The COVID-19 pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. Here, we characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting ~10-fold higher expression than its parental construct and the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A 3.2 Å-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.

1The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated 2 efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health 3 emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion 4 protein that is metastable and difficult to produce recombinantly in large quantities. Here, we 5 designed and expressed over 100 structure-guided spike variants based upon a previously 6 determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical 7 and structural characterization of these variants identified numerous individual substitutions that 8 increased protein yields and stability. The best variant, HexaPro, has six beneficial proline 9 substitutions leading to ~10-fold higher expression than its parental construct and is able to 10 withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 Å-resolution 11 cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-12 yield production of a stabilized prefusion spike protein will accelerate the development of 13 vaccines and serological diagnostics for SARS-CoV-2. 14 3 INTRODUCTION 15 Coronaviruses are enveloped viruses containing positive-sense RNA genomes. Four human 16 coronaviruses generally cause mild respiratory illness and circulate annually. However, SARS-17 CoV and MERS-CoV were acquired by humans via zoonotic transmission and caused outbreaks 18 of severe respiratory infections with high case-fatality rates in 2002 and 2012, respectively 1,2 . 19 SARS-CoV-2 is a novel betacoronavirus that emerged in Wuhan, China in December 2019 and 20 is the causative agent of the ongoing COVID-19 pandemic 3,4 . As of May 26, 2020, the WHO has 21 reported over 5 million cases and 350,000 deaths worldwide. Effective vaccines, therapeutic 22 antibodies and small-molecule inhibitors are urgently needed, and the development of these 23 interventions is proceeding rapidly. 24 Coronavirus virions are decorated with a spike (S) glycoprotein that binds to host-cell 25 receptors and mediates cell entry via fusion of the host and viral membranes 5 . S proteins are 26 trimeric class I fusion proteins that are expressed as a single polypeptide that is subsequently 27cleaved into S1 and S2 subunits by cellular proteases 6,7 . The S1 subunit contains the receptor-28 binding domain (RBD), which, in the case of SARS-CoV-2, recognizes the angiotensin-29 converting enzyme 2 (ACE2) receptor on the host-cell surface [8][9][10] . The S2 subunit mediates 30 membrane fusion and contains an additional protease cleavage site, referred to as S2′, that is 31 adjacent to a hydrophobic fusion peptide. Binding of the RBD to ACE2 triggers S1 dissociation, 32 allowing for a large rearrangement of S2 as it transitions from a metastable prefusion 33 conformation to a highly stable postfusion conformation 6,11 . During this rearrangement, the 34 fusion peptide is inserted into the host-cell membrane after cleavage at S2′, and two h...

is the primary causative agent of pertussis (whooping cough), which is a respiratory infection that leads to a violent cough and can be fatal in infants. There is a need to develop more effective vaccines because of the resurgence of cases of pertussis in the United States since the switch from the whole-cell pertussis vaccines (wP) to the acellular pertussis vaccines (aP; diphtheria-tetanus-acellular-pertussis vaccine/tetanus-diphtheria-pertussis vaccine). Adenylate cyclase toxin (ACT) is a major virulence factor of that is (i) required for establishment of infection, (ii) an effective immunogen, and (iii) a protective antigen. The C-terminal repeats-in-toxin domain (RTX) of ACT is sufficient to induce production of toxin-neutralizing antibodies. In this study, we characterized the effectiveness of vaccines containing the RTX antigen against experimental murine infection with RTX was not protective as a single-antigen vaccine against challenge, and adding RTX to 1/5 human dose of aP did not enhance protection. Since the doses of aP used in murine studies are not proportionate to mouse/human body masses, we titrated the aP from 1/20 to 1/160 of the human dose. Mice receiving 1/80 human aP dose had bacterial burden comparable to those of naive controls. Adding RTX antigen to the 1/80 aP base resulted in enhanced bacterial clearance. Inclusion of RTX induced production of antibodies recognizing RTX, enhanced production of anti-pertussis toxin, decreased secretion of proinflammatory cytokines, such as interleukin-6, and decreased recruitment of total macrophages in the lung. This study shows that adding RTX antigen to an appropriate dose of aP can enhance protection against challenge in mice.

The severe acute respiratory syndrome coronavirus 2 spike protein is a critical component of coronavirus disease 2019 vaccines and diagnostics and is also a therapeutic target. However, the spike protein is difficult to produce recombinantly because it is a large trimeric class I fusion membrane protein that is metastable and heavily glycosylated. We recently developed a prefusion-stabilized spike variant, termed HexaPro for six stabilizing proline substitutions, that can be expressed with a yield of >30 mg/L in ExpiCHO cells. This protocol describes an optimized workflow for expressing and biophysically characterizing rationally engineered spike proteins in Freestyle 293 and ExpiCHO cell lines. Although we focus on HexaPro, this protocol has been used to purify over a hundred different spike variants in our laboratories. We also provide guidance on expression quality control, long-term storage, and uses in enzyme-linked immunosorbent assays. The entire protocol, from transfection to biophysical characterization, can be completed in 7 d by researchers with basic tissue cell culture and protein purification expertise.

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To address the ongoing SARS-CoV-2 pandemic and prepare for future coronavirus outbreaks, understanding the protective potential of epitopes conserved across SARS-CoV-2 variants and coronavirus lineages is essential. We describe a highly conserved, conformational S2 domain epitope present only in the prefusion core of β-coronaviruses: SARS-CoV-2 S2 apex residues 980–1006 in the flexible hinge. Antibody RAY53 binds the native hinge in MERS-CoV and SARS-CoV-2 spikes on the surface of mammalian cells and mediates antibody-dependent cellular phagocytosis and cytotoxicity against SARS-CoV-2 spike in vitro. Hinge epitope mutations that ablate antibody binding compromise pseudovirus infectivity, but changes elsewhere that affect spike opening dynamics, including those found in Omicron BA.1, occlude the epitope and may evade pre-existing serum antibodies targeting the S2 core. This work defines a third class of S2 antibody while providing insights into the potency and limitations of S2 core epitope targeting.

Understanding protein stability is central to combatting protein aggregation diseases and developing new protein therapeutics. At the high concentrations often present in biological systems, purified proteins can exhibit undesirable high solution viscosities and poor solubility mediated by short-range electrostatic and hydrophobic protein-protein interactions. The interplay between protein amino acid sequence, protein structure and solvent conditions to minimize protein-protein interactions is key to design of well-behaved pharmaceutical proteins. However, theoretical approaches have yet to yield a general framework to address these problems. Here, we analyzed the high concentration behavior of super-folder GFP (sfGFP) and two super-charged sfGFP variants engineered to have formal charges of −18 or +15. Under low cosolute conditions, sfGFP and the −18 variant formed a gel or phase separated at ~10 mg/ml. Under conditions that screen surface charges, including formulations with high histidine or high NaCl concentrations, all three variants attained concentrations up to 250 mg/ml with moderate viscosities. Moreover, all three variants exhibited very similar viscosity-concentration profiles over this range. This effect was not mimicked by high sugar concentrations that exert excluded volume effects without shielding charge. Collectively, these data demonstrate that charge shielding not only neutralizes long-range electrostatic interactions but also, surprisingly, short-range electrostatic effects due to surface charge anisotropy. This work shows that supercharged sfGFP behavior under high ionic strength is largely determined by particle geometry, a conclusion that is supported by colloid models and may be applicable to pharmaceutically-relevant proteins.

Pertussis continues to cause considerable infant mortality world-wide, which could be addressed in part by passive immunization strategies. Antibody hu1B7 is a candidate therapeutic that potently neutralizes pertussis toxin in vitro, prevents leukocytosis in mice and treats established disease in weanling baboons as part of an antibody cocktail. Here, we evaluated the potential for hu1B7 and an extended half-life hu1B7 variant to prevent death, leukocytosis and other clinical symptoms in a newborn baboon model that mimics many aspects of human disease. We administered a single antibody dose to newborn baboons five weeks prior to experimental infection. While all animals were heavily colonized with Bordetella pertussis, prophylaxed animals showed significantly greater survival (P < 0.005), delayed and suppressed leukocytosis (P < 0.01) and enhanced clinical outcomes, including coughing (P < 0.01), as compared to controls. Together, this work demonstrates that a single neutralizing anti-PTx antibody is sufficient to prevent clinical pertussis symptoms.