Inhaled SARS-CoV-2 vaccine for single-dose dry powder aerosol immunization.

The COVID-19 pandemic has fostered major advances in vaccination technologies1-4; however, there are urgent needs for vaccines that induce mucosal immune responses and for single-dose, non-invasive administration4-6. Here we develop an inhalable, single-dose, dry powder aerosol SARS-CoV-2 vaccine that induces potent systemic and mucosal immune responses. The vaccine encapsulates assembled nanoparticles comprising proteinaceous cholera toxin B subunits displaying the SARS-CoV-2 RBD antigen within microcapsules of optimal aerodynamic size, and this unique nano-micro coupled structure supports efficient alveoli delivery, sustained antigen release and antigen-presenting cell uptake, which are favourable features for the induction of immune responses. Moreover, this vaccine induces strong production of IgG and IgA, as well as a local T cell response, collectively conferring effective protection against SARS-CoV-2 in mice, hamsters and nonhuman primates. Finally, we also demonstrate a mosaic iteration of the vaccine that co-displays ancestral and Omicron antigens, extending the breadth of antibody response against co-circulating strains and transmission of the Omicron variant. These findings support the use of this inhaled vaccine as a promising multivalent platform for fighting COVID-19 and other respiratory infectious diseases.

Bioinformatic analysis of structures and encoding genes of Escherichia coli surface polysaccharides sheds light on the heterologous biosynthesis of glycans.

BACKGROUND: Surface polysaccharides (SPs), such as lipopolysaccharide (O antigen) and capsular polysaccharide (K antigen), play a key role in the pathogenicity of Escherichia coli (E. coli). Gene cluster for polysaccharide antigen biosynthesis encodes various glycosyltransferases (GTs), which drive the process of SP synthesis and determine the serotype. RESULTS: In this study, a total of 7,741 E. coli genomic sequences were chosen for systemic data mining. The monosaccharides in both O and K antigens were dominated by D-hexopyranose, and the SPs in 70-80% of the strains consisted of only the five most common hexoses (or some of them). The linkages between the two monosaccharides were mostly α-1,3 (23.15%) and ß-1,3 (20.49%) bonds. Uridine diphosphate activated more than 50% of monosaccharides for glycosyltransferase reactions. These results suggest that the most common pathways could be integrated into chassis cells to promote glycan biosynthesis. We constructed a database (EcoSP, http://ecosp.dmicrobe.cn/ ) for browse this information, such as monosaccharide synthesis pathways. It can also be used for serotype analysis and GT annotation of known or novel E. coli sequences, thus facilitating the diagnosis and typing. CONCLUSIONS: Summarizing and analyzing the properties of these polysaccharide antigens and GTs are of great significance for designing glycan-based vaccines and the synthetic glycobiology.

Dual-band complementary metamaterial perfect absorber for multispectral molecular sensing.

Metamaterial perfect absorbers (MPAs) show great potential in achieving exceptional sensing performance, particularly in the realm of surface-enhanced infrared absorption (SEIRA) spectroscopy. To this aim, it is highly desirable for the localized hotspots to be readily exposed and accessible to analyte with strong mode confinement to enhance absorption. Here, we propose a quasi-three-dimensional MPA based on cross-shaped coupled complementary plasmonic arrays for highly sensitive refractive index sensing and molecular vibrational sensing. Dual-band perfect absorption can be approached with the two plasmonic resonances corresponding to the electric dipole-like mode of cross antenna array and the magnetic dipole-like mode of cross hole array, respectively. Large portions of the electric field of the hotspots are exposed and concentrated in the gap between the elevated cross antenna and its complementary structure on the substrate, leading to improved sensing sensitivities. An ultrathin polymethyl methacrylate (PMMA) film induces a significant redshift of the magnetic dipole-like mode with an 11.8 nm resonance shift per each nanometer polymer thickness. The value is comparable to the reported sensitivity of single molecule layer sensors. Additionally, the simultaneous detection of the C = O and C-H vibrations of PMMA molecules is enabled with the two plasmonic resonances adjusted by changing the lengths of the two cross branches. Remarkably, the observed mode splitting and anti-crossing behavior imply the strong interaction between plasmonic resonance and molecular vibration. Our dual-band MPA based on coupled complementary plasmonic arrays opens a new avenue for developing highly sensitive sensors for the detection of refractive index and multispectral molecular vibrations.

Dual color infrared photodetector with superconducting metamaterials.

Superconducting photodetection offers a wide spectral coverage ranging from the microwave to X-ray, and in the short wavelength range, single photon sensitivity can be achieved. However, in the longer wavelength infrared region, the system detection efficiency is low due to the lower internal quantum efficiency and weak optical absorption. Here, we utilized the superconducting metamatieral to enhance the light coupling efficiency and reach nearly perfect absorption at dual color infrared wavelengths. Dual color resonances arise from hybridization of local surface plasmon mode of the metamaterial structure and the Fabry-Perot-like cavity mode of metal (Nb)-dielectric (Si)-metamatieral (NbN) tri-layer structure. We demonstrated that, at the working temperature of 8 K slightly below TC ∼8.8 K, this infrared detector exhibits the peak responsivity of 1.2 × 106V/W and 3.2 × 106V/W at two resonant frequencies 36.6 THz and 104 THz, respectively. The peak responsivity is enhanced about ∼8 and ∼22 times, respectively, compared to that of non-resonant frequency (67 THz). Our work provides a way to harvest infrared light efficiently and hence improve the sensitivity of superconducting photodetectors in multispectral infrared range, which may find promising applications in thermal image and gas sensing etc.

Preparation of a Klebsiella pneumoniae conjugate nanovaccine using glycol-engineered Escherichia coli.

BACKGROUND: Engineered strains of Escherichia coli have been used to produce bioconjugate vaccines using Protein Glycan Coupling Technology (PGCT). Nanovaccines have also entered the vaccine development arena with advances in nanotechnology and have been significantly developed, but chassis cells for conjugate nanovaccines have not been reported. RESULTS: To facilitate nanovaccine preparation, a generic recombinant protein (SpyCather4573) was used as the acceptor protein for O-linked glycosyltransferase PglL, and a glycol-engineered Escherichia coli strain with these two key components (SC4573 and PglL) integrated in its genome was developed in this study. The targeted glycoproteins with antigenic polysaccharides produced by our bacterial chassis can be spontaneously bound to proteinous nanocarriers with surface exposed SpyTag in vitro to form conjugate nanovaccines. To improve the yields of the targeted glycoprotein, a series of gene cluster deletion experiments was carried out, and the results showed that the deletion of the yfdGHI gene cluster increased the expression of glycoproteins. Using the updated system, to the best of our knowledge, we report for the first time the successful preparation of an effective Klebsiella pneumoniae O1 conjugate nanovaccine (KPO1-VLP), with antibody titers between 4 and 5 (Log10) after triple immunization and up to 100% protection against virulent strain challenge. CONCLUSIONS: Our results define a convenient and reliable framework for bacterial glycoprotein vaccine preparation that is flexible and versatile, and the genomic stability of the engineered chassis cells promises a wide range of applications for biosynthetic glycobiology research.

Disruption of SpoIIID decreases sporulation, increases extracellular proteolytic activity and virulence in Bacillus anthracis.

Endospores are important for maintenance of the B. anthracis lifecycle and necessary for its effective spread between hosts. Our experiments with B. anthracis showed that disruption of SpoIIID results in a spore formation defect, as determined by heat resistance assays and microscopic assessment. We further found complete engulfment by the ΔspoIIID mutant strain by membrane morphology staining but no synthesis of the clarity coat and exosporium by transmission electron microscopy. Reduced transcription and expression of small acid-soluble spore protein(sasP-2) and the spore development associated genes (σK, gerE and cotE) in the mother cell were found in the ΔspoIIID strain, suggesting that the spore formation defect in B. anthracis A16R is related to decreased transcription and expression of these genes. Extracellular protease and virulence enhancement in the ΔspoIIID strain may be related to the elevation of metalloproteinases (TasA and Camelysin) levels. Our findings pave the way for further research on the regulation network of sporulation, survival and virulence in these two morphological forms of B. anthracis.

Genome sequence of Bacillus anthracis typing phage AP631.

AP631, a virulent bacteriophage of Bacillus anthracis, is widely used in China to identify anthrax bacteria. In this study, we report the complete AP631 phage genome sequence as well as comparative genomic analysis with other bacteriophages of B. cereus and related species. The double-stranded circular DNA genome of phage AP631 was 39,549 bp in length with 35.01% G + C content. The phage genome contained 56 putative protein-coding genes but no rRNA or tRNA genes. Comparative phylogenetic analysis of the phage major capsid proteins and terminase large subunits showed that phage AP631 belongs to the B. cereus sensu lato phage clade II. Comparative genomic analysis revealed a high degree of sequence similarity between phage AP631 and B. anthracis phages Wbeta, Gamma, Cherry, and Fah, as well as three AP631-specific genes bearing no significant similarity to those of other phages.

Effect of Environmental Conditions on the Degradation of Florasulam in Typical Soils of Northern China.

The widespread use of florasulam in China makes residues from this herbicide in soil a serious concern due to their potential to pollute the soil environment and groundwater. Accelerating the degradation of these residues will reduce their pollution potential. In this study, we investigated the degradation and adsorption of florasulam in four typical soils in northern China and examined the degradation of florasulam in Inceptisols at different temperatures, soil moisture contents, and pH values, as well as the influence of microorganisms and the use of organic matter and biogas slurry as soil amendments. The half-lives of florasulam in the four soils were 13.6 d (Ultisols), 13.9 d (Vertisols), 15.1 d (Alfisols), and 19.3 d (Inceptisols), and the adsorption ability of the four soils followed the order Inceptisols > Alfisols > Vertisols > Ultisols. Florasulam degradation rates increased as temperature and soil moisture increased and as soil pH decreased (from 8.0 to 6.0). Adding a small amount of organic matter to the soil increased the florasulam degradation rates. The use of biogas slurry also increased the degradation rates. Florasulam half-life in unsterilized soil ( = 19.3 d) was significantly shorter than in sterilized soil ( = 113.4 d). These results provide agricultural producers and environmental managers useful information for reducing the environmental risk associated with florasulam use.

Analysis of Soluble protein complexes in Shigella flexneri reveals the influence of temperature on the amount of lipopolysaccharide.

Shigella flexneri, which is closely related to Escherichia coli, is the most common cause of the endemic form of shigellosis. In this study, 53 homomultimeric protein complexes and nine heteromultimeric protein complexes from S. flexneri 2a strain 2457T were separated and identified. Among these, three potential homomultimeric protein complexes had not been previously described. One complex, thought to be composed of 12 PhoN1 subunits, is a periplasmic protein with an unknown physiological role encoded on the virulence plasmid of S. flexneri. The abundance of the protein complexes was compared following growth at 37 or 30°C, and the abundance of three protein complexes (PyrB-PyrI, GlmS, and MglB) related to the synthesis of lipopolysaccharides (LPS) appeared to be temperature-dependent. Many studies have shown that LPS is essential to the virulence of S. flexneri. Here, we report the influence of temperature on the amount of LPS.

The effect of quorum sensing system for growth competitiveness on Shigella flexneri.

Quorum sensing (QS) regulates the onset of bacterial social responses related to cell density. Comparison between the gene sequences of all components of QS system of Escherichia coli and Shigella strains, shows that the QS system is generally lost or mutated in Shigella. Since AI-2 is produced and processed by the lsr operon, we analyzed the potential function of the lsr operon. We first detected AI-2 in Shigella flexneri 2a strain 301 through the reporter bacteria Vibrio harveyi BB170, indicating that S. flexneri can produce AI-2. Then, the lsr operon of E. coli MG1655 was cloned into S. flexneri using the Golden Gate method. Colony counting experiments showed that the QS system recovery strain had growth advantage over the wild-type strain when they were mixed and cultured. The preliminary comparative proteomics analysis showed that the lsr operon could be expressed and the abundance of stress response proteins also changed when the QS system was introduced into S. flexneri.

Preparation and immunogenicity-evaluation of typhoid O-specific polysaccharides bio-conjugate vaccines.

Typhoid fever caused by Salmonella Typhi is still a major public health problem in developing countries. In this study, we constructed a genetically modified Salmonella Typhi strain expressing O-specific polysaccharides (OPS) antigen conjugated to a carrier, recombinant Pseudomonas aeruginosa exotoxin A(rEPA N29). The conjugates (OPS-rEPA N29) were further purified and evaluated for their immunogenicity. The results of ELISA showed that the conjugates evoked higher titers of IgG than OPS, suggesting that rEPAN29 increased immunogenicity of OPS significantly as a carrier. Moreover, three injections with 3-week interval evoked slightly higher titers of IgG than three injections with 2-week interval. However, injection of excess conjugates could not evoke higher titers of IgG against lipid polysaccharide (LPS). In summary, our study provides a new strategy for preparing polysaccharides-protein conjugate vaccines as well as similar bio-conjugate vaccines of other Gram-negative pathogens.

The function of PlcR in Bacillus anthracis vaccine strain A16R.

Bacillus anthracis, B. thuringiensis and B. cereus are members of the B. cereus group. They share high genetic similarity. Whereas plcR (Phospholipase C regulator) usually encodes a functional pleiotropic activator protein in B. cereus and B. thuringiensis isolates, a characteristic nonsense mutation is found in all B. anthracis strains investigated, making the gene dysfunctional. To study the function of PlcR in B. anthracis, we used the B. cereus CMCC63301 genome as a template and constructed a recombinant expression plasmid pBE2A-plcR, and introduced it into the B. anthracis vaccine strain A16R, and then analyzed the activity of the hemolysin and sphingomyelinase. The results showed that transformation of B. anthracis with plasmid pBE2A-plcR carrying the native B. cereus plcR gene active the expression of sphingomyelinase gene, but did not activate expression of hemolysin genes of B. anthracis A16R.

[Clustered regularly interspaced short palindromic repeats (CRISPR) site in Bacillus anthracis].

OBJECTIVE: To investigate the polymorphism of clustered regularly interspaced short palindromic repeats (CRISPR) in Bacillu santhracis and the application to molecular typing based on the polymorphism of CRISPR in B. anthracis. METHODS: We downloaded the whole genome sequence of 6 B. anthracis strains and extracted the CRISPR sites. We designed the primers of CRISPR sites and amplified the CRISPR fragments in 193 B. anthracis strains by PCR and sequenced these fragments. In order to reveal the polymorphism of CRISPR in B. anthracis, wealigned all the extracted sequences and sequenced results by local blasting. At the same time, we also analyzed the CRISPR sites in B. cereus and B. thuringiensis. RESULTS: We did not find any polymorphism of CRISPR in B. anthracis. CONCLUSION: The molecular typing approach based on CRISPR polymorphism is not suitable for B. anthracis, but it is possible for us to distinguish B. anthracis from B. cereus and B. thuringiensis.

Genome sequences of two Bacillus anthracis strains utilized as veterinary vaccines in China.

In this report, we present the complete genome sequences of two Bacillus anthracis strains utilized as veterinary vaccines in China. The sequencing was conducted using a hybrid assembly methodology that combined Illumina short reads and PacBio long reads. This approach provides a high-quality representative sequence for the strains mentioned above.

Production of Promising Heat-Labile Enterotoxin (LT) B Subunit-Based Self-Assembled Bioconjugate Nanovaccines against Infectious Diseases.

Nanoparticles (NPs) have been widely utilized in vaccine design. Although numerous NPs have been explored, NPs with adjuvant effects on their own have rarely been reported. We produce a promising self-assembled NP by integrating the pentameric Escherichia coli heat-labile enterotoxin B subunit (LTB) (studied as a vaccine adjuvant) with a trimer-forming peptide. This fusion protein can self-assemble into the NP during expression, and polysaccharide antigens (OPS) are then loaded in vivo using glycosylation. We initially produced two Salmonella paratyphi A conjugate nanovaccines using two LTB subfamilies (LTIB and LTIIbB). After confirming their biosafety in mice, the data showed that both nanovaccines (NP(LTIB)-OPSSPA and NP(LTIIbB)-OPSSPA) elicited strong polysaccharide-specific antibody responses, and NP(LTIB)-OPS resulted in better protection. Furthermore, polysaccharides derived from Shigella or Klebsiella pneumoniae were loaded onto NP(LTIB) and NP(LTIIbB). The animal experimental results indicated that LTIB, as a pentamer module, exhibited excellent protection against lethal infections. This effect was also consistent with that of the reported cholera toxin B subunit (CTB) modular NP in all three models. For the first time, we prepared a novel promising self-assembled NP based on LTIB. In summary, these results indicated that the LTB-based nanocarriers have the potential for broad applications, further expanding the library of self-assembled nanocarriers.

Efficient Production of Self-Assembled Bioconjugate Nanovaccines against Klebsiella pneumoniae O2 Serotype in Engineered Escherichia coli.

Nanoparticles (NPs) have been surfacing as a pivotal platform for vaccine development. In our previous work, we developed a cholera toxin B subunit (CTB)-based self-assembled nanoparticle (CNP) and produced highly promising bioconjugate nanovaccines by loading bacterial polysaccharide (OPS) in vivo. In particular, the Klebsiella pneumoniae O2 serotype vaccine showcased a potent immune response and protection against infection. However, extremely low yields limited its further application. In this study, we prepared an efficient Klebsiella pneumoniae bioconjugate nanovaccine in Escherichia coli with a very high yield. By modifying the 33rd glycine (G) in the CNP to aspartate (D), we were able to observe a dramatically increased expression of glycoprotein. Subsequently, through a series of mutations, we determined that G33D was essential to increasing production. In addition, this increase only occurred in engineered E. coli but not in the natural host K. pneumoniae strain 355 (Kp355) expressing OPSKpO2. Next, T-cell epitopes were fused at the end of the CNP(G33D), and animal experiments showed that fusion of the M51 peptide induced high antibody titers, consistent with the levels of the original nanovaccine, CNP-OPSKpO2. Hence, we provide an effective approach for the high-yield production of K. pneumoniae bioconjugate nanovaccines and guidance for uncovering glycosylation mechanisms and refining glycosylation systems.

Rapid Identification of Brucella Genus and Species In Silico and On-Site Using Novel Probes with CRISPR/Cas12a.

Human brucellosis caused by Brucella is a widespread zoonosis that is prevalent in many countries globally. The high homology between members of the Brucella genus and Ochrobactrum spp. often complicates the determination of disease etiology in patients. The efficient and reliable identification and distinction of Brucella are of primary interest for both medical surveillance and outbreak purposes. A large amount of genomic data for the Brucella genus was analyzed to uncover novel probes containing single-nucleotide polymorphisms (SNPs). GAMOSCE v1.0 software was developed based on the above novel eProbes. In conjunction with clinical requirements, an RPA-Cas12a detection method was developed for the on-site determination of B. abortus and B. melitensis by fluorescence and lateral flow dipsticks (LFDs). We demonstrated the potential of these probes for rapid and accurate detection of the Brucella genus and five significant Brucella species in silico using GAMOSCE. GAMOSCE was validated on different Brucella datasets and correctly identified all Brucella strains, demonstrating a strong discrimination ability. The RPA-Cas12a detection method showed good performance in detection in clinical blood samples and veterinary isolates. We provide both in silico and on-site methods that are convenient and reliable for use in local hospitals and public health programs for the detection of brucellosis.

Multi-color two-laser super-resolution structured illumination microscopy for the visualization of multi-organelle in living cells.

In this study, we introduced a novel dual-laser multi-color imaging system. Integrated with a multi-channel filter wheel, this system compared three spectral decontamination algorithms (nonnegative matrix factorization [NMF], RCAN, and PICASSO) showcasing its efficacy in achieving four-color imaging with only two laser sources. Combined with a reliable image reconstruction algorithm, the spatial resolution of four channels super-resolution four-color images reached 130, 125, 133, and 132 nm, respectively. Lipid droplets, mitochondria, lysosomes, and nuclei from the mouse hepatocytes (AML12), human neuroblastoma cells (SH-SY5Y), mouse hippocampal neuronal cells (HT-22), and immortalized murine bone marrow-derived macrophages were imaged. At the same time, the chromatin condensation, nuclear contraction, DNA fragmentation, apoptotic body formation, as well as the fusion of Mito and Lyso involved in mitochondrial autophagy were observed in HT-22 and SH-SY5Y cells suffering oxidative stress. Our multi-color SIM imaging system establishes a powerful platform for dynamic organelle studies and other high-resolution investigations in live cells.

[Screening and identification of glycoproteins from Campylobacter jejuni NCTC 11168 based on specific affinity between lectins and glycoproteins].

OBJECTIVE: To screen glycoproteins from Campylobacter jejuni NCTC 11168. METHODS: We introduced a screening strategy based on specific affinity between lectins and glycoproteins. First, the specific affinity between Soybean Agglutinin (SBA) and glycoproteins in C. jejuni was confirmed by western blotting. Then, magnetic beads coating with Soybean Agglutinin (SBA) were used to capture the putative glycoproteins. Putative glycoproteins were competitively eluted by N-acetylgalactosamine and separated by two-dimensional gel electrophoresis. Spots in gels were further analyzed by mass spectrometry. RESULTS: A total of 22 proteins were identified by mass spectrometric analysis, of which 5 have ever been identified to be glycoproteins. Others including Cj0633 have not been proved to be glycosylated so far. CONCLUSION: The method could be used to screen glycoproteins in bacteria.

Genetic Engineering of Klebsiella pneumoniae ATCC 25955 for Bioconjugate Vaccine Applications.

Vaccination is considered the most effective means to fight against the multidrug-resistant strains of Klebsiella pneumoniae. In recent years, a potential protein glycan coupling technology has been extensively used in the production of bioconjugated vaccines. Here, a series of glycoengineering strains derived from K. pneumoniae ATCC 25955 were designed for protein glycan coupling technology. The capsule polysaccharide biosynthesis gene cluster and the O-antigen ligase gene waaL were deleted via the CRISPR/Cas9 system to further weaken the virulence of host stains and block the unwanted endogenous glycan synthesis. Particularly, the SpyCatcher protein in the efficient protein covalent ligation system (SpyTag/SpyCatcher) was selected as the carrier protein to load the bacterial antigenic polysaccharides (O1 serotype), which could covalently bind to SpyTag-functionalized nanoparticles AP205 to form nanovaccines. Furthermore, two genes (wbbY and wbbZ) located in the O-antigen biosynthesis gene cluster were knocked out to change the O1 serotype of the engineered strain into the O2 serotype. Both KPO1-SC and KPO2-SC glycoproteins were successfully obtained as expected using our glycoengineering strains. Our work provides new insights into the design of nontraditional bacterial chassis for bioconjugate nanovaccines against infectious diseases.