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Establishment of the cross-clade antigen detection system for H5 subtype influenza viruses using peptide monoclonal antibodies specific for influenza virus H5 hemagglutinin
Biochemical and Biophysical Research Communications 498 (2018) 758e763
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Establishment of the cross-clade antigen detection system for H5 subtype influenza viruses using peptide monoclonal antibodies specific for influenza virus H5 hemagglutinin Hitoshi Takahashi, Shiho Nagata, Takato Odagiri, Tsutomu Kageyama* Influenza Virus Research Center, National Institute of Infectious Diseases, Tokyo, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 February 2018 Accepted 7 March 2018 Available online 15 March 2018
The H5 subtype of highly pathogenic avian influenza (H5 HPAI) viruses is a threat to both animal and human public health and has the potential to cause a serious future pandemic in humans. Thus, specific and rapid detection of H5 HPAI viruses is required for infection control in humans. To develop a simple and rapid diagnostic system to detect H5 HPAI viruses with high specificity and sensitivity, we attempted to prepare monoclonal antibodies (mAbs) that specifically recognize linear epitopes in hemagglutinin (HA) of H5 subtype viruses. Nine mAb clones were obtained from mice immunized with a synthetic partial peptide of H5 HA molecules conserved among various H5 HPAI viruses. The antigen-capture enzyme-linked immunosorbent assay using the most suitable combination of these mAbs, which bound specifically to lysed H5 HA under an optimized detergent condition, was specific for H5 viruses and could broadly detect H5 viruses in multiple different clades. Taken together, these peptide mAbs, which recognize linear epitopes in a highly conserved region of H5 HA, may be useful for specific and highly sensitive detection of H5 HPAI viruses and can help in the rapid diagnosis of human, avian, and animal H5 virus infections. © 2018 Elsevier Inc. All rights reserved.
Keywords: Influenza Diagnostics H5 subtype Monoclonal antibody Antigen-capture ELISA
1. Introduction The H5 subtype of highly pathogenic avian influenza (H5 HPAI) virus has spread from Southeast Asia to China, Russia, Europe, Middle East, and Africa after 2003 [1,2]. It is still prevalent in wild birds and poultry in many areas including Japan, and these areas have suffered enormous economic damage as a result of H5 HPAI infections [3]. A high mortality rate of H5N1 HPAI virus infection in humans has also been confirmed, resulting in 454 deaths of 860 infected people in 16 countries as of December 7, 2017 [4]. Recently, several H5N1 HPAI viruses have acquired NA genes from unrelated avian influenza viruses via reassortment. H5N2, H5N3, H5N6, and H5N8 reassortant viruses isolated from ducks and poultry in China were reported during or after 2010 [5]. H5N6 viruses have also caused sporadic human infections in China; at least 17 human
Abbreviations: HPAI, highly pathogenic avian influenza; mAbs, monoclonal antibodies; HA, hemagglutinin; ELISA, enzyme-linked immunosorbent assay. * Corresponding author. Influenza Virus Research Center, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan. E-mail addresses: [email protected] (H. Takahashi), [email protected] (S. Nagata), [email protected] (T. Odagiri), [email protected] (T. Kageyama). https://doi.org/10.1016/j.bbrc.2018.03.054 0006-291X/© 2018 Elsevier Inc. All rights reserved.
infections have been reported since 2014 [6]. H5 HPAI virus transmission from human to human is still limited, however, there is concern about the emergence of mutated or reassortant viruses that are easily propagated in humans. Therefore, establishment of a rapid and specific diagnostic test for H5 HPAI virus infection is necessary. A number of commercially available rapid influenza diagnostic tests have been developed, which are immunochromatography assays for influenza A and B virus infections targeting the viral nucleoprotein. However, these tests cannot distinguish HA subtypes of influenza A virus such as H1, H3, H5, and H7; thus, human seasonal influenza A viruses and avian influenza viruses are indistinguishable [7]. The antigen-capture enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (mAbs), which specifically recognize H5 HA antigen, prepared by immunization of virus particles or recombinant HA protein has been used to diagnose H5 virus infection in humans [8e12]. Although the produced mAbs recognized HA antigen of the clade of the immunized virus, it is difficult to obtain mAbs that recognize clades of other H5 viruses. Preparation of mAbs using a synthetic partial peptide as an immunizing antigen has also been reported [13,14]. By using this method, it is possible to obtain specific mAbs that recognize the
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target amino acid sequence of the synthetic partial peptide. All recently disseminated H5 viruses are classified into HA clades 1 or 2. These clades have been further diverged into several subclades. H5N1 clade 1 virus was introduced and caused the first wave of major outbreaks in Vietnam in 2003 [2]. Thereafter, the WHO/OIE/FAO H5 Evolution Working Group identified three actively circulating H5 clades (2.1.3.2a, 2.2.1, and 2.3.4) [5]. H5N1 clade 2.1.3.2a viruses circulated in Indonesia from 2009 to 2014. H5N1 clade 2.2.1 viruses continued to evolve through 2013 and 2014 in Egypt. H5N1 clade 2.3.4 viruses have circulated in Asia, Europe, and North America since 2005. Furthermore, recently spreading avian H5N6 viruses, which cause sporadic infection in humans, and H5N8 avian viruses were classified into clade 2.3.4.4, which originated from H5N1 clade 2.3.4 viruses [5]. We hypothesized that it is possible to detect multiple clades of H5 viruses by using mAbs prepared from a synthetic partial peptide that contains a conserved amino acid sequence among the above representative H5 HA molecules. This report describes the preparation of mAbs by immunization of a synthetic partial peptide designed to target a highly conserved region in H5 HA molecules and the establishment of a specific and highly sensitive antigen-capture ELISA using these cross-reactive mAbs to H5 viruses.
2. Materials and methods 2.1. Viruses A/Vietnam/1194/2004 (H5N1, NIBRG-14, clade 1) virus, which possesses modified HA and neuraminidase (NA) genes derived from the A/Vietnam/1194/2004 virus on the backbone of six internal genes of A/Puerto Rico/8/34 virus, was provided by the National Institute for Biological Standards and Control (Potters Bar, UK). A/ Indonesia/05/2005 (H5N1, IBCDC-RG2, clade 2.1.3.2), A/Egypt/ N03072/2010 (H5N1, IDCDC-RG29, clade 2.2.1), and A/Anhui/01/ 2005 (H5N1, IBCDC-RG5, clade 2.3.4) were also developed and provided by the United States Centers for Disease Control and Prevention (Atlanta, GA, USA). A/duck/Hyogo/1/2016 (H5N6, NIIDRG-001, clade 2.3.4.4) was developed in our laboratory. All non-H5 viruses used in this study were obtained from a stockpile in our laboratory (Table 1). These viruses were propagated in the allantoic cavity of 10-day-old embryonated chicken eggs at 34 C
Table 1 Influenza A viruses used in this study. Subtype H5N1 (Clade H5N1 (Clade H5N1 (Clade H5N1 (Clade H5N6 (Clade H1N1pdm H2N3 H3N8 H4N6 H6N2 H7N9 H8N4 H9N2 H10N7 H11N6 H12N5 H13N6 H14N5 H15N8 a
1) 2.1.3.2) 2.2.1) 2.3.4) 2.3.4.4)
Virusa
HA titer
A/Vietnam/1194/2004 (NIBRG-14) A/Indonesia/05/2005 (IBCDC-RG2) A/Egypt/N03072/2010 (IDCDC-RG29) A/Anhui/1/2005 (IBCDC-RG5) A/duck/Hyogo/1/2016 (NIIDRG-001) A/California/7/2009 (X-179A) A/duck/Germany/1215/73 A/duck/Ukraine/1/63 A/duck/Czechoslovakia/56 A/turkey/Massachusetts/3740/65 A/Anhui/1/2013 (NIBRG-268) A/turkey/Ontario/6118/68 A/turkey/Wisconsin/1/66 A/chicken/Germany/N/49 A/duck/England/56 A/duck/Alberta/60/76 A/gull/Maryland/704/77 A/mallard/Gurjev/263/82 A/duck/Australia/341/83
1024 256 128 512 64 256 128 256 256 128 256 128 256 1024 256 256 128 256 128
Names in parentheses indicate candidate vaccine viruses.
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for 48 h and HA titer was determined using 0.5% turkey red blood cells. A/Vietnam/1194/2004 (NIBRG-14) virus was purified through a 20e60% sucrose gradient by ultracentrifugation. The virus was then resuspended in phosphate-buffered saline (PBS) and inactivated by treatment with 0.05% b-propiolactone at 4 C for 18 h and then at 37 C for 90 min. Total protein amount of purified NIBRG-14 virus was determined using BCA protein assay kit (Pierce) and then adjusted to a final concentration of 1 mg/mL.
2.2. Preparation and screening of mAbs specific for H5 HA antigen BALB/c mice were immunized with the synthetic partial peptide, which contains a region conserved among various H5 HA molecules, to obtain mAbs specific for H5 HA antigen. To conjugate the synthetic partial peptide with keyhole limpet hemocyanin, cysteine was added at the C-terminal of the peptide. The mAbproducing hybridomas, in which mouse myeloma cells (P3U1) were fused with fibula lymphocytes harvested from immunized mice, were cloned by BEX Co., Ltd. mAb screening was conducted by ELISA using A/Vietnam/1194/2004 (NIBRG-14) virus antigen, which was lysed with 1% Triton X-100. The lysate was diluted with ELISAcoating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) and coated onto an ELISA plate (MaxiSorp; Thermo Fisher Scientific) at 4 C overnight. After washing with PBS-Tween, the microplate was blocked with BlockAce (DS Pharma Biomedical Co., Ltd.) for 1 h at room temperature. The microplate was then washed and hybridoma culture supernatants were added and incubated for 1 h at room temperature. After another wash, horseradish peroxidase (HRP)-conjugated anti-mouse IgG mAb (1:8000; Invitrogen) was added to the wells and incubated for 1 h at room temperature. The plate was then washed and TMB substrate (ScyTek Laboratories, Inc.) was added. The reaction was stopped by adding TMB Stop Buffer (ScyTek Laboratories, Inc.), and the optical density at 450 nm (OD450) was measured using a multi-well plate reader (SpectraMax Plus 384; Molecular Devices). After screening, mouse ascites that contained mAb were prepared and purified by BEX Co., Ltd. according to the conventional method.
2.3. Optimization of detergent condition for detection of H5 viruses Sodium dodecyl sulfate (SDS) and Empigen BB [n-dodecyl-N,Ndimethylglycine] (Sigma-Aldrich) detergent were selected to determine the optimum detergent condition for the ELISA. A/ Vietnam/1194/2004 (NIBRG-14) virus antigen was lysed with these detergents at several concentrations (0.1e0.0025%), diluted with ELISA-coating buffer, and incubated on coated ELISA plates at 4 C overnight. The ELISA protocol was the same as that described above.
2.4. Antigen-capture ELISA Antigen-capturing mAb (10 mg/mL) was coated onto an ELISA plate and incubated with ELISA-coating buffer at 4 C overnight. The ELISA plate was then blocked with BlockAce, washed with PBSTween, and viral antigen was added to the wells with 0.025% SDS for 1 h at room temperature. After washing, HRP-conjugated mAb (10 mg/mL), for detection of viral antigen, was added to the wells with 0.025% SDS for 1 h at room temperature. After washing, TMB substrate was added, the reaction was stopped by adding TMB Stop Buffer, and the OD450 was measured using a multi-well plate reader.
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3. Results 3.1. Preparation and screening of mAbs specific for H5 HA antigen The amino acid sequence of H5-2 synthetic partial peptide used as an immunizing antigen for preparation of mAbs was determined from a highly conserved amino acid sequence among various H5 HA molecules. We selected representative HA genetic clades of H5 viruses: clades 1, 2.1.3.2, 2.2.1, 2.3.4, and 2.3.4.4 (Fig. 1A). H5-2 peptide consists of 21 amino acid residues (107e127 amino acid position in H5 numbering) and is located on the surface of the globular head. Mice were immunized with H5-2 peptide, and we obtained nine mAb clones that showed avidity to A/Vietnam/1194/ 2004 (NIBRG-14) virus antigen by ELISA screening. However, the avidity of these mAb clones to H5 HA antigen was not strong (Fig. 1B). The avidity of antibody can be affected by the kind and concentration of the detergent used for antigen detection by ELISA [15]. It was thought that 1% Triton X-100 was insufficient to lyse H5 HA antigen and bind mAbs to the antigen in this ELISA. Therefore, we attempted to optimize the detergent condition for detection of H5 viruses by ELISA.
3.2. Optimization of detergent condition for detection of H5 viruses A/Vietnam/1194/2004 (NIBRG-14) virus antigen was lysed with SDS or Empigen at several concentrations and detected by ELISA using three mAb clones (5A7, 7D4, and 7D6), which showed intermediate and high avidity to the viral antigen among the nine mAb clones. The avidity of the mAbs to the antigen increased after
using these detergents at 0.05e0.01% SDS or 0.025e0.0025% Empigen (Fig. 2A and B). From these results, the optimal composition of the detergent was determined to be 0.025% SDS.
3.3. Establishment of the antigen-capture ELISA for detection of H5 viruses We attempted to establish the antigen-capture ELISA using 5A7, 7D4, and 7D6 mAbs. These mAbs were used to capture and detect A/Vietnam/1194/2004 (NIBRG-14) virus antigen. The viral antigen was lysed with 0.025% SDS. As a result, the antigen-capture ELISA for detection of H5 viruses was established (Fig. 3). The most suitable combination of mAbs was 7D6 as the antigen capture mAb and HRP-labeled 5A7 as the antigen detection mAb for sensitive detection of H5 HA. Specificity and sensitivity for detection of H5 viruses using this antigen-capture ELISA were examined, as shown in Fig. 4. Several H5 clade viruses could be detected, including A/Vietnam/1194/ 2004 (H5N1, NIBRG-14, clade 1), A/Indonesia/05/2005 (H5N1, IBCDC-RG2, clade 2.1.3.2), A/Egypt/N03072/2010 (H5N1, IDCDCRG29, clade 2.2.1), A/Anhui/01/2005 (H5N1, IBCDC-RG5, clade 2.3.4), and A/duck/Hyogo/1/2016 (H5N6, NIIDRG-001, clade 2.3.4.4) (Fig. 4A and B). Although other influenza A virus subtypes were also tested using viruses at 64 HA titer to check for non-specific binding to each antigen, reactivity was not confirmed in all non-H5 viruses (Fig. 4A). Furthermore, the detection threshold of H5 virus A/Vietnam/1194/2004 (NIBRG-14) was 0.3 mg/mL (Fig. 4C). These results indicate that the selected mAbs are cross-reactive to and specific and highly sensitive for H5 viruses in the antigen-capture ELISA.
Fig. 1. The amino acid sequence of H5-2 synthetic partial peptide and ELISA screening results. (A) The amino acid sequence of H5-2 synthetic partial peptide was compared with that of H5 HA (107e127 amino acids position in H5 numbering) from representative genetic clades of H5 viruses. The amino acid positions of S121 and D124 are underlined as the reported epitope. (B) The avidity of mAb clones to A/Vietnam/1194/2004 (NIBRG-14) virus antigen by ELISA. The viral antigen was coated onto an ELISA plate at 1 mg/mL. NC: no mAb clone.
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Fig. 2. Optimization of detergent condition for detection of H5 viruses. A/Vietnam/ 1194/2004 (NIBRG-14) virus antigen was lysed with several concentrations (0.1e0.0025%) of (A) SDS or (B) Empigen detergent and detected by ELISA using 5A7, 7D4, and 7D6 mAb clones. The viral antigen was coated onto an ELISA plate at 2.5 mg/ mL. NC: no mAb clone.
Fig. 3. Establishment of the antigen-capture ELISA. Indicated mAbs (10 mg/mL) were used for capturing and detecting A/Vietnam/1194/2004 (NIBRG-14) virus antigen. The viral antigen was used at 2.5 mg/mL and lysed with 0.025% SDS.
4. Discussion Specific and rapid detection of H5 HPAI viruses is required for infection control in birds, animals, and humans. In this study, we prepared mAbs by immunization with a synthetic partial peptide, which was designed to cover a highly conserved region of various
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H5 HA molecules. Using these mAbs, we established the antigencapture ELISA to detect multiple clades of H5 viruses in an optimized detergent condition of 0.025% SDS. We presume that under the lysis buffer conditions, the viral antigen was sufficiently recognized by the mAbs without decreasing the avidity of the mAbs to antigen. To increase the avidity of mAb to antigen in the ELISA, the kind and concentration of detergent must be optimized. Use of a synthetic partial peptide as an immunizing antigen for mAb preparation has the major advantage of peptide mAbs that specifically recognize regions of the peptide antigen can be easily obtained. However, it has been reported that the peptide mAb prepared using a synthetic partial peptide may not be able to recognize the three-dimensional structure of the target antigen [16]. In this study, we demonstrated that the prepared peptide mAb recognized and bound to H5 viral antigen lysed with 0.025% SDS. Furthermore, we established a diagnostic system (antigen-capture ELISA) for detecting multiple clades of H5 viruses using peptide mAbs that do not recognize the three-dimensional structure of the viral antigens. Therefore, preparation of mAbs that recognize the three-dimensional structure of H5 viral antigens is not absolutely necessary for establishment of a diagnostic system (antigen-capture ELISA) for detecting clades of H5 viral antigens. H5N6 and H5N8 viruses are circulating in Asian and European countries in birds, which are classified as H5 HA clade 2.3.4.4 [5]. The H5 HA amino acid sequence of clade 2.3.4.4 viruses is slightly different from that of other clades of H5 viruses. mAbs prepared by immunization of virus particles recognize HA antigen of viruses with the same clade as the immunized virus but tend to have low specificity for other distant clades. Actually, mAbs prepared to recognize HA antigens of several clades of H5 viruses do not strongly recognize HA antigens of clade 2.3.4.4 viruses [12,17]. In the case of peptide mAbs, if there is a difference between the sequence of the immunized peptide and the target antigen, then mAbs may not recognize the antigen. We compared the HA amino acid sequence of H5-2 synthetic partial peptide (21 amino acids) with that of a clade 2.3.4.4 virus, A/duck/Hyogo/1/2016 (H5N6), which showed a 5-amino acid difference (Fig. 1A). However, we could detect A/duck/Hyogo/1/2016 (H5N6, clade 2.3.4.4) by the antigen-capture ELISA using the prepared mAbs (Fig. 4B). H5 HA epitopes have been mapped [18], and the amino acid positions of S121 and D124 (in H5 numbering) were shown to be the epitope in the amino acid sequence corresponding to H5-2 synthetic partial peptide. We deduced that the peptide mAbs used in this study recognize the 116e122 amino acid position, which includes S121, or 107e114 amino acid position, which is common to each clade. We also considered that the peptide mAbs do not recognize the 123e127 amino acid position, including D124, which has amino acid differences among each clade (Fig. 1A). Taken together, it may be possible to use the prepared mAbs for a long time unless amino acid changes occur in the conserved region between each clade of H5 viruses. HA amino acid sequences (107e127 amino acid position) of 15 H5N6 and H5N8 strains each, which were collected from April 2017 as recently circulating viruses, were compared with that of A/duck/ Hyogo/1/2016. The HA amino acid sequences of these viruses were consistent with that of A/duck/Hyogo/1/2016, except that the position at 114 is isoleucine (data not shown). This finding suggests that these viruses could be detected by the antigen-capture ELISA using the prepared mAbs. Additionally, when a new H5 virus emerges, we can easily predict the avidity of the mAb by examining these conserved HA amino acid sequences of the emerging virus. Although PCR-based molecular tests are one of the most sensitive methods to detect influenza virus, viral antigen detection using antibody is useful as an easier and quicker diagnostic test in clinical hospitals. The immunochromatography (IC) assay has been widely
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Fig. 4. Specificity and sensitivity of the antigen-capture ELISA for H5 viruses. (A) Various influenza viruses were tested at 64 HA titer by the antigen-capture ELISA. A/Vietnam/ 1194/2004 (H5N1, NIBRG-14, clade 1), A/Indonesia/05/2005 (H5N1, IBCDC-RG2, clade 2.1.3.2), A/Egypt/N03072/2010 (H5N1, IDCDC-RG29, clade 2.2.1), and A/Anhui/01/2005 (H5N1, IBCDC-RG5, clade 2.3.4) viruses were used as H5 viruses. Non-H5 viruses, as described in Table 1, were also tested. (B) A/duck/Hyogo/1/2016 (H5N6, NIIDRG-001, clade 2.3.4.4) virus was tested by the antigen-capture ELISA. A/Anhui/01/2005 (H5N1, IBCDC-RG5, clade 2.3.4) virus was used as a comparative control. Each virus was tested at 64 HA titer. (C) Serially diluted A/Vietnam/1194/2004 (NIBRG-14) virus was tested by the antigen-capture ELISA. The dotted line indicates the lowest detection threshold, which was determined by adding a standard deviation to the average of seven measurements of the no antigen control.
employed as a simple antibody-based diagnostic test. However, in recent years, the development of a simple and high sensitivity diagnostic system based on the principle of antigen-capture ELISA has been reported. The POCube is a fully automated and compact immunological analyzer, which is intended to support point-ofcare testing [19]. This method could detect several clades of H5N1 viruses in humans. The fluorescent immunochromatography (FLIC) assay detects H5N1 and H5N2 viruses by using fluorescent beads [20]. This method has an improved sensitivity of 10e100-fold higher than traditional IC. The detection limit of the antigencapture ELISA in this study was 0.3 mg/mL (Fig. 4C). This detection limit is comparable to that of another reported antigen-capture ELISA using different H5 HA-specific mAbs [10]. By incorporating our prepared mAbs into the above methods, it may be possible to detect H5 viruses more readily and sensitively. In conclusion, we prepared H5 HA-specific mAbs by using a synthetic partial peptide, which covers a highly conserved amino
acid sequence among various H5 HA molecules, as the immunizing antigen and established the antigen-capture ELISA using these mAbs to detect multiple clades of H5 viruses under an optimized detergent condition. These peptide mAbs may be useful for specific and highly sensitive diagnosis of H5 HPAI virus infection in birds, animals, and humans. Acknowledgments We thank Ikuyo Takayama, Mina Nakauchi, and Kiyohiko Matsui for technical assistance. We also thank Yasushi Suzuki and Eri Nobusawa for generating the A/duck/Hyogo/1/2016 (NIIDRG-001) virus. This study was partly supported by a grant from the Ministry of Health, Labour, and Welfare (MHLW) of Japan (H26 ShinkoIppan-003), and by the Research Program on Emerging and Reemerging Infectious Diseases from the Japan Agency for Medical Research and Development (AMED) (JP17fk0108103).
H. Takahashi et al. / Biochemical and Biophysical Research Communications 498 (2018) 758e763
Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.03.054.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Establishment of the cross-clade antigen detection system for H5 subtype influenza viruses using peptide monoclonal antibodies specific for influenza virus H5 hemagglutinin Hitoshi Takahashi, Shiho Nagata, Takato Odagiri, Tsutomu Kageyama* Influenza Virus Research Center, National Institute of Infectious Diseases, Tokyo, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 February 2018 Accepted 7 March 2018 Available online 15 March 2018
The H5 subtype of highly pathogenic avian influenza (H5 HPAI) viruses is a threat to both animal and human public health and has the potential to cause a serious future pandemic in humans. Thus, specific and rapid detection of H5 HPAI viruses is required for infection control in humans. To develop a simple and rapid diagnostic system to detect H5 HPAI viruses with high specificity and sensitivity, we attempted to prepare monoclonal antibodies (mAbs) that specifically recognize linear epitopes in hemagglutinin (HA) of H5 subtype viruses. Nine mAb clones were obtained from mice immunized with a synthetic partial peptide of H5 HA molecules conserved among various H5 HPAI viruses. The antigen-capture enzyme-linked immunosorbent assay using the most suitable combination of these mAbs, which bound specifically to lysed H5 HA under an optimized detergent condition, was specific for H5 viruses and could broadly detect H5 viruses in multiple different clades. Taken together, these peptide mAbs, which recognize linear epitopes in a highly conserved region of H5 HA, may be useful for specific and highly sensitive detection of H5 HPAI viruses and can help in the rapid diagnosis of human, avian, and animal H5 virus infections. © 2018 Elsevier Inc. All rights reserved.
Keywords: Influenza Diagnostics H5 subtype Monoclonal antibody Antigen-capture ELISA
1. Introduction The H5 subtype of highly pathogenic avian influenza (H5 HPAI) virus has spread from Southeast Asia to China, Russia, Europe, Middle East, and Africa after 2003 [1,2]. It is still prevalent in wild birds and poultry in many areas including Japan, and these areas have suffered enormous economic damage as a result of H5 HPAI infections [3]. A high mortality rate of H5N1 HPAI virus infection in humans has also been confirmed, resulting in 454 deaths of 860 infected people in 16 countries as of December 7, 2017 [4]. Recently, several H5N1 HPAI viruses have acquired NA genes from unrelated avian influenza viruses via reassortment. H5N2, H5N3, H5N6, and H5N8 reassortant viruses isolated from ducks and poultry in China were reported during or after 2010 [5]. H5N6 viruses have also caused sporadic human infections in China; at least 17 human
Abbreviations: HPAI, highly pathogenic avian influenza; mAbs, monoclonal antibodies; HA, hemagglutinin; ELISA, enzyme-linked immunosorbent assay. * Corresponding author. Influenza Virus Research Center, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan. E-mail addresses: [email protected] (H. Takahashi), [email protected] (S. Nagata), [email protected] (T. Odagiri), [email protected] (T. Kageyama). https://doi.org/10.1016/j.bbrc.2018.03.054 0006-291X/© 2018 Elsevier Inc. All rights reserved.
infections have been reported since 2014 [6]. H5 HPAI virus transmission from human to human is still limited, however, there is concern about the emergence of mutated or reassortant viruses that are easily propagated in humans. Therefore, establishment of a rapid and specific diagnostic test for H5 HPAI virus infection is necessary. A number of commercially available rapid influenza diagnostic tests have been developed, which are immunochromatography assays for influenza A and B virus infections targeting the viral nucleoprotein. However, these tests cannot distinguish HA subtypes of influenza A virus such as H1, H3, H5, and H7; thus, human seasonal influenza A viruses and avian influenza viruses are indistinguishable [7]. The antigen-capture enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (mAbs), which specifically recognize H5 HA antigen, prepared by immunization of virus particles or recombinant HA protein has been used to diagnose H5 virus infection in humans [8e12]. Although the produced mAbs recognized HA antigen of the clade of the immunized virus, it is difficult to obtain mAbs that recognize clades of other H5 viruses. Preparation of mAbs using a synthetic partial peptide as an immunizing antigen has also been reported [13,14]. By using this method, it is possible to obtain specific mAbs that recognize the
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target amino acid sequence of the synthetic partial peptide. All recently disseminated H5 viruses are classified into HA clades 1 or 2. These clades have been further diverged into several subclades. H5N1 clade 1 virus was introduced and caused the first wave of major outbreaks in Vietnam in 2003 [2]. Thereafter, the WHO/OIE/FAO H5 Evolution Working Group identified three actively circulating H5 clades (2.1.3.2a, 2.2.1, and 2.3.4) [5]. H5N1 clade 2.1.3.2a viruses circulated in Indonesia from 2009 to 2014. H5N1 clade 2.2.1 viruses continued to evolve through 2013 and 2014 in Egypt. H5N1 clade 2.3.4 viruses have circulated in Asia, Europe, and North America since 2005. Furthermore, recently spreading avian H5N6 viruses, which cause sporadic infection in humans, and H5N8 avian viruses were classified into clade 2.3.4.4, which originated from H5N1 clade 2.3.4 viruses [5]. We hypothesized that it is possible to detect multiple clades of H5 viruses by using mAbs prepared from a synthetic partial peptide that contains a conserved amino acid sequence among the above representative H5 HA molecules. This report describes the preparation of mAbs by immunization of a synthetic partial peptide designed to target a highly conserved region in H5 HA molecules and the establishment of a specific and highly sensitive antigen-capture ELISA using these cross-reactive mAbs to H5 viruses.
2. Materials and methods 2.1. Viruses A/Vietnam/1194/2004 (H5N1, NIBRG-14, clade 1) virus, which possesses modified HA and neuraminidase (NA) genes derived from the A/Vietnam/1194/2004 virus on the backbone of six internal genes of A/Puerto Rico/8/34 virus, was provided by the National Institute for Biological Standards and Control (Potters Bar, UK). A/ Indonesia/05/2005 (H5N1, IBCDC-RG2, clade 2.1.3.2), A/Egypt/ N03072/2010 (H5N1, IDCDC-RG29, clade 2.2.1), and A/Anhui/01/ 2005 (H5N1, IBCDC-RG5, clade 2.3.4) were also developed and provided by the United States Centers for Disease Control and Prevention (Atlanta, GA, USA). A/duck/Hyogo/1/2016 (H5N6, NIIDRG-001, clade 2.3.4.4) was developed in our laboratory. All non-H5 viruses used in this study were obtained from a stockpile in our laboratory (Table 1). These viruses were propagated in the allantoic cavity of 10-day-old embryonated chicken eggs at 34 C
Table 1 Influenza A viruses used in this study. Subtype H5N1 (Clade H5N1 (Clade H5N1 (Clade H5N1 (Clade H5N6 (Clade H1N1pdm H2N3 H3N8 H4N6 H6N2 H7N9 H8N4 H9N2 H10N7 H11N6 H12N5 H13N6 H14N5 H15N8 a
1) 2.1.3.2) 2.2.1) 2.3.4) 2.3.4.4)
Virusa
HA titer
A/Vietnam/1194/2004 (NIBRG-14) A/Indonesia/05/2005 (IBCDC-RG2) A/Egypt/N03072/2010 (IDCDC-RG29) A/Anhui/1/2005 (IBCDC-RG5) A/duck/Hyogo/1/2016 (NIIDRG-001) A/California/7/2009 (X-179A) A/duck/Germany/1215/73 A/duck/Ukraine/1/63 A/duck/Czechoslovakia/56 A/turkey/Massachusetts/3740/65 A/Anhui/1/2013 (NIBRG-268) A/turkey/Ontario/6118/68 A/turkey/Wisconsin/1/66 A/chicken/Germany/N/49 A/duck/England/56 A/duck/Alberta/60/76 A/gull/Maryland/704/77 A/mallard/Gurjev/263/82 A/duck/Australia/341/83
1024 256 128 512 64 256 128 256 256 128 256 128 256 1024 256 256 128 256 128
Names in parentheses indicate candidate vaccine viruses.
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for 48 h and HA titer was determined using 0.5% turkey red blood cells. A/Vietnam/1194/2004 (NIBRG-14) virus was purified through a 20e60% sucrose gradient by ultracentrifugation. The virus was then resuspended in phosphate-buffered saline (PBS) and inactivated by treatment with 0.05% b-propiolactone at 4 C for 18 h and then at 37 C for 90 min. Total protein amount of purified NIBRG-14 virus was determined using BCA protein assay kit (Pierce) and then adjusted to a final concentration of 1 mg/mL.
2.2. Preparation and screening of mAbs specific for H5 HA antigen BALB/c mice were immunized with the synthetic partial peptide, which contains a region conserved among various H5 HA molecules, to obtain mAbs specific for H5 HA antigen. To conjugate the synthetic partial peptide with keyhole limpet hemocyanin, cysteine was added at the C-terminal of the peptide. The mAbproducing hybridomas, in which mouse myeloma cells (P3U1) were fused with fibula lymphocytes harvested from immunized mice, were cloned by BEX Co., Ltd. mAb screening was conducted by ELISA using A/Vietnam/1194/2004 (NIBRG-14) virus antigen, which was lysed with 1% Triton X-100. The lysate was diluted with ELISAcoating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) and coated onto an ELISA plate (MaxiSorp; Thermo Fisher Scientific) at 4 C overnight. After washing with PBS-Tween, the microplate was blocked with BlockAce (DS Pharma Biomedical Co., Ltd.) for 1 h at room temperature. The microplate was then washed and hybridoma culture supernatants were added and incubated for 1 h at room temperature. After another wash, horseradish peroxidase (HRP)-conjugated anti-mouse IgG mAb (1:8000; Invitrogen) was added to the wells and incubated for 1 h at room temperature. The plate was then washed and TMB substrate (ScyTek Laboratories, Inc.) was added. The reaction was stopped by adding TMB Stop Buffer (ScyTek Laboratories, Inc.), and the optical density at 450 nm (OD450) was measured using a multi-well plate reader (SpectraMax Plus 384; Molecular Devices). After screening, mouse ascites that contained mAb were prepared and purified by BEX Co., Ltd. according to the conventional method.
2.3. Optimization of detergent condition for detection of H5 viruses Sodium dodecyl sulfate (SDS) and Empigen BB [n-dodecyl-N,Ndimethylglycine] (Sigma-Aldrich) detergent were selected to determine the optimum detergent condition for the ELISA. A/ Vietnam/1194/2004 (NIBRG-14) virus antigen was lysed with these detergents at several concentrations (0.1e0.0025%), diluted with ELISA-coating buffer, and incubated on coated ELISA plates at 4 C overnight. The ELISA protocol was the same as that described above.
2.4. Antigen-capture ELISA Antigen-capturing mAb (10 mg/mL) was coated onto an ELISA plate and incubated with ELISA-coating buffer at 4 C overnight. The ELISA plate was then blocked with BlockAce, washed with PBSTween, and viral antigen was added to the wells with 0.025% SDS for 1 h at room temperature. After washing, HRP-conjugated mAb (10 mg/mL), for detection of viral antigen, was added to the wells with 0.025% SDS for 1 h at room temperature. After washing, TMB substrate was added, the reaction was stopped by adding TMB Stop Buffer, and the OD450 was measured using a multi-well plate reader.
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3. Results 3.1. Preparation and screening of mAbs specific for H5 HA antigen The amino acid sequence of H5-2 synthetic partial peptide used as an immunizing antigen for preparation of mAbs was determined from a highly conserved amino acid sequence among various H5 HA molecules. We selected representative HA genetic clades of H5 viruses: clades 1, 2.1.3.2, 2.2.1, 2.3.4, and 2.3.4.4 (Fig. 1A). H5-2 peptide consists of 21 amino acid residues (107e127 amino acid position in H5 numbering) and is located on the surface of the globular head. Mice were immunized with H5-2 peptide, and we obtained nine mAb clones that showed avidity to A/Vietnam/1194/ 2004 (NIBRG-14) virus antigen by ELISA screening. However, the avidity of these mAb clones to H5 HA antigen was not strong (Fig. 1B). The avidity of antibody can be affected by the kind and concentration of the detergent used for antigen detection by ELISA [15]. It was thought that 1% Triton X-100 was insufficient to lyse H5 HA antigen and bind mAbs to the antigen in this ELISA. Therefore, we attempted to optimize the detergent condition for detection of H5 viruses by ELISA.
3.2. Optimization of detergent condition for detection of H5 viruses A/Vietnam/1194/2004 (NIBRG-14) virus antigen was lysed with SDS or Empigen at several concentrations and detected by ELISA using three mAb clones (5A7, 7D4, and 7D6), which showed intermediate and high avidity to the viral antigen among the nine mAb clones. The avidity of the mAbs to the antigen increased after
using these detergents at 0.05e0.01% SDS or 0.025e0.0025% Empigen (Fig. 2A and B). From these results, the optimal composition of the detergent was determined to be 0.025% SDS.
3.3. Establishment of the antigen-capture ELISA for detection of H5 viruses We attempted to establish the antigen-capture ELISA using 5A7, 7D4, and 7D6 mAbs. These mAbs were used to capture and detect A/Vietnam/1194/2004 (NIBRG-14) virus antigen. The viral antigen was lysed with 0.025% SDS. As a result, the antigen-capture ELISA for detection of H5 viruses was established (Fig. 3). The most suitable combination of mAbs was 7D6 as the antigen capture mAb and HRP-labeled 5A7 as the antigen detection mAb for sensitive detection of H5 HA. Specificity and sensitivity for detection of H5 viruses using this antigen-capture ELISA were examined, as shown in Fig. 4. Several H5 clade viruses could be detected, including A/Vietnam/1194/ 2004 (H5N1, NIBRG-14, clade 1), A/Indonesia/05/2005 (H5N1, IBCDC-RG2, clade 2.1.3.2), A/Egypt/N03072/2010 (H5N1, IDCDCRG29, clade 2.2.1), A/Anhui/01/2005 (H5N1, IBCDC-RG5, clade 2.3.4), and A/duck/Hyogo/1/2016 (H5N6, NIIDRG-001, clade 2.3.4.4) (Fig. 4A and B). Although other influenza A virus subtypes were also tested using viruses at 64 HA titer to check for non-specific binding to each antigen, reactivity was not confirmed in all non-H5 viruses (Fig. 4A). Furthermore, the detection threshold of H5 virus A/Vietnam/1194/2004 (NIBRG-14) was 0.3 mg/mL (Fig. 4C). These results indicate that the selected mAbs are cross-reactive to and specific and highly sensitive for H5 viruses in the antigen-capture ELISA.
Fig. 1. The amino acid sequence of H5-2 synthetic partial peptide and ELISA screening results. (A) The amino acid sequence of H5-2 synthetic partial peptide was compared with that of H5 HA (107e127 amino acids position in H5 numbering) from representative genetic clades of H5 viruses. The amino acid positions of S121 and D124 are underlined as the reported epitope. (B) The avidity of mAb clones to A/Vietnam/1194/2004 (NIBRG-14) virus antigen by ELISA. The viral antigen was coated onto an ELISA plate at 1 mg/mL. NC: no mAb clone.
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Fig. 2. Optimization of detergent condition for detection of H5 viruses. A/Vietnam/ 1194/2004 (NIBRG-14) virus antigen was lysed with several concentrations (0.1e0.0025%) of (A) SDS or (B) Empigen detergent and detected by ELISA using 5A7, 7D4, and 7D6 mAb clones. The viral antigen was coated onto an ELISA plate at 2.5 mg/ mL. NC: no mAb clone.
Fig. 3. Establishment of the antigen-capture ELISA. Indicated mAbs (10 mg/mL) were used for capturing and detecting A/Vietnam/1194/2004 (NIBRG-14) virus antigen. The viral antigen was used at 2.5 mg/mL and lysed with 0.025% SDS.
4. Discussion Specific and rapid detection of H5 HPAI viruses is required for infection control in birds, animals, and humans. In this study, we prepared mAbs by immunization with a synthetic partial peptide, which was designed to cover a highly conserved region of various
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H5 HA molecules. Using these mAbs, we established the antigencapture ELISA to detect multiple clades of H5 viruses in an optimized detergent condition of 0.025% SDS. We presume that under the lysis buffer conditions, the viral antigen was sufficiently recognized by the mAbs without decreasing the avidity of the mAbs to antigen. To increase the avidity of mAb to antigen in the ELISA, the kind and concentration of detergent must be optimized. Use of a synthetic partial peptide as an immunizing antigen for mAb preparation has the major advantage of peptide mAbs that specifically recognize regions of the peptide antigen can be easily obtained. However, it has been reported that the peptide mAb prepared using a synthetic partial peptide may not be able to recognize the three-dimensional structure of the target antigen [16]. In this study, we demonstrated that the prepared peptide mAb recognized and bound to H5 viral antigen lysed with 0.025% SDS. Furthermore, we established a diagnostic system (antigen-capture ELISA) for detecting multiple clades of H5 viruses using peptide mAbs that do not recognize the three-dimensional structure of the viral antigens. Therefore, preparation of mAbs that recognize the three-dimensional structure of H5 viral antigens is not absolutely necessary for establishment of a diagnostic system (antigen-capture ELISA) for detecting clades of H5 viral antigens. H5N6 and H5N8 viruses are circulating in Asian and European countries in birds, which are classified as H5 HA clade 2.3.4.4 [5]. The H5 HA amino acid sequence of clade 2.3.4.4 viruses is slightly different from that of other clades of H5 viruses. mAbs prepared by immunization of virus particles recognize HA antigen of viruses with the same clade as the immunized virus but tend to have low specificity for other distant clades. Actually, mAbs prepared to recognize HA antigens of several clades of H5 viruses do not strongly recognize HA antigens of clade 2.3.4.4 viruses [12,17]. In the case of peptide mAbs, if there is a difference between the sequence of the immunized peptide and the target antigen, then mAbs may not recognize the antigen. We compared the HA amino acid sequence of H5-2 synthetic partial peptide (21 amino acids) with that of a clade 2.3.4.4 virus, A/duck/Hyogo/1/2016 (H5N6), which showed a 5-amino acid difference (Fig. 1A). However, we could detect A/duck/Hyogo/1/2016 (H5N6, clade 2.3.4.4) by the antigen-capture ELISA using the prepared mAbs (Fig. 4B). H5 HA epitopes have been mapped [18], and the amino acid positions of S121 and D124 (in H5 numbering) were shown to be the epitope in the amino acid sequence corresponding to H5-2 synthetic partial peptide. We deduced that the peptide mAbs used in this study recognize the 116e122 amino acid position, which includes S121, or 107e114 amino acid position, which is common to each clade. We also considered that the peptide mAbs do not recognize the 123e127 amino acid position, including D124, which has amino acid differences among each clade (Fig. 1A). Taken together, it may be possible to use the prepared mAbs for a long time unless amino acid changes occur in the conserved region between each clade of H5 viruses. HA amino acid sequences (107e127 amino acid position) of 15 H5N6 and H5N8 strains each, which were collected from April 2017 as recently circulating viruses, were compared with that of A/duck/ Hyogo/1/2016. The HA amino acid sequences of these viruses were consistent with that of A/duck/Hyogo/1/2016, except that the position at 114 is isoleucine (data not shown). This finding suggests that these viruses could be detected by the antigen-capture ELISA using the prepared mAbs. Additionally, when a new H5 virus emerges, we can easily predict the avidity of the mAb by examining these conserved HA amino acid sequences of the emerging virus. Although PCR-based molecular tests are one of the most sensitive methods to detect influenza virus, viral antigen detection using antibody is useful as an easier and quicker diagnostic test in clinical hospitals. The immunochromatography (IC) assay has been widely
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Fig. 4. Specificity and sensitivity of the antigen-capture ELISA for H5 viruses. (A) Various influenza viruses were tested at 64 HA titer by the antigen-capture ELISA. A/Vietnam/ 1194/2004 (H5N1, NIBRG-14, clade 1), A/Indonesia/05/2005 (H5N1, IBCDC-RG2, clade 2.1.3.2), A/Egypt/N03072/2010 (H5N1, IDCDC-RG29, clade 2.2.1), and A/Anhui/01/2005 (H5N1, IBCDC-RG5, clade 2.3.4) viruses were used as H5 viruses. Non-H5 viruses, as described in Table 1, were also tested. (B) A/duck/Hyogo/1/2016 (H5N6, NIIDRG-001, clade 2.3.4.4) virus was tested by the antigen-capture ELISA. A/Anhui/01/2005 (H5N1, IBCDC-RG5, clade 2.3.4) virus was used as a comparative control. Each virus was tested at 64 HA titer. (C) Serially diluted A/Vietnam/1194/2004 (NIBRG-14) virus was tested by the antigen-capture ELISA. The dotted line indicates the lowest detection threshold, which was determined by adding a standard deviation to the average of seven measurements of the no antigen control.
employed as a simple antibody-based diagnostic test. However, in recent years, the development of a simple and high sensitivity diagnostic system based on the principle of antigen-capture ELISA has been reported. The POCube is a fully automated and compact immunological analyzer, which is intended to support point-ofcare testing [19]. This method could detect several clades of H5N1 viruses in humans. The fluorescent immunochromatography (FLIC) assay detects H5N1 and H5N2 viruses by using fluorescent beads [20]. This method has an improved sensitivity of 10e100-fold higher than traditional IC. The detection limit of the antigencapture ELISA in this study was 0.3 mg/mL (Fig. 4C). This detection limit is comparable to that of another reported antigen-capture ELISA using different H5 HA-specific mAbs [10]. By incorporating our prepared mAbs into the above methods, it may be possible to detect H5 viruses more readily and sensitively. In conclusion, we prepared H5 HA-specific mAbs by using a synthetic partial peptide, which covers a highly conserved amino
acid sequence among various H5 HA molecules, as the immunizing antigen and established the antigen-capture ELISA using these mAbs to detect multiple clades of H5 viruses under an optimized detergent condition. These peptide mAbs may be useful for specific and highly sensitive diagnosis of H5 HPAI virus infection in birds, animals, and humans. Acknowledgments We thank Ikuyo Takayama, Mina Nakauchi, and Kiyohiko Matsui for technical assistance. We also thank Yasushi Suzuki and Eri Nobusawa for generating the A/duck/Hyogo/1/2016 (NIIDRG-001) virus. This study was partly supported by a grant from the Ministry of Health, Labour, and Welfare (MHLW) of Japan (H26 ShinkoIppan-003), and by the Research Program on Emerging and Reemerging Infectious Diseases from the Japan Agency for Medical Research and Development (AMED) (JP17fk0108103).
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Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.03.054.
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