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. 2021 Oct 4:12:729914.
doi: 10.3389/fmicb.2021.729914. eCollection 2021.

Tetrameric Neuraminidase of Influenza A Virus Is Required to Induce Protective Antibody Responses in Mice

Xiren Deng  1 , Qimin Wang  1 , Mei Liu  1 , Qinwen Zheng  1 , Fan Wu  1 , Jinghe Huang  1
Affiliations

Tetrameric Neuraminidase of Influenza A Virus Is Required to Induce Protective Antibody Responses in Mice

Xiren Deng et al. Front Microbiol. .

Abstract

Influenza neuraminidase (NA) is able to induce cross-subtype immunity and is considered as a promising target for the development of universal influenza vaccines. However, commercial influenza vaccines only induced low NA-specific immune responses due to the low amounts and the denatured conformation of NA proteins in current inactivated or split influenza vaccines. Here we investigated the protective efficacy of recombinant tetrameric and monomeric NA proteins to determine whether the conformation contributed to induce protective immunity. We found that H1N1 P R 8NA tetramer (NA tet ) could provide complete homologous protection against A/PR8 (H1N1) virus infection in mice, while the protection of H1N1 P R 8NA monomer (NA mono ) was moderate. Higher levels of NA-reactive binding and inhibition antibodies and less weight loss were observed in the H1N1 P R 8NA tet -vaccinated group. Similarly, H5N1 V N NA tet immunization exhibited a preferable heterologous protection than H5N1 V N NA mono , but neither H7N9 S H NA tet nor H7N9 S H NA mono vaccination showed heterosubtypic protection. We also compared the effect of three adjuvants, aluminum, 3'3'-cGAMP (cGAMP), and Poly(I:C), on the humoral response and protective efficacy induced by H1N1 P R 8NA tet . H1N1 P R 8NA tet protein adjuvanted with aluminum was observed to exhibited better capacity in inducing NA-specific humoral immunity and preventing weight loss than with cGAMP or Poly(I:C). In conclusion, our data demonstrate that tetrameric NA with natural conformation is required to induce protective anti-NA immunity. The NA tetramer could provide homologous protection and subtype-specific cross-protection. In addition, the aluminum adjuvant is preferable in recombinant NA protein vaccination.

Keywords: cross-protection; influenza virus; neuraminidase; protein; vaccine.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Expression, purification, and characterization of recombinant neuraminidase (NA) proteins. Tetrameric and monomeric NA proteins derived from A/Puerto Rico/8/1934 (H1N1), A/Shanghai/37T/2009 (H1N1), A/Hong Kong/16/68 (H3N2), A/Vietnam/1204 (H5N1), and A/Shanghai/4664T/2013 (H7N9) were expressed by the Expi293F cell expression system and purified by Ni-nitrilotriacetic acid (NTA). (A,B) Purified NA proteins were confirmed by Western blotting. (C,D) Cross-linking SDS-PAGE of tetrameric and monomeric recombinant NA proteins. (E,F) MUNANA-based enzymatic activity assays were used to determine the enzymatic activity of recombinant NA proteins. (G,H) Fetuin-based enzymatic activity assays were used to determine the enzymatic activity of recombinant NA proteins.
FIGURE 2
FIGURE 2
Protective efficacy of tetrameric neuraminidase (NA) vs. monomeric NA. (A) The experimental design for immunization and challenge studies. Six- to eight-week-old BALB/c mice (n = 8 in each group) were immunized twice at 2-week interval with 20 μg of H1N1PR8NAtet, H5N1VNNAtet, H7N9SHNAtet, H1N1PR8NAmono, H5N1VNNAmono, and H7N9SHNAmono proteins adjuvanted with aluminum (i.p.), respectively. Sera were collected at 1 week after the final immunization; then, the mice were challenged with 5 LD50 of A/PR8 (H1N1) virus intranasally (i.n.) at 2 weeks after the final immunization. (B–G) Survival rates (B,D,F) and weight loss (C,E,G) were monitored for 14 days post-infection.
FIGURE 3
FIGURE 3
Humoral response induced tetrameric and monomeric neuraminidase (NA) proteins. The experimental design for immunization and challenge studies was identical to that detailed in the legend of Figure 2A. Serum were collected at 1 week after the final immunization. (A–C) The sera of mice vaccinated, respectively, with H1N1PR8NAtet and H1N1PR8NAmono (A), H5N1VNNAtet and H5N1VNNAmono (B), and H7N9SHNAtet and H7N9SHNAmono (C) proteins were tested for NA-specific binding antibody levels to the respective immunogen via ELISA. (D–F) The same sera whose results are shown in panels (A–C) were tested for cross-binding activity to H1N1PR8NAtet protein via ELISA, respectively. (G–I) The same sera mentioned above were tested for NA inhibition (NAI) activity against NA of A/PR8 (H1N1) via enzyme-linked lectin assay (ELLA), respectively.
FIGURE 4
FIGURE 4
Effect of three adjuvants—aluminum, cGAMP, and Poly(I:C)—on the protective efficacy and humoral response induced by H1N1PR8NAtet tetramer. (A) Six- to eight-week-old BALB/c mice were immunized twice at 2-week interval with 20 μg of H1N1PR8NAtet protein adjuvanted with aluminum (n = 8, i.p.), cGAMP (n = 4, i.c.), or Poly(I:C) (n = 4, i.p.), respectively. The mice were immunized with phosphate-buffered saline (PBS) as control (n = 4, i.p.). Sera were collected at 1 week after the final immunization; then, the mice were challenged with 5 LD50 of A/PR8 (H1N1) virus intranasally (i.n.) at 2 weeks after the final immunization. (B,C) The survival rates (B) and weight loss (C) were monitored for 14 days post-infection. (D) The serum from individual mice in each experimental group was tested for neuraminidase (NA)-specific antibody levels against H1N1PR8NAtet protein via ELISA. (E) The NAI activity of mice serum against NA of A/PR8 (H1N1) was tested via enzyme-linked lectin assay (ELLA).
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