Stanley Ress

The Effect of Bacille Calmette-Guérin Vaccine Strain and Route of Administration on Induced Immune Responses in Vaccinated Infants

Vaccination with Mycobacterium bovis bacille Calmette-Guerin (BCG) has variable efficacy in preventing tuberculosis. Both BCG strain and route of administration have been implicated in determining efficacy; however, these variables are not considered in current clinical recommendations for vaccine choice. We evaluated antigen-specific immunity after percutaneous or intradermal administration of Japanese BCG or intradermal administration of Danish BCG. Ten weeks after vaccination of neonates, percutaneous Japanese BCG had induced significantly higher frequencies of BCG-specific interferon- gamma -producing CD4(+) and CD8(+) T cells in BCG-stimulated whole blood than did intradermal Danish BCG. Similarly, percutaneous vaccination with Japanese BCG resulted in significantly greater secretion of the T helper 1-type cytokines interferon- gamma, tumor necrosis factor- alpha , and interleukin-2; significantly lower secretion of the T helper 2-type cytokine interleukin-4; and greater CD4(+) and CD8(+) T cell proliferation. Thus, BCG strain and route of neonatal vaccination confer different levels of immune activation, which may affect the efficacy of the vaccine.

Neonatal mycobacterial specific cytotoxic T-lymphocyte and cytokine profiles in response to distinct BCG vaccination strategies.

This study evaluated whether different bacillus Calmette–Gue´rin (BCG) strains, routes of administration, vaccination age and percutaneous tools influenced immune responses to BCG vaccination in infants. Proliferative responses, cytokine production and cell‐mediated cytotoxicity obtained in post‐vaccinated children were compared to baseline cord bloods and unvaccinated 10‐week‐old infants. BCG vaccination generally induced strong lymphoproliferative and T helper type 1 (Th1)‐type cytokine responses. There was a trend for greater responsiveness following the intradermal route of vaccination, with Japanese‐172 strain and with delaying vaccination until 10 weeks. Cord mononuclear cells differentially stimulated the Th2‐type cytokines interleukin‐5 (IL‐5) and IL‐10 selectively in response to BCG, as compared to H37Rv or purified protein derivative stimulation. We document for the first time the generation of mycobacterium‐specific cytotoxic T lymphocytes in neonates, following BCG vaccination. Cytotoxic activity correlated with the ratio of interferon‐γ to IL‐5, aside from a single instance where use of the Biovac® tool resulted in a striking dissociation selectively against H37Rv targets. These data have implications for correlates of protective immunity in design of vaccine studies.

Immunomodulation with Recombinant Interferon-γ1b in Pulmonary Tuberculosis

Background Current treatment regimens for pulmonary tuberculosis require at least 6 months of therapy. Immune adjuvant therapy with recombinant interferon-γ1b (rIFN-γb) may reduce pulmonary inflammation and reduce the period of infectivity by promoting earlier sputum clearance. Methodology/Principal Findings We performed a randomized, controlled clinical trial of directly observed therapy (DOTS) versus DOTS supplemented with nebulized or subcutaneously administered rIFN-γ1b over 4 months to 89 patients with cavitary pulmonary tuberculosis. Bronchoalveolar lavage (BAL) and blood were sampled at 0 and 4 months. There was a significant decline in levels of inflammatory cytokines IL-1β, IL-6, IL-8, and IL-10 in 24-hour BAL supernatants only in the nebulized rIFN-γ1b group from baseline to week 16. Both rIFN-γ1b groups showed significant 3-fold increases in CD4+ lymphocyte response to PPD at 4 weeks. There was a significant (p = 0.03) difference in the rate of clearance of Mtb from the sputum smear at 4 weeks for the nebulized rIFN-γ1b adjuvant group compared to DOTS or DOTS with subcutaneous rIFN-γ1b. In addition, there was significant reduction in the prevalence of fever, wheeze, and night sweats at 4 weeks among patients receiving rFN-γ1b versus DOTS alone. Conclusion Recombinant interferon-γ1b adjuvant therapy plus DOTS in cavitary pulmonary tuberculosis can reduce inflammatory cytokines at the site of disease, improve clearance of Mtb from the sputum, and improve constitutional symptoms. Trial Registration ClinicalTrials.gov NCT00201123

The human macrophage cell line U937 as an in vitro model for selective evaluation of mycobacterial antigen-specific cytotoxic T-cell function

Despite strong evidence for CD8+ T‐cell function in murine mycobacterial infections, their corresponding role in human tuberculosis has proven more difficult to demonstrate. We have evaluated the human macrophage (Mφ) cell line U937 as an in vitro model for human leucocyte antigen (HLA) class I‐restricted presentation of mycobacterial antigens, as HLA class I is constitutively expressed at high levels by U937 cells in the absence of detectable HLA class II or CD1 molecules. U937 cells were evaluated for their ability to phagocytose Mycobacterium tuberculosis and for their ability to present mycobacterial antigens to human HLA class I‐matched cytotoxic T lymphocytes (CTLs). Differentiated U937 cells were capable of efficient phagocytosis of M. tuberculosis but did not generate a subsequent respiratory burst response, and were permissive for intracellular growth of both bacillus Calmette–Guérin (BCG) and the virulent M. tuberculosis H37Rv strain. CTL activity was restricted to live mycobacterial organisms and was shown to be mediated by M. tuberculosis‐specific, HLA class I‐matched, purified CD8+ CTL lines and CD8+ T‐cell clones. Furthermore, M. tuberculosis‐infected U937 targets were more rapidly and strongly lysed by CD8+ CTLs than were infected autologous Mφ. Finally, M. tuberculosis‐infected U937 cells simultaneously provided a sensitive indicator for detection of mycobacterial‐specific, HLA‐unrestricted γδ+ CTL activity.

Exposure of Cord Blood to Mycobacterium bovis BCG Induces an Innate Response but Not a T-Cell Cytokine Response

ABSTRACT Despite routine vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) soon after birth, tuberculosis in babies and adults remains epidemic in South Africa. The immune responses of the naïve newborn child and how they are affected by vaccination with BCG are as yet not fully understood. Immunity during pregnancy and in healthy human newborns may be skewed toward type 2 cytokine production; however, it is type 1 cytokines that are required for protection against M. tuberculosis infection. To better understand neonatal cytokine responses prior to and following exposure to mycobacteria, we have collected cord blood and peripheral blood samples and evaluated the cytokine response following ex vivo incubation with BCG. Gamma interferon (IFN-γ), interleukin 10 (IL-10), IL-12, and low levels of IL-13 and IL-5 but no IL-4 were secreted into the culture supernatant of cord blood mononuclear cells. Intracellular staining showed that IL-10 and IL-12 were produced by monocytes and that IFN-γ was produced by natural killer (NK) cells but not by CD4+ or CD8+ T cells. In contrast, in the peripheral blood samples collected from babies 13 weeks post-BCG vaccination, IFN-γ was detected within CD4+ and CD8+ cells. Taken together, the data suggest a central role for Th1 cytokines in naïve as well as BCG-vaccinated neonates in the protective immune response to tuberculosis. NK cell-derived IFN-γ produced in naïve neonates likely plays a key protective role via monocyte activation and the priming of a subsequent adaptive Th1 response.

Induction of granulysin and perforin cytolytic mediator expression in 10-week-old infants vaccinated with BCG at birth.

Background. While vaccination at birth with Mycobacterium bovis Bacilli Calmette-Guérin (BCG) protects against severe childhood tuberculosis, there is no consensus as to which components of the BCG-induced immune response mediate this protection. However, granulysin and perforin, found in the granules of cytotoxic T lymphocytes and Natural Killer (NK) cells, can kill intracellular mycobacteria and are implicated in protection against Mycobacterium tuberculosis. Methods. We compared the cellular expression of granulysin and perforin cytolytic molecules in cord blood and peripheral blood from 10-week-old infants vaccinated at birth with either Japanese or Danish BCG, administered either intradermally or percutaneously. Results. In cord blood, only CD56+ NK cells expressed granulysin and perforin constitutively. These cytolytic mediators were upregulated in CD4+ and CD8+ cord blood cells by ex vivo stimulation with BCG but not with PPD. Following BCG vaccination of neonates, both BCG and PPD induced increased expression of granulysin and perforin by CD4+ and CD8+ T cells. There was no difference in expression of cytolytic molecules according to vaccination route or strain. Conclusions. Constitutive expression of perforin and granulysin by cord blood NK-cells likely provides innate immunity, while BCG vaccination-induced expression of these cytolytic mediators may contribute towards protection of the neonate against tuberculosis.

Cellular immunity in tuberculous pleural effusions: evidence of spontaneous lymphocyte proliferation and antigen-specific accelerated responses to purified protein derivative (PPD).

The kinetics of in vitro cellular proliferation against a PPD of Mycobacterium tuberculosis or streptococcal antigen (streptokinase‐streptodornase) was evaluated in pleural fluid and peripheral blood mononuclear cells (PBMC) from patients with tuberculous and non‐tuberculous pleuritis. The peak proliferative response to PPD by mononuclear cells from pleural fluid occurred earlier (day 3) in 65% of patients with tuberculosis, a finding not seen in non‐tuberculous effusions. Spontaneous lymphocyte proliferation of both peripheral blood lymphocytes and pleural effusion lymphocytes was frequently observed, irrespective of etiology. However, 20 of 21 tuberculous patients manifesting spontaneous lymphocyte proliferation had accelerated kinetics of proliferation to PPD, which was antigen‐specific. These results suggest that spontaneous lymphocyte proliferation occurs as a response to antigen stimulation at the site of disease, and is not a non‐specific response to inflammation. Furthermore, enhanced reactivity against mycobacterial antigen, manifested by accelerated kinetics of proliferation, has diagnostic potential in patients with pleural effusions.

Dose-dependent immune response to Mycobacterium bovis BCG vaccination in neonates.

ABSTRACT In 10-week-old infants vaccinated at birth with Japanese Mycobacterium bovis BCG, the number of dermal needle penetrations correlated positively with frequency of proliferating CD4+ T cells in whole blood following BCG stimulation for 6 days but did not correlate with secreted cytokine levels after 7 h or interferon CD4+ T-cell frequency after 12 h of BCG stimulation.

HLA class II induction by interferon-γ in K562 variant cell line: inhibition by serum lipid

Abstract The induction of class II antigen (Ag) by interferon-γ (IFN-γ) in variants of the K562 cell line has been examined in this study. Following incubation of K562A cells with IFN-γ, surface expression of HLA-DR molecules was demonstrated by indirect immunofluorescence, confirmed by immunoblotting, and HLA-DR3 Ag specificity identified. HLA-DP and HLA-DRW52 Ag were co-expressed, but no HLA-DQ expression occurred. A variant of this line, designated K1A, spontaneously developed resistance to IFN-γ induction of class II Ag, but continued to express class I Ag. In a third K562 variant, designated K562B, no class II gene products could be induced. Northern blot analysis indicated that mRNA levels correlated with surface class I and II Ag expression in all of the K562 cell lines. Resista IFN-γ inducible class II Ag expression in K1A cells did not involve changes in IFN-γ receptor affinity or number but was shown to be due to an inhibitory effect of serum lipid. These results indicate that cells derived from a common parental line may differ in susceptibility to a regulatory mechanism affecting IFN-γ inducible class II Ag expression