Recombinant canarypoxvirus vaccine carrying the prM/E genes of West Nile virus protects horses against a West Nile virus-mosquito challenge.

An ALVAC (canarypoxvirus)-based recombinant (vCP2017) expressing the prM and E genes derived from a 1999 New York isolate of West Nile virus (WNV) was constructed and assessed for its protective efficacy in horses in two different experiments. In the first trial, a dose titration study was conducted to evaluate both serum neutralising antibody responses to WNV and duration of immunity. In the second trial the onset of protection was determined. Twenty-eight adult horses received two doses of vCP2017 administered intramuscularly at 5-week intervals and sixteen horses comprised age-matched non-vaccinated controls. Individual sera were taken periodically and tested for neutralising antibodies against WNV. Horses were challenged by allowing WNV-infected Aedes albopictus mosquitoes to feed on them two weeks (second trial) or one year (first trial) after the second vaccination. After challenge, horses were monitored for clinical signs of disease, and blood samples were collected for detection of WNV viremia and antibody. In both trials, all vaccinated horses developed neutralising antibodies against WNV. None of the vaccinated or control horses developed clinical signs of WNV disease upon challenge. None of the nine horses challenged 2 weeks after primary vaccination and only one of the ten vaccinated horses challenged 1 year after vaccination developed detectable viremia after challenge, whereas more than 80% of the controls became infected. Results from these studies demonstrated that a primary course of two doses of vCP2017 provides both antibody response and an early immunity in horses against WNV viremia.

Properties of recombinant HtrA: an otitis media vaccine candidate antigen from non-typeable Haemophilus influenzae.

Non-encapsulated or non-typable Haemophilus influenzae (NTHi) is a major cause of middle ear infections in young children. HtrA has been identified as a vaccine candidate antigen from NTHi; therefore physicochemical characterization of this antigen is important for vaccine development. Recombinant NTHi HtrA has been expressed in E. coli and shown to have serine protease activity. Several mutant, recombinant HtrA proteins were expressed and purified to obtain suitable vaccine antigens lacking protease activity. Two mutants with alterations at the putative active site His91 and Ser197, designated H91A and S197A were examined by circular dichroic spectropolarimetry (CD) to evaluate secondary structure. The S197A mutant had a more random secondary structure compared to wild-type rHtrA or H91A. It is likely that improper folding of S197A accounts for its lack of immunoprotective properties in a chinchilla model of otitis media.

Disruption of the mycobacterial cell entry gene of Mycobacterium bovis BCG results in a mutant that exhibits a reduced invasiveness for epithelial cells.

Mycobacteria belonging to the Mycobacterium tuberculosis complex have the ability to invade and replicate in non-phagocytic cells, an event that requires the presence of bacterial surface components capable of triggering a cell response and the subsequent internalization of the microorganism. In this study, we report the sequencing of the mycobacterial cell entry gene (mce) of Mycobacterium bovis bacillus Calmette-Guérin (BCG) and the generation and characterization of a mutant BCG strain with an inactivated mce gene, by homologous recombination with double cross-over. This mutant strain does not express the mycobacterial cell entry protein (Mce) and exhibits a reduced ability to invade the non-phagocytic epithelial cell line HeLa as compared to wild-type BCG.

Characterization of Moraxella (Branhamella) catarrhalis lbpB, lbpA, and lactoferrin receptor orf3 isogenic mutants.

Pathogenic members of the family Neisseriaceae produce specific receptors to acquire iron from their host's lactoferrin and transferrin. Recently, putative Moraxella catarrhalis lactoferrin receptor genes and a third open reading frame (lbpB, lbpA, and orf3) were cloned and sequenced. We describe the preliminary characterization of isogenic mutants deficient in LbpB, LbpA, or Orf3 protein.

The transferrin binding protein B of Moraxella catarrhalis elicits bactericidal antibodies and is a potential vaccine antigen.

The transferrin binding protein genes (tbpA and tbpB) from two strains of Moraxella catarrhalis have been cloned and sequenced. The genomic organization of the M. catarrhalis transferrin binding protein genes is unique among known bacteria in that tbpA precedes tbpB and there is a third gene located between them. The deduced sequences of the M. catarrhalis TbpA proteins from two strains were 98% identical, while those of the TbpB proteins from the same strains were 63% identical and 70% similar. The third gene, tentatively called orf3, encodes a protein of approximately 58 kDa that is 98% identical between the two strains. The tbpB genes from four additional strains of M. catarrhalis were cloned and sequenced, and two potential families of TbpB proteins were identified based on sequence similarities. Recombinant TbpA (rTbpA), rTbpB, and rORF3 proteins were expressed in Escherichia coli and purified. rTbpB was shown to retain its ability to bind human transferrin after transfer to a membrane, but neither rTbpA nor rORF3 did. Monospecific anti-rTbpA and anti-rTbpB antibodies were generated and used for immunoblot analysis, which demonstrated that epitopes of M. catarrhalis TbpA and TbpB were antigenically conserved and that there was constitutive expression of the tbp genes. In the absence of an appropriate animal model, anti-rTbpA and anti-rTbpB antibodies were tested for their bactericidal activities. The anti-rTbpA antiserum was not bactericidal, but anti-rTbpB antisera were found to kill heterologous strains within the same family. Thus, if bactericidal ability is clinically relevant, a vaccine comprising multiple rTbpB antigens may protect against M. catarrhalis disease.

Cloning and expression of the Moraxella catarrhalis lactoferrin receptor genes.

The lactoferrin receptor genes from two strains of Moraxella catarrhalis have been cloned and sequenced. The lfr genes are arranged as lbpB followed by lbpA, a gene arrangement found in lactoferrin and transferrin receptor operons from several bacterial species. In addition, a third open reading frame, orf3, is located one nucleotide downstream of lbpA. The deduced lactoferrin binding protein A (LbpA) sequences from the two strains were found to be 99% identical, the LbpB sequences were 92% identical, and the ORF3 proteins were 98% identical. The lbpB gene was PCR amplified and sequenced from a third strain of M. catarrhalis, and the encoded protein was found to be 77% identical and 84% similar to the other LbpB proteins. Recombinant LbpA and LbpB proteins were expressed from Escherichia coli, and antisera raised to the purified proteins were used to assess antigenic conservation in a panel of M. catarrhalis strains. The recombinant proteins were tested for the ability to bind human lactoferrin following gel electrophoresis and electroblotting, and rLbpB, but not rLbpA, was found to bind lactoferrin. Bactericidal antibody activity was measured, and while the anti-rLbpA antiserum was not bactericidal, the anti-rLbpB antisera were found to be weakly bactericidal. Thus, LbpB may have potential as a vaccine candidate.

A 20-kilodalton N-terminal fragment of the D15 protein contains a protective epitope(s) against Haemophilus influenzae type a and type b.

A conserved 80-kDa minor outer membrane protein, D15, of Haemophilus influenzae has been shown to be a protective antigen in laboratory animals against H. influenzae type a (Hia) or type b (Hib) infection. To localize the protective B-cell epitope(s) within the D15 protein and to further explore the possibility of using synthetic peptides as vaccine antigens, a 20-kDa N-terminal fragment of D15 protein (truncated D15 [tD15]) was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The tD15 moiety was cleaved from glutathione S-transferase by using thrombin and purified to homogeneity. The purified soluble tD15 appeared to contain immunodominant protective epitope(s) against Hia and Hib, since rabbit antisera directed against tD15 were capable of protecting infant rats from Hia or Hib bacteremia. The ease of purification of soluble tD15, therefore, makes it a better candidate antigen than the full-length recombinant D15 which is produced as inclusion bodies in E. coli. Furthermore, both the purified tD15 fragment and a mixture of tD15-derived peptides spanning amino acid residues 93 to 209 of the mature D15 protein were capable of inhibiting the protection against Hib conferred on infant rats by rabbit anti-tD15 antiserum, indicating that the protective epitopes of D15 may not be conformational. However, the administration of pooled rabbit immune sera raised against the same panel of peptides failed to protect infant rats from Hib infection.

Nasopharyngeal colonization with nontypeable Haemophilus influenzae in chinchillas.

Colonization of the nasopharynx by a middle ear pathogen is the first step in the development of otitis media in humans. The establishment of an animal model of nasopharyngeal colonization would therefore be of great utility in assessing the potential protective ability of candidate vaccine antigens (especially adhesins) against otitis media. A chinchilla nasopharyngeal colonization model for nontypeable Haemophilus influenzae (NTHI) was developed with antibiotic-resistant strains. This model does not require coinfection with a virus. There was no significant difference in the efficiency of NTHI colonization between adult (1- to 2-year-old) and young (2- to 3-month-old) animals. However, the incidence of middle ear infection following nasopharyngeal colonization was significantly higher in young animals (83 to 89%) than in adult chinchillas (10 to 30%). Chinchillas that had recovered either from a previous middle ear infection caused by NTHI or from an infection by intranasal inoculation with NTHI were completely protected against nasopharyngeal colonization with a homologous strain and were found to be the best positive controls in protection studies. Systemic immunization of chinchillas with inactivated whole-cell preparations significantly protected animals not only against homologous NTHI colonization but also partially against heterologous NTHI infection. In all protected animals, significant serum anti-P6 and anti-HMW antibody responses were observed. The outer membrane P6 and high-molecular-weight (HMW) proteins appear to be promising candidate vaccine antigens to prevent nasopharyngeal colonization and middle ear infection caused by NTHI.

The Haemophilus influenzae HtrA protein is a protective antigen.

The htrA gene from two strains of nontypeable Haemophilus influenzae has been cloned and sequenced, and the encoded approximately 46-kDa HtrA proteins were found to be highly conserved. H. influenzae HtrA has approximately 55% identity with the Escherichia coli and Salmonella typhimurium HtrA stress response proteins, and expression of the H. influenzae htrA gene was inducible by high temperature. Recombinant HtrA (rHtrA) was expressed from E. coli, and the purified protein was found to have serine protease activity. rHtrA was found to be very immunogenic and partially protective in both the passive infant rat model of bacteremia and the active chinchilla model of otitis media. Immunoblot analysis indicated that HtrA is antigenically conserved in encapsulated and nontypeable H. influenzae species. Site-directed mutagenesis was performed on the htrA gene to ablate the endogenous serine protease activity of wild-type HtrA, and it was found that eight of nine recombinant mutant proteins had no measurable residual proteolytic activity. Two mutant proteins were tested in the animal protection models, and one, H91A, was found to be partially protective in both models. H91A HtrA may be a good candidate antigen for a vaccine against invasive H. influenzae type b disease and otitis media and is currently in phase I clinical trials.

Outer membrane protein D15 is conserved among Haemophilus influenzae species and may represent a universal protective antigen against invasive disease.

We have cloned and sequenced the d15 gene from two strains of Haemophilus influenzae type b (Hib) and two strains of nontypeable H. influenzae (NTHI). The nucleotide and deduced protein sequences of d15 are highly conserved, with only a small variable region identified near the carboxyl terminus of the protein. Analysis of upstream sequences revealed that the H. influenzae d15 gene may be part of a large potential operon of closely spaced open reading frames, including one with significant homology to the Escherichia coli cds gene encoding CDP-diglyceride synthetase. Southern blot analysis demonstrated that the d15 gene is also present in H. influenzae types a, c, d, e, and f and in Haemophilus parainfluenzae. A recombinant D15 (rD15) protein was expressed in good quantity in E. coli from the inducible T7 promoter, and monospecific anti-rD15 antibodies were raised. Immunoblot analysis of H. influenzae serotypes a, b, c, d, e, and f, NTHI, and H. parainfluenzae lysates revealed that they all expressed a cross-reactive D15-like protein. Purified rD15 was found to be highly immunogenic in mice, guinea pigs, and rabbits, and passive transfer of anti-rD15 antibodies protected infant rats from challenge with H. influenzae type b or type a in infant rat models of bacteremia. Thus, D15 is a highly conserved antigen that is protective in animal models and it may be a useful component of a universal subunit vaccine against Haemophilus infection and disease.

Cloning and expression of the Haemophilus influenzae transferrin receptor genes.

The genomic transferrin receptor genes (tbpA and tbpB) from two strains of Haemophilus influenzae type b (Hib) and two strains of non-typable H. influenzae (NTHi) have been cloned and sequenced. The deduced protein sequences of the H. influenzae tbpA genes were 95-100% conserved and those of the tbpB genes were 66-100% conserved. The tbpB gene from one strain of NTHi was found to encode a truncated Tbp2 protein. The tbpB genes from four additional NTHi strains were amplified by the polymerase chain reaction (PCR) utilizing primers derived from the conserved N-terminal sequences of Tbp1 and Tbp2 and were found to encode full-length proteins. Although several bacterial species express transferrin receptors, when the Tbp1 and Tbp2 sequences from different organisms were compared, there was only limited homology. Recombinant Tbp1 and Tbp2 proteins were expressed from Escherichia coli and antisera were raised to the purified proteins. There was significant antigenic conservation of both Tbp1 and Tbp2 amongst H. influenzae strains, as determined by Western blot analysis. In a passive model of bacteraemia, infant rats were protected from challenge with Hib after transfer of anti-rTbp2 antiserum, but not after anti-rTbp1 antiserum.

Sequences of the genes encoding the A, B and C subunits of the Haemophilus influenzae dimethylsulfoxide reductase complex.

The genes (dms) encoding the dimethylsulfoxide reductase protein complex have been cloned and sequenced from Haemophilus influenzae (Hi) type b (Hib) strain Eagan. The Hib dms genes are arranged as an operon whose genomic organization is similar to that of the Escherichia coli (Ec) dmsABC operon. The deduced Hib DmsA, and DmsB and DmsC amino-acid sequences are highly homologous to their Ec counterparts and nearly identical to the recently published sequences of the Hi type-d strain Rd Dms proteins. Hi dimethylsulfoxide reductase appears to be a new member of the superfamily of oxidoreductase enzymes.

Identification and characterization of genes encoding the human transferrin-binding proteins from Haemophilus influenzae.

Haemophilus influenzae, a strict human pathogen, acquires iron in vivo through the direct binding and removal of iron from human transferrin by an as yet uncharacterized process at the bacterial cell surface. In this study, the tbpA and tbpB genes of H. influenzae, encoding the transferrin-binding proteins Tbp1 and Tbp2, respectively, were cloned and sequenced. Alignments of the H. influenzae Tbp1 and Tbp2 protein sequences with those of related proteins from heterologous species were analyzed. On the basis of similarities between these and previously characterized proteins, Tbp1 appears to be a member of the TonB-dependent family of outer membrane proteins while Tbp2 is lipid modified by signal peptidase II. Isogenic mutants deficient in expression of Tbp1 or Tbp2 or both proteins were prepared by insertion of the Tn903 kanamycin resistance cassette into cloned sequences and reintroduction of the interrupted sequences into the wild-type chromosome. Binding assays with the mutants showed that a significant reduction in transferrin-binding ability resulted from the loss of either of the Tbps and a complete loss of binding was evident when neither protein was expressed. Loss of either Tbp2 or both proteins correlated with an inability to grow on media supplemented with transferrin-bound iron as the sole source of iron, whereas the Tbp1+ Tbp2- mutant was able to grow only at high transferrin concentrations.

The sequencing of the 80-kDa D15 protective surface antigen of Haemophilus influenzae.

The 80-kDa D15 antigen (D-15-Ag) has previously been shown to be a target for protective immunity and conserved amongst typeable and nontypeable Haemophilus influenzae. Here, the gene encoding D-15-Ag is shown to encode a 797-aa polypeptide which, after cleavage of the predicted signal peptide, would have a molecular mass of 85,632 Da.

Hybrid genes over-express pertactin from Bordetella pertussis.

Pertactin is a surface adhesin of Bordetella pertussis which is produced in small quantities when expressed from the native prn promoter. Hybrid genes were constructed in which the prn promoter was replaced by either the fha or tox promoter. Recombinant B. pertussis strains containing chromosomally integrated hybrid tox promoter/prn (toxpprn) or fha promoter/prn (fhapprn) genes expressed pertactin at approximately 5- and 8-fold the wild-type level, respectively. The pertactin was correctly processed and secreted and was biochemically and antigenically comparable to its wild-type counterpart, as determined by N-terminal sequence analysis, immunoblotting, peptide mapping, circular dichroism and antigenicity studies. In an adherence assay, a strain over-expressing pertactin was no more adherent than the wild-type strain, but a pertactin-deficient strain was less adherent.

Characterization of pertussis toxin analogs containing mutations in B-oligomer subunits.

The S2, S3, and S4 subunit genes of pertussis toxin (PT) from Bordetella pertussis were subjected to site-directed mutagenesis, and the resultant PT analogs were assayed for altered biological properties. PT analogs S2(T91,R92,N93) delta and S2(Y102A,Y103A) exhibited reduced binding to fetuin. Several PT analogs with mutations in the S2, S3, or S4 subunit showed reduced in vitro toxicity, as measured in the Chinese hamster ovary (CHO) cell clustering assay. In particular, PT analogs S3(Y82A) and S3(I91,Y92,K93) delta retained 10% or less residual toxicity. These mutants also exhibited significantly lower mitogenic and hemagglutinating activities and reduced in vivo activities, as measured by the histamine sensitization and leukocytosis assays. The S4(K54A,K57A) PT analog had significantly reduced CHO cell clustering activity, though other biological activities remained unaffected. PT analogs S1(E129G)/S3(Y82A) and S1(E129G)/S3(I91,Y92,K93) delta displayed a cumulative effect of the S1 and S3 mutations for both in vitro and in vivo toxic activities. These PT analogs, as well as S1(R9K,E129G)/S3(K82A) and S1(R9K,E129G)/S3(I91,Y92,K93) delta, still expressed an epitope which elicits a neutralizing antitoxin antibody and were protective in the mouse intracerebral challenge test. Recombinant pertussis vaccines based on PT analogs with detoxifying mutations in multiple subunits may thus represent the next generation of improved whooping cough vaccines.

Identification of T- and B-cell epitopes of the S2 and S3 subunits of pertussis toxin by use of synthetic peptides.

To design an optimized synthetic vaccine against whooping cough, we have studied the biological and immunological properties of three peptides of the S2 subunit and nine overlapping synthetic peptides covering the entire sequence of the S3 subunit of pertussis toxin (PT). Synthetic peptides corresponding to sequences 18 to 41, 78 to 108, 134 to 154, and 149 to 176 of S3 were found to be consistently capable of stimulating the proliferation of PT-specific T-cell lines primed with pertussis toxoid in both BALB/c and A/J strains of mice. All synthetic peptides were recognized by rabbit antisera raised against PT or pertussis toxoid. Both S2 and S3 peptide-keyhole limpet hemocyanin (KLH) conjugates in the presence of complete Freund's adjuvant induced peptide-specific antibody responses in rabbits, and the antisera raised against S2(1-23), S3(18-41), S3(37-64), and S3(149-176) peptide-KLH conjugates cross-reacted with both subunits in the immunoblots. All antisera except those against S2(123-154) and S3(103-127) reacted with native PT in an enzyme-linked immunosorbent assay (ELISA) with PT directly coated onto microtiter wells. In contrast, antisera raised against S2(123-154), S3(1-23), S3(18-41), S3(37-64), S3(60-87), and S3(103-127) peptide-KLH conjugates recognized native PT in a fetuin-PT capture ELISA. S2(78-98), S3(1-23), and S3(149-176) peptide-KLH conjugates elicited good PT-neutralizing antibody responses as judged by the antitoxin CHO cell assay. Identification of these B-cell neutralization epitopes and T-cell immunodominant determinants represents a first step towards the rational design of a synthetic vaccine against whooping cough.

Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin.

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.