Malaria immunoassemblins : A novel combinatorial vaccine approach against plasmodium falciparum based on highly improved Fc-fusion proteins

Research towards the development of malaria vaccines has been pursued since the 1960s, but still there is no licensed malaria vaccine available yet. This PhD thesis entitled “Malaria Immunoassemblins – A novel combinatorial vaccine approach against Plasmodium falciparum based on highly improved Fc-fusion proteins” aimed at overcoming challenges and limitations in malaria vaccine development that are deriving from the complex parasite life-cycle and the high antigenic diversity, while expanding mainstream Fc-fusion technology to develop new strategies for making complex multi-stage and multi-component subunit vaccines. By using the Agrobacterium-based transient expression platform, N. benthamina plants were utilized as green factories for recombinant fusion proteins. Malaria antigens and fusion proteins thereof, were N- and C-terminally fused to the human IgG1 constant domain (CH1-hinge-CH2-CH3), and upstream the human kappa light chain (CL), to benefit from natural heavy and light chain assembly. Additional, engineered heavy chain units were generated to further enable hetero-dimerization between heavy chain units. Successfully generated fusion proteins were named malaria immunoassemblins, or MIAs in short, to distinguish them from prior art immunoadhesins. More than 40 MIA pairs were produced, purified by protein-A chromatography, and efficiently assembled into IgG-like hetero-multimeric proteins. Malaria antigens reacted with specific monoclonal antibodies and immunized mice mounted high and balanced responses against each single component, including antigens that by themselves are only poorly immunogenic. The MIA approach was also successfully translated to endosperm specific expression in transgenic barley. Based on expression performance (i.e. yield and integrity) and immunological relevance, a single MIA vaccine candidate, ARC25, was produced and used to immunize rabbits. It was highly immunogenic and comprised native epitopes, as anti-ARC25 sera successfully immuno-labelled sporozoites and merozoites. Analysis of stage-specific ARC25 performance revealed low effectivity against the pre-eryhtocytic stage (22 - 26%), moderate efficiency towards the blood-stage (44%), and high sexual-stage transmission blocking activity (92%). Taken together, the MIA approach was successfully established for transient and transgenic production, and demonstrated promising results for further investigation as a platform for multi-component, multi-stage subunit vaccine candidates

Antibodies from plants for bionanomaterials

Antibodies are produced as part of the vertebrate adaptive immune response and are not naturally made by plants. However, antibody DNA sequences can be introduced into plants, and together with laboratory technologies that allow the design of antibodies recognizing any conceivable molecular structure, plants can be used as green factories' to produce any antibody at all. The advent of plant-based transient expression systems in particular allows the rapid, convenient, and safe production of antibodies, ranging from laboratory-scale expression to industrial-scale manufacturing. The key features of plant-based production include safety, speed, low cost, and convenience, allowing newcomers to rapidly master the technology and use it to its full advantage. Manufacturing in plants has recently achieved significant milestones and offers more than just an alternative to established microbial and mammalian cell platforms. The use of plants for product development in particular offers the power and flexibility to easily coexpress many different genes, allowing the plug-and-play construction of novel bionanomaterials, perfectly complementing existing approaches based on plant virus-like particles. As well as producing single antibodies for applications in medicine, agriculture, and industry, plants can be used to produce antibody-based supramolecular structures and scaffolds as a new generation of green bionanomaterials that promise a bright future based on clean and renewable nanotechnology applications

The stage-specific in vitro efficacy of a malaria antigen cocktail provides valuable insights into the development of effective multi-stage vaccines

Multicomponent vaccines targeting different stages of Plasmodium falciparum represent a promising, holistic concept towards better malaria vaccines. Additionally, an effective vaccine candidate should demonstrate cross-strain specificity because many antigens are polymorphic, which can reduce vaccine efficacy. A cocktail of recombinant fusion proteins (VAMAX-Mix) featuring three diversity-covering variants of the blood-stage antigen PfAMA1, each combined with the conserved sexual-stage antigen Pfs25 and one of the pre-erythrocytic-stage antigens PfCSP TSR or PfCelTOS, or the additional blood-stage antigen PfMSP1 19, was produced in Pichia pastoris and used to immunize rabbits. The immune sera and purified IgG were used to perform various assays determining antigen specific titers and in vitro efficacy against different parasite stages and strains. In functional in vitro assays we observed robust inhibition of blood-stage (up to 90%), and sexual-stage parasites (up to 10 0%) and biased inhibition of pre-erythrocytic parasites (0-40%). Cross-strain blood-stage efficacy was observed in erythrocyte invasion assays using four different P. falciparum strains. The quantification of antigen-specific IgGs allowed the determination of specific IC50 values. The significant difference in antigen-specific IC50 requirements, the direct correlation between antigen-specific IgG and the relative quantitative representation of antigens within the cocktail, provide valuable implementations for future multi-stage, multi-component vaccine designs

Malaria vaccine candidate antigen targeting the pre-erythrocytic stage of Plasmodium falciparum produced at high level in plants

Plants have emerged as low-cost production platforms suitable for vaccines targeting poverty-related diseases. Besides functional efficacy, the stability, yield, and purification process determine the production costs of a vaccine and thereby the feasibility of plant-based production. We describe high-level plant production and functional characterization of a malaria vaccine candidate targeting the pre-erythrocytic stage of Plasmodium falciparum. CCT, a fusion protein composed of three sporozoite antigens (P. falciparum cell traversal protein for ookinetes and sporozoites [PfCelTOS], P. falciparum circumsporozoite protein [PfCSP], and P. falciparum thrombospondin-related adhesive protein [PfTRAP]), was transiently expressed by agroinfiltration in Nicotiana benthamiana leaves, accumulated to levels up to 2 mg/g fresh leaf weight (FLW), was thermostable up to 80 degrees C and could be purified to >95% using a simple two-step procedure. Reactivity of sera from malaria semi-immune donors indicated the immunogenic conformation of the purified fusion protein consisting of PfCelTOS, PfCSP TSR, PfTRAP TSR domains (CCT) protein. Total IgG from the CCT-specific mouse immune sera specifically recognized P. falciparum sporozoites in immunofluorescence assays and induced up to 35% inhibition in hepatocyte invasion assays. Featuring domains from three promising sporozoite antigens with different roles (attachment and cell traversal) in the hepatocyte invasion process, CCT has the potential to elicit broader immune responses against the pre-erythrocytic stage of P. falciparum and represents an interesting new candidate, also as a component of multi-stage, multi-subunit malaria vaccine cocktails

Heat-Precipitation Allows the Efficient Purification of a Functional Plant-Derived Malaria Transmission-Blocking Vaccine Candidate Fusion Protein

Malaria is a vector-borne disease affecting more than two million people and accounting for more than 600,000 deaths each year, especially in developing countries. The most serious form of malaria is caused by Plasmodium falciparum. The complex life cycle of this parasite, involving pre-erythrocytic, asexual and sexual stages, makes vaccine development cumbersome but also offers a broad spectrum of vaccine candidates targeting exactly those stages. Vaccines targeting the sexual stage of P. falciparum are called transmission-blocking vaccines (TBVs). They do not confer protection for the vaccinated individual but aim to reduce or prevent the transmission of the parasite within a population and are therefore regarded as an essential tool in the fight against the disease. Malaria predominantly affects large populations in developing countries, so TBVs need to be produced in large quantities at low cost. Combining the advantages of eukaryotic expression with a virtually unlimited upscaling potential and a good product safety profile, plant-based expression systems represent a suitable alternative for the production of TBVs. We report here the high level (300g/g fresh leaf weight (FLW)) transient expression in Nicotiana benthamiana leaves of an effective TBV candidate based on a fusion protein F0 comprising Pfs25 and the C0-domain of Pfs230, and the implementation of a simple and cost-effective heat treatment step for purification that yields intact recombinant protein at >90% purity with a recovery rate of >70%. The immunization of mice clearly showed that antibodies raised against plant-derived F0 completely blocked the formation of oocysts in a malaria transmission-blocking assay (TBA) making F0 an interesting TBV candidate or a component of a multi-stage malaria vaccine cocktail

Detailed functional characterization of glycosylated and nonglycosylated variants of malaria vaccine candidate Pf AMA1 produced in Nicotiana benthamiana and analysis of growth inhibitory responses in rabbits

One of the most promising malaria vaccine candidate antigens is the Plasmodium falciparum apical membrane antigen 1 (PfAMA1). Several studies have shown that this blood-stage antigen can induce strong parasite growth inhibitory antibody responses. PfAMA1 contains up to six recognition sites for N-linked glycosylation, a post-translational modification that is absent in P. falciparum. To prevent any potential negative impact of N-glycosylation, the recognition sites have been knocked out in most PfAMA1 variants expressed in eukaryotic hosts. However, N-linked glycosylation may increase efficacy by improving immunogenicity and/or focusing the response towards relevant epitopes by glycan masking. We describe the production of glycosylated and nonglycosylated PfAMA1 in Nicotiana benthamiana and its detailed characterization in terms of yield, integrity and protective efficacy. Both PfAMA1 variants accumulated to high levels (>510 μg/g fresh leaf weight) after transient ex pression, and high-mannose-type N-glycans were confirmed for the glycosylated variant. No significant differences between the N. benthamiana and Pichia pastoris PfAMA1 variants were detected in conformation-sensitive ligand-binding studies. Specific titres of >2 × 106 were induced in rabbits, and strong reactivity with P. falciparum schizonts was observed in immunofluorescence assays, as well as up to 100% parasite growth inhibition for both variants, with IC50 values of ~35 μg/mL. Competition assays indicated that a number of epitopes were shielded from immune recognition by N-glycans, warranting further studies to determine how glycosylation can be used for the directed targeting of immune responses. These results highlight the potential of plant transient expression systems as a production platform for vaccine candidates

Kombinierte Trochleaplastik und Rekonstruktion des MPFL bei patellofemoraler Instabilität

Combining key antigens from the different stages of the P. falciparum life cycle in the context of a multi-stage-specific cocktail offers a promising approach towards the development of a malaria vaccine ideally capable of preventing initial infection, the clinical manifestation as well as the transmission of the disease. To investigate the potential of such an approach we combined proteins and domains (11 in total) from the pre-erythrocytic, blood and sexual stages of P. falciparum into a cocktail of four different components recombinantly produced in plants. After immunization of rabbits we determined the domain-specific antibody titers as well as component-specific antibody concentrations and correlated them with stage specific in vitro efficacy. Using purified rabbit immune IgG we observed strong inhibition in functional in vitro assays addressing the pre-erythrocytic (up to 80%), blood (up to 90%) and sexual parasite stages (100%). Based on the component-specific antibody concentrations we calculated the IC50 values for the pre-erythrocytic stage (17-25 μg/ml), the blood stage (40-60 μg/ml) and the sexual stage (1.75 μg/ml). While the results underline the feasibility of a multi-stage vaccine cocktail, the analysis of component-specific efficacy indicates significant differences in IC50 requirements for stage-specific antibody concentrations providing valuable insights into this complex scenario and will thereby improve future approaches towards malaria vaccine cocktail development regarding the selection of suitable antigens and the ratios of components, to fine tune overall and stage-specific efficacy

The purified antibody preparations used for all <i>in vitro</i> assays.

<p>The total IgG concentrations and CFCA results are listed for each component. Nomenclature is based on the number of the rabbit (R1, R2 or R3) followed by the sampling day (35, 63 or 91). The PlasmoMix-specific antibody concentration is the sum of the specific antibody response against the four components. The antigen-specific antibody concentration is given in mg/ml and % of total IgG.</p

<i>In vitro</i> growth inhibition assay (GIA) of asexual <i>P</i>. <i>falciparum</i> 3D7 parasites with purified PlasmoMix-specific rabbit IgG.

<p>The rabbit IgGs were purified from the serum of three rabbits (R1, R2 and R3) collected on days 35, 63 and 91 post-immunization with PlasmoMix. Nomenclature of the sample first features the number of the rabbit (R1, R2 or R3) followed by the sampling day (35, 63 or 91). (A) Four serial 1/1 dilutions from 6–0.75 mg/ml of total IgGs were used to demonstrate the concentration dependency of the assay and to calculate the IC_<b>50</b> values (the total IgG concentrations needed for 50% inhibition). (B) The same GIA but instead of total IgG, the gAMA1-specifc antibody concentration (based on CFCA and calculated from total IgG, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131456#sec002" target="_blank">methods</a> section) was used and the IC_<b>50</b> values for gAMA1-specific antibodies were calculated. Each data point represents the mean of technical triplicates.</p

Antigen-specific antibody titers of rabbit immune sera determined by ELISA.

<p>Three rabbits (R1, R2 and R3) were immunized six times with PlasmoMix and serum samples were collected on days 0 (pre-immune), 35, 63 and 91. (A) The antibody titer against the immunization mixture (PlasmoMix) as well as against the four protein-based components (CCT, E3, gAMA1 and F0) were determined. (B) To further dissect the immune response, the specific antibody response against each individual domain was analyzed. Therefore, the domains were C-terminaly fused to DsRed (fluorescent reporter protein), and expressed and purified as described in the methods section. Antigen domains comprising a fusion protein are connected by a bracket. Antibody titers are shown for each rabbit (R1: open black square, R2: open red circle, R3: open blue triangle) as well as the geometric mean (black line) of three rabbits.</p