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Lack of Detectable Allergenicity in Genetically Modified Maize Containing “Cry” Proteins as Compared to Native Maize Based on In Silico & In Vitro Analysis

Background Genetically modified, (GM) crops with potential allergens must be evaluated for safety and endogenous IgE binding pattern compared to native variety, prior to market release. Objective To compare endogenous IgE binding proteins of three GM maize seeds containing Cry 1Ab,1Ac,1C transgenic proteins with non GM maize. Methods An integrated approach of in silico & in vitro methods was employed. Cry proteins were tested for presence of allergen sequence by FASTA in allergen databases. Biochemical assays for maize extracts were performed. Specific IgE (sIgE) and Immunoblot using food sensitized patients sera (n = 39) to non GM and GM maize antigens was performed. Results In silico approaches, confirmed for non sequence similarity of stated transgenic proteins in allergen databases. An insignificant (p> 0.05) variation in protein content between GM and non GM maize was observed. Simulated Gastric Fluid (SGF) revealed reduced number of stable protein fractions in GM then non GM maize which might be due to shift of constituent protein expression. Specific IgE values from patients showed insignificant difference in non GM and GM maize extracts. Five maize sensitized cases, recognized same 7 protein fractions of 88-28 kD as IgE bindng in both GM and non-GM maize, signifying absence of variation. Four of the reported IgE binding proteins were also found to be stable by SGF. Conclusion Cry proteins did not indicate any significant similarity of >35% in allergen databases. Immunoassays also did not identify appreciable differences in endogenous IgE binding in GM and non GM maize.

Results
In silico approaches, confirmed for non sequence similarity of stated transgenic proteins in allergen databases. An insignificant (p> 0.05) variation in protein content between GM and non GM maize was observed. Simulated Gastric Fluid (SGF) revealed reduced number of stable protein fractions in GM then non GM maize which might be due to shift of constituent protein expression. Specific IgE values from patients showed insignificant difference in non GM and GM maize extracts. Five maize sensitized cases, recognized same 7 protein fractions of 88-28 kD as IgE bindng in both GM and non-GM maize, signifying absence of variation. Four of the reported IgE binding proteins were also found to be stable by SGF.

Introduction
If the modified plant is from a common allergenic source (e.g. soybean), recommended guidelines suggest testing for increase in the expression of endogenous allergens [14]. Some organizations in Japan and Europe have opted for evaluating endogenous allergens for rarely allergenic plants e.g. maize and rice [15].
Maize is not known to contain endogenous toxins or antinutritional factors till date as per literature search and reports on allergy to maize and its genetic variants in Indian context are still lacking.
The major objective of this study has been to assess the GM maize seeds containing Bt-Cry 1Ab, Cry 1Ac and Cry 1C protein sequences for allergenicity by in silico bioinformatic searches as well as by in vitro methods involving determination of specific IgE binding pattern to GM as well as non-GM maize antigens using maize sensitized patients sera.

Materials & Methods
Three GM maize seeds containing Cry 1Ab, Cry 1Ac and Cry 1C transgenic proteins along with non-GM maize seed were procured from the developer, Metahelix Pvt. Ltd., Bangalore, India as per DBT guidelines.
The transgenes (events) incorporated in seeds were obtained from sources with no allergenic history and have been approved by regulatory agency RCGM-DBT, Govt. of India.

In silico Analysis
Selection of the query protein sequences. Three insecticidal proteins of Bt-Cry 1Ab, Cry 1Ac, Cry 1C (Accession No-Uniprot P0A370,P05068, Q58FM0) which are preferable for development of genetically modified maize as mentioned in IGMORIS database were selected for present investigation.
Sequence Database Selection. All sequence homology search were performed against two allergen specific databases-Allergen Online of Food Allergy Research and Resource Program (FARRP) and Structural Database of Allergenic Proteins (SDAP) [16].
Full Length FASTA Search. FASTA comparisons are initiated by aligning the first match of a specific word size (k-tuple parameter, ktup = 2), followed by extending the alignment. Specific parameters used for this analysis included an expectation threshold of 10, a gap creation penalty of 12 and gap extension penalty of two. The BLOSUM matrix series was derived from a set of aligned, ungapped regions from protein families, called the BLOCKS database. The BLOSUM50 matrix identified blocks of conserved residues that are at least 50% identical. The extent of similarity was calculated as percent similarity and E score (expectation score). The E score reflected the degree of similarity between a pair of sequences based on matches of identical or functionally similar amino acids. A large E score indicated a low degree of similarity between the sequences under study and the sequence from the database and vice versa.
80 amino acid Sliding window approach. FASTA alignment was performed to compare all possible contiguous amino acid segments of each of the 3 proteins against the sequences listed in the databases. Each contiguous 80 amino acid sequence of individual protein was searched starting with 1-80 aa, then 2-81 aa, and so on until the last 80 amino acid segment of each protein is compared with database. In this search, % (percent) identities were calculated to evaluate possible cross allergenicity. An alignment of query sequence if showing >35% similarity over segments as short as 80 amino acids with known allergen(s) depicted that the query sequence might be a potential allergen and should be subjected to further in vivo and in vitro testing [10].

Allergen Database
AllergenOnline Database version 14.0. The Food Allergy Research and Resource Program (FARRP) administered by University of Nebraska, Lincoln provides access to a peer reviewed allergen list, with 1076 protein sequences and sequence searchable database intended for the identification of protein that may present a potential risk of allergenic cross-reactivity. The objective was to identify proteins which might require additional tests, such as serum IgE binding, basophil histamine release or in vivo challenge to evaluate potential cross-reactivity. The database is updated annually and has added 66-85 known or putative allergen sequences (many of which are homologues of proteins al-ready included in the database) per year since 2007 [10]. The scoring matrix used on the AllergenOnline is BLOSUM 50 [17] that is weighted to favor identical matches between amino acids. Output of the FASTA was given as E-score.
Structural Database of Allergenic Proteins (SDAP). SDAP is a Web server that links to the major protein (PDB, SWISS-PROT, PIR, GenBank) and literature (PubMed, MEDLINE) servers with 1526 Allergens and isoallergens. It uses an original algorithm based on conserved properties of amino acid side chains to identify regions of known allergens similar to usersupplied peptides or selected from the SDAP database of IgE epitopes. The major SDAP source of allergens is the IUIS list, http://www.allergen.org and alignments are made with FASTA v 3.45. The alignments between the query sequence and all SDAP allergens are reflected in terms of E score. Sequences with E values < 0.01 are almost always homologous.

Bioassay
Qualitative Evaluation of Transgenic proteins in GM maize seeds. Qualitative GMO Check Bt test kit (KRISHILISA,Krishgen Biosystems, India) was used to evaluate the presence/absence of Cry 1Ab, Cry 1Ac as well as Cry 1C proteins in GM maize. The kit contained precoated microtitre plate containing antibodies to Bt-Cry1Ab, Cry 1Ac or Cry 1C protein along with positive control and extraction buffer. The kit employs the principle of sandwich ELISA of Antibody-Toxin-Enzyme labeled antibody. One twenty five milligrams of respective GM and non GM seed powder was resuspended in 1 ml of Extraction buffer (1X) of the kit provided. The seed suspension was mixed thoroughly and the solids was allowed to settle for 30 minutes and the supernatant was used for the assay. The manufacturer's instructions were followed thereafter as described-50 μl of sample supernatant along with positive control and extraction buffer as blank were added to the precoated microtitre plate. The microtitre plate was mixed slightly and incubated for 15 min, at 37°C followed by addition of 50 μl of enzyme conjugate and incubation for 45 min at 37°C. The plate was then aspirated and washed 4 times with wash buffer (1X). Substrate solution 100 μl was added to all the wells in the plate and incubated for 15-30 min at ambient temperature in dark. Finally, stop solution (100 μl) was added to all the wells and absorbance was measured at 450 nm.
Preparation of Maize Seed Extracts. GM seed powders of maize containing Cry 1Ab, Cry 1Ac and Cry 1C protein along with the non-GM seed powder were suspended in 1:20 (w/v) ammonium bicarbonate buffer (50 mM, pH 8.0) with 5mM ethylene diamine tetra acetate and 1mM phenyl methyl sulfonyl fluoride, followed by continuous stirring for 6 h at 4°C as per standard protocol [18]. The extracts were centrifuged at 10,000g and the supernatant was filtered through a 0.45μm membrane filter. The filterate thus obtained, was lypohilized and stored in small aliquots at -70°C for further use.
The lyophilized extracts were reconstituted in 50% glycerinated phosphate buffered saline (PBS) (1:10 w/v) for use in SPT. A drop of the extract was placed on the volar aspect of the forearm and the skin was pricked by a 26 1/2@ G sterile needle. The SPT reactions were observed after 20 minutes and wheal diameter ! 3 mm were considered positive and graded from 1+ to 3+ based on wheal diameter [18].
Protein Content and Profiling. Protein content in the extracts of GM and non GM maize seeds were estimated by modified Lowrys method by precipitation of proteins using phosphotungstic acid [19].
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% reducing gel was employed for obtaining protein profile of all the antigenic extracts followed by staining with Coomassie Brilliant Blue R-250, CBB [20].
Simulated Gastric Fluid Digestion (SGF). Pepsin digestion assay was carried out to find out the stability of food protein under gastric fluid environment. The protocol of Astwood et al. [21] and Ofori-Anti AO et al. [22] with slight modification was followed. Briefly, a protein extract (680 μg) of each of the four maize seed extracts was treated with 200 μL of prewarmed SGF containing 0.32 w/v percentage of pepsin [23] in 0.03MNaCl, pH-1.2. Digestion was proceeded at 37°C with continuous shaking, and an aliquot (20 μL) of this digest was periodically withdrawn at 0, 1, 5, 15, 30, 45, and 60 minutes for analysis. These aliquots were quickly mixed with 26 μL of a sample buffer (containing 2% β-mercaptoethanol and 4% SDS) for SDS-PAGE together with 6 μL of Na 2 CO 3 solution (200 mmol/L). As control, each protein sample was treated with SGF that did not contain pepsin and processed as described above. Breakdown of GM and non GM seed proteins was evaluated by SDS-PAGE using 12% acrylamide gel.

ImmunoAssay
Ethics Statement. The study protocol was approved by Institutional Human Ethics Committee. Informed written consent was obtained from patients and controls subjects for participating in the study.
Study Subjects. A total of 39 patients of suspected food allergy by history and Skin Prick Test (SPT) from two allergy clinics located in North India (PC) were selected for the immunoassay. The mean age of patients was recorded as 35 ± 2.3 yrs. All the patients recruited in the study showed SPT positivity to one or the other common food allergens including five foods selected for detailed study (Table A in S1 File). Blood samples were drawn from all food sensitised patients (n = 39) as well as healthy volunteers (n = 11) who served as control.
Estimation of Specific IgE (sIgE) to Maize antigens. Serum IgE against three transgenic maize as well non-transgenic maize extracts, was estimated in the sera of all 39 patients as well as 11 healthy volunteers by Enzyme linked Immunosorbent Assay (ELISA) following the protocol of Sepulveda et al. [24]. Protein extract(s) in carbonate buffer (0.2 μg/100 μl per well) was coated in a microtitre plate (Nunc, USA) and kept overnight at 4°C. After repeated washing with phosphate buffer, the nonspecific sites were blocked with 1% Non-fat dry milk for 2 hr. at 37°C. The plate was washed and incubated with 1:10 v/v diluted sera of patients, overnight at 4°C. The plate was washed with phosphate buffer saline-Tween 20 (PBST) usually four times (each wash 3 min) and incubated with monoclonal antihuman IgE-horse radish peroxidase (1:1000 v/v; Southern Biotech, USA) in 0.05% non-fat dry milk in PBST for 4 h at 37°C. Colour was developed with orthophenylene diamine after 4-6 min. The reaction was stopped by adding 50 μl 2.5 M H 2 SO 4 and the absorbance was read at 492 nm and the value of different serum samples was analysed according to the criteria of Kauffman et al [25].
Immunoblot. The SDS resolved proteins of non GM and GM maize antigenic extracts on 12% acrylamide gels were transferred on to nitrocellulose membrane (NCM) as described by Towbin et al [26]. The unbound sites were blocked by 3% Non fat dry milk for 3 hr at 37°C. The NCM strips were washed and incubated with 1:10 v/v food allergic sera at 4°C overnight. Healthy serum pool was taken as control. The strips were washed with PBST and incubated with 1:1000 diluted antihuman IgE-peroxidase. The IgE binding was detected by diaminobenzidine (DAB) with hydrogen peroxide in sodium acetate buffer (pH 5.0). The detection was finally stopped using 0.2N HCl.

Statistical Analysis
All statistical analysis were performed using MS-Excel and Prism V software (Graph Pad Prism, San Diego, California, USA). Values are represented as Mean±SEM(standard error mean). The significance level was considered to be p<0.05 (two-tailed).
Qualitative Interpretation for confirmation of Cry proteins in transgenic/GM seed powder was based on absorbance/Optical Density greater than the cutoff value (Mean for Blank + 0.1) obtained in ELISA.
ELISA based specific IgE values of sera samples were analysed as per Criteria of Kauffman et al. [25] with slight modifications. Serum IgE percent binding for each of the 39 sample relative to control sera(pool) and positivity were calculated as follows: Percent Binding ð%Þ ¼ OD of individual patient sera-OD of control sera OD of highly positive sera-OD of control sera Â 100 OD values depending on per cent binding were categorized into 4 groups i.e < 15, 15-30, 30-60 and > 60%. Sera showing more than 30% binding were considered to have significantly raised (p < 0.05) specific IgE against respective non GM and GM maize antigens.

In silico Analysis
A comparative bioinformatic approach of sequence homology have been used, for assessment of allergenicity of Cry proteins. The length of three transgenic Cry protein sequences is-Cry 1Ab 1155 amino acid(aa), Cry 1Ac 1178 aa while Cry 1C 1044 aa. The sequences of the proteins have been tested against the AllergenOnline and SDAP databases. The result of bioinformatic searches are compiled and analysed as provided in Table 1.
The Bioinformatics Assessment of Cry proteins using FASTA 35 against the FARRP AllergenOnline. The full length FASTA alignment as well as sliding 80 mer window approach for the protein sequence of Cry 1Ab, Cry 1Ac as well as Cry 1C in AllergenOnline database was assessed. Both the tests were performed using the default settings of E value (< 1) and Z score for full length FASTA and a similarity of ! 35% over 80 amino acid stretch ( Table 1). The results obtained on assessment for the proteins revealed that transgenic protein Cry1Ab and 1Ac did not reflected any sequence similarity with any known allergens due to E score >1. On the other hand, the Cry 1C protein exhibited an E score = 0.9, and only 26% full length identity with a fungal protein of Candida albicans (as in Table 1), which is well below the threshold of greater than 50% to be assumed a allergenic sequence as suggested by Aalbrese (2000) [27].
Sliding 80 mer window approach of the FARRP didn't presented any segment in any of the three Cry protein sequences with over 35% similarity to any known allergen. This results also did not adhere to the criteria as laid down by Codex for positivity of allergenic cross reactivity by any of three transgenic proteins (Table 1).
Bioinformatic assessment of the Cry proteins in Structural Database of Allergenic Proteins (SDAP). Protein sequences of Cry 1Ab, Cry 1Ac and Cry 1C were inspected for presence of allergenic segment in another allergen specific database-SDAP. Full FASTA (v 3.45) alignment for the Cry protein sequences was analysed for sequence homology against all the allergens in SDAP with the default parameter to show sequences with E score 0.01. FASTA alignments using sliding 80 mer window for identifying !35% similarity region of amino acids was also observed for three Cry proteins under study in SDAP.
The maximum similarity in full length alignment for the three Cry proteins was obtained as percentage similarity in the range of 6-4%, as described below and depicted in Table 1.
Cry1Ab. Full FASTA alignment of the protein yielded sequence similarity of the range 6-2% with allergens of fungal origin such as Asp f 5 (Aspergillus fumigates) and Pench 20 (Penicillium chrysogenum) as well as food allergen Tria gliadin from Triticum aestivum. Under the 80 amino acid sliding window, a highest of 32% identity with Mala s 1 a reported allergen from Malassezia synpodialis was observed, followed by 27% sequence homology with Glycine max. allergen Gly m 1 and 24% with Bos d 8 from Bos Taurus. Plant derived allergens were more frequent in the compiled matching sequences.
Cry 1Ac. The highest scoring identity for the protein under Full FASTA alignment was found to be~3-2% with different proteins-Tri a gliadin, Anac 2 (Bromelain) from Ananas comosus, globin named Chit 6 from Chironmus thummi, Ves v 2.0, an insect origin Hyaluronidase and timothy grass allergen named Phl p 5. The same protein sequence (Cry 1Ac) under 80 amino acid window showed a highest of thirty percent identity with Asp f 13, a fungal alkaline proteinase from Aspergillus fumigatus followed by 25% identity with Lig v1(pollen allergen) and 23% with Tria gliadin (food allergen).
Cry 1C. The best scoring identity observed under Full FASTA alignment was found to be 4.7% with reported allergen Hev b 9 from Hevea brasiliensis, 4% with fungal peroxisomal protein, Cand a 3 as well as food allergen Tri a 12 from wheat and~3% with Pasn 1,a grass allergen and Bos d 8 (animal allergen). Under the 80 aa window approach highest identity of 33.7% with Cor a1, pollen allergen from Corylus avellana was recorded, 27% with Cand a 3 and 26% with Chaf 1, crab tropomyosin as well as Pasn 1, a plant protein was observed. Specifically comparison of Cry 1C analysis for sequence homology in FARRP and SDAP database presented similarity to a common known allergen from Candida albicans. But the E score was almost equal to the threshold in Full FASTA alignment and percentage similarity was below than required, the probability of assuming Cry 1C protein as allergenic was nullified. Several sequences with decreasing similarities in all the above cases were observed which are not described here.
Quality Analysis of Cry proteins in procured maize seeds. Presence of Transgenic proteins Cry 1Ab, Cry 1Ac and Cry 1C in respective GM maize powder extracts were detected by Qualitative ELISA Kit as stated in methodology. Extraction buffer served as blank while Non GM maize seed, serving as negative control was also assessed for absence of any transgenic protein, by the same protocol. The higher the values for absorbance in ELISA for GM maize, above the described cutoff under section material & methods, the greater the concentration of respective transgenic protein. The absorbance values for GM and blank in each of three cases recorded were as follows-GM with Cry 1Ac and blank were 0.2 ± 0.002, 0.07 ± 0.09; GM with Cry 1Ab and blank were 0.23 ± 0.01, 0.07± 0.05; GM with Cry 1C and blank were 0.34± 0.01, 0.07± 0.09. Since, the absorbance values for GM sample in each of the three cases recorded were greater than the desired criteria, this leads to confirmation for presence of respective transgenic proteins, Fig. 1.
Protein Assay. Protein content in three GM maize antigens was estimated as-Cry 1Ac with 18 mg/gm dry wt., Cry 1Ab with 19 mg/gm dry wt., Cry 1C with 15 mg/gm dry wt. and and in non GM maize as 20 mg/gm dry wt. Thus, there was no significant variation in protein content of three GM maize extracts and non GM extract (p> 0.05).  Maize Specific IgE (sIgE) in Patients Sera. In the sera of 39 suspected food allergic patients and 11 control subjects, specific IgE against non-GM as well as three GM maize extracts was analyzed by ELISA in terms of absorbance/ optical density (OD) and further categorized according to percent binding criteria of Kauffman et al [25] (Fig. 3, Table B in S1 File).
The mean OD values of specific IgE of patients & control subjects against non GM maize were 0.27± 0.02 & 0.03±0.007, in GM maize with Cry 1Ab as 0.25± 0.02 & 0.02 ± 0.008, 0.3 ± 0.02 & 0.033 ± 0.014 against GM maize with Cry 1Ac and in GM maize containing Cry 1C as 0.26 ± 0.02 & 0.02 ± 0.008. A statistically significant difference of specific IgE values was observed in patients v/s control (p < 0.001) with respect to described four groups. Thereby signifying raised IgE levels in patient group against non GM and GM maize antigens under study (Fig. 3).
Further, on the basis of specific IgE values against non GM and GM maize antigens of patient group only, variation among maize antigens (intra species) viz non GM v/s GM maize with Cry 1Ab, non GM v/s GM with Cry 1Ac, non GM v/s GM with Cry 1C was observed to be statistically non significant (p > 0.5). Thus, similarity of specific IgE values of the patients was observed against the non GM as well as GM maize antigens.
In Non GM maize, OD values of the patients ranged from 0.03-0.83 while in the control subjects it ranged from 0.004-0.08. Among the 39 patient sera, 16 showed IgE binding of < 15% as compared to control and were considered showing low specific IgE. Non-GM maize specific IgE of 15-30% was observed in 15 cases and were considered moderately positive. However, serum samples from 9 cases showed more than 30-60% specific IgE binding and selected for further immunoassay studies.
GM maize with Cry 1Ab. All the 39 patients reflected OD values of specific IgE in the range of 0.04-0.6 while in the control subjects the range was 0.0-0.10. Out of the total 39 cases Lack of Allergenicity Assessment in GM Maize evaluated, 19 showed IgE binding of < 15% as compared to control and were considered showing low specific IgE. GM containing Cry 1Ab maize specific IgE of 15-30% was observed in 11 cases and were considered moderately positive. However, serum samples from 9 cases showed 30-60% specific IgE binding.
GM maize with Cry 1Ac. The OD values for specific IgE for 39 patients ranged from 0.03-0.60 while in the control group, the range was 0.008-0.16. Amongst the 39 patients, 16 showed IgE binding of < 15% as compared to control and were considered showing low specific IgE. GM maize specific IgE of 15-30% was observed in 12 cases and were considered moderately positive. However, serum samples from 11 cases showed 30-60% specific IgE binding.
GM with Cry 1C. All the 39 cases reflected OD values of specific IgE in the range of 0.10-0.64 while in the control subjects the range was 0.001-0.09. Out of the sera of 39 cases evaluated, 21 showed IgE binding of < 15% as compared to control and were considered showing low specific IgE. GM maize containing Cry 1C specific IgE of 15-30% was observed in 9 cases and were considered to be moderately positive. However, serum samples from 9 cases reflected 30-60% specific IgE binding while no single sera sample showed greater than 60% binding and a total of 9 sera were selected for further immunoassay.
Thus, out of the 39 sera samples analysed for specific IgE, the same sera of 9 cases reflecting significantly high specific IgE binding of 30-60% against non GM and GM maize antigens were selected. Out of these nine, selective five maize sensitized cases with SPT to maize as well as suspected food allergy were considered for immunoblot. The five patients were all adults varying from 18-53 yrs.(mean age 33 ± 6.3) of age with 1:4 male/female ratio. They suffered either from nasobronchial allergy, urticaria, diarrohea or oral allergy syndrome etc. These patients had recurrent symptoms whenever they consumed corn "roti" or roasted maize preparations. Their symptomatic treatment included antihistamines, bronchodilators and corticosteroids etc. (Table 2).
IgE Immunoblot Reactivity Assay. The identification of IgE binding proteins in non GM and GM maize antigenic extracts was carried out by Immunoblot. Out of the 39 suspected food allergy/sensitized patients sera analysed for specific IgE so far, sera from five shortlisted maize sensitized cases (specific IgE positive) along with pooled sera of control subjects were further used to perform Immunoblot against non GM maize as well as GM maize antigenic extracts. Comparative analysis of IgE binding among non GM and GM maize. In non GM maize extract, 7 protein fractions of 28 (smear), 33, 41, 48,68, 78 and 88 kD approx. were recognized as IgE binding by selected five sera samples on Immunoblot. These same seven fractions were also observed as IgE binding by the same five cases in GM maize extracts of Cry 1Ab, Cry 1AC and Cry 1C respectively. This signifies absence of variation in IgE binding by selected sera samples against non GM and GM maize antigen extracts (Fig. 4).

Discussion
Food is one of the basic requirement without which survival is at stake. In India, food production capacity has to be substantially raised to meet growing demand for human food and a falling ratio of arable land population. The major concern for farmers is to protect the crops both during pre and post harvesting conditions. To address these major concerns, varieties of crops are successfully engineered through genetic engineering i.e GM crops to improve crop quality, enhanced pest resistance, reduced dependence on pesticides, increased nutrient component, and production of pharmaceuticals etc. The same desirable traits may also be achieved through conventional breeding but it has limitations in terms of long duration for development, species barrier, concern for pesticide residue in crops and as a result genetic engineering has taken advantage for development of GM crops. This "gene revolution" as compared to "green revolution" is poised to benefit both poor and rich farmers equally and has an immense potential in transforming global and local agriculture.
Cereals are the major energy constituents of Indian food diet with maize being the third most important cereal after wheat and rice. In the past, efforts to increase maize production have been productive but there have been reports on annual loss of maize [28] of about 13.2% due to diseases. In India, spotted stem borer (Chilo partellus) and Sesamia inferens are serious pests causing diseases as stalk and ear rots of maize. It is very difficult to control this pest using contact insecticides since it bores into the stem and feeds on tissue affecting the growth of plant [29]. The establishment of the All India Coordinated Maize Improvement Project (AICMIP) in 1957, had led to significant progress in maize breeding with development of a large number of hybrids and composites with high grain yield, appropriate seed maturity, pesticide resistance etc.
However,the progress in breeding for insect resistance has been slow due to lack of effective insect pest rearing methods and germplasm screening technique [29]. This issue is being addressed and resolved through the approach of biotechnology, leading to development of GM maize containing traits such as insect resistance due to expression of Bt cry genes (Cry proteins) and herbicide tolerance through enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase transgene by Indian seed developers. Cry proteins in GM crops are expressed as large insoluble proteins aggregates, in normal conditions and are safe for humans, higher animals and most insects but become solubilized as active toxin in reducing conditions of high pH = 9.5, as found in mid gut of lepidopteran larvae feeding on Bt Crops which leads to killing of these pests. These characteristics favour long term environmental friendly use as transgenic proteins [30].
Batista et al. [31] studied the allergenicity of three GM maize varieties expressing different levels of Cry 1Ab protein, based on SPT and Immunoblot reactivity assay in two groups of sensitive population with the assumption that participating population were consuming maize derived products from a GM maize source. Their study was carried in 2004 while the selected GM materials were commercialized by European Union since 1998. The same group concluded for safe nature of Cry 1Ab because of absence of detectable differences in IgE reactivity between GM and non maize. Buzoianu et al. [32] and Walsh et al. [33], have reported for non significant effect on growth and immune response of animals feed with GM maize (Bt-MON810) expressing Cry 1Ab and ruled out for any possibility of cry1Ab gene or protein translocation to the organs and blood at any stage of life cycle.
As per the guidelines of national and international scientific agencies such as ICMR-DBT, Codex, GM crops (before commercialization) need to go through a safety and allergenicity assessment for considering it is as safe as its traditional counterpart. In India, Bt cotton is the only commercialized crop till date. As per the data of IGMORIS [29], many GM maize crops with Cry proteins are under field trials and need to be evaluated for safety aspects.
To fill the lacunae of limited information on allergenicity to maize, the present work is targeted at comparing the IgE binding profile of GM maize containing Cry proteins with the non-GM maize using selected maize sensitized sera of Indian patients. The objective of the study has been achieved with a combination of in silico and in vitro tools. Cry protein sequences have been assessed for sequence similarity to known allergens using FASTA based bioinformatics tools for both full length sequence similarity as well as using short stretch of 80 amino acid window with cutoff criteria of > 35% sequence similarity [2,5,27] and found that these proteins are of non-allergenic source. Our findings are in accordance with an earlier study by Verma et al. [34] reporting for validation of the safety aspect of Cry 1Ab and Cry 2Ab inserted proteins sequences using in silico tools and predicted them as safe for genetic incorporation in plant kingdom. Similarly, Randhawa et al. [35], also reported that in various Cry group of proteins viz Cry1Ac, Cry1Ab, Cry2Ab, Cry1Ca, Cry1fa/Cry1Ca which are being incorporated in Bt Crops indicated no significant alignment and similarity at domain level with allergens, thus revealing no potential risk of allergenic cross-reactivity. Thus, our findings by in silico analysis on three Cry proteins confirm the findings of earlier workers [34][35] for incorporation in GM maize.
The second aspect of our study has focused on in vitro analysis to compare for changes in endogenous protein expression level due to incorporation of Cry 1Ab, Cry 1Ac and Cry 1C transgenes in maize seeds.
Melinda et al. [36] reported on Glyphosate-Tolerant Soybeans and its parental variety for determination of basic nutritional and biologically active components over three years of field testing. Analytes such as fat, protein, carbohydrates, isoflavones were determined and it was reported that the genetically engineered soybean with glyphosate tolerance was substantially equivalent to the conventional soybean. Similarly Noteborn et al. [37] studied safety and toxicity assessment of transgenic tomatoes expressing Bt Cry 1Ab protein, and observed no major changes in nutrient composition by chemical analysis between the transgenic and the native variety.
The variability in protein content among the three GM maize varieties and the non GM variety evaluated by us did not show any statistically significant variation (p> 0.05). Thus, absence of significant variability in protein content among GM and parental variety of maize are in synchrony with earlier reported studies [36][37].
Codex and the European Food Safety Authority have stressed on the potential that a newly expressed protein might become an allergen should be evaluated based on resistance to digestion in pepsin and abundance in food fractions [14]. However, each country has modified the testing criteria as per their regulations and policies but still adhere to Codex guidelines for food safety to facilitate international trade.
The simulated gastric model also provides a method to assess allergenic potential of endogenous proteins upon introduction and expression of foreign gene (with no allergenic history) such as Bt Cry genes, CP4 EPSPS, into food plants. Purified proteins and stained gel have been emphasized for analysis for stability in SGF by Astwood et al. [21]. Purified commercial preparations of various potential transgenes have been analyzed for IgE binding using sensitized human sera samples also. Batista et al. [31] have performed for SPT and IgE immunoblot reactivity assay on pure Cry 1Ab and CP4EPSPS proteins using selected maize and soybean sensitized patients sera and observed for absence of binding to these control proteins by any of the sera.
The relative stability profile on SDS-PAGE of pepsin digested antigenic extracts of GM and non GM maize seeds revealed different stable fractions. Higher molecular weight protein fractions mainly above 70 kD in all the three GM maize antigens were found to be more susceptible, as lesser time duration was required for digestion as compared to proteins above > 70 kD present in non GM maize which were observed to be partially digested as evident from faint appearance even after 1 hr. of digestion (Fig. 2). Protein fractions which were reluctant to digestion and interpreted as "stable" in the non GM and GM maize extracts were observed to be in the range of 68-9 kD. Six stable protein fractions of the range 60, 38, 28, 19, 14 & 10 kD were observed to be stable in non GM maize antigen. In the GM maize with Cry 1Ac, 5 protein fractions of the range 66-9 kD were observed as stable fractions while in the GM maize with Cry 1Ab five stable protein fractions-68, 36, 30, 25 & 16 kD were observed. Also, in GM with Cry 1C, five stable protein fraction of the range 67, 31, 28, 17, 14 kD were recorded. Thus, there seems to be no overall difference in protein profile of the three pepsin digested GM maize antigens. Presence of a extra protein fraction in addition to five stable protein fractions in non GM maize antigens as against five in GM maize antigens suggest that incorporation of transgene in maize genome might have resulted in change of the constituent expression of protein fraction of the range 68-9 kD which has led to alteration (reduction) in number of protein fractions within similar molecular weight range.
No identifable differences in allergic potency by specific IgE and Immunoblot as well as changes in endogenous proteins of genetically engineered soybean varieties containing glyphosate tolerant genes as against wild varieties have been reported by Burks and Fuchs [38]. In a study by Satoh et al. [15] on salt soluble protein extracts of non-transgenic and transgenic rice, patients revealed few qualitative differences between the IgE-binding proteins of the two extracts by 1D immunoblot. The antigen-specific-animal sera show no qualitative or quantitative differences in two known allergens of rice, RAG2 and glyoxalase I as well as novel proteins at 19, 52, and 63 kD. Metcalfe et al. [5] recognized that for less commonly allergic sources such as rice or corn, five test sera may be sufficient for an adequate safety test. In the present investigation, the variation in the specific IgE values against the transgenic and non transgenic maize antigens from the sera of 39 food sensitized patients were statistically non significant (p >0.05). This signifies that three GM maize antigens under analysis does not differ in the ability to bind serum IgE as presented by non GM maize, similar to earlier reported observations [15,31,38]. Based on Kauffman et al. [25] criteria, out of 39 cases analysed for maize specific IgE, nine (23%) patients reflected greater than 30% binding to the GM and non GM maize antigens and were considered having significantly high maize specific IgE. Further in the analysis, using sera of five cases with clinical sensitization to maize, seven protein fractions in the range 88-28 kD were recognized as IgE binding in non GM maize antigen. Simultaneously, these 7 fractions in GM maize antigens with Cry 1Ab, Cry 1Ac and Cry 1C were also recognized as IgE binding. Thus, an overall similarity in IgE binding pattern to non GM and GM maize antigens was observed. This finding are in accordance with reported guidelines by Taylor suggesting for low levels of Cry proteins expression in transgenic foods, with allergic sensitization being more likely to occur to major or constituent proteins that exists in foods [39].
Pastorello et al. [40] have reported two major maize allergens as-an alpha-amylase inhibitor and a 9-kDa LTP, lipid transfer protein showing 36% and 86% binding in Italian population respectively. Other relevant allergens such as 30-kDa endochitinases A and B, 19 kDa zein-β precursor, and 26 kDa zein-α precursor have also been reported. High frequency of maize allergy in Italy has been due to high exposure to airborne maize dust, due to its high production of about 10.5 million tons per year (ISTAT official data 2008, www.istat.it).
Fasloli et al. [41] have identify broadly 10 protein fractions in maize extract as vicilin like embryo storage proteins, globulin-2 precursor, 50 kDa gamma-zein, endo-chitinase, thioredoxin and trypsin inhibitor. With respect to present observations, of the detected 7 IgE binding proteins on Immunoblot, it is suspected that 4 low molecular weight proteins of 28-48 kD could possibly be classified as zeins and remainder three higher molecular weight could be seed storage proteins as vicilin [40][41].
A correlation between the allergenicity of a protein and its digestibility in SGF has been suggested [21]. Out of the seven IgE binding protein fractions obtained by us, four fractions of approx. 28, 33, 41 & 62 kD have also been found to be stable proteins in the GM and non GM maize extracts as observed in the simulated gastic fluid digestion even after 1 hour.
In conclusion, based on in silico tools, it is reconfirmed that Cry 1Ab, Cry 1Ac and Cry 1C protein sequences are non allergenic sequences with no cross reactivity to known allergens and incorporation with these transgene protein sequences presents no appreciable changes in endogenous protein expression of GM and non GM maize seeds as analysed by specific IgE and Immunoblot using native maize allergic patients sera.
Supporting Information S1 File. Table A. Food Sensitization details based on SPT and specific IgE positivity of the enrolled 39 patients. Legends-Ã Only three patients showed specific IgE positivity to given antigens. Table B. Data for specific IgE values of 39 patients and 11 healthy controls against GM and non GM maize antigens. (DOCX)


extfieldsofstudy: Medicine Biology

text:
The relative magnitude of transgene-specific adaptive immune responses induced by human and chimpanzee adenovirus vectors differs between laboratory animals and a target species

Highlights • HAdV-5 (HAdV-C) vectors are more immunogenic than AdC68 or ChAdOx1 (HAdV-E) vectors in mice.• In mice, CD8+ T cell responses peak later, and are more durable after HAdV-5 vaccination.• In cattle, ChAdOx1 is at least as immunogenic as HAdV-5.

Introduction
Replication-defective recombinant adenoviruses are now widely employed as vectors in the development of both human and veterinary vaccines [14,16,40]. Pre-existing neutralising antibodies to common human adenoviruses such as human adenovirus serotype 5 (HAdV-5) may have contributed to the lack of clinical efficacy of some vaccine vectors based on this serotype in human trials [21]. Adenoviruses isolated from chimpanzees and other great apes group phylogenetically within the human adenovirus species [32] but the seroprevalence of neutralising antibodies against these serotypes in humans is considerably lower than against HAdV-5, prompting the development of chimpanzee adenoviruses (ChAds) as vaccine vectors [8,42]. ChAd vectors have primed unprecedented frequencies of antigen-specific CD8 + T cells in recent human clinical vaccine trials [26,35,36].
For veterinary applications, HAdV-5 is still the most commonly used human adenovirus serotype. Indeed, a new foot-and-mouth disease virus (FMDV) vaccine based on this serotype has recently been licensed in the United States for use in cattle [27]. Some studies have successfully used serotypes originating from the target species, such as bovine adenovirus vectors in cattle, despite pre-existing immunity to the vector [46]. However, the use of alternative adenovirus serotypes has yet to be fully explored.
Vector serotypes for both human and veterinary applications are currently screened and selected largely on the basis of pre-clinical studies in mice [8]. However, it remains unclear to what extent these pre-clinical studies predict immunogenicity and efficacy in other mammalian species. In this study we compared antigenspecific immune responses elicited by vaccination with a HAdV-5 vector (Human adenovirus C, here referred to as species C) or a new chimpanzee adenovirus vector ChAdOx1 [13] (Human adenovirus E, here referred to as species E), in mice and cattle. In cattle, we tested vectors encoding mycobacterial antigen 85A to represent bovine tuberculosis vaccine candidates in the target species [12,43]. This is the first time, to our knowledge, that a chimpanzee adenovirus vector, or any vector derived from species E, has been tested in a ruminant species. In both species, we demonstrate differences in the ability of vectors of different serotypes to elicit cellular and humoral immune responses. Furthermore, by comparing studies in laboratory animals and cattle, we demonstrate that comparative immunogenicity studies in mice do not predict the hierarchy of vector performance in a target species.

Mice and immunizations
Female BALB/c or C57BL/6J mice (Harlan, UK) above 6 weeks of age were immunized intramuscularly (i.m.). Viral vectors were formulated in phosphate buffered saline (PBS, Sigma, UK) in a total volume of 50 l and injected into the tibialis anterior muscle of each animal. All mouse procedures were performed in accordance with the terms of the UK Animals (Scientific Procedures) Act Project Licence (PPL 30/2414 or PPL 30/2889) and were approved by the University of Oxford Animal Care and Ethical Review Committee.

Mouse immunology
Spleen ex vivo interferon-gamma (IFN-␥) ELISpot was performed as described previously [19]. To measure vaccine antigen specific responses, cells were re-stimulated for 18-20 h with peptides (Supplementary Table 1) at a final concentration of 1 g/mL. Spot forming cells (SFC) were measured using an automated ELISpot reader system (AID). Flow cytometry on peripheral blood mononuclear cells (PBMC) was performed as described previously [37], except that peptide re-stimulation was with 1 g/mL Pb9. All fluorophore-conjugated antibodies were obtained from eBiosciences. Data were acquired on a CyAn-ADP flow cytometer (Dako) and analysed using FlowJo (Treestar). The program SPICE was used to generate graphical representations of functional T cell responses using backgrounddeducted data [31]. IgG endpoint ELISA was performed as described previously [6,15] except plates were coated with recombinant GFP protein (Millipore, UK) at 1 g/mL.

Cattle immunization
MHC-defined, weight-and age-matched, conventionally reared Friesian Holstein cattle from The Pirbright Institute's (Pirbright) MHC-defined herd were vaccinated i.m. with viral vectors formulated in PBS.

Statistical analyses
Statistical analyses as indicated in figure legends were performed using Prism (GraphPad Software, Inc.).

HAdV-5 vectors elicit higher frequencies of vaccine antigen specific T cells and antibodies in mice than AdC68 and ChAdOx1 based vectors
The ability of AdC68 and ChAdOx1 to induce transgene product specific T cell and antibody responses was compared with HAdV-5 in a series of dose-response experiments in mice with vectors encoding the model antigen TIPeGFP [1]. Responses were assessed after two weeks, identified previously to be at or near the peak of the antigen specific CD8 + T cell response after adenovirus vector administration [4,38]. HAdV-5 elicited higher frequencies of splenic transgene product specific IFN-␥ + CD8 + and CD4 + T cell responses than either of the chimpanzee adenovirus vectors in the 10 5 -10 8 infectious units (ifu) dose range ( Fig. 1A-C). The two chimpanzee adenovirus vectors induced similar CD8 + and CD4 + T cell frequencies, consistent with our previous studies [13]. GFP-specific total IgG antibody titers at the maximum 10 8 ifu dose were approximately two orders of magnitude higher after HAdV-5 than after ChAdOx1 or AdC68 (Fig. 1D). Titers were below the limit of detection after administration of either ChAd vector at doses lower than 10 8 ifu, though detectable titers were still observed after vaccination with 10 6 ifu HAdV-5 (data not shown). Higher transgene specific CD8 + T cell frequencies (at 10 6 ifu) and IgG endpoint titers (at 10 8 ifu) were also observed in C57BL/6 mice after vaccination with HAdV-5 TIPeGFP than with ChAdOx1 TIPeGFP ( Supplementary  Fig. 1). We compared the CD8 + T cell responses elicited by HAdV-5 and ChAdOx1 vectors encoding antigen constructs that have been employed in clinical trials: 85A [12,39,43] and NP + M1 [2]. IFN-␥ ELISpot responses to the epitope P11 were of higher frequency after HAdV-5 85A administration than after ChAdOx1 85A at a dose of 10 8 ifu (p < 0.001) ( Fig. 2A). Recombinant HAdV-5 NP + M1 elicited a higher frequency of CD8 + T cells against the immunodominant epitope NP 147-155 than ChAdOx1 NP + M1 at a dose of 10 6 (p < 0.001) but not at doses of 10 7 or 10 8 ifu (Fig. 2B).
3.2. The IFN-+ CD8 + T cell response after HAdV-5 vaccination exhibits a later peak magnitude and contraction to memory than after AdC68 and ChAdOx1 vaccination IFN-␥ + CD8 + T cell responses were monitored longitudinally by intracellular cytokine staining (ICS) of peripheral blood mononuclear cells (PBMCs). Fig. 3A shows that, after an intramuscular dose of 10 8 ifu, HAdV-5 TIPeGFP elicited a higher frequency of Pb9 specific CD8 + T cell responses throughout the eight-week duration of study than AdC68 and ChAdOx1 vectors expressing the same recombinant antigen. The peak magnitude of the response after HAdV-5 administration was at the three-week time-point, while the peak of the response after vaccination with either ChAd vector was at, or prior to, the two-week time-point.
To assess memory phenotype, PBMCs were stained for the surface markers CD62L and CD127 to define effector T cells (T EFF ), effector memory T cells (T EM ), and central memory T cells (T CM ) as described previously [3] (Fig. 3B). Although the percentage of Pb9 specific CD8 + T cells displaying a T EFF phenotype decreased with time in all three groups (consistent with a gradual differentiation to T EM ), HAdV-5 vaccination elicited an increased T EFF response compared with AdC68 and ChAdOx1 up to 35 days post-vaccination. However, 56 days post-vaccination, the relative proportions of T EFF and T EM cells were comparable between all three groups, despite the overall frequency of Pb9 specific IFN-␥ + CD8 + T cells being statistically significantly higher in the HAdV-5-vaccinated group (Fig. 3B).
At a 100-fold lower dose (10 6 ifu), there was a trend towards higher responses after HAdV-5 vaccination, but IFN-␥ + CD8 + T cell frequencies were not significantly different from those in the AdC68-vaccinated group (Fig. 3C). Memory phenotypes could not be determined reliably owing to the limited size of the Pb9-specific CD8 + T cell population.  Pb9 specific IFN-␥ + CD8 + T cells as a percentage of total CD8 + T cells. Graphs display mean and SEM. Statistical analysis by two-way repeated measures ANOVA with Bonferroni post tests. HAdV-5 versus AdC68; *** p < 0.001, ** p < 0.01. HAdV-5 vs ChAdOx1; † † † p < 0.001 (B) Pb9 specific CD8 + IFN-␥ + T cells from the same mice as in A were identified as TEFF (CD127 − CD62L − ) or TEM (CD127 + CD62L − ), and the frequency of each subset was calculated as a percentage of the total response. Frequencies of CD8 + TCM cells (CD127 + CD62L + ) were <1% of the total IFN-␥ + CD8 + T cell response at all time points. Stacked bars display mean and SEM; statistical analysis by one-way ANOVA at each time point. ** p < 0.01, * p < 0.05. (C) The experiment in A-B was repeated using doses of 10 6 ifu. HAdV-5 vs ChAdOx1; † † p < 0.01, † p < 0.05. No statistically significant differences between HAdV-5 and AdC68 groups.

Cytokine secretion by vaccine antigen-specific CD8 + T cells varies with time after vaccination with HAdV-5 versus ChAdOx1
Production of IFN-␥, TNF-␣ and IL-2 by Pb9 specific CD8 + T cells was assessed by ICS of PBMCs collected from the same animals used in the experiment shown in Fig. 3. Twelve days post vaccination, a significantly higher percentage of cells produced more than one of these three cytokines after HAdV-5 vaccination than after AdC68 or ChAdOx1 (Fig. 4). The percentage of cells expressing all three cytokines was also significantly higher in the HAdV-5  Fig. 3A and B) for IFN-␥, IL-2, and TNF-␣. The proportion of CD8 + T cells producing all three cytokines (orange), any combination of two cytokines (yellow), or any one cytokine only (green), in response to peptide re-stimulation is show in the pie charts below. Statistical analyses on pie chart segments were performed by one-way ANOVA with Bonferroni post-tests. Segments from ChAdOx1 and AdC68 groups that differ statistically significantly in percentage from the corresponding segment in the HAdV-5 group are indicated. *** p < 0.001,** p < 0.01, * p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) vaccinated group. At 21 days and 35 days post-vaccination, the percentage of cells expressing more than one cytokine remained significantly higher in the HAdV-5 vaccinated group. At 56 days post-vaccination, cytokine production profiles were comparable in groups vaccinated with HAdV-5 and ChAdOx1, though an elevated percentage of cells expressing only one of the three cytokines was maintained in the AdC68 vaccinated group (Fig. 4).

ChAdOx1 elicits higher vaccine antigen specific T cell frequencies and antibody titers than HAdV-5 after intramuscular administration in cattle
Transgene product-specific T cell and antibody responses were compared after intramuscular injection of cattle with 10 9 ifu of HAdV-5 85A or ChAdOx1 85A vectors. Fig. 5A shows that 85Aspecific IFN-␥ + CD8 + T cell frequencies in the blood were highest at the two-week time-point and were statistically significantly higher at this time-point after ChAdOx1 than after HAdV-5 vaccination. 85A-specific IFN-␥ + CD4 + T cell responses followed a similar kinetic, albeit at lower frequencies than the CD8 + T cell responses, and the difference in frequency between vectors was not statistically significant at any time-point (Fig. 5B). 85A-specific serum IgG antibody titers continued to rise during the first four weeks after vaccination with both vectors (Fig. 5C). There was a trend toward higher anti-85A endpoint titers after ChAdOx1 vaccination throughout the six-week duration of study, but statistical significance was reached at the two-week time-point only.

Cytokine production profiles of vaccine antigen specific CD8 + T cells are comparable after vaccination with HAdV-5 and ChAdOx1 vectors in cattle
Production of IFN-␥, TNF-␣ and IL-2 by 85A-specific CD8 + T cells at the two-week and three-week time-points was assessed by ICS Fig. 6. Similar profiles of expression of three cytokines by CD8 + T cells in cattle vaccinated with HAdV-5 and ChAdOx1. ICS of 85A-specific CD8 + T cells (from the same experiment as Fig. 5) for IFN-␥, IL-2, and TNF-␣ at (A) two weeks or (B) three weeks post-vaccination. The proportion of CD8 + T cells producing all three cytokines (orange), any combination of two cytokines (yellow), or any one cytokine only (green), in response to peptide re-stimulation is shown in the pie charts. Bar charts indicate the percentages of total CD8 + T cells secreting each combination of cytokines. Statistical analyses were performed by two-tailed t-tests between groups at each time-point; there were no significant differences at either time-point. There were also no statistically significant differences in the percentages of 85A-specific CD8 + T cells producing one, two or three cytokines between the two and three week time points by one-way ANOVA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) of PBMCs collected from the same animals used in the experiment shown in Fig. 5. The data shown in Fig. 6 indicate that the profiles of cytokine secretion of induced CD8 + T cells were similar between ChAdOx1 and HAdV-5 vaccinated animals at both these time points. There were no statistically significant differences in the percentages of 85A -specific CD8 + T cells producing one, two or three cytokines between the two and three week time points.

Discussion
This study revealed marked differences in the relative magnitude of transgene product specific T cell and antibody responses elicited by human and chimpanzee adenovirus vectors in two mammalian species. In mice, HAdV-5 elicited T cell and antibody responses were higher in frequency than HAdV-E chimpanzee adenovirus vectors after intramuscular immunization. This relationship was observed using three antigens and in two mouse strains, suggesting that it is independent of the transgene or inbred mouse strain. In cattle, an important target of veterinary vaccination, a different relationship was observed; ChAdOx1 was as immunogenic as HAdV-5.
In mice, the observation that HAdV-5 vectors are more immunogenic than HAdV-E ChAd vectors is consistent with some published studies [7,8,29] but is not supported by others [28,30,38]. Disagreements within the published literature may result from differences in the method used to determine vaccine dose [13]. Previous publications that are not consistent with the current study based vaccine doses on viral particle estimation rather than on infectious titers.
The relationship between vector dose and the magnitude of CD8 + T cell responses varied considerably with different transgene antigens or epitopes, highlighting the importance of conducting comparative studies with multiple antigens across a range of doses ( Figs. 1 and 2). Mechanisms responsible for differences in the magnitude of responses against different transgene antigens are not well understood, but likely involve differences in the efficiency of antigen expression, peptide processing and presentation on MHC molecules, and the hierarchy of immunodominant epitopes present within both the viral vector and antigen construct [33,45]. Previous studies comparing vectors in mice have typically administered vaccine doses in the order of 10 10 viral particles (equivalent to 5 × 10 8 -5 × 10 9 ifu): at high doses, potential differences in immunogenicity between vectors may be undetectable as the magnitudes of responses reach a plateau. At a dose of 10 8 ifu, Pb9 specific IFN-␥ + CD8 + T cell responses reached peak magnitude later and were more durable after HAdV-5 vaccination compared to ChAdOx1 and AdC68. However, at a 100-fold lower dose, the magnitude and kinetic of the Pb9 specific CD8 + T cell response was comparable between all three vectors (Fig. 3C) demonstrating a dependence on vector dose, as previously suggested [29]. We also observed a later contraction to memory after HAdV-5, with a significantly higher proportion of CD8 + T cells retaining an effector (T EFF ) phenotype compared to the ChAd vectors. After eight weeks, the proportion of T EFF relative to effector memory CD8 + T cells (T EM ) was similar for all three vectors, with a majority adopting a T EM phenotype as observed previously [30,44].
Few previous studies have assessed the kinetics of vaccine antigen specific T cell responses after adenovirus vector administration in cattle [43], and none to our knowledge have compared the immunogenicity of vectors derived from members of different human adenovirus species. In the current study, IFN-␥ + CD8 + and CD4 + T cell responses were highest two weeks post-vaccination (Fig. 5), similar to observations in mice at the lower 10 6 ifu dose (Fig. 3C). We have not investigated the effect of dose on the timing of peak CD8 + T cell frequency in cattle. Both HAdV-5 and ChAdOx1 induced higher CD8 + T cell responses than CD4 + T cell responses, in agreement with data in mice from this study and the consensus in the literature [18,40]. Antibody titers continued to increase at least six weeks after immunization of both vectors. The continued elevation could suggest that expression of the transgene product is persistent, as in mice [41], although studies in cattle with different antigens have observed antibody titers reaching a plateau after just two weeks [24] and a marked reduction in transgene expression at the injection site after 24 h [23]. The kinetics of CD8 + T cell, CD4 + T cell, and antibody responses were similar after HAdV-5 and ChAdOx1 vaccination, perhaps a function of the equivalence in magnitude of these responses in cattle.
Recent studies have suggested that the ability of primed CD8 + and CD4 + T cells to secrete multiple cytokines may be an important requirement for establishing a protective immune response [5,11,34]. In mice, a higher proportion of CD8 + T cells primed after HAdV-5 vaccination secreted two or more of the three cytokines IFN-␥, TNF-␣ and IL-2 during the first 35 days post vaccination compared to AdC68 and ChAdOx1 vaccinated animals (Fig. 4). In cattle, however, there were no statistically significant differences in cytokine secretion profiles between CD8 + T cells primed after HAdV-5 or ChAdOx1 vaccination (Fig. 6).
It is unclear whether the differences in frequency and phenotype of vaccine antigen specific T cells observed in this study are due to differences in intrinsic vector biology including activation of distinct innate signalling pathways, or simply due to differences in the effective antigen dose delivered by HAdV-5 and ChAd vectors. In mice, a previous study has shown that HAdV-5 elicits higher transgene expression in vivo after vaccination and within murine DCs transduced in vitro compared to AdC68 [17]. In this study, the kinetic of the CD8 + T cell response in mice was dose dependent, and HAdV-5 responses at 10 6 ifu followed a similar kinetic to ChAd induced responses at 10 8 ifu. Persistence of antigen after HAdV-5 administration at the 10 8 ifu dose may be responsible for the observed differences in kinetic and for the delayed contraction of CD8 + T cells to a T EM phenotype. Indeed, the proportion of transgene specific CD8 + T cells expressing CD127 has been shown to decrease with increasing dose of HAdV-5 [29]. Differences in the proportion of T cells secreting multiple cytokines may also be a function of the overall magnitude of the response. In cattle, CD8 + T cell responses were similar in magnitude between HAdV-5 and ChAdOx1, and no significant differences in T cell 'multi-functionality' were observed.
At present, selection of adenovirus vectors for human and veterinary applications is largely based on comparative studies in mice [8]. Here, we show that the hierarchy of vector immunogenicity in mice does not predict relative immunogenicity in cattle. Vectors derived from members of species E, including those isolated from chimpanzees, represent promising alternatives to current HAdV-5 based bovine vaccine platforms.

Conflict of interest statement
MDJD, SCG, AVSH, and MGC are named inventors on a patent application describing the ChAdOx1 vector (PCT Application No. PCT/GB2012/000467).


extfieldsofstudy: Biology Medicine

text:
d-Amino Acids Do Not Inhibit Biofilm Formation in Staphylococcus aureus

Bacteria can either exist in the planktonic (free floating) state or in the biofilm (encased within an organic framework) state. Bacteria biofilms cause industrial concerns and medical complications and there has been a great deal of interest in the discovery of small molecule agents that can inhibit the formation of biofilms or disperse existing structures. Herein we show that, contrary to previously published reports, d-amino acids do not inhibit biofilm formation of Bacillus subtilis (B. subtilis), Staphylococcus aureus (S. aureus), and Staphylococcus epidermis (S. epidermis) at millimolar concentrations. We evaluated a diverse set of natural and unnatural d-amino acids and observed no activity from these compounds in inhibiting biofilm formation.

Introduction
Most bacteria escape environmental chemical and physical stresses by forming complex matrix encapsulated aggregates known as biofilms [1]. The biofilm matrix consists of a polymeric conglomeration of exo-polysaccharides, proteins and extracellular DNA [2,3]. Biofilm formation is crucial in the lifecycle of bacteria and is tightly regulated with both spatial and temporal control. In bacterial cultures, biofilm develops either at the liquid-air interface (e.g., B. subtilis) [4] or at the solid-liquid interface (e.g., S. aureus and S. epidermis) [4,5,6,7]. With these models in hand, great strides have been made in terms of obtaining key insight into the steps that go into the formation of biofilms, the identities of the macromolecules contained within these matrices, and the phenotypic changes that occur during this process.
Microbial biofilms are of significant environmental and biomedical importance [8,9]. Formation of biofilms can have severe detrimental and life-threatening impact in clinical settings [10]. For example, bacterial biofilms frequently develop on surgical implants, which may lead to deteriorating performance and further complications [8,9,11]. Most significantly, over 80 percent of all incidences of microbial infections are related to biofilms [11]. Biofilm-associated bacterial cells are inherently more tolerant to antibiotic treatment and can lie dormant to resist the actions of the antimicrobial agents [11,12]. The development of effective small molecule agents that can disrupt biofilm formation and disperse existing biofilm is of prime importance [13,14,15,16,17]. Recently, the field was invigorated by the demonstration that unnatural Damino acids can effectively inhibit and disperse bacterial biofilms [4,5,18]. Media from aged disrupted biofilm cultures were shown to contain significant levels of D-amino acids and the same exhausted media was sufficient to prevent biofilm formation in fresh cultures. Importantly, the enantiomeric L-amino acid counterparts failed to show any activity in biofilm disruption and disassembly, highlighting the specificity and hinting at a mechanism of inhibition. It was hypothesized that D-amino acid inhibition of biofilm formation proceeded via the insertion of the unnatural D-amino acids in place of the terminal D-alanine in the stem peptide of the bacterial peptidoglycan [19]. While single amino acid treatment proved to be effective, synergistic effects were also observed with the concurrent incubation of three unnatural D-amino acids.
The promising anti-biofilm activities reported for D-amino acids led us to investigate a large series of unnatural D-amino acids in search of more potent D-amino acid agents. We hypothesized that by exploring a larger and more diverse chemical space for the side chain of the D-amino acid, we would discover potent inhibitors of bacterial biofilm. Instead, we found that, contrary to published reports, none of the unnatural D-amino acids we evaluated displayed specific inhibition of biofilm formation against strains of B. subtilis, S. epidermis, and S. aureus at millimolar concentrations.

General Methods
All D-amino acids were purchased from either ChemImpex or PepTech (98%+ purity). NaCl, MnCl 2 and glycerol were purchase from Fischer. Luria broth (LB) and Tryptic soy broth (TSB) were purchase from Sigma and BD respectively. Costar 3628 96-well and Costar 3337 24-well sterile polystyrene, flat bottom tissue culture treated plates with low evaporation lid were purchased from corning. Absorbance was recorded on a Tecan Infinite F200 plate reader at 595 nm. Images were recorded on a Canon Powershot ELPH 100HS digital camera.

Preparation of D-amino acid Stock Solution
Individual stock solutions of 100 mM of D-amino acids were prepared by dissolving appropriate amounts of each D-amino acids into 0.2 M NaOH.
S. aureus (SC01 and ATTC 12228) biofilm assay (with 1 mM of D-amino acids) S. aureus SC01 were grown in LB overnight at 37°C with shaking and diluted 1:100 in TSB medium supplemented with NaCl (3%), glucose (0.5%), appropriate D-amino acid stocks in 0.2 M NaOH (1 mM), HCl (4 mM) and PBS (0.5X). This bacterial culture was then transferred (200 μL) into 96-well plates and incubated either 24 h or 48 h with low evaporation lid at 37°C without shaking and covered with aluminum foil to protect from light. The supernatant were discarded and the plates were washed twice with PBS (1X, 200 μL) and dried at 65°C for 1 h. The plates were then cooled to room temperature and stained with 1% crystal violet for 10 min. The plates were then washed twice with double distilled water (200 μL) and dried overnight. The crystal violet stained biofilms were dissolved by adding 95% ethanol (200 μL) to each well and shaking for 2 h. The contents were diluted 20 times and then the absorbance was recorded at 595 nm.
S. aureus SC01 biofilm assay (with 5 mM of D-amino acids) S. aureus SC01 were grown in LB overnight at 37°C with shaking and diluted 1:100 in TSB medium supplemented with NaCl (3%), glucose (0.5%), appropriate D-amino acid (D-Trp, D-Tyr, D-Tyr/ D-Pro/ D-Phe mix) stocks in 0.2 M NaOH (5 mM), HCl (20 mM) and PBS (0.5X). For untreated cells, double distilled water was added instead of the D-amino acids. These bacterial cultures were then transferred (800 μL) into a 24-well plate and incubated for 24 h with a low evaporation lid at 37°C without shaking. The supernatant was discarded and the plate was washed twice with PBS (1X, 1 mL) and dried at 65°C for 1 h. The plate was then allowed to cool to room temperature and stained with 1% crystal violet (500 μL) for 10 min. The plate was then washed twice with double distilled water (1 mL) and dried overnight. Photographs of the stained wells were recorded at this time. The crystal violet stained biofilms were dissolved by adding 95% ethanol (500 μL) to each well and shaking for 2 h. The contents were diluted 20 times and then the absorbance was recorded at 595 nm.
S. epidermis ATTC 12228 biofilm assay (with 1 mM of D-amino acids) S. epidermis ATCC 12228 were grown in LB overnight at 37°C with shaking and diluted 1:100 in TSB medium supplemented with NaCl (3%), glucose (0.5%), appropriate D-amino acid stocks in 0.2 M NaOH (1 mM), HCl (4 mM) and PBS (0.5X). The bacterial cultures was then transferred (200 μL) into 96-well plates and incubated for 48 h with a low evaporation lid at 37°C without shaking and covered with aluminum foil to protect from light. The supernatant was discarded and the plates were washed twice with PBS (1X, 200 μL) and dried at 65°C for 1 h. The plated were then allowed to cool to room temperature and stained with 1% crystal violet for 10 min. The plates were then washed twice with double distilled water (200 μL) and dried overnight. The crystal violet stained biofilms were dissolved by adding 33% glacial acetic acid (200 μL) to each well and shaking for 2 h. The contents were diluted 20 times and then the absorbance was recorded at 595 nm.

B. subtilis 3610 biofilm assay (with 1 mM of D-amino acids)
B. subtilis 3610 were grown in LB overnight at 37°C with shaking and diluted 1:1000 in LB supplemented with glycerol (3% v/v), MnCl 2 (0.1 mM), appropriate D-amino acid stocks in 0.2 M NaOH (1 mM), HCl (4 mM) and PBS (0.5X). These bacterial cultures were then transferred (200 μL) into 96-well plates and incubated for 72 h with a low evaporation lid at room temperature without shaking and covered with aluminum foil to protect from light. The biofilms formation was analyzed by recording images.

Results and Discussion
In order to evaluate the effect of D-amino acids on biofilm formation we initially used S. aureus (SC01 strain), the same strain that had been previously shown to be sensitive to D-amino acids [5]. In the initial report, D-tyrosine was reported to fully inhibit biofilm formation of S. aureus at 50 μM and the mixture of D-leucine/D-methionine/D-tyrosine/D-tryptophan at 15 nM each [4]. In a later report, the potency of D-tyrosine was revised. They found that D-phenylalanine, D-proline, and D-tyrosine were individually able to fully inhibit biofilm formation at 500 μM [5]. The combination of these three D-amino acids led to significant impairment at 10 μM and full inhibition at 100 μM [5]. Initially, we set out to reproduce these results as control compounds in inhibiting the formation of S. aureus biofilm as previously reported. We observed that D-tyrosine, D-tryptophan, and the equimolar mixture of D-tyrosine/D-proline/D-phenylalanine failed to inhibit biofilm formation at 1 mM (Fig. 1). Visual inspection of the biofilm confirmed that none of the compounds disrupted biofilm formation. Next, we doubled the incubation time to 48 hours to explore the possible inhibition by D-amino acids at longer incubation times. Clearly, the biofilms became denser at the 48-hour period as seen by the higher crystal violet-associated absorbance for all the compounds evaluated. Next, we evaluated the ability of D-amino acids to inhibit bacterial biofilm formation at 5 mM, which is ten times higher than the reported revised concentration for full inhibition and a hundred times higher than the initially reported concentration. For this series, only the reported biofilm inhibitors (D-tyrosine, D-tryptophan, and the equimolar mixture of D-tyrosine/D-proline/D-phenylalanine) were assessed at 5 mM. Following a 24 hour incubation period, there was no biofilm inhibition based on visual inspection and no significant inhibition was observed following staining (Fig. 2). Our data indicates that the D-amino acids evaluated herein show no activity in modulating the formation of S. aureus biofilm.
Next, we compiled a diverse library of 96 unnatural D-amino acids, including D-tyrosine as the reference positive control compound (compounds 1-96, Table A in S1 File). On average, the compounds were approximately 203 Da in molecular weight and all contained the free termini and D-stereochemistry. We had predicted that aromatic side chains on D-amino acids (similar to D-tyrosine) derivatized with either electron donating or electron withdrawing groups would help us to determine the appropriate structural elements required for effective biofilm disruption. Therefore, a large number of the D-amino acids included in the library contained elements that resemble D-tyrosine such as hydrogen bond donors and acceptors (e.g., nitrogen atoms). Additionally, a number of derivatives contained halogenated aromatic sidechains. The placement of these heteroatoms were diversified by their positioning along the aromatic ring (ortho, para, meta positions). We also included several non-aromatic side chains within our library of D-amino acids to probe a diversified chemical space. Following a 24-hour incubation period to induce S. aureus biofilm formation, no inhibition was observed for the entire set of compounds that were screened at 1 mM ( Fig. 3 and see additional figures in S1 File for larger version of the same data). As a positive control, we included the established S. aureus biofilm inhibitor carvacrol (Fig. C in S1 File). [20] Based on the stringent cut-off of 5 standard deviations from the mean, none of the D-amino acids or their combinations displayed biofilm inhibition. Extending the incubation to 48 h led to overall increased signals but did not change the results (Fig. 4). We believe that this is the largest reported screen of D-amino acids to date against biofilm formation and it is consistent with the negative results observed for the control compounds. From this data, we conclude that D-amino acids do not inhibit S. aureus biofilm formation at the concentrations tested. Next, we were interested in exploring the effect of D-amino acids on the biofilm formation of a different strain of S. aureus. In this screen, the same set of unnatural D-amino acids was evaluated along with the previously reported positive controls against S. aureus (ATCC 25923). This particular strain of S. aureus is widely used as a model biofilm-forming strain and should d-Amino Acids Do Not Inhibit Staphylococcus aureus Biofilm complement our initial screen using SC01 [19,21,22]. Each unnatural D-amino acid was evaluated at 1 mM for a 48-hour incubation period. None of the compounds from the library were found to be effective at inhibiting biofilm formation (see Fig. D in S1 File). Once again, D-tyrosine and D-tryptophan displayed no significant inhibition even at the elevated concentration Fig. 4. Diverse set of D-amino acids effect on biofilm formation. S. aureus (SC01) biofilm formation was evaluated after 48 h in the presence of specified unnatural D-amino acids at a concentration of 1 mM or the positive control carvacrol (2 mM). The absorbance was recorded at 595 nm following crystal violet staining. All points are significantly different from the positive control carvacrol (****p<0.0001 relative to positive control, unpaired t-test.). of 1 mM. Together, we concluded that all the unnatural D-amino acids evaluated show no activity in inhibiting S. aureus biofilm formation in two different strains.
In further evaluating our panel of unnatural D-amino acids, we explored the effects of our compounds with another biofilm forming microorganism. S. epidermis is a pathogen that frequently causes complications with implants due to their ability to form biofilms at the interface with human tissue [23,24,25,26]. We investigated the effect of our panel of unnatural D-amino acids at 1 mM on the biofilm development of S. epidermis (ATCC 12228) strain. Similar to our results with S. aureus, we observe no inhibition of biofilm development with any of the unnatural D-amino acids in our library after 48 hours of incubation in biofilm medium (See Fig. E in S1 File). Of note, neither D-tyrosine nor D-tryptophan led to a decrease in biofilm formation based on visual inspection and crystal violet staining.
Finally, we were interested in determining the effect of our unnatural D-amino acid library on biofilm formation for the widely used model organism B. subtilis. B. subtilis are capable of forming distinct biofilms at the air-liquid interface in liquid culture [27,28,29,30]. We evaluated the effect of each unnatural D-amino acid against B. subtilis (3610 strain) at 1 mM. Previously, it had been demonstrated that various D-amino acids were potent inhibitors of B. subtilis biofilm formation, with D-tyrosine showing low micromolar activity [4]. Recently, the same group discovered that the inhibitory effects by D-tyrosine were an artifact due to a mutation in the gene encoding D-tyrosyl-tRNA deacylase [18]. Upon repair of the gene, B. subtilis were no longer sensitive to the actions of D-tyrosine, a clear indication that biofilm inhibition was being mediated by a mechanism distinct from the originally proposed mechanism. In agreement with their findings, we observe a similar trend for the D-amino acids within our panel of compounds upon incubation for three days at concentrations of 1 mM (see Fig. F in S1 File). As expected, we observed that D-tyrosine and the D-tyrosine/D-proline/D-phenylalanine mixture inhibited biofilm formation in B. subtilis after three days whereas D-tryptophan inhibited biofilm formation to a moderate extent. The sensitivity to D-tryptophan is consistent with other organisms whose D-tyrosyl-tRNA deacylase activity has been inactivated [18,31]. It should be noted that for some of the compounds, the solution became orange during the incubation period with the cells. The coloration was a result of the inherent absorbance properties of the D-amino acid variants. The same color was also observed in the absence of bacterial cells. For example, the treatment of mutated Escherichia coli with D-tryptophan led to toxicity at levels that were absent in the wildtype cells [32]. The finding that B. subtilis are not sensitive to any of the molecules within our panel of D-amino acids suggests that this may not be a viable strategy for the inhibition and dispersion of biofilm of this organism.

Conclusion
The complications brought on by the development of bacterial biofilms can be immense. In industrial settings, the removal of biofilms requires remediation steps that are often highly costly and procedurally difficult. In medical settings, bacterial biofilms require interventions that can be life-threatening. There is a clear unmet need for the prevention of biofilm formation and the dispersion of existing biofilm. The use of anti-biofilm small molecules could potentially provide a strategy to combat biofilm formation. However, it has been difficult to discover compounds that are potent anti-biofilm agents. It was recently reported that unnatural D-amino acids could inhibit the formation and disperse existing biofilms for three distinct types of bacteria. In contrast to the published reports, we determined that the reported active D-amino acids (D-tyrosine, D-tryptophan, and combinations of D-amino acids) failed to inhibit biofilm formation in three different types of bacteria: S. aureus, S. epidermis, and B. subtilis. Likewise, none of the D-amino acids from our panel of compounds displayed anti-biofilm activity.
Supporting Information S1 File. This file contains Table A