Plant‐based, adjuvant‐free, potent multivalent vaccines for avian influenza virus via Lactococcus surface display

Abstract

Influenza epidemics frequently and unpredictably break out all over the world, and seriously affect the breeding industry and human activity. Inactivated and live attenuated viruses have been used as protective vaccines but exhibit high risks for biosafety. Subunit vaccines enjoy high biosafety and specificity but have a few weak points compared to inactivated virus or live attenuated virus vaccines, especially in low immunogenicity. In this study, we developed a new subunit vaccine platform for a potent, adjuvant-free, and multivalent vaccination. The ectodomains of hemagglutinins (HAs) of influenza viruses were expressed in plants as trimers (tHAs) to mimic their native forms. tHAs in plant extracts were directly used without purification for binding to inactivated Lactococcus (iLact) to produce iLact-tHAs, an antigen-carrying bacteria-like particle (BLP). tHAs BLP showed strong immune responses in mice and chickens without adjuvants. Moreover, simultaneous injection of two different antigens by two different formulas, tHA H5N6 + H9N2 BLP or a combination of tHA H5N6 BLP and tHA H9N2 BLP, led to strong immune responses to both antigens. Based on these results, we propose combinations of plant-based antigen production and BLP-based delivery as a highly potent and cost-effective platform for multivalent vaccination for subunit vaccines.

The B1 Domain of Streptococcal Protein G Serves as a Multi-Functional Tag for Recombinant Protein Production in Plants

“…GFP is well-known to have good solubility and expression in plants. To further test the functionality of the GB1 domain in increasing the expression levels of recombinant proteins, we selected two proteins, human interleukin 6 (hIL-6) ( Islam et al, 2019 ) and hemagglutinin (HA) of H9N2 ( Song et al, 2021 ). The recombinant constructs, BiP-MP-CBM3-SUMO-hIL6-HDEL (hIL6 in short) and BiP-HA H 9 N 2 -mCor1-LysM-His-HDEL (HA H 9 N 2 in short) were tested in N .…”

“…Both two recombinant proteins have been quantified in the previous studies. The yield of purified hIL6 was approximately 18.5 μg/g FW leaf tissues at near homogeneity ( Islam et al, 2019 ), and the expression level of trimeric HA H 9 N 2 was 150 μg/g FW leaf tissues ( Song et al, 2021 ). We estimated that the expression level of GB1-hIL6 and GB1-HA H 9 N 2 is approximately 23.7 μg/g FW and 222 μg/g fresh weight, respectively, according to the relative ratio with hIL6 and HA H 9 N 2 .…”

“…The B1 domain (amino acid positions from 1st to 56th) of Streptococcal protein G (GB1) was inserted after the ER leader sequence or RbcS tp of BiP-EK-GFP or RbcS tp -EK-GFP to yield BiP-GB1-EK-GFP-HDEL, or C-terminally fused to BiP-EK-GFP to yield BiP-EK-GFP-GB1. The DNA fragments encoding hIL6 and HA H 9 N 2 were prepared from recombinant constructs BiP-MP-CBM3-SUMO-hIL6-HDEL and BiP-HA H 9 N 2 - mCor1-LysM-His-HDEL , respectively ( Islam et al, 2019 ; Song et al, 2021 ), by digesting with Bam HI and Xho I, and ligated into BiP-GB1-EK-GFP-HDEL digested with Bam HI and Xho I to yield BiP-GB1- MP-CBM3-SUMO-hIL6-HDEL and BiP-GB1-HA H 9 N 2 - mCor1-LysM-His-HDEL , respectively. CTB with Bam HI and Xho I restriction sites at N- and C-terminal ends, respectively, was chemically synthesized (Gene Universal, Inc., Newark, United States).…”

“…CTB with Bam HI and Xho I restriction sites at N- and C-terminal ends, respectively, was chemically synthesized (Gene Universal, Inc., Newark, United States). All the constructs were placed under the MacT promoter ( Song et al, 2021 ) and accompanied by RD29B Terminator of Arabidopsis RD29b . Primers used in this study are shown in Supplementary Table 1 .…”

Production of Recombinant Active Human TGFβ1 in Nicotiana benthamiana

“…TGF β 1 was amplified with PCR from LAPTGF β 1 using Bam HI_6xHis_TGFβ1 forward primer and Xho I_LAP_TG_R2 reverse primer ( Supplementary Table 1 ). The PCR product was digested by Bam HI and Xho I restriction endonucleases and ligated to pTEX1-BiP:HA:mCor1:LysM:His:HDEL ( Song et al, 2021 ) to generate pTEX1-BiP-6xHis-TGF β 1 . The M domain and CBM3 were amplified with PCR from 1300-BMC-SazCA ( Kumari et al, 2020 ).…”