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Preparation of egg yolk antibodies against BoNT/B and their passive protection in mouse models a
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Zherong You , Hao Yang , Wenwen Xin , Lin Kang , Shan Gao , Junhong Wang , Tao Zhang & a
Jinglin Wang a
State Key Laboratory of Pathogen and Biosecurity; Institute of Microbiology and Epidemiology; Fengtai District, Beijing, PR China Published online: 19 Jun 2014.
Click for updates To cite this article: Zherong You, Hao Yang, Wenwen Xin, Lin Kang, Shan Gao, Junhong Wang, Tao Zhang & Jinglin Wang (2014) Preparation of egg yolk antibodies against BoNT/B and their passive protection in mouse models, Human Vaccines & Immunotherapeutics, 10:8, 2321-2327, DOI: 10.4161/hv.29433 To link to this article: http://dx.doi.org/10.4161/hv.29433
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Research Paper
Research Paper
Human Vaccines & Immunotherapeutics 10:8, 2321–2327; August 2014; © 2014 Landes Bioscience
Preparation of egg yolk antibodies against BoNT/B and their passive protection in mouse models Zherong You, Hao Yang, Wenwen Xin, Lin Kang, Shan Gao, Junhong Wang, Tao Zhang, and Jinglin Wang* State Key Laboratory of Pathogen and Biosecurity; Institute of Microbiology and Epidemiology; Fengtai District, Beijing, PR China
Abbreviations: BoNTs, botulinum neurotoxins; IgY, egg yolk antibodies or yolk immunoglobulin; BHc, C-terminal heavy chain of BoNT/B; pAb, polyclonal antibody; E. coli, Escherichia coli; IPTG, sopropyl-B-D-thiogalactopyranoside; PBS, phosphate-buffered saline solution; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; CFA, Freund’s Complete Adjuvant; IFA, Freund’s Incomplete Adjuvant; PBST, 10 mM PBS, 0.05%Tween 20, pH 7.2; GPB, 50 mM PBS, 0.2% gelatin, pH 6.8; LD50, 50% lethal dose values; i.p., intraperitoneal injection; i.g., intragastric administration; SEC, size-exclusion chromatography; HIC, hydrophobic interaction chromatography.
Botulism in human is a devastating intoxication caused mainly by type A, B, and E botulinum neurotoxins (BoNTs). The most effective treatment of botulism is injection of BoNT antiserum in the first 24 h. In this study, a recombinant C-terminal heavy chain of BoNT/B (BHc) was successfully expressed in E. coli. The soluble BHc was used as an antigen to immunize laying hens for yolk immunoglobulin (IgY) production. The purified IgY against BHc subunit, preincubated with the BoNT/B, was predominantly involved in the neutralization of BoNT/B toxicity. Furthermore, both intraperitoneal and intragastric administration of the IgY could protect mice from death caused by injection of toxin at a lethal dose. Our results therefore suggest that anti-BHc IgY directed to the Hc domain is effectively involved in the neutralization of BoNT/B toxin and may be considered as preventive and therapeutic intervention in the case of botulism.
Introduction Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum, contain 7 structurally similar yet antigenic distinct proteins divided into serotypes A to G.1 These toxins are released as a inactive 150 kDa single polypeptide chain and then (endogenously or exogenously) cleaved into a heavy chain (MW 100 kDa) and a light chain (MW 50 kDa) joined by a single disulphide bond, resulting in the biologically active toxin acknowledged as the most toxic substances in nature.2 Among these active BoNTs, serotypes A, B, and E commonly cause human botulism, which includes food-borne and infant botulism (gastrointestinal tract), inhalation botulism (pulmonary tract), and wound botulism (mucus membranes of wounds) based on portals of entry for BoNTs.3 Injection of the antidote in the first 24 h can neutralize BoNT directly and has been accepted for clinical use. Currently, the most effective botulism antidote for therapy is serum-derived equine polyclonal antibodies (pAb). The only 2 non-infant therapeutic products available for clinical therapy are the heptavalent BoNT F(ab’)2 anti-toxin (HBAT) available from the CDC, and the anti-ABE pAb BotulismusAntitoxin Behring from.4 However, the antitoxin serum intended to protect against primary BoNTs can result in serious
complications, such as serum sickness, complement induction and occasionally anaphylactic shock. Besides, the preparation method for antitoxin serum is time-consuming and dangerous during detoxication of large quantities of toxin. To overcome the limitations of these serum-derived equine antitoxins, specific yolk immunoglobulin (IgY) against BoNTs has been investigated. As we know, IgY is considered a useful candidate to conventional mammalian antibodies.5 Researchers have successfully produced IgY against a number of antigens, including microbial species and biotoxins. Furthermore, IgY has unique binding characteristics and is more useful in many applications, including immunotherapy and immunodiagnostics. For instance, IgY induces less hypersensitivity reaction and serum sickness, because IgY does not activate the mammalian complement system or bind rheumatoid factors or human Fc-receptors.6,7 In addition, oral administration of the specific IgY has been proved to be effective against a variety of gastrointestinal intoxication.8 Thus, the specific IgY has been recognized as an economical source for the clinical treatment. Recently, some researchers have successfully improved protocol for producing chicken IgY with highly toxic BoNT/A.9 However, few researchers have immunized hens with recombinant subunit of BoNTs since then. The recombinant C-terminal heavy chain of BoNT/B (BHc) expressed in E. coli was used to immunize the
*Correspondence to: Jinglin Wang; Email: [email protected] Submitted: 04/09/2014; Revised: 05/26/2014; Accepted: 06/02/2014; Published Online: 06/19/2014 http://dx.doi.org/10.4161/hv.29433 www.landesbioscience.com Human Vaccines & Immunotherapeutics 2321
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Keywords: botulinum neurotoxin, Hc fragment, botulism, IgY, passive protection
hens, and the neutralization effect of the purified anti-BHc IgY was tested in mouse model.
Results The purification of the expressed BHc The synthesized gene was cloned into expression plasmid pTIG-Trx. Recombinant BHc (MW 50 kDa) was expressed in E. coli (BL21) and the product was partly soluble. After purification by Ni-NTA affinity chromatography, the proteins were identified by SDS-PAGE (Fig. 1A) and western blot (probed with antiBoNT/B antiserum) (Fig. 1B). The identification of purified IgY and its ELISA titers The partly purified IgY showed 2 typical protein bands with relative molecular masses of 68kDa (heavy chain) and 25kDa (light chain) coinciding with the commercial control. Their molecular masses matched the theoretical prediction. In addition, one hybridprotein band (43 kDa) was observed. IgY was identified as about 85% pure by Bandscan 5.0 software (Fig. 2). Also, the approximate yield of IgY was 110 mg per egg yolk determined by BCA protein assay. To determine the level of anti-BoNT/B antibodies throughout the immunization, both serum and yolk samples were measured by ELISA. The titer of anti-BHc IgY was at a low level (1:800) after primary immunization. An obvious increase of IgY titer was observed gradually after 3 boosting immunizations. The titer of IgY antibody response reached the maximum level of 105. (Fig. 3). The kinetic of the serum in response to the introduction of BoNT/B was similar to what was observed in the IgY in the yolks (Fig. 3).
Lethal toxicity of BoNT/B complex The LD50 /i.p. of BoNT/B toxin using Karber method was 4.363 ng/kg (about 0.0873 ng/mouse) and the detailed data are shown as Kaplan–Meier plots. Deaths occurred in most cases between 12 and 48 h after injection, and mortalities also observed at lower doses (2 and 4 ng/kg) after 48 h (Fig. 4A), as was described before.10 The LD50 /i.g. of BoNT/B complex was 1.135 μg/kg (about 22.7 ng/mouse), nearly 260 times LD50 /i.p.. Most of the mice died between 24 and 60 h after injection, and mortalities were also observed at high doses after 60 h (Fig. 4B). In addition, the mice that were given dosage of BoNT/B complex displayed typical symptoms of botulism 3–6 h after challenge, including wasp-waist, ruffled fur, hind limb paralysis, and labored breathing.11 Neutralization effect of IgY by pre-incubation In a mouse neutralization assay via i.p., dose dependency of IgY treatment was investigated. Challenged with 4 × LD50/i.p., all the mice in the control group died between 6 and 36 h after injection(Fig. 5A). After injection of toxin-antidote incubated mixtures, the intoxication symptoms of botulism were lessened and the survival rate was increased. IgY provided a partial protection in mouse groups treated with 12.5 and 25 ng/mouse. The data showed that the increasing dose of IgY greatly enhanced the neutralization effect, and the surviving mice increased from 25% (2 out of 8) to 50% (4 out of 8) (Fig. 5A). Finally, we observed that all the mice treated with 50 and 100 ng/mouse of IgY survived. The results were significantly different from those of the BoNT/B control group. The detailed data are shown as Kaplan–Meier plots. Furthermore, the results of serial IgY solutions premixed with 40 × LD50/i.p. and 400 × LD50/i.p. (Fig. 5B and C) respectively were similar to the result described before. The neutralization capacity of anti-BHc IgY was approximately 4 × LD50 doses per 25 ng/ mouse IgY, or nearly 160 000 LD50 doses (namely 16 IU) per mg
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Figure 1. SDS-PAGE (A) and western blot (B) analysis of recombinant soluble BHc. Lane 1, low-molecular-weight protein markers; lane 2, the lysate from cells transformed with pTIG-Trx-BHc vector; lanes 3 and 4, purified BHc. Molecular weights of the protein standards are indicated on the left (×103). An arrow indicates the position of the BHc.
Figure 2. SDS–PAGE analysis of the purified IgY. Lane 1, low-molecularweight protein markers; lane 2, IgY purified from the eggs after last vaccine; lane 3, IgY purified from eggs before the first vaccine. The upper arrow shows a heavy chain of IgY (65 kDa), and the lower arrow shows a light chain of IgY (25 kDa).
IgY.4 Based on the calculation of IgY concentration, we calculated that one egg collected in this study contained a the neutralization capacity of 1760 IU. The time dependency of treatment with IgY Time dependency of treatment with IgY was investigated (Table 1). After successive injection of anti-BHc IgY and BoNT/B complex, the mortality and intoxication symptoms of botulism varied. All the mice survived in groups that were treated i.p. with anti-BHc IgY before, simultaneously with or 1.5 h after BoNT/B challenge via i.g. route. In contrast, all 8 mice died in the group which IgY was given 3 h via i.g. after the challeng of BoNT/B intraperitoneally. In the control group, all the mice challenged i.p. with BoNT/B died. The dose dependency of treatment with IgY In a mouse neutralization assay via i.g. route in vivo, dose dependency of treatment with IgY was investigated (Fig. 6A). In the mice challenged with BoNT/B via i.p. route, all the mice treated with PBS or low level of IgY (1.25 ng/mouse) via i.g. route died between 12 and 48 h after injection. The groups treated with an increasing dose of IgY (from2.5 to 5 ng/mouse) were partially protected from 25% (2 out of 8) to 50% (4 out of 8), and 10 ng/mouse of IgY protected mice from BoNT/B complex intoxication completely. A similar result was detected in groups treated with IgY via i.g. route (Fig. 6B). The mice were partially under protection from 25% (2 out of 8) to 50% (4 out of 8) in groups treated with an increasing dose of IgY (from 25 to 50 μg/mouse), and the mice treated with 100 μg/mouse of IgY all survived (Fig. 6B). The detailed data are shown as Kaplan–Meier plots.
Discussion In previous studies, specific-pathogen-free laying hens were immunized with BoNT or denatured BoNT (toxoid), and the production of egg yolk IgY specific to these agent can be high-risk threat.4 While, no one has immunized hens with carboxyl-terminal of BoNT heavy chain (Hc domain). The soluble BHc subunit proteins, synthesized in E. coli constructed vectors, have shown a higher degree of purity and posed less of a hazard than the traditional inactivated toxin.12 In addition, the concentrated antigencity may increase immunogenicity and
Figure 4. Survival curves of the mice treated with BoNT/B complex via different routes. BoNT/B complex was treated i.p. (A) and i.g. (B). Percentage of survival was plotted over time. Groups of 8 mice were used at each dosage level and five-dose levels were tested per experiment as described in Materials and Methods. The animals were observed for death over a period of 96 h.
stimulate protection efficacy in animal models. In this study, specific-pathogen-free laying hens were housed to determine the usefulness of an egg antibody platform for producing antiBHc IgY. We expressed soluble BHc as an appropriate antigen to generate toxin-specific IgY for immunotherapy. Hence, the first specific objective of this study was to develop an improved protocol for producing egg yolk IgY against BoNT/B with BHc protein as a new immunogen. The IgY platform, capable of providing a rapid method for commercial-scale antitoxin production, may serve as an inexpensive alternative source of antitoxin for neutralization purposes. The second specific objective was to detect the capacity of anti-BHc IgY to neutralize BoNT/B by pre-incubation before injection of testing mice. The third specific objective was to test the ability of anti-BHc IgY to neutralize BoNT/B as a neutralizing reagent by injecting IgY and toxin via different routes respectively in a mouse bioassay. The ELISA data analysis demonstrated that the anti-BHc IgY reacted well with the corresponding toxin BoNT/B. The anti-BHc IgY was obtained by injecting laying hens with the recombinant BHc proteins, and was prepared from eggs by water dilution method. An obvious increase of IgY titer was observed in both serum and yolk samples on the seventh day after the second inoculations (21st day). We also observed stably high levels of IgY after the last immunization.13 Our results showed that 4 immunizations are necessary for maintaining the levels of IgY for a period of nearly 1 mo.12 As the ‘gold standard’ for the measurement of BoNTs, the intraperitoneal mouse bioassay was used to test LD50 of the purified toxin complex. The data indicated that a higher dose of BoNT/B killed more sufficiently and effectively than a lower dose, and the LD50 of BoNT/B calculated by the Karber method was 4.363 ng/ kg, which was consistent with our previous study.10 These results
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Figure 3. Levels of IgY and serum titer by ELISA. Arrows indicate the day of each vaccination.
provided information for the toxicity of BoNT/B given by intraperitoneal injection, but there are also reports that the toxicity of BoNT/B is higher than BoNT/A. This discrepancy may be attributed to the purity of BoNT and strains of mice (ddY, CD-1, or BALB/c) used in the experiments. Furthermore, the intragastric administration mouse bioassay was used to test LD50 of BoNT/B of the purified toxin complex. As for food-borne and infant botulism, the gastrointestinal tract is an important portal of entry for BoNTs. The LD50 /i.g. calculated by the Karber method was 1.135 μg/kg in the mouse model. We chose 4 × LD50 /i.p. and 4 × LD50 /i.g. as a potentially lethal dose for almost all the mice that died within 24 h after administration. As for the neutralization activity of mice experiments via intraperitoneal injection, lower doses of IgY (12.5 and 25 ng/ mouse) could provide partial neutralization against 4 × LD50 / i.p. BoNT/B, while higher doses of IgY (50 and 100 ng/mouse) could protect the mice from any BoNT/B complex intoxication (Fig. 5A). Neutralization activity tested again with higher doses, namely 40 × LD50 /i.p. and 400 × LD50 /i.p., yielded a similar result. Lower doses of IgY could provide partial protection, while higher doses of IgY could protect the mice from BoNT/B complex intoxication completely (Fig. 5B and C). As Hc has been proved to contain an important epitopes to generate neutralizing
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Figure 5. Neutralization effect of anti-BHc IgY. (A) Mixture of 4 × LD50/i.p. and serial IgY, (B) mixture of 40 × LD50/i.p. and serial IgY, and (C) mixture of 400 × LD50/i.p. and serial IgY were administered i.p. to BALB/c mice respectively. The control mice were given PBS instead of IgY. Groups of 8 mice were used at each IgY dosage level and four-dose levels were tested per experiment as described in Materials and Methods. There was a significant difference at ***P < 0.001 and ***P < 0.001 vs. the control (log rank test).
antibodies, anti-BHc IgY could bind with the protein receptor binding domain of BoNT/B efficiently in our study, for the Hc is responsible for toxin binding to the sialoganglioside receptor and the putative protein receptor on presynaptic neurons. Thus, we can predict that IgY of chickens immunized with recombinant BHc subunit neutralized the bioactivity of BoNT/B sufficiently.9 The current horse serum treatment accepted for clinical use, HBAT for instance, represents 5500 IU of anti-toxin B per 10 mL.4 In our study, the neutralizing capacity of one egg was approximately 1760 IU, much higher than other study before.9 We calculated that 3.125 toxin-specific eggs is equal to the 10mL-treatment vial of horse serum. Therefore, anti-BHc IgY can be considered a useful alternative to mammalian antibodies. In the experiment for determination of LD50 /i.p., typical symptoms of botulism were displayed 3 h after challenge, suggesting that BoNT/B entered the bloodstream from the intestine nearly at this point. It is likely that BoNT/B toxin takes almost 3 h to enter circulation after i.g. injection. The i.p. administration of the IgY in survived groups may precede to enter of BoNT/B to the bloodstream in this mouse model.14 Thus, we can conclude that anti-BHc IgY can neither activate neutralizing capacities until BoNT/B binds to target receptors nor inhibit toxicity after toxins are transported to the neuromuscular junctions of motor neurons where its destructive action occurs.13,14 From the perspective of botulism therapy, anti-BHc IgY injected i.p. has the capacity to inactivate BoNT/B in the blood stream. Partly due to the unique physiochemical characteristics of IgY, several researchers have demonstrated the efficacy of IgY as an oral treatment or prophylactic for gastrointestinal disease. The neutralizing activity of IgY injected i.g. against death in mice was investigated. According to the data (Table 1), all the mice survived in the group that was treated i.g. with anti-BHc IgY 3 h before BoNT/B challenge via i.p. route. Anti-BHc IgY at the dosage of 100 μg/mouse showed complete protection following i.p. administration in mice challenged i.g. with 4 × LD50 /i.p. BoNT/B (Fig. 6B). Given orally, IgY showed its neutralization ability after reaching the large intestine.15 Thus, anti-BHc IgY was measured in the feces within 24 h to detect the duration of antibody activity in mice. From the perspective of botulism prevention, anti-BHc IgY injected i.g. before has the capacity to inactivate BoNT/B for at least 4 h.16 Hence, IgY antidote may serve as a useful oral therapy in botulism. Since BoNTs can be weaponized as aerosols as well as contaminate food and water supplies, the efficacy of anti-BHc IgY has not yet been determined in an aerosol intoxication model in mice. Furthermore, animal models are needed to investigate the safety of anti-BHc IgY as a human therapeutic and prevention before preclinical human trials.7 Limited researches have been conducted on the safety of peripherally administered IgY as a mouse therapeutic in previous studies.9 In addition, egg yolk antibodies can be expanded against other types of BoNTs in the future. In conclusion, we have demonstrated that chickens immunized with the recombinant BHc can produce IgY against BoNT/B, which can neutralize the bioactivity of neurotoxins via intraperitoneal injection and can help reduce the risk of botulism
Table 1. Mortality of the mice administered anti-BHc IgY after challenge with BoNT/B
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Timing of IgY administration (h)
(A) BoNT/B i.g.
(B) BoNT/B i.p.
IgY i.p.
IgY i.g.
Control
8/8
8/8
-3
0/8***
0/8***
-1.5
0/8***
7/8
0
0/8***
8/8
1.5
0/8***
8/8
3
8/8
8/8
(A) BoNT/B (4 × LD50/i.g.) was given via i.g. and 0.25mg/mouse IgY were administered i.p. at various timing. (B) BoNT/B (4 × LD50/i.p.) was given via i.p. and 0.25mg/mouse IgY were administered i.g. at various timing. Groups of 8 mice were used at each time level and 5 different groups were tested per experiment as described in Materials and Methods; ***There is a statistically significant difference at P < 0.001 vs. the control (log rank test).
by the intragastric route. The anti-BHc IgY can be potentially used in biodefense and is generally applicable to the field of infectious botulism diseases.
Materials and Methods Bacterial strain and production of BHc The C. botulinum serotype Danish (B1 subtype, GenBank Accession number M81186) that could produce BoNT/B toxin was provided by the State Key Laboratory of Pathogens and Biosecurity. The strain stored at -80 °C was inoculums (0.1 mL) for 10 mL of cooked meat media (peptone 3%, yeast extract 0.5%, beef extract 0.5%, glucose 0.3%, NaH2PO4 0.5%, pH 7.2) incubated at 37 °C. One L of an anaerobic medium TPYG (5% trypticase peptone, 0.5% bacto peptone, 0.4% glucose, 2% yeast extract and 0.1% l-cysteine, pH 7.4) was inoculated with 10 mL of active culture growing in the cooked meat medium and incubated undisturbed for 96 h at 30 °C. Then, BoNT/B was extracted from cultured media using PEG precipitation. The following purification was accomplished by sizeexclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) techniques using SephacrylTM S-100 and phenyl HP columns. This new platform for purification of BoNT/B was developed by Yao Zhao.10 The synthesized BHc DNA fragment of C.botulinum serotype Danish (1314bp: 2616–3929) was cloned into an modified pET22b expression vector (named pTIG-Trx) to create recombinant plasmid pTIG-Trx-BHc, and then was transformed into E.coli strain BL21 (DE3) (TransGen).17 Then the expressed strain was cultured with shaking in L-Broth (LB) medium to prepare the recombinant protein BHc. In the mid-logarithmic phase of growth, 0.2 mmol/L isopropyl-B-D-thiogalactopyranoside (IPTG, Promega) was added to the culture to induce BHc production, followed by cultivation for an additional 6 h. The culture supernatant was purified through a HiTrap Chelating high performance 5-mL column pre-packed with a precharged Ni2+ column (GE Healthcare), and the purified protein was analyzed by SDS-PAGE and western blots using hyperimmune horse neurotoxin B antiserum. The antiserum was provided by
the Lanzhou Institute of Biological Products. The identified BHc was used as antigens in this study. Animals Kept in an individual cage, two 20-wk-old specific-pathogenfree laying hens were housed as the primary egg to produce the anti-BoNT/B IgY. BALB/c mice (18–22 g) under pathogen free conditions were used to determine lethality of BoNT/B and passive protection capacity of anti-BoNT/B IgY. These mice with free access to food and water were obtained from the Laboratory Animal Center, Academy of Military Medical and Sciences. All experiments were conducted strictly in accordance with the guidelines of the Chinese Association for the Accreditation of Laboratory Animals Care (CAALAC), including the relevant local animal welfare bodies in China. In addition, the permit number of all animal work was SCXK-(JUN) 2013–005 approved by the animal ethics committee of Beijing Institute of Microbiology and Epidemiology. Hens immunization To prepare anti-BHc IgY, two laying hens were housed in individual cages. The purified recombinant BHc (0.5 mg in 0.1 mL PBS) was pre- mixed with an equal volume of Freund’s Complete Adjuvant (CFA, Sigma). The mixture was given intramuscularly at 4–6 sites of the pectoral muscle. In the same manner, hens were boosted every 2 wk after the first immunization i.e., on days 14, 28, and 42 and CFA was substituted with Freund’s Incomplete Adjuvant (IFA, Sigma). The hen’s blood was sampled the day prior to the first immunization and 7 d after each injection; furthermore, eggs were collected and labeled accordingly during the experiment. All the samples were stored at 4 °C. The preparation and identification of IgY Under acidic conditions, IgY was prepared by dilution method with modification. 18 Eighteen eggs for obtaining IgY were collected from 50 to 65 d after initial immunization. The yolks were separated, pooled and well mixed with 9 times volume of distilled water (pH 2.5–2.8). The pH of the suspension was then adjusted with 0.1 mol/L HCl to 5.0. After being kept at 4 °C overnight, the aggregate lipoproteins of the yolk were separated by centrifugation (10 000 g for 30 min at 4 °C). The supernatant was added with solid ammonium sulfate to 19% (m/v). The overnight precipitate was dissolved
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Mortality (Dead/Total)
at the original volume of the yolk in PBS (pH 7.2) and finally identified by SDS–PAGE. The digital image was analyzed using Bandscan 5.0 software and the protein concentrations were estimated using BCA protein assay (Sigma) according to the manufacturer’s protocol. To obtain the anti-BoNT/B IgY titer throughout the immunization, the eggs prior to the first vaccination and seventh day after each vaccination were purified separately as described before. ELISA for antibody titer monitoring The titers of both the egg and the serum samples were tested by a standard ELISA. The ELISA plates were coated with BoNT/B (10 ng/mL in 10 mM PBS, pH 9.6, 100 µL/well) for 1 h at 37 °C and blocked with 3% BSA in PBS and were washed with PBST (10 mM PBS, 0.05%Tween 20, pH 7.2) between all incubations. The eggs and serum samples were applied in serial 2-fold dilution from 1:200 and incubated for 1 h at 37 °C. After washing, HRP-conjugated goat anti-chicken IgG (SouthernBiotech) was added at a dilution of 1:4000–1:8000. Anti-BoNT/B reactivity was visualized by adding TMB and H2O2 substrates, and the colorimetric reaction was stopped with 2 M sulfuric acid before absorbance at 450 nm was read using a multifunctional micro plate reader (MDC, USA). Antibody titers were estimated as the reciprocal of the maximum dilution giving an absorbance reading greater than 0.5 units following subtraction of nonspecific binding detected in the control sample and 2-fold greater than that of the matched dilution of the control sample.19
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Figure 6. The protective effect of anti-BHc IgY in vivo given via different routes. (A) BoNT/B (4 × LD50/i.g.) was given via i.g. and serial IgY was administered i.p. at 1.5h after challenge. (B) BoNT/B (4 × LD50/i.p.) was given via i.p. and serial IgY was administered i.g. at 3 h before challenge. Groups of 8 mice were used at each dosage level and four-dose levels were tested per experiment as described in Materials and Methods. There was a significant difference at ***P < 0.001 and ***P < 0.001 vs. the control (log rank test).
Determining the lethal toxicity of BoNT/B 50% lethal dose (LD50) of biologically active BoNT/B complex was determined in BALB/c mice. The toxin complex was purified using the modified Karber method.10 2-fold serial dilutions of purified complex toxin were diluted in gelatin-phosphate-buffer (GPB: 50 mM PBS, 0.2% gelatin, pH 6.8), and then were injected via 2 different routes, intraperitoneal injection (i.p.) and intragastric administration (i.g.). Before injection, the mice in all groups were under an adaptation for 3 d. In addition, the mice via i.g. route were under starvation and water deprivation for an additional 8 h. The number of survived mice in each group was recorded over a period of 96 h. The experiments were repeated 3 times. 50% lethal dose values (LD50/i.p. and LD50/i.g.) were calculated using the Karber method.10 A survival curve of mice was measured using the Prism 5 statistics software (Graph Pad Software Inc.). The neutralization capacity of IgY by pre-incubation The neutralization activity of the anti-BHc IgY by preincubating with BoNT/B was evaluated in mouse assay via intraperitoneal injection. The purified anti-BHc IgY samples were followed by 2-fold serial dilutions in GPB. The serial dilutions (0.25 mL) were premixed with the same volume 4 × LD50 /i.p., 40 × LD50 /i.p., and 400 × LD50 /i.p. BoNT/B solution respectively. Incubated at room temperature for 0.5 h, the mixtures were injected i.p. in all groups tested. The mice in control group were treated with PBS instead of IgY. Mouse survival in all groups was observed for 96 h after administration and the data were calculated. The neutralization capacity of anti-BHc IgY was expressed in international units (IU) of antidotes per milliliter of yolk.20 In the mouse neutralization assay, the capacity of antiBHc IgY was estimated in IU. One IU of anti-BoNT products protects a mouse against 10 000 mouse LD50 of A–F or 1000 LD50 of E BoNTs by definition.4 The time dependency of IgY treatment To further support the application in treatment of botulinum, the protective effect of IgY against BoNT/B complex was detected in mouse assay by injecting IgY and toxin via 2 different routes respectively. First, time dependency of IgY treatment via i.p. was detected. BALB/c mice in these groups were challenged with 4 × LD50 /i.g. and overdose IgY (0.25 mg/mouse ) was given via i.p. at various timing. Then, time dependency of IgY treatment via i.g., another different administered route, was detected. These mice were challenged with 4 × LD50 /i.g. and overdose IgY was given via i.p. route at various timing. The dose dependency of IgY treatment After the time dependency experiment, different dosages of IgY (2-fold serial dilutions in GPB) were given to the mice via i.p. route certain timing before or after challenge with a dose of 4 × LD50 /i.g.. In the dose dependency of IgY treatment via i.g., the 2-fold serial IgY dilutions in GPB were given to the mice via i.g. route certain time before or after challenge with 4 × LD50 /i.p.. In control groups, the same volume of PBS was substituted with IgY solution. The data were collected and calculated as described before.
1. Turton K, Chaddock JA, Acharya KR. Botulinum and tetanus neurotoxins: structure, function and therapeutic utility. Trends Biochem Sci 2002; 27:5528; PMID:12417130; http://dx.doi.org/10.1016/ S0968-0004(02)02177-1 2. Luvisetto S, Rossetto O, Montecucco C, Pavone F. Toxicity of botulinum neurotoxins in central nervous system of mice. Toxicon 2003; 41:47581; PMID:12657317; http://dx.doi.org/10.1016/ S0041-0101(02)00370-7 3. Smith LA. Botulism and vaccines for its prevention. Vaccine 2009; 27(Suppl 4):D33-9; PMID:19837283; http://dx.doi.org/10.1016/j.vaccine.2009.08.059 4. Froude JW, Stiles B, Pelat T, Thullier P. Antibodies for biodefense. MAbs 2011; 3:517-27; PMID:22123065; http://dx.doi.org/10.4161/mabs.3.6.17621 5. Shimizu M, Nagashima H, Sano K, Hashimoto K, Ozeki M, Tsuda K, Hatta H. Molecular stability of chicken and rabbit immunoglobulin G. Biosci Biotechnol Biochem 1992; 56:2704; PMID:1368302; http://dx.doi.org/10.1271/ bbb.56.270 6. Spillner E, Braren I, Greunke K, Seismann H, Blank S, du Plessis D. Avian IgY antibodies and their recombinant equivalents in research, diagnostics and therapy. Biologicals 2012; 40:31322; PMID:22748514; http://dx.doi.org/10.1016/j. biologicals.2012.05.003 7. Dias da Silva W, Tambourgi DV. IgY: a promising antibody for use in immunodiagnostic and in immunotherapy. Vet Immunol Immunopathol 2010; 135:173-80; PMID:20083313; http://dx.doi. org/10.1016/j.vetimm.2009.12.011 8. Xu Y, Li X, Jin L, Zhen Y, Lu Y, Li S, You J, Wang L. Application of chicken egg yolk immunoglobulins in the control of terrestrial and aquatic animal diseases: a review. Biotechnol Adv 2011; 29:8608; PMID:21787857; http://dx.doi.org/10.1016/j. biotechadv.2011.07.003
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16. Chen C, Wang S, Wang H, Mao X, Zhang T, Ji G, Shi X, Xia T, Lu W, Zhang D, et al. Potent neutralization of botulinum neurotoxin/B by synergistic action of antibodies recognizing protein and ganglioside receptor binding domain. PLoS One 2012; 7:e43845; PMID:22952786; http://dx.doi.org/10.1371/ journal.pone.0043845 17. Yu YZ, Sun ZW, Wang S, Yu WY. High-level expression of the Hcc domain of Clostridium botulinum neurotoxin serotype A in Escherichia coli and its immunogenicity as an antigen. Sheng Wu Gong Cheng Xue Bao 2007; 23:812-7; PMID:18051857; http://dx.doi.org/10.1016/S1872-2075(07)60052-1 18. Wang Q, Hou XJ, Cai K, Li T, Liu YN, Tu W, Xiao L, Bao SZ, Shi J, Gao X, et al. Passive protection of purified yolk immunoglobulin administered against Shiga toxin 1 in mouse models. Can J Microbiol 2010; 56:1003-10; PMID:21164570; http://dx.doi. org/10.1139/W10-087 19. Mukamoto M, Maeda H, Kohda T, Nozaki C, Takahashi M, Kozaki S. Production of a neutralizing mouse-human chimeric antibody against botulinum neurotoxin serotype E. Jpn J Infect Dis 2013; 66:4650; PMID:23429085; http://dx.doi.org/10.7883/ yoken.66.46 20. Jones RG, Corbel MJ, Sesardic D. A review of WHO International Standards for botulinum antitoxins. Biologicals 2006; 34:223-6; PMID:16490362; http://dx.doi.org/10.1016/j.biologicals.2005.11.009
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References
Human Vaccines & Immunotherapeutics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/khvi20
Preparation of egg yolk antibodies against BoNT/B and their passive protection in mouse models a
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Zherong You , Hao Yang , Wenwen Xin , Lin Kang , Shan Gao , Junhong Wang , Tao Zhang & a
Jinglin Wang a
State Key Laboratory of Pathogen and Biosecurity; Institute of Microbiology and Epidemiology; Fengtai District, Beijing, PR China Published online: 19 Jun 2014.
Click for updates To cite this article: Zherong You, Hao Yang, Wenwen Xin, Lin Kang, Shan Gao, Junhong Wang, Tao Zhang & Jinglin Wang (2014) Preparation of egg yolk antibodies against BoNT/B and their passive protection in mouse models, Human Vaccines & Immunotherapeutics, 10:8, 2321-2327, DOI: 10.4161/hv.29433 To link to this article: http://dx.doi.org/10.4161/hv.29433
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Research Paper
Research Paper
Human Vaccines & Immunotherapeutics 10:8, 2321–2327; August 2014; © 2014 Landes Bioscience
Preparation of egg yolk antibodies against BoNT/B and their passive protection in mouse models Zherong You, Hao Yang, Wenwen Xin, Lin Kang, Shan Gao, Junhong Wang, Tao Zhang, and Jinglin Wang* State Key Laboratory of Pathogen and Biosecurity; Institute of Microbiology and Epidemiology; Fengtai District, Beijing, PR China
Abbreviations: BoNTs, botulinum neurotoxins; IgY, egg yolk antibodies or yolk immunoglobulin; BHc, C-terminal heavy chain of BoNT/B; pAb, polyclonal antibody; E. coli, Escherichia coli; IPTG, sopropyl-B-D-thiogalactopyranoside; PBS, phosphate-buffered saline solution; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; CFA, Freund’s Complete Adjuvant; IFA, Freund’s Incomplete Adjuvant; PBST, 10 mM PBS, 0.05%Tween 20, pH 7.2; GPB, 50 mM PBS, 0.2% gelatin, pH 6.8; LD50, 50% lethal dose values; i.p., intraperitoneal injection; i.g., intragastric administration; SEC, size-exclusion chromatography; HIC, hydrophobic interaction chromatography.
Botulism in human is a devastating intoxication caused mainly by type A, B, and E botulinum neurotoxins (BoNTs). The most effective treatment of botulism is injection of BoNT antiserum in the first 24 h. In this study, a recombinant C-terminal heavy chain of BoNT/B (BHc) was successfully expressed in E. coli. The soluble BHc was used as an antigen to immunize laying hens for yolk immunoglobulin (IgY) production. The purified IgY against BHc subunit, preincubated with the BoNT/B, was predominantly involved in the neutralization of BoNT/B toxicity. Furthermore, both intraperitoneal and intragastric administration of the IgY could protect mice from death caused by injection of toxin at a lethal dose. Our results therefore suggest that anti-BHc IgY directed to the Hc domain is effectively involved in the neutralization of BoNT/B toxin and may be considered as preventive and therapeutic intervention in the case of botulism.
Introduction Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum, contain 7 structurally similar yet antigenic distinct proteins divided into serotypes A to G.1 These toxins are released as a inactive 150 kDa single polypeptide chain and then (endogenously or exogenously) cleaved into a heavy chain (MW 100 kDa) and a light chain (MW 50 kDa) joined by a single disulphide bond, resulting in the biologically active toxin acknowledged as the most toxic substances in nature.2 Among these active BoNTs, serotypes A, B, and E commonly cause human botulism, which includes food-borne and infant botulism (gastrointestinal tract), inhalation botulism (pulmonary tract), and wound botulism (mucus membranes of wounds) based on portals of entry for BoNTs.3 Injection of the antidote in the first 24 h can neutralize BoNT directly and has been accepted for clinical use. Currently, the most effective botulism antidote for therapy is serum-derived equine polyclonal antibodies (pAb). The only 2 non-infant therapeutic products available for clinical therapy are the heptavalent BoNT F(ab’)2 anti-toxin (HBAT) available from the CDC, and the anti-ABE pAb BotulismusAntitoxin Behring from.4 However, the antitoxin serum intended to protect against primary BoNTs can result in serious
complications, such as serum sickness, complement induction and occasionally anaphylactic shock. Besides, the preparation method for antitoxin serum is time-consuming and dangerous during detoxication of large quantities of toxin. To overcome the limitations of these serum-derived equine antitoxins, specific yolk immunoglobulin (IgY) against BoNTs has been investigated. As we know, IgY is considered a useful candidate to conventional mammalian antibodies.5 Researchers have successfully produced IgY against a number of antigens, including microbial species and biotoxins. Furthermore, IgY has unique binding characteristics and is more useful in many applications, including immunotherapy and immunodiagnostics. For instance, IgY induces less hypersensitivity reaction and serum sickness, because IgY does not activate the mammalian complement system or bind rheumatoid factors or human Fc-receptors.6,7 In addition, oral administration of the specific IgY has been proved to be effective against a variety of gastrointestinal intoxication.8 Thus, the specific IgY has been recognized as an economical source for the clinical treatment. Recently, some researchers have successfully improved protocol for producing chicken IgY with highly toxic BoNT/A.9 However, few researchers have immunized hens with recombinant subunit of BoNTs since then. The recombinant C-terminal heavy chain of BoNT/B (BHc) expressed in E. coli was used to immunize the
*Correspondence to: Jinglin Wang; Email: [email protected] Submitted: 04/09/2014; Revised: 05/26/2014; Accepted: 06/02/2014; Published Online: 06/19/2014 http://dx.doi.org/10.4161/hv.29433 www.landesbioscience.com Human Vaccines & Immunotherapeutics 2321
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Keywords: botulinum neurotoxin, Hc fragment, botulism, IgY, passive protection
hens, and the neutralization effect of the purified anti-BHc IgY was tested in mouse model.
Results The purification of the expressed BHc The synthesized gene was cloned into expression plasmid pTIG-Trx. Recombinant BHc (MW 50 kDa) was expressed in E. coli (BL21) and the product was partly soluble. After purification by Ni-NTA affinity chromatography, the proteins were identified by SDS-PAGE (Fig. 1A) and western blot (probed with antiBoNT/B antiserum) (Fig. 1B). The identification of purified IgY and its ELISA titers The partly purified IgY showed 2 typical protein bands with relative molecular masses of 68kDa (heavy chain) and 25kDa (light chain) coinciding with the commercial control. Their molecular masses matched the theoretical prediction. In addition, one hybridprotein band (43 kDa) was observed. IgY was identified as about 85% pure by Bandscan 5.0 software (Fig. 2). Also, the approximate yield of IgY was 110 mg per egg yolk determined by BCA protein assay. To determine the level of anti-BoNT/B antibodies throughout the immunization, both serum and yolk samples were measured by ELISA. The titer of anti-BHc IgY was at a low level (1:800) after primary immunization. An obvious increase of IgY titer was observed gradually after 3 boosting immunizations. The titer of IgY antibody response reached the maximum level of 105. (Fig. 3). The kinetic of the serum in response to the introduction of BoNT/B was similar to what was observed in the IgY in the yolks (Fig. 3).
Lethal toxicity of BoNT/B complex The LD50 /i.p. of BoNT/B toxin using Karber method was 4.363 ng/kg (about 0.0873 ng/mouse) and the detailed data are shown as Kaplan–Meier plots. Deaths occurred in most cases between 12 and 48 h after injection, and mortalities also observed at lower doses (2 and 4 ng/kg) after 48 h (Fig. 4A), as was described before.10 The LD50 /i.g. of BoNT/B complex was 1.135 μg/kg (about 22.7 ng/mouse), nearly 260 times LD50 /i.p.. Most of the mice died between 24 and 60 h after injection, and mortalities were also observed at high doses after 60 h (Fig. 4B). In addition, the mice that were given dosage of BoNT/B complex displayed typical symptoms of botulism 3–6 h after challenge, including wasp-waist, ruffled fur, hind limb paralysis, and labored breathing.11 Neutralization effect of IgY by pre-incubation In a mouse neutralization assay via i.p., dose dependency of IgY treatment was investigated. Challenged with 4 × LD50/i.p., all the mice in the control group died between 6 and 36 h after injection(Fig. 5A). After injection of toxin-antidote incubated mixtures, the intoxication symptoms of botulism were lessened and the survival rate was increased. IgY provided a partial protection in mouse groups treated with 12.5 and 25 ng/mouse. The data showed that the increasing dose of IgY greatly enhanced the neutralization effect, and the surviving mice increased from 25% (2 out of 8) to 50% (4 out of 8) (Fig. 5A). Finally, we observed that all the mice treated with 50 and 100 ng/mouse of IgY survived. The results were significantly different from those of the BoNT/B control group. The detailed data are shown as Kaplan–Meier plots. Furthermore, the results of serial IgY solutions premixed with 40 × LD50/i.p. and 400 × LD50/i.p. (Fig. 5B and C) respectively were similar to the result described before. The neutralization capacity of anti-BHc IgY was approximately 4 × LD50 doses per 25 ng/ mouse IgY, or nearly 160 000 LD50 doses (namely 16 IU) per mg
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Figure 1. SDS-PAGE (A) and western blot (B) analysis of recombinant soluble BHc. Lane 1, low-molecular-weight protein markers; lane 2, the lysate from cells transformed with pTIG-Trx-BHc vector; lanes 3 and 4, purified BHc. Molecular weights of the protein standards are indicated on the left (×103). An arrow indicates the position of the BHc.
Figure 2. SDS–PAGE analysis of the purified IgY. Lane 1, low-molecularweight protein markers; lane 2, IgY purified from the eggs after last vaccine; lane 3, IgY purified from eggs before the first vaccine. The upper arrow shows a heavy chain of IgY (65 kDa), and the lower arrow shows a light chain of IgY (25 kDa).
IgY.4 Based on the calculation of IgY concentration, we calculated that one egg collected in this study contained a the neutralization capacity of 1760 IU. The time dependency of treatment with IgY Time dependency of treatment with IgY was investigated (Table 1). After successive injection of anti-BHc IgY and BoNT/B complex, the mortality and intoxication symptoms of botulism varied. All the mice survived in groups that were treated i.p. with anti-BHc IgY before, simultaneously with or 1.5 h after BoNT/B challenge via i.g. route. In contrast, all 8 mice died in the group which IgY was given 3 h via i.g. after the challeng of BoNT/B intraperitoneally. In the control group, all the mice challenged i.p. with BoNT/B died. The dose dependency of treatment with IgY In a mouse neutralization assay via i.g. route in vivo, dose dependency of treatment with IgY was investigated (Fig. 6A). In the mice challenged with BoNT/B via i.p. route, all the mice treated with PBS or low level of IgY (1.25 ng/mouse) via i.g. route died between 12 and 48 h after injection. The groups treated with an increasing dose of IgY (from2.5 to 5 ng/mouse) were partially protected from 25% (2 out of 8) to 50% (4 out of 8), and 10 ng/mouse of IgY protected mice from BoNT/B complex intoxication completely. A similar result was detected in groups treated with IgY via i.g. route (Fig. 6B). The mice were partially under protection from 25% (2 out of 8) to 50% (4 out of 8) in groups treated with an increasing dose of IgY (from 25 to 50 μg/mouse), and the mice treated with 100 μg/mouse of IgY all survived (Fig. 6B). The detailed data are shown as Kaplan–Meier plots.
Discussion In previous studies, specific-pathogen-free laying hens were immunized with BoNT or denatured BoNT (toxoid), and the production of egg yolk IgY specific to these agent can be high-risk threat.4 While, no one has immunized hens with carboxyl-terminal of BoNT heavy chain (Hc domain). The soluble BHc subunit proteins, synthesized in E. coli constructed vectors, have shown a higher degree of purity and posed less of a hazard than the traditional inactivated toxin.12 In addition, the concentrated antigencity may increase immunogenicity and
Figure 4. Survival curves of the mice treated with BoNT/B complex via different routes. BoNT/B complex was treated i.p. (A) and i.g. (B). Percentage of survival was plotted over time. Groups of 8 mice were used at each dosage level and five-dose levels were tested per experiment as described in Materials and Methods. The animals were observed for death over a period of 96 h.
stimulate protection efficacy in animal models. In this study, specific-pathogen-free laying hens were housed to determine the usefulness of an egg antibody platform for producing antiBHc IgY. We expressed soluble BHc as an appropriate antigen to generate toxin-specific IgY for immunotherapy. Hence, the first specific objective of this study was to develop an improved protocol for producing egg yolk IgY against BoNT/B with BHc protein as a new immunogen. The IgY platform, capable of providing a rapid method for commercial-scale antitoxin production, may serve as an inexpensive alternative source of antitoxin for neutralization purposes. The second specific objective was to detect the capacity of anti-BHc IgY to neutralize BoNT/B by pre-incubation before injection of testing mice. The third specific objective was to test the ability of anti-BHc IgY to neutralize BoNT/B as a neutralizing reagent by injecting IgY and toxin via different routes respectively in a mouse bioassay. The ELISA data analysis demonstrated that the anti-BHc IgY reacted well with the corresponding toxin BoNT/B. The anti-BHc IgY was obtained by injecting laying hens with the recombinant BHc proteins, and was prepared from eggs by water dilution method. An obvious increase of IgY titer was observed in both serum and yolk samples on the seventh day after the second inoculations (21st day). We also observed stably high levels of IgY after the last immunization.13 Our results showed that 4 immunizations are necessary for maintaining the levels of IgY for a period of nearly 1 mo.12 As the ‘gold standard’ for the measurement of BoNTs, the intraperitoneal mouse bioassay was used to test LD50 of the purified toxin complex. The data indicated that a higher dose of BoNT/B killed more sufficiently and effectively than a lower dose, and the LD50 of BoNT/B calculated by the Karber method was 4.363 ng/ kg, which was consistent with our previous study.10 These results
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Figure 3. Levels of IgY and serum titer by ELISA. Arrows indicate the day of each vaccination.
provided information for the toxicity of BoNT/B given by intraperitoneal injection, but there are also reports that the toxicity of BoNT/B is higher than BoNT/A. This discrepancy may be attributed to the purity of BoNT and strains of mice (ddY, CD-1, or BALB/c) used in the experiments. Furthermore, the intragastric administration mouse bioassay was used to test LD50 of BoNT/B of the purified toxin complex. As for food-borne and infant botulism, the gastrointestinal tract is an important portal of entry for BoNTs. The LD50 /i.g. calculated by the Karber method was 1.135 μg/kg in the mouse model. We chose 4 × LD50 /i.p. and 4 × LD50 /i.g. as a potentially lethal dose for almost all the mice that died within 24 h after administration. As for the neutralization activity of mice experiments via intraperitoneal injection, lower doses of IgY (12.5 and 25 ng/ mouse) could provide partial neutralization against 4 × LD50 / i.p. BoNT/B, while higher doses of IgY (50 and 100 ng/mouse) could protect the mice from any BoNT/B complex intoxication (Fig. 5A). Neutralization activity tested again with higher doses, namely 40 × LD50 /i.p. and 400 × LD50 /i.p., yielded a similar result. Lower doses of IgY could provide partial protection, while higher doses of IgY could protect the mice from BoNT/B complex intoxication completely (Fig. 5B and C). As Hc has been proved to contain an important epitopes to generate neutralizing
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Figure 5. Neutralization effect of anti-BHc IgY. (A) Mixture of 4 × LD50/i.p. and serial IgY, (B) mixture of 40 × LD50/i.p. and serial IgY, and (C) mixture of 400 × LD50/i.p. and serial IgY were administered i.p. to BALB/c mice respectively. The control mice were given PBS instead of IgY. Groups of 8 mice were used at each IgY dosage level and four-dose levels were tested per experiment as described in Materials and Methods. There was a significant difference at ***P < 0.001 and ***P < 0.001 vs. the control (log rank test).
antibodies, anti-BHc IgY could bind with the protein receptor binding domain of BoNT/B efficiently in our study, for the Hc is responsible for toxin binding to the sialoganglioside receptor and the putative protein receptor on presynaptic neurons. Thus, we can predict that IgY of chickens immunized with recombinant BHc subunit neutralized the bioactivity of BoNT/B sufficiently.9 The current horse serum treatment accepted for clinical use, HBAT for instance, represents 5500 IU of anti-toxin B per 10 mL.4 In our study, the neutralizing capacity of one egg was approximately 1760 IU, much higher than other study before.9 We calculated that 3.125 toxin-specific eggs is equal to the 10mL-treatment vial of horse serum. Therefore, anti-BHc IgY can be considered a useful alternative to mammalian antibodies. In the experiment for determination of LD50 /i.p., typical symptoms of botulism were displayed 3 h after challenge, suggesting that BoNT/B entered the bloodstream from the intestine nearly at this point. It is likely that BoNT/B toxin takes almost 3 h to enter circulation after i.g. injection. The i.p. administration of the IgY in survived groups may precede to enter of BoNT/B to the bloodstream in this mouse model.14 Thus, we can conclude that anti-BHc IgY can neither activate neutralizing capacities until BoNT/B binds to target receptors nor inhibit toxicity after toxins are transported to the neuromuscular junctions of motor neurons where its destructive action occurs.13,14 From the perspective of botulism therapy, anti-BHc IgY injected i.p. has the capacity to inactivate BoNT/B in the blood stream. Partly due to the unique physiochemical characteristics of IgY, several researchers have demonstrated the efficacy of IgY as an oral treatment or prophylactic for gastrointestinal disease. The neutralizing activity of IgY injected i.g. against death in mice was investigated. According to the data (Table 1), all the mice survived in the group that was treated i.g. with anti-BHc IgY 3 h before BoNT/B challenge via i.p. route. Anti-BHc IgY at the dosage of 100 μg/mouse showed complete protection following i.p. administration in mice challenged i.g. with 4 × LD50 /i.p. BoNT/B (Fig. 6B). Given orally, IgY showed its neutralization ability after reaching the large intestine.15 Thus, anti-BHc IgY was measured in the feces within 24 h to detect the duration of antibody activity in mice. From the perspective of botulism prevention, anti-BHc IgY injected i.g. before has the capacity to inactivate BoNT/B for at least 4 h.16 Hence, IgY antidote may serve as a useful oral therapy in botulism. Since BoNTs can be weaponized as aerosols as well as contaminate food and water supplies, the efficacy of anti-BHc IgY has not yet been determined in an aerosol intoxication model in mice. Furthermore, animal models are needed to investigate the safety of anti-BHc IgY as a human therapeutic and prevention before preclinical human trials.7 Limited researches have been conducted on the safety of peripherally administered IgY as a mouse therapeutic in previous studies.9 In addition, egg yolk antibodies can be expanded against other types of BoNTs in the future. In conclusion, we have demonstrated that chickens immunized with the recombinant BHc can produce IgY against BoNT/B, which can neutralize the bioactivity of neurotoxins via intraperitoneal injection and can help reduce the risk of botulism
Table 1. Mortality of the mice administered anti-BHc IgY after challenge with BoNT/B
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Timing of IgY administration (h)
(A) BoNT/B i.g.
(B) BoNT/B i.p.
IgY i.p.
IgY i.g.
Control
8/8
8/8
-3
0/8***
0/8***
-1.5
0/8***
7/8
0
0/8***
8/8
1.5
0/8***
8/8
3
8/8
8/8
(A) BoNT/B (4 × LD50/i.g.) was given via i.g. and 0.25mg/mouse IgY were administered i.p. at various timing. (B) BoNT/B (4 × LD50/i.p.) was given via i.p. and 0.25mg/mouse IgY were administered i.g. at various timing. Groups of 8 mice were used at each time level and 5 different groups were tested per experiment as described in Materials and Methods; ***There is a statistically significant difference at P < 0.001 vs. the control (log rank test).
by the intragastric route. The anti-BHc IgY can be potentially used in biodefense and is generally applicable to the field of infectious botulism diseases.
Materials and Methods Bacterial strain and production of BHc The C. botulinum serotype Danish (B1 subtype, GenBank Accession number M81186) that could produce BoNT/B toxin was provided by the State Key Laboratory of Pathogens and Biosecurity. The strain stored at -80 °C was inoculums (0.1 mL) for 10 mL of cooked meat media (peptone 3%, yeast extract 0.5%, beef extract 0.5%, glucose 0.3%, NaH2PO4 0.5%, pH 7.2) incubated at 37 °C. One L of an anaerobic medium TPYG (5% trypticase peptone, 0.5% bacto peptone, 0.4% glucose, 2% yeast extract and 0.1% l-cysteine, pH 7.4) was inoculated with 10 mL of active culture growing in the cooked meat medium and incubated undisturbed for 96 h at 30 °C. Then, BoNT/B was extracted from cultured media using PEG precipitation. The following purification was accomplished by sizeexclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) techniques using SephacrylTM S-100 and phenyl HP columns. This new platform for purification of BoNT/B was developed by Yao Zhao.10 The synthesized BHc DNA fragment of C.botulinum serotype Danish (1314bp: 2616–3929) was cloned into an modified pET22b expression vector (named pTIG-Trx) to create recombinant plasmid pTIG-Trx-BHc, and then was transformed into E.coli strain BL21 (DE3) (TransGen).17 Then the expressed strain was cultured with shaking in L-Broth (LB) medium to prepare the recombinant protein BHc. In the mid-logarithmic phase of growth, 0.2 mmol/L isopropyl-B-D-thiogalactopyranoside (IPTG, Promega) was added to the culture to induce BHc production, followed by cultivation for an additional 6 h. The culture supernatant was purified through a HiTrap Chelating high performance 5-mL column pre-packed with a precharged Ni2+ column (GE Healthcare), and the purified protein was analyzed by SDS-PAGE and western blots using hyperimmune horse neurotoxin B antiserum. The antiserum was provided by
the Lanzhou Institute of Biological Products. The identified BHc was used as antigens in this study. Animals Kept in an individual cage, two 20-wk-old specific-pathogenfree laying hens were housed as the primary egg to produce the anti-BoNT/B IgY. BALB/c mice (18–22 g) under pathogen free conditions were used to determine lethality of BoNT/B and passive protection capacity of anti-BoNT/B IgY. These mice with free access to food and water were obtained from the Laboratory Animal Center, Academy of Military Medical and Sciences. All experiments were conducted strictly in accordance with the guidelines of the Chinese Association for the Accreditation of Laboratory Animals Care (CAALAC), including the relevant local animal welfare bodies in China. In addition, the permit number of all animal work was SCXK-(JUN) 2013–005 approved by the animal ethics committee of Beijing Institute of Microbiology and Epidemiology. Hens immunization To prepare anti-BHc IgY, two laying hens were housed in individual cages. The purified recombinant BHc (0.5 mg in 0.1 mL PBS) was pre- mixed with an equal volume of Freund’s Complete Adjuvant (CFA, Sigma). The mixture was given intramuscularly at 4–6 sites of the pectoral muscle. In the same manner, hens were boosted every 2 wk after the first immunization i.e., on days 14, 28, and 42 and CFA was substituted with Freund’s Incomplete Adjuvant (IFA, Sigma). The hen’s blood was sampled the day prior to the first immunization and 7 d after each injection; furthermore, eggs were collected and labeled accordingly during the experiment. All the samples were stored at 4 °C. The preparation and identification of IgY Under acidic conditions, IgY was prepared by dilution method with modification. 18 Eighteen eggs for obtaining IgY were collected from 50 to 65 d after initial immunization. The yolks were separated, pooled and well mixed with 9 times volume of distilled water (pH 2.5–2.8). The pH of the suspension was then adjusted with 0.1 mol/L HCl to 5.0. After being kept at 4 °C overnight, the aggregate lipoproteins of the yolk were separated by centrifugation (10 000 g for 30 min at 4 °C). The supernatant was added with solid ammonium sulfate to 19% (m/v). The overnight precipitate was dissolved
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Mortality (Dead/Total)
at the original volume of the yolk in PBS (pH 7.2) and finally identified by SDS–PAGE. The digital image was analyzed using Bandscan 5.0 software and the protein concentrations were estimated using BCA protein assay (Sigma) according to the manufacturer’s protocol. To obtain the anti-BoNT/B IgY titer throughout the immunization, the eggs prior to the first vaccination and seventh day after each vaccination were purified separately as described before. ELISA for antibody titer monitoring The titers of both the egg and the serum samples were tested by a standard ELISA. The ELISA plates were coated with BoNT/B (10 ng/mL in 10 mM PBS, pH 9.6, 100 µL/well) for 1 h at 37 °C and blocked with 3% BSA in PBS and were washed with PBST (10 mM PBS, 0.05%Tween 20, pH 7.2) between all incubations. The eggs and serum samples were applied in serial 2-fold dilution from 1:200 and incubated for 1 h at 37 °C. After washing, HRP-conjugated goat anti-chicken IgG (SouthernBiotech) was added at a dilution of 1:4000–1:8000. Anti-BoNT/B reactivity was visualized by adding TMB and H2O2 substrates, and the colorimetric reaction was stopped with 2 M sulfuric acid before absorbance at 450 nm was read using a multifunctional micro plate reader (MDC, USA). Antibody titers were estimated as the reciprocal of the maximum dilution giving an absorbance reading greater than 0.5 units following subtraction of nonspecific binding detected in the control sample and 2-fold greater than that of the matched dilution of the control sample.19
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Figure 6. The protective effect of anti-BHc IgY in vivo given via different routes. (A) BoNT/B (4 × LD50/i.g.) was given via i.g. and serial IgY was administered i.p. at 1.5h after challenge. (B) BoNT/B (4 × LD50/i.p.) was given via i.p. and serial IgY was administered i.g. at 3 h before challenge. Groups of 8 mice were used at each dosage level and four-dose levels were tested per experiment as described in Materials and Methods. There was a significant difference at ***P < 0.001 and ***P < 0.001 vs. the control (log rank test).
Determining the lethal toxicity of BoNT/B 50% lethal dose (LD50) of biologically active BoNT/B complex was determined in BALB/c mice. The toxin complex was purified using the modified Karber method.10 2-fold serial dilutions of purified complex toxin were diluted in gelatin-phosphate-buffer (GPB: 50 mM PBS, 0.2% gelatin, pH 6.8), and then were injected via 2 different routes, intraperitoneal injection (i.p.) and intragastric administration (i.g.). Before injection, the mice in all groups were under an adaptation for 3 d. In addition, the mice via i.g. route were under starvation and water deprivation for an additional 8 h. The number of survived mice in each group was recorded over a period of 96 h. The experiments were repeated 3 times. 50% lethal dose values (LD50/i.p. and LD50/i.g.) were calculated using the Karber method.10 A survival curve of mice was measured using the Prism 5 statistics software (Graph Pad Software Inc.). The neutralization capacity of IgY by pre-incubation The neutralization activity of the anti-BHc IgY by preincubating with BoNT/B was evaluated in mouse assay via intraperitoneal injection. The purified anti-BHc IgY samples were followed by 2-fold serial dilutions in GPB. The serial dilutions (0.25 mL) were premixed with the same volume 4 × LD50 /i.p., 40 × LD50 /i.p., and 400 × LD50 /i.p. BoNT/B solution respectively. Incubated at room temperature for 0.5 h, the mixtures were injected i.p. in all groups tested. The mice in control group were treated with PBS instead of IgY. Mouse survival in all groups was observed for 96 h after administration and the data were calculated. The neutralization capacity of anti-BHc IgY was expressed in international units (IU) of antidotes per milliliter of yolk.20 In the mouse neutralization assay, the capacity of antiBHc IgY was estimated in IU. One IU of anti-BoNT products protects a mouse against 10 000 mouse LD50 of A–F or 1000 LD50 of E BoNTs by definition.4 The time dependency of IgY treatment To further support the application in treatment of botulinum, the protective effect of IgY against BoNT/B complex was detected in mouse assay by injecting IgY and toxin via 2 different routes respectively. First, time dependency of IgY treatment via i.p. was detected. BALB/c mice in these groups were challenged with 4 × LD50 /i.g. and overdose IgY (0.25 mg/mouse ) was given via i.p. at various timing. Then, time dependency of IgY treatment via i.g., another different administered route, was detected. These mice were challenged with 4 × LD50 /i.g. and overdose IgY was given via i.p. route at various timing. The dose dependency of IgY treatment After the time dependency experiment, different dosages of IgY (2-fold serial dilutions in GPB) were given to the mice via i.p. route certain timing before or after challenge with a dose of 4 × LD50 /i.g.. In the dose dependency of IgY treatment via i.g., the 2-fold serial IgY dilutions in GPB were given to the mice via i.g. route certain time before or after challenge with 4 × LD50 /i.p.. In control groups, the same volume of PBS was substituted with IgY solution. The data were collected and calculated as described before.
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References
