Alphavirus capsid proteins self-assemble into core-like particles in insect cells: A promising platform for nanoparticle vaccine development

2015 ◽  
Vol 11 (2) ◽  
pp. 266-273 ◽  
Author(s):  
Mia C. Hikke ◽  
Corinne Geertsema ◽  
Vincen Wu ◽  
Stefan W. Metz ◽  
Jan W. van Lent ◽  
...  
Keyword(s):  
Insect Cells ◽  
Vaccine Development ◽  
Capsid Proteins

scholarly journals Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

2017 ◽  
Vol 91 (20) ◽  
Author(s):  
Stefanie Grosse ◽  
Magalie Penaud-Budloo ◽  
Anne-Kathrin Herrmann ◽  
Kathleen Börner ◽  
Julia Fakhiri ◽  
...  
Keyword(s):  
Protein Stability ◽  
Capsid Protein ◽  
Insect Cells ◽  
Critical Factor ◽  
Capsid Proteins ◽  
Wild Type ◽  
Particle Assembly ◽  
Adeno Associated Virus ◽  
Recombinant Vectors

ABSTRACT The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans-complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production.

scholarly journals Bioinformatics analysis of rhinovirus capsid proteins VP1-4 sequences for cross-serotype vaccine development

2021 ◽  
Vol 14 (11) ◽  
pp. 1603-1611
Author(s):  
Ahmed S. Alshrari ◽  
Shuaibu A. Hudu ◽  
Syed M.B. Asdaq ◽  
Alreshidi M. Ali ◽  
Chin V. Kin ◽  
...  
Keyword(s):  
Bioinformatics Analysis ◽  
Vaccine Development ◽  
Capsid Proteins

scholarly journals Production of SARS-CoV-2 virus-like particles in insect cells

2021 ◽  
Author(s):  
Youjun Mi ◽  
Tao Xie ◽  
Bingdong Zhu ◽  
Jiying Tan ◽  
Xuefeng Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Human Health ◽  
Insect Cells ◽  
Vaccine Development ◽  
Recombinant Baculovirus ◽  
Expression Plasmid ◽  
Virus Like Particles ◽  
Recombinant Bacmid ◽  
Multiple Cloning Sites ◽  
S Genes

ABSTRACTCoronavirus disease (COVID-19) causes a serious threat to human health. To production of SARS-COV-2 virus-like particles (VLPs) in insect cells for vaccine development and scientific research. The E, M and S genes were cloned into multiple cloning sites of the new triple expression plasmid with one p10 promoter, two pPH promoters and three multiple cloning sites. The plasmid was transformed into DH10 BacTMEscherichia coli competent cells to obtain recombinant bacmid. Then the recombinant bacmid was transfected in ExpiSf9™ insect cells to generate recombinant baculovirus. After ExpiSf9™ infected with the recombinant baculovirus, the E, M, and S protein co-expressed in insect cells. Finally, SARS-CoV-2 VLPs were self-assembled in insect cells after infection. The morphology and the size of SARS-CoV-2 VLPs are similar to the native virions.

scholarly journals Hepatitis C Virus Structural Proteins Assemble into Viruslike Particles in Insect Cells

1998 ◽  
Vol 72 (5) ◽  
pp. 3827-3836 ◽  
Author(s):  
Thomas F. Baumert ◽  
Susumu Ito ◽  
David T. Wong ◽  
T. Jake Liang
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Insect Cells ◽  
Vaccine Development ◽  
Cultured Cells ◽  
Recombinant Baculovirus ◽  
Structural Proteins ◽  
Particle Assembly ◽  
Viruslike Particles ◽  
Hcv Structural Proteins

ABSTRACT Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of HCV has been hampered by the low level of viral particles in infected individuals, the inability to propagate efficiently the virus in cultured cells, and the lack of a convenient animal model. Due to these obstacles, neither the structure of the virus nor the prerequisites for its assembly have been clearly defined. In this report, we describe a model for the production and purification of HCV-like particles in insect cells using a recombinant baculovirus containing the cDNA of the HCV structural proteins. In insect cells, expressed HCV structural proteins assembled into enveloped viruslike particles (40 to 60 nm in diameter) in large cytoplasmic cisternae, presumably derived from the endoplasmic reticulum. Biophysical characterization of viruslike particles by CsCl and sucrose gradient centrifugation revealed biophysical properties similar to those of putative virions isolated from infected humans. The results suggested that HCV core and envelope proteins without p7 were sufficient for viral particle formation. Analysis of particle-associated nucleic acids demonstrated that HCV RNAs were selectively incorporated into the particles over non-HCV transcripts. The synthesis of HCV-like particles in insect cells may provide an important tool to determine the structural requirements for HCV particle assembly as well as to study viral genome encapsidation and virus-host interactions. The described system may also represent a potential approach toward vaccine development.

Architecture of African swine fever virus and implications for viral assembly

Science ◽  
2019 ◽  
Vol 366 (6465) ◽  
pp. 640-644 ◽  
Author(s):  
Nan Wang ◽  
Dongming Zhao ◽  
Jialing Wang ◽  
Yangling Zhang ◽  
Ming Wang ◽  
...  
Keyword(s):  
Vaccine Development ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Capsid Proteins ◽  
Dna Viruses ◽  
Capsid Shell ◽  
Nucleocytoplasmic Large Dna Viruses ◽  
Swine Disease ◽  
Capsid Stability

African swine fever virus (ASFV) is a giant and complex DNA virus that causes a highly contagious and often lethal swine disease for which no vaccine is available. Using an optimized image reconstruction strategy, we solved the ASFV capsid structure up to 4.1 angstroms, which is built from 17,280 proteins, including one major (p72) and four minor (M1249L, p17, p49, and H240R) capsid proteins organized into pentasymmetrons and trisymmetrons. The atomic structure of the p72 protein informs putative conformational epitopes, distinguishing ASFV from other nucleocytoplasmic large DNA viruses. The minor capsid proteins form a complicated network below the outer capsid shell, stabilizing the capsid by holding adjacent capsomers together. Acting as core organizers, 100-nanometer-long M1249L proteins run along each edge of the trisymmetrons that bridge two neighboring pentasymmetrons and form extensive intermolecular networks with other capsid proteins, driving the formation of the capsid framework. These structural details unveil the basis of capsid stability and assembly, opening up new avenues for African swine fever vaccine development.

scholarly journals Immunogenicity and protective potency of Norovirus GII.17 virus-like particle-based vaccine

2018 ◽  
Author(s):  
Wei Chen ◽  
Tao Kang ◽  
Rongliang Yuan ◽  
Yuyang Zhang ◽  
Siqi Xin ◽  
...  
Keyword(s):  
Insect Cells ◽  
Vaccine Development ◽  
Recombinant Baculovirus ◽  
Spherical Particles ◽  
Effective Vaccine ◽  
Viral Gastroenteritis ◽  
Blood Group Antigens ◽  
Electron Microscopic ◽  
Capsid Protein Vp1 ◽  
Norovirus Gii

Noroviruses (NoVs) are a major cause of acute viral gastroenteritis in adults and children worldwide. Lacking of cell culture system and animals models that must be considered the virus like particles (VLPs) used as an effective vaccine development. In the present study, we investigated the expression of the major capsid protein (VP1) of Genogroup II, genotype 17 (GII.17) NoV using recombinant baculovirus system in insect cells and saliva binding blockade assay to detect their protective potency. Our results showed that GII.17 VLPs could be successfully generated in sf9 insect cells and electron microscopic revealed that GII.17 VLPs was visualized as spherical particles of -35nm in diameter. Immunized mouse with purified VLPs produced GII.17 specific sera and could efficiently block GII.17 VLPs binding to saliva histo-blood group antigens (HBGAs). Together, these results suggested that GII.17 VLPs represent a promising vaccine candidate against NoV GII.17 infection and strongly support further preclinical and clinical studies.

scholarly journals Assembly of Marek's disease virus (MDV) capsids using recombinant baculoviruses expressing MDV capsid proteins

2004 ◽  
Vol 85 (4) ◽  
pp. 769-774 ◽  
Author(s):  
E. Kut ◽  
D. Rasschaert
Keyword(s):  
Disease Virus ◽  
Insect Cells ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Capsid Proteins ◽  
Spherical Particles ◽  
Recombinant Baculoviruses ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Thin Sections

The genes UL18, UL19, UL26, UL26.5, UL35 and UL38 of Marek's disease virus 1 (MDV-1) strain RB1B, encoding the homologues of herpes simplex virus type 1 (HSV-1) capsid proteins VP23, VP5, VP21–VP24, preVP22a, VP26 and VP19C, were identified and sequenced. Recombinant baculoviruses were used to express the six capsid genes in insect cells. Coexpression of the six genes or of UL18, UL19, UL26.5 and UL38 in insect cells resulted in the formation of capsids with a large core. In addition, electron microscopy of thin sections clearly revealed the presence of large numbers of small spherical particles. Experimental coinfection demonstrated that these small particles were associated with production of the preVP22a protein.

scholarly journals Establishment of a Recombinant AAV2/HBoV1 Vector Production System in Insect Cells

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 439
Author(s):  
Xuefeng Deng ◽  
Wei Zou ◽  
Ziying Yan ◽  
Jianming Qiu
Keyword(s):  
Production System ◽  
Insect Cells ◽  
Capsid Proteins ◽  
Hek293 Cells ◽  
Sf9 Cells ◽  
Human Bocavirus ◽  
Transmission Electron Microscopy Analysis ◽  
Hek 293 ◽  
Highly Efficient ◽  
Baculovirus Expression Vector

We have previously developed an rAAV2/HBoV1 vector in which a recombinant adeno-associated virus 2 (rAAV2) genome is pseudopackaged into a human bocavirus 1 (HBoV1) capsid. Recently, the production of rAAV2/HBoV1 in human embryonic kidney (HEK) 293 cells has been greatly improved in the absence of any HBoV1 nonstructural proteins (NS). This NS-free production system yields over 16-fold more vectors than the original production system that necessitates NS expression. The production of rAAV with infection of baculovirus expression vector (BEV) in the suspension culture of Sf9 insect cells is highly efficient and scalable. Since the replication of the rAAV2 genome in the BEV system is well established, we aimed to develop a BEV system to produce the rAAV2/HBoV1 vector in Sf9 cells. We optimized the usage of translation initiation signals of the HBoV1 capsid proteins (Cap), and constructed a BEV Bac-AAV2Rep-HBoV1Cap, which expresses the AAV2 Rep78 and Rep52 as well as the HBoV1 VP1, VP2, and VP3 at the appropriate ratios. We found that it is sufficient as a trans helper to the production of rAAV2/HBoV1 in Sf9 cells that were co-infected with the transfer Bac-AAV2ITR-GFP-luc that carried a 5.4-kb oversized rAAV2 genome with dual reporters. Further study found that incorporation of an HBoV1 small NS, NP1, in the system maximized the viral DNA replication and thus the rAAV2/HBoV1 vector production at a level similar to that of the rAAV2 vector in Sf9 cells. However, the transduction potency of the rAAV2/HBoV1 vector produced from BEV-infected Sf9 cells was 5–7-fold lower in polarized human airway epithelia than that packaged in HEK293 cells. Transmission electron microscopy analysis found that the vector produced in Sf9 cells had a high percentage of empty capsids, suggesting the pseudopackage of the rAAV2 genome in HBoV1 capsid is not as efficient as in the capsids of AAV2. Nevertheless, our study demonstrated that the rAAV2/HBoV1 can be produced in insect cells with BEVs at a comparable yield to rAAV, and that the highly efficient expression of the HBoV1 capsid proteins warrants further optimization.

scholarly journals Structures of Coxsackievirus A16 Capsids with Native Antigenicity: Implications for Particle Expansion, Receptor Binding, and Immunogenicity

2015 ◽  
Vol 89 (20) ◽  
pp. 10500-10511 ◽  
Author(s):  
Jingshan Ren ◽  
Xiangxi Wang ◽  
Ling Zhu ◽  
Zhongyu Hu ◽  
Qiang Gao ◽  
...  
Keyword(s):  
High Resolution ◽  
Insect Cells ◽  
Vaccine Development ◽  
Enterovirus 71 ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Vaccine Antigen ◽  
Coxsackievirus A16 ◽  
Structure Based Drug Design ◽  
Foot And Mouth

ABSTRACTEnterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the primary causes of the epidemics of hand-foot-and-mouth disease (HFMD) that affect more than a million children in China each year and lead to hundreds of deaths. Although there has been progress with vaccines for EV71, the development of a CVA16 vaccine has proved more challenging, and the EV71 vaccine does not give useful cross-protection, despite the capsid proteins of the two viruses sharing about 80% sequence identity. The structural details of the expanded forms of the capsids, which possess nonnative antigenicity, are now well understood, but high resolution information for the native antigenic form of CVA16 has been missing. Here, we remedy this with high resolution X-ray structures of both mature and natural empty CVA16 particles and also of empty recombinant viruslike particles of CVA16 produced in insect cells, a potential vaccine antigen. All three structures are unexpanded native particles and antigenically identical. The recombinant particles have recruited a lipid moiety to stabilize the native antigenic state that is different from the one used in a natural virus infection. As expected, the mature CVA16 virus is similar to EV71; however, structural and immunogenic comparisons highlight differences that may have implications for vaccine production.IMPORTANCEHand-foot-and-mouth disease is a serious public health threat to children in Asian-Pacific countries, resulting in millions of cases. EV71 and CVA16 are the two dominant causative agents of the disease that, while usually mild, can cause severe neurological complications, leading to hundreds of deaths. EV71 vaccines do not provide protection against CVA16. A CVA16 vaccine or bivalent EV71/CVA16 vaccine is therefore urgently needed. We report atomic structures for the mature CVA16 virus, a natural empty particle, and a recombinant CVA16 virus-like particle that does not contain the viral genome. All three particles have similar structures and identical antigenicity. The recombinant particles, produced in insect cells (a system suitable for making vaccine antigen), are stabilized by recruiting from the insect cells a small molecule that is different from that used by the virus in a normal infection. We present structural and immunogenic comparisons with EV71 to facilitate structure-based drug design and vaccine development.

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