Analysis of host responses to Mycobacterium tuberculosis antigens in a multi-site study of subjects with different TB and HIV infection states in sub-Saharan Africa

Abstract

Background: Tuberculosis (TB) remains a global health threat with 9 million new cases and 1.4 million deaths per year. In order to develop a protective vaccine, we need to define the antigens expressed by Mycobacterium tuberculosis (Mtb), which are relevant to protective immunity in high-endemic areas.

Methods: We analysed responses to 23 Mtb antigens in a total of 1247 subjects with different HIV and TB status across 5 geographically diverse sites in Africa (South Africa, The Gambia, Ethiopia, Malawi and Uganda). We used a 7-day whole blood assay followed by IFN-γ ELISA on the supernatants. Antigens included PPD, ESAT-6 and Ag85B (dominant antigens) together with novel resuscitation-promoting factors (rpf), reactivation proteins, latency (Mtb DosR regulon-encoded) antigens, starvation-induced antigens and secreted antigens.

Results: There was variation between sites in responses to the antigens, presumably due to underlying genetic and environmental differences. When results from all sites were combined, HIV- subjects with active TB showed significantly lower responses compared to both TST(-) and TST(+) contacts to latency antigens (Rv0569, Rv1733, Rv1735, Rv1737) and the rpf Rv0867; whilst responses to ESAT-6/CFP-10 fusion protein (EC), PPD, Rv2029, TB10.3, and TB10.4 were significantly higher in TST(+) contacts (LTBI) compared to TB and TST(-) contacts fewer differences were seen in subjects with HIV co-infection, with responses to the mitogen PHA significantly lower in subjects with active TB compared to those with LTBI and no difference with any antigen.

Conclusions: Our multi-site study design for testing novel Mtb antigens revealed promising antigens for future vaccine development. The IFN-γ ELISA is a cheap and useful tool for screening potential antigenicity in subjects with different ethnic backgrounds and across a spectrum of TB and HIV infection states. Analysis of cytokines other than IFN-γ is currently on-going to determine correlates of protection, which may be useful for vaccine efficacy trials.

Figure 1. Heat map of IFN-γ responses to secreted, latent, and reactivation Mtb antigens stratified according to HIV status, TB status and location.

Median levels of IFN-γ are shown (pg/mL). Red indicates relatively high levels of IFN-γ and blue indicates relatively low levels. HIV⁺ = human immunodeficiency virus positive; HIV^- = human immunodeficiency virus negative; TB = active tuberculosis; LTBI = latently TB infected; n/d = not done; SUN = Stellenbosch University, South Africa; MRC = Medical Research Council, The Gambia; AHRI = Armauer Hansen Research Institute, Ethiopia; KPS = Karonga Prevention Study, Malawi; MAK = Makerere University, Uganda. The right column indicates the number of groups (out of a possible 24) who responded to a particular antigen. PPD was not used at MAK nor was Ag85b used at SUN, so the maximum number for these is 20.

Figure 2. IFN-γ secretion in response to secreted, latent, and reactivation Mtb antigens in active TB and LTBI subjects, from five African sites.

IFN-γ ELISA was performed on supernatants collected after 7-day antigen stimulation of diluted blood from TB cases (grey) and TST⁺ (LTBI) controls (white) from five sites in Africa. Line indicates median, whiskers indicate 5–95% range and dots indicate outliers. Data were analysed by Mann-Whitney U-test within sites for HIV^– and/or HIV⁺ subjects. Significant differences are indicated: *=p<0.05; **=p<0.01; ***=P<0.001.

Figure 3. IFN-γ secretion in response to secreted, latent, and reactivation Mtb antigens in active TB and LTBI subjects, from Ethiopia.

IFN-γ ELISA was performed on supernatants collected after 7 day antigen stimulation of diluted blood from TB cases (black) and TST⁺ (LTBI) controls (white) from AHRI (Ethiopia). Line indicates median, whiskers indicate 5–95% range and dots indicate outliers. Data were analysed by Mann-Whitney U-test for HIV^– and/or HIV⁺ subjects. Significant differences are indicated.

Figure 4. IFN-γ secretion in response to secreted, latent, and reactivation Mtb antigens in active TB and LTBI subjects, from Malawi.

IFN-γ ELISA was performed on supernatants collected after 7-day antigen stimulation of diluted blood from TB cases (black) and TST+ (LTBI) controls (white) from KPS (Malawi). Line indicates median, whiskers indicate 5–95% range and dots indicate outliers. Data were analysed by Mann-Whitney U-test for HIV^– and/or HIV⁺ subjects. Significant differences are indicated.

Figure 5. IFN-γ secretion in response to secreted, latent, and reactivation Mtb antigens in active TB and LTBI subjects, from Uganda.

IFN-γ ELISA was performed on supernatants collected after 7-day antigen stimulation of diluted blood from TB cases (black) and TST⁺ (LTBI) controls (white) from MAK (Uganda). Line indicates median, whiskers indicate 5–95% range and dots indicate outliers. Data were analysed by Mann-Whitney U-test for HIV^– and/or HIV⁺ subjects. Significant differences are indicated.

Figure 6. IFN-γ secretion in response to secreted, latent, and reactivation Mtb antigens in active TB and LTBI subjects, from South Africa.

IFN-γ ELISA was performed on supernatants collected after 7-day antigen stimulation of diluted blood from TB cases (black) and TST⁺ (LTBI) controls (white) from SUN (South Africa). Line indicates median, whiskers indicate 5–95% range and dots indicate outliers. Data were analysed by Mann-Whitney U-test for HIV^– and/or HIV⁺ subjects. Significant differences are indicated. n/d = antigen not analysed.

Figure 7. IFN-γ secretion in response to secreted, latent, and reactivation Mtb antigens in active TB and LTBI subjects, from The Gambia.

IFN-γ ELISA was performed on supernatants collected after 7-day antigen stimulation of diluted blood from TB cases (black) and TST⁺ (LTBI) controls (white) from MRC (The Gambia). Line indicates median, whiskers indicate 5–95% range and dots indicate outliers. Data were analysed by Mann-Whitney U-test for HIV^– subjects. Significant differences are indicated.

Figure 8. Heat map of combined responses from all sites for TST^–, TST⁺ (LTBI) and active TB with or without HIV infection in response to secreted, latent, and reactivation Mtb antigens.

Median levels of IFN-γ are shown (pg/mL). Red indicates relatively high levels of IFN-γ and blue indicates relatively low levels. HIV⁺ = human immunodeficiency virus positive; HIV^- = human immunodeficiency virus-negative; TB = active tuberculosis; LTBI = latently TB infected; SUN = Stellenbosch University, South Africa; MRC = Medical Research Council, The Gambia; AHRI = Armauer Hansen Research Institute, Ethiopia; KPS = Karonga Prevention Study, Malawi; MAK = Makerere University, Uganda.