Anti-SARS-CoV-2 IgG profile of protein subunit, adenovector and mRNA vaccines (preprint)

Different vaccine platforms were developed in 2019 and 2020 to provide immunization for protection against the SARS-CoV-2-caused disease COVID-19. The majority of vaccinated individuals will develop antibodies against SARS-CoV-2. The isotype (subclass) and Fc-glycosylation of IgG antibodies determine their affinity for secondary effector functions. For protein subunit vaccines in COVID-19, the IgG profile of the subclass and glycosylation are unknown. Therefore, we measured the IgG subclass and N-297 Fc glycosylation by ELISA and LC-MS/MS of anti-spike IgG from individuals vaccinated with the Taiwanese protein subunit vaccine Medigen, the mRNA vaccines (BNT, Moderna), and the adenovector Astrazeneca. Samples were taken after the first and second doses. For all vaccine types, the main IgG response was dominated by IgG1 and IgG3 as subclasses. For glycosylation, mRNA vaccines presented with an afucosylation after the first dose and a constant significant higher galactosylation and sialylation than non-mRNA vaccines.

Emergence and Persistent Dominance of Omicron BA.2.3.7 Variant in Community Outbreaks in Taiwan (preprint)

Using high-throughput sequencing of the SARS-CoV-2 genome, we have analyzed 2405 PCR-positive swab samples from 2339 individuals and identified the Omicron BA.2.3.7 variant as a major lineage of the recent community outbreak in Taiwan. Since April 2022, a series of new waves of Omicron cases have surfaced in Taiwan and spread throughout the island. We conducted genomic surveillance with a high success rate and have submitted 1966 full-length Omicron sequences to GISAID. This has permitted identification of signature amino acid changes (ORF1a:L631F, S:K97E, N:M322I) in 1584 (80.6%) of the translated SARS-CoV-2 sequences. The newly established BA.2.3.7 lineage, which is relatively common in Asian countries, is now persistently present in Taiwan. By June 2022 this dominant strain had established a substantial existence in the sequence pool, resulting in additional mutations. The rapid spread and expansion of the Omicron BA.2.3.7 lineage in Asia has had an important socioeconomic impact on health policy.

Titers and capacity of neutralizing antibodies against SARS-CoV-2 variants after heterologous booster vaccination in health care workers primed with two doses of ChAdOx1 nCov-19: a single-blinded, randomized clinical trial (preprint)

Background Booster vaccination is important because of waning immunity and variant immune evasion. We conducted a single-blinded, randomized trial to evaluate the safety, reactogenicity, and immunogenicity of heterologous booster vaccination in health care workers (HCW) who had received two doses of ChAdOx1 nCov-19. Methods and findings HCW at least 90 days after the second dose were enrolled to receive one of the four vaccines: BNT162b2, half-dose mRNA-1273, mRNA-1273, and MVC-COV1901. The primary outcomes were humoral and cellular immunogenicity and the secondary outcomes safety and reactogenicity 28 days post-booster. 340 HCW were enrolled: 83 received BNT162b2 (2 excluded), 85 half-dose mRNA-1273, 85 mRNA-1273, and 85 MVC-COV1901. mRNA vaccines had more reactogenicity than protein vaccine. Anti-spike IgG increased by a fold of 8.4 for MCV-COV1901, 32.2 for BNT162b2, 47.6 for half-dose mRNA-1273 and 63.2 for mRNA1273. The live virus microneutralization assay (LVMNA) against the wild type, alpha and delta variants were consistent with anti-spike IgG for all booster vaccines. The LVMNA in the four groups against omicron variant were 6.4 to 13.5 times lower than those against the wild type. Serum neutralizing antibody against omicron variant was undetectable in 60% of the participants who received MCV-COV1901 as a booster by LVMNA. By using pseudovirus neutralizing assay, we found that neutralization activity in the four groups against omicron variant were 4.6 to 5.2 times lower than that against the D614G. All booster vaccines induced comparable T cell response. Conclusions Third dose booster not only increases neutralizing antibody titer but also enhances antibody capacity against SARS-CoV-2 variants. mRNA vaccines are preferred booster vaccines for those after primary series of ChAdOx1 nCov-19.

Anti-spike antibody response to natural infection with SARS-CoV-2 and its activity against emerging variants (preprint)

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has substantially impacted human health globally. Spike-specific antibody response plays a major role in protection against SARS-CoV-2. Here, we demonstrated that acute SARS-CoV-2 infection elicits rapid and robust spike-binding and ACE2-blocking antibody responses, which wane approximately 11 months after infection. Serological responses were found to be correlated with the frequency of spike-specific memory B cell responses to natural infections. Further, significantly higher spike-binding, ACE2-blocking, and memory B cell responses were detected in patients with fever and pneumonia. Spike-specific antibody responses were found to be greatly affected by spike mutations in emerging variants, especially the Beta and Omicron variants. These results warrant continued surveillance of spike-specific antibody responses to natural infections and highlight the importance of maintaining functional anti-spike antibodies through immunization.

The reactogenicity and immunogenicity of a booster dose after the second dose of a protein subunit vaccine MVC-COV1901. (preprint)

Abstract: In this extension of the phase 1 clinical study, we report the immunogenicity and reactogenicity of the booster dose of a COVID-19 vaccine, MVC-COV1901, administered six months after the completion of the primary two dose schedule. Antibody persistence was detected at 6 months after the second dose of MVC-COV1901, albeit at reduced levels. At 28 days after the booster dose, the neutralizing antibody titer was 1.7-fold higher compared to the previous peak at 2 weeks after the second dose. These data demonstrated the safety and immunogenicity of booster shot of MVC-COV1901 after the primary schedule of the vaccine.

Safety and Immunogenicity of CpG 1018 and Aluminium Hydroxide-Adjuvanted SARS-CoV-2 S-2P Protein Vaccine MVC-COV1901: A Large-Scale Double-Blinded, Randomised, Placebo-Controlled Phase 2 Trial (preprint)

BackgroundWe have assessed the safety and immunogenicity of the COVID-19 vaccine MVC-COV1901, a recombinant protein vaccine containing prefusion-stabilized spike protein S-2P adjuvanted with CpG 1018 and aluminium hydroxide. MethodsThis is a phase 2, prospective, randomised, double-blind, placebo-controlled, and multi-centre study to evaluate the safety, tolerability, and immunogenicity of the SARS-CoV-2 vaccine candidate MVC-COV1901. The study comprised 3,844 participants of [≥] 20 years who were generally healthy or with stable pre-existing medical conditions. The study participants were randomly assigned in a 6:1 ratio to receive either MVC-COV1901 containing 15 g of S-2P protein or placebo containing saline. Participants received two doses of MVC-COV1901 or placebo, administered 28 days apart via intramuscular injection. The primary outcomes were to evaluate the safety, tolerability, and immunogenicity of MVC-COV1901 from Day 1 (the day of first vaccination) to Day 57 (28 days after the second dose). Immunogenicity of MVC-COV1901 was assessed through geometric mean titres (GMT) and seroconversion rates (SCR) of neutralising antibody and antigen-specific immunoglobulin. This clinical trial is registered at ClinicalTrials.gov: NCT04695652. FindingsFrom the start of this phase 2 trial to the time of interim analysis, no vaccine-related Serious Adverse Events (SAEs) were recorded. The most common solicited adverse events across all study participants were pain at the injection site (64%), and malaise/fatigue (35%). Fever was rarely reported (<1%). For all participants in the MVC-COV1901 group, at 28 days after the second dose against wild type SARS-CoV-2 virus, the GMT was 662{middle dot}3 (408 IU/mL), the GMT ratio was 163{middle dot}2, and the seroconversion rate was 99{middle dot}8%. InterpretationMVC-COV1901 shows good safety profiles and promising immunogenicity responses. The current data supports MVC-COV1901 to enter phase 3 efficacy trials and could enable regulatory considerations for Emergency Use Authorisation (EUA). FundingMedigen Vaccine Biologics Corporation and Taiwan Centres for Disease Control.

Antibody titers measured by commercial assays are correlated with neutralizing antibody titers calibrated by international standards (preprint)

The World Health Organization (WHO) has highlighted the importance of an international standard (IS) for SARS-CoV-2 neutralizing antibody titer detection, with the aim of calibrating different diagnostic techniques. In this study, IS was applied to calibrate neutralizing antibody titers (IU/mL) and binding antibody titers (BAU/mL) in response to SARS-CoV-2 vaccines. Serum samples were collected from participants receiving the Moderna (n = 20) and Pfizer (n = 20) vaccines at three time points: pre-vaccination, after one dose, and after two doses. We obtained geometric mean titers of 1404.16 and 928.75 IU/mL for neutralizing antibodies after two doses of the Moderna and Pfizer vaccines, respectively. These values provide an important baseline for vaccine development and the implementation of non-inferiority trials. We also compared three commercially available kits from Roche, Abbott, and MeDiPro for the detection of COVID-19 antibodies based on binding affinity to S1 and/or RBD. Our results demonstrated that antibody titers measured by commercial assays are highly correlated with neutralizing antibody titers calibrated by IS.

Plasmablast-derived antibody response to acute SARS-CoV-2 infection in humans (preprint)

Plasmablast responses and derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients. An average of 13.7% and 13.0% of plasmablast-derived IgG MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. Of thirty-two antibodies specific for the spike glycoprotein, ten recognised the receptor-binding domain (RBD), thirteen were specific for non-RBD epitopes on the S1 subunit, and nine recognised the S2 subunit. A subset of anti-spike antibodies (10 of 32) cross-reacted with other betacoronaviruses tested, five targeted the non-RBD S1, and five targeted the S2 subunit. Of the plasmablast-derived MAbs reacting with nucleocapsid, over half of them (19 of 35) cross-reacted with other betacoronaviruses tested. The cross-reactive plasmablast-derived antibodies harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. We identified 14 of 32 anti-spike MAbs that neutralised SARS-CoV-2 in independent assays at [≤] 133 nM (20 g/ml) (five of 10 anti-RBD, three of 13 anti-non-RBD S1 subunit, six of nine anti-S2 subunit). Six of 10 anti-RBD MAbs showed evidence of blockade of ACE2 binding to RBD, and five of six of these were neutralising. Non-competing pairs of neutralising antibodies were identified, which offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2.

Comparative study of a 3CLpro inhibitor and remdesivir against both major SARS-CoV-2 clades in human airway models (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19), a pandemic that has claimed over 700,000 human lives. The only SARS-CoV-2 antiviral, for emergency use, is remdesivir, targeting the viral polymerase complex. PF-00835231 is a pre-clinical lead compound with an alternate target, the main SARS-CoV-2 protease 3CLpro (Mpro). Here, we perform a comparative analysis of PF-00835231 and remdesivir in A549+ACE2 cells, using isolates of two major SARS-CoV-2 clades. PF-00835231 is antiviral for both clades, and, in this assay, statistically more potent than remdesivir. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps and validates PF-00835231s time of action. Both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 in human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective antiviral for SARS-CoV-2, addresses concerns from non-human in vitro models, and supports further studies with this compound.

Prunella vulgaris extract and suramin block SARS-coronavirus 2 virus Spike protein D614 and G614 variants mediated receptor association and virus entry in cell culture system (preprint)

Until now, no approved effective vaccine and antiviral therapeutic are available for treatment or prevention of SARS-coronavirus 2 (SCoV-2) virus infection. In this study, we established a SCoV-2 Spike glycoprotein (SP), including a SP mutant D614G, pseudotyped HIV-1-based vector system and tested their ability to infect ACE2-expressing cells. This study revealed that a C-terminal 17 amino acid deletion in SCoV-2 SP significantly increases the incorporation of SP into the pseudotyped viruses and enhanced its infectivity, which may be helpful in the design of SCoV2-SP-based vaccine strategies. Moreover, based on this system, we have demonstrated that an aqueous extract from the Chinese herb Prunella vulgaris (CHPV) and a compound, suramin, displayed potent inhibitory effects on both wild type and mutant (G614) SCoV-2 SP pseudotyped virus (SCoV-2-SP-PVs)-mediated infection. The 50% inhibitory concentration (IC50) for CHPV and suramin on SCoV-2-SP-PVs are 30, and 40 g/ml, respectively. To define the mechanisms of their actions, we demonstrated that both CHPV and suramin are able to directly interrupt SCoV-2-SP binding to its receptor ACE2 and block the viral entry step. Importantly, our results also showed that CHPV or suramin can efficiently reduce levels of cytopathic effect caused by SARS-CoV-2 virus (hCoV-19/Canada/ON-VIDO-01/2020) infection in Vero cells. Furthermore, our results demonstrated that the combination of CHPV/suramin with an anti-SARS-CoV-2 neutralizing antibody mediated more potent blocking effect against SCoV2-SP-PVs. Overall, this study provides evidence that CHPV and suramin has anti-SARS-CoV-2 activity and may be developed as a novel antiviral approach against SARS-CoV-2 infection.

Replication dynamics and cytotoxicity of SARS-CoV-2 Swedish isolate in commonly used laboratory cell lines (preprint)

We assessed the infectivity, replication dynamics and cytopathogenicity of the first Swedish isolate of SARS-CoV-2 in six different cell lines of human origin and compared their growth characteristics. High replication kinetics in absence of cytopathic-effect observed in many cell lines provided important clues on SARS-CoV-2 pathogenesis.

Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates throughout human airways. The polarized human airway epithelium (HAE) cultured at an airway-liquid interface (HAE-ALI) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of SARS-CoV-2. However, previous studies only characterized short-period SARS-CoV-2 infection in HAE. In this study, continuously monitoring the SARS-CoV-2 infection in HAE-ALI cultures for a long period of up to 51 days revealed that SARS-CoV-2 infection was long lasting with recurrent replication peaks appearing between an interval of approximately 7-10 days, which was consistent in all the tested HAE-ALI cultures derived from 4 lung bronchi of independent donors. We also identified that SARS-CoV-2 does not infect HAE from the basolateral side, and the dominant SARS-CoV-2 permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detectable. Notably, virus infection immediately damaged the HAE, which is demonstrated by dispersed Zonula occludens-1 (ZO-1) expression without clear tight junctions and partial loss of cilia. Importantly, we identified that SARS-CoV-2 productive infection of HAE requires a high viral load of 2.5 x 105 virions per cm2 of epithelium. Thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of HAE with SARS-CoV-2.

Identification of 14 Known Drugs as Inhibitors of the Main Protease of SARS-CoV-2 (preprint)

A consensus virtual screening protocol has been applied to ca. 2000 approved drugs to seek inhibitors of the main protease (Mpro) of SARS-CoV-2, the virus responsible for COVID-19. 42 drugs emerged as top candidates, and after visual analyses of the predicted structures of their complexes with Mpro, 17 were chosen for evaluation in a kinetic assay for Mpro inhibition. Remarkably 14 of the compounds at 100-M concentration were found to reduce the enzymatic activity and 5 provided IC50 values below 40 M: manidipine (4.8 M), boceprevir (5.4 M), lercanidipine (16.2 M), bedaquiline (18.7 M), and efonidipine (38.5 M). Structural analyses reveal a common cloverleaf pattern for the binding of the active compounds to the P1, P1, and P2 pockets of Mpro. Further study of the most active compounds in the context of COVID-19 therapy is warranted, while all of the active compounds may provide a foundation for lead optimization to deliver valuable chemotherapeutics to combat the pandemic.

Development of CpG-adjuvanted stable prefusion SARS-CoV-2 spike antigen as a subunit vaccine against COVID-19 (preprint)

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 is a worldwide health emergency. The immense damage done to public health and economies has prompted a global race for cures and vaccines. In developing a COVID-19 vaccine, we applied technology previously used for MERS-CoV to produce a prefusion-stabilized SARS-CoV-2 spike protein by adding two proline substitutions at the top of the central helix (S-2P). To enhance immunogenicity and mitigate the potential vaccine-induced immunopathology, CpG 1018, a Th1-biasing synthetic toll-like receptor 9 (TLR9) agonist was selected as an adjuvant candidate. S-2P was combined with various adjuvants, including CpG 1018, and administered to mice to test its effectiveness in eliciting anti-SARS-CoV-2 neutralizing antibodies. S-2P in combination with CpG 1018 and aluminum hydroxide (alum) was found to be the most potent immunogen and induced high titer of spike-specific antibodies in sera of immunized mice. The neutralizing abilities in pseudotyped lentivirus reporter or live wild-type SARS-CoV-2 were measured with reciprocal inhibiting dilution (ID50) titers of 5120 and 2560, respectively. In addition, the antibodies elicited were able to cross-neutralize pseudovirus containing the spike protein of the D614G variant, indicating the potential for broad spectrum protection. A marked Th-1 dominant response was noted from cytokines secreted by splenocytes of mice immunized with CpG 1018 and alum. No vaccine-related serious adverse effects were found in the dose-ranging study in rats administered single- or two-dose regimens with up to 50 g of S-2P combined with CpG 1018 alone or CpG 1018 with alum. These data support continued development of CHO-derived S-2P formulated with CpG 1018/alum as a candidate vaccine to prevent COVID-19 disease.

Relative COVID-19 viral persistence and antibody kinetics (preprint)

ImportanceThe COVID-19 antibody response is a critical indicator for evaluating immunity and also serves as the knowledge base for vaccine development. The picture is still not clear because of many limitations including testing tools, time of sampling, and the unclear impact of varying clinical status. In addition to these problems, antibody levels may not be equivalent to protective capacity. ObjectiveTo define the key factor for the different patterns of COVID-19 antibody response. DesignWe elucidated the antibody response with time-series throat and serum samples for viral loads and antibody levels, then used a neutralization test to evaluate protectiveness. SettingA medical center that typically cares for patients with moderate to severe diseases. Because of the low prevalence of COVID-19 in Taiwan and local government policy, however, we also admit COVID-19 patients with mild disease or even those without symptoms for inpatient care. ParticipantsRT-PCR-confirmed COVID-19 patients. ResultsWe found that only patients with relative persistence of virus at pharynx displayed strong antibody responses that were proportional to the pharyngeal viral load. They also had proportional neutralization titers per unit of serum. Although antibody levels decreased around 2 weeks after symptom onset, the neutralization efficacy per unit antibody remained steady and even continued to increase over time. The antibody response in patients with rapid virus clearance was weak, but the neutralization efficacy per unit antibody in these patients was comparable to those with persistent presence of virus. The deceased were with higher viral load, higher level of antibody, and higher neutralization titers in the serum, but the neutralization capacity per unit antibody is relatively low. Conclusions and RelevanceStrong antibody response depends on the relative persistence of the virus, instead of the absolute virus amount. The antibody response is still weak if large amount of virus is cleared quickly. The neutralization efficacy per unit antibody is comparable between high and low antibody patterns. Strong antibody response contains more inefficient and maybe even harmful antibodies. Low antibody response is also equipped with a capable B cell pool of efficient antibodies, which may expand with next virus encounter and confer protection. Key pointsO_ST_ABSQuestionC_ST_ABSThe key factor for the different "patterns" of COVID-19 antibody response. FindingsStrong antibody response depends on the relative persistence of the virus, instead of the absolute virus amount. The antibody response is still weak if large amount of virus is cleared quickly. The neutralization efficacy per unit antibody is comparable between high and low antibody patterns. High antibody level contains more inefficient antibodies. MeaningStrong response contains inefficient and maybe harmful antibodies. Low antibody response is also equipped with a capable B cell pool of efficient antibodies, which may expand with next virus encounter and confer protection.

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient (preprint)

The COVID-19 pandemic has had unprecedented health and economic impact, but currently there are no approved therapies. We have isolated an antibody, EY6A, from a late-stage COVID-19 patient and show it neutralises SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds tightly (KD of 2 nM) the receptor binding domain (RBD) of the viral Spike glycoprotein and a 2.6[A] crystal structure of an RBD/EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues of this epitope are key to stabilising the pre-fusion Spike. Cryo-EM analyses of the pre-fusion Spike incubated with EY6A Fab reveal a complex of the intact trimer with three Fabs bound and two further multimeric forms comprising destabilized Spike attached to Fab. EY6A binds what is probably a major neutralising epitope, making it a candidate therapeutic for COVID-19.

Sequence variation among SARS-CoV-2 isolates in Taiwan (preprint)

Taiwan experienced two waves of imported cases of coronavirus disease 2019 (COVID-19), first from China in January to late February, followed by those from other countries starting in early March. Additionally, several cases could not be traced to any imported cases and were suspected as sporadic local transmission. Twelve full viral genomes were determined in this study by Illumina sequencing either from virus isolates or directly from specimens, among which 5 originated from clustered infections. Phylogenetic tree analysis revealed that these sequences were in different clades, indicating that no major strain has been circulating in Taiwan. A deletion in open reading frame 8 was found in one isolate. Only a 4-nucleotide difference was observed among the 5 genomes from clustered infections.