Chimeric hemagglutinin-based universal influenza mRNA vaccine induces protective immunity and bone marrow plasma cells in rhesus macaques

Abstract

A universal influenza vaccine that elicits a strong and lasting stalk-specific antibody response is advantageous. We utilize nucleoside-modified mRNA in lipid nanoparticles (mRNA-LNP) and unmodified self-amplifying mRNA in modified dendritic nanoparticles (sam-MDNP), expressing chimeric hemagglutinin (cHA) antigens to induce stalk-specific humoral immunity in non-human primates with pre-existing influenza virus immunity. mRNA-LNP immunization induces strong stalk-specific binding antibodies capable of protecting mice from lethal heterologous influenza virus challenges and bone marrow plasma cells (BMPCs) that persist for up to 8 months. sam-MDNP vaccine induces lower humoral immunity, despite showing strong innate activation. Transcriptomic and cytokine analyses reveal a more persistent induction of interferon responses, interleukin (IL)-1β signaling, and IL-6 production in the mRNA-LNP group, correlating with the induction of serum antibody responses and BMPCs. These results identify a transcriptional signature associated with induction of BMPCs following mRNA vaccination and highlight the utility of cHA-based mRNA-LNP vaccines in inducing persistent stalk-directed protective antibody responses.

Keywords: bone marrow plasma cells; chimeric hemagglutinin; mRNA-LNP; rhesus macaques; self-amplifying mRNA; transcriptional signatures; universal influenza vaccine.

Graphical abstract

Figure 1

Chimeric H8 (cH8/1) mRNA-LNP immunization results in robust activation of the innate immune response (A) Vaccine schematic. (B) Representative gating strategy for monocyte and DC subsets. (C) Frequency of classical monocytes and intermediate monocytes. (D) Expression of CD80 on classical monocytes and intermediate monocytes. (E) Frequency of pDCs, CD11c myeloid DCs, BDCA1+, BDCA1+BDCA3+, BDCA3+, and BDCA1−BDCA3− DCs. (F) Expression of CD80 on pDCs, CD11c myeloid DCs, BDCA1+, BDCA1+BDCA3+, BDCA3+, and BDCA1−BDCA3− DCs. (G) Representative gating strategy for NK cells and CD69 expression. (H) Frequency of NK cells and CD69 expression on NK cells. (I) Representative gating strategy for neutrophils. (J) Frequency of neutrophils. n = 5 biological replicates. Statistical significance defined as a p value of < 0.05.

Figure 2

cH8/1 and cH5/1 mRNA-LNP immunization significantly enhance HA stalk-specific antibody responses (A) Representative ELISpot responses for total IgG, cH8/1, cH5/1, and cH6/1 from blood (in triplicate). (B) Longitudinal cH8/1-specific plasmablast responses. (C) Longitudinal cH5/1-specific plasmablast responses. (D) Longitudinal cH6/1-specific plasmablast responses. (E) Longitudinal serum-binding antibody responses against cH8/1. (F) Longitudinal serum-binding antibody responses against cH5/1. (G) Longitudinal serum-binding antibody responses against cH6/1. (H) Longitudinal serum-binding antibody responses against headless HA. (I) Longitudinal HAI titers against influenza virus strains. (J) Top panel: matrix of negative-stain EM reconstructions of pAbs in complex with recombinant H1 (A/California/04/09) from each RM at all time points listed. Due to limited particle representation, Fab graphics with dashed outlines are predicted placements. For samples with immune complexes in low abundance, example 2D class averages with labels are shown. Bottom panel: summary of epitopes targeted by pAbs. Each circle represents a Fab specificity from the corresponding RM and time point. (NA, not available; ND, not detectable). (K) Blocking of MEDI8852 by mRNA-LNP hyperimmune serum from 38, 48, and 64 weeks post-vaccination. See also Figure S1. n = 5 biological replicates. Statistical significance defined as a p value of < 0.05.

Figure 3

cH8/1 immunization induces expansion of cTfh and non-Tfh in the blood and results in influenza-specific BMPC and MBC (A) Representative CXCR5+ and CXCR5- CD4 T cells at week 0 and week 1 post-cH8/1 immunization. (B) Longitudinal CXCR5+ and CXCR5− CD4 T cell frequencies post-cH8/1 immunization. (C) Boolean pie graphs of CCR4+/CCR6/CXCR3 on Ki-67+ CXCR5^+/− CD4s week 0 and week 1 post-cH8/1 immunization. (D) CXCR3 expression on Ki-67+ CXCR5+ and CXCR5− CD4 T cells week 0 and week 1 post-cH8/1 immunization. (E) Representative ELISpot responses for total IgG, cH8/1, cH5/1, and cH6/1 from bone marrow of mRNA-LNP-immunized RMs (in triplicate). (F) Longitudinal cH8/1 BMPC frequencies. (G) Longitudinal cH5/1 BMPC frequencies. (H) Longitudinal cH6/1 BMPC frequencies. (I) Representative ELISpot responses for total IgG, cH8/1, cH5/1, and cH6/1 from PBMCs of mRNA-LNP-immunized RMs (in triplicate). (J) Longitudinal cH8/1-specific MBC frequencies. (K) Longitudinal cH5/1-specific MBC frequencies. (L) Longitudinal cH6/1-specific MBC frequencies. See also Figure S2 and Table S1. n = 5 biological replicates. Statistical significance defined as a p value < 0.05.

Figure 4

Chimeric mRNA-LNP serum completely protects mice against lethal influenza challenge (A) Passive antibody transfer schematic. (B) Weight loss of mice passively immunized with serum from naive RMs, week 14 serum (8 weeks post-QIV boost), week 38 serum (2 weeks post-cH5/1 immunization), week 48 (12 weeks post cH5), and week 64 serum (7 months post-cH5/1 boost) and challenged with A/Netherlands/602/2009. (C) Survival curve of mice passively immunized with serum from naive RMs, week 14 serum, week 38 serum, week 48, and week 64 serum and challenged with A/Netherlands/602/2009. (D) Weight loss of mice passively immunized with serum from naive RMs, week 14 serum, week 38 serum, week 48, and week 64 serum and challenged with cH6/1N5. (E) Survival curve of mice passively immunized with serum from naive RMs, week 14 serum, week 38 serum, week 48, and week 64 serum and challenged with cH6/1N5. See also Figure S3. n = 5 mice/group. Statistical significance defined as a p value < 0.05.

Figure 5

sam-MDNP vaccine responses (A) sam-MDNP vaccine schematic. (B) Longitudinal CD80 expression on intermediate monocytes, CD11c+ myeloid DCs, BDCA1+BDCA3+ mDCs, and BDCA3+ mDCs. (C) Longitudinal serum-binding antibody responses against cH8/1. (D) Longitudinal serum-binding antibody responses against cH5/1. (E) Longitudinal serum-binding antibody responses against cH6/1. (F) Longitudinal serum-binding antibody responses against headless HA. (G) Longitudinal expression of ICOS on total Ki-67+ CD4 T cells, CXCR5+ Ki-67+ CD4-T cells, and CXCR5- Ki-67+ CD4 T cells. (H) CXCR3 expression on Ki-67+ CXCR5+ and CXCR5− CD4 T cells week 0 and week 1 post-cH8/1 immunization. (I) IFNγ+ CD4 T cell responses before and after cH8/1 and cH5/1 immunization. (J) IFNγ+ CD8 T cell responses before and after cH8/1 and cH5/1 immunization. (K) Longitudinal cH6/1 MBC frequencies. (L) Longitudinal cH6/1 BMPC frequencies. See also Figures S4 and S5 and Table S1. n = 5 biological replicates. Statistical significance defined as a p value < 0.05.

Figure 6

Immune activation post-sam-MDNP immunization peaked at day 1 post-immunization and subsided rapidly, while activation in the mRNA-LNP immunized group was more persistent (A) Vaccine schematic. (B) Principal-component analysis (PCA) for mRNA-LNP and sam-MDNP groups day 0–7 post-cH8/1 immunization. (C) Number of upregulated and downregulated genes days 0, 1, and 2 post-cH8/1 immunization. (D) Gene ontology enrichment analysis (GSEA) of differentially altered pathways at day 1 (D1) and day 2 (D2) post-cH8/1 immunization. (E) Heatmaps of the IFNα response, IL-6/JAK/STAT3 signaling, and E2F targets genes at day 1 and day 2 post-cH8/1 immunization. (F) Longitudinal gene expression of IRF7, IRF9, ISG15, MX1, and IFIT2. (G) Longitudinal gene expression of IL-1β, IL-1R1, IFNGR1, CXCL10, and IL-15RA. (H) Plasma concentration of IL-6, IL-1RA, and CXCL10. See also Figure S6. n = 5 biological replicates. Statistical significance defined as a p value < 0.05.

Figure 7

Induction of type I and II interferon pathways is important for persistent BMPC responses (A) Association between humoral immune responses and specific transcriptional profiles day 1 and 2 post-cH8/1 immunization. (B) Correlation between IL-6 and IL-1ra protein expression and cH6/1-specific peak plasmablasts, binding antibody, and BMPC responses. (C) Graphical representation of genes that correlated with vaccine responses and over-representation analysis (ORA) of the pathways most represented by those correlated genes. (D) List of pathways that significantly correlated with cH6/1-specific plasmablast, binding antibody, and BMPC responses without prior down-selection. n = 5 biological replicates. Statistical significance defined as a p value of < 0.05.