<p> Traditionally, seasonal forcing has been considered to be the major cause of the influenza seasonality. However, Andreasen [2003] showed that repetitive introductions of new strains can lead to cyclic dynamics. The cyclic dynamic produced by his model is not seasonal, because the length of seasons cannot be defined in his model. In this report, we develop a model that combines a stochastic mutation process with a two-strain competition process governing the spread of the mutant strain. This model can produce stable seasonal dynamics. If we introduce a small seasonal forcing to the transmission rate, the length of a season can be regulated to one year if the unforced system oscillates with a period close to one year. If the system has a period that is far from one year, then the forced system may behave chaotically.</p> / Thesis / Master of Science (MSc)

Emerging influenza viruses continue to challenge public health. The problem is public health science professionals have been battling emerging human influenza diseases with tactile and reactionary methods because there is a lack of knowledge and data at the human-animal interface. This research was a baseline study of the proportion of influenza A virus (IAV) in urban and rural communities in California. The population was artificial recirculating water ponds in the geographic locations of rural and urban Californian communities. Surface water samples [N = 182] were collected from artificial recirculating ponds in California. Positivity for IAV was verified by real time RT-PCR, MDCK cells for virus infectivity, nucleotide sequencing of the RNA genome, and phylogenic analysis of IAV H5N1 strains. The proportion of IAV in rural and urban ponds favored the greater burden of IAV in urban ponds over rural ponds. The presence of waterfowl and IAV M gene sequence positivity were found not to be significantly related. The geochemical properties--pH, salinity, and water temperature at time of collection--were not predictors of IAV infectivity. This baseline research study validated these water ponds as resource sites for IAV surveillance and monitoring. The social change implications of this study can be recognized at the national and international levels, to the population level, and to the individual level by providing geospatial analysis and spatial-temporal data for IAV surveillance, initiating biosecurity measures to protect poultry industries in the United States and Brazil, and contributing to the current IAV strain library. Contributions to the IAV strain library may be used to develop vaccines against human pandemics.

This thesis investigates antigenic variation and its evolution in Plasmodium falciparum, the cause of the most deadly form of human malaria. Antigenic variation is a strategy for evading immunity by switching between antigenic variants during infection. In P. falciparum, such variable antigens confer different binding phenotypes that may affect parasite survival and have also been linked to pathology. Here, a new statistical method is described for determining the switching patterns that underlie antigenic variation. This method is then applied to experimental data to yield a full description of an antigenic switching network in P. falciparum. In light of the findings, theoretical modelling is used to show how immune selection and binding phenotypes may have contributed to the evolution of antigenic repertoire structure, expression order and virulence. Related models are also used to investigate parasite population diversity, providing possible explanations for observations reported here and elsewhere, with implications for vaccine design. Together, these chapters advance understanding of P. falciparum immune evasion and how it relates to pathology. This work further reinforces the role of host immunity in shaping pathogen population diversity at multiple levels.

T. pallidum can survive a primary immune response and continue growing in the host for an extended period of time. T. pallidum is thought to bind serum fibronectin (FN) through Tp0483 on the surface to obscure antigens. A Tp0483 fragment (rTp0483) was adsorbed onto functionalized self-assembled monolayers (SAMs) with FN. FN capture by adsorbed rTp0483 depended greatly on surface chemistry with COO- groups being best for FN binding. Hemocompatibility was determined by analysis of plasma protein adsorption, intrinsic pathway activation, and platelet activation. rTp0483+FN bound an equal or lesser amount of fibrinogen (Fg), human serum albumin (HSA), and factor XII (FXII) compared to rTp0483 or FN alone and adsorption of rTp0483 prior to FN greatly decreased platelet activation. Inhibition of protein binding and platelet activation suggested an attenuated hematological response. Biocompatibility of rTp0483 and FN coated surfaces was characterized by macrophage uptake of protein coated polystyrene microspheres (PSMs), macrophage adsorption onto protein coated surfaces, cytotoxic effects of adsorbed rTp0483 and FN, and TNF-α and NO2- release in macrophages stimulated with rTp0483 and FN adsorbed and in solution. Addition of FN to rTp0483 on plain and COO- PSMs reduced phagocytosis compared to rTp0483 alone and on plain PSMs compared to FN alone. On plain PSMs addition of FN to adsorbed rTp0483 decreased TNF-α generation. Adsorption of rTp0483 before FN on large, flat COO- surfaces decreased macrophage adsorption and TNF-α and NO2- generation. High concentrations of rTp0483 were mildly cytotoxic to macrophages. FN binding by Tp0483 on T. pallidum likely plays a role in antigenic disguise and rTp0483+FN coatings may potentially inhibit FN and rTp0483 specific interactions with macrophages. Molecularly imprinted polymer coatings were also examined for biomaterial development. Fouling resistant 2-methacryloyloxyethyl phosphorylcholine (MPC) was imprinted with bovine serum albumin (BSA) protein templates to facilitate BSA specific binding. The BSA template was constructed and verified and BSA specific binding quantified using quartz crystal microbalance (QCM) and enzyme linked immunosorbent assay (ELISA). BSA imprinted coatings were determined to bind significantly more BSA than nonfouling MPC controls demonstrating the feasibility of targeted protein capture.

<p>Evolution by natural selection can occur at multiple biological scales. This is particularly the case for host-pathogen systems, where selection occurs both within each infected host as well as through transmission between hosts. Despite there being established mathematical models for understanding evolution at a single biological scale, fewer tractable models exist for multiscale evolutionary dynamics. Here I present mathematical approaches using tools from probability and stochastic processes as well as dynamical systems to handle multiscale evolutionary systems. The first problem I address concerns the antigenic evolution of influenza. Using a combination of ordinary differential equations and inhomogeneous Poisson processes, I study how immune selection pressures at the within-host level impact population-level evolutionary dynamics. The second problem involves the more general question of evolutionary dynamics when selection occurs antagonistically at two biological scales. In addition to host-pathogen systems, such situations arise naturally in the evolution of traits such as the production of a public good and the use of a common resource. I introduce a model for this general phenomenon that is intuitively visualized as a a stochastic ball-and-urn system and can be used to systematically obtain general properties of antagonistic multiscale evolution. Lastly, this ball-and-urn framework is in itself an interesting mathematical object which can studied as either a measure-valued process or an interacting particle system. In this mathematical context, I show that under different scalings, the measure-valued process can have either a propagation of chaos or Fleming-Viot limit.</p> / Dissertation

This thesis focuses on the population dynamics of three antigenically diverse RNA viruses: dengue, influenza and HIV-1. It comprises a set of studies highlighting the roles of structural constraints on critical antigenic determinants, interactions between immune responses to different antigenic types, host lifespan, and the degree of mixing between different host populations in determining the epidemiology and within-host dynamics of these pathogen systems. Dengue exists in humans as a collection of four antigenically related serotypes. Although infection by one serotype appears to convey life-long protection to homologous infection, it is believed to be a risk factor for severe disease manifestations upon secondary, heterologous infection due to the phenomenon of Antibody-Dependent Enhancement (ADE). It is not clear if third or fourth infections are possible, and if so, how they contribute to dengue epidemiology. In this thesis, I investigate the effect of third and fourth infections on the transmission dynamics of dengue. By contrast with dengue, human influenza viruses are known to be in rapid antigenic flux, manifesting in the sequential replacement of antigenic types. This pattern of evolution does not appear to be the same in shorter-lived hosts such as swine and birds. In this thesis, I have used a simple multi-locus model to explore the relationship between host lifespan and viral evolution, as well as to elucidate the effects of transmission between hosts of different lifespan in effort to capture the cross-species element of influenza transmission. My final chapter concerns the within-host evolution of HIV-1. I propose a new model for the pathogenesis of HIV-1 where the transition to AIDS is primarily linked to the gradual loss of the ability to make new antibody responses as the CD4+ population declines. Together these studies emphasise that it is the changing profile of immune responses – either at the population level or within the host – that is the principal determinant of the dynamics of the pathogen, rather than the mode and tempo of antigenic innovation.

A major virulence factor of the human malaria parasite Plasmodium falciparum is Plasmodium falciparum erythrocyte membrane protein 1(PfEMP-1). This protein is inserted into the erythrocyte membrane, giving cytoadherence properties. A family of genes called var, located sub-telomerically and in chromosome central clusters encode this protein. Var genes are expressed in a mutually exclusive manner, how this is controlled is unclear. A non-coding RNA (ncRNA) termed the GC-rich element (GRE) had been identified that is only located at the central clusters and is transcribed throughout the parasite lifecycle. A screen of the P. falciparum genome for novel ncRNAs identified ncRNAs from known classes. Novel transcripts were identified, but none in the proximity of var genes. We have investigated the role of the GRE in var gene regulation. A set of qRT-PCR primers have been designed and tested to follow var gene expression in the HB3 isolate, these are not cross-reactive with a published set for the 3D7 isolate. Alterations were made to the 3D7 set to remove cross-reactivity with HB3. Var gene expression was studied in 31 HB3 clones and progeny of the 3D7xHB3 genetic cross. Following var switching over five months in eleven HB3 clones showed that all of the clones ended up expressing var genes from the same central cluster on chromosome 4. GRE Transcription in these clones is linked to a specific class of var gene. Transcription from a single GRE locus occurs only when a var gene of the central UpsC class is expressed from the same cluster. Expression of other classes of var gene gives multiple transcripts from different GRE loci. Investigations into the in vitro binding properties of the GRE revealed an RNA:protein complex that can be resolved by electrophoresis. Proteomic analysis of the complex revealed predominantly ribosome proteins and translation factors.

Protozoan parasites of the genus Plasmodium are the causative agent of malaria. The most severe form of human malaria is caused by P. falciparum, responsible for approximately three quarters of a million deaths each year. One major problem in the treatment of malaria is resistance to existing chemotherapies. Consequently, there is an urgent need to identify and validate novel drug targets. A possible recently identified drug target is the PfNitA protein of P. falciparum which contains orthologues in other Plasmodium species but is absent from humans. The gene is annotated as a putative formate-nitrite transporter and orthologues are found in a range of prokaryotes as well as the lower eukaryotes algae and fungi. To determine the biological function of the protein, pfnita was expressed in Escherichia coli strains lacking the endogenous formate and nitrite transporters. In order to analyse the essentiality of the gene a reverse genetics approach was taken and the data discussed. Results indicate that the PfNitA protein is located in the plasma membrane and digestive vacuole of intraerythrocytic parasites suggesting a role in the uptake or excretion of metabolites. A second complexity with regard to treatment is the lack of a vaccine. A problem in crating a vaccine is antigenic variation. The PIR family of proteins contain a so-called hypervariable domain that has led to the suggestion that the family may play a role in antigenic variation. The objective of the work carried out in this thesis was to investigate the topology and structure of the PcCir2 protein of Plasmodium chabaudi, using E. coli as the expression host. The topology of Cir2 has been examined by means of reporter fusions and overexpression/purification studies undertaken towards crystallisation. As the PcCir2 amino acid sequence does not show significant homology to other proteins, structural data may provide insights into potential functional or binding domains.

Anticorpos monoclonais (AcMo) contra proteínas do vírus da raiva (RABV) foram produzidos para adequar a caracterização antigênica dos isolados no Brasil. Foram selecionados dois isolados de morcegos insetívoros, sendo um de Nyctinomops laticaudatus e outro de Eptesicus furinalis que apresentaram perfis não compatíveis (NC) com os pré-estabelecidos. As suspensões virais foram adaptadas para crescimento em cultura de células N2A. Para o preparo de AcMo foram utilizadas como antígeno as ribonucleoproteínas dos isolados selecionados. Foram obtidos dois AcMo, o 3A7 e o 4E10. Analisando 57 isolados de RABV com esses AcMo, o 3A7 reagiu com 21 (36,84%) e o 4E10 com 25 (43,85%). Dos 13 isolados caracterizados como variante antigênica 3 (Desmodus rotundus) o 3A7 reagiu com 8 (61,53%) e o 4E10 com 11 (84,61%). Dos 9 isolados com perfil NC em morcegos o 3A7 reagiu com 5 (55,55%) e o 4E10 com 4 (44,44%). Os anticorpos produzidos poderão auxiliar na complementação do painel existente de caracterização antigênica o que poderá aprimorar a vigilância epidemiológica da doença. / Monoclonal antibodies (MAb) against the rabies virus (RABV) proteins were produced to improve the antigenic characterization of the isolates in Brazil. Two isolates from insectivorous bats were selected; one was from the species Nyctinomops laticaudatus and the other from Eptesicus furinalis, which showed non-compatible (NC) profiles from pre-established ones. The viral suspensions were adapted for growth in N2A cells. Ribonucleoproteins from selected isolates were used as antigen for the preparation of Mab. We obtained two Mab, the 3A7 and the 4E10. Of the 57 RABV isolates analyzed with these MAb, the 3A7 reacted with 21 (36.84%) and 4E10 with 25 (43.85%). Of the 13 isolates characterized as antigenic variant 3 (Desmodus rotundus), the 3A7 MAb reacted with 8 (61.53%) and 4E10 with 11 (84.61%). Of the nine isolates with the profile NC of bats the 3A7 reacted with 5 (55.55%) and the 4E10 with 4 (44.44%). The antibodies produced may help to complement the existing panel to antigenic characterization which could improve the disease epidemiological surveillance.

A partir de uma biblioteca de cDNA de glândulas de veneno de Micrurus corallinus (cobra-coral), uma serpente da Família Elapidae bastante representada no Brasil e muito comum em áreas florestais tropicais, foram gerados 1.438 Expressed Sequences Tags (ESTs), agrupados em 611 clusters. O banco representa os genes mais expressos na glândula de veneno de M. corallinus. Os transcritos relacionados às toxinas apresentaram ao redor de 46% de representação nesse banco de seqüências. A composição geral das toxinas inclui: toxinas de três dígitos (3FTx) (24%), fosfolipases A2 (PLA2) (16%), lectinas do tipo C (5%), entre outros. O banco permitiu não somente a identificação de possíveis toxinas, mas também de transcritos celulares, sendo a maioria envolvida nas funções fisiológicas de células da glândula de veneno. A maior parte dessas moléculas apresenta um envolvimento na expressão gênica e protéica, o que reflete uma alta especialização do tecido para a síntese de toxinas. A análise do transcriptoma de glândulas de veneno de M. corallinus possibilitou a identificação de alguns candidatos antigênicos para um anti-soro antielapídico alternativo. Cinco candidatos antigênicos foram selecionados por meio da análise do transcriptoma obtido: Atg1 (Grupo das neurotoxinas Homolog 8), Atg2 (Grupo das neurotoxinas Homolog 7/3/1), Atg3 (Outras neurotoxinas 1), Atg4 (Outras neurotoxinas 2) e Atg5 (fosfolipase do tipo A2). Avaliamos a viabilidade de imunização com o DNA desses candidatos. Para isso, os cinco grupos de antígenos foram clonados, primeiramente em pGEM-T e, posteriormente, em pSecTag2A, que é um vetor de expressão em células de mamíferos. As clonagens foram inicialmente testadas em células do tipo COS (transfecção transiente), entretanto não ficou clara a capacidade dessas células em expressar os antígenos. Para a análise da resposta imunológica da vacina de DNA, proteínas recombinantes produzidas em E. coli foram utilizadas para o coating de ELISA para detectar anticorpos presentes no soro primário proveniente da imunização com DNA. Os resultados mostraram que o soro dos animais imunizados foi capaz de reconhecer os antígenos recombinantes. Isso indica que a imunização por DNA em camundongos poderia ser uma boa alternativa em relação à imunização do veneno puro de serpente, que é custosa e muito depende da disponibilidade do veneno. Apesar da necessidade de testes complementares, esse é um resultado promissor, já que a produção de anticorpos pode ser alcançada por via de imunização intramuscular, mais prática para objetivos de produção. / Micrurus corallinus(coral snake) is a tropical forest snake belonging to the Elapidae Family, and is very common in Brazil. From the cDNA library of its venom glands, 1.438 Expressed Sequences Tags (ESTs) were generated and grouped into 611 clusters. This database contains the most expressed genes in the M. corallinus venom glands. The transcripts related to toxins represent approximately 46% of the total genes in this database. The toxin compound consists of: three finger toxins (24%), phospholipases A2 (PLA2s) (16%), type-C lectins (5%), among others. This database allowed not only the identification of possible toxins, but also the identification of cellular transcripts, most of which seems to be involved in physiological functions of venom gland cells. The majority of these molecules are involved in gene and protein expression, revealing the high level of specialization of the tissue for toxin synthesis. The analysis of the M. corallinus venom gland transcriptome allowed the identification of some antigenic candidates for an alternative antielapidic antiserum. Five antigenic candidates were selected after analysing the transcriptome: Atg1 (Homolog group 8), Atg2 (Homolog group 7/3/1), Atg3 (Other neurotoxins 1), Atg5 (A2-type phospholipase). These five antigenic groups were used for DNA immunization. Then they were first cloned in pGEM-T and, after, in pSecTag2A, which is an expression vector in mammal cells. The cloning was tested in COS-type cells (transient transfection), without signs of expression. To analyze the immunological response, recombinant proteins were produced in E. coli and used for ELISA coating to react with the primary serum deriving from the DNA immunization. The results showed that the serum from the immunized animals was able to recognize the recombinant antigens, indicating that the DNA immunization in mice could be a feasible alternative regarding the traditional immunization with crude snake venom, which is costly and heavily dependent on the availability of the venom. Regardless the need for additional tests, this is a promising result, because the antibody production can be achieved by intramuscular immunization, a more effective method when aiming for downstream production.