Application of Mycobacterial Proteomics to Vaccine Design: Improved Protection by Mycobacterium bovis BCG Prime-Rv3407 DNA Boost Vaccination against Tuberculosis

Abstract

Information from comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin (BCG) principally allows prediction of potential vaccine candidates. Thirty-six M. tuberculosis DNA vaccine candidates identified by comparative proteome analysis were evaluated in the mouse model for protection against low-dose aerosol M. tuberculosis infection. We identified the DNA vaccine candidate Rv3407 as a protective antigen and analyzed putative major histocompatibility complex class I epitopes by computational predictions and gamma interferon Elispot assays. Importantly, we discovered that the DNA vaccine Rv3407 improved the efficacy of BCG vaccination in a heterologous prime-boost vaccination protocol. Our data demonstrate the rationale of a combination of proteomics, epitope prediction, and broad screening of putative antigens for identification of novel DNA vaccine candidates. Furthermore, our experiments show that heterologous prime-boost vaccination with a defined antigen boost "on top" of a BCG primer provides superior protection against tuberculosis over vaccination with BCG alone.Together with AIDS and malaria, tuberculosis remains a major health threat, particularly in developing countries (50). Frequently, chemotherapy with at least three drugs over a period of several months is hampered by insufficient compliance, resulting in dramatically increasing incidences of multidrug-resistant strains of Mycobacterium tuberculosis (9). The currently available vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is the most widely used viable vaccine worldwide (2,11,12,43). BCG apparently fails to protect against the most common form of the disease, pulmonary tuberculosis in adults, but it prevents miliary tuberculosis in newborns and toddlers (11,25). This raises two issues: first, novel tuberculosis vaccine candidates, which protect against adulthood tuberculosis, are urgently needed (25, 38); second, BCG vaccination of newborns should be continued and not be given up prematurely. A solution which fulfills both requirements can be offered by heterologous prime-boost vaccination strategies comprised of priming with BCG and boosting with a novel vaccine candidate (4, 14).In preclinical trials, genetic immunization with naked DNA has proven to be a powerful antigen discovery tool and vaccination protocol (8, 16). DNA vaccines use episomal vectors expressing antigens under eukaryotic promoters in the vaccinated host and therefore can elicit potent humoral and cellular immune responses comprising both CD4 and CD8 T cells (8,16). Several mycobacterial DNA vaccines have been shown to elicit protective immune responses in mice (7,10,21,23,31,35,(45)(46)(47)(48). However, considerable amounts of DNA are required, particularly in nonhuman primates and humans. In clinical trials, multiple high doses of DNA were required for the induction of immune responses (5, 27, 49).Elucidation of the complete genome of M. tuberculosis has provided a blueprint for the search for novel vaccine ...

Two‐dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry

“…The newly identified antigens are being used to develop vaccines, e.g. for pathogenic bacteria, such as H. pylori and M. tuberculosis [121][122][123][124]. New data on cell differentiation and on cell regulation can be obtained using the total cell protein approach, and new insights in functional genomics can be derived using these data.…”

“…From the mycobacterial unique spots, 36 were evaluated in a mouse model as DNA vaccines and one vaccine improved the efficacy of BCG vaccination [124]. In comparison with the proteome from H. pylori samples from gastric cancer and ulcer patients were examined and candidate antigens for serological detection found [125].…”

Comparative study of Mycobacterium tuberculosis and Mycobacterium bovis protein profiles

“…These strains were cultured in Middlebrook 7H9 medium for 4 weeks. The supernatants of stationary-phase liquid cultures were collected and used to extract proteins [10]. In order to extract membrane and secretory proteins, strains were cultured into Middle Brook 7H9 broth at 37 ° C and maintained for 4 weeks in order to get in logarithmic growth phase.…”

The Impact of Bioinformatics on Vaccine Design and Development

“…One strategy applied for vaccine design is to identify the structures present only in M. tuberculosis and absent in Mycobacterium bovis BCG [43]. In addition, the vaccine candidates studied presented the characteristics described previously, such as nonhuman homology, adhesins [44], secreted or membrane structures [45,46] with low transmembrane helix, and in addition, the proteins expressed in the latent or active state of the microorganism [47].…”

“…Some of these have been expressed and proven in vitro and in vivo, demonstrating their immunogenicity and protective efect. Among these are highlighted the ESAT-6, PE and PPE protein family group [51], and the Ag85 protein family, which obtained beter immune response than the BCG vaccine in an animal model [43].…”