The authors have declared that no competing interests exist.
Conceived and designed the experiments: FH SS ST JI. Performed the experiments: FH SS RI JI NID. Analyzed the data: FH EE. Contributed reagents/materials/analysis tools: FH ST JI EE. Wrote the paper: FH EE.
Available avian influenza (AIV) serological diagnostic tests cannot distinguish vaccinated from naturally infected birds. Differentiation of vaccinated from infected animals (DIVA) is currently advocated as a means of achieving the full control of H5N1. In this study, for the first time, recombinant ectodomain of M2 protein (M2e) of avian influenza virus (H5N1 strain) was used for the DIVA serology test. M2e was cloned into pMAL-P4X vector and expressed in
Highly pathogenic avian influenza virus (AIV) of H5N1 subtype has become endemic in poultry in some countries, especially in Southeast Asian Countries
Multiple vaccinations are expensive and in many instances not entirely effective enabling H5N1 to persist in the environment and mutate through the process known as “antigenic drift”
To overcome this limitation, several DIVA strategies have been attempted; the most feasible approach is the use of subunit-based strategy which targets differential rate of propagated avian influenza proteins between killed virus (vaccine) and naturally infected birds. Hemagglutinin (H) is the most used target subunits
A major improvement was the use of nonstructural protein 1 (NS1) as the target subunit which has zero copy number per mature virion
Another surface segment of influenza virus, the matrix protein 2 (M2), is a transmembrane integral protein where it exists as homotetramer, each monomer contains 96 amino acid with 3 domains: a small external domain (M2e) comprising 23 amino acids, a transmembrane domain (19 amino acids) and a cytoplasmic domain (54 amino acid)
The matrix protein 2 (M2) is receiving increased attention since unlike HA and NA, the extracellular domain of the M2 protein (M2e) is not subjected to severe immune selection pressure and is very well conserved
The extracellular domain of the M2 protein (M2e protein) is abundantly expressed on the surface of infected cells, while it is present in small quantities in the mature virions (20–60 molecules per vision)
Only a limited number of studies have examined M2e as a diagnostic marker in DIVA test. Lambrecht, et al. (2007) and Kim et al (2010) utilized the M2e synthetic peptide in ELISA and were successful in discrimination of infected and vaccinated birds indicating the potential of M2e for DIVA test
In the present study, the M2e from a H5N1 Indonesian strain expressed in
We selected consensus open reading frame of the M2e domain of M2 protein based on the multiple alignment of available H5N1 sequences of H5N1 Indonesian strains in GeneBank (
M2-NT1 vector was transformed into BL21 strain of
For expression of recombinant M2e-MBP protein, a single colony of transformed BL21 cells was cultured in a rich Luria-Bertani Medium (LB) (10 g Tryptone, 10 g NaCl, 5 g yeast extract and 0.1% D-glucose per liter of distilled water) containing 30 µg/ml of filtered sterile Ampicillin. The 10 ml overnight culture was transferred in 1 L of LB medium containing Ampicillin and incubated at 37°C until optical density at 600 nm (OD600) reached to about 0.5, then the production of M2e-pMAL protein was induced by application of 0.3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma, St Louis, MO, USA) with subsequent incubation for 3 h at 250 rpm at 37°C. A non-induced culture was used as a negative control. The culture fluid was centrifuged at 5,000×g for 20 min at 4°C in (Sorvall RC 5B centrifuge). The supernatant was discarded, and the cells were resuspended in 400 ml Tris/sucrose buffer (30 mM Tris-HCl, 20% sucrose, 1 mM EDTA, pH 8.0). Then, the suspension incubated for 5–10 minutes at room temperature with shaking, or stirring, and centrifuged at 8000×g at 4°C for 10 minutes. The pelleted cells were resuspended in 400 ml ice-cold 5 mM MgSO4, incubated for 10 minutes in an ice-water bath and centrifuged as mentioned above. The supernatant was termed “cold osmotic shock fluid”.
The “cold osmotic shock fluid” containing M2e-MBP loaded onto a column of amylose affinity resin (New England Biolabs, Beverly, Mass., USA), washed overnight at 4°C with 5 volumes of column buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1 mM NaN3 and 1 mM Dithiothreitol). Then, the M2e-MBP protein eluted with column buffer containing 10 mM maltose (Sigma, St Louis, MO, USA). The first 10 fractions were collected and protein concentration of each fraction monitored by measurement of OD280 on a NanoDrop system (Thermo Scientific, DE, USA). The fractions that contained measurable protein were desalted and concentrated by ultrafiltration in Vivaspin size exclusion centrifugal membrane tube with cut-off 30,000 Dalton (Sartorius Stedim Biotech, Goettingen Germany). Protein concentration of the purified protein was measured using NanoDrop, adjusted at 800 µg/ml and stored in aliquots at −70°C.
Purified protein was analysed by
Purified recombinant M2e-MBP protein was ran on 12.5% SDS-PAGE and transferred to a nitrocellulose membrane. Molecular weight markers (New England Biolabs, Beverly, Mass, USA) were also included. After transferring, the membrane was blocked using 10% bovine serum albumen (BSA) in PBS containing 0.5% Tween 20 for 2 h at room temperature. Test sera (
Avian influenza strain used for immunization | Type | HI Titre log2 |
|
1 | A/Chicken/Scotland/1959 | H5N1 | 7 |
2 | A/Ostrich/Denmark/72420/1996 | H5N2 | 7 |
3 | A/Turkey/Wisconsin/1/1966 | H9N2 | 9 |
4 | A/Tky/England/96 | H3N2 | 7 |
5 | A/Duck/Alberta/35/76 | H1N1 | 9 |
6 | A/African starling/England-Q/983/79 | H7N1 | 8 |
7 | A/Duck/England/1/1956 | H11N6 | 9 |
8 | A/Duck/Ukraine/1/1963 | H3N8 | 6 |
9 | A/Duck/Germany/1215/1973 | H2N3 | 6 |
10 | A/Turkey/England/647/77 | H7N7 | 7 |
11 | A/Ck/Viet Nam/8/2004 | H5N1 | ND |
12 | A/Chicken/Konawi Selatan/8/2004 H5N1 | H5N1 | ND |
Homologous HI Titre.
Source of challenged and vaccinated antibodies.
ND: HI Titre not detected.
Two synthetic peptides corresponding to the M2e recombinant sequences were made; the M2e-23 peptide, corresponding to amino acid position 2 to 24, with the sequence SLLTEVETPTRNEWECKCSDSSD, and the M2e-18 peptide, amino acid position 2 to 18, with the sequence SLLTEVETPTRNEWECKC. Both peptides were synthesized by PEPTIDE 2.0 (Chantilly, VA, USA) with a minimum of 85% purity as measured by high performance liquid chromatography. Both peptides had high solubility in distilled water.
Antisera against different strains and subtypes of AIV were obtained from the Veterinary Laboratory Agency (New Haw, Addlestone, UK) and CSIRO Australian Animal Health Laboratory, Geelong, Victoria (
A checkerboard titration of the M2e-MBP antigen and AIV positive and negative sera were initially performed to determine the optimal OD at 450 nm (OD450) of each positive and negative serum. Briefly, purified M2e-MBP protein, diluted in 0.1 M carbonate– bicarbonate buffer (pH 9.6), at concentrations of 200 to 0.82 µg/ml, was used to coat the 96-well flat bottom microtitre plate (
Both M2e-23 and M2e-18 synthetic peptides were used in ELISA as coating antigens. Briefly, 1 mg of each lyophilized peptide was dissolved in 1 ml of sterile distilled water, and 2-fold serial dilutions of 160 to 0.31 µg/ml of the peptide made in 0.1 M carbonate–bicarbonate buffer, pH 9.6. Diluted peptides were used to coat the 96-well flat bottom microtiter plates (Maxisorp, NUNC). All steps, including the checkerboard titration, were performed as described for M2e-MBP ELISA.
In both M2e peptides and M2e-MBP ELISAs, for each serum sample, 4 wells were used: 2 wells were coated by the antigen whereas 2 wells were not coated with any antigen and served as an internal control for each serum. For each serum, the mean OD450 of antigen negative wells subtracted from the mean OD450 of antigen positive wells and the obtained OD450 was termed as “corrected OD450 value”. A serum was considered positive when its corrected OD450 value was greater than the mean corrected OD450 for negative sera plus 2 times standard deviation (cut-off value).
The aim of this experiment was to simulate the condition and measure the sensitivity of the presented recombinant M2e-based ELISA method when the vaccinated chickens infect with the live virus. In addition, the efficiency of our method was compared with the HI titre (common HA-based method).
Chicken sera resulting from an experimental vaccination/challenge experiment were provided by the Indonesian Research Centre for Veterinary Science, Bogor. Twenty chicks, 21 days old, were sourced from non-vaccinated AI free broiler farm and divided into 2 groups, each with 10 chicks. Chickens in the first group were vaccinated with a single dose of an experimental inactivated vaccine prepared from A/Ck/West Java/Pwt-Wij/2006 strain of H5N1 (Accession No; EU124148). Chickens in the second group were remained unvaccinated and kept in the same condition but in a separate isolation units.
Three weeks after vaccination, all birds were tested for HI antibody titres using the homologous HA antigen (A/Ck/West Java/Pwt-Wij). Then, chicks in both groups were transferred to isolation units, housed within the PC3 facility. After that, chicks in the first group were challenged with 106 ELD50 in 0.1 ml via intranasal inoculation with A/Ck/West Java/Pwt-Wij/2006. Chicks in the second group were remained unvaccinated and unchallenged. Two weeks after challenge, sera were collected from all chicks in both groups and tested individually for HI antibody titres using the HA antigen A/Ck/West Java/Pwt-Wij and also M2e ELISA, to compare the ability of these methods in monitoring the infection of vaccinated chickens. In addition, in first group, the responses of M2e ELISA and HI Titre methods to challenged infection were compared by T-test statistics using Minitab 16 package (
The goal of this experiment was to evaluate the robustness of the recombinant M2e ELISA method in the field scale. Three test groups were examined, including (a) Negative group (non-infected and non-vaccinated): 204 field sera from commercial broiler and layer flocks in Australia and Indonesia which were confirmed to be AIV antibody free by an IDEXX AIV antibody test (IDEXX Laboratories, Inc) and were obtained from the diagnostic laboratory, School of Veterinary Science, the University of Melbourne, (b) Vaccinated group, sera (334 in total) were collected from vaccinated commercial broiler and layer flocks in Indonesia, and (c) Infected group: 56 sera were collected from infected chickens from different farms in Indonesia.
Corrected ODs (450 nm) of each serum in recombinant M2e ELISA were recorded individually and compared between different test groups. Furthermore, analysis of variance mean comparisons by Tukey test was performed to evaluate the ability of M2e-based ELISA in distinguishing the infected sera from the vaccinated and non-vaccinated sera (
Mean comparison | |||||
Test group | Mean | Standard Deviation | Mean comparison by Tukey method at 99% Confidence level |
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Infected | 0.9269 | 0.2711 | A | ||
Vaccinated | 0.2124 | 0.1981 | B | ||
Negative | 0.1955 | 0.0108 | B | ||
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Test group | 2 | 13.197 | 355.44 | 0.0001 | |
Error | 591 | 0.037 |
Means that do not share a letter are significantly different.
Highly significant.
The cut-off values for the ELISAs were calculated from the results of sera of H5N1 infected (standard and challenge experiment groups) and vaccinated chickens (field and challenge experiment groups) using the two-graph receiver operating characteristic (TG-ROC) analysis
All animal experiments were performed at the Indonesian Research Centre for Veterinary Science, Bogor, Indonesia. The study proposal was approved by the institutional Research Committee.
The animals were managed by a veterinarian who specializes in animal studies based on the guidelines of the National Health and Medical Research Council of Australia. All birds were bleed via brachial vein during the experiment and cardiac puncture at the terminal step just after CO2 euthanization.
In total, 64 nucleotide sequences of the M2e gene of deposited Indonesian H5N1 strains in GeneBank (with human and avian origin) were aligned. The obtained consensus M2e sequence from this alignment was that of A/Indonesia/CDC540/2006 H5N1 strain since it shared the highest identity matrix, >95%, with other Indonesian H5N1 strains. The length of sequence was 72 nucleotides and also had the highest hydrophilicity profile among the compared M2e amino acid sequences (result not shown). The hydrophilicity is one of the factors that influence both antigenicity and antibody binding avidity. High hydrophilicity increases the chance of detection of the specific antibodies. We optimized the codons of this 72 bp long M2e sequence, cloned and expressed in
Maximum expression level of M2e-MBP was detected at 3 h after induction. SDS-PAGE analysis of the purified M2e-MBP protein demonstrated the presence of two 45.1 and 42.5 kd bands (
A) Purified recombinant M2e-MBP (lane 1) and MBP (lane 2) proteins and pre-stained molecular marker (M) analysed by SDS–PAGE (12.5%) and stained using Coomassie Brilliant Blue. (B & C) Western blot analysis of purified M2e-MBP (1B) and MBP (1C) with reference AIV antisera to: (1) live A/Chicken/Scotland/1959 (H5N1); (2) inactivated H5N1; (3) live A/Ck/Viet Nam/8/2004 (H5N1); (4) live A/Turkey/Wisconsin/1/1966 (H9N2); (5) live A/Ostrich/Denmark/72420/1996 (H5N2); (6)sera from commercial non-vaccinated non-infected; (7) SPF chicken sera.
The purified M2e-MBP fusion protein reacted in Western blot with AIV chicken sera that included antisera to live H5N1 (A/Chicken/Scotland/1959 and A/Ck/Viet Nam/8/2004), live H5N2 (A/Ostrich/Denmark/72420/1996), and live H9N2 (A/Turkey/Wisconsin/1/1966) (
Pool of field sera from AIV free, non-vaccinated chicks, did not react with the M2e-MBP protein in Western blot (
Since Western blot analysis confirmed that the recombinant M2e-MBP fusion protein is antigenic and can differentiate live infected from vaccinated antisera, feasibility of using this antigen in ELISA was assessed. In addition, the synthetic M2e-18 peptide ELISA was used for comparison as was previously shown to be able to discriminate between infected and vaccinated chicks
Anti-H5N1 live, anti-H5N1 killed, SPF and sera from non-vaccinated commercial chickens (field sera) were diluted 1∶100 and incubated with M2e-MBP or M2e-18 used for coating at various concentration. Reactivity of sera with coating antigen was detected by rabbit anti-chicken IgG-HRP. Absorbance (OD) of conjugate control (CC) was measured for each plate.
Reactivity of sera from chicks immunized with 11 different subtypes of AIV, with HI titres between 6 to 9 (log 2 base) were compared in recombinant M2e-MBP and synthetic M2e peptide ELISA (
All sera were generated against live AIV of indicated subtypes, except for H5N2 killed virus.
As presented in
Of interest was that antisera to live AIV of subtypes other than H5N1, including H11N6, H7N7, H1N1, H7N1, H2N3, H9N2, H3N8 and H5N2, all reacted in ELISA with both M2e-MBP and M2e peptide while M2e sequence was derived from H5N1 strain. In line with this finding, De Filette et al., (2008) showed that vaccines based on M2e can induce broad-spectrum immunity against influenza in mice and is the best candidate for M2e-based universal influenza vaccine
Specificity of M2e-MBP and M2e peptide ELISA were also evaluated using chicken sera from non-vaccinated and vaccinated commercial layers and non-vaccinated broilers as described in materials and methods. Sera from non-vaccinated layers were negative in both M2e-MBP and M2e peptide ELISA, except for 6 sera that were positive in M2e-MBP ELISA with the OD450 value greater than 0.58 (false positive rate = 2.9%) (
Cut-off value (0.58) calculated as mean corrected OD for negative samples plus 2 standard deviations (0.364).
As it can be inferred from
Vaccination is the method of choice to control AIV in poultry industry in many countries. Fast, efficient, and inexpensive DIVA test has a vital role in the success of this strategy. To address this issue, we expressed the M2e peptide as a recombinant M2e-MBP protein in
The expressed recombinant M2e-MBP protein had an expected size of 45.1 kd corresponded to 2.6 kd M2e and 42.5 kd MBP. Another minor protein of 42.5 kd corresponding to truncated M2e-MBP, was also co-purified with M2e-MBP and was not possible to remove from the preparation of M2e-MBP by additional purification.
Western blotting recognised the M2e-MBP protein of 45.1 kd length only by antisera to live AIV whereas antisera to inactivated H5N1 did not react with the M2e-MBP. This reaction in Western blotting was demonstrated to be due to M2e only and not due to MBP.
The comparison of M2e-MBP and peptide ELISA using reference sera indicted that M2e-MBP antigen was equally capable of detecting M2e positive sera without any significant background noise, or non-specific reaction. All sera that were positive by peptide ELISA were also positive in M2e-MBP ELISA, and in full agreement with the results obtained by M2e-MBP based Western blotting, indicated that M2e expressed as fusion M2eMBP protein is antigenically functional. The recombinant M2e-MBP was expressed in large quantities and allowed sera to be tested for M2e antibodies at a relatively high dilution of 1/100. The signal to noise ratio in ELISA was significantly different and allowed clear differentiation of M2e positive reference sera.
UP to now, M2e peptide has been expressed in
The comparison of the results of M2e-MBP ELISA and HI (HA-based) tests demonstrated the excellent ability of M2e-MBP antigen to discriminate between antibodies produced against live virus challenge and killed virus vaccination for DIVA serological test. M2e-MBP ELISA was highly sensitive to live virus infection as the ELISA OD of vaccinated chickens greatly increased by more than 8 time when vaccinated chickens were infected with the live AIV virus (
Furthermore, the robustness of the method reinforced by large scale M2e-MBP ELISA in differentiation of infected chickens from non-infected and vaccinated chickens (
The newly designed M2e-MBP ELISA system using as a DIVA tool has some limitations in its application in old chicken and the serum samples that were haemolysed or lipemic. Some of the field serum samples from old flocks (older than 44 weeks) had different degrees of non-specific reactions with M2e-MBP antigen. Western blot analysis using MBP purified fusion protein revealed that these samples had different reactivity just with MBP fusion protein but not with M2e.
Also some of the serum samples from layer or free range chicks had non-specific reactions to MBP fusion protein and/or just the ELISA plates alone. To overcome these problems using fresh, non-haemolysed and preferably non-lipemic serum samples would be an advantageous. Where it is necessary the results can be confirmed by Western blotting or subtracting the MBP value from the whole antigen. Second critical item in reliability of the M2e-MBP ELISA is purity of the antigen, in some cases during purification procedure of the antigen some of
H5N1-originated recombinant M2e-MBP protein or synthetic M2e peptide reacts with a wide range of other AIV strains antibodies such as H5N2, H9N2, H7N7, H11N6, etc. While the major surface influenza glycoproteins, HA and NA undergo major antigenic changes resulting in short-time effectiveness of available vaccines, the extracellular domain of matrix protein 2 (M2e) is strongly invariable and conserved. Therefore, vaccines based on extracellular domain of M2e are capable in inducing broad-spectrum immunity and inhibit virus replication up to 90–100% protection in mice and heterosubtypic immunity in pigs
In addition to the above-mentioned advantages, extracellular domain of M2e opens a new vista in avian influenza management via DIVA test. Hydrophilic structure of M2e as well as its invariability and broad-spectrum reactivity show that M2e is an appropriate candidate for both vaccine production and DIVA test. The high performance of M2e subunit for DIVA test can be explained by considerable lower number of M2 molecules per virion (20–60) comparing to HA and NA. Due to larger amount of HA molecule per virion. HA can induce a very strong immune response in both vaccinate (killed virus) and infected chickens. In contrast, the amount of M2e subunit in vaccine is very low, and immune response can be detected after live virus infection as presented in
The authors would like to thank Indonesian Research Centre for Veterinary Science (Balitvet) for their assistance. We thank Mr. Gary Anderson from school of veterinary science, Melbourne University who statistically analysed the data.