The high incidence of TB around the world and the inability of BCG to protect certain populations clearly indicate that an improved vaccine against TB is needed. Currently, many vaccines are under development and there is a desire to simplify vaccination schedules by decreasing the number of doses. With this purpose, various substances have been added to vaccines and certain formulations have been devised in an attempt to render vaccines more effective . Despite of success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amount of plasmid for effective immunization, which could lead to an exacerbated inflammatory reaction in the lungs of challenged mice or guinea pigs. To optimize this DNA vaccine, we used an approach where adjuvants with targeting and immunostimulatory properties prepared by microencapsulation techniques are administered in conjunction with the DNA-encoding antigen. BALB/c mice immunized with a single dose of Me-DNAhsp65/TDM-loaded microspheres produced high levels of IgG2a subtype antibody and high amounts of IFN-γ in mice as previously described [20, 24]. Here we show that Me-DNAhsp65/TDM-loaded microspheres were also able to confer protection as effective as that attained after three doses of naked DNA administration either in mice or guinea pigs. This new formulation also allowed a ten-fold reduction in the DNA dose when compared to naked DNA as well as a significant reduction in the cellular infiltrate in the lung parenchyma of mice and guinea pigs. Thus, this combination of DNA vaccine and adjuvants with immunomodulatory and carrier properties holds the potential for an improved vaccine against TB. PLGA biodegradable microspheres also have the potential to act as mediators of DNA transfection targeted to phagocytic cells such as macrophages or dendritic cells, and to protect against biological degradation by nucleases [28, 29]. We previously show that DNAhsp65-loaded microsphere without adjuvant was unable to protect mice against challenge . Thus, the entrapment of DNA plus an immunostimulant compound into PLGA microspheres could be an interesting strategy for vaccine formulation. The adjuvant effect of purified TDM on immune response has been recognized long ago . The immunostimulatory activities made TDM an attractive candidate for adjuvant use in vaccine formulation. Moreover, the polymer has an established clinical safety record and its slow degradation permits sustained delivery of antigen . Hence, if the quality of the immunity is dependent on antigen persistency, or if compliance is compromised due to socio-economic or demographic circumstances, PLGA-like microspheres offer a potential advantage for the vaccines.
A more recently devised tool for generating a protective and long-lasting immune response involves combining different vehicles carrying the same immunogen in heterologous prime-boost protocols . These prime-boost vaccination strategies consist of using two different vaccines, each encoding the same antigen, administered some weeks apart. Most prime-boost protocols currently under evaluation include priming with DNA and boosting with viral vectors [32, 33]. This strategy resurrects questions concerning the safety of using live attenuated viruses that have been replaced by subunit or DNA vaccines alone. In the prevention of TB, the prime-boost strategy of combining DNA priming and boosting with BCG or recombinant proteins has been evaluated by various authors [17, 19]. Such protocols typically require more than one DNA dose for priming, followed by a booster with live vectors, thereby necessitating the use of large quantities of DNA. As shown above, the encapsulation of antigen into PLGA microspheres allows the development of controlled-release delivery systems, in which the release profile of the encapsulated material can be tailored to specific purposes. Taking advantage of this fact, we evaluated the use of a vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNA-hsp65 and the rhsp65 (Figure 1). Our aim was to achieve DNA priming and protein boosting after a single-dose vaccination. We demonstrated previously in mice , that the Me-Prime/boost formulation induced high levels of anti-hsp65 antibodies and IFN-γ, which remained high 90 days after vaccination, whereas the Me-DNAhsp65/TDM formulation was unable to sustain antibody levels in the same fashion. Here we show that mice or guinea pigs challenged with a virulent strain of M. tuberculosis 30 days after vaccination, we observed significantly lower numbers of CFUs in the lungs of those vaccinated with Me-Prime/boost formulation than in the lungs of the controls. Moreover, we showed that the infection remained under control and the lung parenchyma unaffected only in the group immunized with the Me-Prime/boost formulation. These data suggest that Me-Prime/boost is a formulation capable of sustaining the protective response in mice and guinea pigs. Therefore, using biodegradable microspheres in a single dose seems to be a promising strategy for stimulating long-lasting immune responses in large animals.
In this study, we set out to overcome significant obstacles currently faced in for the field of DNA vaccine development. We described, for the first time, the development of a single dose/prime-boost DNA vaccine formulation for immunizing mice and guinea pigs against mycobacterial challenge. This new technology allows radically different approaches to the problems of immunization with DNA vaccines. Furthermore, using combinations of vaccines and alternative routes of administration will allow researchers to customize vaccination programs. Moreover, this new technique may increase veterinarian and patient acceptance of vaccination by reducing the number of injections and avoiding the use of boosters containing live vectors. Using this technology, vaccinologists could develop many DNA vaccines that would induce specific forms of immunity, access new routes of delivery, provide increased safety when necessary, be more stable and lower costs. We believe that this strategy can be applied to vaccines for humans and to other veterinary vaccines, thereby having a tremendous impact on the control of infectious diseases in humans and in large animals.