https://orcid.org/0000-0002-2996-1548
Figures
Abstract
Bacillus thuringiensis insecticidal proteins have been safely and effectively used for several decades as tools for insect pest control either as active ingredients in biopesticides or via transgenic expression in crop plants (GM crops). While the first generations of GM crops in the 1990s expressed only three-domain Cry proteins, GM corn and cotton varieties expressing vegetative insecticidal proteins from the Vip3 class began to be cultivated in the late 2000s (Vip3A). Here we describe a comprehensive food and feed safety assessment of Vip3Cb1, the first member of the Vip3C class to be expressed in commercial varieties of GM crops. Bioinformatic protein safety screens reiterate the placement of Vip3Cb1 as an insecticidal member of the Vip3 protein class and demonstrate a lack of homology to known allergens or toxins. In vitro characterization of the thermal stability and digestive fate demonstrates that dietary exposure to an intact, functional form of Vip3Cb1 is highly unlikely. Vip3Cb1 also showed no measurable effects in an in vivo acute oral toxicity study in mice. By weight of evidence, the bioinformatics assessments, in vitro and in vivo studies, history of safe use, and the previously reported mechanism of action similar to Vip3A collectively demonstrate no indication of a risk of Vip3Cb1 to the health of humans or animals and reinforces the overall safety of the Vip3 class of insecticidal proteins.
Citation: McKinnon LJ, Wang R, Wang C, Saracco S, Kessenich C, Johnson VM, et al. (2026) Food and feed safety of a novel Vip3C insecticidal protein derived from Paenibacillus spp. for protection of crop plants against key Lepidopteran pests. PLoS One 21(3): e0344572. https://doi.org/10.1371/journal.pone.0344572
Editor: Allah Bakhsh, Nigde Omer Halisdemir University, TÜRKIYE
Received: July 29, 2025; Accepted: February 22, 2026; Published: March 18, 2026
Copyright: © 2026 McKinnon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data or URLs are within the manuscript and its Supporting Information files.
Funding: The authors are employees of Bayer CropScience, a manufacturer of crop seeds developed through conventional plant breeding or biotechnology. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
Competing interests: The authors are employees of Bayer CropScience, a manufacturer of crop seeds developed through conventional plant breeding or biotechnology. This does not alter our adherence to PLOS ONE policies in sharing data and conducting robust research. To adhere to PLOS One’s policy around material sharing, materials described in this work can be provided upon establishment of a research agreement with appropriate material transfer terms. Certain materials described in the study have patent protection, but can be provided upon establishment of a research agreement with material transfer terms, in order to adhere to PLOS ONE’s policies.
Introduction
Insect pests pose a prominent threat to modern agriculture, causing significant yield losses and presenting challenges to global food security. The Food and Agriculture Organization of the United Nations estimates that global crop yield losses due to insect pests amount to at least $70 billion annually [1]. Chemical insecticides are established tools which have been successfully used for decades to abate crop loss due to insect pests. However, evolution of pest resistance which can limit the long-term efficacy of chemical insecticides as well as the potential environmental impact if they are overused fuel the need for additional solutions as part of an integrated pest management system. In the mid-1990s, genetically modified (GM), crops expressing one or more insecticidal proteins that can control susceptible target insect pests were introduced as a new safe and effective solution to control insect pests in agriculture [2]. Since then, insect-protected GM crops have been widely adopted by farmers across the globe and have enabled significant crop yield and farm income gains [3]. In addition to being relatively specific for the target pest compared to many broad spectrum insecticides, the benefits of insect control (IC) proteins in GM crops also include the ability to be combined (pyramided) with other IC proteins for enhanced resistance management and the potential to reduce exposure to the IC protein in food, feed and the environment via tissue-specific gene expression [4–6] Despite the many benefits of insect-resistant GM crops, their efficacy is also subject to the evolution of pest resistance [7]. Therefore, continual identification of new IC protein candidates is still necessary to develop new generations of insect-protected GM crops with the same benefits as older products.
The most common IC proteins expressed in insect-resistant GM crops belong to three-domain crystal (Cry) proteins derived from Bacillus thuringiensis (Bt) [8,9]. Decades before being expressed in GM crops, Cry proteins served as primary active ingredients in many biological insecticides [8,10]. Several different Cry proteins derived from Bt have been expressed in commercial varieties of corn, cotton, and soybeans for the successful protection of these crops from major Lepidopteran and Coleopteran pests [11–14]. Other classes of insecticidal proteins have also been identified from Bt, other microbes, fungi, and even certain plants [9,15–22]. Vegetative insecticidal proteins were first identified as a distinct class of proteins produced as soluble rather than crystalline forms and secreted during the vegetative growth phase of Bacillus species [23,24]. Vegetative insecticidal proteins were initially divided into multiple families but have been recently reclassified into three groups (i.e., Vpa, Vpb, and Vip3) based on structural homologies [21]. Thus, only the Vip3 class now retains the abbreviation “Vip” [21], and it is restricted to vegatative insecticidal proteins with Lepidopteran activity.
Vegetative insecticidal proteins are relatively recently expressed in GM crop varieties intended for commercialization with recent examples including Vip3Aa19 in cotton and Vip3Aa20 in maize [25,26]. While Vip3 proteins exhibit notable differences in sequence and three-dimensional structure from other classes of insecticidal proteins, the general molecular mechanism by which they cause insect death is quite similar. Like other classes of IC proteins, Vip3 proteins are synthesized as inactive protoxins. They are then proteolytically processed in the target insect midgut to form an active core which recognizes host-specific receptors, leading to pore formation in cell membranes, midgut epithelium disruption, and eventually insect stunting or death [22,27,28]. Differences in activity spectra between Vip3 and other classes of IC proteins are ultimately related to differences in three dimensional structures and domain organization, the fate of each protein in the biochemical milieu of different insect gut systems, the identity of the specific midgut receptor molecule (s) they recognize, specific residues and structural features involved in receptor binding, and other factors [24,27,29–31].
Vip3 proteins are by far the most numerous and well-studied among the vegetative insecticidal proteins [24]. The Vip3 family consists of three sub-families (Vip3A-Vip3C) with well over 100 currently reported members and are known for their insecticidal effect on several Lepidopteran pests in major row crops [24,32]. Recently, a new Vip3 protein encoded in the genome of a bacterial strain from the Paenibacillus popilliae-containing clade was discovered [22]. This new Vip3C protein, named Vip3Cb1, confers robust in-field tolerance to economically important Lepidopteran pests when expressed in the cytosol in GM cotton (i.e., MON 89151) and when targeted to chloroplasts (i.e., with a chloroplast transit peptide) in maize plants [22]. Vip3Cb1 shares 84% sequence identity to three Vip3Ca proteins derived from Bt [33] (S1-S3 Figs) and 66% amino acid sequence identity to the Vip3Aa19 and Vip3Aa20 proteins expressed in COT102 cotton and MIR162 maize, respectively [22]. Like other Vip3 proteins, Vip3Cb1 contains two alpha helical domains in the N-terminal region (DI and DII) and three beta-sheet domains in the C-terminal region (DIII, DIV, and DV) [22,28]. Furthermore, experimental data indicate that Bt-derived Vip3Aa, Vip3Ca1 as well as this new Vip3Cb1 function by the same general mechanism of action described above [22,24,28]. Therefore, as Vip3Cb1 has sequence and structural similarity with Vip3Aa and Vip3Ca1, and Vip3Aa already has an established history of safe use, a comprehensive safety assessment of Vip3Cb1 may not be necessary. Nonetheless, this study aimed to assess whether the ~ 30% amino acid sequence difference between Vip3Aa and Vip3Cb1 impacts food and feed safety considerations. Here we employ a series of in vitro and in vivo studies along with bioinformatics and in combination with key mechanistic data [22] demonstrating, by weight of evidence, the food and feed safety of the Vip3Cb1 protein.
Materials and methods
Bioinformatic analysis
The Vip3Cb1 protein sequence was screened for similarity against known allergens, toxins, and protein databases in accordance with established methods as described and utilized previously [34–36].
The Allergen Database used (herein described as AD_2024) was the “COMprehensive Protein Allergen Resource” (COMPARE) database as provided by the Health and Environmental Sciences Institute (HESI; http://db.comparedatabase.org/) and is composed of 2,748 sequences [37].
The toxin database (TOX_2024) used was a keyword selected subset of sequences found in the Swiss-Prot database (https://www.uniprot.org/). This keyword process is used to isolate likely toxins by utilizing the search terms “(keyword:toxin) AND (reviewed:true)”, followed by a subsequent counter screen to remove unlikely toxins with annotations such as “antitoxin” and/or “non-toxic”. The final TOX_2024 database is composed of 7,338 sequences.
The all protein (PRT_2024) database is the ncbi-asn1 all protein fasta data from GenBank release 259 (https://ftp.ncbi.nlm.nih.gov/ncbi-asn1/protein_fasta/; accessed 01-10-2024) and is comprised of 286,068,982 sequences.
Alignments were generated with FASTA v36.3.5d run with an E-score cutoff of 1, however, a threshold of ≤1e − 5 (1 × 10−5) was used for alignment significance. This is an established threshold used previously [35,36,38] and is a conservative threshold for identifying homologous proteins against databases of these sizes [39,40].
Cloning, expression, and purification of recombinant Vip3Cb1
The vip3Cb1 coding sequence was amplified from plasmid DNA by PCR using specific primers, was fused with an N-terminal 6His-SUMO tag into a pET-SUMO vector and was transformed into chemically competent E. coli DH5-α cells for plasmid propagation and isolation. The isolated pET-SUMO-vip3Cb1 plasmid was then transformed into E. coli Rosetta 2 cells for recombinant protein expression. The His-SUMO-Vip3Cb1 fusion protein was expressed by fermentation of E. coli at 16–20°C. The presence of the Vip3Cb1 expression construct in the harvested E. coli cell paste was confirmed by DNA sequencing, and the cell paste was frozen and stored at −80°C until needed.
For protein isolation, the E. coli cell paste was thawed and suspended in 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole supplemented with benzonase and lysozyme and homogenized. The homogenized cell paste was lysed using a microfluidizer and the supernatant was recovered by centrifugation. The recovered supernatant was diluted and subjected to Ni-NTA-agarose (QIAGEN, Venlo, Netherlands) packed into a column (BPG 200/500, Cytiva, Marlborough, Massachusetts) to enrich for His-SUMO-Vip3Cb1 protein. The His-SUMO tag was then removed by proteolysis using His-SUMO protease. The tag-free Vip3Cb1 protein was then purified by subjecting the sample after protease treatment to a second Ni-NTA-agarose column to remove the cleaved His-SUMO tag and His-SUMO protease. The resulting flow-through containing tag-free Vip3Cb1 was collected, buffer-exchanged into 25 mM sodium carbonate/bicarbonate (pH 10) and 25 mM NaCl by continuous diafiltration, and aliquoted and frozen at −80°C until needed.
Purification of Vip3Cb1 from cotton seed
A total of 4 kg of defatted cotton seed was processed to yield sufficient Vip3Cb1 material for characterization. In brief, defatted cotton seed was extracted in 50 mM Tris-HCl pH 8, and the clarified extract was subject to Q Sepharose Fast Flow anion exchange chromatography (Cytiva, Marlborough, Massachusetts). Proteins were eluted with a continuous gradient of NaCl (0–500 mM), and the fractions containing Vip3Cb1 were pooled and exchanged into 50 mM Tris-HCl pH 8 by continuous diafiltration. The buffer-exchanged sample was subjected to immunoaffinity chromatography using a Vip3Cb1-specific mouse monoclonal antibody (isotype IgG2a, Envigo) conjugated to MabSelect Protein A (Cytiva). Vip3Cb1 was eluted from the immunoaffinity column using 100 mM triethanolamine (pH 10.5), immediately quenched with 1 M Tris-HCl pH 8, concentrated, and exchanged into 25 mM sodium carbonate/bicarbonate pH 10, 25 mM NaCl. The final purified sample was aliquoted and frozen at −80°C until needed.
Characterization of Vip3Cb1 proteins
Vip3Cb1 proteins purified from cotton seed and E. coli were characterized by several biochemical assays and an insect bioassay to determine their functional activity. Total protein concentration was determined by amino acid analysis and a colorimetric assay with Bio-Rad Protein Assay Dye for the recombinant and plant-produced Vip3Cb1, respectively. For quantification of the plant-produced protein, recombinant Vip3Cb1 was used to generate a standard curve. The purity of Vip3Cb1 from each source was determined by SDS-PAGE and Colloidal Blue G staining followed by analysis of gels with ImageLab software. The identity of each was confirmed by (i) western blotting using a monoclonal antibody described above and (ii) Nano LC-MS/MS using an Orbitrap Fusion instrument [41]. To assess the glycosylation status of plant-produced and recombinant Vip3Cb1, a carbohydrate staining method was employed using transferrin as a positive control. Briefly, proteins were subject to SDS-PAGE, were electrotransferred to PVDF membranes, and carbohydrate moieties were oxidized and biotinylated. Biotinylated proteins were then detected by probing the membrane with a streptavidin-HRP conjugate followed by incubation with enhanced chemiluminescence substrate.
Insect bioassays of Vip3Cb1 proteins
Functional activity of Vip3Cb1 was measured in a 7-day diet incorporation bioassay and reported as an EC50 value (the mean concentration that leads to 50% growth inhibition relative to the control group). Activity was determined against a susceptible laboratory colony of Spodoptera frugiperda (Bayer Crop Science, Union City, TN) using seven concentrations ranging from 0.003125–0.2 µg Vip3Cb1/mL to generate a dose-response. Three separate replicates were conducted on different days with different batches of insects. 25 mM sodium carbonate/bicarbonate pH 10, 25 mM NaCl was used as a negative control. For each replicate, agar-based diet (Southland Products Inc.) containing Vip3Cb1 was dispensed in 1.0 ml aliquots into 16 wells of a 128-well tray (Frontier Scientific, Inc., Newark, DE) for each concentration. Each replicate also included a buffer control dispensed into 48 wells of a 128-well tray for a total of 16 insects per concentration and 48 insects per control in each of the three bioassay replicates. Larvae were individually placed in wells and incubated at 27°C. After seven days, the number of larvae surviving in each treatment group and their mass was recorded. SAS PROC MEANS [42] was used to calculate the sample mean and standard error of the average insect mass for each treatment and concentration. Dose-response data were analyzed by logistic regression to determine the EC50 value. The mean EC50 values were statistically compared between the MON 89151-produced and E. coli-produced Vip3Cb1 protein treatments using a t-test at the 5% level of significance within the procedure of PROC NLMIXED [42]. All bioassays were accepted by meeting the criteria including (1) ≤ 20% mean mortality for the buffer control treatment at bioassay termination, and (2) the dose response curve bracketed the EC50 value.
Susceptibility to pepsin and pancreatin
The susceptibility of Vip3Cb1 protein to degradation by pepsin was assessed following a standardized protocol and as described previously [36,43]. All test and control reactions were carried out at pH 1.2. Bovine Serum Albumin (BSA) and Soybean Trypsin Inhibitor (STI) were included as controls for pepsin labile and pepsin resistant proteins, respectively [43] and were subject to the same digestion protocol. The susceptibility of Vip3Cb1 to degradation by pancreatin was also assessed as described previously [36]. Beta-Lactoglobulin (BLG) and STI were included as controls for pancreatin labile and pancreatin resistant proteins, respectively [44] and were subject to the same digestion protocol.
Digestion assay samples were analyzed by SDS-PAGE using Tricine 10–20% gels and Coomassie staining and/or western blot analysis. The limits of detection of SDS-PAGE or western blot were also assessed using zero-minute samples for pepsin and pancreatin digestion.
Temperature lability
To determine the stability and functional activity of Vip3Cb1 as a function of temperature, the E. coli-produced protein was subject to a range of temperatures for defined time periods followed by SDS-PAGE analysis and an insect diet bioassay. Briefly, the Vip3Cb1 protein was incubated for 15 and 30 minutes at 25°C, 37°C, 55°C, 75°C, 95°C, or 0°C (wet ice) as a control for the temperature incubations (11 samples total). Immediately after incubation at each temperature and amount of time, the designated aliquot of Vip3Cb1 was placed on wet ice and then prepared for SDS-PAGE or an insect bioassay on the same day. The stability of Vip3Cb1 at each time point and temperature was evaluated by analyzing the band patterns on Colloidal Blue G-stained SDS-PAGE gels. The functional activity in each sample was assessed using an insect bioassay with S. frugiperda and six concentrations ranging from 0.003–0.1 µg Vip3Cb1/mL. For each of the eleven heat-treated samples, a single replicate was prepared at each Vip3Cb1 concentration (16 wells per concentration in the bioassay), and one buffer control was prepared in triplicate (48 wells in total). The seven-day bioassay was then carried out as described above. All bioassays were accepted if ≤ 20% mean mortality for the buffer control treatment was observed at bioassay termination. If a given sample had no clear dose-response in the bioassay then an EC50 value could not be estimated, and no activity was therefore observed at the corresponding dose level.
Acute oral toxicity study in mice
An acute oral (gavage) toxicity study with purified, recombinant Vip3Cb1 was conducted in CD-1 mice at Charles River Laboratories, (Spencerville, Ohio) according to EPA guideline EPA-OPPTS (870.1100) [45]. The Vip3Cb1 dose solution was formulated in 10 mM sodium carbonate/bicarbonate buffer, pH 10 (vehicle buffer) at a dose concentration of 78.9 mg Vip3Cb1 protein per ml to enable a targeted dose level of 5,000 mg Vip3Cb1 protein/kg body weight. In addition to the test dosing solution, bovine serum albumin (BSA) as a control protein was formulated in vehicle buffer to a concentration of 92.3 mg/ml to enable a 5,000 mg protein/kg body weight dose level. Stability of the proteins over the course of dosing was confirmed by SDS-PAGE analysis, and functional activity of Vip3Cb1 was confirmed using an insect bioassay with Spodoptera frugiperda.
Two dose groups of ten male and ten female mice, at approximately 8 weeks of age at dosing, were administered the formulated Vip3Cb1 or BSA proteins (four dose groups in total). In addition, ten male and ten female mice of the same age were administered the vehicle buffer. Mice from all six groups (60 mice in total) were given split doses (two separate doses given ~4 hrs apart on the same day) of the Vip3Cb1 protein, BSA protein control, or vehicle buffer via oral gavage administration to achieve the target dose level. Due to an insufficient quantity of the formulated Vip3Cb1 protein, three of the female mice did not receive the second dose and were therefore omitted from the statistical analyses of the study. A 14-day observation period was conducted following dosing (i.e., day 0). Endpoints evaluated during the dosing and observation periods included survival (monitored twice daily), body weights (monitored on days 0, 7 and 14), and food consumption (monitored on days 0, 7 and 14). In addition, the health and behavior of each individual in the study was monitored on a daily basis by the technical and veterinary staff to assess clinical signs and evidence of significant health concerns. These included prostration, weight loss, immobility, and other severe injuries. Based on the veterinary and study director assessment, treatment options or unscheduled euthanasia would be determined. Because no adverse effects were expected from any of the dose solutions, euthanasia by carbon dioxide inhalation followed by exsanguination was scheduled for all surviving animals prior to study initiation. All animals survived during the 14-day observation period and were euthanized as described above rotating across groups such that similar numbers of animals from each group were necropsied throughout the day. On the same day, euthanized animals were subjected to a complete necropsy examination, including evaluation of the carcass and musculoskeletal system, all external orifices and surfaces, cranial cavity and external brain surfaces, and the abdominal, pelvic, and thoracic cavities and their associated organs and tissues under the supervision of a board-certified veterinary pathologist. All work was conducted in an American Association for the Accreditation of Laboratory Animal Care (AAALAC) accredited laboratory (Charles River Laboratories, Spencerville, Ohio), and the study protocols were reviewed and approved by the test facility IACUC committee prior to study initiation to ensure animal welfare.
Statistical analyses of acute oral toxicity study data
Statistical analysis was conducted on body weight, body weight changes, and food consumption data obtained in the acute toxicology study. Data were subjected to a statistical decision tree. Levene’s test was used to assess the homogeneity of group variances parametric assumption at the 5% significance level. Datasets with three groups (vehicle buffer, BSA, and Vip3Cb1) were compared using an overall one-way ANOVA F-test when Levene’s test was not significant or the Kruskal-Wallis test when it was significant. Datasets with two groups were compared using a two-sided t-test when Levene’s test was not significant or Wilcoxon Rank-Sum test when it was significant. All significant pairwise comparisons were reported at the 1% and 5% significance levels.
Results
Bioinformatic analysis of Vip3Cb1 does not reveal homology to known toxins or allergens
The sequence similarity of Vip3Cb1 to known proteins was evaluated using FASTA (v36.3.5d) search of all proteins in PRT_2024 (Table 1). Notably this search resulted in the identification of Vip3Cb1, which represents self-identification, and is labeled as “Sequence 4 from patent US 10155960” and described as an insect inhibitory protein (NCBI Accession QBC81278.1) consistent with its discovery and previous classification [22].
A bioinformatic analysis against the AD_2024 (Allergen) database utilizing the Vip3Cb1 protein sequence returned no alignments displaying an E-score of ≤1e-5 indicative of homology, nor any alignments surpassing the WHO/FAO Codex Alimentarius [34] recommended linear 8 amino acid match and/or alignment of ≥35% identity in a sliding 80 amino acid window (Table 1). Likewise in a search against the TOX_2024 sequence database, no alignments exceeded the E-score threshold of ≤1e-5, which could indicate a level of homology that may result in toxicity (Table 1).
Recombinant Vip3Cb1 is a suitable surrogate for the protein as expressed in cotton
Large quantities of highly purified protein are required to assess the safety of any new protein expressed in GM crops. Purification of a protein from plant material of sufficient quality and in sufficient quantity to satisfy all global regulatory study requirements is impractical due to low in planta expression levels (e.g., Vip3Cb1 = ~0.8 ppm d.w. in cotton seed) of these proteins. Therefore, a recombinant form expressed in and purified from a heterologous host such as E. coli is routinely used. To establish equivalency between E. coli and plant-produced Vip3Cb1 the protein was purified from E. coli and GM cotton seed. Large quantities of highly purified (96% pure, Table 2) Vip3Cb1 protein were successfully obtained from E. coli. A band representing the full-length form of plant-produced Vip3Cb1 was identified by SDS-PAGE followed by Coomassie staining (Fig 1). Side-by-side comparisons using Coomassie-stained SDS-PAGE gels and western blotting showed that the proteins purified from each source have a comparable apparent MW and immunoreactivity towards a Vip3Cb1-specific antibody (Fig 1). Mass spectrometry analysis also confirmed the identities of E. coli and plant-produced Vip3Cb1 with ≥72% sequence coverage and showed they exhibit identical N-terminal sequences (Table 2) with the exception of the missing N-terminal Met residue in plant-produced Vip3Cb1 (Table 2). Lack of the N-terminal Met residue has been reported in other plant-produced proteins [38,46] and is likely due to the activity of a cytosolic enzyme in plants known as methionine aminopeptidase [47,48]. As protein glycosylation can impact physicochemical properties [49], demonstrating that plant-produced and recombinant forms of a protein have the same glycosylation status is important in the equivalence determination. A carbohydrate staining method demonstrated that both recombinant and plant-produced Vip3Cb1 lack detectable glycosylation (Fig 2). Finally, insect diet bioassays demonstrated that plant-produced and recombinant Vip3Cb1 have comparable insecticidal activity when measured as an EC50 against Spodoptera frugiperda (Fig 3), also confirming that the removal of the N-terminal Met residue does not impact the Vip3Cb1 functional activity. The above-mentioned protein characterization results establish functional equivalency between recombinant and plant-produced Vip3Cb1 and justify the use of E. coli-produced Vip3Cb1 as a suitable surrogate in all safety testing.
Purified Vip3Cb1 expressed in cotton seed and E. coli were subjected to SDS-PAGE and western blot analysis to determine the purity, MW and immunoreactivity of each protein. (A) Representative SDS-PAGE gel stained with Brilliant Blue G-Colloidal stain of the E. coli protein (Lane 2; ~ 1.0 μg total protein) and cotton seed protein (Lane 3, ~ 2.0 μg total protein). BioRad Precision Plus molecular weight markers are loaded in lane 1. Approximate MWs (kDa) are shown on the left. The arrow denotes the full-length form of Vip3Cb1. (B) Representative western blot after electrotransfer of cotton seed protein (Lanes 1-6) and E. coli protein (Lanes 7-12, ~ 8 ng of Vip3Cb1) to a nitrocellulose membrane. Gel loadings were as follows: ~ 8 ng of Vip3Cb1 (Lanes 1, 2, 7, & 8), ~ 10 ng of Vip3Cb1 (Lanes 3, 4, 9, & 10), and ~12 ng of Vip3Cb1 (Lanes 5, 6, 11, & 12). Proteins were detected using an anti-Vip3Cb1 monoclonal antibody and visualized using HRP-conjugated secondary antibodies and an ECL system. MagicMark™ XP Western Protein Standard was used as a MW standard, and approximate MWs (kDa) are shown on the left. The arrow denotes the full-length form of Vip3Cb1.
Purified Vip3Cb1 expressed in cotton seed (A and B, Lanes 9 and 10, ~ 100 ng and 200 ng, respectively) and E. coli (A and B, Lanes 6 and 7, ~ 100 ng and 200 ng, respectively), and transferrin (positive control, A and B, Lanes 3 and 4) were subjected to SDS-PAGE and electrotransferred to a PVDF membrane. Lane 1 was loaded with BioRad Precision Plus MW Marker, and Lane 2 was empty. (A) Carbohydrate moieties were detected by addition of streptavidin conjugated HRP followed by luminol-based detection using ECL reagents and visualization with a Bio-Rad ChemiDoc Imager. (B) An equivalent blot was stained with Coomassie Blue R-250 to confirm the presence of proteins.
Sixteen insects were used per test concentration, and forty-eight insects were used per control (diet lacking Vip3Cb1) sample in each of the three bioassay replicates.
Vip3Cb1 loses functional activity when incubated at elevated temperatures
Vip3Cb1 was characterized after incubation at temperatures ranging from 0–95°C to assess the thermal stability. The intactness and insecticidal activity of Vip3Cb1 were unaffected after incubation for 15 or 30 min at 0–37°C as judged by SDS-PAGE analysis (Figs 4A and 4B) and an insect bioassay (Table 3). In contrast, incubation of Vip3Cb1 for 15 or 30 min at 55°C and higher led to a loss of detectable activity (Table 3) and varying amounts of aggregation products or degradation of the full-length protein (Fig 4).
The Vip3Cb1 protein was incubated at 0°C (Lane 2), 25°C (Lane 3), 37°C (Lane 4), 55°C (Lane 5), 75°C (Lane 6), and 95°C (Lane 7) for either 15 min (A) or 30 min (B) prior to SDS-PAGE and Coomassie staining to assess the structural integrity at elevated temperatures. Lanes 8 and 9 are approximately 3 μg (equivalent total protein load) and 0.3 μg (10% of the total protein load) of untreated Vip3Cb1 as a control. Lanes 1 and 10 were loaded with BioRad Precision Plus MW Marker, and approximate MWs (kDa) are shown on the left. Arrows denote the full-length form of Vip3Cb1.
Intact Vip3Cb1 is readily degraded in the presence of pepsin and pancreatin
To understand the stability of Vip3Cb1 in the human digestive system, the digestive fate in simulated gastric and intestinal environments was assessed. SDS-PAGE and western blot analyses indicate that nearly all (~99%) of full-length Vip3Cb1 is degraded within 0.5 min of incubation with pepsin (Fig 5A and S4 Fig). A ~ 10-kDa peptide fragment was observed within the first 5 minutes of incubation with pepsin based on SDS-PAGE and Coomassie staining (Fig 5A), and this fragment was detectable by western blotting only within the first 0.5 min likely due to the lower amounts of Vip3Cb1 loaded on the gel for western blotting (Fig 5B). An ~ 8-kDa peptide fragment was observed by Coomassie staining and western blotting through the 60 minutes of incubation with pepsin (Figs 5A and 5B). In the first 30 sec of incubation with pepsin, minor fragments of ~20 and ~30-kDa were observed by Coomassie staining and western blotting, respectively (Figs 5A and 5B). As expected for the control proteins, BSA was rapidly degraded, and STI remained intact during the 60 min incubation with pepsin (S5 Fig.). For the pancreatin incubation experiment, nearly all (~99%) of the full-length Vip3Cb1 protein was degraded within 5 minutes when analyzed by western blot (Fig 5C and S4 Fig). Multiple lower molecular weight fragments of Vip3Cb1 were observed throughout 24 hours of incubation with pancreatin alone (Fig 5C). As expected for the control proteins, BLG was completely degraded within 5 minutes, and STI remained intact after 24 hours of incubation with pancreatin (S5 Fig.). These results show that the full-length Vip3Cb1 protein is rapidly degraded by pepsin and pancreatin.
Stability of the Vip3Cb1 protein in the presence of pepsin (A and B) was assessed by SDS-PAGE (A) and western blot analysis (B). Stability of the Vip3Cb1 protein in the presence of pancreatin (C) was assessed by western blot analysis. Based on pre-reaction protein concentrations, 1 μg or 10 ng of Vip3Cb1 were loaded into each well on SDS-PAGE gels for electrophoresis followed by Coomassie staining or western blot analysis, respectively. Lane designations are as follows: A and B: Lane 1, BioRad Precision Plus MW Marker (A) or MagicMark™ XP Western Protein Standard (B) were used as MW standards, Lane 2, pepsin only at time zero, Lane 3, Vip3Cb1 only at time zero, Lanes 4–11, time course of Vip3Cb1 incubation with pepsin for 0, 0.5, 2, 5, 10, 20, 30, and 60 min, respectively, Lane 12, Vip3Cb1 only at 60 min, Lane 13, Pepsin only at 60 min. C: Lane 1, MagicMark™ XP Western Protein Standard was used as a MW standard, Lane 2, pancreatin only at time zero, Lane 3, Vip3Cb1 only at time zero, Lanes 4–12, time course of Vip3Cb1 digestion by pancreatin at time zero, 5 min, 15 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr, and 24 hr, respectively, Lane 13, Vip3Cb1 only at 24 hr, Lane 14, pancreatin only at 24 hours.
Vip3Cb1 displays no toxicity to mice at a high dose level
To experimentally assess the potential toxicity of Vip3Cb1 towards mammals, an acute oral gavage toxicity study in CD-1 mice was conducted at a dose level of 5000 mg protein per kg of body weight. After gavage administration of vehicle buffer, Vip3Cb1, or BSA as the control protein, several parameters and endpoints were evaluated over a 14-day period followed by a gross necropsy. There were no Vip3Cb1-related effects on body weight changes (Tables 4 and 5) or food consumption (Table 6) in both male and female mice. In male mice dosed with Vip3Cb1, a slight but statistically significant increase in food consumption was observed from Days 7–14 (Table 6). However, this change was not considered to be related to Vip3Cb1 as there was no correlation to body weight changes. After termination of the in-life phase of the study, the gross pathology observations did not reveal any lesions in male or female mice dosed with Vip3Cb1 (Table 7). Because no concerns or adverse effects were observed during the clinical endpoint and gross pathological examinations, further analyses (e.g., histopathology) were neither justified nor required by EPA test guidelines which follow a tiered approach [45]. In summary, there were no effects of Vip3Cb1 protein when administered by oral gavage at a high dose of 5000 mg protein/kg body weight in male and female CD-1 mice. Therefore, the No Adverse Effect Level (NOAEL) for Vip3Cb1 protein was determined to be greater than or equal to 5000 mg protein/kg body weight.
Discussion
The guidelines for assessing the food and feed safety of newly expressed proteins (NEP) in GM crops are described in the Codex Alimentarius [34] and have been thoroughly described elsewhere [11,50]. To briefly reiterate here, different approaches are taken to identify potential hazards of a NEP including review of scientific literature and other public information, bioinformatic analyses, and biochemical characterization. If any potential hazards are identified, then a characterization of those hazards in the context of likely exposure scenarios would be warranted. Since proteins are complex macronutrients and are subject to food processing and digestion, no single method alone can give conclusive evidence regarding the safety in the context of food and animal feed. Thus, the overall safety assessment of an NEP is then conducted within a well-established “weight-of-evidence” (WOE) risk assessment approach [34,50–52], where each study contributes specific information to the overall conclusion of safety. In the following sections we describe a WOE risk assessment approach utilized to assess the safety of Vip3Cb1 in alignment with international guidelines, including those from the Codex Alimentarius Commission, the Organisation for Economic Co-operation and Development (OECD), and the European Food Safety Authority (EFSA) [34,51,53].
History of safe use
The concept of history of safe use is an important aspect of the risk assessment of NEPs in GM crops and is described in detail elsewhere [36,38,54,55]. The Vip3Cb1 protein expressed in MON 89151 cotton is derived from a gene present in a bacterial strain in the Paenibacillus popilliae-containing clade and represents the first Vip3 protein identified in this genus [22]. At the 16S RNA sequence level, the specific Paenibacillus strain from which Vip3Cb1 is sourced is 98.7% and 99.2% identical to P. popilliae and P. dendritiformis, respectively [22]. The genus of Paenibacillus, from which the vip3Cb1 gene is derived, has a well-documented history of safe use. Paenibacillus species in general are not regarded as human pathogens [56] and have only been associated with limited opportunistic infections in immunocompromised individuals [57–61]. Current evidence therefore indicates that (i) infections caused by Paenibacillus species are rare and opportunistic in nature and (ii) Paenibacillus species do not present a risk to public health.
One member of the Paenibacillus genus (P. popilliae) also has a history of safe use as a biological control agent against the Japanese beetle (Popillia japonica) in which it causes so-called Milky diseases [56,62–64]. According to Deans and Krischik, P. popilliae was first detected in New Jersey in 1933, and it became commercially available as spore formulations for use on turfgrass in 1948 [65]. A large-scale campaign to inoculate new areas with milky spore disease ran from 1939–1953 and was declared successful in suppression of Japanese beetle populations [65]. Since then, additional spore formulations of P. popilliae intended to control beetle populations in various soil settings have also been approved and registered by the U.S. EPA for use in the lawn and garden industry [66]. It is also important to note that P. popilliae spores, such as those found in formulations registered in the U.S., are not considered harmful to humans or other mammals by the U.S. EPA [67].
Three families of Vip3 proteins (i.e., Vip3A, Vip3B, and Vip3C) have been identified to date [24,28]. However, Vip3A proteins are the only Vip3 members to be expressed as plant-incorporated protectants in commercialized GM crops to date [24,28,68]. Members of the Vip3A and Vip3C protein families share between 45% and 78% identity at the amino acid sequence level [24] as well as a high degree of similarity at the tertiary and quaternary structure level [22]. Therefore, what is known about the safety of one group can provide meaningful insights into the safety of another. Vip3Cb1 expressed in MON 89151 shares 66% amino acid sequence identity to the Vip3Aa19 and Vip3Aa20 proteins expressed in COT102 cotton and MIR162 maize [22], respectively. As shown by Ciche et al [22], the three-dimensional structure of Vip3Cb1 is highly similar to other Vip3 proteins including Vip3Aa, and superimposition of the three-dimensional structures further illustrates this similarity (calculated RMSD values of ~1 Å) [22].
Vip3Aa19 and Vip3Aa20, which differ by one amino acid [26], have a strong history of safe use as plant-incorporated protectants as well as strong safety profiles from risk assessment data [69]. Safety assessments submitted to the U.S. EPA show that Vip3Aa19 and Vip3Aa20 do not have significant homology to known toxins or allergens [25,26], which is consistent with the fact that Vip3 proteins do not display significant homology to any other group of proteins [24]. Like many Cry proteins [55,70], Vip3Aa proteins are susceptible to degradation by gastrointestinal proteases [25,26] indicating that digestion of proteins present in foods and/or feeds is expected to diminish exposure to functional, intact Vip3Aa proteins. Results of Vip3Aa toxicity studies also showed no evidence of acute toxicity to mammals when administered at very high dose levels [25,26]. Similarly, Vip3Aa proteins were found to pose a negligible risk to non-target organisms from highly diverse taxa including fish, rodents, and birds [68]. Finally, crops expressing Vip3Aa19 (COT102) and Vip3Aa20 (MIR162) received regulatory approvals beginning in the U.S. in 2005 and 2008, respectively, and have to date been approved for food, feed, and/or cultivation in 15 and 26 different countries/regions, respectively [71,72]. Therefore, the strong safety profile of Vip3A proteins along with their high degree of sequence similarity to Vip3C proteins supports the safety of Vip3Cb1 expressed in MON 89151.
Structure and function of Vip3Cb1
An assessment of the structure and function of Vip3Cb1 compared to other Vip3 family proteins provides further information supporting the safety of this protein. Although Vip3 proteins have distinct tertiary structure and domain organization compared to three domain Cry proteins [22,24,73], current evidence indicates that members from both classes of proteins exert their insecticidal action by virtually the same general sequence of events [22,24,27,28,74,75]. Structural and functional studies of Vip3Cb1 indicate that this protein also behaves like other Vip3 proteins from a mechanistic standpoint. For example, Vip3Cb1 and Vip3Aa19 have a higher degree of sequence similarity (~79%) in pore forming domains I and II and are more divergent in specific receptor binding domains III-V [22]. The high degree of similarity between the experimental structures of the protoxin and processed forms of Vip3Cb1 and those of Vip3A indicate that the overall fold and conformational changes leading to activation are quite similar between these two Vip3 subfamilies [22]. Like Vip3Aa [24], Vip3Cb1 is synthesized as an approximately 90-kDa protoxin and contains two trypsin-resistant fragments (~20- and ~68-kDa) which non-covalently associate to form one subunit of the tetrameric activated complex [22]. As shown in the digestibility study data (Fig 5), full-length Vip3Cb1 is rapidly degraded, and neither of these fragments are stably formed in the presence of simulated gastric fluid. Thus, since the precursors of a stable Vip3Cb1 toxin core are not expected to form in the human gut, this protein is not expected to be capable of eliciting an adverse reaction in humans or animals. Immunohistochemical experiments from mouse oral acute toxicity studies with Vip3Aa also suggest that Vip3 proteins are unable to bind to the mammalian gut [69]. In summary, structural and functional data indicate that Vip3Cb1 exerts its insecticidal activity through a similar molecular mechanism compared to all other insecticidal proteins safely introduced into broadly cultivated GM crops to date.
Assessment of potential hazards and exposure to functional Vip3Cb1
Bioinformatic analysis to determine if an introduced protein is structurally, and therefore potentially functionally, similar to known toxins or allergens is a fundamental component in the WOE assessment of GM crop protein safety [34,76,77]. The analysis presented in this study for Vip3Cb1 demonstrates that no structurally relevant similarity exists with any known toxic or allergenic proteins that would be harmful to human or animal health. The absence of homology between Vip3Cb1 and known allergens and protein toxins supports the conclusion that there is no safety concern with Vip3Cb1 in relation to humans or animals and reiterates Vip3Cb1 as an insecticidal member of the Vip3 family, which has been described previously [22].
Dietary proteins are typically degraded in the human digestive tract by gastrointestinal proteases [78]. Furthermore, commodities derived from GM crops often undergo one or more processing steps including being subject to elevated temperatures expected to lead to loss of protein structure and activity. Early research related to protein allergenicity reported a correlation between a protein’s resistance to digestion and allergenicity towards humans [79]. However, subsequent research in the last two decades show this to be a weak correlation [80]. Nevertheless, susceptibility/resistance to proteolytic digestion remains a valid assessment of the potential exposure to intact proteins. As such, the potential exposure to intact Vip3Cb1 in food or animal feed was assessed by evaluating the digestive fate of this protein in simulated gastric and intestinal environments as well as by assessing the thermal stability. Based on the digestibility experiments (Fig 5), the full-length form of Vip3Cb1 is expected to be rapidly degraded in the human gut. Two persistent peptide fragments (~10-kDa and ~8-kDa) were detected after pepsin digestion (Fig 5). The observation of persistent fragments from Vip3 proteins is not unprecedented, however. Digestion of Vip3Aa expressed in commercial cotton and corn events by pepsin or pancreatin yielded multiple persistent fragments with similar apparent molecular weights to those observed after in vitro digestion of Vip3Cb1 (Fig 5) [26,69]. Nevertheless, it is important to reiterate that bioinformatic analyses already indicate a lack of homology of the entire Vip3Cb1 sequence to any known human allergens or toxins (Table 1), and rapid degradation of the full-length Vip3Cb1 protein by gut proteases indicates that exposure to intact Vip3Cb1 protein is highly unlikely. Thermal stability experiments (Fig 4) also indicate that, like many other proteins including those expressed in GM crops [26,35,36,55,69], Vip3Cb1 exhibits a predictable tendency towards loss of structure and functional activity at elevated temperatures and is expected to lose structure and activity following typical commodity processing steps such as cooking. These studies together show that the digestive fate and temperature lability of Vip3Cb1 is comparable to other insecticidal proteins [26,36,55] and support the conclusion that dietary exposure to intact, functional Vip3Cb1 protein is unlikely to occur. Thus, expression of this protein in GM crop plants should not pose a safety hazard to human or animal health.
Extensive publications and regulatory submissions demonstrate that in vivo toxicity studies function as secondary assessments in the safety evaluation of NEPs in GM crops and are only necessary if initial bioinformatics or in vitro assays, such as heat stability and in vitro digestion analyses, indicate potential risks [25,26,35,54,55,69,77]. In addition, it is important to interpret acute toxicity studies with consideration for the mechanisms by which protein toxins act. Specifically, most known protein toxins exert their effects acutely [ 77,81–83], with certain antinutritional factors such as lectins manifesting toxicity over short-term periods in specific feeding studies [84]. International regulatory guidance concurs that if a protein demonstrates no acute toxicity, is readily digestible, and has an established history of safe use, further long-term studies on the isolated protein are generally unwarranted [34,51,85].
Despite the absence of any identified hazard for Vip3Cb1, an acute oral toxicity study using purified protein was still conducted in mice at the limit dose (i.e., the highest recommended test dose) consistent with established scientific standards [45]. Administering a limit dose of the Vip3Cb1 protein using whole cottonseed is technically and practically unfeasible. The concentration of the Vip3Cb1 protein in cottonseed is very low (~0.8 μg/g cottonseed). To deliver a dose of 5000 mg/kg to a CD-1 mouse (~30 g body weight), the animal would have to consume ~188 kg of cottonseed in a single day. This would introduce severe and unavoidable confounding factors, including nutritional imbalances, presence of antinutritional factors in cottonseed (e.g., gossypol, cyclopropenoid fatty acids) that can be toxic at high concentrations [86], and the physical impracticability of requiring a mouse to consume the unrealistic quantity of cottonseed required to meet the limit dose. Hence, using purified protein presented the most appropriate method to achieve a limit dose and is standard practice for the safety assessment of NEPs in GM crops [35,55,69]. Importantly, limit dose studies are intended to assess the intrinsic hazard of the protein using animal models and to ensure that any observed effects can be directly and unambiguously attributed to the protein being studied. Furthermore, the oral gavage administration ensures precise and complete delivery of the target dose to each animal. This study, as one part of the overall WOE safety assessment, provides foundational evidence for the safety of the Vip3Cb1 protein.
Exposure of mice to Vip3Cb1 protein at the limit dose level (5000 mg/kg b.w.) did not cause any detectable adverse effects (Tables 4–Table 7) and indicates that the no adverse effect level (NOAEL) of this protein is similar to that for Vip3Aa (NOAEL > 3675 mg/kg b.w.) and other insecticidal proteins expressed in GM crops [36,55]. Furthermore, exposure in humans to Vip3Cb1 derived from MON 89151 cotton, especially at this high dose level is highly unlikely. Refined cotton seed oil and linters (i.e., nearly pure cellulose) are two processed cotton products that are used in various human food products [87,88]. Both processed cotton products contain undetectable amounts of protein [89–91]. Importantly, reaching a 5000 mg/kg b.w. exposure in the diet is highly unlikely even for insecticidal proteins present in protein-containing products consumed by humans and animals due to the extremely high grain consumption amounts required to achieve this level of exposure in a 24-hour period [35,36,55].
Conclusion
Insecticidal proteins remain an important tool in integrated pest management programs and have been used successfully in this context in GM crops since the 1990s. The first non-three domain insecticidal protein to be used as a plant-incorporated protectant (i.e., Vip3A) presented beneficial properties both from a pest control and food and feed safety standpoint. Despite Vip3Cb1 being derived from a bacterium other than Bt, the high degree of sequence and structural similarity to Vip3A, as well as results of the above-described safety studies, present a robust weight-of-evidence for the food and feed safety of this protein. The safety conclusions for Vip3Cb1 in this study are in line with earlier findings related to Vip3Aa, which is expressed in commercially available corn and cotton varieties. Therefore, the approximately 30% amino acid sequence difference between Vip3Cb1 and Vip3Aa does not have an impact on the food and feed safety of these proteins. This reinforces the concept that when two proteins display high sequence and structural similarity, safety data from one protein can be applicable to the other.
Supporting information
S1 Fig. Alignment of the Vip3Cb1 and Vip3Ca1 amino acid sequences.
https://doi.org/10.1371/journal.pone.0344572.s001
(DOCX)
S2 Fig. Alignment of the Vip3Cb1 and Vip3Ca2 amino acid sequences.
https://doi.org/10.1371/journal.pone.0344572.s002
(DOCX)
S3 Fig. Alignment of the Vip3Cb1 and Vip3Ca3 amino acid sequences.
https://doi.org/10.1371/journal.pone.0344572.s003
(DOCX)
S4 Fig. Vip3Cb1 limit of detection in digestibility assays.
(A) Limit of detection on SDS-PAGE and Coomassie staining. (B) Limit of detection by western blotting with Vip3Cb1-specific monoclonal antibody.
https://doi.org/10.1371/journal.pone.0344572.s004
(DOCX)
S5 Fig. In vitro digestibility of control proteins.
(A) Pepsin digestion of BSA. (B) Pepsin digestion of STI. (C) Pancreatin digestion of BLG. (D) Pancreatin digestion of STI.
https://doi.org/10.1371/journal.pone.0344572.s005
(DOCX)
Acknowledgments
We thank Najma Sherzoy for supporting mass spectrometric analyses of Vip3Cb1, Jianguo Tan and David Dyck for conducting insect bioassays, and Gregory Brown for characterization of recombinant Vip3Cb1. We also thank Yong Yin, David Caldwell, William Moar, and Aster Beyene for their critical reviews of this manuscript.
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