<i>C</i>. <i>parvum</i> priming enhances Th1-type cytokine responses to re-challenge in protein malnourished mice.

<p>(A) Ileal inflammatory mediators and chemokines and (B) cytokines measured three days after 10⁷ <i>C</i>. <i>parvum</i> challenge in previously uninfected (PBS) compared with mice primed with 10⁶ <i>C</i>. <i>parvum</i> (Cp10^<i>6</i>) 20 days prior to re-challenge. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

<i>C</i>. <i>parvum</i> priming leads to sustained changes in ileal tissue chemokine and cytokine profiles during protein malnutrition.

<p>Mice were conditioned on PD for 5 days prior to infection with 10⁶ <i>C</i>. <i>parvum</i> (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 <i>C</i>. <i>parvum</i> 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 <i>C</i>. <i>parvum</i> challenge. There was a relative decrease in all Th2-type cytokines through 23 days post-<i>C</i>. <i>parvum</i> compared with uninfected controls. (n = 3-4/group). *<i>P</i><0.05 for PBS vs Cp10⁶ as indicated; #<i>P</i><0.05 for Cp10⁶ D3 vs Cp10⁶ D23 as indicated.</p

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

<p>Strategies to enhance immune defenses against <i>Cryptosporidium</i> 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 <i>Cryptosporidium</i> with little evidence of a secondary immune response (dark blue), mucosal vaccination with <i>Cryptosporidium</i> antigens expressed in an <i>S</i>. Typhi vector can elicit strongly boosted IFNγ-predominant immune responses to subsequent challenge (light blue). Vaccine attenuates the mild disease caused by <i>Cryptosporidium</i> 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 <i>C</i>. <i>parvum</i> 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 <i>C</i>. <i>parvum</i> challenge, but rather the <i>S</i>. Typhi vector alone drives increased IL17A and partially attenuates disease severity similar to the TLR9 agonist CpG (yellow). <i>C</i>. <i>parvum</i> priming, however, leverages a robust secondary Th1-type response to <i>C</i>. <i>parvum</i> 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).</p

Protein malnutrition alters basal immune responses to primary <i>C</i>. <i>parvum</i> exposure, but secondary responses are intact.

<p>Immunologic responses to two different recombinant <i>Cryptosporidium</i> sporozoite antigens (CApy and Cp15) were performed at 13–15 days post <i>C</i>. <i>parvum</i> challenge in mice fed either control-diet (<i>C</i>. <i>parvum</i>^CD) or protein-deficient diet (<i>C</i>. <i>parvum</i>^pd) and results were compared with naïve age and diet-matched uninfected controls (PBS^CD and PBS^pd). Mice began respective diets 12 days prior to <i>C</i>. <i>parvum</i> 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 <i>Cryptosporidium</i> antigens. (B) Serum antibody production as anti-CApy or anti-Cp15 IgG titer (<i>*P<</i>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. (*<i>P</i><0.05 as indicated). Data is representative of pooled individual responses from two separate tissue harvests (n = 4-5/group).</p

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

<p>(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 <i>Cryptosporidium parvum</i> oocysts, heat-inactivated <i>C</i>. <i>parvum</i> oocysts (Δ<i>C</i>. <i>parvum</i>) 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 (**<i>P</i><0.01, ***<i>P</i><0.001 <i>C</i>. <i>parvum</i>^PD vs. <i>C</i>. <i>parvum</i> or <i>C</i>. <i>parvum</i>^RBD), fecal parasite shedding (**<i>P</i><0.01 PD or RBD vs. CD day 2, *<i>P</i><0.05 PD vs. RBD or CD day 3), tissue parasite burden (*<i>P</i><0.05 PD vs CD or RBD ileum, *<i>P</i><0.05 PD vs CD colon), and ileum villus:crypt ratios (**<i>P</i><0.01 PBS^CD vs PBS^RBD, ***<i>P</i><0.001 PBS^CD vs PBS^PD, ****<i>P</i><0.0001 <i>C</i>. <i>parvum</i>^CD or <i>C</i>. <i>parvum</i>^RBD vs <i>C</i>. <i>parvum</i>^PD, ^<i>P</i><0.05 PBS^CD vs. <i>C</i>. <i>parvum</i>^CD, ^##<i>P</i><0.01 PBS^PD vs. <i>C</i>. <i>parvum</i>^PD (n = 3-7/group). (C) Growth through three days post-challenge with either <i>C</i>. <i>parvum</i> or Δ<i>C</i>. <i>parvum</i>. (N = 5-10/group, **<i>P</i><0.01, ***<i>P</i><0.001 (<i>C</i>. <i>parvum</i> vs. either PBS or Δ<i>C</i>. <i>Parvum</i>) and fecal parasite shedding (n = 5-10/group, *<i>P</i><0.05, **<i>P</i><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, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ****<i>P</i><0.0001 <i>C</i>. <i>parvum</i> 10⁷ vs <i>C</i>. <i>parvum</i> 10⁶; and ^####<i>P</i><0.0001 <i>C</i>. <i>parvum</i> vs PBS) and (F) fecal parasite shedding (**<i>P</i><0.01, ****<i>P</i><0.0001) through 21 days post-challenge. Data is representative of 2 replicate experiments.</p

<i>C</i>. <i>parvum</i> priming protects against re-challenge despite continuous protein malnutrition.

<p>Growth (A) and parasite fecal shedding (B) following challenge with 10⁷ <i>C</i>. <i>parvum</i> oocysts in either naïve (PBS-<i>C</i>. <i>parvum</i> 10⁷) PD-fed mice, or mice previously exposed to either 10⁶ (Cp 10⁶) or 10⁷ (Cp 10⁷) <i>C</i>. <i>parvum</i> oocysts 21 days prior as indicated (n = 5/group). (A) *<i>P</i><0.05, **<i>P</i><0.01 for PBS-PBS vs PBS-<i>C</i>. <i>parvum</i> 10⁷, ^#<i>P</i><0.05, ^### <i>P</i><0.001 ^#### <i>P</i><0.0001 for Cp 10^6-<i>C</i>. <i>parvum</i> 10⁷ vs. PBS-<i>C</i>. <i>parvum</i> 10⁷, <i>^</i><i>P</i><0.05, and ^^<i>P</i><0.01, ^^^^<i>P</i><0.0001 for Cp 10^7-<i>C</i>. <i>parvum</i> 10⁷ vs. PBS-<i>C</i>. <i>parvum</i> 10⁷. (B) *<i>P</i><0.05, **<i>P</i><0.01 for Cp 10^6-<i>C</i>. <i>parvum</i> 10⁷ or Cp 10⁷-<i>C</i>. <i>parvum</i> 10⁷ vs. PBS-<i>C</i>. <i>parvum</i> 10⁷.</p

Viable <i>C</i>. <i>parvum</i> priming provides greater protection against re-challenge than either CpG-ODN or <i>S</i>. Typhi.

<p>(A, B) Comparison of protective immunity following priming with either viable and heat-inactivated (Δ) <i>C</i>. <i>parvum</i> 10⁶. (A) Growth of PD-fed mice through 23 days post-priming with either 4x10⁶ viable (<i>C</i>. <i>parvum</i>) or 4x10⁶ heat-inactivated (Δ<i>C</i>. <i>parvum</i>). Mice were challenged with viable 4<i>x</i>10⁷ <i>C</i>. <i>parvum</i> oocysts on day 20 post-priming. *<i>P</i><0.05 for Δ<i>C</i>. <i>parvum-C</i>. <i>parvum</i> vs. <i>C</i>. <i>parvum</i>-<i>C</i>. <i>parvum</i> (d3 and d23); ^^<i>P</i><0.01 for <i>C</i>. <i>parvum</i>-PBS vs PBS-<i>C</i>. <i>parvum</i> (d23); ^###<i>P</i><0.001 for <i>C</i>. <i>parvum</i>-<i>C</i>. <i>parvum</i> vs. PBS-<i>C</i>. <i>parvum</i> (d23). (B) RT-PCR of <i>Cryptosporidium</i> stool shedding on experimental days 21 and 23 (day 1 and day 3 after <i>C</i>. <i>parvum</i> 10⁷ challenge, respectively). *<i>P</i><0.05 and *<i>P</i><0.01 for <i>C</i>. <i>parvum</i>-<i>C</i>. <i>parvum</i> vs either PBS-<i>C</i>. <i>parvum</i> or Δ<i>C</i>. <i>parvum-C</i>. <i>parvum</i>. (C,D) 3-week-old C57Bl/6 mice were conditioned on PD for 7 days prior to orogastric inoculation with 10⁶ <i>C</i>. <i>parvum</i>, intranasal (i.n.) 10⁹ <i>S</i>. Typhi 908<i>htr</i>, 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 <i>C</i>. <i>parvum</i> 10⁷. (C) Growth as percentage of initial weight, normalized to the day of 10⁷ <i>C</i>. <i>parvum</i> challenge (Day 0). The group labeled “All uninfected” includes animals that received either PBS during both inoculations, CpG followed by PBS, or <i>S</i>. Typhi followed by PBS (n = 5/group x 3 = 15) given all three groups grew similarly and were never exposed to <i>C</i>. <i>parvum</i> (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004820#pntd.0004820.s004" target="_blank">S4 Fig</a>). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 for PBS–<i>C</i>.<i>p</i>.10⁷ (red) vs <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷, ^<i>P</i><0.05 for CpG-<i>C</i>.<i>p</i>.10⁷ (yellow) vs <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷, and ^#<i>P</i><0.05 for <i>S</i>. Typhi-<i>C</i>.<i>p</i>.10⁷ (green) vs <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷). Horizontal lines designate significant differences at <i>P</i><0.05 between CpG-<i>C</i>.<i>p</i>.10⁷ (yellow), <i>S</i>. Typhi-<i>C</i>.<i>p</i>.10⁷ (green), and PBS-<i>C</i>.<i>p</i>.10⁷ (red) vs. All uninfected controls, respectively. (D) Parasite fecal shedding in serial fecal pellets collected on indicated experimental days post <i>C</i>. <i>parvum</i> 10⁷ challenge. *<i>P</i><0.05 for PBS–<i>C</i>.<i>p</i>.10⁷ vs. <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷, ^<i>P</i><0.05 for CpG-<i>C</i>.<i>p</i>.10⁷ vs <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷, and ^#<i>P</i><0.05 for <i>S</i>. Typhi-<i>C</i>.<i>p</i>.10⁷ vs <i>C</i>.<i>p</i>.10^6-<i>C</i>.<i>p</i>.10⁷. Data is representative of two replicate experiments.</p

Protein malnutrition interferes with vaccine-boosted immunity, but the <i>S</i>. Typhi vector improves recovery after <i>C</i>. <i>parvum</i> challenge.

<p>(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 <i>S</i>. <i>enterica</i> Typhi 908<i>htr</i> vector expressing either of two recombinant sporozoite antigens, ClyA-Cp15 (<i>S</i>. Typhi^Cp15 (aqua)) or ClyA-CApy (<i>S</i>. Typhi^CApy (blue)) was administered at two-week intervals. The <i>S</i>. Typhi vector alone (<i>S</i>. Typhi (green)) and a PBS-only (red) treatment served as a double-sham control. Intramuscular injection with rCp15 (<i>S</i>. Typhi^Cp15), rCApy (<i>S</i>. Typhi^CApy), the inert NUS peptide (for <i>S</i>. 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 (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004820#pntd.0004820.s001" target="_blank">S1 Fig</a>). On day 107 (9 weeks after <i>S</i>. 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 <i>C</i>. <i>parvum</i> (<i>Cp</i>) on day 119 and followed for 13–15 days post-challenge. (B) Serum geometric mean IgG titers (GMT) in <i>C</i>. <i>parvum</i> challenged groups. (C) IFN-γ and (D) IL-17A cytokine secretion recall responses to homologous vaccinogen as indicated. For (B-D), <i>*P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, One-way ANOVA, Tukey post-test analysis, ^# <i>P</i><0.05 by Student’s <i>t</i>-test. (E) Growth as percentage of weight change on the day of <i>C</i>. <i>parvum</i> infection beginning on the day of transition to either CD (left) or PD (right) diets. (left: *<i>P</i><0.05, <i>S</i>. Typhi^Cp15 vs PBS-<i>Cp</i>; right: colored bars indicate <i>P</i><0.05 for PBS-<i>Cp</i> (red), <i>S</i>. Typhi-<i>Cp</i> (green), <i>S</i>. Typhi^Cp15 (aqua), and <i>S</i>. Typhi^CApy (blue) vs. uninfected controls, *<i>P</i><0.05 for individual vaccine groups [<i>S</i>. Typhi-<i>Cp</i> (green), <i>S</i>. Typhi^Cp15 (aqua), and <i>S</i>. Typhi^CApy (blue)] vs. PBS-<i>Cp</i>. (F) Parasite shedding for infected groups: PBS (red), <i>S</i>. Typhi (green, <i>S</i>. Typhi^Cp15 (aqua), and <i>S</i>. Typhi^CApy (blue). (G) Ileal villus:crypt for CD-fed (left) and PD-fed (right) mice in each <i>C</i>. <i>parvum</i> infected group or combined uninfected controls as indicated. Left: ***<i>P</i><0.001 PBS-<i>Cp</i> vs uninfected controls, ^###<i>P</i><0.001 <i>S</i>. Typhi^Cp15 or <i>S</i>. Typhi^CApy vs <i>S</i>. Typhi; Right: ***<i>P</i><0.001 for PBS-<i>Cp</i>, <i>S</i>. Typhi, or <i>S</i>. Typhi^Cp15 vs uninfected controls, ^###<i>P</i><0.001 <i>S</i>. Typhi^CApy vs PBS-<i>Cp</i>. ^<i>P</i><0.05 for PD-PBS and PD-<i>S</i>. Typhi^Cp15 vs DD-PBS and DD-<i>S</i>. Typhi^Cp15. ns = not significant vs. uninfected controls.</p