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II.TILE(lcloe ecriy Jaufiaton Ue f 61102 .3M161102BS15 AK-l DA3139551 1 TTL (ndce~cuit~lsstictin)Us o avaccinya const-ruct expressing r-ne c icuI1pl~ 'L

protein in the auialysis of protective immunity to Plasmodium Yoelii

12. PERSONAL AO',OR(S)Sedegah M, Beaudoin -RL, De la Vega F, Leer I N, ofiT ASones E,

Chnrnpnvit Y.. Yuan LF, Gross M, Majarian WR, Robey. FA, Weiss W, 0 ~manS

13a. TYPE OF REPORT 13b. TIME COVERED j14-1.E OF REPORT (Year, month, Day) 115. PAGE COUNT

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16. SUPPLEMENTARY NOTATION in Technological advance SIn VdLLIL'u development: riew 'SeL t~t Vol.84

Editor: Laurence Lasky. N.Y.: Alan R. Liss. 1988. pp. 295-309

17. COSATI CODES 18 UJC EM (Cniu ifve iecss trenri~pi b ~ f'~a

FIEL GROP SU.GROaccinia.iis icm ion, ant 0o y Speii~tMjurine ma iar ia, inMmun~~zaecicy

19. ABSTRACT (Continue on reverse if necessary and identify by block number')

DTICMAR 13 31990

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All other edition$ are obsol ete. UN~CASIFIEDJ

Technological Advances in Vaccine Development. pages 295-309Z 1988 Aan R. Liss. Inc.

USE OF A VACCINIA CONSTRUCT EXPRESSING THECIRCUMSPOROZOITE PROTEIN IN THE ANALYSIS OF PROTECTIVE

IMMUNITY TO P.LASMODiT YQEIL

Martha Sedegah, 1 Richard L. Beaudoin, 1

Patricia De la Vega,1 Mary F. Leef,

1 M. Ali Ozcel,1 , 4

Elaine Jones, 2 Yupin Charoenvit, 1 Leo F. Yuan,1 Mitchell

Orc'ss,2 William R. Majarian,

2 ,5 Frank A. Robey,3

Walter Weiss,1 and Stephen L. Hoffman

1

lInfectious Diseases Department, Naval Medical ResearchInstitute, Bethesda, Maryland 20814

2 Smith Kline and French Laboratories, 709 Swedeland

Road, King of Prussia, Pennsylvania 19406

National Institute of Dental Research, Bldg. 30, Rm.21TB,Natiznal institutes of Health, Bethesda, Maryland

20892

ABSTFACT ntibodies to the circumsporozoite (CS)protein may play a role in the protective immunity S CRA&|induced by immunization with irradiated sporozoites. DTIC TAB flHowever, the observation that protection against a iarae Unnnot,:;cod(5000 sporozoites) inoculum is dependent on T cells of Juslrf'c.ji l-he suppressor/cytotoxic phenotype (CD8) has clearlyestablished the importance of cellular mechanisms in Bythis -odel system. In an attempt to stimulate acrCtec:.ive cellu.ar immune response, Balb/CByJ mice were Oistribiitin"!rmunzed intraperitoneally with one to 4 doses of a'acc-n;i recombinant construct expressing the entire C3 "prcein of D'=dvum U . The mice were challenaec- weeks after the last dose of vaccine with 200 or10,,CO soorozoites. Mice vaccinated with irradiated

4Present address: Department of Parasitology, Medical I,1_Facultv, Ege University, Bornova-Izmir, Turkey5Present address: Praxis Biologics, 300 E. River Road,

Rochester, New York 14623

rv

f0

903 03' 13 052

296 Sedeph et al

sporozoites were protected, but all of the animalsimmunized with the recombinant construct becameinfected, even though the anti-sporozoite IFA titers ofboth groups of vaccinated mice were comparable. Todetermine if antibodies alone might be responsible forprotecting mice immunized with irradiated sporozoitesagainst a low dose challenge, we depleted them of CD8+ Tcells, and found that they were no longer protectedagainst even a 200 sporozoite challenge . It is unclearwhether failure to protect mice with the vacciniaCS construct results from antigen presention inadequateto induce the required cell mediated immune response, orwhether the CS protein is not the appropriate antigen. " . . .'c\c3Tt dc- apnear however, that antibodies induced by theirradiated sporozoite vaccine are not an adequate -explanation for the protection observed against even alow dose challenge.

J

:NTRODUCTION -

Malaria is transmitted to humans by anophelinemosquitoes that inject sporozoites while feeding.

initial attempts to develop subunit sporooitP vaccinesfocused on developing immunogens that induced highlevels of antibodies to the repeat regions of the:ircumsporczcite (CS) protein of the sporozoite. Thizapproach has thus far proved only marginally success-fuin humans (,2). Furthermore, synthetic peptides ana:. on' produced reccmbinant vaccines have been shownt induce protection against moderate (500-1000), zutnot large (10,000) sporozoite challenge in the-' -odiu erghei rodent model (3,4), but nocrotection in the Z. y system (5). This is neturprisina since several studies have shown that the

:rotective immunity developed after immunization wit--fradiated socrozoites can be independent of antihoa"

7), an is dependent on CDB+ T lymphocytes (S, 3)7rotective im-munity after immunization with irradlazea:pcrazoites Is also dependent on immunization with i:e-ccrozoites suggesting that the sporozoites must enter-lis :to induce the appropriate imrmune response. -"

.cc known if this immune response is directed againstntiens excresea Ly t-.e poioite or nec-antiocns

f:zst exuressed by the parasite within hepatocYtes.

Analysing Protective Immunity to Plasmodium yoeLi 297

in an attempt to develop a vaccine that wouldinduce a protective T cell response by presentingantigen expressed within host cells, we constructed avaccinia recombinant that includes the entire geneencoding the £. v CS protein. We now report theresults of the first immunization experiments with thisconstruct.

MATERIALS AND METHODS

Construction of the vaccinia F. v.iL. CS Recombinant(VCV).

A 1.3 Kb frarment encoding the entire _. vnjj CS

crotein coding region (Fig. 1) (10) was isolated by PAGEand subcloned into the Sma I site of pGS20 (11) such-na- tne 7.5 Kt gene pt.MoLer directs transcription of:.he inserted DNA. Vaccinia virus recombinants were thenzenerated as referenced previously. Briefly, monolayers

'V-1 cells were infected with wild type vaccinia"rus (TK- Wyeth strain) at a multiplicity of infection

:.C5 plaque forming units (pfu) per cell. Two hoursinfection, recombinant plasmid DNA was introduced

cv cransfection of calcium phosphate precipitated DNA.- -s were harvested 48 hrs later, and virus wasreleased bv 3 freeze-thaw cycles. TK- recombinant virus

was selected from cell lysates by plaque assay on TK-:.!in tne presence of 25 pg/ml 5-bromodecxyuridine

.Z7ZR) . :soiated TK- plaques wer o_:ked and used to

_.ie:: fresh monoiayers of TK-143 F orty eight

lr5 later, when significant cytc, lic effect (cpe)• as evident, cells were harvested, subjected to 3 cycles:f freezing and thawing, and cell lysates were then

screened for expression of the p. v CS protein cyc7 unonlot analysis. Lysates were applied tonczroellulose sheets with the aid of a Dot-blot

7nnlfold (Minifold I; Schleicher & Schuell, Keene NIH)

a.unoccupied sites blocked by incubation for 1 hr in-:PA buffer (20 mM Tris, pH 7.5, 150 mM NaC1, 1 mM EZTA,:." q7S, C.5% Triton X-10C) supplemented with 3% BSAiwv .Filters .,re thpn incubated overnight at room

temperature in the presence of NYSI, a mnnoclcnalanticody specific for the Z. vjjJ. CS protein (12)ociuted 1:1000 in RIPA buffer. These were then washedo:,-es with RIPA buffer, and

29 Sedeh eta&

further incubated for 1 hr at room temperature with RIPAcontaining 1 ICi 1251-protein A. Filters were againwashed three times with RIPA and positive spots

visualized by autoradiography. Recombinant viruses

which were positive in this assay were subject to 3rounds of plaque purification. Finally, plaque purifiedvirus was used to infect monolayers of CV-1 cells and

cell lysates were screened by western blotting toconfirm the presence of the full length E. .ePii£ CSprotein.

I --------- Hinc II fragment ------- IHinc II Hinc II

Site Site

Conserved Conserved

Region 1 8 Asn Region 2_ I /

/ I IRepeat I Repeat 2 Repeat 3

(QGPGAP) X 19 (QQPP) x 8

FIGURE 1. Ziagramatic structure of the P cci-

': gene ercoding the circumsporozoite (CS) prote~n.

'.alaria Parasites.

The §-X (NL) strain of E. y 4 was used

il experiments. Sporozoites were isolated byu s cnt nucus gradient centrifugation as previousLy

scr bed (12) and adjusted to required concentratlcn

.... edium 199 suppienmented with 10% mouse serum.

raciate2 sporozoites (:RRspz) used for i.munizati-..ere isolated from infected mosquitoes that had been:x. eta o. Krads from a 3Te cnrce.

Analysing Protective Immunity to P=smodium yoei 299

Immunogens.

The following were using as immunogens:a) The Vpy vaccinia CS construct encoding the full

length CS protein of _. mii (108 pfu/dose) inoculated

intraperitoneally (ip).

b) A control vaccinia construct (Vgalk) encoding

an unrelated E. zoIi galactokinase (108 pfu/dose)

inoculated ip.c) IRRspz injected intravenously (iv). The first

dose was 5 x I4 sporozoites, and subsequent doses w~-re3 x 104 sporozoites.

Immunization and Challenge Schedule.

Groups of 8 female Balb/CByJ mice (Charles RiverLabs), 8 weeks old, received 1, 2, 3 or 4 doses ofvaccine prior to challenge. Vaccine doses wereadwinistered at 2 week intervals. Two weeks after thelast immunizing dose, blood for serum was obtained and

the mice were challenged with infective sporozoites.Mice that received 1, 2 and 3 doses were challenged with

04 sporozoites, while mice that received 4 doses of%accine were challenged with 200 sporozoites. Mice were.ten monitored for parasitemia as an indicator ofcrctective immunitv.

2etection cf Antibodies to Sporozoite Epitopes.

Pooled sera were analyzed for reactivity with-porozoite antigens using the following techniques:

nret f e n anibodv test (IFAT) . The

:FAT was carried cut using air dried whole sporozoites

3s previously described (12).

Enzv"p linked immunn-rtent assay (ELIA) . ELISAwas used to analyze the epotopes on the CS proteinreccanized by sera from the different groups ofvacc:nated mice. Briefly, the test consisted of a soiinase assay in wnicn synthetic peptides or recombinantrcrteins were coated to the wells of 96 well flat b:tt=c.mulcn :7 micrctlter plates (Dynatech Laboratories,

.lexandria,VA) . ,ells were coated with one of thefollcwing: a) a recombinant fusion protein produced

the ?inc 71 fragment CS gene in L. coli that includesthe 3 P. vf-li; repeat domains and the conserved Reozr

1 sequence fused to 81 amino acids from the

300 Sedegah et al

non-structural protein of viral influenza; b) a

synthetic peptide containing 2 copies of repeat #2,Gln.Gly.Pro.Gly.Ala.Pro [QGPGAP] 2; and c) a synthetic

peptide containing 3 copies of repeat #3,Cln Gln.Pro.Pro [QQPP] 3. Each well was then blocked

with casein and incubated overnight. The wells werethen reacted with the appropriate dilution of a testserum, washed 3x with buffer after which the revealingantibody, a horseradish peroxidase (HRP) labelled goatanti-mouse Ig was added followed by the peroxidasesubstrate consisting of equal volumes of H202 and ABTS

(2.2'-azino-di[3-ethyl- benzothiazoline sulfonate])(Kirkegaard and Perry Laboratories, Inc., GaithersburgMD). Color development was measured photo-metrically at405 nm using Microelisa Autoreader MR 580 (DynatechLaboratories).

Depletion of CD8+ T Lymphocytes.

Mice immunized with 4 doses of irradiatedsporozoites and shown to be protected against challenge

with F-.veli sporozoites 17 days previously were given

two I mg ip injections of monoclonal antibody 19/178 at24 hour intervals to deplete them of CD8+ T lymphocytes

as previously described (8). They were challenged with

eitner 200 or 1-0,000 sporozoites 48 hours later.:reatment with Mab 19/178 was continued every third dayafter challenge until blood parasites appeared. Bloodf .ms were examined for parasites until patency or for14 days, whichever occurred sooner. When parasites were4etected in the blood, mice were killed and spleen cellsprepared and analysed for depletion of the subpopuiation

-arrving the CD8 marker by fluorescent activated cellacrier. (FACS).

RESULTS

7trtecticn Against Challenge.

Results Df the challenge of mice immunized with '.'py

ire su.mmarized in Table 1. Forty percent of the mice

-hat received a single Jose of irradiated sporezcites

were protected against challenge with 104 sporozoltes,

ind this level .cf protection increased to 100% in the

Analysing Protective Immunity to Plasmodium voe4i 301

three groups of mice that received 2, 3, or 4 doses ofirradiated sporozoites. In contrast, all mice immunizedwith the recombinant Vpy construct became infected afterchallenge including the group that had received 4 dosesof the construct and was challenged with only 200

sporozoites.

TABLE 1Results of challenging mice immunized with vaccine

preparations Vpy, Vgalk and IRRspz.

Expt No.of Vaccine IFAT Ratio of MiceDoses Preparation Titer Prot./Chall. U

Challenged with 104 sporozoites/mouse.

A Vpy 32 0/8Vgalk <8 0/8IRRspz 16 3/8Control <8 0/8

Vpy 512 0/8Vgalk <8 0/8IRRspz 256 8/8Control <8 0/,8

C 3 Vpy 2,048 0/8

Vgalk <8 0/8IRRspz 2,048 8/8Control <8 0/8

thal-enged with 200 sporozoites/mouse.

4 Vpy !,024 0/8Vqaik <8

!RRsoz 2,048 8

Ccr.rci <8 2

302 sedepet al

Antibody levels.

Anti-sporozoite antibody titers as measured by IFATwere comparable in the groups that were vaccinated withIRRspz and those that received the Vpy construct.Results obtained by ELISA using the Hinc II fusionprotein containing all the repeat domains were alsoconsistent with the IFAT findings. However, althoughboth groups had made comparable levels of antibodies toHinc II peptide (Fig.2), there was a significantdifference in the epitopes recognized by each at theendpoint titer.

4 Doses vs Hinc 11 Fusion Peptide2000-

rr~

0

y 0 0 0 0 0NN0

Serum Oliulon - N V

z'IGURE 2. ELISA determination of the anti-body

resnonse to the Hinc II fusion peptide of mice i runrzea

wit h vaccine preparations IRRspz, Vpy and Vgalk.

Mice immunized with irradiated sporozoites producec

3icn'ificantly higher levels of antibodies to repeat 12

(frQ32GAP17) while mice immunized with the Voy construct

7n-e hiaher levels of antibodies to repeat #3 (QP>

(7iasL.3 and 4 respectively).

Analysing Protective Immunity to Plasmodjum yoelui 30.3

4 Doses vs Repeat 82 (OGPGAPix2

0 1 OWN0 ~

FIGtIPE 3. ELISA determination of the antibodyresoonse to Repeat #2 [QGPGAPJ2 of mice immunized with.

~a~ne preparations IRRspz, Vpy and Vgaik.

4 Doses vs Repeat 83 (OOPP) x 32000

IRR soz[3l Vqa1

a 1000 -

0

yW to V CO N CY

Serumilution - N 0 V 1o ~ 1 0 - Y-~ ~ M .

FIGURE 4. ELISA determination of the ant-todvresponse to Repeat #3 (QQPP) 3 of mice immunized with~

,accne preparations IRRspz, Vpy and Vgaik.

304 Sedegah et al

Protection After CD8 T Cell Depletion.

We knew that a Mab to repeat #2 protected mice inpassive transfer experiments, whereas a second Mabreacting with repeat #3 did not (Charoenvit, inpreparation) and we wondered if the difference inantib'ody specificity between the IRRspz and Vpyimunized groups might account for the differences inprotection observed in each, especially for lowchallenge doses.

TABLE 2Results of rechallenging protected ice

depleted of CD8+ T-lymphocytes

Mouse No. IFAT Titer Day after Challenge,t challenge Blood Infection Detected

+3 +4 5

17'en d ith 200 s;2r-zoitesimnuse.

CDS+ T cell depleted.512 - -

2 1,024-- 512 - -

-. 2,048 -Thallence controls.

- <S - -

'-<8 - -

3 <8 -

4 <8 -

2:S- zell depleted.:,024 -

2,048

Thaiience :ontrois.

- '8

Analysing Protective Immunity to Plasmodium yoelii 305

However, when mice immunize-_ with 4 doses of irradiated

sporozoites were depleted of CD8+ T cells (verified by'FACS analysis; data not shown) and challenged with

either 200 or l04 sporozoites, they all became infectedindicating that the antibodies induced by immunizationwith !RRspz were not sufficient to protect these mi(

against challenge with 104 sporozoites or even the '.erv1-w challenge dose of 200 (Table 2).

*DISCUSSION

h Observations in the rodent malaria model systemsP-ae provided -he foundation for development of human

7 -alaria sporozoite vaccines. Murine studies now* :--early demonstrate the importance of cell-mediated

immune mechanisms in the protective immunity elicited by

inmunization with irradiated sporozoites (3,7,8,9). In

an attempt to induce a protective cellular immune

response directed against the CS protein, we constructed

a E. ojei-CS vaccinia recombinant. The faiIre cf_his vaccine to protect against even a minimal

sccrozoite challenge indicate3 that either the CS

orotein is not the target of such irnmunity, or that.ntriperitoneal immunization with Vpy did not induce therequired cellular immune response. Recent studies -rno.e Z. berqhei system (Sadoff J; personal cD~rmunication)

-n.cate that the CS nrotein may be the target ot sucnan mmune response. I so, this vaccinia recombinant

-nay _! an inadequate vector for proper-y uelivering che

-r'.uncgc, or the ru-te of adminictration may nave beenaopprrpriate for induction of the cytotoxic T cells

ohso we believe are involved in a protective response.

Further studies are in progress to explore thesepcssiblities.

<'

306 Setsh e Ii

MICE PROTECTED AFTER 2 DOSES OF IRRspz

000

2900- ImIRR spz

2000

Ci

In0 1000-

0

VCctoI N 4 I D N N co'D Nq in -o to C

D lutlon M U 000-MIN4 WT 4 W

FIGURE 5. Comparison of the ELISA response to

._--eat 42 [:'GPGAPI 2 in mice vaccinated with the :3Rso:

d Vuy vaccine preparations. The IRRspz group was-:Cmpietely protected after 2 vaccine doses, whereas :. e"py group wag totally suscpetible after 4 doses of "hy.

Analysing Protective 1mwunity to PMsmodium yoeti 307

Mice immunized with 4 doses of Vpy were not protectedagainst challenge with even 200 sporozoites, althoughthey had levels of antibody against CS protein andsporozoite epitopes comparable to the levels in miceirn.unized with 2 doses of IRRspz that were protectedagainst challenge with 10,000 sporozoites (Fig.5).

The results of the CD8+ depletion study (Table 2)demonstrate that circulating antibody levels in miceimmunized with 4 doses of irradiated sporozoites are notadequate to protect against even a low dose challenge.Protective immunity in IRRspz immunized mice appears tobe wholly dependent on CD8+ T cells regardless of thesize of challenge.

Although IRRspz immunized mice are not protected byantibodies, we have shown that passive transfer of amonoclonal antibody, NYSI directed against repeat #2(QGPGAP) can protect against challenge with a largeinoculum of sporozoites (Charoenvit,in preparation). itis therefore theoretically possible that vaccineseliciting antibodies against the CS antigen will stillprove protective, if the levels of antibody they achieveexceed those reached with the IRRspz and Vpypreparations used in this study. Our data provide thefirst indication that the immunodominance of B cellepitopes on the CS protein may be dependent on how thenimunogen is delivered. 1 ntibodies measured by IFATwere similar in the IRRspz and Vpy immunized groups.However, immunization witn the IRRspz vaccine inducedthe highest levels of antibodies against (QGPGAP] incontrast to immunization with Vpy which produced higherlevels to (QQPP] . The protective Mab, NYSI, is directed3aainst an epitope in the [QGPGAP] domain, whereas asecond Mab, NYS3 reacting with a different epitopeczontained in the [QQPP] domain is not protective(Charoenvit, in preparation). It would appear thatuture studies of subunit vaccines in this model systemwould be well advised to consider the epitopes3ecificity of vaccine induced antibodies and not rely

,n antibodies to the entire sporozoite as the sole:neasure of irmmunogenicity.

308 Sedegahet a]

ACKNOWLEDG-MENTS

We wish to thank Ms. B.J. Leckey for editing themanuscriat. M. Sedegah and P.de la Vega were supportedin part by OMP. Grant No. N00014'-85-G-0255 to the PanAmerican Health Organization. This project wassupported in part by NNR&DC work unit 3M1611O2BS1OAK11I.

The opinions and assertions herein are the privateones of the authors and are not to be construect asofficial or as reflecting the views of the NavyDepartment or the naval service at large. Theexperiments reported herein were conducted according t-'the principles set forth in the current edition of theGuide fnr the Care and Use of Laboratory Animals,Institute of Laboratory Animal Resources, NationalResearch Council, DHHS Pub. No. (NIH) 86-23.

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Plamoiu ipa,, sporozoite vaccine. Lancet--11277.

2. H-errington DA, Clyde DF, Losonsky G, Cortesia m,Murphy JR, Davis J, Bagar S, Felix AM, ?Heimer EP,3;1esen D, Nardin E,Nussenzweig RS5, Nussenzweic V1,"M;llingdale MR, Levine MM (1987) . Safety andin-nunogenicity in man of a synthetic peptide maiariavaccine agai.nst Plsoi fac;au sporozoites.Nature (London) 328:257.

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S. L,311 AA, Je !a Cruz V, Good MF, Weiss WR, Lunde M,Maloy W, Welsh JA, McCutchan TF (1987) . in vivo

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6. Spitalny GL, Nussenzweig RS (1973). Relationshipbetween protective immunity and antisporozoite (CSP)antibody in mice. Exp Parasitol 33:168.

7. Chen DH, Tigelaar RE, Weinbaum FI (1977). Immunity tosporozoite-induced malaria infection in mice. I. Theeffect of immunization of T and B cell deficientmice. J Immunol 118:1322.

8. Weiss WR, Sedegah M, Beaudoin RL, Miller LH, Good MF(1988). CD8+ T cells (cytotoxic/suppressors) arerequired for protection in mice immunized withmalaria sporozoites. Proc Natl Acad Sci USA 85:573.

9. Schofield L, Villaquiran J, Ferreira A, SchellekensH, Nussenzweig R, Nussenzweig V (1987). y Interferon,CC8+ T cells and antibodies required for immunity tomalaria sporozoites. Nature 330:664.

-o. Theismn T, Gross M, Young JF, Charoenvit Y, BeaudoinRL (1987). Cloning, sequencing and expression of theP voslii circumsporozoite gene. Fed Proc46:2185.

iMackett M, Smith GL, Moss B (1984). General methodfor production and selection of infectious vacciniavirus recombinants expressing foreign genes. J Viroi49:857.

!2.Charoenvit Y, Leef MF, Yuan LF, Sedegah M, BeaudoinRL (1987). Characterization of P jjjrmonoclcnal antibodies directed against stage-specificsporozoite antigens. inf & Imm 55:604.

:3.Pacheco ND, Strome CPA, Mitchell F, Bawden MP,Beaudoin RL (1979) . Rapid, large-scale isolation of_ asmodium x-g.Lhi. sporozoites from infectedmosquitoes. 3 Parasitol 65:414.