A natural oil increases specific anti-OVA IgG levels and induces a cellular immune response combined with aluminum hydroxide

Abstract

The efficacy of certain vaccines is improved by the use of adjuvants. Nowadays, the development of new, effective, and safe adjuvants that stimulate the innate immune response is researched. In this context, medicinal plants appear as a suitable alternative. Minthostachys verticillata essential oil (EO) has demonstrated the ability to modulate mechanisms of the innate immune response. Thus, the present work aimed to evaluate the EO adjuvant effect on humoral and cellular immunity, coadministered with OVA as antigen. The chemical analysis of EO by gas chromatography–mass spectrometry revealed a predominant pulegone–menthone chemotype. EO (1.25, 2.5, or 5.0 mg/ml) did not alter the viability of murine fibroblasts (3T3 cell line) neither showed signs of toxicity in Balb/c mice inoculated subcutaneously. The serum of mice immunized with OVA + EO showed increased levels of anti-OVA-specific antibodies of IgG1 subclass compared with the mice immunized with OVA alone revealing an adjuvant effect of EO. The delayed type hypersensitivity showed that the combination OVA + Al(OH)₃ + EO was the best to induce a cellular immune response that extended until 48 h postinjection of OVA. M. verticillata EO appears as a new, safe, and effective adjuvant, which should continue to be studied for their possible future incorporation into vaccine formulations.

Introduction

Adjuvants improve the efficacy of vaccines formulated with inactivated microorganisms or highly purified antigens, allowing the reduction in the number of doses and the maintenance of long-term memory.¹ The reference adjuvant considered for use in human and animal vaccines continues to be currently aluminum hydroxide (Al(OH)₃) despite its multiple adverse effects related to local toxicity, delayed hypersensitivity reactions or autoimmunity, among others.² In addition, the strong limitation of Al(OH)₃ is the inefficient elicitation of Th1-dependent cellular immunity, which limited their use in a vaccine against some pathogens.³

For some years, there has been a trend in the design of adjuvant candidates that induce a protective immune response with an adequate Th1/Th2 balance and safety profile. In this sense, a large number of adjuvant strategies have been studied; however, their toxicity has been the main disadvantage for introducing them as new candidates in the clinic.¹ The aim of vaccination is the activation of adaptive immunity (antigen specific). However, this would not be possible if the mechanisms of the innate immune response are not activated.⁴ For this reason, the stimulation of the innate immune response using pattern recognition receptors (PRRs) constitutes a key in the design of new adjuvants that would achieve an appropriate balance between immunogenicity and toxicity.⁵ In this context, medicinal plants with immunomodulatory properties appear as a suitable alternative.⁶  Minthostachys verticillata (Griseb.) Epling (Lamiaceae) known as “peperina” is one of the 17 aromatic species of Minthostachys genus, which are distributed throughout South America. The content of essential oil is the key to the ethnobotanic, pharmacologic, and commercial value of these plants. Therefore, they are used in folk medicine as well as to flavor foods and drinks becoming internationally demanded natural products.^7,8 The M. verticillata essential oil (EO) has been well studied and shown different biologic activities related to its chemical composition. It has been reported that the percentage of monoterpenes of M. verticillata EO varies depending on the geographic area where the plant is located. According to this, 3 chemotypes can be found: (i) carvone, (ii) thymol–carvacrol, and (iii) pulegone–menthone.⁹ Other monoterpenes as limonene, α-pinene, β-pinene, among others, are present in a lower percentage.¹⁰ Our research group has demonstrated the immunomodulatory effect of M. verticillata EO (pulegone–menthone chemotype). This oil has shown in vitro immunostimulatory effect on human CD4⁺ and CD8⁺ T cells by increasing the IFN-γ production as well as the inhibition of proinflammatory substances both in vitro as in vivo in Balb/c mice.¹¹ Recent studies have reported an in vitro modulator effect of this oil on murine macrophages by activation of phagocytosis and reactive oxygen species production. In addition, M. verticillata EO stimulated in vivo the innate immune response in mammary glands of female Balb/c mice generating an inflammatory microenvironment with mild production of proinflammatory cytokines and polymorphonuclear neutrophils infiltration.¹² Take in count, the ability of M. verticillata EO to modulate the innate immune response; the present work aimed to evaluate its adjuvant effect, on humoral and cellular immunity, coadministered with OVA as antigen.

Material and Methods

EO extraction and chemical analysis

The plant material was obtained in a commercial herb store in February 2017. EO extraction and gas chromatography–mass spectrometry (GC–MS) analysis were carried out following the methodology described by Montironi et al.¹³

Cytotoxicity assay in mouse fibroblasts

Before performing the inoculation of EO in mice, its cytotoxic effect was evaluated in the 3T3 cell line (derivatives of Mus musculus embryo-fibroblast) by the colorimetric method of reduction of MTT.¹⁴ Cells were exposed to different concentrations of EO (1.25, 2.5, 5.0, 10.0, 20.0, and 40.0 mg/ml) and incubated at 37°C with 5% CO₂ and humidity during 24 h. Cells with complete DMEM medium alone were used as control.

Experimental animals

Male Balb/c mice aged 6–8-weeks (weighing 20–25 g), supplied from Bioterio of the Universidad Nacional de Río Cuarto, were used. All experimental procedures were conducted in accordance with recent legislation and following the ARRIVE guidelines. The study was approved by the Comité de Ética de la Investigación Científica (CoEdI), Universidad Nacional de Río Cuarto (121/2015).

Assessment of the adjuvant dose of EO

For this assay, mice were divided into 9 groups of 4 animals each. (i) Group 1 (negative control): received 100 µl saline solution. (ii) Group 2: received 100 µl OVA (0.2 mg/ml). (iii) Group 3 (positive control): received 100 µl Al(OH)₃ (0.5 mg/ml) coadministered with OVA (0.2 mg/ml). (iv) Groups 4–9: received 100 µl EO (1.25, 2.5, and 5.0 mg/ml) coadministered with OVA (0.2 mg/ml).

All groups were inoculated subcutaneously according to the following scheme:

To perform the assay, stock solutions of OVA (1 mg/ml), Al(OH)₃ (1 mg/ml), and EO (50 mg/ml) were prepared in PBS. The dilutions assayed were prepared from stock solutions before experimental immunizations. The different formulations were performed by mixing with OVA at room temperature for 2 h in a shaker.¹⁵ Experimental immunizations were performed 3 times with intervals of 2 weeks.

Mice examination and monitoring

The mice were examined daily in order to observe local and generalized reactions. The weight was also recorded at the beginning of the experiment and at the end.

Measurement of OVA-specific IgG antibodies

At day 35, the animals were lightly anesthetized¹⁶ and blood was taken by cardiac puncture. The serum was separated by centrifugation and pools from each group were performed. Anti-OVA antibodies were measured by an indirect ELISA assay following the protocol described by Hu et al.¹⁷

OVA-specific IgG1 and IgG2a titers

The OVA-specific antibody titers were measured by ELISA as described by Chiodetti et al.¹⁸ Titer was considered to be the reciprocal of the last serum dilution that yielded an absorbance value 490 nm greater than that of twice the mean value of blank. The sera from nonimmunized mice were not reactive to OVA.

Delayed type hypersensitivity

For this assay, mice were divided into 5 groups of 2 animals each group. (i) Group 1 (negative control): received 100 µl saline solution. (ii) Group 2: received 100 µl OVA (0.2 mg/ml). (iii) Group 3 (positive control): received 100 µl Al(OH)3 (0.5 mg/ml) coadministered with OVA (0.2 mg/ml). (iv) Group 4: received 100 µl EO (2.5 mg/ml) coadministered with OVA (0.2 mg/ml). (v) Group 5: received 100 µl EO (2.5 mg/ml) + Al(OH)₃ (0.5 mg/ml) coadministered with OVA (0.2 mg/ml).

All groups were inoculated subcutaneously according to the following scheme:

Seven days after the second immunization, mice were inoculated intradermally in the plantar pads of the right hind legs using a challenge with 0.05 ml of OVA (0.4 mg/ml) according to described in Verza et al.¹⁹ Injection of each animal with sterile saline solution in the plantar pad of the left hind leg served as a control. The thickness of the hind legs was measured with a caliber before the challenge with the antigen and after 24 and 48 h. The results were expressed as the difference between the thickness of the plantar pads before and after inoculation.

Statistical analysis

The values obtained were expressed as averages with standard deviations. The GraphPad Prism version 5.00.288 (San Diego, USA; 2007) was used. Data were compared with ANOVA and the Tukey multiple comparison test. A P < 0.05 difference was considered statistically significant.

Results and Discussion

Chemical analysis of M. verticillata EO

Table 1 and Figs. 1A and 1B show the identified compounds in EO by GC–MS. The chemical analysis of EO revealed the same chemotype previously reported as an immunostimulant by Cariddi et al.¹¹ and Montironi et al.¹² with pulegone and menthone as main compounds. Sample components of less than 1% are not reported.

Effect of the EO on the viability of murine fibroblasts

Prior to studying the adjuvant effect of EO in Balb/c mice, its toxicity on murine fibroblasts cell line 3T3 was evaluated, being the first study with EO in this type of cell. The results showed that EO concentrations of 1.25, 2.5, and 5.0 mg/ml did not alter cell viability. Concentrations greater than 5.0 mg/ml were toxic to murine fibroblasts (P < 0.001) (Fig. 2). In previous studies carried out by our research group, the toxic effect of M. verticillata EO has been evaluated on other cell types. Sutil et al.²⁰ and Escobar et al.²¹ revealed a noncytotoxic EO concentration of 1000 µg/ml on Vero and Hep-2 cells. According to Cariddi et al.¹¹ and Escobar et al.,²¹  M. verticillata EO did not alter the viability of human lymphocytes in concentrations of up to 1000 µg/ml. Montironi et al.²² reported safe concentrations of M. verticillata EO on bovine mammary epithelial cells (MAC-T) of up to 500 µg/ml. The results obtained in the present study showed that M. verticillata EO was less toxic on murine fibroblasts than in other cells types and suggested a range of safe concentrations, which could be used to inoculate into tissues, such as skin, without risks to the host.

Examination and mice monitoring

The in vivo experiences were performed by subcutaneous inoculation in Balb/c mice. No deaths were recorded in any experimental group. There were no changes in animal weight or in locomotion. No signs of inflammation, redness, induration, or loss of hair in the inoculation zone were evidenced; generalized reactions such as bristling hairs, weakness, and hunched appearance were not observed. This result is consistent with those obtained in the in vitro assay in which the same concentrations of EO (2.5 and 5.0 mg/ml) were not toxic on murine fibroblasts. Al(OH)₃ subcutaneous inoculation also did not show toxicity in mice in our experimental model. However, other authors have reported adverse effects as local swelling and loss of hair in mice inoculated with Al(OH)₃ combined with OVA.^15,17 Oily adjuvants, such as Freund’s adjuvant (water-in-mineral oil emulsion), are also characterized as inducing local reactions as important abscesses and granulomas of various sizes,²³ so its use is only allowed in research trials. The present study demonstrated that EO in spite of being an oily substance did not present local adverse effects inoculated subcutaneously. In addition, the toxicity of M. verticillata EO has already been studied in vivo by our research group using other routes of administration. The intraperitoneal administration of EO in concentrations of up to 500 mg/kg body weight was safe in Balb/c mice as was reported by Cariddi et al.¹¹ and Escobar et al.²¹ Other study demonstrated that the administration of EO at concentrations of up to 7 g/kg feed on Wistar rats diet did not exert cytogenotoxic effects.²⁴ Montironi et al.¹² revealed that the intramammary inoculation of EO in female Balb/c mice at concentrations of up to100 µg/ml did not alter the mammary tissue. The present study and the previous ones demonstrate the safety of this natural product enhancing its pharmacologic potential.

OVA-specific antibodies in immunized mice

Figure 3 shows the levels of IgG antibodies in the serum of immunized mice. As expected, a significant increase in antibody levels was observed in the serum of mice inoculated with OVA + Al(OH)₃ compared with the group of animals inoculated with OVA alone (P < 0.001). Interestingly, a significant increase in antibody levels was also observed in groups inoculated with OVA + EO (2.5 or 5.0 mg/ml) compared with the OVA group (P < 0.001), with no statistical differences between both concentrations. The antibody levels were similar to those observed with Al(OH)₃, revealing that this natural product acted as an adjuvant to the humoral immune response. In a previous study, Montironi et al.¹² showed that M. verticillata EO modulated the innate immune response in a murine model of mastitis increasing the expression of proinflammatory cytokines as TNF-α and IL-1β. These results indicated that EO was sensed directly or indirectly by PRRs, leading to cell activation. At the moment, we know that TLR2 is not involved. Although it has not yet been elucidated which PRRs would be involved in the activation of the innate immune system by EO, the results obtained in the present study could be related to those of Montironi et al.¹² The increase in antibody levels in mice induced by EO could indicate that it could have stimulated APCs at the level of innate immunity. Therefore, APCs activated the adaptive immunity for anti-OVA-specific antibodies production. Similarly, other natural products as chitosan and Quil-A saponin act as immunostimulant adjuvants activating the innate immune response through PRRs.²⁵ According to the mode of action of M. verticillata EO on the innate immune response demonstrated in previous studies, we can hypothesize that this natural compound could be acting as an immunostimulant adjuvant.

The development of new immunostimulant adjuvants derived from medicinal plants is suitable considering that natural products are less expensive, effective, and safe.²⁶ In this way, Freitas et al.²⁷ determined the adjuvant activity of peanut, cottonseed, or rice vegetable oils in Swiss mice using OVA (10 mg/ml) as antigen. All oil formulations significantly increased the production of anti-OVA antibodies on day 42 after the first immunization with respect to OVA alone. These results demonstrate the interest and importance of vegetable oils as biotechnological tools that could be used in vaccine formulations.

OVA-specific IgG1 and IgG2a titers

Figure 4 shows the OVA-specific titers IgG1 and IgG2a in the serum of immunized mice. The groups inoculated with OVA, OVA + Al(OH)₃, or OVA + EO showed a humoral response mediated by IgG1. Although no significant statistical differences were observed between them, the group inoculated with OVA + Al(OH)₃ showed higher titers. For the IgG2a, only the group inoculated with OVA + Al(OH)₃ induced a detectable immune response. The IgG1 and IgG2a are subclasses of IgG immunoglobulin in Balb/c mice that are regulated by different cytokines. IFN-γ or TNF-α are cytokines that increase the production of IgG2a and IgG2b. The increased levels in these subclasses of immunoglobulins are indicative of a primarily cell-mediated immune response. On the other hand, cytokines such as IL-4 increase the production of IgG1. Therefore, increased levels of IgG1 denote a primarily humoral immune response.²⁸ In this study, Al(OH)₃ triggered a strong humoral immune response mainly mediated by IgG1 and a weak induction of cell-mediated immune response with low IgG2a levels. This result is consistent with the mechanism of action reported for aluminum-based adjuvants.^3,29 On the other hand, M. verticillata EO also increased IgG levels of IgG1 subclass, stimulating mainly a humoral response in similar way to Al(OH)₃, without increasing the IgG2a levels.

Delayed type hypersensitivity

The results of delayed type hypersensitivity (DTH) are shown in Figs. 5A and 5B. At 24 h after OVA injection into the plantar pad, a significant increase in the thickness of the pad was observed in the groups of animals immunized with OVA + Al(OH)₃, OVA + EO, and OVA + EO + Al(OH)₃ regarding the group of animals inoculated with OVA alone (P < 0.001 and P < 0.05). Although the effect observed in the mice immunized with OVA + EO was significantly lower compared with the OVA + Al(OH)₃ group (P < 0.001), the combination EO + Al(OH)₃ did not alter the efficacy of Al(OH)₃ at 24 h. The effect on cellular immunity of Al(OH)₃ decreased significantly at 48 h compared with the effect found at 24 h (P < 0.001). A significant increase in the plantar pads of the group of animals immunized with OVA + EO + Al(OH)₃ was observed compared with the animals group immunized with OVA alone (P < 0.001) or OVA + Al(OH)₃ (P < 0.01) 48 h after the OVA injection (Figs. 5A and 5B). The DTH is a cellular response attributed to memory Th1 cells that can be detected when the antigen is inoculated into the skin, which appears hours or days after intradermal injection. Thus, DTH is routinely used as the first approach to evaluate the activation of the cell-mediated immune response.³⁰ Given the low cellular response induced by both EO and Al(OH)₃, not only the effect of each substance alone by DTH was evaluated, but also the possible potentiating effect of its combination. The results obtained in this assay were related to the levels of IgG subclasses observed in the serum of all groups of mice. The DTH observed in the group inoculated with OVA + Al(OH)₃ at 24 h postinjection of OVA was indicative of an activated cellular immune response and was consistent with the low but detectable IgG2a levels in serum. The group of mice inoculated with OVA + EO showed a significantly lower cellular response at 24 h postinjection of OVA compared with the animals group inoculated with OVA + Al(OH)₃. This result was also consistent with the levels of IgG2a, which were not detectable in serum. However, the effect on cellular immunity of Al(OH)₃ decreased significantly at 48 h. This result could be due, as described above, to the fact that aluminum-based adjuvants generate a weak induction of cell-mediated immune response.^3,29 Interestingly, the combination of M. verticillata EO and Al(OH)₃ was more effective than both compounds alone to induce T-cell-mediated immunity in vivo since the effect lasted up to 48 h, suggesting an enhancer effect between both. Similarly, Khorshidvand et al.³¹ immunized Balb/c mice using Toxoplasma gondii lysate (ATL) as antigen in combination with Alum (aluminum phosphate gel) and drug mixtures as naloxone (an opioid receptor antagonist) and naltrexone (a nonselective opioid antagonist). They observed a significant increase in plantar pad thickness in the groups that received the ATL + Alum + drug mixtures, compared with ATL + Alum or ATL alone. The authors suggested a potentiating effect between the commercial adjuvant and the compounds tested, as observed in our study. The combination of EO + Al(OH)₃ could be beneficial to activate the cellular immune response against certain antigens.

In conclusion, M. verticillata EO combined with OVA acted as a safe adjuvant mainly to the humoral immune response by increasing IgG levels of IgG1 subclass in a similar way that the commercial Al(OH)₃ adjuvant. In addition, the combination with Al(OH)₃ enhanced the cellular immune response induced by both. This natural product appears as a new, safe, and effective adjuvant, which should continue to be studied for their possible future incorporation into vaccine formulations either alone or combined with other adjuvants. Although in this study OVA was used as a model antigen, the results obtained serve as a basis to perform assays with other antigens.

Authorship

L.N.C. and E.B.R. worked on the design of the study; N.A.C. and J.R. did the animal experiments; N.A.C., I.D.M., B.M., F.R.M., and L.N.C. performed the ELISA assays and statistical analysis; N.A.C. and I.D.M. delayed hypersensitivity test and statistical analysis; M.J.R., L.N.C., E.B.R., and N.A.C. drafted the manuscript. The revision of the manuscript was done by all the authors.

Abbreviations

 
• Al(OH)₃

  aluminum hydroxide

 
• DTH

  delayed-type hypersensitivity

 
• EO

  essential oil

 
• PRRs

  pattern recognition receptors

Acknowledgments

Part of this work was presented at LXIV Annual Meeting of The Argentine Society of Immunology (SAI) held in Buenos Aires, Argentina on November 13–17, 2017. Mic. N. Campra has Fellowship from CONICET. Dr. L. Cariddi, Dr. E. Reinoso, and Dr. B Maletto are the Members of the Research Career of CONICET.

This work was supported by the grants from PICT 2268/13, CONICET PIP-2014-2016, and PID 2014–2016.

Disclosures

The authors declare no conflicts of interest.

References