Sequential Immunizations with Influenza Neuraminidase Protein Followed by Peptide Nanoclusters Induce Heterologous Protection

Abstract

Enhancing cross-protections against diverse influenza viruses is desired for influenza vaccinations. Neuraminidase (NA)-specific antibody responses have been found to independently correlate with a broader influenza protection spectrum. Here, we report a sequential immunization regimen that includes priming with NA protein followed by boosting with peptide nanoclusters, with which targeted enhancement of antibody responses in BALB/c mice to certain cross-protective B-cell epitopes of NA was achieved. The nanoclusters were fabricated via desolvation with absolute ethanol and were only composed of composite peptides. Unlike KLH conjugates, peptide nanoclusters would not induce influenza-unrelated immunity. We found that the incorporation of a hemagglutinin peptide of H2-d class II restriction into the composite peptides could be beneficial in enhancing the NA peptide-specific antibody response. Of note, boosters with N2 peptide nanoclusters induced stronger serum cross-reactivities to heterologous N2 and even heterosubtypic N7 and N9 than triple immunizations with the prototype recombinant tetrameric (rt) N2. The mouse challenge experiments with HK68 H3N2 also demonstrated the strong effectiveness of the peptide nanocluster boosters in conferring heterologous protection.

Keywords: cross-protection; influenza vaccine; nanocluster; neuraminidase; sequential immunization; serum cross-reactivity.

Figure 1

Construction and characterization of rtN1 and rtN2. (A) The diagram of protein construction depicts the compositions of rtN1 and rtN2. (B,C) Western blot and Coomassie blue staining analyses of rtN1 and rtN2. The hexahistidine-specific mAb was used for blotting rtNA protein bands. (D) SEC analysis of rtN1 and rtN2 proteins. The rtNA samples were collected at the major peak between the elution volumes of 10 and 11 for later protein characterization and immunization. (E,F) Antigenicity analysis of rtN1 and rtN2 using the ELISA method. Immunoplates coated with bovine serum albumin (BSA) were used as the negative control. Error bars represent the standard derivations (SDs) of the values of triplicate samples (n = 3 per group).

Figure 2

Preparation and characterization of peptide nanoclusters. (A) The peptide construction diagram depicts the amino acid sequences of composite peptides. N1P2TP peptide consists of N1P2, HA2 (166–180), and a SGGS linker in between; N2P2TP1 peptide consists of N2P2, HA1(183–199), and a SGGS linker in between; and N2P2TP2 peptide consists of N2P2, HA2 (166–180), and a SGGS linker in between. (B) The diagram depicts the preparation process of desolvated peptide nanoclusters. (C) Size-distribution analysis of N2P2TP2 nanoclusters based on DLS.

Figure 3

The schematic diagram of the immunization regimen and serum cross-reactivity analysis. (A) The schematic diagram depicts the time schedule of immunization, bleeding, and viral infection. (B–G) Serologic analysis. The hyper-immune serum samples collected on day 56 from each group (n = 6) were pooled and tested in triplicate using ELISA to evaluate serum reactivities to homologous (B) rtN1 and (C) rtN2 proteins, and (D–G) NA proteins from other strains expressed and displayed on the plasma membrane of transiently transfected HEK293T cells, (D) A/Japan/305/1957 H2N2, (E) A/Philippines/2/1982 (Phi) H3N2, (F) N7 from A/Netherlands/219/2003 H7N7, and (G) N9 from A/Shanghai/02/2013 H7N9. Error bars represent the SD of the results of triplicate samples. The red dash lines represent the values of mean + 2 × SD from the PBS group. *, **, ***, and **** indicate significant difference between the compared groups and represent p-values less than 0.05, 0.01, 0.005, and 0.001, respectively. The abbreviation ns indicates not significant.

Figure 4

Mouse challenge experiment with HK68 H3N2 virus. Body weights from five groups of mice with N2 immunities were recorded on the day of intranasal infection and on the 5th day post infection. Statistical significance between the compared groups was determined using an unpaired t-test. *, **, and *** indicate significant difference between the compared groups and represent p-values less than 0.05, 0.01, and 0.005, respectively. The abbreviation ns indicates not significant.

Figure 5

The alignment of NA amino acid sequences. The N2 head domain of rtN2 immunogen is derived from KS17 N2. The amino acid sequence of N2P2 is selected based on the consensus amino acid sequence of N2 [8] and is also identical to the corresponding peptide from KS17 N2. The HK68 H3N2 virus was used in the mouse challenge experiment. The eukaryotic expression plasmids encoding NA from PH82, JP57, NL03, and SH13 were used for transfection in cell-based ELISA.