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Divergence of Immune Responses To BCG vaccine In Childhood

February 2023

February 2023

Publisher: Services For Sciene ANd Education ,UK.

Authors:

University of Qasim,College of Biotechnology

The book undertake the theme of divergence of the immune responses to BCG vaccine in childhood.It was practical approach to this theme tackling both humoral and cellular immune responses to this vaccine in child of 6 to 60 months age.

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Ibrahim M S Shnawa

Tiba Ahmed Karim

The Divergence Of The Immune Responses To BCG Vaccine In

Childhood

SERVICES FOR SCIENCE AND EDUCATION, UNITED KINGDOM

Ibrahim M S Shnawa

Tiba Ahmed Karim

The Divergence Of The Immune Responses To BCG Vaccine In

Childhood

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permission in this book appear on appropriate pages within text.

Society for Science and Education – United Kingdom (SSE-UK) applies the Creative

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DOI: 10.14738/eb.325.2023

Published by:

Services for Science and Education

Stockport, Cheshaire, SK4 2BT

United Kingdom

The Divergence Of The Immune Responses To BCG Vaccine

In Childhood*

IBRAHIM M S SHNAWA

Professor Emeritus Doctor, College of Biotechnology

University of Qasim And Hilla University College,

Babylon Province/IRAQ.

AND

TIBA AHMED KARIM

MSC Medical Biotechnology,

Diwanyah Teaching Hospital

Diawnyah Board of Health,ALQadisyah Province/IRAQ.

*Based Upon MSC thesis of the second Author

PREFACE

BCG vaccination programs in children is eligible and mandatory in most of world countries.

Though their immune protection efficacy is of variable degrees corresponds to different

geographic niches ,nutrition and socio-economic status of the vaccinated child .Some

vaccinated children forms a scar other are not .BCG scar among vaccinated child may fade up

as the child age scale up. The objective of the present book was to investigate the divergence

of the immune responses to BCG vaccine in childhood at ALQadisyah Province/IRAQ.

SHNAWA AND KARIM

Jan.2023

ACKNOWLEDGMENTS

The authors wish to express their gratitude to the padiatric specialist DR. Haneen A H AL-

Abdly Diwanyah Gynecology and Childhood Hospital for interviewing the test child, control

child and filling the information case sheets. Thanks extended to the health care providers

and nursing personals helping us in some technical matters during the study.

III

CONTENTS

Chapter One: Introduction…………………………………………………………………………………….1-3

Chapter Two: Basic Platform…………………………………………………………………………………4-32

Chapter Three : Investigative Approaches…………………………………………………………… 33-48.

Chapter Four : Results And Discussion………………………………………………………………. 49-66

Chapter Five : Conclusions And Recommendations…………………………………………….. 67-68

References………………………………………………………………………………………………………… 69-101

Appendix…………………………………………………………………………………………………………… 102-103

Summary…………………………………………………………………………………………………………… 104-105

Chapter One

Introduction

Introduction 1

1. Introduction

More than 90% of infants worldwide receive the (BCG) vaccine shortly

after birth to protect them against tuberculosis (TB). The vaccine was

created by Bacille ,Calmette and Guerin, and it was given to people for the

first time in 1921. Only BCG protects against TB (Okafor et al., 2022). Its

effectiveness against pulmonary tuberculosis in adults is low and varies

from 0 to 80% depending on a number of variables such geographic

location and prior exposure to environmental mycobacteria (Mangtani et

al., 2014).

The live bacteria was used in the BCG vaccine have been weakened

(attenuated) in order to boost the immune system without harming healthy

individuals(Covián et al., 2019).In nations with higher mortality rates, BCG

scarification has been linked to increased survival and a stronger

immunological response in children who have received the

vaccine(Tabatabaei & Hassan, 2019) . A scar that develops after

immunization with the vaccine is one of the distinctive signs of the

effectiveness of the vaccine and a crucial signal of immunity. The size of

the resulting scar could be a useful gauge of the immune system's reaction

to the BCG immunization (Park et al., 2015).

The lack of a scar may have unknown origins, although several factors,

such as the infant's immature immunity, the improper way the vaccine was

administered, or it could be the vaccination has no impact (Birk et al., 2017)

.It is thought that primarily cellular-immunity and humoral immunity is the

mechanism by which the BCG vaccine protects against TB(Rao et al.,

2015). In the fight against M. tuberculosis, cellular immunity predominates,

and alveolar macrophages and T lymphocytes play crucial roles in the

Introduction 2

elimination of infection. Release of various cytokines and the balance of

Th1 and Th2 responses directly influence the cellular immune

response.(Mansouri et al., 2018). The link between T-cell cytokine

responses and tuberculosis-protective immunity has received a lot of

attention(Domingo-Gonzalez et al., 2016). The host's antimicrobial defenses

and stress response include the cytokine macrophage migration inhibitory

factor (MIF), which supports both innate and acquired immune systems'

pro-inflammatory function. After being exposure to M. tuberculosis

antigens or after contracting virulent M. tuberculosis, macrophages release

MIF( Li et al., 2012). After receiving the BCG vaccine, pro-inflammatory

cytokines including IL-2 were produced, which promote the activation of

Th1 cells and CD8 T cells(Lalor et al., 2010). BCG primes both IL-4 and

Th1 responses(Mansouri et al., 2018). IL-4 can be produced by a variety of

cell types. IL-4 may be responsible for triggering Th2 reactions., but it can

also be present at later stages and down-regulate Th1 responses (Domingo-

Gonzalez et al., 2016). Immunoglobulin E (IgE) antibodies' function in

regulating allergic reactions (Sutton et al., 2019).In other word the immune

responses to BCG among children may expresses a sort of divergence.

1.1 Aim of the Research

The study aims to determining the extending fade-up of the gross

manifestations and cellular immune reaction to the BCG vaccination scar in

children of different ages and indicate the extent of children's immunity to

tuberculosis exposure. This in itself is of great importance for the growth

welfare of children.

Introduction 3

This aim was carried out by the following objects:

1-Investigating the extent of the scar in children and studying and

estimating its size and photographing specific cases of it .

2-Collecting blood samples from children to determining the levels of IL-2,

IL-4,IgE and MIF in children to match the extent of immune divergence in

these cytokine responses.

Chapter Two

Basic Platform

Basic Platform 4

2. Basic Platform

2.1 Mycobacterium tuberculosis

An overview

Almost one and a half million deaths due to tuberculosis infection around

the world have been reported, and it ranks among the top ten global causes

of death at the level of 2017, and with ten million cases of infection, the

proportion of infected children was about 10% (de Martino et al., 2019). In

most cases 90% treatment is provided. However, tuberculosis remains a

major and global health problem. There are new infections that occur at a

rate of 1% of the population each year(Chapple et al., 2012) .The fact that

Mycobacterium tuberculosis that causes tuberculosis in humans has its own

characteristics of being aerobic and acid-fast, it is a chronic infection of the

respiratory tract. It has many types, including: M. bovis BCG, M. canettii,

M. africanum, M. pinnipedii, M. microti, M. caprae (van Ingen et al., 2012).

Tuberculosis has evolved the evasion to avoid the immune cells and

immune system of the host. Although the host recovers from infection

frequently, its strength lies in its ability to spread to a sufficient number of

people to support and develop itself. The difficulty of creating a treatment

for it lies in the strength of its interaction with the host(Blanc et al., 2017).

Depending on how the disease develops, the clinical signs of active

tuberculosis (TB) can range from a mild cough to more serious symptoms

including irreparable lung damage and, eventually, death(Luies & Preez,

2020). The laboratory diagnosis of the presence of tuberculosis infection is

by using a chest x-ray, but methods such as microscopy and microbiological

culture are also used after taking samples of body fluids such as sputum. In

the case of latent tuberculosis, where the presence of the bacteria does

nether pose a threat nor activate and it does not cause any symptoms

Basic Platform 5

(Esmail et al., 2014), the Tuberculin Mantoux skin test (Gualano et al.,

2019) and/or Interferon-gamma release measures (IGRAs) for blood

samples are used(Luies & Preez, 2020). Europe has seen an upsurge in the

prevalence of TB, North and South America, to a wide campaign called the

White Plague. This led to the establishment of sanatoriums scattered around

the countries, which are still common and well-known in the media (Martini

et al., 2018).

Anton Ghon. The Australian pathologist and the first to describe the

center of infection that can occur during the early stages of infection. His

name is taken and used generally in the concentration of Ghon . He

describes how this primary infection can progress to involve nearby lymph

nodes, a group of infections called the Ghon complex, which often calcifies

in the course of the disease to form a distinct form of infection(Donald et

al., 2021) . It was believed that this form of infection is the distinctive form

or known feature of tuberculosis in childhood, but in our current and

modern times, the possibility of adults catching tuberculosis is low,

especially in countries where the infection rate is weak. Therefore, the

development of the disease is more in people with known immune

weakness And those who have not had a previous immunity against

mycobacteria(Lodden kemper et al., 2016).

Most infections from tuberculosis occur due to inhalation of aerosols

that contain tuberculosis bacteria. As for infection outside the lung, it

occurs as a result of the spread of tuberculosis bacteria from the lung to the

surrounding lymphoid tissue, and then distributed throughout the body

through the circulatory system. These advances in the disease can be

avoided by early vaccination or early detection and treatment of the

Basic Platform 6

disease(Schepers et al., 2015). The best way to avoid infection is to get a

vaccine against the disease. The BCG vaccination is still the most reliable

defense against many Mycobacterium TB strains. A dependable immune

defense of the host may be reestablished by comprehending the vaccine's

mode of action(Ranaivomanana et al., 2015).

2.2 Mycobacterium tuberculosis

Antigenic structure epitopes

An epitope is a term that means a small portion of an antigen

identified and recognized by an antibody, coined by Nils Jerne in 1960.

Antigenic variation is frequently present in pathogens that resist adaptive

immunity. The TB pathogen M.tuberculosis causes acute ,subacute and

chronic human tuberculosis (TB) (Coscolla et al., 2015). Numerous studies

on human ,pathogenic microbial , have shown that certain antigen-

encoding genes are more highly changeable as a consequence of the host

immune system's resistance to and evasion of these antigens (Karls &

Perkins-Balding, 2013).Tuberculosis bacteria express a wide range of

proteins known as antigens at different stages of infection. It triggers a

stage-specific immune response(Demissie et al., 2006; Goletti et al., 2010).

The understanding of antigenic epitopes of M. tuberculosis greatly

helped in a broad understanding of diseases immunity and also helped in

controlling the disease. Also, the characterization of the immunogenic

epitopes displayed genetically affected the development of the relationship

between the host body and bacteria. It also contributed to the development

of immunoprotective vaccines and tests that are used In immunodiagnostics

(Schepers et al., 2015). Because of the discovery of only a small part of the

antigens of the tuberculosis bacteria express is used to identify antigens that

Basic Platform 7

can activate T cells in the human immune system, although it is seriously

and critically needed to create a plan and strategies for stronger vaccination

against tuberculosis (Tameris et al., 2013) . There are several techniques for

detecting antigens, including expression library, biochemical, genetic, and

T-cell peptide epitope-based strategies. Antigens having protective

properties against TB bacteria were found via the use of animal research. A

few of them were of used in Human tests .(Goletti et al., 2010).

The T cell epitopes were present in just 7% of the 4000 open reading

frame (ORF) from the TB bacteria. The highest containing 30 important

protein antigens became the subject of frequent study and contained 65% of

the currently known epitopes(Arlehamn et al., 2014).Tuberculosis bacteria

dramatically alter their gene expression profile within the host's

macrophages during intracellular stress. Because of this, the repertoire of

antigens produced and presented to the host's immune system changes

significantly as the infection progresses, and the host's immunological

defense systems are also put under stress. It is crucial to have a thorough

understanding of the genuine M.tuberculosis antigenome produced during

different stages of M.tuberculosis infection, especially in the lung, the main

target organ of M.tuberculosis, in order to design more effective

tuberculosis vaccines and TB correlates of immunity(Mandalakas et al.,

2022). The detection of antigens for tuberculosis vaccine depends mainly

on antigens of M.tuberculosis . It has been investigated how infected cells,

wherever they are situated, express and offer to macrophages. T cells have

to recognize it and carry out an appropriate immune response. These T cells

have to help the phagocytes control the living pathogen or eliminate it

through different intracellular pathways such as [ Oropharyngeal maturation

Basic Platform 8

and pharyngeal fusion; oxidizing /nitrate intermediates; oxygen/ nutrient

deprivation; activity defensing and other antimicrobial peptides and

enzymes; autophagy] (Ottenhoff et al., 2012). Epitope sequence variation is

rarely used by M. tuberculosis as a survival strategy against human T cells

as a result of the identification of human T cell epitopes and the

overwhelming evidence that antigenic epitope conservation predominate in

this bacteria. Since T cells are the mainstay of protective adaptive immunity

in TB and peptide epitopes represent the pathogen's primary molecular

interface with them, these bacteria have evolved to adopt methods other

than an antigenic mutation to circumvent T cell defense (Coscolla et al.,

2015).

A substantial body of research suggests that the host microbiome has

a significant impact on innate and adaptive immunity, as well as host

reactivity and immunological functioning. Given the circumstances, it

seems fair to assume that the sequence similarity of antigens recognized by

the host immune system (whether they be pathogens, allergens, or

autoantigens) may possibly affect adaptive immunity (Bresciani et al.,

2016). The immune system's response to peptide epitopes originating from

infections and allergens that are similar may be changed by exposure to

microbial peptide sequences. These studies particularly postulated that

immune systems would identify these microbiome-derived peptides as self

and be tolerogenic, resulting in the deletion or functional suppression of

potentially reactive T cell clones(Carrasco Pro et al., 2018). According to

the biological theory, excessive of inducing the immune response using

mycobacterial antigens may boost bacterial death. The immunological

control of TB in humans, however, may be negatively impacted by some

Basic Platform 9

types of nature immune response, according to experimental findings(Sakai

et al., 2016).

2.3 Mycobacterium tuberculosis vaccine

Children who receive the live attenuated tuberculosis vaccine BCG are

protected from developing the disease (Gupta, 2020) The failure of the

present BCG vaccination, to provide protection against the disease's

respiratory form necessitates the urgent need for the creation of a new

tuberculosis vaccine (Arbues et al., 2013). The BCG vaccine is subject to

numerous restrictions and regulations. Because of this, numerous

tuberculosis vaccines have been produced and developed, some of which

are currently undergoing clinical trials. The potential TB vaccines fall into

the following categories:

1. A vaccination that protects against infection before exposure.

Newborns receive this kind. It is referred to as the initial vaccine.

2. The vaccination is preventive in nature and given after exposure.

which concentrates on adults and teenagers. also known as the

booster shot.

3. With regard to the therapeutic type used in tuberculosis treatments,

particularly for those who have experienced ongoing and recurrent

infections(Kaufmann et al., 2017).

A heat-inactivated whole-cell vaccine produced from Mycobacterium

vaccae (also known as "Vaccae") is now being researched for the protection

from o TB after being approved in China for the supplemental treatment of

TB. There is presently no more publicly accessible information on the

Basic Platform 10

preventive study, which was initiated in 2013 with the aim of recruiting

10,000 individuals who had a positive tuberculin skin test (Weng et al.,

2016). Whole-cell vaccines are defined as viable live-attenuated and

inactivated vaccinations. These polyantigenic vaccines are more likely to

contain the essential epitopes needed for protective effectiveness than

subunit vaccinations because it is unknown which antigens will cause

protective immunity in people(Scriba et al., 2016).

To reduce the use of antibiotics while simultaneously enhancing the

outcomes of the treatment of both latent tuberculosis infection (LTBI) and

TB disease, a vaccine known RUTI is vaccine currently being talked . It

functions by inducing a poly antigenic cellular response in non-replicating

bacilli in M. tuberculosis detoxified cell wall nano-fragments. In a phase I

examination, volunteers with LTBI who were HIV-negative or HIV-

positive showed safety and immunogenicity, and a phase II trial confirmed

these results.In a phase II trial, the immunogenicity and safety of the RUTI

therapeutic vaccine for patients with MDR-TB will be evaluated(Nell et al.,

2014). Clinical trials have been conducted on several novel tuberculosis

(TB) vaccines, some of which use genetically altered mycobacteria, viral

vectors that transport mycobacterial antigens, or adjuvants that contain

mycobacterial antigens. While some of these vaccines are intended to

replace the current BCG shot, others will be administered as a booster shot

after the BCG shot, which is typically given shortly after

delivery(Kaufmann et al., 2017).

Basic Platform 11

2.4 Bacillus Calmette-Guerin (BCG) current view

The Bacillus Calmette-Guerin (BCG) vaccine was developed by Drs.

Calmette and Guerin in 1921, some 40 years after the discovery of

M.tuberculosis. After a century of usage, BCG is still the only TB vaccine

that is currently deemed effective. It is the most commonly used vaccination

in the world, having been administered to over 4 billion individuals. It is the

most extensively used vaccine and is typically included in the standard

infant vaccination regimen. Leprosy and Buruli ulcer are two non-

tuberculous mycobacterial illnesses that are protected by the BCG vaccine

(Okafor et al., 2022).In young children, BCG provides significant

protection against disseminated TB, but its effectiveness is highly variable

in both children and adults with pulmonary TB is restricted and varies from

0 to 80% (Moorlag et al., 2019) .Although the cause of varied efficacy in

adolescents and adults is unknown, exposure to environmental

mycobacteria may already provide some protection against M.tuberculosis,

which BCG fails to enhance(Mangtani et al., 2014).

In actuality, little exposure to environmental mycobacteria or the

absence of prior M.tuberculosis infection is associated with increased BCG

efficacy(Kuan et al., 2020). The WHO does not currently advocate for

repeated BCG immunization due to insufficient evidence supporting its

efficacy . Despite a wide range of claimed efficacy in preventing primary

TB, the vaccine's favorable safety profile—measured as 1 significant

adverse event per 1 million doses administered to immunocompetent

people—may help explain why it is still used in TB-endemic areas. (Lyon

& Rossman, 2017). One reason for the broad efficacy range of the BCG

vaccination is the genotypic variability within it. In 1924, Once the efficacy

Basic Platform 12

of their newly discovered vaccine was proved, Calmette and Guerin

distributed their strain to all nations. Geographic location eventually

allowed independent mutations to occur between samples, boosting

genotypic heterogeneity across strains that were previously identical(Ritz

et al., 2008).

Additional 14 distinct sub-strains were discovered following further

research on the strains, each named for the area in which it was

disseminated. Six of the sub-strains that have been the subject of the

greatest research are BCG Tokyo, BCG TICE, BCG Danish, BCG Pasteur,

BCG China, and BCG Prague. BCG strains are often localized in their

distribution, and UNICEF, which gets its BCG vaccination from four

providers, controls the majority of the global vaccination market. Three

vaccine strains are produced by these four suppliers: BCG Denmark, BCG

Russia, and BCG Japan(Abdallah et al., 2015). A significant effort has also

been made to change the current intradermal (ID) BCG vaccination into an

oral vaccine (Luca & Mihaescu, 2013).

Oral delivery is more convenient for most individuals, less expensive,

and requires no medical expertise to administer.It is believed that direct

mucosal contact with the vaccination is highly immunogenic (Derrick et al.,

2014), but developing vaccines in forms that can with stand the mucosa's

deteriorating characteristics, particularly in the stomach, has restricted their

usage(Bussi & Gutierrez, 2019).It is common knowledge that giving

newborns and young children one injection of BCG will dramatically

reduce the risk of them developing severe types of pediatric

tuberculosis(Shnawa, 2019). The WHO and UNICEF coverage figures are

based on coverage at 12 months of age, with the caveat that BCG is

sometimes postponed in low-income countries. Delaying getting the

Basic Platform 13

vaccination may decrease its validity (Thysen et al., 2020). BCG as

standard biologics may act as a vaccine, immune adjuvants, and nonspecific

immune stimulant because its safety has been shown in neonates, children,

and adults who have received the vaccine for about 100 years (Rey-Jurado

et al., 2018).

2.4.1 Attenuation of BCG Strains

A newborn infant received the first dosage of the vaccine in the year

1921, and this strain of the vaccine was subsequently delivered to all

laboratories throughout the world under various settings based on the

laboratory. Numerous strains, including BCG Pasteur, BCG Tokyo, and

BCG Russia, as a result, emerged(Joung & Ryoo, 2013). Several genomic

regions eliminated in BCG but present in M. bovis, have been found by

pioneering investigations testing the genomic basis of BCG attenuation.

Most likely as a consequence of the original M. bovis strain not being

exposed to the host's immunological vigor , which led it to progressively

lose virulence components, these deletions evolved during subcultivation in

the lab. Different BCG "daughter" strains appeared as a result of BCG's

ongoing passage in the lab after the lyophilization procedure was devised.

These substrains exhibit varying effectiveness and have different genomic

make up (Tran et al., 2014; Zhang et al., 2016). The first stage (1921–

1908) is made up of 230 lab iterations that Calmete and Guérin carried out

to create the vaccine for the first time. After the second stage, which ended

in the year (1924), the vaccine was widely used for several decades, and

many people survived. The attenuated led to loss of genomic sections

during passage in the culture, but it also destroyed some of the key

protective antigens and some of the virulence components, to which BCG

ineffective attributed (Joung & Ryoo, 2013b).

Basic Platform 14

2.5 Mycobacterium tuberculosis immunity

Mycobacterium tuberculosis causes tissue-resident transcriptional

macrophages, which emerged during embryonic development, to become

infected(Cohen et al., 2018; Huang et al., 2018). During TB infection DCs

take up , progenitors present epitope in conjugated MHC molecule to name

T cell within the lymph nodes . This cycle lasts for a number of weeks.

However, when a successful adaptive immune response takes place, through

the development of T cells, B cells, and activated macrophages, bacterial

control is achieved and characteristic granulomas formed. Sometimes the

bacterial reproduction is contained and turns into a latent infection once the

inflammatory response subsides. This type of infection does not show any

symptoms in patients, but they have an adaptive immune response. The

consequences of latent tuberculosis include bacterial eradication and

subclinical disease (Boom et al., 2021).

To halt the spread of progressive, disseminated TB, an efficient adaptive

immune response is necessary. The histology sign of M. tuberculosis

infection is granulomas. A well-organized collection of lymphocytes,

neutrophils, and macrophages makes up mature granulomas. Granulomas

become sclerotic and calcified when an adaptive immune response is

effective, as contrast to active TB granulomas, which are necrotic and have

a caseating look. They also regulate and even sterilize the infection. For the

creation of well-organized granulomas and host defense, CD4+ T cells and

TNF are essential(Caruso et al., 1999). Infection with M. tuberculosis also

activates CD8+ T lymphocytes, which can be found in the blood of TB

patients(Chávez‐Galán et al., 2019). According to recent evidence, CD4+ T

Basic Platform 15

cells and CD8+ T cells work together to inhibit the infection of M.

tuberculosis( Lu et al., 2021). It is well recognized in this context that

adaptive immune cells, such as CD4 and CD8 T cells, support the

antimycobacterial defense by releasing IFN-y, which may activate infected

myeloid cells and inhibit bacterial growth. M. tuberculosis may withstand

the strains of host immunity and persist within the host(Sia & Rengarajan,

2019).

In humans, tuberculosis bacteria are detectable 3-8 weeks after infection.

The adaptive immune response to the mutated bacteria is the antigen-

specific response of CD4 + T cells. The type Ⅳ hypersensitivity reaction is

more recognizable in the majority of people with tuberculosis and is driven

by CD4 + T cells. Its main feature is visible scleroderma after 1 to 3 days.

From ingestion of purified protein from Mycobacterium or from the

tuberculin skin test (TST)(Coscolla et al., 2015). The quantity of circulating

CD4 + T cells has been shown to be adversely correlated with the

susceptibility of HIV-positive people to TB infection because they worsen

active tuberculosis infection(Shafer & Edlin, 1996; Shnawa, 2019) .Those

infected with tuberculosis develop a specific immune response by CD4 + T

and also CD8 + T, which contributes to the killing of bacteria inside the cell

(Bruns et al., 2009; Lewinsohn et al., 2007) .

2-6 Bacillus Calmette-Guerin (BCG) vaccine immunity

Overall, compared to adult humans or adult animal models, newborns'

immune responses are different and cannot be generalized (Saso &

Kampmann, 2017). During the first 2 years of life, neonates and babies'

immune systems are continually evolving, and they exhibit a number of

Basic Platform 16

innate and adaptive immunological limits that have an impact on how well

they will respond to an infection or a vaccination. Neonatal immune

abnormalities can affect phagocytosis, bactericidal activity, and cell

migration. Restricted elements of the innate response include dendritic cell

(DC) maturation, the range of DC subpopulations, including lower

plasmacytoid DC numbers and MYD 88 expression, as well as the

activation of Toll-like receptors. (TLRs)(Alexander-Miller, 2014). Neonates

have low complement levels and high quantities of distributing

antimicrobial peptides at birth. In comparison to adults, babies have a

distinct profile of innate leukocytes' produced cytokines(Kollmann et al.,

2017).

According to the limited research on neonatal immune cells, Tumor

necrosis factor - alpha (often abbreviated as TNF- α) levels in newborn cells

are much lower, comparable, perhaps much greater than in adult cells.

These studies, however, have produced inconsistent and divergent results

(Kollmann et al., 2009). Additionally, it is generally believed that while

neonates may elicit both a primary T-helper 1 (Th1) and a secondary T-

helper 2 (Th2) immune response, the latter is biased more towards

Th2.(Zaghouani et al., 2009). Newborns have diminished CD4+ T cell

synthesis of interferon-gamma (IFN-γ), which may reduce the effectiveness

of their Th1 response (Kollmann et al., 2017). For pediatric immunizations

intended to stimulate Th1 responses to fight pathogens, neonates must not

have a secondary Th2 immune response(Zaghouani et al., 2009).In contrast

to past vaccines, BCG contains ligands for five different TLRs (TLR 1,

TLR 2, TLR 4, TLR 6 and TLR 9)(Randhawa & Hawn, 2014), leading to

the hypothesis that the ability to cause a long-term strengthening of innate

Basic Platform 17

immune responses, also known as trained immunity, is influenced by the

engagement of several TLRs(Netea & Van Crevel, 2014).

Figure (2.1): Important elements of newborn immune responses and protective

properties of BCG. The BCG germs attach to and activate Toll-like receptors (TLR

1, TLR 2, TLR 4, TLR 6, and TLR 9) upon vaccination of a baby, it causes innate

immune responses to be strongly stimulated. To stop pathogens from receiving this

nutrient, the antigens in the immunization also encourage the activity of monocytes

and macrophages and iron sequestration in these cells. In addition to widespread B

and T cell responses, BCG also induces particular Th1 IFN-γ driven responses. The

several layers of innate and adaptive immunological activation brought on by BCG

work as a slender line of defense against tuberculosis. Additionally, BCG is

currently being researched for its potential role as an adjuvant for other

vaccinations as well as its ability to promote trained innate immunity against

common baby and pediatric respiratory illnesses. This figure is adopted from

author Ramos et al., 2020.

. The CD4+ effector T cell phenotypes, comprising the CD3+CD4+ and

CD3+CD4+CD8a+ populations, were the most common T cell subsets. The

highest CD4+ T cell response was reached in neonates 10 weeks after

receiving the BCG immunization; during this time, these CD4+ T cells

Basic Platform 18

released significant quantities of IFN, TNF, and IL-2(Soares et al.,

2008).From weeks 6 to 40, newborns appeared to maintain constant states

of TNF expression in CD4+ T cells tend to ranging from 50 to 60 percent,

but around one year of age, these levels started to decline.(Soares et al.,

2013)Figure (2.1)above .

Infant BCG vaccinations have varying immunogenicities depending on

the population. Infants have been shown to produce cytokine-expressing T

cell responses of the same magnitude as adult responses, typically with a T

helper Th-1 bias, after receiving the BCG vaccine. These responses are

measurable in terms of their immune responses to mycobacterial antigens,

such as purified protein derivatives of M

tuberculosis.(Covián et al., 2019)

.It is not dependent on memory T or B cells for innate immune cells, such

as monocytes and natural killer (NK) cells, to provide this non-specific

immune defense. Epigenetic reprogramming through histone modification

in the regulatory elements of specific genes has been reported as one of the

mechanisms associated with the induction of trained immunity in both

humans and mice. This phenomenon is known as "trained immunity," and it

is characterized by a memory-like response in innate immune

cells(Bekkering et al., 2021).

In fact, it has been demonstrated that the BCG vaccine causes "trained"

condition in circulating monocytes by altering the methylation pattern of

histones linked to particular genes. Importantly, these changes can result in

the expression and/or suppression of genes that are linked to greater

pathogen identification, quicker inflammatory responses, and increased

protection against secondary infections following vaccination. Different

pattern recognition receptors (PRRs) interact with pathogen-associated

Basic Platform 19

molecular patterns (PAMPs), such as peptidoglycan, arabinogalactan, and

mycolic acids present at the bacterial cell wall, to recognize BCG by

immune cells. Toll-like receptors (TLRs) TLR2 and TLR4, which are found

on the cell surface membrane, are among the receptors that recognize BCG

(Kaufmann, et al., 2017).

It has been demonstrated that a variety of mycobacterial proteins can act

as TLR agonists, promoting DC and macrophage maturation as well as the

release of pro-inflammatory cytokines. (Kumar et al., 2019). Similarly,

complement receptors CR3 and CR4 play a role in DCs' ability to recognize

opsonized mycobacteria. Nucleotide-binding oligomerization domain

(NOD)-like receptors, such as NOD2, which are located in the cytoplasm of

innate immune cells and connect with a particular portion of the bacterial

peptidoglycan, are another class of cell receptors that identify BCG PAMPs.

Additionally, C-type lectins, like DC-specific intercellular adhesion

molecule-3-grabbing nonintegrin (DC-SIGN), engage with parts of the

bacterial wall and are crucial for the detection and internalization of BCG.

(Gagliardi et al.,2005).

The mycobacterium can survive for up to 2 weeks inside DCs after

internalization. (Jiao et al.,2002). A rise in the expression of co-stimulatory

molecules including CD40, CD80, CD83, and CD86 is one of the

characteristics of the DC maturation and migration that are brought on by

this interaction. (Tsuji et al.,2000). One of the antigens found in the BCG

cell wall is similar to antigen (Ag), which is found in M. tuberculosis and

induces the production of TNF-α, Interleukin-1β, and IL-6. (,Bertholet et al

.,2008 ,Joosten et al .,2018). which are able to produce an inflammatory

state that encourages immune cell activation (Tsuji et al.,2000).

Basic Platform 20

Figure (2.2) : The immunological reaction brought on by the newborn's BCG

vaccine. Neutrophils, macrophages, and DCs at the injection site recognizing the

BCG (A). To activate adaptive immune cells, activated cutaneous DCs go to the

draining lymph nodes (Figure B). (C) Activation of CD4+ and CD8+ T cells with a

TH1 profile that are specific for Mycobacteria and secrete high levels of IFN-y and

granzymes. (D) When BCG antigens are present, activation of B cells causes the

development of memory and plasma cells as well as the production of antigen-specific

antibodies. Memory T and B cells stay in lymph nodes after activation. This figure is

adopted from auther (Covián et al., 2019)

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Antigen-presenting cells (APCs), such as DCs, macrophages, and B cells,

deliver antigenic peptides on MHC molecules and prime T cells in the

nearby secondary lymphoid tissues or the spleen to initiate an adaptive

immune response. (Kaufmann.,2013).

BCG-infected cutaneous DCs move to the draining lymph nodes,

where they release TNF-α, IL-6, and IL-12 and activate both CD4+ and

CD8+ T cells, according to in vitro and in vivo investigations. (Bollampalli et

al.,2015) (Figure 2-2). It's interesting to note that BCG-infected human

neutrophils reportedly work with infected DCs to activate T cell responses

specific to antigens. The BCG vaccine induces an adaptive immunological

response that involves the activation of both CD4+ and CD8+ T

cells(Andersen et al.,2014).

With increased IFN-γ production, which boosts macrophages' anti-

mycobacterial activity. (Kaufmann et al.,2013). This cytokine also helps B

cells become activated and plasma cells produce antigen-specific antibodies

as a result. Up to 10 weeks after BCG vaccination, a pool of mycobacteria-

specific CD8+ T cells grow in the early post-immunization period and are

present in peripheral blood(Hanekom et al.,2005). These CD8+ T cells were

able to produce IFN-γ, express granzymes, and perforins, supporting their

capacity for cytotoxicity. (Murray et al.,2014). TH1 CD4+ T cells that are

activated have also been seen. (Soares et al.,2010), which have high levels

of IFN-γ, TNF-α, and IL-2 production. In infants, BCG-specific CD4+ T

cells were found in the peripheral blood three weeks following vaccination,

peaking at ten weeks. (Su H et al.,2019).

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2.7 Bacillus Calmette-Guerin (BCG) scar

A vital index in the immunization schedule, the BCG scar serves as a

substitute vaccination indication. Children who develope a scar after

receiving a BCG vaccination had reduced morbidity and greater survival

rates than children who do not develop a scar.A papule usually develops

after intradermal BCG vaccination, ulcerates, and then spontaneously heals,

leaving a permanent scar within a few months(Storgaard et al., 2015).

With a single 0.05 ml injection with a 26 G needle, the critical

intradermal layer of the left upper arm's lateral side is treated at the level of

the shoulder muscle insertion(Schaltz-Buchholzer et al., 2017). A tiny,

permanent scar called the "BCG Scar", is formed at the injection site about

two weeks after receiving the BCG vaccine. This scar size has an average

diameter of about 10mm, is usually indurated, then swells, suppurates, and

spontaneously ulcerates. figure (2.3) The crust eventually falls off on its

own after healing in about six to twelve weeks. After getting the BCG

vaccine, it takes two to three months for a "BCG scar" to form without any

systemic side effects ( Li et al., 2021). Despite evidence demonstrating that

scar formation is not a good predictor of the immune response to BCG, the

existence of a BCG scar is the only simple and precise way to prove prior

vaccination in clinical scenarios as well as in health surveys to evaluate

vaccine uptake(Storgaard et al., 2015). An infant's immune characteristics

may affect how a scar develops prior to immunization (Dhanawade et al.,

2015). Post-vaccination scarring is still a distinctive feature of BCG because

it is the only prophylactic vaccine against TB that is legally permitted to be

administered anywhere in the globe (Birk et al., 2017).

Basic Platform 23

Since the administered amount can be precisely measured and

controlled, intradermal injection with a syringe and needle is often seen to

be the most precise method and results in more reliable mycobacteria-

specific immunity(Lee et al., 2012). One could hypothesize that the positive

effects of having a BCG scar were simply indicators of some kids' greater

survival due to stronger immune systems. BCG scarring depends on type of

vaccine and vaccination technique(E. J. Anderson et al., 2012).

2.8 Bacillus Calmette-Guerin (BCG) scar fade up

Not all kids who get the BCG immunization get a BCG scar.(Storgaard

et al., 2015). Unknown reasons of scarring fade up include inadequate

immune system development, poor administration techniques, and

inefficient immunizations (Kheir et al., 2011). When given the BCG vaccine

in vivo, close to 12 to 15% of babies show a positive Cell-Mediated Immune

Response (CMIR), but do not develop scars. The relatively low prevalence

of scar formation in children who received BCG as soon as delivery may be

due to immunocompetent cell immaturity or the condition's prenatal

Basic Platform 24

occurrence (Yanamandala et al.,2017). There is no doubt that some of the

kids without a BCG scar were still cytokine-producing in response to

PPD(Djuardi et al., 2010). Due to the fact that BCG vaccinations took place

in various years, variations in scar prevalence may potentially be attributed

to modifications in vaccination methods, vaccine types, the vaccination

nurse, or a shortage of BCG vaccine at particular times that led to a delayed

vaccination ,all of these elements may affect BCG scarring, as seen(Jensen

et al., 2021). For newborns without scars, receiving the BCG vaccine again

is not yet universally advised. It's important to consider whether monitoring

these infants who don't develop a scar is necessary(Dhanawade et al., 2015)

2.8 Bacillus Calmette-Guerin (BCG) allergy

Some parents are reluctant to give their atopic children early vaccines.

However, this is uncommon when contrasted to the possibility that

vaccination with vaccine may exacerbate the atopic disorder and cause a

delay in the infants immune protectively . The numerous reports and studies

on this subject have increased any doubts that may be warranted(Barbacariu,

2014). Early childhood vaccinations have been linked to the development of

atopy, Infections that would otherwise trigger a favored Th1-type immune

response and bias the stability of cytokines away from atopy can be avoided

directly by providing drugs that trigger a Th2-type immune response or

indirectly by doing the same (Kiraly et al., 2016). In some groups, there has

been evidence of an antagonistic association between atopic disease and

M.bovis (BCG) vaccination, as well as Atopic disease risk may be lowered by

early infancy BCG immunization, according to a number of extensive

research(Zhao et al., 2021). The incubation period between the initial

exposure (sensitization) and the development of symptoms (elicitation),

which may entail IgE and/or non-IgE-mediated reactions, is what

Basic Platform 25

distinguishes atopic illnesses.Th2 cytokines are produced during an IgE-

mediated allergic reaction, also known as immediate-type hypersensitivity

(type I), which triggers the production of IgE by B cells ( Anderson et al.,

2019).

The divergence observed in epidemiological studies can be due to the

possibility that a relationship is not as direct as first thought. For instance, it

might be altered by elements that vary between populations and

environments, such as genetic diversity, timing of exposure, amount, and

length of exposure, kind of infection, burden of environmental allergens, and

other mycobacteria(Barbacariu, 2014). Reducing exposure to microbes early

in life of children prevents the auxiliary immune response from shifting to the

T helper1 (Th1) that is common in newborns to the T helper 2 (Th2) response

that develops allergic disease (Freyne & Curtis, 2014). Strong

immunomodulatory characteristics of the BCG vaccine cause Th1 cytokines

to be stimulated. also has a major impact on innate immunity (Kleinnijenhuis

et al., 2012; Rook, 2009).

2.10 Bacillus Calmette-Guerin (BCG) induced cytokines

The T - cell , neutrophils, and macrophages are responsible for

producing the majority of the low molecular weight extracellular

polypeptides and glycoproteins known as cytokines that promote and regulate

immune response (i.e. activity, differentiation, proliferation and production of

cells and other cytokines).To drive signaling molecules, cells, key

lymphocyte development factors, and other biological processes toward

injury, infection, and inflammatory sites, these polypeptides operate on them.

Cytokines can have three different types of actions: autocrine activity,

Basic Platform 26

paracrine action, and distant action (endocrine action). For the control and

growth of immune system cells, they are crucial. There are several distinct

cytokines, such as lymphokines, tumor necrosis factor, interferons (IFN), and

interleukins (IL)(Turner et al., 2014) . It is believed that hematopoietic cells,

lymphoid cells, and other pro- and anti-inflammatory cells contribute to the

development of inflammatory and immune responses, and cytokines are

thought to mediate the intricate interactions of these cells. The immune

system's intercellular messengers, or cytokines, combine the actions of many

cell types in diverse bodily compartments to produce a immunological

response(Gulati et al., 2016).

As a result of years of evolution, they currently belong to the

interferon, interleukin, chemokine family, tumor necrosis factor family, and

adipokine groups. All cells, with the exception of red blood cells, create and

respond to cytokines, which were formerly classified as "soluble

factors"(Owen et al., 2013). The BCG-mediated primary immunological

response often includes the generation of chemokines and cytokines, which

further activate the immune cells at the site in the microenvironment. These

mediators are believed to function in a typical manner or pattern. The primary

cytokines that BCG activates are (IL-2), (TNF), and (IFN-γ), which are

produced when CD4+ T cells are stimulated (Li et al., 2011).

2.10.1 Bacillus Calmette-Guerin (BCG) induce macrophage migration

inhibitory factor(MIF).

In 1966, The first lymphokine associated with delayed-type

hypersensitivity was discovered as macrophage migration inhibitory factor

(MIF), which is generated by activated T cells(Wen et al., 2021). Numerous

diseases have been associated with a pleiotropic cytokine known as (MIF).

The bulk of studies that can be found in the literature classify MIF as a

Basic Platform 27

proinflammatory cytokine implicated in chronic inflammatory illnesses,

Despite the fact that new research indicates that many of MIF's main effects

are not primarily related to inflammation. Since MIF is constitutively

produced in the majority of human tissues, frequently at high levels, it does

not really display the expression pattern of a typical proinflammatory

cytokine. Additionally, MIF is strongly expressed throughout embryonic

development, and as we become older, its expression decreases(Florez-

Sampedro et al., 2020). Its relationship to illness, wide variety of actions,

receptors, and downstream signaling cascades have all been studied by

several scientists. The role of MIF in metabolic and acute inflammatory

processes is now known to be critical(Wen et al., 2021), autoimmune and

infectious (Leaver et al., 2010) . After being exposed to M. tuberculosis

antigens or after obtaining virulent M. tuberculosis, human macrophages

release MIF. The MIF generated by activated macrophages, which

functions in an autocrine manner to prevent the production of pathogenic

mycobacteria, is a critical component of human innate immune defenses

against M. tuberculosis. Studies have also shown that TB patients had

considerably greater MIF levels than healthy controls( Li et al., 2012).

2.10.2 Bacillus Calmette-Guerin (BCG) induce Interleukin-2 (IL-2)

A crucial cytokine that affects the immune system in a variety of

ways known is interleukin-2 (IL-2) (Jiang et al., 2016). and CD8 T cell

activation is promoted by IL-2, after receiving the BCG vaccination, pro-

inflammatory cytokines such IL-2 were produced, which encourage the

stimulation of TH1 cells and CD8 T cells. the stimulation of IL-2 is more

than that of the unvaccinated (Lalor et al., 2010). Ten weeks following

BCG vaccination, babies' peripheral blood contains a sizable number of

Basic Platform 28

BCG-specific CD4+ and CD8+ T cells. TNF- α , I L-2, granzyme, IFN-γ

blood levels may also be elevated(J. Li et al., 2021). After receiving the

BCG vaccination, pro-inflammatory cytokines including IL-2 were

produced, which promote the activation of TH1 cells and CD8 T cells

(Lalor et al., 2010).

The IL-2 serves as the foundation for various vaccinations and is

required for the development of cytotoxic T-cells (CTL) (Dinarello, 2007).

(IL-2), (TNF), and (IFN-γ), which are released during the activation of

CD4+ T cells, are the main cytokines activated by BCG(L. Li et al., 2011).

2.10.3 Bacillus Calmette-Guerin (BCG) induce Interleukin-4 (IL-4)

A type I cytokine is interleukin-4 (IL-4), with a four-α-helical bundle

that has broad pleiotropic effects on many lineages. Although the main

functions of IL-4 were first identified in B and T cells, more than a dozen

other target cells of this cytokine are active in both the innate and adaptive

immune systems. It is also produced by a variety of distinct cellular sources.

Even though IL-4 was identified in 1982, just under 40 years have passed

since its discovery, and new information about it is still being found. There

have been significant new developments in its biological effects and

signaling processes, including key genes and downstream targets in a variety

of cell types (T cells, eosinophils, basophils, mast cells, NK cells and some

APCs ). IL-4 has a crucial role in the modulation of immunoglobulin

synthesis as well as in the inflammatory, fibrotic, allergy, and anticancer

processes(Keegan et al., 2021). Consequently, there is currently a

substantial amount of research that supports that, in addition to Th 1

cytokines, human TB also induces an IL-4 response (Pooran et al., 2019).

Basic Platform 29

2. 14 Bacillus Calmette-Guerin (BCG) induce Immunoglobulin-E

(IgE)

Specific Immunoglobulins are glycoproteins known as antibodies that are

made by B-cells as membrane-bound receptors or in a secretory form (Bilal

et al., 2021). Human immunoglobulins fall into five categories: IgM, IgG,

IgA, IgE, and IgD (Sheikh et al., 2022). natural IgM's position as the body's

initial line of defense against invasive microorganisms(Kaveri et al., 2012).

Although it is thought that humoral immunity has a role in the BCG

vaccine's ability to protect against TB, there is mounting evidence indicating

the contrary (Rao et al., 2015). patients with attenuated antibody responses

to be at higher risk of developing active infection if humoral immunity is a

factor in the TB defense mechanism.

Vaccines have been developed to offer protection against natural or

experimental infection challenge (Jacobs et al., 2016). The capacity of

innate cells, particularly macrophages, to phagocytosis and eradicate M.

tuberculosis may be impacted by variations in antibody isotype and

glycosylation patterns(L. L. Lu et al., 2016; Zimmermann et al., 2016). The

BCG vaccine results in long-lasting B-cell responses in healthy people, This

is in line with the production of antibody reactions(Sebina et al., 2012). The

immunological response to TB infection may be influenced by the kind and

number of antibodies produced in response to BCG vaccination. New

strategies based on these differences may be helpful for the creation of

novel TB vaccines, TB diagnosis (including differentiating between active

and latent TB), and monitoring treatment response. Additionally, the

potential of maternal transfer of anti-BCG IgG during pregnancy impacting

Basic Platform 30

the neonatal humoral response to BCG may have an impact on the

development of novel TB vaccines as well as BCG vaccination procedures

(Bright et al., 2021).

Immunoglobulin-E (IgE) antibodies have a well-established function

in mediating allergic responses(Sutton et al., 2019). Mast cells, basophils,

and innate granulocytic effector cells of anaphylaxis are all coated with

these IgE antibodies. These cells release the contents of their granules when

they interact with allergens and cell-bound IgE, and when these mediators

are joined with the quickly generated prostaglandin and leukotriene

mediators by the same cells, they work on several target organs to trigger

physiologic reactions(Jimenez-Rodriguez et al., 2018).

The BCG vaccine, used to protect against tuberculosis, is a

potentially helpful model for examining the impact of TH1 cell stimulation

in newborns. This is since to the fact that BCG has a TH1 stimulatory

impact, which causes it to change cytokine response patterns in a way that

inhibits the TH2 immunologic response, combating atopy. based on this, it

has been hypothesized that the BCG vaccine given to infants may have a

preventative impact on the emergence of atopic illnesses(Arnoldussen et al.,

2011). T-helper (Th) cells may be split into the Th1 and Th2 subsets based

on the cytokines they release. As a result of the release of the interleukins

IL-4 and IL-5 by Th-2 type lymphocytes, B cells produce more class E

immunoglobulin (IgE), eosinophils are produced, and other allergic

illnesses such as asthma are brought on(Rehman & Ullah, 2009).

It has been demonstrated that (BCG) therapy can change pathogenic

Th2 responses into protective Th1 and/or regulatory T cell responses,

preventing or lowering an established allergen-driven inflammatory

Basic Platform 31

response. The effectiveness of BCG against allergic illnesses could be

improved by utilizing it as an adjuvant or by researching the effects of

recombinant BCG strains. When administered after the newborn stage, the

BCG vaccine failed to stop the emergence of atopic disease or the creation

of IgE. This does not, however, refute the hypothesis that BCG has a

preventative effect in risk populations with lower genetic

susceptibility(Kowalewicz-Kulbat & Locht, 2021).

2.15 Bacillus Calmette-Guerin (BCG) induce anergy VS tolerance

The T cells' self-tolerance mechanisms can be categorized broadly as

central or peripheral. T cell progenitors developed were in the thymus,

central tolerance mechanisms either kill high-affinity self-reactive T cells or

cause them to differentiate into the regulatory T (Treg) cell lineage(Klein et

al., 2014).However, it appears that peripheral tolerance mechanisms are

required as central tolerance does not appear to be adequate to eliminate all

self-reactive T cells. The basis for peripheral tolerance could be (Davis,

2015; Mueller, 2010):

1-ignorance, when autoreactive T cells never come into contact with

their target antigen.

2-deletion, wherein peripheral T lymphocytes that are directed against

oneself are eliminated after TCR interaction.

3-Anergy, a state of functional immobility brought on by self-Ag

awareness.

One of the mechanisms in the adaptive immune system that controls

responses to target self-antigens is what is known as anergy, one of the

processes that develop tolerance and alter immune cells to stop self-

destruction(Rosenspire & Chen, 2015). The anergy process is mainly

occurring in lymphocytes(Valdor & Macian, 2010). If self-reactive T cells

Basic Platform 32

are able to avoid being negatively selected in the thymus, they may launch

damaging attacks against self-tissues. At several stages of B-cell maturation

and differentiation, including the immature B-cell stage (central tolerance)

and the germinal center (GC) B - cell stage, self-reactive B cells are

tolerated. B-cell tolerance is mediated by a variety of mechanisms,

including deletion, anergy, and receptor modification.

The apoptosis-regulated systems of peripheral anergy and central

tolerance may tolerate self-reactive B lymphocytes produced by random

rearrangements of the immunoglobulin variable genes. GC response-

induced somatic mutations that result in self-reactive B cells are also

removed(Tsubata, 2017). BCG has also been proven to provide benefits in

autoimmune diseases by inducing a state of tolerance (Basak et al.,

2020). investigations on newborns and young children in Guinea-Bissau

have shown observational studies that the BCG vaccine has a protective

effect against anergy (non-responsiveness to common (heterologous)

vaccine antigens)(Garly et al., 2001)

Chapter Three

Investigative Approaches

Investigative Approaches 33

3.Investigative Approaches

3.1 Child subject

The test child population includes 90 child subjects. The study group did

not include the child who had hereditary disorders and concomitant

conditions. Thirty normal subjects served as the control group, followed by

another 30 normal subjects without BCG scars and a final 30 normal

subjects with BCG scars. This study was carried out in the period of

"October 2021 to February 2022 " at Al-Qasim Green University / College

of Biotechnology/Department of Biotechnology. Samples were collected

from the Women's and Children's Hospital in Al-Diwaniyah.

Several questions have been asked and recorded from parents regarding

the child's health, age, place of birth and date of vaccination. Either the

presence or absence of scars and their size were diagnosed visually.

3.2 Scar size determination

During the collection of samples, all children were examined to ensure

the presence of a scar or not, and to ensure that children who did not have a

scar had received the vaccine completely by asking their parents and their

knowledge of the vaccine and using their answers to ensure that the children

who received the vaccine did not get the scar. In this study, we examined

the left and right upper arms or other areas to check up BCG scars.The

scar's measurements were measured using a plastic ruler. The scar's longest

diameter or length was measured twice, and the perpendicular diameter or

length was then measured a third time (because not all scars were circular).

Investigative Approaches 34

The average of these four measurements was used to determine the

approximate scar size.

Figure 3.1: Scar size measurement

3.2 Material

.2.1 Laboratory equipment and instrument:

Table (3.1) shows the laboratory equipment and instrument utilized in this

study and the company, as well as their origins .

Table 3.1 : This study's laboratory equipment and instrument

laboratory equipment and instrument

Company

Country

Blue tips (1000 µL)

JRL

LEBANON

Centrifuge

HETTICH

GERMANY

ELISA system (reader,washer)

BIOTECH READER

GERMANY

Eppendorf tubes 1.5ml

ATACO

CHINA

Gloves

(powder-free)

ADWIC

EGYPT

Incubator

BINDER

GERMANY

Syringe 5 mL

MEDECO

UAE

Yellow tips (100µL)

JRL

LEBANON

3.2.2 Immune reagent and test kits

Investigative Approaches 35

The table(3.2) lists the Immunoassay compounds that were used and

their origins. All immunological tests are conducted at Al-Qasim Green

University's College of Biotechnology utilizing the Biotek ELx808 Manual

12 mode l0, 1997, USA.

Table 3.2 : The Immunoassay Kits were utilized in the study.

Immunoassay Kits

Company Country

Human Immunoglobulin E ELISA Kit

BT LAB

CHINA

Human Interleukin 2 ELISA Kit

BT LAB

CHINA

Human Interleukin 4 ELISA Kit

BT LAB

CHINA

Human Macrophage Migration Inhibitory

Factor ELISA Kit

BT LAB

CHINA

3.3 Methods

3.3.1 Study group

Samples were collected for immunological examination from all groups

of healthy children. 5ml of blood was taken through the intravenous

puncture and placed in a gel tube. the serum was separated into 4 parts and

stored at -20°C in a deep freeze. Thirty normal subjects served as the

control group, followed by another 30 normal subjects without BCG scars

and a final 30 normal subjects with BCG scars

3.3.2 Cellular Immunoassay

3.3.2.1 Human Macrophage Migration Inhibitory Factor(MIF)

Principle of Assay

The pre-coated plate has been treated with anti-MIF antibody. MIF from

the sample binds to antibodies that have been coated on the wells when it is

Investigative Approaches 36

added. Then a human MIF antibody that has been biotinylated is included,

and it binds to the MIF present in the sample. After that, streptavidin-HRP

binds to the biotinylated MIF antibody. Any unbound Streptavidin-HRP is

taken out by washing after incubation. After incorporating the substrate

solution, the quantity of human MIF is subsequently regulated, and color is

produced, when an acidic stop solution is added, the reaction is stopped,

after which the absorbance is calculated at (450 nm).

Prepare the Reagent

1- Before the experiment ,all of the chemicals were ready and kept at room

temperature..

2-To create a 24 ng/ml standard stock solution, 120 µL of standard

(48ng/ml) and 120 µL of standard diluent were combined. Before dilutions

were prepared, it was allowed to rest for 15 minutes while being gently

stirred. The standard stock solution (24 ng/ml) was serially diluted 1:2 with

of standard diluent to provide solutions of (12 ng/ml), (6 ng/ml), (3 ng/ml),

and (1.5 ng/ml). The 0 standard (0 ng/ml) is diluted using a standard

solution. At -20 °C, each remaining solution was frozen. The cited standard

solutions have been diluted as follows:

Table 3.3: Preparation of the (MIF) Standards

24 ng/ml

Standard No.5

120μl Original Standard + 120μl Standard

Diluent

12 ng/ml

Standard No.4

120μl Standard No.5 + 120μl Standard

Diluent

6 ng/ml

Standard No.3

120μl Standard No.4 + 120μl Standard

Investigative Approaches 37

Diluent

3 ng/ml

Standard No.2

120μl Standard No.3 + 120μl Standard

Diluent

1.5 ng/ml

Standard No.1

120μl Standard No.2 + 120μl Standard

Diluent

0 ng/ml

Standard No.0

120μl Standard Diluent

Figure 3.2: Dilution of the (MIF) Standards

3- A 20 ml concentrated wash solution was made, and 500 ml of it was

created by diluting it 25 times in distilled deionized water.

Assay Procedure

1. The examination is conducted in a lab environment after gathering all

relevant reagents.

2. After determining how many strips would be required for the inspection,

the extras were returned and properly kept.

Investigative Approaches 38

3. A 50 µ l of the standard solution were poured into the standard well..

4. Only sample wells added the 40µ L of sample serum and 10 µL of anti-

MIF antibody, whereas the standard wells and sample wells added 50µ L of

streptavidin-HRP. The panel was covered by a sealant treatment before

being incubated for 1 hour at 37 °C.

5- After the incubation time, the lid was removed from the plate and washed

5 time in a specialist washer..

6. For all wells received 50.0 µl of (substrate A) and 50.0µ l of (substrate B).

The plate was re-covered and incubated at 37 °C for 10 minutes.

7. After incubation, blue plate wells turned yellow after adding 50 µL of

the stop solution.

8. Ten minutes after employing the stop solution, the optical density of the

wells was assessed using an ELISA reader set to 450 nm.

Construct the Standard Curve

The human MIF standard value has been verified on each standard vial,

which is how the standard curve was created. Depending on the lot, this

figure could change. Plotting each MIF standard's absorbance against its

concentration along a horizontal axis has resulted in the best curve possible

Table 3.4: Standard concentration of the (MIF)

Standard concentration

Absorbance at (450 nm)

0(0 ng/ml)

0.021

1(1.5ng/ml)

0.547

2(3ng/ml)

0.594

3(6ng/ml)

0.957

4(12ng/ml)

1.526

5(24ng/ml)

2.043

Investigative Approaches 39

Figure 3.3: The Human Macrophage Migration Inhibitory Factor(MIF) standard curve.

3.3.2.2 Human Interleukin 2 (IL-2)

Assay Principle

The plate has been pre-coated with human IL-2 antibody. IL-2

present in the sample is added and binds to antibodies coated on

the wells. And then biotinylated human IL-2 Antibody is added

and binds to IL-2 in the sample. Then Streptavidin-HRP is added

0.5

1.5

2.5

0 5 10 15 20 25 30

bsorbence at

450 mm

Concentration ng/ml

MIF standard curve

Investigative Approaches 40

and binds to the Biotinylated IL-2 antibody. After incubation

unbound Streptavidin-HRP is washed away during a washing

step. Substrate solution is then added and color develops in

proportion to the amount of human IL-2. The reaction is

terminated by addition of acidic stop solution and absorbance is

measured at 450 nm.

Reagent Preparation

1-Prior to use, all reagents must reach room temperature.

2-A 1200 ng/l standard stock solution was created by combining 120µ L

of standard (2400 ng /L) and 120µ L of standard diluent. It was allowed to

settle for 15 minutes with gentle stirring before dilutions were made. To

create solutions at concentrations, the stock standard solution (1200ng/L)

was serially diluted 1:2 with the standard diluent of (600ng/L), (300

ng/L), (150ng/L), and (75ng/L). The 0 standards (0 ng/l) are diluted using

the standard diluent. At -20 °C, each remaining solution was frozen. The

cited standard solutions have been diluted as follows:

Table 3.5: Preparation of the Human Interleukin 2 (IL-2) Standards

1200 ng/l

Standard No.5

120μl Original Standard + 120μl Standard

Diluent

600 ng/l

Standard No.4

120μl Standard No.5 + 120μl Standard

Diluent

Investigative Approaches 41

300 ng/l

Standard No.3

120μl Standard No.4 + 120μl Standard

Diluent

150 ng/l

Standard No.2

120μl Standard No.3 + 120μl Standard

Diluent

75 ng/l

Standard No.1

120μl Standard No.2 + 120μl Standard

Diluent

0 ng/l

Standard No.0

120μl Standard Diluent

Figure 3.4: Dilution of the Human Interleukin 2 (IL-2) Standards

3- Wash Buffer preparation: For 500m l of Wash, prepare 20 ml of Buffer

was diluted with 25 times as much deionized distilled water.

Assay Procedure

1. Prepared each and all reagent, standard solution, and sample in

accordance with the instructions. All reagents were at room temperature

prior to use. The experiment was carried out in a temperature-neutral

environment

2- The surplus strips were returned and stored at 2 to 8°C when determined

the quantity of strips would needed for the experiment.

3 -Standard well received 50 µl of standard.

Investigative Approaches 42

4-The 40 μl of sample and 10 μl of anti-IL-2 antibody were added to the

sample wells and added 50 μl of streptavidin-HRP for the standard wells

and sample.Combined well, covered, and let aside for an hour. at 37°C

5- Following incubation, the cover was taken out and subjected to five

washings in the washing machine.

6. Substrates A and B poured into each well in amounts of 50μ l each. and

the dish sited at 37 °C for 10 minutes.

7. Each well received 50 µ l of Stop Solution, which quickly changed the

blue tint of the wells to yellow..

8. Using a microplate reader set to 450 nm, the (OD value) of each well was

determined ten minutes after adding the stop solution.

Construct the Standard Curve

The following is how the standard curve was made: The human IL-2

standard value has been verified on each standard vial. Depending on the

lot, this figure could change. The best curve was created by graphing the

absorbance for each IL-2 standard against the IL-2 standard concentrations..

Table 3.6: Standard concentration of the Human Interleukin 2 (IL-2)

Standard concentration

Absorbance at (450 nm)

0(0 ng/l)

0.012

1(75ng/l)

0.344

2(150ng/l)

0.478

3(300ng/l)

0.85

4(600ng/l)

1.365

5(1200ng/l)

1.958

Investigative Approaches 43

Figure 3.5: The Human Interleukin 2 (IL-2) standard curve.

3.3.2.3 Human Interleukin 4 (IL-4)

Assay Principle

On the plate, there is a pre-coated human IL-4 antibody. When IL-4 from

the sample is introduced. It attaches to the coated antibodies on the wells.

IL-4 antibody that has been biotinylated is then added, and it binds to the

sample's IL-4. The biotinylated IL-4 antibody is then bound by the addition

of streptavidin-HRP. After incubation, unbound streptavidin-HRP washed

away via a washing process. Following the addition of the substrate

solution, color changes according to the concentration of human IL-4. By

adding an acidic stop solution, the process is stopped, and absorbance is

measured at 450 nm.

Prepare the Reagent

1- Before use, each and every reagent has to be brought to room

temperature.

0.5

1.5

2.5

0200 400 600 800 1000 1200 1400

Absorbance at

450nm

IL-2 Standard curve

Investigative Approaches 44

2- A 120 µ l of the standard (1280 ng/L) and 120 µ l of the standard diluent

were mixed to make a 640 ng /L stock standard solution. The solution was

allowed to stand for 15 minutes while being gently agitated before the

dilutions were made. The stock standard solution was progressively diluted

to create duplicate standard points. (640 ng /L) 1:2 with the standard diluent

to make solutions of 320 ng /L, 160 ng /L, 80 ng /L, and 40 ng/L. The

standard diluent supplies zero standards (0 ng /L). Whatever solution was

left over and frozen at -20°C. The following dilutions have been made to

the stated standard solutions:

Table 3.7: Preparation of the Human Interleukin 4 (IL-4) Standards

640 ng/l

Standard No.5

120μl Original Standard + 120μl Standard

Diluent

320 ng/l

Standard

No.4

120μl Standard No.5 + 120μl Standard

Diluent

160 ng/l

Standard

No.3

120μl Standard No.4 + 120μl Standard

Diluent

80 ng/l

Standard

No.2

120μl Standard No.3 + 120μl Standard

Diluent

40 ng/l

Standard

120μl Standard No.2 + 120μl Standard

Investigative Approaches 45

No.1

Diluent

0 ng/l

Standard

No.0

120μl Standard Diluent

Figure 3.6: Dilution of the Human Interleukin 4 (IL-4) Standards

3- Wash Buffer preparation: The concentrated washing buffer 20 ml

diluted 25 times with distilled water to form a washing solution with a

volume of 500 ml.

Assay Procedure

1. The test is carried out at room temperature, with every reagent, standard,

and samples being ready for use as instructed.

2. It was decided how many test strips would be needed..

3. The standard well received an addition of 50 µl.

4.Along with 40µ l of the sample, 10µ l of anti-IL-4 antibody, and 50µl of

streptavidin, samples and standard wells were treated with streptavidin-

HRP. A sealer was used to seal the panel. At 37 °C, incubate for one hour.

5. The plate was rinsed with wash solution five times after the sealer was

taken off.

6. Each well got 50 µl for each of the substrates A and B. The plate was

incubated at 37°C for 10 minutes with a fresh sealer.

Investigative Approaches 46

7. As soon as 50µ l of Stop Solution were applied, each well became

yellow.

8. Right away, ascertain the (OD value) of each well.

Construct the Standard Curve

The human IL-4 standard value has been verified on each standard vial,

which is how the standard curve is formed. This number might change to be

more . The absorbance for each IL-4 standard was plotted against the

standard concentrations (horizontal axis) to create the standard curve, and

the best curve has drowned.

Table 3.8: Standard concentration of the Human Interleukin 4 (IL-4)

Figure 3.7: The

Human

Interleukin 4 (IL-4) standard curve

Standard concentration

Absorbance at (450 nm)

0(0ng/l)

0.021

1(40ng/l)

0.427

2(160ng/l)

1.045

3(320ng/l)

1.566

4(640ng/l)

2.007

0.5

1.5

2.5

0200 400 600 800

Absorbd at 450

concentration ng/l

IL-4 standard curve

Investigative Approaches 47

3.3.3 Humoral Immunoassay

3.3.3.1 Human Immunoglobulin E

Assay Principle

The plate has previously been pre-coated with the Human IgE antibody.

The antibodies that have been coated on the wells are promptly bound to by

the sample's IgE. The IgE in the serum is subsequently bound by an

additional biotinylated human IgE antibody. The biotinylated IgE antibody

is then bound by the addition of streptavidin-HRP. Unbound Streptavidin-

HRP is removed by washing after incubation. The concentration of human

IgE is then related to how color develops when the substrate solution is

present. After stopping the process using an acidic stop solution, absorbance

is measured at 450 nm.

Prepare the Reagent

1- All reagents must be room temperature prior to use.

2- A 1200 ng /l standard stock solution was created by combining 120 µl of

the standard (2400 ng /l) with 120 µl of the standard diluent. Give the

standard a gentle shake for a quarter hour before diluting. By effectively

diluting the standard stock solution (1200 ng/l) by 1:2 with the standard

diluent to obtain (600ng/l),( 300 ng /l), (150 ng /l), and( 75ng /l) solutions,

duplicate standard points may be produced. The 0 standard (0 ng /l) is a

typical diluent. At -20°C, the remaining solution was frozen. The following

were the suggested standard solution dilutions:

Investigative Approaches 48

Table 3.9: Preparation of the Human Immunoglobulin E(IgE)Standards

1200ng/ml

Standard No.5

120μl Original Standard + 120μl Standard

Diluent

600ng/ml

Standard

No.4

120μl Standard No.5 + 120μl Standard

Diluent

300ng/ml

Standard

No.3

120μl Standard No.4 + 120μl Standard

Diluent

150ng/ml

Standard

No.2

120μl Standard No.3 + 120μl Standard

Diluent

75ng/ml

Standard

No.1

120μl Standard No.2 + 120μl Standard

Diluent

0 ng/ml

Standard No.0

120μl Standard Diluent

Figure 3.8: Dilution of the Human Immunoglobulin-E (IgE)

3- To create 500 ml of Wash Buffer, carefully blend 20m l of Wash Buffer

with 25 times the amount of deionized distilled water.

Assay Method

Investigative Approaches 49

1- Prior to use, all reagents, standard , and samples were prepared in

accordance with the directions. It was done to warm up the reactants. The

setting in which the experiment was carried out was temperature-neutral.

2- The number of strepss required for the test was determined, and the

remaining ones were returned and stored at a temperature between 2 to 8

°C.

3- A 50µl of standard prepared were added to the standard well.

4-The 40 μl of sample and 10 μl of anti-IgE antibody were added to the

sample wells and added 50 μl of streptavidin-HRP for the standard wells

and sample. The panel was covered by a sealant treatment before being

incubated for 1 hour at 37 °C.

5- The sealing cover was taken off after an hour of incubation, and the plate

was then washed with the ELISA washing machine five times.

6- A 50µ l of substrate solution A was given to each well, followed by 50 µl

of substrate solution B. The plate must be incubated at 37 degrees Celsius in

the dark for 10 minutes.

7- The color of each well will instantly change from blue to yellow when 50

µl of Stop Solution are added.

8- The microplate reader with a 450 nm setting read the plate within ten

minutes after adding the stop solution.

Construct the Standard Curve

The human IgE standard value has been verified on each standard vial,

which is how the standard curve is formed. This number could change more

and more . The absorbance for each IgE standard was plotted (vertical axis)

against the IgE standard concentrations (horizontal axis) to create the

standard curve, and the best curve drowned.

Investigative Approaches 50

Table 3.10:-

Standard

Concentration of Human Immunoglobulin E

Figure 3.9: Human Immunoglobulin E standard curve.

Standard concentration

Absorbance at (450 nm)

0(0ng/ml)

0.016

1(75ng/ml)

0.526

2(150ng/ml)

0.953

3(300ng/ml)

1.412

4(600ng/ml)

2.057

0.5

1.5

2.5

0100 200 300 400 500 600 700

Absorbent at

450nm

Concentration ng/ml

IgE Standard curve

Investigative Approaches 51

3.3.4 Statistical Analysis

SPSS Win software version 28 was used for data analysis and entered the

data into a Microsoft Excel worksheet. Determine the mean ,median

,minimum, maximum ,rang, Two-sample t-tests, ONE WAY ANOVA,

using Pearson correlation for data that were normally distributed, the

correlation between two continuous data sets was performed., findings from

the investigations, and outcomes between the children who had and did not

have a BCG scar and between vaccinated and nonvaccinated (Deeks and

Higgins, 2010).

Chapter Four

Results and Discussion

Results and Discussion 49

4. Results and Discussion

4.1 Scar fade

The development of scars following BCG is especially significant since

the presence of a scar on the left upper deltoid region may indicate adequate

BCG immunity throughout infancy. Among the infants who are vaccinated

immediately after their birth, there are those whose vaccination scars

disappear after a while or do not consist of the foundation, and this made it

difficult to know and distinguish whether these children were actually

vaccinated or not.

4.2 Scar size and age of children

Among those children who had been vaccinated with BCG (15 children)

by the intradermal method , the overall mean of the BCG scar size was( 4.7

mm).The age of about one year developed a mean scar size of 5.3 mm. While

that of about two years was with scar size mean 6.1 mm. Age range of 3-5

years have shown scar size means of 3-3.1mm.Therefore ,the scar size

decreased as the age increase up to five years old.Table-1.Figure – 1.Simple

linear regression analysis has shown moderate inverse correlation between age

and scar size.

Table ( 4-1 ) ;Age range and scar size correlation

Age range in months

Mean scar size to the

nearest mm

No. of subjects

1-11

5.3

12-23

7.5

24-35

3.1

36-47

3.0

48-59

2.5

Results and Discussion 50

Figure (4.1): Relationship between (scar size) and the (age) of the children.

Study of (Gudjónsdóttir et al., 2016) indicated that the age had

caused the scar to fade, another study by (Kumar et al., 2005) indicated that

the BCG scar may deteriorate with time. Studies by (Tabatabaei & Hassan,

2019),(Storgaard et al., 2015) and (Kheir et al., 2011) , They also showed

that there is a large group of children who did not have scars after

vaccination.

On the other hand In a study by (Trollfors et al., 2021), the results of

which showed that the effectiveness of the vaccine is not affected by the

regression of the scar with age. Similar results by (Sarfraz et al., 2020)

reported that age or gender had no bearing on the formation of BCG scars.

4.3 Immunoassay Parameters

4.3.1 Macrophage migration inhibitory factor(MIF)

The Minimum MIF concentration for the nonscar subject were higher than

the control. While those scar subject were less than those nonscar.

5.3

6.1

7.5

3.5

3 3

2.5

010 20 30 40 50 60 70

Scar size mm

Age (month)

Results and Discussion 51

Vaccinated children had greater mean MIF than nonvaccinated children.

The maximum concentration was higher in vaccines than in nonvaccine

Table (4.2).

Table(4.2): Features between vaccinated children and nonvaccinated (control) in

MIF .

Features

Vaccinated children

MIF concentration (ng/ml)

Nonvaccinated

(ng/ml)

Scar ( ng/ml)

No scar (ng/ml)

Minimum

0.309

0.954

0.863

Mean

9.483

12.341

6.454

Maximum

26.189

29.219

8.773

Range

0.309-26.189

0.945-29.219

0.8-8.413

Table 4.3:Compare between vaccinated children and nonvaccinated (control) in

MIF.

Groups

Parameter

Nonvaccinated

Vaccination

Mean ± S.E

MIF 6.454 ± 1.46 11.2±1.67

p-value 0.05*

* Significant

The difference between the vaccine and nonvaccine means was statistically

significant(p-value=0.05) at (p-value ≤ 0.05). This means that vaccinated

children have a much higher MIF level compared to unvaccinated children.

Results and Discussion 52

Table 4.4: Compare between vaccinated children with scar and without scar in

MIF

The difference between scars and non-scars was statistically non-

significant (p-value=0.4) at(p-value ≤ 0.05). This means that the level of

MIF of vaccinated people who have a scar and those who do not have it is

equal.

Table 4.5 :MIF Study population plot response

The MIF cytokine herd response plot was: low, Moderate, and high

response. The herd immune plot figure (4.2) ,(4.3) were of Gausion

distribution curve type .

Groups

Parameter

Vaccination with scar

Vaccination

without scar

Mean ± S.E

MIF 9.483 ± 2.12 12.341 ± 2.47

p-value 0.4 NS

NS :Non Significant

Herd Response Scar( ng/ml) NO. Nonscar

( ng/ml) NO. Nonvaccinated

( ng/ml)

NO.

1. Low

response

0.3-4 4 0.945-5 5 0.865-3 5

2. Moderated

response

5-9 7 6-9 6 4-7 8

3. High

response

10-27 4 10-29 4 8-21 2

Results and Discussion 53

Figure( 4.2) : Normal Distribution of MIF concentration in non-vaccinated

children and vaccinated.

Figure (4.3) : Normal Distribution of MIF concentration in non-vaccinated

children ,scar and without scar.

These results are similar to the study by (Hecht et al., 2021),Following

BCG vaccination, increased MIF levels were observed. The process by

which the vaccination modifies macrophages role as a mediator of tissue

invasion and pain modulation is the cause of this.

Contrary to a study conducted by(Freyne et al., 2018) Where the results of

the study indicated that there was no effect of the BCG vaccine alone on the

level of MIF. As the rise in MIF was a result of infection or stress.

0123456

Number of children

MIF response ng/ml

Vaccinated

control

012345

Number of children

MIF response ng/ml

non scar

scar

Novaccineted

Results and Discussion 54

4.3.2 Interleukin 2 (IL-2)

The IL-2 cytokine means and maximum value BCG vaccine in table (4.6)

were higher than control .Scar BCG vaccine Minimum were higher than

control , nonscar BCG vaccine were lower than control.

Table 4.6 : Features of interleukin2 (IL-2) in both vaccinated and nonvaccinated

children

Table 4.7 : Compare between vaccinated children and nonvaccinated (control) in

IL-2

The difference between the vaccine and nonvaccine means was statistically

significant (p-value=0.01) at(p-value ≤ 0.05). This means that the level of

IL-2 in vaccinated children is higher compared to unvaccinated children.

Features

BCG Vaccinee

Il-2 concentration ng/l

Control

ng/l

Scar

No scar

Minimum

304.86

179.50

187.36

Mean

925.14

980

562.61

Maximum

1887

1839.86

1488.43

Range

304.86-1887

179.50-1839.86

187.36-1488.43

Groups

Parameter

Control Vaccination

Mean ± S.E

IL-2 562.61 ± 97.33 952.27±114.137

p-value 0.01*

*Significant

Results and Discussion 55

Table(4-8):Compare between vaccinated children with scar and vaccinated

children without scar in IL-2

The distinction between scars and non-scars was statistically non-significant

(p-value=0.8) at the (p-value ≤ 0.05). This means that the level of IL-2 in

vaccinated children whether they have a scar or not, there is no difference.

Table 4.9:IL-2 Study population plot response.

The BCG vaccinated children IL-2 cytokine herd plot was of skewness type

while those of nonvaccinated was of Gaussian distribution plot type figure

(4.4), (4.5).

Groups

Parameter

Vaccination with

scar

Vaccination without scar

Mean ± S.E

IL-2 925.14± 147.8 980.0± 161.48

p-value 0.8 NS

NS: Non Significant

Herd

Response

Scar

( ng/l)

NO.

Non scar

( ng/l)

NO.

Control

( ng/l)

NO.

1.Low response

300-499

100-499

100-399

2.Moderated

response

500-699

500-999

400-599

3.High

response

700-1999

1000-1999

600-1999

Results and Discussion 56

Figure 4.4 : Normal Distribution of IL-2 concentration in non-vaccinated children

and vaccinated..

Figure 4.5: Normal Distribution of IL-2 concentration in non-vaccinated children ,scar and

without scar.

Study in 2021 by (Bitencourt et al., 2021) this study indicated that IL-2

levels increased after vaccination. Another study by( Lalor et al., 2010 )

reveals the level of IL-2 was higher in vaccinated children than in

unvaccinated children. Which is similar , a study conducted in (Zajac et al.,

2016) indicated that IL-2 with other cytokines increased their response level

after vaccination with BCG vaccine, that their levels are higher compared to

the unvaccinated children.

012345

Number of children

IL-2 response ng/l

unvaccinated

vaccinated

012345

Number of children

IL-2 response ng/l

unvaccinated

Scar

noscar

Results and Discussion 57

4-3-3 Interleukin 4 (IL-4)

The minimum ,mean and maximum value of IL-4 cytokine in BCG

vaccinated children concentration in table (4.10) were higher than in

control children .The nonscar BCG IL-4 mean concentration was higher

than that of scar children .

Table 4.10: Features in Interleukin 4 (IL-4) in the vaccinated children and

nonvaccinated.

Table 4.11 :Compare between vaccinated children and nonvaccinated (control) in

IL-4

Groups

Parameter

Nonvaccinated Vaccination

Mean ± S.E

IL-4 224.28 ± 41.45 356.72±44.199

p-value 0.03*

* Significant

The differences between the vaccinated and the unvaccinated were

significant statistical differences (P-value= 0.03) at (p-value ≤ 0.05). This

means that the level of IL-4 in vaccinated children is higher compared to

unvaccinated children.

Features

BCG Vaccinated

IL-4 concentration ng/l

Nonvaccinated

ng/l

scar

No scar

Minimum

78.59

57.03

51.88

Mean

333.3

380.0

224.28

Maximum

773.59

753.28

631.25

Range

78.59-773.59

57.03-753.28

51.88-631.25

Results and Discussion 58

Table 4-12 : Compare between vaccinated children with scar and without scar in

IL-4

The differences between the scar and nonscar, there were no statistically

significant differences (p-value= 0.6) at (p-value ≤ 0.05). This means that

the level of IL-4 in vaccinated children whether they have a scar or not,

there is no difference.

Table 4.13 :IL-4 Study population plot response

Herd Response

Scar

(ng/l) NO.

Without scar

(ng/l)

NO.

Control

( ng/l)

NO.

1.Low response

70-199

50-299

50-199

2.Moderated

response

200-599

300-599

200-399

3.High response

600-799

400-699

The IL-4 herd responses were found as low ,moderated and high response

table (4.13).BCG vaccination children and nonvaccinated children were

Gaussian distribution type figure (4.6).Nonscar BCG vaccine children and

nonvaccinated were Gaussian distribution type plot ,while scar BCG vaccine

was skewness plot type figure (4.7).

Groups

Parameter

Vaccination with

scar

Vaccination

without scar

Mean ± S.E

IL-4 333.37± 65.52 380.08± 61.65

p-value 0.6 NS

NS : Non Significant

Results and Discussion 59

Figure 4.6: Normal Distribution of IL-4 concentration in non-vaccinated children

and vaccinated.

Figure (4-7): Normal distribution of IL-4 concentration in non-vaccinated children

,scar and without scar.

Study from a (Rook et al., 2004) suggested that the BCG vaccination

stimulates cells to secrete interleukin-4, and that some peripheral blood

mononuclear cells from healthy children immunized with BCG vaccine

release interleukin-4 in response to M. tuberculosis antigens. Previous

study by( Lalor et al., 2010) reveals the level of IL-4 was higher in

vaccinated children than in unvaccinated children. Study by (Jason et al.,

012345

Number of children

IL-4 response ng/l

nonvacinated

scar

noscar

012345

Number of children

IL-4 response ng/l

nonvacinated

vaccinated

Results and Discussion 60

2002 reveals )that the scarring itself appeared to be linked to reduced

median proportions of lymphocytes producing induced IL-4, as opposed to

inflammation at the immunization site or the lack of a BCG-associated

lesion. Contrary to that .study by (Li et al., 2012) showed that IL-4 levels

decreased after BCG vaccination.

4-2-4 Immunoglobulin E(IgE)

The minimum IgE concentration for the control were higher than the

vaccinated . While those with subjected scar were low than the nonscar.

Vaccinated children had greater mean IgE concentrations than unvaccinated

children, but the scar have the lower mean than without scar . The

maximum concentration limit was higher in vaccinee than in nonvaccine

table (4.14).

Table 4.14 : Features between vaccinated children and nonvaccinated (control) in

Immunoglobulin E(IgE)

Features

BCG Vaccinee

IgE Concentration ng/ml

Control

ng/ml

scar

No scar

Minimum

88.56 93.40 184.40

Mean

538.75 672.15 382.83

Maximum

1138.70 1138.40 1105.65

Range

88.56-1138.40 93.40-1138.40 184.40-1105.65

Results and Discussion 61

Table 4.15 : compare between vaccination children and nonvaccinated (control) in

IgE.

The difference between the vaccinated and nonvaccinated means was

statistically significant ( p-value =0.01) at (P ≤ 0.05). This means that the

level of IgE in vaccinated children is higher compared to unvaccinated

children.

Table 4.16 :compare between vaccination children with scar and without scar in

IgE.

The distinction between scar and non-scar was statistically non-

significant(p-value=0.3) at (P ≤ 0.05). This means that the level of IgE in

vaccinated children whether they have a scar or not, there is no difference in

the level.

The herd IgE response in BCG table (4.17) vaccine children was of lower,

moderated and high response .Herd IgE response plot of vaccine children

Groups

Parameter

Control

Vaccination

Mean ± S.E

IgE 382.8±60.11 605.45±62.23

p-value 0.01*

* Significant

Groups

Parameter

Vaccination with

scar

Vaccination

without scar

Mean ± S.E

IgE 538.75± 84.98 672.15± 102.48

p-value 0.3 NS

NS : Non Significant

Results and Discussion 62

and control were of Gaussian distribution curve figure (4.8).The scar BCG

vaccine IgE herd plot and nonvaccination were of Gaussian plot type .Non

scar BCG vaccine children was of skewness plot type figure (4.9).

Table 4.17: IgE Study population plot response

Figure 4.8: Normal Distribution of IgE concentration in normal non-vaccinated

children and normal vaccinated.

Figure 4.9: Normal distribution of IgE concentration in non-vaccinated children

,scar and without scar.

Herd Response Scar

( ng/ml)

NO.

Non scar

( ng/ml)

NO.

Control

( ng/ml)

NO.

1.Low response 80-399 6 90-699 6 100-299 7

2.Moderated

response

400-799 6 700-999 4 300-799 7

3.High response 800-1999 3 1000-1999 5 800-1999 1

6 6

7 7

012345

scar

nonscar

nonvaccinated

IgE responce ng/ml

12 11

0123456

Number of children

IgE responce ng/ml

Unvaccinated

Vaccinated

Results and Discussion 63

study by (Peleteiro et al., 2018) provide that A typical child's IgE

allergic responses increase after exposure to environmental allergens,

children who received the BCG vaccine elicited stronger IgE allergic

reactions. Study by (Carrasco et al., 2018) and (Ivanyi, 2014) provided that

the lower limits of IgE concentration in BCG-vaccinated subjects were

lower than those in kid subjects who had not received the vaccine. Based on

the possibility that the BCG antigenic epitopes contain a lower threshold

toleragenic epitope.

Opposite results found in a study by Krause et al 2003 found no

difference in total IgE levels between BCG-vaccinated and unvaccinated

children . The study of Giovanni et al 2002 provided that the total IgE and

allergen-specific IgE levels were significantly decreased after BCG

vaccination.

The herd immune plot of MIF ,IL-2 ,IL-4 and IgE herd immune response in

BCG vaccine children were of both Gaussian and skewness herd plot type

.These finding were in line with what have been reported in other human

infectious disease in this area( Shnawa, 2014) .

4.4 Correlation between immune parameters

4.4.1 Parameters correlation in vaccinated children with scar

The correlation between the parameters was examined and the results

showed a strong correlation between them as shown in table(4-18).

Whereas, IL-2 strongly binds with IL-4 (r=0.971), and with MIF

(r=0.952),also with IgE (r=0.914) . MIF strongly binds with IgE (r=0.858).

Results and Discussion 64

Table 4.18: Parameters correlation between Vaccinated children with scar

formation.

Parameters

IgE

IL-2

IL-4

MIF

IgE

R=1

IL-2

R=0.914*

P=0

R=1

IL-4

R=0.884*

P=0

R=0.971*

P=0

R=1

MIF

R=0.858*

P=0

R=0.952*

P=0

R=0.973*

P=0

R=1

*. Correlation is significant at the 0.05 level (2-tailed).

4-4-2 Parameters correlation in vaccinated children without scar

The correlation between the parameters in vaccinated children without scar

was examined and the results showed a strong correlation between them as

shown in table(4-19). Whereas, IL-2 strongly binds with IL-4 (r=0.808),

and with MIF (r=0.947),also with IgE (r=0.840) . MIF strongly binds with

IgE (r=0.870).

Table 4 .19: Parameters correlation between vaccinated children without scar

Parameters

IgE

IL-2

IL-4

MIF

IgE

R=1

IL-2

R=0.840*

P=0

R=1

IL-4

R=0.897*

P=0

R=0.808*

P=0

R=1

MIF

R=0.870*

P=0

R=0.947*

P=0

R=0.729*

P=0

R=1

*. Correlation is significant at the 0.05 level (2-tailed).

Results and Discussion 65

4-4-3 Parameters correlation in unvaccinated children

The correlation between the parameters in nonvaccinated children was

examined and the results showed some the correlation between parameters,

as seen in table(4-20). Whereas, IL-2 strongly binds with MIF (r=0.852).

IgE moderated binds with IL-4 (r=0.563).As for interleukin-2 and

interleukin-4, there was no correlation(r=0.303), as well as for IL-2 with

IgE (r= 0.320).

Table 4.20 : Parameters correlation between unvaccinated children

Parameters

IgE

IL-2

IL-4

MIF

IgE

R=1

IL-2

R=0.320

P=0.245

R=1

IL-4

R=0.563*

P=0.029

R=0.303

P=0.273

R=1

MIF

R=0.158

P=0.574

R=0.852*

P=0

R=0.293

P=0.288

R=1

*. Correlation is significant at the 0.05 level (2-tailed).

A considerable increase in the Th1/Th2-type cytokine ratio would be

predicted to be the greatest predictor of BCG vaccination-induced

protective immunity since Th1-type and Th2-type cytokines are cross-

regulatory and mutually inhibitory. BCG vaccination led to the production

of pro-inflammatory cytokines such IFN and TNF, which are known to

activate macrophages infected with M.tuberculosis , and IL-2, which

encourages the activation of Th1 cells and CD8 T cells. Both IL-2 and IL-4

were strongly and positively associated with each of the vaccinated

Results and Discussion 66

children, whether they had a scar or had no scar, and this indicates that the

immune response stimulated by the vaccine includes both cellular response

and humoral response. IgE was associated with a strong correlation in

vaccinated children, in contrast to unvaccinated children, its correlation was

moderate, and this indicates that the vaccine stimulates an allergic response

more.

On the contrary, in unvaccinated children, there was no correlation between

Th1 and Th2 in terms of stimulating cytokines, as there was no correlation

between IL-2 and IL-4.

The cellular immune response to BCG vaccine and /or

M.tuberculosis pulmonary infection have been described as divergent

(Shnawa et al., 2022) . The findings show that higher and lower MIF ,IL-

2 ,IL-4 and IgE .Individual result curing both scar and nonscar being BCG

vaccine children . Hence both vaccine groups mounts cellular and IgE

responses to variable degrees.A situation that indicate a given child vaccine

mount BCG vaccine response whether produce scar then fade up or

produce scare and reserve its size to a variable degrees as age progressed.

Therefore it is an indirect proof from immune deviation or immune

divergence(Baha et al., 2019; Shnawa et al., 2020).

Chapter Five

Conclusions and

Recommendations

Conclusions and Recommendations 67

5. Conclusions And recommendations

5.1 eConclusions

1- e Results showed a moderate inverse relationship between the size of

the scar and the age of the child. That is, the older the child, the

smaller the diameter of the scar size.

2- Statistical results showed an increase in the level of MIF, IL-2 and

IL-4 in vaccinated healthy children compared to healthy

unvaccinated children, and this indicates that BCG induces a change

in the levels of cellular immunity of children after vaccination .The

BCG vaccine caused a greater change in IgE concentrations healthy

vaccinated children than in unvaccinated children, signifying a

condition of humoral immunological conversion caused by the BCG.

3- Children who have received BCG but have a scar from the vaccine

experience the same outcomes as non-scar. which were evaluated for

MIF, IL-2, IL-4, and IgE as well, the findings demonstrating that the

scar's existence or absence has been either associated or dissociated

with the child's immunity. This indicated the limits of divergence in

child cellular immune responses to BCG vaccines.

Conclusions and Recommendations 68

5.2 Recommendations

1- Investigate the subjects who have no scar formation at all after BCG

vaccination using the same study parameters.

2- Study the role of IL17 and IFN cytokine families in this experimental

setting.

3-Flow cytometeric determination studies for the role of T and B cells

using this experimental setting.

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Appendix

Appendix 102

Appendix (1)

A questionnaire form used to collect information about children.

Age

Place of birth

Presence

or absence

of scars

Date of

vaccination

Child's health

8 month

City center

absence

Unknown

Diarrhea

8 month

City center

absence

First week

Diarrhea

4 year

City center

absence

First week

Malnutrition

3 year

City center

absence

After 2 days

head injury

3 year

city side

absence

Unknown

infections

8 year

City center

absence

Less than month

healthy

4 year

City center

absence

In 2019

healthy

1 year

City center

Presence

First week

healthy

7 year

City center

absence

After 10 days

healthy

10 year

City center

absence

After 3 days

healthy

2 year

City center

absence

First week

Diarrhea

5 year

City center

absence

First week

diabetic

Appendix 103

Appendix 2

BCG vaccine used in Women's and Children's Hospital in Al-

Diwaniyah city.

104

Summary

Bacille Calmette-Guérin (BCG) vaccine, often produces a surface scar

after proper administration. The size of the scar that developed is

frequently regarded as a sign of immunity. Lack of immune system

development, improper administration, and ineffective vaccines are a few

causes of scarring that don't develop. Recognition of these common

mycobacterial components improves the innate and adaptive immune

response against Mycobacterium tuberculosis, which is how BCG

protects against TB. The current study aims at determining the gross

manifestations and cellular immune response to the BCG vaccination scar

and scar fade up in children of various ages can help us determine how

much immunity children have against exposure to tuberculosis. This

alone is crucial for children's development and welfare.

Ninety children made up the test group in the current study. This study

was carried out in the period of "October 2021 to February 2022 " and the

Samples were collected from the Women's and Children's Hospital in Al-

Diwaniyah cite .The children who had inherited illnesses and concurrent

diseases were not a part of the study group. Among which 30 were

control (non-vaccinated),30 vaccinated without scar and 30 vaccinated

with scar.

The Enzyme-Linked Immunosorbent Assay (ELISA) assay was used to

measure the levels of cytokines represented by Macrophage Migration

Inhibitory Factor (MIF), Interleukin 2 (IL-2) and Interleukin 4 (IL-4), as

well as to measure the concentrations of Immunoglobulin E (IgE).

105

A plastic ruler was used to measure the size and dimensions of the

vaccine scar.

The results showed a moderate inverse relationship between age

and scar size. The present results also showed that the group of

vaccinated healthy children showed a higher response than the

healthy unvaccinated children at MIF (P = 0.05). Also, IL-4

showed an increase in response levels after vaccination (P=0.03).

As for the IL-2, it also witnessed a high rise in its levels after

vaccination, compared to healthy non-vaccinated children

(P=0.01). Also, in the level of IgE, the response level in the

vaccinated children was higher than that of the non-vaccinated

children (P=0.01). As for the vaccinated children and those who

were non scar vaccinated and those who were scar vaccinated

There were no significant differences, according to the findings :

"MIF (P = 0.4)", "IL-2" (P = 0.8) ,"IL-4" (P = 0.6) " IgE (P =

0.3)".

The study concluded that , The size of the scar decreases with the age

of the children . And that the vaccinated children showed an increase in

the levels of the cellular and IgE humoral immune response, and that

there was no statistical significant difference between the immunity of

the children who had the vaccine scar from others who did not have the

scar .

More than 90% of infants worldwide receive the (BCG) vaccine shortly after birth to protect them against

tuberculosis (TB). The vaccine was created by Bacille ,Calmette and Guerin, and it was given to people for

the first time in 1921. Only BCG protects against TB (Okafor et al., 2022). Its effectiveness against pulmonary

tuberculosis in adults is low and varies from 0 to 80% depending on a number of variables such geographic

location and prior exposure to environmental mycobacteria (Mangtani et al., 2014).

The live bacteria was used in the BCG vaccine have been weakened (attenuated) in order to boost the

immune system without harming healthy individuals(Covián et al., 2019).In nations with higher mortality

rates, BCG scarification has been linked to increased survival and a stronger immunological response in

children who have received the vaccine(Tabatabaei & Hassan, 2019). A scar that develops after immunization

with the vaccine is one of the distinctive signs of the effectiveness of the vaccine and a crucial signal of

immunity. The size of the resulting scar could be a useful gauge of the immune system's reaction to the BCG

immunization (Park et al., 2015).

Ibrahim M S Shnawa

Professor Emeritus Doctor, College of Biotechnology

University of Qasim And Hilla University College,

Babylon Province/IRAQ.

Tiba Ahmed Karim

MSC Medical Biotechnology,

Diwanyah Teaching Hospital

Diawnyah Board of Health,ALQadisyah Province/IRAQ.

ResearchGate has not been able to resolve any citations for this publication.

Mycobacterium tuberculosis Transmission in High-Incidence Settings–New Paradigms and Insights

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INT J INFECT DIS

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Abstract

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