Green Biologics: Harnessing the Power of Plants to Produce Pharmaceuticals.

Plants are increasingly used for the production of high-quality biological molecules for use as pharmaceuticals and biomaterials in industry. Plants have proved that they can produce life-saving therapeutic proteins (Elelyso™-Gaucher's disease treatment, ZMapp™-anti-Ebola monoclonal antibodies, seasonal flu vaccine, Covifenz™-SARS-CoV-2 virus-like particle vaccine); however, some of these therapeutic proteins are difficult to bring to market, which leads to serious difficulties for the manufacturing companies. The closure of one of the leading companies in the sector (the Canadian biotech company Medicago Inc., producer of Covifenz) as a result of the withdrawal of investments from the parent company has led to the serious question: What is hindering the exploitation of plant-made biologics to improve health outcomes? Exploring the vast potential of plants as biological factories, this review provides an updated perspective on plant-derived biologics (PDB). A key focus is placed on the advancements in plant-based expression systems and highlighting cutting-edge technologies that streamline the production of complex protein-based biologics. The versatility of plant-derived biologics across diverse fields, such as human and animal health, industry, and agriculture, is emphasized. This review also meticulously examines regulatory considerations specific to plant-derived biologics, shedding light on the disparities faced compared to biologics produced in other systems.

The Re-Emergence of Hepatitis E Virus in Europe and Vaccine Development.

Hepatitis E virus (HEV) is one of the leading causes of acute viral hepatitis. Transmission of HEV mainly occurs via the fecal-oral route (ingesting contaminated water or food) or by contact with infected animals and their raw meat products. Some animals, such as pigs, wild boars, sheep, goats, rabbits, camels, rats, etc., are natural reservoirs of HEV, which places people in close contact with them at increased risk of HEV disease. Although hepatitis E is a self-limiting infection, it could also lead to severe illness, particularly among pregnant women, or chronic infection in immunocompromised people. A growing number of studies point out that HEV can be classified as a re-emerging virus in developed countries. Preventative efforts are needed to reduce the incidence of acute and chronic hepatitis E in non-endemic and endemic countries. There is a recombinant HEV vaccine, but it is approved for use and commercially available only in China and Pakistan. However, further studies are needed to demonstrate the necessity of applying a preventive vaccine and to create conditions for reducing the spread of HEV. This review emphasizes the hepatitis E virus and its importance for public health in Europe, the methods of virus transmission and treatment, and summarizes the latest studies on HEV vaccine development.

First Insight into the Seroepidemiology of Hepatitis E Virus (HEV) in Dogs, Cats, Horses, Cattle, Sheep, and Goats from Bulgaria.

In recent years, hepatitis E virus (HEV) infection has been found to be widespread among different animal species worldwide. In Bulgaria, high HEV seropositivity was found among pigs (60.3%), wild boars (40.8%), and East Balkan swine (82.5%). The aim of the present study was to establish the seroprevalence of HEV among dogs, cats, horses, cattle, sheep, and goats in Bulgaria. In total, 720 serum samples from six animal species were randomly collected: dogs-90 samples; cats-90; horses-180; cattle-180; sheep-90; and goats-90. The serum samples were collected from seven districts of the country: Burgas, Kardzhali, Pazardzhik, Plovdiv, Sliven, Smolyan, and Stara Zagora. The animal serum samples were tested for HEV antibodies using the commercial Wantai HEV-Ab ELISA kit (Beijing, China). The overall HEV seroprevalence among different animal species from Bulgaria was as follows: dogs-21.1%; cats-17.7%; horses-8.3%; cattle-7.7%; sheep-32.2%; and goats-24.4%. We found the lowest overall HEV seropositivity in Plovdiv district (6.2%; 4/64; p = 0.203) and Smolyan district (8.8%; 4/45; p = 0.129), vs. the highest in Pazardzhik district (21.6%; 29/134; p = 0.024) and Burgas district (28.8%; 26/90; p = 0.062). To the best of our knowledge, this is the first serological evidence of HEV infection in dogs, cats, horses, cattle, sheep, and goats from Bulgaria. We found high HEV seropositivity in small ruminants (sheep and goats), moderate seropositivity in pets (dogs and cats), and a low level of seropositivity in large animals (horses and cattle). Previous Bulgarian studies and the results of this research show that HEV infection is widespread among animals in our country. In this regard, the Bulgarian health authorities must carry out increased surveillance and control of HEV infection among animals in Bulgaria.

The Plant Viruses and Molecular Farming: How Beneficial They Might Be for Human and Animal Health?

Plant viruses have traditionally been studied as pathogens in the context of understanding the molecular and cellular mechanisms of a particular disease affecting crops. In recent years, viruses have emerged as a new alternative for producing biological nanomaterials and chimeric vaccines. Plant viruses were also used to generate highly efficient expression vectors, revolutionizing plant molecular farming (PMF). Several biological products, including recombinant vaccines, monoclonal antibodies, diagnostic reagents, and other pharmaceutical products produced in plants, have passed their clinical trials and are in their market implementation stage. PMF offers opportunities for fast, adaptive, and low-cost technology to meet ever-growing and critical global health needs. In this review, we summarized the advancements in the virus-like particles-based (VLPs-based) nanotechnologies and the role they played in the production of advanced vaccines, drugs, diagnostic bio-nanomaterials, and other bioactive cargos. We also highlighted various applications and advantages plant-produced vaccines have and their relevance for treating human and animal illnesses. Furthermore, we summarized the plant-based biologics that have passed through clinical trials, the unique challenges they faced, and the challenges they will face to qualify, become available, and succeed on the market.

Plant-Derived Recombinant Vaccines against Zoonotic Viruses.

Emerging and re-emerging zoonotic diseases cause serious illness with billions of cases, and millions of deaths. The most effective way to restrict the spread of zoonotic viruses among humans and animals and prevent disease is vaccination. Recombinant proteins produced in plants offer an alternative approach for the development of safe, effective, inexpensive candidate vaccines. Current strategies are focused on the production of highly immunogenic structural proteins, which mimic the organizations of the native virion but lack the viral genetic material. These include chimeric viral peptides, subunit virus proteins, and virus-like particles (VLPs). The latter, with their ability to self-assemble and thus resemble the form of virus particles, are gaining traction among plant-based candidate vaccines against many infectious diseases. In this review, we summarized the main zoonotic diseases and followed the progress in using plant expression systems for the production of recombinant proteins and VLPs used in the development of plant-based vaccines against zoonotic viruses.

Development and Optimization of an Enzyme Immunoassay to Detect Serum Antibodies against the Hepatitis E Virus in Pigs, Using Plant-Derived ORF2 Recombinant Protein.

Hepatitis E is an emerging global disease, mainly transmitted via the fecal-oral route in developing countries, and in a zoonotic manner in the developed world. Pigs and wild boar constitute the primary Hepatitis E virus (HEV) zoonotic reservoir. Consumption of undercooked animal meat or direct contact with infected animals is the most common source of HEV infection in European countries. The purpose of this study is to develop an enzyme immunoassay (EIA) for the detection of anti-hepatitis E virus IgG in pig serum, using plant-produced recombinant HEV-3 ORF2 as an antigenic coating protein, and also to evaluate the sensitivity and specificity of this assay. A recombinant HEV-3 ORF2 110-610_6his capsid protein, transiently expressed by pEff vector in Nicotiana benthamiana plants was used to develop an in-house HEV EIA. The plant-derived HEV-3 ORF2 110-610_6his protein proved to be antigenically similar to the HEV ORF2 capsid protein and it can self-assemble into heterogeneous particulate structures. The optimal conditions for the in-house EIA (iEIA) were determined as follows: HEV-3 ORF2 110-610_6his antigen concentration (4 µg/mL), serum dilution (1:50), 3% BSA as a blocking agent, and secondary antibody dilution (1:20 000). The iEIA developed for this study showed a sensitivity of 97.1% (95% Cl: 89.9-99.65) and a specificity of 98.6% (95% Cl: 92.5-99.96) with a Youden index of 0.9571. A comparison between our iEIA and a commercial assay (PrioCHECK™ Porcine HEV Ab ELISA Kit, ThermoFisher Scientific, MA, USA) showed 97.8% agreement with a kappa index of 0.9399. The plant-based HEV-3 ORF2 iEIA assay was able to detect anti-HEV IgG in pig serum with a very good agreement compared to the commercially available kit.

Efficient Production of Chimeric Hepatitis B Virus-Like Particles Bearing an Epitope of Hepatitis E Virus Capsid by Transient Expression in Nicotiana benthamiana.

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.

A plant-based transient expression system for the rapid production of highly immunogenic Hepatitis E virus-like particles.

OBJECTIVE: Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. The aim of the study is the development of plant expression system for the production of virus-like particles formed by HEV capsid and the characterization of their immunogenicity. RESULTS: Open reading frame (ORF) 2 encodes the viral capsid protein and possesses candidate for vaccine production. In this study, we used truncated genotype 3 HEV ORF 2 consisting of aa residues 110 to 610. The recombinant protein was expressed in Nicotiana benthamiana plants using the self-replicating potato virus X-based vector pEff up to 10% of the soluble protein fraction. The yield of HEV 110-610 after purification was 150-200 µg per 1 g of green leaf biomass. The recombinant protein formed nanosized virus-like particles. The immunization of mice with plant-produced HEV 110-610 protein induced high levels of HEV-specific serum antibodies. CONCLUSIONS: HEV ORF 2 (110-610 aa) can be used as candidate for the development of a plant-produced vaccine against Hepatitis E.

Increasing Hepatitis E Virus Seroprevalence in Domestic Pigs and Wild Boar in Bulgaria.

(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.

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.

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.