Browsing by Subject "vaccine"
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Item Characterization of single-cycle flavivirus particles for use as a vaccine to prevent West Nile disease and to examine immune responses to flavivirus infection(2009-08-28) Douglas Gregory Widman; Peter W. Mason; Stanley M. Lemon; Nigel Bourne; Mark Heise; Gustavo ValbuenaWest Nile virus (WNV) is responsible for the largest outbreak of viral encephalitis in the history of North America, yet there are no vaccines available to prevent this disease. To address these needs we have developed RepliVAX WN, a single-cycle flavivirus (SCFV)-based vaccine to prevent West Nile disease. RepliVAX WN contains a C-deleted WNV genome, and is produced in trans-complementing cell lines that express WNV C. When used for vaccination, RepliVAX WN infects a single cell where the genome replicates and drives the production of highly antigenic subviral particles (SVPs) and NS1 without producing infectious virions. Thus, RepliVAX is expected to be highly potent yet exhibit a safety profile superior to traditional live-attenuated viral vaccines.\r\nHere we demonstrate that RepliVAX WN can be safely passaged in C-expressing cell lines and that this blind passage selected for mutations used to engineer a second-generation RepliVAX WN with an enhanced in vitro growth phenotype. When evaluated in mouse and hamster models of WN disease, this second-generation RepliVAX was safe, exhibited 100% protective efficacy, and induced significantly higher antibody levels than the parental virus. Furthermore, we observed that RepliVAX WN-induced antibody levels remain steadily at high levels for at least 6 months after vaccination of hamsters, and all animals were protected from lethal WNV challenge at this time. Evaluation in non-human primates indicated that one or two doses of RepliVAX WN was safe, induced WNV-specific antibody responses, and protected animals from WNV viremia. \r\nHaving demonstrated the usefulness of RepliVAX WN as a vaccine to prevent WN disease, we were interested in the immunological mechanisms underlying vaccine immunity. We observed that although RepliVAX WN vaccination induces high levels of interferon (IFN) alpha, the ability to respond to either type-I or type-II IFNs was not required for the development of activated B cells, IgG, IgM, or neutralizing antibody titers. Type-I IFN signaling did, however, play a role in viral gene expression, as in vivo imaging of animals inoculated with luciferase-expressing SCFVs revealed 1000-fold greater bioluminescence in the absence of a type-I IFN response. The affect of this IFN response on gene expression was dramatic, but short lived and did not appear to play a role in SCFV persistence, as SCFV gene expression was detectable for at least 18 days after SCFV inoculation. Taken together these results demonstrate the usefulness of SCFVs like RepliVAX WN as vaccines to prevent flavivirus disease, and tools with which to examine immune responses to viral infection.Item Effects of Cytosine-phosphate-Guanosine Oligodeoxynucleotides (CpG-ODN) on vaccination and immunization of neonatal chickens(Texas A&M University, 2005-02-17) Barri, AdrianaThe objective of this investigation was to evaluate the effects of administering CpG-ODN to commercial strain chickens as a potential adjuvant to vaccination against Salmonella, Eimeria spp., and Newcastle disease virus, or immunization to bovine serum albumin (BSA). During Experiment 1, which evaluated the dual application of CpG-ODN and a Newcastle disease virus vaccine, in the first of three replicate trials, on day 28 of the experiment, animals in the Vaccine + CpG 1& 14 experimental group were observed to have the highest levels of (p<0.05) anti-NDV IgG in serum. These levels were elevated above levels in animals from all other experimental groups. This suggestion for an adjuvant effect associated with CpG-ODN administration was not supported in the remaining two trials of experiment 1. Experiment 2 evaluated the potential for CpG-ODN to adjuvant a commercial live oocyst coccidial vaccine when applied by an oral route to neonatal broiler chickens. Overall, when body weight gain during challenge, development of intestinal lesions, and anti-Eimeria IgG levels were evaluated, vaccine administration alone was demonstrated to provide the best measure of protection among animals in all experimental groups, including those receiving either CpG-ODN or Non CpG-ODN. Experiment 3 investigated the simultaneous administration of CpG-ODN or Non-CpG ODN and a commercially acquired Salmonella typhimurium vaccine to SCWL chickens. Similar to experiments 1 and 2, antigen specific IgG responses in serum and indices of protection against field strain Salmonella challenge were variable and inconsistent. Anti-BSA IgG levels were compared in broiler and SCWL chickens immunized against BSA by a drinking water route of administration alone, or in combination with two different concentrations of CpG-ODN or Non CpG-ODN in experiment 4. The only observation where CpG-ODN and BSA co-administration resulted in anti-BSA IgG levels that were elevated above BSA alone immunized chickens was measured in broilers at day 19 post-final immunization. Taken together, given the variable results reported in this investigation related to the co-administration of ODN and vaccine or protein antigen, these data are largely inconclusive for suggesting that CpG-ODN can effectively adjuvant humoral immune responses in commercial strain chickens.Item Evaluation of unmarked deletion mutants as improved Brucella vaccine strains in the mouse and goat models(Texas A&M University, 2006-10-30) Kahl, Melissa MarieHistorical data suggests that prolonged survival of Brucella vaccine organisms in the target host enhances immune protection. Recent research has focused upon the development of rough vaccine strains to avoid interference with standard diagnostic tests. Rough organisms are rapidly cleared from the host, however. In an effort to develop improved vaccine strains, we have screened signature tagged mutagenesis banks to identify mutants with varying survival characteristics. We hypothesize that in order for a vaccine to be efficacious, it must survive in the host. In order to test this, we constructed marked and unmarked deletion mutants of B. abortus and B. melitensis in genes previously demonstrated by transposon mutagenesis to attenuate in vivo and in vitro virulence. Survival and efficacy of these novel deletion mutants were then evaluated in the mouse model. The asp24 mutants, which persist for extended periods in vivo, appear superior as a vaccine candidate compared to approved vaccine strains S19 and Rev1 in the mouse model against either homologous or heterologous challenges. Once enhanced protection against infection was demonstrated in the mouse, components of immune function that appeared to be most important were identified to correlate the immune response with the observed protection. We demonstrated that the most persistent mutant, delta-asp24, affords the greatest protection in mice against virulent challenge. In order to evaluate safety of the novel vaccine strains as well as protection against infection and abortion, we tested selected B. melitensis unmarked deletion mutants in a natural host, the goat. The delta-asp24 mutant was shown to be safe in pregnant goats while providing significant protection against infection and abortion.Item Extensive investigation of reticuloendotheliosis virus in the endangered Attwater's prairie chicken(Texas A&M University, 2007-09-17) Bohls, Ryan LanierReticuloendotheliosis virus (REV) is a retrovirus that causes a neoplastic disease in a wide range of avian hosts including chickens, turkeys, and ducks. In 1993, REV was detected in the endangered Attwater's prairie chicken (Tympanachus cupido attwateri), a subspecies of Tympanachus cupido. Subsequent infections of this prairie chicken have been identified at captive breeding facilities throughout Texas. The implications of these infections have severely hindered repopulation efforts by these facilities. This study focused on investigating REV infection of captive Attwater'????????s prairie chicken in order to better understand the disease affecting these endangered birds. The overall objective was to develop a means of eliminating this threat to the repopulation of the Attwater's prairie chicken. Several aspects of virus infection were investigated. Reagents capable of recognizing prairie chicken IgY and viral gag polypeptides were developed for use in assays for detection of antibody responses and titration of viral concentrations. Sequencing data of genomes collected from isolates of Texas prairie chickens and domestic chickens, as well as three REV prototype viruses, were compared to determine relationships among strains and identify the potential origin of the REV infecting Attwater'????????s prairie chicken. Additionally, a flow cytometry technique of segregating the lymphocyte population from peripheral blood mononuclear cells (PBMC) using a pan leukocyte monoclonal antibody was developed to more accurately measure changes within lymphocyte populations. This technique combined with intracellular labeling was used to deduce the target cells of REV infection. A nested polymerase chain reaction (PCR) test was developed for greater sensitivity in detecting infection in birds than the previous method of single amplification PCR. This greater sensitivity results in earlier identification of the virus in infected birds, which allows for earlier removal of infected birds to minimize transmission of the virus throughout the flock. The sensitivity of the nested PCR diagnostic test was determined in a dose response pathogenesis study, which was conducted on hybrid greater/Attwater's prairie chicken to observe the experimental development of disease in these birds. Finally, a vaccine was developed using plasmid DNA with REV encoded genes and tested on naturally infected prairie chickens to determine its efficacy in reducing viral load. Although no reduction in viral load was detected, the vaccine may be effective in providing prophylactic protection in future studies.Item Identification and Evaluation of Brucella Recombinant Outer Membrane Proteins as Subunit Vaccinogen Candidates in the Mouse Model of Brucellosis(2013-02-26) Gomez, GabrielDespite being amongst the most common zoonotic diseases in the world, brucellosis is a neglected disease for which an approved vaccine for human use does not exist. Thus far, the traditional approaches to Brucella antigen selection for subunit vaccine development have yielded unacceptable results. In this work, we evaluated the predictive ability of a multistep Brucella antigen selection process with in vitro immunological and invasion assays and in vivo protection experiments. Initial in silico screening for antigens was performed via genomic sequence analysis where 27 Brucella melitensis open reading frames (ORF) coding for outer membrane proteins bearing MHC epitopes, adhesin and conserved properties were identified. Evidence for a role in any aspect of Brucella virulence (i.e., invasion, co-regulation/expression with known Brucella virulence factors, intracellular adaptation) was then used to narrow the list of candidate antigens. To further increase confidence in the candidate ORF putative role in Brucella pathogenesis, differential expression of candidate ORF was evaluated using previously generated global transcriptomics data in in vitro HeLa and in vivo bovine models of acute Brucella infection. Protein expression in the E. coli heterologous system resulted in the successful expression of OmpW, BtuB, Omp22, Hia, and FlgK. With regards to virulence, the two proteins with the highest predicted adhesin scores conferred an invasive phenotype to the non-invasive BL-21 E. coli strain in alveolar epithelial cells. From an immunogenicity standpoint, all proteins elicited IgG production in Brucella-exposed goats, mouse and humans. Antigen-specific recall responses in splenocytes from C57BL/6 mice immunized with a cocktail of the three proteins with highest MHC scores revealed a mixed Th1/Th2 response with a comparatively greater Th1 response. In protection studies, subcutaneous (SQ) immunization with BtuB, Hia and FlgK, individually, promoted bacterial clearance following a robust intraperitoneal challenge dose of Brucella melitensis 16M. In addition, single SQ inoculation of FlgK enhanced protective efficacy of the vaccine strain B. abortus S19. In contrast, immunization of mice with the three protective antigens in a cocktail formulation elicited immune responses but no protection against intraperitoneal challenge with Brucella melitensis 16M in the spleen and liver. In conclusion, our results indicate that our combinatorial in silico, in vitro and in vivo antigen selection and identification modeling approach provides strong evidence for prediction of Brucella protective antigens, and represent a novel strategy with broad application to other major pathogens.Item Role of Vaccination in the Control of Turkey Coccidiosis: Vaccine Associated Oocyst Shedding, Lesions, and Mucosal Gene Expression(2012-07-11) Behl, Michelle 1983-Coccidiosis vaccine associated side effects, oocyst shedding patterns, intestinal lesions, and mucosal gene expression in the turkey were studied. The first study examined vaccine associated side effects and oocyst shedding patterns under experimental conditions. Peak oocyst shedding occurred on days 5-6, 13-17, and 19-20 days post vaccination. Throughout the course of the study, several poults exhibited clinical coccidiosis. Based on body weights, growth was correlated with vaccine cycling. The second study examined coccidiosis vaccine induced lesions and changes in mucosal gene expression on day 5, 10, 13, 17, and 20 days post vaccination. Poults were gavaged the equivalent of 0x, 1/2x, 1x, and 2x the available vaccine dose. Intestinal sections adjacent to the Meckel's diverticulum, ileocecal junction, and middle of the ceca were collected for histological analysis and gene expression. Measurements from the tip of the villus to the base of the lamina propria, villus width, and the muscularis mucosae thickness were acquired from the histological sections. Interleukin-10, IL-1beta, and GAPDH gene expression were measured by extracting mRNA in the tissues and quantified using real-time RT-qPCR. Starting on day five post vaccination, the control group weighed significantly more than the group that received the 2x dose. Body weight and oocyst dose were inversely related through day 17. Intestinal measurements did not necessarily correlate with the vaccine dose, although there appears to be some correlation on day five. There were no significant changes in the mucosal gene expression of IL-10 and IL-1beta in the intestinal tissue adjacent to the Meckel's diverticulum throughout the course of the study. On day five post vaccination, IL-10 and IL-1beta were significantly upregulted in the ileocecal junction. Interleukin-10 was significantly upregulated on day 17 and IL-1beta was significanlty down regulated on day 20 in the ileocecal junction. Both IL-10 and IL-1beta were significantly upregulated in the ceca days 5, 10, and 13 post vaccination. Interleukin-10 was significnalty upregulated in the ceca on day 17 and significantly down regulated on day 20. Individual variation among poults in the same group merits further attention.Item The Effects of Probiotic and Eimeria on Gut Morphology and Humoral Immunity in Broilers(2012-02-14) Horrocks, Sadie LynCoccidiosis 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.Item Virulence associated functions and vaccine potential of the burkholderia mallei type three secretion effector BopA(2008-08-15) Gregory Whitlock; Katherine Brown; Gustavo Valbuena; Ashok ChopraThe detailed mechanism(s) of Burkholderia mallei pathogenesis is virtually unknown, with the production of a polysaccharide capsule and presence of secretion systems as the only known mechanisms associated with virulence. Various gram-negative pathogens utilize a secretion system known as type III, which is used to deliver bacterial (effector) proteins into host cells to modulate immunological and cellular responses. The intracellular survival of B. mallei within murine macrophage J774.2 cells requires the type III secretion system (TTSS), although the effector protein(s) involved are unknown. Additionally, previous reports have documented that B. mallei TTSS is required for phagosomal membrane lysis and bacterial escape into the macrophage cytoplasm. While progress has been made in demonstrating the importance of the TTSS contribution to B. mallei pathogenicity, additional work is needed to identify the potential secreted (effector) molecule(s) involved. BopA, a predicted effector protein of B. mallei, shares 24% amino acid homology to the TTS effector IcsB of Shigella flexneri, which is responsible for intercellular spread and host cell invasion. Characterization of the involvement of this putative TTS protein with bacterial survival strategies will provide crucial information for the development and assessment of a candidate vaccine. The work presented herein identified an N-terminal portion of BopA sufficient to drive secretion throughout a TTS apparatus and characterized the potential virulence-associated functions of B. mallei TTS protein BopA to subvert the host cell and promote intracellular replication and survival utilizing an in vitro respiratory cell model. Furthermore, in vivo studies assessed the attenuation of virulence offered by the B. mallei bopA mutant. Additionally, evaluation of the immune effectors involved in the protective host response to B. mallei infection identified key cellular and humoral components. Finally, a recombinant BopA sub-unit vaccine was utilized to assess the efficacy as a potential vaccine candidate.