Optimization, production, purification and characterization of HIV-1 Gag VLPs functionalized with SARS-CoV-2 Spike glycoprotein

Virus-like particles (VLPs) constitute a promising approach for recombinant vaccine development. They are robust, safe, versatile and high immunogenic supra-molecular structures that closely mimic the native conformation of the viruses without carrying its genetic material. HIV-1 Gag VLPs share similar characteristics with wild type Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus, making them a suitable platform to express the Spike membrane protein to generate a potential vaccine candidate for COVID-19. This work proposes a methodology for the generation of SARS-CoV-2 VLPs by its co-expression with HIV-1 Gag protein by transient transfection of HEK 293 cultures. We first evaluated the cellular co-expression of SARS-CoV-2 Spike glycoprotein with HIV-1 Gag: confocal microscopy analysis showed that after its expression, native envelope Spike glycoprotein travels to the plasmatic membrane of the HEK 293 producer cells, where it co-localizes with Gag::eGFP (Figure 1.A,B) and Spike-functionalized VLPs (S-VLP) generation occurs. Please click Download on the upper right corner to see the full abstract

Development of a purification process for HIV-1 VLPs, from supernatant to lyophilization

HIV-1-based virus-like particles (VLPs) have high potential as scaffold for the development of chimeric or multivalent vaccines by functionalizing them with specific antigens [1], [2]. The obtention of vaccine formulations independent of cold chain is desirable to facilitate transportation and administration worldwide [3]. Recently, efforts are being made to develop cost-effective scalable processes to obtain these particles. The present study aims to compare some of the most used downstream processing (DSP) technologies for capture and purification of VLPs, taking especial consideration in using technologies enabling operation at large scale [4]. First, suspension adapted HEK 293 cells cultured in chemically defined cell culture media were used to produce the Gag-eGFP VLPs. Then, some steps of the purification process were studied, including a primary and secondary clarification by depth filtration and filtration respectively, an intermediate step by tangential flow filtration (TFF) or multimodal chromatography (MC), a capture step by ion exchange (IEC), heparin affinity (AC) and hydrophobic interaction chromatography (HIC), a polishing step by size exclusion chromatography (SEC) and a finally lyophilization step by freeze-drying process. Different operation units were tested for each step. Finally, a complete DSP train was implemented using the best results obtained in each stage. A concentration of 2.2 ± 0.8·109 VLPs/mL in the lyophilized samples was obtained after its storage at room temperature for 2 months. The morphology and structural integrity were further assessed by cryo-TEM. These first results in enveloped VLP lyophilization offer great promise to overcome the difficulty to distribute vaccines in poorly served remote rural areas and increase vaccine stability until their administration. Likewise, the purification methodologies proposed here could be easily scaled up and applied to purify similar enveloped viruses and vesicles. Please click Additional File below for the presentation

The role of extracellular vesicles in the modulation of the cell density effect

Please click Additional Files below to see the full abstract

Generation and efficacy assessment of a chimeric antigen E2-CD154 as a marker Classical Swine Fever Virus subunit vaccine produced in HEK 293 and CHO K1 mammalian cells

The E2 glycoprotein is the major antigen that induces neutralizing and protective antibodies in CSFV infected pigs, thus a marker vaccine based on this antigen appears to be the most promising alternative to induce a protective immune response against CSFV. However, the structural characteristics of this protein state the necessity to produce glycoprotein E2 in more complex expression systems such as mammalian cells. In this study, we use a lentivirus-based gene delivery system to establish a stable recombinant HEK 293 and CHO K1 cell line for the expression of E2 fused to porcine CD154 as immunostimulatory molecule. In a first experiment, E2his and E2-CD154 were compared in an immunization trial. The average antibody titers in E2his immunized pigs was in the range of 30-40% of blocking and the average antibody titers for E2-CD154 are above 40% at day 14, meaning that the chimeric antigen is able to raise antibodies at positive levels in a shorter time. Additionally, the blocking rate of E2his vaccinated group in ELISA ranged between 66-88% and in the E2-CD154- vaccinated groups ranged between 86-92%, one week after booster immunization. The NPLA antibody titers also increased greatly. Later on, the protective capacity of purified E2-CD154 glycoprotein was demonstrated in a challenge experiment in pigs using a biphasic immunization schedule with 25 and 50 μg. The immunized animals developed neutralizing antibodies that were protective when the animals were faced to a challenge with 105 LD50 of ‘‘Margarita’’ CSFV highly pathogenic strain. No clinical signs of the disease were detected in the vaccinated pigs. Unvaccinated pigs in the control group exhibited symptoms of CSF at 3–4 days after challenge and were euthanized from 7–9 days when the pigs became moribund. These results indicate that E2-CD154 produced in recombinant HEK 293 and CHOK1cell line is a high quality candidate for the development of a safe and effective CSFV subunit vaccine. In the next steps, pilot and production scale, E2-CD154 expression levels should be increased in 10 to 50 fold, arriving to a very attractive productive platform for an implementation of a commercial subunit vaccine against CSF

Optimization, Production, Purification and Characterization of HIV-1 GAG-Based Virus-like Particles Functionalized with SARS-CoV-2

Virus-like particles (VLPs) constitute a promising approach to recombinant vaccine development. They are robust, safe, versatile and highly immunogenic supra-molecular structures that closely mimic the native conformation of viruses without carrying their genetic material. HIV-1 Gag VLPs share similar characteristics with wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, making them a suitable platform for the expression of its spike membrane protein to generate a potential vaccine candidate for COVID-19. This work proposes a methodology for the generation of SARS-CoV-2 VLPs by their co-expression with HIV-1 Gag protein. We achieved VLP functionalization with coronavirus spike protein, optimized its expression using a design of experiments (DoE). We also performed the bioprocess at a bioreactor scale followed by a scalable downstream purification process consisting of two clarifications, an ion exchange and size-exclusion chromatography. The whole production process is conceived to enhance its transferability at current good manufacturing practice (cGMP) industrial scale manufacturing. Moreover, the approach proposed could be expanded to produce additional Gag-based VLPs against different diseases or COVID-19 variants

A four-step purification process for gag vlps : from culture supernatant to high-purity lyophilized particles

Gag-based virus-like particles (VLPs) have high potential as scaffolds for the development of chimeric vaccines and delivery strategies. The production of purified preparations that can be preserved independently from cold chains is highly desirable to facilitate distribution and access worldwide. In this work, a nimble purification has been developed, facilitating the production of Gag VLPs. Suspension-adapted HEK 293 cells cultured in chemically defined cell culture media were used to produce the VLPs. A four-step downstream process (DSP) consisting of membrane filtration, ion-exchange chromatography, polishing, and lyophilization was developed. The purification of VLPs from other contaminants such as host cell proteins (HCP), double-stranded DNA, or extracellular vesicles (EVs) was confirmed after their DSP. A concentration of 2.2 ± 0.8 × 10 VLPs/mL in the lyophilized samples was obtained after its storage at room temperature for two months. Morphology and structural integrity of purified VLPs was assessed by cryo-TEM and NTA. Likewise, the purification methodologies proposed here could be easily scaled up and applied to purify similar enveloped viruses and vesicles