Novel Iscoms from Quillaja Brasiliensis Saponins Induce Mucosal And

Vaccine 34 (2016) 1162–1171

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Vaccine

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Novel ISCOMs from Quillaja brasiliensis saponins induce mucosal and

systemic antibody production, T-cell responses and improved antigen uptake

a,b c a

Samuel Paulo Cibulski , Gustavo Mourglia-Ettlin , Thais Fumaco Teixeira ,

d b e d,∗

Lenora Quirici , Paulo Michel Roehe , Fernando Ferreira , Fernando Silveira

FEPAGRO Saúde Animal, Instituto de Pesquisas Veterinárias Desidério Finamor, Laboratório de Virologia, Eldorado do Sul, RS, Brazil

Departamento de Microbiologia Imunologia e Parasitologia, Laboratório de Virologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

Cátedra de Inmunología, Departamento de Biociencias, Facultad de Ciencias/Química, Universidad de la República (UdelaR), Av. Alfredo Navarro 3051,

Montevideo CP. 11600, Uruguay

Laboratorio de Carbohidratos y Glicoconjugados, Departamento de Desarrollo Biotecnológico, Facultad de Medicina. Universidad de la República (UdelaR),

Av. Alfredo Navarro 3051, Montevideo CP. 11600, Uruguay

Laboratorio de Carbohidratos y Glicoconjugados, Departamento de Desarrollo Biotecnológico, Facultad de Medicina, Departamento de Química Orgánica,

Facultad de Química, Universidad de la República (UdelaR), Av. Alfredo Navarro 3051, Montevideo CP. 11600, Uruguay

a r t i c l e i n f o a b s t r a c t

Article history: In the last decades, signiﬁcant efforts have been dedicated to the search for novel vaccine adjuvants. In

Received 15 October 2015

this regard, saponins and its formulations as “immunostimulating complexes” (ISCOMs) have shown to be

Received in revised form

capable of stimulating potent humoral and cellular immune responses, enhanced cytokine production and

15 December 2015

activation of cytotoxic T cells. The immunological activity of ISCOMs formulated with a saponin fraction

Accepted 17 January 2016 ®

extracted from Quillaja brasiliensis (QB-90 fraction) as an alternative to classical ISCOMs based on Quil A

Available online 28 January 2016

(IQA) is presented here. The ISCOMs prepared with QB-90, named IQB-90, typically consist of 40–50 nm,

spherical, cage-like particles, built up by QB-90, cholesterol, phospholipids and antigen (ovalbumin, OVA).

Keywords:

These nanoparticles were efﬁciently uptaken in vitro by murine bone marrow-derived dendritic cells.

Quillaja brasiliensis

ISCOM Subcutaneously inoculated IQB-90 induced strong serum antibody responses encompassing speciﬁc IgG1

Uptake and IgG2a, robust DTH reactions, signiﬁcant T cell proliferation and increases in Th1 (IFN-␥ and IL-2)

Subcutaneously cytokine responses. Intranasally delivered IQB-90 elicited serum IgG and IgG1, and mucosal IgA responses

Intranasally delivered at distal systemic sites (nasal passages, large intestine and vaginal lumen). These results indicate that IQB-

Humoral and cell responses

90 is a promising alternative to classic ISCOMs as vaccine adjuvants, capable of enhancing humoral and

cellular immunity to levels comparable to those induced by ISCOMs manufactured with Quillaja saponaria

saponins.

1. Introduction Subunit vaccines, whose immunogenicity relies essentially on a

particular protein or peptide, are generally less immunogenic than

Infectious diseases are major causes of morbidity and mortality vaccines based on live attenuated or whole inactivated microor-

worldwide, especially in poor and developing countries. Amongst a ganisms. Therefore, subunit vaccines usually require addition of

plethora of preventive measures available in attempting to reduce adjuvants in order to improve its immunogenicity. These have been

such burden, vaccines stand out as highly efﬁcacious and cost effec- used in order to either induce more rapid and robust immune

tive tools, which have been successfully used in the control or responses that correlate with increased protection or to allow dose

eradication of some of the most impacting infectious diseases in sparing in the context of antigens which can be in limited supply

humans and animals [1]. or problematic to manufacture [2].

A crucial aspect addressing the challenges in vaccine develop-

ment is antigen delivery, which encompasses administration of

drugs to speciﬁc sites of the body, as well as the delivery of the

∗ antigen provide the necessary signals for activation and maturation

Corresponding author. Tel.: +598 2 4871288x1124; fax: +598 2 4873073.

E-mail address: [email protected] (F. Silveira). of relevant antigen presenting cells (APCs) [3,4]. Most pathogens

http://dx.doi.org/10.1016/j.vaccine.2016.01.029

S.P. Cibulski et al. / Vaccine 34 (2016) 1162–1171 1163

invade the host or establish infection at mucosal surfaces. In this ISCOMs derived from QB-90 (IQB-90) and ISCOMs from QA (IQA)

regard, antigen delivery in mucosal surfaces may mimicry natural were obtained.

infection and induce local and remote speciﬁc immune responses

[5–7]. 2.2. Transmission electron microscopy (TEM)

Most vaccine adjuvants in clinical use for human and veterinary

vaccines (mainly parenteral injections) of which alum compounds An aliquot (10 ␮L) of an aqueous solution of ISCOMs was placed

are the main representatives, induce protection by enhancing on formvar carbon grids (200 mesh) and negatively stained with 2%

antibody responses. However, for some infections, particularly phosphotungstic acid (pH 7.2) for 2 min at room temperature and

those caused by intracellular pathogens, speciﬁc antibodies are dried. The samples were examined with a JEOL (JEMM 10.10) trans-

not sufﬁcient to induce protection. In such cases, stimulation of mission electron microscope (JEOL, Japan) operated at an 80 kV

+ +

antigen-speciﬁc CD4 or CD8 T-cell is not only required but essen- accelerating voltage.

tial for eliciting protective responses [8].

Many natural products with potential for use as adjuvants are 2.3. In vitro and in vivo toxicity assays

currently under investigation [9,10]. In veterinary medicine, triter-

penoid saponins extracted from Quillaja saponaria Molina have a Hemolytic activities of QA, QB-90 and IQB-90 were assessed

long usage history as vaccine adjuvants. In fact, a partially puri- as previously described [24], over a range of 10–200 ␮g/mL and

ﬁed mixture of saponins from Q. saponaria, named Quil A [11], using a concentration of 0.5% rabbit red blood cells. Saline and Q.

is the most widely used saponin-based vaccine adjuvant and it is saponaria saponins (250 ␮g/mL) were used for 0% and 100% hemol-

known to stimulate both humoral and cellular responses against ysis, respectively. Samples were tested in triplicates. Hemolytic

co-administered antigens, with the generation of T helper 1 (Th1) activity was expressed as the sample concentration producing 50%

and cytotoxic cells (CTLs) responses [12]. However, Quil A use in of the maximum hemolysis (HD50).

human vaccines has been restricted due to undesirable side effects Cytotoxicity on VERO cells (ATCC CCL-81) was determined

such as local reactions, hemolytic activity and occasional events through MTT assay and by determination of lactate dehydroge-

of systemic toxicity [11,13]. A similar saponin fraction has been nase (LDH) release into the supernatant media with samples of

extracted from Quillaja brasiliensis leaves, named QB-90 [14], which QA and QB-90. Brieﬂy, cells were cultured in Eagle’s minimal

was found to possess adjuvant potential into levels comparable essential medium (E-MEM) supplemented with 10% fetal bovine

to those of Quil A . In addition, QB-90 was less toxic than Quil serum (FBS, GIBCO) and antibiotics (penicillin 100 UI/mL; strepto-

® ® 4

A [15–17]. Both QB-90 and Quil A showed similar patterns of mycin 100 ␮g/mL) (E-MEM/FBS) at 4.0 × 10 cells/well on 96-well

◦

antibody induction (IgG and subclasses) and stimulation of cellular microplates and incubated for 18 h at 37 C in a humid atmosphere

immunity by generation of Th1 responses [15,17,18]. with 5% CO2. Afterwards, the medium was removed and cells

An improvement to the use of saponins as adjuvants was were further incubated for 24 h with 100 ␮L/well of E-MEM/FBS

introduced by the development of immune stimulating complexes containing different concentrations of either QA or QB-90. For

(ISCOMs). These have been used as antigen delivery systems MTT assays, 50 ␮L/well of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-

that proved to exert powerful immune stimulating activities, yet diphenyltetrazolium bromide; Invitrogen, USA) at 2 mg/mL was

◦

displaying reduced toxicity in several animal models [7,19,20]. added. Cells were incubated during 4 h at 37 C. After centrifugation

Physicochemical properties of ISCOMs include the cage-like struc- (1400 × g for 5 min), supernatants were removed and 100 ␮L/well

tures with about 40 nm in diameter, composed by aggregates of an of dimethyl sulfoxide (DMSO) were added. Optical density (OD)

antigen (usually proteic), cholesterol, phospholipids and saponins was measured in a microplate reader (Dynex MMXII, USA) at

from Q. saponaria. ISCOMs were shown to up-regulate both Th1- 570 nm. Results were expressed as the percentage of each culture

and Th2-like immune responses as well as to stimulate strong OD related to the OD of untreated cells. General cytotoxicity was

humoral responses (IgG1, IgG2b and IgG2a) with cytotoxic T cell reported as the concentrations that decreased viability by 50%. For

induction [19,21]. In view of these ﬁndings, ISCOMs are considered the LDH release assay, LDH released from damaged cells into cul-

promising innate immune cell-stimulating adjuvants [2,22]. ture media was quantiﬁed with the aid of a LDH assay kit (Labtest,

In this study, for the very ﬁrst time, ISCOM formulations were Brazil). Survival ratio was determined by comparing the absorbance

constructed by replacing the Quil A component by QB-90. This in test wells with those of positive (complete cell destruction)

formulation was assessed on its adjuvant capacity using a model and negative (spontaneous cell destruction) control wells. Values

protein antigen. Safety and efﬁciency analyses were performed and were expressed as the highest dilution of sample concentration

its use as an effective alternative to Quil A -based ISCOMs has been which caused 50% LDH activity release compared to positive con-

successfully developed. trols (EC50). Cytotoxicity assays were not performed with IQB-90

because ISCOMs formulation are well known formulations retained

the adjuvant activity of the saponins, while increasing its stability,

2. Material and methods reducing its hemolytic activity, and producing less toxicity [20,25].

Acute toxicity was evaluated as previously described [26] with

2.1. Adjuvant and vaccine preparation some modiﬁcations. Brieﬂy, groups of CD-1 male mice (8 weeks of

age, n = 5) were given a subcutaneous administration of 100 ␮L of

Q. brasiliensis (A. St.-Hil. et Tul) Mart. leaves were collected in QB-90 or QA in PBS (31.25, 62.5 and 125 ␮g/dose) on the scapu-

Parque Battle (Montevideo, Uruguay). Extraction and puriﬁcation lar region. Mice were monitored during 3 days in search for signs

of saponins were carried out as previously described [14]. ISCOMs of toxicity (lethality, local swelling, loss of hair, and piloerection).

with ovoalbumin (OVA-ISCOMs) were prepared by the modiﬁed Control mice were inoculated with 100 ␮L of PBS.

ethanol injection technique [23]. Brieﬂy, ovoalbumin (OVA, Sigma,

USA) solution (1 mg/mL in TBS, pH 7.4) was added either to a mix- 2.4. Antigen uptake by murine bone marrow-derived dendritic

ture of Q. brasiliensis saponins fraction (QB-90, 1 mg/mL) or to Quil cells

A (QA) (Brenntag, Denmark) (1 mg/mL). Ethanol-dissolved choles-

terol (Sigma, USA) and di-palmitoyl phosphatidyl choline (Avanti Bone marrow-derived dendritic cells (BMDCs) were obtained

Polar Lipids, USA) were immediately injected into the mixture, by differentiation of bone marrow precursors from 8 to 10 weeks

◦

which were finally stirred during 48 h at 4 C. After that procedure, old C57Bl/6 mice [27]. Briefly, femurs and tibia were flushed out

1164 S.P. Cibulski et al. / Vaccine 34 (2016) 1162–1171

◦

and the cells were cultured on Petri plates in RPMI 1640 culture through the nostrils and stored at −80 C. Vaginal washes were

medium (Gibco), complemented with 10% FBS (Gibco), 50 ␮M 2- performed with a micropipette. PBS (75 ␮L) were ﬂushed 10 times

mercaptoethanol (Sigma), 1% HEPES (Sigma), 1% sodium pyruvate through vaginas, and samples were centrifuged 5 min at 16.000 × g.

◦

(Gibco), 1% non-essential amino acids (Gibco) and 20 ng/mL of Supernatants were stored at −80 C.

recombinant mouse GM-CSF (PeproTech). Medium was replaced

on day 3 and BMDCs were obtained on day 10. Phenotype was 2.6. Splenocyte proliferation assay

routinely checked and 90–95% cells were CD11c .

Antigen (Ag) uptake was performed according to protocols Six-weeks-old female Rockfeller mice were divided into 5

already reported [28]. Brieﬂy, BMDCs (2 × 10 ) were incubated with groups, each consisting of six mice. Animals were immunized sub-

2 ␮g of FITC-labeled OVA (OVA:FITC, Molecular Probes ) in 200 ␮L cutaneously with OVA 10 ␮g alone or with OVA 10 ␮g dissolved in

◦ ®

of RPMI media for 120 min at 37 C in the presence of 10 ␮g/mL of saline containing QB-90 (10 ␮g), Quil A (10 ␮g), IQB-90 (10 ␮g)

QB-90, vehicle or IQB-90 formulated with 2 ␮g of OVA:FITC. One and IQA (10 ␮g) on day 0. Saline-treated animals were included as

parallel experiment was performed on ice to inhibit intracellu- controls. A boosting injection was given 2 weeks later.

lar uptake (cell surface binding controls). Then, cells were washed Spleens were collected 28 days after the second immunization

twice with ice-cold PBS, and resuspended in FACS buffer (PBS with under aseptic conditions, immersed in RPMI 1640 medium (Gibco),

2% FBS and 0.1% sodium azide). Cell staining was performed with minced, and mechanically dissociated to obtain a homogeneous

PE-Cy7-conjugated anti-mouse CD11c (clone HL3, BD) and ﬂow cell suspension. Erythrocytes were lysed with ACK (Ammonium-

cytometry was performed on a FACS Canto II (BD) cytometer. Fluo- Chloride-Potassium) lysis buffer. After centrifugation (380 × g at

◦

rescence values were reported as mean ﬂuorescence intensity (MFI) 4 C for 10 min), pelleted cells were washed three times in

of FITC in CD11c cells. RPMI 1640 and resuspended in the same medium supplemented

For ﬂuorescence microscopy, 1.0 × 10 BMDCs were plated on with 0.05 mM 2-mercaptoethanol, 100 IU/mL penicillin, 100 ␮g/mL

24-well plates (Costar) and after 18 h of culture, OVA:FITC alone, streptomycin, 2 mM l-glutamine, and 10% fetal bovine serum

with QB-90 or formulated as IQB-90 was added. After 120 min, cells (RPMI complete media). By trypan blue dye exclusion, cells count-

were washed with ice-cold PBS, ﬁxed with 4% paraformaldehyde ing revealed >95% viability. Splenocytes were seeded at 2.5 × 10

and then stained with DAPI (4 ,6-diamidino-2-phenylindole, Dilac- cells/mL in 100 ␮L of RPMI complete medium into each well of a 96-

◦

tate, Invitrogen) for 10 min at 37 C. Fluorescence microscopy was well ﬂat-bottom microtiter plate (Nunc). Subsequently, 100 ␮L OVA

carried out with a Spot insight camera (model no. 3.1.0; Diagnostic (10 ␮g/mL, Sigma) or medium only was added. Plates were then

◦

Instruments Inc, Sterling Heights, MI) mounted over an Axiovert incubated at 37 C in a humid atmosphere with 5% CO2. After 68 h,

S100 microscope (Zeiss, Göttingen, Germany). Image acquisition 50 ␮L of MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan

was performed with Meta Imaging Series 6.1 software (Universal Sigma) solution (2 mg/mL) was added to each well and incubated

Imaging Corporation, Downington, PA). for 4 h. The plates were centrifuged at 1400 × g for 5 min and the

untransformed MTT was removed carefully by pipetting. Next, a

2.5. Immunizations and sample collection DMSO solution (192 ␮L of DMSO with 8 ␮L of 1 N HCl) was added

to wells in volumes of 100 ␮L. After 15 min of incubation, the

Experimental work involving animals was carried out follow- absorbance was measured in an ELISA reader at 550 nm with wave-

ing international guides on use and care of laboratory animals. length reference ﬁxed at 620 nm. The stimulation index (SI) was

Protocols were performed in accordance with CHEA guidelines calculated as the absorbance ratio of mitogen-stimulated cultures

(Comisión Honoraria de Experimentación Animal) and were and the non-mitogen-stimulated cultures.

approved by the Uruguayan University Research Ethics Commit-

tee (approval number 070153-000531-13). Animals were properly 2.7. Quantiﬁcation of cytokine levels in spleen culture

◦

housed under controlled temperature (22 ± 2 C) and humidity in supernatants

a 12/12 h light/dark cycle, with food and water ad libitum.

Female Rockfeller mice of the CF-1 breed (5–6 weeks old) were Spleens were aseptically removed, rinsed and mechanically dis-

purchased from Fundac¸ ão Estadual de Produc¸ ão e Pesquisa em rupted in RPMI media and isolated spleen cells were pelleted and

Saúde (FEPPS, Porto Alegre, RS, Brazil) and were immunized on incubated in RBC lysis solution. After washing with RPMI, spleno-

days 0 and 14 with OVA through subcutaneous (s.c.) or intranasal cytes (5 × 10 cells) were re-stimulated for 3 days with OVA antigen

(i.n.) routes. Subcutaneous immunizations (n = 6) (in the hind neck) (10 ␮g/mL). The supernatants were harvested and IL-2 and IFN-

␥

were performed with 10 ␮g/dose of IQB-90, IQA or OVA alone cytokines were measured by capture ELISA using commercial

(unadjuvanted). For intranasal immunization, mice (n = 7) were kits from Novex (USA) and following the manufacturers’ instruc-

anesthetized with ketamine–xylazine and received 2 ␮g of antigen tions. Sensitivities were 8 pg/mL and 2 pg/mL for IL-2 and IFN-␥

per dose. Bleedings were performed immediately before inocula- kits, respectively.

tions (days 0 and 14) and 2 weeks after the second immunization

◦

(day 28). Sera were stored at −20 C. 2.8. Determination of antigen-speciﬁc antibodies

The samples, including nasal and vaginal washes as well as feces,

were collected from euthanized animals on 28 day post priming. Anti-OVA IgG, IgG1 and IgG2a antibodies were determined

Fecal samples were obtained from 3 to 4 freshly voided pellets from by ELISA as described [24]. Brieﬂy, ELISA plates (Greiner Bio-

each animal, which were weighed and collected into a 15 mL conical One, Germany) were coated with OVA (5 ␮g/mL) in acetate buffer

◦

tube. A 10 × volume (per gram of wet feces) of extraction buffer (100 ␮L/well, pH 5.0) overnight at 4 C. Then, plates were washed

(PBS with 5% FBS and 0.02% sodium azide) was added to each tube, three times with PBS containing 0.05% Tween 20 (Sigma, USA)

® ◦

which were vigorously vortexed and centrifuged at 16,000 × g for (PBS-T20) and blocked with 1% Tween 20 in PBS at 37 C for 2 h.

◦

20 min. Supernatants were collected and stored at −80 C. Appropriately diluted samples in PBS-T20 were added in dupli-

◦

For nasal washes, a 1 cm incision was performed parallel to the cate (100 ␮L/well) and incubated for 1 h at 37 C. After washing

trachea through the skin, and a midline incision was made on the three times with PBS-T20, HRP-conjugated anti-mouse IgG (Sigma,

ventral aspect of the trachea slightly superior to the thoracic inlet USA), IgG1 (Invitrogen, USA) and IgG2a (Invitrogen, USA) diluted

with a scalpel. A 25G needle was tied at the top of the trachea and in PBS-T20 (1:5000, 1:5000 and 1:2000, respectively) were added

0.5 mL of PBS was slowly injected. Nasal washes were collected to each well (100 ␮L/well) and plates were incubated for 1 h at

S.P. Cibulski et al. / Vaccine 34 (2016) 1162–1171 1165

◦

37 C. After ﬁve washings, 100 ␮L of OPD (ortho-phenylenediamine;

Sigma, USA) with 0.003% H2O2 were added to wells, and plates were

◦

further incubated for 30 min at 25 C. Reactions were stopped with

30 ␮L/well of 1 N HCl. Optical densities (OD) were measured in an

ELISA plate reader (Anthos 2020) at 492 nm. A pool of positive sera

was used as standard curve, and antibody titers were expressed in

arbitrary units per mL (AU/mL).

Anti-OVA IgA determinations were similarly performed. After

◦

incubation of samples for 1 h at 37 C, plates were washed ﬁve times

and 100 ␮L/well of goat anti-mouse IgA antibodies were added to

◦

each well (1:4000 dilution; Sigma, USA). After incubation at 37 C

for 1 h and ﬁve washings, 100 ␮L/well of HRP-conjugated anti-

goat antibodies (1:5000, Zimed, USA) were added and incubated

◦

for 1 h at 37 C and reveled as above. OVA-speciﬁc IgA titers were

expressed as OD values for samples diluted 1:10 (fecal, vaginal and

nasal samples).

2.9. Delayed-type hypersensitivity (DTH) assay

Delayed type hypersensitivity (DTH) responses were tested 28

days post-priming. Brieﬂy, mice were intradermally injected with

1 ␮g of OVA in one footpad of the hind limb. Thickness of the

injected footpads was measured 24 h later with a caliper. Swelling

in mice inoculated with saline revealed basal conditions. OVA-

speciﬁc DTH responses in each animal were determined as the

thickness of injected footpad minus average basal swelling.

2.10. Statistical analyses

Statistical signiﬁcance was assessed by one-way-ANOVA with

Dunnet’s post test correction (GraphPad Prism 5.01, GraphPad Soft-

ware, USA). Signiﬁcance was assigned at P-value < 0.05.

Fig. 1. Transmission electron microscopy (TEM) of IQB-90. Microphotography of IQB-

90 prepared with puriﬁed fraction of saponins from Quillaja brasiliensis (QB-90) and

OVA by the ethanol injection technique. QB-90 or QA formulation with 3:2:5 relative

proportions of saponins:cholesterol:phosphatidylcholine rendered mostly ISCOM

particles with an average diameter of 47 nm (overall range of 40–50 nm).

Fig. 2. In vitro toxicity assays. (A) Haemolytic activity of QB-90, QA and IQB-90.

Haemolysis was expressed as percent referred to saline and Q. saponaria saponins

␮

Table 1 (250 g/mL), which were used as 0% and 100% of haemolysis, respectively. (B) Cyto-

In vivo toxicity assays for QB-90 and QA. Results are expressed as the percentage toxicity of QB-90 and QA on VERO cells. Cell viability was measured by MTT 24 h

of mortality in each group of mice inoculated subcutaneously with QB-90 or QA after treatment with the indicated saponin concentrations. (C) LDH release. QB-

saponins within 72 h. 90 and QA on VERO cells was measured 24 h after treatment with the indicated

saponin concentrations. Results are presented as the mean value ± SD (A) and ± SEM

Sample Dose

(B and C).

125 ␮g 62.5 ␮g 31.25 ␮g

Saline (n = 5) 0% (0) 0% (0) 0% (0)

QB-90 (n = 5) 60% (3) 40% (2) 0% (0)

Quil A (n = 5) 100% (5) 80% (4) 40% (2)

() Number of death animals in each group.

1166 S.P. Cibulski et al. / Vaccine 34 (2016) 1162–1171

3. Results electron microscopy (TEM), where the average diameter of the

cage-like structures was about 47 nm (overall range: 40–50 nm)

3.1. Formulation of ISCOMs with Quillaja brasiliensis saponins (Fig. 1).

Puriﬁed fraction of saponins from Q. brasiliensis (QB-90) and

3.2. QB-90 showed lower toxicity than QA in vitro and in vivo

OVA were combined with cholesterol and phospholipid under

controlled conditions to obtain IQB-90, which formed cage-like Hemolytic activities of QB-90 and QA showed HD50 values

structures. IQB-90 structures were conﬁrmed by transmission of 88.83 ± 0.16 ␮g/mL and 40.43 ± 0.10 ␮g/mL, respectively. No

Fig. 3. Uptake of model antigen (OVA) by BMDCs. (A) BMDCs were loaded with 2 ␮g of ﬂuorescein isothiocyanate (FITC)-conjugated OVA (OVA:FITC) in the presence of 10 ␮g/mL

of QB-90, vehicle or as IQB-90 and their uptake was detected by ﬂow cytometry after 120 min. The mean ﬂuorescence increase (MFI) ± standard deviation of three experiments

is shown. (B) BMDCs were treated as in (A) and examined by ﬂuorescence microscopy (600×). Data is shown as mean ± SEM and one-way ANOVA with Dunnet’s post-test

was used to investigate signiﬁcant statistical differences (*** P < 0.001).

S.P. Cibulski et al. / Vaccine 34 (2016) 1162–1171 1167

signiﬁcant hemolytic activity was observed at the concentra-

tion used in the vaccine formulations (i.e. 10 ␮g/mL) (Fig. 2A).

Interestingly, no hemolytic activity was determined for ISCOMs

formulated with QB-90 in any tested concentration (Fig. 2A).

Similar results were obtained from the cytotoxicity

assays. Results shown in Fig. 2B revealed that QA was more

toxic to VERO cells (EC50 = 50.6 ± 0.38 ␮g/mL) than QB-90

(EC50 = 70.8 ± 0.03 ␮g/mL). Indeed, at 50 ␮g/mL, more than

90% of cells exposed to QB-90 were viable, whereas cell viability of

QA-exposed cells was only 55% (P ≤ 0.001).

The LDH assay (Fig. 2C) revealed that QA promoted a more

extensive cytoplasmic content release than QB-90 (EC50 values

of 56.59 ± 2.37 ␮g/mL and 72.76 ± 3.93 ␮g/mL for QA and QB-

90, respectively). In fact, VERO cells exposed to 50 ␮g/mL of QA

released approximately 40% of LDH, whereas those exposed to

QB-90 released similar levels of LDH as unexposed control cells

(P ≤ 0.001).

Finally, acute toxicity assay at the lowest dose tested (31.25 ␮g)

showed no lethality or signs of local toxicity (local swelling, loss

of hair and piloerection) within the mice group inoculated with

QB-90, but 40% lethality in the group treated with QA (Table 1).

Moreover, at the highest dose tested (125 ␮g) lethality showed to

be 60% and 100% in mice inoculated with QB-90 and QA, respec-

tively (Table 1). Summing up, the results presented here provide

evidence that QB-90 is signiﬁcantly less toxic than QA, both in vivo

and in vitro, suggesting that QB-90 could be used as an alternative

to QA.

3.3. BMDCs take up IQB-90 more efﬁciently than OVA formulated

with soluble QB-90

In order to determine whether IQB-90 enhances antigen inter-

nalization, the uptake of OVA:FITC by BMDCs in vitro in the presence

of soluble QB-90 or IQB-90 was analyzed (Fig. 3). Results in Fig. 3A

showed that BMDCs internalized OVA:FITC at the same extent

either in the presence or in absence of soluble QB-90 (10 ␮g/mL)

(P ≥ 0.05). However, OVA:FITC as IQB-90 was more efﬁciently inter-

nalized by BMDCs than OVA:FITC in the presence or absence of

soluble QB-90 (P < 0.001). In order to conﬁrm that detected ﬂu-

orescence in BMDCs was due to internalization of OVA:FITC and

not an artifact of OVA:FITC binding to cell surface, similar exper-

◦

iments were performed at 4 C showing a FITC signal abrogation

independently of the test conditions. Interestingly, similar results

were obtained by ﬂuorescence microscopy (Fig. 3B). Therefore, the

results presented here showed that OVA uptake by BMDCs is largely

more efﬁcient when formulated as IQB-90 than a mixture with

soluble QB-90.

3.4. Subcutaneous administration of QB-90 or

IQB-90-adjuvanted vaccine signiﬁcantly increases humoral and

cellular responses in mice

Immune-stimulating activities of OVA formulated in ISCOMs

from QB-90 or QA were studied in immunized mice, both at

the humoral (i.e. antibody) and cellular (i.e. DTH and prolifera-

tion assay) levels. Administration of OVA formulated with soluble

saponins was also analyzed. Modulation of antibody responses

(Fig. 4A and B) show that anti-OVA IgG as well as IgG1 levels were

Fig. 4. OVA-speciﬁc antibodies response of subcutaneously immunized mice. Serum

titers of anti-OVA total IgG (A), IgG1 (B) and IgG2a (C) 2 weeks after the second

immunization. Data is shown as geometric mean titers (n = 5–6). One-way ANOVA

with Dunnet’s post-test was used to investigate signiﬁcant statistical differences (**

P < 0.01 and *** P < 0.001).

1168 S.P. Cibulski et al. / Vaccine 34 (2016) 1162–1171

Fig. 5. Evaluation of delayed type hypersensitivity (DTH) and splenocyte proliferation responses in OVA-immunized mice. (A) DTH; (B) splenocyte proliferation assay. Data is

shown as mean SEM (n = 3 or 6 mice, DTH reaction or cell proliferation assay, respectively). One-way ANOVA with Dunnet’s post-test was used to investigate any signiﬁcant

statistical differences (* P < 0.05, ** P < 0.01 and *** P < 0.001).

signiﬁcantly enhanced in respect to control group (P < 0.001), inde- similar signiﬁcantly increase of IFN-␥ (P < 0.01 and P < 0.05, respec-

pendently of the saponin (QB-90 or QA) or the formulation type tively) and IL-2 (P < 0.05) cytokines levels in the supernatant than

(ISCOMs or soluble). However, anti-OVA IgG2a levels were only those obtained for the control group (Fig. 6).

increased in mice immunized with ISCOM formulations with either

IQB-90 or IQA (P < 0.001) (Fig. 4C).

3.5. Intranasal immunization with IQB-90 induces serum and

In order to determine immune-stimulating activity on cellu-

mucosal speciﬁc antibody responses

lar immune responses, we evaluated the induction of in vivo DTH

reactions and in vitro cell proliferation in response to OVA. Sig- Results in Fig. 7 illustrate the serum levels of OVA-speciﬁc IgG,

niﬁcant DTH reactions were observed in mice immunized with IgG1 and IgA in i.n. immunized mice determined 2 weeks after

both ISCOM formulations (IQB-90 and IQA) and with soluble QA, the last immunization. Delivery of IQB-90 and IQA by intranasal

while no DTH reactions were observed for soluble QB-90 (Fig. 5A). route induced signiﬁcant serum levels of OVA-speciﬁc IgG, IgG1 and

␮

On the other hand, animals vaccinated with QB-90 (10 g) or QA IgA (P < 0.001). However, no signiﬁcant increases in speciﬁc IgG2a

␮

(10 g) saponins signiﬁcantly enhanced the proliferative responses antibodies and DTH reactions were observed (P ≥ 0.05) (data not

(P < 0.05, P < 00.1, respectively). The highest proliferation was also shown). Interestingly, no antibody induction was observed after i.n.

observed in the groups vaccinated with IQB-90 and IQA (P < 0.01) administration of OVA formulated with either soluble QB-90 or QA

which was not signiﬁcantly different between them (Fig. 5B). Alto- (Fig. 7). Results on production of speciﬁc antibodies at mucosal sites

gether, results showed that IQB-90 induces strong humoral and 2 weeks after i.n. immunizations are shown in Fig. 8. While adminis-

cellular immune responses. Interestingly, Th1 and Th2 modulation tration of IQA induced signiﬁcant OVA-speciﬁc sIgA responses only

of antibody responses seem to be induced by IQB-90. at the nasal level, IQB-90 enhanced speciﬁc sIgA responses at sev-

␥

The proﬁle of major Th1 cytokines (IFN- and IL-2) was obtained eral distal mucosal sites (Fig. 8). Once again, no antibody production

from antigen stimulated and unstimulated splenocytes cultures. at mucosal sites was observed after i.n. administration of OVA for-

Spleen cells from OVA, QB-90, IQB-90, QA and IQA-treated mice mulated with either soluble QB-90 or QA. In summary, these results

were cultured in vitro in presence of antigen and cytokine pro- indicate that intranasal delivery of IQB-90 induces signiﬁcant spe-

duction was measured in the supernatant after 3 days of culture ciﬁc antibody responses both at the systemic and mucosal level,

using ELISA. Mice immunized with either IQB-90 or IQA presented a even at distal mucosal sites.

Fig. 6. Analysis of IL-2 and IFN- patterns in immunized mice. Mice were immunized with OVA alone or adjuvanted with QB-90, IQB-90, QA or IQA and a booster immunization

was administered at day 14. Splenocytes were prepared 2 weeks after second antigen dose. The cell supernatant was analyzed after 72 h of stimulation with OVA antigen for

IFN-␥ and IL-2 using ELISA (n = 3). Data is shown as mean ± SEM and one-way ANOVA with Dunnet’s post-test was used to investigate any signiﬁcant statistical differences

(* P < 0.05, ** P < 0.01 and *** P < 0.001).

S.P. Cibulski et al. / Vaccine 34 (2016) 1162–1171 1169

Fig. 8. OVA-speciﬁc antibodies response of intranasal immunized mice in mucosal sites.

Mucosal titers of anti-OVA sIgA in nasal, fecal and vaginal mucosae 2 weeks after the

second immunization. Data is shown as media ±SEM (n = 6–7) and one-way ANOVA

with Dunnet’s post-test was used to investigate any signiﬁcant statistical differences

(* P < 0.05 and ** P < 0.01).

4. Discussion

Adjuvants are deﬁned as any substance usually added to vaccine

antigens in order to enhance and/or modulate their immuno-

genicity. Remarkably, saponins have been shown to activate the

mammalian immune system, turning them into interesting sub-

stances in the ﬁeld of potential vaccine adjuvants [12]. Quil A

have been widely used as adjuvants in veterinary vaccines [29] and

have the exclusive capacity of stimulating Th1 immune responses

as well as inducing production of CTLs against exogenous anti-

gens [30,31]. This fact makes them ideal for subunit vaccines and

vaccines directed against intracellular pathogens or cancer [13,32].

In the present study a saponin fraction from Q. brasiliensis known

as QB-90 [14] – which is structurally similar to Quil A – showed

lower toxic effects in vitro and in vivo. In this regard, hemolytic

activity and cytotoxicity on VERO cells of QB-90 were lower than

QA saponins, a fact previously reported [15]. Here, complementary

toxicity assays were performed and showed that QB-90 induced-

release of LDH is lower than QA. Consistent results were obtained

in vivo through acute toxicity assays, which showed no lethality or

signs of local toxicity in mice after s.c. administration of 31.25 ␮g

of QB-90 while 40% lethality with the same dose of QA. Therefore,

as reported elsewhere [14,15] this work reinforces that saponins in

QB-90 show signiﬁcantly less toxicity. This toxicity has been associ-

ated with saponins high-afﬁnity for cholesterol, and consequently

tend to form pores in mammalian cell membranes [33]. Concern-

ing this matter, ISCOMs–as well as other micellar formulations

built up by saponins and cholesterol–prevent interactions between

saponins and membranes, thus preventing hemolysis [34]. In this

work we were able to show that IQB-90 displays no hemolysis in

comparison with soluble QA or QB-90.

Particulate formulations of saponins – like Matrix-M and

Fig. 7. OVA-speciﬁc serum antibodies response from intranasal immunized mice. Serum

titers of anti-OVA IgG (A), IgG1 (B) and IgA (C) 2 weeks after the second immunization ISCOMs – are known to enhance cell trafﬁcking and activate innate

administered either with no adjuvant or formulated with QB-90, IQB-90, QA or IQA. immune cells [22,35]. BMDCs were evaluated for their capacity

Data is shown as geometric mean (n = 6–7) and one-way ANOVA with Dunnet’s

of uptaking FITC-labeled OVA formulated with IQB-90 or mixed

post-test was used to investigate signiﬁcant statistical difference (*** P < 0.001).

with soluble QB-90. OVA:FITC was taken up more efﬁciently by

BMDCs as IQB-90 than mixed with soluble QB-90, supporting the

idea that saponin-based formulations as ISCOMs represent the best

1170 S.P. Cibulski et al. / Vaccine 34 (2016) 1162–1171

choice for activation of innate immunity [20,36]. Likewise, ISCOMs IQB-90 reported here demonstrate their potential as a candidate

as well as Quil A formulations are known to induce potent Th1 and for further development of prophylactic and therapeutic vaccines,

Th2 responses, to activate CTLs and to enhance antibody responses as an alternative to the classic ISCOMs derived from Quil A .

including high levels of IgG1, IgG2b and IgG2a [20,30]. Here, the

effects of IQB-90 and QB-90 on humoral and cellular immune Conﬂict of interest statement

responses were analyzed. The results showed that ISCOMs (IQB-90

and IQA) and soluble saponins (QB-90 and QA) promoted signiﬁ- The authors declare that they have no conﬂict of interest.

cant Ag-speciﬁc splenocyte proliferation and strong DTH reactions.

Regarding humoral responses, ISCOMs, as well as saponin formula- Acknowledgements

tions, efﬁciently enhanced the systemic production of Ag-speciﬁc

antibodies after s.c. immunizations. Remarkably, ISCOMs – unlike

This work was supported by the Programa de Desarrollo de las

soluble saponin formulations – were able to induce signiﬁcant titers

Ciencias Básicas (PEDECIBA) and Agencia Nacional de Innovación

of serum OVA-speciﬁc IgG2a. The results indicated that IQB-90 is

e Investigación (ANII) from Uruguay and the Conselho Nacional de

an efﬁcient and potent modulator of T and B cells functions, a fact

Desenvolvimento Cientíﬁco e Tecnológico (CNPq), Financiadora de

previously reported for ISCOMs containing Quil A [20,37,38]. Fur-

Estudos e Projetos (FINEP) and Coordenac¸ ão de Aperfeic¸ oamento de

thermore, the immune potentiation of Q. brasiliensis saponins in a

Pessoal de Nível Superior (CAPES) from Brazil to a CAPES/UDELAR

OVA antigen model are in agreement with other reports [15,17]. project.

The Th1 immune response is characterized by production of the

␤ ␥

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