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*
Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, NY 12201;
Department of Diagnostic Medicine/Pathology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506; and
Wadsworth Center, New York State Department of Health, Albany, NY 12201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the role of Abs during intracellular bacterial infection in more detail, this study has used Ehrlichia chaffeensis, an obligate intracellular pathogen that infects cells of the monocyte/macrophage lineage (10). The bacterium, which is tick transmitted, is the agent of human monocytic ehrlichiosis. The factors influencing disease susceptibility in humans have not been defined, although immunocompromised individuals appear to be particularly susceptible to serious disease (11). In previous studies, which used a mouse model for E. chaffeensis infection, it was demonstrated that susceptibility was correlated with immunodeficiency (12, 13). Infected SCID mice developed severe and fatal disease that bore resemblance to human monocytic ehrlichiosis (12). Studies of the immunological basis of disease resistance revealed that passive transfer of polyclonal Abs provided significant protection to the susceptible SCID mice (9). Most remarkable was the ability of Abs to mediate bacterial clearance from the livers of SCID mice, even when the Abs were administered well after infection had been established in this tissue (9). To begin to understand the mechanism(s) whereby Abs could mediate intracellular bacterial clearance, two mAbs that protect SCID mice from infection and disease have been generated. Both Abs recognized an outer membrane protein (OMP)3 that is immunodominant in both humans and mice, indicating that OMPs are likely to be major targets of the protective immune response. Moreover, the ability of Abs to mediate clearance of these intracellular bacteria in the absence of T or B lymphocytes indicates that Abs alone can play a significant role in host defense during intracellular bacterial infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6, C57BL/6-scid, and BALB/c-scid mice were obtained from The Jackson Laboratory (Bar Harbor, ME), or were bred in the Wadsworth Center Animal Care Facility (Albany, NY) under institutional guidelines for animal care and use. The mice were routinely infected with 1–2 x 106 E. chaffeensis-infected DH82 cells via the peritoneum, as described previously (12). Institutional animal care and use guidelines did not permit the use of death as an experimental endpoint in these studies. In some experiments body weight was used to monitor animal health and Ab efficacy.
Bacteria and cell lines and PCR analyses
E. chaffeensis was cultured, as described previously (12), in the canine monocytic cell line DH82. The Arkansas isolate of E. chaffeensis was used for all mouse infections in this study, and was cultured from an early passage obtained from the Centers for Disease Control (Atlanta, GA). The bacteria were quantitated by semiquantitative PCR assay, as described previously (12, 9), using E. chaffeensis-specific 16S rDNA oligonucleotide primers (14). PCR amplification of the mouse glucose-6 phosphate dehydrogenase gene was performed using the oligonucleotide primers 5'-GACCTGCAGAGCTCCAATCAAC-3' (sense) and 5'-CACGACCCTCAGTACCAAAGGG-3' (antisense). Genomic DNA (200 ng) was amplified for 40 cycles (94°/30s; 52°/45s; 72°/60s) using 0.5 U AmpliTaq polymerase (Perkin-Elmer, Wellesley, MA) in reaction buffer containing 1.5 mM MgCl2.
The PCR products were electrophoresed in 1.5% agarose gels and
visualized with ethidium bromide. The limit of bacteria detection by
PCR has been estimated to be 
1 x 106
organisms per gram of liver tissue (12). The
JAX and St. Vincent isolates of E.
chaffeensis were generously provided by Dr. Christopher Paddock
(Centers for Disease Control), and were also cultured in the DH82
cells.
Hybridoma production
C57BL/6 mice were infected i.p. with E. chaffeensis-infected DH82 cells, in the absence of adjuvant, two to four times at 2- to 4-wk intervals. Splenocytes were harvested and fused to the myeloma cell line SP2/0 using standard protocols. The hybridoma supernatants were screened for reactivity to E. chaffeensis by immunofluorescence assay, as described previously (9), and cells that produced specific Abs were expanded and subcloned by limiting dilution. Heavy and light chain Ab isotypes were determined by ELISA using isotype-specific polyclonal reagents (Southern Biotechnology Associates, Birmingham, AL).
Ab purification and administration
Abs were purified by fast performance liquid chromatography from hybridoma culture supernatants using protein A- or protein G-Sepharose (for IgGs; Amersham Pharmacia Biotech, Piscataway, NJ) or IgM purification columns (Amersham Pharmacia Biotech), following the instructions of the manufacturer. Abs were administered to infected mice via the peritoneum (100–200 µg/mouse). Ab concentrations were determined by measurement of absorbance at 280 nm. Irrelevant isotype control Abs used were KJ1-26 (IgG2a; anti-TCR; Ref. 15 ; purified as described above), and a mouse myeloma IgG3 (Sigma, St. Louis, MO).
Production and purification of rOMPs
Production and purification of the full-length rOMP-1g and
OMP-1d have been described previously (16). To generate
truncated forms of OMP-1g, the OMP-1g gene was also cloned into the
expression vector pET32-LIC (Novagen, Madison, WI). The full-length
OMP-1g was first isolated by PCR from E. chaffeensis
DNA obtained from infected DH82 cells using the oligonucleotides
5'-GACGACGACAAGATGGACCCAGCAGGTAGT-3' (sense) and
5'-GAGGAGAAGCCCGGTTTAGAAAGCAAACCTTCC-3' (antisense), and the PCR
product was cloned using a ligation independent cloning strategy, as
described by the manufacturer (Novagen). The three-step cycling PCR
conditions were as follows: an initial 3-min denaturation at 94°C; 30
cycles of a 1-min denaturation at 94°C, 1-min annealing at 55°C,
and 1-min polymerization at 72°C; followed by a 10-min extension at
72°C. The truncated forms of OMP-1g were subsequently generated by
PCR using the cloned OMP-1g plasmid as a template, by pairing the
OMP-1g sense oligonucleotide with the following antisense
oligonucleotides: 5'-GAGGAGAAGCCCGGTTTATATGTCAACTAATCC-3' (to generate
OMP
3), 5'-GAGGAGAAGCCCGGTTTAAAACGGGTTGTTTTC-3' (OMP2/3), and
5'-GAGGAGAAGCCCGGTTTACTTCAGTCCAAAC-3' (OMP1/2/3). The positions
of the resulting truncations are shown in Fig. 4
. OMP1 was generated
using the sense oligonucleotide 5'-GACGACGACAAGATGTTAGGTTTTGCAGGA-3',
paired with the OMP-1g antisense oligonucleotide. This construct
introduced an additional methionine at the immediate amino terminus of
the OMP-1g 1 coding sequence, which began at amino acid residue 101
of the wild-type protein. The OMPs were expressed in pET32-LIC as
thioredoxin fusion proteins, and contained, in addition, a 6X histidine
peptide tag. The nucleotide sequences of the cloned genes were
verified. The predicted sizes of these fusion proteins are as follows:
OMP-1g, 48.2 kDa; 3, 32.9 kDa; 2/3, 25.2 kDa; 1/2/3, 21.7 kDa;
and 1, 40.2 kDa. The OMP expression plasmids were transfected into
E. coli BL21(DE3) competent cells (Novagen), and protein
expression was induced for 4 h at 37°C using 100 mM
isopropyl-
-D-thiogalactopyranoside (Amresco,
Solon, OH). Purification of the rOMPs was performed using nickel
chelate chromatography. The bacteria were lysed in 8 M urea buffer
containing 50 mM Tris-HCl (pH 7.5), and the rOMPs were purified from
cleared lysates by fast performance liquid chromatography using a
HiTrap chelating chromatography column (Amersham Pharmacia Biotech).
The bound proteins were eluted from the column with an elution buffer
containing 8 M urea, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 50 mM
EDTA. The purified proteins were dialyzed in 1 M urea and 50 mM
Tris-HCl (pH 7.5), and stored at 4°C. In some cases, the proteins
were further dialyzed in PBS.
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Western analyses of lysates of bacteria-infected cells were performed as described previously (9). SDS-PAGE sample buffer containing 2-ME (2% SDS, 2% 2-ME, 10% glycerol, 50 mM Tris-HCl (pH 6.9)) was added to 0.1 µg of each rOMP, the proteins were boiled and electrophoresed in an 8–20% gradient SDS-PAGE gel, and transferred to polyvinyl difluoride blotting membranes. The membranes were blocked with 1% nonfat dry milk in PBS, and probed with C57BL/6 normal or immune serum at a 100-fold dilution, or mAbs at 10 µg/ml in blocking solution. Bound Abs were detected using HRP-conjugated anti-mouse Ig secondary reagents (at a concentration of 1:200; Southern Biotechnology Associates), and the blots were developed using a chemiluminescent substrate (ECL Plus; Amersham Pharmacia Biotech).
ELISA
Purified rOMPs were adsorbed overnight to 96-well microtiter plates (Dynex Technologies, Chantilly, VA) at a concentration of 3 µg/ml in PBS. Peptides were adsorbed in sodium carbonate buffer (pH 9.6), at a concentration of 10 µg/ml. The peptides were synthesized by Genemed Synthesis (South San Francisco, CA; peptide 61–90), or by the Wadsworth Center Peptide Synthesis Core Facility. The microtiter plates were blocked with 1% nonfat dry milk in PBS. Bound Abs were detected using alkaline phosphatase-conjugated subclass-specific anti-mouse IgG secondary Abs (Southern Biotechnology Associates), or alkaline phosphatase-conjugated anti-human Ig (heavy plus light) secondary Abs (Southern Biotechnology Associates), followed by p-nitrophenyl phosphate (Sigma), a colorimetric substrate for alkaline phosphatase. The absorbance was read at 405 nm using a ThermoMax microplate reader (Molecular Devices, Sunnyvale, CA).
Statistical analysis
A single cubic polynomial "growth curve" was initially
employed to model the time dependency of weight simultaneously for all
four experimental groups shown in Fig. 2c. Parameter
estimates were determined by classical least squares regression and a
stepwise model selection technique proceeded as follows. Groups of four
parameters, one for each experimental group, were sequentially added to
the initial model if they produced a significant contribution: four
intercepts, four slopes, and four quadratic terms. The group of four
cubic terms was not significant. A backwards elimination procedure was
then employed to sequentially remove or merge the individual most
insignificant parameter until only significant terms remained. The
validity of using least squares was investigated with Bartlett’s test
for homoscedasticity (17), the Kolomogrovo-Smirnov test
for normal residuals (18), and by comparing the results to
a robust regression (19). Because the cubic term was
nonsignificant, the final set of significant parameters potentially
included up to four quadratic equations in 12 parameters. However,
three parameters not significantly different from zero and matches
between six other parameters reduced this number by half. The final
models contain linear growth for Ec56.5 and three similar parabolas in
the remaining groups. The final model showed homoscedastic errors,
p = 0.34; normal residuals, p = 0.07;
and estimates closely similar to those obtained from robust regression.
No outliers were present.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous studies demonstrated that polyclonal Abs mediated
clearance of E. chaffeensis from the livers of SCID mice
(9). To identify and characterize mAbs that could mediate
ehrlichial immunity, mAbs were generated from infected immunocompetent
C57BL/6 mice. E. chaffeensis-specific Abs produced by 27
hybridomas were tested in vivo for their abilities to mediate bacterial
elimination from the livers of infected SCID mice. Bacteria were
quantitated in liver tissue because this organ is a major site of
ehrlichia infection (12). Of nine Abs that were found to
mediate bacterial clearance (i.e., partial to apparently complete
elimination of bacterial infection), two Abs were chosen for in depth
study. In vivo administration of the Abs Ec56.5 (IgG2a) or Ec18.1
(IgG3) provided significant protection from infection when administered
to C57BL/6-scid mice either before infection, or 10 days
postinfection (Fig. 1a).
Bacterial infection was decreased, often to below detectable levels,
within 4 days of Ab administration. Administration of Ec56.5 resulted
in nearly complete bacterial elimination from infected liver tissue.
Administration of Ec18.1 was also effective, but perhaps less
efficient, because clearance was not always complete (Fig. 1a). Abs were also effective in BALB/c-scid mice,
and isotype matched irrelevant Abs did not mediate bacterial clearance
(Fig. 1b). The timing of Ab administration was not critical
because bacterial clearance was observed when administration was
performed before, or well after infection had been established. These
data extend our previous studies by demonstrating that both monoclonal
and polyclonal Abs could provide effective immunity during E.
chaffeensis infection in SCID mice.
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a). An additional group
of Ec56.5-treated mice that continued to receive weekly Ab treatment
showed no signs of disease 70 days postinfection. PCR analyses revealed
undetectable or very low levels of bacterial infection in the livers of
these mice (Fig. 2a). Repeated administration of Ec18.1 also
enhanced survival and resulted in significantly lower levels of
infection when examined on day 42 postinfection (Fig. 2b).
However, long term immunity mediated by Ec18.1 was somewhat less
effective because the onset of morbidity was delayed, but was not
prevented, in the Ec18.1-treated mice. The reason for the delayed
appearance of morbidity relative to earlier published experiments
(9) is not known, but may have been attributable to
attenuation of the bacteria in culture. These observations were
supported by analyses of mouse body weight, which provided an
independent measure of Ab efficacy (Fig. 2c). Mice that did
not receive Abs exhibited a pronounced loss of body weight beginning
about 3 wk postinfection. Weight loss was delayed in the immune serum
or Ec18.1-treated mice, and was completely abrogated in the
Ec56.5-treated mice. However, bacterial infection was reduced in the
livers of the Ec18.1-treated mice on day 59 (Fig. 2b,
panel 4), indicating that decreased bacterial infection in
the liver did not always correlate with loss of morbidity. The data
provided further evidence that mAb administration effectively cleared
the ehrlichiae from infected livers, and identified one Ab, Ec56.5,
that protected SCID mice from an otherwise lethal infection for at
least 35 days. Ab recognition of E. chaffeensis OMPs
To begin to understand the mechanism(s) whereby Abs mediate
bacterial clearance, the Ag(s) recognized by the protective mAbs were
identified. Both Ec56.5 and Ec18.1 detected by Western analysis an Ag
of 28 kDa in detergent extracts of infected DH82 cells, and the
Ag(s) comigrated with an immunodominant Ag identified previously using
polyclonal mouse sera (Fig. 3a; 9). The 28 kDa Ag was only
found in infected cells, and was not recognized by normal sera, sera
raised against uninfected DH82 cells, or irrelevant isotype matched Abs
(Fig. 3a). The molecular size of the 28-kDa Ag was
characteristic of a previously described E. chaffeensis OMP
(20, 21, 22). To determine whether the mAbs also recognized
OMPs, Western analysis was performed using rOMP-1g (also known as open
reading frame (ORF)5). OMP-1g is an expressed OMP in the E.
chaffeensis Arkansas isolate used in these studies
(16). Both mAbs recognized the rOMP-1g (Fig. 3b, top), thus identifying OMP-1g as a target of
the Abs, and confirming OMP-1g as an immunodominant Ag in the mouse
(9).
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b). OMP-1g and OMP-1d
differ largely in three regions, known as hypervariable regions (HVRs),
that are divergent among all of the E. chaffeensis OMPs
(21, 22, 23). Therefore, the data suggested that the relevant
mAb epitopes were located within the HVRs of OMP-1g (Fig. 4a). The polyclonal Ab
response was also largely directed at HVRs, because OMP-1d was
recognized only weakly by the immune serum (Fig. 3b).
Thus, the HVRs are highly antigenic, which probably explains why
these regions also exhibit the greatest degree of diversity.
E. chaffeensis OMPs exhibit allelic differences within the
HVRs among clinical isolates (23), so Ab recognition of
OMPs putatively expressed by two additional isolates was also examined.
Both mAbs recognized putative OMPs from the Arkansas, St.
Vincent, and JAX isolates, which indicated that the Ab
epitope(s) were conserved among the OMPs expressed by the three
isolates (Fig. 3c). The apparently weaker recognition of the
JAX OMP(s) was due to lower levels of bacterial infection in
the cultures. It is not known whether OMP-1g is the only OMP expressed
by these isolates because it is possible that the Abs also recognized
other OMPs expressed by the isolates. However, the data from these
analyses, combined with the above finding that the Ec18.1 and Ec56.5
epitope(s) were found in OMP-1g, but not OMP-1d, indicated that
the epitopes were likely to be located within the HVRs, in
particular, at residues in the HVRs that were conserved among the
clinical isolates, and yet differed between OMP-1g and OMP-1d (Fig. 4a).
To identify the OMP-1g HVR(s), which encoded the Ab epitopes, truncated
forms of rOMP-1g were produced and purified (Fig. 4, b and
c). ELISAs indicated that both Abs recognized epitopes
within HVR1 (residues 70–100) of OMP-1g (Fig. 5). This was evident because deletion of
HVR3 and HVR2 had no effect, but deletion of HVR1 (in both 1/2/3 and
1) abrogated nearly all mAb recognition (Fig. 5). Moreover, a
significant portion of the reactivity of the immune sera could
similarly be attributed to recognition of HVR1 (Fig. 5), indicating
that within OMP-1g HVR1 was immunodominant.
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a). A peptide containing
residues 71–90 of OMP-1g was recognized by neither Ab, but peptides
61–90 and 65–78 were recognized by both Abs, indicating that critical
portions of the epitopes were contained within or adjacent to residues
65–70 (Fig. 6b). The weaker binding of Ec18.1 to peptide
65–78 relative to peptide 61–90 suggested that residues flanking
peptide 65–78 also contributed to recognition by this Ab. Within
peptide 65–70, only two residues, L68 and K69, differed between
OMP-1d, which was not recognized, and OMP-1g expressed by the
Arkansas, and presumably the JAX and St.
Vincent isolates (Fig. 6; Ref. 23). The conservative
leucine to isoleucine substitution at position 68 between OMP-1g and
OMP-1d was not required for recognition. However, substitution of the
OMP-1d-encoded glutamic acid for lysine at position 69 in OMP-1g
(peptide K69E) abolished reactivity by Ec18.1, but not Ec56.5,
indicating that K69 was critical for Ab recognition by Ec18.1. Note
that although K69 was present in truncation 1/2/3, which was not
recognized by Ec18.1, the location of this residue at the carboxyl
terminus of this truncation probably abrogated Ab recognition. Ec56.5
recognition of the peptide containing the K69E substitution was only
partially affected (Fig. 6), so the contribution of other nearby
residues was also examined. Ec56.5, as well as Ec18.1, recognized N71D,
as expected, because this residue was encoded by the JAX and
St. Vincent OMP-1gs. However, neither Ab recognized the
doubly substituted peptide Q70N/N71D, indicating that residue Q70 in
OMP-1g was critical for recognition by both Abs (Fig. 6). Residue Q70
was essential, but not sufficient for Ec56.5 binding, because only very
weak binding of Ec56.5 to OMP-1d was detected by Western analysis (Fig. 3b). Because Ec56.5 required Q70, but not K69, and because
some reactivity was detected using the truncation mutant 1/2/3 (Fig. 5), the data suggested that additional residues amino-terminal to the
truncation breakpoint in 1/2/3, excluding K69, probably also
contributed to Ag recognition. In contrast, Ec18.1 required as part of
its epitope both K69 and Q70 (Fig. 6). Thus, although subtle
differences in specificity were observed, two independently isolated
protective Abs recognized nearly identical epitopes in OMP-1g
HVR1.
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Humans also mount an immunodominant OMP Ab response (24, 25). To determine whether similar OMP epitopes might be
recognized by both mice and humans, acute and convalescent sera from
E. chaffeensis-infected human patients was examined by ELISA
using the Arkansas OMP-1g peptide 61–90. Patient sera
exhibited significantly higher levels of anti-peptide reactivity
when compared with normal human sera (Fig. 7). Although the bacteria that infected
the human patients had not been characterized, it is likely that OMPs
containing epitopes conserved with OMP-1g were expressed in the
patients. Therefore, the data indicated that similar HVR1 epitopes were
recognized by both mouse and man.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously demonstrated that passive transfer of immune serum not only protected SCID mice from E. chaffeensis infection, but also ameliorated disease, even when administered during a well-established infection (9). Here, these findings have been extended by demonstrating that similar protection could be observed using either of two mAbs. The mAbs demonstrated equal or better efficacy than that achieved using immune serum, as determined by direct assay for bacterial infection, and by analyses of body weight. Repeated administration of one Ab (Ec56.5) rescued SCID mice from an otherwise fatal infection for at least 5 wk, and may do so indefinitely. To our knowledge, this is the first report of such efficacy of a mAb in an immunocompromised host during an ehrlichia infection. The data confirm and extend our and others’ previous studies that demonstrated the efficacy of Abs during some ehrlichia infections (7, 8, 9), and support the assertion that, in contrast to prevailing dogma, Abs can play a significant role during host defense against an obligate intracellular bacterium.
The role of OMPs in ehrlichia immunity
Both protective mAbs recognized immunodominant E. chaffeensis OMPs. Moreover, of a total of 39 mAbs recovered from three independent B cell fusions, 22 Abs recognized OMP-1g, and in preliminary studies, nine of these OMP Abs were protective after administration to SCID mice (J.S.L. and G.M.W., unpublished data). The effectiveness of the OMP immune response is supported by a previous study that demonstrated that immunization of immunocompetent mice with purified OMP-1g protected them from infection (21). Thus, the OMPs are major targets of the protective immune response in the mouse, and are also likely to be important in human immunity (26).
The OMPs in E. chaffeensis and related ehrlichiae are encoded by multigene families (10, 21, 22, 27, 28, 29, 30), so it is likely that variation in the expression of different OMPs contribute to immune evasion in the natural host for E. chaffeensis, the white tailed deer (31). Indeed, cyclic rickettsemia has been reported to occur in cattle infected with the related erythrocyte tropic ehrlichial pathogen Anaplasma marginale (32, 33). However, a role for antigenic variation during opportunistic infection by E. chaffeensis has not yet been demonstrated, and antigenic variation has not been reported to occur during ehrlichial infection of either mice or humans. The potential for OMP variation suggests, nevertheless, that the bacteria have evolved mechanisms to avoid protective humoral immune responses in the natural hosts.
Epitope characterization
Epitope characterization studies indicated that the protective Abs Ec18.1 and Ec56.5 recognized nearly identical regions of HVR1, because the glutamine residue at residue 70 in OMP-1g was critical for recognition by both Abs. Of the 22 OMP-1g-specific mAbs recovered during hybridoma analyses, at least 15 recognized HVR1 epitopes (J.S.L and G.M.W., unpublished data), indicating that among the OMP-1g HVRs, HVR1 was immunodominant. Moreover, a significant component of the polyclonal Ab response was directed at HVR1 in OMP-1g. Therefore, epitope use within OMP-1g may be critical for Ab efficacy. The high antigenicity of HVR1 suggests that this region of OMP-1g is particularly exposed on the bacterium, and is likely to be a strong candidate for the development of vaccines designed to elicit protective humoral responses.
Comparative studies indicated that infected humans produced Abs that recognized epitopes in OMP-1g similar to those recognized by the mouse Abs. These data suggest that mice and humans generate similar Ab responses to these Ags. It is not yet known whether Abs will show similar efficacy in infected humans, but it is likely that similar immune mechanisms will be used by both mice and humans.
The epitope characterization studies presented here suggested that changes in OMP expression might have been sufficient to allow the bacteria to avoid elimination by the mAbs. However, upon repeated Ab treatment mice were protected for at least 70 days postinfection, which suggested that significant numbers of bacterial escape variants did not arise during this time. Low levels of infection were observed in the liver on day 70 postinfection in some of the Ec56.5-treated mice, but it is not yet known whether these possibly resistant bacteria resulted from the expression of variant OMPs. Of the five OMPs for which sequences are currently available, four likely encode the Ec56.5 epitope, but it is nevertheless possible that expression of other uncharacterized E. chaffeensis OMPs might have contributed to resistance to mAb-mediated elimination in the long term protection studies.
Mechanisms of humoral immunity
Of particular interest is the observation that humoral immunity was effective in SCID mice in the absence of an adaptive cell-mediated immune response. This finding contrasts with other examples of humoral immunity to intracellular pathogens such as Cryptococcus neoformans, where Abs were effective only in the presence of T cells (34). Therefore, the mechanism(s) of Ab-mediated protection is likely to differ during the different infections, probably because of differences in the pathogenesis and lifecycles of the different organisms.
The data also suggest that Ab isotype is critical for Ab efficacy.
Although protective Abs of IgG2a and IgG3 isotypes were recovered, in
similar experiments several IgMs failed to provide significant
protection in SCID mice (J.S.L. and G.M.W., unpublished data).
Moreover, greater efficacy was achieved with Ec56.5 (IgG2a) than Ec18.1
(IgG3) in the extended protection experiments, even though Ec56.5 and
Ec18.1 are both high-affinity Abs and recognize very similar OMP-1g
epitopes. IgG2a are more effective at complement fixation and FcR
binding than IgG3 (35), so complement fixation and
FcR-mediated IgG recognition are likely to be important for Ab-mediated
bacterial clearance. Protective immunity was similarly correlated with
the development of a T cell-dependent IgG2 major surface protein Ab
response during infection of cattle by the related ehrlichia
Anaplasma marginale (36). However,
the efficacy of IgG3 suggests that alternative mechanisms may be
involved, because phagocytosis mediated by IgG2a and IgG3 Abs probably
occurs via different FcRs (37). Ab FcR interactions are
likely to be critical because of the failure of OMP-1g-specific IgM Abs
(some which recognized HVR1), which do not bind murine FcRs, to provide
protection. One hypothesis is that ehrlichia-IgG immune complexes
mediate an FcR-dependent oxidative burst in phagocytes that contributes
to bacterial clearance. Mouse macrophages express the high-affinity
type I, and the low-affinity type II and type III Fc
R
(38), and immune complex mediated oligomerization of some
of these receptors has been demonstrated to activate intracellular
signaling pathways associated with microbicidal activity (39, 40).
Why are Abs so effective against this obligate intracellular bacterium? The details of the E. chaffeensis lifecycle in the infected host have not been well characterized, and it is possible that during part of their lifecycle, the bacteria are exposed to Abs in the extracellular milieu, perhaps during intercellular transfer. Alternatively, immune complex might trigger effector functions in macrophages or other phagocytes and thereby directly or indirectly mediate clearance of resident intracellular organisms. Kupffer cells, a principal target of the Abs, may be particularly sensitive to such stimuli. The mouse model of ehrlichia infection will provide the tools for dissecting the mechanism(s) involved.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gary Winslow, Wadsworth Center, 120 New Scotland Avenue, Albany, NY 12208.
3 Abbreviations used in this paper: OMP, outer membrane protein; ORF, open reading frame; HVR, hypervariable region.
Received for publication March 20, 2000. Accepted for publication October 31, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C. Bitsaktsis, B. Nandi, R. Racine, K. C. MacNamara, and G. Winslow T-Cell-Independent Humoral Immunity Is Sufficient for Protection against Fatal Intracellular Ehrlichia Infection Infect. Immun., October 1, 2007; 75(10): 4933 - 4941. [Abstract] [Full Text] [PDF]
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K. A. Nethery, C. K. Doyle, X. Zhang, and J. W. McBride Ehrlichia canis gp200 Contains Dominant Species-Specific Antibody Epitopes in Terminal Acidic Domains Infect. Immun., October 1, 2007; 75(10): 4900 - 4908. [Abstract] [Full Text] [PDF]
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N. Ismail, E. C. Crossley, H. L. Stevenson, and D. H. Walker Relative Importance of T-Cell Subsets in Monocytotropic Ehrlichiosis: a Novel Effector Mechanism Involved in Ehrlichia-Induced Immunopathology in Murine Ehrlichiosis Infect. Immun., September 1, 2007; 75(9): 4608 - 4620. [Abstract] [Full Text] [PDF]
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Y. Ge and Y. Rikihisa Surface-Exposed Proteins of Ehrlichia chaffeensis Infect. Immun., August 1, 2007; 75(8): 3833 - 3841. [Abstract] [Full Text] [PDF]
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 A. M. Cardenas, C. K. Doyle, X. Zhang, K. Nethery, R. E. Corstvet, D. H. Walker, and J. W. McBride Enzyme-Linked Immunosorbent Assay with Conserved Immunoreactive Glycoproteins gp36 and gp19 Has Enhanced Sensitivity and Provides Species-Specific Immunodiagnosis of Ehrlichia canis Infection Clin. Vaccine Immunol., February 1, 2007; 14(2): 123 - 128. [Abstract] [Full Text] [PDF]
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E. Yager, C. Bitsaktsis, B. Nandi, J. W. McBride, and G. Winslow Essential Role for Humoral Immunity during Ehrlichia Infection in Immunocompetent Mice Infect. Immun., December 1, 2005; 73(12): 8009 - 8016. [Abstract] [Full Text] [PDF]
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J. E. Lopez, W. F. Siems, G. H. Palmer, K. A. Brayton, T. C. McGuire, J. Norimine, and W. C. Brown Identification of Novel Antigenic Proteins in a Complex Anaplasma marginale Outer Membrane Immunogen by Mass Spectrometry and Genomic Mapping Infect. Immun., December 1, 2005; 73(12): 8109 - 8118. [Abstract] [Full Text] [PDF]
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J.-z. Zhang, H. Guo, G. M. Winslow, and X.-j. Yu Expression of Members of the 28-Kilodalton Major Outer Membrane Protein Family of Ehrlichia chaffeensis during Persistent Infection Infect. Immun., August 1, 2004; 72(8): 4336 - 4343. [Abstract] [Full Text] [PDF]
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M. L. Levin, D. J. Coble, and D. E. Ross Reinfection with Anaplasma phagocytophilum in BALB/c Mice and Cross-Protection between Two Sympatric Isolates Infect. Immun., August 1, 2004; 72(8): 4723 - 4730. [Abstract] [Full Text] [PDF]
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H.-M. Feng, T. Whitworth, J. P. Olano, V. L. Popov, and D. H. Walker Fc-Dependent Polyclonal Antibodies and Antibodies to Outer Membrane Proteins A and B, but Not to Lipopolysaccharide, Protect SCID Mice against Fatal Rickettsia conorii Infection Infect. Immun., April 1, 2004; 72(4): 2222 - 2228. [Abstract] [Full Text] [PDF]
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H.-M. Feng and D. H. Walker Mechanisms of Immunity to Ehrlichia muris: a Model of Monocytotropic Ehrlichiosis Infect. Immun., February 1, 2004; 72(2): 966 - 971. [Abstract] [Full Text] [PDF]
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N. Ismail, L. Soong, J. W. McBride, G. Valbuena, J. P. Olano, H.-M. Feng, and D. H. Walker Overproduction of TNF-{alpha} by CD8+ Type 1 Cells and Down-Regulation of IFN-{gamma} Production by CD4+ Th1 Cells Contribute to Toxic Shock-Like Syndrome in an Animal Model of Fatal Monocytotropic Ehrlichiosis J. Immunol., February 1, 2004; 172(3): 1786 - 1800. [Abstract] [Full Text] [PDF]
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R. R. Ganta, C. Cheng, M. J. Wilkerson, and S. K. Chapes Delayed Clearance of Ehrlichia chaffeensis Infection in CD4+ T-Cell Knockout Mice{dagger} Infect. Immun., January 1, 2004; 72(1): 159 - 167. [Abstract] [Full Text] [PDF]
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J. S.-y. Li and G. M. Winslow Survival, Replication, and Antibody Susceptibility of Ehrlichia chaffeensis outside of Host Cells Infect. Immun., August 1, 2003; 71(8): 4229 - 4237. [Abstract] [Full Text] [PDF]
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J. W. McBride, R. E. Corstvet, S. D. Gaunt, C. Boudreaux, T. Guedry, and D. H. Walker Kinetics of Antibody Response to Ehrlichia canis Immunoreactive Proteins Infect. Immun., May 1, 2003; 71(5): 2516 - 2524. [Abstract] [Full Text] [PDF]
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 C. Ramakrishna, C. C. Bergmann, R. Atkinson, and S. A. Stohlman Control of Central Nervous System Viral Persistence by Neutralizing Antibody J. Virol., April 15, 2003; 77(8): 4670 - 4678. [Abstract] [Full Text] [PDF]
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 C. D. Paddock and J. E. Childs Ehrlichia chaffeensis: a Prototypical Emerging Pathogen Clin. Microbiol. Rev., January 1, 2003; 16(1): 37 - 64. [Abstract] [Full Text] [PDF]
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W. C. Brown, T. C. McGuire, W. Mwangi, K. A. Kegerreis, H. Macmillan, H. A. Lewin, and G. H. Palmer Major Histocompatibility Complex Class II DR-Restricted Memory CD4+ T Lymphocytes Recognize Conserved Immunodominant Epitopes of Anaplasma marginale Major Surface Protein 1a Infect. Immun., October 1, 2002; 70(10): 5521 - 5532. [Abstract] [Full Text] [PDF]
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J. S.-y. Li, F. Chu, A. Reilly, and G. M. Winslow Antibodies Highly Effective in SCID Mice During Infection by the Intracellular Bacterium Ehrlichia chaffeensis Are of Picomolar Affinity and Exhibit Preferential Epitope and Isotype Utilization J. Immunol., August 1, 2002; 169(3): 1419 - 1425. [Abstract] [Full Text] [PDF]
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R. R. Ganta, M. J. Wilkerson, C. Cheng, A. M. Rokey, and S. K. Chapes Persistent Ehrlichia chaffeensis Infection Occurs in the Absence of Functional Major Histocompatibility Complex Class II Genes Infect. Immun., January 1, 2002; 70(1): 380 - 388. [Abstract] [Full Text] [PDF]
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