Suppression of major histocompatibility complex (MHC) class II antigen presentation is believed to be among the major mechanisms used by Mycobacterium tuberculosis to escape protective host immune responses. Through a genome-wide screen for the genetic loci of M. tuberculosis that inhibit MHC class II-restricted antigen presentation by mycobacteria-infected dendritic cells, we identified the PE_PGRS47 protein as one of the responsible factors. Targeted disruption of the PE_PGRS47 (Rv2741) gene led to attenuated growth of M. tuberculosis in vitro and in vivo, and a PE_PGRS47 mutant showed enhanced MHC class II-restricted antigen presentation during in vivo infection of mice. Analysis of the effects of deletion or over-expression of PE_PGRS47 implicated this protein in the inhibition of autophagy in infected host phagocytes. Our findings identify PE_PGRS47 as a functionally relevant, non-redundant bacterial factor in the modulation of innate and adaptive immunity by M. tuberculosis, suggesting strategies for improving antigen presentation and the generation of protective immunity during vaccination or infection.

Solids dispersion due to the simultaneous diffusion (random particle motion) and classification (segregation) of fluidized spheres has been investigated. The model developed permits calculation of the mean concentration of spheres of both sizes in a mixture of two sizes of fluidized spheres os a function of bed length. Fick's law has been applied to the diffusional phenomenon.A hypothesis has been advanced and confirmed which permits calculation [Equation (511 of the classification velocity for each size of sphere in a fluidized mixture. Calculation of the classification velocities in a mixture of spheres is based on the relation of void fraction to superficial velocity for the individual sizes of spheres. To facilitate carrying out these calculations, a generalized equation has been developed for the relation of void fraction to superficial velocity in terms of the Galileo number (d3gAppf/p2) and sphere-to-column diameter ratio. Measurement of the bead size grodients (change in bead size with bed length) at steady state fluidization was used to investigate dispersion. Closely sized glass beads of 0.1 and 0.2 cm. diameters were fluidized with liquids of 1 and 15 cps. a t void fractions of 0.5 to 0.8 in 2.5 and 5.0 cm. columns. Lead beads of 0.12 cm. diameter were also used. Further, several experiments were conducted by a method which is analogous to that used in molecular diffusion cells.The unique transport behavior which is observed in a fluidized bed is, of course, due to the mobility of the solid phase in addition to that of the fluid phase. The work reported here is an investigation of the diffusion and classification of spheres which have been fluidized with a liquid. Diffusion is used to describe the random mixing of the particles which is observed in fluidized beds. Classifcation refers to the tendency of a mixture of fluidized particles of different sizes or densities to separate into layers which are of the same size or density. These two mechanisms are the sources of dispersion which are considered here. As will be explained in more detail, since only the average concentration variations in the direction of fluid flow were measured, consideration is given only to dispersion in the axial direction. PREVIOUS WORKRichards ( 1 ) has shown in a remarkable photograph the importance of both classification and diffusion in the dispersion occurring in a fluidized bed of quartz and galena. Classification is clear in that the larger and denser particles tend to move to the bottom of the bed. Diffusion is observed since a wider range of particles is found in each section of the bed than would be possible if classification were not offset by mixing of the particles in the axial direction. In his work it is pointed out that the greater the terminal velocity of a single particle in an infinite media, the lower is the equilibrium level to which the particle tends to move, as a result of classification. This observation was observed regardless of the void fraction of the fluidized bed.Brotz ( 2 ) used a method somewha...

Tuberculosis (TB) due to Mycobacterium tuberculosis remains a major global infectious disease problem, and a more efficacious vaccine is urgently needed for the control and prevention of disease caused by this organism. We previously reported that a genetically modified strain of Mycobacterium smegmatis called IKEPLUS is a promising TB vaccine candidate. Since protective immunity induced by IKEPLUS is dependent on antigen-specific CD4 ϩ T cell memory, we hypothesized that the specificity of the CD4 ϩ T cell response was a critical feature of this protection. Using in vitro assays of interferon gamma production (enzyme-linked immunosorbent spot [ELISPOT] assays) by splenocytes from IKEPLUS-immunized C57BL/6J mice, we identified an immunogenic peptide within the mycobacterial ribosomal large subunit protein RplJ, encoded by the Rv0651 gene. In a complementary approach, we generated major histocompatibility complex (MHC) class II-restricted T cell hybridomas from IKEPLUS-immunized mice. Screening of these T cell hybridomas against IKE-PLUS and ribosomes enriched from IKEPLUS suggested that the CD4 ϩ T cell response in IKEPLUS-immunized mice was dominated by the recognition of multiple components of the mycobacterial ribosome. Importantly, CD4 ϩ T cells specific for mycobacterial ribosomes accumulate to significant levels in the lungs of IKEPLUSimmunized mice following aerosol challenge with virulent M. tuberculosis, consistent with a role for these T cells in protective host immunity in TB. The identification of CD4 ϩ T cell responses to defined ribosomal protein epitopes expands the range of antigenic targets for adaptive immune responses to M. tuberculosis and may help to inform the design of more effective vaccines against tuberculosis.

Summary The development of more effective vaccines against Mycobacterium tuberculosis (Mtb) remains a major goal in the effort to reduce the enormous global burden of disease caused by this pathogen. Whole-cell vaccines based on live mycobacteria with attenuated virulence represent an appealing approach, providing broad antigen exposure and intrinsic adjuvant properties to prime durable immune responses. However, designing vaccine strains with an optimal balance between attenuation and immunogenicity has proven to be extremely challenging. Recent basic and clinical research efforts have broadened our understanding of Mtb pathogenesis and created numerous new vaccine candidates that are designed to overcome different aspects of immune evasion by Mtb. In this review, we provide an overview of current efforts to create improved vaccines against tuberculosis based on modifications of live attenuated mycobacteria. In addition, we discuss the use of such vaccine strains as vectors for stimulating protective immunity against other infectious diseases and cancers.

remains a threat to global health, and a more efficacious vaccine is needed to prevent disease caused by We previously reported that the mycobacterial ribosome is a major target of CD4 T cells in mice immunized with a genetically modified strain (IKEPLUS) but not in mice immunized with BCG. Two specific ribosomal proteins, RplJ and RpsA, were identified as cross-reactive targets of , but the breadth of the CD4 T cell response to ribosomes was not determined. In the present study, a library of ribosomal proteins and -predicted peptide libraries were used to screen CD4 T cell responses in IKEPLUS-immunized mice. This identified 24 out of 57 ribosomal proteins distributed over both large and small ribosome subunits as specific CD4 T cell targets. Although BCG did not induce detectable responses against ribosomal proteins or peptide epitopes, the ribosomal protein RplJ produced a robust and multifunctional Th1-like CD4 T cell population when administered as a booster vaccine to previously BCG-primed mice. Boosting of BCG-primed immunity with the RplJ protein led to significantly reduced lung pathology compared to that in BCG-immunized animals and reductions in the bacterial burdens in the mediastinal lymph node compared to those in naive and standard BCG-vaccinated mice. These results identify the mycobacterial ribosome as a potential source of cryptic or subdominant antigenic targets of protective CD4 T cell responses and suggest that supplementing BCG with ribosomal antigens may enhance protective vaccination against .