Supplementary Materials for

Route of self-amplifying mRNA vaccination modulates the establishment of

pulmonary resident memory CD8 and CD4 T cells
Marco Künzli et al.

Corresponding author: David Masopust, masopust@umn.edu

Sci. Immunol. 7, eadd3075 (2022)

DOI: 10.1126/sciimmunol.add3075

The PDF file includes:

Figs. S1 to S6
Tables S1 and S2

Other Supplementary Material for this manuscript includes the following:

Data file S1
MDAR Reproducibility Checklist
Fig. S1: Impact of ipsilateral versus contralateral intramuscular mRNA-vaccination on

CD8 T cell, CD4 T cell, and humoral immunity. (A) Representative flow cytometry plot of

CD8 T cell NP366+ tetramer staining in various tissues. (B) Representative flow cytometry plot

of CD4 T cell NP261+ tetramer staining in various tissues. (C-D) Representative flow cytometry

plot (C) and quantification (D) of NP261-specific CD4+ TFH identified by high expression of PD-

1 and CXCR5 in SLOs. (E) Representative flow cytometry plot of NP261+ CD4+ T cells (grape)

and endogenous IV-neg CD44+ CD4 T cell compartment (gray). 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. N.D. = not determined.

Fig S2. MDNP-encapsulated mRNA induces less acute inflammation than LNPs

composed of DLin-MC3-DMA or SM-102.

(A) BALB/cJ mice (n = 4 per group) 6-10 weeks of age were administered 6 µg of the indicated

mRNA-loaded nanoparticles (encoding secreted embryonic alkaline phosphatase [SEAP]

reporter gene) by IN instillation, and survival was monitored over a 24-hour period. (B)

Cumulative clinical score of the same animal groups shown in (A) according to the scoring

rubric set forth in Table S1. Asterisk indicates time of euthanasia for 2 animals of the DLin-

MC3-DMA group resulting in an artifactual drop in cumulative score before remaining mice

were euthanized (dashed line). Asterisk in the SM-102 group indicates time of euthanasia for

1 animal. (C) In a separate experiment modeling conventional IM vaccine administration,

circulating proinflammatory cytokines were measured by ELISA in serum of IM-injected

BALB/cJ mice (n = 3 for PBS, n = 5 for MDNP and LNP) 24 hours post-treatment with 5 µg

doses of SEAP mRNA encapsulated in MDNP or LNP composed of DLin-MC3-DMA. (D-E)

Alkaline phosphatase (D) and alanine aminotransferase (E) in BALB/cJ mice (n = 3 per group)

were measured at the indicated times post-injection with a 2X dose of the Cal/09 NP MDNP

formula used in this study. A 2X dose was selected for this experiment to maximize the

likelihood of serum ALP and ALT elevation in response to vaccination. Data are shown as

mean ± SD.
Fig. S3. Longevity of vaccine-elicited CD8 T cells in the respiratory tract.

(A-C) Quantification of total IV-neg (A), CD69+CD103- (B) and CD69+CD103+ (C) antigen-

specific CD8+ T cells in the lung, bronchoalveolar lavage fluid (BAL), nasal associated

lymphoid tissue (NALT) and trachea over time. Data are pooled from N = 3 experiments with

n = 14-15 total mice per group (3-4 mice per time point). Symbols represent individual mice

and the solid-colored lines represent a fitted linear regression with confidence intervals

(shaded area).
Fig S4. mRNA vaccination generates long-lived TFH in the draining LN but not in the

lung.

(A-B) Representative flow cytometry plot (A) and quantification (B) of NP261-specific CD4+ TFH

identified by high expression of PD-1 and CXCR5 in SLOs. (C) Representative flow cytometry

plot of NP261+ CD4+ T cells (green) and endogenous IV-neg CD44+CD69+ CD4 T cell

compartment (gray). (D) Representative flow cytometry plot of NP261-specific CD4+ T cell

subsets in the lung tissue (left) and expression of PD-1 and CXCR6 of the indicated subsets

(right). 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. (E) Quantification of IV-neg (left) and CD69+ (middle)

antigen-specific CD4+ T cells and NP-specific IgG antibody titers in BAL fluid over time.

Symbols represent individual mice and the solid-colored lines represent a fitted linear

regression with confidence intervals (shaded area). Data represent N = 2 independent

experiments with n = 4-5 mice per group except for E where data are pooled from N = 3

experiments with n = 14-15 mice per group.

Fig S5. Anatomic distribution of mRNA vaccine elicited CD8 and CD4 memory T cells.
Fig S6. Cal/09 nucleoprotein expression mediated by self-amplifying mRNA vectors in

BHK cells.

Cal/09 nucleoprotein self-amplifying RNA was synthesized by in vitro transcription and

enzymatic capping. BHK cells were transfected with 1 µg capped RNA from two

independently manufactured lots, and 24 hr later cells were lysed and subjected to

immunoblot using primary antibody IC5-1B7 followed by HRP-conjugated anti-mouse IgG

secondary. The indicated band between the 50 and 70 kDa markers corresponds to the

anticipated mass of nucleoprotein (predicted mass = ~56 kDa). After development, blots

were stripped and probed with an HRP-conjugated anti-GAPDH antibody as loading control.
 Variable Score and description
Appearance/Attitude 0- active, smooth coat
1- mild hunched posture +/- piloerection
2- moderate hunched posture, “puffy” from piloerection
3- decreased body condition (thin) +/- hunched posture, piloerection,
dehydration based on low skin elasticity from dorsal pinch test
4- Low activity level (decreased responsiveness to stimuli),
decreased body condition +/- piloerection
Respiration quality 0- Normal
1- Increased respiratory rate, normal effort
2- Increased respiratory rate, some effort (abdominal)
3- Increased abdominal effort, intermittent gasping
4- High effort, gasping

Table S1. Clinical scoring rubric for inflammation and respiratory distress.

Humane endpoints: If a mouse has either an Appearance or Respiration score of 4, the

mouse will be euthanized. If a cumulative score of >4 is observed, the mouse will be

euthanized.

MDNP Sample Diameter (nm) Polydispersity Encapsulation (%)

Index
Cal/09 NP MDNP 115 ± 5 0.06 ± 0.03 90

Table S2. Nanoparticle size, polydispersity, and encapsulation efficiency.

Average of 3 independent measurements ±SD shown for diameter and PDI as measured by

DLS.