key: cord-0293567-iljksx2f
authors: Limonta-Fernandez, M.; Chinea-Santiago, G.; Martin-Dunn, A. M.; Gonzalez-Roche, D.; Bequet-Romero, M.; Marquez-Perera, G.; Gonzalez-Moya, I.; Canaan-Haden-Ayala, C.; Cabrales-Rico, A.; Espinosa-Rodriguez, L. A.; Ramos-Gomez, Y.; Andujar-Martinez, I.; Gonzalez-Lopez, L. J.; Perez de la Iglesia, M.; Zamora-Sanchez, J.; Cruz-Sui, O.; Lemos-Perez, G.; Cabrera-Herrera, G.; Valdes-Hernandez, J.; Martinez-Diaz, E.; Pimentel-Vazquez, E.; Ayala-Avila, M.; Guillen-Nieto, G.
title: The SARS-CoV-2 receptor-binding domain expressed in Pichia pastoris as a candidate vaccine antigen
date: 2021-07-03
journal: nan
DOI: 10.1101/2021.06.29.21259605
sha: d78dbb8649b80b35d3a3b82e9d48764eab6dedba
doc_id: 293567
cord_uid: iljksx2f

The effort to develop vaccines based on economically accessible technological platforms available by developing countries vaccine manufacturers is essential to extend the immunization to the whole world population and to achieve the desired herd immunity, necessary to end the COVID-19 pandemic. Here we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed in yeast Pichia pastoris. The RBD was modified with the addition of flexible N- and C-terminal amino acid extensions aimed to modulate the protein/protein interactions and facilitate protein purification. Fermentation with yeast extract culture medium yielded 30-40 mg/L. After purification by immobilized metal ion affinity chromatography and hydrophobic interaction chromatography, the RBD protein was characterized by mass-spectrometry, circular dichroism, and binding affinity to angiotensin-converting enzyme 2 (ACE2) receptor. The recombinant protein shows high antigenicity with convalescent human sera and also with sera from individuals vaccinated with the Pfizer-BioNTech mRNA or Sputnik V adenoviral-based vaccines. The RBD protein stimulates IFN{gamma}, IL-2, IL-6, IL-4, and TNF in mice secreting splenocytes from PBMC and lung, CD3+ enriched cells. Immunogenicity studies with 50 g of the recombinant RBD formulated with alum, induce high levels of binding antibodies in mice and non-human primates, assessed by ELISA plates covered with RBD protein expressed in HEK293T cells. The mouse sera inhibited the RBD binding to ACE2 receptor in an in-vitro test and show neutralization of SARS-CoV-2 infection of Vero E6 cells. These data suggest that the RBD recombinant protein expressed in yeast P. pastoris is suitable as a vaccine candidate against COVID-19.

previously [16] . 220

The interaction between mFc-ACE2 fusion protein and the recombinant C-RBD-H6 PP 222 was monitored by SPR using a BIACORE X (GE Health-care) at 25 C in a multi-cycle 223 mode. Briefly, mFc-ACE2 was immobilized on a Protein A biosensor chip (GE Health-224 care) according to the manufacturer's protocol through the flow cell 1 (FC1). The FC2 225 was used as the reference cell. The real-time response of the C-RBD-H6 PP over the 226 immobilized mFc-ACE2 was recorded by duplicate in a concentration range from 15 to 227 2000 nM, at 10 µL/min flow rate for 120 s, while the dissociation took place for another 228 120 s. The running buffer was PBS (pH = 7.2). After each cycle the chip was 229 regenerated using pH = 2.0 glycine buffer. The equilibrium dissociation constant 230 (binding affinity, K D ) was estimated with the BIAevaluation® software (GE Healthcare) 231 using the Langmuir 1:1 interaction model. At least five curves were taken into account 232 for kinetics calculations. 233

CD spectra were acquired in a Jasco J-1500 CD spectrometer (Jasco, Japan). All 235 measurements were carried out at 24 C, the far UV CD spectra were studied at 100 236 µg/ml protein concentration (a 10 fold dilution in water of the stock solution) using a 237 1mm quartz cuvette. The near UV CD spectra were studied at the protein concentration 238

of the stock solution in 20 mM pH 7.4 Tris buffer using a quartz cuvette of 10 mm path 239 length. The spectra of the corresponding solution were subtracted. The far UV CD 240 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.29.21259605 doi: medRxiv preprint structure content of the protein and the results were compared with the values derived 242 from the 3D coordinates of the crystallographic structure of the spike protein of SARS-243

CoV-2 (PDB file 6yla) using DSSP method [23] implemented in Whatif program package 244 [24] . 245

Three different animal species were used for evaluation of immunogenicity of the C-247 RBD-H6 PP protein: BALB/c mice, Sprague-Dawley (SD) rats, and African green 248 monkeys (Chlorocebus aethiops sabaeus). Six-to eight-week-old female BALB/c mice, 249 and male and female SD rats were used for the study and housed in the animal facility. 250

The experimental protocols were approved by the Ethical Committee on Animal 251

Experimentation of the Center for Genetic Engineering and Biotechnology (CIGB, 252

Havana, Cuba) and the Center for Production of Laboratory Animals (CENPALAB, 253

Bejucal, Cuba). 254

The immunogen content per 500 µL: 50 µg of C-RBD-H6 PP protein adjuvated with 0.3 255 mg of aluminum hydroxide gel (Alhydrogel ®) in phosphate buffer (0.28 mg of disodium 256 hydrogen phosphate, 0.31 mg of sodium dihydrogen phosphate dihydrate, 4.25 mg of 257 sodium chloride). 258 BALB/c mice: Immunogenicity in mice was evaluated using a three-dose schedule with 259 a 50 µg dose by intraperitoneal route with a 7 and 14 days interval before the second 260 and third doses respectively. Blood was collected a week after the first boost and 7 and 261 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.29.21259605 doi: medRxiv preprint rats (10 male and 10 female) during chronic toxicology study, with a 9 µg dose 265 administered by the intramuscular route once a week, for 10 consecutive weeks for a 266 total of 90 µg. Animals were bled three days after the last dose. 267

Chlorocebus aethiops sabaeus non-human primates: NHP ages between 3 to 6 years 268 and with 2-7 kg of weight were kept at the animal's facility at the CENPALAB. A total of 269 20 NHP were randomly assigned to 3 groups including the placebo (2 animals/gender, 270 total 4), the low dose (50 µg, 3 animals/gender, total 6), and the high dose (100 µg, 5 271 animals/gender, total 10). Seven days post-1st and 2nd boosting and after overnight 272 fasting, monkeys were sedated by intramuscular injection of ketamine hydrochloride (10 273 mg/kg) and bled from the femoral vein. 274

Specific anti-RBD titers and the inhibition of its interaction with ACE2 receptor were 275 evaluated using ELISA. Live SARS-CoV-2 neutralization was assessed using a 276 microneutralization assay. 277

Antibody detection by ELISA 279

The reactivity of sera from immunized animals was determined by ELISA. Briefly, 0.25 280 µg of RBD protein produced in HEK293T cells (Center for Molecular Immunology, 281

Havana) was used to coat 96-well microtiter plates (Corning Costar, Acton, MA) in 0.1 282 M sodium carbonate buffer (pH 9.6) at 4 °C overnight. After the plates were blocked 283 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 3, 2021. human IgG (1:10000, Jackson, USA) at 37 °C for 1 h, followed by washes. The reaction 290 was detected after the addition of 3,3-5,5-tetramethylbenzidine and quantified using a 291 microplate reader at 450 nm (BMG Labtech, Germany). This assay was also used for 292 the evaluation of RBD antigenicity using sera from immunized animals, and from 293 subjects that received Pfizer-BioNTech or Sputnik V vaccines, or are COVID-19 294 convalescents. 295 RBD to ACE2 plate-based binding assay 296 A competitive ELISA was performed to determine the inhibitory activity of the anti-RBD 297 polyclonal sera on the binding of the hFc-ACE2 coated plates to an hFc-RBD-HRP 298 conjugate. Briefly, the wells of ELISA plates were coated with 0.25 µg of recombinant 299 hFc-AEC2 as described above. A mixture containing an hFc-RBD-HRP conjugate and 300 serial dilutions of the sera were pre-incubated for 1h at 37 °C. A hundred microliters of 301 the mixture were added to hFc-ACE2 coated plates and further incubated for 90 min at 302 37 °C. The binding of the HRP tagged RBD to the receptor was detected after the 303 addition of 3,3-5,5-tetramethylbenzidine and reading at 450 nm. A similar assay was 304 used to characterize the ability of the C-RBD-H6 PP and C-RBD-H6 HEK proteins to 305 block the interaction of hFc-RBD-HRP with coated hFc-ACE2. 306 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 3, 2021. 

Long-term cellular immune response was evaluated in BALB/c mice using 325 subcutaneous administration of a formulation containing an equal antigen to alum ratio. 326

Animals receive 25 µg of the C-RBD-H6 PP antigen by the subcutaneous route in a 100 327 µL volume in a 0-14-35 days schedule. Blood samples were evaluated two weeks after 328 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 3, 2021. For the re-stimulation assays, splenocyte or lung selected CD3+ suspensions were 339 diluted to 10x10 6 CD3+ live cells/mL and 50 µL of each sample was seeded in two 96-a high accessible hexa-Histidine tag (His 232 -His 237 ). Topologically, the extensions are 372 located in the opposite site of the protein respect to the receptor binding motif and its 373 presence should sterically hinder potential aggregation problems associated to the 374 presence of the exposed and disulfide bonded Cys 76 and Cys 210 . (The C-RBD-H6 PP 375 protein sequence is included as a supplemental material S1). 376

A construct for the expression in P. pastoris of the RBD from SARS-CoV-2 under 378 control of the AOX1 promoter, denominated pPICZα-CtagRBDH6, was prepared as 379 described in Materials and Methods and used to obtain RBD-expressing yeast clones. 380 Based on signal intensity in Western blotting, clone C was denominated X33-23 and 390 selected for further work (Fig. 1) . The C-RBD-H6 PP protein was purified from the 391 supernatant following the procedures described in Materials and Methods, by IMAC, 392 followed by polishing using a RP to obtain a final preparation with high purity. After 393 purification, the C-RBD-H6 PP was obtained with a final yield between 30-40 mg/L of 394 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 3, 2021. Table) . The four disulfide bonds (C 336 -C 361 , 416 C 379 -C 432 , C 391 -C 525 and C 480 -C 488 ) present in the native S protein of SARS-CoV-2 were 417 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.29.21259605 doi: medRxiv preprint peptides confirmed that N-glycans in its structure increased considerably its molecular 421 mass by SDS-PAGE analysis. Table 2 the secondary structure content 437 of the protein estimated by BeStSel (7.9% helix, 28.7% beta -antiparallel, relaxed and 438 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.29.21259605 doi: medRxiv preprint protein. The IgG antibody response in mice at days 21 and 35 shows that the C-RBD-505 H6 PP protein was able to induce antibody responses in ELISA coated with the RBD 506 protein produced in mammalian HEK293T cells. Seroconversion was achieved a week 507 after the first boost (Fig. 10.A) . The administration of a second booster at day 21 508 significantly increases both RBD-specific IgG titers and the inhibitory potential of the 509 sera to reduce the protein binding to its cognate receptor ACE2 a fact that correlates 510 with live SARS-CoV-2 neutralization (Fig. 10.B,C) . 511

The C-RBD-H6 PP protein was also tested in SD rats using intramuscular 512 administration of 9 µg every 7 days for 10 weeks. Assessment of IgG and RBD-ACE2 513 receptor binding inhibition three days after the last immunization indicates high RBD-514 specific antibody titers that correlate with the inhibitory titer. The former also display a 515 high correlation with the neutralization titer of live SARS-CoV-2 virus in Vero E6 cells 516 ( Fig. 10.D,F) . 517

The C-RBD-H6 PP protein evaluation in NHP using short intramuscular administration 518 schedule on days 0-14-28, and two dose levels indicate a dose-response effect with 519 seroconversion of 83 % (5 of 6 animals) and 100% (the 10 animals) for the 50 µg and 520 100 µg dose respectively after the first booster and a 100 % seroconversion in both 521 schedules after the second booster. Total IgG titers increased to 44,240 StdU/mL and 522 62,435 StdU/mL respectively for monkeys included in the low and high-dose groups. In 523 both cases, the titers were significantly higher than those detected in the convalescent 524 panel of sera. The geometric median ACE2 binding inhibition titer for 50 µg dose was 525 1:230, while a significantly higher value of 1:705 was detected for the animals receiving 526 the 100 µg dose. Both values were higher than those detected in a panel of COVID-19 527 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.29.21259605 doi: medRxiv preprint correlate with and enhancement in the inhibition of RBD-ACE2 binding for this group. 529

The analysis of the neutralization titer of live SARS-CoV-2 virus in Vero E6 cells 530 corroborates these findings, indicating that 50µg dose is sufficient to induce ACE2 531 binding inhibition titers with an EC50 geometric mean of 1:66 serum dilution, a value 6 532 times higher than the reported for the convalescent panel. extend TNFα and IL-4 (Fig. 11 ). Our findings in the systemic compartment (Fig. 11 .A) 547 were similar to those found for CD3+ cells enriched from mice lungs (Fig. 11.B) , 548

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 3, 2021. Optimal conditions for the production of a recombinant protein in P. pastoris expression 566 system differ according to the target protein. Indeed, we were able to obtain 30-40 mg/L 567 of the RBD with more than 98% of purity, close to the yield obtained in previous report 568 by Arbeitman C.R., et al [8] , an essential condition to develop a vaccine candidate. In 569 addition to the protein yield, it is important the sugar composition. In P. pastoris the 570 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 3, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.29.21259605 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 3, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 3, 2021. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 3, 2021. 

Nt: N-terminal end, Ct-His 6 : His-tag C-terminal end. C # -C # corresponds to tryptic 733 peptides linked either by intermolecular disulfide bonds or a tryptic peptide that contains 734 an intramolecular disulfide bond in its structures. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.29.21259605 doi: medRxiv preprint

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