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. 2022 Jun 14;14(6):1304.
doi: 10.3390/v14061304.

Neuraminidase (NA) 370-Loop Mutations of the 2009 Pandemic H1N1 Viruses Affect NA Enzyme Activity, Hemagglutination Titer, Mouse Virulence, and Inactivated-Virus Immunogenicity

Ting-Hsuan Chen  1 , Chung-Chu Chen  2   3 , Suh-Chin Wu  1   4   5
Affiliations

Neuraminidase (NA) 370-Loop Mutations of the 2009 Pandemic H1N1 Viruses Affect NA Enzyme Activity, Hemagglutination Titer, Mouse Virulence, and Inactivated-Virus Immunogenicity

Ting-Hsuan Chen et al. Viruses. .

Abstract

Hemagglutinin (HA) and neuraminidase (NA) are the two major envelope proteins of influenza viruses. The spatial organization of HA and NA on the virus surface needs to be optimized to promote viral fitness, host specificity, transmissibility, infectivity, and virulence. We previously demonstrated that the recombinant NA protein of the 2009 pandemic H1N1 (pH1N1) with the I365T/S366N mutation in the NA 370-loop elicited higher NA-inhibition antibody titers against the homologous pH1N1 virus and three heterologous H5N1, H3N2, and H7N9 viruses in mice. In this study, we used PR8-based reverse genetics (RG) by replacing the HA and NA genes of A/Texas/05/2009 pH1N1 virus to obtain the wild-type pH1N1 and three NA 370-loop mutant viruses of pH1N1 (I365T/S366N), RG pH1N1 (I365E/S366D), and RG pH1N1 (I365T/S366A). Our results revealed that the viral NA enzyme activity increased for the RG pH1N1(I365T/S366N) and RG pH1N1 (I365E/S366D) viruses but reduced for the RG pH1N1 (I365T/S366A) virus. The increased or decreased NA enzyme activity was found to correlate with the increase or decrease in HA titers of these NA 370-loop mutant viruses. All of these three NA 370-loop mutant RG pH1N1 viruses were less virulent than the wild-type RG pH1N1 virus in mice. Immunizations with the inactivated viruses carrying the three NA 370-loop mutations and the wild-type RG pH1N1 virus were found to elicit approximately the same titers of NA-inhibition antibodies against H1N1 and H5N1 viruses. These results may provide information for developing NA-based influenza virus vaccines.

Keywords: 370-loop; influenza virus; neuraminidase; vaccine.

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Conflict of interest statement

The authors have declared that no competing interests exist. S.-C.W. is an employee of Adimmune Corporation, and the company had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish.

Figures

Figure 1
Figure 1
NA 370-loop amino acid sequence analysis. (A) WebLogo of 370-loop was generated based on 31,185 sequences of N1NA from HXN1 viruses from NCBI using the WebLogo3 website. (B) Amino acid sequence alignment of NA sequences from A/Texas/05/2009 (H1N1), A/Vietnam/1203/2004 (H5N1), A/Udorn/307/1972 (H3N2), and A/Shanghai/02/2013 (H7N9). (C) 3D structure of a monomer of N1NA of pH1N1 (PDB: 4b7r) The 370-loop is shown in cyan. The 365, 366, and 369 residues are shown in red, yellow, and green, respectively.
Figure 2
Figure 2
Generation and characterization of pH1N1 RG viruses. Viral plaques of (A) RG pH1N1, (B) RG pH1N1(I365T/S366N), (C) RG pH1N1(I365E/S366D), and (D) RG pH1N1(I365T/S366A). TEM images of (E) RG pH1N1, (F) RG pH1N1(I365T/S366N), (G) RG pH1N1(I365E/S366D), and (H) RG pH1N1(I365T/S366A). (I) Viral growth curves in MDCK cells infected at MOI = 0.1 at different hours post-infection. (J) Viral NA enzyme activity on monovalent MUNANA substrates. (K) Viral NA enzyme activities on O-linked SIA of multivalent fetuin substrate. (L) Viral NA enzyme activities on the N-linked SIA of multivalent fetuin substrate.
Figure 3
Figure 3
Viral NA enzyme kinetics. To determine the viral NA enzyme activities in real-time, the emission signals from different concentrations of MUNANA cleaved by NA of (A) RG pH1N1, (B) RG pH1N1(I365T/S366N), (C) RG pH1N1(I365E/S366D), and (D) RG pH1N1(I365T/S366A) were measured every 60 s for 1 h. (E) Michaelis–Menten plots of viral NA enzyme activities. (F) Lineweaver–Burk plots of viral NA enzyme activity. (G) Vmax, (H) Km, and (I) Kcat/Km of each viral NA calculated using Lineweaver–Burk plots. Statistical tests for multiple comparisons in (G–I) were analyzed using one-way ANOVA with Tukey’s or Holm–Sidak’s multiple comparison tests. (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001). Error bars are plotted as standard deviation from the mean value.
Figure 4
Figure 4
HA titers of RG pH1N1, RG pH1N1 (I365T/S366N), RG pH1N1 (I365E/S366D, and RG pH1N1 (I365T/S366A). (A) HA titers of RG pH1N1, RG pH1N1 (I365T/S366N), RG pH1N1 (I365E/S366D) and RG pH1N1 (I365T/S366A) with or without oseltamivir carboxylate treatment determined by hemagglutination assays using 0.5% turkey RBCs at 4 °C and 37 °C. (B) The HA titers plotted as a bar chart.
Figure 5
Figure 5
Virulence of the RG pH1N1, RG pH1N1(I365T/S366N), RG pH1N1 (I365E/S366D), and RG pH1N1(I365T/S366A) viruses. Five mice from each group were intranasally infected with RG pH1N1, RG pH1N1(I365T/S366N), RG pH1N1(I365E/S366D), or RG pH1N1(I365T/S366A). (A) Survival rates of mice inoculated with 102 viruses. (B) Body weight of mice inoculated with 102 viruses. (C) Survival rates of mice infected with 103 viruses. (D) Body weight of mice infected with 103 viruses.
Figure 6
Figure 6
NA-inhibition, HA-inhibition, and virus-neutralizing antibodies elicited by the inactivated RG pH1N1 viruses with NA 370-loop mutations. (A) Groups of BALB/c mice (n = 5 per group) were intramuscularly immunized with each group of inactivated viruses containing 10 µg total protein plus alum adjuvant using a two-dose regimen. Antisera were collected on week 5. NA-inhibition antibody curves against (B) pH1N1 (NA activity on O-linked SIA), (C) pH1N1 (NA activity on N-linked SIA), (D) H5N1 (NA activity on O-linked SIA), and (E) H5N1 (NA activity on N-linked SIA). (F) IC50 titers of NA-inhibition antibodies against pH1N1 and H5N1 viruses. (G) HI titers against pH1N1 virus. (H) Virus-neutralization inhibition for pH1N1 virus.
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