Enhanced potency of an IgM-like nanobody targeting conserved epitope in SARS-CoV-2 spike N-terminal domain.

Almost all the neutralizing antibodies targeting the receptor-binding domain (RBD) of spike (S) protein show weakened or lost efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged or emerging variants, such as Omicron and its sub-variants. This suggests that highly conserved epitopes are crucial for the development of neutralizing antibodies. Here, we present one nanobody, N235, displaying broad neutralization against the SARS-CoV-2 prototype and multiple variants, including the newly emerged Omicron and its sub-variants. Cryo-electron microscopy demonstrates N235 binds a novel, conserved, cryptic epitope in the N-terminal domain (NTD) of the S protein, which interferes with the RBD in the neighboring S protein. The neutralization mechanism interpreted via flow cytometry and Western blot shows that N235 appears to induce the S1 subunit shedding from the trimeric S complex. Furthermore, a nano-IgM construct (MN235), engineered by fusing N235 with the human IgM Fc region, displays prevention via inducing S1 shedding and cross-linking virus particles. Compared to N235, MN235 exhibits varied enhancement in neutralization against pseudotyped and authentic viruses in vitro. The intranasal administration of MN235 in low doses can effectively prevent the infection of Omicron sub-variant BA.1 and XBB in vivo, suggesting that it can be developed as a promising prophylactic antibody to cope with the ongoing and future infection.

Broad protective RBD heterotrimer vaccines neutralize SARS-CoV-2 including Omicron sub-variants XBB/BQ.1.1/BF.7.

SARS-CoV-2 variants with severe immune evasion are a major challenge for COVID-19 prevention, especially the circulating Omicron XBB/BQ.1.1/BF.7 strains. Thus, the next-generation of broad-spectrum vaccines are urgently needed. Previously, we developed a COVID-19 protein subunit vaccine, ZF2001, based on the RBD-homodimer as the immunogen. To adapt SARS-CoV-2 variants, we developed chimeric RBD-heterodimers to induce broad immune responses. In this study, we further explored the concept of tandem RBD homotrimer and heterotrimer. Prototype SARS-CoV-2 RBD-homotrimer, prototype-Delta-BA.1 (PDO) RBD-heterotrimer and Delta-BA.2-BA.5 (DBA2BA5) RBD-heterotrimer were designed. Biochemical and cryo-EM structural characterization demonstrated total epitope exposure of the RBD-trimers. In mouse experiments, PDO and DBA2BA5 elicited broad SARS-CoV-2 neutralization. Potent protection against SARS-CoV-2 variants was observed in challenge assays and was correlated with neutralizing antibody titer. This study validated the design strategy of tandem RBD-heterotrimers as multivalent immunogens and presented a promising vaccine candidate, DBA2BA5, eliciting broad-spectrum immune responses, including against the circulating XBB/BF.7/BQ.1.1.

A pan-coronavirus peptide inhibitor prevents SARS-CoV-2 infection in mice by intranasal delivery.

Coronaviruses (CoVs) have brought serious threats to humans, particularly severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), which continually evolves into multiple variants. These variants, especially Omicron, reportedly escape therapeutic antibodies and vaccines, indicating an urgent need for new antivirals with pan-SARS-CoV-2 inhibitory activity. We previously reported that a peptide fusion inhibitor, P3, targeting heptad repeated-1 (HR1) of SARS-CoV-2 spike (S) protein, could inhibit viral infections. Here, we further designed multiple derivatives of the P3 based on structural analysis and found that one derivative, the P315V3, showed the most efficient antiviral activity against SARS-CoV-2 variants and several other sarbecoviruses, as well as other human-CoVs (HCoVs). P315V3 also exhibited effective prophylactic efficacy against the SARS-CoV-2 Delta and Omicron variants in mice via intranasal administration. These results suggest that P315V3, which is in Phase II clinical trial, is promising for further development as a nasal pan-SARS-CoV-2 or pan-CoVs inhibitor to prevent or treat CoV diseases.

Rational design of an influenza-COVID-19 chimeric protective vaccine with HA-stalk and S-RBD.

Highly contagious respiratory illnesses like influenza and COVID-19 pose serious risks to public health. A two-in-one vaccine would be ideal to avoid multiple vaccinations for these diseases. Here, we generated a chimeric receptor binding domain of the spike protein (S-RBD) and hemagglutinin (HA)-stalk-based vaccine for both SARS-CoV-2 and influenza viruses. The S-RBD from SARS-CoV-2 Delta was fused to the headless HA from H1N1 (H1Delta), creating a chimera that forms trimers in solution. The cryo-electron microscopy structure of the chimeric protein complexed with the RBD-targeting CB6 and the HA-stalk-targeting CR9114 antibodies shows that the trimeric protein is stable and accessible for neutralizing antibody binding. Immunization with the vaccine elicited high and long-lasting neutralizing antibodies and effectively protected mice against the challenges of lethal H1N1 or heterosubtypic H5N8, as well as the SARS-CoV-2 Delta or Omicron BA.2 variants. Overall, this study offers a two-in-one universal vaccine design to combat infections caused by both SARS-CoV-2 variants of concern and influenza viruses.

Low levels of neutralizing antibodies against XBB Omicron subvariants after BA.5 infection.

The COVID-19 response strategies in Chinese mainland were recently adjusted due to the reduced pathogenicity and enhanced infectivity of Omicron subvariants. In Chengdu, China, an infection wave was predominantly induced by the BA.5 subvariant. It is crucial to determine whether the hybrid anti-SARS-CoV-2 immunity following BA.5 infection, coupled with a variety of immune background, is sufficient to shape the immune responses against newly emerged Omicron subvariants, especially for XBB lineages. To investigate this, we collected serum and nasal swab samples from 108 participants who had been infected in this BA.5 infection wave, and evaluated the neutralization against pseudoviruses. Our results showed that convalescent sera from individuals, regardless of vaccination history, had remarkably compromised neutralization capacities against the newly emerged XBB and XBB.1.5 subvariants. Although post-vaccination with BA.5 breakthrough infection slightly elevated plasma neutralizing antibodies against a part of pseudoviruses, the neutralization activities were remarkably impaired by XBB lineages. Furthermore, we analyzed the impacts of the number of vaccinations, age, and sex on the humoral and cellular immune response after BA.5 infection. Our findings suggest that the neutralization against XBB lineages that elicited by current hybrid immunity after BA.5 infection, are remained at low levels, indicating an urgent need for the development of next-generation of COVID-19 vaccines that designed based on the XBB sub-lineages and other future variants.

Stability of SARS-CoV-2 and persistence of viral nucleic acids on common foods and widely used packaging material surfaces.

SARS-CoV-2 is still spreading globally. Studies have reported the stability of SARS-CoV-2 in aerosols and on surfaces under different conditions. However, studies on the stability of SARS-CoV-2 and viral nucleic acids on common food and packaging material surfaces are insufficient. The study evaluated the stability of SARS-CoV-2 using TCID50 assays and the persistence of SARS-CoV-2 nucleic acids using droplet digital polymerase chain reaction on various food and packaging material surfaces. Viral nucleic acids were stable on food and material surfaces under different conditions. The viability of SARS-CoV-2 varied among different surfaces. SARS-CoV-2 was inactivated on most food and packaging material surfaces within 1 day at room temperature but was more stable at lower temperatures. Viruses survived for at least 1 week on pork and plastic at 4°C, while no viable viruses were detected on hairtail, orange, or carton after 3 days. There were viable viruses and a slight titer decrease after 8 weeks on pork and plastic, but titers decreased rapidly on hairtail and carton at -20°C. These results highlight the need for targeted preventive and disinfection measures based on different types of foods, packaging materials, and environmental conditions, particularly in the cold-chain food trade, to combat the ongoing pandemic.

Surveillance of SARS-CoV-2 at the Huanan Seafood Market.

SARS-CoV-2, the causative agent of COVID-19, emerged in December 2019. Its origins remain uncertain. It has been reported that a number of the early human cases had a history of contact with the Huanan Seafood Market. Here we present the results of surveillance for SARS-CoV-2 within the market. From January 1st 2020, after closure of the market, 923 samples were collected from the environment. From 18th January, 457 samples were collected from 18 species of animals, comprising of unsold contents of refrigerators and freezers, swabs from stray animals, and the contents of a fish tank. Using RT-qPCR, SARS-CoV-2 was detected in 73 environmental samples, but none of the animal samples. Three live viruses were successfully isolated. The viruses from the market shared nucleotide identity of 99.99% to 100% with the human isolate HCoV-19/Wuhan/IVDC-HB-01/2019. SARS-CoV-2 lineage A (8782T and 28144C) was found in an environmental sample. RNA-seq analysis of SARS-CoV-2 positive and negative environmental samples showed an abundance of different vertebrate genera at the market. In summary, this study provides information about the distribution and prevalence of SARS-CoV-2 in the Huanan Seafood Market during the early stages of the COVID-19 outbreak.

Safety and immunogenicity of a mosaic vaccine booster against Omicron and other SARS-CoV-2 variants: a randomized phase 2 trial.

An ongoing randomized, double-blind, controlled phase 2 trial was conducted to evaluate the safety and immunogenicity of a mosaic-type recombinant vaccine candidate, named NVSI-06-09, as a booster dose in subjects aged 18 years and older from the United Arab Emirates (UAE), who had administered two or three doses of inactivated vaccine BBIBP-CorV at least 6 months prior to enrollment. The participants were randomly assigned with 1:1 to receive a booster dose of NVSI-06-09 or BBIBP-CorV. The primary outcomes were immunogenicity and safety against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant, and the exploratory outcome was cross-immunogenicity against other circulating strains. Between May 25 and 30, 2022, 516 adults received booster vaccination with 260 in NVSI-06-09 group and 256 in BBIBP-CorV group. Interim results showed a similar safety profile between two booster groups, with low incidence of adverse reactions of grade 1 or 2. For immunogenicity, by day 14 post-booster, the fold rises in neutralizing antibody geometric mean titers (GMTs) from baseline elicited by NVSI-06-09 were remarkably higher than those by BBIBP-CorV against the prototype strain (19.67 vs 4.47-fold), Omicron BA.1.1 (42.35 vs 3.78-fold), BA.2 (25.09 vs 2.91-fold), BA.4 (22.42 vs 2.69-fold), and BA.5 variants (27.06 vs 4.73-fold). Similarly, the neutralizing GMTs boosted by NVSI-06-09 against Beta and Delta variants were also 6.60-fold and 7.17-fold higher than those by BBIBP-CorV. Our findings indicated that a booster dose of NVSI-06-09 was well-tolerated and elicited broad-spectrum neutralizing responses against divergent SARS-CoV-2 variants, including Omicron and its sub-lineages.

Two pan-SARS-CoV-2 nanobodies and their multivalent derivatives effectively prevent Omicron infections in mice.

With the widespread vaccinations against coronavirus disease 2019 (COVID-19), we are witnessing gradually waning neutralizing antibodies and increasing cases of breakthrough infections, necessitating the development of drugs aside from vaccines, particularly ones that can be administered outside of hospitals. Here, we present two cross-reactive nanobodies (R14 and S43) and their multivalent derivatives, including decameric ones (fused to the immunoglobulin M [IgM] Fc) that maintain potent neutralizing activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after aerosolization and display not only pan-SARS-CoV-2 but also varied pan-sarbecovirus activities. Through respiratory administration to mice, monovalent and decameric R14 significantly reduce the lung viral RNAs at low dose and display potent pre- and post-exposure protection. Furthermore, structural studies reveal the neutralizing mechanisms of R14 and S43 and the multiple inhibition effects that the multivalent derivatives exert. Our work demonstrates promising convenient drug candidates via respiratory administration against SARS-CoV-2 infection, which can contribute to containing the COVID-19 pandemic.

Immune response and homeostasis mechanism following administration of BBIBP-CorV SARS-CoV-2 inactivated vaccine.

The BBIBP-CorV severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inactivated vaccine has been authorized for emergency use and widely distributed. We used single-cell transcriptome sequencing to characterize the dynamics of immune responses to the BBIBP-CorV inactivated vaccine. In addition to the expected induction of humoral immunity, we found that the inactivated vaccine induced multiple, comprehensive immune responses, including significantly increased proportions of CD16+ monocytes and activation of monocyte antigen presentation pathways; T cell activation pathway upregulation in CD8+ T cells, along with increased activation of CD4+ T cells; significant enhancement of cell-cell communications between innate and adaptive immunity; and the induction of regulatory CD4+ T cells and co-inhibitory interactions to maintain immune homeostasis after vaccination. Additionally, comparative analysis revealed higher neutralizing antibody levels, distinct expansion of naive T cells, a shared increased proportion of regulatory CD4+ T cells, and upregulated expression of functional genes in booster dose recipients with a longer interval after the second vaccination. Our research will support a comprehensive understanding of the systemic immune responses elicited by the BBIBP-CorV inactivated vaccine, which will facilitate the formulation of better vaccination strategies and the design of new vaccines.

A mosaic-type trimeric RBD-based COVID-19 vaccine candidate induces potent neutralization against Omicron and other SARS-CoV-2 variants.

Large-scale populations in the world have been vaccinated with COVID-19 vaccines, however, breakthrough infections of SARS-CoV-2 are still growing rapidly due to the emergence of immune-evasive variants, especially Omicron. It is urgent to develop effective broad-spectrum vaccines to better control the pandemic of these variants. Here, we present a mosaic-type trimeric form of spike receptor-binding domain (mos-tri-RBD) as a broad-spectrum vaccine candidate, which carries the key mutations from Omicron and other circulating variants. Tests in rats showed that the designed mos-tri-RBD, whether used alone or as a booster shot, elicited potent cross-neutralizing antibodies against not only Omicron but also other immune-evasive variants. Neutralizing antibody ID50 titers induced by mos-tri-RBD were substantially higher than those elicited by homo-tri-RBD (containing homologous RBDs from prototype strain) or the BIBP inactivated COVID-19 vaccine (BBIBP-CorV). Our study indicates that mos-tri-RBD is highly immunogenic, which may serve as a broad-spectrum vaccine candidate in combating SARS-CoV-2 variants including Omicron.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to pose a serious threat to public health and has so far resulted in over six million deaths worldwide. Mass vaccination programs have reduced the risk of serious illness and death in many people, but the virus continues to persist and circulate in communities across the globe. Furthermore, the current vaccines may be less effective against the new variants of the virus, such as Omicron and Delta, which are continually emerging and evolving. Therefore, it is urgent to develop effective vaccines that can provide broad protection against existing and future forms of SARS-CoV-2. There are several different types of SARS-CoV-2 vaccine, but they all work in a similar way. They contain molecules that induce immune responses in individuals to help the body recognize and more effectively fight SARS-CoV-2 if they happen to encounter it in the future. These immune responses may be so specific that new variants of a virus may not be recognized by them. Therefore, a commonly used strategy for producing vaccines with broad protection is to make multiple vaccines that each targets different variants and then mix them together before administering to patients. Here, Zhang et al. took a different approach by designing a new vaccine candidate against SARS-CoV2 that contained three different versions of part of a SARS-CoV2 protein ­ the so-called spike protein ­ all linked together as one molecule. The different versions of the spike protein fragment were designed to include key features of the fragments found in Omicron and several other SARS-CoV-2 variants. The experiments found that this candidate vaccine elicited a much higher immune response against Omicron and other SARS-CoV-2 variants in rats than an existing SARS-CoV-2 vaccine. It was also effective as a booster shot after a first vaccination with the existing SARS-CoV-2 vaccine. These findings demonstrate that the molecule developed by Zhang et al. induces potent and broad immune responses against different variants of SARS-CoV-2 including Omicron in rats. The next steps following on from this work are to evaluate the safety and immunogenicity of this vaccine candidate in clinical trials. In the future, it may be possible to use a similar approach to develop new broad-spectrum vaccines against other viruses.

Immunogenicity and safety of NVSI-06-07 as a heterologous booster after priming with BBIBP-CorV: a phase 2 trial.

The increased coronavirus disease 2019 (COVID-19) breakthrough cases pose the need of booster vaccination. We conducted a randomised, double-blinded, controlled, phase 2 trial to assess the immunogenicity and safety of the heterologous prime-boost vaccination with an inactivated COVID-19 vaccine (BBIBP-CorV) followed by a recombinant protein-based vaccine (NVSI-06-07), using homologous boost with BBIBP-CorV as control. Three groups of healthy adults (600 individuals per group) who had completed two-dose BBIBP-CorV vaccinations 1-3 months, 4-6 months and ≥6 months earlier, respectively, were randomly assigned in a 1:1 ratio to receive either NVSI-06-07 or BBIBP-CorV boost. Immunogenicity assays showed that in NVSI-06-07 groups, neutralizing antibody geometric mean titers (GMTs) against the prototype SARS-CoV-2 increased by 21.01-63.85 folds on day 28 after vaccination, whereas only 4.20-16.78 folds of increases were observed in control groups. For Omicron variant, the neutralizing antibody GMT elicited by homologous boost was 37.91 on day 14, however, a significantly higher neutralizing GMT of 292.53 was induced by heterologous booster. Similar results were obtained for other SARS-CoV-2 variants of concerns (VOCs), including Alpha, Beta and Delta. Both heterologous and homologous boosters have a good safety profile. Local and systemic adverse reactions were absent, mild or moderate in most participants, and the overall safety was quite similar between two booster schemes. Our findings indicated that NVSI-06-07 is safe and immunogenic as a heterologous booster in BBIBP-CorV recipients and was immunogenically superior to the homologous booster against not only SARS-CoV-2 prototype strain but also VOCs, including Omicron.

Safety and immunogenicity of a hybrid-type vaccine booster in BBIBP-CorV recipients in a randomized phase 2 trial.

NVSI-06-08 is a potential broad-spectrum recombinant COVID-19 vaccine that integrates the antigens from multiple SARS-CoV-2 strains into a single immunogen. Here, we evaluate the safety and immunogenicity of NVSI-06-08 as a heterologous booster dose in BBIBP-CorV recipients in a randomized, double-blind, controlled, phase 2 trial conducted in the United Arab Emirates (NCT05069129). Three groups of healthy adults over 18 years of age (600 participants per group) who have administered two doses of BBIBP-CorV 4-6-month, 7-9-month and >9-month earlier, respectively, are randomized 1:1 to receive either a homologous booster of BBIBP-CorV or a heterologous booster of NVSI-06-08. The incidence of adverse reactions is low, and the overall safety profile is quite similar between two booster regimens. Both Neutralizing and IgG antibodies elicited by NVSI-06-08 booster are significantly higher than those by BBIBP-CorV booster against not only SARS-CoV-2 prototype strain but also multiple variants of concerns (VOCs). Especially, the neutralizing antibody GMT against Omicron variant induced by heterologous NVSI-06-08 booster reaches 367.67, which is substantially greater than that boosted by BBIBP-CorV (GMT: 45.03). In summary, NVSI-06-08 is safe and immunogenic as a booster dose following two doses of BBIBP-CorV, which is immunogenically superior to the homologous boost with another dose of BBIBP-CorV.

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.

Intrahost variation and evolutionary dynamics of adenoviruses correlate to neutrophils in infected patients.

With deep sequencing of virus genomes within the hosts, intrahost single nucleotide variations (iSNVs) have been used for analyses of virus genome variation and evolution, which is indicated to correlate with viral pathogenesis and disease severity. Little is known about the features of iSNVs among DNA viruses. We performed the epidemiological and laboratory investigation of one outbreak of adenovirus. The whole genomes of viruses in both original oral swabs and cell-cultured virus isolates were deeply sequenced. We identified 737 iSNVs in the viral genomes sequenced from original samples and 46 viral iSNVs in cell-cultured isolates, with 33 iSNVs shared by original samples and cultured isolates. Meanwhile, we found these 33 iSNVs were shared by different patients, among which, three hot spot areas 6367-6401, 9213-9247, and 10 584-10 606 within the functional genes of the adenovirus genome were found. Notably, the substitution rates of iSNVs were closely correlated with the clinical and immune indicators of the patients. Especially a positive correlation to neutrophils was found, indicating a predictable biomarker of iSNV dynamics. Our findings demonstrated the neutrophil-correlated dynamic evolution features of the iSNVs within adenoviruses, which indicates a virus-host interaction during human infection of a DNA virus.

An engineered bispecific human monoclonal antibody against SARS-CoV-2.

The global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires effective therapies against coronavirus disease 2019 (COVID-19), and neutralizing antibodies are a promising therapy. A noncompeting pair of human neutralizing antibodies (B38 and H4) blocking SARS-CoV-2 binding to its receptor, ACE2, have been described previously. Here, we develop bsAb15, a bispecific monoclonal antibody (bsAb) based on B38 and H4. bsAb15 has greater neutralizing efficiency than these parental antibodies, results in less selective pressure and retains neutralizing ability to most SARS-CoV-2 variants of concern (with more potent neutralizing activity against the Delta variant). We also selected for escape mutants of the two parental mAbs, a mAb cocktail and bsAb15, demonstrating that bsAb15 can efficiently neutralize all single-mAb escape mutants. Furthermore, prophylactic and therapeutic application of bsAb15 reduced the viral titer in infected nonhuman primates and human ACE2 transgenic mice. Therefore, this bsAb is a feasible and effective strategy to treat and prevent severe COVID-19.

A novel mRNA vaccine, SYS6006, against SARS-CoV-2.

The development of vaccines that can efficiently prevent the infection of SARS-CoV-2 is necessary to fight the COVID-19 epidemic. mRNA vaccine has been proven to induce strong humoral and cellular immunity against SARS-CoV-2. Here, we studied the immunogenicity and protection efficacy of a novel mRNA vaccine SYS6006. High expression of mRNA molecules in 293T cells was detected. The initial and boost immunization with a 21-day interval was determined as an optimal strategy for SYS6006. Two rounds of immunization with SYS6006 were able to induce the neutralizing antibodies against the SARS-CoV-2 wild-type (WT) strain, and Delta and Omicron BA.2 variants in mice or non-human primates (NHPs). A3rd round of vaccination could further enhance the titers of neutralization against Delta and Omicron variants. In vitro ELISpot assay showed that SYS6006 could induce memory B cell and T cell immunities specifically against SARS-CoV-2 in mice. FACS analysis indicated that SYS6006 successfully induced SARS-CoV-2-specific activation of T follicular helper cell (Tfh) and Th1 cell, and did not induce CD4+Th2 response in NHPs. SYS6006 vaccine could significantly reduce the viral RNA loads and prevent lung lesions in Delta variant infected hACE2 transgenic mice. Therefore, SYS6006 could provide significant immune protection against SARS-CoV-2.