A clinically validated Drosophila S2 based vaccine platform for production of malaria vaccines

Drosophila S2 insect cell expression is less known than the extensively used Spodoptera or Trichoplusia ni (Hi-5) insect cell based Baculovirus expression system (BEVS). Nevertheless it has been used in research for almost 40 years. The cell line was derived from late stage Drosophila melanogaster (Fruit fly) embryos by Schneider in the 1970s, who named the cell line Drosophila Schneider line 2 (synonyms: S2, SL2, D.mel. 2). The system has been widely applied to fundamental research, where the availability of the whole genome sequence of Drosophila melanogaster (1, 2) and the S2 cells’ susceptibility to RNA interference methods (3, 4) have enabled genome wide RNAi screening and whole genome expression analysis techniques to be used to great effect. S2 cells have proved to be highly effective for the production of proteins from a great variety of protein classes (5), such as: viral proteins, toxins, membrane proteins, enzyme, etc. Recent publications have also shown the strength of the S2 system in expression of Virus Like Particles (VLPs) (6). ExpreS2ion has developed the ExpreS2, Drosophila S2 platform to achieve improved yields for difficult to express proteins. Furthermore, several technologies have been developed to improve the ease of use of the system, as well as enable fast and efficient screening of multiple constructs. S2 based production processes for two malaria vaccine clinical trails with The Jenner Institute, Oxford University (Rh5 (7,8), blood-stage malaria) and Copenhagen University (VAR2CSA (9) pregnancy associated malaria) have been developed. The placental malaria vaccine is currently in a phase Ia trail in Germany, and a Phase Ib trial in Benin. The blood-stage malaria vaccine is currently in Phase IIa trial and is expecting results by the end of 2018. Several transmission-blocking candidates have been identified over the years with some of the most prominent being pfs48/45, Pfs230C and Pfs25(10). Other vaccine targets focus on blood-stage malaria such as Rh5, PfRIPR and CyrPA. We will present data on the development of a high producing Pfs25 monoclonal cell line and the purification from said cell line,as well as expression data on a range of other malaria vaccine targets. This present the clinically validated ExpreS2 platform as a complete system for a wide range of malaria targeting vaccines. (1) Adams M.D. et al. Science 2000 287:2185-2195 (2) Ashburner M, et al. Genome Res. 2005 Dec;15(12):1661-7 (3) Neumüller RA, et al. Wiley Interdiscip Rev Syst Biol Med. 2011 Jul-Aug; 3(4):471-8 (4) D’Ambrosio M.V. et al. J. Cell Biol. Vol. 191 No. 3 471–478 (5) Schetz J.A. et al. Protein Expression in the Drosophila Schneider 2 Cell System, Current Protocols in Neuroscience, 2004 (6) Yang L. et al. J Virol. 2012, Jul;86(14):7662-76. (7) Wright K.E. et al. Nature, 2014 Nov 20;515(7527):427-30 (8) Hjerrild K.A. et al. Sci Rep. 2016 Jul 26;6:30357 (9) Nielsen M.A. et al. PLoS One. 2015 Sep 1;10(9):e0135406 (10) Chaturvedi N et al. Indian J Med Res. 2016 Jun;143(6):696-71

Current and cumulative malaria infections in a setting embarking on elimination: Amhara, Ethiopia.

BACKGROUND: Since 2005, Ethiopia has aggressively scaled up malaria prevention and case management. As a result, the number of malaria cases and deaths has significantly declined. In order to track progress towards the elimination of malaria in Amhara Region, coverage of malaria control tools and current malaria transmission need to be documented. METHODS: A cross-sectional household survey oversampling children under 5 years of age was conducted during the dry season in 2013. A bivalent rapid diagnostic test (RDT) detecting both Plasmodium falciparum and Plasmodium vivax and serology assays using merozoite antigens from both these species were used to assess the prevalence of malaria infections and exposure to malaria parasites in 16 woredas (districts) in Amhara Region. RESULTS: 7878 participants were included, with a mean age of 16.8 years (range 0.5-102.8 years) and 42.0% being children under 5 years of age. The age-adjusted RDT-positivity for P. falciparum and P. vivax infection was 1.5 and 0.4%, respectively, of which 0.05% presented as co-infections. Overall age-adjusted seroprevalence was 30.0% for P. falciparum, 21.8% for P. vivax, and seroprevalence for any malaria species was 39.4%. The prevalence of RDT-positive infections varied by woreda, ranging from 0.0 to 8.3% and by altitude with rates of 3.2, 0.7, and 0.4% at under 2000, 2000-2500, and >2500 m, respectively. Serological analysis showed heterogeneity in transmission intensity by area and altitude and evidence for a change in the force of infection in the mid-2000s. CONCLUSIONS: Current and historic malaria transmission across Amhara Region show substantial variation by age and altitude with some settings showing very low or near-zero transmission. Plasmodium vivax infections appear to be lower but relatively more stable across geography and altitude, while P. falciparum is the dominant infection in the higher transmission, low-altitude areas. Age-dependent seroprevalence analyses indicates a drop in transmission occurred in the mid-2000s, coinciding with malaria control scale-up efforts. As malaria parasitaemia rates get very low with elimination efforts, serological evaluation may help track progress to elimination

Plasmodium falciparum serology: A comparison of two protein production methods for analysis of antibody responses by protein microarray.

The evaluation of protein antigens as putative serologic biomarkers of infection has increasingly shifted to high-throughput, multiplex approaches such as the protein microarray. In vitro transcription/translation (IVTT) systems-a similarly high-throughput protein expression method-are already widely utilised in the production of protein microarrays, though purified recombinant proteins derived from more traditional whole cell based expression systems also play an important role in biomarker characterisation. Here we have performed a side-by-side comparison of antigen-matched protein targets from an IVTT and purified recombinant system, on the same protein microarray. The magnitude and range of antibody responses to purified recombinants was found to be greater than that of IVTT proteins, and responses between targets from different expression systems did not clearly correlate. However, responses between amino acid sequence-matched targets from each expression system were more closely correlated. Despite the lack of a clear correlation between antigen-matched targets produced in each expression system, our data indicate that protein microarrays produced using either method can be used confidently, in a context dependent manner, though care should be taken when comparing data derived from contrasting approaches

Rapid high-yield expression and purification of fully post-translationally modified recombinant clusterin and mutants

The first described and best known mammalian secreted chaperone, abundant in human blood, is clusterin. Recent independent studies are now exploring the potential use of clusterin as a therapeutic in a variety of disease contexts. In the past, the extensive post-translational processing of clusterin, coupled with its potent binding to essentially any misfolded protein, have meant that its expression as a fully functional recombinant protein has been very difficult. We report here the first rapid and high-yield system for the expression and purification of fully post-translationally modified and chaperone-active clusterin. Only 5–6 days is required from initial transfection to harvest of the protein-free culture medium containing the recombinant product. Purification to near-homogeneity can then be accomplished in a single affinity purification step and the yield for wild type human clusterin is of the order of 30–40 mg per litre of culture. We have also shown that this system can be used to quickly express and purify custom-designed clusterin mutants. These advances dramatically increase the feasibility of detailed structure–function analysis of the clusterin molecule and will facilitate identification of those specific regions responsible for the interactions of clusterin with receptors and other molecules

The potential of clusterin to act as an immune modulator

Clusterin (CLU) was the first described extracellular mammalian chaperone and can interact with misfolded proteins to stabilise them in a soluble form, until the bound protein can be re-folded or degraded. In the last twenty years there has been an interest in using chaperones as adjuvants of the immune system. Numerous, mainly intracellular, chaperones have been investigated in this context and some are able to elicit a specific, cytotoxic T lymphocyte (CTL) response and in some cases a humoral response against bound antigens. The first aim of this project was to develop a chaperone-active form of recombinant CLU (rCLU), as forms of rCLU described in published reports or currently commercially available have not been thoroughly characterised or tested for chaperone activity, and in many cases are impure. A bacterial expression system was first investigated which yielded high molecular weight aggregates of rCLU; the protein was not glycosylated or cleaved into α- and β-chains, and lacked chaperone activity. A mammalian expression system was next trialled; previous attempts in our laboratory to produce pure rCLU by mammalian expression failed due to the chaperone activity of CLU resulting in it binding to misfolded proteins in the culture medium, which then co-purified with the rCLU. Therefore an innovative approach was required to overcome this problem, whereby a polyclonal culture of HEK293 cells expressing rCLU were allowed to adhere to the surface of tissue culture flasks in medium containing foetal calf serum (FCS), before replacement with FCS-free media. Under these conditions cells remained attached to the flask surface and secreted rCLU into the medium until dead. Using this approach, rCLU was successfully expressed and purified, and shown to have chaperone activity comparable to that of wild type (wt)CLU; there were only minor client protein-specific differences in activity between the rCLU and wtCLU that may be attributable to differences in glycosylation between the two forms of CLU. The mechanism by which chemically linked complexes formed between CLU and ovalbumin (i.e. CLU-OVA) were bound and internalised by a range of cells was next investigated. CLU-OVA and wtCLU showed significantly higher binding to murine bone marrow derived macrophages, human granulocytes, monocytes and to a lower extent lymphocytes, than OVA or control ligands. Confocal microscopy studies indicated that CLU-OVA was rapidly internalised by murine bone marrow derived macrophages, in less than 15 min, and subsequently co-localised with both early endosomes and lysosomes. The binding of CLU-OVA was inhibitable by fucoidan and polyinosinic acid, implicating scavenger receptors in the uptake CLU-OVA into these cell types. The final aim of the project was to determine whether CLU could act as an adjuvant of the immune system, to elicit both a CTL response and a humoral response when bound to the model antigen OVA. The results obtained from an in vivo cytolysis assay suggest that injection with CLU-OVA or CLU-OVA and a strong adjuvant (Complete Freund’s Adjuvant; CFA) is able to stimulate an OVA-specific CTL response. Relative to mice immunised with OVA or PBS alone, the levels of interleukin 2 and interferon gamma contained within CD8+ T cells isolated from the draining lymph nodes and secreted from cells obtained from the draining lymph nodes or spleen were significantly greater in mice immunised with CLU-OVA + CFA, suggesting that the CD8+ T cells from this treatment group were undergoing the highest levels of proliferation and had the highest gain of (CTL) effector function. These experiments, however, need to be repeated with more replicates to confirm these interpretations. Following immunisation with CLU-OVA or CLU/OVA (complexes formed using the chaperone action of CLU), mice mounted a mature immune response to OVA of a predominantly IgG1 isotype. Immunisation with CLU-OVA and CLU/OVA also significantly enhanced the production of OVA-specific IgM antibodies compared to mice immunised with OVA, or other controls. The results from this study provide a practical method to successfully express and purify chaperone active, correctly post-translationally modified rCLU, thereby opening the path for future studies to identify functional sites within CLU. It also provides a theoretically unlimited supply of rCLU which may find future application in the development of therapeutics. Similar to the results obtained for intracellular chaperones that act as adjuvants of the immune system, CLU appears to direct bound proteins for uptake by a wide range of cells via scavenger receptors. Further work is required to definitively establish whether CLU can induce an antigen specific CTL response, as is suggested by the results of this study. If CLU is able to activate both a CTL and an antibody response to bound antigen, then CLU may provide a useful vector for the development of vaccines to overcome the current limitations of vaccines based on the use of intracellular chaperones

Roles of extracellular chaperones in amyloidosis

Extracellular protein misfolding and aggregation underlie many of the most serious amyloidoses including Alzheimer\u27s disease, spongiform encephalopathies and type II diabetes. Despite this, protein homeostasis (proteostasis) research has largely focussed on characterising systems that function to monitor protein conformation and concentration within cells. We are now starting to identify elements of corresponding systems, including an expanding family of secreted chaperones, which exist in the extracellular space. Like their intracellular counterparts, extracellular chaperones are likely to play a central role in systems that maintain proteostasis; however, the precise details of how they participate are only just emerging. It is proposed that extracellular chaperones patrol biological fluids for misfolded proteins and facilitate their clearance via endocytic receptors. Importantly, many amyloidoses are associated with dysfunction in rates of protein clearance. This is consistent with a model in which disruption to, or overwhelming of, the systems responsible for extracellular proteostasis results in the accumulation of pathological protein aggregates and disease. Further characterisation of mechanisms that maintain extracellular proteostasis will shed light on why many serious diseases occur and provide us with much needed strategies to combat them

Extracellular chaperones

The maintenance of the levels and correct folding state of proteins (proteostasis) is a fundamental prerequisite for life. Life has evolved complex mechanisms to maintain proteostasis and many of these that operate inside cells are now well understood. The same cannot yet be said of corresponding processes in extracellular fluids of the human body, where inappropriate protein aggregation is known to underpin many serious diseases such as Alzheimer\u27s disease, type II diabetes and prion diseases. Recent research has uncovered a growing family of abundant extracellular chaperones in body fluids which appear to selectively bind to exposed regions of hydrophobicity on misfolded proteins to inhibit their toxicity and prevent them from aggregating to form insoluble deposits. These extracellular chaperones are also implicated in clearing the soluble, stabilized misfolded proteins from body fluids via receptor-mediated endocytosis for subsequent lysosomal degradation. Recent work also raises the possibility that extracellular chaperones may play roles in modulating the immune response. Future work will better define the in vivo functions of extracellular chaperones in proteostasis and immunology and pave the way for the development of new treatments for serious diseases

Expression and Purification of Chaperone-Active Recombinant Clusterin

<div><p>Clusterin was the first described secreted mammalian chaperone and is implicated as being a key player in both intra- and extracellular proteostasis. Its unique combination of structural features and biological chaperone activity has, however, previously made it very challenging to express and purify the protein in a correctly processed and chaperone-active form. While there are multiple reports in the literature describing the use of recombinant clusterin, all of these reports suffer from one or more of the following shortcomings: details of the methods used to produce the protein are poorly described, the product is incompletely (if at all) characterised, and purity (if shown) is in many cases inadequate. The current report provides the first well validated method to economically produce pure chaperone-active recombinant clusterin. The method was developed after trialling expression in cultured bacterial, yeast, insect and mammalian cells, and involves the expression of recombinant clusterin from stably transfected HEK293 cells in protein-free medium. The product is expressed at between 7.5 and 10 µg/ml of culture, and is readily purified by a combination of immunoaffinity, cation exchange and size exclusion chromatography. The purified product was shown to be glycosylated, correctly proteolytically cleaved into α- and β-subunits, and have chaperone activity similar to that of human plasma clusterin. This new method creates the opportunity to use mutagenesis and metabolic labelling approaches in future studies to delineate functionally important sites within clusterin, and also provides a theoretically unlimited supply of recombinant clusterin which may in the future find applications in the development of therapeutics.</p></div

Images of Coomassie blue stained SDS-PAGE gels analysing b-rCLU.

<p>(A) Lane 1: Molecular weight markers with size indicated in kDa. Lane 2: Sample of b-rCLU after SEC purification in solubilisation buffer containing 8 M urea and 5 mM DTT. (B) Lane 1: Molecular weight markers with size indicated in kDa. Lane 2: Sample of b-rCLU after step-wise dialysis into PBS/Az.</p

Chaperone assays of m-rCLU and wtCLU using 3 client proteins.

<p>(A) BSA (1.5 mg/ml) was induced to precipitate using 20 mM DTT. The A₃₆₀ at 30 min intervals is plotted (mean ± standard deviation; n  = 3). (B) CS (200 µg/ml) and (C) CPK (1.12 mg/ml) were heated at 43°C to induce precipitation; the A₃₆₀ (mean ± standard deviation; n  = 3) is plotted at intervals of 5 min (CS) or 10 min (CPK). In all cases, m-rCLU and wtCLU were tested at a range of concentrations and representative plots shown, and OVA was used as a negative control (at the highest concentration of CLU tested or greater).</p