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The core antigen of hepatitis B virus (HBcAg) is capable of self-assembly into virus-like particles (VLPs) when expressed in a number of heterologous systems. Such VLPs are potential carriers of foreign antigenic sequences for vaccine design. In this study, we evaluated the production of chimeric HBcAg VLPs presenting a foreign epitope on their surface, the 551–607 amino acids (aa) immunological epitope of the ORF2 capsid protein of hepatitis E virus. A chimeric construct was made by the insertion of 56 aa into the immunodominant loop of the HBcAg. The sequences encoding the chimera were inserted into the pEAQ-HT vector and infiltrated into Nicotiana benthamiana leaves. The plant-expressed chimeric HBcHEV ORF2 551–607 protein was recognized by an anti-HBcAg mAb and anti-HEV IgG positive swine serum. Electron microscopy showed that plant-produced chimeric protein spontaneously assembled into “knobbly” ~34 nm diameter VLPs. This study shows that HBcAg is a promising carrier platform for the neutralizing epitopes of hepatitis E virus (HEV) and the chimeric HBcAg/HEV VLPs could be a candidate for a bivalent vaccine.
Gergana Zahmanova; Milena Mazalovska; Katerina Takova; Valentina Toneva; Ivan Minkov; Hadrien Peyret; George Lomonossoff. Efficient Production of Chimeric Hepatitis B Virus-Like Particles Bearing an Epitope of Hepatitis E Virus Capsid by Transient Expression in Nicotiana benthamiana. Life 2021, 11, 64 .
AMA StyleGergana Zahmanova, Milena Mazalovska, Katerina Takova, Valentina Toneva, Ivan Minkov, Hadrien Peyret, George Lomonossoff. Efficient Production of Chimeric Hepatitis B Virus-Like Particles Bearing an Epitope of Hepatitis E Virus Capsid by Transient Expression in Nicotiana benthamiana. Life. 2021; 11 (1):64.
Chicago/Turabian StyleGergana Zahmanova; Milena Mazalovska; Katerina Takova; Valentina Toneva; Ivan Minkov; Hadrien Peyret; George Lomonossoff. 2021. "Efficient Production of Chimeric Hepatitis B Virus-Like Particles Bearing an Epitope of Hepatitis E Virus Capsid by Transient Expression in Nicotiana benthamiana." Life 11, no. 1: 64.
(1) Background: Hepatitis E virus (HEV) is a causative agent of acute viral hepatitis, predominantly transmitted by the fecal–oral route. In developed countries, HEV is considered to be an emerging pathogen since the number of autochthonous cases is rising. Hepatitis E is a viral disease with a proven zoonotic potential for some of its genotypes. The main viral reservoirs are domestic pigs and wild boar. Consumption of undercooked meat, as well as occupational exposure, are key factors for the spread of HEV. In order to evaluate the risks of future viral evolution, a detailed examination of the ecology and distribution of the virus is needed. The aim of the present study is to investigate the prevalence of anti-HEV IgG Ab in domestic pigs and wild boar in Bulgaria; (2) Methods: In this study, during the period of three years between 2017 and 2019, 433 serum samples from 19 different pig farms and 1 slaughterhouse were collected and analyzed. In addition, 32 samples from wild boar were also collected and analyzed during the 2018–2019 hunting season. All samples were analyzed by commercial indirect ELISA; (3) Results: Overall, HEV seroprevalence was 60% (95% CI 42.7–77.1) in domestic pigs and 12.5% (4/32) in wild boar. The observed seroprevalence of the slaughter-aged pigs was 73.65% (95% Cl 58.7–87.3). Prevalence in domestic pigs was significantly higher in the samples collected during 2019 (98% (95% Cl 96.1–99.9)) compared to those collected during 2017 (45.33% (95% CI 2.7–87.3)) and 2018 (38.46% (95% CI 29.1–49.7.); (4) Conclusions: Our findings suggest that domesticated pigs and wild boar might be the reason for the increased HEV transmission across Bulgaria. The genotypic characterization of HEV found in pigs, wild boar and humans will give a more accurate view of the zoonotic transmission of this virus.
Katerina Takova; Tsvetoslav Koynarski; Ivan Minkov; Zdravka Ivanova; Valentina Toneva; Gergana Zahmanova. Increasing Hepatitis E Virus Seroprevalence in Domestic Pigs and Wild Boar in Bulgaria. Animals 2020, 10, 1521 .
AMA StyleKaterina Takova, Tsvetoslav Koynarski, Ivan Minkov, Zdravka Ivanova, Valentina Toneva, Gergana Zahmanova. Increasing Hepatitis E Virus Seroprevalence in Domestic Pigs and Wild Boar in Bulgaria. Animals. 2020; 10 (9):1521.
Chicago/Turabian StyleKaterina Takova; Tsvetoslav Koynarski; Ivan Minkov; Zdravka Ivanova; Valentina Toneva; Gergana Zahmanova. 2020. "Increasing Hepatitis E Virus Seroprevalence in Domestic Pigs and Wild Boar in Bulgaria." Animals 10, no. 9: 1521.
The Hepatitis E virus (HEV) is a causative agent of acute hepatitis, mainly transmitted by the fecal-oral route or zoonotic. Open reading frame (ORF) 2 encodes the viral capsid protein, which is essential for virion assembly, host interaction, and inducing neutralizing antibodies. In this study, we investigated whether full-length and N- and C-terminally modified versions of the capsid protein transiently expressed in N. benthamiana plants could assemble into highly-immunogenic, virus-like particles (VLPs). We also assessed whether such VLPs can act as a carrier of foreign immunogenic epitopes, such as the highly-conserved M2e peptide from the Influenza virus. Plant codon-optimized HEV ORF2 capsid genes were constructed in which the nucleotides coding the N-terminal, the C-terminal, or both parts of the protein were deleted. The M2e peptide was inserted into the P2 loop after the residue Gly556 of HEV ORF2 protein by gene fusion, and three different chimeric constructs were designed. Plants expressed all versions of the HEV capsid protein up to 10% of total soluble protein (TSP), including the chimeras, but only the capsid protein consisting of aa residues 110 to 610 (HEV 110–610) and chimeric M2 HEV 110–610 spontaneously assembled in higher order structures. The chimeric VLPs assembled into particles with 22–36 nm in diameter and specifically reacted with the anti-M2e antibody.
Gergana G. Zahmanova; Milena Mazalovska; Katerina H. Takova; Valentina Toneva; Ivan N. Minkov; Eugenia S. Mardanova; Nikolai V. Ravin; George P. Lomonossoff. Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope. Plants 2019, 9, 29 .
AMA StyleGergana G. Zahmanova, Milena Mazalovska, Katerina H. Takova, Valentina Toneva, Ivan N. Minkov, Eugenia S. Mardanova, Nikolai V. Ravin, George P. Lomonossoff. Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope. Plants. 2019; 9 (1):29.
Chicago/Turabian StyleGergana G. Zahmanova; Milena Mazalovska; Katerina H. Takova; Valentina Toneva; Ivan N. Minkov; Eugenia S. Mardanova; Nikolai V. Ravin; George P. Lomonossoff. 2019. "Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope." Plants 9, no. 1: 29.
We describe a method for the production of HEV ORF2 protein in N. benthamiana and the usefulness of this protein for the serological detection of anti-HEV antibodies in both humans and swine.
Milena Mazalovska; Nikola Varadinov; Tsvetoslav Koynarski; Ivan Minkov; Pavel Teoharov; George P. Lomonossoff; Gergana Zahmanova. Detection of Serum Antibodies to Hepatitis E Virus Based on HEV Genotype 3 ORF2 Capsid Protein Expressed in Nicotiana benthamiana. Annals of Laboratory Medicine 2017, 37, 313 -319.
AMA StyleMilena Mazalovska, Nikola Varadinov, Tsvetoslav Koynarski, Ivan Minkov, Pavel Teoharov, George P. Lomonossoff, Gergana Zahmanova. Detection of Serum Antibodies to Hepatitis E Virus Based on HEV Genotype 3 ORF2 Capsid Protein Expressed in Nicotiana benthamiana. Annals of Laboratory Medicine. 2017; 37 (4):313-319.
Chicago/Turabian StyleMilena Mazalovska; Nikola Varadinov; Tsvetoslav Koynarski; Ivan Minkov; Pavel Teoharov; George P. Lomonossoff; Gergana Zahmanova. 2017. "Detection of Serum Antibodies to Hepatitis E Virus Based on HEV Genotype 3 ORF2 Capsid Protein Expressed in Nicotiana benthamiana." Annals of Laboratory Medicine 37, no. 4: 313-319.
Gergana Zahmanova; Milena Mazalovska; Valentina Toneva; Ivan Minkov; George Lomonossoff. Production of chimeric virus-like particles bearing M2e influenza epitope in Nicotiana benthamiana plants. Journal of Biotechnology 2015, 208, S109 .
AMA StyleGergana Zahmanova, Milena Mazalovska, Valentina Toneva, Ivan Minkov, George Lomonossoff. Production of chimeric virus-like particles bearing M2e influenza epitope in Nicotiana benthamiana plants. Journal of Biotechnology. 2015; 208 ():S109.
Chicago/Turabian StyleGergana Zahmanova; Milena Mazalovska; Valentina Toneva; Ivan Minkov; George Lomonossoff. 2015. "Production of chimeric virus-like particles bearing M2e influenza epitope in Nicotiana benthamiana plants." Journal of Biotechnology 208, no. : S109.
Advances in transient expression technologies have allowed the production of milligram quantities of proteins within a matter of days using only small amounts (tens of grams) of plant tissue. Among the proteins that have been produced using this approach are the structural proteins of viruses which are capable of forming virus-like particles (VLPs). As such particulate structures are potent stimulators of the immune system, they are excellent vaccine candidates both in their own right and as carriers of additional immunogenic sequences. VLPs of varying complexity derived from a variety of animal viruses have been successfully transiently expressed in plants and their immunological properties assessed. Generally, the plant-produced VLPs were found to have the expected antigenicity and immunogenicity. In several cases, including an M2e-based influenza vaccine candidate, the plant-expressed VLPs have been shown to be capable of stimulating protective immunity. These findings raise the prospect that low-cost plant-produced vaccines could be developed for both veterinary and human use.
Eva Thuenemann; Paolo Lenzi; Andrew Love; Michael Taliansky; Martina Becares; Sonia Zuñiga; Luis Enjuanes; Gergana Zahmanova; Ivan Minkov; Slavica Matic; Emanuela Noris; Ann Meyers; Alta Hattingh; Edward Rybicki; Oleg Kiselev; Nikolai Ravin; Michael Eldarov; Konstantin Skryabin; George Lomonossoff. The Use of Transient Expression Systems for the Rapid Production of Virus-like Particles in Plants. Current Pharmaceutical Design 2013, 19, 5564 -5573.
AMA StyleEva Thuenemann, Paolo Lenzi, Andrew Love, Michael Taliansky, Martina Becares, Sonia Zuñiga, Luis Enjuanes, Gergana Zahmanova, Ivan Minkov, Slavica Matic, Emanuela Noris, Ann Meyers, Alta Hattingh, Edward Rybicki, Oleg Kiselev, Nikolai Ravin, Michael Eldarov, Konstantin Skryabin, George Lomonossoff. The Use of Transient Expression Systems for the Rapid Production of Virus-like Particles in Plants. Current Pharmaceutical Design. 2013; 19 (31):5564-5573.
Chicago/Turabian StyleEva Thuenemann; Paolo Lenzi; Andrew Love; Michael Taliansky; Martina Becares; Sonia Zuñiga; Luis Enjuanes; Gergana Zahmanova; Ivan Minkov; Slavica Matic; Emanuela Noris; Ann Meyers; Alta Hattingh; Edward Rybicki; Oleg Kiselev; Nikolai Ravin; Michael Eldarov; Konstantin Skryabin; George Lomonossoff. 2013. "The Use of Transient Expression Systems for the Rapid Production of Virus-like Particles in Plants." Current Pharmaceutical Design 19, no. 31: 5564-5573.