CA2175081A1 - Vaccine and method for treatment of chlamydial infections - Google Patents

Abstract

A vaccine for treatment of chlamydial infections made up of a major outer membrane protein (MOMP) preparation and a lipopolysaccharide (LPS) preparation from a Chlamydia organism is provided. Methods of treating and immunizing animals against chlamydial infections are also provided.

Description

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

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.

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 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 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).

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 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.

Claims (24)

What is claimed is:

1. A vaccine for treatment of chlamydial infections comprising a MOMP
preparation and a LPS preparation from a Chlamydia organism.

2. The vaccine of claim 1 wherein the MOMP preparation is selected from a group consisting of MOMP-E, MOMP-C, and MOMP in the context of EBs or COMCs.

3. The vaccine of claim 2 wherein the MOMP preparation comprises MOMP in the context of EBs.

4. The vaccine of claim 2 wherein the MOMP preparation comprises MOMP-E.

5. The vaccine of claim 1 wherein the Chlamydia organism comprises Chlamydia psittaci.

6. The vaccine of claim 5 wherein the Chlamydia psittaci comprises a Baker strain.

7. The vaccine of claim 1 further comprising an adjuvant.

8. A vaccine for treatment of chlamydial infections comprising MOMP in the context of EBs, LPS and an adjuvant.

9. A method for treatment of chlamydial infections comprising administering to an animal infected with a Chlamydia organism an effective amount of a vaccine comprising a MOMP preparation and a LPS preparation from a Chlamydia organism.

10. The method of claim 9 wherein the MOMP preparation in the vaccine is selected from a group consisting of MOMP-E, MOMP-C, and MOMP in the context of EBs or COMCs.

11. The method of claim 10 wherein the MOMP preparation in the vaccine comprises MOMP in the context of EBs.

12. The method of claim 10 wherein the MOMP preparation in the vaccine comprises MOMP-E.

13 The method of claim 9 wherein the Chlamydia organism comprises Chlamydia psittaci.

14. The method of claim 13 wherein the Chlamydia psittaci comprises a Baker strain.

15. The method of claim 9 wherein the vaccine further comprises an adjuvant.

16. A method for treatment of chlamydial infections comprising administering to an animal infected with a Chlamydia organism an effective amount of a vaccine comprising MOMP in the context of EBs, LPS and an adjuvant.

17. A method for immunizing an animal against chlamydial infections comprising administering to a healthy animal a vaccine comprising a MOMP preparation and a LPS preparation from a Chlamydia organism.

18. The method of claim 17 wherein the MOMP preparation in the vaccine is selected from a group consisting of MOMP-E, MOMP-C and MOMP in the context of EBs or COMCs.

19. The method of claim 18 wherein the MOMP preparation in the vaccine comprises MOMP in the context of EBs.

20. The method of claim 18 wherein the MOMP preparation in the vaccine comprises MOMP-E.

21. The method of claim 17 wherein the Chlamydia organism comprises Chlamydia psittaci.

22. The method of claim 21 wherein the Chlamydia psittaci comprises a Baker strain.

23. The method of claim 17 wherein the vaccine further comprises an adjuvant.

24. A method for immunizing an animal against chlamydial infections comprising administering to a healthy animal a vaccine comprising MOMP in the context of EBs, LPS and an adjuvant.