Safety and immunogenicity of a ferritin nanoparticle H2 influenza vaccine in healthy adults: a phase 1 trial

Abstract

Currently, licensed seasonal influenza vaccines display variable vaccine effectiveness, and there remains a need for novel vaccine platforms capable of inducing broader responses against viral protein domains conserved among influenza subtypes. We conducted a first-in-human, randomized, open-label, phase 1 clinical trial ( NCT03186781 ) to evaluate a novel ferritin (H2HA-Ferritin) nanoparticle influenza vaccine platform. The H2 subtype has not circulated in humans since 1968. Adults born after 1968 have been exposed to only the H1 subtype of group 1 influenza viruses, which shares a conserved stem with H2. Including both H2-naive and H2-exposed adults in the trial allowed us to evaluate memory responses against the conserved stem domain in the presence or absence of pre-existing responses against the immunodominant HA head domain. Fifty healthy participants 18-70 years of age received H2HA-Ferritin intramuscularly as a single 20-μg dose (n = 5) or a 60-μg dose either twice in a homologous (n = 25) prime-boost regimen or once in a heterologous (n = 20) prime-boost regimen after a matched H2 DNA vaccine prime. The primary objective of this trial was to evaluate the safety and tolerability of H2HA-Ferritin either alone or in prime-boost regimens. The secondary objective was to evaluate antibody responses after vaccination. Both vaccines were safe and well tolerated, with the most common solicited symptom being mild headache after both H2HA-Ferritin (n = 15, 22%) and H2 DNA (n = 5, 25%). Exploratory analyses identified neutralizing antibody responses elicited by the H2HA-Ferritin vaccine in both H2-naive and H2-exposed populations. Furthermore, broadly neutralizing antibody responses against group 1 influenza viruses, including both seasonal H1 and avian H5 subtypes, were induced in the H2-naive population through targeting the HA stem. This ferritin nanoparticle vaccine technology represents a novel, safe and immunogenic platform with potential application for pandemic preparedness and universal influenza vaccine development.

Extended Data Fig. 1 |. Summary of 20 μg H2HA-Ferritin immunogenicity results.

Sera from H2-naïve participants who received a single dose of H2HA-Ferritin (red circles) were analyzed for vaccine-induced antibody responses. Individual participant results are displayed for (a) hemagglutination inhibition assay (HAI), (b-f) HA stem-binding ECLIAs, (g,h) reporter microneutralization assays, and (I) Fc-mediated antibody-dependent cell-mediated cytotoxicity ADCC reporter assays. n = 5 for each assay. Dotted lines indicate the lower limit of detection for each assay, arrows indicate vaccination timepoints. Negative samples were reported and calculated as half the limit of detection.

Extended Data Fig. 2 |. H2HA-Ferritin vaccination did not result in notable antibody development against human ferritin, despite developing a response to H. pylori ferritin.

Participant sera were tested by ELISA for antibodies against human ferritin (heavy and light chains) as well as H. pylori ferritin at baseline and four weeks after each vaccination. Sera from four NHPs (cynomolgus macaques) vaccinated with an influenza ferritin vaccine at weeks 0, 4, and 10 were included in the H. pylori analysis as positive controls. Arrows indicate vaccination time points.

Extended Data Fig. 3 |. Statistical comparison between groups at key timepoints following H2HA-Ferritin vaccination.

Sera from H2-naïve (blue circles) and H2-exposed (green circles) participants who received H2HA-Ferritin in either homologous (closed circles) or heterologous (open circles) prime-boost regimens were compared at select timepoints, including baseline, four weeks after the prime vaccination, two weeks after the boost vaccination (four weeks after the boost for the ADCC assay), and at 40 weeks after the prime vaccination. Results are shown for (a) hemagglutination inhibition assays (HAI), (b-f) HA stem-binding ECLIAs, (g-i) reporter microneutralization assays, (j-k) pseudotyped lentivirus neutralization assays, and (l) Fc-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assays. Values displayed represent the group geometric mean titers, with whiskers indicating the 95% confidence intervals. Dotted lines indicate the lower limit of detection. Negative samples were reported and calculated as half the limit of detection. Comparisons between treatment groups were made using two-sided two-sample t-tests, after log₁₀ (for the neutralization and ADCC assays) transformations of the raw titers. Number of participant sera analyzed at each time point is summarized in Supplementary Table 7.

Extended Data Fig. 4 |. H2HA-Ferritin vaccination induces broadly neutralizing antibodies in H2-naïve adults.

Sera from H2-naïve (blue circles) and H2-exposed (green circles) participants who received H2HA-Ferritin in either homologous (closed circles) or heterologous (open circles) prime-boost regimens were analyzed with a pseudotyped lentivirus neutralization assay. Individual results, group geometric means, and geometric mean fold increases over baseline are shown for A) H2N2 A/Singapore/1/57 and B) H5N1 A/Vietnam/1203/04. Whiskers indicate 95% confidence intervals. Dotted lines indicate the lower limit of detection, arrows indicate vaccination timepoints. Negative samples were reported and calculated as half the limit of detection. Number of participant sera analyzed at each time point is summarized in Supplementary Table 7.

Extended Data Fig. 5 |. Preexisting and vaccine-induced antibody titers against H2N2 virus compared to age.

The neutralizing antibody titers from the reporter microneutralization assay at two weeks after the boost (week 18) for H2-naïve (blue circles, n = 13) and H2-exposed (green circles, n = 9) participants that received two doses of H2HA-Ferritin were compared to the age of the participants at time of enrollment. Two-sided Spearman’s rank correlation coefficient (ρ) and p values for all age groups were ρ=0.39 and p = 0.064. For the H2-naïve adults only, the values were ρ=−0.52 and p = 0.072. For the H2-exposed adults only, the values were ρ=0.6 and p = 0.067.

Extended Data Fig. 6 |. The neutralizing H2HA-Ferritin vaccine-induced antibodies are directed against the HA stem.

Sera from H2-naïve (blue circles) and H2-exposed (green circles) participants who received H2HA-Ferritin in either homologous (closed circles) or heterologous (open circles) prime-boost regimens were evaluated by a competition microneutralization assay against homologous H2N2 A/Singapore/1/57 virus at two weeks after the boost vaccination (week 18). Competing antigens include the H2 HA stem and a negative control (DSCav-1). Lines represent group geometric means and whiskers indicate 95% confidence intervals. Dotted lines indicate the lower limit of detection for each assay.

Extended Data Fig. 7 |. The vaccine-induced antibodies display Fc-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

Sera from H2-naïve (blue circles) and H2-exposed (green circles) participants who received H2HA-Ferritin in either homologous (closed circles) or heterologous (open circles) prime-boost regimens were analyzed for Fc-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) activity. Group geometric means, geometric mean fold increases over baseline, and individual values are shown for against the recombinant H6/1N5 virus. Whiskers indicate 95% confidence intervals. Dotted lines indicate the lower limit of detection for each assay, arrows indicate vaccination time points. Number of participant sera analyzed at each time point is summarized in Supplementary Table 7.

Fig. 1 |. CONSORT diagram for the clinical trial.

Participants 18–47 years of age (born after 1969) were considered ‘H2-naive’, and those 52–70 years of age (born before 1966) were considered ‘H2-exposed’, based on potential historical exposure to H2N2 influenza. The interval between prime and boost vaccinations was 16 weeks. Participants who had altered or discontinued vaccination schedules were monitored for safety and were included in the immunogenicity analysis until their vaccination schedules were changed.

Fig. 2 |. Maximum local and systemic solicited reactogenicity.

Percent of participants (x axis) who reported a local or systemic symptom (y axis) in the 7 d after prime (week 0) or boost (week 16) vaccination. Participants 18–47 years of age (born after 1969) were considered ‘H2-naive’, and those 52–70 years of age (born before 1966) were considered ‘H2-exposed’, based on potential historical exposure to H2N2 influenza. *Local solicited symptoms of swelling or redness were not observed after vaccination.

Fig. 3 |. Vaccine-induced binding antibodies are observed by HAI assay after H2HA-Ferritin vaccination.

Sera from H2-naive (blue circles) and H2-exposed (green circles) participants who received H2HA-Ferritin in either homologous (closed circles) or heterologous (open circles) prime-boost regimens were analyzed with an HAI assay with H2N2 A/Singapore/1/57. a, Individual results, group geometric means and geometric mean fold changes over baseline are shown. b, The seroconversion rate for each group is displayed as a percentage, with the number of participants per group seroconverting included above. Whiskers indicate 95% confidence intervals. Dotted lines indicate the lower limit of detection; arrows indicate vaccination time points. Negative samples were reported and calculated as half the limit of detection. The number of participant sera analyzed at each time point is summarized in Supplementary Table 7. GMT, geometric mean titer.

Fig. 4 |. H2HA-Ferritin vaccine platform induces heterosubtypic group 1 HA stem-targeting antibodies in H2-naive adults.

Sera from H2-naive (blue circles) and H2-exposed (green circles) participants who received H2HA-Ferritin in either homologous (closed circles) or heterologous (open circles) prime-boost regimens were analyzed for HA stem-binding antibodies against both group 1 and group 2 influenza HA antigens. Individual results, group geometric means and geometric mean fold changes over baseline are shown for group 1 HA stabilized stem (ss) or full-length (FL) antigens, including H2ss (a), H5ss (b), H6FL (c), H1ss (d) and the group 2 viruses represented by H7ss (e). Whiskers indicate 95% confidence intervals. Dotted lines indicate the lower limit of detection for each assay; arrows indicate vaccination time points. The number of participant sera analyzed at each time point is summarized in Supplementary Table 7. AUC, area under the curve; GMT, geometric mean titer.

Fig. 5 |. H2HA-Ferritin vaccination induces broadly neutralizing antibodies against group 1 viruses in H2-naive adults.

Sera from H2-naive (blue circles) and H2-exposed (green circles) participants who received H2HA-Ferritin in either homologous (closed circles) or heterologous (open circles) prime-boost regimens were analyzed for neutralizing activity (IC₈₀) by a reporter-based microneutralization assay. Individual values, group geometric means and geometric mean fold changes over baseline are shown for H2N2 A/Singapore/1/57 (a) and H5N1 A/Vietnam/1203/04 (b) viruses. Whiskers indicate 95% confidence intervals. Dotted lines indicate the lower limit of detection for each assay; arrows indicate vaccination time points. Negative samples were reported and calculated as half the limit of detection. The number of participant sera analyzed at each time point is summarized in Supplementary Table 7. GMT, geometric mean titer.

Fig. 6 |. The neutralizing H2HA-Ferritin vaccine-induced antibodies are directed against the HA stem.

Sera from H2-naive (blue circles) and H2-exposed (green circles) participants who received H2HA-Ferritin in either homologous (closed circles) or heterologous (open circles) prime-boost regimens were evaluated by a competition microneutralization assay against heterologous H5N1 A/Vietnam/1203/04 virus at 2 weeks after the boost vaccination (week 18). Competing antigens include the full-length H2 HA, H2 HA stem and a negative control (DSCav-1). Lines represent group geometric means, and whiskers indicate 95% confidence intervals. Dotted lines indicate the lower limit of detection for the assay.