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COVID-19 pandemic caused approximately 750,000 deaths and over 20 million confirmed cases of infection by SARS-CoV-2 within 8 months since the emergence of the virus. While there are no vaccines approved and considering the difficulty in meeting the large vaccination demand worldwide, the potential use of passive immunization should be considered based on existing successful therapies against many diseases. Here we demonstrate that hyperimmune globulin preparations raised in horses against the recombinant trimeric spike (S) glycoprotein of SARS-CoV-2 in the prefusion conformation provide very high ELISA titers as well as highly potent neutralizing activity against SARS-CoV-2. Five horses were subcutaneously inoculated for 6 weeks with the recombinant S protein (ectodomain, residues 1-1208). Four out of the 5 horses presented a strong immune response. Considering the average of all 5 horses, ELISA titers above 1:1,000,000 and neutralizing titers (PRNT90) reaching 1:14,604 were observed. When compared with the plasma of three convalescent COVID-19 patients, sera of immunized horses displayed approximately 140-fold higher neutralizing titers measured as PRNT90. To prevent eventual side effects caused by horse antiserum, IgG was digested with pepsin and purified by fractional salt precipitation to eliminate Fc fragments, a process that is industrially used for the production of passive immunization F(ab')2 concentrates against rabies, tetanus and snake venoms. The high neutralizing titers against SARS-CoV-2 obtained for the unprocessed sera were confirmed for the F(ab')2 fragments and were 150-fold higher than the PRNT90 neutralizing titers of plasma of three COVID-19 convalescent patients. The great advantage of using the recombinant trimeric S glycoprotein is that it is safe and provides quick adaptive immunity in horses. Our data show the perspective of using hyperimmune anti-SARS-CoV-2 F(ab')2 preparations as a passive immunization therapy in humans, similar to therapies that have been safely used for decades against rabies, tetanus and snake venoms.

Yellow fever (YF) is a life‐threatening viral disease endemic in parts of Africa and Latin America. Although there is a very efficacious vaccine since the 1930s, YF still causes 29,000–60,000 annual deaths. During recent YF outbreaks there were issues of vaccine shortage of the current egg‐derived vaccine; rare but fatal vaccine adverse effects occurred; and cases were imported to Asia, where the circulating mosquito vector could potentially start local transmission. Here we investigated the production of YF virus‐like particles (VLPs) using stably transfected HEK293 cells. Process intensification was achieved by combining sequential FACS (fluorescence‐activated cell sorting) rounds to enrich the stable cell pool in terms of high producers and the use of perfusion processes. At shaken‐tube scale, FACS enrichment of cells allowed doubling VLP production, and pseudoperfusion cultivation (with daily medium exchange) further increased VLP production by 9.3‐fold as compared to batch operation mode. At perfusion bioreactor scale, the use of an inclined settler as cell retention device showed operational advantages over an ATF system. A one‐step steric exclusion chromatography purification allowed significant removal of impurities and is a promising technique for future integration of upstream and downstream operations. Characterization by different techniques confirmed the identity and 3D‐structure of the purified VLPs.

Despite the intramuscular route being the most used vaccination strategy against SARS-CoV-2, the intradermal route has been studied around the globe as a strong candidate for immunization against SARS-CoV-2. Adjuvants have shown to be essential vaccine components that are capable of driving robust immune responses and increasing the vaccination efficacy. In this work, our group aimed to develop a vaccination strategy for SARS-CoV-2 using a trimeric spike protein, by testing the best route with formulations containing the adjuvants AddaS03, CpG, MPL, Alum, or a combination of two of them. Our results showed that formulations that were made with AddaS03 or CpG alone or AddaS03 combined with CpG were able to induce high levels of IgG, IgG1, and IgG2a; high titers of neutralizing antibodies against SARS-CoV-2 original strain; and also induced high hypersensitivity during the challenge with Spike protein and a high level of IFN-γ producing CD4+ T-cells in mice. Altogether, those data indicate that AddaS03, CpG, or both combined may be used as adjuvants in vaccines for COVID-19.