Safety and immunogenicity of SW-BIC-213, a modified COVID-19 Lipo-Polyplex mRNA vaccine, in Laotian healthy adults aged 18 years and above: a Phase 1/2 trial (preprint)

Background The mRNA vaccine against SARS-CoV-2 has demonstrated remarkable efficacy in protecting against coronavirus disease 2019 (COVID-19), including providing high protection against severe disease during the emergence of variant waves. In this study, we aimed to investigate the safety and immunogenicity of a 2-dose regimen of the LPP-based mRNA vaccine, SW-BIC-213, in Laos. Methods For this phase 1/2 clinical trial, we recruited healthy adults aged 18-60 years (phase 1) or [≥]18 years (phase 2) from Mahosot Hospital (Vientiane) and Champhone District Hospital (Savannakhet). Participants with SARS-CoV-2 infection, previous COVID-19 vaccination, known allergies to any vaccine component, or pregnancy were excluded. In the phase 1 trial, 41 eligible participants were sequentially assigned to either the 25 g dose group (25 g) or the 45 g dose group (45 g) in accordance with their enrollment order, with 21 participants in 45 g dose group and 20 participants in 25 g dose group. In the phase 2 trial, 480 participants were randomly allocated (2:2:1 ratio) to either the 25 g dose group, 45 g dose group, or placebo group. The primary endpoints for the phase 1 trial were the incidence of local/systemic solicited adverse reactions/events (0-6 days after each vaccination dose), unsolicited adverse events (0-21 days and 0-28 days after the first and second dose of immunization, respectively), and serious adverse events from the first dose of vaccination to 28 days after completing the full course of immunization. In the phase 2 trial, the primary endpoints were the seroconversion rate and geometric mean titer (GMT) of SARS-CoV-2 S-protein specific IgG antibodies and neutralizing antibodies 14 days after the second dose in participants. As for neutralizing antibodies, we detected pseudo-virus neutralizing antibody against wild type (WT), Delta, BA.1 and BA.2. We also detected live viral neutralizing antibody against WT strain 14 days after the second dose. Furthermore, the safety endpoints were also measured during the trial. This seamless phase 1/2 trial was registered with ClinicalTrials.gov under the identifier NCT05144139. Results Between December 3, 2021, and March 31, 2022, a total of 41 participants were recruited in the phase 1 trial, while the phase 2 trial enrolled 480 participants from January 20 to July 6, 2022. In the phase 1 trial, a total of 32 subjects (80.0%) reported 103 cases of adverse reactions. All adverse reactions were limited to Grade 1-2. In the phase 2 trial, a total of 479 subjects, 372 subjects (77.7%) reported 929 cases of adverse reactions. All adverse reactions in severity of Grade 3 were manifested as fever (3.4%, 2.1% and 2.9% in 45 g dose, 25 g dose and placebo group respectively, only observed in adults), except which all other reactions were limited to Grade 1-2. All adverse reactions noted during the study were tolerable, predominantly transient, and resolved spontaneously. No serious adverse events (SAEs) related to vaccination were observed. In Phase 2 study, SW-BIC-213 could elicit a high level of seroconversion rate of pseudo-virus neutralizing antibody against WT (100.0% in 25 g dose group, 99.3% in 45 g dose group), Delta (99.2% in 25 g dose group, 98.0% in 45 g dose group), Omicron BA.1 (84.1% in 25 g dose group, 84.7% in 45 g dose group) and Omicron BA.2 (96.0% in 25 g dose group, 88.8% in 45 g dose group) at 14 days after the second dose. The pseudo-virus neutralizing antibody titer against WT, Delta, BA.1 and BA.2 was all significant higher (P<0.0001) in both 45 g dose group (1175.02, 620.62, 72.39 and 172.80) and 25 g dose group (885.80, 579.40, 47.24 and 101.96) compared with the placebo group (9.67, 10.66, 13.99 and 29.53) at 14 days after the second dose. As for live viral neutralizing antibodies against WT strain, the seroconversion rate could reach more than 94% at 14 days after second dose. The neutralizing antibody titer against WT strain was significantly higher (P<0.0001) in both 45 g dose group (315.00) and 25 g dose group (323.18) compared with the placebo group (8.51) at 14 days after second dose. Conclusion: COVID-19 mRNA vaccine SW-BIC-213 manifests a favorable safety profile and is highly immunogenic in eligible subjects aged [≥]18 years.

SARS-CoV-2 breakthrough infections induce somatically hypermutated broadly neutralizing antibodies against heterologous variants (preprint)

Currently circulating SARS-CoV-2 Omicron variants feature highly mutated spike proteins with extraordinary abilities in evading acute-infection-induced germline antibodies isolated earlier in the pandemic. We identified that memory B cells from Delta variant breakthrough-infection patients expressed antibodies with more extensive somatic hypermutations (SHMs) allowing isolation of a number of broadly neutralizing antibodies with activities against heterologous variants of concerns (VOCs) including Omicron variant. Structural studies identified that SHM introduced altered amino acids and highly unusual HCDR2 insertions respectively in two representative broadly neutralizing antibodies - YB9-258 and YB13-292. Previously, insertion/deletion were rarely reported for antiviral antibodies except for those induced by HIV-1 chronic infections. Identified SHMs involved heavily in epitope recognition, they broadened neutralization breadth by rendering antibodies resistant to VOC mutations highly detrimental to previously isolated antibodies targeting similar epitopes. These data provide molecular mechanisms for enhanced immunity to heterologous SARS-CoV-2 variants after repeated antigen exposures with implications for future vaccination strategy.

A population antibody response against SARS-CoV-2 with differential neutralization activities towards the Delta and Omicron variants (preprint)

Population antibody response is believed to be important in selection of new variant viruses. We identified that SARS-CoV-2 infections elicit a population immune response mediated by a lineage of VH1-69 germline antibodies. The representative antibody R1-32 targets a novel semi-cryptic epitope defining a new class of RBD targeting antibodies. Binding to this non-ACE2 competing epitope leading to spike destruction impairing virus entry. Based on epitope location, neutralization mechanism and analysis of antibody binding to spike variants we propose that recurrent substitutions at 452 and 490 are associated with immune evasion of this population antibody response. These substitutions, including L452R found in the Delta variant, disrupt interaction mediated by the VH1-69 specific hydrophobic HCDR2 to impair antibody-antigen association allowing variants to escape. Lacking 452/490 substitutions, the Omicron variant is sensitive to this class of antibodies. Our results provide new insights into SARS-CoV-2 variant genesis and immune evasion.

Immunogenicity and safety of the homogenous booster shot of a recombinant fusion protein vaccine (V-01) against COVID-19 in healthy adult participants primed with a two-dose regimen (preprint)

Background: Rising concerns over waning immunity and reduction in neutralizing activity against variants of concern (VOCs) have contributed to deploying booster doses by different strategies to tackle the COVID-19 pandemic. Preliminary findings from Phase I and II have shown that V-01, a recombinant fusion protein vaccine against COVID-19, exhibited favorable safety and immunogenicity profiles in 1060 adult participants of both younger and senior age. Herein, we aimed to assess the immunogenicity and safety for a booster dose in participants previously primed with a two-dose 10mcg V-01 regimen (day 0, 21) from phase I trial, providing reassuring data for necessity and feasibility of a homogenous booster dose. Methods: We conducted a single-arm, open-label trial at the Guangdong Provincial Center for Disease Control and Prevention (Gaozhou, China). Forty-three eligible participants who were previously primed 4-5 months earlier with two-dose 10mcg V-01 regimen from phase I trial received booster vaccination. We primarily assessed the immunogenicity post-booster vaccination, measured by RBD-binding antibodies using ELISA and neutralizing activity against wild-type SARS-CoV-2 and emerging variants of concern (VOCs) using neutralization assays. We secondarily assessed the safety and reactogenicity of the booster vaccination. Results: The third dose of V-01 exhibited significant boosting effects of humoral immune response in participants primed with two-dose 10g V-01 regimen regarding both wild-type SARS-CoV-2 and VOCs. We observed a 60.4-folds increase in neutralizing titres against SARS-CoV-2 of younger adults, with GMTs of 17 (95%CI: 12-23) prior to booster vaccination in comparison to 1017 (95%CI: 732-1413) at day 14 post booster vaccination; and a 53.6-folds increase in that of older adults, with GMTs of 14 (95%CI: 9-20) before booster vaccination in comparison to 729(95%CI: 397-1339) at day 14 post-booster vaccination. The neutralizing titres against SARS-CoV-2 Delta strain also demonstrated a sharp increase from the day of pre booster vaccination to day 14 post booster vaccination, with GMTs of 11 (95%CI:8-15) versus 383 (95%CI:277-531) in younger adults (35.4-folds increase), and 6.5(95%CI: 5-8) versus 300(95%CI:142-631) in older adults (46.0-folds increase), respectively. We also observed a considerable and consistent increase of pseudovirus neutralizing titres against emerging VOCs from day 28 post second vaccination to day 14 post booster vaccination, with GMTs of 206 (95%CI:163-259) versus 607 (95%CI: 478-771) for Alpha strain, 54 (95%CI:38-77) versus 329 (95%CI: 255-425) for Beta strain, 219 (95%CI:157-306) versus 647 (95%CI: 484-865) for Delta strain. Our preliminary findings indicate a homogenous booster dose of V-01 was safe and well-tolerated, with overall adverse reactions being absent or mild-to-moderate in severity, and no grade 3 or worse AEs were related to booster vaccination. Conclusions: A homogenous booster immunization in participants receiving a primary series of two-dose V-01 elicited a substantial humoral immune response against wild-type SARS-CoV-2 and emerging VOCs, along with a favorable safety and reactogenicity profile. Our study provided promising data for a homogenous prime-boost strategy using recombinant protein vaccine to tackle the ongoing pandemic, potentially providing broad protection against emerging VOCs and overcoming waning immunity.

Structural characterization of cocktail-like targeting polysaccharides from Ecklonia kurome Okam and their anti-SARS-CoV-2 activities in vitro (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for the worldwide coronavirus disease 2019 (COVID-19) outbreak. Investigation has confirmed that polysaccharide heparan sulfate can bind to the spike protein and block SARS-CoV-2 infection. Theoretically, similar structure of nature polysaccharides may also have the impact on the virus. Indeed, some marine polysaccharide has been reported to inhibit SARS-Cov-2 infection in vitro, however the convinced targets and mechanism are still vague. By high throughput screening to target 3CLpro enzyme, a key enzyme that plays a pivotal role in the viral replication and transcription using nature polysaccharides library, we discover the mixture polysaccharide 375 from seaweed Ecklonia kurome Okam completely block 3Clpro enzymatic activity (IC50, 0.48 {micro}M). Further, the homogeneous polysaccharide 37502 from the 375 may bind to 3CLpro molecule well (kD value : 4.23 x 10-6). Very interestingly, 37502 also can potently disturb spike protein binding to ACE2 receptor (EC50, 2.01 {micro}M). Importantly, polysaccharide 375 shows good anti-SARS-CoV-2 infection activity in cell culture with EC50 values of 27 nM (99.9% inhibiting rate at the concentration of 20 {micro}g/mL), low toxicity (LD50: 136 mg/Kg on mice). By DEAE ion-exchange chromatography, 37501, 37502 and 37503 polysaccharides are purified from native 375. Bioactivity test show that 37501 and 37503 may impede SARS-Cov-2 infection and virus replication, however their individual impact on the virus is significantly less that of 375. Surprisingly, polysaccharide 37502 has no inhibition effect on SARS-Cov-2. The structure study based on monosaccharide composition, methylation, NMR spectrum analysis suggest that 375 contains guluronic acid, mannuronic acid, mannose, rhamnose, glucouronic acid, galacturonic acid, glucose, galactose, xylose and fucose with ratio of 1.86 : 9.56 : 6.81 : 1.69 : 1.00 : 1.75 : 1.19 : 11.06 : 4.31 : 23.06. However, polysaccharide 37502 is an aginate which composed of mannuronic acid (89.3 %) and guluronic acid (10.7 %), with the molecular weight (Mw) of 27.9 kDa. These results imply that mixture polysaccharides 375 works better than the individual polysaccharide on SARS-Cov-2 may be the cocktail-like polysaccharide synergistic function through targeting multiple key molecules implicated in the virus infection and replication. The results also suggest that 375 may be a potential drug candidate against SARS-CoV-2.

Distinct Patterns of Emergence of SARS-CoV-2 Spike Variants including N501Y in Clinical Samples in Columbus Ohio (preprint)

Following the worldwide emergence of the p.Asp614Gly shift in the Spike (S) gene of SARS-CoV-2, there have been few recurring pathogenic shifts occurring during 2020, as assessed by genomic sequencing. This situation has evolved in the last several months with the emergence of several distinct variants (first identified in the United Kingdom and South Africa, respectively) that illustrate multiple changes in the S gene, particularly p.Asn501Tyr (N501Y), that likely have clinical impact. We report here the emergence in Columbus, Ohio in December 2020 of two novel SARS-CoV-2 clade 20C/G variants. One isolate, that has become the predominant virus found in nasopharyngeal swabs in the December 2020-January 2021 period, harbors S p.Gln677His, membrane glycoprotein (M) p.Ala85Ser (Q677H) and nucleocapsid (N) p.Asp377Tyr (D377Y) mutations. The other isolate contains S N501Y and ORF8 Arg52Ile (R52I), which are two markers of the UK-B.1.1.7 (clade 20I/501Y.V1) strain, but lacks all other mutations from that virus. It is also from a different clade and shares multiple mutations with the clade 20C/G viruses circulating in Ohio prior to December 2020. These two SARS-CoV-2 viruses emerging now in the United States add to the diversity of S gene shifts occurring worldwide and support multiple independent acquisition of S N501Y (in likely contrast to the unitary S D614G shift) occurring first during this period of the pandemic.

CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells (preprint)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly contagious presenting a significant public health issue. Current therapies used to treat coronavirus disease 2019 (COVID-19) include monoclonal antibody cocktail, convalescent plasma, antivirals, immunomodulators, and anticoagulants, though the current therapeutic options remain limited and expensive. The vaccines from Pfizer and Moderna have recently been authorized for emergency use, which are invaluable for the prevention of SARS-CoV-2 infection. However, their long-term side effects are not yet to be documented, and populations with immunocompromised conditions (e.g., organ-transplantation and immunodeficient patients) may not be able to mount an effective immune response. In addition, there are concerns that wide-scale immunity to SARS-CoV-2 may introduce immune pressure that could select for escape mutants to the existing vaccines and monoclonal antibody therapies. Emerging evidence has shown that chimeric antigen receptor (CAR)- natural killer (NK) immunotherapy has potent antitumor response in hematologic cancers with minimal adverse effects in recent studies, however, the potentials of CAR-NK cells in preventing and treating severe cases of COVID-19 has not yet been fully exploited. Here, we improve upon a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2 and its D614G mutant. CAR-NK cells were generated using the scFv domain of S309 (henceforward, S309-CAR-NK), a SARS-CoV and SARS-CoV-2 neutralizing antibody that targets the highly conserved region of SARS-CoV-2 spike (S) glycoprotein, therefore would be more likely to recognize different variants of SARS-CoV-2 isolates. S309-CAR-NK cells can specifically bind to pseudotyped SARS-CoV-2 virus and its D614G mutant. Furthermore, S309-CAR-NK cells can specifically kill target cells expressing SARS-CoV-2 S protein in vitro and show superior killing activity and cytokine production, compared to that of the recently published CR3022-CAR-NK cells. Thus, these results pave the way for generating off-the-shelf S309-CAR-NK cells for treatment in high-risk individuals as well as provide an alternative strategy for patients unresponsive to current vaccines.

Enhanced Cholesterol-dependent Hemifusion by Internal Fusion Peptide 1 of SARS Coronavirus-2 Compared to its N-terminal Counterpart (preprint)

Membrane fusion is an important step for the entry of the lipid-sheathed viruses into the host cells. The fusion process is being carried out by fusion proteins present in the viral envelope. The class I viruses contains a 20-25 amino acid sequence at its N-terminal of the fusion domain, which is instrumental in fusion, and is termed as fusion peptide. However, Severe Acute Respiratory Syndrome Coronavirus (SARS) coronaviruses contain more than one fusion peptide sequences. We have shown that the internal fusion peptide 1 (IFP1) of SARS-CoV is far more efficient than its N-terminal counterpart (FP) to induce hemifusion between small unilamellar vesicles. Moreover, the ability of IFP1 to induce hemifusion formation increases dramatically with growing cholesterol content in the membrane. Interestingly, IFP1 is capable of inducing hemifusion, but fails to open pore.

Human Embryonic Stem Cell-derived Lung Organoids: a Model for SARS-CoV-2 Infection and Drug Test (preprint)

The coronavirus disease 2019 (COVID-19) pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is spread primary via respiratory droplets and infects the lungs. Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines (transformed or cancer cells) and species differences between animals and humans. Organoids are stem cell-derived self-organized three-dimensional culture in vitro and model the physiological conditions of natural organs. Here we demonstrated that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids. Ciliated cells, alveolar type 2 (AT2) cells and rare club cells were virus target cells. Electron microscopy captured typical replication, assembly and release ultrastructures and revealed the presence of viruses within lamellar bodies in AT2 cells. Virus infection induced more severe cell death in alveolar organoids than in airway organoids. Additionally, RNA-seq revealed early cell response to SARS-CoV-2 infection and an unexpected downregulation of ACE2 mRNA. Further, compared to the transmembrane protease, serine 2 (TMPRSS2) inhibitor camostat, the nucleotide analog prodrug Remdesivir potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model for SARS-CoV-2 infection and drug discovery.

Airborne SARS-CoV-2 and the Use of Masks for Protection against Its Spread in Wuhan, China (preprint)

The outbreak of COVID-19 has caused a global public health crisis. The spread of SARS-CoV-2 by contact is widely accepted, but the relative importance of aerosol transmission for the spread of COVID-19 is controversial. Here we characterize the distribution of SARA-CoV-2 in 123 aerosol samples, 63 masks, and 30 surface samples collected at various locations in Wuhan, China. The positive percentages of viral RNA included 21% of the aerosol samples from an intensive care unit and 39% of the masks from patients with a range of conditions. A viable virus was isolated from the surgical mask of one critically ill patient while all viral RNA positive aerosol samples were cultured negative. The SARS-CoV-2 detected in masks from patients, ambient air, and respirators from health workers compose a chain of emission, transport, and recipient of the virus. Our results indicate that masks are effective in protecting against the spread of viruses, and it is strongly recommended that people throughout the world wear masks to break the chain of virus transmission and thus protect themselves and others from SARS-CoV-2.

Mycophenolate Mofetil Is Active Against SARS-CoV-2 in Vero E6 Cells (preprint)

Mycophenolate mofetil was reported to have broad in vitro activity against different viruses and had been tried in combination with IFN-β in treating MERS infection. We tested the pharmacological activity of mycophenolate mofetil using SARS-CoV-2 infected Vero cells. The half-maximal effective concentration (EC50) of mycophenolate mofetil against SARS-CoV-2 was 0.47 μM while that of remdesivir was 0.77 μM. Molecular docking results of mycophenolate mofetil to potential target proteins of COVID-19 suggested that mycophenolate mofetil might inhibit SARS-CoV-2 mainly by interacting with DHODH and IMPDH2. Furthermore, mycophenolate mofetil as an immunosuppressant may be a good therapeutic option for the management of hyperinflammation in patients with severe COVID-19. Based on its high potency against SARS-CoV-2 in Vero E6 cells, its good pharmacokinetics and clinical safety profile, mycophenolate mofetil deserves further exploration as potential treatment for COVID-19.