Epitope mapping and fine specificity of human T and B cell responses for novel candidate blood-stage malaria vaccine P27A

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

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

International audiencePlacental 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 adjuvant GLA-SE promotes human Tfh cell expansion and emergence of public TCRβ clonotypes

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.</jats:p

Cloning, expression and purification of recombinant cotton rat interferon-gamma.

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.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Purification and characterization of recombinant varicella-zoster virus glycoprotein gpII, secreted by Chinese hamster ovary cells.

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.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Immunogenicity and safety of an investigational AS02(v)-adjuvanted hepatitis B vaccine in patients with renal insufficiency who failed to respond or to maintain antibody levels after prior vaccination results of two open, randomized, comparative trials

An investigational AS02(v)-adjuvanted hepatitis B (HB-AS02) was compared with a licensed conventional recombinant hepatitis B vaccine (HBVAXPRO™; Sanofi Pasteur MSD, Lyon, France) in pre-dialysis, peritoneal dialysis and hemodialysis patients aged ≥18 years who had failed either to respond to prior vaccination with a conventional hepatitis B vaccine (Study A; n=251) or to maintain protective antibody concentrations after prior hepatitis B vaccination (Study B; n=181). These were open, randomized, comparative trials. Mean (range) age was 65.9 (31-92) and 64.6 (29-92) years in the two studies, respectively. In Study A, two doses of HB-AS02 given one month apart were found to be superior to two doses of the licensed vaccine in terms of seroprotection rate (76.9% versus 37.6%) and anti-HBs geometric mean antibody concentration (GMC; 139.3 versus 6.9mIU/ml), with antibody concentrations ≥100mIU/ml in 61.1% and 15.4% of subjects in the two groups, respectively. In Study B, one month after administration of a single booster dose, seroprotection rates were 89.0% in the HB-AS02 group and 90.8% in the licensed vaccine group, 81.3% and 60.9% of subjects had antibody concentrations ≥100mIU/ml, and anti-HBs GMCs were 1726.8 and 189.5mIU/ml. HB-AS02 was found to be more reactogenic than the licensed vaccine. In summary, the investigational HB-AS02 vaccine induced higher seroprotection rates and anti-HBs GMCs than a licensed conventional hepatitis B vaccine in uremic patients who had failed to respond or to maintain protective antibody titers after prior hepatitis B vaccination

The Influence of Sub-Unit Composition and Expression System on the Functional Antibody Response in the Development of a VAR2CSA Based Plasmodium falciparum Placental Malaria Vaccine

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

Comparison of the induction of binding inhibitory antibodies against antigens expressed in High-Five or S2 cells.

<p>Bars indicate the mean binding inhibition of 3D7 (A) or FCR3 (B) <i>Plasmodium falciparum</i> infected erythrocytes (IE). The values are the percentages of released IEs in wells with test serum compared to wells with control serum from non-immunized animals. Serum was used in a one to ten dilution. Error bars indicate the coefficients of variation of the mean of triplicate measurements (100*(SD/(mean of control binding)).</p