Efficacy of a ML336 Derivative Against Venezuelan and Eastern Equine Encephalitis Viruses

Currently, there are no licensed human vaccines or antivirals for treatment of or prevention from infection with encephalitic alphaviruses. Because epidemics are sporadic and unpredictable, and endemic disease is common but rarely diagnosed, it is difficult to identify all populations requiring vaccination; thus, an effective post-exposure treatment method is needed to interrupt ongoing outbreaks. To address this public health need, we have continued development of ML336 to deliver a molecule with prophylactic and therapeutic potential that could be relevant for use in natural epidemics or deliberate release scenario for Venezuelan equine encephalitis virus (VEEV). We report findings from in vitro assessments of four analogs of ML336, and in vivo screening of three of these new derivatives, BDGR-4, BDGR-69 and BDGR-70. The optimal dosing for maximal protection was observed at 12.5 mg/kg/day, twice daily for 8 days. BDGR-4 was tested further for prophylactic and therapeutic efficacy in mice challenged with VEEV Trinidad Donkey (TrD). Mice challenged with VEEV TrD showed 100% and 90% protection from lethal disease when treated at 24 and 48 h post-infection, respectively. We also measured 90% protection for BDGR-4 in mice challenged with Eastern equine encephalitis virus. In additional assessments of BDGR-4 in mice alone, we observed no appreciable toxicity as evaluated by clinical chemistry indicators up to a dose of 25 mg/kg/day over 4 days. In these same mice, we observed no induction of interferon. Lastly, the resistance of VEEV to BDGR-4 was evaluated by next-generation sequencing which revealed specific mutations in nsP4, the viral polymerase

Visualization of Murine Intranasal Dosing Efficiency Using Luminescent Francisella tularensis: Effect of Instillation Volume and Form of Anesthesia

Intranasal instillation is a widely used procedure for pneumonic delivery of drugs, vaccine candidates, or infectious agents into the respiratory tract of research mice. However, there is a paucity of published literature describing the efficiency of this delivery technique. In this report we have used the murine model of tularemia, with Francisella tularensis live vaccine strain (FTLVS) infection, to evaluate the efficiency of pneumonic delivery via intranasal dosing performed either with differing instillation volumes or different types of anesthesia. FTLVS was rendered luminescent via transformation with a reporter plasmid that constitutively expressed the Photorhabdus luminescens lux operon from a Francisella promoter. We then used an IVIS Spectrum whole animal imaging system to visualize FT dissemination at various time points following intranasal instillation. We found that instillation of FT in a dose volume of 10 µl routinely resulted in infection of the upper airways but failed to initiate infection of the pulmonary compartment. Efficient delivery of FT into the lungs via intranasal instillation required a dose volume of 50 µl or more. These studies also demonstrated that intranasal instillation was significantly more efficient for pneumonic delivery of FTLVS in mice that had been anesthetized with inhaled (isoflurane) vs. parenteral (ketamine/xylazine) anesthesia. The collective results underscore the need for researchers to consider both the dose volume and the anesthesia type when either performing pneumonic delivery via intranasal instillation, or when comparing studies that employed this technique

A DNA Vaccine against Chikungunya Virus Is Protective in Mice and Induces Neutralizing Antibodies in Mice and Nonhuman Primates

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus indigenous to tropical Africa and Asia. Acute illness is characterized by fever, arthralgias, conjunctivitis, rash, and sometimes arthritis. Relatively little is known about the antigenic targets for immunity, and no licensed vaccines or therapeutics are currently available for the pathogen. While the Aedes aegypti mosquito is its primary vector, recent evidence suggests that other carriers can transmit CHIKV thus raising concerns about its spread outside of natural endemic areas to new countries including the U.S. and Europe. Considering the potential for pandemic spread, understanding the development of immunity is paramount to the development of effective counter measures against CHIKV. In this study, we isolated a new CHIKV virus from an acutely infected human patient and developed a defined viral challenge stock in mice that allowed us to study viral pathogenesis and develop a viral neutralization assay. We then constructed a synthetic DNA vaccine delivered by in vivo electroporation (EP) that expresses a component of the CHIKV envelope glycoprotein and used this model to evaluate its efficacy. Vaccination induced robust antigen-specific cellular and humoral immune responses, which individually were capable of providing protection against CHIKV challenge in mice. Furthermore, vaccine studies in rhesus macaques demonstrated induction of nAb responses, which mimicked those induced in convalescent human patient sera. These data suggest a protective role for nAb against CHIKV disease and support further study of envelope-based CHIKV DNA vaccines

Cardiopulmonary Injury in the Syrian Hamster Model of COVID-19

The Syrian hamster has proved useful in the evaluation of therapeutics and vaccines for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). To advance the model for preclinical studies, we conducted serial sacrifice of lungs, large pulmonary vessels, and hearts from male and female Syrian hamsters for days 1–4, and 8 post-infection (dpi) following infection with a high dose of SARS-CoV-2. Evaluation of microscopic lung histopathology scores suggests 4 and 8 dpi as prime indicators in the evaluation of moderate pathology with bronchial hyperplasia, alveolar involvement and bronchiolization being key assessments of lung disease and recovery, respectively. In addition, neutrophil levels, red blood cell count and hematocrit showed significant increases during early infection. We present histological evidence of severe damage to the pulmonary vasculature with extensive leukocyte transmigration and the loss of endothelial cells and tunica media. Our evidence of endothelial and inflammatory cell death in the pulmonary vessels suggests endothelialitis secondary to SARS-CoV-2 epithelial cell infection as a possible determinant of the pathological findings along with the host inflammatory response. Lastly, pathological examination of the heart revealed evidence for intracardiac platelet/fibrin aggregates in male and female hamsters on 8 dpi, which might be indicative of a hypercoagulative state in these animals

Kinetic <i>in vivo</i> localization of luminescent FTLVS following intranasal dosing in titrated volumes of inocula.

<p>BALB/c mice (3/group) were challenged via the intranasal route with 1×10⁶ CFU of FTLVS-lux suspended in a volume of 10 µl, 20 µl, 50 µl, or 100 µl of sterile PBS. <b>Panel A</b>: All mice were then subjected to whole animal imaging using an IVIS Spectrum Imaging system at the indicated time points. Scaling intensity of all images was normalized and data are reported as photons/sec/cm∧2/sr. <b>Panel B</b>: All mice were weighed daily as a measure of disease-state. Statistical analysis was performed via 2-way ANOVA with Bonferroni post-tests. Significant differences between the 10 µl instillation volume group and all other groups are indicated toward the top of the graph and are color-coded. Significant differences between the 100 µl instillation volume group and either the 20 µl or 50 µl dose volume groups are indicated toward the bottom of the graph and are color-coded. The calculated p values are indicated as follows: p<0.05 (*), p<0.01 (**), and p<0.001 (***).</p

Pulmonary delivery of FTLVS-lux was more efficient under inhaled vs. parenteral anesthesia.

<p>BALB/c mice (5/group) were anesthetized using either inhaled isoflurane or parenterally-administered ketamine/xylazine and then challenged with either 1×10⁵ CFU FTLVS-lux in a volume of 50 µl (<b>Panel A</b>) or 1×10⁶ CFU FTLVS-lux in a volume of 100 µl (<b>Panel B</b>). Dissemination of FTLVS was monitored 24 hrs later using an IVIS Spectrum whole animal imaging system. Lungs were collected after imaging was completed for bacterial burden determination via dilution plating. All IVIS images were normalized to reflect photons per second per cm∧2/sr. Statistical analyses were performed using the student t test.</p

Genetic Map of pXB173-lux.

<p>pXB173-lux constitutively expresses the <i>Photorhabdus luminescens</i> luciferace (<i>lux AB</i>) and luciferase substrate (<i>luxCDE</i>) from the <i>Francisella groE</i> promoter. This vector also encodes a selectable marker for kanamycin resistance (<i>aph3′</i>).</p

Correlation between whole animal <i>in vivo</i> imaging with viable bacterial counts 24 hours after challenge.

<p>BALB/c mice (5/group) were challenged with 1×10⁶ CFU of FTLVS bearing the pXB173-lux reporter plasmid via the intranasal route in a total bolus volume of either 10 µl, 20 µl, 50 µl, or 100 µl. <b>Panel A</b>: Dissemination of FTLVS was then monitored 24 hrs later using an IVIS Spectrum whole animal imaging system. Images were collected at the indicated time points post-infection and were normalized to reflect photons per second per cm∧2/sr. (<b>Panel B</b>): Lungs were collected after imaging was completed for bacterial burden determination via dilution plating. Statistical analyses were performed via one-way ANOVA using a Bonferroni multiple comparisons posttest. Statistically significant differences are indicated as follows: p<0.05 (*) and p<0.01 (**).</p

Isolation and identification of CHIKV.

<p>The microphotographs show normal uninfected Vero cells (A) and the Vero cells infected with CHIKV virus isolate. CHIKV infection in Vero cells causes characteristic foamy cytopathic effect (CPE) 48 hours p.i. as seen with the isolate. (B) RT-PCR analysis of CHIKV viral isolates. Agarose gel photograph showing the RT-PCR amplified product (305 bp) of the CHIKV positive patient isolates (Lane 1&2). The uninfected negative control (Lane 3) shows no amplification. (C) Electron micrographs of CHIKV viral isolates (D) Phylogenetic Tree generated with E2 amplicon from CHIKV Isolate. * Indicates the PC-08 CHIKV strain.</p