Publications

SARS-CoV-2 infection results in viral burden in the respiratory tract, enabling transmission and leading to substantial lung pathology. The 1212C2 fully human monoclonal antibody was derived from an IgM memory B cell of a COVID-19 patient, has high affinity for the Spike protein receptor binding domain, neutralizes SARS-CoV-2, and exhibits in vivo prophylactic and therapeutic activity in hamsters when delivered intraperitoneally, reducing upper and lower respiratory viral burden and lung pathology. Inhalation of nebulized 1212C2 at levels as low as 0.6 mg/kg, corresponding to 0.03 mg/kg lung-deposited dose, reduced the viral burden below the detection limit and mitigated lung pathology. The therapeutic efficacy of an exceedingly low dose of inhaled 1212C2 supports the rationale for local lung delivery for dose-sparing benefits, as compared to the conventional parenteral route of administration. These results suggest that the clinical development of 1212C2 formulated and delivered via inhalation for the treatment of SARS-CoV-2 infection should be considered.

The current pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights an urgent need to develop a safe, efficacious, and durable vaccine. Using a measles virus (rMeV) vaccine strain as the backbone, we developed a series of recombinant attenuated vaccine candidates expressing various forms of the SARS-CoV-2 spike (S) protein and its receptor binding domain (RBD) and evaluated their efficacy in cotton rat, IFNAR-/-mice, IFNAR-/–hCD46 mice, and golden Syrian hamsters. We found that rMeV expressing stabilized prefusion S protein (rMeV-preS) was more potent in inducing SARS-CoV-2-specific neutralizing antibodies than rMeV expressing full-length S protein (rMeV-S), while the rMeVs expressing different lengths of RBD (rMeV-RBD) were the least potent. Animals immunized with rMeV-preS produced higher levels of neutralizing antibody than found in convalescent sera from COVID-19 patients and a strong Th1-biased T cell response. The rMeV-preS also provided complete protection of hamsters from challenge with SARS-CoV-2, preventing replication in lungs and nasal turbinates, body weight loss, cytokine storm, and lung pathology. These data demonstrate that rMeV-preS is a safe and highly efficacious vaccine candidate, supporting its further development as a SARS-CoV-2 vaccine.

With continued expansion of the coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome 2 (SARS-CoV-2), both antiviral drugs as well as effective vaccines are desperately needed to treat patients at high risk of life-threatening disease. Here, we present in vitro evidence for significant inhibition of SARS-CoV-2 by oleandrin and a defined extract of N. oleander (designated as PBI-06150). Using Vero cells, we found that prophylactic (pre-infection) oleandrin (as either the pure compound or as the active principal ingredient in PBI-06150) administration at concentrations as low as 0.05 µg/ml exhibited potent antiviral activity against SARS-CoV-2, with an 800-fold reduction in virus production, and a 0.1 µg/ml concentration resulted in a greater than 3000-fold reduction in infectious virus production. The half maximal effective concentration (EC50) values were 11.98 ng/ml when virus output was measured at 24 h post-infection, and 7.07 ng/ml measured at 48 h post-infection. Therapeutic (post-infection) treatment up to 24 h after SARS-CoV-2 infection of Vero cells also reduced viral titers, with 0.1 µg/ml and 0.05 µg/ml concentrations causing greater than 100-fold reduction as measured at 48 h, and the 0.05 µg/ml concentration resulting in a 78-fold reduction. Concentrations of oleandrin up to 10 µg/ml were well tolerated in Vero cells. We also present in vivo evidence of the safety and efficacy of defined N. oleander extract (PBI-06150), which was administered to golden Syrian hamsters in a preparation containing as high as 130 µg/ml of oleandrin. In comparison to administration of control vehicle, PBI-06150 provided a statistically significant reduction of the viral titer in the nasal turbinates (nasal conchae). The potent prophylactic and therapeutic antiviral activities demonstrated here, together with initial evidence of its safety and efficacy in a relevant hamster model of COVID-19, support the further development of oleandrin and/or defined extracts containing this molecule for the treatment of SARS-CoV-2 and associated COVID-19 disease and potentially also for reduction of virus spread by persons diagnosed early after infection.

The influenza A virus (IAV) is able to infect multiple mammalian and avian species, and in humans IAV is responsible for annual seasonal epidemics and occasional pandemics of respiratory disease with significant health and economic impacts. Studying IAV involves laborious secondary methodologies to identify infected cells. Therefore, to circumvent this requirement, in recent years, multiple replication-competent infectious IAV expressing traceable reporter genes have been developed. These IAVs have been very useful for in vitro and/or in vivo studies of viral replication, identification of neutralizing antibodies or antivirals, and in studies to evaluate vaccine efficacy, among others. In this report, we describe, for the first time, the generation and characterization of two replication-competent influenza A/Puerto Rico/8/1934 H1N1 (PR8) viruses where the viral non-structural protein 1 (NS1) was substituted by the monomeric (m)Cherry fluorescent or the NanoLuc luciferase (Nluc) proteins. The ΔNS1 mCherry was able to replicate in cultured cells and in Signal Transducer and Activator of Transcription 1 (STAT1) deficient mice, although at a lower extent than a wild-type (WT) PR8 virus expressing the same mCherry fluorescent protein (WT mCherry). Notably, expression of either reporter gene (mCherry or Nluc) was detected in infected cells by fluorescent microscopy or luciferase plate readers, respectively. ΔNS1 IAV expressing reporter genes provide a novel approach to better understand the biology and pathogenesis of IAV, and represent an excellent tool to develop new therapeutic approaches against IAV infections.

The influenza A virus (IAV) PA-X protein is a virulence factor that selectively degrades host mRNAs leading to protein shutoff. This function modulates host inflammation, antiviral responses, cell apoptosis, and pathogenesis. In this work we describe a novel approach based on the use of bacteria and plasmid encoding of the PA-X gene under the control of the bacteriophage T7 promoter to identify amino acid residues important for A/Brevig Mission/1/1918 H1N1 PA-X's shutoff activity. Using this system, we have identified PA-X mutants encoding single or double amino acid changes, which diminish its host shutoff activity, as well as its ability to counteract interferon responses upon viral infection. This novel bacteria-based approach could be used for the identification of viral proteins that inhibit host gene expression as well as the amino acid residues responsible for inhibition of host gene expression.

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. As of 19 May 2021, John Hopkins University's COVID-19 tracking platform reported 3.3 million deaths associated with SARS-CoV-2 infection. Currently, the World Health Organization has granted emergency use listing (EUL) to six COVID-19 vaccine candidates. However, much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse-genetics system approach to successfully engineer recombinant SARS-CoV-2 (rSARS-CoV-2) constructs; we removed individual viral ORF3a, -6, -7a, -7b, and -8 proteins from them, and we characterized the resulting recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF-deficient (ΔORF) viruses producing smaller plaques than those of the wild type (rSARS-CoV-2/WT). However, growth kinetics of ΔORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mice with the ΔORF rSARS-CoV-2s identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while ΔORF7a, ΔORF7b, and ΔORF8 rSARS-CoV-2s induced pathology comparable to that of rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse-genetics system to generate rSARS-CoV-2 constructs and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19. IMPORTANCE Despite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and the contribution of viral proteins to disease outcome remain elusive. Our study aims (i) to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins to viral pathogenesis and disease outcome and (ii) to develop a synergistic platform combining our robust reverse-genetics system to generate recombinant SARS-CoV-2 constructs with a validated rodent model of infection and disease. We demonstrate that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as a foundation for generating attenuated forms of the virus to develop live attenuated vaccines for the treatment of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus-2 (SAR-CoV-2) causes coronavirus disease 2019 (COVID19) that is responsible for short and long-term disease, as well as death, in susceptible hosts. The receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) protein binds to cell surface angiotensin converting enzyme type-II (ACE2) to initiate viral attachment and ultimately viral pathogenesis. The SARS-CoV-2 S RBD is a major target of neutralizing antibodies (NAbs) that block RBD - ACE2 interactions. In this report, NAb-RBD binding epitopes in the protein databank were classified as C1, C1D, C2, C3, or C4, using a RBD binding profile (BP), based on NAb-specific RBD buried surface area and used to predict the binding epitopes of a series of uncharacterized NAbs. Naturally occurring SARS-CoV-2 RBD sequence variation was also quantified to predict NAb binding sensitivities to the RBD-variants. NAb and ACE2 binding studies confirmed the NAb classifications and determined whether the RBD variants enhanced ACE2 binding to promote viral infectivity, and/or disrupted NAb binding to evade the host immune response. Of 9 single RBD mutants evaluated, K417T, E484K, and N501Y disrupted binding of 65% of the NAbs evaluated, consistent with the assignment of the SARS-CoV-2 P.1 Japan/Brazil strain as a variant of concern (VoC). RBD variants E484K and N501Y exhibited ACE2 binding equivalent to a Wuhan-1 reference SARS-CoV-2 RBD. While slightly less disruptive to NAb binding, L452R enhanced ACE2 binding affinity. Thus, the L452R mutant, associated with the SARS-CoV-2 California VoC (B.1.427/B.1.429-California), has evolved to enhance ACE2 binding, while simultaneously disrupting C1 and C2 NAb classes. The analysis also identified a non-overlapping antibody pair (1213H7 and 1215D1) that bound to all SARS-CoV-2 RBD variants evaluated, representing an excellent therapeutic option for treatment of SARS-CoV-2 WT and VoC strains.

Influenza B viruses (IBV) circulate annually, with young children, the elderly and immunocompromised individuals being at high risk. Yearly vaccinations are recommended to protect against seasonally influenza viruses, including IBV. Live attenuated influenza vaccines (LAIV) provide the unique opportunity for direct exposure to the antigenically variable surface glycoproteins as well as the more conserved internal components. Ideally, LAIV Master Donor Viruses (MDV) should accurately reflect seasonal influenza strains. Unfortunately, the continuous evolution of IBV have led to significant changes in conserved epitopes compared to the IBV MDV based on B/Ann Arbor/1/1966 strain. Here, we propose a recent influenza B/Brisbane/60/2008 as an efficacious MDV alternative, as its internal viral proteins more accurately reflect those of circulating IBV strains. We introduced the mutations responsible for the temperature sensitive (ts), cold adapted (ca) and attenuated (att) phenotype of B/Ann Arbor/1/1966 MDV LAIV into B/Brisbane/60/2008 to generate a new MDV LAIV. In vitro and in vivo analysis demonstrated that the mutations responsible of the ts, ca, and att phenotype of B/Ann Arbor/1/1966 MDV LAIV were able to infer the same phenotype to B/Brisbane/60/2008, demonstrating its potential as a new MDV for the development of LAIV to protect against contemporary IBV strains.