Development of an engineered extracellular vesicles-based vaccine platform for combined delivery of mRNA and protein to induce functional immunity.

mRNA incorporated in lipid nanoparticles (LNPs) became a new class of vaccine modality for induction of immunity against COVID-19 and ushered in a new era in vaccine development. Here, we report a novel, easy-to-execute, and cost effective engineered extracellular vesicles (EVs)-based combined mRNA and protein vaccine platform (EV vaccine) and explore its utility in proof-of-concept immunity studies in the settings of cancer and infectious disease. As a first example, we engineered EVs, natural nanoparticle carriers shed by all cells, to contain ovalbumin mRNA and protein (EV vaccine) to serve as cancer vaccine against ovalbumin-expressing melanoma tumors.

KRAS inhibition reprograms the microenvironment of early and advanced pancreatic cancer to promote FAS-mediated killing by CD8 T cells.

The KRAS mutation is present in nearly half of pancreatic adenocarcinomas (PDAC). We investigated the effects of inhibiting the KRAS mutant protein with MRTX1133, a non-covalent small molecule inhibitor of KRAS, on early and advanced PDAC and its influence on the tumor microenvironment. Employing 16 different models of KRAS-driven PDAC, we demonstrate that MRTX1133 reverses early PDAC growth, increases intratumoral CD8 effector T cells, decreases myeloid infiltration, and reprograms cancer-associated fibroblasts.

Engineered exosomes targeting MYC reverse the proneural-mesenchymal transition and extend survival of glioblastoma.

Dysregulated Myc signaling is a key oncogenic pathway in glioblastoma multiforme (GBM). Yet, effective therapeutic targeting of Myc continues to be challenging. Here, we demonstrate that exosomes generated from human bone marrow mesenchymal stem cells (MSCs) engineered to encapsulate siRNAs targeting Myc (iExo-Myc) localize to orthotopic GBM tumors in mice.

Oncogenic Kras specific non-covalent inhibitor reprograms tumor microenvironment to prevent and reverse early pre-neoplastic pancreatic lesions and in combination with immunotherapy regresses advanced PDAC in a CD8 T cells dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is associated with mutations in Kras, a known oncogenic driver of PDAC; and the mutation is present in nearly half of PDAC patients. Recently, a non-covalent small molecule inhibitor (MRTX1133) was identified with specificity to the Kras mutant protein. Here we explore the impact of Kras inhibition by MRTX1133 on advanced PDAC and its influence on the tumor microenvironment.

Identification of Functional Heterogeneity of Carcinoma-Associated Fibroblasts with Distinct IL6-Mediated Therapy Resistance in Pancreatic Cancer.

Unlabelled: The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) involves a significant accumulation of fibroblasts as part of the host response to cancer. Using single-cell RNA sequencing, multiplex immunostaining, and several genetic mouse models, we identify carcinoma-associated fibroblasts (CAF) with opposing functions in PDAC progression. Depletion of fibroblast activation protein (FAP)+ CAFs results in increased survival, in contrast to depletion of alpha smooth muscle actin (αSMA)+ CAFs, which leads to decreased survival.

αSMA fibroblasts suppress Lgr5 cancer stem cells and restrain colorectal cancer progression.

The development and progression of solid tumors is dependent on cancer cell autonomous drivers and the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) in the TME possess both tumor-promoting and tumor-restraining functions. In the current study, we interrogated the role of αSMA CAFs in a genetic mouse model of metastatic colorectal cancer (CRC).

Dissemination of non-typhoidal Salmonella during Plasmodium chabaudi infection affects anti-malarial immunity.

Malaria-associated bacteremia accounts for up to one-third of deaths from severe malaria, and non-typhoidal Salmonella (NTS) has been reported as a major complication of severe malarial infection. Patients who develop NTS bacteremia during Plasmodium infection show higher mortality rates than individuals with malaria alone. Systemic bacteremia can be caused by a wound or translocation from epithelial or endothelial sites.

Combating Multidrug-Resistant Pathogens with Host-Directed Nonantibiotic Therapeutics.

Earlier, we reported that three Food and Drug Administration-approved drugs, trifluoperazine (TFP; an antipsychotic), amoxapine (AXPN; an antidepressant), and doxapram (DXP; a breathing stimulant), identified from an murine macrophage cytotoxicity screen, provided mice with 40 to 60% protection against pneumonic plague when administered at the time of infection for 1 to 3 days. In the present study, the therapeutic potential of these drugs against pneumonic plague in mice was further evaluated when they were administered at up to 48 h postinfection. While the efficacy of TFP was somewhat diminished as treatment was delayed to 24 h, the protection of mice with AXPN and DXP increased as treatment was progressively delayed to 24 h.

Identification of New Virulence Factors and Vaccine Candidates for .

Earlier, we reported the identification of new virulence factors/mechanisms of using an signature-tagged mutagenesis (STM) screening approach. From this screen, the role of , which encodes an ATP-binding protein of ribose transport system, and , an essential component of the type VI secretion system (T6SS), were evaluated in mouse models of plague and confirmed to be important during infection. However, many of the identified genes from the screen remained uncharacterized.

A Bivalent Anthrax-Plague Vaccine That Can Protect against Two Tier-1 Bioterror Pathogens, and .

Bioterrorism remains as one of the biggest challenges to global security and public health. Since the deadly anthrax attacks of 2001 in the United States, and , the causative agents of anthrax and plague, respectively, gained notoriety and were listed by the CDC as Tier-1 biothreat agents. Currently, there is no Food and Drug Administration-approved vaccine against either of these threats for mass vaccination to protect general public, let alone a bivalent vaccine.

Phospholipase A2-activating protein is associated with a novel form of leukoencephalopathy.

Leukoencephalopathies are a group of white matter disorders related to abnormal formation, maintenance, and turnover of myelin in the central nervous system. These disorders of the brain are categorized according to neuroradiological and pathophysiological criteria. Herein, we have identified a unique form of leukoencephalopathy in seven patients presenting at ages 2 to 4 months with progressive microcephaly, spastic quadriparesis, and global developmental delay.

New Insights into Autoinducer-2 Signaling as a Virulence Regulator in a Mouse Model of Pneumonic Plague.

The family members, including the infamous , the causative agent of plague, have a highly conserved interbacterial signaling system that is mediated by the autoinducer-2 (AI-2) quorum-sensing molecule. The AI-2 system is implicated in regulating various bacterial virulence genes in diverse environmental niches. Deletion of the gene encoding the synthetic enzyme for the AI-2 substrate, , leads to either no significant change or, paradoxically, an increase in bacterial virulence.

Protective Immunity Elicited by Oral Immunization of Mice with Serovar Typhimurium Braun Lipoprotein (Lpp) and Acetyltransferase (MsbB) Mutants.

We evaluated the extent of attenuation and immunogenicity of the Δ and Δ Δ mutants of serovar Typhimurium when delivered to mice by the oral route. These mutants were deleted either for the Braun lipoprotein genes ( and ) or in combination with the gene, which encodes an acetyltransferase required for lipid A modification of lipopolysaccharide. Both the mutants were attenuated (100% animal survival) and triggered robust innate and adaptive immune responses.

Immunisation of two rodent species with new live-attenuated mutants of CO92 induces protective long-term humoral- and cell-mediated immunity against pneumonic plague.

We showed recently that the live-attenuated Δ Δ Δ and Δ Δ mutants of CO92 provided short-term protection to mice against developing subsequent lethal pneumonic plague. These mutants were either deleted for genes encoding Braun lipoprotein (Lpp), an acetyltransferase (MsbB) and the attachment invasion locus (Ail) (Δ Δ Δ) or contained a modified version of the gene with diminished virulence (Δ Δ). Here, long-term immune responses were first examined after intramuscular immunisation of mice with the above-mentioned mutants, as well as the newly constructed Δ Δ Δ mutant, deleted for the plasminogen-activator protease () gene instead of .

A Replication-Defective Human Type 5 Adenovirus-Based Trivalent Vaccine Confers Complete Protection against Plague in Mice and Nonhuman Primates.

Currently, no plague vaccine exists in the United States for human use. The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS) components, the low-calcium-response V antigen (LcrV) and the needle protein YscF, represent protective antigens of Yersinia pestis We used a replication-defective human type 5 adenovirus (Ad5) vector and constructed recombinant monovalent and trivalent vaccines (rAd5-LcrV and rAd5-YFV) that expressed either the codon-optimized lcrV or the fusion gene designated YFV (consisting of ycsF, caf1, and lcrV). Immunization of mice with the trivalent rAd5-YFV vaccine by either the intramuscular (i.

New Role for FDA-Approved Drugs in Combating Antibiotic-Resistant Bacteria.

Antibiotic resistance in medically relevant bacterial pathogens, coupled with a paucity of novel antimicrobial discoveries, represents a pressing global crisis. Traditional drug discovery is an inefficient and costly process; however, systematic screening of Food and Drug Administration (FDA)-approved therapeutics for other indications in humans offers a rapid alternative approach. In this study, we screened a library of 780 FDA-approved drugs to identify molecules that rendered RAW 264.

Cross-talk among flesh-eating Aeromonas hydrophila strains in mixed infection leading to necrotizing fasciitis.

Necrotizing fasciitis (NF) caused by flesh-eating bacteria is associated with high case fatality. In an earlier study, we reported infection of an immunocompetent individual with multiple strains of Aeromonas hydrophila (NF1-NF4), the latter three constituted a clonal group whereas NF1 was phylogenetically distinct. To understand the complex interactions of these strains in NF pathophysiology, a mouse model was used, whereby either single or mixed A.

High-throughput, signature-tagged mutagenic approach to identify novel virulence factors of Yersinia pestis CO92 in a mouse model of infection.

The identification of new virulence factors in Yersinia pestis and understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a high-throughput, signature-tagged mutagenic approach, we created 5,088 mutants of Y. pestis strain CO92 and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 50% lethal doses (LD50) of wild-type (WT) CO92.

Combinational deletion of three membrane protein-encoding genes highly attenuates yersinia pestis while retaining immunogenicity in a mouse model of pneumonic plague.

Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants.

Functional genomic characterization of virulence factors from necrotizing fasciitis-causing strains of Aeromonas hydrophila.

The genomes of 10 Aeromonas isolates identified and designated Aeromonas hydrophila WI, Riv3, and NF1 to NF4; A. dhakensis SSU; A. jandaei Riv2; and A.

Deletion of Braun lipoprotein and plasminogen-activating protease-encoding genes attenuates Yersinia pestis in mouse models of bubonic and pneumonic plague.

Currently, there is no FDA-approved vaccine against Yersinia pestis, the causative agent of bubonic and pneumonic plague. Since both humoral immunity and cell-mediated immunity are essential in providing the host with protection against plague, we developed a live-attenuated vaccine strain by deleting the Braun lipoprotein (lpp) and plasminogen-activating protease (pla) genes from Y. pestis CO92.

The effects of modeled microgravity on growth kinetics, antibiotic susceptibility, cold growth, and the virulence potential of a Yersinia pestis ymoA-deficient mutant and its isogenic parental strain.

Previously, we reported that there was no enhancement in the virulence potential (as measured by cell culture infections) of the bacterial pathogen Yersinia pestis (YP) following modeled microgravity/clinorotation growth. We have now further characterized the effects of clinorotation (CR) on YP growth kinetics, antibiotic sensitivity, cold growth, and YP's virulence potential in a murine model of infection. Surprisingly, none of the aforementioned phenotypes were altered.

Mutated and bacteriophage T4 nanoparticle arrayed F1-V immunogens from Yersinia pestis as next generation plague vaccines.

Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy.

Characterization of Aeromonas hydrophila wound pathotypes by comparative genomic and functional analyses of virulence genes.

Unlabelled: Aeromonas hydrophila has increasingly been implicated as a virulent and antibiotic-resistant etiologic agent in various human diseases. In a previously published case report, we described a subject with a polymicrobial wound infection that included a persistent and aggressive strain of A. hydrophila (E1), as well as a more antibiotic-resistant strain of A.

Evaluation of the roles played by Hcp and VgrG type 6 secretion system effectors in Aeromonas hydrophila SSU pathogenesis.

Aeromonas hydrophila, a Gram-negative bacterium, is an emerging human pathogen equipped with both a type 3 and a type 6 secretion system (T6SS). In this study, we evaluated the roles played by paralogous T6SS effector proteins, hemolysin co-regulated proteins (Hcp-1 and -2) and valine glycine repeat G (VgrG-1, -2 and -3) protein family members in A. hydrophila SSU pathogenesis by generating various combinations of deletion mutants of the their genes.