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Protective vaccines are crucial for preventing and controlling coronavirus disease 2019 (COVID-19). Updated vaccines are needed to confront the continuously evolving and circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. These vaccines should be safe, effective, amenable to easily scalable production, and affordable. Previously, we developed receptor binding domain (RBD) dimer-based protein subunit vaccines (ZF2001 and updated vaccines) in mammalian cells. In this study, we explored a strategy for producing RBD-dimer immunogens in Pichia pastoris. We found that wild-type P. pastoris produced hyperglycosylated RBD-dimer protein containing four N-glycosylation sites in P. pastoris. Therefore, we engineered the wild type P. pastoris (GS strain) into GSΔOCH1pAO by deleting the OCH1 gene (encoding α-1,6-mannosyltransferase enzyme) to decrease glycosylation, as well as by overexpressing the HIS4 gene (encoding histidine dehydrogenase) to increase histidine synthesis for better growth. In addition, RBD-dimer protein was truncated to remove the R328/F329 cleavage sites in P. pastoris. Several homogeneous RBD-dimer proteins were produced in the GSΔOCH1pAO strain, demonstrating the feasibility of using the P. pastoris expression system. We further resolved the cryo-EM structure of prototype-Beta RBD-dimer complexed with the neutralizing antibody CB6 to reveal the completely exposed immune epitopes of the RBDs. In a murine model, we demonstrated that the yeast-produced RBD-dimer induces robust and protective antibody responses, which is suitable for boosting immunization. This study developed the yeast system for producing SARS-CoV-2 RBD-dimer immunogens, providing a promising platform and pipeline for the future continuous updating and production of SARS-CoV-2 vaccines.

ABSTRACTThe SARS-CoV-2 Omicron variants of concern (VOCs) showed severe resistance to the early-approved COVID-19 vaccines-induced immune responses. The breakthrough infections by the Omicron VOCs are currently the major challenge for pandemic control. Therefore, booster vaccination is crucial to enhance immune responses and protective efficacy. Previously, we developed a protein subunit COVID-19 vaccine ZF2001, based on the immunogen of receptor-binding domain (RBD) homodimer, which was approved in China and other countries. To adapt SARS-CoV-2 variants, we further developed chimeric Delta-Omicron BA.1 RBD-dimer immunogen which induced broad immune responses against SARS-CoV-2 variants. In this study, we tested the boosting effect of this chimeric RBD-dimer vaccine in mice after priming with two doses of inactivated vaccines, compared with a booster of inactivated vaccine or ZF2001. The results demonstrated that boosting with bivalent Delta-Omicron BA.1 vaccine greatly promoted the neutralizing activity of the sera to all tested SARS-CoV-2 variants. Therefore, the Delta-Omicron chimeric RBD-dimer vaccine is a feasible booster for those with prior vaccination of COVID-19 inactivated vaccines.

Abstract Current attempts in vaccine delivery systems concentrate on replicating the natural dissemination of live pathogens, but neglect that pathogens evolve to evade the immune system rather than to provoke it. In the case of enveloped RNA viruses, it is the natural dissemination of nucleocapsid protein (NP, core antigen) and surface antigen that delays NP exposure to immune surveillance. Here, we report a multi-layered aluminum hydroxide-stabilized emulsion (MASE) to dictate the delivery sequence of the antigens. In this manner, the receptor-binding domain (RBD, surface antigen) of the spike protein was trapped inside the nanocavity, while NP was absorbed on the outside of the droplets, enabling the burst release of NP before RBD. Compared with the natural packaging strategy, the inside-out strategy induced potent type I interferon-mediated innate immune responses and triggered an immune-potentiated environment in advance, which subsequently boosted CD40+ DC activations and the engagement of the lymph nodes. In both H1N1 influenza and SARS-CoV-2 vaccines, rMASE significantly increased antigen-specific antibody secretion, memory T cell engagement, and Th1-biased immune response, which diminished viral loads after lethal challenge. By simply reversing the delivery sequence of the surface antigen and core antigen, the inside-out strategy may offer major implications for enhanced vaccinations against the enveloped RNA virus.

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.

Safe, efficacious, and deployable vaccines are urgently needed to control COVID-19 in the large-scale vaccination campaigns. We report here the preclinical studies of an approved protein subunit vaccine against COVID-19, ZF2001, which contains tandem-repeat dimeric receptor-binding domain (RBD) protein with alum-based adjuvant. We assessed vaccine immunogenicity and efficacy in both mice and non-human primates (NHPs). ZF2001 induced high levels of RBD-binding and SARS-CoV-2 neutralizing antibody in both mice and non-human primates, and elicited balanced TH1/TH2 cellular responses in NHPs. Two doses of ZF2001 protected Ad-hACE2-transduced mice against SARS-CoV-2 infection, as detected by reduced viral RNA and relieved lung injuries. In NHPs, vaccination of either 25 μg or 50 μg ZF2001 prevented infection with SARS-CoV-2 in lung, trachea, and bronchi, with milder lung lesions. No evidence of disease enhancement was observed in both animal models. ZF2001 has been approved for emergency use in China, Uzbekistan, Indonesia, and Columbia. The high safety, immunogenicity, and protection efficacy in both mice and NHPs found in this preclinical study was consistent with the results in human clinical trials.

Abstract A safe and effective vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is urgently needed to tackle the COVID-19 global pandemic. Here, we describe the development of chimpanzee adenovirus serotypes 6 and 68 (AdC6 and AdC68) vector-based vaccine candidates expressing the full-length transmembrane spike glycoprotein. We assessed the vaccine immunogenicity, protective efficacy, and immune cell profiles using single-cell RNA sequencing in mice. Mice were vaccinated via the intramuscular route with the two vaccine candidates using prime-only regimens or heterologous prime-boost regimens. Both chimpanzee adenovirus-based vaccines elicited strong and long-term antibody and T cell responses, balanced Th1/Th2 cell responses, robust germinal center responses, and provided effective protection against SARS-CoV-2 infection in mouse lungs. Strikingly, we found that heterologous prime-boost immunization induced higher titers of protective antibodies, and more spike-specific memory CD8+ T cells in mice. Potent neutralizing antibodies produced against the highly transmissible SARS-CoV-2 variants B.1.1.7 lineage (also known as N501Y.V1) and B.1.351 lineage (also known as N501Y.V2) were detectable in mouse sera over 6 months after prime immunization. Our results demonstrate that the heterologous prime-boost strategy with chimpanzee adenovirus-based vaccines is promising for further development to prevent SARS-CoV-2 infection.

Accurate identification of the botanical origins of Rhizoma Alismatis (RA) is pivotal to its precise clinical usage. We herein present a strategy, by integrating untargeted metabolomics, data cross validation, absolute quantification, and vector machine model, for species discrimination and source recognition of Rhizoma Alismatis for the first time. An ultra-high-performance liquid chromatography/LTQ-Orbitrap mass spectrometry with precursor ions list-including data-dependent acquisition approach was developed for metabolite profiling. Holistic, continuous, and pattern recognition chemometrics could make it feasible to unveil forty-one markers, with “multi-duplicated and traceability samples comparison” cross validation to narrow down to twelve robust markers, some of which were further validated by absolute quantification. A support vector machine model was eventually developed to distinguish these two species and predict origins of commercially available varieties. This was the first report on systematic comparison and discrimination of two original species of RA. This integral strategy, in contrast to conventional approaches, renders more convincing data supporting for the discovery of multi-source chemical makers of traditional Chinese medicines (TCM).

Jujube fruit was well-loved and praised by the broad masses due to its delicious taste, abundant nutritional value, and medicinal properties. Few studies reported the quality evaluation and gut microbiota regulation effect of polysaccharides of jujube fruits from different producing areas. In the present study, multi-level fingerprint profiling, including polysaccharides, oligosaccharides, and monosaccharides, was established for the quality evaluation of polysaccharides from jujube fruits. For polysaccharides, the total content in jujube fruits ranged from 1.31% to 2.22%, and the molecular weight distribution (MWD) ranged from 1.14 × 105 to 1.73 × 106 Da. The MWD fingerprint profiling of polysaccharides from eight producing areas was similar, but the profile of infrared spectroscopy (IR) showed differentiation. The characteristic signals were screened and used to establish a discrimination model for the identification of jujube fruits from different areas, and the accuracy of identification reached 100.00%. For oligosaccharides, the main components were galacturonic acid polymers (DP, 2–4), and the profile of oligosaccharides exhibited high similarity. The monosaccharides, GalA, Glc, and Ara, were the primary monosaccharides. Although the fingerprint of monosaccharides was semblable, the composing proportion of monosaccharides revealed significant differences. In addition, the polysaccharides of jujube fruits could regulate the gut microbiota composition and possess potential therapeutic effects on dysentery and nervous system diseases.

A safe and effective vaccine is urgently needed to control the unprecedented COVID-19 pandemic. Four adenovirus-vectored vaccines expressing spike (S) protein have been approved for use. Here, we generated several recombinant chimpanzee adenovirus (AdC7) vaccines expressing S, receptor-binding domain (RBD), or tandem-repeat dimeric RBD (RBD-tr2). We found vaccination via either intramuscular or intranasal route was highly immunogenic in mice to elicit both humoral and cellular immune responses. AdC7-RBD-tr2 showed higher antibody responses compared to either AdC7-S or AdC7-RBD. Intranasal administration of AdC7-RBD-tr2 additionally induced mucosal immunity with neutralizing activity in bronchoalveolar lavage fluid. Either single-dose or two-dose mucosal administration of AdC7-RBD-tr2 protected mice against SARS-CoV-2 challenge, with undetectable subgenomic RNA in lung and relieved lung injury. AdC7-RBD-tr2-elicted sera preserved the neutralizing activity against the circulating variants, especially the Delta variant. These results support AdC7-RBD-tr2 as a promising COVID-19 vaccine candidate.

Conventional type 1 dendritic cells (cDC1s) are critical for innate sensing of cancer, yet they are scarce in the tumor microenvironment (TME). Here, we present a protocol to identify and isolate cDC1 subsets from murine implantable tumors for subsequent transcriptomic profiling using a flow sorting-based strategy. We describe steps for cell culture of mouse tumors, tumoral growth, dissociation and isolation of tumoral cells, extracellular staining, and cell sorting. We then detail procedures for RNA isolation, mRNA library preparation, and sequencing. For complete details on the use and execution of this protocol, please refer to Papadas et al.1.

The preparation of rigid polyurethane foam with uniform pore size is extremely challenging. In this work, double-effect baking powder was used as foaming agent, and rigid polyurethane foam with regular morphology was successfully prepared through semi-prepolymerization. The rigid polyurethane foam apertures with regular morphology were controlled by the secondary release of C[O.sub.2] when the content of baking powder is 0.30 part. Combined with theoretical calculation and experimental test, it is found that the particle size distribution, wall thickness, and hole shape are uniform, regular, thin, polygonal, and few broken holes. The compression strength, contact angle, and surface energy of the rigid polyurethane foam are 33.36 kPa, 109.6[degrees], and 21.8 mJ/[m.sup.2] after adding 0.20 part of double-effect baking powder, respectively. By adjusting the amount of double-acting baking powder, the porosity, mechanical properties and foam rate of the polyurethane foams could be significantly improved. Due to the molecular solvation effect, rigid polyurethane foams will deform in the water and oil due phase, but it can remain stable for a long time at room temperature without solvent. It provides a new method for the preparation of rigid polyurethane foam, which is expected to be widely used in transportation and other fields. Highlights * Baking powder was firstly used as a foaming modifier for rigid polyurethane. * Baking powder releases C[O.sub.2] twice to ensure uniform structure of polyurethane cell. * Rigid polyurethane foam has long-term stability and regular, uniform cell holes. * The porosity and mechanical properties of foams could be significantly improved. * Foaming mechanism of rigid polyurethane using baking powder was firstly revealed. KEYWORDS blowing agent, foam, mechanical properties, microstructure, polyurethane, 1 | INTRODUCTION With the rapid development of the transportation and logistics industry, there is an increasing demand for high-performance materials, particularly in enhancing fuel efficiency, safety, and passenger comfort. [...]