Humoral immune responses and protective efficacy of sequential B- and T-cell epitopes of V antigen of Yersinia pestis by intranasal immunization in microparticles.

Capsular F1 and secretory V antigen are the putative vaccine candidates for plague, caused by Yersinia pestis. Contemplating this, we studied the immunogenicity and protective efficacy of collinearly synthesized B- and T-cell epitopes (B-T constructs) of V antigen entrapped in poly (DL-lactide-co-glycolide) microparticles immunized intranasally using single dose immunization schedule in outbred, H-2(b) and H-2(d) mice. High antibody levels were observed in terms of IgG, IgA and SIgA peak titers in sera and mucosal washes to different B-T constructs. The constructs ai, bi and fi especially showed high peak antibody titers ranging from 51,200 to 204,000, which were maintained till day 120 post immunization. IgG/IgA Specific activity in sera and washes correlated well with the peak antibody titers. Moreover, all the B-T constructs showed mixed IgG1 and IgG2a/2b response, variable immunoreactivity as well as memory response with V antigen. B-T constructs, viz ai, ak, bi, fi, di and ik showed comparatively high isotype levels. These constructs showed high immunoreactivity, and good recall response with V antigen. Finally, in vivo protective study in BALB/c mice demonstrated the protective efficacy of three B-T constructs (ai, bi and fi) against lethal doses of Yersinia pestis till day 20 post challenge, while construct 'id' showed partial protection.

Semen characteristics: Advancement in andrological assessment.

Progress in diagnosis of infertility, has been dramatically increased during the past decades with changes occurring in virtually all aspects of infertility research, thus providing innovative diagnostic testing and sophisticated instrumentation for improved management and treatment of infertility. There are about 50% of infertile couples who are suffering because of male infertility. Semen examination is a basic investigation for these infertile couples. It not only reveals the quantity and quality of sperm but also the quality of the seminal plasma, which is essential for normal sperm function. In this review, the recent advancement in investigation procedures has been analyzed which are very important in clinical practice to (a) evaluate the sperm fertilizing ability (Acrosin, aniline blue, HOS), (b) characterization of male accessory sex glands secretions (Fructose, alpha-glucosidase, PSA) and (c) the management of azoospermic patients. It is believed that use of such diagnostic procedures will facilitate wide selection of patients for whom an effective therapy might be then possible.

Asymptomatic memory CD8+ T cells: from development and regulation to consideration for human vaccines and immunotherapeutics.

Generation and maintenance of high quantity and quality memory CD8(+) T cells determine the level of protection from viral, bacterial, and parasitic re-infections, and hence constitutes a primary goal for T cell epitope-based human vaccines and immunotherapeutics. Phenotypically and functionally characterizing memory CD8(+) T cells that provide protection against herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) infections, which cause blinding ocular herpes, genital herpes, and oro-facial herpes, is critical for better vaccine design. We have recently categorized 2 new major sub-populations of memory symptomatic and asymptomatic CD8(+) T cells based on their phenotype, protective vs. pathogenic function, and anatomical locations. In this report we are discussing a new direction in developing T cell-based human herpes vaccines and immunotherapeutics based on the emerging new concept of "symptomatic and asymptomatic memory CD8(+) T cells."

Evaluation of CD4+/CD8+ T-cell expression and IFN-γ, perforin secretion for B-T constructs of F1 and V antigens of Yersinia pestis.

Yersinia pestis is a facultative bacterium that can survive and proliferate inside host macrophages and cause bubonic, pneumonic and systemic infection. Understanding the immune response generated by epitopes recognized by CD4+ and CD8+ T cells is important for the development of safe and effective vaccines designed to promote protective cellular immunity. Apart from humoral response, CD4+ T cells have shown to have a major role in combating the pneumonic form of the disease. In the present study, the secretion of IFN-γ and IL-4 by splenocytes, stimulated by different constructs of B and T cell epitopes of F1 and V antigens, was measured by ELISpot assay. We also measured perforin and IFN-γ expression as a function of cell mediated immunity by flow cytometry. Three B-T constructs of F1 and seven B-T constructs of V antigens produced a high number of IFN-γ secreting cells as compared to native antigen and a low number of IL-4 secreting cells. B-T conjugates of F1 and V antigens showed significantly high (p<0.001) percentage of CD4+ IFN-γ(+) cells as compared to CD8+ IFN-γ(+) cells. Thus, the study highlights the importance of Th1 cytokine and existence of high proportion of CD4+ T cells probably contributing protection in the host. This study proposes a new perspective for the development of vaccination strategies for Y. pestis that trigger T cell immune response.

Identifying B and T cell epitopes and studying humoral, mucosal and cellular immune responses of peptides derived from V antigen of Yersinia pestis.

Yersinia pestis is a Gram negative bacterium which causes bubonic and pneumonic plague. Out of the two protective antigens F1 and V as vaccine candidates, we focused our attention on V antigen as peptide-based immunogen. Eleven peptides of varying lengths were synthesized for mapping major antigenic sites on V antigen. Six peptides showed competition by three complimentary approaches (1) with anti-V sera, (2) with anti-peptide sera and (3) by direct binding assay. Peptides which showed competition were found to be highly immunogenic in terms of peak antibody titres (both IgG and IgA) with longer duration, showed predominantly IgG2a/IgG2b subclass distribution, high affinity/avidity indices with single immunization schedule through intranasal routes. After intranasal immunization, these peptides showed high IgA response in sera and SIgA in intestinal washes and bronchoalveolar lavages while intramuscular immunization failed to give mucosal response. Intramuscular immunization generated high titre antibodies (both IgG and IgA) in sera with negligible SIgA response in washes which required additional booster immunization. All the peptides sera reacted with V antigen in Western blot analysis. During T cell proliferation assay, five peptides showed lymphocyte proliferation response irrespective of how antigen priming and pulsing was done in three strains of mice. These five peptides also showed Th1 cytokine profile whereas rest of the peptides showed mixed Th1/Th2 response. Two peptides exclusively showed high levels of IL-10 production. Mathematical modeling of V antigen indicates that these peptides lie on the surface of the protein. In conclusion, peptides a, g and j are pure B cell epitopes; peptides d and k are pure T cell epitopes and peptides b, f and i showed both B and T cells property on V antigen.