Evaluation of immune responses and allergenic risks to different adjuvants in an OVA-based immunisation model using BALB/c mice

Evaluation of immune responses and allergenic risks to different adjuvants in an OVA-based immunisation model using BALB/c mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of immune responses and allergenic risks to different adjuvants in an OVA-based immunisation model using BALB/c mice Hivda Ulbegi POLAT, Bilal KOCAMAN, Hatice Nur AYDIN This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7004970/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives Adjuvants play a crucial role in enhancing vaccine efficacy by engaging the innate immune system and shaping a robust adaptive response. However, some adjuvants can also influence allergenicity by promoting Th2-biased responses, potentially increasing the risk of allergic sensitization depending on their mode of action. Methods In this study, we investigated the immunogenic properties of various FDA-approved adjuvants using ovalbumin (OVA) as a model antigen. BALB/c mice were immunized with different formulations of OVA combined with Alum, Freund's Adjuvant, and MF59 to assess their immunostimulatory effects. A total of nine experimental groups were evaluated, including two different doses of Alum. To assess the allergenic potential of these formulations, we analysed IgG1, IgG2a, and IgG2b responses, allowing us to evaluate the balance between Th1- and Th2-mediated immunity. Results CFA induced the strongest anti-OVA IgG1 response, while MF59 caused a short-lived response. OVA + 0.1 mg Alum group produced higher IgG1 response than the OVA + 0.2 mg group. CFA also strongly triggered Th1 immunity, with high IgG2a/IgG2b levels, while Alum elicited a low IgG2a/IgG2b response. No IgG2a/IgG2b response was seen in other groups. Conclusion The OVA + CFA group induced the highest IgG1 response, with IgG2a and IgG2b modulating the Th1-biased immune response. MF59 caused a rapid decline in IgG1 and promoted a Th2 response, with no IgG2a or IgG2b detected. The OVA + 0.1 mg Alum group showed a higher IgG1 response than the OVA + 0.2 mg group, highlighting the importance of optimizing the alum/antigen ratio. Alum triggered a strong Th2 response and a weak Th1 response, resulting in low IgG2a and IgG2b levels. Adjuvants OVA BALB/c Allergenicty Immune response Figures Figure 1 Figure 2 Figure 3 Introduction An adjuvant is a substance that does not cause an immunological response on its own but instead enhances the immune response to the relevant antigen present in the medium [ 1 ]. Aluminum compounds, such as aluminum hydroxide, are often used adjuvants in human vaccines to boost humoral immune responses in humans [ 2 , 3 ]. However, in limited instances, adjuvants have been shown to result in immediate-type hypersensitivity mediated by IgE [ 4 ]. The use of antigen alone in vaccines may be minimal for vaccine efficiency, so the adjuvant used in vaccines delivers the antigen to the immune system, increasing vaccine effectiveness [ 5 ]. Therefore, adjuvants are used to enhance the immunogenicity of the antigen [ 6 , 7 ]. Adjuvants are essential to prevent the rapid dispersion of the antigen in the body, ensuring a strong and long-lasting immune response [ 3 ]. They provide several advantages, such as increased vaccine efficacy, the use of less antigen, and reduced dosing requirements [ 4 , 7 , 8 ]. There are several types of adjuvants produced for diverse purposes, but despite significant adjuvant research, only seven (Aluminum hydroxide, MF59, AS01, Virus particles, AS03, AS04, and CpG 1018) have been FDA authorized for use in humans [ 6 , 9 , 10 ]. Aluminum compounds, particularly aluminum phosphate, are the most commonly used adjuvants in humans. Unlike emulsion adjuvants, they bind to antigens through electrostatic forces. Aluminum potassium sulfate is commonly referred to as Alum. Alum-based vaccines rely on the precipitation of the antigen together with the adjuvant. Alum was the first adjuvant to be approved for use, and it has been widely used for nearly a century due to its safety and effectiveness [ 11 , 12 ]. Aluminum adjuvants exhibit a depot effect, whereby the antigen binds to the surface of the aluminum adjuvant and is slowly released, thus triggering a strong antibody response [ 11 , 13 , 14 ]. These adjuvants, made up of aluminum salts, form clusters at the injection site. The clusters are taken up by macrophages via phagocytosis and can remain within macrophages transported to lymph nodes for years [ 12 , 15 ]. Freund's adjuvant consists of two parts: Complete Freund’s Adjuvant (CFA), which is composed of mineral oil, mannite monooleate, and mycobacterial cells ( Mycobacterium tuberculosis ), and Incomplete Freund's Adjuvant (IFA), which does not contain mycobacterial cells [ 9 , 16 ]. CFA is a widely used immunological agent due to its efficacy in stimulating both humoral and cellular immune responses. Its ability to bind or encapsulate antigens on the surface of cells makes them more stable, enabling a slow and controlled release of the antigen. This allows for a prolonged interaction between the antigen and the immune system [ 17 ]. It gains its effectiveness as an adjuvant by forming a stable water-in-oil emulsion [ 18 ]. Due to the inactivated mycobacterial cells in CFA, its immunostimulatory property is much higher compared to other adjuvants [ 18 , 19 ]. However, its use in humans is limited due to side effects such as lesion formation, granulomas at the injection site, hepatic and renal granulomas, and necrotizing dermatitis [ 18 , 20 – 22 ]. MF59 is a water-in-oil emulsion that forms stable micro-vesicles, surrounded by single-layer, non-ionic detergent molecules. It is an emulsion containing squalene, a fat compound. This emulsion facilitates a more efficient presentation of the antigen to the immune system, enhances the immune response, and increases the vaccine's protective effect. Squalene oil improves antigen dispersion and prolongs its exposure to the immune system, allowing for stronger immune recognition and a more robust immune response [ 23 , 24 ]. Studies have shown that squalene-based emulsions do not require additional immunostimulants to elicit an effective immune response. MF59 was approved for human use in 1997 by Italian authorities for an influenza vaccine (Fluad®, Novartis Vaccines and Diagnostics Inc., MA, USA). MF59 adjuvant has been shown to elicit a sufficient immune response even in elderly individuals, where the immune response to influenza antigens is typically insufficient [ 25 – 27 ]. Ovalbumin (OVA) is one of the most widely used food-derived proteins in allergy studies. Allergen responses of this protein have been optimized [ 28 ]. Ovalbumin, with a molecular weight of 45 kDa, denatures at 78–86°C [ 29 ]. Due to its trypsin and chymotrypsin regions, it exhibits antimicrobial properties. For this reason, it can activate the immune system and is commonly used as a model antigen in Immunisation studies [ 30 , 31 ]. In this study, BALB/c mice were immunised using different adjuvants (Alum, Freund’s Adjuvant, and MF59) along with the model antigen ovalbumin, and the differences in immune responses were compared. This study aims to contribute to vaccine development by using an adjuvant that does not induce allergic reactions while yet demonstrating antigen effectiveness. Materials and methods Ethical approval and experimental materials The TUBITAK MRC, Climate and Life Sciences, Biotechnology Unit provided us with 6-8-week-old female BALB/c mice for our study. The Institutional Biosafety Committee and the Institutional Animal Care and Use Committee (HADYEK-16563500-111-103) reviewed and approved all animal-related procedures in this work, and all studies were carried out in line with all applicable ethical regulations. In our study, the antigenic properties and doses of ovalbumin (Genaxxon, Cat No: M3105), a biocompatible, non-toxic biopolymer protein found in chicken eggs, were utilized. Alum adjuvant was obtained from InvivoGen (San Diego, CA). Freund’s complete and incomplete adjuvants were purchased from Sigma and MF59 adjuvant was obtained from InvivoGen, (Cat. No: vac-adx-10). Antibodies, goat anti-mouse polyclonal IgG conjugated alkaline phosphatase obtained from Sigma (Code: A3562); Goat anti-mouse polyclonal IgG1 conjugated alkaline phosphatase obtained from AbD Serotec (Code: STAR81A); Goat anti-mouse polyclonal IgG2a conjugated alkaline phosphatase purchased from AbD Serotec (Code: STAR82A); Goat anti-mouse polyclonal IgG2b conjugated alkaline phosphatase purchased from AbD Serotec (Code: STAR83A). Immunisation study Female BALB/c mice, aged 6-8 weeks (~20g) (n=4 animals per group), were housed in a temperature-controlled environment (25°C ± 2°C) with a 12-hour light-dark cycle. Mice were provided free access to food and water. The mice were divided into 9 groups. The native group received sterile serum physiologic (0.9% NaCl) as a negative control, while the other groups were inoculated with various doses of OVA and antigen-adjuvant formulation. Three different adjuvants used in the study were included as a control group to demonstrate that adjuvants alone are not causing an immunological response on humoral immunity. Freund's adjuvant and MF59 adjuvant were used in a 1:1 ratio with protein. In aluminum adjuvants, the antigen is slowly released after attaching to the adjuvant surface. Also, it is critical to recognize the appropriate effective dose. As a result, two distinct alum doses were tested as long as they remained within the human safety limits in this investigation. The human safety limits for aluminum adjuvant are 1.25mg [32], in this study, doses of 0.2mg and 0.1mg were used [33-36]. Mice received two intraperitoneal immunisations at 14-day intervals. Blood samples were collected at baseline (day 0), on day 14 prior to the second immunisation, and subsequently every two weeks for a total of 14 weeks following the final immunisation. In accordance with the 3R refinement principles, mice were sacrificed under general anesthesia using intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), followed by cervical dislocation. This procedure was conducted in compliance with ethical standards and the ARRIVE guidelines 2.0 (37). Table 1 Immunisation in each mice group (n=4) Mice groups Formulations Route of administration OVA 100µg OVA, 100 µl 0.9%NaCl Intraperitoneal OVA, 0.2mg Alum 100µg OVA, 0.2mg Alum, 80µl 0.9% NaCl Intraperitoneal OVA, 0.1mg Alum 100µg OVA, 0.1mg Alum, 90µl 0.9% NaCl Intraperitoneal OVA, Freund’s Adjuvant 100µg OVA, 100µl Freund’s Adjuvant Intraperitoneal OVA, MF59 100µg OVA, 100µl MF59 Intraperitoneal Alum 0.2mg Alum, 180µl 0.9% NaCl Intraperitoneal Freund’s Adjuvant 100µl Freund’s Adjuvant, 0.9% NaCl Intraperitoneal MF59 100µl MF59, 100µl 0.9% NaCl Intraperitoneal Control 200µl 0.9% NaCl Intraperitoneal ELISA for determination of serum antibody responses Prior to each blood collection, mice were placed under an infrared lamp to promote dilation of the tail vein. The tail was then disinfected with sterile alcohol, and a small incision was made using a sterile lancet. Approximately 30–50 µL of blood was collected using a micropipette and immediately mixed in a 1:1 ratio with sodium citrate. Plasma was separated by centrifugation at 7,000 × g for 10 minutes at room temperature. Hemostasis was achieved by applying gentle pressure and an antiseptic to the puncture site. All plasma samples were stored at –20°C. The levels of OVA specific serum antibodies were analyzed using indirect ELISA method. In summary, 96-well microtiter plate were coated with 1µg/100µl OVA in 50mM sodium carbonate buffer, pH 9.6. The plate was kept at 4°C overnight. The plate was washed three times with PBS-T (100mM PBS with 0.1% Tween20, pH 7.4), followed by incubation with 100µl of 1% BSA in PBS-T for 1 h at 37°C. The plate was washed with PBS-T three times and incubated for 1 h at 37°C with 100µl of the serum (x500 and x1000 dilutions) obtained from mice. After the plate was washed 3 times with PBS-T, 100µl of alkaline phosphatase conjugated anti-mouse IgG (Sigma) (1:2000 dilution), goat anti-mouse IgG1, IgG2a and IgG2b (AbD Serotec) (1:2000 dilution). The plate was washed 3 times with PBS-T and 100µl p-Nitrophenyl phosphate (pNPP) (1mg ml -1 ) (Thermo Scientific) in 0.1M diethanolamine buffer (pH 9.8) for 45 min at room temperature. Absorbance was read at 405nm. Statistical analysis A one-way ANOVA was employed to compare full groups. A two-sided p value <0.0001 indicated statistical significance. Statistical analyses and graphs were created using the GraphPad Prism 10.4.1 program. Results In the experiment, IgG titers of mice were compared after Immunisation of nine different groups with ovalbumin combined with different adjuvants and ratios. The sera were collected at 2-week intervals. Indirect ELISA method was used for detecting the IgG titers. There is a significant difference between the treatment groups and the control group. All groups containing OVA (groups 1, 2, 3, 4, and 5) showed an immune response while the groups that formulated with only adjuvants (without OVA) (groups 6, 7 and 8) and control group (group 9) showed no immune responses detected (Fig. 1 ). At the end of the second Immunisation (week 4), the group's immune responses began to develop. In this study, the compatibility of OVA with Freund's adjuvant in Group 4 produced a favorable result. It produced the strongest IgG response until the end of the 14th week when compared to other adjuvants. OVA protein coupled with another oily adjuvant, MF59 (Group 5), resulted in the second strongest response until the sixth week. However, as time passed after Immunisation, Group 5's responses began to decline. After the sixth week, Group 3 demonstrated a strong response to the slow-release Alum adjuvant, depending on the dose. Group 3 (0.1mg Alum) showed the best long-term response after Group 4. The absorbance value for Group 2 (0.2mg Alum) IgG titer was consistently below 2 and never above it. After the sixth week, Ova antigen without adjuvant in Group 1 produced no significant response. Neither the experimental control group nor the adjuvant control group showed any discernible reaction throughout the experiment. To analyze allergic reactions to ovalbumin and adjuvants, we measured IgG subunits (IgG1, IgG2a, IgG2b) levels in the blood, which indicate allergic responses. In our graph, we compared groups according to their allergic responses. IgG1 is responsible for the vast majority of IgG-induced immune responses. Therefore, it has no direct influence on allergic reactions. Allergic responses start within the first 20 to30 days. IgG2a and IgG2b inhibit allergic reactions by blocking IgE from interacting with allergens. This study found allergy responses in Group 3 (Ova, 0.1mg Alum) and Group 4 (Ova, Freund). In Fig. 2 , only IgG2b levels began to rise in Group 4. However, on the 56th day of the research, both IgG2a and IgG2b responses rose in Groups 3 and 4, as seen in Fig. 3 . IgG1 has a limited direct effect on allergy reactions, whereas IgG is responsible for the majority of immunological responses. IgG2a and IgG2b promote tolerance and reduce allergic responses. It is expected that the IgG1 reaction is similar to IgG between Groups 1–5. IgG2a suppresses allergy reactions by blocking IgE from interacting with allergens. Increased IgG2a levels promote allergen tolerance. IgG2b inhibits the interaction of IgE with allergens and modulates allergic responses. Allergic reactions happen within the first 20–30 days. In this study, we detect allergy responses, especially in groups 3 and 4. Figure 2 shows that IgE levels increase in Group 4, but IgG2a and IgG2b levels decrease. However, on the 56th day of the trial, Fig. 3 shows that IgE responses drop while IgG2b increases, particularly in Group 4. The long-term drop in IgE occurs because allergen immunotherapy increases IgG2b levels, which suppress the IgE reaction. Discussion The selection of the appropriate adjuvant is one of the most important aspects in designing an effective vaccine. Adjuvants serve a role in eliciting long-lasting and safe humoral immune responses, as well as allergic immunological responses. The optimum adjuvant is believed to reduce adverb effects, including allergens [ 38 , 39 ]. So, in this study, the most common adjuvants (CFA, Alum, and MF59) were tested to see how they affected IgG, IgG1, IgG2a, and IgG2b responses to OVA. According to our results, all OVA-containing groups predominantly generated IgG and IgG1 responses to OVA, regardless of the presence or type of adjuvant. The OVA-only group, without an adjuvant, showed a low anti-OVA IgG response, which declined rapidly as expected [ 40 ]. Additionally, only adjuvant groups showed no IgG response. OVA in CFA group produced the highest anti-OVA IgG titer compared to the other groups. This strong humoral response can be attributed to the potent immunostimulatory properties of CFA, primarily due to its mycobacterial components. Although CFA is widely recognized for promoting Th1-biased immune responses by enhancing cell-mediated immunity and pro-inflammatory cytokine production, studies have also shown that the mycobacterial content within CFA contributes significantly to the induction of robust antibody responses, including elevated levels of total IgG and IgG1 isotype production against the administered antigen [ 18 , 41 ]. This dual effect makes CFA the gold standard of adjuvants; no other adjuvants have surpassed CFA's immunostimulatory capability, however, its use in humans is limited due to side effects [ 18 ]. The Th1 response plays a crucial role in the defense against intracellular pathogens (anti-viral, anti-intracellular bacteria [ 42 ]. CFA triggers a Th1-mediated immune response, which is more likely associated with IgG2a and IgG2b production. IgG2a can suppress allergic reactions by preventing the interaction of IgE with allergens and supporting the development of tolerance against allergens. IgG2b plays an anti-inflammatory role and can regulate allergic reactions by increasing immune tolerance and preventing IgE-allergen interactions [ 5 ]. In our experiment, on day 56, the OVA, CFA group generated the highest anti-OVA IgG2a and IgG2b responses compared to the other groups (Fig. 3 ). This shows that IgG2a and IgG2b antibodies generated in response to OVA may contribute to the modulation of the Th1-biased immune response elicited by CFA. Given their effector functions, IgG2a and IgG2b could engage inhibitory Fc gamma receptors (FcγRIIB) on antigen-presenting cells or other immune cells, thereby downregulating pro-inflammatory cytokine production and limiting further Th1 cell activation [ 43 ]. This potential negative feedback mechanism highlights the role of these antibody subclasses not only as markers of a Th1-biased response but also as active regulators that help prevent excessive or prolonged Th1-driven inflammation. Unlike Alum, oil-in-water adjuvants like MF59 rapidly release the antigen. This shows that IgG2a and IgG2b in OVA inhibit the Th1-mediated immune response triggered by CFA, resulting in short-term protection [ 44 ]. This explains the rapid rise and subsequent decline in the anti-OVA IgG response of OVA, MF59. MF59 is an adjuvant that promotes a Th2-based immune response [ 45 ]. Therefore, no IgG2a or IgG2b response was observed in the OVA, MF59 group. The humoral response induced by the MF59 adjuvant with the OVA antigen is not as strong as that observed in the influenza vaccine [ 46 ]. This indicates that the interaction between the oil-based (squalene-containing) MF59 adjuvant and the OVA antigen does not provide sufficient compatibility compared to that in the influenza vaccine. One possible explanation for this difference lies in the distinct structural and biochemical properties of the two antigens. Influenza virus surface proteins, such as hemagglutinin (HA) and neuraminidase (NA), possess complex glycosylation patterns and membrane-associated domains that may interact more favorably with MF59's emulsion structure, enhancing antigen uptake and presentation [ 47 , 48 ]. In contrast, ovalbumin is a soluble, non-glycosylated protein with a different molecular conformation and physicochemical properties, potentially limiting its ability to benefit from MF59’s adjuvant effects. This highlights the importance of compatibility between MF59 and antigen. Alum adjuvants show higher immunogenicity than oil-in-water adjuvants [ 49 ]. In our result, OVA, MF59 induced a lower anti-OVA IgG response than OVA, 0.1mg of Alum but a higher response than OVA, 0.2mg of Alum. The adsorption strength resulting from the interaction between the antigen and adjuvant is one of the key factors in designing an ideal vaccine. The concentration of radicals within Alum influences adsorption strength. Therefore, the optimal Alum amount is crucial for ensuring proper physical and chemical interactions with the antigen [ 50 ]. Based on this information, we formulated two different Alum concentrations (0.1mg and 0.2mg) with OVA, as detailed in Table 1 . Our results showed that 100µg OVA, 0.1mg Alum induced a higher anti-OVA IgG response compared to 100µg OVA, 0.2mg Alum. These findings indicate that the optimal alum/antigen ratio should be determined based on the physicochemical properties of the antigen and the adjuvant. Alum stimulates cellular signaling pathways through dendritic cells, macrophages, and directly induces Th2 immune responses. It also triggers humoral immunity [ 51 – 53 ]. Alum not only induces a strong Th2 response but also a weak Th1 response [ 54 – 55 ]. In the OVA, 0.1mg Alum group, a strong IgG1 response and a weak IgG2a and IgG2b response were detected. However, in the OVA, 0.2mg Alum group, no IgG2a or IgG2b was detected. As additional information, the Th2 response can be detected with IgG4, as well as IgG1, due to their production under the influence of IL-4 and IL-13, cytokines central to Th2 immunity [ 56 ]. Conclusion Among all tested groups, CFA elicited the strongest anti-OVA IgG1 response, confirming its potency in inducing long-term humoral immunity. In contrast, MF59, due to its rapid antigen release, led to a short-lived immune response. Alum, benefiting from a depot effect, triggered a sustained but dose-dependent response. The OVA, 0.1mg Alum group exhibited a higher IgG1 response compared to the OVA, 0.2mg Alum group, reinforcing the necessity of antigen-adjuvant dose optimization. Regarding allergenic responses, CFA, which strongly induces Th1 immunity, generated the highest IgG2a and IgG2b levels, while Alum, which weakly activates Th1 pathways, resulted in a low but detectable IgG2a/IgG2b response in the OVA, 0.1mg Alum group. No IgG2a/IgG2b response was observed in other groups. Overall, adjuvant selection significantly impacts both immunogenicity and allergenicity, and these findings contribute to the optimization of vaccine formulations for future applications. To be efficient in antigen presentation, the adjuvant must be compatible with the antigen. The MF59 adjuvant, which is an oil-in-water emulsion, did not present antigens well enough with OVA in our study, but it did well with influenza. Alternatively, it should be noted that with adjuvants such as Alum, it is critical to select the appropriate dose compatible with the antigen, and thus adjuvant antigen dose studies are undoubtedly required in Alum investigations. While conducting all of these trials, we also sought to demonstrate that the adjuvant activates allergenicity to some level. Declarations Acknowledgments The authors acknowledge the financial support and infrastructure provided by the TÜBİTAK Marmara Research Center, funded by the Presidency of Turkey, Strategy and Budget Directorate. Author Contributions All in vivo studies were completed out by H.U.P, with assistance from B.K. and H.N.A. B.K. and H.N.A. contributed to the experimental design. B.K. and H.N.A. authored the manuscript. H.U.P. supervised and co-directed the research, as well as edited the manuscript. All authors reviewed and approved the final manuscript. Funding This work was supported by the TUBITAK Marmara Research Center (MRC) through funding provided by the Presidency of Turkey, Presidency of Strategy and Budget. Availability of data and materials All data generated or analyzed during this study are included in this published article. Declarations Conflict of interest The authors declare no conflicts of interest. Ethics Approval and Consent to Participate All animal experiments were approved by the Local Animal Ethics Committee (HADYEK) under permit number 16563500-111-103 and conducted in accordance with the European Union Directive 2010/63/EU on the protection of animals used for scientific purposes. Consent to Participate Not applicable. This study did not involve human participants; all ethical procedures related to animal use were approved by HADYEK. Orcid Hivda Ulbegi POLAT https://orcid.org/0000-0001-8424-6849 Bilal KOCAMAN https://orcid.org/ 0009-0008-0226-7899 Hatice Nur AYDIN https://orcid.org/0000-0002-7975-2189 References Facciolà A, Visalli G, Laganà A, Di Pietro A. An Overview of Vaccine Adjuvants: Current Evidence and Future Perspectives. Vaccines (Basel). 2022;10:819. Tafalla C, Bøgwald J, Dalmo RA. Adjuvants and immunostimulants in fish vaccines: Current knowledge and future perspectives. Fish Shellfish Immunol. 2013;35:1740–50. Sivakumar SM, Safhi MM, Kannadasan M, Sukumaran N. Vaccine adjuvants – Current status and prospects on controlled release adjuvancity. Saudi Pharm J. 2011;19:197–206. Schijns VE, Lavelle EC. Trends in vaccine adjuvants. Expert Rev Vaccines. 2011;10:539–50. Yanase N, Toyota H, Hata K, Yagyu S, Seki T, Harada M, et al. OVA-bound nanoparticles induce OVA-specific IgG1, IgG2a, and IgG2b responses with low IgE synthesis. Vaccine. 2014;32:5918–24. Zhao T, Cai Y, Jiang Y, He X, Wei Y, Yu Y, et al. Vaccine adjuvants: mechanisms and platforms. Signal Transduct Target Ther. 2023;8:283. Moni S, Abdelwahab S, Jabeen S, Elmobark A, Aqaili M, Ghoal D. G, Advancements in Vaccine Adjuvants: The Journey from Alum to Nano Formulations. Vaccines (Basel). 2023;11. Wang Z, Xu J. Better Adjuvants for Better Vaccines: Progress in Adjuvant Delivery Systems, Modifications, and Adjuvant–Antigen Codelivery. Vaccines (Basel). 2020;8:128. Nanishi E, Dowling D, Levy O. Toward precision adjuvants: Optimizing science and safety. Curr Opin Pediatr. 2020;32:1. Wan X, Yin Y, Zhou C, Hou L, Cui Q, Zhang X, et al. Polysaccharides derived from Chinese medicinal herbs: A promising choice of vaccine adjuvants. Carbohydr Polym. 2021;276:118739. Danielsson R, Eriksson H. Aluminium adjuvants in vaccines – A way to modulate the immune response. Semin Cell Dev Biol. 2021;115:3–9. Zhang T, He P, Guo D, Chen K, Hu Z. Zou 烨宁. Research Progress of Aluminum Phosphate Adjuvants and Their Action Mechanisms. Pharmaceutics. 2023;15:1756. Dewangan HK, Singh S, Mishra R, Dubey RK. A review on application of nanoadjuvant as delivery system. Int J Appl Pharm. 2020;12:24–33. Smith WJ, Thompson R, Egan PM, Zhang Y, Indrawati L, Skinner JM, et al. Impact of aluminum adjuvants on the stability of pneumococcal polysaccharide-protein conjugate vaccines. Vaccine. 2023;41:5113–25. Gherardi R, Coquet M, Cherin P, Belec L, Moretto P, Dreyfus PA, et al. Macrophagic myofascitis lesions assess long-term persistence of vaccine-derived aluminum hydroxide in muscle. Brain. 2001;124:1821–31. Gül B, Yurdakök Dikmen B. AŞI ADJUVANLARI ve İSTENMEYEN ETKİLERİ. Veteriner Farmakoloji ve Toksikoloji Derneği Bülteni. 2019;10:91–105. Bassi N, Luisetto R, Ghirardello A, Gatto M, Bottazzi B, Shoenfeld Y, et al. Vaccination of mice for research purpose: Alum is as effective as and safer than complete Freund adjuvant. Reumatismo. 2012;64:380–7. Stils H. Adjuvants and Antibody Production: Dispelling the Myths Associated with Freund’s Complete and Other Adjuvants. ILAR J. 2005;46:280–93. Smith DE, O’Brien ME, Palmer VJ, Sadowski JA. The selection of an adjuvant emulsion for polyclonal antibody production using a low-molecular-weight antigen in rabbits. Lab Anim Sci. 1992;42:599–601. Leenaars PPAM, Hendriksen CFM, Angulo AF, Koedam MA, Claassen E. Evaluation of several adjuvants as alternatives to the use of Freund’s adjuvant in rabbits. Vet Immunol Immunopathol. 1994;40:225–41. Broderson JR. A retrospective review of lesions associated with the use of Freund’s adjuvant. Lab Anim Sci. 1989;39:400–5. Leenaars M, Koedam M, Wester P, Baumans V, Claassen E, Hendriksen C. Assessment of side effects induced by injection of different adjuvant/antigen combinations in rabbits and mice. Lab Anim. 1998;32:387–406. Schultze V, D’Agosto V, Wack A, Novicki D, Zorn J, Hennig R. Safety of MF59 adjuvant. Vaccine. 2008;26:3209–22. Sahly H, El. MF59™ as a vaccine adjuvant: a review of safety and immunogenicity. Expert Rev Vaccines. 2010;9:1135–41. O’Hagan D. MF59 is a safe and potent vaccine adjuvant that enhances protection against influenza virus infection. Expert Rev Vaccines. 2007;6:699–710. Coleman B, Sanderson R, Haag M, McGovern I. Effectiveness of the MF59-adjuvanted trivalent or quadrivalent seasonal influenza vaccine among adults 65 years of age or older, a systematic review and meta‐analysis. Influenza Other Respir Viruses. 2021;15. Villa M, Black S, Groth N, Rothman KJ, Apolone G, Weiss NS, et al. Safety of MF59-Adjuvanted Influenza Vaccination in the Elderly: Results of a Comparative Study of MF59-Adjuvanted Vaccine Versus Nonadjuvanted Influenza Vaccine in Northern Italy. Am J Epidemiol. 2013;178:1139–45. Helm RM, Burks AW. Animal models of food allergy. Curr Opin Allergy Clin Immunol. 2002;2:541–6. Xiao J-X, Wang L-H, Xu T-C, Huang G-Q. Complex coacervation of carboxymethyl konjac glucomannan and chitosan and coacervate characterization. Int J Biol Macromol. 2018;123. Pellegrini A, Hülsmeier A, Hunziker P, Thomas U. Proteolytic fragments of ovalbumin display antimicrobial activity. Biochim Biophys Acta. 2004;1672:76–85. Rostamabadi H, Chaudhary V, Chhikara N, Sharma N, Nowacka M, Demirkesen Mert I, et al. Ovalbumin, an outstanding food hydrocolloid: Applications, technofunctional attributes, and nutritional facts, A systematic review. Food Hydrocoll. 2023;139:108514. Vecchi S, Bufali S, Skibinski DAG, O’hagan DT, Singh M. Aluminum adjuvant dose guidelines in vaccine formulation for preclinical evaluations. J Pharm Sci. 2012;101:17–20. Sun Y, Liu J. Adjuvant effect of water-soluble polysaccharide (PAP) from the mycelium of Polyporus albicans on the immune responses to ovalbumin in mice. Vaccine. 2008;26:3932–6. Bansal V, Kumar M, Dalela M, Brahmne HG, Singh H. Evaluation of synergistic effect of biodegradable polymeric nanoparticles and aluminum based adjuvant for improving vaccine efficacy. Int J Pharm. 2014;471:377–84. Dong N, Zhang GQ, Jia PY, Wu JH, Li S, Shan JJ, et al. Adjuvant activities of seven natural polysaccharides on immune responses to ovalbumin in mice. Chin J Pharmacol Toxicol. 2013;27:307–13. Ma B, Fu X, Zhu P, Niu J, Lu F. Allergenicity, assembly and applications of ovalbumin in egg white: a review. Crit Rev Food Sci Nutr. 2023;64:1–17. Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, et al. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol. 2020;14(7):18. Allison AC, Byars NE. Immunological adjuvants: Desirable properties and side-effects. Mol Immunol. 1991;28:279–84. Petrovsky N, Aguilar J, Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and future trends. Immunol Cell Biol 82: 488–496. Immunol Cell Biol. 2004;82:488–96. Persiyanova EV, Kuznetsova TA, Silchenko AS. Effect of Sulfated Polysaccharides from Marine Hydrobionts on Humoral Immune Response to Ovalbumin in Mice. Bull Exp Biol Med. 2020;169:246–8. Oscherwitz J, Hankenson FC, Yu F, Cease KB. Low-dose intraperitoneal Freund’s adjuvant: Toxicity and immunogenicity in mice using an immunogen targeting amyloid-β peptide. Vaccine. 2006;24:3018–25. Fontes JA, Barin JG, Talor MV, Stickel N, Schaub J, Rose NR, et al. Complete Freund’s adjuvant induces experimental autoimmune myocarditis by enhancing IL-6 production during initiation of the immune response. Immun Inflamm Dis. 2017;5:163–76. Nimmerjahn F, Ravetch JV. Fcγ receptors as regulators of immune responses. Nat Rev Immunol. 2008;8:34–47. Radmehri M, Talebi A, Ameghi Roudsari A, Mousaviyan SM, Gholipour MAJ, Taghizadeh M. Comparative Study on the Efficacy of MF 59, ISA70 VG, and Nano-Aluminum Hydroxide Adjuvants, Alone and with Nano-Selenium on Humoral Immunity Induced by a Bivalent Newcastle + Avian Influenza Vaccine in Chickens. Arch Razi Inst. 2021;76:1213–20. Lin P-H, Yang H-C. The adjuvant effects of MF59 on antigen-specific regulatory and effector T cells. J Immunol. 2019;202 1_Supplement:196.15-196.15. Fang J-H, Hora M. The Adjuvant MF59: A 10-Year Perspective Gary Ott, Ramachandran Radhakrishnan. In: O’Hagan DT, editor. Vaccine Adjuvants: Preparation Methods and Research Protocols. Totowa, NJ: Springer New York; 2000. pp. 211–28. Caton AJ, Brownlee GG, Yewdell JW, Gerhard W. The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). Cell. 1982;31:417–27. Krammer F. The human antibody response to influenza A virus infection and vaccination. Nat Rev Immunol. 2019;19:1. Banihashemi SR, Baradaran B, Tebianian M, Majidi J, Jabbari AR, Jalali S, et al. Development of Leptospira Killed Whole Culture Vaccine Using Different Adjuvants and Evaluation of Humoral Immune Response in Hamsters. J Vaccines Vaccin. 2013;4:1–5. He P, 烨宁 Z, Hu Z. Advances in aluminum hydroxide-based adjuvant research and its mechanism. Hum Vaccin Immunother. 2015;11. Sasaki E, Asanuma H, Momose H, Furuhata K, Mizukami T, Hamaguchi I. Nasal alum-adjuvanted vaccine promotes IL-33 release from alveolar epithelial cells that elicits IgA production via type 2 immune responses. PLoS Pathog. 2021;17:e1009890. Gogoi H, Mani R, Bhatnagar R. Re-inventing traditional aluminum-based adjuvants: Insight into a century of advancements. Int Rev Immunol. 2024;44:1–24. Hogenesch H. Mechanism of Immunopotentiation and Safety of Aluminum Adjuvants. Front Immunol. 2013;3:406. HogenEsch H, O’Hagan DT, Fox CB. Optimizing the utilization of aluminum adjuvants in vaccines: you might just get what you want. NPJ Vaccines. 2018;3:51. Kool M, Soullie T, Nimwegen M, Willart M, Muskens F, Jung S, et al. Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells. J Exp Med. 2008;205:869–82. van de Veen W, Akdis M. Role of IgG4 in IgE-mediated allergic responses. J Allergy Clin Immunol. 2016;138:1434–5. Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstractImage.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7004970","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":486200064,"identity":"ba7e9519-7574-4d77-bd43-20b3614d062b","order_by":0,"name":"Hivda Ulbegi POLAT","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIie3RMUvDQBjG8SccXJaXZr3SkM+QkqEUC/kqKYW4OknAxSJkKviVrhxkCnaNkMFSiIuDBYcMDl6CpSiJcRS8/3QH9+Pu5QCT6Q/mM0A0ixFgPQGSAP5LwsGYLzWhE6E+gjPhQhMMkplN06JG6XF7l90c09IN7Syy3hKFcCI7yfyOgvkGVcBpxYttWhFRLJmbK9Ao6n6Y4rEgqGWKlig9y+UtG6cxqOdlDRm/N8Q58KuWOM9DhGWT9hax4miJiKV1TBc/EXXh+noWcQhE/qBnKapIIV8Q5T1kt10/viSl5zjL/WtyXYb2fTzd14nw7E03+YTy615R/7ec+kaseuC8yWQy/as+ABwfVlJCS3fUAAAAAElFTkSuQmCC","orcid":"","institution":"TÜBİTAK Marmara Research Center","correspondingAuthor":true,"prefix":"","firstName":"Hivda","middleName":"Ulbegi","lastName":"POLAT","suffix":""},{"id":486200069,"identity":"4ce4c004-4fbc-4416-86d7-15763d56d8ae","order_by":1,"name":"Bilal KOCAMAN","email":"","orcid":"","institution":"Gebze Technical University","correspondingAuthor":false,"prefix":"","firstName":"Bilal","middleName":"","lastName":"KOCAMAN","suffix":""},{"id":486200075,"identity":"e8f030e9-e836-41cf-81b9-ae8a103dc444","order_by":2,"name":"Hatice Nur AYDIN","email":"","orcid":"","institution":"Istanbul Medeniyet University","correspondingAuthor":false,"prefix":"","firstName":"Hatice","middleName":"Nur","lastName":"AYDIN","suffix":""}],"badges":[],"createdAt":"2025-06-29 22:53:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7004970/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7004970/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87050853,"identity":"81d8cb0b-c89f-42ec-a02f-6f1636df4832","added_by":"auto","created_at":"2025-07-18 14:59:54","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":240682,"visible":true,"origin":"","legend":"\u003cp\u003eSerum OVA-specific IgG levels in mice groups immunised with different adjuvant formulations. A one-way ANOVA was used for statistical analysis. The contrasted results were statistically significant (P\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7004970/v1/17195c3559989f868175dddc.jpeg"},{"id":87052300,"identity":"7ad7fb84-8084-48cc-a849-a42a7afcd5a9","added_by":"auto","created_at":"2025-07-18 15:07:54","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":212206,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of allergic responses on day 28.\u003cstrong\u003e \u003c/strong\u003eA one-way ANOVA was used for statistical analysis. The contrasted results were statistically significant (P\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7004970/v1/e4e62c8d271faecc0cac9ebc.jpeg"},{"id":87053708,"identity":"3db73553-f967-40ec-8d7d-29fe58055954","added_by":"auto","created_at":"2025-07-18 15:15:54","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":220109,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of allergic responses on day 56. A one-way ANOVA was used for statistical analysis. The contrasted results were statistically significant (P\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7004970/v1/bcc9f34d4a32aa039926f0ff.jpeg"},{"id":87459052,"identity":"f5b16419-38b8-401c-8f0c-32e38ae14196","added_by":"auto","created_at":"2025-07-24 05:32:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1236465,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7004970/v1/65d71851-5758-4363-b72e-7306e46ccf9b.pdf"},{"id":87050861,"identity":"f9df8195-e5c0-443a-ab99-1c99520b892e","added_by":"auto","created_at":"2025-07-18 14:59:54","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":121850,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstractImage.docx","url":"https://assets-eu.researchsquare.com/files/rs-7004970/v1/310bf7530247c107b24dcc3e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of immune responses and allergenic risks to different adjuvants in an OVA-based immunisation model using BALB/c mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAn adjuvant is a substance that does not cause an immunological response on its own but instead enhances the immune response to the relevant antigen present in the medium [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Aluminum compounds, such as aluminum hydroxide, are often used adjuvants in human vaccines to boost humoral immune responses in humans [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, in limited instances, adjuvants have been shown to result in immediate-type hypersensitivity mediated by IgE [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The use of antigen alone in vaccines may be minimal for vaccine efficiency, so the adjuvant used in vaccines delivers the antigen to the immune system, increasing vaccine effectiveness [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, adjuvants are used to enhance the immunogenicity of the antigen [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAdjuvants are essential to prevent the rapid dispersion of the antigen in the body, ensuring a strong and long-lasting immune response [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. They provide several advantages, such as increased vaccine efficacy, the use of less antigen, and reduced dosing requirements [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. There are several types of adjuvants produced for diverse purposes, but despite significant adjuvant research, only seven (Aluminum hydroxide, MF59, AS01, Virus particles, AS03, AS04, and CpG 1018) have been FDA authorized for use in humans [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAluminum compounds, particularly aluminum phosphate, are the most commonly used adjuvants in humans. Unlike emulsion adjuvants, they bind to antigens through electrostatic forces. Aluminum potassium sulfate is commonly referred to as Alum. Alum-based vaccines rely on the precipitation of the antigen together with the adjuvant. Alum was the first adjuvant to be approved for use, and it has been widely used for nearly a century due to its safety and effectiveness [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Aluminum adjuvants exhibit a depot effect, whereby the antigen binds to the surface of the aluminum adjuvant and is slowly released, thus triggering a strong antibody response [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These adjuvants, made up of aluminum salts, form clusters at the injection site. The clusters are taken up by macrophages via phagocytosis and can remain within macrophages transported to lymph nodes for years [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFreund's adjuvant consists of two parts: Complete Freund\u0026rsquo;s Adjuvant (CFA), which is composed of mineral oil, mannite monooleate, and mycobacterial cells (\u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e), and Incomplete Freund's Adjuvant (IFA), which does not contain mycobacterial cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. CFA is a widely used immunological agent due to its efficacy in stimulating both humoral and cellular immune responses. Its ability to bind or encapsulate antigens on the surface of cells makes them more stable, enabling a slow and controlled release of the antigen. This allows for a prolonged interaction between the antigen and the immune system [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It gains its effectiveness as an adjuvant by forming a stable water-in-oil emulsion [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Due to the inactivated mycobacterial cells in CFA, its immunostimulatory property is much higher compared to other adjuvants [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, its use in humans is limited due to side effects such as lesion formation, granulomas at the injection site, hepatic and renal granulomas, and necrotizing dermatitis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMF59 is a water-in-oil emulsion that forms stable micro-vesicles, surrounded by single-layer, non-ionic detergent molecules. It is an emulsion containing squalene, a fat compound. This emulsion facilitates a more efficient presentation of the antigen to the immune system, enhances the immune response, and increases the vaccine's protective effect. Squalene oil improves antigen dispersion and prolongs its exposure to the immune system, allowing for stronger immune recognition and a more robust immune response [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Studies have shown that squalene-based emulsions do not require additional immunostimulants to elicit an effective immune response. MF59 was approved for human use in 1997 by Italian authorities for an influenza vaccine (Fluad\u0026reg;, Novartis Vaccines and Diagnostics Inc., MA, USA). MF59 adjuvant has been shown to elicit a sufficient immune response even in elderly individuals, where the immune response to influenza antigens is typically insufficient [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOvalbumin (OVA) is one of the most widely used food-derived proteins in allergy studies. Allergen responses of this protein have been optimized [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Ovalbumin, with a molecular weight of 45 kDa, denatures at 78\u0026ndash;86\u0026deg;C [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Due to its trypsin and chymotrypsin regions, it exhibits antimicrobial properties. For this reason, it can activate the immune system and is commonly used as a model antigen in Immunisation studies [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In this study, BALB/c mice were immunised using different adjuvants (Alum, Freund\u0026rsquo;s Adjuvant, and MF59) along with the model antigen ovalbumin, and the differences in immune responses were compared. This study aims to contribute to vaccine development by using an adjuvant that does not induce allergic reactions while yet demonstrating antigen effectiveness.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and experimental materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe TUBITAK MRC, Climate and Life Sciences, Biotechnology Unit provided us with 6-8-week-old female BALB/c mice for our study. The Institutional Biosafety Committee and the Institutional Animal Care and Use Committee (HADYEK-16563500-111-103) reviewed and approved all animal-related procedures in this work, and all studies were carried out in line with all applicable ethical regulations. In our study, the antigenic properties and doses of ovalbumin (Genaxxon, Cat No: M3105), a biocompatible, non-toxic biopolymer protein found in chicken eggs, were utilized.\u0026nbsp;Alum adjuvant was obtained from InvivoGen (San Diego, CA). Freund\u0026rsquo;s complete and incomplete adjuvants were purchased from Sigma and MF59 adjuvant was obtained from InvivoGen, (Cat. No: vac-adx-10). Antibodies, goat anti-mouse polyclonal IgG conjugated alkaline phosphatase obtained from Sigma (Code: A3562); Goat anti-mouse polyclonal IgG1 conjugated alkaline phosphatase obtained from AbD Serotec (Code: STAR81A); Goat anti-mouse polyclonal IgG2a conjugated alkaline phosphatase purchased from AbD Serotec (Code: STAR82A); Goat anti-mouse polyclonal IgG2b conjugated alkaline phosphatase purchased from AbD Serotec (Code: STAR83A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunisation study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale BALB/c mice, aged 6-8 weeks (~20g) (n=4 animals per group), were housed in a temperature-controlled environment (25\u0026deg;C \u0026plusmn; 2\u0026deg;C) with a 12-hour light-dark cycle. Mice were provided free access to food and water. The mice were divided into 9 groups. The native group received sterile serum physiologic (0.9% NaCl) as a negative control, while the other groups were inoculated with various doses of OVA and antigen-adjuvant formulation. Three different adjuvants used in the study were included as a control group to demonstrate that adjuvants alone are not causing an immunological response on humoral immunity. Freund\u0026apos;s adjuvant and MF59 adjuvant were used in a 1:1 ratio with protein. In aluminum adjuvants, the antigen is slowly released after attaching to the adjuvant surface. Also, it is critical to recognize the appropriate effective dose. As a result, two distinct alum doses were tested as long as they remained within the human safety limits in this investigation. The human safety limits for aluminum adjuvant are 1.25mg [32], in this study, doses of 0.2mg and 0.1mg were used [33-36]. Mice received two intraperitoneal immunisations at 14-day intervals. Blood samples were collected at baseline (day 0), on day 14 prior to the second immunisation, and subsequently every two weeks for a total of 14 weeks following the final immunisation. In accordance with the 3R refinement principles, mice were sacrificed under general anesthesia using intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), followed by cervical dislocation. This procedure was conducted in compliance with ethical standards and the ARRIVE guidelines 2.0 (37).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Immunisation in each mice group (n=4)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"96%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMice groups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFormulations\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRoute of administration\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eOVA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e100\u0026micro;g OVA, 100 \u0026micro;l 0.9%NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003eIntraperitoneal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eOVA, 0.2mg Alum\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e100\u0026micro;g OVA, 0.2mg Alum, 80\u0026micro;l 0.9% NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003eIntraperitoneal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eOVA, 0.1mg Alum\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e100\u0026micro;g OVA, 0.1mg Alum, 90\u0026micro;l 0.9% NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003eIntraperitoneal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eOVA, Freund\u0026rsquo;s Adjuvant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e100\u0026micro;g OVA, 100\u0026micro;l Freund\u0026rsquo;s Adjuvant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003eIntraperitoneal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eOVA, MF59\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e100\u0026micro;g OVA, 100\u0026micro;l MF59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003eIntraperitoneal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eAlum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e0.2mg Alum, 180\u0026micro;l 0.9% NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003eIntraperitoneal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eFreund\u0026rsquo;s Adjuvant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e100\u0026micro;l Freund\u0026rsquo;s Adjuvant, 0.9% NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003eIntraperitoneal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eMF59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e100\u0026micro;l MF59, 100\u0026micro;l 0.9% NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003eIntraperitoneal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eControl\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e200\u0026micro;l 0.9% NaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003eIntraperitoneal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eELISA for determination of serum antibody responses \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrior to each blood collection, mice were placed under an infrared lamp to promote dilation of the tail vein. The tail was then disinfected with sterile alcohol, and a small incision was made using a sterile lancet. Approximately 30\u0026ndash;50 \u0026micro;L of blood was collected using a micropipette and immediately mixed in a 1:1 ratio with sodium citrate. Plasma was separated by centrifugation at 7,000 \u0026times; g for 10 minutes at room temperature. Hemostasis was achieved by applying gentle pressure and an antiseptic to the puncture site. All plasma samples were stored at \u0026ndash;20\u0026deg;C. The levels of OVA specific serum antibodies were analyzed using indirect ELISA method. In summary, 96-well microtiter plate were coated with 1\u0026micro;g/100\u0026micro;l OVA in 50mM sodium carbonate buffer, pH 9.6. The plate was kept at 4\u0026deg;C overnight. The plate was washed three times with PBS-T (100mM PBS with 0.1% Tween20, pH 7.4), followed by incubation with 100\u0026micro;l of 1% BSA in PBS-T for 1 h at 37\u0026deg;C. The plate was washed with PBS-T three times and incubated for 1 h at 37\u0026deg;C with 100\u0026micro;l of the serum (x500 and x1000 dilutions) obtained from mice. After the plate was washed 3 times with PBS-T, 100\u0026micro;l of alkaline phosphatase conjugated anti-mouse IgG (Sigma) (1:2000 dilution), goat anti-mouse IgG1, IgG2a and IgG2b (AbD Serotec) (1:2000 dilution). The plate was washed 3 times with PBS-T and 100\u0026micro;l p-Nitrophenyl phosphate (pNPP) (1mg ml\u003csup\u003e-1\u003c/sup\u003e) (Thermo Scientific) in 0.1M diethanolamine buffer (pH 9.8) for 45 min at room temperature. Absorbance was read at 405nm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA one-way ANOVA was employed to compare full groups. A two-sided p value \u0026lt;0.0001 indicated statistical significance. Statistical analyses and graphs were created using the GraphPad Prism 10.4.1 program.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn the experiment, IgG titers of mice were compared after Immunisation of nine different groups with ovalbumin combined with different adjuvants and ratios. The sera were collected at 2-week intervals. Indirect ELISA method was used for detecting the IgG titers. There is a significant difference between the treatment groups and the control group.\u003c/p\u003e\u003cp\u003eAll groups containing OVA (groups 1, 2, 3, 4, and 5) showed an immune response while the groups that formulated with only adjuvants (without OVA) (groups 6, 7 and 8) and control group (group 9) showed no immune responses detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). At the end of the second Immunisation (week 4), the group's immune responses began to develop. In this study, the compatibility of OVA with Freund's adjuvant in Group 4 produced a favorable result. It produced the strongest IgG response until the end of the 14th week when compared to other adjuvants. OVA protein coupled with another oily adjuvant, MF59 (Group 5), resulted in the second strongest response until the sixth week. However, as time passed after Immunisation, Group 5's responses began to decline. After the sixth week, Group 3 demonstrated a strong response to the slow-release Alum adjuvant, depending on the dose. Group 3 (0.1mg Alum) showed the best long-term response after Group 4. The absorbance value for Group 2 (0.2mg Alum) IgG titer was consistently below 2 and never above it. After the sixth week, Ova antigen without adjuvant in Group 1 produced no significant response. Neither the experimental control group nor the adjuvant control group showed any discernible reaction throughout the experiment.\u003c/p\u003e\u003cp\u003eTo analyze allergic reactions to ovalbumin and adjuvants, we measured IgG subunits (IgG1, IgG2a, IgG2b) levels in the blood, which indicate allergic responses. In our graph, we compared groups according to their allergic responses. IgG1 is responsible for the vast majority of IgG-induced immune responses. Therefore, it has no direct influence on allergic reactions. Allergic responses start within the first 20 to30 days. IgG2a and IgG2b inhibit allergic reactions by blocking IgE from interacting with allergens. This study found allergy responses in Group 3 (Ova, 0.1mg Alum) and Group 4 (Ova, Freund). In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, only IgG2b levels began to rise in Group 4. However, on the 56th day of the research, both IgG2a and IgG2b responses rose in Groups 3 and 4, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eIgG1 has a limited direct effect on allergy reactions, whereas IgG is responsible for the majority of immunological responses. IgG2a and IgG2b promote tolerance and reduce allergic responses. It is expected that the IgG1 reaction is similar to IgG between Groups 1\u0026ndash;5. IgG2a suppresses allergy reactions by blocking IgE from interacting with allergens. Increased IgG2a levels promote allergen tolerance. IgG2b inhibits the interaction of IgE with allergens and modulates allergic responses. Allergic reactions happen within the first 20\u0026ndash;30 days. In this study, we detect allergy responses, especially in groups 3 and 4. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows that IgE levels increase in Group 4, but IgG2a and IgG2b levels decrease. However, on the 56th day of the trial, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that IgE responses drop while IgG2b increases, particularly in Group 4. The long-term drop in IgE occurs because allergen immunotherapy increases IgG2b levels, which suppress the IgE reaction.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe selection of the appropriate adjuvant is one of the most important aspects in designing an effective vaccine. Adjuvants serve a role in eliciting long-lasting and safe humoral immune responses, as well as allergic immunological responses. The optimum adjuvant is believed to reduce adverb effects, including allergens [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. So, in this study, the most common adjuvants (CFA, Alum, and MF59) were tested to see how they affected IgG, IgG1, IgG2a, and IgG2b responses to OVA. According to our results, all OVA-containing groups predominantly generated IgG and IgG1 responses to OVA, regardless of the presence or type of adjuvant. The OVA-only group, without an adjuvant, showed a low anti-OVA IgG response, which declined rapidly as expected [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Additionally, only adjuvant groups showed no IgG response.\u003c/p\u003e\u003cp\u003eOVA in CFA group produced the highest anti-OVA IgG titer compared to the other groups. This strong humoral response can be attributed to the potent immunostimulatory properties of CFA, primarily due to its mycobacterial components. Although CFA is widely recognized for promoting Th1-biased immune responses by enhancing cell-mediated immunity and pro-inflammatory cytokine production, studies have also shown that the mycobacterial content within CFA contributes significantly to the induction of robust antibody responses, including elevated levels of total IgG and IgG1 isotype production against the administered antigen [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This dual effect makes CFA the gold standard of adjuvants; no other adjuvants have surpassed CFA's immunostimulatory capability, however, its use in humans is limited due to side effects [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The Th1 response plays a crucial role in the defense against intracellular pathogens (anti-viral, anti-intracellular bacteria [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. CFA triggers a Th1-mediated immune response, which is more likely associated with IgG2a and IgG2b production. IgG2a can suppress allergic reactions by preventing the interaction of IgE with allergens and supporting the development of tolerance against allergens. IgG2b plays an anti-inflammatory role and can regulate allergic reactions by increasing immune tolerance and preventing IgE-allergen interactions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In our experiment, on day 56, the OVA, CFA group generated the highest anti-OVA IgG2a and IgG2b responses compared to the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This shows that IgG2a and IgG2b antibodies generated in response to OVA may contribute to the modulation of the Th1-biased immune response elicited by CFA. Given their effector functions, IgG2a and IgG2b could engage inhibitory Fc gamma receptors (FcγRIIB) on antigen-presenting cells or other immune cells, thereby downregulating pro-inflammatory cytokine production and limiting further Th1 cell activation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. This potential negative feedback mechanism highlights the role of these antibody subclasses not only as markers of a Th1-biased response but also as active regulators that help prevent excessive or prolonged Th1-driven inflammation.\u003c/p\u003e\u003cp\u003eUnlike Alum, oil-in-water adjuvants like MF59 rapidly release the antigen. This shows that IgG2a and IgG2b in OVA inhibit the Th1-mediated immune response triggered by CFA, resulting in short-term protection [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. This explains the rapid rise and subsequent decline in the anti-OVA IgG response of OVA, MF59. MF59 is an adjuvant that promotes a Th2-based immune response [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Therefore, no IgG2a or IgG2b response was observed in the OVA, MF59 group. The humoral response induced by the MF59 adjuvant with the OVA antigen is not as strong as that observed in the influenza vaccine [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. This indicates that the interaction between the oil-based (squalene-containing) MF59 adjuvant and the OVA antigen does not provide sufficient compatibility compared to that in the influenza vaccine. One possible explanation for this difference lies in the distinct structural and biochemical properties of the two antigens. Influenza virus surface proteins, such as hemagglutinin (HA) and neuraminidase (NA), possess complex glycosylation patterns and membrane-associated domains that may interact more favorably with MF59's emulsion structure, enhancing antigen uptake and presentation [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In contrast, ovalbumin is a soluble, non-glycosylated protein with a different molecular conformation and physicochemical properties, potentially limiting its ability to benefit from MF59\u0026rsquo;s adjuvant effects. This highlights the importance of compatibility between MF59 and antigen.\u003c/p\u003e\u003cp\u003eAlum adjuvants show higher immunogenicity than oil-in-water adjuvants [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In our result, OVA, MF59 induced a lower anti-OVA IgG response than OVA, 0.1mg of Alum but a higher response than OVA, 0.2mg of Alum. The adsorption strength resulting from the interaction between the antigen and adjuvant is one of the key factors in designing an ideal vaccine. The concentration of radicals within Alum influences adsorption strength. Therefore, the optimal Alum amount is crucial for ensuring proper physical and chemical interactions with the antigen [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Based on this information, we formulated two different Alum concentrations (0.1mg and 0.2mg) with OVA, as detailed in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Our results showed that 100\u0026micro;g OVA, 0.1mg Alum induced a higher anti-OVA IgG response compared to 100\u0026micro;g OVA, 0.2mg Alum. These findings indicate that the optimal alum/antigen ratio should be determined based on the physicochemical properties of the antigen and the adjuvant.\u003c/p\u003e\u003cp\u003eAlum stimulates cellular signaling pathways through dendritic cells, macrophages, and directly induces Th2 immune responses. It also triggers humoral immunity [\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Alum not only induces a strong Th2 response but also a weak Th1 response [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In the OVA, 0.1mg Alum group, a strong IgG1 response and a weak IgG2a and IgG2b response were detected. However, in the OVA, 0.2mg Alum group, no IgG2a or IgG2b was detected. As additional information, the Th2 response can be detected with IgG4, as well as IgG1, due to their production under the influence of IL-4 and IL-13, cytokines central to Th2 immunity [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAmong all tested groups, CFA elicited the strongest anti-OVA IgG1 response, confirming its potency in inducing long-term humoral immunity. In contrast, MF59, due to its rapid antigen release, led to a short-lived immune response. Alum, benefiting from a depot effect, triggered a sustained but dose-dependent response. The OVA, 0.1mg Alum group exhibited a higher IgG1 response compared to the OVA, 0.2mg Alum group, reinforcing the necessity of antigen-adjuvant dose optimization. Regarding allergenic responses, CFA, which strongly induces Th1 immunity, generated the highest IgG2a and IgG2b levels, while Alum, which weakly activates Th1 pathways, resulted in a low but detectable IgG2a/IgG2b response in the OVA, 0.1mg Alum group. No IgG2a/IgG2b response was observed in other groups. Overall, adjuvant selection significantly impacts both immunogenicity and allergenicity, and these findings contribute to the optimization of vaccine formulations for future applications. To be efficient in antigen presentation, the adjuvant must be compatible with the antigen. The MF59 adjuvant, which is an oil-in-water emulsion, did not present antigens well enough with OVA in our study, but it did well with influenza. Alternatively, it should be noted that with adjuvants such as Alum, it is critical to select the appropriate dose compatible with the antigen, and thus adjuvant antigen dose studies are undoubtedly required in Alum investigations. While conducting all of these trials, we also sought to demonstrate that the adjuvant activates allergenicity to some level.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the financial support and infrastructure provided by the T\u0026Uuml;BİTAK Marmara Research Center, funded by the Presidency of Turkey, Strategy and Budget Directorate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll in vivo studies were completed out by H.U.P, with assistance from B.K. and H.N.A. B.K. and H.N.A. contributed to the experimental design. B.K. and H.N.A. authored the manuscript. H.U.P. supervised and co-directed the research, as well as edited the manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the TUBITAK Marmara Research Center (MRC) through funding provided by the Presidency of Turkey, Presidency of Strategy and Budget.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Local Animal Ethics Committee (HADYEK) under permit number 16563500-111-103 and conducted in accordance with the European Union Directive 2010/63/EU on the protection of animals used for scientific purposes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. This study did not involve human participants; all ethical procedures related to animal use were approved by HADYEK.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOrcid\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHivda Ulbegi POLAT\u0026nbsp;\u003c/em\u003e\u003cem\u003ehttps://orcid.org/0000-0001-8424-6849\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBilal KOCAMAN\u0026nbsp;\u003c/em\u003e\u003cem\u003ehttps://orcid.org/ 0009-0008-0226-7899\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHatice Nur AYDIN\u0026nbsp;\u003c/em\u003e\u003cem\u003ehttps://orcid.org/0000-0002-7975-2189\u003c/em\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFacciol\u0026agrave; A, Visalli G, Lagan\u0026agrave; A, Di Pietro A. An Overview of Vaccine Adjuvants: Current Evidence and Future Perspectives. Vaccines (Basel). 2022;10:819.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTafalla C, B\u0026oslash;gwald J, Dalmo RA. Adjuvants and immunostimulants in fish vaccines: Current knowledge and future perspectives. Fish Shellfish Immunol. 2013;35:1740\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSivakumar SM, Safhi MM, Kannadasan M, Sukumaran N. Vaccine adjuvants \u0026ndash; Current status and prospects on controlled release adjuvancity. Saudi Pharm J. 2011;19:197\u0026ndash;206.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchijns VE, Lavelle EC. Trends in vaccine adjuvants. Expert Rev Vaccines. 2011;10:539\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYanase N, Toyota H, Hata K, Yagyu S, Seki T, Harada M, et al. OVA-bound nanoparticles induce OVA-specific IgG1, IgG2a, and IgG2b responses with low IgE synthesis. Vaccine. 2014;32:5918\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao T, Cai Y, Jiang Y, He X, Wei Y, Yu Y, et al. Vaccine adjuvants: mechanisms and platforms. Signal Transduct Target Ther. 2023;8:283.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoni S, Abdelwahab S, Jabeen S, Elmobark A, Aqaili M, Ghoal D. G, Advancements in Vaccine Adjuvants: The Journey from Alum to Nano Formulations. Vaccines (Basel). 2023;11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang Z, Xu J. Better Adjuvants for Better Vaccines: Progress in Adjuvant Delivery Systems, Modifications, and Adjuvant\u0026ndash;Antigen Codelivery. Vaccines (Basel). 2020;8:128.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNanishi E, Dowling D, Levy O. Toward precision adjuvants: Optimizing science and safety. Curr Opin Pediatr. 2020;32:1.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWan X, Yin Y, Zhou C, Hou L, Cui Q, Zhang X, et al. Polysaccharides derived from Chinese medicinal herbs: A promising choice of vaccine adjuvants. Carbohydr Polym. 2021;276:118739.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDanielsson R, Eriksson H. Aluminium adjuvants in vaccines \u0026ndash; A way to modulate the immune response. Semin Cell Dev Biol. 2021;115:3\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang T, He P, Guo D, Chen K, Hu Z. Zou 烨宁. Research Progress of Aluminum Phosphate Adjuvants and Their Action Mechanisms. Pharmaceutics. 2023;15:1756.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDewangan HK, Singh S, Mishra R, Dubey RK. A review on application of nanoadjuvant as delivery system. Int J Appl Pharm. 2020;12:24\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSmith WJ, Thompson R, Egan PM, Zhang Y, Indrawati L, Skinner JM, et al. Impact of aluminum adjuvants on the stability of pneumococcal polysaccharide-protein conjugate vaccines. Vaccine. 2023;41:5113\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGherardi R, Coquet M, Cherin P, Belec L, Moretto P, Dreyfus PA, et al. Macrophagic myofascitis lesions assess long-term persistence of vaccine-derived aluminum hydroxide in muscle. Brain. 2001;124:1821\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eG\u0026uuml;l B, Yurdak\u0026ouml;k Dikmen B. AŞI ADJUVANLARI ve İSTENMEYEN ETKİLERİ. Veteriner Farmakoloji ve Toksikoloji Derneği B\u0026uuml;lteni. 2019;10:91\u0026ndash;105.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBassi N, Luisetto R, Ghirardello A, Gatto M, Bottazzi B, Shoenfeld Y, et al. Vaccination of mice for research purpose: Alum is as effective as and safer than complete Freund adjuvant. Reumatismo. 2012;64:380\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStils H. Adjuvants and Antibody Production: Dispelling the Myths Associated with Freund\u0026rsquo;s Complete and Other Adjuvants. ILAR J. 2005;46:280\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSmith DE, O\u0026rsquo;Brien ME, Palmer VJ, Sadowski JA. The selection of an adjuvant emulsion for polyclonal antibody production using a low-molecular-weight antigen in rabbits. Lab Anim Sci. 1992;42:599\u0026ndash;601.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLeenaars PPAM, Hendriksen CFM, Angulo AF, Koedam MA, Claassen E. Evaluation of several adjuvants as alternatives to the use of Freund\u0026rsquo;s adjuvant in rabbits. Vet Immunol Immunopathol. 1994;40:225\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBroderson JR. A retrospective review of lesions associated with the use of Freund\u0026rsquo;s adjuvant. Lab Anim Sci. 1989;39:400\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLeenaars M, Koedam M, Wester P, Baumans V, Claassen E, Hendriksen C. Assessment of side effects induced by injection of different adjuvant/antigen combinations in rabbits and mice. Lab Anim. 1998;32:387\u0026ndash;406.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchultze V, D\u0026rsquo;Agosto V, Wack A, Novicki D, Zorn J, Hennig R. Safety of MF59 adjuvant. Vaccine. 2008;26:3209\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSahly H, El. MF59\u0026trade; as a vaccine adjuvant: a review of safety and immunogenicity. Expert Rev Vaccines. 2010;9:1135\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eO\u0026rsquo;Hagan D. MF59 is a safe and potent vaccine adjuvant that enhances protection against influenza virus infection. Expert Rev Vaccines. 2007;6:699\u0026ndash;710.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eColeman B, Sanderson R, Haag M, McGovern I. Effectiveness of the MF59-adjuvanted trivalent or quadrivalent seasonal influenza vaccine among adults 65 years of age or older, a systematic review and meta‐analysis. Influenza Other Respir Viruses. 2021;15.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVilla M, Black S, Groth N, Rothman KJ, Apolone G, Weiss NS, et al. Safety of MF59-Adjuvanted Influenza Vaccination in the Elderly: Results of a Comparative Study of MF59-Adjuvanted Vaccine Versus Nonadjuvanted Influenza Vaccine in Northern Italy. Am J Epidemiol. 2013;178:1139\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHelm RM, Burks AW. Animal models of food allergy. Curr Opin Allergy Clin Immunol. 2002;2:541\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXiao J-X, Wang L-H, Xu T-C, Huang G-Q. Complex coacervation of carboxymethyl konjac glucomannan and chitosan and coacervate characterization. Int J Biol Macromol. 2018;123.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePellegrini A, H\u0026uuml;lsmeier A, Hunziker P, Thomas U. Proteolytic fragments of ovalbumin display antimicrobial activity. Biochim Biophys Acta. 2004;1672:76\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRostamabadi H, Chaudhary V, Chhikara N, Sharma N, Nowacka M, Demirkesen Mert I, et al. Ovalbumin, an outstanding food hydrocolloid: Applications, technofunctional attributes, and nutritional facts, A systematic review. Food Hydrocoll. 2023;139:108514.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVecchi S, Bufali S, Skibinski DAG, O\u0026rsquo;hagan DT, Singh M. Aluminum adjuvant dose guidelines in vaccine formulation for preclinical evaluations. J Pharm Sci. 2012;101:17\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun Y, Liu J. Adjuvant effect of water-soluble polysaccharide (PAP) from the mycelium of Polyporus albicans on the immune responses to ovalbumin in mice. Vaccine. 2008;26:3932\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBansal V, Kumar M, Dalela M, Brahmne HG, Singh H. Evaluation of synergistic effect of biodegradable polymeric nanoparticles and aluminum based adjuvant for improving vaccine efficacy. Int J Pharm. 2014;471:377\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDong N, Zhang GQ, Jia PY, Wu JH, Li S, Shan JJ, et al. Adjuvant activities of seven natural polysaccharides on immune responses to ovalbumin in mice. Chin J Pharmacol Toxicol. 2013;27:307\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa B, Fu X, Zhu P, Niu J, Lu F. Allergenicity, assembly and applications of ovalbumin in egg white: a review. Crit Rev Food Sci Nutr. 2023;64:1\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePercie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, et al. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol. 2020;14(7):18.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAllison AC, Byars NE. Immunological adjuvants: Desirable properties and side-effects. Mol Immunol. 1991;28:279\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePetrovsky N, Aguilar J, Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and future trends. Immunol Cell Biol 82: 488\u0026ndash;496. Immunol Cell Biol. 2004;82:488\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePersiyanova EV, Kuznetsova TA, Silchenko AS. Effect of Sulfated Polysaccharides from Marine Hydrobionts on Humoral Immune Response to Ovalbumin in Mice. Bull Exp Biol Med. 2020;169:246\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOscherwitz J, Hankenson FC, Yu F, Cease KB. Low-dose intraperitoneal Freund\u0026rsquo;s adjuvant: Toxicity and immunogenicity in mice using an immunogen targeting amyloid-β peptide. Vaccine. 2006;24:3018\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFontes JA, Barin JG, Talor MV, Stickel N, Schaub J, Rose NR, et al. Complete Freund\u0026rsquo;s adjuvant induces experimental autoimmune myocarditis by enhancing IL-6 production during initiation of the immune response. Immun Inflamm Dis. 2017;5:163\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNimmerjahn F, Ravetch JV. Fcγ receptors as regulators of immune responses. Nat Rev Immunol. 2008;8:34\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRadmehri M, Talebi A, Ameghi Roudsari A, Mousaviyan SM, Gholipour MAJ, Taghizadeh M. Comparative Study on the Efficacy of MF 59, ISA70 VG, and Nano-Aluminum Hydroxide Adjuvants, Alone and with Nano-Selenium on Humoral Immunity Induced by a Bivalent Newcastle\u0026thinsp;+\u0026thinsp;Avian Influenza Vaccine in Chickens. Arch Razi Inst. 2021;76:1213\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin P-H, Yang H-C. The adjuvant effects of MF59 on antigen-specific regulatory and effector T cells. J Immunol. 2019;202 1_Supplement:196.15-196.15.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFang J-H, Hora M. The Adjuvant MF59: A 10-Year Perspective Gary Ott, Ramachandran Radhakrishnan. In: O\u0026rsquo;Hagan DT, editor. Vaccine Adjuvants: Preparation Methods and Research Protocols. Totowa, NJ: Springer New York; 2000. pp. 211\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCaton AJ, Brownlee GG, Yewdell JW, Gerhard W. The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). Cell. 1982;31:417\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKrammer F. The human antibody response to influenza A virus infection and vaccination. Nat Rev Immunol. 2019;19:1.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBanihashemi SR, Baradaran B, Tebianian M, Majidi J, Jabbari AR, Jalali S, et al. Development of Leptospira Killed Whole Culture Vaccine Using Different Adjuvants and Evaluation of Humoral Immune Response in Hamsters. J Vaccines Vaccin. 2013;4:1\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHe P, 烨宁 Z, Hu Z. Advances in aluminum hydroxide-based adjuvant research and its mechanism. Hum Vaccin Immunother. 2015;11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSasaki E, Asanuma H, Momose H, Furuhata K, Mizukami T, Hamaguchi I. Nasal alum-adjuvanted vaccine promotes IL-33 release from alveolar epithelial cells that elicits IgA production via type 2 immune responses. PLoS Pathog. 2021;17:e1009890.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGogoi H, Mani R, Bhatnagar R. Re-inventing traditional aluminum-based adjuvants: Insight into a century of advancements. Int Rev Immunol. 2024;44:1\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHogenesch H. Mechanism of Immunopotentiation and Safety of Aluminum Adjuvants. Front Immunol. 2013;3:406.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHogenEsch H, O\u0026rsquo;Hagan DT, Fox CB. Optimizing the utilization of aluminum adjuvants in vaccines: you might just get what you want. NPJ Vaccines. 2018;3:51.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKool M, Soullie T, Nimwegen M, Willart M, Muskens F, Jung S, et al. Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells. J Exp Med. 2008;205:869\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan de Veen W, Akdis M. Role of IgG4 in IgE-mediated allergic responses. J Allergy Clin Immunol. 2016;138:1434\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Adjuvants, OVA, BALB/c, Allergenicty, Immune response","lastPublishedDoi":"10.21203/rs.3.rs-7004970/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7004970/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e\u003cp\u003eAdjuvants play a crucial role in enhancing vaccine efficacy by engaging the innate immune system and shaping a robust adaptive response. However, some adjuvants can also influence allergenicity by promoting Th2-biased responses, potentially increasing the risk of allergic sensitization depending on their mode of action.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eIn this study, we investigated the immunogenic properties of various FDA-approved adjuvants using ovalbumin (OVA) as a model antigen. BALB/c mice were immunized with different formulations of OVA combined with Alum, Freund's Adjuvant, and MF59 to assess their immunostimulatory effects. A total of nine experimental groups were evaluated, including two different doses of Alum. To assess the allergenic potential of these formulations, we analysed IgG1, IgG2a, and IgG2b responses, allowing us to evaluate the balance between Th1- and Th2-mediated immunity.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eCFA induced the strongest anti-OVA IgG1 response, while MF59 caused a short-lived response. OVA\u0026thinsp;+\u0026thinsp;0.1 mg Alum group produced higher IgG1 response than the OVA\u0026thinsp;+\u0026thinsp;0.2 mg group. CFA also strongly triggered Th1 immunity, with high IgG2a/IgG2b levels, while Alum elicited a low IgG2a/IgG2b response. No IgG2a/IgG2b response was seen in other groups.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThe OVA\u0026thinsp;+\u0026thinsp;CFA group induced the highest IgG1 response, with IgG2a and IgG2b modulating the Th1-biased immune response. MF59 caused a rapid decline in IgG1 and promoted a Th2 response, with no IgG2a or IgG2b detected. The OVA\u0026thinsp;+\u0026thinsp;0.1 mg Alum group showed a higher IgG1 response than the OVA\u0026thinsp;+\u0026thinsp;0.2 mg group, highlighting the importance of optimizing the alum/antigen ratio. Alum triggered a strong Th2 response and a weak Th1 response, resulting in low IgG2a and IgG2b levels.\u003c/p\u003e","manuscriptTitle":"Evaluation of immune responses and allergenic risks to different adjuvants in an OVA-based immunisation model using BALB/c mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 14:59:49","doi":"10.21203/rs.3.rs-7004970/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e00ac699-88d3-4255-9651-94f253cc0826","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-24T05:08:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-18 14:59:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7004970","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7004970","identity":"rs-7004970","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}