Synthetic vaccine update: Applying lessons learned from recent SPf66 malarial vaccine physicochemical, structural and immunological characterization

Vaccine 25 (2007) 4487–4501

Synthetic vaccine update: Applying lessons learned from recent SPf66 malarial vaccine physicochemical, structural and immunological characterization Adriana Berm´udez a , Claudia Reyes b , Fanny Guzm´an b , Magnolia Vanegas b , Jaiver Rosas c,f , Roberto Amador c,f , Raul Rodr´ıguez c , Manuel Alfonso Patarroyo d,f , Manuel Elkin Patarroyo e,f,∗ a

Nuclear Magnetic Resonance Department, Fundaci´on Instituto de Inmunolog´ıa de Colombia, Bogota, Colombia b Chemical Synthesis Department, Fundaci´ on Instituto de Inmunolog´ıa de Colombia, Bogota, Colombia c Vaccinology Department, Fundaci´ on Instituto de Inmunolog´ıa de Colombia, Bogota, Colombia d Molecular Biology Department, Fundaci´ on Instituto de Inmunolog´ıa de Colombia, Bogota, Colombia e Fundaci´ on Instituto de Inmunolog´ıa de Colombia (FIDIC), Cra. 50 No. 26-00 Bogota, Colombia f Universidad Nacional de Colombia, Bogota, Colombia Received 31 October 2006; accepted 7 March 2007 Available online 26 March 2007

Abstract The SPf66 synthetic malaria vaccine, developed and obtained almost 2 decades ago, represents the first approach towards developing a multi-antigenic, multi-stage synthetic malarial vaccine composed of subunits derived from different Plasmodium falciparum stage proteins. It is shown here that batches 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15 and 16 produced from a few milligrams to kilogram amounts and used in assays on monkeys and humans showed high reproducibility in physicochemical analysis. 1 H NMR two-dimensional studies also revealed high similarity, even in non-oxidized batches. Reproducibility was also high, especially in preclinical studies carried out on Aotus, clinical trials Phase I, IIa and IIb and field-studies carried out in La Tola, Rio Rosario (Colombia), Majadas (Venezuela), La Te (Ecuador), Ifakara (Tanzania) in which there was high antibody titer production, having similar population distribution when done with different batches. These results provide great support for peptide-synthesized vaccines containing minimal epitopes from protection-inducing antigens which have several advantages, such as low cost, safety, reproducibility, stability, being straightforwardly scaled-up from milligram to kilogram amounts; make them the vaccines of choice for the future in a worldwide attempt to scourge diseases such as malaria. © 2007 Elsevier Ltd. All rights reserved. Keywords: SPf66; Malaria; MALDI-TOF; DC; HPLC; NMR; Immunogenicity

Abbreviations: FVO, falciparum Vietnam Oak noll; HPLC, high performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; GMP, good manufacturing practices; GLP, good laboratory practices; FCA, Freund’s complete adjuvant; FIA, Freund’s incomplete adjuvant; FAST-ELISA, Falcon assay screening test enzyme-linked immunosorbent assay; IPV, Imovax Polio Vaccine; ␣-CCA, ␣-cyano-4-hydroxycinnamic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of flight; CD, circular dichroism; DQF-COSY, double-quantum-filtered correlated spectroscopy; TOCSY, total correlation spectroscopy; NOESY, nuclear overhauser enhancement spectroscopy; QS-21, Quillaja saponaria-21 ∗ Corresponding author at: Fundaci´ on Instituto de Inmunolog´ıa de Colombia (FIDIC), Cra. 50 No. 26-00 Bogota, Colombia. Tel.: +57 1 4815219; fax: +57 1 4815269. E-mail address: [email protected] (M.E. Patarroyo).

0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.03.016

4488

A. Berm´udez et al. / Vaccine 25 (2007) 4487–4501

1. Introduction There are many approaches towards developing an effective anti-malarial vaccine. The paradigm regarding how a vaccine may be used efficiently is that its design includes economic methods for developing it, valid physicochemical characterization techniques, scenarios for field-studies in conditions close to those of the real world where determining clinical cases must be as exact as possible (absolute absence of parasites in the blood and the total absence of fever), the target population must be genetically compatible and mature from the immune point of view and a there must be a single dose for achieving large-scale coverage. SPf66 was the first chemically synthesized vaccine (more than 18 years ago) against the causal agent of human malaria, Plasmodium falciparum having the greatest prevalence and lethality [1,2]. Extensive basic, preclinical and clinical studies, as well as field-trials, have been carried out aimed at determining its protective efficacy [1]. The 45-amino-acidlong SPf66 molecule is a synthetic hybrid chimera, composed of the amino-acid sequence from 3 merozoite protein-derived peptides plus the PNANP sporozoite repeat sequence intercalated twice, to which glycine and cysteine (GC) were added at their amino- and carboxy-terminal ends to allow their polymerization by oxidation [2]. Pre-clinical immunogenicity and protection studies in the experimental Aotus monkey model (Phase 0) indicated that this synthetic molecule was highly immunogenic and totally protective (as no parasites appeared in blood) in ∼40% of the immunized monkeys exposed to experimental challenge with a highly infective, Aotus monkey-adapted, heterologous P. falciparum strain (FVO) [3]. Clinical safety and immunogenicity studies in large groups of humans (Phase I and II) showed that this synthetic vaccine was safe and highly immunogenic, since it was administered exclusively with aluminum hydroxide [Al(OH)3 ] as adjuvant (the only immunopotentiator permitted at this time), inducing high antibody titers as determined by ELISA and recognizing merozoite lysate proteins by Western blot. Different batches of chemically synthesized SPf66, analyzed in large-scale field-studies, were produced throughout the 1980s when vaccine production was scaled-up from a few milligrams to 1.2 kg. It was the first and is the only chemically synthesized vaccine with which thousands of people were vaccinated and with which physical–chemical, biological and structural studies have since been carried out. These studies have been carried out with different batches produced and used in both Aotus and humans for identifying reproducibility, scaling-up possibilities and improving immunogenicity by adding new adjuvants for developing new generations of chemically-synthesized vaccines. This data has provided new knowledge regarding developing synthetic vaccines, particularly against malaria caused by P. falciparum which is desperately needed due to extremely high annual morbidity (∼500 million sick) and mortality (∼2

million deaths, mainly in children aged less than 5) around the world [4].

2. Methods 2.1. Synthetic malarial vaccine SPf66 production Large-scale SPf66 production involved synthesizing it by using standard t-Boc solid-phase peptide methodology, previously described by Merrifield [5] and modified by Houghten [6], in polypropylene bags with p-methylbenzhydrylamine resin HCl (Bachem, Torrance CA, USA). The polypeptide’s primary structure is: CGDELEAETQNVYAAPNANPYSLFQKEKMVLPNANPPANKKNAGC. The resin was deprotonated by adding 5% diisopropylethylamine (Merck) in methylene chloride before introducing the first amino acid. The coupling cycle was initiated by submerging the bags in a solution containing equimolecular amounts of t-Boc amino acid (BachemPeninsula, Torrance, CA, USA) and diisopropylcarbodiimide (Merck), in a 10-fold molar excess over available amine in the bag. The reaction was allowed to proceed for 60 min and the product was washed with methylene chloride. The t-Boc groups of the newly coupled amino acids were removed with 55% trifluoracetic acid (Pierce, Rockford, IL, USA) in methylene chloride. Reaction products were washed and amino groups deprotonated with diisopropylethylamine. asparagine (Asn) and glutamine (Gln) coupling was carried out by adding 1-hydroxy-benzotriazole hydrate (Aldrich) in dimethylformamide. Protected amino acids were liberated from the resin by treatment with 10% anisole in anhydrous hydrogen fluoride (Air Products, Allentown, PA, USA) for 60 min at 0 ◦ C. The hydrogen fluoride was distilled from the reaction and the product was washed five times with ethyl ether (Merck). The peptides were subsequently extracted in 5% acetic acid (Merck) and then lyophilized and analyzed by different techniques. All batches were synthesized with Cys-Gly in peptide’s N- and C-terminus to allow polymerization by an oxidation reaction forming disulfide bonds. This procedure has been carefully standardized to guarantee the inclusion of high-molecular-mass polymers for immunization purposes. For this purpose each batch was dissolved in distilled water (4 mg/ml), pH was adjusted to 7.4 and blowing of medicinal oxygen was carried out for 12 h until obtaining the final product. Ellman’s test [7] was then used for identifying free thiols and thus verifying the complete formation of disulphide bridges (polymer). Dialysis was then carried out using membranes with a 6–8 kDa pore-size for removing monomers which might have cycled and low molecular weight contaminants, thereby being used for immunological studies. HPLC, SDS-PAGE, amino acid content and amino acid sequence were used for testing each vaccine batch following synthesis. Sterility, cytotoxicity and pyrogenicity tests

A. Berm´udez et al. / Vaccine 25 (2007) 4487–4501

(in rabbits) were performed on the final product before its use on humans. Three subcutaneously administered doses in a final volume of 0.5 ml, each consisting of 1 or 2 mg of the synthetic polymerized peptide adsorbed onto 1 mg of alum hydroxide Al(OH)3 (provided by the Instituto Nacional de Salud de Colombia which they had obtained from Biosector, Denmark) were used for all human trials. The dose was decreased two-fold for the Tumaco E1 , E2 and E3 trial, although maintaining the 2:1 ratio between peptide and adjuvant. Different SPf66 batches (batch 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15 and 16) were produced in the Instituto de Inmunolog´ıa del Hospital San Juan de Dios’ Chemistry Laboratory (Bogot´a) and then at the Fundaci´on Instituto de Inmunolog´ıa de Colombia (FIDIC, Bogot´a) under good manufacturing procedures (GMP) and good laboratory practices (GLP) for studies in humans. 2.2. Pre-clinical assays or Phase O studies 2.2.1. Animals Previously unused, spleen-intact Aotus monkeys from the Amazon jungle were used in our Leticia trials; they were kept in our monkey colony in Leticia, Amazonas (Colombia). The monkeys were captured in the jungle by experienced local trackers. Field information was compiled (source, family group size, capturing conditions, etc.) on arrival and the monkeys were weighed and housed singly in stainless steel cages (80 cm × 80 cm × 60 cm) in different isolation rooms with ≤20 animals/room. Temperature, relative humidity, climatic changes and other environmental factors in which the monkeys were kept resembled their natural habitat, since Leticia is located in the middle of the Amazon jungle. All monkeys were phenotypically characterized and clinically studied. Blood obtained from the femoral vein (3–4 ml/monkey) was used for immunological, chemical, hematological and parasitological tests. The monkeys were maintained in quarantine before any trials. Those included in this report weighed an average of 800 g and had >45% hematocrite and negative serological and parasitological tests for Plasmodium. Members of the same family were randomized into different groups to avoid immunological phenomena attributable to immune response genes [3]. The presence of antibodies against P. falciparum was assessed prior to the first immunization via immunofluorescence antibody tests starting at 1:20 dilution. Those monkeys proving positive were immediately released back into the jungle without further manipulation. All monkeys were handled according to NIH and CORPOAMAZONIA guidelines for animal handling. 2.2.2. Monkey immunization for Leticia trials Monkeys were immunized with SPf66 (batch 03); 250 ␮g of this synthetic polymeric protein were dissolved in 0.25 ml distilled water emulsified in an equal volume of Freund’s complete adjuvant (FCA) containing 1 mg/ml of dead Mycobacterium tuberculosis for each monkey for the first

4489

immunization and Freund’s incomplete adjuvant (FIA) for the next two immunizations on days 20 and 40. They were intra-dermally immunized in multiple places (≥16) in the abdominal region. Control monkeys were immunized with the same volume of FCA and FIA in saline solution, using the same immunization conditions as in the experimental groups [3]. 2.2.3. Immunizations at the CDC in Atlanta Primary immunization consisted of 250 ␮g SPf66 (batch 03) synthetic polymeric vaccine emulsified with an equal volume of FCA. The FCA contained 0.1 mg/ml of dead Mycobacterium tuberculosis. A total 0.4 ml volume was injected intra-muscularly in 4–8 doses of 0.05 ml each dose in the right and left deltoid muscles and triceps and right and left hamstring and gluteus muscles. The second to fifth immunizations were administered 28, 42, 56 and 70 days after the primary immunization. The antigens were emulsified in FIA for these immunizations and administered intramuscularly in 4 doses of 0.1 ml each in both arms and both legs [8]. 2.3. Phase I and II clinical trials 2.3.1. Volunteers Several trials were performed with volunteers from the Colombian Armed Forces to determine the best immunization regime in humans and to assess toxicity, immunogenicity and reactogenicity. Volunteers for the Tumaco A to G trials were all young men, aged 18–22, in excellent physical condition and having had at least 6 years of schooling. They were all Colombian soldiers who had previously been living in low endemic areas for malaria (i.e. having no previous clinical history of being exposed to any type of malaria). All individuals were clinically evaluated before being allowed to participate in the study. Written consent was obtained from each individual [9]. After each injection, all volunteers, those being vaccinated with SPf66 + Al(OH)3 or controls receiving only Al(OH)3 were observed for 60 min and examined for local or systemic side-effects by expert physicians; clinical examinations were repeated 24 and 48 h later and 5 and 20 days later to detect late hypersensitive reactions. Blood samples were drawn pre-immunization and 1 day, 5 days and 20 days after each dose had been administered to determine changes induced by immunization during their clinical laboratory test (32 parameters were assessed) and thus determine the toxicity of the SPf66 anti-malarial vaccine. The same blood samples were also used to determine the presence of antibodies (thoroughly described in this manuscript) against the vaccine by FAST-ELISA and against parasite lysate by Western blot. Since only a brief description of antibody levels (geometric mean titer) was shown in corresponding studies at the time [1,2,9–17] a thorough analysis of these sera was performed in this manuscript for the presence of these antibodies and their distribution in the different trials.

4490

A. Berm´udez et al. / Vaccine 25 (2007) 4487–4501

2.3.2. Human trials The study groups called Tumaco A, B, C, E, E2 , E3 and G, their formation, immunization schedule and specific objectives are described below: Tumaco A. This study involved 193 volunteers split into two groups. The first group had 63 individuals who were immunized on days 0, 20 and 220 with 2 mg of the SPf66 anti-malarial vaccine (batch 04) adsorbed on Al(OH)3 ; the second group of 130 individuals each received saline solution in alum hydroxide (Al(OH)3 ) as a placebo [10]. Tumaco B. The interval between the second and third doses was reduced in this trial, relative to the Tumaco A trial, and immunizations were performed on days 0, 20 and 90. This group consisted of 122 volunteer soldiers who received 2 mg SPf66 vaccine (batch 04), adsorbed as before, and 84 who received saline solution in Al(OH)3 as a placebo [10]. Tumaco C. The 38 volunteers in this group received the SPf66 vaccine (batch 06) adsorbed on Al(OH)3 and 38 more received the SPf66 vaccine (batch 06) adsorbed on calcium phosphate Ca3 (PO4 )2 (Superfos Biosector, Denmark) on days 0, 30 and 150 [10]. Fifty volunteers participated per group in Tumaco E1 , E2 and E3 , receiving 2, 1 and 0.5 mg of the vaccine (batch 07) adsorbed on 1, 0.5 and 0.25 mg Al(OH)3, respectively, on days 0, 30 and 180. No controls were included in this group. Tumaco G. An immunization regime and characteristics’ confirmatory group consisting of 93 adult volunteers, described here for the first time, was immunized with 2 mg of the vaccine (batch 08) adsorbed on Al(OH)3 on days 0, 30 and 180. 2.3.3. Safety and immunogenicity in children aged 1–15 The vaccine was administered to 292 children aged 1–14 following the same procedure as in confirmatory group (unpublished results). The schedule used was 0, 30 and 180 days, day 0 being defined as the day on which the first dose was applied by subcutaneous administration of 1 mg of the SPf66 vaccine (batch 05) adsorbed on alum hydroxide and dissolved in saline solution, for children <5 years old, and 2 mg of the vaccine for children over this age. A thorough medical examination was carried out on each of the children before administering each dose of the vaccine to detect the presence of any side-effects. Written permission was obtained from the parents [11]. 2.3.4. Macrotumaco–immunization and safety precautions A large field-trial involving 15,351 people >1-year-old named Macrotumaco was started in the same mesoendemic area for malaria on the Pacific Coast of Colombia to determine the safety, immunogenicity delivery conditions, operability, logistics for administration, etc. The SPf66 vaccine (0.5 ml) was subcutaneously injected into each individual, as previously described. Children <5 years old received half this dose. The first dose was given intradermically in the left deltoid

area, the second in the right deltoid and the third again in the left deltoid to evaluate any possible local reaction associated with the injections. The vaccination schedule was 0, 30, and 180 days; vaccine from a mixture of batches 6 and 7 was given for the first two doses and from batch 09 for the third dose. Ten vaccination posts, each having four internal areas, were constructed in each geographical region. One area in each post was for registration procedures, another for vaccination and a third for general observations. The fourth was a critical care management area for treating patients in case of any severe immediate hypersensitivity reaction. Each post was staffed as follows: one intensive-care specialist, three medical interns from the Universidad Nacional de Colombia, one pediatrician, several nurses and staff from the Colombian Malaria Eradication Service. Adverse hypersensitivity reactions and any general discomfort which could be ascribed to the vaccine were recorded 30 min and 48 h after each immunization. These were evaluated and registered according to the criteria established by physicians specializing in internal medicine and intensive care. A 9957 people completed the full immunization regime 6 months later as this is a very spread-out and poor rural area [9]. A representative sample of 757 subjects was selected for logistical reasons from the total vaccinated population according to age and sex distribution for analyzing the immunogenicity of this trial. Sera from these vaccinees were obtained before applying the first dose and 20–30 days after the third dose when the highest antibody titers were observed [18]. The samples were kept at −20 ◦ C in polypropylene tubes, without preserving agents, until use. 2.4. Phase III studies 2.4.1. Clinical field-trials regarding immunogenicity and protection conferred by SPf66 in different parts of the world 2.4.1.1. Vaccination for immunogenicity and protection studies in La Tola, Colombia [12]. A field-study was mounted for determining the protective efficacy of the best vaccination regimens in an area which was mesoendemic for malarial in Colombia (La Tola) based on prior studies. Seven hundred and thirty-eight people participated in it who were vaccinated with SPf66; 631 aged over 5 received 2 mg of synthetic vaccine SPf66 whilst 107 children aged 1–5 included in the study received 1 mg SPf66 adsorbed on Al(OH)3 on days 1, 30 and 180. Another 810 people aged 1–78 participated as controls, receiving tetanus toxoid in the first dose and Al(OH)3 in the second and third doses as placebos. Batch 08 was used for this study. Passive and active monitoring was begun 30 days later (lasting 1 year) by doctors and expert personal in malaria (from the Colombian Malaria Eradication Service) for detecting cases presenting ≥37.5 ◦ C fever and having a positive diagnosis of malaria. A single parasite, appearing in three consecutive samples was considered to be a positive sign of malarial infection (such criteria was used in all trials performed in South America).

A. Berm´udez et al. / Vaccine 25 (2007) 4487–4501

2.4.1.2. Rio Rosario efficacy trial in Colombia [17]. A new study was carried out for determining the time during which protection lasted (≥2 years) induced by synthetic vaccine SPf66 in another area which was mesoendemic for malaria on Colombia’s Pacific coast; this was called R´ıo Rosario, lying 100 km distant from the other areas but having similar climatic and epidemiological malarial conditions to those found in Tumaco and La Tola. Six hundred and thirty-four people aged more than 1-year-old received the vaccine (batch 10) adsorbed as described previously; another 534 people received tetanus toxoide and Al(OH)3 as placebo, following the same immunization regimens as described before. The criteria for determining safety, immunogenicity and protective efficacy were the same as those described for the studies carried out in Macrotumaco and La Tola. 2.4.1.3. La Te, Ecuador, efficacy trial [14]. This trial was carried out in an area which was mesoendemic for malaria on Ecuador’s Pacific coast; 230 Indoamerican/European volunteers aged ≥1-year-old participated in the study. They received SPf66 (batch 09) adsorbed on Al(OH)3 brought from Colombia and 234 having the same ethnic background received the placebo in the form described above. 2.4.1.4. A population-based clinical trial in Majadas, Venezuela [15]. A 1422 people >11 years old received the SPf66 vaccine (batch 09) formulated as described above on days 0, 20 and 112; however, 160 individuals received the third dose on day 156 due to logistical problems involving transport. Following the third dose, a comparative group of 938 people having the same characteristics paired by similar gender, age, occupation and geographical distribution were included as controls without receiving the placebo; however, their clinical and hematological situation was followed-up in the same conditions as the vaccinated people. 2.4.1.5. Efficacy field-trial in Tanzania [13]. Based on the positive results obtained in Colombia, Venezuela and Ecuador, an immunogenicity and protective efficacy fieldtrial was performed in Tanzania in an area of intense perennial malarial transmission (the Kilombero valley) in children aged 1–5; 274 received 1 mg of the vaccine (batch 09) adsorbed on Al(OH)3 (Llorente SA batch 10/92, 16679/1/10) whilst 312 children received tetanus toxoid in the first dose and Al(OH)3 in the second and third doses as placebo control on days 0, 30 and 180. 2.4.1.6. Protective efficacy trial in children aged 6–11 months old in Gambia [16]. SPf66 batch 09 was used in this study carried out by the Gambian Medical Research Council and the London School of Tropical Medicine and Hygiene. A mistake in the trial occurred when investigators mixed up the coding for the syringes when the third dose was delivered; 154 children consequently received the wrong injection, half of them receiving the placebo and the other half Imovax Polio Vaccine (IPV), which was being used as control, instead of

4491

SPf66. The children who thus received SPf66 instead of the third dose of control IPV were thus excluded from the analysis. Those receiving IPV instead of SPf66 were immunized later on with just the third dose of the synthetic malarial vaccine. Three hundred and sixteen children aged 6–11 months thus received SPf66 versus 231 who were immunized with IPV. The above has provided some historical background on the immunological and clinical work done with SPf66, displaying the immunological result briefly presented in their corresponding previous papers. A thorough description of these trials is presented in Figs. 1 and 2. Furthermore some comments must now be made regarding recent research (1996 onwards) into characterizing SPf66 by SEC-HPLC, MALDITOF, CD and 1 H NMR to demonstrate the reproducibility of this synthetic vaccine at the deepest levels independent of the amounts produced and recent emphasis on quality control. 2.4.2. Immunological studies 2.4.2.1. Serum samples. Venous blood was drawn in each trial to perform all immunogenicity studies according to a clearly defined bleeding schedule. The sera were separated and aliquots frozen and stored at −20 ◦ C without conserving agents. 2.4.2.2. Falcon assay screening test (FAST-ELISA). Vaccinated volunteers’ IgG antibody levels determined by FAST-ELISA (Becton Dickinson Labware, Oxrand, CA) which is briefly described below. This is a modification of the previous method reported by Campbell et al. [19]. A 10 ␮g of the peptide per ml, diluted in phosphate-buffered saline (PBS), pH 7.2, were placed in microtiter plates and incubated with constant shaking at room temperature for 30 min. Antigen-coated lids were then washed by spraying with PBS0.5% Tween 20 and rinsed with deionized water. These lids were left to dry at room temperature and then stored in jars with a drying agent. After being thoroughly dried, the coated lids were immersed in microtiter wells containing appropriate serum dilutions and incubated with constant shaking for 10 min at room temperature. The lids were then incubated with goat affinity purified anti-human IgG peroxidase conjugate with shaking for 1 h. They were then incubated with the substrate for 5 min in the same conditions. The lids were washed and rinsed after every step. When the lids were removed, optical densities were then determined at 630 nm wavelength using an ELISA plate reader. The antibody titers were expressed as maximum sera dilution in which optical density value was higher than 3 standard deviations above the mean of values for pre-immune sera. All ELISA tests were calibrated with the same negative (pre-immune serum) and positive (hyper-immune serum) controls. 2.4.2.3. Parasite lysate for Western blotting. This was obtained from late stage schizonts from continuous P. falciparum cultures [20], exhibiting 20% parasitaemia, collected,

4492

A. Berm´udez et al. / Vaccine 25 (2007) 4487–4501

Fig. 1. Monkey trials in Atlanta and Leticia. (A) Antibody titers against SPf66 and corresponding to merozoite-derived peptides 83.1, 35.1 and 55.1. (B1) Western blot analysis of P. falciparum schizont solubilized antigens showing monkey sera weak reactivity with only 137 kDa precursor and normally with 115 kDa cleavage products (precursor of the 35.1 peptide) in the CDC trial in monkeys. (B2) Leticia trial in monkeys showing very strong reactivity with 137, 115 and 83 kDa cleavage products (MSP-1 fragment from which the 83.1 peptide amino acid sequence was derived) with all the monkeys’ sera and some at 55 kDa (from which the 55.1 peptide sequence was derived). Pre-immune as well as non-protected sera are shown.

washed in sterile PBS and lysed in 0.2% saponine solution with vigorous vortexing for 45 s. The pellet was washed twice with large volumes of PBS to remove haemoglobin and erythrocyte debris. The lysate was separated in a discontinuous SDS-PAGE system using 7.5–15% acrylamide (w/v) gradient, and transferred to nitrocellulose membranes then blocked with TBS-T (0.02 M Tris–HCl, pH 7.5, 0.05 M NaCl, 1% Tween-20) and 5% skimmed milk (blocking solution) for 1 h and then cut into strips. Each one of these strip was individually incubated with monkey sera diluted 1:200 in blocking solution, washed several times with TBS-T and then incubated with goat anti Aotus IgG, alkaline phosphatase (AP)

conjugated at 1:1000 dilution and developed with NBT/BCIP [21]. 2.4.2.4. Isolating PBMCs and lympho-proliferation assay. Peripheral blood mononuclear cells (PBMCs), isolated 20 days after the third immunization from 30 randomly chosen vaccinated individuals and 6 controls from whom a 10 ml blood sample was drawn, were obtained by Ficoll-Hypaque gradient centrifugation (1.077 density) (Lymphoprep, Nycomed Pharma AS, Oslo, Norway). A 1 × 105 PBMCs per well were used. A 10 ␮g ml−1 each peptide (monomer and polymer) were used as antigen. Phy-

A. Berm´udez et al. / Vaccine 25 (2007) 4487–4501

4493

Fig. 2. Antibody titer distribution in phase II and III trials performed with SPf66. (a) Tumaco A, B and C trials (b) Tumaco E1, E2, E3 and G. (c) Antibody titer distribution in children aged 1–5, 5–10 and older than 10 (d) Macrotumaco trial. (e) Field-trials (Phase III) in La Tola, Rio Rosario, Venezuela and Tanzania. Note the similar binomial distribution of antibody titers in all different trials performed with the different batches. (f) Lymphoproliferative response of SPf66 vaccines.

tohemagglutinin M (Difco, Michigan, USA) (PHA) was used as positive control for cellular proliferation. Cell proliferation was carried out in triplicate and cells were cultured in 200 ␮l complete medium. After 72 h of culture at 37 ◦ C and 5%

CO2 , supernatant was harvested (cytokine production test) and 100 ␮l per well fresh complete medium, containing 1 ␮Ci [3 H]-thymidine (Amersham-Pharmacia, Buckinghamshire, UK), were added to pulse the cultures for the last 16 h.

4494

A. Berm´udez et al. / Vaccine 25 (2007) 4487–4501

The cells were then harvested by liquid scintillation in a beta counter to determine specific incorporation. The results are expressed as stimulation index (SI): mean counts per minute (cpm) of culture stimulated with peptides/mean cpm of culture without antigen. SI greater than 2 represented a statistically significant difference. 2.5. Recent physical–chemical studies of SPf66 synthetic vaccine 2.5.1. Analytical chromatography Analytical RP-HPLC was run on a Merck Hitachi Autosampler. A Lichrospher-C18 column was used ˚ particle size). H2 O and CH3 CN (4.0 mm × 125 mm, 100 A were used as solvents, both at 0.05% (v/v) TFA. UV absorbance was recorded at 210 nm. The polymers were analyzed by size-exclusion chromatography; their molecular masses ranged from 8 to 24 kDa. 2.5.2. MALDI-TOF spectrometry—preparing samples A saturated ␣-cyano-4-hydroxycinnamic acid (␣-CCA) matrix solution was prepared (weighing around 70 mg). A 1.5 ml distilled water:acetonitryl (2:1) and 0.01% triphluoroacetic acid (TFA) were added. One part of the respective SPf66 batch weighed around 0.2 mg and was dissolved in 1 ml deionized water. A 18 ␮l of matrix solution and 2 ␮l of batch solution were then mixed, obtaining an approximate 10,000:1 matrix:analyte molar ratio. A 1.0 ␮l of this mixture were then taken and placed in the wells of an AnchorChip target; this procedure was carried out for each of the batches to be analyzed. The spectra were taken on a Bruker Protein MALDI-TOF mass spectrometer (Billerica, MA, USA). This instrument uses a 337 nm N2 radiation laser with 3 ns pulses. Acceleration voltage was +17.5 kV and reflector voltage +20 kV, giving 20–30 laser pulses and 30–40 attenuation, thereby maintaining homogeneous conditions when taking all the spectra. 2.5.3. Circular dichroism (CD) A JASCO J720 spectropolarimeter was used to take spectra for SPf66 batches (07, 08, 09, 10, 11, 12, 13, 14 and 15). The instrument was calibrated using d-10-camphorsulfonic acid. The spectra were smoothed using JASCO software. The peptide sample consisted of 50 mM concentration in phosphate buffer, pH 7.0, and 500 ␮l H2 O mixtures with a 1 mm path-length quartz cuvette. Measurements were taken at 20 ◦ C and expressed in terms of mean residue elipticity (deg cm2 dmol−1 ). The spectra were measured between 190 and 260 nm using a 0.2 nm spectra bandwidth and 10 nm/min scan speed [22]. 2.5.4. Nuclear magnetic resonance (NMR) experiments Polymerized batches 09 and 10 used for NMR experiments came from stock samples existing as already-oxidized batches, whilst batches 15 and 16 came from stock samples

still anchored to the resin, meaning that both protector groups and the resin had to be removed. NMR experiment samples were prepared by dissolving 10 mg of each batch in 600 ␮l D2 O. All NMR spectra were run on a 500 MHz Bruker DRX spectrometer and processed on an Indy computer equipped with updated XWINNMR software (Bruker). Spin systems were assigned by Double-QuantumFiltered-Correlation (DQF-COSY) [23] and total correlation spectroscopy (TOCSY) experiments using Mlev17 pulse sequences [24] and Nuclear Overhauser Effect SpectroscopY (NOESY) 1 H–1 H two-dimensional experiments [25]. Standard spectrum procedure was used for sequential assignment [26]. Some batches were not analyzed by NMR due to the lack of an appreciable amount of samples. 2.6. Further immunological studies 2.6.1. Immunization with QS21 After finalizing safety, immunogenicity and protectioninducing studies for the synthetic anti-malarial SPf66 vaccine exclusively adsorbed on Al(OH)3 , another group of 200 volunteers participated in a study in which a new, potent adjuvant authorized for human use was added to SPf66. This was derived from a saponin obtained from Quillaja saponaria, known as QS-21 [27], volunteers were divided into 4 groups; 25 of them received the three doses of 2 mg of SPf66 adsorbed on Al(OH)3 following the classical immunization regime. Another 25 volunteers received 2 mg SPf66 plus 50 ␮g in QS-21, 25 were immunized with 2 mg SPf66 + 100 mg QS21 and a further group (125) received 2 ␮g SPf66 absorbed on 1 ␮g Al(OH)3 + 100 ␮g QS-21 [28].

3. Results As Figures can state more than pages of writing, then the description of the results obtained will be brief as the Figures used to reflect the data presented throughout this manuscript are most explicit. 3.1. Preclinical studies (Phase 0)—monkey trials When Aotus nancymaee monkeys from the Amazon were vaccinated (using the regime described in experimental procedures), batch 03 of SPf66 synthetic vaccine induced very high antibody titers as assessed by FAST-ELISA against the SPf66 molecule and its constituent peptides 83.1 (YSLFQKEKMVL), 35.1 (YGGPANKKNAG) and 55.1 (DELEAETQNVYAA) derived from the different merozoite proteins from which the sequence of amino acids included in this synthetic vaccine was obtained [1]. However, totally different results were achieved in the assay carried out at CDC in Atlanta (USA) where the sera from these monkeys vaccinated with the same SPf66 batch 03 exclusively recognized peptide 35.1 (Fig. 1A).

A. Berm´udez et al. / Vaccine 25 (2007) 4487–4501

Sera from Aotus immunized in Atlanta weakly recognized 137 and 115 kDa molecules (precursors of the 35.1 peptide) by Western blot whilst those immunized in Leticia strongly recognized 137, 115 and 83 kDa molecules (195 kDa MSP1 molecule cleavage product from which the 83.1 peptide amino acid sequence was derived) and some of them the 55 kDa protein (from which the 55.1 peptide sequence was obtained). These results agreed with studies of antibodies against the SPf66 molecule and peptides 83.1, 55.1 and 35.1 conforming it, as determined by FAST-ELISA, in which only peptide 35.1 was recognized in the study carried out in Atlanta [19], whilst both SPf66 and the peptides constituting it were strongly recognized in the study carried out in Leticia (Fig. 1B). There was a total lack of protection against experimental challenge in the 6 Aotus immunized in the CDC Atlanta trial, contrasting with the results obtained in Leticia where 3 out of the 8 Aotus vaccinated with the same SPf66 batch 03 became totally protected. Antibody titers against the individual peptides composing SPf66 were totally different; the monkeys immunized in Atlanta produced high antibody titers exclusively against peptide 35.1 (Fig. 1) whilst those vaccinated in Leticia produced extremely high titers against the 3 peptides making up SPf66 (83.1, 55.1 and 35.1). The respective sera’s reactivity in Western blot was totally different in the two trials since the monkeys immunized in Leticia presented very strong reactivity with de 137, 115, 83 and 55 kDa proteins whilst those from Atlanta only presented weak reactivity with just 137 and 116 kDa ones (Fig. 1B).

4495

3.3. Lymphoproliferation responses Early lymphoproliferation response studies (performed in 1990) showed that SPf66 was able to stimulate a strong cellular immune response in vaccinees, independent of their high, medium or low antibody titers, contrasting with unvaccinated controls, displaying clear stimulation of both parts of the immune response (humoral and cellular) (Fig. 2f). 3.4. Characterizing SPf66 batches by analytical HPLC, MALDI-TOF spectrometry and circular dichroism (CD) Analytical chromatography results from different batches of SPf66 are shown in Fig. 3A. Batches 03, 04, 07, 08, 09, 10, 11, 13 and 16 presented the typical behavior attributed to a polymer specie, having a maximum absorbance peak presenting a 26.72–29.86 min retention time, showing that they all had a similar chromatographic profile. Mass spectra for batches 07, 10, 13, 14, 15 and 16 shown in Fig. 3B presented similar patterns, having an average 4844.82 mz molecular weight, corresponding to their expected mass and extra signals, since the spectra were obtained for unpurified batches (crude specie), i.e. they were polymeric. Fig. 4 shows the circular dichroism for batches 07, 08, 09, 10, 11, 12, 13, 14 and 15 which were produced in H2 O, a similar pattern being presented for all batches having a minimum length of around 198 nm, this being representative of a random structure. 3.5. Nuclear magnetic resonance (NMR) experiments

3.2. Clinical studies (Phases I, IIa and IIb) SPf66 reactogenicity was extremely low, contralateral induration only being presented at the inoculation site in <5% of volunteers; this only occurred with the administration of the third dose. When only QS-21 was added, reactogenicity increased to 20% with the third dose. Antibody titers determined by FAST-ELISA in Tumaco A–G studies (Fig. 2a and b) using different batches (04, 06, 07) and different immunization regimens for vaccinating children aged less than 15 years old (batch 05) always followed a binominal distribution similar to that obtained with batch 05 (Fig. 2c). Batches 06, 07, 09 were used in vaccination in Macrotumaco (Fig. 2d) in which 15,351 people received SPf66 as anti-malarial vaccine (Amador et al. [10]), 230 people were vaccinated in Ecuador with batch 09 [14], 1422 individuals were immunized in Venezuela with batch 08 [15] and 738 received the vaccine in La Tola with batch 08 [12] (Fig. 2e). Batch 09 was used for immunizing 152 children aged 6–12 months in Gambia and 274 children aged 1–5 in Tanzania (Fig. 2e).

Fig. 5A–D shows NOESY spectra in the C␣H-NH region corresponding to batches 09, 10, 15 and 16, respectively. The first two, which were oxidized, polymerized and dialyzed, were used in immunization field-studies in different parts of the world, whilst samples from the latter two were used as reference (their protector groups were removed, as was the resin and they were analyzed immediately without being oxidized). Similar behavior was revealed for COSY, TOCSY and NOESY spectra in batches 09 and 10 in different regions of the spectrum: C␣H-NH; C␤, C␥, C␦-NH; C␥, C␦-C␣; NHi –NHi+1 , etc. (they did not possess signals revealing possible specific structural conformations but just random ones). This data was confirmed in CD spectra which did not present special conformation but rather a random structure. Similar behavior was seen for C (batch 15) and D (batch 16) spectra, even though presenting a shift in signal protons, possibly due to these batches not being oxidized and cysteine and glycine signals being more prominent. Similar behavior was seen in non-oxidized batches 09 and 10. a high degree of overlapping was observed in all batches (09, 10, 15 and 16), especially in the NOESY fingerprint region and ␣H-␤H, ␥H, ␦H region, etc. This led to the safe and complete assignment

4496

A. Berm´udez et al. / Vaccine 25 (2007) 4487–4501

Fig. 3. Characterization of different batch of SPf66. (A) Analytical RP-HPLC for batches 03, 04, 07, 08, 09, 10, 11, 13 and 16. Carried out on a RP-18 Lichrosorb column using a 0–70% ACN linear gradient (containing 1% TFA) for 30 min (B) MALDI-TOF mass Spectra for polymerized batches 07, 10, 13 and 14 and monomeric batches 15 and 16.

of all amino acid residues. All cross-peaks were correlated to the different diagonal peaks. The results presented here show the clear reproducibility of batches of SPf66 synthesized in Colombia. 3.6. QS-21 immunopotentiator action on SPf66 synthetic vaccine

Fig. 4. Structural features by CD. Batches 07, 08, 09, 10, 11, 13, 14 and 15 were analyzed.

Antibody titers determined by FAST-ELISA (Fig. 6A) induced by immunization with 2 mg SPf66 (batch 15) adsorbed on Al(OH)3 alone revealed the classical binomial distribution observed in all previous studies. However, adding QS-21 notably increased antibody titers (up to 150×) 20 days after the second immunization when compared to immunizing with SPf66 absorbed just on Al(OH)3 . The tendency to decrease antibody titers in all groups when bleeding was done 4 months after the second immunization can be observed in Fig. 6A. However, such titers rose again with the third immunization (but were not as high with the second dose in all groups). The antibody titers determined 4 months after the third dose also revealed a descending trend.

A. Berm´udez et al. / Vaccine 25 (2007) 4487–4501

4497

Fig. 5. NOESY fingerprint of 09 (A), 10 (B), 15 (C) and 16 (D) batches (15 and 16 were non-oxide batches). All uni/bidimensional experiments were carried out in D2 O at 298 K, with 400 ms mixing time at 500 MHz.

Western blot studies of a random sample of sera, taken 20 days after the second (II20) and 20 days after the third doses (III20), revealed strong reactivity with 195, 83 and 74 kDa proteins (MSP1 and their cleavage products) and a 115 kDa protein in II20 and III20 bleeding samples from most sera (79, 84, 77, 48), having high antibody titers determined by FAST-ELISA. However, such strong reactivity with the same proteins fell dramatically in some of the III20 bleeding sera samples, independently of the very high anti-SPf66 antibody titers (volunteers 80, 60, 74). A group of individuals (shown as 0 in the right-hand column in Fig. 6B) was observed having low antibody titers by ELISA (≤1:800), no merozoite lysate proteins being recognized by Western blot.

4. Discussion 4.1. Characterization The present work clearly shows the reproducibility of the batches needed for obtaining similar immunological and protective results. Their thorough physicochemical characterization and analysis provides a guarantee for future

multi-epitopic, multi-stage, subunit-based synthetic vaccines. The great similarity of the different batches synthesized over 18 years and used in a large number of studies has been shown here. HPLC chromatography, MALDI-TOF spectrometry and CD support the different batches’ similarity, confirmed by the similar behavior of different profiles obtained by 1 H NMR. Chemically synthesized vaccines are thus easily scaledup from a few milligrams to kilogram amounts (1.2 kg in batch 16), they can be reproductively produced, their chemical and 3D structures are both easily analyzable and their reactogenicity, immunogenicity and homogeneity from batch to batch can be easily assessed, as shown here. This paper has been aimed at demonstrating the advantages of using chemical synthesized vaccines by giving thoroughly analyzed examples, thereby opening a new way forward for developing desperately needed vaccines (i.e. the SPf66 synthetic, anti-malarial vaccine). 4.2. Monkey trials Repeated studies on Aotus monkeys can predict subsequent results in human trials having inherent problems

4498

A. Berm´udez et al. / Vaccine 25 (2007) 4487–4501

Fig. 6. Antibody reactivity of SPf66 vaccinees sera with or without QS-21. In all trials 2 mg of SPf66 (batch 15) were used for immunization. (A) Antibody titers for SPf66 adsorbed in 1 mg AlOH3 (group A); SPf66 plus 50 ␮g QS-21 (group B); SPf66 plus 100 ␮g QS-21 (group C) and SPf66 adsorbed in 1 mg AlOH3 plus 100 ␮g QS-21 (group D) (B) Western blot analysis of P. falciparum protein lysate. The reactivity with 195, 115, 83 and 74 kDa proteins is prominent. Note the absence of reactivity with these proteins in volunteers 80, 60 and 74 in the b lane in spite the very high antibody titers determined by the FAST-ELISA test. a = 20 day post-second, b = 20 day post-third.

A. Berm´udez et al. / Vaccine 25 (2007) 4487–4501

regarding robustness, differences in trial design, vaccine production, population age and epidemiological settings, as shown for SPf66 in the different Phase III trials. It also means that extensive field-trials on humans aimed at determining efficacy can also be avoided. Using a relatively small number of Aotus monkeys (∼200) might validate in vitro assays and field-trials’ protective efficacy as well as determining the potency of different vaccine batches [29]. In spite of batch 03 being used for monkey studies performed at both CDC (Atlanta, Georgia, USA) and Leticia (Colombia), striking differences were presented between the two trials that may explain their differences. The immunization route used in Atlanta was subcutaneous or intramuscular in the legs, whilst it was intra-dermal in the abdominal area in the Leticia trial. Four injections were performed in Atlanta per immunization whilst >16 small injections were performed in Leticia. Mycobacterium tuberculosis included in the CFA in Atlanta was 1/10 of that in Leticia to avoid granulomas being formed. Confined Aotus monkeys were used in Atlanta while Aotus which had been recently caught from the Amazon jungle were used in Leticia. A lower treatment threshold for when parasitaemia developed was taken as being a failure in Atlanta. In spite of the numerous differences, the authors concluded that SPf66 was not protective in Aotus monkeys, although procedural differences could partly explain these two trials’ discordant results [8,9]. 4.3. Clinical trials SPf66 was shown to have induced total protection in 2/5 individuals when a small group of volunteer soldiers were vaccinated with the synthetic vaccine (SPf66, batch 03) adsorbed on Al(OH)3 and when a study of protection against experimental challenge was carried out by intravenously inoculating 1 million erythrocytes infected with a heterologous P. falciparum strain (Phase IIb). Another 5 volunteer soldiers vaccinated with another synthetic chimerical, polymeric molecule (called SPf105) did not develop any protection against experimental infection, just like the 4 individuals who received Al(OH)3 as placebo [2]. This data, added to that provided by Aotus monkey studies regarding experimental challenge with the FVO strain, suggested that the first chemically produced vaccine against P. falciparum had been developed. Vaccinology studies with wide-ranging investigations into SPf66 safety and immunogenicity carried out on more than 1000 volunteer soldiers with batches 04–09 of the synthetic anti-malarial SPf66 vaccine in the search for a better immunization route, the number of doses, the time to be taken between each administration, the amount administered, etc. (some already published, i.e. Tumaco A, B, C) (Fig. 2a) and others described in the current work, i.e. Tumaco E1 , E2 , E3 and G) (Fig. 2b) have shown that SPf66 was extremely safe and immunogenic in both adults and children aged 1–14 [11] (Fig. 2c). It induced antibodies against SPf66 whose titers fol-

4499

lowed binominal distribution, moderate (higher titers at 1:200 dilution) or high (some of them being 1:12,800) antibody production predominating over the lack of this synthetic vaccine’s immunogenicity. The above suggests genetic control of the immune response regarding SPf66 (Fig. 2a–c). The field-study called Macrotumaco, where 15,351 people aged over 1 were vaccinated with SPf66 (batches 06, 07 and 09), 9957 of whom completed the full regime 6 months later, confirmed chemically-produced vaccines’ safety and immunogenicity when used on wide-ranging population groups, making it one of the greatest field-studies involving an anti-malarial vaccine and a synthetic vaccine [9] (Fig. 2d). The Phase III, double-blind, placebo-controlled field-trial carried out in Colombia in a meso-endemic area (La Tola trial) showed a 38.8% protective efficacy which lasted for at least 1 year. There was 33.6% protective capacity for the first episode and 50.5% for second episodes during the same space of time, showing the vaccination’s boosting effect. The criterion for inducing protection was the ABSOLUTE absence of parasites in the blood and the total absence of fever in protected individuals. In this study, SPf66 showed that its greatest efficacy was achieved in children aged 1–4 (77%) and individuals aged over 45 (67%) with suitable confidence intervals, all of them being statistically significant. A later study carried out in another meso-endemic area on the Colombian Pacific coast (the R´ıo Rosario trial) with another group of 1256 individuals revealed that SPf66 synthetic vaccine (batch 10) had 35.2% protective efficacy for a period of ∼2 years. SPf66 was shown to be equally safe and highly immunogenic in a population-based clinical trial carried out in Venezuela with 1422 individuals vaccinated with SPf66 (batch 09) and 938 subjects who were not vaccinated. All were followed-up for a period of 18 months; 73.6% vaccinated individuals sero-converted and the vaccine presented 55% statistically significant protective efficacy, having suitable confidence intervals [15] (Fig. 2e). Double-blind, placebo-controlled studies in Ecuador on 468 individuals, showed that the SPf66 synthetic vaccine (batch 09) induced 60.2% protective efficacy. However, even though the difference was statistically significant, no significant confidence interval was found due to the small number of individuals enrolled in the study. In studies carried out by the MRC group in Gambia on infants aged 6–11 months, the authors described 8% protective efficacy in a very limited number of children, as 154 out of the 630 children who received the first dose were excluded when those carrying out the study confused the vaccine with the placebo. Only 266 children received the third dose of SPf66 (batch 09) and 203 the placebo [16]. Vaccinating 586 children aged 1–5 in another hyperendemic African area where 274 children received the SPf66 vaccine (batch 09) in aluminum hydroxide and 312 just aluminum hydroxide as placebo displayed 31% protective efficacy when a clinical case of malaria was defined as being an individual having ≥20,000 parasites ␮l and fever higher

4500

A. Berm´udez et al. / Vaccine 25 (2007) 4487–4501

than 37.5 ◦ C [16]. This protective efficacy was maintained at around 25% 18 months later [30]. All this data clearly suggests that the chemical synthesis of the different batches of SPf66 anti-malarial vaccine produced in Colombia is completely reproducible. Antibody titers did not increase after the second immunization in children in Thailand who were given Al(OH)3 as a placebo and SPf66 had a null protective effect. This batch of SPF66 had been produced in the USA by another group; it presented lesser polymerization, as described by the authors, and a greater amount of Al(OH)3 was required for it to become adsorbed [31]. Unfortunately, we were not allowed access to this batch of SPf66 produced in the USA and used by the US army nor to the sera from the children vaccinated in Thailand. The synthetic vaccine’s protective ability in people aged more than 1 vaccinated with SPf66 absorbed on Al(OH)3 as sole inmunopotentiator has shown ∼35% protective efficacy for up to 2 years in different ethnic groups having different malarial transmission patterns, something which has not been achieved by any other vaccine candidate against this disease. Differing trial results could have been due to methodological differences, such as those observed in Aotus, in Atlanta at the CDC and in Leticia, Colombia, to differences in Al(OH)3 adsorption as a consequence of a greater monomer concentration as reported from Thailand, to different genetic variants of the P. falciparum parasite and/or to clear genetic differences in the population being vaccinated. It has been found that there is strong genetic control of the immune response to malaria (being clearer in the response of a vaccine having few epitopes, such as SPf66). This has been found to be associated with Major Histocompatibility Complex Class II molecules since most (∼70%) non-responders to immunization with SPf66 were typed as being HLADR␤1*04 and their T-lymphocyte receptors (TCR) from the V␤3, V␤10 and V␤14 families were preferentially used in the immune response [32]. Equally important data shown in the present work is related to the enormous increase in antibody titers against SPf66 (up to 150×) when immunization is carried out with SPf66 in collaboration with an adjuvant as potent as QS21 when compared to the results obtained with the only immunopotentiator available at that time for use in humans: Al(OH)3 (Fig. 6A). However, a note of caution should be introduced regarding these studies as, following the second immunization (even when antibody titers against SPf66 remained high), the reactivity of some sera by Western blot with merozoite lysate proteins totally disappeared, suggesting that what continued to be detected was high peptide–anti-peptide reactivity and not anti-parasite reactivity. The studies carried out with SPf66 malaria vaccine have made the fine-tuning model for developing synthetic vaccines into a paradigm which began when designing the molecule and lasted up to large-scale field-trials. It also became the

prototype for FDA and WHO guidelines and clinical study designs for being applied since then to other types of vaccines during the last 20 years. SPf66 demonstrated synthetic vaccines’ feasibility; today we wish to show its reproducibility at molecular level and emphasize that its production can be easily and safely scaled-up from a few milligrams to kilogram amounts per batch. Large-scale Phase I and II trials [2] and field-studies (Phase III) carried out more than 12 years ago in populations from different ethnic and age groups, including thousands of people >1-year-old carried out in Colombia [12], Ecuador [14], Venezuela [15] and Tanzania [13] have shown that SPf66 produced in Colombia was safe, immunogenic and able to protect ∼35% of the vaccinated population for a minimum of 2 years [17]. SPf66 is therefore the first successful, subunit-based, multi-component, multistage, chemically-synthesized vaccine and the first and only antimalarial one to be tested in large-scale human field-trials showing a high degree of protective efficacy against this deadly disease. SPf66 has shown synthetic and anti-malarial vaccines’ feasibility and the possibility of scaling-up their production to obtain a vaccine having identical physicochemical conditions from milligrams to kilogram amounts, in GMP and GLP conditions, even in small laboratories like ours, showing the tremendous advantages of chemicallysynthesized vaccines. The well-documented SPf66 experience has highlighted synthetic vaccines representing a feasible opening for the pathway leading towards designing, subunit-based, multiantigenic, multi-stage, tailor-made synthetic vaccines, malaria one of them.

Acknowledgement This research has been supported by the Instituto Colombiano para el Desarrollo de la Ciencias y la Tecnolog´ıa “Francisco Jos´e de Caldas” COLCIENCIAS contract #RC 060-2006.

References [1] Patarroyo ME, Romero P, Torres ML, Clavijo P, Moreno A, Martinez A, et al. Induction of protective immunity against experimental infection with malaria using synthetic peptides. Nature 1987;328:629–32. [2] Patarroyo ME, Amador R, Clavijo P, Moreno A, Guzman F, Romero P, et al. A synthetic vaccine protects humans against challenge with asexual blood stages of Plasmodium falciparum malaria. Nature 1988;332:158–61. [3] Rodr´ıguez R, Moreno A, Guzm´an F, Calvo M, Patarroyo ME. Studies in owl monkeys leading to the development of a synthetic vaccine against the asexual blood stages of Plasmodium falciparum. Am J Trop Med Hyg 1990;43:339–54. [4] White N, Nosten F, Bjorkman A, Marsh K, Snow RW. WHO, the Global Fund, and medical malpractice in malaria treatment. Lancet 2004;363:1160. [5] Merrifield RB. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J Am Chem Soc 1963;85:2149–54.

A. Berm´udez et al. / Vaccine 25 (2007) 4487–4501 [6] Houghten RA. General method for the rapid solid phase synthesis of large numbers of peptides: specificity of antigen antibody interaction at the level of individual amino acids. Proc Natl Acad Sci USA 1985;82:5131–5. [7] Bodanszky M. Principles of Peptide Synthesis. Berlin/Heildelberg/NY: Wiley InterScience; 1984. [8] Ruebush 2nd TK, Campbell GH, Moreno A, Patarroyo ME, Collins WE. Immunization of owl monkeys with a combination of Plasmodium falciparum asexual blood-stage synthetic peptide antigens. Am J Trop Med Hyg 1990;43:355–66. [9] Amador R, Moreno A, Murillo LA, Sierra O, Saavedra D, Rojas M, et al. Safety and immunogenicity of the synthetic malaria vaccine SPf66 in a large field trial. J Infect Dis 1992;166:139–44. [10] Amador R, Moreno A, Valero V, Murillo L, Mora AL, Rojas M, et al. The first field trials of the chemically synthesized malaria vaccine SPf66: safety, immunogenicity and protectivity. Vaccine 1992;10:179–84. [11] Patarroyo G, Franco L, Amador R, Murillo LA, Rocha CL, Rojas M, et al. Study of the safety and immunogenicity of the synthetic malaria SPf66 vaccine in children aged 1–14 years. Vaccine 1992;10:175–8. [12] Valero MV, Amador LR, Galindo C, Figueroa J, Bello MS, Murillo LA, et al. Vaccination with SPf66, a chemically synthesised vaccine, against Plasmodium falciparum malaria in Colombia. Lancet 1993;341:705–10. [13] Alonso PL, Smith T, Armstrong Schellenberg JRM, Masanja H, Mwankusye S, Urassa H, et al. Randomised trial of efficacy of SPf66 vaccine against Plasmodium falciparum malaria in children in southern Tanzania. Lancet 1994;344:1175–81. [14] Sempertegui F, Estrella B, Moscoso J, Piedrahita L, Hernandez D, Gaybor J, et al. Safety, immunogenicity and protective effect of the SPf66 malaria synthetic vaccine against Plasmodium falciparum infection in a randomized double-blind placebo-controlled field trial in an endemic area of Ecuador. Vaccine 1994;12:337–42. [15] Noya O, Berti YG, Alarcon de Noya B, Borges R, Zerpa N, Urbaez JD, et al. A population-based clinical trial with the SPf66 synthetic Plasmodium falciparum malaria vaccine in Venezuela. J Infect Dis 1994;170:396–402. [16] Leach A, Drakeley CJ, D’Alessandro U, Fegan GW, Bennett S, Ballou WR, et al. A pilot safety and immunogenicity study of the malaria vaccine SPf66 in Gambian infants. Parasite Immunol 1995;17:441–4. [17] Valero MV, Amador R, Aponte JJ, Narvaez A, Galindo C, Silva Y, et al. Evaluation of SPf66 malaria vaccine during a 22-month follow-up field trial in the Pacific coast of Colombia. Vaccine 1996;14:1466–70. [18] Salcedo M, Barreto L, Rojas M, Moya R, Cote J, Patarroyo ME. Studies on the humoral immune response to a synthetic vaccine against Plasmodium falciparum malaria. Clin Exp Immunol 1991;84:122–8.

4501

[19] Campbell GH, Aley SB, Ballou WR, Hall T, Hockmeyer WT, Hoffman SL, et al. Use of synthetic and recombinant peptides in the study of host-parasite interactions in the malarias. Am J Trop Med Hyg 1987;37:428–44. [20] Lambros C, Vanderberg JP. Synchronization of Plasmodium falciparum erythrocyte stages in culture. J Parasitol 1979;65:418–20. [21] Blake MS, Johnston KH, Rusell-Jones GJ, Gotschlich EC. A rapid sensitive method for detection of alkaline Phosphatase conjugated antibody on Western Blot. Anal Biochem 1984;136:175–9. [22] Alder AJ, Greenfield NJ, Fasman GD. Circular dichroism and optical rotary dispersion of proteins and polypeptides. Meth Enzymol 1973;27:675. [23] Rance M, Soressen OW, Bodenhausen G, Wagner G, Ernst RR, W¨uthrich K. Improved spectral resolution in cosy 1H NMR spectra of proteins via double quantum filtering. Biochem Biophys Res Commun 1983;117:479–85. [24] Bax A, Davis DG. MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy. J Magn Reson 1985;65:355–60. [25] Jeener J, Meier BH, Backman P, Ernst RR. Investigation of exchange processes by two-dimensional NMR spectroscopy. J Chem Phys 1979;71:4546–53. [26] W¨uthrich K. NMR of Proteins and Nucleic Acids. New York: John Wiley & Sons; 1986. p. 117–29. [27] Kensil CR. Saponins as vaccine adjuvants. Crit Rev Ther Drug Carrier Syst 1996;13:1–55. [28] Kashala O, Amador R, Valero MV, Moreno A, Barbosa A, Nickel B, et al. Safety, tolerability and immunogenicity of new formulations of the Plasmodium falciparum malaria peptide vaccine SPf66 combined with the immunological adjuvant QS-21. Vaccine 2002;20:2263– 77. [29] Heppner DG, Cummings JF, Ockenhouse C, Kester KE, Lyon JA, Gordon DM. New World monkey efficacy trials for malaria vaccine development: critical path or detour? Trends Parasitol 2001;17:419–25. [30] Alonso PL, Smith TA, Armstrong-Schellenberg JR, Kitua AY, Masanja H, Hayes R, et al. Duration of protection and age-dependence of the effects of the SPf66 malaria vaccine in African children exposed to intense transmission of Plasmodium falciparum. J Infect Dis 1996;174:367–72. [31] Nosten F, Luxemburger C, Kyle DE, Ballou WR, Wittes J, Wah E, et al. Randomised double-blind placebo-controlled trial of SPf66 malaria vaccine in children in north-western Thailand. Shoklo SPf66 Malaria Vaccine Trial Group. Lancet 1996;348:701–7. [32] Murillo LA, Rocha CL, Mora AL, Kalil J, Goldenberg AK, Patarroyo ME. Molecular analysis of HLA DR4-beta 1 gene in malaria vaccinees. Typing and subtyping by PCR technique and oligonucleotides. Parasite Immunol 1991;13:201–10.