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

Marco Künzli; Stephen D O’Flanagan; Madeleine LaRue; Poulami Talukder; Thamotharampillai Dileepan; J Michael Stolley; Andrew G Soerens; Clare F Quarnstrom; Sathi Wijeyesinghe; Yanqi Ye; Justine S McPartlan; Jason S Mitchell; Christian W Mandl; Richard Vile; Marc K Jenkins; Rafi Ahmed; Vaiva Vezys; Jasdave S Chahal; David Masopust

doi:10.1126/sciimmunol.add3075

. Author manuscript; available in PMC: 2023 Dec 11.

Published in final edited form as: Sci Immunol. 2022 Dec 2;7(78):eadd3075. doi: 10.1126/sciimmunol.add3075

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

Marco Künzli ^1,^*, Stephen D O’Flanagan ¹, Madeleine LaRue ¹, Poulami Talukder ³, Thamotharampillai Dileepan ¹, J Michael Stolley ¹, Andrew G Soerens ¹, Clare F Quarnstrom ¹, Sathi Wijeyesinghe ¹, Yanqi Ye ², Justine S McPartlan ³, Jason S Mitchell ¹, Christian W Mandl ³, Richard Vile ⁴, Marc K Jenkins ¹, Rafi Ahmed ², Vaiva Vezys ¹, Jasdave S Chahal ³, David Masopust ^1,^†

PMCID: PMC9832918 NIHMSID: NIHMS1861568 PMID: 36459542

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 lung and draining lymph nodes. Contralateral, compared to 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. Of note, 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.

One Sentence summary:

Intramuscular mRNA vaccination induces pulmonary resident memory T cells that can be further expanded with intranasal boosting.

Introduction

Despite the successful control and in some cases eradication of numerous infectious diseases, classical vaccination approaches have failed to eliminate endemic pathogens such as HIV, malaria, tuberculosis, and influenza, highlighting a need for innovative vaccine design. To combat the emergent threat of SARS-CoV-2, novel mRNA vaccine technologies were rapidly developed, adopted, and shown to prevent severe disease outcomes (1-4).

Standard immunization with a SARS-CoV-2 mRNA vaccine includes two intramuscular (IM) immunizations at 21 (BNT162b2) or 28 day (mRNA-1273) intervals, followed by one or more additional boosts five months later. Advantages of mRNA vaccination include the relatively potent immunogenicity for CD8 T cells as well as CD4 T cells and antibodies, the absence of vector immunity which permits homologous boosting, and the rapidity of production at scale. mRNA vaccines are being considered for diverse disease conditions, including universal influenza vaccines and cancer, both of which likely depend on regional cell-mediated immunity (5, 6). This furthers the impetus to better characterize the differentiation and distribution of memory CD8 and CD4 T cells after mRNA vaccination.

mRNA vaccine-elicited T cells have been proposed to contribute to protection upon infection of SARS-CoV-2 variants of concern (7, 8). In contrast to neutralizing antibodies, T cells recognize partially conserved epitopes and hence may not be completely evaded by mutations in the spike protein of SARS-CoV-2. Similarly, T cells recognize epitopes in the nucleoprotein of influenza viruses that may be conserved despite antigenic drift and antigenic shift, unlike neutralizing antibody targets on hemagglutinin (HA) and neuraminidase (NA) (9, 10). However, mRNA vaccine-elicited T cells have been best characterized in blood, and remain underexplored in the lung, the local site of influenza and SARS-CoV-2 infections. Indeed, even in mouse studies, little work has been done on antigen-specific T cells in tissues, including their migration properties, following mRNA vaccination.

Unlike circulating memory T cell populations that patrol blood and lymph, resident memory T cells (T_RM) are the predominant surveyors of nonlymphoid tissues and accelerate pathogen control in the event of local infection (11, 12). Upon reactivation, CD8⁺ T_RM are poised for rapid cytotoxic and innate-like ‘sensing and alarm’ functions which establish an antiviral tissue microenvironment that is further supported by the IFN-γ production of CD4⁺ T_H1 TRM (13-16). In addition, lung resident CD4⁺ T follicular helper cells (T_FH) support local antibody production after heterosubtypic reinfection (15, 17). T_RM have also been identified in lung-draining lymph nodes (LNs), and local LN memory T cells may make independent contributions to respiratory immunity (18, 19). Because of the known protective role of pulmonary T_RM in mice and humans, whether mRNA prime-boost vaccination elicits bona fide T_RM in the lung or regional LNs is clinically relevant (16, 19-35). Induction of pulmonary T_RM might represent a supplementary strategy to bolster the efficacy of vaccines designed to elicit neutralizing antibodies or to induce protective immunity in immunocompromised individuals that have an impaired ability to generate antibodies (16, 20, 22, 24, 36). However, previous reports indicate that local antigen expression within the lung is required to establish pulmonary T_RM (20, 23, 37-41). Therefore, IM mRNA vaccinations may not establish T_RM in the respiratory mucosa.

Here, we characterized how antigen-specific CD8 and CD4 T cell and serum antibody responses related to immunization routes after vaccination with modified dendron-based nanoparticle (MDNP)-encapsulated self-amplifying mRNA encoding an influenza nucleoprotein (NP). RNA encapsulation in MDNPs is driven by charge-based interactions between the ionizable dendritic material and the RNA phosphate backbone, and hydrophobic interactions with additional lipid excipients. This is in contrast to ionizable lipids currently used in the clinic, which are monocationic. In comparison to previously-reported lipid nanoparticles (LNPs) used for self-amplifying RNA, the vaccine platform used in this study allows delivery of greater masses of self-amplifying mRNA per unit mass of delivery compounds with minimal acute reactogenicity and systemic toxicity (42).

Results

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

To assess memory T cell populations after mRNA vaccination, we primed C57BL/6J mice with 5 μg of mRNA encoding the Cal/09 influenza nucleoprotein (NP) in the right hamstring. ≥28 days later, we administered an additional 5 μg dose of mRNA. To examine whether the administration site of the booster impacted the regionalization of the memory T cell response, we boosted in either the right (ipsilateral) or the left (contralateral) hamstring (Fig. 1A). We used MHC I (H-2K^b NP_366-375) and MHC II (I-A^b NP_261-277) tetramers to track antigen-specific CD8 and CD4 T cell responses in various tissues ≥28 days after the last immunization (Fig S1 A-B) (43). Compared to a single immunization, both ipsilateral and contralateral boosting increased the number of H-2K^b NP_366-375⁺ CD8 memory T cells in the spleen (Fig. 1B). Contralateral immunization was required for establishing memory CD8 T cells in the contralateral LN (Fig. 1C-D). This unexpected result might be explained by the lack of CD62L expression on most memory CD8 T cells (Fig. 1C). We also observed that IM immunization induced a trackable extravascular (IV-neg) CD69⁻ and CD69⁺ H-2K^b NP_366-375⁺ CD8 T cell population in the lung that was bolstered upon boosting (Fig 1E-G).

mRNA prime-boost vaccination also induced regionalization of antigen-specific memory CD4 T cells (Fig 1H). We used the marker combinations Ly6C⁺FR4^lo and Ly6C⁻FR4^hi to define T_H1 and T_FH memory subsets, respectively (44-46). T_H1 memory cells were mostly restricted to the spleen, whereas long-lived T_FH were essentially exclusive to the draining LNs (Fig 1 I-J, Fig S1 C-D). Spleen contained a population of cells expressing intermediate levels of FR4 (FR4^int). FR4 is also a known T_reg marker, however FR4^int I-A^b NP_261-277 tetramer⁺ CD4 T splenocytes do not express Foxp3 (47) (Fig. S1E). IM prime-boost vaccination also induced I-A^b NP_261-277 tetramer⁺ CD4 memory T cells in the lung parenchyma (Fig 1 K-M). We next investigated the humoral response and found that NP-specific IgG titers were higher in ipsilaterally boosted mice compared to both unboosted and contralateral boosted mice (Fig. 1N). These data indicate that the side of boosting impacts the quality of the immune response, where contralateral boosting established CD69⁺ CD8 and CD4 T_FH memory T cells in the left iliac LN, but ipsilateral boosting generated slightly higher antibody levels.

We next tested whether ipsilateral prime-boost IM vaccination conferred protection. Mice were IM prime-boost vaccinated, rested for 38 or 39 additional days, depending on the experiment, then anesthetized with ketamine/xylazine. Mice were then challenged intranasally (IN) with influenza A/PR8. Weight loss and survival were compared alongside contemporaneously challenged age-matched naïve C57BL/6J mice. Cal/09-NP mRNA vaccinated mice were largely protected from lethal influenza A/PR8 challenge (Fig. 1O).

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

Human mRNA vaccination is administered by IM injection, though immunization via alternative routes may be an approach to induce specific immunological outcomes (48). Conventional RNA lipid nanoparticles (LNPs) cause material-induced inflammatory responses which can cause morbidity and mortality upon IN instillation in mice due to the sensitivity of the respiratory airway to acute inflammation (Fig. S2A-B, Table S1) (49). The MDNP formulation selected for this study resulted in complete survival following IN RNA vaccination. Moreover, compared to equivalent RNA doses of conventional LNP, this delivery formula induced negligible material-induced inflammation measured by the induction of IP-10, IL-6, MCP-1, CXCL1, and RANTES after IM immunization (Fig S2C). Liver toxicity studies of this MDNP formulation containing the self-amplifying Cal/09 influenza NP mRNA was assessed by serum alkaline phosphatase (ALP) and alanine transaminase (ALT) activity measurement at a high dose (10μg), and while a transient increase in ALT was observed, levels remained within acceptable ranges. (Fig. S2D-E). Given this opportunity, we tested how IM, IN, and intravenous (IV) routes of immunization would compare with respect to memory T cell differentiation and distribution to the respiratory mucosa and lung-draining mediastinal LN (medLN) (Fig. 2A). We observed that the magnitude of H-2K^b NP_366-375 tetramer⁺ T cells following IM and IV vaccination were largely comparable in secondary lymphoid organs (Fig. 2B). In contrast to IM immunization, IV immunization did not robustly establish CD62L⁻CD69⁺ memory CD8 T cells in the muscle-draining iliac LN (Fig 2C-D). IN vaccination resulted in fewer memory T cells in the spleen and iliac LN but generated more in the medLN (Fig. 2B). After IN vaccination, medLN H-2K^b NP_366-375 tetramer⁺ T cells phenocopied T cells in the iliac LN of IM immunized mice (CD62L⁻CD69⁺), which is consistent with a SLO-T_RM phenotype (Fig. 2C-D)(50). These data demonstrate that vaccination route influences the regionalization and phenotype of mRNA vaccine-elicited T cells.

Fig 2. — (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.

While IM and IV vaccinations induced demonstrable T cells within the extravascular compartments of the lung, most cells were labeled with iv antibody. In comparison, IN prime-boost vaccination established ~10x more extravascular NP366⁺ T cells within the lung (Fig. 2E). Unlike IM and IV elicited CD8 T cells, most influenza-specific memory CD8 T cells following IN mRNA administration were CD69⁺CD103⁺, a phenotype previously associated with localization in proximity to airways (Fig. 2F-G) (18, 20, 51-53). Independent of route, mRNA vaccination induced an NP-specific memory CD8 T cell population in the nasal-associated lymphoid tissues (NALT), trachea, and bronchoalveolar lavage fluid (BAL) (Fig. S3A-C). These populations persisted for at least 123 and 160 days after the last IN and IM immunization, respectively, in both the NALT and trachea. While CD8 T cells in the BAL following IN vaccination contracted over time, IM prime-boost was less effective at inducing memory T cells in the BAL fluid (Fig. S3A-C). Together, these data highlight that all routes of immunization established broadly distributed memory T cells, but that IN immunization increased the magnitude of CD69⁺CD103⁺ CD8 T cells in the lung and medLN.

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

The impact of IN mRNA delivery on the CD8 memory compartment prompted us to investigate whether the route also impacted antigen-specific CD4 memory T cell magnitude and phenotype in the lung and draining LN (Fig. 3A). In contrast to CD8 T cells, the frequency of antigen-specific CD4 T cells in the spleen following IN or IM vaccination was comparable, whereas the abundance of I-A^b NP_261-277⁺ T cells in the different LNs correlated with the vaccination route (Fig. 3B). Again, T_H1 cells were enriched in spleen and T_FH were restricted to the draining LN (Fig. 3 C-D, Fig. S3A-B). NP-specific IgG serum antibody titers were not affected by vaccination route (Fig. 3E). We observed a significant increase in I-A^b NP_261-277⁺ CD4s in the lung after IN vaccination which was completely accounted for by an increase in extravascular cells (Fig. 3F), many of which were CD69⁺ (Fig. 3G-H). In contrast to NP366⁺ CD8 memory T cells, CD103 upregulation was minimal, consistent with previously reported phenotypes of CD4 T cells in non-lymphoid tissues (54). In addition to well-described pulmonary T_H1 resident CD4 T cells (PSGL1^hi FR4^lo), a T_FH-like memory population (PSGL1^lo FR4^hi) was recently identified in the lung following influenza infection that promoted local antibody production upon reactivation (14, 15). In contrast to influenza infection, mRNA vaccination did not induce I-A^b NP_261-277⁺ PSGL1^lo FR4^hi cells in the lung (Fig. 3I-J, Fig. S4C). However, IN vaccination reproducibly induced an FR4 intermediate population that otherwise phenocopied T_H1-like resident memory T cells (Fig. S4D) (14, 15). I-A^b NP_261-277⁺ CD4 T cells in the lung and NP-specific IgG antibodies in BAL fluid persisted for at least 123 days post immunization after both IM and IN immunization (Fig. S4E). Taken together, IN vaccination induced long-lived T_FH in the medLN and in contrast to IM vaccination generated a robust pulmonary CD4 T cell memory population.

Fig 3. — (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.

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

Upon IM mRNA prime-boost vaccination, we noted a population of CD8 and CD4 memory T cells in the lung, some of which expressed CD69. To further validate the presence of H-2K^b NP_366-375 tetramer⁺ CD8 T cells in the lung after IM immunization, we performed in situ tetramer staining and found influenza-specific memory T cells in the lung parenchyma (Fig. 4A). Although CD69 is often used as a proxy to denote resident memory T cells, many T_RM do not express CD69 and not all CD69⁺ cells are resident (11, 50). Moreover, the establishment of T_RM in the lung often requires local antigen recognition (20, 23, 37-41). Therefore, we wondered whether or not IM vaccine-induced T cells were resident or recirculating. To assess migration of mRNA vaccine-elicited pulmonary T cells, we conjoined CD45.2⁺ mRNA primed-boosted mice to congenically mismatched naive CD45.1⁺ C57BL/6J mice via parabiosis surgery (Fig. 4B). While circulating bloodborne populations equilibrate between the two mice, the failure of cells to equilibrate in corresponding tissues is strong evidence for residency. Three weeks after surgery, the blood contained equal proportions of CD45.2⁺ and CD45.1⁺ CD8 T cells, demonstrating successful anastomosis between the immune and naive parabionts (Fig. 4C). To calculate the proportion of resident T cells, we compared the number of antigen-specific T cells in the immune versus naive parabiont using a previously reported equation (11). Unexpectedly, extravascular pulmonary NP366⁺ CD8 T cells were preferentially present in the immune parabiont – demonstrating that IM mRNA vaccination elicits bona fide resident memory T cells (Fig. 4D-E). The few NP366⁺ CD8 T cells found in the lung of the naive parabiont were >90% CD69⁻ CD103⁻. The proportion of resident cells increased stepwise from 40% in the CD69⁻CD103⁻ compartment to 95% and 100% residence in the CD69⁺CD103⁻ and CD69⁺CD103⁺ subsets, respectively. To test whether local antigen deposition impacts the formation of pulmonary T_RM, we repeated parabiosis with IN prime-boosted mice. Not only did IN vaccination boost the number of extravascular pulmonary T cells by ~10 fold, the fraction of resident T cells also increased from ~70% to ~90% (Fig. 2E, Fig. 4 D-E). We also assessed residence within the CD4 compartment. Our analysis was limited to the IN vaccination route due to insufficient recovery of antigen-specific CD4 T cells upon parabiosis after IM vaccination. Following IN mRNA vaccination, ~90% of I-A^b NP_261-277⁺ CD4 T cells resided in the lung for at least three weeks and this proportion increased to 100% if the analysis was limited to CD69⁺ cells (Fig. 4 F-G). In conclusion, mRNA vaccination induces bona fide T_RM independent of vaccination route.

Fig 4. — (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.

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

While IM vaccination resulted in a large population of NP366⁺ CD8 T cells in the circulation, IN immunization biased differentiation towards a higher number of pulmonary T_RM. We next tested if 1) antigen-specific memory T cells retain the potential to generate abundant lung T_RM populations and 2) whether one could generate a large pool of both circulating and pulmonary T cells by giving IM prime-boosted mice a secondary IN boost (Fig 5A). Compared to IM prime-boost alone, IN boosting increased the number of antigen-specific CD8 and CD4 T cells in the spleen, medLN, and lung (IV-neg), but not in the iliac LN (Fig. 5B-C). IN boosting expanded the proportion and number of CD69⁺CD103⁺ CD8 T cells and CD69⁺ CD4 T cells in the extravascular compartment of the lung, comparable to the IN prime-boost regimen (Fig 2F-G, 3G-H, Fig.5D-G). These data demonstrate that combining IM prime-boost immunizations with a third IN mRNA immunization achieved high levels of both circulating and lung memory T cells.

Fig 5. — (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.

Discussion

In this study, we characterized the abundance, phenotype, and anatomic distribution of mRNA vaccine-elicited antigen-specific CD8 and CD4 memory T cells (Fig. S5). Independent of route of administration, mRNA vaccination induced T_RM in the lung, where frontline cellular immunity is known to contribute to protection against certain respiratory infections (16, 19-35). Thus, mRNA vaccines may fulfill an important criterion in the pursuit of a universal influenza vaccine: establishing cellular immune memory at the primary site of infection. Importantly, we found that prime-boosted memory T cells elicited by IM vaccinations retain the potential to generate abundant pulmonary T_RM upon an additional IN boost. These data could inform a strategy to leverage pre-existing memory T cells to bolster frontline immunity at the site of infection without compromising the circulating compartment. This could have implications for future iterations of SARS-CoV-2 vaccination, or vaccines for other respiratory pathogens. Including conserved T cell epitopes in vaccines that also establish humoral immunity may provide immunity in immunocompromised individuals that have impaired ability to efficiently generate antibodies or against pathogens that evolve to evade vaccine-elicited neutralizing antibodies. With that said, this study focused primarily on the differentiation and distribution of mRNA vaccine-elicited memory CD4 and CD8 T cells. However, we demonstrated that IM mRNA prime-boost vaccination solely encoding Cal/09 NP was still sufficient to protect mice against lethal influenza A virus, PR8 strain. NP is not thought to be a significant target of neutralizing antibodies, but contains conserved T cell epitopes that could elicit other mechanisms of immunity that could also cross-react with serological variants. Future studies will be needed to determine how to best integrate cell-mediated immunity with other vaccine-elicited mechanisms (which may include various classes of antibodies) to optimize long-term protective potential. This is a complex issue, and will depend on the route, dose, virulence, and antigenic profile of the pathogens in question, as well as availability of animal models that adequately predict human outcomes.

Lazcko and colleagues demonstrated the presence of activated CD69⁺ IV-neg T cells in the lung 10 days after IM mRNA-LNP vaccination and a recent study from Mao et al. showed persistence of antigen-specific CD8 T cells in the lung for at least 56 days, raising the question of whether T_RM are established (55, 56). Here, we report the persistence of vaccine-elicited antigen-specific CD8 and CD4 T cells in the lung for at least 188 days after IM priming. Numerous studies have shown that establishment of pulmonary CD8 TRM requires local antigen recognition (20, 23, 37-41). To our surprise, based on migration assays in parabiotic mice most CD8 memory T cells, including CD69⁻ cells, were T_RM. Although we would not expect antigen to be present in the lung after IM immunization, we cannot exclude this possibility. Nevertheless, IN immunization was much more effective at establishing pulmonary T_RM compared to IV and IM immunization. IN vaccination also induced persisting memory CD8 T cells in BAL fluid and in the upper respiratory tract. However, NP-specific serum IgG titers were similar in the BAL fluid between IM and IN vaccine delivery. Another group using an IM mRNA prime followed by a protein boost reported that IN protein delivery is particularly efficient in inducing IgA in BAL fluid (55). While we were not able to detect NP-specific IgA, that might be consistent with the absence of vaccine-induced pulmonary T_FH or due to technical limitations.

We found it surprising that IN and IV immunizations were effective at establishing memory CD8 and CD4 T cells, as the vaccine is tailored for IM administration. A previous report of self-amplifying RNA delivery by the IN route using multiple delivery systems including LNPs showed poor immunogenicity relative to more typical IM or intradermal injections, and was associated with rapid clearance (57). For the most part, IN delivery of mRNA vaccines have been attempted using well-established polymer-based transfection reagents, though such polymer-based approaches have not historically reached clinical application successfully, possibly in part due to toxicity issues (58-61). Moreover, IN LNP can induce lethal inflammatory responses in mice, complicating studies of alternative administration routes (49). The MDNP formulation here did not have that effect and induced reduced levels of proinflammatory cytokines compared to commonly used LNPs. It is unclear whether this could be translated to humans. Of note however, we found that IN immunization of mice that had previously received two IM immunizations resulted in particularly robust memory CD8 T cell responses in both the pulmonary and circulating compartments. CD4 T cells were augmented in lung as well. These data add to the possibility that future mRNA vaccinations, perhaps combined with systemic immunizations, might be tailored to focus immunity at specific sites including the respiratory mucosa based on modulating the site of boosting.

A common question concerning mRNA vaccines is whether one should get a booster vaccine in the same (ipsilateral) or opposite (contralateral) arm. In the mouse model, we found moderate differences in the immunological parameters that we measured. Serum antibody titers were higher after ipsilateral versus contralateral boosting. A recent report indicated that affinity maturation was also enhanced by same-site boosting, suggesting that ipsilateral boosting may offer advantages for humoral immunity (62). Opposite-site boosting readily affected T cell responses. Like Lederer et al., we showed that IM mRNA elicits T_FH memory cells in the vaccine-site draining LNs, however, we extended these data and showed that T_FH memory cells were restricted to the immunization-site draining LNs (and absent from the contralateral LN, spleen, and lung) (63). Moreover, we demonstrated that IN vaccination was sufficient to establish T_FH in the medLN and contralateral IM immunization broadened distribution of this population. This observation also extended to CD69⁺CD62L⁻ memory CD8 T cells, a phenotype previously shown to correlate with LN residence (50). LN TRM are putatively derived from retrograde migration from upstream nonlymphoid tissues, so this population may reflect ex-T_RM derived from muscle (18). However, preliminary attempts to examine muscle tissue did not yield viable lymphocyte populations that could be analyzed. We also observed that T_FH and T_RM-phenotype CD8 T cells were uniquely established in the lung-draining LN after IN immunization.

RNA-based vaccines are rapidly scalable against essentially any protein antigen and are not vulnerable to vector immunity that could otherwise limit opportunities for boosting or reusing the same vector, encoding different antigens, in future vaccines. The prospects are exciting for combatting emerging pathogens, antigenically variable pathogens that might be addressed by megavalent vaccination, and perhaps non-infection conditions such as personalized tumor vaccines. For these reasons, it is important to more fully characterize RNA vaccine immunogenicity. The establishment of T_RM is of obvious importance, but difficult to assess in humans, despite COVID-19 providing intense interest and subject availability. Genetically defined mice and parabiosis surgery provided an opportunity to address this fundamental question and suggest that RNA vaccines may be effectively exploitable for establishing T_RM.

Materials and Methods

Study design

The aim of this study was to characterize the self-amplifying mRNA-MDNP vaccine-elicited CD8 and CD4 T cell responses against the Cal/09 nucleoprotein of the Influenza virus in secondary lymphoid organs and the lung. We used MHC I and II tetramers to identify polyclonal antigen-specific CD8 and CD4 memory T cells. Analysis of experimental data was conducted unblinded. Detailed description of experimental replicates, sample sizes, and statistical analysis can be found in the figure captions or in the “Statistical analysis” section.

Mice

Female C57BL/6J (B6) and B6.SJL-Ptprc^aPepc^b/BoyJ (B6 CD45.1), were purchased from Jackson Laboratory and maintained under specific pathogen-free conditions at the University of Minnesota according to the Institutional Animal Care and Use Committees guidelines. All B6 mice used in the experiments were female and 6-9 weeks of age at the timepoint of the first immunization. For inflammatory response testing, BALB/cJ mice were purchased from Jackson Laboratory and maintained according to Tiba Biotech’s Institutional Animal Care and Use Committee guidelines. All BALB/cJ mice were 6-10 weeks of age at time of experimentation.

MDNP vaccine production

The Cal/09 nucleoprotein coding sequence was cloned into a DNA plasmid encoding a self-amplifying mRNA template based on the genome of Venezuelan equine encephalitis virus, followed by RNA synthesis by run-off in vitro transcription from an upstream T7 promoter and subsequent enzymatic capping essentially as reported previously (64). Expression potency of each lot of self-amplifying mRNA produced for this work was validated by transfection of BHK cells and subsequent immunoblot using anti-influenza A virus nucleoprotein (NP) antibody (clone 1C5-1B7; NR-43899) obtained through BEI Resources Repository, NIAID, NIH. A representative blot is shown in Fig. S6. The capped self-amplifying RNA was dissolved in 10 mM citrate buffer and mixed using a NanoAssemblr Ignite microfluidic mixer (Precision NanoSystems) with a proprietary ethanolic solution of an ionizable modified dendron provided by Tiba Biotech (at an N:P ratio of 4), plus cholesterol, DOPE, and DMG-PEG2000 (Avanti Polar Lipids) in a 100:288:60:1 molar ratio respectively. Nanoparticles were dialyzed against sterile, endotoxin-free PBS using 20,000 Da molecular weight cutoff dialysis cassettes and sterile filtered using 0.2 micron polyethersulfone filters (CELLTREAT Scientific Products). Nanoparticle diameter and polydispersity index were assessed by dynamic light scattering and encapsulation efficiency measured by RiboGreen^® (Thermo) dye-exclusion assay. Representative nanoparticle characteristics of the final Cal/09 nucleoprotein self-amplifying mRNA vaccine are provided in Table S2.

ALP and ALT measurement

ALP and ALT were measured in serum samples using the QuantiChrom^™ Assay Kit (BioAssay Systems) or Colorimetric Activity Assay Kit (Cayman Chemical Company), respectively.

Immunizations

Mice were immunized with 5 μg (50 μl at 0.1 mg/ml) of mRNA encapsulated in MDNP encoding for the nucleoprotein of the Influenza A H1N1 Cal/09 strain per dose for all vaccine delivery routes. Vaccine was either delivered IM (right or left hamstring), IN, or IV. For injections, mice were either anaesthetized with vaporized isoflurane (IM) or ketamine and xylazine (IN).

Lymphocyte isolation from spleen, LNs, and lung

Mice were injected intravenously (tail vein) 15 minutes prior to sacrifice with NAD-induced cell death (NICD) protector (25 μg, BioLegend, #149802) to prevent NAD-induced cell death during tissue processing (46). Intravascular staining was used to discriminate cells present in the vasculature from cells present in the tissues parenchyma as previously described (65). Briefly, 3 minutes prior to euthanasia mice were injected retroorbitally with 3 μg of fluorescently conjugated αThy1.2 antibody (BV650, BD, #740443). Where indicated, bronchoalveolar lavage fluid was collected in three 1 ml lavages with cold PBS before the lung was excised. Lymphocyte isolation from spleen, LNs, trachea, nasal associated lymphoid tissues and lung was performed as described (11, 21). Spleen and LN single cell suspensions were generated via dissociation through 70μm filters (Falcon, #352350). For lung and trachea cell isolations, tissues were excised, minced, and digested in Collagenase I (Worthington, #LS004197) for 1 hour at 37°C shaking at 200 rpm. Tissue homogenates were then mechanically dissociated using a gentleMACS Dissociator (Miltenyi Biotec) and subsequently passed through a 70 μm filter. Finally, lung lymphocytes were enriched on a Percoll gradient. Cell enumerations were determined using PKH26 Reference Microbeads (Sigma-Aldrich, # P7458-100ML) as previously described (66).

Flow cytometry

To identify influenza-specific CD8⁺ T cells, isolated lymphocytes were surface-stained with homemade PE-conjugated H-2K^b NP_366-375 tetramer for 1 hour at room temperature in the presence of viability dye (Ghost Dye Red 780, Tonbo Biosciences, #13-0865-T500), dasatinib (50 nM), Fc Shield (Tonbo, #70-0161-M001), and indicated antibodies. Splenic influenza-specific CD4⁺ T cells were isolated by staining single cell suspensions with homemade APC-conjugated I-A^b NP_261-277 tetramer for 1 hour at room temperature in the presence of dasatinib (50 nM) followed by tetramer enrichment (66, 67). CXCR5 (BV421, BioLegend, #145512) was added at the time of tetramer staining. Further surface staining was performed for 30 minutes on ice in the presence of viability dye (Ghost Dye Red 780, Tonbo Biosciences, #13-0865-T500). Lung and LN-derived I-A^b NP_261-277 tetramer⁺ CD4 T cells were identified by staining single cell suspensions with I-A^b NP₂₆₁₂₇₇ APC tetramer for 1 hour at room temperature in the presence of viability dye (Ghost Dye Red 780, Tonbo Biosciences, #13-0865-T500), Dasatinib (50nM), Fc Shield (Tonbo, #70-0161-M001), and indicated antibodies. All stained samples were acquired with LSR Fortessa flow cytometers (BD) and analyzed with FlowJo software (TreeStar).

Isolated H-2K^b NP_366-375 tetramer⁺ lymphocytes were stained with the following antibodies: CD8a (BUV496, BD, #750024), CD45.2 (AF700, BioLegend, #109822), CD69 (BV421, BD, #562920), CD103 (BV510, BD, #563087), CD44 (BV785, BioLegend, #103041), CD62L (BUV737, BD, #612833), and were indicated CD45.1 (APC, BioLegend, # 110714)

Isolated I-A^b NP_261-277 tetramer+ lymphocytes were stained with the following antibodies: CD4 (BUV496, BD, #612952), CD45.2 (AF700, BioLegend, #109822), CD103 (BV510, BD, #563087), CD44 (BV785, BioLegend, #103041), CD62L (BUV737, BD, #612833), Ly6C (FITC, BD, #553104), CD69 (PE-Dazzle, BioLegend, #104536), FR4 (PE-Cy7, eBioscience, #25-5445-82), PSGL1 (BV605, BD, #740384), CXCR6 (BV711, BioLegend , #151111), PD-1 (BUV395, BD, #744549), and were indicated CD45.1 (PE, BD, #553776).

Enzyme-linked immunosorbent assay (ELISA)

96-well plates (ThermoFisher, #44-2404-21) were coated with the Cal/09 influenza nucleoprotein (1 μg/ml, Sino Biological, #40205-V08B-100) and incubated overnight at 37°C. Using the Invitrogen ELISA Kit (#88-50400-88), plates were blocked for 2 hours at 37°C before diluted sera were added and incubated for 1 hour at 37°C. HRP-conjugated goat anti-mouse IgG detection antibody (Jackson ImmunoResearch, #115-035-062) was incubated for 1 hour at 37°C before plates were developed with 1x TMB for 15 min followed by addition of stop solution (Biofix, #NC9141468). Optical density was measured at 450 nm wavelength using a Tecan Infinite M Plex.

Immunohistochemistry

Lung tissues were manually cut into sections using surgical blades and incubated with PE-conjugated-H-2K^b NP_366-375 overnight at 4°C. Sections were fixed in 2% paraformaldehyde in PBS for 2 hours at 4°C and incubated in 30% sucrose overnight at 4°C before freezing in Tissue-Tek O.C.T. compound. 20 μM thick sections were generated using the Leica Cryostat and subsequently stained with CD8 BV480 (BD, #566096), CD31 AF488 (BioLegend, #102514), EpCAM AF594 (BioLegend, #118222) and anti-PE (Novus Biologicals, #NB120-7011). Tetramer staining was further amplified using Cy3-donkey anti-rabbit (Jackson ImmunoResearch, #711-165-152). Images were acquired with a 20x immersion objective (set to glycerol NA 0.75) and voxel size of 0.568 μm on a Leica Stellaris 8 using laser scanning confocal microscopy with a 1 AU pinhole. Grouped sequential scans with excitation wavelengths of 405, 440, 498, 550, 594, and 647 nm were used with spectral HyD-S detectors with emission ranges set to 410-25, 445-510, 505-550, 555-585, 600-630, and 655-690 nm respectively.

Parabiosis surgery

Parabiosis surgeries were performed as previously described (13). In brief, mice were anaesthetized with Ketamine and Xylazine and fur was depilated on each mouse along the opposite lateral flank via electric clippers. The skin was wiped clean and sterilized with alcohol prep pads, Betadine solution, and 70% alcohol. Identical incisions were made on the lateral aspect of each mouse from the hip to shoulder and 9 mm wound clips (BD, #427631) were used to approximate and join the dorsal and ventral skins of adjacent mice. Conjoined mice were then allowed to rest for 21 days before sacrifice and analysis. Equilibration was confirmed in the peripheral blood prior to euthanasia.

Influenza virus infection

Vaccinated and age matched naïve C57BL/6 mice were anesthetized with ketamine and xylazine followed by IN infection with a lethal dose (30 PFU) of influenza A/PR8 virus. Mice were monitored by daily weighing. A euthanization endpoint was defined at a weight loss in excess of 25% of the initial body weight.

Statistical analysis

Data were analyzed and visualized using Prism 9 (GraphPad). Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparisons test (more than two groups) or unpaired two-tailed Student’s t test (two groups), or with log-rank Mantel-Cox test for survival with P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are shown as mean ± SEM or SD as indicated in figure legends. Fitted linear regression models in Fig S3 A-C and S4E were computed in R (version 4.1.3) using the packages ggplot2 and ggpmisc. For visualization purposes only, values equal to zero were adjusted to 1 to appear on the axis of log-scaled data.

Supplementary Material

reproducibility checklist

NIHMS1861568-supplement-reproducibility_checklist.docx^{(67.9KB, docx)}

data file S1

Data file S1: Raw data file (Excel spreadsheet)

NIHMS1861568-supplement-data_file_S1.xlsx^{(129.4KB, xlsx)}

main supplementary materials

Fig. S1: Impact of ipsilateral versus contralateral intramuscular mRNA-vaccination on CD8 T cell, CD4 T cell, and humoral immunity.

Fig. S2: MDNP-encapsulated mRNA induces less acute inflammation than LNPs composed of DLin-MC3-DMA or SM-102.

Fig. S3: Longevity of vaccine-elicited CD8 T cells in the respiratory tract.

Fig. S4: mRNA vaccination generates long-lived T_FH in the draining LN but not in the lung.

Fig S5.: Anatomic distribution of mRNA vaccine elicited CD8 and CD4 memory T cells.

Fig. S6: Cal/09 nucleoprotein expression mediated by saRNA vectors in BHK cells.

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

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

NIHMS1861568-supplement-main_supplementary_materials.docx^{(1.8MB, docx)}

Acknowledgments

We thank the Masopust and Vezys group members for helpful discussions. We thank the Dr. Ryan Langlois for generously providing influenza virus. Funding: 75N93019C00051 Collaborative Influenza Vaccine Innovation Centers (CIVICs) (D.M., R.A.), Minnesota Partnership for Biotechnology and Medical Genomics (D.M., R.V.), National Institutes of Health grant 3R01AI084913 (D.M.), Swiss National Science Foundation (SNSF) grant P2BSP3_200187 (M.K.)

Footnotes

Competing interests: P.T, J.S.M., C.W.M., J.S.C. are employees of Tiba Biotech LLC and have equity interests. Tiba Biotech has filed a patent on the MDNP RNA delivery system (patent application PCT/US21/25542), naming P.T. and J.S.C. as inventors. J.S.C. is also an inventor on US Patent No. 10,548,959 entitled “Compositions and Methods for Modified Dendrimer Nanoparticle Delivery.” C.W.M. has worked as a freelance consultant advising commercial and non-profit organizations working in the fields of vaccines and viral vectors. The other authors declare no competing interests.

Data and Materials Availability:

The self-amplifying mRNA vaccine construct used in this study is available through a Material Transfer Agreement with Tiba Biotech. Requests should be directed to J.S.C. at Tiba Biotech.

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Supplementary Materials