DOI: 10.5061/dryad.98sf7m0sg
title: Data from: A multivalent mRNA-LNP vaccine protects against Clostridioides difficile infection

description: Clostridioides difficile infection (CDI) is an urgent public health threat
 with limited therapeutic options. In this work, we developed a messenger
 RNA (mRNA)-lipid nanoparticle (LNP) vaccine targeting C. difficile toxins
 and virulence factors. This multivalent vaccine elicited robust and
 long-lived systemic and mucosal antigen-specific humoral and cellular
 immune responses across animal models and independent of changes to the
 intestinal microbiota. Vaccination protected mice from lethal CDI in both
 primary and recurrent infection models, and inclusion of non-toxin
 cellular and spore antigens improved decolonization of toxigenic C.
 difficile from the gastrointestinal tract. Our studies demonstrate
 mRNA-LNP vaccine technology as a promising platform for the development of
 novel C. difficile therapeutics with potential for limiting acute disease
 and promoting bacterial decolonization.
description: mRNA design and production The amino acid
 sequence of the TcdA and TcdB receptor binding domain (RBD) containing the
 combined repetitive oligopeptides (CROP) motifs were obtained from GenBank
 accession numbers P16154.2 and P18177.3 respectively. The amino acid
 sequences of PPEP-1 and CdeM was obtained from GenBank accession number
 Q183R7, and WP_009893169.1, respectively.  The putative N-glycosylation
 sites were disrupted by substituting the asparagine residue at the
 predicted N-glycolysation site with a glutamine (N to Q) to prevent
 posttranslational epitopes masking in eukaryotic cells. The sequences
 underwent codon optimization and GC enrichment using our proprietary
 algorithm to improve expression and reduce potential immunogenicity of the
 in vitro transcribed mRNA. The codon-optimized sequences were gene
 synthesized by Genscript with an optimized and modified IL-2 secretion
 signal, and cloned into our proprietary in vitro transcription template
 containing an optimized T7 promoter, 3’UTR, 5’UTR and a 100-adenine tail.
 The TcdA, TcdB, PPEP-1, and CdeM nucleoside modified mRNA sequences were
 prepared using the MegaScript transcription kit (ThermoFisher Scientific),
 co-transcriptionally capped using the CleanCap™ system (TriLink
 Biotechnologies) and purified using a modified cellulose based
 chromatography method (36), precipitated, eluted in nuclease free water,
 and quantified using the NanoDrop One system. Length and integrity were
 determined using the Agilent BioAnalyzer 2100 system. Endotoxin content
 was measured using the GenScript Toxisensor chromogenic assay, and values
 were below detection levels (0.1 EU/mL). mRNA was frozen at -20oC until
 formulation. Production and characterization of
 mRNA-LNP vaccines The hydrodynamic size,
 polydispersity index (PDI) and zeta potential of mRNA-LNPs were measured
 using a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). The mRNA
 encapsulation efficiency was determined using a modified Quant-iT
 RiboGreen RNA assay (Invitrogen). Endotoxin levels were determined using
 the Limulus Amebocyte Lysate (LAL) chromogenic assay and found to be
 &lt;0.5 endotoxin unit (EU)/mL. Phylogenomic
 analyses Genomic analyses were conducted on 137
 <em>C. difficile</em> isolates sourced from the work of
 Bushman, Frederic D., et al(12). Initial quality control was implemented
 with FastQC v0.12.1 and MultiQC v1.14 (37). Subsequently, de novo assembly
 was performed on the quality-assessed raw reads utilizing Shovill v1.1.0
 (38). The genome assemblies were subjected to quality evaluation with
 CheckM v1.1.6 and BUSCO v5.4.7(39, 40). Genome annotations were then
 performed employing bakta v1.8.1(41). The core-genome alignment was
 carried out using Roary v3.13.0(42),  which subsequently was used to infer
 the maximum likelihood phylogeny in RAxML-NG v1.1(43) using the general
 time-reversible (GTR) substitution model (44) accounting for among-site
 rate heterogeneity using the Γ distribution and four rate categories(45)
 for 10 individual searches with maximum parsimony random-addition starting
 trees and random topology starting tree. Node support was evaluated with
 100 nonparametric bootstrap pseudoreplicates (46). The phylogeny was
 rooted at the midpoint using the Tree Of Life (iTOL) v6.8 interactive
 tool(47). For subsequent visualization and annotation of the phylogeny,
 ggplot2, and ggtree were utilized (48, 49).
 Comparative Genomic Analysis of Protein Domains
 The amino acid sequences for the TcdA CROP domain, TcdB CROP
 domain, PPEP-1, and CdeM (excluding the IL-2 secretion signal domain) were
 extracted from UniProt Knowledgebase with primary accession numbers of
 P16154 from position 1840 to position 2710, P18177 from position 1851 to
 position 2366, Q183R7 from position 27 to position 220, WP_009893169.1
 from position 2 to position 164, respectively. These sequences served as
 the seed sequences for constructing a protein-type BLAST database.
 Subsequently, BlastX was applied to execute a comparative analysis of the
 amino acid sequences in the database against the 137 genomes included in
 the phylogenetic assessment. The hits with the highest bit score from
 BlastX were utilized to determine the percentage of coverage and
 identity(50-57). Identification of Toxigenic Strains
 via Comparative Genomic Analysis The coding sequences
 for tcdA, tcdB, tcdC, tcdE, tcdR, cdu1, and cdd1 were sourced from the
 comprehensively annotated complete genome of <em>C.
 difficile</em> 630 (NCBI Reference Sequence: NC_009089.1). The
 nucleotide sequence for the PaLoc region was inferred and extracted by the
 locations of the flank genes cdu1 and cdd1. These nucleotide sequences
 served to construct an original nucleotide-specific BLAST database. To
 enhance the likelihood of detecting diverse toxin genes, additional
 sequences of toxin genes and the PaLoc region were extracted from 54
 representative toxigenic strains and incorporated into the database. The
 raw sequencing reads from 54 representative toxigenic strains were
 downloaded and assembled (58). The initial round of blastn was conducted
 against the genomes of these 54 toxigenic strains using the original
 database, setting the parameters to accommodate more gaps and mismatches
 to boost the coverage of hits. The sequences from the highest-scoring hits
 were subsequently extracted and used as seed sequences in a new BLAST
 database. Genomes of good quality were also used to infer and extract
 sequences from the PaLoc region using flanking genes cdd1 and cdu1. A
 conserved non-coding region of 115 bp was employed as a marker for
 non-toxigenic strains (59). A second round of blastn was performed against
 the 137 genomes included in the previous phylogenetic analysis, using
 sequences from the new BLAST database. An arbitrary threshold of 80%
 coverage and 80% identity was applied to the highest-scoring hits and used
 as criteria for determining the presence or absence of genes. Strains were
 classified as non-toxigenic if they exhibited the 115 bp conserved region
 and lacked toxin genes or the PaLoc region, and vice versa.
 <em>In vitro</em> transfection of mRNA-LNP and
 Western blot Neuro2a cells were seeded in 12-well
 plates at a density of 0.2x106 cells per well and transfected 24 hours
 post-seeding at a confluence of around 80%. mRNA-LNPs (1 mg/mL) were
 diluted (1:10) in PBS and added to each well at a final dose of 2.5
 µg/well. Medium was aspirated, and cells were directly lysed 24 hours
 post-transfection using the EZLys tissue protein extraction reagent
 (BioVision #8002-500) supplemented with 1X protease inhibitors
 (Sigma-Aldrich, #0493116001). Untreated cells were used as controls.
 Lysate was clarified at 15,000 rpm for 5 minutes using a refrigerated
 centrifuge to pellet cell debris, and protein concentration in the
 supernatant was determined using the Pierce microBCA assay. A total of 12
 μg of cell lysate was loaded into a 4-15% polyacrylamide gel (Bio-Rad
 #4561083) and transferred to a PVDF membrane using the ThermoFisher iBlot
 system (dry transfer). Membranes were blocked with 5% skimmed milk
 extract, washed, and incubated for 2 hours with primary antibodies against
 TcdA and TcdB (Abcam 19953 and 252712) at a final concentration of 4
 µg/mL. PPEP-1 was detected using a polyclonal serum from immunized mice
 (Boosted sera at 1:100 dilution, incubation 4 hours). Membranes were then
 washed three times using 1X TBST and incubated for 1 hour in the presence
 of an HRP-conjugated donkey anti-goat IgG (Abcam 97040) to detect TcdA and
 TcdB. PPEP-1 was detected using mouse polyclonal sera. Proteins were
 visualized using ECL detection reagent (Cytiva #RPN2209) on a GE
 ImageQuant™ system. Cell viability
 measurements Cells that are relevant for toxicity
 assessment and representative of different tissues that come in to contact
 with the expressed immunogens encoded on our mRNA-LNPs were used for in
 vitro transfection and assessment of viability post-transfection. HUVEC
 (CRL-1730), Caco-2 (HTB-37), and human primary skeletal muscle cells
 (PCS-950-010) were cultured as per ATCC using F12K Medium (ATCC 30-2004),
 EMEM (ATCC 30-2003), and Mesenchymal Stem Cell Basal medium (ATCC
 PCS-500-030) respectively. The muscle cells were cultured in the presence
 of the Primary Skeletal Muscle Growth Kit (ATCC PCS-950-040). Cells were
 seeded in 96-well plates at a density of 10,000 cells per well and
 transfected with 1 or 3 µg TcdA or TcdB mRNA-LNP.  Full-length recombinant
 TcdA (8619-GT-020) or TcdB (6246-GT-020), recombinant Luciferase mRNA-LNP
 (Luc LNP), and untreated cells were used as controls. After incubation for
 24 or 48 hours, 50 µL XTT cell proliferation reagent (Roche 11465015001)
 was added to each well, and plates were incubated for an additional 5
 hours before measuring absorbance at 492 nm using the Thermofisher
 Varioskan plate reader. Cell viability was calculated relative to the
 untreated cells. Generation of recombinant TcdA, TcdB,
 PPEP-1, and CdeM proteins The region encoding the TcdA
 and TcdB receptor binding domains (including CROP domain), the full length
 PPEP-1 coding sequence, and the full length CdeM coding sequence was
 codon-optimized for expression in E. coli, gene synthesized at Genscript,
 and cloned into the pET30a vector. An N-terminal 6XHIS tag followed by the
 TEV recognition/cleavage site was introduced after the start (ATG) codon
 of all three constructs to allow for purification using affinity
 chromatography. E. coli BL21(DE3) was transformed, and positive clones
 were used to inoculate 1 L cultures. Recombinant proteins from the
 supernatant were concentrated and purified using the NI-IDA column on an
 AKTA Avant 150 system. Purified proteins were cleaved with TEV protease,
 buffer-exchanged into PBS and 5% sucrose (pH 7.4), filter-sterilized,
 quantified using the micro-BCA assay (Pierce), and aliquoted and stored at
 -80ºC. Aliquots were tested for precipitation following multiple freeze
 and thaw cycles and the purity was determined using densitometric analysis
 of a Coomassie blue stained SDS-PAGE gel under reducing conditions. All
 proteins displayed more than 90% purity and had the predicted molecular
 weights using Western blot. Spleen and lymph node
 harvests Spleens were collected, processed as single
 cells, filtered using 70 µm cell strainers in complete RPMI 1640,
 centrifuged, and red blood cells lysed in ACK lysis buffer to obtain a
 clear single cell suspension. Splenocytes were resuspended in 1 mL
 complete RPMI 1640 media, counted, and used immediately for studies.
 Draining lymph nodes (inguinal and popliteal) were collected and processed
 as described above (without ACK). Cells from the dLNs were resuspended in
 250µL RPMI 1640 media, counted, and used immediately.
 Production of fluorescently labeled recombinant proteins for
 antigen-specific B cells Fluorescently-labeled
 recombinant TcdA, TcdB, and PPEP-1 protein were prepared using the
 Lightning-Link R-Phycoerythrin (R-PE) and Lightning-Link (R) Rapid Alexa
 Fluor 647 conjugation reagents (Novus Biologicals, 703-0010 and 336-0005).
 TcdA, TcdB, and PPEP-1 were diluted to 0.5 mg/mL in PBS and 50μg reacted
 in the presence of 1:10 (v/v) of LL modifier for 3 hours at room
 temperature. The labeling reaction was stopped in the presence of 1:10
 (v/v) LL quencher for 30 minutes and stored at 4°C until use.
  Flow cytometry analysis of T and B cells
 <strong>Tfh cells</strong>: 2 million cells were
 stained with anti-mouse CD16/32 antibody for 20 minutes and stained with
 an anti-CXCR5- biotin for 30 minutes on ice, washed twice and incubated
 with Streptavidin BV-421 in the presence of surface antibodies (Table S5)
 for 30 minutes, washed, fixed and permeabilized with the
 FoxP3/Transcription Factor Staining Kit (eBioScience) according to the
 manufacturer instructions and stained for Bcl6. Following intracellular
 staining, cells were washed twice, fixed in 300 µL 1% paraformaldehyde for
 acquisition. The gating strategy, as well as the antibody list and catalog
 numbers are provided (Table S5; fig. S6A).
 <strong>Germinal Center (GC) and Memory B
 cells</strong>: 2 million cells per sample incubated with anti-mouse
 CD16/32 antibody for 20 min at 4°C. Cells were then washed with FACS
 buffer (2% FBS in PBS) and stained for 1 h using antibodies (Table S3).
 Following staining, cells were washed twice, and fixed in 300 µL 1%
 paraformaldehyde for acquisition. The gating strategy, as well as the
 antibody list, fluorescent TcdA and TcdB RBD probes, and catalog numbers
 are provided (Table S3, fig. S6B-D). <strong>T
 cells</strong>: 2 million splenocytes were stimulated with 2.5 µg/mL
 of TcdA CROP, or TcdB CROP, or PPEP-1 peptide pools (15 mers, 4 amino acid
 overlapping peptide pool) in a FACS tube for 6 hours at 37oC, 5% CO2 with
 2 mg/mL anti-CD28 (Tonbo, 40-0281-M001) providing co-stimulation.
 Stimulations proceeded for 1 hour before adding 5 mg/mL brefeldin A
 (Biolegend, 420601), 2 mM monensin (Biolegend, 420701), and 5 mg/mL
 anti-CD107a (Biolegend, 121610) Alexa Fluor 647 for 5 hours. DMSO served
 as a negative control and the combination of 50 mg/mL phorbol 12-myristate
 13-acetate and 1 mg/mL ionomycin served as a positive control. After a
 total of 6 hours, samples were washed with PBS, stained with Live/Dead
 Aqua for 5 minutes, blocked using anti-mouse CD16/32 antibody for 20
 minutes, and stained extracellularly for 30 minutes using antibodies
 (Table S4). Cells were washed in FACS buffer, fixed and permeabilized
 using the Cytofix/Cytoperm kit (BD Biosciences, 554714), and stained
 intracellularly using antibodies for 30 min (Table S4). Following
 intracellular staining, cells were washed twice, and fixed in 300 µL 1%
 paraformaldehyde for acquisition. The gating strategy, as well as the
 antibody list and catalog numbers are provided (Table S4; fig.
 S9A). All samples were acquired on a BD LSR II equipped
 with 4 laser lines and 18 PMTs and data were analyzed in FlowJo
 v10. <em>In vivo</em> toxin
 neutralization assays To determine the LD100 of
 intraperitoneally injected TcdA and TcdB, recombinant toxins were prepared
 at doses of 5, 10, 25, 50, 100, and 125ng in 200μL of sterile PBS.  Naïve
 7-week-old C57BL/6J mice were randomly assigned into groups of 5.
 Recombinant proteins were injected i.p. and mice were monitored for
 behavior, body condition, and mortality every 4 hours post-injection.
 Doses were selected for use in subsequent studies by their ability to kill
 100% of animals within 4 hours after injection. To determine
 neutralization capacity of vaccine-elicited immune response, mice were
 immunized twice i.m. with 1 or 5mg of TcdA or TcdB monovalent mRNA-LNP or
 monovalent recombinant protein vaccine with or without alum adjuvant
 (details on vaccine preparation below). Two weeks after last immunization,
 immunized mice were either challenged i.p. with five times the previously
 determined LD100 (625ng rTcdA, 125ng rTcdB) and monitored for behavior,
 body condition, and mortality every 4 hours post-injection or terminally
 bled. Serum from immunized mice was diluted 1:20 in PBS and incubated with
 rTcdA or rTcdB LD100 (150 or 25 ng respectively) for 1 hour at 37 °C.
 Naïve mice were then challenged i.p. with recombinant toxin alone,
 monovalent immunized serum + recombinant toxin, or control (unvaccinated)
 serum + recombinant toxin and monitored for behavior, body condition, and
 mortality every 4 hours post-injection.  Mouse
 immunization and <em>C. difficile</em> infection
 5-week-old male and female C57BL/6J mice (Jackson Laboratories,
 strain no. 000664) were used in most studies and BALB/c (Jackson
 Laboratories, strain no. 000651) were used where indicated. All mice were
 maintained under specific pathogen-free conditions at either the
 Children's Hospital of Philadelphia or the University of
 Pennsylvania. Multivalent mRNA-LNPs (1-5 mg) were mixed at 1:1 w/w ratio,
 diluted up to 50μL in PBS, and administered intra-muscularly into the hind
 leg within two hours of thaw. Recombinant protein vaccines were mixed 1:1
 with alhydrogel (aluminum hydroxide 2% w/v). 28 days later, mice were
 either euthanized for analysis of vaccine-induced humoral and cellular
 immune responses after single immunization or they were boosted with the
 same dose. 14 days later, mice were either euthanized for analysis of
 vaccine-induced immune responses after second immunization or they were
 infected with <em>C. difficile</em> as previously described
 (28). Antibiotic treatment was administered by providing 0.5g/L
 cefoperazone in their drinking water ad libitum for 5 days, followed by a
 2-day recovery period before <em>C. difficile</em> infection
 via oral gavage. Two different <em>C. difficile</em> strains
 were utilized where indicated: VPI 10463 at a dose of 1x106 spores and
 CD196 at a dose of 1x105 spores. Mice were monitored daily for survival
 and weight loss, and mice were euthanized when weight loss exceeded 20% of
 their original body weight. For studies to analyze
 immune responses vaccination after <em>C. difficile</em>
 infection, 5-week-old C57BL/6J mice were infected with 1x105 spores
 <em>C. difficile</em> CD196 as described above. 14 days
 post-infection, mice were immunized with 1mg mRNA-LNPs. 28 days later,
 mice received a second immunization. 14 days later, serum and feces were
 collected for analysis of vaccine-induced humoral immune
 responses. For long-term studies, C57BL/6J mice were
 immunized as described above. 40 days after last immunization, mice were
 either euthanized for analysis of vaccine-induced immune responses or
 infected with <em>C. difficile</em> as described
 above. For re-challenge studies, C57BL/6J mice were
 re-infected with <em>C. difficile</em> 200+ days after primary
 infection as described above. Hamster
 studies Syrian hamsters (HsdHan: AURA, Envigo, catalog
 no. 8901M) were maintained at University of Texas Medical Branch.
 4-5-week-old male golden Syrian hamsters were pre-bled and vaccinated
 intra-muscularly with trivalent mRNA-LNP vaccine (1:1:1 w/w ratio),
 trivalent recombinant protein + alhydrogel, or empty lipid nanoparticles
 at week 0 and 3. Body weight and clinical signs were collected at days 0,
 1, 3, 5, 7, and 14 post-prime and at day 1, 2, 3, 7, and 14 post-boost.
 Feces were collected 14 days after each immunization. Serum was collected
 at days 0, 21, 28, and 45 post-prime. Hamsters were sacrificed as per
 university protocols and observed for gross abnormalities – no findings
 were recorded. Detection of antibodies in serum and
 feces 96-well High Bind StripwellTM plates (Corning)
 were coated overnight with 1 mg/mL of purified TcdA (CROP), TcdB (CROP),
 PPEP-1, or CdeM. Plates were washed once with wash buffer (0.5% Tween-20
 in PBS) and blocked for two hours at room temperature using a solution of
 heat-inactivated, IgG-depleted, protease-free bovine serum albumin (2% w/v
 BSA in PBS). After blocking, plates were washed three times with wash
 buffer. Feces were homogenized in 1 mL PBS and centrifuged for 10 minutes
 at 10,000 x g. Sera or fecal supernatants were serially diluted in the
 blocking solution, added to plates, and incubated for two hours at room
 temperature. Plates were washed three times before the addition of
 horseradish peroxidase-conjugated anti-mouse secondary antibody specific
 to total mouse IgG (1:10,000) and IgA (1:5,000), anti-rhesus IgG
 (1:20,000), or anti-hamster IgG (1:8,000) in blocking buffer. Plates were
 incubated for 1.5 hours and washed three times before the addition of
 tetramethylbenzidine (TMB) substrate solution. The reaction was stopped by
 adding 2 N sulfuric acid, and the absorbance was measured at 450 nm using
 a SpectraMaxTM 190 microplate reader. Antigen-specific antibody end-point
 dilution titer was defined as the highest dilution of serum or feces to
 give an OD greater than the cut-off OD value determined using the Frey
 Method (60). Fecal antibody titers were normalized to gram of
 feces. <em>C. difficile</em> enumeration
 and toxin titers <em>C. difficile</em>
 burdens were quantified by collecting fecal samples at indicated
 timepoints and plating on taurocholate cycloserine cefoxitin fructose agar
 (TCCFA). <em>C. difficile</em> toxin titers were quantified in
 the stool using a previously described Vero cell cytotoxicity assay (28).
 Briefly, fecal samples were homogenized in sterile PBS, pelleted, and
 supernatant was filtered through a 0.2µm filter. Supernatant was diluted
 serially and incubated overnight with Vero monolayers (ATCC-CCL-81). Toxin
 titers in stool were calculated as the reciprocal value of the highest
 dilution that rounded 100% of the cells and normalized per gram of
 feces. Histology and scoring
 Samples were fixed in 10% neutral buffered formalin (NBF)
 prefilled HistoTainer™ II (Simport, M96160FW), dehydrated in graded
 ethanol series, cleared with Xylene, and embedded in paraffin. Sections (5
 µm) were collected on Superfrost™ Plus stain slides (Fisher Scientific,
 Ottawa, ON, Canada), and stained with Hematoxylin and Eosin. Slides were
 scanned using a NanoZoomer digital slide scanner (Hamamatsu, Boston, MA,
 USA) and visualized using the NDP® view 2.0 software (Hamamatsu, Boston,
 MA, USA). Sections were scored in a blind manner by a pathologist based on
 previously described criteria (30). Histological scores were reported as a
 cumulative score of three independent criteria: inflammation, edema, and
 epithelial cell damage. DNA extraction from
 stool Mice were co-housed for one week before
 experimental manipulation. Stool samples were collected at indicated
 timepoints for microbiota analysis. Microbial genomic DNA was extracted
 using DNeasy Power Soil Kit (Qiagen) according to manufacturer’s
 instructions. 16S rRNA gene library prep 
 Barcoded PCR primers annealing to the V4 region of the 16S rRNA
 gene were used for library generation.  PCR reactions were carried out in
 duplicate using Q5 High-Fidelity DNA Polymerase (NEB, Ipswich, MA).  Each
 PCR reaction contained 0.5μM of each primer, 0.34 U Q5 Pol, 1X Buffer, 0.2
 mM dNTPs, and 5.0μl DNA in a total volume of 50μl.  Cycling conditions
 were as follows: 1 cycle of 98ºC for 1 minute; 20 cycles of 98ºC for 10
 seconds, 56ºC for 20 seconds, and 72ºC for 20 seconds; and 1 cycle of 72ºC
 for 8 minutes.  After amplification, duplicate PCR reactions were pooled
 and then purified using a 1:1 volume of SPRI beads.  DNA in each sample
 was then quantified using PicoGreen and pooled in equal molar amounts.
  The resulting library was sequenced on the Illumina MiSeq using 2x250 bp
 chemistry.  Extraction blanks and DNA-free water were subjected to the
 same amplification and purification procedure to allow for empirical
 assessment of environmental and reagent contamination. Positive controls,
 consisting of five artificial 16S gene fragments synthesized in gene
 blocks and combined in known abundances, were also included.
 Bioinformatics processing and statistical analysis
 Sequence data were processed using QIIME2 (61). Read pairs were
 processed to identify amplicon sequence variants with DADA2 (62).
  Taxonomic assignments were generated by comparison to the Silva reference
 database version 132 (63), using the naïve Bayes classifier implemented in
 scikit-bio (64). A phylogenetic tree was inferred from the sequence data
 using MAFFT (65).  Similarity between samples were assessed by weighted
 and unweighted UniFrac distance (66, 67). Data files from QIIME were
 analyzed in R environment for statistical computing. Linear mixed effects
 models were used at each time point to estimate the mean difference
 between study groups. Cage information was added to the models as the
 random effect to account for the coprophagic nature of mice. Relative
 abundances of bacteria were log10 transformed and modeled as the outcome.
 Only the bacteria with at least 1% mean relative abundance across samples
 were tested. Community-level differences between sample groups were
 assessed using the PERMANOVA test (24, 68). When multiple tests were done,
 p-values were corrected for false discovery rate using Benjamini-Hochberg
 method.  Non-human primate studies
 An 18-year-old male rhesus macaque (N = 1) was injected i.m. in
 the right deltoid with 200 μg of tetravalent mRNA-LNP vaccine (TcdA, TcdB,
 PEPP-1, and CdeM) in 250μL total volume (1:1:1:1 w/w or 50µg/immunogen). A
 second age-matched male rhesus monkey (N = 1) was i.m. injected in the
 right deltoid with 200 μg of an eDHFR-tagged SARS-CoV-2 S2P mRNA-LNP
 vaccine. NHPs received two immunizations separated by 21 days, and blood
 was collected at day 1, 8, 21, and 35 post injection. Plasma and PBMCs
 were separated and stored until use. Statistical
 analysis Data are presented as mean ± SD or mean ± SEM
 where indicated. Graph schematics and illustrations were created using
 iTOL v6.8 (47), SPICE 6 (69) and Graphpad prism V10.0.2
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description: # Data from: A multivalent mRNA-LNP vaccine protects against
 Clostridioides difficile infection ## Description of the data and file
 structure We have submitted our raw data for each figure as it appears in
 the paper (adn4955_SourceData_FigureX.csv). File names indicate the
 specific figure from the manuscript (ie. FigureX indicates a main figure,
 where X equals 1-4; FigureSX indicates a supplemental figure, where X
 equals 1-16). Sheets in each file represent panels in an indicated figure
 (ie. adn4955_SourceData_Figure1.csv includes sheets labeled "Figure
 1b", "Figure 1c", "Figure 1d", "Figure
 1e", "Figure 1f", and "Figure 1g"). We have also
 submitted core genome alignments
 (adn4955_SourceData_Figure1A_CoreGeneAlignment), and a zip file included
 with Shovill v1.1.0 used for phylogenomic analysis as described in the
 Materials and Methods.  #### Variable definitions for immune response to
 vaccination datasets * *Antigen-specific IgG titers:* Concentration of IgG
 antibodies specific to *C. difficile* toxin A (TcdA), toxin B (TcdB),
 Pro-Pro endopeptidase 1 (PPEP1), or *C. difficile* exosporium morphogenic
 protein (CdeM) in serum samples. Units: Arbitrary units (AU) *
 adn4955_SourceData_Figure1.csv ; *Figure 1B* *
 adn4955_SourceData_Figure1.csv ; *Figure 3A* *
 adn4955_SourceData_Figure4.csv ; *Figure 4B* *
 adn4955_SourceData_Figure4.csv ; *Figure 4J* *
 adn4955_SourceData_FigureS2.csv ; *figure S2A-C* *
 adn4955_SourceData_FigureS3.csv ; *figure S3A-B* *
 adn4955_SourceData_FigureS4.csv ; *figure S4* *
 adn4955_SourceData_FigureS5.csv ; *figure S5A-B* *
 adn4955_SourceData_FigureS8.csv ; *figure S8C* *
 adn4955_SourceData_FigureS15.csv ; *figure S15* * *Number of Tfh per
 100000 cells (dLN):* Count of T follicular helper cells (Tfh) per 100,000
 total cells in draining lymph nodes (dLN). Units: Cells per 100,000 *
 adn4955_SourceData_Figure1.csv ; *Figure 1C* * *Number of GC B cells
 (spleen):* Total count of germinal center (GC) B cells in the spleen.
 Units: Absolute cell count * adn4955_SourceData_Figure1.csv ; *Figure 1D*
 * adn4955_SourceData_FigureS7.csv ; *figure S7A* * *Number of
 antigen-specific B cells (spleen):* Count of B cells specific for TcdA,
 TcdB, or PPEP-1 in the spleen. Units: Absolute cell count *
 adn4955_SourceData_Figure1.csv ; *Figure 1E* *
 adn4955_SourceData_FigureS7.csv ; *figure S7B* * *Number of
 antigen-specific memory cells (spleen)*: Count of memory B cells specific
 for TcdA, TcdB, or PPEP-1 in the spleen. Units: Absolute cell count *
 adn4955_SourceData_Figure3.csv ; *Figure 3A* *
 adn4955_SourceData_Figure3.csv ; *Figure 3C* * *Percent viability*:
 Percentage of cells that remained viable after transfection relative to
 the untreated cells. Cell lines: HUVEC, VERO, Caco-2, human primary
 skeletal muscle cells. Treatments: untransfected, recombinant TcdA or
 TcdB, Luciferase or TcdA or TcdB mRNA encapsulated in LNP.  *
 adn4955_SourceData_FigureS1.csv ; *figure S1C* #### Variable definitions
 for *C. difficile* T cell responses datasets All measurements are reported
 as percentages (%) of CD8+ or CD4+ T cells expressing the specified marker
 or cytokine in response to stimulation with the indicated peptide pool. *
 *CD8+*: CD8-positive T cells * *CD4+*: CD4-positive T cells * *IFNγ*:
 Interferon-gamma * *IL-2*: Interleukin-2 * *TNFα*: Tumor Necrosis
 Factor-alpha * *CD107a*: Cluster of Differentiation 107a (marker of
 degranulation) * adn4955_SourceData_FigureS9.csv ; *figure S9B-E* Boolean
 gating was used to analyze the expression of IL-2, IFN-γ, and TNF-α in T
 cells. This method allows for the identification of all possible
 combinations of cytokine expression. Units (value): percent of cells in
 each combination of cytokine expression * *Subject*: Unique identifier for
 each sample that includes vaccine formulation (bivalent mRNA, trivalent
 mRNA, recombinant trivalent, and naive), dose (1ug or 5ug), and mouse
 identified (A-E). * *IL2*: Expression of Interleukin-2 (- = negative, + =
 positive) * *IFNg*: Expression of Interferon-gamma (- = negative, + =
 positive) * *TNFa*: Expression of Tumor Necrosis Factor-alpha (- =
 negative, + = positive) * adn4955_SourceData_FigureS10.csv  #### Variable
 definitions for genomic analysis datasets * *Genome*: Unique identifier
 for each bacterial genome analyzed. * adn4955_SourceData_Figure1A.csv *
 *Coverage*: Percentage of the genome covered by sequencing reads. Unit:
 Percentage (%) * adn4955_SourceData_Figure1A.csv * *Identity*: Percentage
 of sequence identity to a reference genome. Unit: Percentage (%) *
 adn4955_SourceData_Figure1A.csv * adn4955_SourceData_Figure4.csv ; *Figure
 4A* * *Toxin Status*: Description: Indicates whether the strain has the
 genetic machinery to produce toxins. Categories: "Positive":
 Strain produces toxins. "Negative": Strain does not produce
 toxins * Relevant files and sheets * adn4955_SourceData_Figure1A.csv ####
 Variable definitions for *C. difficile* infection study datasets *
 *Survival*: Binary measure (0 = deceased, 1 = alive) recorded daily for
 each mouse. * adn4955_SourceData_Figure1.csv ; *Figure 1F* *
 adn4955_SourceData_Figure1.csv ; *Figure 1G* *
 adn4955_SourceData_Figure2.csv ; *Figure 2B* *
 adn4955_SourceData_Figure3.csv ; *Figure 3E* *
 adn4955_SourceData_Figure3.csv ; *Figure 3I* *
 adn4955_SourceData_Figure4.csv ; *Figure 4C* *
 adn4955_SourceData_FigureS11.csv * adn4955_SourceData_FigureS13.csv ;
 *figure S13A* * adn4955_SourceData_FigureS13.csv ; *figure S13C* *
 adn4955_SourceData_FigureS13.csv ; *figure S13E* *
 adn4955_SourceData_FigureS14.csv ; *figure S14A* - *Percent weight loss*:
 Calculated as [current weight / initial weight] * 100. Recorded daily and
 expressed as a percentage (%). * adn4955_SourceData_Figure2.csv ; *Figure
 2C* * adn4955_SourceData_Figure3.csv ; *Figure 3F* *
 adn4955_SourceData_Figure3.csv ; *Figure 3J* *
 adn4955_SourceData_Figure4.csv ; *Figure 4D* *
 adn4955_SourceData_FigureS8.csv ; *Figure S8B* *
 adn4955_SourceData_FigureS13.csv ; *Figure S13B* *
 adn4955_SourceData_FigureS13.csv ; *Figure S13D* *
 adn4955_SourceData_FigureS13.csv ; *Figure S13F* *
 adn4955_SourceData_FigureS14.csv ; *Figure S14B* * *Clinical sickness
 score*: A composite score ranging from 0-4 based on observed symptoms:
 Weight loss, stool consistency, behavior * adn4955_SourceData_Figure2.csv
 ; *Figure 2D* * adn4955_SourceData_Figure3.csv ; *Figure 3G* *
 adn4955_SourceData_Figure3.csv ; *Figure 3K* *
 adn4955_SourceData_Figure4.csv ; *Figure 4E* *
 adn4955_SourceData_FigureS14.csv ; *figure S14C* - *Pathology score*: A
 cumulative score of three independent criteria: inflammation (0-4), edema
 (0-4), and epithelial cell damage (0-4). * adn4955_SourceData_Figure2.csv
 ; *Figure 2E* - *C. difficile CFU per gram feces*: Bacterial load measured
 in colony-forming units (CFU) per gram of feces. Reported on a log10 scale
 * adn4955_SourceData_Figure2.csv ; *Figure 2F* *
 adn4955_SourceData_Figure4.csv ; *Figure 4F* *
 adn4955_SourceData_Figure4.csv ; *Figure 4H* * *Toxin titer per gram
 feces*: Concentration of *C. difficile* toxins in feces, measured using
 ELISA. Reported as reciprocal of the highest dilution giving a positive
 result per gram of feces. * adn4955_SourceData_Figure2.csv ; *Figure 2G* *
 adn4955_SourceData_Figure4.csv ; *Figure 4G* *
 adn4955_SourceData_Figure4.csv ; *Figure 4I* - *Immunoglobulin/g feces*:
 Concentration of Immunoglobulin A (IgA) or IgG in feces, measured using
 ELISA. Reported in Arbitrary Units of IgA per gram of feces. *
 adn4955_SourceData_Figure2.csv ; *Figure 2H* *
 adn4955_SourceData_Figure2.csv ; *Figure 2I* *
 adn4955_SourceData_Figure3.csv ; *Figure 3B* *
 adn4955_SourceData_FigureS8.csv ; *figure S8D* *
 adn4955_SourceData_FigureS16.csv ; *figure S16B* ## Sharing/Access
 information The data from 16S rRNA gene sequencing are publicly available
 in the NCBI SRA under BioProject ID PRJNA104103, and the *C.
 difficile* genome sequences are publicly available under BioProject ID
 PRJNA524299.