Expression of rabies VLPs in adherence and suspension conditions: a flexible platform for rabies vaccine production

Rabies is a zoonotic viral disease with a mortality by close to 100%. As there is not an efficacious treatment available, post-exposure vaccination is recommended for individuals in contact with the virus. On the other hand, the most common source of virus transmission is saliva of infected animals, mostly dogs, whereby mass vaccination of pets is the most cost‑effective way to reduce human infections. In this context, availability of both human and veterinary vaccines is critical. In previous works1-2, our group developed immunogenic rabies VLPs, expressing the virus glycoprotein in HEK293 cells. We obtained a producer clone capable of growing in adherence (adhP2E5) and then adapted to suspension conditions (sP2E5). In this work, we analyzed the production of VLPs in both conditions, using two different platforms. On the one hand, adhP2E5 was cultured in 850 cm2 roller bottles (GBO) using medium with 5% FCS, that was exchanged every 48 h during the first 10 days and every 24 h during the last 5 days. RV-VLPs were continuously produced and the harvest obtained (2.5 L per bottle) was analyzed by sandwich ELISA, using the 6th International Standard for rabies vaccine that quantify the glycoprotein content (NIBSC), presenting a value of 19 IU.ml‑1 in average. On the other hand, we cultured sP2E5 in a 5 L bioreactor during 15 days, using EX‑CELL293 SFM (SAFC). The culture reached densities of 2x107 cel.ml-1 and VLPs were continuously secreted to the supernatant. The obtained harvest (28.5 L) presented a glycoprotein content of 28 IU.ml-1, a results that is comparable with the previous one taking into account the number of cells presents in both conditions. These results showed that our clone could be cultured in both platforms depending on the objectives and characteristics of the desired product. For the production of the rabies veterinary vaccine, RV-VLPs can be produced in adherent conditions using medium supplemented with FCS and, for human vaccine production, RV-VLPs can be produced in bioreactors using SFM. 1 Fontana et al. Rabies virus-like particles expressed in HEK293 cells. Vaccine 32 (2014) 2799-2804. 2 Fontana et al. Immunogenic virus-like particles continuously expressed in mammalian cells as a veterinary rabies vaccine candidate. Vaccine 33 (2015) 4238-4246

Rational design, expression and characterization of chimeric rabies VLPs displaying the major antigenic site of Foot and Mouth Disease Virus

Virus-like Particles (VLPs) are supramolecular arrangements of one or more viral proteins that resemble the structure of the native virus but are not infective, due the fact that they lack the viral genome. They have become very important in the field of novel recombinant vaccines, because of their biosafety and improved immunogenicity over subunit vaccines due to their particulate nature and highly repetitive epitope display. Moreover, they can elicit immune responses against heterologous antigens by the incorporation of epitopes by genic fusion to the viral proteins, constituting chimeric VLPs (cVLPs). In a previous work, by recombinant expression of the rabies G glycoprotein (RVG) in HEK293 cells, we stablished a platform to produce rabies VLPs as a new generation vaccine candidate 1–3. The main goal of this work was to generate a platform for heterologous antigen presentation based on RVG cVLPs. The heterologous epitope chosen for this study was the immunodominant site of Foot-and-Mouth Disease Virus (FMDV), named G-H loop (that is part of the capsid protein VP1), which is responsible of virus entry into the cell 4. Please click Download on the upper right corner to see the full abstract

Study of rabies VLPs expression in BHK-21 cell line for vaccine applications

In the last decades, virus-like particles (VLPs) have played an essential role in the development of novel vaccines due the fact that they trigger robust and balanced immune responses and, as they lack viral genome, are biosafe. Nowadays, several VLPs are commercially available for human use and one veterinary product was licensed. Besides, other VLP‑based vaccine candidates are in the stages of clinical trials or preclinical evaluation. Our group had previously developed a rabies glycoprotein based-VLP (RV-VLPs) expressed in HEK293 cells. These RV-VLPs were fully characterized and their capacity to induce a protective response and neutralizing antibodies production was confirmed. As inactivated veterinary vaccines for rabies are usually produced using BHK‑21, the goal of the present work was to develop a RV-VLPs expressing BHK‑21 cell line to analyze the characteristics of the VLPs produced using this cell substrate. Therefore, by lentivirus vector-mediated transduction, we generated a rabies virus glycoprotein expressing a stable cell line. The cellular expression of the recombinant protein was analyzed by flow cytometry and the membrane localization was confirmed by fluorescent microscopy. Later, RV-VLPs budding to the supernatant was analyzed by sandwich ELISA. After that, VLPs were purified by density gradient ultracentrifugation and the hydrodynamic diameter of the particles was analyzed by DLS. In a western blot assay, the particles were recognized by specific antibodies present in a rabies polyclonal serum. Finally, the recombinant cell line was cultured in 850 cm2 roller bottles producing RV-VLPs continuously during 25 days of culture. Thus, these results encourage further studies to confirm if BHK-21 is a good cell substrate for the production of RV-VLPs as a veterinary rabies vaccine candidate

RHABDO-LIKE RECOMBINANTE (VLPs), a novel veterinary rabies vaccine: Safety and efficacy trials in pets and cattle

Rabies is a viral fatal disease transmitted to humans from bites of infected animals. Due to nearly 95 % of human infections are related with dog bites, the WHO, OIE and FAO agreed in the implementation of a worldwide plan for the elimination of dog-mediated human rabies death by 2030. On the other hand, in some Latin-American countries paralytic bovine rabies transmitted by vampire bats causes economic losses to the farming industry. In this context, it is important to develop an economic vaccine that could be produced in a short period of time. Our research group have been working in the development and validation of a recombinant rabies vaccine based on virus‑like particles (RV-VLPs), expressing the rabies glycoprotein in HEK293 cells1. Performing a rational design, we carried out the expression vectors construction, stable cell line development and the full biochemical and morphological characterization of the expressed RV-VLPs. On the other hand, the immune response triggered by these VLPs in mice was analysed, demonstrating that this new antigen is able to induce a potent humoral immune response with high titers of neutralizing antibodies. Furthermore, protection against rabies virus challenge was confirmed in NIH potency test assays, demonstrating that RV-VLPs are an excellent new generation and virus-free vaccine candidate. Please click Download on the upper right corner to see the full abstract

Rational design of novel fusion rabies glycoproteins displaying a major antigenic site of foot-and-mouth disease virus for vaccine applications

Chimeric virus-like particles are self-assembling structures composed of viral proteins that had been modified to incorporate sequences from different organisms, being able to trigger immune responses against the heterologous sequence. However, the identification of suitable sites for that purpose in the carrier protein is not an easy task. In this work, we describe the generation of rabies chimeric VLPs that expose a major antigenic site of foot-and-mouth disease virus (FMDV) by identifying suitable regions in rabies glycoprotein (RVG), as a proof of concept of a novel heterologous display platform for vaccine applications. To identify adequate sites for insertion of heterologous sequences without altering the correct folding of RVG, we identified regions that were evolutionally non-conserved in Lyssavirus glycoproteins and performed a structural analysis of those regions using a 3D model of RVG trimer that we generated. The heterologous sequence was inserted in three different sites within RVG sequence. In every case, it did not affect the correct folding of the protein and was surface exposed, being recognized by anti-FMDV antibodies in expressing cells as well as in the surface of VLPs. This work sets the base for the development of a heterologous antigen display platform based on rabies VLPs.Fil: Garay, Ernesto Sergio. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Fontana, Diego Sebastian. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Leschiutta, Lautaro. Universidad Nacional del Litoral; ArgentinaFil: Kratje, Ricardo Bertoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Prieto, Claudio. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentin

Recombinant human granulocyte-macrophage colony-stimulating factor: effect of glycosylation on pharmacokinetic parameters

The pharmacokinetic behaviour of the non-glycosylated, bacterially-derived recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and the glycosylated mammalian product was studied after intra and extra vascular administration of a single dose in rodents. Each route of administration gave a different rhGM-CSF concentration-time profile. After extra vascular administration of equivalent doses, a higher peak concentration and faster elimination were observed in the group treated with the E. coli-derived cytokine. The faster elimination resulted in a return to pre-treatment plasma levels after 12 hrs, versus 48 hrs following the administration of glycosylated rhGM-CSF. After intravascular administration, clearance of rhGM-CSF was significantly decreased by the presence of carbohydrates. Non-significant differences in the terminal phase of the biphasic kinetics were found, but the distribution phase was significantly longer for the glycosylated form

Chimeric VLPs Based on HIV-1 Gag and a Fusion Rabies Glycoprotein Induce Specific Antibodies against Rabies and Foot-and-Mouth Disease Virus

Foot and mouth disease is a livestock acute disease, causing economic losses in affected areas. Currently, control of this disease is performed by mandatory vaccination campaigns using inactivated viral vaccines. In this work, we describe the development of a chimeric VLP-based vaccine candidate for foot-and-mouth disease virus (FMDV), based on the co-expression of the HIV-1 Gag protein and a novel fusion rabies glycoprotein (RVG), which carries in its N-term the FMDV main antigen: the G-H loop. It is demonstrated by confocal microscopy that both Gag-GFP polyprotein and the G-H loop colocalize at the cell membrane and, that the Gag polyprotein of the HIV virus acts as a scaffold for enveloped VLPs that during the budding process acquires the proteins that are being expressed in the cell membrane. The obtained VLPs were spherical particles of 130 ± 40 nm in diameter (analyzed by TEM, Cryo-TEM and NTA) carrying an envelope membrane that efficiently display the GH-RVG on its surface (analyzed by gold immunolabeling). Immunostainings with a FMDV hyperimmune serum showed that the heterologous antigenic site, genetically fused to RVG, is recognized by specific G-H loop antibodies. Additionally, the cVLPs produced expose the G-H loop to the liquid surrounding (analyzed by specific ELISA). Finally, we confirmed that these FMD cVLPs are able to induce a specific humoral immune response, based on antibodies directed to the G-H loop in experimental animals

Screening of CHO-K1 endogenous promoters for expressing recombinant proteins in mammalian cell cultures

For the production of recombinant protein therapeutics in mammalian cells, a high rate of gene expression is desired and hence strong viral-derived promoters are commonly used. However, they usually induce cellular stress and can be susceptible to epigenetic silencing. Endogenous promoters, which coordinates their activity with cellular and bioprocess dynamics while at the same time they maintain high expression levels, may help to avoid such drawbacks. In this work, new endogenous promoters were discovered based on high expression levels in RNA-seq data of CHO-K1 cells cultured in high density. The promoters of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were selected for generating new expression vectors for the production of recombinant proteins in mammalian cells. The in silico-derived promoter regions were experimentally verified and the majority showed transcriptional activity comparable or higher than CMV. Also, stable expression following a reduction of culture temperature was investigated. The characterized endogenous promoters (excluding Rps18) constitute a promising alternative to CMV promoter due to their high strength, long-term expression stability and integration into the regulatory network of the host cell. These promoters may also comprise an initial panel for designing cell engineering strategies and synthetic promoters, as well as for industrial cell line development.Fil: Tossolini, Ileana del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Gugliotta, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: López Díaz, Fernando. Salk Institute for Biological Studies; Estados UnidosFil: Kratje, Ricardo Bertoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Prieto, Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin