Inflammation and disintegration of intestinal villi in an experimental model for Vibrio parahaemolyticus-induced diarrhea.

Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis in many parts of the world, but there is limited knowledge of the pathogenesis of V. parahaemolyticus-induced diarrhea. The absence of an oral infection-based small animal model to study V. parahaemolyticus intestinal colonization and disease has constrained analyses of the course of infection and the factors that mediate it. Here, we demonstrate that infant rabbits oro-gastrically inoculated with V. parahaemolyticus develop severe diarrhea and enteritis, the main clinical and pathologic manifestations of disease in infected individuals. The pathogen principally colonizes the distal small intestine, and this colonization is dependent upon type III secretion system 2. The distal small intestine is also the major site of V. parahaemolyticus-induced tissue damage, reduced epithelial barrier function, and inflammation, suggesting that disease in this region of the gastrointestinal tract accounts for most of the diarrhea that accompanies V. parahaemolyticus infection. Infection appears to proceed through a characteristic sequence of steps that includes remarkable elongation of microvilli and the formation of V. parahaemolyticus-filled cavities within the epithelial surface, and culminates in villus disruption. Both depletion of epithelial cell cytoplasm and epithelial cell extrusion contribute to formation of the cavities in the epithelial surface. V. parahaemolyticus also induces proliferation of epithelial cells and recruitment of inflammatory cells, both of which occur before wide-spread damage to the epithelium is evident. Collectively, our findings suggest that V. parahaemolyticus damages the host intestine and elicits disease via previously undescribed processes and mechanisms.

A case study of a bispecific antibody manufacturability assessment and optimization during discovery stage and its implications.

The manufacturability assessment and optimization of bispecific antibodies (bsAbs) during the discovery stage are crucial for the success of the drug development process, impacting the speed and cost of advancing such therapeutics to the Investigational New Drug (IND) stage and ultimately to the market. The complexity of bsAbs creates challenges in employing effective evaluation methods to detect developability risks in early discovery stage, and poses difficulties in identifying the root causes and implementing subsequent engineering solutions. This study presents a case of engineering a bsAb that displayed a normal solution appearance during the discovery phase but underwent significant precipitation when subjected to agitation stress during 15 L Chemistry, Manufacturing, and Control (CMC) production Leveraging analytical tools, structural analysis, in silico prediction, and wet-lab validations, the key molecular origins responsible for the observed precipitation were identified and addressed. Sequence engineering to reduce protein surface hydrophobicity and enhance conformational stability proved effective in resolving agitation-induced aggregation. The refined bsAb sequences enabled successful mass production in CMC department. The findings of this case study contribute to the understanding of the fundamental mechanism of agitation-induced aggregation and offer a potential protein engineering procedure for addressing similar issues in bsAb. Furthermore, this case study emphasizes the significance of a close partnership between Discovery and CMC teams. Integrating CMC's rigorous evaluation methods with Discovery's engineering capability can facilitate a streamlined development process for bsAb molecules.

D3S-001, a KRAS G12C Inhibitor with Rapid Target Engagement Kinetics, Overcomes Nucleotide Cycling, and Demonstrates Robust Preclinical and Clinical Activities.

First-generation KRAS G12C inhibitors, such as sotorasib and adagrasib, are limited by the depth and duration of clinical responses. One potential explanation for their modest clinical activity is the dynamic "cycling" of KRAS between its guanosine diphosphate (GDP)- and guanosine triphosphate (GTP)-bound states, raising controversy about whether targeting the GDP-bound form can fully block this oncogenic driver. We herein report that D3S-001, a next-generation GDP-bound G12C inhibitor with faster target engagement (TE) kinetics, depletes cellular active KRAS G12C at nanomolar concentrations. In the presence of growth factors, such as epithelial growth factor and hepatocyte growth factor, the ability of sotorasib and adagrasib to inhibit KRAS was compromised whereas the TE kinetics of D3S-001 was nearly unaffected, a unique feature differentiating D3S-001 from other GDP-bound G12C inhibitors. Furthermore, the high covalent potency and cellular TE efficiency of D3S-001 contributed to robust antitumor activity preclinically and translated into promising clinical efficacy in an ongoing phase 1 trial (NCT05410145). Significance: The kinetic study presented in this work unveils, for the first time, that a GDP-bound conformation-selective KRAS G12C inhibitor can potentially deplete cellular active KRAS in the presence of growth factors and offers new insights into the critical features that drive preclinical and clinical efficacy for this class of drugs.

Reactogenicity of live-attenuated Vibrio cholerae vaccines is dependent on flagellins.

Cholera is a severe diarrheal disease caused by the motile Gram-negative rod Vibrio cholerae. Live-attenuated V. cholerae vaccines harboring deletions of the genes encoding cholera toxin have great promise for reducing the global burden of cholera. However, development of live vaccines has been hampered by the tendency of such strains to induce noncholeric reactogenic diarrhea in human subjects. The molecular bases of reactogenicity are unknown, but it has been speculated that reactogenic diarrhea is a response to V. cholerae's flagellum and/or the motility that it enables. Here, we used an infant rabbit model of reactogenicity to determine what V. cholerae factors trigger this response. We found that V. cholerae ctx mutants that produced flagellins induced diarrhea, regardless of whether the proteins were assembled into a flagellum or whether the flagellum was functional. In contrast, approximately 90% of rabbits infected with V. cholerae lacking all five flagellin-encoding genes did not develop diarrhea. Thus, flagellin production, independent of flagellum assembly or motility, is sufficient for reactogenicity. The intestinal colonization and intraintestinal localization of the nonreactogenic flagellin-deficient strain were indistinguishable from those of a flagellated motile strain; however, the flagellin-deficient strain stimulated fewer mRNA transcripts coding for proinflammatory cytokines in the intestine. Thus, reactogenic diarrhea may be a consequence of an innate host inflammatory response to V. cholerae flagellins. Our results suggest a simple genetic blueprint for engineering defined nonreactogenic live-attenuated V. cholerae vaccine strains.

Longitudinal high-dimensional analysis identifies immune features associating with response to anti-PD-1 immunotherapy.

Response to immunotherapy widely varies among cancer patients and identification of parameters associating with favourable outcome is of great interest. Here we show longitudinal monitoring of peripheral blood samples of non-small cell lung cancer (NSCLC) patients undergoing anti-PD1 therapy by high-dimensional cytometry by time of flight (CyTOF) and Meso Scale Discovery (MSD) multi-cytokines measurements. We find that higher proportions of circulating CD8+ and of CD8+CD101hiTIM3+ (CCT T) subsets significantly correlate with poor clinical response to immune therapy. Consistently, CD8+ T cells and CCT T cell frequencies remain low in most responders during the entire multi-cycle treatment regimen; and higher killer cell lectin-like receptor subfamily G, member 1 (KLRG1) expression in CCT T cells at baseline associates with prolonged progression free survival. Upon in vitro stimulation, CCT T cells of responders produce significantly higher levels of cytokines, including IL-1ß, IL-2, IL-8, IL-22 and MCP-1, than of non-responders. Overall, our results provide insights into the longitudinal immunological landscape underpinning favourable response to immune checkpoint blockade therapy in lung cancer patients.

Discovery of JNJ-64264681: A Potent and Selective Covalent Inhibitor of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK) is a Tec family kinase that plays an essential role in B-cell receptor (BCR) signaling as well as Fcγ receptor signaling in leukocytes. Pharmacological inhibition of BTK has been shown to be effective in treating hematological malignancies and is hypothesized to provide an effective strategy for the treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. We report the discovery and preclinical properties of JNJ-64264681 (13), a covalent, irreversible BTK inhibitor with potent whole blood activity and exceptional kinome selectivity. JNJ-64264681 demonstrated excellent oral efficacy in both cancer and autoimmune models with sustained in vivo target coverage amenable to once daily dosing and has advanced into human clinical studies to investigate safety and pharmacokinetics.

Identification of a luxO-regulated extracellular protein Pep and its roles in motility in Vibrio alginolyticus.

The fish pathogen Vibrio alginolyticus virulence factors and regulation mechanism are barely known except the quorum sensing regulated extracellular products. In the present study, the cleavable isotope-coded affinity tags (cICATs) were used to analyze the differentially expressed extracellular proteins of V. alginolyticus wild-type and a ΔluxO mutant, and a putative peptidase Pep was identified. Quantitative real-time reverse transcription PCR confirmed that the transcription of pep was down-regulated by LuxO. Furthermore, western blotting demonstrated that the expression of Pep was cell-density dependent and quorum sensing regulated in V. alginolyticus. The pep in-frame mutant abolished the swimming and swarming motility, suggesting that it was involved in motility regulation in V. alginolyticus. In liquid medium, the polar flagellum was observed intact in ∆pep mutant while the chemotaxis related gene cheY, which is a response regulator causing change in direction of flagellar rotation, was down-regulated in ∆pep mutant. When ∆pep mutant was grown on hard agar plate, the lateral flagella disappeared and the transcriptions of flagellar biogenesis genes flaK, fliS, lafK, and lafA were down-regulated in ∆pep mutant. These results suggested that Pep was a luxO-regulated extracellular protein and involved in the motility of V. alginolyticus.

Role of alkaline serine protease, asp, in vibrio alginolyticus virulence and regulation of its expression by luxO-luxR regulatory system.

The alkaline serine protease asp, which was shown to be a virulence factor of Vibrio alginolyticus as a purified protein, was cloned from V. alginolyticus EPGS, a strain recently isolated from moribund Epinephelus coioides in an outbreak of vibriosis in a mariculture farm of Shenzhen. The asp null mutant was constructed by homologous recombination with suicide plasmid pNQ705-1. Compared with the wild-type strain, the asp null mutant exhibited a significant decrease of total extracellular protease activity, and caused a 15-fold decrease in virulence of V. alginolyticus. In our previous study, the luxO and luxR(val) genes from V. alginolyticus MVP01 were cloned and identified, and the luxO-luxR(val) regulatory couple was shown to regulate various genes expression, suggesting that it played a central role in the quorum sensing system of V. alginolyticus. In this study, the regulation of the asp gene was analyzed by using RT-PCR and quantitative real-time PCR methods; we proved that its transcription was greatly induced at the late large stage of growth and was regulated by luxO-luxR(val) regulatory system.

Roles of LuxR in regulating extracellular alkaline serine protease A, extracellular polysaccharide and mobility of Vibrio alginolyticus.

In marine Vibrio species, the Vibrio harveyi-type LuxR protein, a key player in a quorum-sensing system, controls the expression of various genes. In this study, the luxR homologue in Vibrio alginolyticus was identified and named luxR(val), whose expression was greatly induced by the increase of cell number. The luxR(val) in-frame deletion mutant showed a significant downregulation of total extracellular protease activity, and especially caused a 70% decrease in the transcript levels of extracellular alkaline serine protease A (proA), which was an important virulent factor of V. alginolyticus. Complementation in trans with luxR(val) could restore the expression of proA to the level of the wild-type strain. Deletion of the luxR(val) gene also resulted in changes of colony morphology, extracellular polysaccharide production and mobility. Therefore, another member of the V. harveyi-type LuxR regulator family has been characterized in V. alginolyticus.

Back to the future: studying cholera pathogenesis using infant rabbits.

Cholera is a severe diarrheal disease, caused by Vibrio cholerae, for which there has been no reproducible, nonsurgical animal model. Here, we report that orogastric inoculation of V. cholerae into 3-day-old rabbits pretreated with cimetidine led to lethal, watery diarrhea in virtually all rabbits. The appearance and chemical composition of the rabbit diarrheal fluid were comparable to those of the "rice-water stool" produced by cholera patients. As in humans, V. cholerae mutants that do not produce cholera toxin (CT) and toxin-coregulated pilus (TCP) did not induce cholera-like disease in rabbits. CT induced extensive exocytosis of mucin from intestinal goblet cells, and wild-type V. cholerae was predominantly found in close association with mucin. Large aggregates of mucin-embedded V. cholerae were observed, both attached to the epithelium and floating within the diarrheal fluid. These findings suggest that CT-dependent mucin secretion significantly influences V. cholerae's association with the host intestine and its exit from the intestinal tract. Our model should facilitate identification and analyses of factors that may govern V. cholerae infection, survival, and transmission, such as mucin. In addition, our results using nontoxigenic V. cholerae suggest that infant rabbits will be useful for study of the reactogenicity of live attenuated-V. cholerae vaccines.