Combo variants were produced of the best performing substitutions. The combo variants included two disulfide bonds, one disulfide bond and one proline variant, one disulfide bond with one hydrophobic residue, and one hydrophobic residue with one proline substitution. The best performing combo variant was named HexaPro and contained all four beneficial proline substitutions. The expression was increased 9x, the prefusion conformation was conserved, and the melting point increased by 5 degrees C. HexaPro could be used as a vaccine antigen or as a diagnostic reagent. HexaPro is easily produced by cell cultures, the binding ability to human ACE2 is comparable to the wild-type, and the prefusion conformation is maintained through freezing, thawing, and storage at room temperature. Further investigation is needed to determine why the presence of a two-RBD-up intermediate conformation was detected using cryo-EM.