mteb/CovidQA huggingface dataset:
Immunization of chickens with the control plasmid (CMV/R) without an HA gene insert elicited minimal neutralizing antibody titers compared to HA-immunized animals 1 week after 3 DNA immunizations. Nearly all chickens immunized with either monovalent or multivalent HA DNA vaccines generated significant neutralization titers ( Fig. 4 and Table S1 ). In general, there was a progressive increase in the amount of neutralization after each successive DNA vaccination (data not shown) with maximal response at 1 week after the 3 rd DNA immunization, with highest and most consistent levels in the trivalent vaccine group delivered with the Agro-JetH device. Neutralization of the Indonesia HA strain was the most robust, with neutralization nearing 100% at titers greater than 1:3200. Both the monovalent and multivalent vaccines elicited robust homologous ( vaccine (Fig. 4 ). Even though one chicken (238) in the multivalent vaccine group produced almost the same degree of neutralization at each
time point and was protected, it did not produce a high neutralizing antibody titer for reasons that were uncertain but possibly related to a non-specific inhibitor in the sera.
To determine whether chickens immunized with single or multiple DNA vaccines were protected from a lethal challenge of a heterologous HPAI H5N1 virus, vaccinated chickens were In panels B and C, mice (n = 10) were immunized with 15 mg of plasmid (3 mg each) three times at 3 week intervals. Serum pools from the immunized animals were collected 14 days after the third immunization. The antisera were tested against the 12 indicated pseudotyped lentiviral vectors at varying dilutions. Error bars at each point indicate the standard deviation; each sample was evaluated in triplicate. In general, the immunized serum neutralized all tested pseudotyped lentiviruses at low dilutions while differences were often observed at high dilution. doi:10.1371/journal.pone.0002432.g002 inoculated with 20 LD 50 of highly pathogenic A/Vietnam/1203/ 2004 heterologous virus intranasally using standard methods [25, 40] and monitored for morbidity, mortality, viral shedding and serum antibodies. While all the
control animals died within 2 days of infection, 100% survival was noted in the rest of the chickens (Fig. 5A ). The animals that were healthy, showing no signs of clinical disease or malaise, were euthanized on day 14. There was no evidence for viral shedding monitored via tracheal and cloacal swabs of infected chickens 2-14 days after challenge as determined by embryonal inoculation (data not shown: egg infectious dose 50 (EID 50 ) limit of detection ,100 virus particles).
To compare the relative efficacy of DNA vaccines delivered IM by needle and syringe versus the needle-free Agro-JetH device injection, a dose-response study was performed with amounts of DNA vaccine ranging from 500 to 0.5 mg with two inoculations. In these experiments, the HA derived from A/chicken/Nigeria/641/ 2006 was substituted for A/Vietnam/1203/2004 since it represented a more contemporary isolate. The observed rate of protection was higher among the animals receiving 5 mg by Agro-Jet (8/8) than by IM injection (6/8) (Fig. 5, B vs. C). Both modes provided complete protection for all animals at doses higher than this, and 25% protection for the animals receiving 0.5 mg doses (Fig. 5B, C) . Survival differences between consecutive doses were assessed using a log-rank test on the survival curves for pairs of groups. A test was deemed significant if the p-value was ,.01, and marginally significant if the p-value was ,.05 but ..01. Chickens injected IM showed a marginally
significant difference between 0.5 and 5 mg (p = .047). In the same group there was a significant difference between control and 5, 50 and 500 mg (p,.001 for all comparisons) and the difference between control and 0.5 mg was marginally significant (p = .016). Chickens that were injected using Agro-JetH showed a significant difference between 0.5 and 5 mg (p = .004) and between control and 5, 50, and 500 mg (p,.001 for all comparisons). There were no differences between control and 0.5 mg or between 5, 50, and 500 mg. Lastly, the survival differences between Agro-JetH and IM for each dose group were not significant. The neutralizing antibody response to homologous and heterologous HAs corresponded with protection and correlated with dose, with higher titers elicited by injection with Agro-JetH compared to needle (Table S2) . We assessed viable viral shedding after inoculation by chick embryo inoculation three days after virus challenge (Week 8). While we noted some embryonic lethality
at the 0.5 mg dose, there was no embryonic lethality at 5, 50 or 500 mg groups (data not shown).
Since the HPAI H5N1 virus first appeared ten years ago, this highly pathogenic avian influenza virus has shown increasing diversification and dissemination in Asia, Africa, and Europe [28, [41] [42] [43] [44] . In addition to its effects on human health by crossspecies transmission [28, 45, 46] and ability to compromise food sources, it poses a continuing threat to public health as it evolves and adapts in different species. The pandemic potential of this virus, especially as it relates to the poultry industry and for reservoir avian hosts, underscores the need for a vaccine that offers broad spectrum immunity and protection against lethal viral challenge. While the virus remains restricted in its ability to infect humans and undergo efficient human-to-human transmission [28, 47] , its persistence and spread in poultry increases the risk of the emergence of a pandemic strain. One approach to pandemic risk reduction is to limit the propagation of the virus in poultry and other relevant
avian species.
We have previously reported that DNA vaccines encoding HA can confer protection against a highly lethal human pandemic influenza virus, the 1918 H1N1 virus, in mice [36] . DNA vaccines offer several advantages, including the ability to express diverse antigens, tolerability in various hosts, ease of delivery, and stability for storage and distribution without the necessity of maintaining a cold chain; they have been shown to be safe and efficacious in a variety of animal models [2, 4, 12, 22, 48] . Because they do not contain other viral proteins used to screen for infection, they also address the need to differentiate vaccinated from infected animals. There is evidence that DNA vaccination elicits cell-mediated immunity against influenza HA in addition to inducing an antibody response [36] , an effect that could significantly contribute to protective immunity as viruses show genetic drift and reduced susceptibility to neutralization.
Ideally, a highly effective influenza vaccine should not only be able to let the host develop a protective immune response against a matching live virus challenge but also elicit robust protective immune responses against a broad range of homologous and heterologous H5 influenza strains. A multivalent H5 vaccine containing diverse serotypes could expand the antigenic breadth sufficiently to provide protection against heterologous challenge and may preclude the emergence of vaccine-resistant strains that may arise due to evolutionary vaccine pressure on the virus. Due to the antigenic drift and shift of the influenza virus genome, it has been very difficult to predict the next dominant strain of an avian endemic outbreak. DNA vaccines can be synthesized in a relatively short period of time, and the targeted mutations can be tailored to specific viral serotypes. The mutations promote a focused and enhanced immune response [3, 49, 50] that may be particularly important in the event of an
outbreak where specificity is the key to epidemic control. The use of modified codons ensures maximal expression in the host and eliminates the possibility of recombination with influenza viruses that might potentially generate new strains.
A more broadly protective murine vaccine was developed here by including more HAs from varying strains in the multivalent vaccine (Figs. 2 and 3) . However, it is less practical to include large numbers of different HAs in one vaccine due to the cost and complexity of manufacturing such a vaccine. Therefore, we simplified the vaccine regimen based on cross-neutralization studies and phylogenetic relationships. A trivalent vaccine was subsequently identified for further studies. Due [51] .
While three DNA immunizations were used initially to demonstrate protective immunity and have been used previously to elicit protection in mice [36] , we found that effective protective immunity could be induced with two DNA vaccinations and as little as 5 mg trivalent DNA immunization using the ID/SC route with the Agro-JetH device. In addition, based on the chick embryo inoculation data, we believe that there is effective neutralization of the virus and lack of infectious viral shedding in chicken vaccinated with as little as 5 mg of DNA. The device's capacity for rapid repetitive injection and the lower quantity and stability of DNA enhance the practicality and utility of this approach for vaccination of endangered species in captivity or administration to poultry or other animals.
A/Vietnam/1203/2004 (H5N1) (A/VN/1203/04) was obtained from the repository at the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia. The virus was propagated in 10-day old embryonated chicken eggs at 35uC and stored at 270uC until use. The virus was titrated by the Reed and Muench method to determine EID 50 [52] . GenBank ABD28180) were synthesized using human-preferred codons (GeneArt, Regensburg, Germany) [36] . HA cDNAs from diverse strains of influenza viruses were then inserted into plasmid expression vectors, pCMV/R or pCMV/R 8kB, which mediates high level expression and immunogenicity in vivo [34, 35, 36] . For initial trivalent immunizations in chickens, the A/Vietnam/1203/ 2004, A/Anhui/1/2005 and A/Indonesia/05/2005 strains were used and in the dose response study, the Vietnam strain was replaced with A/chicken/Nigeria/641/2006. The immunogens used in DNA vaccination contained a cleavage site mutation (PQRERRRKKRG to PQRETRG) as previously described [35, 36]
. This mutation was generated by site-directed mutagenesis using a QuickChange kit (Stratagene, La Jolla, CA).
DNA immunization of mice [6] [7] [8] week old female BALB/c mice were purchased from The Jackson Laboratory and maintained in the AAALAC-accredited Vaccine Research Center Animal Care Facility (Bethesda, MD) under specific pathogen-free conditions. All experiments were approved by the Vaccine Research Center Animal Care and Use Committee. The mice were immunized as previously described [5] . Briefly, mice (10 animals for all test groups, 20 animals for the
The study was carried out in the AAALAC-accredited animal facility at the University of Maryland School of Medicine. Six groups of 8 one-day-old male and female SPAFAS White Leghorn Chickens, Gallus domesticus, were obtained from Charles River Laboratories (Connecticut). The animals were housed in brooder and grower cages (McMurray Hatcheries, Iowa). Feed (Teklad Japanese Quail Diet -3050, Harlan-Teklad, WI) and water were provided to the animals ad libitum. The study was performed in strict accordance with the ''Guide'' after approvals from the Animal Care and Use Committees of the Vaccine Research Center, NIH and the University of Maryland. DNA immunizations were performed as described at 0, 3 and 6 weeks. A total dose of 500 mg of one or a combination of the following DNA plasmids in a volume of 250 ml was administered to each animal: pCMV/ R, pCMV/R-HA
Agro-JetH is a needle-free device used for mass delivery of vaccines and drugs in livestock and poultry. The device is semiautomatic and requires a small CO 2 tank or compressed air for low pressure delivery. Upon trigger activation, CO 2 disperses the injectate at a precise dose into the muscle, dermis or subcutaneous tissue depending on the setting that was standardized for our use. We used an effective volume of 0.1 ml in our injectate [39] . In this study we were able to effectively deliver 0.1 ml of injectate into the animal's dermis/subcutaneous tissue at a pressure of 48-55 psi.
Sixty-eight weeks after the last immunization, female BALB/c mice were lightly anesthetized with Ketamine/Xylazine and inoculated intranasally with 10 LD 50 of A/Vietnam/1203/2004 virus diluted in phosphate-buffered saline in a 50 ul volume. Mice were monitored daily for morbidity and measured for weight loss and mortality for 21 days post infection. Any mouse that had lost more than 25% of its body weight was euthanized. All experiments involving the HPAI virus were conducted in an AAALAC accredited facility (BioQual Inc., Gaithersburg, MD) under BSL 3 conditions that included enhancements required by the USDA and the Select Agent Program.
White Leghorn chickens were challenged one week after the last immunization with 20 lethal dose 50 (LD 50 ) of A/Vietnam/1203/04 (H5N1) influenza A virus, equivalent to 2610 4 EID 50 based on previous challenges [53] . Chickens were infected with 200 ml virus intranasally. Tracheal and cloacal swabs were collected days 3 and 5 post-challenge and stored in glass vials containing BHI medium (BBL TM Brain Heart Infusion, Becton Dickinson) at 280uC. Blood was collected 14 days post-challenge and serum was titered by microneutralization assay. Chickens were observed and scored daily for clinical signs of infection, morbidity and mortality. Chickens that survived the study were bled and humanely euthanized at day 14 post-challenge. Lungs, heart, intestine and kidney were collected and samples were stored in formalin for histopathology. Experiments were carried out under BSL3+ conditions with investigators wearing appropriate protective equipment and compliant with all Institutional Animal
Care and Use Committee-approved protocols and under Animal Welfare Act regulations at the University of Maryland, College Park, Maryland.
Representative tracheal and cloacal swabs were chosen to run an EID 50 assay for comparison and virus titers were determine by the method of Reed and Meunch [52] . Briefly, swabs were used to infect 10 day-old embryonated chicken eggs in 10-fold dilutions. Three eggs were inoculated per dilution and incubated for 48 hours before titration.
Neutralizing antibodies were titrated from serum samples collected week 5 and 7 post-vaccination and day 14 post-challenge. The microneutralization assay was performed using a 96-well plate format. Serum was treated with receptor-destroying enzyme (Denka Seiken Co.) and treated at 37uC per the manufacturer's instructions. After an overnight incubation and subsequent inactivation samples were brought to a final dilution of 1:10 using PBS and each sample was serially diluted and virus, diluted to 100 TCID 50 , was added to each well. The plates were then incubated at 37uC, 5% CO 2 for 1-2 hours. Following incubation, supernatants were used to infect a second 96-well plate of MDCK cells. Microplates were incubated at 4uC for 15 minutes and then 37uC, 5% CO 2 for 45 minutes. Supernatants of serum and virus were then discarded and 200 ml of OptiMEM (containing 1X antibiotics/antimycotics, 1 mg/ml TPCK-trypsin) was added and incubated at 37uC, 5% CO 2 for 3 days. After 3 days, 50 ml of the
supernatant from each well was transferred into a new 96-well microplate, and an HA assay was performed to calculate the antibody titers. Virus and cell controls were included in the assay.
Two-fold dilutions of heat-inactivated sera were tested in a microneutralization assay as previously described [54] for the presence of antibodies that neutralized the infectivity of 100 TCID 50 (50% tissue culture infectious dose) of the A/Vietnam/ 1203/2004 H5N1 virus on MDCK cell monolayers by using two wells per dilution on a 96-well plate.
The recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described [35, 36] . For the neutralization assay, antisera from immunized animals were heat-inactivated at 55uC for 30 minutes and mixed with 50 ml of pseudovirus at various dilutions. The sera/virus mixture was then added to 293A cells in 96-well B&W TC Isoplates (Wallac, Turku, Finland; 12,000 cells/well). Two hours later, the plates were washed and fresh medium was added. Cells were lysed in mammalian cell lysis buffer (Promega, Madison, WI) 24 hrs after infection and luciferase activity was measured using the Luciferase Assay System (Promega, Madison, WI).

The following strains were used for the production of pseudotyped viruses: for HA we used A/Thailand/1(KAN-
The HA/HI titers were determined as previously described [54] . Briefly, HA titers were calculated using 50 ml of 0.5% chicken red blood cell suspension in PBS added to 50 ml of twofold dilutions of virus in PBS. This mix was incubated at room temperature for 30 minutes. The HA titers were calculated as the reciprocal value of the highest dilution that caused complete hemagglutination. HI titers were calculated by titrating 50 ml of antiserum treated with receptor-destroying enzyme and an equivalent amount of A/Vietnam/1203/2004 virus (four hemagglutinating doses) was added to each well. Wells were incubated at room temperature for 30 minutes and 50 ml of a 0.5% suspension of chicken red blood cells was added. HI titers were calculated after 30 minutes as the reciprocal of the serum dilution that inhibited hemagglutination.
Table S1 Hemagglutination inhibition (HI), microneutralization titer (NT), and LAI of sera from individual chickens immunized with different vaccines. Sera from immunized animals were obtained at week 5 or 7, a week before or after the final boost, and neutralization was assessed by HI, microneutralization (NT) and LAI (shown as IC 50 ). Individual animal serum of each group is shown and was analyzed as described in the Materials and Methods section. Figure S1 Characterization of needle-free (Agro-JetH) DNA immunization in chickens. To evaluate the distribution of fluid into superficial or deep layers of subcutaneous tissues after delivery by AgroJetH, 4 or 7 week old chickens were injected with a solution containing India ink with this needle-free device at various pressures, ranging from 45 to 55 mm Hg. Three sites (thigh, wing and breast) were used, and biopsies were taken for routine hematoxylin and eosin staining. Representative sections of thigh injections are shown from 7 week
old chickens and were similar at 4 weeks (data not shown). While the 48 mm Hg pressure deposited the injectate into the dermis/subcutaneous region (left), the higher pressure injections, 52 and 58 mm Hg, deposited the injectate into the subcutaneous and muscle layers (middle, right). 48 mm Hg consistently provided an optimal pressure to deposit the injectate into the dermis and subcutaneous tissue and was chosen for all AgroJetH immunizations. Found at: doi:10.1371/journal.pone.0002432.s003 (10.74 MB DOC)
Metabolic engineering of Escherichia coli into a versatile glycosylation platform: production of bio-active quercetin glycosides

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573293/

SHA: f4cd52975e6aa33e8c947082eda9b261952b0f8f

Authors: De Bruyn, Frederik; Van Brempt, Maarten; Maertens, Jo; Van Bellegem, Wouter; Duchi, Dries; De Mey, Marjan
Date: 2015-09-16
DOI: 10.1186/s12934-015-0326-1
License: cc-by
Abstract: BACKGROUND: Flavonoids are bio-active specialized plant metabolites which mainly occur as different glycosides. Due to the increasing market demand, various biotechnological approaches have been developed which use Escherichia coli as a microbial catalyst for the stereospecific glycosylation of flavonoids. Despite these efforts, most processes still display low production rates and titers, which render them unsuitable for large-scale applications. RESULTS: In this contribution, we expanded a previously developed in vivo glucosylation platform in E. coli W, into an efficient system for selective galactosylation and rhamnosylation. The rational of the novel metabolic engineering strategy constitutes of the introduction of an alternative sucrose metabolism in the form of a sucrose phosphorylase, which cleaves sucrose into fructose and glucose 1-phosphate as precursor for UDP-glucose. To preserve these intermediates for glycosylation purposes, metabolization reactions were
knocked-out. Due to the pivotal role of UDP-glucose, overexpression of the interconverting enzymes galE and MUM4 ensured the formation of both UDP-galactose and UDP-rhamnose, respectively. By additionally supplying exogenously fed quercetin and overexpressing a flavonol galactosyltransferase (F3GT) or a rhamnosyltransferase (RhaGT), 0.94 g/L hyperoside (quercetin 3-O-galactoside) and 1.12 g/L quercitrin (quercetin 3-O-rhamnoside) could be produced, respectively. In addition, both strains showed activity towards other promising dietary flavonols like kaempferol, fisetin, morin and myricetin. CONCLUSIONS: Two E. coli W mutants were engineered that could effectively produce the bio-active flavonol glycosides hyperoside and quercitrin starting from the cheap substrates sucrose and quercetin. This novel fermentation-based glycosylation strategy will allow the economically viable production of various glycosides. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article
(doi:10.1186/s12934-015-0326-1) contains supplementary material, which is available to authorized users.
Text: Flavonoids are a class of plant secondary metabolites, which are chemically characterized by a 15-carbon backbone that consists of two phenyl rings and a heterocyclic ring. To date, over 10,000 flavonoids have been characterized from various plants, which are classified according to their chemical structure, i.e., the number and presence of hydroxyl groups and further functional group modifications into various subgroups, such as anthoxanthins, flavanones, and flavanonols [1, 2] .
In recent years flavonoids have garnered much attention from various application domains because of the various beneficial effects on human health that have been attributed to them, such as anticancer [3] and antioxidant [4] to anti-inflammatory [5] , antimicrobial [6] and antiviral [6, 7] effects. As final step in their biosynthesis, flavonoids are often glycosylated which has a profound effect on their solubility, stability and bio-activity [8, 9] . For example, the best studied flavonol quercetin, which makes up to 75 % of our daily flavonoid intake, predominantly occurs as different glycosides. Over 350 different quercetin glycoforms have been reported to date with varying pharmacological properties [10, 11] . In this context, hyperoside (quercetin 3-O-galactoside) and quercitrin (quercetin 3-O-rhamnoside) ( Fig. 1) have gained a lot of attention as valuable products for the pharmaceutical industry e.g., as powerful antioxidants with cytoprotective effects [12] [13] [14] [15] and
as promising antiviral agents that block replication of the influenza virus [16] or inhibit the viruses hepatitis B [17] and SARS [18] . Furthermore, they have been attributed with anti-inflammatory [19, 20] , antidepressant [21, 22] , apoptotic [23] and antifungal [24] activities, rendering them interesting therapeutics resulting in a steadily increasing market demand.
To date, the majority of quercetin and its glycosides are extracted from plant material, which is generally a laborious and low-yielding process requiring many purification steps [25] . In vitro plant cell cultures or engineered plants can be used to overcome the low yields and improve production [26] [27] [28] , however since metabolic engineering of plants is both very controversial and still in its infancy [29] , this approach is often restricted to small-scale production. Although chemical synthesis of quercetin (glycosides) has proven to be feasible [30] [31] [32] , stereoselective formation of glycosidic linkages is often hampered by the presence of various reactive groups [33] , which requires many protecting and deprotecting steps [34] . In addition, the generation of toxic waste and a low atomefficiency [35] render these production processes neither sustainable nor economically viable.