A A212T Substitution in Hemagglutinin Impacts on the Rescue of Influenza A(H1N1)/B Chimeric Live-Attenuated Vaccine Candidate

doi:10.21203/rs.3.rs-2563420/v1

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A A212T Substitution in Hemagglutinin Impacts on the Rescue of Influenza A(H1N1)/B Chimeric Live-Attenuated Vaccine Candidate

https://doi.org/10.21203/rs.3.rs-2563420/v1

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The influenza virus, a viral respiratory pathogen, causes major morbidity and mortality worldwide. In this study, we compared and analyzed the effectiveness of the rescued two chimeric H1N1 vaccine strains rA/B-California/04/2009 and rA/B-California/07/2009, respectively, which expressed the hemagglutinin of A/California/07/2009 or A/California/04/2009 based on an influenza B virus master donor. The rA/B-California/07/2009 strain was rescued successfully. However, the rA/B-California/04/2009, which expressed the HA of A/California/07/2009 could hardly be generated. In substitution for alanine at residue 212 in the chimeric HA protein of rA/B-California/04/2009 virus, threonine was identified as a deleterious change. The free Gibbs energy change of mutating residue 212 reflected that threonine in site 212 caused a reduction in the stability of rA/B-California HAs. Additionally, with the analysis of binding free energy data and RMSD values of receptor analogs and two chimeric HAs, substitution 212T also conferred decreased receptor-binding avidity. These results suggested that the substitution A212T may enhance the stability and binding affinity of the chimeric H1N1 vaccine strains. This finding may help to design a live-attenuated influenza vaccine with more efficacy.

live attenuated influenza vaccine

Chimeric hemagglutinin

reverse genetics

Influenza, an infectious respiratory disease caused by influenza viruses[1], contributes to high morbidity and mortality in avians or humans[2]. Seasonal influenza affects 5–15% of the human population and results in ∼500,000 deaths annually[3], in which these effects are attributed to the continued evolution of seasonal influenza viruses[4]. Notably, vaccination is the most effective way to prevent and control influenza infections[5]. The CFDA (China Food and Drug Administration)-approved influenza vaccines include three types: traditional inactivated, subunit, and live-attenuated [6]. Live-attenuated influenza vaccine (LAIV), mimicking a natural infection, creates broad protection at the mucosal surfaces by inducing secretory IgA and T-cell immunity. It has been found to protect children more broadly than the inactivated influenza vaccines[7]. Furthermore, there is a concern about the possibility that the attenuated vaccine strains will recombine themselves with wild types to generate a novel strain[8].

The live attenuated influenza vaccines were generated by reverse genetics methods. To match the circulating strains and influenza vaccines, the influenza vaccine strains need to be prepared and generated before the vaccine is manufactured. Influenza A virus and influenza B virus belong to the same orthomyxovirus family, both of which are single-stranded RNA viruses whose genomes are divided into 8 segments[9]. However, reassortment or genetic exchange between influenza A and influenza B viruses has never been observed in nature[10]. Hai Rong et al[11] designed a chimeric A/B hemagglutinin virus as a LAIV candidate to avoid the risk of genetic recombination. Chimeric A/B viruses harbor chimeric hemagglutinin (HA) molecules with the influenza B HAs’ transmembrane (TM) and cytoplasmic tail (CT) domains, and segment-specific packaging signals present in both the 3′- and 5′-terminal regions of type B HA segments[11, 12]. It is demonstrated that immunizations with a chimeric live attenuated influenza vaccine can protect fully against lung replication and death from influenza A virus in mouse models[11].

HA glycoproteins, trimer proteins distributed on the surface of influenza viruses, have vital functions in host cell virus attachment and uncoating [13]. Influenza virus infection begins with the binding of influenza HA to cellular receptors - sialic acid (SA), in which SA is present on the cell membrane and is involved in host-pathogen interactions[14]. The terminal SA links with galactose residues in a linkage of SAα2-3Gal or SAα2-6Gal[15]. The receptor binding site (RBS), located on the globular head of HA, comprises the 130-loop, the 150-loop, the 190-helix, and the 220-loop[16, 17]. Notably, residues close to the RBS may influence virus-receptor binding avidity[18–20]. Although chimeric viruses are promising live attenuated vaccine candidates, they have some limitations. The replication of the chimeric A/B virus may not be as optimal as the wild type[12]. Despite it is known that a reduced receptor-binding affinity generally leads to the low growth of influenza viruses[21], it remains unclear whether there are mutations of the RBS that may result in decreased or inadmissibility to chimeric CVVs replication.

In this study, we attempted to construct a panel of influenza B-based vaccine candidates with H1N1 hemagglutinin using a reverse genetics system. A substitution near RBS-A212T was identified. Thus, we produced mutants with altered 212 sites between two chimeric CVVs to reveal that position 212 is a critical amino acids in the generation of H1N1 chimeric CVVs and performed a structure prediction of chimeric HAs with substitution A212T. Our study illustrated the selectivity of the 212 position and assessed its impact on the stability of H1N1 chimeric HAs.

Construction and biological characteristic of influenza B virus-based vaccine candidates with H1N1 hemagglutinin

We constructed a series of chimeric A/B HA expressing the HA ectodomain of various seasonal influenza H1N1 viruses. We then harbored the chimeric H1N1 HA molecules with the influenza B HAs’ TM and CT domains and segment-specific packaging signals presented in the 3′- and 5′-terminal regions of influenza B virus as detailed in Fig. 1a. Cloning the chimeras into the HA plasmid of B/Yamagata/16/1988 using the In-Fusion technology. Chimeric H1-HA plasmids were transfected jointly with seven other genomic segments from B/Yamagata/16/1988 (with a truncated NS1) into 293T cells.

By applying plasmid-based reverse genetics methods, we have generated the recombinant viruses with the chimeric A/Hawaii/70/2019(H1N1)-HA and the chimeric A/Victoria/2570/2019(H1N1)-HA. Notably, this chimeric vaccine candidate could grow to 10^3.75 TCID₅₀/mL and 10^5.25EID₅₀/mL. We also produced a chimeric A/California/07/2009 CVV and named it rA/B-California/07/2009 (R_07). Additionally, the nucleotide sequences of rescued CVVs were confirmed by sanger sequencing, and the chimeric candidates' virus particles were identified using a Transmission electron microscope (TEM).

Adaptive mutations on H1N1 influenza viruses near the receptor site may influence the growth of virus as previously described[22]. To determine the viral growth capability of the chimeric CVV, MDCK cells were infected with the virus with a multiplicities of infection(MOI)of 0.01(Fig. 1c). The growth curve indicates that the replication capability of rA/B-California/07/2009 was significantly lower than wild type A or wild type B viruses.

Alternation Of The 212 Site Between Two Chimeric Cvvs

We present the capacity of producing chimeric A/California/07/2009 CVV. The chimeric A/California/04/2009 CVV, named rA/B-California /04/2009 (R_04), could hardly be generated. We deemed two chimeras, R_07 HA protein and R_04 HA protein, similar. However, the production of two chimeric CVVs strikingly diverged. We then conducted pair-wise alignment of two chimeras to expound the divergence. By aligning the sequence of R_07 HA protein and R_04 HA protein in pairs, a A212T substitution was observed (Fig. 2).

To confirm the A212T substitution, we designed a rescue scheme for an alternation at site 212 between two chimeric CVVs to verify the significance of a generated H1N1 chimeric CVV of site 212. Mutant virus of rA/B-California/07/2009, carrying 212T, failed to generate chimeric H1N1 CVVs as before. These results indicated that the 212T substitution had a deleterious effect on the rA/B-California/04/ 2009 vaccine strain. All results are summarized in Table 1.

Table 1

Rescue scheme for an alternation at site 212
	Chimeric virus	Mutant chimeric virus with altered site 212
rA/B-California/07	√	-
rA/B-California/04	-	√
* “√” indicates the rescued virus generated successfully;

“-” indicates the rescued virus failed to be generated.

With a revelation that the generation of chimeric A/California /07/2009 and A/California/04/2009 CVVs related to a substitution A212T, we attempted to undertake single nucleotide polymorphism analysis to calculate the scope of variation belonging to amino acid sequences of influenza H1N1 HA. Single Nucleotide Polymorphism(SNP) data for the H1N1 HA proteins of human and avian influenza viruses were selected from Influenza Research Database (IRD). In both human and avian H1N1 AIVs, alanine (A) was more prevalent than threonine (T) in residue 212. About 94% of human H1N1 IAVs had A at residue 212, followed by 5% having T. Serine (S), glycine (G), valine (V), and glutamic acid (E) at residue 212 were far lower than 1% (Fig. 3a). Furthermore, about 99% of avian H1N1 IAVs had A at residue 212, followed by T at 1% (Fig. 3b). Therefore, A212 was overwhelmingly dominant in the HA of the H1N1 subtypes.

Structural Characterization Of Chimeric Has And Site 212

To predict the structural features of CVV chimeric HA proteins and properties of substitution A212T, we submitted the sequences of R_07 and R_04 proteins to Alphafold2 (AF2) to predict the 3D structures of chimeric proteins (Fig. 4). The theoretical R_07 structure was generated based on multiple sequence alignments and available experimental protein structures [23].

As the 3D predictions were established, phylogenetic analysis describing sequence conservation grades could be conducted to demonstrate the evolutionary conservation at residues of chimeric HAs. To investigate whether residue 212 was conserved, we calculated the evolutionary degree of all sites in chimeric HA proteins using the ConSurf web server. Residue color values, a conservation score the ConSurf provided for each residue in 3D models of proteins, assigned a score of 6 to the residue 212 in the R_07 structure (Fig. 5). Although the key functional positions had a bias towards being highly conserved, residue 212 of chimeric H1N1 HAs was intermediate.

212t Substitution Induces A Reduction In The Stability Of Chimeric H1 Has

To examine how the substitution A212T affects the generation of recombinant influenza viruses, we performed a range of mutations on each amino acid in predicted chimeric HA models. FoldX, a frequently used algorithm for protein stabilization experiments, can be used as a plugin in the protein structure visualizer YASARA[24]. We used the predicted 3D structure of chimeric HA proteins to generate possible single amino acid substitutions in the R_04 sequence and calculate the energy difference between the single amino acid 212T and 212A of H1N1 chimeric HAs. The point of the predictions was to evaluate the change in thermodynamic stability regarding the R_04 protein. The corresponding free Gibbs energy change of mutating all non-A residues of R_04 protein to an A residue (ΔΔG) was obtained using FoldX. Mutations with negative ΔΔG values of AlaScan indicated that they were more stable than the wild-type R_04 protein, yet those with positive values indicated that they were less stable than the wild-type. The ΔΔG values of residue T212 were − 1.64 kcal/mol, which suggested the T212A substitution stabilized the chimeric H1N1 HA proteins. This value corroborated the residue 212T destabilizing the chimera R_04. Following the PositionScan operations of the software FoldX for mutating every amino acid in the R_07 sequence to the other 5 amino acids that might appear at this site, we excluded other variants (all ΔΔG༞0 kcal/mol, listed in Table2). Furthermore, we confirmed A was the optimum amino acid for the chimeric CVVs at position 212.

Table 2

The free Gibbs energy change (G) of mutating residue 212A of R_07 protein to the other 5 amino acids
ΔΔ
Residue 212	Mutants	Free Gibbs energy change (kcal/mol)
212A	212T	1.83067
	212S	0.99211
	212G	1.05901
	212V	0.98729
	212E	11.1011

212t Substitution Induces A Decreased Sa-binding Avidity

The chimeric H1N1 HAs shared a large component of California HA ectodomains that made up 89% of the size of the H1N1 HA protein. Thus, the globular domain of the R_07 chimeric HA protein was deemed similar to that of the H1N1 HA protein. It was demonstrated previously that the residue 212 of the chimeric HA adjoined to the 190-helix, a component of the receptor binding site, in both protein primary sequence and tertiary structure, as it appeared in California HAs. Human influenza viruses were attached to α2,3-linked sialylated glycans, and avian influenza viruses had a partiality for α2,6-linked sialylated glycans[25]. To further test the contribution of the substitution A212T to the receptor-binding avidity of chimeric H1N1 HAs, we used molecular docking technology to perform the interactions between receptors–chimeric hemagglutinins and ligands–SA analogs. Sialylneolacto-N-tetraose c (LSTc, PubChem CID: 53477861) was a sialooligosaccharide, and has been suggested for a human glycan receptor analog. Additionally, sialyllacto-N-tetraose (LSTa, PubChem CID: 53477859) has been suggested as an avian glycan receptor analog [26]. AutoDock was used to dock the receptor analogs LSTc and LSTa with R_07 and R_04 HA proteins and predict the optimal conformations. Optimal conformations of ligands were selected from different poses according to the docking scores. Considering that docking in the water environment could significantly improve the estimation accuracy of binding energy[27], water molecules were added to the ligand during the docking process[28]. Among the predicted R_07 HA-LSTa (Fig. 6a) and R_04 HA-LSTa (Fig. 6b) complexes, the best poses of LSTa had SIA directed to the 130-loop. Instead, the optimal pose of the R_04 HA-LSTc (Fig. 6c) complex had a SIA toward 130-loop. In contrast, the R_07 HA-LSTc (Fig. 6d) complex had SIA turn toward 220-loop. Furthermore, the two-dimensional interaction diagrams of ligand-receptor interaction were drawn using MOE.

It has been reported that the CA04 H1 HA showed a receptor-binding property of preferential recognition to α2-6-linked glycans. As anticipated, the binding affinities of chimeric H1 HAs showed a partiality for LSTc, but the ability to bind both analogs LSTc and LSTa declined. We recorded 4 binding free energy data of each optimal structure after docking (Table 3). Detection using AutoDock algorithm about the binding energy showed whether a T212 substitution increased the binding free energy between R_04 HA protein and analogs. The R_04- analogs complexes displayed higher binding energy values than R_07 complexes, and thus T in site 212 appeared to decrease the chimeric H1 HAs’ SA-binding avidity.

Table 3

Binding free energy data (G) of receptor analogs and chimeric HAs
Δ
Ligands	Complexes	Binding Free Energy (kJ/mol)
LSTc	R_07 HA-LSTc	-8.9408
LSTc	R_04 HA-LSTc	-8.6752
LSTa	R_07 HA-LSTa	-7.5533
LSTa	R_04 HA-LSTa	-6.8546

To further analyze the receptor binding characterization of the substitution A212T, we visualized the receptor-ligand interactions in the optimal complexes with their binding pockets (Fig. 7). Root-mean-square deviation (RMSD) was calculated for measuring the conformational change of ligands and related pockets through pair-wise comparison by PyMol software. The RMSD value for R_07-LSTa-binding pockets and R_04-LSTa-binding pockets indicated the convergence of structure (RMSD value: 0.265, Fig. 7a). While comparing R_07-LSTc-binding pockets and R_04-LSTc-binding pockets, the RMSD value still showed a slight distinction (RMSD value:0.816, Fig. 7b). In comparison, the rA/B-California viruses with substitutions in 212 could affect the conformation of LSTc-chimeric H1N1 HAs.

Cells and viruses

Human embryonic kidney cells (293T) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Corning) with 10% fetal bovine serum (Gibco) and 1× antibiotic-antimycotic (Gibco). Madin-Darby canine kidney (MDCK) cells were grown in Dulbecco’s modified Eagle’s medium with 5% fetal bovine serum and 1× antibiotic-antimycotic. Chimeric H1N1 viruses were inoculated into 7-day-old specific pathogen-free (SPF) chicken embryos for 72 hours at 33℃.

Construction Of Plasmids

The 8 plasmids reverse genetics system of the B/Yamagata/16/1988 strain was constructed by a bidirectional transcription vector pBD as described previously[29]. Plasmids encoding the chimeric H1N1 HAs were constructed using the strategy mentioned above. The chimeric H1N1 HAs were constructed by swapping the influenza B HA for H1N1 HA segments using an In-Fusion cloning kit (Takara). To introduce the ectodomain of A/California/07/2009 HA protein into the B/Yamagata/16/1988 HA segment, an insert fragment containing H1 ectodomain was generated with forward primer 5’- GTAACATCCAACGCAGACACATTATGTATAGGTTATCATG-3’ and reverse primer 5’- AGTATGATTATCCAATGATTCCAGCTTTACCCC-3’. The respective linearized vector, composed of the influenza B HAs’ transmembrane and cytoplasmic tail domains, was amplified from pBD-YA1688-HA transfection plasmid using forward primer 5’- TTGGATAATCATACTATACTGCTCT-3’ and reverse primer 5’- TGCGTTGGATGTTACTACCATGAGT. Furthermore, both fragments were purified using Wizard® SV Gel and PCR Clean-Up Start-Up Kit (Promega).

Rescue Of Chimeric H1n1 Cvvs

To generate influenza B virus-based vaccine candidate viruses with H1N1 HA, we transfected 500ng of each of eight B/Yamagata/16/1988 plasmids into 293T cells, which was mediated by Lipofectamine 3000 (Invitrogen). Plasmids encoding chimeric H1N1 HA took over from the wild-type HA plasmid as a chimeric A/B H1N1 virus generator. Replacing the cell culture medium with Opti-MEM containing 0.2µg/ml TPCK (tosylsulfonyl phenylalanyl chloromethyl ketone)- treated trypsin (Sigma) at 72 hours post-transfection, supernatant fluid containing chimeric viruses were inoculated into 7-day-old SPF chicken embryos. 72 hours post-inoculation, allantoic fluid was harvested and detected by the HA test using 1% chicken erythrocyte cells. .

Viral Titration And Growth Kinetics

The virus titer of the chimeric H1N1 virus, rA/B-California/07/2009, was determined using a 50% tissue culture infective dose (TCID₅₀) assay and 50% egg infective dose (EID₅₀) assay. The TCID₅₀ and EID₅₀ results were calculated by the Reed-Muench method[30].

MDCK cells were infected with rA/B-California/07/2009 virus by 10-fold serially dilution assay at 33℃ for 72h. The 7-day-old embryonated eggs were inoculated with rA/B-California/07/2009 virus by 10-fold serially dilution assay at 33℃ for 72h. MDCK cells were infected with rA/B-California/07/2009 virus at a MOI of 0.01 and cultured in DMEM containing 1.5µg/ml TPCK-trypsin at 33℃ to analyze viral replication. To evaluate the growth kinetics, supernatants were sampled and titrated at 0, 12, 24, 36, 48, 60, and 72 hours post-infection (hpi).

Sequence Alignment And Single Nucleotide Polymorphism Analysis

Two sequences of R_07 and R_04 HA were aligned using ClustalW (MEGA11)[31]. Sequence alignment was viewed and managed using the GeneDoc program. We then queried the Influenza Research Database (IRD) and retrieved encoded protein sequences of human and avian influenza H1N1 HA[32]. Furthermore, SNP was predicted using a visualization tool available in IRD: Analyze Sequence Variation/SNP.

Structure Predictions Of Chimeric Ha Proteins

The 3D structures of the R_07 monomer, R_04 monomer, and R_07 trimer were all predicted by AlphaFold2. AlphaFold2 was a machine-learning algorithm applied to protein structure prediction[33, 34]. It used evolutionarily conserved regions across protein sequences starting from multiple sequence alignment. To assess the confidence of the predicted model quantitively, two evaluation metrics, the predicted local distance difference test (pLDDT) scores and the predicted aligned error (PAE), were obtained from AlphaFold2[35]. Additionally, pLDDT was a score for each residue in the 3D structure prediction and scaled between 0 and 100. If pLDDT > 90, it indicated very high confidence in the predicted residue; if 90 > pLDDT > 70, it indicated confidence; if 70 > pLDDT > 50, low confidence; and it was deemed very low confidence if pLDDT < 50[36]. PAE values indicated the error in the position of each amino acid in the predicted structure. If PAE values were low, it suggested the relative positions and orientations of the predicted domains were reliable. If the PAEs were high, the relative position and orientation of the predicted domains were considered unreliable[37]. We evaluated five models ranked by pLDDT and PAE scores and got the predicted R_07 and R_04 structures in the protein data bank (PDB) format. Furthermore, PyMol Molecular Graphics System was used as the molecular visualization software, in which all 3D structures were presented by PyMol software.

Evolutionary Conservation Analysis Of Amino Acid 212

The evolutionary conservation of amino acid 212 in chimeric H1N1 HA was calculated with the ConSurf web server (http://consurf.tau.ac.il). ConSurf extracted the sequence from the 3D structure and computed position-specific conservation scores based on a multiple sequence alignment of R_07 homologs. The conservation scores were divided into nine grades, from turquoise, which indicated the most variable region (grade 1), to maroon, which indicated the most conserved region (grade 9), and intermediate residues (grade 5) were colored white. The colored 3D structures of chimeric HAs were visualized using PyMol software[38].

Predictions Of Residue 212 Mutations On Stability

FoldX (http://foldx.embl.de/) was an algorithm developed for free energy calculations, and it enabled a fast means to evaluate the effectiveness of chimeric HA protein mutations on stability. FoldX read the R_04 PDB file, performed an Alascan to mutate each residue except alanine to Alanine, and obtained difference values of energy (ΔΔG, in kcal/mol) based on free energy change of the mutant from the wildtype[39].

Molecular Docking

All molecular docking calculations were performed using AutoDock software [40], and the docking experiments were validated with MOE (Molecular Operating Environment) program[41, 42]. Our previous section predicted the 3D structure of chimeric influenza HA proteins R_07 and R_04 and used them as receptors in the docking process. We then retrieved the 3D structure of LSTc and LSTa as ligands from the Protein Data Bank (PBD ID: 3NXD and 4QN5). We optimized the receptors and ligands, including hydrogen atom additions and Gasteiger atomic charges additions, using the AutoDock tool (MGLTools 1.5.7). We adopted a grid-based scoring method, a flexible ligand docking to a rigid receptor, which allowed the SA analogs to rearrange structurally in return for the attachment to the chimeric HAs. AutoDock usually removes all water from the 3D structures of receptors before docking. However, some water molecules are conserved as a part of a ligand-receptor docking structure[43, 44]. After creating two files, ligand. pdbqt and protein. pdbqt for docking, we modified the AutoGrid map to add water molecules to the ligand in the hydrated docking experiment[45]. For higher accuracy, in our molecular docking process, the size of grid dimensions was set to X, Y, Z: 100, 100, 100, and the number of Genetic Algorithms was 500. Therefore, complexes with the lowest free binding energy of molecular docking and optimal conformations were selected and visualized by PyMol software.

The live attenuated influenza vaccines are generated by reverse genetics methods, and the candidate vaccine strains are updated annually. However, some studies indicated that some key amino acids result in the failure of the rescued influenza viruses. Here, we tried to generate two H1N1 seasonal influenza vaccines using a chimeric A/B hemagglutinin strategy[11]. These LAIVs possessed reassortments incompatible with wild-type influenza A strains, which prevented vaccine strains from reassort with circulating A viruses. The intertypic vaccine candidates with intertypic hemagglutinin flanked by transmembrane, cytoplasmic tail domains, and packaging signals from the HA protein of the parent virus produced lower growth characteristics than the wild-type virus.

In this study, reverse genetics methods generated a panel of influenza B virus-based vaccine candidates with H1N1 hemagglutinin such as rA/B-California/07/2009. Yet, it left us little margin for generating the rA/B-California/04/2009 virus. Through pair-wise alignment of two chimeric HAs as well as amino acids exchange and rescue of the mutant viruses, we assumed that substitution of T212 would not be tolerable for rA/B-California/04/2009 virus. With suppositional rA/B-California/07/2009 virus, we predicted the 3D structure of HA of rA/B-California viruses and did a single nucleotide polymorphism analysis to calculate the scope of variation at site 212 using computational approaches as several studies conducted[46]. The results showed that residue 212 was not highly conserved in HA RBS. With computational approaches, we also carried out 3D structure predictions based on influenza HA protein co-evolution information. We exchanged every amino acid in chimeric H1 hemagglutinins with 20 amino acid residues by the algorithm to evaluate the impact of substitution at site 212. The substitution 212T destabilized chimeric H1 HAs, proving that T in residue 212 was deleterious for generating the rA/B-California/04/2009 strain.

Molecular docking between chimeric HAs and receptor analogs was applied to find how the substitution A212T affects the receptor-binding avidity of chimeric H1N1 HAs. Compared with wild-type influenza H1N1 viruses, molecular docking results suggested that the chimeric HA of rA/B-California viruses decreased the binding with human and avian receptors. The binding properties of influenza virus to α2,3 and α2,6 linked sialylated glycans were routinely evaluated using solid-phase binding assay[47]. To detect whether the mutants in site 212 took part in regulating receptor binding affinity and binding specificity, we extracted and aligned both LSTa-binding pocket and LSTc-binding pocket conformations from the docking structure. We combined the binding-free energy value with the RMSD value, and it was concluded that chimeric H1N1 viruses carrying T at residue 212 displayed a detectable decrease in receptor binding avidity. The R_07 HA protein made the optimal interactions between chimeric HAs and analogs with A in site 212. With amino acid A in 212 as the absolute maximum in chimeric H1N1 HAs, it brings more stability and binding affinity to the generation of chimeric H1N1 vaccines.

In summary, we identified a key A212T substitution in H1N1 HA that may impact the rescue of the live attenuated influenza A virus. Notably, these findings may benefit the updated influenza vaccines in the future.

Contributions

WANG Tiecheng, XIA Xianzhu and GAO Yuwei conceived and designed of the study; ZHAO Menglin, LI Dongxu, SUN Yue and ZHU Menghan performed research; ZHAO Menglin, LIU Xiawei Analyzed data; ZHAO Menglin and SUN Weiyang wrote the paper. All authors read and approved the final manuscript.

Competing Interests

The authors have no conflicts of interest to disclose.

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No competing interests reported.

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A A212T Substitution in Hemagglutinin Impacts on the Rescue of Influenza A(H1N1)/B Chimeric Live-Attenuated Vaccine Candidate

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Abstract

Figures

Introduction

Results

Construction and biological characteristic of influenza B virus-based vaccine candidates with H1N1 hemagglutinin

Alternation Of The 212 Site Between Two Chimeric Cvvs

Structural Characterization Of Chimeric Has And Site 212

212t Substitution Induces A Reduction In The Stability Of Chimeric H1 Has

212t Substitution Induces A Decreased Sa-binding Avidity

Materials And Methods

Cells and viruses

Construction Of Plasmids

Rescue Of Chimeric H1n1 Cvvs

Viral Titration And Growth Kinetics

Sequence Alignment And Single Nucleotide Polymorphism Analysis

Structure Predictions Of Chimeric Ha Proteins

Evolutionary Conservation Analysis Of Amino Acid 212

Predictions Of Residue 212 Mutations On Stability

Molecular Docking

Discussion

Declarations

References

Additional Declarations

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