Biology subjects

Kobie, J.

Publications and source records attributed to Kobie, J..

11 recordsLinked to original sources

bioRxiv2026

A One-Shot Multivalent Live-Attenuated Candidate Influenza Vaccine against Divergent Zoonotic H5N1 Clades

The continued emergence of genetically diverse high pathogenicity avian influenza (HPAI) H5N1 viruses with zoonotic potential highlights the urgent need for developing vaccines capable of providing broad protection against multiple circulating clades. Here, we developed a one-shot, multivalent, live-attenuated influenza vaccine (LAIV) based on the temperature-sensitive (ts), cold-adapted (ca), and attenuated (att) influenza A/Ann Arbor/6/1960 master donor virus (MDV) that incorporates the hemagglutinin (HA) and neuraminidase (NA) glycoproteins from representative clades 2.3.4.4b (A/Louisiana/12/2024), 2.3.2.1a (A/Victoria/149/2024), and 2.3.2.1e (A/Cambodia/2302009/2023) H5N1 viruses. A single intranasal (IN) immunization of C57BL/6 mice with the multivalent LAIV elicited robust humoral immune responses, with immune sera exhibiting broad cross-reactivity against antigens from all three H5N1 clades included in the vaccine. Following homologous viral challenge, vaccinated C57BL/6 mice were completely protected from disease, demonstrating the immunogenicity and protective efficacy of the multivalent LAIV. By simultaneously targeting antigenically distinct H5N1 lineages with pandemic potential, this strategy expands antigenic coverage within a single LAIV to confirm pan-H5N1 protection. Together, these findings support the development and implementation of this multivalent LAIV as a broadly protective pan-H5N1 LAIV for pandemic preparedness. SignificanceThe increasing genetic diversity of zoonotic H5N1 viruses complicates vaccine design. We developed a multivalent live-attenuated influenza vaccine (LAIV) based on the temperature-sensitive, cold-adapted, and attenuated (ts, ca, att) master donor virus (MDV) influenza A/Ann Arbor/6/1960 backbone that expresses the hemagglutinin (HA) and neuraminidase (NA) glycoproteins of H5N1 clades 2.3.4.4b, 2.3.2.1a, and 2.3.2.1e. A single intranasal (IN) immunization with the multivalent LAIV induced broadly cross-reactive neutralizing antibody (NAb) responses and protected experimental vaccinated animals against homologous lethal viral challenge, demonstrating the feasibility of the multivalent LAIV to protect against H5N1 clades of highest concern to humans. These findings demonstrate the feasibility of developing and implementing this multivalent LAIV as a broad protective pan-H5N1 LAIV against divergent H5N1 viruses for human use.

microbiology↗

bioRxiv2026

A bireporter recombinant SARS-CoV-2 Omicron BA.5 for in vitro and in vivo studies

The continuous emergence of variants of concern (VoC) represent a significant challenge to effectively control SARS-CoV-2. Although FDA-approved vaccines and antivirals have been successfully developed and implemented for the prophylactic and therapeutic intervention of SARS-CoV-2 infection, recent VoC could escape protection garnered by previous vaccine and antiviral approaches. Determining the efficacy of prophylactics and/or therapeutics against recent VoC will assist in efficiently controlling currently circulating SARS-CoV-2 strains. We used our previously described bacterial artificial chromosome (BAC)-based reverse genetics approach for Omicron BA.5 to generate a recombinant SARS-CoV-2 BA.5 encoding a fusion of ZsGreen to Nanoluciferase (rBA.5 ZsG-Nluc) from the locus of the viral nucleocapsid (N) protein separated by the porcine teschovirus-1 (PTV-1) 2A proteolytic cleavage site. The rBA5 ZsG-Nluc replicates to levels comparable to recombinant BA.5 wild-type (rBA.5 WT) and expresses high levels of ZsG and Nluc in cultured cells. This facilitates tracking viral infection and the identification of antivirals and neutralizing antibodies (NAbs) with EC50 and NT50 values, respectively, similar to those obtained with rBA.5 WT. Importantly, in K18 hACE2 mice, rBA.5 ZsG-Nluc retains the same pathogenicity and ability to replicate in the lungs of infected mice as rBA.5 WT. Using rBA.5 ZsG-Nluc, we detected Nluc activity systemically and Nluc and ZsG expression in the lungs of infected mice using an in vivo imaging system (IVIS). Our results demonstrate the feasibility of using rBA.5 ZsG-Nluc to track viral infections and identify prophylactics and therapeutics against recent SARS-CoV-2 VoC in vitro, ex vivo, and in vivo. IMPORTANCESARS-CoV-2, the causative virus of the COVID-19 pandemic, is continually evolving to escape immunity acquired by previous natural infections or vaccinations. Moreover, recent SARS-CoV-2 variants of concern (VoC) have acquired antiviral resistant mutations to FDA-approved drugs. The emergence of these VoC highlights the importance of identifying new prophylactics and therapeutics against currently circulating SARS-CoV-2 strains. We generated a recombinant bireporter Omicron BA.5 SARS-CoV-2 (rBA.5 ZsG-Nluc) that expresses reporter proteins, which are useful for cellular and whole animal studies and has similar viral replication and pathogenicity to a wild-type (WT) recombinant Omicron BA.5 SARS-CoV-2 (rBA.5 WT). In K18 hACE2 mice, rBA.5 ZsG-Nluc infection can be tracked systemically or in the lungs of infected mice using an in vivo imaging system (IVIS). We establish a proof-of-concept platform of rBA.5 ZsG-Nluc in combination with an ancestral SARS-CoV-2 strain expressing mCherry to simultaneously identify antivirals and neutralizing antibodies against original and recent SARS-CoV-2 strains.

microbiology↗

bioRxiv2025

A cocktail of SARS-CoV-2 spike stem helix domain and receptor binding domain human monoclonal antibodies prevent the emerge of viral escape mutants

Neutralizing antibodies (NAbs) targeting the spike (S) glycoprotein remain a crucial therapeutic strategy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, emerging viral variants have escaped all Food and Drug Administration (FDA)-approved NAb treatments, underscoring the urgent need for effective therapeutic alternatives. Using a nanoluciferase (Nluc)-expressing attenuated recombinant SARS-CoV-2 lacking the open reading frames (ORF) 3a and 7b ({Delta}3a7b-Nluc), we characterized resistance profiles of two broadly protective NAbs targeting the S receptor binding domain (RBD) in S1 (1301B7) and the stem helix domain (SH) in S2 (1249A8). Serial passaging of {Delta}3a7b-Nluc under selective pressure identified a 1301B7 antibody-resistant mutants (ARM-B7) harboring an RBD mutation (S371F) that conferred resistance to 1301B7 and other RBD-directed NAbs (Casirivimab, SC27 and Sotrovimab). In contrast, no ARM emerged under treatment with 1249A8, or an antibody cocktail of 1301B7 (RBD) + 1249A8 (SH). These findings demonstrate that S2 SH-targeting NAbs shows higher genetic barrier to resistance than S1 RBD-targeting NAbs, and that a NAbs cocktail therapy targeting the SARS-CoV-2 S1 RBD and S2 SH offers the most effective strategy to prevent the emergence of escape mutations. Together, our findings provide critical insights into developing next-generation resistance-evading NAb therapies against SARS-CoV-2, and potentially other coronaviruses, and demonstrate the value of using our attenuated viral platforms for the safe identification of ARM without the potential biosafety concerns of doing these experiments using wild-type (WT) forms of SARS-CoV-2. SIGNIFICANCEThe clinical efficacy of early SARS-CoV-2 NAbs has been challenged by the emergence of escape viral variants, highlighting an urgent need to anticipate resistance. Using a luminescent attenuated SARS-CoV-2 platform, we profiled resistant mutations against two broadly protective SARS-CoV-2 NAbs. Passage of {Delta}3a7b-Nluc in the presence of a NAb targeting the RBD S1 domain (1301B7) readily selected for an ARM, whereas passage in the presence of an SH S2 domain NAb (1249A8) did not. Notably, a cocktail of 1301B7 and 1249A8 created a high barrier of selecting SARS-CoV-2 ARM, preventing the emergence of resistant variants. We identified an S371F mutation in the S1 RBD of ARM-B7 that confers resistance to 1301B7 and other S1 RBD-targeting NAbs. These results highlight the importance of combination therapies targeting both variable RBD S1 and conserved SH S2 domain for the efficient treatment of SARS-CoV-2 and to prevent the emerge of NAb-induced escape mutations.

microbiology↗

bioRxiv2025

Bioluminescent reporter influenza A viruses to track viral infections

Influenza A viruses (IAV) infect a wide range of mammal and bird species and are responsible for seasonal outbreaks and occasional pandemics. Studying IAV requires methods to detect the presence of the virus in infected cells or animal models. Recombinant IAV expressing fluorescent proteins have allowed monitoring viral infection in cultured cells and ex vivo in the organs of infected animals. However, fluorescent-expressing IAV are often attenuated and are not suited for the imaging of infected animals using in vivo imaging systems (IVIS). To overcome this limitation, we generated a recombinant A/California/04/2009 H1N1 (pH1N1) expressing nanoluciferase (Nluc) from the non-structural (NS) viral segment (pH1N1-Nluc) that replicates efficiently in vitro, with growth kinetics and plaque morphology comparable to wild-type pH1N1 (pH1N1-WT). We used this pH1N1-Nluc to demonstrate its ability to identify neutralizing antibodies and antivirals, with neutralization and inhibition results comparable to pH1N1-WT. In mice, pH1N1-Nluc was able to induce similar body weight loss and mortality, and viral titers comparable to pH1N1-WT, results that were recapitulated in a ferret model of IAV infection. Using IVIS, pH1N1-Nluc enabled non-invasive, real-time tracking of viral infection in vivo and ex vivo following infection of mice with viral titers comparable to pH1N1-WT. The flexibility of this approach was further demonstrated by the generation of a Nluc-expressing recombinant A/Puerto Rico/8/1934 H1N1 (PR8-Nluc). Altogether, our results demonstrate that Nluc-expressing recombinant IAV represent a valuable tool for in vitro and in vivo studies, including the identification of antivirals and/or neutralizing antibodies, and to assess protective efficacy of vaccines. IMPORTANCEDespite the availability of recombinant influenza A viruses (IAV) expressing fluorescent reporter genes to track viral infections in vitro and ex vivo, these viruses are often attenuated and do not represent the best option for imaging entire animals using in vivo imaging systems (IVIS). To solve this limitation, we generated recombinant influenza pandemic A/California/04/2009 H1N1 expressing nanoluciferase (pH1N1-Nluc) from the viral non-structural (NS) segment and demonstrate how expression of Nluc does not affect viral replication in vitro or viral pathogenesis in vivo. Importantly, we demonstrate the feasibility of detecting pH1N1-Nluc infection in vivo using IVIS. We also validate the flexibility of this approach by generating an influenza A/Puerto Rico/8/1934 H1N1 (PR8-Nluc). Our results support the feasibility of using these recombinant IAVs expressing Nluc from the NS segment for in vitro and in vivo studies, including the identification of neutralizing antibodies and/or antivirals, and to assess protective efficacy of vaccines.

microbiology↗

bioRxiv2022

Structure and Epitope of a Neutralizing Monoclonal Antibody that Targets the Stem Helix of β Coronaviruses

Monoclonal antibodies (MAbs) that retain neutralizing activity against distinct coronavirus (CoV) lineages and variants of concern (VoC) must be developed to protect against future pandemics. These broadly neutralizing MAbs (BNMAbs) may be used as therapeutics and/or to assist in the rational design of vaccines that induce BNMAbs. 1249A8 is a BNMAb that targets the stem helix (SH) region of CoV spike (S) protein and neutralizes Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) original strain, delta, and omicron VoC, Severe Acute Respiratory Syndrome CoV (SARS-CoV) and Middle East Respiratory Syndrome CoV (MERS-CoV). To understand its mechanism of action, the crystal structure of 1249A8 bound to a MERS-CoV SH peptide was determined at 2.1[A] resolution. BNMAb 1249A8 mimics the SARS-CoV-2 S loop residues 743-749, which interact with the C-terminal end of the SH helix in the S postfusion conformation. The crystal structure shows that BNMAb 1249A8 disrupts SH secondary structure and packing rearrangements required for CoV S to adopt its prefusion conformation that mediates membrane fusion and ultimately infection. The mechanisms regulating BNMAb 1249A8 CoV S specificity are also defined. This study provides novel insights into the neutralization mechanisms of SH-targeting CoV BNMAbs that may inform vaccine development and the design of optimal BNMAb therapeutics.

immunology↗

bioRxiv2022

Monitoring SARS-CoV-2 infection using a double reporter-expressing virus

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the highly contagious agent responsible for the coronavirus disease 2019 (COVID-19) pandemic. An essential requirement for understanding SARS-CoV-2 fundamental biology and the impact of anti-viral therapeutics are robust methods to detect for the presence of the virus in infected cells or animal models. Despite the development and successful generation of recombinant (r)SARS-CoV-2 expressing fluorescent or luciferase reporter genes, knowledge acquired from their use in in vitro assays and/or in live animals are limited to the properties of the fluorescent or luciferase reporter genes. Herein, for the first time, we engineered a replication-competent rSARS-CoV-2 that expresses both fluorescent (mCherry) and luciferase (Nluc) reporter genes (rSARS-CoV-2/mCherry-Nluc) to overcome limitations associated with the use of a single reporter gene. In cultured cells, rSARS-CoV-2/mCherry-Nluc displayed similar viral fitness as rSARS-CoV-2 expressing single reporter fluorescent and luciferase genes (rSARS-CoV-2/mCherry and rSARS-CoV-2/Nluc, respectively), or wild-type (WT) rSARS-CoV-2, while maintaining comparable expression levels of both reporter genes. In vivo, rSARS-CoV-2/mCherry-Nluc has similar pathogenicity in K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice than rSARS-CoV-2 expressing individual reporter genes, or WT rSARS-CoV-2. Importantly, rSARS-CoV-2/mCherry-Nluc facilitates the assessment of viral infection and transmission in golden Syrian hamsters using in vivo imaging systems (IVIS). Altogether, this study demonstrates the feasibility of using this novel bireporter-expressing rSARS-CoV-2 for the study SARS-CoV-2 in vitro and in vivo. IMPORTANCEDespite the availability of vaccines and antivirals, the coronavirus disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to ravage health care institutions worldwide. Previously, we have generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent or luciferase reporter proteins to track viral infection in vitro and/or in vivo. However, these rSARS-CoV-2 are restricted to express only a single fluorescent or a luciferase reporter gene, limiting or preventing their use to specific in vitro assays and/or in vivo studies. To overcome this limitation, we have engineered a rSARS-CoV-2 expressing both fluorescent (mCherry) and luciferase (Nluc) genes and demonstrated its feasibility to study the biology of SARS-CoV-2 in vitro and/or in vivo, including the identification and characterization of neutralizing antibodies and/or antivirals. Using rodent models, we visualize SARS-CoV-2 infection and transmission through in vivo imaging systems (IVIS).

microbiology↗

bioRxiv2022

Immunization with recombinant accessory protein-deficient SARS-CoV-2 protects against lethal challenge and viral transmission

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to a worldwide Coronavirus Disease 2019 (COVID-19) pandemic. Despite high efficacy of the authorized vaccines, protection against the surging variants of concern (VoC) was less robust. Live-attenuated vaccines (LAV) have been shown to elicit robust and long-term protection by induction of host innate and adaptive immune responses. We sought to develop a COVID-19 LAV by generating 3 double open reading frame (ORF)-deficient recombinant (r)SARS-CoV-2 simultaneously lacking two accessory open reading frame (ORF) proteins (ORF3a/ORF6, ORF3a/ORF7a, and ORF3a/ORF7b). Here, we report that these double ORF-deficient rSARS-CoV-2 have slower replication kinetics and reduced fitness in cultured cells as compared to their parental wild-type (WT) counterpart. Importantly, these double ORF-deficient rSARS-CoV-2 showed attenuation in both K18 hACE2 transgenic mice and golden Syrian hamsters. A single intranasal dose vaccination induced high levels of neutralizing antibodies against different SARS-CoV-2 VoC, and also activated viral component-specific T-cell responses. Notably, the double ORF-deficient rSARS-CoV-2 were able to protect, as determined by inhibition of viral replication, shedding, and transmission, against challenge with SARS-CoV-2. Collectively, our results demonstrate the feasibility to implement these double ORF-deficient rSARS-CoV-2 as safe, stable, immunogenic and protective LAV for the prevention of SARS-CoV-2 infection and associated COVID-19 disease.

microbiology↗

bioRxiv2021

A bifluorescent-based assay for the identification of neutralizing antibodies against SARS-CoV-2 variants of concern in vitro and in vivo

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and has been responsible for the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Prophylactic vaccines have been authorized by the United States (US) Food and Drug Administration (FDA) for the prevention of COVID-19. Identification of SARS-CoV-2 neutralizing antibodies (NAbs) is important to assess vaccine protection efficacy, including their ability to protect against emerging SARS- CoV-2 variants of concern (VoC). Here we report the generation and use of a recombinant (r)SARS-CoV-2 USA/WA1/2020 (WA-1) strain expressing Venus and a rSARS-CoV-2 expressing mCherry and containing mutations K417N, E484K, and N501Y found in the receptor binding domain (RBD) of the spike (S) glycoprotein of the South African (SA) B.1.351 (beta, {beta}) VoC, in bifluorescent-based assays to rapidly and accurately identify human monoclonal antibodies (hMAbs) able to neutralize both viral infections in vitro and in vivo. Importantly, our bifluorescent-based system accurately recapitulated findings observed using individual viruses. Moreover, fluorescent- expressing rSARS-CoV-2 and the parental wild-type (WT) rSARS-CoV-2 WA-1 had similar viral fitness in vitro, as well as similar virulence and pathogenicity in vivo in the K18 human angiotensin converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 infection. We demonstrate that these new fluorescent-expressing rSARS- CoV-2 can be used in vitro and in vivo to easily identify hMAbs that simultaneously neutralize different SARS-CoV-2 strains, including VoC, for the rapid assessment of vaccine efficacy or the identification of prophylactic and/or therapeutic broadly NAbs for the treatment of SARS-CoV-2 infection.

microbiology↗

bioRxiv2021

Visualization of SARS-CoV-2 infection dynamic

Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing rSARS-CoV-2 have jeopardized their use to monitor the dynamics of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the viral nucleocapsid gene followed by a 2A cleavage peptide. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and K18 hACE2 transgenic mice. Importantly, real-time viral infection was readily tracked using a non-invasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2 retained wild-type virus like pathogenicity in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

microbiology↗

bioRxiv2020

Rapid in vitro assays for screening neutralizing antibodies and antivirals against SARS-CoV-2

Towards the end of 2019, a novel coronavirus (CoV) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), genetically similar to severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), emerged in Wuhan, Hubei province of China, and has been responsible of coronavirus disease 2019 (COVID-19) in humans. Since its first report, SARS-CoV-2 has resulted in a global pandemic, with over 10 million human infections and over 560,000 deaths reported worldwide at the end of June 2020. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines and/or antivirals licensed against SARS-CoV-2, and the high economical and health impact of SARS-CoV-2 has placed global pressure on the scientific community to identify effective prophylactic and therapeutic treatments for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. While some compounds have been already reported to reduce SARS-CoV-2 infection and a handful of monoclonal antibodies (mAbs) have been described that neutralize SARS-CoV-2, there is an urgent need for the development and standardization of assays which can be used in high through-put screening (HTS) settings to identify new antivirals and/or neutralizing mAbs against SARS-CoV-2. Here, we described a rapid, accurate and highly reproducible plaque reduction microneutralization (PRMNT) assay that can be quickly adapted for the identification and characterization of both neutralizing mAbs and antivirals against SARS-CoV-2. Importantly, our MNA is compatible with HTS settings to interrogate large and/or complex libraries of mAbs and/or antivirals to identify those with neutralizing and/or antiviral activity, respectively, against SARS-CoV-2.

microbiology↗

bioRxiv2020

A broad and potent H1-specific human monoclonal antibody produced in plants prevents influenza virus infection and transmission in guinea pigs

Although seasonal influenza vaccines block most predominant influenza types and subtypes, humans still remain vulnerable to waves of seasonal and new potential pandemic influenza viruses for which no immunity may exist because of viral antigenic drift and/or shift, respectively. Previously, we have described a human monoclonal antibody (hMAb), KPF1, which was produced in human embryonic kidney 293T cells (KPF1-HEK) with broad and potent neutralizing activity against H1N1 influenza A viruses (IAV) in vitro, and prophylactic and therapeutic activities in vivo. In this study, we produced hMAb KPF1 in tobacco plants (KPF1-Antx) and demonstrate how the plant-produced KPF1-Antx hMAb possesses similar biological activity compared with the mammalian produced KPF1-HEK hMAb. KPF1-Antx hMAb shows broad binding to recombinant HA proteins and H1N1 IAV, including A/California/04/2009 (pH1N1) in vitro, that are comparable to those observed with KPF1-HEK hMAb. Importantly, prophylactic administration of KPF1-Antx hMAb to guinea pigs prevented pH1N1 infection and transmission in both prophylactic and therapeutic experiments, substantiating its clinical potential to prevent and treat H1N1 infections. Collectively, this study demonstrates, for the first time, that plant-produced influenza hMAbs have similar in vitro and in vivo biological properties to those produced in mammalian cells. Because of the many advantages of plant-produced hMAbs, such as rapid batch production, low cost, and the absence of mammalian cell products, they represent an alternative strategy for the production of immunotherapeutics for the treatment of influenza viral infections, including emerging seasonal and/or pandemic strains.

immunology↗