A microarray patch SARS-CoV-2 vaccine induces sustained antibody responses and polyfunctional cellular immunity.

Sustainable global immunization campaigns against COVID-19 and other emerging infectious diseases require effective, broadly deployable vaccines. Here, we report a dissolvable microarray patch (MAP) SARS-CoV-2 vaccine that targets the immunoresponsive skin microenvironment, enabling efficacious needle-free immunization. Multicomponent MAPs delivering both SARS-CoV-2 S1 subunit antigen and the TLR3 agonist Poly(I:C) induce robust antibody and cellular immune responses systemically and in the respiratory mucosa. MAP vaccine-induced antibodies bind S1 and the SARS-CoV-2 receptor-binding domain, efficiently neutralize the virus, and persist at high levels for more than a year. The MAP platform reduces systemic toxicity of the delivered adjuvant and maintains vaccine stability without refrigeration. When applied to human skin, MAP vaccines activate skin-derived migratory antigen-presenting cells, supporting the feasibility of human translation. Ultimately, this shelf-stable MAP vaccine improves immunogenicity and safety compared to traditional intramuscular vaccines and offers an attractive alternative for global immunization efforts against a range of infectious pathogens.

Tick extracellular vesicles enable arthropod feeding and promote distinct outcomes of bacterial infection.

Extracellular vesicles are thought to facilitate pathogen transmission from arthropods to humans and other animals. Here, we reveal that pathogen spreading from arthropods to the mammalian host is multifaceted. Extracellular vesicles from Ixodes scapularis enable tick feeding and promote infection of the mildly virulent rickettsial agent Anaplasma phagocytophilum through the SNARE proteins Vamp33 and Synaptobrevin 2 and dendritic epidermal T cells. However, extracellular vesicles from the tick Dermacentor andersoni mitigate microbial spreading caused by the lethal pathogen Francisella tularensis. Collectively, we establish that tick extracellular vesicles foster distinct outcomes of bacterial infection and assist in vector feeding by acting on skin immunity. Thus, the biology of arthropods should be taken into consideration when developing strategies to control vector-borne diseases.

Entamoeba histolytica Cyclooxygenase-Like Protein Regulates Cysteine Protease Expression and Virulence.

The intestinal protozoan parasite Entamoeba histolytica (Eh) causes amebiasis associated with severe diarrhea and/or liver abscess. Eh pathogenesis is multifactorial requiring both parasite virulent molecules and host-induced innate immune responses. Eh-induced host pro-inflammatory responses plays a critical role in disease pathogenesis by causing damage to tissues allowing parasites access to systemic sites. Eh cyclooxygenase (EhCox) derived prostaglandin E2 stimulates the chemokine IL-8 from mucosal epithelial cells that recruits neutrophils to the site of infection to exacerbate disease. At present, it is not known how EhCox is regulated or whether it affects the expression of other proteins in Eh. In this study, we found that gene silencing of EhCox (EhCoxgs) markedly increased endogenous cysteine protease (CP) protein expression and virulence without altering CP gene transcripts. Live virulent Eh pretreated with arachidonic acid substrate to enhance PGE2 production or aspirin to inhibit EhCox enzyme activity or addition of exogenous PGE2 to Eh had no effect on EhCP activity. Increased CP enzyme activity in EhCoxgs was stable and significantly enhanced erythrophagocytosis, cytopathic effects on colonic epithelial cells and elicited pro-inflammatory cytokines in mice colonic loops. Acute infection with EhCoxgs in colonic loops increased inflammation associated with high levels of myeloperoxidase activity. This study has identified EhCox protein as one of the important endogenous regulators of cysteine protease activity. Alterations of CP activity in response to Cox gene silencing may be a negative feedback mechanism in Eh to limit proteolytic activity during colonization that can inadvertently trigger inflammation in the gut.

Identification of S-Nitrosylated (SNO) Proteins in Entamoeba histolytica Adapted to Nitrosative Stress: Insights into the Role of SNO Actin and In vitro Virulence.

We have recently reported that Entamoeba histolytica trophozoites can adapt to toxic levels of the nitric oxide (NO) donor, S-nitrosoglutathione (GSNO). Even if the consequences of this adaptation on the modulation of gene expression in NO-adapted trophozoites (NAT) have been previously explored, insight on S-nitrosylated (SNO) proteins in NAT is missing. Our study aims to fill this knowledge gap by performing a screening of SNO proteins in NAT. Employing SNO resin-assisted capture (RAC), we identified 242 putative SNO proteins with key functions in calcium binding, enzyme modulation, redox homeostasis, and actin cytoskeleton. Of the SNO proteins in NAT, proteins that are associated with actin family cytoskeleton protein are significantly enriched. Here we report that the formation of actin filaments (F-actin) is impaired in NAT. Consequently, the ability of NAT to ingest erythrocytes and their motility and their cytopathic activity are impaired. These phenotypes can be imitated by treating control parasite with cytochalasin D (CytD), a drug that binds to F-actin polymer and prevent polymerization of actin monomers. Removal of GSNO from the culture medium of NAT restored the sensitivity of the parasite to nitrosative stress (NS) and its ability to form F-actin formation and its virulence. These results establish the central role of NO in shaping the virulence of the parasite through its effect on F-actin formation and highlight the impressive ability of this parasite to adapt to NS.

N-acetyl ornithine deacetylase is a moonlighting protein and is involved in the adaptation of Entamoeba histolytica to nitrosative stress.

Adaptation of the Entamoeba histolytica parasite to toxic levels of nitric oxide (NO) that are produced by phagocytes may be essential for the establishment of chronic amebiasis and the parasite's survival in its host. In order to obtain insight into the mechanism of E. histolytica's adaptation to NO, E. histolytica trophozoites were progressively adapted to increasing concentrations of the NO donor drug, S-nitrosoglutathione (GSNO) up to a concentration of 110 µM. The transcriptome of NO adapted trophozoites (NAT) was investigated by RNA sequencing (RNA-seq). N-acetyl ornithine deacetylase (NAOD) was among the 208 genes that were upregulated in NAT. NAOD catalyzes the deacetylation of N-acetyl-L-ornithine to yield ornithine and acetate. Here, we report that NAOD contributes to the better adaptation of the parasite to nitrosative stress (NS) and that this function does not depend on NAOD catalytic activity. We also demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is detrimental to E. histolytica exposed to NS and that this detrimental effect is neutralized by NAOD or by a catalytically inactive NAOD (mNAOD). These results establish NAOD as a moonlighting protein, and highlight the unexpected role of this metabolic enzyme in the adaptation of the parasite to NS.

Proteomic Identification of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to Oxidative Stress.

Entamoeba histolytica is an obligate protozoan parasite of humans, and amebiasis, an infectious disease which targets the intestine and/or liver, is the second most common cause of human death due to a protozoan after malaria. Although amebiasis is usually asymptomatic, E. histolytica has potent pathogenic potential. During host infection, the parasite is exposed to reactive oxygen species that are produced and released by cells of the innate immune system at the site of infection. The ability of the parasite to survive oxidative stress (OS) is essential for a successful invasion of the host. Although the effects of OS on the regulation of gene expression in E. histolytica and the characterization of some proteins whose function in the parasite's defense against OS have been previously studied, our knowledge of oxidized proteins in E. histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the oxidized proteins in oxidatively stressed E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry. We detected 154 oxidized proteins (OXs) and the functions of some of these proteins were associated with antioxidant activity, maintaining the parasite's cytoskeleton, translation, catalysis, and transport. We also found that oxidation of the Gal/GalNAc impairs its function and contributes to the inhibition of E. histolytica adherence to host cells. We also provide evidence that arginase, an enzyme which converts L-arginine into L-ornithine and urea, is involved in the protection of the parasite against OS. Collectively, these results emphasize the importance of OS as a critical regulator of E. histolytica's functions and indicate a new role for arginase in E. histolytica's resistance to OS.

Proteomic identification of S-nitrosylated proteins in the parasite Entamoeba histolytica by resin-assisted capture: insights into the regulation of the Gal/GalNAc lectin by nitric oxide.

Entamoeba histolytica is a gastrointestinal protozoan parasite that causes amebiasis, a disease which has a worldwide distribution with substantial morbidity and mortality. Nitrosative stress, which is generated by innate immune cells, is one of the various environmental challenges that E. histolytica encounters during its life cycle. Although the effects of nitric oxide (NO) on the regulation of gene expression in this parasite have been previously investigated, our knowledge on S-nitrosylated proteins in E.histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale detection of S-nitrosylated (SNO) proteins in E.histolytica trophozoites that were treated with the NO donor, S-nitrosocysteine by resin-assisted capture (RAC). We found that proteins involved in glycolysis, gluconeogenesis, translation, protein transport, and adherence to target cells such as the heavy subunit of Gal/GalNac lectin are among the S-nitrosylated proteins that were enriched by SNO-RAC. We also found that the S-nitrosylated cysteine residues in the carbohydrate recognition domain (CRD) of Gal/GalNAc lectin impairs its function and contributes to the inhibition of E.histolytica adherence to host cells. Collectively, these results advance our understanding of the mechanism of reduced E.histolytica adherence to mammalian cells by NO and emphasize the importance of NO as a regulator of key physiological functions in E.histolytica.

Growth and development of Aedes aegypti larvae at limiting food concentrations.

Mosquitoes have a complex life-cycle with dramatic changes in shape, function, and habitat. Aedes aegypti was studied by growing individual larvae at different concentrations of a defined rich food source. At higher food concentrations, rate of larval growth was faster, but the time required for 4th instar larvae to molt into the pupal stage was unexpectedly extended. These opposite tendencies resulted in constant times from hatching to pupation and up to adult eclosion at permissive food concentrations. The results demonstrate that nutritional conditions of 4th instar larvae impact initiation of the first metamorphic molt.

The basis for rootstock resilient to Capnodis species: screening for genes encoding δ-endotoxins from Bacillus thuringiensis.

BACKGROUND: Conventional methods often fail to control the flatheaded borers Capnodis spp., major pests of stone fruit trees; the larvae are protected from insecticides and predation because they feed deep in the roots. A potential solution is transgenic trees producing in their roots toxic compounds such as Cry proteins of Bacillus thuringiensis (Bt). RESULTS: Toxicities against Capnodis larvae were demonstrated by exploiting a recently designed artificial larval diet and an available collection of field isolated Bt. An isolate of Bt tenebrionis (Btt) from commercial bioinsecticide (Novodor) displayed LC50 and LC95 values of 3.2 and 164 mg g(-1) , respectively, against neonates of Capnodis tenebrionis, whereas values of the most toxic field isolate K-7 were 1.9 and 25.6 mg g(-1) respectively. Weights of surviving larvae after 1 month on diets containing low concentrations of K-7 (0.1-1.0 mg g(-1) ) were lower than on Btt or untreated larvae. K-7 was also toxic against larvae of C. cariosa and C. miliaris and found to harbour genes encoding Cry9Ea-like and Cry23Aa/Cry37Aa binary toxins. CONCLUSION: Larvae of Capnodis spp. are susceptible to Bt Cry toxins. Expressing cry genes active against these pests thus seems a feasible solution towards production of transgenic rootstock trees resilient to the pest.