Two-Component Nanoparticle Vaccine Displaying Glycosylated Spike S1 Domain Induces Neutralizing Antibody Response against SARS-CoV-2 Variants.

Vaccines pave the way out of the SARS-CoV-2 pandemic. Besides mRNA and adenoviral vector vaccines, effective protein-based vaccines are needed for immunization against current and emerging variants. We have developed a virus-like particle (VLP)-based vaccine using the baculovirus-insect cell expression system, a robust production platform known for its scalability, low cost, and safety. Baculoviruses were constructed encoding SARS-CoV-2 spike proteins: full-length S, stabilized secreted S, or the S1 domain. Since subunit S only partially protected mice from SARS-CoV-2 challenge, we produced S1 for conjugation to bacteriophage AP205 VLP nanoparticles using tag/catcher technology. The S1 yield in an insect-cell bioreactor was ∼11 mg/liter, and authentic protein folding, efficient glycosylation, partial trimerization, and ACE2 receptor binding was confirmed. Prime-boost immunization of mice with 0.5 µg S1-VLPs showed potent neutralizing antibody responses against Wuhan and UK/B.1.1.7 SARS-CoV-2 variants. This two-component nanoparticle vaccine can now be further developed to help alleviate the burden of COVID-19. IMPORTANCE Vaccination is essential to reduce disease severity and limit the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Protein-based vaccines are useful to vaccinate the world population and to boost immunity against emerging variants. Their safety profiles, production costs, and vaccine storage temperatures are advantageous compared to mRNA and adenovirus vector vaccines. Here, we use the versatile and scalable baculovirus expression vector system to generate a two-component nanoparticle vaccine to induce potent neutralizing antibody responses against SARS-CoV-2 variants. These nanoparticle vaccines can be quickly adapted as boosters by simply updating the antigen component.

Environmental flow regimes for Dysidea avara sponges.

The aim of our research is to design tank systems to culture Dysidea avara for the production of avarol. Flow information was needed to design culture tanks suitable for effective production. Water flow regimes were characterized over a 1-year period for a shallow rocky sublittoral environment in the Northwestern Mediterranean where D. avara sponges are particularly abundant. Three-dimensional Doppler current velocities at 8-10-m depths ranged from 5 to 15 cm/s over most seasons, occasionally spiking to 30-66 cm/s. A thermistor flow sensor was used to map flow fields in close proximity ( approximately 2 cm) to individual sponges at 4.5-, 8.8-, and 14.3-m depths. These "proximal flows" averaged 1.6 cm/s in calm seas and 5.9 cm/s during a storm, when the highest proximal flow (32.9 cm/s) was recorded next to a sponge at the shallowest station. Proximal flows diminished exponentially with depth, averaging 2.6 cm/s +/- 0.15 SE over the entire study. Flow visualization studies showed that oscillatory flow (0.20-0.33 Hz) was the most common regime around individual sponges. Sponges at the 4.5-m site maintained a compact morphology with large oscula year-around despite only seasonally high flows. Sponges at 8.8 m were more erect with large oscula on tall protuberances. At the lowest-flow 14.3-m site, sponges were more branched and heavily conulated, with small oscula. The relationship between sponge morphology and ambient flow regime is discussed.

Evaluation of baculovirus expression vectors with enhanced stability in continuous cascaded insect-cell bioreactors.

Continuous protein production with baculovirus expression vectors in insect-cell bioreactors is characterized by a dramatic drop in heterologous protein production within a few weeks. This is mainly due to the spontaneous deletion of the heterologous gene(s) from the baculovirus genome and/or to the rapid accumulation of defective interfering baculoviruses (DIs). Cell culture experiments with bacmid-derived baculoviruses showed that spontaneous deletions in the foreign bacterial artificial chromosome (BAC) sequences readily occurred. These deletions correlated with a low density of baculovirus homologous (repeat) regions (hrs), which are located dispersed throughout the baculovirus genome and are believed to act as origins of viral DNA replication (oris). To test the hypothesis that deletions are more likely to occur in regions with a low ori density, the properties of bacmid-derived baculoviruses with an additional hr in the unstable BAC sequences were compared to the standard bacmid-derived baculovirus in a continuous cascaded insect-cell bioreactor configuration. All viruses were equipped with a green fluorescent protein (GFP) gene and a gene encoding the classical swine fever virus E2 glycoprotein (CSFV-E2). The insertion of an extra hr in the BAC vector led to improved genetic stability of adjacent sequences, resulting in prolonged protein expression. The maintenance of the BAC sequences appeared to be dependent on the orientation of the inserted hr. The advantages of the utilization of hrs to improve the stability of baculovirus expression vectors for the large-scale protein production in insect-cell bioreactors are discussed.