Structure and computation-guided design of a mutation-integrated trimeric RBD candidate vaccine with broad neutralization against SARS-CoV-2 (preprint)

The spike (S) protein receptor-binding domain (RBD) of SARS-CoV-2 is an attractive target for COVID-19 vaccine developments, which naturally exists in a trimeric form. Here, guided by structural and computational analyses, we present a mutation-integrated trimeric form of RBD (mutI tri-RBD) as a broadly protective vaccine candidate, in which three RBDs were individually grafted from three different circulating SARS-CoV-2 strains including the prototype, Beta (B.1.351) and Kappa (B.1.617). The three RBDs were then connected end-to-end and co-assembled to possibly mimic the native trimeric arrangements in the natural S protein trimer. The recombinant expression of the mutI tri-RBD, as well as the homo-tri-RBD where the three RBDs were all truncated from the prototype strain, by mammalian cell exhibited correct folding, strong bio-activities, and high stability. The immunization of both the mutI tri-RBD and homo-tri-RBD plus aluminum adjuvant induced high levels of specific IgG and neutralizing antibodies against the SARS-CoV-2 prototype strain in mice. Notably, regarding to the immune-escape Beta (B.1.351) variant, mutI tri-RBD elicited significantly higher neutralizing antibody titers than homo-tri-RBD. Furthermore, due to harboring the immune-resistant mutations as well as the evolutionarily convergent hotspots, the designed mutI tri-RBD also induced strong broadly neutralizing activities against various SARS-CoV-2 variants, especially the variants partially resistant to homo-tri-RBD. Homo-tri-RBD has been approved by the China National Medical Products Administration to enter clinical trial (No. NCT04869592), and the superior broad neutralization performances against SARS-CoV-2 support the mutI tri-RBD as a more promising vaccine candidate for further clinical developments.

Rapid development of SARS-CoV-2 receptor binding domain-conjugated nanoparticle vaccine candidate (preprint)

The ongoing of coronavirus disease 2019 (COVID-19) pandemic caused by novel SARS-CoV-2 coronavirus, resulting in economic losses and seriously threating the human health in worldwide, highlighting the urgent need of a stabilized, easily produced and effective preventive vaccine. The SARS-COV-2 spike protein receptor binding region (RBD) plays an important role in the process of viral binding receptor angiotensin-converting enzyme 2 (ACE2) and membrane fusion, making it an ideal target for vaccine development. In this study, we designed three different RBD-conjugated nanoparticles vaccine candidates, RBD-Ferritin (24-mer), RBD-mi3 (60-mer) and RBD-I53-50 (120-mer), with the application of covalent bond linking by SpyTag-SpyCatcher system. It was demonstrated that the neutralizing capability of sera from mice immunized with three RBD-conjugated nanoparticles adjuvanted with AddaVax or Sigma Systerm Adjuvant (SAS) after each immunization was ~8- to 120-fold greater than monomeric RBD group in SARS-CoV-2 pseudovirus and authentic virus neutralization assay. Most importantly, sera from RBD-conjugated NPs groups more efficiently blocked the binding of RBD to ACE2 or neutralizing antibody in vitro, a further proof of promising immunization effect. Besides, high physical stability and flexibility in assembly consolidated the benefit for rapid scale-up production of vaccine. These results supported that our designed SARS-CoV-2 RBD-conjugated nanoparticle was competitive vaccine candidate and the carrier nanoparticles could be adopted as universal platform for future vaccine development.

COVID-19 risk haplogroups differ between populations, deviate from Neanderthal haplotypes and compromise risk assessment in non-Europeans (preprint)

Recent genome wide association studies (GWAS) have identified genetic risk factors for developing severe COVID-19 symptoms. The studies reported a 1bp insertion rs11385942 on chromosome 3 and furthermore two single nucleotide variants (SNVs) rs35044562 and rs67959919, all three correlated with each other. Zeberg and Paabo subsequently traced them back to Neanderthal origin. They found that a 49.4 kb genomic region including the risk allele of rs35044562 is inherited from Neanderthals of Vindija in Croatia. Here we add a differently focused evaluation of this major genetic risk factor to these recent analyses. We show that (i) COVID-19-related genetic factors of Neanderthals deviate from those of modern humans and that (ii) they differ among world-wide human populations, which compromises risk prediction in non-Europeans. Currently, caution is thus advised in the genetic risk assessment of non-Europeans during this world-wide COVID-19 pandemic.

Computational prediction of SARS-CoV-2 encoded miRNAs and their putative host targets (preprint)

Background: Over the past two decades, there has been a continued research on the role of small non-coding RNAs including microRNAs (miRNAs) in various diseases. Studies have shown that viruses modulate the host cellular machinery and hijack its metabolic and immune signaling pathways by miRNA mediated gene silencing. Given the immensity of coronavirus disease 19 (COVID-19) pandemic and the strong association of viral encoded miRNAs with their pathogenesis, it is important to study Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) miRNAs. Results: To address this unexplored area, we identified 8 putative novel miRNAs from SARS-CoV-2 genome and explored their possible human gene targets. A significant proportion of these targets populated key immune and metabolic pathways such as MAPK signaling pathway, maturity-onset diabetes of the young, Insulin signaling pathway, endocytosis, RNA transport, TGF-{beta} signaling pathway, to name a few. The data from this work is backed up by recently reported high-throughput transcriptomics datasets obtains from SARS-CoV-2 infected samples. Analysis of these datasets reveal that a significant proportion of the target human genes were down-regulated upon SARS-CoV-2 infection. Conclusions: The current study brings to light probable host metabolic and immune pathways susceptible to viral miRNA mediated silencing in a SARS-CoV-2 infection, and discusses its effects on the host pathophysiology.

Antiviral activity of plant juices and green tea against SARS-CoV-2 and influenza virus in vitro (preprint)

Respiratory viruses initially infect the naso- and oropharyngeal regions, where they amplify, cause symptoms and may also be transmitted to new hosts. Preventing initial infection or reducing viral loads upon infection might soothe symptoms, prevent dissemination into the lower airways, or transmission to the next individual. We here analyzed the potential of plant derived products to inactivate SARS-CoV-2 and influenza virus. We found that black chokeberry (Aronia melanocarpa) juice, pomegranate (Punica granatum) juice, and green tea (Camellia sinensis) have virucidal activity against both viruses, suggesting that oral rinsing may reduce viral loads in the oral cavity thereby lowering virus transmission.

Potential Opportunity Of Antisense Therapy Of COVID-19 on an in vitro model (preprint)

Data on potential effectiveness and prospects of treatment of new coronavirus infection of COVID-19 caused by virus SARS-CoV-2 with the help of antisense oligonucleotides acting against RNA of virus on an in vitro model are given. The ability of antisense oligonucleotides to suppress viral replication in diseases caused by coronaviruses using the example of SARS and MERS is shown. The identity of the initial regulatory section of RNA of various coronaviruses was found within 50 - 100 nucleotides from the 5'-end, which allows using antisense suppression of this RNA fragment. A new RNA fragment of the virus present in all samples of coronovirus SARS-CoV-2 has been identified, the suppression of which with the help of an antisense oligonucleotide can be effective in the treatment of COVID-19. The study of the synthesized antisense oligonucleotide 5`-AGCCGAGTGACAGCC ACACAG, complementary to the selected virus RNA sequence, was carried out. The low toxicity of the preparations of this group in the cell culture study and the ability to reduce viral load at high doses according to real time-PCR data are shown. The cytopathogenic dose exceeds 2 mg/ml. At a dosage of 1 mg/ml, viral replication is reduced by 5 - 13 times. Conclusions are made about the prospects of this direction and the feasibility of using the inhalation way of drug administration into the body.

Modelling the active SARS-CoV-2 helicase complex as a basis for structure-based inhibitor design (preprint)

Having claimed over 1 million lives worldwide to date, the ongoing COVID-19 pandemic has created one of the biggest challenges to develop an effective drug to treat infected patients. Among all the proteins expressed by the virus, RNA helicase is a fundamental protein for viral replication, and it is highly conserved among the coronaviridae family. To date, there is no high-resolution structure of helicase bound with ATP and RNA. We present here structural insights and molecular dynamics (MD) simulation results of the SARS-CoV-2 RNA helicase both in its apo form and in complex with its natural substrates. Our structural information of the catalytically competent helicase complex provides valuable insights for the mechanism and function of this enzyme at the atomic level, a key to develop specific inhibitors for this potential COVID-19 drug target.

The immunodominant and neutralization linear epitopes for SARS-CoV-2 (preprint)

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) becomes a tremendous threat to global health. Although vaccines against the virus are under development, the antigen epitopes on the virus and their immunogenicity are poorly understood. Here, we simulated the three-dimensional structures of SARS-CoV-2 proteins with high performance computer, predicted the B cell epitopes on spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins of SARS-CoV-2 using structure-based approaches, and then validated the epitope immunogenicity by immunizing mice. Almost all 33 predicted epitopes effectively induced antibody production, six of which were immunodominant epitopes in patients identified via the binding of epitopes with the sera from domestic and imported COVID-19 patients, and 23 were conserved within SARS-CoV-2, SARS-CoV and bat coronavirus RaTG13. We also found that the immunodominant epitopes of domestic SARS-CoV-2 were different from that of the imported, which may be caused by the mutations on S (G614D) and N proteins. Importantly, we validated that eight epitopes on S protein elicited neutralizing antibodies that blocked the cell entry of both D614 and G614 pseudo-virus of SARS-CoV-2, three and nine epitopes induced D614 or G614 neutralizing antibodies, respectively. Our present study shed light on the immunodominance, neutralization, and conserved epitopes on SARS-CoV-2 which are potently used for the diagnosis, virus classification and the vaccine design tackling inefficiency, virus mutation and different species of coronaviruses.

SARS-CoV-2 Nucleocapsid protein is decorated with multiple N- and O-glycans. (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19) started at the end of 2019 in Wuhan, China has spread rapidly and became a pandemic. Since there is no therapy available that is proven as fully protective against COVID-19, a vaccine to protect against deadly COVID-19 is urgently needed. Nucleocapsid protein (N protein), is one of the most abundant proteins in coronaviruses and is a potential target for both vaccine development and point of care diagnostics. The variable mass of N protein (45 to 60 kDa), suggests the presence of post-translational modifications (PTMs), and it is critical to clearly define these PTMs to gain the structural understanding necessary for further vaccine research. There have been several reports suggesting that the N protein is phosphorylated but lacks glycosylation. Our comprehensive glycomics and glycoproteomics experiments confirm that the N protein is highly O-glycosylated and also contains significant levels of N-glycosylation. We were able to confirm the presence of O-glycans on seven sites with substantial glycan occupancy, in addition to less abundant O-glycans on four sites. We also detected N-glycans on two out of five potential N-glycosylation sites. Moreover, we were able to confirm one phosphorylation site. Recent studies have indicated that the N protein can serve as an important diagnostic marker for coronavirus disease and a major immunogen by priming protective immune responses. Thus, detailed structural characterization of the N protein may provide useful insights for understanding the roles of glycosylation on viral pathogenesis and also in vaccine design and development.

Designing of Epitope-Based Vaccine from the Conserved Region of the Spike Glycoprotein of SARS-CoV-2 (preprint)

The emergence of COVID-19 as a pandemic with a high morbidity rate is posing serious global concern. There is an urgent need to design a suitable therapy or vaccine that could fight against SARS-CoV-2 infection. As spike glycoprotein of SARS-CoV-2 plays a crucial role in receptor binding and membrane fusion inside the host, it could be a suitable target for designing of an epitope-based vaccine. SARS-CoV-2 is an RNA virus and thus has a property to mutate. So, a conserved peptide region of spike glycoprotein was used for predicting suitable B cell and T cell epitopes. 4 T cell epitopes were selected based on stability, antigenicity, allergenicity and toxicity. Further, MHC-I were found from the immune database that could best interact with the selected epitopes. Population coverage analysis was also done to check the presence of identified MHC-I, in the human population of the affected countries. The T cell epitope that binds with the respective MHC-I with highest affinity was chosen. Molecular dynamic simulation results show that the epitope is well selected. This is an in-silico based study that predicts a novel T cell epitope from the conserved spike glycoprotein that could act as a target for designing of the epitope-based vaccine. Further, B cell epitopes have also been found but the main work focuses on T cell epitope as the immunity generated by it is long lasting as compared to B cell epitope.

Rethinking Remdesivir: Synthesis of Lipid Prodrugs that Substantially Enhance Anti-Coronavirus Activity (preprint)

The FDA has granted Remdesivir (RDV, GS-5734) an emergency use authorization on the basis of an acceleration of clinical recovery in hospitalized patients with COVID-19. Unfortunately, the drug must be administered intravenously, restricting its use to those with relatively advanced disease. RDV is also unstable in plasma and has a complex activation pathway which may contribute to its highly variable antiviral efficacy in SARS-CoV-2 infected cells. A potent orally bioavailable antiviral for early treatment of SARS-CoV-2 infection is needed. We focused on making simple orally bioavailable lipid analogs of Remdesivir nucleoside (RVn, GS-441524) that are processed to RVn-monophosphate, the precursor of the active RVn-triphosphate, by a single step intracellular cleavage. In addition to likely improved oral bioavailability and simpler metabolic activation, two of the three new lipid prodrugs of RVn had anti-SARS-CoV-2 activity 9 to 24 times greater than that of RDV in Vero E6 cells

Compositional Variability and Mutation Spectra of Monophyletic SARS-CoV-2 Clades (preprint)

COVID-19 and its causative pathogen SARS-CoV-2 have rushed the world into a staggering pandemic in a few months and a global fight against both is still going on. Here, we describe an analysis procedure where genome composition and its variables are related, through the genetic code, to molecular mechanisms based on understanding of RNA replication and its feedback loop from mutation to viral proteome sequence fraternity including effective sites on replicase-transcriptase complex. Our analysis starts with primary sequence information and identity-based phylogeny based on 22,051 SARS-CoV-2 genome sequences and evaluation of sequence variation patterns as mutation spectrum and its 12 permutations among organized clades tailored to two key mechanisms: strand-biased and function-associated mutations. Our findings include: (1) The most dominant mutation is C-to-U permutation whose abundant second-codon-position counts alter amino acid composition toward higher molecular weight and lower hydrophobicity albeit assumed most slightly deleterious. (2) The second abundance group includes: three negative-strand mutations U-to-C, A-to-G, G-to-A and a positive-strand mutation G-to-U generated through an identical mechanism as C-to-U. (3) A clade-associated and biased mutation trend is found attributable to elevated level of the negative-sense strand synthesis. (4) Within-clade permutation variation is very informative for associating non-synonymous mutations and viral proteome changes. These findings demand a bioinformatics platform where emerging mutations are mapped on to mostly subtle but fast-adjusting viral proteomes and transcriptomes to provide biological and clinical information after logical convergence for effective pharmaceutical and diagnostic applications. Such thoughts and actions are in desperate need, especially in the middle of the War against COVID-19.

SARS-CoV-2 lineage B.6 is the major contributor to transmission in Malaysia (preprint)

BackgroundAs of June 30, 2020, Malaysia had confirmed 8,639 cases of COVID-19. About 39% of these were associated with a religious mass gathering event held in Kuala Lumpur between February 27 and March 1, 2020, which drove community transmission during Malaysias main wave. We analysed genome sequences of SARS-CoV-2 from Malaysia to understand the molecular epidemiology. MethodsWe obtained whole genome sequences of SARS-CoV-2 from 58 COVID-19 patients in Kuala Lumpur, Malaysia, and performed phylogenetic analyses on these and a further 50 Malaysian sequences available in the GISAID database. Malaysian lineage B.6 sequences were further analysed with all available worldwide lineage B.6 sequences. ResultsNine different SARS-CoV-2 lineages (A, B, B.1, B.1.1, B.1.1.1, B.1.36, B.2, B.3 and B.6) were detected in Malaysia. The B.6 lineage was first reported a week after the mass gathering and became predominant (63%) despite being relatively rare (1.4%) among available global sequences. Increases in reported cases and community-acquired B.6 lineage strains were temporally linked. Non-B.6 lineages were mainly associated with travel and showed limited onward transmission. There were also temporally-correlated increases in B.6 sequences in other Southeast Asian countries, India and Australia, linked to participants returning from this event. We also report the presence of a nsp3-C6310A substitution found in 40.5% of global B.6 sequences which has associated with reduced sensitivity in a commercial assay. ConclusionLineage B.6 became the predominant cause of community transmission in Malaysia after likely introduction during a religious mass gathering. This event also contributed to spikes of lineage B.6 in other countries in the region. Author SummaryThe COVID-19 pandemic in Malaysia was driven mainly by transmission following a religious mass gathering held in Kuala Lumpur at the end of February. To study the genetic epidemiology of SARS-CoV-2 in Malaysia, we analysed 50 available and 58 newly-generated Malaysian whole genome virus sequences. We found that lineage B.6, rare (1.4%) globally, first appeared after the mass gathering and became the most predominant (62.9%) in Malaysia. Increases in COVID-19 cases and locally-acquired B.6 strains were temporally linked. Non-B.6 viruses were mainly associated with travel and showed limited spread. Increases in B.6 viruses in Southeast Asian countries, India and Australia were linked to participants returning from this mass gathering. Altogether, 95.3% of global B.6 sequences originated in Asia or Australia. We also report a mutation in the virus nsP3 gene found in 40.5% of global B.6 sequences and associated with reduced detection by a commercial diagnostic test. In conclusion, the religious mass gathering in Kuala Lumpur was associated with the main wave of COVID-19 cases of predominantly B.6 lineage in Malaysia, and subsequent spread of B.6 viruses regionally. Genome sequence data provides valuable insight into virus spread and is important for monitoring continued accuracy of diagnostic kits.

Clinical, Immunological and Virological Characterization of COVID-19 Patients that Test Re-Positive for SARS-CoV-2 by RT-PCR (preprint)

Background: COVID-19 pandemic is underway. Some COVID-19 cases re-tested positive for SARS-CoV-2 RNA after discharge raising the public concern on their infectivity. Characterization of re-positive cases are urgently needed for designing intervention strategies. Methods: Clinical data were obtained through Guangdong COVID-19 surveillance network. Neutralization antibody titre was determined using a microneutralization assay. Potential infectivity of clinical samples was evaluated after the cell inoculation. SARS-CoV-2 RNA was detected using three different RT-PCR kits and multiplex PCR with nanopore sequencing. Findings: Among 619 discharged COVID-19 cases, 87 were re-tested as SARS-CoV-2 positive in circumstance of social isolation. All re-positive cases had mild or moderate symptoms in initial diagnosis and a younger age distribution (mean, 30·4). Re-positive cases (n=59) exhibited similar neutralization antibodies (NAbs) titre distributions to other COVID-19 cases (n=150) parallel-tested in this study. No infective viral strain could be obtained by culture and none full-length viral genomes could be sequenced for all re-positive cases. Interpretation: Re-positive SARS-CoV-2 was not caused by the secondary infection and was identified in around 14% of discharged cases. A robust Nabs response and a potential virus genome degradation were detected from nearly all re-positive cases suggesting a lower transmission risk, especially through a respiratory route. Funding: This work was supported by grants from Guangdong Provincial Novel Coronavirus Scientific and Technological Project (2020111107001), Science and Technology Planning Project of Guangdong(2018B020207006), National Science and Technology Project(2020YFC0846800).Declaration of Interests: All authors: No reported conflicts of interest.Ethics Approval Statement: This study was reviewed and approved by the Medical Ethical Committee of Guangdong Provincial Center for Disease Control and Prevention. Data collection and analysis of cases were determined by the Health Commission of Guangdong province to be part of a continuing public health outbreak investigation during the emergency response and were thus considered exempt from institutional review board approval.