Whole Yeast Vaccine Displaying ZIKV B and T Cell Epitopes Induces Cellular Immune Responses in the Murine Model

Improving antigen presentation is crucial for the success of immunization strategies. Yeasts are classically used as biofactories to produce recombinant proteins and are efficient vehicles for antigen delivery, in addition to their adjuvant properties. Despite the absence of epidemic outbreaks, several vaccine approaches continue to be developed for Zika virus infection. The development of these prophylactic strategies is fundamental given the severity of clinical manifestations, mainly due to viral neurotropism. The present study aimed to evaluate in vivo the immune response induced by P. pastoris recombinant strains displaying epitopes of the envelope (ENV) and NS1 ZIKV proteins. Intramuscular immunization with heat-attenuated yeast enhanced the secretion of IL-6, TNF-α, and IFN-γ, in addition to the activation of CD4+ and CD8+ T cells, in BALB/c mice. P. pastoris displaying ENV epitopes induced a more robust immune response, increasing immunoglobulin production, especially IgG isotypes. Both proposed vaccines showed the potential to induce immune responses without adverse effects, confirming the safety of administering P. pastoris as a vaccine vehicle. Here, we demonstrated, for the first time, the evaluation of a vaccine against ZIKV based on a multiepitope construct using yeast as a delivery system and reinforcing the applicability of P. pastoris as a whole-cell vaccine.


Introduction
Biotechnological-relevant yeast species such as Saccharomyces cerevisiae, Pichia pastoris (Komagataella phaffii), Hansenula polymorpha, and Kluyveromyces lactis are conventionally employed in the synthesis of immunobiological products [1]. These species have GRAS (Generally Recognized as Safe) status, guaranteeing safety in their application as a biofactory and as a vehicle for vaccine antigens [2][3][4]. The most attractive aspects of this vaccine delivery system are the ability to induce specific immune responses against the recombinant antigen and the yeast adjuvant properties [5]. Whole yeast vaccines can induce higher antigen-specific responses than those promoted by inactivated virus vaccines or proteins conjugated to traditional adjuvants, such as aluminum salts [6][7][8]. The first phase of the study included the selection of immunogenic epitopes from the envelope and NS1 proteins, the construction of the expression cassettes, and the obtaining of the recombinant yeasts [25]. The sets of Env and Env + NS1 epitopes were fused to the α-agglutinin anchor protein, allowing the display of the recombinant proteins on the P. pastoris surface. PGK1: promoter; MF-α: signal peptide for protein secretion; 6H: 6x His-tag for immunodetection. (B) The concentration of yeasts cells was 10 YU, and these cells were heatinactivated before immunization procedures. The BALB/c mice received two doses of each yeast preparation, via intramuscular, and were monitored for weight and activity throughout the 21 days of the experiment. Blood collection, euthanasia, and removal of the spleen were performed on the last day of the schedule.

Yeast Preparation
After 72 h of cultivation in a YPD medium at 28 °C under agitation (150 rpm), yeast cells were harvested after centrifugation (4500 rpm; 10 min) and washed twice with 1x PBS. Cells were resuspended in sterile 1x PBS to adjust the final concentration to OD600 = 10 in 50 µL. The yeasts were subjected to heat treatment by incubation at 60 °C for 1 h for metabolic inactivation, and stored at 4 °C until the moment of use. The first phase of the study included the selection of immunogenic epitopes from the envelope and NS1 proteins, the construction of the expression cassettes, and the obtaining of the recombinant yeasts [25]. The sets of Env and Env + NS1 epitopes were fused to the α-agglutinin anchor protein, allowing the display of the recombinant proteins on the P. pastoris surface. PGK1: promoter; MF-α: signal peptide for protein secretion; 6H: 6x His-tag for immunodetection. (B) The concentration of yeasts cells was 10 YU, and these cells were heatinactivated before immunization procedures. The BALB/c mice received two doses of each yeast preparation, via intramuscular, and were monitored for weight and activity throughout the 21 days of the experiment. Blood collection, euthanasia, and removal of the spleen were performed on the last day of the schedule.

Yeast Preparation
After 72 h of cultivation in a YPD medium at 28 • C under agitation (150 rpm), yeast cells were harvested after centrifugation (4500 rpm; 10 min) and washed twice with 1x PBS. Cells were resuspended in sterile 1x PBS to adjust the final concentration to OD600 = 10 in 50 µL. The yeasts were subjected to heat treatment by incubation at 60 • C for 1 h for metabolic inactivation, and stored at 4 • C until the moment of use.

Mice, Ethical Parameters, and Immunization Protocol
Female immunocompetent BALB/c mice, 6-8 weeks old, were raised and maintained in the bioterium of the Aggeu Magalhães Institute (Oswaldo Cruz Foundation-Recife, PE, Brazil) under sterile, pathogen-free conditions. All experiments involving mice strictly Pharmaceutics 2023, 15, 1898 4 of 13 followed the standards established by the institutional Ethics Committee for the Use of Animals (protocol No. 110/2017). The immunization schedule was performed in two doses, one week apart, with intramuscular injection ( Figure 1B). The mice were divided into three groups (n = 5) defined as G1-mice inoculated with non-recombinant P. pastoris (nr); G2-mice vaccinated with P. pastoris:ENV; and G3-mice vaccinated with P. pastoris:ENVNS1. Before each immunization, all mice were anesthetized with xylazine hydrochloride (10 mg·Kg −1 ) and ketamine (115 mg·Kg −1 ). Each experimental group received doses of 50 µL with yeast cells in an OD 600 = 10. Twenty-one days after the first dose, all animals were anesthetized for blood collection and subsequent euthanasia. Spleens were removed for isolation and culture of lymphocytes.

In Vitro Culture and Stimulation of Isolated Spleen Lymphocytes
Splenocytes from vaccinated animals were isolated and the mononuclear immune cells were isolated by separation with Ficoll-Paque PLUS 1.077 g·mL −1 (GE Healthcare Life Sciences, Uppsala, Sweden) and distributed in 48-well plates, 10 6 cells/well. The isolated cells were restimulated in vitro with the yeasts P. pastoris:nr (for G1), P. pastoris expressing ENV epitopes (for G2), and ENVNS1 epitopes (for G3), at a concentration of 10 5 cells/well. The cells were incubated in an RPMI medium (Sigma-Aldrich) containing 10% FBS at 37 • C (5% CO 2 ), at the experimental times of 24 h, 48 h, and 72 h.

Immunological Analysis
Lymphocytes isolated from blood and the spleen were characterized for the presence of surface markers CD4, CD8, and CD16 by labeling the cells with the corresponding antibodies (anti-CD4-FITC, anti-CD8-PE, and anti-CD16/32-FITC; BD TM Bioscience, Franklin Lakes, NJ, USA). Serum and culture supernatant were analyzed for cytokine dosage of TNF-α, IFN-γ, IL-2, IL-4, IL-6, IL-10, and IL-17A using the BD CBA Mouse Th1/Th2/Th17 Kit (BD TM Bioscience), following the manufacturer's instructions. IgG, IgM, IgA, and IgE immunoglobulins were measured in the serum of vaccinated mice using the Mouse Immunoglobulin Isotyping Kit (BD TM Bioscience). All acquisitions for immunological assays were performed by flow cytometry (BD ACCURI C6).

Hematological and Biochemical Analyses
Blood samples were collected using a cardiac puncture, placed into EDTA-K2 tubes, and centrifuged at 3600 rpm for 10 min to separate serum and plasma. The hematological evaluation included global counts of red blood cells, leukocytes, platelets, determination of hematocrit, and hemoglobin concentration. The values of red blood cells, hematocrit, and hemoglobin allowed the calculation of the mean corpuscular volume and the mean corpuscular hemoglobin concentration. The blood cell count was performed in a Neubauer chamber, with differential counting of the slides stained by the Rapid Panoptic method [27]. The platelet number was determined using the Fonio method [28]. Hemoglobin was measured by colorimetry using a spectrophotometer. The biochemical analyses were performed with Labtest Diagnostic kits (Lagoa Santa, MG, Brazil). The levels of glucose, urea, and creatinine were measured with end-point colorimetric enzymatic assays. Alkaline phosphatase was detected by a modified Roy's method, and the liver transaminases by the Reitman-Frankel method.

Statistical Analysis
Graphs and statistical analyses were generated by GraphPad Prism version 7.04. The analysis of variance (ANOVA) was applied to assess statistical differences between groups. Results with a p-value < 0.05 were considered statistically significant.

P. pastoris Can Surface Display Vaccine Antigens
The recombinant yeasts were generated and characterized in a previous study [25]. A Yeast-ELISA was performed to confirm the accessibility of anchored antigens. P. pastoris:ENV and P. pastoris:ENVNS1 showed higher immunoreactivity compared to nonrecombinant yeast (NR), confirming protein anchoring ( Figure 2).

P. pastoris Can Surface Display Vaccine Antigens
The recombinant yeasts were generated and characterize A Yeast-ELISA was performed to confirm the accessibility of toris:ENV and P. pastoris:ENVNS1 showed higher immunoreac combinant yeast (NR), confirming protein anchoring (Figure 2

Recombinant P. pastoris Strains Induce Increased Secretion of S
The vaccine constructs promoted different cytokine patter kines, P. pastoris:ENV caused an increase in the levels of IL-2, T other hand, P. pastoris:ENVNS1 increased all analyzed cytokin recombinant yeast and P. pastoris:ENV, mainly in IL-2, IL-4, IL The response elicited by both recombinant yeasts was equiva lease. The profile of cytokines secreted by splenic lymphocytes more restricted than observed in animal serum, with a great pattern. P. pastoris:ENV caused an increase in IL-6 (48 h) and IFN 4A,B), while P. pastoris:ENVNS1 induced IL-6 and TNF-α in 72 There was no significant production of the other cytokines (T yeast did not cause a significant stimulus compared to P. pasto gens.

Recombinant P. pastoris Strains Induce Increased Secretion of Serum and Splenic Cytokines
The vaccine constructs promoted different cytokine patterns. Regarding serum cytokines, P. pastoris:ENV caused an increase in the levels of IL-2, TNF-α, and IL-17A. On the other hand, P. pastoris:ENVNS1 increased all analyzed cytokines, overcoming both non-recombinant yeast and P. pastoris:ENV, mainly in IL-2, IL-4, IL-10, and IL-1A ( Figure 3). The response elicited by both recombinant yeasts was equivalent only to the TNF-α release. The profile of cytokines secreted by splenic lymphocytes, restimulated in vitro, was more restricted than observed in animal serum, with a greater tendency toward a Th1 pattern. P. pastoris:ENV caused an increase in IL-6 (48 h) and IFN-γ at 24 h and 48 h ( Figure 4A,B), while P. pastoris:ENVNS1 induced IL-6 and TNF-α in 72 h cultures ( Figure 4A-C). There was no significant production of the other cytokines (Table S1). Non-recombinant yeast did not cause a significant stimulus compared to P. pastoris expressing vaccine antigens.

P. pastoris:ENV and P. pastoris:ENVNS1 Enhance Antibody Production
The levels of different types and isotypes of immunoglobulins were assessed to evaluate the humoral response. Overall, the P. pastoris:ENV vaccine stood out compared to ENVNS1 and non-recombinant yeast ( Figure 5A-D). P. pastoris:ENV induced production of all of the IgG isotypes, especially IgG3. Both vaccine constructs elicited a similar profile for IgA and IgM ( Figure 5E,G), superior to P. pastoris:nr. The two vaccines led to an increase in IgE, mainly P. pastoris:ENV ( Figure 5F). The ratio of IgG2a/IgG1 indicates the response profile type (Th1 or Th2) and was similar for all three yeast strains. Although the mean for P. pastoris:nr (1.27) was higher than the recombinants (1.14 and 1.03), the difference was not statistically significant ( Figure 5H). Values greater than 1 indicate a tendency toward a Th1 response pattern. However, the proximity between the averages of IgG1 and IgG2a for all groups suggested a balance between Th1 and Th2 responses.

Recombinant Yeasts Stimulate Cellular Responses
The evaluation of the cellular response induced by vaccination was based on the analysis of CD4 + , CD8 + , and CD16 + T lymphocyte populations present in the blood and spleen after immunization. Regarding circulating lymphocytes, mice inoculated with nonrecombinant P. pastoris promoted a higher stimulus to CD4 + T lymphocytes. No difference in the number of CD4 + T cells between the recombinant yeasts was observed ( Figure 6A). On the other hand, regarding the number of CD8 + T cells, P. pastoris:nr and P. pastoris:ENVNS1 showed a similar pattern, superior to that induced by P. pastoris:ENV ( Figure 6B). To Natural Killer (NK) CD16 + T lymphocytes, P. pastoris:ENV and P. pastoris:ENVNS1 promoted an increase in the number of cells compared to the non-recombinant ( Figure 6C).

P. pastoris:ENV and P. pastoris:ENVNS1 Enhance Antibody Production
The levels of different types and isotypes of immunoglobulins were assessed to eva uate the humoral response. Overall, the P. pastoris:ENV vaccine stood out compared ENVNS1 and non-recombinant yeast ( Figure 5A-D). P. pastoris:ENV induced productio of all of the IgG isotypes, especially IgG3. Both vaccine constructs elicited a similar profi for IgA and IgM ( Figure 5E,G), superior to P. pastoris:nr. The two vaccines led to an i crease in IgE, mainly P. pastoris:ENV ( Figure 5F). The ratio of IgG2a/IgG1 indicates t response profile type (Th1 or Th2) and was similar for all three yeast strains. Although t mean for P. pastoris:nr (1.27) was higher than the recombinants (1.14 and 1.03), the diffe ence was not statistically significant ( Figure 5H). Values greater than 1 indicate a tenden toward a Th1 response pattern. However, the proximity between the averages of IgG1 an IgG2a for all groups suggested a balance between Th1 and Th2 responses.

Recombinant Yeasts Stimulate Cellular Responses
The evaluation of the cellular response induced by vaccination was based on the ana ysis of CD4 + , CD8 + , and CD16 + T lymphocyte populations present in the blood and sple after immunization. Regarding circulating lymphocytes, mice inoculated with non-recom binant P. pastoris promoted a higher stimulus to CD4 + T lymphocytes. No difference in t number of CD4 + T cells between the recombinant yeasts was observed ( Figure 6A). On t other hand, regarding the number of CD8 + T cells, P. pastoris:nr and P. pastoris:ENVN showed a similar pattern, superior to that induced by P. pastoris:ENV ( Figure 6B). To Na ural Killer (NK) CD16 + T lymphocytes, P. pastoris:ENV and P. pastoris:ENVNS1 promot Lymphocytes isolated from the spleen of the immunized animals were restimulated with the respective strain and incubated for periods of 24 h, 48 h, and 72 h. The most significant stimulus was observed for the CD4 + T lymphocyte subpopulation. The immunization with the recombinant yeasts, expressing ENV and ENVNS1, induced an increase in the amount of CD4 + T cells, especially when observing the 72 h culture, where both were superior to non-recombinant P. pastoris ( Figure 7A). The levels of CD8 + T cells were lower than the expression of CD4 + and CD16 + and substantially reduced throughout the culture period. The proportion of CD8 + T cells stimulated by P. pastoris:ENV stood out at times of 24 h and 48 h. After 72 h of stimulus, the culture plates of splenocytes from animals inoculated with P. pastoris:ENVNS1 had a higher number of CD8 + T lymphocytes ( Figure 7B). Regarding the CD16 + T cell profile, P. pastoris:ENV induced a significant increase in the first 24 h; however, it declined at 48 h and 72 h. Meanwhile, P. pastoris:ENVNS1 caused a more significant expansion at 48 h and 72 h, even though it also diminished at 72 h ( Figure 7C). Lymphocytes isolated from the spleen of the immunized animals were resti with the respective strain and incubated for periods of 24 h, 48 h, and 72 h. T significant stimulus was observed for the CD4 + T lymphocyte subpopulation. The ization with the recombinant yeasts, expressing ENV and ENVNS1, induced an in the amount of CD4 + T cells, especially when observing the 72 h culture, wh were superior to non-recombinant P. pastoris ( Figure 7A). The levels of CD8 + T ce lower than the expression of CD4 + and CD16 + and substantially reduced throug culture period. The proportion of CD8 + T cells stimulated by P. pastoris:ENV stoo times of 24 h and 48 h. After 72 h of stimulus, the culture plates of splenocytes f mals inoculated with P. pastoris:ENVNS1 had a higher number of CD8 + T lymp ( Figure 7B). Regarding the CD16 + T cell profile, P. pastoris:ENV induced a signifi crease in the first 24 h; however, it declined at 48 h and 72 h. Meanwhile, P. past VNS1 caused a more significant expansion at 48 h and 72 h, even though it also dim at 72 h ( Figure 7C).   Lymphocytes isolated from the spleen of the immunized animals were restimulated with the respective strain and incubated for periods of 24 h, 48 h, and 72 h. The most significant stimulus was observed for the CD4 + T lymphocyte subpopulation. The immunization with the recombinant yeasts, expressing ENV and ENVNS1, induced an increase in the amount of CD4 + T cells, especially when observing the 72 h culture, where both were superior to non-recombinant P. pastoris ( Figure 7A). The levels of CD8 + T cells were lower than the expression of CD4 + and CD16 + and substantially reduced throughout the culture period. The proportion of CD8 + T cells stimulated by P. pastoris:ENV stood out at times of 24 h and 48 h. After 72 h of stimulus, the culture plates of splenocytes from animals inoculated with P. pastoris:ENVNS1 had a higher number of CD8 + T lymphocytes ( Figure 7B). Regarding the CD16 + T cell profile, P. pastoris:ENV induced a significant increase in the first 24 h; however, it declined at 48 h and 72 h. Meanwhile, P. pastoris:EN-VNS1 caused a more significant expansion at 48 h and 72 h, even though it also diminished at 72 h ( Figure 7C).

The Whole Yeast Vaccines Do Not Cause Significant Side Effects
Animals were weighed on the first day of the immunization regimen, on the booster dose day, and 21 days after the initial dose (Table S2). Throughout the vaccine schedule, there was no weight loss or significant behavioral changes. There were no points of inflammation, swellings, or adverse effect in the injection site. Overall, there were no biochemical alterations that would qualify as clinical disturbances ( Table 1). The hematological changes were relative to leukocytosis and lymphopenia, in addition to neutrophilia in the three experimental groups.

The Whole Yeast Vaccines Do Not Cause Significant Side Effects
Animals were weighed on the first day of the immunization regimen, on the booster dose day, and 21 days after the initial dose (Table S2). Throughout the vaccine schedule, there was no weight loss or significant behavioral changes. There were no points of inflammation, swellings, or adverse effect in the injection site. Overall, there were no biochemical alterations that would qualify as clinical disturbances ( Table 1). The hematological changes were relative to leukocytosis and lymphopenia, in addition to neutrophilia in the three experimental groups.

Discussion
In this study, we evaluated a multiepitope vaccine based on B and T cell epitopes from Env and NS1 ZIKV proteins in a yeast surface display strategy. The whole yeast vaccines evaluated were developed in a previous study that assessed, in vitro, the potential of P. pastoris yeast as an adjuvant and vaccine platform [25]. The immune analyses showed the induction of a Th1-type immune response based on the profile of cytokines (IL-6, TNF-α) and activated T cells (CD4 + and CD8 + ). Interestingly, in this same study, the construction P. pastoris:ENVNS1 showed the induction of an immunological profile similar to that promoted by the virus when incubated with splenocytes from BALB/c mice. Together, the results enabled the continuation of the evaluation using a pre-clinical test in a murine model.
After a two-dose schedule, the immune response was assessed by cytokine dosage, immunophenotyping, and immunoglobulin production. BALB/c mice immunized with recombinant P. pastoris showed a serum elevation of IL-2 and TNF-α for the two tested vaccines, in addition to the induction of IL-17A. Noteably, in addition to its conventional regulatory role, IL-17A may play pro-inflammatory functions in some viral, fungal, and cancer infections [32]. Moreover, high levels of IFN-γ, TNF-α, and IL-2 secreted by CD4 + and CD8 + T lymphocytes are also detected in immunocompetent C57BL/6 mice infected with ZIKV, exhibiting a Th1 pattern [33,34].
Despite eliciting Th1 cytokines, the P. pastoris:ENVNS1 vaccine induced an increase in IL-4 and IL-10, which may be a consequence of follicular T cell (Thf) activation. In Zika virus infection, Tfh cells enhanced the production of cytokines, such as IL-4 and IL-21, and also acted in a Th1-like manner, producing IFN-γ. Additionally, this lymphocyte subpopulation influences the development of neutralizing antibodies against ZIKV [35]. The detection of cytokines such as IL-2, IL-4, and IL-17 in individuals in the acute phase of ZIKV infection suggests a polyfunctional response profile characterized by Th1, Th2, and Th17 responses [36]. Concerning the splenic immune cells, the cytokine dosage indicated a Th1 profile, where P. pastoris:ENV induced an increase in IL-6 and IFN-γ levels, while P. pastoris:ENVNS1 stimulated IL-6 and TNF-α production. These pro-inflammatory cytokines compose the anti-ZIKV immune response and were also observed, in vitro, in assays performed with these recombinant yeasts in previous studies [25,37]. The role of IL-6 resulting from the administration of whole yeast vaccines has been linked to the generation of CD4 + T cells [38]. Furthermore, increased production of TNF-α and IFN-γ is related to the activation of CD4 + and CD8 + T cells that mediate effective responses against ZIKV-infected cells [39].
Regarding circulating lymphocytes, the recombinant antigens seem to modulate the CD8 + T cells since there was a significant difference between the vaccines. Nonrecombinant P. pastoris and expressing ENVNS1 induced a greater stimulus than P. pastoris:ENV. The NS1 epitopes may have influenced the activation of the CD8 + T lymphocytes, which reinforces the importance of including immunodominant epitopes present in non-structural proteins in the design of vaccines for flaviviruses [39]. In addition, both recombinant yeasts elicited the expansion in the number of CD16 + T lymphocytes, mainly P. pastoris:ENVNS1 construction.
Analysis of splenic lymphocytes reflects the induction of immune memory responses [40]. The stimulus promoted by recombinant yeasts was greater for the CD4 + T cell population than CD8 + T cells, overall. P. pastoris:ENV stimulated CD4 + (48 h and 72 h), CD8 + (24 h and 48 h), and CD16 + (24 h and 72 h) cells. P. pastoris:ENVNS1 elicited an increase in the number of CD4 + cells (24 h and 72 h), CD8 + (72 h), even in low levels, and CD16 + (48 h and 72 h). The vaccine responses reported are in line with other studies about the importance of T cells in the context of vaccine development, indicating that a coordinated balance among the action of antibodies, Tfh CD4 + , Th1 CD4 + , and CD8 + T cells are essential to the infection control and long-term protection [39,41].
In addition to the cellular immune response, the recombinant yeasts induced significant production of different classes of immunoglobulins. The highest levels were observed for IgG3, IgA, and IgM. The P. pastoris:ENV vaccine promoted a significant increase in the production of all IgG isotypes. Elevations in the generation of IgG1 and IgG2a were observed in mice challenged with ZIKV PE243 [42] and immunized with VLPs [43]. The IgG2 isotype is predominant among neutralizing antibodies that induce protection and is important for ZIKV infection clearance [35,42]. Additionally, there were elevations in IgG3 and IgM, commonly observed in the acute phase of viral infections, which were involved in pro-inflammatory effector mechanisms [43,44].
Both recombinant yeasts caused an increase in IgE. Antiviral functions performed by IgE have been investigated, but there is no consistent data regarding the role of this immunoglobulin in ZIKV infection [45]. Once there were no changes in eosinophil levels or signs of anaphylaxis, the contribution of IgE was possibly related to non-allergic defense mechanisms induced by the vaccine. The two vaccine constructs also promoted an increase in IgA and IgM at equivalent levels. These immunoglobulins are frequent in recent ZIKV infections, making them helpful for diagnostic assays [46].
All tested yeasts induced neutrophilia in inoculated mice. In addition to acting as critical cells of innate immunity, neutrophils also contribute to adaptive immunity in the transport and presentation of antigens and the regulation of antigen-specific responses [47]. Although neutrophil activation is involved in the pathogenesis of ZIKV infection, the role of these cells in the antiviral immune response generated by vaccination is unclear [48].
Overall, the results obtained suggest the involvement of Th1 and Th2 responses, providing a polyfunctional immune response profile also observed in the ZIKV infection [33]. Similar patterns are observed in vaccine studies for ZIKV based on different platforms, such as EDIII-based subunit vaccines [49] and VLPs [50]. This study explored the biotechnological potential of P. pastoris regarding its applicability as a biofactory and antigen carrier. We observed that although previous studies show yeast immunostimulatory properties, the expression of vaccine antigens can modulate the immune response. Recombinant P. pastoris promoted an increase in immunoglobulins production and cellular immune response activation. There were no deaths, diarrhea, or weight loss in the animals vaccinated with yeasts during the vaccination schedule, which was in agreement with previous studies using P. pastoris and other species [51].
In vivo evaluations of whole yeast vaccines are essential to validate this biotechnological platform highlighted as an alternative vaccine antigen delivery system. The biotechnological potential of P. pastoris was explored in different ways: as a biofactory, as an antigen carrier, and as a vaccine adjuvant, showing its versatility. P. pastoris is pointed out as a cost-effective platform for vaccine production, favoring production and distribution in low-and middle-income countries [52].

Conclusions
Despite the several immunoinformatic-based studies for predicting ZIKV epitopes, data at the preclinical level are still scarce. This study demonstrates, for the first time, the evaluation of a multi-epitope construct as a vaccine antigen for ZIKV using yeast as a vehicle with the surface display system. As the main product, P. pastoris:ENV proved to be a promising vaccine candidate, inducing the activation of T CD4 + and T CD8 + lymph;ocytes and effector cytokines, as well as producing immunoglobulins, compared to the other experimental groups. Together, the results point to the generation of immune responses that should be tested to verify the induction of protection against ZIKV infection.