The Current and Future State of Vaccines, Antivirals and Gene Therapies Against Emerging Coronaviruses

Emerging coronaviruses (CoV) are constant global public health threats to society. Multiple ongoing clinical trials for vaccines and antivirals against CoVs showcase the availability of medical interventions to both prevent and treat the future emergence of highly pathogenic CoVs in human. However, given the diverse nature of CoVs and our close interactions with wild, domestic and companion animals, the next epidemic zoonotic CoV could resist the existing vaccines and antivirals developed, which are primarily focused on Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS CoV). In late 2019, the novel CoV (SARS-CoV-2) emerged in Wuhan, China, causing global public health concern. In this review, we will summarize the key advancements of current vaccines and antivirals against SARS-CoV and MERS-CoV as well as discuss the challenge and opportunity in the current SARS-CoV-2 crisis. At the end, we advocate the development of a “plug-and-play” platform technologies that could allow quick manufacturing and administration of broad-spectrum countermeasures in an outbreak setting. We will discuss the potential of AAV-based gene therapy technology for in vivo therapeutic antibody delivery to combat SARS-CoV-2 outbreak and the future emergence of severe CoVs

Inducing circular RNA formation using the CRISPR endoribonuclease Csy4

Circular RNAs (circRNAs) are highly stable, covalently closed RNAs that are regulated in a spatiotemporal manner and whose functions are largely unknown. These molecules have the potential to be incorporated into engineered systems with broad technological implications. Here we describe a switch for inducing back-splicing of an engineered circRNA that relies on the CRISPR endoribonuclease, Csy4, as an activator of circularization. The endoribonuclease activity and 3' end-stabilizing properties of Csy4 are particularly suited for this task. Coexpression of Csy4 and the circRNA switch allows for the removal of downstream competitive splice sites and stabilization of the 5' cleavage product. This subsequently results in back-splicing of the 5' cleavage product into a circRNA that can translate a reporter protein from an internal ribosomal entry site (IRES). Our platform outlines a straightforward approach toward regulating splicing and could find potential applications in synthetic biology as well as in studying the properties of different circRNAs

Genomewide CRISPR knockout screen identified PLAC8 as an essential factor for SADS-CoVs infection

Zoonotic transmission of coronaviruses poses an ongoing threat to human populations. Endemic outbreaks of swine acute diarrhea syndrome coronavirus (SADS-CoV) have caused severe economic losses in the pig industry and have the potential to cause human outbreaks. Currently, there are no vaccines or specific antivirals against SADS-CoV, and our limited understanding of SADS-CoV host entry factors could hinder prompt responses to a potential human outbreak. Using a genomewide CRISPR knockout screen, we identified placenta-associated 8 protein (PLAC8) as an essential host factor for SADS-CoV infection. Knockout of PLAC8 abolished SADS-CoV infection, which was restored by complementing PLAC8 from multiple species, including human, rhesus macaques, mouse, pig, pangolin, and bat, suggesting a conserved infection pathway and susceptibility of SADS-CoV among mammals. Mechanistically, PLAC8 knockout does not affect viral entry; rather, knockout cells displayed a delay and reduction in viral subgenomic RNA expression. In a swine primary intestinal epithelial culture (IEC) infection model, differentiated cultures have high levels of PLAC8 expression and support SADS-CoV replication. In contrast, expanding IECs have low levels of PLAC8 expression and are resistant to SADS-CoV infection. PLAC8 expression patterns translate in vivo; the immunohistochemistry of swine ileal tissue revealed high levels of PLAC8 protein in neonatal compared to adult tissue, mirroring the known SADS-CoV pathogenesis in neonatal piglets. Overall, PLAC8 is an essential factor for SADS-CoV infection and may serve as a promising target for antiviral development for potential pandemic SADS-CoV

Therapeutic treatment with an oral prodrug of the remdesivir parental nucleoside is protective against SARS-CoV-2 pathogenesis in mice

The coronavirus disease 2019 (COVID-19) pandemic remains uncontrolled despite the rapid rollout of safe and effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. Additionally, the emergence of SARS-CoV-2 variants of concern, with their potential to escape neutralization by therapeutic monoclonal antibodies, emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parent nucleoside of remdesivir, which targets the highly conserved virus RNA-dependent RNA polymerase. GS-621763 exhibited antiviral activity against SARS-CoV-2 in lung cell lines and two different human primary lung cell culture systems. GS-621763 was also potently antiviral against a genetically unrelated emerging coronavirus, Middle East Respiratory Syndrome CoV (MERS-CoV). The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 administration reduced viral load and lung pathology; treatment also improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral which has recently received EUA approval, proved both drugs to be similarly efficacious in mice. These data support the exploration of GS-441524 oral prodrugs for the treatment of COVID-19

Tissue-Dependent Expression and Translation of Circular RNAs with Recombinant AAV Vectors In Vivo

Circular RNAs (circRNAs) are long-lived, covalently closed RNAs that are abundantly expressed and evolutionarily conserved across eukaryotes. Possible functions ranging from microRNA (miRNA) and RNA binding protein sponges to regulators of transcription and translation have been proposed. Here we describe the design and characterization of recombinant adeno-associated viral (AAV) vectors packaging transgene cassettes containing intronic sequences that promote backsplicing to generate circularized RNA transcripts. Using a split GFP transgene, we demonstrate the capacity of vectors containing different flanking intronic sequences to efficiently drive persistent circRNA formation in vitro. Further, translation from circRNA is efficiently driven by an internal ribosomal entry site (IRES). Upon injecting AAV vectors encoding circRNA in mice, we observed robust transgene expression in the heart, but low transduction in the liver for the intronic elements tested. Expression in the murine brain was restricted to astrocytes following systemic or intracranial administration, while intravitreal injection in the eye yielded robust transgene expression across multiple retinal cell layers. These results highlight the potential for exploiting AAV-based circRNA expression to study circRNA function and tissue-specific regulation in animal models, as well as development of therapeutic platforms using this approach. Keywords: adeno-associated virus, circular RNA, gene therap

Ring finger protein 121 is a potent regulator of adeno-associated viral genome transcription.

Adeno-associated viruses (AAV) are Dependoparvoviruses that have shown promise as recombinant vectors for gene therapy. While infectious pathways of AAV are well studied, gaps remain in our understanding of host factors affecting vector genome expression. Here, we map the role of ring finger protein 121 (RNF121), an E3 ubiquitin ligase, as a key regulator of AAV genome transcription. CRISPR-mediated knockout of RNF121 (RNF121 KO) in different cells markedly decreased AAV transduction regardless of capsid serotype or vector dose. Recombinant AAV transduction is partially rescued by overexpressing RNF121, but not by co-infection with helper Adenovirus. Major steps in the AAV infectious pathway including cell surface binding, cellular uptake, nuclear entry, capsid uncoating and second strand synthesis are unaffected. While gene expression from transfected plasmids or AAV genomes is unaffected, mRNA synthesis from AAV capsid-associated genomes is markedly decreased in RNF121 KO cells. These observations were attributed to transcriptional arrest as corroborated by RNAPol-ChIP and mRNA half-life measurements. Although AAV capsid proteins do not appear to be direct substrates of RNF121, the catalytic domain of the E3 ligase appears essential. Inhibition of ubiquitin-proteasome pathways revealed that blocking Valosin Containing Protein (VCP/p97), which targets substrates to the proteasome, can selectively and completely restore AAV-mediated transgene expression in RNF121 KO cells. Expanding on this finding, transcriptomic and proteomic analysis revealed that the catalytic subunit of DNA PK (DNAPK-Cs), a known activator of VCP, is upregulated in RNF121 KO cells and that the DNA damage machinery is enriched at sites of stalled AAV genome transcription. We postulate that a network of RNF121, VCP and DNA damage response elements function together to regulate transcriptional silencing and/or activation of AAV vector genomes

Evolution of a functionally intact but antigenically distinct DENV fusion loop

A hallmark of dengue virus (DENV) pathogenesis is the potential for antibody-dependent enhancement, which is associated with deadly DENV secondary infection, complicates the identification of correlates of protection, and negatively impacts the safety and efficacy of DENV vaccines. Antibody-dependent enhancement is linked to antibodies targeting the fusion loop (FL) motif of the envelope protein, which is completely conserved in mosquito-borne flaviviruses and required for viral entry and fusion. In the current study, we utilized saturation mutagenesis and directed evolution to engineer a functional variant with a mutated FL (D2-FL), which is not neutralized by FL-targeting monoclonal antibodies. The FL mutations were combined with our previously evolved prM cleavage site to create a mature version of D2-FL (D2-FLM), which evades both prM- and FL-Abs but retains sensitivity to other type-specific and quaternary cross-reactive (CR) Abs. CR serum from heterotypic (DENV4)-infected non-human primates (NHP) showed lower neutralization titers against D2-FL and D2-FLM than isogenic wildtype DENV2 while similar neutralization titers were observed in serum from homotypic (DENV2)-infected NHP. We propose D2-FL and D2-FLM as valuable tools to delineate CR Ab subtypes in serum as well as an exciting platform for safer live-attenuated DENV vaccines suitable for naïve individuals and children

Dengue virus 4/2 envelope domain chimeric virus panel maps type-specific responses against dengue serotype 2

ABSTRACT The DENV envelope (E) and pre-membrane (prM) glycoproteins are primary targets of serologic immunity after infection and vaccination. Of these, serotype-specific (TS) antibodies typically target E domains, while serotype cross-reactive (CR) antibodies typically the target prM protein and conserved E regions. To identify and quantify E-domain TS neutralizing antibody responses in polyclonal sera, we developed a panel of chimeric DENV4/2 viruses that incorporate DENV2 envelope domain I, II, and III (DENV4/2-EDI, EDII, EDIII) into the DENV4 E glycoprotein. Chimeric DENV4/2 viruses were recovered, replicated efficiently, and displayed similar maturation states as parental viruses. The recovery of viable DENV4/2-EDII recombinants required the inclusion of chimeric DENV4/2 prM that maintained critical interactions with chimeric E. To assess structural integrity and epitope display of chimeric viruses, we examined neutralization of mature virions by monoclonal antibodies (mAbs) and heterotypic polyclonal sera. The ED-chimeric virions preserved epitopes of TS and envelope-dimer-epitope CR mAbs and had similar sensitivity to CR polyclonal responses as parental strains. Primary sera from natural infection and human challenge target a region centered on EDIII and secondarily target EDII and EDI. Sera from natural infection had a unique neutralization pattern compared to sera from human challenge, which included greater frequency and higher titer of responses against DENV EDII. In summary, DENV4/2 E recombinant viruses delineate the subdomain targets of TS antibodies after vaccination and primary infection, which may provide new correlates of protection or identify epitopes of neutralizing monoclonal antibodies. IMPORTANCE The four dengue virus (DENV) serotypes infect several hundred million people each year. Although primary infection is generally mild, subsequent infection by differing serotypes increases the risk for symptomatic disease ranging from fever to life-threatening shock. Despite the availability of licensed vaccines, a comprehensive understanding of antibodies that target the viral envelope protein and protect from infection remains incomplete. In this manuscript, we develop a panel of recombinant viruses that graft each envelope domain of DENV2 onto the DENV4 envelope glycoprotein, revealing protein interactions important for virus viability. Furthermore, we map neutralizing antibody responses after primary DENV2 natural infection and a human challenge model to distinct domains on the viral envelope protein. The panel of recombinant viruses provides a new tool for dissecting the E domain-specific targeting of protective antibody responses, informing future DENV vaccine design

SARS-CoV-2 variant of concern fitness and adaptation in primary human airway epithelia

Summary: The severe acute respiratory syndrome coronavirus 2 pandemic is characterized by the emergence of novel variants of concern (VOCs) that replace ancestral strains. Here, we dissect the complex selective pressures by evaluating variant fitness and adaptation in human respiratory tissues. We evaluate viral properties and host responses to reconstruct forces behind D614G through Omicron (BA.1) emergence. We observe differential replication in airway epithelia, differences in cellular tropism, and virus-induced cytotoxicity. D614G accumulates the most mutations after infection, supporting zoonosis and adaptation to the human airway. We perform head-to-head competitions and observe the highest fitness for Gamma and Delta. Under these conditions, RNA recombination favors variants encoding the B.1.617.1 lineage 3′ end. Based on viral growth kinetics, Alpha, Gamma, and Delta exhibit increased fitness compared to D614G. In contrast, the global success of Omicron likely derives from increased transmission and antigenic variation. Our data provide molecular evidence to support epidemiological observations of VOC emergence

Positive-strand RNA viruses-a Keystone Symposia report

Positive-strand RNA viruses have been the cause of several recent outbreaks and epidemics, including the Zika virus epidemic in 2015, the SARS outbreak in 2003, and the ongoing SARS-CoV-2 pandemic. On June 18-22, 2022, researchers focusing on positive-strand RNA viruses met for the Keystone Symposium "Positive-Strand RNA Viruses" to share the latest research in molecular and cell biology, virology, immunology, vaccinology, and antiviral drug development. This report presents concise summaries of the scientific discussions at the symposium