The "humanized" N-glycosylation pathway in CRISPR/Cas9-edited Nicotiana benthamiana significantly enhances the immunogenicity of a S/preS1 Hepatitis B Virus antigen and the virus-neutralizing antibody response in vaccinated mice.

The recent SARS-CoV-2 pandemic has taught the world a costly lesson about the devastating consequences of viral disease outbreaks but also, the remarkable impact of vaccination in limiting life and economic losses. Vaccination against human Hepatitis B Virus (HBV), a major human pathogen affecting 290 million people worldwide, remains a key action towards viral hepatitis elimination by 2030. To meet this goal, the development of improved HBV antigens is critical to overcome non-responsiveness to standard vaccines based on the yeast-produced, small (S) envelope protein. We have recently shown that combining relevant immunogenic determinants of S and large (L) HBV proteins in chimeric antigens markedly enhances the anti-HBV immune response. However, the demand for cost-efficient, high-quality antigens remains challenging. This issue could be addressed by using plants as versatile and rapidly scalable protein production platforms. Moreover, the recent generation of plants lacking ß-1,2-xylosyltransferase and α-1,3-fucosyltransferase activities (FX-KO), by CRISPR/Cas9 genome editing, enables production of proteins with "humanized" N-glycosylation. In this study, we investigated the impact of plant N-glycosylation on the immunogenic properties of a chimeric HBV S/L vaccine candidate produced in wild-type and FX-KO Nicotiana benthamiana. Prevention of ß-1,2-xylose and α-1,3-fucose attachment to the HBV antigen significantly increased the immune response in mice, as compared with the wild-type plant-produced counterpart. Notably, the antibodies triggered by the FX-KO-made antigen neutralized more efficiently both wild-type HBV and a clinically relevant vaccine escape mutant. Our study validates in premiere the glyco-engineered Nicotiana benthamiana as a substantially improved host for plant production of glycoprotein vaccines.

RBD-VLP Vaccines Adjuvanted with Alum or SWE Protect K18-hACE2 Mice against SARS-CoV-2 VOC Challenge.

The ongoing COVID-19 pandemic has contributed largely to the global vaccine disparity. Development of protein subunit vaccines can help alleviate shortages of COVID-19 vaccines delivered to low-income countries. Here, we evaluated the efficacy of a three-dose virus-like particle (VLP) vaccine composed of hepatitis B surface antigen (HBsAg) decorated with the receptor binding domain (RBD) from the Wuhan or Beta SARS-CoV-2 strain adjuvanted with either aluminum hydroxide (alum) or squalene in water emulsion (SWE). RBD HBsAg vaccines were compared to the standard two doses of Pfizer mRNA vaccine. Alum-adjuvanted vaccines were composed of either HBsAg conjugated with Beta RBD alone (ß RBD HBsAg+Al) or a combination of both Beta RBD HBsAg and Wuhan RBD HBsAg (ß/Wu RBD HBsAg+Al). RBD vaccines adjuvanted with SWE were formulated with Beta RBD HBsAg (ß RBD HBsAg+SWE) or without HBsAg (ß RBD+SWE). Both alum-adjuvanted RBD HBsAg vaccines generated functional RBD IgG against multiple SARS-CoV-2 variants of concern (VOC), decreased viral RNA burden, and lowered inflammation in the lung against Alpha or Beta challenge in K18-hACE2 mice. However, only ß/Wu RBD HBsAg+Al was able to afford 100% survival to mice challenged with Alpha or Beta VOC. Furthermore, mice immunized with ß RBD HBsAg+SWE induced cross-reactive neutralizing antibodies against major VOC of SARS-CoV-2, lowered viral RNA burden in the lung and brain, and protected mice from Alpha or Beta challenge similarly to mice immunized with Pfizer mRNA. However, RBD+SWE immunization failed to protect mice from VOC challenge. Our findings demonstrate that RBD HBsAg VLP vaccines provided similar protection profiles to the approved Pfizer mRNA vaccines used worldwide and may offer protection against SARS-CoV-2 VOC. IMPORTANCE Global COVID-19 vaccine distribution to low-income countries has been a major challenge of the pandemic. To address supply chain issues, RBD virus-like particle (VLP) vaccines that are cost-effective and capable of large-scale production were developed and evaluated for efficacy in preclinical mouse studies. We demonstrated that RBD-VLP vaccines protected K18-hACE2 mice against Alpha or Beta challenge similarly to Pfizer mRNA vaccination. Our findings showed that the VLP platform can be utilized to formulate immunogenic and efficacious COVID-19 vaccines.

A Novel DNA Vaccine Against SARS-CoV-2 Encoding a Chimeric Protein of Its Receptor-Binding Domain (RBD) Fused to the Amino-Terminal Region of Hepatitis B Virus preS1 With a W4P Mutation.

A coronavirus SARS-CoV-2, which has caused the pandemic viral pneumonia disease COVID-19, significantly threatens global public health, highlighting the need to develop effective and safe vaccines against its infection. In this study, we developed a novel DNA vaccine candidate against SARS-CoV-2 by expressing a chimeric protein of its receptor-binding domain (RBD) fused to a 33-bp sequence (11 aa) from the hepatitis B virus (HBV) preS1 region with a W4P mutation (W4P-RBD) at the N-terminal region and evaluated its immunogenicity. In vitro transfection experiments in multiple cell lines demonstrated that W4P-RBD vs. wild-type RBD protein (W-RBD) led to enhanced production of IL-6 and TNFα at the transcription and translation levels, suggesting the adjuvant potential of N-terminal HBV preS1 sequences for DNA vaccines against SARS-CoV-2. W4P-RBD also led to enhanced production of IgG and IgA, which can neutralize and block SARS-CoV-2 infection in both blood sera and bronchoalveolar lavage (BAL) fluid from the lung in vaccinated mice. Additionally, W4P-RBD led to an enhanced T-cell-mediated cellular immune response under S1 protein stimulation. In summary, W4P-RBD led to robust humoral and cell-mediated immune responses against SARS-CoV-2 in vaccinated mice, highlighting its feasibility as a novel DNA vaccine to protect against SARS-CoV-2 infection.