S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is a highly contagious virus infecting pigs of all ages with high morbidity and mortality among newborn piglets. Currently, there is no effective vaccine available to protect the pigs from PEDV. The N-terminal subunit of spike protein (S1) is responsible for virus binding to the cellular receptor and contains a number of neutralizing antibody epitopes. Thus, we expressed and produced recombinant S1 protein to protect newborn piglets by immunization of sows. METHODS: Affinity tagged PEDV S1 protein was expressed in a secretory form in yeast, insect and mammalian cells to identify the most suitable production system. Purified recombinant protein was analysed by SDS-PAGE, Western blot and deglycosylation assay. A pregnant sow was intramuscularly immunized three times with adjuvanted recombinant protein prior to farrowing. PEDV-specific immune responses in sera and colostrum of the sow and piglets were assayed by ELISA and virus neutralization assays. Piglets were challenged orally with PEDV, and clinical parameters were monitored for 6 days post-challenge. RESULTS AND CONCLUSION: Of three eukaryotic expression systems tested (yeast, insect-cell, and mammalian), expression by HEK-293 T cells gave the highest yield of protein that was N-glycosylated and was the most appropriate candidate for vaccination. Administration of the subunit vaccine in a sow resulted in induction of S1-specific IgG and IgA that were passively transferred to the suckling piglets. Also, high virus neutralization titres were observed in the serum of the vaccinated sow and its piglets. After PEDV challenge, piglets born to the vaccinated sow exhibited less severe signs of disease and significantly lower mortality compared to the piglets of a control sow. However, there were no significant differences in diarrhea, body weight and virus shedding. Thus, vaccination with S1 subunit vaccine failed to provide complete protection to suckling piglets after challenge exposure, and further improvements are needed for the development of a subunit vaccine that fully protects against PEDV infection.

Proteomic analysis of purified turkey adenovirus 3 virions.

Turkey adenovirus 3 (TAdV-3) causes high mortality and significant economic losses to the turkey industry. However, little is known about the molecular determinants required for viral replication and pathogenesis. Moreover, TAdV-3 does not grow well in cell culture, thus detailed structural studies of the infectious particle is particularly challenging. To develop a better understanding of virus-host interactions, we performed a comprehensive proteomic analysis of proteinase K treated purified TAdV-3 virions isolated from spleens of infected turkeys, by utilizing one-dimensional liquid chromatography mass spectrometry. Our analysis resulted in the identification of 13 viral proteins associated with TAdV-3 virions including a novel uncharacterized TaV3gp04 protein. Further, we detected 18 host proteins in purified virions, many of which are involved in cell-to cell spread, cytoskeleton dynamics and virus replication. Notably, seven of these host proteins have not yet been reported to be present in any other purified virus. In addition, five of these proteins are known antiviral host restriction factors. The availability of reagents allowed us to identify two cellular proteins (collagen alpha-1 (VI) chain and haemoglobin) in the purified TAdV-3 preparations. These results represent the first comprehensive proteomic profile of TAdV-3 and may provide information for illustrating TAdV-3 replication and pathogenesis.

Volatile resistance states in electrochemical metallization cells enabling non-destructive readout of complementary resistive switches.

Redox-based resistive memory cells exhibit changes of OFF or intermediate resistance values over time and even ON states can be completely lost in certain cases. The stability of these resistance states and the time until resistance loss strongly depends on the materials system. On the basis of electrical measurements and chemical analysis we found a viable explanation for these volatile resistance states (VRSs) in Ag-GeSx-based electrochemical metallization memory cells and identified a technological application in the field of crossbar memories. Complementary resistive switches usually suffer from the necessity of a destructive read-out procedure increasing wear and reducing read-out speed. From our analysis we deduced a solution to use the VRS as an inherent selector mechanism without the need for additional selector devices.

Physical origins and suppression of Ag dissolution in GeS(x)-based ECM cells.

Electrochemical metallisation (ECM) memory cells potentially suffer from limited memory retention time, which slows down the future commercialisation of this type of data memory. In this work, we investigate Ag/GeSx/Pt redox-based resistive memory cells (ReRAM) with and without an additional Ta barrier layer by time-of-flight secondary ion mass spectrometry (ToF-SIMS), X-ray absorption spectroscopy (XAS) and synchrotron high-energy X-ray diffractometry (XRD) to investigate the physical mechanism behind the shift and/or loss of OFF data retention. Electrical measurements demonstrate the effectiveness and high potential of the diffusion barrier layer in practical applications.

Innate immune cocktail partially protects broilers against cellulitis and septicemia.

Antimicrobial/host defense peptides (A/HDP) are natural compounds that are found in leucocyte cells and on the skin and bodily fluids of birds, reptiles, and mammals. Not only do they possess antibacterial, antiviral, and antiparasitic characteristics but they also stimulate the host immune system to combat infectious diseases and may play a role in the promotion of wound repair. Gamma-amino butyric acid (GABA) is an amino acid-based inhibitory neurotransmitter in the brain that has also been shown to promote wound healing on skin. The objective of this study was to establish a therapeutic cocktail that protects birds against Escherichia coli-related disease and lesions in broilers. We injected a cocktail of six A/HDPs with or without GABA into 3-wk-old broilers by a subcutaneous or intramuscular route followed 24 hr later by challenge with a field isolate of serogroup O2 E. coli. Birds were examined for 5-6 days post-E. coli challenge and clinical, pathologic, and bacteriologic assessments were conducted. Birds that were subcutaneously injected with an A/HDP plus GABA cocktail had significantly higher survival rates and lower levels of bacteremia (P < 0.05), but a similar percentage of the surviving birds had large cellulitis lesions compared to the surviving phosphate-buffered saline-injected control birds. When this cocktail was administered intramuscularly, there was a trend towards protection against E. coli-related death, although the results were not statistically significant and there was no reduction in bacteremia. A significant number of birds had a reduced bacterial load on cellulitis lesions but no reduction in lesion size, which suggests that when the cocktail was administered intramuscularly it failed to protect against cellulitis. These results suggest that the route of administration of the cocktail influences disease outcome. Gene expression analysis was performed to investigate whether the cocktail induced immunomodulatory functions in avian cells that complemented their antimicrobial and anti-endotoxic effects. A/HDP plus GABA mediated temporal induction of pro-inflammatory cytokines but no induction of any of the chemokines under investigation. This cocktail shows potential to protect against E. coli-related death, which is a major economic burden to the poultry industry.

The bovine viral diarrhea virus E2 protein formulated with a novel adjuvant induces strong, balanced immune responses and provides protection from viral challenge in cattle.

Bovine viral diarrhea virus (BVDV) is still one of the most serious pathogens in cattle, meriting the development of improved vaccines. Recently, we developed a new adjuvant consisting of poly[di(sodium carboxylatoethylphenoxy)]-phosphazene (PCEP), either CpG ODN or poly(I:C), and an immune defense regulator (IDR) peptide. As this adjuvant has been shown to mediate the induction of robust, balanced immune responses, it was evaluated in an E2 subunit vaccine against BVDV in lambs and calves. The BVDV type 2 E2 protein was produced at high levels in a mammalian expression system and purified. When formulated with either CpG ODN or poly(I:C), together with IDR and PCEP, the E2 protein elicited high antibody titers and production of IFN-γ secreting cells in lambs. As the immune responses were stronger when poly(I:C) was used, the E2 protein with poly(I:C), IDR and PCEP was subsequently tested in cattle. Robust virus neutralizing antibodies as well as cell-mediated immune responses, including CD8(+) cytotoxic T cell (CTL) responses, were induced. The fact that CTL responses were demonstrated in calves vaccinated with an E2 protein subunit vaccine indicates that this adjuvant formulation promotes cross-presentation. Furthermore, upon challenge with a high dose of virulent BVDV-2, the vaccinated calves showed almost no temperature response, weight loss, leukopenia or virus replication, in contrast to the control animals, which had severe clinical disease. These data suggest that this E2 subunit formulation induces significant protection from BVDV-2 challenge, and thus is a promising BVDV vaccine candidate; in addition, the adjuvant platform has applications in bovine vaccines in general.

Ag/GeSx/Pt-based complementary resistive switches for hybrid CMOS/nanoelectronic logic and memory architectures.

Complementary resistive switches based on two anti-serially connected Ag/GeSx/Pt devices were studied. The main focus was placed on the pulse mode properties as typically required in memory and logic applications. A self-designed measurement setup was applied to access each CRS part-cell individually. Our findings reveal the existence of two distinct read voltage regimes enabling both spike read as well as level read approaches. Furthermore, we experimentally verified the theoretically predicted kinetic properties in terms of pulse height vs. switching time relationship. The results obtained by this alternative approach allow a significant improvement of the basic understanding of the interplay between the two part-cells in a complementary resistive switch configuration. Furthermore, from these observations we can deduce a simplified write voltage scheme which is applicable for the considered type of memory cell.

Two doses of bovine viral diarrhea virus DNA vaccine delivered by electroporation induce long-term protective immune responses.

Bovine viral diarrhea virus (BVDV) is a pathogen of major importance in cattle, so there is a need for new effective vaccines. DNA vaccines induce balanced immune responses and are relatively inexpensive and thus promising for both human and veterinary applications. In this study, newborn calves with maternal antibodies were vaccinated intramuscularly (i.m.) with a BVDV E2 DNA vaccine with the TriGrid Delivery System for i.m. delivery (TDS-IM). Two doses of this vaccine spaced 6 or 12 weeks apart were sufficient to induce significant virus-neutralizing antibody titers, numbers of activated T cells, and reduction in viral shedding and clinical presentations after BVDV-2 challenge. In contrast to the placebo-treated animals, the vaccinated calves did not lose any weight, which is an excellent indicator of the well-being of an animal and has a significant economic impact. Furthermore, the interval between the two vaccinations did not influence the magnitude of the immune responses or degree of clinical protection, and a third immunization was not necessary or beneficial. Since electroporation may enhance not only the magnitude but also the duration of immunity after DNA immunization, the interval between vaccination and challenge was extended in a second trial, which showed that two doses of this E2 DNA vaccine again significantly reduced clinical disease against BVDV for several months. These results are promising and support this technology for use against infectious diseases in cattle and large species, including humans, in general.

Direct observation of charge transfer in solid electrolyte for electrochemical metallization memory.

X-ray absorption spectroscopy study on an electrochemical metallization cell of GeS(x) :Ag shows clear experimental evidence of chemical ionization of the active metal atoms (Ag) and consequent transfer of charge to the electrolyte (GeS(x) ). The valence electron density and its change upon the Ag intercalation are depicted schematically as transparent waves on the Ge-S bond structure in amorphous GeS(x) .

Electroporation enhances immune responses and protection induced by a bovine viral diarrhea virus DNA vaccine in newborn calves with maternal antibodies.

Bovine viral diarrhea virus (BVDV) is one of the major pathogens in cattle. In this study, newborn calves with maternal antibodies were vaccinated with a BVDV DNA vaccine, either by conventional intramuscular (IM) injection or with the TriGrid™ Delivery System for IM delivery (TDS-IM). The calves vaccinated with the TDS-IM developed more rapidly and effectively BVDV-specific humoral and cell-mediated immune responses in the presence of maternal antibodies. Overall, the immune responses induced by delivery with the TDS-IM remained stronger than those elicited by conventional IM injection of the BVDV DNA vaccine. Accordingly, electroporation-mediated delivery of the BVDV DNA vaccine resulted in close to complete protection from clinical signs of disease, while conventional IM administration did not fully prevent morbidity and mortality following challenge with BVDV-2. These results demonstrate the TDS-IM to be effective as a delivery system for a BVDV DNA vaccine in newborn calves in the presence of maternal antibodies, which supports the potential of electroporation as a delivery method for prophylactic DNA vaccines.

Priming with DNA encoding E2 and boosting with E2 protein formulated with CpG oligodeoxynucleotides induces strong immune responses and protection from Bovine viral diarrhea virus in cattle.

The objective of this study was to develop an optimal vaccination strategy for Bovine viral diarrhea virus (BVDV). The E2 protein of BVDV plays a major protective role against BVDV infection. In order to be able to compare DNA, protein and DNA prime-protein boost regimens, a plasmid was constructed encoding a secreted form of the NADL strain E2 protein (pMASIA-tPAsDeltaE2). Furthermore, a pure secreted recombinant DeltaE2 (rDeltaE2) protein was produced. The rDeltaE2 protein was formulated with a combination of Emulsigen and CpG oligodeoxynucleotide. Groups of calves were immunized with pMASIA-tPAsDeltaE2 or with rDeltaE2, or first with pMASIA-tPAsDeltaE2 and then with rDeltaE2. To evaluate the protection against BVDV, calves were challenged with BVDV strain NY-1 after the last immunization. Although all immunized calves developed humoral and cellular immune responses, the antibody responses in the DNA prime-protein boost group were stronger than those elicited by either the DNA vaccine or the protein vaccine. In particular, E2-specific antibody titres were enhanced significantly after boosting the DeltaE2 DNA-primed calves with rDeltaE2 protein. Moreover, protection against BVDV challenge was obtained in the calves treated with the DNA prime-protein boost vaccination regimen, as shown by a significant reduction in weight loss, viral excretion and lymphopenia, compared with the unvaccinated calves and the animals immunized with the DNA or protein only. These results demonstrate the advantage of a DNA prime-protein boost vaccination approach in an outbred species.

Immunization with plasmid DNA encoding a truncated, secreted form of the bovine viral diarrhea virus E2 protein elicits strong humoral and cellular immune responses.

The major protective antigen of bovine viral diarrhea virus (BVDV), the E2 protein, is cell-associated and not expressed on the cell surface. In this study we evaluated a DNA vaccine encoding various secreted versions of E2. In vitro analysis demonstrated that deletion of the transmembrane anchor and addition of the signal sequence of bovine herpesvirus-1 (BHV-1) (gDsDeltaE2) resulted in efficient secretion of E2 into the culture medium. In contrast, full-length E2, either without or with gDs (gDsE2), as well as truncated E2 without gDs (DeltaE2), remained entirely cell-associated. Mice immunized with plasmid encoding gDsDeltaE2 developed significantly higher IgG and virus neutralizing antibody titres compared to animals vaccinated with plasmid encoding E2, DeltaE2 or gDsE2. To optimize secretion of E2, the efficiency of gDs was compared with that of the tissue plasminogen activator signal (tPAs) sequence. In addition, the effect of the plasmid backbone was assessed by comparing two vectors. Four plasmids, pMASIA-gDsDeltaE2, pMASIA-tPAsDeltaE2, pSLKIA-gDsDeltaE2 and pSLKIA-tPAsDeltaE2, were constructed and administered intradermally to mice. The mice immunized with pMASIA-tPAsDeltaE2 developed the strongest and most balanced immune responses. Vaccination of cattle confirmed that pMASIA-tPAsDeltaE2 elicited both strong humoral and cellular immune responses and thus could be a candidate DNA vaccine against BVDV.