Targeting the Mincle and TLR3 receptor using the dual agonist cationic adjuvant formulation 9 (CAF09) induces humoral and polyfunctional memory T cell responses in calves
Targeting the Mincle and TLR3 receptor using the dual agonist cationic adjuvant formulation 9 (CAF09) induces humoral and polyfunctional memory T cell responses in calves
Aneesh Thakur 0 1 2
Athina Andrea 0 2
Heidi Mikkelsen 0 1 2
Joshua S. Woodworth 0 2
Peter Andersen 0 2
Gregers Jungersen 0 1 2
Claus Aagaard 0 2
0 a Current address: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark ¤b Current address: Department of Science and Environment, Roskilde University, Roskilde, Denmark ¤c Current address: Analytical Development, ALK-AbelloÂ , Hoersholm, Denmark ¤d Current address: DTU Bioengineering , Technical University of Denmark , Kgs. Lyngby , Denmark
1 Section for Immunology and Vaccinology, National Veterinary Institute, Technical University of Denmark , Kgs. Lyngby , Denmark , 2 Department of Infectious Disease Immunology, Statens Serum Institut , Copenhagen , Denmark
2 Editor: Paulo Lee Ho, Instituto Butantan , BRAZIL
There is a need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as Mycobacterium tuberculosis and Mycobacterium avium subsp. paratuberculosis in a number of species. One of the main challenges for vaccine development is the lack of safe adjuvants that induce protective immune responses. Cationic Adjuvant Formulation 01 (CAF01)Ðan adjuvant based on trehalose dibehenate (TDB) and targeting the Mincle receptorÐhas entered human trials based on promising preclinical results in a number of species. However, in cattle CAF01 only induces weak systemic immune responses. In this study, we tested the ability of three pattern recognition receptors, either alone or in combination, to activate bovine monocytes and macrophages. We found that addition of the TLR3 agonist, polyinosinic:polycytidylic acid (Poly(I:C)) to either one of the Mincle receptor agonists, TDB or monomycoloyl glycerol (MMG), enhanced monocyte activation, and calves vaccinated with CAF09 containing MMG and Poly(I:C) had increased cell-mediated and humoral immune response compared to CAF01
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This work was supported by
PARATBVACCINE, PARAVAC from the FP7 EMIDA
ERA-NET-EMIDA programme (PA, GJ). http://
animal-health/. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
vaccinated animals. In contrast to the highly reactogenic Montanide ISA 61 VG,
CAF09primed T cells maintained a higher frequency of polyfunctional CD4+ T cells (IFN-γ+ TNF-α+
IL-2+). In conclusion, CAF09 supports the development of antibodies along with a
high-quality cell-mediated immune response and is a promising alternative to oil-in-water adjuvant in
cattle and other ruminants.
Vaccines are the most efficient tool for preventing diseases caused by infectious pathogens.
Many of the current vaccines were developed fifty or more years ago and are based on live
attenuated forms of the pathogen. For intracellular mycobacterial infections, there is a strong
need for modern vaccines not only for humans but also for a number of other species
including, cattle, goat, sheep, buffalo, and deer. The current challenge is to achieve a potent
vaccination effect specific for the intracellular mycobacterial infection while avoiding reactogenicity
and toxicity typically associated with the most potent adjuvants, and without interfering with
the diagnostic tests currently in place for these infections [
]. Subunit vaccines based on
adjuvant formulations such as cationic adjuvant formulation 01 (CAF01) combined with selected
antigens seems well suited for this. CAF01 is based on the cationic lipid DDA
(dimethyldioctadecylammonium) and TDB (α, αÂ trehalose dibehenate). DDAÂs function is to create a
longlasting depot at the site of injection and increase cellular uptake of antigens. TDB stabilizes
DDA liposomes and is an agonist of the macrophage inducible C-type lectin (Mincle) receptor
that activates antigen-presenting cells through the TLR-independent Syk-CARD9 pathway [
Mouse models have shown that CAF01 induces a Th1- and Th17-biased CD4 T cell response
combined with a humoral immune response [
] and confers protective immunity against
tuberculosis (TB) in mice, guinea pig and non-human primate models when formulated with
antigens from Mycobacterium tuberculosis [4±6]. Furthermore, CAF01-adjuvanted vaccines
have shown promising results against chlamydia, malaria, and influenza infections in animal
models [4, 7±9]. CAF01 has been tested successfully in Phase I clinical trials where the safety,
tolerability, and immunogenicity profile of the adjuvant was investigated when administered
in combination with both a protein TB vaccine (ClinicalTrials.gov identifier NCT00922363)
and a peptide based HIV-1 cocktail (ClinicalTrials.gov identifier NCT01141205). Thus, the use
of CAF01 seems to be quite versatile. Unfortunately, immunization studies in cattle have
shown that CAF01 only induces a weak CD4 T cell response against the same TB subunit
vaccine (Ag85B-ESAT6) that gave strong responses in other species [
]. Using other
mycobacterial antigens, we have confirmed the low immunogenicity of CAF01 in cattle. However, we
also found that both the age of the animal at immunization and the antigen itself had a major
influence on the magnitude of the specific response [
]. In larger animals, there is very
limited studies regarding vaccine dose and ratio of antigen and adjuvant and it is possible that
increasing the dose or changing the ratio could improve immunogenicity but may be at an
inhibitory cost. With the aim of improving vaccine efficacy in cattle, we test the ability of
selected immune modulators to activate monocytes isolated from blood samples from naïve
animals. We find that by combining TDB or MMG with a double stranded RNA analog
polyinosinic:polycytidylic acid (Poly(I:C)) that activates APCs via toll like receptor 3 (TLR3) we
obtain a synergistic effect on the in vitro activation of bovine monocytes. This translates into
increased humoral and cell-mediated responses in calves immunized with the newly developed
CAF09 adjuvant containing DDA, MMG, and poly(I:C), and we compare the responses to a
water-in-oil based adjuvant developed for veterinary use (Montanide™ ISA 61 VG) [
Materials and methods
Male jersey calves (n = 12) were obtained from a dairy farm proven to have a true prevalence
equal or close to zero by the Danish paratuberculosis surveillance program [
] and were
included at two months of age. All animals used in the study were kept under appropriate bio
containment facilities (biosafety level 2 [BSL-2]) in a community pen with straw bedding.
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Animals were fed pellets and hay ad libitum and bedding was changed every day. The use of
animals in this study was approved by the Danish National Experiment Inspectorate and work
was carried out in accordance with their regulations and policies, following institutional ethical
Isolation of bovine CD14 positive peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation
using blood collected aseptically in heparinized tubes. Briefly, red blood cells were lysed by
adding 10 ml 0.84% w/v NH4Cl to 20 ml blood in 50 ml tubes. After 2 min. on ice, 20 ml ice
cold PBS was added and the cells were pelleted (10 min, 1800 rpm, 4ÊC). Cell pellet was
re-suspended in 35 ml PBS and layered over 15 ml Lympholyte1 Mammal (Cedarlane Laboratories,
Burlington, NC, USA) at room temperature. After centrifugation (10 min, 2500 rpm, 15ÊC)
the PBMC interface between Lympholyte1 and PBS was harvested and washed twice before
being re-suspended in 1 ml PBS. CD14 (cluster of differentiation 14) positive cells were
purified by positive selection using the human CD14 MACS1 cell separation kit (Miltenyi Biotec
GmbH), according to the manufacturer's protocol. In brief, a single cell suspension of PBMCs
was generated by filtering through a 30 μm pre-separation filter whereafter cells were
incubated with magnetic beads conjugated to mouse anti-human CD14 monoclonal antibodies,
which cross react with the bovine CD14 molecules. The CD14+ monocytes were retained on a
TDB and MMG lipid layers
Solutions of α,α`-trehalose 6,6'- dibehenate and (TDB) and Monomycolyl glycerols (MMG)
lipids were prepared in methanol/chloroform and chloroform respectively to a concentration
of 1 mg/ml, in glass vials (1 mg/vial). To evaporate the solvent, the solutions were exposed to a
stream of nitrogen gas for one hour followed by overnight incubation in the fume hood. The
dry lipids were stored at -20ÊC or used directly. Serial dilutions of the dry lipid stock were
performed in 96 well, flat bottom plates using isopropanol as solvent. After evaporation of the
isopropanol, the lipid-coated plates were used for cell stimulation.
In vitro stimulation of monocytes with immunostimulators
Purified CD14 positive monocytes (5 104 cells/well) were added to 96 well flat bottom plates,
that were either pre-coated with lipid layers of TDB or MMG or supplemented with 5 or
50 μg/ml Poly(I:C) or 15, 5 or 1.7 μg/ml CAF09 [
]. All combinations of immunostimulators
were tested in triplicates. After 3 days stimulation, the interleukin-6 levels (IL-6) were
determined in cell supernatants by enzyme-linked immunosorbent assay (ELISA) (Thermo Fisher
Scientific, US) according to the manufacturer's protocol (data in S1 Table).
Antigens, adjuvants and immunization
All animals were immunized with antigenic Mycobacterium avium subsp. paratuberculosis
(MAP) proteins in a mixture comprising of MAP3694c (20 μg/vaccination) and a fusion
protein (30 μg/vaccination) consisting of the proteins: MAP1507, MAP1508, MAP3783 and
MAP3784. The vaccine antigens were produced as recombinant proteins in E. coli and purified
by metal affinity and anion columns as previously reported [
]. One hour prior to
vaccination, the antigens were formulated with adjuvant. For Montanide™ ISA 61 VG (Seppic,
France), a mineral water-in-oil based adjuvant, antigens, sterile Tris buffer pH 7.8 and
adjuvant were mixed in the recommended ratio and the formulation passed 20 times slowly and
3 / 19
then 60 times at high speed through a syringe-connector-syringe apparatus supplied with the
adjuvant. The cationic-liposome adjuvants CAF01 (DDA, 2500 μg/ml and TDB, 500 μg/ml)
and CAF09 (2500 μg/ml DDA, 500 μg/ml TDB and 500 μg/ml Poly(I:C) were prepared as
previously described [
4, 11, 15
] and mixed with antigens in Tris buffer at room temperature one
hour before use. All vaccines were administered as 2 ml subcutaneous injections in the
midneck region at experimental week 0 and 4. Control groups were immunized twice with Tris
buffer containing antigens alone. Two independent immunization studies were performed
Isolation of peripheral blood mononuclear cells
PBMCs for restimulation assays described below were isolated from heparinized cattle blood
by Ficoll1 Paque Plus 1.077 (GE Healthcare Life Sciences, US) gradient density centrifugation.
For ex vivo IFN-γ ELISPOT assay, cells were washed and resuspended in serum-free medium
(AIM-V1, Invitrogen). For intracellular cell staining, cells were washed twice in RPMI 1640
media (Life Technologies, UK) supplemented with 2% heat-inactivated fetal calf serum (Sigma
Aldrich). After washing, cells were resuspended in RPMI 1640 medium supplemented with
10% heat-inactivated fetal calf serum, 100 U/ml penicillin (Gibco, UK), and 100 μg/ml
streptomycin sulphate (Gibco, UK).
Whole-blood IFN-γ assay
Cytokine IFN-γ production in antigen-stimulated whole-blood cultures was determined by
culturing whole-blood in the presence of antigens as described previously [
]. Briefly, 0.5 ml
whole-blood was cultured in the presence of 1 μg/ml of fusion protein, MAP3694c, MAP2487c
(non-vaccine control protein), 10 μg/ml of Johnine purified protein derivative (PPDj), PBS, or
positive control stimulation with 1 μg/ml superantigen Staphylococcus enterotoxin B (SEB).
Secreted IFN-γ was measured in cell supernatants using an in-house monoclonal sandwich
ELISA as previously described [
]. To define the antigen-specific IFN-γ response the
background level (IFN-γ response to PBS) was subtracted from the response to the vaccine antigens
(data in S2 Table).
Ex vivo IFN-γ ELISPOT assay
An in-house developed and standardized ex vivo IFN-γ ELISPOT assay was used to enumerate
effector T cells as a function of IFN-γ production. Briefly, ELISPOT plates (EMD Millipore)
were coated overnight at 4ÊC with bovine IFN-γ-specific monoclonal antibody (clone 6.19) at
a concentration of 5 μg /ml. After washing with sterile PBS, wells were blocked with
serum4 / 19
free medium (AIM-V1, Life Technologies) and PBMCs suspended in serum-free medium
were added (1 x 106 PBMC/well) and cultured in the presence of fusion protein and
MAP3694c antigen or mitogen SEB or a non-vaccine control protein MAP2487c at 1 μg/ml
for 20 hours in a humified 37ÊC, 5% CO2 incubator. Plates were washed twice with distilled
water and three times with PBS-0.01% Tween 20 (PBS-T). An anti-IFN-γ biotinylated
monoclonal antibody (clone cc302) was added at a concentration of 1 μg /ml in PBS-0.1% bovine
serum albumin (1 hour at room temperature). After washing four times with PBS-T, plates
were incubated with streptavidin-alkaline phosphatase (Roche; 1:2000 dilution, 1 hour at
room temperature) followed by washing three times with PBS-T and twice with sterile PBS.
Spots were visualized using BCIP/NBT substrate (Sigma) and counted using an ELISPOT
reader (AID GmbH)(data in S2 Table).
Antigen-specific IgG1 ELISA
Serum levels of antigen-specific immunoglobulin G1 (IgG1) were measured by an in-house
developed and standardized ELISA as previously described [
]. Briefly, plates were coated
with 1 μg/ml of each of the five vaccine antigens or a non-vaccine MAP protein (MAP2487c,
negative control). Serum samples were plated at 1:40 dilution. IgG1 was detected with
HRPconjugated mouse anti-bovine IgG1 mAb (clone IL-A60; Bio-Rad) diluted 1:500. Substrate
was o-phenylenediamine dihydrochloride and optical density was read at 493 nm with a
649-nm reference subtraction (data in S3 Table). The results are presented as calibrated OD
(ODc) as previously described [
Cell stimulation, antibody staining and flow cytometry
Purified PBMCs (2 x 106 cells/ml) were cultured in the presence of 1 μg/ml of fusion protein
and 1 μg/ml MAP3694c protein for 6 hours followed by 16 hours in the presence of Brefeldin
A (10 μg/ml; Sigma) and Monensin (2 μM/ml; Sigma). Control cultures containing either
medium alone or mitogen (1 μg/ml of SEB) were run in parallel. After washing cultured
PBMCs with PBS (supplemented with EDTA and sodium azide) the cells were stained.
Anti-CD4 antibody (clone IL-A11; Veterinary Medical Research & Development, VMRD,
Pullman, WA, USA) was used for surface stain in combination with violet dead cell stain
(Invitrogen) before the cells were intracellularly stained with antibodies specific for IFN-γ
(PE-conjugated, clone CC302; Bio-Rad), TNF-α (AF488-conjugated, clone CC327; Bio-Rad), and IL-2
(DyLight649-conjugated, Clone 86; a kind gift from Adam Whelan, APHA, UK) [
]. Using a
BD FACSCanto II analyser equipped with 405, 488 and 633 nm lasers (BD Bioscience, USA)
flow data were acquired and analyzed with BD FACSDiva software vs. 6.1.2 (data in S4 Table).
ID Screen1 paratuberculosis indirect ELISA
For detection of anti-MAP antibodies, a commercial indirect ELISA kit (ID Vet, France)
developed for the surveillance of MAP infection was used. Sera samples collected 7½ weeks after
first vaccination were tested for total IgG production following manufacturer instructions.
Results were reported as sample to positive ratio (S/P ratio) calculated using the formula S/P
ratio = ((ODSample−ODNegative control) (ODPositive control−ODNegative control) × 100). Animals
were assigned MAP status as seropositive or seronegative according to manufacturer's
interpretations, with serum samples interpreted as negative if S/P ratio is 60, doubtful if between
60 and 70, and positive if 70 percent.
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Comparative tuberculin skin test
To rule out cross-reactivity with surveillance testing for bovine tuberculosis, comparative
intradermal tuberculin testing was performed 8 weeks after first vaccination as previously
] and results were interpreted according to standard protocol (European
Communities Commission regulation 141 number 1226/2002) [
Data were analyzed using the Graphpad Prism 7 statistical package (GraphPad Software Inc.,
USA). Immune responses in all animal groups were compared by two-way analysis of variance
(ANOVA) using time and vaccination as independent variables. For multiple comparison we
used Tukey's multiple comparison test, testing for both vaccine effects measured over the
entire length of the experiment as well as effects at the individual time points. P values < 0.05
were considered significant.
Synergistic effect on cell activation by combining receptor agonists is augmented by liposome formulation
Using IL-6 secretion as a marker for monocyte and macrophage activation, we investigated the
ability of three different immune modulators to activate CD14-purified bovine monocytes and
macrophages in vitro. The immune modulators included two lipids, TDB and MMG, and a
synthetic analog of double-stranded RNA, Poly(I:C). These lipids and the RNA analog are
being exploited as immune stimulators in the CAF01, CAF04, and CAF09 adjuvants. CAF01 is
based on the surfactant DDA and the glycolipid TDB. CAF04 is based on DDA and
mycobacterial cell wall lipid MMG while CAF09 is based on DDA/MMG and poly(I:C). Both TDB and
MMG lipids were capable of stimulating the CD14+ cells (Fig 1A and 1B). The stimulatory
effect of the MMG lipid was quite small and only detectable at one MMG lipid coating density
(2 μg/well). For TDB, the strongest response was measured at the same lipid coating density,
but the level of released IL-6 cytokine was almost seven times higher than for MMG with
monocyte activation for TDB lipid coating densities detected above and below the optimum.
For soluble Poly(I:C), the lowest concentration tested (5 μg/ml) did not activate the CD14+
cells to produce and release IL-6, but increasing the Poly(I:C) concentration ten-fold (50 μg/
ml) resulted in cell expression and secretion of relatively large quantities of IL-6 (1605 ± 384
pg/ml)(Fig 1C). To identify a possible synergistic effect from simultaneously stimulating the
cells with two agonist binding to distinct receptors and activating different signaling pathways
we combined the TLR3 activator, Poly(I:C), with either one of the two Mincle activators, TDB
and MMG. When co-stimulated, the low concentration of Poly(I:C) (5 μg/ml) in the culture
medium was sufficient to boost the cell activation and extend the activation range for both
TDB and MMG lipids (Fig 1D and 1E). The co-stimulation of increasing TDB lipid with fixed
Poly(I:C) concentration gave an increasing dose-response curve with IL-6 release reaching the
same level as the high Poly(I:C) concentration for the highest TDB density (1542 ± 436 pg/ml).
MMG and Poly(I:C) co-stimulation resulted in more than a doubling of IL-6 release into the
medium, relative to TDB and Poly(I:C), for the highest lipid coating densities (3988 ±2701 pg/
ml). Because MMG and Poly(I:C) co-stimulation induced a strong cell activation and CAF09
can induce both CD4+ and CD8+ cell mediated immunity combined with a humoral response
in mice, we decided to test the ability of CAF09 adjuvant to activate bovine monocytes and
macrophages in vitro (Fig 1F). CAF09 stimulation of bovine monocytes and macrophages
resulted in a dose-response curve with the maximum activation (3090 ± 954 pg/ml IL-6) at the
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Fig 1. Agonists have synergistic effect on monocyte and macrophage activation in vitro. CD14+ cells were purified from peripheral blood
mononuclear cells (PBMCs) and transferred to cell culture plates pre-coated with lipid layers of TDB (trehalose dibehenate) or MMG (monomycoloyl
glycerol) or supplemented with Poly(I:C) (polyinosinic:polycytidylic acid) or CAF09. Levels of IL-6 (pg/ml) were measured in culture supernatants after
three days of incubation by enzyme-linked immunosorbent assay (ELISA). Bars represent mean ± SD of three calves.
highest concentration tested (15 μg/ml CAF09). For comparison, this corresponds to an MMG
dose of 0.43 μg and a Poly(I:C) concentration of 2.14 μg/ml, significantly below the MMG
density (2 μg/well) or Poly(I:C) concentration (5 or 50 μg/ml) needed for cell activation when
these were used individually or in combination without DDA liposomes. Based on the
promising results we decided to test CAF09 as an adjuvant for use in cattle and compare it against
CAF01 and the water-in-oil emulsion Montanide ISA 61 VG.
Specific cell-mediated responses in calves after vaccination with the liposome based CAF09 adjuvant
To monitor the development of cell-mediated responses with the different adjuvants, calves
were immunized with mycobacterial protein antigens (a fusion protein consisting of
MAP1507-MAP1508-MAP3783-MAP3784 and MAP3694c as a single protein) in the
waterin-oil emulsion Montanide ISA 61 VG or the cationic liposome adjuvant formulations CAF01
and CAF09. At different time points, up to 7½ weeks after first immunization, blood was
drawn and stimulated in vitro with vaccine antigens, whereafter the frequency of
antigen-specific cells was measured by IFN-γ ELISPOT, and total IFN-γ secretion into the cell medium
was measured by ELISA (Fig 2A±2F). Vaccination with antigens in Montanide ISA 61 VG
resulted in a statistically significant and rapid induction of specific cells after one
immunization (Fig 2A±2C). The number of IFN-γ producing cells were statistically higher than in
CAF01 or non-adjuvant vaccinated animals regardless if we compared for whole fusion
protein- or MAP3694c-specific cells (p<0.001). In CAF09 vaccinated animals the number of
fusion protein-specific cells was higher than in the non-adjuvant group (p<0.05) but there was
7 / 19
Fig 2. CAF09 and ISA 61 VG adjuvants induce a specific cell-mediated immune response. IFN-γ ELISPOT analysis (A-C) and IFN-γ ELISA (D-F)
were performed on peripheral blood mononuclear cells (PBMCs) from vaccinated animals up to 7½ weeks after first immunization. Animals were
immunized twice by the subcutaneous route with recombinant antigens and one of the indicated adjuvants. Vaccination time points are indicated by
dotted lines. The frequency of vaccine-induced IFN-γ cells and released cytokine IFN-γ into the cell medium was determined after 24 hours of in vitro
stimulation of PBMCs with either the vaccine fusion protein, MAP3694c or PPDj. Shown are means ± SD of three calves. Statistical analysis: two-way
analysis of variance (ANOVA) and Tukey's post-test. Black stars indicate significant difference relative to the non-adjuvant group. Circles and squares
indicate differences between the curves for the adjuvanted and non-adjuvanted groups p < 0.05, p < 0.01, p < 0.001.
no statistical difference when compared to the CAF01 vaccination group. In the Montanide
ISA 61 VG and CAF09 comparison, there was only a significant difference at the first time
point after one vaccination where the CAF09-vaccinated animals had not yet developed a
vaccine response. We found one animal in each of the CAF01 and Montanide ISA 61 VG adjuvant
groups with a high PPDj response relative to the other animals. The secretion of IFN-γ in
culture supernatants after antigen stimulation confirmed the patterns of vaccine-induced
antigen-specific responses. Montanide ISA 61 VG induced statistically significant IFN-γ levels
compared to the CAF adjuvants (p<0.01) and the non-adjuvant control group (p<0.05) before
the second vaccination (Fig 2D and 2E). Interestingly, after a booster immunization the
CAF09-induced IFN-γ level increased significantly and was nearly on par with the Montanide
ISA 61 VG-promoted response throughout the rest of the study for both the fusion and
MAP3694c protein. Accordingly, CAF09-induced IFN-γ responses measured at time points
after the second immunization were statistically different from the responses in the
non-adjuvanted control group (p<0.01) but, as with the ELISPOT data, were not statistically
distinguishable from the other vaccination groups. IFN-γ release in response to PPDj was high in
two animals vaccinated with Montanide™ ISA 61 VG adjuvanted vaccination when compared
to the other adjuvant groups (Fig 2F).
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Significant vaccine antigen-specific IgG1 after Montanide ISA 61 VG and
Using serum samples collected in parallel with the blood samples above, we measured the level
of IgG1 in serum from immunized animals that was specific for the five individual vaccine
antigens and included the MAP2487c protein as a negative control (Fig 3A±3F). The IgG1
serum data support and strengthen the cell-mediated pattern found in the vaccination groups
by ELISPOT and whole blood IFN-γ assays. Antigen-specific IgG1 levels in Montanide ISA 61
VG group were significantly higher than in the non-adjuvant control, the CAF01-, or the
CAF09-vaccinated group (p<0.01) for all vaccine antigens tested except MAP1507, which
seems to be the least immunogenic of the vaccine proteins. As observed with the cell-mediated
response, the IgG1 levels in the CAF09-adjuvanted group increased significantly after the
second immunization. This was especially true for MAP1508 and MAP3694c where the specific
IgG1 levels were significantly higher than for the non-adjuvant control group (p<0.05).
Cationic liposomes induces polyfunctional memory CD4+ T cells
Having shown that protein immunization with Montanide ISA 61 VG and CAF09, and to a
lesser degree CAF01, can induce a sustained cell-mediated and humoral immune response we
subsequently characterized the phenotype of the antigen-specific CD4+ T cells generated after
immunization, as polyfunctional CD4+ T cells have been associated with vaccine-induced
protection in mycobacterial infections [
]. After staining for surface markers and intracellular
cytokines we used flow cytometry and a gating strategy to measure the number and frequency
Fig 3. Strong humoral immune responses after CAF09 and ISA61 VG immunization. The single-antigen specific serum IgG1 level was measured in
individual immunized animals. After capturing IgG1 specific for the vaccine antigens MAP1507, MAP1508, MAP3694c, MAP3783, MAP3784 or the
control antigen MAP2487c the bound amount of IgG1 was measured by an in-house enzyme-linked immunosorbent assay ELISA. (A-F). The
calibrated OD means ± SD for three calves per group are shown for all time points. Statistical analysis: two-way analysis of variance (ANOVA) and
Tukey's post-test. Black stars indicate significant difference relative to the non-adjuvant group. Circles and squares indicate differences between the
curves for the adjuvanted and non-adjuvanted groups p < 0.05, p < 0.01, p < 0.001.
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Fig 4. Large frequency of memory CD4+ T cells after CAF09 immunization. PBMCs isolated from vaccinated animals 7½ weeks after first
immunization were stimulated with vaccine antigens (fusion protein + MAP3694c) and surface stained for the CD4 receptor and intracellular localized
IFN-γ, TNF-α and IL-2 cytokines. Boolean gating was used to determine the frequencies of the seven possible CD4+ T cell subpopulations expressing
any combination of the IFN-γ, TNF-α and IL-2 cytokines. The frequencies are shown for each of the three vaccination groups after subtraction of
background from non-stimulated adjuvant controls. Pie charts illustrate the fraction of CD4+ T cell expressing three cytokines (3+), any two cytokines
(2+), or any one cytokine (1+) for each vaccination group (CAF01, CAF09, and ISA 61 VG). Pie chart color-coding and the subpopulation association
for each color is shown below the bar graph (A). The mean fluorescence intensity (MFI) for the IFN-γ-, TNF-α-, or IL-2 producing CD4+ T cells in each
vaccination group was calculated as a mean of all cytokine producing cell regardless if a cell produce one, two or three of the measured cytokines. Bars
represent mean ± SD of three calves. Statistical analysis: unpaired t test p < 0.001 (B).
of antigen-specific CD4+ T cells and characterized their ability to secrete one or more of the
effector cytokines IFN-γ, TNF-α, and IL-2 at the single cell level (Gating in S1 Fig). With this
gating strategy, it is possible to determine the functionality of antigen-specific CD4+ T cells
with respect to their expression of IFN-γ, TNF-α, and IL-2 in each of the vaccination groups
(Fig 4A). Immunization with antigens formulated in CAF01 or CAF09 adjuvant induced
primarily polyfunctional T cell population consisting of triple cytokine producing memory CD4+
T cells (IFN-γ+ TNF-α+ IL-2+) and a lower frequency of double (IFN-γ+ TNF-α+) and single
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(IFN-γ+ or TNF-α+) cytokine positive effector CD4+ T cells. In agreement with the stronger
responses measured above, there was a higher frequency of vaccine-specific CD4+ T cells in
the CAF09-immunized compared to CAF01-immunized animals, but also a relatively larger
portion of more differentiated single and double cytokine positive effector cells. Following
Montanide ISA 61 VG immunization, we found the opposite. The animals had a high
frequency of terminally differentiated effector CD4+ T cells, and especially cells producing two
cytokines (IFN-γ+ TNF-α+), but there was also a considerable frequency of IFN-γ single
producers and triple positive memory T cells. To compare the average amount of each cytokine
that was produced by an individual cell, regardless if the cell was a single- or multi-cytokine
producer, we measured the median fluorescence intensities (MFI) for each of the cytokines.
For all three cytokines, CD4+ T cells from CAF09-immunized animals produced the most
cytokine per cell (Fig 4B). This was also reflected in the results when we compared the level of
IFN-γ-, TNF-α-, or IL-2 per cell in each of the CD4+ T cell cytokine producing subgroups
(data in S2 Fig). In the triple positive polyfunctional T cells, cytokine production per cell was
highest in T cells from CAF09 immunized animals for all three cytokines measured and
particularly for TNF-α in the TNF-α IL-2 double producing T cell population. Thus, the flow
cytometry data confirms the IFN-γ ELISPOT and ELISA data showing that Montanide ISA61 VG
predominantly induces a strong response because it activates a high number of CD4+ T cells.
In contrast, CAF09 activates fewer CD4+ T cells, but these produce more cytokines and a
higher amount of each individual cytokine per cell.
Subunit immunization does not interfere with tuberculosis or paratuberculosis diagnosis
The majority of the commercially available vaccines against mycobacterial infections interfere
with skin test and ELISA-based diagnoses. To address this issue we tested all animals used in
the study with standard tuberculin skin test as well as with a commercial indirect ELISA kit
measuring anti-MAP antibodies. Using the standard guidelines for interpreting the reactors in
tuberculin skin test, we found that all animals from the vaccinated groups were negative (<2
mm increase in skin thickness 72 hours post injection; data not shown). Furthermore, all
animals used in the study were seronegative in a commercial ID Screen1 paratuberculosis assay
using indirect ELISA to measure IgG responses in serum. For serum samples collected 7½
weeks after the first vaccination we found an S/P ratio range between 1.36 and 17.76 with a
mean value of 2.33 for all animals, far below the cut-off value of 70 percent.
Lymphocytes, in particular CD4 T cells, are believed to be critically required for host defense
against intracellular mycobacterial pathogens in both man and animals [19±21]. The
liposome-based CAF01 adjuvant can induce strong CD4 T cell responses in several species
including humans and pigs, but in cattle the systemic response has been quite low and a significant
increase in dose will make a future vaccine prohibitively expensive [
quality and strength of the vaccine-mediated immune response can be directed by the nature and
dose of immune-stimulating component(s) in the vaccine formulation [22±24]. Thus, with the
aim of improving the immunogenicity of liposome-based adjuvants, we evaluated
immunostimulators using bovine monocytes and macrophages in vitro to reinforce the potential use of
next generation liposome-based adjuvants in cattle.
Macrophages and dendritic cells are located throughout the body to capture and internalize
invading pathogens. As part of the innate immune system, they are first line of defense against
infections and activators of the adaptive immune system through antigen presentation and
co11 / 19
stimulation of lymphocytes. Circulating naïve monocytes and macrophages present pattern
recognition receptors (PRRs), which recognize molecules (pathogen-associated molecular
patterns, PAMPs) that are broadly shared by pathogens but distinguishable from host molecules.
PRRs include Toll-like receptors (TLR), RIG like receptors (RLR), C-type lectin receptors
(CLR), and NOD-like receptors (NLR). PRR engagement leads to monocyte and macrophage
activation, increased antigen presentation, and release of cytokines and chemokines that
orchestrate the ensuing adaptive immune response. A trend in modern vaccine development
is to mimic the pathogen danger signal by incorporating one or more PRR ligands into the
adjuvant formulation, thereby providing the immunogenicity of a live-attenuated vaccine
without the associated safety issues. In vitro studies have shown that PRR engagement affects
the APC's secretion of cytokines, including IL-12, IL-6, and TNF-α. In this study, we have
used secretion of cytokine IL-6 after incubation with ligands as a proxy for
monocyte/macrophage activation. TDB (trehalose 606-dibehenate) is a synthetic analogue of mycobacterial cord
factor or trehalose dimycolate (TDM) with less toxicity but retained adjuvanticity in vivo [
TDB is a PRR-ligand that activates cells through the TLR-independent Syk-Card9 signaling
pathway by binding to and upregulating the Mincle receptor on the cell surface [
TDB activation of bovine monocytes and macrophages followed an inverted U-shaped
doseresponse curve with a narrow range of lipid densities activating the cells. The highest coating
density did not give the strongest signal most likely because, even though less toxic than TDM,
TDB is toxic to APCs at high lipid densities. Monomycolyl glycerol (MMG) is another
mycobacterial cell-wall lipid that signals via the Mincle receptor. MMG has been reported to induce
a Th1 immune response  and, based on enhanced expression of activation markers and the
release of proinflammatory cytokines, to be a better stimulator of human DCs than TDB [
When bovine monocytes and macrophages were stimulated with an MMG lipid layer the cells
were specifically activated but cytokine release was lower than for TDB and only one coating
density resulted in measurable cell activation. Due to a very low solubility in aqueous solutions,
the polystyrene surface was coated with TDB and MMG lipids only allowing a 2-D surface for
]. We might have obtained higher cytokine IL-6 release by sonicating the lipids
in the medium. Nevertheless, the in vitro stimulation data shows that both TDB and MMG has
immunostimulatory activity on bovine monocytes and macrophages and the Mincle receptor
and SykÐCard9 pathway is a rational target for vaccine development in cattle. Poly(I:C) is a
synthetic analog that is structurally similar to double-stranded RNA and a stimulant of the
endosomal located TLR3 and cytosolic MDA-5 and RIG-1 [
]. Poly(I:C) targets a different
signaling pathway than TDB and MMG, but Poly(I:C) and its derivatives have consistently
been shown to be among the strongest Th1-inducing immunomodulators [
signaling is MyD88-independent and utilizes adaptor protein toll/interleukin-1 receptor (TIR)
domain-containing adaptor inducing IFN-β (TRIF) to induce the release of inflammatory
cytokinesis and a type 1 IFN response [
]. We tested Poly(I:C) in two in vitro
concentrations, and only the highest concentration (50 μg/ml) activated the monocytes and
monocytederived macrophages. While this is a high Poly(I:C) concentration the specific cell activation
showed that also the TLR3/TRIF pathway can be exploited in bovine vaccine development.
During natural infections, microbial derived PRR agonists do not function individually; rather
they synergize to manipulate the immune response via activation of APCs. In vivo and in vitro
studies have found that triggering of several PRRs simultaneously can induce diverse innate
immune responses. Some combinations enhanced cytokine and chemokine production by
APCs others impaired APCs activation [
23, 36, 37
]. Specifically it has been shown that
combining the TLR3 agonist Poly(I:C) with the TLR6/TLR2 agonist MALP-2 increased the
number of activated T cells . Adding a third agonist, the TLR9 agonist CpG, and a HIV peptide,
induced higher-functional avidity T cells than with single or double ligand combinations and
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an effective response against viral challenge [
]. Using a mycobacterial vaccine, it was shown
that combining a TLR4 and TLR9 agonist enhanced the vaccine-specific T cell response and
increased protection against challenge [
]. These synergistic effects of multiple receptor
ligations have primarily been reported when TLR agonists activating the TRIF pathway have been
combined with TLR agonists activating the MyD88 pathway [
]. Our results extend these
observations by demonstrating a synergistic effect on in vitro activation of bovine monocytes
and macrophages after simultaneously targeting two different types of PRRs and their
signaling pathways. Our in vitro data do not select between TDB+Poly(I:C) and MMG+Poly(I:C).
Previous studies have shown that DDA-Poly(I:C) combined with TDB or MMG-1 can be
formulated to stable adjuvants (CAF05 and CAF09, respectively) which both can induce strong
CD4+ T cell responses of the Th1/Th17 phenotype [39±41]. However, MMG-1 is structurally
simpler and easier to manufacture than TDB, the CAF09 liposomes are smaller, less
polydispersed and more stable than CAF05 liposomes, and CAF09 was therefore selected for further
]. In vitro, bovine monocytes and macrophages were activated by CAF09 doses
containing significantly less MMG and Poly(I:C) than needed for activation without the DDA
liposomes. We suggest this is due to a dual function of the DDA liposomes. They keep the
MMG glycolipid in dispersion for better presentation to the Mincle receptor on the cell surface
and increase the cellular uptake of the liposomes for optimal Poly(I:C) activation via the
endosomal located TLR3. Because of the significant increase in activation efficacy, we decided to
test CAF09 in cattle.
Several Montanide adjuvants have been shown to induce strong and long-lasting humoral
and cell mediated immune responses in cattle, and Montanide ISA 61 VG was therefore
included as a positive control for vaccine specific IgGs and cell-mediated immune responses
after immunization [
]. Montanide ISA 61 VG consists of squalene emulsified with
mannide mono-oleate and belongs to the group of incomplete Freund's adjuvants (IFA). Despite
well advanced into Phase I and II vaccine trials, the only information regarding the mechanism
of action for Montanide based vaccines is a depot effect at the site of injection, enabling slow
antigen release. However, new insights have been made into the mode of action of IFA which
could also explain the action mechanisms of Montanide, including more effective transport of
the antigens to the lymphatic system and providing a complex set of signals to the innate
immune system, thereby directing and orchestrating development and function of
antigenspecific T and B lymphocytes . As discussed above, CAF01's activation mechanism via
TDB is well described and, like Montanide, it creates a long-lasting depot at the site of
injection and increases the monocyte and macrophage influx to the site of injection [
]. In cattle,
CAF01-adjuvanted vaccines induce low immune responses, which is surprising since the
Mincle receptor is phylogenetic conserved and expressed on bovine antigen-presenting cells.
However, in transfected macrophages expressing either the human or the murine Mincle receptor a
few amino acid differences between the Mincle sequences determined if the cells were
effectively activated by the Mincle ligand MMG (Monomycoloyl glycerol) or not [
]. Thus, it is
possible that sequence polymorphism is the main culprit for the lack of strong cellÐmediated
immune responses in CAF01 vaccinated cattle and potentially other ruminants. In this study,
CAF01 served as a benchmark for the CAF09 based vaccine and in line with previous studies it
promoted low humoral and cell mediated vaccine specific responses. Montanide ISA 61 VG
induced both a strong cell-mediated and humoral vaccine-specific immune responses after
one immunization but the second immunization did not boost the number of IFN-γ
producing cells or the level of released IFN-γ cytokine. CAF09-induced humoral responses were
stronger than for CAF01 but weaker than for Montanide ISA 61 VG and detectable only after
the second immunization. After one immunization, CAF09-induced approximately the same
number of vaccine-specific IFN-γ secreting cells as Montanide ISA 61 VG but, based on
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secreted IFN-γ levels, the cells produced less IFN-γ per cell, and it took a second immunization
before they reached the same cytokine secretion level as in the Montanide ISA 61 VG group.
We know that polyfunctional T cells produce a higher amount of cytokine per cell than single
functional T cells and it was thus possible that repeated CAF09 immunization improved the
quality of the immune response [
]. To determine the quality and multifunctionality of
vaccine-specific CD4+ T cells, we measured IFN-γ, TNF-α and IL-2 cytokine production in
individual cells. IFN-γ is a key effector molecule that synergizes with TNF-α in activating infected
APCs, allowing the cells to better control intracellular infection [
]. IL-2 is needed for T
cell expansion, differentiation, and long-term cell survival and is used as a marker for less
differentiated central memory T cells [
46, 49, 50
]. In mice, the CD4+ T cell population induced
by CAF01- or CAF09-adjuvanted vaccines have been shown to be dominated primarily by
TNF-α+ IL-2+ and secondarily by IFN-γ+ TNF-α+ IL-2+ multifunctional CD4+ T cells. These
T cells have a large proliferative potential, they are efficiently maintained for more than one
year, and provide long-term protective immunity [
]. Although TDB and MMG derives
from Mycobacterium tuberculosis, the CAF01 and CAF09 induced CD4+ memory T cell
population differ significantly from the effector T cell population (IFN-γ+ and TNF-α+ single or
double cytokine producers) dominant in Mycobacterium tuberculosis-infected animals [
cattle, the cytokine expression profile in CD4+ T cells from Montanide ISA 61 VG-immunized
animals was very similar to the effector populations seen after a Mycobacterium tuberculosis
infection in mice, with a large population of IFN-γ+ TNF-α+ effector cells. In the CAF01 and
CAF09 immunized groups, the vaccine-specific CD4+ T cells were primarily triple positive T
cells (IL-2+ IFN-γ+ TNF-α+) but, although less dominant, there was an equally fraction of
these polyfunctional T cells in the Montanide ISA 61 VG-immunized animals. Cytokine
production per cell in this important subgroup of CD4+ T cells was highest in CAF09 immunized
animals for each of the three cytokines. In mice the dominant vaccine specific T cell
subpopulation is the less differentiated IL-2+ TNF-α+ cells but this subgroup was less prominent in our
study most likely because murine cells typically are isolated from lymph nodes, spleens or the
site of infection whereas the bovine data are from peripheral blood cells [
One important consideration for mycobacterial vaccines intended for use in cattle is that
the vaccine does not interfere with standard tuberculin-based skin testing but allows
concurrent vaccination and stamping-out procedures, i.e. it has to be possible to discriminate infected
from vaccinated animals (DIVA). Here and in previous work with recombinant MAP
antigens, we did not find any animals in any of the vaccination groups that were positive to the
tuberculin skin tests or an ELISA-based paratuberculosis test detecting IgG antibodies [
We did however find higher PPDj-specific IFN-γ responses in blood from animals vaccinated
with Montanide ISA 61 VG adjuvanted antigens relative to the other adjuvant groups and that
could potentially interfere with PPDj based blood CMI assays. Currently, we do not have a
correlate of protection against mycobacterial infection in any species, but it appears that CAF09 is
an attractive alternative to oil-in-water based adjuvants in cattle, not only for mycobacterial
infections but also for a broader use.
The demand for a more precise tailoring of the immune response will drive adjuvant
development based on combining PAMPs. In this regard, the two-step in vitro/in vivo approach is
relevant for preselecting and testing agonist combinations and adjuvants. Combined with a
dissection of the signaling pathways and their effects on host immune cells in different species,
it will advance our understanding of the molecular mechanisms involved in the induction of
adaptive immune responses.
The next generation CAF09 based adjuvant, could e.g. include a TLR agonist that
signals through MyD88 or PRRs such as DC-SIGN, BDCA2, DCIR and MICL that induces
signaling pathways modulating TLR-induced gene expression at the transcriptional or
post14 / 19
transcriptional level. These receptors do not induce immune responses on their own, but
modulate signaling pathways induced by other PRRs and could provide the flexibility and
variability in cytokine expression that is needed to combat different pathogens [52±54].
CD14 is a cofactor for several TLRs and activation of CD14 could synergistically stimulate
multiple TLR for a more potent adaptive immunity. However, since, CD14 induces an
endocytosis pathway that delivers TLR4 to endosomes and evasion of CD14-dependent
endocytosis is a survival strategy for intracellular bacterial pathogens CD14 activation could be
especially relevant to include in a therapeutic vaccine [
In summary, combining a distinct TLR and CLR agonist into an adjuvanted subunit
vaccine increased the magnitude of both the cellular Th1 response and the humoral antibody
response directly in a vaccine target species without compromising the polyfunctionality of the
vaccine-specific CD4+ T cells in the blood.
New vaccines that harness these powerful signaling properties of PRRs will allow us to tailor
immune responses against a specific pathogen or disease.
S1 Fig. Gating strategy for cytokine producing CD4 cells.
S2 Fig. Level of cytokine production in subgroups of cytokine producing CD4 T cells. The
mean fluorescent intensity (MFI) was measured in CD4 T cell subgroupsÐgrouped based on
their cytokine expression profilesÐfor each of the three cytokines in PBMCs isolated from ISA
61VG (blue), CAF01 (red) or CAF09 (green) vaccinated animals. Blood was drawn 7½ weeks
after first immunization.
S1 Table. IL-6 production after in vitro stimulation. The concentration of cytokine IL-6 in
the supernatant is given in pg/ml.
S2 Table. Immune responses in blood from vaccinated and control animals. IFN-γ
production or the number of IFN-γ producing cells was measured by ELISA or ELISpot in blood
from vaccinated and non-vaccinated control animals. Numbers of IFN-γ positive cells are
given per 106 PBMCs and cytokine production as pg/ml.
S3 Table. IgG1 levels against single antigens in blood from vaccinated and control animals.
Optical densities (493 nm) are given after a reference subtraction (649-nm).
S4 Table. Flow cytometry data. Frequencies of specific CD4 T cells divided into subgroups
based on cytokine expression profiles and MFIs for the three cytokines measured.
We are grateful for the animal husbandry services provided by the staff of DTU-Vet animal
facilities. We thank Jeanne Toft Jakobsen, Panchale Olsen, Rune Fledelius Jensen and Vivi
Andersen for their technical assistance and Dennis Christensen and Frank Follmann for
critically reading the manuscript.
15 / 19
Conceptualization: Peter Andersen, Claus Aagaard.
Data curation: Aneesh Thakur, Athina Andrea, Heidi Mikkelsen, Gregers Jungersen.
Formal analysis: Aneesh Thakur, Athina Andrea, Joshua S. Woodworth.
Funding acquisition: Peter Andersen, Gregers Jungersen.
Investigation: Aneesh Thakur.
Methodology: Athina Andrea, Heidi Mikkelsen.
Project administration: Gregers Jungersen, Claus Aagaard.
Supervision: Joshua S. Woodworth, Gregers Jungersen, Claus Aagaard.
Writing ± original draft: Aneesh Thakur, Claus Aagaard.
16 / 19
paratuberculosis subunit vaccine at different ages. Clin Vaccine Immunol. 2013; 20(4):551±8. Epub
2013/02/08. https://doi.org/10.1128/CVI.05574-11 PMID: 23389934.
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Card9-dependent innate immune activation. J Exp Med. 2009; 206(1):89±97. Epub 2009/01/14. https://
doi.org/10.1084/jem.20081445 PMID: 19139169.
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