A multivalent nucleoside-modified mRNA vaccine against all known influenza virus subtypes

Abstract

Seasonal influenza vaccines offer little protection against pandemic influenza virus strains. It is difficult to create effective prepandemic vaccines because it is uncertain which influenza virus subtype will cause the next pandemic. In this work, we developed a nucleoside-modified messenger RNA (mRNA)-lipid nanoparticle vaccine encoding hemagglutinin antigens from all 20 known influenza A virus subtypes and influenza B virus lineages. This multivalent vaccine elicited high levels of cross-reactive and subtype-specific antibodies in mice and ferrets that reacted to all 20 encoded antigens. Vaccination protected mice and ferrets challenged with matched and mismatched viral strains, and this protection was at least partially dependent on antibodies. Our studies indicate that mRNA vaccines can provide protection against antigenically variable viruses by simultaneously inducing antibodies against multiple antigens.

Fig. 1.. 20-HA mRNA-LNP vaccine elicits long-lived antibody responses that react to all 20 HAs.

(A) Mice were simultaneously vaccinated i.m. with 20 different HA mRNA-LNPs (a combined total dose of 50 μg mRNA-LNP including 2.5 μg of each individual HA mRNA-LNP). Other groups of mice were vaccinated i.m. with 50 μg of H1 mRNA-LNP (B), 50 μg of H3 mRNA-LNP (C), 50 μg of IBV HA mRNA-LNP (D), or PBS (E). Sera were collected 28 days (A to E) or 118 days (F) later and antibody reactivity to different HAs were quantified using ELISAs coated with recombinant proteins. Seven or eight mice were included for each experimental group and in some instances data points overlap. Group 1 HAs are shown in blue and group 2 HAs are shown in red. AUC; area under the curve. Data are representative of two independent experiments and are shown as mean ± SEM. Raw ELISA data curves are shown in fig S2.

Fig. 2.. 20-HA mRNA-LNP vaccine elicits diverse antibodies targeting both conserved and variable epitopes.

Serum samples were collected from mice 28 days after (A) H1, (B) H3, (C) IBV, or (D) 20-HA mRNA-LNP vaccination. Samples were absorbed with magnetic beads coupled to recombinant H1, H3, or no HA (mock), and antibody levels remaining in the unabsorbed fraction were quantified by ELISA (A to D). Focus reduction neutralization tests (FRNT) were completed using (E) A/Michigan/45/2015 H1, (F) A/Singapore/INFIMH-16-0019/2016 H3, (G) A/Vietnam/1203/2004 H5, or (H) A/Shanghai/02/1013 H7. Titers are reported as the inverse of the highest dilutions of serum amount required to inhibit 50% of virus infection are reported. HA stalk-reactive antibodies were quantified by ELISA using “headless” (I) group 1 (H1) and (J) group 2 (H3) recombinant proteins. AUC; area under the curve. (A to H) Six mice were included for each experimental group. (I and J) Twelve mice were included for each experimental group. Data are representative of two or three independent experiments and are shown as mean ± SEM. Data in panels A to D are shown as mean. Data in panels E to H are shown as geometric mean ± 95% CI and were compared using a one-way ANOVA with Tukey’s post-test on log-transformed values. *P<0.05.

Fig. 3.. The 20-HA mRNA-LNP vaccine protects mice from challenge with antigenically matched and mismatched distinct H1N1 strain.

Mice were vaccinated with mRNA-LNPs encoding H1 (blue), H3 (red), IBV (gray), luciferase (green) or 20 HAs (purple) and then 28 days later they were infected i.n. with A/California/7/2009 (5 LD50) or A/Puerto Rico/8/1934 H1N1 (2 LD50) influenza virus respectively. (A) Weight loss, (B) clinical scores, and (C) survival were monitored for 14 days following A/California/7/2009 infection. Virus levels in lung homogenate samples isolated 2 (D) and 5 (E) days following infection were quantified using TCID₅₀ assays. (F) Weight loss, (G) clinical scores, and (H) survival were monitored for 14 days following A/Puerto Rico/8/1934 H1N1 infection. Virus levels in lung homogenate samples isolated 2 (I) and 5 (J) days following infection were quantified using TCID₅₀ assays. Horizontal dotted lines in D, E, I, and J denote limit of detection of the assay and samples with no detectable titers were assigned a titer at this limit of detection. Five mice were included per group. Data in A, B, F, and G are shown as mean ± SEM and were analyzed by mixed model ANOVA with Geisser-Greehouse correction and Sidak’s multiple comparisons. For animals that died, weight on day prior to death was carried forward for statistical analyses. Differences compared to Luciferase mRNA vaccination are indicated in A, B, F, and G; *P<0.05. Data in C and H were analyzed using a log-rank test *P<0.05. Data in D, E, I, and J are shown as mean ± 95% CI and titers were compared using a one-way ANOVA with Tukey’s post-test; * P <0.05.

Fig. 4.. 20-HA mRNA-LNP vaccination protects ferrets from challenge with an antigenically distinct H1N1 strain.

Ferrets were primed with 60 μg of the 20-HA mRNA-LNP vaccine (3 μg of each HA mRNA-LNP) and then boosted with the same vaccine dose 28 days later. (A) Sera were collected 28 days after the first and second vaccinations and antibody reactivity to different HAs were quantified using ELISAs coated with recombinant proteins. Twenty-eight days after the second vaccination, ferrets were infected i.n. with 10⁶ TCID₅₀ of A/Ruddy turnstone/Delaware/300/2009 H1N1 influenza virus. As a control, unvaccinated animals were also infected with the virus. (B) Weight loss (C) survival and (D) signs of disease were monitored for 14 days following infection. The same animal can make multiple contributions to the graph in (D). (E) Virus levels in nasal wash samples isolated 1-7 days following infection were quantified using TCID₅₀ assays. Horizontal dashed line indicates limit of detection. 4 ferrets were included for each experimental group. Data shown are means ± SEM (A, B, and E). Data in B and E are shown as mean ± SEM and were analyzed by mixed model ANOVA with Geisser-Greehouse correction and Sidak’s multiple comparisons; * P <0.05. For animals that died, weight on day prior to death was carried forward for statistical analyses. Data in C were analyzed using a Mantel-Cox log-rank test.