development of more efficacious acellular vaccines is an important problem to prevent Whooping Cough. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0010]    This invention contributes to solve the above mentioned problems, and comprises the engineering of the prnA gene, coding for the outer membrane protein of  B. pertussis  denominated Pertactin (Prn). This invention suffices the needs evidenced in the state of the art, making possible obtaining different variants of engineered Prn, in such a way that they comprise in their structure two different polymorphic domains of the Prn R1 region. The versatility of the invention also covers the engineering of new Prn molecules, additionally comprising three or more different polymorphic domains of the Prn R1 region. 
         [0011]    Is subject of the present invention a polynucleotidic sequence coding for an engineered Prn protein, wherein said protein comprises up to the first 300 amino acids proximal to the N-terminal end of a natural, mature Prn of a given type (PrnX300), and an aminoacidic sequence comprising up to 620 amino acids proximal to the C-terminal end of a natural, mature Prn of given type (PrnY620), resulting in an engineered PrnX300-PrnY620Prn protein. 
         [0012]    In the context of the present invention, the term ‘engineered Prn’ refers to a protein resulting from coupling, adjacently or not, of a fragment comprising up to the first 300 amino acids proximal to the N-terminal end of a given natural, mature Prn protein, to another fragment comprising the last 620 amino acids proximal to the C-terminal end of a natural, mature Prn protein. 
         [0013]    The new Prn engineered variants are obtained by molecular mutagenesis, by adjacent coupling of sequences comprising up to the first 300 amino acids proximal to the N-terminal end of a natural, mature Prn of a given type, to sequences comprising up to the last 620 amino acids proximal to the C-terminal end of a natural, mature Prn of a given type. The new variants of engineered Prn comprise sequences from the same or different type of Prn in a single molecule, without affecting the protective immune response. 
         [0014]    In a preferred embodiment of the present invention, different variants of Prn engineered variants are obtained, encoded by the nucleic acid sequences identified as SEQ ID Nr. 1-SEQ ID Nr. 6. Highly significantly protection levels and opsonophagocytic activities were obtained by immunizing mice with the different variants of engineered Prn, higher than those obtained with natural Prn molecules formulated alone or combined in mixes. The immune response generated with the engineered Prn was equally effective against strains expressing different types of Prn. 
         [0015]    In a preferred embodiment of the present invention, the fragment comprising the first 300 amino acids proximal to the N-terminal end of a natural, mature Prn of a given type, named PrnX300, corresponds to Prn from the genera  Bordetella . In another preferred embodiment of the present invention, this fragment corresponds to Prn molecules from  B. pertussis  or  B. parapertussis , preferably Prn1, Prn2 and Prn3 variants of  B. pertussis.    
         [0016]    In a preferred embodiment of the invention, the las 620 amino acids proximal to the C-terminal end of a natural, mature Prn of a given type, named PrnY620, corresponds to Prn from the genera  Bordetella . In another preferred embodiment of the present invention, this fragment corresponds to Prn molecules from  B. pertussis  or  B. parapertussis , preferably Prn1, Prn2 and Prn3 variants of  B. pertussis.    
         [0017]    The polynucleotidic sequence of the present invention codes for a polypeptidic sequence comprising any possible combination of Prn types in the format PrnX300-PrnY620. 
         [0018]    The amino acid sequences PrnX300 and PrnY620 coded by the polynucleotide sequence of the present invention are adjacently coupled, or by using the amino acid sequences IDNATWVMTDN or IDNATWVMTDNIDNATWVMTDN. 
         [0019]    In the present invention, the amino acid sequences PrnX300 and PrnY620 can be devoid of repetitive sequences, preferably of GGXXP and PQP sequences of the R1 and R2 regions. The evidences supporting this design are the following: the Region 1 (R1), comprising the repetitive sequence GGXXP is weakly recognized by human and rabbit sera, indicating that it is not an immunodominant region [Hijnen, M., F. R. Mooi, et al. (2004). Infect Immun 72(7): 3716-23]. On the other hand, recent work reported Prn mutants where the repetitive GGXXP and PQP sequences or regions containing these sequences were deleted. GGXXP deletions did not affect the physicochemical properties of the mutant Prn molecules obtained, as evidenced in the similar methods used for expression and purification of mutant and non-mutant Prn proteins [Hijnen, M., P. G. van Gageldonk, et al. (2005). Protein Expr Purif 41(1): 106-12]. Similarly, deletions of the GGXXP sequences did not significantly affected structural properties, since Prn molecules mutated in R1 were well recognized by MAbs generated against conformational epitopes in natural Prn molecules, and also not recognized by anti-GGXXP MAbs directed against linear GGXXP epitopes. Additionally, it was observed that certain mutations inside R1 can enhance the binding capacity to certain MAbs against conformational epitopes. Finally, there were evidences indicating that R1 (GGXXP) and R2 (PQP) form a single epitope [Hijnen, M., R. de Voer, et al. (2007). Vaccine 25(31): 5902-14] 
         [0020]    In another preferred embodiment of the present invention, the said polynucleotidic sequence codes for an engineered Prn, wherein said amino acid PrnX300 and PrnY620 sequences comprise heterologous peptides able to function as T helper cell epitopes isolated from Diphtheria, Tetanus, the hepatitis B virus (HBV), Polioviruses, Vaccinia, the human immunodeficiency virus (HIV) or the human Influenza virus. It is well known among people skilled in the art that the immune response against a given antigen can be enhanced by including this type of epitopes. 
         [0021]    An additional preferred embodiment of the present invention comprises the polynucleotidic sequences according to claim  1 , wherein said polynucleotide sequences could be optimized for optimal codon usage, to increase the expression of the encoded protein in bacteria, yeast, insect or mammalian cells. The resulting increase in the expression of the molecules encoded by recombinant procedures is widely know by people skilled in the art in this particular technical field. 
         [0022]    In another preferred embodiment, the new protein subject of the present invention can be one of the multiple components of a new combined vaccine, emphasizing that none of the precedent inventions comprised obtaining the minimal number of molecular entities satisfying the existing requirements of this technical field. 
         [0023]    Finally, the demands for vaccine preparations able to generate cross-protection between  B. pertussis  and  B. parapertussis  are more than evident in the state of the art. The present invention also comprises generating engineered Prn molecules comprising in a single structure different polymorphic regions of different  Bordetella  species, based on the high homology levels existing between Prn proteins of the different  Bordetella  species. 
         [0024]    Unexpectedly, the engineered Prn subject of the present invention was not only capable of inducing an effective immune response against different Prn1- and Prn2-expressing  B. pertussis  strains, but also generated antibody responses more effective than that generated by other non-engineered recombinant Prn proteins, as evidenced in the mice respiratory challenge model and the opsonophagocytic assay. Surprisingly, the immune response induced by the engineered Prn was superior to that induced by an equimolar mix of Prn1 and Prn2 (Prn1+Prn2). 
         [0025]    Vaccine compositions made by mixing different Prn proteins of the same or different species, although covering polymorphisms, lead to technical difficulties associated to the new production processes, such as the undesired increase in the concentration of contaminants and the productive inconsistency between lots. This is an essential aspect to develop combined vaccines, composed of multiple antigens with quite different characteristics, which can compromise the systemic immunogenicity of the formulation. On the other hand, it is expected that strategies based on synthetic peptides of the R1 region could lead to vaccines less effective than vaccines currently available, by excluding other epitopes present in the natural Prn from the antigen, relevant to develop a protective response. 
         [0026]    To meet this unsolved requirement of this technical field, the present invention comprises a pharmaceutical composition comprising one or more engineered Prn, coded by polynucleotidic sequences from Claims  1  to  13 , in amounts sufficient to generate humoral and cellular immune responses effective against  Bordetella  species, when administered through immunization procedures in mammals, and preferably, in humans. In a preferred embodiment of the present invention, the pharmaceutical composition comprising one or more Prn engineered variants generates humoral and cellular immune responses effective against  B. pertussis.    
         [0027]    It is also the aim of the present invention a life or attenuated vaccine comprising one or more Prn engineered variants, coded by sequences from Claims  1  to  13 , wherein said Prn engineered variants are expressed in the outer membrane of the life or attenuated organism. In this live or attenuated vaccine, said polynucleotidic sequences from Claims  1  to  13  are included in a plasmid vector or a bacterial chromosome. 
         [0028]    In another embodiment of the present invention, said polynucleotidic sequences from Claims  1  to  13 , which code for Prn engineered variants, are included in a vector for expression in mammalian cells. In another embodiment of the present invention, said expression vector which contains the polynucleotidic sequences from Claims  1  to  13  is the basis for a nucleic acids vaccine. 
         [0029]    In another embodiment of the invention, the polypeptidic sequences coded by said polynucleotidic sequences from Claims  1  to  13 , can be used to detect  Bordetella  infections. Is also the aim of the present invention a diagnostic kit to detect the presence or absence of antibodies against  Bordetella , comprising polypeptidic sequences coded by the polynucleotidic sequences referred on Claims  1  to  13 . 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0030]      FIG. 1 . Protection experiment in Balb/c mice vaccinated with different recombinant Prn engineered variants. Strains of  B. pertussis  Tohama I (Prn1) and the clinical isolate CH53 (Prn2) were used as challenge. Barrs represent the mean logarithm of the reduction of viable bacterial cells in lungs. 
           [0031]      FIG. 2 . Opsonophagocytosis mediated by sera from Balb/c mice vaccinated with the different recombinant Prn engineered variants. The chart shows the difference of fluorescence (phycoerithrin, PE) in arbitrary units (AU) of cells stained with fluorescein isothiocyanate (FITC) in two incubation conditions (PE 4° C.-PE 37° C.). 
           [0032]      FIG. 3 . Humoral IgG immune response against Prn1 and Prn2CCPrn1 generated in mice immunized with plasmids expressing the Prn1, Prn2, Prn2CCPrn1 and Prn2CLPrn1 engineered variants. 
       
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS/EXAMPLES 
     Example 1 
     Construction of Vectors for the Intracellular Expression in  Escherichia coli  of the Different Prn Engineered Variants and its Purification 
       [0033]    The prnA1 and prnA2 genes from  Bordetella  strains  B. pertussis  Tohoma I (Prn1) and CH53 (Prn2) were amplified by Polymerase Chain Reaction (PCR) from genomic DNA by using the previously reported oligonucleotides 1 and 2 [Hijnen, M., P. G. van Gageldonk, et al. (2005). Protein Expr Purif 41(1): 106-12]. 
         [0034]    The fragments obtained were cloned into the vector pET-28 a  (Novagen) using the sites Nde I and BamH I. The Prn engineered variants were obtained by using the reverse PCR method previously reported by Imai and co-workers in 1991 [Imai, Y., et al. Nucleic Acids Res, 1991. 19(10): p. 2785]. Nucleotides used to amplify the different polynucleotidic sequences are shown in Table 1. The oligonucleotide pair 1,2 was used to linearize vector pET28aprn1 and pETaprn2, corresponding to Prn1 and Prn2, respectively. The DomR1 fragments were obtained by amplification with oligonucleotides 3 and 4. Additionally, this region was amplified by using the nucleotide pairs 3,5 and 3,6 to add sequences coding for the short and long linkers, respectively. The conditions used for PCR amplification of the fragments used in the present invention are summarized in Table 2. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Oligonucleotides used for amplification of the different sequences. 
               
             
          
           
               
                   
                 Oligonucleotide 
                   
                 Result of the PCR 
               
               
                 Number 
                 name 
                 Sequence 5′→3′ 
                 amplification 
               
               
                   
               
             
          
           
               
                 1 
                 pET28aprn1 1401- 
                 AGCGTGGAGCTCGCCCA 
                 Linearized vector with 
               
               
                   
                 30 LinVect 
                 GTCGATCGTCGAG 
                 blunt ends 
               
               
                   
               
               
                 2 
                 pET28aprn1 1431- 
                 GGAGCCCGATACGTCCA 
                   
               
               
                   
                 60 LinVect 
                 CGCCATACCAGCC 
                   
               
               
                   
               
               
                 3 
                 pET28aprn1 1975- 
                 GTCAAGGCCGGCAAGCT 
                 Domain R1 (DomR1) of 
               
               
                   
                 97 DomR1 
                 GGTCGC 
                 any type of Prn 
               
               
                   
               
               
                 4 
                 pET28aprn1 1431- 
                 GGAGCCCGATACGTCCA 
                   
               
               
                   
                 53 DomR1 
                 CGCCAT 
                   
               
               
                   
               
               
                 5 
                 pET28aprn1 1431- 
                 ATCGACAACGCCACCTG 
                 Amplifies DomR1 from 
               
               
                   
                 53 DomR1 CC-Nt 
                 GGTCATGACGGACAACG 
                 any type of Prn, also 
               
               
                   
                   
                 TCAAGGCCGGCAAGCTG 
                 adding a linker of 11 
               
               
                   
                   
                 GTCGC 
                 amino acids to the N- 
               
               
                   
                   
                   
                 term. 
               
               
                   
               
               
                 6 
                 pET28aprn1 1431- 
                 ATCGACAACGCCACCTG 
                 Amplifies DomR1 from 
               
               
                   
                 53 DomR1 CL-Nt 
                 GGTCATGACGGACAACA 
                 any type of Prn, also 
               
               
                   
                   
                 TCGACAACGCCACCTGG 
                 adding a linker of 22 
               
               
                   
                   
                 GTCATGACGGACAACGT 
                 amino acids to the N- 
               
               
                   
                   
                 CAAGGCCGGCAAGCTG 
                 term. 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Conditions for PCR amplification of the different fragments used in 
               
               
                 the present invention 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                   
                 Size of 
               
               
                   
                   
                   
                   
                   
                   
                   
                 the 
               
               
                   
                   
                   
                   
                   
                   
                   
                 amplific. 
               
               
                 Oligonucl 
                 Hybridation 
                 Template 
                 Extension 
                 Polymerase 
                 No. of 
                 Amplific. 
                 product 
               
               
                 pair 
                 temp (° C.). 
                 DNA (μg) 
                 time (min) 
                 (Units) 
                 cycles 
                 product 
                 (bp) 
               
               
                   
               
             
          
           
               
                 1, 2 
                 65 
                 pET28aprn1 
                 7.5 
                 Pfx (2.5) 
                 5 
                 Vector 
                 7370 
               
               
                   
                   
                 (1) 
                   
                   
                   
                 Lineal 
               
               
                 1, 2 
                 65 
                 pET28aprn2 
                 7.5 
                 Pfx (2.5) 
                 5 
                 Vector 
                 7385 
               
               
                   
                   
                 (1) 
                   
                   
                   
                 Lineal 
               
               
                 3, 4* 
                 67 
                 pET28aprn1 
                 0.6 
                 Pfu (2.5) 
                 30 
                 DomR1 
                 567 
               
               
                   
                   
                 (0.1) 
                   
                   
                   
                 prn1 
               
               
                 3, 4* 
                 67 
                 pET28aprn2 
                 0.6 
                 Pfu (2.5) 
                 30 
                 DomR1 
                 582 
               
               
                   
                   
                 (0.1) 
                   
                   
                   
                 prn2 
               
               
                 3, 5* 
                 67 
                 DomR1 prn1 
                 0.6 
                 Pfu (2.5) 
                 30 
                 CC- 
                 600 
               
               
                   
                   
                 (0.1) 
                   
                   
                   
                 DomR1 
               
               
                   
                   
                   
                   
                   
                   
                 prn1 
               
               
                 3, 6* 
                 67 
                 DomR1 prn1 
                 0.6 
                 Pfu (2.5) 
                 30 
                 CL- 
                 633 
               
               
                   
                   
                 (0.1) 
                   
                   
                   
                 DomR1 
               
               
                   
                   
                   
                   
                   
                   
                 prn1 
               
               
                 3, 5* 
                 67 
                 DomR1 prn2 
                 0.6 
                 Pfu (2.5) 
                 30 
                 CC- 
                 620 
               
               
                   
                   
                 (0.1) 
                   
                   
                   
                 DomR1 
               
               
                   
                   
                   
                   
                   
                   
                 prn2 
               
               
                 3, 6* 
                 67 
                 DomR1 prn2 
                 0.6 
                 Pfu (2.5) 
                 30 
                 CL- 
                 648 
               
               
                   
                   
                 (0.1) 
                   
                   
                   
                 DomR1 
               
               
                   
                   
                   
                   
                   
                   
                 prn2 
               
               
                   
               
               
                 *Phosphorylated oligonucleotides, 
               
               
                 CC: Short linker, 
               
               
                 CL: Long linker 
               
             
          
         
       
     
         [0035]    The linearized pET28aprn1 and pET28aprn2 vectors, obtained by reverse PCR, were ligated to the different fragments coding for domains containing region 1 from Prn1 and Prn2. In these vectors, the new engineered genes are under the transcriptional control of the T7 inducible promoter. Clones bearing the correct sequences were introduced into the BL21-Codonplus(DE3)-RP  E. coli  strain, for the expression of the corresponding proteins as inclusion bodies [Hijnen, M., et al. Protein Expr Purif, 2005. 41(1): p. 106-12]. 
         [0036]    The expression levels of the recombinant Prn1 and Prn2, as well as for the other variants, reached between 15 and 20% of total proteins, as evidenced by densitometry in polyacrylamide gels stained with Coomassie blue. 
         [0037]    The different proteins were purified by suspending the bacterial paste for each variant in rupture buffer (at a cell concentration of 100 mg/mL) and cells were lysed with ultrasound. The cellular pellets were solubilized in 8 M Urea and fractionated by Sodium Dodecyl Sulphate polyacrylamide gel electrophoresis (SDS-PAGE, 12.5%). The gel was stained by reverse Zinc-Imidazol staining, and the slice containing the band corresponding to the protein of interest was passed through a stainless steel mesh of 100 μm in the presence of extraction buffer. The protein was further extracted, and renatured and concentrated by ultrafiltration through an Amicon concentration cell, with a membrane of 50 kDa, and the final concentration was determined by the Bicinchoninic acid method. No contaminants were detected by assaying 15 μg of each protein purified from the analytical SDS-PAGE gels stained with Coomassie blue, evidencing that protein preparations were more than 95% pure. The characteristics of the different constructs and the Prn engineered variants obtained are summarized in Table 3. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Characteristics of the different Prn constructs and Prn engineered 
               
               
                 variants 
               
             
          
           
               
                   
                   
                   
                   
                 Characteristics 
               
               
                   
                 Linker 
                   
                   
                 of the 
               
               
                 Plasmid 
                 between 
                 Name of the 
                 Prn 
                 engineered 
               
               
                 name 
                 DomR1 
                 engineered Prn 
                 type 
                 Prn 
               
               
                   
               
               
                 pET28aprn1 
                 No 
                 Prn1 
                 1 
                 — 
               
               
                 pET28aprn2 
                 No 
                 Prn2 
                 2 
                 — 
               
               
                 pETprn 
                 No 
                 Prn1-Prn2 
                 1, 2 
                 Nt . . . DomR1 
               
               
                 DomR1 
                   
                   
                   
                 (Prn1)- 
               
               
                 (1-2) 
                   
                   
                   
                 DomR1(Prn2) . . . 
               
               
                   
                   
                   
                   
                 Ct 
               
               
                 pETprn 
                 IDNATWVMT 
                 Prn1-CC-Prn2 
                 1, 2 
                 Nt . . . DomR1 
               
               
                 DomR1 
                 DN 
                   
                   
                 (Prn1)-CC- 
               
               
                 (1-CC-2) 
                   
                   
                   
                 DomR1(Prn2) . . . 
               
               
                   
                   
                   
                   
                 Ct 
               
               
                 pETprn 
                 IDNATWVMT 
                 Prn1-CL-Prn2 
                 1, 2 
                 Nt . . . DomR1 
               
               
                 DomR1 
                 DNIDNATWV 
                   
                   
                 (Prn1)-CL- 
               
               
                 (1-CL-2) 
                 MTDN 
                   
                   
                 DomR1(Prn2) . . . 
               
               
                   
                   
                   
                   
                 Ct 
               
               
                 pETprn 
                 — 
                 Prn2-Prn1 
                 1, 2 
                 Nt . . . DomR1 
               
               
                 DomR1 
                   
                   
                   
                 (Prn2)- 
               
               
                 (2-1) 
                   
                   
                   
                 DomR1(Prn1) . . . 
               
               
                   
                   
                   
                   
                 Ct 
               
               
                 pETprn 
                 IDNATWVMT 
                 Prn2-CC-Prn1 
                 1, 2 
                 Nt . . . DomR1 
               
               
                 DomR1 
                 DN 
                   
                   
                 (Prn2)-CC- 
               
               
                 (2-CC-1) 
                   
                   
                   
                 DomR1(Prn1) . . . 
               
               
                   
                   
                   
                   
                 Ct 
               
               
                 pETprn 
                 IDNATWVMT 
                 Prn2-CL-Prn1 
                 1, 2 
                 Nt . . . DomR1 
               
               
                 DomR1 
                 DNIDNATWV 
                   
                   
                 (Prn2)-CL- 
               
               
                 (2-CL-1) 
                 MTDN 
                   
                   
                 DomR1(Prn1) . . . 
               
               
                   
                   
                   
                   
                 Ct 
               
               
                   
               
             
          
         
       
     
       Example 2 
     Active Immunization, Antibody Response and Protection in a Mice Model 
       [0038]    Mice were immunized with 0.2 μg or 0.02 μg of the recombinant Prn1 and Prn2, PBS, an equimolar mix of Prn1 and Prn2 (Prn1+Prn2), and six of the Prn engineered variants (shown in Table 3). All the proteins were administered formulated in alum. Doses corresponded to 1/40 and 1/400 fractions of the dose commonly employed in humans (Infanrix®, 8 μg). Mice were immunized by the subcutaneous route, with a volume of 100 μL. Sera from the immunized mice were evaluated by an ELISA type immunoenzymatic assay. The antibody titers reached mean values from 1.2×10 3  to 4.6×10 4 . The mean values of the titers for corresponding to the highest doses significantly differed from the titers reached with the lowest doses used, for all the cases (p&lt;0.05, Kruskal Wallis-Dunns). No differences were observed in the antibody response generated with Prn1, Prn2 or the equimolar mix Prn1+Prn2. Similarly, there were no differences between the mean titers of the different Prn engineered variants. Surprisingly, the titers obtained with the Prn engineered variants were significantly higher than those generated by the non-engineered recombinant Prn proteins (p&lt;0.01, Kruskal Wallis-Dunns). 
         [0039]    The strain Tohama I (Prn1) and the clinical isolate CH 53  (Prn2) were used for the intranasal challenge. Bacteria were cultures in plates containing Bordet-Gengou-Agar media (Sigma) supplemented with 1% glycerol and 14% defibrinated goat blood. Plates were incubated for 24 h at 37° C. and the resulting colonies were suspended in Stainer-Scholte medium at a 10 8  cells/mL concentration. This suspension was used for the intranasal challenge. Mice immunized were challenged 15 days after the last immunization, by instillation of 50 μL of the bacterial suspension (5×10 6  cells). Five days after challenge, mice were sacrificed and lungs aseptically extracted and homogenized to measure the bacterial burden [Denoel, P., et al. Vaccine, 2005. 23(46-47): p. 5333-41]. The different variants showed protection levels significantly higher than the non-vaccinated controls (p&lt;0.001). Unexpectedly, the engineered Prn variants showed higher protection levels when compared with the recombinant Prn proteins or the equimolar Prn1+Prn2 mix for both strains (p&lt;0.001). 
         [0040]    The Prn engineered variants showed similar protection levels against both strains at the lowest administered dose, an effect unattained with the Prn1 or Prn2 proteins. These results evidence that these Prn engineered variants bear immunological properties different from, and superior to, those showed by the recombinant Prn1 and Prn2 proteins assayed both separately or as equimolar mixes ( FIG. 1 ). 
       Example 3 
     Opsonophagocytic Activity in Sera 
       [0041]    The opsonophagocytic activity mediated by anti-Prn sera has been shown as a crucial parameter in the response of people vaccinated with acellular vaccines [Hellwig, S. M., et al. J Infect Dis, 2003. 188(5): p. 738-42]. The present invention shows that the different Prn engineered variants were capable of inducing antibodies resembling these properties. The opsonophagocytic activity was studied by the previously mentioned method, adapted to the mice model. Strains Tohama I and CH 53  of  B. pertussis  were grown in Bordet-Gengou-Agar and the cells stained with FITC (2×10 6  colony-forming units). Afterwards, the labeled bacteria were opsonized for 30 min at 37° C., in a plate shaker, with sera from mice immunized with the recombinant Prn1, Prn2, Prn1+Prn2 and two variants of the engineered Prn proteins (Prn2-CC-Prn1 and Prn2-CL-Prn1). During the adhesion step, the opsonized bacteria and the non-opsonized control were incubated with polymorphonuclear cells (PMN). Samples were further divided into two equal subgroups of cells, one incubated for another 45 min at 4° C. and the other at 37° C. All the samples were finally incubated for another 30 min at 4° C. with the goat anti-mouse-PE labeled conjugate. Samples were analyzed by flow cytometry (PARTEC PAS III). The fluorescence intensities of green- and red-stained cells incubated at 4° C. were used as adhesion controls. The difference of red fluorescence in green-stained cells was used to evidence the phagocytic activity mediated by sera. 
         [0042]    The Prn engineered variants showed opsonophagocytic activity ( FIG. 2 ). Surprisingly, there were significant differences only in among the groups immunized with the Prn engineered variants when compared with mice inoculated with PBS (p&lt;0.05, Kruskal Wallis-Dunns). The opsonophagocytic activity of sera generated by the recombinant, non-engineered Prn protein alone or in combination reached values 6-fold higher than the PBS control, although these differences were not significant. Finally, these results evidenced that the Prn engineered variants are able to induce antibodies with significant opsonophagocytic activity, irrespective of the type of Prn present in the bacterium. 
       Example 4 
     Construction of Vectors for the Expression in Mammals of Prn Engineered Variants and Evaluation of the Humoral Immune Response Generated 
       [0043]    The genes prnA1 and prnA2, and the gene variants prn2CCprn1 and prn2CLprn1, were amplified by PCR from their respective expression vectors (see Table 3) by using the previously reported oligonucleotides 1 and 2 [Hijnen, M., P. G. van Gageldonk, et al. (2005). Protein Expr Purif 41(1): 106-12]. In this case, the oligonucleotide 1 was modified, substituting the Nde I by a BamH I restriction site. The fragments obtained were cloned into the BamH I restriction site of the pAEC-SPE3 plasmid vector [Herrera A M, Rodriguez E G, et al. (2000) BBRC, 279, 548-551]. This vector is designed for the extracellular expression of antigens in mammalian cells. The resulting constructs were purified by using the commercial plasmid DNA purification kit Endo-free plasmid Giga kit (Qiagen). Groups of female 6-to-7-week-old Balb/c mice were immunized thrice with 100 μg of DNA in 100 μL of PBS, at three-week intervals by the intraperitoneal route. The control group was immunized with the empty vector without insert (pAEC-SPE3). Fifteen days after the last immunization, mice were sacrificed and blood collected for the evaluation of sera. The specific IgG antibody responses were evaluated by the ELISA technique, at a 1/1000 dilution and coating plates with equimolar amounts of the Prn1 (2 μg/mL) and Prn2CCPrn1 (2.4 μg/mL) proteins. As shown in  FIG. 3 , animals immunized with the different plasmids expressing Prn1, Prn2, Prn2CCPrn1 and Prn2CLPrn1 generated specific IgG antibody responses, significantly higher (p&lt;0.001) than animals immunized with the empty pAEC-SPE3 vector. Similarly to sera generated in mice immunized with protein and alum (data not shown), sera from immunized mice preferentially recognized the Prn engineered variant Prn2CCPrn1 (p&lt;0.05), more than the natural Prn1 protein, what could be due to a better exposure of the shared epitopes in Prn2CCPrn1 than in Prn1.