As June wraps up Pride Month, it is still important to address LGBTQ+ health and risk factors for hepatitis B. Many resources are available regarding gay and bisexual men’s risk factors for hepatitis B, but information discussing lesbian, bisexual women, and transgender folx for hepatitis B is lacking.
Gay, bisexual, and men who have sex with men (MSM) have a higher chance of getting hepatitis B. It can be spread through body fluids like semen or blood from an infected person to an uninfected person during unprotected sex.
A research study found that lesbian, bisexual women, and womxn who have sex with womxn (WSW) had significantly higher rates of hepatitis B than the control group due to risk factors like multiple sexual partners, injection drug use, and sex work1. Additionally, potential mothers need to know their hepatitis B status because it can easily transmit from mother-to-child during childbirth.
Being transgender is not a risk factor for hepatitis B (HBV), but some transgender folx may have a higher risk due to discrimination surrounding their gender identity. Discrimination in workplaces or health care facilities can lead transgender individuals to engage in risky behaviors like sex work and exposure to unsterile needles which can put some transgender individuals more at risk than others2. While there is insufficient information regarding hepatitis B and transgender folx, much information exists about hepatitis C (HCV) and its co-infection with hepatitis B. Since both viruses have similar modes of transmission it is not uncommon for someone to be co-infected with HCV and HBV. It is important to get tested for HBV because hepatitis C can become a dominant liver disease which leaves HBV levels virtually undetectable and can cause further liver damage if hepatitis B is not addressed3. This is especially true for individuals being treated with hepatitis C curative Direct Acting Antivirals (DAAs), which can lead to hep B reactivation.
For LGBTQ+ individuals living in the United States and who want to know their hepatitis B status, here is a list of LGBTQ+ friendly healthcare providers. If you identify as LGBTQ+, ask your provider to be tested for hepatitis B today. The great news is that if you are not infected, there is a safe and effective vaccine that can prevent you from getting hepatitis B in the future!
On the other side; healthcare professionals have a duty to provide culturally competent care to LGBTQ+ individuals and encourage hepatitis B testing and vaccinations. The Centers for Disease Control and Prevention (CDC) has recommendations and guidelines for health professionals here.
Citations:
Fethers, K., Marks, C., Mindel, A., & Estcourt, C. S. (2000). Sexually transmitted infections and risk behaviours in women who have sex with women. Sexually transmitted infections, 76(5), 345–349.https://doi.org/10.1136/sti.76.5.345
June is Pride Month! As we celebrate our differences and recognize the rights of the LQBTQ+ community, it is important to highlight the health disparities that they face, and ways to overcome such difficulties. To help spread awareness about the impacts of hepatitis B within this group, we‘ve interviewed Thaddeus Pham, Co-Director of Hep Free Hawaii!
Hi Thaddeus! So to start off with, can you tell us a little about who you are, and why this topic is important to you?
Thanks, Michaela! Well, I am currently the Co-Director for Hep Free Hawaii, our statewide coalition dedicated to eliminating hepatitis and related harms on our islands. I am also the Viral Hepatitis Prevention Coordinator for the Hawaii State Department of Health, although most folks in the community have also dubbed me the “Queen of Hepatitis” hahaha.
This work is important to me personally because I am a cisgender, gay man whose parents were born in Vietnam. As such, my work in public health has always been informed by the intersection of multiple identities, in this case, people who are LGBTQ+ and also foreign-born Asians or Pacific Islanders. That’s why I was so excited you asked me to chat about the impact of hepatitis B on gay and bisexual men.
As the Queen of Hepatitis, can you explain more about hepatitis B & how it impacts members of the LGBTQ+ community?
Sure thing! First of all, hepatitis B is a highly infectious blood-borne disease that can be transmitted through sex, injection drug use, and from mother-to-child during childbirth. The hepatitis B virus attacks the liver and causes an acute (short-term) or chronic (life-long) infection. If untreated, it can lead to liver disease, liver cancer, and even death.
In the United States, gay and bisexual men are at high risk for hepatitis B infection, usually through sex. According to the CDC, about 20% of new hepatitis B infections occur among this community. Think about that: 1 out of 5 new cases of hep B is a gay or bisexual man. To my community, I say: get tested!
Good point! What are some additional reasons to get tested for hepatitis B?
Hepatitis B is called a “silent infection”; there are usually no symptoms until it gets pretty bad (e.g., serious liver damage or even liver cancer). Liver damage can be happening even if you don’t notice any symptoms. Also, the virus can be spread even if you are asymptomatic. Testing is the only way to know for sure that you are not living with hepatitis B.
Co-infections with hepatitis B can be dangerous. People living with hepatitisC and HIV/AIDS are at higher risk of contracting hepatitis B, and will also suffer more serious complications. Even a co-infection with hepatitis A, which is short-term, can cause liver damage. Knowing your hepatitis B status can help your healthcare providers treat you properly and lower your risk of liver disease and liver cancer!
Acute infections can have future consequences. About 90% of hepatitis B infections in adults are acute. This means that your body will recover from the virus in 6 months or less. The virus will no longer be in your bloodstream, but it will be “sleeping” in your liver. Even though the hepatitis B virus is not causing any damage and you are not infectious, theinfection can be reactivated by certain medications and treatments. That’s why it is important to know that both you and your healthcare provider are 100% sure of your hepatitis B status.
Wow, so is hepatitis B preventable?
Hepatitis B can be prevented with a vaccine! If you get tested for hepatitis B and learn that you have no infection and no immunity, you can get the 2-dose hepatitis B vaccine, which protects you in a month, or the 3-dose vaccine, which can offer protection in six months. If possible, get tested first because the vaccine will only protect you if you don’t have the virus yet. Also, remember to get ALL the recommended doses of the vaccine series so you can be fully protected. Finally, if you are unsure of your status, it is important to use a condom. A condom is effective in preventing transmission of hepatitis B as well as other STIs, including HIV.
Great! So, where can someone get tested or vaccinated for hepatitis B?
Your healthcare provider can provide hepatitis B testing and vaccination services. If you do not have a doctor, federally qualified health centers, community health clinics like Planned Parenthood, and your local health department can test and vaccinate you for hepatitis B. The CDC lists LGBTQ+-friendly health centers here. The vaccine is covered by most insurance providers, as well as Medicare part B for high-risk groups.
Is there anything I can do to help raise awareness?
Let’s talk about it! Even though it is just as harmful as HIV, hepatitis B is not as widely discussed among gay and bisexual men. This is scary because the U.S. is seeing an increase in adult acute hepatitis B cases, and studies show that hepatitis B vaccination rates are low amongst gay and bisexual men.
Talk to your friends, your partners, and your community to know their status, and to take action to protect themselves! The CDC has free resources that can help promote vaccination, as well as information that can help you get the discussion started.
Thanks Thaddeus! Any final thoughts you would like to share with our readers?
Thank YOU, Michaela! I really appreciate this opportunity to chat about the impact of viral hepatitis on gay and bisexual men. I think it is important to point out the LGBTQ+ community also encompasses our lesbian and bisexual sisters as well as our transgender and gender nonconforming siblings, who could also benefit from hepatitis vaccinations and care.
Finally, I can’t help but think about Pride month in the context of the COVID-19 pandemic and Black Lives Matter protests. I am super grateful to work with Hepatitis B Foundation, who has always aligned with one of the core concepts of our hepatitis efforts in Hawaii: public health work is social justice work!
This month, researchers at Jilin University in Changchun, China have discovered an anti-HBV role of the HIV-1 host restriction factor SERINC5. At Seoul National University in South Korea, HBV researchers have elucidated a mechanism by which HBV hijacks host transcription regulation. Researchers from the Paul Ehrlich Institute in Langen, Germany have demonstrated that HBV DNA can be sensed by the cGAS/STING pathway, but is not in the context of natural hepatocyte infection.
SERINC5 Inhibits the Secretion of Complete and Genome-Free Hepatitis B Virions Through Interfering with the Glycosylation of the HBV Envelope – Frontiers in Microbiology
This paper from Jilin University in Changchun, China reveals the protein serine incorporator 5 (SERINC5) as a host restriction factor for HBV virion secretion. The SERINC family of proteins facilitate lipid biosynthesis and transport in mammalian cells. SERINC5 was recently shown to restrict the replication of HIV-1 and other retroviruses by incorporating into the membrane of budding virions and preventing their entry into target cells. Additionally, the HIV-1 protein NEF as well as the structurally unrelated murine leukemia virus (MLV) protein glycogag have been shown to down-regulate SERINC5 expression on cell surfaces. In this paper, the role of SERINC5 in HBV replication was examined. SERINC5 was found to inhibit HBV virion secretion but not affect intracellular core particle-associated DNA or RNA. Furthermore, the group found that SERINC5 decreased the glycosylation levels of the HBV surface antigens (HBsAg) LHB, MHB, and SHB (large, medium, and small). In order to determine the possible role of SERINC proteins in HBV replication, SERINC proteins 1, 3, and 5, were each transfected into cells alongside an HBV expression vector using Lipofectamine 2000. Transfection of SERINC plasmids was performed in a dose-responsive manner and was confirmed using Western blot. Transfected cell supernatants were then analyzed using an ELISA for HBsAg. Cells transfected with SERINC5 showed a reduction of HBsAg in the supernatant with increasing amounts of SERINC5. Extracellular HBsAg levels in cells transfected with SERINC1 or SERINC3 were unaffected. Furthermore, compared to cells transfected with a control vector, cells transfected with SERINC5 had less HBV virion DNA in the supernatant as measured by qPCR following immunoprecipitation with an anti-HBsAg antibody. Those cells transfected with SERINC1 or SERINC3 showed no change in extracellular HBV virion DNA compared to the control. Interestingly, intracellular levels of HBV DNA and HBV RNA as measured by Southern blot and Northern blot respectively, showed no change between cells transfected with the control vector or any of the SERINC proteins. Additionally, siRNA knockdown of SERINC5 in HepG2 cells concomitantly transfected with an HBV expression vector yielded increased secretion of HBsAg as measured by ELISA and HBV viron DNA as measured by qPCR following immunoprecipitation with an anti-HBsAg antibody. Next, in order to understand the mechanism of SERINC5-mediated HBV secretion inhibition, flag-tagged LHB, MHB, or SHB were transfected into HepG2 cells alongside either a plasmid expressing HA-tagged SERINC5 or a control vector. Interestingly, the glycosylated forms of all three HBsAg proteins were reduced in cells co-transfected with SERINC5 as measured by Western blot. The group then found that SERINC5 colocalizes with LHB in the Golgi apparatus. This was accomplished by co-transfecting HepG2 cells with LHB fused to enhanced cyan fluorescent protein (LHB-ECFP) alongside HA-tagged SERINC5. Cells were then subjected to immunofluorescence dual staining with an antibody against HA as well as an antibody against GM130, a resident protein of the Golgi. These three signals overlapped, implying that SERINC5 interacts with LHB in the Golgi. This finding was further validated by co-immunoprecipitation experiments showing the interaction of SERINC5 with LHB, MHB, and SHB. The group also found, using mutagenesis studies that the fourth to sixth domains of SERINC5 are required for inhibition of HBV secretion. These domains are different than those involved in HIV-1 inhibition, and the group has concluded that SERINC5 inhibits HBV by a completely different mechanism than it does HIV-1. While SERINC5 inhibits HIV-1 by inducing conformational changes on the viral envelope, it inhibits HBV secretion by preventing glycosylation of HBsAg. This publication demonstrates that SERINC5 is a potential anti-HBV host factor. Stimulation of SERINC5 may be a possible treatment for chronic HBV and SERINC5 may prove useful as a diagnostic marker if it is found to correlate with HBV viral load and chronicity.
Viral hijacking of the TENT4–ZCCHC14 complex protects viral RNAs via mixed tailing – Nature Structural & Molecular Biology
This paper from Seoul National University in South Korea identifies the TENT4-ZCCHC14 complex as a host factor which protects viral messenger RNA (mRNA) transcripts from degradation. Terminal nucleotidyltransferases (TENTs) are noncanonical poly(A) polymerases. These enzymes add many adenine residues as well as occasional non-adenosine residues to the 3′ end of mRNA molecules. TENT4A and TENT4B (also known as PAPD7 and PAPD5) extend mRNA poly(A) tails with the occasional non-adenosine residue which is typically a guanosine. The results are mRNAs bearing “mixed tails”. Deadenylases are enzymes which trim poly(A) tails to initiate mRNA degradation. The carbon catabolite repression 4–negative on TATA-less (CCR4-NOT or CNOT) complex is the main cytoplasmic deadenylase complex. CNOT trims mRNA poly(A) tails, but its activity is hindered when it encounters a guanosine reside. Therefore, mixed tails protect mRNAs from being targeted for degradation. Interestingly, the inhibitor of HBV called DHQ-1 was recently found to interact with TENT4A and TENT4B. The protein called zinc finger CCHC domain-containing protein 14 (ZCCHC14) was previously found to be an essential host factor for HBV surface antigen production in a genome-wide CRISPR screen. This publication demonstrates that ZCCHC14 recognizes a pentaloop motif in the HBV post-transcriptional regulatory element (PRE) of HBV mRNAs and in turn recruits TENT4A or TENT4B which provide the mRNAs with a protective mixed tail. Additionally, it was demonstrated that viral mRNAs of the human cytomegalovirus (HCMV) contain a similar pentaloop motif and also receive protective mixed tails. This group used a method which they developed previously called TAIL-seq. This method allows for sequencing of 3′ tails on mRNAs as well as identification of the transcript. First, total RNA is extracted from cells. Ribosomal RNA (rRNA) is removed using an rRNA depletion kit in which ssDNA probes are specifically bound to rRNA which are then digested by RNase H. Next, a biotinylated adaptor sequence is ligated to the 3′ end of RNAs. A low concentration of RNase T1 is then used to partially digest the transcripts. Next, the RNAs are pulled down, using streptavidin, phosphorylated, and gel purified to obtain fragments which are 500-1000 nucleotides in length. This size fractionation step removes small non-coding RNAs such as tRNA, snRNA, snoRNA, and miRNA. Next, a second adaptor sequence is added to the 5′ end of the mRNAs. Finally, the mRNAs are subjected to next generation sequencing (NGS) on an Illumina HiSeq 2500 platform. Two reads are obtained for each mRNA, one from the 3′ adaptor and one from the 5′ adaptor. Sequence information derived from these reads reveals the specific composition of mRNA poly(A) tails. In this publication, TAIL-seq was employed to investigate viral mRNA tailing. HepG2.2.15 cells which express the HBV genome, as well as human foreskin fibroblasts (HEF) infected with HCMV were subjected to TAIL-seq. mRNA 3′ tails of both viruses were found to be guanylated significantly more than cellular mRNAs. Additionally, viral mRNA 3′ tails were longer than cellular ones, indicating slower net deadenylation. To check the mechanism of viral mixed tailing, the noncanonical poly(A) polymerases TENT4A and TENT4B were knocked down using siRNA. TAIL-seq showed a significant reduction of viral mRNA 3′ tail guanylation in TENT4-knockdown cells. Additionally, the half-lives of HBV mRNAs were shown to decrease in TENT4-knockdown HepG2.2.15 cells as measured by RT-qPCR at intervals following the addition of the transcription blocker actinomycin D. In order to determine how HBV mRNAs recruit TENT4A and TENT4B, formaldehyde-based crosslinking and immunoprecipitation sequencing (fCLIP-seq) was employed on HepG2.2.15 cells. fCLIP-seq reveals what RNA sequences proteins bind to. In fCLIP-seq, formaldehyde is used to crosslink RNA-protein interactions. RNA-protein complexes are then “pulled down” using an antibody and run on a gel. The protein may then be degraded using proteinase K and RNA molecules may be sequenced. RNA sequencing reads from fCLIP-seq of the HBV genome were enriched in lysates pulled down using antibodies against TENT4A or TENT4B compared to input cell lysate and that pulled down using normal mouse IgG. Importantly, the greatest enrichment occurred specifically in the PRE region of HBV mRNAs. The group goes on to show that the sterile alpha motif (SAM) of ZCCHC14 binds to the stem loop ⍺ region of the PRE and recruits TENT4 proteins. This publication demonstrates that both HBV and HCMV have taken advantage of host mRNA transcription regulation to prolong transcript half-life. ZCCHC14, TENT4A, and TENT4B may be possible host targets for HBV or HCMV antiviral treatments.
Hepatitis B Virus DNA is a Substrate for the cGAS/STING Pathway but is not Sensed in Infected Hepatocytes – Viruses This paper from the Paul Ehrlich Institute in Langen, Germany shows that HBV DNA is sensed by cGAS, but not in natural HBV infection of hepatocytes. Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS), is a pattern recognition receptor (PRR) that senses cytoplasmic double-stranded DNA (dsDNA). In response to dsDNA binding, cGAS catalyzes the production of 2’3′-cGAMP, a cyclic dinucleotide (CDN) which activates stimulator of interferon genes (STING) by direct binding. Once activated, STING signaling results in the activation of transcription factors promoting the production of type I interferons (IFN-I) and proinflammatory cytokines including tumor necrosis factor alpha (TNFα). IFN-I production and secretion lead to the activation of numerous IFN-stimulated genes (ISGs) which induce a robust antiviral state in the cell. The cGAS/STING pathway is a key component of innate immunity, protecting cells from bacterial and viral infections. How viruses interact with host innate immune sensors such as cGAS is important for understanding their pathogenesis. While the innate immune mechanisms activated by HBV infection remain disputed, HBV is largely considered to be a stealth virus in that it bypasses host innate immunity. Some groups have postulated that the HBV X protein (HBx) or HBV polymerase may inhibit innate immune responses. In this publication it is demonstrated that HBV RNAs are not immunostimulatory, however HBV DNA does elicit an innate immune response mediated by the cGAS/STING pathway. In order to test the immunostimmulatory potential of HBV nucleic acids, they were transfected at multiple concentrations into monocyte-derived dendritic cells (MDDCs) generated from primary human peripheral blood mononuclear cells (PBMCs). Following transfection, mRNA of the gene ISG54 was measured by RT-qPCR. ISG54 was selected as the read-out for innate immune signaling because it is a direct target of the transcription factor IRF3 which is activated downstream of both RIG-I (RNA-sensing) and cGAS/STING (DNA-sensing) pathways. HBV nucleic acids were extracted from HBV virions and quantified prior to transfection. Some groups of nucleic acids were subjected to either DNase or RNase digestion, leaving only HBV RNA or DNA respectively. Total HBV nucleic acids stimulated ISG54 transcription in a dose-dependent manner. Similarly, HBV DNA also stimulated ISG54 transcription. However, transfection of HBV RNA alone did not activate ISG54 transcription, implying that only HBV DNA elicits an innate immune response. In order to test which specific innate immune pathway senses HBV DNA, the human monocytic leukemia cell line THP-1 was used. CRISPR/Cas9 genome editing was used in THP-1 cells to knockout (KO) cGAS, STING, or mitochondrial antiviral-signaling protein (MAVS), which is a key node downstream of the RNA-sensing RIG-I-like receptor (RLR) protein family. Transfection with HBV nucleic acids caused a high level of ISG54 transcription in wild type (WT) and MAVS KO cells which was abrogated when HBV nucleic acids were treated with DNase prior to transfection. However, HBV nucleic acids caused no measurable ISG54 transcription in either cGAS KO or STING KO cells. Next, the group wanted to determine if HBV activates the cGAS/STING pathway in its natural infection of hepatocytes. The levels of cGAS, STING, and other PRRs in a panel of cells were determined using RT-qPCR. The hepatocellular carcinoma cell line HepG2 as well as primary human hepatocytes (PHH) were shown to express less cGAS and STING than Kupffer cells, MDDCs, THP-1 cells, or monocyte derived macrophages (MDMs). Next, HepG2 cells expressing the human sodium taurocholate cotransporting polypeptide used for HBV cell entry (HepG2-hNTCP) and PHHs were transfected with HBV nucleic acids. Both hepatocyte types showed a dose-responsive increase in ISG54 transcription when transfected. Finally, HepG2-hNTCP cells and PHHs were infected with HBV and HBV RNA and ISG54 mRNA were quantified by RT-qPCR. Although both cell types were efficiently infected, they showed no induction of ISG54 across several days. These results indicate that although hepatocytes are capable of sensing transfected HBV genomic DNA via cGAS, they are not able to do so in the context of a natural infection. One possible explanation for the failure of hepatocytes to sense HBV nucleic acids is that they are shielded by the viral nucleocapsid upon infection and during the formation of replication intermediates. Another possibility is that the level of HBV nucleic acids in a natural infection is too low to activate cGAS/ STING, given that these proteins are sparse in hepatocytes. This publication demonstrated for the first time that HBV RNAs are not immunostimulatory, while HBV DNAs activate the cGAS/STING pathway. This finding shows that it may be possible to utilize the cGAS/STING pathway in order to eradicate chronic HBV infection. Perhaps small molecules which destabilize HBV nucleocapsids may be used to expose the DNA of intracellular HBV virions, leading to the activation of the cGAS/STING pathway and an innate antiviral response.
Meet our guest blogger, David Schad, B.Sc., Junior Research Fellow at the Baruch S. Blumberg Institute studying programmed cell death such as apoptosis and necroptosis in the context of hepatitis B infection under the direction of PI Dr. Roshan Thapa. David also mentors high school students from local area schools as part of an after-school program in the new teaching lab at the PA Biotech Center. His passion is learning, teaching and collaborating with others to conduct research to better understand nature.
The short answer is, possibly. Although there is extensive research to support the role of hepatitis delta in accelerating the risk for progression to cirrhosis (liver scarring) compared to hepatitis B infection (1,2) only, strong data directly linking an increase in risk for hepatocellular carcinoma (HCC) is lacking. It is known that coinfection promotes continually progressing inflammation within the liver by inducing a strong immune response within the body; where it essentially attacks itself (3), but the specific role of hepatitis delta in HCC isn’t fully understood. It gets complicated because although cirrhosis is usually present in hepatitis B patients who also have HCC, but scientists have not pinpointed a specific way that the virus may impact cancer development (4). There have been some small studies that have documented a correlation between hepatitis delta and an increase in HCC, but some analysis’s have even called the extent of its involvement in HCC as ‘controversial’ (5). However, other scientific studies may suggest the contrary.
Because hepatitis delta cannot survive without hepatitis B, and doesn’t integrate into the body the same way, it may not be directly responsible for cancer development, but it has been suggested that the interactions between the two viruses may play a role (6). It has also been suggested that hepatitis delta may play a role in genetic changes, DNA damage, immune response and the activation of certain proteins within the body – similarly to hepatitis B and may amplify the overall cancer risk (7,8). One of these theories even suggests that hepatitis delta inactivates a gene responsible for tumor suppression, meaning it may actually promotes tumor development, a process that has been well-documented in HCC cases (9,10).
Regardless of the specific impact or increase in risk for HCC due to the hepatitis delta virus, hepatitis B is known to increase someone’s risk, with 50-60% of all HCC globally attributable to hepatitis B (11). People with hepatitis delta coinfection still need to be closely monitored by a liver specialist, as 70% of people with both viruses will develop cirrhosis within 5-10 years (12). Monitoring may be blood testing and a liver ultrasound to screen for HCC every 6 months. Closer monitoring may be required if cirrhosis is already present, or to monitor response to treatment (interferon).
Manesis EK, Vourli G, Dalekos G. Prevalence and clinical course of hepatitis delta infection in Greece: A 13-year prospective study. J Hepatol. 2013;59:949–956.
Coghill S, McNamara J, Woods M, Hajkowicz K. Epidemiology and clinical outcomes of hepatitis delta (D) virus infection in Queensland, Australia. Int J Infect Dis. 2018;74:123–127.
Zhang Z, Filzmayer C, Ni Y. Hepatitis D virus replication is sensed by MDA5 and induces IFN-β/λ responses in hepatocytes. J Hepatol. 2018;69:25–35.
Nault JC. Pathogenesis of hepatocellular carcinoma according to aetiology. Best Pract Res Clin Gastroenterol. 2014;28:937–947.
Puigvehí, M., Moctezuma-Velázquez, C., Villanueva, A., & Llovet, J. M. (2019). The oncogenic role of hepatitis delta virus in hepatocellular carcinoma. JHEP reports: innovation in hepatology, 1(2), 120–130.
Romeo R, Petruzziello A, Pecheur EI, et al. Hepatitis delta virus and hepatocellular carcinoma: an update. Epidemiol Infect. 2018;146(13):1612‐1618.
Majumdar A, Curley SA, Wu X. Hepatic stem cells and transforming growth factor β in hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol. 2012;9:530–538.
Mendes M, Pérez-Hernandez D, Vázquez J, Coelho AV, Cunha C. Proteomic changes in HEK-293 cells induced by hepatitis delta virus replication. J Proteomics. 2013;89:24–38.
Chen M, Du D, Zheng W. Small Hepatitis Delta Antigen Selectively Binds to Target mRNA in Hepatic Cells: A Potential Mechanism by Which Hepatitis D Virus Down-Regulates Glutathione S-Transferase P1 and Induces Liver Injury and Hepatocarcinogenesis. Biochem Cell Biol. August 2018.
Villanueva A, Portela A, Sayols S. DNA methylation-based prognosis and epidrivers in hepatocellular carcinoma. 2015;61:1945–1956.
Hayashi PH, Di Bisceglie AM. The progression of hepatitis B- and C-infections to chronic liver disease and hepatocellular carcinoma: epidemiology and pathogenesis. Med Clin North Am. 2005;89(2):371‐389.
Abbas, Z., Abbas, M., Abbas, S., & Shazi, L. (2015). Hepatitis D and hepatocellular carcinoma. World journal of hepatology, 7(5), 777–786.
A common question among people living with hepatitis B and their families is, “What happened to the cure for hepatitis B?” You can find answers in a new commentary by Dr. Timothy Block, HBF president and co-founder; Dr. Chari Cohen, senior vice president; and Maureen Kamischke, our director of international engagement.
The Hepatitis B Foundation’s Commentaries on the Cure is a new series written by hepatitis B experts. The series will feature thoughts and updates about the progress being made towards a cure for hepatitis B. Many of you have been awaiting a cure for years, and we understand that the wait can be frustrating. In addition to providing a look into the drug development process, we hope this series will serve as a source of information and hope for individuals living with hepatitis B.
Over the last 10 years, great strides have been made in hepatitis B cure research. The number of therapies in clinical trial stages has more than doubled, and four potential treatments for hepatitis Delta are in development! We believe that at least a “functional” cure is on it’s way, but it is extremely difficult to predict when one will be available. According to the Pharmaceutical Research and Manufacturers of America, it takes an average of 12-15 years to bring a drug from research to market. New treatments must undergo a rigorous testing process to ensure that it is both safe and effective for a large population. This process is extremely expensive – costing around $800 million USD per drug – and can be influenced by numerous factors, such as the number of volunteers for a clinical trial.
In recent years, we have seen an increase in interest and investments in a cure for hepatitis B, but more funding and support are needed to complete the journey. The Hepatitis B Foundation will continue to give the hepatitis B community a platform to share their voice, and advocate for the resources needed for the cure.
This month, research from Melbourne, Australia indicates that the kinases TBK1 and IKKε act redundantly to initiate STING-induced, NF-kB-mediated transcription of proinflammatory cytokines. Nearby researchers also working in Melbourne have demonstrated that an HBV vaccine composed of glycosylated HBV surface protein outperforms those currently in use. Also, researchers at St. Jude Children’s Research Hospital in Memphis, Tennessee have elucidated the role of caspase-6 in influenza A virus host defense.
TBK1 and IKKε Act Redundantly to Mediate STING Induced NF-kB Responses in Myeloid Cells – Cell Reports
This paper from The Walter and Eliza Hall Institute of Medical Research in Melbourne, Australia deciphers the role of the kinases TBK1 and IKKε in STING-induced, NF-kB-mediated cytokine production. Stimulator of Interferon Genes (STING) protein is a vital component of the innate immune system. Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS), is a pattern recognition receptor (PRR) that senses cytoplasmic double-stranded DNA (dsDNA). In response to dsDNA binding, cGAS catalyzes the production of 2’3′-cGAMP, a cyclic dinucleotide (CDN) which activates STING by direct binding. Once bound to 2’3′-cGAMP, STING dimers undergo a conformational change and translocate from the endoplasmic reticulum (ER) to the Golgi apparatus. At the Golgi, the serine-threonine protein kinase TANK-binding kinase 1 (TBK1) phosphorylates STING at residues in its C-terminal tail (CTT). This phosphorylation causes the recruitment of interferon regulatory factor 3 (IRF3) to STING which is also phosphorylated by TBK1. Phosphorylated IRF3 forms dimers and translocates to the nucleus where it induces the expression of type I interferons (IFN-I) such as IFN-β. IFN-I production and secretion lead to the activation of numerous IFN-stimulated genes (ISGs) which induce a robust antiviral state in the cell. Concomitant to IFN-I induction, STING activation is also known to induce a set of proinflammatory cytokines through the transcription factor called nuclear factor-kB (NF-kB). These cytokines include tumor necrosis factor alpha (TNFα) and interleukins (IL) IL-1β and IL-6. While TBK1 and to a much lesser extent IkB kinase ε (IKKε) are needed for IRF3-mediated IFN-I transcription, several lines of evidence indicate that they may be unnecessary for STING-induced NF-kB activity. For instance, the CTT region of STING, critical to IFN induction, is observed only in vertebrates. While STING activation in the invertebrate species Drosophila melanogaster and Nematostella vectensis results in NF-kB-mediated transcription of cytokines, it does not induce IFN-I transcription. Additionally, ubiquitination of STING at lysine residues K244 and K288 which is required for its trafficking from the ER to the Golgi is essential for IFN-I induction, but not for NF-kB activation. Finally, phosphorylation of STING at serine residues S358 and S366 in the CTT is required for IRF3 activation but is unnecessary for NF-kB activity. This publication reports that while TBK1 kinase activity is critical for IRF3 activation, TBK1 and IKKε act redundantly and in a kinase-independent manner to activate NF-kB signaling. To determine this, conditional TBK1-knockout mice were generated. These mice were the offspring of mice “floxed” for TBK1 and “RosaCre” mice (ROSA26-CreERT2). The floxed mice were mutated to have their TBK1 gene sandwiched between two lox P sites (Tbk1fl/fl). The RosaCre mice were mutated to constituatively produce a fusion protein of the Cre recombinase and the estrogen receptor (CreER). The TBK1 conditional knockout mice (Tbk1fl/fl x RosaCre) transcribe TBK1 until they are treated with the synthetic steroid tamoxifen. Tamoxifen binds the the CreER fusion protein (CreERT) and causes its translocation to the nucleus where it binds to lox P sites and its recombinase activity causes the deletion of the TBK1 gene. Conditional knockout mice had to be used to study TBK1 because complete constituative TBK1 knockout is lethal to mice. Primary bone marrow-derived macrophages (BMDM) were obtained from both tamoxifen-treated wild-type Tbk1fl/fl (WT) and Tbk1fl/fl x RosaCre (TBK1 knockout) mice. When subjected to the STING agonist 2’3′-cGAMP, BMDMs from WT mice showed phosphorylation of IRF3 by Western blot and secretion of IFN-β by ELISA. Under the same treatment, BMDMs derived from TBK1 knockout mice showed drastically reduced IRF3 phosphorylation and IFN-β secretion. Interestingly, BMDMs derived from both WT and TBK1 knockout mice secreted similar levels of TNFα when treated with 2’3′-cGAMP. Next, BMDCs from normal mice were immortalized and CRISPR/Cas9 was used to knockout expression of TBK1, IKKε, or both. Significantly, while TNFα secretion upon 2’3′-cGAMP treatment was modestly reduced by the knockout of either TBK1 or IKKε, it was almost completely ablated by the knockout of both genes. Interestingly, knockout of both genes had no effect on the secretion of TNFα in response to treatment with lipopolysaccharide (LPS). Finally, in order to determine the upstream signaling responsible for STING-mediated NF-kB activity, two proteins were investigated: transforming growth factor b-activated kinase 1 (TAK1) and inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ). Small molecule inhibitors were used to inhibit TAK1 and IKKβ prior to treatment with the mouse STING agonist DMXAA. Inhibition of both TAK1 and IKKβ resulted in diminished NF-kB activity, implicating their role as kinase activators of NF-kB downstream of STING. Taken together, these results indicate that TBK1 and IKKε act redundantly to carry out STING-mediated NF-kB activity. Additionally, it is likely that TAK1 acts downstream of TBK1 and IKKε to activate the IKK complex, resulting in NF-kB activity. This finding has direct therapeutic significance for STING-driven autoimmune disorders such as chronic polyarthritis. Many strategies for overcoming such diseases only target the IFN-I-producing pathway, while pro-inflammatory cytokine production may go unchecked. This finding elucidates a less-studied arm of STING signaling which is important for basic science and future therapies.
Glycoengineered Hepatitis B Virus-Like Particles with Enhanced Immunogenicity – Vaccine
This paper from the Royal Melbourne Institute of Technology University in Melbourne, Australia shows that an HBV vaccine using glycosylated HBV surface protein may have better efficacy than the current vaccine. HBV encodes three surface proteins (large, medium, and small) which are truncated forms of the same protein. The small HBV surface protein (HBsAgS) contains the major antigenic determinants of the protein. In the absence of other viral proteins, HBsAgS will self-assemble into non-infectious particles termed subviral particles (SVP), also known as virus-like particles (VLP). VLPs are the major species of HBV viral particle secreted from infected hepatocytes. When grown in mammalian cells in vivo, approximately half of HBsAgS molecules receive N-glycosylation at asparagine residue N146. N-glycosylation is the addition of an oligosacharide molecule to the nitrogen atom of an asparagine residue within a protein. These modifications occur in the endoplasmic reticulum (ER) and are important for the function of proteins and for signaling within the cell. The current HBV vaccines are composed of HBsAgS VLPs grown in yeast. In contrast to VLPs grown in mammalian cells, yeast-derived VLPs have no N-glycosylation. Additionally, HBV vaccines contain adjuvants which aid in immune system stimulation. The widely-used HBV vaccines Engerix-B (GlaxoSmithKine) and Recombivax HB (Merck) contain the adjuvants aluminum hydroxide and aluminum hydroxyphosphate respectively. Aluminum salts stimulate the immune system by causing activation of the NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome pathway. Upon vaccination, aluminum salt crystals are taken into local dendritic cells via phagocytosis where they rupture the lysosome, causing activation of the NLRP3 inflammasome which includes active caspase 1. The catalytic activity of caspase 1 cleaves pro-interleukin 1β (IL-1β) as well as gasdermin D into their active forms. Cleaved gasdermin D forms pores in the cell membrane resulting in the rapid release of pro-inflammatory IL-1β and ultimately causing pyroptosis, an immunogenic form of cell death. This publication shows that using glycosylated HBsAgS VLPs in the presence of aluminum hydroxide may result in a more immunogenic vaccine than that which is currently used. To study the effect of HBsAgS glycosylation, first N-terminal FLAG-tagged wild-type (WT) HBsAgS and point-mutated variants were expressed in HEK 293 cells. Variants used were threonine-to-asparagine mutant T116N and asparagine-to-glutamine mutant N146Q. The T116N mutant contained an additional asparagine available for glycosylation on the domain of HBsAgS which faces the lumen of the ER. On the other hand, the N146Q mutant lacked the asparagine which is typically N-glycosylated. SDS-PAGE followed by Coomassie staining revealed that about 50% of WT HBsAgS was glycosylated, running as two distinct bands at 27 kDa (glycosylated) 24 kDa (non-glycosylated). However, HBsAgS mutant T116N ran as two predominant bands at 27 kDa (monoglycosylated) and 29 kDa (diglycosylated). HBsAgS mutant N146Q ran as a single band at 24 kDa, indicating no glycosylation. This result confirmed that about half of HBsAgS produced in mammalian cells are N-glycosylated at N146 and no other amino acid. Both HBsAgS mutants formed VLPs similar to WT as viewed by transmission electron microscopy. VLPs were mostly spherical with some elongated in shape. Next, following removal of N-glycans using the enzyme peptide:N-glycosidase F (PNGase), quantitative N-glycome profiling was conducted using an advanced spectrometry technique called porous graphitized carbon liquid chromatography-electrospray ionization-tandem mass spectrometry (PGC-LC-ESIMS/MS). The T116N mutant was found to have a greater N-glycan density than WT HBsAgS, but a similar distribution of N-glycan types. Finally, the immunogenicity of glycoengineered HBsAg was tested using a mouse model of vaccination. BALB/c mice were immunized at weeks 1, 3, 5, and 7 with purified WT or T116N HBsAgS in the presence or absence of aluminum hydroxide. Some mice were immunized with Engerix-B as a control group. Serum samples were taken at weeks 2, 4, 6, 8, and 18 post-vaccination and analyzed by an ELISA assay against yeast-derived VLPs. Mice immunized with T116N HBsAgS combined with aluminum hydroxide had the highest titer of anti-HBsAgS antibodies at every time point tested. This indicates that hyper-glycosylated HBsAg is more effective than non-glycosylated HBsAg in mounting an immune response. The authors propose that hyper-glycosylated HBsAgS is more readily taken into antigen-presenting cells (APCs) due to an increased affinity for manose-binding lectin receptors expressed on those cells. Additionally, hyper-glycosylation of HBsAgS may lower its strength of adsorption with aluminum hydroxide, making it more prone to release and antigen processing. Taken together, these results demonstrate that glycoengineered HBsAgS formed VLPs and when combined with aluminum hydroxide, exhibited increased immunogenicity in BALB/c mice in comparison to a currently used vaccine. This publication shows one way in which molecular cloning techniques may be used to improve the efficiency and reliability of HBV vaccines.
Caspase-6 Is a Key Regulator of Innate Immunity, Inflammasome Activation, and Host Defense – Cell
This paper from St. Jude Children’s Research Hospital in Memphis, Tennessee shows that caspase-6 mediates inflammasome activation and plays a role in the activation of the programmed cell death (PCD) pathways pyroptosis, apoptosis, and necroptosis (PANoptosis). The caspase family of proteins are cysteine-aspartic proteases which cleave proteins between cysteine and aspartic acid residues. Caspases play essential rolls in inflammation and PCD pathways. Caspases exist as inactive zymogens (pro-forms) within the cell until they are cleaved, resulting their active form. Caspases are grouped as being either inflammatory (caspase-1, -4, -5, and -11) or apoptotic (caspase-3, -6, -7, -8, -9 and -10). However, emerging evidence has demonstrated crosstalk between these groups under certain conditions. Inflammatory caspases can play a role in PCD pathways and apoptotic caspases can play a role in inflammatory pathways. While caspase-6 has long been considered an executioner caspase in the apoptotic pathway, its major functions have remained unknown. This publication demonstrates that caspase-6 is an essential upstream component of Z-DNA binding protein 1 (ZBP1)-mediated inflammasome activation and subsequent PANoptosis. The NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome is a multimeric structure consisting of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase 1 subunits. NLRP3 inflammasome activation results in caspase-1 mediated cleavage of pro-interleukin 1β (IL-1β) as well as gasdermin D into their active forms. Cleaved gasdermin D forms pores in the cell membrane resulting in the rapid release of pro-inflammatory IL-1β and ultimately causing pyroptosis. The NLRP3 inflammasome can be activated by a variety of stimuli including canonical stimuli (pore-forming toxins, ATP) and non-canonical stimuli (intracellular LPS sensed by caspase-4/5). Additionally, this group has previously demonstrated that the NLRP3 inflammasome can also be activated by ZBP1 sensing of influenza A virus (IAV). In order to discern if caspase-6 is involved in NLRP3 inflammasome activation, bone marrow-derived macrophages (BMDMs) were derived from caspase-6 knockout (Casp6–/–) mice. Caspase-6 was shown to be dispensable for both canonical and non-canonical activation of the NLRP3 inflammasome, as caspase-1 cleavage was shown via Western blot and secretion of both IL-1β and IL-18 was shown via ELISA. However, when infected with IAV, Casp6–/– BMDMs failed to display caspase-1 cleavage and cytokine release compared to the wild-type (WT) control. This indicates that caspase-6 plays an essential role in IAV-induced NLRP3 inflammasome activation and pyroptosis. As this group and others have shown that ZBP1 regulates various forms PCD in response to IAV infection, next the roll of caspase-6 in PCD pathways was investigated. Overall cell death 12 hours following IAV infection was reduced by about 50% in Casp6–/– BMDMs as measured by SYTOX Green nucleic acid stain and high-content imaging. To investigate this phenomenon further, CRISPR-Cas9 was used to generate caspase-6 knockout (Casp6KO) mouse embryonic fibroblasts (MEFs). IAV-induced cell death was largely ablated in Casp6KO MEFs compared to WT MEFs as measured by SYTOX Green nucleic acid stain and high-content imaging. Furthermore, Casp6KO MEFs showed highly reduced IAV-induced cleavage of apoptotic caspases-3, -7, and -8 as measured by Western blot. Additionally, Casp6–/– BMDMs showed highly reduced cleavage of the pyroptosis effector gasdermin D and phosphorylation of the necroptosis effector pseudokinase mixed lineage kinase domain-like (MLKL) upon IAV infection. Taken together, these results indicate that caspase-6 plays a critical role in the IAV-induced PCD pathways pyroptosis, apoptosis, and necroptosis. Interestingly, Casp6–/– BMDMs were still susceptible to necroptosis by the classical trigger of TNFα plus zVAD, indicating an IAV-specific necroptotic function of caspase-6. In a mouse model, the authors found that caspase-6 deficiency increased susceptibility to IAV infection. Upon IAV infection, ZBP1 recruits RIPK1 and RIPK3 via the receptor-interacting protein homotypic interaction motif (RHIM) to form a cell death complex. It has been demonstrated that from this complex, RIPK3 activates parallel pathways of apoptosis and necroptosis. In order to explore if this complex directly regulates caspase-6 cleavage, Ripk3–/– and Zbp1–/– BMDMs were utilized. Both Ripk3–/– and Zbp1–/– BMDMs showed reduced cleavage of caspase-6, -8, -7, -3 and gasdermin D as well as reduced MLKL phosphorylation. This result confirms the previous finding that in response to IAV infection, ZBP1 and RIPK3 mediate both apoptotic and necroptotic pathways and suggests a third role for RIPK3 in IAV-induced, ZBP1-mediated pyroptosis. This result also indicates that caspase-6 is regulated at the level of the ZBP1-RIPK3 complex when taken together with the finding that caspase-6 deletion affected all three forms of PCD. Additionally, similar experiments using BMDMs lacking either gasdermin D or NLRP3 both showed no change in caspase-6 cleavage. To determine which protein in the ZBP1-RIPK3 complex interacts with caspase-6, components of the complex (RIPK1, RIPK3, ZBP1, caspase-8) were individually over-expressed in HEK293T cells via transfection alongside a catalytically dead, FLAG-tagged caspase-6, followed by co-immunoprecipitation (Co-IP) using an anti-FLAG antibody. Only RIPK3 was pulled down alongside FLAG-caspase-6, indicating that caspase-6 interacts with RIPK3. Further Co-IP experiments in immortalized BMDMs utilizing a doxycycline-inducible FLAG-caspase-6 showed that increased levels of caspase-6 improved the ability of RIPK3 to interact with ZBP1. This indicates that caspase-6 may promote IAV-induced PANoptosis by facilitating the interaction of ZBP1 with RIPK3. This paper identifies a previously unknown role for caspase-6 in regulating ZBP1-mediated inflammasome activation and PANoptosis. Additionally, caspase-6 was shown to be essential for host defense against AIV in a mouse model. The results presented here further elucidate the complex interactions of cell death effectors in the context of IAV infection. These findings may help in the development of novel IAV therapies as well as treatments for diseases with abnormally regulated cell death pathways.
Meet our guest blogger, David Schad, B.Sc., Junior Research Fellow at the Baruch S. Blumberg Institute studying programmed cell death such as apoptosis and necroptosis in the context of hepatitis B infection under the direction of PI Dr. Roshan Thapa. David also mentors high school students from local area schools as part of an after-school program in the new teaching lab at the PA Biotech Center. His passion is learning, teaching and collaborating with others to conduct research to better understand nature.
Join HepBUnited, NASTAD,National Viral Hepatitis Roundtable (NVHR) and CDC’s Division of Viral Hepatitis for a Twitter Chat on Hepatitis Testing Day, May 19th at 2 P.M. EDT. The chat will highlight hepatitis events and allow partner organizations to share their successes, challenges and lessons learned from their efforts, particularly during this unique time. Partners will also highlight innovative strategies for outreach during COVID-19. This twitter chat serves to keep us all informed, raise awareness and share messaging. All are encouraged to join the twitter chat conversation with the hashtag #HepChat20, and to keep partners posted throughout the month about events and messaging with the hashtag #HepAware2020.
In 2019, the hepatitis B community successfully advocated for the introduction of U.S. House and Senate resolutions to designate April 30th as National Adult Hepatitis B Vaccination Awareness Day for the first time!
Why is Awareness about Adult Hep B Vaccination Needed?
Adults in the United States have extremely low rates of vaccination, primarily because many were born before the vaccine became a healthcare standard and mandated for school. Just 25% of all U.S.adults have completed their vaccine series. Without completing the series, individuals are still vulnerable to potential exposures; one dose of the vaccine is not enough. Coupled with the recent increasein injection drug use, low vaccination rates among adults have been driving arise in acute hepatitis B casesacross the nation. The new cases that are linked to injection drug use are particularly prevalent among adults aged 30 to 49. Unfortunately, newly infected women may be unaware of their status and may pass the virus on to their infants during birth, putting them at significantly higher risk of chronic infection and liver cancer.
Image Courtesy of National Foundation for Infectious Diseases
Immunization rates remain low among vulnerable populations including those living with other chronic conditions such as hepatitis C, HIV, kidney disease, or diabetes. In fact, just 12% of diabetic adults 60 years old or older are fully vaccinated, and 26% of diabetic adults ages 19-59 have received the complete vaccine series. Healthcare workers are an under-vaccinated vulnerable population as well. According to the Centers for Disease Control and Prevention, just 60% of healthcare personnel have completed their vaccine series.
National Adult Hepatitis B Vaccine Awareness Day Resolution
This resolution is an opportunity to raise awareness about the importance of the hepatitis B vaccine for providers and community members, as well as providing support for testing, vaccination, and linkage to care for individuals. In addition, the resolution helps encourage a commitment to increasing hepatitis B vaccination rates for adults while maintaining high childhood vaccination rates.
Hepatitis B Vaccine
Themodern hepatitis B vaccinehas been widely used – with over 1 billion doses given – since it was created in 1985, and has been proven to be one of the safest and most effective vaccines in the world! The 3-dose vaccine is given over the span of 6 months, and provides lifelong protection once completed. Adults can also be fully vaccinated with a new 2-dose vaccine called Heplisav-B!Heplisav-B can be completed in just one month and has been proven to be highly effective in populationsthat may be hard to vaccinate, such as older adults and people living with diabetes.
Raising awareness about adult hepatitis B vaccination is a small, but essential step in the journey towards the elimination of hepatitis B. With national support and resources, the U.S. can protect vulnerable communities from serious liver damage and even liver cancer.
You can show your support for National Adult Hepatitis B Vaccine Day by using the hashtag #AdultHepBVaxDay on April 30th and when discussing the hepatitis B vaccine on social media! Graphics are also available to share throughout your networks.
Please see the below links to access additional resources on adult hepatitis B vaccination:
Being diagnosed with hepatitis B can be a confusing experience and may leave you with many questions. Understanding your diagnosis is essential for your health, and understanding how hepatitis B is transmitted can help prevent transmission to others.
How is it Spread?
Hepatitis B is transmitted through direct contact with infected blood. This can happen through direct blood-to-blood contact, unprotected sex, unsterile needles, and unsterile medical or dental equipment. Globally, hepatitis B is most commonly spread from an infected mother to her baby due to the blood exchange during childbirth. It can also be transmitted inadvertently by the sharing of personal items such as razors, toothbrushes, nail clippers, body jewelry, and other personal items that have small amounts of blood on them.
Hepatitis B is not transmitted casually by sneezing or coughing, shaking hands, hugging or sharing or preparing a meal. In fact, hepatitis B is not contracted during most of life’s daily activities. You don’t need to separate cups, utensils, or dishes. You can eat a meal with or
prepared by someone with hep B. Hugging, or even kissing won’t cause infection unless there are bleeding gums or open sores during the exchange. As an infection that is spread through the blood, standard precautions such as covering all wounds tightly, practicing safe sex (using a condom), and cleaning up all blood spills with gloves and a solution of one part bleach to nine parts water will protect against transmission. The best tool we have to prevent transmission is the hepatitis B vaccine!
Most of those who are newly infected have no notable symptoms. This is why it is important to encourage family members and sexual partners to get tested if you test positive. Often, it remains undetected until it is caught in routine blood work, blood donation, or later in life after there is liver inflammation or disease progression.
Dealing with a Possible Exposure:
One important factor for those that may have been exposed is thetiming. There is a 4-6 week window period between an exposure to hepatitis B and when the virus shows up in the blood (positive HBsAg test result). If you go for immediate testing, please understand that you will need to be re-tested 9 weeks later to confirm whether or not you have been infected. It is essential to practice safe sex and follow general precautions until everyone is sure of their status –both the known and potentially infected.
You may still be in a waiting period trying to determine if you are acutely or chronically infected. It is possible that you have not had symptoms with your hepatitis B. It’s also very likely you are unsure as to when you were infected. Not knowing the details of your infection can be stressful and confusing, but the most important thing to do now is to educate yourself so that you can take the proper steps to protect your liver and prevent transmission.
Preventing Future Transmission:
Always cover open wounds. Keep cuts, bug bites – anything that bleeds or oozes – covered with a bandage. It’s also a good idea to carry a spare bandage.
Be sure to practice safe sex (use a condom) until you are sure your partner has completed their hepatitis B vaccine series. Be aware that multiple sex partners and non-monogamous relationships can expose you to the potential of more health risks and even the possibility of a co-infection, so it is best to use a condom. Co-infections are when someone has more than one serious chronic condition (like HBV andHCV , HBV and HIV or HBVand HDV). Co-infections are complicated health conditions that you want to avoid. Therefore, practice safe sex by using a latex or polyurethane condom if you have multiple partners.
Keep personal items personal. Everyday items that are sharp may contain small amounts of blood. This includes things like razors, nail clippers, files, toothbrushes and other personal items where microscopic droplets of blood are possible. This is good practice for everyone in the house. Simple changes in daily habits keep everyone safe!
If a person has been tested and their results show that they are not already vaccinated or have not recovered from a past infection, then they should start the series as soon as possible. This includes sexual partners and close household contacts and family members. The HBV vaccine is a safe and effective 2 or 3-shot series.
If you wish to confirm protection, the timing of the antibody titre test should be 4-8 weeks following the last shot of theseries. If titers are equal to or above 10 mIU/mL, then there is protection for life. If someone has been previously vaccinated a titer test may show that their titers have waned and dipped below the desired reading. There is no reason to panic, as a booster shot can be administered and then a repeated titer test 1-2 months later can ensure adequate immunity. Once you know you have generated adequate titers, there is no need for concern of transmission!
When recovering from an acute infection, if your follow up blood test results read: HBsAg negative, HBcAb positive and HBsAb positive then you have resolved your HBV infection and are no longer infectious to others and you are no longer at risk for infection by the HBV virus again.
However if your follow up blood tests show that you are chronically infected or your infection status is not clear, you will want to take the precautionary steps to prevent transmitting your HBV infection to others. You will also need to talk to your doctor to be sure you have the appropriate blood work to determine your HBV status and whether or not you are chronically infected.
Please be sure to talk to your doctor if you are unsure, and don’t forget to get copies of all of your lab results!
Welcome to the Hepatitis B Research Review! This monthly blog shares recent scientific findings with members of Baruch S. Blumberg Institute (BSBI) labs and the hepatitis B (HBV) community. Technical articles concerning HBV, Hepatocellular Carcinoma, and STING protein will be highlighted as well as scientific breakthroughs in cancer, immunology, and virology. For each article, a brief synopsis reporting key points is provided as the BSBI does not enjoy the luxury of a library subscription. The hope is to disseminate relevant articles across our labs and the hep B community.
Summary: This month, researchers at Fudan University in Shanghai, China have identified activation of the cGAS/STING pathway by extracellular DNA as a mediator of radiation-induced liver disease. At the Pennsylvania State University College of Medicine in Hershey, PA, HBV researchers have elucidated the role of the host kinase protein CDK2 in phosphorylating the HBV core protein, leading to new cccDNA formation. Researchers from the University of Charlottesville in Virginia have characterized the “apoptotic metabolite secretome”, a select group of molecules released from cells undergoing apoptosis.
DNA sensing and associated type 1 interferon signaling contributes to progression of radiation-induced liver injury – Cellular & Molecular Immunology
This paper from Fudan University in Shanghai, China reveals the role of the cGAS/STING pathway in radiation-induced liver disease (RILD). Either radiation therapy (RT) or accidental exposure to ionizing radiation may cause RILD. RT is used to treat various cancers, including hepatocellular carcinoma (HCC). The dose of radiation used when treating HCC and gastrointestinal malignancies is limited by the risk of RILD as the liver is a highly radiosensitive organ. RILD is associated with a high mortality in patients with HCC and typically occurs within four months of receiving RT. RILD is characterized by hepatic injury due to the deposition of fibrin into the central veins and sinusoids of the liver. While the exact mechanism of RILD development is not well understood, it has been shown that hepatic nonparenchymal cells (NPCs) such as Kupffer cells, sinusoidal endothelial cells, and hepatic stellate cells play an important role. NPCs are cells in the liver that are not hepatocytes; they consist of immune cells, endothelial cells, pericytes, and other cell types. The cGAS/STING pathway is a component of the innate immune system in cells responsible for sensing double-stranded DNA (dsDNA) in the cytoplasm and subsequently initiating the expression and secretion of type 1 interferons (IFN-I). This publication identifies the cGAS/STING-mediated production of IFN-I by NPCs as a key mediator of RILD. The authors propose that RT induces massive hepatocyte apoptosis, resulting in a large amount of ectopic dsDNA which is then taken up by liver NPCs, resulting in the activation of cGAS and subsequently STING. In order to determine this, the group exposed wild-type (WT), cGAS knockout, and STING knockout mice to 30Gy of radiation. While livers of WT mice subjected to radiation showed increased steatosis (retention of lipids), mice lacking either cGAS or STING showed less at 48 hours as measured by histological staining. The knockout mice also showed reduced apoptosis in liver tissue at 48 hours as measured by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay of histological sections. Additionally, histological staining of mouse liver tissues six weeks after radiation showed that the knockout mice had less veno-occlusive inflammation, an indicator of RILD. Next, the group showed that hepatocytes extracted from mice 24 hours following irradiation secrete much more dsDNA in vitro than NPCs extracted from the same liver. Furthermore, levels of cGAS, STING, IFN-α, IFN-β, and TLR9 mRNA transcripts were found to increase dramatically in liver NPCs but not in hepatocytes following radiation as measured by RT-qPCR. Additionally, expression levels of cGAS/STING-related genes TBK1, IRF3, ISG15, JAK1, TYK2, AKT1, AGBL5, TRIM32, RSAD2, and TTL4 were all increased in liver NPCs but not in hepatocytes following radiation. The group then showed that DNase treatment of mice during and after RT prevented increased expression levels of cGAS, STING, IFN-α, and IFN-β mRNAs. This result indicates that extracellular DNA is a trigger for RT-induced IFN-I secretion. Finally, the group showed that knockout of the IFNα and IFNβ receptors in mice reduced the amount of liver steatosis and apoptosis caused by RT. Additionally, blockade of IFN-I signaling with an interferon alpha and beta receptor subunit 1 (IFNAR1)-specific antibody did not negatively affect the tumor-reducing properties of RT in a mouse HCC model. This paper indicates that cGAS/STING-signaling in liver NPCs is a major cause of RILD. Extracellular DNA from hepatocytes killed during RT is taken up by NPCs where it activates cGAS/STING signaling to produce IFN-I. This finding could help scientists and clinicians devise ways to prevent RILD in patients undergoing RT for HCC or other cancers. Perhaps short-term immune modulators may be used in tandem with RT to prevent an excessive response of the innate immune system.
Role of Hepatitis B Virus Capsid Phosphorylation in Nucleocapsid Disassembly and Covalently Closed Circular DNA Formation – PLOS Pathogens
This paper from Dr. Jianming Hu’s laboratory at the Pennsylvania State University College of Medicine in Hershey, PA outlines the role of phosphorylation of the HBV core protein (HBc) in the HBV life cycle. HBV has a relaxed circular (RC) DNA genome which it delivers to the nucleus of hepatocytes. In the nucleus, the RC DNA is converted into covalently closed circular (CCC) DNA which is the viral transcriptional template for all HBV mRNA species including pregenomic RNA (pgRNA). Along with the viral reverse transcriptase (RT), pgRNA is packaged by HBc into newly formed nucleocapsids (NC) where it is reverse-transcribed to form RC DNA resulting in mature NCs. Mature NCs may either be enveloped and secreted as infectious virions or uncoat within the cell and further contribute to CCC DNA formation. Because CCC DNA is the reservoir of HBV in infected hepatocytes, its eradication is highly sought after and is required to achieve a true cure for the virus. This publication reports a model wherein HBc phosphorylation by the host protein cyclin-dependent kinase 2 (CDK2) facilitates the uncoating of newly formed NCs and their subsequent formation of CCC DNA. Previously, this group has found that CDK2 is a host kinase which is incorporated into HBV NCs. CDK2 is a highly conserved kinase (phosphorylating protein) which is essential during the G1, S, and G2 phases of the cell cycle. First, the group identified two S-P (serine-proline) motifs on the globular N-terminal domain (NTD) of HBc, S44 and S49 which are potential CDK2 substrates that are on the interior surface of assembled NCs. In order to mimic constitutive phosphorylation or to block phosphorylation of the serine residues, they were mutated to glutamic acid residues (N2E) or alanine residues (N2A) respectively. The phospho-mimetic mutant N2E showed decreased levels of pgRNA packaging into NCs as measured by native agarose gel electrophoresis (NAGE) and Southern blot following transfection of the constructs into HepG2 cells. After release from NCs into the nucleus, the RC DNA HBV genome takes the form of protein free (PF) RC DNA lacking the RT protein, prior to forming CCC DNA. The phospho-mimetic N2E mutant yielded more PF-RC DNA and CCC DNA than wild type (WT) HBV and conversely, the phospho-null N2A mutant yielded less of both species than WT HBV. These results show that while NCs phosphorylated at both S44 and S49 are less efficient at packaging pgRNA, they are more likely to uncoat and release their genomes into the nucleus. Next, PhoenixBio (PXB) primary human hepatocytes harvested from human-liver chimeric mice were infected with HBV and treated with two CDK2 small molecule inhibitors. PF DNA was then extracted from the cells and analyzed via Southern blot. Both CDK2 inhibitors dramatically reduced the level of CCC DNA formation as compared to the mock control. This result indicates that CDK2 activity within NCs modulates their stability causing them to uncoat and deliver their genomes to the nucleus as opposed to being exported as virions. This publication sheds light on the exact stages of HBc phosphorylation and how they affect CCC DNA formation. This work is important because understanding the molecular mechanisms of CCC DNA formation will help in the development HBV antivirals. Small molecules which interfere with specific stages of HBc phosphorylation and dephosphorylation may prove efficacious in preventing CCC DNA formation in individuals chronically infected with HBV.
Metabolites released from apoptotic cells act as tissue messengers – Nature
This paper from the University of Charlottesville in Virginia investigates the “apoptotic metabolite secretome” and its effect on neighboring cells. Apoptosis is a highly regulated form of programmed cell death (PCD) which accounts for approximately 90% of homeostatic cell turnover. Metabolites are small molecules that are the intermediates or end products of metabolism. Here, a panel of conserved apoptotic metabolites was identified in the supernatants of apoptotic cells using advanced spectroscopy techniques (spectroscopy-based metabolomics). Six metabolites were found to be secreted across a variety of cell types in response to various apoptosis inducers. These six metabolites are: adenosine monophosphate (AMP), guanosine 5′-monophosphate (GMP), creatine, spermidine, glycerol-3-phosphate (G3P), and adenosine triphosphate (ATP). These metabolites were all found in the supernatants of Jurkat cells (acute T cell leukemia) following exposure to UV irradiation as well as following treatment with anti-Fas antibody. These metabolites were also released from primary mouse bone-marrow-derived macrophages (BMDMs) treated with anthrax and primary mouse thymocytes treated with anti-Fas antibody. Additionally, lung and colon cancer cell lines A549 and HCT116 released four of these metabolites (ATP, spermadine, G3P, and creatine) when subjected to the BH3-mimetic ABT-737 (induces mitochondrial outer membrane permeabilization) as measured using commercial kits. Secretion of these metabolites was prevented by pretreatment of cells with the pan-caspase inhibitor zVAD, indicating apoptosis as the mechanism of release. The metabolites alanine, pyruvate, and creatinine were retained within apoptotic cells, showing that metabolite release was organized and not due to nonspecific rupture of apoptotic bodies. Because only specific metabolites were released during apoptosis, the group hypothesized that the opening of plasma membrane channels may determine the apoptotic secretome. Pannexin 1 (PANX1) is a membrane channel activated by caspase 3 and 7 cleavage during apoptosis. Previously, this group has demonstrated that PANX1 activation is responsible for the secretion of ATP and UTP from apoptotic cells, which function as “find me” signals to recruit phagocytes to perform efferocytosis. In order to determine the role of PANX1 activation in the apoptotic secretome, prior to UV irradiation, PANX1 was inhibited in Jurkat cells using two methods: pharmacological inhibition with the drugs trovafloxacin (Trovan) or spironolactone and generation of a cell line bearing a dominant-negative PANX1 mutation at the caspase cleavage site. Jurkat cells with inhibited or nonfunctional PANX1 showed less secretion of 25 metabolites released from UV-treated Jurkat cells as measured by spectroscopy-based metabolomics. Spermidine, GMP, AMP, and G3P were all secreted dependent upon PANX1 activation. Next, to test whether metabolic activity within the dying cell affects its secretome, the group chose to focus on the release of spermidine. Spermidine released from apoptotic cells naturally reduces local inflammation and counteracts autoimmunity. Interestingly, while spermidine was heavily secreted from apoptotic cells, its precursor molecule putrescine was not released at all. As the starting product of spermidine synthesis is arginine, the isotope carbon-13 (13C)-containing argenine was administered to Jurkat cells one minute prior to UV irradiation. Apoptotic cells showed 40% and 25% more incorporation of 13C label into putrescine and spermidine respectively than live cells at one hour post-UV. This indicates that in addition to the caspase-dependent opening of membrane channels, apoptotic cells also maintain or even upregulate certain metabolic pathways to contribute to the apoptotic secretome. Next, in order to test the effect of the apoptotic secretome on neighboring cells, supernatant from apoptotic Jurkat cells was administered to LR73 cells (phagocytic, Chinese hamster ovary). RNA-sequencing analysis of the LR73 cells after four hours in the apoptotic supernatant revealed altered transcription of programs linked to cytoskeletal rearrangements, inflammation, wound healing or tissue repair, antiapoptotic functions, metabolism and the regulation of cell size within the phagocyte. Finally, the group used two concoctions of PANX1-dependent metabolites to treat mouse models of inflammatory arthritis and lung-transplant rejection. Treatment with the metabolite mixtures resulted in significantly reduced inflammation and better clinical outcomes in both inflammatory disease models. This publication shows that apoptotic cells affect their microenvironment by secreting anti-inflammatory metabolites. It also demonstrates that apoptosis may be harnessed to ameliorate inflammatory diseases. Once fully elucidated, other forms of PCD may also prove useful in treating other diseases such as cancer and viral infections.
Meet our guest blogger, David Schad, B.Sc., Junior Research Fellow at the Baruch S. Blumberg Institute studying programmed cell death such as apoptosis and necroptosis in the context of hepatitis B infection under the direction of PI Dr. Roshan Thapa. David also mentors high school students from local area schools as part of an after-school program in the new teaching lab at the PA Biotech Center. His passion is learning, teaching and collaborating with others to conduct research to better understand nature.
