Construction and preliminary immunobiological characterization of a novel, non-reverting, intranasal live attenuated whooping cough vaccine candidate.

We describe the construction and immunobiological properties of a novel whooping cough vaccine candidate, in which the aroQ gene, encoding 3-dehydroquinase, was deleted by insertional inactivation using the kanamycin resistance gene cassette and allelic exchange using a Bordetella suicide vector. The aroQ B. pertussis mutant required supplementation of media to grow but failed to grow on an unsupplemented medium. The aroQ B. pertussis mutant was undetectable in the trachea and lungs of mice at days 6 and 12 post-infection, respectively. Antigen-specific antibody isotypes IgG1 and IgG2a, were produced, and cell-mediated immunity [CMI], using interleukin-2 and interferon-gamma as indirect indicators, was induced in mice vaccinated with the aroQ B. pertussis vaccine candidate, which were substantially enhanced upon second exposure to virulent B. pertussis. Interleukin- 12 was also produced in the aroQ B. pertussis-vaccinated mice. On the other hand, neither IgG2a nor CMI-indicator cytokines were produced in DTaP-vaccinated mice, although the CMI-indicator cytokines became detectable post-challenge with virulent B. pertussis. Intranasal immunization with one dose of the aroQ B. pertussis mutant protected vaccinated mice against an intranasal challenge infection, with no pathogen being detected in the lungs of immunized mice by day 7 post-challenge. B. pertussis aroQ thus constitutes a safe, non-reverting, metabolite-deficient vaccine candidate that induces both humoral and cellmediated immune responses with potential for use as a single-dose vaccine in adolescents and adults, in the first instance, with a view to disrupting the transmission cycle of whooping cough to infants and the community.

Evaluation of immune response to recombinant potential protective antigens of Mycoplasma hyopneumoniae delivered as cocktail DNA and/or recombinant protein vaccines in mice.

Intramuscular immunization of mice with DNA cocktail vaccines, comprising potential protective antigens P36, P46, NrdF, and P97or P97R1 of Mycoplasma hyopneumoniae, induced strong Th1-polarized immune responses against each antigen, with only P46 eliciting a serum IgG response. Subcutaneous immunization with protein cocktail vaccines, surprisingly, induced both Th1-polarized immune response as well as antibody response whereas mice immunized with DNA cocktail vaccines followed by boosting with protein cocktail vaccines generated strong Th1-polarized and humoral immune responses. P97 was not recognized by serum antibodies from commercial bacterin-immunized mice indicating potential lack of expression of this important antigen in inactivated whole-cell vaccines.

Parenteral immunization of mice with a genetically inactivated pertussis toxin DNA vaccine induces cell-mediated immunity and protection.

The immunogenicity and protective efficacy of a DNA vaccine encoding a genetically inactivated S1 domain of pertussis toxin was evaluated using a murine respiratory challenge model of Bordetella pertussis infection. It was found that mice immunized via the intramuscular route elicited a purely cell-mediated immune response to the DNA vaccine, with high levels of gamma interferon (IFN-gamma) and interleukin (IL)-2 detected in the S1-stimulated splenocyte supernatants and no serum IgG. Despite the lack of an antibody response, the lungs of DNA-immunized mice were cleared of B. pertussis at a significantly faster rate compared with mock-immunized mice following an aerosol challenge. To gauge the true potential of this S1 DNA vaccine, the immune response and protective efficacy of the commercial diphtheria-tetanus-acellular pertussis (DTaP) vaccine were included as the gold standard. Immunization with DTaP elicited a typically strong T-helper (Th)2-polarized immune response with significantly higher titres of serum IgG than in the DNA vaccine group, but a relatively weak Th1 response with low levels of IFN-gamma and IL-2 detected in the supernatants of antigen-stimulated splenocytes. DTaP-immunized mice cleared the aerosol challenge more efficiently than DNA-immunized mice, with no detectable pathogen after day 7 post-challenge.

Targeted expression of redesigned and codon optimised synthetic gene leads to recrystallisation inhibition and reduced electrolyte leakage in spring wheat at sub-zero temperatures.

Antifreeze proteins (AFPs) adsorb to ice crystals and inhibit their growth, leading to non-colligative freezing point depression. Crops like spring wheat, that are highly susceptible to frost damage, can potentially be made frost tolerant by expressing AFPs in the cytoplasm and apoplast where ice recrystallisation leads to cellular damage. The protein sequence for HPLC-6 alpha-helical antifreeze protein from winter flounder was rationally redesigned after removing the prosequences in the native protein. Wheat nuclear gene preferred amino acid codons were used to synthesize a recombinant antifreeze gene, rAFPI. Antifreeze protein was targeted to the apoplast using a Murine leader peptide sequence from the mAb24 light chain or retained in the endoplasmic reticulum using C-terminus KDEL sequence. The coding sequences were placed downstream of the rice Actin promoter and Actin-1 intron and upstream of the nopaline synthase terminator in the plant expression vectors. Transgenic wheat lines were generated through micro projectile bombardment of immature embryos of spring wheat cultivar Seri 82. Levels of antifreeze protein in the transgenic lines without any targeting peptide were low (0.06-0.07%). The apoplast-targeted protein reached a level of 1.61% of total soluble protein, 90% of which was present in the apoplast. ER-retained protein accumulated in the cells at levels up to 0.65% of total soluble proteins. Transgenic wheat line T-8 with apoplast-targeted antifreeze protein exhibited the highest levels of antifreeze activity and provided significant freezing protection even at temperatures as low as -7 degrees C.

Evaluation of the immunogenicity of the P97R1 adhesin of Mycoplasma hyopneumoniae as a mucosal vaccine in mice.

The immunogenicity of P97 adhesin repeat region R1 (P97R1) of Mycoplasma hyopneumoniae, an important pathogenesis-associated region of P97, was evaluated in mice as a mucosal vaccine. Mice were immunized orally with attenuated Salmonella typhimurium aroA strain CS332 harbouring a eukaryotic or prokaryotic expression vector encoding P97R1. Local and systemic immune responses were analysed by ELISA on mouse sera, lung washes and splenocyte supernatants following splenocyte stimulation with specific antigens in vitro. Although no P97R1-specific antibody responses were detected in serum and lung washes, significant gamma interferon was produced by P97R1-stimulated splenocytes from mice immunized orally with S. typhimurium aroA harbouring either expression system, indicating induction of a cell-mediated immune response. These results suggested that live bacterial vectors carrying DNA vaccines or expressing heterologous antigens preferentially induce a Th1 response. Surprisingly, however, mice immunized with the vaccine carrier S. typhimurium aroA CS332 induced serum IgG, but not mucosal IgA, against P97R1 or S. typhimurium aroA CS332 whole-cell lysate, emphasizing the importance of assessing the suitability of attenuated S. typhimurium antigen-carrier delivery vectors in the mouse model prior to their evaluation as potential vaccines in the target species, which in this instance was pigs.

Comparative immunogenicity of M. hyopneumoniae NrdF encoded in different expression systems delivered orally via attenuated S. typhimurium aroA in mice.

The Mycoplasma hyopneumoniae ribonucleotide reductase R2 subunit (NrdF) gene fragment was cloned into eukaryotic and prokaryotic expression vectors and its immunogenicity evaluated in mice immunized orally with attenuated Salmonella typhimurium aroA CS332 harboring either of the recombinant expression plasmids. We found that NrdF is highly conserved among M. hyopneumoniae strains. The immunogenicity of NrdF was examined by analyzing antibody responses in sera and lung washes, and the cell-mediated immune (CMI) response was assessed by determining the INF-gamma level produced by splenocytes upon in vitro stimulation with NrdF antigen. S. typhimurium expressing NrdF encoded by the prokaryotic expression plasmid (pTrcNrdF) failed to elicit an NrdF-specific serum or secretory antibody response, and IFN-gamma was not produced. Similarly, S. typhimurium carrying the eukaryotic recombinant plasmid encoding NrdF (pcNrdF) did not induce a serum or secretory antibody response, but did elicit significant NrdF-specific IFN-gamma production, indicating induction of a CMI response. However, analysis of immune responses against the live vector S. typhimurium aroA CS332 showed a serum IgG response but no mucosal IgA response in spite of its efficient invasiveness in vitro. In the present study we show that the DNA vaccine encoding the M. hyopneumoniae antigen delivered orally via a live attenuated S. typhimurium aroA can induce a cell-mediated immune response. We also indicate that different live bacterial vaccine carriers may have an influence on the type of the immune response induced.

Late-onset and recurrent neonatal Group B streptococcal disease associated with breast-milk transmission.

The purpose of the study was to determine the epidemiological relationships in three unrelated cases of neonatal late-onset Group B streptococcal (GBS) disease and maternal breast-milk infection with GBS. All deliveries were by cesarean section; case 1 was at term, and cases 2 and 3 were at 32- and 33-wk gestation, respectively. Case 1 relates to a mother with clinical mastitis and recurrent GBS infection in a 20-day-old male infant. Following antibiotic therapy and cessation of breast-feeding, the infant recovered without sequelae. Case 2 refers to a mother with clinical mastitis and the occurrence of late-onset GBS disease in 5-wk-old male twins. Despite intervention, one infant died and the second became ill. Following antibiotic therapy and cessation of breast-feeding, the surviving infant recovered without sequelae. Case 3 refers to a mother with sub-clinical mastitis and late-onset GBS infection occurring in a 6-day-old female twin. Following intervention, the infant recovered but suffered a bilateral thalamic infarction resulting in developmental delay and a severe seizure disorder. Following recovery of GBS from an inapparent mastitis and cessation of breast-feeding, the second infant remained well. Blood cultures from all affected infants and maternal breast milk were positive for GBS. Epidemiological relationships between neonatal- and maternal-derived GBS isolates were confirmed by a random amplified polymorphic DNA polymerase chain reaction assay (RAPD-PCR). This study is significant in that it has demonstrated that maternal milk (in cases of either clinical or sub-clinical mastitis) can be a potential source of infection resulting in either late-onset or recurrent neonatal GBS disease.