Evaluation of a broadly protective Chlamydia-cholera combination vaccine candidate.

Abstract

The need to simultaneously target infections with epidemiological overlap in the population with a single vaccine provides the basis for developing combination vaccines. Vibrio cholerae ghosts (rVCG) offer an attractive approach for developing vaccines against a number of human and animal pathogens. In this study, we constructed a multisubunit vaccine candidate co-expressing the serovar D-derived Porin B and polymorphic membrane protein-D proteins of Chlamydia trachomatis and evaluated its ability to simultaneously induce broad-based chlamydial immunity and elicit a vibriocidal antibody response to the Vibrio carrier envelope. Intramuscular (IM) immunization with the vaccine candidate elicited high levels of antigen-specific genital mucosal and systemic Th1 cell-mediated and humoral immune responses against heterologous serovars and strains, including serovars E-H and L. Also, in addition to the multisubunit vaccine, the single subunit constructs conferred significant cross protection against the heterologous mouse strain, Chlamydia muridarum. Furthermore, all mice immunized with rVCG vaccine constructs responded with a significant rise in vibriocidal antibody titer, the surrogate marker for protection in cholera. These findings demonstrate the ability of the multisubunit vaccine to induce cross protective chlamydial as well as vibriocidal immunity and establish the possibility of developing a broadly efficacious Chlamydia-cholera combination vaccine.

Full text

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Vaccine 29 (2011) 3802–3810
Contents lists available at ScienceDirect
Vaccine
journal homepage: www.elsevier.com/locate/vaccine
Evaluation of a broadly protective Chlamydia–cholera combination vaccine
candidate
F.O. Ekoa,∗, D.N. Okenua, U.P. Singha,b, Q. Hea, C. Blackc, J.U. Igietsemea,c
aMorehouse School of Medicine, Atlanta, GA, United States
bUniversity of South Carolina School of Medicine, Columbia, SC, United States
cCenters for Disease Control and Prevention (CDC), Atlanta, GA, United States
a r t i c l ei n f o
Article history:
Received 27 January 2011
Received in revised form 18 February 2011
Accepted 5 March 2011
Available online 21 March 2011
Keywords:
Chlamydia
Cross-protection
Combination vaccine
Cholera
a b s t r a c t
Theneedtosimultaneouslytargetinfectionswithepidemiologicaloverlapinthepopulationwithasingle
vaccine provides the basis for developing combination vaccines. Vibrio cholerae ghosts (rVCG) offer an
attractive approach for developing vaccines against a number of human and animal pathogens. In this
study, we constructed a multisubunit vaccine candidate co-expressing the serovar D-derived Porin B and
polymorphic membrane protein-D proteins of Chlamydia trachomatis and evaluated its ability to simul-
taneously induce broad-based chlamydial immunity and elicit a vibriocidal antibody response to the
Vibrio carrier envelope. Intramuscular (IM) immunization with the vaccine candidate elicited high lev-
els of antigen-specific genital mucosal and systemic Th1 cell-mediated and humoral immune responses
againstheterologousserovarsandstrains,includingserovarsE–HandL.Also,inadditiontothemultisub-
unitvaccine,thesinglesubunitconstructsconferredsignificantcrossprotectionagainsttheheterologous
mouse strain, Chlamydia muridarum. Furthermore, all mice immunized with rVCG vaccine constructs
responded with a significant rise in vibriocidal antibody titer, the surrogate marker for protection in
cholera. These findings demonstrate the ability of the multisubunit vaccine to induce cross protective
chlamydialaswellasvibriocidalimmunityandestablishthepossibilityofdevelopingabroadlyefficacious
Chlamydia–cholera combination vaccine.
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Chlamydiatrachomatisgenitalinfectionsconstituteamajorpub-
lic health challenge due to the significant morbidity that includes
pelvicinflammatorydisease,ectopicpregnancyandinfertility[1,2].
Ofthe15serovarsofC.trachomatis,serovarsD–Karechieflyrespon-
sible for urogenital infections and of these serovars, D–F account
for approximately 60–70% of these infections worldwide [3–6].
Although Chlamydia genital infection can be treated with antibi-
otics, the frequent asymptomatic infection especially in women
precludes early diagnosis and treatment, making clinical presen-
tation of sequelae often the first indication of infection. In the USA
alone more than $2 billion is spent annually in the management
of chlamydial genital infections [7]. Consequently, it has been sug-
gested that the development and administration of a prophylactic
or therapeutic vaccine capable of protecting against infection or
even ameliorating severe disease would be the most promising
∗Corresponding author at: Department of Microbiology, Biochemistry and
Immunology Morehouse School of Medicine, 720 Westview Dr., S.W., Atlanta, GA
30310, United States. Tel.: +1 404 752 1584; fax: +1 404 752 1179.
E-mail address: feko@msm.edu (F.O. Eko).
and effective strategy to control Chlamydia [7,8]. However, there
is currently no licensed vaccine against Chlamydia.
The current immunologic paradigms for designing and evalu-
ating chlamydial vaccines include the obligatory requirement for
a T-helper Type 1 (Th1) immune response [7,9] and an accessory
antibody response. Additional requirements include the selection
of a suitable vaccine candidate capable of inducing the required
immune effectors and the development of an effective delivery
system with adequate immunostimulatory properties to boost
immuneresponses.Theuseofwholechlamydialagentsasvaccines
isunattractiveduetothepotentialexistenceofimmunopathogenic
components [10]. Also, there are currently no tools to genetically
modify chlamydiae to produce safe, attenuated vaccine strains.
Therefore, the development of vaccines based on chlamydial sub-
unit components is the current focus of chlamydial vaccine design.
In this regard, the major outer membrane protein (MOMP), a
key determinant of chlamydial genus and species specificity [9],
has been one of the leading subunit vaccine candidates to date.
However,experiencewithpurifiedorrecombinantMOMPasapro-
tective antigen in several animal models [11–13] indicated that
MOMP alone is inadequate, suggesting a need for a multisubunit
approach or a more effective delivery system that will optimize
the protective immune response. Besides, the sequence diversity
0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2011.03.027

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F.O. Eko et al. / Vaccine 29 (2011) 3802–3810
3803
in the surface-exposed variable domains of MOMP from differ-
ent C. trachomatis serovars imposes restrictions on the latitude of
protective immunity elicited against heterologous serovars [14].
Moreover, since a broadly protective subunit chlamydial vaccine
would be preferred, additional subunit vaccine candidates that will
elicit both optimal and broad-based immunity are being sought.
In this respect, among the recently predicted immunogenic pro-
teins [15,16], the polymorphic outer membrane proteins (POMPs
or Pmps) [17,18] and the conserved porin B (PorB) family of mem-
brane proteins [19] are likely vaccine candidates. PmpD and PorB
are evolutionarily conserved antigens on the surface of chlamydial
elementary (EBs) and reticulate (RBs) bodies [10,19,20] each with
potential to generate broad-based protective immunity [21]. We
have previously shown that the novel recombinant Vibrio cholerae
ghost(rVCG)platformisaneffectivecarrieranddeliverysystemfor
cloned C. trachomatis proteins, accommodating multiple subunits,
and supporting the elicitation of protective chlamydial-specific
immune responses [12,21,22].
Cholera is an acute diarrheal disease caused by V. cholerae and is
still a major cause of morbidity and mortality in many parts of the
world [23]. The recent cholera outbreak in Haiti with an attendant
highcasefatalityrate[24]underlinestheurgentneedforappropri-
ateinterventionstrategiesthatcanbeusedinbothendemicregions
and during epidemics. Although access to safe drinking water and
provision of adequate sanitation systems are essential to the long-
term control of cholera, this is a distant goal for many developing
countries[25].Theavailabilityofaneffectivecholeravaccinewould
therefore, be a realistic means to control the disease in endemic
regions. The problems associated with live attenuated oral cholera
vaccines[26]andthefindingthatinhumans,protectionisafforded,
primarilybyantibodiestoLPS[27]havefueledthequesttodevelop
nonliving oral cholera vaccines. This is supported by the fact that,
whether immune responses to V. cholerae O1 are generated by vac-
cination or environmental exposure, the best indicator of immune
status is the level of serum bactericidal antibody [28]. VCG possess
the normal array of cell surface antigens of live bacteria, in partic-
ular those of greatest vaccine significance and have been proposed
as an alternative to heat or chemically killed cholera vaccines [29].
Indeed, previous studies have shown that anti-VCG sera produced
in rabbits were highly protective against challenge with live bac-
teria [30,31]. Considering the epidemiological overlap of endemic
cholera and incidence of oculogenital chlamydial infections, the
developmentofaneffectivecombinationvaccineagainstChlamydia
and cholera will be highly desirable.
In this communication we report the development of a self-
adjuvanting chlamydial vaccine candidate that was designed to
include multiple antigens (PmpD and PorB) each with capacity
to induce adequate protective immunity against infection. We
demonstrated that the immune effectors generated in mice fol-
lowing IM immunization cross-reacted with different chlamydial
serovars and protected mice against heterotypic infection with
Chlamydia muridarum. Furthermore, immunized mice responded
withasignificantriseinvibriocidalantibodytiteragainsttheVibrio
carrier envelope. These results may have major implications in the
rational design and development of broadly protective chlamydial
as well as combination vaccines targeted for human use.
2. Materials and methods
2.1. Construction of vaccine vectors and expression of vaccine
antigens
PmpD and PorB cDNA were obtained from serovar D Chlamydia
genomic DNA by PCR. The pKS–PmpD and pKS–PorB single vac-
cine vectors harboring the PmpD or PorB coding sequences were
constructed by inserting the amplified PmpD or PorB PCR product,
respectively between the E?and L?anchors of vector pKSEL5-
2 using restriction sites incorporated into the primer sets. The
resultant plasmids were designated as pKS–PmpD and pKS–PorB,
respectively. Also, the pKS–PmpD/PorB vaccine vector harboring
the PmpD and PorB coding sequences was constructed by sequen-
tially inserting the amplified PmpD and PorB PCR products into
vector, pKSEL5-2 to generate plasmid pKS–PmpD/PorB (Fig. 1a) in
whichthePmpDproteinisexpressedfromtheC-terminalE?anchor
and PorB is expressed as an N-terminal-L?fusion protein. For the
expression of PorB and PmpD, sequences were subcloned into the
pMAL-p2x and pRSET-A vectors, respectively, and protein expres-
sion was detected by SDS–PAGE and immunoblotting analysis.
2.2. Production of rVCG vaccines
The multisubunit vaccine candidate consisted of recombinant
VCG expressing the chlamydial porin B (PorB) and polymorphic
membrane (PmpD) proteins while the single candidates contained
either PmpD or PorB. An rVCG construct expressing glycoprotein D
from HSV-2, a chlamydial irrelevant antigen (rVCG–gD2) was used
as an antigen control. Production of the rVCG vaccines was carried
out by gene E-mediated lysis essentially as described previously
[12]. Lyophilized VCGs were weighed and the number of CFU per
milligram of VCG was estimated based on the total number of CFU
in the culture medium at the highest absorbance attained before
lysis. Ghost preparations were stored at room temperature until
use.
2.3. Chlamydia stocks and antigens
In stock preparations of C. trachomatis serovars (D–H, L and
MoPn) were used in this study. All stocks were previously titrated
on HeLa cell monolayers followed by purification of EBs over
renografin gradients and stored at −70◦C. Chlamydial antigens
werepreparedbyUV-inactivationofEBsfor3handstoredat−70◦C
until used.
2.4. Mice
AllmiceusedinthesestudieswereoftheC57BL/6strain(female,
aged 6–8weeks) from The Jackson Laboratory (Bar Harbor, ME).
They were housed in the animal facility of Morehouse School of
Medicine and animal study protocols were performed in compli-
ance with institutional IACUC and federal guidelines.
2.5. Immunization, challenge and analysis of protective immunity
Groups of mice were immunized intramuscularly (IM) with
50?l PBS containing 2mg of lyophilized rVCG–PmpD/PorB or
rVCG–gD2 control (rVCG expressing glycoprotein D from HSV-2;
a chlamydial irrelevant antigen) on days 0, 14 and 28 as previously
described [22]. Also, two groups of mice were similarly immu-
nizedwithrVCG–PmpDorrVCG–PorB.Adoseof1mgoflyophilized
rVCG corresponds to about 2×109cfu. Animals were anesthetized
by intraperitoneal injection of 200?l of a 5% sodium pento-
barbital solution (Sigma–Aldrich, Milwaukee, WI) before vaccine
administration. Two weeks after the last immunization, animals
designated for efficacy studies were administered Depo Provera
(2.5mg/mouse; UpJohn Co., Kalamazoo, MI) to stabilize the estrous
cycleandfacilitateaproductiveinfectionandchallengedintravagi-
nallyoneweeklaterwith1.0×104inclusionformingunits(IFUs)of
live C. muridarum (MoPn), the heterologous mouse strain, to assess
cross protection. After challenge, mice were observed twice daily
to monitor health status, such as clinical signs of adverse reaction
to infection. To assess the level of infection, cervicovaginal swabs

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F.O. Eko et al. / Vaccine 29 (2011) 3802–3810
Fig. 1. Construction of membrane targeting plasmids pKS–PmpD/pPorB and pKS–gD2 and expression of recombinant chlamydial proteins. (a) The amplified PmpD and PorB
genes were genetically fused to and in frame with the membrane spanning domains of genes E?and L?of phages PhiX174 and MS2, respectively. The HSV-2 gD2 gene was
similarly inserted between LacZ?and L?in pKSEL5-2. In both constructs, PmpD/PorB and gD2 are under the transcriptional control of the lacPO promoter. Restriction and
sequencing analysis confirmed the correct orientation and size of the cloned genes. (b) For the expression of PorB and PmpD, sequences were subcloned into the pMAL-p2x
and pRSET-A vectors, respectively, and protein expression was detected using mAb to MBP (PorB–MBP) and Anti-XpressTM(PmpD–XE), respectively due to the absence of
antibodies to these proteins. (A). Lanes 1 and 2, rPorB–MBP expressed from plasmid pMAL–PorB at 2 and 3h, respectively; lane 3, purified MBP; (B). Lane 1, MW markers;
lane 2, rPmpD–XE fusion protein expressed from plasmid, pRSET–PmpD.
were collected from each animal every 3days following the chal-
lenge and chlamydiae were isolated from swabs in tissue culture
by standard methods [11]. Experiments were repeated to contain
10–12 mice per group.
For immunogenicity studies, additional sets of 8 mice per
group were intramuscularly immunized with the multisubunit
vaccine construct and control and challenged 2weeks after with
1.0×105IFU of serovar D. Ninety-eight days after initial primary
challenge, a time when mice vaginally infected with live chlamy-
diae are usually susceptible to reinfection, mice were rechallenged
with2.5×104IFUofserovarDpermouse.Serumandvaginallavage
samples were collected at different time points and stored as pre-
viously described [32].
2.6. Purification of CD4+ T cells
Eight weeks after immunization, animals designated for
immunogenicity studies were sacrificed and the genital tract (GT),
iliac lymph nodes (ILN) draining the genital tract and spleens (SPL)
forsystemicdrainingwereharvested.ImmuneTcell-enrichedcells
were prepared from tissues of immunized and control mice by
the nylon wool enrichment procedure described previously [33].
T cells were then purified by the Midi magnetic bead-activated
cell sorting (MidiMACS) purification system, by positive selection
of CD4+ T cells, using CD4-specific MACS microbeads (Miltenyi
Biotech, Auburn, CA). The purity of the CD4+ T cell population
was determined to be at least >95% by flow cytometry using an
APC-conjugated anti-CD4 monoclonal antibody (Pharmingen, San
Diego, CA). A separate pool of splenocytes prepared from naive ani-
mals and treated with mitomycin C (2.5?g/107cells) for 20min or
?-irradiated (3000rad) was used as a source of antigen-presenting
cells (APCs).
2.7. Detection of cytokine production by ELISA
The level of Chlamydia-specific Th1 and Th2 response was
assayed by measuring the antigen-specific Th1 (IFN-?) and Th2 (IL-
4, IL-5) cytokine production by each cell population, respectively.
Briefly, purified CD4+ T cells were plated in triplicate wells of 96-
well tissue culture plates at 2×105cells/well and cultured with
APCs (2×105) and 10?g/ml of the respective chlamydial serovar
antigenfor5days.ControlculturescontainedAPCsandTcellswith-
out antigen. At the end of the incubation period, supernatants were
harvested and assayed for cytokines using the Bio-Plex cytokine
assay kit in combination with the Bio-Plex Manager software (Bio-
Rad, Hercules, CA). The mean and SD of all replicate cultures were
calculated. The experiment was repeated three times.
2.8. Measurement of T cell proliferation
Purified immune T cells were assessed for their ability to prolif-
erate in response to in vitro restimulation in culture with MoPn
or serovar D (positive control) chlamydial antigen using the 5-
bromo-2?-deoxy-uridine (BrdU) cell proliferation assay procedure
describedpreviously[34].Tcellsculturedintheabsenceofchlamy-
dial antigen served as internal control.

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F.O. Eko et al. / Vaccine 29 (2011) 3802–3810
3805
2.9. Antibody and antibody isotype determinations
Blood samples were collected by retro-orbital plexus puncture
and vaginal lavage was obtained by flushing the vaginal vault with
100?l of PBS before immunization and on week 8 after the last
boost. The amount of specific antibodies (IgG2a and secretory IgA)
in sera and vaginal washes against different chlamydial serovar
antigens was measured by a standard ELISA procedure described
previously [32]. Plates were incubated with HRP-conjugated goat
anti-mouse IgA or IgG isotype (Southern Biotechnology Associates,
Inc., Birmingham, AL) for 1h and developed with 2,2?-azino-bis
(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). The optical den-
sity was measured at 490nm on a microplate reader. Results,
generated simultaneously with a standard curve, display data
sets corresponding to absorbance values as mean concentrations
(ng/ml)±SD and represent the mean values from triplicate exper-
iments.
2.10. Bactericidal (vibriocidal antibody titer)
Serum samples from mice immunized with rVCG–PmpD/PorB
and rVCG–gD2 or PBS controls were tested for the presence of IgG
antibodies and antibodies with complement-dependent bacterici-
dal activity, using an assay described previously [30]. Bactericidal
endpoints(vibriocidaltiters)representtheserumdilutionscapable
of lysing 50% of the indicator bacteria.
2.11. Statistical analysis
The levels of Th1/Th2 cytokines, secretory IgA, and IgG2a in
the ILN and serum samples from different experiments as well
as the level of protection conferred by the two vaccine constructs
were compared by Student’s t test. The results were expressed as
mean±standard deviation (SD). Tests were performed using Sig-
maStat software (SPSS Inc.). A value of p≤0.05 was considered
significant.
3. Results
3.1. Construction of vaccine vectors and expression of vaccine
antigens
The single vaccine vectors, pKS–PmpD and pKS–PorB were con-
structed to contain the coding sequences for the mature PmpD
and PorB proteins and in frame with the E?and L?membrane
anchors, respectively. Also, the multisubunit vaccine expression
vector, pKS–PmpD/PorB, was constructed such that the respec-
tive antigen coding regions were inserted between and in frame
with the E?and L?membrane anchors under the transcriptional
control of the lac promoter (Fig. 1a). Sequencing results confirmed
that the cloned genes were in frame with the fusion anchors and
that the integrity of the newly generated plasmid constructs was
maintained. Following transformation of V. cholerae 01 strain H1
with the subcloned plasmid vectors, expression of the recom-
binant proteins (rPmpD and rPorB) was confirmed by Western
immunoblotting analysis using mAb to MBP (PorB–MBP) and Anti-
XpressTM(PmpD–OM), respectively (Fig. 1b) due to the absence of
antibodiestotheseproteins.ProductionofrVCGexpressingvaccine
antigens was achieved by gene E-mediated lysis as described pre-
viously [30]. Lyophilized ghost preparations were stored at room
temperature until used.
CYTOKINE
CYTOKINE CONC. (ng/ml)
0
200
400
600
800
1000
1200
1400
1600
1800
IL-10
IFN-γ γ
IL-17 IL-5
MoPn
Ser D
Ser E
Ser H
Ser L
Fig. 2. Cross-protective chlamydial-specific genital mucosal Th1/Th2 cytokine
responses. Groups of mice were immunized and boosted as described in Section
2. At week 8 post immunization, T cells were purified and pooled from the ILNs
of immunized mice and controls and restimulated in vitro with MoPn and a panel
of human chlamydial serovar antigens (UV-irradiated EBs; 10mg/ml). The amount
of local mucosal Th1 (IFN-?) and Th2 (IL-5) as well as IL-10 and IL-17 cytokines
contained in supernatants of culture-stimulated cells was measured using Bio-Plex
cytokine assay kit in combination with the Bio-Plex Manager software. The con-
centration of the cytokines in each sample was obtained by extrapolation from a
standard calibration curve generated simultaneously. Data were calculated as the
meanvalues(±SD)fortriplicateculturesforeachexperiment.Thecontrols(cultures
without antigen and from rVCG–gD2 antigen control) did not contain detectable
levels of cytokine and so the data were excluded from the results. The results are
from two independent experiments. Significant differences between Th1 and Th2
cytokines (IFN-? and IL-5) are indicated by asterisk (p<0.05).
3.2. Induction of cross-reactive chlamydial-specific genital
mucosal Th1/Th2 cytokine responses by the multisubunit vaccine
candidate
Immune T cells purified from the genital tract draining iliac
lymph nodes (ILN) ILN of immunized mice at week 8 after
immunization were analyzed for chlamydial-specific Th1/Th2 cell
responses by measuring the amount of Th1 and Th2 cytokine
secretion upon restimulation with a panel of different chlamy-
dial serovar antigens. The results revealed that the multisubunit
vaccine candidate induced the secretion of high levels of genital
mucosal Th1-type cytokine responses against the different C. tra-
chomatis serovar antigens tested; IFN-? levels were significantly
higher (p<0.05) than IL-5 levels, indicating the preferential induc-
tion of antigen-specific Th1 cells in the genital mucosa (Fig. 2).
Significantly higher (p<0.05) amounts of Chlamydia-specific IFN-
? were produced by immune T cells following restimulation with
all the serovar antigens tested compared to T cells derived from
rVCG–gD2 (control)-immunized mice (data not shown). Also, com-
paratively higher levels of the inflammatory cytokine, IL-17 were
produced by immune T cells in relation to IL-10 after restimulation
withthedifferentchlamydialserovarantigens,suggestingthatthis
inflammatorycytokinemaybeafactorinprotectionagainstgenital
chlamydial infection.
To confirm the cross-reactivity of serovar D-activated T cells
with other chlamydial strains, purified immune T cells from vac-
cinated mice were also assessed for their ability to proliferate in
response to in vitro restimulation with the heterologous C. muri-
darum by the BrdU incorporation assay. The result showed that
the serovar D multisubunit vaccine-derived immune T cells pro-
liferated in response to restimulation with both the homologous
serovar D and the heterologous C. muridarum (MoPn) antigens,
indicating cross reactivity (Fig. 3). Also, the vaccine immunized-
micehadsignificantlyhigher(p<0.05)Tcellproliferativeresponses
in both the mucosal and systemic compartments evaluated com-
pared to controls [SI, 2.58–4.06 (serovar D) and 1.69–2.08 versus
0.354–0.47 (MoPn)].

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F.O. Eko et al. / Vaccine 29 (2011) 3802–3810
STIMULATION INDEX
0
1
2
3
4
5
SPLEEN
ILN
rVCG-
PmpD/PorB
rVCG-
PmpD/PorB
rVCG-
gD2
rVCG-
gD2
Serovar D
MoPn
*
*
Fig.3. Antigen-specificproliferativeresponse.Eightweeksafterthelastboost,gen-
italtractandsplenicCD4+Tcellsfromgroupsofimmunizedmicewererestimulated
invitrowithC.trachomatisserovarDorC.muridarum(MoPn)antigenfor5days.The
antigen-specific proliferative response was determined using the BrdU incorpora-
tionassay;incorporationwasdetectedbyadditionofABTSsubstrateandtheoptical
density was read at 405nm. Results are expressed as the stimulation index (SI),
the ratio between absorbance values of stimulated and non-stimulated cells and
the bars represent the mean and SD of three independent experiments. *p<0.05
(rVCG–PmpD/PorB versus rVCG–gD2 control).
3.3. Induction of cross-reactive chlamydial-specific genital
mucosal IgA and gG2a antibody responses by the multisubunit
vaccine candidate
Specific antibody responses, elicited 8weeks after immuniza-
tionwithrVCG–PmpD/PorBorrVCG–gD2(control),weremeasured
by titrating the serum and vaginal secretions of vaccinated mice
against a panel of different chlamydial serovar antigens, using
an ELISA assay. Significant (p<0.05) levels of IgA and the Th1-
associated IgG2a antibodies to the different chlamydial serovars
were detected in vaginal wash and serum of mice immunized with
the multisubunit vaccine candidate compared to controls (Fig. 4).
In addition, IgG2a levels in serum against all the serovar antigens
tested were always higher than those from vaginal secretions.
To assess the specific memory antibody responses, the levels
of mucosal and systemic IgA and IgG2a antibody responses were
measured two weeks after challenge (2.5×104IFU/mouse) of pre-
viously immunized mice. The results (Fig. 5) showed that high
levelsofsecretoryIgAandIgG2aantibodiestothedifferentchlamy-
dial serovar antigens tested were detected in vaginal washes and
serum 2weeks after challenge, indicating that the infection driven
recall responses were cross-reactive as well. Although in general,
post-challengemucosalandsystemicIgAandIgG2aantibodylevels
were comparable to those obtained at week 8 post immunization,
the systemic IgG2a responses to serovars D and F elicited 2weeks
postchallenge were lower than post-week 8 levels (Figs. 4 and 5).
In addition, the amount of the Th1-associated IgG2a antibodies
secreted in serum 2weeks after the primary challenge was signifi-
cantly higher (p<0.05) than that secreted into the vaginal mucosa.
To further establish if the long-term mucosal and systemic
humoral recall responses are cross reactive, serum and vaginal
secretions obtained from immune mice rechallenged 98days after
the initial primary challenge were evaluated for the magnitude of
antibodies elicited. The results showed that high levels of secre-
toryIgAandIgG2aantibodiestodifferentchlamydialserovarswere
detectedinvaginalwashandserum,14daysafterrechallengeindi-
cating that the recall responses were cross-reactive (Fig. 6). Also,
thereweresignificantly(p<0.05)higherlevelsofbothmucosaland
systemic IgA and IgG2a antibodies compared to controls (Fig. 6).
Mean Antibody concentration (ng/ml)
VACCINE REGIMEN/ANTIBODY ISOTYPE
0
200
400
600
800
1000
rVCG-
PmpD/PorB
rVCG-
gD2
rVCG-
PmpD/PorB
rVCG-
gD2
IgA
IgG2a
Serovar D
Serovar E
Serovar F
Serovar G
1200
B
100
200
300
400
500
600
700
Serovar D
Serovar F
Serovar G
A
0
*
*
*
*
Fig. 4. Chlamydial-specific IgA and IgG2a antibody responses induced 8weeks post
immunization. Groups of mice were immunized and boosted as described above.
Vaginal lavage and serum samples were obtained 8weeks post immunization and
pooled for each group. The concentration of antibodies elicited in genital lavage
(mucosal, A) and serum (systemic, B) samples was assessed by an antibody ELISA.
Results generated simultaneously with a standard curve, display data sets corre-
sponding to absorbance values as mean concentrations (ng/ml)±SD of triplicate
cultures for each experiment. The results are from two independent experiments.
*Statistically significant (p<0.05) differences between IgG2a and IgA antibodies.
Furthermore, both mucosal and systemic IgG2a levels were signif-
icantly higher (p<0.05) than IgA levels (Fig. 6).
3.4. Immunization with serovar D-derived chlamydial antigens
induces cross protection against heterologous MoPn (murine
strain) challenge infection
Since the ultimate goal of vaccination is to provide vaccinees
protection against infection by different chlamydial serovars, ani-
mals immunized with the serovar D-derived vaccine constructs
werechallengedwiththeheterologousC.muridarum(MoPn)strain
two weeks after the last immunization and periodically monitored
forclearance.Fig.7showsthatmiceimmunizedwiththesingleand
multisubunit vaccine constructs were protected against intravagi-
nalchallengewiththeheterologousC.muridarumstrain.Theresults
alsoshowedthatmiceimmunizedwiththemultisubunitcandidate
vaccine shed about 2.5-log lower chlamydial IFUs than the controls
(rVCG–gD2)asearlyas3dayspostchallengeandatleast1-loglower
IFUsthanthesinglesubunitconstructsupto15dayspostchallenge.
Thedegreeofpreventionofbacterialcolonizationbythetwosingle
subunit vaccines was comparable.
3.5. Induction of bactericidal antibodies
Pre- and post-immunization sera from rVCG vaccine- and
control-immunized mice were titrated for Vibrio-specific IgG anti-
bodies against heat-inactivated whole cell vibrios (WCV) and
bactericidal activity against the homologous H1strain of V. cholerae
O1. IgG antibodies to WCV were detected in the sera of all