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1.
J Infect Dis ; 2021 Jan 28.
Article in English | MEDLINE | ID: covidwho-1853084

ABSTRACT

BACKGROUND: REGN3048 and REGN3051 are human monoclonal antibodies (mAb) targeting the Spike (S) glycoprotein on the Middle Eastern Respiratory Syndrome coronavirus (MERS-CoV), which binds to the receptor dipeptidyl peptidase-4 (DPP4) and is necessary for infection of susceptible cells. METHODS: Preclinical study: REGN3048, REGN3051 and isotype immunoglobulin G (IgG) were administered to huDPP4 mice 1 day prior to and 1 day after infection with MERS-CoV (Jordan strain). Virus titers and lung pathology were assessed. Phase 1 study: healthy adults received the combined mAb (n = 36) or placebo (n = 12) and followed for 121 days. Six dose levels were studied. Strict safety criteria were met prior to dose escalation. RESULTS: Preclinical study: REGN3048 plus REGN3051 prophylactically or therapeutically, is substantially more effective for reducing viral titer, lung inflammation and pathology in huDPP4 mice compared with control antibodies and to each antibody monotherapy. Phase 1 study: REGN3048 plus REGN3051 was well tolerated with no dose-limiting adverse events, deaths, serious adverse events, or infusion reactions. Each mAb displayed pharmacokinetics expected of human IgG1 antibodies; it was not immunogenic. CONCLUSIONS: REGN3048 and REGN3051 in combination were well-tolerated. The clinical and preclinical data support further development for the treatment or prophylaxis of MERS-CoV infection.

2.
Front Physiol ; 12: 634839, 2021.
Article in English | MEDLINE | ID: covidwho-1526786

ABSTRACT

Though the current preponderance of evidence indicates the toxicity associated with the smoking of tobacco products through conventional means, less is known about the role of "vaping" in respiratory disease. "Vaping" is described as the use of electronic cigarettes (E-Cigarettes or E-Cigs), which has only more recently been available to the public (∼10 years) but has quickly emerged as a popular means of tobacco consumption worldwide. The World Health Organization (WHO) declared the SARS-CoV-2 outbreak as a global pandemic in March 2020. SARS-CoV-2 can easily be transmitted between people in close proximity through direct contact or respiratory droplets to develop coronavirus infectious disease 2019 (COVID-19). Symptoms of COVID-19 range from a mild flu-like illness with high fever to severe respiratory distress syndrome and death. The risk factors for increased disease severity remain unclear. Herein, we utilize a murine-tropic coronavirus (beta coronavirus) MHV-A59 along with a mouse model and measures of pathology (lung weight/dry ratios and histopathology) and inflammation (ELISAs and cytokine array panels) to examine whether vaping may exacerbate the pulmonary disease severity of coronavirus disease. While vaping alone did result in some noted pathology, mice exposed with intranasal vaped e-liquid suffered more severe mortality due to pulmonary inflammation than controls when exposed to coronavirus infection. Our data suggest a role for vaping in increased coronavirus pulmonary disease in a mouse model. Furthermore, our data indicate that disease exacerbation may involve calcium (Ca2+) dysregulation, identifying a potential therapeutic intervention.

3.
Elife ; 102021 02 02.
Article in English | MEDLINE | ID: covidwho-1513045

ABSTRACT

Evolutionary medicine argues that disease can arise because modern conditions do not match those in which we evolved. For example, a decline in exposure to commensal microbes and gastrointestinal helminths in developed countries has been linked to increased prevalence of allergic and autoimmune inflammatory disorders (the hygiene hypothesis). Accordingly, probiotic therapies that restore 'old friend' microbes and helminths have been explored as Darwinian treatments for these disorders. A further possibility is that loss of old friend commensals also increases the sterile, aging-associated inflammation known as inflammaging, which contributes to a range of age-related diseases, including cardiovascular disease, dementia, and cancer. Interestingly, Crowe et al., 2020 recently reported that treatment with a secreted glycoprotein from a parasitic nematode can protect against murine aging by induction of anti-inflammatory mechanisms. Here, we explore the hypothesis that restorative helminth therapy would have anti-inflammaging effects. Could worm infections provide broad-spectrum protection against age-related disease?


Subject(s)
Helminthiasis/immunology , Immunosenescence , Inflammation/immunology , Aging , Animals , Autoimmune Diseases/physiopathology , Helminths , Host-Parasite Interactions/immunology , Humans
4.
J Nucl Med ; 62(11): 1631-1637, 2021 11.
Article in English | MEDLINE | ID: covidwho-1496930

ABSTRACT

In this study, we developed angiotensin-converting enzyme 2 (ACE2)-specific, peptide-derived 68Ga-labeled radiotracers, motivated by the hypotheses that ACE2 is an important determinant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) susceptibility and that modulation of ACE2 in coronavirus disease 2019 (COVID-19) drives severe organ injury. Methods: A series of NOTA-conjugated peptides derived from the known ACE2 inhibitor DX600 were synthesized, with variable linker identity. Since DX600 bears 2 cystine residues, both linear and cyclic peptides were studied. An ACE2 inhibition assay was used to identify lead compounds, which were labeled with 68Ga to generate peptide radiotracers (68Ga-NOTA-PEP). The aminocaproate-derived radiotracer 68Ga-NOTA-PEP4 was subsequently studied in a humanized ACE2 (hACE2) transgenic model. Results: Cyclic DX-600-derived peptides had markedly lower half-maximal inhibitory concentrations than their linear counterparts. The 3 cyclic peptides with triglycine, aminocaproate, and polyethylene glycol linkers had calculated half-maximal inhibitory concentrations similar to or lower than the parent DX600 molecule. Peptides were readily labeled with 68Ga, and the biodistribution of 68Ga-NOTA-PEP4 was determined in an hACE2 transgenic murine cohort. Pharmacologic concentrations of coadministered NOTA-PEP (blocking) showed a significant reduction of 68Ga-NOTA-PEP4 signals in the heart, liver, lungs, and small intestine. Ex vivo hACE2 activity in these organs was confirmed as a correlate to in vivo results. Conclusion: NOTA-conjugated cyclic peptides derived from the known ACE2 inhibitor DX600 retain their activity when N-conjugated for 68Ga chelation. In vivo studies in a transgenic hACE2 murine model using the lead tracer, 68Ga-NOTA-PEP4, showed specific binding in the heart, liver, lungs and intestine-organs known to be affected in SARS-CoV-2 infection. These results suggest that 68Ga-NOTA-PEP4 could be used to detect organ-specific suppression of ACE2 in SARS-CoV-2-infected murine models and COVID-19 patients.


Subject(s)
Angiotensin-Converting Enzyme 2 , Gallium Radioisotopes/chemistry , Peptides, Cyclic , Animals , Male , Mice , Positron-Emission Tomography , Tissue Distribution
5.
J Am Soc Nephrol ; 32(1): 99-114, 2021 01.
Article in English | MEDLINE | ID: covidwho-1496673

ABSTRACT

BACKGROUND: C3 glomerulopathy (C3G) is characterized by the alternative-pathway (AP) hyperactivation induced by nephritic factors or complement gene mutations. Mice deficient in complement factor H (CFH) are a classic C3G model, with kidney disease that requires several months to progress to renal failure. Novel C3G models can further contribute to understanding the mechanism behind this disease and developing therapeutic approaches. METHODS: A novel, rapidly progressing, severe, murine model of C3G was developed by replacing the mouse C3 gene with the human C3 homolog using VelociGene technology. Functional, histologic, molecular, and pharmacologic assays characterize the presentation of renal disease and enable useful pharmacologic interventions in the humanized C3 (C3hu/hu) mice. RESULTS: The C3hu/hu mice exhibit increased morbidity early in life and die by about 5-6 months of age. The C3hu/hu mice display elevated biomarkers of kidney dysfunction, glomerulosclerosis, C3/C5b-9 deposition, and reduced circulating C3 compared with wild-type mice. Administration of a C5-blocking mAb improved survival rate and offered functional and histopathologic benefits. Blockade of AP activation by anti-C3b or CFB mAbs also extended survival and preserved kidney function. CONCLUSIONS: The C3hu/hu mice are a useful model for C3G because they share many pathologic features consistent with the human disease. The C3G phenotype in C3hu/hu mice may originate from a dysregulated interaction of human C3 protein with multiple mouse complement proteins, leading to unregulated C3 activation via AP. The accelerated disease course in C3hu/hu mice may further enable preclinical studies to assess and validate new therapeutics for C3G.


Subject(s)
Complement C3/genetics , Disease Models, Animal , Glomerulonephritis, Membranoproliferative/genetics , Kidney Diseases/genetics , Animals , Complement C3/metabolism , Complement Pathway, Alternative/genetics , Exons , Gene Expression Regulation , Glomerulonephritis, Membranoproliferative/metabolism , Humans , Kidney Diseases/metabolism , Liver/metabolism , Male , Mice , Mice, Knockout , Microscopy, Fluorescence , Phenotype , Polymorphism, Single Nucleotide , Renal Insufficiency/genetics , Renal Insufficiency/metabolism
6.
J Virol ; 95(15): e0053021, 2021 07 12.
Article in English | MEDLINE | ID: covidwho-1486507

ABSTRACT

Elicitation of lung tissue-resident memory CD8 T cells (TRMs) is a goal of T cell-based vaccines against respiratory viral pathogens, such as influenza A virus (IAV). C-C chemokine receptor type 2 (CCR2)-dependent monocyte trafficking plays an essential role in the establishment of CD8 TRMs in lungs of IAV-infected mice. Here, we used a combination adjuvant-based subunit vaccine strategy that evokes multifaceted (TC1/TC17/TH1/TH17) IAV nucleoprotein-specific lung TRMs to determine whether CCR2 and monocyte infiltration are essential for vaccine-induced TRM development and protective immunity to IAV in lungs. Following intranasal vaccination, neutrophils, monocytes, conventional dendritic cells (DCs), and monocyte-derived dendritic cells internalized and processed vaccine antigen in lungs. We found that basic leucine zipper ATF-like transcription factor 3 (BATF3)-dependent DCs were essential for eliciting T cell responses, but CCR2 deficiency enhanced the differentiation of CD127hi, KLRG-1lo, OX40+ve CD62L+ve, and mucosally imprinted CD69+ve CD103+ve effector and memory CD8 T cells in lungs and airways of vaccinated mice. Mechanistically, increased development of lung TRMs induced by CCR2 deficiency was linked to dampened expression of T-bet but not altered TCF-1 levels or T cell receptor signaling in CD8 T cells. T1/T17 functional programming, parenchymal localization of CD8/CD4 effector and memory T cells, recall T cell responses, and protective immunity to a lethal IAV infection were unaffected in CCR2-deficient mice. Taken together, we identified a negative regulatory role for CCR2 and monocyte trafficking in mucosal imprinting and differentiation of vaccine-induced TRMs. Mechanistic insights from this study may aid the development of T-cell-based vaccines against respiratory viral pathogens, including IAV and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IMPORTANCE While antibody-based immunity to influenza A virus (IAV) is type and subtype specific, lung- and airway-resident memory T cells that recognize conserved epitopes in the internal viral proteins are known to provide heterosubtypic immunity. Hence, broadly protective IAV vaccines need to elicit robust T cell memory in the respiratory tract. We have developed a combination adjuvant-based IAV nucleoprotein vaccine that elicits strong CD4 and CD8 T cell memory in lungs and protects against H1N1 and H5N1 strains of IAV. In this study, we examined the mechanisms that control vaccine-induced protective memory T cells in the respiratory tract. We found that trafficking of monocytes into lungs might limit the development of antiviral lung-resident memory T cells following intranasal vaccination. These findings suggest that strategies that limit monocyte infiltration can potentiate vaccine-induced frontline T-cell immunity to respiratory viruses, such as IAV and SARS-CoV-2.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunity, Mucosal , Immunologic Memory , Influenza A virus/immunology , Influenza Vaccines/immunology , Orthomyxoviridae Infections/immunology , Receptors, CCR2/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , Influenza A virus/genetics , Influenza Vaccines/genetics , Influenza Vaccines/pharmacology , Lung/immunology , Mice , Mice, Knockout , Orthomyxoviridae Infections/genetics , Orthomyxoviridae Infections/prevention & control , Receptors, CCR2/genetics
7.
Med Sci Monit ; 26: e922281, 2020 Mar 31.
Article in English | MEDLINE | ID: covidwho-1453382

ABSTRACT

BACKGROUND Acute respiratory distress syndrome (ARDS) is a sudden and serious disease with increasing morbidity and mortality rates. Phosphodiesterase 4 (PDE4) is a novel target for inflammatory disease, and ibudilast (IBU), a PDE4 inhibitor, inhibits inflammatory response. Our study investigated the effect of IBU on the pathogenesis of neonatal ARDS and the underlying mechanism related to it. MATERIAL AND METHODS Western blotting was performed to analyze the expression levels of PDE4, CXCR4, SDF-1, CXCR5, CXCL1, inflammatory cytokines, and proteins related to cell apoptosis. Hematoxylin-eosin staining was performed to observe the pathological morphology of lung tissue. Pulmonary edema score was used to assess the degree of lung water accumulation after pulmonary injury. Enzyme-linked immunosorbent assay (ELISA) was used to assess levels of inflammatory factors (TNF-alpha, IL-1ß, IL-6, and MCP-1) in serum. TUNEL assay was used to detect apoptotic cells. RESULTS Increased expression of PDE4 was observed in an LPS-induced neonatal ARDS mouse model, and IBU ameliorated LPS-induced pathological manifestations and pulmonary edema in lung tissue. In addition, IBU attenuated the secretion of inflammatory cytokines by inactivating the chemokine axis in the LPS-induced neonatal ARDS mouse model. Finally, IBU significantly reduced LPS-induced cell apoptosis in lung tissue. CONCLUSIONS IBU, a PDE4 inhibitor, protected against ARDS by interfering with pulmonary inflammation and apoptosis. Our findings provide a novel and promising strategy to regulate pulmonary inflammation in ARDS.


Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Inflammation/drug therapy , Phosphodiesterase 4 Inhibitors/pharmacology , Pyridines/pharmacology , Respiratory Distress Syndrome, Newborn/drug therapy , Animals , Animals, Newborn , Apoptosis/drug effects , Apoptosis/immunology , Bronchoalveolar Lavage Fluid , Disease Models, Animal , Humans , Inflammation/diagnosis , Inflammation/immunology , Inflammation/pathology , Injections, Intraperitoneal , Lipopolysaccharides/immunology , Lung/drug effects , Lung/immunology , Lung/pathology , Mice , Phosphodiesterase 4 Inhibitors/therapeutic use , Pyridines/therapeutic use , Respiratory Distress Syndrome, Newborn/diagnosis , Respiratory Distress Syndrome, Newborn/immunology , Respiratory Distress Syndrome, Newborn/pathology , Signal Transduction/drug effects , Signal Transduction/immunology
8.
Curr Top Med Chem ; 21(14): 1235-1250, 2021 Oct 05.
Article in English | MEDLINE | ID: covidwho-1441869

ABSTRACT

BACKGROUND: Virus-like Particles (VLPs) are non-genetic multimeric nanoparticles synthesized through in vitro or in vivo self-assembly of one or more viral structural proteins. Immunogenicity and safety of VLPs make them ideal candidates for vaccine development and efficient nanocarriers for foreign antigens or adjuvants to activate the immune system. AIMS: The present study aimed to design and synthesize a chimeric VLP vaccine of the phage Qbeta (Qß) coat protein presenting the universal epitope of the coronavirus. METHODS: The RNA phage Qß coat protein was designed and synthesized, denoted as Qbeta. The CoV epitope, a universal epitope of coronavirus, was inserted into the C-terminal of Qbeta using genetic recombination, designated as Qbeta-CoV. The N-terminal of Qbeta-CoV was successively inserted into the TEV restriction site using mCherry red fluorescent label and modified affinity purified histidine label 6xHE, which was denoted as HE-Qbeta-CoV. Isopropyl ß-D-1-thiogalactopyranoside (IPTG) assessment revealed the expression of Qbeta, Qbeta-CoV, and HE-Qbeta-CoV in the BL21 (DE3) cells. The fusion protein was purified by salting out using ammonium sulfate and affinity chromatography. The morphology of particles was observed using electron microscopy. The female BALB/C mice were immunized intraperitoneally with the Qbeta-CoV and HE-Qbeta-- CoV chimeric VLPs vaccines and their sera were collected for the detection of antibody level and antibody titer using ELISA. The serum is used for the neutralization test of the three viruses of MHV, PEDV, and PDCoV. RESULTS: The results revealed that the fusion proteins Qbeta, Qbeta-CoV, and HE-Qbeta-CoV could all obtain successful expression. Particles with high purity were obtained after purification; the chimeric particles of Qbeta-CoV and HE-Qbeta-CoV were found to be similar to Qbeta particles in morphology and formed chimeric VLPs. In addition, two chimeric VLP vaccines induced specific antibody responses in mice and the antibodies showed certain neutralizing activity. CONCLUSION: The successful construction of the chimeric VLPs of the phage Qß coat protein presenting the universal epitope of coronavirus provides a vaccine form with potential clinical applications for the treatment of coronavirus disease.


Subject(s)
Antibodies, Neutralizing/immunology , Capsid Proteins/immunology , Coronavirus/immunology , Vaccines, Virus-Like Particle/immunology , Animals , Antigens, Viral/genetics , Antigens, Viral/immunology , Enzyme-Linked Immunosorbent Assay , Female , Mice, Inbred BALB C , Microscopy, Electron, Scanning , Phylogeny , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Vaccines, Virus-Like Particle/genetics , Viral Proteins/genetics
9.
Vaccines (Basel) ; 9(1)2021 Jan 18.
Article in English | MEDLINE | ID: covidwho-1389565

ABSTRACT

In this concise review, we summarize the concepts behind mRNA vaccination. We discuss the innate and adaptive immune response generated by mRNA vaccines in different animal models and in humans. We give examples of viral infections where mRNA vaccines have shown to induce potent responses and we discuss in more detail the recent SARS-CoV-2 mRNA vaccine trials in humans.

10.
Nat Commun ; 12(1): 3587, 2021 06 11.
Article in English | MEDLINE | ID: covidwho-1387350

ABSTRACT

There is a great need for the development of vaccines that induce potent and long-lasting protective immunity against SARS-CoV-2. Multimeric display of the antigen combined with potent adjuvant can enhance the potency and longevity of the antibody response. The receptor binding domain (RBD) of the spike protein is a primary target of neutralizing antibodies. Here, we developed a trimeric form of the RBD and show that it induces a potent neutralizing antibody response against live virus with diverse effector functions and provides protection against SARS-CoV-2 challenge in mice and rhesus macaques. The trimeric form induces higher neutralizing antibody titer compared to monomer with as low as 1µg antigen dose. In mice, adjuvanting the protein with a TLR7/8 agonist formulation alum-3M-052 induces 100-fold higher neutralizing antibody titer and superior protection from infection compared to alum. SARS-CoV-2 infection causes significant loss of innate cells and pathology in the lung, and vaccination protects from changes in innate cells and lung pathology. These results demonstrate RBD trimer protein as a suitable candidate for vaccine against SARS-CoV-2.


Subject(s)
Adjuvants, Immunologic/administration & dosage , COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/prevention & control , Heterocyclic Compounds, 3-Ring/administration & dosage , Stearic Acids/administration & dosage , Alum Compounds/administration & dosage , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody Formation/immunology , COVID-19 Vaccines/administration & dosage , Disease Models, Animal , Heterocyclic Compounds, 3-Ring/immunology , Humans , Macaca mulatta , Mice , Protein Binding , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/immunology , Stearic Acids/immunology
11.
Nat Commun ; 12(1): 2790, 2021 05 13.
Article in English | MEDLINE | ID: covidwho-1387341

ABSTRACT

SARS-CoV-2 is of zoonotic origin and contains a PRRA polybasic cleavage motif which is considered critical for efficient infection and transmission in humans. We previously reported on a panel of attenuated SARS-CoV-2 variants with deletions at the S1/S2 junction of the spike protein. Here, we characterize pathogenicity, immunogenicity, and protective ability of a further cell-adapted SARS-CoV-2 variant, Ca-DelMut, in in vitro and in vivo systems. Ca-DelMut replicates more efficiently than wild type or parental virus in Vero E6 cells, but causes no apparent disease in hamsters, despite replicating in respiratory tissues. Unlike wild type virus, Ca-DelMut causes no obvious pathological changes and does not induce elevation of proinflammatory cytokines, but still triggers a strong neutralizing antibody and T cell response in hamsters and mice. Ca-DelMut immunized hamsters challenged with wild type SARS-CoV-2 are fully protected, with little sign of virus replication in the upper or lower respiratory tract, demonstrating sterilizing immunity.


Subject(s)
COVID-19/diagnosis , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Virus Replication/genetics , Animals , COVID-19/immunology , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Cricetinae , Cytokines/immunology , Cytokines/metabolism , Female , Host-Pathogen Interactions , Humans , Male , Mesocricetus , Mice, Inbred BALB C , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Vero Cells , Virulence/genetics , Virulence/immunology
12.
Nat Commun ; 12(1): 250, 2021 01 11.
Article in English | MEDLINE | ID: covidwho-1387324

ABSTRACT

Understanding the mechanism for antibody neutralization of SARS-CoV-2 is critical for the development of effective therapeutics and vaccines. We recently isolated a large number of monoclonal antibodies from SARS-CoV-2 infected individuals. Here we select the top three most potent yet variable neutralizing antibodies for in-depth structural and functional analyses. Crystal structural comparisons reveal differences in the angles of approach to the receptor binding domain (RBD), the size of the buried surface areas, and the key binding residues on the RBD of the viral spike glycoprotein. One antibody, P2C-1F11, most closely mimics binding of receptor ACE2, displays the most potent neutralizing activity in vitro and conferred strong protection against SARS-CoV-2 infection in Ad5-hACE2-sensitized mice. It also occupies the largest binding surface and demonstrates the highest binding affinity to RBD. More interestingly, P2C-1F11 triggers rapid and extensive shedding of S1 from the cell-surface expressed spike glycoprotein, with only minimal such effect by the remaining two antibodies. These results offer a structural and functional basis for potent neutralization via disruption of the very first and critical steps for SARS-CoV-2 cell entry.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Viral/immunology , Binding Sites , COVID-19/drug therapy , COVID-19/virology , Disease Models, Animal , Epitopes , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Models, Molecular , Protein Binding , Protein Conformation , Receptors, Virus/immunology , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
13.
Mol Ther ; 29(6): 1970-1983, 2021 06 02.
Article in English | MEDLINE | ID: covidwho-1386766

ABSTRACT

A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell-mediated immunity produced a strong viral antigen-specific CD8+ T lymphocyte response. Assaying for intracellular cytokine staining for interferon (IFN)γ and interleukin-4 (IL-4)-positive CD4+ T helper (Th) lymphocytes as well as anti-spike glycoprotein immunoglobulin G (IgG)2a/IgG1 ratios supported a strong Th1-dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 µg and 10 µg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of LUNAR-COV19 as a single-dose vaccine.


Subject(s)
Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Synthetic/administration & dosage , Alphavirus/genetics , Alphavirus/immunology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Animals , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , COVID-19/immunology , COVID-19/pathology , COVID-19/virology , COVID-19 Vaccines/biosynthesis , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , Female , Gene Expression , Humans , Immunity, Cellular/drug effects , Immunity, Humoral/drug effects , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-4/genetics , Interleukin-4/immunology , Mice , Mice, Transgenic , Replicon/immunology , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Th1 Cells/drug effects , Th1 Cells/immunology , Th1 Cells/virology , Transgenes , Treatment Outcome , Vaccination/methods , Vaccines, Synthetic/biosynthesis , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology
14.
Sci Bull (Beijing) ; 66(9): 925-936, 2021 May 15.
Article in English | MEDLINE | ID: covidwho-1386590

ABSTRACT

The SARS-CoV-2 infection is spreading rapidly worldwide. Efficacious antiviral therapeutics against SARS-CoV-2 is urgently needed. Here, we discovered that protoporphyrin IX (PpIX) and verteporfin, two Food and Drug Administration (FDA)-approved drugs, completely inhibited the cytopathic effect produced by SARS-CoV-2 infection at 1.25 µmol/L and 0.31 µmol/L, respectively, and their EC50 values of reduction of viral RNA were at nanomolar concentrations. The selectivity indices of PpIX and verteporfin were 952.74 and 368.93, respectively, suggesting a broad margin of safety. Importantly, PpIX and verteporfin prevented SARS-CoV-2 infection in mice adenovirally transduced with human angiotensin-converting enzyme 2 (ACE2). The compounds, sharing a porphyrin ring structure, were shown to bind viral receptor ACE2 and interfere with the interaction between ACE2 and the receptor-binding domain of viral S protein. Our study suggests that PpIX and verteporfin are potent antiviral agents against SARS-CoV-2 infection and sheds new light on developing novel chemoprophylaxis and chemotherapy against SARS-CoV-2.

15.
JCI Insight ; 5(19)2020 10 02.
Article in English | MEDLINE | ID: covidwho-1388620

ABSTRACT

The emergence of SARS-CoV-2 has created an international health crisis, and small animal models mirroring SARS-CoV-2 human disease are essential for medical countermeasure (MCM) development. Mice are refractory to SARS-CoV-2 infection owing to low-affinity binding to the murine angiotensin-converting enzyme 2 (ACE2) protein. Here, we evaluated the pathogenesis of SARS-CoV-2 in male and female mice expressing the human ACE2 gene under the control of the keratin 18 promoter (K18). In contrast to nontransgenic mice, intranasal exposure of K18-hACE2 animals to 2 different doses of SARS-CoV-2 resulted in acute disease, including weight loss, lung injury, brain infection, and lethality. Vasculitis was the most prominent finding in the lungs of infected mice. Transcriptomic analysis from lungs of infected animals showed increases in transcripts involved in lung injury and inflammatory cytokines. In the low-dose challenge groups, there was a survival advantage in the female mice, with 60% surviving infection, whereas all male mice succumbed to disease. Male mice that succumbed to disease had higher levels of inflammatory transcripts compared with female mice. To our knowledge, this is the first highly lethal murine infection model for SARS-CoV-2 and should be valuable for the study of SARS-CoV-2 pathogenesis and for the assessment of MCMs.


Subject(s)
Cause of Death , Coronavirus Infections/pathology , Disease Progression , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral/pathology , Severe Acute Respiratory Syndrome/pathology , Angiotensin-Converting Enzyme 2 , Animals , COVID-19 , Coronavirus Infections/physiopathology , Disease Models, Animal , Female , Humans , Lung/pathology , Male , Mice , Mice, Transgenic , Pandemics , Pneumonia, Viral/physiopathology , Severe Acute Respiratory Syndrome/physiopathology , Severity of Illness Index , Survival Rate , Virus Replication/genetics
16.
Front Immunol ; 12: 624703, 2021.
Article in English | MEDLINE | ID: covidwho-1354863

ABSTRACT

Accumulating evidence suggests that the breakdown of immune tolerance plays an important role in the development of myocarditis triggered by cardiotropic microbial infections. Genetic deletion of immune checkpoint molecules that are crucial for maintaining self-tolerance causes spontaneous myocarditis in mice, and cancer treatment with immune checkpoint inhibitors can induce myocarditis in humans. These results suggest that the loss of immune tolerance results in myocarditis. The tissue microenvironment influences the local immune dysregulation in autoimmunity. Recently, tenascin-C (TN-C) has been found to play a role as a local regulator of inflammation through various molecular mechanisms. TN-C is a nonstructural extracellular matrix glycoprotein expressed in the heart during early embryonic development, as well as during tissue injury or active tissue remodeling, in a spatiotemporally restricted manner. In a mouse model of autoimmune myocarditis, TN-C was detectable before inflammatory cell infiltration and myocytolysis became histologically evident; it was strongly expressed during active inflammation and disappeared with healing. TN-C activates dendritic cells to generate pathogenic autoreactive T cells and forms an important link between innate and acquired immunity.


Subject(s)
Autoimmune Diseases/metabolism , Autoimmunity , Cardiomyopathies/metabolism , Inflammation Mediators/metabolism , Myocarditis/metabolism , Myocardium/metabolism , Tenascin/metabolism , Animals , Autoimmune Diseases/immunology , Autoimmune Diseases/pathology , Cardiomyopathies/immunology , Cardiomyopathies/pathology , Cellular Microenvironment , Humans , Myocarditis/immunology , Myocarditis/pathology , Myocardium/immunology , Myocardium/pathology , Self Tolerance , Signal Transduction
17.
Transbound Emerg Dis ; 68(4): 1995-2004, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1331772

ABSTRACT

This study reports outbreak of a new disease caused by Staphylococcus pseudintermedius (S. pseudintermedius) in raccoon dogs. The disease occurred in a breeding farm of raccoon dogs in Guan County of Shandong Province in China in August of 2019. 47% (425/896) of the raccoon dogs showed some abnormal symptoms; 17.6% (75/425) of which had severe skin and soft tissue infections (SSTIs), dyspnoea and severe pathological lesions in lungs, livers, etc; and 4.2% (18/425) of which died within 4 weeks. The pathogen of the disease was identified as S. pseudintermedius by mass spectrometer detection, animal pathogenicity tests, microscopic examination and biochemical reaction tests. Its nucleotide homology of 16S rRNA gene was 100% with that of other published strains, and its genotype was between the American and Brazilian strains from other animals. The isolated S. pseudintermedius strain from the diseased raccoon dogs could cause ulceration and suppuration in the skins and severe pathological lesions not only in raccoon dogs, but also in mice; and it is confirmed as a methicillin-resistant S. pseudintermedius (MRSP) strain by the amplification of mecA gene; and 12 sensitive drugs were screened by drug sensitivity tests. Full attention should be paid to the great economic loss and the potential zoonotic risk caused by the S. pseudintermedius in raccoon dogs, and this study can provide a reference for the clinical diagnosis and treatment of this new disease.


Subject(s)
Dog Diseases , Raccoon Dogs , Staphylococcal Infections , Animals , Anti-Bacterial Agents/pharmacology , Disease Outbreaks/veterinary , Dog Diseases/epidemiology , Dogs , Mice , RNA, Ribosomal, 16S , Rodent Diseases , Staphylococcal Infections/epidemiology , Staphylococcal Infections/veterinary , Staphylococcus
18.
Mol Ther Methods Clin Dev ; 21: 299-314, 2021 Jun 11.
Article in English | MEDLINE | ID: covidwho-1144097

ABSTRACT

Antigen-specific lung-resident memory T cells (TRMs) constitute the first line of defense that mediates rapid protection against respiratory pathogens and inspires novel vaccine designs against infectious pandemic threats, yet effective means of inducing TRMs, particularly via non-viral vectors, remain challenging. Here, we demonstrate safe and potent induction of lung-resident TRMs using a biodegradable polymeric nanoshell that co-encapsulates antigenic peptides and TLR9 agonist CpG-oligodeoxynucleotide (CpG-ODN) in a virus-mimicking structure. Through subcutaneous priming and intranasal boosting, the combinatorial nanoshell vaccine elicits prominent lung-resident CD4+ and CD8+ T cells that surprisingly show better durability than live viral infections. In particular, nanoshells containing CpG-ODN and a pair of conserved class I and II major histocompatibility complex-restricted influenza nucleoprotein-derived antigenic peptides are demonstrated to induce near-sterilizing immunity against lethal infections with influenza A viruses of different strains and subtypes in mice, resulting in rapid elimination of replicating viruses. We further examine the pulmonary transport dynamic and optimal composition of the nanoshell vaccine conducive for robust TRM induction as well as the benefit of subcutaneous priming on TRM replenishment. The study presents a practical vaccination strategy for inducing protective TRM-mediated immunity, offering a compelling platform and critical insights in the ongoing quest toward a broadly protective vaccine against universal influenza as well as other respiratory pathogens.

19.
Methods Mol Biol ; 2099: 161-171, 2020.
Article in English | MEDLINE | ID: covidwho-1292551

ABSTRACT

First identified in 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel virus that can cause acute respiratory distress syndrome (ARDS), multiorgan failure, and death, with a case fatality rate of ~35%. An animal model that supports MERS-CoV infection and causes severe lung disease is useful to study pathogenesis and evaluate therapies and vaccines. The murine dipeptidyl peptidase 4 (Dpp4) protein is not a functional receptor for MERS-CoV; thus, mice are resistant to MERS-CoV infection. We generated human DPP4 knock-in (hDPP4 KI) mice by replacing exons 10-12 at the mouse Dpp4 locus with exons 10-12 from the human DPP4 gene. The resultant human DPP4 KI mice are permissive to MERS-CoV (HCoV-EMC/2012 strain) infection but develop no disease. To generate a mouse model with associated morbidity and mortality from respiratory disease, we serially passaged HCoV-EMC/2012 strain in the lungs of young hDPP4 KI mice. After 30 in vivo passages, an adapted virus clone was isolated and designated MERSMA6.1.2. This virus clone produced significantly higher titers than the parental clone in the lungs of hDPP4 KI mice and caused diffuse lung injury and a fatal respiratory infection. In this chapter, we will describe in detail the procedures used to mouse adapt MERS-CoV by serial passage of the virus in lungs. We also describe the methods used to isolate virus clones and characterize virus infection.


Subject(s)
Coronavirus Infections/virology , Dipeptidyl Peptidase 4/genetics , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Respiratory Distress Syndrome/virology , Animals , Disease Models, Animal , Humans , Lung/virology , Mice , Mice, Inbred C57BL , Serial Passage , Virulence
20.
Methods Mol Biol ; 2099: 137-159, 2020.
Article in English | MEDLINE | ID: covidwho-1292550

ABSTRACT

Since 2012, monthly cases of Middle East respiratory syndrome coronavirus (MERS-CoV) continue to cause severe respiratory disease that is fatal in ~35% of diagnosed individuals. The ongoing threat to global public health and the need for novel therapeutic countermeasures have driven the development of animal models that can reproducibly replicate the pathology associated with MERS-CoV in human infections. The inability of MERS-CoV to replicate in the respiratory tracts of mice, hamsters, and ferrets stymied initial attempts to generate small animal models. Identification of human dipeptidyl peptidase IV (hDPP4) as the receptor for MERS-CoV infection opened the door for genetic engineering of mice. Precise molecular engineering of mouse DPP4 (mDPP4) with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology maintained inherent expression profiles, and limited MERS-CoV susceptibility to tissues that naturally express mDPP4, notably the lower respiratory tract wherein MERS-CoV elicits severe pulmonary pathology. Here, we describe the generation of the 288-330+/+ MERS-CoV mouse model in which mice were made susceptible to MERS-CoV by modifying two amino acids on mDPP4 (A288 and T330), and the use of adaptive evolution to generate novel MERS-CoV isolates that cause fatal respiratory disease. The 288-330+/+ mice are currently being used to evaluate novel drug, antibody, and vaccine therapeutic countermeasures for MERS-CoV. The chapter starts with a historical perspective on the emergence of MERS-CoV and animal models evaluated for MERS-CoV pathogenesis, and then outlines the development of the 288-330+/+ mouse model, assays for assessing a MERS-CoV pulmonary infection in a mouse model, and describes some of the challenges associated with using genetically engineered mice.


Subject(s)
Coronavirus Infections/virology , Dipeptidyl Peptidase 4/genetics , Disease Models, Animal , Mice/genetics , Middle East Respiratory Syndrome Coronavirus/physiology , Respiratory Distress Syndrome/virology , Animals , CRISPR-Cas Systems , Coronavirus Infections/pathology , Dipeptidyl Peptidase 4/metabolism , Disease Susceptibility , Female , Genetic Engineering , Humans , Lung/virology , Male , Mice, Inbred C57BL , Respiratory Distress Syndrome/pathology
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