Immunostimulant plant proteins: Potential candidates as vaccine adjuvants.

The COVID-19 pandemic is shaking up global scientific structures toward addressing antibiotic resistance threats and indicates an urgent need to develop more cost-effective vaccines. Vaccine adjuvants play a crucial role in boosting immunogenicity and improving vaccine efficacy. The toxicity and adversity of most adjuvant formulations are the major human immunization problems, especially in routine pediatric and immunocompromised patients. The present review focused on preclinical studies of immunoadjuvant plant proteins in use with antiparasitic, antifungal, and antiviral vaccines. Moreover, this report outlines the current perspective of immunostimulant plant protein candidates that can be used by researchers in developing new generations of vaccine-adjuvants. Future clinical studies are required to substantiate the plant proteins' safety and applicability as a vaccine adjuvant in pharmaceutical manufacturing.

INTRODUCTION

Vaccines represent one of the greatest public health advances of the 20th century, the vaccinations make a remarkable difference in reducing morbidity and mortality worldwide (Schijns et al., 2020). According to the world health organization (WHO), it is estimated that it prevents between two and three million deaths annually https://www.who.int/immunization/global_vaccine_action_plan/GVAP_doc_2011_2020/en/. The rational vaccine construction is shaped by antigen selection, type of vaccine adjuvants, delivery route, linkers, and histidine tags (Coffman, Sher, & Seder, 2010). Vaccine adjuvant is an important vaccine component that could enhance overall vaccine immunogenicity and decline the likelihood of initiating immune tolerance (Saylor, Gillam, Lohneis, & Zhang, 2020).

Effective vaccines induce adaptive immunity in response to future infections or to treat an already diagnosed disease (Schijns et al., 2021). Many infectious diseases are prevented by vaccination such as diphtheria, haemophilus influenza type b, hepatitis B, human papillomavirus (HPV), measles, mumps, pertussis, pneumococcal diseases, poliovirus, rotavirus, rubella, tetanus, and tuberculosis (Rappuoli, Pizza, Del Giudice, & De Gregorio, 2014). Moreover, the therapeutic vaccines propose paradigm shifts in oncology healthcare and target breast, colorectal, lung, pancreatic, and prostate cancers (Boukhebza, Bellon, Limacher, & Inchauspé, 2012). Besides, peptide cancer vaccines (PCVs) are designed to target tumor‐specific antigens and tumor‐associated antigens (Tsung & Norton, 2016). The PCVs possess numerous advantages since they lack significant toxicity associated with chemotherapy and radiotherapy (Sotillo et al., 2015).

Despite these vaccines' success and recent advances in vaccinology, there are many diseases for which the development of safe and effective vaccine remains elusive, such as AIDS, arboviral disease, bird flu, chickenpox, Ebola, herpes, Zika, malaria, and hepatitis C other than different cancer types. Regulatory/economic/ethical factors associated with clinical trials and the unique characters of the individual pathogens could be attributed to the unavailability of vaccines for these emerging pathogens and intractable diseases (Tannock, Kim, & Xue, 2020). In addition, traditional design methods to produce a functionally protective vaccine are still a simple reason that hinders production. Hence, it is important to consider new technologies for the development and design of vaccine formulas (Vrba, Kirk, Brisse, Liang, & Ly, 2020).

Plant‐based immunoadjuvants have the potential to optimize immune responses, indeed they can be considered a promising adjuvant candidate for the development of novel vaccines (Lakshmi, Kumar, Pawar, Sudheesh, & Pawar, 2018).

The present review illuminates the critical role of adjuvants in vaccination strategies, as well as the recent contribution of plant protein immunomodulators to preclinical immunization protocols, with an emphasis on promising plant proteins that could impact future vaccine applications.

ADJUVANTS

Since the early 1920s, vaccine adjuvants have been used as chemical substances that strengthen and maintain immune responses to antigens (Gupta & Chaphalkar, 2015a, 2015b; Schijns et al., 2020). Although high‐purity vaccines showed a wide therapeutic safety index, an impaired immunogenic potential has been recorded. Consequently, there is a growing demand for using immune potentiators in vaccination protocols (Fendler et al., 2022; Rüthrich, Giesen, Mellinghoff, Rieger, & von Lilienfeld‐Toal, 2022).

Numerous vaccine types induce artificial adaptive immunity such as live attenuated, inactivated “killed,” subunit peptide or polysaccharide, RNA vaccines, DNA vaccines, recombinant viral vector vaccines, and bacterial vector vaccines. Though live attenuated vaccines mimic the closest form of natural infection, they usually induce mild or moderate adverse effects, however, they are still highly effective vaccines. The potential for virulence reversion highlights a severe threat that emphasizes the importance of rational vaccine strain composition (Christensen, 2016).

In contrast to attenuated live vaccines, dead subunit vaccines (inactivated “killed” subunit peptide or polysaccharide), present weaker efficiency and shorter duration of immunity. Therefore, adjuvant booster immunizations are required (Gupta & Chaphalkar, 2015a, 2015b). Alternatively, DNA and recombinant viral vector vaccines significantly promoted antigen‐specific cellular and humoral immune responses without adjuvants. Despite the advantages of the recombinant viral vector vaccine, few vaccines are licensed (Moingeon, Haensler, & Lindberg, 2001). Moreover, the immunogenicity of DNA vaccines in humans still needs to lot of effort to certify their immunological efficiency and safety (Hobernik & Bros, 2018).

The mRNA is a very large molecule, prone to degradation by nucleases, and intrinsically unstable. Hence the stability of mRNA vaccines is considered the main challenge that impedes successful translation into drugs. However, mRNA chemical modification partially solved such a hurdle, but intracellular delivery of mRNA still represents a major demand (Wadhwa, Aljabbari, Lokras, Foged, & Thakur, 2020). This limitation of DNA/RNA‐based vaccines highlights the need for novel vaccine adjuvant constructs.

Adjuvants play a significant role in immune responses and they are mentioned below:

Boosting the immunogenicity of weak antigens: to elicit adequate antibody responses (Wang & Xu, 2020).

Dose‐sparing: decrease antigen doses, which in consequence lower vaccine production costs and makes it affordable worldwide. The adjuvant use can reduce the incidents of any global vaccination shortages. The BCG tuberculosis vaccine was an example of a worldwide demand‐deficient incidence in 2015 when 180 million doses were required, and only 107 million doses were available (Nicholls, Madera, & Hancock, 2010).

Enhancing the onset and duration of the immune response: facilitating phagocytosis and antigen detection by immune cells, which prolongs the immunological memory (Schijns et al., 2021).

Modulation of antibody specificity, avidity, and isotope distribution: adjuvants expand B cell diversity release and improve the durability and quality of antibody responses to destroy pathogens (Reed, Orr, & Fox, 2013).

Initiation of mucosal immune responses: although most pathogens enter through mucosal channels such as the nose, mouth, or genital tract, vaccination is primarily applied to parenteral modes that stimulate the development of distinct isotype antibodies. Adjuvant oral immunization allows access to the intestine, which is the body's largest immune organ (Smith et al., 2020).

Improving vaccine efficacy for weak responders: immunostimulant adjuvants are able to activate both innate and adaptive immunity. An attenuated microorganism may cause life‐threatening health risks to people with a compromised immune system such as children, the elderly, and those patients who have a weak immune system (Reyna‐Margarita et al., 2019). Therefore, the administration of non‐traditional vaccines, along with immunostimulant adjuvants, might help in enhancing its efficacy in weak responders.

ADJUVANT TYPES AND CHALLENGES

The immune potentiator adjuvants can activate innate immunity directly as cytokines or indirectly through pattern‐recognition receptors (PRRs) (Ong, Lian, Kawasaki, & Kawai, 2021), while the use of mucosal adjuvants can induce local mucosal immunity (Savelkoul, Ferro, Strioga, & Schijns, 2015). The recent approach to optimize vaccine immune responses is the use of different adjuvant combinations that could trigger different signaling pathways (Lee & Nguyen, 2015).

Adjuvants can be primarily classified into two main types based on their particle size (Makwana et al., 2018) (a) particulate (liposomes, w/o emulsions, aluminum salts, nanoparticles, and microparticles) and (b) nonparticulate (protein, polysaccharide, saponin, lipid, bacterial toxins, and cytokines) (Gupta & Chaphalkar, 2015b; Schijns et al., 2020, 2021). Further classification based on mechanism of action, can be divided into delivery systems adjuvants, immunostimulatory, mucosal adjuvants, and adjuvants combination (Apostólico et al., 2016).

The delivery systems cover a wide range of materials such as mineral salts (alum), emulsions, and microparticles (virus‐like particles) (Pashine, Valiante, & Ulmer, 2005). Until now aluminum hydroxide and aluminum phosphate (alum) are the most commonly used adjuvants in human vaccinations, though calcium phosphate and oil emulsions have also been used (Gupta & Siber, 1995). Alum has a fair safety record, but comparative studies have indicated that it is a poor adjuvant for cell‐mediated immunity and a weak adjuvant of antibody production to protein subunits. Furthermore, its (IgE) antibody response has been linked to some allergic reactions (Singh & O'Hagan, 2002).

Emulsion adjuvants have a long history of use, the water‐in‐oil (w/o) emulsion is classified into two forms; complete Freund's adjuvant (CFA) (mineral oil, emulsifier, and killed bacteria) and incomplete Freund's adjuvant, which has the same composition as CFA, but lacks the bacteria (Valverde et al., 2017). The virus‐like particle adjuvant is formed from capsid or envelope structural viral proteins that mimic intact virus size and shape. While liposomes adjuvant is characterized by their biodegradability and biocompatibility. However, manufacturing costs and the generation of a weak immune response are still the major limitations of their application (Ali, Singh, & Datusalia, 2021).

Only a few adjuvants, such as aluminum salts, virosomes, MF59TM, AS01TM, AS03TM, AS04TM, and CpG, have been licensed for use in human vaccinations (Schijns et al., 2020). The incorporation of an adjuvant into a new or licensed vaccine is still a challenge that could take many years (Apostólico et al., 2016). Indeed, several factors contribute to the relatively slow development of new adjuvanted vaccines, where the assessment of adjuvant safety is critical. This necessitates large phase III studies with adequate sample sizes, which can take several years (Del Giudice, Rappuoli, & Didierlaurent, 2018).

Immunoadjuvants are a subcategory of immunomodulators that can increase the effectiveness of vaccines (Smith et al., 2020). Cytokines, which come in the form of interleukins (ILs), interferons, chemokines, and other soluble extracellular proteins or glycoproteins, are essential for both innate and acquired immunity. These cytokines maintain physiological stability in all nucleated cells (Mbawuike, Wyde, & Anderson, 1990). Based on the induction of cytokines signal to the bloodstream, several neurochemicals, neuroendocrine, and neuroimmune substances can be delivered (Jakobsen, Saeland, Gizurarson, Schulz, & Jónsdóttir, 1999; Morel & Turner, 2010).

Different cytokines were reported as adjuvants that could induce antigen‐specific serum/mucosal antibody and cell‐mediated immunity. The most notable cytokine adjuvants are granulocyte/macrophage colony‐stimulating factor (GM‐CSF) (Faries, Hsueh, Ye, Hoban, & Morton, 2009), IFN (Le Bon et al., 2001), IL‐1 (Staats & Ennis, 1999), IL‐2 (Shah & Abraham, 1992), IL‐6 (Kishimoto, 2006), IL‐12 (Bermúdez‐Humarán et al., 2005), IL‐15 (Yang & Lundqvist, 2020), IL‐18 (Mountforda & A., 2000), and chemokines. Moreover, activating major histocompatibility complex (MHC), costimulatory signals, or related intracellular signaling is also considered a different adjuvant‐reported mechanism (Mohan, Verma, & Nageswara Rao, 2013).

Compatibility of adjuvant‐vaccine components is critical and evaluation tests of immunogenicity/safety for the formulation are essential (Apostólico et al., 2016). The demonstration of the added value of adjuvant action over plain antigen necessitates the generation of additional evidence to validate the use of an adjuvant, thus increasing the time required for vaccine development (Del Giudice et al., 2018). The selection of adjuvants is determined by balancing the need for adjuvanticity and an acceptable low level of side effects (Gupta & Siber, 1995). The design of potential and less toxic adjuvants with optimal matching for specific antigens has typically been an empirical fact (Awate, Babiuk, & Mutwiri, 2013).

A better understanding of adjuvant mechanisms likely has sped up their development. New technologies, such as systems vaccinology, are being used earlier in the development of novel vaccine adjuvant formulations in the hopes of speeding up their development and introduction into clinical practice (O'Hagan, Friedland, Hanon, & Didierlaurent, 2017). Moreover, non‐biodegradability, instability, and large‐scale production costs are the main factors affecting adjuvant development (Wallis, Shenton, & Carlisle, 2019). Advancement in these areas could lead to the availability of economically feasible, potent vaccines that could not only aid in managing clinical autoimmune‐based manifestations but also created new platform against viral pathogens and diseases for which no interventions currently exist.

CURRENT HYPOTHESIS MECHANISM OF ADJUVANTS

Immunomodulatory peptides act directly on immune system‐specific cells, however, to date, the mechanisms behind these interactions are unclear. Even the currently available adjuvants included in the licensed, function of vaccines' remains underdeveloped. However, advances that have occurred during the past decade are beginning to yield deeper insights into the mechanism of action of adjuvants and are revitalizing the process of adjuvant discovery and development (Pulendran, Arunachalam, & O'Hagan, 2021).

Although the major focus of adjuvant discovery during the past decade has been to target the Toll‐like receptor (TLR) pathway, the dendritic cells (DCs) have long been considered the primary cellular targets of vaccine adjuvants (Pulendran & Ahmed, 2011). Recent reports evidenced that stimulation of TLR ligands, PRRs, and NLRs could be targeted by adjuvants to induce an immunostimulant effect (Ho, Huis In't Veld, Raaijmakers, & Adema, 2018). Additionally, there is emerging evidence that adjuvants that are already in use in the clinic may activate immune responses via tissue damage pathway, where the release of a plethora of damage‐associated molecular patterns, such as ATP or uric acid, or fragments of DNA or RNA or high mobility group box 1, could activate DCs to stimulate adaptive immunity (De Lorenzo, Ferrari, Cervone, & Okun, 2018). The adjuvants' main molecular targets for currently licensed vaccines are represented in Figure 1.

FIGURE 1

Molecular targets of adjuvants in currently licensed vaccines

Peptide adjuvant with either arginine in the N‐ or C‐terminal regions, tryptophan chain, phosphoserine residues, or glutamine units, could recognize by immunological cells' opioid receptors. Such interaction with δ‐, 𝜇‐, or 𝜅‐type opioid receptors can regulate the peripheral immune system (Haque, Chand, & Kapila, 2008). Additionally, the presence of arginine at the C‐terminal is one of the structural characteristics of ACE inhibitory peptides that also may be related to the immunomodulatory activity. Besides, the membrane‐bound receptors of T cells could recognize the N‐ or C‐terminal of peptides and stimulate charge‐changing lymphokines (Santiago‐López, Hernández‐Mendoza, Vallejo‐Cordoba, Mata‐Haro, & González‐Córdova, 2016). The peptides could also activate TLRs—which are typically expressed on DCs. They can sense highly conserved pathogen‐associated molecular patterns (Hartmann & Meisel, 2007) in microbes—resulting in activation of DCs, which consequently stimulates antigen‐specific T and B cell responses (Kitts & Weiler, 2003; Udenigwe & Aluko, 2012).

In addition, the coordinated action of the RIPK1‐dependent cell death pathway and NF‐κB‐dependent inflammation synergize to promote enhanced cross‐priming of CD8+ T cells (Yatim et al., 2015). Taken together, these findings suggest that an adjuvant that targeted lymph node macrophages transiently induces RIPK3‐mediated pathways, along with cell death, and stimulates CD8+ T cell response. Summary for different types of adjuvants mechanism according to Pulendran et al., 2021 shown in Figure 2.

FIGURE 2

Summary of different adjuvants' mechanisms of action according to Pulendran et al. (2021). Abbreviations: pattern recognition receptors (PRRs), tissue‐resident memory t cells (TRM), nucleotide‐binding oligomerization domain (noD)‐like receptors (nlRs), retinoic acid‐inducible gene I (RIG‐I), cGAS‐stimulator of interferon genes (STING), C‐type lectin receptors (CLRs), receptor‐interacting serine/threonine protein kinase 3 (RIPK3), nF‐κB‐dependent inflammation, nuclear factor kappa B (NF‐κB), damage‐associated molecular patterns (DAMPs), pathogen‐associated molecular patterns (PAMPs)

PLANTS AND IMMUNOSTIMULATION

Plants are bio‐factories of diverse active compounds that can significantly contribute to vaccine immunomodulation (Bhuiyan, Howlader, Raihan, & Hasan, 2020). Many studies revealed the efficacy of herbal medicine to induce the release of different cytokines (Burns, Zhao, Taylor, & Spelman, 2010; Woods et al., 2017). The plant phenolics that bind to the carrier protein (purified antigen) and plant proteins (e.g., lectins based on binding to carbohydrate cellular receptors, stimulating cell signaling, and triggering the immunological response) play an important role as a natural vaccine adjuvant (Gupta & Chaphalkar, 2015a, 2015b).

Several plant‐based vaccine adjuvants have been purified from Boswellia serata, Picrorhiza kurroa (Khajuria et al., 2007), and Emblica officinalis (Gupta & Chaphalkar, 2015b). Furthermore, medicinal plants including Asparagus racemosus, Emblica officinalis, Withania somnifera, Panax notoginseng, and Tinospora cordifolia have shown significant immunostimulatory activity at the humoral level (Gupta & Chaphalkar, 2015b). Aristolochia longa, Datura stramonium, Marrubium vulgare, Sinapis nigra, Delphynium staphysagria, Lepidium sativum, Ammi visnaga, and Tetraclinis articulata extracts markedly alter the proliferation of immune cells (Daoudi, Aarab, & Abdel‐Sattar, 2013; Gupta & Chaphalkar, 2015b).

Polypodium leucotomos stimulates interleukin IL‐1α, IL‐1β, and TNFα release in vitro (Bernd et al., 1995). The immune‐modulating effects of astragalus root and elderberry fruit extracts were examined in bone marrow‐derived murine DCs. The ELISA and RT‐PCR tests indicated that both extracts enhanced IFN‐ß production, increased endocytosis in immature DCs, upregulated toll‐like receptor 3, and enhanced the IL‐12, IL‐6, IL‐1b, and TNFα cytokines release (Aldahlawi, 2016). Also, induction, maturation, and differentiation of DCs were reported by Mucuna pruriens extract (Kurokawa et al., 2011). The Abelmoschus esculentus increased the expression of IL‐12, interferon IFNγ, and stimulated DCs by modulating the expression of class II‐MHC and costimulatory CD+80/86 molecules (Aldahlawi, 2016; Sheu & Lai, 2012).

Garlic is one of the most used flavoring plants in cooking, the plant and its formulations have been extensively used as an immunomodulator in vitro and in vivo. Modulation of cytokine profile, in addition to direct stimulation of immune cells, has been reported as the main mechanism of action (Moutia, Habti, & Badou, 2018). Furthermore, the biological studies of Aloe vera have shown its ability to improve the production of TNFα and IL‐6 levels in human peripheral blood macrophages (Ali et al., 2021).

PLANT IMMUNOMODULATOR PROTEINS

According to the food and agriculture organization of the United Nations, the latest forecast for world cereal production in 2021 stands at 2.793 million tonnes, 0.8% higher year on year (Canton, 2021). Grains are important sources of diet proteins, and for some populations, they are the major protein sources (Duranti, 2006). Up to 20% of legumes' dry weight represents their protein, therefore it may be a potential source of peptides with varying biological activities (Clemente, MacKenzie, Johnson, & Domoney, 2004). Nevertheless, the immunomodulatory activity of peptides derived from plant proteins that have been not fully explored (Santiago‐López et al., 2016).

The composition, sequence, hydrophobicity, and length of plant protein amino acids are all vital parameters that influence the immunomodulatory activity of a hydrolysate (Chalamaiah, Yu, & Wu, 2018). Short peptides have immense potential for vaccine adjuvant development as they play a significant immunomodulatory role by stimulating the natural killer cells (Dong & Kobinger, 2013).

Various plant proteins and peptides have been used in a vaccination strategy against influenza virus, Neospora lysate, Trypanosoma cruzi, leishmania, and Paracoccidioides brasiliensi (Figure 3). There are many reports that the immune enhancer vegetable proteins like the peptides hydrolysate of rice, soybean, and pea can enhance lymphocyte proliferation and improve macrophage phagocytosis (Anderson, 1997). The chickpea peptides with an active sequence Met‐Ile‐Thr‐Leu‐Ala‐Ile‐Phe‐Val‐Asn‐Lys‐Phe‐Gly‐Arg that derived from microbial proteases (Dominguez‐Vega, Kotkowska, Garcia, Crego, & Marina, 2011) and a bioactive peptide derived from rice albumin with a sequence, Gly‐Tyr‐Pro‐Met‐Tyr‐Pro‐LeuPro‐Arg stimulated the immune system through phagocytosis (Takahashi, Moriguchi, Yoshikawa, & Sasaki, 1994).

FIGURE 3

Finding of preclinical studies for some plant proteins as vaccine adjuvants

Soy protein hydrolysate has been found to induce IgG and IgA, and enhance B‐cell differentiation and antibody production (Kiewiet, Faas, & de Vos, 2018). The soybean tridecapeptide isolated by a trypsin digest, common bean peptides (Vital, De Mejia, Dia, & Loarca‐Piña, 2014), and lupine protein hydrolysate (Cruz‐Chamorro et al., 2019), are reported as stimulants to phagocytosis of human neutrophils (Tsuruki et al., 2003). Furthermore, in a clinical examination of a small group of volunteers, an increase in granulocytes, NK, CD11b+, CD56+, and a change in leukocyte count was also observed (Yimit, Hoxur, Amat, Uchikawa, & Yamaguchi, 2012).

Gluten is the main protein of wheat as it accounts for about 80% of their total protein content. Peptides derived from hydrolysates of gluten activate NK cells in humans and act as immunostimulants (Horiguchi, Horiguchi, & Suzuki, 2005). Also, the peptides showed modulation of lymphocytes, monocytes, and granulocytes (Sawaki, Takaoka, Sakuraba, & Suzuki, 2004). Furthermore, the ex vivo immunomodulatory effect of alcalase‐generated wheat gluten protein hydrolysates showed an increase in IL‐10/IFNγ ratio in treated cultured human peripheral blood mononuclear cells (Cruz‐Chamorro et al., 2020). Several reports screened the plant proteins' immunostimulatory effects in vitro are summarized and shown in Table 1, along with the in vivo models in Table 2.

TABLE 1

In vitro immunostimulant assays of plant proteins

Source	Protein	Concentration	Finding/mechanism	References
Momordica charantia Phytolacca americana	Momordica Charantia inhibitor (MCI) protein pokeweed antiviral protein (PAP‐S)	0.1–1 μg/mL range	50% inhibition of macrophage protein synthesis	Spreafico et al. (1983)
		MCI; 5 μg/mL (1.6 × 10−7 M) PAP‐S; 10 μg/mL (1.6 × 10−7 M)	Inhibition of both ConA and PHA responsiveness
Triticum aestivum (Wheat)	Wheat gluten protein hydrolysates (WGPHs)	0.5 mg/mL	Potent anti‐proliferative effect	Cruz‐Chamorro et al. (2020)
			Decreased IFNγ, IL‐17, IL‐10, increased ratios of IL‐4/IFNγ, IL‐4/IL‐17, and IL‐10/IFNγ, IL‐10 mRNA
Pseudostellaria heterophylla	P. heterophylla peptide (PPH <1,000 Da)	100 μg/mL−1	SI was 1.53 for 48 h/promoted TNFα, IFNγ, IL‐10 Activated spleen lymphocytes via the Ca2+/CaN/NFATc1/IFN‐signaling pathway	Yang et al. (2019)
	YG‐9 (YGPSSYGYG) C44H55N9O15	50 μg/mL	Proliferation index = 1.19 Promoted pinocytosis activity, TNFα Activated RAW264.7 cells via the TLRs/NF‐kB/TNFα signaling pathway	Yang et al. (2020a)
	RGPPP Proline—molecular weight 522.29 Da 47.0% β‐sheet, 10.6% β‐turn, and 42.4% random coil	Spleen lymphocyte proliferation rate = 1.27 at 100 μg/mL. RP‐5 activates RAW264.7 cells to secrete NO, ROS, and TNFɑ. A signaling pathway, TLR2/NF‐κB, revealed	Promoted proliferation of spleen lymphocytes Arginine in the N‐ or C‐terminal regions, phosphoserine, glutamine, or tryptophan, recognized by opioid receptors on the immunological cells and resulted in immunomodulation The stimulation index = 1.27 at 100 μg/mL, subsequently dropped to 1.04 at 400 μg/mL. Proliferation index = 1.52 in spleen lymphocytes	Yang et al. (2020b)
Oryza sativa (Rice protein)	Tyr‐Gly‐Ile‐Tyr‐Pro‐Arg (YGIYPR) protein hydrolysate	12.5 μg/mL	Enhanced macrophage proliferation SI value = 1.324	Xu et al. (2019)
Berberis hispanica	Protein fractions F1 = 75 kDa, F2; 20 KDa F3; 5 KDa	1 mg/mL	Stimulated the thymocyte and splenocyte proliferation Enhanced complement activity Promoted allogenic mixed lymphocyte	El Youbi et al. (2012)
Allium sativum (Garlic)	Three protein components of 13 kD (QR‐1, QR‐2, and QR‐3 in the ratio 7:28:1)	1 μg/mL	Mitogenic activity towards human peripheral blood lymphocytes, murine splenocytes, and thymocytes QR‐2 > QR‐1, QR‐3. Mannose‐binding activities	Clement, Pramod, and Venkatesh (2010)
Allium sativum (Aged garlic)	Three major proteins (12–14 kDa) QA‐1, QA‐2, and QA‐3	4 μg/mL	QA‐2 showed the highest mitogenic activity QA‐3 exhibits mitogenic activity, mannose‐binding activity	Chandrashekar and Venkatesh (2009)
Viscum album var. (coloratum)	Korean mistletoe lectin (KML)	2–20 ng/mL	Enhanced macrophage responses Induced cytokines (IL‐3, IL‐23, and TNFα), phagocytic uptake Up‐regulation of functional activation of adhesion molecules	Lee et al. (2007)
Anoectochilus formosanus	Immunomodulatory protein (IPAF)	8 μg/mL	Induced the cell proliferation in mouse splenic B lymphocytes	Kuan et al. (2011)

TABLE 2

In vivo immunostimulatory screening of plant proteins

Source	Extract	Tests	Animal	Dose/day	Duration	Induction	Finding	References
Seeds of Momordica charantia, Leaves of Phytolacca americana	Momordica Charantia inhibitor (MCI) protein Pokeweed antiviral protein (PAP‐S)	Spleen hemolytic plaque‐forming cells (PFC)	CD2F1	MCI (4.3 mg/kg) PAP‐S (3.2 mg/kg)	5 days	4 × 108 sheep erythrocytes (SRBC) i.p. on day 0, (PFC were counted on day 4) Type IlI pneumococcal polysaccharide was injected i.p. at 0.5 μg (PFC specific for S III were counted on day 5)	For 200 μg/kg—2 days before an antigen, producing over 90% reductions from control values For 400 μg/kg—6 days before SRBC were associated with over 90% inhibition of PFC counts	Spreafico et al. (1983)
		Skin grafting	C57B1/6 mice		Grafts were inspected daily starting on day 7	Skin grafting from male C3H donors	400 μg/kg given 2 days before transplant significantly delayed graft rejection by approximately 3 days
Cocos nucifera L (Coconut)	Protein extract	Hematological test	Swiss albino mice	Orally 120 g/35 mL	30 days protein intake	Cyclophosphamide 20 mg/kg/day for 10 days	Increased levels of RBC, WBC, platelet counts	Vigila and Baskaran (2008)
Glycine max (Soy)	Soya protein isolate (SPI)	Measurement of serum total and soya protein‐specific antibodies	Sprague–Dawley rats	20% alcohol–SPI (age before 28 days)	For 70, 190, or 310 days	–	Increased serum total IgA and IgM, induced the production of SPI‐specific IgA, IgG, IgM, and IgE antibodies	Cornish et al. (2011)
Anoectochilus formosanus	Protein (IPAF)	Production of anti‐IPAF monoclonal antibody	BALB/c mice	50 μg	30 days	I.P. injection IPAF + aluminum potassium sulfate (10% w/v).	Induced B cells, CD69, MHC class II & IgM production	Kuan et al. (2011)

The major drawback of plant peptide production using conventional chemical synthesis was the high costs involved, which ultimately hinders the scale‐up. Over the last years, bioactive and immunogenic peptides have been economically synthesized in mass production using a genetic fusion of the corresponding nucleotide sequence of a carrier protein, followed by stable nuclear plastid transformation, or transient expression using bacterial or viral vectors (Lico, Santi, Twyman, Pezzotti, & Avesani, 2012). This process can lower the cost per vaccine and allow for an abundance of more doses, which is particularly important during a pandemic and epidemic situation. Hence, mass production represents the advantageous use of plant peptides, and the peptides can be quickly and cheaply synthesized (Patel et al., 2012). The clinical assessment of immunomodulatory peptides and understanding of their molecular structure permits the establishment of their structure–activity relationships, which is important for developing next‐generation of adjuvants (Azmi, Fuaad, Skwarczynski, & Toth, 2014).

PLANT LECTINS

Various plant lectins have been proposed as potential antigen‐delivery agents (Massa & Franconi, 2012). The reported immunomodulatory effect of various lectins has prompted the screening for their adjuvant potential in pharmaceutical applications (Sander, Corigliano, & Clemente, 2019). The characteristic features of plant lectins rely on their interaction with the mucosal epithelium and their translocation across the gut (Lavelle, Grant, Pusztai, Pfüller, & O'Hagan, 2001). Hence, they are considered a targeting agent to induce potent systemic and mucosal antibody responses, that could be exploited in vaccine formulations to enhance the efficacy of orally administered vaccines (Lavelle, Grant, Pfuller, & O'Hagan, 2004).

The aromatic amino acids found in the lectin‐sheet protein structures interact with the chiral ligand center, resulting in the binding processes between the lectins and their ligands (Teixeira et al., 2006). In terms of the plant lectins' action mechanisms, it is reported to interact with glycosylated TLR on macrophages and/or DCs. Indeed, several plant lectins act as TLR agonists, polyclonal cellular activators, enhance T lymphocytes proliferation, and are involved also in the amplification of IL‐12 and IFNγ production (Unitt & Hornigold, 2011; Venkatalakshmi, Vadivel, & Brindha, 2016). According to Daoudi, Abdel‐Satter, & Aarab, 2014 lectins of Datura stramonium, Lepidium sativum, and Delphinium staphisagria seeds possess a proliferation stimulatory effect on immune cells (Bousfiha et al., 2016).

Lectins have been reported to be an effective immunoadjuvant candidate (Sander et al., 2019). It is used against coccidial infections, as the lectins from wheat germ agglutinin (WGA), Phaseolus vulgaris (PHA), Viscum album (mistletoe lectin 1; ML‐1), Ulex europaeus (UEA‐1), and Lycopersicum esculentum (tomato lectin; LEA), enhanced the production of specific ovalbumin antigen (OVA), and serum IgA and IgG antibodies when assessed as adjuvants with immunization protocols (Lavelle, Grant, Pusztai, Pfüller, & O'hagan, D. T., 2000). Additionally, vaccine formulations containing U. europaeus UEA‐1 conjugated to kill Helicobacter pylori and Campylobacter jejuni showed induction of immune responses in orally immunized mice (Chionh, Wee, Every, Ng, & Sutton, 2009; Daoudi et al., 2014). According to Lavelle et al. (2004) mistletoe lectins (ribosome‐inactivating proteins (RIPs); (ML) I, II, and II (MLI, MLII, and MLIII)) are potent immunogens when administered through the nasotracheal route and could be used as a platform for the generation of effective mucosal adjuvants. In addition, Proteus vulgaris lectin possesses potent lymphocyte stimulation effect (Daoudi et al., 2014).

ArtinM, a mannosyl‐binding lectin derived from the seeds of Artocarpus heterophyllus; activates innate immune cells and in consequence T helper type 1 (Th1) is induced (Da Silva, Oliveira‐Brito, de Oliveira Thomaz, & Roque‐Barreira, 2020). The ArtinM's interactions with TLR2 N‐glycans on the surface of DCs, neutrophils, mast cells, and macrophages are primarily responsible for its potent immunomodulatory activity (Sander et al., 2019). This activity is distinguished by the induction of IL‐12, and the ability to provide in vivo protection against intracellular pathogens, such as Leishmania spp and Paracoccidioides brasiliensis (Souza, Carvalho, Ruas, Ricci‐azevedo, & Roque‐barreira, 2013).

Allium sativum agglutinins (ASAs, mannose‐binding lectins) showed in vitro potent immunomodulatory effects (Clement et al., 2010). Besides, garlic lectins (ASA I and ASA II) were co‐administered with weak antigen ovalbumin (OVA) in BALB/c mice at a 30 μg dose (50 days duration, intranasal). The adjuvanticity character was indicated by the induction of anti‐OVA IgG, also anti‐lectin IgG response increased by threefold and 2.4‐fold for ASA I and ASA II, respectively. Besides, the intradermal administration of ASA I and ASA II had shown the same fold increase (four and twofold anti‐lectin IgG response) for only 14 days. Moreover, the spleen and thymus stimulation index were determined and the results indicate the potential use of garlic lectins as a mucosal and oral adjuvant (Padiyappa et al., 2022; Smart, Ryan, Holdworth, & Preston, 1978).

Viscum album coloratum lectins (KML‐C) at a dose of 50 ng/mouse induce humoral and cellular immune responses against an immunogenic protein antigen (keyhole limpet hemocyanin; KLH). The assay revealed that KML‐C augmented KLH‐specific antibody titers of IgG1, IgG2a, IgG2b, specific Th1, IL‐2, IFN, as well as Th‐2 type cytokine IL‐4 (Yoon et al., 2001). In BALB/c mice, the purified Chinese mistletoe lectin ACML‐55 showed activation of innate lymphocytes, the increased cell number of NK, and γδT cells, and enhanced both antigen‐specific activation and proliferation of CD4+ and CD8+ T cells (Ma et al., 2008). Korean mistletoe lectin (KML) enhanced macrophage responses, phagocytosis, and induce cytokines (IL‐3, IL‐23, and TNFα) (Lee et al., 2007). Furthermore, the Viscum album (mistletoe 1; ML‐1) lectin combined with herpes simplex virus vaccine antigen glycoprotein D2 (gD2), and administrated by a nasotracheal route, the results indicated an induction in mucosal and systemic responses (Leavy, Mcneela, & Mills, 2002).

Narcissus tazetta lectin (26 kDa) (NTL)‐induced cytokines gene expression in vivo, where after 10‐day consecutive peritoneal injections of 5 mg NTL/kg/day, the expression levels of IL‐1β, TNFα, IFNγ, and TGF‐β were markedly increased, and the levels of IL‐2–IL‐4 were up‐regulated in splenocytes. While at the onset of 12–24 h, the TGF‐β was expressed in both macrophages and splenocytes (Ooi, Liu, Ooi, Ng, & Fung, 2002).

Tomato lectin (LEA) delivered intranasally elicited a potent systemic and mucosal antibody response, while, wheat germ agglutinin (WGA) and Ulex europaeus lectin 1 (UEA‐I) when combined with ovalbumin (OVA), a specific serum IgG response to OVA was induced (Lavelle et al., 2001). Additionally, soybean, wheat germ, and peanut agglutinin lectins are reported as extracellular TLRs agonist and induce cytokine gene expression (Sander et al., 2019; Venkatalakshmi et al., 2016).

It worthy noted that lectins are the most common plant immunomodulatory proteins and plant heat‐shock proteins (hsps) have been considered less attention as a biological modifier agent, although the bacteria Hsps have been used as an adjuvant in current vaccine production (Zininga, Ramatsui, & Shonhai, 2018).

Heat‐shock proteins or stress‐induced proteins (Hsps) play an important cytoprotective role in cells exposed to stressful conditions. Hsps induction are a common phenomenon in bacteria, plants, and human beings (Al‐Whaibi, 2011). Plant Hsps vary in size from 15 to 30 kDa and are grouped into five classes according to their molecular weight (small Hsps [sHsps], Hsp60, Hsp70, Hsp90, and Hsp100). The transcription is controlled by the heat stress transcription factors (Hsfs) regulatory gene, and encoded by three classes of cytosolic sHSPs (classes CI, CII, and CIII) and three classes of sHSPs targeted to intracellular organelles (Miroshnichenko et al., 2005). Future screening of the immunomodulator potential of plant Hsps could open up a new area for further exploration in this regard.

PLANT PEPTIDE AS A DELIVERY VEHICLE FOR RECOMBINANT PROTEINS

Biopharming is the use of a host living system for the manufacture of biological drugs that are non‐natively produced using plant cells as bio‐factories allow the economical production of candidate vaccines at large scales that are unavailable with the current in vitro synthesis. The biopharming technologies involve the integration of the desired genes encoding the vaccine antigen protein into the genome of plant tissues (Laere et al., 2016).

Many challenges facing the upstream processes include the selection of antigen and plant expression host, consistency of dosage, and manufacturing of vaccines according to good manufacturing practice procedures. The use of plant peptides offers a simple and flexible way to deal with much of this complexity by acting as a delivery vehicle for recombinant proteins. Moreover, peptides display more drug‐like properties and therefore attract increasing interest from the pharmaceutical industry as a vaccine delivery agent (Purcell, McCluskey, & Rossjohn, 2007).

Schwestka et al., 2020 fused the N‐terminal sequence of the maize storage prolamine protein (γ‐zein) as delivery vehicles for bioencapsulate recombinant proteins. The resultant protein bodies (PBs) showed an internalization into the intestinal epithelial cells at a higher rate than synthetic polystyrene beads. In addition, the PBs showed a pivotal role in the initiation of a humoral response with a potential immunostimulatory effect, resulting in an induced secretion of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) chemoattractant molecules. The GM‐CSF is involved in the differentiation of granulocytes, macrophages, and also in the proliferation of neutrophils, macrophages, and DCs (Hamilton, 2002). Furthermore, GM‐CSF increased antigen‐specific antibody production (Okada et al., 1997), promotes IL‐6 secretion, and accordingly IL‐6 levels were also elevated (Evans, Shultz, Dranoff, Fuller, & Kamdar, 1998).

The use of plant protein as a delivery vehicle could open new avenues as an adjuvant for the next‐generation of vaccines, including the design of functionalized multicomponent PBs with defined structures and uptake kinetics for the pharmaceutical industry (Schillberg, Raven, Spiegel, Rasche, & Buntru, 2019).

HURDLES AND SOLUTIONS IN THE PRODUCTION OF NEW PROTEIN ADJUVANTS

The key hurdle in formulating plant‐based adjuvant vaccines is the choosing of formulation components, to prevent vaccine denaturation due to hydrophobic interactions, as well as preventing chemical degradation during storage (Wakankar & Borchardt, 2006). The excipients may interact with adjuvants or antigen proteins which could alter immunogenicity positively or negatively. Also, during the formulation process, the antigen release and quality control assays could prove to be enormous challenges. Where partial antigen release, inefficient entrapment, and/or degradation after nanoparticle encapsulation leads to high production cost due to wasted antigens (Jain, O'Hagan, & Singh, 2011).

Moreover, interactions between therapeutic protein products and the container closure polyethylene glycol (PEG) chains may negatively affect product quality and immunogenicity. Where the pegylation of vaccine products has been found to diminish the immunogenicity via inducing product solubility, inhibiting product aggregation, and in consequence immunogenicity diminished (Harris, Martin, & Modi, 2001). Moreover, the PEG itself may cause a loss of product efficacy and adverse safety risk (Liu et al., 2011). The glass and air interfaces of prefilled syringes can denature therapeutic protein products and chemical modification could also be observed by leached materials from the container closure system (Fradkin, Carpenter, & Randolph, 2011).

To overcome such challenges excipients and adjuvant‐antigen protein interactions should be carefully evaluated, especially in terms of protein‐excipient adducts formation. Moreover, to confirm and maintain product quality, appropriate in‐use product shelf‐life stability studies should be performed, and a risk assessment must be conducted. Besides, multiple analytical techniques need to be applied to assess the ability of the container closure system to interact and/or degrade protein products (Pulendran et al., 2021), Food and Drug Administration, and Center for Drug Evaluation and Research, 2014.

In using plant protein adjuvants, antibodies can develop not only to the desired antigen protein but also to adjuvant and any other foreign protein components that are potentially present in the product. Such proteins may contain regions of homology to endogenous human proteins. Clinical trials face a challenge in evaluating the capacity of the adjuvant protein to break tolerance and induce antibody responses to the homologous human factor (Pulendran et al., 2021).

The unknown mechanistic hypothesis is one of the challenges that face new development of adjuvant protein, and the use of systems vaccinology could help to answer different questions and aid in clarifying different aspects that could be hindered to demonstrate the clinical trials. The information from vaccinology could indicate (i) mechanistic insights, (ii) rational design of optimal formulations for vaccine delivery, (iii) underlying mechanisms by which formulations work, and (iv) underlying mechanisms of adverse reactions (Petitdemange et al., 2019). Moreover, novel adjuvants can be rapidly tested in small phase I (phase 0) human trials, the results obtained from such phase 0/I studies will create the mechanistic hypothesis about adjuvant antigen formulation (Wagar et al., 2021).

The polymorphisms (the presence of two variant forms of a specific DNA sequence) that could be resulted from the mismatching between the sequence of the patient endogenous protein and the therapeutic product protein are considered a risk factor for the development of immune response (Viel et al., 2009). Additionally, glycosylation may strongly modulate vaccine product immunogenicity by enhancing product solubility, minimizing protein aggregation, and diminishing immunogenicity as well as by shielding immunogenic protein epitopes from the immune system (Cole, Steckbeck, Rowles, Desrosiers, & Montelaro, 2004).

Hence, careful consideration should be given to the primary sequences of adjuvant plant proteins, antigen proteins, and especially of protein product counterparts of endogenous proteins, because of potential polymorphisms across human populations. For proteins that are normally glycosylated, it is recommended to use appropriate manufacturing methods that glycosylate the protein product in a nonimmunogenic manner.

REGULATORY AND COMPLIANCE REQUIREMENTS

For the acceptance of new vaccines/adjuvants, safety and a lack of universality appear to be the major critical factors, the current attitude regarding the risk–benefit of vaccination puts a large emphasis on safety. The cautious approach taken by manufacturers and authorities at the labor of new vaccines often leads to an impasse and explains why aluminum is still the predominant adjuvant used despite its limitations as an immunostimulator, with poor biodegradability and limited use in terms of delivery routes. However, the picture is different for therapeutic vaccines, such as tumor vaccines that are usually administered to seriously ill patients, a higher level of risk is considered acceptable (Goldenthal, Cavagnaro, Alving, & Vogel, 1993).

European union (EU) regulatory developments for the assessment of adjuvants include different stages, (i) development stages where laboratory studies are performed, (ii) development stages/preclinical evaluation, (iii) small‐scale clinical trial, (iv) large‐scale clinical studies, and (v) batch‐to‐batch consistency, where after the product is given marketing approval it monitored to provide ongoing evaluation of the product using the same assays of preclinical and clinical studies (Harandi, Medaglini, & Shattock, 2010).

Preclinical safety studies are also required not only for adjuvant safety, the qualitative and quantitative composition of the adjuvant, physical characteristics, and manufacturing process parameters but also for the adjuvant–antigen compatibility, dose ratio, repeat dose toxicity, characterization of immune response, proof of consistent antigen adjuvant adsorption, demonstration that no significant desorption during the shelf‐life period, biochemical purity and pyrogenicity, systemic toxicity, reproduction toxicity, and carcinogenicity (Aguilar & Rodriguez, 2007).

Clinical studies include the final evaluation of a developed vaccine formulation that requires controlled studies of the adjuvant–antigen combination, which should deliver the following information: (i) justification for adjuvant inclusion, (ii) enhancement of immune response without undue increase in systemic reactions, and (iii) risk–benefit assessment on a case‐by‐case basis and for a modified product this should be at least as favorable as for the existing product (Sesardic, Rijpkema, & Patel, 2007).

Before licensure, the vaccine indication (therapeutic and prophylactic), the disease profile, route of administration, number of doses, and the population targeted to receive the vaccine should be established as the basis for safety assessment.

Post‐licensure safety evaluation should provide safety information in specific populations that were not included in the pre‐registration studies. Moreover, the adverse effects that increase in these populations relative to what was seen in clinical trials should be demonstrated (Da Silva, Di Pasquale, Yarzabal, & Garçon, 2015).

Among the biggest regulatory hurdles is the required population size that needs to be tested to prove the efficacy and particularly safety of a new adjuvant or vaccine. Market withdrawal could happen if a new vaccine is associated with hazardous adverse reactions, which challenged the public perception of safety and have an impact on the regulatory field (Aguilar & Rodriguez, 2007).

CONCLUSION AND FUTURE PROSPECTIVE

Currently, the use of different types of adjuvants to optimize the vaccine immune response has received less attention. When preliminary trials with a “standard” adjuvant do not reveal the expected response, researchers typically seek another type of antigen rather than looking for an appropriate adjuvant that may produce the preferred immune response (Patel et al., 2012).

Adjuvants cannot receive FDA approval as standalone products, but as part of a registered vaccine adjuvant–antigen combination. Hence, only a few adjuvants are approved by regulatory authorities. Using plant protein as a safer immunomodulator adjuvant candidate could overcome the cost hinder of developing novel adjuvant formulations (Massa & Franconi, 2012).

Future advances in new adjuvants are more likely to be driven by better knowledge of the action mechanisms. Technological progress are expected to cut vaccine production costs, as well as novel prophylactic/therapeutic vaccine‐adjuvant discoveries will be available (Singh & O'Hagan, 2002). A graphical presentation of plants that possess immunomodulation and can be considered as a promising bio‐source candidate for future novel adjuvants is represented in Figure 4 and its corresponding mechanisms are demonstrated in Figure 5.

FIGURE 4

Graphical presentation for plants‐derived immunomodulator proteins as promising natural vaccine adjuvant agents

FIGURE 5

Summary of immunomodulator plant proteins mechanisms of action according to previous reports

Improved purification and high‐throughput screening procedures can enhance the yield of proteins and reduce decades of research from chemical elucidation to commercial development. Drug carrier technology increases immunomodulator delivery of polypeptides and proteins, enhances pharmacokinetics, and introduces the optimum absorption rates that may allow effective interactions with the immune system components.

It is worthy to mention that there is the possibility that some of the studies in the present review, may not be performed according to the recent best practices/bioactive plant preparations/pharmacological research (Izzo et al., 2020). To the best of our knowledge, there are no clinical reports that investigated the immunomodulatory effect of plant proteins, and further high‐quality studies are needed to firmly establish the clinical efficacy of the plant proteins hydrolysates (Izzo, Hoon‐Kim, Radhakrishnan, & Williamson, 2016; Kiewiet et al., 2018).

CONFLICT OF INTEREST

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.