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

doi:10.12211/2096-8280.2023-058

合成生物学 ›› 2024, Vol. 5 ›› Issue (2): 385-395.DOI: 10.12211/2096-8280.2023-058

• 研究论文 • 上一篇

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

刘泽众¹^,², 周洁¹, 朱赟³, 陆路¹, 姜世勃¹

^1.复旦大学基础医学院，上海 200032
^2.复旦大学药学院药理系，上海 201203
^3.中国科学院生物物理研究所，北京 100101

收稿日期:2023-08-19 修回日期:2023-11-07 出版日期:2024-04-30 发布日期:2024-04-28
通讯作者: 陆路,姜世勃
作者简介:刘泽众（1990—），男，青年研究员，博士生导师。研究方向为微生物学。E-mail：zezhongliu@fudan.edu.cn
陆路（1982—），男，研究员，博士生导师。研究方向为微生物学。E-mail：lul@fudan.edu.cn
姜世勃（1953—），男，教授，博士生导师。研究方向为微生物学。E-mail：shibojiang@fudan.edu.cn
基金资助:
国家重点研发计划(2022YFC2305800);国家自然科学基金(92169112)

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

Zezhong LIU¹^,², Jie ZHOU¹, Yun ZHU³, Lu LU¹, Shibo JIANG¹

^1.School of Basic Medical Sciences，Fudan University，Shanghai 200032，China
^2.Department of Pharmacology，School of Pharmacy，Fudan University，Shanghai 201203
^3.Institute of Biophysics，Chinese Academy of Sciences，Beijing 100101，China

Received:2023-08-19 Revised:2023-11-07 Online:2024-04-30 Published:2024-04-28
Contact: Lu LU, Shibo JIANG

摘要/Abstract

摘要：

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。

关键词: 三聚体抗原, 人胶原蛋白, 新冠病毒, 流感病毒, 疫苗设计

Abstract:

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. {L-End}

Key words: trimeric antigen, humanized collagen, SARS-CoV-2, influenza virus, vaccine design

中图分类号:

R373

刘泽众, 周洁, 朱赟, 陆路, 姜世勃. 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[J]. 合成生物学, 2024, 5(2): 385-395.

Zezhong LIU, Jie ZHOU, Yun ZHU, Lu LU, Shibo JIANG. 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[J]. Synthetic Biology Journal, 2024, 5(2): 385-395.

图/表 4

图1 基于Rh3C连接的SARS-CoV-2 RBD融合蛋白作为三聚体疫苗抗原的设计、构建和鉴定［（a）Ⅲ型胶原蛋白Rh3C同SARS-CoV-2 RBD融合蛋白的设计。（b）利用AlphaFold2预测的Rh3C-RBD三聚体结构。整个结构由多次的分段预测结果拼接而成。放大的图分别展示了串联重复的胶原三螺旋基序结构和RBD三聚体结构。（c）Ⅲ型人源化胶原蛋白Rh3C同RBD融合表达后的三聚体结构模式图。（d）Ⅲ型人源化胶原蛋白Rh3C同RBD融合表达后的SDS-PAGE图片。（e）凝胶过滤色谱分析Rh3C-RBD分子量。（f）粒径分析Rh3C-RBD融合蛋白获得蛋白分子量］

Fig. 1 Design, construction and identification of the Rh3C-conjugated RBD in the S protein of SARS-CoV-2 as a trimeric vaccine antigen[(a) Schematic representation of the design of Rh3C-conjugated SARS-CoV-2 RBD (Rh3C-RBD) as a trimeric vaccine antigen.(b) Predicted trimeric structure of Rh3C-RBD using AlphaFold2. The entire structure is assembled from multiple segment predictions. The magnified images show the triple-helix motifs of the collagen tandem repeat and the trimeric conformation of RBD. (c) Schematic illustration of the Rh3C-RBD structure. (d) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis for the visualization of the Rh3C-RBD protein. (e) Representative elution chromatograph of the Rh3C-RBD protein using a calibrated Superose 6 Increase 10/300 column. (f) Determination of the molecular weight of Rh3C-RBD through the particle size analysis.]

图2 Rh3C-RBD和RBD免疫小鼠后体液免疫应答的评价［（a）小鼠的免疫程序，分别在第0和14天进行免疫，在第21天收集小鼠血清；（b）免疫两次后第7天检测小鼠血清中针对RBD特异性的IgG、IgG1和IgG2a抗体；（c）RBD特异性的IgG、IgG1和IgG2a抗体滴度。One-way ANOWA用于分析统计学差异。***P<0.0001。每组小鼠n=6］

Fig. 2 Evaluation of the humoral immunity induced by Rh3C-RBD and RBD in the mice[(a) Overview of the immunization protocol. Mice were vaccinated at day 0 and day 14, with serum collected at day 21; (b) Quantification of the SARS-CoV-2 RBD-specific IgG, IgG1, and IgG2a in different serum dilutions from day 21 using the enzyme-linked immunosorbent assay (ELISA); (c) Determination of the RBD-specific IgG, IgG1, and IgG2a titers in sera collected at day 21. Statistical analysis was conducted using one-way ANOWA. *** indicating the statistical significance at P<0.0001.]

图3 免疫后小鼠血清中针对SARS-CoV-2假病毒中和抗体的评价（每组小鼠n=6）［（a）以CF501为佐剂，Rh3C-RBD和RBD免疫小鼠后，小鼠血清针对SARS-CoV-2（D614G）假病毒中和抗体滴度。（b）以Alum为佐剂，Rh3C-RBD和RBD免疫小鼠后，小鼠血清针对SARS-CoV-2（D614G）假病毒中和抗体的评价。（c）以CF501为佐剂，Rh3C-RBD和RBD免疫小鼠后，小鼠血清针对SARS-CoV-2（BA.2.2）假病毒中和抗体滴度。（d）以Alum为佐剂，Rh3C-RBD和RBD免疫小鼠后，小鼠血清针对SARS-CoV-2（BA.2.2）假病毒中和抗体的评价。t检验用于分析统计学差异。**P<0.001］

Fig. 3 Evaluation of the neutralizing antibodies against the pseudovirus SARS-CoV-2 (D614G) in the sera of immunized mice[Assessment of the neutralizing antibody titers against SARS-CoV-2 (D614G) in the sera of mice immunized with CF501/RBD or CF501/Rh3C-RBD (a), Alum/RBD or Alum/Rh3C-RBD (b), CF501/RBD or CF501/Rh3C-RBD (c), and Alum/RBD or Alum/Rh3C-RBD (d). Statistical analysis was performed using the Student t test. ** indicating the statistical significance at P<0.001.]

图4 基于Rh3C连接的H1N1 HA1融合蛋白作为三聚体流感疫苗抗原的设计构建及其免疫原性评价［（a）基于Rh3C连接的H1N1 HA1重组融合蛋白（Rh3C-HA1）的设计。（b）SDS-PAGE图片验证Rh3C-HA1蛋白的表达。（c）小鼠的免疫程序。（d）以CF501为佐剂免疫小鼠后血清中针对HA1特异性的IgG抗体水平。（e）以Alum为佐剂免疫小鼠后血清中针对HA1特异性的IgG抗体水平。利用Two-way ANOWA进行统计学分析。***P<0.0001，*P<0.05］

Fig.4 Design and construction of the Rh3C-conjugated influenza virus HA1 (Rh3C-HA1) and evaluation of its immunogenicity[(a) Design of the Rh3C-conjugated influenza virus HA1 (Rh3C-HA1); (b) SDS-PAGE analysis for the expressed Rh3C-HA1 protein; (c) Overview of the immunization protocol. Mice were vaccinated at day 0 and day 14, with serum collection at day 21; (d and e) ELISA analyses of the HA1-specific IgG in the serum collected at day 21 from mice immunized with CF501/RBD or CF501/Rh3C-RBD and Alum/RBD or Alum/Rh3C-RBD. Statistical analysis was performed using the two-way ANOWA. *** and * indicating the statistical significance at P<0.0001 and P<0.05, respectively.]

参考文献 33

1	DU L Y, HE Y X, ZHOU Y S, et al. The spike protein of SARS-CoV—a target for vaccine and therapeutic development[J]. Nature Reviews Microbiology, 2009, 7(3): 226-236.
2	XIA S A, LIU M Q, WANG C, et al. Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion[J]. Cell Research, 2020, 30(4): 343-355.
3	ZHOU J, LIU Z Z, ZHANG G X, et al. Development of variant-proof severe acute respiratory syndrome coronavirus 2, pan-sarbecovirus, and pan-β-coronavirus vaccines[J]. Journal of Medical Virology, 2023, 95(1): e28172.
4	LIU Z Z, XU W, XIA S, et al. RBD-Fc-based COVID-19 vaccine candidate induces highly potent SARS-CoV-2 neutralizing antibody response[J]. Signal Transduction and Targeted Therapy, 2020, 5: 282.
5	WRAPP D, WANG N S, CORBETT K S, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation[J]. Science, 2020, 367(6483): 1260-1263.
6	ZHANG N R, JI Q T, LIU Z Z, et al. Effect of different adjuvants on immune responses elicited by protein-based subunit vaccines against SARS-CoV-2 and its delta variant[J]. Viruses, 2022, 14(3): 501.
7	ROUTHU N K, CHEEDARLA N, BOLLIMPELLI V S, et al. SARS-CoV-2 RBD trimer protein adjuvanted with Alum-3M-052 protects from SARS-CoV-2 infection and immune pathology in the lung[J]. Nature Communications, 2021, 12: 3587.
8	YAMAYOSHI S, KAWAOKA Y. Current and future influenza vaccines[J]. Nature Medicine, 2019, 25(2): 212-220.
9	GÜTHE S, KAPINOS L, MÖGLICH A, et al. Very fast folding and association of a trimerization domain from bacteriophage T4 fibritin[J]. Journal of Molecular Biology, 2004, 337(4): 905-915.
10	TAO Y Z, STRELKOV S V, MESYANZHINOV V V, et al. Structure of bacteriophage T4 fibritin: a segmented coiled coil and the role of the C-terminal domain[J]. Structure, 1997, 5(6): 789-798.
11	LI T T, ZHANG Z Y, ZHANG Z Q, et al. Characterization of native-like HIV-1 gp140 glycoprotein expressed in insect cells[J]. Vaccine, 2019, 37(11): 1418-1427.
12	RINGE R P, YASMEEN A, OZOROWSKI G, et al. Influences on the design and purification of soluble, recombinant native-like HIV-1 envelope glycoprotein trimers[J]. Journal of Virology, 2015, 89(23): 12189-12210.
13	YANG X Z, LEE J, MAHONY E M, et al. Highly stable trimers formed by human immunodeficiency virus type 1 envelope glycoproteins fused with the trimeric motif of T4 bacteriophage fibritin[J]. Journal of Virology, 2002, 76(9): 4634-4642.
14	AHN J Y, LEE J, SUH Y S, et al. Safety and immunogenicity of two recombinant DNA COVID-19 vaccines containing the coding regions of the spike or spike and nucleocapsid proteins: an interim analysis of two open-label, non-randomised, phase 1 trials in healthy adults[J]. The Lancet Microbe, 2022, 3(3): e173-e183.
15	VOGEL A B, KANEVSKY I, CHE Y, et al. BNT162b vaccines protect rhesus macaques from SARS-CoV-2[J]. Nature, 2021, 592(7853): 283-289.
16	SLIEPEN K, VAN MONTFORT T, MELCHERS M, et al. Immunosilencing a highly immunogenic protein trimerization domain[J]. The Journal of Biological Chemistry, 2015, 290(12): 7436-7442.
17	CAO Y L, JIAN F C, WANG J, et al. Imprinted SARS-CoV-2 humoral immunity induces convergent Omicron RBD evolution[J]. Nature, 2023, 614(7948): 521-529.
18	LI H, YOU S, YANG X, et al. Injectable recombinant human collagen-derived material with high cell adhesion activity limits adverse remodelling and improves pelvic floor function in pelvic floor dysfunction rats[J]. Biomaterials Advances, 2022, 134: 112715.
19	GORDON M K, HAHN R A. Collagens[J]. Cell and Tissue Research, 2010, 339(1): 247-257.
20	LIU X, WU H, BYRNE M, et al. Type Ⅲ collagen is crucial for collagen Ⅰ fibrillogenesis and for normal cardiovascular development[J]. Proceedings of the National Academy of Sciences of the United States of America, 1997, 94(5): 1852-1856.
21	HUA C, ZHU Y, XU W, et al. Characterization by high-resolution crystal structure analysis of a triple-helix region of human collagen type Ⅲ with potent cell adhesion activity[J]. Biochemical and Biophysical Research Communications, 2019, 508(4): 1018-1023.
22	LIU Z Z, ZHOU J, WANG X L, et al. A pan-sarbecovirus vaccine based on RBD of SARS-CoV-2 original strain elicits potent neutralizing antibodies against XBB in non-human primates[J]. Proceedings of the National Academy of Sciences of the United States of America, 2023, 120(11): e2221713120.
23	LIU Z Z, ZHOU J, XU W, et al. A novel STING agonist-adjuvanted pan-sarbecovirus vaccine elicits potent and durable neutralizing antibody and T cell responses in mice, rabbits and NHPs[J]. Cell Research, 2022, 32(3): 269-287.
24	JUMPER J, EVANS R, PRITZEL A, et al. Highly accurate protein structure prediction with AlphaFold[J]. Nature, 2021, 596(7873): 583-589.
25	LIU Z Z, CHAN J F W, ZHOU J, et al. A pan-sarbecovirus vaccine induces highly potent and durable neutralizing antibody responses in non-human primates against SARS-CoV-2 Omicron variant[J]. Cell Research, 2022, 32(5): 495-497.
26	RAINHO-TOMKO J N, PAVOT V, KISHKO M, et al. Immunogenicity and protective efficacy of RSV G central conserved domain vaccine with a prefusion nanoparticle[J]. NPJ Vaccines, 2022, 7: 74.
27	DAI L P, ZHENG T Y, XU K, et al. A universal design of betacoronavirus vaccines against COVID-19, MERS, and SARS[J]. Cell, 2020, 182(3): 722-733. e11.
28	YOU S, LIU S B, DONG X J, et al. Intravaginal administration of human type Ⅲ collagen-derived biomaterial with high cell-adhesion activity to treat vaginal atrophy in rats[J]. ACS Biomaterials Science & Engineering, 2020, 6(4): 1977-1988.
29	YANG L, WU H S, LU L, et al. A tailored extracellular matrix (ECM)-Mimetic coating for cardiovascular stents by stepwise assembly of hyaluronic acid and recombinant human type Ⅲ collagen[J]. Biomaterials, 2021, 276: 121055.
30	HU C, LIU W Q, LONG L Y, et al. Microenvironment-responsive multifunctional hydrogels with spatiotemporal sequential release of tailored recombinant human collagen type Ⅲ for the rapid repair of infected chronic diabetic wounds[J]. Journal of Materials Chemistry B, 2021, 9(47): 9684-9699.
31	LONG L Y, LIU W Q, LI L, et al. Dissolving microneedle-encapsulated drug-loaded nanoparticles and recombinant humanized collagen type Ⅲ for the treatment of chronic wound via anti-inflammation and enhanced cell proliferation and angiogenesis[J]. Nanoscale, 2022, 14(4): 1285-1295.
32	WANG J, QIU H, XU Y, et al. The biological effect of recombinant humanized collagen on damaged skin induced by UV-photoaging: an in vivo study[J]. Bioactive Materials, 2021, 11: 154-165.
33	YOU S, ZHU Y, LI H, et al. Recombinant humanized collagen remodels endometrial immune microenvironment of chronic endometritis through macrophage immunomodulation[J]. Regenerative Biomaterials, 2023, 10: rbad033.

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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

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