Evaluation of the safety and adjuvant effect of a detoxified listeriolysin O mutant on the humoral response to dengue virus antigens.

Listeriolysin O (LLO) has been proposed as a potential carrier or adjuvant molecule in the vaccination field. However, the cytotoxic and pro-apoptotic effects of LLO are the major limitations for this purpose. Here, we have performed a preclinical safety evaluation and characterized a new potential adjuvant application for a non-cytolytic LLO mutant (dtLLO) to enhance and modulate the immune response against the envelope (E) protein from dengue virus. In addition, we have studied the adjuvant effects of dtLLO on human immune cells and the role of membrane cholesterol for the binding and proinflammatory property of the toxoid. Our in-vivo results in the murine model confirmed that dtLLO is a safer molecule than wild-type LLO (wtLLO), with a significantly increased survival rate for mice challenged with dtLLO compared with mice challenged with wtLLO (P < 0·001). Histopathological analysis showed non-toxic effects in key target organs such as brain, heart, liver, spleen, kidney and lung after challenge with dtLLO. In vitro, dtLLO retained the capacity of binding to plasma membrane cholesterol on the surface of murine and human immune cells. Immunization of 6-8-week-old female BALB/c mice with a combination of dtLLO mixed with E protein elicited a robust specific humoral response with isotype diversification of immunoglobulin (Ig)G antibodies (IgG1 and IgG2a). Finally, we demonstrated that cholesterol and lipid raft integrity are required to induce a proinflammatory response by human cells. Taken together, these findings support a potential use of the dtLLO mutant as a safe and effective adjuvant molecule in vaccination.

Comparative analysis of zygotic developmental genes in Rhodnius prolixus genome shows conserved features on the tracheal developmental pathway.

Most of the in-depth studies on insect developmental genetic have been carried out in the fruit fly Drosophila melanogaster, an holometabolous insect, so much more still remains to be studied in hemimetabolous insects. Having Rhodnius prolixus sequenced genome available, we search for orthologue genes of zygotic signaling pathways, segmentation, and tracheogenesis in the R. prolixus genome and in three species of Triatoma genus transcriptomes, concluding that there is a high level of gene conservation. We also study the function of two genes required for tracheal system development in D. melanogaster - R. prolixus orthologues: trachealess (Rp-trh) and empty spiracles (Rp-ems). From that we see that Rp-trh is required for early tracheal development since Rp-trh RNAi shows that the primary tracheal branches fail to form. On the other hand, Rp-ems is implied in the proper formation of the posterior tracheal branches, in a similar way to D. melanogaster. These results represent the initial characterization of the genes involved in the tracheal development of an hemimetabolous insect building a bridge between the current genomic era and V. Wigglesworth's classical studies on insects' respiratory system physiology.

Conserved peptide sequences bind to actin and enolase on the surface of Plasmodium berghei ookinetes.

The description of Plasmodium ookinete surface proteins and their participation in the complex process of mosquito midgut invasion is still incomplete. In this study, using phage display, a consensus peptide sequence (PWWP) was identified in phages that bound to the Plasmodium berghei ookinete surface and, in selected phages, bound to actin and enolase in overlay assays with ookinete protein extracts. Actin was localized on the surface of fresh live ookinetes by immunofluorescence and electron microscopy using specific antibodies. The overall results indicated that enolase and actin can be located on the surface of ookinetes, and suggest that they could participate in Plasmodium invasion of the mosquito midgut.

Prostaglandin E2 modulates the expression of antimicrobial peptides in the fat body and midgut of Anopheles albimanus.

Prostaglandins (PGs) participate in the regulation of vertebrate and in at least six insect orders' immune responses. We identified PGE2 in midgut, fat body, Malpighian tubules, and ovarioles of Anopheles albimanus (Aa) mosquitoes. Our data indicate that PGE2 synthesis in cultured midguts responds to the presence of two bacterial species, Micrococcus luteus and Klebsiella pneumoniae. The production of mRNA coding for antimicrobial peptides Aa-Attacin, Aa-Cecropin, and Aa-Gambicin was observed in cultured fat bodies and midguts. The production of these messengers was reduced in the presence of dexamethasone, and this effect was reversed by arachidonic acid. Adding PGE2 to cultures resulted in increased Aa-cecropin mRNA and decreased Aa-attacin and Aa-gambicin mRNAs.

GP35 ANOAL, an abundant acidic glycoprotein of female Anopheles albimanus saliva.

Salivary glands of female mosquitoes produce proteins, not completely described yet, that participate in carbohydrate and blood feeding. Here, we report an acidic glycoprotein of 35 kDa (GP35 ANOAL) secreted in the saliva of the malaria vector mosquito Anopheles albimanus. GP35 ANOAL is produced exclusively in the distal lateral lobes of adult female salivary glands, it has a pI of 4.45 and is negatively stained by regular silver stain. An 888 bp cDNA clone encoding a predicted product of 240 amino acids has a signal peptide, potential post-translational modification sites, and a disintegrin signature RGD. The GP35 ANOAL sequence depicts high similarities with the 30 kDa saliva allergen of Aedes aegypti, 30 kDa allergen-like hypothetical proteins, and GE-rich proteins present in several Anopheles species, as well as in Ae. albopictus and Culex pipiens quinquefasciatus. The function of this protein family is still unknown.

Induction of DNA synthesis in Anopheles albimanus tissue cultures in response to a Saccharomyces cerevisiae challenge.

DNA synthesis was detected by the incorporation of 5-bromo-2' deoxy-uridine (BrdU) in adult Anopheles albimanus organs in culture in response to a challenge with Saccharomyces cerevisiae. Abdomens of mosquitoes inoculated with Roswell Park Memorial Institute medium (RPMI, control) or yeast were cultivated in RPMI plus ConA and BrdU for 5 days. DNA was obtained by phenolic extraction and the incorporated BrdU was quantified by ELISA using anti-BrdU peroxidase-labeled antibodies. Abdomen tissues of mosquitoes inoculated with yeast showed higher DNA synthesis than controls. Organs from untreated mosquitoes cultured in the presence of zymosan also synthesized DNA but at a lower level than tissues from yeast-inoculated mosquitoes. In similar experiments, DNA synthesis was inhibited by the addition of colchicine. DNA synthesis, evidenced by epifluorescence using an anti-BrdU fluorescein-labeled antibody, occurred in fat body, epithelial cells in pleural membranes, and the dorsal vessel. Pleural membranes showed the highest number of labeled cells. These tissues were also labeled with anti-PCNA (proliferating cell nuclear antigen) antibodies, two of which were able to produce polytene chromosomes under yeast stimulation. These results demonstrate that different An. albimanus tissues undergo DNA synthesis in response to foreign particles.