Standardization of Whole Yeast Cell Vaccines

Version	Summary	Created by	Modification	Content Size	Created at	Operation
1		Antonio Carlos de Freitas	--	2083	2022-12-22 14:20:32	\|
2	format	Jason Zhu	Meta information modification	2083	2022-12-23 04:10:54	\|

This entry is adapted from the peer-reviewed paper 10.3390/pharmaceutics14122792

In the context of vaccine development, improving antigenic presentation is critical for the activation of specific immune responses and the success of immunization, in addition to selecting an appropriate target. In this sense, different strategies have been developed and improved. Among them is the use of yeast cells as vehicles for the delivery of recombinant antigens. These vaccines, named whole yeast vaccines (WYVs), can induce humoral and cellular immune responses, with the additional advantage of dispensing with the use of adjuvants due to the immunostimulatory properties of their cell wall components.

whole cell vaccine antigenic delivery biotechnology vaccine improvement

1. Yeast Genera

The discussions are related to the main yeast genera of biotechnological interest: S. cerevisiae, Pichia pastoris (now named Komagataella phaffii), Hansenula polymorpha, Yarrowia lipolytica, Schizosaccharomyces pombe, and Kluyveromyces lactis. The choice of the yeast species used as a host cell can interfere not only with the production of recombinant proteins but also with the vaccine-induced immune response ^[1]^[2]. Bazan et al. (2018) observed differences in the level of stimulus provoked for the activation of dendritic cells concerning the expression of surface markers such as CD40, CD58, CD80, CD83, and CD86 and the cytokines released by these cells ^[3]. These differences were observed between distinct species and among strains of the same species.

It is essential to know the profile of the immune response induced by the yeast chosen as a carrier and to understand the immunological properties that characterize it as an adjuvant. The cell wall of yeast is the main element responsible for its adjuvant activity. However, it is worth mentioning that this organelle is not static, and its architecture can undergo dynamic changes depending on growth conditions as well as culture media and also vary according to the yeast genus or species used ^[4]. The proportion and arrangement of the cell wall components of each species can influence yeast recognition by the immune system and the uptake by antigen-presenting cells (APCs) ^[1]. While the distribution of mannan polymers appears to be homogeneous among different genera such as S. cerevisiae, P. pastoris, Sz. pombe and K. lactis, the positioning of β-glucans can differ and show variated patterns between budding and fission yeasts ^[5]. Furthermore, depending on the yeast used, the tools available for optimizing the delivery system may differ. Some species, such as S. cerevisiae and P. pastoris, have a broader set of vectors, well-characterized promoters, and knowledge about the best culture conditions for the expression of vaccine antigens ^[6].

2. Yeast Cell Inactivation

The yeasts used in the WYV are neither pathogenic nor toxic. Despite this, they are usually inactivated before immunization to minimize eventual risks regarding vaccine biosafety. The aims of yeast inactivation are reproductive capacity loss and the elimination of cell metabolic activities. The employed protocols are simpler than those used for the inactivation of viruses or bacteria because they do not require chemical processes. Yeast inactivation involves heat treatment, starting with incubation at temperatures ranging from 56–95 °C, which is sometimes accompanied by lyophilization (often freeze-drying). Determining the most suitable heat temperature can be influenced by several factors, from cell concentration to the heating method or yeast species used ^[7]. The efficiency of this procedure can be evaluated through viability tests that include visualization of the yeasts under an optical microscope after incubation with vital dyes such as methylene blue and trypan blue or seeding the treated yeasts in a suitable culture medium for growth evaluation ^[8].

Despite affecting the viability of yeasts in terms of their reproductive capacity, different studies have found no effect on vaccine antigen stability and functionality or yeast immunoreactivity concerning their recognition and uptake by APCs ^[5]^[9]. Although there may be differences due to the cellular architecture of the chosen yeast species, it has been suggested that heat treatment may favor the exposure of the β-1,3-glucan layer on the cell surface. Once this is one of the most immunogenic components of the yeast wall, this exposure may facilitate the binding to receptors such as Dectin-1 present in APCs and M cells ^[5].

In the process of preparing and standardizing doses, it is important to include the verification of protein levels present in the yeast cell suspension after heat treatment. This evaluation can be performed through SDS-PAGE and western blot, as executed by Arnould et al. 2012 who observed that the recombinant protein remained intact after the inactivation procedures ^[10]. Similarly, Kumar and Kharbikar (2021) observed that the protein levels remained stable through freeze (−80 °C)/thaw cycles and lyophilization ^[11]. Further studies should evaluate how long WYVs can be stored without losing stability and efficiency, and which temperatures can be adopted.

Although it is still understudied, the influence of heat treatment or the difference between administering heat-killed or live WYV can be related to the administration pathway adopted in the vaccination schedule. Capilla et al. (2009) observed differences in vaccine-induced responses between the intramuscular and oral routes and between live and heat-killed yeast cells administered orally ^[12]. Several factors could influence this result, including the inactivation protocol, the dose concentration, and the type of delivery (intracellular or surface displayed). Moreover, after heat inactivation and lyophilization, the yeast cells lose their replicative capacity and classification as an organism (Directive 2001/18/EC). This can also change their classification as genetically modified organisms (GMO), which can facilitate adherence to the WYV and simplify their licensing ^[7]^[13].

3. Definition of Doses and Quantification

The unit of measurement of the vaccine doses broadly varies in the different clinical and preclinical studies performed. One of the definitions considers the number of cells per vaccine preparation, adopting “yeast units” (YU), where 1 YU corresponds to 10⁷ cells (approximately equivalent to OD₆₀₀ = 1) or the dry weight in mg or g (1 mg corresponding to approximately OD₆₀₀ = 2). Cell counting can be done in a relative manner by measuring the optical density or by counting through a Neubauer chamber, or using more accurate techniques such as conventional flow cytometry or micro-flow imaging (MFI) ^[14]. The dry weight is evaluated, in general, after the lyophilization process. There are some concerns when the yeast unit is adopted instead of the dry weight because some protocols that rely on optical density, for example, may not be as accurate as ones that use automated equipment to determine the number of cells and their viability. This eventual imprecision may have an impact on the reproducibility of vaccine trials and the accuracy of dose preparation.

There are variations in the minimum threshold suitable for inducing immune responses and in establishing a concentration of cells per preparation. There is considerable variation between studies that use yeast as vaccines, although it is known that there is a correlation between the amount of yeast used and possible immunological effects such as the induction of neutralizing antibodies, for example ^[15]^[16]. In clinical studies, a maximum dose of 10 to 12 YU per injection has been adopted, with at least four administrations at different sites. In a clinical trial conducted by Cohn et al. (2019) aimed at patients with tumors with mutations in the Ras oncogene, it was observed that the subcutaneous administration of up to 10 YU did not lead to significant adverse reactions, even with four applications totaling 40 YU ^[17].

The number and concentration of doses may also differ depending on whether the vaccination is prophylactic or therapeutic. Therapeutic approaches may require more applications to achieve the purpose, which is often related to generating cytotoxic responses and eliminating infected or tumor cells. Preclinical and clinical cancer vaccine studies point to a dose-dependent process where multiple applications seem to lead to an optimal antitumor effect ^[18]. Multiple-site injections can amplify the immune response by targeting multiple peripheral lymph nodes. Thus, depending on the vaccination strategy, inoculation sites that reach the inguinal, axillary, and subclavicular lymph node beds are chosen ^[19]^[20].

Regarding the amount of vaccine antigen per dose, the main criteria are concentration, immunogenicity, and administration route. Some optimizations can help to improve the production of the recombinant antigen and the number of yeast units sufficient to induce protection or treat some pre-existing diseases. Considering the yeast adjuvant properties and, depending on the immunogenicity of the antigen, an adequate immune response can be obtained even with a low concentration of the recombinant protein, only increasing the number of yeast cells per dose ^[10]. Furthermore, an ideal antigen concentration to reach an optimal dose can be influenced by the route of administration ^[12]. Oral vaccines may require higher amounts of antigen in terms of concentration or number of cells than parenterally administered vaccines.

The antigen concentration in WYVs, which carry proteins, depends on the yeast species and strain chosen as biofactories and their ability to produce heterologous proteins, as well as the expression vector, which is also subject to optimization. The application of methods capable of detecting and quantifying the presence of the antigen in a defined number of cells includes approaches such as western blot, Yeast ELISA, and flow cytometry ^[14]^[21]. Flow cytometry is the most widely used method for quantifying the percentage of positive cells (expressing and exposing the antigen) present in the set of cells that comprise the vaccine dose in WYV that uses the surface display system ^[13]^[22].

However, few studies show a correlation between the dose and the actual concentration of the delivered antigen. Bian et al. (2009) quantified the antigen (Hepatitis B virus proteins) and tested concentrations from 0.75 to 1.25 μg in 1 × 10⁸ H. polymorpha cells ^[15]. Similarly, Arnould et al. (2012) assessed the protein concentration per dose through SDS-PAGE and Western blot, estimating a concentration of 0.7 fg of heterologous protein per K. lactis cell ^[10]. The quantification process when the antigen carried is a nucleic acid vaccine is even less explored, so there are no records of well-established methodologies to define the amount of DNA, mRNA, or siRNA carried by the yeast. In these cases, the concentration of the genetic material used to transform the yeast cells can be decisive, but it is important to find out how much has been assimilated by the cell. The most suitable methodologies for these processes may involve conventional PCR to check for the presence of the antigen and qPCR.

4. Culture Scaling-Up

Culture and protein production parameters may influence vaccine dose setting and clinical efficacy. The high cell densities achieved in the yeast cultivation process make it possible to obtain thousands of doses per liter of culture. However, some parameters can optimize this production, such as the yeast species used as the host cell, the period established for the culture, the promoter present in the expression vector, and the number of copies of the recombinant DNA per cell ^[23]^[24]^[25]. A fundamental point for the establishment of a biofactory or vaccine platform is the ability and ease of scaling up cultures, starting from pilot protocols and reaching large fermenters for high biomass generation ^[26]. This ability has been demonstrated for different yeast species such as S. cerevisiae, P. pastoris, and Y. lipolytica and is crucial to indicating the commercial applicability of the vaccine to be produced ^[24]^[27].

The employment of bioreactors for biomass generation makes it possible to reach higher levels of expression than from culture flasks commonly used in the first stages of laboratory cultivation ^[10]. The factors that can be controlled and optimized for better efficiency include culture medium composition, pH control, oxygen availability (dissolved oxygen level), cultivation time, and volume ^[6].The optimization in the scaling steps involves the choice of the bioreactor, the kinetic features of the yeast, and the mode of operation (continuous, discontinuous, and discontinuous-fed) ^[24]. In this scaling process, the culture usually starts in the batch process and, further, is adapted to fed-batch, allowing better monitoring and control of nutrient input throughout the production phases ^[27]. Continuous cultivation has more caveats due to the greater propensity for contamination, mutations in the strains, and instability of the products ^[24]. Regarding kinetics, it is important to promote a balance between growth and protein synthesis and to pay attention to the fact that this parameter can be affected by the promoter and the number of gene copies. Furthermore, the culture conditions may depend on the study objective, whether it is to recover and purify the antigen or to use recombinant cells as a vaccine vehicle.

The demand for culture scaling up also depends on the immunobiological to be produced. Prophylactic and therapeutic strategies have different requirements regarding the number of doses and individuals to be immunized. Overall, prophylactic vaccines aim at mass vaccination, while therapeutic vaccines comprise more personalized immunotherapy with a smaller target population. Thus, the scale of production of a therapeutic vaccine using yeast as a biofactory and vehicle requires less accumulation of biomass. This particularity of therapeutic approaches even favors nucleic acid-carrying strategies, whose scaling-up procedures can be more complex and are not yet well established.

References

Bazan, S.B.; Geginat, G.; Breinig, T.; Schmitt, M.J.; Breinig, F. Uptake of various yeast genera by antigen-presenting cells and influence of subcellular antigen localization on the activation of ovalbumin-specific CD8 T lymphocytes. Vaccine 2011, 29, 8165–8173.
Kim, H.; Lee, J.; Kang, H.; Lee, Y.; Park, E.-J. Oral immunization with whole yeast producing viral capsid antigen provokes a stronger humoral immune response than purified viral capsid antigen. Lett. Appl. Microbiol. 2014, 58, 285–291.
Bazan, S.B.; Walch-Rückheim, B.; Schmitt, M.J.; Breinig, F. Maturation and cytokine pattern of human dendritic cells in response to different yeasts. Med. Microbiol. Immunol. 2018, 207, 75–81.
Stewart, G.G. The Structure and Function of the Yeast Cell Wall, Plasma Membrane and Periplasm. In Brewing and Distilling Yeasts; Springer International Publishing: Cham, Switzerland, 2017; pp. 55–75.
Bazan, S.B.; Breinig, T.; Schmitt, M.J.; Breinig, F. Heat treatment improves antigen-specific T cell activation after protein delivery by several but not all yeast genera. Vaccine 2014, 32, 2591–2598.
Gomes, A.M.V.; Carmo, T.S.; Carvalho, L.S.; Bahia, F.M.; Parachin, N.S. Comparison of Yeasts as Hosts for Recombinant Protein Production. Microorganisms 2018, 6, 38.
Soutter, F.; Werling, D.; Nolan, M.; Küster, T.; Attree, E.; Marugán-Hernández, V.; Kim, S.; Tomley, F.M.; Blake, D.P. A Novel Whole Yeast-Based Subunit Oral Vaccine Against Eimeria tenella in Chickens. Front. Immunol. 2022, 13, 809711.
Jacob, D.; Ruffie, C.; Dubois, M.; Combredet, C.; Amino, R.; Formaglio, P.; Gorgette, O.; Pehau-Arnaudet, G.; Guery, C.; Puijalon, O.; et al. Whole Pichia pastoris Yeast Expressing Measles Virus Nucleoprotein as a Production and Delivery System to Multimerize Plasmodium Antigens. PLoS ONE 2014, 9, e86658.
Kiflmariam, M.G.; Yang, H.; Zhang, Z. Gene delivery to dendritic cells by orally administered recombinant Saccharomyces cerevisiae in mice. Vaccine 2013, 31, 1360–1363.
Arnold, M.; Durairaj, V.; Mundt, E.; Schulze, K.; Breunig, K.D.; Behrens, S.-E. Protective Vaccination against Infectious Bursal Disease Virus with Whole Recombinant Kluyveromyces lactis Yeast Expressing the Viral VP2 Subunit. PLoS ONE 2012, 7, e42870.
Kumar, R.; Kharbikar, B.N. Lyophilized yeast powder for adjuvant free thermostable vaccine delivery. Appl. Microbiol. Biotechnol. 2021, 105, 3131–3143.
Capilla, J.; Clemons, K.V.; Liu, M.; Levine, H.B.; Stevens, D.A. Saccharomyces cerevisiae as a vaccine against coccidioidomycosis. Vaccine 2009, 27, 3662–3668.
Patterson, R.; Eley, T.; Browne, C.; Martineau, H.M.; Werling, D. Oral application of freeze-dried yeast particles expressing the PCV2b Cap protein on their surface induce protection to subsequent PCV2b challenge in vivo. Vaccine 2015, 33, 6199–6205.
Wang, J.; Stenzel, D.; Liu, A.; Liu, D.; Brown, D.; Ambrogelly, A. Quantification of a recombinant antigen in an immuno-stimulatory whole yeast cell-based therapeutic vaccine. Anal. Biochem. 2018, 545, 65–71.
Bian, G.; Cheng, Y.; Wang, Z.; Hu, Y.; Zhang, X.; Wu, M.; Chen, Z.; Shi, B.; Sun, S.; Shen, Y.; et al. Whole recombinant Hansenula polymorpha expressing hepatitis B virus surface antigen (yeast-HBsAg) induces potent HBsAg-specific Th1 and Th2 immune responses. Vaccine 2009, 28, 187–194.
Bal, J.; Luong, N.N.; Park, J.; Song, K.-D.; Jang, Y.-S.; Kim, D.-H. Comparative immunogenicity of preparations of yeast-derived dengue oral vaccine candidate. Microb. Cell Factories 2018, 17, 1–14.
Cohn, A.; Morse, M.A.; O’Neil, B.; Whiting, S.; Coeshott, C.; Ferraro, J.; Bellgrau, D.; Apelian, D.; Rodell, T.C. Whole Recombinant Saccharomyces cerevisiae Yeast Expressing Ras Mutations as Treatment for Patients With Solid Tumors Bearing Ras Mutations: Results From a Phase 1 Trial. J. Immunother. 2018, 41, 141–150.
Roohvand, F.; Shokri, M.; Abdollahpour-Alitappeh, M.; Ehsani, P. Biomedical applications of yeast- a patent view, part one: Yeasts as workhorses for the production of therapeutics and vaccines. Expert Opin. Ther. Pat. 2017, 27, 929–951.
Wansley, E.K.; Chakraborty, M.; Hance, K.W.; Bernstein, M.B.; Boehm, A.L.; Guo, Z.; Quick, D.; Franzusoff, A.; Greiner, J.W.; Schlom, J.; et al. Vaccination with a Recombinant Saccharomyces cerevisiae Expressing a Tumor Antigen Breaks Immune Tolerance and Elicits Therapeutic Antitumor Responses. Clin. Cancer Res. 2008, 14, 4316–4325.
King, T.H.; Kemmler, C.B.; Guo, Z.; Mann, D.; Lu, Y.; Coeshott, C.; Gehring, A.J.; Bertoletti, A.; Ho, Z.Z.; Delaney, W.; et al. A Whole Recombinant Yeast-Based Therapeutic Vaccine Elicits HBV X, S and Core Specific T Cells in Mice and Activates Human T Cells Recognizing Epitopes Linked to Viral Clearance. PLoS ONE 2014, 9, e101904.
Tang, Y.Q.; Han, S.Y.; Zheng, H.; Wu, L.; Ueda, M.; Wang, X.N.; Lin, Y. Construction of cell surface-engineered yeasts displaying antigen to detect antibodies by immunofluorescence and yeast-ELISA. Appl. Microbiol. Biotechnol. 2008, 79, 1019–1026.
Liu, D.-Q.; Lu, S.; Zhang, L.; Huang, Y.-R.; Ji, M.; Sun, X.-Y.; Liu, X.-G.; Liu, R.-T. Yeast-Based Aβ1-15 Vaccine Elicits Strong Immunogenicity and Attenuates Neuropathology and Cognitive Deficits in Alzheimer’s Disease Transgenic Mice. Vaccines 2020, 8, 351.
Mariz, F.C.; Coimbra, E.C.; Jesus, A.L.S.; Nascimento, L.M.; Torres, F.A.G.; Freitas, A.C. Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using the Pichia pastoris PGK1 Promoter. BioMed Res. Int. 2015, 2015, 594120.
Vandermies, M.; Fickers, P. Bioreactor-Scale Strategies for the Production of Recombinant Protein in the Yeast Yarrowia lipolytica. Microorganisms 2019, 7, 40.
De, S.; Mattanovich, D.; Ferrer, P.; Gasser, B. Established tools and emerging trends for the production of recombinant proteins and metabolites in Pichia pastoris. Essays Biochem. 2021, 65, 293–307.
Magalhães, S.d.S.; Keshavarz-Moore, E. Pichia pastoris (Komagataella phaffii) as a Cost-Effective Tool for Vaccine Production for Low- and Middle-Income Countries (LMICs). Bioengineering 2021, 8, 119.
Liu, W.-C.; Inwood, S.; Gong, T.; Sharma, A.; Yu, L.-Y.; Zhu, P. Fed-batch high-cell-density fermentation strategies for Pichia pastoris growth and production. Crit. Rev. Biotechnol. 2019, 39, 258–271.

© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license; additional terms may apply. By using this site, you agree to the Terms and Conditions and Privacy Policy.

Upload a video for this entry

Information

Subjects: Biotechnology & Applied Microbiology

Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :

Anna Jéssica Duarte Silva

Crislaine Kelly da Silva Rocha

Antonio Carlos de Freitas

View Times: 434

Entry Collection: Biopharmaceuticals Technology

Update Date: 23 Dec 2022

Table of Contents

Video Upload Options

Confirm