Molecular engineering of a cryptic epitope in Spike RBD improves manufacturability and neutralizing breadth against SARS-CoV-2 variants.

There is a continued need for sarbecovirus vaccines that can be manufactured and distributed in low- and middle-income countries (LMICs). Subunit protein vaccines are manufactured at large scales at low costs, have less stringent temperature requirements for distribution in LMICs, and several candidates have shown protection against SARS-CoV-2. We previously reported an engineered variant of the SARS-CoV-2 Spike protein receptor binding domain antigen (RBD-L452K-F490W; RBD-J) with enhanced manufacturability and immunogenicity compared to the ancestral RBD. Here, we report a second-generation engineered RBD antigen (RBD-J6) with two additional mutations to a hydrophobic cryptic epitope in the RBD core, S383D and L518D, that further improved expression titers and biophysical stability. RBD-J6 retained binding affinity to human convalescent sera and to all tested neutralizing antibodies except antibodies that target the class IV epitope on the RBD core. K18-hACE2 transgenic mice immunized with three doses of a Beta variant of RBD-J6 displayed on a virus-like particle (VLP) generated neutralizing antibodies (nAb) to nine SARS-CoV-2 variants of concern at similar levels as two doses of Comirnaty. The vaccinated mice were also protected from challenge with Alpha or Beta SARS-CoV-2. This engineered antigen could be useful for modular RBD-based subunit vaccines to enhance manufacturability and global access, or for further development of variant-specific or broadly acting booster vaccines.

Passive immunization with equine RBD-specific Fab protects K18-hACE2-mice against Alpha or Beta variants of SARS-CoV-2.

Emergence of variants of concern (VOC) during the COVID-19 pandemic has contributed to the decreased efficacy of therapeutic monoclonal antibody treatments for severe cases of SARS-CoV-2 infection. In addition, the cost of creating these therapeutic treatments is high, making their implementation in low- to middle-income countries devastated by the pandemic very difficult. Here, we explored the use of polyclonal EpF(ab')2 antibodies generated through the immunization of horses with SARS-CoV-2 WA-1 RBD conjugated to HBsAg nanoparticles as a low-cost therapeutic treatment for severe cases of disease. We determined that the equine EpF(ab')2 bind RBD and neutralize ACE2 receptor binding by virus for all VOC strains tested except Omicron. Despite its relatively quick clearance from peripheral circulation, a 100µg dose of EpF(ab')2 was able to fully protect mice against severe disease phenotypes following intranasal SARS-CoV-2 challenge with Alpha and Beta variants. EpF(ab')2 administration increased survival while subsequently lowering disease scores and viral RNA burden in disease-relevant tissues. No significant improvement in survival outcomes or disease scores was observed in EpF(ab')2-treated mice challenged using the Delta variant at 10µg or 100µg doses. Overall, the data presented here provide a proof of concept for the use of EpF(ab')2 in the prevention of severe SARS-CoV-2 infections and underscore the need for either variant-specific treatments or variant-independent therapeutics for COVID-19.

SARS-CoV-2 receptor binding domain displayed on HBsAg virus-like particles elicits protective immunity in macaques.

Authorized vaccines against SARS-CoV-2 remain less available in low- and middle-income countries due to insufficient supply, high costs, and storage requirements. Global immunity could still benefit from new vaccines using widely available, safe adjuvants, such as alum and protein subunits, suited to low-cost production in existing manufacturing facilities. Here, a clinical-stage vaccine candidate comprising a SARS-CoV-2 receptor binding domain-hepatitis B surface antigen virus-like particle elicited protective immunity in cynomolgus macaques. Titers of neutralizing antibodies (>104) induced by this candidate were above the range of protection for other licensed vaccines in nonhuman primates. Including CpG 1018 did not significantly improve the immunological responses. Vaccinated animals challenged with SARS-CoV-2 showed reduced median viral loads in bronchoalveolar lavage (~3.4 log10) and nasal mucosa (~2.9 log10) versus sham controls. These data support the potential benefit of this design for a low-cost modular vaccine platform for SARS-CoV-2 and other variants of concern or betacoronaviruses.

Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor-binding domain in engineered Komagataella phaffii.

Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply ongoing demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii (Pichia pastoris). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor-binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5X by alleviating protein folding stress. Removal of methanol from the production process enabled to scale up to a 1200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.

Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor binding domain in engineered Komagataella phaffii.

Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply on-going demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii ( Pichia pastoris ). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol-induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5x by alleviating protein folding stress. Removal of methanol from the production process enabled scale up to a 1,200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.

Development of candidate combination vaccine for hepatitis E and hepatitis B: a liposome encapsulation approach.

To reduce extra injections, cost and ensure better coverage, use of combination vaccines is preferable. An attempt was made to evaluate the encapsulation of hepatitis E virus neutralizing epitope (NE) region and hepatitis B virus surface antigen (HBsAg) in liposomes as DNAs, proteins and DNA+protein. Mice groups were immunized with different liposome-encapsulated formulations and monitored for anti-HEV and anti-HBs titres, IgG subtypes, antigen-specific lymphocyte proliferation and cytokine levels. The protective levels of anti-HBs and in vitro virus-binding capacity of anti-HEV antibodies were assessed. Liposome-encapsulated DNA either singly or in combination did not elicit antibody response. Anti-HEV and anti-HBs IgG titres of individual component of protein alone (Lipo-E-P/Lipo-B-P) or DNA+protein formulations (Lipo-E-DP/Lipo-B-DP) were comparable to respective titres in combination vaccine of protein (Lipo-BE-P) and DNA+protein formulations (Lipo-BE-DP). IgG1 levels were significantly higher in Lipo-BE-P group whereas, equivalent levels of IgG1 and IgG2a were observed in Lipo-BE-DP group against both components of the vaccine. Combination vaccine group showed mixed Th1/Th2 cytokine profile. Liposome entrapped NE and HBsAg in protein and DNA+protein formats induce excellent immune response to both the components and need to be evaluated in higher animals.

Challenge studies in Rhesus monkeys immunized with candidate hepatitis E vaccines: DNA, DNA-prime-protein-boost and DNA-protein encapsulated in liposomes.

Complete ORF2 gene (1983bp) of hepatitis E virus (HEV) and the 450bp region within ORF2 containing neutralizing epitope (NE) cloned in pVAX1 and corresponding proteins expressed in baculovirus and prokaryotic systems respectively were evaluated as vaccine candidates. Two doses of liposome encapsulated DNA plus corresponding protein with both ORF2 and NE regions (Lipo-ORF2-DP and Lipo-NE-DP) showed 100% seroconversion and comparable anti-HEV titres in Swiss albino mice. These vaccine candidates were further evaluated as DNA, DNA-prime-protein-boost (DPPB) and liposome formulations in Rhesus monkeys. Monkeys receiving ORF2/NE DNA seroconverted after fourth dose while those immunized employing ORF2-DPPB format seroconverted at 7 weeks post third dose. In view of the delayed weak antibody response, these monkeys were not challenged. Though Lipo-ORF2-DP was immunogenic, 2 of the 4 monkeys developed HEV infection following homologous virus challenge of 100 Monkey Infectious Dose(50). Both monkeys immunized with Lipo-NE-DP and 1 of the 2 monkeys immunized with NE-DPPB showed complete protection, the second monkey being protected from hepatitis with limited viral replication. Irrespective of the type of immunogen, all challenged monkeys were protected from hepatitis. The results document Lipo-NE-DP to be a promising vaccine candidate needing further evaluation.