OpenUCT :: Browsing by Subject "Enzyme-linked immunoassays"

Browsing by Subject "Enzyme-linked immunoassays"

Now showing 1 - 20 of 26

Open Access
Altered ratio of IFN-γ/IL-10 in patients with drug resistant Mycobacterium tuberculosis and HIV-tuberculosis immune reconstitution inflammatory syndrome
(Public Library of Science, 2012) Skolimowska, Keira H; Rangaka, Molebogeng X; Meintjes, Graeme; Pepper, Dominique J; Seldon, Ronnett; Matthews, Kerryn; Wilkinson, Robert J; Wilkinson, Katalin A
We have described a clinical relationship between HIV-Tuberculosis Immune Reconstitution Inflammatory Syndrome (TB-IRIS) and anti-tubercular drug resistance. Here we studied the immune response of TB-IRIS patients from whom a drug-resistant (n = 11) or drug-susceptible (n = 25) Mycobacterium tuberculosis (MTB) strain was isolated after presenting with TB-IRIS. ELISpot analysis and multiplex cytokine analysis of the supernatant collected from peripheral blood mononuclear cells stimulated overnight with the heat-killed H37Rv MTB laboratory strain was used. Although there was no statistical difference in IFN-gamma ELISpot responses between the two groups, the results point towards higher bacterial load in the drug-resistant patients, possibly due to failed therapy. The ratio between secreted IFN-gamma/IL-10 and IL-2/IL-10 was significantly lower in TB-IRIS patients in whom the cause of TB was a drug-resistant strain compared to those with a fully sensitive strain (p = 0.02). Since host immune responses are dependent on the bacterial load, we hypothesise that the impaired cytokine balance is likely to be caused by the poorly controlled bacterial growth in these patients.
Open Access
Anaemia in acute HIV-1 subtype C infection
(Public Library of Science, 2008) Mlisana, Koleka; Auld, Sara C; Grobler, Anneke; Loggerenberg, Francois van; Williamson, Carolyn; Iriogbe, Itua; Sobieszczyk, Magdalena E; Karim, Salim S Abdool; Team, for the CAPRISA Acute Infection Study
BACKGROUND: The high prevalence of anaemia and the increased morbidity and mortality associated with anaemia during AIDS has been well described yet there has been little information about anaemia and changes in haemoglobin levels during acute and early HIV-1 infection. METHODS: HIV-negative women (n = 245) were enrolled into an observational cohort as part of the Centre for the AIDS Programme of Research in South Africa (CAPRISA) Acute Infection Study. Acute infection was diagnosed following a positive HIV RNA PCR in the absence of antibodies, or detection of HIV-1 antibodies within 3 months of a previously negative antibody test. Haemotologic parameters were assessed before infection and at regular intervals in the first twelve months of HIV infection. RESULTS: Fifty-seven participants with acute HIV infection were identified at a median of 14.5 days post-infection (range 10-81) and were enrolled in the CAPRISA Acute Infection cohort at a median of 41 days post-infection (range 15-104). Mean haemoglobin prior to HIV-1 infection was 12.7 g/dL, with a mean decline of 0.46 g/dL following infection. The prevalence of anaemia increased from 25.0% prior to HIV-1 infection to 52.6% at 3 months post-infection, 61.1% at 6 months post-infection, and 51.4% at 12 months post-infection. CONCLUSIONS: Haematologic derangements and anaemia with a trend towards iron deficiency are common with acute HIV-1 subtype C infection in this small cohort. The negative impact of anaemia concurrent with established HIV infection upon morbidity and mortality has been well documented but the prognostic potential and long-term effects of anaemia during acute HIV-1 infection remain unknown.
Open Access
Comparison of a clinical prediction rule and a LAM antigen-detection assay for the rapid diagnosis of TBM in a high HIV prevalence setting
(Public Library of Science, 2010) Patel, Vinod B; Singh, Ravesh; Connolly, Cathy; Kasprowicz, Victoria; Zumla, Allimudin; Ndungu, Thumbi; Dheda, Keertan
Background/Objective: The diagnosis of tuberculous meningitis (TBM) in resource poor TB endemic environments is challenging. The accuracy of current tools for the rapid diagnosis of TBM is suboptimal. We sought to develop a clinical-prediction rule for the diagnosis of TBM in a high HIV prevalence setting, and to compare performance outcomes to conventional diagnostic modalities and a novel lipoarabinomannan (LAM) antigen detection test (Clearview-TB®) using cerebrospinal fluid (CSF). METHODS: Patients with suspected TBM were classified as definite-TBM (CSF culture or PCR positive), probable-TBM and non-TBM. RESULTS: Of the 150 patients, 84% were HIV-infected (median [IQR] CD4 count = 132 [54; 241] cells/µl). There were 39, 55 and 54 patients in the definite, probable and non-TBM groups, respectively. The LAM sensitivity and specificity (95%CI) was 31% (17;48) and 94% (85;99), respectively (cut-point ≥0.18). By contrast, smear-microscopy was 100% specific but detected none of the definite-TBM cases. LAM positivity was associated with HIV co-infection and low CD4 T cell count (CD4<200 vs. >200 cells/µl; p = 0.03). The sensitivity and specificity in those with a CD4<100 cells/µl was 50% (27;73) and 95% (74;99), respectively. A clinical-prediction rule ≥6 derived from multivariate analysis had a sensitivity and specificity (95%CI) of 47% (31;64) and 98% (90;100), respectively. When LAM was combined with the clinical-prediction-rule, the sensitivity increased significantly (p<0.001) to 63% (47;68) and specificity remained high at 93% (82;98). CONCLUSIONS: Despite its modest sensitivity the LAM ELISA is an accurate rapid rule-in test for TBM that has incremental value over smear-microscopy. The rule-in value of LAM can be further increased by combination with a clinical-prediction rule, thus enhancing the rapid diagnosis of TBM in HIV-infected persons with advanced immunosuppression.
Open Access
Correlation of mycobacterium tuberculosis specific and non-specific quantitative Th1 T-cell responses with bacillary load in a high burden setting
(Public Library of Science, 2012) Theron, Grant; Peter, Jonny; Lenders, Laura; van Zyl-Smit, Richard; Meldau, Richard; Govender, Ureshnie; Dheda, Keertan
BACKGROUND: Measures of bacillary load in patients with tuberculosis (TB) may be useful for predicting and monitoring response to treatment. The relationship between quantitative T-cell responses and mycobacterial load remains unclear. We hypothesised that, in a HIV-prevalent high burden setting, the magnitude of mycobacterial antigen-specific and non-specific T-cell IFN-γ responses would correlate with (a) bacterial load and (b) culture conversion in patients undergoing treatment. METHODS: We compared baseline (n = 147), 2 (n = 35) and 6 month (n = 13) purified-protein-derivative (PPD) and RD1-specific (TSPOT.TB and QFT-GIT) blood RD1-specific (TSPOT.TB; QFT-GIT) responses with associates of sputum bacillary load in patients with culture-confirmed TB in Cape Town, South Africa. RESULTS: IFN-γ responses were not associated with liquid culture time-to-positivity, smear-grade, Xpert MTB/RIF-generated cycle threshold values or the presence of cavities on the chest radiograph in patients with culture-confirmed TB and irrespective of HIV-status. 2-month IGRA conversion rates (positive-to-negative) were negligible [<11% for TSPOT.TB (3/28) and QFT-GIT (1/29)] and lower compared to culture [60% (21/35); p<0.01]. CONCLUSIONS: In a high burden HIV-prevalent setting T-cell IFN-γ responses to M. tuberculosis- specific and non-specific antigens do not correlate with bacillary load, including Xpert MTB/RIF-generated C T values, and are therefore poorly suited for monitoring treatment and prognostication.
Open Access
A cross-reactive monoclonal antibody to nematode haemoglobin enhances protective immune responses to Nippostrongylus brasiliensis
(Public Library of Science, 2013) Nieuwenhuizen, Natalie E; Meter, Jeanne M; Horsnell, William G; Hoving, J Claire; Fick, Lizette; Sharp, Michael F; Darby, Matthew G; Parihar, Suraj P; Brombacher, Frank; Lopata, Andreas L
Background: Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg) against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection. Methodology/Principal Findings: Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four –HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens. Conclusion: The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm infection.
Open Access
The diagnostic accuracy of urine-based Xpert MTB/RIF in HIV-infected hospitalized patients who are smear-negative or sputum scarce
(Public Library of Science, 2012) Peter, Jonathan G; Theron, Grant; Muchinga, Tapuwa E; Govender, Ureshnie; Dheda, Keertan
BACKGROUND: Hospitals in sub-Saharan Africa are inundated with HIV-infected patients and tuberculosis (TB) is the commonest opportunistic infection in this sub-group. Up to one third of TB-HIV co-infected patients fail to produce a sputum sample (sputum scarce) and diagnosis is thus often delayed or missed. We investigated the sensitivity of urine-based methods (Xpert MTB/RIF, LAM strip test and LAM ELISA) in such patients. METHODOLOGY/PRINCIPAL FINDINGS: 281 HIV-infected hospitalised patients with clinically suspected TB provided a spot urine sample. The reference standard was culture positivity for Mycobacterium tuberculosis on ≥1 sputum or extra-pulmonary sample. MTB/RIF was performed using 1 ml of both unprocessed and, when possible, concentrated urine. Each unconcentrated urine sample was also tested using the Clearview LAM ELISA and Alere LAM strip test. 42% (116/242) of patients had culture-proven TB. 18% (20/54) were sputum scarce. In sputum-scarce patients, the sensitivity of urine MTB/RIF and LAM ELISA was 40% (95%CI: 22-61) and 60% (95%CI: 39-78), respectively. Urine MTB/RIF specificity was 98% (95%CI: 95-100). Combined sensitivity of urine LAM ELISA and MTB/RIF was better than MTB/RIF alone [MTB/RIF and LAM: 70% (95%CI: 48-85) vs. MTB/RIF: 40% (95%CI: 22-61), p = 0.03]. Significant predictors of urine MTB/RIF positivity were CD4<50 cells/ml (p = 0.001), elevated protein-to-creatinine ratio (p<0.001) and LAM ELISA positivity (p<0.001). Urine centrifugation and pelleting significantly increased the sensitivity of MTB/RIF over unprocessed urine in paired samples [42% (95%CI: 26-58) vs. 8% (95%CI: 0-16), p<0.001]. Urine MTB/RIF-generated C T values correlated poorly with markers of bacillary burden (smear grade and time-to-positivity). Conclusions/Significance This preliminary study indicates that urine-based MTB/RIF, alone or in combination with LAM antigen detection, may potentially aid the diagnosis of TB in HIV-infected patients with advanced immunosuppression when sputum-based diagnosis is not possible. Concentration of urine prior to MTB/RIF-testing significantly improves sensitivity.
Open Access
Feasibility and diagnostic utility of antigen-specific interferon-gamma responses for rapid immunodiagnosis of tuberculosis using induced sputum
(Public Library of Science, 2010) Cashmore, Tamaryn J; Peter, Jonathan G; Van Zyl-Smit, Richard N; Semple, Patricia L; Maredza, Alice; Meldau, Richard; Zumla, Alimuddin; Nurse, Barbara; Dheda, Keertan
BACKGROUND: The diagnosis of smear-negative or sputum-scarce tuberculosis (TB) is problematic as culture takes several weeks and representative biological samples are difficult to obtain. RD-1 antigen-specific interferon-γ release assays (IGRAs) are sensitive and specific blood-based tests for the diagnosis of M. tuberculosis infection. The feasibility and diagnostic utility of this rapid immunodiagnostic assay, using cells from induced sputum, is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Cells isolated from induced sputum were co-cultured with ESAT-6 and CFP-10 antigens using a standardized enzyme-linked immunospot (ELISPOT) assay (T-SPOT®. TB ) in 101 consecutively recruited TB suspects or non-TB controls. An optimization phase using 28 samples was followed by a validation phase using samples from 73 participants (20 with definite or probable TB, and 48 with non-TB). Despite optimization of sputum processing 65/73 (89%) of the IGRAs in the validation phase were inconclusive. 44/73 (60%) tests failed due to sputum induction-related factors [sputum induction-related adverse events (n = 5), inadequate sputum volume (n = 8), non-homogenisable sputum (n = 7), and insufficient numbers of cells to perform the assay (n = 24)], whilst 20/73 (27%) tests failed due T-SPOT®. TB assay-related factors [excessive debris precluding reading of spots in the ELISPOT well (n = 6), failure of the positive control (n = 11), or high spot count in the negative control (n = 3)]. Only 8/73 (11%) of the available samples could therefore be correctly categorized (7 definite or probable TB, and 1 non-TB patient). Thus, 13/20 (65%) of the definite or probable TB cases remained undiagnosed. Conclusions/Significance: Rapid immunodiagnosis of pulmonary TB by antigen-specific IFN-γ ELISPOT responses, using cells from induced sputum, is possible. However, the test, in its current ELISPOT format, is not clinically useful because the majority of the assays are inconclusive.
Open Access
The global epidemiology of syphilis in the past century - a systematic review based on antenatal syphilis prevalence
(Public Library of Science, 2016) Kenyon, Chris Richard; Osbak, Kara; Tsoumanis, Achilleas
Author Summary: Syphilis rates have varied tremendously between different populations around the world. We conducted a systematic review of syphilis prevalence in pregnant women in 13 populations with available data for the last 100 years. Our findings were that in most populations syphilis prevalence dropped to under 1% before 1960. In the 2 populations from sub Saharan Africa, the syphilis prevalence remained around 6% until 50 years after the introduction of penicillin. Other systematic reviews were utilized to provide syphilis prevalence estimates for all countries with available data for the periods 1990-1999 and 2008. We assessed if there was a correlation between national syphilis prevalence in these periods and five explanatory factors. Only residence in sub-Saharan Africa was associated with syphilis prevalence in both time periods. These findings, considered in conjunction with other types of evidence we review, such as the strong correlations at population level between syphilis prevalence and those of Herpes Simplex Virus-2 prevalence and HIV prevalence, suggest that common risk factors may underpin the spread of all three of these sexually transmitted diseases. Establishing what these factors are is of great importance to improve the health of highly affected populations such as those in sub-Saharan Africa.
Open Access
HIV-1 subtype C mosaic Gag expressed by BCG and MVA elicits persistent effector t cell responses in a prime-boost regimen in mice
(Public Library of Science, 2016) Jongwe, Tsungai Ivai; Chapman, Ros; Douglass, Nicola; Chetty, Shivan; Chege, Gerald; Williamson, Anna-Lise
Over 90% of HIV/AIDS positive individuals in sub-Saharan Africa are infected with highly heterogeneous HIV-1 subtype C (HIV-1C) viruses. One of the best ways to reduce the burden of this disease is the development of an affordable and effective prophylactic vaccine. Mosaic immunogens are computationally designed to overcome the hurdle of HIV diversity by maximizing the expression of potential T cell epitopes. Mycobacterium bovis BCG Δ panCD auxotroph and modified vaccinia Ankara (MVA) vaccines expressing HIV-1C mosaic Gag (Gag M ) were tested in a prime-boost regimen to demonstrate immunogenicity in a mouse study. The BCG-Gag M vaccine was stable and persisted 11.5 weeks post vaccination in BALB/c mice. Priming with BCG-Gag M and boosting with MVA-Gag M elicited higher Gag-specific IFN-γ ELISPOT responses than the BCG-Gag M only and MVA-Gag M only homologous vaccination regimens. The heterologous vaccination also generated a more balanced and persistent CD4 + and CD8 + T cell Gag-specific IFN-γ ELISPOT response with a predominant effector memory phenotype. A Th1 bias was induced by the vaccines as determined by the predominant secretion of IFN-γ, TNF-α, and IL-2. This study shows that a low dose of MVA (10 4 pfu) can effectively boost a BCG prime expressing the same mosaic immunogen, generating strong, cellular immune responses against Gag in mice. Our data warrants further evaluation in non-human primates. A low dose vaccine would be an advantage in the resource limited countries of sub-Saharan Africa and India (where the predominating virus is HIV-1 subtype C).
Open Access
Humoral immune responses to Pneumocystis jirovecii antigens in HIV-infected and uninfected young children with pneumocystis pneumonia
(Public Library of Science, 2013) Djawe, Kpandja; Daly, Kieran R; Levin, Linda; Zar, Heather J; Walzer, Peter D
BACKGROUND: Humoral immune responses in human immunodeficiency virus (HIV)-infected and uninfected children with Pneumocystis pneumonia (PcP) are poorly understood. METHODS: Consecutive children hospitalized with acute pneumonia, tachypnea, and hypoxia in South Africa were investigated for PcP, which was diagnosed by real-time polymerase chain reaction on lower respiratory tract specimens. Serum antibody responses to recombinant fragments of the carboxyl terminus of Pneumocystis jirovecii major surface glycoprotein (MsgC) were analyzed. RESULTS: 149 children were enrolled of whom 96 (64%) were HIV-infected. PcP occurred in 69 (72%) of HIV-infected and 14 (26%) of HIV-uninfected children. HIV-infected children with PcP had significantly decreased IgG antibodies to MsgC compared to HIV-infected patients without PcP, but had similar IgM antibodies. In contrast, HIV-uninfected children with PcP showed no change in IgG antibodies to MsgC, but had significantly increased IgM antibodies compared to HIV-uninfected children without PCP. Age was an independent predictor of high IgG antibodies, whereas PcP was a predictor of low IgG antibodies and high IgM antibodies. IgG and IgM antibody levels to the most closely related MsgC fragments were predictors of survival from PcP. CONCLUSIONS: Young HIV-infected children with PcP have significantly impaired humoral immune responses to MsgC, whereas HIV-uninfected children with PcP can develop active humoral immune responses. The children also exhibit a complex relationship between specific host factors and antibody levels to MsgC fragments that may be related to survival from PcP.
Open Access
Hypoxia induces an immunodominant target of tuberculosis specific T cells absent from common BCG vaccines
(Public Library of Science, 2010) Gideon, Hannah Priyadarshini; Wilkinson, Katalin Andrea; Rustad, Tige R; Oni, Tolu; Guio, Heinner; Kozak, Robert Andrew; Sherman, David R; Meintjes, Graeme; Behr, Marcel A; Vordermeier, Hans Martin
Author Summary Mycobacterium tuberculosis (the cause of tuberculosis) can persist for many years in humans without causing disease but has the potential to reactivate. One of the conditions the bacterium must survive in these circumstances is hypoxia. In order to do so, the bacterium uses a characteristic set of genes that help alter its metabolism. It follows that the products of such genes may encode protein antigens that can be recognized by the immune response. We therefore analyzed gene response patterns of tuberculosis subject to prolonged hypoxia as a guide to the discovery of new antigens that might be useful as vaccines or diagnostic agents. Amongst the genes most strongly increased by low oxygen levels, one was identified (known as Rv1986) that is missing from most strains of the tuberculosis vaccine Mycobacterium bovis BCG. When we analyzed human immune responses to this protein in tuberculosis infected people our experiments showed it was particularly well recognized by cells that produce a chemical messenger (cytokine) called interleukin-2. Interleukin-2 is important for long-term immunological memory. The BCG vaccine is only partially effective and our experiments therefore suggest one of the reasons could be that an important immunological target is missing from many strains. Further evaluation of BCG strains in which Rv1986 is present or absent is therefore warranted in the hope that this might improve the efficacy of existing or new tuberculosis vaccines.
Open Access
Identification of Antigens Specific to Non-Tuberculous Mycobacteria: The Mce Family of Proteins as a Target of T Cell Immune Responses
(Public Library of Science, 2011) Checkley, Anna M.; Wyllie, David H.; Scriba, Thomas J.; Golubchik, Tanya; Hill, Adrian V. S.; Hanekom, Willem A.; McShane, Helen
The lack of an effective TB vaccine hinders current efforts in combating the TB pandemic. One theory as to why BCG is less protective in tropical countries is that exposure to non-tuberculous mycobacteria (NTM) reduces BCG efficacy. There are currently several new TB vaccines in clinical trials, and NTM exposure may also be relevant in this context. NTM exposure cannot be accurately evaluated in the absence of specific antigens; those which are known to be present in NTM and absent from M. tuberculosis and BCG. We therefore used a bioinformatic pipeline to define proteins which are present in common NTM and absent from the M. tuberculosis complex, using protein BLAST, TBLASTN and a short sequence protein BLAST to ensure the specificity of this process. We then assessed immune responses to these proteins, in healthy South Africans and in patients from the United Kingdom and United States with documented exposure to NTM. Low level responses were detected to a cluster of proteins from the mammalian cell entry family, and to a cluster of hypothetical proteins, using ex vivo ELISpot and a 6 day proliferation assay. These early findings may provide a basis for characterising exposure to NTM at a population level, which has applications in the field of TB vaccine design as well as in the development of diagnostic tests.
Open Access
IL-1 and IL-23 mediate early IL-17A production in pulmonary inflammation leading to late fibrosis
(Public Library of Science, 2011) Gasse, Paméla; Riteau, Nicolas; Vacher, Rachel; Michel, Marie-Laure; Fautrel, Alain; di Padova, Franco; Fick, Lizette; Charron, Sabine; Lagente, Vincent; Eberl, Gérard
BACKGROUND: Idiopathic pulmonary fibrosis is a devastating as yet untreatable disease. We demonstrated recently the predominant role of the NLRP3 inflammasome activation and IL-1β expression in the establishment of pulmonary inflammation and fibrosis in mice. METHODS: The contribution of IL-23 or IL-17 in pulmonary inflammation and fibrosis was assessed using the bleomycin model in deficient mice. RESULTS: We show that bleomycin or IL-1β-induced lung injury leads to increased expression of early IL-23p19, and IL-17A or IL-17F expression. Early IL-23p19 and IL-17A, but not IL-17F, and IL-17RA signaling are required for inflammatory response to BLM as shown with gene deficient mice or mice treated with neutralizing antibodies. Using FACS analysis, we show a very early IL-17A and IL-17F expression by RORγt + γδ T cells and to a lesser extent by CD4αβ + T cells, but not by iNKT cells, 24 hrs after BLM administration. Moreover, IL-23p19 and IL-17A expressions or IL-17RA signaling are necessary to pulmonary TGF-β1 production, collagen deposition and evolution to fibrosis. CONCLUSIONS: Our findings demonstrate the existence of an early IL-1β-IL-23-IL-17A axis leading to pulmonary inflammation and fibrosis and identify innate IL-23 and IL-17A as interesting drug targets for IL-1β driven lung pathology.
Open Access
Impairment of IFN-gamma response to synthetic peptides of mycobacterium tuberculosis in a 7-day whole blood assay
(Public Library of Science, 2013) Gideon, Hannah Priyadarshini; Hamilton, Melissa Shea; Wood, Kathryn; Pepper, Dominique; Oni, Tolu; Seldon, Ronnett; Banwell, Claire; Langford, Paul R; Wilkinson, Robert J; Wilkinson, Katalin A
Studies on Mycobacterium tuberculosis (MTB) antigens are of interest in order to improve vaccine efficacy and to define biomarkers for diagnosis and treatment monitoring. The methodologies used for these investigations differ greatly between laboratories and discordant results are common. The IFN-gamma response to two well characterized MTB antigens ESAT-6 and CFP-10, in the form of recombinant proteins and synthetic peptides, was evaluated in HIV-1 uninfected persons in both long-term (7 day) and 24 hour, commercially available QuantiFERON TB Gold in Tube (QFT-GIT), whole blood assays. Our findings showed differences in the IFN-gamma response between 24 hour and 7 day cultures, with recombinant proteins inducing a significantly higher response than the peptide pools in 7 day whole blood assays. The activity of peptides and recombinant proteins did not differ in 24 hour whole blood or peripheral blood mononuclear cell (PBMC) based assays, nor in the ELISpot assay. Further analysis by SELDI-TOF mass spectrometry showed that the peptides are degraded over the course of 7 days of incubation in whole blood whilst the recombinant proteins remain intact. This study therefore demonstrates that screening antigenic candidates as synthetic peptides in long-term whole blood assays may underestimate immunogenicity.
Open Access
Incidence and risk factors for neonatal tetanus in admissions to County Hospital, Kenya
(Public Library of Science, 2015) Ibinda, Fredrick; Bauni, Evasius; Kariuki, Symon M; Fegan, Greg; Lewa, Joy; Mwikamba, Monica; Boga, Mwanamvua; Odhiambo, Rachael; Mwagandi, Kiponda; Seale, Anna C
BACKGROUND: Neonatal Tetanus (NT) is a preventable cause of mortality and neurological sequelae that occurs at higher incidence in resource-poor countries, presumably because of low maternal immunisation rates and unhygienic cord care practices. We aimed to determine changes in the incidence of NT, characterize and investigate the associated risk factors and mortality in a prospective cohort study including all admissions over a 15-year period at a County hospital on the Kenyan coast, a region with relatively high historical NT rates within Kenya. METHODS: We assessed all neonatal admissions to Kilifi County Hospital in Kenya (1999-2013) and identified cases of NT (standard clinical case definition) admitted during this time. Poisson regression was used to examine change in incidence of NT using accurate denominator data from an area of active demographic surveillance. Logistic regression was used to investigate the risk factors for NT and factors associated with mortality in NT amongst neonatal admissions. A subset of sera from mothers (n = 61) and neonates (n = 47) were tested for anti-tetanus antibodies. RESULTS: There were 191 NT admissions, of whom 187 (98%) were home deliveries. Incidence of NT declined significantly (Incidence Rate Ratio: 0.85 (95% Confidence interval 0.81-0.89), P<0.001) but the case fatality (62%) did not change over the study period (P = 0.536). Younger infant age at admission (P = 0.001) was the only independent predictor of mortality. Compared to neonatal hospital admittee controls, the proportion of home births was higher among the cases. Sera tested for antitetanus antibodies showed most mothers (50/61, 82%) had undetectable levels of antitetanus antibodies, and most (8/9, 89%) mothers with detectable antibodies had a neonate without protective levels. CONCLUSIONS: Incidence of NT in Kilifi County has significantly reduced, with reductions following immunisation campaigns. Our results suggest immunisation efforts are effective if sustained and efforts should continue to expand coverage.
Open Access
Investigating avian influenza infection hotspots in old-world shorebirds
(Public Library of Science, 2012) Gaidet, Nicolas; Mamy, Ahmed B Ould El; Cappelle, Julien; Caron, Alexandre; Cumming, Graeme S; Grosbois, Vladimir; Gil, Patricia; Hammoumi, Saliha; de Almeida, Renata Servan; Fereidouni, Sasan R
Heterogeneity in the transmission rates of pathogens across hosts or environments may produce disease hotspots, which are defined as specific sites, times or species associations in which the infection rate is consistently elevated. Hotspots for avian influenza virus (AIV) in wild birds are largely unstudied and poorly understood. A striking feature is the existence of a unique but consistent AIV hotspot in shorebirds (Charadriiformes) associated with a single species at a specific location and time (ruddy turnstone Arenaria interpres at Delaware Bay, USA, in May). This unique case, though a valuable reference, limits our capacity to explore and understand the general properties of AIV hotspots in shorebirds. Unfortunately, relatively few shorebirds have been sampled outside Delaware Bay and they belong to only a few shorebird families; there also has been a lack of consistent oropharyngeal sampling as a complement to cloacal sampling. In this study we looked for AIV hotspots associated with other shorebird species and/or with some of the larger congregation sites of shorebirds in the old world. We assembled and analysed a regionally extensive dataset of AIV prevalence from 69 shorebird species sampled in 25 countries across Africa and Western Eurasia. Despite this diverse and extensive coverage we did not detect any new shorebird AIV hotspots. Neither large shorebird congregation sites nor the ruddy turnstone were consistently associated with AIV hotspots. We did, however, find a low but widespread circulation of AIV in shorebirds that contrast with the absence of AIV previously reported in shorebirds in Europe. A very high AIV antibody prevalence coupled to a low infection rate was found in both first-year and adult birds of two migratory sandpiper species, suggesting the potential existence of an AIV hotspot along their migratory flyway that is yet to be discovered.
Open Access
p53 requires the stress sensor USF1 to direct appropriate cell fate decision
(Public Library of Science, 2014) Bouafia, Amine; Corre, Sébastien; Gilot, David; Mouchet, Nicolas; Prince, Sharon; Galibert, Marie-Dominique
Genomic instability is a major hallmark of cancer. To maintain genomic integrity, cells are equipped with dedicated sensors to monitor DNA repair or to force damaged cells into death programs. The tumor suppressor p53 is central in this process. Here, we report that the ubiquitous transcription factor Upstream Stimulatory factor 1 (USF1) coordinates p53 function in making proper cell fate decisions. USF1 stabilizes the p53 protein and promotes a transient cell cycle arrest, in the presence of DNA damage. Thus, cell proliferation is maintained inappropriately in Usf1 KO mice and in USF1-deficient melanoma cells challenged by genotoxic stress. We further demonstrate that the loss of USF1 compromises p53 stability by enhancing p53-MDM2 complex formation and MDM2-mediated degradation of p53. In USF1-deficient cells, the level of p53 can be restored by the re-expression of full-length USF1 protein similarly to what is observed using Nutlin-3, a specific inhibitor that prevents p53-MDM2 interaction. Consistent with a new function for USF1, a USF1 truncated protein lacking its DNA-binding and transactivation domains can also restore the induction and activity of p53. These findings establish that p53 function requires the ubiquitous stress sensor USF1 for appropriate cell fate decisions in response to DNA-damage. They underscore the new role of USF1 and give new clues of how p53 loss of function can occur in any cell type. Finally, these findings are of clinical relevance because they provide new therapeutic prospects in stabilizing and reactivating the p53 pathway.
Open Access
A phase IIA randomized clinical trial of a multiclade HIV-1 DNA prime followed by a multiclade rAd5 HIV-1 vaccine boost in healthy adults (HVTN204)
(Public Library of Science, 2011) Churchyard, Gavin J; Morgan, Cecilia; Adams, Elizabeth; Hural, John; Graham, Barney S; Moodie, Zoe; Grove, Doug; Gray, Glenda; Bekker, Linda-Gail; McElrath, M Juliana
BACKGROUND: The safety and immunogenicity of a vaccine regimen consisting of a 6-plasmid HIV-1 DNA prime (envA, envB, envC, gagB, polB, nefB) boosted by a recombinant adenovirus serotype-5 (rAd5) HIV-1 with matching inserts was evaluated in HIV-seronegative participants from South Africa, United States, Latin America and the Caribbean. METHODS: 480 participants were evenly randomized to receive either: DNA (4 mg IM by Biojector) at 0, 1 and 2 months, followed by rAd5 (10 10 PU IM by needle/syringe) at 6 months; or placebo. Participants were monitored for reactogenicity and adverse events throughout the 12-month study. Peak and duration of HIV-specific humoral and cellular immune responses were evaluated after the prime and boost. RESULTS: The vaccine was well tolerated and safe. T-cell responses, detected by interferon-γ (IFN-γ) ELISpot to global potential T-cell epitopes (PTEs) were observed in 70.8% (136/192) of vaccine recipients overall, most frequently to Gag (54.7%) and to Env (54.2%). In U.S. vaccine recipients T-cell responses were less frequent in Ad5 sero-positive versus sero-negative vaccine recipients (62.5% versus 85.7% respectively, p = 0.035). The frequency of HIV-specific CD4+ and CD8+ T-cell responses detected by intracellular cytokine staining were similar (41.8% and 47.2% respectively) and most secreted ≥2 cytokines. The vaccine induced a high frequency (83.7%-94.6%) of binding antibody responses to consensus Group M, and Clades A, B and C gp140 Env oligomers. Antibody responses to Gag were elicited in 46% of vaccine recipients. CONCLUSION: The vaccine regimen was well-tolerated and induced polyfunctional CD4+ and CD8+ T-cells and multi-clade anti-Env binding antibodies. Trial Registration: ClinicalTrials.gov NCT00125970
Open Access
Predictors of HVTN 503 MRK-AD5 HIV-1 gag/pol/nef vaccine Induced immune responses
(Public Library of Science, 2014) Hopkins, Kathryn L; Laher, Fatima; Otwombe, Kennedy; Churchyard, Gavin; Bekker, Linda-Gail; DeRosa, Stephen; Nchabeleng, Maphoshane; Mlisana, Koleka; Kublin, James; Gray, Glenda
BACKGROUND: Phambili, the Merck (MRK)-Adenovirus Type 5 (Ad5) HIV-1 gag/pol/nef subtype B vaccine study, conducted in South Africa, suspended enrollment and vaccination when companion study, Step, was found non-efficacious. Although the vaccine did not prevent HIV-1 infection or lower viral-load setpoint, immune responses recognized clades B and C HIV-1 subtypes. We investigated predictors of the vaccine-induced antigen-specific immune responses. METHODS: Vaccine-induced immunogenicity was ascertained by interferon-γ ELISpot assays on the first 186 enrolled participants receiving two vaccinations. Analyses, stratified by study arm/sex, were performed on baseline demographics [sex, age, Body Mass Index (BMI), site, Adenovirus Type-5 (Ad5) titer, Herpes Simplex Virus Type-2 (HSV2) status, heavy drinking]. Multivariate logistic regression determined predictors. RESULTS: Of the 186 participants, 53.7% (n = 100) were female, median BMI was 22.5 [IQR: 20.4-27.0], 85.5% (n = 159) were Ad5 seropositive, and 18.8% (n = 35) drank heavily. All vaccine recipients responded to both clade B (n = 87; 47%) and/or C (n = 74; 40%), p = 0.17. In multivariate analysis, female sex [Adjusted Odds Ratio (AOR): 6.478; p = 0.0159], overweight/obese BMI (AOR: 0.186; p = 0.0452), and heavy drinking (AOR: 0.270; p = 0.048) significantly predicted immune response to clade C for any antigens. A marginally significant predictor of clade C-pol antigen was female sex (AOR: 3.182; p = 0.0500). CONCLUSIONS: Sex, BMI, and heavy drinking affected vaccine-induced HIV-1 specific immune responses to clade C antigens. The role of female sex and overweight/obese BMI boosting and suppressing vaccine-induced HIV-1 specific immune responses, respectively, requires elucidation, including any effect on HIV vaccine efficacy, especially in the era of colliding epidemics (HIV and obesity).
Open Access
Priming with recombinant auxotrophic BCG expressing HIV-1 Gag, RT and Gp120 and boosting with recombinant MVA induces a robust T cell response in mice
(Public Library of Science, 2013) Chapman, Rosamund; Stutz, Helen; Jr, William Jacobs; Shephard, Enid; Williamson, Anna-Lise
In previous studies we have shown that a pantothenate auxotroph of Myocbacterium bovis BCG (BCGΔ panCD ) expressing HIV-1 subtype C Gag induced Gag-specific immune responses in mice and Chacma baboons after prime-boost immunization in combination with matched rMVA and VLP vaccines respectively. In this study recombinant BCG (rBCG) expressing HIV-1 subtype C reverse transcriptase and a truncated envelope were constructed using both the wild type BCG Pasteur strain as a vector and the pantothenate auxotroph. Mice were primed with rBCG expressing Gag and RT and boosted with a recombinant MVA, expressing a polyprotein of Gag, RT, Tat and Nef (SAAVI MVA-C). Priming with rBCGΔ panCD expressing Gag or RT rather than the wild type rBCG expressing Gag or RT resulted in higher frequencies of total HIV-specific CD8 + T cells and increased numbers of T cells specific to the subdominant Gag and RT epitopes. Increasing the dose of rBCG from 10 5 cfu to 10 7 cfu also led to an increase in the frequency of responses to subdominant HIV epitopes. A mix of the individual rBCGΔ panCD vaccines expressing either Gag, RT or the truncated Env primed the immune system for a boost with SAAVI MVA-C and generated five-fold higher numbers of HIV-specific IFN-γ-spot forming cells than mice primed with rBCGΔ panCD containing an empty vector control. Priming with the individual rBCGΔ panCD vaccines or the mix and boosting with SAAVI MVA-C also resulted in the generation of HIV-specific CD4 + and CD8 + T cells producing IFN-γ and TNF-α and CD4 + cells producing IL-2. The rBCG vaccines tested in this study were able to prime the immune system for a boost with rMVA expressing matching antigens, inducing robust, HIV-specific T cell responses to both dominant and subdominant epitopes in the individual proteins when used as individual vaccines or in a mix.