Immunogenicity of liposomal malaria sporozoite antigen in monkeys: adjuvant effects of aluminium hydroxide and non-pyrogenic liposomal lipid A

Papers Immunogenicity of liposomal malaria sporozoite antigen in monkeys: adjuvant effects of aluminium hydroxide and non-pyrogenic liposomal lipid A Roberta L. Richards*, Glenn M. Swartz, Jr*, Clyde Schultz**, Michael D. Hayre *§, George S. Ward ~, W. Ripley Ballout§, Jeffrey D. Chulay t, Wayne T. Hockmeyer t§, Sanford L. Berman** and Carl R. Alving* The immunogenicity of a recombinant protein (R32teta2) containing sequences from the tetrapeptide repeat region of the circumsporozoite protein of Plasmodiumfalciparumwas enhanced by encapsulation in liposomes containing lipid A and adsorption of the liposomes with alum. The toxicities and efficacies of preparations containing different types and doses of lipid A were assessed by studying pyrogenicity in rabbits and adjuvanticity in monkeys. In each case liposomal lipid A was 25-fold to 200-fold less pyrogenic than free lipid A. Monophosphoryl lipid A, whether free or in liposomes, was the least pyrogenic of the three lipid A preparations tested. High antibody levels were obtained after immunization of rhesus monkeys with a formulation consisting of alum-adsorbed liposomes in which the liposomes contained R3 2tet j z and a strongly pyrogenic dose of native lipid A. Excellent antibody levels were also observed in monkeys immunized with a combination of R32tet j 2 encapsulated in alum-adsorbed liposomes containing non-pyrogenic doses of monophosphoryi lipid A and alum. The adjuvant effect was related to the dose of the lipid A in the liposomes, and the adjuvant effect was still strongly expressed despite suppression of the pyrogenic effect of lipid A. Antibody levels were considerably lower in monkeys immunized with liposomes lacking lipid A. It was concluded that a non-pyrogenic formulation of alum-adsorbed liposomes, in which the liposomes contained both lipid A and an encapsulated synthetic sporozoite antigen, shows considerable promise for inducing high titres of antibodies to sporozoites.

Keywords:Malaria sporozoite vaccine;liposomal; adjuvant effect Introduction Identification of the complete structure of the gene encoding the circumsporozoite (CS) protein of Plasmodium falciparum has provided an opportunity for utilizing synthetic antigens as the basis for vaccines against malaria 1. Two types of sporozoite antigens are being explored, namely synthetic peptide-carrier protein conjugates 2'3, and recombinant expression proteins ~6. A recombinant protein (R32tet32) that contains sequences from the repeat region of the CS protein of P. falciparum in combination with aluminium hydroxide (alum) has undergone phase I testing in humans 7. Although slow titres of antibodies were obtained in humans, the feasibility of the approach was demonstrated in that one of two individuals given the highest dose of antigen was protected against sporozoite challenge. Similar results Departments of *Membrane Biochemistry, **Biologics Research, and qmmunology, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA and ~Department of Animal Resources, Naval Aerospace Medical Research Laboratory, Pensacola, Florida 32508, USA. §Present addresses: Michael D. Hayre, Rockefeller University, Laboratory Animal Research Center, New York City, New York 10021; W. Ripley Ballou, Univax Corporation, Rockville, Maryland 20852; Wayne T. Hockmeyer, Molecular Vaccine, Inc., Gaithersburg, Maryland 20878, USA. (Received 25 January 1989; accepted 6 April 1989) 0264~410X/89/060506--o7$03.00 © 1989Butterworth & Co. (Publishers) Ltd 506 Vaccine, Vol. 7, December 1989

were found in an independent trial with a CS peptidetetanus toxoid conjugate s. It is widely believed that synthetic antigens may require the use of adjuvants to achieve optimal immunization 9. Adjuvants that have shown promise include liposomes and liposomes containing lipid A 1°-2°. It had been determined previously that the immunogenicity of R32tet32 in rabbits and monkeys was enhanced by encapsulation of the antigen in liposomes, especially when the liposomes contained lipid A, and by adsorbing the liposomes with alum as an adjuvant ~5. Although lipid A has previously been used a s a n adjuvant for a variety of liposome-associated protein antigens, it also expresses toxicity in the form of pyrogenicity. In the present study a markedly reduced pyrogenicity of lipid A when it is present in liposomes has been observed and the abilities of native lipid A and a 'non-toxic' lipid A fraction (monophosphoryl lipid A) to act as adjuvants for liposome-associated R32teta2 in rhesus monkeys have been compared.

Materials and methods Materials Three types of lipid A were used in this study. Lipid A was prepared from Eseheric:~ia coli 0111 lipopolysaccharide (Difco Laboratories, Detroit, MI) as described previously 21. Salmonella minnesota R595 lipid A (List

Liposoma/ malaria sporozoite vaccine: R.L. Richards et al. Table 1

Pyrogenicity of free native lipid A compared with liposomal

lipid A Pyrogenicity a Liposomal lipid A epitope density (/~g lipid A #mol -~ DMPC)

Total lipid A injected (/~g kg -1)

Free lipid A

0.015 0.022 0.077 0.59 0.71 1.2 1.5 3.0

-- -+ -+ + + + + + +++ +++ +++

0.1

0.3

1.2

3.0

--+ + + +++ +++ +++

a + represents a temperature increase of 0.6°C or greater within 3 h after injection of test solution; -- represents a temperature change of < 0.6°C within 3h after injection of test solution. Each -- or ÷ represents an individual rabbit. Liposomes lacking lipid A had no detectable pyrogenicity at the highest doses tested

Biological Laboratories, Inc., Campbell, CA, USA). Monophosphoryl lipid A (MP lipid A), a monophosphoryl fraction isolated from S. minnesota R595 lipid A, was kindly donated by Dr J.A. Rudbach (Ribi ImmunoChem Research, Inc., Hamilton, MT, USA). Lipids for preparation of liposomes were purchased from the following sources: dimyristoyl phosphatidylcholine from Sigma Chemical Co. (St. Louis, MO, USA), liposomes used to obtain the data in Table 1, or Avanti Polar Lipids, Inc. (Birmingham, AL, USA) for all other liposomes; cholesterol from Calbiochem/Behring (La Jolla, CA, USA); dicetyl phosphate from K and K Laboratories (Plainview, NY, USA) and dimyristoyl phosphatidylglycerol from Avanti Polar Lipids. Aluminium hydroxide absorptive gel was used as the alum source. A synthetic protein (R32tet32) containing 32 tetrapeptide repeats from the CS protein of P. falciparum and 32 amino acids derived from the tetracycline resistance gene of the cloning v e c t o r 4 ' 2 2 w a s used as antigen in the studies reported here. This is the antigen that was employed in a recent clinical trial ~.

1.0:0.75:0.11, and lipid A at the epitope densities indicated in Table I. The liposomes used to obtain the data in Table 2 were composed of dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol and cholesterol in molar ratios of 0.9:0.1:0.75, and lipid A at the epitope densities (in #g per/~mol of phospholipid) indicated in the legend to Table 2. For each of the three lipid A preparations in Table 2 the indicated lipid A weight corresponded to approximately 20 nmol of lipid A phosphate per /~mol of phospholipid. This lipid A phosphate concentration was selected because it was the amount used in our previous studies 14'~5. For comparison between different sources of lipid A, it should be pointed out that different lipid A preparations have different weight-to-phosphate ratios because of differing relative proportions of diphosphoryl and monophosphoryl components.

Pyrogenicity testing Pyrogenicity testing was performed in rabbits as previously described 23. After intravenous injection of each sample (1 ml kg- 1 body weight), the body temperature of each rabbit was recorded at 0 h and every hour thereafter for 3 h following injection into the marginal ear veins of three rabbits. Pyrogenicity was defined as a rise in temperature of ~>0.6°C per rabbit, or a total rise of 1.5°C for the three rabbits tested.

Preparation of liposomes for immunization All liposome preparations used for immunizations were prepared under a protocol that was in accordance with Good Manufacturing Practices standards as promulgated by the US Food and Drug Administration (FDA). The exact details of this procedure will be presented to the FDA in a drug master file. However, from an experimental standpoint the method for preparing liposomes was essentially the same as previously described 15,21. The liposomes were composed of dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol and cholesterol in molar ratios of 0.9:0.1:0.75. Where indicated, lipid A from S. minnesota R595 (native lipid A) was included in the liposomes at 0.12 #g or 12/~g lipid A/#mol liposomal phospholipid, and MP lipid A was included in the liposomes at 0.8/~g lipid A//~mol liposomal phospholipid

Preparation of lipid A and iiposomes for pyrogenicity testing Solutions of free lipid A were prepared by drying aliquots of lipid A in chloroform under a stream of nitrogen and solubilizing the dried lipid A in 0.25-0.5 ml 0.5% triethylamine. The solubilized lipid A was then diluted to the desired concentration with 0.15M NaC1. All glassware used was heated for > 3 h at 180°C to ensure sterility and to remove pyrogens. Liposomes (multilamellar vesicles) were prepared as previously described 2~. Lipids were dried under vacuum from chloroform solutions in pear-shaped flasks. After addition of a small quantity of acid-washed 0.5 mm glass beads, the liposomes were swollen by vigorous vortexing for about 2min in 0.15M NaC1 to give a final phospholipid concentration of 10mu. The 10mM liposomes were diluted with 0.15 M NaCl to give the desired lipid A concentration. The liposomes used to obtain the data in Table I were composed of dimyristoyl phosphatidylcholine, cholesterol, and dicetyl phosphate in molar ratios of

Table 2

Effect on pyrogenicity of incorporating three different lipid A samples in liposomes Maximum non-pyrogenic dose a Decrease in

Lipid A

Free

Liposomal

pyrogenicity b

E. coil 0111 S. minnesota R595 MP lipid A c

0.015 0.008 0.32

0.59 1.7 8.1

40-told 214-fold 25-fold

aMaximum lipid A dose in #gkg -1 that did not cause an increase in temperature of 0.6°C or greater in any of the three rabbits over the 3 h test period. The different doses of liposomal lipid A were obtained by diluting liposomes at 10 mM phospholipid (20 nmol lipid A phosphate per #mol phospholipid) with 0.15M NaCI. The liposomes contained 12#g E. coil 0111 lipid A, 22#g S. minnesota R595 lipid A, or 32#g MP lipid A per #tool phospholipid, bThe decrease in pyrogenicity due to incorporation of the lipid A into liposomes was determined by dividing the maximum non-pyrogenic dose of liposomal lipid A by the maximum non-pyrogenic dose of free lipid A. ~MP lipid A is a monophosphoryl lipid A traction derived from S. minnesota R595 lipid A.

Vaccine, Vol. 7, D e c e m b e r 1989

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Lipoeomal malaria sporozoite vaccine: R.L. Richards et al.

for the experiment shown in Figures I-4 and at 0.8,4.8 or 32/zg lipid A/#mol liposomal phospholipid for the experiment shown in Figure 5. For comparison it should be noted that the 12 #g of native lipid A/#mol phospholipid and the 32/zg MP lipid A each corresponded to 20 nmol lipid A phosphate/#mol phospholipid. Lipids were dried from chloroform solutions on a rotary evaporator and then dried further in a desiccator under high vacuum. The dried lipids were hydrated at approximately 30mr,l phospholipid in sterile pyrogenfree water by shaking until all the lipid film was off the glass. The aqueous liposomes were lyophilized, and sealed under vacuum. The lyophilized liposomes were reconstituted with antigen (R32tet32) in Dulbecco's phosphate-buffered saline (DPBS) lacking CaCI 2 and MgC12-6H20 (GIBCO Laboratories, Grand Island, NY) at 100 mr,l phospholipid. After dilution with DPBS (c. 20-fold), the antigen-containing liposomes were washed by centrifugation at 29 000g. After aspiration of the supernatants, the liposomal pellets were resuspended to c. 40mM phospholipid in DPBS and the amount of R32tet32 encapsulation was determined by a modified Lowry protein assay 24. The liposomes were then diluted with DPBS to 30#g R32tet32 per 0.5ml for the experiment shown in Figures I-4 or 80/~g R32tet32 per 0.5 ml for the experiment shown in Figure 5 and either bottled immediately or incubated with alum (final concentration 1.0mgml-1 aluminium) for 1 h at room temperature before bottling in vaccine vials (3 ml per bottle) and storing at 4°C until further use.

aspirated, the wells were filled with blocking buffer (1.0% BSA, 0.5% casein, 0.01% thimerosal and 0.005% phenol red in PBS) and held for 1 h at room temperature. Monkey sera to be tested were diluted in blocking buffer and aliquots of each dilution added to triplicate wells. After a 2 h incubation at room temperature the contents of the wells were aspirated, the wells were washed three times with PBS-Tween 20, and 50/~g horseradish peroxidase-conjugated goat anti-human IgG (Bio-Rad Laboratories, Richmond, CA) diluted 1/500 with 10% heat-inactivated human serum in PBS were added to each well. After 1 h, the contents of the wells were aspirated, 3-

a

.,,<

°b

2

4

6

0-

_ 2

. 4

I 6

i_

o

Immunization procedures In the first series of immunizations, rhesus monkeys (four animals per group) were immunized intramuscularly at 0 and 4 weeks with 0.5ml formulations containing 30#g doses of R32tet32 either adsorbed with alum (FSV-1; Ref. 7), encapsulated in liposomes lacking lipid A [L(Ag)], adsorbed with alum after encapsulation in liposomes lacking lipid A [L(Ag) + Alum], adsorbed with alum after encapsulation in liposomes containing native lipid A [0.5 #g kg- 1 monkey body weight; L(Ag + Lipid A-0.5) + Alum], adsorbed with alum after encapsulation in liposomes containing native lipid A [44 #g/kg monkey body weight; L(Ag+Lipid A-44)+Alum], or adsorbed with alum after encapsulation in liposomes containing MP lipid A [4/~g/kg monkey body weight; L ( A g + M P Lipid A-4)+ Alum]. The weights of lipid A/kg monkey body weight are estimates based on an approximate weight of 8 kg/monkey- 1 In a second series of immunizations (those used in Figure 5) rhesus monkeys (four animals per group) were immunized with 0.5ml formulations containing 80#g doses of R32tet32 either adsorbed with alum [FSV-1], or adsorbed with alum after encapsulation in liposomes containing either 0.6/~g, 3/~g or 24/~g MP lipid A per kg monkey body weight [L(Ag + MP lipid A-0.6) + alum, or L ( A g + M P lipid A-3)+alum, or L ( A g + M P lipid A-24) + alum].

Time

Enzyme-linked immunosorbent assays were carried out as described previously4. Wells of polystyrene microtitre plates were each'coated with 0.1/zg R32tet32 in 0.01M phosphate buffered saline, pH 7.4 (PBS). Approximately 18 h later the contents of the wells were

508

Vaccine, Vol. 7, December 1989

primary

immunization

(weeks)

Rgure 1 Time course of the immune response to liposomal vaccines in monkeys. Each point represents the mean ELISA activity for four monkeys, after subtraction of the pre-immunization values, at a serum dilution of 1:50 (a) or 1:200 (b). Each monkey was immunized at 0 and 4 weeks with 30/zg R32tet~ either: /k, adsorbed with alum, [FSV-1]; A , encapsulated in liposomes lacking lipid A, [L(Ag)]; I-3, encapsulated in liposomes lacking lipid A and then adsorbed with alum, [L(Ag)+Alum]; . , encapsulated in liposomes containing a non-pyrogenic dose of native lipid A (0.5/zgkg -1) and then adsorbed with alum, [L(Ag+Lipid A-0.5)+Alum]; O, encapsulated in liposomes containing a nonpyrogenic dose of monophosphoryl lipid A (4 #g kg -1) and then adsorbed with alum, [L(Ag + MP Lipid A-4)+ Alum]; O, encapsulated in liposomes containing a pyrogenic dose of native lipid A (44/zgkg -1) and then adsorbed with alum, [L(Ag + Lipid A-44) + Alum]

2400. 2000. 1600. cD <

1200.

--

800.

400" ~

Enzyme-linked immunosorbent assays ( ELISA )

after

0°

a

e

i Time

~ after

~ primary

;4

~

immunization

6

~

~8

(weeks)

Rgure 2 Time course of the immune response to liposomal vaccines in monkeys expressed as ELISA units. Each point represents the mean of the ELISA units for four monkeys after subtraction of the preimmunization values. Each monkey was immunized as described in Figure 1. Symbols see Figure 1

Liposoma/ malaria sporozoite vaccine: R.L. Richards et al.

Monophosphoryl lipid A, either free or in liposomes, was the least pyrogenic lipid A of the three preparations tested.

e-

~" 2

Adjuvanticity o f liposome formulations containing or lacking lipid A

e-

<

0 50

100

200

400

800

1600

3200

Reciprocal of serum dilution Rgure 3 Immune response to liposomal R32tet= in monkeys at 6 weeks. Each point represents the mean ELISA activity at 6 weeks after the primary immunization for four monkeys after subtraction of the preimmunization values. Each monkey was immunized as described in Figure 1. Symbols see Figure 1

the wells were washed three times with PBS-Tween 20, and 150 #1 peroxidase substrate in buffer was then added to each well. ELISA activity was measured as absorbance at 414 nm 1 h after addition of peroxidase substrate using an ELISA plate reader (Titertek Multiskan, Flow Laboratories, Inc., McLean, VA). ELISA units were calculated by multiplying the absorbance at 414 nm at a given dilution by the reciprocal of the dilution. When the data permitted, the mean of the ELISA units obtained from three points on the linear portion of the absorbance vs. dilution curve were used.

Results

Pyrogenicity of liposomal lipid A Table 1 shows the results obtained when the pyrogenicity of liposomal lipid A was compared with that of free lipid A. On the basis of calculations from Table 1, the pyrogenicity of lipid A was reduced in the range of 39-fold to 48-fold by incorporation into liposomes (calculated by comparing the highest non-pyrogenic doses for liposomal lipid A, 0.59-0.71/~g kg-1, with the highest non-pyrogenic dose for free lipid A, 0.015 #g kg- 1). Testing of liposomes containing different amounts of incorporated lipid A revealed that, at the doses used, the epitope density of lipid A in the liposomes had no apparent effect on the pyrogenicity. At total lipid A doses of 0.59-0.71#g kg -t none of the four lipid A epitope densities tested was pyrogenic. At a total lipid A dose of 1.2-3.0 #g kg- 1 each of the three lipid A epitope densities tested was pyrogenic. It was concluded that pyrogenicity was dependent only on the total dose of liposomal lipid A injected rather than on the epitope density of liposomal lipid A. The pyrogenicity of liposomal lipid A was determined for three different lipid A preparations (Table 2). For testing pyrogenicity each of the three lipid A samples, each having a different phosphate:weight ratio, was tested at a molar epitope density of 20nmol lipid A phosphate/#mol liposomal phospholipid. This lipid A concentration in liposomes was chosen because we previously found this epitope density to have good adjuvant activity t*'16'19'25. For all three lipid A samples tested, liposomal lipid A was considerably less pyrogenic (25-fold to 214-fold) than free lipid A (Table 2).

The adjuvant activity of different doses of lipid A in liposomes was investigated by immunizing rhesus monkeys with a synthetic malaria sporozoite antigen, R32tet32, in combination with liposomes containing different concentrations and types of lipid A, The time course of the immune response determined by ELISA is shown in Fi#ure 1 at serum dilutions of 1:50 (Figure la) and 1:200 (Figure lb). Monkeys immunized with liposomes containing the highest dose of native lipid A [L(Ag+Lipid A-44)+Alum] had the highest antibody activities. The second highest activities were obtained when the monkeys were immunized with liposomes containing an intermediate dose of MP, lipid A I-L(Ag + MP Lipid A-4) + Alum]. All five of the liposomal immunogens gave higher mean antibody levels than were obtained with the alum-adsorbed R32tet32 EFSV-1]. Figure 2 shows the same experiment illustrated in Figure I, but the data are expressed as ELISA units. This method of representing the data allows comparison between ELISA experiments performed on different days. When plotted on a linear scale, as in Fi#ure 2, the potent activity of the highest dose of lipid A in liposomes is readily obvious. The average titres of the individual groups, as determined by dilution of serum obtained at 6 weeks after primary immunization, are illustrated in Figure 3. The titres confirm the relative ranking of immunogenic potencies of the groups shown in Figures 1 and 2. Even at a dilution of 3200-fold, sera from the group immunized with [L(Ag + MP lipid A-44) + alum] still had detectable activity.

6000 r 5000 F 4000 V 3000

2o j, e-

<

600 500

u.l

gO0 3O0 20O 100 0

FSVlAg

L(Ag)+alum

L { A g + l i p i d A-0.5)+alum

I I

L(Ag + MP lipid A - 4 ) + a l u m L(Ag + lipid A-44) +alun Figure 4 Immune response of individual monkeys to liposomal R32tet~ at 6 weeks after the primary immunization. Each bar represents the ELISA units 6 weeks after the primary immunization for an individual monkey after subtraction of the pre-immunization values. Each monkey was immunized as described in Figure 1

Vaccine, Vol. 7, December 1989

509

Liposomal malaria sporozoite vaccine: R.L. Richards

et al.

700-

< "-i iii

Discussion

/\

600

400300" 20O

I

2

i

4

I

6

T i m e after primary

I

8

1 ~0

I

12

I

14

16

immunization (weeks}

Figure 5 Time course of the immune response to liposomal vaccines containing different doses of monophosphoryl lipid A in monkeys expressed as ELISA units. Each point represents the mean of the ELISA units for four monkeys after subtraction of the pre-immunization values. Each monkey was immunized at 0, 4, and 8 weeks with 80#g R32te%a either: 17, adsorbed with alum [FSV-1]; II, encapsulated in liposomes containing a 0.6/tg kg -1 dose of MP lipid A and then adsorbed with alum, [L(Ag + MP lipid-A 0.6) + alum]; O, encapsulated in liposomes containing a 3 #g kg-1 dose of MP lipid A and then adsorbed with alum [L(Ag + MP Lipid A-3) +alum]; 0 , encapsulated in liposomes containing a 24#g kg -1 dose of MP lipid A and then adsorbed with alum [L(Ag+MP lipid A-24) + alum]

Analysis o f individual

monkeys

The antibody responses of individual monkeys at 6 weeks after the primary immunization is shown in Figure 4. For the group receiving liposomes having the highest dose of native lipid A [L(Ag + Lipid A-44) + Alum] and for the group receiving liposomes having a nonpyrogenic dose of MP lipid A [ L ( A g + M P Lipid A-4)+Alum], all four monkeys gave high levels of antibody and the mean ELISA responses of these two groups were significantly higher than the mean ELISA response of the monkeys immunized with FSV-1. Although the monkeys receiving the highest dose of native lipid A gave the highest responses, the monkeys receiving a lower dose of MP lipid A still showed a stronger response than any of the remaining four groups.

Effects of different doses of M P lipid A Monkeys (four animals per group) were immunized with liposomes containing one of three different doses of MP lipid A (0.6/tgkg -1, 3#gkg -~, or 24pgkg -1) (Figure 5). The dose of antigen (R32tet31) given to each animal, 80#g, represented a 2.7-fold increase over that used in Figures I-4. The higher dose of antigen was employed to correspond more closely on an antigenweight: body-weight basis (10-12 pg kg- 1) with the highest dose of R32tet32 used previously in humans 7. At this higher dose of antigen the FSV-1 gave detectable antibody titres and the antibody activity was equivalent to that obtained with lipsomes having the lower levels of MP lipid A. However, the most potent formulation consisted of liposomes that contained the highest dose of MP lipid A. The highest dose of MP lipid A in Figure 5 (24 pg kg- 1) was intermediate between the highest dose of native lipid A (44pgkg -~) and the dose of MP lipid A (4#gkg -~) used in Figures 1-4. The impression from this comparison is that the intermediate immune response obtained in Figure 5 indicates that MP lipid A still retains much, and perhaps even most, of the adjuvant activity of native lipid A despite the loss of pyrogenic activity.

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Vaccine, Vol. 7, December 1989

In order for any liposomal preparation to be used as a carrier for a malaria vaccine it must be both safe and efficacious. Among the ingredients in the present formulation, lipid A (also known as endotoxin) has been associated with pyrogenicity, and this property might potentially affect the practical acceptance of lipid A as a therapeutic agent. Although many widely used vaccines manufactured from natural products have varying degrees of pyrogenicity, a pyrogenic response would be considered to be an unattractive property for a synthetic liposomal vaccine containing a genetically engineered antigen. The goal of the present study was to determine whether it would be possible to construct an efficacious synthetic liposomal malaria sporozoite vaccine containing lipid A which also would not be pyrogenic. The major focus of the experiments was to compare both the pyrogenicity and adjuvanticity of free and liposomeassociated lipid A. The data show that the pyrogenicity of lipid A was markedly reduced (25-fold to 214-fold) "upon incorporation into liposomes, and pyrogenicity of liposomal lipid A was reduced further (perhaps as much as eightfold) by adsorbing the liposomes with alum. The pyrogenicity of liposomal lipid A apparently was dependent only on the total dose of lipid A administered, as varying the epitope density of lipid A in the liposomes had no effect on the pyrogenicity. It is well known that certain of the biological effects of lipid A are greatly reduced by incorporation of lipid A into liposomes. Incorporation of lipid A into liposomes suppresses neutropenia 26, Limulus lysate activity 22'27'18, lipid A-induced secretion of interleukin-1 by macrophages/s, macrophage-mediated tumour cytotoxicity 29, and tumour necrosis factor secretion 29. The conclusion that liposomes reduce the pyrogenicity of lipid A is therefore compatible with the concept that liposomes greatly reduce the expression of important toxic biological activities of lipid A. MP lipid A is a monophosphoryl lipid A product that has reduced pyrogenic activity, and has successfully completed phase I testing for parenteral use in humans 3°. The data suggest that of the lipid A samples tested, MP lipid A is the least pyrogenic form of lipid A, and the pyrogenic toxicity of MP lipid A is reduced still further by incorporation into liposomes. The mechanism by which the pyrogenicity of lipid A is reduced by incorporation into liposomes is not yet completely known. It has been reported that increased uptake of endotoxin by Kupffer cells occurs in animals made tolerant to the pyrogenic effects of endotoxin 31. In conjunction with increased uptake of endotoxin there also was decreased release of endogenous pyrogen by the Kupffer cells. In fact, the lack of pyrogenicity of endotoxin in tolerant animals appeared to be due almost entirely to the increased uptake of endotoxin and to the decreased release of endogenous pyrogen by the Kupffer cells. This was supported by the observation that other endogenous pyrogen-producing cells (such as blood leucocytes and lung macrophages) from tolerant animals, when tested in vitro, were able to release the same levels of endogenous pyrogen as cells from normally responsive animals 31. A large percentage of circulating liposomes are taken up rapidly by reticuloendothelial cells, particularly Kupffer cells 32. The reduced pyrogenicity of lipid

Liposomal malaria sporozoite vaccine: R.L. Richards et al.

A when incorporated into liposomes thus may occur by a mechanism similar to that proposed for endotoxin tolerance because of the rapid uptake of the liposomes by the Kupffer cells. It has also been reported that the phosphates attached to lipid A may be important in determining the bioactivity of lipid A 33"34. Murine macrophages dephosphorylate the lipid A portion of E. coli lipopolysaccharide 35, and dephosphorylation of the lipid A therefore may be a contributing factor in the reduced pyrogenicity of liposomal lipid A. In the present study we have also compared the adjuvanticity of pyrogenic vs non-pyrogenic doses of lipid A. As expected from previous experiments 15, high levels of antibodies against the tetrapeptide repeat portion of R32tet32 were obtained in monkeys immunized with the liposomal antigen containing the strongly pyrogenic dose of native lipid A (see Figures 2 and 3). Unfortunately, the adjuvanticity of native lipid A in liposomes was substantially reduced when a non-pyrogenic dose was used (see Figure I). A non-pyrogenic dose of liposomal MP lipid A, however, showed considerable adjuvanticity, resulting in much higher antibody levels than obtained with the non-pyrogenic dose of native lipid A in liposomes (see Figures 2, 3, and 5). We conclude that a non-pyrogenic sporozoite malaria vaccine can be made composed of alum-adsorbed liposomes containing monophosphoryl lipid A and encapsulated R32tet32. It is expected that such a vaccine could be useful in humans.

7

8

9 10

11

12

13

14

15

16

Acknowledgements The authors are grateful to Dr J.A. Rudbach, Ribi ImmunoChem Research, Inc., Hamilton, Montana, for providing the monophosphoryl lipid A and to Smith, Kline & French Laboratories, Swedelande, Pennsylvania for providing the R32tet3z used in this study, and also to Mr Rufus Gore for expert technical assistance with the ELISA, and to Ms Mazer Sessoms for assistance in preparation of the manuscript.

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References 1

2

3

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5

6

Dame, J.B., Williams, J.L., McCutchan, T.F., Weber, J.L., Wirtz, R.A., Hockmeyer, W.T. et al. Structure of the gene encoding the immunodominant surface antigen on the sporozoite of the human malaria parasite Plasmodium falciparum. Science 1984, 225, 593-599 Ballou, W.R., Rothbard, J., Wirtz, R.A., Gordon, D.M., Williams, J.S., Gore, R., Schneider, I. et a/. Immunogenicity of synthetic peptides from circumsporozoite protein of P/asmodium fa/ciparum. Science 1985, 228, 996-999 Zavala, F., Tam, J.P., Hollingdale, M.R., Cochran, A.H., Quakyi, I., Nussenzweig, R.S. and Nussenzweig, V. Rationale for development of a synthetic vaccine against P/asmodium fa/ciparum malaria. Science 1985, 228, 1436-1440 Young, J.F., Hockmeyer, W.T., Gross, M., Ballou, W.R., Wirtz, R.A., Trosper, J.H. et al. Expression of Plasmodium falciparum circumsporozoite proteins in Escherichia coil for potential use in a human malaria vaccine. Science 1985, 228, 958-982 Mazier, D., Mellouk, S., Beaudoin, R.L., Texier, B., Druilhe, P., Hockmeyer, W. et al. Effect of antibodies to recombinant and synthetic peptides on P. falciparum sporozoites in vitro. Science 1986, 231, 156-159 Egan, J.E., Weber, J.L., Ballou, W.R., Hollingdale, M.R., Majarian,

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