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1.
BMC Vet Res ; 18(1): 124, 2022 Apr 01.
Article in English | MEDLINE | ID: covidwho-1770541

ABSTRACT

BACKGROUND: Coronaviruses have the potential to cross species barriers. To learn the molecular intersections among the most common coronaviruses of domestic and close-contact animals, we analyzed representative coronavirus genera infecting mouse, rat, rabbit, dog, cat, cattle, white-tailed deer, swine, ferret, mink, alpaca, Rhinolophus bat, dolphin, whale, chicken, duck and turkey hosts; reference or complete genome sequences were available for most of these coronavirus genera. Protein sequence alignments and phylogenetic trees were built for the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The host receptors and enzymes aminopeptidase N (APN), angiotensin converting enzyme 2 (ACE2), sialic acid synthase (SAS), transmembrane serine protease 2 (TMPRSS2), dipeptidyl peptidase 4 (DPP4), cathepsin L (and its analogs) and furin were also compared. RESULTS: Overall, the S, E, M, and N proteins segregated according to their viral genera (α, ß, or γ), but the S proteins of alphacoronaviruses lacked conservation of phylogeny. Interestingly, the unique polybasic furin cleavage motif found in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) but not in severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome coronavirus (MERS-CoV) exists in several ß-coronaviruses and a few α- or γ-coronaviruses. Receptors and enzymes retained host species-dependent relationships with one another. Among the hosts, critical ACE2 residues essential for SARS-CoV-2 spike protein binding were most conserved in white-tailed deer and cattle. CONCLUSION: The polybasic furin cleavage motif found in several ß- and other coronaviruses of animals points to the existence of an intermediate host for SARS-CoV-2, and it also offers a counternarrative to the theory of a laboratory-engineered virus. Generally, the S proteins of coronaviruses show crossovers of phylogenies indicative of recombination events. Additionally, the consistency in the segregation of viral proteins of the MERS-like coronavirus (NC_034440.1) from pipistrelle bat supports its classification as a ß-coronavirus. Finally, similarities in host enzymes and receptors did not always explain natural cross-infections. More studies are therefore needed to identify factors that determine the cross-species infectivity of coronaviruses.


Subject(s)
COVID-19 , Cattle Diseases , Deer , Dog Diseases , Middle East Respiratory Syndrome Coronavirus , Rodent Diseases , Swine Diseases , Animals , COVID-19/veterinary , Cattle , Dogs , Ferrets , Mice , Middle East Respiratory Syndrome Coronavirus/genetics , Phylogeny , Rabbits , Rats , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Swine
2.
BMC Vet Res ; 18(1): 93, 2022 Mar 10.
Article in English | MEDLINE | ID: covidwho-1770540

ABSTRACT

BACKGROUND: Mycobacteria are found in many environmental conditions and infect a variety of species, including rodents and rabbits. Guinea pigs are used experimentally as a model for Mycobacterium tuberculosis, but natural mycobacteriosis in guinea pigs has not been reported. CASE PRESENTATION: A 1.5-year-old female guinea pig was found acutely deceased with no premonitory illness. On gross post-mortem examination, multifocal to coalescing, raised, firm, pale tan nodules with discrete, irregular margins were noted over the surfaces of all lung lobes. Histopathology revealed nodules composed of clustered foamy macrophages and multinucleated giant cells containing numerous bacterial rods. Similar bacteria-laden macrophages were noted within sections of the liver, heart, palpebral conjunctiva, duodenum, and cecum. Polymerase chain reaction was performed on tissues collected during post-mortem examination. The 16S rRNA gene product was sequenced and was identical to the Mycobacterium genavense type strain. CONCLUSIONS: To the best of the author's knowledge, this report details the first documented case of Mycobacterium genvaense infection in a guinea pig and a follow up investigation of close-contact animals. Given their experimental susceptibility and this clinical case report, mycobacteriosis should be considered as a differential in guinea pigs exhibiting weight loss in the absence of other clinical signs. With the potential for zoonotic transmission in immunosuppressed individuals, precautions should be taken to safeguard human health in cases of guinea pigs with suspected M. genavense infection.


Subject(s)
Mycobacterium Infections, Nontuberculous , Mycobacterium , Animals , Female , Guinea Pigs , Mycobacterium Infections, Nontuberculous/veterinary , Polymerase Chain Reaction/veterinary , RNA, Ribosomal, 16S/genetics , Rabbits
3.
Microbiol Spectr ; 10(1): e0169521, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1752774

ABSTRACT

Global control of COVID-19 will require the deployment of vaccines capable of inducing long-term protective immunity against SARS-CoV-2 variants. In this report, we describe an adjuvanted subunit candidate vaccine that affords elevated, sustained, and cross-variant SARS-CoV-2 neutralizing antibodies (NAbs) in multiple animal models. Alhydroxiquim-II is a Toll-Like Receptor (TLR) 7/8 small-molecule agonist chemisorbed on aluminum hydroxide (Alhydrogel). Vaccination with Alhydroxiquim-II combined with a stabilized, trimeric form of the SARS-CoV-2 spike protein (termed CoVac-II) resulted in high-titer NAbs in mice, with no decay in responses over an 8-month period. NAbs from sera of CoVac-II-immunized mice, horses and rabbits were broadly neutralizing against SARS-CoV-2 variants. Boosting long-term CoVac-II-immunized mice with adjuvanted spike protein from the Beta variant markedly increased levels of NAb titers against multiple SARS-CoV-2 variants; notably, high titers against the Delta variant were observed. These data strongly support the clinical assessment of Alhydroxiquim-II-adjuvanted spike proteins to protect against SARS-CoV-2 variants of concern. IMPORTANCE There is an urgent need for next-generation COVID-19 vaccines that are safe, demonstrate high protective efficacy against SARS-CoV-2 variants and can be manufactured at scale. We describe a vaccine candidate (CoVac-II) that is based on stabilized, trimeric spike antigen produced in an optimized, scalable and chemically defined production process. CoVac-II demonstrates strong and persistent immunity after vaccination of mice, and is highly immunogenic in multiple animal models, including rabbits and horses. We further show that prior immunity can be boosted using a recombinant spike antigen from the Beta variant; importantly, plasma from boosted mice effectively neutralize multiple SARS-CoV-2 variants in vitro, including Delta. The strong humoral and Th1-biased immunogenicity of CoVac-II is driven by use of Alhydroxiquim-II (AHQ-II), the first adjuvant in an authorized vaccine that acts through the dual Toll-like receptor (TLR)7 and TLR8 pathways, as part of the Covaxin vaccine. Our data suggest AHQ-II/spike protein combinations could constitute safe, affordable, and mass-manufacturable COVID-19 vaccines for global distribution.


Subject(s)
Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , COVID-19 Vaccines/immunology , SARS-CoV-2/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , CD4-Positive T-Lymphocytes/immunology , Horses , Mice , Rabbits , T-Lymphocytes/immunology
4.
Microbiol Spectr ; 10(1): e0127121, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1752773

ABSTRACT

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global outbreak and prompted an enormous research effort. Still, the subcellular localization of the coronavirus in lungs of COVID-19 patients is not well understood. Here, the localization of the SARS-CoV-2 proteins is studied in postmortem lung material of COVID-19 patients and in SARS-CoV-2-infected Vero cells, processed identically. Correlative light and electron microscopy on semithick cryo-sections demonstrated induction of electron-lucent, lipid-filled compartments after SARS-CoV-2 infection in both lung and cell cultures. In lung tissue, the nonstructural protein 4 and the stable nucleocapsid N-protein were detected on these novel lipid-filled compartments. The induction of such lipid-filled compartments and the localization of the viral proteins in lung of patients with fatal COVID-19 may explain the extensive inflammatory response and provide a new hallmark for SARS-CoV-2 infection at the final, fatal stage of infection. IMPORTANCE Visualization of the subcellular localization of SARS-CoV-2 proteins in lung patient material of COVID-19 patients is important for the understanding of this new virus. We detected viral proteins in the context of the ultrastructure of infected cells and tissues and discovered that some viral proteins accumulate in novel, lipid-filled compartments. These structures are induced in Vero cells but, more importantly, also in lung of patients with COVID-19. We have characterized these lipid-filled compartments and determined that this is a novel, virus-induced structure. Immunogold labeling demonstrated that cellular markers, such as CD63 and lipid droplet marker PLIN-2, are absent. Colocalization of lipid-filled compartments with the stable N-protein and nonstructural protein 4 in lung of the last stages of COVID-19 indicates that these compartments play a key role in the devastating immune response that SARS-CoV-2 infections provoke.


Subject(s)
COVID-19/metabolism , Lipid Metabolism/physiology , Lipids/analysis , Lung/metabolism , Nucleocapsid/analysis , SARS-CoV-2 , Adolescent , Aged , Animals , COVID-19/pathology , Child, Preschool , Chlorocebus aethiops , Disease Outbreaks , Female , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Lung/cytology , Lung/pathology , Lung/ultrastructure , Male , Microscopy, Immunoelectron , Middle Aged , Nucleocapsid/metabolism , Rabbits , SARS-CoV-2/ultrastructure , Vero Cells/virology
5.
Ups J Med Sci ; 1272022.
Article in English | MEDLINE | ID: covidwho-1743250

ABSTRACT

Background: The development of easy-to-perform diagnostic methods is highly important for detecting current coronavirus disease (COVID-19). This pilot study aimed at developing a lateral flow assay (LFA)-based test prototype to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in saliva samples. Methods: Mice were immunized using the recombinant receptor-binding domain (rRBD) of SARS-CoV-2 virus spike protein. The combinations of the obtained mouse anti-receptor-binding domain (RBD) polyclonal antibodies (PAbs) and several commercial antibodies directed against the SARS-CoV-2 spike protein were used for enzyme-linked immunosorbent assay (ELISA) to select antibody pairs for LFA. The antibody pairs were tested in a LFA format using saliva samples from individuals with early SARS-CoV-2 infection (n = 9). The diagnostic performance of the developed LFA was evaluated using saliva samples from hospitalized COVID-19 patients (n = 111); the median time from the onset of symptoms to sample collection was 10 days (0-24 days, interquartile range (IQR): 7-13). The reverse transcription-polymerase chain reaction (rRT-PCR) was used as a reference method. Results: Based on ELISA and preliminary LFA results, a combination of mouse anti-RBD PAbs (capture antibody) and rabbit anti-spike PAbs (detection antibody) was chosen for clinical analysis of sample. When compared with rRT-PCR results, LFA exhibited 26.5% sensitivity, 58.1% specificity, 50.0% positive prediction value (PPV), 33.3% negative prediction value (NPV), and 38.7% diagnostic accuracy. However, there was a reasonable improvement in assay specificity (85.7%) and PPV (91.7%) when samples were stratified based on the sampling time. Conclusion: The developed LFA assay demonstrated a potential of SARS-CoV-2 detection in saliva samples. Further technical assay improvements should be made to enhance diagnostic performance followed by a validation study in a larger cohort of both asymptomatic and symptomatic patients in the early stage of infection.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Antibodies, Viral , COVID-19/diagnosis , Humans , Mice , Pilot Projects , Rabbits , Saliva , Spike Glycoprotein, Coronavirus
6.
Reprod Toxicol ; 108: 56-61, 2022 03.
Article in English | MEDLINE | ID: covidwho-1720799

ABSTRACT

Nirmatrelvir (PF-07321332; NMV) the antiviral component of PAXLOVID™ is a potent and selective inhibitor of the SARS-CoV-2 main protease (Mpro), which plays a critical role in viral replication. PAXLOVID, comprised of nirmatrelvir and ritonavir (used as a pharmacokinetic enhancer), is an oral therapy currently in development as a therapeutic option for those infected with SARS-CoV-2 to prevent progression to severe disease, hospitalization, and death. PAXLOVID has been shown to be efficacious against hospitalization and death in two Phase 2/3 clinical studies that evaluated non hospitalized patients both with and without high risk factors for progression to severe illness. Given that males and females of reproductive age are included in the intended patient population, we assessed the potential effects of NMV up to the limit dose of 1000 mg/kg/day in ICH guideline embryo-fetal development studies in rats and rabbits, and a fertility and early embryonic development study in rats. There were no effects on male and female fertility or early embryonic development in rats, and no severe manifestations of developmental toxicity in rats or rabbits. The lack of adverse findings reported here in nonclinical species is consistent with the intended therapeutic target of NMV (a virus specific protein not present in mammalian cells), the favorable off-target selectivity profile, and lack of genetic toxicity. The results of these nonclinical studies with NMV along with existing ritonavir safety information indicate that there are no clinically relevant risks associated with PAXLOVID administration during pregnancy and in males and females of reproductive age.


Subject(s)
Antiviral Agents/toxicity , COVID-19/drug therapy , Embryonic Development/drug effects , Fertility/drug effects , Lactams/toxicity , Leucine/toxicity , Nitriles/toxicity , Proline/toxicity , Ritonavir/toxicity , Animals , Drug Combinations , Female , Infertility/chemically induced , Male , Pregnancy , Rabbits , Rats , Rats, Wistar
7.
Molecules ; 27(1)2022 Jan 02.
Article in English | MEDLINE | ID: covidwho-1686893

ABSTRACT

Hypercytokinemia, or cytokine storm, is one of the severe complications of viral and bacterial infections, involving the release of abnormal amounts of cytokines, resulting in a massive inflammatory response. Cytokine storm is associated with COVID-19 and sepsis high mortality rate by developing epithelial dysfunction and coagulopathy, leading to thromboembolism and multiple organ dysfunction syndrome. Anticoagulant therapy is an important tactic to prevent thrombosis in sepsis and COVID-19, but recent data show the incompatibility of modern direct oral anticoagulants and antiviral agents. It seems relevant to develop dual-action drugs with antiviral and anticoagulant properties. At the same time, it was shown that azolo[1,5-a]pyrimidines are heterocycles with a broad spectrum of antiviral activity. We have synthesized a new family of azolo[1,5-a]pyrimidines and their condensed polycyclic analogs by cyclocondensation reactions and direct CH-functionalization and studied their anticoagulant properties. Five compounds among 1,2,4-triazolo[1,5-a]pyrimidin-7-ones and 5-alkyl-1,3,4-thiadiazolo[3,2-a]purin-8-ones demonstrated higher anticoagulant activity than the reference drug, dabigatran etexilate. Antithrombin activity of most active compounds was confirmed using lipopolysaccharide (LPS)-treated blood to mimic the conditions of cytokine release syndrome. The studied compounds affected only the thrombin time value, reliably increasing it 6.5-15.2 times as compared to LPS-treated blood.


Subject(s)
Anticoagulants/pharmacology , Azo Compounds/chemistry , Blood Coagulation/drug effects , Hemorrhage/drug therapy , Pyrimidines/chemistry , Animals , Anticoagulants/chemistry , Hemorrhage/chemically induced , Lipopolysaccharides/toxicity , Male , Rabbits , Rats
8.
Int J Nanomedicine ; 17: 351-379, 2022.
Article in English | MEDLINE | ID: covidwho-1674135

ABSTRACT

Purpose: SARS-CoV-2-infected individuals may be asymptomatic, and therefore, the virus is highly contagious. We aimed to develop an agent to control viral replication in the upper respiratory tract and to prevent progression of the disease into the lower airways as well as inter-individual transmission. For this purpose, we investigated the antibacterial and antiviral activities of our novel nanobubble ozonated hyaluronic acid-decorated liposomal (NOHAL) solution, developed by using nanotechnology. Methods: The MIC levels of NOHAL solution were determined on blood agar cultures of Staphylococcus aureus (ATCC 6538), Streptococcus pneumoniae (ATCC 49619) and Escherichia coli (ATCC 25922). The in vitro anti-viral activity of NOHAL solution was studied using recombinant SARS-CoV-2 copies of the original virus, grown in Vero cells generated by reverse genetic technology. Human primary lung epithelial cells obtained by bronchoscopy or lung resection were used for cell viability tests using flow cytometry analysis. The cytotoxicity testing was performed using the BALB/c 3T3 (CCL-163) cell line. Skin, oral, nasal and ocular irritation tests were performed using New Zealand albino rabbits, Syrian hamsters, BALB c mice and New Zealand albino rabbits of both sexes. Results: Bacterial growth was prevented by NOHAL solution in a time-/dose-dependent manner. In vivo or in vitro experiments did not show any toxicity of NOHAL solution. No cytotoxicity was recorded on cell viability. No skin, oral, nasal or ocular toxicities were recorded. In addition, in a SARS-CoV-2 mouse infection model, NOHAL solution diminished the viral RNA levels effectively in nasopharyngeal and lung samples after its prophylactic intranasal application. Conclusion: NOHAL solution has the potential to reduce or prevent the spread of SARS-CoV-2 through the nose and/or oral cavity. The clinical efficacy of this solution needs to be tested in order to determine its efficacy in the early phase of COVID-19.


Subject(s)
COVID-19 , Ozone , Animals , Anti-Bacterial Agents/pharmacology , Chlorocebus aethiops , Cricetinae , Female , Humans , Hyaluronic Acid , Liposomes , Male , Mice , Rabbits , SARS-CoV-2 , Vero Cells
9.
Arch Toxicol ; 96(3): 859-875, 2022 03.
Article in English | MEDLINE | ID: covidwho-1634984

ABSTRACT

rVSV-ΔG-SARS-CoV-2-S is a clinical stage (Phase 2) replication competent recombinant vaccine against SARS-CoV-2. To evaluate the safety profile of the vaccine, a series of non-clinical safety, immunogenicity and efficacy studies were conducted in four animal species, using multiple doses (up to 108 Plaque Forming Units/animal) and dosing regimens. There were no treatment-related mortalities or any noticeable clinical signs in any of the studies. Compared to unvaccinated controls, hematology and biochemistry parameters were unremarkable and no adverse histopathological findings. There was no detectable viral shedding in urine, nor viral RNA detected in whole blood or serum samples seven days post vaccination. The rVSV-ΔG-SARS-CoV-2-S vaccination gave rise to neutralizing antibodies, cellular immune responses, and increased lymphocytic cellularity in the spleen germinal centers and regional lymph nodes. No evidence for neurovirulence was found in C57BL/6 immune competent mice or in highly sensitive type I interferon knock-out mice. Vaccine virus replication and distribution in K18-human Angiotensin-converting enzyme 2-transgenic mice showed a gradual clearance from the vaccination site with no vaccine virus recovered from the lungs. The nonclinical data suggest that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe and immunogenic. These results supported the initiation of clinical trials, currently in Phase 2.


Subject(s)
COVID-19 Vaccines/toxicity , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Vaccines/immunology , Cricetinae , Female , Membrane Glycoproteins/genetics , Mesocricetus , Mice , Mice, Inbred C57BL , Rabbits , Swine , Vaccination , Vaccines, Synthetic/toxicity , Viral Envelope Proteins/genetics
10.
Cell Res ; 32(3): 269-287, 2022 03.
Article in English | MEDLINE | ID: covidwho-1634806

ABSTRACT

The emergence of SARS-CoV-2 variants and potentially other highly pathogenic sarbecoviruses in the future highlights the need for pan-sarbecovirus vaccines. Here, we discovered a new STING agonist, CF501, and found that CF501-adjuvanted RBD-Fc vaccine (CF501/RBD-Fc) elicited significantly stronger neutralizing antibody (nAb) and T cell responses than Alum- and cGAMP-adjuvanted RBD-Fc in mice. Vaccination of rabbits and rhesus macaques (nonhuman primates, NHPs) with CF501/RBD-Fc elicited exceptionally potent nAb responses against SARS-CoV-2 and its nine variants and 41 S-mutants, SARS-CoV and bat SARSr-CoVs. CF501/RBD-Fc-immunized hACE2-transgenic mice were almost completely protected against SARS-CoV-2 challenge, even 6 months after the initial immunization. NHPs immunized with a single dose of CF501/RBD-Fc produced high titers of nAbs. The immunized macaques also exhibited durable humoral and cellular immune responses and showed remarkably reduced viral load in the upper and lower airways upon SARS-CoV-2 challenge even at 108 days post the final immunization. Thus, CF501/RBD-Fc can be further developed as a novel pan-sarbecovirus vaccine to combat current and future outbreaks of sarbecovirus diseases.


Subject(s)
COVID-19 , Vaccines , Animals , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , COVID-19 Vaccines , Macaca mulatta , Mice , Rabbits , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , T-Lymphocytes
11.
Antiviral Res ; 197: 105232, 2022 01.
Article in English | MEDLINE | ID: covidwho-1588314

ABSTRACT

We report the in vitro antiviral activity of DZNep (3-Deazaneplanocin A; an inhibitor of S-adenosylmethionine-dependent methyltransferase) against SARS-CoV-2, besides demonstrating its protective efficacy against lethal infection of infectious bronchitis virus (IBV, a member of the Coronaviridae family). DZNep treatment resulted in reduced synthesis of SARS-CoV-2 RNA and proteins without affecting other steps of viral life cycle. We demonstrated that deposition of N6-methyl adenosine (m6A) in SARS-CoV-2 RNA in the infected cells recruits heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), an RNA binding protein which serves as a m6A reader. DZNep inhibited the recruitment of hnRNPA1 at m6A-modified SARS-CoV-2 RNA which eventually suppressed the synthesis of the viral genome. In addition, m6A-marked RNA and hnRNPA1 interaction was also shown to regulate early translation to replication switch of SARS-CoV-2 genome. Furthermore, abrogation of methylation by DZNep also resulted in defective synthesis of the 5' cap of viral RNA, thereby resulting in its failure to interact with eIF4E (a cap-binding protein), eventually leading to a decreased synthesis of viral proteins. Most importantly, DZNep-resistant mutants could not be observed upon long-term sequential passage of SARS-CoV-2 in cell culture. In summary, we report the novel role of methylation in the life cycle of SARS-CoV-2 and propose that targeting the methylome using DZNep could be of significant therapeutic value against SARS-CoV-2 infection.


Subject(s)
Adenosine/analogs & derivatives , Genome, Viral/drug effects , Methyltransferases/antagonists & inhibitors , SARS-CoV-2/drug effects , Adenosine/pharmacology , Animals , Chick Embryo , Chlorocebus aethiops , Chromatin Immunoprecipitation Sequencing , DNA Methylation/drug effects , DNA Methylation/physiology , Drug Resistance, Viral/drug effects , Genome, Viral/genetics , Heterogeneous Nuclear Ribonucleoprotein A1/metabolism , Humans , Lethal Dose 50 , Mice , Protein Biosynthesis/drug effects , RNA, Viral/drug effects , RNA, Viral/metabolism , Rabbits , SARS-CoV-2/genetics , Specific Pathogen-Free Organisms , Transcription, Genetic/drug effects , Vero Cells
12.
Eur J Pharmacol ; 915: 174670, 2022 Jan 15.
Article in English | MEDLINE | ID: covidwho-1549763

ABSTRACT

Hydroxychloroquine (HCQ) is a derivative of the antimalaria drug chloroquine primarily prescribed for autoimmune diseases. Recent attempts to repurpose HCQ in the treatment of corona virus disease 2019 has raised concerns because of its propensity to prolong the QT-segment on the electrocardiogram, an effect associated with increased pro-arrhythmic risk. Since chirality can affect drug pharmacological properties, we have evaluated the functional effects of the R(-) and S(+) enantiomers of HCQ on six ion channels contributing to the cardiac action potential and on electrophysiological parameters of isolated Purkinje fibers. We found that R(-)HCQ and S(+)HCQ block human Kir2.1 and hERG potassium channels in the 1 µM-100 µM range with a 2-4 fold enantiomeric separation. NaV1.5 sodium currents and CaV1.2 calcium currents, as well as KV4.3 and KV7.1 potassium currents remained unaffected at up to 90 µM. In rabbit Purkinje fibers, R(-)HCQ prominently depolarized the membrane resting potential, inducing autogenic activity at 10 µM and 30 µM, while S(+)HCQ primarily increased the action potential duration, inducing occasional early afterdepolarization at these concentrations. These data suggest that both enantiomers of HCQ can alter cardiac tissue electrophysiology at concentrations above their plasmatic levels at therapeutic doses, and that chirality does not substantially influence their arrhythmogenic potential in vitro.


Subject(s)
Antimalarials/chemistry , Antimalarials/pharmacology , Heart/drug effects , Hydroxychloroquine/chemistry , Hydroxychloroquine/pharmacology , Ion Channels/drug effects , Action Potentials/drug effects , Animals , Arrhythmias, Cardiac/chemically induced , Electrocardiography , Electrophysiologic Techniques, Cardiac , Ether-A-Go-Go Potassium Channels , Humans , Membrane Potentials/drug effects , Patch-Clamp Techniques , Purkinje Fibers/drug effects , Rabbits , Stereoisomerism
13.
J Virol ; 96(3): e0150421, 2022 02 09.
Article in English | MEDLINE | ID: covidwho-1546442

ABSTRACT

In the age of COVID, nucleic acid vaccines have garnered much attention, at least in part, because of the simplicity of construction, production, and flexibility to adjust and adapt to an evolving outbreak. Orthopoxviruses remain a threat on multiple fronts, especially as emerging zoonoses. In response, we developed a DNA vaccine, termed 4pox, that protected nonhuman primates against monkeypox virus (MPXV)-induced severe disease. Here, we examined the protective efficacy of the 4pox DNA vaccine delivered by intramuscular (i.m.) electroporation (EP) in rabbits challenged with aerosolized rabbitpox virus (RPXV), a model that recapitulates the respiratory route of exposure and low dose associated with natural smallpox exposure in humans. We found that 4pox-vaccinated rabbits developed immunogen-specific antibodies, including neutralizing antibodies, and did not develop any clinical disease, indicating protection against aerosolized RPXV. In contrast, unvaccinated animals developed significant signs of disease, including lesions, and were euthanized. These findings demonstrate that an unformulated, nonadjuvanted DNA vaccine delivered i.m. can protect against an aerosol exposure. IMPORTANCE The eradication of smallpox and subsequent cessation of vaccination have left a majority of the population susceptible to variola virus or other emerging poxviruses. This is exemplified by human monkeypox, as evidenced by the increase in reported endemic and imported cases over the past decades. Therefore, a malleable vaccine technology that can be mass produced and does not require complex conditions for distribution and storage is sought. Herein, we show that a DNA vaccine, in the absence of a specialized formulation or adjuvant, can protect against a lethal aerosol insult of rabbitpox virus.


Subject(s)
/immunology , Orthopoxvirus/immunology , Poxviridae Infections/prevention & control , Vaccinia virus/immunology , Vaccinia/prevention & control , Viral Proteins/immunology , Viral Vaccines/immunology , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Dose-Response Relationship, Immunologic , Electroporation , Female , Immunization/methods , Immunogenicity, Vaccine , Lymphocyte Activation/immunology , Oligodeoxyribonucleotides/administration & dosage , Oligodeoxyribonucleotides/immunology , Rabbits , Vaccines, DNA/immunology , Vaccinia virus/genetics , Viral Vaccines/administration & dosage
14.
Vaccine ; 39(45): 6601-6613, 2021 10 29.
Article in English | MEDLINE | ID: covidwho-1541007

ABSTRACT

AKS-452 is a biologically-engineered vaccine comprising an Fc fusion protein of the SARS-CoV-2 viral spike protein receptor binding domain antigen (Ag) and human IgG1 Fc (SP/RBD-Fc) in clinical development for the induction and augmentation of neutralizing IgG titers against SARS-CoV-2 viral infection to address the COVID-19 pandemic. The Fc moiety is designed to enhance immunogenicity by increasing uptake via Fc-receptors (FcγR) on Ag-presenting cells (APCs) and prolonging exposure due to neonatal Fc receptor (FcRn) recycling. AKS-452 induced approximately 20-fold greater neutralizing IgG titers in mice relative to those induced by SP/RBD without the Fc moiety and induced comparable long-term neutralizing titers with a single dose vs. two doses. To further enhance immunogenicity, AKS-452 was evaluated in formulations containing a panel of adjuvants in which the water-in-oil adjuvant, Montanide™ ISA 720, enhanced neutralizing IgG titers by approximately 7-fold after one and two doses in mice, including the neutralization of live SARS-CoV-2 virus infection of VERO-E6 cells. Furthermore, ISA 720-adjuvanted AKS-452 was immunogenic in rabbits and non-human primates (NHPs) and protected from infection and clinical symptoms with live SARS-CoV-2 virus in NHPs (USA-WA1/2020 viral strain) and the K18 human ACE2-trangenic (K18-huACE2-Tg) mouse (South African B.1.351 viral variant). These preclinical studies support the initiation of Phase I clinical studies with adjuvanted AKS-452 with the expectation that this room-temperature stable, Fc-fusion subunit vaccine can be rapidly and inexpensively manufactured to provide billions of doses per year especially in regions where the cold-chain is difficult to maintain.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19 , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , Immunoglobulin G , Mice , Primates , Rabbits , Recombinant Fusion Proteins/immunology , SARS-CoV-2 , Vaccines, Subunit
15.
J Biomed Sci ; 28(1): 80, 2021 Nov 23.
Article in English | MEDLINE | ID: covidwho-1533257

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an RNA virus with a high mutation rate. Importantly, several currently circulating SARS-CoV-2 variants are associated with loss of efficacy for both vaccines and neutralizing antibodies. METHODS: We analyzed the binding activity of six highly potent antibodies to the spike proteins of SARS-CoV-2 variants, assessed their neutralizing abilities with pseudovirus and authentic SARS-CoV-2 variants and evaluate efficacy of antibody cocktail in Delta SARS-CoV-2-infected hamster models as prophylactic and post-infection treatments. RESULTS: The tested RBD-chAbs, except RBD-chAb-25, maintained binding ability to spike proteins from SARS-CoV-2 variants. However, only RBD-chAb-45 and -51 retained neutralizing activities; RBD-chAb-1, -15, -25 and -28 exhibited diminished neutralization for all SARS-CoV-2 variants. Notably, several cocktails of our antibodies showed low IC50 values (3.35-27.06 ng/ml) against the SARS-CoV-2 variant pseudoviruses including United Kingdom variant B.1.1.7 (Alpha), South Africa variant B.1.351 (Beta), Brazil variant P1 (Gamma), California variant B.1.429 (Epsilon), New York variant B.1.526 (Iota), and India variants, B.1.617.1 (Kappa) and B.1.617.2 (Delta). RBD-chAb-45, and -51 showed PRNT50 values 4.93-37.54 ng/ml when used as single treatments or in combination with RBD-chAb-15 or -28, according to plaque assays with authentic Alpha, Gamma and Delta SARS-CoV-2 variants. Furthermore, the antibody cocktail of RBD-chAb-15 and -45 exhibited potent prophylactic and therapeutic effects in Delta SARS-CoV-2 variant-infected hamsters. CONCLUSIONS: The cocktail of RBD-chAbs exhibited potent neutralizing activities against SARS-CoV-2 variants. These antibody cocktails are highly promising candidate tools for controlling new SARS-CoV-2 variants, including Delta.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/therapeutic use , COVID-19/drug therapy , COVID-19/genetics , Humans , Rabbits , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
16.
RNA Biol ; 18(sup2): 804-817, 2021 11 12.
Article in English | MEDLINE | ID: covidwho-1522048

ABSTRACT

Nsp1 of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of the Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1, eRF1 and ABCE1. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates peptide release and formation of termination complexes. Detailed analysis of Nsp1 activity during translation termination stages reveals that Nsp1 facilitates stop codon recognition. We demonstrate that Nsp1 stimulation targets eRF1 and does not affect eRF3. Moreover, Nsp1 increases amount of the termination complexes at all three stop codons. The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that the biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and remove them from the pool of the active ribosomes.


Subject(s)
Protein Biosynthesis , SARS-CoV-2 , Viral Nonstructural Proteins/physiology , Animals , Cell-Free System , Codon, Terminator/metabolism , GTP Phosphohydrolases/metabolism , HeLa Cells , Humans , Mutation , Peptide Chain Termination, Translational , Peptide Termination Factors/chemistry , Peptide Termination Factors/metabolism , Peptides/chemistry , Protein Binding , Protein Conformation , Protein Domains , RNA, Messenger/metabolism , Rabbits , Ribosomes/metabolism
17.
Emerg Microbes Infect ; 10(1): 2199-2201, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1505680

ABSTRACT

We report pilot studies to evaluate the susceptibility of common domestic livestock (cattle, sheep, goat, alpaca, rabbit, and horse) to intranasal infection with SARS-CoV-2. None of the infected animals shed infectious virus via nasal, oral, or faecal routes, although viral RNA was detected in several animals. Further, neutralizing antibody titres were low or non-existent one month following infection. These results suggest that domestic livestock are unlikely to contribute to SARS-CoV-2 epidemiology.


Subject(s)
COVID-19/veterinary , Host Specificity , Livestock/virology , SARS-CoV-2/pathogenicity , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/virology , Camelids, New World/virology , Cattle/virology , Chlorocebus aethiops , Disease Reservoirs/virology , Goats/virology , Horses/virology , Host Specificity/immunology , Humans , Nasal Cavity/virology , RNA, Viral/analysis , Rabbits/virology , Rectum/virology , Respiratory System/virology , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sheep/virology , Species Specificity , Vero Cells , Virus Shedding , Viscera/virology
18.
Front Immunol ; 12: 689065, 2021.
Article in English | MEDLINE | ID: covidwho-1502324

ABSTRACT

Coronavirus disease 2019 (COVID-19) is an acute respiratory infectious disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The US FDA has approved several therapeutics and vaccines worldwide through the emergency use authorization in response to the rapid spread of COVID-19. Nevertheless, the efficacies of these treatments are being challenged by viral escape mutations. There is an urgent need to develop effective treatments protecting against SARS-CoV-2 infection and to establish a stable effect-screening model to test potential drugs. Polyclonal antibodies (pAbs) have an intrinsic advantage in such developments because they can target rapidly mutating viral strains as a result of the complexity of their binding epitopes. In this study, we generated anti-receptor-binding domain (anti-RBD) pAbs from rabbit serum and tested their safety and efficacy in response to SARS-CoV-2 infection both in vivo and ex vivo. Primary human bronchial epithelial two-dimensional (2-D) organoids were cultured and differentiated to a mature morphology and subsequently employed for SARS-CoV-2 infection and drug screening. The pAbs protected the airway organoids from viral infection and tissue damage. Potential side effects were tested in mouse models for both inhalation and vein injection. The pAbs displayed effective viral neutralization effects without significant side effects. Thus, the use of animal immune serum-derived pAbs might be a potential therapy for protection against SARS-CoV-2 infection, with the strategy developed to produce these pAbs providing new insight into the treatment of respiratory tract infections, especially for infections with viruses undergoing rapid mutation.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/administration & dosage , Antibodies, Viral/administration & dosage , Binding Sites , Bronchi/cytology , COVID-19/genetics , COVID-19/therapy , Epithelial Cells , Gene Expression Profiling , Humans , Immunization, Passive , Mice , Mutation , Neutralization Tests , Organoids , Rabbits , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
19.
Antimicrob Agents Chemother ; 65(7): e0001321, 2021 06 17.
Article in English | MEDLINE | ID: covidwho-1476381

ABSTRACT

The SOS response to DNA damage is a conserved stress response in Gram-negative and Gram-positive bacteria. Although this pathway has been studied for years, its relevance is still not familiar to many working in the fields of clinical antibiotic resistance and stewardship. Under some conditions, the SOS response favors DNA repair and preserves the genetic integrity of the organism. On the other hand, the SOS response also includes induction of error-prone DNA polymerases, which can increase the rate of mutation, called the mutator phenotype or "hypermutation." As a result, mutations can occur in genes conferring antibiotic resistance, increasing the acquisition of resistance to antibiotics. Almost all of the work on the SOS response has been on bacteria exposed to stressors in vitro. In this study, we sought to quantitate the effects of SOS-inducing drugs in vivo, in comparison with the same drugs in vitro. We used a rabbit model of intestinal infection with enteropathogenic Escherichia coli strain E22. SOS-inducing drugs triggered the mutator phenotype response in vivo as well as in vitro. Exposure of E. coli strain E22 to ciprofloxacin or zidovudine, both of which induce the SOS response in vitro, resulted in increased antibiotic resistance to 3 antibiotics: rifampin, minocycline, and fosfomycin. Zinc was able to inhibit the SOS-induced emergence of antibiotic resistance in vivo, as previously observed in vitro. Our findings may have relevance in reducing the emergence of resistance to new antimicrobial drugs.


Subject(s)
Escherichia coli , SOS Response, Genetics , Animals , Anti-Bacterial Agents/pharmacology , Ciprofloxacin/pharmacology , Drug Resistance, Microbial , Escherichia coli/genetics , Mutation , Rabbits
20.
Laryngoscope ; 131(6): E1971-E1979, 2021 06.
Article in English | MEDLINE | ID: covidwho-1453618

ABSTRACT

OBJECTIVE/HYPOTHESIS: To assess the ability of ultra-short echo time (UTE)-MRI to detect subglottic stenosis (SGS) and evaluate response to balloon dilation. To correlate measurements from UTE-MRI with endotracheal-tube (ETT)-sizing and to investigate whether SGS causes change in airway dynamics. STUDY DESIGN: Animal research study. METHODS: Eight adult New-Zealand white rabbits were used as they approximate neonatal airway-size. The airways were measured using ETT-sizing and 3D UTE-MRI at baseline, 2 weeks post-cauterization induced SGS injury, and post-balloon dilation treatment. UTE-MR images were acquired to determine airway anatomy and motion. Airways were segmented from MR images. Cross-sectional area (CSA), major and minor diameters (Dmajor and Dminor ), and eccentricity were measured. RESULTS: Post-injury CSA at SGS was significantly reduced (mean 38%) compared to baseline (P = .003) using UTE-MRI. ETT-sizing correlated significantly with MRI-measured CSA at the SGS location (r = 0.6; P < .01), particularly at the post-injury timepoint (r = 0.93; P < .01). Outer diameter from ETT-sizing (OD) correlated significantly with Dmajor (r = 0.63; P < .01) from UTE-MRI at the SGS location, especially for the post-injury timepoint (r = 0.91; P < .01). Mean CSA of upper trachea did not change significantly between end-expiration and end-inspiration at any timepoint (all P > .05). Eccentricity of the upper trachea increased significantly post-balloon dilation (P < .05). CONCLUSIONS: UTE-MRI successfully detected SGS and treatment response in the rabbit model, with good correlation to ETT-sizing. Balloon dilation increased CSA at SGS, but not to baseline values. SGS did not alter dynamic motion for the trachea in this rabbit model; however, tracheas were significantly eccentric post-balloon dilation. UTE-MRI can detect SGS without sedation or ionizing radiation and may be a non-invasive alternative to ETT-sizing. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E1971-E1979, 2021.


Subject(s)
Laryngostenosis/diagnostic imaging , Magnetic Resonance Imaging/methods , Animals , Disease Models, Animal , Female , Imaging, Three-Dimensional , Intubation, Intratracheal , Laryngoscopy , Rabbits
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