Purified Protein Derivative Of Tuberculin Research Articles

Index of Suspicion * Case 1: Round Opacity on Chest Radiograph, Cough, and Fever in a Child * Case 2: Groin Pain and Limp in a 10-year-old * Case 3: Focal Neurologic Signs in the Presence of Sickle Cell Disease * Case 4: Hypoglycemia and Microphallus in an Infant

A 5-year-old previously healthy boy presents from an outside hospital for further evaluation because of abnormal chest radiography findings. He developed fever, cough, and runny nose 2 weeks ago and was diagnosed as having influenza A. Due to persistence of the symptoms, chest radiography was obtained that showed a round opacity in the lower lobe of the left lung (Fig. 1). Cold agglutinin titers were positive and he was treated with a course of azithromycin. His symptoms improved in 1 week. Follow-up radiograph showed persistence of the opacity without any signs of resolution. CT scan confirmed the presence of an ovoid mass (6×5×4.5 cm) in the posterior aspect of the left lung base (Fig. 2). The boy is from a rural area and has no pets and no significant travel history. He has not lost weight. Figure 1. Chest radiograph showing a round soft-tissue shadow in the left lung base. Figure 2. CT scan of the chest showing a homogenous soft-tissue mass in the left lung. On physical examination, the well-appearing boy has a temperature of 37.0°C and normal additional vital signs. Chest examination reveals reduced air entry in the left lung base with dullness to percussion. Findings on the rest of the examination are unremarkable. Laboratory investigations show Hgb of 9 g/dL (90 g/L), WBC count of 10.5×103/μL (10.5×109/L), and platelet count of 853×103/μL (853×109/L). The ESR is 120 mm/hr and C-reactive protein is 64 mg/L (6.4 mg/dL). Mycoplasma immunoglobulin M titers are positive. Blood culture and purified protein derivative tuberculin test results are negative. Serology for histoplasmosis and coccidioidomycosis also is negative. Tumor markers such as β-human chorionic gonadotropin and α-fetoprotein are undetectable. A surgical procedure with further laboratory investigation helps to establish the diagnosis. A 10-year-old boy presents with groin pain and limp …

Resposta de testes de hipersensibilidade tardia utilizando PPD e outros antígenos em crianças e adolescentes saudáveis e infectados pelo HIV-1 e vacinados com BCG

Among HIV-1-infected patients, CD4+ T cell counts are well-established markers of cell-mediated immunity. Delayed-type hypersensitivity (DTH) skin tests can be used to evaluate in vivo cell-mediated immunity to common antigens. DTH responses to tuberculin purified protein derivative (PPD), sporotrichin, trichophytin, candidin and streptokinase/streptodornase antigens were assessed. Thirty-six HIV-1-infected children/adolescents and 56 age- and sex-matched HIV-1/HIV-2-seronegative participants were tested. All participants had a BCG scar. Fisher's exact test was used to evaluate significant differences between groups (p<0.05). The main characteristics of the HIV-1 patients were as follows: median age 8.1 years; 20/36 were males; 35 were vertical transmission cases; 34 were AIDS cases under antiretroviral therapy; median viral load = 3.04 log10 copies/ml; median CD4+ T cell count = 701 cells/μl. A total of 25% (9/36) and 87.5% (49/56) of HIV-1-infected and healthy participants, respectively, displayed DTH reactivity to at least one antigen (p<0.001). Among HIV-1-infected participants, reactivity to candidin predominated (8/36, 22.2%), while PPD positivity prevailed among healthy participants (40/56, 71.4%). PPD reactivity in the HIV-1-positive group was 8.3% (p<0.01). The median PPD induration was 2.5mm (range: 2-5mm) in the HIV-1 group and 6.0 mm among healthy participants (range: 3-15 mm). There was no correlation between PPD positivity and age. No correlation between CD4+ T cell counts and DTH reactivity was observed among HIV-1-infected patients. DTH skin test responses, including PPD reactivity, were significantly lower among HIV-1-infected participants compared to healthy controls, which likely reflects advanced disease and T cell depletion.

Placenta suppresses experimental autoimmune hypophysitis through soluble TNF receptor 1

Pregnancy modulates autoimmune diseases through diverse and still incompletely defined mechanisms, in part operating at the decidua–placenta interface. To assess the immunological contribution of placenta, we administered mouse placental proteins to a mouse model of autoimmune hypophysitis, a disease known to be strongly associated with pregnancy. Emulsified placental proteins suppressed both the cellular and humoral aspects of hypophysitis. Suppression was specific to self antigens and not seen when two foreign antigens, tetanus toxoid or tuberculin purified protein derivative, were used. Proteomic analysis revealed high levels of soluble TNF receptor 1 in placenta, suggesting that blockade of the TNF-α pathway was a mechanism of disease suppression. Placentas derived from mice deficient in TNF receptor 1 lost the ability to suppress hypophysitis. Notably, hypophysitis suppression was seen only when the TNF-α pathway was blocked locally, at the site of immunization, and not systemically. These findings provide evidence that placenta contributes to the immune tolerance of pregnancy by locally inhibiting the TNF-α pathway.

A novel recombinant &amp;lt;italic&amp;gt;Mycobacterium bovis&amp;lt;/italic&amp;gt; bacillus Calmette&amp;minus;Guerin strain expressing human granulocyte macrophage colony-stimulating factor and &amp;lt;italic&amp;gt;Mycobacterium tuberculosis&amp;lt;/italic&amp;gt; early secretory antigenic target 6 complex augments Th1 immunity

Since Mycobacterium bovis bacillus Calmette-Guerin strain (BCG) fails to protect adults from pulmonary tuberculosis (TB), there is an urgent need for developing a new vaccine. In this study, we constructed a novel recombinant BCG strain (rBCG) expressing human granulocyte macrophage colony-stimulating factor (GM-CSF) and the 6 kDa early secretory antigenic target (ESAT6) of Mycobacterium tuberculosis, named rBCG:GE (expressing GMCSF-ESAT6 complex), and evaluated the immunogenicity of the construct in BALB/c mice. Our results indicated that the rBCG:GE was able to induce higher titer of antibody than the conventional BCG, the rBCG:G (expressing GM-CSF) and the rBCG:E (expressing ESAT6). Moreover, the rBCG:GE also elicited a longer-lasting and stronger Th1 cellular immune responses than the other groups, which was confirmed by the incremental proliferation of splenocytes, the increased percentages of CD4(+) and CD8(+) T cells of spleen, the elevated level of interferon-γ in splenocyte culture after tuberculin-purified protein derivative stimulation, and the increased concentration of GM-CSF in serum. The data presented here suggested the possibility that the recombinant BCG:GE might be a good vaccine candidate to TB.

Blocking TNF in vitro with infliximab determines the inhibition of expansion and interferon gamma production of Vγ9/Vδ2 T lymphocytes from patients with active rheumatoid arthritis. A role in the susceptibility to tuberculosis?

Side effects of TNF neutralisation - mostly infectious complications - were recognized, the most important being pulmonary tuberculosis infections. gamma/ d T cells contribute to protective immune response against mycobacterium tuberculosis. The aim of the present study was to assess the expansion capacity of Vgamma9/Vdelta2 T cells from (tuberculin purified protein derivative (PPD) positive and PPD negative) patients with active rheumatoid arthritis (RA), and to examine the in vitro effect of infliximab on this lymphocyte subset. 28 PPD negative RA patients were studied and compared with 14 PPD positive RA patients, 45 PPD-negative and 110 PPD-positive healthy volunteers. Cell separation, expansion in vitro of Vgamma9/Vdelta2 T lymphocytes (EF) and the expression of tumor necrosis factor receptor II and IFN-gamma content by Vgamma9/Vdelta2 T lymphocytes were studied before and after infliximab in vitro addiction. The EF from PPD positive subjects was higher than that from PPD negatives. Patients with RA have the highest levels. The addition of infliximab to the cultures from PPD-positive patients determined a significant inhibition of cell expansion and TNF RII expression and a significant decrease of IFN gamma content. In this study we have documented that gamma/ delta T lymphocytes from patients with PPD positive rheumatoid arthritis have a high capacity to respond in vitro to phosphoantingens with expansion TNF-RII expression and IFN gamma production that is inhibited by the exposure to infliximab. These results might be of relevance in view of the effect of TNF blocking on the pulmonary tuberculosis infection.

The comparative performance of the single intradermal test and the single intradermal comparative tuberculin test in Irish cattle, using tuberculin PPD combinations of differing potencies

In national bovine tuberculosis (BTB) control programmes, testing is generally conducted using a single source of bovine purified protein derivative (PPD) tuberculin. Alternative tuberculin sources should be identified as part of a broad risk management strategy as problems of supply or quality cannot be discounted. This study was conducted to compare the impact of different potencies of a single bovine PPD tuberculin on the field performance of the single intradermal comparative tuberculin test (SICTT) and single intradermal test (SIT). Three trial potencies of bovine PPD tuberculin, as assayed in naturally infected bovines, namely, low (1192IU/dose), normal (6184IU/dose) and high (12,554IU/dose) were used. Three SICTTs (using) were conducted on 2102 animals. Test results were compared based on reactor-status and changes in skin-thickness at the bovine tuberculin injection site. There was a significant difference in the number of reactors detected using the high and low potency tuberculins. In the SICTT, high and low potency tuberculin detected 40% more and 50% fewer reactors, respectively, than normal potency tuberculin. Furthermore, use of the low potency tuberculin in the SICTT failed to detect 20% of 35 animals with visible lesions, and in the SIT 11% of the visible lesion animals would have been classified as negative. Tuberculin potency is critical to the performance of both the SICTT and SIT. Tuberculin of different potencies will affect reactor disclosure rates, confounding between-year or between-country comparisons. Independent checks of tuberculin potency are an important aspect of quality control in national BTB control programmes.

The comparative performance of the single intradermal comparative tuberculin test in Irish cattle, using tuberculin PPD combinations from different manufacturers

Ireland currently obtains its avian and bovine tuberculin purified protein derivatives (PPDs) from a single source. Because problems of supply or quality cannot be discounted, it is prudent that Ireland identify alternative supplier(s) as part of a broad risk management strategy. Therefore, the aim of this study was to compare the performance of a number of different tuberculin combinations (that is, pairings of bovine and avian PPD; with different manufacturers) in the single intradermal comparative tuberculin test (SICTT), as currently performed in Ireland. The study was randomised, controlled and double-blinded. A total of 2172 cattle were used in the study. Each animal was tested using two SICTTs, the first based on the tuberculin combination in current use, and the second using one of six trial tuberculin combinations. Analyses were conducted to compare both reactor-status and skin increase. For each control/trial tuberculin combination, there was good agreement between the control and trial reactor-status. Differences in skin increases were mainly confined to animals categorised as either negative or severe inconclusive. However, the measured differences were minor, and unlikely to have a significant impact on the actual test outcome, either for individual animals or for herds. In conclusion, while further studies determining sensitivity and specificity in Ireland would have to be done in the event of a change in tuberculin PPD there should be minimal disruption of the national programme if alternative tuberculin PPDs meeting WHO, OIE and EU regulations were used. In this study, the precision of the guinea pig bio-assay to assess tuberculin potency was low and therefore Ireland should maintain its practice of periodically assessing potency in naturally infected cattle, even though this is not currently required under WHO, OIE or EU Regulations.

Effectiveness of Brucella abortus lipopolysaccharide as an adjuvant for tuberculin PPD

Bacterial lipopolysaccharide (LPS) has T-helper 1 (Th1) immunostimulatory activities but because of toxicity and pyrogenicity cannot be used as an adjuvant. Brucella abortus LPS has less toxicity and no pyrogenic properties in comparison to other bacterial LPS. In the current study, the immunostimulatory properties of B. abortus LPS were evaluated for its adjuvant activity. Tuberculin purified protein derivative (PPD) from Mycobacterium tuberculosis was extracted and after anion-exchange chromatography on Q-sepharose column, two fractions (17 and 23), which dominantly contained 30- and 70-kDa antigens, were collected for immunological studies. BALB/c mice were immunized with four different antigen preparations (BCG, PPD, 17th and 23rd PPD fractions) along with complete Freund’s adjuvant or B. abortus LPS. The T-cell immune response of mice was assessed by measurement of Th1-type cytokine (IFN-γ) and Th2-type cytokines (IL-5 and IL-10) levels. Also, the humoral immunity was evaluated by measuring the specific IgG levels. Our results showed that immunization of mice with 17th PPD fraction along with B. abortus LPS can induce a Th1-type cytokine response characterized with a high IFN-γ/IL-5 ratio, while immunization with PPD or 23rd PPD fraction along with the same adjuvant resulted to a mixed Th1/Th2-type cytokine response.

Improved Immunogenicity of Recombinant Mycobacterium bovis Bacillus Calmette‐Guérin Strains Expressing Fusion Protein Ag85A‐ESAT‐6 of Mycobacterium tuberculosis

Early secretory antigen target 6 (ESAT-6) is a dominant target for cell-mediated immunity in the early phase of tuberculosis (TB) in patients with TB, causing T-cell proliferation and gamma interferon (IFN-γ) production, which has been considered to be a protective antigen that can be used for future vaccine development. Ag85A is the most essential component for bacterial survival within macrophages and has been used in numerous vaccine preparations, which can induce strong cellular immune responses. In this study, we constructed a new recombinant bacilli Calmette-Guérin (BCG) strain (rBCG-AE) that could express fusion protein Ag85A-ESAT-6 of Mycobacterium tuberculosis and evaluated its immunogenicity in BALB/c mice. There was no evidence for increased virulence of this rBCG. Our experiments illustrated that the rBCG-AE was able to induce higher titer of antibody and elicit more long-lasting and stronger Th1 type cellular immune responses than the parental BCG strain, or rBCG-A (expressing Ag85A) strain, or rBCG-E (expressing ESAT-6) strain, which are characterized by the strong antibody response, the proliferation rate of splenocytes, the ratio of CD4(+) T and CD8(+) T cells stimulated by tuberculin-purified protein derivative and elevated levels of IFN-γ in antigen-stimulated splenocyte cultures. The results show that rBCG-AE is an improved TB vaccine for further study.

Disseminated Histoplasmosis and Pneumocystis Pneumonia in a Child With Crohn Disease Receiving Infliximab

Disseminated Histoplasmosis and Pneumocystis Pneumonia in a Child With Crohn Disease Receiving Infliximab

Hepatitis C virus NS4 protein impairs the Th1 polarization of immature dendritic cells

Dendritic cells (DCs) in chronic hepatitis C patients display impaired function, although the details remain unclear. To investigate the hepatitis C virus (HCV) protein that has the most impact on DC function, we compared five recombinant proteins and seven HCV protein genes in modulating DC phenotype and function. Immature DCs (iDCs) were established from healthy donor peripheral blood monocytes with granulocyte–macrophage colony stimulating factor (GM-CSF) and IL-4. Lipopolysaccharide was used to establish mature DCs (mDCs). Cells were then pulsed with HCV recombinant proteins or transfected with HCV plasmids and subsequently assayed for cell surface marker expression by flow cytometry. For cytokine and proliferative T-cell response analysis, DCs were cultured with autologous CD4 T cells and tuberculin purified protein derivative (PPD). Mean fluorescent intensity of CD86 was reduced in HCV protein-pulsed iDCs. Proliferative T-cell responses and Th1 cytokine concentrations were reduced with HCV nonstructural proteins (NS), particularly with HCV NS4. HCV nonstructural proteins, particularly NS4, change the iDC phenotype and reduce antigen-specific T-cell stimulatory function with Th1 cytokine reductions.

A 36-Year-Old Woman With Cough, Positive Purified Protein Derivative Tuberculin Skin Test, and Cavitary Upper Lobe Consolidation

A 36-Year-Old Woman With Cough, Positive Purified Protein Derivative Tuberculin Skin Test, and Cavitary Upper Lobe Consolidation

Intradermal tuberculin testing of wild African lions ( Panthera leo) naturally exposed to infection with Mycobacterium bovis

African lions in the southern half of Kruger National Park (KNP) are infected with Mycobacterium bovis. Historically, reliable detection of mycobacteriosis in lions was limited to necropsy and microbiological analysis of lesion material collected from emaciated and ailing or repeat-offender lions. We report on a method of cervical intradermal tuberculin testing of lions and its interpretation capable of identifying natural exposure to M. bovis. Infected lions ( n = 52/95) were identified by detailed necropsy and mycobacterial culture. A large proportion of these confirmed infected lions (45/52) showed distinct responses to bovine tuberculin purified protein derivative (PPD) while responses to avian tuberculin PPD were variable and smaller. Confirmed uninfected lions from non-infected areas ( n = 11) responded variably to avian tuberculin PPD only. Various non-tuberculous mycobacteria (NTM) were cultured from 45/95 lions examined, of which 21/45 were co-infected with M. bovis. Co-infection with M. bovis and NTM did not influence skin reactions to bovine tuberculin PPD. Avian tuberculin PPD skin reactions were larger in M. bovis-infected lions compared to uninfected ones. Since NTM co-infections are likely to influence the outcome of skin testing, stricter test interpretation criteria were applied. When test data of bovine tuberculin PPD tests were considered on their own, as for a single skin test, sensitivity increased (80.8–86.5%) but false positive rate for true negatives (18.75%) remained unchanged. Finally, the adapted skin test procedure was shown not to be impeded by persistent Feline Immunodeficiency Virus Ple co-infection.

Human immunodeficiency virus (HIV) infection during pregnancy induces CD4 T‐cell differentiation and modulates responses to Bacille Calmette‐Guérin (BCG) vaccine in HIV‐uninfected infants

Human immunodeficiency virus (HIV)-negative infants born to HIV-positive mothers frequently exhibit a range of immunological abnormalities. We tested the hypothesis that HIV during pregnancy affects the ability of CD4 T cells of HIV-negative infants to respond to vaccine challenge by recruiting HIV-negative infants born to HIV-negative and HIV-positive mothers and measuring their responses to Bacille Calmette-Guérin (BCG) vaccine given at birth. At 2 weeks, maternal HIV status did not influence CD4 T-cell counts or differentiation, but by 10 weeks CD4 counts of infants born to HIV-positive mothers fell to a level characteristic of HIV-positive infants. Among the CD4 T-cell populations, markers of differentiation (CCR7− CD45RA− CD27−) and senescence (CD57, PD-1) were more common among infants born to HIV-positive mothers than among infants born to HIV-negative mothers. At 2 weeks of age, we assessed the effector response to heat-killed BCG and tuberculin purified protein derivative (PPD) by overnight interferon (IFN)-γ enzyme-linked immunosorbent spot-forming cell assay (ELISpot), but found no measurable effect of maternal HIV status. At 10 weeks, we assessed CD4 T-cell memory by measuring proliferation in response to the same antigens. We observed a bimodal response that allowed infants to be classified as high or low responders and found that fewer infants born to HIV-positive mothers were able to mount a robust proliferative response, suggesting that their reduced CD4 counts and increased differentiation indicated a deficiency in their ability to develop immunological memory.

Antibody response to a sterile filtered PPD tuberculin in M. bovis infected and M. bovis sensitized cattle

BackgroundBovine tuberculosis, caused by Mycobacterium bovis, afflicts approximately 50 million cattle worldwide and is detected by the tuberculin skin test (TST). While it has long been recognized that purified protein derivative (PPD) tuberculin is composed of a mixture of M. bovis derived protein components, little is known about the quality, relative quantity and identity of the proteins that make up PPD tuberculin. We manufactured a sterile filtered PPD tuberculin (SF-PPD) from a nine-week-old M. bovis culture supernatant in order to characterise the culture filtrate proteins (CFP) which make up M. bovis PPD tuberculin and to compare the antibody response of M. bovis infected versus M. bovis sensitized cattle.ResultsSF-PPD resolved into approximately 200 discrete spots using two-dimensional polyacrylamide gel electrophoresis (2-DE) while fewer than 65 spots could be discerned from 2-DE gels of tuberculin derived from autoclaved culture supernatant. Two dimensional Western blot analyses indicated that sera from M. bovis sensitized cattle recognized additional SF-PPD antigens as compared to M. bovis infected cattle at seven weeks post infection/sensitization. However, application of a comparative tuberculin skin test resulted in an antibody boosting response to the same set of M. bovis CFPs in both the M. bovis infected and M. bovis sensitized cattle.ConclusionsWe concluded that it is the heat sterilization of the M. bovis CFPs that causes severe structural changes to the M. bovis proteins. This work suggests that M. bovis infected cattle and cattle artificially sensitized to M. bovis with an injection of heat killed cells exhibit similar antibody responses to M. bovis antigens.

Maternal Infection with Trypanosoma cruzi and Congenital Chagas Disease Induce a Trend to a Type 1 Polarization of Infant Immune Responses to Vaccines

BackgroundWe previously showed that newborns congenitally infected with Trypanosoma cruzi (M+B+) display a strong type 1 parasite-specific T cell immune response, whereas uninfected newborns from T. cruzi-infected mothers (M+B−) are prone to produce higher levels of proinflammatory cytokines than control neonates (M−B−). The purpose of the present study was to determine if such fetal/neonatal immunological environments could alter the response to standard vaccines administered in early life.MethodologyInfants (6–7 months old) living in Bolivia, an area highly endemic for T. cruzi infection, and having received Bacillus Calmette Guerin (BCG), hepatitis B virus (HBV), diphtheria and tetanus vaccines, were enrolled into the M+B+, M+B−, M−B− groups mentioned above. The production of IFN-γ and IL-13, as markers of Th1 and Th2 responses respectively, by peripherical blood mononuclear cells stimulated with tuberculin purified protein derivative of Mycobacterium tuberculosis (PPD) or the vaccinal antigens HBs, diphtheria toxoid (DT) or tetanus toxoid (TT), as well as circulating levels of IgG antibodies against HBsAg, DT and TT were analyzed in infants. Cellular responses to the superantigen SEB were also monitored in M+B+, M+B−, M−B−infants and newborns.Principal FindingsM+B+ infants developed a stronger IFN-γ response to hepatitis B, diphtheria and tetanus vaccines than did M+B− and M−B− groups. They also displayed an enhanced antibody production to HBsAg. This was associated with a type 1-biased immune environment at birth, since cells of M+B+ newborns produced higher IFN-γ levels in response to SEB. M+B− infants produced more IFN-γ in response to PPD than the other groups. IL-13 production remained low and similar in all the three groups, whatever the subject's ages or vaccine status.ConclusionThese results show that: i) both maternal infection with T. cruzi and congenital Chagas disease do not interfere with responses to BCG, hepatitis B, diphtheria and tetanus vaccines in the neonatal period, and ii) the overcoming of immunological immaturity by T. cruzi infection in early life is not limited to the development of parasite-specific immune responses, but also tends to favour type 1 immune responses to vaccinal antigens.

Topical photodynamic therapy is immunosuppressive in humans

Visible light irradiation after application of a photosensitizer (topical photodynamic therapy; PDT) is increasingly used to treat nonmelanoma skin cancers and premalignant actinic keratoses. PDT can provide a cosmetically superior alternative to surgery, but carries failure rates of 10-40%. While some murine studies have suggested immune enhancement by PDT, others reported immunosuppressive effects, which may indicate impaired antitumour immunity and thus compromised tumour clearance. This study aimed to determine the in vivo immune effects of PDT in humans. Using healthy, Mantoux-positive volunteers, we irradiated discrete areas of the back with narrowband red light (630 nm; 37 J cm(-2)), with and without prior application of 5-aminolaevulinic acid (ALA) or methyl aminolaevulinate (MAL). Adjacent, untreated areas served as immunologically intact control sites. Delayed-type hypersensitivity responses to tuberculin purified protein derivative (Mantoux reactions) were then elicited in each of the irradiated, unirradiated and control sites, and the intensity of the reactions was quantitated with an erythema meter and by measurement of Mantoux diameter. By comparing Mantoux intensity at treated and control sites, immunosuppression was determined in each volunteer for each intervention. We found that both MAL-PDT and ALA-PDT significantly suppressed Mantoux erythema (by 30% and 50%, respectively) and diameter (41% and 38%). Red light alone significantly suppressed diameter (22%) but not erythema (13%). Topical PDT induced significant immune suppression, which could impair local antitumour immune responses and may thus contribute to treatment failure.

Identification of proteins from tuberculin purified protein derivative (PPD) by LC-MS/MS

The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis, however it is poorly defined. Most mycobacterial proteins are extensively denatured by the procedure employed in its preparation, which explains previous difficulties in identifying constituents from PPD to characterize their behaviour in B- and T-cell reactions. We here described a proteomics-based characterization of PPD from several different sources by LC-MS/MS, which combines the solute separation power of HPLC, with the detection power of a mass spectrometer. The technique is able to identify proteins from complex mixtures of peptide fragments. A total of 171 different proteins were identified among the four PPD samples (two bovine PPD and two avium PPD) from Brazil and UK. The majority of the proteins were cytoplasmic (77.9%) and involved in intermediary metabolism and respiration (24.25%) but there was a preponderance of proteins involved in lipid metabolism. We identified a group of 21 proteins that are present in both bovine PPD but were not detected in avium PPD preparation. In addition, four proteins found in bovine PPD are absent in Mycobacterium bovis BCG vaccine strain. This study provides a better understanding of the tuberculin PPD components leading to the identification of additional antigens useful as reagents for specific diagnosis of tuberculosis.

Immunological and cytotoxicological characterization of tuberculin purified protein derivative (PPD) conjugated to single-walled carbon nanotubes

Tuberculosis (TB) represents one of the leading killers among all infectious disease. Protection against TB depends on the activation of T-helper type I (Th1) immune response. Carbon nanotubes (CNTs) have attracted considerable attention because of their potential applications as new nanovehicle. In the current study, tuberculin purified protein derivative (PPD) was conjugated to carboxylated single-walled carbon nanotubes (SWCNTs). Cytotoxicity of the carboxylated SWCNT and SWCNT-PPD conjugate was analyzed with MTT assay and by reactive oxygen species (ROS) and nitric oxide (NO) generation. Male BALB/c mice were immunized with BCG, PPD, SWCNT-PPD conjugate and PPD in complete Freund's adjuvant (CFA). Induction of cellular immune response was analyzed by measuring the levels of Th1 cytokines (IFN-γ and IL-12) and Th2 cytokines (IL-10 and IL-5). Immunization with non-conjugated PPD or PPD in Freund's adjuvant induced a Th2 cytokine response while immunization with BCG resulted to a mixed Th1/Th2 cytokine response. In contrast, PPD in conjugation with SWCNT generated preferentially a Th1-type cytokine response in the absence of potential cytotoxic effects.

The candidate tuberculosis vaccine Mtb72F/AS02A: Tolerability and immunogenicity in humans

Tuberculosis (TB) remains uncontrolled in many parts of the world and the development of an effective vaccine against TB represents a high priority unmet medical need. Healthy PPD (tuberculin purified protein derivative)-negative adult volunteers, aged 18-40 years received three doses of the candidate Mtb72F/AS02A vaccine according to a 0-1-2 months schedule in an open-label Phase I study (NCT00730795). Solicited, unsolicited and serious adverse events (AEs), hematological and biochemical laboratory parameters were assessed. Mtb72F-specific humoral responses were assessed by ELISA and cell-mediated immune (CMI) responses by intracellular cytokine staining (ICS) and short-term ELISPOT assays. CMI responses to the component peptides (Mtb39a and the Mtb32a C- and N-terminal antigen domains, Mtb32C and Mtb32N) were also assessed by ICS. The Mtb72F/AS02A vaccine appeared to be mainly locally reactogenic but this was considered acceptable, since these AEs were usually transient and resolved within 1-2 days. Most AEs reported were mild in intensity, no serious AEs occurred, no medically significant biochemical or hematological abnormalities related to vaccination were measured and all AEs resolved without sequelae. The vaccine induced statistically significant changes in humoral and CMI response measures. The Mtb72F antigen induced good production of IL-2 and IFN γ in the ELISPOT assay and CD4+T cells expressing at least 2 activation markers (mainly CD40-L and IL-2) were observed with ICS. A similar CMI profile was observed with Mtb39a and Mtb32N. The induced CMI responses persisted for at least 6 months post-vaccination. All subjects were seropositive for anti-Mtb72F antibodies one month post-dose 2 and 6 months post-dose 3. This first trial in humans found Mtb72F/AS02A to have an, acceptable tolerability, to be immunogenic in healthy adults and warrants further development of the vaccine.