Metabolic Modeling Elucidates Phenformin and Atpenin A5 as Broad-Spectrum Antiviral Drugs (preprint)

Abstract: The SARS-CoV-2 pandemic has reemphasized the urgent need for broad-spectrum antiviral therapies. We developed a computational workflow using scRNA-Seq data to assess cellular metabolism during viral infection. With this workflow we predicted the capacity of cells to sustain SARS-CoV-2 virion production in patients and found a tissue-wide induction of metabolic pathways that support viral replication. Expanding our analysis to influenza A and dengue viruses, we identified metabolic targets and inhibitors for potential broad-45 spectrum antiviral treatment. These targets were highly enriched for known interaction partners of all analyzed viruses. Indeed, phenformin, an NADH:ubiquinone oxidoreductase inhibitor, suppressed SARS-CoV-2 and dengue virus replication. Atpenin A5, blocking succinate dehydrogenase, inhibited SARS-CoV-2, dengue virus, respiratory syncytial virus, and influenza A with high selectivity indices. In vivo, phenformin showed antiviral activity against SARS-CoV-2 in a Syrian hamster model. Our work establishes host metabolism as druggable for broad-spectrum antiviral strategies, providing invaluable tools for pandemic preparedness.

Preclinical efficacy, safety, and immunogenicity of PHH-1V, a second-generation COVID-19 vaccine, in non-human primates (preprint)

SARS-CoV-2 emerged in December 2019 and quickly spread worldwide, continuously striking with an unpredictable evolution. Despite the success in vaccine production and mass vaccination programmes, the situation is not still completely controlled, and therefore accessible second-generation vaccines are required to mitigate the pandemic. We previously developed an adjuvanted vaccine candidate coded PHH-1V, based on a heterodimer fusion protein comprising the RBD domain of two SARS-CoV-2 variants. Here, we report data on the efficacy, safety, and immunogenicity of PHH-1V in cynomolgus macaques. PHH-1V prime-boost vaccination induces high levels of RBD-specific IgG and IgA binding and neutralising antibodies against several SARS-CoV-2 variants of concern, as well as a balanced Th1/Th2 cellular immune response. Remarkably, PHH-1V vaccination prevents SARS-CoV-2 replication in the lower respiratory tract and significantly reduces viral load in the upper respiratory tract after an experimental infection. These results highlight the potential use of the PHH-1V vaccine in humans, currently undergoing Phase III clinical trials.

Interactions between Severe Acute Respiratory Syndrome Coronavirus 2 Replication and Major Respiratory Viruses in Human nasal Epithelium.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), along with extensive non-pharmacological interventions, have profoundly altered the epidemiology of major respiratory viruses. Some studies have described virus-virus interactions, particularly manifested by viral interference mechanisms at different scales. Still, our knowledge of the mutual interactions between SARS-CoV-2 and other respiratory viruses remains incomplete. Here, we studied the interactions between SARS-CoV-2 and several respiratory viruses (influenza, RSV, hMPV, and hRV) in a reconstituted human epithelial airway model, exploring different scenarios affecting the sequence and timing of co-infections. We show that the virus type and the sequence of infections are key parameters of virus-virus interactions, having the impact of primary infections on the regulation of the immune response a determinant role in the outcome of secondary infections.

Molnupiravir combined with different repurposed drugs further inhibits SARS-CoV-2 infection in human nasal epithelium in vitro (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first identified in late 2019, has caused a worldwide pandemic with unprecedented economic and societal impact. Currently, several vaccines are available, and multitudes of antiviral treatments have been proposed and tested. Although many of the vaccines show high clinical efficacy, they are not equally accessible worldwide. Additionally, due to the continuous emergence of new virus variants, and generally short duration of immunity, the development of safe and effective antiviral treatments remains of the utmost importance. Since the emergence of SARS-CoV-2, substantial efforts have been undertaken to repurpose existing and approved drugs for accelerated clinical testing and potential emergency use authorizations. However, drug-repurposing using high throughput screenings in cellular assays, often identify hits that later prove ineffective in clinical studies. Our approach was to evaluate the activity of compounds that have either been tested clinically or already undergone extensive preclinical profiling, using a standardized in vitro model of human nasal epithelium. Secondly, we evaluated drug combinations using sub-maximal doses of each active single compound. Here, we report the antiviral effects of 95 single compounds and 30 combinations. The data show that selected drug combinations including 10 M of molnupiravir, a viral RNA-dependent RNA polymerase (RdRp) inhibitor, effectively inhibit SARS-CoV-2 replication. This indicates that such combinations are worthy of further evaluation as potential treatment strategies against coronavirus disease 2019 (COVID-19).

Role of interfering substances in the survival of coronaviruses on surfaces and their impact on the efficiency of hand and surface disinfection (preprint)

Contaminated environmental surfaces are considered to represent a significant vector for hospital-acquired viral infections. In this study, we have evaluated the impact of interfering substances on SARS-CoV-2 surface stability and virucidal efficiency of hand sanitizers and surface disinfectant. To this end, surface stability of SARS-CoV-2 was measured on stainless steel in different experimental conditions, with or without an artificial mucus/saliva mixture and compared against that of human coronavirus HCoV-229E and feline coronavirus FCoV. The impact of the mucus/saliva mixture on the virucidal efficiency of 3 commercial alcohol hand sanitizers and 1 surface chemical disinfectant against SARS-CoV-2, HCoV-229E and FCoV was then measured. Our results indicate that mucus/saliva mixture did not demonstrate a beneficial effect on the surface survival of tested viruses, with temperature being an important parameter. In addition, we demonstrated that interfering substances may play an important role in the virucidal efficacy of hand sanitizers and disinfectants, highlighting the need for adapted testing protocols that better reflect current - real life -conditions of use.

Viral dynamic modeling of SARS-CoV-2 in non-human primates (preprint)

Non-human primates infected with SARS-CoV-2 exhibit mild clinical signs. Here we used a mathematical model to characterize in detail the viral dynamics in 31 cynomolgus macaques infected with 106 pfu of SARS-CoV-2 for which nasopharyngeal and tracheal viral load were frequently assessed. We identified that infected cells had a large daily viral production (>104 virus) and a within-host reproductive basic number of 6 and 4 in nasopharyngeal and tracheal compartment, respectively. After peak viral load, infected cells were rapidly cleared with a half-life of 9 hours, with no significant association between cytokine elevation and clearance. Translating our model to the context of human-to-human infection, human mild infection may be characterized by a peak occurring 4 days after infection, a viral shedding of ~11 days and a generation time of 4 days. These results improve the understanding of SARS-CoV-2 viral replication and better understand the infection to SARS-CoV-2 in humans.

Bivalent binding of a fully human IgG to the SARS-CoV-2 spike proteins reveals mechanisms of potent neutralization (preprint)

In vitro antibody selection against pathogens from naive combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection1-4. Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction5. Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse naive human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the "up" and the other in the "down" position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.

Transcriptional profiling of immune and inflammatory responses in the context of SARS-CoV-2 fungal superinfection in a human airway epithelial model (preprint)

Superinfections of bacterial/fungal origin are known to affect the course and severity of respiratory viral infections. An increasing number of evidence indicate a relatively high prevalence of superinfections associated with COVID-19, including invasive aspergillosis, but the underlying mechanisms remain to be characterized. In the present study, to better understand the biological impact of superinfection we sought to determine and compare the host transcriptional response to SARS-CoV-2 versus Aspergillus superinfection, using a model of reconstituted humain airway epithelium. Our analyses reveal that both simple infection and superinfection induce a strong deregulation of core components of innate immune and inflammatory responses, with a stronger response to superinfection in the bronchial epithelial model compared to its nasal counterpart. Our results also highlight unique transcriptional footprints of SARS-CoV-2 Aspergillus superinfection, such as an imbalanced type I/type III IFN, and an induction of several monocyte- and neutrophil associated chemokines, that could be useful for the understanding of Aspergillus-associated COVID-19 and but also management of severe forms of aspergillosis in this specific context.

Hydroxychloroquine in the treatment and prophylaxis of SARS-CoV-2 infection in non-human primates (preprint)

COVID-19 has become a pandemic that has caused over 200,000 deaths worldwide, with no antiviral drug or vaccine yet available. Several clinical studies are ongoing to evaluate the efficacy of repurposed drugs that have demonstrated antiviral efficacy in vitro. Among these candidates, hydroxychloroquine (HCQ) has been given to thousands of individuals worldwide but definitive evidence for HCQ efficacy in treatment of COVID-19 is still missing.We evaluated the antiviral activity of HCQ both in vitro and in SARS-CoV-2-infected macaques. HCQ showed antiviral activity in monkey African green monkey kidney (VeroE6) cells but not in a model of reconstituted human airway epithelium. In macaques, we tested different treatment strategies in comparison to placebo, before and after peak viral load, alone or in combination with azithromycin (AZTH). Neither HCQ nor HCQ+AZTH showed a significant effect on the viral load levels in any of the tested compartments. When the drug was used as a pre-exposure prophylaxis (PrEP), HCQ did not confer protection against acquisition of infection.Our findings do not support the use of HCQ, either alone or in combination with AZTH, as an antiviral treatment for COVID-19 in humans.

Timing of antiviral treatment initiation is critical to reduce SARS-Cov-2 viral load (preprint)

We modeled the viral dynamics of 13 untreated patients infected with SARS-CoV-2 to infer viral growth parameters and predict the effects of antiviral treatments. In order to reduce peak viral load by more than 2 logs, drug efficacy needs to be greater than 80% if treatment is administered after symptom onset; an efficacy of 50% could be sufficient if treatment is initiated before symptom onset. Given their pharmacokinetic/pharmacodynamic properties, current investigated drugs may be in a range of 20-70% efficacy. They may help control virus if administered very early, but may not have a major effect in severe patients.

Characterization and treatment of SARS-CoV-2 in nasal and bronchial human airway epithelia (preprint)

In the current COVID-19 pandemic context, proposing and validating effective treatments represents a major challenge. However, the lack of biologically relevant pre-clinical experimental models of SARS-CoV-2 infection as a complement of classic cell lines represents a major barrier for scientific and medical progress. Here, we advantageously used human reconstituted airway epithelial models of nasal or bronchial origin to characterize viral infection kinetics, tissue-level remodeling of the cellular ultrastructure and transcriptional immune signatures induced by SARS-CoV-2. Our results underline the relevance of this model for the preclinical evaluation of antiviral candidates. Foremost, we provide evidence on the antiviral efficacy of remdesivir and the therapeutic potential of the remdesivir-diltiazem combination as a rapidly available option to respond to the current unmet medical need imposed by COVID-19. One Sentence SummaryNew insights on SARS-CoV-2 biology and drug combination therapies against COVID-19.