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.

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.

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.

Particle Dynamics and Bioaerosol Viability of Aerosolized Bacillus Calmette-Guérin Vaccine Using Jet and Vibrating Mesh Clinical Nebulizers.

Background: Bacillus Calmette-Guérin (BCG) is a vaccine used to protect against tuberculosis primarily in infants to stop early infection in areas of the world where the disease is endemic. Normally administered as a percutaneous injection, BCG is a live significantly attenuated bacteria that is now being investigated for its potential within an inhalable vaccine formulation. This study investigates the feasibility and performance of two jet and two vibrating mesh nebulizers aerosolizing BCG and the resulting particle characteristics and residual viability of the bacteria postaerosolization. Methods: A jet nebulizer (Collison), outfitted either with a 3- or 6-jet head, was compared with two clinical nebulizers, the vibrating mesh Omron MicroAir and Aerogen Solo devices. Particle characteristics, including aerodynamic particle sizing, was performed on all devices within a common aerosol chamber configuration and comparable BCG innocula concentrations. Integrated aerosol samples were collected for each generator and assayed for bacterial viability using conventional microbiological technique. Results: A batch lot of BCG (Danish) was grown to titer and used in all generator assessments. Aerosol particles within the respirable range were generated from all nebulizers at four different concentrations of BCG. The jet nebulizers produced a uniformly smaller particle size than the vibrating mesh devices, although particle concentrations by mass were similar across all devices tested with the exception of the Aerogen Solo, which resulted in a low concentration of BCG aerosols. Conclusions: The resulting measured viable BCG aerosol concentration fraction produced by each device approximated one another; however, a measurable decrease of efficiency and overall viability reduction in the jet nebulizer was observed in higher BCG inoculum starting concentrations, whereas the vibrating mesh nebulizer returned a remarkably stable viable aerosol fraction irrespective of inoculum concentration.

Exhaled aerosol increases with COVID-19 infection, age, and obesity.

COVID-19 transmits by droplets generated from surfaces of airway mucus during processes of respiration within hosts infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. We studied respiratory droplet generation and exhalation in human and nonhuman primate subjects with and without COVID-19 infection to explore whether SARS-CoV-2 infection, and other changes in physiological state, translate into observable evolution of numbers and sizes of exhaled respiratory droplets in healthy and diseased subjects. In our observational cohort study of the exhaled breath particles of 194 healthy human subjects, and in our experimental infection study of eight nonhuman primates infected, by aerosol, with SARS-CoV-2, we found that exhaled aerosol particles vary between subjects by three orders of magnitude, with exhaled respiratory droplet number increasing with degree of COVID-19 infection and elevated BMI-years. We observed that 18% of human subjects (35) accounted for 80% of the exhaled bioaerosol of the group (194), reflecting a superspreader distribution of bioaerosol analogous to a classical 20:80 superspreader of infection distribution. These findings suggest that quantitative assessment and control of exhaled aerosol may be critical to slowing the airborne spread of COVID-19 in the absence of an effective and widely disseminated vaccine.

Susceptibility of monkeypox virus aerosol suspensions in a rotating chamber.

Viral aerosols can have a major impact on public health and on the dynamics of infection. Once aerosolized, viruses are subjected to various stress factors and their integrity and potential of infectivity can be altered. Empirical characterization is needed in order to predict more accurately the fate of these bioaerosols both for short term and long term suspension in the air. Here the susceptibility to aerosolization of the monkeypox virus (MPXV), associated with emerging zoonotic diseases, was studied using a 10.7 L rotating chamber. This chamber was built to fit inside a Class three biological safety cabinet, specifically for studying airborne biosafety level three (BSL3) microorganisms. Airborne viruses were detected by culture and quantitative polymerase chain reaction (qPCR) after up to 90 h of aging. Viral concentrations detected dropped by two logs for culture analysis and by one log for qPCR analysis within the first 18 h of aging; viral concentrations were stable between 18 and 90 h, suggesting a potential for the MPXV to retain infectivity in aerosols for more than 90 h. The rotating chamber used in this study maintained viral particles airborne successfully for prolonged periods and could be used to study the susceptibility of other BSL3 microorganisms.

MCP-3/CCL7 production by astrocytes: implications for SIV neuroinvasion and AIDS encephalitis.

Monocyte/macrophages and activated lymphocytes traffic through normal brain, and this trafficking is increased in inflammatory conditions such as HIV encephalitis (HIVE). HIVE is characterized in part by perivascular accumulations of macrophages. The earliest events in this process are poorly understood and difficult or impossible to address in humans. The SIV-infected macaque model of neuroAIDS has demonstrated migration of monocytes into the brain early in disease, coincident with peak SIV viremia. The chemotactic signals that initiate the increased emigration of mononuclear cells into the CNS have not been described. Here, we describe astrocytes as a primary source of chemokines to facilitate basal levels of monocyte trafficking to CNS and that increased chemokine (C-C motif) ligand 7 (CCL7) production may be responsible for initiating the increased trafficking in neuroAIDS. We have previously published complementary in vivo work demonstrating the presence of monocyte chemoattractant protein 3 (MCP-3)/CCL7 within the brain of SIV-infected macaques. Here, we demonstrate that MCP-3/CCL7 is a significant chemokine produced by astrocytes, that basal monocyte migration may be facilitated by astrocyte-derived CCL7, that production of CCL7 is rapidly increased by TNF-α and thus likely plays a critical role in initiating neuroinvasion by SIV/HIV.

Association of FAK activation with lentivirus-induced disruption of blood-brain barrier tight junction-associated ZO-1 protein organization.

Expression of tight junction proteins between brain microvascular endothelial cells (BMECs) of the blood-brain barrier (BBB) is lost during development of human immunodeficiency virus (HIV) encephalitis (HIVE). Although many studies have focused on the strains of virus that induce neurological sequelae or on the macrophages/microglia that are associated with development of encephalitis, the molecular signaling pathways within the BMECs involved have yet to be resolved. We have previously shown that there is activation and disruption of an in vitro BBB model using lentivirus-infected CEMx174 cells. We and others have shown similar disruption in vivo. Therefore, it was of interest to determine if the presence of infected cells could disrupt intact cerebral microvessels immediately ex vivo, and if so, which signaling pathways were involved. The present data demonstrate that disruption of tight junctions between BMECs is mediated through activation of focal adhesion kinase (FAK) by phosphorylation at TYR-397. Inhibition of FAK activation is sufficient to prevent tight junction disruption. Thus, it may be possible to inhibit the development of HIVE by using inhibitors of FAK.