Bordetella Pertussis virulence factors in the continuing evolution of whooping cough vaccines for improved performance.

Despite high vaccine coverage, whooping cough caused by Bordetella pertussis remains one of the most common vaccine-preventable diseases worldwide. Introduction of whole-cell pertussis (wP) vaccines in the 1940s and acellular pertussis (aP) vaccines in 1990s reduced the mortality due to pertussis. Despite induction of both antibody and cell-mediated immune (CMI) responses by aP and wP vaccines, there has been resurgence of pertussis in many countries in recent years. Possible reasons hypothesised for resurgence have ranged from incompliance with the recommended vaccination programmes with the currently used aP vaccine to infection with a resurged clinical isolates characterised by mutations in the virulence factors, resulting in antigenic divergence with vaccine strain, and increased production of pertussis toxin, resulting in dampening of immune responses. While use of these vaccines provide varying degrees of protection against whooping cough, protection against infection and transmission appears to be less effective, warranting continuation of efforts in the development of an improved pertussis vaccine formulations capable of achieving this objective. Major approaches currently under evaluation for the development of an improved pertussis vaccine include identification of novel biofilm-associated antigens for incorporation in current aP vaccine formulations, development of live attenuated vaccines and discovery of novel non-toxic adjuvants capable of inducing both antibody and CMI. In this review, the potential roles of different accredited virulence factors, including novel biofilm-associated antigens, of B. pertussis in the evolution, formulation and delivery of improved pertussis vaccines, with potential to block the transmission of whooping cough in the community, are discussed.

Biofilm forming potential and antimicrobial susceptibility of newly emerged Western Australian Bordetella pertussis clinical isolates.

Whooping cough caused by Bordetella pertussis is increasing in several countries despite high vaccine coverage. One potential reason for the resurgence is the emergence of genetic variants of the bacterium. Biofilm formation has recently been associated with the pathogenesis of B. pertussis. Biofilm formation of 21 Western Australian B. pertussis clinical isolates was investigated. All isolates formed thicker biofilms than the reference vaccine strain Tohama I while retaining susceptibility to ampicillin, erythromycin, azithromycin and streptomycin. When two biofilm-forming clinical isolates were compared with Tohama I, minimum bactericidal concentrations of antimicrobial agents increased. Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis revealed significant differences in protein expression in B. pertussis biofilms, providing an opportunity for identification of novel biofilm-associated antigens for incorporation in current pertussis vaccines to improve their protective efficacy. The study also highlights the importance of determining antibiograms for biofilms to formulate improved antimicrobial therapeutic regimens.

Isolation, Biochemical and Molecular Identification, and In-Vitro Antimicrobial Resistance Patterns of Bacteria Isolated from Bubaline Subclinical Mastitis in South India.

Buffaloes are the second largest source of milk. Mastitis is a major impediment for milk production, but not much information is available about bubaline mastitis, especially subclinical mastitis. The aim of this study was to (a) investigate the application of various tests for the diagnosis of bubaline subclinical mastitis, (b) identify the major bacteria associated with it, and (c) evaluate the antibiotic resistance pattern of the bacteria. To this end, 190 quarter milk samples were collected from 57 domesticated dairy buffaloes from organized (64 samples) and unorganized (126 samples) sectors. Of these, 48.4%, 40.0%, 45.8%, 61.1%, and 61.6% were positive for subclinical mastitis by somatic cell count, electrical conductivity, California mastitis test, bromothymol blue test, and N-acetyl glucosaminidase test, respectively. As compared to the gold standard of somatic cell count, California mastitis test performed the best. However, a combination of the two methods was found to be the best option. Microbiological evaluation, both by biochemical methods as well as by monoplex and multiplex polymerase chain reaction, revealed that coagulase-negative staphylococci were the most predominant (64.8%) bacteria, followed by streptococci (18.1%), Escherichia coli (9.8%) and Staphylococcus aureus (7.3%). Most of the pathogens were resistant to multiple antibiotics, especially to ß-lactam antibiotics. We propose that California mastitis test be combined with somatic cell count for diagnosis of subclinical mastitis in domestic dairy buffaloes. Further, our results reveal high resistance of the associated bacteria to the ß-lactam class of antibiotics, and a possible major role of coagulase-negative staphylococci in causing the disease in India.

Preliminary studies on the development of IgA-loaded chitosan-dextran sulphate nanoparticles as a potential nasal delivery system for protein antigens.

This study describes the development of a biodegradable nanoparticulate system for the intranasal delivery of multiple proteins. Chitosan (CS)-dextran sulphate (DS) nanoparticles were developed and optimised for the incorporation of pertussis toxin (PTX) and a potential targeting ligand (immunoglobulin-A, IgA). In vitro characterization and in vivo uptake studies were performed for the evaluation of developed nanoparticles. The ratio of CS to DS, the order of mixing and pH of nanoparticle suspension were identified as important formulation factors governing the size and zeta potential of nanoparticles. An optimised CS-DS nanoparticle formulation prepared with the CS to DS weight ratio of 3 : 1 was used to load PTX and/or IgA. Entrapment efficiency of >90% was obtained for both. The in vivo uptake of IgA-loaded CS-DS nanoparticles in mice showed a preferential uptake of nanoparticles probably by nasal membranous or microfold cells following intranasal administration. The results of this study indicate the potential application of IgA-loaded CS-DS nanoparticles as a nasal vaccine delivery system.

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.