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Infection and Immunity, February 2009, p. 739-748, Vol. 77, No. 2
0019-9567/09/$08.00+0     doi:10.1128/IAI.00974-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Enhanced Immunogenicity of Plasmodium falciparum Peptide Vaccines Using a Topical Adjuvant Containing a Potent Synthetic Toll-Like Receptor 7 Agonist, Imiquimod {triangledown}

Caroline Othoro,1,{dagger} Dean Johnston,2,{dagger} Rebecca Lee,1 Jonathan Soverow,1 Jean-Claude Bystryn,3 and Elizabeth Nardin1*

Department of Medical Parasitology,1 Department of Dermatology and New York University Cancer Institute, School of Medicine, New York University, New York, New York 10010,3 Hunter College School of Health Sciences, New York, New York 100102

Received 4 August 2008/ Returned for modification 4 October 2008/ Accepted 20 November 2008


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ABSTRACT
 
Plasmodium sporozoites injected into the skin by malaria-infected mosquitoes can be effectively targeted by antibodies that block parasite invasion of host hepatocytes and thus prevent the subsequent development of blood stage infections responsible for clinical disease. Malaria subunit vaccines require potent adjuvants, as they lack known pathogen-associated molecular patterns found in attenuated viral or bacterial vaccines that function as Toll-like receptor (TLR) agonists to stimulate dendritic cells and initiate strong adaptive immune responses. A synthetic TLR7 agonist, imiquimod, which is FDA approved for topical treatment of various skin conditions, can function as a potent adjuvant for eliciting T-cell responses to intracellular pathogens and model protein antigens. In the current studies, the topical application of imiquimod at the site of subcutaneously injected Plasmodium falciparum circumsporozoite (CS) peptides elicited strong parasite-specific humoral immunity that protected against challenge with transgenic rodent parasites that express P. falciparum CS repeats. In addition, injection of a simple linear peptide followed by topical imiquimod elicited strong Th1 CD4+ T-cell responses, as well as high antibody titers. The correlation of high anti-repeat antibody titers with resistance to sporozoite challenge in vivo and in vitro supports use of this topical TLR7 agonist adjuvant to elicit protective humoral immunity. The safety, simplicity, and economic advantages of a topical synthetic TLR7 agonist adjuvant also apply to other vaccines requiring high antibody titers, such as malaria asexual or sexual blood stage antigens to prevent red blood cell invasion and block transmission to the mosquito vector, and to vaccines to other extracellular pathogens.


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INTRODUCTION
 
The control of the Plasmodium parasite, which causes 300 to 500 million malaria infections and more than 1 million deaths each year, will require a multifaceted approach involving insecticides, chemotherapy, and development of an inexpensive, efficacious malaria vaccine. While significant advances have been made in the use of insecticide-impregnated bed nets and new drug combinations, a licensed malaria vaccine is not yet available. Thus far, the attenuated sporozoite provides the most potent malaria vaccine that can fully protect human volunteers against experimental challenge by the P. falciparum-infected mosquito and thus prevent development of blood stage parasites responsible for clinical disease (13, 18, 19, 43).

Significant logistical challenges remain in scale-up production of attenuated parasite vaccines for the 40% of the world's population currently at risk of malaria (28, 44). Sporozoites cannot be grown in vitro and can be obtained only by dissection of salivary glands of mosquitoes that have fed on human blood infected with P. falciparum parasites. Although progress has been made in addressing the regulatory and safety issues related to production of purified P. falciparum sporozoites for vaccines, the requirement for human blood products and the need for cryopreservation at –140°C remain significant hurdles for mass distribution of attenuated sporozoite vaccines.

Alternative efforts in malaria vaccine development have focused on subunit vaccines, which are safer, cheaper, and more amendable to regulatory control. The circumsporozoite (CS) protein covers the surface of the sporozoite and has been shown to be a major target of the protective immune response elicited by the irradiated sporozoite (26, 39). The most advanced CS-based subunit vaccine, currently in phase III trials in Africa, is a virus-like particle comprised of a recombinant hybrid hepatitis B surface antigen fused to the repeat region and C terminus of P. falciparum CS protein (52). This virus-like particle, termed RTS,S, elicited transient sterile immunity in malaria-naïve volunteers and adult Africans and protected against clinical disease in 35 to 65% of immunized African children and infants in phase II trials. Vaccine efficacy depends on a complex adjuvant mixture containing monophosphoryl lipid A and a purified saponin, QS21, in an oil-in water emulsion (47). However, malaria vaccines formulated in oil emulsions have been limited by instability and antigen modification during storage and by reactogenicity in clinical trials (5, 29, 45, 54).

The separate administration of vaccine and adjuvant would address problems of immunogen instability and/or modification noted with oil adjuvants while simplifying vaccine analysis and storage. The rational design of new adjuvants has focused on Toll like receptor (TLR) agonists that trigger maturation of dendritic cells to effectively bridge the innate and adaptive immune response. Topical application of synthetic imiquimod, an imidazoquinoline analog of the natural single-strand RNA ligand of TLR7, can significantly enhance antibody as well as T-cell responses to parenterally administered antigens (1, 14, 22, 42, 46, 58, 59). Consistent with the role of TLR7-single-strand RNA interactions in alerting the immune system to the presence of virally infected cells, imiquimod was shown to function as a potent adjuvant for eliciting protective CD4+ and CD8+ T-cell responses against intracellular pathogens (14, 21, 22, 58). The endosomal localization of TLR7 suggests that TLR7 agonists would also function as adjuvants to enhance antibody responses against endocytosed extracellular antigens, such as subunit vaccines.

A cream formulation of imiquimod (Aldara; 3M, St. Paul, MN) is FDA approved for topical treatment of human dermatologic skin conditions including genital warts, actinic kertoses, and superficial basal and squamous cell carcinomas (16, 37). The skin is of particular interest for malaria vaccinologists, as irradiated sporozoites injected by the bite of infected mosquitoes elicit high levels of sterile immunity in human volunteers (13, 18, 19, 43), thus providing the "gold standard" for vaccine development. Although targeting of sporozoites to the liver was previously thought to be essential for induction of protective immunity, recent studies have shown that dendritic cells in the skin and draining lymph nodes play important roles in the initiation of sporozoite-induced immune responses (12, 23). Sporozoite-pulsed dendritic cells can also elicit protective immunity in murine models (7, 41), indicating that sterile immunity is not dependent on the presence or persistence of liver stages and supporting efforts to design subunit CS vaccines to target skin antigen-presenting cells.

We therefore examined whether the topical application of imiquimod can function as an adjuvant for a P. falciparum CS peptide vaccine injected subcutaneously (s.c.). The peptides used in these studies contain minimal T- and B-cell epitopes of the P. falciparum CS protein, which we have shown in previous studies can elicit sporozoite neutralizing antibodies and gamma interferon (IFN-{gamma})-producing CD4+ T cells in human volunteers comparable to those elicited by irradiated sporozoites (9, 10, 33, 36). In the current studies, we provide the first demonstration that protective antibody-mediated immunity can be elicited using a topical TLR7 agonist, imiquimod, as an adjuvant. Immunization with linear peptide s.c. followed by the application of topical imiquimod elicited high antibody titers, comparable to levels obtained in previous studies using potent oil adjuvant formulations, that effectively neutralized sporozoite infectivity in vitro and inhibited infectivity of sporozoites injected by malaria-infected mosquitoes in vivo.


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MATERIALS AND METHODS
 
Peptides. The tri-epitope T1BT* peptide contains minimal T- and B-cell epitopes of the P. falciparum CS protein that were identified using serum and cells of sporozoite-immunized volunteers (31, 34, 35). The T1 epitope, (DPNANPNV)2 is located in the 5' minor repeat, and the B epitope (NANP)3 is in the 3' major repeat of the P. falciparum CS protein. The universal T* epitope, EYLNKIQNSLSTEWSPCSVT, is located in the C terminus of the CS protein of P. falciparum strain NF54 (8, 31) and overlaps a highly conserved region of the CS protein that functions in targeting the sporozoite to the liver (11). The T- and B-cell epitopes were synthesized in tandem either as a 48-mer linear peptide, T1BT* (AnaSpec, San Jose, CA), or as a tetrabranched peptide (T1BT*)4, as previously described (10).

Immunizations. Female C57BL mice (Jackson Labs, Bar Harbor, ME), 6 to 7 weeks old, were immunized with three to four doses of 50 µg of peptide injected s.c. at 2-week intervals. The TLR7 agonist imiquimod was obtained as a 5% cream containing 12.5 mg/ml of imiquimod (Aldara; 3M, St. Paul, MN). Anesthetized mice were treated with approximately 25 µl (1.25 mg of imiquimod) topically applied to the subscapular region over the site of s.c. injected malaria peptide. For multiple applications of imiquimod, the topical adjuvant was reapplied at 24 and 48 h following peptide immunization. Controls included mice injected either with peptide in phosphate-buffered saline (PBS) without topical adjuvant or receiving topical adjuvant alone or with topical imiquimod with s.c. injection of an unrelated antigen, ovalbumin (OVA) (22). Mice were observed for approximately 6 h postimmunization, and no grooming of the topical site was observed.

Serologic assays. Serum was collected 14 days after each immunization and stored at –80 C until used. Enzyme-linked immunoassays (ELISAs) for CS repeat antibodies was performed as previously described (10). Briefly, 96-well plates were coated overnight with the CS repeat peptide (T1B)4 (1 µg/ml) or bovine serum albumin (BSA) as a control. The plates were washed and blocked with 5% BSA, and twofold dilutions of individual sera were added for 1 h at 37°C. After a washing step, plates were incubated with peroxidase-labeled anti-mouse immunoglobulin G (IgG) and the substrate ABTS (2,2'-azino-di-3-ethylbenzthiazoline sulfonate) and H2O2. Results are expressed as geometric mean titers (GMT), with the endpoint cutoff taken as an optical density (OD) greater than three times the OD of BSA-coated control wells. IgG subtypes were determined in (T1B)4 peptide- or BSA-coated wells using a 1:100 dilution of serum, followed by incubation with antibody specific for murine IgG subtypes (Southern Biotech) and subsequently with horseradish peroxidase-labeled antibody and substrate.

Serum was also tested for reactivity with viable sporozoites by using the CS precipitin (CSP) reaction to measure the ability of antibodies to effectively cross-link and precipitate the surface CS protein (49). These assays utilized a transgenic Plasmodium berghei parasite, termed PfPb, that expresses a hybrid CS protein containing the P. falciparum CS repeat region (40). The CSP endpoint titer was the final dilution at which 2/20 sporozoites had detectable precipitin reactions when examined by phase microscopy. Immune serum was also tested for reactivity with native P. falciparum CS protein by indirect immunofluorescence assay (IFA) using air-dried P. falciparum sporozoites. Twofold dilutions of pooled sera were incubated for 1 h on multiwell slides containing sporozoites dissected from the salivary glands of Anopheles stephensi mosquitoes. After a washing step, slides were reacted with fluorescein isothiocyanate-labeled antibodies specific for murine IgG (KPL, Gaitherburg, MD). Slides were coded to prevent bias, and the endpoint was taken as the last dilution giving positive fluorescent sporozoites.

Sporozoite neutralization assays. The biological activity of vaccine-induced antibodies was measured in vitro and in vivo using viable transgenic PfPb sporozoites expressing P. falciparum CS repeats (40). A transgenic sporozoite neutralization assay (TSNA) was used to measure inhibitory activity in vitro to determine the ability of immune serum to block hepatoma cell invasion (10, 25). Immune or normal serum (1:5 dilution) was incubated with 2 x 104 PfPb sporozoites for 45 min prior to being added to confluent cultures of human hepatoma (HepG2) cells. As a positive control, PfPb sporozoites were incubated with 25 µg/ml of monoclonal antibody (MAb) 2A10 specific for P. falciparum CS repeats or with MAb 3D11 specific for P. berghei CS repeats as a negative control (35, 56). After 48 h of incubation at 37°C in vitro, the number of exoerythrocytic forms was determined by real-time PCR using probes specific for parasite 18S rRNA, as previously described (25). Total RNA from cell culture lysates was reverse transcribed to cDNA using a PTC-100 Programmable Thermal Controller (MJ Research Inc.), and a 1-µg aliquot was used for real-time PCR amplification using primers specific for P. berghei 18S rRNA (6) and a Rotor-Gene RG-3000 (Corbett Research Inc.). Results are expressed as the number of parasite 18S rRNA copies, based on an 18S rRNA plasmid reference standard.

Cellular assays. IFN-{gamma} enzyme-linked immunospot (ELISPOT) assays were carried out using spleen cells obtained following the final immunization (10, 30). Prior to plating the cells for the assay, CD8+- and CD4+-enriched T-cell subpopulations were obtained using immune-magnetic beads coated with anti-CD4+ (MACS; Miltenyi Biotec, CA). Total spleen cells or enriched T-cell populations (3 x 105 cells/well) were stimulated with naïve autologous spleen cells pulsed with the 10 µg/ml of the T1BT* immunogen or individual T1B or T* peptide (AnaSpec, Inc., San Jose, CA). Cells were plated in triplicate wells of a 96-well nitrocellulose plate coated with anti-IFN-{gamma} antibody, according to the manufacturer's instructions (BD Biosciences, San Jose, CA). Cells stimulated with phytohemagglutinin were included as positive controls. After 16 to 24 h, plates were washed and incubated with biotinylated anti-{gamma}-IFN MAb, followed by incubation with streptavidin-conjugated alkaline phosphatase. The presence of IFN-{gamma}-secreting cells was revealed by adding BCIP/NBT (5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium) substrate, and the number of spot-forming cells (SFC) in triplicate wells was counted by an ImmunoSpot Analyzer (CTL, Cleveland, OH). Results were expressed as the mean number of SFC/106 cells ± standard error of the mean.

Sporozoite challenge. Protective immune responses to P. falciparum CS repeats was assayed by exposing anesthetized mice to the bites of PfPb-infected mosquitoes for 15 min. Prior to challenge, the level of PfPb sporozoite infection in the mosquito salivary gland was determined by microscopy or by two-site assay using a MAb to P. falciparum CS repeats (57), and the number of mosquitoes was adjusted so that all mice received approximately 5 to 15 infected bites. To determine the role of T cells, mice were injected intraperitoneally with anti-CD4 MAb GK.15 or anti-CD8 MAb 2.43 for 3 days prior to sporozoite challenge, as previously described (10). Protection was determined by measurement of parasite rRNA in liver extracts obtained at 40 h postchallenge in immune versus naïve mice using real-time PCR, as described above.

Statistical analyses. Differences between groups were determined by using a Student's t test or analysis of variance, with a P value of <0.05 considered significant. Differences in antibody titers greater than fourfold were considered significant.


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RESULTS
 
Topical imiquimod enhances immunogenicity of malaria peptide injected s.c. To determine if topical imiquimod could function as an adjuvant for a parenterally administered malaria peptide vaccine, C57BL mice were immunized with 50 µg of (T1BT*)4 peptide injected s.c., followed by topical application of 1.25 mg of imiquimod at the site of injection. The imiquimod adjuvant was rubbed into the skin for 15 s immediately after each immunization. Mice were bled 2 weeks after each immunization, and anti-repeat antibody titers in individual serum were determined by ELISA.

The kinetics of IgG antibody in mice immunized with three doses of (T1BT*)4 injected s.c, either with or without topical application of imiquimod, is shown in Fig. 1. None of the mice primed with peptide alone had detectable anti-repeat IgG antibodies following priming, while positive responses were detected in all of the mice (5/5) receiving peptide followed by topical imiquimod (GMT, 576). A second s.c. immunization with peptide followed by topical imiquimod elicited a strong boost in anti-repeat titers, which were 28-fold higher than in mice immunized with peptide without imiquimod. Following a third immunization, peak IgG titers in mice receiving peptide plus imiquimod were a log higher than in mice receiving peptide alone, with a GMT of 40,960 (range, 20,480 to 81,960) versus 2,560 (range, 640 to 5,120), respectively. At approximately 2.6 months after the third immunization (day 120), the IgG titers in mice immunized with peptide plus imiquimod remained approximately 17-fold higher than in mice receiving peptide alone (15,521 versus 905, respectively).


Figure 1
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  		FIG. 1. Kinetics of IgG antibody response in mice immunized by s.c. injection of a branched CS peptide followed by topical imiquimod. C57BL mice were immunized s.c. with (T1BT*)4 peptide (50 µg) in PBS with or without application of topical imiquimod at the injection site. Serum from individual mice (n = 5 mice/group) was obtained prior to immunization on days 0, 14, and 28 (arrowheads) and at 120 days after the third immunization. Results shown are the GMT of anti-repeat IgG antibody determined by ELISA.

As reflected in the anti-repeat IgG subtypes, immunization with peptide followed by the use of the topical TLR agonist as an adjuvant elicited a mixed Th1/Th2-type antibody pattern (Fig. 2). Administration of the (T1BT*)4 peptide in PBS without imiquimod elicited low levels of predominantly Th2-type IgG1 antibodies. In contrast, the application of topical imiquimod following s.c. peptide immunization elicited antibodies of all IgG subtypes, including the Th1-type IgG2a/IgG2c antibodies, in addition to IgG1 antibodies.


Figure 2
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  		FIG. 2. Malaria-specific IgG antibody subtypes. Anti-repeat antibodies detected by subtype-specific antibodies in serum (1:100 dilution) obtained 14 days after the second or third s.c. immunization with (T1BT*)4 peptide in PBS, either with or without topical application of imiquimod. Results are shown as CS-specific antibody ODs after subtraction of reactivity with wells coated with unrelated antigen (BSA).

Sporozoite neutralizing antibodies elicited by s.c injection of (T1BT*)4 plus topical imiquimod. (i) In vitro sporozoite neutralization assay. The biological function of the anti-repeat antibodies elicited by s.c. injection of (T1BT*)4 plus topical imiquimod was measured by TSNA (25). Individual sera of five mice, obtained after each immunization, was incubated with viable PfPb sporozoites, which express P. falciparum CS repeats, prior to the addition to hepatoma cells. The immune serum obtained after the third immunization reduced sporozoite infectivity 99% (mean, 726 x 103 rRNA copies) compared to sporozoites without serum (127,097 x 103 rRNA copies) (Fig. 3A). This level of neutralizing activity was comparable to that observed with 25 µg of MAb 2A10 specific for P. falciparum CS repeats. Inhibition was P. falciparum specific as a MAb specific for P. berghei CS repeats (MAb 3D11) did not inhibit the infectivity of the PfPb sporozoites (Fig. 3A).


Figure 3
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  		FIG. 3. Sporozoite-neutralizing activity in mice immunized s.c. with (T1BT*)4 plus topical imiquimod. (A) PfPb sporozoites were incubated with immune serum or MAb prior to addition to human HepG2 hepatoma cells in vitro. Immune serum was obtained from mice 14 days after the first or third s.c. immunization with (T1BT*)4 peptide plus topical imiquimod. MAb 2A10 (25 µg), specific for P. falciparum CS repeats, was used as positive control, and MAb 3D11, specific for P. berghei CS repeats, was a negative control. Parasite 18S rRNA copy numbers in cell extracts obtained following 48 h of culture were measured by real-time PCR. Serum from mice immunized with (T1BT*)4 in PBS without imiquimod gave parasite 18S rRNA copy numbers similar to those of the negative control (data not shown). (B) In vivo sporozoite-neutralizing activity was measured in naïve or immunized mice challenged by exposure to the bites of PfPb-infected mosquitoes. Immunized mice received four s.c. doses of (T1BT*)4 with or without topical application of imiquimod after each dose. Levels of parasite rRNA in liver samples obtained 40 h postchallenge were measured by real-time PCR.

The sporozoite neutralizing activity correlated with high anti-repeat antibody titers in the immune serum obtained after the third dose of peptide plus topical imiquimod (GMT, 40,960). Serum obtained from mice following a single immunization had minimal neutralizing activity (11%), consistent with the presence of only low levels of anti-repeat antibody (GMT, 576). The lack of inhibition with immune serum obtained 14 days postpriming indicates that the topical imiquimod did not elicit nonspecific serum factors that inhibit sporozoite infectivity. Similarly, immune serum obtained after three s.c immunizations with an unrelated protein, OVA, followed by topical imiquimod (22) failed to neutralize sporozoite infectivity (data not shown), indicating that multiple doses of imiquimod had no effect on sporozoite infectivity and that neutralization was P. falciparum specific.

(ii) In vivo sporozoite neutralization. The high levels of neutralizing antibodies detected in the in vitro TSNA suggested that s.c. immunization with peptide plus topical imiquimod could elicit protective levels of immunity. In a second experiment, mice immunized s.c with three doses of (T1BT*)4 plus topical imiquimod were challenged by exposure to the bites of 15 PfPb-infected mosquitoes. The levels of parasite rRNA in livers at 40 h postchallenge were reduced 98% compared to levels in naïve mice (mean, 9,541 versus 548,794 rRNA copies, respectively) (Fig. 3B). The number of parasite rRNA copies detected in livers of mice immunized with peptide alone (478,566 rRNA copies) was not significantly different from levels detected in naïve mice after sporozoite challenge.

Immunogenicity of linear versus branched peptides adjuvanted with topical imiquimod. While using the branched (T1BT*)4 peptide plus topical imiquimod elicited high levels of humoral immunity and resistance to sporozoite challenge, the synthesis of tetrabranched peptides is technically challenging and limited by poor yields that complicate scale-up production of synthetic peptide vaccines. In previous studies, a 48-mer T1BT* linear peptide was found to have comparable immunogenicity as the more complex tetrabranched peptide (T1BT*)4 when formulated in potent water-in-oil adjuvants, such as Freund's or Montanide ISA 51 or ISA 720 (10). The use of oil adjuvants in human vaccines, however, has been limited by unacceptable reactogenicity in nonhuman primates and human volunteers (27, 48, 54).

To determine if topical imiquimod could function as an adjuvant for linear peptide, mice were immunized by s.c. injection of 50 µg of T1BT* linear peptide plus topical imiquimod. Following priming, the anti-repeat antibodies in mice immunized with linear peptide plus imiquimod were lower than those obtained with the branched peptide, with only a single mouse seroconverting. A booster immunization with linear peptide plus topical imiquimod elicited 100% seroconversion, but the GMT remained lower than that obtained with branched peptide plus topical imiquimod (GMT, 1,280 versus 13,512, respectively) (Fig. 4A). Despite these initial differences, an additional booster immunization of linear T1BT* plus topical imiquimod elicited IgG titers that were comparable to those in mice immunized with the branched (T1BT)4 peptide plus imiquimod, with a GMT of 28,963 (range, 2,560 to 40,960) and a GMT of 40,960 (range, 20,480 to 81,960), respectively.


Figure 4
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  		FIG. 4. Immunogenicity of linear T1BT* peptide plus topical imiquimod. (A) ELISA anti-repeat GMT were measured in serum obtained from individual mice (n = 3/group) obtained 14 days after each s.c. injection of either the tetrabranched (T1BT*)4 or linear T1BT* peptide, followed by topical imiquimod. (B) Protection of mice immunized s.c. with T1BT* linear peptide plus topical imiquimod following challenge by exposure to the bites of PfPb-infected mosquitoes. Results are shown as the prepatent period, indicating the number of days to the first detection of parasites in Giemsa-stained blood smears.

To determine if protective antibodies were elicited by immunization with linear peptide plus topical imiquimod, in a second experiment immunized mice were challenged by the bite of PfPb-infected mosquitoes and assayed for the time to development of parasitemia (prepatent period) as determined by microscopic examination of Giemsa-stained blood smears. Of the three mice immunized with linear peptide plus topical imiquimod, one was totally protected while the remaining two immunized mice had delayed prepatent periods of 7 days, compared to a prepatent period of 3 days for naïve mice. The significant 4-day delay in time to patent infection in the immunized mice reflects a >90% reduction in the number of infective sporozoites (4, 38, 51).

Fine specificity of antibodies elicited using topical imiquimod as an adjuvant. A concern regarding use of a topical adjuvant is the potential variability of delivery of the TLR agonist at the site of application. We therefore compared the fine specificity of antibodies elicited by peptide plus topical imiquimod in three separate experiments carried out by three different investigators. Anti-repeat antibody ELISA GMT were similar in three experiments in which either the branched or the linear peptide was used as the immunogen, with peak titers ranging from 3 x 104 to 5 x 104 (Table 1). These findings indicate that the adjuvant effects of topical imiquimod are highly reproducible. Moreover, in all of the experiments, the antibodies elicited by peptide plus topical imiquimod immunization effectively reacted with native CS on viable sporozoites, as measured by the CSP reaction using PfPb sporozoites expressing P. falciparum CS repeats. The CSP assay measures the ability of antibodies to cross-link surface CS protein on viable sporozoites, which leads to formation of a terminal precipitin reaction visible by phase microscopy. Consistent with the similar ELISA antibody titers, high CSP titers (128 to 258) were obtained with serum from mice immunized with either branched or linear peptide in all three experiments, indicating the reproducible production of antibodies that efficiently react with surface CS on viable parasites.


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  		TABLE 1. Fine specificity of antibodies elicited by CS peptide injected s.c. followed by topical application of imiquimod

Antibody titers were also measured by IFA using P. falciparum sporozoites to determine whether reactivity with the repeat peptide in an ELISA, and with transgenic parasites expressing P. falciparum CS repeats in the CSP reaction, correlates with reactivity with native P. falciparum CS. Consistent with both ELISA and CSP titers, serum from all three experiments showed high IFA titers with P. falciparum sporozoites. Importantly, the linear peptide elicited IFA titers comparable to the more complex branched peptide when topical imiquimod was used as adjuvant.

Enhancement of immunogenicity by multiple applications of imiquimod. Previous studies using OVA as a model antigen demonstrated enhanced T-cell responses following multiple applications of topical imiquimod applied at 48-h intervals (22). To determine if immunogenicity of linear peptide could be enhanced, mice were injected with peptide and treated with topical imiquimod immediately following injection and again at 24 and 48 h.

Multiple applications of imiquimod led to more rapid kinetics and higher antibody responses to the linear T1BT* peptide. Priming with linear peptide followed by three applications of imiquimod elicited antibody in 66% (2/3) of mice while none of the mice receiving T1BT* followed by a single application of imiquimod seroconverted. After booster immunization s.c. with linear T1BT* followed by three applications of imiquimod, antibody titers were a log higher than those obtained after peptide injection followed by a single application of imiquimod (Fig. 5A versus B). Antibody titers were further increased, reaching a peak 130,040 GMT (range, 81,920 to 327,680) following the third dose of linear peptide plus three applications of imiquimod, which was sixfold higher than the peak obtained with single application of imiquimod. A fourth immunization did not significantly increase antibody titers further.


Figure 5
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  		FIG. 5. Immunogenicity of branched versus linear peptide injected s.c., followed by single or multiple applications of imiquimod. Kinetics of anti-repeat antibody response in mice immunized s.c. with branched (T1BT*)4 or linear T1BT* peptide followed by a single application of topical imiquimod (1X) (A) or three topical applications of imiquimod at 0, 24, and 48 h (3X) (B). Results are shown as GMT in serum of individual mice (n = 3 mice/group) obtained 14 days after each immunization.

The increased adjuvant effect noted with multiple applications of imiquimod was antigen dependent. In contrast to linear peptide, three applications of imiquimod did not significantly enhance antibody responses to the more immunogenic branched peptide (Fig. 5). No significant difference was noted in the peak antibody titers obtained in mice receiving three doses of (T1BT*)4 plus three applications of imiquimod (GMT of 65,020; range, 40,960 to 81,920) compared to (T1BT*)4 plus a single application of imiquimod (GMT of 51,606; range, 20,480 to 81,920).

Th1-type CD4+ T-cell responses elicited by imiquimod adjuvant. Topical imiquimod has been shown in previous studies to stimulate CD8+ T cells, as well as CD4+ T cells, specific for viral, tumor, and model antigens (1, 14, 21, 22, 42, 46, 58). To examine the malaria-specific cellular responses, spleen cells of mice immunized s.c with the linear T1BT* peptide followed by single or multiple applications of imiquimod were assayed by IFN-{gamma} ELISPOT assay (Fig. 6). In the absence of the topical adjuvant, peptide immunization elicited only low levels of CD4+ T cells that produced IFN-{gamma} following stimulation with the T1BT* immunogen (mean, 17 IFN-{gamma} SFC/106 cells). In contrast, CD4+ T cells from mice immunized with peptide plus imiquimod, applied once or three times, developed high levels of IFN-{gamma} SFC when stimulated with the T1BT* immunogen (6,572 IFN-{gamma} SFC/106 SFC and 4,227 IFN-{gamma} SFC/106 SFC, respectively). The cellular response was comprised of CD4+ T cells specific for CS repeats (4,227 to 3,998 IFN-{gamma} SFC/106 cells), as well as the T* epitope (100 to 53 IFN-{gamma} SFC/106 cells). The presence of high levels of repeat-specific CD4+ T cells is consistent with the H-2b genetic background of C57BL mice, which are high responders to P. falciparum CS repeats (15, 17, 32).


Figure 6
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  		FIG. 6. T-cell responses elicited by linear T1BT* peptide plus imiquimod. An IFN-{gamma} ELISPOT assay was performed using spleen cells obtained from mice immunized s.c. with linear T1BT* peptide plus topical imiquimod applied once (1X) or three times (3X). Enriched CD4+ T cells (solid bars) or CD8+ T cells (hatched bars) were stimulated with 10 µg/ml of T1BT* immunogen or with the individual T* or T1B CS repeat peptides. Results are shown as mean numbers of IFN-{gamma} SFC/106 cells.

Cellular responses were primarily CD4+ T cells, as the CD8+ T-cell enriched population obtained from peptide-immunized mice receiving either one or three applications of imiquimod did not have detectable IFN-{gamma} SFC when cells were stimulated with any of the malaria peptides. Cells from naïve mice also did not have detectable IFN-{gamma} SFC following peptide stimulation (data not shown), indicating that the cytokine results reflect malaria-specific immune T cells elicited by immunization.

Correlation of antibody responses and protection against sporozoite challenge. To assay the biological function of the antibodies elicited by the linear peptide plus topical imiquimod, in vitro and in vivo sporozoite neutralization assays were carried out using PfPb transgenic parasites. In the in vitro sporozoite neutralization assay, serum of mice immunized with linear peptide followed by a single application of imiquimod, with an anti-repeat antibody GMT of 32,510, reduced PfPb parasite burden in hepatoma cells by approximately 80% (Fig. 7A). Serum of mice immunized with T1BT* plus three applications of imiquimod reduced parasite burden 98%, consistent with the higher levels of anti-repeat antibodies obtained in these mice (GMT of 130,040). The level of sporozoite-neutralizing activity in the serum of the mice immunized with peptide plus three applications of imiquimod was comparable to inhibition obtained with 25 µg of P. falciparum repeat-specific MAb 2A10.


Figure 7
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  		FIG. 7. Correlation of protection with antibody levels in mice immunized with T1BT* plus topical imiquimod applied once versus three times. (A) In vitro assay of sporozoite-neutralizing antibody was measured by preincubating PfPb sporozoites with immune serum, obtained from mice immunized with four doses of linear T1BT* peptide plus topical imiquimod applied once (Imiq 1X) or three times (Imiq 3X), prior to addition to hepatoma cells. Levels of parasite rRNA were measured by real-time PCR using extracts of hepatoma cells obtained 48 h after PfPb sporozoite inoculation. (B) Challenge by the bite of PfPb-infected mosquitoes of naïve mice or mice immunized s.c. with T1BT* peptide either without imiquimod (PBS) or with topical imiquimod applied once (Imiq 1X) or three times (Imiq 3X). Levels of parasite rRNA were measured by real-time PCR using extracts of liver cells obtained 40 h postexposure to the bites of PfPb-infected mosquitoes. The bar indicates the mean number of 18S rRNA copies for each group of mice (n = 3), with each symbol representing an individual mouse.

The mean number of parasite rRNA copies in hepatoma cells receiving sporozoites incubated in serum of mice receiving T1BT* peptide without imiquimod (PBS) was comparable to the number observed in cultures receiving sporozoites incubated with naïve mouse serum. This lack of inhibitory activity in serum of mice immunized with peptide alone was consistent with the presence of only low levels of anti-repeat antibody (GMT of 320) in these sera.

To determine whether the responses elicited by the linear T1BT* peptide plus topical imiquimod were protective in vivo, the mice where challenged by exposure to the bites of PfPb-infected mosquitoes (Fig. 7B). In liver samples obtained 40 h postinfection, mice immunized with peptide alone had parasite burdens comparable to naïve, nonimmunized mice (mean, 61,120 rRNA copies versus 37,563 rRNA copies, respectively). Mice immunized with peptide plus a single application of imiquimod had an 84.6% reduction of parasite rRNA detectable in the liver (mean, 5,780 rRNA copies) compared to naïve mice. Consistent with the presence of higher levels of anti-repeat antibodies, mice immunized with peptide plus three applications of imiquimod showed enhanced resistance, with 97.4% inhibition (mean, 994 rRNA copies).

Similar results were obtained in a second in vivo challenge experiment in which mice immunized with T1BT* plus three applications of imiquimod gave 95% inhibition of liver stage burden following exposure to the bites of PfPb-infected mosquitoes. The immunized mice (n = 3) had 12,221 ± 6,854 rRNA copies compared to 204,103 ± 117,711 rRNA copies in naïve mice at 40 h postchallenge. No inhibition was observed in mice receiving only three applications of topical imiquimod (782,205 ± 461,796 rRNA copies), indicating that resistance to sporozoite challenge was mediated by malaria-specific responses. Similar levels of inhibition were observed in mice immunized with T1BT* plus three applications of imiquimod and depleted of T cells by treatment with anti-CD4 MAb or anti-CD8 MAb prior to challenge, with 93% and 96% reductions of liver stage burden, respectively. The finding that T cells did not play a significant role in protection against PfPb sporozoitie challenge in the mice immunized with T1BT* plus imiquimod was consistent with previous studies demonstrating that T-cell depletion did not abrogate resistance to sporozoite challenge in mice immunized with T1BT* peptide administered in potent oil adjuvants (10).


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DISCUSSION
 
A critical mechanism of protective immunity against the infectious sporozoite is the presence of high levels of antibodies that can block sporozoite egress from the skin into the circulation and prevent invasion of target cells in the liver (20, 35, 39, 50). The current studies demonstrate that a topical synthetic TLR7 agonist, imiquimod, can function as a strong adjuvant for P. falciparum CS peptide vaccines and elicit high levels of biologically active sporozoite-neutralizing antibodies. Peak antibody titers obtained following topical application of imiquimod to the site of s.c. injected CS peptides were comparable to the titers (104 to 105) obtained in previous studies using s.c. injection of the same peptides formulated in the potent water-in-oil emulsion Montanide ISA 720 or ISA 51 or Freund's adjuvant (10). The pattern of IgG subtypes elicited by the TLR agonist adjuvant differed from the oil-based adjuvants, with high levels of Th1-type IgG2 antibodies as well as Th2-type IgG1 antibodies in the mice immunized with peptide plus imiquimod (Fig. 2) compared to predominantly IgG1 antibodies elicited by peptide in oil adjuvant formulations (10). Consistent with the higher Th1-type IgG2 responses in mice immunized with topical imiquimod adjuvant, the number of IFN-{gamma} secreting CD4+ T cells obtained following T1BT* stimulation were a log higher (>4,000 SFC/106 spleen cells) (Fig. 6) than those reported in a previous murine study using the same peptides formulated in oil adjuvants (mean SFC, 300 to 350 SFC/106 spleen cells) (10).

As found with potent oil adjuvants, the topical imiquimod adjuvant enhanced immunogenicity of the linear T1BT*peptide, with peak responses comparable to titers obtained with the more complex tetrabranched peptide (Table 1). These findings support efforts to develop low-cost, fully synthetic malaria peptide vaccines utilizing a topical synthetic TLR agonist as an adjuvant. The use of a topical adjuvant simplifies vaccine design and development by eliminating the need to optimize adsorption or emulsion formulations, thus providing significant time and cost savings. Separate delivery of vaccine and topical adjuvant also facilitates direct physiochemical characterization of the final vaccine immunogen, without complications due to presence of a coformulated adjuvant, and eliminates potential vaccine modifications or instability during storage, as reported for several malaria antigens formulated in oil-based adjuvants (5, 29, 45).

The identification of new adjuvants for highly purified subunit vaccines requires a balance between potency and reactogenicity. Although vaccines formulated in oil emulsion adjuvants were immunogenic, they were also reactogenic (3, 27, 54), and efforts to reduce adverse side effects by using single-dose immunization elicited suboptimal immunogenicity and no protective efficacy (53). In contrast, topical imiquimod (Aldara; 3M) is FDA approved and has an extensive safety record for treatment of actinic keratoses, superficial carcinomas, and genital warts (16, 37). In contrast to new adjuvants comprised of complex mixtures of emulsions, detergents, and immunostimulatory components, the imiquimod-TLR7 interactions provide a well-defined cellular target and signaling pathways that can be used to rationally enhance adjuvant potency for vaccines.

In the current studies, topical imiquimod enhanced not only malaria CS peptide antibody and cellular immune responses but also protective efficacy, as reflected in sporozoite neutralization in vitro and following sporozoite challenge in vivo, with reduced parasite burden in the liver resulting in delayed prepatent periods or sterile immunity. Protection in vivo and in vitro directly correlated with the levels of anti-repeat antibody in the mice immunized with peptide plus topical imiquimod. This correlation, along with the finding that T-cell depletion prior to challenge did not abrogate resistance, suggests that antibody-mediated immune mechanisms were functioning in the protection of mice immunized with peptide plus topical imiquimod. These results are consistent with studies with T1BT* peptide formulated in the oil adjuvant Freund's or Montanide ISA 720, in which anti-repeat antibodies and sporozoite neutralizing activity in serum correlated with in vivo resistance, and depletion of T cells prior to sporozoite challenge did not abrogate protection (10). Recent clinical trials with a CS-recombinant protein vaccine, RTS,S, have correlated protection against P. falciparum sporozoite challenge with high levels of anti-repeat antibodies in human volunteers (24).

A topical adjuvant is of particular relevance to malaria preerythrocytic vaccines that target sporozoites injected into the skin by the mosquito vector. In humans, high levels of sterile immunity have thus far been obtained only by immunization of volunteers by exposure to the bites of irradiated malaria-infected mosquitoes (13, 18, 19, 43). Recent intravital microscopy studies using fluorescent parasites have demonstrated that sporozoites remain at the site of the mosquito bite for several hours and can also be found in the draining lymph nodes and thus remain targets of antibodies for prolonged periods (2, 55). Moreover, induction of protective cellular immunity against liver stage parasites requires CD8a+ dendritic cells in the skin-draining lymph node, as shown in recent studies with transgenic mice expressing a CD8+ T-cell receptor specific for a protective CS cytotoxic T lymphocyte epitope (12). Therefore, using topical imiquimod adjuvant to target skin antigen-presenting cells that play important roles in induction of protective immunity may provide more efficacious malaria vaccines.

A topical adjuvant formulation capable of eliciting strong humoral immunity is also applicable to other malaria vaccines that require high levels of inhibitory antibodies, such as MSP-1 vaccines that target merozoite invasion of red blood cells and transmission-blocking vaccines that inhibit parasite development within the mosquito vector. A vaccine comprised of a combination of malaria antigens and a topical adjuvant that contains the well-defined TLR 7 agonist imiquimod would have the potential to elicit antibodies that effectively target different stages of the complex malaria parasite to block infection, clinical disease, and parasite transmission.


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ACKNOWLEDGMENTS
 
We thank Robert Mitchell, Leyda Cabrera, and Rita Altszuler for expert technical assistance.

These studies were supported by NIH grants AI025085 and AI045138 (E.N.) and NIH CA096804 (D.J.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medical Parasitology, New York University School of Medicine, 341 East 25th Street, New York, NY 10010. Phone: (212) 263-6819. Fax: (212) 263-8116. E-mail: Elizabeth.Nardin{at}nyumc.org Back

{triangledown} Published ahead of print on 1 December 2008. Back

Editor: W. A. Petri, Jr.

{dagger} C.O. and D.J. contributed equally as first authors. Back


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Infection and Immunity, February 2009, p. 739-748, Vol. 77, No. 2
0019-9567/09/$08.00+0     doi:10.1128/IAI.00974-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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