History and Toxinology of Palytoxins.

Palytoxins are a group of highly potent and structurally complex marine toxins that rank among some of the most toxic substances known to science. Palytoxins are naturally synthesized by a variety of marine organisms, including Palythoa zoanthids, Ostreopsis dinoflagellates, and Trichodesmium cyanobacteria, and are widely distributed in tropical and temperate regions where they can bioaccumulate in marine life. The evolution of research on palytoxins has been an intricate exchange between interdisciplinary fields, drawing insights from chemistry, biology, medicine, and environmental science in efforts to better understand and mitigate the health risks associated with this family of toxins. In this review, we begin with a brief history covering the discovery of this group of toxins and the events that led to its isolation. We then focus on the chemical structure of these compounds and their proposed mechanism of action. Finally, we review in vitro, ex vivo, and in vivo studies related to their toxicity, with the aim to provide a broad overview of the current knowledge on palytoxin toxinology.

Engineered antibodies targeted to bacterial surface integrate effector functions with toxin neutralization to provide superior efficacy against bacterial infections.

Anti-bacterial monoclonal antibody (mAb) therapies either rely on toxin neutralization or opsonophagocytic killing (OPK). Toxin neutralization protects the host from toxin-induced damage, while leaving the organism intact. OPK inducing antibodies clear the bacteria but leave the released toxins unencountered. Infection site targeted anti-toxin antibodies (ISTAbs) that we report here addresses this binary paradigm by combining both functionalities into a single molecule. ISTAbs consist of cell wall targeting (CWT) domains of bacteriophage endolysins fused to toxin neutralizing mAbs (IgG). CWT governs specific binding to the surface of bacteria while the IgG variable domain neutralizes the toxins as they are released. The complex is then cleared by phagocytic cells. As proof of concept, we generated several ISTAb prototypes targeting major toxins from two Gram-positive spore forming pathogens that have a high clinical significance; Clostridium difficile , causative agent of the most common hospital-acquired infection, and Bacillus anthracis , a Category A select agent pathogen. Both groups of ISTAbs exhibited potent toxin neutralization, binding to their respective bacterial cells, and induction of opsonophagocytosis. In mice infected with B. anthracis , ISTAbs exhibit significantly higher efficacy than parental IgG in both pre- and post-challenge models. Furthermore, ISTAbs fully protected against B. anthracis infection in a nonhuman primate (NHP) aerosol challenge model. These findings establish that as a platform technology, ISTAbs are broadly applicable for therapeutic intervention against several toxigenic bacterial pathogens.

Current vaccine strategies and novel approaches to combatting Francisella infection.

Tularemia is caused by subspecies of Francisella tularensis and can manifest in a variety of disease states, with the pneumonic presentation resulting in the greatest mortality. Despite decades of research, there are no approved vaccines against F. tularensis in the United States. Traditional vaccination strategies, such as live-attenuated or subunit vaccines, are not favorable due to inadequate protection or safety concerns. Because of this, novel vaccination strategies are needed to combat tularemia. Here we discuss the current state of and challenges to the tularemia vaccine field and suggest novel vaccine approaches going forward that might be better suited for protecting against F. tularensis infection.

MVA-based vaccines are protective against lethal eastern equine encephalitis virus aerosol challenge in cynomolgus macaques.

MVA-based monovalent eastern equine encephalitis virus (MVA-BN-EEEV) and multivalent western, eastern, and Venezuelan equine encephalitis virus (MVA-BN-WEV) vaccines were evaluated in the cynomolgus macaque aerosol model of EEEV infection. Macaques vaccinated with two doses of 5 × 108 infectious units of the MVA-BN-EEEV or MVA-BN-WEV vaccine by the intramuscular route rapidly developed robust levels of neutralizing antibodies to EEEV that persisted at high levels until challenge at day 84 via small particle aerosol delivery with a target inhaled dose of 107 PFU of EEEV FL93-939. Robust protection was observed, with 7/8 animals receiving MVA-BN-EEEV and 100% (8/8) animals receiving MVA-BN-WEV surviving while only 2/8 mock vaccinated controls survived lethal challenge. Complete protection from viremia was afforded by both vaccines, with near complete protection from vRNA loads in tissues and any pathologic evidence of central nervous system damage. Overall, the results indicate both vaccines are effective in eliciting an immune response that is consistent with protection from aerosolized EEEV-induced disease.

Mid-titer human convalescent plasma administration results in suboptimal prophylaxis against SARS-CoV-2 infection in rhesus macaques.

Introduction: ARS-CoV-2 is a respiratory pathogen currently causing a worldwide pandemic, with resulting pathology of differing severity in humans, from mild illness to severe disease and death. The rhesus macaque model of COVID-19 was utilized to evaluate the added benefit of prophylactic administration of human post-SARS-CoV-2 infection convalescent plasma (CP) on disease progression and severity. Methods: A pharmacokinetic (PK) study using CP in rhesus monkeys preceded the challenge study and revealed the optimal time of tissue distribution for maximal effect. Thereafter, CP was administered prophylactically three days prior to mucosal SARS-CoV-2 viral challenge. Results: Results show similar viral kinetics in mucosal sites over the course of infection independent of administration of CP or normal plasma, or historic controls with no plasma. No changes were noted upon necropsy via histopathology, although there were differences in levels of vRNA in tissues, with both normal and CP seemingly blunting viral loads. Discussion: Results indicate that prophylactic administration with mid-titer CP is not effective in reducing disease severity of SARS-CoV-2 infection in the rhesus COVID-19 disease model.

Elevated soluble urokinase plasminogen activator receptor is associated with renal dysfunction in a Chlorocebus atheiops COVID-19 model.

Increases of soluble urokinase plasminogen activator receptor (suPAR) were measured in both urine and plasma of a Chlorocebus aethiops (African green monkey; AGM) mucosal infected with SARS-CoV-2. The data indicate that elevated suPAR may be associated with renal dysfunction and pathology in the context of COVID-19.

Serum antibody profiling identifies vaccine-induced correlates of protection against aerosolized ricin toxin in rhesus macaques.

Inhalation of the biothreat agent, ricin toxin (RT), provokes a localized inflammatory response associated with pulmonary congestion, edema, neutrophil infiltration, and severe acute respiratory distress. The extreme toxicity of RT is the result of the toxin's B chain (RTB) promoting rapid uptake into alveolar macrophages and lung epithelial cells, coupled with the A chain's (RTA) potent ribosome-inactivating properties. We previously reported that intramuscular vaccination of rhesus macaques with a lyophilized, alum-adsorbed recombinant RTA subunit vaccine (RiVax®) was sufficient to confer protection against a lethal dose of aerosolized RT. That study implicated RT-specific serum IgG, toxin-neutralizing activity (TNA), and epitope-specific responses as being associated with immunity. However, it was not possible to define actual correlates of protection (COP) because all vaccinated animals survived the RT challenge. We addressed the issue of COP in the current study, by vaccinating groups of rhesus macaques with RiVax® following the previously determined protective regimen (100 µg on study days 0, 30 and 60) or one of two anticipated suboptimal regimens (100 µg on study days 30 and 60; 35 µg on study days 0, 30, and 60). Two unvaccinated animals served as controls. The animals were challenged with ~5 × LD50s of aerosolized RT on study day 110. We report that all vaccinated animals seroconverted prior to RT challenge, with the majority also having measurable TNA, although neither antibody levels nor TNA reached statistical significance with regard to a correlation with protection. By contrast, survival correlated with pre-challenge, epitope-specific serum IgG levels, derived from a competitive sandwich ELISA using a panel of toxin-neutralizing monoclonal antibodies directed against distinct epitopes on RiVax®. The identification of a species-neutral, competitive ELISA that correlates with vaccine-induced protection against RT in nonhuman represents an important advance in the development of medical countermeasures (MCM) against a persistent biothreat.

The dynamics of γδ T cell responses in nonhuman primates during SARS-CoV-2 infection.

Although most SARS-CoV-2 infections are mild, some patients develop systemic inflammation and progress to acute respiratory distress syndrome (ARDS). However, the cellular mechanisms underlying this spectrum of disease remain unclear. γδT cells are T lymphocyte subsets that have key roles in systemic and mucosal immune responses during infection and inflammation. Here we show that peripheral γδT cells are rapidly activated following aerosol or intra-tracheal/intra-nasal (IT/IN) SARS-CoV-2 infection in nonhuman primates. Our results demonstrate a rapid expansion of Vδ1 γδT cells at day1 that correlate significantly with lung viral loads during the first week of infection. Furthermore, increase in levels of CCR6 and Granzyme B expression in Vδ1 T cells during viral clearance imply a role in innate-like epithelial barrier-protective and cytotoxic functions. Importantly, the early activation and mobilization of circulating HLA-DR+CXCR3+ γδT cells along with significant correlations of Vδ1 T cells with IL-1Ra and SCF levels in bronchoalveolar lavage suggest a novel role for Vδ1 T cells in regulating lung inflammation during aerosol SARS-CoV-2 infection. A deeper understanding of the immunoregulatory functions of MHC-unrestricted Vδ1 T cells in lungs during early SARS-CoV-2 infection is particularly important in the wake of emerging new variants with increased transmissibility and immune evasion potential.

SARS-CoV-2 Epitopes following Infection and Vaccination Overlap Known Neutralizing Antibody Sites.

Identification of epitopes targeted following virus infection or vaccination can guide vaccine design and development of therapeutic interventions targeting functional sites, but can be laborious. Herein, we employed peptide microarrays to map linear peptide epitopes (LPEs) recognized following SARS-CoV-2 infection and vaccination. LPEs detected by nonhuman primate (NHP) and patient IgMs after SARS-CoV-2 infection extensively overlapped, localized to functionally important virus regions, and aligned with reported neutralizing antibody binding sites. Similar LPE overlap occurred after infection and vaccination, with LPE clusters specific to each stimulus, where strong and conserved LPEs mapping to sites known or likely to inhibit spike protein function. Vaccine-specific LPEs tended to map to sites known or likely to be affected by structural changes induced by the proline substitutions in the mRNA vaccine's S protein. Mapping LPEs to regions of known functional importance in this manner may accelerate vaccine evaluation and discovery of targets for site-specific therapeutic interventions.

Exposure modality influences viral kinetics but not respiratory outcome of COVID-19 in multiple nonhuman primate species.

The novel coronavirus SARS-CoV-2 emerged in late 2019, rapidly reached pandemic status, and has maintained global ubiquity through the emergence of variants of concern. Efforts to develop animal models have mostly fallen short of recapitulating severe disease, diminishing their utility for research focusing on severe disease pathogenesis and life-saving medical countermeasures. We tested whether route of experimental infection substantially changes COVID-19 disease characteristics in two species of nonhuman primates (Macaca mulatta; rhesus macaques; RM, Chlorocebus atheiops; African green monkeys; AGM). Species-specific cohorts were experimentally infected with SARS-CoV-2 by either direct mucosal (intratracheal + intranasal) instillation or small particle aerosol in route-discrete subcohorts. Both species demonstrated analogous viral loads in all compartments by either exposure route although the magnitude and duration of viral loading was marginally greater in AGMs than RMs. Clinical onset was nearly immediate (+1dpi) in the mucosal exposure cohort whereas clinical signs and cytokine responses in aerosol exposure animals began +7dpi. Pathologies conserved in both species and both exposure modalities include pulmonary myeloid cell influx, development of pleuritis, and extended lack of regenerative capacity in the pulmonary compartment. Demonstration of conserved pulmonary pathology regardless of species and exposure route expands our understanding of how SARS-CoV-2 infection may lead to ARDS and/or functional lung damage and demonstrates the near clinical response of the nonhuman primate model for anti-fibrotic therapeutic evaluation studies.

Reversion of Ebolavirus Disease from a Single Intramuscular Injection of a Pan-Ebolavirus Immunotherapeutic.

Intravenous (IV) administration of antiviral monoclonal antibodies (mAbs) can be challenging, particularly during an ongoing epidemic, due to the considerable resources required for performing infusions. An ebolavirus therapeutic administered via intramuscular (IM) injection would reduce the burdens associated with IV infusion and allow rapid treatment of exposed individuals during an outbreak. Here, we demonstrate how MBP134, a cocktail of two pan-ebolavirus mAbs, reverses the course of Sudan ebolavirus disease (Gulu variant) with a single IV or IM dose in non-human primates (NHPs) as late as five days post-exposure. We also investigate the utility of adding half-life extension mutations to the MBP134 mAbs, ultimately creating a half-life extended cocktail designated MBP431. When delivered as a post-exposure prophylactic or therapeutic, a single IM dose of MBP431 offered complete or significant protection in NHPs challenged with Zaire ebolavirus. In conjunction with previous studies, these results support the use of MBP431 as a rapidly deployable IM medical countermeasure against every known species of ebolavirus.

Breakthrough Gastrointestinal COVID-19 and Intrahost Evolution Consequent to Combination Monoclonal Antibody Prophylaxis.

Breakthrough gastrointestinal COVID-19 was observed after experimental SARS-CoV-2 upper mucosal infection in a rhesus macaque undergoing low-dose monoclonal antibody prophylaxis. High levels of viral RNA were detected in intestinal sites contrasting with minimal viral replication in upper respiratory mucosa. Sequencing of virus recovered from tissue in 3 gastrointestinal sites and rectal swab revealed loss of furin cleavage site deletions present in the inoculating virus stock and 2 amino acid changes in spike that were detected in 2 colon sites but not elsewhere, suggesting compartmentalized replication and intestinal viral evolution. This suggests suboptimal antiviral therapies promote viral sequestration in these anatomies.

Neuropathology and virus in brain of SARS-CoV-2 infected non-human primates.

Neurological manifestations are a significant complication of coronavirus disease (COVID-19), but underlying mechanisms aren't well understood. The development of animal models that recapitulate the neuropathological findings of autopsied brain tissue from patients who died from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are critical for elucidating the neuropathogenesis of infection and disease. Here, we show neuroinflammation, microhemorrhages, brain hypoxia, and neuropathology that is consistent with hypoxic-ischemic injury in SARS-CoV-2 infected non-human primates (NHPs), including evidence of neuron degeneration and apoptosis. Importantly, this is seen among infected animals that do not develop severe respiratory disease, which may provide insight into neurological symptoms associated with "long COVID". Sparse virus is detected in brain endothelial cells but does not associate with the severity of central nervous system (CNS) injury. We anticipate our findings will advance our current understanding of the neuropathogenesis of SARS-CoV-2 infection and demonstrate SARS-CoV-2 infected NHPs are a highly relevant animal model for investigating COVID-19 neuropathogenesis among human subjects.

Response to Hypoxia and the Ensuing Dysregulation of Inflammation Impacts Mycobacterium tuberculosis Pathogenicity.

Rationale: Different Mycobacterium tuberculosis (Mtb) strains exhibit variable degrees of virulence in humans and animal models. Differing stress response strategies used by different strains of Mtb could influence virulence. Objectives: We compared the virulence of two strains of Mtb with use in animal model research: CDC1551 and Erdman. Methods: Rhesus macaques, which develop human-like tuberculosis attributes and pathology, were infected with a high dose of either strain via aerosol, and virulence was compared by bacterial burden and pathology. Measurements and Main Results: Infection with Erdman resulted in significantly shorter times to euthanasia and higher bacterial burdens and greater systemic inflammation and lung pathology relative to those infected with CDC1551. Macaques infected with Erdman also exhibited significantly higher early inflammatory myeloid cell influx to the lung, greater macrophage and T cell activity, and higher expression of lung remodeling (extracellular matrix) genes, consistent with greater pathology. Expression of NOTCH4 (neurogenic locus notch homolog 4) signaling, which is induced in response to hypoxia and promotes undifferentiated cellular state, was also higher in Erdman-infected lungs. The granulomas generated by Erdman, and not CDC1551, infection appeared to have larger regions of necrosis, which is strongly associated with hypoxia. To better understand the mechanisms of differential hypoxia induction by these strains, we subjected both to hypoxia in vitro. Erdman induced higher concentrations of DosR regulon relative to CDC1551. The DosR regulon is the global regulator of response to hypoxia in Mtb and critical for its persistence in granulomas. Conclusions: Our results show that the response to hypoxia is a critical mediator of virulence determination in Mtb, with potential impacts on bacillary persistence, reactivation, and efficiency of therapeutics.

A Miniaturized Electrostatic Precipitator Respirator Effectively Removes Ambient SARS-CoV-2 Bioaerosols.

The inhalation of ambient SARS-CoV-2-containing bioaerosols leads to infection and pandemic airborne transmission in susceptible populations. Filter-based respirators effectively reduce exposure but complicate normal respiration through breathing zone pressure differentials; therefore, they are impractical for long-term use. OBJECTIVES: We tested the comparative effectiveness of a prototyped miniaturized electrostatic precipitator (mEP) on a filter-based respirator (N95) via the removal of viral bioaerosols from a simulated, inspired air stream. Methods: Each respirator was tested within a 16 L environmental chamber housed within a Class III biological safety cabinet within biosafety level 3 containment. SARS-CoV-2-containing bioaerosols were generated in the chamber, drawn by a vacuum through each respirator, and physical particle removal and viral genomic RNA were measured distal to the breathing zone of each device. MEASUREMENTS AND MAIN RESULTS: The mEP respirator removed particles (96.5 ± 0.4%), approximating efficiencies of the N95 (96.9 ± 0.6%). The mEP respirator similarly decreased SARS-CoV-2 viral RNA (99.792%) when compared to N95 removal (99.942%), as a function of particle removal from the airstream distal to the breathing zone of each respirator. CONCLUSIONS: The mEP respirator approximated the performance of a filter-based N95 respirator for particle removal and viral RNA as a constituent of the SARS-CoV-2 bioaerosols generated for this evaluation. In practice, the mEP respirator could provide equivalent protection from ambient infectious bioaerosols as the N95 respirator without undue pressure drop to the wearer, thereby facilitating its long-term use in an unobstructed breathing configuration.

Phenotypic and Kinetic Changes of Myeloid Lineage Cells in Innate Response to Chikungunya Infection in Cynomolgus Macaques.

Chikungunya (CHIKV) is an emerging worldwide viral threat. The immune response to infection can lead to protection and convalescence or result in long-term sequelae such as arthritis. Early innate immune events during acute infection have been characterized for some cell types, but more must be elucidated with respect to cellular responses of monocytes and other myeloid lineage cells. In addition to their roles in protection and inflammation resolution, monocytes and macrophages are sites for viral replication and may also act as viral reservoirs. These cells are also found in joints postinfection, possibly playing a role in long-term CHIKV-induced pathology. We examined kinetic and phenotypic changes in myeloid lineage cells, including monocytes, in cynomolgus macaques early after experimental infection with CHIKV. We found increased proliferation of monocytes and decreased proliferation of myeloid dendritic cells early during infection, with an accompanying decrease in absolute numbers of both cell types, as well as a simultaneous increase in plasmacytoid dendritic cell number. An increase in CD16 and CD14 was seen along with a decrease in monocyte Human Leukocyte Antigen-DR isotype expression within 3 days of infection, potentially indicating monocyte deactivation. A transient decrease in T cells, B cells, and natural killer cells correlated with lymphocytopenia observed during human infections with CHIKV. CD4+ T cell proliferation decreased in blood, indicating relocation of cells to effector sites. These data indicate CHIKV influences proliferation rates and kinetics of myeloid lineage cells early during infection and may prove useful in development of therapeutics and evaluation of infection-induced pathogenesis.

CRISPR-based assay reveals SARS-CoV-2 RNA dynamic changes and redistribution patterns in non-human primate model.

Mounting evidence indicates that SARS-CoV-2 can infect multiple systemic tissues, but few studies have evaluated SARS-CoV-2 RNA dynamics in multiple specimen types due to their reduced accessibility and diminished performance of RT-qPCR with non-respiratory specimens. Here, we employed an ultrasensitive CRISPR-RT-PCR assay to analyze longitudinal mucosal (nasal, buccal, pharyngeal, and rectal), plasma, and breath samples from SARS-CoV-2-infected non-human primates (NHPs) to detect dynamic changes in SARS-CoV-2 RNA level and distribution among these specimens. We observed that CRISPR-RT-PCR results consistently detected SARS-CoV-2 RNA in all sample types at most time points post-infection, and that SARS-CoV-2 infection dose and administration route did not markedly affect the CRISPR-RT-PCR signal detected in most specimen types. However, consistent RT-qPCR positive results were restricted to nasal, pharyngeal, and rectal swab samples, and tended to decrease earlier than CRISPR-RT-PCR results, reflecting lower assay sensitivity. SARS-CoV-2 RNA was detectable in both pulmonary and extrapulmonary specimens from early to late infection by CRISPR-RT-PCR, albeit with different abundance and kinetics, with SARS-CoV-2 RNA increases detected in plasma and rectal samples trailing those detected in upper respiratory tract samples. CRISPR-RT-PCR assays for SARS-CoV-2 RNA in non-respiratory specimens may thus permit direct diagnosis of suspected COVID-19 cases missed by RT-PCR, while tracking SARS-CoV-2 RNA in minimally invasive alternate specimens may better evaluate the progression and resolution of SARS-CoV-2 infections.

Bioaerosols and airborne transmission: Integrating biological complexity into our perspective.

There is broad consensus that airborne disease transmission continues to be the thematic focus of COVID-19, the complexities and understanding of which continues to complicate our attempts to control this pandemic. Masking used as both personal protection and source reduction predominates our society at present and, other than vaccination, remains the public health measure that will faithfully reduce aerosol transmission and overall disease burden (Gandhi and Marr, 2021). Early in the advent of the COVID-19 pandemic, and especially after preliminary recognition of airborne transmission, there was considerable efforts in the application of computational fluid dynamics (CFD) modeling aerosols as well as risk models calculations, the products of which were detailed in the literature (Morawska et al., 2020; Buonanno et al., 2020a) and even disseminated in media destined for the public. As the respiratory pathway emerged as the dominant exposure pathway for SARSCoV-2 transmission, much of what was promoted from CFD was applied to risk models to estimate community infection and in some cases expected clinical outcome. COVID-19 proved to fit the profile of an obligate respiratory-transmitted pathogen, and the plausibility of using aerosol modeling when silhouetted with emerging COVID-19 epidemiology provided ample evidence for promotion of masking and ventilation optimization as a required public health measure. Masking is often included as a factor in developed risk models and it remains an essentially important part of our response to this airborne threat, and ultimately will agnostically reduce disease burden although efforts to improve ventilation in indoor spaces remain a challenge. Arguably the most important concept in the airborne transmission of infectious agents is the biologically active componentry that comprises the aerosol particle and the functional dynamic nature of particle contents. Specifically, the innate generation, transport, and ultimate deposition/disposition of bioaerosols; the aerosol particles that nearly exclusively harbor bioactive components, including viruses, when disease agents are transmitted through the air.

Intra-Host SARS-CoV-2 Evolution in the Gut of Mucosally-Infected Chlorocebus aethiops (African Green Monkeys).

In recent months, several SARS-CoV-2 variants have emerged that enhance transmissibility and escape host humoral immunity. Hence, the tracking of viral evolutionary trajectories is clearly of great importance. Little is known about SARS-CoV-2 evolution in nonhuman primate models used to test vaccines and therapies and to model human disease. Viral RNA was sequenced from rectal swabs from Chlorocebus aethiops (African green monkeys) after experimental respiratory SARS-CoV-2 infection. Two distinct patterns of viral evolution were identified that were shared between all collected samples. First, mutations in the furin cleavage site that were initially present in the virus as a consequence of VeroE6 cell culture adaptation were not detected in viral RNA recovered in rectal swabs, confirming the necessity of this motif for viral infection in vivo. Three amino acid changes were also identified; ORF 1a S2103F, and spike D215G and H655Y, which were detected in rectal swabs from all sampled animals. These findings are demonstrative of intra-host SARS-CoV-2 evolution and may identify a host-adapted variant of SARS-CoV-2 that would be useful in future primate models involving SARS-CoV-2 infection.