Induction of cytolytic and antibody responses using Plasmodium falciparum repeatless circumsporozoite protein encapsulated in liposomes

Katherine White*, Urszula Krzych* Daniel M Gordon*, Terence G Porter °~, Roberta L Rlchards t, Carl R Alvln~ Carolyn D. Deal *, Michael Holhngdalell, Carol Sllverman ~, Daniel R Sylvester ~°, W Rlpley Ballou* and Mitchell Gross °~

Plasmodmm ctrcumsporozolte ( CS) protein-induced antibody and T-cell responses are considered to be important in protective immunity Since the key repeat determinant o f the CS protein may actually restrict the recognmon of other potential T- and B-cell sites, a modified Plasmodlum fa lopa rum CS protein lackm9 the central repeat region, RLF, was expressed m Escherachm cola On purification, R L F was encapsulated into hposomes [L(RLF)] and used for the xn wvo induction o f cytolyttc T lymphocytes (CTL) and antibodies Immunization o f BlO Br ( H-2 k) mice with L( RLF), but not with RLF, Induced CD8 + CTL specific for the P falclparum CS protem CTL epttope, amino acid residues 368-390 Antl-L( RLF) serum reacted with antigens on intact sporozoltes and inhibited sporozotte invasion o f hepatoma cells Antibody specificity studies in New Zealand White rabbits revealed new B-cell sites locahzed In amino acid residues 84-94, 91-99, 97-106 and 367-375 Although the mechanisms by which hposomes enhance cellular and humoral immune responses remain unknown, hposome-formulated vaccines have been well tolerated m humans, hence, their use in vaccines, when efficacy depends on antibody and CTL responses, may be broadly applicable

Keywords Malaria, repeatless clrcumsporozolte protein, cytolytlc T cells, antibody, hposomes

Immunization with radiation-attenuated Plasmodtum sporozoltes is a highly effective but impractical method for inducing protective immunity against malaria in humans 1 Attenuated sporozoltes infect hepatocytes but fail to develop normally into mature liver stage schlzonts, and do not release blood stage parasites into the circulation Antigens expressed on sporozoites and attenuated liver schizonts are thought to be critical for the induction and maintenance of immunity The circumsporozolte (CS) protein has been identified as an important parasite antigen capable of inducing both antibodies 2'3 and cytolytic T lymphocytes (CTL) 4 that participate in protective immunity The CS protein is

Departments of *Immunology, tMembrane B)ochemistry and Bacterial D~seases, Walter Reed Army Institute of Research,

Washington DC 20307-5100, USA °°Departments of Macro- molecular Sc)ences and Protein B)ochemlstry, SmlthKhne Beecham Pharmaceuticals, King of Prussia, PA 19406, USA IIBiomed)cal Research Institute, RockviIle, MD 20852, USA ¶To whom correspondence should be addressed at Department of Immunology, WRAIR-WRAMC, Washington DC 20307-5100, USA (Received 10 October 1992, rev)sed 19 March 1993, accepted 19 March 1993)

carried into hepatocytes during sporozolte invasion and can be detected in liver stage schlzonts for at least 48 hours The CS protein of the human malaria parasite Plasmodtumfalctparum has been cloned and sequenced s, and the CTL epltope mapped to the C-terminal region, amino acid ( a a ) residues 368-3906 Neutralizing monospeclfiC antibodies raised against intact sporozoltes are principally directed against an lmmunodomlnant central CS repeat region Subunit vaccines that target only these repeat epltopes have shown hmlted efficacy in human volunteers 7'8 Because the repeat epitopes may act to divert immune responses away from other important regions of the CS, we engineered and expressed a modified P falclparum CS protein lacking the central repeat region, referred to as RLF (Figure I ) Studies have shown that amputation of certain lmmunogenlc regions can expose new determinants previously unrecognized by immune effector cells 9 In the case of malaria, modifications of CS antigens might lead to the induction of more effective responses We report here data demonstrating that RLF, formulated as a hposome vaccine, induced both CTL, which have been implicated in protective immunity, and neutralizing antibodies directed against previously unrecognized B-cell epitopes

0264-410X/93/13/1341~6 (, 1993 Butterworth-Hememann Ltd Vaccine, Vol 11, Issue 13, 1993 1341

Ltposome-encapsulated malaria vaccme K Whtte et al

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Ftgure 1 Structure of RLF A 320 a a single chain polypeptlde with calculated M r of 35200 Da The a a sequence of NS181 is underhned An eight residue vector sequence (m ttahcs) hnks NS181 to a a 19 from the 7G8 CS of P faloparum A glycme residue not found m the authentic protein (double underhned) was inserted between the N-terminal and C-terminal repeatless flanking sequences to facdttate mserhon of alternatwe sequences The numbers on the right refer to the a a m the pubhshed sequence"

MATERIALS AND M E T H O D S

Animals

Adult female B10 Br (H-2 k) mice used in this study were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and used between 8 and 12 weeks of age The New Zealand White (NZW) female rabbits were purchased from Hazelton Laboratories (Lenexa, KS, USA )

RLF construction

A 1216 bp Stu-RsaI fragment of Impf containing the entire CS gene of P Jalctparum strain 7G8 was cloned into the BamHI filled site of plasmlc pAS1 and the resulting plasmld (pCSP) was digested with BamHI-FokI to release a 314 bp fragment Plasmld pCSP was also digested with TthIII1-SalI, releasing a 667 bp fragment that included 275 bp of pAS1 The fragments along with the synthetic DNA linker (5'-ATCCCGGGAAT- AAAAACAACCAAGGTAATGGACA-3' 3'-GCCCTT- ATTTTTGTTGGTTCCATTACCTGTT-5') were hgated into the BamHI-SalI sites of pUC 18 The synthetic DNA linker joins the 314bp BamHI-FokI fragment to the 667 bp TthIII1-SalI fragment and, as a by-product of a created Sinai restriction site, adds a glycme residue between the flanking sequences The resulting plasmld pRLF, containing the region and the initial 57 bp signal sequence, was digested with BamHI, end-filled with DNA polymerase I (Klenow), and digested with SalI The 1013 bp fragment was cloned into pMG 1 which expresses the repeatless CS fused with NS18~ Escherwhta ~olt-expressed RLF was isolated from cell lysates by centrlfugatlon and chaotropic extraction and purified by cation exchange, hydroxyapatite, reversed phase and size exclusion chromatography

Llposomal encapsulation

Liposomes containing encapsulated RLF were prepared as described previously l° except for the following changes Due to the hmlted solubdlty of RLF at physio- logical pH, the lyoph]hzed hposomes were reconstituted with RLF in acetate-phosphate-buffered saline, pH 3 5 After washing in the same buffer to remove unencapsulated antigen, hposomes were washed and then resuspended

in acetate-phosphate-buffered saline, pH 6 5 L]posomes were composed of dlmyristoyl phosphatidylchollne, dimyrlstoyl phosphatidylglycerol, cholesterol and mono- phosphoryl lipid A (MPL) in molar ratios of 0 9 0 1 075 0026 Mice received 25#g and rabbits received 280/~g of MPL per injection

Reagents

RPMI 1640 containing 10% fetal bovine serum, non-essential amino acids (10raM), sodium pyruvate (10 raM), pemcilhn-streptomycln (100 U m1-1), and L-glutamine (4raM) (Glbco, Grand Island, NY, USA) was used as complete medium for lymphocyte cultures Monoclonal antibody (mAb) (HB3) directed against routine IA d was purchased from American Type Culture Collection (Rockvllle, MD, USA) and murlne mAb 8312 5, specific for CD8, was a gift from Dr Richard Hodes (National Cancer Institute, National Institutes of Health, Bethesda, MD, USA)

Immunizations

BI0 Br (H-2 k) mice were immunized intraperltoneally with a single dose of either 10 or 40/zg of RLF or an equivalent dose of L(RLF) Control mice received either an equivalent dose of hposomes based on their content of monophosphoryl lipid A (MPL) or remained naive NZW rabbits received three 100#g doses of L(RLF) adsorbed to alum at weeks 0, 4 and 7

CTL assay

Immune spleen cells were analysed for cytolytlc activity at 2, 4, 6 and 8 weeks after lmmumzation Five to seven days before the assay, splenic lymphocytes were cultured m vitro in the presence of RLF (10/~gml-1), P falctparum CS protein synthetic peptldes a a residues 368-390 or 331-350 (10#gml- l ) , or without any antigen Target cells consisted of syngene]c (H-2 k) L929 cells pulsed with P falctpmum CS protein peptides a a residues 368 390 or 331 350, or L929 cells transfected with P falctparum CS protein gene (LPF), or control transfected L929 cells contammg the plasmld without the CS protein gene (L28) (both gifts from Dr S Kumar, National Institutes of Health, Bethesda, MD, USA) or unmo&fied L929 cells or allogeneic (H-2 d ) NIH-3T3 cells alone or transfected with the Pla~modmm berghet CS gene 11 After labelling for 1 h with 5XCr (Na2CrO4, New England Nuclear, Boston, MA, USA) (100/~CI per 10 6

cells), the target cells were added to different numbers of effector cells at ratios ranging from 12 5 1 to 100 1 The cell mixtures were incubated in 96-well round- bottom tissue culture plates (Costar, Cambridge, MA, USA) m 0 2 ml complete medium for 4 h at 37°C m a 5% CO2 humidified atmosphere During the 4 h assay a 1 4 final concentration of either mAb HB3 or 8312 5 culture supernatant fraction was added to the appropriate wells At the termination of the 4 h culture the super- natants were absorbed by cotton wicks and processed for the determination of 51Cr release by 7-counter The cytolyt]c responses were defined as the percentage specific lysls calculated by the following formula

(experimental release - spontaneous release) × 100

(control release - spontaneous release )

1342 Vaccine, Vol 11, Issue 13, 1993

ELISA, IFA and ISI assays

Sera from immune rabbits were collected at weeks 1, 5 and 8 for antibody determination Antibody levels were determined by enzyme-linked immunosorbent assay (ELISA) as described 12 with the exception that the capture antigen was a recombinantly expressed protein containing 306 residues (a a 155-461) from the cAMP strain of P falctparum gp195 fused to the N terminus of the repeatless CS protein The molecule was constructed by isolating a 980 bp fragment (BstXI-BglII) containing the gp195 coding sequences and a 1027 bp BamHI-HlndlII fragment that encodes repeatless CS These fragments were hgated to the expression vector pMG15 and expressed in E coh AR58 as a 540 a a protem The protein was purified by electroelution from sodium dodecyl sulfate-polyacrylamlde gels ( S D S - P A G E ) for use in the ELISA Immunofluorescent assay (IFA) using 5-week immune sera was performed as previously described 1° Inhibition of sporozolte invasion (ISI) of HepG2 cells by rabbit sera was performed accordmg to the methodology described 13 ISI activity is the percentage reduction of the mean number of invaded sporozoltes per sample compared with the number of sporozoltes that invaded in the presence of the respective control serum For ISI, sera were assayed in duplicate on two separate occasions and media alone as well as each rabbit's pre-lmmunlzatlon sera were run as negative controls

B-cell epitope mapping

The assays were performed according to the protocol described by Geysen et al 1,, Predcrivatizcd polyethylene pins were deprotected, washed, neutralized, washed and ammo-acylated with blocked 9-fluorenomcthoxycarbonyl amino acids repeatedly until the octamers were completed The peptIdes were then acetylated, deblocked and washed The pins were subjected to three rounds of ultrasonic disruption before ELISA was performed The pins were blocked using an ovalbumln buffer for 1 h at room temperature Antibody diluted in phosphate- buffered saline (PBS) with azlde was placed into each well of blocked 96-well mlcrotItre plates and pins were incubated overnight at 4°C The pins were washed three times with PBS/0 1% Twecn 20 Alkaline phosphatase conjugated secondary antibody was added for 1 h at room temperature The pins were washed three times and phosphatase substratc added Absorbance (A414) was determined using an ELISA reader

R E S U L T S A N D D I S C U S S I O N

Induction of CTL responses by immunization with recombinant RLF incorporated in liposomes [ L ( R L F ) ]

Splenic lymphocytes from L(RLF)-immunlzed B10 Br (H-2 k) mice were examined for memory cytolyttc responses after m vttro stimulation with selected antigens Representative data shown in Figure 2 demonstrate the presence of CTL in splenic cultures obtained from mice immunized six weeks previously with L (RLF ) and stimulated with either RLF or peptlde a a residues 368-390, representing the P falclparum CS protein CTL determinant 6 Positive cytolytlc responses reaching 50% specific lysls were recorded using syngenelc L929 target cells ezther pulsed with the stlmulatory peptide or

L~posome-encapsulated malaria vaccine K White et al

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Figure 2 CTL assays Data in the figure represent cytolyhc responses from splenic lymphocytes obtained from mice immunized s~x weeks previously with 10 #g of RLF in the indicated formulations RLF (A, D, G), L(RLF) (B, E, H), L (C, F, I) Splenic lymphocytes were cultured for 5-7 days in the presence of 10#g RLF (A, B,C), 10/~g of synthetic pephde a a residue 368-390 (D,E,F) and 331-350 (G,H,I) or medium alone (data not shown) Standard 4 h cytolyhc assays were performed using a panel of syngenelc target cells labelled with 51Cr at the indicated effector target ratio ranging from 12 5 to 100 1 (/k), LPF, (A), L28, (O), L929 cells pulsed with pephde 368-390, or (O), wdh pepbde 331-350, or (R) L929 cells alone

transfected with the P falctparum CS protein gene (LPF) Another P falclparum CS protein peptlde, a a residues 33 l -350, which marginally stimulated peripheral blood lymphocytes from persons of malaria endemic areas 15, did not recall detectable CTL activity Immune splenic lymphocytes cultured m the presence of medium alone were also negative (data not shown) CTL activity was evident as early as two weeks after immunization and persisted for up to eight weeks CTL activity was not detected in mice Immunized with ezther soluble RLF or llposomes alone or in naive animals (data not shown)

Mediation of L(RLF)-lnduced CTL responses by CD8+T cells

The cytolytlc activity was abrogated on addition of antl-CD8 mAbs to the culture system during the 4 h assay, demonstrating that CD8 + T lymphocytes mediated the lysls (Table 1) The cytolytlc activity was restricted by class I MHC molecules since only a a 368-390-pulsed L929 cells expressmg H-2 k, but not peptlde-pulsed NIH-3T3 cells expressing H-2 d, were lysed The antigen specificity of the CTL response was also tested using L929 cells transfected with the P falclparum gene versus NIH-3T3 cells transfected with the

Vaccine, Vol 11, Issue 13, 1993 1343

L/posome-encapsula ted malar ia vaccme K Whtte et al

Table 1 L(RLF)-mduced CTL are CD8 ÷ and MHC-restncted

Percentage specff)c lysls

LPF L + Pf368 390 A4 3T3 + Pf368-39( In vitro stimulation Treatment (H-2 k) (H-2 k) (H_2 d) (H_2 d)

RLF

Pf368 390

None 71 69 0 0 Ant)-CD8 0 15 0 5 Control mAb 100 55 n d n d

None 52 95 0 10 Antt-CD8 0 0 0 0 Control mAb 50 100 n d n d

L(RLF)-tmmune splemc lymphocytes were tested for cytolytlc acttwty after m vttro stlmulaUon with anUgen The targets consmtmg of syngenem (H-2 ~) and allogene~c (H-2 k) cells were added to the effector cell cultures at ratios ranging from 12 5 1 to 100 1 (effector target, E T) CD8-specffm mAb or control mAb specific for A d were added to the E T mtxture at final concentrations ranging from 1 4 to 1 20 (v/v) Representatwe data from a single experiment deptct CTL responses at a 50 1 E T ratio m the presence of a 1 4 final dilution of mAb

P beryhel CS gene, and hkewtse only (H-2 k) target cells expressing P falctparum anhgens were lysed Therefore, tt ts apparent that the CTL response induced by RLF Rabbtt

incorporated into hposomes is mediated by CD8 +T cells number recogmzlng a pepttde complexed to a syngene]c class I molecule However, the mechanism of the m vtvo CTL 1

2 mductIon by hposome-encapsulated antigens remams 3 unknown

Preservation of the CTL determinant in the RLF

Removal of the repeat region from the CS protein did not cause any detectable shifts m the posttlon of the CTL epltope The specificity of the CTL response Induced wtth L (RLF) was preserved within the same a a residues that have prevmusly been reported for human and murlne CTL induced with attenuated plasmodm 4 16 or a live vector system containing a generically-engineered CS protein11 17 It may be that this site is naturally msensltlVe to degradation by proteases or that its a a constellation and peptlde configuration exhibit an unusually high affimty for the MHC class I molecules

Possible mechanism of CTL induction by liposome-encapsulated malaria antigens

According to previously reported studies 18--21, liposome- encapsulated viral antigens with or without syngenelc or allogenelc H-2 molecules were successfully used for the mductton of secondary CTL responses, possibly by facilitating antigen assocmtlon wtth major hIsto- compattblllty complex (MHC) class I molecules However, m those instances priming with the intact virus was required for the generanon of CTL responses Recently, several laboratories have reported m vtvo generanon of CTL by ]mmumzatlon with protein antigens such as ovalbumln 22 and fl-galactosldase 23 as well as free pepttdes 24 and peptldes incorporated mto hposomes 25

In this study we have clearly demonstrated m vtvo mducnon of cytolytlc responses by immunizing BI0 Br mice w~th RLF encapsulated into hposomes As expected, ]mmumzatlon w~th RLF alone was meffecnve, because exogenously administered antigens are usually taken up by anngen-presentmg cells and processed m endosomes for export in association with class II molecules 26 In studies reported by other investigators, cytolytlc responses were also elicited following immunization with an influenza A CTL pepnde covalently hnked to hpoprotem, but not with the pepUde alone 27 These

Table 2 Ant~-RLF antibody responses

ELISA IFA

Week 0 Week 1 Week 5 Week 8 Week 5

8 52 11164 11187 1600 0 83 22151 32192 > 12800 5 84 2 399 45408 6400

NZW rabbtts were immunized wtth 100 #g doses of L(RLF) vaccine at weeks 0, 4 and 7 Sera were collected and assayed by ELISA and IFA against sahvary gland NF54 sporozo)tes as described TM IFA results are the highest dduUon at whtch a uniform fluorescence was observed over the surface of the sporozo~te ELISA data are reported m absorbance umts whtch represent the ddut)on at whtch an A414 of 1 000 m obtained

observations underscore the need for an Inducer/helper determinant linked to a CTL epItope and support the concept that hposomes, hke the lipid component of the hpoprotem, facilitate the channelling of an antigen to associate with MHC class I molecules

The precise mechanisms by which llposomes facilitate the Induction of cytolytic T cells have not been elucidated The mternahzation and intracellular targeting of hposome-encapsulated anttgens have been reported to be a function of hposome composttton and structure 28, factors which can profoundly affect immune responses reduced by hposomal vaccines 29 It has recently been demonstrated that after phagocytos]s of hposomes by cultured macrophages, hposome-encapsulated anngens can be delivered both to the cytoplasm and to the cell surface 3° We further speculate that fusion of L(RLF) with the cell membrane of antigen-presenting cells results in sufficient modtficat~on of the antigen promoting the formation of a complex between MHC class I molecules and the CTL ep]tope on RLF While this interpretanon does not address the need for antigen processmg, expertmental condmons m which antigen processing was not reqmred for presentation of hposomal antigen to T cells have been prevtously described 31

Induction of antibody responses by L(RLF)

Pronounced antibody responses have been observed m other hposome-encapsulated vaccines formulated with the adjuvant monophosphoryl lipid A (MPL) 32 Likewise, in the present study, high tltres of RLF-reactlve serum antibodies ranging from 1 2399 to 1 45 408 were detected by ELISA m NZW rabbits Immunized with L(RLF) containing MPL 12 at one week after each boost (weeks 5 and 8) (Table 2) Immune serum from each

1344 Vacc ine , Vol 11, I ssue 13, 1993

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Ltposome-encapsulated malarta vaccme K Whtte et al

Reg I Reg II

~ / / / / / / / / / / / / / / / / / / / A W ~ / / / / / / / ~ l J ~ / / / / / / / / / / / / / A

I • ,..=,,.,,-,, ,,=, ,-,, ....... ...........

Figure 3 Epitope mapping of RLF antibodies Overlapping octamer peptldes on pros were prepared as descnbed 1'~ The figure shows a schematic representation of RLF including the locations of the conserved reg=ons I and II The h=stogram below indicates the ELISA reactivity (A.I.) of rabbut RLF antisera for each of the overlapping octamers Each vert=cal bar represents the mean of tnphcate readings for a single pin The numbers above the peaks identdy the amino acids included on the pros hawng greatest reactwlty

rabbit recognized intact P falctparum sporozoltes by immunofluorescence (Table 2) and inhibited tn vttro sporozolte invasion ofhepatoma cells by up to 58% The effect of hposomes containing MPL is multifactorlal and may include efficient phagocytosis of hposomes by antigen-presenting macrophages 29 and subsequent MHC class II-dlrected processing and presentation to T-helper (Th) cells, or preferential handling of hposome- encapsulated antigens by B cells, a mechanism that is considered to be associated with the induction of Th2 cells participating in secondary IgG responses 33

B-cell epltope mapping using P falctparum CS protein overlapping octapeptides 14 revealed seven discrete regions recognized by antI-RLF antibodies (Floure 3), four of which (aa residues 84-94, 91-99, 97-106, 367-375) have not previously been Identified One of the regions (a a residues 367-375), overlaps with the CTL epitope (a a residues 368-390) and the clustering of si tes between (a a residues 84-106) suggests the presence of a configuratlonal B-cell epltope Whether this B-cell site is a successor to the lmmunodominant central repeat region is unknown, nonetheless, its presence implies a nelghbourlng Th site, the identification of which may be crucial for an effective sporozoite-stage vaccine inducing cognate B-Th interactions Two of the three C terminus epltopes (a a residues 298-309, 307-315) and one N terminus epitope (a a residue 113-122) localized within region I, a proposed hgand for sporozoite binding to hepatocytes 34'35, have previously been identified

Because hposome-formulated vaccines induce excellent

antibody and T-cell responses and have been well tolerated in humans 36, this approach may be broadly apphcable for other viral and parasitic diseases in which vaccine efficacy may depend upon induction of both antibody and CTL Llposomes offer the further advantage of being capable of incorporating discontinu- ous vaccine determinants with a variety of functions, making the construction of a multistage subunlt malaria vaccine a practical consideration

A C K N O W L E D G E M E N T S

The authors are grateful to Dr M Carter, Ms C Holland and Mr M Sequin for their contributions to various aspects of this project and to Dr M Rao and Ms H Link for critically reading the manuscript The Investigators adhered to the 'Guide for the Care and Use of Laboratory Animals', as promulgated by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council The views of the authors do not purport to reflect the position of the Department of the Army or the Department of Defense

R E F E R E N C E S

Clyde, DF, McCarthy, V C, Miller, R M and Woodward, WE Charactermtlcs of Plasmodlurn falclparum from the Solomon Islands J Trop Med Hyg 1974, 77, 9-12

Vacc)ne, Vol 11, Issue 13, 1993 1345

Liposome-encapsula ted malar ia vaccine K White et al

2 Potocnjak, P, Yoshida, P and Nussenzwelg, V Monovalent fragments (Fab) of monoclonal antibodies to a sporozolte surface antigen (Pb44) protect mice against malarial refection J Exp Med 1980, 151, 1504-1513

3 Egan, J, Weber, J L, Ballou, W R, Holhngdale, M R, Majanan, W R, Gordon, D M e t al Efficacy of murme malaria sporozoite vaccines =mphcations for human vaccine development Science 1987, 336, 435-437

4 Romero, P, Maryanskl, J L, Corradm, G, Nussenzwelg, R S and Nussenzwelg, V Cloned cytotoxlc T cells recognize an epltope in the c~rcumsporozo~te protein and protect against malaria Nature 1989, 341,323-325

5 Dame, J B, Wdhams, T F, McCutchan, T F, Weber, J L, Wlrtz, R A, Hockmeyer, W T et al Structure of the gene encoding the ~mmunodommant surface antigen on the sporozolte of the human malana parasite Plasmodlum falclparum Science 1985, 225, 593-597

6 Kumar, S, Miller, L H, Quaky=, I A, Ke=ster, D B, Houghten, R A, Malloy, W L e t al Cytotoxlc T cells specific for the c~rcumsporozo=te protein of Plasmod~um falc~parum Nature 1988, 334, 268-365

7 Ballou, W R, Hoffman, S L, Sherwood, J A, Holhngdale, M R, Neva, FA , Hockmeyer, WT et al Safety and efficacy of a recombinant DNA Plasmodtum falctparum sporozo=te vaccine Lancet 1987, ,, 1277 1281

8 Herrington, D A, Clyde, D F, Losonsky, G, Cortes=a, M, Daws, J, Murphy, J R et al Safety and ~mmunogenic=ty m man of a synthetic pepbde malaria vaccine against Plasmod~um falctparum sporo- zo~tes Nature 1987, 320, 257-259

9 Sercarz, E E and Krzych, U The dlstmctwe specificity of antigen-specific suppressor T cells Immunol Today 1991,4, 111-114

10 R=chards, R L, Swartz, G M, Shultz, C, Hayre, M D, Ward, G S, Ballou, W R et al Immunogenicity of hposomal malaria sporozo=te antigen m monkeys adjuvant effects of aluminium hydroxide and non-pyrogenic hposomal hp~d A Vaccme 1989, 7, 506-512

11 Krzych, U, White, K, Link, HT, Sadoff, J, Lanar, D, Ballou, WR et al Role of c~rcumsporozo~te protein spemfic T cells m protectwe immumty against Plasmod~um falclparum Bull WHO 1990, 83(Suppl ), 68-93

12 Gordon, D M, Cosgriff, T M, Schneider, I, Wasserman, G F, Majarian, W R, Holhngdale, M R and Chulay, J D Safety and ~mmunogenic=ty of a Plasmod/um v/vax sporozo~te vaccine Am J o Trop Med Hyg 1990, 42, 527-531

13 Holhngdale, M R, Nardm, E H, Tharavanij, S, Swarlz, A L and Nussenzweig, R S Inhibit=on of entry of Plasmod~um falc~parum and Plasmod~um wvax sporozo=tes into cultured cells an m vitro assay of protectwe antibodies J Immunol 1984, 132, 909-913

14 Geysen, H M, Rodda, S J, Tr=bb=ck, G and Schoofs, P G Strategies for ep~tope analys~s using peptlde synthesis J Immunol Methods 1987, 102, 259-274

15 Good, M F, Pombo, D, Quaky=, IS , Rdey, E M, Houghten, RA , Menon, A et al Human T-cell recogmtlon of the clrcumsporozoite protein of Plasmod~um falclparum Immunodommant T-cell domains map to the polymorphlc regions of the molecule Proc NaU Acad Sc~ USA 1988, 85, 1199-1203

16 Mahk, A, Egan, J, Houghten, R A, Sadoff, J C and Hoffman, S L Human cytolytic T lymphocytes against Plasmodlum falc~parum c~rcumsporozolte protein Proc Natl Acad Sc~ USA 1991, 88, 3300 3304

17 Aggarwal, A, Kumar, S, Jaffe, R, Hone, D, Gross, M and Sadoff, J Salmonella-malaria c~rcumsporozo=te recombinant vaccines reduce specific CD8 + cytotoxlc T cells J Exp Med 1990, 172, 1083 1090

18 Hackett, C J, Taylor, P M and Askonas, B Stimulation of cytotox~c

T cells by hposomes containing influenza virus or ~ts components Immunology 1983, 49, 255-259

19 Loh, D, Ross, A H , Hale, AH , Baltimore, D and Eisen, H N Synthetic phosphohpld vesicles containing a purified viral antigen and cell membrane proteins stimulate the development of cytotoxlc T lymphocytes J Exp Med 1979, 150, 1067-1074

20 Lawman, M J P, Naylor, P T, Huang, L, Courtney, R J and Rouse, B T Cell-mediated immunity to Herpes simplex virus Induction of cytotoxlc T lymphocyte responses by viral antigens incorporated into hposomes J Immunol 1981, 126, 304-308

21 Duprez, V, Mescher, M F and Burakoff, S J Stimulation of secondary anti-MSV cytolytic T lymphocytes with MBL-2 reconsti- tuted membranes J Immunol 1983, 150, 493-495

22 Reddy, R, Zhou, F, Nalr, S, Huang, L and Rouse, BT In vwo cytotoxlc T lymphocyte mduct~on with soluble proteins administered m hposomes J Immunol 1992, 148, 1585-1589

23 Lopes, L M and Chain, B M Liposome-med~ated deliverystamulates a class I-restricted cytotoxlc T cell response to soluble antigen Eur J Immunol 1992, 22, 287-290

24 Aichele, P, Hengartner, H, Zmkernagel, R M and Schultz, M Antw~ral cytotoxlc T cell response reduced by m wvo priming with a free synthebc pept~de J Exp Med 1990, 171, 1815 1820

25 Alwng, C, White, K, Cassatt, D, White, W I, Gordon, D M, Gross, M e t al Liposomes as careers of vaccines Induction of cytolytic T lymphocytes against malaria and HIV epltopes Presented at the Fifth Annual Meebng of the National Cooperatwe Vaccine Development Groups for AIDS, January 1993, Arhe, VA, USA

26 Germam, R The ms and outs of antigen processing Nature 1986, 322, 687-689

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32 Wassef, N M and Alvlng, C R Complement-dependent phagocytosms of hposomes by macrophages Methods Enzymol 1987, 149, 124-134

33 Garson, N M J and Six, H R Universal vaccine carrier hposomes that provide T-dependent help to weak antigens J Imrnunol 1991, 146, 3897-3702

34 Alley, S B, Bates, M D, Tam, J P and Holhngdale, M R Synthetic peptldes from the clrcumsporozoite proteins of Plasmodtum falctparum and Plasmodlum knowlesl recognize the human hepatoma cell hne HepG2-A16 m vitro J Exp Med 1986, 146, 1915-1922

35 Stuber, D, Bannwarth, W, Pink, J R L, Meloen, R H and Mat~le, H New B cell epltopes m the Plasmodlum falctparum malaria circumsporozolte protein Eur J Immunol 1990, 20, 819-824

36 Fries, L F, Gordon, D M, Richards, R L, Egan, J E, Holhngdale, M R, Gross, M et al Liposomal malaria vaccine m humans A novel, safe, and potent adjuvant strategy Proc Natl Acad Scl U S A 1992, 89, 358-362

1346 Vaccine, Vol 11, Issue 13, 1993