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Contains fulltext : 287660.pdf (Publisher’s version ) (Open Access) 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.

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 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 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

Contains fulltext : 248587.pdf (Publisher’s version ) (Open Access) 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).

Highlights • Models capturing key malaria life-cycle stages can help us evaluate vaccine candidates. • Model fitting revealed antibody avidity to be an important determinant of RTS,S vaccine efficacy. • High avidity and titre were associated with increased levels of vaccine efficacy. • Did not identify any thresholds of protection for either immune marker., 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.

International audience; Background: Malaria control remains a challenge in many parts of the Sahel and sub-Sahel regions of Africa.Methods: We conducted an individually randomized, controlled trial to assess whether seasonal vaccination with RTS,S/AS01E was noninferior to chemoprevention in preventing uncomplicated malaria and whether the two interventions combined were superior to either one alone in preventing uncomplicated malaria and severe malaria-related outcomes.Results: We randomly assigned 6861 children 5 to 17 months of age to receive sulfadoxine-pyrimethamine and amodiaquine (2287 children [chemoprevention-alone group]), RTS,S/AS01E (2288 children [vaccine-alone group]), or chemoprevention and RTS,S/AS01E (2286 children [combination group]). Of these, 1965, 1988, and 1967 children in the three groups, respectively, received the first dose of the assigned intervention and were followed for 3 years. Febrile seizure developed in 5 children the day after receipt of the vaccine, but the children recovered and had no sequelae. There were 305 events of uncomplicated clinical malaria per 1000 person-years at risk in the chemoprevention-alone group, 278 events per 1000 person-years in the vaccine-alone group, and 113 events per 1000 person-years in the combination group. The hazard ratio for the protective efficacy of RTS,S/AS01E as compared with chemoprevention was 0.92 (95% confidence interval [CI], 0.84 to 1.01), which excluded the prespecified noninferiority margin of 1.20. The protective efficacy of the combination as compared with chemoprevention alone was 62.8% (95% CI, 58.4 to 66.8) against clinical malaria, 70.5% (95% CI, 41.9 to 85.0) against hospital admission with severe malaria according to the World Health Organization definition, and 72.9% (95% CI, 2.9 to 92.4) against death from malaria. The protective efficacy of the combination as compared with the vaccine alone against these outcomes was 59.6% (95% CI, 54.7 to 64.0), 70.6% (95% CI, 42.3 to 85.0), and 75.3% (95% CI, 12.5 to 93.0), respectively.Conclusions: Administration of RTS,S/AS01E was noninferior to chemoprevention in preventing uncomplicated malaria. The combination of these interventions resulted in a substantially lower incidence of uncomplicated malaria, severe malaria, and death from malaria than either intervention alone. (Funded by the Joint Global Health Trials and PATH; ClinicalTrials.gov number, NCT03143218.).

We previously demonstrated that RTS,S/AS0149 participants (P-Fx: 25; NP-Fx: 24) received a fractional (1/5th dose-volume) RTS,S/AS01Overall 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.A fractional RTS,S/AS01NCT03824236. linked to this article can be found on the Research Data as well as Figshare https://figshare.com/s/ee025150f9d1ac739361.

Objectives To evaluate the efficacy of two doses of the adsorbed vaccine COVID-19 (inactivated) produced by Sinovac in symptomatic individuals, with virological confirmation of COVID-19, two weeks after the completion of the two-dose vaccination regimen, aged 18 years or older who work as health professionals providing care to patients with possible or confirmed COVID-19. To describe the occurrence of adverse reactions associated with the administration of each of two doses of the adsorbed vaccine COVID-19 (inactivated) produced by Sinovac up to one week after vaccination in Adults (18-59 years of age) and Elderly (60 years of age or more). Trial design This is a Phase III, randomized, multicenter, endpoint driven, double-blind, placebo-controlled clinical trial to assess the efficacy and safety of the adsorbed vaccine COVID-19 (inactivated) produced by Sinovac. The adsorbed vaccine COVID-19 (inactivated) produced by Sinovac (product under investigation) will be compared to placebo. Voluntary participants will be randomized to receive two intramuscular doses of the investigational product or the placebo, in a 1: 1 ratio, stratified by age group (18 to 59 years and 60 years or more) and will be monitored for one year by active surveillance of COVID-19. Two databases will be established according to the age groups: one for adults (18-59 years) and one for the elderly (60 years of age or older). The threshold to consider the vaccine efficacious will be to reach a protection level of at least 50%, as proposed by the World Health Organization and the FDA. Success in this criterion will be defined by sequential monitoring with adjustment of the lower limit of the 95% confidence interval above 30% for the primary efficacy endpoint. Participants Healthy participants and / or participants with clinically controlled disease, of both genders, 18 years of age or older, working as health professionals performing care in units specialized in direct contact with people with possible or confirmed cases of COVID-19. Participation of pregnant women and those who are breastfeeding, as well as those intending to become pregnant within three months after vaccination will not be allowed. Participants will only be included after signing the voluntary Informed Consent Form and ensuring they undergo screening evaluation and conform to all the inclusion and exclusion criteria. All the clinical sites are located in Brazil. Intervention and comparator Experimental intervention: The vaccine was manufactured by Sinovac Life Sciences (Beijing, China) and contains 3 μg/0.5 mL (equivalent to 600 SU per dose) of inactivated SARS-CoV-2 virus, and aluminium hydroxide as adjuvant. Control comparator: The placebo contains aluminium hydroxide in a 0.5 mL solution The schedule of both, experimental intervention and placebo is two 0.5 mL doses IM (deltoid) with a two week interval. Main outcomes The primary efficacy endpoint is the incidence of symptomatic cases of virologically confirmed COVID-19 two weeks after the second vaccination. The virological diagnosis will be confirmed by detection of SARS-CoV-2 nucleic acid in a clinical sample. The primary safety endpoint is the frequency of solicited and unsolicited local and systemic adverse reactions during the period of one week after vaccination according to age group in adult (18-59 years old) and elder (60 years of age or older) subjects. Adverse reactions are defined as adverse events that have a reasonable causal relationship to vaccination. Randomisation There will be two randomization lists, one for each age group, based on the investigational products to be administered, i.e., vaccine or placebo at a 1: 1 ratio. Each randomization list will be made to include up to 11,800 (18-59 year-old) adults, and 1,260 elderly (60 y-o and older) participants, the maximum number of participants needed per age group. An electronic central randomization system will be used to designate the investigational product that each participant must receive. Blinding (masking) This trial is designed as a double-blind study to avoid introducing bias in the evaluation of efficacy, safety and immunogenicity. The clinical care team, the professionals responsible for the vaccination and the participants will not know which investigational product will be administered. Only pharmacists or nurses in the study who are responsible for the randomization, separation and blinding of the investigational product will have access to unblinded information. The sponsor's operational team will also remain blind. Numbers to be randomised (sample size) The total number of participants needed to evaluate efficacy, 13,060 participants, satisfies the needed sample size calculated to evaluate safety. Therefore, the total number obtained for efficacy will be the number retained for the study. Up to 13,060 participants are expected to enter the study, with up to 11,800 participants aged 18 to 59 years and 1,260 elderly participants aged 60 and over. Half of the participants of each group will receive the experimental vaccine and half of them will receive the placebo. The recruitment of participants may be modified as recommended by the Data Safety Monitoring Committee at time of the interim unblinded analysis or blind assessment of the COVID-19 attack rate during the study. Trial Status Protocol version 2.0 – 24-Aug-2020. Recruitment started on July 21st, 2020. The recruitment is expected to conclude in October 2020. Trial registration ClinicalTrials.gov Identifier: NCT0445659. Registry on 2 July 2020 Full protocol The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

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.

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 PfCSP (PbVac), and we now report the clinical evaluation of its biological activity against controlled human malaria infection (CHMI). This first-in-human trial shows that PbVac is safe and well tolerated, when administered by a total of ~300 PbVac-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). PbVac elicits dose-dependent cross-species cellular immune responses and functional PfCSP-dependent antibody responses that efficiently block Pf sporozoite invasion of liver cells in vitro. This study demonstrates that PbVac 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.

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.

Significance The Plasmodium falciparum circumsporozoite protein (CSP) has been studied for decades as a potential immunogen, but little structural information is available on how antibodies recognize the immunodominant NANP repeats within CSP. The most advanced vaccine candidate is RTS,S, which includes multiple NANP repeats. Here, we analyzed two functional antibodies from an RTS,S trial and determined the number of repeats that interact with the antibody Fab fragments using isothermal titration calorimetry and X-ray crystallography. Using negative-stain electron microscopy, we also established how the antibody binds to the NANP repeat region in a recombinant CSP construct. The structural features outlined here provide a rationale for structure-based immunogen design to improve upon the efficacy of the current RTS,S vaccine., 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.

For some diseases, successful vaccines have been developed using a nonpathogenic counterpart of the causative microorganism of choice. The nonpathogenicity of the rodent

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, Plasmodium falciparum gametocyte carriage was induced in malaria-naive volunteers by mosquito bite or blood-stage inoculum. Gametocyte densities were associated with preceding gametocyte commitment transcript numbers. After blood-stage malaria, gametocyte densities were sufficiently high to allow transmission to mosquitoes.

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 (PfCS), and showed that this parasite line (PbVac) 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 PbVac in detail and developed tools necessary for its use in clinical studies. A microbiological contaminant-free Master Cell Bank of PbVac 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 PbVac 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 PbVac-related toxicity. In vivo drug sensitivity assays employing rodent models of infection showed that both the liver and the blood stage forms of PbVac were completely eliminated by Malarone® treatment. Collectively, our pre-clinical safety assessment demonstrates that PbVac possesses all characteristics necessary to advance into clinical evaluation.

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.

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

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 (PfCS), and showed that this parasite line (PbVac) 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 PbVac in detail and developed tools necessary for its use in clinical studies. A microbiological contaminant-free Master Cell Bank of PbVac 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 PbVac 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 PbVac-related toxicity. In vivo drug sensitivity assays employing rodent models of infection showed that both the liver and the blood stage forms of PbVac were completely eliminated by Malarone® treatment. Collectively, our pre-clinical safety assessment demonstrates that PbVac possesses all characteristics necessary to advance into clinical evaluation., Malaria: Pre-clinical characterization of transgenic malaria vaccine PbVac is a transgenic malaria parasite expressing circumsporozoite antigen from the human parasite Plasmodium falciparum. PbVac elicits neutralizing P. falciparum antibodies and can infect human hepatocytes but not erythrocytes, suggesting that humans would be non-permissive. Miguel Prudêncio and colleagues at the Institute of Molecular Medicine in Lisbon perform a detailed in vivo analysis and toxicology of PbVac. Extensive biodistribution analysis using a highly sensitive qPCR in non-permissive rabbit hosts shows PbVac are present at the initial bite site early on with later appearance in the liver, but by day 10 is undetectable. Importantly no PbVac could be detected in the blood at any time-point. PbVac was well tolerated with no apparent pathological signatures. In permissive mouse hosts PbVac could be effectively eliminated from both the blood and liver and could thereby act as a potential clinical ‘safety net’ in the event of an erythrocytic stage or persistence in the liver.

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., Host immune responses exert selective pressure on Plasmodium falciparum. Here, the authors show that allele-specific immunity impacts the antigenic diversity of individual malaria infections. This process partially explains the extreme amino acid diversity of many parasite antigens and suggests that vaccines should account for allele-specific immunity.

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.

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 (ClinicalTrials.gov, NCT02836002). 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. Findings 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 PATH Malaria Vaccine Initiative (MVI), eLife digest The parasite that causes malaria, named Plasmodium falciparum, has a life cycle that involves both humans and mosquitoes. Starting in the saliva of female Anopheles mosquitoes, it enters a person’s bloodstream when the insects feed. It then moves to the person’s liver, where it infects liver cells and matures into a stage known as schizonts. The schizonts then divide to form thousands of so-called merozoites, which burst out of the liver cells and into the bloodstream. The merozoites infect red blood cells, producing more schizonts and yet more merozoites, which continue the infection. To complete its life cycle, the parasite must return to a mosquito. Some of the parasites in the person’s blood transform into male and female cells called gametocytes that are taken up by a mosquito when it feeds on that person. Inside the mosquito, male and female parasites reproduce to create the next generation of parasites. The new parasites then move to the mosquito’s salivary glands, ready to begin another infection. Stopping the parasite being transmitted from humans to mosquitoes will stop the spread of malaria in the population. Yet it has proven difficult to study this part of the life cycle from natural infections. Here, Reuling et al. report a new method for generating gametocytes in human volunteers that will enable closer study of the biology of malaria transmission. The method is developed using the Controlled Human Malaria Infection (CHMI) model. Healthy volunteers without a history of malaria are bitten by mosquitoes infected with malaria parasites. Shortly afterwards, the volunteers are given a drug treatment to control and reduce their symptoms. The gametocytes form during this phase of the infection. At the end of the experiment, all the volunteers receive a final treatment that completely cures the infection. Reuling et al. recruited 16 volunteers and assigned them to four groups at random. Each group received a different drug regime. Roughly a week after the mosquito bites, all participants showed malaria parasites in their blood, and between 8.5 and 12 days later, mature gametocytes started to appear. This early appearance suggests that the parasites start to transform into gametocytes when they first emerge from the liver. The experiment also revealed that female gametocytes stay in the blood for a longer period than their male counterparts. These results are proof of principle for a new way to investigate malaria infection. The new model provides a controlled method for studying P. falciparum gametocytes in people. In the future, it could help to test the impact of drugs and vaccines on gametocytes. Understanding more about these parasites’ biology could lead to treatments that block malaria transmission.

Host immunity exerts strong selection on pathogens, but it does not act in a uniform manner across individual hosts. By providing a direct approach for understanding host-specific selection pressures, patterns of intra-host pathogen diversity complement population genetic analyses performed on broad geographic scales. Here, we perform a combined analysis of inter- and intra-host diversity for the malaria parasitePlasmodium falciparumwith haplotype sequences of three antigens sampled from over 4,500 natural infections in sub-Saharan Africa using targeted deep sequencing. We find that multi-strain infections in young children contain non-random combinations of parasite genotypes, and identify individual amino acid positions that may contribute to strain-specific blocking of infections. These results demonstrate for the first time that natural host defenses toPlasmodiumdetectably impact which infections proceed to the blood stage within a given host. This selection partially explains the extreme amino acid diversity observed at many parasite antigens and suggests that vaccines targeting such proteins should account for the impact of allele-specific immunity.

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.

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.

RTS,S is an advanced malaria vaccine candidate and confers significant protection against Plasmodium falciparum infection in humans. Little is known about the molecular mechanisms driving vaccine immunity. Here, we applied a systems biology approach to study immune responses in subjects receiving three consecutive immunizations with RTS,S (RRR), or in those receiving two immunizations of RTS,S/AS01 following a primary immunization with adenovirus 35 (Ad35) (ARR) vector expressing circumsporozoite protein. Subsequent controlled human malaria challenge (CHMI) of the vaccinees with Plasmodium-infected mosquitoes, 3 wk after the final immunization, resulted in ∼50% protection in both groups of vaccinees. Circumsporozoite protein (CSP)-specific antibody titers, prechallenge, were associated with protection in the RRR group. In contrast, ARR-induced lower antibody responses, and protection was associated with polyfunctional CD4+ T-cell responses 2 wk after priming with Ad35. Molecular signatures of B and plasma cells detected in PBMCs were highly correlated with antibody titers prechallenge and protection in the RRR cohort. In contrast, early signatures of innate immunity and dendritic cell activation were highly associated with protection in the ARR cohort. For both vaccine regimens, natural killer (NK) cell signatures negatively correlated with and predicted protection. These results suggest that protective immunity against P. falciparum can be achieved via multiple mechanisms and highlight the utility of systems approaches in defining molecular correlates of protection to vaccination.

Background Since 1986, investigators at Walter Reed Army Institute of Research (WRAIR) have been using controlled human malaria challenge (CHMI) in malaria-naive adults in order to define the protective efficacy of a malaria vaccine and thus guide programmatic decisions on vaccine candidates. Adapting this model to the dengue field could provide similar evidential support for a vaccine or therapeutic product. Methods After completing a vaccine regimen, volunteers are bitten by 5 malaria-infected female Anopheles mosquitoes in a controlled environment. Volunteers are then monitored daily for peripheral parasitemia in a hotel setting with 24-hour access to a nurse and physician. If a single verified parasite is detected, effective antimalarials are promptly administered. Results The vast majority of the over 1000 volunteers having participated in CHMI clinical studies have done so at US military research centers. Numerous pre-erythrocytic and erythrocytic vaccine candidates have been evaluated safely and without any related serious adverse events using this model, including the soon-to-be licensed RTS,S malaria vaccine. Conclusion The lessons learned from over 25 years of experience in consistent, careful preparation and execution of the CHMI model at WRAIR can provide a foundation from which the dengue field can begin to develop a rigorous and safe "CHDI" model.

Background Immunization with genetically engineered, attenuated malaria parasites (GAP) that arrest during liver infection confers sterile protection in mouse malaria models. A first generation Plasmodium falciparum GAP ( Pf p52 − /p36 − GAP) was previously generated by deletion of two pre-erythrocytic stage-expressed genes (P52 and P36) in the NF54 strain. Methods A first-in-human, proof-of-concept, safety and immunogenicity clinical trial in six human volunteers was conducted. Exposure consisted of delivery of Pf p52 − /p36 − GAP sporozoites via infected Anopheles mosquito bite with a five-bite/volunteer exposure followed by an approximately 200-bite exposure/volunteer one month later. Results The exposures were well tolerated with mild to moderate local and systemic reactions. All volunteers remained blood stage negative after low dose exposure. Five volunteers remained blood stage negative after high dose exposure. One volunteer developed peripheral parasitemia twelve days after high dose exposure. Together the findings indicate that Pf p52 − /p36 − GAP was severely but not completely attenuated. All six volunteers developed antibodies to CSP. Furthermore, IFN-γ responses to whole sporozoites and multiple antigens were elicited in 5 of 6 volunteers, with both CD4 and CD8 cell cytokine production detected. Conclusion Severe attenuation and favorable immune responses following administration of a first generation Pf p52 − /p36 − GAP suggests that further development of live-attenuated strains using genetic engineering should be pursued.

The development of vaccine candidates against Plasmodium vivax —the most geographically widespread human malaria species—is challenged by technical difficulties, such as the lack of in vitro culture systems and availability of animal models. Chimeric rodent Plasmodium parasites are safe and useful tools for the preclinical evaluation of new vaccine formulations. We report the successful development and characterization of chimeric Plasmodium berghei parasites bearing the type I repeat region of P. vivax circumsporozoite protein (CSP). The P. berghei-P. vivax chimeric strain develops normally in mosquitoes and produces highly infectious sporozoites that produce patent infection in mice that are exposed to the bites of as few as 3 P. berghei-P. vivax -infected mosquitoes. Using this transgenic parasite, we demonstrate that monoclonal and polyclonal antibodies against P. vivax CSP strongly inhibit parasite infection and thus support the notion that these antibodies play an important role in protective immunity. The chimeric parasites we developed represent a robust model for evaluating protective immune responses against P. vivax vaccines based on CSP.

The Plasmodium falciparum circumsporozoite (CS) protein (CSP) is a major vaccine target for preventing malaria infection. Thus, developing strong and durable antibody and T cell responses against CSP with novel immunogens and potent adjuvants may improve upon the success of current approaches. Here, we compare four distinct full-length P. falciparum CS proteins expressed in Escherichia coli or Pichia pastoris for their ability to induce immunity and protection in mice when administered with long-chain poly(I·C) [poly(I·C)LC] as an adjuvant. CS proteins expressed in E. coli induced high-titer antibody responses against the NANP repeat region and potent CSP-specific CD4 + T cell responses. Moreover, E. coli -derived CS proteins in combination with poly(I·C)LC induced potent multifunctional (interleukin 2-positive [IL-2 + ], tumor necrosis factor alpha-positive [TNF-α + ], gamma interferon-positive [IFN-γ + ]) CD4 + effector T cell responses in blood, in spleen, and particularly in liver. Using transgenic Plasmodium berghei expressing the repeat region of P. falciparum CSP [ Pb -CS( Pf )], we showed that there was a 1- to 4-log decrease in malaria rRNA in the liver following a high-dose challenge and ∼50% sterilizing protection with a low-dose challenge compared to control levels. Protection was directly correlated with high-level antibody titers but not CD4 + T cell responses. Finally, protective immunity was also induced using the Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) as the adjuvant, which also correlated with high antibody titers yet CD4 + T cell immunity that was significantly less potent than that with poly(I·C)LC. Overall, these data suggest that full-length CS proteins and poly(I·C)LC or GLA-SE offer a simple vaccine formulation to be used alone or in combination with other vaccines for preventing malaria infection.

Vaccination with the pre-erythrocytic malaria vaccine RTS,S induces high levels of antibodies and CD4(+) T cells specific for the circumsporozoite protein (CSP). Using a biologically-motivated mathematical model of sporozoite infection fitted to data from malaria-naive adults vaccinated with RTS,S and subjected to experimental P. falciparum challenge, we characterised the relationship between antibodies, CD4(+) T cell responses and protection from infection. Both anti-CSP antibody titres and CSP-specific CD4(+) T cells were identified as immunological surrogates of protection, with RTS,S induced anti-CSP antibodies estimated to prevent 32% (95% confidence interval (CI) 24%-41%) of infections. The addition of RTS,S-induced CSP-specific CD4(+) T cells was estimated to increase vaccine efficacy against infection to 40% (95% CI, 34%-48%). This protective efficacy is estimated to result from a 96.1% (95% CI, 93.4%-97.8%) reduction in the liver-to-blood parasite inoculum, indicating that in volunteers who developed P. falciparum infection, a small number of parasites (often the progeny of a single surviving sporozoite) are responsible for breakthrough blood-stage infections.