Acquisition of resistance to wild-type spike-immune sera by emerging SARS-CoV-2 variants (preprint)

Breakthrough infection is often observed for the SARS-CoV-2 Delta variant, and neutralizing antibody levels are associated with vaccine efficiency 1 . Recent studies revealed that not only anti-receptor binding domain (RBD) antibodies 2 but also antibodies against the N-terminal domain (NTD) play important roles in positively 3,4 or negatively 4-8 controlling SARS-CoV-2 infectivity. Here, we found that the Delta variant completely escaped from anti-NTD neutralizing antibodies, while increasing responsiveness to anti-NTD infectivity-enhancing antibodies. Cryo-EM analysis of the Delta spike revealed that epitopes for anti-NTD neutralizing antibodies are structurally divergent, whereas epitopes for enhancing antibodies are well conserved with wild-type spike protein. Although Pfizer-BioNTech BNT162b2-immune sera neutralized the original Delta variant, when major anti-RBD neutralizing antibody epitopes remaining in the Delta variant were disrupted, some BNT162b2-immune sera not only lost neutralizing activity but became infection-enhanced. The enhanced infectivity disappeared when the Delta NTD was substituted with the wild-type NTD. Sera of mice immunized by Delta spike, but not wild-type spike, consistently neutralized the Delta variant lacking anti-RBD antibody epitopes without enhancing infectivity. Importantly, SARS-CoV-2 variants with similar mutations in the RBD have already emerged according to the GISAID database and their pseudoviruses were resistant to some BNT162b2-immune sera. These findings demonstrate that mutations in the NTD, as well as the RBD, play an important role in antibody escape by SARS-CoV-2. Development of effective vaccines against emerging variants will be necessary, not only to protect against infection, but also to prevent further mutation of SARS-CoV-2.

The SARS-CoV-2 Delta variant is poised to acquire complete resistance to wild-type spike vaccines (preprint)

mRNA-based vaccines provide effective protection against most common SARS-CoV-2 variants. However, identifying likely breakthrough variants is critical for future vaccine development. Here, we found that the Delta variant completely escaped from anti-N-terminal domain (NTD) neutralizing antibodies, while increasing responsiveness to anti-NTD infectivity-enhancing antibodies. Although Pfizer-BioNTech BNT162b2-immune sera neutralized the Delta variant, when four common mutations were introduced into the receptor binding domain (RBD) of the Delta variant (Delta 4+), some BNT162b2-immune sera lost neutralizing activity and enhanced the infectivity. Unique mutations in the Delta NTD were involved in the enhanced infectivity by the BNT162b2-immune sera. Sera of mice immunized by Delta spike, but not wild-type spike, consistently neutralized the Delta 4+ variant without enhancing infectivity. Given the fact that a Delta variant with three similar RBD mutations has already emerged according to the GISAID database, it is necessary to develop vaccines that protect against such complete breakthrough variants.

Preclinical Study of DNA Vaccines Targeting SARS-CoV-2 (preprint)

As potential pandemic vaccines, DNA/RNA vaccines, viral vector vaccines and protein-based vaccines have been rapidly developed to prevent pandemic spread worldwide. In this study, we designed plasmid DNA vaccine targeting the SARS-CoV-2 Spike glycoprotein (S protein) as pandemic vaccine, and the humoral, cellular, and functional immune responses were characterized to support proceeding to initial human clinical trials. After intramuscular injection of DNA vaccine encoding S protein with alum adjuvant (three times at 2-week intervals), the humoral immunoreaction, as assessed by anti-S protein or anti-receptor-binding domain (RBD) antibody titers, and the cellular immunoreaction, as assessed by antigen-induced IFNγ expression, were up-regulated. In IgG subclass analysis, IgG2b was induced as the main subclass. Based on these analyses, DNA vaccine with alum adjuvant preferentially induced Th1-type T cell polarization. We confirmed the neutralizing action of DNA vaccine-induced antibodies by a binding assay of RBD recombinant protein with angiotensin-converting enzyme 2 (ACE2), a receptor of SARSCoV-2, and pseudo-virus assay, TCID assay with live SARS-CoV-2. Further B cell epitope mapping analysis using a peptide array showed that most vaccine-induced antibodies recognized the S2 and RBD subunits. Finally, DNA vaccine protected hamsters form SARSCoV-2 infection. In conclusion, DNA vaccine targeting the spike glycoprotein of SARS-CoV-2 might be an effective and safe approach to combat the COVID-19 pandemic.Funding: This study was supported by Project Promoting Support for Drug Discovery grants(JP20nk0101602 and JP21nf0101623h102) from the Japan Agency for Medical Research andDevelopment and Panasonic Co. (Japan). To fight against the worldwide COVID-19 pandemic, the development of an effective and safe The Department of Health Development and Medicine is an endowed department supported by Anges, Daicel, and FunPep. The Department of Clinical Gene Therapy is financially supported by Novartis, AnGes, Shionogi, Boeringher, Fancl, Saisei Mirai Clinics, Rohto and Funpep. Declaration of Interest: R.M. is a stockholder of FunPep and Anges. T.O. T.K. and Y.S. are employees of Anges. R.I, A.T, H.K, S.K, E.T, S.M, and H.T are employees of FunPep. R.M, H.T, and A.T. are FunPep stockholders. All other authors declare no competing interests.Ethical Approval: All experiments were approved by the Ethical Committee for Animal Experiments of the Osaka University Graduate School of Medicine.

Anti-CoVid19 plasmid DNA vaccine induces a potent immune response in rodents by Pyro-drive Jet Injector intradermal inoculation (preprint)

There is an urgent need to limit and stop the worldwide coronavirus disease 2019 (COVID-19) pandemic via quick development of efficient and safe vaccination methods. Plasmid DNA vaccines are one of the most remarkable vaccines that can be developed in a short term. pVAX1-SARS-CoV2-co, which is a plasmid DNA vaccine, was designed to express severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein. The produced antibodies lead to Immunoreactions against S protein, anti-receptor-binding-domain, and neutralizing action of pVAX1-SARS-CoV2-co, as confirmed in a previous study. To promote the efficacy of the pVAX1-SARS-CoV2-co vaccine, a pyro-drive jet injector (PJI) was employed. PJI is an injection device that can adjust the injection pressure depending on various target tissues. Intradermally-adjusted PJI demonstrated that pVAX1-SARS-CoV2-co vaccine injection caused a strong production of anti-S protein antibodies, triggered immunoreactions and neutralizing actions against SARS-CoV-2. Moreover, a high dose of pVAX1-SARS-CoV2-co intradermal injection via PJI did not cause any serious disorders in the rat model. Finally, virus infection challenge in mice, confirmed that intradermally immunized (via PJI) mice were potently protected from COVID-19 infection. Thus, pVAX1-SARS-CoV2-co intradermal injection via PJI is a safe and promising vaccination method to overcome the COVID-19 pandemic.

Impacts of 203/204: RG>KR mutation in the N protein of SARS-CoV-2 (preprint)

We present a structure-based model of phosphorylation-dependent binding and sequestration of SARS-CoV-2 nucleocapsid protein and the impact of two consecutive amino acid changes R203K and G204R. Additionally, we studied how mutant strains affect HLA-specific antigen presentation and correlated these findings with HLA allelic population frequencies. We discovered RG>KR mutated SARS-CoV-2 expands the ability for differential expression of the N protein epitope on Major Histocompatibility Complexes (MHC) of varying Human Leukocyte Antigen (HLA) origin. The N protein LKR region K203, R204 of wild type (SARS-CoVs) and (SARS-CoV-2) observed HLA-A*30:01 and HLA-A*30:21, but mutant SARS-CoV-2 observed HLA-A*31:01 and HLA-A*68:01. Expression of HLA-A genotypes associated with the mutant strain occurred more frequently in all populations studied. ImportanceThe novel coronavirus known as SARS-CoV-2 causes a disease renowned as 2019-nCoV (or COVID-19). HLA allele frequencies worldwide could positively correlate with the severity of coronavirus cases and a high number of deaths.

SNPnexus COVID: Facilitating the analysis of COVID-19 host genetics (preprint)

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has presented a crisis for global healthcare systems. Human SARS-CoV-2 infection can result in coronavirus disease 2019 (COVID-19), which has been characterised as an acute respiratory illness, with most patients displaying flu-like symptoms, such as a fever, cough and dyspnoea. However, the range and severity of individual symptoms experienced by patients can vary significantly, indicating a role for host genetics in impacting the susceptibility and severity of COVID-19 disease. Whilst most symptomatic infections are known to manifest in mild to moderate respiratory symptoms, severe pneumonia and complications including cytokine release syndrome, which can lead to multi-organ dysfunction, have also been observed in cases worldwide. Global initiatives to sequence the genomes of patients with COVID-19 have driven an expanding new field of host genomics research, to characterise the genetic determinants of COVID-19 disease. The functional annotation and analysis of incoming genomic data, within a clinically relevant turnaround time, is therefore imperative given the importance and urgency of research efforts to understand the biology of SARS-CoV-2 infection and disease. To address these requirements, we developed SNPnexus COVID. This is a web-based variant annotation tool, powered by the SNPnexus software.

assayM: a web application to monitor mutations in COVID-19 diagnostic assays. (preprint)

Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR) is the gold standard as diagnostic assays for the detection of COVID-19 and the specificity and sensitivity of these assays depend on the complementarity of the RT-PCR primers to the genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the virus mutates over time during replication cycles, there is an urgent need to continuously monitor the virus genome for appearances of mutations and mismatches in the PCR primers used in these assays. Here we present assayM, a web application to explore and monitor mutations introduced in the primer and probe sequences published by the World Health Organisation (WHO) or in any custom-designed assay primers for SARS-CoV-2 detection assays in globally available SARS-CoV-2 genome datasets.

An infectivity-enhancing site on the SARS-CoV-2 spike protein is targeted by COVID-19 patient antibodies (preprint)

SARS-CoV-2 infection causes severe symptoms in a subset of patients, suggesting the presence of certain unknown risk factors. Although antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike have been shown prevent SARS-CoV-2 infection, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from COVID-19 patients, and found that some of antibodies against the N-terminal domain (NTD) dramatically enhanced the binding capacity of the spike protein to ACE2, and thus increased SARS-CoV2 infectivity. Surprisingly, mutational analysis revealed that all the infectivity-enhancing antibodies recognized a specific site on the surface of the NTD. The antibodies against this infectivity-enhancing site were detected in all samples of hospitalized COVID-19 patients in the study. However, the ratio of infectivity-enhancing antibodies to neutralizing antibodies differed among patients. Furthermore, the antibodies against the infectivity-enhancing site were detected in 3 out of 48 uninfected donors, albeit at low levels. These findings suggest that the production of antibodies against SARS-CoV-2 infectivity-enhancing site could be considered as a possible exacerbating factors for COVID-19 and that a spike protein lacking such antibody epitopes may be required for safe vaccine development, especially for individuals with pre-existing enhancing antibodies.

The N-terminal domain of spike glycoprotein mediates SARS-CoV-2 infection by associating with L-SIGN and DC-SIGN (preprint)

The widespread occurrence of SARS-CoV-2 has had a profound effect on society and a vaccine is currently being developed. Angiotensin-converting enzyme 2 (ACE2) is the primary host cell receptor that interacts with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Although pneumonia is the main symptom in severe cases of SARS-CoV-2 infection, the expression levels of ACE2 in the lung is low, suggesting the presence of another receptor for the spike protein. In order to identify the additional receptors for the spike protein, we screened a receptor for the SARS-CoV-2 spike protein from the lung cDNA library. We cloned L-SIGN as a specific receptor for the N-terminal domain (NTD) of the SARS-CoV-2 spike protein. The RBD of the spike protein did not bind to L-SIGN. In addition, not only L-SIGN but also DC-SIGN, a closely related C-type lectin receptor to L-SIGN, bound to the NTD of the SARS-CoV-2 spike protein. Importantly, cells expressing L-SIGN and DC-SIGN were both infected by SARS-CoV-2. Furthermore, L-SIGN and DC-SIGN induced membrane fusion by associating with the SARS-CoV-2 spike protein. Serum antibodies from infected patients and a patient-derived monoclonal antibody against NTD inhibited SARS-CoV-2 infection of L-SIGN or DC-SIGN expressing cells. Our results highlight the important role of NTD in SARS-CoV-2 dissemination through L-SIGN and DC-SIGN and the significance of having anti-NTD neutralizing antibodies in antibody-based therapeutics.

Structural basis for the inhibition of the SARS-CoV-2 RNA-dependent RNA polymerase by favipiravir-RTP (preprint)

The RNA polymerase inhibitor, favipiravir, is currently in clinical trials as a treatment for infection with SARS-CoV-2, despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 Ang. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, non-productive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV2 RdRp which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.

Preclinical study of DNA vaccines targeting SARS-CoV-2 (preprint)

To fight against the worldwide COVID-19 pandemic, the development of an effective and safe vaccine against SARS-CoV-2 is required. As potential pandemic vaccines, DNA or RNA vaccines, viral vector vaccines and protein-based vaccines have been rapidly developed to prevent pandemic spread worldwide. In this study, we designed plasmid DNA vaccine targeting the SARS-CoV-2 Spike glycoprotein (S protein) as pandemic vaccine, and the humoral, cellular, and functional immune responses were characterized to support proceeding to initial human clinical trials. After intramuscular injection of DNA vaccine encoding S protein with alum adjuvant (three times at 2-week intervals), the humoral immunoreaction, as assessed by anti-S protein or anti-receptor-binding domain (RBD) antibody titers, and the cellular immunoreaction, as assessed by antigen-induced IFN-g expression, were up-regulated. In IgG subclass analysis, IgG2b was induced as the main subclass. Based on these analyses, DNA vaccine with alum adjuvant preferentially induced Th1-type T cell polarization. We confirmed the neutralizing action of DNA vaccine-induced antibodies via two different methods, a binding assay of RBD recombinant protein with angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, and pseudovirus assay. Further B cell epitope mapping analysis using a peptide array showed that most vaccine-induced antibodies recognized the S2 and RBD subunits, but not the S1 subunit. In conclusion, DNA vaccine targeting the spike glycoprotein of SARS-CoV-2 might be an effective and safe approach to combat the COVID-19 pandemic.

SARS-CoV-2-induced humoral immunity through B cell epitope analysis and neutralizing activity in COVID-19 infected individuals in Japan (preprint)

The aim of this study is to understand adaptive immunity to SARS-CoV-2 through the analysis of B cell epitope and neutralizing activity in coronavirus disease 2019 (COVID-19) patients. We obtained serum from thirteen COVID-19 patients. Most individuals revealed neutralizing activity against SARS-CoV-2 assessed by a pseudotype virus-neutralizing assay. The antibody production against the spike glycoprotein (S protein) or receptor-binding domain (RBD) of SARS-CoV-2 was elevated, with large individual differences, as assessed by ELISA. In the analysis of the predicted the linear B cell epitopes, two regions (671-690 aa. and 1146-1164 aa.), which were located in S1 and S2 but not in the RBD, were highly reactive with the sera from patients. In the further analysis of the B cell epitope within the S protein by utilizing a B cell epitope array, a hot spot in the N-terminal domain of the S protein but not the RBD was observed in individuals with neutralizing activity. Overall, the analysis of antibody production and B cell epitopes of the S protein from patient serum may provide a novel target for the vaccine development against SARS-CoV-2.