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More so than any other infectious disease, malaria has all too often affected the conduct of military operations in war and in some cases has disproportionately influenced the outcome. From Napoleon's defensive action at Walcheren, to the Union Army's attempts to take control of the Mississippi River at Corinth and Vicksburg, to the dreadful numbers of malaria casualties suffered by U.S. Marines on the islands of Efate and Guadalcanal during World War II and more recently in Liberia in 2003, malaria has extracted a heavy toll. In this article, we summarize a few of the significant contributions to malaria control by U.S. military personnel throughout its history. We review examples of scientific achievements, medical breakthroughs, and lessons learned from preceding wars that continue to drive the quest for effective antimalarial therapies and preventive vaccines. This review is by no means comprehensive or complete but serves as a testament to the skill, courage, self-sacrifice, and devotion to duty of the many who have faithfully served their country in the past and to those today who continue the struggle against this disease. [ABSTRACT FROM AUTHOR]

Background: The first licensed malaria vaccine, RTS,S/AS01 E , confers moderate protection against symptomatic disease. Because many malaria infections are asymptomatic, we conducted a large-scale longitudinal parasite genotyping study of samples from a clinical trial exploring how vaccine dosing regimen affects vaccine efficacy., Methods: Between Sept 28, 2017, and Sept 25, 2018, 1500 children aged 5-17 months were randomly assigned (1:1:1:1:1) to receive four different RTS,S/AS01 E regimens or a rabies control vaccine in a phase 2b open-label clinical trial in Ghana and Kenya. Participants in the four RTS,S groups received two full doses at month 0 and month 1 and either full doses at month 2 and month 20 (group R012-20); full doses at month 2, month 14, month 26, and month 38 (group R012-14); fractional doses at month 2, month 14, month 26, and month 38 (group Fx012-14; early fourth dose); or fractional doses at month 7, month 20, and month 32 (group Fx017-20; delayed third dose). We evaluated the time to the first new genotypically detected infection and the total number of new infections during two follow-up periods (12 months and 20 months) in more than 36 000 dried blood spot specimens from 1500 participants. To study vaccine effects on time to the first new infection, we defined vaccine efficacy as one minus the hazard ratio (HR; RTS,S vs control) of the first new infection. We performed a post-hoc analysis of vaccine efficacy based on malaria infection status at first vaccination and force of infection by month 2. This trial (MAL-095) is registered with ClinicalTrials.gov, NCT03281291., Findings: We observed significant and similar vaccine efficacy (25-43%; 95% CI union 9-53) against first new infection for all four RTS,S/AS01 E regimens across both follow-up periods (12 months and 20 months). Each RTS,S/AS01 E regimen significantly reduced the mean number of new infections in the 20-month follow-up period by 1·1-1·6 infections (95% CI union 0·6-2·1). Vaccine efficacy against first new infection was significantly higher in participants who were infected with malaria (68%; 95% CI 50-80) than in those who were uninfected (37%; 23-48) at the first vaccination (p=0·0053)., Interpretation: All tested dosing regimens blocked some infections to a similar degree. Improved vaccine efficacy in participants infected during vaccination could suggest new strategies for highly efficacious malaria vaccine development and implementation., Funding: GlaxoSmithKline Biologicals SA, PATH, Bill & Melinda Gates Foundation, and the German Federal Ministry of Education and Research., Competing Interests: Declaration of interests The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the US Centers for Disease Control and Prevention or the US Department of Health and Human Services. LDM received grants from the Bill and Melinda Gates Foundation and the German Federal Ministry of Education and Research through the KfW Development Bank through her institution. CKL received a grant from the German Federal Ministry of Education and Research through the KfW Development Bank and a grant from the Bill and Melinda Gates Foundation. DFW acted as a principal investigator on the MAL-095 study funded by a PATH grant paid to Harvard University, which also supported DEN, AME, BLM, SFS, and AK. DFW is also Chair of the Malaria Policy Advisory Group that advises the WHO on all malaria policy. PBG discloses a PATH subaward from Harvard for statistical analysis contributing to salary support for PBG, MJ, and LL. AME, LL, AK, BS, NSH, DB, SaA, TA, ScA, DA, DKB, PBYB, SE, NF, JG, SKK, KO, AMS, NW, and CFO declare no conflict of interest. ML, FR, OO-A are employees of GSK. ML, FR, and OO-A own shares in GSK., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: To improve the efficacy of Plasmodium falciparum malaria vaccine RTS,S/AS02, we conducted a study in 2001 in healthy, malaria-naïve adults administered RTS,S/AS02 in combination with FMP1, a recombinant merozoite surface-protein-1, C-terminal 42kD fragment., Methods: A double-blind Phase I/IIa study randomized N = 60 subjects 1:1:1:1 to one of four groups, N = 15/group, to evaluate safety, immunogenicity, and efficacy of intra-deltoid half-doses of RTS,S/AS02 and FMP1/AS02 administered in the contralateral (RTS,S + FMP1-separate) or same (RTS,S + FMP1-same) sites, or FMP1/AS02 alone (FMP1-alone), or RTS,S/AS02 alone (RTS,S-alone) on a 0-, 1-, 3-month schedule. Subjects receiving three doses of vaccine and non-immunized controls (N = 11) were infected with homologous P. falciparum 3D7 sporozoites by Controlled Human Malaria Infection (CHMI)., Results: Subjects in all vaccination groups experienced mostly mild or moderate local and general adverse events that resolved within eight days. Anti-circumsporozoite antibody levels were lower when FMP1 and RTS,S were co-administered at the same site (35.0 µg/mL: 95 % CI 20.3-63), versus separate arms (57.4 µg/mL: 95 % CI 32.3-102) or RTS,S alone (62.0 µg/mL: 95 % CI: 37.8-101.8). RTS,S-specific lymphoproliferative responses and ex vivo ELISpot CSP-specific interferon-gamma (IFN-γ) responses were indistinguishable among groups receiving RTS,S/AS02. There was no difference in antibody to FMP1 among groups receiving FMP1/AS02. After CHMI, groups immunized with a RTS,S-containing regimen had ∼ 30 % sterile protection against parasitemia, and equivalent delays in time-to-parasitemia. The FMP1/AS02 alone group showed no sterile immunity or delay in parasitemia., Conclusion: Co-administration of RTS,S and FMP1/AS02 reduced anti-RTS,S antibody, but did not affect tolerability, cellular immunity, or efficacy in a stringent CHMI model. Absence of efficacy or delay of patency in the sporozoite challenge model in the FMP1/AS02 group did not rule out efficacy of FMP1/AS02 in an endemic population. However, a Phase IIb trial of FMP1/AS02 in children in malaria-endemic Kenya did not demonstrate efficacy against natural infection., Clinicaltrials: gov identifier: NCT01556945., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Evelina Angov has patent #7,550275 issued to United States Government. Evelina Angov has patent #7,595,191 issued to United States Government. Coauthor, Nadia Tornieporth, previously employee of GlaxoSmithKline Biologics., (Published by Elsevier India Pvt Ltd.)

Background: Whole sporozoite immunization under chemoprophylaxis (CPS regime) induces long-lasting sterile homologous protection in the controlled human malaria infection model using Plasmodium falciparum strain NF54. The relative proficiency of liver-stage parasite development may be an important factor determining immunization efficacy. Previous studies show that Plasmodium falciparum strain NF135 produces relatively high numbers of large liver-stage schizonts in vitro. Here, we evaluate this strain for use in CPS immunization regimes., Methods: In a partially randomized, open-label study conducted at the Radboudumc, Nijmegen, the Netherlands, healthy, malaria-naïve adults were immunized by three rounds of fifteen or five NF135-infected mosquito bites under mefloquine prophylaxis (cohort A) or fifteen NF135-infected mosquito bites and presumptive treatment with artemether/lumefantrine (cohort B). Cohort A participants were exposed to a homologous challenge 19 weeks after immunization. The primary objective of the study was to evaluate the safety and tolerability of CPS immunizations with NF135., Results: Relatively high liver-to-blood inocula were observed during immunization with NF135 in both cohorts. Eighteen of 30 (60%) high-dose participants and 3/10 (30%) low-dose participants experienced grade 3 adverse events 7 to 21 days following their first immunization. All cohort A participants and two participants in cohort B developed breakthrough blood-stage malaria infections during immunizations requiring rescue treatment. The resulting compromised immunizations induced modest sterile protection against homologous challenge in cohort A (5/17; 29%)., Conclusions: These CPS regimes using NF135 were relatively poorly tolerated and frequently required rescue treatment, thereby compromising immunization efficiency and protective efficacy. Consequently, the full potential of NF135 sporozoites for induction of immune protection remains inconclusive. Nonetheless, the high liver-stage burden achieved by this strain highlights it as an interesting potential candidate for novel whole sporozoite immunization approaches., Trial Registration: The trial was registered at ClinicalTrials.gov under identifier NCT03813108., (© 2023. The Author(s).)

Background: Malaria elimination requires interruption of the highly efficient transmission of Plasmodium parasites by mosquitoes. TB31F is a humanised monoclonal antibody that binds the gamete surface protein Pfs48/45 and inhibits fertilisation, thereby preventing further parasite development in the mosquito midgut and onward transmission. We aimed to evaluate the safety and efficacy of TB31F in malaria-naive participants., Methods: In this open-label, first-in-human, dose-escalation, phase 1 clinical trial, healthy, malaria-naive, adult participants were administered a single intravenous dose of 0·1, 1, 3, or 10 mg/kg TB31F or a subcutaneous dose of 100 mg TB31F, and monitored until day 84 after administration at a single centre in the Netherlands. The primary outcome was the frequency and magnitude of adverse events. Additionally, TB31F serum concentrations were measured by ELISA. Transmission-reducing activity (TRA) of participant sera was assessed by standard membrane feeding assays with Anopheles stephensi mosquitoes and cultured Plasmodium falciparum gametocytes. The trial is registered with Clinicaltrials.gov, NCT04238689., Findings: Between Feb 17 and Dec 10, 2020, 25 participants were enrolled and sequentially assigned to each dose (n=5 per group). No serious or severe adverse events occurred. In total, 33 grade 1 and six grade 2 related adverse events occurred in 20 (80%) of 25 participants across all groups. Serum of all participants administered 1 mg/kg, 3 mg/kg, or 10 mg/kg TB31F intravenously had more than 80% TRA for 28 days or more, 56 days or more, and 84 days or more, respectively. The TB31F serum concentration reaching 80% TRA was 2·1 μg/mL (95% CI 1·9-2·3). Extrapolating the duration of TRA from antibody kinetics suggests more than 80% TRA is maintained for 160 days (95% CI 136-193) following a single intravenous 10 mg/kg dose., Interpretation: TB31F is a well tolerated and highly potent monoclonal antibody capable of completely blocking transmission of P falciparum parasites from humans to mosquitoes. In areas of seasonal transmission, a single dose might cover an entire malaria season., Funding: PATH's Malaria Vaccine Initiative., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

The timelines for developing vaccines against infectious diseases are lengthy, and often vaccines that reach the stage of large phase 3 field trials fail to provide the desired level of protective efficacy. The application of controlled human challenge models of infection and disease at the appropriate stages of development could accelerate development of candidate vaccines and, in fact, has done so successfully in some limited cases. Human challenge models could potentially be used to gather critical information on pathogenesis, inform strain selection for vaccines, explore cross-protective immunity, identify immune correlates of protection and mechanisms of protection induced by infection or evoked by candidate vaccines, guide decisions on appropriate trial endpoints, and evaluate vaccine efficacy. We prepared this report to motivate fellow scientists to exploit the potential capacity of controlled human challenge experiments to advance vaccine development. In this review, we considered available challenge models for 17 infectious diseases in the context of the public health importance of each disease, the diversity and pathogenesis of the causative organisms, the vaccine candidates under development, and each model's capacity to evaluate them and identify correlates of protective immunity. Our broad assessment indicated that human challenge models have not yet reached their full potential to support the development of vaccines against infectious diseases. On the basis of our review, however, we believe that describing an ideal challenge model is possible, as is further developing existing and future challenge models.

Background: Fever and inflammation are a hallmark of clinical Plasmodium falciparum (Pf) malaria induced by circulating asexual parasites. Although clinical manifestations of inflammation are associated with parasite density, this relationship is influenced by a complex network of immune-modulating factors of both human and parasite origin., Methods: In the Controlled Human Malaria infection (CHMI) model, we compared clinical inflammation in healthy malaria-naïve volunteers infected by either Pf-infected mosquito bites (MB, n=12) or intravenous administration of Pf-infected red blood cells (BS, n=12)., Findings: All volunteers developed patent parasitaemia, but both the incidence and duration of severe adverse events were significantly higher after MB infection. Similarly, clinical laboratory markers of inflammation were significantly increased in the MB-group, as well as serum pro-inflammatory cytokine concentrations including IFN-γ, IL-6, MCP1 and IL-8. Parasite load, as reflected by maximum parasite density and area under the curve, was similar, but median duration of parasitaemia until treatment was longer in the BS-group compared to the MB-group (8 days [range 8 - 8 days] versus 5·5 days [range 3·5 - 12·5 days]). The in vitro response of subsets of peripheral blood mononuclear cells showed attenuated Pf-specific IFNγ production by γδ T-cells in the BS-arm., Interpretation: In conclusion, irrespective the parasite load, Pf-infections by MB induce stronger signs and symptoms of inflammation compared to CHMI by BS infection. The pathophysiological basis remains speculative but may relate to induced immune tolerance., Funding: The trial was supported by PATH's Malaria Vaccine Initiative; the current analyses were supported by the AMMODO Science Award 2019 (TB)., Competing Interests: Declaration of interests RS received consulting fees from Biomedical Primate Research Centre, Rijswijk, The Netherlands and has stocks in TropIQ Health Sciences, TropIQ, the Netherlands. MA was supported by PATH's Malaria Vaccine Initiative. ZY was supported by the AMMODO science award 2019 awarded to TB. All other authors: No reported conflicts., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: We previously demonstrated that RTS,S/AS01 B and RTS,S/AS01 E vaccination regimens including at least one delayed fractional dose can protect against Plasmodium falciparum malaria in a controlled human malaria infection (CHMI) model, and showed inferiority of a two-dose versus three-dose regimen. In this follow-on trial, we evaluated whether fractional booster vaccination extended or induced protection in previously protected (P-Fx) or non-protected (NP-Fx) participants., Methods: 49 participants (P-Fx: 25; NP-Fx: 24) received a fractional (1/5th dose-volume) RTS,S/AS01 E booster 12 months post-primary regimen. They underwent P. falciparum CHMI three weeks later and were then followed for six months for safety and immunogenicity., Results: Overall vaccine efficacy against re-challenge was 53% (95% CI: 37-65%), and similar for P-Fx (52% [95% CI: 28-68%]) and NP-Fx (54% [95% CI: 29-70%]). Efficacy appeared unaffected by primary regimen or previous protection status. Anti-CS (repeat region) antibody geometric mean concentrations (GMCs) increased post-booster vaccination. GMCs were maintained over time in primary three-dose groups but declined in the two-dose group. Protection after re-challenge was associated with higher anti-CS antibody responses. The booster was well-tolerated., Conclusions: A fractional RTS,S/AS01 E booster given one year after completion of a primary two- or three-dose RTS,S/AS01 delayed fractional dose regimen can extend or induce protection against CHMI., Clinical Trial Registration: NCT03824236. linked to this article can be found on the Research Data as well as Figshare https://figshare.com/s/ee025150f9d1ac739361., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lievens, Marc; Morelle, Danielle; Debois, Muriel and Ofori-Anyinam, Opokua; are employees of the GSK group of companies. Debois, Muriel; Morelle, Danielle; Lievens, Marc; Ofori-Anyinam, Opokua have restricted shares in the GSK group of companies. Greenleaf, Melissa; Duncan, Elizabeth; Chuang, Ilin; Angov, Evelina; Ivinson, Karen; Komisar, Jack; Lee, Cynthia; Moon, James; Ockenhouse, Christian; Regules, Jason; Garver, Lindsey; Sedegah, Martha; Sikaffy, April K; Ngauy, Viseth declare no competing interests., (Copyright © 2021 GlaxoSmithKline Biologicals S.A. Published by Elsevier Ltd.. All rights reserved.)

Background: For malaria elimination efforts, it is important to better understand parasite transmission to mosquitoes and develop models for early-clinical evaluation of transmission-blocking interventions., Methods: In a randomized open-label trial, 24 participants were infected by bites from Plasmodium falciparum 3D7-infected mosquitoes (mosquito bite [MB]; n = 12) or by induced blood-stage malaria (IBSM) with the same parasite line (n = 12). After subcurative piperaquine treatment, asexual parasite and gametocytes kinetics were assessed, and mosquito feeding experiments were performed., Results: Study procedures were well tolerated. The median peak gametocyte density was 1304/mL (interquartile range, 308-1607/mL) after IBSM, compared with 14/mL (10-64/mL) after MB inoculation (P < .001), despite similar peak asexual parasite densities (P = .48). Peak gametocyte density was correlated with preceding pfap2-g transcripts, indicative of gametocyte commitment (ρ = 0.62; P = .002). Direct feeding assays resulted in mosquito infections from 9 of 12 participants after IBSM versus 0 of 12 after MB inoculation (P < .001)., Conclusions: We observed a striking effect of inoculation method on gametocyte production, suggesting higher gametocyte commitment after IBSM. Our direct comparison of MB and IBSM establishes the controlled human malaria infection transmission model, using intravenous administration of P. falciparum-infected erythrocytes as a model for early-clinical evaluation of interventions that aim to interrupt malaria transmission., Clinical Trial Registration: NCT03454048., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Anti-circumsporozoite antibody titres have been established as an essential indicator for evaluating the immunogenicity and protective capacity of the RTS,S/AS01 malaria vaccine. However, a new delayed-fractional dose regime of the vaccine was recently shown to increase vaccine efficacy, from 62.5% (95% CI 29.4-80.1%) under the original dosing schedule to 86.7% (95% CI, 66.8-94.6%) without a corresponding increase in antibody titres. Here we reanalyse the antibody data from this challenge trial to determine whether IgG avidity may help to explain efficacy better than IgG titre alone by adapting a within-host mathematical model of sporozoite inoculation. We demonstrate that a model incorporating titre and avidity provides a substantially better fit to the data than titre alone. These results also suggest that in individuals with a high antibody titre response that also show high avidity (both metrics in the top tercile of observed values) delayed-fractional vaccination provided near perfect protection upon first challenge (98.2% [95% Credible Interval 91.6-99.7%]). This finding suggests that the quality of the vaccine induced antibody response is likely to be an important determinant in the development of highly efficacious pre-erythrocytic vaccines against malaria., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ‘ACG has a data-transfer agreement with GSK Vaccines to cover ongoing analysis of trial data related to the RTS,S vaccine. She does not receive any funding from GSK for this work. All other authors declare no competing interests.’., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

For some diseases, successful vaccines have been developed using a nonpathogenic counterpart of the causative microorganism of choice. The nonpathogenicity of the rodent Plasmodium berghei ( Pb ) parasite in humans prompted us to evaluate its potential as a platform for vaccination against human infection by Plasmodium falciparum ( Pf ), a causative agent of malaria. We hypothesized that the genetic insertion of a leading protein target for clinical development of a malaria vaccine, Pf circumsporozoite protein (CSP), in its natural pre-erythrocytic environment, would enhance Pb 's capacity to induce protective immunity against Pf infection. Hence, we recently generated a transgenic Pb sporozoite immunization platform expressing Pf CSP ( Pb Vac), and we now report the clinical evaluation of its biological activity against controlled human malaria infection (CHMI). This first-in-human trial shows that Pb Vac is safe and well tolerated, when administered by a total of ~300 Pb Vac-infected mosquitoes per volunteer. Although protective efficacy evaluated by CHMI showed no sterile protection at the tested dose, significant delays in patency (2.2 days, P = 0.03) and decreased parasite density were observed after immunization, corresponding to an estimated 95% reduction in Pf liver parasite burden (confidence interval, 56 to 99%; P = 0.010). Pb Vac elicits dose-dependent cross-species cellular immune responses and functional Pf CSP-dependent antibody responses that efficiently block Pf sporozoite invasion of liver cells in vitro. This study demonstrates that Pb Vac immunization elicits a marked biological effect, inhibiting a subsequent infection by the human Pf parasite, and establishes the clinical validation of a new paradigm in malaria vaccination., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The RTS,S/AS01 vaccine provides partial protection against Plasmodium falciparum infection but determinants of protection and/or disease are unclear. Previously, anti-circumsporozoite protein (CSP) antibody titers and blood RNA signatures were associated with RTS,S/AS01 efficacy against controlled human malaria infection (CHMI). By analyzing host blood transcriptomes from five RTS,S vaccination CHMI studies, we demonstrate that the transcript ratio MX2/GPR183, measured 1 day after third immunization, discriminates protected from non-protected individuals. This ratiometric signature provides information that is complementary to anti-CSP titer levels for identifying RTS,S/AS01 immunized people who developed protective immunity and suggests a role for interferon and oxysterol signaling in the RTS,S mode of action., (Copyright © 2020 Du, Thompson, Muller, Valvo, Braun, Shankar, van den Berg, Jongert, Dover, Sadoff, Hendriks, Gardner, Ballou, Regules, van der Most, Aderem, Ockenhouse, Hill, Wille-Reece and Zak.)

Introduction : RTS,S/AS01 is currently the most advanced malaria vaccine but provides incomplete, short-term protection. It was developed for use within the expanded program on immunizations (EPI) for African children. Another use could be adding mass RTS,S/AS01 vaccination to the integrated malaria elimination strategy in the Greater Mekong Subregion (GMS), where multidrug-resistant P.falciparum strains have emerged and spread. Prior to evaluating RTS,S/AS01 in large-scale trials we assessed whether the vaccine, administered with and without antimalarial drugs, is safe and immunogenic in Asian populations. Methods : An open-label, randomized, controlled phase 2 trial was conducted in healthy, adult Thai volunteers. Seven vaccine regimens with and without antimalarial drugs (dihydroartemisinin-piperaquine plus a single low dose primaquine) were assessed. Antibody titres against the Pf CSP full-length (NANP) 6, Pf CSP anti-C-term, Pf CSP full-length (N + C-Terminal) were measured by standard enzyme-linked immunosorbent assays. Liquid chromatography was used to measure piperaquine, primaquine and carboxy-primaquine concentrations. Results : 193 volunteers were enrolled and 186 study participants completed the 6 months follow-up period. One month after the last vaccination all study participants had seroconverted to the Pf CSP (NANP)6, and the Pf CSP Full Length (N + C-Terminal). More than 90% had seroconverted to the Pf anti-C-Term CSP. There was no indication that drug concentrations were influenced by vaccine regimens or the antibody levels by the drug regimens. Adverse events were similarly distributed between the seven treatment groups. No serious adverse events attributable to the study interventions were detected. Conclusion : This study found that RTS,S/AS01 with and without dihydroartemisinin-piperaquine plus a single low dose primaquine was safe and immunogenic in a healthy, adult Asian population.

Background: The only licensed malaria vaccine, RTS,S/AS01, has been developed for morbidity-control in young children. The potential impact on transmission of deploying such anti-infective vaccines to wider age ranges, possibly with co-administration of antimalarial treatment, is unknown. Combinations of existing malaria interventions is becoming increasingly important as evidence mounts that progress on reducing malaria incidence is stalling and threatened by resistance., Methods: Malaria transmission and intervention dynamics were simulated using OpenMalaria, an individual-based simulation model of malaria transmission, by considering a seasonal transmission setting and by varying epidemiological and setting parameters such as transmission intensity, case management, intervention types and intervention coverages. Chemopreventive drugs and anti-infective vaccine efficacy profiles were based on previous studies in which model parameters were fitted to clinical trial data. These intervention properties were used to evaluate the potential of seasonal mass applications of preventative anti-infective malaria vaccines, alone or in combination with chemoprevention, to reduce malaria transmission, prevent resurgence, and/or reach transmission interruption., Results: Deploying a vaccine to all ages on its own is a less effective intervention strategy compared to chemoprevention alone. However, vaccines combined with drugs are likely to achieve dramatic prevalence reductions and in few settings, transmission interruption. The combined mass intervention will result in lower prevalence following the intervention compared to chemoprevention alone and will increase chances of interruption of transmission resulting from a synergistic effect between both interventions. The combination of vaccine and drug increases the time before transmission resurges after mass interventions cease compared to mass treatment alone. Deploying vaccines and drugs together requires fewer rounds of mass intervention and fewer years of intervention to achieve the same public health impact as chemoprevention alone., Conclusions: Through simulations we identified a previously unidentified value of deploying vaccines with drugs, namely the greatest benefit will be in preventing and delaying transmission resurgence for longer periods than with other human targeted interventions. This is suggesting a potential role for deploying vaccines alongside drugs in transmission foci as part of surveillance-response strategies.

Whole-sporozoite vaccination/immunization induces high levels of protective immunity in both rodent models of malaria and in humans. Recently, we generated a transgenic line of the rodent malaria parasite P. berghei ( Pb ) that expresses the P. falciparum ( Pf ) circumsporozoite protein ( Pf CS), and showed that this parasite line ( Pb Vac) was capable of (1) infecting and developing in human hepatocytes but not in human erythrocytes, and (2) inducing neutralizing antibodies against the human Pf parasite. Here, we analyzed Pb Vac in detail and developed tools necessary for its use in clinical studies. A microbiological contaminant-free Master Cell Bank of Pb Vac parasites was generated through a process of cyclic propagation and clonal expansion in mice and mosquitoes and was genetically characterized. A highly sensitive qRT-PCR-based method was established that enables Pb Vac parasite detection and quantification at low parasite densities in vivo. This method was employed in a biodistribution study in a rabbit model, revealing that the parasite is only present at the site of administration and in the liver up to 48 h post infection and is no longer detectable at any site 10 days after administration. An extensive toxicology investigation carried out in rabbits further showed the absence of Pb Vac-related toxicity. In vivo drug sensitivity assays employing rodent models of infection showed that both the liver and the blood stage forms of Pb Vac were completely eliminated by Malarone ® treatment. Collectively, our pre-clinical safety assessment demonstrates that Pb Vac possesses all characteristics necessary to advance into clinical evaluation., Competing Interests: A.M.M. and M.P. are inventors on a patent or patent application issued, allowed, or filed internationally, covering parts of this work. The remaining authors declare no competing interests.

Malaria continues to be one of the world's most devastating infectious tropical diseases, and alternative strategies to prevent infection and disease spread are urgently needed. These strategies include the development of effective vaccines, such as malaria transmission blocking vaccines (TBV) directed against proteins found on the sexual stages of Plasmodium falciparum parasites present in the mosquito midgut. The Pfs25 protein, which is expressed on the surface of gametes, zygotes and ookinetes, has been a primary target for TBV development. One such vaccine strategy based on Pfs25 is a plant-produced malaria vaccine candidate engineered as a chimeric non-enveloped virus-like particle (VLP) comprising Pfs25 fused to the Alfalfa mosaic virus coat protein. This Pfs25 VLP-FhCMB vaccine candidate has been engineered and manufactured in Nicotiana benthamiana plants at pilot plant scale under current Good Manufacturing Practice guidelines. The safety, reactogenicity and immunogenicity of Pfs25 VLP-FhCMB was assessed in healthy adult volunteers. This Phase 1, dose escalation, first-in-human study was designed primarily to evaluate the safety of the purified plant-derived Pfs25 VLP combined with Alhydrogel® adjuvant. At the doses tested in this Phase 1 study, the vaccine was generally shown to be safe in healthy volunteers, with no incidence of vaccine-related serious adverse events and no evidence of any dose-limiting or dose-related toxicity, demonstrating that the plant-derived Pfs25 VLP-FhCMB vaccine had an acceptable safety and tolerability profile. In addition, although the vaccine did induce Pfs25-specific IgG in vaccinated patients in a dose dependent manner, the transmission reducing activity of the antibodies generated were weak, suggesting the need for an alternative vaccine adjuvant formulation. This study was registered at www.ClinicalTrials.gov under reference identifier NCT02013687., (Copyright © 2018. Published by Elsevier Ltd.)

Host immunity exerts strong selective pressure on pathogens. Population-level genetic analysis can identify signatures of this selection, but these signatures reflect the net selective effect of all hosts and vectors in a population. In contrast, analysis of pathogen diversity within hosts provides information on individual, host-specific selection pressures. Here, we combine these complementary approaches in an analysis of the malaria parasite Plasmodium falciparum using haplotype sequences from thousands of natural infections in sub-Saharan Africa. We find that parasite genotypes show preferential clustering within multi-strain infections in young children, and identify individual amino acid positions that may contribute to strain-specific immunity. Our results demonstrate that natural host defenses to P. falciparum act in an allele-specific manner to block specific parasite haplotypes from establishing blood-stage infections. This selection partially explains the extreme amino acid diversity of many parasite antigens and suggests that vaccines targeting such proteins should account for allele-specific immunity.

Although RTS,S remains the most advanced malaria vaccine, the factors influencing differences in vaccine immunogenicity or efficacy between individuals or populations are still poorly characterised. The analyses of genetic determinants of immunogenicity have previously been restricted by relatively small sample sizes from individual trials. Here we combine data from six Phase II RTS,S trials and evaluate the relationship between HLA allele groups and RTS,S-mediated protection in controlled human malaria infections (CHMI), using multivariate logistic or linear regression. We observed significant associations between three allele groups (HLA-A ∗ 01, HLA-B ∗ 08, and HLA-DRB1 ∗ 15/ ∗ 16) and protection, while another three allele groups (HLA-A ∗ 03, HLA-B ∗ 53, and HLA-DRB1 ∗ 07) were significantly associated with lack of protection. It is noteworthy that these 'protective' allele groups are thought to be at a lower prevalence in sub-Saharan African populations than in the UK or USA where these Phase II trials occurred. Taken together, the analyses presented here give an indication that HLA genotype may influence RTS,S-mediated protective efficacy against malaria infection., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Malaria elimination strategies require a thorough understanding of parasite transmission from human to mosquito. A clinical model to induce gametocytes to understand their dynamics and evaluate transmission-blocking interventions (TBI) is currently unavailable. Here, we explore the use of the well-established Controlled Human Malaria Infection model (CHMI) to induce gametocyte carriage with different antimalarial drug regimens., Methods: In a single centre, open-label randomised trial, healthy malaria-naive participants (aged 18–35 years) were infected with Plasmodium falciparum by bites of infected Anopheles mosquitoes. Participants were randomly allocated to four different treatment arms (n = 4 per arm) comprising low-dose (LD) piperaquine (PIP) or sulfadoxine-pyrimethamine (SP), followed by a curative regimen upon recrudescence. Male and female gametocyte densities were determined by molecular assays., Results: Mature gametocytes were observed in all participants (16/16, 100%). Gametocytes appeared 8.5–12 days after the first detection of asexual parasites. Peak gametocyte densities and gametocyte burden was highest in the LD-PIP/SP arm, and associated with the preceding asexual parasite biomass (p=0.026). Male gametocytes had a mean estimated circulation time of 2.7 days (95% CI 1.5–3.9) compared to 5.1 days (95% CI 4.1–6.1) for female gametocytes. Exploratory mosquito feeding assays showed successful sporadic mosquito infections. There were no serious adverse events or significant differences in the occurrence and severity of adverse events between study arms (p=0.49 and p=0.28)., Conclusions: The early appearance of gametocytes indicates gametocyte commitment during the first wave of asexual parasites emerging from the liver. Treatment by LD-PIP followed by a curative SP regimen, results in the highest gametocyte densities and the largest number of gametocyte-positive days. This model can be used to evaluate the effect of drugs and vaccines on gametocyte dynamics, and lays the foundation for fulfilling the critical unmet need to evaluate transmission-blocking interventions against falciparum malaria for downstream selection and clinical development., Funding: Funded by PATH Malaria Vaccine Initiative (MVI)., Clinical Trial Number: NCT02836002., Competing Interests: IR, Lv, LC, KL, LM, WG, Gv, KT, RS, Mv, Qd, Av, KI, CH, Sd, JB, KC, CO, JM, RS, TB No competing interests declared, (© 2018, Reuling et al.)

Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development in vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA) 3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I β-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP-Fab311 complex forming a highly organized helical structure. Together, these structural insights may aid in the design of a next-generation malaria vaccine., Competing Interests: Conflict of interest statement: W.V. and D.E. are employees of and own equity in Atreca, Inc., (Copyright © 2017 the Author(s). Published by PNAS.)

The RTS,S candidate malaria vaccine can protect against controlled human malaria infection (CHMI), but how protection is achieved remains unclear. Here, we have analyzed longitudinal peripheral blood transcriptome and immunogenicity data from a clinical efficacy trial in which healthy adults received three RTS,S doses 4 weeks apart followed by CHMI 2 weeks later. Multiway partial least squares discriminant analysis (N-PLS-DA) of transcriptome data identified 110 genes that could be used in predictive models of protection. Among the 110 genes, 42 had known immune-related functions, including 29 that were related to the NF-κB-signaling pathway and 14 to the IFN-γ-signaling pathway. Post-dose 3 serum IFN-γ concentrations were also correlated with protection; and N-PLS-DA of IFN-γ-signaling pathway transcriptome data selected almost all (44/45) of the representative genes for predictive models of protection. Hence, the identification of the NF-κB and IFN-γ pathways provides further insight into how vaccine-mediated protection may be achieved.

Background: The need for a highly efficacious vaccine against Plasmodium falciparum remains pressing. In this controlled human malaria infection (CHMI) study, we assessed the safety, efficacy and immunogenicity of a schedule combining 2 distinct vaccine types in a staggered immunization regimen: one inducing high-titer antibodies to circumsporozoite protein (RTS,S/AS01B) and the other inducing potent T-cell responses to thrombospondin-related adhesion protein (TRAP) by using a viral vector., Method: Thirty-seven healthy malaria-naive adults were vaccinated with either a chimpanzee adenovirus 63 and modified vaccinia virus Ankara-vectored vaccine expressing a multiepitope string fused to TRAP and 3 doses of RTS,S/AS01B (group 1; n = 20) or 3 doses of RTS,S/AS01B alone (group 2; n = 17). CHMI was delivered by mosquito bites to 33 vaccinated subjects at week 12 after the first vaccination and to 6 unvaccinated controls., Results: No suspected unexpected serious adverse reactions or severe adverse events related to vaccination were reported. Protective vaccine efficacy was observed in 14 of 17 subjects (82.4%) in group 1 and 12 of 16 subjects (75%) in group 2. All control subjects received a diagnosis of blood-stage malaria parasite infection. Both vaccination regimens were immunogenic. Fourteen protected subjects underwent repeat CHMI 6 months after initial CHMI; 7 of 8 (87.5%) in group 1 and 5 of 6 (83.3%) in group 2 remained protected., Conclusions: The high level of sterile efficacy observed in this trial is encouraging for further evaluation of combination approaches using these vaccine types., Clinical Trials Registration: NCT01883609., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: Three full doses of RTS,S/AS01 malaria vaccine provides partial protection against controlled human malaria parasite infection (CHMI) and natural exposure. Immunization regimens, including a delayed fractional third dose, were assessed for potential increased protection against malaria and immunologic responses., Methods: In a phase 2a, controlled, open-label, study of healthy malaria-naive adults, 16 subjects vaccinated with a 0-, 1-, and 2-month full-dose regimen (012M) and 30 subjects who received a 0-, 1-, and 7-month regimen, including a fractional third dose (Fx017M), underwent CHMI 3 weeks after the last dose. Plasmablast heavy and light chain immunoglobulin messenger RNA sequencing and antibody avidity were evaluated. Protection against repeat CHMI was evaluated after 8 months., Results: A total of 26 of 30 subjects in the Fx017M group (vaccine efficacy [VE], 86.7% [95% confidence interval [CI], 66.8%-94.6%]; P < .0001) and 10 of 16 in the 012M group (VE, 62.5% [95% CI, 29.4%-80.1%]; P = .0009) were protected against infection, and protection differed between schedules (P = .040, by the log rank test). The fractional dose boosting increased antibody somatic hypermutation and avidity and sustained high protection upon rechallenge., Discussions: A delayed third fractional vaccine dose improved immunogenicity and protection against infection. Optimization of the RTS,S/AS01 immunization regimen may lead to improved approaches against malaria., Clinical Trials Registration: NCT01857869., (Published by Oxford University Press on behalf of the Infectious Diseases Society of America, 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Plasmodium falciparum malaria is a deadly pathogen. The invasion of red blood cells (RBCs) by merozoites is a target for vaccine development. Although anti-merozoite antibodies can block invasion in vitro, there is no efficacy in vivo. To explain this discrepancy we hypothesized that complement activation could enhance RBC invasion by binding to the complement receptor 1 (CR1). Here we show that a monoclonal antibody directed against the merozoite and human polyclonal IgG from merozoite vaccine recipients enhanced RBC invasion in a complement-dependent manner and that soluble CR1 inhibited this enhancement. Sialic acid-independent strains, that presumably are able to bind to CR1 via a native ligand, showed less complement-dependent enhancement of RBC invasion than sialic acid-dependent strains that do not utilize native CR1 ligands. Confocal fluorescent microscopy revealed that complement-dependent invasion resulted in aggregation of CR1 at the RBC surface in contact with the merozoite. Finally, total anti-P. berghei IgG enhanced parasite growth and C3 deficiency decreased parasite growth in mice. These results demonstrate, contrary to current views, that complement activation in conjunction with antibodies can paradoxically aid parasites invade RBCs and should be considered in future design and testing of merozoite vaccines., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Models of controlled human malaria infection (CHMI) initiated by mosquito bite have been widely used to assess efficacy of preerythrocytic vaccine candidates in small proof-of-concept phase 2a clinical trials. Efficacy testing of blood-stage malaria parasite vaccines, however, has generally relied on larger-scale phase 2b field trials in malaria-endemic populations. We report the use of a blood-stage P. falciparum CHMI model to assess blood-stage vaccine candidates, using their impact on the parasite multiplication rate (PMR) as the primary efficacy end point., Methods: Fifteen healthy United Kingdom adult volunteers were vaccinated with FMP2.1, a protein vaccine that is based on the 3D7 clone sequence of apical membrane antigen 1 (AMA1) and formulated in Adjuvant System 01 (AS01). Twelve vaccinees and 15 infectivity controls subsequently underwent blood-stage CHMI. Parasitemia was monitored by quantitative real-time polymerase chain reaction (PCR) analysis, and PMR was modeled from these data., Results: FMP2.1/AS01 elicited anti-AMA1 T-cell and serum antibody responses. Analysis of purified immunoglobulin G showed functional growth inhibitory activity against P. falciparum in vitro. There were no vaccine- or CHMI-related safety concerns. All volunteers developed blood-stage parasitemia, with no impact of the vaccine on PMR., Conclusions: FMP2.1/AS01 demonstrated no efficacy after blood-stage CHMI. However, the model induced highly reproducible infection in all volunteers and will accelerate proof-of-concept testing of future blood-stage vaccine candidates., Clinical Trials Registration: NCT02044198., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Background: Recent vaccine studies have shown that the magnitude of an antibody response is often insufficient to explain efficacy, suggesting that characteristics regarding the quality of the antibody response, such as its fine specificity and functional activity, may play a major role in protection. Previous studies of the lead malaria vaccine candidate, RTS,S, have shown that circumsporozoite protein (CSP)-specific antibodies and CD4(+) T cell responses are associated with protection, however the role of fine specificity and biological function of CSP-specific antibodies remains to be elucidated. Here, the relationship between fine specificity, opsonization-dependent phagocytic activity and protection in RTS,S-induced antibodies is explored., Methods: A new method for measuring the phagocytic activity mediated by CSP-specific antibodies in THP-1 cells is presented and applied to samples from a recently completed phase 2 RTS,S/AS01 clinical trial. The fine specificity of the antibody response was assessed using ELISA against three antigen constructs of CSP: the central repeat region, the C-terminal domain and the full-length protein. A multi-parameter analysis of phagocytic activity and fine-specificity data was carried out to identify potential correlates of protection in RTS,S., Results: Results from the newly developed assay revealed that serum samples from RTS,S recipients displayed a wide range of robust and repeatable phagocytic activity. Phagocytic activity was correlated with full-length CSP and C-terminal specific antibody titres, but not to repeat region antibody titres, suggesting that phagocytic activity is primarily driven by C-terminal antibodies. Although no significant difference in overall phagocytic activity was observed with respect to protection, phagocytic activity expressed as 'opsonization index', a relative measure that normalizes phagocytic activity with CS antibody titres, was found to be significantly lower in protected subjects than non-protected subjects., Conclusions: Opsonization index was identified as a surrogate marker of protection induced by the RTS,S/AS01 vaccine and determined how antibody fine specificity is linked to opsonization activity. These findings suggest that the role of opsonization in protection in the RTS,S vaccine may be more complex than previously thought, and demonstrate how integrating multiple immune measures can provide insight into underlying mechanisms of immunity and protection.

Background: A vaccine to prevent infection and disease caused by Plasmodium vivax is needed both to reduce the morbidity caused by this parasite and as a key component in efforts to eradicate malaria worldwide. Vivax malaria protein 1 (VMP001), a novel chimeric protein that incorporates the amino- and carboxy- terminal regions of the circumsporozoite protein (CSP) and a truncated repeat region that contains repeat sequences from both the VK210 (type 1) and the VK247 (type 2) parasites, was developed as a vaccine candidate for global use., Methods: We conducted a first-in-human Phase 1 dose escalation vaccine study with controlled human malaria infection (CHMI) of VMP001 formulated in the GSK Adjuvant System AS01B. A total of 30 volunteers divided into 3 groups (10 per group) were given 3 intramuscular injections of 15 μg, 30 μg, or 60 μg respectively of VMP001, all formulated in 500 μL of AS01B at each immunization. All vaccinated volunteers participated in a P. vivax CHMI 14 days following the third immunization. Six non-vaccinated subjects served as infectivity controls., Results: The vaccine was shown to be well tolerated and immunogenic. All volunteers generated robust humoral and cellular immune responses to the vaccine antigen. Vaccination did not induce sterile protection; however, a small but significant delay in time to parasitemia was seen in 59% of vaccinated subjects compared to the control group. An association was identified between levels of anti-type 1 repeat antibodies and prepatent period., Significance: This trial was the first to assess the efficacy of a P. vivax CSP vaccine candidate by CHMI. The association of type 1 repeat-specific antibody responses with delay in the prepatency period suggests that augmenting the immune responses to this domain may improve strain-specific vaccine efficacy. The availability of a P. vivax CHMI model will accelerate the process of P. vivax vaccine development, allowing better selection of candidate vaccines for advancement to field trials.

Background: The RTS,S/AS01 vaccine targets the circumsporozoite protein of Plasmodium falciparum and has partial protective efficacy against clinical and severe malaria disease in infants and children. We investigated whether the vaccine efficacy was specific to certain parasite genotypes at the circumsporozoite protein locus., Methods: We used polymerase chain reaction-based next-generation sequencing of DNA extracted from samples from 4985 participants to survey circumsporozoite protein polymorphisms. We evaluated the effect that polymorphic positions and haplotypic regions within the circumsporozoite protein had on vaccine efficacy against first episodes of clinical malaria within 1 year after vaccination., Results: In the per-protocol group of 4577 RTS,S/AS01-vaccinated participants and 2335 control-vaccinated participants who were 5 to 17 months of age, the 1-year cumulative vaccine efficacy was 50.3% (95% confidence interval [CI], 34.6 to 62.3) against clinical malaria in which parasites matched the vaccine in the entire circumsporozoite protein C-terminal (139 infections), as compared with 33.4% (95% CI, 29.3 to 37.2) against mismatched malaria (1951 infections) (P=0.04 for differential vaccine efficacy). The vaccine efficacy based on the hazard ratio was 62.7% (95% CI, 51.6 to 71.3) against matched infections versus 54.2% (95% CI, 49.9 to 58.1) against mismatched infections (P=0.06). In the group of infants 6 to 12 weeks of age, there was no evidence of differential allele-specific vaccine efficacy., Conclusions: These results suggest that among children 5 to 17 months of age, the RTS,S vaccine has greater activity against malaria parasites with the matched circumsporozoite protein allele than against mismatched malaria. The overall vaccine efficacy in this age category will depend on the proportion of matched alleles in the local parasite population; in this trial, less than 10% of parasites had matched alleles. (Funded by the National Institutes of Health and others.).

Methods: In an observer blind, phase 2 trial, 55 adults were randomized to receive one dose of Ad35.CS.01 vaccine followed by two doses of RTS,S/AS01 (ARR-group) or three doses of RTS,S/AS01 (RRR-group) at months 0, 1, 2 followed by controlled human malaria infection., Results: ARR and RRR vaccine regimens were well tolerated. Efficacy of ARR and RRR groups after controlled human malaria infection was 44% (95% confidence interval 21%-60%) and 52% (25%-70%), respectively. The RRR-group had greater anti-CS specific IgG titers than did the ARR-group. There were higher numbers of CS-specific CD4 T-cells expressing > 2 cytokine/activation markers and more ex vivo IFN-γ enzyme-linked immunospots in the ARR-group than the RRR-group. Protected subjects had higher CS-specific IgG titers than non-protected subjects (geometric mean titer, 120.8 vs 51.8 EU/ml, respectively; P = .001)., Conclusions: An increase in vaccine efficacy of ARR-group over RRR-group was not achieved. Future strategies to improve upon RTS,S-induced protection may need to utilize alternative highly immunogenic prime-boost regimens and/or additional target antigens., Trial Registration: ClinicalTrials.gov NCT01366534.

Background: The malaria vaccine RTS,S induces antibodies against the Plasmodium falciparum circumsporozoite protein (CSP) and the concentration of Immunoglobulin G (IgG) against the repeat region of CSP following vaccination is associated with protection from P. falciparum malaria. So far, only the quantity of anti-CSP IgG has been measured and used to predict vaccination success, although quality (measured as avidity) of the antigen-antibody interaction shall be important since only a few sporozoites circulate for a short time after an infectious mosquito bite, likely requiring fast and strong binding., Methods: Quantity and avidity of anti-CSP IgG in African infants who received RTS,S/AS01E in a 0-1-2-month or a 0-1-7-month schedule in a phase 2 clinical trial were measured by enzyme-linked immunosorbent assay. Antibody avidity was defined as the proportion of IgG able to bind in the presence of a chaotropic agent (avidity index). The effect of CSP-specific IgG concentration and avidity on protective efficacy was modelled using Cox proportional hazards., Results: After the third dose, quantity and avidity were similar between the two vaccination schedules. IgG avidity after the last vaccine injection was not associated with protection, whereas the change in avidity following second and third RTS,S/AS01E injection was associated with a 54% risk reduction of getting malaria (hazard ratio: 0.46; 95% confidence interval (CI): 0.22-0.99) in those participants with a change in avidity above the median. The change in anti-CSP IgG concentration following second and third injection was associated with a 77% risk reduction of getting malaria (hazard ratio: 0.23, 95% CI: 0.11-0.51)., Conclusions: Change in IgG response between vaccine doses merits further evaluation as a surrogate marker for RTS,S efficacy., Trial Registration: ClinicalTrials.gov Identifier NCT00436007 .

In an attempt to improve the efficacy of the candidate malaria vaccine RTS,S/AS02, two studies were conducted in 1999 in healthy volunteers of RTS,S/AS02 in combination with recombinant Plasmodium falciparum thrombospondin-related anonymous protein (TRAP). In a Phase 1 safety and immunogenicity study, volunteers were randomized to receive TRAP/AS02 (N=10), RTS,S/AS02 (N=10), or RTS,S+TRAP/AS02 (N=20) at 0, 1 and 6-months. In a Phase 2 challenge study, subjects were randomized to receive either RTS,S+TRAP/AS02 (N=25) or TRAP/AS02 (N=10) at 0 and 1-month, or to a challenge control group (N=8). In both studies, the combination vaccine had an acceptable safety profile and was acceptably tolerated. Antigen-specific antibodies, lymphoproliferative responses, and IFN-γ production by ELISPOT assay elicited with the combination vaccine were qualitatively similar to those generated by the single component vaccines. However, post-dose 2 anti-CS antibodies in the RTS,S+TRAP/AS02 vaccine recipients were lower than in the RTS,S/AS02 vaccine recipients. After challenge, 10 of 11 RTS,S+TRAP/AS02 vaccinees, 5 of 5 TRAP/AS02 vaccinees, and 8 of 8 infectivity controls developed parasitemia, with median pre-patent periods of 13.0, 11.0, and 12.0 days, respectively. The absence of any prevention or delay of parasitemia by TRAP/AS02 suggests no apparent added value of TRAP/AS02 as a candidate vaccine. The absence of significant protection or delay of parasitemia in the 11 RTS,S+TRAP/AS02 vaccine recipients contrasts with previous 2 dose studies of RTS,S/AS02. The small sample size did not permit identifying statistically significant differences between the study arms. However, we speculate, within the constraints of the challenge study, that the presence of the TRAP antigen may have interfered with the vaccine efficacy previously observed with this regimen of RTS,S/AS02, and that any future TRAP-based vaccines should consider employing alternative vaccine platforms., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Controlled human malaria infection (CHMI) studies which recapitulate mosquito-borne infection are a critical tool to identify protective vaccine and drug candidates for advancement to field trials. In partnership with the Walter Reed Army Institute of Research, the CHMI model was established at the Seattle Biomedical Research Institute's Malaria Clinical Trials Center (MCTC). Activities and reagents at both centers were aligned to ensure comparability and continued safety of the model. To demonstrate successful implementation, CHMI was performed in six healthy malaria-naïve volunteers., Methods: All volunteers received NF54 strain Plasmodium falciparum by the bite of five infected Anopheles stephensi mosquitoes under controlled conditions and were monitored for signs and symptoms of malaria and for parasitemia by peripheral blood smear. Subjects were treated upon diagnosis with chloroquine by directly observed therapy. Immunological (T cell and antibody) and molecular diagnostic (real-time quantitative reverse transcriptase polymerase chain reaction [qRT-PCR]) assessments were also performed., Results: All six volunteers developed patent parasitemia and clinical malaria. No serious adverse events occurred during the study period or for six months post-infection. The mean prepatent period was 11.2 days (range 9-14 days), and geometric mean parasitemia upon diagnosis was 10.8 parasites/µL (range 2-69) by microscopy. qRT-PCR detected parasites an average of 3.7 days (range 2-4 days) earlier than blood smears. All volunteers developed antibodies to the blood-stage antigen merozoite surface protein 1 (MSP-1), which persisted up to six months. Humoral and cellular responses to pre-erythrocytic antigens circumsporozoite protein (CSP) and liver-stage antigen 1 (LSA-1) were limited., Conclusion: The CHMI model was safe, well tolerated and characterized by consistent prepatent periods, pre-symptomatic diagnosis in 3/6 subjects and adverse event profiles as reported at established centers. The MCTC can now evaluate candidates in the increasingly diverse vaccine and drug pipeline using the CHMI model., Trial Registration: ClinicalTrials.gov NCT01058226.

The availability of a highly purified and well characterized circumsporozoite protein (CSP) is essential to improve upon the partial success of recombinant CSP-based malaria vaccine candidates. Soluble, near full-length, Plasmodium falciparum CSP vaccine antigen (CS/D) was produced in E. coli under bio-production conditions that comply with current Good Manufacturing Practices (cGMP). A mouse immunogenicity study was conducted using a stable oil-in-water emulsion (SE) of CS/D in combination with the Toll-Like Receptor 4 (TLR4) agonist Glucopyranosyl Lipid A (GLA/SE), or one of two TLR7/8 agonists: R848 (un-conjugated) or 3M-051 (covalently conjugated). Compared to Alum and SE, GLA/SE induced higher CS/D specific antibody response in Balb/c mice. Subclass analysis showed higher IgG2:IgG1 ratio of GLA/SE induced antibodies as compared to Alum and SE. TLR synergy was not observed when soluble R848 was mixed with GLA/SE. Antibody response of 3M051 formulations in Balb/c was similar to GLA/SE, except for the higher IgG2:IgG1 ratio and a trend towards higher T cell responses in 3M051 containing groups. However, no synergistic enhancement of antibody and T cell response was evident when 3M051 conjugate was mixed with GLA/SE. In C57Bl/6 mice, CS/D adjuvanted with 3M051/SE or GLA/SE induced higher CSP repeat specific titers compared to SE. While, 3M051 induced antibodies had high IgG2c:IgG1 ratio, GLA/SE promoted high levels of both IgG1 and IgG2c. GLA/SE also induced more potent T-cell responses compared to SE in two independent C57/BL6 vaccination studies, suggesting a balanced and productive T(H1)/T(H2) response. GLA and 3M-051 similarly enhanced the protective efficacy of CS/D against challenge with a transgenic P. berghei parasite and most importantly, high levels of cytophilic IgG2 antibodies were associated with protection in this model. Our data indicated that the cGMP-grade, soluble CS/D antigen combined with the TLR4-containing adjuvant GLA/SE warrants further evaluation for protective responses in humans.

We have previously reported that Vivax Malaria Protein 001 (VMP001), a vaccine candidate based on the circumsporozoite protein of Plasmodium vivax, is immunogenic in mice and rhesus monkeys in the presence of various adjuvants. In the present study, we evaluated the immunogenicity and efficacy of VMP001 formulated with a TLR9 agonist in a water-in-oil emulsion. Following immunization, the vaccine efficacy was assessed by challenging Aotus nancymaae monkeys with P. vivax sporozoites. Monkeys from both the low- and high-dose vaccine groups generated strong humoral immune responses to the vaccine (peak median titers of 291,622), and its subunits (peak median titers to the N-term, central repeat and C-term regions of 22,188; 66,120 and 179,947, respectively). 66.7% of vaccinated monkeys demonstrated sterile protection following challenge. Protection was associated with antibodies directed against the central repeat region. The protected monkeys had a median anti-repeat titer of 97,841 compared to 14,822 in the non-protected monkeys. This is the first report demonstrating P. vivax CSP vaccine-induced protection of Aotus monkeys challenged with P. vivax sporozoites.

Background: The use of quantitative real-time PCR (qPCR) has allowed for precise quantification of parasites in the prepatent period and greatly improved the reproducibility and statistical power of controlled human malaria infection (CHMI) trials. Parasitological data presented here are from non-immunized, control-challenged subjects who participated in two CHMI trials conducted at the Walter Reed Army Institute of Research (WRAIR)., Methods: Standardized sporozoite challenge was achieved through the bite of five Anopheles stephensi mosquitoes infected with the 3D7clone of the NF54 strain of Plasmodium falciparum. Blood smears were scored positive when two unambiguous parasites were found. Analysis of parasitological PCR data was performed on log-transformed data using an independent sample t-test when comparing the two studies. The multiplication rate of blood-stage parasites was estimated using the linear model., Results: On average, parasites were detected 4.91 days (95% CI = 4.190 to 5.627) before smears. The earliest parasites were detected within 120 hours (5.01 days) after challenge. Parasite densities showed consistent cyclic patterns of blood-stage parasite growth in all volunteers. The parasite multiplication rates for both studies was 8.18 (95% CI = 6.162 to 10.20). Data showed that at low parasite densities, a combination of sequestration and stochastic effects of low copy number DNA may impact qPCR detection and the parasite detection limit., Conclusion: Smear positive is an endpoint which antimalarial rescue is imperative whereas early detection of parasitological data by qPCR can allow for better anticipation of the endpoint. This would allow for early treatment to reduce clinical illness and risk for study participants. To use qPCR as the primary endpoint in CHMI trials, an algorithm of two positives by qPCR where one of the positives must have parasite density of at least 2 parasites/μL is proposed.

Background: Microscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular methods in detection of malaria. Some of the challenges that continue to limit the widespread utilization of qPCR include lack of assay standardization, assay variability, risk of contamination, and the need for cold-chain. Lyophilization of molecular assays can overcome some of these limitations and potentially enable widespread qPCR utilization., Methods: A recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-Ready™ format (MMSR). MMSR assay contained all the required reagents for qPCR including primers and probes, requiring only the addition of water and sample to perform qPCR. The performance of the MMSR assay was compared to the non-freeze dried, "wet" assay. Stability studies were done by maintaining the MMSR assays at four different ambient temperatures of 4°C, room temperature (RT), 37°C and 42°C over a period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two different concentrations. The CT values and the standard deviations (SD) were used in the analysis of the assay performance., Results: The limit of detection for the MMSR assay was 0.244 parasites/μL for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to "wet" assay which was 0.39 and 3.13 parasites/μL for PLU and FAL assay targets, respectively. The MMSR assay performed with high efficiencies similar to those of the "wet" assay and was stable at 37°C for 42 days, with estimated shelf-life of 5 months. When used to analyse field clinical samples, MMSR assay performed with 100% sensitivity and specificity compared to the "wet" assay., Conclusion: The MMSR assay has the same robust performance characteristics as the "wet" assay and is highly stable. Availability of MMSR assay allows flexibility and provides an option in choosing assay for malaria diagnostics depending on the application, needs and budget.

Background: An effective malaria vaccine remains elusive. The most effective experimental vaccines confer only limited and short-lived protection despite production of protective antibodies. However, immunization with irradiated sporozoites, or with live sporozoites under chloroquine cover, has resulted in long-term protection apparently due to the generation of protective CD8+ T cells. The nature and function of these protective CD8+ T cells has not been elucidated. In the current study, the phenotype of CD8+ T cells generated after immunization of C57BL/6 mice with live Plasmodium berghei sporozoites under chloroquine cover was investigated., Methods: Female C57BL/6 mice, C57BL/6 mice B2 macroglobulin -/- [KO], or invariant chain-/- [Ic KO] [6-8 weeks old] were immunized with P. berghei sporozoites and treated daily with 800 μg/mouse of chloroquine for nine days. This procedure of immunization is referred to as "infection/cure". Mice were challenged by inoculating intravenously 1,000 infectious sporozoites. Appearance of parasitaemia was monitored by Giemsa-stained blood smears., Results: By use of MHC I and MHC II deficient animals, results indicate that CD8+ T cells are necessary for full protection and that production of protective antibodies is either CD4+ T helper cells dependent and/or lymphokines produced by CD4 cells contribute to the protection directly or by helping CD8+ T cells. Further, the phenotype of infection/cure P. berghei responsive CD8+ T cells was determined to be KLRG1high CD27low CD44high and CD62Llow., Conclusion: The KLRG1high CD27low CD44high and CD62Llow phenotype of CD8+ T cells is associated with protection and should be investigated further as a candidate correlate of protection.

We have designed a pre-erythrocytic vaccine candidate based on the Plasmodium vivax circumsporozoite (CSV) protein, which includes its N- and C-terminal parts and a truncated region containing repeat sequences from both the VK210 and the VK247 P. vivax subtypes. Two versions of this vaccine candidate were made: a soluble recombinant protein expressed in Escherichia coli, designated VMP001 and a particulate antigen expressed in Saccharomyces cerevisiae, designated CSV-S,S. The latter is composed of CSV-S, a fusion protein between VMP001 and hepatitis B surface antigen (HBsAg), and free HBsAg co-expressed in yeast and self-assembling into mixed particles. Both antigen versions, adjuvanted with AS01, were shown to be immunogenic in rhesus monkeys. CSV-S,S/AS01 induced higher levels of VMP001-specific antibodies than did VMP001/AS01. Antibody responses against the N- and C-terminal regions of CSV and the VK210 repeat motif were of a similar magnitude following immunization with either the soluble or the particulate antigen. However, antibodies against the AGDR region, a potentially protective B cell epitope, were only detected after immunization with CSV-S,S. Analysis of the induced CD4(+) T cells highlighted different cytokine profiles depending on the antigen form. These results warrant further clinical evaluation of these two vaccine candidates to assess the added value of a particulate versus soluble form of CSV, in terms of both immunogenicity and protective efficacy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Malaria is responsible for up to a 600,000 deaths per year; conveying an urgent need for the development of a malaria vaccine. Studies with whole sporozoite vaccines in mice and non-human primates have shown that sporozoite-induced CD8+ T cells targeting liver stage antigens can mediate sterile protection. There is a need for a direct method to identify and phenotype malaria vaccine-induced CD8+ T cells in humans., Methods: Fluorochrome-labelled tetramers consisting of appropriate MHC class I molecules in complex with predicted binding peptides derived from Plasmodium falciparum AMA-1 were used to label ex vivo AMA-1 epitope specific CD8+ T cells from research subjects responding strongly to immunization with the NMRC-M3V-Ad-PfCA (adenovirus-vectored) malaria vaccine. The identification of these CD8+ T cells on the basis of their expression of early activation markers was also investigated., Results: Analyses by flow cytometry demonstrated that two of the six tetramers tested: TLDEMRHFY: HLA-A*01:01 and NEVVVKEEY: HLA-B*18:01, labelled tetramer-specific CD8+ T cells from two HLA-A*01:01 volunteers and one HLA-B*18:01 volunteer, respectively. By contrast, post-immune CD8+ T cells from all six of the immunized volunteers exhibited enhanced expression of the CD38 and HLA-DRhi early activation markers. For the three volunteers with positive tetramer staining, the early activation phenotype positive cells included essentially all of the tetramer positive, malaria epitope- specific CD8+ T cells suggesting that the early activation phenotype could identify all malaria vaccine-induced CD8+ T cells without prior knowledge of their exact epitope specificity., Conclusions: The results demonstrated that class I tetramers can identify ex vivo malaria vaccine antigen-specific CD8+ T cells and could therefore be used to determine their frequency, cell surface phenotype and transcription factor usage. The results also demonstrated that vaccine antigen-specific CD8+ T cells could be identified by activation markers without prior knowledge of their antigen-specificity, using a subunit vaccine for proof-of-concept. Whether, whole parasite or adjuvanted protein vaccines will also induce {CD38 and HLA-DRhi}+ CD8+ T cell populations reflective of the antigen-specific response will the subject of future investigations.