The International Journal of Mycobacteriology

: 2022  |  Volume : 11  |  Issue : 4  |  Page : 378--383

Culture filtrate protein 32 as a potential target to attenuate the heterogeneous antibody response against Mycobacterium tuberculosis Antigens in different endemic settings

Chaouki Benabdessalem1, Rym Ouni1, Wafa Ben Hamouda1, Jihene Bettaieb1, Dahmani Mohamed Fathallah2, Mohamed-Ridha Barbouche1,  
1 Department of Immunology, Laboratory of Transmission, Control and Immunobiology of Infection, Institut Pasteur de Tunis; Department of Immunology, University Tunis El Manar, Tunis, Tunisia
2 Department of Immunology, Laboratory of Transmission, Control and Immunobiology of Infection, Institut Pasteur de Tunis, Tunis, Tunisia; Department of Life Sciences, Health Biotechnology Program, College of Graduate Studies, Arabian Gulf University, Manama, Kingdom of Bahrain

Correspondence Address:
Chaouki Benabdessalem
Laboratory of Transmission, Control and Immunobiology of Infection, Institut Pasteur de Tunis, 13 Place Pasteur BP74, 1002 Tunis


Background: We previously reported the development of an enzyme-linked immunosorbent assay for the detection of the immunoglobulin G (IgG) response to Mycobacterium tuberculosis virulence factor – culture filtrate protein 32 (CFP32). The assay achieved high performance in comparing healthy Bacillus Calmette–Guerin-vaccinated controls with active tuberculosis (TB) patients from the Tunisian population. Herein, we aimed to assess the anti-CFP32 IgG response in suspected or confirmed active pulmonary TB individuals in different endemic settings. Methods: Serum samples were obtained from 224 donors from African and Latin American countries with variable levels of TB endemicity and different ethnical origins. Receiver operating characteristic curve was used to evaluate the performance of the serological assay. Results: The area under the curve was 0.70. The use of a cutoff level of 0.65 gave 67% and 68% sensitivity and specificity, respectively, regardless of ethnicity and endemicity. Except for the suspected Latin American group, overall multiple comparisons of medians pointed out the stability of the anti-CFP32 IgG response across the different endemic settings. Therefore, endemicity and ethnicity seem not to affect anti-CFP32 IgG response, mainly for detecting confirmed active TB individuals. Conclusions: These findings suggest that the inclusion of CFP32 epitopes in multi-antigen TB assay could attenuate serological differences related to heterogeneous endemicity and ethnicity. For this purpose, we further identified B-cell epitopes belonging to CFP32 by an in silico analysis.

How to cite this article:
Benabdessalem C, Ouni R, Hamouda WB, Bettaieb J, Fathallah DM, Barbouche MR. Culture filtrate protein 32 as a potential target to attenuate the heterogeneous antibody response against Mycobacterium tuberculosis Antigens in different endemic settings.Int J Mycobacteriol 2022;11:378-383

How to cite this URL:
Benabdessalem C, Ouni R, Hamouda WB, Bettaieb J, Fathallah DM, Barbouche MR. Culture filtrate protein 32 as a potential target to attenuate the heterogeneous antibody response against Mycobacterium tuberculosis Antigens in different endemic settings. Int J Mycobacteriol [serial online] 2022 [cited 2023 Mar 21 ];11:378-383
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Full Text


Overtaken by the coronavirus disease 2019 pandemic, human tuberculosis (TB) became currently the second leading cause of death worldwide due to a single infectious agent, Mycobacterium tuberculosis (Mtb).[1]

Culture filtrate protein 32 (CFP32) is a culture filtrate antigen restricted to the Mtb complex.[2],[3],[4] It is associated with the virulence of mycobacterial strains and, thus, weakly expressed by the Bacillus Calmette–Guerin (BCG) vaccine strain.[4],[5] The DNA-based booster vaccine based on CFP32 showed durable Th1 and strong humoral responses in mice.[6] In previous studies, we detected anti-CFP32 immunoglobulin (Ig) G in infected human sera, thus we developed a serological enzyme-linked immunosorbent assay (ELISA) using Pichia (P) pastoris recombinant CFP32 (rCFP32) antigen.[7],[8] Interestingly, we showed that CFP32 exhibited higher immunoreactivity when produced in the yeast P. pastoris in comparison to Escherichia coli.[8]

We showed that the rCFP32 produced in the yeast P. pastoris retains protein posttranslational events and homodimeric folding which may better mimic the protein's native conformation.[9]

The developed serological test using rCFP32 showed high diagnostic performance in the Tunisian population with an 85% sensitivity and a nearly perfect specificity when comparing sera from active TB (aTB) patients to sera from BCG-vaccinated healthy controls.[8] Indeed, several antigens displayed high performances when comparing aTB to healthy controls, but these performances decreased when comparing aTB to latent TB infection (LTBI), which is an asymptomatic infection status in high-endemic settings.[10] Furthermore, it has been demonstrated that significant antibody responses are not restricted to aTB disease but can reflect latent infection, particularly in regions with high-endemic settings.[11] Moreover, heterogeneity of antibody responses could be attributed to variable ethnic origins, thus affecting the discriminatory power of serodiagnostic tests. Therefore, what is needed in routine clinical practices is a test that discriminates, between aTB cases and suspected but nonactive TB individuals, regardless of endemic settings.

In this study, we evaluated the anti-CFP32 IgG response in suspected versus aTB patients in different endemic settings. We also sought to assess whether the developed CFP32-based ELISA assay maintains similar performances across different ethnic backgrounds and endemic settings. The observed stability of the anti-CFP32 response, mainly in aTB groups, prompted us to identify, using in silico analysis, specific B-cell epitopes that might be useful for the design of a multiepitope antigen to attenuate the heterogeneous antibody response against Mtb antigens.


Study population

Ethics statement

Sera samples were collected within the framework of a previous study that was approved by the Institut Pasteur de Tunis Ethics Committee (N°14/06/I/LR11IPT02).[12] Written informed consent, regarding the use of banked sera, was obtained from all participants included in the Tunisian group (n = 60). The remaining samples (n = 164) were kindly provided by the TDR TB Specimen Bank.[13]


This is a study of a screening and diagnostic test. We used available banked sera in the laboratory and had access to WHO/TDR banked sera. Sera were categorized as follows: sera from patients with (i) active TB (n = 98) confirmed based on clinical symptoms, chest radiography, and sputum smear microscopic examination and culture and (ii) sera from suspected (n = 126). Suspected individuals included: (a) sera samples from recent close contacts of Tunisian aTB patients (n = 34) with TST higher than 15 mm, negative AFB, and normal chest radiography upon recruitment and 3 months later and (b) sera samples from patients (n = 92), enrolled in health clinics collaborating with WHO/TDR, who showed symptoms suggestive of pulmonary TB and for whom TB was excluded based on smear microscopy, bacterial culture, radiography, and clinical follow-up.

Recombinant protein expression and purification

The recombinant protein CFP32 was produced in the yeast P. pastoris and purified as previously described.[7]

Measurement of anti-culture filtrate protein 32 immunoglobulin G by enzyme-linked immunosorbent assay

The ELISA test was performed as previously reported.[8] Briefly, 96-well plates were coated with 100 μl of 2 μg/ml P. pastoris produced rCFP32. Blocking is performed with phosphate-buffered saline (PBS) (pH 7.2) – 0.05% Tween 20%–4% bovine serum albumin. Serum samples were diluted at 1:200 in PBS and then incubated for 2 h at room temperature (RT). Anti-human IgG coupled with horseradish peroxidase (Sigma) is diluted at 1:8000 in PBS-T and then incubated for 1h at RT. The optical density (OD) was measured at 450 nm.

Prediction of B-cell epitopes

Predictions of epitopes were performed for the identification of B-cell binding types of epitopes achieved using Bepipred Linear Epitope Prediction (version 2.0) (PMID: 28472356). The epitopes were mapped on the structure of CFP32 obtained from AlphaFold database.

Statistical analysis

Nonparametric tests were used given the unequally sized groups and the low size of some study samples (<30). In fact, median OD values of TB patients and suspected groups were compared using the nonparametric Mann–Whitney test. The Kruskal–Wallis analysis and Dunn's multiple comparison test were used for data from more than two groups.

The receiver operating characteristic (ROC) curve was performed to illustrate the performance of OD450 test discriminating two categories (aTB patients and suspected groups) for all three groups. The optimal cutoff level identified from the ROC curve was used for the estimation of sensitivity and specificity using standard methods for each group separately. The sensitivities and specificities of the three groups were compared by the Chi-square test.

Differences between groups were considered statistically significant if the P < 0.05. All statistical analyses and graphs were performed using GaphPadPrsim v 5.0 software (GraphPad by Dotmatics Co., San Diego, California).


Study population

[Table 1] summarizes the demographic characteristics of the study population. Age medium and sex ratio matched well in suspected groups as well as in aTB patients' groups. However, heterogeneity regarding BCG vaccination coverage and history of TB was observed in Tunisian groups versus WHO-TDR groups.{Table 1}

Culture filtrate protein 32-based serodiagnostic test potential to discriminate suspected from active tuberculosis individuals

Anti-CFP32 IgG capture ELISA test previously showed a sensitivity of 85% and a specificity of 98% when comparing sera from aTB patients and BCG-vaccinated healthy controls from Tunisia.[8] We here sought to assess the test potential to discriminate between aTB and suspected groups regardless of endemicity and ethnic origins. Thus, we used sera samples from aTB patients (n = 126) and sera from suspected (n = 98). [Figure 1] shows that the median level of anti-CFP32 IgG in the suspected group was significantly (P < 0.001) lower as compared to the aTB group. The area under the curve (AUC) was 0.70 (95% confidence interval [CI], 0.62–0.78) [Figure 2]. The cutoff level was set at 0.65, giving the optimal combination of sensitivity (67%, CI 95%: 57–77) and specificity (68%, CI 95%: 59–77).{Figure 1}{Figure 2}

Culture filtrate protein 32-based serodiagnostic test performance depending on population variation

We then compared the CFP32-based serologic ELISA performance in different groups based on population variation in terms of TB endemicity and genetic background. Using the fixed universal cutoff, [Table 2] shows sensitivity and specificity in different groups. Samples from the Tunisian population (North Africa) showed a sensitivity of 65% and a specificity of 76%. Despite differences in endemicity and ethnical background, the sub-Saharan African sera groups showed sensitivity and specificity nearly identical to the Tunisia patients (65% and 77%, respectively). The assessment of the ELISA test performance using sera from the Latin American groups showed higher sensitivity (69%) but lower specificity (53%) compared to both other groups [Table 2]. Interestingly, the anti-CFP32 ELISA test presented stability of performance, especially in Africa when samples from Tunisia and samples from sub-Saharan regions were used despite the variability of endemicity and ethnicity. The performance of the test is slightly different in the Latin American groups as a whole.{Table 2}

Multiple comparisons of culture filtrate protein 32-specific immunoglobulin G levels

To assess whether population variation, related to endemicity and genetic background, affects circulating anti-CFP32 IgG levels, we performed multiple comparisons between the different groups [Figure 3]. Mann–Whitney test showed that antibody response against CFP32 was significantly higher in aTB groups compared to suspected individuals for all three groups. Kruskal–Wallis test and Dunn's multiple comparison test showed no differences between anti-CFP32 IgG median of aTB groups from different populations with different ethnic origins and with different degrees of TB incidence (P = 0.18). For suspected groups, no significant difference has been shown between IgG anti-CFP32 medians for African groups from Tunisia and sub-Saharan regions despite the high difference in terms of endemicity and ethnic origin variability. The only significant (P = 0.01) difference was seen between the Latin American suspected group in comparison to the Tunisian suspected group but not in comparison to the sub-Saharan group.{Figure 3}

In silico prediction of B-cell epitopes belonging to culture filtrate protein 32

Using the Bepipred Linear Epitope Prediction (version 2.0), [Figure 4] shows the conformational positions and sequences of six putative epitopes belonging to CFP32 identified to bind to B-cell receptors. The corresponding prediction scores are moderate (not more than 0.61). All the predictive epitopes are located on the surface of CFP32 either on loop regions or beta-strand segments.{Figure 4}


TB is an immunological disease.[14] Knowledge of the immune status of the patient could be obtainable by serological measurements, which gives valuable prognostic and diagnostic clues. At the beginning of the last decade, the WHO banned the use of serological tests based on a meta-analysis study, but there is criticism, mainly because of the statistical analysis adopted in the study.[13],[15] Thus, serology has been abandoned in favor of molecular diagnostic tools which have had several uses in the fight against TB.[16] Although a negative Xpert MTB (MTB/RIF) result does not mean excluding TB, the WHO policy guideline recommends the Xpert MTB/RIF test to be used as an initial diagnostic test in individuals suspected of MDR- or HIV-associated TB.[17] Moreover, molecular tools are far from being used in rural areas, where they are most needed. Serological assays are easy and cost-effective tools to assess immune responses to TB and can offer early diagnosis of smear-negative pulmonary TB. Serology is a good alternative to conventional diagnostic tools in extrapulmonary TB, a public health problem in Tunisia and its neighborhood, as well as pediatric TB cases, due to the inability to access site-specific specimens.[18],[19]

Several studies focused on circulating antibodies to evaluate them as biomarkers of active disease.[20],[21],[22],[23],[24],[25] Indeed, antibody-based diagnosis of aTB could be an excellent approach, particularly multi-antigen tests[20],[21],[26],[27] to be evaluated in countries with variable endemicity.

The CFP32-based ELISA assay we previously developed showed both high sensitivity and specificity in the Tunisian population when comparing sera from aTB patients to sera from BCG-vaccinated healthy controls.[8] Indeed, a test that discriminates between aTB cases and suspected but non-aTB individuals is needed in routine clinical practices. This is particularly important considering that people who are contacts of aTB cases or patients (eventually with a history of TB) with suggestive clinical symptoms but TB negative were considered suspected individuals. Furthermore, it is well known that a major hurdle to the development of a reliable serological test for TB is the heterogeneous antibody response against Mtb antigens in different individuals and populations.[11],[28]

Therefore, we assessed the rCFP32-based ELISA assay potential in discriminating suspected individuals from aTB in people with various genetic backgrounds from different endemic regions. Our study included samples from countries representing environments with different TB settings. By combining the anti-CFP32 IgG levels in all suspected groups as compared to all aTB groups, our data showed that the median level of anti-CFP32 IgG in aTB group was significantly higher as compared to the suspected group (P < 0.001). This is despite it has been reported that antibody responses, in general, are not restricted to aTB disease but can reflect latent infection, mainly in high-burden settings of TB.[11] We used ROC in order to assess the test performance independently of TB setting curves. Based on that, our results showed – as expected – a decrease in the CFP32 serology test performance as compared to our previous study, mainly in terms of specificity and sensitivity.[8] Indeed, it has been reported that the very high prevalence of LTBI in countries where TB is endemic may reduce the performance of any antibody-based test intended to identify aTB.[11] Nonetheless, in a comparative study by Wang et al., three out of four commercial serological assays showed similar AUC as anti-CFP32 ELISA assays in discriminating between aTB patients and subjects with LTBI.[21]

To improve the accuracy of the serodiagnosis of TB, several studies have attempted[35] to use multiple mycobacterial antigens instead of a single antigen.[29],[30],[31],[32] However, the selection of antigens to be used in such cocktails should consider mitigating the heterogeneity of specific antibody responses depending on endemic settings.

To assess whether population variation, regarding endemicity and genetic background, affects circulating anti-CFP32 IgG levels, we performed multiple comparisons between the median of different groups. KW test and Dunn's multiple comparison tests showed no significant differences between anti-CFP32 IgG median of aTB groups from different populations with different ethnic origins and with different degrees of TB incidence. The only significant difference observed was the higher median of anti-CFP32 IgG observed in the Latin American group as compared to the Tunisian group. It seems that the universal cutoff value of 0.65 applied across the three geographic areas may not necessarily be optimal for Latin America to best distinguish between the groups. However, the fact that we have no access to healthy control sera from endemic regions did not allow us to set a specific cutoff for each region which is a limitation. Otherwise, this difference might be also related to the Mtb strain circulating in this geographical area since it has been shown that clinical isolates of Mtb may express different levels of CFP32 antigen.[33] Indeed, it has been reported that Mtb genotype may affect the profile of antigen-specific antibody response.[34]

Finally, the observed stability of the anti-CFP32 response prompted us to identify, by in silico analysis, six specific B-cell epitopes. These mapped epitopes could be used for the development of a multi-epitope antigen that might be useful, mainly for the screening of aTB cases.


In summary, we herein demonstrated that neither variable endemicity nor genetic background does significantly affect the anti-CFP32 IgG response, mainly in aTB cases. Despite the limited diagnostic value of the CFP32 single-antigen serological test, its use might be promising to design a multi-antigen cocktail or fusion protein to improve the detection accuracy for aTB cases.



Ethical statement

The study was approved by the Institut Pasteur de Tunis Ethics Committee (N°14/06/I/LR11IPT02).

Financial support and sponsorship

The Ministry of Higher Education and Scientific Research in Tunisia supported this work through the IPT-LR11_02 grant.

Conflicts of interest

There are no conflicts of interest.


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