Senior honors Thesis--University of Michigan, 1999. / Includes bibliographical references (p. 103-106).

La polyarthrite rhumatoïde (PR) est le rhumatisme inflammatoire le plus fréquent. Cette maladie s’accompagne d’une hyperplasie de la membrane synoviale qui entoure les articulations. La formation du pannus synovial est sous la dépendance de cytokines pro-inflammatoires et pro-angiogéniques. Les immunothérapies anti-TNF utilisées dans le traitement de la PR présentent des inconvénients (perte d’efficacité, risque infectieux), ce qui laisse la place pour le développement d’une stratégie vaccinale anti-TNF. Dans la première partie de mes travaux, nous avons développé des vaccins anti-VEGF afin d’étudier les liens entre angiogenèse et inflammation dans l’arthrite expérimentale au collagène (AEC). Dans la deuxième partie, nous avons développé un vaccin anti-TNF de souris afin d’évaluer les conséquences de la neutralisation du TNF-α par la vaccination dans des modèles infectieux. Le ciblage du VEGF avec un vaccin constitué de VEGF entier ou de peptides du VEGF couplés à la KLH, a permis une protection clinique et histologique dans l’AEC. Dans notre deuxième axe de recherche, nous avons développé un vaccin anti-TNF de souris (TNF-KLH). Ce vaccin est aussi efficace que l’etanercept dans l’AEC, mais n’augmente pas le risque infectieux dans un modèle d’infection à Mycobacterium tuberculosis. Dans le modèle d’infection à Listeria monocytogenes, TNF-KLH n’augmente pas la charge bactérienne et n’induit pas de mortalité, contrairement à l’etanercept. Mes travaux de thèse montrent que la stratégie vaccinale anti-cytokine est efficace dans l’arthrite, et que le ciblage du TNF par une telle stratégie ne semble pas altérer la réponse anti-infectieuse dans nos modèles. / Rheumatoid arthritis (RA) is the most frequent inflammatory rheumatism. This disease is accompanied by hyperplasia of the synovial membrane surrounding the joint. Pannus formation is controlled by pro-inflammatory and pro-angiogenic cytokines. Anti-TNF immunotherapies used in the treatment of RA presents many drawbacks (loss of efficacy, infections), which leaves some place for the development of an anti-TNF immunization strategy. In the first part of my work, we developed an anti-VEGF vaccine to study the links between angiogenesis and inflammation in collagen-induced arthritis (CIA) model. In the second part, we developed a mouse anti-TNF vaccine to assess the consequences of the neutralization of TNF-α by vaccination in infectious models. Inhibition of VEGF with a vaccine consisting of whole VEGF or VEGF peptide coupled to KLH, showed a clinical and histological protection in the CIA model. In the second part of my work, we developed a mouse anti-TNF vaccine (TNF-KLH). This vaccine is as effective as etanercept in CIA, but does not increase the risk of infection in Mycobacterium tuberculosis model. In Listeria monocytogenes model, unlike etanercept, immunization with TNF-KLH does not increase the bacterial burden and mortality. My work contributed to the development of active anti-VEGF vaccine and our results show a partial protection with this strategy. Also, we demonstrate that targeting TNF by active immunotherapy does not alter the immune response in our models of infections.

Orientador: Alessandro Francisco Talamini do Amarante / Banca: Fernando de Almeida Borges / Banca: Renato de Oliveira Orsi / Resumo: Neste estudo avaliou-se a eficácia de uma vacina constituída de glicoproteínas obtidas da membrana do intestino de Haemonchus contortus em bezerros desafiados com H. contortus ou H. placei. Bezerros holandeses machos, criados livres de infecções por helmintos, foram distribuídos em quatro grupos com nove animais cada. Dois grupos foram vacinados com 50 μg do imunógeno diluído no adjuvante QuilA, enquanto os outros dois grupos foram os controles, receberam apenas adjuvante. A vacina foi administrada três vezes com intervalo de 21 dias entre as aplicações. Os bezerros foram artificialmente infectados sete dias após a última imunização e sacrificados para contagem dos vermes 43 dias depois. Os bezerros de um dos grupos vacinados receberam 8000 larvas infectantes (L3) de H. contortus enquanto os do outro grupo foram infectados com o mesmo número de L3 de H. placei. Os controles foram infectados na mesma ocasião com o mesmo número de L3 de H. contortus ou H. placei. A vacinação reduziu significativamente a contagem de ovos por grama de fezes (OPG) e a carga parasitária (P<0,01). A titulação de anticorpos nos animais controle permaneceu perto de zero enquanto nos animais vacinados verificou-se elevada titulação de anticorpos no soro. Os bezerros vacinados e desafiados com H. contortus não eliminaram ovos nas amostras de fezes, enquanto os controles apresentaram média máxima (± erro padrão) de 61,1 (±42,3) OPG 31 dias após a infecção. A partir deste dia, a contagem de OPG diminuiu progressivamente neste grupo controle com apenas um animal eliminando ovos nas fezes 42 dias após a infecção. Nos controles de H. placei, o ápice na contagem de OPG ocorreu 35 dias após a infecção (61,1 ± 21,7) e permaneceu relativamente constante até o final do estudo, enquanto no grupo vacinado apenas dois animais eliminaram ovos nas fezes, na última coleta. Os animais controle... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study evaluated the efficacy of a vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus in calves challenged with H. contortus or Haemonchus placei. Males Holstein calves, raised worm free, were distributed into four groups with nine animals each. Two groups were vaccinated with 50 μg of antigen diluted on QuilA adjuvant, while the others two groups were the controls and received only adjuvant. The vaccine was administered three times 21 days apart. Either vaccinated or not and challenged with either 8,000 H. contortus or H. placei infective larvae. The calves were challenged 7 days after the last immunization and killed for worm counts 43 days later. Vaccination significantly reduced faecal egg counts (FEC) and worm burdens (P< 0.01). Antibody titres in the calves control stayed close to zero meanwhile in the calves vaccinated was observed high antibody titres in the serum. Calves vaccinated and challenged with H. contortus did not shed eggs in faecal samples, while the controls showed a maximum mean (± standard error) FEC of 61.1 (±42.3) at 31 days post infection. Then, FEC progressively declined in this group with only one animal shedding eggs in faeces 42 days post infection. With H. placei the controls FEC peaked at 35 days post infection (61.1 ±21.7) and remained relatively constant until the end of the study, while in the vaccinated group only two animals shed eggs and only on the last collection date. With H. placei the controls contained a mean of 551.1 (±93.7) parasites, while the vaccinates had 174.4 (±56.3) worms. The establishment rate of H. contortus was lower than that of H. placei (P< 0.01) with an average of 163.9 (±39.4) and 74.4 (±20.4) specimens in the control and vaccinated groups, respectively. It was concluded that vaccination of calves with antigens obtained from H. contortus conferred protection against both H. placei and H. contortus / Mestre

Despite progress through expanded use of bed nets and anti-malarial drugs, Plasmodium falciparum (Pf) malaria caused about 200 million cases and 500,000 deaths in 2015. An ideal vaccine would reduce the burden of disease and interrupt transmission. Despite decades of effort, there is no vaccine that can adequately address the global burden of malaria. This thesis focuses on two potential weaknesses in the parasite life-cycle. First, I investigate two vaccination strategies aimed at improving the antibody response to RH5, an essential and conserved protein for erythrocyte invasion. Due to instability of the resultant recombinant vaccine constructs, these efforts have required re-engineering of the vaccine platform, which remains an ongoing effort. Second, the immunogenicity and mechanism of protection of a live-attenuated whole sporozoite vaccine, PfSPZ Vaccine, was assessed. In a study that examined PfSPZ Vaccine at intravenous (IV) doses between 1.35 &tiles; 10⁵ to 4.5 &tiles; 10⁵ PfSPZ, I demonstrate that PfSPZ antibody responses correlated with durable sterile protection against controlled human malaria infection (CHMI). Surprisingly, the pre-vaccine frequency of V&gamma;9⁺V&delta;2⁺ T cells, an innate T cell that recognizes conserved Plasmodium phosphoantigens, also correlated with durable sterile protection. Regarding the mechanism of protection, PfSPZ-specific antibodies as well as CD8 and CD4 T cells in the blood decreased substantially over time, yet sterile protection was maintained. In non-human primates, the CD8 T cell response in the liver at a memory time point was measured to be about 100-fold higher than found in the blood. Collectively, these data suggest that PfSPZ Vaccine confers durable protection in humans by long-lived, tissue-resident CD8 T cells. These findings were extended with a study using 9.0 x 10⁵ PfSPZ, wherein I demonstrate that T cell responses peaked immediately after the first vaccination with minimal T cell activation despite additional immunizations. This suggests that anti-PfSPZ immunity may be limiting the effectiveness of subsequent immunizations. Finally, I examined the T cell response to PfSPZ attenuated by chloroquine (termed PfSPZ-CVac). T cell responses were substantially higher than achieved with comparable PfSPZ Vaccine doses. Additionally, a significantly higher proportion of PfSPZ-specific CD4 T cells were polyfunctional, simultaneously expressing IFN-&gamma;, IL-2, and TNF-&alpha;, in subjects that were protected from CHMI. In sum, these studies provide insight into the immunobiology of a protective immune response that may guide future malaria vaccine development efforts.

BACKGROUND: Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease affecting people of various ages across the globe. Treatment for the disease is in the form of immunosuppressive drugs. Due to either the treatment of the disease or the disease itself, SLE patients have been shown to have abnormal immune function. This dysfunction accounts for an increased rate of infections in this population. Reactivation of the varicella zoster virus (VZV), herpes zoster (HZ), has been shown to occur in the SLE population at higher rates than the general, healthy population. Recently a vaccine for HZ was approved by the Food and Drug Administration for individuals 50 years of age and older. STUDY: In this study we examined the safety and immunogenicity of the HZ vaccine, Zostavax, in a small sample of SLE patients. This study was a case-control with a ratio of 1:1, SLE patients to healthy controls. The total sample size was 20 with an average age of 57.9. All study participants were seen in a clinical setting at the Oklahoma Medical Research Foundation and signed informed consents. Subjects were seen for an initial baseline visit, and were administered the vaccine. Follow-up visits were scheduled at 2, 6 and 12 weeks. RESULTS: The notable finding of this study is a lack of significant differences between SLE patients and healthy individuals with one exception. At the 6-week point a significant difference was found (P=0.03) between SLE patients and healthy controls, with regards to the number of VZV-specific cells stimulated to produce interferon gamma. No vaccine-induced illness was evident and there was no sign of an increase in SLE disease activity in patients.

The Ebola virus disease is one of the most dangerous diseases to develop into a major health concern in the modern era, largely because of the ZEBOV outbreak that has devastated West Africa from 2014 to 2016. The outbreak has compelled many countries and organizations to prioritize finding a vaccine for Ebola, which is key to preventing a similar outbreak on a global scale. As a result, studies on Ebola vaccines have increased in frequency since 2014. This thesis will focus on three vaccine candidates that could potentially be developed into a future vaccine for Ebola: chAd3, rVSV, and rAd5. Each of the vaccines has been the focus of several studies on both animals and humans, which have provided information and understanding of the vaccines’ characteristics in terms of reactogenicity and immunogenicity. All of the vaccines demonstrate safety and immunogenicity profiles that offer promise for the vaccines as future candidates, which at first makes them seem very similar to each other. However, they each differ substantially in their flaws and ability to generate an immunogenic response. More specifically, the chAd3 vaccine requires a boost of MVA to reach its full potential, the rVSV vaccine has expressed a higher level of reactogenicity and adverse effects than the other two vaccines, and the rAd5 vaccine’s efficacy is weakened by the presence of pre-existing immunity against Ad5 in the human population.

Two cysteine and two serine protease inhibitors from a tick Ixodes ricinus saliva were overexpressed using a prokaryotic overexpression system and refolded to their native state. Both cysteine protease inhibitors were tested as potential antigens for an anti-tick vaccine showing no effect on tick feeding or reproduction. Various immunological methods were employed to test the potential immunomodulatory function of these proteins without success.

Master of Agribusiness / Department of Agricultural Economics / Vincent Amanor-Boadu / The development of vaccines and similar pharmaceutical products in the animal health industry are expensive and follow very specific pathways to comply with regulatory requirements for product licensure and registration. The vaccine development process is complex and involves numerous individuals, assets and departments within and outside the organization, and is a long process. The study stage of this long and complex process allows a company to confirm particular solutions to particular health incidents can be efficacious. The study stage involves executives who decide on new products that may be developed, managers who oversee the development of the products and scientists who develop protocols to undertake animal studies to test various aspects of the new product. It also involves clinical study personnel and laboratory personnel who conduct the experiments and collect data for analyses about the new products being studied. The number of people and time sensitivity of the processes contribute to the complexity, making effective communication critical to getting new products developed on time and on budget. The objective of this research is to identify perceived gaps in communication among people in the different roles with the view to finding solutions to address these gaps. Data were collected using an industry-focused online survey instrument. The instrument was designed to have both closed and open-ended questions. Survey participants were purposefully selected from across the global animal health industry, focusing on those directly involved in the study stage of new product development. The results showed the majority of respondents were satisfied with their company’s processes and systems for study development, initiation and execution, but people resources were viewed as the highest contributor to bottlenecks, which could demonstrate gaps in the communication links between groups. However, perceptions about challenges and gaps in communication seem to be influenced by who is providing information and who is receiving it. The different roles perceived the effect of timeliness, accuracy and clarity of communication on product development costs differently, with scientists presenting the highest cost of communication challenges and executives the lowest. On average, the perception was that these communication challenges increased the cost at the study stage of new product development by about 84% for biologicals and over 100% for pharmaceuticals.

abstract: Immunotherapy has been revitalized with the advent of immune checkpoint blockade treatments, and neo-antigens are the targets of immune system in cancer patients who respond to the treatments. The cancer vaccine field is focused on using neo-antigens from unique point mutations of genomic sequence in the cancer patient for making personalized cancer vaccines. However, we choose a different path to find frameshift neo-antigens at the mRNA level and develop broadly effective cancer vaccines based on frameshift antigens. In this dissertation, I have summarized and characterized all the potential frameshift antigens from microsatellite regions in human, dog and mouse. A list of frameshift antigens was validated by PCR in tumor samples and the mutation rate was calculated for one candidate – SEC62. I develop a method to screen the antibody response against frameshift antigens in human and dog cancer patients by using frameshift peptide arrays. Frameshift antigens selected by positive antibody response in cancer patients or by MHC predictions show protection in different mouse tumor models. A dog version of the cancer vaccine based on frameshift antigens was developed and tested in a small safety trial. The results demonstrate that the vaccine is safe and it can induce strong B and T cell immune responses. Further, I built the human exon junction frameshift database which includes all possible frameshift antigens from mis-splicing events in exon junctions, and I develop a method to find potential frameshift antigens from large cancer immunosignature dataset with these databases. In addition, I test the idea of ‘early cancer diagnosis, early treatment’ in a transgenic mouse cancer model. The results show that ii early treatment gives significantly better protection than late treatment and the correct time point for treatment is crucial to give the best clinical benefit. A model for early treatment is developed with these results. Frameshift neo-antigens from microsatellite regions and mis-splicing events are abundant at mRNA level and they are better antigens than neo-antigens from point mutations in the genomic sequences of cancer patients in terms of high immunogenicity, low probability to cause autoimmune diseases and low cost to develop a broadly effective vaccine. This dissertation demonstrates the feasibility of using frameshift antigens for cancer vaccine development. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2018

Made available in DSpace on 2014-06-11T19:23:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-21Bitstream added on 2014-06-13T19:08:43Z : No. of bitstreams: 1 bassetto_cc_me_botib.pdf: 208428 bytes, checksum: c0a49413b34832ecc2265e91d1560b39 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Neste estudo avaliou-se a eficácia de uma vacina constituída de glicoproteínas obtidas da membrana do intestino de Haemonchus contortus em bezerros desafiados com H. contortus ou H. placei. Bezerros holandeses machos, criados livres de infecções por helmintos, foram distribuídos em quatro grupos com nove animais cada. Dois grupos foram vacinados com 50 μg do imunógeno diluído no adjuvante QuilA, enquanto os outros dois grupos foram os controles, receberam apenas adjuvante. A vacina foi administrada três vezes com intervalo de 21 dias entre as aplicações. Os bezerros foram artificialmente infectados sete dias após a última imunização e sacrificados para contagem dos vermes 43 dias depois. Os bezerros de um dos grupos vacinados receberam 8000 larvas infectantes (L3) de H. contortus enquanto os do outro grupo foram infectados com o mesmo número de L3 de H. placei. Os controles foram infectados na mesma ocasião com o mesmo número de L3 de H. contortus ou H. placei. A vacinação reduziu significativamente a contagem de ovos por grama de fezes (OPG) e a carga parasitária (P<0,01). A titulação de anticorpos nos animais controle permaneceu perto de zero enquanto nos animais vacinados verificou-se elevada titulação de anticorpos no soro. Os bezerros vacinados e desafiados com H. contortus não eliminaram ovos nas amostras de fezes, enquanto os controles apresentaram média máxima (± erro padrão) de 61,1 (±42,3) OPG 31 dias após a infecção. A partir deste dia, a contagem de OPG diminuiu progressivamente neste grupo controle com apenas um animal eliminando ovos nas fezes 42 dias após a infecção. Nos controles de H. placei, o ápice na contagem de OPG ocorreu 35 dias após a infecção (61,1 ± 21,7) e permaneceu relativamente constante até o final do estudo, enquanto no grupo vacinado apenas dois animais eliminaram ovos nas fezes, na última coleta. Os animais controle... / This study evaluated the efficacy of a vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus in calves challenged with H. contortus or Haemonchus placei. Males Holstein calves, raised worm free, were distributed into four groups with nine animals each. Two groups were vaccinated with 50 μg of antigen diluted on QuilA adjuvant, while the others two groups were the controls and received only adjuvant. The vaccine was administered three times 21 days apart. Either vaccinated or not and challenged with either 8,000 H. contortus or H. placei infective larvae. The calves were challenged 7 days after the last immunization and killed for worm counts 43 days later. Vaccination significantly reduced faecal egg counts (FEC) and worm burdens (P< 0.01). Antibody titres in the calves control stayed close to zero meanwhile in the calves vaccinated was observed high antibody titres in the serum. Calves vaccinated and challenged with H. contortus did not shed eggs in faecal samples, while the controls showed a maximum mean (± standard error) FEC of 61.1 (±42.3) at 31 days post infection. Then, FEC progressively declined in this group with only one animal shedding eggs in faeces 42 days post infection. With H. placei the controls FEC peaked at 35 days post infection (61.1 ±21.7) and remained relatively constant until the end of the study, while in the vaccinated group only two animals shed eggs and only on the last collection date. With H. placei the controls contained a mean of 551.1 (±93.7) parasites, while the vaccinates had 174.4 (±56.3) worms. The establishment rate of H. contortus was lower than that of H. placei (P< 0.01) with an average of 163.9 (±39.4) and 74.4 (±20.4) specimens in the control and vaccinated groups, respectively. It was concluded that vaccination of calves with antigens obtained from H. contortus conferred protection against both H. placei and H. contortus