Production, quality control, stability, and potency of cGMP-produced Plasmodium falciparum RH5.1 protein vaccine expressed in Drosophila S2 cells

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 highproducing 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

Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization

Virus-like particles (VLPs) are non-infectious self-assembling nanoparticles, useful in medicine and nanotechnology. Their repetitive molecularly-defined architecture is attractive for engineering multivalency, notably for vaccination. However, decorating VLPs with target-antigens by genetic fusion or chemical modification is time-consuming and often leads to capsid misassembly or antigen misfolding, hindering generation of protective immunity. Here we establish a platform for irreversibly decorating VLPs simply by mixing with protein antigen. SpyCatcher is a genetically-encoded protein designed to spontaneously form a covalent bond to its peptide-partner SpyTag. We expressed in E. coli VLPs from the bacteriophage AP205 genetically fused to SpyCatcher. We demonstrated quantitative covalent coupling to SpyCatcher-VLPs after mixing with SpyTag-linked to malaria antigens, including CIDR and Pfs25. In addition, we showed coupling to the VLPs for peptides relevant to cancer from epidermal growth factor receptor and telomerase. Injecting SpyCatcher-VLPs decorated with a malarial antigen efficiently induced antibody responses after only a single immunization. This simple, efficient and modular decoration of nanoparticles should accelerate vaccine development, as well as other applications of nanoparticle devices

The S. aureus 4-oxalocrotonate tautomerase SAR1376 enhances immune responses when fused to several antigens

A persistent goal of vaccine development is the enhancement of the immunogenicity of antigens while maintaining safety. One strategy involves alteration of the presentation of the antigen by combining antigens with a multimeric scaffold. Multi-antigen vaccines are under development, and there are presently far more candidate antigens than antigen scaffolding strategies. This is potentially problematic, since prior immunity to a scaffold may inhibit immune responses to the antigen-scaffold combination. In this study, a series of domains from S. aureus which have been shown to crystallise into multimeric structures have been examined for their scaffolding potential. Of these domains, SAR1376, a 62 amino acid member of the 4-oxalocrotonate tautomerase (4-OT) family, was pro-immunogenic in mice when fused to a range of pathogen antigens from both S. aureus and P. falciparum, and delivered by either DNA vaccination, viral vector vaccines or as protein-in-adjuvant formulations. The adjuvant effect did not depend on enzymatic activity, but was abrogated by mutations disrupting the hexameric structure of the protein. We therefore propose that SAR1376, and perhaps other members of the 4-OT protein family, represent very small domains which can be fused to a wide range of antigens, enhancing immune responses against them

Nanoassembly routes stimulate conflicting antibody quantity and quality for transmission-blocking malaria vaccines.

Vaccine development efforts have recently focused on enabling strong immune responses to poorly immunogenic antigens, via display on multimerisation scaffolds or virus like particles (VLPs). Typically such studies demonstrate improved antibody titer comparing monomeric and nano-arrayed antigen. There are many such studies and scaffold technologies, but minimal side-by-side evaluation of platforms for both the amount and efficacy of antibodies induced. Here we present direct comparison of three leading platforms displaying the promising malaria transmission-blocking vaccine (TBV) target Pfs25. These platforms encompass the three important routes to antigen-scaffold linkage: genetic fusion, chemical cross-linking and plug-and-display SpyTag/SpyCatcher conjugation. We demonstrate that chemically-conjugated Qβ VLPs elicited the highest quantity of antibodies, while SpyCatcher-AP205-VLPs elicited the highest quality anti-Pfs25 antibodies for transmission blocking upon mosquito feeding. These quantative and qualitative features will guide future nanoassembly optimisation, as well as the development of the new generation of malaria vaccines targeting transmission

The S. aureus 4-oxalocrotonate tautomerase SAR1376 enhances immune responses when fused to several antigens

A persistent goal of vaccine development is the enhancement of the immunogenicity of antigens while maintaining safety. One strategy involves alteration of the presentation of the antigen by combining antigens with a multimeric scaffold. Multi-antigen vaccines are under development, and there are presently far more candidate antigens than antigen scaffolding strategies. This is potentially problematic, since prior immunity to a scaffold may inhibit immune responses to the antigen-scaffold combination. In this study, a series of domains from S. aureus which have been shown to crystallise into multimeric structures have been examined for their scaffolding potential. Of these domains, SAR1376, a 62 amino acid member of the 4-oxalocrotonate tautomerase (4-OT) family, was pro-immunogenic in mice when fused to a range of pathogen antigens from both S. aureus and P. falciparum, and delivered by either DNA vaccination, viral vector vaccines or as protein-in-adjuvant formulations. The adjuvant effect did not depend on enzymatic activity, but was abrogated by mutations disrupting the hexameric structure of the protein. We therefore propose that SAR1376, and perhaps other members of the 4-OT protein family, represent very small domains which can be fused to a wide range of antigens, enhancing immune responses against them

A universal plug-and-display vaccine carrier based on HBsAg VLP to maximize effective antibody response

Development of effective malaria vaccines requires delivery platforms to enhance the immunogenicity and efficacy of the target antigens. This is particularly challenging for transmission-blocking malaria vaccines (TBVs), and specifically for those based on the Pfs25 antigen, that need to elicit very high antibody titers to stop the parasite development in the mosquito host and its transmission. Presenting antigens to the immune system on virus-like particles (VLPs) is an efficient way to improve the quantity and quality of the immune response generated. Here we introduce for the first time a new VLP vaccine platform, based on the well-established hepatitis B surface antigen (HBsAg) fused to the SpyCatcher protein, so that the antigen of interest, linked to the SpyTag peptide, can be easily displayed on it (Plug-and-Display technology). As little as 10% of the SpyCatcher::HBsAg VLPs decorated with Pfs25::SpyTag (molar ratio) induces a higher antibody response and transmission-reducing activity in mice compared to the soluble protein, with 50 and 90% of the VLP coupled to the antigen further enhancing the response. Importantly, using this carrier that is a vaccine antigen itself could be beneficial, as we show that anti-HBsAg IgG antibodies are induced without interfering with the Pfs25-specific immune response generated. Furthermore, pre-existing anti-HBsAg immunity does not affect the antigen-specific response to Pfs25::SpyTag-SpyCatcher::HBsAg, suggesting that these VLPs can have a broad use as a vaccine platform

A universal plug-and-display vaccine carrier based on HBsAg VLP to maximize effective antibody response

Development of effective malaria vaccines requires delivery platforms to enhance the immunogenicity and efficacy of the target antigens. This is particularly challenging for transmission-blocking malaria vaccines (TBVs), and specifically for those based on the Pfs25 antigen, that need to elicit very high antibody titers to stop the parasite development in the mosquito host and its transmission. Presenting antigens to the immune system on virus-like particles (VLPs) is an efficient way to improve the quantity and quality of the immune response generated. Here we introduce for the first time a new VLP vaccine platform, based on the well-established hepatitis B surface antigen (HBsAg) fused to the SpyCatcher protein, so that the antigen of interest, linked to the SpyTag peptide, can be easily displayed on it (Plug-and-Display technology). As little as 10% of the SpyCatcher::HBsAg VLPs decorated with Pfs25::SpyTag (molar ratio) induces a higher antibody response and transmission-reducing activity in mice compared to the soluble protein, with 50 and 90% of the VLP coupled to the antigen further enhancing the response. Importantly, using this carrier that is a vaccine antigen itself could be beneficial, as we show that anti-HBsAg IgG antibodies are induced without interfering with the Pfs25-specific immune response generated. Furthermore, pre-existing anti-HBsAg immunity does not affect the antigen-specific response to Pfs25::SpyTag-SpyCatcher::HBsAg, suggesting that these VLPs can have a broad use as a vaccine platform

Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization.

Virus-like particles (VLPs) are non-infectious self-assembling nanoparticles, useful in medicine and nanotechnology. Their repetitive molecularly-defined architecture is attractive for engineering multivalency, notably for vaccination. However, decorating VLPs with target-antigens by genetic fusion or chemical modification is time-consuming and often leads to capsid misassembly or antigen misfolding, hindering generation of protective immunity. Here we establish a platform for irreversibly decorating VLPs simply by mixing with protein antigen. SpyCatcher is a genetically-encoded protein designed to spontaneously form a covalent bond to its peptide-partner SpyTag. We expressed in E. coli VLPs from the bacteriophage AP205 genetically fused to SpyCatcher. We demonstrated quantitative covalent coupling to SpyCatcher-VLPs after mixing with SpyTag-linked to malaria antigens, including CIDR and Pfs25. In addition, we showed coupling to the VLPs for peptides relevant to cancer from epidermal growth factor receptor and telomerase. Injecting SpyCatcher-VLPs decorated with a malarial antigen efficiently induced antibody responses after only a single immunization. This simple, efficient and modular decoration of nanoparticles should accelerate vaccine development, as well as other applications of nanoparticle devices

Enhancing immunogenicity and transmission-blocking activity of malaria vaccines by fusing Pfs25 to IMX313 multimerization technology.

Transmission-blocking vaccines (TBV) target the sexual-stages of the malaria parasite in the mosquito midgut and are widely considered to be an essential tool for malaria elimination. High-titer functional antibodies are required against target antigens to achieve effective transmission-blocking activity. We have fused Pfs25, the leading malaria TBV candidate antigen to IMX313, a molecular adjuvant and expressed it both in ChAd63 and MVA viral vectors and as a secreted protein-nanoparticle. Pfs25-IMX313 expressed from viral vectors or as a protein-nanoparticle is significantly more immunogenic and gives significantly better transmission-reducing activity than monomeric Pfs25. In addition, we demonstrate that the Pfs25-IMX313 protein-nanoparticle leads to a qualitatively improved antibody response in comparison to soluble Pfs25, as well as to significantly higher germinal centre (GC) responses. These results demonstrate that antigen multimerization using IMX313 is a very promising strategy to enhance antibody responses against Pfs25, and that Pfs25-IMX313 is a highly promising TBV candidate vaccine