Plant-based vaccines against viral hepatitis: A panoptic review

Abstract

The traditional vaccines against hepatitis have been instrumental in reducing the incidence of some types of viral hepatitis; however, the need for cost-effective, easily distributable, and needle-free vaccine alternatives has led to the exploration of plant-based vaccines. Plant-based techniques offer a promising avenue for producing viral hepatitis vaccines due to their low-cost cultivation, scalability, and the potential for oral administration. This review highlights the successful expression of hepatitis B surface antigens in plants and the subsequent formation of virus-like particles, which have shown immunogenicity in preclinical and clinical trials. The challenges such as achieving sufficient antigen expression levels, ensuring consistent dosing, and navigating regulatory frameworks, are addressed. The review considers the potential of plant-based vaccines to meet the demands of rapid vaccine deployment in response to outbreaks and their role in global immunization strategies, particularly in resource-limited settings. This review underscores the significant strides made in plant molecular farming and the potential of plant-based vaccines to complement existing immunization methods against viral hepatitis.

Key Words: Plant-based therapeutics; Plant vaccines; Edible vaccines; Viral hepatitis; Phytopharmacology and molecular pharming

Core Tip: The review article “Plant-based vaccines against viral hepatitis” explores the innovative approach of using plant-based systems to produce vaccines for viral hepatitis, particularly focusing on hepatitis B and C. Past and recent articles were identified and highlighted using MeSH terminologies on platforms such as PubMed Central, Google Scholar, Scopus, Web of Science, and Research Gate.

INTRODUCTION

The term “viral hepatitis” denotes liver inflammation caused by a viral infection. Hepatitis viruses are a group of viruses that primarily affect the liver[1]. Hepatitis A, B, C, D, and E are the different types of viral hepatitis, each resulting from a distinct virus. Each type has distinct pathways of transmission, clinical manifestations, geographical distribution, and prevention strategies[2]. Hepatitis B, C, and D are primarily transmitted by contaminated blood or bodily fluids, frequently through sexual contact or sharing needles. Hepatitis A and E are typically caused by ingesting contaminated food or water. Hepatitis B and C are major causes of chronic liver disease and liver cancer, leading to significant morbidity and mortality[3].

The World Health Organization (WHO) estimated in 2019 that 296 million people were living with chronic hepatitis B and 58 million with chronic hepatitis C globally, which causes more than 1.3 million deaths every year[4]. According to a study published in the Indian Journal of Medical Research, the prevalence of hepatitis B surface antigen (HBsAg), indicating chronic hepatitis B infection, in India ranges from 2% to 8% between regions, while hepatitis C virus (HCV) infection in India is estimated to be around 1% to 2%[5,6]. These numbers show how prevalent chronic hepatitis infections are, which, if untreated, can have a permanent adverse impact on health.

To avoid major problems from viral hepatitis, prevention is essential. The WHO’s global hepatitis strategy aims to eliminate viral hepatitis as a public health threat by 2030, aiming to reduce new infections by 90% and deaths by 65%[7]. Chronic hepatitis B and C impose a substantial economic burden due to direct healthcare costs and indirect costs such as loss of income and reduced productivity[8]. The WHO and the centers for disease control and prevention have initiated various programs for the surveillance, prevention, and control of hepatitis infections. Despite these efforts, hepatitis remains a global health challenge due to factors such as asymptomatic infections, lack of awareness, and limited access to vaccination and treatment in some regions.

CURRENT HEPATITIS VACCINES

A vital weapon in the fight against viral hepatitis is vaccination. Hepatitis A and B vaccinations, safe sex practices, refraining from sharing needles, and good hygiene are examples of preventive methods. Hepatitis A and B vaccinations are the two most often used hepatitis immunizations[9]. Given its high rate of infection prevention, the hepatitis A vaccination is advised for all children and adults who may be at higher risk. Similarly, highly effective is the hepatitis B vaccination, which is often administered in three or four doses. It is advised for all newborns and people who are more vulnerable[10]. For hepatitis C, there is currently no vaccine available, but research is ongoing to develop one. Vaccination against hepatitis E is also available in some regions where the disease is common, but its use is limited.

These vaccines have significantly reduced the incidence of new infections and the subsequent health complications associated with hepatitis. However, despite the success, there are ongoing challenges related to vaccine formulations, cost, storage requirements, and accessibility that necessitate exploring alternative vaccine strategies[11]. The numerous genotypes and subtypes of the virus that cause hepatitis, their complexity, and the requirement for long-term protection have made developing vaccines against the illness extremely difficult.

Existing hepatitis vaccines and their formulations

Hepatitis B: First-generation vaccines were initially developed from the plasma of chronic hepatitis B virus (HBV) carriers; These vaccines were effective but raised concerns about safety and supply sustainability. Second-generation vaccines are recombinant vaccines produced using yeast cells. They include brands such as Engerix-B and Recombivax HB. These vaccines are safer and more acceptable but still require cold chain storage. Third-generation vaccines include vaccines with adjuvants to enhance immunogenicity, such as the AS04-adjuvanted vaccines used in some hepatitis B and HPV vaccines[12].

Hepatitis A: Havrix and Vaqta are inactivated vaccines requiring refrigeration. They are administered in a two-dose schedule and provide long-lasting immunity[13].

Limitations of current hepatitis vaccines

The cost of hepatitis vaccines can be prohibitive, especially in low-resource settings. Most hepatitis vaccines require refrigeration, which poses a significant challenge in regions lacking adequate infrastructure[14]. The need for cold chain storage limits the reach of these vaccines to remote or underdeveloped areas. The cost and storage requirements compound accessibility issues. Additionally, the need for multiple doses (as in the case of hepatitis A and B vaccines) complicates the completion of the vaccination schedule, particularly in areas with limited healthcare access[15].

Need for alternative vaccine strategies

Developing a vaccine against HCV has been challenging due to the high genetic diversity of the virus and its ability to evade the immune system. There is currently no vaccine available for hepatitis D virus (HDV), and developing a vaccine presents unique challenges due to the dependence of the virus on HBV[16,17].

Given these limitations, there is a pressing need for alternative vaccine strategies. Developing hepatitis vaccinations has been a challenging but necessary effort to decrease the prevalence of viral hepatitis across the world. For managing viral hepatitis, thermostable formulations, single-dose vaccines, vaccines for non-responders, and innovative delivery system vaccines such as plant-based vaccinations provide a viable substitute to conventional vaccine manufacturing techniques[18]. These vaccines are being developed and produced using various technologies, improving their scalability, safety, and efficacy. With this review, we wish to raise awareness of the fact that vaccines derived from plants have a lot of potential for fighting viral hepatitis and will likely be the primary source of easily administered, inexpensive vaccinations in developing countries in the future.

Molecular pharming/farming

Molecular farming, often called biopharming or plant molecular farming, is the process of using plants to produce valuable proteins, peptides, or small molecules for various uses, such as industrial enzymes, medicines, and vaccinations[19,20]. The concept of using plants as bioreactors for vaccine production was pioneered in the late 1980s and has since evolved significantly, offering a viable alternative to traditional vaccine production methods that rely on microbial fermentation or animal cell cultures.

Examples: The synthesis of the anti-cancer drug paclitaxel in the leaves of the Pacific yew tree is among the most well-known examples of pharming. Two further examples are the production of the human immunodeficiency virus-neutralizing antibody 2G12 in tobacco plants and the lactoferrin enzyme in rice[21].

ADVANTAGES OF MOLECULAR FARMING

Cost and scalability of plant-based production systems are two important advantages. Large-scale plant cultivation is feasible in agricultural environments, making it possible to produce significant amounts of target molecules at a reasonable cost[22,23]. The production costs associated with plant-based systems are generally lower than those of traditional systems because they require less energy and fewer high-tech inputs. Plants do not require sterile conditions to the same extent as microbial or mammalian cell cultures, reducing the overall investment and operational costs[24].

Another significant advantage of plant-based vaccines is the potential for oral delivery. Vaccines produced in edible parts of plants could be administered orally, simplifying the vaccination process by eliminating the need for needles and syringes[24]. Plant-based vaccines often exhibit enhanced stability, reducing the need for cold chain logistics[25]. Proteins expressed in plant tissues, especially seeds, can remain stable at room temperature for extended periods. This is crucial for the distribution of vaccines in regions where cold storage facilities are inadequate or non-existent.

The safety profile of plant-based vaccines is potentially superior to traditional vaccines. As plants are free of human pathogens and the risk of contamination with animal viruses and prions is absent, the vaccines produced are inherently safer[26]. Furthermore, plants are very customizable and flexible in their production since they may be readily altered to generate particular drugs or proteins. Tobacco, maize, and rice are among the plant species effectively employed in molecular farming. It is possible to genetically modify these plants to express foreign genes that code for the desired chemical or protein. The target protein is then produced by the plant cells and may be extracted, refined, and used in various ways[27].

Plant-based vaccines allow for the formulation of multi-component vaccines by blending seeds from different transgenic lines expressing various antigens and showing versatility. Plant-based vaccines are biocompatible and provide enhanced mucosal immunity. Public acceptance is also good.

Technologies in developing plant-based vaccines

Plant-based vaccinations against viral hepatitis are made using various methods, each with distinctive advantages and uses. Listed here are a few important technologies:

Genetic engineering: Plant cells are genetically engineered to have genes expressing viral antigens. The primary techniques include codon optimization, subcellular targeting of proteins, and viral vectors. This enables the plants to produce the antigens, which, when delivered as a vaccine, may be utilized to elicit an immune response[19,28].

Transient expression: Transient expression is the process of inserting the antigen-encoding genetic material into the plant for a brief length of time, which causes the antigen to be generated rapidly. One common technique for transient expression is agroinfiltration, where Agrobacterium tumefaciens, a bacterium that naturally transfers DNA to plant cells, is used to deliver genetic material into plant tissues[29,30].

Plant transformation techniques: To transfer foreign genes into plants, methods such as biolistic and Agrobacterium-mediated transformation are employed. Stable transformation involves the integration of the desired genes into the plant genome, allowing the plant to express the vaccine antigen continuously over its lifetime and pass the trait to its progeny[31]. This method is used for long-term production of vaccines[32,33].

Downstream processing technologies: Purification and formulation techniques are examples of downstream processing technologies that ensure plant vaccines’ safety, stability, and effectiveness. These technological advancements have been made to guarantee that plant-based vaccinations are safe for human use and to satisfy regulatory requirements[34].

Plant breeding and cultivation practices: The development of vaccines based on plants has also benefited from improvements in plant breeding and growing techniques. These techniques provide a consistent and affordable supply of antigens by optimizing the development and output of plants that produce vaccines[33,34].

DEVELOPMENT OF PLANT-BASED HEPATITIS VACCINES

Selection of appropriate antigens

The selection of appropriate hepatitis antigens for plant-based vaccine development involves addressing several challenges, that are informed by immunogenicity, antigenicity, and conservation across viral strains. These challenges are critical to ensure that the resulting vaccine is effective, safe, and capable of providing broad protection against viral strains.

The key challenges in appropriate selection are achieving high immunogenicity and maintaining antigenicity. For example, the envelope protein 2 (E2) glycoprotein of HCV is a primary target for neutralizing antibodies, and its antigenic regions must be conserved in the plant-derived vaccine[35]. Due to the virus’s enormous genetic diversity, with seven known genotypes and more than 80 subtypes, and its ability to elude the immune system, producing an HCV vaccine has proven difficult[36]. Selecting antigens that are conserved across these strains is essential for a vaccine that can provide broad protection.

The stability of antigens during processing and storage is essential for maintaining vaccine efficacy. Ensuring that plant-derived antigens are stable and that production methods yield consistent results is challenging. Controlling the dose for self-administered oral vaccines is also difficult and remains to be addressed[37,38].

Studies evaluating immunogenicity, safety and efficacy of plant-based vaccines

Hepatitis A: In recent years, hepatitis A has witnessed promising development in plant-based vaccinations that use genetic engineering and plant transformation methods. Compared to conventional vaccinations, these have benefits, including cost-effectiveness, scalability, and even enhanced stability. Numerous investigations have examined various techniques for creating plant-based vaccinations against hepatitis A, emphasizing antigen expression, effectiveness, and safety.

According to a study by Chuang et al[39], the hepatitis A virus (HAV) structural protein virus particle (VP1) was successfully expressed in transgenic tobacco plants. The study results demonstrated that the VP1 protein generated from plants was immunogenic and may induce a specific immune response in mice. This strategy emphasizes the possibility of developing hepatitis A vaccinations using plants.

Mason et al[40] used lettuce plants to produce the HAV capsid protein VP1 in a different study. The researchers showed the VP1 protein produced from plants to be structurally and antigenically identical to the natural protein. Mice immunized with the plant-derived VP1 protein developed a particular immunological response, indicating lettuce plants might be used as a platform to produce hepatitis A vaccine.

Hepatitis B: The HBsAg was chosen as the target antigen for vaccination in the process of developing a plant-based hepatitis B vaccine. Using genetic engineering methods, the gene encoding HBsAg was then introduced into the genome of a plant, such as potatoes or tobacco. After that, the plant was grown in carefully regulated environments to manufacture the HBsAg protein.

The efficacy of plant-based hepatitis B vaccinations in preclinical and clinical trials has been shown in several studies. For example, research published in the journal Vaccine in 2005 found that mice could produce a potent immune response to a plant-based hepatitis B vaccine made in tobacco plants[41]. According to another investigation, a plant-based hepatitis B vaccine was successfully produced in lettuce plants[42]. The vaccine also proved to elicit an immunological response in mice.

Hepatitis C: One viable strategy for addressing the worldwide severe health concern of HCV infection is developing a plant-based hepatitis C vaccine. Plants are used as bioreactors to generate HCV antigens, making plant-based vaccinations a scalable and affordable option.

The development of a plant-based hepatitis C vaccine usually entails a few crucial phases. Initially, scientists identify appropriate HCV antigens, such as envelope glycoproteins (E1 and E2), that might trigger a potent immune reaction[43]. Next, using recombinant DNA technology, these antigens are genetically inserted into the genome of a plant, such as a potato, tomato, or tobacco plant. The plant produces the HCV antigens under strict growth conditions, which may then be refined and combined into a vaccine.

Plant-based hepatitis C vaccinations were found to be both feasible and productive in a number of experiments. For instance, research published in 2011 described how tobacco plants could produce HCV envelope glycoproteins successfully. Plant-derived glycoproteins have been demonstrated to elicit particular antibodies against HCV in mice, indicating their immunogenicity. The researchers genetically modified tobacco plants to generate a fusion protein that included many HCV antigens. Mice immunized with the fusion protein produced from plants developed significant humoral and cellular immune responses against HCV[44].

Hepatitis D: The peculiarity of the virus and its dependence on HBV have made the development of a hepatitis D vaccination difficult. Conventional vaccination strategies targeting the HBsAg have not demonstrated efficacy against HDV. However, in recent years, there have been some promising advancements.

A potential strategy for creating a hepatitis D vaccine is to utilize recombinant DNA technology to generate the hepatitis delta antigen, the HDV antigen, in yeast or mammalian cells[45]. Using this recombinant antigen can then provoke an immunological reaction against HDV.

HIGHLIGHTS OF LANDMARK EVENTS IN THE DEVELOPMENT OF HEPATITIS B AND HEPATITIS C PLANT-BASED VACCINES

These are mentioned in Table 1[46-52].

Table 1 Landmark events in the development of hepatitis B and hepatitis C plant-based vaccines.

Virus	Plant species	Antigen	Platforms used	Ref.
Hepatitis B	Tobacco	Surface antigen	Transformation	Mason et al[46]
	Lettuce, lupin	Surface antigen	Expression	Kapusta et al[47]
	Potato	Surface antigen	Genetic engineering	Kong et al[48]
	Banana	Surface antigen	Expression	Kumar et al[49]
Hepatitis C	Tobacco	Sequence HVR1 from E2 protein	Codon optimization	Piazzolla et al[50]
Hepatitis E	Tomato	ORF 2 partial gene	Agroinfiltration	Ma et al[51]
	Potato	Capsid protein	Genetic engineering	Maloney et al[52]

HVR1: Hypervariable region 1; E2: Envelope protein 2; ORF: Open reading frame.

FUTURE DIRECTIONS

The integration of newer biotechnological techniques, such as transient expression systems and the use of novel plant species, may enhance antigen yield and stability, addressing some of the current limitations. Additionally, establishing international regulatory frameworks and guidelines specific to plant-based vaccine production will be crucial in advancing these vaccines from the laboratory to the bedside.

CONCLUSION

Developing plant-based vaccines against viral hepatitis represents a promising frontier in immunization strategies, particularly for hepatitis B. Over the past decades, significant advancements have been made in plant molecular farming, which has paved the way for producing virus-like particles and other antigenic proteins in plant systems. Recent advancements have also seen the expression of these antigens in different plant systems, such as potatoes and rice, which promise ease of scalability and the potential for edible vaccines, simplifying the administration process and enhancing accessibility. However, despite these promising developments, the transition from laboratory success to clinical and commercial application has been slow. Challenges such as low yields, regulatory hurdles, and the need for consistent and stable expression of antigens in plants remain significant obstacles. Moreover, the immunogenicity and efficacy of these plant-derived vaccines need to be validated in human clinical trials, which are still limited.