Vaccination is one of the major strategies available for combating viral infections in humans. However, currently available vaccines are not without pitfalls; they are laborious to produce, could potentially be unsafe, and in the case of the highly variable influenza virus need to be reformulated each season. The use of synthetic peptides thus represents an exciting alternative to traditional vaccines. However, these synthetic peptides are not highly immunogenic without the use of potent adjuvants. The lack of immunogenicity might be addressed by conjugation between T or B cell epitopes with universal or immunodominant T-helper epitopes. The construction of branched peptides, lipidated peptides, or designs combining both of these elements might also enhance the immunogenicity, as they might target Toll-like receptors and/or mimic the 3-dimensional structure of epitopes within the native protein. In this study, a recognized T-B peptide based on the hemagglutinin protein of the A/Puerto Rico/8/34 influenza virus was chosen as a backbone and modified to evaluate if the construction of branched peptides, lipidation, the addition of cysteine residues, or mutations could indeed alter reactivity. Screening the different designs with various antibody binding and cellular assays revealed that combining a branched design with the addition of lipid moieties leads to a greatly enhanced activity as compared to other similar T-B diepitope constructs.

Tuberculosis is a re-emerging global health problem. Bacille Calmette-Guerin (BCG), the available vaccine against the disease, is only effective short term and is associated with adverse reactions clinically. The development of new effective vaccines will require an understanding of virulence, immunogenic factors and the beneficial immune responses induced in the human host. My thesis investigates phoP and whiB3, two genes associated with virulence and immunogenicity in Mycobacterium tuberculosis. Study of PhoP in a natural phoP mutant, BCG-Prague, and in the clinically safe BCG-Japan, shows that over-expression of PhoP increases the immunogenicity of these vaccine strains. In addition, I found that WhiB3 impacts carbon metabolism in BCG-Birkhaug and BCG-Sweden, although the effect of this on virulence in vivo is still unclear. The characterization of genes involved in virulence and immunogenicity allows us to develop novel approaches for improving the efficacy of BCG, which has important implications for future TB vaccine development.

Tuberculosis is a re-emerging global health problem. Bacille Calmette-Guerin (BCG), the available vaccine against the disease, is only effective short term and is associated with adverse reactions clinically. The development of new effective vaccines will require an understanding of virulence, immunogenic factors and the beneficial immune responses induced in the human host. My thesis investigates phoP and whiB3, two genes associated with virulence and immunogenicity in Mycobacterium tuberculosis. Study of PhoP in a natural phoP mutant, BCG-Prague, and in the clinically safe BCG-Japan, shows that over-expression of PhoP increases the immunogenicity of these vaccine strains. In addition, I found that WhiB3 impacts carbon metabolism in BCG-Birkhaug and BCG-Sweden, although the effect of this on virulence in vivo is still unclear. The characterization of genes involved in virulence and immunogenicity allows us to develop novel approaches for improving the efficacy of BCG, which has important implications for future TB vaccine development.

Vaccination is one of the major strategies available for combating viral infections in humans. However, currently available vaccines are not without pitfalls; they are laborious to produce, could potentially be unsafe, and in the case of the highly variable influenza virus need to be reformulated each season. The use of synthetic peptides thus represents an exciting alternative to traditional vaccines. However, these synthetic peptides are not highly immunogenic without the use of potent adjuvants. The lack of immunogenicity might be addressed by conjugation between T or B cell epitopes with universal or immunodominant T-helper epitopes. The construction of branched peptides, lipidated peptides, or designs combining both of these elements might also enhance the immunogenicity, as they might target Toll-like receptors and/or mimic the 3-dimensional structure of epitopes within the native protein. In this study, a recognized T-B peptide based on the hemagglutinin protein of the A/Puerto Rico/8/34 influenza virus was chosen as a backbone and modified to evaluate if the construction of branched peptides, lipidation, the addition of cysteine residues, or mutations could indeed alter reactivity. Screening the different designs with various antibody binding and cellular assays revealed that combining a branched design with the addition of lipid moieties leads to a greatly enhanced activity as compared to other similar T-B diepitope constructs.

Bovine herpesvirus 1 (BHV-1) is a member of the Alphaherpesvirinae subfamily and is the prototype ruminant herpesvirus. BHV-1 causes a number of complications in cattle including upper respiratory tract disorders, conjunctivitis, genital disorders, abortions, and immune suppression. Like all herpesviruses, reactivation from latency can occur throughout the animal’s life. Of particular economic importance is the bovine respiratory disease complex (BRDC) or ‘shipping fever’, in which BHV-1 plays a major role. BRDC is an enormous economic concern as it costs the US cattle industry approximately one billion dollars annually. In order to generate improved gene-deleted vaccines against BHV-1, there is a need to understand the contributions of viral gene products during infection. US3 is a serine/threonine kinase present in BHV-1 and is thought to play major roles during viral infection. As in other herpesviruses, US3 in BHV-1 is expected to phosphorylate several cellular and/or viral proteins. We recently presented evidence that BHV-1 US3 phosphorylates both VP8 and VP22; however, further functional characteristics of BHV-1 US3 during viral infection have not been elucidated. The hypothesis of this project is that the deletion of the US3 gene leads to reduced BHV-1 fitness. To explore this hypothesis, we generated a US3-deleted (ΔUS3) and subsequent US3-rescued (US3R) BHV-1 virus. Using these viral mutants, we characterized the growth properties of the viruses, evaluated the effect of the US3 deletion on major structural BHV-1 proteins, characterized the protein composition of the mature virions, and, identified viral processes that were impaired in the deletion mutant. Initially, the ∆US3 virus was generated through a 3-step PCR strategy which replaced the gene of interest with an antibiotic resistance cassette. Following this, the US3 gene was rescued via a two-step en passant mutagenesis strategy which has been previously used to generate insertions, deletions, and substitutions in herpesvirus-containing bacterial artificial chromosome (BAC) DNA. In vitro characterization of ∆US3 BHV-1 has demonstrated that US3 deletion affects BHV-1 growth characteristics, expression kinetics of major structural proteins, mature virion composition, cell to cell spread, and the subcellular localization of key viral proteins during infection. Growth kinetics of ∆US3 BHV-1 were impaired compared to wild-type (WT) BHV-1, especially at late times post-infection. Plaque sizes formed by ∆US3 BHV-1 were significantly smaller than those formed by either WT or US3R BHV-1, demonstrating that US3 is important for cell to cell spread. The expression kinetics of major structural and regulatory BHV-1 proteins were different between cells infected with ∆US3 or WT BHV-1, and incorporation of these proteins into the mature viruses differed, demonstrating that US3 is instrumental in ensuring proper protein expression and mature virus composition in vitro. Of particular importance, glycoprotein B (gB), was shown to be expressed in higher quantities earlier during infection in the absence of US3, and that this protein was incorporated in significantly higher amounts in mature virions which lacked US3. Qualitative analysis of ∆US3 BHV-1 infected monolayers suggested the abolishment of cell to cell projections characteristic of WT BHV-1 infection. Finally, the disruption of gB in ∆US3 BHV-1 infected cells was confirmed by confocal microscopy and fluorescence-activated cell sorting (FACS) analysis. Through confocal microscopy, evidence was provided that infection with ∆US3 BHV-1 possibly results in earlier expression of gB on the surface of cells and less intracellular accumulation of this protein during late stages of infection. The observed effect on the localization of intracellular gB in ∆US3 BHV-1 infected cells was quantified by flow cytometry. ∆US3 BHV-1 infected cells had approximately 25% higher gB expression on the surface of cells and a corresponding 25% decrease in intracellular gB. Although these differences have not yet been demonstrated to be statistically significant and not confirmed through infection with US3R BHV-1, this suggests that US3 may influence the synthesis and cellular trafficking of gB in vitro.

Introduction: Equine influenza virus, an influenza type A virus, belongs to the family of Orthomyxoviridae. Equine influenza is a major cause of respiratory disease in horses and outbreaks have severe economical repercussions for the horse industry. It is considered to be endemic in Sweden and between 1997 and 2006 there have been around 10 to 40 outbreaks every year. The objective of this study was to do a phylogenetic characterization of equine influenza outbreaks that occurred in Sweden during a twenty year period. Methods: The haemagglutinin and neuraminidase gene of 14 samples and the complete genome of three samples collected over the span of 20 years were sequenced. The viral RNA were extracted, amplified with OneStep RT-PCR and sequenced. Results & Discussion: The phylogenetic tree and deduced amino acid sequence of HA1 illustrated that different lineages of equine influenza virus has circulated simultaneously in the Swedish horse population. The isolates mainly belonged to pre-divergence-, Eurasian- and American lineages. To characterize equine influenza viruses is important for vaccine strain selection, to fully understand the disease and how the virus evolves.

The immunogenicity and protective efficacy of a DNA vaccine encoding a truncated groEL heat shock gene (pcDNA3/groEL) from Burkholderia pseudomallei was evaluated in vaccinated BALB/c mice infected with B. pseudomallei or B. cenocepacia. After vaccination, the levels of anti-GroEL total IgG and IgG2a were increased in mouse sera. The clonal expansion of the spleen cells increased, and the GroEL protein induced IFN-£^ production by spleen cells. The anti-GroEL antibody-mediated opsonic killing effect was not able to eliminate the growth of B. pseudomallei but was able to eliminate the growth of B. cenocepacia. After intravenous challenge of the vaccinated Balb/c mice with B. pseudomallei, the number of bacteria colonizing the in liver and/or spleen was not reduced. Over 50% of vaccinated mice infected with B. pseudomallei died within 7 days post-infection. By contrast, the bacterial loads in organs were significantly reduced if the vaccinated mice were infected with B. cenocepacia. All of vaccinated mice were alive 7 days post-infection. Liver damage, as assessed by histological observation, and abnormalities in the levels of liver enzymes rapidly resolved in vaccinated mice. We suggest that B. pseudomallei groEL plasmid DNA immunization of Balb/c mice induces a Th1-type immune response and provides cross-protection against B. cenocepacia but not against B. pseudomallei infection.

Coccidiosis has a negative economic impact on the commercial poultry industry, and probiotics are beneficial bacteria that aid in maintaining healthy gut microflora. We hypothesized that probiotic administration would positively affect gut morphology and increase IgG secretion during an Eimeria challenge, which was evaluated by measuring total chicken IgG and gut morphology (villus height, villus width, villus surface area, crypt depth, villus height to crypt depth ratio and lamina propria thickness). On day-of-hatch, broilers were placed into floor pens with 50 percent pine shavings and 50 percent used litter. The broilers were exposed to Eimeria oocysts via the feed on day 14 and challenged on day 36. On days 6, 22, 36, and 43, tissue samples from the intestine were collected for morphological evaluation, and blood samples were taken to quantify chicken IgG from serum. Data were measured using a factorial ANOVA and main effect means were deemed significant at P ≤ 0.05. In cases where significant interactions were observed, data was subjected to a one-way ANOVA. All means were separated using a Duncan’s Multiple Range Test. On day 6 in the duodenum, a significant interaction was observed regarding vaccination and probiotic administration (Coccivac®-B, Intervet/Schlering-Plough Animal Health/Merck and Co., Inc., Whitehouse Station, NJ). Villus height to crypt depth ratio decreased in ionophore treated birds compared to control birds in the duodenum and lower ileum on day 6, 36, and 43. Villus crypt depth in vaccinated birds decreased in the duodenum after the challenge. On day 43, the ionophore treated birds had less villus height and surface area compared to control and vaccinated birds, while lamina propria thickness increased in the duodenum, and non probiotic birds had longer villi than probiotic birds. On day 22, vaccinated birds had significantly increased chicken IgG levels compared to the control and ionophore birds, and the non probiotic birds had significantly increased IgG secretion compared to probiotic fed birds. On day 36, the ionophore birds had significantly increased levels of IgG compared to the control birds, which could also support that the ionophore delayed exposure to the parasite. These results suggest that gut morphology and humoral immunity are affected by probiotic administration, coccidiosis vaccination, ionophore application and Eimeria challenge. Both the day 43 morphology results and day 36 chicken IgG results for the ionophore treated birds demonstrates that ionophore administration delays exposure of the avian gut to invasive coccidia. More research is necessary to evaluate how probiotics influence coccidiosis vaccination and humoral immunity, so that probiotics may be used to improve the effectiveness of coccidiosis vaccination and to evaluate if probiotics aid in ameliorating the effects of an Eimeria infection.

Salmonella continues to threaten public health as well as negatively impact dairy producers on multiple levels. Efficacious solutions to control Salmonella among dairy cattle have long been sought to alleviate these problems. A novel vaccine technology has been developed based on purified siderophore receptors and porin proteins (SRP®) derived from Salmonella Newport. When vaccinated with these SRP® cattle are stimulated to produce antibodies which act in concert with host defenses to disrupt iron acquisition of pathogenic bacteria. To evaluate the effectiveness of this technology, a prospective cohort study was designed utilizing herds (n = 11) that practiced whole herd vaccination with the SRP® vaccine (vaccinated cohort) and herds (n = 11) that had not used the SRP® vaccine. Samples were collected during four rounds at approximately six week intervals from June through October 2009. Samples were transported to the laboratory at West Texas A&M University and cultured for the prevalence of Salmonella using selective enrichment methods. Salmonella isolates were evaluated for antimicrobial susceptibility and serotype. Data was analyzed using commercially available software to evaluate the herd-level effects of vaccination. Salmonella was ubiquitous throughout the Texas Panhandle and Eastern New Mexico, within-herd animal level estimates of prevalence ranged from 0.0 – 92%, over the length of the study period. Overall all rounds vaccinated herds had decreased (P = 0.012) Salmonella prevalence (15.3 vs. 27.5%). Vaccinated herds had numerically fewer Salmonella isolates belonging to the Newport serotype. Salmonella Typhimurium isolates were recovered approximately equally from vaccinated and non-vaccinated herds. Isolates from vaccinated herds were resistant to fewer antimicrobials throughout the study period. The ACSSuT(resistant to ampicillin, chloramphenicol, streptomycin, sulphisoxazole, and tetracycline) and MDR-AmpC (ACSSuT resistance plus resistance to ceftiofur and amoxicillin/clavulanate) resistant phenotypes were more frequently observed among non-vaccinated herds and none of the isolates from vaccinated or non-vaccinated herds were resistant to nalidixic acid, gentamicin, ciprofloxacin, or amikacin. These findings indicate vaccine efficacy for the reduction of Salmonella prevalence. Dairy operators along with herd veterinarians are encouraged to utilize this data with other herd specific factors in determining whether to use this specific vaccine.

Schistosomiasis is one of the most widespread parasite diseases in the world, whereas Schistosoma mansoni is a major schistosome species in Africa, America, and the Caribbean islets. Many antigenic vaccine candidates have been postulated, including sm22.6 and GST. Although the lower level of re-infection of human schistosomiasis is related to the higher level of IgE against rsm22.6, unfortunately, the observation of the experimental vaccination in mice finds some difficulties in further development of vaccine. In addition, the biochemical and biophysical properties of the antigen are virtually unknown, thus the present study intends to characterize sm22.6 from biochemistry and cell biology. To do this, sm22.6 was expressed in E. coli (BL21DE3) and purified to homogeneity. Analyses of the recombinant protein showed that the antigen was highly hydrophobic and formed polymers readily as judged by both native and denatured electrophoreses. Because various technologies including NMR and DNA binding which had been applied to the study of the antigen generated vague results, we decided to express the antigen in human breast cancer cell (MDA-MB-435s) to locate in the subcellular compartments where the antigen is situated. Results showed that the antigen, not like the recombinant expressed in E. coli, located in both cellular fluids and membrane, suggesting that the antigen might not be a skeleton protein as predicted by proteomics.