基于重组人Ⅲ型胶原蛋白的三聚体抗原疫苗策略在新冠和流感疫苗中的应用

Applications of the recombinant human collagen type Ⅲ-based trimerization motif in the design of vaccines to fight against SARS-CoV-2 and influenza virus

新冠病毒等多种包膜病毒的糖蛋白在天然状态下呈现三聚体形式。三聚体蛋白通常较单体蛋白具有更优的免疫原性。目前使用非人蛋白来源的Foldon等三聚体基序可促使目的蛋白形成稳定三聚体,但这些基序的强免疫原性会削弱目的蛋白的免疫应答,限制了其在疫苗抗原设计中的应用。在本研究中,发现并利用重组人源化Ⅲ型胶原蛋白(Rh3C)作为新型三聚体基序,利用合成生物学方法,与新冠病毒刺头(spike)蛋白受体结合域(RBD)融合表达后促使RBD形成三聚体。这种胶原-RBD(Rh3C-RBD)三聚体蛋白在免疫小鼠后,较RBD单体蛋白诱导产生了更高滴度的结合抗体和中和抗体。此外,我们还进一步验证了该Rh3C基序与流感病毒HA1蛋白融合后,较HA1单体蛋白可在小鼠体内诱导更强效的抗体应答。因此,这种新型的Rh3C基序,有望广泛用于三聚体疫苗抗原的设计和优化。

Glycoproteins with enveloped viruses,such as severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),influenza virus,and human immunodeficiency virus(HIV),display a trimeric conformation.Different from the monomeric form,the trimeric proteins exhibit superior immunogenicity.Several trimerization motifs,such as Foldon derived from phage T4 fibritin,have been used to promote the formation of trimeric proteins with natural conformations.Although the Foldon-induced trimeric proteins are stable,their high immunogenicity limits applications in the development of vaccine antigens.In a previous study,we developed a recombinant human collagen type Ⅲ protein and determined its crystal structure,revealing a triple-helix conformation.However,the potential of this recombinant protein as a trimerization motif remained unknown.In this study,we demonstrated that the recombinant humanized type Ⅲ collagen(Rh3C)was able to act as a trimerization motif,facilitating the spontaneous trimer formation of the Rh3C-conjugated receptor-binding domain(RBD)within the spike(S)protein of SARS-CoV-2.This trimeric protein could induce a stronger SARS-CoV-2 RBD-specific IgG,IgG1,and IgG2a immune response,when compared with the monomeric RBD protein in the immunized mice.Notably,the Rh3C-RBD protein,when adjuvanted with the novel STING agonist CF501,also elicited significantly higher neutralizing antibody responses against both the pseudotyped SARS-CoV-2(D614G)and its variant Omicron(BA.2.2)in the immunized mice.To showcase the broad applications of the Rh3C trimerization motif,we further demonstrated that the Rh3C-conjugated HA1 of the influenza virus could also elicit a stronger antibody response than free HA1.Considering the wide distribution of the Rh3C protein in human bodies,its use as a trimerization motif would not induce an immune response due to immune tolerance,thereby allowing the immune response to concentrate on targeted viral proteins.Therefore,this Rh3C-based trimerization motif holds great potential for the design and optimization of vaccines consisting of trimeric protein antigens.