An influenza mRNA vaccine protects ferrets from lethal infection with highly pathogenic avian influenza A(H5N1) virus

Abstract

The global spread of the highly pathogenic avian influenza (HPAI) A(H5N1) virus poses a serious pandemic threat, necessitating the swift development of effective vaccines. The success of messenger RNA (mRNA) vaccine technology in the COVID-19 pandemic, marked by its rapid development and scalability, demonstrates its potential for addressing other infectious threats, such as HPAI A(H5N1). We therefore evaluated mRNA vaccine candidates targeting panzootic influenza A(H5) clade 2.3.4.4b viruses, which have been shown to infect a range of mammalian species, including most recently being detected in dairy cattle. Ferrets were immunized with mRNA vaccines encoding either hemagglutinin alone or hemagglutinin and neuraminidase, derived from a 2.3.4.4b prototype vaccine virus recommended by the World Health Organization. Kinetics of the immune responses, as well as protection against a lethal challenge dose of A(H5N1) virus, were assessed. Two doses of mRNA vaccination elicited robust neutralizing antibody titers against a 2022 avian isolate and a 2024 human isolate. Further, mRNA vaccination conferred protection from lethal challenge, whereas all unvaccinated ferrets succumbed to infection. It also reduced viral titers in the upper and lower respiratory tracts of infected ferrets. These results underscore the effectiveness of mRNA vaccines against HPAI A(H5N1), showcasing their potential as a vaccine platform for future influenza pandemics.

Fig. 1.. H5 mRNA vaccines elicited protective immunity in ferrets.

(A) Study design. Three ferrets per group were immunized with PBS mock (group 1), 50 μg of mRNA vaccine encoding H5-WT (group 2) or H5-del (group 3), 50 and 100 μg of H5-del mRNA vaccine together with the same amount of mRNA vaccine encoding N1 NA (groups 4 and 5, respectively), or recombinant H5-del protein adjuvanted with AddaVax (group 6). animals were vaccinated intramuscularly three times. Seven weeks after the third immunization, ferrets were challenged with HPAI BE/FL/22 virus. nasal wash samples were collected on days 1, 3, 5, 7, 9, and 12 after challenge. one of the ferrets in group 6 had an experiment-unrelated issue and was euthanized in week 2 after prime vaccination. (B) HI titers were measured after vaccination using AW/SC/21 CVV antigen with 1% HRBC. (C) The body weights of ferrets were monitored for 14 days after challenge. all animals in the unvaccinated group succumbed by day 9 after infection because of severe disease progression. (D) Virus titers in nasal wash samples were determined on MDCK cells by TCID₅₀ assay. Because all animals in the unvaccinated group succumbed by day 9 after infection, no nasal wash titers on day 12 after infection are shown for this group. Data are presented as geometric mean and geometric SD [(B) and (D)] or arithmetic mean and SD (C). Dotted lines indicate the lower limit of detection. AUC analysis was used for (B) and (C), and differences were tested using a one-way ANOVA with Tukey’s post hoc test. Viral titers were analyzed by pairwise t tests with Tukey-Kramer’s adjustment for multiple comparisons on the log titers (D). Significant differences are indicated by *P < 0.05.

Fig. 2.. Selected mRNA vaccines protected ferrets from lethal challenge.

(A) Study design. Ten ferrets per group were not vaccinated (group 1) or vaccinated with 50 μg of mRNA vaccine encoding H5-WT with or without 50 μg of mRNA vaccine encoding N1 NA (groups 2 and 3) or 50 μg of recombinant H5-del protein mixed with AddaVax (group 4). One of the ferrets in group 2 was eliminated for study-unrelated reasons before virus challenge. Four weeks after the second immunization, ferrets were challenged with BE/FL/22 virus. nasal wash samples were collected on days 1, 3, 5, 7, 9, and 12 after challenge. Three animals per group were euthanized on days 3 and 6 after challenge to collect the organs for viral load quantification and histopathology analysis. (B) HI titers were measured after vaccination using AW/SC/21 CVV antigen with 0.5% TRBC (left) or 1% HRBC (right). (C) Virus neutralization antibody titers were determined by Fra using the AW/SC/21 CVV. (D) NAI titers were measured after vaccination using the recombinant NA protein of AW/SC/21. (E) Body weights were monitored for 14 days after challenge. One ferret in group 1 and one in group 3 demonstrated neurological symptoms on days 5 and 7 after challenge, respectively, and were euthanized. The averages of body weight and the SDs were calculated on the remaining three or four ferrets, excluding the ferrets used for organ sampling. (F) Virus titers in nasal wash samples were determined on MDCK cells by TciD₅₀ assay. Because all animals in the unvaccinated group succumbed by day 7 after infection, no nasal wash titers on days 9 and 12 after infection are shown for this group. Data are presented as the geometric mean with the geometric SD [(B) to (D) and (F)] or arithmetic mean with SD (E). Dotted lines indicate the lower limit of detection. AUC analysis was used for (B), (C), and (E), and differences were tested in a one-way ANOVA with Tukey’s post hoc test. Neutralizing antibody titers and viral titers were analyzed by pairwise t tests with Tukey-Kramer’s adjustment for multiple comparisons on the log titers in (D) and (F). Significant differences are indicated by *P < 0.05.

Fig. 3.. H5-based mRNA vaccines reduced viral RNA load in organs of challenged ferrets.

Organ samples were collected from three ferrets in each group on days 3 and 6 after challenge and homogenized for quantification of viral RNA. Raw copy per microliter numbers for the reaction mixture were converted to copy (cp) per microgram of tissue. Data are presented as geometric mean with geometric SD. Viral RNA copy numbers were analyzed by pairwise t tests with Tukey-Kramer’s adjustment for multiple comparisons on the log titers. Significance values between vaccinated groups and the unvaccinated groups are indicated by *P < 0.05.

Fig. 4.. Serum samples from vaccinated ferrets neutralized a human isolate related to the 2024 H5N1 outbreak in dairy cattle.

Serum samples were collected from ferrets 2 weeks after booster, corresponding to week 7 serum (Fig. 2B). Neutralizing antibody (Ab) titers were measured against the vaccine virus A/American wigeon/South Carolina/22–000345-001/2021 (AW/SC/21 CVV), the challenge virus A/bald eagle/Florida/22–006544-004/2022 (BE/FL/22), and the human isolate A/Texas/37/2024 (TX/37), using an FRA. Data are presented as geometric mean with geometric SD. Neutralizing titers were analyzed by pairwise t tests with Tukey-Kramer’s adjustment for multiple comparisons on the log titers. Significance values between the different viruses are indicated by *P < 0.05.