Serological analysis reveals an imbalanced IgG subclass composition associated with COVID-19 disease severity.

Coronavirus disease 2019 (COVID-19) is associated with a wide spectrum of disease presentation, ranging from asymptomatic infection to acute respiratory distress syndrome (ARDS). Paradoxically, a direct relationship has been suggested between COVID-19 disease severity and the levels of circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies, including virus-neutralizing titers. A serological analysis of 536 convalescent healthcare workers reveals that SARS-CoV-2-specific and virus-neutralizing antibody levels are elevated in individuals that experience severe disease. The severity-associated increase in SARS-CoV-2-specific antibody is dominated by immunoglobulin G (IgG), with an IgG subclass ratio skewed toward elevated receptor binding domain (RBD)- and S1-specific IgG3. In addition, individuals that experience severe disease show elevated SARS-CoV-2-specific antibody binding to the inflammatory receptor FcÉ£RIIIa. Based on these correlational studies, we propose that spike-specific IgG subclass utilization may contribute to COVID-19 disease severity through potent Fc-mediated effector functions. These results may have significant implications for SARS-CoV-2 vaccine design and convalescent plasma therapy.

Sensitization of Airway Epithelial Cells to Toxin-Induced Death by TNF Superfamily Cytokines.

The TNF superfamily of proinflammatory and proapoptotic cytokines influence tissue-wide responses to molecular insults such as small molecules, toxins, and viral infections that perturb cellular homeostasis at the level of DNA replication, transcription, and translation. In the context of acute lung injury, for example, TNF superfamily members like TNF-α and TRAIL can severely exacerbate disease pathophysiology. This chapter describes a systematic approach to optimization of mammalian cell viability assays and transcriptional profiling through nCounter® Technology to permit a detailed examination of how TNF-α and TRAIL modulate programmed cell death pathways in concert with ricin toxin, a ribosome-inactivating protein (RIP) and a potent inducer of acute respiratory distress. We compare two widely used luciferase- and colorimetric-based cell viability assays and provide optimization protocols for adherent and non-adherent cell lines. We provide a computational workflow to facilitate downstream analysis of datasets generated from nCounter® gene expression panels. While combined treatment with ricin toxin and TRAIL serves as the exemplar, the methodologies are applicable to any TNF superfamily member in combination with any biological agent of interest.

Endpoint and epitope-specific antibody responses as correlates of vaccine-mediated protection of mice against ricin toxin.

The successful licensure of vaccines for biodefense is contingent upon the availability of well-established correlates of protection (CoP) in at least two animal species that can be applied to humans, without the need to assess efficacy in the clinic. In this report we describe a multivariate model that combines pre-challenge serum antibody endpoint titers (EPT) and values derived from an epitope profiling immune-competition capture (EPICC) assay as a predictor in mice of vaccine-mediated immunity against ricin toxin (RT), a Category B biothreat. EPICC is a modified competition ELISA in which serum samples from vaccinated mice were assessed for their ability to inhibit the capture of soluble, biotinylated (b)-RT by a panel of immobilized monoclonal antibodies (mAbs) directed against four immunodominant toxin-neutralizing regions on the enzymatic A chain (RTA) of RT. In a test cohort of mice (n = 40) vaccinated with suboptimal doses of the RTA subunit vaccine, RiVax®, we identified two mAbs, PB10 and SyH7, which had EPICC inhibition values in pre-challenge serum samples that correlated with survival following a challenge with 5 × LD50 of RT administered by intraperitoneal (IP) injection. Analysis of a larger cohort of mice (n = 645) revealed that a multivariate model combining endpoint titers and EPICC values for PB10 and SyH7 as predictive variables had significantly higher statistical power than any one of the independent variables alone. Establishing the correlates of vaccine-mediated protection in mice represents an important steppingstone in the development of RiVax® as a medical countermeasure under the United States Food and Drug Administration's "Animal Rule."

Sensitivity of Kupffer cells and liver sinusoidal endothelial cells to ricin toxin and ricin toxin-Ab complexes.

Ricin toxin is a plant-derived, ribosome-inactivating protein that is rapidly cleared from circulation by Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs)-with fatal consequences. Rather than being inactivated, ricin evades normal degradative pathways and kills both KCs and LSECs with remarkable efficiency. Uptake of ricin by these 2 specialized cell types in the liver occurs by 2 parallel routes: a "lactose-sensitive" pathway mediated by ricin's galactose/N-acetylgalactosamine-specific lectin subunit (RTB), and a "mannose-sensitive" pathway mediated by the mannose receptor (MR; CD206) or other C-type lectins capable of recognizing the mannose-side chains displayed on ricin's A (RTA) and B subunits. In this report, we investigated the capacity of a collection of ricin-specific mouse MAb and camelid single-domain (VH H) antibodies to protect KCs and LSECs from ricin-induced killing. In the case of KCs, individual MAbs against RTA or RTB afforded near complete protection against ricin in ex vivo and in vivo challenge studies. In contrast, individual MAbs or VH Hs afforded little (<40%) or even no protection to LSECs against ricin-induced death. Complete protection of LSECs was only achieved with MAb or VH H cocktails, with the most effective mixtures targeting RTA and RTB simultaneously. Although the exact mechanisms of protection of LSECs remain unknown, evidence indicates that the Ab cocktails exert their effects on the mannose-sensitive uptake pathway without the need for Fcγ receptor involvement. In addition to advancing our understanding of how toxins and small immune complexes are processed by KCs and LSECs, our study has important implications for the development of Ab-based therapies designed to prevent or treat ricin exposure should the toxin be weaponized.

Rescue of rhesus macaques from the lethality of aerosolized ricin toxin.

Ricin toxin (RT) ranks at the top of the list of bioweapons of concern to civilian and military personnel alike, due to its high potential for morbidity and mortality after inhalation. In nonhuman primates, aerosolized ricin triggers severe acute respiratory distress characterized by perivascular and alveolar edema, neutrophilic infiltration, and severe necrotizing bronchiolitis and alveolitis. There are currently no approved countermeasures for ricin intoxication. Here, we report the therapeutic potential of a humanized mAb against an immunodominant epitope on ricin's enzymatic A chain (RTA). Rhesus macaques that received i.v. huPB10 4 hours after a lethal dose of ricin aerosol exposure survived toxin challenge, whereas control animals succumbed to ricin intoxication within 30 hours. Antibody intervention at 12 hours resulted in the survival of 1 of 5 monkeys. Changes in proinflammatory cytokine, chemokine, and growth factor profiles in bronchial alveolar lavage fluids before and after toxin challenge successfully clustered animals by treatment group and survival, indicating a relationship between local tissue damage and experimental outcome. This study represents the first demonstration, to our knowledge, in nonhuman primates that the lethal effects of inhalational ricin exposure can be negated by a drug candidate, and it opens up a path forward for product development.

Differential Effects of Temperature and Mosquito Genetics Determine Transmissibility of Arboviruses by Aedes aegypti in Argentina.

Aedes aegypti (L.) (Diptera: Culicidae) have a global distribution and are the primary vector of a number of mosquito-borne viruses responsible for epidemics throughout the Americas. As in much of South America, the threat from pathogens including dengue virus (DENV; Flaviviridae, Flavivirus) and chikungunya virus (CHIKV; Togaviridae, Alphavirus) has increased in Argentina in recent years. The complexity of transmission cycles makes predicting the occurrence and intensity of arbovirus outbreaks difficult. To gain a better understanding of the risk of DENV and CHIKV in Argentina and the factors influencing this risk, we evaluated the role of population and temperature in the vector competence and vectorial capacity (VC) of Ae. aegypti from geographically and ecologically distinct locations. Our results demonstrate that intrinsic and extrinsic factors including mosquito population, viral species, and temperature significantly influence both vector competence and overall VC of Ae. aegypti in Argentina, yet also that the magnitude of these influences is highly variable. Specifically, results suggest that CHIKV competence is more dependent on mosquito genetics than is DENV competence, whereas temperature has a greater effect on DENV transmission. In addition, although there is an overall positive correlation between temperature and competence for both viruses, there are exceptions to this for individual virus-population combinations. Together, these data establish large variability in VC for these pathogens among distinct Ae. aegypti populations in Argentina and demonstrate that accurate assessment of arbovirus risk will require nuanced models that fully consider the complexity of interactions between virus, temperature, mosquito genetics, and hosts.

Effects of Zika Virus Strain and Aedes Mosquito Species on Vector Competence.

In the Western Hemisphere, Zika virus is thought to be transmitted primarily by Aedes aegypti mosquitoes. To determine the extent to which Ae. albopictus mosquitoes from the United States are capable of transmitting Zika virus and the influence of virus dose, virus strain, and mosquito species on vector competence, we evaluated multiple doses of representative Zika virus strains in Ae. aegypti and Ae. albopictus mosquitoes. Virus preparation (fresh vs. frozen) significantly affected virus infectivity in mosquitoes. We calculated 50% infectious doses to be 6.1-7.5 log 10 PFU/mL; minimum infective dose was 4.2 log 10 PFU/mL. Ae. albopictus mosquitoes were more susceptible to infection than Ae. aegypti mosquitoes, but transmission efficiency was higher for Ae. aegypti mosquitoes, indicating a transmission barrier in Ae. albopictus mosquitoes. Results suggest that, although Zika virus transmission is relatively inefficient overall and dependent on virus strain and mosquito species, Ae. albopictus mosquitoes could become major vectors in the Americas.

High levels of local inter- and intra-host genetic variation of West Nile virus and evidence of fine-scale evolutionary pressures.

West Nile virus (WNV; Flaviviridae, Flavivirus) has been endemic in New York State (NYS) since its 1999 introduction, yet prevalence in Culex mosquitoes varies substantially over small spatial and temporal scales. It is unclear if viral genetics plays a role in this variability, as genetic and phenotypic characterization on local scales has generally been lacking. In addition, intrahost diversity of circulating strains have not been fully characterized despite the documented role of minority variants in viral fitness and virulence. In an effort to characterize WNV variability within epidemiologically relevant scales, we performed phylogenetic analyses on NYS isolates from 1999 to 2012. In addition, we performed full-genome, deep-sequencing and genetic analyses on 15 WNV strains isolated in 2012 from Cx. pipiens in an endemic focus of Suffolk County, NY. Our results indicate continued evolution and seasonal maintenance in NYS, yet also widespread mixing and high levels of genetic diversity within geographic foci and individual seasons. Well supported local clusters with shared amino acid differences were identified and suggest local evolutionary pressures and the potential for phenotypic variability. Intrahost diversity of focal isolates was also high, with polymorphism at levels >1.0% identified in approximately 10% of the WNV genome. Although most minority mutations were unique, mutational hotspots shared among local isolates were identified, particularly in C, NS1 and NS2A genes. The most polymorphic region, positions 3198-3388 of the NS1 gene, was comprised predominately of non-synonymous mutations, suggesting a selective advantage for amino acid diversity in this region.

Vertical Transmission of Zika Virus by Aedes aegypti and Ae. albopictus Mosquitoes.

To determine the potential role of vertical transmission in Zika virus expansion, we evaluated larval pools of perorally infected Aedes aegypti and Ae. albopictus adult female mosquitoes; ≈1/84 larvae tested were Zika virus-positive; and rates varied among mosquito populations. Thus, vertical transmission may play a role in Zika virus spread and maintenance.

Increased replicative fitness of a dengue virus 2 clade in native mosquitoes: potential contribution to a clade replacement event in Nicaragua.

UNLABELLED: The four dengue virus (DENV) serotypes (DENV serotype 1 [DENV-1] to DENV-4) are transmitted by Aedes aegypti and A. albopictus mosquitoes, causing up to 390 million DENV infections worldwide each year. We previously reported a clade replacement of the DENV-2 Asian-American genotype NI-1 clade by the NI-2B clade in Managua, Nicaragua. Here, we describe our studies of the replicative ability of NI-1 and NI-2B viruses in an A. aegypti cell line (Aag2) and A. aegypti mosquitoes reared from eggs collected in Managua. In coinfection experiments, several different pairs of NI-1 and NI-2B clinical isolates were used to infect Aag2 cells or blood-fed A. aegypti mosquitoes. Results consistently showed a significant replicative advantage of NI-2B over NI-1 viruses early after infection in vitro, and in mosquitoes, NI-2B viruses attained a higher replicative index than NI-1 isolates 3 to 7 days postinfection (dpi). At 7 dpi, NI-2B viruses displayed a significantly higher replicative index in legs and salivary glands; however, this advantage was lost by 14 and 21 dpi. We also found that the percentage of mosquitoes in which NI-2B viruses were dominant was significantly higher than that in which NI-1 viruses were dominant on day 7 but not at later time points. Taken together, these data demonstrate that clade NI-2B holds a replicative advantage over clade NI-1 early in infection that wanes at later time points. This early fitness advantage of NI-2B viruses over NI-1 viruses in the native vector, A. aegypti, suggests a shorter extrinsic incubation period for NI-2B viruses, which could have contributed to the clade replacement event in Managua. IMPORTANCE: Dengue virus (DENV), one of the most medically important arthropod-borne viruses, is transmitted to humans by Aedes aegypti and A. albopictus mosquitoes in tropical and subtropical regions worldwide. Dengue epidemics continue to increase in frequency, geographic range, and severity and are a major public health concern. This is due to globalization, unplanned urbanization, and climate change, as well as host genetics and immune responses and viral genetic changes. DENV consists of four serotypes, in turn composed of genotypes and genetically distinct clades. What drives the frequent replacement of a previously circulating DENV clade by another is unclear. Here, we investigate the replicative fitness of two clades of DENV serotype 2 in Aedes aegypti cells and mosquitoes collected from the region where the viruses circulated and conclude that increased replicative fitness could have contributed to a DENV clade replacement event in Nicaragua. These findings provide insight into vector-driven evolution of DENV epidemics.

The evolution of virulence of West Nile virus in a mosquito vector: implications for arbovirus adaptation and evolution.

BACKGROUND: Virulence is often coupled with replicative fitness of viruses in vertebrate systems, yet the relationship between virulence and fitness of arthropod-borne viruses (arboviruses) in invertebrates has not been evaluated. Although the interactions between vector-borne pathogens and their invertebrate hosts have been characterized as being largely benign, some costs of arbovirus exposure have been identified for mosquitoes. The extent to which these costs may be strain-specific and the subsequent consequences of these interactions on vector and virus evolution has not been adequately explored. RESULTS: Using West Nile virus (WNV) and Culex pipiens mosquitoes, we tested the hypothesis that intrahost fitness is correlated with virulence in mosquitoes by evaluating life history traits following exposure to either non-infectious bloodmeals or bloodmeals containing wildtype (WNV WT) or the high fitness, mosquito-adapted strain, WNV MP20 derived from WNV WT. Our results demonstrate strain-specific effects on mosquito survival, fecundity, and blood feeding behavior. Specifically, both resistance to and infection with WNV MP20, but not WNV WT, decreased survival of Cx. pipiens and altered fecundity and bloodfeeding such that early egg output was enhanced at a later cost. CONCLUSIONS: As predicted by the trade-off hypothesis of virulence, costs of infection with WNV MP20 in terms of survival were directly correlated to viral load, yet resistance to infection with this virulent strain was equally costly. Taken together, these results demonstrate that WNV MP20 infection decreases the transmission potential of Cx. pipiens populations despite the increased intrahost fitness of this strain, indicating that a virulence-transmission trade-off in invertebrates could contribute significantly to the adaptive and evolutionary constraint of arboviruses.

Cooperative interactions in the West Nile virus mutant swarm.

BACKGROUND: RNA viruses including arthropod-borne viruses (arboviruses) exist as highly genetically diverse mutant swarms within individual hosts. A more complete understanding of the phenotypic correlates of these diverse swarms is needed in order to equate RNA swarm breadth and composition to specific adaptive and evolutionary outcomes. RESULTS: Here, we determined clonal fitness landscapes of mosquito cell-adapted West Nile virus (WNV) and assessed how altering the capacity for interactions among variants affects mutant swarm dynamics and swarm fitness. Our results demonstrate that although there is significant mutational robustness in the WNV swarm, genetic diversity also corresponds to substantial phenotypic diversity in terms of relative fitness in vitro. In addition, our data demonstrate that increasing levels of co-infection can lead to widespread strain complementation, which acts to maintain high levels of phenotypic and genetic diversity and potentially slow selection for individual variants. Lastly, we show that cooperative interactions may lead to swarm fitness levels which exceed the relative fitness levels of any individual genotype. CONCLUSIONS: These studies demonstrate the profound effects variant interactions can have on arbovirus evolution and adaptation, and provide a baseline by which to study the impact of this phenomenon in natural systems.

Quantification of intrahost bottlenecks of West Nile virus in Culex pipiens mosquitoes using an artificial mutant swarm.

Mosquito-borne viruses are predominantly RNA viruses which exist within hosts as diverse mutant swarms. Defining the way in which stochastic forces within mosquito vectors shape these swarms is critical to advancing our understanding of the evolutionary and adaptive potential of these pathogens. There are multiple barriers within a mosquito which a viral swarm must traverse in order to ultimately be transmitted. Here, using artificial mutant swarms composed of neutral variants of West Nile virus (WNV), we tracked changes to swarm breadth over time and space in Culex pipiens mosquitoes. Our results demonstrate that all variants have the potential to survive intrahost bottlenecks, yet mean swarm breadth decreases during both midgut infection and transmission when starting populations contain higher levels of minority variants. In addition, WNV swarms are subject to temporal sweeps which act to significantly decrease intrahost diversity over time. Taken together, these data demonstrate the profound effects that stochastic forces can have in shaping arboviral mutant swarms.

Temporal and spatial alterations in mutant swarm size of St. Louis encephalitis virus in mosquito hosts.

St. Louis encephalitis virus (SLEV; Flaviviridae; Flavivirus) is a member of the Japanese encephalitis serocomplex and a close relative of West Nile virus (WNV). Although SLEV remains endemic to the US, both levels of activity and geographical dispersal are relatively constrained when compared to the widespread distribution of WNV. In recent years, WNV appears to have displaced SLEV in California, yet both viruses currently coexist in Texas and several other states. It has become clear that viral swarm characterization is required if we are to fully evaluate the relationship between viral genomes, viral evolution, and epidemiology. Mutant swarm size and composition may be particularly important for arboviruses, which require replication not only in diverse tissues but also divergent hosts. In order to evaluate temporal, spatial, and host-specific patterns in the SLEV mutant swarm, we determined the size, composition, and phylogeny of the intrahost swarm within primary mosquito isolates from both Texas and California. Results indicate a general trend of decreasing intrahost diversity over time in both locations, with recent isolates being highly genetically homogeneous. Additionally, phylogenic analyses provide detailed information on the relatedness of minority variants both within and among strains and demonstrate how both geographic isolation and seasonal maintenance have shaped the viral swarm. Overall, these data generally provide insight into how time, space, and unique transmission cycles influence the SLEV mutant swarm and how understanding these processes can ultimately lead to a better understanding of arbovirus evolution and epidemiology.