Chiral coordination polymer nanowires boost radiation-induced in situ tumor vaccination.

Radiation-induced in situ tumor vaccination alone is very weak and insufficient to elicit robust antitumor immune responses. In this work, we address this issue by developing chiral vidarabine monophosphate-gadolinium nanowires (aAGd-NWs) through coordination-driven self-assembly. We elucidate the mechanism of aAGd-NW assembly and characterize their distinct features, which include a negative surface charge, ultrafine topography, and right-handed chirality. Additionally, aAGd-NWs not only enhance X-ray deposition but also inhibit DNA repair, thereby enhancing radiation-induced in situ vaccination. Consequently, the in situ vaccination induced by aAGd-NWs sensitizes radiation enhances CD8+ T-cell-dependent antitumor immunity and synergistically potentiates the efficacy immune checkpoint blockade therapies against both primary and metastatic tumors. The well-established aAGd-NWs exhibit exceptional therapeutic capacity and biocompatibility, offering a promising avenue for the development of radioimmunotherapy approaches.

A rapid and sensitive fluorescent chromatography with cloud system for MPXV point-of-care diagnosis.

Monkeypox (mpox) is spreading around the world, and its rapid diagnosis is of great significance. In the present study, a rapid and sensitive fluorescent chromatography assisted with cloud system was developed for point-of-care diagnosis of mpox. To screen high affinity antibodies, nanoparticle antigen AaLS-A29 was generated by conjugating A29 onto scaffold AaLS. Immunization with AaLS-A29 induced significantly higher antibody titers and monoclonal antibodies were generated with the immunized mice. A pair of monoclonal antibodies, MXV 14 and MXV 15, were selected for fluorescence chromatography development. The Time-Resolved Fluorescence Immunoassay (TRFIA) was used to develop the chromatography assay. After optimization of the label and concentration of antibodies, a sensitive TRFIA assay with detection limit of 20 pg/mL and good repeatability was developed. The detection of the surrogate Vaccinia virus (VACA) strain Tian Tan showed that the TRFIA assay was more sensitive than the SYBR green I based quantitative PCR. In real samples, the detection result of this assay were highly consistent with the judgement of Quantitative Real-Time PCR (Concordance Rate = 90.48%) as well as the clinical diagnosis (Kappa Value = 0.844, P < 0.001). By combining the portable detection and online cloud system, the detection results could be uploaded and shared, making this detection system an ideal system for point-of-care diagnosis of mpox both in field laboratory and outbreak investigation.

Rapid and highly potent humoral responses to mpox nanovaccine candidates adjuvanted by thermostable scaffolds.

Monkeypox (mpox) is a zoonotic disease caused by monkeypox virus (MPXV) of the orthopoxvirus genus. The emergence and global spread of mpox in 2022 was declared as a public health emergency by World Health Organization. This mpox pandemic alarmed us that mpox still threaten global public health. Live vaccines could be used for immunization for this disease with side effects. New alternative vaccines are urgently needed for this re-emerging disease. Specific antibody responses play key roles for protection against MPXV, therefore, vaccines that induce high humoral immunity will be ideal candidates. In the present study, we developed thermostable nanovaccine candidates for mpox by conjugating MPXV antigens with thermostable nanoscafolds. Three MPXV protective antigens, L1, A29, and A33, and the thermostable Aquafex aeolicus lumazine synthase (AaLS), were expressed in E. coli and purified by Ni-NTA methods. The nanovaccines were generated by conjugation of the antigens with AaLS. Thermal stability test results showed that the nanovaccines remained unchanged after one week storage under 37℃ and only partial degradation under 60℃, indicating high thermostability. Very interesting, one dose immunization with the nanovaccine could induce high potent antibody responses, and two dose induced 2-month high titers of antibodes. In vitro virus neutralization test showed that nanovaccine candidates induced significantly higher levels of neutralization antibodies than monomers. These results indicated that the AaLS conjugation nanovaccines of MPXV antigens are highly thermostable in terms of storage and antigenic, being good alternative vaccine candidates for this re-emerging disease.

Potent immune responses against thermostable Foot-and-Mouth disease virus VP1 nanovaccine adjuvanted with polymeric thermostable scaffold.

Foot-and-mouth disease (FMD) is an acute zoonosis causes significant economic losses. Vaccines able to stimulate efficient protective immune responses are urgently needed. In this study, Escherichia coli-derived recombinant VP1 of serotype A and O FMD virus (FMDV) was conjugated to thermostable scaffold lumazine synthase (LS) or Quasibacillus thermotolerans encapsulin (QtEnc) using a robust plug-and-display SpyTag/SpyCatcher system to generate multimeric nanovaccines. These nanovaccines induced highly potent antibody responses in vaccinated mice. On day 14 after the first immunisation, antibody titres were approximately 100 times higher than those of monomer antigens. Both vaccines induced high and long-term IgG antibody production. Moreover, the QtEnc-VP1 nanovaccine induced higher antibody titres than the LS-VP1 nanovaccine. The nanovaccines also induced Th1-biased immune responses and higher levels of neutralising antibodies. These data indicated that FMDV nanovaccines generated by conjugating VP1 with a thermostable scaffold are highly immunogenic and ideal candidates for FMDV control in low-resource areas.

Structural basis of Tom20 and Tom22 cytosolic domains as the human TOM complex receptors.

Mitochondrial preproteins synthesized in cytosol are imported into mitochondria by a multisubunit translocase of the outer membrane (TOM) complex. Functioned as the receptor, the TOM complex components, Tom 20, Tom22, and Tom70, recognize the presequence and further guide the protein translocation. Their deficiency has been linked with neurodegenerative diseases and cardiac pathology. Although several structures of the TOM complex have been reported by cryoelectron microscopy (cryo-EM), how Tom22 and Tom20 function as TOM receptors remains elusive. Here we determined the structure of TOM core complex at 2.53 Å and captured the structure of the TOM complex containing Tom22 and Tom20 cytosolic domains at 3.74 Å. Structural analysis indicates that Tom20 and Tom22 share a similar three-helix bundle structural feature in the cytosolic domain. Further structure-guided biochemical analysis reveals that the Tom22 cytosolic domain is responsible for binding to the presequence, and the helix H1 is critical for this binding. Altogether, our results provide insights into the functional mechanism of the TOM complex recognizing and transferring preproteins across the mitochondrial membrane.

A novel and unique ATP hydrolysis to AMP by a human Hsp70 Binding immunoglobin protein (BiP).

Hsp70s are ubiquitous and highly conserved molecular chaperones. They play crucial roles in maintaining cellular protein homeostasis. It is well established that Hsp70s use the energy of ATP hydrolysis to ADP to power the chaperone activity regardless of the cellular locations and isoforms. Binding immunoglobin protein (BiP), the major member of Hsp70s in the endoplasmic reticulum, is essential for protein folding and quality control. Unexpectedly, our structural analysis of BiP demonstrated a novel ATP hydrolysis to AMP during crystallization under the acidic conditions. Our biochemical studies confirmed this newly discovered ATP to AMP hydrolysis in solutions. Unlike the canonical ATP to ADP hydrolysis observed for Hsp70s, this ATP hydrolysis to AMP depends on the substrate-binding domain of BiP and is inhibited by the binding of a peptide substrate. Intriguingly, this ATP to AMP hydrolysis is unique to BiP, not shared by two representative Hsp70 proteins from the cytosol. Taken together, this novel and unique ATP to AMP hydrolysis may provide a potentially new direction for understanding the activity and cellular function of BiP.

Conformation transitions of the polypeptide-binding pocket support an active substrate release from Hsp70s.

Cellular protein homeostasis depends on heat shock proteins 70 kDa (Hsp70s), a class of ubiquitous and highly conserved molecular chaperone. Key to the chaperone activity is an ATP-induced allosteric regulation of polypeptide substrate binding and release. To illuminate the molecular mechanism of this allosteric coupling, here we present a novel crystal structure of an intact human BiP, an essential Hsp70 in ER, in an ATP-bound state. Strikingly, the polypeptide-binding pocket is completely closed, seemingly excluding any substrate binding. Our FRET, biochemical and EPR analysis suggests that this fully closed conformation is the major conformation for the ATP-bound state in solution, providing evidence for an active release of bound polypeptide substrates following ATP binding. The Hsp40 co-chaperone converts this fully closed conformation to an open conformation to initiate productive substrate binding. Taken together, this study provided a mechanistic understanding of the dynamic nature of the polypeptide-binding pocket in the Hsp70 chaperone cycle.

A disulfide-bonded DnaK dimer is maintained in an ATP-bound state.

DnaK, a major Hsp70 molecular chaperones in Escherichia coli, is a widely used model for studying Hsp70s. We recently solved a crystal structure of DnaK in complex with ATP and showed that DnaK was packed as a dimer in the crystal structure. Our previous biochemical studies supported the formation of a specific DnaK dimer as observed in the crystal structure in solution in the presence of ATP and suggested an important role of this dimer in efficient interaction with Hsp40 co-chaperones. In this study, we dissected the biochemical properties of this DnaK dimer. To restrict DnaK in this dimer form, we mutated two residues on the dimer interface to cysteine, A303C, and H541C. Upon oxidation, this DnaK-A303C-H541C protein formed a specific dimer linked by disulfide bonds formed between A303C and H541C only in the presence of ATP, consistent with the crystal structure. Intriguingly, this disulfide-bond-linked dimer of DnaK-A303C-H541C has reduced ATPase activity and decreased affinity for peptide substrate. More interestingly, unlike wild-type DnaK, the peptide substrate-binding kinetics of this dimer is drastically accelerated even in the absence of ATP, suggesting this dimer is restricted in an ATP-bound conformation regardless of nucleotide bound, which was further supported by our analysis using tryptophan fluorescence and ATP-induced peptide release. Thus, formation of the dimer restricted DnaK in an ATP-bound state and blocked the progression through the chaperone cycle. Productive progression through the chaperone cycle requires the dissociation of this transient dimer. Surprisingly, a significantly compromised interaction with Hsp40 co-chaperone was observed for this disulfide-bond-linked dimer. Thus, dissociation of this DnaK dimer is equally crucial for efficient Hsp40 interaction. An initial interaction between Hsp70 and Hsp40 requires the formation of DnaK dimer; but a stable Hsp70-Hsp40 interaction may follow the dissociation of the dimer.