Pichia pastoris an Ideal Host for the Production of Recombinant Influenza Vaccines

Introduction
Recombinant proteins are frequently made by yeasts, which are unicellular fungi. Pichia pastoris and Saccharomyces cerevisiae are two well-known yeast systems that can be used for this purpose. The ability of yeast systems to carry out post-translational modifications such as acetylation, phosphorylation, glycosylation, proper protein folding, and the absence of endotoxin are among its advantages (Tanaka et al., 2012; Kuruti et al., 2020; De et al., 2021).
P. pastoris is a methylotrophic organism known as an ideal organism for expressing recombinant proteins on an industrial scale (Alizadeh et al., 2013; Barone et al., 2023; De et al., 2021). P. pastoris can use methanol as its only carbon source. During the oxidation process inside the peroxisome, this yeast utilizes the alcohol oxidase enzyme to metabolize methanol (Maleknia et al., 2011; Athmaram et al., 2011; Moridi et al., 2020). Different strains of this yeast have been used to produce recombinant proteins (Mohammadzadeh et al., 2021). It should be noted that all P. pastoris strains, such as auxotrophic mutants (GS115) and protease-free strains (SMD1163, SMD1165 and SMD1168), are derived from the wild strain NRRL-Y 11430 (Tanaka et al., 2012). Influenza A virus, a member of the Orthomyxoviridae family, is the cause of avian influenza. Three avian influenza virus subtypes, H5, H7 and H9N2, are commercially and physiologically significant in the poultry industry (Gholami et al., 2022; Mirzaie et al., 2021; Mohammadi et al., 2021; Abtin et al., 2022). Some researchers considered influenza the next pandemic (Morens et al., 2023). Approximately between 250000 and 500000 individuals die from influenza virus infections worldwide annually (Norouzian et al., 2014; Perdue & Swayne, 2005; Kim et al., 2022).
Avian influenza subtype H9N2 is the most prevalent influenza virus in poultry worldwide. It imposes economic losses on the poultry industry and has zoonotic potential (Alizadeh et al., 2009; Mirzaie et al., 2020; Zhao et al., 2021; Golgol et al., 2023). Nili and Asasi, (2003) demonstrated mortality rates between 20% and 60% on H9N2-infected farms. One possible explanation for this high mortality rate is co-infection with other respiratory diseases. 
The expression of the recombinant protein and subsequent manufacturing of the vaccine in yeast are more suitable in terms of timing and scale of production than insect, mammal, or Escherichia coli expression systems (Athmaram et al., 2011). Genetic engineering technology and veterinary medicine allow us to create novel and effective recombinant vaccines against various diseases such as brucellosis, Clostridium, influenza, tuberculosis, and so on (Soleimanpour et al., 2015; Nouri Gharajalar et al., 2016; Mayahi et al., 2016; Yousefi et al., 2016; Farsiani et al., 2016; Shirdast et al., 2021; Asghari Baghkheirati et al., 2023; Taghizadeh & Dabaghian, 2022). Besides, new subunit vaccines have been made in medicine against SARS-CoV-2, enterovirus, papillomavirus, malaria, etc. using the P. pastoris expression system (Mukhopadhyay et al., 2022; Xu et al., 2023; Noseda et al., 2023; Kingston et al., 2023; Li et al., 2023). Previous literature has emphasized the use of this yeast as a safe, cost-effective and suitable organism for vaccine production in the healthcare industry (Kuruti et al., 2020; Barone et al., 2023; De Sá Magalhães & Keshavarz-Moore, 2021). This review study aims to describe P. pastoris as one of the most efficient expression systems for developing recombinant vaccines for the poultry industry, focusing on avian influenza vaccines.

P. pastoris phenotypes
Depending on the yeast genotype, the presence or absence of the alcohol oxidase genes (AOX1 and AOX2), and the use of methanol, this yeast can be classified into three phenotype categories (Maleknia et al., 2011; Singh & Narang, 2020). Although both genes affect the production of enzymes and consumption of methanol, the alcohol oxidase 1 promoter has a greater impact.
1) Mut+phenotype (X33 and GS115 strains): This group is the natural yeast P. pastoris with AOX1 and AOX2 genes. Compared to the other two phenotypes, these strains use methanol more quickly, consume more oxygen, and express more recombinant protein. For these reasons, most studies have used them with this phenotype as an industrial strain (Cámara et al., 2017; Singh & Narang, 2020).
2) Muts phenotype (KM71 strain): Although the AOX2 gene is present in this group, the AOX1 gene has been eliminated. Due to the deletion of AOX1, these stains cannot be used quickly by methanol. Since these strains use methanol slowly, more complex proteins will have time to acquire their correct conformation before being secreted into the medium (Wollborn et al., 2022).
3) Mut-phenotype (MC100-3 and MC101-1 strains): In this group, both AOX1 and AOX2 promoters have been deleted, so these strains cannot use methanol and are practically unable to grow in an environment containing methanol. The main carbon sources utilized by these strains are glycerol, sorbitol, or mannitol (Singh & Narang, 2020).

The advantages of using P. pastoris
Several reasons exist for using this yeast as an expression system (illustrated in Figure 1 and explained here). 

1) Ease of working: There is no need to have complex culture media or special nutrients for P. pastoris yeast propagation. This yeast can be grown easily using a culture medium containing yeast extract, peptone and dextrose (Kuruti et al., 2020).
2) High cell density: Fermentation is an essential process for recombinant protein production, and its efficiency is highly dependent on cell density. P. pastoris can reach a high cell density in an optimized culture medium and produce more recombinant antigens than other expression systems (Zhang et al., 2020).
3) Eukaryotic expression system: Compared to prokaryotic systems, P. pastoris is a eukaryotic organism that can produce mammalian and avian proteins more similar to their original form (Kuruti et al., 2020).
4) Genomic integration of the desired gene: The desired gene can be integrated into several locations of the yeast chromosome. This characteristic plays an important role in the stability of the gene and increased production of the influenza protein (Wu et al., 2023).
5) High efficiency in recombinant protein production: One of the reasons for the tendency towards this yeast is its high expression level. The recombinant protein produced by this yeast can include more than 80% of the total proteins in the culture medium (Li et al., 2007). The AOX1 promoter, one of the most potent eukaryotic promoters, has been used to produce a variety of recombinant proteins, with documented yields of up to 20–30 g/L (Tanaka et al., 2012).
6) Post-translational modifications: One of the most important processes in protein synthesis, performed after transcription and translation, is glycosylation. The role of glycosylation in protein folding, protein structural stability, specific signal transmission, and secretion processes has been proven. In comparing P. pastoris and S. cerevisiae, it should be stated that the oligosaccharide chains that are attached to proteins and make glycoproteins are more reliable in Pichia (Li et al., 2007). One of the advantages of using P. pastoris yeast is the lack of mannosyltransferase. This enzyme causes the production of α-1, 3-mannosyl bonds, which is seen in S. cerevisiae. These connections differ from those in the mammalian system and may be recognized and rejected by the human immune system. On the other hand, P. pastoris yeast is a better option than S. cerevisiae for producing a recombinant protein because it has a higher capacity for producing heavy proteins and secretes fewer unwanted internal proteins into the extracellular environment (Tanaka et al., 2012).
7) Probiotic properties: Several investigations have been accomplished regarding this yeast’s probiotic features. It was demonstrated that the X-33 strain can survive in food at an appropriate concentration for at least two months. Salmonella spp., Clostridium spp. and E. coli are among the most important bacterial pathogens in the poultry industry that cause significant economic losses (Seyedtaghiya et al., 2021; Daneshmand et al., 2022; Peighambari et al., 2023). P. pastoris can be a probiotic and antibiotic alternative to prevent and control these pathogens. This yeast administration prevented Salmonella typhimurium’s growth in the culture medium and decreased bacterial colonization in the BALB/c mice intestine (Franca et al., 2015). The mice had a higher survival rate in the challenge test with the acute strain of S. typhimurium (50% to 80%) than the control group (20% to 50%). In another study, Gaboardi et al. (2019) found that the administration of P. pastoris X-33 strain in the quail’s diet could increase egg weight, adjust the immune system, and increase the level of antibodies against infectious bronchitis virus (IBV), Newcastle disease (ND) and infectious bursal disease (IBD), compared to the control group. Transgenic or wild-type P. pastoris strains can be used as probiotics in chickens as antibiotic alternatives to control necrotic enteritis (SGil de Los Santos et al., 2018; Kulkarni et al., 2022).
8) Natural adjuvant activity: It has been demonstrated that the yeast cell wall components have inherent adjuvant properties (Franca et al., 2015). In other words, yeast-based vaccines do not need adjuvants like aluminum to stimulate the immune system (Stubbs et al., 2001). Therefore, when administered, expressed recombinant proteins and yeast cell wall components will be more immunogenic (Wasilenko et al., 2010; Asghari Baghkheirati et al., 2023).

P. pastoris transformation
It has been indicated that multiple copies of the desired gene in the P. pastoris genome result in elevated gene expression. Therefore, it is important to choose the best method for efficient transformation. The most efficient way to transform P. pastoris is to use the settings of 25 μF, 200 Ω, and 1500 V for the instrument’s capacitance, resistance, and voltage, respectively (Wu & Letchworth, 2004; Yongkiettrakul et al., 2009; Sulfianti et al., 2015; Pratanaphon et al., 2018). Furthermore, pretreating yeast cells with lithium acetate and dithiothreitol has been shown to boost transformation efficiency significantly 150-fold (Wu & Letchworth, 2004).

P. pastoris vectors
There are two expression vectors for P. pastoris, including pPIC9k and pPICZα (A, B and C). The only difference between pPIC9 and pPIC9K is the kanamycin resistance gene, which gives Pichia resistance to Geneticin®. As the number of integrated copies increases, Pichia becomes resistant to higher concentrations of Geneticin® and the expression level will be higher. pPICZ A, B and C are 3.3-kb expression vectors that express recombinant proteins in P. pastoris. This vector’s multiple cloning sites in three reading frames (A, B and C) make it easier to clone the desired gene in a frame with the C-terminal peptide containing a polyhistidine (6xHis) tag and the c-myc epitope. The characteristics of pPIC9k (Invitrogen, Catalog No. V175–20) and pPICZα vectors (Invitrogen, Catalog No. V190-20) are shown in Figure 2.

P. pastoris usage in the production of avian influenza vaccine candidates
The development of influenza vaccines primarily focuses on the hemagglutinin (HA) protein, the main antigenic protein of the influenza virus. Therefore, most research investigations have focused on selecting HA epitopes and their production in various P. pastoris strains. Researchers have employed P. pastoris yeast to produce various recombinant proteins, including influenza antigens (Sulfianti et al., 2015; Qian et al., 2021). Some scientists delivered these proteins through injections or oral administration to animal models, mainly mice and chickens and then measured the antibody titer. Many investigations have been conducted on the various aspects of influenza virus transmission, clinical symptoms, virology, serology, and the development of a novel vaccine using genetic engineering (Salamatian et al., 2020; Mirzaie et al., 2021; Mohammadi et al., 2021; Sahebnazar et al., 2021).
Several studies indicated that subunit influenza vaccines produced using the P. pastoris expression system can elicit high antibody titers in mice and chickens (Taghizadeh & Dabaghian, 2022; Asghari Baghkheirati et al., 2023). For instance, Pietrzak et al. (2016) transformed two hemagglutinin proteins, one with a cleavage region sequence (H5DH) and one without it (H5DHΔ), in P. pastoris. The recombinant antigens were diluted in PBS and injected subcutaneously in the neck area of SPF Leghorn laying hens twice. It was found that 100% of the chickens injected with H5DHΔ had high titers of neutralizing antibodies in the HI assay. Interestingly, all vaccinated chickens survived the challenge with H5N1, and no clinical symptoms were observed, but the control group chickens died on the fourth day after the challenge. This study shows that using the yeast system to produce recombinant proteins as a subunit vaccine can effectively protect chickens against lethal challenges. In research conducted by Liu et al. (2020), the complete HA gene of the H7N9 subtype (A/Hangzhou/1/2013) was cloned into the pPICZαA plasmid. Then, the resulting recombinant plasmid was linearized by the BglII restriction enzyme and transformed into P. pastoris using the electroporation technique. Recombinant H7 protein led to immunostimulation, high HI titer and 100% protection of mice following challenge with wild virus. Wasilenko et al., (2010) cloned the HA gene of the A/Egret/Hong Kong/757.2/02 (H5N1) strain along with alpha-agglutinin as an anchor into the pPIC9K plasmid. The resulting recombinant plasmid was transformed into the P. pastoris GS115 strain. It was found that the recombinant vaccine can agglutinate red blood cells in the HA test, which indicates the yeast’s correct production of HA protein. In addition, the oral administration of the vaccine to SPF Leghorn chickens resulted in the production of neutralizing antibodies. Nguyen et al. (2014) used the HA1 sequence and cloned it into the pPIC9 vector. They transformed the P. pastoris SMD1168 strain and administered the obtained recombinant antigens into BALB/c mice and chickens. The vaccine produced a high antibody titer in the HI test (6.7 and 7 titers in mice and chickens, respectively). According to reports, M1, one of the main structural proteins in influenza viruses with protected epitopes, can stimulate CD8+ lymphocyte cells and protect chickens against influenza infection and mortality. It is possible to produce multiple subunit antigens using the yeast expression system. During the study of Subathra et al. (2014a), the sequence of M1 and HA genes was obtained from the A/Hatay/2004/H5N1 strain and cloned in the pPICZαC plasmid and transformed into the P. pastoris GS115 strain. Based on their results, HA and M1 proteins can be combined to make faster and less expensive vaccines for influenza. In another study, Ebrahimi et al. (2010) used the KM71H strain and the pPICZαA plasmid to produce the M2e antigen of the H9 subtype. They demonstrated that the subcutaneous injection of antigen could produce polyclonal antiserum in rabbits. 
Moreover, the expressed antigen could also be used to produce commercial ELISA kits. Shehata et al. (2012) prepared an ELISA kit using the P. pastoris GS115 strain to detect H5 influenza infection. The results showed that rHA1-ELISA has high specificity and sensitivity. Studies related to recombinant influenza vaccine production, with and without in vivo tests, were illustrated in Tables 1 and 2, respectively.

P. pastoris usage in the production of other recombinant vaccines
In addition to influenza vaccines, there are so many studies in which researchers have produced recombinant antigens. Several studies used P. pastoris to express Mycobacterium tuberculosis as a novel tuberculosis vaccine candidate and the results of their studies showed that this vaccine could elicit protective immunity in BALB/c mice (Mosavat et al., 2016; Kebriaei et al., 2016; Ravansalar et al., 2016).
In the study of Zhang et al. (2015), one of the outer membrane proteins of Proteus mirabilis called OmpA was expressed in P. pastoris and a high level of protection (80%) was observed in administered chickens. It has been documented that chickens vaccinated with the recombinant reticuloendotheliosis vaccine, produced by the SMD1168 strain, were completely protected against challenge with REV (Li et al., 2012). Oral administration of transgenic P. pastoris cells containing VP2 protein can cause a high level of protection against IBD in chickens (Taghavian et al., 2013). Yeast expression systems have been used in different studies to produce Eimeria (EtMic2) and avian reoviruses (σC and σB) proteins (Zhang et al., 2014; Yang et al., 2010). Furthermore, this strong expression system has been used for recombinant production of antimicrobial peptides that can be considered as antibiotic alternatives (Neshani et al., 2018; Neshani et al., 2019; Ghazvini et al., 2021; Azghandi et al., 2022).

Discussion 
Influenza is one of the most crucial diseases that has resulted in uncompensated losses to the poultry industry worldwide (Nili and Asasi, 2003; Golgol et al., 2023). Today, inactivated influenza vaccines are widely used to prevent influenza disease in poultry. However, these vaccines have serious limitations, and in the event of a pandemic, they will not meet the needs of the poultry industry for vaccines. Due to the advancement of technology, researchers have been attracted to the development of recombinant influenza vaccines (Athmaram et al., 2011; Barone et al., 2023). These vaccines, which utilize biotechnology and molecular biology developments, present a viable substitute for conventional immunization techniques. Recombinant DNA technology is utilized to manufacture and deliver particular influenza viral antigens orally, thereby inducing systemic and mucosal immune responses in vaccinated animals (Wasilenko et al., 2010). It is worth mentioning that some influenza subtypes, such as H9N2, have become endemic in a vast geographical area of the Middle East (Nili and Asasi, 2003; Motamedi Nasab et al., 2023). It has been indicated that influenza viruses can evolve through point mutations and genetic reassortment, which can result in pathogenicity and host preference changes (Gong et al., 2021). Potentially, the H9N2 influenza subtype threatens public health and various researchers have mentioned it as the next global pandemic agent (Perdue & Swayne, 2005; Morens et al., 2023). Therefore, focusing on producing new and effective influenza vaccines is so important. 
P. pastoris yeast has been recognized as a promising host for generating recombinant proteins and recombinant DNA technology has been employed to develop novel vaccines against avian influenza. It has been established that P. pastoris can be safely injected into mice and used as a safe vaccine-development delivery system (Becerril-García et al., 2022). 

P. pastoris is an ideal host for influenza vaccine production that can overcome the drawbacks of inactive vaccines (Barone et al., 2023). In addition to having characteristics similar to mammalian cells, P. pastoris can be easily manipulated genetically, making the production of recombinant proteins in this yeast system economically viable (Wu et al., 2023). In addition, this yeast can rapidly express proteins and their translational and post-translational processing (Li et al., 2007). These factors have made this yeast a promising organism in producing eukaryotic proteins. Also, it is possible to achieve high cell density by using a bioreactor. Besides, P. pastoris has a special secretion system, so it secretes a very small amount of its intrinsic proteins into the culture medium; therefore, the cost of protein purification and subsequent processing is reduced. P. pastoris can form disulfide bonds and O- and N-linked glycosylation (Kuruti et al., 2020). This yeast does not cause hyperglycosylation of glycoproteins because it only adds short oligosaccharide chains to proteins. Recently, a lot of research has been done on this yeast to engineer its genome in a way that makes it more suitable for the production of recombinant proteins at high cell density (Tanaka et al., 2012; Kuruti et al., 2020; Zhang et al., 2020).
In this review, P. pastoris was illustrated as a suitable expression platform for creating recombinant antigens for the veterinary medicine and poultry industry. Some influenza vaccines produced by using this yeast system have been dramatically effective, could elicit high antibody titers and could protect animals from challenges with wild strains. Considering the benefits of P. pastoris, it is necessary to conduct more studies on developing universal recombinant influenza vaccines using this yeast.

Ethical Considerations

Compliance with ethical guidelines
There were no ethical considerations to be considered in this research.

Funding
This research was supported by a research project, approved by the University of Tehran (Grant No.: 7825381).

Authors' contributions
All authors equally contributed to preparing this article.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors wish to express their appreciation to everyone that assists us in this study and also would like to thank the research council of University of Tehran.

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