The H1N1 influenza virus split vaccine (2009; SFDA approval no. S20090015) was obtained from Hualan Biological Bacterin Co., Ltd. Seasonal H1N1 and H3N2 influenza vaccines (veterinary drug production approval no.  150132145), and H5N1 and H9N2 influenza vaccines were produced by Yebio Engineering Co., Ltd.
Two mAbs of the anti-H1N1 influenza virus HA protein (A1-10 and H1-84) were prepared in our laboratory. The mouse myeloma cell SP2/0 was provided by the Department of Immunology, Air Force Military Medical University, and the culture supernatant of SP2/0 cells was used as a negative control for the identification and localization of A1-10 and H1-84 recognition polypeptides. Horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies were obtained from Beijing Zhongshan Golden Bridge Co., Ltd.
DNAMAN software was used to analyse features of the H1N1 influenza virus HA protein sequence and continuous amino acid sequences were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/). Subsequently, nine peptide sequences were defined and synthesised by China Peptides Co., Ltd.
A total RNA extraction kit and cDNA first-strand synthesis kit were purchased from Tiangen Biotech (Beijing) Co., Ltd. Polymerase for polymerase chain reaction (PCR), the pMD19-T vector, and DNA markers were obtained from Takara Bio. Primer synthesis and sequencing were performed by the Beijing Genomics Institute.
An indirect enzyme-linked immunosorbent assay (ELISA) was used to identify cross-reactivity of hybridoma cell culture supernatants of the mAbs A1-10 and H1-84 with the following five HA subtypes: 2009 H1N1 influenza virus split vaccine, the seasonal influenza vaccines H1N1 and H3N2, and the bird flu vaccines H5N1 and H9N2. The culture supernatant of mouse myeloma cell SP2/0 was used as a negative control. Subsequently, the antibodies were categorised into different groups based on the cross-reactivity between the two antibodies and the five HA subtypes.
The sequences of the five subtypes of the influenza virus and related information were downloaded from the NCBI database using GenBank IDs (Table 1). Alignment of multiple sequences was performed for the five subtypes using DNAMAN software (version 5.2.2). Nine continuous amino acid sequences, each 5-7 residues in length, were designed as candidate epitopes from the conserved sequence between the five different influenza virus HA protein subtypes (Table 2). The nine peptides were synthesised with a purity of > 85%, as measured by high-performance liquid chromatography and mass spectrometry methods. The candidate epitopes were stored as freeze-dried powder at -20 ℃.
Table 1. Information about the amino acid sequences of subtype influenza virus.
Table 2. Sequence and position of candiate peptides of HA antigens.
PyMOL and Swiss-PdbViewer software were used to analyse the distribution of peptides on the HA crystal structure. Swiss-PdbViewer was used to generate a HA protein X-ray crystal texture model, and 3LZG (Protein Data Bank [PDB]), the crystal structure of HA from A/California/04/2009 H1N1 virus, which was similar (> 99%) to that of the antigen under study, was used as the reference. Subsequently, the distribution of the peptides was determined using PyMOL according to the manufacturer's instructions.
Using a blocking ELISA, with binding peptides of the two mAbs, the nine candidate epitopes were tested and screened. An ELISA plate was coated with the H1N1 influenza virus HA antigens at a concentration of 2 μg/mL. The synthesised polypeptides were mixed with the mAbs A1-10 and H1-84 separately, and incubated for 1 h at 37 ℃. Subsequently, 100 μL of the mixture was added to each well to coat the ELISA plate containing the HA antigens, and incubated for an additional 1 h at 37 ℃. SP2/0 cell culture supernatant was used as the negative control. After washing three times, 100 μL HRP-labelled goat anti-mouse secondary antibody (1:2500 dilution) was added to each well. The chromagen 3, 3', 5, 5'-tetramethylbenzidine (TMB) was used as the substrate for HRP, with detection of the oxidised product at OD450. The inhibition rate (IR) of binding peptides was calculated using the following formula: IR = (ODCTL - ODTEST)/ODCTL. Correlations between the HA antigen and binding epitopes of antibodies were defined as follows: IR ≤ 0.4, no correlation; 0.4 ≤ IR ≤ 0.8, correlation; and IR ≥ 0.8, strong correlation (Xu et al. 2011).
Polyglutamic acid was added to the carboxy terminus of positive peptides to facilitate their coating of ELISA plates (Li et al. 2018). Each well was coated with 2 μg/mL of the peptide and incubated at 4 ℃ overnight. Subsequently, the two mAbs were added at 37 ℃ and incubated for 1 h. SP2/0 cell culture supernatant was used as the negative control. After washing three times, 100 μL HRP-labelled goat anti-mouse secondary antibody (1:2500 dilution) was added to each well, and incubated at 37 ℃ for 1 h. Subsequently, 100 μL TMB-H2O2 chromogenic solution was added to each well and incubated for 10 min at 37 ℃ in the dark. The reaction was terminated using H2SO4 solution (2 mol/L; 50 μL/well). The proportion of bound antibodies, which was correlated with the colour intensity, was measured with an ELISA reader by absorbance at 450 nm. The ratio of each test sample (OD450 to negative control OD450) was calculated. Samples with a ratio of ≥ 2.1 were classified as exhibiting a positive reaction.
For the localised peptide, each amino acid of the sequence was individually replaced with an alanine residue, resulting in the synthesis of a collection of alanine replacement peptides (Table 3). A 96-well ELISA plate was coated with 100 μL of 2-5 μg/mL H1N1 influenza virus HA antigen. The two mAbs were incubated with the localised peptide and its family of alanine replacement peptides at 37 ℃ for 1 h. The mixtures were then added to the pre-coated ELISA plate and incubated at 37 ℃ for 1 h. After washing, HRP-labelled goat anti-mouse secondary antibody (1:2500 dilution) was added and incubated for 1 h at 37 ℃. After further washing, TMB colouring solution was added as substrate for HRP. The OD450 values were measured with an ELISA reader. IR was calculated according to the aforementioned formula based on the OD450 values of each well, as a reflection of the reactivities of the antibodies with the original and mutated peptides.
Table 3. Sequences of the alanine scan replacement peptides for HA protein.
RNA of the two mAbs was extracted and reverse transcribed to cDNA for cloning. In total, 27 primers were designed by comparing the heavy (H) and light (L) chains of published mouse HA variable regions (Supplementary Table S1). PCR was used to obtain the L-chain gene (approximately 320 bp) of the antibodies with eight primers (including seven forward primers and one reverse primer), and the H-chain gene (approximately 350 bp) of the antibodies with nine primers (including five forward primers and four reverse primers). The PCR-amplified products were subjected to further PCR identification (for the L chain, five primers were designed downstream of the amplification product, and for the H chain, five primers were designed upstream of the amplification product). The positive target L/H-chain sequence was identified (approximately 180 bp for the L chain, and 150 bp for the H chain) and purified by gel electrophoresis. Subsequently, the positive target sequences were ligated into pMD19-T and transformed into competent E. coli (DH5α). Cloned DNAs were confirmed by Sanger sequencing and the corresponding amino acid sequences were determined.
Antigens, Antibodies, and Related Reagents
HA protein synthetic peptide
Indirect ELISA and Antibody Classification
Screening Epitopes of the Influenza A virus HA Protein
Distribution of Epitopes on the HA Crystal Structure
Localization of Heterophilic Epitopes
Verification of indirect ELISA localization
Key Amino Acid Site Identification
Cloning of Heavy/light Chains of the Variable Region of the Two mAbs
Indirect ELISA was performed to characterise the reactivity of the mAbs A1-10 and H1-84 with different subtypes of the influenza virus. As shown in Table 1, both mAbs exhibited broad cross-reactivity with HAs from five subtypes of the influenza virus, including the 2009 influenza A H1N1 influenza virus vaccine, the seasonal influenza virus H1N1 and H3N2 vaccines, and the poultry influenza virus H5N1 and H9N2 vaccines. As a negative control, the supernatant of SP2/0 cell culture showed no reaction with any of the HAs. These results suggested that the epitopes recognised by the mAbs are conserved among the five strains of influenza viruses.
The GenBank accession numbers of the five strains of influenza viruses were used to obtain their HA amino acid sequences from the NCBI database. Multiple sequence alignment analysis was performed on the amino acid sequences of HA using DNAMAN software. The conserved sequences were examined as candidate heterophilic epitopes of the viruses that can cross-react with the mAbs A1-10 and H1-84. The nine synthesised peptides (P1-P9) that were estimated by DNAMAN software are listed in Table 2.
An ELISA blocking experiment was performed to identify heterophilic epitopes recognised by mAbs A1-10 and H1-84. P1-P9 candidate epitopes were tested and screened. As shown in Fig. 1A, the IR of the two mAbs binding to peptide 2 (P2) was > 0.8; however, the IR of the two mAbs binding to the other eight peptides (P1, P3-P9) was < 0.4. Indirect ELISAs were also performed to observe reactivity between the two mAbs and P2+; P2+ was synthesised with a 5-glutamic-acid tail (LVLWGIHHPEEEEE), as shown in Fig. 1B. Both of the mAbs reacted with P2+, while no reaction was observed between P2+ and the supernatant of SP2/0 cell culture. The results suggested that the mAbs can only react with P2 (LVLWGIHHP191-199).
Figure 1. Localization analysis of two mAbs against the HA antigen that recognize heterophilic epitopes. A P2 exhibited a higher inhibition rate with the two mAbs compared to the other 8 candidate polypeptides. B The reaction for the two mAbs was also significant for the P2+ polypeptide (synthesized with a 5-glutamic-acid tail). C P2 segment alignment with the HA amino acid sequences of the 5 flu strains. D Distribution of P2 on the HA crystal structure.
The amino acid sequences corresponding to peptide P2 within HAs of the five influenza strains were aligned, as shown in Fig. 1C; the same colour indicates that although the amino acids differed, they belonged to the same class, with similar structure and function. The results showed that peptide P2 was conserved among the five strains of influenza virus, which was the heterophilic epitope of HA recognised by the two mAbs. PyMOL and Swiss-PdbViewer were also used to analyse the distribution of peptide P2 on the HA crystal structure. As shown in Fig. 1D, 3LZG (PDB) was used as the reference structure, and the distribution of P2 on the HA trimer crystal structure is marked in red and located in the β sheets of the HA head region.
Each amino acid of the heterophilic epitope P2 sequence (LVLWGIHHP191-199) was individually replaced with alanine. As shown in Table 3, there were nine alanine replacement peptides: P2-1A and P2-2A to P2-9A. ELISA blocking experiments were used to identify key amino acid residues for the mAbs A1-10 and H1-84 mapped with different alanine replacement peptides. The IR was calculated by measuring the OD450.
As shown in Fig. 2, when the alanine replacement peptide could not block binding of the two mAbs to HA, the IR was < 0.4, suggesting that the alanine replacement site is a key amino acid site for mAb binding. Conversely, when the alanine replacement peptide could still block binding of the mAbs to HA, the IR was > 0.8, suggesting that the alanine replacement site was not a key amino acid site for mAb binding. Therefore, the amino acids critical for mAb A1-10 recognition were L191, V192, L193, W194, I196, and H197, while the amino acids critical for mAb H1-84 recognition were V192, L193, W194, I196, and P199. We clearly showed that the two mAbs recognise two different epitopes, although the two epitopes have overlapping amino acids.
Molecular biological methods were performed to obtain amino acid sequences of the L- and H-chain variable regions (VL and VH) of mAbs A1-10 and H1-84. The VL and VH sequences of the two mAbs are listed in Table 4. abYsis software was used to analyse the complementarity-determining regions (CDRs) and framework regions (FRs) of VL and VH for mAbs A1-10 and H1-84. As shown in Fig. 3, VL and VH contained three CDRs, namely CDR1, CDR2, and CDR3, and four FR regions, namely FR1, FR2, FR3, and FR4. Comparing the amino acid sequences of the CDRs and FRs of VL and VH for mAbs A1-10 and H1-84, it was found that the amino acids in the FRs did not change significantly; however, amino acid changes in the three CDRs of VL and VH were apparent. The three CDRs of VL and VH together constitute the antigen-binding site of the mAb, which recognises and binds the antigen. These results suggested that mAbs A1-10 and H1-84 are derived from different cell clones, and although both mAbs reacted with peptide P2, they recognised different key amino acid sites; i.e., different epitopes.
Figure 3. Sequence analysis of the L- and H-chain variable regions of the two mAbs. A A1-10VH, B A1-10VL, C H1-84VH, and D H1-84VL.
Table 4. Sequences of heavy chain and light chain variable region amino acid of two mAbs.
Reaction Characteristics of A1-10 and H1-84 with Different Subtypes of the Influenza Virus
Localization of the mAbs against Influenza Virus HA to Identify Heterophilic Epitopes
Identification of Key Amino Acids for the mAbs Recognising the Heterophilic Epitope
Sequence Analysis of L- and H-chain Variable Regions of the mAbs
Table S1. Primers of the variable regions of the heavy and light chains of mouse mAb