Ionizable lipid nanoparticles (LNP) that have enabled the success of messenger RNA (mRNA) vaccines have been shown to be immunostimulatory in the absence of mRNA. However, the mechanisms through which they activate innate immune cells is incompletely understood. Using a monocyte cell line, we compared the ability of three LNP formulations to activate transcription factors Nuclear Factor-kappa B (NF-κB) and Interferon Regulatory Factor (IRF). Comparison of signaling in knockout cell lines illustrated a role for Toll-like receptor (TLR) 4 in initiation of this signaling cascade and the contribution of the ionizable lipid component. Activation induced by empty LNPs was similar to that induced by LNPs containing mRNA, indicating that LNPs may provide the majority of innate stimulation for the mRNA vaccine platform. Our findings demonstrate that ionizable lipids within LNPs signal through TLR4 to activate NF-κB and IRF, identifying a mechanism for innate activation that can be optimized for adjuvant design.

CD103+ and CD11b+ populations of CD11c+MHCIIhi murine dendritic cells (DCs) have been shown to carry antigens from the lung through the afferent lymphatics to mediastinal lymph nodes (MLN). We compared the responses of these two DC populations in neonatal and adult mice following intranasal infection with respiratory syncytial virus. The response in neonates was dominated by functionally-limited CD103+ DCs, while CD11b+ DCs were diminished in both number and function compared to adults. Infecting mice at intervals through the first three weeks of life revealed an evolution in DC phenotype and function during early life. Using TCR transgenic T cells with two different specificities to measure the ability of CD103+ DC to induce epitope-specific CD8+ T cell responses, we found that neonatal CD103+ DCs stimulate proliferation in a pattern distinct from adult CD103+ DCs. Blocking CD28-mediated costimulatory signals during adult infection demonstrated that signals from this costimulatory pathway influence the hierarchy of the CD8+ T cell response to RSV, suggesting that limited costimulation provided by neonatal CD103+ DCs is one mechanism whereby neonates generate a distinct CD8+ T cell response from that of adults. Respiratory syncytial virus (RSV) infection is most severe in infants under six months and the most common cause of hospitalization for lower respiratory tract infection in children under five years. Disease is a consequence of virus- and T-cell-mediated pathology. Adaptive immune responses to viral respiratory infections are initiated by dendritic cells (DCs) that traffic to lymph nodes from the infected lungs. We compared the phenotype and function of two lung-migratory DC populations, identified by high expression of MHC Class II and CD103+ (CD103+DCs) or CD11b+ (CD11b+DCs), in mice infected in early life or as adults. We found that DC subsets undergo dramatic quantitative and qualitative changes during the first weeks of life, and CD103+DCs from neonatal mediastinal lymph nodes induced a fundamentally different CD8+ T-cell response profile than CD103+DCs from adults. The adult response pattern required CD28-mediated costimulatory signals, which is a limiting functional property of neonatal CD103+DCs. Thus, the ability of neonatal CD103+DCs to provide sufficient costimulation to neonatal CD8+ T-cells can influence the immunodominance hierarchy and functional properties of neonatal CD8+ T-cell responses compared to those of adults. A better understanding of deficiencies in early life immunity will guide vaccine approaches that induce disease-sparing immune responses in infants.

Following respiratory syncytial virus infection of adult CB6F1 hybrid mice, a predictable CD8+ T cell epitope hierarchy is established with a strongly dominant response to a K^d-restricted peptide (SYIGSINNI) from the M2 protein. The response to K^dM2_82-90 is ∼5-fold higher than the response to a subdominant epitope from the M protein (NAITNAKII, D^bM_187-195). After infection of neonatal mice, a distinctly different epitope hierarchy emerges with codominant responses to K^dM2_82-90 and D^bM_187-195. Adoptive transfer of naïve CD8+ T cells from adults into congenic neonates prior to infection indicates that intrinsic CD8+ T cell factors contribute to age-related differences in hierarchy. Epitope-specific precursor frequency differs between adults and neonates and influences, but does not predict the hierarchy following infection. Additionally, dominance of K^dM2_82-90 –specific cells does not correlate with TdT activity. Epitope-specific Vβ repertoire usage is more restricted and functional avidity is lower in neonatal mice. The neonatal pattern of codominance changes after infection at 10 days of age, and rapidly shifts to the adult pattern of extreme K^dM2_{82- 90} -dominance. Thus, the functional properties of T cells are selectively modified by developmental factors in an epitope-specific and age-dependent manner. RSV causes yearly winter epidemics of respiratory disease with peak hospitalization rates at 2.5 months of age. Clearance of virus-infected cells depends on CD8 T-cells, and defining mechanisms of CD8 T-cell regulation is essential for understanding RSV disease pathogenesis and guiding therapeutic interventions. CD8 T-cells recognize a virus-infected cell by detecting peptides cleaved from viral proteins that are presented in host cell MHC molecules. The strength of CD8 T-cell response to processed peptide epitopes from the virus commonly assumes a predictable response hierarchy. In adult hybrid mice that have both H-2^b and H-2^d MHC alleles, the majority of the CD8 T-cell response is targeted at a peptide from the M2 protein and presented by the K^d MHC molecule, K^dM2_82-90, and a smaller subset of CD8 T-cells respond to a peptide from the M protein, D^bM_187-195. Interestingly, when infecting neonatal hybrid mice the dominance pattern is not seen and the D^bM_187-195 response is equal to or greater than the K^dM2_82-90 response. We show that the adult dominance pattern emerges at 10 days of age and that T cells are modified by developmental factors in an epitope-specific and age-dependent manner. These observations may influence future vaccine design.

The emergence of variants of concern, some with reduced susceptibility to COVID-19 vaccines underscores consideration for the understanding of vaccine design that optimizes induction of effective cellular and humoral immune responses. We assessed a SARS-CoV-2 spike-ferritin nanoparticle (SpFN) immunogen paired with two distinct adjuvants, Alhydrogel^® or Army Liposome Formulation containing QS-21 (ALFQ) for unique vaccine evoked immune signatures. Recruitment of highly activated multifaceted antigen-presenting cells to the lymph nodes of SpFN+ALFQ vaccinated mice was associated with an increased frequency of polyfunctional spike-specific memory CD4⁺ T cells and K^b spike-(539–546)-specific long-lived memory CD8⁺ T cells with effective cytolytic function and distribution to the lungs. The presence of this epitope in SARS-CoV, suggests that generation of cross-reactive T cells may be induced against other coronavirus strains. Our study reveals that a nanoparticle vaccine, combined with a potent adjuvant that effectively engages innate immune cells, enhances SARS-CoV-2-specific durable adaptive immune T cell responses.

CD8⁺ T cell responses are critical for the control of virus infections. Following infection, epitope-specific responses establish an unpredictable but reproducible pattern of dominance that is dictated by a large number of both positive and negative factors. Immunodomination, or diminution of subdominant epitope-specific responses by dominant epitopes, can play a substantial role in the establishment of epitope hierarchy. To determine the role of a dominant (K^dM2_82–90) and a subdominant (D^bM_187–195) epitope of respiratory syncytial virus in viral control and immunodomination, MHC-binding anchor residues in the two epitopes were mutated individually in recombinant infectious viruses, greatly reducing or deleting the epitope-specific CD8⁺ T cell responses. Neither mutation negatively affected viral clearance in mice, and compensation by the unmutated epitope was seen in both cases, whereas compensation by five other subdominant epitopes was minimal. Mutation of the dominant K^dM2_82–90 response resulted in effective viral clearance by the subdominant epitope with less illness, whereas mutation of the subdominant D^bM_187–195 response resulted in overcompensation of the already dominant K^dM2_82–90 epitope, and increased severity of illness. Increased illness was associated with poor functionality of the abundant population of CD8⁺ T cells specific to the dominant K^dM2_82–90 epitope, as measured by the percentage and magnitude of IFN-γ production. These data demonstrate efficient viral clearance, and a protective effect of subdominant CD8⁺ T cell responses.

Background: Characterizing the longevity and quality of cellular immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enhances understanding of coronavirus disease 2019 (COVID-19) immunity that influences clinical outcomes. Prior studies suggest SARS-CoV-2–specific T cells are present in peripheral blood 10 months after infection. Analysis of the function, durability, and diversity of cellular response long after natural infection, over a range of ages and disease phenotypes, is needed to identify preventative and therapeutic interventions. Methods: We identified participants in our multisite longitudinal, prospective cohort study 12 months after SARS-CoV-2 infection representing a range of disease severity. We investigated function, phenotypes, and frequency of T cells specific for SARS-CoV-2 using intracellular cytokine staining and spectral flow cytometry, and compared magnitude of SARS-CoV-2–specific antibodies. Results: SARS-CoV-2–specific antibodies and T cells were detected 12 months postinfection. Severe acute illness was associated with higher frequencies of SARS-CoV-2–specific CD4 T cells and antibodies at 12 months. In contrast, polyfunctional and cytotoxic T cells responsive to SARS-CoV-2 were identified in participants over a wide spectrum of disease severity. Conclusions: SARS-CoV-2 infection induces polyfunctional memory T cells detectable at 12 months postinfection, with higher frequency noted in those who experienced severe disease.