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Background: Shigella infections remain endemic in places with poor sanitation and are a leading cause of diarrheal mortality globally, as well as a major contributor to gut enteropathy and stunting. There are currently no licensed vaccines for shigellosis but it has been estimated that an effective vaccine could avert 590,000 deaths over a 20-year period. A challenge to effective Shigella vaccine development has been the low immunogenicity and protective efficacy of candidate Shigella vaccines in infants and young children. Additionally, a new vaccine might be less immunogenic in a highly endemic setting compared to a low endemic setting ("vaccine hyporesponsiveness"). The use of a potent adjuvant enhancing both mucosal and systemic immunity might overcome these problems. Invaplex AR-Detox is an injectable Shigella vaccine that uses a novel combination of conserved invasion plasmid antigen proteins and a serotype-specific bacterial lipopolysaccharide attenuated for safe intramuscular administration. The adjuvant dmLT has been shown to enhance Shigella immune responses in mice, has safely been administered intramuscularly, and was shown to enhance immune responses in healthy volunteers when given in combination with other antigens in phase I trials. This article describes the protocol of a study that will be the first to assess the safety, tolerability, and immunogenicity of Invaplex AR-Detox co-administered with dmLT in healthy adults in low-endemic and high-endemic settings., Methods: In a multi-center, randomized, double-blind, and placebo-controlled dose-escalation phase Ia/b trial, the safety, tolerability, and immunogenicity of three intramuscular vaccinations administered 4 weeks apart with 2.5 µg or 10 µg of Invaplex AR-Detox vaccine, alone or in combination with 0.1 µg of the dmLT adjuvant, will first be assessed in a total of 50 healthy Dutch adults (phase Ia) and subsequently in 35 healthy Zambian adults (phase Ib) aged 18-50 years. The primary outcome is safety, and secondary outcomes are humoral and cellular immune responses to the adjuvanted or non-adjuvanted vaccine., Discussion: This trial is part of the ShigaPlexIM project that aims to advance the early clinical development of an injectable Shigella vaccine and to make the vaccine available for late-stage clinical development. This trial addresses the issue of hyporesponsiveness in an early stage of clinical development by testing the vaccine and adjuvant in an endemic setting (Zambia) after the first-in-human administration and the dose-escalation has proven safe and tolerable in a low-endemic setting (Netherlands). Besides strengthening the vaccine pipeline against a major diarrheal disease, another goal of the ShigaPlexIM project is to stimulate capacity building and strengthen global North-South relations in clinical research., Trial Registration: EU CT number: 2023-506394-35-02, ClinicalTrials.gov identifier: NCT05961059.

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.

The estimates of enterotoxigenic Escherichia coli (ETEC) and Shigella burden in developing countries are limited by the lack of rapid, accessible, and sensitive diagnostics and surveillance tools. We used a "Rapid LAMP based Diagnostic Test (RLDT)" to detect ETEC and Shigella in diarrheal and non-diarrheal stool samples from a 12-month longitudinal cohort of children under five years of age in a peri-urban area of Ouagadougou in Burkina Faso (West Africa). To allow comparison with the RLDT- Shigella results, conventional culture methods were used to identify Shigella strains in the stool samples. As conventional culture alone cannot detect ETEC cases, a subset of E. coli -like colonies was tested using conventional PCR to detect ETEC toxins genes. Of the 165 stool samples analyzed for ETEC, 24.9% were positive when using RLDT against 4.2% when using culture followed by PCR. ETEC toxin distribution when using RLDT was STp 17.6% (29/165), LT 11.5% (19/165), and STh 8.5% (14/165). Of the 263 specimens tested for Shigella , 44.8% were positive when using RLDT against 23.2% when using culture. The sensitivity and specificity of the RLDT compared to culture (followed by PCR for ETEC) were 93.44% and 69.8% for Shigella and 83.7% and 77.9% for ETEC, respectively. This study indicates that both Shigella and ETEC are substantially underdiagnosed when using conventional culture and highlights the potential contribution of the new RLDT method to improve enteric disease burden estimation and to guide future efforts to prevent and control bacterial enteric infection and disease.

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., Competing Interests: TH is the inventor of BK-SE36; TH and KI are inventors of BK-SE36/CpG. NP and TH are supported by a research fund from Nobelpharma Co., Ltd NPC, the clinical trial sponsor. TS is an employee and SE is medical adviser of NPC. These involvements did not influence the design of the study, the collection, analyses, access to and interpretation of data, and the writing of this manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Ouédraogo, Bougouma, Palacpac, Houard, Nebie, Sawadogo, Berges, Soulama, Diarra, Hien, Ouedraogo, Konaté, Kouanda, Myoui, Ezoe, Ishii, Sato, D’Alessio, Leroy, Tiono, Cousens, Horii and Sirima.)

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., Competing Interests: TH is the inventor of BK-SE36 and all rights have now been turned over to Nobelpharma Co., Ltd (NPC), Tokyo, Japan. NP served as a contract researcher for NPC, Apr - Sept 2017. SH and FD received support from NPC for salaries, travel, and CRO cost for clinical monitoring. EB also received support from NPC for salaries during the long-term follow-up. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tiono, Palacpac, Bougouma, Nebie, Ouédraogo, Houard, Arisue, D’Alessio, Horii and Sirima.)

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., Competing Interests: TH is the inventor of BK-SE36 and all rights have now been turned over to NPC. NP, EN, AY, BB, BK, and TE received remuneration from BIKEN for the Ugandan trial and follow-up. For the Burkina Faso clinical trial/follow-up study, the following received some support from NPC: EB, AD, SH, FD, OL, and NP. TH and NP are currently supported by a research fund from NPC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Arisue, Palacpac, Ntege, Yeka, Balikagala, Kanoi, Bougouma, Tiono, Nebie, Diarra, Houard, D’Alessio, Leroy, Sirima, Egwang and Horii.)

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., Competing Interests: TH is the inventor of BK-SE36 and all rights have now been turned over to NPC. NP served as contract researcher for NPC, Apr - Sept 2017. SH, FD and OL received support from NPC for salaries, travel and CRO cost for clinical monitoring. EB, SaC, AD, AZO, JY also received support from NPC for salaries during the long term follow-up. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bougouma, Palacpac, Tiono, Nebie, Ouédraogo, Houard, Yagi, Coulibaly, Diarra, Tougan, Ouedraogo, Soulama, Arisue, Yaro, D’Alessio, Leroy, Cousens, Horii and Sirima.)

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a candidate malaria vaccine antigen expressed on merozoites and sporozoites. PfAMA1's polymorphic nature impacts vaccine-induced protection. To address polymorphism, three Diversity Covering (DiCo) protein sequences were designed and tested in a staggered phase Ia/b trial. A cohort of malaria-naive adults received PfAMA1-DiCo adjuvanted with Alhydrogel® or GLA-SE and a cohort of malaria-exposed adults received placebo or GLA-SE adjuvanted PfAMA1 DiCo at weeks 0, 4 and 26. IgG and GIA levels measured 4 weeks after the third vaccination are similar in malaria-naive volunteers and placebo-immunised malaria-exposed adults, and have a similar breadth. Vaccination of malaria-exposed adults results in significant antibody level increases to the DiCo variants, but not to naturally occurring PfAMA1 variants. Moreover, GIA levels do not increase following vaccination. Future research will need to focus on stronger adjuvants and/or adapted vaccination regimens, to induce potentially protective responses in the target group of the vaccine.

P27A is a novel synthetic malaria vaccine candidate derived from the blood stage Plasmodium falciparum protein Trophozoite Exported Protein 1 (TEX1/PFF0165c). In phase 1a/1b clinical trials in malaria unexposed adults in Switzerland and in malaria pre-exposed adults in Tanzania, P27A formulated with Alhydrogel and GLA-SE adjuvants induced antigen-specific antibodies and T-cell activity. The GLA-SE adjuvant induced significantly stronger humoral responses than the Alhydrogel adjuvant. Groups of pre-exposed and unexposed subjects received identical vaccine formulations, which supported the comparison of the cellular and humoral response to P27A in terms of fine specificity and affinity for populations and adjuvants. Globally, fine specificity of the T and B cell responses exhibited preferred recognized sequences and did not highlight major differences between adjuvants or populations. Affinity of anti-P27A antibodies was around 10 -8 M in all groups. Pre-exposed volunteers presented anti-P27A with higher affinity than unexposed volunteers. Increasing the dose of GLA-SE from 2.5 to 5 μg in pre-exposed volunteers improved anti-P27A affinity and decreased the number of recognized epitopes. These results indicate a higher maturation of the humoral response in pre-exposed volunteers, particularly when immunized with P27A formulated with 5 μg GLA-SE., (Copyright © 2020 Geiger, Guignard, Yang, Bikorimana, Correia, Houard, Mkindi, Daubenberger, Spertini, Corradin and Audran.)

Background: Malaria in pregnancy has major impacts on mother and child health. To complement existing interventions, such as intermittent preventive treatment and use of impregnated bed nets, we developed a malaria vaccine candidate with the aim of reducing sequestration of asexual "blood-stage" parasites in the placenta, the major virulence mechanism., Methods: The vaccine candidate PAMVAC is based on a recombinant fragment of VAR2CSA, the Plasmodium falciparum protein responsible for binding to the placenta via chondroitin sulfate A (CSA). Healthy, adult malaria-naive volunteers were immunized with 3 intramuscular injections of 20 μg (n = 9) or 50 μg (n = 27) PAMVAC, adjuvanted with Alhydrogel or glucopyranosyl lipid adjuvant in stable emulsion (GLA-SE) or in a liposomal formulation with QS21 (GLA-LSQ). Allocation was random and double blind. The vaccine was given every 4 weeks. Volunteers were observed for 6 months following last immunization., Results: All PAMVAC formulations were safe and well tolerated. A total of 262 adverse events (AEs) occurred, 94 (10 grade 2 and 2 grade 3) at least possibly related to the vaccine. No serious AEs occurred. Distribution and severity of AEs were similar in all arms. PAMVAC was immunogenic in all participants. PAMVAC-specific antibody levels were highest with PAMVAC-GLA-SE. The antibodies inhibited binding of VAR2CSA expressing P. falciparum-infected erythrocytes to CSA in a standardized functional assay., Conclusions: PAMVAC formulated with Alhydrogel or GLA-based adjuvants was safe, well tolerated, and induced functionally active antibodies. Next, PAMVAC will be assessed in women before first pregnancies in an endemic area., Clinical Trials Registration: EudraCT 2015-001827-21; ClinicalTrials.gov NCT02647489., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

The generation of protective humoral immunity after vaccination relies on the productive interaction between antigen-specific B cells and T follicular helper (Tfh) cells. Despite the central role of Tfh cells in vaccine responses, there is currently no validated way to enhance their differentiation in humans. From paired human lymph node and blood samples, we identify a population of circulating Tfh cells that are transcriptionally and clonally similar to germinal center Tfh cells. In a clinical trial of vaccine formulations, circulating Tfh cells were expanded in Tanzanian volunteers when an experimental malaria vaccine was adjuvanted in GLA-SE but not when formulated in Alum. The GLA-SE-formulated peptide was associated with an increase in the extrafollicular antibody response, long-lived antibody production, and the emergence of public TCRβ clonotypes in circulating Tfh cells. We demonstrate that altering vaccine adjuvants is a rational approach for enhancing Tfh cells in humans, thereby supporting the long-lived humoral immunity that is required for effective vaccines., (© 2019 Hill et al.)

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., Competing Interests: No competing interests were disclosed., (Copyright: © 2019 Gunn A et al.)

Background: P27A is an unstructured 104mer synthetic peptide from Plasmodium falciparum trophozoite exported protein 1 (TEX1), the target of human antibodies inhibiting parasite growth. The present project aimed at evaluating the safety and immunogenicity of P27A peptide vaccine in malaria-nonexposed European and malaria-exposed African adults., Methods: This study was designed as a staggered, fast-track, randomized, antigen and adjuvant dose-finding, multicenter phase 1a/1b trial, conducted in Switzerland and Tanzania. P27A antigen (10 or 50 μg), adjuvanted with Alhydrogel or glucopyranosil lipid adjuvant stable emulsion (GLA-SE; 2.5 or 5 μg), or control rabies vaccine (Verorab) were administered intramuscularly to 16 malaria-nonexposed and 40 malaria-exposed subjects on days 0, 28, and 56. Local and systemic adverse events (AEs) as well as humoral and cellular immune responses were assessed after each injection and during the 34-week follow-up., Results: Most AEs were mild to moderate and resolved completely within 48 hours. Systemic AEs were more frequent in the formulation with alum as compared to GLA-SE, whereas local AEs were more frequent after GLA-SE. No serious AEs occurred. Supported by a mixed Th1/Th2 cell-mediated immunity, P27A induced a marked specific antibody response able to recognize TEX1 in infected erythrocytes and to inhibit parasite growth through an antibody-dependent cellular inhibition mechanism. Incidence of AEs and antibody responses were significantly lower in malaria-exposed Tanzanian subjects than in nonexposed European subjects., Conclusions: The candidate vaccine P27A was safe and induced a particularly robust immunogenic response in combination with GLA-SE. This formulation should be considered for future efficacy trials., Clinical Trials Registration: NCT01949909, PACTR201310000683408.

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., (Copyright © 2018.)

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., (© 2017 Crown copyright. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, three Diversity Covering (DiCo) PfAMA1 ectodomain proteins, designed to overcome the intrinsic polymorphism that is present in PfAMA1, were produced under Good Manufacturing Practice (GMP) in Pichia pastoris. Using identical methodology, the 3 strains were cultivated in 70-L scale fed-batch fermentations and PfAMA1-DiCos were purified by two chromatography steps, an ultrafiltration/diafiltration procedure and size exclusion chromatography, resulting in highly pure (>95%) PfAMA1-DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3, with final yields of 1.8, 1.9 and 1.3 gram, respectively. N-terminal determinations showed that approximately 50% of each of the proteins lost 12 residues from their N-terminus, in accordance with SDS-PAGE (2 main bands) and MS-data. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident. The three proteins quantitatively bound to the mAb 4G2 that recognizes a conformational epitope, suggesting proper folding of the proteins. The lyophilized Drug Product (1:1:1 mixture of PfAMA1-DiCo1, DiCo2, DiCo3) fulfilled all pre-set release criteria (appearance, dissolution rate, identity, purity, protein content, moisture content, sub-visible particles, immuno-potency (after reconstitution with adjuvant), abnormal toxicity, sterility and endotoxin), was stable in accelerated and real-time stability studies at -20°C for over 24 months. When formulated with adjuvants selected for clinical phase I evaluation, the Drug Product did not show adverse effect in a repeated-dose toxicity study in rabbits. The Drug Product has entered a phase Ia/Ib clinical trial., Competing Interests: I have read the journal's policy and four of the authors of this manuscript (BWF, AWT, CHMK and EJR) have the following competing interest: They hold a patent on the products described in this paper (patent US8741305 B2 (Jun 3, 2014), entitled "Protein Composition for Inducing an Immune Response in a Vertebrate Comprising a Plurality of Protein Variants Covering the Heterogeneity of a Single Antigen, AMA1". This does not alter our adherence to PLOS ONE policies on sharing data and materials. One of the authors (RB) is employed by a commercial company (Nova Laboratories Ltd.). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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.

The disease caused by Plasmodium falciparum (Pf) involves different clinical manifestations that, cumulatively, kill hundreds of thousands every year. Placental malaria (PM) is one such manifestation in which Pf infected erythrocytes (IE) bind to chondroitin sulphate A (CSA) through expression of VAR2CSA, a parasite-derived antigen. Protection against PM is mediated by antibodies that inhibit binding of IE in the placental intervillous space. VAR2CSA is a large antigen incompatible with large scale recombinant protein expression. Vaccines based on sub-units encompassing the functionally constrained receptor-binding domains may, theoretically, circumvent polymorphisms, reduce the risk of escape-mutants and induce cross-reactive antibodies. However, the sub-unit composition and small differences in the borders, may lead to exposure of novel immuno-dominant antibody epitopes that lead to non-functional antibodies, and furthermore influence the folding, stability and yield of expression. Candidate antigens from the pre-clinical development expressed in High-Five insect cells using the baculovirus expression vector system were transitioned into the Drosophila Schneider-2 cell (S2) expression-system compliant with clinical development. The functional capacity of antibodies against antigens expressed in High-Five cells or in S2 cells was equivalent. This enabled an extensive down-selection of S2 insect cell-expressed antigens primarily encompassing the minimal CSA-binding region of VAR2CSA. In general, we found differential potency of inhibitory antibodies against antigens with the same borders but of different var2csa sequences. Likewise, we found that subtle size differences in antigens of the same sequence gave varying levels of inhibitory antibodies. The study shows that induction of a functional response against recombinant subunits of the VAR2CSA antigen is unpredictable, demonstrating the need for large-scale screening in order to identify antigens that induce a broadly strain-transcending antibody response.

Background: Three doses of the investigational AS02(v)-adjuvanted hepatitis B virus (HBV) vaccine HB-AS02 have been shown to induce more rapid seroprotection and higher anti-HBs antibody concentrations in patients with renal insufficiency than four doses of FENDrix™ (HB-AS04), an adjuvanted HBV vaccine licensed in Europe for use in this population. This study evaluated persistence of immune response up to 36 months after primary vaccination., Methods: In this open, international, Phase III follow-up study, 151 pre-dialysis, peritoneal dialysis and hemodialysis patients ≥15 years of age received HB-AS02 at 0, 1, 6 months and 149 received HB-AS04 at 0, 1, 2, 6 months. Of these, 99 and 80 returned at Month 36, 76 and 62 of whom were eligible for inclusion in the Long-Term According-To-Protocol (LT-ATP) cohort for descriptive analysis of antibody persistence (mean age: 65.6 years)., Results: At Month 36, 89.5% of subjects in the HB-AS02 group and 72.6% of those in the HB-AS04 group had anti-HBs antibody concentrations ≥10 mIU/ml. Anti-HBs antibody concentrations were ≥100 mIU/ml in 82.9% and 35.5% of subjects, respectively. Anti-HBs geometric mean antibody concentrations were higher in the HB-AS02 group over the 36 months of follow-up. An exploratory "time to boost" analysis confirmed that subjects who received HB-AS02 were 2.54 times more likely than those who received HB-AS04 to have anti-HBs antibody concentrations ≥10 mIU/ml at Month 36 (p=0.013 [95% CI: 1.22, 5.31])., Conclusion: HB-AS02 candidate vaccine induces high and persistent anti-HBs antibody levels in pre-dialysis, peritoneal dialysis and hemodialysis patients, potentially reducing the need for booster doses in this population.

HB-AS02 is an investigational adjuvanted hepatitis B virus (HBV) vaccine for potential use in patients with renal insufficiency and other immunocompromized individuals. In this Phase III lot-to-lot consistency study, 450 healthy adult volunteers who had not previously been vaccinated against HBV were randomized to one of three production lots of HB-AS02 at 0 and 1 month and followed until one month after the last vaccine dose. Lot-to-lot consistency was established. High seroprotection rates were already achieved after the first vaccine dose (75.9%). All subjects were seroprotected (anti-HBs antibody concentrations ≥10 mIU/ml) after two doses, with all but one subject achieving anti-HBs antibody concentrations ≥100 mIU/ml (99.7%). Geometric mean anti-HBs antibody concentration was 4594.5 mIU/ml. Local and general symptoms were reported after 80.7% and 45.5% of doses, respectively. However, these were mainly of mild or moderate severity and no subject withdrew from the study due to adverse events.

Tissue factor (TF) is historically known as the trigger of the coagulation cascade. This integral membrane glycoprotein forms a ternary complex with factor VIIa (FVIIa) and zymogen factor (FX), which is then activated to factor Xa (FXa). The latter cleaves prothrombin into thrombin (FIIa), which in turn activates fibrinogen in fibrin monomers. What is less known is its additional non-haemostatic roles in inflammation, tumour growth and angiogenesis. This aspect will be developed here. TF, as a transmembrane protein, has a signalling effect requiring FVIIa. TF-FVIIa complex activates G protein-coupled receptor protease-activated receptor 2 (PAR-2) and therefore modulates various cellular processes, such as cell proliferation and survival, gene transcription and protein translation. In this review we will first highlight, using recent structural data, the 'potentially' active domain able to modulate the triggered intracellular response. We also will focus on the still emerging and promising results deciphering the diverse locations in which TF appears. We conclude with a description of an emerging and atypical use of tissue factor in platelet gel surgery for sinus augmentation.

Methylotrophic yeasts, named after their ability to grow on methanol as the sole carbon source, have raised large interest as recombinant protein factories. In this review, we explain the basic mechanisms underlying this interest and describe the minimal requirements to transform the two genera recognized as methylotrophic, Pichia and Candida, into a powerful protein production tool. We present a comparison between this group of yeasts and the conventional yeasts used as expression system in view of productivity, level of secretion and quality of post-translational modifications. Selected examples of recombinant protein produced by methylotrophic yeast are also included.

The coding sequence of the hispid cotton rat (Sigmodon hispidus) interleukin-5 (IL-5) was isolated by a combination of reverse transcription (RT)-PCR and RACE protocols from concanavalin A stimulated spleen cells. The open reading frame of 399 bp encodes a polypeptide of 132 amino acids. Comparison with the rat, mouse, gerbil and human counterparts revealed 88, 88, 87 and 75% identity at the nucleotide level and 88, 90, 89 and 70% at the amino acid level, respectively. The entire coding sequence, minus the putative signal peptide sequence, was inserted into an inducible Escherichia coli expression vector. The recombinant protein possessed an expected molecular mass of 14kDa and was located in bacterial inclusion bodies. A purification scheme under reducing and denaturing conditions followed by subsequent successive dialysis steps led to the recovery of a recombinant dimeric cotton rat IL-5. The biological activity of the recombinant protein was demonstrated in a murine cell line proliferation assay. This activity was specifically inhibited by rat monoclonal antibodies directed against mouse IL-5. Together with specific antibodies that can be generated easily, cotton rat IL-5 constitutes a useful tool for extending the use of the cotton rat animal model in the study of various human pathogens.

The hispid cotton rat (Sigmodon hispidus) has proven to be an excellent small animal model; however, immunological studies have been limited due to a lack of available reagents. We report cloning of the cotton rat interferon-gamma (IFN-gamma) cDNA from concanavalin A-stimulated spleen cells using a combination of reverse transcription polymerase amplification reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) protocols. The open reading frame of 513 nucleotides encodes a 170 amino acid (aa) protein followed by a stop codon with a predicted molecular mass of 19548Da. Cotton rat IFN-gamma shares 63, 60, 43 and 43% identity with the hamster, gerbil, mouse and rat counterpart, respectively. IFN-gamma nucleotide sequence corresponding to aa 18-153 was expressed in Escherichia coli under tryptophan promoter control, either fused to a single initiating codon or fused to the thioredoxin coding sequence. Both expression products were found exclusively in bacterial inclusion bodies. Two purification schemes have been developed to purify the product fused to a single methionine. One of them is fast and leads to the recovery of a pure product suitable for use in antibody production. The second protocol, which includes chromatographic steps, allows the use of the purified product for in vitro demonstration of biological activity in a viral cytopathic reduction assay on cotton rat cells.

The effects of endogenously expressed ribozymes directed to the mumps virus nucleocapsid (NP) mRNA were studied during viral infection. To this end, eukaryotic expression vectors encoding ribozymes or controls of passive hybridization effects were constructed and used to transfect mumps permissive Vero cells. Transcripts spanning trans-acting ribozymes of the hammerhead and hairpin types were designed to hydrolyze the first 5'GUC-3' sequence downstream from the initiation site and to hybridize to a 16 base sequence containing the putative cleavage site. Control vectors encoded mutated and catalytically inactive forms of the ribozymes or a 16 base antisense version of the target sequence. When stably expressed in cells, both ribozymes and passive control RNAs reduced viral yields. A ribozyme-mediated effect on viral growth was, however, observed, as both ribozyme types reduced viral titers by approximately 80%, well above the highest inhibition level of approximately 35% found when noncatalytic RNAs were expressed. In addition, levels of NP mRNA were generally lower in cells expressing catalytic RNAs, supporting the observed inhibition of viral growth. Although cleavage in vitro of a synthetic analog of the NP mRNA was demonstrated using RNAs isolated from ribozyme-expressing cells, in vivo cleavage products were not detectable despite the use of sensitive methods, possibly because of degradation phenomena. We also suggest here that additional controls should be conducted when semicompetitive RT-PCR methods are used to evaluate intracellular cleavage by ribozymes, as the results may depend on the initial target RNA concentration.

Recombinant hemagglutinin (H) of the measles virus (MV) expressed in a mammalian high-expression system based on the Semliki Forest virus replicon was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulin M (IgM) and IgG in patients with acute-phase measles. One hundred twelve serum specimens from 70 patients with measles were analyzed. Case definition was based on a commercial IgM ELISA that utilizes MV-infected cells (MV-ELISA) (Enzygnost; Behring Diagnostics); the clinical criteria of the Centers for Disease Control and Prevention (Atlanta, Ga.); and/or the increase in hemagglutinin test titers, neutralization test titers, and levels of MV-specific IgG whenever paired sera were available. The initial time courses of the IgM signal after the onset of rash are similar in the H- and MV-ELISAs. On days 0 to 19, both ELISAs detected IgM in 67 of 68 (98.5%) sera. Average maximal levels of IgM seem to persist, however, about 10 days longer in the MV-ELISA (up to day 25) than in the H-ELISA (day 15). From days 20 to 29 and 30 to 59, the H-ELISA detected only 64.3 (9 of 14) and 19.2% (5 of 26), respectively, of sera that were IgM positive by MV-ELISA. At least up to day 30, the performance of the H-ELISA seemed to be similar to that reported for commercial ELISAs based on whole MV. Our results demonstrate that MV H-specific IgM can be used to diagnose most measles cases from a single serum specimen collected within 19 days after the onset of rash and that the recombinant protein used in this study is suitable for this purpose.

Polytailing a cDNA with terminal deoxynucleotidyltransferase (TdT) results in the addition of a homopolymeric sequence at its 3'-end. Here we describe the use of tailing in competitive RT-PCR assays to evaluate cleavage efficiency of ribozymes. Using a system that perfectly mimics intracellular cleavage, we were able to detect as few as 1% of cleaved moieties. Furthermore, employing primers overlapping the junction between tails and the cleaved RNA moiety in non-competitive assays, the sensitivity of the method could be improved to <10 fg. Using the latter protocol and reactions employing a trans -acting hairpin ribozyme targeting the nucleocapsid mRNA of the mumps virus, we were able to demonstrate ribozyme-induced cleavage.

Recombinant hemagglutinin (H) protein of the measles virus (MV) was produced in mammalian cells with a high-yield expression system based on the Semliki Forest virus replicon. Crude membrane preparations of H protein-transfected BHK-21 cells were used to coat microtiter plates to measure specific immunoglobulin G antibodies in 228 serologically defined serum samples mainly from measles late-convalescent adults. The titers by the enzyme-linked immunosorbent assay for the H protein (H-ELISA) closely correlated with neutralization test (NT) titers (R2 = 0.66), hemagglutination inhibition test (HI) titers (R2 = 0.64), with the titers from a certified commercial ELISA based on whole MV-infected cells (MV-ELISA; R2 = 0.45). The correlations described above were better than those of the commercial MV-ELISA titers with the NT (R2 = 0.52) or HI (R2 = 0.48) titers. By using the 2nd International Standard for anti-measles serum, the detection level of the assay corresponds to 215 mIU/ml for undiluted serum, which corresponds to the estimated threshold for protective immunity. The specificity, accuracy, and positive predictive value were, in general, better for the H-ELISA than for a commercial MV-ELISA, independent of whether HI, NT, or HI and NT were used as "gold standards." In contrast, the H-ELISA proved to be slightly less sensitive than the MV-ELISA (sensitivities, 98.6 versus 99.5%, respectively; P was not significant). The assays did not differ significantly in the number of serum samples with positive HI and NT results (n = 212) which measured false negative (H-ELISA, 2 of 212 [0.94%]; MV-ELISA, 1 of 212 [0.47%]), but the H-ELISA detected significantly more measles-susceptible individuals than the MV-ELISA (10 of 11 versus 3 of 11, respectively; P < 0.05) among the individuals whose sera had negative HI and NT results. Our data demonstrate that the H-protein preparation that we describe could be a cost-effective alternative to current whole-virus-based ELISAs for surveillance for immunity to measles and that such an assay could be more efficient in detecting susceptibility to measles. Furthermore, unlike whole MV-based antigens, H-protein would also be suitable for use in the development of a simple field test for the diagnosis of measles.

In an attempt to develop efficient antiviral agents against Mumps virus, we designed ribozymes targeting the nucleocapsid (NP) mRNA. Transacting catalytic RNAs of the hammerhead and hairpin types were synthesized; they contained specific motifs, shared similar flanking regions and were directed against a 5'GUC3' target immediately downstream to the initiation codon of NP mRNA. Both ribozymes were first assayed on a synthetic 16 bases target RNA and found to catalytically and efficiently cleave the substrate in a sequence specific way. No cleavage, however, occurred when mutated forms of the ribozymes were used. In addition, both ribozyme types, when tested on the full length NP mRNA, were also able to cleave the substrate although turnover could not be demonstrated. As a rule, the hammerhead ribozyme proved more efficient than its hairpin counterpart, as well on the synthetic RNA substrate as on the full length NP mRNA target.

The fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of mumps virus (MuV) have been produced in CV1 cells via vaccinia virus recombinants. Recombinant proteins accumulated in infected cells and were glycosylated. Upon reduction, the F protein product was completely converted into its subunits. Hamsters infected with vaccinia recombinants expressing either the F or HN proteins produced antibodies recognizing MuV antigens and neutralizing MuV infectivity in vitro. These antibodies provided protection against MuV-induced encephalitis in newborn hamsters.

Chinese hamster ovary cells have been engineered to secrete an anchorless form of the varicella-zoster virus gpII protein. Purification of the recombinant product was achieved by a combination of hydrophobic and gel filtration chromatography giving rise to a protein more than 85% pure. Recombinant gpII was composed of several polypeptides which, on the basis of amino-terminal sequence analysis, corresponded to a 93-kDa precursor and to the N- and C-terminal subunits of the molecule (64 and 39/36 kDa, respectively). All polypeptides carried N-linked high-mannose and complex glycosylations, whereas O-glycosylations were carried by the precursor and the N-terminal subunits only. Surprisingly, purified recombinant gpII spontaneously formed large oligomeric structures of variable size. These complexes contained noncovalently linked lipids. Mice inoculated with the recombinant gpII absorbed onto the weak adjuvant, aluminium hydroxide, produced virus neutralizing antibodies. The recombinant gpII may thus constitute a good candidate for the development of a subunit vaccine against varicella-zoster virus.

Oligonucleotide primers have been used to amplify DNA regions of the M. leprae genome by the polymerase chain reaction. A first set of primers, PLp1 and PLp2, identifies a specific 386 bp DNA fragment located in the gene coding for the 65 kDa antigen of M. leprae. A second pair of primers, targetted to the same gene, leads to the amplification of a 154 bp DNA piece conserved in mycobacteria. Primers PLp1 and PLp2 discriminate the pathogenic species from other mycobacteria, detect down to 40 bacilli, and constitute potentially useful tools for the identification of M. leprae in clinical specimens.

Oligonucleotide primers were used to amplify specific DNA regions of the Bordetella pertussis genome by the polymerase chain reaction. One pair of primers, PTp1/PTp2, identified a 191-bp DNA fragment located in the regulatory region of the pertussis toxin operon; a second pair of primers led to amplification of a 121-bp DNA piece located in an insertion-like element specific to B. pertussis. Both sets of primers were able to discriminate between the pathogen and related Bordetella species; they detected down to 6 bacteria and appeared suitable for routine detection of B. pertussis in clinical specimens.

The boundaries of gene conversion in variant-specific antigen genes have been determined in six clones of Trypanosoma brucei. In each clone, antigenic switching involved interaction between two telomeric members of the AnTat 1.1 multigene family, which share extensive homology throughout their coding regions. All conversion events occurred by substitution of faithful copies of donor sequences. Conversion endpoints were nonrandomly distributed. In four clones, the 5' conversion limit was near the antigen translation initiation codon, while in three clones, the 3' conversion limit was located at the "hinge" between the two major antigen domains. In one case, two segmental conversions were involved in antigen switching. These observations reveal that antigen gene conversion can occur without generating point mutations, and suggest that postrecombinational selection may impose a limit on the number of possible rearrangements within antigen genes.

Specific oligodeoxynucleotide probes ranging from 20 to 35 nucleotides were defined to differentiate each of the HPV1a, 5, 6b, 8, 11, 16, 18 and 33. They were chosen using computer programs developed to compare simultaneously several 8000 bp long DNA sequences. Sequences common to all and to specific groups of the HPV DNA were also selected. Specificity of 32P-labelled probes for HPV6b, 11, 16, 18 and 33 was demonstrated and the sensitivity of the assays was evaluated by filter hybridization with viral clones and with DNA from cervical tumor biopsies.