Tuberculosis 133 (2022) 102170

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Tuberculosis
journal homepage: www.elsevier.com/locate/tube

Effective combined antiretroviral therapy provides partial immune

recovery to mycobacterial antigens in vertically infected, BCG-vaccinated
youth living with HIV☆
Mariana Virginello Castelhano a, Paulo César Martins Alves a, Vítor Schandler Macedo a,
Mauro Pedromonico Arrym a, Fernando Guimarães b, Patricia Costa Panunto c,
Taís Nitsch Mazzola a, Renan Marrichi Mauch a, 1, Maria Marluce dos Santos Vilela a, d, 1,
Marcos Tadeu Nolasco da Silva a, d, 1, *
a
Center for Investigation in Pediatrics (CIPED), School of Medical Sciences, University of Campinas, Campinas, São Paulo, Brazil
b
Center of Integral Services for Women’s Health (CAISM), University of Campinas (UNICAMP), Campinas, São Paulo, Brazil
c
Laboratory of Pharmacology and Biochemistry, Department of Pharmacology, Faculty of Pharmaceutical Sciences, University of Campinas, Campinas, São Paulo, Brazil
d
Department of Pediatrics, School of Medical Sciences, University of Campinas, Campinas, São Paulo, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Background: We assessed the cytokine response by PBMC of youth living with HIV (YLHIV) under combined
Mycobacterium tuberculosis antiretroviral therapy (cART) to Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis (BCG) antigens.
HIV Methods: PBMC from 20 Brazilian YLHIV under cART with long-term (≥1 year) virological control, and 20
Cytokine
healthy controls were cultured for 24–96 h under stimulation with BCG, Mtb lysates, ESAT-6 and SEB. We
Young adult
measured TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-10 and IL-17 in culture supernatants using a cytometric bead array.
BCG vaccine
Antiretroviral therapy Results: Controls had higher IFN-γ production at 24, 48, 72 and 96 h upon stimulation with BCG lysate, pla­
Highly active teauing at 48 h (Median = 1991 vs. 733 pg/mL; p = 0.01), and after 48–72 h of stimulation with Mtb lysate,
plateauing at 48 h (3838 vs. 2069 pg/mL; p = 0.049). YLHIV had higher TNF-α production at all time points upon
stimulation with ESAT-6, with highest concentration at 36 h (388 vs. 145 pg/mL; p = 0.02). Within the YLHIV
group, total CD4 T cell count and CD4/CD8 ratio were associated with IFN-γ response to Mtb lysate and ESAT-6,
respectively.
Conclusions: Even under long-term cART, YLHIV seem to have a suboptimal T-helper-1 response to mycobacterial
antigens. This can be explained by early immunodeficiency in vertical infection, with lasting damage.

1. Introduction
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), re­
mains one of the most prevalent and deadly infectious diseases world­
wide, despite the availability of effective treatment. Around two billion
people are currently infected with Mtb. In 2020, approximately 10
million people developed TB, with 5.8 million newly diagnosed cases,
the latter being underreported due to the decreased access to TB diag­
nosis during the Covid-19 pandemic. Also in 2020, around 1.5 million
people died of TB – a figure that was up from 1.4 million in 2019 [1].
People infected with the Human Immunodeficiency Virus (HIV) have
a nearly 20-fold increased risk of developing TB, when compared to non-
infected people as HIV-induced immunosuppression worsens [2]. Nearly
70% of TB cases reported worldwide correspond to HIV-infected people,

Abbreviations: PBMC, Peripheral Mononuclear Blood Cells; BCG, Bacillus Calmette-Guérin; ESAT-6, Mycobacterium tuberculosis Early Secreted Antigenic Target
of 6 kDa; SEB, Staphylococcal Enterotoxin B (positive assay control); TNF-α, Tumor Necrosis Factor-Alpha; IFN-γ, Interferon-Gamma; IL, Interleukin.
☆
Presented in part at the Keystone Symposia Conference “New Developments in Our Basic Understanding of Tuberculosis”, January 14–17, 2017, Vancouver,
British Columbia, Canada (Poster #3034).
* Corresponding author. Center for Investigation in Pediatrics, School of Medical Sciences, University of Campinas, Rua Vital Brasil 80, FCM 04 (CIPED), 13083-
888 Campinas/SP, Brazil. Tel.: +55 19 35218979.
E-mail address: nolasco@unicamp.br (M.T. Nolasco da Silva).
1
The authors contributed equally to the article.

https://doi.org/10.1016/j.tube.2022.102170
Received 10 November 2021; Received in revised form 14 January 2022; Accepted 23 January 2022
Available online 31 January 2022
1472-9792/© 2022 Elsevier Ltd. All rights reserved.
M.V. Castelhano et al.
with 214,000 deaths reported in 2020 – up from 209,000 in 2019 [1].
Additionally, since TB can boost the progress of HIV infection, control of
HIV replication with combined antiretroviral therapy (cART) is crucial,
and not only reduces the odds of Mtb co-infection, but also helps in the
TB treatment [3], besides promoting a good quality of life to people
living with HIV.
Cellular immunity has a well-known importance for Mtb infection
control, with a critical role of cytokines of the Interleukin (IL)-12-
Interferon-gamma (IFN-γ) axis, components of the T-helper 1 (Th1) CD4
T cell response. In the same pathway, IL-2 and Tumor Necrosis Factor-
alpha (TNF-α) have also been shown to play a protective role. Addi­
tionally, cytotoxic CD8 T cell (CD8) and Th17 mechanisms may be
meaningful players [4]. In this context, immunity to Mtb relies on T
lymphocytes as mediators and mononuclear phagocytes as effectors.
Active immunization against TB is currently achieved with the Bacillus
Calmette-Guérin (BCG) vaccine, an attenuated Mycobacterium bovis strain
developed 100 years ago. BCG partially protects infants and young
children against severe extrapulmonary TB, but it has a poor efficacy
against pulmonary TB, notably in adolescents and adults [5]. In Brazil,
BCG vaccination is mandatory and provided as a single dose to all in­
fants in their first month of life, covering around 79% of the population
[6].
There is a paucity of reports about antimycobacterial immunity in
people with vertical, well-controlled HIV infection who were vaccinated
with BCG early in life. In a previous study, we found that the activities of
CD8 and γδ T cells are preserved in these patients [7]. Understanding the
anti-Mtb immunity in this population is imperative, as they have
increasing survival rates due to cART and will face exposure to TB
during their lifetime. In this article, we aimed to assess whether the in
vitro cytokine production by peripheral mononuclear blood cells (PBMC)
under stimulation with Mtb and BCG antigens are comparable between
BCG-vaccinated youth living with HIV (YLHIV) with well-controlled
vertical HIV infection and healthy controls.
2. Methods
2.1. Study participants
In this prospective, cross-sectional study, we enrolled 20 BCG-
vaccinated YLHIV, aged 16–23 years, with vertical HIV infection who
were under cART and had a well-controlled viral replication (unde­
tectable plasma viral loads) for at least one year and were clinically
stable at the moment of enrollment. Adherence to cART was assessed
using a protocol based on pharmacy dispensing records [8]. The patients
were recruited at the Pediatric Immunodeficiency Outpatient Unit at the
University of Campinas Teaching Hospital (HC Unicamp, Campinas,
Brazil) from a cohort of 140 children and adolescents longitudinally
followed and vertically infected with HIV-1, diagnosed, clinically and
immunologically classified according to the criteria of Brazil’s Ministry
of Health, adapted from the Centers for Disease Control and Prevention
(CDC, United States) [9,10]. The clinical classification comprises four
categories: N (not symptomatic), A (mildly symptomatic); B (moderately
symptomatic) and C (severely symptomatic). The immunologic classi­
fication is based on total CD4 T cell counts and comprises categories 1
(no immunosuppression), 2 (moderate immunosuppression) and 3 (se­
vere immunosuppression). After a clinical examination, YLHIV who
were diagnosed with any acute infections were not included in the study.
According to their cART regimen, YLHIV were classified into patients
under low-complexity cART (two nucleoside or nucleotide analogues
and one non-nucleoside analogue) and high-complexity cART (all other
therapeutic schemes). Additionally, 20 BCG-vaccinated healthy volun­
teers were enrolled as controls. BCG vaccination was confirmed after
review of the participants’ vaccination records or by verifying the
presence of the BCG scar in their deltoid area. Latent tuberculosis status
could not be assessed in patients or controls due to a worldwide shortage
of the Mtb Purified Protein Derivative (PPD), which also affected the
2
Tuberculosis 133 (2022) 102170
Brazilian public health system.
Blood collection and laboratory procedures were performed from
October 2015 to March 2017. The study protocol was approved by the
University of Campinas Research Ethics Committee (approval report no.
286.070). Written informed consent was obtained from each volunteer,
or from their legal guardians (for volunteers aged less than 18 years old).
2.2. Peripheral blood immunophenotyping, complete blood count and
HIV-1 RNA viral load
One mL of peripheral blood was collected in tubes with ethylene
diaminetetracetic acid (EDTA) in order to perform a complete white
blood cell (WBC) count, using a KX-21 N automated hematology
analyzer (Sysmex, Mundelein, IL, USA). Immunophenotyping was per­
formed using flow cytometry, with the surface markers CD3/APC-H7
(clone SK7), CD4/BB515 (clone RPA-T4), CD8/PercpCy5.5 and
TCRγδ/PE, in a six-color FACSVerse flow cytometer (BD Biosciences,
Franklin Lakes, NJ, USA). The Abbott Real Time HIV-1 kit and the
Abbott m2000rt instrument (Promega Corporation, Fitchburg, WI, USA)
were used to quantify the plasma HIV-1 RNA, with a lower limit of
detection of 40 RNA copies/mL.
2.3. Cell culture and stimulation
Twenty mL of heparinized peripheral blood were drawn for culture
assays. Fresh PBMC were isolated by density gradient centrifugation
over Ficoll-Paque Plus (GE Healthcare, Amershan, Buckinghamshire,
UK). PBMC were washed, counted and diluted to 2x106 cells/mL in
RPMI 1640 medium (Gibco™, Thermo Fisher Scientific, Norcross, GA,
USA) supplemented with 10% human AB serum (Sigma) and 10 μg/mL
gentamycin, and cultured at 37 ◦ C with 5% CO2 in 12 × 75 mm
cytometry tubes (BD Biosciences, Franklin Lakes, NJ, USA) under
different stimulation conditions, namely: not stimulated (medium alone,
negative control), stimulated with a BCG lysate at 10 μg/mL (BCG
Moreau strain, Ataulpho de Paiva Foundation, Rio de Janeiro, RJ,
Brazil), stimulated with an Mtb lysate at 10 μg/mL (H37RA strain, -
ATCC 25177, Manassas, VA, EUA), stimulated with the Mtb Early
Secretory Antigenic Target of 6 kDa (ESAT-6) recombinant antigen at 15
μg/mL (Statens Seruminstitut, Copenhagen, Denmark), and stimulated
with the mitogenic positive control Staphylococcal Enterotoxin B (SEB)
at 500 ng/mL (Sigma, Sant Louis, MO, USA).
Mycobacterial lysates were prepared using in house protocol. The
BCG Moreau strain and the Mtb H37RA strain were grown in
Löwenstein-Jensen medium. Both bacteria were incubated at 37 ◦ C for
at least 15 days. After growth, cultures were scraped and transferred to
tubes containing distilled water. Strains were heat-inactivated in a 95 ◦ C
dry bath for 30 min. Lysates were obtained after sonication in an ul­
trasonic tip sonicator, using average power and three cycles of 10 s. The
supernatant was filtered in a 0.2 μm membrane at the Laboratory of
Biochemical Pharmacology, Unicamp Faculty of Pharmaceutical Sci­
ences. Protein dosage was performed using the Lowry method [11]. All
lysate samples were from the same batch.
2.4. Mycobacterium-specific cytokine production
Cell culture supernatants were collected after 24, 36, 48, 72, and 96 h
of incubation and were stored at − 80 ◦ C until cytokine dosage. The
concentrations of IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-10, and IL-17 were
measured using a Cytometric Bead Array (CBA, BD Biosciences), ac­
cording to the manufacturer’s protocol, in a six-color FACSVerse flow
cytometer (BD Biosciences) and analyzed using the FCAP Array Software
(version 3.0.1., BD Biosciences). Theoretical limits of detection were 2.6
pg/mL for IL-2, 4.9 pg/mL for IL-4, 1.1 pg/mL for IL-5, 4.5 pg mL for IL-
10, 3.8 pg/mL for TNF-α, 3.7 pg/mL for IFN-γ and 18.9 pg/mL for IL-17.
For statistical purposes, samples below the lower limits of detection
were scored as half of the lower limits.
M.V. Castelhano et al. Tuberculosis 133 (2022) 102170

2.5. Statistical analyses This volunteer was tested for latent and symptomatic TB during previous
clinical follow-up, and the infection was ruled out. Due to the age range
The cytokine concentration values were firstly given as picograms of the patients in the YLHIV group, different treatment protocols were
per milliliter (pg/mL), and the values underwent logarithmic (Log) used, resulting in a wide variation in the age of onset of effective cART
transformation for graphic purposes. The transformed values are shown (Median = 2.55 years; range 0.44–16.43) (Table 2).
as the medians (with first and third quartiles), or as individual values.
We used the t-Student test (normal data distribution) and the Mann- 3.2. Cytokine production in response to different stimuli
Whitney U test (non-normal data distribution) to compare numerical
values between two groups, and the chi-square (χ2) test to compare Of note, all non-transformed, raw values of the IL-2, IL-4, IL-5, IL-10
proportions between groups. The Kruskal-Wallis test, followed by the and IL-17 concentrations in different time points, and upon the five
Mann-Whitney test with the Bonferroni correction was used when more different stimulation conditions, are available. These data are displayed
than two groups were compared. More than two related samples were as the median, minimum, maximum values, and the interquartile range
compared using the Friedman test with paired comparisons. To assess (IQR) (Suppl Tables 1 and 2).
the association between clinical category and cytokine levels, patients in
categories A and B were grouped and compared with patients in cate­ 3.3. Stimulation with BCG, Mtb lysates and ESAT-6
gory C. To assess the association between immunological category and
cytokine levels, patients in immunological categories 1 and 2 were The control group had significantly higher concentrations of IFN-γ in
grouped and compared with patients in category 3. These classifications cultures at 24, 48, 72, and 96 h upon PBMC stimulation with BCG lysate.
were done to achieve suitable sample sizes for statistical analysis. For No significant differences between the groups were seen in the TNF-α
these analyses, we considered the peak of production of each cytokine. concentrations in any time point (Fig. 1, Table 3).
Multiple linear regression analysis was used to investigate variables that YLHIV had an irregular variation in the IFN-γ concentrations upon
could predict the cytokine levels. For all tests, a two-tailed p-value stimulation with Mtb lysate. The IFN-γ production was significantly
≤0.05 indicated statistical significance. Statistical analyses were per­ higher in PBMC cultures from controls at 48 and 72 h. No significant
formed using Excel (Microsoft, Redmond, WA, United States) and SPSS differences between the groups were seen in the TNF-α concentrations in
version 25 (IBM, Endicott, NY, United States). We used GraphPad Prism any time point (Fig. 1, Table 3).
version 5.0 (GraphPad Software, San Diego, CA, United States) to yield Controls also showed significantly higher concentrations of IL-5 at 72
graphics. and 96 h after stimulation with BCG lysate (Suppl Table 1, Suppl Fig. 1).
We did not find significant differences between groups regarding pro­
3. Results duction of IL-2, IL-4, IL-10 and IL-17 in response to BCG and Mtb lysates
(Suppl Table 1, Suppl Fig. 1).
3.1. Study participants After PBMC stimulation with ESAT-6, YLHIV and controls did not
significantly differ in the IFN-γ production in any timepoint. However,
The YLHIV group consisted of nine (45%) female and 11 (55%) male YLHIV had significantly higher TNF-α production in all time points
individuals, with a mean age of 18.6 years (Table 1). The control group (Fig. 1, Table 3). There were no significant differences between groups
consisted of 11 (55%) female and nine (45%) male individuals, with a in the concentrations of IL-2, IL-4, IL-5, IL-10 and IL-17 upon PBMC
median age of 20.44 years, and significantly higher age when compared stimulation with ESAT-6 (Suppl Table 1, Suppl Fig. 1).
to the YLHIV group (Table 1). White cell counts and their sub­
populations are also shown in Table 1. YLHIV showed a significantly 3.4. Stimulation with SEB
higher CD8 T cell count, while the controls had a significantly higher
CD4/CD8 ratio (Table 1). Among YLHIV, 13 (65%) were in clinical In cultures of PBMC stimulated with SEB, YLHIV had significantly
category B, and nine (45%) were in immunological category 3. Only one higher concentrations of IL-5 after 24, 36, and 48 h and IL-4 after 24 h,
patient had a previous history of contact with a person with tuberculosis. while the controls had significantly higher concentrations of IL-17 after
96 h (Fig. 1, Suppl Table 1, Suppl Fig. 1). No other significant differences
were seen.
Table 1
Demographical characteristics and basic laboratory markers of youth living with
HIV (YLHIV) under long-term combined antiretroviral therapy (cART) and non- 3.5. Differences in the cytokine production upon stimulation with BCG,
HIV controls included in the study. Mtb lysates and ESAT-6 in subgroups of YLHIV
YLHIV Controls p-
value Since the IFN-γ and TNF-α responses to all stimuli concentrated the
significant differences between groups, we performed subgroup analyses
Age (years) 18.63 ± 2.16 20.44 ± 2.35 0.02a
Mean ± standard deviation
to assess the association between age, time on and complexity of cART,
Gender 9 F/11 M 11 F/9 M 0.53b immunological and clinical categories, total CD4, CD8 cell count and
Female/Male CD4/CD8 ratio of YLHIV and IFN-γ and TNF-α responses to BCG, Mtb
WBC (*103 cells/μL) 6.15 (2.80–8.20) 6.40 (3.50–10.30) 0.46c lysates and ESAT-6. After multiple linear regression analysis, total CD4
Median (Min-Max)
cell count was shown to be a predictor of the IFN-γ response to Mtb
Lymphocytes (*103 cells/μL) 2.40 (1.19–3.57) 2.11 (0.90–3.32) 0.46c
Median (Min-Max) lysate, and the CD4/CD8 ratio was shown to be a predictor of the IFN-γ
Total CD3 cells (*103 cells/μL) 1.61 (0.45–2.82) 1.42 (0.65–2.59) 0.59c response to ESAT-6. We did not find any other significant associations
Median (Min-Max) (Suppl Table 2).
CD4 T cells (*103 cells/μL) 0.62 (0.34–1.95) 0.74 (0.32–1.67) 0.33c
Median (Min-Max)
CD8 T cells (*103 cells/μL) 0.84 (0.29–1.61) 0.50 (0.18–1.04) 0.04c
4. Discussion
Median (Min-Max)
CD4/CD8 T cell ratio 1.04 (0.30–1.71) 1.51 (0.64–2.54) 0.001c We found that YLHIV with controlled viral replication under cART
Median (Min-Max) who were vaccinated with BCG early in life show CD4 T cell activation,
a
t-Student test (t-test). with significant IFN-γ and TNF-α production upon stimulation of their
b
chi-square (χ2) test. PBMC with BCG and Mtb lysates. However, unlike our previous findings
c
Mann-Whitney (U) test. of cytotoxic granule production by activated CD8 and γδ T cells [7],

3
M.V. Castelhano et al. Tuberculosis 133 (2022) 102170

Table 2
Demographical, clinical, immunological, and therapeutic data of youth living with HIV (YLHIV) under combined antiretroviral therapy (cART) with long-term
virological control included in the study. Age, gender, clinical and immunological classification, age at cART initiation, cART duration and current cART composition.
Patients Age (years) Gender Clinical/immunological classification Age at cART initiation cART duration (years) Current cART scheme
(years)

P1 17.52 F C3 2.50 15.01 3 TC + DRV/r + RALb

P2 15.45 M B3 2.14 13.30 TDF+3 TC + EFV + DRV/r + RALb
P3 19.56 M B1 15.56 3.99 TDF+3 TC + EFVa
P4 16.33 F B1 14.19 2.13 TDF+3 TC + EFVa
P5 16.12 M B1 1.77 14.34 TDF+3 TC + EFV + DRV/r + MVC + T20b
P6 16.45 F B3 1.06 15.38 3 TC + DDI + LPV/rb
P7 22.11 M C3 6.00 16.10 TDF+3 TC + FPV/rb
P8 21.17 M C2 1.82 19.34 ZDV+3 TC + EFV + LPV/rb
P9 19.19 F B3 7.25 11.93 TDF+3 TC + LPV/rb
P10 19.67 M A1 16.43 3.23 TDF+3 TC + EFVa
P11 20.22 F B1 2.61 17.59 TDF+3 TC + EFV + DRV/r + RALb
P12 21.57 M B2 10.46 11.10 TDF+3 TC + EFVa
P13 18.24 F B1 1.71 16.53 TDF+3 TC + FPV/r + RALb
P14 19.26 M C3 2.34 16.91 ZDV+3 TC + EFVa
P15 17.58 F B2 1.08 16.49 TDF+3 TC + LPV/rb
P16 20.99 M C3 5.04 15.93 ZDV +3 TC + EFV + LPV/r + RALb
P17 17.82 F B2 1.46 16.35 D4T+3 TC + NVPa
P18 14.61 M C3 0.44 14.17 TDF+3 TC + EFVa
P19 18.09 F B2 4.03 14.05 TDF+3 TC + ATV/rb
P20 20.73 M B3 7.82 12.90 TDF+3 TC + EFVa

Abbreviations: 3 TC (Lamivudine), ATV/r (Atazanavir/ritonavir), D4T (Stavudine), DDI (Didanosine), DRV/r (Darunavir/ritonavir), EFV (Efavirenz), FPV/r
(Fosamprenavir/ritonavir), MVC (Maraviroc), LPV/r (Lopinavir/ritonavir), RAL (Raltegravir), NVP (Nevirapine), T20 (Enfuvirtide), TDF (Tenofovir disoproxil
fumarate), ZDV (Zidovudine).
a
Low complexity cART.
b
High complexity cART.

Fig. 1. Kinetics of IFN-γ and TNF-α in youth living with HIV (YLHIV, n = 20) and non-HIV healthy controls (n = 20) after PBMC stimulation with BCG lysate, Mtb
lysate, Mtb Early Secretory Antigen Target of 6 kDa (ESAT-6) and Staphylococcal Enterotoxin B (SEB). Non-stimulated PBMC were used as intern negative control
samples. The concentrations of each cytokine (pg/mL, logarithmically transformed) were measured in PBMC culture supernatants in different time points after
stimulation, namely 24, 36, 48, 72, and 96 h *p ≤ 0.05 and **p ≤ 0.01 between the groups.

IFN-γ production is lower when compared to PBMC of BCG-vaccinated
healthy controls, suggesting that, although effective cART provides
some degree of immune recovery in this population of YLHIV, it may not
be enough to fully restore their immune response.
Successful cART has been associated with the restoration of the
polyfunctional response to mycobacterial antigens by previous studies,
with a major role played by effector memory CD4 T cells, increased IFN-
γ production in comparison with non-treated patients, and even with
comparable IFN-γ production between people living with HIV and
healthy controls [12–14]. In our study, as there was no difference
between YLHIV and healthy controls regarding the blood concentration
of CD4 T cells, the reduced Th1 response in YLHIV is likely to be caused
by the early HIV pathogenicity in the vertical infection, resulting in a
chronic impairment of the antimycobacterial immune response, as
shown in other reports [15,16]. This is corroborated by many of the
YLHIV enrolled in our study having initiated cART after two years of
age. In early infection stages, cART has been show to correlate with CD4
T cell normalization and less apoptosis of naïve T cells in lymphoid
tissues, as the structure of the damaged lymphoid tissue will limit im­
mune reconstitution in proportion to the extent of lymphoid tissue

4
M.V. Castelhano et al. Tuberculosis 133 (2022) 102170

Table 3A
Kinetics of Interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) (expressed in picograms/mL) in supernatants of cultured peripheral mononuclear blood
cells (PBMC) of youth living with HIV under combined antiretroviral therapy (cART) in all time points (h). PBMC were grown under different stimulation conditions,
namely non-stimulated (NS, culture medium only), stimulated with Bacille Calmette-Guérin (BCG), Mycobacterium tuberculosis (Mtb) lysates, Mtb Early Secretory
Antigen Target of 6 kDa (ESAT-6) and Staphylococcal Enterotoxin B (SEB). Each value is given as the median, followed by the minimum, maximum values and the
interquartile range (IQR): Median (minimum – maximum; IQR).
Cytokine Stimulus Median concentration in pg/mL (Minimum–Maximum; IQR) and time points (h)

24 h 36 h 48 h 72 h 96 h

IFN-γ NS 1.85 (1.85–1648.33; 5.90 (1.85–2470.02; 43.48) 1.85 (1.85–1934.74; 11.76) 3.16 (1.85–5986.88; 6.27) 3.81 (1.85–26343.34;
14.65) 4.66)
BCG 255.11 (1.85–4126.71; 221.33 (8.01–2470.02; 722.68 (11.74–3871.06; 703.15 (1.85–4711.97; 913.30 (1.85–25728.63;
1050.00) 1332.00) 1459.00) 2104.00) 1563.00)
Mtb 1996.21 503.13 (1.85–26855.10; 2068.74 (9.72–23692.85; 944.37 (1.85–51593.08; 3426.77
(1.85–24530.16; 3852.00) 6165.00) 4160.00) (12.16–35568.15;
3345.00) 8044.00)
ESAT-6 1.85 (1.85–2307.80; 1.85 (1.85–2892.75; 9.44) 1.85 (1.85–3592.26; 24.06) 1.85 (1.85–6014.52; 22.22) 4.79 (1.85–7749.30;
6.76) 25.66)
SEB 3016.00 6160.02 10141.34 27543.03 27931.20
(1.85–17403.65; (316.37–31956.38; (1341.86–36337.73; (801.03–105973.63; (1.85–84369.43;
4125.00) 12939.00) 19087.00) 31757.00) 14763.00)

TNF-α NS 5.91 (1.85–148.01; 6.18 (1.85–8147.79; 25.84) 3.26 (1.85–96.28; 18.07) 1.85 (1.85–59.05; 5.19) 1.85 (1.85–63.04; 4.03)
48.07)
BCG 318.15 (1.90–2612.05; 546.88 (13.72–2636.29; 121.95 (5.91–2733.34; 152.74 (1.90–2435.33; 159.61 (1.90–1717.70;
1419.00) 1442.00) 593.60) 455.30) 430.20)
Mtb 506.49 (1.90–4417.69; 979.72 (1.90–7472.66; 379.43 (1.90–3517.51; 385.87 (1.90–5488.43; 423.55 (3.93–6087.09;
1263.00) 3182.00) 1032.00) 930.00) 1413.00)
ESAT-6 292.59 (1.90–1931.64; 347.60 (40.88–3051.23; 191.89 (1.90–1565.82; 198.80 (1.90–769.25; 151.11 (1.90–809.91;
575.70) 513.80) 426.20) 300.40) 228.60)
SEB 674.55 (1.90–4687.62; 2206.01 (20.33–12250.42; 1822.10 (10.28–4753.55; 2283.84 (580.26–4823.91; 1726.55 (1.90–3932.85;
1698.00) 1597.00) 1964.00) 1828.00) 1741.00)

Table 3B
Kinetics of Interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) (expressed in picograms/mL) in supernatants of cultured peripheral mononuclear blood
cells (PBMC) of non-HIV controls in all time points (h). PBMC were grown under different stimulation conditions, namely non-stimulated (NS, culture medium only),
stimulated with Bacille Calmette-Guérin (BCG), Mycobacterium tuberculosis (Mtb) lysates, Mtb Early Secretory Antigen Target of 6 kDa (ESAT-6) and Staphylococcal
Enterotoxin B (SEB). Each value is given as the median, followed by the minimum, maximum values and the interquartile range (IQR): Median (minimum – maximum;
IQR).
Cytokine Stimulus Median concentration in pg/mL (Minimum–Maximum; IQR) and time points (h)

24 36 48 72 96

IFN-γ NS 1.85 (1.85–125.54; 3.20) 9.06 (1.85–247.13; 42.63) 1.85 (1.85–1402.01; 7.45) 1.85 (1.85–23.11; 3.56) 1.85 (1.85–20.73; 3.97)
BCG 968.03 (100.40–5903.54; 1302.47 (53.55–7000.97; 1990.99 2065.85 (164.21–34419.02; 2032.49 (57.81–81804.87;
2065.00) 3309.00) (166.48–22052.57; 3989.00) 3501.00)
4029.00)
Mtb 2425.88 (40.91–33732.10; 1915.05 (63.18–34917.82; 3837.66 4026.42 (145.59–85909.35; 4942.44
3500.00) 3540.00) (136.32–91116.43; 15263.00) (136.27–158526.82;
15896.00) 27285.00)
ESAT-6 1.85 (1.85–578.23; 0.00) 1.85 (1.85–2892.75; 2.61) 1.85 (1.85–3592.26; 1.40) 1.85 (1.85–15473.56; 2.36) 1.85 (1.85–7691.45; 8.52)
SEB 3796.64 7712.02 12378.43 32895.90 34549.87
(917.85–47960.67; (612.35–36270.85; (783.46–51368.38; (1027.59–84765.20; (646.40–88376.46;
13567.00) 13918.00) 18464.00) 37961.00) 44591.00)

TNF-α NS 1.90 (1.90–105.12; 13.87) 8.13 (1.90–7573.50; 1.90 (1.90–96.28; 11.19) 1.90 (1.90–59.05; 0.00) 1.90 (1.90–63.04; 0.00)
72.69)
BCG 318.15 (13.85–3061.97; 642.28 (52.14–4341.84; 191.03 (8.62–2733.34; 115.58 (5.27–2989.73; 186.90 (18.77–4355.83;
1419.00) 1442.00) 593.60) 455.30) 430.20)
Mtb 731.41 (11.63–5535.76; 2161.17 (26.38–8116.09; 661.93 (8.45–5263.84; 592.49 (4.03–4634.03; 817.50 (6.83–5791.91;
1490.00) 3430.00) 1773.00) 1600.00) 1816.00)
ESAT-6 105.76 (1.90–900.83; 144.55 (1.90–6753.15; 71.47 (1.90–742.56; 56.68 (1.90–605.66; 157.50) 55.54 (1.90–723.26;
139.00) 329.30) 127.90) 130.10)
SEB 619.50 (93.71–4333.44; 2155.12 (36.90–5905.65; 1395.32 (10.28–4152.64; 1767.75 (253.86–4944.94; 1625.20 (151.97–3762.65;
922.50) 2363.00) 1329.00) 2874.00) 1389.00)

damage [17,18]. Unfortunately, treatment protocols were not
completely clear when YLHIV included in the present study were born,
and only in 2014 did the Brazilian guidelines recommend that cART
should be started immediately upon diagnosis of either vertical or hor­
izontal HIV infection [19].
On the other hand, we found similar IFN-γ production and increased
TNF-α production in response to ESAT-6 by PBMC of YLHIV when
compared to healthy controls. This is in agreement with a recent study
assessing the response of people living with HIV to novel, specific Mtb
antigens, including Rv2346/2347c, which is associated to the ESAT-6
secretion system, in response to which patients who developed TB had
an increased TNF-α production up to two years before TB diagnosis,
when compared to patients who did not develop TB [20]. Another study,
assessing non-IFN-γ-mediated responses of people living with HIV found
that several novel Mtb antigens expressed in vivo, including
latency-associated antigens of the Mtb DosR-regulon pathway and
resuscitation-promoting factors (Rpf)-state-specific antigens, elicited
intense immune response characterized by increased production of

5
M.V. Castelhano et al.
multiple cytokines, including TNF-α, in the absence of IFN-γ production
[21]. After reviewing individual data of YLHIV in our study, we
observed that seven patients had TNF-α levels above the highest value of
the control group. We thought that these patients could either have LTBI
or memory response to ESAT-6 resulting from previous exposure to
M. tuberculosis, which is carried by 21% of the Brazilian population [22].
Unfortunately, we were not able to search for LTBI in these patients
during the study, due to a global shortage of the M. tuberculosis purified
protein derivate (PPD) at that time. These patients were further
reevaluated after the PPD was available again, and we found that none
of them had LTBI, suggesting that the increased TNF-α levels result from
immunological memory caused by previous exposure to M. tuberculosis.
An important feature of our study is that all individuals in both
YLHIV and control groups received the BCG vaccine in their first month
of life, as per Brazil’s national immunization program [6]. We have
previously shown that this vaccine induces potent Th1 and γδ T-cell
responses [23]. BCG vaccine is well known to be less efficient for TB
prevention in children and adolescents living with HIV [24]. This was
also the case in our study, and the dysfunctional Th1 response of YLHIV
to BCG and Mtb therefore suggests that effective cART is not enough to
restore the efficacy of the BCG vaccine in the long term.
Incomplete recovery of anti-mycobacterial immunity is also shown
by lower CD4/CD8 ratio of YLHIV when compared to healthy controls in
our study. In a 15-year follow-up, low CD4/CD8 ratio was independently
associated with increased risk of TB despite viral suppression. The same
study showed that only about 10% of the patients normalized their CD4/
CD8 ratio [25]. In our study, on the other hand, more than half of the
patients had a CD4/CD8 ratio higher than 1. However, it is noteworthy
that, in the multivariate analysis of the YLHIV group, total CD4 T cell
count and CD4/CD8 ratios were associated with IFN-γ levels in response
to Mtb and ESAT-6, respectively. Although this might indicate that cART
can help to maintain an adequate CD4/CD8 ratio, studying a larger
sample size is necessary before drawing any conclusions. A recent report
from a Canadian cohort showed that earlier initiation of cART and
higher baseline CD4 levels were associated with faster normalization of
CD4/CD8 ratios [26].
Higher levels of IL-4 and IL-5 by PBMC of YLHIV in response to SEB
at specific time points suggest a non-specific shift of the lymphocyte
response towards a Th2 pattern, although the response to this super­
antigen tended to be of the Th1 type. A Th2-skewed response has been
previously shown in another study in progressive stages of HIV infection
[27]. Longitudinal analyses are needed to better evaluate any possible
Th1–Th2 shift in YLHIV followed at our service.
Our study has several limitations, especially its cross-sectional design
with a small sample size. We did not measure the cytokine production to
the single cell level, which would have told us more about the activity of
CD4 and CD8 T cells. Flow cytometry-based assays for assessment of
lymphocyte proliferation and a broader assessment of cell sub­
populations were also lacking. A major drawback was the unavailability
of the PPD reagent during the time of our field activity, which precluded
the categorization of latent tuberculosis. Still, the results are original
and bring new findings on the efficacy of long-term cART regarding the
immunological characteristics of YLHIV. Further longitudinal analyses
using advanced laboratory techniques should guide our next steps.
5. Conclusion
We found that long-term cART is effective for controlling the HIV
replication; however, its effect in the cellular immune response to
mycobacterial infections of YLHIV is suboptimal, as they still have lower
production of the Th1-associated cytokine IFN-γ in response to both Mtb
and BCG antigens, when compared to controls, although both patients
and controls have received the BCG vaccine. These results suggest a
long-lasting impairment of the activity of CD4 T cells, which is likely to
be driven early in life by vertical infection. A combined strategy can be
used to overcome these limitations, including prevention of vertical
6
Tuberculosis 133 (2022) 102170
transmission, early detection and early treatment, which are already
well established in most countries [1], and a better knowledge of the
immunological aspects of the protection against TB by considering
revaccination of YLHIV either with BCG or next generation vaccines [2].
Funding
This study was supported by the São Paulo Research Foundation
(FAPESP, grant number 2013/26862–9).
Authors’ contributions
• MVC: data curation, formal analysis, investigation, methodology,
project administration, validation, visualization, writing (original
draft, review and editing);
• PCMA: formal analysis, investigation, methodology;
• VSM: investigation, methodology;
• MPA: data curation, investigation, methodology;
• FG: formal analysis, validation, visualization, writing (original draft,
review and editing);
• PCP: methodology;
• TNM: formal analysis, validation, visualization, writing (original
draft, review and editing);
• RMM: data curation, formal analysis, validation, visualization,
writing (original draft, review and editing);
• MMSV: formal analysis, validation, visualization, writing (original
draft, review and editing);
• MTNS: conceptualization, data curation, formal analysis, funding
acquisition, investigation, project administration, resources, super­
vision, software, validation, visualization, writing (original draft,
review and editing).
Declaration of competing interest
The authors have no conflicts of interest to disclose.
Acknowledgements
We thank all volunteers and their families for agreeing to participate
in the study. We also thank Dr. Tânia Zaccariotto (HC Unicamp, Division
of Clinical Pathology, Laboratory of Microbiology) for kindly providing
us with the Mtb and BCG lysates used in the study.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.tube.2022.102170.
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