Tumor subunit vaccines have great potential in personalized cancer immunotherapy. They are usually administered with adjuvant owing to their low immunogenicity. Cholera toxin (CT) is a biological adjuvant with diverse biological functions and a long history of use. Our earlier study revealed that a CT-like chimeric protein co-delivered with murine granulocyte-macrophage colony stimulating factor (mGM-CSF) and prostate cancer antigen epitope could co-stimulate dendritic cells (DCs) and enhance cross presentation of tumor epitope. To further study the molecular mechanism of CT-like chimeric protein in cross presentation, major histocompatibility complex class I (MHC I)-restricted epitope 257-264 of ovalbumin (OVAT) was used as a model antigen peptide in this study. Recombinant A subunit and pentameric B subunit of CT protein were respectively genetically constructed and purified. Then both assembled into AB5 chimeric protein in vitro. Three different chimeric biomacromolecules containing mGM-CSF and OVAT were constructed according to the different fusion sites and whether the endoplasmic reticulum (ER) retention sequence was included. It was found that A2 domain and B subunit of CT were both available for loading epitopes and retaining GM1 affinity. The binding activity of GM1 was positively correlated with antigen endocytosis. Once internalized, DCs became mature and cross-presented antigen. KDEL helped the whole molecule to be retained in the ER, and this improved the cross presentation of antigen on MHC I molecules. In conclusion, hexameric CT-like chimeric protein with dual effects of GM1 affinity and ER retention sequence were potential in improvement of cross presentation. The results laid a foundation for designing personalized tumor vaccine based on CT-like chimeric protein molecular structure.

Cholera is an exceptionally aggressive infectious disease characterized by the potential to induce acute, copious, watery diarrhea of considerable severity and renal inflammation. Diabetic nephropathy is a serious complication of diabetes mellitus that can lead to kidney failure through inflammation; thus, anti-inflammatory agents are promising therapies for diabetic nephropathy. Previous studies have shown that the essential oil of Zanthoxylum myriacanthum var. pubescens Huang, Maqian essential oil (MQEO), exhibits potent antibacterial, anti-inflammatory, and renoprotective activities in diabetic mice and has emerged as a potential therapeutic drug for the treatment of diabetic nephropathy complications. Therefore, the present study was carried out to screen the potential inhibition of cholera toxin and the diabetic renoprotective activity of MQEO through computational approaches. Twelve chemical constituents derived from MQEO were docked with cholera toxin and the target proteins involved in diabetic nephropathy, namely, TXNIP, Nrf2, and DPP IV, and, subsequently, the predictions of molecular dynamic simulations, the drug-likeness properties, and the ADMET properties were performed. α-terpineol showed high binding affinities toward the cholera toxin protein. For TXNIP, among all the chemical constituents, α-phellandrene and p-cymene showed strong binding affinities with the TXNIP protein and displayed relatively stable flexibility at the hinge regions of the protein, favorable physicochemical properties in the absence of hepatotoxicity, and low cytotoxicity. For Nrf2, α-terpineol exhibited the highest binding affinity and formed a very stable complex with Nrf2, which displayed high pharmacokinetic properties. All compounds had low free-binding energies when docked with the DPP IV protein, which suggests potent biological activity. In conclusion, based on a computational approach, our findings reveal that MQEO constituents have inhibitory activity against cholera toxin and are promising therapeutic agents for suppressing diabetic inflammation and for the treatment of diabetic nephropathy complications.

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.

Peripheral nerve injury can cause changes in microglial cells on the spinal dorsal and ventral horns. This region-specific response implies that central microglial cells could be activated through both sensory and motor pathways. In order to further determine how peripheral nerve injury activates central microglial cells through neural pathways, the sciatic nerve was selected as the target for neural tract tracing and demyelination. Firstly, we used cholera toxin subunit B (CTB) to map the central sensory and motor territories of the sciatic nerve. Secondly, we applied lysophosphatidylcholine to establish the model of sciatic nerve demyelination and examined the distribution of activated microglial cells via immunofluorescence with ionized calcium-binding adapter molecule 1. It was shown that CTB labeling included the transganglionically labeled sensory afferents and retrogradely labeled somata of motor neurons along the sensory and motor pathways of the sciatic nerve ipsilateral to the injection, in which sensory afferents terminated on the gracile nucleus, Clarke\'s nucleus, and spinal dorsal horn, while motor neurons located on the spinal ventral horn. Consistently, after sciatic nerve demyelination, the activated microglial cells were observed in the same territories as CTB-labeling, showing shortened processes and enlarged cell bodies. These results support the idea that central microglia might be activated by signals from the demyelinated sciatic nerve through both sensory and motor pathways.

The widespread prevalence of Helicobacter pylori (H. pylori) infection remains a great challenge to human health. The existing vaccines are not ideal for preventing H. pylori infection; thus, exploring highly effective adjuvants may improve the immunoprotective efficacy of H. pylori vaccines. In a previous study, we found that the outer membrane vesicles (OMVs), a type of nanoscale particle spontaneously produced by Gram-negative bacteria, could act as adjuvants to boost the immune responses to vaccine antigens. In this study, we explored the potential application of OMVs as delivery vectors for adjuvant development. We constructed recombinant OMVs containing eukaryotic expression plasmid of cytokines, including interleukin 17A or interferon-γ, and evaluated their function as adjuvants in combination with inactivated whole-cell vaccine (WCV) or UreB as vaccine antigens. Our results showed that recombinant OMVs as adjuvants could induce stronger humoral and mucosal immune responses in mice than wild-type H. pylori OMVs and the cholera toxin (CT) adjuvant. Additionally, the recombinant OMVs significantly promoted Th1/Th2/Th17-type immune responses. Furthermore, the recombinant OMV adjuvant induced more potent clearance of H. pylori than CT and wild-type OMVs. Our findings suggest that the recombinant OMVs coupled with cytokines may become potent adjuvants for the development of novel and effective vaccines against H. pylori infection.

Cholera, a persistent global public health concern, continues to cause outbreaks in approximately 30 countries and territories this year. The imperative to safeguard water sources and food from Vibrio cholerae, the causative pathogen, remains urgent. The bacterium is mainly disseminated via ingestion of contaminated water or food. Despite the plate method\'s gold standard status for detection, its time-consuming nature, taking several days to provide results, remains a challenge. The emergence of novel virulence serotypes raises public health concerns, potentially compromising existing detection methods. Hence, exploiting Vibrio cholerae toxin testing holds promise due to its inherent stability. Immunobiosensors, leveraging antibody specificity and sensitivity, present formidable tools for detecting diverse small molecules, encompassing drugs, hormones, toxins, and environmental pollutants. This review explores cholera toxin detection, highlighting phage display-based nano immunosensors\' potential. Engineered bacteriophages exhibit exceptional cholera toxin affinity, through specific antibody fragments or mimotopes, enabling precise quantification. This innovative approach promises to reshape cholera toxin detection, offering an alternative to animal-derived methods. Harnessing engineered bacteriophages aligns with ethical detection and emphasizes sensitivity and accuracy, a pivotal stride in the evolution of detection strategies. This review primarily introduces recent advancements in phage display-based nano immunosensors for cholera toxin, encompassing technical aspects, current challenges, and future prospects.

Genetic screen technology has been applied to study the mechanism of action of bacterial toxins-a special class of virulence factors that contribute to the pathogenesis caused by bacterial infections. These screens aim to identify host factors that directly or indirectly facilitate toxin intoxication. Additionally, specific properties of certain toxins, such as membrane interaction, retrograde trafficking, and carbohydrate binding, provide robust probes to comprehensively investigate the lipid biosynthesis, membrane vesicle transport, and glycosylation pathways, respectively. This review specifically focuses on recent representative toxin-based genetic screens that have identified new players involved in and provided new insights into fundamental biological pathways, such as glycosphingolipid biosynthesis, protein glycosylation, and membrane vesicle trafficking pathways. Functionally characterizing these newly identified factors not only expands our current understanding of toxin biology but also enables a deeper comprehension of fundamental biological questions. Consequently, it stimulates the development of new therapeutic approaches targeting both bacterial infectious diseases and genetic disorders with defects in these factors and pathways.

Aeromonas veronii is a zoonotic agent capable of infecting fish and mammals, including humans, posing a serious threat to the development of aquaculture and public health safety. Currently, few effective vaccines are available through convenient routes against A. veronii infection. Herein, we developed vaccine candidates by inserting MSH type VI pili B (MshB) from A. veronii as an antigen and cholera toxin B subunit (CTB) as a molecular adjuvant into Lactobacillus casei and evaluated their immunological effect as vaccines in a crucian carp (Carassius auratus) model. The results suggested that recombinant L. casei Lc-pPG-MshB and Lc-pPG-MshB-CTB can be stably inherited for more than 50 generations. Oral administration of recombinant L. casei vaccine candidates stimulated the production of high levels of serum-specific immunoglobulin M (IgM) and increased the activity of acid phosphatase (ACP), alkaline phosphatase (AKP) superoxide dismutase (SOD), lysozyme (LZM), complement 3 (C3) and C4 in crucian carp compared to the control group (Lc-pPG612 group and PBS group) without significant changes. Moreover, the expression levels of interleukin-10 (IL-10), interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) genes in the gills, liver, spleen, kidney and gut of crucian carp orally immunized with recombinant L. casei were significantly upregulated compared to the control groups, indicating that recombinant L. casei induced a significant cellular immune response. In addition, viable recombinant L. casei can be detected and stably colonized in the intestine tract of crucian carp. Particularly, crucian carp immunized orally with Lc-pPG-MshB and Lc-pPG-MshB-CTB exhibited higher survival rates (48% for Lc-pPG-MshB and 60% for Lc-pPG-MshB-CTB) and significantly reduced loads of A. veronii in the major immune organs after A. veronii challenge. Our findings indicated that both recombinant L. casei strains provide favorable immune protection, with Lc-pPG-MshB-CTB in particular being more effective and promising as an ideal candidate for oral vaccination.

The aim of this work was to develop a simple and feasible method of mapping the neural network topology of the mouse brain. Wild-type C57BL/6 J mice (n = 10) aged 8-10 weeks were injected with the cholera toxin subunit B (CTB) tracer in the anterior (NAcCA) and posterior (NAcCP) parts of the nucleus accumbens (NAc) core and in the medial (NAcSM) and lateral (NAcSL) parts of the NAc shell. The labeled neurons were reconstructed using the WholeBrain Calculation Interactive Framework. The NAcCA receives neuronal projections from the olfactory areas (OLF) and isocortex; the thalamus and isocortex project more fibers to the NAcSL, and the hypothalamus send more fiber projections to the NAcSM. Cell resolution can be automatically annotated, analyzed, and visualized using the WholeBrain Calculation Interactive Framework, making large-scale mapping of mouse brains at cellular and subcellular resolutions easier and more accurate.

Diarrheal cases caused by non-toxigenic Vibrio cholerae have been reported globally. Lineages L3b and L9, characterized as ctxAB-negative and tcpA-positive (CNTP), pose the highest risk and have caused long-term epidemics in different regions worldwide. From 2001 to 2018, two waves (2001-2012 and 2013-2018) of epidemic caused by non-toxigenic V. cholerae occurred in the developed city of Hangzhou, China. In this study, through the integrated analysis of 207 genomes of Hangzhou isolates from these two waves (119 and 88) and 1573 publicly available genomes, we showed that L3b and L9 lineages together caused the second wave as had happened in the first wave, but the dominant lineage shifted from L3b (first wave: 69%) to L9 (second wave: 50%). We further found that the genotype of a key virulence gene, tcpF, in the L9 lineage during the second wave shifted to type I, which may have enhanced bacterial colonization in humans and potentially promoted the pathogenic lineage shift. Moreover, we found that 21% of L3b and L9 isolates had changed to predicted cholera toxin producers, providing evidence that gain of complete CTXφ-carrying ctxAB genes, rather than ctxAB gain in pre-CTXφ-carrying isolates, led to the transition. Taken together, our findings highlight the possible public health risk associated with L3b and L9 lineages due to their potential to cause long-term epidemics and turn into high-virulent cholera toxin producers, which necessitates a more comprehensive and unbiased sampling in further disease control efforts.