Engineered SARS-CoV-2 receptor binding domain improves manufacturability in yeast and immunogenicity in mice

Dalvie, Neil C.; Rodriguez-Aponte, Sergio A.; Hartwell, Brittany L.; Tostanoski, Lisa H.; Biedermann, Andrew M.; Crowell, Laura E.; Kaur, Kawaljit; Kumru, Ozan S.; Carter, Lauren; Yu, Jingyou; Chang, Aiquan; McMahan, Katherine; Courant, Thomas; Lebas, Celia; Lemnios, Ashley A.; Rodrigues, Kristen A.; Silva, Murillo; Johnston, Ryan S.; Naranjo, Christopher A; Tracey, Mary Kate; Brady, Joseph; Whittaker, Charles A.; Yun, Dongsoo; Brunette, Natalie; Wang, Jing Yang; Walkey, Carl; Fiala, Brooke; Kar, Swagata; Porto, Maciel; Lok, Megan; Andersen, Hanné; Lewis, Mark G.; Love, Kerry R.; Camp, Danielle L.; Silverman, Judith M.; Kleanthous, Harry; Joshi, Sangeeta B.; Volkin, David B.; Dubois, Patrice; Collin, Nicolas; King, Neil P.; Barouch, Dan H.; Irvine, Darrell J.; Love, J. Christopher

doi:10.1073/pnas.2106845118

Cited by 76 publications

(164 citation statements)

References 79 publications

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“…Preclinical studies have evaluated the protective efficacy of several RBD-specific monoclonals, as well as RBD as a protective immunogen. Investigators evaluating RBD as a vaccine candidate have delivered the antigen either as DNA 81 , mRNA 7,74,[82][83][84][85] , viral vector 86 , soluble monomer or dimer 6,22,[87][88][89][90][91][92][93] , a fusion protein nanoparticle 16,26,[94][95][96][97][98] , or as a virus-like particle (VLP) 91,[99][100][101][102] . Additional RBD-based vaccine candidates in clinical trials (Table 2)…”

Section: Preclinical Studies Support Rbd As a Potent Vaccine Antigenmentioning

confidence: 99%

“…have not yet published their preclinical work. The RBD sequences selected from the S-glycoprotein as candidate vaccines encode an ~25 kDa (>200 aa) glycosylated protein 103 , produced using either mammalian 26 , baculovirus-infected insect cells 92 , or yeast-based production platforms 5,88,89,91,104 . In general, vaccination strategies have focused on parenteral immunization to elicit high-titer serum nAb, although some preclinical studies support intranasal delivery of S-protein in NHPs and hamsters using a live vector approach 105 , and RBD on chitosan nanoparticles in mice 106 , with evidence for a role of mucosal immunity in reducing viral load in both the upper and lower respiratory tract.…”

Section: Preclinical Studies Support Rbd As a Potent Vaccine Antigenmentioning

confidence: 99%

“…Vaccine researchers around the world have demonstrated preclinical immunogenicity of multimeric RBD vaccine candidates utilizing a variety of presentation methods including the following: ferritin nanoparticles 94,96,98,108,109 , single-component protein nanoparticles 95 , two-component protein nanoparticles either self-assembling 26 or assembled via SpyTag/Catcher technology 91,99,110 , and VLPs 102,111 . Together, these studies support the assertion that RBD displayed on a particle and co-administered with a suitable adjuvant represents a viable vaccine strategy for SARS-CoV-2, including against VOC.…”

Section: Prototype Rbd-np Vaccinementioning

confidence: 99%

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Scientific rationale for developing potent RBD-based vaccines targeting COVID-19

et al. 2021

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Vaccination of the global population against COVID-19 is a great scientific, logistical, and moral challenge. Despite the rapid development and authorization of several full-length Spike (S) protein vaccines, the global demand outweighs the current supply and there is a need for safe, potent, high-volume, affordable vaccines that can fill this gap, especially in low- and middle-income countries. Whether SARS-CoV-2 S-protein receptor-binding domain (RBD)-based vaccines could fill this gap has been debated, especially with regards to its suitability to protect against emerging viral variants of concern. Given a predominance for elicitation of neutralizing antibodies (nAbs) that target RBD following natural infection or vaccination, a key biomarker of protection, there is merit for selection of RBD as a sole vaccine immunogen. With its high-yielding production and manufacturing potential, RBD-based vaccines offer an abundance of temperature-stable doses at an affordable cost. In addition, as the RBD preferentially focuses the immune response to potent and recently recognized cross-protective determinants, this domain may be central to the development of future pan-sarbecovirus vaccines. In this study, we review the data supporting the non-inferiority of RBD as a vaccine immunogen compared to full-length S-protein vaccines with respect to humoral and cellular immune responses against both the prototype pandemic SARS-CoV-2 isolate and emerging variants of concern.

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Section: Preclinical Studies Support Rbd As a Potent Vaccine Antigenmentioning

confidence: 99%

Section: Preclinical Studies Support Rbd As a Potent Vaccine Antigenmentioning

confidence: 99%

Section: Prototype Rbd-np Vaccinementioning

confidence: 99%

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Scientific rationale for developing potent RBD-based vaccines targeting COVID-19

et al. 2021

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“…Vaccine candidates based on protein subunits are also important ones for enabling interventions for the pandemic in low‐ and middle‐income countries due to existing large‐scale manufacturing facilities and less stringent temperature and storage requirements for distribution (Dai et al, 2020 ). We and others have reported vaccine designs based on the receptor‐binding domain (RBD) of the spike protein (Dalvie et al, 2021 ). In these designs, the RBD can be produced independently, and subsequently, displayed on protein or lipid nanoparticles for enhanced immunogenicity (Cohen et al, 2021 ; Walls et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

“…We previously reported the production of the SARS‐CoV‐2 RBD (Wuhan‐Hu‐1 sequence) in an engineered variant of K. phaffii (Brady et al, 2020 ; Dalvie et al, 2021 ). To assess the feasibility of methanol‐free production, we cultivated the strain expressing RBD regulated under the native AOX1 promoter, and induced expression of the recombinant gene with varying amounts of methanol (Figure 1a ).…”

Section: Introductionmentioning

confidence: 99%

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

Dalvie

Biedermann

Rodriguez-Aponte

et al. 2021

Biotech & Bioengineering

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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.

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Minimal purification method enables developability assessment of recombinant proteins

Rodriguez-Aponte

Naranjo

Johnston

et al. 2023

Biotech & Bioengineering

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Analytical characterization of proteins is a critical task for developing therapeutics and subunit vaccine candidates. Assessing candidates with a battery of biophysical assays can inform the selection of one that exhibits properties consistent with a given target product profile (TPP). Such assessments, however, require several milligrams of purified protein, and ideal assessments of the physicochemical attributes of the proteins should not include unnatural modifications like peptide tags for purification. Here, we describe a fast two‐stage minimal purification process for recombinant proteins secreted by the yeast host Komagataella phaffii from a 20 mL culture supernatant. This method comprises a buffer exchange and filtration with a Q‐membrane filter and we demonstrate sufficient removal of key supernatant impurities including host‐cell proteins (HCPs) and DNA with yields of 1–2 mg and >60% purity. This degree of purity enables characterizing the resulting proteins using affinity binding, mass spectrometry, and differential scanning calorimetry. We first evaluated this method to purify an engineered SARS‐CoV‐2 subunit protein antigen and compared the purified protein to a conventional two‐step chromatographic process. We then applied this method to compare several SARS‐CoV‐2 RBD sequences. Finally, we show this simple process can be applied to a range of other proteins, including a single‐domain antibody, a rotavirus protein subunit, and a human growth hormone. This simple and fast developability methodology obviates the need for genetic tagging or full chromatographic development when assessing and comparing early‐stage protein therapeutics and vaccine candidates produced in K. phaffii.

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Engineered SARS-CoV-2 receptor binding domain improves manufacturability in yeast and immunogenicity in mice

Cited by 76 publications

References 79 publications

Scientific rationale for developing potent RBD-based vaccines targeting COVID-19

Scientific rationale for developing potent RBD-based vaccines targeting COVID-19

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

Minimal purification method enables developability assessment of recombinant proteins

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