Sisteré‑Oró et al. Vet Res (2020) 51:57 https://doi.org/10.1186/s13567-020-00770-4 RESEARCH ARTICLE Open Access Influenza NG‑34 T cell conserved epitope adjuvanted with CAF01 as a possible influenza vaccine candidate Marta Sisteré‑Oró1, Gabriel K. Pedersen2, Lorena Córdoba1, Sergi López‑Serrano1, Dennis Christensen2 and Ayub Darji1* Abstract Conserved epitopes are targets commonly researched to be part of universal vaccine candidates against influenza viruses (IV). These conserved epitopes need to be cross-protecting against distinct IV subtypes and to have a strong immunogenic potential. Nevertheless, subunit vaccines generally require a strong adjuvant to enhance their immu‑ nological effects. Herewith, we compare four different adjuvants differing in their immunological signatures that may enhance efficacy of a conserved hemagglutinin (HA)-epitope from IV, the NG-34, to define the most efficient combination of antigen/adjuvant to combat IV infections. Soluble NG-34 was mixed with adjuvants like aluminium hydroxide (AH) and AddaVax, known to induce Th2 and humoral responses; CAF01 which displays a biased Th1/Th17 profile and Diluvac Forte which augments the humoral response. Combinations were tested in different groups of mice which were subjected to immunological analyses. CAF01 + NG-34 induced a complete immune response with the highest IgG1, IgG2c titers and percentages of activated CD4 T cell promoting IFN-γ, IL-2 and TNF-α producing cells. Furthermore, in NG-34 stimulated mice splenocytes, cytokine levels of IFN-γ, IL-1β, IL-6, IL-10, IL-17 and TNF-α were also the highest in the CAF01 + NG-34 mouse group. This complete induced immune response covering the humoral and the cellular arms of the adaptive immunity promoted by CAF01 + NG-34 group suggests that CAF01 could be a good candidate as an adjuvant to combine with NG-34 for an efficacious vaccine against IV. However, more studies performed in IV hosts as well as studies with a challenge model are further required. Introduction delivered [1]. This aspect makes the pursuit of a proper Purified antigens, mostly identified using in silico tools, adjuvant important when developing subunit vaccines. are the constituants of subunit vaccines. These types of The present study takes into account this aspect and vaccines own numerous advantages when compared with combines an influenza virus hemagglutinin antigen (a the conventional inactivated-type vaccines, for instance: conserved HA1-epitope NG-34) with four distinct adju- higher purity, greater safety (no need for special handling vants known to trigger different immune responses in of infectious viruses) and a quicker massive production. order to determine the optimal antigen/adjuvant combi- Nevertheless, soluble antigens are often poorly immu- nation that may function effectively against IV. nogenic and require an adjuvant when the antigen is Adjuvants are defined as substances whose main func- tion is to enhance the ability of the vaccine to induce an appropriate immune response in the absence of adverse *Correspondence: ayub.darji@irta.cat 1 IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA‑UAB), Campus effects. Their mechanisms of adjuvanticity can be catego- de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, rized accordingly to the criteria of O’Hagan and Valiante Spain [2], which classifies them depending on their delivery Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​ mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/publi​cdoma​in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Sisteré‑Oró et al. Vet Res (2020) 51:57 Page 2 of 11 system and their immunopotentiation. However, few specific antibodies as well as CD4 T cells in the pig adjuvants are currently licensed [3–5] and a long delay model has been documented. Recently, it has been exists in licensing new types. This is creating inconven- shown that NG-34 was effective as a pig influenza vac- ience for novel emerging vaccines, mainly since they can- cine in combating against a heterologous challenge by not utilize novel adjuvants focused on exploring novel reducing shedding and inducing neutralizing antibod- strategies, such as generating also robust cell-mediated ies against a SwH3N2 virus [11]. In poultry, another immune (CMI) responses [6–8]. study supported its cross-protective effect against an The seasonal updated Trivalent/Quadrivalent Inacti- H7 HPAIV strain [15]. AddaVax™, cationic adjuvant formulation 01 (CAF01) vated Vaccine (TIV/QIV) commercially available against The four adjuvants tested (Alhydrogel 2.0%, Forte®) were selected in view of their influenza viruses (IV), covers up to 90% of human vac- cines worldwide [9]. Lamentably, immune responses and Diluvac ­ induced are strain-specific and do not cope with possi- dissimilar immunological profiles and mechanisms ble mutations or probable new emerging strains caused of adjuvanticity (O’Hagan and Valiante [2]) summa- AddaVax™ (similar to M ­ F59®) are formulated in human by antigenic shift. For decades, the idea of developing a rized in Table 1. Among them, Alhydrogel 2.0% and universal vaccine which provides a broad-spectrum pro- ment stages [16]. Diluvac ­Forte®, however, is applied tection against a variety of IV has been pursued. In order registered vaccines while CAF01 is in clinical develop- to achieve this challenge, inducing antibodies by immu- nizing with conserved regions of the viral proteins has only in veterinary studies and has been widely utilized been investigated. Nonetheless, these epitope-based vac- in swine protein vaccines [17, 18]. However, knowing cines usually confer short-lived protection and are com- that pigs are also natural influenza hosts and regard- decided to include Diluvac ­Forte® as a suitable porcine promised compared to current licensed vaccines [10]. In ing its safety and its easy mass-vaccination delivery, we the present experimental study, we evaluated a suitable adjuvant candidate (novel and registered) immunologi- influenza adjuvant candidate. cally to be mixed with a well-studied HA-epitope by our Significant differences in the induction of humoral group [11], the NG-34. The study pursued a prototype for as well as cellular immune responses were only a universal vaccine, thus the murine model was used in observed when combinations of AH + NG-34 and preliminary research. CAF01 + NG-34 were used. However, a combination NG-34 peptide, predicted by Informational Spectrum of CAF01 + NG-34 was also effective in upregulating Methodology (ISM) [12, 13], is located within the site a wide array of cytokines as well as IgG2c antibodies, E in the N terminus of HA1 [14], in a domain close to representing the most complete immune response. the receptor binding site (RBS) of the HA, character- Nonetheless, further studies in influenza hosts are vital ized for being relatively conserved. Its role in inducing to evaluate the efficacy and effectiveness of the vaccine combination in natural IV infections. Table 1 Characteristics of each of the four adjuvants employed Adjuvant name Adjuvant type/ Immunological profile Mechanism of action Stage of development References chemical constituents (delivery system/ immunopotentiation) Alhydrogel® referred Aluminum salts (Mineral TH2, humoral Depot effect, delayed Both human and veteri‑ [47–51] as AH salts)/Aluminum clearance/Activate nary licensed vaccines hydroxide Nalp3 inflammasome AddaVax™ (Invivogen); similar to ­MF59® Oil-in-water emulsion/ TH2, humoral Enhance antigen pres‑ Licensed European [52–57] Squalene and poly‑ entation/Induce APC seasonal influenza sorbate mixture maturation vaccines CAF01 Particulate: Cationic lipo‑ TH1, ­TH17, humoral Protect antigen from Phase 1 [8, 19, 34, 58, 59] some/Dimethyldiocta‑ destruction/Induce decylamonium (DDA) APC maturation and α,α′-trehalose-6,6′- dibehenate (TDB) Diluvac Forte™ Oil-in-water emulsion/α- Humoral Enhance antigen pres‑ Approved in Animal [17, 18, 60, 61] tocopherol entation/Induce APC Health maturation TH = T helper; Nalp3 = NACHT, LRR and PYD domains-containing protein 3; APC = antigen presenting cells. Sisteré‑Oró et al. Vet Res (2020) 51:57 Page 3 of 11 Materials and methods CAF01 was freshly prepared in agreement with other Mice handling and experimental design studies and comprised: 250 µg/50 µg dimethyldiocta- Mouse experiments took place at Statens Serum Institut decylammonium (DDA)/trehalose 6,6 V-dibehenate (SSI) animal facilities. For this adjuvant testing research (TDB)/100 µL [8, 19]. study, a total of twenty-seven 7–8 week old female mice of the inbred strain C57BL/6 (Envigo, Huntingdon, UK) Cells, antigens and viruses were employed. Upon arrival, all mice were allowed to Madin-Darby Canine Kidney (MDCK, ATCC CCL- rest for 1 week previous to the first immunization. More- 34) cells were used in the seroneutralization assays. over, they were randomly distributed into six separate They were properly cultured in Dulbecco’s Modified cages according to the number of groups required in the Eagle Medium (DMEM) supplemented with 10% fetal experiment (groups A to F). Animals were provided food bovine serum (FBS), 1% penicillin/streptomycin and 1% and water ad libitum. l-glutamine. Six groups (group A to F) of n = 5 (in exception of NG-34 epitope predicted by ISM was synthesized by group A, n = 3 and group B, n = 4) were immunized twice GL Biochem Ltd, Shanghai, China. Its sequence cor- subcutaneously (s.c) at the base of the tail with a 21-day responds with 34 amino acids in the HA1 region from intervals. In group A, mice were not immunized (control the strain A/Catalonia/63/2009 (pH1N1) [GenBank group). In the rest of the groups (groups B to F), a dose ACS36215] [11]. of 15 µg/animal of conserved HA-epitope NG-34 was Purified HA from A/California/04/09(H1N1)pdm09 injected alone (group B) or adjuvanted with AH (group and from A/Aichi/2/1968(H3N2) were purchased from C), AddaVax (group D), CAF01 (group E) or Diluvac Sino Biological, Beijing, China (cat. no. 40340-V08B and Forte (group F) (Table 2). Three weeks after the second 11707-V08H, respectively). immunization, all animals were euthanized. SwH1N1 (A/swine/Spain/003/2010 H1N1 IV) [Gen- Sampling from each individual of the study was per- Bank JQ319725 and JQ319727] and SwH3N2 (A/swine/ formed 3 weeks after the second immunization, at day Spain/003/2010 H3N2 IV) [GenBank JQ319724 and 42 (termination day) when blood, spleens and inguinal JQ319726] were the viruses used. lymph nodes were collected. Organ preparation ­ orte® (Statens Serum Institut, Copen- Adjuvants and vaccine preparation Spleens and lymph nodes were treated as follows: they hagen, Denmark), AddaVax™ (Invivogen, Toulouse, CAF01, Diluvac F were forced through a 70 μM filter nylon mesh (BD Bio- sciences, San Jose, CA, USA), suspended in Roswell Park France) and aluminium hydroxide (Al(OH)3, Alhydrogel Memorial Institute (RPMI) media without FBS and fur- 2.0%) (Croda Biosector, Frederikssund, Denmark) were ther centrifuged for 5 min at 1800 rpm. At that point, the adjuvants employed. cells were resuspended in 1 mL using complete RPMI Mouse vaccines with an end volume of 200 µL were (cRPMI) (supplemented with HEPES, penicillin–strep- prepared by mixing the antigen (15 µg/animal/dose) dis- tomycin, sodium pyruvate, l-glutamine and non-essen- solved in Tris-buffer with 9% (w/v) trehalose, in a ratio tial amino acids) with 10% FBS (v/v). Finally, all samples of 1:1 with the adjuvant except for Alhydrogel 2.0% (each NucleoCounter® NC-100™ were counted using NucleoCassetes (ChemoMetec A/S, vaccine comprised 500 µg aluminum content/200 µL) AllerØd, Denmark) on the ­ [19, 20] and group A which was not immunized. (reagents and materials from ChemoMetec A/S, AllerØd, Denmark). Table 2 Representation of the six mice groups (Groups Immunoassays in mice for antigen‑specific serum A-F) antibodies Maxisorb Plates (Nunc) were coated with 2 µg/well of Group n Antigen Adjuvant Inoculation route either NG-34 epitope or HA: A/California/04/09 (H1) in buffer carbonate bicarbonate pH = 9.6 and left to A 3 – – – incubate overnight at 4 °C. The next day, the plates were B 4 NG-34 – s.c. blocked during 1 h 30 min at room temperature (RT) C 5 NG-34 Alhydrogel 2.0% s.c. using 2% BSA PBS solution. Mouse sera were analyzed D 5 NG-34 AddaVax™ s.c. in threefold dilution series in PBS with 1% BSA, start- E 5 NG-34 CAF01 s.c. ing with a 30-fold dilution. After a 2-hour incubating F 5 NG-34 Diluvac ­Forte® s.c. period at RT and three washes with 0.2%Tween20 PBS n= number of mice; s.c = subcutaneously. buffer, HRP-conjugated secondary antibodies (rabbit Sisteré‑Oró et al. Vet Res (2020) 51:57 Page 4 of 11 anti-mouse IgG1; Southern Biotech, Birmingham, AL, ­(B220+ ­IgD− ­CD38− ­GL7+) and T follicular helper cells USA; and IgG2c; Invitrogen, San Diego, CA, USA) were (TFH) ­(B220− ­CD4+ PD-1+ ­CXCR5+). The following added with 1%BSA PBS at dilutions: 1:20 000 (IgG1) or antibodies were mixed in PBS 1% FBS: GL7-FITC (BioLe- 1:5000 (IgG2c) and incubated during 1 h at RT. Reitera- gend GmbH, Koblenz, Germany; 144604), IgG1-PE (BD tively, the plates were washed and developed using 3, 3′, Pharmingen, 550083), B220-PerCP-Cy5.5 (BD Pharmin- 5, 5′-tetramethylbenzidine (TMB) substrate (Kem-En- gen, 552771), CD38-PE-CY7 (eBioscience, 25-0381) and Tec Diagnostics, Kuldyssen, Denmark). The reaction was IgD-BV786 (BD Pharmingen, 563618) to stain the B cells later stopped by adding 0.2 M H­ 2SO4 and the plates were in the GC. To stain the TFH cell population, the follow- read using a two-step fully automated ELISA (Hamiltion ing antibody panel was used: CD4-FITC (BD Pharmin- Starlet System, Switzerland) at 450–620 nm wavelength. gen, 553047); CD279(PD1)-PE (BD Pharmingen, 551892); CD45R(B220)-PerCP-Cy5.5 (eBioscience, 65-0865-14), Intra‑cellular flow cytometry staining for mice splenocytes CXCR5-BV421 (BD Pharmingen, 562889) and live/dead- stimulated in vitro with NG‑34 antigen EF780 (BD Pharmingen, 562889). After an incubation In splenocytes, the production of cytokines IFN-γ, TNF- period of 30 min at 4 °C, the cells were acquired on the α, IL-2 and IL-17 was assessed in the T cell population. FACS LSRII instrument using DIVA software (BD Bio- For that purpose, cells were stained similarly as described science, USA) and all the acquired data analyzed using in [21] but with few modifications. Briefly, splenocytes FlowJo software (Tree Star Inc.). Compensation beads ­(106 cells/well) were stimulated with 2 µg/mL of NG-34 (BD Pharmingen, USA) were used to compensate the peptide during 1 h at 37 °C and subsequently, incu- fluorophores. bated 6 h at 37 °C with brefeldin A (2.5 µg/mL) in order to block cytokine production. After this period, cells Mouse cytokine assay remained O/N at 4 °C. Additionally, from each group Splenocytes were seeded (2 × 105/well) and stimulated two pools were differentially made: not stimulated (nega- with the NG-34 epitope (5 µg/mL); media cRPMI (as tive control) or stimulated with PMA/ionomycin (posi- negative control) and concavalin A (1 µg/mL) (as posi- tive control). Next day, all cells were stained utilizing the tive control) (GE Healthcare, Marlborough, MA, USA). antibodies: CD4-BV786 (BD Pharmingen, San Diego, The supernatants were harvested after 72 h incubation CA, USA; 56331), CD44-FITC (eBioscience, Frankfurt, at 37 °C and a 9-plex panel MSD standard Th1/Th2/Th17 Germany; 11-0441), CD8-PerCpCy5.5 (eBioscience, which checks IFN-γ, TNF-α, IL- 5, IL-10, IL-13 and IL-17 45-0081). The cells were then permeabilized using fix/ cytokines together with two U-plex panels for IL-6 and perm (BD) and washed in permeabilization wash (BD). IL-1β detection were ran following the manufacturer’s Subsequently, the cells were stained with IL-2-APC- instructions (MSD, Rockville, MD, USA). Cy7 (BD Pharmingen, 560547), TNF-α-PE (eBioscience, 12-7321), IFN-γ-PE-Cy7 (eBioscience, 25-7311) and IL- Hemagglutination inhibition (HI) assay and serum 17a-APC (eBioscience, 17-7177). The cells were acquired neutralization test (SNT) on a FACS Fortessa instrument using DIVA software (BD Specific hemagglutination inhibition (HI) titers in mice Bioscience, USA) and all the data analyzed using FlowJo sera were determined as the reciprocal of the later dilu- software (Tree Star Inc.). Upon their acquisition, the cells tion of sera that inhibited hemagglutination. The protocol were gated following this pattern: live > singlets > lym- was adjusted to the recommendations provided by the phocytes > CD3 + > CD4 + versus CD8 + . Furthermore, World Organization for Animal Health (OIE) [22]. Sam- cytokine-producing cells (IFN-γ, IL-2, IL-17 and TNF-α) ples from each group were pooled and ran in duplicates. within the CD4 + CD44 high population were measured. A positive and negative reference serum (GD Animal Compensation beads (BD Pharmingen, USA) were used Health, Deventer, The Netherlands) were also included in to compensate the fluorophores. the assays to validate the technique. Specific seroneutralizing antibody titers were evalu- Flow cytometry staining in mice for cells in the inguinal ated. First, samples from each mice group were pooled, lymph nodes heat inactivated (56 °C for 30 min), diluted two-fold Cells from the inguinal lymph nodes were collected from with DMEM (supplemented with 1% penicillin/strep- all individuals; the cells were plated ­(106 cells/well) and treated with Mouse BD Fc Block™ (BD Pharmingen, tomycin and 1% l-glutamine) and incubated for 2 h at 37 °C with 100 T ­ CID50/well with the SwH1N1 (A/ 553142) to block non-antigen-specific bindings of immu- swine/Spain/003/2010 H1N1 IV) and SwH3N2 (A/ nogloblulins to Fcγ III and Fcγ II receptors. Two panels swine/Spain/003/2010 H3N2 IV) previously treated with of antibodies were used to stain cell populations in the porcine-trypsin (Sigma-Aldrich, St. Louis, MO, USA). germinal centers (GC) of the lymph nodes; GC B cells Later on, the mixture of sera-virus was transferred into Sisteré‑Oró et al. Vet Res (2020) 51:57 Page 5 of 11 pre-washed plates with confluent MDCK cells and kept Results at 37 °C with 5% ­CO2. After 7 days, the cytopathic effect Specific humoral immune responses after 2nd vaccination (CPE) was read and the titers were expressed as the IgG1 and IgG2c antibody responses were analyzed reciprocal dilution of serum that neutralized 100 ­TCID50 against the NG-34 epitope and against the complete of the challenged strain in 50% of the cultured replicates. HA from A/California/04/09 (H1N1) (Figure 1). NG34- Furthermore, in each plate there were added media con- specific IgG1 titers were higher in AH + NG-34 and trols (no virus), virus controls (no serum) and positive CAF01 + NG-34 groups, displaying statistically signifi- and negative reference sera from GD Animal Health, The cant differences (P < 0.05) when compared with the non- Netherlands. adjuvanted group (Figure 1A). Regarding NG34-specific IgG2 titers, the CAF01 + NG-34 mice group achieved the highest titers with statistically major significant differ- Statistical analysis ences (P < 0.0001) when compared to the non-adjuvanted For statistical analysis, Prism 6 software (GraphPad v6.01, group (Figure 1B). The rest of the adjuvanted groups San Diego, CA, USA) was used. One-way ANOVA or a (AH + NG-34, Addavax + NG-34, Diluvac Forte + NG34) non-parametric test (Friedman test) followed by Dunnett were barely inducing IgG2c titers. Furthermore, HA1- multiple comparisons test was performed (assigning as specfic IgG1 and IgG2c titers were also in concordance control group: group B, the non-adjuvanted group). Sta- with the NG-34 specific antibody titers (Figures 1C and tistically significant differences detected are illustrated by D). The AH + NG-34 group elicited high HA1-specific asterisks in each figure and explained in the correspond- antibody levels (P < 0.05) but only the CAF01 + NG34 ing figure legends. group achieved significant statistical differences for both Figure 1 Specific humoral responses elicited after immunizations with different adjuvant-NG34 combinations. Serum samples from individual mice from groups A–F were collected after second immunization and analyzed in serial dilutions for IgG1 and IgG2c titers. A NG34-specfic IgG1 and B IgG2c antibody titers and HA1-specific C IgG1 and D IgG2c antibody titers. Adjuvanted groups were compared to group B (the non-adjuvanted group) using a non-parametric test (Friedman test) followed by Dunnett’s post-test. *P < 0.05; **P < 0.01, *** P < 0.001, ****P < 0.0001. Sisteré‑Oró et al. Vet Res (2020) 51:57 Page 6 of 11 of the IgG1 (P < 0.001) and IgG2 (P < 0.01) antibody titers Supernatants of NG34-stimulated spleens harvested against the non-adjuvanted group (P < 0.01). after 72 h were analyzed with a multiplex system for dif- ferent cytokines like IFN-γ, TNF-α, IL-5, IL-10, IL-13 Specific cellular immune responses in the spleen and IL-17 (Th1/Th2/Th17 type). Similarly, IL-6 and IL-1β NG-34 antigen-specific cellular response was meas- levels were also evaluated (Figure 3). Statistically signifi- ured in the spleen. Intracellular CD4 and/or CD8 cant differences in IFN-γ (P < 0.001), TNF-α (P < 0.01), specific cytokines produced upon NG-34 stimula- IL-10 (P < 0.001), IL-17 (P < 0.001), IL-6 (P < 0.01) and tion was analyzed by flow cytometry. While in all the IL-1β (P < 0.001) cytokine release were only detected groups CD4 T cell response was observed, the CD8 T in the CAF01 + NG-34 group when compared to the cell response was merely present. In parallel, percent- non-adjuvanted group (Figures 3A, B, D, F–H). IL-5 ages of CD4 + CD44high T cells in NG34-stimulated in the AH + NG-34 group (Figure 3C) and IL-13 in the spleens were also determined. The AH + NG-34 and CAF01 + NG-34 group (Figure 3E) were detected at low CAF01 + NG-34 groups showed the highest percentages concentrations although no statistically significant differ- (P < 0.05) in comparison to group B (Figure 2A). Only the ences were observed in IL-5 and IL-13 among the groups. CAF01 + NG-34 mice group demonstrated significant differences in intracellular cytokine, IFN-γ (P < 0.001), Alteration in the inguinal lymph node cell population IL-2 (P < 0.05) and TNF-α (P < 0.01) produced by NG-34 in immunized mice stimulated spleens (Figures 2B–D). Additionally, IL-17 Differences in the number of cells present in the GC of producing cells were the highest in the CAF01 + NG-34 lymph nodes were detected. Significantly higher per- group although no statistical differences were observed centages of B cells (P < 0.005) were observed in the among different adjuvant/NG-34 combinations CAF01 + NG-34 group (Figure 4A). In contrast, no sta- (Figure 2E). tistical differences were observed in TFH cells, although Figure 2 Cellular responses in the spleens of immunized mice. NG34-stimulated spleen cells from individual mice (groups A–F) were collected and analyzed for cytokine production by flow cytometry. Percentages of A CD4 + CD44high, B CD4 + CD44high IFN-γ, C CD4 + CD44high IL-2, D CD4 + CD44high TNF-α and E CD4 + CD44high IL-17 (2) are represented. Sisteré‑Oró et al. Vet Res (2020) 51:57 Page 7 of 11 Figure 3 Cellular responses in the supernatants of NG-34 stimulated spleens of immunized mice. Harvested supernatants of NG34 stimulated spleens from individual mice from groups A-F were collected to determine the levels of A IFN-γ, B TNF-α, C IL-5, D IL-10, E IL-13, F IL-17, G IL-6 and H IL-1β. Adjuvanted groups were compared to group B (the non-adjuvanted group) using Ordinary One-way ANOVA test followed by Dunnett’s post-test. *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001. in CAF01 + NG34 and Diluvac Forte + NG-34 groups an Discussion increase in TFH was noticed (Figure 4B). Many immunogens/subunit antigens do not show strong immunogenicity, making the use of adjuvants necessary to reinforce their immune effect. In the present study, Obtained HI and SNT titers in mouse sera we focused on selecting the most suitable adjuvant that HI titers were negative in all mice sera for each of the could better aid immunogenically when administered in tested IV. SNT titers were either negative or low (1:10) combination with the NG-34 conserved HA-epitope. in CAF01 + NG-34 and Diluvac Forte + NG-34 groups Various groups of mice were either not immunized or against SwH1N1 virus. Against SwH3N2 virus, both Forte®. immunized with NG-34 epitope alone or combined with groups (CAF01 + NG-34 and Diluvac Forte + NG-34) adjuvants: AH, AddaVax, CAF01 and Diluvac ­ achieved a 1:40 titer (Table 3). Sisteré‑Oró et al. Vet Res (2020) 51:57 Page 8 of 11 Figure 4 Changes in inguinal lymph node population. Lymph nodes samples from individual mice (groups A-F) were collected and analyzed by flow cytometry A the percentage of B cells in the GC (B) the percentage of TFH. Adjuvanted groups were compared to the non-adjuvanted group using Ordinary One-way ANOVA test followed by Dunnett’s post-test. *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001. Table 3 SNT titers obtained from the six mice groups when using MF59 adjuvant in IV vaccines [25, 26]. Per- (groups A-F) against SwH1N1 and SwH3N2 isolates haps, MF59-like adjuvants (Addavax) function bet- Group Adjuvant SNT titers ter when the complete HA or IV strain are included in the vaccine formulation [27, 28]. Furthermore, the Against SwH1N1 Against SwH3N2 Addavax + NG-34 group did not boost Th1 and Th2 as demonstrated in other studies [25, 26]. In contrast, our A – – – results were concordant with results using CAF01 com- B – – – bined with TIV, which conferred protection in an IHA- C Alhydrogel 2.0% – – independent manner [29]. D AddaVax™ – – To achieve cross-protection between heterolo- E CAF01 1:10 1:10 gous strains, an appropriate T-cell response seems F Diluvac ­Forte® 1:40 1:40 to be necessary [30]. In this work, the highest cel- SNT = serum neutralization test. lular responses were exhibited by the AH + NG-34 and CAF01 + NG-34 groups (Figure 2A). The results obtained in the AH + NG-34 partly correlated with Interestingly, mice vaccinated with CAF01 + NG-34 elic- the trend for a predominant Th2 differentiation and ited specific humoral and cellular immune responses to Th2-type cytokine profile, usually promoted by alu- the conserved NG-34 HA-epitope (Figures 1 and 2), a minum salts [31, 32]. Statistically significant increase dual immune induction widely considered ideal for an IV in CD4 CD44high population and highest IFN- γ, vaccine. IL-2 and TNF-α responses (Th1-type cytokines) were The AH + NG-34 group induced a polarized Th2-type observed in mice from CAF01 + NG34 (Figures 2A response leading to significantly higher NG34-specific and C) as also reported in other studies [6, 19, 33, IgG1 titers (Figures 1A and 2A), and CAF01 combined 34]. Significantly higher titers of IFN-γ, TNF-α, IL-10, with NG-34, promoted the highest levels of IgG1 (Fig- IL-17, IL-6 and IL-1β were also observed in the NG34- ure 1A). The CAF01-NG-34 group also induced an IgG2c stimulated spleen cell supernatants obtained from the response, being the only peptide/adjuvant combination CAF01 + NG-34 vaccinated animals (Figures 3A, B, D, capable of inducing such a response (Figure 1B) which F–H). These results could be associated with the role is in line with other studies published using other anti- of CAF01 in inducing Th1-cytokines and also with the gens and CAF01 [8, 20]. These induced IgG2a/c titers property of one of the CAF01 compounds (the TDB) are regarded as an important component for IV vaccines which leads to the secretion of IL-1β, IL-6 and TNF-α since they mediate protection when virus neutralizing [35]. Furthermore, the rise in IL-17 levels of the mice titers are absent [23, 24]. from CAF01 + NG-34 (Figure 3F) could be related to Strong HI titers were not induced by any of the the Th17 response that CAF01 promotes through the evaluated epitope/adjuvant combinations. Only TLR-independent Syk/Card9-dependent pathway [36] CAF01 + NG-34 and Diluvac Forte + NG-34 groups dis- (Figure 2). Other cytokines not from a Th1/Th17 profile played SNT titers albeit at low levels (Table 3). These were also boosted (IL-10 and IL-6) by CAF01 + NG-34 results did not correlate with the high HI titers induced Sisteré‑Oró et al. Vet Res (2020) 51:57 Page 9 of 11 vaccinated mice (Figures 3D and G). IL-10 induction TDB: trehalose 6,6 V-dibehenate; TFH: T follicular helper cells; Th: T helper; TMB: 3, 3′,5,5′-tetramethylbenzidine; TIV: trivalent inactivated vaccine. could be related to the CD4 TFH cells which might pro- mote B cell proliferation, survival, and differentiation Acknowledgements into antibody-secreting plasma cells [37–42]. Authors thank the animal caretakers’ from SSI animal facilities and Janne Rabech and Rune Fledelius Jensen for their dedication and technical assis‑ The mice immunized with CAF01 + NG-34 displayed tance provided during the experiments. Authors also show gratitude to CESAC the greatest percentages of B cells population in the GC in (Centre de Sanitat Avícola de Catalunya, Reus, Spain) personnel for supplying the draining lymph nodes (Figure 4A). Other studies have the chicken red blood cells (cRBCs) for the HI assays. reported CAF01 as an enhancer of GC reaction in com- Authors’ contributions parison to other adjuvants such as AddaVax, aluminum MSO, GKP, DC and AD planned and designed the study, interpreting the final hydroxide and CpG ODN 1826 [43] and recognized results. DC and AD obtained its funding; MSO wrote the manuscript with detailed inputs and revisions from GKP, DC and AD. MSO and GKP prepared that this reaction is primordial for generating plasma the vaccines, sampled the organs, developed the immunoassays, acquired and memory B cells [44]. In terms of the percentage of the data from the flow cytometry and performed the mouse cytokine assays. TFH, the highest percentage mean was achieved by the MSO, SLS and LC performed and analyzed the HI and SNT data. MSO and GKP performed the statistical analysis. All authors read and approved the final Diluvac Forte + NG-34 but followed immediately by the manuscript. CAF01 + NG-34 group (Figure 4B). Although not statis- tically significant, these data are of great relevance since Funding The present work was developed under the AGL2013-48923-C2-2-R project the TFH cells promote long-lasting humoral immunity funds supported by Ministerio de Economía y Competitividad (MINECO)/ arising from the GC. CAF01 has been reported to have Spanish Government and the grants supported to M.S.O (scholarship n°: a more than a year duration inducing CMI responses due BES-2014-068506 and EEBB-I-17-12462). Also, financial support was received from TRANSVAC (730964). Funding from CERCA Programme/Generalitat de to its DDA compound which forms a depot at the injec- Catalunya to IRTA is also acknowledged. The funders had no role in study tion site [45]. design, data collection and analysis, decision to publish, or preparation of the The TDB component of CAF01 is essential for the manuscript. induction of CMI responses and that it is recognized Availability of data and materials via the C-type lectin receptor MINCLE (CLEC4E). It The data supporting the conclusions of this article is attached within the is important to note that there may be species-specific article and its additional files. differences in recognition and activation of this MIN- Ethics approval and consent to participate CLE receptor. It is well known that CAF01 is a promis- Mouse experiments took place at SSI animal facilities. Vaccination and sam‑ ing adjuvant in humans, inducing both antibody and plings developed strictly complied with the Danish legislation based on EU Directive 63/2010 and the conditions set for the animal experiment permis‑ CMI responses [16, 34] and also that CAF01 combined sion F2440 issued by The Animal Experiments Inspectorate. Animal Research: with Chlamydia trachomatis fusion proteins (Hirep1 and Reporting of In Vivo Experiments (ARRIVE) guidelines were also considered. CTH93) induce strong CMI responses in Göttingen min- Competing interests ipigs [46]. Nevertheless, whether TDB is also an effec- The authors declare that they have no competing interests. tive immunostimulator in poultry remains to be assessed and further studies are therefore required to develop the Author details 1 IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA‑UAB), Campus de la CAF01 + NG-34 as a vaccine for poultry. Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. 2 Virus As a first step, this study may provide insights into what Research and Development Laboratory, Department of Virus and Microbio‑ may be occurring when administering CAF01 + NG-34 logical Special Diagnostics, Statens Serum Institut, Artillerivej 5, 2300 Copen‑ hagen S, Denmark. to influenza hosts (such as pig and poultry). With regards to the released umbrella of cytokines as well as antibod- Received: 8 October 2019 Accepted: 17 February 2020 ies induced, CAF01 ought to be a potential adjuvant to be mixed with the conserved NG-34 epitope. References Abbreviations 1. Di Pasquale A, Preiss S, Tavares Da Silva F, Garçon N (2015) Vaccine adju‑ AH: aluminium hydroxide; ADCC: antibody-dependent cellular cytotoxicity; vants: from 1920 to 2015 and beyond. Vaccines 3:320–343 ARRIVE: Animal Research Reporting of In Vivo Experiments; BSA: bovine serum 2. 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