The usefulness of live attenuated virus vaccines has been limited by suboptimal immunogenicity, safety concerns or cumbersome manufacturing processes and techniques. Here we describe the generation of a live attenuated influenza A virus vaccine using proteolysis-targeting chimeric (PROTAC) technology to degrade viral proteins via the endogenous ubiquitin-proteasome system of host cells. We engineered the genome of influenza A viruses in stable cell lines engineered for virus production to introduce a conditionally removable proteasome-targeting domain, generating fully infective PROTAC viruses that were live attenuated by the host protein degradation machinery upon infection. In mouse and ferret models, PROTAC viruses were highly attenuated and able to elicit robust and broad humoral, mucosal and cellular immunity against homologous and heterologous virus challenges. PROTAC-mediated attenuation of viruses may be broadly applicable for generating live attenuated vaccines.

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While the SARS-CoV-2 pandemic is attracting attention from all over the world, influenza remains a major challenge to global health. Despite the use of existing vaccines, influenza still causes 3-5 million severe cases and 0.3-0.65 million deaths worldwide each year, highlighting the need for new vaccine strategies that can generate improved vaccines and thus expand our antiviral arsenal. In a proof-of-concept study, Si et al. describe the development of a proteolysis-targeting chimeric (PROTAC) vaccine technology by using the host cellular ubiquitin-proteasome system to conditionally degrade influenza viral proteins. The generated PROTAC influenza vaccine is highly attenuated by the host protein degradation machinery and able to elicit robust and broad humoral, mucosal, and cellular immune responses against homologous and heterologous viral challenges in mice and ferrets. While this study focuses on the influenza virus, this approach could be extended to the production of live attenuated vaccines against other pathogens.

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A human-airway-on-a-chip for the rapid identification of candidate antiviral therapeutics and prophylactics The rapid repurposing of antivirals is particularly pressing during pandemics. However, rapid assays for assessing candidate drugs typically involve in vitro screens and cell lines that do not recapitulate human physiology at the tissue and organ levels. Here we show that a microfluidic bronchial-airway-on-a-chip lined by highly differentiated human bronchial-airway epithelium and pulmonary endothelium can model viral infection, strain-dependent virulence, cytokine production and the recruitment of circulating immune cells. In airway chips infected with influenza A, the co-administration of nafamostat with oseltamivir doubled the treatment-time window for oseltamivir. In chips infected with pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant doses of the antimalarial drug amodiaquine inhibited infection but clinical doses of hydroxychloroquine and other antiviral drugs that inhibit the entry of pseudotyped SARS-CoV-2 in cell lines under static conditions did not. We also show that amodiaquine showed substantial prophylactic and therapeutic activities in hamsters challenged with native SARS-CoV-2. The human airway-on-a-chip may accelerate the identification of therapeutics and prophylactics with repurposing potential. Mechanical control of innate immune responses against viral infection revealed in a human lung alveolus chip Mechanical breathing motions have a fundamental function in lung development and disease, but little is known about how they contribute to host innate immunity. Here we use a human lung alveolus chip that experiences cyclic breathing-like deformations to investigate whether physical forces influence innate immune responses to viral infection. Influenza H3N2 infection of mechanically active chips induces a cascade of host responses including increased lung permeability, apoptosis, cell regeneration, cytokines production, and recruitment of circulating immune cells. Comparison with static chips reveals that breathing motions suppress viral replication by activating protective innate immune responses in epithelial and endothelial cells, which are mediated in part through activation of the mechanosensitive ion channel TRPV4 and signaling via receptor for advanced glycation end products (RAGE). RAGE inhibitors suppress cytokines induction, while TRPV4 inhibition attenuates both inflammation and viral burden, in infected chips with breathing motions. Therefore, TRPV4 and RAGE may serve as new targets for therapeutic intervention in patients infected with influenza and other potential pandemic viruses that cause life-threatening lung inflammation.

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Self-assembling short immunostimulatory duplex RNAs with broad spectrum antiviral activity Recognition of duplex RNAs by cellular RNA sensors plays a central role in host response to infections by initiating signaling cascades that induce secretion of interferon (IFN) and subsequent upregulation of hundreds of interferon-stimulated genes (ISGs). This pathway therefore also serves as a potent point of therapeutic intervention in a broad range of viral diseases. Duplex RNAs with various structural features have been identified that are recognized by the three cellular RNA sensors that are responsible for this innate immune response. One of these, toll-like receptor 3 (TLR3), is located on the cell membrane and the endosomal membrane, while the other two-retinoic acid inducible gene I (RIG I) and melanoma differentiation associated gene 5 (MDA5)-are located in the cytosol. Long forms of duplex RNA are recognized by these sensors based on their length (i.e., independently of the structure of their 5’ ends) with TLR3 recognizing duplex RNAs >35 bp and MDA5 sensing duplex RNAs >300 bp. Short stretch of duplex RNA (>19 bp) can be recognized by RIG I, but strong activation is achieved only when a triphosphate or a diphosphate is present at its 5′ end and if the end is blunt with no overhangs. Duplex RNA-mediated innate immune stimulation is a two-edged sword. For example, in the case of respiratory infections, such as those caused by pandemic viruses (e.g., SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza virus), RNA-mediated activation of this innate immune response provides the first line of host defense against the invading pathogen. However, on the other hand, the use of duplex RNAs for RNA interference (RNAi) approaches can result in undesired immunological off-target effects and misinterpretation of experimental results. Thus, gaining greater insight into the mechanism by which cells sense and respond to duplex RNAs could have a broad impact in biology and medicine. In this study, we serendipitously discovered a class of new immunostimulatory RNAs while using >200 small interfering RNAs (siRNAs) to identify influenza infection-associated host genes in human lung epithelial cells. These short duplex RNAs potently induce type I and type III interferons (IFN-I/III) in a wide type of cells but lack any sequence or structure characteristics of known immunostimulatory RNAs. Systematic mechanistic analysis revealed that these immunostimulatory RNAs specifically activate the RIG-I/IRF3 pathway by binding directly to RIG-I, and that this only occurs when these short RNAs have a common overhanging sequence motif (sense strand: 5’-C, antisense strand: 3’-GGG) and a minimum length of 20 bases. Interestingly, the terminal motif is responsible for the self-assembly of end-to-end RNA dimers through Hoogsteen G-G base pairing. In addition, these immunostimulatory RNAs appear to be novel in that they are capable of inducing IFN production regardless of whether they have blunt or overhanging ends, terminal hydroxyl or monophosphate groups, RNA base- or DNA base-ends, in contrast to previously described immunostimulatory RNAs that require 5’-di or triphosphates to activate cellular RNA sensors. The RNA-mediated IFN-I/III production resulted in significant inhibition of infections by multiple human respiratory viruses, including influenza viruses and SARS-CoV-2 in established cell lines, human Lung Airway and Alveolus Chips that have been previously shown to recapitulate human lung pathophysiology, and mouse model. These findings also should facilitate the development of siRNAs that avoid undesired immune activation and may pave the way for the development of a new class of RNA therapeutics for the prevention and treatment of respiratory virus infections.

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Clinically Relevant Influenza Virus Evolution Reconstituted in a Human Lung Airway-on-a-Chip Human-to-human transmission of viruses, such as influenza viruses and coronaviruses, can promote virus evolution and the emergence of new strains with increased potential for creating pandemics. Clinical studies analyzing how a particular type of virus progressively evolves new traits, such as resistance to antiviral therapies, as a result of passing between different human hosts are difficult to carry out because of the complexity, scale, and cost of the challenge. Here, we demonstrate that spontaneous evolution of influenza A virus through both mutation and gene reassortment can be reconstituted in vitro by sequentially passaging infected mucus droplets between multiple human lung airway-on-a-chip microfluidic culture devices (airway chips). Modeling human-to human transmission of influenza virus infection on chips in the continued presence of the antiviral drugs amantadine or oseltamivir led to the spontaneous emergence of clinically prevalent resistance mutations, and strains that were resistant to both drugs were identified when they were administered in combination. In contrast, we found that nafamostat, an inhibitor targeting host serine proteases, did not induce viral resistance. This human preclinical model may be useful for studying viral evolution in vitro and identifying potential influenza virus variants before they appear in human populations, thereby enabling preemptive design of new and more effective vaccines and therapeutics.

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