INFECTION AND IMMUNITY, Apr. 1996, p. 1385–1390 Vol. 64, No.

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0019-9567/96/$04.0010
Copyright q 1996, American Society for Microbiology

Serological Analysis of Antigenic Heterogeneity

of Giardia lamblia Variant Surface Proteins
N. MÜLLER*, S. STÄGER, AND B. GOTTSTEIN
Institute of Parasitology, University of Berne, Berne, Switzerland
Received 10 October 1995/Returned for modification 17 November 1995/Accepted 23 January 1996

Neonatal ZU.ICR mice were infected with trophozoites of Giardia lamblia clone GS/M-83-H7 expressing the
variant surface protein (VSP) H7 and were subsequently investigated for their serum antibody response
directed against VSPH7. Recombinant polypeptides, representing overlapping segments of VSPH7, and native
Giardia proteins were used as antigenic reagents to examine the antigenic substructure of VSPH7 and the
extent of antigenic variation in vivo. VSPH7 proved to be the predominant antigen of the parasite with respect
to serum antibody reactivity. The data indicated that VSPH7 basically consists of two antigenically distinct
parts: (i) a unique, variant-specific 314-amino-acid N-terminal region which elicits a low antibody response
preferentially detectable during the early phase of the infection and (ii) a 171-amino-acid C-terminal region
which elicits a high antibody response during the later phase or after resolution of the infection. The epitopes
of the C-terminal region appear to be shared by other, as yet uncharacterized, variant antigens. The highly
conserved 34-amino-acid stretch at the extreme C terminus of VSPH7 exhibited no immunoreactivity to any of
the sera tested. Further investigations indicated that antigenic variation of the intestinal parasite population
was associated with a diversification into at least six to nine new antigen types. These variant antigens were
extremely heterogeneous in size ranging from approximately 50 to 115 kDa. None of these proteins shared
antigenic epitopes with the 314-amino-acid N-terminal portion, but several of them cross-reacted with anti-
bodies specific to the 171-amino-acid C-terminal portion of the VSP from the original inoculum.

Giardia lamblia is an intestinal protozoan parasite of hu-
mans and various animals. Manifestation of the disease varies
from asymptomatic carriage to severe diarrhea and malabsorp-
tion. Although the development of the disease is thought to be
partially dependent on the current immunological status of an
infected individual, knowledge about the relevant immunolog-
ical processes is rather rudimentary. Several studies have re-
vealed that the immune response to a G. lamblia infection is
strongly influenced by the parasite’s capability to continuously
change the antigenic characteristics on its surface (12). This
antigenic variability of the parasite is mediated by a unique
family of cysteine-rich proteins, the variant surface proteins
(VSPs) (12).
Biochemical and molecular biological studies have demon-
strated that the VSPs from different variant types are ex-
tremely heterogeneous in size, ranging from 30,000 to 200,000
Da (12). Despite the dramatic size differences, these proteins
have several common biochemical characteristics, most nota-
bly a conserved, cysteine-rich motif (1, 2, 14) and a striking
homology in a 34-amino-acid (34-aa) stretch at the extreme C
terminus (10, 14). A subset of VSPs expressed by different
subclones of G. lamblia isolate WB (human origin) also con-
tain a common repeat motif (1, 2) that can be defined by its
immunoreactivity with a specific monoclonal antibody, 6E7
(MAb 6E7) (13). In addition to these biochemically and im-
munologically related proteins, other VSPs that are antigeni-
cally distinct and do not exhibit extended homology on the
peptide sequence level have been identified. A well-character-
ized member of the latter group is VSPH7, which is expressed
by G. lamblia clone GS/M-83-H7 (human origin) and was de-
fined by its immunoreactivity to MAb G10/4 (3).
* Corresponding author. Mailing address: Institute of Parasitology,
P.O. Box 8466, CH-3001 Berne, Switzerland. Phone: (4131) 6312384.
Fax: (4131) 6312622. Electronic mail address: nmueller@ipa.unibe.ch.
Antigenic variability of G. lamblia and variant specific hu-
moral immune responses in the host have been documented in
vitro as well as in experimental infections of humans and ani-
mals (12). In the mouse model, using G. lamblia clone GS/M-
83-H7 for experimental infection, VSPH7-specific serum anti-
body responses were detectable on day 22 postinfection (p.i.).
Antibody production was associated with gradual antigenic
switching of the intestinal parasite population, which resulted
in a complete switch from a VSPH7-positive to a -negative
antigen type (5). In the present study, we demonstrated that
this process includes a diversification of the parasites into at
least six to nine variant antigen types, each producing a VSP
with a distinct peptide length. As determined in a preliminary
epitope mapping experiment, several of the new variants con-
tain a semiconserved antigenic structure which, in the case of
VSPH7, can be localized on the C-terminal part of the peptide
sequence. This antigenic structure is strongly recognized by
antibodies from sera taken during the regressive phase, or even
after resolution, of the parasite infection. This observation
demonstrates the long-term immunostimulatory potential of
the semiconserved region as a consequence of its persistence
during the process of antigenic diversification of the intestinal
G. lamblia population.
MATERIALS AND METHODS
Animals. Gravid outbred ZU.ICR mice were obtained from the Institut für
Labortierkunde, University of Zürich, Zürich, Switzerland. Animals were kept
according to the Swiss guidelines for animal experiments with free access to
germfree food and sterile water.
Parasites and experimental infection. The origin, axenization, and cloning of
the G. lamblia clone GS/M-83-H7 has been described by Aggarwal et al. (3). This
clone expresses a major 72-kDa antigen (VSPH7) on its surface which is recog-
nized by MAb G10/4. G. lamblia trophozoites were cultivated in TYI-S-33 me-
dium with antibiotics as previously described (7).
ZU.ICR mice from entire litters (8 to 12 animals) were infected postpartum by
intragastric inoculation of 50,000 G. lamblia trophozoites (clone GS/M-83-H7) as
described previously (5). Control animals were inoculated with the same volume

1385
1386 MÜLLER ET AL.
of phosphate-buffered saline (PBS). G. lamblia-infected and uninfected offspring
mice from two litters each as well as respective mothers were sacrificed by CO2
euthanasia on days 21 and 32 p.i., respectively. Whole blood (for serum) was
subsequently obtained by cardiac puncture.
The presence of G. lamblia trophozoites in the small intestine and of G.
lamblia cysts in fecal samples was determined by direct microscopical examina-
tion and by standard coprological examination of fecal samples fixed in 15%
sodium acetate, pH 4.3, containing 40% formaldehyde, respectively.
The kinetics of expression of the major surface antigen on trophozoites was
assessed by the use of MAb G10/4 for the immunofluorescent-antibody test as
described elsewhere (5).
Soluble antigens of G. lamblia trophozoites from clone GS/M-83-H7 and from
the intestinal isolates were prepared for Western blot (immunoblot) analysis by
the method of Gottstein et al. (5).
Generation of anti-G. lamblia hyperimmune serum. Soluble antigen (50 mg of
protein in 50 ml of sterile PBS) of G. lamblia clone GS/M-83-H7 was supple-
mented with 50 ml of RIBI (Ribi ImmunChem Research, Inc., Hamilton, Mont.)
and subsequently used as a subcutaneous immunization dose for ZU.ICR mice.
One additional booster was given with the same inoculum 14 days later, and
blood (for serum) was taken 21 days after the first immunization.
Affinity purification of antibodies. Affinity purification of antibodies from sera
of G. lamblia-infected mice on blotted b-galactosidase fusion proteins (see be-
low) was done as described by Müller et al. (11).
Bacterial expression of recombinant VSPH7. Different gene segments from an
original vspH7 cDNA sequence (14) (see Fig. 1), kindly provided by T. Nash
(National Institutes of Health, Bethesda, Md.), were cloned into Bluescript
plasmid KS-Plus (Stratagene, La Jolla, Calif.), plasmid pTRCHisA (Invitrogen,
San Diego, Calif.), and the Escherichia coli expression vector system pSEM
(pSEM1, -2, and -3) (8), producing recombinant protein as a fusion to an
N-terminal portion of the E. coli b-galactosidase and allowing cloning of DNA in
all three reading frames.
Additionally, one of the vspH7 gene segments (see Fig. 1) was cloned into E.
coli vector pMALc2 (New England Biolabs, Beverly, Mass.), producing a recom-
binant protein as a fusion to an N-terminal portion of the E. coli maltose-binding
protein (MBP). Both expression systems contain an isopropyl-b-D-thiogalacto-
pyranoside (IPTG)-inducible promoter. The particular steps for cloning the
different vspH7 gene segments into these expression vectors are shown in Fig. 1.
All recombinant DNA methods, unless otherwise stated, were those of Sam-
brook et al. (15). For DNA cloning, E. coli XL1-Blue (Stratagene) was used as
the bacterial host.
Cells from different bacterial clones were grown at 378C in Luria-Bertani
medium (containing 50 mg of ampicillin per ml) to an optical density at 578 nm
of 0.5. Subsequently, recombinant antigen expression was achieved by addition of
2 mM IPTG and a further 2-h incubation of the cells at 378C. From induced cells
of bacterial clones harboring pSEM/vspH7 constructs, protein extracts were
prepared and analyzed by Western blot (see below). From induced cells harbor-
ing the pMALc2/vspH7 construct, recombinant MBP-VSPH7 fusion protein was
purified from crude extracts by performing an affinity purification on amylose
resin using the pMAL protein fusion and purification kit (New England Biolabs).
The protein concentration of purified MBP-VSPH7 was determined by applying
the Bio-Rad (Hercules, Calif.) protein assay system, and affinity-purified recom-
binant VSPH7 was used for enzyme-linked immunosorbent assay (ELISA) as
described below.
ELISA. Affinity-purified MBP-VSPH7 protein and MBP control antigen were
used for sensitization of micro-ELISA plates (Immunoplates Maxisorp F 96;
Nunc, Roskilde, Denmark) at 5 mg/ml. Further steps of the ELISA were as
described previously (6). Sera from mice and goat anti-mouse immunoglobulin G
(IgG) (heavy- and light-chain-specific) antibodies conjugated to alkaline phos-
phatase (Promega, Madison, Wis.) were diluted 1:50 and 1:500, respectively.
SDS-PAGE and Western blot analysis. For sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE) and Western blot analysis of different
b-galactosidase-VSPH7 fusion proteins, 109 (for Coomassie brilliant blue stain-
ing of proteins) or 108 (for immunostaining of blotted proteins) bacterial cells
were solubilized in 200 ml of sample buffer (9) and subsequently boiled for 5 min.
Proteins from these extracts (10-ml aliquots) were separated by SDS-PAGE
(10%) and subsequently stained with Coomassie brilliant blue (Merck, Darm-
stadt, Germany) or blotted onto nitrocellulose filters (BA 85; Schleicher &
Schuell, Dassel, Germany) as described by Towbin et al. (16). The filters were
subsequently blocked for 1 h at room temperature with TBS (10 mM Tris-HCl
[pH 7.4], 150 mM NaCl) containing 0.05% Tween 20 and 3% skim milk. Western
blot analysis of soluble G. lamblia antigens was done by the method of Gottstein
et al. (5). Mouse anti-G. lamblia hyperimmune serum, sera from mice infected
with G. lamblia, affinity-purified antibodies, and conjugate (alkaline phos-
phatase-conjugated goat anti-mouse IgG [heavy and light chain specific]; Pro-
mega) were diluted 1:200, 1:50, 1:10, and 1:500, respectively.
RESULTS
Anti-VSPH7 serum-antibody production in response to an
infection with G. lamblia clone GS/M-83-H7. An experimental
infection of suckling (3-day-old) ZU.ICR mice with G. lamblia
INFECT. IMMUN.
clone GS/M-83-H7 was the basis for serological investigations
of the immune response directed against the major surface
protein (VSPH7) of this clone (14). Offspring mice from two
litters experimentally infected by intragastric inoculation of the
parasite as well as respective mothers that had not been in-
fected were sacrificed on day 21 and day 32 p.i., respectively.
From these animals, serum anti-VSPH7 antibodies were tested
in an ELISA using affinity-purified MBP-VSPH7 fusion pro-
tein and MBP control protein as antigenic reagents (Fig. 2).
This serological analysis revealed that only a few offspring mice
produced anti-VSPH7 antibodies during the earlier stage (day
21 p.i.) but most of the infected animals produced such anti-
bodies during the later stage (day 32 p.i.) of the experimental
infection. Further on, three of four mothers developed high
anti-VSPH7 serum antibody concentrations, indicating that in
these animals an anti-G. lamblia immune response had oc-
curred upon transmission of the parasite through contact with
cyst-containing feces excreted by the experimentally infected
offspring. Giardia cysts could be detected in intestinal fecal
samples from experimentally infected offspring mice beginning
from day 7 p.i.
Microscopical examination of the parasite population in the
small intestine from neonatal mice showed a large number of
trophozoites on day 21 p.i. (14 of 14 animals), whereas only a
few (8 to 14 animals) or no trophozoites could be detected on
day 32 p.i. In concordance with data from earlier studies (5, 6),
the parasites isolated on days 21 and 32 p.i. from the intestines
of offspring mice had already undergone an antigenic switch
from a G10/4-positive to a -negative antigen type (data not
shown). Biopsies of mothers did not provide any evidence for
a persisting intestinal G. lamblia infection at either time point.
This indicates that the adult animals had already resolved the
parasitic infection at the time points of examination or that
they had never exhibited a proliferative infection upon uptake
of the parasite from the environment, while developing a par-
asite-specific immune response.
Characterization of the antigenic structure of VSPH7. Indi-
vidual sera originating from the experimental G. lamblia infec-
tion described above were tested in Western blots (Fig. 3) for
their immunoreactivities to different recombinant subfrag-
ments of VSPH7 that had been expressed in the E. coli/pSEM
expression system as a fusion to an N-terminal portion of
b-galactosidase (Fig. 1). Immunoreactivities of these sera were
tested with a set of progressively shortened subfragments of a
314-aa portion (part I) of the N-terminal region of VSPH7,
with a downstream 171-aa region (part II), of the protein, and
a 37-aa region (part III) of the protein containing the highly
conserved 34-aa sequence at the very C-terminal end (Fig. 1).
Western blot analysis of a serum taken from an offspring
mouse on day 21 p.i. (serum 7; Fig. 2A, a) showed weak
immunoreactivities to recombinant VSPH7 parts I and II (Fig.
3B), whereas the serum from the mother (serum 1; Fig. 2A, c),
although exhibiting weak immunoreactivity to subfragments of
part I, focused its immunoreactivity on part II (Fig. 3C). In the
case of another offspring serum (serum 14; Fig. 2B, a) and the
mother serum taken on day 32 p.i. (serum 2; Fig. 2B, c), the
immunoreactivity was focused on part II (Fig. 3D and E). On
the other hand, part III was recognized by none of the sera
from infected animals tested in this experimental section. The
Western blots shown in Fig. 3B to E demonstrated that part II
and, to a much lesser extent, part I, but not part III, of VSPH7
contain antigenic epitopes which had an evident preselective
influence on the seroreactivity patterns with respect to day 21
and/or day 32 p.i. The general character of these results was
demonstrated by successfully reproducing data in equivalent
VOL. 64, 1996 ANTIGENIC HETEROGENEITY OF GIARDIA LAMBLIA VSPs 1387

FIG. 1. Construction of vspH7 subclones in E. coli gene expression systems pSEM and pMalc2. Only those restriction sites (B, BamHI; Bg, BglII; E, EcoRI; EV,
EcoRV; H, HindIII; Ha, HaeIII; N, NlaIV; P, PstI; S, SmaI; Sp, SphI; X, XbaI; Xm, XmnI) that are relevant for cloning are shown, and sites used for the particular
cloning steps are boldfaced. (A) Plasmid pNL1 was constructed by cloning the 1,654-bp EcoRI cDNA fragment of gene vspH7 (14) into the EcoRI site of Bluescript
plasmid KS-Plus (BS). The BglII-XbaI vspH7 fragment of pNL1 was subcloned into the BamHI- and XbaI-digested vector pSEM1 (pS1), resulting in pNL23, and the
analogous BglII-EcoRI fragment was subcloned into BglII- and EcoRI-digested vector pTRCHisA (pTHA), resulting in plasmid pNL15. Plasmid pNL15 contains a
vector-derived BamHI cloning site adjacent to the BglII site which was used for in-frame cloning of different vspH7 subfragments into expression vectors pSEM1, -2,
and -3 (pS1, -2, and -3) or pMALc2 (pMc2). Plasmid pNL26, containing the promoter-proximal portion of vspH7, was obtained by cleavage of pNL23 with PstI followed
by religation of the linearized plasmid. Plasmid pNL38 was constructed analogously by excision of an SphI fragment from the promoter-distal region. Plasmids pNL42
and pNL55 were generated by subcloning the promoter-distal PstI-EcoRI and NlaIV-EcoRI fragments into PstI- and EcoRI-cut pSEM2 and SmaI- and EcoRI-cut
pSEM3, respectively. Plasmids pNL37 and pNL39 (1 and 2, respectively) were prepared from pNL15 via subclones pNL28 (BamHI-SmaI) and pNL27 (BamHI-HaeIII)
in order to introduce a 39 HindIII site for subcloning into pSEM. Plasmid pNL16 was obtained by integrating the BamHI-HindIII vspH7 fragment of pNL15 into the
respective sites of pMALc2. (B) The 1,703-bp vspH7 gene region (14) is shown at the top with the start (ATG) and stop (TGA) codons and positions of the restriction
sites from the 1,703-bp sequence which were relevant for cloning. The 1,654-bp cDNA fragment of vspH7 does not include the start codon and a 49-bp sequence at
the 59 end of the gene but contains flanking EcoRI sites (E) artificially introduced during cDNA cloning by Nash and Mowatt (14). The inferred amino acid sequence
of the recombinant VSPH7 is schematically subdivided into a variable 314-aa residue portion close to the N terminus (part I; open bar), a downstream 171-aa portion
which appears to be antigenically semiconserved (part II; shaded bar), and a 37-aa residue portion (part III; solid bar) containing the highly conserved 34-aa C-terminal
domain. Bars represent different portions of VSPH7 encoded by various constructs and synthesized as fusion proteins containing a 375-aa N-terminal portion of
b-galactosidase (b-GAL9) (pSEM constructs) or a 383-aa N-terminal portion of MBP (MBP9) (pMALc2 construct), indicated on the left. The recombinant pSEM and
pMALc2 clones expressing the particular VSPH7 subfragments are listed on the right.

Western blots (not shown) using additional sera from each
group of infected animals specified above.
As a positive control, blotted proteins were also incubated
with a murine hyperimmune serum raised against a soluble
protein extract from G. lamblia clone GS/M-83-H7 (Fig. 3G).
This serum exhibited immunoreactivity to all recombinant
VSPH7 subfragments. This finding indicates that the weak
reactivity or absence of reactivity of sera from infected animals
to part I and the absence of reactivity to part III were appar-
ently not due to either (i) poor intrinsic immunogenicity of
these segments, even as parts of native VSPH7, or (ii) poor
antigenicity of the recombinant peptide fragments caused by
incorrect folding.
Serological investigation of the antigenic diversification of
G. lamblia clone GS/M-83-H7. Testing of sera from different
mice infected with G. lamblia on blotted soluble protein extract
derived from the G. lamblia clone GS/M-83-H7 resulted in an
abundant immunostaining of the 72-kDa VSPH7 band which
was identified on a parallel blot by its immunoreactivity to
MAb G10/4 (Fig. 4A). The serum antibody responses were
almost exclusively directed against the variable surface antigen
and essentially not against invariant components of the para-
1388 MÜLLER ET AL. INFECT. IMMUN.

gested also an obvious lack of antigenicity of the highly con-

served 34-aa C-terminal sequence shared by the two variants.
The general character of the findings obtained by the West-
ern blot analysis shown in Fig. 4 was ascertained by reproduc-
ing respective results in analogous experiments (not shown)
using additional sera from each group of animals specified
above.

DISCUSSION
The present article describes a serological analysis of the
VSP-specific antibody response during a G. lamblia infection
in mice. This investigation provided basic information about
the complexity of VSP molecules with respect to their individ-
ual antigenic structure but also with respect to their antigenic
variability. For experimental infections, we used the G. lamblia
clone GS/M-83-H7/neonatal mouse system, which in previous
studies had proven to be well suited for investigating various
immunological parameters including parasite-specific serum-
antibody responses (5, 6). G. lamblia clone GS/M-83-H7 ex-
presses a 72-kDa VSP (VSPH7) which had already been char-
acterized on the molecular level (14). Previous cloning and
sequence analysis of the vspH7 gene was an important prereq-
uisite for our molecular biological approach to synthesize dif-

FIG. 2. Anti-VSPH7 serum antibody response in mice infected with G. lam-

blia clone GS/M-83-H7. ELISA-based determination of anti-VSPH7 antibodies
in sera from experimentally infected neonatal mice (a), uninfected neonatal
animals (b), and respective mother animals (c and d) was done by using affinity-
purified MBP-VSP fusion protein (solid bars) and MBP control protein (shaded
bars) as antigenic reagents. Standard deviations for three replicate determina-
tions are indicated. Assay results for sera taken on days 21 (A) and 32 (B) p.i. are
shown.

site. On blots containing a G. lamblia isolate derived on day 32

p.i., the staining patterns of sera from infected mice were
extremely heterogeneous and consisted of a set of six to nine
bands migrating in the molecular size range between approx-
imately 50 and 115 kDa (Fig. 4B). This pattern did not include
the MAb G10/4-reactive band of VSPH7. Several of these
immunoreactive bands were also strongly recognized by affin-
ity-purified antibodies specific to part II of VSPH7. In contrast,
antibodies affinity purified on VSPH7 part I did not exhibit any
immunoreactivity in an analogous blot (Fig. 4B).
Taken together, these findings allow the following conclu-
sions. (i) On day 32 p.i. the original inoculum (G. lamblia clone
GS/M-83-H7) had undergone complete antigenic switching as-
sociated with a diversification into at least six to nine different
new-type antigens. (ii) The new-type antigens of the variants
were extremely heterogeneous in size. (iii) A subset of these
antigens shared antigenic epitopes with part II, but none of
these variants displayed any peptide structure antigenically
related to part I of VSPH7. FIG. 3. Identification of antigenic subregions of VSPH7 by Western blot
The collection of anti-VSPH7 sera and antibodies were also analysis. The b-galactosidase–VSPH7 fusion protein sections of the SDS-10%
tested on blots (Fig. 4C) containing soluble protein extract of polyacrylamide gel, stained with Coomassie brilliant blue (A), and corresponding
Western blots incubated with sera taken from offspring mice on day 21 p.i.
G. lamblia isolate WB, which expresses a 170-kDa VSP (CRP (serum 7; Fig. 2A) (B) and day 32 p.i. (serum 14; Fig. 2B) (C), with sera from
170) that is antigenically distinct from VSPH7 but exhibits respective mother animals (sera 2; Fig. 2, c) (D and E), with serum from the
strong homology to the 34-aa stretch at the very C-terminal mother of uninfected animals (serum 2; Fig. 2B, d) (F), and with a mouse anti-
end (13). These analyses proved that all immune sera and G. lamblia (clone GS/M-83-H7) hyperimmune serum (G) are shown. Crude
protein extracts from E. coli XL1-Blue harboring pNL23 (lanes 1), pNL26 (lanes
antibodies lacked any reaction with the exception of the CRP 2), pNL37 (lanes 3), pNL38 (lanes 4), pNL39 (lanes 5), pNL42 (lanes 6), pNL55
170-specific MAb 6E7. This finding pointed not only at the (lanes 7), or pSEM1 (lanes 8) were analyzed. The positions of the recombinant
antigenic heterology between CRP 170 and VSPH7 but sug- b-galactosidase proteins are indicated (dots).
VOL. 64, 1996 ANTIGENIC HETEROGENEITY OF GIARDIA LAMBLIA VSPs
FIG. 4. Western blot analysis of antigenic diversification of G. lamblia clone
GS/M-83-H7 in experimentally infected mice. SDS-10% polyacrylamide gel-
fractionated and blotted soluble protein from the initial G. lamblia clone GS/
M-83-H7 (A), from an isolate obtained on day 32 p.i. (B), or from isolate WB (C)
was incubated with serum taken from a neonatal mouse on day 21 p.i. (serum 7;
Fig. 2A) (lanes 1) or day 32 p.i. (serum 14; Fig. 2B, a) (lane 2), serum from the
respective mother animal (sera 2; Fig. 2, c) (lanes 3 and 4), serum from the
mother of uninfected animals (serum 2; Fig. 2B, d) (lanes 5), affinity-purified
antibodies against b-galactosidase fusion protein containing parts II and III
(encoded by pNL42) (lanes 6) or b-galactosidase fusion protein containing part
I (encoded by pNL26) (lanes 7) of VSPH7 (Fig. 1B), or MAb G10/4 (lanes 8) or
6E7 (lanes 9).
ferent subfragments of VSPH7 by bacterial overexpression.
These recombinant proteins were applied as antigenic reagents
for ELISA- or Western blot-based serology in order to dem-
onstrate their potential to bind anti-VSPH7 antibodies and to
define possible antigenically active subregions of the protein.
In our serological investigations it became evident that ex-
perimentally infected offspring mice as well as their primarily
uninfected mother animals produced anti-VSPH7 antibodies.
This clearly indicated that offspring mice had been excreting
parasite cysts (as shown by coprological analyses) that had
subsequently caused a natural secondary infection and a con-
comitant anti-Giardia immune response in the mothers. How-
ever, none of the mothers demonstrated any active infection at
the time points of examination (on days 21 and 32 p.i.), al-
though the respective offspring animals represented a perma-
nent source for reinfection during patency. These findings are
in agreement with data from earlier studies which suggested
that, in the case of the mouse strain ZU.ICR and some other
strains, susceptibility to G. lamblia infections relies potentially
on an age-dependent maturation of immunological effector
mechanisms which generate immunoprotection against the
parasite infection (5, 6). In the present study, the stage of
maturation of the immune system was to some extent reflected
by the degree of the relative anti-VSPH7 serum antibody re-
activity, which on day 21 p.i. was much more pronounced in
mother animals than in respective offspring.
Furthermore, we demonstrated that VSP represents the pre-
dominant target for the systemic antibody response in mice
and that the appearance of systemic anti-VSPH7 antibodies is
reciprocal to the intensity of the parasite infection. Further
analysis of these antibodies showed complex kinetics exhibiting
low-abundance specificities against the 314-aa N-terminal re-
gion (part I; Fig. 1B) and the 171-aa C-terminal region of
VSPH7 (part II; Fig. 1B) during the active phase of the infec-
tion but low-abundance specificities against the N-terminal
region and high-abundance specificities against the C-terminal
region during the regressive phase or after resolution of the
infection. The observation of this differential anti-VSPH7 an-
tibody response in mice suggests that VSP consists of a highly
variable N-terminal part which stimulates a transient and con-
sequently low-level antibody response during the initial phase
of the G. lamblia infection. This is contrasted by the relatively
conserved C-terminal part, which stimulates a persistent and
consequently high-level antibody response during the later
phase of the infection, when the antigenic diversification of the
parasite has already occurred. This hypothesis was confirmed
in a Western blot (Fig. 4) demonstrating that the original
1389
parasite inoculum differentiated into at least six to nine new
variants which had completely exchanged the antigenically rel-
evant structures of the 314-aa N-terminal portion of VSPH7
but several of which had essentially conserved the antigenic
properties of the 171-aa C-terminal portion of the protein.
The findings described above raise the question of whether
G. lamblia infections in mice generate intestinal antibody spec-
ificities equivalent to those of the complex serum anti-VSPH7
antibody response, which may have directly interactive conse-
quences for the course of the parasite infection. In this respect,
early antibodies directed against the N-terminal region of
VSPH7 may have an immunological trigger function with re-
gard to the process of antigenic variation, whereas late anti-
bodies directed against the semiconserved C-terminal region
of the protein may have influence on those immunological
processes that mediate parasite clearance. This scenario tack-
les the question, raised in earlier experiments (4, 5), of whether
specific anti-VSP antibodies may be essential for controlling
the course of the parasite infection. In this respect, the exper-
imental conclusions mentioned above need to be substantiated
by further detailed exploration of the immunological effector
mechanisms directly at the site of the parasite infection.
Moreover, our results provided experimental evidence that
the highly conserved 34-aa stretch at the very C-terminal end
of VSPH7 has no significant antigenic potential. Because of the
lack of binding to anti-VSPH7 antibodies (monoclonal anti-
bodies and serum antibodies from infected animals), this por-
tion does not seem to have any immunostimulatory functions
which could contribute to immunoprotection against the par-
asite infection. This finding does not support the hypothesis
formulated in a report of Nash and Mowatt (14) that the 34-aa
C-terminal portion of VSP might elicit protective immune re-
sponses to many, if not all, variants appearing during the
course of an infection. In order to further evaluate this ques-
tion, we are currently investigating the local immune response
of infected mice in terms of its potential to produce antibodies
against the highly conserved part of the protein and in terms of
a possible immunoprotective function of such antibody speci-
ficities.
In the Western blot analysis shown in Fig. 4, all reactive
infection sera showed antibody-binding activity with an almost
identical set of different new-type antigens expressed by an
individual intestinal parasite population derived on day 32 p.i.
Although the detected number of different antigen types may
not reflect the whole number of variants appearing at a par-
ticular time point of infection, it indicates the parasite’s rela-
tively limited capacity to undergo antigenic variation. Accord-
ing to data of Nash and Mowatt (14), the entire repertoire of
vsp genes encodes only about 30 to 40 different VSPs which
could theoretically be expressed during the process of anti-
genic variation. This restriction of the parasite may facilitate
the development of antibody-mediated immunity in the host
and thus may be the reason for the abortive course of G.
lamblia infections in mice.
ACKNOWLEDGMENTS
We acknowledge V. Zimmermann for excellent technical assistance,
R. Felleisen and A. Hemphill for many fruitful discussions, E. Amann
(Behringwerke AG, Marburg, Germany) for providing pSEM vectors,
and T. E. Nash for his gift of MAbs G10/4 and 6E7, vspH7 cDNA
fragment, and G. lamblia GS/M-83-H7 and WB.
This work was supported by a grant (31-37607.93) from the Swiss
National Science Foundation.
1390 MÜLLER ET AL.
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