Swine T-Cells and Specific Antibodies Evoked by Peptide Dendrimers Displaying Different FMDV T-Cell Epitopes

Swine T-Cells and Specific Antibodies Evoked by Peptide Dendrimers Displaying Different FMDV T-Cell Epitopes Patricia de Leo ´n 1† , Rodrigo Cañas-Arranz 1† , Sira Defaus 2† , Elisa Torres 3 , Mar Forner 2 , Marı ´a J. Bustos 1 , Concepcio ´n Revilla 4 , Javier Dominguez 4 , David Andreu 2 , Esther Blanco 3 *and Francisco Sobrino 1 * 1 Microbes in Health and Welfare Unit, Centro de Biologı ´a Molecular “Severo Ochoa”(CSIC-UAM), Madrid, Spain, 2 Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain, 3 Centro de Investigacio ´n en Sanidad Animal (CISA-INIA), Madrid, Spain, 4 Departamento de Biotecnologı a, Instituto Nacional de Investigacio ´n y Tecnologı a Agraria y Alimentaria (INIA), Madrid, Spain Dendrimeric peptide constructs based on a lysine core that comprises both Band T-cell epitopes of foot-and-mouth disease virus (FMDV) have proven a successful strategy for the development of FMD vaccines. Specifically, B 2 T dendrimers displaying two copies of the major type O FMDV antigenic B-cell epitope located on the virus capsid [VP1 (140– 158)], covalently linked to a heterotypic T-cell epitope from either non-structural protein 3A [3A (21–35)] or 3D [3D (56–70)], named B 2 T-3A and B 2 T-3D, respectively, elicit high levels of neutralizing antibodies (nAbs) and IFN-g-producing cells in pigs. To assess whether the inclusion and orientation of T-3A and T-3D T-cell epitopes in a single molecule could modulate immunogenicity, dendrimers with T epitopes juxtaposed in both possible orientations, i.e., constructs B 2 TT-3A3D and B 2 TT-3D3A, were made and tested in pigs. Both dendrimers elicited high nAbs titers that broadly neutralized type O FMDVs, although B 2 TT-3D3A did not respond to boosting, and induced lower IgGs titers, in particular IgG2, than B 2 TT-3A3D. Pigs immunized with B 2, a control dendrimer displaying two B-cell epitope copies and no T-cell epitope, gave no nABs, confirming T-3A and T-3D as T helper epitopes. The T-3D peptide was found to be an immunodominant, as it produced more IFN-gexpressing cells than T-3A in the in vitro recall assay. Besides, in pigs immunized with the different dendrimeric peptides, CD4 + T-cells were the major subset contributing to IFN-gexpression upon in vitro recall, and depletion of CD4 + cells from PBMCs abolished the production of this cytokine. Most CD4 + IFN-g + cells showed a memory (CD4 + 2E3 − ) and a multifunctional phenotype, as they expressed both IFN-gand TNF-a, suggesting that the peptides induced a potent Th1 pro-inflammatory response. Furthermore, not only the presence, but also the orientation of T-cell epitopes influenced the T-cell response, as B 2 TT-3D3A and B 2 groups had fewer cells expressing both cytokines. These results help understand how B 2 T-type dendrimers triggers T-cell populations, highlighting their potential as next-generation FMD vaccines. Keywords: foot-and-mouth disease virus, vaccines, dendrimer peptide, T cell phenotype, cytokine Frontiers in Immunology | www.frontiersin.org February 2021 | Volume 11 | Article 6215371 Edited by: Pedro A. Reche, Complutense University of Madrid, Spain Reviewed by: Rupsa Basu, TechnoVax Inc, United States Gabriel M. Gutierrez, Leidos, United States Haiwei Wang, Chinese Academy of Agricultural Sciences, China *Correspondence: Esther Blanco [email protected] Francisco Sobrino [email protected] † These authors have contributed equally to this work Specialty section: This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology Received: 26 October 2020 Accepted: 18 December 2020 Published: 03 February 2021 Citation: de Leo ´nP,Cañas-Arranz R, Defaus S, Torres E, Forner M, Bustos MJ, Revilla C, Dominguez J, Andreu D, Blanco E and Sobrino F (2021) Swine T-Cells and Specific Antibodies Evoked by Peptide Dendrimers Displaying Different FMDV T-Cell Epitopes. Front. Immunol. 11:621537. doi: 10.3389/fimmu.2020.621537 ORIGINAL RESEARCH published: 03 February 2021 doi: 10.3389/fimmu.2020.621537 INTRODUCTION Foot-and-mouth disease (FMD), an acute and systemic vesicular disease that affects cloven-hoofed animals, is caused by FMD virus (FMDV) from the Aphthovirus genus within the Picornaviridae family (1). FMD is included in the list of notifiable terrestrial and aquatic animal diseases of the World Organization for Animal Health (OIE), as the fatal impact of recurring FMD outbreaks causes huge economic losses in affected countries (2–4). Vaccination remains the most effective method to control FMD (5,6), with the current OIE-approved vaccine types consisting of chemically inactivated whole viruses emulsified with different adjuvants (7). Although these conventional vaccines have demonstrated their success in eliciting protective immunity against the disease in endemic countries, they have shortcomings such the need for a coldchain to preserve antigenicity, high-containment biosafety facilities, and difficulties to distinguish infected from vaccinated animals (DIVA capability), among others. These drawbacks underlie non-vaccination policies in some countries (8). In the face of these limitations, alternative strategies, for instance peptide-based subunit vaccines targeting FMDV protein VP1 have been successfully used to induce anti-FMDV neutralizing antibodies (9). Advantages of such peptide vaccines include: (i) safety, as a non-infectious material is required, and no reversion to virulence is possible; (ii) DIVA condition; (iii) easy handling and storage, with no cold chain needed; (iv) chemical stability, and (v) efficient, affordable large scale production. However, early reports of livestock immunization with linear peptides showed modest levels of protection in livestock, lower than required for use as commercial vaccines (10,11) and interest in peptide-based vaccines temporarily waned. However, with the advent of so-called multiple antigenic peptides (MAPs) pioneered by Tam (12), an effective approach to increase peptide immunogenicity was demonstrated, and peptide vaccines staged a comeback. In the context of FMD, our own research has focused on dendrimeric constructions, generically termed B n T, where several copies of a FMDV B-cell epitope from the G-H loop of VP1 protein in the FMDV capsid (13,14) are covalently linked through a Lys core matrix to a FMDV T-cell epitope from a non-structural protein (i.e., originally 3A protein, residues 21–35) (15). The selected Bcell epitope shows amino acid variations among different serotypes while the T-cell epitope is highly conserved and therefore can evoke heterologous responses in swine. Interestingly, two doses of a dendrimeric peptide named B 4 T3A, displaying four copies of a B cell epitope from type C FMDV linked to T-cell epitope 3A (21–35), was able to protect pigs against homologous FMDV challenge (16). Subsequently, a downsized version, i.e. B 2 T-3A, bearing only two copies of a type O FMDV B-cell epitope and being stable in serum for several hours (17), afforded full protection in swine, even upon a single dose (18–20). These protective responses of B 2 T dendrimers are correlated with the induction of high and longlasting titers of nAbs and the activation of specific lymphocytes providing T-cell help (19,21). Besides, such T-cell epitopes can also stimulate T-cell subsets leading to the expression of IFN-g,a cytokine with a relevant role in the antiviral response (22). In another endeavor, a T-cell epitope at the 3D FMDV protein [3D (56-70)], previously shown to be promiscuous and heterotypic in swine (23), displayed as a B 2 T-3D construct, elicited nAbs titers and IFN-g-producing cells in similar levels to B 2 T-3A (24). As previous results in our groups with linear FMDV peptides had shown that not only the presence but also the orientation of the T-cell epitope influenced the immune response (25), in this work the T-3A and T-3D T-cell epitopes have been juxtaposed in B 2 TT constructs with the two possible orientations, to assess how tandem display and orientation modulate immunogenicity, specifically the T-cell response, a crucial information for improving vaccine design. Also, the requirement of T-cell epitope inclusion in the peptide vaccine was again confirmed, by comparison with ineffective immunizations with construct B 2 , lacking a T-cell epitope. MATERIALS AND METHODS Peptides The B-cell epitope from FMDV O UKG/11/2001, VP1 (residues 140–158), and the T-cell epitopes 3A (residues 21–35) and 3D (residues 56–70) were synthesized by Fmoc-solid phase synthesis. Bivalent branched dendrimers were prepared by conjugation in solution of two B-cell peptides containing an extra C-terminal Cys (free thiol form) to (one or tandem of two) T-cell epitope moieties N-terminally elongated with Lys residues defining a branching point further derivatized by maleimide units (Table 1). A Lys-Lys motif, defining a putative cleavage site for cathepsin D, a protease involved in in vivo antigen processing for presentation to the MHC class II molecules, was included in the constructs at one (N-terminal of the T-cell epitope in B 2 T, before the branching) or two positions (as above, plus between the T-cell epitope sequences in B 2 TT tandems). The end products were obtained via thiol–maleimide ligation at pH 6.0, purified by reverse-phase liquid chromatography and characterized by mass spectrometry (16–18). Final immunogenic peptide dendrimers were water-soluble and stable either lyophilized or in freeze-dried form. Formulation of peptide vaccines was done at the moment of administration. Virus FMDV stocks O UKG/11/2001, O/SKR, O 1 Manisa, O 1 BFS (The Pirbright Institute, UK), and O 1 Campos (OPS-PanAftosa) were amplified in IBRS-2 cells and type C CS8-c1 virus (26) was amplified in BHK-21 cells. Animals and Experimental Design Landrace x Large White female pigs, 9 to 12-week-old (20 Kg), were maintained in a conventional farm facility at the Departamento de Reproduccion Animal, INIA, Madrid. Groups of four pigs were immunized with the B 2 T-3A (pigs 49, 54, 65, and 74), B 2 T-3D (pigs 75, 76, 77, 78), B 2 TT-3A3D (pigs 79, 90, 91, and 92) or B 2 TT-3D3A (pigs 93, 94, 95, and 96) constructs at day 0, with 2 ml of Montanide ISA 50V2 emulsion containing 2 mg of the corresponding peptide, and boosted de Leo ´n et al. T-Cells Evoked by FMDV Dendrimers Frontiers in Immunology | www.frontiersin.org February 2021 | Volume 11 | Article 6215372 likewise at day 21 pi. In addition, two pigs were immunized and boosted with B 2 (pigs 97 and 98) and another two PBSinoculated pigs were maintained as controls (pigs 99 and 100). Because of animal welfare and space requirements, half of the animals (10) corresponding to the lower T-cell responders, were euthanized at day 70 pi. The remaining pigs 10 were housed until the end of the experiment at day 183 pi. Blood samples were collected at days 0, 7, 14, 21, 29, 36, 49, 121, 157, and 183 pi to obtain serum and peripheral blood mononuclear cells (PBMCs). The study was approved (CBS2014/015 and CEEA2014/018) by the INIA Committees on Ethics of Animal Experiments and Biosafety, and by the National Committee on Ethics and Animal Welfare (PROEX 218/14). Virus Neutralization Test (VNT) Neutralization assays were performed in 96-well culture plates. Serial 2-fold dilutions of each serum sample (in DMEM containing 2% fetal bovine serum) were incubated with 100 infection units—50% tissue culture infective doses (TCID 50 )—of FMDV O UKG/11/2001 for 1 h at 37°C. Then, a cell suspension of IBRS-2 cells in DMEM was added and plates were incubated for 72 h. Monolayers were controlled for development of cytopathic effect (cpe), fixed and stained. End-point titers were calculated as the reciprocal of the final serum dilution that neutralized 100 TCID 50 of homologous FMDV in 50% of the wells (18). For cross neutralization assays, incubation of sera with the panel of FMD viruses (O/SKR, O 1 Manisa, O 1 BFS and O 1 Campos) that belonged to different type O topotypes was performed in parallel to that of the homologous isolate O UKG/ 11/2001 and the type C CS8-c1 virus as a negative control. Detection of Anti-FMDV Antibodies by ELISA Total anti-FMDV antibodies were determined by means of an indirect ELISA (25). Briefly, 96-well plates coated with peptide B (1µg) were incubated with three-fold dilutions of serum and detected using HRP-conjugated protein A. Plates were read at 450 nm and titers were expressed as the reciprocal of the last serum dilution given an absorbance range of two standard deviations above the background (serum at day 0). Specific IgG1 and IgG2 titers to peptide B were measured and expressed as above using isotype-specific mAbs (Serotec) that were detected with HRP-labeled anti-mouse IgGs (Thermo Fisher). Antibodies against the non-structural protein precursor 3ABC were detected basically as described (18,27). Purification of PBMCs Porcine PBMCs were isolated from blood samples collected in Vacutainer tubes EDTA-K2, diluted 1:1 in PBS and then used to obtain PBMC by density-gradient centrifugation with Histopaque 1077 (Sigma) and Leucosep tubes (Greiner Bio-One) as described (27).ThenumberoflivePMBCswascalculatedusingaNeubauer chamber (Immune Systems) and cell staining with 0.4% Trypan Blue (Sigma) in PBS. In general, fresh cells were used in the experiments and those remaining were cryo-preserved in 90% FBS and 10% DMSO in liquid nitrogen. The minimum number of cells used for freezing was 2x10 7 /vial. IFN-gDetection by ELISPOT Quantification of IFN-gsecreting cells from immunized and control animals was performed using an ELISPOT assay. Briefly, 2.5 × 10 5 PBMCs were shed in triplicate wells of Immobilon-P plates (Merck Millipore) coated as reported (18) and in vitro stimulated with 50 µg/ml of their respective dendrimers or with T or B peptides. As positive or negative controls, cells were incubated with 10 µg/ml of phytohaemagglutinin (Sigma) or only with medium, respectively. After 48 h at 37°C and 5% CO 2, plates were washed and incubated with a biotinylated mouse anti-pig IFN-gantibody (clone P2C11, BD) followed by HRPstreptavidin (BD). The frequency of peptide-specific T-cells was expressed as the mean number of spot-forming cells/10 6 PBMCs, with background values (number of spots in negative control wells) subtracted from the respective counts of stimulated cells. These experiments were performed using outbred domestic pigs TABLE 1 | Peptide-based vaccine constructs used in this study. General structure a B 2 TB 2 TT B 2 Bacetyl-PVTNVRGDLQVLAQKAARTC-amide T-3A AAIEFFEGMVHDSIK-amide T-3D IFSKHRGDTKMSAED-amide B scrambled TGRTVQANVLQDLPAKRA T-3A scrambled HMAFESFDGIVKIAE Peptides B 2 T-3A B 2 T-3D B 2 TT-3A3D B 2 TT-3D3A B 2 MW b 6742.8 Da 6770.8 Da 8708.1 Da 5066.9 Da HPLC c 6.9 min (98%) 5.1 min (98%) 6.2 min (98%) 6.0 min (97%) 5.2 min (99%) a Bivalent-branched B-cell epitope immunogens conjugated to none, one or two T-cell epitopes (T-3A or T-3D) in tandem via thiol-maleimide linkages at both aand ϵ-amino ends of a branched Lys core. b Experimental mass determined by LC/MS. c HPLC retention time (Phenomenex Luna C 18 column, 4.6×50 mm, 3 mm) eluted with a 20–60% linear gradient of solvent B (0.036% TFA in MeCN) into solvent A (0.045% TFA in H 2 O) over 15 min. c In parenthesis, HPLC homogeneity of purified material. de Leo ´n et al. T-Cells Evoked by FMDV Dendrimers Frontiers in Immunology | www.frontiersin.org February 2021 | Volume 11 | Article 6215373 with different individual genetic backgrounds. In any case, the levels of animal-to-animal variation did notexceedthose observed in other related studies (11). Immunoreagents for Flow Cytometry and MACS Cell Sorting The following mouse anti-pig monoclonal antibodies (mAbs) were used as primary immunoreagents: anti-CD4 (74-12-4, IgG2b) (28), anti-CD8b(PG164A, IgG2a, Eurovet Veterinaria), anti-2E3 (IgM) (29), and anti-CD172a (BL1H7, IgG1) (30). Goat polyclonal Abs (pAbs) specific for mouse Ig subclasses labeled with Alexa647, Alexa488 (Fisher Scientific) or FITC (Bionova) were used as secondary antibodies. For cytokine staining, PEconjugated anti-pig IFN-g(P2G10, IgG1) or APC-conjugated anti-human TNF-a(Mab11, IgG1, BD Biosciences) were used. All immunoreagents were titrated for optimal signal/noise ratios. Primary and secondary isotype-matched antibodies were used as negative controls. No background or false staining was observed. Intracellular Cytokine Staining (ICS) To detect the phenotype of cytokine-producing cells, intracellular staining of purified PBMCs was performed using 5x10 6 PBMCs from each pig that were in vitro stimulated or not for 18 h at 37°C with their respective peptide at a final concentration of 25 µg/ml. During the last 10 h, brefeldin A (Sigma) was present in cultures at a final concentration of 5 µg/ml. Mediumor PHA-M-incubated cultures (25 µg/ml) served as negative (non-stimulated) or positive controls, respectively, in a non-immunized animal of each experiment. Cells were washed in PBS-0.05% EDTA-5% FBS and surface stained by incubation with cell-surface primary antibodies for 30 min at 4°C in PBS-0.05% EDTA5% swine serum. After three washes in PBS-0.05% EDTA-5% FBS, cells were incubated with the adequate secondary labeled antibodies for 30 min at 4°C. Cells were washed three times, fixed and permeabilized for 20 min at 4°C using Cytofix/Cytoperm buffer (BD). Then, samples were washed in PERM-WASH buffer (BD), free binding sites of secondary antibodies were blocked with whole mouse IgG molecules, and samples were incubated with PEand APCconjugated antibodies for the detection of intracellular IFN-gor TNF-a, respectively, for 30 min at 4°C. Finally, cells were washed three times in PERM-WASH buffer and fixed using a solution containing 2% paraformaldehyde (Electron Microscopy Science). After a further wash, cells were analyzed in a flow cytometer FACS CantoA (BD Biosciences). Data was processed using FACSDiva software (BD Biosciences) or FlowJo software (www.flowjo.com/, Tree Star, Ashland, OR, USA), and transferred to Microsoft Excel for further calculations and preparation of graphs with GraphPad Prism Software 5.0. MACS Cell Sorting For isolation of CD4 + T cells, PBMC were incubated with mAb 7412-4 for 30 min and then washed 2 times with PBS containing 2% FBS and 2.5 mM EDTA and 0.1% sodium azide (MACS buffer). Thereafter, cells were incubated with anti-mouse IgG magnetic microbeads (Miltenyi Biotec, Germany) for 15 min. After a further wash in MACS buffer, the cell suspension was passed through the autoMACS cell sorter and the labeled (CD4 + ) and non-labeled (CD4−) fractions were collected. The purity of each fraction was determined by flow cytometry. Cell staining with anti-CD172a antibody revealed the presence of around 4% of APCs in the CD4 + T-cell fraction (Supplemental Figure 1). Finally, cells were processed for IFN-gdetection by ELISPOT as described before. RESULTS Analysis of the Immune Response Induced in Pigs by B 2 T Dendrimers Displaying Two T-Cell Epitopes in Tandem We first addressed the effect on peptide immunogenicity of including both 3A and 3D T-cell epitopes and their orientation in the same molecule. To this end, T-3A and T-3D T-cell epitopes were combined in a B 2 T-type platform in the two possible orientations, to render peptides B 2 TT-3A3D and B 2 TT-3D3A (Table 1). Groups of four pigs were immunized and boosted at day 21 pi, with B 2 T-3A, B 2 T-3D, B 2 TT-3A3D, or B 2 TT-3D3A. Although previous studies with linearpeptideshadshownthatthe inclusion of a T-cell epitope was required for inducing optimal immune responses in pigs (31), this requirement for a T-cell epitope in the context of more complex platforms such as our dendrimeric peptides remained to be clarified. Thus, two additional pigs were immunized and boosted with a B 2 peptide that harbored only two copies of the B-cell epitope without any additional porcine T-cell epitope. Two PBS-immunized animals were included as negative controls. Since in previous work T-cell responses to B 2 T decreased over time, being more short-lived than antibody responses (24), the frequency of IFN-g-secreting cells was only analyzed at early times pi (days 7, 14, and 21) and post-boost (days 28 and 35), and by the end of the experiment at day 183 (6 months pi) for those animals that remained alive. Similar Levels of Total IgG Antibodies are Elicited by Dendrimers Harboring One or Two T-Cell Epitopes Total IgGs in sera from peptide-immunized pigs were determined by ELISA. All the dendrimer-immunized animals elicited similar levels of specific antibodies, first detected at day 14 pi (about 3.6 log 10 ) and slightly increased at day 21 pi (about 4 log 10 ). Upon boosting, an increase in IgG titers was observed in the B 2 Timmunized groups at day 28 pi (about 5 log 10 )thatremained stable up to day 49 pi, except in pigs immunized with B 2 TT-3D3A, whose titers showed a slight decrease. Interestingly, at day 121 (4 months pi) a sustained antibody response was still observed, with average titers about 4.5 log 10 . This long-lasting antibody response was consistently observed up to the end of the experiment at day 183 (6 months pi) (about 4 log 10 ). No antibodies were detected in B 2 and PBS-immunized groups (Figure 1). Neutralizing Activity is Determined by the Presence and Orientation of the T-Cell Epitopes in the Dendrimer Neutralizing activity against FMDV was tested in sera from pigs at different times pi (Figure 2); nAbs were not detected until day 14 pi de Leo ´n et al. T-Cells Evoked by FMDV Dendrimers Frontiers in Immunology | www.frontiersin.org February 2021 | Volume 11 | Article 6215374 (Figure 2B). At this time, slightly higher average VNTs were observed for constructions encompassing two T-cell epitopes in tandem (B 2 TT-3A3D: 1.6 ± 0.5; B 2 TT-3D3A: 1.5 ± 0.5 log10) in comparison with those with a single T-cell motif (B 2 T-3A: 1.3 ± 0.6; B 2 T-3D: 1 ± 0.4) (Figure 2B). At day 21 pi, an average increment was observed in all dendrimer-immunized groups, reaching similar average titers (B 2 T-3A: 1.9 ± 0.3; B 2 T-3D: 1.9 ± 0.4; B 2 TT-3A3D: 1.6 ±0.7;B 2 TT-3D3A: 1.7 ± 0.5 log10) (Figure 2C). After the boost, at day 28 pi, an increment in VNTs was noticed in pigs immunized with B 2 T-3A (2.5 ± 0.6 log10), B 2 T-3D (2.5 ± 0.4 log10), and B 2 TT-3A3D (2.6 ± 0.3 log10), but not in animals from the B 2 TT-3D3A group (2 ± 0.6 log10) (Figure 2D). Titers remained similar over time with minor differences among groups in subsequent days (B 2 T-3A: 2.4 ± 0.5; B 2 T3D: 2.5 ± 0.5; B 2 TT-3A3D: 2.5 ± 0.3; B 2 TT-3D3A: 1.9 ± 0.5 log10, at day 36 pi; B 2 T-3A: 2.3 ± 0.4; B 2 T-3D: 2.1 ± 0.6; B 2 TT-3A3D: 2.2 ± 0.3; B 2 TT-3D3A: 1.6 ± 0.3 log10, at day 49 pi) (Figures 2E, F).These differences were not statistically significant. These results suggested that the presence of T-cell epitopes and their orientation within the dendrimer has an impact in the duration of neutralizing antibody responses against the FMDV G-H loop (B-cell epitope). Indeed, animals that were not euthanized—pigs 49 and 65 (B 2 T-3A), 76, 77 and 78 (B 2 T-3D), 91 and 92 (B 2 TT-3A3D), 93 and 94 (B 2 TT-3D3A)—showed at days 121, 157 and 183 pi (4, 5, and 6 months pi, respectively) VNTs similar to those observed at day 49 pi (data not shown). This result is consistent with previous observations on the longlasting neutralizing responses elicited by B 2 T-dendrimers (19). Remarkably, no nAbs were detected at any time point in pigs immunized with the B 2 peptide, with the branched B-cell epitopes but lacking a T-cell motif. This lack of response confirms the functional role of T-3A and T-3D as T helper epitopes, i.e., inclusion of at least one of them is essential for nAbs to be elicited in pigs. Dendrimers Elicit nAbs Against a Broad Spectrum of Type O FMDVs Since type O FMDVs are responsible for many current FMD outbreaks in endemic countries, a broad-spectrum response is necessary for effective vaccines against this serotype (32). Therefore, we were interested in assessing the neutralization range afforded by B 2 T-dendrimers. Recently, we observed that sera from pigs immunized with B 2 T-3A or B 2 T-3D were able to neutralize a panel of type O FMDVs belonging to various spatiotemporal locations (24). We decided to extend the study to animals vaccinated with B 2 T-dendrimers harboring two different T-cell epitopes in tandem, as well as with B 2 , to assess again the relevance of T-cell epitopes in the induction of neutralizing activity. As shown in Figure 3, both constructions with two T-cell epitopes induced nAbs against the panel of FMDVs, showing similar titers before boosting (Figure 3A), while only B 2 TT-3A3D had a VNT increase afterwards (Figure 3B). Neutralizing activity against viruses such as O 1 Campos and O/SKR appeared to be significantly higher, in some cases, than that against the homologous virus O UKG/11/2001; factors inherent to the assay, such as differential thermal stability among viral isolates, might contribute to explain these observations. As expected, no nAbs were detected in B 2 - immunized animals, confirming our previous results with homologous O UKG/11/2001 virus, nor in all animals tested against isolate CS8-c1 that belongs to a different serotype (Figure 3). Thus, B 2 TT-3A3D and B 2 TT-3D3A behave just like previously tested B 2 T-3A and B 2 T-3D, being able to induce a broad anti-FMDV immunity within a serotype. Isotype-Specific IgG1 and IgG2 Antibodies Elicited by Dendrimers B 2 TT-3A3D and B 2 TT-3D3A To test whether the inclusion of two consecutive T-cell epitopes in the dendrimer and their orientation could favor expression of a specific IgG isotype and influence the class switching, the anti Bpeptide IgG1 and IgG2 profile was analyzed by ELISA in serum samples from pigs immunized with the different constructs at day 21 pi. All dendrimers induced specificIgG1andIgG2albeitto different extents (Figure 4). Three animals from the B 2 T-3A group showed high titers of both IgG1 and IgG2 (about 5 log10) and only one pig did not develop high IgG2 titers (< 3 log 10 )(Figure 4A). In the same day, all animals immunized with B 2 T-3D reached similar IgG1 and IgG2 titers (about 4 log10) that were, in average, lower than those of B 2 T-3A group (Figure 4B). Moreover, pigs immunized with B 2 TT-3A3D reached isotype titers similar to those of the B 2 T-3D group suggesting that T-3D was modulating the isotype switching (Figure 4C). On the other hand, the construction B 2 TT-3D3A induced low (titers < 3 log10) IgG2 titers, in two of the four animals (Figure 4D). T-Cell Responses Elicited by B 2 TT-3A3D and B 2 TT3D3A: Immunodominance of the 3D T-Cell Epitope The T-cell responses elicited by dendrimers B 2 TT-3A3D, B 2 TT3D3A, B 2 T-3A, B 2 T-3D, and B 2 were analyzed using PBMCs collected at different days pi. Cells were in vitro stimulated with the homologous peptide as well as with the heterologous one (B 2 TT-3A3D/B 2 TT-3D3A or B 2 T-3A/B 2 T-3D) and with the Band the 3A and 3D T-cell peptides, in an attempt to further understand the antigenic specificity of the elicited responses (Figure 5). FIGURE 1 | Time course of specific IgG antibodies elicited by B 2 T constructions combining different T-cell epitopes. Each point represents the mean of sera from the indicated group at different days pi (n = 4; except for B 2 and PBS groups in which n = 2) ± SD. Data on day 121 pi corresponds to two pigs per group. Arrow points the day of peptide boost. de Leo ´n et al. T-Cells Evoked by FMDV Dendrimers Frontiers in Immunology | www.frontiersin.org February 2021 | Volume 11 | Article 6215375 When T-3D and T-3A were joined together in dendrimers B 2 TT-3A3D and B 2 TT-3D3A, a consistent T-cell response was observed in pigs immunized with these constructions. Similar high levels of IFN-gproducing cells were found upon in vitro stimulation with the homologous or the heterologous dendrimeric peptides that only differed in the T-cell epitope orientation. Interestingly, the response was higher when cells were in vitro stimulated with peptide T-3D than with peptide T3A, suggesting an immunodominance of T-3D T-cell epitope over T-3A. Low levels of IFN-gsecreting cells were noticed when the B-cell peptide was used as stimulus, unlike what observed in the responses induced in pigs from the B2T-3A and B 2 T-3D groups (Figure 5A). Significant frequencies of IFN-gexpressing cells were detected in the B 2 T-3A group upon in vitro recall with the homologous dendrimer. In general, responses were high in all the animals. The number of IFN-gsecreting cells detected was stimulus-dependent, the highest values being induced by the homologous dendrimer A B FIGURE 3 | Sera from pigs immunized with dendrimers can neutralize a panel of different FMDVs type O topotypes. Sera recovered from animals immunized with the different peptides after the first (day 21 pi or pre-boost) (A) and the second immunization (day 28 pi or post-boost) (B), were tested to neutralize a panel of different type O FMDVs. Individual columns represent the mean of each group (n = 4) ± SD. Values are expressed as the reciprocal log 10 of the last serum dilution that neutralized 100 TCID50 of each FMDV. Statistically significant differences are indicated by one (*) or two (**) asterisks for p < 0.05 or p < 0.005, respectively. A B DEF C FIGURE 2 | VNTs are influenced by the presence and orientation of the T-cell epitopes in the dendrimer. Sera samples were collected after one dose of each dendrimeric peptide (pre-boost) panels (A–C) and after peptide boost [panels (D–F). Each point represents the VNT of one single pig and the mean of each group is shown with a horizontal bar. Dotted line depicts the detection limit. de Leo ´n et al. T-Cells Evoked by FMDV Dendrimers Frontiers in Immunology | www.frontiersin.org February 2021 | Volume 11 | Article 6215376 B 2 T-3A. Responses to T-3A were also significant, confirming that this peptide was recognized as a T-cell epitope. The specificity of the T-cell response was confirmed as no IFN-gspots were detected upon stimulation with the heterologous T-cell epitope T-3D. However, a heterologous response against B 2 T-3D peptide was also noticed, similar to that elicited against B-cell epitope. These results point to a contribution of the B-cell epitope present in the B 2 T dendrimers to the effector cellular responses (Figure 5A), although we cannot rule out that the overall configuration of the dendrimer platform itself may also play a role in T-cell activation. Peptide B 2 T-3D was also able to induce similar frequencies of IFN-gexpressing cells in response to peptide T-3D in all the animals. This result confirms that T-3D can be considered a Tcell epitope able to induce an IFN-geffector immune response. As observed in animals immunized with B 2 T-3A, the B-cell peptide was also able to induce IFN-gsecretion, suggesting, again, that this peptide may be recognized as a T-cell epitope (Figure 5A). Low levels of IFN-gexpressing cells were also detected in animals immunized with peptide B 2. This poor recognition of B 2 as a T-cell epitope highlights the decisive contribution of 3A and 3D epitopes to the cellular immune response elicited by the dendrimer peptides. As expected, non-immunized pigs from the control group did not show specific T-cell responses against any of the peptides (Figure 5A). In order to rule out whether the induction of T-cell responses could be influenced by non-specific mechanisms, a dendrimer with a scrambled amino acid sequence (src) in both the Band the T-3A epitopes (B 2 scr-Tscr) was used to stimulate PBMCs from day 183. No IFN-gsecreting cells were detected neither in response to the whole scrambled dendrimeric peptide (B 2 scrTscr) nor to the scrambled T-cell peptide alone (Tscr) (Figure 5B), showing that a branched architecture is not enough to induce an effector T-cell response, and that specific T-cell epitope sequences are required. Characterization of T-Cell Populations Elicited Upon Immunization With B 2 T Dendrimers Flow cytometry assays, including both surface marker staining and intracellular cytokine staining (ICS), were performed in dendrimer-stimulated PBMCs from immunized pigs at day 21 pi, in order to evaluate the specific T-cell populations induced upon immunization with B 2 T peptides. To make the assay feasible, the two best responders from each group, in terms of levels of IFN-gproducing cells determined by ELISPOT assays, A B CD FIGURE 4 | IgG1 and IgG2 profiles elicited by B 2 T constructions combining different T-cell epitopes. Anti-B peptide IgG1 and IgG2 profiles in sera from pigs collected at 21 days pi with: B 2 T-3A (A),B 2 T-3D (B),B 2 TT-3A3D (C), and B 2 TT-3D3A (D). Each point represents the value of a duplicate of an individual animal. Endpoint titers are expressed as the reciprocal of serum dilution (log 10 ) giving the absorbance recorded in control wells (sera collected at day 0) + 2 x SD. Each symbol represents the IgG1 and IgG2 titers (X and Y values respectively) for an individual pig. In the lower right quadrant, the percentage of pigs with titers above 3 log 10 is shown. No individual spontaneous reactivity was observed in the titers determined at day 0. de Leo ´n et al. T-Cells Evoked by FMDV Dendrimers Frontiers in Immunology | www.frontiersin.org February 2021 | Volume 11 | Article 6215377 A B FIGURE 5 | T-cell responses elicited by B 2 TT-3A3D and B 2 TT-3D3A unveil 3D peptide as an immunodominant T-cell epitope. PBMCs were isolated from individual animals of each group, at different days pi. Cells were in vitro stimulated for 48 h with dendrimers and the frequency of IFN-gproducing cells was determined by ELISPOT. (A) Frequency of IFN-gsecreting cells in the groups of pigs immunized as indicated, in response to the homologous dendrimer and the different constructions indicated. Each line corresponds to an individual animal and arrows show the day of the boost. (B) IFN-gproduction was stimuli-specific and sequence-dependent. The values corresponded to PBMC recovered at day 183 and are presented as IFN-gspots per 10 6 cells). Each column represents the mean (n = 4) ± SD. de Leo ´n et al. T-Cells Evoked by FMDV Dendrimers Frontiers in Immunology | www.frontiersin.org February 2021 | Volume 11 | Article 6215378 were selected: pigs 49 and 65 (B 2 T-3A), pigs 76 and 77 (B 2 T-3D), pigs 91 and 92 (B 2 TT-3A3D), pigs 93 and 94 (B 2 TT-3D3A), pig 98 (B 2 ) and pig 100 (PBS). ICS Analysis Unveils CD4 + T-Cells as the Major Population Involved in T-Cell Response PMBCs were stained for CD4 or CD8bexpression, followed by intracellular cytokine staining for IFN-gin CD4 + (helper) or CD8b + (cytotoxic) T-cells. The percentages of CD4 + and CD8b + populations differed among pigs (17.4 ± 3.7% and 10.1 ± 3.2%, respectively) (Figure 6A). Upon specific stimulation, IFN-gwas preferentially expressed by CD4 + T-cells in all immunized animals (n = 2). The percentage of CD4 + IFN-g + cells was variable, being higher in B 2 T-3D group (5.2 ± 0.3%), followed by B 2 TT-3A3D (3.6 ± 1.9%), B 2 TT-3D3A (3 ± 1.1%), and B 2 T3A (2.8 ± 2%) groups. Notably, the B 2 -immunized animal (pig 98) hardly induced CD4 + IFN-g + T-cells (0.4%), further supporting the need for a T-cell epitope to induce a cellular response, as observed by ELISPOT (Figure 5). IFN-gproduction was less evident in CD8b + T-cells, where the highest response was found in the B 2 TT-3D3A group (1.7 ± 0.1%), followed by B 2 T-3D (1 ± 0.1%), B 2 TT-3A3D (0.9 ± 0.1), B 2 [pig 98 (0.8%)] and B 2 T-3A (0.7 ± 0.4%) groups. These results point to CD4 + Tcells as the main population involved in dendrimer-induced Tcell immunity (Figure 6B). Depletion of CD4 + T-Cells from PBMCs Abolishes IFN-gProduction To confirm the phenotype of the T-cell subset responsible for IFN-gexpression, CD4 + T-cells were depleted of PBMCs isolated from a B 2 T-3D immunized pig (pig #77), which was selected for being a high IFN-gproducer when tested in ELISPOT and ICS assays (Figures 5 and 6). The magnetically isolated positive (CD4 + ) and negative (CD4 − ) fractions were in vitro stimulated with B 2 T-3D or T-3D peptides and IFN-gexpression was determined by ELISPOT. Unfractioned PBMCs and PBMCs reconstituted with the CD4 + and CD4 - fractions (CD4 + /CD4 − ) were included as controls. The efficiency of the isolation process was verified by flow cytometry. As shown in Figure 7, 21.1% of initial PBMCs were CD4 + . After the sorting, a drastic reduction in the number of CD4 + cells (0.1%) was found in the eluted negative fraction (CD4 − ) while the positive fraction (CD4 + ) showed a notable enrichment in CD4 + (95.6%) (Figure 7A). No fluorescence was detected in cells incubated with secondary antibodies alone (data not shown). Both unsorted PBMCs and magnetically sorted fractions were stimulated with B 2 T-3D and T-3D peptides to quantify IFN-gexpression by ELISPOT. As shown in Figure 7B only the CD4 + cells produce IFN-gupon stimulation with either the dendrimer or the T cell epitope, while no IFN-gspots were detected in the CD4 − fraction. As expected, total PBMCs and the reconstituted fraction CD4 + /CD4 − showed similar levels of IFN-gsecreting cells. These results confirm that CD4 + T-cells are the main population responsible for specific IFN-grelease. Memory CD4 + T-Cells Are the Responsible Subset of the IFN-gSecretion 2E3 expression has been used in swine immunological studies to differentiate naïve and memory CD4 + T-cell subpopulations. This marker is expressed on the surface of naïve CD4 + cells in swine PBMCs but not in memory CD4 + cells (29,33). Dendrimer-stimulated PBMCs from immunized pigs were stained for the expression of CD4 in combination with 2E3, and ICS for IFN-gwas performed in both gated CD4 + T cell subpopulations (i.e., CD4 + 2E3 + and CD4 + 2E3 − ). The percentage of CD4 + 2E3 + and CD4 + 2E3 − cells in the pigs varied, being higher for the latter subset (3.3 ± 1.3% and 8.9 ± 3.2%, respectively) (Figure 8A). CD4 + 2E3 + subset showed a low percentage of IFNg-expressing cells, with higher levels in the B 2 T-3A group (1.5 ± 0.6%), followed by B 2 TT-3A3D (0.9 ± 0.6%), B 2 T-3D (0.4 ± 0.3%) and B 2 TT-3D3A and B 2 (0.2 ± 0.2%) groups. A clear induction of IFN-gwas observed in the CD4 + 2E3 − subset from all immunized groups, which was higher in B 2 T-3D (4.6 ± 0.8%), B 2 TT-3A3D (4.2 ± 1.4%), and B 2 T-3A (3.6 ± 0.5%) groups than in B 2 TT-3D3A (1.2 ± 0%) and B 2 (1.3%) groups (Figure 8B). No fluorescence signal was detected in non-stimulated cells (data not shown). These results suggest that memory CD4 + T-cells are the major subset involved in IFN-gproduction and in the T-cell response elicited by B 2 T dendrimers. Multifunctional CD4 + T-Cells Are Activated by the B 2 T Dendrimers Studies on multifunctional T-cells, i.e., T-cells performing several functionslikecytokineproduction or degranulation, have revealed their central role in protective immune responses (34). IFN-gis the main effector cytokine expressed in Th1 T-cells, but other pro-inflammatory cytokines are also involved in Th1 effector responses and are important in T-cell immunity. Therefore, in order to evaluate whether CD4 + T-cells activated by the B 2 T dendrimers were producing effector cytokines other than IFN-g, ICS was performed for IFN-gand TNF-aexpression (Figure 9A; a representative plot from pig #91 is shown). When the expression of IFN-gand TNF-awas analyzed in the gated CD4 + T-cell population, a strong fluorescence signal was detected in PBMCs stimulated with homologous peptides (Figure 9B). Most of the activated CD4 + T-cells expressed both cytokines, which is consistent with a multifunctional CD4 + IFN-g + TNF-a + phenotype. This was particularly evident in pigs immunized with B 2 T-3A (2.7 ± 1.9%), B 2 T-3D (2 ± 0.6%), and B 2 TT-3A3D (1.5 ± 0.8%). In contrast, in B 2 TT-3D3A (0.8 ± 0.1%) and B 2 [pig 98 (0.5%)] groups showed lower levels of cells expressing both cytokines (Figure 9B). 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Nat Med (2013) 19:1305–12. doi: 10.1038/nm.3350 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2021 de Leo n, Cañas-Arranz, Defaus, Torres, Forner, Bustos, Revilla, Dominguez, Andreu, Blanco and Sobrino. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. de Leo ´n et al. T-Cells Evoked by FMDV Dendrimers Frontiers in Immunology | www.frontiersin.org February 2021 | Volume 11 | Article 62153716