Single-dose avian influenza A(H5N1) Clade 2.3.4.4b hemagglutinin-Matrix-M® nanoparticle vaccine induces neutralizing responses in nonhuman primates

Abstract

With the recent rise in cases of highly pathogenic avian influenza (HPAI) A(H5N1) clade 2.3.4.4b infection in humans and animals, there is an associated increase in the risk of human-to-human transmission. In this study, we characterize a recombinant A(H5N1) A/American Wigeon/South Carolina/22/000345-001/2021 (A/AW/SC/2021) clade 2.3.4.4b vaccine. Purified recombinant A/AW/SC/2021 HA trimers formulated with Matrix-M^® adjuvant, saponin-cholesterol-phospholipid combination arranged in cage-like particles, are found to non-covalently anchor to the vertices of the Matrix-M and form A(H5N1) HA-Matrix-M nanoparticles (H5-MNPs). In naïve female mice, two intranasal (IN) or intramuscular (IM) doses of A/AW/SC/2021 H5-MNP vaccine induces robust antibody- and cell-mediated immune responses, including neutralizing antibodies against A(H5N1). In non-human primates (NHPs) primed with seasonal influenza vaccine, a single IM or IN dose of the A/AW/SC/2021 H5-MNP vaccine induces geometric mean serum A(H5N1) clade 2.3.4.4b pseudovirus neutralizing titers of 1:1160 and 1:54, respectively; above the generally accepted seroconverting neutralizing titer of 1:40. Immunization with H5-MNP vaccine induces antibody responses against conserved epitopes in the A(H5N1) HA stem, vestigial esterase subdomain, and receptor binding site. This A(H5N1) H5-MNP IN and IM vaccine is immunogenic in female rodents and NHPs as a potential A(H5N1) pandemic single-dose vaccine.

Fig. 1. Characterization of the recombinant influenza A/American Wigeon/South Carolina/22-000345-001/2021 (A/AW/SC/2021) hemagglutinin (HA) antigen.

a Linear diagram of the full-length HA from A(H5N1) clade 2.3.4.4b A/AW/SC/2021 with the polybasic cleavage site deletion ΔKRRK. The diagram shows the HA construct from N- to C-terminus including the globular head region containing the signal peptide (SP, tan), and the stalk/stem region containing the fusion peptide (FP, red), linker (orange), transmembrane domain (TMD, yellow), and cytoplasmic tail (CT, white) structural elements. b Reduced SDS-PAGE gel with Coomassie blue staining of purified recombinant A(H5N1) A/AW/SC/2021 HA and western blot using an anti-H5N1 HA primary antibody to confirm the identity of the main protein product (experiment repeated 3 times). c Representative electron micrographs of negative-stained A(H5N1) A/AW/SC/2021 HA proteins as pre-fusion HA-trimers (yellow arrows) anchored in detergent (PS80; white arrows) micelles. Negative staining TEM-2D class average images of A/AW/SC/2021 HA nanoparticles shown with one or two HA trimers associated with a detergent micelle. d Representation of a 3D-reconstructed model of A/AW/SC/2021 HA from cryoEM map at 4.2 Å (left). A molecular model for A/AW/SC/2021 HA was generated using PDB ID 6HJR as a template and superimposed into EM density to represent an intact full-length A/AW/SC/2021 HA nanoparticle. Source data are provided in a Source Data file.

Fig. 2. Structural and biophysical characterization of A(H5N1) A/AW/SC/2021 HA–Matrix nanoparticle (MNP) formation.

NS-TEM 3D reconstructions of (a) an A(H5N1) A/AW/SC/2021 HA nanoparticle and (b) a Matrix-M nanoparticle. c High-performance size-exclusion chromatography (HP-SEC) analysis of a mixture of 30 µg/mL A/AW/SC/2021 HA nanoparticles with 150 µg/mL Matrix-M at time 0 and after incubation at room temperature for 2–12 h (hr), showing the percentage of H5-MNP formation as % bound across time. d, e NS-TEM landscape characterizing H5N1 interaction with Matrix-M after co-mixing HA:Matrix-M (120 µg/mL and 75 µg/mL) and the formation of H5-MNPs. Representative micrographs are shown at T0 and T24 along with 2D class average (below) at respective time points (experiment repeated 3 times). Yellow arrows indicate HA-PS80 nanoparticles, purple arrows indicate H5-MNP formation. In (d), we show a micrograph of H5N1-nanoparticle (NP) distribution at time 0 (T0). In (e), we show a micrograph of H5N1-MNP formation at time 24 h (T24hr). f A 3D model of an H5-MNP, with Matrix-M cryoEM map shown in grayscale and H5 trimers (PDB 6HJR shown in blue) docked onto the Matrix-M to mimic the MNP formation from 2D classification. Source data are provided as a Source Data file.

Fig. 3. A/AW/SC/2021 (A/AW) H5-MNP vaccine elicits seroconverting antibody- and cell-mediated immune responses in mice when administered IM or IN.

a Female mice (n = 3–10/group) were immunized on Study Days 0 and 21 with a two-dose series of 1 μg A/AW/SC/2021 HA with 5 μg Matrix-M adjuvant intramuscularly (IM), 1 μg A/AW/SC/2021 HA with 5 μg Matrix-M adjuvant intranasally (IN), or 10 μg A/AW/SC/2021 HA with 5 μg Matrix-M adjuvant (IN). b Hemagglutinin inhibiting (HAI) antibody titers (H(k) = 7.208, n = 30 (10 for 1 μg (IM), 1 μg (IN), and 10 μg (IN)), p = 0.0272) and (c) pseudovirus neutralizing titers (H(k) = 3.254, n = 30 (10 for 1 μg (IM), 1 μg (IN), and 10 μg (IN)), p = 0.1965) against A/AW/SC/2021 were determined in serum collected on Day 34/35 (2 weeks after completion of the primary series). d Anti-A/AW IgA and IgG titers were determined in bronchoalveolar lavage (BAL) fluid on Day 34/35 (IgA: U = 8, n = 12 (6 for 1 μg (IN) and 10 μg (IN)), p = 0.1320; IgG: H(k) = 7.152, n = 17 (5 for 1 μg (IM), 6 for 1 μg (IN) and 10 μg (IN), and 3 for Placebo), p = 0.0200). Polyfunctional Triple Th1 cytokine⁺ (IFN-ɣ⁺ IL-2⁺ TNF-α⁺) CD4⁺ T-cell responses were evaluated in (e) spleen tissue (H(k) = 14.66, n = 21 (6 for 1 μg (IM), 1 μg (IN), and 10 μg (IN), and 3 for Placebo), p = 0.0021) and (f) lung tissue collected on Day 34/35 from groups that received the intranasal H5-MNP vaccine (U = 1, n = 12 (6 for 1 μg (IN) and 10 μg (IN)), p = 0.0043). Symbols represent individual data points, bars represent group geometric mean titers, error bars represent 95% confidence intervals, and the horizontal dashed line represents the assay limit of quantification (LOQ) or limit of detection (LOD) or the seroconverting threshold titer of 1:40. Differences between groups were evaluated by the Kruskal–Wallis multiple comparisons test or the Mann–Whitney U Test (two-tailed). Source data are provided as a Source Data file. Created by Microsoft^® Office Visio Web App 2025.

Fig. 4. A/AW/SC/2021 (A/AW) H5-MNP vaccine elicits seroconverting antibody- and cell-mediated immune responses when administered IM or IN in NHPs primed with qNIV.

a Rhesus macaques (nonhuman primates; NHPs) were immunized by a two-dose quadrivalent nanoparticle seasonal influenza vaccine (qNIV; 60 µg HA/strain) with 75 µg Matrix-M adjuvant (IM) on Study Days 0 and 21 (n = 10). NHPs (n = 5/group) were then administered two sequential booster doses of H5-MNP vaccine (either two IM 60 μg doses of HA or one IN dose of 240 μg HA, followed by one IN dose of 60 μg HA; all with 75 μg Matrix-M adjuvant) on Study Days 83 and 139. Hemagglutinin inhibiting (HAI) antibody titers (n = 10 on Day 0, 35, and 83, and 5 on Day 97, 139, and 153 (IM or IN)) (b) and pseudovirus neutralizing antibody titers (c) against A/AW/SC/2021 were evaluated in sera collected on Study Days indicated on the X-axis (n = 10 on Day 0, 35, and 83, and 5 on Day 97, 139, and 153 (IM or IN)). d Triple Th1⁺ CD4⁺ T-cell responses were evaluated in PBMCs collected on Study Days indicated on the X-axis (n = 10 on Day 0 and 35, 9 on Day 83, 5 on Day 97 (IM or IN), and 4 or 5 on Day 153 for IM or IN, respectively). The cells were stimulated with A/AW HA. Colored symbols represent individual data points, and bars represent group geometric mean titers. Error bars represent 95% confidence intervals, and the horizontal dashed line represents the assay limit of detection (LOD) or quantification (LOQ) or the seroconverting threshold titer of 1:40. Differences between groups were evaluated by the Mann–Whitney U Test (two-tailed). Source data are provided as a Source Data file. Created by Microsoft^® Office Visio Web App 2025.

Fig. 5. Model of A(H5N1) A/AW/SC/2021 HA and its vestigial esterase (VE) and receptor binding site (RBS) neutralizing epitopes where Novavax monoclonal antibodies bind.

The in silico molecular model of A(H5N1) A/AW/SC/2021 HA with mapping where neutralizing mAbs interact with the protein. a HA-antigen modeled with NVX.73.2 mAb (LC-light chain in blue and HC-heavy chain in purple) showing its interaction with the vestigial esterase (VE) subdomain (cyan) of A(H5N1) HA. V_HC is the heavy chain variable domain, and V_LC is the variable light chain variable domain. The modeled 73.2 mAb represents binding of critical residues in the VE subdomain (green spheres). b HA-antigen modeled with NVX.361.4 mAb (Fab-variable heavy chain in yellow and light chain in cyan) showing its interaction with the receptor binding site (RBS) of HA (in purple). The modeled 361.4 mAb represents binding of critical residues of the RBS in the head region (cyan and orange spheres) directly with HC-CDR2 and 3 (yellow). c Competitive antibody equivalent (CAE) for mAbs CR6261 (stem), NVX.73.2 (VE), and NVX.361.4 (RBS) in NHP sera collected on Study Days indicated on the X axes (both IM- and IN-immunized groups). Colored symbols represent individual animal data points, and bars represent group geometric mean titers. Error bars represent 95% confidence intervals, and the horizontal dashed line represents the assay limit of detection (LOD). Differences among groups were evaluated by Kruskal–Wallis tests (CR6261: H(k) = 22.65, n = 31 (1 Day 0, 10 Day 35, 5 Day 97 (IM or IN), and 5 Day 97 (IM or IN) samples), p = 0.0004; NVX.73.2: H(k) = 25.59, n = 30 (1 Day 0, 9 Day 35, 5 Day 97 (IM or IN), and 5 Day 97 (IM or IN) samples), p = 0.0001; NVX.361.4: H(k) = 26.86, n = 31 (1 Day 0, 10 Day 35, 5 Day 97 (IM or IN), and 5 Day 97 (IM or IN) samples), p < 0.0001). Source data are provided as a Source Data file.