Detection and genetic analysis of bovine rhinitis B virus in Japan.

Bovine rhinitis B virus (BRBV) (genus Aphthovirus, family Picornaviridae) is a significant etiological agent of the bovine respiratory disease complex. Despite global reports on BRBV, genomic data for Japanese strains are not available. In this study, we aimed to obtain genomic information on BRBV in Japan and analyze its genetic characteristics. In nasal swabs from 66 cattle, BRBV was detected in 6 out of 10 symptomatic and 4 out of 56 asymptomatic cattle. Using metagenomic sequencing and Sanger sequencing, the nearly complete genome sequences of two Japanese BRBV strains, IBA/2211/2 and LAV/238002, from symptomatic and asymptomatic cattle, respectively, were determined. These viruses shared significant genetic similarity with known BRBV strains and exhibited unique mutations and recombination events, indicating dynamic evolution, influenced by regional environmental and biological factors. Notably, the leader gene was only approximately 80% and 90% identical in its nucleotide and amino acid sequence, respectively, to all of the BRBV strains with sequences in the GenBank database, indicating significant genetic divergence in the Japanese BRBV leader gene. These findings provide insights into the genetic makeup of Japanese BRBV strains, enriching our understanding of their genetic diversity and evolutionary mechanisms.

Genomic diversity of group A rotaviruses from wild boars and domestic pigs in Japan: wide prevalence of NSP5 carrying the H2 genotype.

Group A rotavirus (RVA) sequences were detected in 10.8% (23/212) and 20.7% (87/421) of fecal samples collected in 2017-2022 from wild boars and domestic pigs, using next-generation sequencing. Complete genome sequence analysis of one wild boar and 13 domestic pig RVAs revealed that six of them carried the rare H2 NSP5 genotype. Out of the 39 samples for which the NSP5 genotype could be determined, 23 (59.0%) were of genotype H2. H2 porcine RVAs consist exclusively of Japanese porcine RVAs and exhibit sequence diversity in each segment, suggesting that H2 porcine RVAs may have evolved through reassortment within the Japanese pig population.

First report of the whole-genome sequence analysis of avian rotavirus A from Japanese chickens.

Rotavirus A infects many mammalian species, including humans and causes diarrhea and gastrointestinal diseases. The virus also infects various bird species, including chickens, although information of avian rotavirus A (ARVA) infection in chicken populations in Japan is scarce. In this study, we report for the first time the whole-genome sequences of ARVA strains from Japanese chicken populations. The virus strains were inoculated to MA104 cells and cultured viruses were used to obtain the sequences with the MiSeq system, and genetic analysis demonstrated the genotype constellation of G19-P[30]-I11-R6-C6-M7-A16-N6-T8-E10-H8 of the Japanese chicken ARVA isolates. Phylogenetic analyses demonstrated that the VP1, VP2, VP3, VP4, VP7, NSP2, and NSP4 coding gene sequences of the Japanese strains were closer to those of Korean than the European ARVA strains, although such relationship was not clear for other genes. The data suggest that the Japanese ARVA strains and the ones in Korea have genetically close relationship, although the origin is not clear at this point. Further information including the whole-genome sequences of the Korean strains and sequences of other Japanese chicken ARVA strains will be necessary for elucidation of their origin.

Potent immunogenicity and neutralization of recombinant adeno-associated virus expressing the glycoprotein of severe fever with thrombocytopenia virus.

Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease caused by a tick-borne virus called severe fever with thrombocytopenia syndrome virus (SFTSV). In recent years, human infections through contact with ticks and through contact with the bodily fluids of infected dogs and cats have been reported; however, no vaccine is currently available. SFTSV has two glycoproteins (Gn and Gc) on its envelope, which are vaccine-target antigens involved in immunogenicity. In the present study, we constructed novel SFTS vaccine candidates using an adeno-associated virus (AAV) vector to transport the SFTSV glycoprotein genome. AAV vectors are widely used in gene therapy and their safety has been confirmed in clinical trials. Recently, AAV vectors have been used to develop influenza and SARS-CoV-2 vaccines. Two types of vaccines (AAV9-SFTSV Gn and AAV9-SFTSV Gc) carrying SFTSV Gn and Gc genes were produced. The expression of Gn and Gc proteins in HEK293T cells was confirmed by infection with vaccines. These vaccines were inoculated into mice, and the collected sera produced anti-SFTS antibodies. Furthermore, sera from AAV9-SFTSV Gn infected mice showed a potent neutralizing ability, similar to previously reported SFTS vaccine candidates that protected animals from SFTSV infection. These findings suggest that this vaccine is a promising candidate for a new SFTS vaccine.

Challenges for Precise Subtyping and Sequencing of a H5N1 Clade 2.3.4.4b Highly Pathogenic Avian Influenza Virus Isolated in Japan in the 2022-2023 Season Using Classical Serological and Molecular Methods.

The continuous evolution of H5Nx highly pathogenic avian influenza viruses (HPAIVs) is a major concern for accurate diagnosis. We encountered some challenges in subtyping and sequencing a recently isolated H5N1 HPAIV strain using classical diagnostic methods. Oropharyngeal, conjunctival, and cloacal swabs collected from a dead white-tailed eagle (Haliaeetus albicilla albicilla) were screened via real-time RT-PCR targeting the influenza A virus matrix (M) gene, followed by virus isolation. The hemagglutination inhibition test was applied in order to subtype and antigenically characterize the isolate using anti-A/duck/Hong Kong/820/80 (H5N3) reference serum or anti-H5N1 cross-clade monoclonal antibodies (mAbs). Sequencing using previously reported universal primers was attempted in order to analyze the full-length hemagglutinin (HA) gene. Oropharyngeal and conjunctival samples were positive for the M gene, and high hemagglutination titers were detected in inoculated eggs. However, its hemagglutination activity was not inhibited by the reference serum or mAbs. The antiserum to a recently isolated H5N1 clade 2.3.4.4b strain inhibited our isolate but not older strains. A homologous sequence in the previously reported forward primer and HA2 region in our isolate led to partial HA gene amplification. Finally, next-generation sequencing confirmed the isolate as H5N1 clade 2.3.4.4b HPAIV, with genetic similarity to H5N1 strains circulating in Japan since November 2021.

Natto extract inhibits infection caused by the Aujeszky's disease virus in mice.

Aujeszky's disease virus (ADV), also known as Suid alphaherpesvirus 1, which mainly infects swine, causes life-threatening neurological disorders. This disease is a serious global risk factor for economic losses in the swine industry. The development of new anti-ADV drugs is highly anticipated and required. Natto, a traditional Japanese fermented food made from soybeans, is a well-known health food. In our previous study, we confirmed that natto has the potential to inhibit viral infections by severe acute respiratory syndrome coronavirus 2 and bovine alphaherpesvirus 1 through their putative serine protease(s). In this study, we found that an agent(s) in natto functionally impaired ADV infection in cell culture assays. In addition, ADV treated with natto extract lost viral infectivity in the mice. We conducted an HPLC gel-filtration analysis of natto extract and molecular weight markers and confirmed that Fraction No. 10 had ADV-inactivating ability. Furthermore, the antiviral activity of Fraction No. 10 was inhibited by the serine protease inhibitor 4-(2-Aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF). These results also suggest that Fraction No. 10, adjacent to the 12.5 kDa peak of the marker in natto extract, may inactivate ADV by proteolysis. Our findings provide new avenues of research for the prevention of Aujeszky's disease.

Antisera Produced Using an E. coli-Expressed SARS-CoV-2 RBD and Complemented with a Minimal Dose of Mammalian-Cell-Expressed S1 Subunit of the Spike Protein Exhibits Improved Neutralization.

E. coli-expressed proteins could provide a rapid, cost-effective, and safe antigen for subunit vaccines, provided we can produce them in a properly folded form inducing neutralizing antibodies. Here, we use an E. coli-expressed SARS-CoV-2 receptor-binding domain (RBD) of the spike protein as a model to examine whether it yields neutralizing antisera with effects comparable to those generated by the S1 subunit of the spike protein (S1 or S1 subunit, thereafter) expressed in mammalian cells. We immunized 5-week-old Jcl-ICR female mice by injecting RBD (30 µg) and S1 subunit (5 µg) according to four schemes: two injections 8 weeks apart with RBD (RBD/RBD), two injections with S1 (S1/S1), one injection with RBD, and the second one with S1 (RBD/S1), and vice versa (S1/RBD). Ten weeks after the first injection (two weeks after the second injection), all combinations induced a strong immune response with IgG titer > 105 (S1/RBD < S1/S1 < RBD/S1 < RBD/RBD). In addition, the neutralization effect of the antisera ranked as S1/RBD~RBD/S1 (80%) > S1/S1 (56%) > RBD/RBD (42%). These results indicate that two injections with E. coli-expressed RBD, or mammalian-cell-produced spike S1 subunit alone, can provide some protection against SARS-CoV-2, but a mixed injection scheme yields significantly higher protection.

Novel ollusvirus detected in a solitary wild bee species (Osmia taurus) in Japan.

Pathogens of wild bees in Japan remain largely unknown. We examined viruses harbored by solitary wild Osmia bees, including Osmia cornifrons and Osmia taurus. Interestingly, the full-length genome of a novel virus (designated as "Osmia-associated bee chuvirus", OABV) was identified in three Osmia taurus bees collected in Fukushima prefecture. The sequences and genomic features are similar to those of Scaldis River bee virus. Phylogenetic analysis based on RNA-dependent RNA polymerase, glycoprotein, and nucleoprotein sequences showed that OABV formed a subcluster within ollusviruses and was closely related to strains identified in European countries. This study extends our knowledge of wild bee parasites in Japan.

Zinc deficiency is associated with the development of ovarian endometrial cysts.

Ovarian cancers derived from endometrial cysts, also known as endometriosis in ovaries, are widespread histological types in Japan. Several studies suggest that zinc deficiency plays a role in endometriosis; however, the biological mechanism of zinc deficiency and endometrial cyst remains unknown. Thus, we investigated the association between zinc status and endometrial cysts. We measured the serum zinc levels in patients who had undergone surgery for endometrial cysts (n=19) and non-endometrial benign cysts (n=36). We analyzed cell proliferation, microarray data, and gene expression using N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a zinc chelator, in human immortalized endometrial epithelial cells (EMosis). The endometrial cyst group had considerably lower serum zinc levels than the non-endometrial benign cyst group. After adjusting for age, body mass index, alcohol consumption, smoking, and supplement use, endometrial cysts were markedly associated with serum zinc levels. EMosis cells treated with 5 µM TPEN demonstrated extensively increased proliferation compared to untreated cells. In the microarray analysis of EMosis cells treated with 5 µM TPEN, the enriched cellular components contained nucleoplasm, nuclear parts, and nuclear lumen. The upregulated biological processes included responses to hypoxia and decreased oxygen levels. The upregulated Kyoto Encyclopedia of Genes and Genomes pathway included the hypoxia-inducible factor-1 signaling pathway. EMosis cells treated with 5 µM TPEN demonstrated increased activator 1 (SRA1) expression and decreased AT-rich interaction domain 1A (ARID1A) expression. Protein-protein interaction network analysis indicated that ARID1A and SRA1 were associated with SMARCD1 and ATF1 among the differentially expressed genes in the microarray. EMosis cells treated with 5 µM TPEN revealed increased SRA1 mRNA levels and decreased ARID1A mRNA expression, whereas EMosis cells treated with 5 µM TPEN together with 10 µM zinc did not reveal changes in the mRNA levels of SRA1 or ARID1A compared with those without TPEN. These results suggest that zinc deficiency contributes to endometrial cyst development. Accordingly, zinc supplementation may suppress endometrial cyst development.

Phylogeny of Shrew- and Mole-Borne Hantaviruses in Poland and Ukraine.

Earlier, we demonstrated the co-circulation of genetically distinct non-rodent-borne hantaviruses, including Boginia virus (BOGV) in the Eurasian water shrew (Neomys fodiens), Seewis virus (SWSV) in the Eurasian common shrew (Sorex araneus) and Nova virus (NVAV) in the European mole (Talpa europaea), in central Poland. To further investigate the phylogeny of hantaviruses harbored by soricid and talpid reservoir hosts, we analyzed RNAlater®-preserved lung tissues from 320 shrews and 26 moles, both captured during 1990-2017 across Poland, and 10 European moles from Ukraine for hantavirus RNA through RT-PCR and DNA sequencing. SWSV and Altai virus (ALTV) were detected in Sorex araneus and Sorex minutus in Boginia and the Bialowieza Forest, respectively, and NVAV was detected in Talpa europaea in Huta Dlutowska, Poland, and in Lviv, Ukraine. Phylogenetic analyses using maximum-likelihood and Bayesian methods showed geography-specific lineages of SWSV in Poland and elsewhere in Eurasia and of NVAV in Poland and Ukraine. The ATLV strain in Sorex minutus from the Bialowieza Forest on the Polish-Belarusian border was distantly related to the ATLV strain previously reported in Sorex minutus from Chmiel in southeastern Poland. Overall, the gene phylogenies found support long-standing host-specific adaptation.

Diverse Molecular Mechanisms Underlying Microbe-Inducing Male Killing in the Moth Homona magnanima.

Male killing (MK) is a type of reproductive manipulation induced by microbes, where sons of infected mothers are killed during development. MK is a strategy that enhances the fitness of the microbes, and the underlying mechanisms and the process of their evolution have attracted substantial attention. Homona magnanima, a moth, harbors two embryonic MK bacteria, namely, Wolbachia (Alphaproteobacteria) and Spiroplasma (Mollicutes), and a larval MK virus, Osugoroshi virus (OGV; Partitiviridae). However, whether the three distantly related male killers employ similar or different mechanisms to accomplish MK remains unknown. Here, we clarified the differential effects of the three male killers on the sex-determination cascades and development of H. magnanima males. Reverse transcription-PCR demonstrated that Wolbachia and Spiroplasma, but not OGVs, disrupted the sex-determination cascade of males by inducing female-type splice variants of doublesex (dsx), a downstream regulator of the sex-determining gene cascade. We also found that MK microbes altered host transcriptomes in different manners; Wolbachia impaired the host dosage compensation system, whereas Spiroplasma and OGVs did not. Moreover, Wolbachia and Spiroplasma, but not OGVs, triggered abnormal apoptosis in male embryos. These findings suggest that distantly related microbes employ distinct machineries to kill males of the identical host species, which would be the outcome of the convergent evolution. IMPORTANCE Many microbes induce male killing (MK) in various insect species. However, it is not well understood whether microbes adopt similar or different MK mechanisms. This gap in our knowledge is partly because different insect models have been examined for each MK microbe. Here, we compared three taxonomically distinct male killers (i.e., Wolbachia, Spiroplasma, and a partiti-like virus) that infect the same host. We provided evidence that microbes can cause MK through distinct mechanisms that differ in the expression of genes involved in sex determination, dosage compensation, and apoptosis. These results imply independent evolutionary scenarios for the acquisition of their MK ability.

Polyunsaturated fatty acids-rich dietary lipid prevents high fat diet-induced obesity in mice.

Diet is the primary factor affecting host nutrition and metabolism, with excess food intake, especially high-calorie diets, such as high-fat and high-sugar diets, causing an increased risk of obesity and related disorders. Obesity alters the gut microbial composition and reduces microbial diversity and causes changes in specific bacterial taxa. Dietary lipids can alter the gut microbial composition in obese mice. However, the regulation of gut microbiota and host energy homeostasis by different polyunsaturated fatty acids (PUFAs) in dietary lipids remains unknown. Here, we demonstrated that different PUFAs in dietary lipids improved host metabolism in high-fat diet (HFD)-induced obesity in mice. The intake of the different PUFA-enriched dietary lipids improved metabolism in HFD-induced obesity by regulating glucose tolerance and inhibiting colonic inflammation. Moreover, the gut microbial compositions were different among HFD and modified PUFA-enriched HFD-fed mice. Thus, we have identified a new mechanism underlying the function of different PUFAs in dietary lipids in regulating host energy homeostasis in obese conditions. Our findings shed light on the prevention and treatment of metabolic disorders by targeting the gut microbiota.

Development of a one-run real-time PCR detection system for pathogens associated with poultry infectious diseases.

Infectious diseases are an important issue in the poultry industry, requiring early diagnosis and countermeasures. To address this, we present a system based on TaqMan real-time PCR to detect pathogen genome in specimens collected from chickens. We designed 12 primer-probe sets for pathogens causing respiratory or systemic symptoms. In field samples, we detected three viruses, including DNA and RNA viruses, and three bacteria. The chicken anemia virus and Avibacterium paragallinarum were detected only in young and laying hens, respectively. Bacteria were detected only in throat swabs, and gallid alphaherpesvirus 2 was detected in different specimens at each developmental stage. Our novel TaqMan real-time PCR system effectively detects pathogen's gene in chickens, while taking age into account.

The First Nationwide Surveillance of Severe Fever with Thrombocytopenia Syndrome in Ruminants and Wildlife in Taiwan.

Since the first discovery of severe fever with thrombocytopenia syndrome virus (SFTSV) in China in 2009, SFTSV has rapidly spread through other Asian countries, including Japan, Korea, Vietnam and Pakistan, in chronological order. Taiwan reported its first discovery of SFTSV in sheep and humans in 2020. However, the prevalence of SFTSV in domestic and wildlife animals and the geographic distribution of the virus within the island remain unknown. A total of 1324 animal samples, including 803 domestic ruminants, 521 wildlife animals and 47 tick pools, were collected from March 2021 to December 2022 from 12 counties and one terrestrial island. The viral RNA was detected by a one-step real-time reverse transcription polymerase chain reaction (RT-PCR). Overall, 29.9% (240/803) of ruminants showed positive SFTSV RNA. Sheep had the highest viral RNA prevalence of 60% (30/50), followed by beef cattle at 28.4% (44/155), goats at 28.3% (47/166), and dairy cows at 27.5% (119/432). The bovine as a total of dairy cow and beef cattle was 27.8% (163/587). The viral RNA prevalence in ticks (predominantly Rhipicephalus microplus) was similar to those of ruminants at 27.7% (13/47), but wild animals exhibited a much lower prevalence at 1.3% (7/521). Geographically the distribution of positivity was quite even, being 33%, 29.1%, 27.5% and 37.5% for northern, central, southern and eastern Taiwan, respectively. Statistically, the positive rate of beef cattle in the central region (55.6%) and dairy cattle in the eastern region (40.6%) were significantly higher than the other regions; and the prevalence in Autumn (September-November) was significantly higher than in the other seasons (p < 0.001). The nationwide study herein revealed for the first time the wide distribution and high prevalence of SFTSV in both domestic animals and ticks in Taiwan. Considering the high mortality rate in humans, surveillance of other animal species, particularly those in close contact with humans, and instigation of protective measures for farmers, veterinarians, and especially older populations visiting or living near farms or rural areas should be prioritized.

Prevalence and genetic diversity in bovine parechovirus infecting Japanese cattle.

The first bovine parechovirus (Bo_ParV) was reported in 2021, and currently, only two nearly complete genome sequences of Bo_ParV are available. In this study, we detected Bo_ParVs in 10 out of 158 bovine fecal samples tested using real-time RT-PCR, and Bo_ParVs were isolated from three of these samples using MA104 cells. Analysis of the P1 region revealed that Bo_ParVs shared high pairwise amino acid sequence similarity (≥ 95.7% identity), suggesting antigenic similarity among Bo_ParVs, whereas nucleotide sequence identity values (≥ 84.8%) indicated more variability. A recombination breakpoint was identified in the 2B region, which may influence the evolution of this virus.

Novel recombinant porcine enterovirus G viruses lacking structural proteins are maintained in pig farms in Japan.

Type 1 recombinant enterovirus G (EV-G), which carries the papain-like cysteine protease (PLCP) gene of torovirus between its 2C/3A regions, and type 2 recombinant EV-G, which carries the torovirus PLCP gene with its flanking regions having non-EV-G sequences in place of the viral structural genes, have been detected in pig farms in several countries. In a previous study, we collected 222 fecal samples from 77 pig farms from 2104 to 2016 and detected one type 2 recombinant EV-G genome by metagenomics sequencing. In this study, we reanalyzed the metagenomic data and detected 11 type 2 recombinant EV-G genomes. In addition, we discovered new type 2 recombinant EV-G genomes of the two strains from two pig farms samples in 2018 and 2019. Thus, we identified the genomes of 13 novel type 2 recombinant EV-Gs isolated from several pig farms in Japan. Type 2 recombinant EV-G has previously been detected only in neonatal piglets. The present findings suggest that type 2 recombinant EV-G replicates in weaning piglets and sows. The detection of type 1 recombinant EV-Gs and type 2 recombinant EV-Gs at 3-year and 2-year intervals, respectively, from the same pig farm suggests that the viruses were persistently infecting or circulating in these farms.

YAP/TAZ-TEAD is a novel transcriptional regulator of genes encoding steroidogenic enzymes in rat granulosa cells and KGN cells.

Steroidogenesis in ovarian granulosa cells is regulated by the follicle-stimulating hormone (FSH) via transcriptional regulation of its related genes. We herein showed the involvement of the Hippo pathway in this regulation. In KGN granulosa cell, repression of YAP/TAZ activity induced the expression of CYP11A1, HSD3B2, and CYP19A1 in a TEAD-dependent manner without cAMP stimulation. A selective inhibitor of p38 MAP kinase, suppressed YAP/TAZ knockdown-indued the expression of these genes, suggesting this signal could be involved. The expression of these genes was induced by 8Br-cAMP, whereas that of CYR61 and ADATS1, typical YAP/TAZ-TEAD target genes, was suppressed, suggesting that the cellular signaling of cAMP reduced YAP/TAZ-TEAD activity. The constitutively active mutant YAP canceled the FSH- and 8Br-cAMP-mediated induction of these genes in primary rat granulosa and KGN cells, respectively. Moreover, regulation of steroidogenesis-related genes by YAP/TAZ-TEAD was independent of steroidogenic factor 1, a master gene regulator of steroidogenesis. These results suggest that YAP/TAZ-TEAD is a negative regulator of steroidogenesis and that suppression of YAP/TAZ-TEAD activity by FSH is involved in ovarian steroidogenesis.

Development of a new quantification method of Sarcocystis cruzi through detection of the acetyl-CoA synthetase gene.

Sarcocystis cruzi is a member of the genus Sarcocystis, infecting bovine animals such as cattle and bison as intermediate hosts, and canids such as dogs and raccoon dogs as definitive hosts. Acute sarcocystosis of S. cruzi causes occasional symptoms in cattle, including weight loss, reduced milk production, abortions, and death, and similar to other Sarcocystis species can potentially cause food poisoning in humans when raw or undercooked infected cattle meat is consumed. Despite these issues, genetic information on S. cruzi is scarce, and there is no specific quantitative method for the detection and quantification of the parasite in infected cattle. In this study, we aimed to develop a method based on high-throughput sequencing of S. cruzi genome and transcriptome that specifically and quantitatively detects the S. cruzi acetyl-CoA synthetase gene (ScACS). Cardiac muscles were collected from slaughterhouses in Saitama Prefecture to obtain sarcocysts from which DNA and RNA were extracted for the high-throughput sequencing. Using the sequences, we developed a specific quantitative PCR assay which could distinguish S. cruzi ACS from that of Toxoplasma gondii by taking advantage of the differences in their exon/intron organizations and validated the assay with the microscopic counting of the S. cruzi bradyzoites. Thus, this assay will be useful for future studies of S. cruzi pathogenesis in cattle and for the surveillance of infected animals, thereby easing public health concerns.

Chronic low-grade inflammation and ovarian dysfunction in women with polycystic ovarian syndrome, endometriosis, and aging.

The ovarian microenvironment is critical for follicular development and oocyte maturation. Maternal conditions, including polycystic ovary syndrome (PCOS), endometriosis, and aging, may compromise the ovarian microenvironment, follicular development, and oocyte quality. Chronic low-grade inflammation can induce oxidative stress and tissue fibrosis in the ovary. In PCOS, endometriosis, and aging, pro-inflammatory cytokine levels are often elevated in follicular fluids. In women with obesity and PCOS, hyperandrogenemia and insulin resistance induce ovarian chronic low-grade inflammation, thereby disrupting follicular development by increasing oxidative stress. In endometriosis, ovarian endometrioma-derived iron overload can induce chronic inflammation and oxidative stress, leading to ovarian ferroptosis and fibrosis. In inflammatory aging (inflammaging), senescent cells may secrete senescence-associated secretory phenotype factors, causing chronic inflammation and oxidative stress in the ovary. Therefore, controlling chronic low-grade inflammation and fibrosis in the ovary would present a novel therapeutic strategy for improving the follicular microenvironment and minimizing ovarian dysfunction.

An Escherichia coli Expressed Multi-Disulfide Bonded SARS-CoV-2 RBD Shows Native-like Biophysical Properties and Elicits Neutralizing Antisera in a Mouse Model.

A large-scale Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 could yield a versatile and low-cost antigen for a subunit vaccine. Appropriately folded antigens can potentially elicit the production of neutralizing antisera providing immune protection against the virus. However, E. coli expression using a standard protocol produces RBDs with aberrant disulfide bonds among the RBD's eight cysteines resulting in the expression of insoluble and non-native RBDs. Here, we evaluate whether E. coli expressing RBD can be used as an antigen candidate for a subunit vaccine. The expressed RBD exhibited native-like structural and biophysical properties as demonstrated by analytical RP-HPLC, circular dichroism, fluorescence, and light scattering. In addition, our E. coli expressed RBD binds to hACE2, the host cell's receptor, with a binding constant of 7.9 × 10-9 M, as indicated by biolayer interferometry analysis. Our E. coli-produced RBD elicited a high IgG titer in Jcl:ICR mice, and the RBD antisera inhibited viral growth, as demonstrated by a pseudovirus-based neutralization assay. Moreover, the increased antibody level was sustained for over 15 weeks after immunization, and a high percentage of effector and central memory T cells were generated. Overall, these results show that E. coli-expressed RBDs can elicit the production of neutralizing antisera and could potentially serve as an antigen for developing an anti-SARS-CoV-2 subunit vaccine.