Determining Immune and miRNA Biomarkers Related to Respiratory Syncytial Virus (RSV) Vaccine Types

Respiratory Syncytial Virus (RSV) causes serious respiratory tract illness and substantial morbidity and some mortality in populations at the extremes of age, i.e., infants, young children, and the elderly. To date, RSV vaccine development has been unsuccessful, a feature linked to the lack of biomarkers available to assess the safety and efficacy of RSV vaccine candidates. We examined microRNAs (miR) as potential biomarkers for different types of RSV vaccine candidates. In this study, mice were vaccinated with a live attenuated RSV candidate that lacks the small hydrophobic (SH) and attachment (G) proteins (CP52), an RSV G protein microparticle (GA2-MP) vaccine, a formalin-inactivated RSV (FI-RSV) vaccine or were mock-treated. Several immunological endpoints and miR expression profiles were determined in mouse serum and bronchoalveolar lavage (BAL) following vaccine priming, boost, and RSV challenge. We identified miRs that were linked with immunological parameters of disease and protection. We show that miRs are potential biomarkers providing valuable insights for vaccine development.


INTRODUCTION
Respiratory Syncytial Virus (RSV) is a cause of lower respiratory tract infection (LRTI) worldwide and is responsible for >30 million new LRTI episodes and up to 199,000 deaths in children under 5 years old resulting in more than 3.4 million hospital admission associated with severe RSV disease (1,2). The elderly population is also markedly affected by RSV (3). Currently, the only approved RSV prophylactic is palivizumab which is used for high-risk patients, but such treatment has limited applicability due to cost and treatment logistics (4)(5)(6)(7)(8)(9). Unfortunately, all efforts to develop a safe and effective RSV vaccine have been unsuccessful (10)(11)(12)(13)(14)(15)(16). Attempts in the 1960s to develop a formalin-inactivated RSV (FI-RSV) vaccine candidate were hampered by several factors, including lack of protection against RSV infection in infants and young children, and an association with vaccine enhanced disease that resulted in two deaths upon natural RSV infection of vaccinees (10,(17)(18)(19). Efforts to develop live attenuated RSV vaccine candidates using cold-passaging, chemical mutagenesis, or reverse genetics have also been unsuccessful largely due to over-or underattenuation, which currently cannot be precisely predicted (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30), and natural RSV infection does not provide long-term protective immunity. Several other RSV vaccine platforms have been developed including subunit (31-38), vectored (39-46), particle-based (47-57), or nucleic acid-based (58-63), but none are FDA-approved a feature linked to our incomplete understanding of the host immune response to RSV (10). There are several target populations for RSV vaccines: infants, young children, pregnant women, and the elderly (10). Due to the differences in these target populations, vaccine safety, efficacy, and platform strategies will need to be different (10,11,18,33).
By establishing measures of vaccine protection and disease, a wide range of promising vaccine candidates can be evaluated early in development.
The host immune response is important in the outcome of RSV infections (14,60,64,65), and an imbalance between Th1and Th2-type cytokines is understood to be responsible for a variety of inflammatory disorders (66,67). Biomarkers can be surrogates for clinical endpoints and are needed to improve vaccine design and efficacy. Small regulatory microRNAs (miRs) have fundamental roles in regulating the expression and function of key immunological mediators such as cytokines (68)(69)(70). miR expression profiles have been identified and shown to be useful predictors for several allergic inflammatory diseases (71)(72)(73)(74). In addition, specific miRs have been shown to function in regulating key pathogenic mechanisms in asthma and airway hyperresponsiveness, including polarization of adaptive immune responses, activation of T cells (75)(76)(77)(78), regulation of eosinophil development (79)(80)(81)(82)(83)(84) and modulation of cytokinedriven responses (68)(69)(70). miRs are stable, in a variety of tissues, bodily fluids, and sera allowing for sensitive and accurate measurements regarding the physiological state of the individual (72)(73)(74). miRs govern host gene expression by inducing mRNA degradation or translation inhibition and have a prominent role in determining the level of protein expression of host gene targets (85)(86)(87)(88)(89)(90). Several miRs can also upregulate target gene expression via regulation of promoter function (91,92). It has been shown that miR patterns of expression vary for numerous physiological processes that have been deemed useful for diagnosis of neurodegenerative disorders, autoimmune diseases, cardiovascular disease, and cancers; likewise miRs have also been implicated in infectious diseases (42, [93][94][95][96][97][98]. Assessing circulating miRs in the sera of patients has supported miR profiling as a powerful non-invasive biomarker tool. Previously, it was shown that RSV infection of normal tracheal epithelial cells (NTECs) with GFP-expressing RSV (rgRSV) downregulated the expression of multiple miRNAs (99). Of the 24 miRNAs, miR-221 was shown to regulate nerve growth factor (NGF), a key neurokinin that prevents apoptosis in respiratory cells (99). Later, RSV infection of type II respiratory epithelial cells was shown to induce expression of five and down-regulation of three microRNAs via an RSV G protein regulated mechanism (100). RSV deregulated miRNAs were demonstrated to regulate several key immunological pathways. In a follow-up study, RSV infection of normal human bronchoepithelial (NHBE) cells, miRNA deregulation was tied to mechanisms involving IFN beta and the transcription factor NF-κβ (101). We showed that RSV G and NS1/NS2 proteins can modulate miRNA expression (102,103). Several studies have investigated differential expression of miRNAs in clinical RSV infections and shown deregulated patterns that can be used as potential biomarkers of infections (104)(105)(106)(107)(108)(109). While these data show miRNA deregulation during infection, miRNA expression following vaccination with different RSV vaccine candidates under investigation is not well-understood and has the potential to identify safe vs. unsafe vaccine candidates.
As miRs regulate host gene responses, it is important to determine if miR profiles serve to predict safe, efficacious, or diseased vaccine outcomes, particularly since RSV lacks a licensed vaccine. To determine if patterns of miR expression may serve as a surrogate of RSV immunity or disease requires proof of biological relevance. Therefore, we identified miR biomarkers and immune correlates associated with RSV vaccination to establish baselines for biomarker expression across different vaccination types and strategies. Since serum miR profiles provide indications of how miRs may regulate the immune response induced by RSV vaccination or infection (50), serum miR profiles may also suggest vaccine disease outcomes. We hypothesized that RSV infection or vaccination would alter the pattern and tempo of miR expression and that this would be reflected in changes by the host immune or disease response. In the present study, we examined serum miRs in BALB/c mice at various time-points post-RSV vaccination or RSV challenge using several RSV vaccine types. A miR PCR array was used to identify miRs post-vaccination, post-boost, or post-RSV challenge, and correlated with immune parameters and markers of disease.

Mice
Specific-pathogen-free, 6-to-8 weeks old female BALB/c mice (The Jackson Laboratory) were used. Mice were maintained in microisolator cages with sterilized water and food ad libitum. All experiments were approved by and performed in accordance with the guidelines of the University of Georgia Institutional Animal Care and Use Committee (IACUC).

Viruses and Cell Culture
CP52 was a gift from Stephen Whitehead and Brian Murphy at LID, National Institute of Allergy and Infectious Diseases, Bethesda, MD. CP52 is a cold-passaged live attenuated vaccine strain that lacks the RSV G and SH genes and is derived from RSV B1. RSV A2 and CP52 were propagated in mycoplasma-free Vero E6 cells (ATCC CRL-1586) using DMEM (Gibco) containing 5% FBS (Hyclone) at 37 • C/5% CO 2 and 32 • C/5% CO 2 , respectively (110). Viral titers were determined by plaque assay on Vero E6 cells, and plaques were enumerated by an anti-F protein (clone 131-2A) immunostaining assay (111,112). Infections were performed in serum-free DMEM (SF-DMEM).

FI-RSV Preparation
The preparation of formalin-inactivated RSV (FI-RSV) vaccine was adapted from the FI-RSV Lot 100 method (113). Briefly, strain A2 was used to infect Vero E6 cells (MOI = 0.1), and at day 4 pi, the cells were lysed following scraping, sonicated, and clarified by centrifugation at 600 x g for 15 min at 4 • C.
The supernatant was transferred to a tube and filter sterilized using a 2 µm filter; the final protein concentration (determined by BCA) was adjusted to 1 mg/ml. Viral stocks were inactivated by the addition of 37% formalin (final dilution 1:4,000) and incubated at 37 • C for 3 days in agitation. FI-RSV was pelleted by ultracentrifugation for 2 h at 25,000 rpm, re-suspended in SF-DMEM at 1/25th of the original volume and adsorbed overnight at room temperature in 4 mg/mL aluminum hydroxide. The compound material was pelleted by centrifugation and the pellet was suspended in SF-DMEM and total virus inactivation was confirmed via plaque assay on Vero cells. This procedure resulted in an FI-RSV vaccine that is concentrated 100-fold and contains 16 mg/ml alum. The vaccine was aliquoted in 1 ml volumes and stored at 4 • C.

RSV GA2 Microparticle-Based Vaccine
A microparticle-based RSV G protein vaccine consisting of 3 µm CaCO 3 cores was prepared using alternating poly-I-glutamic acid (PGA, negative charge) and poly-I-lysine (PLL, positive charge) layering to build up to seven layers with an RSV G peptide CX3C motif linked to a cationic sequence added as the outermost layer (4). The composition of the seven-layer film was determined using amino acid analysis, which showed that a comparable amount of the peptide component was present in each vaccine batch. Endotoxin levels by limulus amebocyte lysate (LAL) assay were <0.1 EU/µg. The dispersity of the particle vaccines was monitored by dynamic light scattering (DLS). DLS is used to determine the size distribution profile of small particles in suspension or polymers in solution. This layer-by-layer microparticle vaccine has an apparent diameter of ∼150 nm for uncoated particles to about 400-500 nm for fully coated particles. Some particle aggregation was detected in each batch with a second population of particles in the 1,500-2,000 nm range.

Lung Virus Titers and Disease Endpoints
Lung virus titers were determined in treatment and control mice by plaque assay on Vero E6 cells (111). Briefly, lungs were aseptically removed from mice at day 5 post-RSV (10 6 PFU/mouse) challenge, and individual lung specimens were homogenized at 4 • C in 1 mL of SF-DMEM using a gentleMACS TM Dissociator (Miltenyi Biotec). Samples were clarified by centrifugation for 10 min at 200 × g and supernatants were transferred and stored at −80 • C. For the plaque assay, 10-fold serial dilutions of the lung homogenates were adsorbed to 90% confluent Vero E6 cell monolayers for 2 h, at 37 • C, overlaid with 1% methylcellulose medium and incubated at 37 • C for 5 days. RSV plaques were enumerated by immunostaining with monoclonal antibodies against RSV F protein (clone 131-2A) as previously described (112). Lungs from vaccinated and challenged mice were examined for disease pathogenesis, and as anticipated (54, 114,115), only the lungs from FI-RSV vaccinated mice challenged with RSV showed substantially enhanced disease (data not shown).

Indirect ELISA
RSV A2-specific and B1-specific IgG antibodies were detected by ELISA using 96-well-high binding plates (Corning) coated with 10 6 PFU/mL A2 or B1 in 0.05 M carbonate-bicarbonate buffer, pH 9.6. Sera were added to plates in serial dilutions. RSV-specific antibodies were detected with HRP-conjugated antibodies specific for mouse IgG (Southern Biotech) followed by the addition of SureBlue TMB-peroxidase substrate (KPL, Inc.) for 15 min. Antibody titers were determined as the last sample dilution that generated an OD450 reading of >0.2 (mean OD value of background plus 2 standard deviations of the mean).

Quantification of Cytokines
At day 3 post-A2 challenge, a subset of mice from each group was sacrificed and BAL and sera were collected. The mouse lungs were flushed three times with 1 ml of PBS and the retained BAL was centrifuged at 400 × g for 5 min at 4 • C. The recovered supernatants were collected and stored at −80 • C until assessed for cytokine concentration, and the cell pellet was suspended in 200 µL of FACS staining buffer (PBS containing 1% BSA). Total cell numbers were counted using a hemocytometer. The Luminex R xMAP system using a MILLIPLEX MAP mouse cytokine immunoassay (Millipore) was used to quantitate cytokines in cell-free BAL supernatants and sera according to the manufacturer protocol. Briefly, beads coupled with anti-IFNγ, anti-IL1α, anti-IL2, anti-IL4, anti-IL5, anti-IL6, anti-IL9, anti-IL10, anti-IL12p40, anti-IL13, anti-IL15, anti-IL17A, anti-MCP1, anti-RANTES, anti-TNFα, and anti-eotaxin monoclonal antibodies were sonicated, mixed, and diluted 1:50 in assay buffer. For the assay, 25 µL of beads were mixed with 25 µL of PBS (for BAL samples) or serum matrix (for serum samples), 25 µL of assay buffer and 25 µL of BAL supernatant or serum and incubated overnight at 4 • C. After washing, beads were incubated with biotinylated detection antibodies for 1 h and the reaction mixture was then incubated with streptavidin-phycoerythrin (PE) conjugate for 30 min at room temperature, washed, and suspended in PBS. The assay was analyzed on a Luminex 200 instrument (Luminex Corporation, Austin, TX) using Luminex xPONENT 3.1 software.

RNA Isolation
Blood was collected from mice via axillary vessels in 1.5 ml microcentrifuge tubes (Fisher), allowed to clot for 30 min at room temperature, and centrifuged at 900 × g for 10 min and 4 • C. Serum layer was transferred to a new microcentrifuge tube and centrifuged for 10 min at 16,000 × g and 4 • C, and the cleared supernatant was transferred to a new microcentrifuge tube without disturbing the pellet. One hundred microliter of serum sample per mouse was processed for RNA isolation using miRNeasy Serum/Plasma Kit (Qiagen) as per manufacturer's recommended protocol or stored at −80 till processing. Serum/Plasma C. elegans miR-39 Spike-In Control (Qiagen) was spiked into each sample prior to RNA purification as an internal control for miR expression profiling in serum to allow for monitoring of RNA recovery and purity, and reverse transcription efficiency. The RNA concentration and purity were determined by Qubit RNA assay broad range (Qubit RNA assay BR, Invitrogen) fluorometry. This reagent specifically binds to RNA only and does not detect DNA, protein or free nucleotides. Additionally, spectrophotometric analysis of all samples using Epoch Gen 5 spectrophotometer (Biotek) showed that all RNA samples had A260/280 ratios ≥1.8.

miR PCR Arrays and Data Analysis
First-strand cDNA synthesis was performe4d with 200 ng/total RNA from each sample using the miScript II RT kit with miScript HiSpec Buffer (Qiagen) following manufacturer protocol. Briefly, cDNA synthesis was performed at 37 • C for 60 min followed by inactivation at 95 • C for 5 min. First-strand cDNA was diluted 1:10 in molecular grade water and expression of 84 miRs having a role in T or B cell function was assessed using a miScript miR PCR Array Mouse T cell and B cell Activation (Qiagen) array following the manufacturer's protocol on a Stratagene Mx3000P/3005P real-time instrument. Each array plate contains oligos specific to 84 mature miRs validated to regulate T cell or B cell development and function as well as oligos for spike in (C. elegans miR-39), six housekeeping genes [small nucleolar/nuclear RNA (snoRNAs) SNORD61, SNORD95, SNORD96A, SNORD68, SNORD72, and RNU6B] and positive and negative controls for reverse transcription and PCR. Data obtained were analyzed with miScript miR PCR analysis template (Qiagen) using the C T method (116). miRs with fold change ≥2 were considered upregulated while miRs with fold change ≤0.5 were considered downregulated. RT-qPCR using miR-specific primers was then performed on differentially expressed miRs between treatment groups using a PCR array. The specificity of amplification was validated by dissociation curve analysis.

Statistical Analysis
All statistical analyses were performed using GraphPad Prism (ver 5.0; GraphPad). Statistical significance was determined using a one-way ANOVA or two-way ANOVA followed by Bonferroni's post-hoc comparisons tests; a p ≤ 0.05 was considered significant.

Pathway Analysis
Analysis of pathways regulated by differentially expressed miRs was carried out using DIANA miRPath ver 3.0 (117-119) using the microT-CDS database. The significance of pathway association was determined using a p-value threshold ≤0.05 and microT threshold of 0.8 using Fisher's exact test. When multiple miRs were analyzed together, data were filtered to identify pathway intersections instead of unions to identify common core pathways regulated by the miRs. Pathway hits were corrected for false discovery rate.

RESULTS
To assess the serum miR profiles in RSV-vaccinated or challenged mice, the mice were vaccinated (primed) with a live attenuated vaccine (CP52), an inactivated vaccine (FI-RSV), or a microparticle peptide-based vaccine carrying the G CX3C motif (GA2-MP) and boosted 3 weeks later. Three weeks post-boost vaccinated mice were i.n. challenged with 10 6 PFU A2, and the sera and lung tissues were collected from vaccinated and mocktreated mice at several time-points. Antibody responses to RSV were assessed to confirm that antigens induced a recall response upon vaccination and challenge. RSV A2 or B1-specific serum IgG was determined at 2 weeks post-boost and 5 days post-RSV challenge, and the levels of neutralizing antibody determined at day 5 pi. Lung viral titers were determined at 5 days postchallenge. Broadly, vaccination with CP52 or FI-RSV elicited a cross-reactive humoral response to either A2 or B1 relative to mock (Figures 1A,C, respectively). CP52 (but not FI-RSV) vaccination followed by A2 challenge elicited a strong antibody response (Figures 1B,D, respectively), which was neutralizing ( Figure 1E) and correlated with a statistically significant (p ≤ 0.05) reduction in lung viral titers ( Figure 1F). Despite the G protein CX3C motif having intra-strain conservation (120), it was less immunogenic as indicated by the lower anti-A2 and B1 IgG serum levels. These data show the prototypical responses associated with CP52, GA2-MP, and FI-RSV vaccination in mice (121,122).

RSV Vaccine Types and the Th1-and Th2-Type Response
Th1-or Th2-type responses were assessed by ELISPOT assays, and levels of IFNγ or IL4 expression (representing Th1-or Th2-type responses, respectively) were evaluated at day 14 postboost vaccination by re-stimulated splenocytes with RSV M2, F or G peptides. As expected, CP52 vaccinated mice had the highest frequency of IFNγ expressing cells compared to IL4 expressing cells (Figure 2A), while splenocytes from FI-RSV vaccinated mice had the highest frequency of IL4 expressing cells compared to IFNγ expressing cells ( Figure 2B). GA2-MP vaccinated mice had higher levels of G protein-specific IL4 secreting cells than IFNγ expressing cells, however this difference was not statically significant (p > 0.005). MCP1 and RANTES are chemokines involved in leukocyte recruitment to the airway, and to sites of inflammation in response to RSV infection (123)(124)(125). Given the role of these immune cell types in disease pathogenesis, levels of MCP1 ( Figure 2C) and RANTES ( Figure 2D) in sera and BAL were determined by multiplex cytokine/chemokine assays. MCP1 was localized to the lungs, with the highest level in the BAL from FI-RSV vaccinated mice although the level of expression was not substantially different between CP52 and GA2-MP vaccinated mice. In contrast, RANTES was expressed systemically, as evident by a higher level in the sera compared to BAL, with higher levels for FI-RSV and CP52 immune mice compared to GA2-MP vaccinated mice at day 3 post-RSV challenge. As anticipated, no detectable cytokine expression occurred following mock (PBS) treatment (data not shown). These data indicate that CP52 or FI-RSV vaccinated mice have an overall higher level of inflammation than mice vaccinated with GA2-MP upon RSV challenge. Other Th2-specific cytokines (IL4, IL5, IL6, IL10, and IL3) were higher in the sera (data not shown) and BAL of FI-RSV vaccinated  The data are presented as ELISPOTS/10 6 splenocytes. Three weeks after the boost mice were i.n. challenged with 10 6 PFU of A2. The level of (C) MCP1 and (D) RANTES were measured in sera and BAL supernatant by multiplex cytokine/chemokine assay and the data are presented as pg/mL of cytokine in BAL supernatant at day 3 post-challenge (n = 4-6 mice/group). The dashed line indicates the limit of detection (LOD) = 3.2 pg/ml. Error bars represent the SEM from n = 4 mice/group and results were considered significant with a *p ≤ 0.05, **p ≤ 0.01, and ****p ≤ 0.0001 as determined by two-way ANOVA and Bonferroni's test using GraphPad Prism ver. 8.0. mice compared to all other vaccinated groups which further confirm the biased Th2-type cytokine response associated with FI-RSV vaccination.

The Type of Vaccination Is Linked With Different miR Expression Patterns
Evaluating the Th1-or Th2-type cytokine response using accompanying assays is not efficient for testing of multiple vaccine candidates; however, the examination of miR biomarkers as correlates of the host immune response may aid vetting of safe or disease vaccine types, and considerably accelerate RSV vaccine research. The pattern of miRs can be readily evaluated using PCR (126) in a variety of fluids and tissues (127)(128)(129)(130)(131)(132)(133), there is sequence conservation across species (134), and miRs regulate key immunological processes (135). These features can be used to determine baseline data that may differentiate safe from disease risk vaccine types to aid the development of vaccine candidates. Since the memory T cell response is pivotal to development of immunity and disease, we analyzed 84 key miRs connected with T cell function in the sera from vaccinated mice, pre-and post-RSV A2 challenge, at several time-points, e.g., 1-week postprime/boost or 3d post-challenge. Total RNA was isolated from sera, used for cDNA synthesis, and miR expression was assayed using optimized primer-probes. Fold-changes in miR expression was plotted after normalization.
Analysis of the 84 miRs across all treatments identified miR expression signatures unique to prime-boost vaccination and RSV challenged mice, and those miR signatures conserved among all treatments (Figure 3). In general, each vaccine type, i.e., CP52, FI-RSV, or GA2-MP induced temporal and vaccine-specific miR expression patterns (Figure 3) where miR expression levels were heightened post-boost relative to prime and challenge. Given the differences in vaccine type and vaccination routes, differences were expected and emphasize miR patterns for their utility as potential vaccination biomarkers. Analogously, miR responses following A2 challenge of CP52 vaccinated mice compared to FI-RSV vaccinated mice correlated with safe vs. disease phenotypes of Th1-vs. Th2-type cytokines. Intergroup comparisons identified 58, 70 and 65 miRs differentially expressed in sera from CP52, FI-RSV, and GA2-MP vaccinated mice, respectively. Within groups, 8, 18, and 11 miRs were conserved between prime, boost and 3 days post-challenge for CP52, FI-RSV, and GA2-MP vaccinated mice, respectively. Of the miRs examined, let-7a-5p expression was upregulated >2SD in all vaccinated mice, thus this is likely not a biomarker for distinguishing vaccine-specific responses, but instead a general inflammatory biomarker ( Table 1).
The results indicate that the miR profiles and their tempos of expression are adjusted to the type of vaccine and challenge, an effect linked to both non-specific responses (e.g., inflammation) and specific immune responses (e.g., T cell activation or memory). It is important to note that some miRs are unaffected by vaccination while others undergo a global up-or down-regulation upon vaccination or challenge. For reasons of brevity, we have focused on understanding those miR expression patterns induced ≥2SD above the control. It is important to note that several immune regulatory molecules are miR targets. Specifically, cytokines/chemokines are immune effector molecules and are integrated in the net responses to Pathways regulated by miRs in Table 1 were analyzed using DIANA miRPath (117)(118)(119) using the microT-CDS database. E-values designate statistical confidence ascribed to gene hits for pathways using standard hypergeometric distribution and meta-analysis statistics (p < 0.05, Fisher's exact t-test). Pathways regulated by miRs in Table 1 were analyzed using DIANA miRPath (117)(118)(119) using the microT-CDS database. E-values designate statistical confidence ascribed to gene hits for pathways using standard hypergeometric distribution and meta-analysis statistics (p < 0.05, Fisher's exact t-test). NA, not applicable.

DISCUSSION
The development of safe and effective RSV vaccine candidates can be assisted by a better understanding of biomarker expression. Biomarkers may allow for the prediction of probable vaccine candidate outcomes. Additional analyses are needed to further aid decisions regarding vaccine candidates, but ways to improve RSV vaccine candidate selection has become paramount after more than 5 decades of unsuccessful research efforts. We hypothesized that assessment of miR profiles with general Th1/Th2 cytokine responses would enable correlations with safe, live vaccines (CP52), subunit vaccines (GA-M2), and diseaseenhancing vaccines (FI-RSV) to help develop baselines for a better understanding of prospective RSV vaccine candidates. In this study, show vaccine-specific and temporal miRNA expression profiles relating to efficacy or vaccine-associated disease. We examined miR expression profiles of vaccinated mice pre-and post-RSV challenge were determined for 84 miRs associated with T cell responses and function. We showed that while both CP52 and FI-RSV vaccination induce a humoral response, only CP52 induced a neutralizing antibody response leading to reduction in RSV replication ( Figure 1E). Further, splenocytes from CP52, FI-RSV, or GA2-MP vaccinated mice were stimulated with RSV-specific peptides then assayed for Th1type or Th2-type cytokines. The cytokine and miR expression showed that M2 82−90 re-stimulation of splenocytes from CP52 but not FI-RSV vaccinated mice led to a strong induction of IFNg which is characteristic of a Th1 response. In contrast, peptide stimulation of splenocytes from FI-RSV vaccinated mice led to a strong induction of IL4, a cytokine characteristic of a Th2-type response. Peptide stimulation of splenocytes from GA2-MP vaccinated mice led to a higher number of G 183−198 IL4-and IFN-specific secreting cells, characteristic of a balanced Th1-/Th2-type response to the G protein. Taken together, these results led to the assessment that CP52 vaccination primes for a safe response while FI-RSV primes for disease following vaccination and GA2-MP primes for a mostly balanced cytokine response. The miR PCR array showed differential expression of a conserved set of miRs across prime-boost vaccination and RSVchallenge, more specifically 11 miRNAs in GA2-MP vaccinated mice, 18 miRs in FI-RSV vaccinated mice, and 8 miRs in RSV CP52 vaccinated mice. Several of these miRs have been shown to participate in the regulation of the immune response, and in some cases are associated with RSV infection. FI-RSV vaccinated mice had let-7d-5p, let-7f-5p, and let-7g-5p miR expression at post-prime, post-boost, and post-challenge. GA2-MP vaccinated mice had similar results with let-7e-5p and let-7f expression. Members of the let-7 family target IL-6 expression, and has an extensive list of other experimentally validated targets including SOCS4, caspase-3, p27, TLR4, IL-13, and IL10 (101). Let-7 could be a mechanism of IL-6 regulation during RSV infection (101). RSV infection induces secretion of numerous pro-inflammatory cytokines, including type I and types II IFNs, TNFα, IL-12, and IL-6 (101, 154-156). Mice vaccinated with CP52 or GA2-MP induced differential miR-467f expression during prime-boost vaccination and post-challenge. Previous miR screens for respiratory viruses have not previously identified miR-467f; however, a microarray-based approach to evaluate the miR profile of HIV-associated nephropathy in a mouse model showed that treatment with rapamycin (an mTOR inhibitor) to halt disease progression induced upregulation of miR-467f expression (157). Interestingly, rapamycin inhibits RSV-induced mTOR activation and increases the frequency of RSV-specific CD8 T cells and RSV-specific memory T cell precursors in mice (158). Therefore, miR-467f may have a role in cellular immunity during vaccination and RSV infection. miR-106a-5p and miR-106b-5p expression levels were upregulated in FI-RSV vaccinated mice during prime-boost vaccination and post-challenge with RSV. Interestingly, allergic airway inflammation in mice has been associated with increased miR-106a expression and decreased IL-10, suggesting that miR-106a may regulate IL-10 expression and Th2-type responses (159,160). Expression levels of miR-30c-5p and miR-30e-5p (from the miR-30/384-5p family) were upregulated in FI-RSV vaccinated mice at prime and boostvaccination, and post-challenge. Upregulation of miR-30c-5p expression in the airway wall has been shown in a BALB/c mouse model of chronic asthma (161). The miR profiles identified for vaccine-induced protection and vaccine-enhanced disease appear to correlate with protective immune functions and airway inflammation, respectively. Although this study produced a list of miRs that may regulate RSV vaccine efficacy, additional studies are warranted to clarify the mechanisms behind how these miRs mediate host-virus interactions.
Overall, the results from these studies show that vaccine candidates associated safe or disease responses exhibited differential miR profiles following boosting which were higher in magnitude compared to priming or RSV challenge sera specimens. The results demonstrate that a considerable number of miRs are different between vaccine types, and a common set of miRs is expressed for all vaccine treatments. Pathway analysis of miR targets identified pathways correlated with inflammation particularly those that may contribute to airway inflammation, leukocyte recruitment and alveolar infiltration (12,162,163). The miR profiles from vaccinated mice were linked to cytokine phenotypes of protection or disease and appear to correlate with miRs that regulate protective immune functions or airway inflammation. Additional studies are warranted to validate miR phenotypes to determine the mechanisms of action linked to host gene regulation, and the associated immune response to determine their value as predictive biomarkers. These studies show that serum miR profiles may offer a proxy to assist vaccine development and facilitate a better understanding of vaccine studies.

ETHICS STATEMENT
Specific-pathogen-free, 6-to-8 weeks old female BALB/c mice (The Jackson Laboratory) were used. Mice were maintained in microisolator cages with sterilized water and food ad libitum. All experiments were performed in accordance with the guidelines of the University of Georgia Institutional Animal Care and Use Committee (IACUC).

AUTHOR CONTRIBUTIONS
LA, PJ, AB, and RT conceived, designed, performed, analyzed the experiments, and wrote the manuscript. RT contributed reagents, materials, and analysis tools.