Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis

Vaccine xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

Vaccine journal homepage: www.elsevier.com/locate/vaccine

Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis Gérald Larrouy-Maumus a, Emilie Layre a, Simon Clark b, Jacques Prandi a, Emma Rayner b, Marco Lepore c, Gennaro de Libero c, Ann Williams b, Germain Puzo a, Martine Gilleron a,⇑ a b c

Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, France Public Health England, National Infections Service, Porton Down, Wiltshire SP4 0JG, UK Experimental Immunology, Department of Biomedicine, University Hospital and University of Basel, Basel 4031, Switzerland

a r t i c l e

i n f o

Article history: Received 16 November 2016 Received in revised form 12 January 2017 Accepted 30 January 2017 Available online xxxx Keywords: Glycolipid CD1b Guinea pig Vaccine Tuberculosis Liposome

a b s t r a c t The bacillus Calmette Guérin (BCG) vaccine, the only licensed vaccine against TB, displays partial and variable efficacy, thus making the exploitation of novel vaccination strategies a major priority. Most of the current vaccines in pre-clinical or clinical development are based on the induction of T cells recognizing protein antigens. However, a large number of T cells specific for mycobacterial lipids are induced during infection, suggesting that lipid-based vaccines might represent an important component of novel subunit vaccines. Here, we investigated whether immunization with defined mycobacterial lipid antigens induces protection in guinea pigs challenged with M. tuberculosis. Two purified mycobacterial lipid antigens, the diacylated sulfoglycolipids (Ac2SGL) and the phosphatidyl-myo-inositol dimannosides (PIM2) were formulated in biophysically characterized liposomes made of dimethyl-dioctadecyl-ammonium (DDA) and synthetic trehalose 6,60 -dibehenate (TDB). In three protection trials, a reduction of bacterial load in the spleen of inoculated animals was consistently observed compared to the unvaccinated group. Moreover, a reduction in the number of lesions and severity of pathology was detected in the lungs and spleen of the lipid vaccine group compared to unvaccinated controls. As the degree of protection achieved is similar to that observed using protein antigens in the same guinea pig model, these promising results pave the way to future investigations of lipid antigens as subunit vaccines. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction The only tuberculosis (TB) vaccine currently available is BCG (bacillus Calmette-Guerin), an attenuated Mycobacterium bovis strain, given soon after birth to protect against severe forms of TB in childhood. However, BCG’s efficacy against pulmonary TB in adults is highly variable and depends on, amongst other factors, ethnicity and geographical location [1]. Therefore, a more efficient vaccine is needed, particularly one offering greater protection to adults. Among novel strategies under investigation, one aims at replacing the BCG with an improved whole-organism prime vaccine, which could be either a recombinant BCG or an attenuated strain of Mycobacterium tuberculosis. An alternative strategy consists of developing a subunit post-exposure boosting vaccine, designed to enhance and prolong the protection already provided by BCG [2,3]. ⇑ Corresponding author at: Institut de Pharmacologie et de Biologie Structurale, Toulouse, France. E-mail address: [email protected] (M. Gilleron).

What constitutes an essential and sufficient immune response for vaccination-induced protection against TB infection is still unclear [2]. Many vaccine candidates are based on the induction of conventional MHC-restricted Th1 cytokine producing T cells, although there is no clear proof that such vaccines are efficacious in humans. Whilst some studies have questioned the long-held belief that IFN-c suffices as biomarker of protection [4], recent data have re-affirmed the role of antigen specific IFN- c secreting cells in reducing the risk of developing TB disease in BCG vaccinated infants [5]. There is a need to develop new vaccine concepts that exploit immunological diversity, and which target unconventional immunity, such as immunity mediated by antibodies, CD1restricted ab T cells, cd T cells and MAIT cells [2]. CD1-restricted ab T cells are stimulated by lipids, which are presented by the non-polymorphic group 1 CD1 proteins (CD1a, CD1b and CD1c isoforms) expressed by dendritic cells, B lymphocytes and epithelial cells [6–8]. These T cells have essential effector functions, including cytolytic capacity, secretion of proinflammatory cytokines such as IFN- c and bactericidal activity. The M. tuberculosis cell wall is very rich in lipids and many of these

http://dx.doi.org/10.1016/j.vaccine.2017.01.079 0264-410X/Ó 2017 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Larrouy-Maumus G et al. Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.01.079

2

G. Larrouy-Maumus et al. / Vaccine xxx (2017) xxx–xxx

have been characterized as antigens of CD1b-restricted T cells [9– 13]. The analysis of the T cell response during human M. tuberculosis infection clearly indicates that infected patients have increased CD1restricted T cell responses to lipid antigens [10,11,14]. Moreover, a study described the existence of lipid-recognizing T cells displaying memory phenotype over one year after curative treatment [15]. The important role of lipids in the adaptive immune response to M. tuberculosis infection was also provided using animal models. Vaccination of cattle, which naturally express CD1b, with an adjuvanted (DDA) mycobacterial glycolipid resulted in a specific T cell response comparable in strength to that mounted against a model protein adjuvanted in the same way [16]. Immunization of guinea pigs with mycobacterial lipid mixtures was shown to induce a lipid-specific CD1-restricted response [17]. Moreover, vaccination of guinea pigs with M. tuberculosis total lipids before a challenge infection with M. tuberculosis led to the reduction of the bacterial load and improved pathology [18]. Mice cannot be used as animal models to evaluate lipid antigen vaccines because they only express the group 2 CD1 isoform (CD1d), which so far has not been found to present mycobacterial glycolipid antigens. In contrast, the guinea pig is a suitable animal model as they express homologs of human CD1 proteins, including four CD1b (i.e., guinea pig CD1B1 to B4), three CD1c (CD1C1 to C3) and one CD1e orthologs [8]. Moreover, guinea pigs are sensitive to M. tuberculosis infection and their patterns of granulomatous inflammation are similar to humans [19]. In this study, we evaluated in M. tuberculosis challenged guinea pigs a combination of purified mycobacterial lipid antigens, diacylated sulfoglycolipids (Ac2SGL) and phosphatidyl-myo-inositol dimannosides (PIM2), formulated in biophysically characterized liposomes. We selected these lipids because of their important properties. They are respectively able to activate CD8 and CD4 ab T cells [10,13,20]. Ac2SGL is specific to M. tuberculosis, while PIM2 is ubiquitous to all mycobacterial species. PIM2 also have adjuvant properties [21,22]. 2. Materials and methods 2.1. Animals – Compliance with regulations on animal welfare and ethics Dunkin Hartley guinea pigs free from pathogen-specific infection were used according to UK Home Office Legislation for animal experimentation and studies were approved by a local ethical committee at Public Health England. Individual animals were randomly assigned to vaccine groups and identified using subcutaneously implanted microchips (PLEXX BV, The Netherlands). Group sizes were determined by statistical power calculations (Minitab version 16) with the aim to reliably detect a difference between the median colony forming units (CFU) per ml of 1.0 log10. 2.2. Purification of lipid antigens Ac2SGL was purified as previously described [10,23] from a MmpL8::hyg mutant of M. tuberculosis H37Rv Pasteur strain [24]. PIM2 was prepared from M. bovis BCG and corresponds to ‘‘fraction A” described in [25]. Purity of molecules was assessed by TLC and MALDI-Tof-MS, as previously described [26]. 2.3. Liposomes preparation The liposomes were prepared by the thin film method as previously described [27]. Briefly, the four components, DDA (Avanti polar lipids), TDB (Cayla-Invivogen), PIM2, natural or synthetic sulfoglycolipids (Ac2SGL and SL37, respectively) were added in the ratio 1250 mg/250 mg/250 mg/250 mg. DDA and TDB were solubi-

lized in chloroform/methanol 8:2 (by vol.), PIM2 in chloroform/ methanol/water 60:35:8 (by vol.) and sulfoglycolipids (SGL) in chloroform/methanol 9:1 (by vol.). The solvents were next removed under N2, allowing the formation of a lipid film. This film was dried overnight under vacuum and hydrated in 10 mM Tris HCl pH 7.4 to a final concentration of 1.25 mg/ml/dose of DDA by heating at 60 °C and vortexing 30 s every 5 min for 30 min. LUV were prepared at 60 °C using the mini-extruder set (Avanti Polar Lipids) using polycarbonate membranes (0.1 mm). Size and zeta potential measurements were performed on samples diluted 1:200 (N = 3) at 25 °C using a DynaPro Nanostar (Wyatt Technology) and a NanoZS (Malvern Instruments) respectively. For viscosity and refractive index, the value of pure water, i.e. 1.0, was used. 2.4. Phase transition temperature determination by steady-state fluorescence polarization 1,6-Diphenyl hexatriene (DPH) (Molecular probes) dissolved in dimethylformamide (12.5 mM) was incorporated to the liposomes preparation without exceeding 0.5% of the total volume and 200:1 lipid/probe ratio. Fluorescence anisotropy measurements were performed using the protocol described by Carayon et al. [28]. 2.5. T Cell activation assays Dendritic cells (3  104/well) were incubated for 2 h at 37 °C with different concentrations of liposomes before addition of T cells (105/well in triplicate). Supernatants were harvested after 36 h of incubation, and GM-CSF release was measured by using enzyme-linked immunosorbent assay kits. Data are expressed as mean ng/mL ± standard deviation (SD) of triplicates. 2.6. Immunization of guinea pigs and challenge Groups of eight female Dunkin-Hartley guinea-pigs (250 g) were intramuscularly inoculated three times at 3 weeks intervals with PIM2/SGL in DDA:TDB (1 mL/guinea-pig divided between the two hind quadriceps muscles). A positive control group of animals was given 250 ml of BCG Danish 1331 (Statens Serum Institut, Denmark) (5  104 CFU per dose) sub-cutaneously at week 0. A negative control group consisted of unvaccinated animals. Animals were aerosolchallenged, 6 weeks after the final inoculations with a low dose (10–50 CFU/animal) of M. tuberculosis H37Rv (NCTC 7416), generated from a suspension at 3  106 CFU/ml using a modified Henderson apparatus and AeroMP control unit, as previously described [29]. 2.7. Bacterial load and histopathology analysis Four weeks post-challenge, animals were euthanized by intraperitoneal injection of sodium pentobarbital (Dolethal, Vetoquinol UK Ltd) and lungs and spleen were removed aseptically. The spleen minus a small apical section and the combined left apical, cardiac, right cardiac and right diaphragmatic lung lobes were homogenized in 5 and 10 ml sterile water, respectively. Serial dilutions were plated (0.1 ml per plate, in duplicate) on Middlebrook 7H11 selective agar (bioMerieux UK Ltd). After 3–4 weeks incubation at 37 °C, colonies were counted to measure CFU/ml of homogenate. Total CFU was calculated by multiplying CFU/ml by the homogenate volume. Where no colonies were observed, a minimum detection limit was set by assigning a count of 0.5 colonies, equating to 5 CFU/ml. Samples for histopathology were processed and analysed as described in [30]. 2.8. Statistical analysis Pair-wise analysis of the log transformed CFU values was performed using the Mann-Whitney non-parametric test to compare

Please cite this article in press as: Larrouy-Maumus G et al. Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.01.079

3

G. Larrouy-Maumus et al. / Vaccine xxx (2017) xxx–xxx

between the groups. The histopathology scores for the lung were the product of a subjective scoring system. Therefore, statistical analysis was not performed on these data, but a two-sample T-test was used to compare the number of lesions in the spleen. 3. Results 3.1. Selection of mycobacterial lipid antigens for vaccination assays Di-acylated sulfoglycolipids (Ac2SGL) and phosphatidylmyoinositol dimannosides (PIM2) were chosen. Ac2SGL is one of the components of the sulfoglycolipid family, which is specifically

A

100

Ac2SGL

produced by the virulent M. tuberculosis species (Fig. 1A). We previously demonstrated that Ac2SGL-specific T cells are present in patients previously exposed to M. tuberculosis and that these cells are CD8+ and capable to kill intracellular mycobacteria [10]. The mass spectrum of the purified molecule shows the expected mixture of molecular species differing by the fatty acids present on both positions of the glucose unit [10,23]. Beside the natural molecule, a synthetic analogue (SL37) was also used (Fig. 1B). SL37 proved to be the most active analogue among all synthesized ones when tested on the Ac2SGL-specific T cell clone [31,32], even more active than the natural compound [33]. As expected, the negative mode MALDI mass spectrum indicates a single molecular species

1248.6

HO HO HO

80

O

-O SO 3

% Intensity

O

O

1276.7

60

OH OH

O

OH HO

40

O

*

n O

20 0 1000

800

1200

1400

1600

Mass m/z

B

100

1175.8 HO HO HO

SL37

% Intensity

80

O

- O SO 3

60

O

O OH OH

O

O

40

O O

20 0 1000

800

1200

1400

1600

Mass m/z

C

100

OH

HO HO HO

1413.9

PIM 2

O

% Intensity

O

HO HO

1175.7 60

O

O

80

851.6

O O

40

1694.2

HO HO O

20

O

P O

O O

O OH

0 600

1000

1400

1800

2200

Mass m/z Fig. 1. Negative-ion mode MALDI-Tof MS analysis of the antigens used for guinea pig vaccination. (A) Natural di-acylated sulfoglycolipids (Ac2SGL) produced by MmpL8::hyg mutant of M. tuberculosis H37Rv Pasteur strain. The natural Ac2SGL is acylated in position 2 of the glucose unit by a palmitic or stearic acid and in position 3 by a phthioceranoic or an hydroxyphthioceranoic acid with different chain length, from C25 to C54 (2 < n < 11). (B) Synthetic di-acylated sulfoglycolipids analogue SL37. SL37 differs from the natural Ac2SGL by the presence in position 3 of the glucose unit of a tetra-methyl-ab unsaturated C32 fatty acid instead of the hydroxyphthioceranoic acid present on the natural Ac2SGL. A palmitic acid esterifies the position 2 of the glucose. (C) Natural phosphatidyl-myoinositol dimannosides (PIM2) isolated from M. bovis BCG. In di-acylated PIM2 (PIM2) (m/z 1175.7) R1 = C19, R2 = C16, R3, R4 = H In tri-acylated PIM2 (Ac1PIM2) (m/z 1413.9) R1 = C19, R2 = C16, R3 = C16, R4 = H In tetra-acylated PIM2 (Ac2PIM2) (m/z 1694.2) R1 = C19, R2 = C16, R3 = C16, R4 = C19 m/z 851.6 corresponds to phosphatidyl-myoinositol (PI) acylated by C16/C19.

Please cite this article in press as: Larrouy-Maumus G et al. Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.01.079

4

G. Larrouy-Maumus et al. / Vaccine xxx (2017) xxx–xxx

The stability of the formulations was monitored over a period of 30 days (Fig. 2). Ac2SGL-containing GUV progressively increased in size from 369 nm to 1090 nm, while the LUV reached 3 mm showing that these liposomes were not stable at 4 °C over this timeframe. Concerning SL37-containing liposomes, their size at 4 °C increased from 326 nm to 742 nm for GUV, while LUV diameter gradually increased from 139 nm to 262 nm. These observations were consistent with the fact that SL37, as a unique molecular species, was expected to generate more stable liposomes than liposomes containing the mixture of Ac2SGL acylforms. Taken together it should be noted that the different formulations (GUV/ LUV) with Ac2SGL or SL37 appeared to be stable over 1 week as there was less than 50% change in particle size. As a result, the formulations were always prepared extemporaneously before animal immunization. Finally, the capacity of the formulations to activate T cells was tested in vitro using the SGL-specific [10] and PIM-specific [20] T cell clones. Both liposomes (GUV and LUV) were able to induce activation of the specific T cell clones (Fig. 3), showing that both antigens retained their immunogenicity upon formulation in liposomes and that the size of the liposomes had no effect on T cell activation. As previously observed for purified antigens, liposomes containing the synthetic SL37 were more potent that Ac2SGL liposomes in activating SGL specific T cells (Fig. 3A and B), confirming this synthetic compound as more immunogenic than the natural one [31–33]. Due to their high stability and because their preparation requires less material, GUV were chosen for vaccination studies.

at m/z 1175.8. PIM2 are abundant and ubiquitous components of the mycobacterial cell walls. They are not only antigens of T cells but have also adjuvant properties, as they were described as ligands of Toll like receptor 2 [21,22]. They were isolated from M. bovis BCG and also exist as a mixture of acyl-forms [25] (Fig. 1C). 3.2. Formulation of the lipid antigens in DDA:TDB liposomes We chose to formulate PIM2 and natural or synthetic SGL (Ac2SGL and SL37, respectively) antigens inside cationic liposomes made of dimethyl-dioctadecyl-ammonium (DDA) with the synthetic trehalose 6,60 -dibehenate (TDB), a vehicle described to induce a powerful cell-mediated immune response with a particular emphasis on Th1 immunity [27]. Eleven mol% of TDB were inserted in DDA liposomes, as this ratio was shown to induce the highest immune response in mice in terms of cytokines release in blood [27]. Then, vesicles containing DDA:TDB:PIM2:SGL (75/11/7/8 mol%) were generated by the lipid film hydratation method, with or without extrusion at 100 nm, to generate GUV and LUV, respectively. We observed by cryo-TEM that the vesicles were predominantly unilamellar (not shown), as expected in view of the high content of positively charged DDA lipids [27]. 3.3. Characterization of the liposome formulations

GUV

100

DDA/TDB

3.4. Protection studies in the guinea pig model The experimental schedules for the protection studies are summarized in Fig. 4. In the study evaluating Ac2SGL/PIM2-containing GUV (LipVac1), there was a significant reduction of CFU in the spleen of LipVac1-inoculated animals compared to the unvaccinated group (Fig. 5A), of approximately 1 Log10. The BCG positive control vaccine group showed significant protection with 1.5 and 3.7 Log10 decrease of CFU in lungs and spleen, respectively, compared to the negative control group. In the lungs, no difference

C Zeta potential (mV)

A

Zeta potential (mV)

Lipid antigens insertion was evaluated by measuring the phase transition temperature (Tm) and the zeta potential of the formulations. The gel-liquid transition temperature was evaluated by fluorescence polarization measurements. We observed that the insertion of PIM2 and SGL led to a decrease of the Tm of DDA: TDB liposomes (Suppl. Table 1). The zeta potential analysis of the formulations showed that all liposomes had a high net positive charge (70 mV for GUV and 50 mV for LUV), clearly resulting from the cationic nature of DDA. As expected, the adjunction of anionic lipids such as SGL and PIM2 decreased the zeta potential of the liposomes (Fig. 2). Moreover, the zeta potential of DDA: TDB:PIM2:Ac2SGL decreased markedly from 62.5 mV to 11.4 mV for GUV and from 44.8 mV to 17.7 mV for LUV revealing aggregations or fusion events over time.

DDA/TDB/PIM2

80

DDA/TDB/PIM2/SL37 DDA/TDB/PIM2/Ac2SGL

60 40 20 0

0

10

20

DDA/TDB/PIM2 DDA/TDB/PIM2/SL37 DDA/TDB/PIM2/Ac2SGL

60 40 20 0

Time (Days) 2500

DDA/TDB

2100

DDA/TDB/PIM2 DDA/TDB/PIM2/SL37

1700

DDA/TDB/PIM2/Ac2SGL

1300 900 500 100

D

10

20

Time (Days)

20

30

10000

30

DDA/TDB DDA/TDB/PIM DDA/TDB/PIM/SL37

1000

DDA/TDB/PIM/Ac2SGL

100

10 0

10

Time (Days) Particle size (nm)

Particle size (nm)

B

DDA/TDB

80

0

30

LUV

100

0

10

20

30

Time (Days)

Fig. 2. Characterization of the formulations. Time development of average zeta potential (A and C) and vesicle size (B and D) of GUV (A and B, respectively) and LUV (C and D, respectively). Liposome size and zeta potential were measured in Tris buffer (10 mM pH 7.4). Results represent mean ± SD of triplicate experiments.

Please cite this article in press as: Larrouy-Maumus G et al. Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.01.079

5

G. Larrouy-Maumus et al. / Vaccine xxx (2017) xxx–xxx

Ac2 SGL-specific T cells 8

DDA/TDB/PIM2/SL37

6

DDA/TDB/PIM2/Ac2SGL

4 2 0

C

PIM-specific T cells DDA/TDB/Ac2SGL

DDA/TDB

10-1

100

101

GM-CSF (ng/ml)

GM-CSF (ng/ml)

A

2.0

1.0 0.5

DDA/TDB DDA/TDB/PIM2/SL37

10

DDA/TDB/PIM2/Ac2SGL

8 6 4 2 100

101

100

101

102

D GM-CSF (ng/ml)

GM-CSF (ng/ml)

B

10-1

10-1

GUV Liposomes ( g/ml)

GUV Liposomes ( g/ml)

0

DDA/TDB/PIM2/Ac2SGL

1.5

0.0

102

DDA/TDB/PIM2

DDA/TDB/Ac2SGL

2.0

DDA/TDB/PIM2

1.5

DDA/TDB/PIM2/Ac2SGL

1.0 0.5 0.0

102

LUV Liposomes ( g/ml)

10-1

100

101

102

LUV Liposomes ( g/ml)

Fig. 3. Stimulation of Ac2SGL-specific (Z4B27) and PIM-specific (Z5B11) T cell clones by different formulations. T cell activation is expressed as GM-CSF released in culture supernatants (mean ± s.d. of duplicate cultures; **P 6 0.01, ***P 6 0.001, by two-tailed Student’s t test). Graphs are representative of three independent experiments.

between the CFU in the LipVac1 group and the unvaccinated control group was observed. These results were reproduced in a second trial of vaccination (Suppl. Fig. 1). Analysis of the severity of the microscopic lesions in the lungs revealed an equivalent score for consolidated lesions in the LipVac1 and BCG Danish groups. The extent of caseation and necrosis in the LipVac1 group was intermediate between the BCG Danish and unvaccinated groups. No differences in splenic lesion morphology, but a significantly reduced number of lesions were observed between the LipVac1 vaccinated group compared to the unvaccinated control group (P = 0.013) (Figs. 5B, 6A and C). Overall, the pathology in the lungs and spleen of the vaccinated and control groups reflected that of the bacterial load in the respective tissues. A third trial was performed using the SL37/PIM2-containing GUV (LipVac2) which also gave in the spleen significantly improved protection compared to the unvaccinated control group,

Time (weeks) 0

3

6

12

LipVac1 or 2 LipVac1 or 2 LipVac1 or 2 Challenge (H37Rv) 0

12

16

CFU and Pathology 16

BCG Challenge (H37Rv)

CFU and Pathology

LipVac1: Ac2 SGL/PIM2 in DDA -TDB GUV LipVac2: SL37/PIM2 in DDA-TDB GUV Fig. 4. Immunization and challenge schedule. Guinea pigs were intramuscularly immunized three times at three weeks interval with formulations containing Ac2SGL/PIM2 in DDA-TDB (LipVac1) or SL37/PIM2 in DDA-TDB (LipVac2). In parallel at time 0, guinea pigs were vaccinated subcutaneously with BCG. Six weeks after the last immunization, the guinea pigs were aerosol-challenged with M. tuberculosis H37Rv (NCTC 7416), using an estimated retained dose of 10–50 CFU/lungs. Four weeks after challenge, they were euthanized and their spleens and lungs collected for CFU quantification and histopathological analysis.

but no improved protection in terms of CFU in the lungs (Fig. 5C). The LipVac2 group also had lower lesion scores in both lung and spleen (P = 0.0056) compared to the unvaccinated group but the scores were higher than the BCG group (Figs. 5D, 6B and D).

4. Discussion The TB vaccine portfolio comprises recombinant live vaccines for prime instead of BCG, as well as recombinant viral vectors expressing- or adjuvanted formulations of- M. tuberculosis antigenic proteins for boost following the prime with live vaccine (such as BCG) [34,35]. In this context, current research is focused on optimal protein antigen selection for enhancement of T cell response allowing long-term vaccine-induced protection against TB. Besides proteins, antigenic lipids are also potential candidates but their use in TB vaccine strategies has been underinvestigated. Whereas extensive studies have established the way by which lipids are able to activate T lymphocytes through CD1 presentation, very few studies have addressed the critical question of the role of CD1-restricted T cells in host defense against infection in vivo [17,18]. We investigated in this study whether immunization with lipid antigens may provide protection against M. tuberculosis H37Rv challenge of guinea pigs. Two antigenic lipids, i.e. SGL and PIM2, were formulated in liposomes and the protective capacity of these formulations was assessed by measuring the induced reduction of the mycobacterial load and the severity of pathology in target organs (lungs and spleen). The guinea pig model offers the advantage of testing lipid antigens presented by group I CD1 molecules, but is limited by the poor availability of immunological reagents, thus preventing a detailed analysis of the immune responses [36]. In the three trials we performed, a 1 Log10 reduction of CFU in the spleen of inoculated animals was consistently observed compared to the unvaccinated group. Bacterial burdens and the degree of histopathology were compared among BCG vaccinated animals, lipid vaccinated animals, and unvaccinated animals. We did not include an adjuvant-only control in the study because previous experiments performed in our laboratory and by others have shown that DDA/TDB liposomes do not induce protection if given alone without antigen [37–39]. Then, an intermediate number of

Please cite this article in press as: Larrouy-Maumus G et al. Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.01.079

6

G. Larrouy-Maumus et al. / Vaccine xxx (2017) xxx–xxx

A

lungs

spleen

B

lungs

spleen

Consolidation Caseation/necrosis

C

D

Fig. 5. Protection efficacy of LipVac1 and LipVac2 vaccines in the guinea pig low dose infection model. Guinea pigs were vaccinated with a formulation containing Ac2SGL/ PIM2 in DDA-TDB (LipVac1) (A and B) or containing SL37/PIM2 in DDA-TDB (LipVac2) (C and D) using the procedure described in Fig. 4. Four weeks after infection, the bacterial load in lungs and spleen (A and C) were determined. At the same time point, histological analysis of consolidation in lungs and spleen (B and D) of infected guinea pigs was performed. The nature and severity of the lesions were assessed blind using a subjective scoring system as previously described [44,45]. For the spleen, a score based on number and size of lesions, presence of necrosis and calcification, was calculated. For the lung, each lobe was assigned a score as follows: 0-normal; 1-very few or small lesions, 0–10% consolidation; 2-few or small lesions, 10–20% consolidation; 3-medium sized lesions, 20–33% consolidation; 4-moderate sized lesions, 33–50% consolidation; 5–50–80% consolidation; extensive pneumonia; > 80% consolidation; plus number of foci of necrosis. Scores from each lobe were combined. Group mean differences were compared to BCG and unvaccinated control groups.

lesions and severity of pathology was observed in the lipid vaccine group compared to BCG and unvaccinated groups, both in lungs and spleen. So, in spite of there being no reduction in bacillary load in the lungs, less pathology and also less necrosis were observed in

this organ. A statistically significant decrease in the bacillary load observed in the spleen could be interpreted as there having been less dissemination from the lung to the spleen, because of immune control mediated in the lung, or that the vaccine-induced suppres-

Fig. 6. Macroscopic images, representative of the group mean. Reduced numbers of splenic lesions were observed in guinea pigs vaccinated with LipVac1 (A) and LipVac2 (B) compared to the respective study unvaccinated control groups (C and D, respectively).

Please cite this article in press as: Larrouy-Maumus G et al. Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.01.079

G. Larrouy-Maumus et al. / Vaccine xxx (2017) xxx–xxx

sion of bacterial growth was more potent in the spleen than the lung. These results are very promising, as they indicate that harnessing the CD1-restricted T cells response using purified and synthetic lipids may activate a protective mechanism during M. tuberculosis infection. Interestingly, the degree of protection achieved was similar to that observed for protein antigens in the guinea pig model [30]. In a study comparing vaccine testing in three laboratories using the guinea pig model of TB, Grover et al. tested two proteic subunit vaccines, the ‘‘culture filtrate protein” fraction (CFP) and the recombinant antigen 85A [40], both emulsified in DDA: Monophosphoryl Lipid A. In the lung, a marginal reduction in CFU of approximately 0.5 Log10 was observed for both of the vaccine groups. Analysis of the number of CFU in the spleen showed a significant reduction of more than 1 Log10 CFU for only CFP inoculated animals. Comparison of glycolipid-induced protection with those obtained using recombinant proteins support the potential of non-protein antigens as components of a TB vaccine and paves the way for new studies in which combinations of both types of antigens might be used. The nature of the cellular immune response of the guinea pigs to glycolipids should now be deciphered in order to confirm that a specific CD1-restricted response against the microbial antigenic lipid was actually generated in vivo. Ideally, the antigen-specific T-cells should be identified in lung, spleen and blood compartments using CD1 tetramers and phenotypically characterized (CD4 or 8). This methodology would provide direct detection of the CD1-reactive T cell repertoire. Unfortunately, only human (but not guinea pig) CD1 tetramer methodology is available to date. The encouraging protection data generated here indicate the protective potential of T cells recognizing mycobacterial lipid antigens. Many aspects in lipid vaccine development now need to be further explored, such as (i) the most appropriate mycobacterial lipid(s) to be selected for the formulation; (ii) the delivery system; (iii) the nature of the adjuvant to be used, particularly in order to boost a CD1-restricted T cell response. In this study, the two mycobacterial lipid antigens tested were selected according to their capacity to stimulate specific T cells from M. tuberculosis-infected individuals. However, specific experiments evaluating which mycobacterial lipids are most immunogenic during infection remain to be made. The mycobacterial cell wall is very rich in many different types of lipids and only some have been tested as T cell antigens so far [6,8]. A second important outcome of our study is that synthetic lipid antigens can be considered as subunit vaccines. Natural mycobacterial lipids are not unique molecules, for instance, Ac2SGL is composed of more than 100 molecules that mainly differ by the length of their fatty acids [23]. Our findings revealed that the observed protection was similar when using the synthetic analogue SL37 instead of the natural Ac2SGL. Moreover, the use of SL37 avoids complex extraction and purification steps, potential contamination, molecular heterogeneity and finally unstable liposomes. Thus, in the process of developing lipid subunit vaccines, synthetic glycolipids should be preferred to natural molecules and synthesis of lipid antigen analogues should be a priority. A final important aspect of our studies is that liposomes are amenable to be used as delivery systems of lipid antigens in vivo. Liposomes are more potent sub-unit vaccine adjuvants when compared to microspheres [41] and they are well suited to incorporate in their bilayer amphipathic lipid antigens. In DDA:TDB liposomes, the adjuvant activity of DDA was previously ascribed to a ‘depot effect’ with retention and slow release of the antigen at the injection site [42]. In addition, TDB, a synthetic analogue of the mycobacterial cord factor was found to stimulate the Mincle receptor expressed by antigen-presenting cells, thus facilitating their

7

maturation and increasing presentation capacity. Its adjuvant activity was ascribed to its capacity to trigger IL-1 production which is preliminary to induction of strong Th1 and Th17 immune responses in vivo [43]. In the future, it could be of value to target additional immune receptors, such as Toll like receptors, which are highly abundant on monocyte/macrophage and dendritic cell populations. Indeed, the combination of several pathogenassociated molecular patterns that activate different signaling pathways could induce different types of adaptive immune responses, hence enhancing vaccine efficacy.

Conflict of interest All the authors declare no conflict of interest. Acknowledgments This work was supported by the Centre National de la Recherche Scientifique, the European Community’s Seventh Framework Program under grant agreement HEALTH-F7-2009241745 (EU-FP7), NEWTBVAC, Research Grant from Mérieux Alliance (23/4/2009–22/04/2011), the Swiss National Foundation (310030-149571), the BMRC-SERC Diagnostic grant 1121480006 and the Department of Health, UK. The views expressed in this publication are those of the authors and not necessarily those of the Department of Health. We thank the staff of the Biological Investigations Group at PHE Porton for assistance in conducting studies and to Emma Rayner and her PHE Pathology team for the provision of histopathology data. We also thank Dr Julien Vaubourgeix and Dr Diane Cala for purification of natural Ac2SGL and Dr Alexiane Decout for formulation preparation.

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2017.01. 079. References [1] Mangtani P, Abubakar I, Ariti C, et al. Protection by BCG vaccine against tuberculosis: a systematic review of randomized controlled trials. Clin Infect Dis 2014;58:470–80. [2] Karp CL, Wilson CB, Stuart LM. Tuberculosis vaccines: barriers and prospects on the quest for a transformative tool. Immunol Rev 2015;264:363–81. [3] Kaufmann SH. Tuberculosis vaccines: time to think about the next generation. Semin Immunol 2013;25:172–81. [4] Kagina BM, Abel B, Scriba TJ, et al. Specific T cell frequency and cytokine expression profile do not correlate with protection against tuberculosis after bacillus Calmette-Guerin vaccination of newborns. Am J Respir Crit Care Med 2010;182:1073–9. [5] Fletcher HA, Snowden MA, Landry B, et al. T-cell activation is an immune correlate of risk in BCG vaccinated infants. Nat Commun 2016;7:11290. [6] Mori L, Lepore M, De Libero G. The immunology of CD1- and MR1-restricted T cells. Annu Rev Immunol 2016;34:479–510. [7] De Libero G, Mori L. The T-cell response to lipid antigens of mycobacterium tuberculosis. Front Immunol 2014;5:219. [8] Van Rhijn I, Moody DB. CD1 and mycobacterial lipids activate human T cells. Immunol Rev 2015;264:138–53. [9] Beckman EM, Porcelli SA, Morita CT, et al. Recognition of a lipid antigen by CD1-restricted alpha beta+ T cells. Nature 1994;372:691–4. [10] Gilleron M, Stenger S, Mazorra Z, et al. Diacylated sulfoglycolipids are novel mycobacterial antigens stimulating CD1-restricted T cells during infection with Mycobacterium tuberculosis. J Exp Med 2004;199:649–59. [11] Layre E, Collmann A, Bastian M, et al. Mycolic acids constitute a scaffold for mycobacterial lipid antigens stimulating CD1-restricted T cells. Chem Biol 2009;16:82–92. [12] Moody DB, Reinhold BB, Guy MR, et al. Structural requirements for glycolipid antigen recognition by CD1b-restricted T cells. Science 1997;278:283–6. [13] Sieling PA, Chatterjee D, Porcelli SA, et al. CD1-restricted T cell recognition of microbial lipoglycan antigens. Science 1995;269:227–30.

Please cite this article in press as: Larrouy-Maumus G et al. Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.01.079

8

G. Larrouy-Maumus et al. / Vaccine xxx (2017) xxx–xxx

[14] Moody DB, Ulrichs T, Muhlecker W, et al. CD1c-mediated T-cell recognition of isoprenoid glycolipids in Mycobacterium tuberculosis infection. Nature 2000;404:884–8. [15] Montamat-Sicotte DJ, Millington KA, Willcox CR, et al. A mycolic acid-specific CD1-restricted T cell population contributes to acute and memory immune responses in human tuberculosis infection. J Clin Investig 2011;121:2493–503. [16] Nguyen TK, Koets AP, Santema WJ, et al. The mycobacterial glycolipid glucose monomycolate induces a memory T cell response comparable to a model protein antigen and no B cell response upon experimental vaccination of cattle. Vaccine 2009;27:4818–25. [17] Hiromatsu K, Dascher CC, LeClair KP, et al. Induction of CD1-restricted immune responses in guinea pigs by immunization with mycobacterial lipid antigens. J Immunol 2002;169:330–9. [18] Dascher CC, Hiromatsu K, Xiong X, et al. Immunization with a mycobacterial lipid vaccine improves pulmonary pathology in the guinea pig model of tuberculosis. Int Immunol 2003;15:915–25. [19] Williams A, Hall Y, Orme IM. Evaluation of new vaccines for tuberculosis in the guinea pig model. Tuberculosis (Edinb) 2009;89:389–97. [20] de la Salle H, Mariotti S, Angenieux C, et al. Assistance of microbial glycolipid antigen processing by CD1e. Science 2005;310:1321–4. [21] Gilleron M, Quesniaux VF, Puzo G. Acylation state of the phosphatidylinositol hexamannosides from Mycobacterium bovis bacillus Calmette Guerin and Mycobacterium tuberculosis H37Rv and its implication in Toll-like receptor response. J Biol Chem 2003;278:29880–9. [22] Jones BW, Means TK, Heldwein KA, et al. Different Toll-like receptor agonists induce distinct macrophage responses. J Leukoc Biol 2001;69:1036–44. [23] Layre E, Paepe DC, Larrouy-Maumus G, et al. Deciphering sulfoglycolipids of Mycobacterium tuberculosis. J Lipid Res 2011;52:1098–110. [24] Domenech P, Reed MB, Dowd CS, et al. The role of MmpL8 in sulfatide biogenesis and virulence of Mycobacterium tuberculosis. J Biol Chem 2004;279:21257–65. [25] Gilleron M, Ronet C, Mempel M, et al. Acylation state of the phosphatidylinositol mannosides from Mycobacterium bovis bacillus Calmette Guerin and ability to induce granuloma and recruit natural killer T cells. J Biol Chem 2001;276:34896–904. [26] Cala-De Paepe D, Layre E, Giacometti G, et al. Deciphering the role of CD1e protein in mycobacterial phosphatidyl-myo-inositol mannosides (PIM) processing for presentation by CD1b to T lymphocytes. J Biol Chem 2012;287:31494–502. [27] Davidsen J, Rosenkrands I, Christensen D, et al. Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from M. tuberculosis (trehalose 6,6’-dibehenate)-a novel adjuvant inducing both strong CMI and antibody responses. Biochim Biophys Acta 2005;1718:22–31. [28] Carayon K, Chaoui K, Ronzier E, et al. Proteolipidic composition of exosomes changes during reticulocyte maturation. J Biol Chem 2011;286:34426–39. [29] Williams A, Davies A, Marsh PD, et al. Comparison of the protective efficacy of bacille calmette-Guerin vaccination against aerosol challenge with

[30]

[31]

[32]

[33]

[34] [35] [36] [37]

[38] [39]

[40]

[41]

[42]

[43]

[44]

[45]

Mycobacterium tuberculosis and Mycobacterium bovis. Clin Infect Dis 2000;30(Suppl 3):S299–301. Grover A, Troudt J, Arnett K, et al. Assessment of vaccine testing at three laboratories using the guinea pig model of tuberculosis. Tuberculosis (Edinb) 2012;92:105–11. Guiard J, Collmann A, Garcia-Alles LF, et al. Fatty acyl structures of Mycobacterium tuberculosis sulfoglycolipid govern T cell response. J Immunol 2009;182:7030–7. Guiard J, Collmann A, Gilleron M, et al. Synthesis of diacylated trehalose sulfates: candidates for a tuberculosis vaccine. Angew Chem Int Ed Engl 2008;47:9734–8. Gau B, Lemetais A, Lepore M, et al. Simplified deoxypropionate acyl chains for Mycobacterium tuberculosis sulfoglycolipid analogues chain length is essential for high antigenicity. Chembiochem Eur J Chem Biol 2013;14:2413–7. Kaufmann SH, McElrath MJ, Lewis DJ, Del Giudice G. Challenges and responses in human vaccine development. Curr Opin Immunol 2014;28:18–26. Ottenhoff TH, Kaufmann SH. Vaccines against tuberculosis: where are we and where do we need to go? PLoS Pathog 2012;8:e1002607. Orme IM, Ordway DJ. Mouse and guinea pig models of tuberculosis. Microbiol Spectrum 2016;4. Aagaard C, Hoang T, Dietrich J, et al. A multistage tuberculosis vaccine that confers efficient protection before and after exposure. Nat Med 2011;17:189–94. Derrick SC, Yabe IM, Yang A, et al. Immunogenicity and protective efficacy of novel Mycobacterium tuberculosis antigens. Vaccine 2013;31:4641–6. Vipond J, Clark SO, Hatch GJ, et al. Re-formulation of selected DNA vaccine candidates and their evaluation as protein vaccines using a guinea pig aerosol infection model of tuberculosis. Tuberculosis (Edinb) 2006;86:218–24. De Bruyn J, Huygen K, Bosmans R, et al. Purification, characterization and identification of a 32 kDa protein antigen of Mycobacterium bovis BCG. Microb Pathog 1987;2:351–66. Kirby DJ, Rosenkrands I, Agger EM, et al. Liposomes act as stronger sub-unit vaccine adjuvants when compared to microspheres. J Drug Target 2008;16:543–54. Henriksen-Lacey M, Bramwell VW, Christensen D, et al. Liposomes based on dimethyldioctadecylammonium promote a depot effect and enhance immunogenicity of soluble antigen. J Control Release 2010;142:180–6. Desel C, Werninghaus K, Ritter M, et al. The Mincle-activating adjuvant TDB induces MyD88-dependent Th1 and Th17 responses through IL-1R signaling. PLoS ONE 2013;8:e53531. Williams A, James BW, Bacon J, et al. An assay to compare the infectivity of Mycobacterium tuberculosis isolates based on aerosol infection of guinea pigs and assessment of bacteriology. Tuberculosis (Edinb) 2005;85:177–84. Bottai D, Frigui W, Clark S, et al. Increased protective efficacy of recombinant BCG strains expressing virulence-neutral proteins of the ESX-1 secretion system. Vaccine 2015;33:2710–8.

Please cite this article in press as: Larrouy-Maumus G et al. Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.01.079