Route of self-amplifying mRNA vaccination modulates the establishment of pulmonary resident memory CD8 and CD4 T cells

Abstract

Respiratory tract resident memory T cells (T_RM), typically generated by local vaccination or infection, can accelerate control of pulmonary infections that evade neutralizing antibody. It is unknown whether mRNA vaccination establishes respiratory T_RM. We generated a self-amplifying mRNA vaccine encoding the influenza A virus nucleoprotein that is encapsulated in modified dendron-based nanoparticles. Here, we report how routes of immunization in mice, including contralateral versus ipsilateral intramuscular boosts, or intravenous and intranasal routes, influenced influenza-specific cell-mediated and humoral immunity. Parabiotic surgeries revealed that intramuscular immunization was sufficient to establish CD8 T_RM in the lung and draining lymph nodes. Contralateral, compared with ipsilateral, intramuscular boosting broadened the distribution of lymph node T_RM and T follicular helper cells but slightly diminished resulting levels of serum antibody. Intranasal mRNA delivery established modest circulating CD8 and CD4 T cell memory but augmented distribution to the respiratory mucosa. Combining intramuscular immunizations with an intranasal mRNA boost achieved high levels of both circulating T cell memory and lung T_RM. Thus, routes of mRNA vaccination influence humoral and cell-mediated immunity, and intramuscular prime-boosting establishes lung T_RM that can be further expanded by an additional intranasal immunization.

Fig 1.. Impact of ipsilateral versus contralateral intramuscular mRNA-vaccination on CD8 T cell, CD4 T cell, and humoral immunity.

(A) Mice were primed in the right hamstring then boosted in either the right (ipsilateral) or left (contralateral) hamstring. ≥28 days post boost, spleen, iliac LN, medLN and lungs were examined. (B) Quantification of antigen-specific CD8⁺ T cells in the indicated SLOs. (C-D) Representative flow cytometry plot (C) and quantification (D) of NP366-specific CD8⁺ CD69⁺CD62L⁻ SLO T_RM. (E) Enumeration of antigen-specific CD8⁺ T cells in the indicated lung compartments. (F-G) Representative flow cytometry plot (F) and quantification (G) of NP366⁺ CD8 T cell subsets in the lung parenchyma (IV-neg). (H) Enumeration of antigen-specific CD4⁺ T cells in the indicated SLOs. (I-J) Representative flow cytometry plot (I) and quantification (J) of NP261-specific CD4⁺ T_H1 and T_FH in SLOs. (K) Quantification of antigen-specific CD4⁺T cells in the indicated lung compartments. (L-M) Representative flow cytometry plot (L) and quantification (M) of NP261-specific CD4⁺ T cell subsets in the lung parenchyma (IV-neg). (N) NP IgG specific serum antibody titers. Open and closed circles represent data from two independent experiments. (O) Ipsilaterally prime-boosted mice were challenged with Influenza A/PR8 IN. Weight relative to initial bodyweight over time (left, middle), and survival (right). Data represent N = 2 independent experiments with n = 4-5 mice per group except for (N-O) where data was pooled from N = 2 independent experiments with n = 8-10 mice. Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 as determined by one-way ANOVA and Tukey’s multiple comparisons test (for 3 groups) or unpaired two-tailed Student’s t test (2 groups) or with log-rank Mantel-Cox test for survival. N.D. = not determined.

Fig 2.. Comparing routes of immunization reveals that intranasal prime-boost vaccination induces more CD103+ CD8 T cells in the lung parenchyma.

(A) Mice were either IM, IV or IN prime-boosted and spleen, iliac LN, medLN and lungs were examined ≥28 days post boost. (B) Quantification of antigen-specific CD8⁺ T cells in the indicated SLOs. (C-D) Representative flow cytometry plot (C) and quantification (D) of NP366-specific CD8⁺ CD69⁺CD62L⁻ SLO T_RM. (E) Quantification of antigen-specific CD8⁺ T cells in the indicated lung compartments. (F-G) Representative flow cytometry plot (F) and quantification (G) of NP366⁺ CD8 T cell subsets in the lung parenchyma (IV-neg). Data represent N = 2 independent experiments with n = 4-5 mice per group. Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 as determined by one-way ANOVA and Tukey’s multiple comparisons test.

Fig 3.. mRNA vaccination generates long-lived T_FH in the draining LN but not in the lung.

(A) Mice were either IM or IN prime-boosted and spleen, iliac LN, medLN and lungs were examined ≥28 days post boost. (B) Quantification of antigen-specific CD4⁺ T cells in the indicated SLOs. (C-D) Representative flow cytometry plot (C) and quantification (D) of NP261-specific CD4⁺ T_H1 and T_FH in SLOs. (E) NP IgG specific serum antibody titers. Open and closed circles represent data from two independent experiments. (F) Quantification of antigen-specific CD4⁺ T cells in the indicated lung compartments. (G-H) Representative flow cytometry plot (G) and quantification (H) of NP261-specific CD4⁺ T cell subsets in the lung parenchyma (IV-neg). (I-J) Quantification (I) and representative flow cytometry plot (J) of NP261-specific CD4⁺ T cell subsets in the lung parenchyma (IV-neg). Data represent N = 2 independent experiments with n = 4-5 mice per group. Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 as determined by one-way ANOVA and Tukey’s multiple comparisons test (for 3 groups) or unpaired two-tailed Student’s t test (2 groups). N.D. = not determined.

Fig 4.. All mRNA immunization routes were sufficient to induce pulmonary resident memory CD8 T cells.

(A) Representative image of in situ NP366-tetramer staining in the lung upon IM prime-boost. (B) CD45.2⁺ mice were either IM or IN prime-boosted and then conjoined to CD45.1⁺ naïve hosts. After 21 days, lungs were harvested to examine recirculating and resident populations antigen-specific CD8 and CD4 T cells. (C) Representative flow cytometry plots depicting CD45.1⁺ and CD45.2⁺ CD8 T cells to demonstrate equilibration in the blood. (D-E) Representative flow cytometry plot (D) and quantification (E) of NP366-specific CD8 T cell subsets in the lung parenchyma. (F-G) Representative flow cytometry plot (F) and quantification (G) of NP261-specific CD4 T cell subsets in the lung parenchyma. Data represent N = 2 independent experiments with n = 4-5 mice per group, except for (A) which represents N = 1 independent experiment with n = 3 mice. Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001as determined by unpaired two-tailed Student’s t test.

Fig 5.. Combining intramuscular immunizations with an intranasal mRNA boost achieves high levels of both circulating memory and lung T_RM.

(A) IM prime-boosted mice received a second booster via IN route and spleen, iliac LN, medLN and lungs were examined ≥28 days post secondary boost. (B-C) Quantification of antigen-specific CD8⁺ T cells (B) and CD4⁺ T cells (C) in the indicated compartments. (D-E) Representative flow cytometry plot (D) and quantification (E) of IV-neg NP366-specific CD8⁺ T cell subsets in the lung. (F-G) Representative flow cytometry plot (F) and quantification (G) of IV-neg NP261-specific CD4⁺ T cell subsets in the lung. Data represent N = 2 independent experiments with n = 4-5 mice per group. Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 as determined by unpaired two-tailed Student’s t test.