CFP10 is a small mycobacterial protein that is recognized by most persons who are infected with M. tuberculosis
], in part because it contains promiscuous epitopes that bind with high affinity to multiple HLA alleles [19
]. However, CFP10 has no epitopes for C57Bl/6 mice [20
], the most commonly used strain for the study of the immune response to M. tuberculosis
. By evaluating the response to CFP10 in DR4 C57Bl/6 mice, the current report demonstrated several important findings. First, we found that the course of infection with aerosolized M. tuberculosis
was similar in DR4 and C57Bl/6 mice and that CD4+ cells from DR4 mice produce IFN-γ in response to CFP10 during natural M. tuberculosis
infection, indicating that they recognize epitopes in the context of human DRB1*0401. Second, immunization with a DNA vaccine encoding CFP10 elicited production of IFN-γ by systemic CD4+ cells which was enhanced by lysosomal targeting using a vaccine construct encoding a fusion protein of CFP10 and LIMP-2. Finally, a single intravenous dose of the CFP10-based DNA vaccine coated with PEI was the only strategy that yielded a high frequency of IFN-γ-producing CD4+ cells in the lungs and a significant reduction in the pulmonary bacillary burden. These findings indicate that CFP10 elicits protective immunity by CD4+ T cells and is a potential vaccine candidate. In addition, we conclude that coating vaccines with PEI can enhance the local pulmonary immune response to mycobacterial antigens.
CD4+ T cells play a pivotal role in the human immune response to tuberculosis, and optimal vaccination strategies hinge on a comprehensive understanding of the capacity of specific mycobacterial Ags and epitopes to elicit protective T cell responses. To address this issue, we took advantage of a transgenic mouse that expresses human HLA-DRB1*0401, which has previously been used to study human metabolic diseases. DR4 mice express chimeric MHC class II molecules on their APCs, with the Ag-binding domains of DRB1*0401, and the remaining domains from murine IEd
–α and -β chains [21
], allowing recognition of Ag in the context of DRB1*0401, but signaling and interactions with murine CD4 through murine portions of the MHC molecule. To our knowledge, this is the first report in which human HLA-transgenic animals have been used to evaluate the potency of a vaccine candidate and to evaluate the effects of different vaccine delivery systems on the systemic and local immune response to a pulmonary pathogen. T cells from C57Bl/6 mice have previously been reported not to recognize CFP10 or its peptides [20
], and we confirmed that CD4+ cells from M. tuberculosis
-infected C57Bl/6 mice did not produce IFN-γ in response to CFP10 (). In contrast, approximately 0.3–0.5% of all CD4+ splenic T-cells from infected DR4 mice produced IFN-γ in response to CFP10 or its CFP1071-90
peptide (), indicating that these animals recognized Ag in the context of DRB1*0401.
Local immunity is critical for protection against infection by gastrointestinal and pulmonary pathogens [28
]. Vaccination with live attenuated oral polio virus, influenza A virus, and Salmonella
results in effective mucosal immunity, as well as systemic humoral and cellular immune responses [29
]. Also, establishment of M. tuberculosis
infection in the lungs has been attributed to delayed initiation of adaptive immunity, especially by CD4+ T cells [16
]. Therefore, local immunity in the lung is likely to play a critical role in protecting against tuberculosis. Previous studies have demonstrated that intranasal DNA vaccination efficiently deposits DNA plasmid in lungs [30
] and can generate potent cell mediated immune responses in murine models [31
]. However, in our hands immunization with naked DNA intranasally induced no response.
As an alternative means to enhance the local pulmonary immune response, we coated our DNA vaccine constructs with PEI, a cationic polymer that compacts DNA by electrostatic interactions, protecting it from enzymatic degradation and providing an overall positive charge that facilitates uptake by cells and enhances transfection efficiency [32
]. PEI is particularly effective at delivering complexed DNA to pulmonary tissue [33
]. In the current report, PEI induced a high frequency of IFN-γ-producing CFP10-specific splenic CD4+ T-cell after a single intravenous immunization, comparable to results obtained with two subcutaneous immunizations with the non-PEI-coated LIMP-2 construct ( and ). Furthermore, intravenous administration of PEI-coated DNA was the only method that elicted a vigorous CFP10-specific pulmonary response, with approximately 1% of lung CD4+ cells producing IFN-γ, compared to 0.2% of splenic CD4+ cells (). This potent local response was only induced by intravenous, but not by subcutaneous, administration of the PEI-coated CFP10-based DNA vaccine (), as subcutaneous administration would not be expected to deliver CFP10 to the lung. It was notable that intranasal immunization with the PEI-coated vaccine yielded a minimal pulmonary and systemic CD4+ IFN-γ response (data not shown) perhaps because through the intranasal route vaccine did not reach the lungs and/or it was taken up by nasal associated lymphoid tissues, rather than being deposited in the lower airways.
For several antituberculosis vaccines, the magnitude of the systemic IFN-γ response does not correlate with the capacity to lower the burden of organisms after challenge with M. tuberculosis
]. Similarly, subcutaneous administration of the LIMP-2 CFP10 fusion construct did not elicit protective immunity (), despite a substantial splenic CD4+ T-cell IFN-γ response (). The PEI-coated DNA vaccine, the only preparation to induce a strong pulmonary immune response, significantly reduced the lung and lymph node bacillary burdens (). This vaccine also increased the percentage of CD4+ effector memory cells in the lungs (), suggesting that these cells contribute to inhibition of bacterial growth in the early stages of infection. The PEI-coated vaccine did not reduce the splenic bacillary burden, indicating that a mucosal response alone does not prevent bacterial dissemination and replication in other organs. We speculate that combining a PEI-coated vaccine with other strategies to foster strong systemic immune responses could reduce extrapulmonary bacillary growth. Alternatively, or in addition, coating vaccines that encode several immunogenic M. tuberculosis
Ags, such as ESAT-6, Ag 85 and CFP10 with PEI, may elicit a more potent mucosal immune response that could drastically lower the pulmonary and systemic burden of organisms. Additional studies are needed to evaluate these strategies. Although PEI-coated DNA vaccines in their current form cannot readily be used in humans, our data provide proof of principle that mechanisms that enhance pulmonary delivery of DNA vaccines can protect against pulmonary tuberculosis.
In summary, we demonstrated that DR4 mice transgenic for human HLA DRB1*0401 could be used to demonstrate the capacity of vaccination with CFP10 to elicit production of IFN-γ by Ag-reactive CD4+ cells. Lysosomal targeting by fusion of CFP10 to LIMP-2, combined with PEI coating to deliver the vaccine to the lungs, yielded a potent pulmonary immune response and reduced the bacillary burden after challenge with M. tuberculosis. These findings indicate that CFP10 is a potential vaccine candidate, and that coating vaccines with PEI is an effective means to enhance the local mucosal immune response to mycobacterial infection.