Next-Generation Sequencing of the Intrahepatic Antibody Repertoire Delineates a Unique B-Cell Response in HBV-Associated Acute Liver Failure

Abstract

Hepatitis B virus (HBV) is a major cause of acute liver failure (ALF) worldwide. While liver damage in classic acute hepatitis B is believed to be T-cell mediated, the pathogenesis of HBV-associated associated ALF remains largely unknown. Access to liver specimens from well-characterized patients with HBV-associated ALF provided us with the opportunity to perform next-generation sequencing (NGS) of the entire VH repertoires of IgM and IgG from the livers of four ALF patients, a control liver donor and a patient with chronic HBV infection.We found that ALF is not associated with expansion of specific B-cell lineages. However, NGS showed that the intrahepatic VH repertoires from ALF patients were characterized by the abundant presence of antibodies in germline configuration in contrast to their marginal prevalence in controls. Moreover, NGS identified a large number of VH genes in germline configuration with identical VDJ sequences in the IgM and IgG repertoires in all four ALF patients, indicating that isotype switch from IgM to IgG had occurred without somatic hypermutation.The results of this study indicate that the presence of intrahepatic antibodies in unmutated germline configuration is a broad phenomenon in the global antibody repertoire generated from total RNA derived from whole liver tissue that is strongly associated with ALF, suggesting a major role of T cell-independent humoral immunity in the pathogenesis of ALF.

1. INTRODUCTION

Infection with hepatitis B virus (HBV) causes a wide spectrum of liver diseases ranging from acute self-limited hepatitis to acute liver failure (ALF, also known as fulminant hepatitis), chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HBV-associated ALF, which results in death or liver transplantation in up to 80% of the cases, occurs in approximately 1% of patients with acute HBV infection.¹ The pathogenesis of ALF remains largely unknown although some studies have suggested an association with pre-core or basal-core mutant virus strains with enhanced replicative capacity^2,3 and/or an exuberant immune response of the host.^4,5 Most studies on the pathogenesis of ALF have been based on samples derived from serum rather than liver, which is the anatomical site where the disease occurs.

Our recent comprehensive studyon liver specimens obtained from four well-characterized cases of ALF identified several host and viral factors that wereuniquely associated with the development of ALF.⁵ By phage display libraries we documented massive intrahepatic production of unmutated, germline antibodies with high affinity to hepatitis B core antigen (HBcAg) in all ALF cases. Moreover, we showed that these intrahepatic anti-HBc antibodies can bind HBcAg on the cell surface and trigger complement-mediated cell lysis, which may lead to massive liver necrosis.⁵

In the present study, the entire intrahepatic antibody repertoires from four patients with HBV-associated ALF were characterized by next-generation sequencing (NGS) and the data were compared with the repertoires from the liver of a healthy liver donor and a patient with chronic HBV infection. The results of this study showed that a unique humoral immune response is associated with the pathogenesis of HBV-related ALF.

2. METHODS

2.1. Patients

We examined liver specimensobtained from four patients with well-documented HBV-associated ALF, whose clinical, serologic, virologic and histopathologic features were previously reported (supplemental Table 1).⁵ None of them had a history of intravenousdrug addiction, alcohol abuse or evidence of coinfection with HCV or HDV.⁶ In addition, we studied one liver donor (LD) with histologicallydocumented normal liver, and one patient with chronic HBV infection (CHB). Written informed consent was obtained from each patient or the next of kin. Our study was approved by the Review Board of the Hospital Brotzu, Cagliari, Italy, and by the NIH Office of Human Subjects Research, granted on the condition that all samples were de-identified.

bioinformatics analysis of Illumina paired-end sequencing

To amplify genes coding for Fd (VH + CH1) by PCR, we used a set of 5’-end VH family specific primers and 3’-end IgM-or IgG1-specific CH1 primers that we previously used in the construction of the phage display library.⁷ NGS of IgM and IgG from liver RNA extracted from four ALF patients 241, 31, 32, 219, and a liver donor with histologically normal liver (donor 37) were downloaded from the database (accession number SRP126953) we previously deposited.⁵ In addition, we generated 2 amplicons (IgG and IgM) from a patient with chronic HBV infection (Patient 7). The amplicons were purified and subjected to Illumina 2X300 bp paired end sequencing and then analyzed. Next-generation sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) (accession number PRJNA422423).

2.3. NGS data analysis

Data analysis, including the new additional control represented by a patient with chronic hepatitis B, were extensively reanalyzed by using Antibodyomics1 pipeline to determine:1) the entire VH IgM and IgG repertoires detected in the liver; 2) the frequency of somatic hypermutation; and 3) the comparison of the intrahepatic global VH IgM and IgG repertoires with the repertoire obtained from antibodies specific to HBcAg,⁵ cloned from the liver by phage display technologies. Paired-end Illumina sequencing reads were merged using FLASH (Fast Length Adjustment of Short reads).⁸ Primer sequences were removed using FLEXBAR with a 0.3 threshod.⁹ The Stand-alone IgBLAST¹⁰ running on the NIH HPC Biowulf Linux cluster (https://hpc.nih.gov/) was used for V(D)J germline gene assignments and an in-house developed python script was applied to process IgBLAST outputs.¹¹ Non-Ig reads, non-duplicate and non-production reads were filtered out. Phred scores of less than 20 occurring over 80% of the V(D)J region were also discarded from all NGS samples. Only sequences assigned FR1-FR4 were retained in the data set. Unique antibody sequences were extracted from reads passing the above filters and were subjected to further analysis. Unique sequences were parsed into clones, which means that the clone antibody had sequences that have identical amino acids on CDR H3 and use the same V, J germline gene.¹² Antibody sequences with equal to or fewer than 3 mutations were considered to be in germline configuration. The first nucleotide to the second conserved cysteine codon of the V region was used to evaluate somatic hypermutations. Antibody sequences shared between IgG and IgM were evaluated at the V(D)J region.

3. RESULTS

Total RNA was isolated from the livers of 4 HBV-ALF cases, one liver donor and one individual with chronic hepatitis B, and the heavy-chain Fd fragments (VH+CH1) of IgM and IgG were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). The ampliconswere sequenced using the Illumina MiSeq platform, which revealedan average of 10⁷ raw reads and 2–5×10⁵ unique VDJ sequences for each sample after data processing (supplemental Table 2). Both the IgM and IgG repertoires were highly diverse in all cases studied, being composed of 51 functionalVH genes (Figure 1A). The VH IgM and IgG repertoires detected in the liver were compared with the repertoires obtained from peripheral blood B cells of 10 healthy donors in a recently reported study.¹³ This comparison showed that the VH gene expression pattern in the liver resembles that observed in peripheral blood B cells from healthy donors (supplemental Figure1). Although V and J gene usage in the liver didnot clearly separate ALF patients from controls, there was a trend toward an increase in VH gene 3-30-3 usage in both the IgM and IgG repertoires derived from ALF patients (Figure 1 A–B). In line with recent data from human peripheral blood IgG repertoires,¹³ JH4 was the most frequently used J gene in both ALF and controls (Figure 1B). Interestingly, VH3-30-3 and JH4 were also the most represented genes among HBcAg-specific Fabs isolated from the liver of the same ALF patients by phage display libraries.⁵ Thus, it seems that ALF is not associated with expansion of a specific lineage of B-cell populations. Similarly, the comparison of VH repertoires between intrahepatic and peripheral blood B cells showed that the VH gene expression pattern in the liver resembles that observed in circulating B cells from healthy donors.

FIGURE1. Characteristics of antibody (IgM and IgG) repertoires in the livers of four patients with HBV-associated acute liver failure (ALF, 241, 31, 32 and 219), a normal liver donor (LD, 37), and a patient with chronic hepatitis B (CHB, 7).

(A) V gene usage by individual subjects. Upper panel indicates IgM and lower panel indicates IgG. Panel A includes data previously published in reference 5. (B) J gene usage by individual subjects. Upper panel indicates IgM and lower panel indicates IgG. Increased color intensity indicates higher frequency of gene usage. Subjects are colored as in panel C. (C) HCDR3 length distribution of IgM and IgG for each subject. (D) V gene somatic mutations in IgM and IgG for each subject. V genes with 0–3 nucleotide substitutions are defined as unmutated antibodies in germline configuration.Numbers of VH genes in each mutation category, calculated as percentage of total VH genes analyzed, are shown in different shades of blue within the pies.

Analysis of HCDR3 length distribution showed an overall similar pattern in both IgM and IgG with the major peak around 15 aa, although an additional narrow peak of 25 aa was detected in one ALF patient (219) and one of 23 aa in the CHBpatient (Figure1C).

Striking differences in the frequency of somatic hypermutation of the VH genes were detected between ALF patients and controls (normal liver donorand CHB patient). In both controlsonly 4% of V genes in the IgM repertoire and 1% in the IgG repertoire were in germline configuration. In contrast, in all four patients with ALF 20–54% of V genes in the IgM repertoires and 20–61% of V genes in the IgG repertoireswere in unmutated, germline configuration (Figure1D). Thus, NGS confirmed the presence and abundance of antibodies in germline configuration in ALF in contrast to their marginal prevalence in controls, consistent with our previous finding of specific anti-HBc Fabsisolated from the same ALF patientsby phage display libraries.⁵ Moreover, a large number of VH genes (>6,000) in germline configuration with identical VDJ sequences in the IgM and IgG repertoires were identified in all four ALF patients, indicating that isotype switch from IgM to IgGhad occurred despite the lack of affinity maturation. In contrast, only 30 or feweridentical VDJ sequences in IgM and IgG were found in controls (LD and CHB) (Table 1).

Table 1.

Number of shared germline-like heavy chain sequences in the IgG and IgM repertoires

IgM╲IgG	7	37	241	31	219	32
7	24*	22	40	22	35	23
37	46	30	162	182	881	515
241	177	70	50,542	480	636	239
31	33	20	282	12,732	460	1127
219	40	24	226	237	6,629	888
32	43	26	184	692	1,608	51,963

^*Numbers within green bars indicate identical VDJ sequences in germline configuration shared by IgM and IgG repertoires for each subject

4. DISCUSSION

Most studies on the pathogenesis of HBV-associated ALF have been based on samples derived from serum rather than liver, which is the anatomical site where the disease occurs. Moreover, the liver harbors one of the largest populations of immune cells in the body, which can be rapidly and dramatically altered as a consequence of inflammation and disease.¹⁴ Our in-depth analysis of the intrahepatic IgM and IgG VH repertoires associated with ALF using NGS showed striking differences in the frequency of somatic hypermutation, shedding new insights on the unique B-cell immune response against HBV in ALF that may have implications for the pathogenesis of this disease, which is one of the most dramatic clinical syndromes in medicine.¹ The major findings from this study are that an unusually large proportion of intrahepatic VH genes from both the IgM and IgG repertoires of ALF patients lack somatic hypermutation. Furthermore, many unmutated, identical VH genes were found in both the IgM and IgG repertoires of ALF patients, indicating that isotype switch occurred without somatic hypermutation. These two features are apparently linked together and exclusively associated with ALF because each of the four ALF patients studied exhibited the same pattern, which is profoundly different from the findings in control individuals, including a liver donor negative for HBV markers and a patient with chronic HBV infection. These results highlight the role of the humoral immunity in one of the most dramatic clinical syndromes affecting the liver, suggesting the importance of extending the study of the intrahepatic antibody repertoire of a large number of patients with chronic hepatitis B as well as in acute liver failure during an exacerbation of chronic HBV infectionby using phage display library and NGS technologies.

It is well known that Ig isotype switch and somatic hypermutation of V genes take place in the germinal center and require T cell help.¹⁵ Therefore, the lack of somatic hypermutation of V genes in ALF could denote that B-cell immunity develops in a T-cell independent manner. The swift activation of B-cell genes that we documented hereinis in line with the extremely rapid clinical course of ALF, which leads to death or liver transplantation within a few days of disease onset. The hypothesis of a T-cell independent humoral response as a key factor in ALF pathogenesis is further corroborated by the upregulation of negative regulators of T-cell immunity, such as CTLA4, that we recently described in the liver of ALF patients.⁵ T-cell independent antibody responses characterized by isotype switch without somatic hypermutation have been reported in other settings, including viral infections.^15,16 Although the role of a delayed or impaired T-cell response in ALF remains to be elucidated, it is plausible that it might play a role in the pathogenesis of this disease.

Our previous study based on phage display technology demonstrated that antibodies cloned from the liver of ALF patients targeted exclusively a single viral antigen, the HBcAg, with subnanomolar affinity, indicating that HBcAg is the main antigenic target of the extensive intrahepatic B-cell response documented in ALF. Strikingly, the vast majority of these anti-HBc antibodies were in germline configuration, with several pairs of IgM and IgG in each patient sharing identical variable regions, indicating that isotype switch had occurred without affinity maturation,⁵ a characteristic of T-cell-independent B-cell response. In the present study we used NGS technology to examine the global liver IgM and IgG repertoires generated from total RNA derived from whole liver tissue. Our results demonstrated that a high prevalence of germline antibodies with IgM to IgG switch without somatic hypermutations is a broad phenomenon typically associated with ALF. Thus, this study reinforces and expands our prior findings, providing further evidence that a robust T-cell independent B-cell response appears to be a hallmark of HBV-associated ALF.