Mechanisms of GII.4 norovirus antigenic variation and evolution

Noroviruses infect an estimated 21 million people annually in the United States, resulting in ~70,000 hospitalizations and ~800 deaths. These viruses are easily transmitted among people in close proximity, including healthcare and educational settings, cruise ships, military environments, and restaurants. Norovirus symptoms include vomiting and diarrhea over a period of 24-72 hours, but life-threatening or chronic infections can develop in infants and very young children, the elderly and immunocompromised individuals. Unfortunately, there are no licensed norovirus therapeutics or vaccines currently available. One factor complicating vaccine and therapeutic design for noroviruses is antigenic variation in GII.4 norovirus, which account for over 70% of all outbreaks. Every 2-4 years the predominant circulating GII.4 strain is replaced by a new emergent strain. In predominant GII.4 strains, genetic changes are most prevalent in continuously evolving areas of the capsid P2 domain and correlate with antigenic changes. This suggests that GII.4 norovirus strain emergence is driven by changes in neutralization epitopes as a result of escaping herd immunity to the previous strain. Therefore, effectively designed therapeutics and vaccines must be broadly active or easily reformulated to account for the antigenic properties of emergent viruses. Our work aims to define the mechanisms that drive genetic changes leading to antigenic changes in GII.4 noroviruses. In Chapter 2, we use structure-guided approaches to map epitope A, the immunodominant GII.4 blockade (potential neutralizaton) epitope. Chapter 3 characterizes antigenic changes between two successive GII.4 strains, GII.4-2009 New Orleans and GII.4-2012 Sydney. In Chapter 4, we investigate the antigenic change that occurs within an individual chronically infected with norovirus over time, map the varying epitopes and compare the degree of change with population-wide changes. We also propose that broadly-blocking GII.4 human monoclonal antibody 71.4 is a potential treatment for chronic norovirus infection. Chapter 5 proposes a VLP-based vaccine platform that utilizes chimeric particles to induce a broadly-blocking immune response against multiple GII.4 strains. These GII.4 norovirus studies have allowed us to identify major determinants of antigenic change in GII.4 noroviruses that will allow for rapid diagnostic identification of new epidemic strains and direct the rational development of norovirus therapeutics and vaccines.Doctor of Philosoph

Low Birth Weight Inequities in Context: Racial Segregation, Neighborhood Factors, and Preconception Care.

Black women experience disproportionately higher rates of low birth weight, preterm delivery, and infant mortality. At least some amount of this disparity results from differences in exposure to detrimental social factors. Segregation is one social force shaping the distribution of power and resources and, therefore, women’s exposure to negative social environments. Using data from California Vital Records and the US Census, paper 1 evaluates the relationship between racial isolation and low birth weight for black, Hispanic, and Asian/Pacific Islander women. Isolation from whites predicts higher risk of low birth weight for black women specifically through an increased risk of small for gestational age births. For Hispanic women, isolation from whites predicts and increase in low birth weight for US-born women, but not foreign-born women, suggesting that ethnic enclave effects may not persist in this group. Finally, for Asian/Pacific Islander women, isolation predicts lower risk of low birth weight as a result of decreased risk of appropriate for gestational age preterm birth. Social capital is one mechanism through which segregation may impact birth outcomes. In Paper 2, analyses using data from California Vital Records and LAFANS suggest that higher levels of perceived social reciprocity are associated with lower risk of low birth weight for foreign-born women, but not for US-born women. Reciprocity predicts higher rates of low birth weight for Hispanic women, indicating that social reciprocity may be a unique stressor for US-born Hispanic women. For black women, social capital measures did not predict low birth weight outcomes. Paper 3 explores the potential for preconception care – a proposed public health intervention – to alleviate racial inequities in pregnancy outcomes. Preconception care may have positive impacts on individual women’s health. However, preconception care utilizes a clinical, individualistic, non-specific approach that does not address either the clinical or social drivers of racial inequities in birth outcomes, and should be reconsidered as a strategy for reducing these gaps. Taken together, these analyses provide insight into inequities in birth outcomes for women of color living in segregated neighborhoods, and evaluate policies aimed at mitigating this disparity.PHDHealth Services Organization & PolicyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/97930/1/mdebbink_1.pd

The State of Norovirus Vaccines

Noroviruses represent the most important cause of acute gastroenteritis worldwide; however, currently no licensed vaccine exists. Widespread vaccination that minimizes overall norovirus disease burden would benefit the entire population, but targeted vaccination of specific populations such as healthcare workers may further mitigate the risk of severe disease and death in vulnerable populations. While a few obstacles hinder the rapid development of efficacious vaccines, human trials for virus-like particle (VLP)-based vaccines show promise in both immune response and protection studies, with availability of vaccines being targeted over the next 5–10 years. Ongoing work including identification of important norovirus capsid antigenic sites, development of improved model systems, and continued studies in humans will allow improvement of future vaccines. In the meantime, a better understanding of norovirus disease course and transmission patterns can aid healthcare workers as they take steps to protect high-risk populations such as the elderly and immunocompromised individuals from chronic and severe disease

Coronavirus non-structural protein 16: Evasion, attenuation, and possible treatments

The recent emergence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV), nearly a decade after the Severe Acute Respiratory Syndrome (SARS) CoV, highlights the importance of understanding and developing therapeutic treatment for current and emergent CoVs. This manuscript explores the role of NSP16, a 2′O-methyl-transferase (2′O-MTase), in CoV infection and the host immune response. The review highlights conserved motifs, required interaction partners, as well as the attenuation of NSP16 mutants, and restoration of these mutants in specific immune knockouts. Importantly, the work also identifies a number of approaches to exploit this understanding for therapeutic treatment and the data clearly illustrate the importance of NSP16 2′O-MTase activity for CoV infection and pathogenesis

Immunogenetic Mechanisms Driving Norovirus GII.4 Antigenic Variation

Noroviruses are the principal cause of epidemic gastroenteritis worldwide with GII.4 strains accounting for 80% of infections. The major capsid protein of GII.4 strains is evolving rapidly, resulting in new epidemic strains with altered antigenic potentials. To test if antigenic drift may contribute to GII.4 persistence, human memory B cells were immortalized and the resulting human monoclonal antibodies (mAbs) characterized for reactivity to a panel of time-ordered GII.4 virus-like particles (VLPs). Reflecting the complex exposure history of the volunteer, human anti-GII.4 mAbs grouped into three VLP reactivity patterns; ancestral (1987–1997), contemporary (2004–2009), and broad (1987–2009). NVB 114 reacted exclusively to the earliest GII.4 VLPs by EIA and blockade. NVB 97 specifically bound and blocked only contemporary GII.4 VLPs, while NBV 111 and 43.9 exclusively reacted with and blocked variants of the GII.4.2006 Minerva strain. Three mAbs had broad GII.4 reactivity. Two, NVB 37.10 and 61.3, also detected other genogroup II VLPs by EIA but did not block any VLP interactions with carbohydrate ligands. NVB 71.4 cross-neutralized the panel of time-ordered GII.4 VLPs, as measured by VLP-carbohydrate blockade assays. Using mutant VLPs designed to alter predicted antigenic epitopes, two evolving, GII.4-specific, blockade epitopes were mapped. Amino acids 294–298 and 368–372 were required for binding NVB 114, 111 and 43.9 mAbs. Amino acids 393–395 were essential for binding NVB 97, supporting earlier correlations between antibody blockade escape and carbohydrate binding variation. These data inform VLP vaccine design, provide a strategy for expanding the cross-blockade potential of chimeric VLP vaccines, and identify an antibody with broadly neutralizing therapeutic potential for the treatment of human disease. Moreover, these data support the hypothesis that GII.4 norovirus evolution is heavily influenced by antigenic variation of neutralizing epitopes and consequently, antibody-driven receptor switching; thus, protective herd immunity is a driving force in norovirus molecular evolution

Emergence of New Pandemic GII.4 Sydney Norovirus Strain Correlates With Escape From Herd Immunity

Background. GII.4 noroviruses are a significant source of acute gastroenteritis worldwide, causing the majority of human norovirus outbreaks. Evolution of the GII.4 major capsid protein occurs rapidly, resulting in the emergence of new strains that produce successive waves of pandemic disease. A new pandemic isolate, GII.4 2012 Sydney, largely replaced previously circulating strains in late 2012. We compare the antigenic properties of GII.4 2012 Sydney with previously circulating strains. Methods. To determine whether GII.4-2012 Sydney is antigenically different from recently circulating strains GII.4-2006 Minerva and GII.4-2009 New Orleans in previously identified blockade epitopes, we compared reactivity and blockade profiles of GII.4-2006, GII.4-2009, and GII.4-2012 virus-like particles in surrogate neutralization/blockade assays using monoclonal antibodies and human polyclonal sera. Results. Using monoclonal antibodies that map to known blockade epitopes in GII.4-2006 and GII.4-2009 and human outbreak polyclonal sera, we demonstrate either complete loss or significantly reduced reactivity and blockade of GII.4.2012 compared to GII.4-2006 and GII.4-2009. Conclusions. GII.4-2012 Sydney is antigenically different from GII.4-2006 Minerva and GII.4-2009 New Orleans in at least 2 key blockade epitopes. Viral evolution in key potential neutralization epitopes likely allowed GII.4-2012 to escape from human herd immunity and emerge as the new predominant strai

Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus

ABSTRACT The evolutionary origins of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) are unknown. Current evidence suggests that insectivorous bats are likely to be the original source, as several 2c CoVs have been described from various species in the family Vespertilionidae . Here, we describe a MERS-like CoV identified from a Pipistrellus cf. hesperidus bat sampled in Uganda (strain PREDICT/PDF-2180), further supporting the hypothesis that bats are the evolutionary source of MERS-CoV. Phylogenetic analysis showed that PREDICT/PDF-2180 is closely related to MERS-CoV across much of its genome, consistent with a common ancestry; however, the spike protein was highly divergent (46% amino acid identity), suggesting that the two viruses may have different receptor binding properties. Indeed, several amino acid substitutions were identified in key binding residues that were predicted to block PREDICT/PDF-2180 from attaching to the MERS-CoV DPP4 receptor. To experimentally test this hypothesis, an infectious MERS-CoV clone expressing the PREDICT/PDF-2180 spike protein was generated. Recombinant viruses derived from the clone were replication competent but unable to spread and establish new infections in Vero cells or primary human airway epithelial cells. Our findings suggest that PREDICT/PDF-2180 is unlikely to pose a zoonotic threat. Recombination in the S1 subunit of the spike gene was identified as the primary mechanism driving variation in the spike phenotype and was likely one of the critical steps in the evolution and emergence of MERS-CoV in humans. IMPORTANCE Global surveillance efforts for undiscovered viruses are an important component of pandemic prevention initiatives. These surveys can be useful for finding novel viruses and for gaining insights into the ecological and evolutionary factors driving viral diversity; however, finding a viral sequence is not sufficient to determine whether it can infect people (i.e., poses a zoonotic threat). Here, we investigated the specific zoonotic risk of a MERS-like coronavirus (PREDICT/PDF-2180) identified in a bat from Uganda and showed that, despite being closely related to MERS-CoV, it is unlikely to pose a threat to humans. We suggest that this approach constitutes an appropriate strategy for beginning to determine the zoonotic potential of wildlife viruses. By showing that PREDICT/PDF-2180 does not infect cells that express the functional receptor for MERS-CoV, we further show that recombination was likely to be the critical step that allowed MERS to emerge in humans

Chimeric GII.4 Norovirus Virus-Like-Particle-Based Vaccines Induce Broadly Blocking Immune Responses

There is currently no licensed vaccine for noroviruses, and development is hindered, in part, by an incomplete understanding of the host adaptive immune response to these highly heterogeneous viruses and rapid GII.4 norovirus molecular evolution. Emergence of a new predominant GII.4 norovirus strain occurs every 2 to 4 years. To address the problem of GII.4 antigenic variation, we tested the hypothesis that chimeric virus-like particle (VLP)-based vaccine platforms, which incorporate antigenic determinants from multiple strains into a single genetic background, will elicit a broader immune response against contemporary and emergent strains. Here, we compare the immune response generated by chimeric VLPs to that of parental strains and a multivalent VLP cocktail. Results demonstrate that chimeric VLPs induce a more broadly cross-blocking immune response than single parental VLPs and a similar response to a multivalent GII.4 VLP cocktail. Furthermore, we show that incorporating epitope site A alone from one strain into the background of another is sufficient to induce a blockade response against the strain donating epitope site A. This suggests a mechanism by which population-wide surveillance of mutations in a single epitope could be used to evaluate antigenic changes in order to identify potential emergent strains and quickly reformulate vaccines against future epidemic strains as they emerge in human populations

Engineering Liver-detargeted AAV9 Vectors for Cardiac and Musculoskeletal Gene Transfer

We report the generation of a new class of adeno-associated virus serotype 9 (AAV9)-derived vectors displaying selective loss of liver tropism and demonstrating potential for cardiac and musculoskeletal gene transfer applications. Random mutagenesis of residues within a surface-exposed region of the major AAV9 capsid protein yielded a capsid library with mutations clustered at the icosahedral threefold symmetry axis. Using a combination of sequence analysis, structural models, and in vivo screening, we identified several functionally diverse AAV9 variants. The latter were classified into three functional subgroups, with respect to parental AAV9 displaying: (i) decreased transduction efficiency across multiple tissues; (ii) a selective decrease in liver transduction, or (iii) a similar transduction profile. Notably, variants 9.45 and 9.61 (subgroup II) displayed 10- to 25-fold lower gene transfer efficiency in liver, while transducing cardiac and skeletal muscle as efficiently as AAV9. These results were further corroborated by quantitation of vector genome copies and histological analysis of reporter (tdTomato) gene expression. The study highlights the feasibility of generating AAV vectors with selectively ablated tissue tropism, which when combined with other targeting strategies could allow sharply segregated gene expression. Liver-detargeted AAV9 variants described herein are excellent candidates for preclinical evaluation in animal models of cardiac and musculoskeletal disease