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

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

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

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

Cross-modulation of pathogen-specific pathways enhances malnutrition during enteric co-infection with <i>Giardia lamblia</i> and enteroaggregative <i>Escherichia coli</i>

<div><p>Diverse enteropathogen exposures associate with childhood malnutrition. To elucidate mechanistic pathways whereby enteric microbes interact during malnutrition, we used protein deficiency in mice to develop a new model of co-enteropathogen enteropathy. Focusing on common enteropathogens in malnourished children, <i>Giardia lamblia</i> and enteroaggregative <i>Escherichia coli</i> (EAEC), we provide new insights into intersecting pathogen-specific mechanisms that enhance malnutrition. We show for the first time that during protein malnutrition, the intestinal microbiota permits persistent <i>Giardia</i> colonization and simultaneously contributes to growth impairment. Despite signals of intestinal injury, such as IL1α, <i>Giardia</i>-infected mice lack pro-inflammatory intestinal responses, similar to endemic pediatric <i>Giardia</i> infections. Rather, <i>Giardia</i> perturbs microbial host co-metabolites of proteolysis during growth impairment, whereas host nicotinamide utilization adaptations that correspond with growth recovery increase. EAEC promotes intestinal inflammation and markers of myeloid cell activation. During co-infection, intestinal inflammatory signaling and cellular recruitment responses to EAEC are preserved together with a <i>Giardia</i>-mediated diminishment in myeloid cell activation. Conversely, EAEC extinguishes markers of host energy expenditure regulatory responses to <i>Giardia</i>, as host metabolic adaptations appear exhausted. Integrating immunologic and metabolic profiles during co-pathogen infection and malnutrition, we develop a working mechanistic model of how cumulative diet-induced and pathogen-triggered microbial perturbations result in an increasingly wasted host.</p></div

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

<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

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

<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

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