Induction of Cell Cycle and NK Cell Responses by Live-Attenuated Oral Vaccines against Typhoid Fever

Christoph J Blohmke, Jennifer Hill, Thomas C Darton, Matheus Carvalho-Burger, Andrew Eustace, Claire Jones, Fernanda Schreiber, Martin R Goodier, Gordon Dougan, Helder I Nakaya, Andrew J Pollard, Christoph J Blohmke, Jennifer Hill, Thomas C Darton, Matheus Carvalho-Burger, Andrew Eustace, Claire Jones, Fernanda Schreiber, Martin R Goodier, Gordon Dougan, Helder I Nakaya, Andrew J Pollard

Abstract

The mechanisms by which oral, live-attenuated vaccines protect against typhoid fever are poorly understood. Here, we analyze transcriptional responses after vaccination with Ty21a or vaccine candidate, M01ZH09. Alterations in response profiles were related to vaccine-induced immune responses and subsequent outcome after wild-type Salmonella Typhi challenge. Despite broad genetic similarity, we detected differences in transcriptional responses to each vaccine. Seven days after M01ZH09 vaccination, marked cell cycle activation was identified and associated with humoral immunogenicity. By contrast, vaccination with Ty21a was associated with NK cell activity and validated in peripheral blood mononuclear cell stimulation assays confirming superior induction of an NK cell response. Moreover, transcriptional signatures of amino acid metabolism in Ty21a recipients were associated with protection against infection, including increased incubation time and decreased severity. Our data provide detailed insight into molecular immune responses to typhoid vaccines, which could aid the rational design of improved oral, live-attenuated vaccines against enteric pathogens.

Keywords: NK cell; Ty21a; cell cycle regulation; functional genomics; typhoid; typhoid vaccines; vaccine immunity.

Figures

Figure 1
Figure 1
Gene expression (GEX) following oral, live-attenuated typhoid vaccination. (A,B) Study design and sample overview for Ty21a (orange; three doses administered at D-32, D-30, and D-28) and M01ZH09 (green; one dose administered at D-28). Numbers in circles represent N of which samples were taken at each time point/assay. Gray shaded time points indicated where no samples were taken. Samples were collected for GEX, antibody serology (Ab), and in vitro infection [peripheral blood mononuclear cell (PBMC)] analysis. (C,D) Number of differentially expressed (DE) genes over pre-vaccination baseline following Ty21a (C) and M01ZH09 (D) vaccination at time points after vaccination corresponding to panel (A,B). (E) Circos plot indicating the overlap of DE genes between each vaccine arm and time point. Blue links: overlap between M01ZH09 time points. Purple links: overlap between M01ZH09 and Ty21a. Yellow and red links: overlap between Ty21a time points. Red and blue dots indicate up- or downregulation of each gene, respectively. (F) Significant pathways overrepresented by DE genes following Ty21a and M01ZH09 vaccination (increasing bubble size depicts increasing significance level).
Figure 2
Figure 2
Gene Set Enrichment Analysis was performed at each time point following Ty21a and M01ZH09 vaccination. (A) Tile graph with significant blood transcriptional modules (BTMs) (adjusted p < 0.05) following vaccination (blue: negative enrichment; red: positive enrichment). Non-significant BTMs were set to normalized enrichment score (NES) = 0 (gray: not significant). Bar graph to the right indicates the number of significantly enriched BTMs at each time point/group. (B) NESs of BTMs at the D-21 time point following vaccination. Middle: scatter plot displaying NES of significantly enriched BTMs (adjusted p < 0.05) following either Ty21a (x-axis) or M01ZH09 (y-axis) vaccination. If a BTM was not significantly enriched in one of the two groups, the respective NES was set to 0. Colors represent different BTM categories. Dot plots depict enrichment scores of cell cycle (left) and NK cell (right) related modules. Solid dots: p < 0.05.
Figure 3
Figure 3
Single sample GSEA (ssGSEA) following vaccination at time point D-21. (A) Normalized enrichment score (NES) (mean + SEM) of blood transcriptional modules (BTMs) related to NK cells (blue bar), the cell cycle (purple), B cells (pink bar), and T cells (cyan bar) across all participants in the Ty21a (orange) and M01ZH09 (green) arm at time point D-21 after vaccination is plotted. (B) Tile graph representing the NES for all NK cell-related BTMs for each participant (red: positive NES; blue: negative NES). Color bar on the right represents the vaccine group membership. Bar graph along the tile graph signifies the heterogeneity within each participant (depicted by inter-decile range). (C) NK cell modules were combined into one network and superimposed with mean gene expression values at time point D-21 following Ty21a (top) and M01ZH09 (bottom). (D) Mean expression of genes within the NK cell-related BTMs in the Ty21a and M01ZH09 group at time point D-21 following vaccination. (E) Association networks. NESs derived from the ssGSEA at time point D-21 were correlated with antibody responses to vaccination and parameters reflecting outcome following challenge [temperature (Temp); time to diagnosis (ttDx)] in Ty21a (left) and M01ZH09 (right) recipients. BTM colors: purple: cell cycle. Blue: NK cells. Cyan: T cells. Red: inflammation, monocytes, and dendritic cells. Pink: antibodies, B cells, and antigen presentation. Green: platelet activation. Colors of edges represent significance level and Spearman’s rank correlation coefficients (orange: p < 0.05, rho > 0; blue: p < 0.05, rho < 0; gray: n.s.). Gray shaded nodes: BTMs associated with post-challenge outcomes. (F) Correlation plot of maximum Temp following challenge and ttDx. Modules indicated on the plot represent modules inversely associated with Temp and ttDx. Top left: positive and negative correlation with Temp and ttDx, respectively. Bottom right: positive and negative correlation with ttDx and Temp, respectively. Orange: Ty21a recipients; dark green: M01ZH09 recipients.
Figure 4
Figure 4
In vitro infection of peripheral blood mononuclear cells (PBMCs) from study participants. (A) Experimental design. PBMCs harvested from study participants at pre-vaccination (D-32: Ty21a; D-28: M01ZH09), 14 days (D-14), and 28 days after vaccination (D0, day of challenge) were infected in vitro with Ty21a (orange) or M01ZH09 (dark green). (B) Differential induction of activation markers on NK, T, and NKT cells following in vitro stimulation of PBMCs from vaccine-naïve volunteers with Ty21a (orange) and M01ZH09 (green). Multiplicity of infection = 0.1:1. Statistics: paired t-test: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 5
Figure 5
Autologous recall responses in peripheral blood mononuclear cells (PBMCs) from individuals post-vaccination. PBMCs harvested from study participants at 14 days (D-14) and 28 days after vaccination (D0, day of challenge) were infected in vitro with the autologous vaccine strain and NK cell responses measured. Fold-change increases of baseline responses are presented. Statistics: two-sided t-test.

References

    1. Crump JA, Mintz ED. Global trends in typhoid and paratyphoid fever. Clin Infect Dis (2010) 50:241–6.10.1086/649541
    1. Buckle GC, Walker CL, Black RE. Typhoid fever and paratyphoid fever: systematic review to estimate global morbidity and mortality for 2010. J Glob Health. (2012) 2:010401.10.7189/jogh.02.010401
    1. Antillon M, Warren JL, Crawford FW, Weinberger DM, Kurum E, Pak GD, et al. The burden of typhoid fever in low- and middle-income countries: a meta-regression approach. PLoS Negl Trop Dis (2017) 11:e0005376.10.1371/journal.pntd.0005376
    1. de Jong HK, Parry CM, Van Der Poll T, Wiersinga WJ. Host-pathogen interaction in invasive salmonellosis. PLoS Pathog (2012) 8:e1002933.10.1371/journal.ppat.1002933
    1. Dougan G, Baker S. Salmonella enterica serovar Typhi and the pathogenesis of typhoid fever. Annu Rev Microbiol (2014) 68:317–36.10.1146/annurev-micro-091313-103739
    1. Begier EM, Burwen DR, Haber P, Ball R, Vaccine Adverse Event Reporting System Working Group . Postmarketing safety surveillance for typhoid fever vaccines from the Vaccine Adverse Event Reporting System, July 1990 through June 2002. Clin Infect Dis (2004) 38:771–9.10.1086/381548
    1. Levine MM, Ferreccio C, Black RE, Germanier R. Large-scale field trial of Ty21a live oral typhoid vaccine in enteric-coated capsule formulation. Lancet (1987) 1:1049–52.10.1016/S0140-6736(87)90480-6
    1. Simanjuntak CH, Paleologo FP, Punjabi NH, Darmowigoto R, Soeprawoto, Totosudirjo H, et al. Oral immunisation against typhoid fever in Indonesia with Ty21a vaccine. Lancet (1991) 338:1055–9.10.1016/0140-6736(91)91910-M
    1. Anwar E, Goldberg E, Fraser A, Acosta CJ, Paul M, Leibovici L. Vaccines for preventing typhoid fever. Cochrane Database Syst Rev (2014) 3:CD001261.10.1002/14651858.CD001261.pub3
    1. Germanier R, Fuer E. Isolation and characterization of Gal E mutant Ty 21a of Salmonella typhi: a candidate strain for a live, oral typhoid vaccine. J Infect Dis (1975) 131:553–8.10.1093/infdis/131.5.553
    1. Dragunsky EM, Wooden CR, Vargo SA, Levenbook IS. Salmonella typhi vaccine strain in vitro; low infectivity in human cell line U937. J Biol Stand (1989) 17:353–60.10.1016/S0092-1157(89)80006-X
    1. Sizemore DR, Elsinghorst EA, Eck LC, Branstrom AA, Hoover DL, Warren RL, et al. Interaction of Salmonella typhi strains with cultured human monocyte-derived macrophages. Infect Immun (1997) 65:309–12.
    1. Hindle Z, Chatfield SN, Phillimore J, Bentley M, Johnson J, Cosgrove CA, et al. Characterization of Salmonella enterica derivatives harboring defined aroC and Salmonella pathogenicity island 2 type III secretion system (ssaV) mutations by immunization of healthy volunteers. Infect Immun (2002) 70:3457–67.10.1128/IAI.70.7.3457-3467.2002
    1. Kirkpatrick BD, Mckenzie R, O’Neill JP, Larsson CJ, Bourgeois AL, Shimko J, et al. Evaluation of Salmonella enterica serovar Typhi (Ty2 aroC-ssaV-) M01ZH09, with a defined mutation in the Salmonella pathogenicity island 2, as a live, oral typhoid vaccine in human volunteers. Vaccine (2006) 24:116–23.10.1016/j.vaccine.2005.08.008
    1. Lyon CE, Sadigh KS, Carmolli MP, Harro C, Sheldon E, Lindow JC, et al. In a randomized, double-blinded, placebo-controlled trial, the single oral dose typhoid vaccine, M01ZH09, is safe and immunogenic at doses up to 1.7 x 10(10) colony-forming units. Vaccine (2010) 28:3602–8.10.1016/j.vaccine.2010.02.017
    1. Levine MM, Ferreccio C, Black RE, Tacket CO, Germanier R. Progress in vaccines against typhoid fever. Rev Infect Dis (1989) 11(Suppl 3):S552–67.10.1093/clinids/11.Supplement_3.S552
    1. Salerno-Goncalves R, Pasetti MF, Sztein MB. Characterization of CD8(+) effector T cell responses in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine. J Immunol (2002) 169:2196–203.10.4049/jimmunol.169.4.2196
    1. Sztein MB, Salerno-Goncalves R, McArthur MA. Complex adaptive immunity to enteric fevers in humans: lessons learned and the path forward. Front Immunol (2014) 5:516.10.3389/fimmu.2014.00516
    1. Salerno-Goncalves R, Wahid R, Sztein MB. Ex vivo kinetics of early and long-term multifunctional human leukocyte antigen E-specific CD8+ cells in volunteers immunized with the Ty21a typhoid vaccine. Clin Vaccine Immunol (2010) 17:1305–14.10.1128/CVI.00234-10
    1. McArthur MA, Fresnay S, Magder LS, Darton TC, Jones C, Waddington CS, et al. Activation of Salmonella Typhi-specific regulatory T cells in typhoid disease in a wild-type S. Typhi challenge model. PLoS Pathog (2015) 11:e1004914.10.1371/journal.ppat.1004914
    1. Toapanta FR, Bernal PJ, Fresnay S, Darton TC, Jones C, Waddington CS, et al. Oral wild-type Salmonella typhi challenge induces activation of circulating monocytes and dendritic cells in individuals who develop typhoid disease. PLoS Negl Trop Dis (2015) 9:e0003837.10.1371/journal.pntd.0003837
    1. Carreno JM, Perez-Shibayama C, Gil-Cruz C, Lopez-Macias C, Vernazza P, Ludewig B, et al. Evolution of Salmonella Typhi outer membrane protein-specific T and B cell responses in humans following oral Ty21a vaccination: a randomized clinical trial. PLoS One (2017) 12:e0178669.10.1371/journal.pone.0178669
    1. Waddington CS, Darton TC, Jones C, Haworth K, Peters A, John T, et al. An outpatient, ambulant design, controlled human infection model using escalating doses of Salmonella typhi challenge delivered in sodium bicarbonate solution. Clin Infect Dis (2014) 58:1230–40.10.1093/cid/ciu078
    1. Darton TC, Jones C, Blohmke CJ, Waddington CS, Zhou L, Peters A, et al. Using a human challenge model of infection to measure vaccine efficacy: a randomised, controlled trial comparing the typhoid vaccines M01ZH09 with placebo and Ty21a. PLoS Negl Trop Dis (2016) 10:e0004926.10.1371/journal.pntd.0004926
    1. Lynn DJ, Chan C, Naseer M, Yau M, Lo R, Sribnaia A, et al. Curating the innate immunity interactome. BMC Syst Biol (2010) 4:117.10.1186/1752-0509-4-117
    1. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A (2005) 102:15545–50.10.1073/pnas.0506580102
    1. Li S, Rouphael N, Duraisingham S, Romero-Steiner S, Presnell S, Davis C, et al. Molecular signatures of antibody responses derived from a systems biology study of five human vaccines. Nat Immunol (2014) 15:195–204.10.1038/ni.2789
    1. Feenstra B, Pasternak B, Geller F, Carstensen L, Wang T, Huang F, et al. Common variants associated with general and MMR vaccine-related febrile seizures. Nat Genet (2014) 46:1274–82.10.1038/ng.3129
    1. Kampmann B, Jones CE. Factors influencing innate immunity and vaccine responses in infancy. Philos Trans R Soc Lond B Biol Sci (2015) 370(1671):ii:20140148.10.1098/rstb.2014.0148
    1. Horowitz A, Behrens RH, Okell L, Fooks AR, Riley EM. NK cells as effectors of acquired immune responses: effector CD4+ T cell-dependent activation of NK cells following vaccination. J Immunol (2010) 185:2808–18.10.4049/jimmunol.1000844
    1. Ip JY, Tong A, Pan Q, Topp JD, Blencowe BJ, Lynch KW. Global analysis of alternative splicing during T-cell activation. RNA (2007) 13:563–72.10.1261/rna.457207
    1. Martinez NM, Lynch KW. Control of alternative splicing in immune responses: many regulators, many predictions, much still to learn. Immunol Rev (2013) 253:216–36.10.1111/imr.12047
    1. Choi JM, Bothwell AL. The nuclear receptor PPARs as important regulators of T-cell functions and autoimmune diseases. Mol Cells (2012) 33:217–22.10.1007/s10059-012-2297-y
    1. Cipolletta D, Feuerer M, Li A, Kamei N, Lee J, Shoelson SE, et al. PPAR-gamma is a major driver of the accumulation and phenotype of adipose tissue Treg cells. Nature (2012) 486:549–53.10.1038/nature11132
    1. Sinclair LV, Rolf J, Emslie E, Shi YB, Taylor PM, Cantrell DA. Control of amino-acid transport by antigen receptors coordinates the metabolic reprogramming essential for T cell differentiation. Nat Immunol (2013) 14:500–8.10.1038/ni.2556
    1. O’Neill LA, Kishton RJ, Rathmell J. A guide to immunometabolism for immunologists. Nat Rev Immunol (2016) 16:553–65.10.1038/nri.2016.70
    1. Waickman AT, Powell JD. Mammalian target of rapamycin integrates diverse inputs to guide the outcome of antigen recognition in T cells. J Immunol (2012) 188:4721–9.10.4049/jimmunol.1103143
    1. Maciver NJ, Michalek RD, Rathmell JC. Metabolic regulation of T lymphocytes. Annu Rev Immunol (2013) 31:259–83.10.1146/annurev-immunol-032712-095956
    1. Blohmke CJ, Darton TC, Jones C, Suarez NM, Waddington CS, Angus B, et al. Interferon-driven alterations of the host’s amino acid metabolism in the pathogenesis of typhoid fever. J Exp Med (2016) 213(6):1061–77.10.1084/jem.20151025
    1. Pennington SH, Thompson AL, Wright AK, Ferreira DM, Jambo KC, Wright AD, et al. Oral typhoid vaccination with live-attenuated Salmonella Typhi strain Ty21a generates Ty21a-responsive and heterologous influenza virus-responsive CD4+ and CD8+ T Cells at the human intestinal mucosa. J Infect Dis (2016) 213:1809–19.10.1093/infdis/jiw030
    1. Salerno-Goncalves R, Luo D, Fresnay S, Magder L, Darton TC, Jones C, et al. Challenge of humans with wild-type Salmonella enterica serovar Typhi elicits changes in the activation and homing characteristics of mucosal-associated invariant T cells. Front Immunol (2017) 8:398.10.3389/fimmu.2017.00398
    1. Nakaya HI, Clutterbuck E, Kazmin D, Wang L, Cortese M, Bosinger SE, et al. Systems biology of immunity to MF59-adjuvanted versus nonadjuvanted trivalent seasonal influenza vaccines in early childhood. Proc Natl Acad Sci U S A (2016) 113:1853–8.10.1073/pnas.1519690113
    1. Kazmin D, Nakaya HI, Lee EK, Johnson MJ, Van Der Most R, Van Den Berg RA, et al. Systems analysis of protective immune responses to RTS,S malaria vaccination in humans. Proc Natl Acad Sci U S A (2017) 114:2425–30.10.1073/pnas.1621489114
    1. Blanchard Rohner G, Snape MD, Kelly DF, John T, Morant A, Yu LM, et al. The magnitude of the antibody and memory B cell responses during priming with a protein-polysaccharide conjugate vaccine in human infants is associated with the persistence of antibody and the intensity of booster response. J Immunol (2008) 180:2165–73.10.4049/jimmunol.180.4.2165
    1. Clutterbuck EA, Oh S, Hamaluba M, Westcar S, Beverley PC, Pollard AJ. Serotype-specific and age-dependent generation of pneumococcal polysaccharide-specific memory B-cell and antibody responses to immunization with a pneumococcal conjugate vaccine. Clin Vaccine Immunol (2008) 15:182–93.10.1128/CVI.00336-07
    1. Bao S, Beagley KW, France MP, Shen J, Husband AJ. Interferon-gamma plays a critical role in intestinal immunity against Salmonella typhimurium infection. Immunology (2000) 99:464–72.10.1046/j.1365-2567.2000.00955.x
    1. Goodier MR, Rodriguez-Galan A, Lusa C, Nielsen CM, Darboe A, Moldoveanu AL, et al. Influenza vaccination generates cytokine-induced memory-like NK cells: impact of human cytomegalovirus infection. J Immunol (2016) 197:313–25.10.4049/jimmunol.1502049
    1. Hall LJ, Clare S, Dougan G. NK cells influence both innate and adaptive immune responses after mucosal immunization with antigen and mucosal adjuvant. J Immunol (2010) 184:4327–37.10.4049/jimmunol.0903357
    1. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol (2004) 5:R80.10.1186/gb-2004-5-10-r80
    1. Miller JA, Cai C, Langfelder P, Geschwind DH, Kurian SM, Salomon DR, et al. Strategies for aggregating gene expression data: the collapseRows R function. BMC Bioinformatics (2011) 12:322.10.1186/1471-2105-12-322
    1. Kauffmann A, Gentleman R, Huber W. arrayQualityMetrics – a bioconductor package for quality assessment of microarray data. Bioinformatics (2009) 25:415–6.10.1093/bioinformatics/btn647
    1. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res (2015) 43:e47.10.1093/nar/gkv007
    1. Subramaniam KS, Spaulding E, Ivan E, Mutimura E, Kim RS, Liu X, et al. The T-cell inhibitory molecule butyrophilin-like 2 is up-regulated in mild Plasmodium falciparum infection and is protective during experimental cerebral malaria. J Infect Dis (2015) 212:1322–31.10.1093/infdis/jiv217
    1. R Development Core Team. R: A Language and Environment for Statistical Computing. Vienna: the R Foundation for Statistical Computing. Available online at
    1. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res (2003) 13:2498–504.10.1101/gr.1239303

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