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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.

Plasmodium invasion of red blood cells involves malaria proteins, such as reticulocyte-binding protein homolog 5 (RH5), RH5 interacting protein (RIPR), cysteine-rich protective antigen (CyRPA), apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2), all of which are blood-stage malaria vaccine candidates. So far, vaccines containing AMA1 alone have been unsuccessful in clinical trials. However, immunization with AMA1 bound with RON2L (AMA1-RON2L) induces better protection against P. falciparum malaria in Aotus monkeys. We therefore sought to determine whether combinations of RH5, RIPR, CyRPA and AMA1-RON2L antibodies improve their biological activities and sought to develop a robust method for determination of synergy or additivity in antibody combinations. Rabbit antibodies against AMA1-RON2L, RH5, RIPR or CyRPA were tested either alone or in combinations in P. falciparum growth inhibition assay to determine Bliss' and Loewe's additivities. The AMA1-RON2L/RH5 combination consistently demonstrated an additive effect while the CyRPA/RIPR combination showed a modest synergistic effect with Hewlett’s $$S=1.07 \left[95\% \mathrm{C}\mathrm{I}: 1.03, 1.19\right].$$ S = 1.07 95 % C I : 1.03 , 1.19 . Additionally, we provide a publicly-available, online tool to aid researchers in analyzing and planning their own synergy experiments. This study supports future blood-stage vaccine development by providing a solid methodology to evaluate additive and/or synergistic (or antagonistic) effect of vaccine-induced antibodies.

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 clearly defined relationship 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.Statement of significance of the studyProtein microarray technology is increasingly being realised as a powerful tool in disease biomarker identification. Protein-based, serologic arrays are already well utilised in the characterisation of antibody responses to the malarial Plasmodium spp., drastically improving throughput and efficiency compared to more typical experimental assays. Such approaches have commonly made use of in vitro transcription/translation (IVTT) protein expression systems, though other protein expression methods are regularly employed. In this study we have directly compared antibody responses in a malaria endemic population, to matched protein antigens derived from both an IVTT and purified bacterial recombinant system in the same protein microarray. We demonstrated that the magnitude of measured antibody response to matched protein antigens tends to be greater to purified recombinants, rather than IVTT products. Further, our analysis showed highly variable levels of correlation of response between antigen-matched targets derived from each expression system, although relationships in antibody response were stronger between proteins with overlapping amino acid sequences. This study highlights the importance of considering the strengths and weaknesses of each expression system in the context of a protein microarray according to experimental hypotheses, and illustrates the need for attention when comparing data generated by these different methodologies.

Summary Interactions between B cells and CD4+ T follicular helper (Tfh) cells are key determinants of humoral responses. Using samples from clinical trials performed with the malaria vaccine candidate antigen Plasmodium falciparum merozoite protein (PfRH5), we compare the frequency, phenotype, and gene expression profiles of PfRH5-specific circulating Tfh (cTfh) cells elicited by two leading human vaccine delivery platforms: heterologous viral vector prime boost and protein with AS01B adjuvant. We demonstrate that the protein/AS01B platform induces a higher-magnitude antigen-specific cTfh cell response and that this correlates with peak anti-PfRH5 IgG concentrations, frequency of PfRH5-specific memory B cells, and antibody functionality. Furthermore, our data indicate a greater Th2/Tfh2 skew within the polyfunctional response elicited following vaccination with protein/AS01B as compared to a Th1/Tfh1 skew with viral vectors. These data highlight the impact of vaccine platform on the cTfh cell response driving humoral immunity, associating a high-magnitude, Th2-biased cTfh response with potent antibody production., Graphical abstract, Highlights CD4 Tfh comparison in malaria vaccine trials using leading human vaccine platforms Protein/AS01B drives stronger antigen-specific Tfh responses than viral vectors Greater T(f)h2 skewing of antigen-specific CD4 T cells in protein/AS01B vaccinees Antigen-specific CD4 T(fh) cell parameters correlate with functional antibody, Nielsen et al. evaluate the induction of antigen-specific T follicular helper cell responses in human malaria vaccine trials using protein/adjuvant (AS01B) or heterologous viral vector platforms. They demonstrate that protein/AS01B drives a higher-magnitude polyfunctional response with greater skewing toward a Th2 phenotype, associated with superior production of neutralizing antibodies.

Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is a leading asexual blood-stage vaccine candidate for malaria. In preparation for clinical trials, a full-length PfRH5 protein vaccine called “RH5.1” was produced as a soluble product under cGMP using the ExpreS2 platform (based on a Drosophila melanogaster S2 stable cell line system). Following development of a high-producing monoclonal S2 cell line, a master cell bank was produced prior to the cGMP campaign. Culture supernatants were processed using C-tag affinity chromatography followed by size exclusion chromatography and virus-reduction filtration. The overall process yielded >400 mg highly pure RH5.1 protein. QC testing showed the MCB and the RH5.1 product met all specified acceptance criteria including those for sterility, purity, and identity. The RH5.1 vaccine product was stored at −80 °C and is stable for over 18 months. Characterization of the protein following formulation in the adjuvant system AS01B showed that RH5.1 is stable in the timeframe needed for clinical vaccine administration, and that there was no discernible impact on the liposomal formulation of AS01B following addition of RH5.1. Subsequent immunization of mice confirmed the RH5.1/AS01B vaccine was immunogenic and could induce functional growth inhibitory antibodies against blood-stage P. falciparum in vitro. The RH5.1/AS01B was judged suitable for use in humans and has since progressed to phase I/IIa clinical trial. Our data support the future use of the Drosophila S2 cell and C-tag platform technologies to enable cGMP-compliant biomanufacture of other novel and “difficult-to-express” recombinant protein-based vaccines., Malaria: Successful clinical trial preparation for blood-stage vaccine A vaccine candidate for blood-stage malaria has overcome previous hurdles to enter clinical trials. The protein PfRH5 is an essential blood-stage infection facilitator of malarial parasite Plasmodium falciparum, and a promising target for vaccine strategies. Unfortunately, efforts to produce the protein in an immunogenic, clinically-viable way have been met with difficulty. Here, researchers led by Simon Draper, from the UK’s Jenner Institute, used a fruit fly expression system to produce over 400 mg of high-purity protein. Formulated with an immunity-boosting adjuvant, the vaccine elicited antibodies in mice that proved inhibitory to blood-stage P. falciparum during in vitro assays. The PfRH5 vaccine candidate and its adjuvant have been approved for a clinical trial in the UK, and the authors hope that the expression system used may be beneficial in the expression of other ‘difficult’ proteins.

Extracellular protein misfolding and aggregation underlie many of the most serious amyloidoses including Alzheimer's 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.

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's 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.

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.