Immunophenotyping in RSV Infected Pediatric Patients:

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1 LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

MEDICAL ACADEMY FACULTY OF MEDICINE DEPARTMENT OF PAEDIATRICS

Immunophenotyping in RSV Infected Pediatric Patients:

A Literature Review

Student: Samer Najeh Mustafa Abu Dayeh

Supervisor: Lina Jankauskaitė MD, PhD

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ABSTRACT

Aims and Objectives: The aim of this review is to analyze the different phenotypes in an RSV infection in a specific group of patients 1-18 Y/O, and to determine whether or not a correlation to disease severity exists.

Methods: The article search was performed in Medline and Google Scholar. Articles collected for this literature review were found according to the following terms: ((Acute viral respiratory infection) OR upper respiratory infection) AND RSV) OR respiratory syncytial virus) OR influenza) AND pediatric) OR pediatric) OR child*) AND lymphocyte) OR immunopheno*) OR phenotype*) OR character*)). All articles were published within the last five years, as of the year 2020, in English, and included only pediatric patients.

Results: The search yielded 17,827 articles on Medline (PubMed). Using our inclusion and exclusion criteria, 440 articles were retrieved. 422 of them did not meet further specific criteria, yielding 18 articles. A further of 13 articles were unfit for this review after careful inspection. 2 more articles were later found on a Google Scholar search. This resulted in a total of 7 articles for the purposes of this review. The RSV-induced infection was analyzed with regards to CD4+ T-cells, CD8+ T-T-cells, IFN- γ, IgG and IgA antibodies, and interleukins. Results indicate CD4+ cells to be increased as well as CD8+ T-cells during an RSV infection. Regarding disease severity, some studies showed CD4+ cells to be a better correlating marker, while one study indicated that CD8+ T-cells are a better candidate. Also, in almost all studies, IFN- γ always increased in relation to infection and disease severity. Interleukins were variably increased/decreased during RSV infection.

Conclusion: Many conflicting results were obtained after analyzing the studies in this review. Firstly, all studies had small samples sizes that could have resulted in different conclusions. As complete blood count is not recommended in bronchiolitis cases, it could be an issue to further analyse RSV-induced diseases (such as bronchiolitis) phenotypes. A comprehensive understanding of perhaps other biomarkers must be analyzed in order to better understand the role of these markers in RSV-induced disease severity and outcomes.

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ACKNOWLEDGMENT

I would like to sincerely thank and recognize Dr. Lina Jankauskaite’s utmost generosity in advice and for all her help throughout the completion of this review.

CONFLICT OF INTEREST

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ABBREVIATIONS

• APRIL - A Proliferation Inducing Ligand • BAFF – B-cell Activating Factor

• BCL6 - B-Cell Lymphoma (transcription repressor) • CCL - Chemokine Ligand

• CD - Cluster of Differentiation

• CDSS - Clinical Disease Severity Score • CXCL - Chemokine Ligand

• CXCR - Chemokine Receptor • DC - Dendritic Cells

• FDA - Federal Drug Administration • GATA - G-A-T-A (core sequence) • GC - Germinal Center

• ICOS - Inducible Co-Stimulatory • IFN - Interferon

• IgE - Immunoglobulin E • IL - Interleukin

• JAK - Janus Kinases

• LRT - Lower Respiratory Tract

• LRTI - Lower Respiratory Tract Infection • MHC - Major Histocompatibility Complex • MPO - Myeloperoxidase

• NET - Neutrophil Extracellular Traps

• NLR - Nucleotide Oligomerization Domain Like Receptors • PAMP - Pathogen Associated Molecular Patterns

• PD-1 - Programmed Cell Death • PRR - Pattern Recognition Pattern • RIG - Retinoic Acid Inducing Gene • RNA - Ribonucleic Acid

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6 • RSV - Respiratory Syncytial Virus

• SD - Standard Deviation

• STAT - Signal Transducer and Activator of Transcription proteins • Tfh - T follicular helper

• TGFβ - Transforming Growth Factor beta • Th - T helper cells

• TLR - Toll Like Receptor

• TNF α - Tumor Necrosis Factor alpha • Tregs - T regulatory cell

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INTRODUCTION

RSV is a negative sense single-stranded RNA enveloped virus of the family Paramyxoviridae and is one of the most common causes of virus-induced upper respiratory tract infections (1). In 1901, “acute catarrhal bronchitis” was the first description of respiratory syncytial virus (RSV) in a clinical manner (2). Later on in 1956, RSV was recognized first by Robert Chanock (3).

RSV causes infections worldwide, mainly in temperate areas in times of winter. RSV can induce bronchiolitis or pneumonia and lead to asthma exacerbations. The clinical symptoms include cough, wheezing, low grade fever, and dyspnea in some cases (4). Mainstay of treatment is supportive care (i.e. hydration, nasal suctioning with saline solution, etc.). In addition, no current vaccine exists (5).

Mortality associated with RSV is generally low, estimated to be lower than 1% in healthy subjects; higher in children with health risk (immune deficits, cardiovascular disease, etc.) (2). However, due to high infectivity and morbidity, the burden RSV carries on the US economy equates to about 340-450 million dollars annually (6).

As no current vaccine or treatment exists (5), research focusing on the molecular phenotypes of the human immune system in relation to RSV infections can be very beneficial in

1_{grasping a better understanding of the RSV,}2_{how our immune system responds, and}3_{how it}

manifests in clinical severity. With a lot of research conducted in recent years on RSV and the intricate mechanisms in which the immune system responds to it, advancements have been made in better understanding this virus. This surely provided an opportunity for new targeted medications with fewer side effects and better efficacy. It could also allow for more effective or even prophylactic handling of the potential RSV induced complications.

Thus, the aim of this review was to identify certain cell types in RSV infected patients that correlate with the disease severity. This can provide a chance to notice certain markers that can predict an increasing severity in disease, aiding in treatment and prophylaxis towards possible complications in the RSV infected pediatric population.

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AIMS AND OBJECTIVES

Aim: To find out the different immunological phenotypes in RSV infected pediatric patients and their relation to disease severity.

Objectives:

1. To perform a comprehensive research on RSV and establish a full understanding of the different immunophenotypes it induces in RSV infected pediatric patients.

2. To correctly categorize different and specific lymphocytic phenotypes in RSV infected patients.

3. To analyze the relationship between the various immunophenotypes and disease severity in RSV infected children.

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1. LITERATURE REVIEW

1.1 Respiratory Syncytial Virus

1.1.1 RSV Structure

RSV virion is made up of a nucleocapsid embedded within a nuclear envelope (7). This nuclear envelope contains 3 integral transmembrane proteins: G, F, and SH, with G protein as the special attachment glycoprotein, the F protein being the fusion protein, and SH the hydrophobic protein (Figure 1. on the following page). However, the main way this virus infects the host is by attaching to airway epithelial cells and the process is mediated by glycoproteins G and F (8). These two glycoproteins are of importance, specifically as protein G allows the virus to attach to host cells and modulate an immune response, while protein F paves entry of the virus into the cell (9).

Although all surface glycoproteins are important in understanding the pathophysiologic and virologic characteristics of this virus, the glycoprotein F is of special importance. Studies have shown that antibodies to F protein demonstrated the best results in neutralizing the virus when compared to G or SH protein antibodies (10). This could be mainly due to the difference in the two proteins’ structure: fusion protein F is conserved in structure while attachment protein G is highly variable. Therefore, as protein G not being an ideal target for therapy, the only FDA approved prophylactic treatment for RSV infection in children is Palivizumab, a monoclonal RSV-F protein antibody (11).

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10 Figure 1. Structure of RSV virus

Adapted from: Jung et al. (12). ssRNA: single stranded RNA. L: Large polymerase protein. F: Fusion protein. SH: Small Hydrophobic protein. P: Phosphoprotein. G: attachment protein. N: Nucleoprotein. M: Matrix protein

1.1.2 Virus Tropism

RSV tropism can be summed up as cell and receptor specific. Firstly, with the help of its glycoproteins F and G, RSV can evade the immune system and ease its way into the superficial cells of the respiratory epithelium. The virus has a particular affinity towards the ciliated columnar cells of the upper respiratory tract epithelium (7). And as it travels towards the lower respiratory tract, the other cell lines that it mainly infects are the alveolar epithelial cells (AEC) (8).

Another factor that can affect RSV tropism is tissue receptor tropism. This can be determined by cells having virus specific receptors/co-receptors that help viral attachment/entry. The surface protein F on RSV is an example: protein F binds to a specific receptor found on cells, nucleolin (13). Furthermore, with regards to G protein, it is known to bind to CX3CR1 (a fractalkine receptor) to begin infection (14). One study by Johnson SM et al. has found that this same receptor, CX3CR1, is also expressed on the ciliated cells of the respiratory epithelium. This could explain the virus’ affinity and tropism to these particular cell lines (15).

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11 1.1.3 RSV Epidemiology

Despite extensive research on this virus, severe complications and hospital admissions are still as high as 3.8 million cases worldwide per year (16). It is estimated that nearly all children will be affected by RSV by the age of two, with up to 2% ending up hospitalized and of which, up to 90% will suffer from complications such as RSV induced bronchiolitis (17).

A study in the US analyzed 5067 children with bronchiolitis. They observed that 18% of the cases were caused by RSV and 20% of these children were hospitalized for observation and treatment. They concluded that RSV infections carry a heavy morbidity profile in the US (18).

In Canada, RSV causes up to 12 thousand children to be hospitalized every year, while the US faces around 77 thousand admissions annually, making it the leading cause of admission in children younger than 1 year of age (17).

Another study in Germany demonstrated that the total expenditure was 66 million euros for lower respiratory tract infections (LRTI) including RSV induced diseases in children admitted to the hospital (19). Globally, RSV is estimated to cause 33 million LRTI annually, 3 million hospitalizations, and around 200 thousand deaths among children every year (20).

1.1.4 Clinical Picture

Several risk factors exist and have been documented in numerous studies. Some of which are the following: close contact areas (i.e. daycare centers), established chronic disease (i.e. asthma, atopy, immune system disorders, congenital anomalies, etc.), prematurity, immunosuppressant use in the elderly, and particularly children with atopic disease as IgE is known to increase disease severity (21).

Almost all children by the age of 2 are infected at some point with RSV. Young children, specifically younger than 12 months, are much more prone to complications such as bronchiolitis, caused by this virus when compared to older children. This was shown in a survey where a total of 81% of children hospitalized with RSV were younger than 1 year (22). In addition, one study demonstrated that children with a previous history of RSV infection had a 10-fold increase in risk of asthma. RSV was rated as the highest independent risk ratio for asthma development (23).

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12 RSV can cause a range of respiratory illnesses and complications such as bronchiolitis, bronchospasms, asthma exacerbation, respiratory failure, and pneumonia. The symptoms vary from wheezing, cough, to mild fever, and in severe cases, apnea. Apnea can be the presenting symptom and cause of death (sudden infant death syndrome) in hospitalized children due to RSV (24).

Rhinorrhea and cough being the most common presenting complaint, the course of disease is generally mild and self-limited. In some cases, it can cause exacerbation of previously diagnosed asthma, sometimes extending to cause pneumonia and leading to symptoms such as fever, purulent cough, and dyspnea (24).

RSV induced bronchiolitis comprises 40% of RSV childhood respiratory illnesses (25). Bronchiolitis is a lower respiratory tract infection that is usually caused by a virus (RSV being the most common in children younger than 2 years). Symptoms, ranging from mild to severe are nasal congestion, cough, mild fever, wheezing, and dyspnea. In very severe cases, signs of respiratory failure could be seen such as nasal grunting, sternal retractions, grunting, and in some cases cyanosis (26). Figure 2., shows the radiographic signs, which include patchy atelectasis, lung hyperinflation, and bronchial infiltration that is bilateral.

Figure 2. Radiographic (X-ray) signs of RSV induced bronchiolitis in children in anterior/posterior and lateral positions.

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1.2 Molecular Mechanisms of RSV-Induced Infection (Host Immune

Response To RSV)

According to numerous studies, the severity of an RSV infection is directly related to the viral load (27). Hence, it’s the underlying inflammatory process that dictates the severity of disease and possibly its complications as well (28).

Once infected with RSV, a pro neutrophil inflammation of both the upper and lower respiratory tracts occurs (29). This has been demonstrated in numerous studies, one of which showed that neutrophils in children infected with RSV comprised 73-90% of total inflammatory cells, with CXCL8 (chemokine ligand 8) as one of its main chemoattractant (30). Thus, neutrophils encompass the highest number of leukocytes in RSV infected lungs. This can both improve or worsen the infection progress. Other than eliminating infected cells and limiting viral spread, neutrophils also release enzymes, such as neutrophil elastase, that may damage lung tissue leading to possible long-term consequences (31).

This finding supports that in infants with RSV infection, neutrophils were documented to be the main source of release of IL-9. IL-9 is a proinflammatory cytokine associated with bronchial hyperresponsiveness and asthma exacerbation (32). Although the exact mechanism is not well understood, it is hypothesized that neutrophils function during infection through the following suggested mechanisms: oxidative burst, NETosis (Neutrophil Extracellular Traps), inflammatory mediator release, and degranulation (31). This has been established in a few studies where, for example, MPO (myeloperoxidase) related NETosis levels were elevated in children that had acute RSV bronchiolitis (33).

Furthermore, the levels of certain T-cells such as CD4+ and CD8+ correlate with disease severity (30). CD8+ cells have shown to be inversely proportional to mortality and severity in RSV respiratory infections (34).

Once RSV enters the lungs and infects host cells, dendritic cells (DCs) found throughout the respiratory system are activated. DCs are specialized cells that are essential in linking the innate and adaptive immune systems (35). The host immune cells have many Pattern Recognition Receptor (PRRs), namely: Toll Like Receptors (TLR), Retinoic acid Inducing Gene 1 (RIG-1),

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14 and Nucleotide oligomerization domain Like Receptors (NLR); these PRRs help in initiation of the innate immune response (36).

Figure 3. CD4+ T-Cell Differentiation and Pathway Signaling

As adapted from: Heinonen Santtu et al. (46). IL: Interleukin; CD: cluster of differentiation; IFN: interferon; TNF: tumor necrosis factor; Th: T helper cell; T-reg: Regulatory T-cell; Tfh: Follicular helper T-cell; ICOS: inducible T-cell costimulatory; TGF-β: transforming growth factor beta.

DCs function by obtaining viral antigens by either engulfing virus infected cells (phagocytosis) or by infection. RSV virus promotes DC maturation by the PRRs expressed on DC surface recognizing PAMPs, thus activating a downstream signaling pathway, inducing DC maturation and cytokine production (37). Moreover, MHC class II expression on DCs is promoted

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15 by RSV infection which leads to further production of pro-inflammatory cytokines and type 1 interferon (IFN-1) - (IFN α,β) (38).

Once viral antigens are acquired by the DCs, they migrate to their respective draining lymph nodes to initiate a T-cell response that is virus specific by presenting the antigen bound MHC class I or II (39). There, further cytokine production (IFN-1 IFN- γ) by T-cells occurs, including Th1 CD4+ cells and CD8+ cytotoxic cells (40). Other cytokines that are involved in the early innate immune response to RSV infection are tumor necrosis factor-⍺ (TNF-⍺), IL-6, IL-9, IL-10, CXC chemokine ligand 10 (CXCL10), CXCL8 (IL-8), CC chemokine ligand 2 (CCL2), and CCL3 (macrophage inflammatory protein) (41).

After the innate system is activated, mostly CD8+ cells travel to the respiratory tract for viral clearance. Regarding CD4+ T-cells, after activation of the T-cell receptors, they differentiate into specific T-helper subsets, each with a specific function, including Th1, Th2, Th17, regulatory T-cells (Tregs), and T follicular helper (Tfh) cells (Figure 3.). Th1 cells are important in the initial stages of infection and are mediated primarily by IFN-γ; however, other cytokines are also involved such as IL-1, IL-2, IL-12, IL-18, and TNF-α (42). Regarding Th2 cells response, it is regulated by IL-4, 5, 9, and 13 and is involved in antibody production and class switching making them very important in the long-term consequences that RSV infection holds (e.g. persistent wheezing in infants with a predominant Th2 response) (43).

Th17 produces mainly IL-17A/F and IL-22, giving it an important role in protection against extra-cellular pathogens. This is achieved by Th17 functioning to increase mucus production (primary protections), enhancing Th2 response, and modulating CD8+ cells (44). Another function worth noting is its enhancement of lung neutrophil infiltration which could be connected to its relation to asthma exacerbation and recurrent wheezing as previously mentioned. Tregs function to regulate and maintain the innate and cellular immune response and tissue homeostasis during an acute infection. They are associated with multiple cytokines, mainly IL-10 and transforming growth factor-beta (TGF-β). While IL-12 favors differentiation into Th1 lineage, IL-10 inhibits it. Thus, leading to a more Th2 centered response, which as previously noted, can result in recurrent wheezing and asthma exacerbation (45).

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16 Despite limited research on Tfh cells, they characteristically express CXCR5 (chemokine receptor), BCL6 (a transcription factor), and PD-1 (an inhibitory molecule). They are collectively involved with B-cell class switching, affinity maturation, and memory B-cell development. Early differentiation of these cells is mediated by IL-6, inducible co-stimulator (ICOS-induces directional migration of CD4+ cells), and IL-2. The second stage of its differentiation occurs when they contact antigen presenting B-cells finally becoming a GC (germinal center) Tfh cell, mainly expressing CXCR5 on its surface and interacting with GC B-cells for memory B-cell production (46).

A general pathway then follows as such: a T-cell independent response activates B-cells, B-cell co-stimulating factors (BAFF, APRIL, etc.), and the release of IgA and IgG antibodies to help fight off the virus over the course of days to weeks (35). Higher levels of pre-inoculated IgA have been shown to reduce the viral load in RSV infection (47). While on the other hand, pro inflammatory markers such as IFN-γ, IL-1, IL-2, IL-12, IL-18, and TNF-α, have been documented to possibly be deleterious to some patients regardless of their initial adequate immune response (48). A summary of the process can be found in Figure 4 on the following page.

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17 Figure 4. A summary of the immune response to RSV infection in human beings

As adapted from Russell et al. (36). RSV: Respiratory syncytial virus; IgE: immunoglobulin E; aRSV-IgG/IgA: alpha RSV immunoglobulin G/A; BAFF: B-cell activating factor; APRIL: A proliferation inducing ligand; IFN: interferon; NK: natural killer; HLA-I/II: Human leukocyte antigen I/II; DC: dendritic cell; IL-12/18: Interleukin-12/18; CCL-5: chemokine ligand 5; TNF-α: Tumor necrosis factor alpha; CXCL-19: chemokine ligand-19; mTOR: mammalian target of rapamycin.

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1.3 Treatment and Vaccination

Treatment is generally supportive in the form of giving fluids, controlling fever, patient monitoring, and providing symptomatic care. In severe cases of bronchiolitis, supplemental oxygen and IV fluids may be administered. The entirety of this disease’s course is mild and lasts around a week, with children recovering within 5 days of symptom onset. However, some patients maintain wheezing up to an additional week (49).

Additionally, in the US, there is a drug that is FDA approved for use against RSV infection. Given in aerosol form, Ribavirin is an antiviral drug that has shown activity against the virus in vitro (50). However, Ribavirin has been shown to have some teratogenic adverse effects (51), and therefore, the subject of doubt in its cost-effectiveness in RSV infected patients.

In particular cases, passive immunization to RSV is an option in combatting RSV infection. Palivizumab, previously mentioned in the RSV structure section, is a monoclonal antibody that can be given monthly throughout the RSV infection season. However, it is an expensive option and up until recently has been debated over for its cost-effectiveness (20). It should be mentioned that Synagis (Palivizumab), a monoclonal antibody, is approved for use in Lithuania. It is mostly given as a preventive method against RSV for those born prematurely, or children born with cardiac or respiratory tract disorders such as bronchopulmonary dysplasia (52). It has been proved to be effective in preventing severe RSV-induced respiratory tract disorders such as severe bronchiolitis in children less than 24 months of age. However, it is not used as a treatment method.

Regarding RSV, no current vaccine exists. Nevertheless, there are many potential vaccine candidates that are currently under trial, including other monoclonal antibodies as prophylaxis (53).

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2. METHODOLOGY

This literature review was conducted in accordance with the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. The search was performed in Medline (PubMed) and Google Scholar. The following search strategy was implemented: ((Acute viral respiratory infection) OR upper respiratory infection) AND RSV) OR respiratory syncytial virus) OR influenza) AND pediatric) OR pediatric) OR child*) AND lymphocyte) OR immunopheno*) OR phenotype*) OR character*)).

The last PubMed search was performed on January 15th, 2020. This resulted in 17,827 citations at the time. There were no duplicates removed as none could be found. The articles in this review were conducted in English, limited to human studies, published within the past five years (2015-2020), and centered around children between birth – 18 years old (except neonates). Articles that focused on adults as the sample to study, were a review or commentary, and did not meet all the inclusion criteria, were excluded. Of the 17,827 citations, 440 articles were retrieved after the first title/abstract screening. A further 422 more articles were excluded after a full text screening as well as another 13 articles after data extraction. 5 Articles met the inclusion criteria and a further of 2 more articles were identified via Google Scholar.

Table 1. A list of the inclusion/exclusion criteria adapted in this study

Inclusion Criteria Exclusion Criteria

Children (1 month –18 years) Adults Publications within the last 5 years

(2015-2020)

Systematic Reviews; Literature Reviews or Meta-Analysis

Human Studies (human trials, clinical studies, etc.)

Infection as a sequela of other condition/disease

English language-based studies Animal or in-vitro based study

RSV specific Not RSV-specific (TB, Rhinovirus, etc.)

Free full text studies Chronic disease (cancer, immunodeficiency, chronic viral infections)

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3. RESULTS

The article search was implemented according to the following terms: ((Acute viral respiratory infection) OR upper respiratory infection) AND RSV) OR respiratory syncytial virus) OR influenza) AND pediatric) OR pediatric) OR child*) AND lymphocyte) OR immunopheno*) OR phenotype*) OR character*)). The resulting search yielded 17,827 articles on Medline (PubMed). Upon application of our inclusion/exclusion criteria, we ended up with 440 articles. Furthermore, upon inspection of these articles, 422 of them were unfit after a full text screening, while 13 more were deemed ineligible in accordance with the thesis criteria leaving with 5 articles identified to be suitable. Two more articles were added later through a Google Scholar search, bringing to a total of 7 articles for use in this review.

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21 Table 2. A general outlook highlighting the main differences between the articles picked for this review

Authors Study Type Publishing Year Country Number of participants Type of virus Cell type and/or protein analyzed Results 1. Noyola Et al. (54) Prospective Cohort 18 May 2015 Mexico 64 Total 55 RSV infected 9 Control

RSV NK Cell The amount of NK cells was not significantly different when compared between infants infected with RSV and the Control Group.

2. Green Et al. (55) Prospective Cohort 21 Aug 2018 UK 65 Total 30 Adults 35 Children RSV IgA memory cell IFN-γ IL-4 IL-13 IL-17 Anti-F IgG Anti-G IgG

Children 3-6 years old had Anti-F IgG memory cells while infants <12 months old conferred little to no immunity. Only the 3-6 Y/O pediatric pop. was found to have IFN- γ that was RSV specific.

None of the pediatric pop. recorded Anti-F IgA memory cells.

Childhood exposure to RSV generates antibodies and confers cellular immunity to RSV. 3. Fedele Et al. (56) Prospective Cohort 30 Apr 2018 Italy 56 Total 43 RSV infected 13 HRV infected RSV HRV IFN-γ IL-4 CD3+ CD4+ CD8+

RSV-bronchiolitis triggered a type 1 immune response predominantly, IFN-γ, CD3+ and CD4+ (Th1). While HRV-bronchiolitis triggered a type 2 immune response predominantly, IL-4, CD3+, and CD4+ (Th2).

There was no relation that is statistically significant found between Th1/Th2

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22 response to the disease severity in RSV bronchiolitis.

The study demonstrated that infants with HRV-bronchiolitis display a different acquired immunological response compared to infants with RSV bronchiolitis. 4. Raiden et al. (57) Prospective Cohort 12 Feb 2017 Argentin a 55 Total 35 RSV infected 20 Control RSV IFN-γ CD4+ CD8+

IL-2 and IFN-γ production by CD4+ cells was impaired by RSV infection.

CD4+ and CD8+ cells are permissive to RSV entry.

CD4+ cells expressing RSV antigens can be used to correlate disease severity. 5. Connors et al. (58) Prospective Cohort 30 Nov 2015 USA 54 Total 34 VRTI 20 non-infectious lung injury Multi ple Neutrophil s IL-6 IFN- γ CD4+ CD8+

Main cell pop. detected from aspiration were neutrophils in both study groups. Difference of (ANC) was higher in infected patients.

Airway CD3+ levels increased throughout the course of infection in infected sample while blood CD3+ were comparatively low.

Increased CD8+/CD4+ ratio in the airway of infected patients.

IL-6 and IFN- γ levels correlated with acute lung injury in infected patients. It detected an increased ratio of CD8+_to

CD4+ T-cells in the airways of virally infected children with acute lung injury compared to those infected without lung injury.

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23 6. Mariani et al. (59) Prospective Cohort 15 Oct 2017 USA 46 Total 23 Mild 23 Severe

RSV CD4+ Genetic expression of CD4+ cell activity was increased in severely ill patients. JAK/STAT pathway associated with clinical severity.

CD4+ cells were found to correlate with clinical severity. 7. Caballero et al. (60) Prospective Cohort 2 Feb 2015 Argentin a 418 Total 246 Severe 172 Mild (control) RSV IFN-γ IL-4 IL-6 IL-8 IL-1β TNF-α

IFN-γ were low in patients with severe disease while IL-4 levels were high. IL-4:IFN-γ ratio was high in severely ill patients when compared to the control. Levels of IL-6, IL-8, IL-1β, and TNF-α did not differ when compared between the two study groups.

n: number of patients; RSV: Respiratory Syncytial Virus; NK cell: Natural killer cell; UK: United Kingdom; Ig: Immunoglobulin; IFN- γ: interferon gamma; IL: interleukin; HRV: Human Rhinovirus; CD: Cluster of differentiation; Th1: T helper cell; ANC: absolute neutrophil count; TNF- α: tumor necrosis factor alpha; pop.: population.

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4. DISCUSSION

4.1 CD3+, CD4+, and CD8+ cells

A total of 4 studies included clusters of differentiation as their subject of study to relate to disease severity (56-59). Cluster of differentiation, or CD, is a type of nomenclature that is commonly given to certain groups of lymphocytes and/or leukocytes. The positive sign (+) that is in front of the nomenclature, CD, is to indicate whether or not that molecule, CD3 or CD4 for example, is present on the cell (61).

In a study by Fedele et al. (56), the difference between RSV and HRV induced bronchiolitis and the immune response to it was compared. It was found that RSV bronchiolitis held a mainly Th1 immune response (type 1 immune response). Researchers grouped their patients according to a clinical severity score, with 0 being the lowest and 8 the highest and subdivided into low and high severity groups (ones that scored less than 5 were the low risk low severity while more than 5 meant high risk high severity). Comparing the T-cell frequencies between the first and second group in RSV bronchiolitis patients yielded no difference in the CD3+ and CD4+ levels. They concluded statistically no significant difference in the T-cell subsets in low and high-risk patients and thus, no reliability in the use of CD4+ levels as a method to determine clinical severity. One limitation of this specific study is that the data of the T-cell frequencies for the 2 groups was not provided.

Another study by Raiden et al. (57) analyzed blood samples from 35 children mostly under the age of 2 with RSV bronchiolitis. 21 males and 14 females were recruited with average blood panel values of as follows: WBC: 12263 ± 6437 units/mm3 and lymphocytes = 30.8 ± 10.1% [mean ± SD]. To determine clinical severity in their RSV infected patients, they used the CDSS (clinical disease severity score), adapted in accordance with the Tal score. It was found that, of the infected patients, who mostly had a CDSS score >7 (severe), there were variable results in RSV+ cells in CD4+ and CD8+ T-cells ranging between 0-20% and averaging 2.36%. Almost all patients with more than 1% of RSV+ CD4+ T-cells reported in the study as being admitted to the ICU. They found a positive correlation between the clinical severity and the RSV+CD4+ T-cells. However, the positive correlation, as mentioned in the study, was primarily driven by patients with an RSV+ distribution among CD4+ cells of >1%. When these values were excluded, interestingly, a positive

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25 correlation was still found. This strengthens the study’s conclusion that RSV+CD4+ cells can be used as a marker to assess disease severity. No relation between CD8+ cells and disease severity was found. One possible limitation of this particular study is that only patients with severe RSV bronchiolitis were included and were compared to a control uninfected group. The question remains, how and if lymphocyte phenotype differed between mild or moderate RSV bronchiolitis. Connors et al. (58) attempted to compare CD4+ and CD8+ levels between the respiratory and circulatory systems and how they differ. They recruited 54 patients under the age of 4 that required mechanical ventilation; 34 of which were due to viral respiratory tract infections and the remaining 20 due to non-infectious causes. They found that in infected patients, when compared to non-infected patients, the CD3+ levels in the blood comprised more as a percentage of mononuclear cells, as shown in Table 3. Furthermore, CD3+ levels in the airways were lower in all samples as a percentage of total cellular composition.

Table 3. CD3+ T-cell levels, median and range, from blood and airway samples in two groups (infected and non-infected)

Blood Airways

Infected Non-infected Infected Non-infected

Median (%) 8.9 1.9 0.11 0.12

Range (%) 3.9–23.6 0.1–10.9 0.01-4.8 0.01-0.9

In both infected and non-infected patients, levels of CD4+ in the blood comprised around 80% while CD8+ was 20% with a ratio that is similarly low between both groups (median of 0.45 and range of 0.26–0.97 in infected group versus median of 0.45 and range of 0.17–1.9 in non-infected patients). However, in the airways, interestingly, the CD8+:CD4+ ratio was recognizably higher when compared to what was found in the blood samples, especially in the infected group. This parameter was analyzed every day to determine whether or not it increased. Table 4. on the following page shows the results obtained in this study.

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26 Table 4. Results obtained from airway samples comparing CD8+:CD4+ T-cell ratio between two groups (infected and non-infected)

Airways

Infected Non-infected

Median (%) 2.8 0.4

Range (%) 0.1–25.2 0.1–2.4

The results revealed that over the course of infection, airway samples from infected patients showed a significant increase in CD8+ cells and thus a CD8+:CD4+ ratio, when compared to the non-infected group, which showed a minimal increase of CD8+ cells throughout the period of time. This means that CD8+ cells have a major role in the respiratory tracts during viral infections. Further, patients with viral-induced acute lung injury (ALI) were compared to patients without ALI. ALI was considered as a measure of disease severity. Majority of the viral ALI were RSV induced. It was found that in ALI, CD8+:CD4+ ratio had a higher median peak when compared to those without ALI. Over the course of disease, the peak increased in ALI, while it did not, to a significant degree, change in those without ALI (median peak in ALI: 5.9 versus median peak in those without ALI: 0.8). The results showed that CD8+ cells not only respond to infection but also to the severity of infection as well as the viral agent (RSV). However, the study by Raiden et al. (57) showed that CD8+ cells did not pose a significant role in determining disease severity. These two results reveal contradictory values that must be investigated further.

Another study by Mariani et al. (59) focused mostly on genetic expression of intracellular signaling molecules and pathways that regulate CD4+ T-cell activity. It demonstrated that in RSV infected individuals, some genetic factors are upregulated causing CD4+ T-cells to change in accordance to disease severity. These particular genes that were associated with disease severity activated the JAK/STAT signaling pathway and GATA3 transcription factor. This, along with another signaling pathway, results in the early differentiation of Th1/Th2 and thus, an immune response. Aforementioned study by Fedele et al. (56) deducted that the immune response to RSV is mainly of type 1 (Th1). Similarly, Mariani et al. (59) indicated that the genetic changes, resulting in CD4+ activity changes too, are related to disease severity, particularly the GATA3 transcription factor; the STAT pathway was found to be statistically insignificant (P > .05). Knowing that the

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27 GATA3 transcription factor is needed for Th2 differentiation, when comparing the two studies, it is clear that there are some conflicting results; Fedele et al. (56) elaborated that RSV induced Th1 immune response while Mariani et al. (59) revealed, through genetic sequencing, that the most statistically significant (P = 1.62E-05) relation to disease severity is the GATA3 transcription factor (which differentiates Th2 cell, type 2 response, as previously mentioned). However, Fedele et al. (56) did mention that the more severe cases had a stronger type 2 immune response, rather than a type 1 response. Therefore, despite the contradictory results, there could be a relation built between the two studies. In conclusion, further investigation is needed to properly assess the relationship between RSV and the type of immune response it induces.

4.2 Protein Markers

A total of 5 studies included immune markers as a subject of study in their relation to disease severity (55-58,60).

Firstly, Green et al. (55) found negligible amounts of Th2 associated cytokines (IL-4, 13, and 17) in RSV infected children. However, they did find a strong IFN-γ producing T-cell response in the children that are 4-6-year-old with 41% of the patients showing elevated levels of IFN -γ. This is corresponding to Fedele’s study (56),with findings that suggest a more Th1 oriented immune (type 1) response in RSV bronchiolitis and predominantly IFN-γ, CD3+ and CD4+ T-cells. Both studies show that RSV induces a more Th1 oriented immune response with cytokines such as IL-4 (the common cytokine investigated between the two studies), not having a dominant and/or important role in the immune response towards RSV pathogen in children.

Another study by Raiden et al. (57), compared IFN-γ, IL-2, IL-5, and IL-17 production by CD4+ cells in RSV infected children. They found that IFN-γ and IL-2 production was inhibited during infection in RSV infected cells which could provide means of immune evasion for the virus, while IL-5 and IL-17 were not affected. This agrees partly with both Green’s (55) and Fedele’s (56) studies; both Green (55) and Raiden (57) agreed that IL-17 was not a dominant factor in the immune response, while Fedele (56) and Raiden (57) confirmed that IFN-γ has an important role and its inhibition helps RSV infected cells evade the immune system's mechanisms.

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28 A study performed by Connors et al. (58) analyzed IFN-γ, IL-4, IL-6, IL-10, IL-17, and TNF-α and their relation to severity by comparing between patients with ALI and those without. From all the cytokines investigated, only IL-6 showed a significant increase in patients with ALI. IFN-γ and TNF-α increased as well, but not to significantly different levels as compared to IL-6. This, when compared to the results of the aforementioned studies, argues that while IFN-γ plays an important role in fighting RSV infection, it does not play as a major role as other cytokines, possibly IL-6 for example, in assessing disease severity.

In contrast, Caballero et al. (60), examined IL-6, IL-8, TNF-α, and IL-1β and their different levels between mildly and severely ill patients, but no difference was observed. When comparing Connor’s study (58) with Caballeor’s (60), it is clear that a contradiction exists. Further investigation regarding this cytokine should be carried out to determine the true role of IL-6 in RSV infected patients.

Caballero’s study (60) further investigated IFN-γ in a Th1 mediated response and 4, IL-5, IL-9, IL-13 and IL-17 cytokines in a Th2 mediated response and their relation to disease severity. They found that IFN-γ was low in severely ill patients while IL-4 was inversely very high, with a high IL-4/IFN-γ ratio with severe RSV bronchiolitis when compared to mildly diseased patients. There was no difference in IL-9 and IL-13 between the 2 groups and IL-17 was not detectable. These results correspond to the findings of studies by Green and Raiden (55,57).

5. STUDY LIMITATIONS

This study has several limitations. To begin with, only 2 sources, PubMed and Google Scholar were used in finding articles for the purpose of this review. Moreover, only one individual carried out the research behind this thesis. This could potentially cause errors in overlooking essential articles that could have been implemented in this study. Needless to say, the use of multiple sources and multiple investigators in research could yield a more comprehensive review. Another limitation is the lack of statistical data provided by one of the studies, Fidele et al. (56) as the P value could not be found within the article provided.

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6. CONCLUSION

The aim of this study was to analyse the different cellular phenotypes of RSV infected pediatric patients and their possible relation to disease severity. The various phenotypes and immune markers included: CD3+, CD4+, CD8+, various interleukins and interferons, and some other markers. The results obtained were analyzed and compared to each other in order to view their relativity to RSV‘s effect on the immune system and the severity of the disease in these children.

There were various conflicting results. Some studies, such as Raiden‘s (57) concluded that CD4+ T-cells are positively correlating with disease sseverity while CD8+ T-cells are not. On the other hand, Connor‘s study (58) shows that while CD4+ cells rise with disease severity, CD8+ cells increase even more, as seen with an increased CD8+:CD4+ ratio throughout the course of disease. One study by Fedele et al. (56) indicated statistically insignificant relation between CD4+ cells and disease severity.

Most of the research groups focused on IFN-γ. Almost all the studies, excluding Connor‘s (58), identified IFN-γ to play a major role in disease and its relation to severity. Another mentionable marker was IL-4, which according to Caballero‘s findings (60), suggest that IL-4 has a role in disease severity; indicated with a high IL-4:IFN-γ ratio in severely ill patients. Two studies contradicted with findings on IL-6, in that Connor‘s study (58) concluded a positive correlation with disease severity and Caballeor‘s study (60) determined an insignificant relationship between IL-6 and disease severity.

In conclusion, it is clear that a lot of contradictory findings exist. Therfore, a more thorough, wide scale meta-analysis is required to properly assess which immune markers and cells can be used as indicators of disease severity in RSV infected pediatric patients. However, it should be noticed that following evidence-based guidelines, complete blood count is not performed in most of the cases of bronchiolitis. Thus, it could be a limitation for future studies. Furthermore, additional studies are needed to analyse and find possible non-invasive markers to predict bronciolitis severity and its outcomes.

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Immunophenotyping in RSV Infected Pediatric Patients: