BACKGROUND:Malaria vaccines that target parasite development in mosquitoes offer a strategy to block disease transmission and support control, elimination, and eradication. In this article, we evaluate Pfs230D1-exoprotein A (EPA) and Pfs25-EPA for safety, immunogenicity, and field efficacy in Malian adults. METHODS:We first conducted a comparator-controlled, dose-escalating pilot safety trial, assessing Pfs25-EPA (16 vs. 47 μg) and Pfs230D1-EPA (13 vs. 40 μg), and their combinations, each formulated in the adjuvant AS01, at a 0-, 1-, and 6-month schedule. We then conducted a randomized, double-blind, comparator-controlled main trial to evaluate two Pfs230D1-EPA/AS01 regimens on a 0-, 1-, 4-, 16-month schedule. Pfs230D1-full, consisting of 40 μg of Pfs230D1-EPA plus 50 μg of AS01 for each dose, versus Pfs230D1-fractional, identical to Pfs230D1-full except for the third dose that used 8 μg of Pfs230D1-EPA and 10 μg of AS01. Primary end points were safety and reactogenicity (as-treated population), and secondary end points (as-randomly-assigned population) were immunogenicity by enzyme-linked immunosorbent assay, serum activity by mosquito standard membrane feeding assay (SMFA), and efficacy by direct skin feeding assay (DSF). RESULTS:In the pilot safety trial, 65 participants received injections (45 Pfs230D1 and/or Pfs25; 20 comparator). In the main phase, 236 participants received injections (56 Pfs230D1-full; 61 Pfs230D1-fractional; 119 comparator). No serious adverse events (SAEs) occurred in vaccinees in the pilot or main phase. Pfs230D1-full and Pfs230D1-fractional regimens induced antibody responses and transmission-reducing activity (based on SMFA) detectable up to approximately 1 year post-vaccination 3. Primary efficacy analysis showed combined Pfs230D1-full and Pfs230D1-fractional groups were not associated with reductions in mosquito positivity rate in the first 6 weeks of year 1 (efficacy, -1.55; 95% confidence interval [CI], -11.05 to 0.46). In the Pfs230D1-full group, DSF positivity was lower by 72.5% (95% CI, 30.4 to 89.1), and the proportion of infected mosquitoes was lower by 77.3% (95% CI, 19.5 to 93.6) over two transmission seasons. CONCLUSIONS:In this trial, Pfs230D1-EPA/AS01 regimens did not result in SAEs and generated antibody responses and functional activity that persisted for up to 1 year postvaccination. Although the primary efficacy estimate did not demonstrate a reduction in parasite transmission during the first 6 weeks of follow-up, the full dosing regimen was associated with reduced transmission events and infected mosquitoes over 2 years. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov ID, NCT02942277.).

Background Malaria transmission-blocking vaccines target mosquito-stage parasites and will support elimination programmes. Gamete vaccine Pfs230D1-EPA/Alhydrogel induced superior activity to zygote vaccine Pfs25-EPA/ Alhydrogel in malaria-naive US adults. Here, we compared these vaccines in malaria-experienced Malians.Methods We did a pilot safety study then double-blind, block-randomised, comparator-controlled main-phase trial in malaria-intense Bancoumana, Mali. 18-50-year-old healthy non-pregnant, non-breastfeeding consenting adult residents were randomly assigned (1:1:1:1) to receive four doses at months 0, 1, 4 center dot 5, and 16 center dot 5 of either 47 mu g Pfs25, 40 mu g Pfs230D1 or comparator (Twinrix or Menactra)-all co-administered with normal saline for blinding-or 47 mu g Pfs25 plus 40 mu g Pfs230D1 co-administered. We documented safety and tolerability (primary endpoint in the as-treated populations) and immunogenicity (secondary endpoint in the as-treated populations: ELISA, standard-membrane-feeding assay, and mosquito direct skin feed assay). This trial is registered at ClinicalTrials.gov, NCT02334462.Findings Between March 19, and June 2, 2015, we screened 471 individuals. Of 225 enrolled for the pilot and main cohorts, we randomly assigned 25 participants to pilot safety cohort groups of five (20%) to receive a two-dose series of Pfs25-EPA/Alhydrogel (16 mu g), Pfs230D1-EPA/Alhydrogel (15 mu g) or comparator, followed by Pfs25-EPA/ Alhydrogel (16 mu g) plus Pfs230D1-EPA/Alhydrogel (15 mu g) or comparator plus saline. For the main cohort, we enrolled 200 participants between May 11 and June 2, 2015, to receive a four-dose series of 47 mu g Pfs25-EPA/Alhydrogel plus saline (n=50 [25%]; Pfs25), 40 mu g Pfs230D1-EPA/Alhydrogel plus saline (n=49 [25%]; Pfs230D1), 47 mu g Pfs25-EPA/ Alhydrogel plus 40 mu g Pfs230D1-EPA/Alhydrogel (n=50 [25%]; Pfs25 plus Pfs230D1), or comparator (Twinrix or Menactra) plus saline (n=51 [25%]). Vaccinations were well tolerated in the pilot safety and main phases. Most vaccinees became seropositive after two Pfs230D1 or three Pfs25 doses; peak titres increased with each dose thereafter (Pfs230D1 geometric mean: 77 center dot 8 [95% CI 56 center dot 9-106 center dot 3], 146 center dot 4 [108 center dot 3-198 center dot 0], and 410 center dot 2 [301 center dot 6-558 center dot 0]; Pfs25 geometric mean 177 center dot 7 [130 center dot 3-242 center dot 4] and 315 center dot 7 [209 center dot 9-474 center dot 6]). Functional activity (mean peak transmission-reducing activity) appeared for Pfs230D1 (74 center dot 5% [66 center dot 6-82 center dot 5]) and Pfs25 plus Pfs230D1 (68 center dot 6% [57 center dot 3-79 center dot 8]), after the third dose and after the fourth dose (88 center dot 9% [81 center dot 7-96 center dot 2] for Pfs230D1 and 85 center dot 0% [78 center dot 4-91 center dot 5] Pfs25 plus Pfs230D1) but not for Pfs25 (58 center dot 2% [49 center dot 1-67 center dot 3] after the third dose and 58 center dot 2% [48 center dot 5-67 center dot 9] after the fourth dose). Pfs230D1 transmission-reducing activity (73 center dot 7% [64 center dot 1-83 center dot 3]) persisted 10 weeks after the fourth dose. Transmission-reducing activity of 80% was estimated at 1659 ELISA units for Pfs25, 218 for Pfs230D1, and 223 for Pfs230D1 plus Pfs25. After 3850 direct skin feed assays, 35 participants (12 Pfs25, eight Pfs230D1, five Pfs25 plus Pfs230D1, and ten comparator) had transmitted parasites at least once. The proportion of positive assays in vaccine groups (Pfs25 33 [3%] of 982 [-0 center dot 013 to 0 center dot 014], Pfs230D1 22 [2%] of 954 [-0 center dot 005 to 0 center dot 027], and combination 11 [1%] of 940 [-0 center dot 024 to 0 center dot 002]) did not differ from that of the comparator (22 [2%] of 974), nor did Pfs230D1 and combination groups differ (-0 center dot 024 to 0 center dot 001).Interpretation Pfs230D1 but not Pfs25 vaccine induces durable serum functional activity in Malian adults. Direct skin feed assays detect parasite transmission to mosquitoes but increased event rates are needed to assess vaccine effectiveness.Funding Intramural Research Program of the National Institute of Allergy and Infectious Diseases and US National Institutes of Health.Copyright (c) 2023 Published by Elsevier Ltd. All rights reserved.

Rhoptry neck protein 2 (RON2) binds to the hydrophobic groove of apical membrane antigen 1 (AMA1), an interaction essential for invasion of red blood cells (RBCs) by Plasmodium falciparum (Pf) parasites. Vaccination with AMA1 alone has been shown to be immunogenic, but unprotective even against homologous challenge in human trials. However, the AMA1‐RON2L (L is referred to as the loop region of RON2 peptide) complex is a promising candidate, as preclinical studies with Freund's adjuvant have indicated complete protection against lethal challenge in mice and superior protection against virulent infection in Aotus monkeys. To prepare for clinical trials of the AMA1‐RON2L complex, identity and integrity of the candidate vaccine must be assessed, and characterization methods must be carefully designed to not dissociate the delicate complex during evaluation. In this study, we developed a native Tris‐glycine gel method to separate and identify the AMA1‐RON2L complex, which was further identified and confirmed by Western blotting using anti‐AMA1 monoclonal antibodies (mAbs 4G2 and 2C2) and anti‐RON2L polyclonal Ab coupled with mass spectrometry. The formation of complex was also confirmed by Capillary Isoelectric Focusing (cIEF). A short‐term (48 h and 72 h at 4°C) stability study of AMA1‐RON2L complex was also performed. The results indicate that the complex was stable for 72 h at 4°C. Our research demonstrates that the native Tris‐glycine gel separation/Western blotting coupled with mass spectrometry and cIEF can fully characterize the identity and integrity of the AMA1–RON2L complex and provide useful quality control data for the subsequent clinical trials.

BACKGROUNDVaccines that block human-to-mosquito Plasmodium transmission are needed for malaria eradication, and clinical trials have targeted zygote antigen Pfs25 for decades. We reported that a Pfs25 protein-protein conjugate vaccine formulated in alum adjuvant induced serum functional activity in both US and Malian adults. However, antibody levels declined rapidly, and transmission-reducing activity required 4 vaccine doses. Functional immunogenicity and durability must be improved before advancing transmission-blocking vaccines further in clinical development. We hypothesized that the prefertilization protein Pfs230 alone or in combination with Pfs25 would improve functional activity.METHODSTransmission-blocking vaccine candidates based on gamete antigen Pfs230 or Pfs25 were conjugated with Exoprotein A, formulated in Alhydrogel, and administered to mice, rhesus macaques, and humans. Antibody levels were measured by ELISA and transmission-reducing activity was assessed by the standard membrane feeding assay.RESULTSPfs25-EPA/Alhydrogel and Pfs230D1-EPA/Alhydrogel induced similar serum functional activity in mice, but Pfs230D1-EPA induced significantly greater activity in rhesus monkeys that was enhanced by complement. In US adults, 2 vaccine doses induced complement-dependent activity in 4 of 5 Pfs230D1-EPA/Alhydrogel recipients but no significant activity in 5 Pfs25-EPA recipients, and combination with Pfs25-EPA did not increase activity over Pfs230D1-EPA alone.CONCLUSIONThe complement-dependent functional immunogenicity of Pfs230D1-EPA represents a significant improvement over Pfs25-EPA in this comparative study. The rhesus model is more predictive of the functional human immune response to Pfs230D1 than is the mouse model.TRIAL REGISTRATIONClinicalTrials.gov NCT02334462.FUNDINGIntramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Background Pfs25H-EPA is a protein-protein conjugate transmission-blocking vaccine against Plasmodium falciparum that is safe and induces functional antibodies in malaria-naive individuals. In this field trial, we assessed Pfs25H-EPA/Alhydrogel for safety and functional immunogenicity in Malian adults. Methods This double-blind, randomised, comparator-controlled, dose-escalation trial in Bancoumana, Mali, was done in two staggered phases, an initial pilot safety assessment and a subsequent main phase. Healthy village residents aged 18-45 years were eligible if they had normal laboratory results (including HIV, hepatitis B, hepatitis C tests) and had not received a previous malaria vaccine or recent immunosuppressive drugs, vaccines, or blood products. Participants in the pilot safety cohort and the main cohort were assigned (1:1) by block randomisation to a study vaccine group. Participants in the pilot safety cohort received two doses of Pfs25H-EPA/Alhydrogel 16 mu g or Euvax B (comparator vaccine), and participants in the main cohort received Pfs25H-EPA/Alhydrogel 47 mu g or comparator vaccine (Euvax B for the first, second, and third vaccinations and Menactra for the fourth vaccination). Participants and investigators were masked to group assignment, and randomisation codes in sealed envelopes held by a site pharmacist. Vials with study drug for injection were covered by opaque tape and labelled with a study identification number. Group assignments were unmasked at final study visit. The primary outcomes were safety and tolerability for all vaccinees. The secondary outcome measure was immunogenicity 14 days after vaccination in the per-protocol population, as confirmed by the presence of antibodies against Pfs25H measured by ELISA IgG and antibody functionality assessed by standard membrane feeding assays and by direct skin feeding assays. This trial is registered with ClinicalTrials.gov, number NCT01867463. Findings Between May 15, and Jun 16, 2013, 230 individuals were screened for eligibility. 20 individuals were enrolled in the pilot safety cohort; ten participants were assigned to receive Pfs25H-EPA/Alhydrogel 16 mu g, and ten participants were assigned to receive comparator vaccine. 100 individuals were enrolled in the main cohort; 50 participants were assigned to receive Pfs25H-EPA/Alhydrogel 47 mu g, and 50 participants were assigned to receive comparator vaccine. Compared with comparator vaccinees, Pfs25H vaccinees had more solicited adverse events (137 events vs 86 events; p=0.022) and treatment-related adverse events (191 events vs 126 events, p=0.034), but the number of other adverse events did not differ between study vaccine groups (792 vs 683). Pfs25H antibody titres increased with each dose, with a peak geometric mean of 422.3 ELISA units (95% CI 290-615) after the fourth dose, but decreased relatively rapidly thereafter, with a half-life of 42 days for anti-Pfs25H and 59 days for anti-EPA (median ratio of titres at day 600 to peak, 0.19 for anti-Pfs25H vs 0.29 for anti-EPA; p=0.009). Serum transmission-reducing activity was greater for Pfs25H than for comparator vaccine after the fourth vaccine dose (p<0.001) but not after the third dose (p=0.09). Repeated direct skin feeds were well tolerated, but the number of participants who infected at least one mosquito did not differ between Pfs25H and comparator vaccinees after the fourth dose (p=1, conditional exact). Interpretation Pfs25H-EPA/Alhydrogel was well tolerated and induced significant serum activity by standard membrane feeding assays but transmission blocking activity was not confirmed by weekly direct skin feed. This activity required four doses, and titres decreased rapidly after the fourth dose. Alternative antigens or combinations should be assessed to improve activity. Copyright (C) 2018 Elsevier Ltd. All rights reserved.

Pfs25, a Plasmodium falciparum surface protein expressed during zygote and ookinete stages in infected mosquitoes, is a lead transmission-blocking vaccine candidate against falciparum malaria. To enhance immunogenicity, recombinant Pfs25 was chemically conjugated to recombinant nontoxic Pseudomonas aeruginosa ExoProtein A (rEPA) in conformance with current good manufacturing practices (cGMP), and formulated with the alum adjuvant Alhydrogel. In order to meet the regulatory requirements for a phase 1 human clinical trial, the vaccine product was extensively evaluated for stability at an initial time point and through the clinical trial period annually. Because basic quality control methods to characterize alum-based vaccines remain unavailable, a thermal forced degradation study was performed prior to the initial evaluation to identify the methods suitable to detect the quality of vaccine formulations. Our results show that the vaccine product Pfs25-EPA formulated on Alhydrogel is in conformance with regulatory guidelines and suitable for human trials.

The Plasmodium falciparum protein, apical membrane antigen 1 forms a complex with another parasite protein, rhoptry neck protein 2, to initiate junction formation with the erythrocyte and is essential for merozoite invasion during the blood stage of infection. Consequently, apical membrane antigen 1 has been a target of vaccine development but vaccination with apical membrane antigen 1 alone in controlled human malaria infections failed to protect and showed limited efficacy in field trials. Here we show that vaccination with AMA1–RON2L complex in Freund’s adjuvant protects Aotus monkeys against a virulent Plasmodium falciparum infection. Vaccination with AMA1 alone gave only partial protection, delaying infection in one of eight animals. However, the AMA1–RON2L complex vaccine completely protected four of eight monkeys and substantially delayed infection (>25 days) in three of the other four animals. Interestingly, antibodies from monkeys vaccinated with the AMA1–RON2L complex had significantly higher neutralizing activity than antibodies from monkeys vaccinated with AMA1 alone. Importantly, we show that antibodies from animals vaccinated with the complex have significantly higher neutralization activity against non-vaccine type parasites. We suggest that vaccination with the AMA1–RON2L complex induces functional antibodies that better recognize AMA1 as it appears complexed with RON2 during merozoite invasion. These data justify progression of this next generation AMA1 vaccine towards human trials.

Aluminum based adjuvants (alum), including aluminum hydroxide (Alhydrogel) and aluminum phosphate are the most commonly used adjuvant in the US. In order to ensure quality of vaccines, regulatory authorities require evaluation of antigen content in final vaccine products. Currently, there are no generic methods available for the determination of protein content in alum-based vaccines. Aluminum hydroxide gels exist as particles in solution, which interfere with direct quantitation of protein content in formulations using assays such as Lowry, BCA or Bradford protein assay. The present study adapts a simple fluorescent assay to directly (without the need for antigen extraction) determine antigen content on Alhydrogel with accuracy and sensitivity using the o-phthalaldehyde (OPA) reagent. Malaria vaccine candidates AMA1-C1/Alhydrogel, AMA1-C2/Alhydrogel, MSP1(42)-3D7/Alhydrogel, MSP1(42)-C1/Alhydrogel or BSAM-2/Alhydrogel were used as model formulations. The results of the present study show that the OPA assay is highly accurate (87-100%), reproducible, and simple with a linear detection range of 25-400 microg/mL for Alhydrogel vaccines (except for MSP1(42)-C1, which has a linear detection range of 31.25-500 microg/mL). This assay has proven to be highly useful in our laboratory and been used in routine vaccine quality control processes.

Transmission-blocking vaccines (TBVs) that target sexual stage parasite development could be an integral part of measures for malaria elimination. Pfs25 is a leading TBV candidate, and previous studies conducted in animals demonstrated an improvement of its functional immunogenicity after conjugation to EPA, a recombinant, detoxified ExoProtein A from Pseudomonas aeruginosa. In this report, we describe results of an open-label, dose-escalating Phase 1 trial to assess the safety and immunogenicity of Pfs25-EPA conjugates formulated with Alhydrogel®. Thirty malaria-naïve healthy adults received up to four doses of the conjugate vaccine, with 8, 16, or 47 μg of conjugated Pfs25 mass, at 0, 2, 4, and 10 months. Vaccinations were generally well tolerated. The majority of solicited adverse events were mild in severity with pain at the injection site the most common complaint. Anemia was the most common laboratory abnormality, but was considered possibly related to the study in only a minority of cases. No vaccine-related serious adverse events occurred. The peak geometric mean anti-Pfs25 antibody level in the highest dose group was 88 (95% CI 53, 147) μg/mL two weeks after the 4th vaccination, and declined to near baseline one year later. Antibody avidity increased over successive vaccinations. Transmission blocking activity demonstrated in a standard membrane feeding assay (SMFA) also increased from the second to the third dose, and correlated with antibody titer and, after the final dose, with antibody avidity. These results support the further evaluation of Pfs25-EPA/Alhydrogel® in a malaria-endemic population.

QS-21 is one of the active fractions of the bark of Chilean tree, Quillaja saponaria, purified using a reverse-phase chromatography (RP-HPLC) [1–3]. QS denotes its source as Q. saponaria and the number 21 as the identity of the RP-HPLC peak [2]. QS-21 is an acylated 3, 28-bisdesmodic triterpene glycosides (1,3) or “saponin” with a molecular formula of C92O46H148 and molecular weight of 1990 Da [1]. It is one of the most potent immunological adjuvants that has been widely used [4–8]. The adjuvant effect of saponins was first reported in 1925, when it was shown that the addition of bread crumbs, tapioca, saponin and “starch oil” to antigenic preparations greatly enhanced antibody responses to diphtheria or tetanus [7,9–11]. In 1951, Espinet used a crude commercially available saponin preparation to increase the potency of foot-and-mouth disease vaccines [10,12]. Further in 1974, Dalsgaard successfully isolated saponin Quil A from the cortex of the South-American Tree Quillaja saponaria Molina [3,13–15] and found that Quil A stimulated both humoral and cellular immunity, as well as induced differential antibody isotypes [3, 6,15–17]. Since then, the Quil A has been commercialized and has gained widespread use in veterinary vaccines and pre-clinical studies [3,6,10,14,17–20]. Additional studies showed its effects when co-formulated with aluminum salts, liposomes and oil-in-water emulsions, and with amphipathic proteins and lipids forming detergent/lipid/saponin complexs termed immune-stimulating complexes (ISCOMs) [3,20–23]. However, Quil A is still a heterogeneous product, consisting of up to 23 different saponin peaks detectable by HPLC and its toxicity precluded its use in human vaccines [2–3,6,15,24]. Due to the fact that Quil A is a mixture, a further study was performed by Kensil et al. in 1991[2,6], in which 10 of RP-HPLC fractions from aqueous extract of Q. saponaria bark treated by ultrafiltration were tested and found the fractions QS-7, QS-17, QS-18 and QS-21 to be particularly potent. However, their toxicity varies considerably. QS-18, the major component of Q saponaria, was found to be highly toxic in mice, while QS-7 and QS- 21 shows far less toxicity [2,6,25]. QS-21 was further extensive studied because it is more abundant than QS-7 [7]. Studies showed that QS-21 promoted high antigen-specific antibody responses and CD8+ T-cell response in mice [2–6,17,26] and favored a balanced production of both IgG1 and IgG2a [1,2,27]; in contrast, aluminum hydroxide mainly promotes IgG1 production [2,6,27]. Further studies showed that QS-21 stimulated the production of cytotoxic T-lymphocytes (CTLs), induces Th1cytokines, interleukin-2 and interferon-gamma (IL-2 and IFN-γ), and antibodies of the IgG2a isotype to protein antigens [1,6,26,28,29]. A number of comparative studies has demonstrated that QS-21 significantly outperformed the other classes of adjuvants including glucan formulations, peptidoglycans, amphophilic block copolymers, bacterial nucleosides and bacterial lipopolysaccharides, in augmenting antibody responses as well as T-cell responses against target antigens [2,7]. Due to the high potency, QS-21 has been used as an effective adjuvant with a recombinant retroviral subunit vaccine against feline leukemia virus (FeLV), which is commercially available now [3,6,30]. However, the toxicity and the undesirable haemolytic effect still remain for human use [2,6,7]. QS-21 caused 50% haemolysis of sheep red blood cells (SRBC) at concentrations as low as 7–9 µg/ml (2, 29), and apparently, the side effects associated with QS-21 limit doses to about 50 µg with exception of cancer patient (melanoma, breast and prostate) [1,7]. It is clear that if the higher doses of this adjuvant could be safely used, its immunogenicity should be further increased [2,7]. The Adjuvant System (AS) developed by GlaxoSmithKline demonstrated that the apparent adverse events of QS-21 in rat skeletal muscle could be significantly reduced if QS-21 was formulated in AS01 (composed of liposomes, MPL and QS-21) at a dose of 5 µg, when compared to formulations containing QS-21 alone at the same dose, suggesting that the toxicity of QS-21 can be reduced when formulated with other appropriate adjuvants [5]. QS-21 containing adjuvants such as AS01, AS02 are currently tested in human clinical trials for various vaccine candidates and infectious agents, including influenza, malaria, hepatitis B virus (HBV), human papillomavirus (HPV), HIV/AIDS, tuberculosis (TB), non-small-cell lung carcinoma (NSCLC) and melanoma [1,4,5,8,17,28]. Over the past two decades, the saponin adjuvants have emerged as one of the leading adjuvant candidates. QS-21 containing complex adjuvants in particular have been widely popular in its application to develop vaccines platforms [4,5,7]. It is promising that these QS-21 containing complex adjuvants may be one of the first to be approved as alternative adjuvants in human use in the US in the future.