CLINICAL AND VACCINE IMMUNOLOGY, June 2010, p. 954–965
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 17, No. 6
Cloning and Characterization of the Acidic Ribosomal Protein P2 of
Cryptosporidium parvum, a New 17-Kilodalton Antigen?
Jeffrey W. Priest,1* James P. Kwon,1,2Joel M. Montgomery,1Caryn Bern,1Delynn M. Moss,1
Amanda R. Freeman,1Cara C. Jones,1,2Michael J. Arrowood,1Kimberly Y. Won,1
Patrick J. Lammie,1Robert H. Gilman,3,4,5and Jan R. Mead6,7
Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia1; Atlanta Research and Education Foundation,
Decatur, Georgia2; Department of International Health, Johns Hopkins University School of Public Health and Hygiene,
Baltimore, Maryland3; Asociacio ´n Bene ´fica Proyectos en Informa ´tica, Salud, Medicina, y Agricultura, Lima, Peru4;
Universidad Peruana Cayetano Heredia, Lima, Peru5; Department of Pediatrics, Emory University, Decatur,
Georgia6; and Atlanta Veterans Medical Center, Decatur, Georgia7
Received 23 February 2010/Returned for modification 1 April 2010/Accepted 9 April 2010
Cryptosporidium infection is commonly observed among children and immunocompromised individuals in
developing countries, but large-scale outbreaks of disease among adults have not been reported. In contrast,
outbreaks of cryptosporidiosis in the United States and Canada are increasingly common among patients of
all ages. Thus, it seems likely that residents of regions where Cryptosporidium is highly endemic acquire some
level of immunity, while residents of the developed world do not. A new immunodominant Cryptosporidium
parvum antigen in the 15- to 17-kDa size range was identified as the Cryptosporidium parvum 60S acidic
ribosomal protein P2 (CpP2). We developed a recombinant protein-based enzyme-linked immunosorbent assay
for serologic population surveillance for antibodies that was 89% sensitive and 92% specific relative to the
results of the large-format Western blot assay. The human IgG response is directed almost exclusively toward
the highly conserved, carboxy-terminal 15 amino acids of the protein. Although IgG antibody cross-reactivity
was documented with sera from patients with acute babesiosis, the development of an anti-CpP2 antibody
response in our Peru study population correlated better with Cryptosporidium infection than with infection by
any other parasitic protozoan. In Haiti, the prevalence of antibodies to CpP2 plateaus at 11 to 20 years of age.
Because anti-CpP2 IgG antibodies were found only among residents of countries in the developing world where
Cryptosporidium infection occurs early and often, we propose that this response may be a proxy for the intensity
of infection and for acquired immunity.
Cryptosporidium parvum and C. hominis are enteric proto-
zoan parasites that commonly cause outbreaks of diarrheal
disease in the developed world (for reviews, see references 24
and 26). All age groups are affected, and the disease is usually
self-limiting in immunocompetent individuals (5, 13). Out-
breaks have been linked to public water system treatment
failures, recreational exposure to contaminated water, contam-
ination of unpasteurized fresh-squeezed juices, and contami-
nation of food products by infected food handlers (14, 28, 35,
37, 39, 58). In the developing world, where potential sources of
food and water contamination are widespread, acute crypto-
sporidiosis is usually limited to young children and to immu-
nocompromised populations (4, 5, 48, 50, 59). In a longitudinal
serologic study of enteric parasites in Peru, we reported that
repeated infection was common among young children and
that Cryptosporidium-specific IgG antibody levels increased
with age and with experience of infection (54). Large-scale
outbreaks of overt illness among immunocompetent adults in
these regions where cryptosporidiosis is highly endemic have
not been reported. These observations suggest that some level
of immunity to disease (although not necessarily to infection)
may eventually develop upon repeated exposure to the parasite
In previous work (68), we noted that sera from individuals
who live in Haiti often contain IgG antibodies to several C.
parvum antigens, in addition to the immunodominant 27- and
17-kDa antigens. In the current work, we demonstrate that one
of these novel antigens, located in the 17-kDa-molecular-mass
range but distinct from the C. parvum 17-kDa antigen family
(56), is the C. parvum acidic ribosomal protein P2 (CpP2).
Several acidic ribosomal proteins (P0, P1, P2, or variants) have
been described as prominent antigens in leishmaniasis (69, 70),
Chagas’ disease (32, 65, 67), malaria (10), Brucella abortus
infection (6), Babesia bovis infection (12), and systemic lupus
erythematosus (SLE) (16, 17, 62). In particular, ribosomal pro-
teins P0 and P2 from Leishmania spp., Plasmodium falciparum,
and Trypanosoma cruzi have been reported to be immuno-
stimulatory, as sera from infected animals and humans recog-
nize these antigens (10, 65, 66, 67 69, 70). Although the acidic
ribosomal proteins are classically associated with the cytoplas-
mic ribosomes, they have also been localized to the cell surface
of some parasites. Chatterjee et al. (9) used antibody fluores-
cence to demonstrate the presence of the P0 protein on the
surface of P. falciparum merozoites, and Sehgal et al. (63) used
transiently transfected Toxoplasma gondii cells to demonstrate
the translocation of tagged P0 to the parasite surface.
Because of their surface localization and immunogenicity, it
has been suggested that P proteins may be possible vaccine
* Corresponding author. Mailing address: Division of Parasitic Dis-
eases, Centers for Disease Control and Prevention, 4770 Buford High-
way, NE, Mail Stop F-13, Atlanta, GA 30341. Phone: (770) 488-4587.
Fax: (770) 488-4108. E-mail: email@example.com.
?Published ahead of print on 21 April 2010.
candidates. In recent reports, immunization with the P-domain
peptide of ribosomal protein P0 provided protection against P.
falciparum challenge (60), immunization with Babesia gibsoni
P0 protein was cross-protective for infection with Babesia
microti (73), and antibodies against Neospora caninum P0 in-
hibited infection with T. gondii in vitro (79). Furthermore, a
Leishmania infantum ribosomal protein DNA vaccine con-
ferred protective immunity against Leishmania major infection
in mice (22). The strong anti-CpP2 antibody responses ob-
served for most of the Haitians who were also antibody positive
for the 27-kDa antigen suggest that the CpP2 antigen may play
a role in the generation of immune responses against C. par-
vum in areas where it is highly endemic and, therefore, might
be a potential vaccine target.
MATERIALS AND METHODS
Protein extraction and Western blot assay. The Maine isolate of C. parvum
was maintained by passage in Holstein calves, as described previously (3, 39). A
crude antigen supernatant fraction was generated by sonication and freeze-
thawing of purified Maine isolate oocysts, followed by centrifugation at 24,000 ?
g for 30 min (42). A membrane-associated protein fraction was isolated from the
crude antigen by Triton X-114 detergent phase partitioning, as described previ-
ously, collected by acetone precipitation, and dissolved in buffer containing 0.5%
SDS and 20 mM HEPES at pH 7.4 (55). Protein concentrations were determined
by the bicinchoninic acid microassay (Pierce, Rockford, IL) with bovine serum
albumin as the standard. Triton X-114-extracted proteins (140 ?g) were
resolved by preparative polyacrylamide gradient gel electrophoresis on a 3 to
25% discontinuous gel (44), and the proteins in the 17-kDa region were
excised and recovered from the gel by electroelution (Elutrap; Schleicher &
Schuell, Keene, NH).
Crude oocyst antigens were resolved on 10 to 22.5% SDS-polyacrylamide gels
by using the buffer system of Laemmli (29) and electrotransferred onto a poly-
vinylidene difluoride (PVDF) membrane (Immobilon P; Millipore Corp., Bed-
ford, MA). IgG Western blot assays were conducted on 2-mm-wide membrane
strips with the biotinylated mouse monoclonal anti-human IgG and streptavidin-
alkaline phosphatase system described previously (55). Rabbit and mouse IgG
antibodies were detected by using biotinylated goat anti-rabbit IgG and mono-
clonal rat anti-mouse IgG, respectively (Zymed, South San Francisco, CA).
Rabbit immunization and screening of expression library. A female New
Zealand White rabbit was immunized three times at 4-week intervals with ap-
proximately 0.6 ?g of the electroeluted 17-kDa region protein and an equal
volume of TiterMax adjuvant per immunization (TiterMax USA, Inc., Norcross,
GA). Serum was collected 2 weeks after the final immunization. No antibodies to
C. parvum antigens were detected prior to immunization (data not shown). All
animal work was approved by the Animal Use Committee of the Centers for
Disease Control and Prevention (CDC).
An Iowa strain sporozoite cDNA library constructed from poly(A)?mRNA in
the bacteriophage ? UniZAP XR vector (CpLib3; kindly provided by N. J.
Pieniazek, M. J. Arrowood, S. B. Slemenda, and J. R. Mead, CDC, Atlanta, GA,
and Emory University, Atlanta, GA) was screened for IgG antibody-reactive
clones by using the rabbit serum at a dilution of 1:50 in buffer containing 0.85%
NaCl and 10 mM Na2HPO4at pH 7.2 (phosphate-buffered saline [PBS]) with
0.3% Tween 20. Positive plaques were identified by using the biotinylated goat
anti-rabbit IgG and streptavidin-alkaline phosphatase system. The plasmids were
excised from the bacteriophage ? phage cultures in XL1-Blue Escherichia coli
cells by using R408 helper phage (Stratagene, La Jolla, CA) and were plated on
LB agar in the presence of ampicillin. The plasmids were isolated from the
resulting clones by using a Wizard plasmid purification kit (Promega, Madison,
WI) and were sequenced by using primers T3 and T7. Deduced protein se-
quences were aligned by use of the ClustalW (version 2) program (30) and were
manually optimized in the carboxy-terminal region.
CpP2 cloning and expression. The following deoxyoligonucleotide pair was
used to amplify the CpP2-coding sequence (GenBank accession number
AF099744) by PCR for directional cloning into the KpnI and HindIII restriction
sites of vectors pQE-41 and pQE-81 (Qiagen, Valencia, CA): 5?-GGG GTA
CCC CTG GTT CCG CGT GGA TCC ATG GGT ATG AAA TAC GTT GC-3?
and 5?-CGC CCA AGC TTA TTT AAT TAG TCA AAC AAT GAG AAA
CC-3?. The forward primer was designed so that the 6? His tag (pQE-81) or the
6? His-dihydrofolate reductase (DHFR) fusion partner (pQE-41) could be
removed with thrombin protease. In these primer sequences and those presented
below, the restriction sites are underlined, and the thrombin site-coding se-
quences are italicized. AmpliTaq Gold DNA polymerase (Perkin-Elmer Cetus,
Foster City, CA) was used as directed by the manufacturer to amplify the target
sequence from genomic sporozoite DNA (55).
The 6? His fusion proteins were expressed in E. coli DH5?(pQE-81) (Invitro-
gen, Carlsbad, CA) or E. coli JM-109(pQE-41) (Promega). The recombinant
proteins were purified by nickel-affinity chromatography (HiTrap Chelating HP
1-ml column; GE Healthcare, Piscataway, NJ) in PBS and cleaved with thrombin
to remove the amino-terminal tag, as directed by the manufacturer. Uncleaved
protein and 6? His-tagged DHFR were removed by reapplication to the nickel
Cloning and expression of other apicomplexan P proteins. The C. parvum
protein P1 (CpP1)-coding sequence (GenBank accession number XM_626174)
was amplified from genomic C. parvum DNA by PCR with the following forward
and reverse deoxyoligonucleotide pairs: 5?-GGG GTA CCC CTG GTT CCG
CGT GGT TCC ATG GCA GCT GTT TCA ATG AAT G-3? and 5?-CGC CCA
AGC TTA TTT AAT TAG TCA AAC AAT GAG AAA CC-3?, respectively.
The Plasmodium falciparum CpP2-coding sequence (GenBank accession number
U78753) was amplified from genomic DNA (from strain FCR3F86, kindly pro-
vided by N. Lang-Unnash, University of Alabama at Birmingham, Birmingham,
AL) by PCR with the following forward and reverse deoxyoligonucleotide pairs:
5?-GGG GTA CCC CTG GTT CCG CGT GGA TCC ATG GCT ATG AAA
TCA GTT GC-3? and 5?-CGC CCA AGC TTA ACC AAA TAA GGA AAA
TCC-3?, respectively. The Toxoplasma gondii CpP2-coding sequence (GenBank
accession number XM_002364187) was amplified from cDNA (from strain RH,
kindly provided by J. Boothroyd, Stanford University, Stanford, CA) by PCR
with the following forward and reverse deoxyoligonucleotide pairs: 5?-GGG
GTA CCC CTG GTT CCG CGT GGA TCC ATG GCA ATG AAA TAC GTC
GC-3? and 5?-CGC CCA AGC TTA GTC GAA GAG CGA GAA GCC-3? PCR,
respectively. The PCR conditions were those described above for the amplifica-
tion of CpP2. The products were cloned into the KpnI- and HindIII-digested
pQE-81 plasmid vector and expressed in E. coli DH5? cells (Invitrogen). The C.
parvum protein P0 (CpP0)-coding sequence (GenBank accession number
XM_625816) was amplified from genomic C. parvum DNA by PCR with the
following forward and reverse deoxyoligonucleotide pairs: 5?-GGG GTA CCC
CTG GTT CCG CGT GGT TCC ATG CCA TCT CCA GAG AAA GC-3? and
5?-CGC CCC TGC AGT CAG TCA AAT AAT GAA AAA CC-3?, respectively.
This product was cloned into the KpnI- and PstI-digested pQE-81 plasmid vector
for expression. Recombinant proteins were purified on a nickel-affinity chroma-
tography, digested with thrombin, and dialyzed against 25 mM Tris at pH 7.5
(Spectra/Por3; 3,500-Da cutoff; Spectrum Laboratories, Rancho Dominguez,
CA). The proteins were bound on a Mono Q HR 5/5 strong anion-exchange
column (GE Healthcare) and eluted with a linear gradient of from 0 to 1 M NaCl
in 25 mM Tris at pH 7.5. The protein-containing fractions were concentrated
with Centricon-10 centrifugal filter devices (Millipore Corporation, Bedford,
MA), and the 6? His-tagged, uncleaved proteins were removed by reapplication
onto nickel columns in PBS buffer.
Monoclonal antibody production and purification of monospecific polyclonal
antibody. A mouse monoclonal antibody (monoclonal antibody 1D6) to recom-
binant CpP2 (rCpP2) was generated by Southern Biotech (Birmingham, AL).
This IgG1 isotype antibody was shown to react specifically with a linear epitope
located between CpP2 residues 43 and 60 (VLISNMSGKLSHEVIASG) (data
not shown). Anti-CpP2 antibodies were purified from human serum by using
Western-blotted rCpP2 antigen and the MgCl2elution method of Tsang and
Wilkins (75). The eluted antibodies were desalted on G-25 M size-exclusion
columns (PD-10; GE Healthcare) that were preequilibrated with buffer contain-
ing PBS with 0.3% Tween 20.
Epitope mapping. A series of 33 overlapping biotinylated peptides (15-mers,
12-residue overlap) were synthesized on the basis of the CpP2 gene sequence.
The peptides were in the format of biotin-SerGlySerGly-peptide sequence-
amide. All peptides were dissolved in 0.05% Tween 20–PBS at a concentration
of 2 ?g/ml. The diluted peptides (50 ?l/well) were incubated overnight at 4°C in
streptavidin-coated 96-well microtiter plates (200 ng protein/well; Immulon 2
HB; Thermo Electron Corp., Milford, MA). After four washes with 0.05%
Tween 20–PBS, diluted serum (50 ?l/well, diluted 1:100 in 0.3% Tween 20–PBS
with 1% casein) was added and the plates were incubated for 2 h at room
temperature. The plates were washed four times, and a peroxidase-labeled, goat
anti-human IgG antibody was added (50 ?l/well, diluted 1:2,000 in 0.3% Tween
20–PBS with 1% casein). After 1 h of incubation at room temperature, the plates
were washed four times and 50 ?l of 2,2?-azino-di-[3-ethylbenzthiazoline sulfo-
nate (6)] (ABTS; Kirkegaard & Perry, Gaithersburg, MD) substrate was added
to each well. The absorbances were read at 405 nm with a Molecular Dynamics
VOL. 17, 2010 ANTIBODY RECOGNITION OF CRYPTOSPORIDIUM P2 PROTEIN955
UVmax kinetic microplate reader (Sunnyvale, CA). Positive- and negative-con-
trol peptides were included on each plate and were incubated with a strong
positive-control serum. The absorbances of the CpP2 peptide wells were ex-
pressed as a percentage of the positive-control absorbance.
Measurement of antibody responses by ELISA. Test sera were diluted 1:50
with buffer containing PBS with 0.05% Tween 20 and were assayed in duplicate.
Assays for the detection of IgG antibodies to the recombinant C. parvum 27-kDa
antigen have been described previously (11, 54). Antibody values of ?116 arbi-
trary units were considered a positive result (11).
Assays for the detection of IgG antibodies to the recombinant CpP2 antigen
were conducted by using the same blocking, assay, and development conditions
used for the detection of the C. parvum 27-kDa antigen. Immulon 2HB flat-
bottom microtiter plates (Thermo Electron Corp.) were sensitized overnight at
4°C with 50 ?l of purified, recombinant CpP2 antigen per well at a concentration
of 0.4 ?g/ml in 0.1 M sodium bicarbonate buffer (pH 9.6). Each CpP2 enzyme-
linked immunosorbent assay (ELISA) plate included three positive controls,
three negative controls, as well as a 2-fold serial dilution series (9 dilutions, 1:50
to 1:12,800) of a strong CpP2-positive serum sample. The test sera were assigned
a unit value on the basis of a four-parameter curve fit of the positive-control
dilution series absorbance values (405 nm) in which the value for the 1:50
dilution was arbitrarily set equal to 6,400 units. Antibody values ?136 were
considered positive on the basis of a receiver operating characteristic curve by
use of the Western blot results as the “gold standard” (194 samples; 89%
sensitivity and 92% specificity with 91% correct results) (81).
The definition of a CpP2 antibody response applied to the analysis of longi-
tudinal serum samples from a Peruvian birth cohort used the 136-arbitrary-unit
cutoff and the interval criteria previously described for the 27- and 17-kDa C.
parvum antigens (53, 54). Briefly, a P2 serologic response was identified when
two consecutive serum samples were collected ?180 days apart, the P2 response
of the second serum sample was above the cutoff, and the P2 response of the
second serum sample increased ?50% relative to that for the first serum sample.
If the interval between consecutive responses was ?90 days, they were consid-
ered separate events. Other characteristics and analyses of the cohort have been
described previously (5, 53, 54, 78).
The levels of antibodies to other P proteins (20 ng antigen/well) were assayed
by ELISA under the conditions described above. Absorbance values (405 nm)
were expressed as a percentage of the CpP2 positive-control value (1:50 dilu-
tion). The secondary antibody used for assays containing hamster sera was
biotinylated rabbit anti-hamster IgG (Zymed).
Human serum specimens. Expired plasma samples from 30 anonymous Haiti
residents were obtained from a local hospital in Leogane, Haiti, in 1998. Banked
sera were available for analysis from 30 U.S. citizens who had no history of
foreign travel (55). Serum specimens from 30 individuals who were involved in a
food-borne outbreak of cryptosporidiosis in 1997 were collected approximately 8
weeks after exposure (7, 45). Twenty-five of the serum specimens were from
symptomatic individuals, and 6 of these were from individuals whose infections
were confirmed by the analysis of stool samples. Written informed consent was
obtained from the study participants, and the study was approved by the Centers
for Disease Control and Prevention Institutional Review Board. Pools of sera (10
serum samples per pool) from patients with acute infections caused by Crypto-
sporidium spp., Cyclospora cayetanensis, T. gondii, P. falciparum, and B. microti
were made. The Cryptosporidium pool contained high-titer sera from patients
involved in two U.S. cryptosporidiosis outbreaks whose infections were con-
firmed by analysis of stool samples. Sera were also obtained from patients with
cyclosporiasis involved in a single U.S. outbreak of cyclosporiasis whose infec-
tions were confirmed by analysis of stool samples (27). The titers of antibodies to
C. cayetanensis antigens were not determined. Sera were also obtained from
patients with acute toxoplasmosis involved in a U.S. toxoplasmosis outbreak and
were positive for both IgM and IgG with titers of ?1:4,096, determined by
immunofluorescence assay (74). Sera were collected from patients with sporadic
cases of malaria and babesiosis in the United States, but demographic informa-
tion on the patients was not available. A pool of sera was made from 10 U.S.
residents with chronic T. gondii infections who were IgM negative and who had
IgG titers of ?1:1,024.
Serum samples (n ? 533) from a subset of 61 children from a longitudinal birth
cohort study of diarrheal disease in Peru were tested for antibodies to the CpP2
antigen (5, 53, 54). Reports describing the detection of parasites by stool mi-
croscopy and analysis of the serologic responses to the 17- and 27-kDa C. parvum
antigens in a larger subset of the cohort have been published previously (5, 53,
54). Written informed consent was obtained from the parent or guardian of each
participating child. The original cohort study was approved by the Institutional
Review Boards of the Johns Hopkins University School of Public Health and the
Asociacio ´n Bene ´fica Proyectos en Informa ´tica, Salud, Medicina, y Agricultura.
The Cryptosporidium serology study was approved by the Centers for Disease
Control and Prevention Institutional Review Board.
Sera from 441 inhabitants of Miton, Haiti, were collected in 1998 as part of a
community-wide study of lymphatic filariasis and other parasitic infections (19).
The protocol for this study was approved by the Centers for Disease Control and
Prevention Institutional Review Board and by the Ethics Committee of the Saint
Croix Hospital, Leogane, Haiti. Written consent was required for participation in
Data analysis. Frequencies were compared by the chi-square test with Yates’
correction for continuity. Antibody responses to the CpP2 and 27-kDa antigens
among the different age groups were compared by the Kruskal-Wallis test. The
distributions of positive results for the two antigens among Miton, Haiti, resi-
dents were compared by the chi-square goodness-of-fit test. Statistical analyses
were conducted with the SigmaStat for Windows (version 2.03.0) program (SPSS,
Inc., Chicago, IL) or the SAS (version 9.0) program. Statistical significance was
set at an alpha level of 0.05.
Identification, cloning, and characterization of the CpP2
antigen. Infections with various species of Cryptosporidium
have been shown to result in the development of characteristic
IgG antibody responses to families of antigens in the 27- and
17-kDa regions (43, 46, 55). These families are composed of
both soluble proteins that lack modifications and membrane-
associated proteins that are posttranslationally modified by the
addition of fatty acids or a glycosylphosphatidylinositol (GPI)
anchor (52, 57; J. W. Priest et al., unpublished observations).
The lower-molecular-mass, unmodified proteins remain in the
aqueous phase, while the modified proteins readily partition
into the detergent phase during Triton X-114 detergent extrac-
tion. Large-format Western blot studies of sera from healthy
Haitian adults (normal human sera ([NHS]) by use of a total C.
parvum antigen preparation showed that all serum samples had
antibodies to the 27-kDa antigen, 93% had antibodies to the
17-kDa antigen, and 70% had antibodies to two novel antigens
in the 17-kDa size range. Although our panels of NHS from
healthy U. S. adults and sera from U.S. adults involved in a
cryptosporidiosis outbreak also had high levels of antibodies to
the 27- and 17-kDa antigens (93% and 57%, respectively, for
NHS and 100% and 93%, respectively, for outbreak sera) the
two new antigens in the 17-kDa region were not detected.
Representative blots of NHS samples from 9 Haitian adults
(Haiti NHS), 10 U.S. adults (U.S. NHS), and 10 individuals
involved in a cryptosporidiosis outbreak in the United States
(U.S. outbreak) are shown in Fig. 1. We wondered whether
antibodies to these two antigens were a marker for Cryptospo-
ridium infection or were a marker for infection with some other
agent not commonly found in the United States.
Triton X-114 detergent-soluble C. parvum proteins were
eluted from the 17-kDa region of a 3 to 25% SDS-polyacryl-
amide gel and were used to immunize a rabbit. Instead of
the expected pattern of five related 17-kDa antigen bands, the
rabbit IgG antibodies recognized three new proteins in the
17-kDa region, and two of these had the same apparent mo-
lecular masses as the two new antigens observed by use of the
Haitian sera. The antigens were present in the total crude C.
parvum antigen preparation (Fig. 2, lane 1) as well as in the
Triton X-114 extract (Fig. 2, lane 2). We used the rabbit sera
to screen a C. parvum sporozoite cDNA bacteriophage lambda
expression library and obtained two clones containing the C.
parvum acidic ribosomal protein P2 sequence (Fig. 3). CpP2
shares between 48% and 55% identity with other reported
956 PRIEST ET AL.CLIN. VACCINE IMMUNOL.
apicomplexan P2 proteins and with the human P2 protein, but
it is less similar to the kinetoplastid P2 proteins that have been
used in diagnostic assays (40% identity with Leishmania infan-
tum; 39% identity with Trypanosoma cruzi) (65, 69).
As with nearly all ribosomal P proteins, the CpP2 carboxy
terminus contains elements of the human systemic lupus ery-
thematosus P-protein consensus sequence (underlined in the
human [Homo sapiens] sequence in Fig. 3) (32). In some SLE
patients, this 11-amino-acid sequence (and especially the car-
boxy-terminal 6 residues of the sequence) elicits a strong au-
toimmune IgG antibody response (16, 32, 36). CpP2 contains 6
of the conserved-motif residues at Asp-104, Gly-106, Phe-108,
Leu-109, Phe-110, and Asp-111, as well as 2 conserved substi-
tutions at Glu-102 (for Asp) and Leu-105 (for Met). All of the
apicomplexan P2 proteins reported thus far contain a Gly-to-
Ser substitution at the third position of the critical GFGLFD
hexapeptide sequence (Ser-108 in the CpP2 sequence). Amino
acid replacement studies with anti-ribosomal protein P-posi-
tive sera from patients with SLE have suggested that a Gly-to-
Ser substitution at the third position eliminates most of the
IgG antibody binding to the hexapeptide (36).
From our expression library screening with the rabbit anti-
body, we also obtained two clones that encode CpP0. This
probably occurred because the carboxy-terminal 16 amino ac-
ids of CpP0 are identical to the CpP2 terminal sequence (data
not shown). Although the same is also true for CpP1, we did
not obtain any clones containing the P1 sequence.
In order to identify which protein band on the Western blot
was the CpP2 band, monoclonal and monospecific polyclonal
antibodies were generated with rCpP2 as the antigen. Serum
from a BALB/c mouse that was immunized with rCpP2 recog-
nized two major bands in the 17-kDa region (Fig. 4A, lane 1)
in a pattern that was reminiscent of that observed with the
Haitian sera in Fig. 1. However, monoclonal antibody 1D6, an
antibody that recognized a CpP2 sequence that was not shared
with either CpP0 or CpP1 (italicized and underlined in Fig. 3),
recognized only the lower-molecular-mass band (Fig. 4A, lane
2). Polyclonal IgG antibodies affinity purified from a Haitian
FIG. 1. Representative Western blots of NHS samples collected from 9 healthy Haitian blood donors (Haiti NHS), 10 healthy U.S. citizen blood
donors (US NHS), and 10 U.S. cryptosporidiosis outbreak patients (US outbreak). A crude oocyst antigen preparation was resolved on 10 to 22.5%
SDS-polyacrylamide gels and electrotransferred to a PVDF membrane. Individual serum samples were incubated with strips at a dilution of 1:100,
and bound IgG antibodies were visualized by using the streptavidin-alkaline phosphatase system described in Materials and Methods. The locations
of the 27- and 17-kDa antigens are indicated. Two new immunodominant antigens in the 15- to 17-kDa size range (indicated by arrows) were
observed on some blots of the Haitian serum samples but were not evident on the blots of sera from the United States.
VOL. 17, 2010 ANTIBODY RECOGNITION OF CRYPTOSPORIDIUM P2 PROTEIN 957
blood donor by using rCpP2 reacted with the same two bands
as the mouse serum (Fig. 4B). Taken together, these results
identify the lower band in the Western blots as CpP2 and
strongly suggest that the higher-molecular-mass band that
shares one or more antibody epitopes with CpP2 is CpP1. A
protein band corresponding to CpP0 (predicted molecular
mass, 33.5 kDa) was not conclusively identified on the blot.
This protein may be underrepresented in our extract.
The apparent molecular masses of native CpP1 and CpP2
(approximately 14.5 and 13.8 kDa, respectively; Fig. 2) are
greater than the predicted molecular masses of 12.1 and 11.5
kDa, respectively. Because recombinant CpP2 also migrated
more slowly than expected, the size discrepancy may simply
result from an unusual secondary structure induced by the
alanine- and acidic residue-rich sequence of the carboxy-ter-
minal one-third of the protein (61, 77).
Epitope mapping of CpP2. The antibody cross-reactivity
noted above led us to make a closer examination of the anti-
genic characteristics of CpP2. Using a series of overlapping,
biotinylated peptides, we determined that only the carboxy-
terminal 15-amino-acid peptide (EEEEEEGDLGFSLFD) was
recognized by IgG antibodies from P2-positive Haitian sera
(Fig. 5). A 3-amino-acid upstream shift in the peptide se-
quence eliminated all antibody recognition in the human sera
tested (Fig. 5 and data not shown). These results are similar to
those reported for anti-ribosomal protein P sera from SLE
patients by Mahler et al. (36) and for immune sera from pa-
tients with Chagas’ disease by Skeiky et al. (67). Interestingly,
sera from mice immunized with recombinant CpP2 strongly
recognized two internal epitopes, in addition to the carboxy-
terminal one (data not shown). One of these epitopes is rec-
ognized by our 1D6 mouse monoclonal antibody (underlined
and italic sequence in Fig. 3).
Anti-P2 antibody cross-reactivity. The exclusive concentra-
tion of the human immune response on the highly conserved
carboxy-terminal 15 amino acids of CpP2 raised the possibility
that antibodies from patients with various apicomplexan par-
asitic infections might cross-react at this epitope. We expressed
recombinant CpP0, CpP1, the P. falciparum P2 protein (PfP2),
and the T. gondii P2 protein (TgP2) and used these along with
CpP2 to screen high-titer serum pools from humans infected
with Cryptosporidium, P. falciparum, T. gondii, and B. microti.
As predicted from their identical carboxy-terminal sequences,
all of the C. parvum P proteins reacted with the P2 positive-
control serum sample (sample Haiti NHS 14) (Table 1). This
serum sample also reacted with the T. gondii P2 protein but did
not react significantly with the P. falciparum protein. In con-
trast to the Haiti NHS 14 sample, another P2-positive Haitian
serum sample, Haiti NHS 1, did not discriminate between PfP2
and the other P proteins. This result suggests that there is some
variability between individuals in the exact antibody recogni-
tion sequence within the conserved, carboxy-terminal 15-amino-
As demonstrated by Western blotting (Fig. 1), acute-phase,
high-titer sera from patients involved in U.S. outbreaks of
cryptosporidiosis did not react with the P proteins (Table 1).
Similarly, significant levels of antibody to either the homolo-
gous or the heterologous P proteins were not evident in pools
of 10 serum samples from U.S. patients with acute malaria,
acute cyclosporiasis, acute toxoplasmosis, or chronic toxoplas-
mosis. In contrast, pooled sera from patients with acute babe-
siosis did react with most of the P proteins. Because clinical
data were not available for the patients with babesiosis, we
assayed a serum sample from an experimentally infected ham-
ster to confirm that the antibody response was the result of B.
microti infection rather than exposure to some other unchar-
acterized parasite. High levels of IgG antibodies to all of the P
proteins except PfP2 were detected in a hamster 28 days after
inoculation. These results confirm that CpP2 cross-reactive
antibodies can develop during infection with another apicom-
plexan parasite, but because human babesiosis has not been
reported in Haiti, the data do not definitively link a particular
parasite to our observations.
Many P2 responses are temporally linked to Cryptospo-
ridium sp. infection. To determine whether the CpP2 antibody
responses were coincident with Cryptosporidium infection, we
FIG. 2. Generation of antibodies to the 17-kDa antigen fraction. A
partially purified C. parvum 17-kDa antigen fraction, resolved and
electroeluted as described in Materials and Methods, was used to
immunize a New Zealand White rabbit. Rabbit serum was incubated
with a total C. parvum antigen PVDF strip (lane 1) and with a Triton
X-114-extracted C. parvum antigen PVDF strip (lane 2). Bound IgG
antibodies were visualized by use of a biotinylated goat anti-rabbit IgG
secondary antibody and developed as described in the legend to Fig. 1.
The locations of the molecular mass markers are indicated on the left.
958 PRIEST ET AL.CLIN. VACCINE IMMUNOL.
evaluated 533 longitudinal serum specimens collected from 61
children who participated in a Peruvian birth cohort study of
diarrheal disease. These 61 children, part of a larger cohort
subset (n ? 74) that was previously analyzed for antibody
responses to the 17- and 27-kDa antigens by ELISA (53, 54),
had 101 serologic responses to the 17- and 27-kDa antigens
and had 90 discrete infections, detected by stool microscopy.
However, only 71 of the oocyst detection events had appropri-
ate paired serum samples for CpP2 antibody analysis. Repre-
sentative profiles showing the antibody responses to rCpP2 and
the 27-kDa antigen from three of the children are shown in
Fig. 6. The child whose findings are represented in Fig. 6A had
a very intense CpP2 antibody response (indicated by a closed
star) during the first of two separate Cryptosporidium-specific
antibody responses (indicated by asterisks). Oocysts were de-
tected by microscopic stool assay at 29 and 30 months of age
(indicated by arrows). Figure 6B represents the findings for a
child who had two CpP2 responses, a weak response at 7
months of age (indicated by the open star) and a moderate
response at 30 months of age, that correlated with a Crypto-
sporidium-specific antibody response. Interestingly, the P2 an-
tibody levels decreased to the baseline level when the child was
33 months of age, even though subsequent Cryptosporidium
infections were detected by both microscopy and serologic
antibody assay. The child whose findings are represented in
Fig. 6C had two CpP2 responses that coincided with Crypto-
sporidium-specific antibody responses at 4 and 11 months of
age and one CpP2 response detected at 16 months of age,
when the level of antibody to the 27-kDa antigen was in de-
cline. None of the CpP2 responses were temporally associated
with the single oocyst detection event at 5 months of age. Four
additional Cryptosporidium-specific antibody responses were
marked either by a declining CpP2 antibody level (at 7 months
of age) or by CpP2 antibody levels below the positive cutoff
value (at 27, 30, and 38 months of age).
By using the antibody response definition described in Ma-
terials and Methods, 45 CpP2 antibody responses were iden-
tified among 428 serum intervals representing 115.1 child-years
of surveillance (Table 2). All of the CpP2 responses were
transient, and most (82%) occurred while the children were
also positive for antibodies to both the 17- and 27-kDa antigens
(54). Twenty-five (56%) of the responses occurred among in-
dividuals in whom a previous Cryptosporidium infection event
was documented either by microscopy or by serologic antibody
assay. A total of 8 CpP2 responses (18%) were coincident with
Cryptosporidium-specific antibody responses to the 27- and 17-
kDa antigens in the presence of oocyst shedding (as defined in
reference 54), 19 CpP2 responses (42%) were associated with
an antibody response in the absence of detectable oocyst shed-
ding, 2 CpP2 responses (4%) were coincident with the detec-
tion of Cryptosporidium oocysts by stool microscopy in the
absence of an antibody response to the 17- and 27-kDa anti-
gens, and 16 responses (36%) could not be linked to Crypto-
sporidium-specific antibody responses (according to our defi-
nition) or to oocyst shedding (Table 2). Thus, 10 of 71 (14%)
oocyst detection events obtained with serum samples appro-
priately spaced for analysis were associated with a CpP2 re-
sponse and 27 of 101 (27%) serologic responses to the 17- and
27-kDa antigens were associated with a CpP2 response. The P2
antibody responses were significantly related to serologic an-
tibody status (chi-square analysis of a two-by-two contingency
table with Yates’ correction, P ? 0.001) but were not related to
stool microscopy status (P ? 0.435).
Given that the P2 protein from C. cayetanensis, a commonly
FIG. 3. ClustalW sequence alignment of apicomplexan, kinetoplastid, and human acidic ribosomal P2 proteins. Predicted ribosomal protein P2
amino acid sequences from T. gondii (GenBank accession number XM_002364187), Sarcocystis neurona (GenBank accession number BQ784279),
P. falciparum (GenBank accession number U78753), Eimeria tenella (GenBank accession number AAK38885), C. parvum (GenBank accession
number AF099744), B. bovis (GenBank accession number XP_001611755), Theileria parva (GenBank accession number XP_764936), Homo
sapiens (GenBank accession number AAA36472), L. infantum (GenBank accession number XP_001467169), and T. cruzi (GenBank accession
number CAA52946) were aligned by using the ClustalW2 sequence alignment program (30). The human SLE P-protein consensus sequence is
underlined in the human sequence (32). The carboxy-terminal 15-amino-acid peptide (EEEEEEGDLGFSLFD) recognized by IgG antibodies
from P2-positive Haitian sera is indicated in boldface with underlining in the C. parvum P2 sequence. The linear epitope recognized by mouse
monoclonal antibody 1D6 is underlined in italics in the C. parvum P2 sequence.
VOL. 17, 2010 ANTIBODY RECOGNITION OF CRYPTOSPORIDIUM P2 PROTEIN959
reported enteric parasite in both Peru and Haiti (5, 15, 34), is
likely to have the same conserved carboxy-terminal antibody
recognition sequence as the other apicomplexan proteins
whose sequences are shown in Fig. 3, we wanted to determine
whether Cyclospora infection might be associated with the
CpP2 antibody response. In our particular subset of 61 cohort
children, 37 Cyclospora infections with durations of between 1
and 64 days (median, 11 days) were identified by stool micros-
copy, and for 27 of these infections serum sampling was done
at appropriately spaced intervals for CpP2 antibody analysis.
Thirty-six of the children (59%), including the three whose
findings are shown in Fig. 6, had no evidence of Cyclospora
infection at any time during the multiyear study. Only 5 of the
45 CpP2 responses (11%) were temporally associated with
Cyclospora infection: three CpP2 responses occurred during
intervals that also included Cryptosporidium-specific 17- and
27-kDa antibody responses, one occurred during an interval
that included a Cryptosporidium oocyst excretion episode, and
one occurred during an interval that did not have either a
Cryptosporidium oocyst detection event or a Cryptosporidium-
specific antibody response. Although the result is not defini-
tive, it would appear that Cyclospora infection is not a major
contributor to the CpP2 antibody response.
Age-specific distribution of CpP2 antibodies in a Haitian
population. Serum samples (n ? 441; age range, 1 month to 90
years) collected from a convenience sample of the population
of Miton, Haiti (population, approximately 2,000), as part of a
FIG. 4. Identification of CpP2 band. (A) Total antigen C. parvum
PVDF strips were incubated with serum from a mouse that was im-
munized with purified rCpP2 (lane 1), monoclonal antibody 1D6 raised
against rCpP2 (lane 2), or buffer alone (lane 3). Bound IgG antibodies
were visualized by use of a biotinylated rat anti-mouse IgG monoclonal
antibody and developed as described in the legend to Fig. 1. (B) Total
antigen C. parvum PVDF strips were incubated either with serum from
a Haitian donor (lane 1) or with human serum antibodies that were
eluted from purified rCpP2 (lane 2). Bound IgG antibodies were vi-
sualized as described in the legend to Fig. 1. The location of the CpP2
band is indicated by the double-headed arrow.
FIG. 5. Map of CpP2 epitopes recognized by human serum IgG
antibodies. A series of 33 overlapping biotinylated peptides (15-mers,
12-residue overlap) were synthesized on the basis of the CpP2 cloned
sequence. Peptides (100 ng/well) were incubated in streptavidin-coated
96-well microtiter plates (200 ng protein/well) overnight at 4°C. The
plates were then washed and incubated with 1:100-diluted Haiti blood
donor sera for 2 h at room temperature. The plates were washed and
then developed by using peroxidase-labeled goat anti-human IgG and
ABTS, as described in Materials and Methods. The CpP2 peptide
responses are expressed as a percentage of the value for the positive-
TABLE 1. Analysis of serum antibody responses to
apicomplexan P proteins
Relative ELISA responsea
CpP2 CpP0 CpP1PfP2TgP2
Haiti NHS 14
Haiti NHS 1
Acute Cryptosporidium infectionc,d
Acute P. falciparum infectionc
Acute T. gondi infectionc
Chronic T. gondii infectione
Acute C. cayetanensis infectionf
Acute B. microti infectionc
B. microti-infected hamsterg,h
98105 96 99
aThe absorbances recorded at 405 nm were divided by the absorbance ob-
served for the CpP2 protein by use of the Haiti NHS 14 control serum sample,
and the results are expressed in percent.
bThe value was arbitrarily set equal to 100%.
cPools of serum (n ? 10 each) from patients with acute infection and high-
titer IgG antibody responses to the organism of interest.
dSera from patients with acute cryptosporidiosis were demonstrated to be
negative for antibodies to CpP2 and CpP1 by Western blotting.
ePooled sera from 10 patients with chronic toxoplasmosis who were IgM
negative but who had IgG titers of ?1:1,024.
fPooled sera from 10 U.S. patients confirmed to have cyclosporiasis by analysis
of stool samples. The parasite-specific titers were not determined.
gHamster serum assays used a different secondary antibody than the human
antibody assays: biotinylated rabbit anti-hamster IgG (Zymed).
hSerum collected 28 days after parasite inoculation.
960 PRIEST ET AL.CLIN. VACCINE IMMUNOL.
1998 trial of a drug used to control of lymphatic filariasis were
tested by ELISA for the presence of IgG antibodies to Cryp-
tosporidium antigens. The prevalence of Giardia was reported
to be 11% in this community at the time of sample collection
(19), but stool samples were not tested for the presence of
Cryptosporidium or Cyclospora oocysts at the time of sample
collection. In 2001, a sample of people between 1 and 86 years
of age from a nearby community in Haiti was reported to have
Giardia, Cyclospora, and Cryptosporidium prevalence rates, de-
tected by stool microscopy, of 27.4%, 6.0%, and 0.7%, respec-
As would be expected in a setting where Cryptosporidium is
highly endemic, much of the population of Miton had IgG
responses to the 27-kDa antigen (Table 3): 55% of individuals
?21 years of age and 83% of adults were positive. Adults also
had significantly higher median antibody levels than young
children. The response to the CpP2 antigen showed a different
profile from that of the 27-kDa antigen: young children (ages
0 to 5 years) were largely negative and had antibody levels
significantly lower than those in individuals in all other age
categories. Both the median level of antibody and the antibody
prevalence increased sharply in individuals in the 6- to 10-year-
old and 11- to 20-year old age categories but did not change
significantly among older adults. The distributions of antibody-
positive results across the age groups were significantly differ-
ent for the two antigens by the chi-square goodness-of-fit test
(P ? 0.001).
The ELISA results for the CpP2 antigen for adults are con-
sistent with the Western blot results that we obtained for adult
Haitian blood donors (Fig. 1). However, the results of the
27-kDa antigen ELISA are somewhat lower than expected and
probably underestimate the true antibody prevalence, as re-
flected by the Western blot results. The sensitivity of the 27-
kDa antigen ELISA is known to be lower in the region of the
cutoff value (J. W. Priest, unpublished observation), and 44%
of the samples from Miton fell within ?50% of the cutoff
values. Even with this limitation, 77% of the individuals who
had a CpP2 response also had antibodies to either the 27-kDa
or the 17-kDa antigen by ELISA.
FIG. 6. Representative profiles showing longitudinal IgG responses
to the 27-kDa antigen and CpP2 in three Peruvian children (A to C,
respectively). Longitudinal serum samples collected from the children
were assayed for IgG antibodies by using the recombinant 27-kDa
protein and CpP2, as described in Materials and Methods. Responses
are presented in arbitrary units on the basis of a standard curve with a
maximum value of 6,400. Asterisks, intervals that had serologic anti-
body responses to both the 17- and 27-kDa antigens, according to our
response definition; closed stars, intervals during which a CpP2 anti-
body response occurred in conjunction with a 17- and 27-kDa antibody
response; open stars, CpP2 responses during intervals that did not
meet our Cryptosporidium-specific antibody response definition for the
17- and 27-kDa antigens. Cryptosporidium oocyst detection events are
shown by black arrows.
TABLE 2. Summary of stool microscopy and serologic antibody
results for a subset of children from the Peruvian
birth cohort study
No. (%) of samples with
the following result for P2
aSerologic antibody responses to the 27- and 17-kDa C. parvum antigens
determined as described by Priest et al. (53).
bPercentage of the total number of P2 antibody responses (n ? 45) that were
present in each category.
cTwo oocyst detection events overlapped with separate antibody responses to
the P2 protein and to the 27- and 17-kDa antigens.
dIncludes one consecutive antibody response to the 27- and 17-kDa antigens,
as defined previously (53).
VOL. 17, 2010ANTIBODY RECOGNITION OF CRYPTOSPORIDIUM P2 PROTEIN961
We have previously demonstrated that specific antibody re-
sponses to the 17- and 27-kDa sporozoite surface antigens
usually increase in parallel following infection with Cryptospo-
ridium (38, 43, 46, 51). Here we report that IgG antibodies to
two additional low-molecular-mass antigens, the acidic ribo-
somal proteins P1 and P2, are frequently detected in sera from
individuals who live in communities where potential environ-
mental sources of oocyst contamination are widespread. Be-
cause detergent extraction of C. parvum sporozoites yielded a
fraction that contained both CpP1 and CpP2 as well as the
membrane-bound 17- and 27-kDa antigen species, we believe
that CpP1 and CpP2, as with the P0 proteins of P. falciparum
(2, 9, 10) and T. gondii (64), are likely to be associated with the
cell surface. However, preliminary attempts at C. parvum
sporozoite cell surface staining with our anti-CpP2 monoclonal
antibody have so far been unsuccessful (data not shown).
Because antibodies to CpP2 are not found in sera from U.S.
(55; this work) or Canadian (51) cryptosporidiosis outbreak
patients and because the target of the human immune re-
sponse is a highly conserved carboxy-terminal peptide epitope
with a demonstrated potential for cross-reactivity (this work),
the challenge has been to determine whether the observed
CpP2 responses resulted from infections with Cryptosporidium
or from infections with some as-yet-unidentified organism that
is more prevalent in the developing world than in the North
America. In support of Cryptosporidium being the causative
agent, we observed that many Haitians who had IgG antibodies
to CpP2 had concurrent evidence of past Cryptosporidium in-
fection by serologic ELISA. Furthermore, the CpP2 responses
in Peruvian children were often temporally associated with
Cryptosporidium infection. We did, however, also note that
36% of the CpP2 responses did not correlate with serologic
assay-defined Cryptosporidium infection. Four of these re-
sponses (9%), like the third response in Fig. 6C, occurred after
a strong antibody response to both the 17- and 27-kDa antigens
and in the presence of persistent and high levels of antibody to
both antigens (more than four times the cutoff). Eight addi-
tional CpP2 responses (17.7%), like the first response in Fig.
6B, occurred during intervals when the titer of one of the
antigens (usually the 27-kDa antigen) demonstrated an in-
crease that met our serologic response definition but the titer
of the other one (usually the 17-kDa antigen) did not. Thus,
the correlation between the CpP2 and Cryptosporidium-specific
17- and 27-kDa antigen serologic responses that we reported is
likely to be conservative and may be limited by our working
definitions of antibody responses.
Even though the acidic ribosomal proteins are well con-
served, we were not conclusively able to link any other infec-
tious protozoan to the observed CpP2 antibody responses.
Although Giardia intestinalis is ubiquitous in Haiti and other
settings in the developing world (8, 19, 34, 47), it lacks the
conserved carboxy-terminal epitope that is critical for P2 pro-
tein immune recognition (G. intestinalis P2; GenBank acces-
sion number XP_001707951) (41). Isospora belli infection was
very rare in the Peruvian cohort study (V. Cama, CDC, per-
sonal communication). T. gondii is cosmopolitan in distribution
and has a prevalence in the United States of approximately
16% (23), but we did not detect CpP2 antibodies among U.S.
citizens, nor did we find CpP2 antibodies in sera from U.S.
patients with acute or chronic toxoplasmosis. In addition, one
of the four strongly CpP2 antibody-positive serum samples
used in the epitope mapping study whose results are shown in
Fig. 5 (sample Haiti NHS 1) was negative for antibodies to
Toxoplasma (data not shown). From our study, it is clear that
sera from patients with babesiosis in the United States react
with most apicomplexan P proteins. In fact, the Babesia P0
protein has been reported to be an immunodominant antigen
shared between Babesia species (72, 73). However, Babesia is
not likely to be the agent responsible for the observed CpP2
responses, because no species capable of infecting humans has
ever been reported in either of our study areas.
Similarly, we do not believe that P. falciparum infection can
explain our CpP2 results, as we saw no evidence of P-protein
antibodies in sera from patients with acute malaria who were
U.S. residents. Malaria is not endemic in the region around
Lima, Peru, where our child cohort study was conducted, nor is
it common in the Leogane commune of Haiti, where Miton is
located. However, antibodies to the P. falciparum P0 protein
have been detected among clinically immune patients with
malaria from India (33), and our results do not specifically
address the potential impact of repeated or chronic infections
with P. falciparum on the P-protein antibody response. We did
test a serum sample from a laboratory-reared rhesus monkey
TABLE 3. Serum antibody responses to Cryptosporidium parvum antigens among residents of Miton, Haiti
Response to 27-kDa antigenResponse to CpP2
No. (%) of
Median no. of
No. (%) of
aSome samples had insufficient volume for multiple antigen testing.
cSignificantly different by the Kruskal-Wallis test (P ? 0.05).
dSignificantly different by the Kruskal-Wallis test (P ? 0.05).
eSignificantly different from all other age ranges by the Kruskal-Wallis test (P ? 0.05).
fSignificantly different from all other age ranges by the Kruskal-Wallis test (P ? 0.05).
gSignificantly different by the Kruskal-Wallis test (P ? 0.05).
962 PRIEST ET AL.CLIN. VACCINE IMMUNOL.
that had been infected multiple times with various Plasmodium
parasite species, but we were unable to detect IgG antibodies
to any of the apicomplexan P proteins (data not shown). We
would suggest that P-protein serologic results for patients with
malaria should be interpreted with some caution, if the donors
reside in regions of the developing world where infection with
Cryptosporidium is frequent.
C. cayetanensis is the one parasite commonly found in our
study areas that is difficult to rule out as a contributor to the
CpP2 response. In a cross-sectional survey in Haiti, Cyclospora
oocysts were detected by stool microscopy in 13% of children
0 to 10 years of age (34), and 33% of the children in the Peru
cohort study had at least one episode of cyclosporiasis during
follow-up (5). However, only one of the CpP2 responses found
among our Peruvian study children was associated with a
Cyclospora oocyst detection event in the absence of Cryptospo-
ridium infection. Further work to conclusively rule out any
contributions from Cyclospora infection would be greatly aided
by the development of a Cyclospora-specific serologic antibody
On the basis of the results presented here, we believe that
Cryptosporidium infection is the most likely cause of the CpP2
antibodies observed in sera from Haiti and Peru, and we hy-
pothesize that repeated, early infection may be required for
the development of a persistent CpP2 antibody response. Pop-
ulations in developing regions of the world, like Haiti and
Peru, are certainly exposed to Cryptosporidium at a young age
and suffer repeated infections. The prevalence of antibodies to
the 27-kDa antigen was 2- to 3-fold higher for Haitian children
?10 years of age (52 to 63%) (this work) than for U.S. children
(21.3%) (21), and although the method of assay has varied
between studies, similarly high seroprevalence values (40 to
100%) have been reported for children in other developing
countries around the world (71, 76, 80). Incidence rates based
on stool microscopy are also much higher among children in
the developing world. Laupland and Church (31) reported an
incidence of approximately 0.0003 infections per child-year
among children 1 to 9 years of age in Calgary, Alberta, Canada,
whereas the rate reported among Peruvian children ?12 years
of age was 0.22 infections per child-year (5). Repeated infec-
tions were seen in 11.7% of the Peruvian children during that
study. In addition, the infection rate determined by stool mi-
croscopy may significantly underestimate the true rate in Peru:
we previously reported that an infection rate of 0.34 infections
per child-year was determined by serologic antibody assay
among children who were consistently stool negative for oocyst
Two of our more intriguing observations are, first, that most
Cryptosporidium infections in children do not elicit a CpP2
antibody response and, second, that the seroprevalence profile
for CpP2 is different from that observed for the 27-kDa anti-
gen. In Peru, we found that 86% of oocyst detection events
with appropriately spaced serum samples and 73% of the se-
rologic responses to the 17- and 27-kDa antigens were not
associated with a CpP2 response. Whether the first observation
is a reflection of some characteristic of the species or dose of
Cryptosporidium, of the immune status (i.e., previous infection
experience) or the nutritional status of the child, or of some
other factor, such as the time interval between serum sample
collections, is not known. We attempted to examine the effects
of the strain or species of Cryptosporidium on the CpP2 re-
sponse, but we had available too few infections for a meaning-
ful analysis by DNA extraction.
There is evidence showing that acute cryptosporidiosis in
children from developing countries is associated with intestinal
inflammation and malnutrition. Kirkpatrick et al. (25) demon-
strated that symptomatic children in Haiti ?2 years of age had
higher levels of fecal lactoferrin, interleukin-8, and tumor ne-
crosis factor alpha receptor I than healthy control or noncryp-
tosporidiosis diarrheal control children. Increased levels of
lactoferrin, a proxy marker for leukocytes and intestinal in-
flammation, were also detected in a Brazilian study of crypto-
sporidiosis in children but were not apparent during experi-
mental infections of healthy adults in the United States (1).
Recent studies have shown that malnutrition significantly in-
creases the risk for Cryptosporidium infection in children from
Bangladesh (40). We do not yet know whether malnutrition
can downregulate the immune response to Cryptosporidium as
it does in the case of malaria (18) or promote inflammation
and shift the Th1/Th2 dynamics of the immune response. Ad-
ditional work is needed to determine the conditions necessary
for the development of a CpP2 response in young children.
With respect to the seroprevalence profiles, we observed
that the CpP2 responses in Peruvian children ?4 years of age
were invariably transient and that Haitian children ?5 years
old were mostly (78%) negative for anti-CpP2 antibodies. In
contrast to the CpP2 response, persistent responses to the
27-kDa antigen were often observed among children, and
?50% were positive by serologic ELISA by the age of 3 years
(54). Age-related differences in the seroprevalence profiles for
specific antigens from the same parasite have previously been
reported for both Cryptosporidium and P. falciparum. We have
shown that antibody responses to the 17-kDa antigen are more
transient than those to the 27-kDa antigen, even though the
half-lives of the responses to both antigens are similar (51). In
fact, a significant proportion of the U.S. population is positive
for antibodies to the 27-kDa antigen but negative for antibod-
ies to the 17-kDa antigen, and the proportion of the population
with detectable antibodies to both antigens increases with age
(21). Similarly, the seroprevalence of antibodies to specific
malarial antigens varies depending upon the age of the popu-
lation and the intensity of transmission (49). In a stable and
high-transmission area where the population has acquired
some level of immunity to malaria, the prevalence of antibod-
ies to apical membrane antigen 1 reached a plateau at 6 years
of age, while the prevalence of antibodies to the circumsporo-
zoite protein continued to increase with age to levels of ?70%.
However, in the unstable and low-transmission area where no
immunity was acquired and epidemics occurred frequently, the
seroprevalence of the circumsporozoite protein remained low
(?10%) and the seroprevalence of apical membrane antigen 1
continued to increase with age. Thus, seroprevalence can vary
between antigens from the same organism and can serve as a
proxy for the development of immunity upon repeated infec-
tion. Given the Miton cross-section results, children and young
adults between 6 and 20 years of age would probably be more
appropriate for inclusion in a longitudinal study of the devel-
opment of a CpP2 response than children under 4 years old.
Further studies will be required to determine if the CpP2
antibody response is related to the acquisition of immunity to
VOL. 17, 2010ANTIBODY RECOGNITION OF CRYPTOSPORIDIUM P2 PROTEIN963
Cryptosporidium infection or disease. We are currently exam-
ining this antigen as a potential vaccine target in an animal
We thank J. B. Phu and D. Sara for their assistance with the Peru
cohort study and Jean Marc Brissau for program support in Haiti. P.
falciparum genomic DNA was kindly provided by N. Lang-Unnash,
University of Alabama at Birmingham, Birmingham, AL. T. gondii
cDNA was kindly provided by J. Boothroyd, Stanford University, Stan-
ford, CA. N. J. Pieniazek and S. B. Slemenda (CDC) contributed to the
creation of the Iowa strain C. parvum sporozoite cDNA library. We
thank B. Collins and J. Sullivan (Malaria Branch, CDC) for access to
sera from Plasmodium-infected monkeys and J. M. Roberts (DPD,
CDC) for support with statistical analysis.
We acknowledge the support of NIH grant R21A1059661 (to
The use of trade names is for identification only and does not imply
endorsement by the Public Health Service or by the U.S. Department
of Health and Human Services. The findings and conclusions in this
report are those of the authors and do not necessarily represent the
official position of the Centers for Disease Control and Prevention.
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