Qs 21

SARS-CoV-2 different recombinant S and receptor proteins were obtained from Sino Biologicals (Wayne, PA, USA): Full-length S (S1–S2) ectodomain amino acid (aa) residues 16-1213 (40589-V08B1, 134.36 kDa, expressed in baculovirus-insect cells), S1 subunit (aa 16-685) with RBD domain (40591-V08H, 76.5 kDa, expressed in HEK293 cells); S2 subunit (aa 686-1213) with fusion domain (40589-V08B1, 59.36 kDa, expressed in baculovirus-insect cells); human angiotensin-converting enzyme 2 (hACE2) protein (aa 1-740) fused to Fc tag (10108-H02H, expressed in HEK293 cells). Heat-inactivated (65 °C, 30 min; NR-52286) and gamma-irradiated SARS-CoV-2 (NR-52287), and human embryonic kidney cells expressing hACE2 (HEK-293T-hACE2) were provided from BEI/ATCC resources. The SARS-CoV-2 spike pseudoviral particles were purchased from eEnzyme (Gaithersburg, MD). The monophosphoryl lipid A (MPL) and the saponin QS-21 were purchased from Sigma-Aldrich and Desert King, respectively, and used as AS01-like combined vaccine adjuvant (MPL 1 µg + QS-21 10 µg). AS01 (QS-21 + MPL) liposome adjuvant is licensed and included in herpes Zoster vaccination [24 (link),25 (link),26 (link),27 (link)].

BALB/c mice (Female, 6 to 8 weeks old) were intramuscularly (i.m.) immunized in a prime-boost schedule (3-week interval) in the hind legs. M2e-H3 stalk proteins (5–20 μg) were used for prime with adjuvants [10 μg QS-21 (cat no: 34-6-07-2, Desert King International) plus 1 μg monophosphoryl lipid A (MPL, cat no: L6895, Sigma Aldrich)] and boost with half dose adjuvant (5 μg QS-21 + 0.5 μg MPL). For aged mice, 16-month-old BALB/c mice were i.m. immunized. Blood samples were collected after 2 weeks of prime and boost immunization. Four to eight weeks after boost, mice were challenged intranasally with a lethal dose of influenza A viruses. Weight loss of >20% was considered as the IACUC endpoint. Group 1 influenza A viruses were as follows: A/Puerto Rico/8/1934 H1N1 (A/PR8/H1N1), A/California/04/2009 H1N1 (A/Cal/H1N1), A/WSN/1933 H1N1 (A/WSN/H1N1), mouse-adapted A/Fort Monmouth/1/1947 H1N1 (A/FM/H1N1), and reverse genetic (rg) reassortant A/Vietnam/1203/2004 H5N1 with A/PR8 backbone (A/Viet/H5N1), and A/Hong Kong/1073/1999 H9N2 (A/HK/H9N2). Group 2 viruses used include A/Philippine/2/1982 H3N2 (A/Phil/H3N2), A/Hong Kong/1/1968 H3N2 (A/HK/H3N2), reassortants A/Shanghai/11/2013 H7N9 with A/PR8 backbone (A/Sha/H7N9), and A/Nanchang/933/1995 H3N2 with A/PR8 backbone (A/Nanchang/H3N2).