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Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / Influenza A virus (IAV) is a negative sense, single-stranded, segmented RNA virus with eight gene segments. It is an important respiratory pathogen which causes annual epidemics and occasional pandemics worldwide in humans and leads to considerable economic problems for the livestock industry. To control and prevent this significant disease, understanding the pathogenesis of IAVs is critical. Although some molecular mechanisms regarding virulence have been determined, IAV pathogenesis is not completely understood and is difficult to predict. The eight viral gene segments of IAV were thought to encode for 10 viral proteins. Since 2001, eight additional viral proteins have been identified, including PB1-F2, PB1-N40, PA-X, NS3, PA-N155, PA-N182, M42, and PB2-S1. However, the functions of these novel proteins in influenza virus replication as well as pathogenesis have not been fully elucidated. Although PB1-F2 protein is an important virulence factor of IAV, the effects of this protein on viral pathogenicity of swine influenza virus (SIV) remain unclear. In Chapter 2, we investigated the contribution of the PB1-F2 protein to viral pathogenicity of a virulent triple-reassortant (TR) H1N1 SIV in different hosts, pigs and mice. Our data indicate that PB1-F2 expression in virulent TR H1N1 SIV modulates virus replication and pathogenicity in the natural host, pigs, but not in mice. In addition, single amino acid (aa) substitution at position 66 (N/S) in the PB1-F2 has a critical role in virulence in mice but no effect was found in pigs. A novel IAV protein, PA-X consists of the N-terminal 191aa of PA protein and a unique C-terminal 41 (truncated form) or 61 (full-length form) aa residues encoded by +1 ribosomal frameshifting. Although several studies have demonstrated the PA-X protein as an important immune modulator and virulence factor, the impact of different expressions of PA-X protein including full-length, truncated or PA-X deficient forms on viral pathogenicity and host response remains unclear. In Chapter 3, we showed that expression of either truncated or full-length PA-X protein in 2009 human pandemic H1N1 (pH1N1) viruses suppresses host antiviral response by host shutoff activity which promotes viral growth and virulence in mice when compared to loss of PA-X expression. Furthermore, full-length PA-X expression displayed stronger impact on viral pathogenicity and host immune response compared to truncated PA-X expression. Taken together, our results provide new insights into the impact of PB1-F2 and PA-X proteins on virus replication, pathogenicity and modulation of host immune responses. This knowledge is important for better understanding of IAV pathogenesis.

Cotesia flavipes (Cameron) (Hym.: Braconidae), como outros cenobiontes, é capaz de regular seu hospedeiro, criando um ambiente que sustenta e promove o desenvolvimento de suas larvas, comumente em detrimento do hospedeiro. Substâncias derivadas do trato reprodutivo das fêmeas (proteínas ovarianas, veneno e polidnavírus) são injetadas no hospedeiro, afetando a resposta imune e outros processos fisiológicos com o propósito de regular os níveis hormonais, nutrição e comportamento. O presente trabalho teve por objetivo avaliar o papel dessas substâncias no crescimento e desenvolvimento de Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae), e avaliar como o parasitismo afeta a resposta imune do hospedeiro. Todas as substâncias derivadas da fêmea foram obtidas após a dissecação do parasitóide, através da coleta do reservatório de veneno ou dos ovários (proteínas ovarianas e polidnavírus) em tampão resfriado. As secreções foram processadas adequadamente e injetadas logo após a coleta. O veneno e as proteínas ovarianas + polidnavírus (PDV) foram injetados juntos ou separadamente em lagartas entre 0-12h do 6º instar. O efeito de cada um dos componentes isolados do parasitóide no desenvolvimento e crescimento do hospedeiro foi avaliado através de observações no ganho de peso, duração e viabilidade da fase larval e pupal. Os efeitos do parasitismo na resposta imune do hospedeiro foram avaliados tanto ao nível celular, através da contagem do número total de hemócitos e capacidade de encapsulação, como ao nível bioquímico, medindo-se a ativação da profenoloxidase e produção de óxido nítrico na hemolinfa das lagartas de D. saccharalis em diferentes estágios de desenvolvimento do parasitóide (0, 1, 3, 5, 7 e 9 dias após o parasitismo). As proteínas ovarianas do parasitóide e o PDV sozinho, ou co-injetado com o veneno, suspenderam o desenvolvimento larval do hospedeiro, enquanto que o veneno, sozinho, afetou o processo de metamorfose. A resposta imune do hospedeiro também foi afetada por C. flavipes, de maneira dependente do tempo. Lagartas parasitadas apresentaram declínio no número total de hemócitos a partir do 3º dia e a capacidade de encapsulação foi afetada ao longo do desenvolvimento do parasitóide. A atividade da fenoloxidase do hospedeiro foi alterada apenas no final do desenvolvimento imaturo do parasitóide, enquanto que o óxido nítrico foi afetado nas 24 h iniciais após parasitismo. / Cotesia flavipes (Cameron) (Hym., Braconidae), as other koinobionts, is capable of regulating the host development to produce an suitable host environment to sustain and promote its own larval development at the host expenses. Female-derived substances from the reproductive tract (ovarian proteins, venom, polydnavirus) are injected into the host, affecting the host immune response and other physiological processes aiming to regulate the host hormone levels, nutrition and behavior. Our goal was to evaluate the role of these substances on Diatraea saccharalis (F.) (Lepidoptera: Crambidae) growth and development, and how the parasitism affects the host immune response. All female-derived substances were collected after parasitoid dissection by collecting the venom reservoir or the ovaries (ovarian proteins and polydnavirus). Dissections were carried out in ice-cold buffer, collected tissues were processed accordingly and the desired substances injected immediately after collection. Venom and ovarian proteins+polydnavirus (PDV) were injected jointly and separated in 0-12 hold 6th instars of D. saccharalis. The effect of these substances on host development and growth was evaluated by measuring the host weight gain, larval and pupal survivorship and developmental time. The effects of the parasitism on the host immune response was evaluated either at the cellular level, by measuring the total hemocyte count and the encapsulation capacity, and at the biochemical level, by measuring the prophenoloxidase activity and nitric oxide levels at different stages of parasitoid development (0, 1, 3, 5, 7 and 9 days after parasitism). Parasitoid ovarian proteins and PDV alone or co-injected with the venom arrested the host larval development, while the venom by itself only affected the host metamorphosis process. The host immune response was also affected by C. flavipes at a time-dependent manner. The total hemocyte count dropped at day 3 of parasitism, while the host encapsulation capacity was reduced during parasitoid development. The host prophenoloxidase activity was also affected mainly towards the end of parasitoid larval development, while the nitric oxide at the first 24 h after parasitism.

This thesis aimed to understand the mechanisms underlying within-host interactions among coinfecting parasites in wild rodents, how they are affected by the host immune response, and how they contribute to shape disease dynamics in nature. Coinfection is ubiquitous in human, domestic and wild animal populations, and can consist of both microparasites (viruses, bacteria and protozoa) and macroparasites (parasitic helminths). Moreover, coinfecting parasites can interact with each other in a number of ways (positive or negative, direct or indirect), which affects disease severity and progression, parasite transmission, the response of target and non-target parasites to treatment and, ultimately, the epidemiology of each coinfecting parasite species. While previous work on laboratory animals has generated detailed knowledge of the cellular components of the host immune response involved during coinfection, we still mostly lack a conceptual understanding of the role of the host immune response in mediating within-host interactions in nature. I used a known within-host interaction between two important intestinal parasites (the nematode Heligmosomoides polygyrus and the protozoan Eimeria hungaryensis) of wild wood mice (Apodemus sylvaticus) to study the underlying causes and consequences of this interaction for both parasite dynamics and host health. I first investigated if specific and total antibody levels can explain natural burdens and infection of H. polygyrus and Eimeria spp. in the context of other parasites and variation in host demography in a cross-sectional field study. I found that H. polygyrus-specific IgG1 and total faecal IgA were the strongest predictors of both H. polygyrus infection and burden and Eimeria spp. infection. Further, Eimeria spp. infection was associated with lower antibody levels, suggesting an interaction between Eimeria spp. and anti-helminth immunity. Next, I tested the causative relationship between antibody levels and parasite infection. Over the course of a longitudinal anthelmintic treatment study in the field, I measured infection and burden of both target and non-target parasites, as wells as specific and general antibody levels. I found that treatment successfully reduced H. polygyrus burden, wild led to a change in both antibody levels and E. hungaryensis dynamics. Further, H. polygyrus-specific IgG1 levels were predicted by pre-treatment H. polygyrus burden, suggesting that helminth infection induces antibody production, rather than vice versa. Following from this, I explored if treatment of single or multiple parasite groups (helminths, coccidia or both) had an effect on host survival. I used data from a longitudinal field study spanning an entire season of A. sylvaticus (April-December), where animals were given either Ivermectin (anthelmintic), Vecoxan (anti-coccidial), a mix of both drugs or water every fortnight. Ivermectin treatment led to a consistent reduction in H. polygyrus prevalence and burden, as well as a steady increase in E. hungaryensis prevalence, whereas Vecoxan treatment failed to show any effect on either target or non-target parasites. Interestingly, anthelmintic treatment led to a reduction in survival at intermediate H. polygyrus burdens, suggesting that anti-parasite treatments might not always be beneficial for the host. By bringing this wild coinfection system into the lab, I examined if the interaction between H. polygyrus and E. hungaryensis could be re-created under controlled laboratory condition, and if the lack of environmental variation had an effect on parasite and/or antibody dynamics. I found that coinfection led to a delay in H. polygyrus expulsion, and decreased E. hungaryensis shedding during chronic helminth infection. However, coinfection did not affect antibody dynamics. This not only demonstrated that the interaction between the two parasites was reciprocal, but also showed that coinfection can significantly affect parasite transmission dynamics. In an ongoing bioinformatic analysis, I investigated the level of genetic diversity in wild Eimeria spp. populations in order to uncover the mechanism underlying a common lack of protective immunity towards Eimeria spp. infections in wild and domestic animal populations. I found that there were multiple genetically distinct strains circulating within all populations tested, but homologous re-infection was not less likely than heterologous reinfection. This suggests that the lack of protective immunity in wild Eimeria spp. Populations cannot solely be explained by high levels of genetic diversity. This thesis provides several important insights into the mechanisms underlying parasite within-host interactions. Importantly, it highlights that, whilst host immunity plays a crucial role in determining the outcome of coinfection, other factors such as host demography have to be taken into account in order to understand the interplay between immunity and coinfection. I further show that anti-parasite treatments in the wild can be successful, but the benefits of such treatments can be context dependent. More broadly, my findings can have important implications for the planning and evaluation of treatment programs targeted at both single and coinfected animals and humans in their natural environment.

HIV-1 genetic diversity is a major obstacle for developing an effective vaccine. My hypothesis is that HIV-1 genetic diversity can be characterized and that cross-clade immunogens can be predicted at the population level. I systematically investigated positive selection (PS) pressures on HIV-1 Env and Gag proteins based on the analysis of the sequences collected from the Los Alamos Sequence Database. I identified PS sites, investigated PS patterns, correlated PS with the known functional sites of the two proteins, calculated frequencies of HLA alleles targeting CTL epitopes, and compared PS patterns among major subtypes. The results showed that PS pressure was widely dispersed across the entire regions of both HIV-1 Env and Gag proteins, suggesting the conserved regions are under host immune response pressure. The neutralizing antibody, non-neutralizing antibody, and CTL responses were found to be the major forces driving genetic diversity of HIV-1 env and gag genes at population level. However, PS pressures on both Env and Gag proteins remain stable over time, suggesting genetic diversity of HIV-1 driven by host immune responses changed very little over the last 29 years. Furthermore, the results also demonstrated that up to 70% PS sites were shared among the major HIV-1 clades, implying the existence of cross-clade immunogenicity. A number of potential cross-clades immunogens were predicted to elicit CTL or neutralizing antibody responses from Env and Gag proteins. I also detected a significant correlation between HLA allele frequencies and host CTL responses elicited by Accessory/Regulator’s proteins at population level. Moreover, I detected an association between the frequency of HLA-B7 supertype and the number of identified optimal CTL epitopes. The results suggest HLA class I allele frequencies in a population influence the evolution of HIV-1. I also systematically evaluated the utility of ultra-deep pyrosequencing to characterize genetic diversity of HIV-1 gag genes within quasispecies. The results showed that ultra-deep pyrosequencing of amplified HIV genes is a better method than the traditional Sanger-clone-based method in the comprehensive characterization of genetic diversity of HIV-1 quasispecies, especially in detecting low frequency variations. In conclusion, my thesis provides important information for rational design of an effective HIV-1 vaccine.

HIV-1 genetic diversity is a major obstacle for developing an effective vaccine. My hypothesis is that HIV-1 genetic diversity can be characterized and that cross-clade immunogens can be predicted at the population level. I systematically investigated positive selection (PS) pressures on HIV-1 Env and Gag proteins based on the analysis of the sequences collected from the Los Alamos Sequence Database. I identified PS sites, investigated PS patterns, correlated PS with the known functional sites of the two proteins, calculated frequencies of HLA alleles targeting CTL epitopes, and compared PS patterns among major subtypes. The results showed that PS pressure was widely dispersed across the entire regions of both HIV-1 Env and Gag proteins, suggesting the conserved regions are under host immune response pressure. The neutralizing antibody, non-neutralizing antibody, and CTL responses were found to be the major forces driving genetic diversity of HIV-1 env and gag genes at population level. However, PS pressures on both Env and Gag proteins remain stable over time, suggesting genetic diversity of HIV-1 driven by host immune responses changed very little over the last 29 years. Furthermore, the results also demonstrated that up to 70% PS sites were shared among the major HIV-1 clades, implying the existence of cross-clade immunogenicity. A number of potential cross-clades immunogens were predicted to elicit CTL or neutralizing antibody responses from Env and Gag proteins. I also detected a significant correlation between HLA allele frequencies and host CTL responses elicited by Accessory/Regulator’s proteins at population level. Moreover, I detected an association between the frequency of HLA-B7 supertype and the number of identified optimal CTL epitopes. The results suggest HLA class I allele frequencies in a population influence the evolution of HIV-1. I also systematically evaluated the utility of ultra-deep pyrosequencing to characterize genetic diversity of HIV-1 gag genes within quasispecies. The results showed that ultra-deep pyrosequencing of amplified HIV genes is a better method than the traditional Sanger-clone-based method in the comprehensive characterization of genetic diversity of HIV-1 quasispecies, especially in detecting low frequency variations. In conclusion, my thesis provides important information for rational design of an effective HIV-1 vaccine.

Cotesia flavipes (Cameron) (Hym.: Braconidae), como outros cenobiontes, é capaz de regular seu hospedeiro, criando um ambiente que sustenta e promove o desenvolvimento de suas larvas, comumente em detrimento do hospedeiro. Substâncias derivadas do trato reprodutivo das fêmeas (proteínas ovarianas, veneno e polidnavírus) são injetadas no hospedeiro, afetando a resposta imune e outros processos fisiológicos com o propósito de regular os níveis hormonais, nutrição e comportamento. O presente trabalho teve por objetivo avaliar o papel dessas substâncias no crescimento e desenvolvimento de Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae), e avaliar como o parasitismo afeta a resposta imune do hospedeiro. Todas as substâncias derivadas da fêmea foram obtidas após a dissecação do parasitóide, através da coleta do reservatório de veneno ou dos ovários (proteínas ovarianas e polidnavírus) em tampão resfriado. As secreções foram processadas adequadamente e injetadas logo após a coleta. O veneno e as proteínas ovarianas + polidnavírus (PDV) foram injetados juntos ou separadamente em lagartas entre 0-12h do 6º instar. O efeito de cada um dos componentes isolados do parasitóide no desenvolvimento e crescimento do hospedeiro foi avaliado através de observações no ganho de peso, duração e viabilidade da fase larval e pupal. Os efeitos do parasitismo na resposta imune do hospedeiro foram avaliados tanto ao nível celular, através da contagem do número total de hemócitos e capacidade de encapsulação, como ao nível bioquímico, medindo-se a ativação da profenoloxidase e produção de óxido nítrico na hemolinfa das lagartas de D. saccharalis em diferentes estágios de desenvolvimento do parasitóide (0, 1, 3, 5, 7 e 9 dias após o parasitismo). As proteínas ovarianas do parasitóide e o PDV sozinho, ou co-injetado com o veneno, suspenderam o desenvolvimento larval do hospedeiro, enquanto que o veneno, sozinho, afetou o processo de metamorfose. A resposta imune do hospedeiro também foi afetada por C. flavipes, de maneira dependente do tempo. Lagartas parasitadas apresentaram declínio no número total de hemócitos a partir do 3º dia e a capacidade de encapsulação foi afetada ao longo do desenvolvimento do parasitóide. A atividade da fenoloxidase do hospedeiro foi alterada apenas no final do desenvolvimento imaturo do parasitóide, enquanto que o óxido nítrico foi afetado nas 24 h iniciais após parasitismo. / Cotesia flavipes (Cameron) (Hym., Braconidae), as other koinobionts, is capable of regulating the host development to produce an suitable host environment to sustain and promote its own larval development at the host expenses. Female-derived substances from the reproductive tract (ovarian proteins, venom, polydnavirus) are injected into the host, affecting the host immune response and other physiological processes aiming to regulate the host hormone levels, nutrition and behavior. Our goal was to evaluate the role of these substances on Diatraea saccharalis (F.) (Lepidoptera: Crambidae) growth and development, and how the parasitism affects the host immune response. All female-derived substances were collected after parasitoid dissection by collecting the venom reservoir or the ovaries (ovarian proteins and polydnavirus). Dissections were carried out in ice-cold buffer, collected tissues were processed accordingly and the desired substances injected immediately after collection. Venom and ovarian proteins+polydnavirus (PDV) were injected jointly and separated in 0-12 hold 6th instars of D. saccharalis. The effect of these substances on host development and growth was evaluated by measuring the host weight gain, larval and pupal survivorship and developmental time. The effects of the parasitism on the host immune response was evaluated either at the cellular level, by measuring the total hemocyte count and the encapsulation capacity, and at the biochemical level, by measuring the prophenoloxidase activity and nitric oxide levels at different stages of parasitoid development (0, 1, 3, 5, 7 and 9 days after parasitism). Parasitoid ovarian proteins and PDV alone or co-injected with the venom arrested the host larval development, while the venom by itself only affected the host metamorphosis process. The host immune response was also affected by C. flavipes at a time-dependent manner. The total hemocyte count dropped at day 3 of parasitism, while the host encapsulation capacity was reduced during parasitoid development. The host prophenoloxidase activity was also affected mainly towards the end of parasitoid larval development, while the nitric oxide at the first 24 h after parasitism.

Staphylococcus aureus (SA) is a major human pathogen that colonizes the anterior nares in 30% of the human population. Though nasal carriage of SA is a known risk factor for the subsequent spread of SA infections, the dynamics of SA nasal colonization is poorly understood. Our research focuses on understanding the host and bacterial factors that might contribute to the human nasal colonization by SA. In an attempt to elucidate the host response to SA, we performed an autologous human in vivo nasal colonization study, which showed decreased survival rates of SA in hosts who elicited a robust immune response. We also identified a significant correlation between SA nasal colonization and the expression of host proinflammatory, chemotactic and growth factors. Additionally, we functionally disrupted a major autolysin, atl a surface expressed bacterial protein that plays multiple roles in cell separation, adhesion and biofilm formation of SA. Microscopic analysis of the ∆atl strains showed phenotypic differences, including cell clumping and cluster formation due to defective cell separation, which confirmed the functional loss of atl. Subsequent analysis of the ∆atl and wild-type strains revealed that there was no significant difference in their ability to adhere to human nasal epithelial cells (hNEC) in an ex vivo hNEC model. Similarly, our competitive in vivo human nasal colonization study, in which equal colony-forming units of each wild-type and ∆atl SA strain were inoculated in the anterior nares of donors, showed similar survival rates between wild-type and ∆atl. These results suggest that Atl might not be directly involved in the adherence and colonization of SA to the anterior nares. Furthermore, our study suggests that host factors might play a predominant role in determining SA colonization to human anterior nares.

No description available.

Periodontal disease affects 47% of Americans over 30 and is a growing global concern. Current treatments for periodontal disease focus on the mechanical elimination of periodontal biofilms. Very few treatments are available that target the rampant, unregulated host immune response that is ultimately responsible for tissue degradation. BET proteins have been shown to play critical roles in inflammatory gene regulation and are therefore potentially ideal therapeutic targets for treating periodontal disease. RVX-208 is a selective BET-inhibitor with a high affinity for Bromodomain 2 (BD2) as compared to BD1 in BET proteins. Our previous studies have shown that RVX-208 inhibits inflammatory cytokine production and suppresses osteoclast differentiation. Cell culture assays have provided proof of concept for RVX-208 and its feasibility as a treatment for periodontal disease. As such, our long term goal is to develop RVX-208 as a front-line treatment for periodontitis. The objectives of this study were to determine the ability of RVX-208 to reduce bone loss in a ligature-induced periodontitis model, and to further investigate the mechanisms through which RVX-208 mediates its anti-inflammatory and osteoclastogenesis-suppressive effects. The specific aims of this study were: 1) To further validate the in vivo effects of RVX-208 on a ligature-induced periodontitis model in rats, and 2) To determine the molecular mechanisms of RVX-208 on preventing alveolar bone loss in periodontal disease. To investigate, a ligature-induced periodontitis model was created in rodents. Those rodents were treated with increasing dosages of RVX-208 (0-2.5 mM) by subgingival injection every other day. After 2 weeks, the maxillae were harvested and analyzed via a micro-CT protocol that had been created and validated through statistical analyses. To study the ability of RVX-208 to suppress osteoclastogenesis, RAW264.7 cells were induced into osteoclasts by RANKL and then treated with RVX-208. To ensure RVX-208 was not species specific, THP-1 cells were challenged with either E. coli-LPS or P. gingivalis bacteria and then treated with RVX-208. Linear and volumetric micro-CT analysis showed that RVX-208 could significantly ameliorate bone loss in a ligature-induced periodontitis model. RVX-208 was shown to prevent osteoclast differentiation by suppressing the expression of genes closely associated with osteoclast differentiation and maturation. RVX-208 was found to not be species specific, as it was able to mediate its effects on a human cell line, and had consistent anti-inflammatory effects regardless of whole pathogen or LPS-induced inflammatory response. Therefore, RVX-208 is a promising therapeutic for treatment of periodontal diseases.