A single immunization with CAF08 provides newborns with Th1-mediated protection against Respiratory Syncytial Virus infection

Respiratory Syncytial Virus (RSV) is a leading cause of morbidity and mortality in children, due in part to their distinct immune system, characterized by impaired induction of T-helper 1 (Th1)-immunity. Here we describe cationic adjuvant formulation (CAF)-08, a liposomal vaccine formulation tailored to induce Th1 immunity in early life via synergistic engagement of Toll-like Receptor 7/8 and the C-type lectin receptor Mincle. Quantitative phosphoproteomics applied to human dendritic cells revealed a key role for Protein Kinase C-δ for enhanced Th1 cytokine production in neonatal dendritic cells and identied signaling events resulting in antigen cross-presentation. In vivo, a single immunization at birth with CAF08-adjuvanted RSV pre-fusion antigen protected newborn mice from RSV infection through induction of antigen-specic CD8 + and Th1 cells. Overall, we describe a novel pediatric adjuvant formulation and characterize its mechanism of action providing a promising avenue for development of early life vaccines against RSV and other intracellular pathogens. IL-10 were measured using a multiplexing bead array (Millipore). Sample uorescence was acquired on a Flexmap 3D analyzer with xPONENT version 4.2 software (Luminex). Results were converted into pg/ml values by 4-point log curve tting on kit-provided standards using Milliplex Analyst version 3.5.5.0 software. a frequency of RSV tetramer-positive cells (closed circles) that was signicantly higher than the frequency of control tetramer-positive cells (open circles, paired Student’s t-test). (B). No signicant presence of RSV tetramer-specic T cells as compared to control tetramer-positive T cells was observed in the spleen. Analysis of the relative percentage of T-bet+ Th1 cells and GATA3+ Th2 cells within the tetramer-positive gate revealed an increase in Th1 cells and a relatively lower abundance of Th2 cells in CAF08-immunized animals (C) Statistical comparisons are Mann-Whitney U-test comparisons to saline groups. (D) Draining LN cultures were stimulated with 1 mg/ml pre-F antigen for 24 hours in the presence of Brefeldin A. Quantication of (IFN-g) cells conrmed a statistically signicant generation of Th1 cells in CAF08-immunized mice. Detection of CD8+CD44+ Cytokine+ (IFN-g and/or TNF-a) cells indicates induction of RSV-specic CD8+ T cells in CAF08-treated mice. Cytokine analysis indicates prominent production of Th2/anti-inammatory cytokines (IL-5, -13, -10) in newborn mice as compared to adult mice. CAF08b reduced production of Th2/anti-inammatory cytokines and increased that of IFN-g, indicating enhancement of a Th1 response. All statistical comparisons are Mann-Whitney U-test comparisons to saline groups.


Introduction
Infectious diseases are the leading global cause of death in children under 5 years of age 1 . Among the viral pathogens of early life is Respiratory Syncitial Virus (RSV), an enveloped, non-segmented negativestrand RNA virus of the family Pneumoviridae that is a common viral cause of serious respiratory illness in children under 5 years of age. Indeed, RSV is estimated to cause 3.2 million hospitalizations and at least 59,000 deaths worldwide, predominantly in children under the age of 6 months 2 rendering it the second most likely single pathogen to cause death in children under 1 year of age 3 . RSV infection is a particular threat in the very young due to limited airway size as well distinct features of the infant innate immune system 4 . Speci cally, the Th2-biased response observed in newborns is likely associated with more severe manifestations of RSV infection 5,6 , as it correlates with airway eosinophilia 7 . Similarly, the propensity of newborns to produce CXCL8 leads to in ltration of neutrophils, which also contributes to disease exacerbation 8,9 . A key approach to protect infants from viral pathogens such as RSV is immunization. Unfortunately, although immunization of infants with a formalin-inactivated preparation of RSV (FI-RSV) induced antibody (Ab) responses, it led to enhanced disease progression and in some cases even death after subsequent RSV infection 10 . Lung and blood pathology of these immunized children, as well as mechanistic insight from murine studies, revealed that both antibody and cell-mediated immune responses associated with severe disease. Induction of Abs with poor neutralizing activity led to immune complex deposition and complement activation in small airways and Th2-biased T cell response coincided with excess in ltration of neutrophils and eosinophils into the lungs 11,12 .
In addition to a predominant Th1 response, potential immunization against RSV would require the generation of Abs capable of neutralizing the virus. The RSV fusion (F) glycoprotein is the target of most RSV-neutralizing Abs, speci cally in its metastable prefusion conformation (pre-F) 13 . Recent studies of the F protein in complex with Abs have led to structure-guided stabilization of a recombinant F antigen variant that retains its pre-F state 14,15 . This variant, also known as DS-Cav1, is a promising vaccine candidate, eliciting high a nity Abs with strong neutralizing ability in mice, non-human primates, and antigen-experienced humans 16,17 . However, given the aforementioned history of enhanced disease in infants, there understandably remains a challenge to trial such RSV vaccinal antigens in early life 18 .
A key approach to enhance and shape immune responses in distinct populations is the use of adjuvants. This approach may be particularly important in newborns and infants as they often do not generate a su ciently protective immune response when vaccinated with a single dose of vaccinal antigen at birth [19][20][21] . Studies modeling the function and phenotype of the immune system in neonates in vitro and ex vivo have demonstrated a distinct neonatal innate immune system that responds differently to pathogenassociated molecular patterns (PAMPs), as compared to adults or elderly [22][23][24][25][26][27] . In this context, as many vaccine adjuvants are designed to enhance the immunostimulatory effect of vaccines via activation of pattern recognition receptors (PRRs) 28 recognizing such PAMPs, these adjuvants may not optimally induce protective immune responses through vaccination at birth. The most well studied PRRs in newborns are Toll-like receptors (TLRs), type I transmembrane glycoproteins expressed on the cell surface or intracellularly. Recognition of microbial products by TLRs is a central event in the host response to pathogens. As compared to their adult counterparts, hallmark features of TLR activation in newborn monocytes or dendritic cells (DCs) include reduced NF-κB signaling 29,30 , reduced production of T-helper 1 (Th1)-polarizing cytokines TNF-a and IL-12p70 31−34 , and increased production of Th2/anti-in ammatory cytokines IL-6 and IL-10, and of neutrophil chemoattractant CXCL8 33,35−39 . Activation of human neonatal leukocytes via imidazoquinoline agonists of TLR7/8, however, can enhance the magnitude of the newborn immune response in vitro 40,41 through involvement of the NLRP3 in ammasome 40 , and can dramatically accelerate and enhance immunogenicity of pneumococcal conjugate vaccine (PCV13) in newborn non-human primates 42 . However, studied in vitro, TLR7/8 agonists alone were not able to su ciently change the type of MoDC immune response to a predominantly Th1 phenotype that is likely needed for protection against intracellular pathogens such as respiratory syncytial virus (RSV) 43 .
We have previously conducted a screen of TLR and CLR adjuvant combinations that demonstrated that newborn DCs can be activated to instruct a Th1 response by combined stimulation through TLR7/8 and the C-type Lectin Receptor (CLR) Mincle, using R848 and trehalose-6,6-dibehenate (TDB), respectively 43 . This combined adjuvantation system results in NF-κB activation, as well as activation of the NLRP3 in ammasome and induces production of TNF-α, IL-1β and IL-18, cytokines that contribute to Th1 polarization. Furthermore, production of Th2/anti-in ammatory cytokines IL-10 and IL-12p40, and of neutrophil chemoattractant CXCL8 was reduced relative to each of the components tested alone. As a result, these DCs induce the differentiation of newborn naïve CD4 + T cells into Th1 cells instead of Th2 43 . The highly synergistic and age-speci c fashion in which the R848 + TDB combination acts on newborn DCs makes it suitable for potential development of an early-life vaccine against RSV. Our prior studies also showed that formulation of liposomes with TDB enables e cient induction of germinal center formation 44 as well as polyfunctional T cells 45 in neonatal mice.
Herein, we evaluated the ability of liposomal formulations containing a TLR7/8 agonist, a Mincle agonist and the RSV pre-F antigen, to induce a potent and balanced Th1/Th2 immune response in newborn mice. We also dissected the molecular mechanism of synergistic activation of newborn human DCs by R848 + TDB, using label-free quantitative phosphoproteomics. Phosphoproteomic analysis of DCs indicated that synergistic activation of cytokine signaling pathways in newborn DCs by R848 + TDB is driven by TDBinduced endocytosis and prolongation of phosphorylation of the δ-isoform of protein kinase C (PKC-δ). In addition, it revealed marked synergistic enhancement of antigen cross-presenting ability, which was con rmed in human CD8 + T cell activation studies in vitro as well as in newborn immunization model in vivo. Immunization of newborn mice demonstrated the ability of CAF08, a liposomal formulation consisting of combined TLR7-and Mincle-agonists and RSV pre-F, to induce strongly neutralizing antibodies of IgG2a/c isotype as well as pre-F -speci c Th1 and CD8 + T cell responses and to protect against RSV infection. This novel adjuvant platform for neonatal vaccines represents a promising approach to enhance the e cacy of a future vaccine against RSV.

Results
Phosphoproteomic analysis of human moDCs reveals a link between endocytosis and the magnitude of cytokine induction in newborn cells.
We have previously shown that combined in vitro stimulation of newborn monocyte-derived dendritic cells (MoDCs) with R848 and TDB synergistically enhances the production of Th1-polarizing cytokines, such as TNF-α, in an age-speci c manner, such that the effect of this combination was only additive in infants/adults and actually antagonistic in elderly 43 . Key roles for NF-κB, Syk and the NLRP3in ammasome in overcoming the intrinsic impairment of newborn cells to instruct Th1-differentiation of CD4 + T cells were identi ed 43 . However, the extent of synergistically induced TNF-α and the age-speci c nature of this synergy imply that additional signaling events may occur. Therefore, we utilized a mass spectrometry-based method for label-free quantitation of phosphorylation events induced by stimulation of MoDCs. This method was optimized to identify and quantitate hundreds of phosphorylation events in only 3-5 million cells, thus enabling application of this technique to human newborn blood-derived primary leukocytes. Between 250 and 900 phosphorylation events were identi ed and quanti ed per sample. In a sample size of nine newborn donors and ve adult donors, the quantity of 186 to 467 phosphorylation sites changed their phosphorylation status in a statistically signi cant manner, depending on the treatment and age group ( Supplementary Fig. 1). Pathway overrepresentation analysis, mining multiple protein function databases (KEGG 46 , REACTOME 47 , PID NCI 48 , BioCarta 49 , NetPath 50 and INOH 51 ), for increased con dence, con rmed the synergistic induction of cytokine secretion signaling pathways by R848 + TDB in newborn MoDCs, as compared to stimulation with each individual agonist (Fig. 1A). This synergy is absent in adult MoDCs, where treatment with single agonists results in the greatest induction of phosphorylation events in cytokine signaling pathways (Fig. 1B). In addition, a marked synergy in phosphorylation in endocytosis-related signaling pathways was observed in newborn MoDCs, but not in adult MoDCs. As R848 induces activation through endosomal receptors (TLR7/8), we hypothesized that increased endocytosis could be the cause of the observed synergy in cytokine signaling induction. The activation of one phosphorylated protein that was categorized in multiple endocytosis and cytokine signaling pathways, Protein Kinase C delta (PKC δ, phosphorylated on serine 645), was previously identi ed as a key component of both endosomal tra cking and signaling 52,53 . It has also been identi ed as an important downstream signaling component of Syk and can induce phosphorylation of Signal transducer and activator of transcription 1 (STAT1) on serine 727 54,55 . Both phosphorylations, PKCD S645 and STAT1 S727, were synergistically induced in newborn moDCs by R848 + TDB, as demonstrated with phosphoproteomics ( Fig. 1C + D), as well as by western blotting (Fig. 1E). Indeed, a correlation between endocytosis-induced PKCD phosphorylation and production of cytokines was observed after inhibition of PKCD S645 phosphorylation by clathrin inhibitors (Fig. 1G) and inhibition of TNF-α secretion after treatment with a PKCD inhibitor (Fig. 1F). In addition, inhibition of endocytosis with clathrin inhibitors reduced (R848 + TDB)-induced TNF-α production in newborn MoDCs to R848-like levels ( Fig. 1H), further indicating the role of endocytosis in enhancement of cytokine production in newborn DCs.
R848 and TDB synergistically induce antigen cross-presentation in newborn and adult moDCs.
In addition to synergistic induction of phosphorylation in cytokine and endocytosis pathways in newborn MoDCs, R848 + TDB also synergistically induced the phosphorylation of nine proteins in antigen processing and cross-presentation in both adult and newborn MoDCs. In total, 17 phosphorylated proteins in the MHC I antigen processing and presentation pathway were identi ed, nine of which showed increased phosphorylation upon treatment with R848 + TDB ( Fig. 2A-C). STRING analysis 56 illustrates the type and degree of interactions between the identi ed proteins within this pathway ( Fig. 2A). Several of the proteins in which we identi ed phosphorylation upon R848 + TDB treatment (shown in bold) are known to physically interact with each other. The phosphorylation of four key proteins in the process of antigen cross-presentation, including Sec22b 57− 59 and Syntaxin-4 (STX4) 57,60 , was con rmed in adult MoDCs by western blotting after electrophoresis using phos-tag™ reagent to retard the migration of phosphorylated proteins (Fig. 2B). Signi cant R848 + TDB-induced phosphorylation of additional crosspresentation-related proteins are shown in Fig. 2C.
The functional implications of these ndings were investigated using a MoDC:CD8 co-culture in which adult MoDCs were pulsed with a soluble protein antigen, to which our study participants had predetermined pre-existing CD4-and CD8-T cell immunity. Contrary to a CD4 + T cell recall response, a CD8 + recall response in this assay would require cross-presentation of the internalized and processed protein on MHC I. Indeed, robust induction of a CD8 + recall response to any of the four proteins tested was observed only when MoDCs were also activated with R848 + TDB (Fig. 2D). These observations con rm the ability of combined R848 + TDB stimulation to synergistically induce antigen cross-presentation.
Immunization at birth with the pre-F antigen in combination with liposomal adjuvant formulation incorporating TLR7/8 + Mincle agonist (CAF08) improves vaccine immunogenicity.
A range of imidazoquinoline congeners were formulated into the liposomal adjuvant CAF01 consisting of dimethyldioctadecylammonium (DDA) and TDB for evaluation of optimal incorporation e cacy and adjuvant stability ( Supplementary Fig. 2). 3M-052, a lipidated congener of R848 with potent TLR7/8-and in ammasome-activating ability in vitro and in vivo 42,61,62 , incorporated most e ciently. Liposomes containing both TDB and 3M-052 demonstrated stability in terms of size and surface charge over several weeks ( Supplementary Fig. 2). We therefore decided to use this formulation for the in vivo studies in mice. Formulations with varying combinations of TDB and 3M-052 incorporated into DDA liposomes were therefore prepared and combined with 20 mg/ml RSV pre-F antigen 15,16 to evaluate the adaptive immune response induced by synergistic activation via TLR7/8 and Mincle in newborn and adult mice. Adult doses of 100 mL therefore consisted of 2 mg of pre-F antigen. Newborn mice were given half the adult dose. Immunization with DDA liposomes containing RSV pre-F and either 3M-052 (DDA052), or TDB (CAF01), or TDB and a low dose of 3M-052 (CAF08 low), or TDB and a high dose of 3M-052 (CAF08 high), as well as pre-F alone or formulated in an MF-59-like oil-in-water emulsion (Addavax) all elicited a high titer of pre-F -speci c IgG antibodies in newborn as well as adult mice (Fig. 3A). Newborn mice reached comparable levels of anti-pre-F antibodies to adults. Notably, in liposome formulations containing 3M-052, alone or in combination with TDB, the antibody response was skewed to IgG2a/c and less to IgG1, as compared to the other vaccine formulations. In mice, an antibody response skewed to IgG2a/c isotypes may indicate a Th1-driven immune response rather than a Th2-driven response 63 , or, alternatively, a T cell-independent immune response 64 . Serum from immunized newborn mice was able to neutralize the infection of HEp-2 cells by RSV in vitro (Fig. 3A). The highest neutralization titers were observed in groups that received pre-F formulated in either CAF01 or CAF08. Phenotypic analysis of draining lymph node B-and T-cells con rmed the formation of B cell germinal centers upon immunization with pre-F -containing formulations ( Supplementary Fig. 3). In accordance with the RSV-speci c antibody titers in Fig. 3, these germinal centers contained predominantly IgG1 + B cells in pre-F only, CAF01 and Addavax groups, and B cells of alternate IgG isotypes in DDA052 and CAF08 groups. A signi cant increase in T follicular helper cells (Tfh) was also seen in DDA052, CAF01 and CAF08 groups, but only in adult mice. There was no apparent increase in Tfh cells in draining lymph nodes or spleens from newborn mice.
Immunization with CAF08 induces RSV F-speci c Th1 and CD8 + T cells.
To evaluate RSV F-speci c CD4 + T cells, we developed an MHC II tetramer presenting RSV pre-F peptide 235-249 (REFSVNAGVTTPVST). The tetramer speci city was validated on lymph node cell suspensions from immunized CB6F1 mice. Staining of CD4 + cells from newborn mice immunized with pre-Fcontaining formulations consistently stained positive (1-2%) with the RSV F235-249 tetramer, but not the CLIP tetramer ( Fig. 4A/B). Cells from saline-injected mice did not stain positive with the RSV F235-249 tetramer. In addition, single cell sorting of tetramer-positive cells and clonal expansion with anti-CD3/CD28-coated microbeads (Thermo Fisher) generated a tetramer-positive CD4 + T cell clone, further con rming tetramer staining speci city ( Supplementary Fig. 4E). CD4 + T cells from newborn mice in all treatment groups except the saline group exhibited similar frequencies of tetramer-positive CD4 + T cells in the draining lymph node, but less in the spleen (Fig. 4B). The difference between treatment groups was evident when the relative percentage of T-bet expressing tetramer-positive cells (Th1) and GATA-3 expressing tetramer-positive cells (Th2) was determined (Fig. 4C). The relative ratio of antigen-speci c Th1 cells over Th2 cells was signi cantly increased after immunization with CAF08 high only, although CAF01 and CAF08 low also showed non-signi cant trends towards enhanced Th1 responses. Furthermore, T-bet and GATA-3 staining on tetramer-negative cells clearly indicates that the induction of RSV-speci c Th1 cells in newborn mice, which are intrinsically skewed to a Th2 response, is selective and antigen-speci c, as no change in T-helper ratios was observed in tetramer-negative cells ( Supplementary   Fig. 4). Ex vivo re-stimulation of cell suspensions derived from draining lymph nodes or spleens with pre-F antigen con rmed the presence of IFN-γ secreting CD4 + Th1 cells in newborn mice immunized with CAF08, but not any of the other formulations (Fig. 4D). Analysis of intracellular cytokines by ow cytometry demonstrated a signi cant percentage of RSV F-speci c Th1 cells, as well as CD8 + T cells, induced only in newborn mice immunized with CAF08. The nding of antigen-speci c CD8 + T cells after immunization with CAF08, which activates newborn cells through TLR7/8 and Mincle, further documents induction of antigen cross-presentation as noted in human MoDCs in vitro (Fig. 2). Analysis of secreted cytokines upon re-stimulation con rmed a higher preference for secretion of Th2/anti-in ammatory cytokines IL-5, IL-13 and IL-10 by cells from newborn mice as compared to adult mice (Fig. 4D). Secretion of IFN-γ was most pronounced in adult mice after immunization with CAF01 or CAF08, whereas in newborn mice the greatest IFN-γ secretion was observed in mice immunized with CAF08, coinciding with a reduction in secretion of IL-5 and IL-10 as compared to other antigen-containing immunization groups. These observations con rm the ability of combined TLR7/8 and Mincle stimulation in newborn leukocytes to induce Th1 polarization and reduce the intrinsic neonatal Th2 bias 43 .
RSV pre-F protein combined with CAF08 protects mice from RSV infection.
We compared the ability of Alum, CAF01, CAF08, or Addavax, when added to RSV pre-F, to enhance protection against RSV challenge in neonatal mice. Post-F adsorbed to Aluminum hydroxide (AlOH), was included as a control condition to emulate the Th2-driven immune stimulating ability of formalininactivated RSV. DDA052 was not a stable formulation and was hence excluded. Mice were immunized at birth with a single immunization and subsequently infected with live RSV A2 strain (Fig. 5). These data revealed that 4/6 mice in the CAF01 group and 6/6 mice in the CAF08 group did not have detectable levels of the virus in the lungs and 2-log reduced viral levels in the nose. Although a reduction in viral load was observed in the Addavax and Post-F + AlOH groups, CAF01-and CAF08-adjuvanted vaccines were signi cantly more effective. Infected mice had minimal weight loss, and histopathological evaluation of the lungs demonstrated an increase in in ammation, mucus, and eosinophil recruitment for all immunization groups compared to PBS control, with no signi cant difference between treatment groups-i.e., the vaccinated groups did not demonstrate any enhancement of disease compared to RSV infection of unimmunized mice. (Supplementary Fig. 5).
Altogether, the in vitro human phosphoproteomics platform and the murine immunization model presented here provide novel insight into age-speci c features of PRR-mediated innate immune recognition and the adaptive immune bias towards a Th2-driven immune response also observed in humans in vitro and in vivo. Moreover, it reveals that combined stimulation of newborn DCs via TLR7/8 and Mincle results in protection against infection with RSV by induction of antigen-speci c Th1 cells, CD8 + T cells, and neutralizing antibodies.

Discussion
The present work provides further insight into the mechanism of action and in vivo applicability of a previously identi ed adjuvant combination, R848 and TDB, that acts in an age-speci c manner to activate balanced Th1/Th2 polarizing responses by neonatal antigen-presenting cells 43 . We demonstrate for the rst time that synergistic TLR7/8 and Mincle stimulation activates antigen cross-presenting pathways in vitro and protects against RSV infection in newborn mice, correlating with Th1-skewed CD4 + and CD8 + T cell responses towards the RSV pre-F antigen in vivo. The major challenge with the induction of early life immunity against RSV is the innate propensity of the immune system in early life to be skewed towards a Th2 phenotype, where a Th1 phenotype would be preferred for host defense against intracellular pathogens such as respiratory viruses. A Th2-mediated response may even worsen disease symptoms upon encounter of RSV, due to excessive in ltration of polymorphonuclear leukocytes (PMNs) into the lungs as well as Th2 cytokine-promotion of mucus production and airway hypersensitivity, both of which increase airway obstruction. Airway obstruction is thought to be the major factor causing severe illness and hypoxemia, especially in young infants with small airways.
Synergistic stimulation through the receptors TLR7/8 and Mincle enhances the production of Th1favoring cytokines TNF-a, IL-18 and IL-1β in newborn MoDCs through restoration of NF-κB signaling and activation of the NLRP3 in ammasome, while also reducing the propensity of newborn MoDCs to secrete Th2/anti-in ammatory cytokines IL-10 and IL-12p40 43 . Given the age-speci c nature of MoDC activation with R848 and TDB, we investigated whether additional signaling pathways were induced upon stimulation with R848 + TDB. We leveraged state-of-the-art label-free quantitative proteomics to determine changes in phosphorylation in newborn and adult MoDCs. Pathway overrepresentation analysis revealed age-speci c synergy in cytokine signaling pathways such as TLR signaling, MAPK signaling and JAK-STAT pathways (Fig. 1), as well as synergistic induction of endocytic pathways, suggesting a link between endocytosis and cytokine induction in newborn MoDCs. Indeed, synergistic TNF-a induction appeared dependent on endocytosis, with a proposed key role for PKC-δ. One possible explanation for these ndings would be that cytokine signaling pathways are activated over a prolonged period as a result of prolonged delivery of R848 to endosomes. Future studies investigating the kinetics of signal transduction may shed further light on the role of the individual proteins identi ed.
In addition to establishing a link between endocytosis and cytokine production in newborn MoDCs, phosphoproteomic studies also revealed synergistic induction of antigen cross-presentation on MHC class I by R848 + TDB, both in newborn and adult cells (Fig. 2). This is in accordance with the observation in our murine immunization study that RSV-speci c CD8 + T cells were only induced in mice immunized with the liposomal adjuvant combining 3M-052 and TDB (CAF08, Fig. 4).
A Although the vaccine formulations studied induced a similar quantity of RSV-speci c CD4 + T cells, the phenotype of these cells differed markedly between the formulations. In this study we describe the development of a novel MHC class II tetramer for the detection of H2-IAb-restricted CD4 + T cell responses against an epitope in the F protein (Fig. 4). This validated tetramer detects very robust frequencies of CD4 + T cells against a previously undescribed epitope. Recent mapping of T cell epitopes in the RSV proteome in C57BL/6 mice did not report this epitope 69 , emphasizing the novelty of this tetramer and its value for future murine immunization studies. Using this tetramer, we showed that immunization with CAF08 redirects the CD4 + T cell response to a Th1 phenotype in newborn mice, as opposed to the Th2 response that is typical for newborn mice and which was observed with single adjuvant-containing formulations. These observations are consistent with prior studies employing C-type lectin receptor agonists as adjuvants 44,45 . Other adjuvant combinations, such as (Alum + STING agonist) and IC31 (antimicrobial peptide + CpG-ODN), may also be able to elicit a Th1 response in newborn mice 70,71 .
Our results suggest that the quality of the induced immune response is a critical parameter. Although all formulations studied, either with single adjuvants or with adjuvant combination (3M-052 + TDB), induced similar levels of antibodies or CD4 + T cells, marked differences were noted in antibody isotype and T cell phenotype. The induction of RSV-speci c Th1 cells as well as CD8 + T cells by CAF08 is relevant for protection against respiratory disease, as both Th2-driven pathology and lack of CD8 + T cells have been associated with enhanced disease after infection with RSV or after immunization with FI-RSV 72,73 .
Most importantly, this study has provided evidence that an adjuvanted protein subunit vaccine administered to newborns can protect from RSV infection. A single immunization with CAF08-adjuvanted pre-F vaccine at birth was able to prevent infection in the lungs and signi cantly reduce carriage in the nose (Fig. 5). A limitation of the challenge model is that the mice are no longer newborns when challenged post immunization. In addition, the murine RSV challenge model is semi-permissive, and carriage can occur without disease symptoms. Given their ability to mount different types of T-helper subsets relatively equally without intrinsic bias, CB6F1 mice were employed to enable determination of the T-helper phenotype induced by our formulations. However, CB6F1 experience less weight loss after RSV infection than Th2-prone BALB/c 74 . Although it is unclear if directing immune responses away from the neonatal Th2 bias could ameliorate lung pathology following infection, the robust ability of CAF08 to enhance Th1 responses and diminish viral carriage suggests that this adjuvantation system is well-suited for an RSV vaccine. In ltration of eosinophils and other immune cells was observed in all immunization groups except the saline group, which did receive viral challenge. This is in accordance with prior literature 75 , and likely contributes minimally to pathology, as evidenced by lack of weight loss. Excessive lung pathology in Post-F + Alum groups and Pre-F + Addavax groups may have been anticipated, but was not observed, possibly because CB6F1 mice were used rather than BALB/c. Future studies to further assess safety and e cacy in early life would therefore bene t from utilizing a non-human primate model for RSV infection 76, 77 .
In conclusion, our study describes a novel adjuvanted vaccine formulation that overcomes the neonatal bias towards Th2-mediated immunity and provide robust Th1 and CD8-mediated protective immunity against RSV. Using state-of-the-art phosphoproteomics, this study also sheds light on the molecular basis of age-speci c signal transduction upon receptor ligation and adjuvant synergy. The CAF08-adjuvanted pre-F vaccine induced an RSV-speci c, Th1-polarized immune response in newborn animals and therefore has great potential for future vaccine development for protection against RSV and other intracellular pathogens in early life.

Materials And Methods
Agonists and inhibitors R848 (TLR7/8 agonist; InvivoGen) was reconstituted in sterile water at a concentration of 3mM. TDB (Mincle agonist; Avanti Polar Lipids) was reconstituted in dimethyl sulfoxide (DMSO; Sigma-Aldrich) at a concentration of 20 mg/mL, heated at 60 ºC for 30 seconds, then brought to 2 mg/mL using sterile All agonists and inhibitors were veri ed to be endotoxin-free (< 1 EU/ml), as measured by Limulus amebocyte lysate (LAL) assay per the manufacturer's instructions (Charles River). Sterile DPBS or DMSO were included in experiments at appropriate concentrations as a negative control when indicated.
Liposome formulation Liposomes comprised of Dimethyldioctadecylammonium (DDA; Clauson-Kaas A/S) were prepared with or without adjuvants as indicated by using the thin lm method combined with high shear mixing. Liposome formulations were characterized with respect to the average intensity-weighted hydrodynamic diameter (z-average), polydispersity index (PDI) and zeta-potential (Laser-doppler electrophoresis) essentially as previously described 78  The conformation of pre-F after incorporation into DDA liposome formulations was evaluated by binding to monoclonal antibodies AM-14, 5C4 and Motavizumab by dot blot (Supplementary Fig. 2). The binding of each monoclonal to pre-F after incorporation con rmed the intact presence of three major antigenic sites 15,16 . The number of study participants used for each experimental approach is described in the text.

Isolation of human mononuclear cells and moDC generation
Heparinized blood from newborns and adults was centrifuged for 10 min at 500x g, then the upper layer of clear yellow plasma was removed. This platelet-rich plasma was then centrifuged for 15 min at 3000x g, and platelet-poor plasma was collected from the top and stored at + 4˚C. The remaining blood was reconstituted to its original volume by resuspending in DPBS. Then, reconstituted blood was layered on to Ficoll-Hypaque gradients (Ficoll-Paque Premium; GE Healthcare) and centrifuged for 30 min at 500x g.
After Ficoll separation, the mononuclear cell fraction was collected. Monocytes were then isolated from mononuclear cell fractions by positive selection with magnetic CD14 MicroBeads, performed according to the manufacturer's instructions (Miltenyi Biotec). Purity was routinely checked by ow cytometry and was always more than 98%. Isolated monocytes were seeded in 75 cm2 tissue culture dishes for 5 d at 37˚C in a humidi ed incubator at 5% CO2 with 10 6 cells/ml medium. Medium consisted of Cellgro DC medium (Cellgenix) supplemented with 1% penicillin-streptomycin (Life Technologies) and 10% autologous plasma. Cultures were supplemented with 50 ng/ml recombinant human IL-4 and 100 ng/ml recombinant human GM-CSF (R&D Systems). After 5 days, immature MoDCs were harvested by gently pipetting the loosely adherent fraction.

Phosphoproteomics
MoDCs were seeded in 6-well plates at 3-5 All adult formulations contained 2 µg pre-F antigen. Blood was drawn for antibody titer determinations 14 days after each immunization. Newborn cohorts were immunized in the rst week of life with half the adult dose (50 µL) s.c formulation and again 21 days later. Blood was also drawn for antibody titer determinations 14 days after each immunization.

RSV challenge
For the challenge study, CB6F1 mice were immunized in the rst week of life with a single dose of each formulation as indicated, and subsequently challenged intranasally with 5x10 6 PFU of wild-type RSV A2 in a volume of 100 µL under iso urane (2-3%) anesthesia at 8 weeks of age. These studies, conducted at the Vaccine Research Center (VRC) at NIH, were approved by the VRC Animal Care and Use Committee (protocol VRC-17-716). Several animals were euthanized on days 5 and 8 post-challenge to assess viral titer in the nose and lungs (day 5), and lung histopathology (day 8). The remaining 5 animals per group were monitored for weight loss through day 14. To measure viral load, noses and lungs were homogenized (using chilled mortar and pestle with ground glass for noses, and gentleMACS dissociator for lungs), and clari ed homogenates were titrated by serial dilution on HEp-2 cells as previously described (PMID 22144888). Virus titers are reported as the log 10 TCID50/g of tissue or per nose.

Lung Histopathology and Immunohistochemistry
Lung samples from mice were processed per a standard protocol. Brie y, the tissues were xed in 10% neutral buffered formalin, processed with Leica ASP6025 tissue processor (Leica Microsystems), embedded in para n, sectioned at 5 µm, and blocked in para n for histological analysis. Tissue sections were stained with hematoxylin and eosin (H&E) or PAS (Periodic Acid-Schiff) histochemical stains for routine histopathology and the for the location of Goblet cells and presence of airway mucin accumulation, respectively. Sections were examined, in a blinded manner, by a boarded-certi ed veterinary pathologist using an Olympus BX51 light microscope and photomicrographs were taken using an Olympus DP73 camera.
For immunohistochemical (IHC) evaluation, formalin-xed para n-embedded tissues sections (5 µm) obtained from mice, were used to perform immunohistochemical staining using an antibody which identi es eosinophils in tissue; A Goat Polyclonal EMBP (Santa Cruz; sc-33938) antibody at a dilution of 1:500. Staining was carried out on the Bond RX (Leica Biosystems) platform according to manufacturersupplied protocols. Brie y, 5µm-thick sections were depara nized and rehydrated. Heat-induced epitope retrieval (HIER) was performed using Epitope Retrieval Solution 1, pH 6.0, heated to 100 o C for 20 minutes. The specimen was then incubated with hydrogen peroxide to quench endogenous peroxidase activity prior to applying the primary antibody. Detection with DAB chromogen was completed using the Bond Polymer Re ne Detection kit (Leica Biosystems CAT# DS9800). Slides were nally cleared through gradient alcohol and xylene washes prior to mounting and placing cover slips.
Sections were examined by a boarded-certi ed veterinary pathologist using an Olympus BX51 light microscope and photomicrographs were taken using an Olympus DP73 camera.
Anti-pre-F antibody titer determination Microtiter plates were coated with 1 µg/mL pre-F. Serum from immunized mice was incubated in 10-fold serial dilutions onto coated plates at room temperature for two hours. Pre-F -speci c antibody isotypes were detected using HRP-labeled anti-mouse IgG (AH Diagnostics), IgG1 (Southern Biotech), IgG2a (AH Diagnostics), or IgG2c (Southern Biotech). Endpoint titers were subsequently derived from dilution curves using adaptation of Frey et al 83 . Brie y, titer was de ned as the reciprocal of the highest serum dilution generating a reading above a cutoff de ned as: where µX is the mean of independent control sera readings, SD is the standard deviation, n is the number of independent controls, t is the (1-a)th percentile of the one-tailed t-distribution with v=n-1 degrees of freedom. As an adaptation, all individual curves were t to 4-point log curve using nonlinear regression analysis. The approximate serum dilution (Y) that intersected with the above calculated cutoff was then calculated using: Cutoff=Bottom + (Span)/(1+10^((LogIC50-Y) *Slope)).
High-throughput uorescence plate reader neutralization assay cells from mice immunized with pre-F -containing formulations consistently stained positive (1-2%) with the RSV F235-249 tetramer, but not the CLIP tetramer. Cells from saline-injected mice did not stain positive with the RSV F235-249 tetramer. In addition, as further evidence of tetramer speci city, single cell sorting of tetramer-positive cells and clonal expansion with anti-CD3/CD28-coated microbeads (Thermo Fisher) generated a fully tetramer-positive CD4 + T cell clone ( Supplementary Fig. 4), further evidence of tetramer speci city.

Ex vivo recall assay
Cell suspensions from murine brachial lymph nodes and spleens were generated by mashing through a 70 µM strainer (Thermo Fisher). In spleen suspensions, erythrocytes were lysed by 2 minutes of incubation in ammonium chloride-based lysis buffer (BD Biosciences). Cells were then counted and  STRING-analysis illustrates the order of interaction between these identi ed proteins. Proteins signi cantly phosphorylated upon treatment are indicated in bold. (B-C) The phosphorylation of nine of these proteins was signi cantly increased upon stimulation (paired Student's t-test, all comparisons to vehicle condition) (B) The treatment-speci c phosphorylation of four proteins was con rmed by western blotting after separation of phosphorylated and unphosphorylated species of each protein using phos-tag gel electrophoresis. (C) Comparative intensity of the ve remaining analytes with signi cant differences in phosphorylation (D) Cross-presentation was con rmed in a MoDC:CD8 co-culture assay. MoDCs were pulsed with 1 µg/ml soluble protein antigen as indicated, in the presence or absence of adjuvants as indicated on the axes, and subsequently cultured with autologous CD4+ or CD8+ T cells for 4 days before evaluation of antigen-speci c T cell activation (N=5 adults, 2-way ANOVA with Tukey post-hoc test).   RSV pre-F protein combined with CAF08 protects mice from RSV infection. Newborn CB6F1 mice were challenged with live RSV A2 strain after single immunization at birth with the formulations indicated.
Viral load was measured in lungs and nose as described in the Methods. Adjuvantation of pre-F with CAF01 or CAF08 resulted in the greatest reduction in viral carriage in the lungs and nose.