Correlation of IL-15 with viral load of hepatitis B and its role in the clearance of the virus

Introduction and Aim: Hepatitis B virus (HBV) causes hepatitis B infection resulting in acute and chronic liver damage . Diagnosis of HBV involves blood tests that can distinguish acute and chronic infections . Molecular tests are also available that aid in viral detection and quantification. HBV infected individuals exhibit increasing levels of cytokines throughout the progress of infection which is thought to play an important role in the virus clearance process. Hence in this study we aim to evaluate the role of IL-15 in clearance of hepatitis B virus in acute and chronic infection. Materials and Methods: This cross-sectional hospital-based investigation conducted in Baghdad, Iraq comprised 34 patients with HBV infection and 30 apparently healthy individuals aged between 20-75 years. Blood drawn from each individual was subjected to ELISA and RT-PCR assays for detection of the virus. The serum obtained was assayed for IL-15 levels. Statistical analysis was performed on the data obtained. Results: HBsAg was detected in all acute hepatitis B patients. The study revealed that 85.29 % of acute hepatitis B patients with HBsAg by ELISA were positive by RT-PCR. The viral load was highest in patients aged 40-49 years. Patients with acute hepatitis B but detected negative by RT-PCR had the highest mean IL-15 level (22.17 pg/ml) compared to patients positive by RT-PCR and the control group (11.16 and 4.21 pg/ml, respectively). A strong negative association (R = -0.8657) existed between IL-15 level and viral load in patients with acute hepatitis B. Conclusion: High levels of IL-15 are associated with the HBV clearance in patients with acute hepatitis.


INTRODUCTION
iral hepatitis B diagnosis has progressed from simple HBsAg detection to more advanced antibody detection of particular viral proteins, and finally to viral DNA detection and quantification (1).The application of HBV DNA quantification techniques that are ever more sensitive has been of tremendous assistance in both the diagnosis and management of disease (2).The evaluation of viral load is critical throughout therapy because the majority of guidelines mention that one of the primary therapeutic goals is to suppress HBV replication (3).Thus, the protective immunological response to HBsAg in conjunction with the creation of a neutralizing antibody specific to the antigen is critical for its treatment (4).
Interleukin-15 (IL-15) is a powerful stimulatory cytokine that plays a crucial role in preserving the homeostasis of lymphocytes as well as their activity.It plays a part in the activation, proliferation, and differentiation of CD8+ T cells, NK cells, and CD4+ T cells, among other actions involving these cell types (5).It has been observed that IL-15 is capable of resurrecting tolerant CD8+ T cells used in adoptive immunotherapy of established tumors.Additionally, when IL-15 is coupled with retinoic acid, it is able to abrogate tolerance to dietary antigens.Both of these effects are dependent on the presence of IL-15 (6).A recent study has demonstrated that an elevated amount of IL-15 in the liver can initiate an anti-HBV response, probably by functioning as a mediator in the production of IFN- (7).Hence, in this study we aimed to investigate the function of the IL-15 cytokine in the elimination of the hepatitis B virus during an acute infection.

MATERIALS AND METHODS
This hospital-based cross-sectional study was undertaken during October 2019 to February 2020, in Baghdad, Iraq.Healthy individuals (controls n=30) and patients (n=34) with acute hepatitis B ranging in age from 20 to 75 years old were included in the study.Venous blood (5 ml) was drawn from each participant and distributed into two tubes, one containing anticoagulant EDTA and other without EDTA.The tubes were centrifuged at 3000 rpm for 15 minutes at 37oC.The plasma from the first tube was aspirated using a micropipette, transferred to Eppendorf tubes, and stored at -20 o C for further molecular tests.Serum obtained from the second tube was also stored at -20 o C and used in determination of the IL-15 levels.

V
Isolation and quantification of HBV-DNA HBV genomic DNA was isolated using the The ZR viral DNA kit TM (Zymo Research, Germany).The HBV DNA extracted from plasma was subjected to quantitative detection of Hepatitis B virus and the simultaneous detection of a HBV-specific Internal Control (HBV-IC) using the real-time PCR assay kit (HBV Real-TM Quant, Italy).The presence of quantitative HBV IC allows for checking possible PCR inhibition and DNA loss during the extraction procedure, enabling the precise calculation of the viral load.The protocol used was based on the manufacturer's instructions with detection based on fluorescent reporter dye probes specific for HBV or HBV IC.

Determination of cytokine IL-15 level
The cytokine IL-15 was detected in serum using the sandwich ELISA assay.The assay was using an antibody pre-coated 96 well microplates as per the manufacturer's instructions (Koma Bioteck, USA).

Statistical analysis
Statistical analysis was performed using the Minitab version 17 statistic program.Comparison was carried out using chi-square (X 2 ), t-test probability, and analysis of variance (ANOVA) and interpreted as P value > 0.01 : Highly significant (HS); 0.1 ≤ (P value) ≤ 0.5: significant (S); and P value> 0.05 : nonsignificat (NS).

RESULTS
In this study, all patients tested were diagnosed with acute hepatitis B and observed to possess different serological markers (Table 1).Serologically all patients were positive for anti-HBc IgM and HBsAg.None of the samples was positive for anti-HBs Ab (Table 1).Results also showed that 85.29 % of acute hepatitis B patients who tested positive for HBsAg by ELISA also tested positive for HBV by RT-PCR which was significant in comparison (Table 2).The viral load in each of the acute hepatitis B patients was estimated and has been presented in Table 3.According to Table 3, 47.06% of patients diagnosed with acute hepatitis B had viral loads that ranged from 100,000 to 1,000,000 IU/ml.The RT-PCR assay used to detect HBV in all 34 subjects revealed that only 29 were positive for the virus (Table 5).Interleukin-15 levels were also measured in all subjects, and the mean+SD IL-15 levels in PCR-positive HBV patients was 11.16+4.1.Participants who were negative for HBV by PCR assay had a higher mean IL-15 level (Table 5), a comparison of which was found statistically significant (Table 5).Further, a graphical plot for the hepatitis B viral load and the IL-15 level detected in each participant showed an inverse association (R value = -0.8657) to exist between the two (Fig. 1).

DISCUSSION
Although the presence of HBsAg in the blood signifies the onset of an acute hepatitis B infection and active replication of the virus, there may be mutant strains of HBV that could replicate without producing HBsAg (1).Elimination of HBsAg in individuals with persistent HBV infection is common, estimated to be between 0.5 and 1%, and not always associated with the production of new anti-HBs (8).Studies show that anti-HBc IgM may be possibly detectable for up to two years after an acute infection, while in cases of chronic infections the titer of anti-HBc IgM may rise to detectable levels sooner (9).Recently have seen an increase applied of polymerase chain reaction (PCR) in the diagnosis of numerous diseases, including CML (10), Adenocarcinoma (11), and dangerous microorganisms (12) nevertheless, there is a possibility studies report RT-PCR assay as an important diagnostic tool in detecting HBV (13).However, in this study it was seen that all samples that tested HBsAg positive by the ELISA test, were not consistent in RT-PCR assay.Only 85.29% of samples were seen to be positive for HBV by RT-PCR assay in contrast to 1005 positive by HBsAg ELISA test; it is the same appearance in detection of SARS-Cov-2 ( 14).This probably could be attributed to low or undetectable levels of DNA in the samples, and the infection considered as 'inactive' (15).
Quantitative serum HBV DNA levels during different stages of chronic hepatitis B infection has been studied and it has been shown that a viral load of >20,000 IU/ml is indicative of an infection (16).
In this study, a viral load of 10 4 to >10 6 could be detected, with the highest load being prevalent among patients in the age group 40-49 years.This result assumes significance as the risk of developing hepatocellular carcinoma and liver cirrhosis associated in patients with hepatitis B infection has been shown to increase proportionately with increasing viral load beginning at 10 4 copies/ml (17).Studies pertaining to the age of HBV patients with the highest risk of infection were shown to be 41-60 years and 40-49 years (18).
Patients infected with HBV have higher levels of a number of cytokines and chemokines throughout the duration of the infection.These cytokines and chemokines include interleukins, proinflammatory cytokines, IL-10, IL-15, and IL-8 (6).In this study, the hepatitis B viral load was observed to be inversely proportional to the IL-15 levels.Our findings also agree with an earlier study, wherein IL-15 level was reported to dramatically increase during HBV infection (19).Yin et al., (20) reported that an elevated level of IL-15 production in the liver suppresses HBV replication via IFN-β production while, Cheng et al., (21)  independently of the widespread IL-2 receptor ( 22).An IL-2 dependent murine cell line's ability to proliferate was used to evaluate the biologically active form of IL-15's ability to sustain cell division (23).Enhanced proliferation, survival, and differentiation of cell types, such as NK, T, and B cells are some of the significant functions that IL-15 and IL-2 have in common.This is one of the unique characteristics of IL-15, which also has these important functions with IL-2 (24).It has been hypothesized that this contradictory function of IL-15 may make it possible for a rapid development of a pro-inflammatory response to a pathogen while at the same time allowing for the generation of TREG cells that are ready to bring about a reduction in inflammation once the infection has been eradicated (25).

CONCLUSION
High levels of IL-15 were associated with the clearance of HBV in patients with acute hepatitis, and the highest percentage of patients with acute hepatitis B had HBsAg as detected by ELISA were positive by RT-PCR.

Fig. 1 :
Fig. 1: Graph showing the association between Hepatitis B viral load and the IL-15 level

Table 1 :
Frequency of HBV serological markers in patients with acute hepatitis B

Table 2 :
Samples tested positive for HBV by HBsAg ELISA and RT-PCR assay

Table 3 :
Classification of patients based on hepatitis B viral load An analysis for age and hepatitis B viral load in patients studied, showed the highest load to be prevalent among the age group 40-49 years in comparison to HBV patients in other age groups (Table 4).Similarly, patients in the age group 60-70 years had the lowest level of viral load.

Table 4 :
Comparison of age and viral load in patients diagnosed with acute hepatitis B

Table 5 :
Interleukin-15 level in patients and control tested positive and negative by PCR assay