Gastrointestinal microbiota and metabolites possibly contribute to distinct pathogenicity of SARS-CoV-2 proto or its variants in rhesus monkeys.

Gastrointestinal (GI) infection is evidenced with involvement in COVID-19 pathogenesis caused by SARS-CoV-2. However, the correlation between GI microbiota and the distinct pathogenicity of SARS-CoV-2 Proto and its emerging variants remains unclear. In this study, we aimed to determine if GI microbiota impacted COVID-19 pathogenesis and if the effect varied between SARS-CoV-2 Proto and its variants. We performed an integrative analysis of histopathology, microbiomics, and transcriptomics on the GI tract fragments from rhesus monkeys infected with SARS-CoV-2 proto or its variants. Based on the degree of pathological damage and microbiota profile in the GI tract, five of SARS-CoV-2 strains were classified into two distinct clusters, namely, the clusters of Alpha, Beta and Delta (ABD), and Proto and Omicron (PO). Notably, the abundance of potentially pathogenic microorganisms increased in ABD but not in the PO-infected rhesus monkeys. Specifically, the high abundance of UCG-002, UCG-005, and Treponema in ABD virus-infected animals positively correlated with interleukin, integrins, and antiviral genes. Overall, this study revealed that infection-induced alteration of GI microbiota and metabolites could increase the systemic burdens of inflammation or pathological injury in infected animals, especially in those infected with ABD viruses. Distinct GI microbiota and metabolite profiles may be responsible for the differential pathological phenotypes of PO and ABD virus-infected animals. These findings improve our understanding the roles of the GI microbiota in SARS-CoV-2 infection and provide important information for the precise prevention, control, and treatment of COVID-19.

Infectious hepatitis E virus excreted into the vagina.

Hepatitis E virus (HEV) persists in the male genital tract that associates with infertility. However, the presence of HEV in the female genital tract is unreported. Vaginal secretions, cervical smears, and cervix uteri were collected to explore the presence of HEV in the female genital tract. HEV RNA and/or antigens were detected in the vaginal secretions, cervical smears, and the cervix uteri of women. The infectivity of HEV excreted into vaginal secretions was further validated in vitro. In addition, HEV replicates in the female genital tract were identified in HEV-infected animal models by vaginal injection or vaginal mucosal infection to imitate sexual transmission. Serious genital tract damage and inflammatory responses with significantly elevated mucosal innate immunity were observed in women or animals with HEV vaginal infection. Results demonstrated HEV replicates in the female genital tract and causes serious histopathological damage and inflammatory responses.

Single-cell transcriptomic atlas of distinct early immune responses induced by SARS-CoV-2 Proto or its variants in rhesus monkey.

Immune responses induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection play a critical role in the pathogenesis and outcome of coronavirus disease 2019 (COVID-19). However, the dynamic profile of immune responses postinfection by SARS-CoV-2 variants of concern (VOC) is not fully understood. In this study, peripheral blood mononuclear cells single-cell sequencing was performed to determine dynamic profiles of immune response to Prototype, Alpha, Beta, and Delta in a rhesus monkey model. Overall, all strains induced dramatic changes in both cellular subpopulations and gene expression levels at 1 day postinfection (dpi), which associated function including adaptive immune response, innate immunity, and IFN response. COVID-19-related genes revealed different gene profiles at 1 dpi among the four SARS-CoV-2 strains, including genes reported in COVID-19 patients with increased risk of autoimmune disease and rheumatic diseases. Delta-infected animal showed inhibition of translation pathway. B cells, T cells, and monocytes showed much commonality rather than specificity among the four strains. Monocytes were the major responders to SARS-CoV-2 infection, and the response lasted longer in Alpha than the other strains. Thus, this study reveals the early immune responses induced by SARS-CoV-2 Proto or its variants in nonhuman primates, which is important information for controlling rapidly evolving viruses.

Differential Transcriptomic Landscapes of SARS-CoV-2 Variants in Multiple Organs from Infected Rhesus Macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the persistent coronavirus disease 2019 (COVID-19) pandemic, which has resulted in millions of deaths worldwide and brought an enormous public health and global economic burden. The recurring global wave of infections has been exacerbated by growing variants of SARS-CoV-2. In this study, the virological characteristics of the original SARS-CoV-2 strain and its variants of concern (VOCs; including Alpha, Beta, and Delta) in vitro, as well as differential transcriptomic landscapes in multiple organs (lung, right ventricle, blood, cerebral cortex, and cerebellum) from the infected rhesus macaques, were elucidated. The original strain of SARS-CoV-2 caused a stronger innate immune response in host cells, and its VOCs markedly increased the levels of subgenomic RNAs, such as N, Orf9b, Orf6, and Orf7ab, which are known as the innate immune antagonists and the inhibitors of antiviral factors. Intriguingly, the original SARS-CoV-2 strain and Alpha variant induced larger alteration of RNA abundance in tissues of rhesus monkeys than Beta and Delta variants did. Moreover, a hyperinflammatory state and active immune response were shown in the right ventricles of rhesus monkeys by the up-regulation of inflammation- and immune-related RNAs. Furthermore, peripheral blood may mediate signaling transmission among tissues to coordinate the molecular changes in the infected individuals. Collectively, these data provide insights into the pathogenesis of COVID-19 at the early stage of infection by the original SARS-CoV-2 strain and its VOCs.

SARS-CoV-2 Omicron (B.1.1.529) infection in rhesus macaques, hamsters, and BALB/c mice with severe lung histopathological damage.

Since the first SARS-CoV-2 outbreak in late 2019, the SARS-CoV-2 genome has harbored multiple mutations, especially spike protein mutations. The currently fast-spreading Omicron variant that manifests without symptoms or with upper respiratory diseases has been recognized as a serious global public health problem. However, its pathological mechanism is largely unknown. In this work, rhesus macaques, hamsters, and BALB/C mice were employed as animal models to explore the pathogenesis of Omicron (B.1.1.529). Notably, Omicron (B.1.1.529) infected the nasal turbinates, tracheae, bronchi, and lungs of hamsters and BALB/C mice with higher viral loads than in those of rhesus macaques. Severe histopathological damage and inflammatory responses were observed in the lungs of Omicron (B.1.1.529)-infected animals. In addition, viral replication was found in multiple extrapulmonary organs. Results indicated that hamsters and BALB/c mice are potential animal models for studies on the development of drugs/vaccines and therapies for Omicron (B.1.1.529).

Quantification of host proteomic responses to genotype 4 hepatitis E virus replication facilitated by pregnancy serum.

BACKGROUND: Hepatitis E virus (HEV) infection is a common cause of acute hepatitis worldwide and causes approximately 30% case fatality rate among pregnant women. Pregnancy serum (PS), which contains a high concentration of estradiol, facilitates HEV replication in vitro through the suppression of the PI3K-AKT-mTOR and cAMPK-PKA-CREB signaling pathways. However, the proteomics of the complex host responses to HEV infection, especially how PS facilitates viral replication, remains unclear. METHODS: In this study, the differences in the proteomics of HEV-infected HepG2 cells supplemented with fetal bovine serum (FBS) from those of HEV-infected HepG2 cells supplemented with serum from women in their third trimester of pregnancy were quantified by using isobaric tags for relative and absolute quantification technology. RESULTS: A total of 1511 proteins were identified, among which 548 were defined as differentially expressed proteins (DEPs). HEV-infected cells supplemented with PS exhibited the most significant changes at the protein level. A total of 328 DEPs, including 66 up-regulated and 262 down-regulated proteins, were identified in HEV-infected cells supplemented with FBS, whereas 264 DEPs, including 201 up-regulated and 63 down-regulated proteins, were found in HEV-infected cells supplemented with PS. Subsequently, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that in HEV-infected cells, PS supplementation adjusted more host genes and signaling pathways than FBS supplementation. The DEPs involved in virus-host interaction participated in complex interactions, especially a large number of immune-related protein emerged in HEV-infected cells supplemented with PS. Three significant or interesting proteins, including filamin-A, thioredoxin, and cytochrome c, in HEV-infected cells were functionally verified. CONCLUSIONS: The results of this study provide new and comprehensive insight for exploring virus-host interactions and will benefit future studies on the pathogenesis of HEV in pregnant women.

Genotype 4 Hepatitis E virus replicates in the placenta, causes severe histopathological damage, and vertically transmits to fetuses.

BACKGROUND: Hepatitis E virus (HEV) infection in pregnant women causes adverse pregnancy outcomes, including maternal death, premature delivery, stillbirth, and fetal infection. However, the pathogenesis of maternal and fetal HEV infection is unclear. METHODS: Placenta and placental appendixes were collected from HEV-4 infected pregnant women to explore the vertical transmission of HEV from mothers to fetuses. RESULTS: HEV-4 replicated in the placenta, placental membrane, and umbilical cord and was vertically transmitted from mothers to fetuses. HEV-4 placental infection resulted in serious histopathological damage, such as fibrosis and calcification, and severe inflammatory responses. Adverse maternal outcomes were observed in 38.5% of HEV-4 infected pregnant women. The distinct cytokine/chemokine expression patterns of HEV-infected pregnant women and nonpregnant women may contribute to the adverse pregnancy outcomes. Furthermore, the impaired maternal and fetal innate immune responses against HEV-4 facilitated viral replication during pregnancy. CONCLUSION: HEV-4 replicates in the placenta and is vertically transmitted from mothers to fetuses, causing severe histopathological damage.

Aryl hydrocarbon receptor is a proviral host factor and a candidate pan-SARS-CoV-2 therapeutic target.

The emergence of a series of SARS-CoV-2 variants has necessitated the search for broad-spectrum antiviral targets. The aryl hydrocarbon receptor (AhR) senses tryptophan metabolites and is an immune regulator. However, the role of AhR in SARS-CoV-2 infection and whether AhR can be used as the target of antiviral therapy against SARS-CoV-2 and its variants are yet unclear. Here, we show that infection with SARS-CoV-2 activates AhR signaling and facilitates viral replication by interfering with IFN-I-driven antiviral immunity and up-regulating ACE2 receptor expression. The pharmacological AhR blockade or AhR knockout reduces SARS-CoV-2 and its variants' replication in vitro. Drug targeting of AhR with AhR antagonists markedly reduced SARS-CoV-2 and its variants' replication in vivo and ameliorated lung inflammation caused by SARS-CoV-2 infection in hamsters. Overall, AhR was a SARS-CoV-2 proviral host factor and a candidate host-directed broad-spectrum target for antiviral therapy against SARS-CoV-2 and its variants, including Delta and Omicron, and potentially other variants in the future.

An mRNA-based T-cell-inducing antigen strengthens COVID-19 vaccine against SARS-CoV-2 variants.

Herd immunity achieved through mass vaccination is an effective approach to prevent contagious diseases. Nonetheless, emerging SARS-CoV-2 variants with frequent mutations largely evaded humoral immunity induced by Spike-based COVID-19 vaccines. Herein, we develop a lipid nanoparticle (LNP)-formulated mRNA-based T-cell-inducing antigen, which targeted three SARS-CoV-2 proteome regions that enriched human HLA-I epitopes (HLA-EPs). Immunization of HLA-EPs induces potent cellular responses to prevent SARS-CoV-2 infection in humanized HLA-A*02:01/DR1 and HLA-A*11:01/DR1 transgenic mice. Of note, the sequences of HLA-EPs are highly conserved among SARS-CoV-2 variants of concern. In humanized HLA-transgenic mice and female rhesus macaques, dual immunization with the LNP-formulated mRNAs encoding HLA-EPs and the receptor-binding domain of the SARS-CoV-2 B.1.351 variant (RBDbeta) is more efficacious in preventing infection of SARS-CoV-2 Beta and Omicron BA.1 variants than single immunization of LNP-RBDbeta. This study demonstrates the necessity to strengthen the vaccine effectiveness by comprehensively stimulating both humoral and cellular responses, thereby offering insight for optimizing the design of COVID-19 vaccines.

Aggravated pneumonia and diabetes in SARS-CoV-2 infected diabetic mice.

Multiple clinical and epidemiological studies have shown an interconnection between coronavirus disease 2019 (COVID-19) and diabetes, but experimental evidence is still lacking. Understanding the interplay between them is important because of the global health burden of COVID-19 and diabetes. We found that C57BL/6J mice were susceptible to the alpha strain of SARS-CoV-2. Moreover, diabetic C57BL/6J mice with leptin receptor gene deficiency (db/db mice) showed a higher viral load in the throat and lung and slower virus clearance in the throat after infection than C57BL/6J mice. Histological and multifactor analysis revealed more advanced pulmonary injury and serum inflammation in SARS-CoV-2 infected diabetic mice. Moreover, SARS-CoV-2 infected diabetic mice exhibited more severe insulin resistance and islet cell loss than uninfected diabetic mice. By RNA sequencing analysis, we found that diabetes may reduce the collagen level, suppress the immune response and aggravate inflammation in the lung after infection, which may account for the greater susceptibility of diabetic mice and their more severe lung damage after infection. In summary, we successfully established a SARS-CoV-2 infected diabetic mice model and demonstrated that diabetes and COVID-19 were risk factors for one another.

Implantation underneath the abdominal anterior rectus sheath enables effective and functional engraftment of stem-cell-derived islets.

Human pluripotent stem cell-derived islets (hPSC islets) are a promising alternative to primary human islets for the treatment of insulin-deficient diabetes. We previously demonstrated the feasibility of this approach in nonhuman primates; however, the therapeutic effects of hPSC islets can be limited by the maladaptive processes at the transplantation site. Here, we demonstrate successful implantation of hPSC-derived islets in a new transplantation site in the abdomen, the subanterior rectus sheath, in eight nonhuman primates (five male and three female). In this proof-of-principle study, we find that hPSC islets survive and gradually mature after transplantation, leading to improved glycemic control in diabetic primates. Notably, C-peptide secretion responds to meal challenge from 6 weeks post-transplantation (wpt), with stimulation indices comparable to those of native islets. The average post-prandial C-peptide level reaches approximately 2.0 ng ml-1 from 8 wpt, which is five times higher than the peak value we previously obtained after portal vein infusion of hPSC islets and was associated with a decrease of glycated hemoglobin levels by 44% at 12 wpt. Although additional studies in larger cohorts involving long-term follow-up of transplants are needed, our results indicate that the subanterior rectus sheath supports functional maturation and maintenance of hPSC islets, suggesting that it warrants further exploration as a transplantation target site in the context of for hPSC-based cell-replacement therapies.

Severe maternal-fetal pathological damage and inflammatory responses contribute to miscarriage caused by hepatitis E viral infection during pregnancy.

BACKGROUND: Hepatitis E virus (HEV) infection causes serious adverse pregnancy outcomes during pregnancy. However, the maternal and fetal damage induced by HEV infection is rarely reported. METHODS: A BALB/c pregnant mouse model was established to explore the maternal and fetal pathological damage and inflammatory responses caused by HEV infection. RESULTS: Notably, miscarriages and stillbirths were observed in HEV-infected pregnant mice. HEV infections were identified by qRT-PCR, immunohistochemical analysis and immunofluorescence assay in the uterus, placenta, umbilical cords and livers and brains of fetuses. Serious inflammatory responses and pathological damage were triggered in the uterus and placenta of HEV-infected pregnant mice. Vertical transmission of HEV resulted in severe pathological damage and inflammatory responses in the livers and brains of fetuses, as well as emerging apoptosis cells in the brains of fetuses. Most of the cytokines/chemokines in the sera were significantly increased in the HEV-infected pregnant mice. Remarkably, cytokines/chemokines were significantly different between HEV-infected pregnant and miscarriage mice; IL9, GM-CSF and IL1α were the most important three cytokines/chemokines in determining the pregnancy outcomes. CONCLUSION: HEV infections cause serious maternal/fetal pathological damage, inflammatory responses and apoptosis, which may be responsible for adverse pregnancy outcomes.

Prevalence of hepatitis E virus and its association with adverse pregnancy outcomes in pregnant women in China.

BACKGROUND: Hepatitis E virus (HEV) infection has become a global concern, especially in pregnant women. However, the association between HEV prevalence and age, gravidity and parity of pregnant women remains unclear. METHODS: Pregnant women (n=19,762) were enrolled for HEV prevalence and associated adverse pregnancy outcomes investigation in Qujing City, Yunnan Province of China from May 2019 to December 2020. RESULTS: The seroprevalence of HEV was 11.6% (2,297/19,762; 95% CI:11.2%-12.1%). About 11.4% (2,247/19,762; 95% CI:10.9%-11.8%) were positive for anti-HEV IgG antibody, 0.1% (22/19,762; 95% CI:0.1%-0.2%) were positive for anti-HEV IgM antibody, and 0.1% (28/19,762; 95% CI:0.1%-0.2%) were positive for both anti-HEV IgM and IgG antibodies. Sixty-one out of 2,297 anti-HEV-antibodies-positive pregnant women were positive for HEV RNA. Phylogenetic analysis revealed that all HEV isolates from pregnant women belong to genotype 4. Age, gravidity and parity are associated with increased prevalence of HEV. Pregnant women positive for HEV-IgG antibody bear a higher risk for an adverse pregnancy history and liver injury with elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels than anti-HEV-negative pregnant women. Furthermore, seropositive pregnant women suffered a higher adverse maternal outcomes risk (crude odds ratio [cOR]=1.29; 95% CI: 1.16-1.43; adjusted odds ratio [aOR]=1.40, 95% CI: 1.25-1.55 for anti-HEV-IgG-positive pregnant women and cOR=1.38, 95% CI: 1.02-1.86; aOR=1.43, 95% CI: 1.05-1.95 for anti-HEV-IgM-positive pregnant women) and fetal outcomes risk (cOR=1.80, 95% CI: 1.61-2.01; aOR=1.77, 95% CI: 1.57-1.99) than anti-HEV-negative pregnant women. Adverse pregnancy outcomes of HEV infection are aggravated by age, gravidity and parity. CONCLUSION: In this study, we demonstrated high prevalence of HEV in pregnancy women in China, and HEV infection can cause various adverse maternal and neonatal outcomes.

Splicing factor SF3B3, a NS5-binding protein, restricts ZIKV infection by targeting GCH1.

Zika virus (ZIKV), a positive-sense single-stranded RNA virus, causes congenital ZIKV syndrome in children and Guillain-Barré Syndrome (GBS) in adults. ZIKV expresses nonstructural protein 5 (NS5), a large protein that is essential for viral replication. ZIKV NS5 confers the ability to evade interferon (IFN) signalling; however, the exact mechanism remains unclear. In this study, we employed affinity pull-down and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and found that splicing factor 3b subunit 3 (SF3B3) is associated with the NS5-Flag pull-down complex through interaction with NS5. Functional assays showed that SF3B3 overexpression inhibited ZIKV replication by promoting IFN-stimulated gene (ISG) expression whereas silencing of SF3B3 inhibited expression of ISGs to promote ZIKV replication. GTP cyclohydrolase I (GCH1) is the first and rate-limiting enzyme in tetrahydrobiopterin (BH4) biosynthesis. NS5 upregulates the expression of GCH1 during ZIKV infection. And GCH1 marginally promoted ZIKV replication via the IFN pathway. Additionally, GCH1 expression is related to the regulation of SF3B3. Overexpression of the SF3B3 protein effectively reduced GCH1 protein levels, whereas SF3B3 knockdown increased its levels. These findings indicated that ZIKV NS5 binding protein SF3B3 contributed to the host immune response against ZIKV replication by modulating the expression of GCH1.

A self-assembled trimeric protein vaccine induces protective immunity against Omicron variant.

The recently emerged Omicron (B.1.1.529) variant has rapidly surpassed Delta to become the predominant circulating SARS-CoV-2 variant, given the higher transmissibility rate and immune escape ability, resulting in breakthrough infections in vaccinated individuals. A new generation of SARS-CoV-2 vaccines targeting the Omicron variant are urgently needed. Here, we developed a subunit vaccine named RBD-HR/trimer by directly linking the sequence of RBD derived from the Delta variant (containing L452R and T478K) and HR1 and HR2 in SARS-CoV-2 S2 subunit in a tandem manner, which can self-assemble into a trimer. In multiple animal models, vaccination of RBD-HR/trimer formulated with MF59-like oil-in-water adjuvant elicited sustained humoral immune response with high levels of broad-spectrum neutralizing antibodies against Omicron variants, also inducing a strong T cell immune response in vivo. In addition, our RBD-HR/trimer vaccine showed a strong boosting effect against Omicron variants after two doses of mRNA vaccines, featuring its capacity to be used in a prime-boost regimen. In mice and non-human primates, RBD-HR/trimer vaccination could confer a complete protection against live virus challenge of Omicron and Delta variants. The results qualified RBD-HR/trimer vaccine as a promising next-generation vaccine candidate for prevention of SARS-CoV-2, which deserved further evaluation in clinical trials.

B.1.351 SARS-CoV-2 Variant Exhibits Higher Virulence but Less Viral Shedding than That of the Ancestral Strain in Young Nonhuman Primates.

We investigated the distribution, virulence, and pathogenic characteristics of mutated SARS-CoV-2 to clarify the association between virulence and the viral spreading ability of current and future circulating strains. Chinese rhesus macaques were infected with ancestral SARS-CoV-2 strain GD108 and Beta variant B.1.351 (B.1.351) and assessed for clinical signs, viral distribution, pathological changes, and pulmonary inflammation. We found that GD108 replicated more efficiently in the upper respiratory tract, whereas B.1.351 replicated more efficiently in the lower respiratory tract and lung tissue, implying a reduced viral shedding and spreading ability of B.1.351 compared with that of GD108. Importantly, B.1.351 caused more severe lung injury and dramatically elevated the level of inflammatory cytokines compared with those observed after infection with GD108. Moreover, both B.1.351 and GD108 induced spike-specific T-cell responses at an early stage of infection, with higher levels of interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in the B.1.351 group and higher levels of interleukin 17 (IL-17) in the GD108 group, indicating a divergent pattern in the T-cell-mediated inflammatory "cytokine storm." This study provides a basis for exploring the pathogenesis of SARS-CoV-2 variants of concern (VOCs) and establishes an applicable animal model for evaluating the efficacy and safety of vaccines and drugs. IMPORTANCE One of the priorities of the current SARS-CoV-2 vaccine and drug research strategy is to determine the changes in transmission ability, virulence, and pathogenic characteristics of SARS-CoV-2 variants. In addition, nonhuman primates (NHPs) are suitable animal models for the study of the pathogenic characteristics of SARS-CoV-2 and could contribute to the understanding of pathogenicity and transmission mechanisms. As SARS-CoV-2 variants continually emerge and the viral biological characteristics change frequently, the establishment of NHP infection models for different VOCs is urgently needed. In the study, the virulence and tissue distribution of B.1.351 and GD108 were comprehensively studied in NHPs. We concluded that the B.1.351 strain was more virulent but exhibited less viral shedding than the latter. This study provides a basis for determining the pathogenic characteristics of SARS-CoV-2 and establishes an applicable animal model for evaluating the efficacy and safety of vaccines and drugs.

Protective prototype-Beta and Delta-Omicron chimeric RBD-dimer vaccines against SARS-CoV-2.

Breakthrough infections by SARS-CoV-2 variants become the global challenge for pandemic control. Previously, we developed the protein subunit vaccine ZF2001 based on the dimeric receptor-binding domain (RBD) of prototype SARS-CoV-2. Here, we developed a chimeric RBD-dimer vaccine approach to adapt SARS-CoV-2 variants. A prototype-Beta chimeric RBD-dimer was first designed to adapt the resistant Beta variant. Compared with its homotypic forms, the chimeric vaccine elicited broader sera neutralization of variants and conferred better protection in mice. The protection of the chimeric vaccine was further verified in macaques. This approach was generalized to develop Delta-Omicron chimeric RBD-dimer to adapt the currently prevalent variants. Again, the chimeric vaccine elicited broader sera neutralization of SARS-CoV-2 variants and conferred better protection against challenge by either Delta or Omicron SARS-CoV-2 in mice. The chimeric approach is applicable for rapid updating of immunogens, and our data supported the use of variant-adapted multivalent vaccine against circulating and emerging variants.

Circular RNA vaccines against SARS-CoV-2 and emerging variants.

As the emerging variants of SARS-CoV-2 continue to drive the worldwide pandemic, there is a constant demand for vaccines that offer more effective and broad-spectrum protection. Here, we report a circular RNA (circRNA) vaccine that elicited potent neutralizing antibodies and T cell responses by expressing the trimeric RBD of the spike protein, providing robust protection against SARS-CoV-2 in both mice and rhesus macaques. Notably, the circRNA vaccine enabled higher and more durable antigen production than the 1mΨ-modified mRNA vaccine and elicited a higher proportion of neutralizing antibodies and distinct Th1-skewed immune responses. Importantly, we found that the circRNARBD-Omicron vaccine induced effective neutralizing antibodies against the Omicron but not the Delta variant. In contrast, the circRNARBD-Delta vaccine protected against both Delta and Omicron or functioned as a booster after two doses of either native- or Delta-specific vaccination, making it a favorable choice against the current variants of concern (VOCs) of SARS-CoV-2.

Histones released by NETosis enhance the infectivity of SARS-CoV-2 by bridging the spike protein subunit 2 and sialic acid on host cells.

Neutrophil extracellular traps (NETs) can capture and kill viruses, such as influenza viruses, human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV), thus contributing to host defense. Contrary to our expectation, we show here that the histones released by NETosis enhance the infectivity of SARS-CoV-2, as found by using live SARS-CoV-2 and two pseudovirus systems as well as a mouse model. The histone H3 or H4 selectively binds to subunit 2 of the spike (S) protein, as shown by a biochemical binding assay, surface plasmon resonance and binding energy calculation as well as the construction of a mutant S protein by replacing four acidic amino acids. Sialic acid on the host cell surface is the key molecule to which histones bridge subunit 2 of the S protein. Moreover, histones enhance cell-cell fusion. Finally, treatment with an inhibitor of NETosis, histone H3 or H4, or sialic acid notably affected the levels of sgRNA copies and the number of apoptotic cells in a mouse model. These findings suggest that SARS-CoV-2 could hijack histones from neutrophil NETosis to promote its host cell attachment and entry process and may be important in exploring pathogenesis and possible strategies to develop new effective therapies for COVID-19.