Development of an Optimized Process for Functional Recombinant SARS-CoV-2 Spike S1 Receptor-Binding Domain Protein Produced in the Baculovirus Expression Vector System.

To map the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and evaluate immune response variations against this virus, it is essential to set up efficient serological tests locally. The SARS-CoV-2 immunogenic proteins were very expensive and not affordable for lower- middle-income countries (LMICs). For this purpose, the commonly used antigen, receptor-binding domain (RBD) of spike S1 protein (S1RBD), was produced using the baculovirus expression vector system (BEVS). In the current study, the expression of S1RBD was monitored using Western blot under different culture conditions. Different parameters were studied: the multiplicity of infection (MOI), cell density at infection, and harvest time. Hence, optimal conditions for efficient S1RBD production were identified: MOI 3; cell density at infection 2-3 × 106 cells/mL; and time post-infection (tPI or harvest time) of 72 h and 72-96 h, successively, for expression in shake flasks and a 7L bioreactor. A high production yield of S1RBD varying between 4 mg and 70 mg per liter of crude cell culture supernatant was achieved, respectively, in the shake flasks and 7L bioreactor. Moreover, the produced S1RBD showed an excellent antigenicity potential against COVID-19 (Wuhan strain) patient sera evaluated by Western blot. Thus, additional serological assays, such as in-house ELISA and seroprevalence studies based on the purified S1RDB, were developed.

Evaluation of the Immunogenicity in Mice Orally Immunized with Recombinant Lactobacillus casei Expressing Porcine Epidemic Diarrhea Virus S1 Protein.

Porcine epidemic diarrhea (PED), characterized by diarrhea, vomiting, and dehydration, is an acute enteric infectious disease of pigs. The disease is caused by porcine epidemic diarrhea virus (PEDV), which infects the intestinal mucosal surface. Therefore, mucosal immunization through the oral route is an effective method of immunization. Lactic acid bacteria, which are acid resistant and bile-salt resistant and improve mucosal immunity, are ideal carriers for oral vaccines. The S1 glycoprotein of PEDV mediates binding of the virus with cell receptors and induces neutralizing antibodies against the virus. Therefore, we reversely screened the recombinant strain pPG-SD-S1/Δupp ATCC 393 expressing PEDV S1 glycoprotein by Lactobacillus casei deficient in upp genotype (Δupp ATCC 393). Mice were orally immunized three times with the recombinant bacteria that had been identified for expression, and the changes of anti-PEDV IgG and secreted immunoglobulin A levels were observed over 70 days. The results indicated that the antibody levels notably increased after oral administration of recombinant bacteria. The detection of extracellular cytokines on the 42nd day after immunization indicated high levels of humoral and cellular immune responses in mice. The above results demonstrate that pPG-SD-S1/Δupp ATCC 393 has great potential as an oral vaccine against PEDV.

Functional responsiveness of memory T cells from COVID-19 patients.

The presence of memory T cells in COVID-19 patients has been acknowledged, however the functional potency of memory responses is critical for protection. In this study, naïve, effector, effector memory, and central memory CD4+ and CD8+ T cells obtained from the COVID-19 survivors were re-exposed to autologous monocyte-derived DCs that were loaded with SARS-CoV-2 spike glycoprotein S1. Proliferation capacity, CD25, 4-1BB, and PD-1 expression, and IFN-γ, IL-6, granzyme, granulysin, and FasL secretion were enhanced in CD4+ and CD8+ effector memory and central memory T cells. Albeit being at heterogeneous levels, the memory T cells from the individuals with COVID-19 history possess functional capacities to reinvigorate anti-viral immunity against SARS-CoV-2.

A Potential Peptide From Soy Cheese Produced Using Lactobacillus delbrueckii WS4 for Effective Inhibition of SARS-CoV-2 Main Protease and S1 Glycoprotein.

The COVID-19 pandemic caused by novel SARS-CoV-2 has resulted in an unprecedented loss of lives and economy around the world. In this study, search for potential inhibitors against two of the best characterized SARS-CoV-2 drug targets: S1 glycoprotein receptor-binding domain (RBD) and main protease (3CLPro), was carried out using the soy cheese peptides. A total of 1,420 peptides identified from the cheese peptidome produced using Lactobacillus delbrueckii WS4 were screened for antiviral activity by employing the web tools, AVPpred, and meta-iAVP. Molecular docking studies of the selected peptides revealed one potential peptide "KFVPKQPNMIL" that demonstrated strong affinity toward significant amino acid residues responsible for the host cell entry (RBD) and multiplication (3CLpro) of SARS-CoV-2. The peptide was also assessed for its ability to interact with the critical residues of S1 RBD and 3CLpro of other ß-coronaviruses. High binding affinity was observed toward critical amino acids of both the targeted proteins in SARS-CoV, MERS-CoV, and HCoV-HKU1. The binding energy of KFVPKQPNMIL against RBD and 3CLpro of the four viruses ranged from -8.45 to -26.8 kcal/mol and -15.22 to -22.85 kcal/mol, respectively. The findings conclude that cheese, produced by using Lb. delbrueckii WS4, could be explored as a prophylactic food for SARS-CoV-2 and related viruses. In addition, the multi-target inhibitor peptide, which effectively inhibited both the viral proteins, could further be used as a terminus a quo for the in vitro and in vivo function against SARS-CoV-2.

Epidemiological investigation of avian infectious bronchitis and locally determined genotype diversity in central China: a 2016-2018 study.

Infectious bronchitis (IB), caused by avian IB virus (IBV), is an acute and highly contagious disease of chickens. From 2016 to 2018, 56 IBV strains were isolated and identified from clinical samples obtained from various chicken farms located in central China. The S1 sequencing of these strains revealed nucleotide and amino acid identities of 70.2 to 100% and 62.6 to 100%, respectively, compared with those of reference strains. Phylogenetic analysis indicated that the genotypes of the isolates included GI-13 (4/91), GI-7 (TW-I), GI-24 (Mass), GI-19 (QX), and GI-18 (LDT3-A), with GI-19 (QX) being the predominant genotype. Meanwhile, GI-13 (4/91) was the second most dominant genotype in Henan Province, whereas it was GI-7 (TW-I) in Hunan and Hubei provinces. Recombination analysis of 3 variant strains showed that CK/CH/HeN/20160113 might be a recombination of LDT3-A- and QX-type strains and that CK/CH/HeN/20160316 might be a recombination of Italy-02-type strain and CK-CH-LJS08II. The predicted tertiary structure between CK/CH/HeN/20160113 and LDT3-A-type strain revealed that the novel 336 (L-P) and 455 (S-A) mutations changed the structure from an alpha helix to a random crimp. In addition, the 275 (Y-F) site reduced the length of the ß-sheet, whereas the site 353 (A-T) extended the ß-sheet. These findings suggested that GI-19 (QX) remains the predominant genotype in central China, and a locally determined complex genotype associated with variable clinical symptoms exists related to gene recombination and mutations.

Sequencing and In Silico Multi-aspect Analysis of S1 Glycoprotein in 793/B Serotype of Infectious Bronchitis Virus Isolated From Iran in 2003 and 2011.

Infectious bronchitis (IB) is an acute, highly contagious, and economically important viral disease of chickens. The S1 subunit from Spike (S) protein plays the major role in protective immunity and is involved in the host-virus interactions, as well as infectious bronchitis virus (IBV) serotyping. Aim of the present study was multi-aspect analysis of the molecular and immunological features of 5' part belonging to the S1 glycoprotein sequence of Iranian 793/B IBV strain isolates. This might ideally help in characterization, prevention, and vaccine development. The tissue samples were prepared, followed by virus isolation, reverse transcription polymerase chain reaction and restriction fragment length polymorphism analysis. In addition, sequencing and registration of the sequences in the National Center for Biotechnology Information were performed. Moreover, 12 sequences were retrieved from Fars province, Iran. The next steps included evaluation of conservation/variability along the sequences, phylogenetic analysis, estimation of the average evolutionary divergence over all the sequence pairs, predicting the phosphorylation/N-glycosylation/palmitoylation sites, and the final analysis of antigenicity. The findings of alignment, entropy plot, and pairwise similarity analysis revealed 17 hypervariable regions. The isolates belonging to Tehran were clustered in phylogenetic tree, and the most similar isolates to them were ADW11182 and ADW11183. Location of some of the N-glycosylation/phosphorylation/palmitoylation points indicated that these sites were conserved among the isolates. Furthermore, the frequency of epitopes and their scores reflect the high immunogenicity of S1 protein in 793/B serotype. Analysis of the primary and secondary structures demonstrated that their parameters had variable values and were different regarding the number and location of α-helix, β-strand, and coils. According to our findings, the Iranian isolates of 793/B serotype change their molecular characteristics during time and in different geographical regions. These alterations might account for failure in prevention programs and differences in virulence and pathogenicity.

Analysis of the S1 gene of the avian infectious bronchitis virus (IBV) reveals changes in the IBV genetic groups circulating in southern Thailand.

The new variants of the avian infectious bronchitis virus (IBV) produce a range of symptoms and cause global economic losses to the poultry industry. We investigated the S1 glycoprotein of 24 recent IBV isolates from chickens and demonstrated that two predominant genetic groups were circulating in southern Thailand between 2008 and 2013. Seven IBV variants, isolated from 2008 to 2009, were clustered in the Thailand THA001 group I while 15 IBV variants, isolated from 2009 to 2013, were classified into the QX-like group II. Moreover, a single isolate from a broiler was categorized into the Massachusetts-type, and an isolate from a layer belonged to the 4/91 type virus. Interestingly, both the IBV groups I and II were isolated from native chickens (62.5%) and caused a range of symptoms. Our results indicate that the QX-like viruses were predominant after 2009, replacing the THA001 type viruses. Furthermore, native chickens may contribute to the epidemiology of IB.

Clonagem e expressão do gene da glicoproteína S1 do vírus da bronquite infecciosa das galinhas em Pichia pastoris / Cloning and expression in Pichia pastoris of the S1 glycoprotein gene of the chicken infectious bronchitis virus

Variações genética e antigênica são observadas com frequência elevada entre estirpes do VBIG e envolvem principalmente a glicoproteína S1. Com o objetivo de contribuir com a disponibilidade de ferramentas para o imunodiagnóstico e a imunoprofilaxia da bronquite infecciosa das galinhas foi desenvolvida uma metodologia para expressão recombinante da glicoproteína S1 na levedura Picchia pastoris. O cDNA do gene codificador dessa proteína foi obtido a partir de RNA viral de ovos embrionados infectados com a estirpe M41 do VBIG submetido à transcrição reversa (RT) e reação em cadeia da polimerase (PCR), amplificando-se a sequência codificadora de S1 acrescida de extremidades compatíveis com a clonagem no vetor usado na transformação de leveduras. A indução com metanol resultou na produção de uma proteína detectada como banda única do tamanho previsto, em western-blot, no lisado celular das leveduras transformadas. A expressão em P. pastoris mostrou ser um método eficaz para a produção recombinante da proteína S1 do VBIG, com potencial para utilização em técnicas de imunodiagnóstico da bronquite infecciosa das galinhas.

Genetic and antigenic variation are very frequently observed among IBV strains and affect mainly the S1 glycoprotein. In order to contribute to the availability of tools for immunodiagnosis and immunoprophylaxis of chicken infectious bronchitis we developed an expression system for production of recombinant S1 glycoprotein in Pichia pastoris. We obtained the cDNA from viral RNA on embryonated eggs infected with the M41 strain of IBV, by reverse transcription (RT) and polymerase chain reaction (PCR), amplifying the S1 coding sequence with extremities compatible with the vector used to transform yeast. Induction with methanol led to the production of a protein with the predicted molecular weight that was detected by Western blot in the cell lysate of transformed yeast. Expression in P. pastoris proved to be an effective method for recombinant production of S1 protein from IBV, with potential for use in immuno-diagnosis of chicken infectious bronchitis virus.

Sequence analysis of the S1 glycoprotein gene of infectious bronchitis viruses: identification of a novel phylogenetic group in Korea

Twelve Korean infectious bronchitis viruses (IBVs) were isolated in the field from chickens suspected of being carriers of infectious bronchitis between 2001 and 2003. The S1 glycoprotein genes of these IBV isolates were amplified by reverse transcriptase-polymerase chain reaction (RTPCR) and analyzed by restriction fragment length polymorphism (RFLP) analysis. These Korean IBV isolates were classified into three groups according to their RFLP patterns obtained using the restriction enzyme HaeIII. Half of the twelve isolates were similar to the KM91 RFLP pattern, which is a common pattern in Korea. Three more isolates were related to the Arkansas strain pattern, but with some unique variations. The other three viruses showed variant RFLP patterns. For a comparison with the published sequences for non-Korean IBV strains, amplified PCR products from the twelve isolates were cloned and sequenced. The Korean IBV field isolates had 71.2-99.7% nucleotide sequence homology with each other and 45.9-80.7% nucleotide sequence homology with non-Korean IBV strains. With respect to the deduced amino acid sequence, the Korean IBV isolates had 71.5-99.3% similarity with each other and 44.9-80.3% similarity with non-Korean IBV strains. Phylogenetic tree analysis revealed that some of the IBV isolates appear to belong to a new group, different from the non-Korean IBV strains or from previously isolated Korean IBV strains. Specifically, the new Korean IBV isolates K10217-03, K3-3 and K1255-03 represented a separate group. These findings suggest that the Korean IBVs appear to be continuously evolving.