Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 95112411 - PCT/US9~12626
217~
Vaccine and Method for Treatment of Chlamydial Infec*ons
Background of Invention
Chlamydia ~ is the most prevalently sexually transmitted bacterial
pathogen in the United States today. The c(lmrlir~ir,n~ resul*ng from chlamydialinfec*ons can be quite serious. Those infected may suffer from pelvic
'' y disease, urethritis, urethral syndrome and urinary tract infcctions. It
has also been confirmed that infec*on may result in ~l,n,.l",. u ~ abortion in
pregnant woman. In addition, chlamydial Cu~ cLiviLi~ and chlamydial pneumonia
may occur in infants infected from their mothers as they pass through the brrth
canal.
Chlamydial infecùons in animals and humans are quite similar. The
organism C psittaci in animals primarily affects the mucosal epithelial cells of the
eye and genital tract. After infection, a chronic carricr state typically develops with
symptoms c ~ during stress. A~yll~Lv~ LiC female animals carrying C
psittaci in epithelial cells of the distal genital tract have been shown to infect therr
newborn during patt~ri*r~n ('hl~rnydial infec.*on in sheep is an '~y
devastating disease in many countries. Ovine chlamydial abor*on, also referred to
as ovine enzoo.*c abortion (OEA), results from infection by the C. psittaci pathogen.
This organism causes a necro*zing placen.*tis in sheep and consequent abortion of
the lamb. Vaccines prepared from egg-grown Chlamydia psittaci inac*vated with
Formalin induced immunity in ewes against ovine chlamydial abortion. This
vaccine and similar products have been used successfully for decades in sheep toprotect against OEA sttains of the pathogen. Recently, however, the efficacy of this
vaccine has been quite vatiable, with outbreaks of chlamydial OEA infection
occurring in vaccinated flocks. HCLC-UIV~;UU~ challenge c.~, ` have indicated
possible strain variation to be the cause.
More recently, protection of sheep against OEA has been ,~. ,"~",~I,,.t .~1 using
a subcellular vaccine containing major outer membrane protein (MOMP) and a
cllhfr:lr*tm vaccine containing elementary bodies (EBs) frûm an OEA chlamydial
strain (Tan et al., 1990, ~nfect. Immun., 58:3101-3108). Tan et al. used a modified
procedure for isola*ng chla--m-ydial outer membrane complexes (COMCs) to producea subcellular vaccine highly enriched in u~d~ Lul~, i MOMP. This I~lc~J~uaLiull,given as a single dûse containing 20 yg of protein, protected sheep against OEA. A
single dose of a vaccine prepared from purified EBs, which contained 160 ,ug
protein, also provided protec*on against OEA in sheep. Tan et al. iden.*fied MOMP
--1--
WO 95/12411 PCTIUS9~112626
21~nsl
as the major protective component in OEA vaccines and suggested using a
IC~ DNA approach to protect against OEA because they be~ieved a 40 kDa
MOMP antigen alone was ~sufficiènt. While the vaecine disclosed by Tan et al.
contained residual amounts of lilJu~Oly~a~,Clla i~c (LPS), this component was not
5 considercd important in dc~cl.~ .,..L of a vaccine against OEA chlamydial
infections beeause serum containing antibodies against LPS did not confer
protectioninpassivetransfer.Al,..;.". ~ andthe~.,."l,l..,.. -~ fixingantibodies
thought to be directed against genus-specific epitopes of LPS did not corre~ate with
protection against ovine abortion strains of C. psittaci.
Attempts to vaccinate against other strains of chlamydial infections in
humans and in animals have been even less successful. Several vaecines prepared
from attenuatcd-live or inactivated organisms are available for prevention of C.psittaci infections. However, these vaccines have only resulted in reduction of
symptom severity, not in the prevention of disease or elimination of the organism.
Vaccination against C IIA- 1111111 ~I;C in humans using killcd, whole elementary bodies
has proven to be somewhat protective, however, a delaycd-type lly ~ cll~;Liv;~y has
oecurrcd in some eases, o~a1c~b~Li~lg the disease. Oeular all~ l l A l ;l ll l of a 57 kDa
heat shoek protein has been shown to induce a ' cellular r~ . y
rcsponse in animals. In addition to the heat shoek protein, the li~Ju~c~ly~al,~llal;lic0 (LPS) component of Chlamydia has also been shown to contribute to the
c of the ocular disease causcd by the Chlamydia organisms.
While whole virus Chlamydia vaceines have been shown to be somewhat
protective, they have also been shown to cause deleterious effects. This has led to
the evaluation of individual - r for use in a subunit or .c~ - l vaccine.
25 Polyelonal and m~nnrlt~l antibodies to the C. ~ major outer membrane
protein (MOMP) have been shown to neutralize the oeular infectivity of this
organism in a primate model. Oral ` ` ` of monkeys with purified MOMP
from C. I I A 1 1~ has only resultcd in partial protection from subsequent ocular
ehallenge. Due to its proteetive eapacity and the ability of antibodies specific for
30 MOMP to neutralize infectivity, researeh has foeused on the ' ~ of B- and
T-cell epitopes on this protein.
It has now been found that the ~JIC ' " of MOMP from Chlamydia
organisms, either alone or in the context of elementary bodies or outer membranecomplex, in ~ I I with LPS, is an effective vaccine against chlamydial
35 infeetions in animals. This ....",l.: -~i., is more efficient in the induction of a
protective response against chlamydial infection than denatured MOMP alone.
W095112411 PCTIU594/12626
2175081
Summary of Invention
In one aspect, the present invention provides a vaccine for treatment of
chlamydial infections comprising major outer membrane protein and
lipopoly ~d~a,llalhlc from a Chlamydia organism.
In another aspect, the present invention provides a method for treatment of
chlamydiai infections comprising ~.l."~.,;-~ . ;"~ to an infected animai an effective
amount of a vaccine comprising major outer membrane protein and
l;lJu~.al~i~a~a,lldliJe from a Chlamydia organism.
In yet another aspect, the present invention provides a method for
; ., ., ., ,; ; - ~ a healthy animai against chlamydiai infections Ct mrriein~
to a heaithy animal a vaccine comprising major outer membrane
proiein and li~u~oly~d~,lldlidc from a Chlamydia organism.
Detailcd Description
The present invention provides a vaccine for treatment and ;.. " . ,.. ;, ~
against chlamydiai infections in animais. The vaccine is compriscd of major outer
membrane protein (MOMP) either aione in purified fomm or in the context of
elementary bodies (EBs) or outer membrane complexes (COMCs), with
li~u~ul~a.,~ alide (LPS) from a Chlamydia organism, wherein the Chlamydia
organism is preferably C. psittaci or C. ~ more preferably C. psittaci of
the Bai~er strain. The effect of the vaccine may be enhanced by addition of an
adjuvant.
The MOMP in the vaccine is provided in purified form or in the context of
EBs or COMCs. MOMP can be putified by various methods including but not
limited to t ', O , ~ lly or e~ lu~,l.ulc;i.,~ily.
Ei~ u~,i.u.c.ic ~ ;.... of MOMP (MOMP-E) is .~ h- l in the
following manner. Chiamydia harvest fluid is c~nt-~fi-~eA and the resulting pellet is
/1 in water. Samples are then resolved by gel cl~,uu~llu~ is, preferably
sodium dodccyl sulphate pul,~a~,l yldllti i~ gel CI~LIU~ U-~ (SDS-PAGE). Proteins
30 in the samples are: ' ' ' 1, preferably by heating at about 95C for
~pluAilllat~,ly 5 minutes in buffer, preferably Tris buffer, pH 6.8, containing SDS,
2~ l glycerol,and iJlUill~ .llUl blue. El~ ulJllu~e~i~iscarriedout
using pol~à~,.yldl..idc gels. The gels are stained, then briefiy destained. The 40 kDa
band is then excised, placed in a diaiysis bag, and the protein is ch,~ ~h..~i out of
35 the gel.
Cil., - .6,~hi~ 1' i ri- -~ ;.... of MOMP (MOMP-C) is performed in the
following manner. Non-MOMP proteins are extracted from EBs, preferably by
WO 95/12411 PCT/I~S94/12626
21~an8~ ~
treatmGnt with N-lauroyl-sarcosine in phosphate buffered saline (PBS) containingEDTA for I hour at 37C. Following extraction, the solution is r~ntrifil~Prl Theresulting pellet is washed and ~ in buffer, preferably sodium phosphate
buffer containing MgCl, d~".y, ;~ - and ~ A. The suspension is
5 then incubated at about 37C for ayyl~ ly 2 hours and spun. The resulting
pellet is washed and .~ .1 in buffer, preferably PBS containing sodium
dodecyl sulfate and EDTA. The suspension is incubated again at about 37C and
then spun. The resulting ~ is dialyzed against buffer, preferably sodium
phosphate buffer, containing diLll;ull~lc~tul and SDS, then loaded onto a
10 llJLu~yl~ c column previously ~p.ilil ' with the same buffer.~ The column
is washed and a linear gradient from about 0.1 to about 0.6 M sodium phosphate, pH
6.4, containing Aithi. ' .,.~ul and SDS is run. The pellet from the SDS extraction is
re-extracted. Fractions fi om the ~ and the pellet containing MOMP are
pooled and then dialyzed against water.
The MOMP ylCy~LliUII in the vaccine can also be provided in the context of
EBs or COMCs.
The EB subfraction ~)ICIJ~liUll is isolated from Chlamydia harvest fluid.
Chlamydia organisms, preferably Chlamydia psittaci, more preferably Baker strain,
is propagated in msmm~lisn cells. The harvest fluid from the cells is -- - 1,
20 layered on top of 35% Renografin-76, and c~nh~fil~A The pellet is ~
layered on top of a fl . ~.l l l ;, ....,. ,~ gradient of diatrizoate , ' and diatrizoate
sodium and ~- :lirl L...l The band at the 44-52% interface which contains the EBs
is collected, washed and . ~ l in buffer, preferably a phosphate buffer, more
preferably 0.0l M phosphate buffer, pH 8.0, containing 0 l5 M NaCl (PBS). The
25 EBs are then inactivated, preferably with binary Glll,yl~ (BEI), ~-
Yl~ r ' . formalin or glutaraldehyde. If BEI is used in ill~,liv~Liull the
solution must be ' 1, preferably by addition of sodium thiosulfate.
To isolate the COMC preparation, the inactivated EBs are c~ntrifil~A and
the resulting pellet is solllhili7~A. preferably using PBS containing N-lauroyl-
30 sarcosine and EDTA. The solution is then c~ ntrifil~i. the resulting pellet beingwashed and ~ . 1..1 in PBS.
Lil~ulJol~ ,llafidc (LPS) is also added to the vaccine. LPS is isolated via
el~llul,llu.c~is. Samples of the harvest fluid pellet are prepared for ele~.l.u~l.u.c,,;,
as described for the MOMP-E. El~ u~ ulG~i~ is carried out on puly.l~;lylalllidc
35 gels. It is prGferred to use either a Tris/tricine buffer system or a Tris/glycine buffer
system with interior resolution. The portion of the gel below the 6 kDa marker is
cut off and placed in a basic solution, preferably 0.1 M glycine-NaOH, pH l l.0 and
-- 4--
WO 9S/12411 PCTIU59~112626
217.S~l
incubated. The liquid is separated from the gel. the pH is adjusted to neutrality and
then dialyzed.
The identity of the antigen is confirmed and the protein ~ .,". -., ~ are
~l.ot~rrnin-~l MOMP-C, MOMP-E, EBs, COMCs and LPS are placed in vials and
5 Iyophilized. The amount of LPS is deter~nined by weighing the Iyophilized
samples. Vials are rehydrated with a l-l.- " ., - .. ,l ;. ~lly acceptable carrier. Such
carriers include normal isotonic saline, standard 5% dextrose in water or water,preferably adjuvanted. Examples of adjuvants include, but are not limited to, Quil
A, Alhydrogel, and Quil A and 5% Alhydrogel in tissue culture media. Vials
containing LPS arc rehydrated first. These solutions are then used to rehydrate thc
MOMP ~ Liu--~ resulting in a vaccine containing both the MOMP IJl~u~Liu-~
and the LPS ~q~ Liùl~.
The vaccine is ad.l i-,;~L.,.cd to an animal suffering from a Chlamydial
infection. The vaccine is also ~ r/l to healthy animals as ;.,~
against infection by a Chlatnydia organism. The vaccine can be a~ t~cd
_L ~, ir~rnllcr~ rly, ;~ lly, intravitreally, orally, intranasally
or by ~ rr y at doses ranging from 0.01-100 ,ug/dose of MOMP and LPS each.
As used within the ~ a "MOMP ~ ioll" refers to any
vaccine l~ having purified MOMP, including, but not limited to, MOMP-E
and MOMP-C, and MOMP in the context of EBs or COMCs. "LPS preparation"
refe~stoavaccinel"c~ havingpurifiedlil~upul~ "1.,,;dc. "Effective
amount" refers to that amount of vaccine which invokes in an animal infected by a
Chlamydia organism an immune response sufficient to kill the organism.
"Adjuvant" refers to materials which when injected on their own produce a state of
~ ;c immunity expressed as a heightened resistance to infection. An
exarnple is Quil A in 5% Alhydrogel in tissue culture media.
This invenion is further illustrated by the following ~ examples.
Example 1: Sllhfr~rtinn Antigen A~ iulls
Chlamydia harvest fluid. Chlamydia psittaci, Baker strain was propagated in
dog kidney (DK) cells in DMEM with 2% fetal bovine serum. The harvest fluid
was inactivated with 1% BEI and the soluion neutralized by addition of 0.25%
sodium thiosulfate.
Chlamydia elementary bodies (EBs). Non-inacivated Chlamydia harvest
fluid was . - - ' using a stirred cell CUII~,. ' ' or by ~ ~ The
" ~ or pellet was layered on top of 35% Renografin-76 (Squibb Diagnosics,
New Brunswick, NJ 08903), and r r ntrifl~ed at 43,000 g for I hour. The pellet was
W095112411 PCT/IJS94/12626
217~
, ,, ,, ,~1,~ . ,.1~ d and layered on top of a .1; ~., .,, I;, ....~, ,~ gradient of 40, 44 and 52%
Renografin-76 ~Squibb Diagnostics, New Brunswick, NJ 08903) and cf n~nfi~f~1 at
43,000 g fo} I hour. The band at the 44-52% interface containing the EBs was
collected, washed and ~ d in 0.01 M phosphate buffer, pH 8.0, containing
5 0.15 M NaCI (PBS). The EBs were inactivated with 1% BEI and the solution
neutralized by addition of 0.25% sodium thiosulfate.
Chlamydia outer membrane complexes (COMCs). Inactivated EBs were
cf n-nf ~A at 100,000 g for I hour at 10C, and the resulting pellets solubilized
with PBS containing 2% N-lauroyl-sarcot~ine and 1.5 mM EDTA for I hour, at
37C The solution was subjected to n~nhifilosl~ n at 100,000 g for 1 hour and the
pellet washed once in PBS and then .~ 1 in PBS.
Example 2: Subunit Antigen Preparation
Cluu~ lly purified MOMP (MOMP-C). The MOMP was
15 clu~ lly purified using a " .~ i. ., . of the method described by
Caldwell et al. (1981) Infect. ~nmun., 31:1161-1176. Briefly, EBs prepared as
above were treated with 2% N-lauroyl-sarcosine in PBS containing 1.5 mM EDTA
for I hour at 37C to extract non-MOMP proteins from the outer membrane
complex. The solution was c~n~rifi~A at 100,000 g for I hour, and the resulting
20 pellet was washed in PBS then ~ F.i in 3-5 ml of 0.02 M sodium phosphate
containing 10 mM MgC12 and 25 ,ug each of d~,v~ylil)ulluclu~ c I and ~ 1
A. This suspension was incubated at 37C for 2 hours then rf n~nfil~d at 100,000 g
for I hour. The pellet was washed in PBS, then ~ ..1f.l in 2% sodium dodecyl
sulfate (SDS) in PBS with 1.5 mM EDTA and incubated for 1 hour at 37C. The
suspension was ç~nmfi.~A at 100,000 g for 1 hour. The resulting ~u~. was
dialyzed again 0.01 M sodium phosphate, pH 6.4, containing I mM lilll;uLI~, .ul and
0.1% SDS (start buffer) and loaded onto a lly~u~.yl~l~aliLc column which had been
...l;l.,,.l~d in start buffer. The column was washed with start buffer, and then a
150 ml linear gradient of 0.1 to 0.6 M sodium phosphate, pH 6.4, containing 1 mM' ' ' and 0.1% SDS was initiated and I ml fractions were collected. The
pellet from the SDS extraction was re-extracted for 30 minutes each at 37C withperiodic sonication sequentially using each of the following buffers: 1% N-lauroyl-
sarcosine in 0.01 M sodium phosphate, pH 7.4; 1% N-lauroyl-sarcosine and 10 mM
diLl~iutl~,;Lul in 0.01 M sodium phosphate, pH 7.4; and 1% uclyll;lu~Osidc and 10
mM dithiotreitol in 0.01 M sodium phosphate, pH 7.4 according to the method of
Bavoil et al. (1984) Infect. Immuno., 44:479-485. Fractions firom the ~
and the pellet containing MOMP were finally pooled and dialyzed against water.
W095112411 PCTIUS9~112626
217~081
El~,uulJholcli~,ally purified MOMP (MOMP-E). Chlamydia harvest fluid
was c~ntrifi~ l at 25,000 g and the resulting p~llets were ~ in distillcd
water. Samples were resolved by sodium dodecyl sulphate ~ulya~ lal~lide gel
cle~uU~IIul~s;~ (SDS-PAGE). Proteins were solubilized by heating at 95~C for 5
5 minules in a buffer containing 2% (w/v) SDS, 5% 2-UI.,.~ 1 (V/V)~ 3%
(w/v) glycerol, 0.002% (w/v) 1~. u-~lul ' - I blue, and 50 mM Tris (pH 6.8).
El~ upll~ is was carlied out using 10% (w/v) pOlya~,lyl~ lid~ gels (1.5 mm
thick) in the ~1icrrntinl-ollc buffer system of Laemmli (1970) Nature (London),
227:680-685. The gels were stained with Coomassie Blue G in 50% methanol and
10% acetic acid, and briefly destained in 50% methanol and 10% acetic acid. The
40 I~Da band was then excised, placed in a 12-14 kDa molecular weight cut-off
dialysis bag and the protein elc~,uu~,"..~,d out of the gel for I hour at 50 V.
Li~ol,c.l.~i,a~,clldride (LPS). Samples of the hatvest fluid pellet were
prcpared for cl~."uu~l-u-c~;~ as described for the MOMP-E. EI.,~ II.U.~ was
carried out on 12.5% pOl~a~,lylalll;dc gels using the Tris/tticine buffer system of
Schagger and von Jagow (1987) Anal. Biochem. 166:368-379, wherein the cathode
buffer was 0.1 M Tris, 0.1M Tricine, 0.1% SDS, pH 8.25 and the anode buffer was
0.2M Tris, pH 8.9. The portion of the gel below the 6 kDa marker was cut off andplaced in a solution of 0.1 M glycine-NaOH, pH 11.0 and incubated at 37C for 3
hours. The liquid was then removed from the gel, the pH adjusted to neutrality and
then dialyzed against distilled water using I kDa molecular weight cut-off dialysis
membrane.
Example 3: Quantitaion and Evaluation of Protein and LPS
Protein was quantitated using a BCA assay kit (Pierce, P.O. Box lA,
Rockford, ~linois 61105). The amount of LPS was tl~ r~rrnin~ by weighing
, ' '- ' samples. Prior to vaccine ~ ual~lliull~ antigens were evaluated for
purity by staining SDS-~ul.~a~,lylàull~ gels with Coomassie Blue G in 50%
methanol and 10% acetic acid, and briefly destained in 50% methanol and 10%
acetic acid. The identity of the antigens was confirmed by western blot analysisusing polyclonal antiserum specific for C. psittaci generated in cats. The procedure
involved cl.,~,u.,~ lly ll~..,f~ fractioned antigens from SDS gels onto
T.. ~--l.;l.,.. ~ 11 b. (Millipore t~nrrrlr~ m. Bedford, MA 01730). The
.5 were then blocked with 5% instant non-fat dry milk in PBS followed by
35 incubation with the specific antibody for I hour at room t~,ull~ L..c. Blots were
then washed in PBS containing 0.3% Tween (v/v) and incubated with goat anti-cat
alkaline l.l~ -labeled antibody (Kirkegaard & Perry 1 ~ lnc., 2
WO 95/12411 PCTIUS9~/12626
2l~snsl -
Cessna Court, Gaithesburg, Maryland 20879). After extensive washing of the
".. ."1" ,.". c color was developed using BCIP/NBT substrate (Kirkegaard & PerryT -~ Inc., 2 Cessna Court, Gaithesburg, Maryland 20879). Stained gels and
blots were analyzed by optical image scanning using the Bio Image System
(Millipore Corporation, Bedford, MA 01730). ,~
Example 4: Vaccine Preparation
S-lhfr~--ti-7n antigen ~ iUII for studies I-III were formulated as follows
fom~ ldliull of mice. For study I, a specific lot of ~ d infected tissue
10 culture fluid was used as a positive control since previous studies ~1~ ' itsefficacy. To prepare this vaccine Iyophilized fluid was rehydrated with 200 ,ug/ml
Quil A in 59~o Alhydrogel in RPMI at a dilution that had been shown to prooect mice
in past ~ The total protein present in this dose was 8.1 ,ug/100 ,ul. The
quantity of EBs in the vaccine was determined by ~ ~d;~."g to the western blot
15 intensity of the MOMP band in the harvest fluid. Based on this, the protein
c~ l l in the EB vaccine was 0.575 ,ug/100 pl. Three lO-fold serial dilutions
of both harvest fiuid and EB vaccines were also made.
For study II, EBs and COMCs were formulated as follows. The quantity of
COMCs in the vaccine was determined by ,~ .,l;,;,.g to the westem blot intensity20 of the MOMP band in a purified EB rrcp -:lric n The purified EB preparation was
then diluted in PBS to equal the MOMP c ~ i. ,. . in COMCs. These were then
further diluted in adjuvant (2.5 yg/ml Quil A) in 0.85% NaCI to a total protein
,. - - 1ll ..l;. ll~ of 5 ,ug/100 pl. The same procedure was followed to produce the
COMC vaccine resulting in a protein, of 2.8 ug/100 ,ul and 0.28 ~ug/100
25 ,ul.
To formulate subunit antigens 1~l'; " - ~ quantities of each antigen were
aliquoted into vials and Iyophilized. Vials containing the MOMP-C, MOMP-E and
LPS were rehydrated with 200 pg/ml Quil A in 5% Alhydrogel in RPMI medium to
yield a c~ , l ;. .l l of 100 ,ug/100 ,ul. Rehydrated LPS was used to rehydrate a
30 few aliquots of the MOMP-E to provide a MOMP-E+LPS vaccine containing 100
pg of each antigen in a 100 ,ul total volume. Three 10-fold dilutions of the above 4
vaccines were made in 200 ,ug/ml Quil A in 5% Alhydrogel diluted in RPMI. All
vaccines were stored at 4C between the time of preparation and ~.1",;,,;~1..,l;."~ (1
day for the first dose and 14 days for the second dose).
- 8 -
wo 95/12411 217 ~ ~3 81 PCT/US94/12626
Example 5: Vaccination and Challenge
Female Swiss White CF-I mice weighing 12-14 grams (Charles .~iver)
received 2 v~ of 100 ,ul s~ f J"CIy, two weeks apart. There were 8
mice in each group with the exception of the 10 ,ug group for the MOMP-C,
MOMP-E, LPS, MOMP-E+LPS groups, the 0.058 yg group for the EB and the 0.81
æg group for the harvest fluid in which there were 10 mice per group. The 2
additionai mice in these groups were sacrificed and bled on the day the remainder of
the mice were challenged. Vaccinates as well as 10 controls were cha lenged withan ' , I inoculation of C. psittaci, Cello strain (Cello (1967) Am. J.
07-7~h~7mr~1 63:1270-1273) 2 weeks after the second ;, . ." .. . I .;, ~ 1 i,
For each study, 3 additiona. groups of 10 mice were cha lenged with 10-fold
serial diludons of the challenge materia7. to confirm the LDso. At the dilution of the
Chlarnydia organisms used to cha lenge vaccinates and at a 1:10 dilution of thischa7.1enge materia. a l the control mice (10 pe} group) ~ied.
All subunit ~ Liol~s, except LPS, protected 100% of the mice when
j1~l1,.;..;.~. .~,7at IOOugf'dose. Adose-relatedtitrationoftheprotectiveeffectwas
a so observed. ~ mp ~ on of the various MOMP-containing ~I~,pa~ ns
~,l,,,;,,;~r~ .c~1 at the 10 pg dose indicated that both MOMP-C alone and MOMP-Eplus LPS sill protected 100% of the mice, while MOMP-E could only induce a 60%
protective level. Comparable levels of protection were a so obserYed with the 1.0
,ug and 0.1 ,ug doses of the MOM?-C a one and MOMP-E plus LPS IJI~I.lli-)IIS.
These levels were significantly greater than that induced by MOMP-E a one. See
data proYided in Table 1.
WO95/12411 P~TIUS9.t112626
~ nsl ' -
TABLE l
Protection of Mice with Chlamydia Subunit P-c~ iu~
Subunit P~ dliU.I Tûtal Prûtein P. ~ vel (%)
Antigen/Adjuvant , (ug) #~u- ~;vul~ uli~l
MOMP-C ` 100 8/8(100)
2% ALOH, 25 ,ug Quil A 10 8/8 (100)
1.0 6/8 (75)
0.1 0/8 (0)
MOMP-E 100 8/8 (100)
2% ALOH, 25 pg Quil A 10 5/8 (62.5)
1.0 3/8 (37/5)
0.1 0/8 (0)
LPS 100 3/8 (37/5)
2% ALOH, 25 ug Quil A 10 3/8 (37/5)
1.0 0/8 (0)
0.1 2/8 (25)
MOMP-E + LPS 100 8/8 (100)
2% ALOH, 25 ,ug Quil A 10 8/8 (100)
1.0 5/8 (63.5)
0.1 1/8 (12.5)
5 Example 6: Evaluatiûn ûf Immune Respûnse by ELISA
EBs were fixed with methanûl to 96-well round-bottom Immulonp 2 plates
(Dynatech I .qhorqtnri~c 14340 Sullyfield Circle, Chantilly, Virginia) at I ,ug/well.
Plates were washed with distilled water, then blocked with 3% horse serum (Hy-
Clone T .~r.qrt~ri~S, Inc., 1725 South HyClone Road, Logan, Utah 84321) in PBS
10 for I hour at room tUlll~ UlC. Mouse sera was diluted in PBS containing 0.3%
Tween (v/v) and applied to plates After a I hour incubation at room ~ UIC,
plates were washed with PBS containing 0.3% Tween and funher incubated with
peroxidase-labeled antiserum to mouse IgG (Kirkegaard & Perry I . ' Inc.,
2 Cessna Court, Gaithesburg, Maryland 20879). Plates were then washed with PBS
15 containg 0.3% Tween and developed with ABTS substrate (T~irk~g?qrrl & Perry
I r' ' Inc., 2 Cessna Coun, (~lqi~hl-ch~rg, Maryland 20879). After 30
minutes, the OD. 405-490 was measured. Titers are expressed as the dilution of
- 10-
WO 95112411 PCT/[TS94/12626
217~
mouse serum giving an O.D. reading of 0.200. Background values were
s~s,.llly between 0.002-0.004 O.D. units.
Serum titers were ~ rnnin.-(l for two randomly chosen mice from each dose
group prior to being rhslllrn~r~l A correlation between the ability of the vaccines to
5 protect mice from infection and the EB-specific serum response was observed.
Western b~ot analysis from mice vaccinated with MOMP-C alone and MOMP-E
plus LPS indicated that the response was almost exclusively directed to the MOMP.
