Cellular Immune Responses to Nine Mycobacterium tuberculosis Vaccine Candidates following Intranasal Vaccination

BACKGROUND: The identification of Mycobacterium tuberculosis vaccines that elicit a protective immune response in the lungs is important for the development of an effective vaccine against tuberculosis. METHODS AND PRINCIPAL FINDINGS: In this study, a comparison of intranasal (i.n.) and subcutaneous (s.c.) vaccination with the BCG vaccine demonstrated that a single moderate dose delivered intranasally induced a stronger and sustained M. tuberculosis-specific T-cell response in lung parenchyma and cervical lymph nodes of BALB/c mice than vaccine delivered subcutaneously. Both BCG and a multicomponent subunit vaccine composed of nine M. tuberculosis recombinant proteins induced strong antigen-specific T-cell responses in various local and peripheral immune compartments. Among the nine recombinant proteins evaluated, the alanine proline rich antigen (Apa, Rv1860) was highly antigenic following i.n. BCG and immunogenic after vaccination with a combination of the nine recombinant antigens. The Apa-induced responses included induction of both type 1 and type 2 cytokines in the lungs as evaluated by ELISPOT and a multiplexed microsphere-based cytokine immunoassay. Of importance, i.n. subunit vaccination with Apa imparted significant protection in the lungs and spleen of mice against M. tuberculosis challenge. Despite observed differences in the frequencies and location of specific cytokine secreting T cells both BCG vaccination routes afforded comparable levels of protection in our study. CONCLUSION AND SIGNIFICANCE: Overall, our findings support consideration and further evaluation of an intranasally targeted Apa-based vaccine to prevent tuberculosis

Attrition of T-cell functions and simultaneous upregulation of inhibitory markers correspond with the waning of BCG-induced protection against tuberculosis in mice.

Mycobacterium bovis bacille Calmette-Guérin (BCG) is the most widely used live attenuated vaccine. However, the correlates of protection and waning of its immunity against tuberculosis is poorly understood. In this study, we correlated the longitudinal changes in the magnitude and functional quality of CD4(+) and CD8(+) T-cell response over a period of two years after mucosal or parenteral BCG vaccination with the strength of protection against Mycobacterium tuberculosis in mice. The BCG vaccination-induced CD4(+) and CD8(+) T cells exhibited comparable response kinetics but distinct functional attributes in-terms of IFN-γ, IL-2 and TNF-α co-production and CD62L memory marker expression. Despite a near life-long BCG persistence and the induction of enduring CD4(+) T-cell responses characterized by IFN-γ and/or TNF-α production with comparable protection, the protective efficacy waned regardless of the route of vaccination. The progressive decline in the multifactorial functional abilities of CD4(+) and CD8(+) T cells in-terms of type-1 cytokine production, proliferation and cytolytic potential corresponded with the waning of protection against M. tuberculosis infection. In addition, simultaneous increase in the dysfunctional and terminally-differentiated T cells expressing CTLA-4, KLRG-1 and IL-10 during the contraction phase of BCG-induced response coincided with the loss of protection. Our results question the empirical development of BCG-booster vaccines and emphasize the pursuit of strategies that maintain superior T-cell functional capacity. Furthermore, our results underscore the importance of understanding the comprehensive functional dynamics of antigen-specific T-cell responses in addition to cytokine polyfunctionality in BCG-vaccinated hosts while optimizing novel vaccination strategies against tuberculosis

Peripheral Blood and Pleural Fluid Mononuclear Cell Responses to Low-Molecular-Mass Secretory Polypeptides of Mycobacterium tuberculosis in Human Models of Immunity to Tuberculosis

A total of 104 polypeptides were purified from the low-molecular-mass secretory proteome of Mycobacterium tuberculosis H(37)Rv using a combination of anion exchange column chromatography and high resolution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution. The goal of this study was to identify polypeptides from a low-molecular-mass secretory proteome recognized by human subjects infected with M. tuberculosis and to ascertain the differences in specificity of antigen recognition by the peripheral blood mononuclear cells (PBMCs) and pleural fluid mononuclear cells (PFMCs) of these individuals. The study identified CFP-8 (Rv0496), CFP-11 (Rv2433c), CFP-14.5 (Rv2445c), and CFP-31 (Rv0831c) as novel T-cell antigens apart from previously characterized ESAT-6, TB10.4, CFP10, GroES, MTSP14, MTSP17, CFP21, MPT64, Ag85A, and Ag85B on the basis of recognition by PBMCs of tuberculosis contacts and treated tuberculosis patients. Further, polypeptides prominently recognized by PFMCs of tuberculous pleurisy patients were the same as those recognized by PBMCs of healthy contacts and treated tuberculosis patients. The results of our study indicate the homogeneity of antigenic target recognition by lymphocytes at the site of infection and at the periphery in the human subjects studied and the need to evaluate these antigenic targets as components of future antituberculous vaccines

T-cell dysfunction markers are up-regulated during waning of BCG responses.

<p>(A) The expression of KLRG-1 on the pooled spleen T cells of age-matched naïve, i.n. or s.c. BCG-vaccinated mice (<i>n</i> = 4) at week 78. (B) Longitudinal changes in the magnitude of KLRG-1⁺ CD4⁺ and CD8⁺ T cells from the lung and spleen. (C) CTLA-4 and PD-1 expression on the pooled splenic CD4⁺ T cells of vaccinated or age-matched naive mice (<i>n</i> = 4) at week 78. (D) Summary of magnitude of CTLA-4⁺ CD4⁺ and CD8⁺ T cells in the lung and spleen. (E) The WCL-specific IL-10 production by the pooled splenic CD4⁺ T cells of vaccinated or age-matched naïve mice. (F) Summary of magnitude of WCL-specific IL-10⁺ CD4⁺ and CD8⁺ T cells in the lung and spleen. (G–H) The spleen and lung cells isolated from i.n. BCG-vaccinated mice at week 78 were treated with rIL-2 to evaluate potential to reinvigorate WCL-specific CD4⁺ and CD8⁺ T-cell responses. (G) The WCL-specific IFN-γ, IL-4 and IL-17 SFU after treatment with or without rIL-2. (H) The magnitude of WCL-specific cytokine-producing 7 subsets of CD4⁺ and CD8⁺ T cells by flow cytometry. The data in figure B, D, F are mean ± s.e.m. responses of 2 independent experiments using pooled organ cells (<i>n</i> = 4 mice/time point/group). * <i>P</i><0.05, ** <i>P</i><0.01 compared to the corresponding 12-week time point by 1-way ANOVA with Tukey's post-test. 5G, H are mean ± s.e.m responses of pooled cells evaluated in triplicates. *Significant using 1-way ANOVA with Tukey's post-test.</p

Waning of BCG-induced protection against <i>Mtb</i> coincides with decreased T-cell functional capacity.

<p>(A–C) BCG-vaccinated or age-matched naïve mice were infected with <i>Mtb</i> Erdman, and 6 weeks later the spleen and lungs were isolated. (A) Differences in <i>Mtb</i> load between vaccinated and control mice are shown. The data are mean ± s.e.m. <i>Mtb</i> CFU in each organ at eight different time points involving two to four independent experiments each using five individually analyzed mice per time point. (*<i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001, compared to the corresponding age-matched naïve control by Kruskal-Wallis test followed by Dunn's post-test). (B) The longitudinal changes in the frequency of rESAT-6+rCFP-10-specific total CD4⁺ cytokine⁺ (of IFN-γ, IL-2 and TNF-α) T cells from the lung (upper panel) and spleen (lower panel) of vaccinated and naïve mice 6 weeks after challenge (*<i>P</i><0.05, ** <i>P</i><0.01 compared to the corresponding week 32 BCG-vaccinated mice by 1-way ANOVA and Tukey's post-test). At the 32-week time point, rESAT-6+rCFP-10-specific responses in naïve mice are statistically higher compared to the corresponding vaccinated groups (<i>P</i><0.05). (C) The magnitudes of WCL-specific total CD4⁺ cytokine⁺ T cells before and 6 weeks after <i>Mtb</i> challenge. The data are mean ± s.e.m. responses measured by intracellular cytokine staining (ICS) of four mice per time point. (*<i>P</i><0.05 comparing pre- and post-challenge WCL-specific responses using 1-way ANOVA and Tukey's post-test) (D) The schematic summary of longitudinal changes in BCG load in the lung and spleen of i.n. and s.c. BCG-vaccinated mice over a period of 2 years and its correlation with the magnitude and functional capacity of T cells and levels of protection against <i>Mtb</i> infection.</p

BCG-induced T cells predominantly exhibit effector or effector memory phenotype and their proliferative potential wanes with time.

<p>(A–B) The proliferation of CD4⁺ and CD8⁺ T cells from the lung and spleen of the BCG-vaccinated mice was investigated based on CFSE-dilution at 72 h following WCL or no antigen stimulation. (A) Representative plots illustrate the proliferation of pooled lung CD4⁺ and CD8⁺ T cells of vaccinated mice (<i>n</i> = 4) from one experiment at the expansion and contraction phase. (B) The longitudinal changes in the proliferative potential of the lung and spleen CD4⁺ or CD8⁺ T cells from the two vaccinated groups at 4 different time points were summarized and are presented as the fold increase in proliferation compared to unstimulated controls (*<i>P</i><0.05, **<i>P</i><0.01 using 1-way ANOVA with Tukey's post-test compared to corresponding week 12). (C) The CD44 and CD62L expression on the pooled splenic CD4⁺ and CD8⁺ T cells of vaccinated mice (<i>n</i> = 4) from one representative experiment at the expansion and contraction phase are shown. The green dots represent WCL-specific IFN-γ-producing T cells. (D) The magnitudes of WCL-specific IFN-γ-producing CD44⁺CD62L⁺ and CD44⁺CD62L⁻ CD4⁺ and CD8⁺ T cells from the lung and spleen of two groups at week 12 and 52. The data in (B) and (D) are mean ± s.e.m. responses of 2 independent experiments using pooled organ cells (<i>n</i> = 4 mice/time point/group).</p

BCG vaccination induces polyfunctional CD4⁺ but monofunctional CD8⁺ T-cell response.

<p>(A–C) The lung and spleen cells of i.n. and s.c. BCG-vaccinated mice (<i>n</i> = 4/time point/group) were stimulated with WCL, and the magnitudes and polyfunctionality of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells in-terms of IFN-γ, IL-2 and TNF-α production were determined using polychromatic flow cytometry. (A) The longitudinal changes in the magnitudes of WCL-specific individual IFN-γ, IL-2 or TNF-α-producing cells among CD4⁺ or CD8⁺ T cells in the lung and spleen are plotted. (B) Representative dot plots show the frequency of WCL-specific IFN-γ and IL-2 or IFN-γ and TNF-α-producing cells among splenic CD4⁺ and CD8⁺ T cells from one mouse per vaccinated group at week 32 in comparison with age-matched naïve control, and the distributions of single, double and triple-cytokine-producers are illustrated. (C) The magnitudes of 7 possible combinations of WCL-specific cytokine-producing subsets constituting total cytokine⁺ CD4⁺ or CD8⁺ T cells in the lung, spleen, cervical lymph node (CLN) and inguinal lymph node (ILN) are depicted. The data (A, C) are mean ± s.e.m. responses of 2 (at week 3, 78 and 104), 3 (at week 32) or 4 (at week 6 and 12) independent experiments.</p

BCG persists and induces a durable cell-mediated immunity in mice.

<p>(A–B) BALB/c mice were vaccinated with 1×10⁶ CFU of BCG Copenhagen by intranasal (i.n.) or subcutaneous (s.c.) route. At 7 different time points after vaccination at the same time, mice were euthanized and their lungs and spleen were isolated. (A) BCG load in the two organs of individual mice was determined. Detection limit of the assay as shown by dotted line was 30 CFU/organ, and data shown are mean CFU ± s.e.m. in the lung (left) and spleen (right) from two independent experiments using 5 individually analyzed mice per group at each time point. The experiment was not performed at the104-week time point. (*<i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001, **** <i>P</i><0.0001 using 1-way analysis of variance (ANOVA) with Tukey's post-test). (B) In parallel, pooled cells from the lung (left) or spleen (right) of BCG-vaccinated and naïve mice (<i>n</i> = 4/time point/group) were stimulated with WCL in a cultured ELISPOT assay and IFN-γ, IL-4 and IL-17A SFU were enumerated. Data are means ± s.e.m. of individual cytokine SFU constituting the total cytokine responses of 2 (at week 3, 78 and 104), 3 (at week 32) or 4 (at week 6 and 12) independent experiments evaluated in triplicate. Responses are significant (<i>P</i><0.001) in vaccinated compared to naïve mice at 6 time points after week 3 using 1-way ANOVA with Tukey's post-test.</p

Author&apos;s personal copy Bacillus Calmette-Guérin vaccination using a microneedle patch

a b s t r a c t Tuberculosis (TB) caused by Mycobacterium tuberculosis continues to be a leading cause of mortality among bacterial diseases, and the bacillus Calmette-Guérin (BCG) is the only licensed vaccine for human use against this disease. TB prevention and control would benefit from an improved method of BCG vaccination that simplifies logistics and eliminates dangers posed by hypodermic needles without compromising immunogenicity. Here, we report the design and engineering of a BCG-coated microneedle vaccine patch for a simple and improved intradermal delivery of the vaccine. The microneedle vaccine patch induced a robust cell-mediated immune response in both the lungs and the spleen of guinea pigs. The response was comparable to the traditional hypodermic needle based intradermal BCG vaccination and was characterized by a strong antigen specific lymphocyte proliferation and IFN-␥ levels with high frequencies of CD4 + IFN-␥ + , CD4 + TNF-␣ + and CD4 + IFN-␥ + TNF-␣ + T cells. The BCG-coated microneedle vaccine patch was highly immunogenic in guinea pigs and supports further exploration of this new technology as a simpler, safer, and compliant vaccination that could facilitate increased coverage, especially in developing countries that lack adequate healthcare infrastructure