The prevalence and phenotype of hepatitis B virus vaccine escape variants propagated by mother to child transmission in Indonesia.

Lead Research Organisation: University of Nottingham
Department Name: School of Medicine


Hepatitis B virus (HBV) infection is a major cause of ill health and death worldwide. HBV can lead to can lead to cirrhosis, liver failure and liver cancer, and kills more than 780,000 people every year. Across the world 350 million people are chronically infected with the virus, with the majority (75%) occurring in Asia. HBV is therefore a major health and economic burden in Indonesia.

Transmission of HBV is through contact with HBV-infected blood, and can occur during blood transfusions, needle sharing and during childbirth. The prevalence of HBV infection is particularly high in south-east Asia, where mother-to-child transmission (MTCT) is the most common route of infection. Infection with the virus during childhood increases the risk of developing a chronic infection and the chances of developing severe disease and understanding how the virus transmits between mother and infant is therefore of upmost importance. Breaking the chain of mother-to-child transmission is an essential part of the global strategy to limit HBV infections. In Indonesia, universal vaccination of newborns has been introduced to help prevent transmission, but infection remains common in pre-school and school age children and urgent review of strategies to prevent MTCT is required. Current diagnostic tests look for the presence of viral proteins in the blood. However, 'false negative' results can occur in these diagnostic tests. Testing for the presence of the viral genome in infected patients is a far more sensitive way of screening for potential infections. Cheaper, more sensitive tests will need to be deployed in resource-poor countries to enable better clinical intervention. Improved screening and targeted intervention in those cases that are likely to transmit virus to a newborn child would make a major contribution to reduced incidence of chronic infection.

In this research project we aim to investigate the factors involved in MTCT of HBV. The first objective of the project is to screen large numbers of mothers who are likely to be infected with the virus, utilising routine diagnostic tests and high-sensitivity assays that detect the HBV genome. We will then screen for the presence of new infection in infants, both at time of birth and nine months later. In those mother-infant pairs that demonstrate MTCT we will sequence the viral genome using state-of-the-art sequencing techniques to determine if specific types of the virus are transmitted. We will also analyse the properties of the transmitted virus variants and see if they are resistant to current vaccines. We will furthermore establish if specific clinical interventions could be introduced that prevent transmission of these variants.

This research will determine if the current clinical tests do not detect all of the transmission events, and if there are better screening techniques that will permit targeted clinical intervention. The research findings have the potential to demonstrate if there are specific viral determinants of MTCT in areas of high prevalence of HBV infection, and if there are variants which escape vaccination. Taken together, this information has potential to improve the global healthcare management of HBV.

Technical Summary

In order to assess the prevalence of HBsAg positive and HBsAg negative infections, we will screen 4000 maternal serum samples by direct PCR and HBsAg assay. Genotype will be established using routine sequencing methodology and HBV DNA titre estimated using quantitative PCR. Samples which are HBsAg negative and PCR positive will identify occult HBV infection, including candidate vaccine escape mutants. For those women and children in whom it is not possible to store serum, we will use dried blood spots (DBS) as a stable source of DNA.

In order to estimate rates of incident infection and MTCT in the study cohort children we will analyse two sets of samples from children. i) Cord blood samples of mothers found to be HBV positive ii) serum samples taken from all children born to mothers in the study group at between 9 and 12 months of age, analysed by both PCR and HBsAg assays. The purpose of this collection is to identify all children with incident HBV infection. HBV positive samples will stored for further analysis using NGS.

In order to detect, analyse and compare viral diversity in samples from HBV positive mother and child pairs using NGS we will i) conduct in depth sequence analysis of the viral population in mother and child by NGS ii) identify sequence diversity in coding and non-coding regions in HBsAg, including analysis of potential vaccine escape mutants iv) compare viral variants detected by NGS in mother and child to interrogate selection events operating on MTCT

To assess the phenotype of candidate vaccine escape mutants we will clone the majority HBsAg sequence from patient sera and use the results of deep sequencing to inform a strategy of site-directed mutagenesis. The impact of genetic variation on viral phenotype will be assessed in established receptor binding and neutralisation assays. We also plan the use of lentiviral pseudoviruses expressing patient derived HBVsAgs to quantify infectivity in cell lines and primary human hepatocytes

Planned Impact

More than 350 million individuals are chronically infected with Hepatitis B Virus (HBV), with an estimated 4.5 million incident infections annually. The consequences of persistent infection with HBV include cirrhosis, end stage liver disease and hepatocellular carcinoma (HCC) and result in more than 780,000 deaths each year. Seventy-five percent of all chronic HBV infections occur in endemic areas in the Asia-Pacific region. HBV prevalence rates in Indonesia are high with marked regional variation, ranging from 4% in Jakarta to 33% in North Sulawesi, and carry a high economic and social burden

Vertical transmission from mother to child (MTCT) is the most common route for acquisition of the virus worldwide and can be prevented by immunoprophylaxis. HBV vaccination is a core component of the WHO hepatitis B control programme and is an essential part of infant immunisation programmes worldwide. Effective prophylaxis also requires the selective use of passive immunisation with HBV specific immunoglobulin together with the use of anti-viral therapy in mothers with high viral loads. MTCT, however, remains a major route for the propagation of HBV even in the presence of established regimens for immunoprophylaxis and HBV remains endemic in pre-school and school age children in Indonesia. HbsAg negative viruses selected by vaccination are an emerging risk. Understanding the mechanisms and consequences of vaccine failure as a basis for new HBV prevention strategies is therefore a major scientific and public health challenge worldwide, particularly in low and middle income countries.

Our study will enable us to provide answers to key questions of enormous importance to clinical services and Public Health in Indonesia:

i) is MTCT an important cause of incident HBV infection in early life
ii) what is the frequency of HBsAg negative infections in a large cohort of antenatal women in Indonesia
iii) what is the frequency of transmission of HBsAg positive and HBsAg viruses from mother to child
ii) what selection events operate on MTCT and are HBsAg negative/vaccine escape mutants an important cause of incident infections in the new borne
iv) is it possible to identify mothers in whom, either because of high viral load or the presence of pre-existing escape mutants, alternative strategies for prevention of MTCT will be required i.e. early maternal anti-viral therapy in addition to routine infant vaccination.

The knowledge generated from this project will inform the development of new diagnostic tools to detect occult HBV infection in Indonesian populations, and impact on the Public Health policies and the design of new vaccines for the effective prevention of MTCT. These initiatives have the potential to reduce incident HBV infection in and reduce the health and economic burden of HBV infection in Indonesia. The project will also advance our understanding of the virological and immunological events which together set the balance between immune escape and fitness to replicate which may be a critical driver of HBV infection in the era of universal vaccination.


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Title High through put technology for next generation sequencing and viral phenotyping 
Description 1. Establishment of a robust sequencing platform for assessing sequence diversity in HBV infections utilising nanopore sequencing, making use of locally available samples. We have determined the sensitivity of this technique and validated the ability of this approach to detect the presence of variant HBV populations. This has revealed that DNA extracts from HBV-infected sera can harbour surprisingly diverse populations of viruses (manuscript in preparation) in patients with high viral loads, such as those in the mother population investigated in this project. 2. Successful development of methodologies for recovering NGS datasets from dried blood spots. We have further developed methods that, for the first time, enable recovery of NGS datasets from samples stored as dried blood spots, utilising the Oxford Nanopore Technologies (ONT) MinION sequencer (manuscript submitted). These datasets are essential for assessing the genome sequences of viruses present following transmission of HBV in our MTCT cohort, permitting sampling of neonates using microlitre quantities of blood. 3. Deployment of nanopore sequencing technologies in an Indonesian healthcare setting. Having developed the necessary technology and bioinformatics pipelines to interrogate the genomic sequences of HBV in patient samples using the ONT MinION platform, we deployed staff, mobile equipment and technology at the University Hospital Hasanuddin, Makassar, Indonesia. We have used these approaches and technology to sequence HBV populations in high viral titre samples taken from HBV-infected mothers, providing preliminary data on our study cohort. In order to inform and facilitate our subsequent interrogation of Indonesian samples, we plan to analyse samples of MTCT of HBV in the UK (provided by PHE). This cohort will provide data on sequence motifs associated with MTCT, and a framework for the analysis and comparison of Indonesian samples. 4. Development of phenotyping techniques to assess the phenotype of HBV surface antigen (HBsAg) variants. Utilising retroviruses pseudotyped with the HBsAg clones isolated from our patients, we have established assays for high-throughput assessment of the entry phenotype of different viral genotypes relevant to the phenotyping HBV populations infecting the Indonesian population. This has permitted assessment of the requirement of entry receptors of viral variants (manuscript in preparation). We have also demonstrated the ability of HBV hyperimmune globulin to neutralize entry of HBV variants using this assay. This will be essential to assessing the neutralizing potency of antibodies present in vaccine recipients, and assessment of the ability of HBV variants to escape the neutralizing antibody response generated in neonates receiving the Indonesian HBV vaccine. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact We are currently preparing manuscripts decribing the technology we have developed, which can be used in resource poor settings to capture basic and molecular virological data and Nanopore sequencing which can be conducted in situ. 
Description Reducing the health and economic burden of communicable and non-communicable liver disease in Indonesia 
Organisation Eijkman Institute
Country Indonesia 
Sector Public 
PI Contribution Sharing knowledge, experience and technology through regular teleconferences, free exchange of knowledge and visits by scientific staff. The Nottingham PI has visited the Eijkman Institute and met with the Director and senior academic staff aligned with the current and planned future projects. We have worked in collaboration to develop research strategies and contributed to formation of a high level clinical research network in Indonesia with the purpose of addressing the burden of both communicable and non-communicable liver disease.
Collaborator Contribution Contributed in equal partnership to the devlopment of further research programmes and driven formation of a nation wide clinical network centred on chronic liver disease, which is a major health burden in Indonesia. The Eijkman have also contributed expertise in molecular biology and informatics to the current MRC project and will expand this contribution in any future joiunt enterprise.
Impact This is a multi-disciplinary partnership which is currently the basis for an application to the forthcoming MRC Applied Global Health Research Board.
Start Year 2018