Specificity and efficacy of dendritic cell-based vaccination against tuberculosis with complex mycobacterial antigens in a mouse model

Summary

Dendritic cells (DC) likely play important and unique roles in the generation of protective immunity to mycobacteria. In order to clarify their contributions, bone marrow-derived DC loaded with Mycobacterium tuberculosis sonicate antigens were used to stimulate T cell proliferation both in vitro and in vivo and to vaccinate C57BL/6 mice against subsequent challenge with virulent mycobacteria. Antigen-pulsed DC developed in fetal calf serum (FCS-DC), but not DC developed in normal mouse serum (NMS-DC), stimulated significant proliferation of both naïve and immune T cells in vitro. The difference between cell populations developed in FCS and NMS in the content of CD11c⁺ cells and in production of key cytokines indicated that NMS is less supportive for the development of activated DC. However, following adoptive transfer of a single dose of antigen-pulsed DC into naive recipients, NMS-DC induced T cells that proliferated in response to mycobacterial antigen, whereas FCS-DC stimulated strong non-specific proliferation. Vaccination with two doses of antigen-pulsed NMS-DC by the subcutaneous route induced significant protection against intravenous challenge with a moderate dose of virulent M. tuberculosis. DC-vaccinated mice exhibited significant reductions in bacillary loads in the lungs and spleens, and markedly reduced lung pathology. Three doses of antigen-pulsed NMS-DC induced a significant increase in survival time following high dose challenge, which correlated with a significant increase in IFN-γ-producing cells in both lung and lymphoid tissues, as assessed by the ELISPOT assay. Taken together, these results indicate that DC play a critical role in the induction of protective resistance against virulent mycobacterial challenge accompanied by the development of antigen-reactive, IFN-γ-producing T cells, and that their antigenic specificity is influenced by the culture conditions under which the DC are developed.

Introduction

The only vaccine against tuberculosis (TB) presently available for human use is BCG. Although there is convincing evidence that it is highly successful in protecting against miliary, disseminated infection and TB meningitis in children,1, 2 its efficacy against pulmonary infection in adults is quite variable. In certain clinical trials, BCG failed to prevent pulmonary TB,³ indicating an urgent need for more effective vaccines to decrease the transmission of M. tuberculosis. One vaccination approach that has received significant attention is improvement of the existing BCG vaccine by using heterologous prime-boost strategies, and some experimental results look promising.4, 5 Another attractive strategy is genetic modification of BCG in order to improve its performance. There is evidence that the insertion of particular genes, in conjunction with switching off other genes, in BCG may significantly improve its protectivity in a mouse model.6, 7, 8 This approach, however, is complicated by concerns about the safety of BCG in populations infected with HIV,9, 10 and the effects of genetic manipulations on the attenuation and stability of modified BCG strains.

Several purified or recombinant M. tuberculosis proteins, alone or in cocktails, have been shown to induce protective immunity,11, 12 however, powerful adjuvants are required to generate strong immune responses and protection. Importantly, the only adjuvant licensed for use in humans, aluminium hydroxide, biases immunity towards type 2-like responses, which are not protective for TB.¹³ Other powerful “type 1-skewing” adjuvants, including monophosphoryl lipid A,⁹ are currently being considered for human use.

Inaba, Steinman and others14, 15, 16 demonstrated that injection of dendritic cells (DC) loaded with several antigens are capable of initiating T cell responses in the absence of additional adjuvants. There is ample evidence that DC efficiently engulf attenuated, as well as virulent, mycobacteria and are capable of inducing antimycobacterial immunity,15, 17, 18, 19 especially the CD4⁺ T cell response which is generally considered to be an essential element in resistance to TB.20, 21 In the lung, immature DC are strategically located adjacent to pulmonary airways and distal alveoli and are critical for the initiation of the early local cellular immune response against lung pathogens.22, 23 There is substantial interest in the use of DC as a natural, non-inflammatory, adjuvant-replacing TB vaccine component. Although wide practical application of DC-based TB vaccines is unlikely, due to technical and financial difficulties, and the requirement for syngenic administration, their study in experimental systems is of utmost importance to enhance our understanding of the fundamental mechanisms of protective immunity to TB.

Several studies have been published on the use of infected or mycobacterial antigen-loaded DC to protect experimental animals against TB challenge. DC infected in vitro with either M. bovis BCG¹⁷ or M. tuberculosis H37Rv²⁴ were able to confer some degree of protection against TB infection in mice in adoptive transfer assays. This was not surprising, given that BCG vaccine is always protective in murine TB models when administered alone. On the other hand, vaccination with DC loaded with individual antigens derived from mycobacteria provided less convincing results. While the capacity of Ag-loaded DC to induce strong T cell responses in vivo was readily observed,25, 26 protection against subsequent virulent challenge was either absent²⁵ or very weak and non-specific.²⁶

The issue of the specificity of DC-induced immunity is of particular importance. DC usually are developed in vitro in the presence of 10 per cent fetal calf serum (FCS), i.e. high concentrations of foreign serum proteins, and very likely acquire the ability to present these proteins to the recipient's T cells after reaching full activation status in vivo following adoptive transfer. Such non-specific, “bystander” reactions may even have some protective effect due to T cell proliferation and cytokine production, but they lack the most important feature of true vaccination—specific immunological memory. The importance of the lack of specificity of DC-based vaccines that have been developed in FCS was addressed in a tumor-challenge model²⁷ but received little attention in experimental TB models, probably because mycobacterial antigenic stimuli are intuitively considered too strong to be modifiable by serum components. Thus, besides establishing conditions for immunization with antigen-pulsed DC to induce in mice protection against subsequent virulent TB challenge superior to that reported earlier,15, 17, 18, 24 we found it worthy to compare the specificity of anti-mycobacterial immune responses elicited by DC developed in the presence of FCS or autologous serum and to assess protective efficacy of mycobacterial antigen-loaded DC that never encountered other foreign antigenic stimuli. To this end, we evaluated the phenotypic characteristics of bone marrow-derived DC developed under different conditions, investigated their capacity to induce primary and secondary T cell responses to a complex mixture of mycobacterial antigens (M. tuberculosis sonicate) in vitro and in vivo and analysed the specificity of these responses.