The past decade in malaria synthetic peptide vaccine clinical trials

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1 Human Vaccines ISSN: (Print) (Online) Journal homepage: The past decade in malaria synthetic peptide vaccine clinical trials Elizabeth Nardin To cite this article: Elizabeth Nardin (2010) The past decade in malaria synthetic peptide vaccine clinical trials, Human Vaccines, 6:1, 27-38, DOI: /hv To link to this article: Published online: 01 Jan Submit your article to this journal Article views: 179 View related articles Citing articles: 20 View citing articles Full Terms & Conditions of access and use can be found at

2 review Human Vaccines 6:1, 27-38; January 2010; 2010 Landes Bioscience MALARIA REVIEW The past decade in malaria synthetic peptide vaccine clinical trials Elizabeth Nardin Department of Medical Parasitology; New York University School of Medicine; New York, NY USA Key words: P. falciparum, synthetic peptide vaccine, clinical trials, antibody, T cells Abbreviations: Th, T helper; EEF, exoerythrocytic form; s.c., subcutaneous; i.m., intramuscular; i.d., intradermal; MAB, monoclonal antibody; S.I., stimulation index Over the past decade ( ), there have been nine clinical trials of synthetic malaria peptide vaccines designed to target the pre-erythrocytic and erythrocytic stages of the Plasmodium falciparum parasite. Recent advances in parasite immunology and cell biology have been utilized to improve peptide design and adjuvant formulations. The clinical trials demonstrated the potential of second generation peptide vaccines to elicit antibodies that can neutralize sporozoite infectivity and cooperate with monocytes in ADCI to inhibit blood stage parasites. In addition, peptide-induced malaria-specific human CD4 + and CD8 + T cells were shown in vitro to have similar fine specificity and function as parasite-induced T cells. The results of these clinical trials, while encouraging, have emphasized the critical roles of immunological assays, in particular functional assays, for the evaluation of potential vaccine candidates. Additional challenges include the need for potent adjuvants for the development of synthetic peptide vaccines that can effectively target multiple stages of the Plasmodium parasite. Introduction It is remarkable that it was over 20 years ago that the first clinical trial of a parenteral synthetic peptide vaccine against an infectious disease was carried using a malaria peptide-protein conjugate vaccine, (NANP) 3 -TT. 1 Also noteworthy was that it took only three years to progress from the cloning of the P. falciparum circumsporozoite (CS) gene and the identification of the NANP repeat as the target of protective antibodies to the initiation of that first Phase I/II trial. 2-5 The sterile immunity and delayed prepatent period observed in the small number of volunteers who were challenged with P. falciparum infected mosquitoes in this first trial provided proof-of-concept that a synthetic peptide malaria vaccine could elicit protective immune responses. These findings, combined with the well known safety advantages and established industrial standards for large scale production of synthetic peptides for clinical applications, encouraged efforts to develop more efficacious synthetic peptide malaria vaccines. Correspondence to: Elizabeth Nardin; Elizabeth.Nardin@nyumc.org Submitted: 06/12/09; Accepted: 07/23/09 Previously published online: In the intervening years, preclinical and clinical studies have addressed the limitations of the first peptide-protein conjugate vaccine, which included low immunogenicity, lack of parasitederived T helper (Th) epitopes, and the need for more potent adjuvants. The current review focuses on clinical trials of synthetic peptide malaria vaccines carried out over the past decade ( ) to summarize the progress made in the development of new adjuvants and delivery platforms, and in the design of peptide immunogens that can elicit antibody and cell mediated immunity to pre-erythrocytic, as well as erythrocytic, stages of the parasite. This review focuses on P. falciparum, as the majority of clinical trials target this highly lethal plasmodial species, with the expectation that the lessons learned from the iterative process of bench-to-bedside studies over the past ten years will also advance development of vaccines to other Plasmodium species, 6 as well as other pathogens. Pre-erythrocytic Stage Circumsporozoite (CS) Peptide Vaccines The goal of developing a vaccine to eliminate the pre-erythrocytic stages of the parasite, thus preventing the blood stage infections that cause clinical disease, was based on the early studies demonstrating that immunization with attenuated sporozoites could elicit sterile immunity in experimental rodents, monkeys and human volunteers. 7 The CS protein was the first protective antigen identified using immune serum and cells obtained from these experimental hosts and more recent studies in CS transgenic mice confirm that the CS protein is the immunodominant target of protective immune responses elicited by irradiated sporozoites. 8 Sporozoite neutralizing antibodies that target the species-specific central repeat region of the CS protein were the first immune effector mechanism identified in the sporozoite immunized hosts. The anti-repeat antibodies bind to the surface of the sporozoite, inhibit motility and block invasion of the host hepatocytes Recent intravital microscopy demonstrated that anti-repeat antibodies also immobilize sporozoites in the skin at the site of the mosquito bite and prevent their egress into the circulation. 13 Infective sporozoites are now known to remain at the site of the mosquito bite for several hours and thus provide a longer window for antibody mediated attack Human Vaccines 27

3 The induction of high levels of sporozoite neutralizing antibodies is T cell dependent, requiring CD4 + T cell cytokines as helper factors for B cell differentiation, 17 as well as for development of CD8 + memory T cells. 18 T cell cytokines, such as IFNγ, also function directly in protective immunity by inhibiting development of intracellular exoerythrocytic form (EEF), mainly through upregulation of inos and induction of NO in the infected cell. 19,20 Our continuing CS peptide vaccine efforts at NYU have focused on the design of peptide immunogens that elicit high levels of neutralizing antibodies to target the CS repeats and CD4 + Th1 cells that would function both as Th for antibody responses and as a potential source of IFNγ to target the intracellular hepatic stages. The peptide vaccines have been designed to contain only minimal T and B cell epitopes, indentified using sera and cells of humans immunized with irradiated P. falciparum sporozoites, in an effort to focus the host immune responses on functional targets. Branched CS Peptide Vaccines A limitation of the first (NANP) 3 -TT peptide-conjugate vaccine was the dependence on the tetanus toxoid carrier protein for induction of Th cells, thus limiting potential for malaria specific anamnestic responses following exposure to the parasite. Branched peptides, termed Multiple Antigen Peptide (MAP), originally developed as immunogens by Tam and colleagues, overcome this limitation and provided a source of macromolecular peptides that were highly immunogenic in the absence of carrier protein (reviewed in ref. 21). These branched synthetic peptides allowed synthesis of precisely defined malaria T and B cell epitopes on a non-cationic polylysine core matrix constructed by sequential propagation of the α and ε amino groups of lysine (Lys). The branched peptides can be constructed by stepwise synthesis, or by ligation of purified peptidic modules to a branched core peptide. 22,23 Unlike peptide-carrier protein conjugates, the size, number, ratio and stoichiometry of T and B cell epitopes in the branched peptides can be readily modified to design optimal immunogens. Branched peptide comprised of P. falciparum CS repeat epitopes: (T1B) 4 MAP. Design. MAPs containing T and B cell epitopes of rodent malaria CS were shown to elicit high levels of protective sterile immunity against sporozoite induced malaria in mice that was mediated by anti-repeat antibodies. 24 The choice of T cell epitopes for inclusion in the P. falciparum MAP vaccine was determined empirically by testing MAPs constructs containing the (NANP) 3 B cell epitope in combination with several Th epitopes identified in the repeat and non-repeat regions of the P. falciparum CS protein. 25,26 The optimal immunogen was a (T1B) 4 MAP construct containing the (NANP) 3 B cell epitope combined with a T cell epitope from the CS repeat, termed T The T1 epitope, which is located in the P. falciparum CS minor repeat region comprised of alternating NANPNVDP repeats, was originally identified by human CD4 + Th1-type T cells from a volunteer immunized with irradiated P. falciparum sporozoites. 30 The 5' repeat sequence provides an attractive vaccine component as it is conserved in P. falciparum strains of diverse geographical origin. In preclinical studies, the (T1B) 4 MAP elicited antibody responses in three species of Aotus monkeys and in three out of four strains of mice. 27 High responder C57Bl/10 (H-2 b ) mice developed indirect immunofluorescent antibody (IFA) titers of 10 6 against P. falciparum sporozoites, a magnitude of anti-repeat antibody response 1 2 logs higher than those obtained using parasites or recombinant proteins as immunogens. The (T1B) 4 MAP also elicited a strong anamnestic response in P. falciparum sporozoite-primed mice and monkeys, indicating the potential to boost the low sporozoite titers found in naturally infected individuals living in malaria endemic areas. The strong correlation between anti-peptide and anti-sporozoite antibody titers induced by the (T1B) 4 MAP was particularly noteworthy in light of the poor correlation noted with MAPs containing different T helper epitopes from the C terminus of the P. falciparum CS protein. 26 The low titers of sporozoite specific antibodies elicited by these constructs was explained, in part, by the finding that a large proportion of the anti-map antibodies were specific for the P. falciparum T cell epitopes localized in the C terminus. Recent studies have shown that the enzymatic cleavage of CS protein on contact with hepatocytes induces a conformational change in the C terminus. 31 Thus antibodies targeting C terminus epitopes may be limited in ability to access these cryptic epitopes on the migrating sporozoite. Thus far, the CS repeat region remains the optimal target based on high sporozoite neutralizing activity of monoclonal and polyclonal antibodies from irradiated sporozoite immunized hosts. Phase I trial of (T1B) 4 MAP vaccine. As predicted by the restriction of murine responses to (T1B) 4 MAP, 27 in the human studies the T1 epitope was presented in the context of a small number of HLA class II molecules, DQB1*0603, DRB1*0401 and DRB1* A Phase I prospective study was carried out to assess safety and immunogenicity of the (T1B) 4 MAP vaccine in individuals expressing these HLA class II molecules as compared to volunteers of random HLA haplotypes. 33 Volunteers were immunized s.c. on 0, 1 and 2 months with 500 1,000 ug (T1B) 4 MAP adsorbed to alum with, or without, QS21 (50 or 100 ug), a purified saponin derivative, as co-adjuvant (Table 1). The antibody responses in the volunteers immunized with MAP/alum/QS21 demonstrated clear high- and low-responder phenotypes. The high responders developed ELISA and IFA GMT of , an order of magnitude higher than those obtained in the (NANP) 3 -TT immunized volunteers and comparable to peak titers observed in protected volunteers immunized by irradiated P. falciparum sporozoites. 1,34 The high-responder (T1B) 4 MAP vaccinees were of the DQB1*0603, DRB1*0401, or DRB1*1101 genotypes, as predicted by in vitro T1 peptide/hla binding assays. 33 Anti-repeat antibodies were predominantly IgG1 and IgG3, consistent with the adjuvant effect of QS-21 for Th1-type responses. In all (T1B) 4 MAP/alum/QS21 vaccinees, there was a strong correlation between anti-repeat ELISA and IFA reactivity with P. falciparum sporozoites (r = 0.9). The high responder phenotype also correlated with strong Th1 CD4 + T cell responses. Antibody and cellular responses were restricted to the responder class II genotoypes, as only 50% (5/10) of volunteers of random HLA 28 Human Vaccines Volume 6 Issue 1

4 haplotypes seroconverted, with low antibody responses (ELISA GMT 206 and IFA GMT 80). The high antibody responses were dependent not only on genotype but also on adjuvant. In the absence of QS-21, as only 1 of 8 vaccinees of responder genotype seroconverted after immunization with three doses of (T1B) 4 MAP/alum without QS-21. The inclusion of the more potent QS21 adjuvant, however, increased reactogenicity as well as immunogenicity. After the third dose of vaccine, low levels of MAP-specific IgE were observed in the 500 ug dose group and two of the volunteers developed urticaria, suggesting a shift to Th2 type responses with continued immunization with high doses of peptide. Administration of the third 1,000 ug dose was delayed by seven months, at which time all volunteers had negative skin tests and no detectable MAP-specific IgE antibodies. 35 The third dose of vaccine was administered to these volunteers without any adverse reactions, suggesting that increased intervals between peptide immunizations can reduce the potential for reactogenicity. Immunization of a second cohort of seven volunteers of responder genotypes with two injections of the low dose formulation [500 ug (T1B) 4 MAP/ alum/50 ug QS21] confirmed the correlation of class II genotypes and high-responder phenotypes. 33 These studies demonstrated that only two doses of (T1B) 4 MAP vaccine was highly immunogenic (ELISA GMT 10,568; IFA GMT 5,511), with minimal reactogenicity. While a P. falciparum sporozoite experimental challenge model exists for clinical trials, the cost of Phase II trials is high and a small animal model for testing biological function of antibodies elicited during Phase I trials, as well as for preclinical screening, is needed. For this purpose we developed a transgenic P. berghei rodent malaria parasite engineered to express the P. falciparum CS repeats. 36 These transgenic parasites, termed PfPb, are biologically a rodent malaria and develop normally in vivo in mice and in vitro in hepatoma cells. However, they are antigenically P. falciparum, since they express the immunodominant P. falciparum CS repeat region. When incubated with MAB to P. falciparum repeats, the PfPb sporozoites develop the characteristic terminal precipitin reaction (CSP) indicating antibody mediated cross-linking and shedding of the surface CS protein (Fig. 1A). Conversely, WT P. berghei sporozoites, or transgenic parasites complemented with WT P. berghei CS (termed SX Pb), reacted only with MAB specific for P. berghei repeats and did not react with MAB specific for P. falciparum repeats. When the PfPb sporozoites pre-incubated with MAB to P. falciparum repeats were added to hepatoma cell cultures in vitro, invasion of cells was significantly inhibited, as measured by reduction in the levels of parasite rrna in hepatoma extracts detectable by real-time PCR. 12 Figure 1. (A) Wild type P. berghei (WT Pb) sporozoites or transgenic sporozoites expressing P. falciparum CS repeats (PfPb) or as control P. berghei CS repeats (SX-Pb) were incubated with monoclonal antibody specific for P. berghei (MAB 3D11) or P. falciparum (MAB 2A10) CS repeats. The presence of terminal circumsporozoite precipitin (CSP) reactions was assessed by phase microscopy. 36 (B) Pre-immune serum (Day 0), or immune serum (Day 42) obtained following immunization of volunteers with two doses of (T1B) 4 MAP/alum/QS21, was incubated with PfPb sporozoites prior to addition of parasites to HepG2 cells. 12 The levels of parasite 18SrRNA in hepatoma cell extracts obtained 48 hours post infection was determined by real-time PCR and the percent inhibition of immune serum relative to pre-immune serum is indicated. When sera of the (T1B) 4 MAP immunized volunteers was incubated with the viable PfPb sporozoites, sporozoite neutralizing antibodies were detected in the majority of the volunteers which conferred inhibition ranging from 49% 93% when compared to pre-immune Day 0 serum (Fig. 1B). Inhibition was specific for CS repeats as serum of Vol #4, a non-responder who failed to develop antibodies following MAP immunization, did not have inhibitory activity. All of the sera with neutralizing activity had comparable levels of anti-repeat ELISA titers (10,240 20,480), indicating that static ELISA assays do not accurately reflect levels of sporozoite neutralizing activity and emphasizing the importance of functional assays in defining potential efficacy of vaccineinduced antibody responses. This first (T1B) 4 MAP clinical trial demonstrated that a synthetic peptide vaccine comprised of only five amino acids, N, A, P, V and D (exclusive of the nonimmunogenic lysine core), could Human Vaccines 29

5 elicit high levels of parasite specific antibodies and IFNγ producing TH1 type CD4 + cells in humans. As expected for highly purified peptide immunogens, adjuvant formulation was critical. Even in genetically high responder genotypes, little or no immune response was elicited by the (T1B) 4 MAP/alum in the absence of the QS-21 co-adjuvant. These studies caution that a potent antigen may be overlooked as a potential vaccine candidate if tested with a suboptimal adjuvant formulation. Branched peptides comprised of CS repeats and universal T cell epitope: (T1BT*) 4 -P3C. Design: The (T1B) 4 MAP Phase I trial demonstrated that high levels of anti-sporozoite antibody could be elicited in human volunteers immunized with synthetic peptide vaccines. However, the frequency of high responder genotypes was approx % of the population, and an efficacious vaccine must elicit high antibody responses in all vaccinees. To address this limitation, a universal T cell epitope recognized in the context of multiple class II molecules was identified using cells from volunteers immunized with irradiated sporozoites. 37,38 This universal T cell epitope, termed T*, was located in the C terminus of P. falciparum CS and overlapped the highly conserved region RII-plus that functions in sporozoite/host cell interaction in the liver. 39 The T*peptide bound to multiple DR and DQ molecules in peptide competition binding assays and was immunogenic in multiple inbred strains of mice. 32 Consistent with universal epitopes derived from bacterial or viral proteins, the ability of the T*peptide to bind to multiple DR was a function of the presence of numerous overlapping epitopes within the 20mer T* sequence. 38,40 The construction of a MAP containing the universal T* epitope in combination with the T1B repeats, required synthesis of a 48mer sequence on each arm of the tetrabranched core and thus was limited by stepwise solid-phase synthesis. The ability to link purified peptide modules to a branched lysine core via oxime bonds 22,41,42 was therefore utilized to construct a tetrabranched polyoxime malaria vaccine, termed (T1BT*) 4 -P3C. 43 The triepitope T1BT* malaria peptide modules were linked to a core modified with the lipopeptide palmitoyl-s-glyceryl cysteine (P3C), a TLR2 agonist, to function as endogenous adjuvant. Chemoselective ligation of the purified aldehyde-modified 48-mer malaria epitopic module and the reciprocal aminooxyactyl modified tetrabranched lipopeptide core peptide, yielded a homogeneous 23,936 MW peptide which could be characterized by mass spectrometry. The polyoxime (T1BT*) 4 -P3C alone, without further addition of adjuvant or emulsifiers, was highly immunogenic in all inbred strains of mice tested. The high levels of anti-repeat antibodies measured by ELISA (GMT 10 4 ) correlated with high IFA titers with P. falciparum sporozoites (GMT 10 4 ). These antibody titers persisted without significant reduction in titer for over three months after the last immunizing dose. Phase I trial of (T1BT*) 4 -P3C vaccine. A small Phase I trial was carried out in ten volunteers of diverse HLA types to assess safety and immunogenicity of 1 mg of (T1BT*) 4 -P3C polyoxime vaccine administered s.c. on 0, 1 and 3 months (Table 1). 44 All volunteers seroconverted with the majority of the volunteers (8/10) reaching peak titers of following three immunizations. The level of anti-repeat antibodies (GMT 1114) correlated with IFA with P. falciparum sporozoites (GMT 830). Sera of immunized volunteers gave positive CSP reactions, indicating vaccineinduced antibodies could cross-link the surface CS on the viable P. falciparum sporozoites. The anti-peptide antibodies elicited by (T1BT*) 4 -P3C immunization were predominately Th1-type IgG1 and IgG3 subtypes. There was a strong correlation of T*-specific CD4 + T cell responses with high anti-repeat antibody titers, indicating that the T* epitope functioned as a universal Th cell epitope in humans, as predicted by the HLA class II/T*peptide in vitrobinding assays. PBMC and CD4 + T cell lines (TCL) from these volunteers produced IFNγ in response to stimulation with T1BT* and/ or T*peptide, consistent with the Th1-type antibody responses observed in the vaccinees. Cellular responses remained positive when tested 10 months after the final immunization in 57% (4/7) of these volunteers. To characterize the peptide-induced T cell responses in detail, a panel of CD4 + T cell clones were derived from volunteers at various time points after immunization with (T1BT*) 4 -P3C. 45 The majority of the peptide-induced CD4 + T clones (72%) recognized the T* epitope in the context of multiple HLA DR and DQ molecules. In contrast, only 3 clones of a total of 213 clones tested recognized the CS repeat epitopes, consistent with the limited HLA restrictions of T cell epitopes within the CS repeat region. 33 The T cell clones derived from the (T1BT*) 4 -P3C immunized volunteers were polyfunctional, with all of the clones secreting high levels of IFNγ (>1,500 pg/ml), as well as variable levels of TNFα and Th2-type cytokines (IL-4, IL-6). 45 Recent studies have shown an important role of polyfunctional T cells in other infectious diseases. 46 In addition to secretion of cytokines that could potentially inhibit EEF, such as IFNγ, a subset of the human CD4 + T cell clones also directly lysed target cells pulsed with CS peptide. 37,47 The T* specific CD4 + CTL derived from (T1BT*) 4 -P3C peptideimmunized volunteers where comparable to those obtained from P. falciparum sporozoite immunized volunteers in their fine specificity and lytic activity. Videomicroscopy of the peptide-induced and sporozoite-induced human CD4 + CTLs demonstrated formation of typical immunological synapses, which resulted in apoptosis of peptide-pulsed target cells, suggesting that the malaria specific CD4 + CTL utilize cytotoxic mechanisms similar to those reported for CD8 + T cells. The immunodominant T* epitope overlaps a polymorphic region of the circumsporozoite protein hypothesized to function in parasite immune evasion. 48 However, in analyzing a large number of clones from sporozoite or peptide immunized individuals, the T* specific clones were shown to recognize multiple P. falciparum strains. 38,45 Within a single individual, both highly cross-reactive as well as strain-specific T cells were present at the same time point. These findings caution that analysis of single or limited numbers of clones may fail to reflect the strain crossreactivity of cellular responses elicited by vaccines. Importantly, as also found following sporozoite immunization, 38,49 long-lived CD4 + T memory cells specific for the T* epitope were detectable 10 months after peptide immunization. 45 These studies provided the first demonstration that malaria peptide vaccines can elicit 30 Human Vaccines Volume 6 Issue 1

6 human CD4 + T cells with fine specificity and potential effector function comparable to those elicited by attenuated P. falciparum sporozoites. Simplifying CS synthetic peptide vaccines Peptide design. While the (T1B) 4 MAP and (T1BT*) 4 -P3C Polyoxime clinical trials demonstrated the vaccines were safe and immunogenic, the scale-up and synthesis of large quantities of branched peptides proved to be technically challenging. A potential means to overcome this limitation was provided by recent murine studies demonstrating that a simple 48mer linear T1BT*peptide was as immunogenic as the tetrabranched peptides. 50 The 48-mer T1BT* linear synthetic peptide, formulated in adjuvants suitable for human use, elicited high anti-sporozoite antibody of GMT 81, ,680, in BALB/c and C57Bl, respectively. The frequency of malaria specific IFNγ interferon-secreting T-cells elicited by immunization with the linear peptide (300 SFU/10 6 ) was comparable to that elicited by the more complex tetrabranched peptides. The immunogenicity of the linear peptide was dependent on presence of the universal T* epitope, as the T1B peptide, which contains only the weak T1 Th epitope, was immunogenic only as a branched peptide in respondor stains. Importantly, the simple 48mer T1BT* linear peptide was also immunogenic in outbred nonhuman primates. Aotus monkeys immunized with the T1BT* linear peptide formulated in Montanide ISA 51 developed high anti-repeat ELISA titers (GMT 32,510) and anti-p. falciparum sporozoite IFA titers (GMT 51,606). The antibody responses elicited by linear peptide were comparable to those observed in monkeys immunized with (T1BT*) 4 -P3C branched peptide (ELISA GMT 81,920; IFA GMT 28,963). Of particular significance for human vaccines, the 48-mer linear peptide administered in adjuvants suitable for human use elicited protection against viable sporozoite challenge when tested in the PfPb transgenic sporozoite model (Fig. 2). The number of parasite liver stages following challenge by bites of PfPb infected mosquitoes was reduced >90% in mice immunized with linear T1BT*peptide in Montanide ISA 720, as measured by real-time PCR of parasite ribosomal RNA in liver extracts. Depletion of CD4 + or CD8 + T cells prior to PfPb sporozoite challenge did not abrogate immune protection. Resistance correlated with levels of sporozoite neutralizing antibodies detected in the serum of individual mice, as measured by inhibition of PfPb sporozoite invasion of hepatoma cells in vitro. The predominance of Th2 type IgG1, a non-opsonizing antibody subtype, in the mice protected against challenge suggests that direct inhibition of sporozoites, rather than enhanced Fc-dependent phagocytosis, was functioning in immune resistance in the peptide immunized mice. The studies using the PfPb transgenic sporozoite rodent model provide the first demonstration that a simple 48-mer linear peptide can elicit functional antibodies specific for P. falciparum CS repeats that protect against sporozoite challenge. Adjuvant formulations. Potent adjuvants are required for synthetic peptide vaccines, as well as all malaria subunit vaccines, since these highly purified immunogens lack the pathogen-associated molecular patterns (PAMPs) that function as TLR agonists to stimulate innate immune responses that initiate strong adaptive immune Figure 2. Mice immunized with linear T1BT*peptide formulated in Montanide ISA 720 were depleted of CD4 + or CD8 + T cells prior to challenge by exposure to the bites of 15 PfPb infected mosquitoes. Extracts of livers of immunized or naïve mice were obtained 48 hours post challenge and levels of parasite 18S rrna was determined by real-time PCR. 50 Inhibition of >90% was obtained in the T cell depleted, or intact, immunized mice when compared to levels of parasite rrna in naive mice. responses. Although encouraging results were obtained with the TLR 2 lipopeptide agonist P3C as an endogenous adjuvant in the (T1BT*)-P3C Phase I trial, recent efforts have attempted to further simplify the use of TLR agonists as adjuvants. A topical cream containing a synthetic TLR 7 agonist imiquimod (Aldara, 3M, Minnesota, MI), currently FDA approved for topical treatment of several dermatologic conditions, is known to function as a potent T cell adjuvant for intracellular pathogens, tumor and model protein antigens In recent murine studies, topical imiquimod provided a potent adjuvant for parenterally administered T1BT* linear peptide or (T1BT*) 4 peptide. 54 The s.c. injection of either linear or branched peptide followed by topical application of imiquimod elicited peak antibody titers of GMT, comparable to those obtained in previous studies using peptides formulated in potent water-in-oil emulsions Montanide ISA 720, ISA 51 or Freunds adjuvant. High levels of murine Th1- type IgG2 antibodies, as well as Th2-type IgG1 antibodies, were elicited using the topical adjuvant, as compared to predominantly IgG1 antibodies following immunization of mice with peptides emulsified in oil adjuvants. Consistent with increased Th1 type antibody responses, levels of malaria specific IFNγ secreting CD4 + T cells were a log higher (>4,000 SFC/10 6 spleen cells) in mice immunized with peptide + topical imiquimod, as compared to peptide/oil adjuvant formulations (mean SFC SFC/10 6 spleen cells). 50 More importantly, sporozoite neutralizing activity in serum of mice immunized with peptide + topical imiquimod correlated with in vivo protection when the peptide + topical imiquimod immunized mice were challenged by exposure to the bites of PfPb infected mosquitoes. 54 A topical adjuvant such as imiquimod may be of particular relevance to malaria pre-erythrocytic vaccines, since infection is the result of sporozoites injected into the skin by the mosquito Human Vaccines 31

7 Table 1. Clinical trials of malaria synthetic peptide vaccines: Parasite stage Antigen target Nomenclature Peptide structure Composition Adjuvant/ Carrier Clinical trial (# volunteers) route, months Reference Pre-erythrocytic Circumsporozoite (CS) Protein (T1B) 4 MAP branched peptide CS repeat T1 (B/Th epitope) (NANP) 3 alum +/- QS21 Phase 1 (30)(7) s.c 0, 1, 2/8 Nardin (T1BT*) 4 -P3C Polyoxime branched peptide CS repeat T* universal Th epitope (aa NF54) CS aa LSP CD4 + and CD8 + T cell epitopes Pam3Cys phase 1 (10) s.c. 0, 1, 3 alum or ISA 720 phase 1 (16) i.m. 0, 1, 6 Nardin Lopez Erythrocytic Infected RBC SPf66 Polymer peptides from MSP-1, 35 kda, 55 kda proteins alum or QS21 Phase 1 (99) s.c. 0, 1, 6 Kashala Merozoite Glutamine Rich Protein GLURP aa LSP B and CD4 + T cell epitopes alum or ISA 720 Phase 1 (36) s.c, 0, 1, 4 Hermsen Merozoite Surface Protein 3 MSP-3 aa LSP B and CD4 + T cell epitopes alum or ISA 720 Phase 1a (36) s.c. 0, 1, 4 Audran alum Phase 1b (30) s.c. 0, 1, 4 Sirima Combination Apical Membrane Antigen 1 AMA-1 49-C1 (Pev 301) cyclic peptide AMA-1 Domain III ectodomain loop 1 Virosome Phase 1 (46) i.m. 0, 2, 6 Genton, CS CS (Pev 302, UK49) constrained peptide PNANP repeats AMA-1 CS AMA-1-C1 (Pev 301), CS (Pev 302) Cyclic + constrained peptides AMA-1 Domain III ectodomain PNANP repeat virosome Phase 1/IIa (24) i.m./i.d. 0, 1, 2 Thompson, /- +/- Multiple, TRAP ME-TRAP fowl pox/ MVA viral vectors CD4 + and CD8 + T cell epitopes (ME) + TRAP viral vector vector. 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 node. 15,16 Moreover, induction of protective cellular immunity against liver stage parasites recently shown to require CD8a + DC found in the skin draining lymph node. 55 Therefore, the use of a topical imiquimod adjuvant may provide more efficacious malaria vaccines by specifically targeting skin APC. The separate administration of vaccine and adjuvant would also address problems of vaccine instability and/or modification noted with oil adjuvants, while simplifying vaccine analysis and storage. Long synthetic peptides (LSP) containing C-terminus of CS. In addition to antibody mediated immunity, T cells that target the intracellular hepatic stages, via direct cytolytic mechanisms or secretion of inhibitory cytokines such as IFNγ, are an important immune mechanism in protection against sporozoite challenge. Early studies in rodent malaria models demonstrated that CS linear or branched peptides could elicit CD8 + T cells, as well as CD4 + T cells, that can protect against sporozoite challenge These studies, as well as more recent studies using transgenic mice expressing TCR specific for a rodent malaria CD8 + CTL epitope, 18 also demonstrated the requirement for CD4 + Th cells in induction of memory CD8 + CTL. Design. Long synthetic peptides (LSP) (>100 mer) immunogens that contain multiple CD4 + and CD8 + T cell epitopes can elicit strong cellular responses to CS. 62 Mice immunized with a P. falciparum aa LSP, developed CD8 + CTL that lysed target cells transfected with P. falciparum CS protein in a class I restricted manner. 63 In the P. berghei rodent malaria model, CS specific CD8 + T cells induced by a P. berghei CS aa LSP, elicited CD8 + CTL specific for a known K d restricted protective epitope. 64,65 The frequency of IFNγ secreting CD8 + T cells specific 32 Human Vaccines Volume 6 Issue 1

8 for the protective epitope were similar in LSP and sporozoite immunized mice. Immunization with a P. berghei C-terminus LSP in which the four cysteine residues were oxidized, protected % of immunized mice against challenge by the bites of P. berghei infected mosquitoes, in a CD8 + T cell dependent manner. Phase I trial of P. falciparum CS LSP vaccine. In a small Phase 1 trial, an oxidized 102mer P. falciparum CS LSP, formulated in alum or Montanide ISA 720, was tested for safety and immunogenicity. 66 The LSP vaccine (100 or 300 ug dose) was administered i.m. to volunteers at 0, 1 and 6 months (Table 1). Seroconversion, kinetics and magnitude of antipeptide and anti-parasite antibodies were optimal with the Montanide formulation. In contrast, PBMC lymphoproliferation to the 102mer LSP did not correlate with antibody responses and was similar in both adjuvant groups. CD8 + T cells of 13/15 volunteers gave positive responses when stimulated with the 102mer peptide in IFNγ ELISPOT assay. Following the third immunization, HLA-A*0201 positive volunteers specifically recognized two HLA-A*0201 restricted P. falciparum CS epitopes, aa (5/8 volunteers) and aa (2/8 volunteers), epitopes previously identified using cells of naturally infected individuals. 67 CD8 + T cell responses did not correlate with antibody, lymphoproliferation or adjuvant formulation. These studies represent the first proof-of-principle that a synthetic peptide vaccine can elicit CD8 + T cells specific for P. falciparum CS in human volunteers. Erythrocytic Stage Peptide Vaccines Synthetic peptide polymer: SPf66 vaccine. The SPf66 polypeptide was the first synthetic peptide blood stage vaccine to be tested in extensive clinical field trials carried out in the 1990 s. 68,69 SPf66 was comprised of a polymer of peptide sequences derived from three proteins of infected erythrocytes (MSP-1, and unknown proteins of 55 kda and 35 kda MW) that were shown to be protective in Aotus monkeys. The Phase I III trials of SPf66/ alum demonstrated that while first attacks with P. falciparum were reduced by 30% in South America (four trials), the vaccine had limited efficacy in Africa (four trials) and Asia (one trial). 70 The alum formulation required large vaccine doses (2 mg) in adults and elicited short-lived antibody and low T cell responses. A more recent Phase I trial explored SPf66 formulated in the more potent QS21 adjuvant formulation (Table 1). 71 Addition of QS21 elicited anti-immunogen titers 2 logs higher than alum. However, positive IFA against P. falciparum parasites were detected in only 37% of vaccinees, with maximum titers 320 for all groups. Following the third immunization, two individuals had severe vaccine allergy that led to a decision to stop further immunizations. Current efforts on improving polymer vaccine design have focused on optimization of peptide binding to class II molecules to increase immunogenicity of new blood stage peptides for development of asexual stage vaccines. 72 The enhanced binding to class II molecules correlated with enhanced immunogenicity, as assayed in protection of DR typed Aotus monkeys against P. falciparum blood stage challenge. Long synthetic peptides (LSP) blood stage vaccines. Glutamate rich protein (GLURP) LSP: Design. Glutamate-rich protein (GLURP) is a 220 kda antigen found on the surface of merozoites that is the target of antibodies associated with protection in children and adults in Africa. 73 The GLURP LSP vaccine was synthesized to contain the N terminus aa , which is relatively conserved. The N terminus contains multiple Th epitopes as well as a dominant B cell epitope (aa ) that is the target of human IgG1/IgG3 cytophilic antibodies that mediate Antibody Dependent Cellular Inhibition (ADCI) Mice immunized s.c. with GLURP LSP formulated in Montanide ISA 720, elicited anti-peptide ELISA titers of in multiple inbred strains and positive IFA titer of 10 3 with mature schizonts in 60% of F1 mice. 76 Phase I trial GLURP LSP vaccine. Phase I trial of LSP GLURP 85- assessed safety and immunogenicity of alum or Montanide ISA vaccine formulations administered s.c. at 0, 1 and 4 months (Table 1). 77 Vaccinees immunized with 10, 20 and 300 ug GLURP LSP developed a dose dependent cellular and antibody response. Reactogenicity in the Montanide group led to exclusion of 8/18 volunteers, the majority in the high dose group. After two immunizations, PBMC of the majority of volunteers proliferated in response to stimulation with the GLURP LSP. The presence of IFNγ, but not IL-10, was detected in culture supernatants of the peptide stimulated cells. Consistent with a Th1- type cellular response, primarily cytophilic (IgG1) antibodies were elicited. Antibodies specific for the dominant B cell epitope (aa ) were detected, with maximal titers of 10 3 post second or third dose of LSP formulated in Montanide or alum, respectively. Sera of the majority of volunteers (21/28) reacted with P. falciparum (NF54) schizonts, with peak IFA titers Importantly, the GLURP specific antibodies elicited by LSP were shown to be functional in the in vitro ADCI assay. Plasma from each adjuvant formulation with the highest cytophilic antibody titers was shown to inhibit parasite growth in the presence, but not the absence, of human monocytes, similar to inhibition obtained with hyperimmune serum from adult Africans. Merozoite surface protein-3 (MSP 3) LSP. Design. MSP3, a 48 kda protein on the merozoite surface, was the first ADCI target to be identified using cytophilic antibodies from serum of clinically protected African adults. 78 A conserved domain of MSP3 was identified as the target of antibodies mediating protective ADCI, as measured in vivo in a P. falciparum humanized mouse model, Pf-HuRBC. 79,80 In this model, P. falciparum IRBC are engrafted in BXN (Beige, X-linked immunocompromised X nude) mice by continual depletion of phagocytes, through treatment with liposome encapsulated dichloromethylenediphosphonate and anti- PMN MAB every 3 4 days. The engrafted mice are injected with fresh human AB + erythrocytes at 3 4 day intervals to maintain parasitemia. In this in vivo ADCI model, purified IgG (6 mg) from clinically immune African adults, when administered with human CD14 + monocytes, led to a significant reduction in parasite levels. Passive transfer of monocytes and anti-msp3 IgG from the African immune serum which was affinity purified on a 27mer peptide representing a dominant MSP3 B cell epitope, similarly Human Vaccines 33

9 led to a rapid 10-fold decrease in parasitemia in the Pf-HuRBc mice. Additional analysis using affinity-purified hyperimmune serum from clinically immune Senegalese adults identified multiple cytophilic antibody targets and Th1 cell epitopes in a conserved 70aa sequence from the C terminus of MSP3, aa , which was subsequently used for development of MSMP3 LSP for clinical trials. Phase I trial of MSP3 LSP vaccine. A Phase Ia trial examined the safety and immunogenicity of MSP-3 LSP administered s.c. at 0, 1 and 4 months (Table 1). 81 The MSP LSP formulated in Montanide ISA 720 at doses of 10, 30, 100 or 300 ug, was compared to alum-adsorbed 30 and 100 ug peptide doses. A total of 35 volunteers were immunized, but DTH reactions (erythema and/or induration) in Montanide groups led to withdrawal of five volunteers after second injection, and reduction of Montanide dose to 10 ug for subsequent doses. The majority of volunteers (23/30) seroconverted to the immunogen, with median 10 3 ELISA titer following the third dose in all groups. The comparable immunogenicity of the alum and Montanide formulations was in contrast to preclinical murine and monkey studies, in which the alum adsorbed LSP MSP3 formulation was poorly immunogenic. Following the third immunization, 60 70% of the volunteers in both adjuvant groups had antibodies that reacted with mature schizonts of P. falciparum (IFA titers ) and positive western blots, indicating the MSP3 LSP elicited antibody reactive with native protein. Both the alum and the Montanide formulations elicited predominantly cytophilic IgG1 antibody. Noteworthy was the strong T cell proliferative responses (median S.I. 27) and parallel IFNγ production observed in the majority of volunteers, which persisted to month 12 of the study. The biological function of the vaccine-induced anti-msp3 antibodies was examined by in vitro assays and using the Pf-HuRBc ADCI mouse model. 82 The majority of western blot positive sera gave significant levels of inhibition (>30%) in ADCI in vitro. Several serum in both adjuvant groups inhibited growth of parasites comparable to, or exceeding, levels obtained with positive control serum obtained from clinically immune African adult sera. The levels of ADCI activity persisted longer in serum from volunteers immunized with the alum, as compared to the Montanide, adjuvant formulation. In the in vivo ADCI assay, passive transfer of three western blot positive sera in combination with human monocytes significantly inhibited P. falciparum parasitemia in the Pf-HuRBC mice. Transfer of monocytes with pre-immune sera, or with sera from western blot negative individuals, did not reduce parasitemia. In vivo ADCI was also positive in two volunteers retested eight months after the final immunization. There was a significant correlation between ADCI and the presence of IgG1 and IgG3 antibodies, consistent with the ability of the LSP MSP3 vaccine to elicit functional antibody dependent monocyte mediated protection. The encouraging Phase 1a trial results lead to a Phase 1b trial in West African adults immunized with 30 ug of MSP3-LSP/ alum. 83 In contrast to the Phase 1a trials in malaria naïve volunteers, there was no increase in IgM or IgG antibody to MSP3, nor to overlapping peptides spanning the aa sequence, in the African adults. Positive PBMC proliferation and IFNγ production was obtained following stimulation with MSP3-LSP, although the magnitude of the response was significantly lower than in the Phase Ia trial. The lack of a significant antibody increase was attributed to the presence of high levels of pre-existing antibodies in the African adults, suggesting that studies in children who have not yet acquired immunity to MSP3, and who are the primary target for a vaccine, may be more informative. Multistage Malaria Peptide Vaccines Malaria peptide virosomes. Virosomes, also known as IRIV (Immunopotentiating Reconstituted Influenza Virosomes), are 150 nm unilamellar liposomal vesicles containing integrated influenza virus glycoproteins that can function both as vaccine carrier and adjuvant system. 84 The presence of the influenza HA glycoprotein on the surface of the virosomes facilitates uptake and endosomal processing for class II antigen presentation of foreign antigens. In addition, as in the virus infected cell, the viral glycoproteins can mediate fusion with host cell membrane so that antigens encapsulated within the virosome lumen are released into the cytosol for entry into the class I antigen processing pathway. 85,86 Virosome based viral vaccines have been licensed in 40 countries and delivered to more than 20 million people including infants and children. 87 A virosomal hepatitis A vaccine comprised of formalin-inactivated hepatitis A virus non-covalently coupled to the IRIV vesicle, and a virosome influenza vaccine comprised of HA and NA integrated vesicles, are commercially available for i.m. immunization. An intranasal virosome flu vaccine, containing a heat-labile toxin (HLT) derived from enterotoxigenic Escherichia coli as a mucosal adjuvant, was also licensed. 88,89 In contrast to the safety of the parenterally administered virosomes, the intranasal formulation was associated with increased incidence of Bell s Palsy, one sided paralysis of facial muscles, and was subsequently withdrawn from market. 90 Design. A malaria virosome vaccine was designed to contain a combination of pre-erythrocytic and erythrocytic antigens. Structurally constrained peptides from either CS Apical Membrane Antigen 1 (AMA-1) blood stage antigen were conjugated to phosphatidylethanolmine (PE) and integrated into virosomes containing influenza A viral glycoproteins. In mice, the CS virosome (termed PEV302 or UK-39), comprised of a constrained peptide with multiple NPNA repeats, elicited antibodies reactive with P. falciparum sporozoites when administered i.m. to influenza-primed BALB/c mice. 91 MAB (200 ug/ml) derived from the CS peptide (UK 39) virosome immunized mice inhibited % invasion of P. falciparum sporozoites into primary human hepatocytes in vitro. Purified IgG from two UK39 immunized rabbits (1 mg/ml) inhibited 40 70% of sporozoite invasion in vitro. Immunogenicity of the CS peptide virosome required the presence of influenza-specific T and B cells in the primed mice, as immunization of naïve mice, or using peptide/liposomes without viral glycoproteins, elicited minimal antibody, indicating the T help for the anti-cs antibody responses was provided by the viral proteins contained in the virosome Human Vaccines Volume 6 Issue 1

10 The malaria blood stage virosome vaccine was designed to contain a cyclic 49mer peptide representing a semi-conserved domain III of the ectodomain of AMA-1, an 83 kda protein localized in mature blood stage parasites that functions in merozoite invasion of RBC. 93,94 Mice immunized with AMA-1 virosomes containing either a linear or cyclized 49mer peptidomimetic of AMA-1 aa , conjugated via the N terminus to PE and incorporated into IRIV, elicited antibodies reactive with parasites, as measured by IFA and western blot. 95 MABs (300 ug/ml) derived from AMA-1 49-C1 virosome immunized mice reduced P. falciparum blood stage parasite growth 40 95% in vitro. Phase I trial of AMA-1 and CS virosomes. For clinical trials, virosomes comprised of PE modified AMA 49-C1 (PEV301) or CS peptide (PEV302) were incorporated into vesicles containing phosopholipids (100 ug), and glycoproteins HA (10 ug) and NA (2 ug) from influenza A virus. 96 Volunteers received three i.m. injections of either the AMA1 or the CS virosome, or a combination of both virosomes, at 0, 2 and 6 months (Table 1). The CS (UK-39) virosome at the optimal concentration (10 ug), elicited anti-uk 39 peptide and anti-sporozoite antibodies in all volunteers, with peak titers of 10 4 and 10 3 respectively. 91,96 The combination of CS and AMA-1 virosomes did not significantly alter the magnitude of antibody responses to either the UK9 or AMA-1 C49 peptide. Purified total IgG (1 mg/ml) from four vaccinees receiving CS virosome alone, and three receiving the combined virosome formulation, were found to inhibit sporozoite invasion into primary human hepatocytes in vitro by 44% (range 18% 74%). Anti-UK9 peptide ELISA and anti-sporozoite IFA titers remained detectable one year post third immunization. No positive cellular responses to the CS peptide were detected in vaccinees. In volunteers receiving the AMA1 virosomes, positive PBMC lymphoproliferation following stimulation with the AMA1-C1 49mer peptide was detected at at least one time point in 50% (8/16) volunteers (SI range 2 4.5). 92 All volunteers immunized with AMA-1 virosome, either alone or combined with CS virosome, developed anti-peptide antibodies after first or second immunization, respectively, with peak anti AMA-1 peptide ELISA titers of Despite high anti-ama-1 peptide titers in all vaccinees, a minority of the vaccinees had positive IFA with malaria blood stage parasites, 96 and 7/16 had positive western blots with P. falciparum IRBC. 92 In murine studies, pre-existing flu specific immune responses were required for optimal immunogenicity of the malaria virosomes. 92 PBMC of all volunteers had pre-existing CD4 + cellular immunity to influenza HA protein, as measured by IFNγ ELISPOT and lymphoproliferation assay with inactivated influenza virus. 92 Flu-specific cellular responses did not increase following immunization with virosomes. The magnitude of pre-existing influenza cellular immunity did not correlate with enhanced AMA1 proliferative responses. Phase I/II trials of a combined CS/AMA-1 virosomes with or without ME-TRAP. Phase I/II studies were carried out to examine protective efficacy of the optimal CS and AMA-1 virosome formulations, delivered at 0, 1 and 2 months, when challenged by exposure to the bites of P. falciparum infected mosquitoes (Table 1). 97 In an effort to enhance immune responses, protection was also assayed in volunteers receiving virosomes co-delivered with recombinant viral vectors expressing a multi-epitope malaria construct ME-TRAP. The ME-TRAP antigen is comprised of preerythrocytic stage CD4 + and CD8 + T cell epitopes (ME) fused to the Thrombospondin Related Adhesive/anonymous Protein (TRAP). Previous clinical trials of heterologous prime boost immunization schemes of ME-TRAP expressed in fowl pox (FP) or vaccinia virus (MVA) vectors, demonstrated ability to elicit high levels of CD4 + TRAP specific cellular responses and sterile protection in several volunteers. 98,99 Murine studies also demonstrated a synergistic effect of combined CS and ME-TRAP immunization, with the recombinant viral vectors functioning as adjuvants for co-delivered immunogens. 100,101 In the clinical trials, virosomes were delivered i.m., while the recombinant viral vectors were injected i.d. in the contralateral arm. All 24 volunteers who received the full course of three immunizations participated in the challenge study. Anti-UK9 repeat peptide antibody were induced in all vaccinees following second immunization with either virosomes or virosomes + ME-TRAP, but antibodies elicited with virosome + ME-TRAP were of lower avidity and lower IFA titers. All volunteers seroconverted to AMA1 peptide following a single immunization with both virosome formulations, with positive IFA to blood stage parasites in 6/24 vaccinees. Functional inhibitory antibody against blood stage parasites was not detected in vitro in a Growth Inhibition Assay (GIA) which used a parasite strain that was identical to the vaccine AMA-1 sequences. As in the previous Phase I trial, 92,96 IFNγ ELISPOT to either the AMA1 or the CS peptide were negative in vaccinees immunized with either virosome or virosome + ME-TRAP. Positive IFNγ ELISPOT responses to ME peptides or TRAP protein were obtained (50 SFU/10 6 PBMC), however these were a log lower than in previous studies with ME-TRAP viral vectors, suggesting negative interference when combined with virosomes. Following challenge by P. falciparum infected mosquitoes, the time to patent infection in vaccinees was not significantly different from controls. Consistent with low anti-cs IFA and ME-TRAP ELISPOT responses, the estimated numbers of P. falciparum infected hepatocytes were also the same in vaccinees and controls. In the previous Phase 1 trial, 91,96 sporozoite inhibitory activity in vitro was obtained with IgG of volunteers immunized with CS virosomes, but in vitro analysis was not reported in the Phase II trial. Evidence for an anti-blood stage response, however, was suggested by significantly lower rates of blood stage parasite growth in both groups of vaccinees when compared to controls. In addition, the presence of abnormal blood stage parasites, presumably crisis forms, was observed in the blood of all vaccines vs. 30% of controls. Perspectives The clinical trials over the past decade have examined safety and immunogenicity of new malaria peptide vaccine designs, delivery systems and adjuvant formulations (Table 1). In these trials, the peptide vaccines were shown to elicit neutralizing anti-repeat antibodies to target the extracellular sporozoite and Human Vaccines 35