An intranasal adjuvanted, recombinant influenza A/H5 vaccine candidate induces broad priming against diverse influenza A/H5N1 virus clades in a phase I randomized trial in healthy adults

Abstract

We conducted a randomized, controlled phase I trial (NCT05397119) of a novel adjuvanted recombinant influenza A/H5 (A/Indonesia/05/2005, clade 2.1) hemagglutinin vaccine, administered intranasally in two doses 28 days apart at three antigen levels. Control groups received unadjuvanted recombinant H5 or formulation buffer placebo. Six months later, participants received a heterologous unadjuvanted inactivated influenza A/H5N1 (A/Vietnam/1203/2004, clade 1) vaccine intramuscularly. All vaccines were safe and well tolerated. After the primary intranasal series, serum hemagglutination inhibition and microneutralization responses were minimal. Increases in mucosal and serum IgG/IgA, serum surface plasmon resonance antibody binding, memory B cell and CD4 T cell activity, and antibody-dependent cell-mediated cytotoxicity were observed only in recipients primed intranasally with adjuvanted H5 vaccine. Following the inactivated H5N1 boost, robust responses across all immune assays, as well as microneutralization responses against diverse H5N1 clades (including currently circulating clade 2.3.4.4b), occurred in adjuvanted vaccine recipients, demonstrating successful priming and broad responses.

Figure 1. Consort diagram

Forty participants were enrolled and received the first vaccination. The withdrawal of one participant (Group E) before the second vaccination was not related to any safety event. A second participant (Group D) left the study area after receiving the second vaccination and could not complete the trial. A third participant (Group E) did not receive the third vaccination and was lost to follow up. He did not respond to multiple inquiries from the study team, but his emergency contact informed the study team that the participant was well.

Figure 2. Hemagglutination inhibition, microneutralization, and surface plasmon resonance by vaccine strain and group

Antibody responses to influenza A/H5N1 A/Indonesia/05/2005 (clade 2.1) and A/Vietnam/1203/2004 (clade 1) at baseline, Day 57 (28 days post-second intranasal vaccination), Day 197 (immediately before intramuscular H5N1 IIV boost), and Day 225 (28 days post intramuscular IIV boost). Individual serum HAIs (A) and microneutralization (B) titers are shown with lines connecting the geometric mean titers at each timepoint with 95% confidence intervals. Individual Surface Plasmon Resonance (SPR) responses shown with connecting mean and standard deviations (C). HAI assays were also conducted with specimens from Day 204 (seven days post intramuscular IIV boost). For the HAI assay, sera that were negative at the initial dilution were assigned a titer of 5. For the MN assay, sera that were negative at the initial dilution were assigned a titer of 10. Dotted lines in panels A and B show the 1:40 dilution. Low-dose (Group A), medium-dose (Group B) and high-dose (Group C) of rH5-NE are shown in maroon, orange and yellow. Controls, including the unadjuvanted rH5 (Group D) and placebo (Group E) are shown in cyan and gray.

Figure 3. Microneutralization on Day 225 by A/H5N1 virus panel and group

Sera from Day 225 (28 days post intramuscular IIV boost) were tested by MN assay using a panel of the following viruses: A/Indonesia/5/2005 (clade 2.1), A/Vietnam/1194/2004 (clade 1), A/Anhui/1/2000 (clade 2.3.4), A/Egypt/3072/2010 (clade 2.2.1), A/Turkey/15/2006 (clade 2.2), and A/Wigeon/sc/22/2021 (clade 2.3.4.4b). Sera that were negative at the initial dilution were assigned a titer of 10. Individual values shown with geometric mean titers indicated by horizontal bar with 95% confidence intervals. Samples from the placebo group were not tested against the broader panel due to low MN titers against the vaccine strains (ND, not done).

Figure 4. H5N1 clade 2.1 serum and nasal wash binding antibodies by group

Panel A shows the geometric mean concentration with 95% confidence intervals of H5 A/Indonesia (Clade 2.1)-specific serum IgG and IgA responses, as well as H5 stalk-specific IgG responses (EU/mL). Panel B shows nasal wash responses (IgG and IgA) as the median and interquartile range of the ratio of H5-specific IgG or IgA (EU/μg) to total IgG or IgA at each timepoint per group. Inset shows nasal wash responses on an extended y axis.

Figure 5. Antibody-dependent cell-mediated cytotoxicity (ADCC), memory B cell and memory T cell responses by group

Panel A shows the induction of antibodies with ADCC capacity as fold-changes over Day 1. At the bottom of the panel, in blue text, we show the percentage of volunteers that seroconverted (SC) at each timepoint in every group. SC was defined as a 4-fold increase in ADCC titers compared to Day 1 and the dotted line indicates this threshold. Panel B shows the frequency of memory B cells producing anti-H5 IgG antibodies, reported as SFU per 1×10e6 cells. The median and 95% CI are shown. Each dot represents an individual. Panel C shows the frequency of memory CD4 T cells producing IL-2 (net %) upon ex-vivo stimulation with an H5 peptide pool (A/Vietnam/1203/2004 (clade 1)). D shows data from IFN-γ producing cells. In C and D, the data are shown in violin plots denoting the distribution of the data. Each dot represents one individual. In plots A-D data from volunteers vaccinated with the low-dose (Group A), middle-dose (Group B) and high-dose (Group C) of rH5-NE is shown in maroon, orange, and yellow colors. Controls, including the unadjuvanted rH5 (Group D) and placebo (Group E) are shown in cyan and gray colors. Panel E shows the ability of rH5-NE (pooled Groups A-Cs) to induce multifunctional (MF) cells at each timepoint of the study. Data are shown in violin plots, and each dot represents one volunteer. MF cells (IL-2+ & IFN-γ+) are shown by the white circles, IFN-γ-only producing cells are shown by the blue circles and IL-2-only producing cells are shown by the grey circles. IL-2-only and IFN-γ-only cells are referred to as Single Functional (SF) cells. Panel Fdisplays the frequency of MF and SF cells by Groups A-C at days 57 (post-intranasal vaccination) and 255 (post-systemic boost). The data in panel D is presented as percentage of the mean of MF and SF cells. White, blue, and gray areas of the pie show IFN-γ-only, IL-2-only and MF cells, respectively. The yellow semicircle shows the added percentage of SF cells (IL-2-only plus IFN-γ-only). Statistics from panels B-E are derived from Wilcoxon signed-rank tests *p<0.05, **p<0.01, ***p<0.005, **** p<0.0001.

Figure 6. Immediate and seven-day solicited symptoms (excluding immediate events) after the first and second intranasal vaccination by group

There were no reports of the following solicited adverse events after intranasal vaccination: bleeding from the nose, difficulty with hearing, double vision, joint pain, ringing in ears, slurred speech, swelling around the eyes, tightness in the chest, or wheezing. There were no events of severity greater than moderate.