The importance of adjuvant formulation in the development of a TB vaccine

Abstract

An effective protein based vaccine for tuberculosis (TB) will require a safe and effective adjuvant. There are few adjuvants in approved human vaccines, including Alum and the oil-in-water (o/w) based emulsions MF59 (Novartis Vaccines and Diagnostics), AS03 and AS04 (GlaxoSmith Kline Biologics, GSK) AF03 (Sanofi), and liposomes (Crucell). When used with pure, defined proteins, both Alum and emulsion adjuvants are effective at inducing primarily humoral responses. One of the newest adjuvants in approved products is AS04, which combines monophosphoryl lipid A (MPL), a TLR-4 agonist, with Alum. In this study, we compared two adjuvants, an o/w emulsion (SE), and an o/w emulsion incorporating glucopyranosyl lipid adjuvant (GLA), a synthetic TLR-4 agonist, together with a recombinant protein, ID93. Both the emulsion SE and GLA-SE adjuvants induce potent cellular responses in combination with ID93 in mice. ID93/SE induced Th2 biased immune responses, whereas ID93/GLA-SE induced multifunctional CD4⁺ Th1 cell responses (IFN-γ, TNF-α and IL-2). The ID93/GLA-SE vaccine candidate induced significant protection in mice and guinea pigs, whereas no protection was observed with ID93/SE, as assessed by reductions in bacterial burden, survival, and pathology. These results highlight the importance of properly formulating subunit vaccines with effective adjuvants for use against TB.

INTRODUCTION

Approximately 1.3 million deaths per year are associated with Mycobacterium tuberculosis (Mtb) in HIV seronegative populations and an additional 0.38 million deaths occur in people co-infected with HIV (1). Efforts are currently underway to develop vaccines, such as subunit vaccines, that can boost the current BCG vaccine or can be used as a stand-alone prophylactic vaccine in immune-compromised individuals where BCG immunization is not recommended. Vaccination with an antigen alone is frequently insufficient to elicit a protective immune response. Adjuvants are critical for use with subunit vaccines to increase the magnitude and duration of adaptive immunity. Emulsion adjuvants effectively induce humoral responses and are optimal for vaccines requiring dose-sparing of the antigen, such as pandemic influenza vaccines (2). Vaccines that effectively induce a potent Th1 cellular response need additional components in the adjuvant, such as MPL (a TLR-4 agonist). MPL is present in the GSK adjuvants AS01 (liposomal formulation plus QS21; a saponin), AS02 (combined with a water-in-oil emulsion plus QS21), and AS04 (combined with Alum) (3). Of the MPL-containing adjuvants, only AS04 is approved for use in humans for use with HBV (Fendrix) and HPV (Cervarix) vaccines (3). AS02A (GSK), is currently being evaluated in human clinical trials with the TB subunit vaccine M72 (4). We hypothesized that our candidate TB antigen, ID93, would elicit protection against Mtb infection when combined with GLA-SE [a synthetic TLR-4 agonist (GLA), formulated in a stable oil-in-water emulsion (SE)] due to the potent Th1-inducing properties afforded by the TLR-4 component (5, 6).

Previously we published work showing that our lead TB vaccine candidate, ID93 combined with GLA-SE, boosts the effects of BCG and protects mice against a low dose aerosol (LDA) infection with Mtb (7). ID93 is a fusion of four Mtb proteins, including Rv2608, Rv3620, Rv1813 and Rv3619. Each protein is segregated into different Mtb protein categories: Rv2608 falls within the PE/PPE family of proteins, Rv3619 and Rv3620 are in the EsX family of virulence factors, and Rv1813 is associated with latent growth of Mtb and is expressed during hypoxia (8). ID93 was developed following rigorous screening of a large panel of Mtb recombinant proteins (8). Proteins were pre-selected based on their ability to induce IFN-γ from healthy human purified protein derivative [PPD(+)] peripheral blood mononuclear cells (PBMCs). A subset of these proteins was tested further, each individually combined with CpG in the Mtb aerosol mouse model, in order to determine whether they could reduce lung bacterial load in infected mice (8). In the current study, the mouse and guinea pig models were selected to test the prophylactic efficacy of ID93 by measuring bacterial burden within the lungs of mice, and by monitoring survival and lung pathology following Mtb challenge in guinea pigs (9). Guinea pigs develop lung pathology during pulmonary TB that resemble some aspects of pathology observed in infected humans, including necrotic centers within the granulomatous lesions (9). In this study, we report that a Th1 immune response is generated in ID93/GLA-SE immunized mice and bacterial burden is decreased in the lungs of mice aerogenically infected with Mtb. In addition, we show that ID93/GLA-SE induces antigen-specific T-cell proliferative responses and is capable of extending survival and preventing severe lung pathology in guinea pigs following challenge. When ID93 is combined with an emulsion in the absence of the TLR-4 agonist, antigen-specific T-cell proliferative responses are also induced. However this adjuvant formulation fails to increase survival and actually worsens pathology in the lungs compared to untreated animals.

This work demonstrates that our candidate subunit vaccine antigen, ID93, combined with two adjuvant formulations can lead to two different outcomes. ID93 combined with the Th1 adjuvant, GLA-SE (5) enhances the quality of the immune response, leading to protection (functional reduction of bacterial burden in the lungs of Mtb infected mice, increased survival, and decreased lung pathology in guinea pigs); whereas ID93 combined with a Th2 adjuvant, oil-in-water emulsion alone (SE), lacks protection (no significant reduction of bacterial burden in the lungs of Mtb infected mice, accelerated death, and failure to protect against immunopathology in the lungs of guinea pigs).

MATERIALS AND METHODS

ID93

ID93 is a fusion protein that incorporates the three proteins which comprise ID83 (Rv1813, Rv2620 and Rv2608) (10) plus an additional Mtb protein, Rv3619, produced as previously described (7).

Immunization (Mice)

Female C57BL/6 mice, 5–7 weeks old, were purchased from Charles River Laboratories (Wilmington, MA) and were housed in the Infectious Disease Research Institute animal care facility under specific pathogen-free conditions. Ten mice per group were immunized three times, three weeks apart. Injections were administered intramuscularly (i.m.) with saline or ID93 (0.5 μg) plus either a stable oil-in-water emulsion (SE at 2%) or with GLA-SE (a synthetic TLR-4 agonist at 5 μg) formulated in the oil-in-water emulsion. Mice immunized with BCG (Pasteur strain, Sanofi Pasteur) were given a single intradermal (i.d.) dose of 5 × 10⁴ CFU at the base of the tail.

Antibody Endpoint Titers (Mice)

Mice were bled at Days 0, 14 and 56 and ID93-specific endpoint titers for IgG1, IgG2c and total IgG were performed. Briefly, Nunc Polysorp plates were coated with 2 μg/ml of recombinant protein (ID93) in 0.1 M bicarbonate and blocked overnight at 4°C with 0.05% PBS-Tween 20/1% BSA. Plates were washed and developed using SureBlue tetramethylbenzidine (TMB) substrate (Kirkegaard & Perry Laboratories Inc., Gaithersburg MD). The enzymatic reaction was stopped with 1 N H2SO4. Plates were read within 30 min at 450 nm with a reference filter set at 650 nm using a microplate ELISA reader (Molecular Devices, Sunnyvale, CA) and Soft Max Pro5 software, Inc. (San Diego, CA) with a cutoff of 0.1 absorbance unit.

Cytokine ELISPOTs (Mice)

One week after the last immunization, mice (n=3/group) were euthanized to perform IFN-γ and IL-5 ELISPOTs. A MultiScreen 96-well filtration plate (Millipore, Bedford MA) was coated with 10 μg/ml rat anti-mouse IFN-γ or IL-5 capture antibodies (eBioscience) and incubated overnight at 4°C. Plates were washed with PBS, blocked with RPMI 1640 and 10% FBS for at least 1 h at RT, and washed again. Splenocytes were plated in duplicate at 2 × 10⁵ cells/well and stimulated with media, ConA (3 μg/ml), or ID93 (10 μg/ml) for 48 h at 37°C. The plates were then washed with 0.1% PBS-Tween 20 and incubated overnight with a biotin-conjugated rat anti-mouse IFN-γ secondary Ab (eBioscience) or rat anti-mouse IL-5 secondary Ab (eBioscience) diluted 1:250 in 0.1% PBS-Tween 20/0.5% BSA. The filters were developed using the VectaStain ABC avidin peroxidase conjugate and Vectastain AEC substrate kits (Vecter Laboratories, Burlingame, CA) according to the manufacturer’s protocol. The reaction was stopped by washing the plates with deionized water. Plates were dried in the dark and spots were counted on an automated ELISPOT reader (C.T.L. Seri3A Analyzer, Cellular Technology Ltd., Cleveland, OH) and analyzed with ImmunSpot® software (CTL Analyzer LLC).

Flow Cytometry (Intracellular Cytokine Staining, Mice)

One week following the last immunization, splenocytes from 3 individual mice/group were plated at 1–2 × 10⁶ cells/well in 96-well V bottom plates and were stimulated for 12 hs with anti-CD28/CD49d (eBioscience), each at 1 ng/ml, and either media, ID93 (10 μg/ml) or PMA/Ionomycin (1 μg/ml; included as a positive control) in the presence of GolgiStop (eBioscience). The cells were fixed for 10 min with Cytofix/Cytoperm (BD Biosciences, San Jose CA), washed in PBS BSA 0.1%, incubated with Fc Block (anti-CD16/CD32, eBioscience) for 15 min at 4°C. Cells were stained with fluorochrome-conjugated mAbs anti-CD3, CD4, CD44, IFN-γ, TNF, IL-2 (eBioscience) and CD4 mAb (Invitrogen) in Perm/Wash buffer 1X (BD Biosciences) for 30 min at 4°C, washed twice in Perm/Wash buffer, suspended in PBS, and analyzed on a modified 3 laser LSRII flow cytometer (BD Biosciences). Viable lymphocytes were gated by forward and side scatter, and 20,000 CD3⁺/CD4⁺ events were acquired for each sample and analyzed with BD FACSDiva software v5.0.1 (BD Biosciences).

M. tuberculosis Infection (Mice)

Six weeks after the last immunization, mice (n=7/group) were aerogenically infected with a low dose aerosol (LDA) of M. tuberculosis H37Rv (ATCC #35718; American Type Culture Collection, Manassas, VA) using a Glas-Col aerosol generator (Terre Haute, IN) calibrated to deliver 50–100 bacteria into the lungs. A 24 hour CFU was performed (n=3 mice) to confirm the amount of bacteria delivered. Protection was determined 6 weeks after challenge by harvesting the lungs from the infected mice, homogenizing the tissue in 0.05% PBS-Tween 80, and plating five-fold serial dilutions on 7H10 agar plates (Molecular Toxicology, Inc. Boone, NC) for bacterial growth. Bacterial colonies were counted 2–3 weeks later after incubation at 37°C. Reductions in bacterial burden in the lungs were calculated as: the MeanLog10 CFU_saline - MeanLog10 CFU_vaccine.

Immunization (Guinea Pig)

Female Hartley guinea pigs (400–450 g) (Charles River Laboratories) were housed in the Infectious Disease Research Institute animal care facility under specific pathogen-free conditions. All animals were treated in accordance with the regulations and guidelines of the IDRI Animal Care and Use Committee.

Guinea pigs (n=5–7 per group) were immunized intramuscularly (i.m.) three times, three weeks apart. For these immunizations, ID93 (10 μg) was combined with either saline, SE (2%) or GLA-SE (5 μg). Animals in the BCG group were immunized intradermally (i.d.) with a single dose (5 × 10⁴) of live BCG Pasteur (Sanofi Pasteur). A University of Wisconsin-Madison aerosol exposure chamber was then calibrated to deliver 20–100 bacteria (M. tuberculosis H37Rv strain, American Type Culture Collection No. 35718) 6 weeks after the last immunization. Animals were inoculated with 50–100 bacteria in an aerosol, as determined by 24 h CFU. Weight loss and survival was monitored for greater than 200 days post challenge until all saline-treated animals died.

Antibody Endpoint Titers (Guinea Pig)

ID93-specific IgG1, IgG2 or total IgG were measured from immunized guinea pigs at 42 days (2 wks after the second immunization) or 77 days (3 wks after the third immunization). ELISA plates (Costar EIA/RIA 96 well plates, VWR) were coated with ID93 at 2.0 μg/mL in 0.1M bicarbonate coating buffer. Plates were incubated overnight at 4°C and blocked with 0.1% PBS-Tween 20, 5% milk (Carnation milk powder) for 2 h at room temperature (RT). Plates were washed in PBS-tween 0.1% followed by a single wash in 1X PBS. Sera were serially-diluted 5-fold starting at either a 1:10 or 1:100 dilution and then added to the plates for 2 h at RT. Plates were washed and secondary antibody (1:2000 dilution), either goat anti-guinea pig IgG (H+L)-, IgG1- or IgG2-conjugated to HRP (Southern Biotech), was added to the plates for 1 h at RT. Plates were washed and SureBlue TMB substrate solution (100 μL) was added to the plates for 1–2 min. The reaction was stopped with H₂SO₄ and plates were read within 30 min on a microplate ELISA reader (Molecular Devices, Sunnyvale, CA) at 450 nm with a reference filter set at 650 nm using Soft Max Pro5 software. Reciprocal dilutions corresponding to endpoint titers were determined with GraphPad Prism 4 (GraphPad Software, Inc., San Diego, CA) with a 0.1 absorbance cutoff.

Histology

A single lobe of the lung was fixed for at least 7 days in 10% normal buffered formalin. The tissues were examined grossly by a pathologist and trimmed into cassettes and paraffin processed. Tissues were arranged such that any visible gross lesions were on the tissue face to be sectioned. Each tissue block had 3 microscopic sections cut 4 μm thick, which were then mounted on glass microscopic slides and stained with hematoxylin and eosin (H&E), Fite’s Acid-Fast stain and a trichrome stain by the BioGenetics Research Laboratories, Inc. (Greenbank, WA). Qualitative analyses were performed by a board certified veterinary pathologist, Dr. Lawrence L. Kunz, D.V.M., in a blinded fashion. Two to three sections were evaluated per animal. Histological analysis of the lung included the organization of cellular infiltrate, number of granulomas, visual quantification of AFB, and pulmonary fibrosis and the severity was graded and scored by the pathologist (Supplemental Table I). The severity of the lesions were graded according to the referenced standardized grading scheme for non-neoplastic lesions (11).

Lesion grades are defined as follows: 0, normal tissue morphology with no lesion present; 1, lesions involving <10% of the tissue and minimal infiltration of fibroblasts, mononuclear, or polymorphonuclear inflammatory cells; 2, lesions affecting 10 to 20% of the tissue and mild cellular infiltration; 3, lesions covering 21 to 40% of the tissue and moderate cellular infiltration; 4, lesions covering 41 to 100% of the tissue and marked cellular infiltration. The relative number of organisms between treatment groups was determined by counting the number of AFB in 5 high power fields (HPF) (600X) of the granuloma zones containing the highest concentration of bacilli. A total of five HPF counts were averaged. Slides were viewed on a Nikon Labophot-2 microscope. High-resolution digital microscopic images (16x to 1000x magnification) were obtained using a Nikon D5000 digital microscopic camera and a Nikon Super CoolScan 5000 microscopic scanner (1x magnification). The results are summarized in Supplementary Table 1.

PBMC Isolation (Guinea Pig)

Cardiac bleeds were performed on anesthetized guinea pigs for isolation of PBMCs (4 mL). Heparinized blood was obtained from guinea pigs and diluted 1:1 with PBS. Diluted blood was placed over a Ficoll-Paque Premium 1.084 (GE Healthcare, Piscataway, NJ) gradient and spun for 25 min at 2000 rpM. Cells at the interface were collected using a 2 ml pipette and washed several times with PBS. Cells were counted using a Guava ViaCount® assay on a Guava personal cell analysis machine (Guava Technologies, Hayward, CA) and frozen in 90% heat inactivated-FBS/10% DMSO (Sigma, St. Louis, MO).

eFluor670 Cell-Labeling for Proliferation (Guinea Pig)

Frozen guinea pig PBMC were thawed, washed twice with PBS and resuspended in 0.5 ml PBS. Cell Proliferation Dye eFluor670 (4 μM in 0.5 ml) (eBioscience) was added, mixed and incubated at 37°C for 10 min in the dark. Cold RPMI plus 10% FBS, 1% penicillin/streptomycin and 0.1% 2-betamercaptoethanol (cRPMI) (in 12 ml) was added to cells and incubated on ice for 5 min to stop the reaction. Cells were spun down and washed twice with cRPMI.

In Vitro Cell Stimulation (Guinea Pig)

eFluor670-labeled PBMC were cultured for 5 days at 37°C with 5% CO₂ in round-bottom 96 well plates at up to 5 × 10⁵ cells per well for each condition. The following culture conditions were tested: (a) cRPMI only, (b) BSA-coated beads (3 μl/well), (c) ID93-coated beads (3 μl/well), (d) ID93 peptide pool (ID93pp, 0.5 μg/ml), (e) 0.4% DMSO (Sigma Hybri-Max) control for ID93pp (selected samples only) or 1 μg/ml ConA (Sigma-Aldrich). Beads were 3 μm latex Polybeads (Polysciences, Inc., Warrington, PA). 15 μl of beads (21 × 10⁶) in a sterile eppendorf tube were washed 3 times with sterile dH₂O, resuspended in 50 μl of 0.33 mg/mL BSA (Sigma-Aldrich) or ID93 in PBS, and incubated on a rotator at 4°C overnight. 250 μl of cRPMI was added to the bead suspensions before dispensing into cultures.

Flow Cytometry and Antibodies (Guinea Pig)

After 5 days of culture, plates were spun and supernatant removed. Cells were stained with mouse anti-guinea pig CD4-RPE and mouse anti-guinea pig CD8-FITC (both from Serotec, Raleigh, NC) in PBS, 5% HI-FBS, 0.05% sodium azide (Sigma-Aldrich). Stained cells were washed and suspended in PBS, 5% HI-FBS, 0.05% sodium azide. Dead cells were excluded by propidium iodide (Calbiochem, La Jolla, CA) fluorescence. The lymphocyte population of live cells was selected on a side scatter vs. forward scatter plot and displayed as CD4-RPE vs. CD8-FITC. The eFluor670 histograms of CD4⁺ and CD8⁺ populations were displayed, and the percent proliferation was determined by decreased fluorescence intensity. Fluorescence data were acquired using a FACS Calibur cytometer (BD Biosciences, San Jose, CA) with Cell Quest Pro software (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR).

Statistical Analysis

One-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test, was used for statistical analysis of antibody titers. A Mann-Whiney t-test was used for some analyses as indicated. Log-rank test was used for statistical comparisons of median guinea pig survival among the experimental groups. P values ≤0.05 were considered significant.

RESULTS

ID93/GLA-SE induces Th1-biased responses whereas ID93/SE induces Th2-biased responses in mice

Mice were immunized with ID93 adjuvanted with either a standard oil-in-water emulsion (SE) or with a synthetic TLR-4 agonist mixed in an emulsion (GLA-SE). The two vaccine formulations (SE and GLA/SE) had distinct effects on the antibody isotype response, as determined by antibody ELISA (Figure 1). ID93/SE induced significant levels of ID93-specific IgG1 antibody titers following immunization, indicative of a Th2-biased response. In contrast, ID93/GLA-SE immunized mice induced greater ID93-specific IgG2c responses, indicative of a Th1-based response. The cytokine profiles in the mouse model, as determined by ELISPOT assay, are also reflective of the adjuvant-induced biases. Mice immunized with ID93/SE induced a significantly higher frequency of IL-5-secreting T-cells whereas ID93/GLA-SE induced a greater frequency of IFN-γ-secreting T-cells (Figure 2). Immunization with ID93/GLA-SE but not ID93/SE also led to the generation of polyfunctional CD4⁺ T-cells expressing IFN-γ, TNF-α and IL-2 (Figure 3).

Figure 1.

Increased ID93-specific IgG2c endpoint titers are induced following mouse immunization with ID93/GLA-SE; increased IgG1 titers are induced with ID93/SE. Serum was collected two weeks after the first immunization (Day 14) or two weeks after the last immunization (Day 56). Mean reciprocal dilutions are represented as the endpoint titer (Log10) ± SD. Asterisks represent statistical significance, where p<0.05, comparing ID93/SE to ID93/GLA-SE. A two-tailed Mann Whitney t-test was used to determine significance. This is one representative study of at least two independent studies.

Figure 2.

ID93/GLA-SE increases IFN-γ responses whereas ID93/SE increases IL-5 responses. Mice were immunized 3 times, 3 weeks apart and spleens were harvested from individual mice (n=3/group) one week following the last immunization. The frequency of (A) IFN-γ, or (B) IL-5 secreting T-cells was determined by ELISPOT. Results are represented as spot forming units/million cells ± SD. The asterisks indicate statistical significance compared to saline, where p<0.05. This is one representative study of at least two independent studies.

Figure 3.

ID93/GLA-SE induces polyfunctional CD4⁺ T-cells (IFN-γ, TNF-α, IL-2). Cytokine production from ID93-specific CD4⁺ T-cells in immunized mice (n=3 individual mice/group) was measured by flow cytometry. Splenocytes from vaccinated mice were stimulated with ID93 for 12 hs in the presence of GolgiStop. ID93-stimulated splenocytes were identified by ICS based on CD3 and CD4 expression and were further gated on CD44^high cells. The results are represented as the percent frequency of cells expressing triple cytokines (IFN-γ, TNF-α and/or IL-2) double cytokines, or single cytokines ± SD for each of the groups. The asterisks indicate significance by students t-test, where p<0.05 of ID93/SE compared to ID93/GLA-SE. The number sign indicates significance by students t-test compared to saline, where p<0.05. This is one representative study of at least two independent studies.

ID93/GLA-SE reduces bacterial burden in the lungs of mice

A Th1-mediated cellular response is an important factor in the generation of protective immune responses against Mtb. Here we show that the same antigen, ID93, given with either a Th1-inducing adjuvant (GLA-SE) or a Th2-inducing adjuvant (SE) leads to either a significant reduction in lung bacterial burden (as seen with ID93/GLA-SE) or to no significant reduction in lung bacterial load (as observed with ID93/SE) (Table 1). These different outcomes demonstrate the importance of adjuvant formulation with a TB vaccine protein, such as ID93.

Table 1.

CFU reduction in the lungs 6 weeks post 3^rd immunization

Groups	Log10	SD	SE	Log10 Protection vs Saline	p-value vs Saline
Saline	6.38	0.19	0.07
BCG	5.83	0.19	0.07	0.55	p<0.05
ID93/SE	6.15	0.12	0.05	0.23	p>0.05
ID93/GLA-SE	5.94	0.14	0.05	0.44	p<0.05

Immunological responses to vaccination with ID93 vaccines in guinea pigs

Antigen-specific antibody responses were measured and unlike the mouse data, the IgG1 and IgG2 responses were not found to be different in guinea pigs immunized with either ID93/SE or ID93/GLA-SE after the boost immunization (Figure 4). Significant IgG1 and IgG2 antigen-specific antibody responses were observed to ID93 in both vaccine groups compared to the saline injected guinea pigs, representing an IgG1/IgG2 mixed antibody response.

Figure 4.

ID93-specific IgG1, IgG2 and total IgG endpoint antibody titers in guinea pigs following a boost immunization (Day 42). (A) IgG1, (B) IgG2, (C) Total IgG. For statistical analysis, *p<0.05 compared to saline.

To further characterize immune responses, proliferative responses to ID93 were measured following stimulation of PBMCs from immunized guinea pigs. PBMCs were isolated by Ficoll-Paque gradient as previously described (12) and labeled with eFluor670 (a cell proliferation dye). Labeled PBMCs were cultured for five days with either saline, BSA-beads (used as a negative control), ID93-beads, or a peptide pool encompassing the entire ID93 protein (Rv2608, Rv1813, Rv3620, Rv3619). Cells were stained with anti-CD4 and anti-CD8 and gated as shown (Figure 5A). Dead cells were excluded by propidium iodide fluorescence. As expected, CD4⁺ or CD8⁺ T-cell proliferative responses were not observed from guinea pigs injected with saline only (Figure 5B). CD4⁺ T-cell proliferative responses were enhanced in both the ID93/SE and ID93/GLA-SE groups compared to either media or BSA-beads; however, the results were not considered statistically significant (Figure 5B). Statistically significant CD8⁺ T-cell proliferative responses to ID93 were observed in the group given ID93/GLA-SE (Figure 5B).

Figure 5.

ID93-specific proliferative responses in guinea pig PBMCs following immunization with either ID93/SE or ID93/GLA-SE. PBMCs were isolated 3 weeks after the third immunization and frozen. Thawed PBMCs were labeled with Cell Proliferation Dye eFluor670, cultured for 5 days with the indicated stimulators, stained with anti-CD4-RPE and anti-CD8-FITC antibodies and dead cells labeled with propidium iodide (PI). Proliferation of CD4⁺ or CD8⁺ lymphocytes were defined as a decrease in eFluor670 fluorescence intensity in live cells and expressed as either % of live CD4⁺ cells or % of live CD8⁺ cells ± SEM.(A) Gating strategy; (B) Percent proliferation of CD4⁺ or CD8⁺ T-cells from stimulated PBMCs. An additional study was performed in guinea pigs using the same adjuvants with an ID83 subunit vaccine which resulted in similar results.

ID93 plus GLA-SE improves survival in guinea pigs following aerosol challenge with Mtb whereas ID93 plus SE worsens survival

To test the protective efficacy of ID93, weekly weight measurements were performed (data not shown) and long-term survival to Day 210 following challenge with Mtb was included as a readout in guinea pigs. ID93/GLA-SE significantly improved survival in guinea pigs compared to unimmunized guinea pigs (p<0.05) (Figure 6) with a mean survival time of 210 days (Supplemental Table 1). ID93 combined with SE, however, was not protective (Figure 6, Supplemental Table 1). ID93/SE appeared to exacerbate infection, where the animals died slightly earlier (mean survival of 143 days) than animals given saline alone (mean survival of 170 days); however these responses were not statistically different. BCG, similar to ID93/GLA-SE, significantly protected guinea pigs following Mtb challenge (Figure 6).

Figure 6.

ID93/GLA-SE confers long-term protection against Mtb in guinea pigs. Guinea pigs were injected with saline or were immunized with BCG, ID93/SE, or ID93/GLA-SE. The data is represented as percentage survival of guinea pigs over time following challenge with Mtb to Day 210, at which point all saline-treated guinea pigs succumbed to infection. Log-rank test was used for statistical comparisons of median guinea pig survival among the experimental groups. P values ≤0.05 were considered significant. An additional study was performed in guinea pigs using the same adjuvants with an ID83 subunit vaccine which resulted in similar results.

ID93/GLA-SE but not ID93/SE vaccination reduces lung pathology

Histology was performed on the lungs of immunized animals following infection with Mtb and extensively analyzed by a board certified pathologist in a blinded fashion. Lesion scores are based on the percentage of lung comprised by lesions, the nature of the cellular infiltration, and the extent of collagen deposition, as an indication of fibrosis. The presence of acid fast bacilli (AFB) present in the lung was also determined. Three out of five ID93/GLA-SE immunized guinea pigs (which were protected following challenge) had moderate histopathological tissue response grades (grade 3); histology was not performed for one guinea pig which died on Day 136 (Supplemental Table 1). Animals within this group exhibited lesions involving 21–40% of the lung with <1 bacilli/HPF (Figure 7, Supplemental Table 1). One guinea pig from this group had marked lung tissue involvement (grade 4, 41–100%) and a large number of bacilli were present within the lung tissue (21 bacilli/HPF) (Supplemental Table 1). Animals immunized with BCG were assigned scores ranging from 1 to 3: two grade 3’s, two grade 2’s and two grade 1’s. Grade 1 responses are considered minimal, where there is less than 10% lung involvement and minimal infiltration of fibroblast and mononuclear inflammatory cells into the tissue. Grade 2 lesions correspond to mild histological changes, where between 11–20% of the lung tissue was involved. (Figure 7, Supplemental Table 1). Four out of five animals in the saline control group and four out of six animals in the ID93/SE group had grade 4, or marked histological changes, whereby 41–100% of the lung was involved; lung tissue was not available for one ID93/SE-immunized guinea pig (Supplemental Table 1). Marked diffuse or focal infiltration of fibroblast, mononuclear or polymorphonuclear inflammatory cells into the tissue was observed in the lungs (Figure 7, Supplemental Table 1). Interestingly, all lungs contained only 0–2 bacilli/HPF, suggesting that the extensive pathology rather than the lack of bacterial containment may account for mortality in the guinea pigs given saline or ID93/SE (Figure 7, Supplemental Table 1).

Figure 7.

Histology of representative lung lobes from each group. Lungs were stained with Masson’s Trichrome stain (1X magnification). Collagen (indicative of lung fibrosis) is stained blue, the cell nuclei are stained black, and the cytoplasm stains red. The dark purple areas indicate lung consolidation. (A) Saline, lung from guinea pig euthanized on Day 135; (B) BCG, Day 234 (collected at the end of study); (C) ID93/SE, guinea pig euthanized on Day 143; (D) ID93/GLA-SE; Day 234 (collected at the end of study).

DISCUSSION

The imminent threat of multiple drug resistant strains of Mtb and the lack of protection afforded by BCG in adults has led to the development of many new experimental TB vaccines, some of which are currently in human clinical trials (13). These vaccines will likely be used (if approved) to boost the existing response elicited by BCG, which is given at birth. Even so, a vaccine that is effective without the need of BCG priming could also be of clinical use, particularly in immune-compromised populations (such as HIV infected people) where the attenuated BCG vaccine could potentially replicate without containment from the host’s immune response. In the current study, we investigated the protective capacity of the fusion protein, ID93, against Mtb in both the mouse and guinea pig models. The vaccine was combined with either a Th1-inducing adjuvant (GLA-SE) or an emulsion with Th2-inducing properties (SE), in non-BCG primed guinea pigs. In this paper we demonstrate the importance of adjuvant formulation on protective efficacy.

Both ID93 in a heterologous BCG prime/boost guinea pig study, and the closely related ID83 antigen and ID93 in a prophylactic mouse model have been shown to protect against Mtb in the presence of GLA-SE (7, 10). This is the first report in guinea pigs where we show that ID93 combined with GLA-SE is protective as a prophylactic vaccine, without a BCG prime, against Mtb. We also demonstrate the necessity to properly formulate such vaccines to elicit a desired immunological response. When ID93 is combined with SE alone, a Th2 cytokine IL-5 response is elicited that fails to protect against Mtb and is detrimental in both models. In contrast, the addition of GLA to the adjuvant formulation (GLA-SE) has immuno-stimulatory properties that enhance early innate immune responses such as enhanced IL-12 production, which promotes a Th1-mediated immune response, known to be protective in the host response against Mtb. Previously, we demonstrated that GLA stimulates cytokine production specifically through binding TLR-4 on both murine and human monocyte/macrophage derived cells lines (10). In addition, GLA signals through MyD88 and/or TRIF-dependent signaling pathways (5). The stimulation of dendritic cells (DC) with GLA-SE leads to increased immunological functions such as dendritic cell maturation (increased HLA-DR, CD40, CD83, CD86 and CCR7) and upregulation of MHC-II on antigen presenting cells, in addition to the production of many pro-inflammatory cytokines and chemokines (5). Recently, we also demonstrated that mRNA for IL-12 and TNF-α are upregulated following stimulation of guinea pig whole blood, PBMCs, or splenocytes with several different TLR agonists (12), including the TLR-4 agonist GLA. GLA-SE is also promising when combined with vaccines for influenza and malaria. We reported that the inclusion of GLA-SE with a seasonal influenza vaccine, Fluzone, increases both the magnitude and breadth of the antibody response against antigenically drifted influenza virus strains (14). By using a massively parallel signature sequencing approach (MPSS) we demonstrated that immunization with Plasmodium vivax antigen, PvRII, combined with GLA-SE enhances the hosts antibody repertoire to include a much broader recognition of B-cell variable region sequences (15). Furthermore, the Leishmania sp. subunit vaccine antigen, Leish-110f, which has been tested both pre-clinically and clinically, is protective in mice in the presence of GLA-SE, while no efficacy against parasite challenge is observed in the presence of the emulsion without the TLR-4 ligand (16). Agger et al. also showed in mice that combining an Ag85B-ESAT6 subunit vaccine with aluminum hydroxide, Al(OH)3 results in a Th2 response, similar to the oil-in-water emulsion in our study, which provided no protection against Mtb, whereas a strong Th1-inducing adjuvant (cationic liposomes containing monophosphoryl lipid A) combined with the same subunit vaccine elicited a protective Th1 immune response (17).

The results of these studies show that the ID93/GLA-SE vaccine protects against aerosol infection with Mtb in the mouse and guinea pig models. Demonstration of efficacy with ID93/GLA-SE in non-BCG primed guinea pigs was observed by increased long-term survival following Mtb challenge. Histological analysis of lung tissue from animals vaccinated with ID93/GLA-SE suggests that the animals were protected against the severe pathology that was observed in the saline and ID93/SE treated groups. A fine balance exists between control of bacterial load and preservation of host tissue. Some evidence suggests that blocking immunopathology may be more important than inducing a strong Th1 response, especially when the Th1 response is mixed with a pre-existing Th2 response (18). One hypothesis regarding the lack of effectiveness in BCG vaccinated individuals in developing countries is the presence of Th2-type responses driven by helminth infection at the time of immunization (18).

Many of the proteins contained in ID93 are also produced in BCG. This observation is of interest, since vaccination with ID93 may allow for potential boosting in BCG-immunized individuals. Previously, we showed that ID93/GLA-SE is capable of boosting BCG-primed guinea pigs by extending survival following Mtb infection over that seen with BCG alone (7). Interestingly, BCG-immunized guinea pigs responded to the ID93 fusion protein with a delayed-type hypersensitivity (DTH) response (data not shown). Animals immunized with ID93 vaccines did not elicit a DTH response to PPD, but, were able to induce a DTH response to the vaccine antigen (data not shown). These data suggest that the ID93/GLA-SE vaccine is able to generate recall responses to BCG, and phenotypic differences in the responding T-cells are likely responsible for the protection elicited by the ID93/GLA-SE formulated vaccine. We are currently investigating both innate and adaptive responses to ID93/GLA-SE in guinea pigs that could correlate with the observed protective responses.

In conclusion, we demonstrate the efficacy of ID93 when combined with the Th1-inducing adjuvant GLA-SE in both the mouse and guinea pig models. We demonstrate that immunization with this vaccine antigen combined with GLA-SE leads to Th1-biased CD4⁺ T-cell responses and decreases bacterial burden within the lungs of mice infected with Mtb. ID93/GLA-SE also induces antigen-specific T-cell proliferative responses, increases survival against Mtb and preserves the lung tissue in infected guinea pigs. In contrast, ID93 combined with a Th2-biased adjuvant fails to protect against Mtb in either the mouse or guinea pig, and may worsen the outcome of disease in guinea pigs following challenge. These results suggest that careful attention to the formulation of adjuvants should be considered in the development of subunit TB vaccines.