Cationic liposomes formulated with synthetic mycobacterial cordfactor (CAF01): a versatile adjuvant for vaccines with different immunological requirements

Abstract

Background: It is now emerging that for vaccines against a range of diseases including influenza, malaria and HIV, the induction of a humoral response is insufficient and a substantial complementary cell-mediated immune response is necessary for adequate protection. Furthermore, for some diseases such as tuberculosis, a cellular response seems to be the sole effector mechanism required for protection. The development of new adjuvants capable of inducing highly complex immune responses with strong antigen-specific T-cell responses in addition to antibodies is therefore urgently needed.

Methods and findings: Herein, we describe a cationic adjuvant formulation (CAF01) consisting of DDA as a delivery vehicle and synthetic mycobacterial cordfactor as immunomodulator. CAF01 primes strong and complex immune responses and using ovalbumin as a model vaccine antigen in mice, antigen specific cell-mediated- and humoral responses were obtained at a level clearly above a range of currently used adjuvants (Aluminium, monophosphoryl lipid A, CFA/IFA, Montanide). This response occurs through Toll-like receptor 2, 3, 4 and 7-independent pathways whereas the response is partly reduced in MyD88-deficient mice. In three animal models of diseases with markedly different immunological requirement; Mycobacterium tuberculosis (cell-mediated), Chlamydia trachomatis (cell-mediated/humoral) and malaria (humoral) immunization with CAF01-based vaccines elicited significant protective immunity against challenge.

Conclusion: CAF01 is potentially a suitable adjuvant for a wide range of diseases including targets requiring both CMI and humoral immune responses for protection.

Figure 1. Time schedule for the immunization and infection regimen for TB, chlamydia and malaria.

Figure 2. Comparison of CAF01 with other adjuvants.

C57BL/6 mice (n = 4) were immunized three times with 100 µg of OVA in CAF01, Al(OH)3, Montanide, MPL, CFA/2×IFA boosting with a two-week interval. A) Three weeks after the last immunization, the number of OVA-specific cells was assessed by IFN-γ ELISPOT. Mean spot-forming units (SFU) upon stimulation with 5 µg of OVA per million cells ± SEM for each group is shown. B) SFU per million cells ± SEM upon stimulation with 5 µg of OVA CD4 T cell epitope. C) SFU per million cells ± SEM upon stimulation with 5 µg of OVA CD4 T cell epitope. D) Sera were analysed for the presence of OVA-specific IgG1, E) IgG2b, F) IgG2c antibodies by ELISA. Mean EC50 ± SEM is shown. Values marked with an asterisk are significantly different (*, p<0.05; **, p<0.01, p<0.001) compared to naïve controls as assessed by ANOVA and Dunnett's multiple comparison test.

Figure 3. Immune responses and protection induced by CAF01 in a TB model.

C57BL/6 mice were immunized three times with 2 µg of Ag85B-ESAT-6 in CAF01 or Al(OH)₃. A) Three weeks after the last immunization, mice were bled by periorbital puncture and individual sera tested for Ag85B-ESAT-6 IgG1, B) IgG2b or C) IgG2c by ELISA (n = 6). D) Individual cultures of splenocytes (n = 3) were harvested at the same time point and re-stimulated in vitro with different concentrations of the Ag85B-ESAT-6. The release of IFN-γ was determined by ELISA. Six weeks after the last immunization, mice were challenged by the aerosol route with virulent M. tuberculosis. Six weeks postchallenge, mice were sacrificed and the bacterial burden (CFU) measured in the E) lungs or F) spleen (expressed as log₁₀ CFU). As a positive control group, a group of mice received a BCG vaccination ten weeks before challenge. Data shown are mean values of six mice ± SEM. G) At different time points after infection, mice were sacrificed and the CFU measured in the lungs. Values marked with an asterisk are significantly different (*, p<0.05; **, p<0.01, p<0.001) compared to naïve controls as assessed by ANOVA and Dunnett's multiple comparison test.

Figure 4. CAF01-induced responses in TLR2, 3, 4, 7 and MyD88-deficient mice.

A) MyD88-/- and B) TLR2, 3, 4, 7-/- mice as well as WT controls mice (n = 3) were vaccinated twice with 2 µg of Ag85B-ESAT-6 in CAF01 by footpad immunization. Two weeks after the last immunization, the inguinal lymphnodes were harvested and restimulated in vitro with 10 µg of Ag85B-ESAT-6. The release of IFN-γ was determined by ELISA.

Figure 5. Immune responses and protection induced by CAF01 in a malaria blood-stage model.

BALB/c mice were immunized three times with 10 µg of MSP1 in CAF01 or Al(OH)₃. One week after the last immunization, mice were bled by periorbital puncture and individual sera tested for MSP1-specific A) IgG1 or B) IgG2a by ELISA (n = 5). C) Individual cultures of splenocytes (n = 3) were harvested at the same time point and re-stimulated in vitro with different concentrations of MSP1. The release of IFN-γ was determined by ELISA. D and E) Three weeks after the last immunization, mice were challenged by the i.p. route with Plasmodium yoelli and the number of infected red blood cells measured at various time points during infection. Data shown are mean values of five mice ± SEM. Values marked with an asterisk are significantly different (*, p<0.05; **, p<0.01, p<0.001) compared to Al(OH)₃-vaccinated as assessed by t-test.

Figure 6. Immune responses and protection induced by CAF01 in a chlamydia model.

C57BL/6 mice were immunized three times with 5 µg of MOMP in CAF01 or Al(OH)₃. Three weeks after the last immunization, mice were bled by periorbital puncture and individual sera tested for MOMP A) IgG1, B) IgG2b and C) IgG2c by ELISA (n = 6). D) Individual cultures of splenocytes (n = 3–6) were harvested at the same time point and re-stimulated in vitro with 5 µg of MOMP. The release of IFN-γ was determined by ELISA. E) Six weeks after the last immunization, mice were administered a vaginal challenge with 1.5×10⁵ Chlamydia muridarum IFU/mouse and the bacterial load in the vagina monitored at day 4, 7, 10, 14 and 21. Data shown are mean values of 6–7 mice ± SEM. Values marked with an asterisk are significantly different (*, p<0.05; **, p<0.01, p<0.001) compared to naïve controls as assessed by ANOVA and Dunnetts post test.