Plant-Produced Antigen Displaying Virus-Like Particles Evokes Potent Antibody Responses against West Nile Virus in Mice

In this study, we developed a hepatitis B core antigen (HBcAg)-based virus-like particle (VLP) that displays the West Nile virus (WNV) Envelope protein domain III (wDIII) as a vaccine candidate for WNV. The HBcAg-wDIII fusion protein was quickly produced in Nicotiana benthamiana plants and reached a high expression level of approximately 1.2 mg of fusion protein per gram of leaf fresh weight within six days post gene infiltration. Electron microscopy and gradient centrifugation analysis indicated that the introduction of wDIII did not interfere with VLP formation and HBcAg-wDIII successfully assembled into VLPs. HBcAg-wDIII VLPs can be easily purified in large quantities from Nicotiana benthamiana leaves to >95% homogeneity. Further analysis revealed that the wDIII was displayed properly and demonstrated specific binding to an anti-wDIII monoclonal antibody that recognizes a conformational epitope of wDIII. Notably, HBcAg-wDIII VLPs were shown to be highly immunogenic and elicited potent humoral responses in mice with antigen-specific IgG titers equivalent to that of protective wDIII antigens in previous studies. Thus, our wDIII-based VLP vaccine offers an attractive option for developing effective, safe, and low-cost vaccines against WNV.

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A Plant-Produced Antigen Elicits Potent Immune Responses against West Nile Virus in Mice

BioMed Research International ◽

10.1155/2014/952865 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10 ◽

Cited By ~ 16

Author(s):

Junyun He ◽

Li Peng ◽

Huafang Lai ◽

Jonathan Hurtado ◽

Jake Stahnke ◽

...

Keyword(s):

West Nile Virus ◽

Immune Responses ◽

Viral Vector ◽

Specific Binding ◽

Low Cost ◽

Expression System ◽

Conformational Epitope ◽

West Nile ◽

Domain Iii ◽

Subcutaneous Immunization

We described the rapid production of the domain III (DIII) of the envelope (E) protein in plants as a vaccine candidate for West Nile Virus (WNV). Using various combinations of vector modules of a deconstructed viral vector expression system, DIII was produced in three subcellular compartments in leaves ofNicotiana benthamianaby transient expression. DIII expressed at much higher levels when targeted to the endoplasmic reticulum (ER) than that targeted to the chloroplast or the cytosol, with accumulation level up to 73 μg DIII per gram of leaf fresh weight within 4 days after infiltration. Plant ER-derived DIII was soluble and readily purified to > 95% homogeneity without the time-consuming process of denaturing and refolding. Further analysis revealed that plant-produced DIII was processed properly and demonstrated specific binding to an anti-DIII monoclonal antibody that recognizes a conformational epitope. Furthermore, subcutaneous immunization of mice with 5 and 25 μg of purified DIII elicited a potent systemic response. This study provided the proof of principle for rapidly producing immunogenic vaccine candidates against WNV in plants with low cost and scalability.

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A glycosylated peptide in the West Nile virus envelope protein is immunogenic during equine infection

Journal of General Virology ◽

10.1099/vir.0.2008/003731-0 ◽

2008 ◽

Vol 89 (12) ◽

pp. 3063-3072 ◽

Cited By ~ 20

Author(s):

Jody Hobson-Peters ◽

Philip Toye ◽

Melissa D. Sánchez ◽

Katharine N. Bossart ◽

Lin-Fa Wang ◽

...

Keyword(s):

West Nile Virus ◽

Fusion Protein ◽

Synthetic Peptides ◽

Mammalian Cells ◽

Protein Glycosylation ◽

The West ◽

West Nile ◽

High Sequence Identity ◽

Recombinant Peptide ◽

E Protein

Using a monoclonal antibody directed to domain I of the West Nile virus (WNV) envelope (E) protein, we identified a continuous (linear) epitope that was immunogenic during WNV infection of horses. Using synthetic peptides, this epitope was mapped to a 19 aa sequence (WN19: E147–165) encompassing the WNV NY99 E protein glycosylation site at position 154. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognized the recombinant peptide. Failure of most WNV- and MVEV-positive horse sera to recognize the epitope as a deglycosylated fusion protein confirmed that the N-linked glycan was important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain.

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Solution Structure and Antibody Binding Studies of the Envelope Protein Domain III from the New York Strain of West Nile Virus

Journal of Biological Chemistry ◽

10.1074/jbc.m402385200 ◽

2004 ◽

Vol 279 (37) ◽

pp. 38755-38761 ◽

Cited By ~ 78

Author(s):

David E. Volk ◽

David W. C. Beasley ◽

Deborah A. Kallick ◽

Michael R. Holbrook ◽

Alan D. T. Barrett ◽

...

Keyword(s):

New York ◽

West Nile Virus ◽

Envelope Protein ◽

Solution Structure ◽

Antibody Binding ◽

Protein Domain ◽

Binding Studies ◽

West Nile ◽

Domain Iii

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Structural basis for Zika envelope domain III recognition by a germline version of a recurrent neutralizing antibody

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1919269117 ◽

2020 ◽

Vol 117 (18) ◽

pp. 9865-9875

Author(s):

Shannon R. Esswein ◽

Harry B. Gristick ◽

Andrea Jurado ◽

Avery Peace ◽

Jennifer R. Keeffe ◽

...

Keyword(s):

West Nile Virus ◽

Envelope Protein ◽

Yellow Fever Virus ◽

Cross Reactivity ◽

Affinity Maturation ◽

Protein Domain ◽

Structural Basis ◽

Maturation Process ◽

West Nile ◽

Domain Iii

Recent epidemics demonstrate the global threat of Zika virus (ZIKV), a flavivirus transmitted by mosquitoes. Although infection is usually asymptomatic or mild, newborns of infected mothers can display severe symptoms, including neurodevelopmental abnormalities and microcephaly. Given the large-scale spread, symptom severity, and lack of treatment or prophylaxis, a safe and effective ZIKV vaccine is urgently needed. However, vaccine design is complicated by concern that elicited antibodies (Abs) may cross-react with other flaviviruses that share a similar envelope protein, such as dengue virus, West Nile virus, and yellow fever virus. This cross-reactivity may worsen symptoms of a subsequent infection through Ab-dependent enhancement. To better understand the neutralizing Ab response and risk of Ab-dependent enhancement, further information on germline Ab binding to ZIKV and the maturation process that gives rise to potently neutralizing Abs is needed. Here we use binding and structural studies to compare mature and inferred-germline Ab binding to envelope protein domain III of ZIKV and other flaviviruses. We show that affinity maturation of the light-chain variable domain is important for strong binding of the recurrent VH3-23/VK1-5 neutralizing Abs to ZIKV envelope protein domain III, and identify interacting residues that contribute to weak, cross-reactive binding to West Nile virus. These findings provide insight into the affinity maturation process and potential cross-reactivity of VH3-23/VK1-5 neutralizing Abs, informing precautions for protein-based vaccines designed to elicit germline versions of neutralizing Abs.

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