A Click-Type Enzymatic Method for Antigen-Adjuvant Conjugation.

The Toll-like receptor 9 (TLR9) stimulator, CpG oligodeoxynucleotide, has emerged as a potent enhancer of protein subunit vaccines. Incorporating the protein antigen directly with the CpG adjuvant presents a novel strategy to significantly reduce the required dosage of CpG compared to traditional methods that use separate components. In contrast to existing chemical conjugation methods, this study introduces an enzymatic approach for antigen-adjuvant coupling using a recombinant endonuclease DCV fused with SpyTag. This fusion protein catalyzes the covalent linkage between itself and the CpG adjuvant under mild conditions. These conjugates can be further linked with target protein antigens containing the SpyCatcher sequence, yielding stable, covalently-linked antigen-adjuvant complexes. The corresponding complex utilizing the receptor-binding domain (RBD) of SARS-CoV-2 spike protein as the model antigen, elicits high-titer, specific antibody production in mice via both subcutaneous administration and intratracheal inoculation. Notably, the tumor vaccine candidate fabricated by this method has also shown significant inhibition of cancer progression after intratracheal administration. The technique ensures precise, site-specific coupling and preserves the antigen's structural integrity due to the post-purification coupling strategy that simplifies manufacturing and aids in developing inhalable vaccines.

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.

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.

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.