Liposomes Containing Lipid A Serve as an Adjuvant for Induction of Antibody and Cytotoxic T-Cell Responses against RTS,S Malaria Antigen

Liposome components.

Dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, and cholesterol used to prepare liposomes were obtained from Avanti Polar Lipids, Alabaster, Ala. MPL was from Ribi ImmunoChem, Hamilton, Mont.

Antigens.

RTS,S is a yeast-expressed HBsAg that has coexpressed epitopes from the CSP of P. falciparum 7G8 (16). The CSP epitopes contain amino acids 210 to 398 of the C terminus of the CSP, which includes 19 NANP repeats and amino acids 367 to 390, which have been identified as a CSP T-cell epitope (22). RTS,S exists as a lipid-containing particle, since it contains lipids (primarily phospholipid) in addition to protein. RTS,S was kindly provided by SmithKline Beecham Biologicals, Rixensart, Belgium. RTS,S was conjugated with Texas red (TR) (Molecular Probes, Inc., Eugene, Oreg.) according to the procedure of Titus et al. (39).

The peptide containing the 367-to-390 CTL epitope was synthesized by SynPep Corporation, Dublin, Calif.

Antibodies.

Hybridomas producing monoclonal antibodies against CD8 (8312.5) and CD4 (2RL) were kindly provided by R. Hodes, National Institutes of Health, Bethesda, Md. Hybridomas HB 20 (k^kD^k) and HB 6 (I-E^k) were obtained from the American Type Culture Collection, Rockville, Md.

Mice.

B10.Br (H-2^k) mice were purchased from Jackson Laboratories, Bar Harbor, Maine. Each group of three or five mice was housed in an individual cage and given water and food ad libitum. Care and handling of the mice were conducted according to the principles set forth in the Animal Welfare Act of 1985 and reference 19a.

Preparation of liposomes.

Liposomes were prepared essentially as described previously (5, 43). The liposomes were composed of dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, and cholesterol in molar ratios of 0.9:0.1:0.75 and, where indicated, MPL. Lipids were dried from chloroform solution as a thin film under vacuum, suspended in water, aliquoted into vaccine vials, and lyophilized. For the immunizations, the lyophilized liposomes were reconstituted with RTS,S, diluted with buffer, and centrifuged to remove RTS,S not associated with the liposomes. Since unencapsulated RTS,S was recovered in the supernatant fraction after centrifugation, the centrifugation conditions used did not cause free RTS,S to pellet. The washed liposomes were aliquoted into vaccine vials and stored at 2 to 6°C until they were used for immunization. For the trafficking experiments, lyophilized liposomes either containing or lacking MPL were reconstituted with TR-labeled RTS,S (TR-RTS,S).

APCs.

Bone marrow stem cells that were cultured under conditions promoting only the growth of macrophages (BMs) were used as the APCs for the intracellular trafficking experiments. Marrows from the femurs of 8- to 10-week-old B10.Br mice were isolated as described previously (32, 41), and cells were seeded at a density of 2 × 10⁵ on acid-washed circular glass coverslips (no. 1; VWR Scientific, West Chester, Pa.) in macrophage growth medium (RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 10% L-929 cell-conditioned medium, 8 mM l-glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin/ml [all from Gibco-BRL, Life Technologies, Grand Island, N.Y.]). On day 9, the macrophage cultures were supplemented with 10 U of murine gamma interferon (Gibco-BRL) per ml, and they were used as APCs the next day.

Immunofluorescence.

BMs grown on coverslips as described above were washed in Hanks balanced salt solution without phenol red (HBSS), pH 7.4, and then incubated in a total volume of 1 ml of HBSS containing either 200 μg of free TR-RTS,S, 30 μg of TR-RTS,S encapsulated in liposomes lacking lipid A, or 45 μg of TR-RTS,S encapsulated in liposomes containing lipid A for 90 min at 37°C. A greater amount of free TR-RTS,S than of liposomal TR-RTS,S was used, based on previous experiments with antigens not encapsulated in liposomes (31), to ensure that any low-level trafficking to the Golgi could be detected. The doses of liposomal TR-RTS,S were adjusted to give equal amounts of liposomal phospholipid and varied between the liposomes containing and lacking lipid A due to small differences in the TR-RTS,S encapsulation. At the end of the incubation period, the BMs were washed in HBSS and then incubated for an additional 2 h (chase) in HBSS or they were mounted immediately on a depression slide and the live cells were observed under a Leitz Orthoplan microscope (Leica) with an oil immersion 63× objective. Images were collected and processed as described previously (31).

Labeling of the trans-Golgi complex.

At the end of the incubation period, the trans-Golgi was visualized by staining the cells with a green-fluorescent analog of ceramide [N-(ɛ-nitrobenzooxadiazole-aminohexanoyl)-d-erythro-sphingosine (hereafter referred to as C₆-NBD-ceramide); Molecular Probes, Inc.] as described previously (29, 31). Briefly, coverslips containing BMs from B10.Br mice were incubated on ice with 2 nmol of C₆-NBD-ceramide/ml for 30 min. The cells were then washed twice with HBSS and incubated at 37°C for 15 min. After being washed twice more with HBSS, the cells were mounted and viewed as described above.

Immunizations.

B10.Br mice (five per group) were immunized intraperitoneally at 0 and 4 weeks with liposomal RTS,S (0.8, 1.4, 4.2, or 8.5 μg of RTS,S per 0.2-ml dose) or free RTS,S (10 μg of RTS,S per 0.1-ml dose) and bled at weeks 0, 2, 4, and 6 after the primary immunization. Each dose of the liposomal RTS,S formulations contained, in addition, 20 μmol (13.6 mg) of phospholipid, 15 μmol (5.8 mg) of cholesterol, and 40 μg of MPL.

For the experiment to study the effect of liposomal lipid A on CTL induction, B10.Br mice (three per group) were immunized as described above with RTS,S (4 μg per 0.1-ml dose) encapsulated in liposomes containing or lacking MPL. Each dose of these liposomal RTS,S formulations contained, in addition, 5.5 μmol (3.7 mg) of phospholipid, 4 μmol (1.5 mg) of cholesterol, and, where indicated, 18 μg of MPL.

Assay for antigen-specific CTLs.

Splenic lymphocytes were collected 2 weeks after the boosting immunization. The cells were cultured in vitro in complete medium (RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 8 mM l-glutamine, 100 U of penicillin/ml, 100 μg of streptomycin/ml, and 100 μM 2-mercaptoethanol) for 6 days, with the addition of 5% rat concanavalin A supernatant (as a source of interleukin 2), either without any antigen or with RTS,S (10 μg/ml) or the 367-to-390 CTL peptide (10 μg/ml) as an antigen. Target cells consisted of syngeneic (H-2^k) L929 cells, L929 cells transfected with the P. falciparum gene (LPF), L929 cells pulsed with the 367-to-390 CTL peptide, or allogeneic (H-2^b) EL-4 cells pulsed with the 367-to-390 CTL peptide. The target cells were labeled for 1 h with ⁵¹Cr (Na₂CrO₄; Amersham Life Science, Arlington Heights, Ill.) (100 μCi/10⁶ cells), and then they were added to effector cells at ratios ranging from 3:1 to 100:1. The cell mixtures were incubated in 96-well round-bottom tissue culture plates (Costar, Cambridge, Mass.) in 0.2 ml of complete RPMI medium for 4 h at 37°C in a 5% CO₂ humidified atmosphere. During the 4-h assay, a 1:5 final concentration of 8312.5, 2RL, HB 20, or HB 6 culture supernatant was added to the appropriate wells. At the end of the 4-h culture, the supernatants were absorbed by cotton wicks and processed for the determination of ⁵¹Cr release. Specific lysis was determined by the following formula: percent specific lysis = 100 × (experimental release − spontaneous release)/(maximal release − spontaneous release).

ELISA.

Solid-phase enzyme-linked immunosorbent assays (ELISAs) were performed to measure the levels of IgG antibody against the NANP repeat epitopes of the P. falciparum CSP by using R32LR, a recombinant protein with the sequence MDP(NANP)₁₅NVDP(NANP)₁₅NVDPLR (9), as the capture antigen, essentially as described previously (34, 41). Immulon-2 96-well U-bottom polystyrene plates (Dynatech Laboratories, Chantilly, Va.) were incubated with R32LR (2 μg/ml; 50 μl/well) in antigen diluent (4 μg/ml boiled casein in phosphate-buffered saline [PBS]) overnight at 4 to 6°C and then blocked with 0.5% casein in PBS containing 1% Tween 20 (PBS-casein-Tween). Individual mouse sera diluted in PBS-casein-Tween were added to the plates in triplicate wells and incubated overnight at 4 to 6°C. After being washed with 0.05% Tween 20 in PBS (PBS-Tween), the plates were incubated with phosphatase-labeled goat anti-mouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) for 2 h at room temperature and then washed again with PBS-Tween. Substrate (p-nitrophenyl phosphate; Kirkegaard & Perry Laboratories) was added, and the plates were incubated in the dark for 30 min. Absorbance was read at 405 nm with a UVmax plate reader (Molecular Devices, Sunnyvale, Calif.). Antibody levels are expressed as ELISA units. The ELISA units for a given serum sample are defined as the reciprocal of the serum dilution at which the absorbance is 1.0, and this value is determined from the linear portion of an antibody titration curve.

Quantitation of antigen-specific IgG subclass antibodies was performed by solid-phase ELISA as previously described (15). Immulon-2 plates were incubated overnight with antigen in PBS (0.1 μg/50 μl) and blocked with PBS containing 0.5% casein (PBS-casein). Individual mouse sera were diluted in PBS-casein and incubated at room temperature for 4 h. Peroxidase-conjugated goat anti-mouse isotype-specific antibody (The Binding Site, San Diego, Calif.) was utilized as a secondary antibody. Standard curves for each subclass were determined by using mouse myeloma IgG1, IgG2a, IgG2b, and IgG3 (The Binding Site). Both the test sera and myeloma standards were detected by using 2,2′-azino-di(3-ethyl-benzthiazoline) sulfonic acid (Kirkegaard & Perry Laboratories) as a substrate. Absorbance was read at 405 nm. Individual antigen-specific subclasses were quantitated by using the values from the linear titration curve computed against the myeloma standard curve and were reported as micrograms per milliliter.

Trafficking of liposomal RTS,S versus free RTS,S in BMs.

BMs were incubated with TR-RTS,S, either free or encapsulated in liposomes containing or lacking MPL. The resulting fluorescence patterns in BMs were examined to determine the intracellular trafficking of free and liposome-encapsulated RTS,S. When BMs were incubated with free (nonliposomal) TR-RTS,S, the red fluorescence initially appeared to be present throughout the cytoplasm, but after longer incubation times it also accumulated in the area of the Golgi. A similar pattern was observed when TR-RTS,S encapsulated in liposomes either containing or lacking MPL was incubated with BMs. This was verified by incubation of the BMs with C₆-NBD-ceramide, a fluorophore-labeled lipid which is specific for the trans-Golgi (29), following incubation with TR-RTS,S. The green trans-Golgi-specific fluorescence colocalized in BMs with the red TR-RTS,S fluorescence regardless of whether the BMs were incubated with free (Fig. 1A and B) or with liposomal (Fig. 1D, E, G, and H) RTS,S. In most cases, the area of TR fluorescence extended beyond the borders of the C₆-NBD-ceramide fluorescence, suggesting that localization of RTS,S was not limited solely to the trans-Golgi. This concentration in the Golgi has not been observed when free soluble antigens are incubated with BMs (31). The fluorescence patterns in the BMs suggested that RTS,S, both free and liposomal, went initially to the cytoplasm after uptake by the cells and later accumulated in the Golgi. This trafficking pattern is consistent with the intermediate steps that are known to be part of the processing of intracellular antigens through the MHC class I pathway.

To ensure that the localization of TR-RTS,S in the trans-Golgi area was not just an isolated event occurring in only a few cells, BMs incubated with free TR-RTS,S were also examined at a lower magnification. The fluorescence patterns in BMs from multiple fields indicated that in 47% of 38 BMs examined, TR-RTS,S was definitely present in the region of the trans-Golgi. In the other 53% of the cells examined, TR-RTS,S was present either in the general area of the Golgi, but not localized to the trans-Golgi, or only in other areas of the BMs. It is likely that this heterogeneity of response was due to the fact that the cells were not synchronized for antigen processing.

CTL responses in mice immunized with liposomal RTS,S.

Immunization with RTS,S encapsulated in liposomes containing MPL resulted in the induction of CTLs in mice receiving a 4.2-μg dose of liposomal RTS,S (Fig. 2A, C, and E) but not in mice immunized with free RTS,S (Fig. 2B, D, and F). The response was specific for RTS,S, since the specific lysis from L929 cells pulsed with the CSP-derived 367-to-390 CTL peptide was substantially higher when these target cells were cultured with RTS,S than when they were cultured with medium alone (Fig. 2A). Although specific lysis greater than 30% was not observed in these experiments, similar levels of specific lysis have been reported previously as representing a positive CTL response (18, 26, 33, 44). Control L929 cells also gave somewhat higher lysis with effector cells cultured with RTS,S than with effector cells cultured with medium (Fig. 2C), but the lysis of the control L929 cells due to RTS,S-cultured effector cells was always lower than that obtained from CTL peptide-pulsed L929 cells, whereas the lysis levels of both control and peptide-pulsed L929 cells were similar with control effector cells (cultured with medium alone) (Fig. 2A and C). The RTS,S-specific cytolytic activity was restricted by MHC class I molecules, since only peptide-pulsed L929 cells (Fig. 2A), expressing H-2^k, and not peptide-pulsed EL-4 cells (Fig. 2E), expressing H-2^b, were lysed. The MHC class I restriction of the RTS,S-specific cytolytic activity was further confirmed by abrogation of the antigen-specific cytolytic activity when anti-class I antibodies and anti-CD8 antibodies, but not anti-class II antibodies or anti-CD4 antibodies, were added to the cultures (data not shown).

The CTLs induced in the mice immunized with liposomal RTS,S recognized the 367-to-390 CTL epitope of the P. falciparum CSP, since specific lysis was observed when effector cells from the mice immunized with liposomal RTS,S which had been cultured with the 367-to-390 peptide were incubated with L929 cells transfected with the P. falciparum gene (Fig. 3A) but not after incubation with control L929 cells (Fig. 3B). This activity also was MHC class I restricted, since no antigen-specific lysis was observed when EL-4 cells pulsed with the 367-to-390 peptide were the target cells (Fig. 3C).

A CTL response against the 367-to-390 CTL epitope was induced only when lipid A was present in the liposomes (Table 1). Specific lysis was observed when effector cells from mice immunized with RTS,S encapsulated in liposomes containing MPL which had been cultured with the 367-to-390 peptide were incubated with L929 cells pulsed with the 367-to-390 peptide as the targets, but not with effector cells from mice immunized with RTS,S encapsulated in liposomes lacking MPL. No specific lysis was obtained when L929 cells pulsed with medium alone were used as the targets (data not shown).

NANP-specific IgG antibody response to liposomal RTS,S in mice.

Immunization of B10.Br mice with different doses of liposomal RTS,S resulted in the production of NANP-specific IgG antibodies at all four RTS,S doses tested. As can be seen in Fig. 4, the NANP-specific IgG response at 6 weeks after the primary immunization (2 weeks after the secondary immunization) increased with increasing RTS,S doses. All five mice receiving the highest liposomal RTS,S dose (8.5 μg) gave high IgG titers, and these titers were as high as or higher than any obtained in the mice receiving the lower doses of liposomal RTS,S. At both the 4.2- and 1.4-μg doses, all five mice responded, and the titers were similar for the two doses. At the 0.8-μg dose, only four of the five mice responded, and of these four, only one had a high titer. With all four doses of liposomal RTS,S, however, the IgG responses were much higher than those obtained after immunization with a 10-μg dose of free RTS,S, with the exception of one of the five mice immunized with free RTS,S, which had an IgG titer comparable to the titers obtained with the lower doses of liposomal RTS,S.

IgG subclass pattern in mice immunized with liposomal or free RTS,S.

The IgG subclass distribution was also examined in the mice immunized with RTS,S, either free or encapsulated in liposomes containing MPL. The subclass distribution patterns were similar for all four doses of liposomal RTS,S. The predominant subclasses observed after immunization with liposomal RTS,S were IgG1 and IgG2a (Fig. 5). Since the mice immunized with free RTS,S gave much lower antibody titers, the IgG subclass pattern was difficult to determine (Fig. 5). However, the subclass distribution pattern was consistent with that obtained in the mice immunized with liposomal RTS,S.