Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 7, 34
eCollection

Peracetic Acid Treatment Generates Potent Inactivated Oral Vaccines From a Broad Range of Culturable Bacterial Species

Affiliations

Peracetic Acid Treatment Generates Potent Inactivated Oral Vaccines From a Broad Range of Culturable Bacterial Species

Kathrin Moor et al. Front Immunol.

Abstract

Our mucosal surfaces are the main sites of non-vector-borne pathogen entry, as well as the main interface with our commensal microbiota. We are still only beginning to understand how mucosal adaptive immunity interacts with commensal and pathogenic microbes to influence factors such as infectivity, phenotypic diversity, and within-host evolution. This is in part due to difficulties in generating specific mucosal adaptive immune responses without disrupting the mucosal microbial ecosystem itself. Here, we present a very simple tool to generate inactivated mucosal vaccines from a broad range of culturable bacteria. Oral gavage of 10(10) peracetic acid-inactivated bacteria induces high-titer-specific intestinal IgA in the absence of any measurable inflammation or species invasion. As a proof of principle, we demonstrate that this technique is sufficient to provide fully protective immunity in the murine model of invasive non-typhoidal Salmonellosis, even in the face of severe innate immune deficiency.

Keywords: IgA; Salmonella typhimurium; Yersina enterocolytica; inactivated vaccines; oral vaccines.

Figures

Figure 1
Figure 1
Peracetic acid highly efficiently kills bacteria, with minimal lysis. (A–D) Avirulent S. Typhimurium was cultured overnight to stationary phase, concentrated, and inactivated by incubating in 3% H2O2 for 1 h, heating to 60°C for 1 h, incubating in 4% PFA/PBS for 1 h, or incubating in 0.4% peracetic acid for 1 h. All aliquots were subsequently washed three times to remove fixatives/oxidizing agents before resuspending for CFU analysis by plating (A) and total sterility by enrichment culture (B). Lysis was determined by counting bacterial particles by flow cytometry (C). To examine morphology, the bacteria were diluted in PBS containing 0.5 μM Sytox-green and were imaged by bright-field and fluorescence microscopy at 100× magnification (D). One representative experiment of two. (E–H) C57BL/6 LCM mice received 1.0 g/kg streptomycin p.o. 24 h before oral gavage of 5 × 1010 particles of peracetic acid-inactivated wild-type S. Typhimurium SB300 (PA-wild-type), 5 × 1010 particles of peracetic acid-inactivated avirulent S. Typhimurium M2702 (PA-avirulent), or 10 CFU or 100 CFU of wild-type live S. Typhimurium SB300. After 72 h, all animals were analyzed. (E,F) Intestinal pathology as determined by histopathology or fecal Lipocalin 2 (LCN2). Kruskal–Wallis test, P = 0.0023 (G,H). Cecal content (Kruskal–Wallis test, P = 0.0007) and mesenteric lymph node CFU (Kruskal–Wallis test, P = 0.0007). **Dunn’s post-test, P < 0.01. One experiment with three to five mice per group.
Figure 2
Figure 2
Peracetic acid can be used to inactivate a broad range of bacterial species relevant to intestinal immunology research. (A) Dendrogram based on 16S rDNA sequence differences of the species tested so far, highlighting potential pitfalls. Legend refers to the distance score as calculated by ClustalX neighbor-joining multiple alignment. (B) Schematic diagram of oral vaccine production, including homogenization to disrupt bacterial clumps produced during inactivation, and expected brightfield images.
Figure 3
Figure 3
Oral PA-STm is a strong inducer of specific intestinal IgA in the absence of pathology. C57BL/6 SOPF mice were either pre-treated with 1.0 g/kg streptomycin and infected orally with 5 × 107 CFU of the oral vaccination S. Typhimurium strain M556 (SB300 ΔsseD) (“live”) or were gavaged once a week with 1010 particles of peracetic acid-killed S. Typhimurium (“PA-STm”) over 3 weeks. (A) Intestinal lavage IgA titer curves and (B) intestinal lavage IgA titers, as calculated in Figure S3 in Supplementary Material (Kruskal–Wallis test on log-normalized values, P < 0.0001, Pairwise comparisons calculated by Dunn’s post-tests). (C) Serum IgG2b titer curves at day 21 after the first vaccination/infection, as determined by bacterial flow cytometry. (D) Lipocalin 2 in feces at day 21 after the first vaccination/infection (Kruskal–Wallis test, P = 0.0054 with Dunn’s post-test). (E,F) CFU of live S. Typhimurium recovered from the cecal content and mesenteric lymph nodes at the same time-point. One representative experiment of two shown. (G–I) Specific IgA induced by vaccination with peracetic acid-killed vaccines generated from with S. Enteritidis, Yersinia enterocolitica, and Citrobacter rodentium. Titers were determined by flow cytometry and ELISA. N = 5 mice per vaccine tested.
Figure 4
Figure 4
Specific IgA induction by PA-STm is dependent on T cells and the microbiota. (A) TCRβδ−/− and matched heterozygote controls were vaccinated three times over 3 weeks with PA-STm. On day 21, after the first vaccination, all mice were euthanized, and IgA in the intestinal lavage analyzed by bacterial flow cytometry for Salmonella specific IgA, and ELISA for total IgA concentrations. Pooled data from two independent experiments. Mann–Whitney U test P = 0.0079. (B) Female LCM and SPF mice were either co-housed for 3 weeks, or were housed separately under identical conditions. Subsequently, all mice were gavaged three times over 3 weeks with PA-STm. Antibody titers were determined as above on day 21 after the initial vaccination. Pooled data from three independent experiments. Two-way ANOVA P (hygiene effect) = 0.0142, P (Interaction between housing and hygiene) = 0.0104. (C) C57BL/6 SPF mice were pre-treated orally with vehicle only (PBS) or high-dose ampicillin (0.8 g/kg) or gentamycin (1 g/kg) 24 h prior to each PA-STm dose. Three rounds of pre-treatment and vaccination were carried out over 3 weeks. On day 21, antibody titers were determined as in (A). Pooled data from two independent experiments. Kruskal–Wallis P = 0.0140 with Dunn’s post-test.
Figure 5
Figure 5
PA-STm provides protection from non-typhoidal Salmonellosis in an O-antigen and antibody-dependent manner. (A) C57BL/6 SOPF or JH−/− mice recently rederived into an SOPF colony were vaccinated once per week for 3 weeks with the indicated vaccine (PA-STm: killed O-antigen-sufficient vaccine, PA-SKI10: killed O-antigen-deficient “rough” strain). On day 21, all mice were pre-treated with 1.0 g/kg streptomycin p.o. 24 h later, all mice received 10 CFU of wild-type S. Typhimurium SB300 p.o. Mice were euthanized 24-h post-infection. (A,B) Live S. Typhimurium CFU in the mesenteric lymph nodes (Kruskal–Wallis P = 0.0009, with Dunn’s post-tests) and cecal content. (C) Histopathology of the cecum at 24-h post-infection. (Kruskal–Wallis test P = 0.0325) (D) Fecal Lipocalin 2 on day 21 post-vaccination, 24 h post streptomycin treatment and 24 h post-challenge (24 h post-challenge, Kruskal–Wallis P = 0.0006, with Dunn’s post-tests). (E–G) Intestinal IgA titer specific for the surface of wild-type S. Typhimurium, rough S. Typhimurium (ΔwbaP) and deep-rough S. Typhimurium (ΔrfaI), as determined by bacterial surface-specific bacterial flow cytometry. (H–J) IgA± and IgA−/− SOPF littermate mice were vaccinated three times over 3 weeks with PA-STm. All mice were streptomycin pre-treated, followed by infection with 105 CFU wild-type S. Typhimurium. All parameters were assessed 24-h post-infection. (H) CFU of live S. Typhimurium in the mesenteric lymph nodes (Mann–Whitney U P = 0.0358). (I,J) Intestinal pathology as determined by fecal Lipocalin 2 levels (Mann–Whitney U P = 0.0357) and histopathology (Mann–Whitney U P = 0.0336).
Figure 6
Figure 6
Oral PA-STm is safe for vaccination in cybb-deficient mice and provides dose-dependent protection from tissue invasion and pathology up to at least 80-h post-infection in the non-typhoidal Salmonellosis model. C57BL/6 and cybb−/− mice recently re-derived into an identical SOPF foster colony were vaccinated three times over 3 weeks with PA-STm or vehicle alone (PBS). (A,B) On day 21 after the first vaccination, cecal pathology was determined by fecal Lipocalin 2 ELISA [two-way ANOVA P (genotype) = 0.3583, P (vaccination) = 0.3500, P (Interaction) = 0.3515] (A) and intestinal IgA titers were determined by bacterial flow cytometry (Mann–Whitney U test on vaccinated samples only, P = 0.1679) (B). (C–E) Mice vaccinated as in A and B were pre-treated with 1 g/kg streptomycin on day 21 after the first vaccination and subsequently infected with 105 wild-type S. Typhimurium. (C) Live S. Typhimurium CFU recovered from the mesenteric lymph nodes at 24-h post-infection. (D,E). Intestinal inflammation as determined by fecal Lipocalin 2 (D) and histopathology scoring of cecum tissue (E). (C–E) were analyzed by (two-way ANOVA, ***P (vaccination) <0.001) (F–H). Mice vaccinated as in (A,B) were pretreated with 1 g/kg streptomycin on day 21 after the first vaccination and subsequently infected with 50 CFU wild-type S. Typhimurium. (F,G) Live S. Typhimurium CFU recovered from the mesenteric lymph nodes (F) [two-way ANOVA. P (genotype) = <0.0001, P (vaccination) <0.0001, P (interaction) = <0.0001] and spleen (G) (two-way ANOVA. P (genotype) = 0.0098, P (vaccination) = 0.0038, P (interaction) = 0.0098) at 80-h post-infection. (H) Intestinal inflammation as determined by fecal Lipocalin 2 up to 80 h (day 3) post-infection (two-way repeat-measures ANOVA with Bonferroni post-tests on log-normalized data. **P < 0.01, ****P < 0.0001).

Similar articles

See all similar articles

Cited by 7 articles

See all "Cited by" articles

References

    1. Vidarsson G, Dekkers G, Rispens T. IgG subclasses and allotypes: from structure to effector functions. Front Immunol (2014) 5:520.10.3389/fimmu.2014.00520 - DOI - PMC - PubMed
    1. Bournazos S, DiLillo DJ, Ravetch JV. The role of Fc-FcgammaR interactions in IgG-mediated microbial neutralization. J Exp Med (2015) 212(9):1361–9.10.1084/jem.20151267 - DOI - PMC - PubMed
    1. Iwasaki A, Medzhitov R. Control of adaptive immunity by the innate immune system. Nat Immunol (2015) 16(4):343–53.10.1038/ni.3123 - DOI - PMC - PubMed
    1. Wiesel M, Oxenius A. From crucial to negligible: functional CD8(+) T-cell responses and their dependence on CD4(+) T-cell help. Eur J Immunol (2012) 42(5):1080–8.10.1002/eji.201142205 - DOI - PubMed
    1. Tubo NJ, Jenkins MK. CD4+ T Cells: guardians of the phagosome. Clin Microbiol Rev (2014) 27(2):200–13.10.1128/CMR.00097-13 - DOI - PMC - PubMed
Feedback