Infection with Plasmodium species leads to nearly 400,000 deaths a year despite widespread use of mosquito bed nets, insecticides, and anti-malarial drugs. To date, there is not a licensed vaccine capable of providing complete protection from Plasmodium infection to vaccinees. Whole parasite vaccination of humans and rodents can achieve complete protection in vaccines, but the dose of sporozoites, number of administrations, and production concerns in generating these types of vaccines will likely prevent these approaches from achieving worldwide use. However, the protective immunological responses against Plasmodium parasites engendered by these vaccination approaches can be studied and aid in the development of advanced subunit vaccines against Plasmodium. Using rodent models of malaria to elucidate the features of protective immunity engendered by whole parasite vaccination, it has been repeatedly shown that CD8 T cell responses directed against liver-stage parasite antigens can provide complete protection with some contribution by CD4 T cells and antibody responses depending on the model system studied. However, the quantatitive and qualitative requirements for CD8 T cell immunity against Plasmodium remains largely undefined. To enhance our understanding of how to generate protective immunity against Plasmodium, I have utilized rodent models of malaria to study the superior protection afforded from single-dose vaccination with virulent sporozoites administered under prophylatic chloroquine-cover, referred to as chemoprophylaxis sporozoites (CPS) vaccination, compared to the well-studied approach of administering radiation-attenuated Plasmodium sporozoites (RAS). RAS vaccination has long been considered the “gold standard” in vaccination due the ability of RAS vaccination to engender complete protection following sporozoite challenge of vaccinated humans and rodents. However, CPS vaccination is arguably a superior vaccination approach since it can achieve protection through less vaccine administrations relative to RAS vaccination, but the immunological basis of this enhanced CPS vaccine-induced immune response was unclear. In my study, I utilized a stringent host/parasite model to find that C57Bl/6 mice administered CPS vaccination with P. yoelii sporozoites elicit substantially higher parasite-specific CD8 T cell responses than RAS vaccination, but CPS-induced CD8 T cells were not necessary for protection following liver-stage sporozoite or blood-stage parasite challenge. CPS vaccination resulted in a low grade, transient parasitemia shortly following cessation of chloroquine treatment, which lead to the generation of potent antibody responses to blood-stage parasites; this blood-stage parasite-specific antibody response correlated with sterilizing protection in sporozoite challenged CPS-vaccinated mice. Therefore, my data provide a mechanistic basis for enhanced protective immunity elicited by single-dose CPS vaccination in a rodent model that is independent of CD8 T cells. The other portion of my work examines how CD8 T cell specificity impacts protective capacity against Plasmodium. I show that robust CD8 T cell responses of similar phenotype are mounted following prime-boost immunization against three novel Plasmodium berghei protein-derived epitopes in addition to a previously described protective, immunodominant epitope. I show that only CD8 T cells specific to sporozoite surface-expressed protein-derived epitopes, but not the intracellular protein-derived epitopes, are efficiently recognized by sporozoite-infected hepatocytes in vitro. These results suggest that antigenic targets must be efficiently presented by infected hepatocytes for CD8 T cells to eliminate liver-stage Plasmodium infection and proteins expressed on the surface of sporozoites may be good target antigens for protective CD8 T cells. Collectively, my work highlights the ability to generate protective CD8 T cell independent and dependent immunity against Plasmodium infections, whether achieved through potent blood-stage-specific antibody responses, or via numerically large monospecific CD8 T cell responses that target parasite antigens that are efficiently presented during liver-stage infection. These studies are relevant in understanding how to efficiency engender protective immunity against Plasmodium, and could aid in the advancement of subunit vaccination approaches that generate immunity through the priming of responses from multiple arms of the immune response, targeting both the liver- and blood-stages of Plasmodium.

Hispanic and African American women are infected with sexually transmitted diseases more often than are Caucasian women. This racial disparity is also seen in the incidence of human papillomavirus (HPV) and cervical cancer. The medical connection between HPV and cervical cancer is often unknown or misunderstood among women. This study addressed the beliefs and subsequent health decisions of minority parents regarding whether to get their daughters vaccinated against HPV. The theoretical framework for this study was Rosenstock's health belief model (HBM). The specific study design used was Husserl and Heidegher's theory on Phenomenology. This qualitative study utilized focus groups containing mothers of young girls ages 9 to 12 years, who were recruited from local churches in San Antonio, TX. Twenty-seven mothers, African American (9), Hispanic (7), and Caucasian (11), participated in one of two focus groups for each racial group. Each focus group session was audiotaped and NVivo for Mac was used to perform a content analysis and to identify the themes present. Minority parents held stronger cultural and spiritual beliefs against vaccinating their daughters for a sexually transmitted disease more so than believing that their daughters were at risk for being exposed to STDs such as HPV. These beliefs presented as barriers to initiating the desired HPV prevention and screening practices. Gaps in the current knowledge of all parents exist and must be thoroughly addressed for all racial/ ethnic groups. Future educational programs need to not only address the gaps in knowledge but also shape and package public health messages with sensitivity to cultural and spiritual concerns.

The purpose of this study was to collect information regarding healthcare worker (HCW) vaccination policies in Utah family practice clinics. Data sources: The study was conducted in Utah family practice clinics in the most densely populated counties in the state and was a cross-sectional descriptive design. Data were collected from 155 family practice clinic managers. Analyses included frequencies and percentages for quantitative items and a content analysis for open-ended items. Conclusions: HCWs are employed in environments where infectious diseases can be easily spread from person to person, thus, vaccinations can be instrumental in protecting the health of HCWs and patients alike. In Utah, 56.8% of family practice clinics had either no vaccination policy for HCWs or had a policy with no consequences for noncompliance. Utah family practice clinics need to implement changes to create and maintain HCW vaccination policies. Implications for practice: Nurse practitioners can be leaders and change agents by working with their county and state health departments to create state-wide policies that mirror the position statements from the American Nurses Association and the American Association of Nurse Practitioners.

Autism is a childhood developmental disorder characterized by impaired social interaction, difficulty with verbal and nonverbal communication and limited activities. The root cause of autism is unknown. However it has been postulated that administration of the measles, mumps, and rubella (MMR) vaccine may be causally related to the development of autism. MMR vaccination is a requirement for entry into schools, so any increase in adverse events associated with the vaccine carries widespread public health importance. The primary objective of this study was to conduct a meta-analysis of the association between the MMR vaccination and the development of autism. The meta-analysis was limited to studies with an experimental design, unvaccinated control group, and odds ratio or relative risk as the effect measure. Both the fixed effects and random effects models were applied. A total of 29 studies were identified for possible inclusion in the meta-analysis. After applying the inclusion/exclusion criteria seven studies were included in the meta-analysis. The pooled treatment effect was weighted based on the width of the 95% confidence interval for each of the individual studies. The pooled effect measure for the seven studies was 1.052 (95% CI: 0.973, 1.138) (P=0.202). An association between the MMR vaccine and the development of autism was not found in this analysis. Public health initiatives should continue to support mandatory vaccination with MMR for entry into school and steps should be taken to eliminate any barriers to vaccination. Further epidemiological studies are needed to assess the root cause of autism

This thesis examines the results of a hepatitis B vaccination questionnaire study that was completed by medical officers, nurses, carers of the developmentally disabled, and correctional officers in the Greater Western Sydney area of New South Wales, Australia. The main aim of the study was to contrast these four high risk occupational groups for their acceptance of hepatitis B vaccination, seroconversion status, and behavioural, attitudinal, motivational and institutional determinants of their vaccination status. The results of the thesis revealed that medical officers and nurses were the most likely to be tested and vaccinated for seroconversion, while DD carers and correctional officers had the largest number of Not Vaccinated respondents. The findings of the questionnaire are discussed in some detail. It is also suggested that groups differ in the degree of hepatitis risk anxiety they experience as well as the degree of control felt over their health status. A number of indications for personal, institutional and governmental interventions to increase vaccination levels are discussed. / Doctor of Philosophy (PhD)

Includes list of publications arising from the thesis. Bibliography: leaves 327-341. Examines the effect of adverse storage on the immunogenicity of pertussis, diphtheria and tetanus vaccines, the protective efficacy of pertussis vaccines and the effect of premature birth on antibody response to routine childhood immunisations.

Vaccination with immunogenic peptides offers a safe and specific way of inducing protection against pathogens, however as of yet there are no peptide-based vaccines available. The limitations on the therapeutic use of peptides are due to their poor immunogenicity and short life span in vivo. Peptide delivery systems act to circumvent these issues. The aims of this research were to investigate the ability of virus-like particles (VLP) from Rabbit haemmorhagic disease virus (RHDV) to deliver immunogenic peptides, to characterize the immune response to these particles, and to investigate whether baculovirus could also act as a delivery system. The vaccine peptides HAT (representing a T helper cell epitope) and HAB (representing the major B cell epitope) derived from the haemagglutinin antigen of influenza virus A/PR/8/34 were used as a model to investigate the ability of these virus particles to act as delivery vehicles to the immune system. A scheme for the production and purification of RHDV VLP was established. Expression of the capsid protein from RHDV in a serum-free recombinant baculovirus system using suspension cultures of up to 200 ml, and separation by isopycnic centrifugation on cesium chloride gradients led to high yields of purified RHDV VLP. Up to 20 mg of pure VLP could be obtained from an 800 ml culture of insect cells infected with recombinant baculovirus. In vitro testing revealed that RHDV VLP carrying the peptide HAT as a genetic fusion were processed by dendritic cells (DC), and that this peptide could be presented to induce activation of T cells. However, the purified RHDV VLP alone were not able to induce significant upregulation of cell activation markers CD40, CD86, and CD80. A preliminary in vivo study revealed that when RHDV VLP carrying the HAT peptide were delivered by an intraperitoneal injection in the absence of adjuvant, the immune response to the peptide was weak, therefore the route of delivery and the use of immune adjuvants with the VLP were optimised. Five different routes of delivery and two different immune adjuvants were compared. VLP were delivered through subcutaneous, intraperitoneal, transcutaneous, intramuscular and intranasal routes. Delivery of the VLP through each of these routes resulted in potent serum antibody responses. However, the strongest antibody responses were elicited when the VLP were delivered through the intraperitoneal or intranasal routes. Of these two routes, intranasal delivery gave the best mucosal responses at the lung surface, and was therefore chosen as the route of delivery for subsequent trials. CpG DNA and the wild-type baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) were tested as adjuvants for the RHDV VLP. These two adjuvants gave similar results, both acting to enhance a T[H]1 type response against the VLP, characterized by significantly increased levels of serum IgG2a and enhanced IFN-γ production. Two approaches were then tested: using the RHDV VLP as a peptide carrier with a CpG adjuvant, and using baculovirus particles directly as self-adjuvanting carriers for vaccine peptides. HAT and HAB peptides were chemically coupled to RHDV VLP. Mice that were vaccinated with these VLP mixed with a CpG adjuvant were able to raise low levels of specific antibody in the serum against influenza, and specific IgA against influenza was detected in the lung. These results indicated that, though the immune responses raised were modest, the RHDV VLP was able to deliver the vaccine peptides to the immune system. HAT and HAB peptides were chemically coupled to baculovirus particles. When mice were immunized with the baculovirus carrying the vaccine peptides, they raised significant levels of IgG1 (p<0.001) and IgG2a (p<0.05) against influenza in the serum, when compared to peptide delivered alone. A significant level of influenza-specific IgA was also detected in the lung at 10 ng/ml in the mice that received the baculovirus coupled with peptide. Analysis of splenocyte cytokines showed that these mice also responded to restimulation with IFN-γ production at around 100 pg/ml. This research revealed that RHDV VLP are able to act as carriers for vaccine peptides, however there are some limitations to their use with the HAT and HAB model peptides. It also showed that baculovirus can be rapidly modified to carry vaccine peptides by chemical conjugation, and that these peptides can be delivered to induce specific systemic and mucosal immunity, raising both B cell and cell mediated responses. Both virus particles have potential as components for new strategies for vaccination.

Includes list of publications arising from the thesis. / Bibliography: leaves 327-341. / 341, [15] leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the effect of adverse storage on the immunogenicity of pertussis, diphtheria and tetanus vaccines, the protective efficacy of pertussis vaccines and the effect of premature birth on antibody response to routine childhood immunisations. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2002?

This thesis aimed to the identification of immune biomarkers of mycobacterial infection for better diagnosis of tuberculosis (TB) and also focused on new vaccination strategies with a particular emphasis on the immune responses in the respiratory tract using murine models. Since the lung is the natural habitat for the M. tuberculosis, we reasoned that immune responses detected locally in the lungs would be good correlates of infection (Paper I). Likewise, immune responses induced in the respiratory tract following immunization would be more effective against mycobacterial infection. We showed that cytokines (IL-12, TNF, and IFN-γ) and cytokine receptors (sTNFR1 and sTNFR2) together with specific antibodies in the respiratory tract correlated better with the bacterial burden in the organs. In Paper II, we investigated the role of the BCG vaccination as a priming vaccine in a heterologous prime-boost immunization protocol. The results showed that the neonatal BCG vaccination primed the immune system for a relevant antigen and showed a generalized adjuvant effect. Using this immunization protocol, protective immune responses in the lungs were generated independently of the route used for the booster immunization. In Paper III, We showed that exposure to mycobacterial antigens during the gestational period led to antigen transportation from the mother to the fetus and this resulted in an early priming of the fetal immune system. Immunization with the same antigen during the postnatal life increased antigen-specific recall IFN-γ responses and protection against infection. We examined the role of innate immunity for the induction of acquired immune responses upon immunization with mycobacterial antigens using TLR2 deficient mice (Paper IV). Our data indicated that suboptimal innate immune responses in the TLR2-/- mice might compromise the induction of acquired immune responses. Overall, the current findings suggested that a better understanding of the mucosal immunity would be useful for the improvement of diagnostic procedures and the development of efficient vaccines against TB. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript

Vaccination has proven a cost-effective method of managing infectious diseases, but attempts to develop an effective fetal vaccine have proven difficult due to the immaturity of the immune system and the propensity of the developing immune system to induce tolerance to immunizing antigens. This thesis is concerned with the induction of specific immunity in the second trimester ovine fetus using the oral DNA immunization method. In utero oral delivery of naked DNA plasmid was selected as the method of immunization due to previous successes in the third trimester ovine fetus and the immunostimulatory properties of the bacterial DNA backbone, which may help overcome developmental tolerance. Transfection and expression studies in the third trimester ovine fetus revealed the oral mucosal epithelium as the primary site of transgene expression and functionally active antigen was also localized to lymph nodes draining the oral cavity. Efficient transfection and expression of plasmid following oral delivery was specific to the fetus and correlated with a lesser degree of epithelial differentiation. Oral DNA delivery in the second trimester resulted in detection of transgene activity in 100% of treated fetuses and the level of transgene activity was greater than in fetuses treated in the mid-third trimester. Using a plasmid encoding the gene for bovine herpesvirus-1 truncated glycoprotein D (tgD), immunization studies were then conducted in the second trimester fetus. A new lower age limit for fetal immunization was established at 55-60 days gestation (gestation period is 148 days), which coincides with the appearance of lymphocytes in peripheral tissues. Antigen-specific antibody, interferon-× responses and/or neonatal anamnestic responses were detected in 66% of fetuses immunized between 55 and 84 days gestation. The duration of fetal primary immune responses was equivalent to that achieved in young lambs following optimized DNA vaccination, but the magnitude of fetal immune responses was limited. The persistence of immune memory from the second trimester to birth was consistent with experimental data which showed that the duration of immune memory had a stronger correlation to the duration, as compared to the magnitude, of the primary antibody response. Overall, the experiments within showed that oral DNA immunization of the early second trimester fetus is feasible and not associated with the induction of tolerance. These findings suggest that it may be possible to protect against mother-to-child transmission of infectious pathogens by targeting protection at the level of the fetus.