pubmed.ncbi.nlm.nih.gov

Cryptosporidium Priming Is More Effective than Vaccine for Protection against Cryptosporidiosis in a Murine Protein Malnutrition Model - PubMed

  • ️Fri Jan 01 2016

. 2016 Jul 28;10(7):e0004820.

doi: 10.1371/journal.pntd.0004820. eCollection 2016 Jul.

Affiliations

Cryptosporidium Priming Is More Effective than Vaccine for Protection against Cryptosporidiosis in a Murine Protein Malnutrition Model

Luther A Bartelt et al. PLoS Negl Trop Dis. 2016.

Abstract

Cryptosporidium is a major cause of severe diarrhea, especially in malnourished children. Using a murine model of C. parvum oocyst challenge that recapitulates clinical features of severe cryptosporidiosis during malnutrition, we interrogated the effect of protein malnutrition (PM) on primary and secondary responses to C. parvum challenge, and tested the differential ability of mucosal priming strategies to overcome the PM-induced susceptibility. We determined that while PM fundamentally alters systemic and mucosal primary immune responses to Cryptosporidium, priming with C. parvum (106 oocysts) provides robust protective immunity against re-challenge despite ongoing PM. C. parvum priming restores mucosal Th1-type effectors (CD3+CD8+CD103+ T-cells) and cytokines (IFNγ, and IL12p40) that otherwise decrease with ongoing PM. Vaccination strategies with Cryptosporidium antigens expressed in the S. Typhi vector 908htr, however, do not enhance Th1-type responses to C. parvum challenge during PM, even though vaccination strongly boosts immunity in challenged fully nourished hosts. Remote non-specific exposures to the attenuated S. Typhi vector alone or the TLR9 agonist CpG ODN-1668 can partially attenuate C. parvum severity during PM, but neither as effectively as viable C. parvum priming. We conclude that although PM interferes with basal and vaccine-boosted immune responses to C. parvum, sustained reductions in disease severity are possible through mucosal activators of host defenses, and specifically C. parvum priming can elicit impressively robust Th1-type protective immunity despite ongoing protein malnutrition. These findings add insight into potential correlates of Cryptosporidium immunity and future vaccine strategies in malnourished children.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Severe protein malnutrition in mice selectively enhances intestinal disruption and severity of cryptosporidiosis.

(A) Experimental timeline. 3-week-old C57Bl/6 female mice were initiated on experimental malnutrition diets (RBD or PD) or control diet (CD) immediately upon receipt from supplier. Challenge with 106 or 107 Cryptosporidium parvum oocysts, heat-inactivated C. parvum oocysts (ΔC. parvum) or PBS occurred via oral gavage 5 days after initiating diet. Serial weights were collected daily post-challenge, and fecal parasite shedding was determined by RT-PCR. On day 3–4 post-challenge, tissue parasite burden and mucosal injury was assessed by measuring ileum villus:crypt ratios and alterations in epithelial tight-junction proteins. (B) Impact of diet on growth (**P<0.01, ***P<0.001 C. parvumPD vs. C. parvum or C. parvumRBD), fecal parasite shedding (**P<0.01 PD or RBD vs. CD day 2, *P<0.05 PD vs. RBD or CD day 3), tissue parasite burden (*P<0.05 PD vs CD or RBD ileum, *P<0.05 PD vs CD colon), and ileum villus:crypt ratios (**P<0.01 PBSCD vs PBSRBD, ***P<0.001 PBSCD vs PBSPD, ****P<0.0001 C. parvumCD or C. parvumRBD vs C. parvumPD, ^P<0.05 PBSCD vs. C. parvumCD, ##P<0.01 PBSPD vs. C. parvumPD (n = 3-7/group). (C) Growth through three days post-challenge with either C. parvum or ΔC. parvum. (N = 5-10/group, **P<0.01, ***P<0.001 (C. parvum vs. either PBS or ΔC. Parvum) and fecal parasite shedding (n = 5-10/group, *P<0.05, **P<0.01). (D) Immunofluorescence staining of epithelial cell tight-junction proteins (ZO-1, occludin, claudin-2) in ileum of CD and PD-fed infected mice and uninfected controls (n = 4/group). (E) Dose dependent persistent growth faltering (n = 10/group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 C. parvum 107 vs C. parvum 106; and ####P<0.0001 C. parvum vs PBS) and (F) fecal parasite shedding (**P<0.01, ****P<0.0001) through 21 days post-challenge. Data is representative of 2 replicate experiments.

Fig 2
Fig 2. Protein malnutrition alters basal immune responses to primary C. parvum exposure, but secondary responses are intact.

Immunologic responses to two different recombinant Cryptosporidium sporozoite antigens (CApy and Cp15) were performed at 13–15 days post C. parvum challenge in mice fed either control-diet (C. parvumCD) or protein-deficient diet (C. parvumpd) and results were compared with naïve age and diet-matched uninfected controls (PBSCD and PBSpd). Mice began respective diets 12 days prior to C. parvum challenge and remained on the same diets post-challenge. (A) Cytokine secretion in splenocytes of naïve (uninfected) CD or PD-fed mic after stimulation with Cryptosporidium antigens. (B) Serum antibody production as anti-CApy or anti-Cp15 IgG titer (*P<0.05). (C) Cytokines secreted after CApy or Cp15 antigen stimulation in (C) mesenteric lymph nodes. (D) Cytokine secretion in splenocytes expressed as fold change relative to CD-fed uninfected controls. (*P<0.05 as indicated). Data is representative of pooled individual responses from two separate tissue harvests (n = 4-5/group).

Fig 3
Fig 3. C. parvum priming protects against re-challenge despite continuous protein malnutrition.

Growth (A) and parasite fecal shedding (B) following challenge with 107 C. parvum oocysts in either naïve (PBS-C. parvum 107) PD-fed mice, or mice previously exposed to either 106 (Cp 106) or 107 (Cp 107) C. parvum oocysts 21 days prior as indicated (n = 5/group). (A) *P<0.05, **P<0.01 for PBS-PBS vs PBS-C. parvum 107, #P<0.05, ### P<0.001 #### P<0.0001 for Cp 106-C. parvum 107 vs. PBS-C. parvum 107, ^P<0.05, and ^^P<0.01, ^^^^P<0.0001 for Cp 107-C. parvum 107 vs. PBS-C. parvum 107. (B) *P<0.05, **P<0.01 for Cp 106-C. parvum 107 or Cp 107-C. parvum 107 vs. PBS-C. parvum 107.

Fig 4
Fig 4. C. parvum priming promotes a progressive increase in CD3+CD8+CD103+ cells that further expand upon re-challenge.

(A) Experimental timeline of 3 week-old C57Bl/6 mice initiated on the maintained on PD diet beginning 5 days prior to priming with C. parvum 106 (●) or PBS control (grey◦). Day 0 is designated as the day of C. parvum priming. Mice were challenged with C. parvum 107 20 days later indicated as PBS-C.p. 107 (red ◻) or C. p. 106-C.p. 107 (◻). Flow cytometry was obtained on day 3 (D3) and day 23 (D23) after C. parvum priming. (n = 3/group D3; n = 4-5/group D23). Day 23 also corresponds to 3 days after challenge with C.p. 107. (B) Intestinal leukocytes in the ileum decrease between D3 and D23 in uninfected mice, but both B-cell (B220+) and T-cells (CD3ε+) increase in C. parvum primed mice. ^P<0.05 for Cp 106 D3 vs Cp 106 D23, #P<0.05 for PBS D3 vs PBS D23, *P<0.05 for PBS vs. Cp 106. C) Expansion of T-cells in C. parvum-primed mice through D23 after C. parvum priming mice is specific to the ileum and accompanied by a predominance of CD8+ T-cells (left) that further increase 3 days after C. parvum re-challenge (right). *P<0.05 for PBS vs. Cp 106, #P<0.05 between groups as indicated. (D) CD11c+CD103+, CD3ε+CD8+ and CD3ε+CD8+CD103+ cells expand in the ileum through D23 after C. parvum priming. **P<0.01 for PBS vs. Cp 106. (E) C. parvum priming leads to expansion of CD8+CD103+ cells and CD11c+CD103+ but not NK1.1+ cells compared with initial C. parvum 107 challenge. *P<0.05, **P<0.01.

Fig 5
Fig 5. C. parvum priming leads to sustained changes in ileal tissue chemokine and cytokine profiles during protein malnutrition.

Mice were conditioned on PD for 5 days prior to infection with 106 C. parvum (Cp106). Luminex was performed for measurement of chemokines and cytokines in ileal tissues at. day 3 (D3) and day 23 (D23) post challenge compared to uninfected controls (PBS) (n = 3-4/group). (A) Primary C. parvum infection led to increases in CXCL9, CXCL10, CCL-3, CCL-5, and CCL11 on D3. On D23, TNFα, IL1β, and IL-8 were diminished in infected mice relative to uninfected controls, however, CCL-5 continued to be elevated and other chemokines had returned to baseline. (B) Only IL12p40 and IL-13 were modestly elevated three days after primary C. parvum challenge. There was a relative decrease in all Th2-type cytokines through 23 days post-C. parvum compared with uninfected controls. (n = 3-4/group). *P<0.05 for PBS vs Cp106 as indicated; #P<0.05 for Cp106 D3 vs Cp106 D23 as indicated.

Fig 6
Fig 6. C. parvum priming enhances Th1-type cytokine responses to re-challenge in protein malnourished mice.

(A) Ileal inflammatory mediators and chemokines and (B) cytokines measured three days after 107 C. parvum challenge in previously uninfected (PBS) compared with mice primed with 106 C. parvum (Cp106) 20 days prior to re-challenge. *P<0.05, **P<0.01, ***P<0.001.

Fig 7
Fig 7. Protein malnutrition interferes with vaccine-boosted immunity, but the S. Typhi vector improves recovery after C. parvum challenge.

(A) Timeline for immunization, growth monitoring, infection, and analysis of immune responses. 21 day-old mice acclimated for 4 days prior to weight-matched randomization (n = 8-19/group). Intranasal immunization with S. enterica Typhi 908htr vector expressing either of two recombinant sporozoite antigens, ClyA-Cp15 (S. TyphiCp15 (aqua)) or ClyA-CApy (S. TyphiCApy (blue)) was administered at two-week intervals. The S. Typhi vector alone (S. Typhi (green)) and a PBS-only (red) treatment served as a double-sham control. Intramuscular injection with rCp15 (S. TyphiCp15), rCApy (S. TyphiCApy), the inert NUS peptide (for S. Typhi), or PBS-’sham’ (for PBS only group) combined with 1:1 alum adjuvant occurred two weeks after the second intranasal immunization. Serial weights (*) were obtained throughout the vaccination protocol (S1 Fig). On day 107 (9 weeks after S. Typhi exposure), mice were transitioned to either PD or CD diets (n = 4-10/group) and continued on respective diets throughout the remainder of the experiment. Mice were challenged with 5x107 C. parvum (Cp) on day 119 and followed for 13–15 days post-challenge. (B) Serum geometric mean IgG titers (GMT) in C. parvum challenged groups. (C) IFN-γ and (D) IL-17A cytokine secretion recall responses to homologous vaccinogen as indicated. For (B-D), *P<0.05, **P<0.01, ***P<0.001, One-way ANOVA, Tukey post-test analysis, # P<0.05 by Student’s t-test. (E) Growth as percentage of weight change on the day of C. parvum infection beginning on the day of transition to either CD (left) or PD (right) diets. (left: *P<0.05, S. TyphiCp15 vs PBS-Cp; right: colored bars indicate P<0.05 for PBS-Cp (red), S. Typhi-Cp (green), S. TyphiCp15 (aqua), and S. TyphiCApy (blue) vs. uninfected controls, *P<0.05 for individual vaccine groups [S. Typhi-Cp (green), S. TyphiCp15 (aqua), and S. TyphiCApy (blue)] vs. PBS-Cp. (F) Parasite shedding for infected groups: PBS (red), S. Typhi (green, S. TyphiCp15 (aqua), and S. TyphiCApy (blue). (G) Ileal villus:crypt for CD-fed (left) and PD-fed (right) mice in each C. parvum infected group or combined uninfected controls as indicated. Left: ***P<0.001 PBS-Cp vs uninfected controls, ###P<0.001 S. TyphiCp15 or S. TyphiCApy vs S. Typhi; Right: ***P<0.001 for PBS-Cp, S. Typhi, or S. TyphiCp15 vs uninfected controls, ###P<0.001 S. TyphiCApy vs PBS-Cp. ^P<0.05 for PD-PBS and PD-S. TyphiCp15 vs DD-PBS and DD-S. TyphiCp15. ns = not significant vs. uninfected controls.

Fig 8
Fig 8. Viable C. parvum priming provides greater protection against re-challenge than either CpG-ODN or S. Typhi.

(A, B) Comparison of protective immunity following priming with either viable and heat-inactivated (Δ) C. parvum 106. (A) Growth of PD-fed mice through 23 days post-priming with either 4x106 viable (C. parvum) or 4x106 heat-inactivated (ΔC. parvum). Mice were challenged with viable 4x107 C. parvum oocysts on day 20 post-priming. *P<0.05 for ΔC. parvum-C. parvum vs. C. parvum-C. parvum (d3 and d23); ^^P<0.01 for C. parvum-PBS vs PBS-C. parvum (d23); ###P<0.001 for C. parvum-C. parvum vs. PBS-C. parvum (d23). (B) RT-PCR of Cryptosporidium stool shedding on experimental days 21 and 23 (day 1 and day 3 after C. parvum 107 challenge, respectively). *P<0.05 and *P<0.01 for C. parvum-C. parvum vs either PBS-C. parvum or ΔC. parvum-C. parvum. (C,D) 3-week-old C57Bl/6 mice were conditioned on PD for 7 days prior to orogastric inoculation with 106 C. parvum, intranasal (i.n.) 109 S. Typhi 908htr, i.n. CpG-ODN 1668 (100 mcg), or PBS (100 mcl) as indicated (n = 10/group). On day 21, mice were re-challenged with either PBS or C. parvum 107. (C) Growth as percentage of initial weight, normalized to the day of 107 C. parvum challenge (Day 0). The group labeled “All uninfected” includes animals that received either PBS during both inoculations, CpG followed by PBS, or S. Typhi followed by PBS (n = 5/group x 3 = 15) given all three groups grew similarly and were never exposed to C. parvum (S4 Fig). *P<0.05, **P<0.01, ***P<0.001 for PBS–C.p.107 (red) vs C.p.106-C.p.107, ^P<0.05 for CpG-C.p.107 (yellow) vs C.p.106-C.p.107, and #P<0.05 for S. Typhi-C.p.107 (green) vs C.p.106-C.p.107). Horizontal lines designate significant differences at P<0.05 between CpG-C.p.107 (yellow), S. Typhi-C.p.107 (green), and PBS-C.p.107 (red) vs. All uninfected controls, respectively. (D) Parasite fecal shedding in serial fecal pellets collected on indicated experimental days post C. parvum 107 challenge. *P<0.05 for PBS–C.p.107 vs. C.p.106-C.p.107, ^P<0.05 for CpG-C.p.107 vs C.p.106-C.p.107, and #P<0.05 for S. Typhi-C.p.107 vs C.p.106-C.p.107. Data is representative of two replicate experiments.

Fig 9
Fig 9. Schematic of non-specific (S. Typhi and CpG) and specific (C. parvum priming) mucosal exposures that modulate host immunity and protect against cryptosporidiosis during malnutrition.

Strategies to enhance immune defenses against Cryptosporidium infection during malnutrition were investigated in a protein deficient murine model that replicates clinical features of childhood cryptosporidiosis. Whereas the well-nourished host (black) clears Cryptosporidium with little evidence of a secondary immune response (dark blue), mucosal vaccination with Cryptosporidium antigens expressed in an S. Typhi vector can elicit strongly boosted IFNγ-predominant immune responses to subsequent challenge (light blue). Vaccine attenuates the mild disease caused by Cryptosporidium in well-nourished hosts. In protein malnourished hosts (light grey) there is ongoing depletion of mucosal lymphocytes including Th1-type effectors. This results in enhanced disease after primary C. parvum challenge with a response characterized by decreased IFNγ but increased IL13 and tendency toward Th2-type cytokines (red). Unlike in nourished hosts, vaccine does not further enhance IFNγ to primary C. parvum challenge, but rather the S. Typhi vector alone drives increased IL17A and partially attenuates disease severity similar to the TLR9 agonist CpG (yellow). C. parvum priming, however, leverages a robust secondary Th1-type response to C. parvum during protein malnutrition, and even at low-doses in this model establishes a mucosal imprinted population of CD8+ T-cells along with protective immunity to subsequent re-challenge (dark grey).

Similar articles

Cited by

References

    1. Black RE, Victora CG, Walker SP, Bhutta ZA, Christian P, de Onis M, et al. (2013) Maternal and child undernutrition and overweight in low-income and middle-income countries. Lancet: 382(9890):427–51. 10.1016/S0140-6736(13)60937-X - DOI - PubMed
    1. Rytter MJH, Kolte L, Briend A, Friis H, Christensen VB (2014) The immune system in children with malnutritionA systematic review. PLoS One: 9(8):e105017 10.1371/journal.pone.0105017 - DOI - PMC - PubMed
    1. Haque R, Snider C, Liu Y, Ma JZ, Liu L, Nayak U, et al. (2014) Oral polio vaccine response in breast fed infants with malnutrition and diarrhea. Vaccine: 32(4):478–82. 10.1016/j.vaccine.2013.11.056 - DOI - PMC - PubMed
    1. Mølbak K, Højlyng N, Gottschau A, Sá JC, Ingholt L, da Silva AP, Aaby P (1993) Cryptosporidiosis in infancy and childhood mortality in Guinea Bissau, West Africa. BMJ: 307(6901):417–20. - PMC - PubMed
    1. Tumwine JK, Kekitiinwa A, Nabukeera N, Akiyoshi DE, Rich SM, Widmer G, et al. (2003) Cryptosporidium parvum in children with diarrhea in mulago hospital, Kampala, Uganda. Am J Trop Med Hyg: 68(6):710–5. - PubMed

Publication types

MeSH terms

Substances