Lyme disease (LD) is the most prevalent vector borne disease is North America with 300,000-600,000 human cases each year. Preventative strategies for LD in humans are poorly developed and largely inadequate. While preventive vaccines for LD are widely used in veterinary medicine, there are no vaccines available for use in humans. The goal of this study was to develop a human vaccine that can elicit antibody responses that kill spirochetes in both the tick and mammalian environments. The approach applied in this study centered on the development of chimeric epitope proteins, referred to as chimeritopes. Chimeritopes consist of a series of epitopes derived from one or more proteins or protein variants. Three chimeritope proteins designated as Chv1, Chv2 and Chv3 were designed. These proteins harbor the same set of 18 linear epitopes derived from 9 different OspC type proteins. They differ in epitope arrangement or by the presence or absence of linkers between specific protein segments. The immunogenicity of each protein was assessed in multiple animal models including mice, rats, and purpose bred beagles. Immunoblot, ELISA, and IFA analyses using sera from immunized animals demonstrated that the Chv proteins elicit IgG responses that recognize a diverse array of OspC type proteins. Anti-Chv and anti-OspA antisera displayed complement dependent bactericidal activity. To assess protective efficacy, purpose bred beagles were immunized with each vaccine formulation and then challenged by infestation with infected ticks. Efficacy was assessed by monitoring seroconversion, cultivation of tissue biopsies, clinical presentation and histopathological analysis of joints and tissues. All dogs vaccinated with the Chv2-OspA combination were fully protected. All dogs in this group were seronegative for LD, biopsy culture negative and did not develop LD associated symptoms including lameness or lesions in tissues or joints. In light of market concerns centered on the use of full length OspA in a human vaccine, epitope mapping was performed to identify a linear epitope that could be employed in development of a possible OspC-OspA chimeritope. A linear epitope, designated as OspA221-240was identified. Antisera to KLH-OspA221-240displayed potent and broad bactericidal activity. Interestingly, the OspA221-240epitope has homology to residues 244 to 263 of OspB suggesting that OspB may also be a potential candidate for inclusion in a human vaccine. This study establishes proof of principle for the use of OspC chimeritopes in LD subunit vaccines and highlights the need to employ a multi-valent, multi-antigen vaccine approach in development of a human LD vaccine.

The threat and occurrence of terrorist attacks have increased in the United States since September 2011, heightening concerns for weaponized anthrax, other biological pathogens, and epidemics and pandemics. Early decisions and funding levels in local public health agencies can be the first line of defense or first point of failure; yet little is understood about how decisions are made when there are budget cuts before a biological event happens. Using Lindblom's conceptualization of limited rational choice theory, the purpose of this single case study was to understand how a local public health official made decisions after budget cuts in a single public health entity in the mid-Atlantic area of the United States. Data were collected through an interview with 1 public health official and publicly available plans, procedures, and funding documents. These data were inductively coded and then subjected to Braun and Clarke's thematic analysis procedure. Findings indicated that the public health agency's ability to make the best decisions were negatively impacted by limited resources, though adequate planning before a catastrophic event, active and continual communication with stakeholders, and clarity about financial and resource needs can partially offset the impact of budgetary reductions. The implications for social change include recommendations to anticipate and address the needs of the public health system through decision making to protect the health care community and the reduction or elimination of the spread of disease in the wake of a biological incident.

Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS) with characteristic multifocal lesions or ‘ plaques’ of demyelination mainly in the white matter of the brain ( involving cerebral cortex, cerebellar, brain stem and spinal cord). These MS plaques vary in size and shape, and are composed of infiltrates of lymphocytes and macrophages - which contain myelin debris. CD8 T cells are more prevalent in CNS lesions and display oligoclonal expansion. However, their role in disease remains unclear with studies showing both protective and pathogenic roles for myelin-specific CD8 T cells in the experimental autoimmune encephalomyelitis (EAE) model. Our studies have demonstrated a disease-suppressive function for CNS-specific CD8 T cells in a model where the antigen is exogenously administered in vivo and used for in vitro CD8 activation. My studies focus on probing the nature of the CD8 response elicited by endogenously presented myelin antigens in vivo utilizing a novel approach, infection with Listeria monocytogenes (LM) encoding for myelin proteolipid protein peptide PLP178-191 (LM-PLP). I show that LM-PLP infection preferentially induces PLP-specific CD8, but not CD4, T cell responses. Despite this, infection does not result in autoimmunity. In fact, routinely induced EAE is significantly ameliorated in LM-PLP-infected mice, compared to controls. Disease suppression is dependent on the presence of CD8 T cells, and the effector molecules IFN-g and perforin. CNS T cell infiltration and inflammatory responses are reduced in LM-PLP-protected mice, and CD4 T cells from LM-PLP-protected mice are less inflammatory than those from controls. Importantly, infection with LM-PLP ameliorates already established disease. My studies indicate that myelin-specific CD8 T cells induced by endogenous presentation of antigen attenuate CNS autoimmunity in multiple mouse models of EAE, implicating the potential of this approach as a novel immunotherapeutic strategy.

Human γδ T cells expressing the Vγ2Vδ2 T cell antigen receptor play important roles in immune responses to microbial pathogens by monitoring prenyl pyrophosphate isoprenoid metabolites. Most adult Vγ2Vδ2 cells are memory cytotoxic cells that produce interferon-γ (IFN-γ). Recently, murine γδ T cells were found to be major sources of interleukin (IL)-17A in anti-microbial and autoimmune responses. To determine if primate γδ T cells play similar roles, we characterized IL-17A and IL-22 production by Vγ2Vδ2 T cells. IL-17A-producing memory Vγ2Vδ2 T cells exist at low but significant frequencies in adult humans (1:2,762 T cells) and at even higher frequencies in adult rhesus macaques. Higher levels of Vγ2Vδ2 T cells produce IL-22 (1:1,864 T cells) although few produce both IL-17A and IL-22. Unlike adult humans where many IL-17A+ V#947;2Vδ2 T cells also produce IFN-#947; (T#947;δ1/17), the majority of adult macaques IL-17A+ Vδ2 T cells (T#947;δ17) do not produce IFN-#947;. To define the cytokine requirements for T#947;δ17 cells, we stimulated human neonatal V#947;2Vδ2 T cells with the bacterial antigen, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, and various cytokines and mAbs in vitro. We find that IL-6, IL-1β, and transforming growth factor-β (TGF-β) are required to generate T#947;δ17 cells in neonates whereas T#947;δ1/17 cells additionally required IL-23. In adults, memory T#947;δ1/17 and T#947;δ17 cells required IL-23, IL-1β, and TGF-β but not IL-6. IL-22-producing cells showed similar requirements. Both neonatal and adult IL-17A+ V#947;2Vδ2 T cells expressed elevated levels of retinoid-related orphan receptor-#947;t. Our data suggest that, like Th17 αβ T cells, V#947;2Vδ2 T cells can be polarized into T#947;δ17 and T#947;δ1/17 populations with distinct cytokine requirements for their initial polarization and later maintenance.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells identified in mice as Gr-1+CD11b+ cells with the ability to inhibit T cell function. MDSC are emerging as important regulators of T cell mediated immune responses. Current paradigm suggests that despite heterogeneity, all Gr-1+CD11b+ cells are suppressive when exposed to inflammatory stimuli. In vitro evaluation shows MDSC from multiple tissue sites have suppressive activity, and in vivo inhibition of MDSCenhances T cell function. However, the relative capacity of MDSC present at localized inflammatory sites or in peripheral tissues to suppress T cell responses in vivo has not been directly evaluated. Using a tissue specific acute inflammatory prostatitis model, we demonstrate that MDSC inhibition of CD8+ T-cell proliferation is restricted to the inflammatory site.Further, MDSC from inflammatory sites possess immediate capacity to inhibit T-cell function, whereas those isolated from peripheral tissues (spleens and liver) were not suppressive without activation of iNOS by exposure to IFN-_.Using two mouse models of prostate cancer, we extend these findings to thetumor micro-environment. During a chronic inflammatory response induced by tumorgrowth, we show Gr-1+CD11b+ cells from the tumor site possess immediate capacity toregulate effector T cell function whereas those from the spleen do not. In both tumormodels and in our prostatitis model, long term culture of activated T cells with splenicGr-1+CD11b+ cells converted precursor cells into functional MDSC during standard in vitro suppression assays. These data highlight the importance of MDSC in the prostate, and demonstrate the function of MDSC during a localized inflammatory response isrestricted to the site of an ongoing immune responseGrowing evidence suggests that prostatitis associated with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is mediated in part by the loss of T cell and B cell tolerance to prostate antigens. Clinical data demonstrates the presence of T cell proliferative responses to prostate auto-antigens in CP/CPPS patients. However, the mechanisms leading to this loss of tolerance are not clearly understood, largely because of a lack of available animal models. We report the development of a new mouse model for the study of chronic prostate inflammation (CPI), the Prostate Ovalbumin Expressing Transgenic-3 (POET-3) model. Adoptive transfer of antigen specific OT-I T cells induces CPI characterized by infiltration of exogenous (OT-I) and endogenous T cells into the prostate persisting as long as 45 days after transfer. In vitro and in vivo data demonstrate inflammation induced loss of T cell tolerance to prostate auto-antigens. Auto-antibody responses to prostate antigens were detected in POET-3 mice after induction of CPI. These data have important therapeutic implications for treatment of CPI.

Memory CD8 T cells offer increased protection to immune hosts by rapidly eliminating pathogen-infected cells during re-infection. Generating and sustaining a protective memory CD8 T cell response is considered a hallmark of adaptive immunity. Extensive research has been devoted to understanding the parameters affecting memory CD8 T cell generation after infection or immunization in order to design the most effective vaccines. An accepted notion in the field is that increased protection from re-infection is afforded by the generation of a large number of memory CD8 T cells. Consecutive prime-boost immunization strategies that elicit secondary responses are often used to increase the absolute numbers of memory CD8 T cells. While parameters affecting the generation of primary memory CD8 T cells are well known, the factors influencing the development of re-stimulated secondary CD8 T cell responses remain understudied. Here, I addressed the mechanisms involved in the generation and development of secondary memory CD8 T cells. I found that the time at which primary memory CD8 T cells enter into an immune response during re-infection impacts their fate and differentiation into secondary memory CD8 T cells. Late-entry of primary memory CD8 T cells into an immune response (relative to the initiation of infection) not only facilitates expression of transcription factors associated with memory formation in secondary effector CD8 T cells, but also influences the ability of secondary memory CD8 T cells to localize within the lymph nodes, produce interleukin-2 cytokine (IL-2), and undergo robust antigen-driven proliferation. The timing of stimulation of primary memory CD8 T cells also impacts the duration of expression of the high-affinity IL-2 receptor (CD25) on secondary effector CD8 T cells and their sensitivity to IL-2 signaling. Importantly, by blocking or enhancing IL-2 signaling in developing secondary CD8 T cells, I verify the role of IL-2 in controlling the differentiation of secondary CD8 T cell responses. The data I present herein suggest that the process of primary memory-to-secondary memory CD8 T cell differentiation is not fixed and can be manipulated, a notion with implications in the design of future prime-boost vaccination approaches. Although vaccines are designed and intended to benefit a range of individuals, at times the efficacy of a vaccination regime depends on the overall health status of a host. Thus, in another portion of my thesis work I explored the extent to which obesity compromises the differentiation and maintenance of protective memory CD8 T cell responses. I found that diet-induced obesity did not impact the maintenance of pre-existing memory CD8 T cells, including their acquisition of a long-term memory phenotype (i.e., CD27hi, CD62Lhi, KLRG1low) and function (i.e., cytokine production, antigen-driven secondary expansion, and memory CD8 T cell-mediated protection). Additionally, diet-induced obesity did not influence the differentiation and maintenance of newly evoked memory CD8 T cell responses, in inbred and outbred hosts, that were generated in response to different types of systemic (LCMV, L. monocytogenes) and/or localized (influenza virus) infections. Interestingly, I found that the rate of naïve-to-memory CD8 T cell differentiation after a peptide-coated dendritic cell immunization was similar in lean and obese hosts. This suggests that obesity-associated inflammation is unlike pathogen- or adjuvant-induced inflammation, and does not influence the development of an endogenous memory CD8 T cell response. My studies reveal that the obese environment does not influence the development or maintenance of memory CD8 T cell responses that are either primed before or after obesity is established. This is a surprising notion with implications for future studies aiming to elucidate the role of obesity in susceptibility to infection and vaccine efficacy. Collectively, the data presented here further the understanding of memory CD8 T cell responses in contexts of health and disease.

A recent study using human fecal samples in conjunction with a culture-independent approach revealed immense diversity of antibiotic resistance (AR) genes in the human gut microflora. We hypothesize that food animal gut microflora also contain diverse and novel AR genes which could contribute to the emergence and transmission of AR in pathogens important in animal and human health. To test this, we examined AR reservoir in chicken gut microflora using a metagenomic, functional cloning method. Total genomic DNA was extracted from individual cecal contents of two free range chickens and two conventionally raised chickens. The DNAs were physically sheered into 1 to 3 kb fragments, cloned into expression vector pZE21-MCS, and transformed into E. coli TOP10 host strain, resulting in four metagenomic libraries of a total size of 108 base pairs per library. The AR transformants from the libraries were selected on plates containing the specific antibiotic of interest; six antibiotics including ampicillin, tetracycline, chloramphenicol, spectinomycin, ciprofloxacin and norfloxacin were used for screening. Plasmids from selected transformants were extracted and subjected to sequence analysis of inserted fragments. Identified AR genes were annotated and aligned with homologs that have been deposited in GenBank. A total of 12 AR genes and 3 AR genes were identified from the microbiome in conventionally raised chickens and free-range chickens, respectively. Of the identified 15 AR genes, 8 genes that confer resistance to ampicillin, spectinomycin or chloramphenicol shared low sequence similarity (58% - 76% at amino acid level) with the corresponding AR genes previously identified using culture-dependent approaches. Notably, among the 8 novel AR genes identified in this study, 4 genes also shared low sequence similarities (59%-76% at amino acid level) with recently identified AR genes in human gut. An E. coli-Campylobacter shuttle vector bearing the flaA sigma 28 promoter was constructed. Two novel genes conferring resistance to ampicillin (FRAmp1.1) and spectinomycin (FRSpe1.1) were cloned into this new expression vector, respectively. The derived vectors have conferred increased AR in C. jejuni, a leading zoonotic bacterial pathogen causing human gastroenteritidis in many industrialized countries. Together, findings from this study showed the effectiveness of the metagenomic approach for examination of AR reservoir in food animals, revealed novel AR resistance genes in chicken gut microflora, and demonstrated the functionality of such AR genes in foodborne human pathogens.

This thesis represents the first anthropological perspective to be offered on the nature of the Klondike Gold Rush population. In order to better understand the experience of the average gold rusher, morbidity and mortality patterns are examined for the residents of the Yukon Territory following the discovery of gold in the region (1898-1904). Infectious diseases such as measles, pneumonia, smallpox and typhoid fever are the primary focus of this study, however local factors such as the severe climate and the seclusion of the gold fields from the outside world also offers an interesting opportunity to examine the consequences of leading a particularly harsh and physically demanding lifestyle in an inhospitable environment. / October 2008

Many important factors affect the spread of childhood infectious disease. To understand better the fundamental drivers of infectious disease spread, several researchers have estimated seasonal transmission coefficients using discrete-time models. This research addresses several shortcomings of the discrete-time approaches, including removing the need for the reporting interval to match the serial interval of the disease using infectious disease data from three major cities: New York City, London, and Bangkok. Using a simultaneous approach for optimization of differential equation systems with a Radau collocation discretization scheme and total variation regularization for the transmission parameter profile, this research demonstrates that seasonal transmission parameters can be effectively estimated using continuous-time models. This research further correlates school holiday schedules with the transmission parameter for New York City and London where previous work has already been done, and demonstrates similar results for a relatively unstudied city in childhood infectious disease research, Bangkok, Thailand.

Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use against a wide variety of diseases. Alone, this vaccine technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity against the expressed antigen. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our lab recently developed a non-invasive electrode known as the multi-electrode array (MEA), which lies flat on the surface of the skin without penetrating the tissue. This study evaluated the use of the MEA for the development of DNA vaccines. We assessed the appropriate delivery conditions for gene expression and the development of humoral immunity. We used both B. anthracis and HBV as infectious models for our experiments. Our results indicated that the MEA can enhance gene expression in a mouse model with minimal to no tissue damage. Optimal delivery conditions, based on generation of antibodies, were determined to be 125-175V/cm and 150ms with 200ug and a prime boost protocol administered on Day 0 and 14. Under these conditions, end-point titers of 20,000-25,000 were generated. Neutralizing antibodies were noted in 40-60% of animals. Additionally, we utilized a guinea pig model to assess the translation potential of this electrode. The plasmid encoding HBsAg, pHBsAg, was delivered intradermally with the MEA to guinea pig skin. The results show increased protein expression resulting from plasmid delivery using the MEA as compared to injection alone. Within 48 hours of treatment, there was an influx of cellular infiltrate in the experimental groups. Humoral responses were also increased significantly in both duration and intensity as compared to the injection only groups. Results from both experimental models demonstrate that protective levels of humoral immunity can be generated and that this electrode should translate well to the clinic.