Immunogenicity and protective efficacy of an anti-Streptococcus pyogenes vaccine candidate in multiple animal species

Streptococcus pyogenes, also known as Group A Streptococcus (GAS) has been associated with a range of diseases from the mild pharyngitis and pyoderma to more severe invasive infections such as streptococcal toxic shock. GAS also causes a number of non-suppurative post-infectious diseases such as rheumatic fever, rheumatic heart disease and glomerulonephritis. The large extent of GAS disease burden necessitates the need for a prophylactic vaccine that could target the diverse GAS emm types circulating globally. Anti-GAS vaccine strategies have focused primarily on the GAS M-protein, an extracellular virulence factor anchored to GAS cell wall. As opposed to the hypervariable N-terminal region, the C-terminal portion of the protein is highly conserved among different GAS emm types and is the focus of a leading GAS vaccine candidate, J8-DT/alum. The vaccine candidate J8-DT/alum was shown to be immunogenic in mice, rabbits and the non-human primates, hamadryas baboons. Similar responses to J8-DT/alum were observed after subcutaneous and intramuscular immunization with J8-DT/alum, in mice and in rabbits. Further assessment of parameters that may influence the immunogenicity of J8-DT demonstrated that the immune responses were identical in male and female mice and the use of alum as an adjuvant in the vaccine formulation significantly increased its immunogenicity, resulting in a long-lived serum IgG response. Contrary to the previous findings, the data in this thesis indicates that a primary immunization with J8-DT/alum (50ƒÊg) followed by a single boost is sufficient to generate a robust immune response in mice. As expected, the IgG response to J8- DT/alum was a Th2 type response consisting predominantly of the isotype IgG1 accompanied by lower levels of IgG2a. Intramuscular vaccination of rabbits with J8-DT/alum demonstrated that an increase in the dose of J8-DT/alum up to 500ƒÊg does not have an impact on the serum IgG titers achieved. Similar to the immune response in mice, immunization with J8-DT/alum in baboons also established that a 60ƒÊg dose compared to either 30ƒÊg or 120ƒÊg was sufficient to generate a robust immune response. Interestingly, mucosal infection of naive baboons with a M1 GAS strain did not induce a J8-specific serum IgG response. As J8-DT/alum mediated protection has been previously reported to be due to the J8- specific antibody formed, the efficacy of J8-DT antibodies was determined in vitro and in vivo. In vitro opsonization and in vivo passive transfer confirmed the protective potential of J8-DT antibodies. A reduction in the bacterial burden after challenge with a bioluminescent M49 GAS strain in mice that were passively administered J8-DT IgG established that protection due to J8-DT was mediated by antibodies. The GAS burden in infected mice was monitored using bioluminescent imaging in addition to traditional CFU assays. Bioluminescent GAS strains including the ‘rheumatogenic’ M1 GAS could not be generated due to limitations with transformation of GAS, however, a M49 GAS strain was utilized during BLI. The M49 serotype is traditionally a ‘nephritogenic’ serotype associated with post-streptococcal glomerulonephritis. Anti- J8-DT antibodies now have been shown to be protective against multiple GAS strains such as M49 and M1. This study evaluated the immunogenicity of J8-DT/alum in different species of experimental animals in preparation for phase I human clinical trials and provided the ground work for the development of a rapid non-invasive assay for evaluation of vaccine candidates.

Road safety messages tailored for young adults : Using the Internet and encouraging protective passenger behaviour

Young drivers are at higher risk of crashes than other drivers when carrying passengers. Graduated Driver Licensing has demonstrated effectiveness in reducing fatalities however there is considerable potential for additional strategies to complement the approach. A survey with 276 young adults (aged 17-25 years, 64% females) was conducted to examine the potential and importance of strategies that are delivered via the Internet and potential strategies for passengers. Strategies delivered via the Internet represent opportunity for widespread dissemination and greater reach to young people at times convenient to them. The current study found some significant differences between males and females with regard to ways the Internet is used to obtain road safety information and the components valued in trusted road safety sites. There were also significant differences between males and females on the kinds of strategies used as passengers to promote driver safety and the context in which it occurred, with females tending to take more proactive strategies than males. In sum, young people see value in Internet delivery for passenger safety information (80% agreed/ strongly agreed) and more than 90% thought it was important to intervene while a passenger of a risky driver. Thus tailoring Internet road safety strategies to young people may differ for males and females however there is considerable potential for a passenger focus in strategies aimed at reducing young driver crashes.

Long term immunity to live attenuated Japanese encephalitis chimeric virus vaccine : randomized, double-blind, 5-year phase II study in healthy adults.

In a randomized, double-blind study, 202 healthy adults were randomized to receive a live, attenuated Japanese encephalitis chimeric virus vaccine (JE-CV) and placebo 28 days apart in a cross-over design. A subgroup of 98 volunteers received a JE-CV booster at month 6. Safety, immunogenicity, and persistence of antibodies to month 60 were evaluated. There were no unexpected adverse events (AEs) and the incidence of AEs between JE-CV and placebo were similar. There were three serious adverse events (SAE) and no deaths. A moderately severe case of acute viral illness commencing 39 days after placebo administration was the only SAE considered possibly related to immunization. 99% of vaccine recipients achieved a seroprotective antibody titer ≥ 10 to JE-CV 28 days following the single dose of JE-CV, and 97% were seroprotected at month 6. Kaplan Meier analysis showed that after a single dose of JE-CV, 87% of the participants who were seroprotected at month 6 were still protected at month 60. This rate was 96% among those who received a booster immunization at month 6. 95% of subjects developed a neutralizing titer ≥ 10 against at least three of the four strains of a panel of wild-type Japanese encephalitis virus (JEV) strains on day 28 after immunization. At month 60, that proportion was 65% for participants who received a single dose of JE-CV and 75% for the booster group. These results suggest that JE-CV is safe, well tolerated and that a single dose provides long-lasting immunity to wild-type strains

Novice riders and the predictors of riding without motorcycle protective clothing

Background While helmet usage is often mandated, few motorcycle and scooter riders make full use of protection for the rest of the body. Little is known about the factors associated with riders’ usage or non-usage of protective clothing. Methods Novice riders were surveyed prior to their provisional licence test in NSW, Australia. Questions related to usage and beliefs about protective clothing, riding experience and exposure, risk taking and demographic details. Multivariable Poisson regression models were used to identify factors associated with two measures of usage, comparing those who sometimes vs rarely/never rode unprotected and who usually wore non-motorcycle pants vs motorcycle pants. Results Ninety-four percent of eligible riders participated and usable data was obtained from 66% (n = 776). Factors significantly associated with riding unprotected were: youth (17–25 years) (RR = 2.00, 95% CI: 1.50–2.65), not seeking protective clothing information (RR = 1.29, 95% CI = 1.07–1.56), non-usage in hot weather (RR = 3.01, 95% CI: 2.38–3.82), awareness of social pressure to wear more protection (RR = 1.48, 95% CI: 1.12–1.95), scepticism about protective benefits (RR = 2.00, 95% CI: 1.22–3.28) and riding a scooter vs any type of motorcycle. A similar cluster of factors including youth (RR = 1.17, 95% CI: 1.04–1.32), social pressure (RR = 1.32, 95% CI: 1.16–1.50), hot weather (RR = 1.30, 95% CI: 1.19–1.41) and scooter vs motorcycles were also associated with wearing non-motorcycle pants. There was no evidence of an association between use of protective clothing and other indicators of risk taking behaviour. Conclusions Factors strongly associated with non-use of protective clothing include not having sought information about protective clothing and not believing in its injury reduction value. Interventions to increase use may therefore need to focus on development of credible information sources about crash risk and the benefits of protective clothing. Further work is required to develop motorcycle protective clothing suitable for hot climates.

Induction of anti-chlamydial mucosal immunity by transcutaneous immunization is enhanced by topical application of GM-CSF

Transcutaneous immunization (TCI) involves the direct application of antigen plus adjuvant to skin, taking advantage of the large numbers of Langerhans cells and other resident skin dendritic cells, that process antigen then migrate to draining lymph nodes where immune responses are initiated. We have used this form of immunization to protect mice against genital tract and respiratory tract chlamydial infection. Protection was associated with local antibody responses in the vagina, uterus and lung as well as strong Th1 responses in the lymph nodes draining the reproductive tract and lungs respectively. In this study we show that topical application of GM-CSF to skin enhances the numbers and activation status of epidermal dendritic cells. Topical application of GM-CSF also increased the immune responses elicited by TCI. GM-CSF supplementation greatly increased cytokine (IFNgamma and IL-4) gene expression in lymph node and splenic cells compared to cells from animals immunized without GM-CSF. IgG responses in serum, uterine lavage and bronchoalveolar lavage and IgA responses in vaginal lavage were also increased by topical application of GM-CSF. The studies show that TCI induces protection against genital and respiratory tract chlamydial infections and that topical application of cytokines such as GM-CSF can enhance TCI-induced antibody and cell-mediated immunity.

Motorcycle protective clothing : protection from injury or just the weather?

Background Apart from helmets, little is known about the effectiveness of motorcycle protective clothing in reducing injuries in crashes. The study aimed to quantify the association between usage of motorcycle clothing and injury in crashes. Methods and findings Cross-sectional analytic study. Crashed motorcyclists (n = 212, 71% of identified eligible cases) were recruited through hospitals and motorcycle repair services. Data was obtained through structured face-to-face interviews. The main outcome was hospitalization and motorcycle crash-related injury. Poisson regression was used to estimate relative risk (RR) and 95% confidence intervals for injury adjusting for potential confounders. Results Motorcyclists were significantly less likely to be admitted to hospital if they crashed wearing motorcycle jackets (RR = 0.79, 95% CI: 0.69–0.91), pants (RR = 0.49, 95% CI: 0.25–0.94), or gloves (RR = 0.41, 95% CI: 0.26–0.66). When garments included fitted body armour there was a significantly reduced risk of injury to the upper body (RR = 0.77, 95% CI: 0.66–0.89), hands and wrists (RR = 0.55, 95% CI: 0.38–0.81), legs (RR = 0.60, 95% CI: 0.40–0.90), feet and ankles (RR = 0.54, 95% CI: 0.35–0.83). Non-motorcycle boots were also associated with a reduced risk of injury compared to shoes or joggers (RR = 0.46, 95% CI: 0.28–0.75). No association between use of body armour and risk of fracture injuries was detected. A substantial proportion of motorcycle designed gloves (25.7%), jackets (29.7%) and pants (28.1%) were assessed to have failed due to material damage in the crash. Conclusions Motorcycle protective clothing is associated with reduced risk and severity of crash related injury and hospitalization, particularly when fitted with body armour. The proportion of clothing items that failed under crash conditions indicates a need for improved quality control. While mandating usage of protective clothing is not recommended, consideration could be given to providing incentives for usage of protective clothing, such as tax exemptions for safety gear, health insurance premium reductions and rebates.

Innate Immunity in Lobsters: Partial Purification and Characterization of a Panulirus cygnus Anti-A Lectin

A lectin detected in haemolymph from the Australian spiny lobster Panulirus cygnus agglutinated human ABO Group A cells to a higher titre than Group O or B. The lectin also agglutinated rat and sheep erythrocytes, with reactivity with rat erythrocytes strongly enhanced by treatment with the proteolytic enzyme papain, an observation consistent with reactivity via a glycolipid. The lectin, purified by affinity chromatography on fixed rat-erythrocyte stroma, was inhibited equally by N-acetylglucosamine and N-acetylgalactosamine. Comparison of data from gel filtration of haemolymph (behaving as a 1,800,000 Da macromolecule), and polyacrylamide gel electrophoresis of purified lectin (a single 67,000 Da band), suggested that in haemolymph the lecin was a multimer. The purified anti-A lectin autoprecipitated unless the storage solution contained chaotropic inhibitors (125 mmol/L sucrose: 500 mmol/L urea). The properties of this anti-A lectin and other similar lectins are consistent with a role in innate immunity in these invertebrates.

SNAREing immunity : the role of SNAREs in the immune system

The trafficking of molecules and membranes within cells is a prerequisite for all aspects of cellular immune functions, including the delivery and recycling of cell surface proteins, secretion of immune mediators, ingestion of pathogens and activation of lymphocytes. SNARE (soluble-N-ethylmaleimide-sensitive-factor accessory-protein receptor)-family members mediate membrane fusion during all steps of trafficking, and function in almost all aspects of innate and adaptive immune responses. Here, we provide an overview of the roles of SNAREs in immune cells, offering insight into one level at which precision and tight regulation are instilled on immune responses.

Evaluation of an intervention to promote protective infant feeding practices to prevent childhood obesity : outcomes of the NOURISH RCT at 14 months of age and 6 months post the first of two intervention modules

OBJECTIVE: To evaluate a universal obesity prevention intervention, which commenced at infant age 4-6 months, using outcome data assessed 6-months after completion of the first of two intervention modules and 9 months from baseline. DESIGN: Randomised controlled trial of a community-based early feeding intervention. SUBJECTS AND METHODS: 698 first-time mothers (mean age 30±5 years) with healthy term infants (51% male) aged 4.3±1.0 months at baseline. Mothers and infants were randomly allocated to self-directed access to usual care or to attend two group education modules, each delivered over three months, that provided anticipatory guidance on early feeding practices. Outcome data reported here were assessed at infant age 13.7±1.3 months. Anthropometrics were expressed as z-scores (WHO reference). Rapid weight gain was defined as change in weight-for-age z-score (WAZ) > +0.67. Maternal feeding practices were assessed via self-administered questionnaire. RESULTS: There were no differences according to group allocation on key maternal and infant characteristics. At follow up (n=598 [86%]) the intervention group infants had lower BMIZ (0.42±0.85 vs 0.23±0.93, p=0.009) and infants in the control group were more likely to show rapid weight gain from baseline to follow up (OR=1.5 CI95%1.1-2.1, p=0.014). Mothers in the control group were more likely to report using non- responsive feeding practices that fail to respond to infant satiety cues such as encouraging eating by using food as a reward (15% vs 4%, p=0.001) or using games ( 67% vs 29%, p<0.001). CONCLUSIONS: These results provide early evidence that anticipatory guidance targeting the ‘when, what and how’ of solid feeding can be effective in changing maternal feeding practices and, at least in the short term, reducing anthropometric indicators of childhood obesity risk. Analyses of outcomes at later ages are required to determine if these promising effects can be sustained.

A randomised controlled trial of a theory-based intervention to improve sun protective behaviour in adolescents ('you can still be HOT in the shade') : study protocol

Background: Most skin cancers are preventable by encouraging consistent use of sun protective behaviour. In Australia, adolescents have high levels of knowledge and awareness of the risks of skin cancer but exhibit significantly lower sun protection behaviours than adults. There is limited research aimed at understanding why people do or do not engage in sun protective behaviour, and an associated absence of theory-based interventions to improve sun safe behaviour. This paper presents the study protocol for a school-based intervention which aims to improve the sun safe behaviour of adolescents. Methods/design: Approximately 400 adolescents (aged 12-17 years) will be recruited through Queensland, Australia public and private schools and randomized to the intervention (n = 200) or 'wait-list' control group (n = 200). The intervention focuses on encouraging supportive sun protective attitudes and beliefs, fostering perceptions of normative support for sun protection behaviour, and increasing perceptions of control/self-efficacy over using sun protection. It will be delivered during three × one hour sessions over a three week period from a trained facilitator during class time. Data will be collected one week pre-intervention (Time 1), and at one week (Time 2) and four weeks (Time 3) post-intervention. Primary outcomes are intentions to sun protect and sun protection behaviour. Secondary outcomes include attitudes toward performing sun protective behaviours (i.e., attitudes), perceptions of normative support to sun protect (i.e., subjective norms, group norms, and image norms), and perceived control over performing sun protective behaviours (i.e., perceived behavioural control). Discussion: The study will provide valuable information about the effectiveness of the intervention in improving the sun protective behaviour of adolescents.