The impact of host immune status on the within-host and population dynamics of antigenic immune escape.

Antigenically evolving pathogens such as influenza viruses are difficult to control owing to their ability to evade host immunity by producing immune escape variants. Experimental studies have repeatedly demonstrated that viral immune escape variants emerge more often from immunized hosts than from naive hosts. This empirical relationship between host immune status and within-host immune escape is not fully understood theoretically, nor has its impact on antigenic evolution at the population level been evaluated. Here, we show that this relationship can be understood as a trade-off between the probability that a new antigenic variant is produced and the level of viraemia it reaches within a host. Scaling up this intra-host level trade-off to a simple population level model, we obtain a distribution for variant persistence times that is consistent with influenza A/H3N2 antigenic variant data. At the within-host level, our results show that target cell limitation, or a functional equivalent, provides a parsimonious explanation for how host immune status drives the generation of immune escape mutants. At the population level, our analysis also offers an alternative explanation for the observed tempo of antigenic evolution, namely that the production rate of immune escape variants is driven by the accumulation of herd immunity. Overall, our results suggest that disease control strategies should be further assessed by considering the impact that increased immunity--through vaccination--has on the production of new antigenic variants.

Suppression of conformational heterogeneity at a protein-protein interface.

Staphylococcal protein A (SpA) is an important virulence factor from Staphylococcus aureus responsible for the bacterium's evasion of the host immune system. SpA includes five small three-helix-bundle domains that can each bind with high affinity to many host proteins such as antibodies. The interaction between a SpA domain and the Fc fragment of IgG was partially elucidated previously in the crystal structure 1FC2. Although informative, the previous structure was not properly folded and left many substantial questions unanswered, such as a detailed description of the tertiary structure of SpA domains in complex with Fc and the structural changes that take place upon binding. Here we report the 2.3-Å structure of a fully folded SpA domain in complex with Fc. Our structure indicates that there are extensive structural rearrangements necessary for binding Fc, including a general reduction in SpA conformational heterogeneity, freezing out of polyrotameric interfacial residues, and displacement of a SpA side chain by an Fc side chain in a molecular-recognition pocket. Such a loss of conformational heterogeneity upon formation of the protein-protein interface may occur when SpA binds its multiple binding partners. Suppression of conformational heterogeneity may be an important structural paradigm in functionally plastic proteins.

Determination of Biomolecular Interdomain Motions using Nuclear Magnetic Resonance

Biological macromolecules can rearrange interdomain orientations when binding to various partners. Interdomain dynamics serve as a molecular mechanism to guide the transitions between orientations. However, our understanding of interdomain dynamics is limited because a useful description of interdomain motions requires an estimate of the probabilities of interdomain conformations, increasing complexity of the problem.

Staphylococcal protein A (SpA) has five tandem protein-binding domains and four interdomain linkers. The domains enable Staphylococcus aureus to evade the host immune system by binding to multiple host proteins including antibodies. Here, I present a study of the interdomain motions of two adjacent domains in SpA. NMR spin relaxation experiments identified a 6-residue flexible interdomain linker and interdomain motions. To quantify the anisotropy of the distribution of interdomain orientations, we measured residual dipolar couplings (RDCs) from the two domains with multiple alignments. The N-terminal domain was directly aligned by a lanthanide ion and not influenced by interdomain motions, so it acted as a reference frame to achieve motional decoupling. We also applied {\it de novo} methods to extract spatial dynamic information from RDCs and represent interdomain motions as a continuous distribution on the 3D rotational space. Significant anisotropy was observed in the distribution, indicating the motion populates some interdomain orientations more than others. Statistical thermodynamic analysis of the observed orientational distribution suggests that it is among the energetically most favorable orientational distributions for binding to antibodies. Thus, the affinity is enhanced by a pre-posed distribution of interdomain orientations while maintaining the flexibility required for function.

The protocol described above can be applied to other biological systems in general. Protein molecule calmodulin and RNA molecule trans-activation response element (TAR) also have intensive interdomain motions with relative small intradomain dynamics. Their interdomain motions were studied using our method based on published RDC data. Our results were consistent with literature results in general. The differences could be due to previous studies' use of physical models, which contain assumptions about potential energy and thus introduced non-experimental information into the interpretations.

Live Imaging of Host-Parasite Interactions in a Zebrafish Infection Model Reveals Cryptococcal Determinants of Virulence and Central Nervous System Invasion.

UNLABELLED: The human fungal pathogen Cryptococcus neoformans is capable of infecting a broad range of hosts, from invertebrates like amoebas and nematodes to standard vertebrate models such as mice and rabbits. Here we have taken advantage of a zebrafish model to investigate host-pathogen interactions of Cryptococcus with the zebrafish innate immune system, which shares a highly conserved framework with that of mammals. Through live-imaging observations and genetic knockdown, we establish that macrophages are the primary immune cells responsible for responding to and containing acute cryptococcal infections. By interrogating survival and cryptococcal burden following infection with a panel of Cryptococcus mutants, we find that virulence factors initially identified as important in causing disease in mice are also necessary for pathogenesis in zebrafish larvae. Live imaging of the cranial blood vessels of infected larvae reveals that C. neoformans is able to penetrate the zebrafish brain following intravenous infection. By studying a C. neoformans FNX1 gene mutant, we find that blood-brain barrier invasion is dependent on a known cryptococcal invasion-promoting pathway previously identified in a murine model of central nervous system invasion. The zebrafish-C. neoformans platform provides a visually and genetically accessible vertebrate model system for cryptococcal pathogenesis with many of the advantages of small invertebrates. This model is well suited for higher-throughput screening of mutants, mechanistic dissection of cryptococcal pathogenesis in live animals, and use in the evaluation of therapeutic agents. IMPORTANCE: Cryptococcus neoformans is an important opportunistic pathogen that is estimated to be responsible for more than 600,000 deaths worldwide annually. Existing mammalian models of cryptococcal pathogenesis are costly, and the analysis of important pathogenic processes such as meningitis is laborious and remains a challenge to visualize. Conversely, although invertebrate models of cryptococcal infection allow high-throughput assays, they fail to replicate the anatomical complexity found in vertebrates and, specifically, cryptococcal stages of disease. Here we have utilized larval zebrafish as a platform that overcomes many of these limitations. We demonstrate that the pathogenesis of C. neoformans infection in zebrafish involves factors identical to those in mammalian and invertebrate infections. We then utilize the live-imaging capacity of zebrafish larvae to follow the progression of cryptococcal infection in real time and establish a relevant model of the critical central nervous system infection phase of disease in a nonmammalian model.

Drivers of Dengue Within-Host Dynamics and Virulence Evolution

Dengue is an important vector-borne virus that infects on the order of 400 million individuals per year. Infection with one of the virus's four serotypes (denoted DENV-1 to 4) may be silent, result in symptomatic dengue 'breakbone' fever, or develop into the more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Extensive research has therefore focused on identifying factors that influence dengue infection outcomes. It has been well-documented through epidemiological studies that DHF is most likely to result from a secondary heterologous infection, and that individuals experiencing a DENV-2 or DENV-3 infection typically are more likely to present with more severe dengue disease than those individuals experiencing a DENV-1 or DENV-4 infection. However, a mechanistic understanding of how these risk factors affect disease outcomes, and further, how the virus's ability to evolve these mechanisms will affect disease severity patterns over time, is lacking. In the second chapter of my dissertation, I formulate mechanistic mathematical models of primary and secondary dengue infections that describe how the dengue virus interacts with the immune response and the results of this interaction on the risk of developing severe dengue disease. I show that only the innate immune response is needed to reproduce characteristic features of a primary infection whereas the adaptive immune response is needed to reproduce characteristic features of a secondary dengue infection. I then add to these models a quantitative measure of disease severity that assumes immunopathology, and analyze the effectiveness of virological indicators of disease severity. In the third chapter of my dissertation, I then statistically fit these mathematical models to viral load data of dengue patients to understand the mechanisms that drive variation in viral load. I specifically consider the roles that immune status, clinical disease manifestation, and serotype may play in explaining viral load variation observed across the patients. With this analysis, I show that there is statistical support for the theory of antibody dependent enhancement in the development of severe disease in secondary dengue infections and that there is statistical support for serotype-specific differences in viral infectivity rates, with infectivity rates of DENV-2 and DENV-3 exceeding those of DENV-1. In the fourth chapter of my dissertation, I integrate these within-host models with a vector-borne epidemiological model to understand the potential for virulence evolution in dengue. Critically, I show that dengue is expected to evolve towards intermediate virulence, and that the optimal virulence of the virus depends strongly on the number of serotypes that co-circulate. Together, these dissertation chapters show that dengue viral load dynamics provide insight into the within-host mechanisms driving differences in dengue disease patterns and that these mechanisms have important implications for dengue virulence evolution.

Transcriptomic Analysis of the Host Response and Innate Resilience to Enterotoxigenic Escherichia coli Infection in Humans.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a globally prevalent cause of diarrhea. Though usually self-limited, it can be severe and debilitating. Little is known about the host transcriptional response to infection. We report the first gene expression analysis of the human host response to experimental challenge with ETEC. METHODS: We challenged 30 healthy adults with an unattenuated ETEC strain, and collected serial blood samples shortly after inoculation and daily for 8 days. We performed gene expression analysis on whole peripheral blood RNA samples from subjects in whom severe symptoms developed (n = 6) and a subset of those who remained asymptomatic (n = 6) despite shedding. RESULTS: Compared with baseline, symptomatic subjects demonstrated significantly different expression of 406 genes highlighting increased immune response and decreased protein synthesis. Compared with asymptomatic subjects, symptomatic subjects differentially expressed 254 genes primarily associated with immune response. This comparison also revealed 29 genes differentially expressed between groups at baseline, suggesting innate resilience to infection. Drug repositioning analysis identified several drug classes with potential utility in augmenting immune response or mitigating symptoms. CONCLUSIONS: There are statistically significant and biologically plausible differences in host gene expression induced by ETEC infection. Differential baseline expression of some genes may indicate resilience to infection.