The authors have declared that no competing interests exist.
Conceived and designed the experiments: JRC JLA LPV CV BV PFS MAP AM. Performed the experiments: JRC JLA BV. Analyzed the data: JRC JLA LPV MAP AM EO. Contributed reagents/materials/analysis tools: JRC JLA BV EO. Wrote the paper: JRC JLA PFS MAP AM.
Fasciolosis is considered the most widespread trematode disease affecting grazing animals around the world; it is currently recognised by the World Health Organisation as an emergent human pathogen. Triclabendazole is still the most effective drug against this disease; however, resistant strains have appeared and developing an effective vaccine against this disease has increasingly become a priority. Several bioinformatics tools were here used for predicting B- and T-cell epitopes according to the available data for
Fasciolosis is one of the most important helminthiasis worldwide affecting grazing livestock due its widespread geographical distribution and resulting economic loss; it is caused by the common liver fluke
It is well-known that methodological and technical difficulties related to diagnosis have limited progress in combating human fasciolosis globally, including drawbacks in diagnosing infection and assessing drug efficacy and resistance, mainly concerning triclabendazole which is still the most effective drug for combating the disease. Indeed, no commercial vaccine is currently available and developing vaccines for controlling animal and human fasciolosis thus represents a tremendous research opportunity. Many candidate proteins have been tested for a long time now as target antigens in vaccination assays against fluke, including fatty acid-binding proteins, glutathione S-transferases, cathepsin proteases, leucine aminopeptidase, fluke haemoglobin and thioredoxin peroxidase. However, no consensus regarding the factors required for immunological protection has yet emerged and there has been no report to date of a successful field trial concerning a liver fluke vaccine
Public access to an increasing number of pathogen genomes which have been totally or partially sequenced, along with the use of powerful
As epitope-based vaccines only contain small sequences derived from an entire protein known to bind to various major histocompatibility complex (MHC) molecules, predicting peptide-MHC binding and mapping epitopes are crucial in their design
Synthetic peptides have been examined as potential prophylactic vaccines against viral, bacterial and parasitic diseases for many years now
Several peptides have thus been chemically-synthesised and then assessed using
The animal procedures in this study complied with Spanish (Real Decreto RD53/2013) and European Union (European Directive 2010/63/EU) guidelines regarding animal experimentation for the protection and humane use of laboratory animals, and were conducted at the University of Salamanca’s accredited Animal Experimentation Facilities (Register number: PAE/SA/001). University of Salamanca’s Ethics Committee approved procedures used in the present study (protocol approval number 48531). Seven-week-old female BALB/c and CD1 mice (Charles River Laboratories, Barcelona, Spain) weighing 20 to 22 g were used for the experiments. Animals were kept in plastic boxes with food and water
Due to a lack of data regarding the
The BepiPred method was used for predicting linear B-cell epitopes
The SYFPEITHI database
All derived peptides selected on the basis of B- or T-cell epitope predictions for each protein were chemically synthesised (Fundación Instituto de Inmunología, FIDIC, Colombia) by the solid-phase peptide synthesis according to the methodology first described by Merrifield
The J774.2 mouse peritoneal macrophage cell line was used in this study; it was grown in RPMI-1640 culture medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin, at 37°C in humidified 95% air and 5% CO2. J774.2 peritoneal macrophage cells were plated in complete RPMI-1640 culture medium at 1×106 cells/well concentration in 12-well culture plates (Costar, Cambridge, MA), and left to adhere for 2 h at 37°C in 5% CO2. Non-adhering cells were removed by gentle washing with complete RPMI-1640 culture medium. Adherent J774.2 cells were incubated with each synthetic peptide at different concentrations (1–100 µg/mL). After 48 h incubation at 37°C in 5% CO2, supernatants were removed and cell viability was measured on adhered cells by MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) assay, measuring the absorbance at 540 nm. Controls for checking solvent cytotoxicity were also included.
One hundred and sixty-eight female BALB/c mice (56 groups of 3 mice per group) were used in this study. The immune response in mice was studied in two separate experiments (A and B). Experiment A: group 1 (untreated control group; n = 6), group 2 (ADAD and natural immunomodulator PAL [ADADn]; n = 6), groups 3–14 (ADADn together with B1–B12 B-epitope-containing peptides; n = 36) and groups 15–26 (ADADn together with T13–T24 T-epitope-containing peptides; n = 36). Experiment B: group 1 (untreated control group; n = 6), group 2 (ADAD and synthetic immunomodulator AA0029 [ADADs]; n = 6), groups 3–14 (ADADs together with B1–B12 B-epitope-containing peptides; n = 36) and groups 15–26 (ADADs together with T13–T24 T-epitope-containing peptides; n = 36). The mice were subcutaneously immunised using an adjuvant adaptation (ADAD) system
Briefly, the ADAD vaccination system included the vaccine antigen, an immunomodulator (natural or chemically-synthesised), together with non-haemolytic adjuvant
Mice were humanely euthanised 2 weeks after third immunisation. The spleen was aseptically removed during necropsy to obtain splenocytes for
Sera from mice immunised with the aforementioned formulations were analysed by ELISA for measuring total IgG, IgE and IgM levels as well as IgG1 and IgG2a antibody isotype levels. Briefly, 96-well polystyrene microplates (Costar, Corning Costar Corp, Cambridge, Mass) were coated with 1 µg of each peptide in carbonate buffer pH 9.6 (100 µL per well) and incubated overnight at 4°C. The plates were then washed thrice for 5 min with PBS containing 0.05% Tween 20 (PBST). The plates were blocked with 5% skimmed milk (SM) in PBST (200 µL per well) for 1 h at 37°C and then washed again thrice, as described above. Sera samples were appropriately diluted at 1∶100 in dilution buffer (5% SM and PBST) and added to the wells (100 µL per well) in duplicate. After 1 h incubation at 37°C the plates were washed as described above and, according to each assay, goat peroxidase-conjugated anti-mouse IgG, IgE, IgM, IgG1 and IgG2a (1∶1,000 in dilution buffer, 100 µL per well; Sigma) were incubated for 1 h at 37°C. After washing as above, the bound antibodies were detected using H2O2 (0.012%) and ortho-phenylenediamine (0.04%) in 0.1 M citrate/phosphate buffer (100 µL per well). The enzyme reaction was stopped after 15–20 min by adding 3N H2SO4 (100 µL per well) and optical density was measured at 550 nm (OD550) on an Ear400FT ELISA reader (STL Lab Instruments, Groding, Austria). The mean absorbance values for each mouse serum from each group were determined and included in each data point.
The frequencies of antigen specific IFN-γ, IL-1α, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, TNF-α producing T-cells in the spleens were quantified by using a flow cytometry-based methodology. Individual mouse splenocytes were cultured in 6-well plates at 1×106 cells per well concentration in complete medium (RPMI 1640 medium containing 10% heat-inactivated foetal bovine serum and antibiotics, 100 U/mL penicillin and 100 µg/mL streptomycin) and stimulated with each synthetic peptide at final 10 µg/mL concentration for 72 h at 37°C in a humidified atmosphere with 5% CO2. Control wells were prepared containing untreated mouse splenocytes. After the incubation period, splenocyte culture supernatants were recovered for cytokine determination. A FlowCytomix Mouse Th1/Th2 10plex kit (Bender MedSystems GmbH, Vienna, Austria) was used, according to the manufacturer’s instructions. Briefly, different sized fluorescent beads coated with capture antibodies specific for the cytokines mentioned above were incubated with splenocyte supernatant cell culture samples to form sandwich complexes with phycoerythrin (PE)-conjugated secondary antibodies. Flow cytometry data were collected using a FACSCalibur flow cytometer (BD Biosciences) at the University of Salamanca’s Flow Cytometry Central Service. A total of 8,000 events were collected gated by forward and side scatter and data were analysed using FlowCytomix Pro 3.0 software (Bender MedSystems, Vienna, Austria). Each cytokine concentration was determined from standard curves using known concentrations of mouse recombinant cytokines.
An ELISA assay was used for evaluating the presence of each B-cell epitope using sera from infected mice. Briefly, plates were coated with 1 µg of either each synthetic peptide or
A flow cytometry analysis was performed on stained and fixed splenocytes to investigate T-cell populations responding to the immunisation of mice with T-cell epitope-containing peptides. Regarding immunofluorescence staining, 5×105 cells were incubated with fluorescein isothyosanate (FITC)-conjugated mouse monoclonal antibodies (mAb) against CD4, or with phycoerythrin (PE)-conjugated mouse mAb against CD8, or with allophycocyanin (APC)-conjugated mouse mAb against CD62L. All samples were incubated with anti CD16/CD32 blocking monoclonal antibody for 5 min at room temperature. Each specific antibody (BD Biosystems) was incubated in 1/50 dilution factor in PBS plus 2% foetal calf serum (PBS-FCS) for 30 min at 4°C. The cells were washed with PBS-FCS after the incubation period, spun at 1,200 rpm for 5 min and the supernatant discarded. Splenocytes were fixed with 100 µL of a solution containing 2% p-formaldehyde in PBS for no longer than 12 h at 4°C before data acquisition. Data was collected using a FACSCalibur flow cytometer (BD Biosciences) at the University of Salamanca’s Flow Cytometry Central Service. A total of 30,000 events were collected (gated by forward and side scatter) and data were analysed using Gatelogic Flow Cytometry Analysis Software (Inivai Technologies Pty Ltd).
Based on the immune response induced by each of the peptides assayed, those inducing the following immunological patterns were selected for the
All the animals included in this study (except untreated controls) were orally infected with 7
An initial descriptive analysis was made of each group of mice, exploring cytokine and immunoglobulin patterns and cell expression. A linear model with interaction was used to evaluate cytokine and cell expression, including two factors: epitope type (B or T) and the immunomodulator used (AA0029 or PAL). Interactions were plotted for representing this linear model using Plotrix package in R. Furthermore, groups of peptides were compared using a Kruskall Wallis test followed by a Dunn multiple comparisons test. Differences having p<0.05 were considered statistically significant. The results were reported as the mean of each group and the standard deviation (SD). SPSS 20.0 software (SPSS Inc., USA) was used for data analysis. Hierarchical clustering was used to identify sets of cytokines whose expression levels correlated among peptides with B- and T-cell epitopes. Centring and scaling are the previous data transformation steps. The complete linkage clustering method was used, based on a similarity matrix derived from Pearson (rows) and Spearman (columns) moment correlations. The heatmap was visualised using the heatmap.2 function of gplots package in R. Kaplan-Meier survival curves were used for evaluating survival rates.
A total of 269 reported
Epitope | Synthesised peptide sequence | Position | GenBank | Description | Proteinlength (aa) |
B1 | KGAGSSQDACIKFIQYEVDG | 63–82 | AAB02579.1 | Amoebapore homologue | 102 |
B2 | KGAGSSQDATIKFIQYEVDG | 63–82 | AAB02579.1 | Amoebapore homologue | 102 |
B3 | FASFDVPSKQPTIDIDLCDI | 14–33 | AAF88069.1 | Amoebapore-like protein | 101 |
B4 | FASFDVPSKQPTIDIDLTDI | 14–33 | AAF88069.1 | Amoebapore-like protein | 101 |
B5 | ISEIRDQSSTSSTWAVSSAS | 102–121 | ABU62951.1 | Cathepsin B | 337 |
B6 | GVENGVKYWLIANSWNEGWG | 293–312 | ABU62951.1 | Cathepsin B | 337 |
B7 | QTCSPLRVNHAVLAVGYGTQ | 260–279 | AAB41670.2 | Secreted cathepsin L1 | 326 |
260–279 | AAA29136.1 | Cathepsin | 326 | ||
264–283 | AAP49831.1 | Cathepsin L | 326 | ||
260–279 | Q24940.1 | Cathepsin L-like proteinase | 326 | ||
B8 | QTTSPLRVNHAVLAVGYGTQ | 260–279 | AAB41670.2 | Secreted cathepsin L1 | 326 |
260–279 | AAA29136.1 | Cathepsin | 326 | ||
264–283 | AAP49831.1 | Cathepsin L | 326 | ||
260–279 | Q24940.1 | Cathepsin L-like proteinase | 326 | ||
B9 | YTEPRSVTPEERSVFQPMIL | 27–46 | AAV68752.1 | Cystatin | 116 |
B10 | FVPLYSSKSATSVGTPTRVS | 95–114 | AAV68752.1 | Cystatin | 116 |
B11 | VTTNGPPNGKHNDKHTYVEC | 350–369 | CAC85636 | Legumain-like | 419 |
B12 | VTTNGPPNGKHNDKHTYVET | 350–369 | CAC85636 | Legumain-like | 419 |
T13 | TVNLVKRLLQNSVVE | 37–51 | AAB02579.1 | Amoebapore homologue | 102 |
T14 | DYIIDHVDQHNATEI | 80–94 | AAF88069.1 | Amoebapore-like protein | 101 |
T15 | DRNTQRQTVRYSVSE | 69–83 | ABU62925.1 | Cathepsin B | 337 |
T16 | FYMFEDFLVYKSGIY | 260–274 | ABU62925.1 | Cathepsin B | 337 |
T17 | KYLTEMSRASDILSH | 83–97 | AAB41670.2 | Secreted cathepsin L1 | 326 |
83–97 | ABQ95351.1 | Secreted cathepsin L2 | 326 | ||
83–97 | AAA29136.1 | Cathepsin | 326 | ||
83–97 | AAP49831.1 | Cathepsin L | 326 | ||
83–97 | AAR99518.1 | Cathepsin L protein | 326 | ||
83–97 | Q24940.1 | Cathepsin L-like proteinase | 326 | ||
T18 | ISFSEQQLVDTSGPW | 153–167 | AAB41670.2 | Secreted cathepsin L1 | 326 |
153–167 | AAA29136.1 | Cathepsin | 326 | ||
153–167 | AAP49831.1 | Cathepsin L | 326 | ||
153–167 | BAA23743.1 | Cathepsin L | 325 | ||
153–167 | AAR99518.1 | Cathepsin L protein | 326 | ||
153–167 | AAT76664.1 | Cathepsin L1 proteinase | 326 | ||
153–167 | Q24940.1 | Cathepsin L-like proteinase | 326 | ||
T19 | ENAYEYLKHNGLETE | 178–192 | AAC47721.1 | Secreted cathepsin L2 | 326 |
178–192 | ABN50361.2 | Cathepsin L | 326 | ||
178–192 | CAA80446.1 | Cathepsin L-like protease | 326 | ||
53–67 | CAA80445.1 | Cathepsin L-like protease | 166 | ||
T20 | LDPYFNLVSPEVYNY | 29–43 | BAE44988.1 | Cytochrome oxidase subunit I | 145 |
29–43 | BAE45005.1 | Cytochrome oxidase subunit I | 145 | ||
T21 | DLNLPRLNALSAWLL | 76–90 | BAE44988.1 | Cytochrome oxidase subunit I | 145 |
76–90 | BAE45005.1 | Cytochrome oxidase subunit I | 145 | ||
T22 | FAGHGKAYLHGSFDK | 56–70 | AAA29144.1 | Vitelline protein B2 | 272 |
T23 | YEKYEDDYARETPYD | 254–268 | AAA29144.1 | Vitelline protein B2 | 272 |
254–268 | AAA29143.1 | Vitelline protein B1 | 272 | ||
T24 | 101–115 | AAA31753.2 | NADH dehydrogenase subunit 3 | 118 | |
101–115 | Q34522.1 | NADH dehydrogenase subunit 3 | 118 | ||
101–115 | AAG13152.2 | NADH dehydrogenase subunit 3 | 118 |
GenBank accession number and amino acid sequences from each B- and T-cell chemically synthesised epitope are also indicated.
Each peptide was assayed in four different concentrations ranging from 1 to 100 µg/mL for
Immunomodulator (PAL and AA0029), epitope (B or T) and peptide effect on antibody levels were studied. Regarding the immunomodulator effect, it was observed that using PAL induced higher IgG levels than AA0029 ([0.5267]
The bottom and the top of the box indicate the 25th and 75th percentiles, respectively. A). Mice immunised using PAL. B). Mice immunised using AA0029.
A). IgG1 related to IgG. B). IgG2a related to IgG. Red indicates the PAL immunomodulator and blue AA0029. Circles and squares represent B- and T-peptides, respectively.
There were no statistical differences regarding IgE levels when either of the immunomodulators was used. Furthermore, B-epitope-containing peptides showed a stronger IgE response than T-epitope-containing peptides ([0.1588]
The type of immunomodulator and epitope used in the immunisation trial influenced cytokine levels, as can be seen in the three-dimensional scatterplots in
Each peptide was also individually analysed for cytokine quantitation after being injected into the mice using the ADAD vaccination system. The following relationships were described: IL-4 and IL-10 levels (
A). IL-4 related to IL-10. B). IL-5 related to IL-10. Red indicates PAL and blue AA0029. Circles and squares represent B- and T-peptides, respectively.
A). Mice immunised with PAL. B). Mice immunised with AA0029.
A). Mice immunised with PAL. B). Mice immunised with AA0029.
Although analysing individual cytokines identified peptides associated with different cytokine level patterns, such associations did not represent the relationship between the peptides or cytokines included in this study. Two-dimensional cluster analysis was therefore performed to identify sets of cytokines that might have been coordinately expressed induced by immunisation with peptides having a low immune response (compared to a high one) and more strongly correlate them with an effective immune response. Bicluster analysis led to a comprehensive representation of splenocyte state throughout their response to peptides used in this study. The expressed cytokines’ functional concordance gave biological significance to the broad patterns seen in images like the biclusters in
The right-hand margin provides the names of peptide sets. Rows and columns represent clusters of interleukins and peptides having a similar immunological response. A list of cytokines grouped within each cluster is also provided. Groupings having shorter distances (as indicated by the distance to k-means nearest group) had greater similarity. Bicluster analysis for B- and T-cell peptides formulated with the PAL immunomodulator. Five major clusters can be discerned (1, 2, 3, 4 and 5), encompassing peptides having similar cytokine levels.
The right-hand margin provides the name of peptide sets. Rows and columns represent clusters of interleukins and peptides having a similar immunological response. A list of cytokines grouped within each cluster is also provided. Groupings having shorter distances (as indicated by the distance to k-means nearest group) had greater similarity. Bicluster analysis for B- and T-cell peptides formulated with the AA0029 immunomodulator. Seven major clusters can be discerned (1, 2, 3, 4, 5, 6 and 7), encompassing peptides having similar cytokine levels.
Analysis of B-epitopes clustered with the PAL adjuvant (
An ELISA assay was used for evaluating the recognition of peptides containing predicted B-cell epitopes using sera from mice infected with
A). B-cell peptides formulated with PAL. B). B-cell peptides formulated with AA0029.
Three lymphocyte subsets (LT CD4+, LT CD8+ and LT CD62L+) were analysed to better evaluate the cellular immune response of T-cell epitope-containing synthetic peptides formulated with both the PAL and/or AA0029 immunomodulators. No statistically significant differences were detected in either LT CD4+ or LT CD8+ immunophenotypes in mice immunised with the T-cell epitope-containing peptides. However, differences in the CD62L+ immunophenotype were detected in mice immunised with peptides containing T-cell epitopes formulated in either of the immunomodulators (PAL or AA0029) when compared to the non-immunised control group. Different lymphocyte expression patterns were observed when the peptides were individually analysed.
A). T-cell peptides formulated with PAL. B). T-cell peptides formulated with AA0029.
Immunising mice with the synthetic peptides enhanced their survival rate, compared to the unimmunised and infected control group. All mice in the unimmunised and infected control group died between 24 and 34 days pi; mice which died before day 42 pi were considered unprotected in our study.
A). Mice immunised with single peptides containing B-epitopes. B). Mice immunised with single peptides containing T-epitopes. Survival rates of mice belonging to both untreated and infected controls are also represented. Human endpoint was established when an indicator of severe pain, excessive distress, suffering or an impending death was observed in any of the animals and then euthanised with an intraperitoneal injection of pentobarbital at 60 mg/kg using 30 g needles.
Immunising mice with the peptide B5 (group 6) produced the lowest fluke burden compared to the unimmunised and infected control group ([0.7±0.3]
Group | Treatment | Number of flukes in individual mice | Worm recovery (Mean ± SEM) | Reduction (%) | Hepatic lesion in individual mice | Lesion score (Mean ± SEM) | Reduction (%) |
1 | Untreated uninfected | ||||||
2 | Infected | 3, 1, 2, 2, 2 | 2.0±0.3 | 14, 14, 12, 10, 10 | 12.0±1.5 | ||
3 | Adjuvant treated | 0, 2, 0, 2, 2, 0 | 1.0±0.4 | 50 | 10, 14, 5, 11, 14, 2 | 9.3±1.8 | 22 |
4 | B1 | 1, 0, 1, 2, 1, 1, 1 | 1.0±0.2 | 50 | 10, 12, 6, 11, 6, 9, 11 | 9.3±1.5 | 23 |
5 | B2 | 0, 1, 1, 1, 1, 1 | 0.8±0.2 | 58 | 6, 14, 5, 9, 9, 5 | 8.0±1.3 | 33 |
6 | B5 | 1, 0, 1, 0, 2, 0 | 0.7±0.3 | 67 |
4, 1, 12, 12, 7, 8 | 7.3±2.1 | 39 |
7 | B6 | 2, 0, 2, 1, 0, 1 | 1.0±0.3 | 50 | 13, 2, 8, 10, 14, 13 | 10.0±1.7 | 17 |
8 | T14 | 0, 1, 2, 1, 1, 0, 1 | 0.9±0.3 | 57 | 4, 7, 10, 13, 14, 9, 10 | 9.6±1.8 | 20 |
9 | T15 | 0, 2, 0, 1, 2, 0 | 0.8±0.4 | 58 | 6, 14, 5, 4, 14, 10 | 8.8±1.7 | 26 |
10 | T16 | 1, 0, 0, 1, 2, 1, 1 | 0.9±0.3 | 57 | 10, 10, 11, 5, 13, 12 14 | 10.7±1.1 | 11 |
*p<0.05 compared to infected controls.
The present study was designed to bioinformatically predict and experimentally assess both the immune response and protection-inducing ability of
A prolonged Th2 immune response to
Cytokine quantitation in stimulated-splenocyte supernatant cell cultures has indicated that B- or T-cell epitope-containing peptides have been able to induce high levels of Th1 and Th2-associated immune response. Peptides inducing high IL-17 levels were also found in the present study. These results were consistent with those from other immunisation trials, resulting in the generation of cytokines involved in a mixed Th1/Th2 immune response
The specific role of CD4+ and CD8+ T-cells in protection against
Most recent work aimed at developing an effective vaccine against
The present study’s findings have highlighted the immunoprophylactic potential of B- and T-cell epitope-containing synthetic peptides (B2, B5, B6 and T15) formulated in the ADAD vaccination system in a murine model. However, it should be noted that a deeper knowledge of the host-parasite interactions, and understanding the molecular and immunological mechanisms involved in inducing a protective response, might be crucial for better selecting the most appropriate vaccine formulation. Further studies aimed at studying the protection-inducing ability of the aforementioned peptides when formulated in combination, and tested in natural fasciolosis models, are in need.
(TIF)
IgE antibody level detection in mice immunised with the synthetic peptides throughout the immunisation schedule. Data presented as box plots with the bottom and the top of the box indicating the 25th and 75th percentiles, respectively. A). Peptides formulated with the PAL immunomodulator. B). Peptides formulated with AA0029.
(TIF)
IgM antibody level detection in mice immunised with synthetic peptides throughout the immunisation schedule. Data presented as box plots with the bottom and the top of the box indicating the 25th and 75th percentiles, respectively. A). Peptides formulated with the PAL immunomodulator. B). Peptides formulated with AA0029.
(TIF)
Three-dimensional scatterplots represents cytokine levels induced by immunisation of mice with peptides containing B-cell epitopes. The Z-axis represents IFN-γ, IL-4, IL-10 and IL-17 levels for Figure A, B, C and D, respectively. The x and y axis represent CD197 and CD27 memory T-lymphocytes for each Figure. Blue indicates the use of AA0029 and green indicates PAL.
(TIF)
Three-dimensional scatterplots represents cytokine levels induced by immunisation of mice with peptides containing T-cell epitopes. The Z-axis represents IFN-γ, IL-4, IL-10 and IL-17 levels for Figure A, B, C and D, respectively. The x and y axis represent CD197 and CD27 memory T-lymphocytes for each Figure. Blue indicates the use of AA0029 and green indicates PAL.
(TIF)
Interaction plot for regulatory (A. IL-10), Th2 (B. IL-5, C. IL-4), Th17 (D. IL-17), Th1 (E. IL-2, F. IFN-γ) and innate inflammatory cytokine levels (G. IL-6, H. TNFα, I. IL-1α) elicited by epitope effect (B & T) and immunomodulator effect (AA0029 & PAL).
(TIF)
We would like to thank Magnolia Vanegas (Fundación Instituto de Inmunología de Colombia - FIDIC) for peptide synthesis, Dr Arturo San Feliciano (Universidad de Salamanca) for kindly providing us with AA0029, Dr Antonio Martínez-Fernández (Universidad Complutense de Madrid) for supplying PAL and Montanide, and Jason Garry for correcting the manuscript.