key: cord-0934717-m49zlfcp
authors: Rajamanickam, Anuradha; Kumar, Nathella Pavan; Padmapriyadarsini, C; Nancy, Arul; Selvaraj, Nandhini; Karunanithi, Kushiyasri; Munisankar, Saravanan; BM, Shrinivasa; Renji, Rachel Mariam; Ambu, T.C; V, Vijayalakshmi; Babu, Subash
title: Latent tuberculosis co-infection is associated with heightened levels of humoral, cytokine and acute phase responses in seropositive SARS-CoV-2 infection
date: 2021-07-28
journal: J Infect
DOI: 10.1016/j.jinf.2021.07.029
sha: 28a43af8731441b9ca2be46a97a0fe09d7f679e6
doc_id: 934717
cord_uid: m49zlfcp

OBJECTIVES: : Latent Tuberculosis infection (LTBI) is postulated to modulate immune responses and alter disease severity in SARS-CoV-2 co-infection. However, no data exist on the effect of LTBI on the immune responses in SARS-CoV-2 co-infected individuals. METHODS: : We examined the SARS-CoV-2 specific antibody responses, plasma cytokines, chemokines, acute phase proteins and growth factor levels in LTBI positive and negative individuals with SARS-CoV-2 infection. RESULTS: : Our results demonstrated that individuals with LTBI (LTBI+) and seropositive for SARS-CoV-2 infection were associated with elevated SARS-CoV-2 specific IgM, IgG and IgA antibodies, as well as enhanced neutralization activity compared to those negative for LTBI (LTBI-) individuals. Our results also demonstrate that LTBI+ individuals exhibited significantly higher plasma levels of IFNγ, IL-2, TNFα, IL-1α, IL-1β, IL-6, IL-12, IL-15, IL-17, IL-3, GM-CSF, IL-10, IL-25, IL-33, CCL3 and CXCL10 compared to LTBI- individuals. Finally, our results show that LTBI+ individuals exhibit significantly higher levels of C-reactive protein, alpha-2 macroglobulin, VEGF and TGFα compared to LTBI- individuals. CONCLUSIONS: : Thus, our data clearly demonstrates that LTBI+ individuals seropositive for SARS-CoV2 infection exhibit heightened levels of humoral, cytokine and acute phase responses compared to LTBI- individuals. Thus, LTBI is associated with modulation of antibody and cytokine responses as well as systemic inflammation in individuals seropositive for SARS-CoV2 infection.

Latent Tuberculosis infection (LTBI) is known to infect about a quarter of the world's population, while SARS-CoV-2 has thus far infected over 130 million people worldwide(1, 2). Although both LTBI and SARS-CoV-2 are co-prevalent in many parts of the world, there is a paucity of information about the effect of co-infection on the host immune responses.

Tuberculosis (TB) has been postulated to play a role in the development of SARS-CoV-2 infection and exacerbation of COVID-19 disease in small studies conducted in China and India (3) . It has been shown that COVID-19 can occur before, after or simultaneously with the diagnosis of TB and that mortality is likely to be increased in elderly individuals with these co-infections (4) . A recent study showed that indeterminate result in Interferon Gamma

Release Assays was associated with severe lymphocytopenia in COVID-19 patients (5) . In addition, LTBI was not shown to affect the ability to in vitro respond to SARS-CoV-2 (6) .

Various papers have suggested that in areas of high LTBI prevalence, the profound lymphopenia induced by SARS-CoV-2 and use of steroids as a treatment for COVID-19 could (a) predispose patients to TB reactivation as a consequence of a transient suppression of cellular immunity and/or (b) increase the risk of progressive primary TB infection (7) (8) (9) (10) (11) .

The pathogenesis of COVID-19 is driven by immune responses, excessive inflammation and increased coagulation (12) (13) (14) . COVID-19 can manifest as an asymptomatic, mild, moderate or severe disease (15, 16) . Both LTBI and asymptomatic SARS-CoV-2 infections are associated with enhanced cytokine, chemokine, acute phase protein and growth factor responses (17) . Moreover, antibody responses to the spike protein of SARS-CoV-2 and enhanced neutralizing antibody responses are integral hallmarks of SARS-CoV-2 infection (18) . Therefore, to elucidate the interaction between LTBI and seropositive SARS-CoV-2 infection, we examined binding and neutralizing antibody responses to SARS-CoV-2 as well as the systemic levels of cytokines, chemokines, acute phase proteins and growth factors in seropositive SARS-CoV-2 individuals with or without LTBI.

Consenting individuals with age range among 60 -80 years of age, living in hotspots for SARS-CoV-2 infection in Chennai, India between July 2020 and September 2020. Those who were diagnosed with tuberculosis (TB) in the previous 6-months or were currently on anti-TB treatment were not included in the study. The study participants were negative for HIV or malignancy. SARS-CoV-2 seropositivity and prior infection was diagnosed by IgG positivity for spike protein. All the seropositive individuals were asymptomatic and did not report any signs or symptoms of COVID-19 and did not seek any treatment. LTBI was determined on the basis of positivity for QuantiFERON TB Gold in tube (QGIT) test, with no symptoms or signs suggestive of active TB. Individuals with SARS-CoV-2 acute infection (as determined by RT-PCR) were excluded from the study. Individuals with no history of previous TB and normal chest radiographs were included. QGIT was done based on the manufacturer's recommendations (Qiagen). The study groups are defined as those who are positive for both SARS-CoV-2 and LTBI (hereafter LTBI+) and those who are SARS-CoV-2 positive but negative for LTBI (hereafter as LTBI-).

The SARS-CoV-2 serology was measured by an iFLASH 1800 chemiluminescent immunoassay from Shenzhen YHLO Biotech which measures IgM and IgG assays against both SARS-CoV-2 S-and N-proteins. The tests were performed following the manufacturer's protocol (Shenzhen YHLO Biotech Co., Ltd.) The results were determined by chemiluminescent reaction as relative light units (RLUs). IgM and IgG concentrations were obtained using the iFlash 1800 assay, and ≥10 AU/ml were defined as positive and <10.00 AU/mL was considered as non-reactive. Nucleocapsid specific IgA levels were detected using COVID-19 Human IgA ELISA kit (RayBiotech) based on the manufacturer's protocol. The assay was validated by the positive control mean optical density (PC:OD450) greater than 0.5 and negative control mean less than 0.3.

Plasma samples were used to measure the circulating neutralizing antibodies levels using SARS-CoV-2 Surrogate Virus Neutralization Test Kit according to manufacturer's (GenScript) instructions. The cut-off value for SARS-CoV-2 neutralizing antibody detection, according to the manufacturer, the SARS-CoV-2 Surrogate Virus Neutralization more than or equal to 30% was considered as positive and <20% was considered as non-reactive (19) .

Systemic plasma levels of acute phase proteins, cytokines and chemokines were estimated using Luminex Magpix Multiplex Assay system (Bio-Rad, Hercules, CA).

Milliplex MAP Human CVD Panel Acute Phase magnetic bead panel 3 was used to measure the acute phase proteins and Luminex Human Magnetic Assay kit 45 Plex (R & D systems) was used to measure the cytokines and chemokine levels. The minimum detection levels for acute phase proteins was as follows: alpha-2 macroglobulin (-2-M), 0.49 ng/mL; C-reactive protein (CRP), 0.05 ng/mL; haptoglobin, 0.06 ng/mL; and Serum Amyloid A-1 (SAA-1) 0.06 ng/mL; The lowest detection limits for cytokines were as follows: IFN, 5.7 pg/mL; IL-2, 3.6 pg/mL; TNF, 12.4 pg/mL; IL-1, 10.6 pg/mL; IL-1, 3.5 pg/mL; IFN, 3.9 pg/mL; IFN 3.25 pg/mL; IL-6, 9.0 pg/mL; IL-12, 18.5 pg/mL; IL-15, 2.5 pg/mL; IL-17, 9 pg/mL; IL-3, 17 pg/mL; IL-7, 3.5 pg/mL; G-CSF, 8.4 pg/mL; GM-CSF, 18.4 pg/mL; IL-4, 1.1 pg/mL; IL-5, 6.2 pg/mL; IL-13, 31.8 pg/mL; IL-10, 32.2 pg/mL; IL-25, 18.4 pg/mL; IL-33, 13 .8 pg/mL; IL-1Ra, 11.7 pg/mL. The lowest detection limits for chemokines were as follows: CCL2, 5.9 pg/mL; CCL3, 5.1 pg/mL; CCL4, 103.8 pg/mL; CCL5, 297 pg/mL; CCL11, 21.6 pg/mL; CCL19, 3.9 pg/mL; CCL20, 2.4 pg/mL; CXCL1, 19.1 pg/mL; CXCL2, 21.1 pg/mL; CXCL8, 1.4 pg/mL; CXCL10, 2.6 pg/mL and CX3CL1, 188 pg/mL. The lowest detection limits for growth factors was as follows: VEGF, 5.9 pg/mL; EGF, 8.6 pg/mL; FGF-2, 8.7 pg/mL; PDGF-AA, 5.2 pg/mL; PDGF-BB, 7.31 pg/mL; TGF, 8.6 pg/mL; Flt-3L, 22.9 pg/mL; Granzyme B (GZB), 4.9 pg/mL; PDL-1, 69.3 pg/mL; TRAIL, 22.5 pg/mL.

We used the linear regression curve fit model for the consideration of the values below or above the detection limit and further we normalized the data by performing the normality test prior to statistical analysis. Central tendency were measured using Geometric means. Nonparametric Mann-Whitney U test was used to compare the LTBI+ versus LTBIto identify the statistical significant differences. Multiple comparisons were corrected using the Holm's correction. Data analyses were done using GraphPad PRISM version 9 (GraphPad Software, Inc., San Diego, CA, USA). Principle Component Analysis (PCA) was applied to distinguishing possibly significant trends of cytokines, chemokines, acute phase proteins and growth factors, which are dependable for any of the clustering/separation between LTBI+ versus LTBI-groups. JMP14 software was used to plot Principle Component Analysis (PCA).

The study was approved by the Ethics Committees of NIRT (NIRT-INo:2020010).

Informed written consent was received from all study individuals. The study is part of the clinical study entitled, Study to evaluate the effectiveness of the BCG vaccine in reducing morbidity and mortality in elderly individuals in COVID-19 hotspots in India (NCT04475302).

The study population demographics and clinical characteristics are shown in Table I. There was no significant difference in age or sex or clinical characteristics between the study groups.

To estimate the impact of LTBI on humoral immunity in SARS-CoV-2 infection, we 

To estimate the impact of LTBI on cytokine responses in seropositive SARS-CoV-2 infection, we compared the plasma levels of pro and anti-inflammatory cytokines in seropositive SARS-CoV-2 infected individuals with or without LTBI.

As illustrated in Fig. 2A 

To determine the influence of LTBI on systemic levels of acute phase proteins in seropositive SARS-CoV-2 infection, we compared the plasma levels of these markers in seropositive SARS-CoV-2 infected individuals with or without LTBI. As illustrated in 

Principal components analysis (PCA) was used to visualize differences between the groups created on the entire data set. To visualize the clustering pattern of cytokines between SARS-CoV-2 positive with or without LTBI and LTBI+ denoted as red circle and LTBIdenoted as blue circle individuals, we performed PCA analysis with the cytokines which are statistically difference (IFN, IL-2, TNF, IL1, IL1, IFN, IFN, IL-6, IL-12, IL-15, IL-17, IL-3, GM-CSF, IL-4, IL-10, IL-25 

After excluding the factors with commonalities as low as 0.3. We assessed PCA-1 (IFN, IL-2, TNF, IL1, IL1, IFN, IFN, IL-6 

Since both LTBI and asymptomatic SARS-CoV-2 infection are highly prevalent in South India, it provided us the opportunity to examine the impact of LTBI on the humoral and innate immune responses in these coinfected asymptomatic individuals. We first examined the effect of LTBI on SARS-CoV-2 specific IgM, IgG and IgA binding antibodies as well as SARS-CoV-2 induced neutralizing antibodies. Our data clearly demonstrates elevated levels of binding antibodies and enhanced neutralizing antibody capacity in LTBI/SARS-CoV-2 coinfected individuals. Previous studies have shown that SARS-CoV-2 antibodies are induced in asymptomatic individuals but at a lower magnitude (20) . This has been taken as evidence for a weak adaptive humoral immunity in these individuals. Our data demonstrate that the neutralizing antibody capacity of asymptomatic individuals is not dampened but rather enhanced in the presence of LTBI coinfection. A meta-analysis of 23,320 individuals revealed that SARS-CoV-2 co-infection increased the risk of mortality in TB by a factor of 2.1 (Relative risk, 2.10, Confidence interval, 1.75-2.51) (21) . Whether The early onset of protective cytokines responses, which includes Type 1 cytokines such as IFN, TNF and IL-2 as well as Type 17 cytokines such as IL-17 and Type I IFNs such as IFN and IFN appears to be the key event in protection against infection as well as severe disease (23) . Asymptomatic patients appear protected from this manifestation perhaps due to their ability to mount protective cytokine responses early (24) . Our study clearly demonstrates that coexistent LTBI is associated with heightened plasma levels of a variety of protective cytokines, including IFN, IL-2, TNFa, IL-1a, IL-1, IFN, IFN, IL-6, IL-12, IL-17 and GM-CSF. In addition, LTBI is also associated with a concomitant increase in plasma levels of Type 2 and regulatory cytokines such as IL-4, IL-10, IL-25 and IL-33. Whether the increase in these cytokines is a compensatory response to the enhanced levels of proinflammatory cytokines remains to be determined. Of interest, Type 1 IFN levels were also elevated in LTBI/SARS-CoV-2 infected individuals. Thus, the capacity to produce protective cytokines appears to be enhanced in LTBI individuals, which again might be a potential mechanism to combat infection and morbidity.

Protection against COVID-19 is also dependent on the increased and early production of chemokines that exhibit the capacity to attract innate and adaptive immune cells to the lung (25) . Our data reveal that CCL3 and CXCL10 levels were significantly higher in LTBI/SARS-CoV-2 coinfected individuals suggesting that LTBI is associated with enhanced chemokine responses in addition the cytokine responses. Thus, the occurrence of elevated cytokine and chemokine levels might predispose asymptomatic individuals to protection against symptomatic disease in the presence of LTBI.

Heightened levels of acute phase proteins are a major hallmark of COVID-19 (26) .

CRP is both a diagnostic and prognostic marker of morbidity and mortality in COVID-19 (27) . Other acute phase reactants such as a-2m, SAP and Hp are less well studies in SARS-CoV-2 infection. Our data on coinfected individuals suggests that LTBI is associated with elevated levels of CRP and -2-M. Finally, LTBI is also associated with enhanced levels of two other biomarkers of disease pathogenesis in COVID-19 -VEGF and TGF (28) . Our study also reveals that cytokines for the most part (and chemokines, acute phase proteins and growth factors to a lesser extent) are significantly associated with LTBI positivity. The correlation was performed between the plasma levels of SARS-CoV-2 spike protein specific IgM and IgG, N protein specific IgA and neutralizing antibodies and the QGIT

Tuberculosis and novel Wuhan coronavirus infection: Pathological interrelationship

Tuberculosis and COVID-19 interaction: A review of biological, clinical and public health effects

Preliminary observations on IGRA testing for TB infection in patients with severe COVID-19 eligible for immunosuppressive therapy

Coinfection of tuberculosis and COVID-19 limits the ability to in vitro respond to SARS-CoV-2

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Conflict of interest: F. Palmieri has nothing to disclose. Conflict of interest: F. Riviere has nothing to disclose. Conflict of interest: T. Rodrigo has nothing to disclose. Conflict of interest: D.R. Silva has nothing to disclose. Conflict of interest: A. Sanchez-Montalva has nothing to disclose

Risk factors for COVID-19 death in a population cohort study from the Western Cape Province, South Africa

Impact of the COVID-19 pandemic on tuberculosis management in Spain

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Pubmed Central PMCID: PMC7775097 www.icmje.org/coi_disclosure.pdf and declare: no support from any organisation for the submitted work; JM is chief scientific officer, shareholder and scientific founder of Leucid Bio, a spinout company focused on development of cellular therapeutic agents; no other relationships or activities that could appear to have influenced the submitted work. This does not alter our adherence to PLoS ONE policies on sharing data and materials

Occurrence and transmission potential of asymptomatic and presymptomatic SARS-CoV-2 infections: A living systematic review and meta-analysis

Pubmed Central PMCID: PMC7508369 following competing interests: GS has participated in two scientific meetings for Merck and Biogen. NL is a member of the PLOS Medicine editorial board

Cytokine storm and COVID-19: a chronicle of pro-inflammatory cytokines

Active or latent tuberculosis increases susceptibility to COVID-19 and disease severity

The cytokine storm in COVID-19: An overview of the involvement of the chemokine/chemokine-receptor system

Laboratory characteristics of patients infected with the novel SARS-CoV-2 virus

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Pathology of TB/COVID-19 Co-Infection: The phantom menace. Tuberculosis (Edinb)

LTBI with seropositive SARS-CoV-2 infection is associated with heightened systemic levels of pro and anti-inflammatory cytokines (A) The plasma levels of Type 1

The plasma levels of inflammatory cytokines, IL-17 cytokines were measured in LTBI+ (n = 61) and LTBI-(n=72) individuals seropositive SARS-CoV-2 infection. (C) The plasma levels of Type 2 cytokines, and other anti-inflammatory cytokines were measured in LTBI+ (n = 61) and LTBI-(n=72) individuals seropositive SARS-CoV-2 infection. The data are represented as scatter plots with each circle representing a single individual

A) The plasma levels of CC chemokines were measured in LTBI+ (n = 61) and LTBI-(n=72) individuals seropositive SARS-CoV-2 infection. (B) The plasma levels of CXC chemokines were measured in LTBI+ (n = 61) and LTBI-(n=72) individuals seropositive SARS-CoV-2 infection. The data are represented as scatter plots with each circle representing a single individual

LTBI with seropositive SARS-CoV-2 infection is associated with heightened systemic levels of acute phase proteins and growth factors The plasma levels of acute phase proteins were measured in LTBI+ (n = 61) and LTBI-(n=72) individuals seropositive SARS-CoV-2 infection. (B) The plasma levels of growth factors were measured in LTBI+ (n = 61) and LTBI-(n=72) individuals seropositive SARS-CoV-2 infection. The data are represented as scatter plots with each circle representing a single individual

We thank the Director of the ICMR-NIRT, and staff of the Department of Clinical Research, NIRT. We thank the data entry operators Mr. Jaiganesh and Mr.Vigneshwaran, and also all the staff members of the ICER department for the timely help.

values (A) NIL (B) TB Antigen-1 (C) TB Antigen-II and (D) Mitogen levels of SARS-CoV-2 with LTBI+ (n = 61) individuals. p and r values were calculated using the non-parametric spearman correlation. 

Positive Positive

Positive Positive