A needle-free subunit vaccine platform inducing mucosal and systemic antibody immunity (preprint)

While the established route for vaccines against the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is intramuscular, it may be preferable to deliver vaccines intranasally to secure mucosal protection at the site of infection. This will limit the spread of the virus, ease administration and likely improve vaccine acceptance. Here, we report on a subunit vaccine platform, where the antigen is genetically fused to engineered human albumin. Upon intranasal delivery the subunit vaccines target the neonatal Fc receptor (FcRn) and induce both local and systemic antigen-specific antibody responses at magnitudes higher than after intramuscular delivery. We provide evidence that such needle-free vaccination induces production of antibodies with neutralizing capacity against SARS-CoV-2 or influenza A. Thus, the vaccine platform is particularly well suited for design of subunit vaccines against these and other infectious respiratory diseases.

Titers of antibodies the receptor-binding domain (RBD) of ancestral SARS-CoV-2 are predictive for levels of neutralizing antibodies to multiple variants (preprint)

Diagnostic assays currently used to monitor the efficacy of COVID-19 vaccines measure levels of antibodies to the receptor-binding domain of ancestral SARS-CoV-2 (RBDwt). However, the predictive value for protection against new variants of concern (VOCs) has not been firmly established. Here, we used bead-based arrays and flow cytometry to measure binding of antibodies to spike proteins and receptor-binding domains (RBDs) from VOCs in 12,000 sera. Effects of sera on RBD-ACE2 interactions were measured as a proxy for neutralizing antibodies. The samples were obtained from healthy individuals or patients on immunosuppressive therapy who had received two to four doses of COVID-19 vaccines and from COVID-19 convalescents. The results show that anti-RBDwt titers correlate with the levels of binding- and neutralizing antibodies against the Alpha, Beta, Gamma, Delta, Epsilon and Omicron variants. The benefit of multiplexed analysis lies in the ability to measure a wide range of anti-RBD titers using a single dilution of serum for each assay. The reactivity patterns also yield an internal reference for neutralizing activity and binding antibody units per milliliter (BAU/ml). Results obtained with sera from vaccinated healthy individuals and patients confirmed and extended results from previous studies on time-dependent waning of antibody levels and effects of immunosuppressive agents. We conclude that anti-RBDwt titers correlate with levels of neutralizing antibodies against VOCs and propose that our method may be implemented to enhance the precision and throughput of immunomonitoring.

Multiplexed measurement of binding- and neutralizing antibodies to SARS-CoV-2 variants in 12.000 post-vaccine sera (preprint)

Diagnostic assays currently used to monitor the efficacy of COVID-19 vaccines measure levels of antibodies to the receptor-binding domain of ancestral SARS-CoV-2 (RBDwt). However, the predictive value for protection against new variants of concern (VOCs) has not been firmly established. Here, we used bead-based arrays and flow cytometry to measure binding of antibodies to spike proteins and receptor-binding domains (RBDs) from VOCs in 12,000 sera. Effects of sera on RBD-ACE2 interactions were measured as a proxy for neutralizing antibodies. The samples were obtained from healthy individuals or patients on immunosuppressive therapy who had received two to four doses of COVID-19 vaccines and from COVID-19 convalescents. The results show that anti-RBDwt titers correlate with the levels of binding- and neutralizing antibodies against the Alpha, Beta, Gamma, Delta, Epsilon and Omicron variants. The benefit of multiplexed analysis lies in the ability to measure a wide range of anti-RBD titers using a single dilution of serum for each assay. The reactivity patterns also yield an internal reference for neutralizing activity and binding antibody units per milliliter (BAU/ml). Results obtained with sera from vaccinated healthy individuals and patients confirmed and extended results from previous studies on time-dependent waning of antibody levels and effects of immunosuppressive agents. We conclude that anti-RBDwt titers correlate with levels of neutralizing antibodies against VOCs and propose that our method may be implemented to enhance the precision and throughput of immunomonitoring.

Breakthrough infections with the omicron and delta variants of SARS-CoV-2 result in similar re-activation of vaccine-induced immunity (preprint)

Background: The Omicron variant of SARS-CoV-2 is now overtaking the Delta variant in many countries. Results showing that sera from double vaccinated individuals have minimal neutralizing activity against Omicron may indicate that the higher rate of transmission is due to evasion from vaccine-induced immunity. However, there is little information about activation of recall responses to Omicron in vaccinated individuals. Methods: We measured inflammatory mediators, antibodies to the SARS-CoV-2 spike and nucleocapsid proteins, and spike peptide-induced release of interferon gamma in whole blood in 51 vaccinated individuals infected with Omicron, in 14 infected with Delta, and in 18 healthy controls. The median time points for the first and second samples were 7 and 14 days after symptom onset, respectively. Findings: Infection with Omicron or Delta led to a rapid and similar increase in antibodies to the SARS-CoV-2 spike and nucleocapsid proteins and spike peptide-induced interferon gamma in whole blood. Both the Omicron and the Delta infected patients had a mild and transient increase in inflammatory parameters. Interpretation: The results suggest that vaccine-induced immunological memory yields similar coverage for the Omicron and Delta variants.

Nafamostat-interferon-alpha combination suppresses SARS-CoV-2 infection in vitro and in vivo (preprint)

SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here we address these challenges by combining Pegasys (IFNa) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNa and that both Serpin E1 and camostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world.

Preexisting Cross-Reactive T Cells are Boosted and Comprise Significant Immunity in COVID-19 Recovered Patients (preprint)

Most SARS-CoV-2 infected individuals develop symptoms that do not require medical management. We hypothesized that pre-existing cross-reactive T cell responses could protect the majority from severe disease. Here we found that CTL and Th cells specific for seasonal human coronaviruses (HCoV) were significantly expanded in recovered COVID-19 donors and that CTL responses were significantly higher than responses to private SARS-CoV-2 peptides not shared with seasonal HCoV. A third of the SARS-CoV-2 peptide:HLA ligandome was matched by highly similar peptide mimics from seasonal HCoV, constituting a common HCoV peptide pool. CTL immunity was significantly skewed to the common HCoV peptide pool in age groups 20-70y, but not >70y-old donors. Over 40% of recovered donors lacked neutralizing antibodies, highlighting the role of T cell immunity in COVID-19. Results suggest a protective pre-acquired T cell immunity to SARS-CoV-2 and identify epitopes that may help boost vaccine responses and ensure broad protection against this family of viruses.Trial Registration: ClinicalTrials.gov: NCT04320732.Funding: This study was funded by The Health-South East Health Authority (Project 29286), the Research Council of Norway (Project 312693), the Oslo University Hospital, the KG Jebsen Foundation (grant 19), the University of Oslo, The Norwegian Cancer Society.Conflict of Interest: The authors declare that we have no competing interests.

Synergistic Interferon Alpha-based Drug Combinations Inhibit SARS-CoV-2 and other viral Infections in Vitro (preprint)

There is an urgent need for new antivirals with powerful therapeutic potential and tolerable side effects. In the present study, we found that recombinant human interferon-alpha (IFNa) triggered cell intrinsic and extrinsic antiviral responses and reduced replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human lung epithelial Calu-3 cells. However, IFNa alone was insufficient to completely abolish SARS-CoV-2 replication. Combinations of IFNa with camostat, remdesivir, EIDD-2801, cycloheximide or convalescent serum showed strong synergy and effectively inhibited SARS-CoV-2 infection. Additionally, we demonstrated synergistic antiviral activity of IFNa2a with pimodivir against influenza A virus (FluAV) infection in human lung epithelial A549 cells, as well as of IFNa2a with lamivudine against human immunodeficiency virus 1 (HIV-1) infection in human TZM-bl cells. Our results indicate that IFNa2a-based combinational therapies help to reduce drug dose and improve efficacy in comparison with monotherapies, making them attractive targets for further pre-clinical and clinical development.

Rejuveinix Mitigates Sepsis-Associated Oxidative Stress in the Brain of Mice: Clinical Impact Potential in COVID-19 and Nervous System Disorders (preprint)

Here, we demonstrate that our anti-sepsis and COVID-19 drug candidate Rejuveinix (RJX) substantially improves the survival outcome in the LPS-GalN animal model of sepsis and multi-organ failure. One hundred (100) percent (%) of untreated control mice remained alive throughout the experiment. By comparison, 100% of LPS-GalN injected mice died at a median of 4.6 hours. In contrast to the invariably fatal treatment outcome of vehicle-treated control mice, 40% of mice treated with RJX (n=25) remained alive with a 2.4-fold longer median time survival time of 10.9 hours (Log-rank X2=20.60, P<0.0001). Notably, RJX increased the tissue levels of antioxidant enzymes SOD, CAT, and GSH-Px, and reduced oxidative stress in the brain. These findings demonstrate the clinical impact potential of RJX as a neuroprotective COVID-19 and sepsis drug candidate.

Hydroxyzine inhibits SARS-CoV-2 Spike protein binding to ACE2 in a qualitative in vitro assay (preprint)

COVID-19 currently represents a major public health problem. Multiple efforts are being performed to control this disease. Vaccinations are already in progress. However, no effective treatments have been found so far. The disease is caused by the SARS-CoV-2 coronavirus that through the Spike protein interacts with its cell surface receptor ACE2 to enter into the host cells. Therefore, compounds able to block this interaction may help to stop disease progression. In this study, we have analyzed the effect of compounds reported to interact and modify the activity of ACE2 on the binding of the Spike protein. Among the compounds tested, we found that hydroxyzine could inhibit the binding of the receptor-binding domain of Spike protein to ACE2 in a qualitative in vitro assay. This finding supports the reported clinical data showing the benefits of hydroxyzine on COVID-19 patients, raising the need for further investigation into its effectiveness in the treatment of COVID-19 given its well-characterized medical properties and affordable cost.

Interferon alpha-based combinations suppress SARS-CoV-2 infection in vitro and in vivo (preprint)

There is an urgent need for new antivirals with powerful therapeutic potential and tolerable side effects. In the present study, we found that recombinant human interferon-alpha (IFNa) triggers intrinsic and extrinsic cellular antiviral responses, as well as reduces replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. Although IFNa alone was insufficient to completely abolish SARS-CoV-2 replication, combinations of IFNa with remdesivir or other antiviral agents (EIDD-2801, camostat, cycloheximide, or convalescent serum) showed strong synergy and effectively inhibited SARS-CoV-2 infection in human lung epithelial Calu-3 cells. Furthermore, we showed that the IFNa-remdesivir combination suppressed virus replication in human lung organoids, and that its single prophylactic dose attenuated SARS-CoV-2 infection in lungs of Syrian hamsters. Transcriptome and metabolomic analyses showed that the combination of IFNa-remdesivir suppressed virus-mediated changes in infected cells, although it affected the homeostasis of uninfected cells. We also demonstrated synergistic antiviral activity of IFNa2a-based combinations against other virus infections in vitro. Altogether, our results indicate that IFNa2a-based combination therapies can achieve higher efficacy while requiring lower dosage compared to monotherapies, making them attractive targets for further pre-clinical and clinical development.