HIV-1 chronically infected individuals represent a large disease burden, making the development of a therapeutic vaccine for use in chronic infection a priority. However, therapeutic vaccination has not been successful to date. Most approaches have employed viral vectored vaccines encoding full length viral proteins, which aimed to boost pre-existing CD8+ T cell responses by mimicking natural HIV-1 infection. Simply boosting these pre-existing CD8+ T cell responses which have previously failed to control the virus may be insufficient. Although HIV-1 has a huge capacity to diversify, certain regions are less tolerant of mutations due to structural and functional constraints. We therefore hypothesised that it would be necessary to redirect the immune response to more vulnerable regions of the proteome, such as conserved regions. HIVconsv is a rationally designed conserved region vaccine. Vaccination of 19 chronically HIV-1 infected HAART treated patients with MVA.HIVconsv was safe and well tolerated. There was a significant increase in the magnitude of HIVconsv-specific T cell response following vaccination (p = 0.001), as measured by IFN-γ Elispot assay, but changes observed in vaccinees did not reach statistical significance when compared with placebo recipients (p = 0.48). The capacity of CD8+ T cells to inhibit HIV-1 replication in vitro is highly predictive of virus control in vivo and is thus a possible surrogate of vaccine efficacy. There was a trend towards increased CD8+ T cell mediated inhibition following vaccination with 2x10⁸pfu MVA.HIVconsv (17% inhibition pre- vs 54% inhibition post-vaccination, p = 0.06). However, measurement of the latent HIV-1 reservoir by quantification of total HIV-1 DNA in circulating CD4+ T cells by droplet digital PCR showed no reduction in size upon vaccination, indicating CD8+ T cells induced by vaccination with MVA.HIVconsv were not of sufficient potency to impact the reservoir. In a second cohort of HIV-1 infected individuals, antiviral inhibitory activity was measured in 36 HIV-1-infected STEP and Phambili trial participants. Sustained potent CD8+ T cell antiviral inhibitory responses were rare but were strongly correlated with IFN-γ responses to so-called ‘beneficial’ low entropy regions in HIV-1 Gag and Pol, that had been reported previously to be associated with HIV-1 control, (r = 0.69, p = 0.0001). This correlation was still significant after controlling for protective HLA alleles, whereas responses to conserved elements were only weakly correlated with viral inhibition (r = 0.41, p = 0.04). These data indicate that immunogens that are based on the selection of regions within the viral proteome by conservation score alone may not induce the most effective HIV-1-specific T cell responses and they highlight the importance of systematically selecting specific regions associated with HIV-1 control, together with exclusion of immunodominant decoy epitopes.

Human immunodeficiency virus-1 (HIV-1) and tuberculosis (TB) are significant global health issues today and the immunological correlates of protection to both diseases remain poorly defined. HIV-1 is the greatest risk factor for the development of TB and HIV-associated TB contributes significantly to the global TB burden, particularly in sub-Saharan Africa. The host T cell response to Mycobacterium tuberculosis (Mtb) is critical for control and is weakened in HIV-1 disease. However, the extent and precise mechanisms remain incompletely elucidated. Antiretroviral therapy (ART) for HIV-1 reduces TB risk in treated individuals significantly, although not to levels seen in HIV-uninfected individuals. This thesis studies HIV- and Mtb-specific T cell responses in 2 cohorts of individuals from a community with high HIV-1 and TB prevalence in Bloemfontein, South Africa. An analysis of HIV-specific CD8 T cell responses in Chapter 4, confirms the superior role of HIV-1 Gag-specific CD8 responses in controlling HIV-1 viraemia and demonstrates that the loss of such responses contributes to HIV-1 disease progression and likely susceptibility to opportunistic infection. I investigated the impact of HIV-1 infection on the Mtb-specific T cell response through a cross-sectional comparison of T cell responses in HIV-infected and HIV-uninfected individuals (Chapters 5 and 6). HIV-infected individuals had a significant depletion of both Th1 and Th17 CD4 responses to Mtb-specific antigens as well inhibition of Mtb-specific CD8 T cell responses, in comparison to those uninfected with HIV-1. PPD- and Rv2031c-specific responses were particularly reduced in HIV-infected individuals. Over a 12 month period of therapy, ART partially restored the Mtb-specific CD4 T cell response (Chapters 5 and 7). This effect was greater for Th1 than Th17 responses and had no detectable effect on the Mtb-specific CD8 response. However, despite some evident restoration, there remains a significant quantitative deficit in individuals on ART that is likely to contribute to persistent elevated TB risk. Overall, these data contribute to a better understanding of the mechanisms of susceptibility to TB during HIV-1 disease and ART, as well as of the correlates of protective immune responses to both pathogens.

Recent events have thrown the spotlight on infectious disease outbreak response. We developed a data-driven method, EpiGro, which can be applied to cumulative case reports to estimate the order of magnitude of the duration, peak and ultimate size of an ongoing outbreak. It is based on a surprisingly simple mathematical property of many epidemiological data sets, does not require knowledge or estimation of disease transmission parameters, is robust to noise and to small data sets, and runs quickly due to its mathematical simplicity. Using data from historic and ongoing epidemics, we present the model. We also provide modeling considerations that justify this approach and discuss its limitations. In the absence of other information or in conjunction with other models, EpiGro may be useful to public health responders. (C) 2016 The Authors. Published by Elsevier B.V.

Apical membrane antigen 1 (AMA1) is a leading candidate vaccine antigen against blood stage malaria and several clinical trials using mostly protein-in-adjuvant vaccines have shown limited success. This thesis describes the development of recombinant adenoviral (AdHu5) and poxviral (MVA) vectors encoding AMA1 from Plasmodium chabaudi murine parasites. In this murine malaria model, AdHu5 and MVA encoding AMA1 when used in a heterologous prime boost regime showed excellent immunogenicity, both humoral and cellular. The vaccination regime was protective against blood stage challenge and both antibodies and CD4+ T cells found to be important for vaccine induced blood stage protection. In parallel to this novel P. falciparum vaccines encoding AMA1 were also developed and administered in a similar prime boost regime to mice and rabbits. The vaccination regime induced cellular immune response and high titre antibodies against AMA1 and these antibodies showed growth inhibitory activity against the homologous parasite strain. In an effort to overcome the issue of antigenic polymorphism and to circumvent pre-existing immunity to human adenovirus, biallelic simian and human adenoviral vectors and MVA encoding AMA1 vaccines were also developed and administered to mice and macaques. These vectors also induced high titre antibodies and the serum from macaques was found to have growth inhibitory activity. These vaccine candidates are now being taken forward to Phase I/II clinical trials in Oxford. This work also described the attempt to improve MVA as a antibody inducing vector to allow better antibody mediated immunity to blood stage malaria.

Ebolavirus is responsible for a deadly hemorrhagic fever that has claimed thousands of lives in Africa and could become a global health threat. Because of the danger of infection, novel Ebola research is restricted to BSL-4 laboratories; this slows progress due to both the cost and expertise required to operate these laboratories. The development of a safe surrogate would speed research and reduce risk to researchers. Two highly conserved Ebola gene segments—from the glycoprotein and nucleoprotein genes—were designed with modifications preventing expression while maintaining sequence integrity, spliced into high copy number plasmids, cloned into E.coli, and tested for stability, safety, and potential research applications. The surrogates were stable over 2-3 months, had a negligible mutation rate (<0.165% over the experiment), and were detectable in human blood down to 5.8E3-1.17E4 surrogates/mL. These protocols could be used to safely simulate other pathogens and promote infectious disease treatment and detection research. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Next-generation sequencing technologies are revolutionising our ability to characterise and investigate infectious diseases. Utilising the power of high throughput sequencing, this study reports, the development of a sensitive, non-PCR based, unbiased amplification method, which allows the rapid and accurate sequencing of multiple microbial pathogens directly from clinical samples. The method employs Φ29 DNA polymerase, a highly efficient enzyme able to produce strand displacement during the polymerisation process with high fidelity. Problems with DNA secondary structure were overcome and the method optimised to produce sufficient DNA to sequence from a single bacterial cell in two hours. Evidence was also found that the enzyme requires at least six bases of single stranded DNA to initiate replication, and is not capable of amplification from nicks. Φ29 multiple displacement amplification was shown to be suitable for a range of GC contents and bacterial cell wall types as well as for viral pathogens. The method was shown to be able to provide relative quantification of mixed cells, and a method for quantification of viruses using a known standard was developed. To complement the novel molecular biology workflow, a data analysis pipeline was developed to allow pathogen identification and characterisation without prior knowledge of input. The use of de novo assemblies for annotation was shown to be equivalent to the use of polished reference genomes. Single cell Φ29 MDA samples had better assembly and annotation than non-amplification controls, a novel finding which, when combined with the very long DNA fragments produced, has interesting implications for a variety of analytical procedures. A sampling process was developed to allow isolation and amplification of pathogens directly from clinical samples, with good concordance shown between this method and traditional testing. The process was tested on a variety of modelled and real clinical samples showing good application to sterile site infections, particularly bacteraemia models. Within these samples multiple bacterial, viral and parasitic pathogens were identified, showing good application across multiple infection types. Emerging pathogens were identified including Onchocerca volvulus within a CSF sample, and Sneathia sanguinegens within an STI sample. Use of Φ29 MDA allows rapid and accurate amplification of whole pathogen genomes. When this is coupled with the sample processing developed here it is possible to detect the presence of pathogens in sterile sites with a sensitivity of a single genome copy.

This thesis examines the Influenza A/H5N1 virus in action through an ethnographic study focused on the entwined concepts of risk and modernity. The objective is to explain why the response to the virus has been challenged in Indonesia. Concerned with policy formulation, and everyday practice, the thesis argues that assemblages of historical, political, institutional and knowledge‐power processes create multiple hybrid constructions of risk and modernity, which challenge technical responses based on epistemological positions and institutional arrangements that do not allow for such hybridity. The thesis is organised into four sections. The first section (chapters 1 – 3) introduces the virus and its terrain, outlines a constructivist position, and argues that conceptually risk and modernity have multiple, dynamic, power‐laden forms. The second section (chapters 4 – 6) contrasts constructions of risk and modernity among the actors and networks responding to the emergence, spread and persistence of the H5N1 virus, with the constructions of affected people in Indonesia. The third section (chapters 7 – 9) investigates the multi‐directional processes that occur when ‘global' policies and practices encounter ‘local' social and political settings, and vice versa, through three empirical case studies of the response to H5N1 in Indonesia between 2005 and 2010. The final section (chapter 10) provides a set of reflections and conclusions. Given the conceptual plurality of risk and modernity, and the multiple overlapping interacting hybrid constructions that have been empirically demonstrated in the case of H5N1, it is concluded that reductive, science‐based, governmentally‐orientated responses which treat nature as a matter of separate, fixed identity do not allow for such hybridity. The virus in action in Indonesia shows that any divide between nature and society is artificial and deceiving. Technical disease control responses need to incorporate understandings which accept the dynamics of culture, politics, and power.

Orientador: Luís Antonio Mathias / Banca: Samir Issa Samara / Banca: Edvirges Maristela Pituco / Resumo: O presente estudo teve como objetivo avaliar a influência da brucelose, leptospirose, rinotraqueíte infecciosa bovina (IBR), diarreia viral bovina (BVD) e neosporose no indicador de reprodução mais comumente utilizado por pecuaristas brasileiros, a taxa de concepção (ou taxa de prenhez) após a estação de monta. Dois estudos epidemiológicos analíticos foram feitos: um estudo caso-controle, utilizando uma amostra das matrizes do rebanho que foram submetidas à estação de monta durante o período chuvoso, e um estudo de coortes retrospectivo, utilizando todas as novilhas submetidas à estação de monta durante a estação de "inverno". A determinação do estado gestacional foi realizada por palpação retal entre 60 e 90 dias após o término da estação de monta. A pesquisa de anticorpos foi realizada pelos seguintes métodos: antígeno acidificado tamponado (AAT) e reação de fixação de complemento (RFC) para brucelose; soroaglutinação microscópica (SAM) para leptospirose; virusneutralização (VN) para IBR e BVD; e reação de imunofluorescência indireta (RIFI) para neosporose. A significância estatística da associação entre a doença e o fracasso na concepção foi avaliada pelos métodos de qui-quadrado ou teste exato de Fisher (p < 0,05) e pelo intervalo de confiança a 95% para o risco relativo (RR) ou a odds ratio (OR). Associações com relação causal possível foram encontradas entre falha na concepção e: infecção considerando qualquer sorovariedade de Leptospira spp. (Título ≥ 400) em primíparas (OR = 7,3636, IC 95%: 1,3373-40,5479; significativo), todas as quais foram infecções causadas por L. Wollfi; e infecção por L. Autmumnalis (título de ≥ 200), considerando todos os animais da estação de monta de "verão" (OR = 8,4058; IC95%: 1,0377 - 68,0882; significativo). Brucelose, neosporose IBR, BVD... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present study aims to evaluate the influence of brucellosis, leptospirosis, infectious bovine rhinotracheitis (IBR), bovine viral diarrhea (BVD) and neosporosis in the reproductive indicator most commonly used among Brazilian ranchers, the conception rate (or pregnancy rate) after breeding season. Two analytical epidemiological studies were made: a case control study using a sample of matrices of the herd that were submitted to the breeding season during the rainy season, and a retrospective cohort study using all heifers submitted to the breeding season during the dry season. The determination of pregnancy status was performed by rectal palpation between 60 and 90 days after the end of the breeding season. The antibody search was performed by the following methods: rose bengal test and complement fixation test for brucellosis; microscopic agglutination test for leptospirosis, virus neutralization for IBR and BVD, and indirect immunofluorescence assay for neosporosis. The statistical significance of association between the disease and failure in the conception was held by the methods of chi-square or Fisher's exact test (p < 0,05) and by the 95% confidence interval for the relative risk (RR) or odds ratio (OR). Associations with possible causal relationship were found between conception failure and: Infection considering any serovar of Leptospira spp. (titer ≥ 400) in primiparous (OR = 7.3636, 95% CI: 1.3373 to 40.5479; significant), all of which were infections caused by L. Wollfi; and infection by L. Autmumnalis (titer of ≥ 200) considering all the animals of the rainy breeding season (OR = 8.4058; IC95%: 1.0377 - 68.0882; significative). Brucellosis, IBR, BVD, neosporosis and leptospirosis caused by other serovars did not influence the rate of conception in the breeding season... (Complete abstract click electronic access below) / Mestre

Infectious Pancreatic Necrosis (IPN), due to infection with the IPN virus (IPNv), continues to cause heavy mortalities and is endemic across the major Atlantic salmon farming regions of the world. Prevalances of 0.3-0.8 or more at the freshwater stage and 0.05 to 0.3 in the seawater phase of production are typical. Partially effective injectable vaccines are available against seawater IPN but biosecurity measures remain the main methods of control. To explore the feasibility of selecting salmon for resistance to IPN, a selective breeding program was initiated in 1996, including a series of field and experimental trials challenging known full-sib families with IPNv. A total of 404,723 fish faced IPNv challenge (376,541 seawater and 28,182 freshwater) covering 14 years and 17 separate locations across 7 sites. Mortalities and survivors following IPN challenge were counted by full-sib family and analysed as binomial data (alive / dead). Initial heritabilities were obtained from expressions based on the variance and covariance of full-sib family means for the 2001 year-group, indicating heritabilities (h2) of 0.16, range 0.08 to 0.24, and genetic correlations (rg) between replicate families of 0.71 to 0.78. These results were then confirmed by residual maximum likelihood across all seawater challenged data (year-groups 1997-2003), indicating a h2 of 0.43 (s.e.0.02) across all sites, range 0.06 to 0.40 for individual sites, and a range of rg between replicates of 0.70 to 0.87 (s.e. approx 0.05). To accommodate datasets and pedigrees approaching half a million individually identified fish, an implementation of the Reduced Animal model (RAM) was used to obtain these estimates. A similar level of genetic variation for resistance to freshwater IPN (year-groups 2005-2009) was confirmed with a h2 of 0.49, (s.e. 0.03), range 0.31 to 0.59, and rg between replicates ( 0.80 to 0.95, s.e. approx 0.05), using an Individual Animal Model. When all the data were analysed together, assuming seawater and freshwater survival to be the same trait, the heritability increased to 0.67, (s.e. 0.02). On testing this assumption, the genetic correlation between freshwater and seawater survival was found to be 0.68 s.e. 0.09. Both these pooled estimates account better than those for the individual site estimates, for the known selection of superior families that was incorporated at the earliest opportunity (2001) into the selective breeding program. To further investigate if there were favourable or antagonistic relationships operating between traits under active selection, genetic correlations between IPN mortality and a range of performance and harvest traits were obtained. When restricting the harvest data to year-groups where the harvested fish had not experienced an IPN event (2003 for seawater IPN, 2005 for freshwater IPN) : fish length and flesh colour just reached significance with seawater IPN (0.27 to 0.53. s.e. 0.14), while only harvest weight (0.30 s.e 0.11) attained significance with freshwater IPN mortality. All these correlations were antagonistic. When all the data were combined, (ie both IPN and harvest events taken from all yeargroups) these became non-significant. Taken as a whole, these results indicate that selecting salmon for resistance to both seawater and freshwater IPN challenge certainly is feasible, and that adverse effects on selection for other important production traits is not expected. How these medium to high heritabilities relate to the discovery of a major QTL for IPN resistance segregating in these populations, reported in a parallel scheme of work but based on a sub-set of the same families, is discussed.

Photocopy of typescript. / Digitized by Kansas Correctional Industries