Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses

Abstract

T follicular helper (Tfh) cells are required to develop germinal center (GC) responses and drive immunoglobulin class switch, affinity maturation, and long-term B cell memory. In this study, we characterize a recently developed vaccine platform, nucleoside-modified, purified mRNA encapsulated in lipid nanoparticles (mRNA-LNPs), that induces high levels of Tfh and GC B cells. Intradermal vaccination with nucleoside-modified mRNA-LNPs encoding various viral surface antigens elicited polyfunctional, antigen-specific, CD4⁺ T cell responses and potent neutralizing antibody responses in mice and nonhuman primates. Importantly, the strong antigen-specific Tfh cell response and high numbers of GC B cells and plasma cells were associated with long-lived and high-affinity neutralizing antibodies and durable protection. Comparative studies demonstrated that nucleoside-modified mRNA-LNP vaccines outperformed adjuvanted protein and inactivated virus vaccines and pathogen infection. The incorporation of noninflammatory, modified nucleosides in the mRNA is required for the production of large amounts of antigen and for robust immune responses.

Figure 1.

m1Ψ-mRNA-LNPs are translated at higher levels than naked m1Ψ-mRNAs or U-mRNA-LNPs in mice. (A) Representative IVIS image (4 h after injection) of BALB/c mice i.d. injected with 5.0 µg of naked (uncomplexed) or LNP-complexed Luc m1Ψ-mRNA. (B) Quantitation of the bioluminescent signal measured after delivery of mRNA over time. (C) Total amount of protein produced from the mRNA over time. (D) Representative IVIS image (24 h after injection) of BALB/c mice i.d. injected with 5.0 µg of Luc U- or m1Ψ-mRNA-LNPs. (E) Quantitation of the bioluminescent signal measured over time after delivery of mRNA-LNPs. (F) Total amount of protein produced from Luc U- or m1Ψ-mRNA-LNPs over time. Area under the curve was calculated as described in the Materials and methods section. Error bars are SEM.

Figure 2.

Two immunizations with HIV-1 Env m1Ψ-mRNA-LNPs elicit potent antigen-specific CD4⁺ T cell responses. Mice received i.d. immunizations of Luc or HIV Env m1Ψ-mRNA-LNPs at weeks 0 and 4. Splenocytes were stimulated with Env peptides 2 wk after the second immunization, and cytokine production by CD4⁺ T cells was assessed by flow cytometry. (A–C) Percentages of CD4⁺ T cells producing IFN-γ (A), TNF-α (B), and IL-2 (C) are shown. (D) Frequencies of combinations of cytokines produced by CD4⁺ T cells. n = 5–8 mice, and each symbol represents one animal. Experiments were repeated at least two times to achieve statistical significance. Error bars are SEM. Statistical analysis: (A–C) one-way ANOVA with Bonferroni correction, *, P < 0.05; (D) Student’s t test, *, P < 0.05 compared with Luc mRNA–immunized mice.

Figure 3.

A single immunization with PR8 HA m1Ψ-mRNA-LNPs elicits strong antigen-specific CD4⁺ T cell responses. Mice received a single i.d. injection of 30 µg of m1Ψ-mRNA-LNPs or an i.m. immunization with 1,000 hemagglutinating units (HAU) of inactivated PR8 influenza virus. Splenocytes were stimulated 10 d after immunization with HA peptides, and cytokine production by CD4⁺ T cells was assessed. (A–C) The percentages of CD4⁺ T cells producing IFN-γ (A), TNF-α (B), and IL-2 (C) were measured by flow cytometry. (D) Frequencies of combinations of cytokines produced by CD4⁺ T cells. n = 8–10 mice, and each symbol represents one animal. Experiments were repeated three times to achieve statistical significance. Error bars are SEM. Statistical analysis: (A–C) one-way ANOVA with Bonferroni correction, *, P < 0.05; (D) Student’s t test, *, P < 0.05 compared with Luc mRNA–immunized mice.

Figure 4.

Nucleoside-modified Env mRNA-LNPs elicit antigen-specific CD8⁺ T cell responses. Mice received two i.d. injections of Luc or Env m1Ψ-mRNA-LNPs. (A–C) Percentages of CD8⁺ T cells producing IFN-γ (A), TNF-α (B), and CD107a (C) were analyzed. (D) Frequencies of combinations of cytokines and degranulation marker produced by CD8⁺ T cells. Splenocytes were stimulated with Env peptides, and cytokine production by CD8⁺ T cells was assessed by flow cytometry. n = 5–8 mice, and each symbol represents one animal. Error bars are SEM. Statistical analysis: (A–C) one-way ANOVA with Bonferroni correction, *, P < 0.05; (D) Student’s t test, *, P < 0.05 compared with Luc mRNA–immunized mice.

Figure 5.

HA m1Ψ-mRNA-LNP immunization elicits strong antigen-specific B cell responses. Mice were immunized once with 30 µg of m1Ψ-mRNA-LNPs i.d. or 100 HAU of inactivated PR8 influenza virus i.m or infected with a sublethal dose (25 TCID₅₀) of live PR8 influenza virus. (A–C) HA inhibition (HAI) titers from mouse serum were determined over time. The dashed lines indicate that HAI values for the live PR8 virus infection were assayed separately. (D and E) Mice were challenged with a lethal dose of PR8 influenza virus 13 mo after immunization and weight loss (D) and survival (E) were followed. n = 4–8 mice, and each symbol represents values for one animal. Experiments were repeated at least two times. Statistical analysis: (A–B) one-way ANOVA with Bonferroni correction, *, P < 0.05; (C) Student’s t test, comparing HA m1Ψ-mRNA with Luc m1Ψ-mRNA, *, P < 0.05 for each time point.

Figure 6.

Potent Tfh cell responses are elicited by a single immunization with m1Ψ-mRNA-LNPs in mice. Mice were immunized once i.d. with 30 µg of Luc or HA m1Ψ-mRNA-LNPs, a single i.m. injection with 1,000 HAU of inactivated PR8 virus, MF59-adjuvanted recombinant PR8 HA protein, or intranasally infected with 25 TCID₅₀ of live PR8 influenza virus, and immune responses were examined 10 d after immunization (A and B). (A) Total numbers of splenic Tfh cells were determined by staining for TCR⁺CD19⁻CD4⁺CD62L⁻CXCR5⁺PD-1⁺ T cells. (B) IFN-γ, IL-4, and IL-21 transcript levels in sorted Tfh cells from PR8 HA m1Ψ-mRNA-LNP–immunized mice were determined by quantitative real-time RT-PCR. Fold induction of cytokines compared with total universal RNA is shown. (C and D) Mice were immunized with a single i.d. injection of 30 µg of HA or Env m1Ψ-mRNA-LNPs, and immune responses were examined 12 d after immunization. Percentage of IFN-γ producing CD4⁺Bcl-6⁺ Tfh-like cells was measured by flow cytometry after HA (C) or Env (D) peptide stimulation. (E) Mice were immunized with a single i.d. injection of 30 µg of PR8 HA m1Ψ-mRNA-LNPs, and rates of binding to PR8 HA were examined 2, 4, and 8 wk later by biolayer interferometry. The apparent nanomolar affinity of anti–HA antibodies, derived from the mean rates of HA-binding in polyclonal sera, is plotted for each serum sample, with lower values corresponding to higher apparent affinity. n = 5–8 mice, and each symbol represents values for one animal. Experiments were repeated at least two times to achieve sufficient numbers of values for mice in each group. Error bars are SEM. Statistical analysis: one-way ANOVA with Bonferroni correction, *, P < 0.05.

Figure 7.

Potent Tfh cell and sustained neutralizing antibody responses are elicited by a single immunization with m1Ψ-mRNA-LNPs in nonhuman primates. (A and B) Rhesus macaques were immunized with 50 µg of CH505 Env m1Ψ-mRNA-LNPs or 100 µg of Env gp140 protein + poly-ICLC adjuvant, and Tfh cell responses in draining lymph nodes were analyzed 7 d after immunization. (A) The percentage of Tfh cells (CXCR5^hiPD1^hi) among total CD4⁺ T cells in lymph nodes is shown. (B) Lymph node cells were analyzed for Env-specific Tfh cells by activation-induced marker assay. The percentage of OX40⁺CD25⁺ cells in the Tfh gate (CD4⁺CXCR5^hiPD1^hi cells) after stimulation with Env peptide pool + protein is shown. The percentage of Env-specific Tfh cells for each sample was calculated as the percentage of OX40⁺CD25⁺ in Env-stimulated conditions minus the percentage of OX40⁺CD25⁺ in unstimulated conditions. Each point represents one animal. (C) Rhesus monkeys were immunized with 50 µg of 1086C Env m1Ψ-mRNA-LNPs at weeks 0, 4, 20, and 32, and neutralization titers (expressed as the reciprocal serum dilution resulting in ID₅₀) from preimmune and week 34 sera were determined against the MW965.26 (tier 1A) and autologous Ce1086_B2 (tier 2) viruses. Each point represents one animal. (D) Rhesus macaques were immunized with 600 µg (n = 4) or 200 µg (n = 3) of ZIKV prM-E m1Ψ-mRNA-LNPs, and the antibody response was measured by PRNT against ZIKV MR-766. Points represent individual monkeys; horizontal lines indicate the mean. Statistical analysis: (A–B) two-way ANOVA with Bonferroni correction, *, P < 0.05; (D) dose groups were compared by Kruskal-Wallis test, *, P > 0.05 for all comparisons.

Figure 8.

Nucleoside modification induces superior CD4⁺ T cell responses compared with unmodified mRNA. Mice received a single i.d. injection of 30 µg of Luc m1Ψ-mRNA-LNPs, HA U-mRNA-LNPs, HA m1Ψ-mRNA-LNPs or an i.m. immunization with 1,000 HAU of inactivated PR8 influenza virus. Splenocytes were stimulated 10 d after immunization with HA peptides and cytokine production by CD4⁺ T cells was assessed. (A–C) The percentage of CD4⁺ T cells producing IFN-γ (A), TNF-α (B), and IL-2 (C) was measured by flow cytometry. (D) Frequencies of combinations of cytokines produced by CD4⁺ T cells. n = 5–10 mice, and each symbol represents one animal. Experiments were repeated at least two times to achieve statistical significance. Error bars are SEM. Statistical analysis: (A–C) one-way ANOVA with Bonferroni correction, *, P < 0.05; (D) Student’s t test, *, P < 0.05 compared with Luc mRNA–immunized mice.

Figure 9.

A single immunization with m1Ψ HA mRNA-LNPs induces more potent Tfh cell responses, higher splenic GC B and plasma cell numbers, and higher HAI titers compared with unmodified mRNA-LNPs. Mice were immunized with a single i.d. injection of 30 µg of Luc m1Ψ-mRNA-LNP, HA U- or m1Ψ-mRNA-LNPs, or a single i.m. immunization of 1,000 HAU of inactivated PR8 virus, and immune responses were examined 10 d later. (A) The total number of splenic Tfh cells was counted by staining for TCRβ⁺CD19⁻CD4⁺CD62L⁻CXCR5⁺PD-1⁺ T cells. (B) Percentage of IFN-γ producing CD4⁺Bcl6⁺ Tfh-like cells was measured by flow cytometry. (C and D) Total numbers of splenic GC B cells (C) and plasma cells (D) were determined. (E) HAI titers from mouse serum were determined 10 d after immunization. n = 5–10 mice, and each symbol represents values for one animal. Experiments were repeated at least two times to achieve statistical significance. Error bars are SEM. Statistical analysis: one-way ANOVA with Bonferroni correction, *, P < 0.05.

Figure 10.

mRNA-LNPs provide potent adjuvant activity in a protein subunit vaccine. Mice were immunized with a single i.m. injection of 10 µg of recombinant PR8 HA protein alone or in combination with 30 µg of Luc U- or m1Ψ-mRNA-LNPs. (A and B) Total number of splenic Tfh cells (A) and GC B cells (B) were determined 12 d after immunization. (C) HAI titers were measured 4 wk after immunization. Error bars are SEM. n = 5 mice. Statistical analysis: one-way ANOVA with Bonferroni correction, *, P < 0.05.