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A rationally designed oral vaccine induces immunoglobulin A in the murine gut that directs the evolution of attenuated Salmonella variants

Abstract

The ability of gut bacterial pathogens to escape immunity by antigenic variation—particularly via changes to surface-exposed antigens—is a major barrier to immune clearance1. However, not all variants are equally fit in all environments2,3. It should therefore be possible to exploit such immune escape mechanisms to direct an evolutionary trade-off. Here, we demonstrate this phenomenon using Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm). A dominant surface antigen of S.Tm is its O-antigen: a long, repetitive glycan that can be rapidly varied by mutations in biosynthetic pathways or by phase variation4,5. We quantified the selective advantage of O-antigen variants in the presence and absence of O-antigen-specific immunoglobulin A and identified a set of evolutionary trajectories allowing immune escape without an associated fitness cost in naive mice. Through the use of rationally designed oral vaccines, we induced immunoglobulin A responses blocking all of these trajectories. This selected for Salmonella mutants carrying deletions of the O-antigen polymerase gene wzyB. Due to their short O-antigen, these evolved mutants were more susceptible to environmental stressors (detergents or complement) and predation (bacteriophages) and were impaired in gut colonization and virulence in mice. Therefore, a rationally induced cocktail of intestinal antibodies can direct an evolutionary trade-off in S.Tm. This lays the foundations for the exploration of mucosal vaccines capable of setting evolutionary traps as a prophylactic strategy.

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Fig. 1: Vaccine-induced IgA exerts a strong selective pressure on O-antigen variants during murine non-typhoidal salmonellosis.
Fig. 2: O-antigen variants rapidly emerge during S.TmWT infection of vaccinated mice.
Fig. 3: The single-repeat O-antigen confers a selective advantage in the presence of broad-specificity vaccine-induced IgA.
Fig. 4: Single-repeat O-antigen mutants arising during infection of vaccinated mice have attenuated virulence and fitness and diminished resistance to phage predation.

Data availability

All raw flow cytometry data, ordered by figure, are publicly available via the ETH research collection at https://doi.org/10.3929/ethz-b-000477737. All Illumina sequencing data are publicly available from the NCBI BioProject resource (accession PRJNA720270). Source data are provided with this paper.

Code availability

R code used to generate Extended Data Fig. 5 can be downloaded freely from https://github.com/marnoldini/evotrap.

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Acknowledgements

O.H. acknowledges H. Käßner for recording NMR spectra, R. Engel for gas/liquid chromatography–mass spectrometry and K. Jakob and S. Düpow for technical support. We thank M. Schneider, C. Kiessling, E. Schultheiss, R.-M. Vesco and C. Straub for the DNA extraction, library preparations and sequencing of the bacterial isolates. M.D. acknowledges D. Cornillet and the group of D. Bumann for serum resistance measurements. E.S., W.-D.H. and M.D. acknowledge M. Aebi, M. Loessner, J. Cherry and the group of A. Harms for helpful discussion and insight, as well as members of the W.-D.H., E.S. and M.D. groups for comments. E.S., W.-D.H. and M.D. further acknowledge the staff of the ETH Phenomics Center and Rodent Centre HCI for support with animal experimentation. M.D. is supported by a Swiss National Science Foundation professorship (PP00PP_176954) and Gebert Rüf Microbials (PhagoVax GRS-093/20). E.S. acknowledges support from the Swiss National Science Foundation (40B2-0_180953 and 310030_185128; NCCR Microbiomes), a European Research Council Consolidator Grant and Gebert Rüf Microbials (GR073_17). M.D. and E.S. acknowledge the Botnar Research Centre for Child Health Multi-Investigator Project 2020. B.M.S. acknowledges support from R01 AI041239/AI/NIAID NIH HHS/United States. W.-D.H. acknowledges support from the Swiss National Science Foundation (310030B-173338 and 310030_192567; NCCR Microbiomes), ProMedica Foundation, Gebert Rüf Foundation (with A.E.) and Helmut Horten Foundation. E.B. is supported by a Boehringer Ingelheim Fonds PhD fellowship. B.H.M. acknowledges support from the Swiss National Science Foundation (200020_159707).

Author information

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Authors

Contributions

M.D., W.D.H. and E.S. designed the project and wrote the paper. M.D. and E.S. designed and carried out the experiments relating to the vaccination and infection of mice, re-isolation of S.Tm clones, phenotyping of S.Tm clones by flow cytometry and gel electrophoresis, characterization of human monoclonal antibodies, analysis of antibody titres and analysis of the fitness of O-antigen variants of S.Tm in vitro and in vivo. M.W.v.d.W., B.H.M., C.L. and R.M. contributed to experimental design and data interpretation. G.Z. carried out the HR-MAS NMR analysis. O.H. carried out the proton NMR analysis. M.A. generated the mathematical model for O:12 switching. N.D. supplied unpublished Salmonella strains. J.A. carried out and analysed all of the AFM imaging. A.R. and N.A.B. carried out phage sensitivity assays. A.E., F.B. and D.W. carried out the Illumina whole-genome resequencing of re-isolated S.Tm isolates. E.B., V.L., D.H., F.B., K.S.-M. and S.A. carried out S.Tm challenge infections in vaccinated mice and analysed the re-isolated clones. A.H. carried out the microfluidic video microscopy of O:12 switching. P.H.V. and L.F. carried out the methylome analysis of re-isolated S.Tm clones. L.P., A.L. and B.M.S. generated the novel antibody reagents. All authors critically reviewed the manuscript.

Corresponding authors

Correspondence to Médéric Diard or Wolf-Dietrich Hardt or Emma Slack.

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Competing interests

Evolutionary trap vaccines are covered by European patent application number EP19177251, submitted by M.D. W.-D.H. and E.S. All other authors declare no competing interests.

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Peer review information Nature Microbiology thanks Reiko Shinkura and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Surface phenotype of S.Tm mutants.

Atomic force microscopy phase images of S.Tmwt, S.TmΔwzyB (single-repeat O-antigen), and S.TmΔwbaP (rough mutant - no O-antigen) at low magnification (A, uncropped image, scale bar = 1µm) and high magnification (B and C, scale bar main image = 150nm, scale bar inset = 15nm). Invaginations in the surface of S.TmΔwbaP (dark colour, B) show a geometry and size consistent with outer membrane pores63. These are already less clearly visible on the surface of S.TmΔwzyB with a single-repeat O-antigen, and become very difficult to discern in S.Tmwt. One representative image of 3 for each genotype is shown. While arrows point to features with consistent size and abundance to be exposed outer membrane porins. C. Fast-Fourier transform of images shown in ‘B’ demonstrating clear regularity on the surface of S.TmΔwbaP, which is progressively lost when short and long O-antigen is present.

Extended Data Fig. 2 Mutations detected in the oafA gene sequence among several strains of S.Tm.

A. Aligned fractions of the oafA ORF from a natural isolate (from chicken) presenting the same 7 bp deletion detected in mutants of S.Tm SL1344 emerging in vaccinated mice. S.Tm SL1344 was used a reference64. B. Aligned oafA promoter sequences from three natural isolates of human origin (stool or cerebrospinal fluid65) showing variations in the number of 9 bp direct repeats.

Extended Data Fig. 3 Loss of the O:12-0 epitope is a reversible phenotype.

A. Wild type and evolved S.Tm clones were picked from LB plates, cultured overnight, phenotypically characterized by O:12-0 (left panel) and O:5 staining (right panel), plated and re-picked. This process was repeated over 3 cycles with lines showing the descendants of each clone. B and C. Wild type 129S1/SvImJ mice were mock-vaccinated or were vaccinated with PA-S.TmΔoafA ΔgtrC as in Fig. 1. On d28, all mice were pre-treated with streptomycin, and infected with the indicated strain. B. Feces recovered at day 10 post-infection, was enriched overnight by culture in streptomycin, and stained for O:12-0 (human monoclonal STA5). Fraction O:12-0-low S.Tm was determined by flow cytometry. Percentage of S.Tm that are O:12-0-negative was quantified over 10 days and is plotted in panel C. Vaccination selects for S.Tm that have lost the O:12-0 epitope, only if the gtrC gene is intact.

Source data

Extended Data Fig. 4 Loss of the O:12-0 epitope can be driven by adoptive transfer of O:12-0-specific IgA.

C57BL/6 SPF mice received oral streptomycin to deplete the microbiota 23.5h before an intravenous injection with saline only, or with 1mg of recombinant dimeric murine IgA specific for the O:12-0 epitope (STA121). 0.5 h later all mice were orally inoculated with S.TmΔoafA pM965 or S.TmΔoafAΔG4 pM965 (lacking 4 different glucosyl transferases, including gtrC) both carrying pM965 to drive constitutive GFP production. The adoptive transfer was repeated 12h later and all animals were euthanized at 24h post-infection. A. O:12-0 expression on S.Tm enriched from cecum content by overnight culture on 1:1000 dilution LB with selective antibiotics, determined by staining with the monoclonal antibody STA5. Flow cytometry plots shown have been gated on scatter only – see Supplementary Fig. 1 for example. B. Quantification of the O:12-0-high fraction of S.Tm from A. C. Individual clones of S.Tm of the indicated genotype were recovered from the cecal content of mice from A that had received an adoptive transfer of mSTA121 and individual clones, cultured overnight in LB were analysed as in A and B for fraction of O:12-0-high cells.

Source data

Extended Data Fig. 5 Phase-variation and selection, without a shift in switching rate, underly recovery of O:12-2 producing clones from vaccinated mice.

A. Comparison of fractions of O:12-0-positive and O:12-0-negative bacteria (in fact O:12-2) determined by flow cytometry (gating – see Fig.S1) staining with typing sera and by blue-white colony counts using a gtrABC-lacZ reporter strain and overnight cultures from individual clonal colonies. B-D: Results of a mathematical model simulating bacterial growth and antigen switching (see supplementary methods). B. Switching rates from O:12-0 to O:12-2 and from O:12-2 to O:12-0 were varied computationally, and the fraction of O:12-2 was plotted after 16 h of growth. Left-hand plot depicts the results of the deterministic model when starting with 100% O:12-2, right-hand plot depicts the results when starting with 100% O:12-0. C. depicts only the switching rates that comply with the experimentally observed antigen ratios after overnight growth (90% O:12-0 when starting with O:12-0, and 50% O:12-0 when starting with O:12-2). Right-hand plot is a zoomed version showing values for switching rates between 0 – 0.2 h−1 (marked by a grey rectangle). Dashed lines are linear regressions on the values in this range, and their intersection marks the switching rates used for the stochastic simulation in (D). D. Simulation results of bacterial population growth, when starting with only O:12-2 (left-hand plot) or only O:12-0 (right-hand plot). µ = 2.05h−1 was kept constant in all simulations; switching rates were kept constant at s->12-0 = 0.144h−1 and s->12-2 = 0.0365h−1; the starting populations were always individuals of the indicated phenotype; carrying capacity was always K = 109 cells. Time resolution for the simulations is 0.2h.

Extended Data Fig. 6 NMR of purified LPS and HR-MAS 1H-NMR confirms O-antigen structures in evolved clones.

A. Schematic diagram of expected NMR peaks for each molecular species B. HR-MAS 1H-NMR spectra. Spectra show predicted peak positions and observed spectra for C1 protons of the O-antigen sugars. C. 1H NMR of purified LPS from the indicated strains. Note that non-acetylated abequose can be observed in wild type strains due to spontaneous deacetylation at low pH in late stationary phase cultures53. A gtrA mutant strain is used here to over-represent the O:12-2 O-antigen variant due to loss of regulation5.

Extended Data Fig. 7 S.Tm O-antigen variants arise during chronic S.Tm infections, dependent on a specific IgA response.

IgA−/− (A) and Rag1−/− (B) and heterozygote littermate controls (C57BL/6-background) were pre-treated with streptomycin and infected with S.TmΔsseD orally. Fecal S.Tm were enriched overnight by culturing a 1:2500 dilution of feces in LB plus kanamycin. These enrichment cultures were then stained for O:5 and O:12-0 and analysed by flow cytometry (gating as in Supplementary Fig. 14). The fraction of the population that lost O:5 and O:12-0 antisera staining is shown over time, as well as the total CFU/g in feces. Both immunocompetent mouse strains show increased O:5-negative S.Tm in the fecal enrichments from day 14 post-infection: approximately when we expect to see a robust secretory IgA response developing. These changes are not observed in Rag1-deficient or IgA-deficient mice. The kinetics of O:5-loss are likely influenced by development or broader IgA responses as the chronic infection proceeds. Note that lines joining the points are to permit tracking of individual animals through the data set, and may not be representative of what occurs between the measured time-points. C. Titres of intestinal lavage IgA specific for O:4[5] (S.Tmwt, O:4[5], 12-0) and O:4(S.TmΔoafA, O:4,12-0), presented as the dilution of intestinal lavage required to give an IgA-staining MFI=1000 by bacterial flow cytometry, and the ratios of these titres. Samples: d28 post-vaccination with PA-STmwt (n = 12) or d35 post-colonization with live-attenuated S.Tm (n = 8 S.TmΔaroA + n = 8 S.TmΔsseD), This revealed a weaker, but less biased IgA response in mice infected with the live-vaccine strain, when compared to that induced by the inactivated oral vaccine. Results of 2-tailed Mann-Whitney U tests shown.

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Extended Data Fig. 8 Enzymes of the O-antigen synthesis pathway.

Schematic of S.Tm O-antigen synthesis (with reference to ref. 66).

Extended Data Fig. 9 Synthetic and natural deletions of wzyB reduce the fitness of S.Tm in presence of Tris-EDTA, Cholate, SDS and serum complement.

The deletion of wzyB does not affect the growth of S.Tm or S.TmΔoafA ΔgtrC in LB (No stress) (A) but impairs growth in presence of Tris-EDTA (B), 2% cholate (C) and 0.05% SDS (D). Dashed lines represent the range of variations between the n = 4 pooled experiments. (E). Relative fitness of the long versus short O-antigen in the presence of membrane stress as quantified by competitive growth of S.TmGFP against S.TmΔoafA ΔgtrC, S.Tm ΔoafA ΔgtrC ΔwzyB or an evolved S.TmΔwzyB, in LB with or without Tris-EDTA. 2-tailed Mann-Whitney U test. ** p = 0.0013 (F) Loss of complement resistance in evolved and synthetic wzyB mutants revealed by relative CFU recovery after treatment with heat-inactivated and fresh human serum. Mann-Whitney U 2-tailed tests * p = 0.0167.

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Extended Data Fig. 10 Analysis of bacteriophages preferentially infecting short O-antigen S. Tm mutants.

A. Lysis plaques observed on lawns of S.Tm ΔgtrC ΔoafA and S.Tm ΔgtrC ΔoafA ΔwzyB isogenic mutants exposed to wastewater samples. Scale = 1cm. This phenocopies the observation with naturally arising wzyB mutants B. Growth curves of S.Tm ΔgtrC ΔoafA ΔwzyB exposed to purified bacteriophages from Fig. 4D. The re-growing S.Tm clones were isolated for sequencing. The mutations identified and their effects are listed in the table below (C), confirming btuB as the most likely exposed outer-membrane receptor for these phages.

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Supplementary information

Supplementary Information

Text descriptions for Supplementary Tables 1–4 and Supplementary Videos 1 and 2, and Supplementary Figs. 1–10.

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Supplementary Tables

Supplementary Tables 1–4.

Supplementary Video 1

Visualization of O:12 phase variation using live-cell immunofluorescence (loss of antibody binding).

Supplementary Video 2

Visualization of O:12 phase variation using live-cell immunofluorescence (gain of antibody binding).

Supplementary Data 1

Numerical source data for Supplementary Figs. 1–10.

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Uncropped gel image for Fig. 3g.

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Uncropped phage plaque images for Fig. 4g (ancestral strain and evolved clone).

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Uncropped phage plaque images for Extended Data Fig 10a (long and short O-antigens).

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Diard, M., Bakkeren, E., Lentsch, V. et al. A rationally designed oral vaccine induces immunoglobulin A in the murine gut that directs the evolution of attenuated Salmonella variants. Nat Microbiol (2021). https://doi.org/10.1038/s41564-021-00911-1

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