Structural basis for ultrapotent antibody-mediated neutralization of human metapneumovirus.

Human metapneumovirus (hMPV) is a leading cause of morbidity and hospitalization among children worldwide, however, no vaccines or therapeutics are currently available for hMPV disease prevention and treatment. The hMPV fusion (F) protein is the sole target of neutralizing antibodies. To map the immunodominant epitopes on the hMPV F protein, we isolated a panel of human monoclonal antibodies (mAbs), and the mAbs were assessed for binding avidity, neutralization potency, and epitope specificity. We found the majority of the mAbs target diverse epitopes on the hMPV F protein, and we discovered multiple mAb binding approaches for antigenic site III. The most potent mAb, MPV467, which had picomolar potency, was examined in prophylactic and therapeutic mouse challenge studies, and MPV467 limited virus replication in mouse lungs when administered 24 h before or 72 h after viral infection. We determined the structure of MPV467 in complex with the hMPV F protein using cryo-electron microscopy to a resolution of 3.3 Å, which revealed a complex novel prefusion-specific epitope overlapping antigenic sites II and V on a single protomer. Overall, our data reveal insights into the immunodominant antigenic epitopes on the hMPV F protein, identify a mAb therapy for hMPV F disease prevention and treatment, and provide the discovery of a prefusion-specific epitope on the hMPV F protein.

Species-agnostic polymeric formulations for inhalable messenger RNA delivery to the lung.

Messenger RNA has now been used to vaccinate millions of people. However, the diversity of pulmonary pathologies, including infections, genetic disorders, asthma and others, reveals the lung as an important organ to directly target for future RNA therapeutics and preventatives. Here we report the screening of 166 polymeric nanoparticle formulations for functional delivery to the lungs, obtained from a combinatorial synthesis approach combined with a low-dead-volume nose-only inhalation system for mice. We identify P76, a poly-ß-amino-thio-ester polymer, that exhibits increased expression over formulations lacking the thiol component, delivery to different animal species with varying RNA cargos and low toxicity. P76 allows for dose sparing when delivering an mRNA-expressed Cas13a-mediated treatment in a SARS-CoV-2 challenge model, resulting in similar efficacy to a 20-fold higher dose of a neutralizing antibody. Overall, the combinatorial synthesis approach allowed for the discovery of promising polymeric formulations for future RNA pharmaceutical development for the lungs.

G-Protein-Coupled Receptor and Ion Channel Genes Used by Influenza Virus for Replication.

Influenza virus causes epidemics and sporadic pandemics resulting in morbidity, mortality, and economic losses. Influenza viruses require host genes to replicate. RNA interference (RNAi) screens can identify host genes coopted by influenza virus for replication. Targeting these proinfluenza genes can provide therapeutic strategies to reduce virus replication. Nineteen proinfluenza G-protein-coupled receptor (GPCR) and 13 proinfluenza ion channel genes were identified in human lung (A549) cells by use of small interfering RNAs (siRNAs). These proinfluenza genes were authenticated by testing influenza virus A/WSN/33-, A/CA/04/09-, and B/Yamagata/16/1988-infected A549 cells, resulting in the validation of 16 proinfluenza GPCR and 5 proinfluenza ion channel genes. These findings showed that several GPCR and ion channel genes are needed for the production of infectious influenza virus. These data provide potential targets for the development of host-directed therapeutic strategies to impede the influenza virus productive cycle so as to limit infection.IMPORTANCE Influenza epidemics result in morbidity and mortality each year. Vaccines are the most effective preventive measure but require annual reformulation, since a mismatch of vaccine strains can result in vaccine failure. Antiviral measures are desirable particularly when vaccines fail. In this study, we used RNAi screening to identify several GPCR and ion channel genes needed for influenza virus replication. Understanding the host genes usurped by influenza virus during viral replication can help identify host genes that can be targeted for drug repurposing or for the development of antiviral drugs. The targeting of host genes is refractory to drug resistance generated by viral mutations, as well as providing a platform for the development of broad-spectrum antiviral drugs.

Structure, Immunogenicity, and Conformation-Dependent Receptor Binding of the Postfusion Human Metapneumovirus F Protein.

Human metapneumovirus (hMPV) is an important cause of acute viral respiratory infection. As the only target of neutralizing antibodies, the hMPV fusion (F) protein has been a major focus for vaccine development and targeting by drugs and monoclonal antibodies (MAbs). While X-ray structures of trimeric prefusion and postfusion hMPV F proteins from genotype A, and monomeric prefusion hMPV F protein from genotype B have been determined, structural data for the postfusion conformation for genotype B is lacking. We determined the crystal structure of this protein and compared the structural differences of postfusion hMPV F between hMPV A and B genotypes. We also assessed the receptor binding properties of the hMPV F protein to heparin and heparan sulfate (HS). A library of HS oligomers was used to verify the HS binding activity of hMPV F, and several compounds showed binding to predominantly prefusion hMPV F, but had limited binding to postfusion hMPV F. Furthermore, MAbs to antigenic sites III and the 66-87 intratrimeric epitope block heparin binding. In addition, we evaluated the efficacy of postfusion hMPV B2 F protein as a vaccine candidate in BALB/c mice. Mice immunized with hMPV B2 postfusion F protein showed a balanced Th1/Th2 immune response and generated neutralizing antibodies against both subgroup A2 and B2 hMPV strains, which protected the mice from hMPV challenge. Antibody competition analysis revealed the antibodies generated by immunization target two known antigenic sites (III and IV) on the hMPV F protein. Overall, this study provides new characteristics of the hMPV F protein, which may be informative for vaccine and therapy development. IMPORTANCE Human metapneumovirus (hMPV) is an important cause of viral respiratory disease. In this paper, we report the X-ray crystal structure of the hMPV fusion (F) protein in the postfusion conformation from genotype B. We also assessed binding of the hMPV F protein to heparin and heparan sulfate, a previously reported receptor for the hMPV F protein. Furthermore, we determined the immunogenicity and protective efficacy of postfusion hMPV B2 F protein, which is the first study using a homogenous conformation of the protein. Antibodies generated in response to vaccination give a balanced Th1/Th2 response and target two previously discovered neutralizing epitopes.

Innate Antiviral Cytokine Response to Swine Influenza Virus by Swine Respiratory Epithelial Cells.

Swine influenza virus (SIV) can cause respiratory illness in swine. Swine contribute to influenza virus reassortment, as avian, human, and/or swine influenza viruses can infect swine and reassort, and new viruses can emerge. Thus, it is important to determine the host antiviral responses that affect SIV replication. In this study, we examined the innate antiviral cytokine response to SIV by swine respiratory epithelial cells, focusing on the expression of interferon (IFN) and interferon-stimulated genes (ISGs). Both primary and transformed swine nasal and tracheal respiratory epithelial cells were examined following infection with field isolates. The results show that IFN and ISG expression is maximal at 12 h postinfection (hpi) and is dependent on cell type and virus genotype. IMPORTANCE Swine are considered intermediate hosts that have facilitated influenza virus reassortment events that have given rise pandemics or genetically related viruses have become established in swine. In this study, we examine the innate antiviral response to swine influenza virus in primary and immortalized swine nasal and tracheal epithelial cells, and show virus strain- and host cell type-dependent differential expression of key interferons and interferon-stimulated genes.

Silver nanotriangle array based LSPR sensor for rapid coronavirus detection.

A rapid, portable, and cost-effective method to detect the infection of SARS-CoV-2 is fundamental toward mitigating the current COVID-19 pandemic. Herein, a human angiotensin-converting enzyme 2 protein (ACE2) functionalized silver nanotriangle (AgNT) array localized surface plasmon resonance (LSPR) sensor is developed for rapid coronavirus detection, which is validated by SARS-CoV-2 spike RBD protein and CoV NL63 virus with high sensitivity and specificity. A linear shift of the LSPR wavelength versus the logarithm of the concentration of the spike RBD protein and CoV NL63 is observed. The limits of detection for the spike RBD protein, CoV NL63 in buffer and untreated saliva are determined to be 0.83 pM, 391 PFU/mL, and 625 PFU/mL, respectively, while the detection time is found to be less than 20 min. Thus, the AgNT array optical sensor could serve as a potential rapid point-of-care COVID-19 diagnostic platform.

MicroRNAs affect GPCR and Ion channel genes needed for influenza replication.

Influenza virus causes seasonal epidemics and sporadic pandemics resulting in morbidity, mortality, and economic losses worldwide. Understanding how to regulate influenza virus replication is important for developing vaccine and therapeutic strategies. Identifying microRNAs (miRs) that affect host genes used by influenza virus for replication can support an antiviral strategy. In this study, G-protein coupled receptor (GPCR) and ion channel (IC) host genes in human alveolar epithelial (A549) cells used by influenza virus for replication (Orr-Burks et al., 2021) were examined as miR target genes following A/CA/04/09- or B/Yamagata/16/1988 replication. Thirty-three miRs were predicted to target GPCR or IC genes and their miR mimics were evaluated for their ability to decrease influenza virus replication. Paired miR inhibitors were used as an ancillary measure to confirm or not the antiviral effects of a miR mimic. Fifteen miRs lowered influenza virus replication and four miRs were found to reduce replication irrespective of virus strain and type differences. These findings provide evidence for novel miR disease intervention strategies for influenza viruses.

A Potent Neutralizing Site III-Specific Human Antibody Neutralizes Human Metapneumovirus In Vivo.

Human metapneumovirus (hMPV) is a leading cause of viral lower respiratory tract infection in children. The sole target of neutralizing antibodies targeting hMPV is the fusion (F) protein, a class I viral fusion protein mediating virus-cell membrane fusion. There have been several monoclonal antibodies (mAbs) isolated that neutralize hMPV; however, determining the antigenic sites on the hMPV F protein mediating such neutralizing antibody generation would assist efforts for effective vaccine design. In this report, the isolation and characterization of four new human mAbs, termed MPV196, MPV201, MPV314, and MPV364, are described. Among the four mAbs, MPV364 was found to be the most potent neutralizing mAb in vitro Binding studies with monomeric and trimeric hMPV F revealed that MPV364 had the weakest binding affinity for monomeric hMPV F compared to the other three mAbs, yet binding experiments with trimeric hMPV F showed limited differences in binding affinity, suggesting that MPV364 targets an antigenic site incorporating two protomers. Epitope binning studies showed that MPV364 targets antigenic site III on the hMPV F protein and competes for binding with previously discovered mAbs MPE8 and 25P13, both of which cross-react with the respiratory syncytial virus (RSV) F protein. However, MPV364 does not cross-react with the RSV F protein, and the competition profile suggests that it binds to the hMPV F protein in a binding pose slightly shifted from mAbs MPE8 and 25P13. MPV364 was further assessed in vivo and was shown to substantially reduce viral replication in the lungs of BALB/c mice. Overall, these data reveal a new binding region near antigenic site III of the hMPV F protein that elicits potent neutralizing hMPV F-specific mAbs and provide a new panel of neutralizing mAbs that are candidates for therapeutic development.IMPORTANCE Recent progress in understanding the human immune response to respiratory syncytial virus has paved the way for new vaccine antigens and therapeutics to prevent and treat disease. Progress toward understanding the immune response to human metapneumovirus (hMPV) has lagged behind, although hMPV is a leading cause of lower respiratory tract infection in children. In this report, we advanced the field by isolating a panel of human mAbs to the hMPV F protein. One potent neutralizing mAb, MPV364, targets antigenic site III on the hMPV F protein and incorporates two protomers into its epitope yet is unique from previously discovered site III mAbs, as it does not cross-react with the RSV F protein. We further examined MPV364 in vivo and found that it limits viral replication in BALB/c mice. Altogether, these data provide new mAb candidates for therapeutic development and provide insights into hMPV vaccine development.

Probenecid Inhibits Influenza A(H5N1) and A(H7N9) Viruses In Vitro and in Mice.

Avian influenza (AI) viruses cause infection in birds and humans. Several H5N1 and H7N9 variants are highly pathogenic avian influenza (HPAI) viruses. H5N1 is a highly infectious bird virus infecting primarily poultry, but unlike other AIs, H5N1 also infects mammals and transmits to humans with a case fatality rate above 40%. Similarly, H7N9 can infect humans, with a case fatality rate of over 40%. Since 1996, there have been several HPAI outbreaks affecting humans, emphasizing the need for safe and effective antivirals. We show that probenecid potently inhibits H5N1 and H7N9 replication in prophylactically or therapeutically treated A549 cells and normal human broncho-epithelial (NHBE) cells, and H5N1 replication in VeroE6 cells and mice.

Rapid Detection of SARS-CoV-2 Variants Using an Angiotensin-Converting Enzyme 2-Based Surface-Enhanced Raman Spectroscopy Sensor Enhanced by CoVari Deep Learning Algorithms.

An integrated approach combining surface-enhanced Raman spectroscopy (SERS) with a specialized deep learning algorithm to rapidly and accurately detect and quantify SARS-CoV-2 variants is developed based on an angiotensin-converting enzyme 2 (ACE2)-functionalized AgNR@SiO2 array SERS sensor. SERS spectra with concentrations of different variants were collected using a portable Raman system. After appropriate spectral preprocessing, a deep learning algorithm, CoVari, is developed to predict both the viral variant species and concentrations. Using a 10-fold cross-validation strategy, the model achieves an average accuracy of 99.9% in discriminating between different virus variants and R2 values larger than 0.98 for quantifying viral concentrations of the three viruses, demonstrating the high quality of the detection. The limit of detection of the ACE2 SERS sensor is determined to be 10.472, 11.882, and 21.591 PFU/mL for SARS-CoV-2, SARS-CoV-2 B1, and CoV-NL63, respectively. The feature importance of virus classification and concentration regression in the CoVari algorithm are calculated based on a permutation algorithm, which showed a clear correlation to the biochemical origins of the spectra or spectral changes. In an unknown specimen test, classification accuracy can achieve >90% for concentrations larger than 781 PFU/mL, and the predicted concentrations consistently align with actual values, highlighting the robustness of the proposed algorithm. Based on the CoVari architecture and the output vector, this algorithm can be generalized to predict both viral variant species and concentrations simultaneously for a broader range of viruses. These results demonstrate that the SERS + CoVari strategy has the potential for rapid and quantitative detection of virus variants and potentially point-of-care diagnostic platforms.

Antiviral Activity of Probenecid and Oseltamivir on Influenza Virus Replication.

Influenza can cause respiratory infections, leading to significant morbidity and mortality in humans. While current influenza vaccines offer varying levels of protection, there remains a pressing need for effective antiviral drugs to supplement vaccine efforts. Currently, the FDA-approved antiviral drugs for influenza include oseltamivir, zanamivir, peramivir, and baloxavir marboxil. These antivirals primarily target the virus, making them vulnerable to drug resistance. In this study, we evaluated the efficacy of the neuraminidase inhibitor, oseltamivir, against probenecid, which targets the host cells and is less likely to engender resistance. Our results show that probenecid has superior antiviral efficacy compared to oseltamivir in both in vitro replication assays and in vivo mouse models of influenza infection.

Immunogenicity and protective efficacy of an RSV G S177Q central conserved domain nanoparticle vaccine.

Introduction: Respiratory syncytial virus (RSV) can cause lower respiratory tract disease in infants and elderly populations. Despite decades of research, there remains no safe and approved RSV vaccine. Previously, we showed that an RSV G glycoprotein subunit vaccine candidate with a single point mutation within the central conserved domain (CCD), i.e. S177Q, considerably improved immunogenicity. Methods: Here, we examine the development of nanoparticle (NP) vaccines having either an RSV G protein CCD with wild-type sequence (NPWT) or an S177Q mutation (NP-S177Q). The NP vaccine immunogens were adjuvanted with monophosphoryl lipid A (MPLA), a TLR4 agonist to improve Th1- type responses. BALB/c mice were primed with 10 µg of NP-WT vaccine, NPS177Q, or vehicle, rested, and then boosted with a high (25 µg) or low (10 µg) dose of the NP-WT or NP-S177Q homologous candidate and subsequently challenged with RSV A2. Results: The results showed that mice boosted with NP-S177Q developed superior immunogenicity and neutralizing antibodies compared to NP-WT boosting. IgG from either NP-S177Q or NP-WT vaccinated mice did not interfere with fractalkine (CX3CL1) binding to CX3CR1 and effectively blocked G protein CX3C-CX3CR1 binding. Both NP-WT and NP-S177Q vaccination induced similar neutralizing antibodies to RSV in challenged mice compared to vehicle control. NP-S177Q boosting improved correlates of protection including reduced BAL cell infiltration following RSV challenge. However, the NP vaccine platform will require improvement due to the poor solubility and the unexpectedly weaker Th1-type IgG2a response. Discussion: The results from this study support further NP-S177Q vaccine candidate development.

Rapid Detection of SARS-CoV-2 RNA in Human Nasopharyngeal Specimens Using Surface-Enhanced Raman Spectroscopy and Deep Learning Algorithms.

A rapid and cost-effective method to detect the infection of SARS-CoV-2 is fundamental to mitigating the current COVID-19 pandemic. Herein, a surface-enhanced Raman spectroscopy (SERS) sensor with a deep learning algorithm has been developed for the rapid detection of SARS-CoV-2 RNA in human nasopharyngeal swab (HNS) specimens. The SERS sensor was prepared using a silver nanorod array (AgNR) substrate by assembling DNA probes to capture SARS-CoV-2 RNA. The SERS spectra of HNS specimens were collected after RNA hybridization, and the corresponding SERS peaks were identified. The RNA detection range was determined to be 103-109 copies/mL in saline sodium citrate buffer. A recurrent neural network (RNN)-based deep learning model was developed to classify 40 positive and 120 negative specimens with an overall accuracy of 98.9%. For the blind test of 72 specimens, the RNN model gave a 97.2% accuracy prediction for positive specimens and a 100% accuracy for negative specimens. All the detections were performed in 25 min. These results suggest that the DNA-functionalized AgNR array SERS sensor combined with a deep learning algorithm could serve as a potential rapid point-of-care COVID-19 diagnostic platform.

Immune Prophylaxis Targeting the Respiratory Syncytial Virus (RSV) G Protein.

The respiratory syncytial virus (RSV) causes significant respiratory disease in young infants and the elderly. Immune prophylaxis in infants is currently limited to palivizumab, an anti-RSV fusion (F) protein monoclonal antibody (mAb). While anti-F protein mAbs neutralize RSV, they are unable to prevent aberrant pathogenic responses provoked by the RSV attachment (G) protein. Recently, the co-crystal structures of two high-affinity anti-G protein mAbs that bind the central conserved domain (CCD) at distinct non-overlapping epitopes were solved. mAbs 3D3 and 2D10 are broadly neutralizing and block G protein CX3C-mediated chemotaxis by binding antigenic sites γ1 and γ2, respectively, which is known to reduce RSV disease. Previous studies have established 3D3 as a potential immunoprophylactic and therapeutic; however, there has been no similar evaluation of 2D10 available. Here, we sought to determine the differences in neutralization and immunity to RSV Line19F infection which recapitulates human RSV infection in mouse models making it useful for therapeutic antibody studies. Prophylactic (24 h prior to infection) or therapeutic (72 h post-infection) treatment of mice with 3D3, 2D10, or palivizumab were compared to isotype control antibody treatment. The results show that 2D10 can neutralize RSV Line19F both prophylactically and therapeutically, and can reduce disease-causing immune responses in a prophylactic but not therapeutic context. In contrast, 3D3 was able to significantly (p < 0.05) reduce lung virus titers and IL-13 in a prophylactic and therapeutic regimen suggesting subtle but important differences in immune responses to RSV infection with mAbs that bind distinct epitopes.

Rapid and quantitative detection of respiratory viruses using surface-enhanced Raman spectroscopy and machine learning.

Rapid and sensitive pathogen detection is important for prevention and control of disease. Here, we report a label-free diagnostic platform that combines surface-enhanced Raman scattering (SERS) and machine learning for the rapid and accurate detection of thirteen respiratory virus species including SARS-CoV-2, common human coronaviruses, influenza viruses, and others. Virus detection and measurement have been performed using highly sensitive SiO2 coated silver nanorod array substrates, allowing for detection and identification of their characteristic SERS peaks. Using appropriate spectral processing procedures and machine learning algorithms (MLAs) including support vector machine (SVM), k-nearest neighbor, and random forest, the virus species as well as strains and variants have been differentiated and classified and a differentiation accuracy of >99% has been obtained. Utilizing SVM-based regression, quantitative calibration curves have been constructed to accurately estimate the unknown virus concentrations in buffer and saliva. This study shows that using a combination of SERS, MLA, and regression, it is possible to classify and quantify the virus in saliva, which could aid medical diagnosis and therapeutic intervention.

Probenecid Inhibits Respiratory Syncytial Virus (RSV) Replication.

RNA viruses like SARS-CoV-2, influenza virus, and respiratory syncytial virus (RSV) are dependent on host genes for replication. We investigated if probenecid, an FDA-approved and safe urate-lowering drug that inhibits organic anion transporters (OATs) has prophylactic or therapeutic efficacy to inhibit RSV replication in three epithelial cell lines used in RSV studies, i.e., Vero E6 cells, HEp-2 cells, and in primary normal human bronchoepithelial (NHBE) cells, and in BALB/c mice. The studies showed that nanomolar concentrations of all probenecid regimens prevent RSV strain A and B replication in vitro and RSV strain A in vivo, representing a potential prophylactic and chemotherapeutic for RSV.

Efficacy of Parainfluenza Virus 5 (PIV5)-vectored Intranasal COVID-19 Vaccine as a Single Dose Vaccine and as a Booster against SARS-CoV-2 Variants.

Immunization with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines has greatly reduced coronavirus disease 2019 (COVID-19)-related deaths and hospitalizations, but waning immunity and the emergence of variants capable of immune escape indicate the need for novel SARS-CoV-2 vaccines. An intranasal parainfluenza virus 5 (PIV5)-vectored COVID-19 vaccine CVXGA1 has been proven efficacious in animal models and blocks contact transmission of SARS-CoV-2 in ferrets. CVXGA1 vaccine is currently in human clinical trials in the United States. This work investigates the immunogenicity and efficacy of CVXGA1 and other PIV5-vectored vaccines expressing additional antigen SARS-CoV-2 nucleoprotein (N) or SARS-CoV-2 variant spike (S) proteins of beta, delta, gamma, and omicron variants against homologous and heterologous challenges in hamsters. A single intranasal dose of CVXGA1 induces neutralizing antibodies against SARS-CoV-2 WA1 (ancestral), delta variant, and omicron variant and protects against both homologous and heterologous virus challenges. Compared to mRNA COVID-19 vaccine, neutralizing antibody titers induced by CVXGA1 were well-maintained over time. When administered as a boost following two doses of a mRNA COVID-19 vaccine, PIV5-vectored vaccines expressing the S protein from WA1 (CVXGA1), delta, or omicron variants generate higher levels of cross-reactive neutralizing antibodies compared to three doses of a mRNA vaccine. In addition to the S protein, the N protein provides added protection as assessed by the highest body weight gain post-challenge infection. Our data indicates that PIV5-vectored COVID-19 vaccines, such as CVXGA1, can serve as booster vaccines against emerging variants.

Exploring Noncovalent Protease Inhibitors for the Treatment of Severe Acute Respiratory Syndrome and Severe Acute Respiratory Syndrome-Like Coronaviruses.

Over the last 20 years, both severe acute respiratory syndrome coronavirus-1 and severe acute respiratory syndrome coronavirus-2 have transmitted from animal hosts to humans causing zoonotic outbreaks of severe disease. Both viruses originate from a group of betacoronaviruses known as subgroup 2b. The emergence of two dangerous human pathogens from this group along with previous studies illustrating the potential of other subgroup 2b members to transmit to humans has underscored the need for antiviral development against them. Coronaviruses modify the host innate immune response in part through the reversal of ubiquitination and ISGylation with their papain-like protease (PLpro). To identify unique or overarching subgroup 2b structural features or enzymatic biases, the PLpro from a subgroup 2b bat coronavirus, BtSCoV-Rf1.2004, was biochemically and structurally evaluated. This evaluation revealed that PLpros from subgroup 2b coronaviruses have narrow substrate specificity for K48 polyubiquitin and ISG15 originating from certain species. The PLpro of BtSCoV-Rf1.2004 was used as a tool alongside PLpro of CoV-1 and CoV-2 to design 30 novel noncovalent drug-like pan subgroup 2b PLpro inhibitors that included determining the effects of using previously unexplored core linkers within these compounds. Two crystal structures of BtSCoV-Rf1.2004 PLpro bound to these inhibitors aided in compound design as well as shared structural features among subgroup 2b proteases. Screening of these three subgroup 2b PLpros against this novel set of inhibitors along with cytotoxicity studies provide new directions for pan-coronavirus subgroup 2b antiviral development of PLpro inhibitors.

Nebulized mRNA-Encoded Antibodies Protect Hamsters from SARS-CoV-2 Infection.

Despite the success of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines, there remains a clear need for new classes of preventatives for respiratory viral infections due to vaccine hesitancy, lack of sterilizing immunity, and for at-risk patient populations, including the immunocompromised. While many neutralizing antibodies have been identified, and several approved, to treat COVID-19, systemic delivery, large doses, and high costs have the potential to limit their widespread use, especially in low- and middle-income countries. To use these antibodies more efficiently, an inhalable formulation is developed that allows for the expression of mRNA-encoded, membrane-anchored neutralizing antibodies in the lung to mitigate SARS-CoV-2 infections. First, the ability of mRNA-encoded, membrane-anchored, anti-SARS-CoV-2 antibodies to prevent infections in vitro is demonstrated. Next, it is demonstrated that nebulizer-based delivery of these mRNA-expressed neutralizing antibodies potently abrogates disease in the hamster model. Overall, these results support the use of nebulizer-based mRNA expression of neutralizing antibodies as a new paradigm for mitigating respiratory virus infections.

Drug repositioning of Clopidogrel or Triamterene to inhibit influenza virus replication in vitro.

Influenza viruses cause respiratory tract infections and substantial health concerns. Infection may result in mild to severe respiratory disease associated with morbidity and some mortality. Several anti-influenza drugs are available, but these agents target viral components and are susceptible to drug resistance. There is a need for new antiviral drug strategies that include repurposing of clinically approved drugs. Drugs that target cellular machinery necessary for influenza virus replication can provide a means for inhibiting influenza virus replication. We used RNA interference screening to identify key host cell genes required for influenza replication, and then FDA-approved drugs that could be repurposed for targeting host genes. We examined the effects of Clopidogrel and Triamterene to inhibit A/WSN/33 (EC50 5.84 uM and 31.48 uM, respectively), A/CA/04/09 (EC50 6.432 uM and 3.32 uM, respectively), and B/Yamagata/16/1988 (EC50 0.28 uM and 0.11 uM, respectively) replication. Clopidogrel and Triamterene provide a druggable approach to influenza treatment across multiple strains and subtypes.