Persistence of Anti-SE36 Antibodies Induced by the Malaria Vaccine Candidate BK-SE36/CpG in 5-10-Year-Old Burkinabe Children Naturally Exposed to Malaria.

Information on the dynamics and decline/persistence of antibody titres is important in vaccine development. A recent vaccine trial in malaria-exposed, healthy African adults and children living in a malaria hyperendemic and seasonal area (Ouagadougou, Burkina Faso) was the first study in which BK-SE36/CpG was administered to different age groups. In 5- to 10-year-old children, the risk of malaria infection was markedly lower in the BK-SE36/CpG arm compared to the control arm. We report here data on antibody titres measured in this age-group after the high malaria transmission season of 2021 (three years after the first vaccine dose was administered). At Year 3, 83% of children had detectable anti-SE36 total IgG antibodies. Geometric mean antibody titres and the proportion of children with detectable anti-SE36 antibodies were markedly higher in the BK-SE36/CpG arm than the control (rabies) arm. The information obtained in this study will guide investigators on future vaccine/booster schedules for this promising blood-stage malaria vaccine candidate.

Safety and immunogenicity of BK-SE36/CpG malaria vaccine in healthy Burkinabe adults and children: a phase 1b randomised, controlled, double-blinded, age de-escalation trial.

Background: BK-SE36/CpG is a recombinant blood-stage malaria vaccine candidate based on the N-terminal Plasmodium falciparum serine repeat antigen5 (SE36), adsorbed to aluminium hydroxide gel and reconstituted, prior to administration, with synthetic oligodeoxynucleotides bearing CpG motifs. In healthy Japanese adult males, BK-SE36/CpG was well tolerated. This study assessed its safety and immunogenicity in healthy malaria-exposed African adults and children. Methods: A double-blind, randomised, controlled, age de-escalating clinical trial was conducted in an urban area of Ouagadougou, Burkina Faso. Healthy participants (n=135) aged 21-45 years (Cohort 1), 5-10 years (Cohort 2) and 12-24 months (Cohort 3) were randomised to receive three vaccine doses (Day 0, 28 and 112) of BK-SE36/CpG or rabies vaccine by intramuscular injection. Results: One hundred thirty-four of 135 (99.2%) subjects received all three scheduled vaccine doses. Vaccinations were well tolerated with no related Grade 3 (severe) adverse events (AEs). Pain/limitation of limb movement, headache in adults and fever in younger children (all mild to moderate in intensity) were the most frequently observed local and systemic AEs. Eighty-three of BK-SE36/CpG (91%) recipients and 37 of control subjects (84%) had Grade 1/2 events within 28 days post vaccination. Events considered by the investigator to be vaccine related were experienced by 38% and 14% of subjects in BK-SE36/CpG and control arms, respectively. Throughout the trial, six Grade 3 events (in 4 subjects), not related to vaccination, were recorded in the BK-SE36/CpG arm: 5 events (in 3 subjects) within 28 days of vaccination. All serious adverse events (SAEs) (n=5) were due to severe malaria (52-226 days post vaccination) and not related to vaccination. In all cohorts, BK-SE36/CpG arm had higher antibody titres after Dose 3 than after Dose 2. Younger cohorts had stronger immune responses (12-24-month-old > 5-10 years-old > 21-45 years-old). Sera predominantly reacted to peptides that lie in intrinsically unstructured regions of SE36. In the control arm, there were no marked fold changes in antibody titres and participants' sera reacted poorly to all peptides spanning SE36. Conclusion: BK-SE36/CpG was well-tolerated and immunogenic. These results pave the way for further proof-of-concept studies to demonstrate vaccine efficacy. Clinical trial registration: https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=1921, PACTR201701001921166.

Plasmodium falciparum infection coinciding with the malaria vaccine candidate BK-SE36 administration interferes with the immune responses in Burkinabe children.

Background: A vaccine targeting the erythrocyte stages of Plasmodium falciparum could play a role in preventing clinical disease. BK-SE36 is a promising malaria vaccine candidate that has shown a good safety profile and immunological responses during field evaluations. It was observed that repeated natural infections could result in immune tolerance against SE36 molecule. Methods: The primary trial was conducted to assess the safety and immunogenicity of the BK-SE36 in two cohorts of children aged 25-60 months (Cohort 1) and 12-24 months (Cohort 2). Immunization was at full dose (1.0 mL) administered at 0, 1, and 6 months. Blood samples were collected before each vaccination for immunological assessments and detection of Plasmodium falciparum infection by microscopy. Blood samples were further collected one month post each vaccination to evaluate immunogenicity. Results: Of seventy-two (72) subjects that have received BK-SE36 vaccination, 71 had available blood smears during vaccination days. One month post Dose 2, the geometric mean of SE36 antibodies was 263.2 (95% CI: 178.9-387.1) in uninfected individuals compared to 77.1 (95% CI: 47.3-125.7) in infected participants. The same trend was observed one-month post booster dose. Participants uninfected at the time of booster vaccination had significantly higher GMTs compared to those who were infected (424.1 (95% CI: 301.9-595.8) vs. 92.8 (95% CI: 34.9-246.6), p = 0.002. There was a 14.3 (95% CI: 9.7-21.1) and 2.4 (95% CI: 1.3-4.4) fold-change, respectively, in uninfected and infected participants between one-month post Dose 2 and booster. The difference was statistically significant (p < 0.001). Conclusion: Concomitant infection by P. falciparum during BK-SE36 vaccine candidate administration is associated with reduced humoral responses. However, it is to be noted that the BK-SE36 primary trial was not designed to investigate the influence of concomitant infection on vaccine-induced immune response and should be interpreted cautiously. Trial registration: WHO ICTRP, PACTR201411000934120.

Safety and immunogenicity of BK-SE36 in a blinded, randomized, controlled, age de-escalating phase Ib clinical trial in Burkinabe children.

Background: A blood-stage vaccine targeting the erythrocytic-stages of the malaria parasite Plasmodium falciparum could play a role to protect against clinical disease. Antibodies against the P. falciparum serine repeat antigen 5 (SE47 and SE36 domains) correlate well with the absence of clinical symptoms in sero-epidemiological studies. A previous phase Ib trial of the recombinant SE36 antigen formulated with aluminum hydroxyl gel (BK-SE36) was promising. This is the first time the vaccine candidate was evaluated in young children below 5 years using two vaccination routes. Methods: Safety and immunogenicity of BK-SE36 was assessed in a double-blind, randomized, controlled, age de-escalating phase Ib trial. Fifty-four Burkinabe children in each age cohort, 25-60 or 12-24 months, were randomized in a 1:1:1 ratio to receive three doses of BK-SE36 either by intramuscular (BK IM) or subcutaneous (BK SC) route on Day 0, Week 4, and 26; or the control vaccine, Synflorix® via IM route on Day 0, Week 26 (and physiological saline on Week 4). Safety data and samples for immunogenicity analyses were collected at various time-points. Results: Of 108 subjects, 104 subjects (96.3%) (Cohort 1: 94.4%; Cohort 2: 98.1%) received all three scheduled vaccine doses. Local reactions, mostly mild or of moderate severity, occurred in 99 subjects (91.7%). The proportion of subjects that received three doses without experiencing Grade 3 adverse events was similar across BK-SE36 vaccines and control arms (Cohort 1: 100%, 89%, and 89%; and Cohort 2: 83%, 82%, and 83% for BK IM, BK SC, and control, respectively). BK-SE36 vaccine was immunogenic, inducing more than 2-fold change in antibody titers from pre-vaccination, with no difference between the two vaccination routes. Titers waned before the third dose but in both cohorts titers were boosted 6 months after the first vaccination. The younger cohort had 2-fold and 4-fold higher geometric mean titers compared to the 25- to 60-month-old cohort after 2 and 3 doses of BK-SE36, respectively. Conclusion: BK-SE36 was well tolerated and immunogenic using either intramuscular or subcutaneous routes, with higher immune response in the younger cohort. Clinical Trial Registration: https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=934, identifier PACTR201411000934120.

African-specific polymorphisms in Plasmodium falciparum serine repeat antigen 5 in Uganda and Burkina Faso clinical samples do not interfere with antibody response to BK-SE36 vaccination.

BK-SE36, based on Plasmodium falciparum serine repeat antigen 5 (SERA5), is a blood-stage malaria vaccine candidate currently being evaluated in clinical trials. Phase 1 trials in Uganda and Burkina Faso have demonstrated promising safety and immunogenicity profiles. However, the genetic diversity of sera5 in Africa and the role of allele/variant-specific immunity remain a major concern. Here, sequence analyses were done on 226 strains collected from the two clinical trial/follow-up studies and 88 strains from two cross-sectional studies in Africa. Compared to other highly polymorphic vaccine candidate antigens, polymorphisms in sera5 were largely confined to the repeat regions of the gene. Results also confirmed a SERA5 consensus sequence with African-specific polymorphisms. Mismatches with the vaccine-type SE36 (BK-SE36) in the octamer repeat, serine repeat, and flanking regions, and single-nucleotide polymorphisms in non-repeat regions could compromise vaccine response and efficacy. However, the haplotype diversity of SERA5 was similar between vaccinated and control participants. There was no marked bias or difference in the patterns of distribution of the SE36 haplotype and no statistically significant genetic differentiation among parasites infecting BK-SE36 vaccinees and controls. Results indicate that BK-SE36 does not elicit an allele-specific immune response.

Pipeline analysis of a vaccine candidate portfolio for diseases of poverty using the Portfolio-To-Impact modelling tool.

Background: The Portfolio-To-Impact (P2I) P2I model is a recently developed product portfolio tool that enables users to estimate the funding needs to move a portfolio of candidate health products, such as vaccines and drugs, along the product development path from late stage preclinical to phase III clinical trials, as well as potential product launches over time. In this study we describe the use of this tool for analysing the vaccine portfolio of the European Vaccine Initiative (EVI). This portfolio includes vaccine candidates for various diseases of poverty and emerging infectious diseases at different stages of development. Methods: Portfolio analyses were conducted using the existing assumptions integrated in the P2I tool, as well as modified assumptions for costs, cycle times, and probabilities of success based on EVI's own internal data related to vaccine development. Results: According to the P2I tool, the total estimated cost to move the 18 candidates currently in the EVI portfolio along the pipeline to launch would be about US $470 million, and there would be 0.69 cumulative expected launches during the period 2019-2031. Running of the model using EVI-internal parameters resulted in a significant increase in the expected product launches. Conclusions: The P2I tool's underlying assumptions could not be tested in our study due to lack of data available. Nevertheless, we expect that the accelerated clinical testing of vaccines (and drugs) based on the use of controlled human infection models that are increasingly available, as well as the accelerated approval by regulatory authorities that exists for example for serious conditions, will speed up product development and result in significant cost reduction. Project findings as well as potential future modifications of the P2I tool are discussed with the aim to improve the underlying methodology of the P2I model.

Workshop report: Experimental animal models for universal influenza vaccines.

A major challenge in influenza research is the selection of an appropriate animal model that accurately reflects the disease and the protective immune response observed in humans. A workshop organised by the EDUFLUVAC consortium, a European Union funded project coordinated by the European Vaccine Initiative, brought together experts from the influenza vaccine community with the aim to discuss the current knowledge and future perspectives for testing broadly reactive influenza vaccines in animal models. The programme included a diversity of models from well-established and publicly accepted models to cutting edge, newly developed animal models as well as ex-vivo approaches and human models. The audience concluded that different vaccine approaches may require evaluation in different animal models, depending on the type of immune response induced by the vaccine. Safety is the main concern for transition to clinical development and influenza vaccine associated enhanced disease was specifically emphasised. An efficient animal model to evaluate this aspect of safety still needs to be identified. Working with animal models requires ethical compliance and consideration of the 3R principles. Development of alternative approaches such as ex-vivo techniques is progressing but is still at an early stage and these methods are not yet suitable for broader application for vaccine evaluation. The human challenge is the ultimate model to assess influenza vaccines. However this model is expensive and not largely applicable. The currently used pre-clinical models are not yet specifically focused on studying unique aspects of a universal influenza vaccine. Further collaboration, communication and effective networking are needed for success in establishment of harmonised and standardised pre-clinical models for evaluation of new influenza vaccines. This report does not provide a complete review of the field but discusses the data presented by the speakers and discussion points raised during the meeting.

Safety and Immunogenicity of Malaria Vectored Vaccines Given with Routine Expanded Program on Immunization Vaccines in Gambian Infants and Neonates: A Randomized Controlled Trial.

BACKGROUND: Heterologous prime-boost vaccination with chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) encoding multiple epitope string thrombospondin-related adhesion protein (ME-TRAP) has shown acceptable safety and promising immunogenicity in African adult and pediatric populations. If licensed, this vaccine could be given to infants receiving routine childhood immunizations. We therefore evaluated responses to ChAd63 MVA ME-TRAP when co-administered with routine Expanded Program on Immunization (EPI) vaccines. METHODS: We enrolled 65 Gambian infants and neonates, aged 16, 8, or 1 week at first vaccination and randomized them to receive either ME-TRAP and EPI vaccines or EPI vaccines only. Safety was assessed by the description of vaccine-related adverse events (AEs). Immunogenicity was evaluated using IFNγ enzyme-linked immunospot, whole-blood flow cytometry, and anti-TRAP IgG ELISA. Serology was performed to confirm all infants achieved protective titers to EPI vaccines. RESULTS: The vaccines were well tolerated in all age groups with no vaccine-related serious AEs. High-level TRAP-specific IgG and T cell responses were generated after boosting with MVA. CD8+ T cell responses, previously found to correlate with protection, were induced in all groups. Antibody responses to EPI vaccines were not altered significantly. CONCLUSION: Malaria vectored prime-boost vaccines co-administered with routine childhood immunizations were well tolerated. Potent humoral and cellular immunity induced by ChAd63 MVA ME-TRAP did not reduce the immunogenicity of co-administered EPI vaccines, supporting further evaluation of this regimen in infant populations. CLINICAL TRIAL REGISTRATION: The clinical trial was registered on http://Clinicaltrials.gov (NCT02083887) and the Pan-African Clinical Trials Registry (PACTR201402000749217).

Workshop report: Immunoassay standardisation for "universal" influenza vaccines.

The development of broadly reactive influenza vaccines raises the need to identify the most appropriate immunoassays that can be used for the evaluation of so-called universal influenza vaccines and to explore a path towards the standardisation of such assays. More than fifty experts from the global influenza vaccine research and development field met to initiate such discussion at a workshop co-organised by the EDUFLUVAC consortium, a European Union funded project coordinated by the European Vaccine Initiative, and the National Institutes of Health/National Institute of Allergy and Infectious Diseases, USA. The workshop audience agreed that it was not possible to establish a single immunoassay for "universal" influenza vaccines because the current approaches differ in the vaccines' nature and immunogenicity properties. Therefore, different scientific rationales for the immunoassay selection are required. To avoid dilution of efforts, the choice of the primary evaluation criteria (eg serological assays or T-cell assays) should drive the effort of harmonisation. However, at an early phase of clinical development, more efforts on exploratory assessments should be undertaken to better define the immune profile in response to immunisation with new vaccines. The workshop concluded that each laboratory should aim towards validation of the appropriate immunoassays used during the entire process of vaccine development from antigen discovery up to establishment of correlates of protection, including the different steps of quality control (eg potency assays), animal studies and human clinical development. Standardisation of the immunoassays is the ultimate goal, and there is a long way to go.

Evaluation of the Clinical and Microbiological Response to Salmonella Paratyphi A Infection in the First Paratyphoid Human Challenge Model.

Background: To expedite the evaluation of vaccines against paratyphoid fever, we aimed to develop the first human challenge model of Salmonella enterica serovar Paratyphi A infection. Methods: Two groups of 20 participants underwent oral challenge with S. Paratyphi A following sodium bicarbonate pretreatment at 1 of 2 dose levels (group 1: 1-5 × 103 colony-forming units [CFU] and group 2: 0.5-1 × 103 CFU). Participants were monitored in an outpatient setting with daily clinical review and collection of blood and stool cultures. Antibiotic treatment was started when prespecified diagnostic criteria were met (temperature ≥38°C for ≥12 hours and/or bacteremia) or at day 14 postchallenge. Results: The primary study objective was achieved following challenge with 1-5 × 103 CFU (group 1), which resulted in an attack rate of 12 of 20 (60%). Compared with typhoid challenge, paratyphoid was notable for high rates of subclinical bacteremia (at this dose, 11/20 [55%]). Despite limited symptoms, bacteremia persisted for up to 96 hours after antibiotic treatment (median duration of bacteremia, 53 hours [interquartile range, 24-85 hours]). Shedding of S. Paratyphi A in stool typically preceded onset of bacteremia. Conclusions: Challenge with S. Paratyphi A at a dose of 1-5 × 103 CFU was well tolerated and associated with an acceptable safety profile. The frequency and persistence of bacteremia in the absence of clinical symptoms was notable, and markedly different from that seen in previous typhoid challenge studies. We conclude that the paratyphoid challenge model is suitable for the assessment of vaccine efficacy using endpoints that include bacteremia and/or symptomatology. Clinical Trials Registration: NCT02100397.

Clinical development of placental malaria vaccines and immunoassays harmonization: a workshop report.

Placental malaria caused by Plasmodium falciparum infection constitutes a major health problem manifesting as severe disease and anaemia in the mother, impaired fetal development, low birth weight or spontaneous abortion. Prevention of placental malaria currently relies on two key strategies that are losing efficacy due to spread of resistance: long-lasting insecticide-treated nets and intermittent preventive treatment during pregnancy. A placental malaria vaccine would be an attractive, cost-effective complement to the existing control tools. Two placental malaria vaccine candidates are currently in Phase Ia/b clinical trials. During two workshops hosted by the European Vaccine Initiative, one in Paris in April 2014 and the other in Brussels in November 2014, the main actors in placental malaria vaccine research discussed the harmonization of clinical development plans and of the immunoassays with a goal to define standards that will allow comparative assessment of different placental malaria vaccine candidates. The recommendations of these workshops should guide researchers and clinicians in the further development of placental malaria vaccines.

Workshop report: Malaria vaccine development in Europe--preparing for the future.

The deployment of a safe and effective malaria vaccine will be an important tool for the control of malaria and the reduction in malaria deaths. With the launch of the 2030 Malaria Vaccine Technology Roadmap, the malaria community has updated the goals and priorities for the development of such a vaccine and is now paving the way for a second phase of malaria vaccine development. During a workshop in Brussels in November 2014, hosted by the European Vaccine Initiative, key players from the European, North American and African malaria vaccine community discussed European strategies for future malaria vaccine development in the global context. The recommendations of the European malaria community should guide researchers, policy makers and funders of global health research and development in fulfilling the ambitious goals set in the updated Malaria Vaccine Technology Roadmap.

Unbiased RNAi screen for hepcidin regulators links hepcidin suppression to proliferative Ras/RAF and nutrient-dependent mTOR signaling.

The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the "iron-regulated" bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6-triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism.

A multi-scale model of hepcidin promoter regulation reveals factors controlling systemic iron homeostasis.

Systemic iron homeostasis involves a negative feedback circuit in which the expression level of the peptide hormone hepcidin depends on and controls the iron blood levels. Hepcidin expression is regulated by the BMP6/SMAD and IL6/STAT signaling cascades. Deregulation of either pathway causes iron-related diseases such as hemochromatosis or anemia of inflammation. We quantitatively analyzed how BMP6 and IL6 control hepcidin expression. Transcription factor (TF) phosphorylation and reporter gene expression were measured under co-stimulation conditions, and the promoter was perturbed by mutagenesis. Using mathematical modeling, we systematically analyzed potential mechanisms of cooperative and competitive promoter regulation by the transcription factors, and experimentally validated the model predictions. Our results reveal that hepcidin cross-regulation primarily occurs by combinatorial transcription factor binding to the promoter, whereas signaling crosstalk is insignificant. We find that the presence of two BMP-responsive elements enhances the steepness of the promoter response towards the iron-sensing BMP signaling axis, which promotes iron homeostasis in vivo. IL6 co-stimulation reduces the promoter sensitivity towards the BMP signal, because the SMAD and STAT transcription factors compete for recruiting RNA polymerase to the transcription start site. This may explain why inflammatory signals disturb iron homeostasis in anemia of inflammation. Taken together, our results reveal why the iron homeostasis circuit is sensitive to perturbations implicated in disease.

The hemochromatosis proteins HFE, TfR2, and HJV form a membrane-associated protein complex for hepcidin regulation.

BACKGROUND & AIMS: The hereditary hemochromatosis-associated membrane proteins HFE, TfR2, and HJV are required for adequate hepatic expression of the iron hormone hepcidin. While the genetic interactions are clear, it remains elusive how bone morphogenetic protein co-receptor HJV functions together with HFE and TfR2 to activate hepcidin transcription via the BMP-SMAD signaling pathway. Here, we investigate whether HFE, TfR2, and HJV physically interact on the surface of hepatocytes. METHODS: We explore protein-protein interactions by glycerol gradient sedimentation assays and co-immunoprecipitation analyses in transfected HuH7 hepatoma-derived cells. RESULTS: Our data demonstrate that HFE and TfR2 bind HJV in a non-competitive manner. Co-immunoprecipitation analyses provide direct experimental evidence that HFE, TfR2, and HJV form a multi-protein membrane complex. Our experiments show that like TfR2, HJV competes with TfR1 for binding to HFE, indicating that the expression of TfR2 and HJV may be critical for iron sensing. We identify residues 120-139 of the TfR2 extra-cellular domain as the critical amino acids required for the binding of both HFE and HJV. Interestingly, RGMA, a central nervous system homolog, can substitute for HJV in the complex and promote hepcidin transcription, implicating RGMA in the local control of hepcidin in the CNS. CONCLUSIONS: Taken together, our findings provide a biochemical basis for hepcidin control by HFE, TfR2, and HJV.

A novel TMPRSS6 mutation that prevents protease auto-activation causes IRIDA.

IRIDA (iron-refractory iron-deficiency anaemia) is a rare autosomal-recessive disorder hallmarked by hypochromic microcytic anaemia, low transferrin saturation and high levels of the iron-regulated hormone hepcidin. The disease is caused by mutations in the transmembrane serine protease TMPRSS6 (transmembrane protease serine 6) that prevent inactivation of HJV (haemojuvelin), an activator of hepcidin transcription. In the present paper, we describe a patient with IRIDA who carries a novel mutation (Y141C) in the SEA domain of the TMPRSS6 gene. Functional characterization of the TMPRSS6(Y141C) mutant protein in cultured cells showed that it localizes to similar subcellular compartments as wild-type TMPRSS6 and binds HJV, but fails to auto-catalytically activate itself. As a consequence, hepcidin mRNA expression is increased, causing the clinical symptoms observed in this IRIDA patient. The present study provides important mechanistic insight into how TMPRSS6 is activated.