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The study of the immune system has provided insight in the mechanism of protection induced by vaccination; primarily that most clinically protective vaccines are potent in generating neutralizing antibody responses. However, vaccination fails to protect against a wide range of acquired chronic infections caused by viruses, such as HIV, HBV and HCV. One of the major reasons for weak responses to therapeutic vaccine is the impaired function of effector T cells resulting from viral persistence. Although IL-2 can potently increase effect function of viral specific T cells, systemic administration of IL-2 induces organ pathology and expansion of Treg cells. In this study, we have now developed a novel vaccine delivery system IL-2-nanoAPC delivering antigen-MHC complexes (pMHC), co-stimulatory molecules and IL-2 to antigen specific T cells. NanoAPC are derived from the endoplasmic reticulum (ER) membranes of human B cell line 721.221 engineered with selected HLA allele and IL-2 as the ER retention proteins. The IL-2-nanoAPC interacted with antigen specific T cells, induced immune synapses and expression of high affinity IL-2 receptor and enhanced effector function of antigen specific T cells, but did not affect bystander T cells and Foxp3+ Treg cells. Together with pMHC, co-stimulatory molecules, the selective delivery of IL-2 not only increased the CD4 and CD8 T cell responses to viral antigens but also enhanced TCR proximal signalling and suppressed expression of PD1 molecules on IFNγ producing effector CD8 T cells. We also found that the co-induction of T helper responses by IL-2-nanoAPC in a mixed culture could increase CD8 T cell responses to viral antigen. The IL-2-nanoAPC effectively induced responses of CD4 and CD8 T cells from chronic HBV patients. The results demonstrate that selective delivery of IL-2, together with pMHC and co-stimulatory molecules, by nanoAPC to antigen specific T cells has potential to recover anti-viral immune responses in chronic HBV patients.

<p>Tuberculosis is a devastating disease that has been plaguing humankind for millennia. Co-evolution of humans with Mycobacterium tuberculosis, the causative agent of the disease, has allowed for the pathogen to possess an abundance of survival mechanisms. The outcome of this is the ability of the bacterium to create an intracellular niche lifestyle inside host cells where it can successfully evade the host immune system. While there is a vaccine available, named the BCG vaccine, it confers little protection to adults in the pulmonary form of the disease. The lack of an effective vaccine and the rise of Multidrug-Resistant (MDR) and Extensively Drug-Resistant (XDR) tuberculosis highlight the need for more research into combating Mycobacterium tuberculosis. The purpose of this work is to enhance the field of knowledge of how mycobacterial virulence factors affect host cellular pathways so that the interactions can be exploited for novel therapeutics and vaccine development.</p><p>One of the hypotheses for the poor efficacy of the BCG vaccine is that it fails to elicit a strong CD8+ T cell response during infection. Studies have found that vaccinating mice with apoptotic bodies containing mycobacterial antigens were able to protect mice to a greater degree than BCG and that this is dependent on CD8+ T cell activation. Thus, we hypothesized that a pro-apoptotic mutant of M. tuberculosis could be utilized as a novel vaccine candidate. Through screening a library of M. tuberculosis transposon mutants, we identified an Enhanced Cell Death mutant (ECD19) that functions through caspase 3 mediated apoptosis. Sequencing revealed that the mutant has a transposon insertion in Rv2456c, a probable integral membrane transport protein. Immunogenicity testing via Enzyme-Linked ImmunoSpot (ELISPOT) and Intracellular Cytokine Staining (ICS) assays demonstrated that ECD19 induced an altered immune response when compared to the parental strain M. tuberculosis H37Rv. Additionally, ECD19 has reduced survival in an in vitro THP-1 cell model and in an in vivo mouse model. Taken together, our data suggest that Rv2456c is important to the survival of H37Rv in host cells and that deletion of the gene may enhance the immunogenicity of the bacterium.</p><p>Inappropriate dosing and poor adherence to antibiotics in the treatment of tuberculosis has led to MDR and XDR, the highest incidences of which can be found in the KwaZulu-Natal (KZN) province of South Africa. Little is known about the virulence of these strains, but it is hypothesized that the drug resistance mechanisms come at a cost to the bacteria. In an in vitro assay, we have found that clinical isolates from the KZN region induce higher levels of necrosis than virulent laboratory strains of M. tuberculosis. Additionally, our in vivo studies show that the drug-resistant isolates do not disseminate as well as susceptible strains, and in both immunocompetent and immunocompromised mouse models, mice infected with the drug-resistant strains are able to live longer than mice infected with drug-sensitive strains. As all strains are highly related on a genetic level, we can say that the drug-resistant mechanisms acquired by the strains come at a cost of reduced virulence. Thus, it is likely that higher prevalence of the MDR and XDR in the KZN province is due to the high rate of HIV+, immunocompromised individuals living in the region. </p><p>Lastly, we are interested in building on the knowledge that avirulent mycobacteria are able to induce autophagy in a murine macrophage cell line. Through the use of Mammalian Target of Rapamycin (mTOR) inhibitors and autophagy-deficient macrophages, we were able to show that Mycobacterium smegmatis is able to induce both mTOR and autophagy during infection. Additionally, we found that mycobacterial killing occurs in the absence of autophagy when mTOR is inhibited. This effect is not due to a bactericidal effect of the mTOR inhibitors. From these data, we show that there is an underappreciated role in the induction of mTOR after mycobacterial infection. By studying the interplay of mTOR and autophagy, therapies targeted to favoring host defenses could be developed.</p><p>In summary, the insights from this work enhance the knowledge of how mycobacteria are able to be successful pathogens. This data may be useful in the creation of novel vaccine candidates or the identification of potential drug targets to bolster the therapeutic options in treating those afflicted with tuberculosis.</p> / Dissertation

OBJECTIVES: The anti-vaccination movement is prevalent in today's media with claims which continue to create feelings of fear and trepidation in the minds of many parents. The C.A.S.E. Approach (Corroboration, About Me, Science, Explain/Advise) is a method ofcommunication to be used in formulating meaningful, rapid responses to parents hesitant to vaccinating their children. This DNP project assessed the effects of a C.A.S.E. Approach learning module on family nurse practitioner (FNP) students' perceived levels of knowledge and self-efficacy regarding vaccination discussion with vaccine hesitant parents (VHPs). METHODS: This DNP project used a pretest-posttest design to measure the effects of the C.A.S.E. Approach training intervention on both knowledge and self-efficacy levels of FNP students. Fourteen students participated in this study. Each took the 20-question pretest C.A.S.E. Approach Questionnaire, then participated in the C.A.S.E. Approach learning module,and finished by repeating the questionnaire as a posttest following the intervention. The questionnaire was designed using four-item Likert questions scored 1 (strongly disagree) to 4(strongly agree), wherein higher scores reflected better understanding and self-efficacy in the C.A.S.E. Approach. Students were recruited via an online classroom format within a nursing course offered at the University of Arizona: Nursing 612, Introduction to Pediatrics. All testing and module information was accessed online and questionnaire responses were stored at Qualtrics.com, also online. RESULTS: Students' posttest scores following the intervention of the C.A.S.E. Approach learning module were significantly higher than pretest scores. Perceived knowledge (p< 0.001)of the C.A.S.E. Approach increased more significantly than did perceived self-efficacy (p =0.001) of the C.A.S.E. Approach following the module. Mean test scores increased on average 14.29 points in perceived knowledge of the C.A.S.E. Approach following the module, and 7.93 points for perceived self-efficacy following the module. CONCLUSION: Key findings included an observed increase in participating students' perceived knowledge regarding the C.A.S.E. Approach as well as an observed increase inparticipating students' perceived self-efficacy in using the C.A.S.E. Approach. There was strong statistical evidence (p≤0.05) to suggest the learning module increased student knowledge andself-efficacy regarding vaccine discussion.

Human papillomavirus (HPV) is one of the most common sexually transmitted infections in the United States, however vaccination uptake remains low. One of the known barriers of low vaccination rates is lack of a health care provider recommendation. The purpose of this project was to implement a practice change to increase the number of HPV vaccine recommendations provided by primary care providers (PCPs) to patients aged 9-26 years. The setting for this project was the Little Colorado Physician’s Office, a primary care clinic in rural northern Arizona. Four PCPs, including three family physicians and one family nurse practitioner, and members of the QI team participated in the project. The project was designed as a quality improvement project, guided by the Model for Improvement framework. The needs of the individual practice and their population were assessed by a quality improvement (QI) team using a fishbone diagram for root-cause analysis. A practice change was then implemented by the QI team and evaluated for its effectiveness in improving HPV vaccination recommendations. Outcome measures included the number of HPV vaccine recommendations made by a primary care provider to eligible patients and the number of HPV vaccines administered to patients. In a four-week period of practice change implementation, eight patients were considered eligible for the HPV vaccine. Of these patients, 100% were offered the HPV vaccine by their healthcare provider. The practice change was successful in promoting HPV vaccination recommendations by PCPs, and the QI team reported the change was beneficial to their practice.

Modified vaccinia virus Ankara (MVA) has been used extensively as a recombinant vector for delivery of antigens from diverse pathogens. Its ability to generate strong antigen specific CD8+ T cell responses in humans has been shown in clinical trials of novel vaccines against malaria, tuberculosis, HIV I AIDS, influenza and cancer. The work in this thesis describes the use of BAC recombineering technology to harness the endogenous regulatory signal (promoter) that drives the expression of non-essential open reading frames (ORFs) in MVA for immunogenic expression of a recombinant antigen. Replacement of the ORFs of four non-essential genes in MVA; C11R, F11l, A44L and B8R with an epitope tagged luciferase positioned to use the same endogenous promoter showed early transgene expression equal to or slightly higher than traditional p7.5 and short synthetic promoter (SSP) constructs. The frequency of antigen-specific CD8+ T cell induced in mice by single dose MVA or adenovirus-prime, rMVA-boost vaccination showed equivalent or slightly higher responses by the endogenous promoters compared to the traditional p7.5 and SSP constructs. Assessment of the growth rate of these viruses showed they were unimpaired and the insertions were genetically stable. Furthermore, the endogenous promoter driven insertion loci of B8R and C11R were used for the construction of a bivalent MVA expressing an epitope tagged luciferase (rLucPb9) and a Photinus pyralis (pLuc) luciferase. The frequency of antigen-specific CD8+ T cells induced in mice by bivalent MV A was equivalent to single-pLuc and single-Pb9 recombinants co-administered as a mixture, at separate sites or administered alone following single dose MV A vaccination but slightly lower for Pb9-specific CD8+ T cell following adenovirus-prime, rMVA-boost.

Twelve isolates of Ichthyophthirius multifiliis were successfully established and maintained by serial passage through naïve carp, for a maximum of 39 laboratory cycles. The management system employed was such that large numbers of the parasite were available for all investigations. The ability to induce exit of immature trophonts through media incubation was used to confirm events in the initial stages of host colonisation. The normal course of primary infection was also established providing useful criteria for assessing success of the in vitro systems tested. Survival of both theronts and tomonts within selected monophasic media was investigated. Theronts in Eagles Minimum Essential medium (EMEM), survived and were viable for 120 hours, 72 hours longer than water controls. No further development of the theronts was observed. Tomonts also demonstrated an increased survival time in comparison to the controls with tomites surviving within the cyst for 22 days within EMEM-S media diluted 50:50 with sterile distilled water. Division of tomonts was identified as being precystic, post divisional cystic or cystic, and the frequency of such divisions was dependent upon dilution of media. Sterile viable theronts were recovered at 168h from tomonts that had been incubated within EMEM diluted 30:70 with distilled water. Delayed encystment was achieved by incubation in concentrated media, theront production being delayed for 96h, 72h later than seen in the aquatic environment. Cultured cell monolayers were used as associates within culture systems. Behaviour of theronts on introduction into the culture systems indicated recognition of the cultured tissue as potential host material, sustained contact of up to l20hours was observed between the introduced parasite and cells. However, no developmental markers were identified within the cultured parasite and no significant growth was achieved. Attempts to simulate the situation in vivo by use of multilayered systems and crude cell explants were also unsuccessful. Transmission electron microscopy of the parasite within a cell aggregate system was undertaken at daily intervals up to 120h providing evidence that the parasite was attempting to gain nutrients by phagocytosis. However, increased vacuolation of the parasite during the period of culture was clearly evident leading eventually to parasite death. The significance of the results is discussed in relation to the normal course of infection and the future promise of a long term culture method for this important pathogen.

Doctor of Philosophy / Department of Anatomy and Physiology / Jishu Shi / Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important swine diseases worldwide that leads to severe reproductive failure in sows and high mortality in young pigs. Vaccination is currently the most effective way to control this disease. The protection ability provided by vaccines however is limited due to the large diversity of field PRRSV strains. In chapter 2, we compared immune responses induced by vaccination and/or PRRSV infection by using IngelVac® Modified Live PRRSV vaccine (MLV), its parental strain VR-2332, and the heterologous KS-06 strain. Our results showed that MLV provide complete protection to homologous virus and partial protection to heterologous challenge. The protection was associated with the levels of PRRSV neutralizing antibodies at the time of challenge. Besides developing new vaccines to combat PRRSV, adjuvants have been applied to PRRSV MLV vaccines to induce vaccination-mediated cross-protection against genetically dissimilar PRRSV strains. In chapter 3, we demonstrated that a commercial MontanideTM Gel01ST adjuvant provides enhanced protection to homologous PRRSV infection by regulating the production of PRRSV-specific antibodies. In chapter 4, we tested a novel peptide nanofiber hydrogel acting as a potent adjuvant for PRRSV MLV vaccines. We found that the hydrogel adjuvant enhanced vaccine efficacy by developing of higher titers of neutralizing antibodies and stronger IFN-γ cellular immune responses. Chinese highly pathogenic PRRSV (HP-PRRSV) variants were isolated in 2006 and they belong to genotype 2 of PRRSV. Compared with classic PRRSV, HP-PRRSV is characterized by robust proliferation ability and high morbidity/mortality with all ages of pigs. In chapter 5, we compared the difference of immune responses elicited by HV-PRRSV, a Chinese HP-PRRSV, and a US virulent strain of PRRSV NADC-20. Traditional PRRSV MLV vaccines developed in US offer no protection to HP-PRRSV. Vaccines specific to HP-PRRSV strains available in China provide protection to HP-PRRSV. In chapter 6, we demonstrated that pigs challenged with US NADC-20 strain were protected by vaccination with Chinese MLV HP-PRRSV vaccines. The availability of Chinese HP-PRRSV vaccines in North America may act to increase the preparedness of possible transmission of HP-PRRSV to North America.

Thesis (M.Med.(Paediatrics), Faculty of Health Sciences, University of the Witwatersrand, 2009 / Background In settings with a high burden of tuberculosis, children with unrecognised culture-confirmed pulmonary tuberculosis (PTB) may be discharged from hospital before mycobacterial culture results are available; in these cases clinical improvement may have been due to successful treatment of an intercurrent viral or bacterial co-infection. Aim To estimate the burden of tuberculosis in children who were enrolled in a double-blind, placebo-controlled pneumococcal conjugate vaccine (PCV) trial, and to probe for the presence of pneumococcal co-infection in trial participants who had a hospital-based diagnosis of PTB. Methods A retrospective case-finding strategy was adopted in order to define the tuberculosis case load amongst 39 836 children that had been enrolled in a PCV efficacy trial in Soweto, Gauteng Province. The trial follow-up period was 5.3 years. Children with a hospital-based diagnosis of tuberculosis were categorised by strength of evidence for the disease, HIV status and PCV vaccination status. Incidence rates and risk ratio assessments were conducted using standard statistical methods. Results Four-hundred and ninety-two episodes of tuberculosis arose amongst 425 of the 39 836 PCV Study participants. Tuberculosis incidence was 1067 per 100 000 children (95% Confidence Interval [CI], 968 – 1173), with the greatest burden observed amongst HIV-infected children (10 633 per 100 000 children [95% CI, 9411 – 11 969]; Risk Ratio [RR] 27.5 [95% CI, 22.6 – 33.5], P<0.001). The burden of PTB in the cohort was 982 cases per 100 000 children (95% CI, 887 – 1084): 9895 per 100 000 (95% CI, 8718 – 11 187) in the HIV-infected children and 352 per 100 000 (95% CI 294 – 417) in the HIV-uninfected children (RR 28.1; 95% CI, 22.9 – 34.6), P<0.001. PCV recipients exhibited a 44 percent (95% CI, 11 – 65), P=0.010, reduction in incident culture-confirmed PTB compared to placebo recipients; this apparent reduction was demonstrated chiefly in PCV-vaccinated HIV-infected children (RR 0.53; 95% CI, 0.31 – 0.90) compared to HIV-infected placebo recipients, P=0.017. Conclusions A high burden of tuberculosis is carried by children under 5.3 years in the study setting, with HIV-infected children bearing the brunt of the morbidity. Pneumococcal co-infections are common in the context of hospitalised PTB in the study setting.