Molecular engineering and plant expression of an immunoglobulin heavy chain scaffold for delivery of a dengue vaccine candidate.

In order to enhance vaccine uptake by the immune cells in vivo, molecular engineering approach was employed to construct a polymeric immunoglobulin G scaffold (PIGS) that incorporates multiple copies of an antigen and targets the Fc gamma receptors on antigen-presenting cells. These self-adjuvanting immunogens were tested in the context of dengue infection, for which there is currently no globally licensed vaccine yet. Thus, the consensus domain III sequence (cEDIII) of dengue glycoprotein E was incorporated into PIGS and expressed in both tobacco plants and Chinese Ovary Hamster cells. Purified mouse and human cEDIII-PIGS were fractionated by HPLC into low and high molecular weight forms, corresponding to monomers, dimers and polymers. cEDIII-PIGS were shown to retain important Fc receptor functions associated with immunoglobulins, including binding to C1q component of the complement and the low affinity Fcγ receptor II, as well as to macrophage cells in vitro. These molecules were shown to be immunogenic in mice, with or without an adjuvant, inducing a high level IgG antibody response which showed a neutralizing potential against the dengue virus serotype 2. The cEDIII-PIGS also induced a significant cellular immune response, IFN-γ production and polyfunctional T cells in both the CD4+ and CD8+ compartments. This proof-of-principle study shows that the potent antibody Fc-mediated cellular functions can be harnessed to improve vaccine design, underscoring the potential of this technology to induce and modulate a broad-ranging immune response.

Production of anti-LPS IgM by B1a B cells depends on IL-1ß and is protective against lung infection with Francisella tularensis LVS.

The role of IL-1ß and IL-18 during lung infection with the gram-negative bacterium Francisella tularensis LVS has not been characterized in detail. Here, using a mouse model of pneumonic tularemia, we show that both cytokines are protective, but through different mechanisms. Il-18-/- mice quickly succumb to the infection and showed higher bacterial burden in organs and lower level of IFNγ in BALF and serum compared to wild type C57BL/6J mice. Administration of IFNγ rescued the survival of Il-18-/- mice, suggesting that their decreased resistance to tularemia is due to inability to produce IFNγ. In contrast, mice lacking IL-1 receptor or IL-1ß, but not IL-1α, appeared to control the infection in its early stages, but eventually succumbed. IFNγ administration had no effect on Il-1r1-/- mice survival. Rather, Il-1r1-/- mice were found to have significantly reduced titer of Ft LPS-specific IgM. The anti-Ft LPS IgM was generated in a IL-1ß-, TLR2-, and ASC-dependent fashion, promoted bacteria agglutination and phagocytosis, and was protective in passive immunization experiments. B1a B cells produced the anti-Ft LPS IgM and these cells were significantly decreased in the spleen and peritoneal cavity of infected Il-1b-/- mice, compared to C57BL/6J mice. Collectively, our results show that IL-1ß and IL-18 activate non-redundant protective responses against tularemia and identify an essential role for IL-1ß in the rapid generation of pathogen-specific IgM by B1a B cells.

Inflammasome-dependent pyroptosis and IL-18 protect against Burkholderia pseudomallei lung infection while IL-1ß is deleterious.

Burkholderia pseudomallei is a Gram-negative bacterium that infects macrophages and other cell types and causes melioidosis. The interaction of B. pseudomallei with the inflammasome and the role of pyroptosis, IL-1ß, and IL-18 during melioidosis have not been investigated in detail. Here we show that the Nod-like receptors (NLR) NLRP3 and NLRC4 differentially regulate pyroptosis and production of IL-1ß and IL-18 and are critical for inflammasome-mediated resistance to melioidosis. In vitro production of IL-1ß by macrophages or dendritic cells infected with B. pseudomallei was dependent on NLRC4 and NLRP3 while pyroptosis required only NLRC4. Mice deficient in the inflammasome components ASC, caspase-1, NLRC4, and NLRP3, were dramatically more susceptible to lung infection with B. pseudomallei than WT mice. The heightened susceptibility of Nlrp3⁻/⁻ mice was due to decreased production of IL-18 and IL-1ß. In contrast, Nlrc4⁻/⁻ mice produced IL-1ß and IL-18 in higher amount than WT mice and their high susceptibility was due to decreased pyroptosis and consequently higher bacterial burdens. Analyses of IL-18-deficient mice revealed that IL-18 is essential for survival primarily because of its ability to induce IFNγ production. In contrast, studies using IL-1RI-deficient mice or WT mice treated with either IL-1ß or IL-1 receptor agonist revealed that IL-1ß has deleterious effects during melioidosis. The detrimental role of IL-1ß appeared to be due, in part, to excessive recruitment of neutrophils to the lung. Because neutrophils do not express NLRC4 and therefore fail to undergo pyroptosis, they may be permissive to B. pseudomallei intracellular growth. Administration of neutrophil-recruitment inhibitors IL-1ra or the CXCR2 neutrophil chemokine receptor antagonist antileukinate protected Nlrc4⁻/⁻ mice from lethal doses of B. pseudomallei and decreased systemic dissemination of bacteria. Thus, the NLRP3 and NLRC4 inflammasomes have non-redundant protective roles in melioidosis: NLRC4 regulates pyroptosis while NLRP3 regulates production of protective IL-18 and deleterious IL-1ß.

Work preferences and Animal Welfare Perception of Veterinary Medicine and Animal Science Students in Northeastern Mexico.

Veterinary medicine and animal science (VMAS) students coexist in asocial, geographic, and economic context that influences personal and career decisions. The goal of this study was to analyze students' perceptions of Animal Welfare (AW) and World Organization for Animal Health (OIE) topics by gender, religion, and stage of study at the school of veterinary medicine in the northeastern Mexican border area. Survey response rate was 60% of VMAS student enrollment, which was divided in basic, intermediate, and advanced academic levels. Student respondents reported animal production followed by animals for companionship and wildlife appreciation as their job placement expectations after graduation. Students in the basic training stage rated AW in general practice to be more important compared with those in intermediate and advanced training (p < 0.005). Compared with intermediate and advanced level students, students at the basic level considered bioethics, sustainable food production, and OIE animal welfare topics more important (p < 0.05). Regarding gender differences, compared with male students, their female counterparts rated AW more important, depending on areas of work practice and OIE topics (p < 0.05).

Milk Composition of Creole Goats Raised at Different Altitudes in an Extensive Production System in Northeast Mexico.

Goat milk composition is affected by feeding, and in semiarid rangeland, information on Creole goat milk physicochemical composition is lacking. For the fulfillment of this objective, three agroecological regions (AR) considering altitude (lowland 87, highland 779, and mountain 1309 m above sea level) with different botanical compositions were chosen. Every AR analyzed accounted for 30 goat herds, with a total of 90 herds. The results demonstrated that altitude had an influence mainly on density and protein. Milk density increases as altitude increases; conversely, milk protein increases as altitude decreases. On the other hand, in the mountain and lowland ARs, the salts and solids not fat (SNF) percentages were higher compared to that of the highland AR (p < 0.05). The freezing point (FP) was higher at highland altitudes compared to that of mountain and lowland ARs (p < 0.01). In the milk fatty acids (FA) profile, only the C14:1 value was affected by altitude, whereas goat milk at lowland and mountain altitudes had higher values compared to that at highland altitudes (p < 0.05). Additionally, late lactation stage fat, FP, and pH values were higher compared to early lactation values. The opposite effect was observed for salts and SNF. In the FA profile, late lactation values were higher for C10:0 and C8:0 compared to early lactation values. The opposite trend was observed for C18:2n6t. The thrombogenic index was significantly higher at lowland altitudes compared to highland altitudes, and similar to the mountain AR. These goat milk characteristics could be explained as a consequence of animal nutrition, as well as the goat's meat-type phenotype.

Role of the inflammasome, IL-1ß, and IL-18 in bacterial infections.

The inflammasome is an important innate immune pathway that regulates at least two host responses protective against infections: (1) secretion of the proinflammatory cytokines IL-1ß and IL-18 and (2) induction of pyroptosis, a form of cell death. Inflammasomes, of which different types have been identified, are multiprotein complexes containing pattern recognition receptors belonging to the Nod-like receptor family or the PYHIN family and the protease caspase-1. The molecular aspects involved in the activation of different inflammasomes by various pathogens are being rapidly elucidated, and their role during infections is being characterized. Production of IL-1ß and IL-18 and induction of pyroptosis of the infected cell have been shown to be protective against many infectious agents. Here, we review the recent literature concerning inflammasome activation in the context of bacterial infections and identify important questions to be answered in the future.

Bovine Lactoferrin can Decrease the In Vitro Biofilm Production and Show Synergy with Antibiotics Against Listeria and Escherichia coli Isolates.

BACKGROUND: Bovine Lactoferrin (bLf) has been reported as antimicrobial, antiviral, immunomodulatory and anticancer protein. Escherichia coli and Listeria spp. are food-borne bacteria that can produce illness in human being and mammals, the emergent antimicrobial drug resistance has been reported in these pathogens. OBJECTIVE: The aim for this study was to evaluate the bLf effect on in vitro biofilm production and the synergic effect of antibiotics on E. coli and Listeria isolates. METHODS: E. coli and Listeria specimens were isolated from bovine carcasses and slaughterhouses surfaces, respectively. Biofilm formation was analyzed with or without bLf, incubated for 48 h and spectrophotometry, cell viability was analyzed by colony-forming unit (CFU) and the synergistic effect of bLf with ampicillin, oxytetracycline, and streptomycin was evaluated through the fractional concentration index (FCI). RESULTS: Our results show that a low bLf concentration (0.8 µM) can diminish the in vitro biofilm production in Listeria isolates; also improves the in vitro oxytetracycline and streptomycin activity against E. coli, and ampicillin activity against Listeria isolates. CONCLUSION: bLf can affect the biofilm production in Listeria isolates from slaughterhouses surfaces and shown synergic effect with ampicillin. Also has a synergic effect with oxytetracycline and streptomycin against E. coli isolates from bovine carcasses.

Heat shock effect upon dengue virus replication into U937 cells.

The molecules involved in dengue virus entry into human cells are currently unknown. We have previously shown that two surface heat shock proteins (Hsps), Hsp90 and Hsp70 are part of a receptor complex in monocytic cells. In the present report, the effect of heat shock (HS) on dengue virus infection is analyzed. We have documented a more than twofold increase in dengue virus infectivity after HS treatment in monocytic cells U937; this effect correlates mainly with an increase in viral entry due to a major presence of both Hsps on the surface of monocytic cells, particularly in membrane microdomains. Interestingly, since heat shock treatment at 6h post-infection also increased viral yields, it is likely that HS also modulates positively dengue virus replication.

Use of a commercial enzyme immunoassay to monitor dengue virus replication in cultured cells.

Current methods for dengue virus quantitation are either time consuming, technically demanding or costly. As an alternative, the commercial enzyme immunoassay Platelia Dengue NS1 AG (Bio-Rad Laboratories) was used to monitor semiquantitatively dengue virus replication in cultured cells. The presence of NS1 protein was evaluated in supernatants from Vero and C6/36 HT cells infected with dengue virus. The amount of NS1 detected in the supernatants of infected cells was proportional to the initial MOI used and to the time of post infection harvest. This immunoassay was also able to detect the presence of NS1 in the supernatants of infected human macrophages. Inhibition of dengue virus replication in C6/36 HT cells treated with lysosomotropic drugs was readily monitored with the use of this assay. These results suggest that the Platelia Dengue NS1 AG kit can be used as a fast and reliable surrogate method for the relative quantitation of dengue virus replication in cultured cells.

Evidence that the 45-kD glycoprotein, part of a putative dengue virus receptor complex in the mosquito cell line C6/36, is a heat-shock related protein.

Dengue virus (DENV) is transmitted to humans by mosquitoes of the genus Aedes. Although several molecules have been described as part of DENV receptor complex in mosquito cells, none of them have been identified. Our group characterized two glycoproteins (40 and 45 kD) as part of the DENV receptor complex in C6/36 cells. Because identification of the mosquito cell receptor has been unsuccessful and some cell receptors described for DENV in mammalian cells are heat-shock proteins (HSPs), the role of HSPs in DENV binding and infection in C6/36 cells was evaluated. Our results indicate that gp45 and a 74-kD molecule (p74), which interact with DENV envelope protein, are immunologically related to HSP90. Although p74 is induced by heat shock, gp45 apparently is not. However, these proteins are relocated to the cell surface after heat-shock treatment, causing an increase in virus binding without any effect on virus yield.

Immunogenic Subviral Particles Displaying Domain III of Dengue 2 Envelope Protein Vectored by Measles Virus.

Vaccines against dengue virus (DV) are commercially nonexistent. A subunit vaccination strategy may be of value, especially if a safe viral vector acts as biologically active adjuvant. In this paper, we focus on an immunoglobulin-like, independently folded domain III (DIII) from DV 2 envelope protein (E), which contains epitopes that elicits highly specific neutralizing antibodies. We modified the hepatitis B small surface antigen (HBsAg, S) in order to display DV 2 DIII on a virus-like particle (VLP), thus generating the hybrid antigen DIII-S. Two varieties of measles virus (MV) vectors were developed to express DIII-S. The first expresses the hybrid antigen from an additional transcription unit (ATU) and the second additionally expresses HBsAg from a separate ATU. We found that this second MV vectoring the hybrid VLPs displaying DIII-S on an unmodified HBsAg scaffold were immunogenic in MV-susceptible mice (HuCD46Ge-IFNar(k)(o)), eliciting robust neutralizing responses (averages) against MV (1:1280 NT90), hepatitis B virus (787 mIU/mL), and DV2 (1:160 NT50) in all of the tested animals. Conversely, the MV vector expressing only DIII-S induced immunity against MV alone. In summary, DV2 neutralizing responses can be generated by displaying E DIII on a scaffold of HBsAg-based VLPs, vectored by MV.

Novel vaccination approach for dengue infection based on recombinant immune complex universal platform.

Dengue infection is on the rise in many endemic areas of the tropics. Vaccination remains the most realistic strategy for prevention of this potentially fatal viral disease but there is currently no effective vaccine that could protect against all four known serotypes of the dengue virus. This study describes the generation and testing of a novel vaccination approach against dengue based on recombinant immune complexes (RIC). We modelled the dengue RIC on the existing Ebola RIC (Phoolcharoen, et al. Proc Natl Acad Sci USA 2011;108(Dec (51)):20695) but with a key modification that allowed formation of a universal RIC platform that can be easily adapted for use for other pathogens. This was achieved by retaining only the binding epitope of the 6D8 ant-Ebola mAb, which was then fused to the consensus dengue E3 domain (cEDIII), resulting in a hybrid dengue-Ebola RIC (DERIC). We expressed human and mouse versions of these molecules in tobacco plants using a geminivirus-based expression system. Following purification from the plant extracts by protein G affinity chromatography, DERIC bound to C1q component of complement, thus confirming functionality. Importantly, following immunization of mice, DERIC induced a potent, virus-neutralizing anti-cEDIII humoral immune response without exogenous adjuvants. We conclude that these self-adjuvanting immunogens have the potential to be developed as a novel vaccine candidate for dengue infection, and provide the basis for a universal RIC platform for use with other antigens.

JNK phosphorylation, induced during dengue virus infection, is important for viral infection and requires the presence of cholesterol.

Infection with a broad diversity of viruses often activates host cell signaling pathways including the mitogen-activated protein kinase pathway. The present study established that dengue virus infection of human macrophages activates Jun NH(2)-terminal kinase (JNK) and the p38 MAPKs pathways. The activation was observed at early times after infection and occurs when either infectious or UV-inactivated dengue virus was used. The role of these activated kinases in dengue virus infection was evaluated using specific inhibitors. Inhibition of JNK and p38 kinases did result in a significant reduction in viral protein synthesis and in viral yield. Additionally, lipid rafts disruption induced a strong inhibition of JNK activation. These results suggest that, at early stages after dengue virus infection, MAPKs are activated and that activation of JNK and p38 is required for dengue virus infection.