Hepatitis B Core Antigen Research Articles

ATM-Dependent Phosphorylation of Hepatitis B Core Protein in Response to Genotoxic Stress.

Chronic hepatitis caused by infection with the Hepatitis B virus is a life-threatening condition. In fact, 1 million people die annually due to liver cirrhosis or hepatocellular carcinoma. Recently, several studies demonstrated a molecular connection between the host DNA damage response (DDR) pathway and HBV replication and reactivation. Here, we investigated the role of Ataxia-telangiectasia-mutated (ATM) and Ataxia telangiectasia and Rad3-related (ATR) PI3-kinases in phosphorylation of the HBV core protein (HBc). We determined that treatment of HBc-expressing hepatocytes with genotoxic agents, e.g., etoposide or hydrogen peroxide, activated the host ATM-Chk2 pathway, as determined by increased phosphorylation of ATM at Ser1981 and Chk2 at Thr68. The activation of ATM led, in turn, to increased phosphorylation of cytoplasmic HBc at serine-glutamine (SQ) motifs located in its C-terminal domain. Conversely, down-regulation of ATM using ATM-specific siRNAs or inhibitor effectively reduced etoposide-induced HBc phosphorylation. Detailed mutation analysis of S-to-A HBc mutants revealed that S170 (S168 in a 183-aa HBc variant) is the primary site targeted by ATM-regulated phosphorylation. Interestingly, mutation of two major phosphorylation sites involving serines at positions 157 and 164 (S155 and S162 in a 183-aa HBc variant) resulted in decreased etoposide-induced phosphorylation, suggesting that the priming phosphorylation at these serine-proline (SP) sites is vital for efficient phosphorylation of SQ motifs. Notably, the mutation of S172 (S170 in a 183-aa HBc variant) had the opposite effect and resulted in massively up-regulated phosphorylation of HBc, particularly at S170. Etoposide treatment of HBV infected HepG2-NTCP cells led to increased levels of secreted HBe antigen and intracellular HBc protein. Together, our studies identified HBc as a substrate for ATM-mediated phosphorylation and mapped the phosphorylation sites. The increased expression of HBc and HBe antigens in response to genotoxic stress supports the idea that the ATM pathway may provide growth advantage to the replicating virus.

In Vivo Modelling of Hepatitis B Virus Subgenotype A1 Replication Using Adeno-Associated Viral Vectors

The paucity of animal models that simulate the replication of the hepatitis B virus (HBV) is an impediment to advancing new anti-viral treatments. The work reported here employed recombinant adeno-associated viruses (AAVs) to model HBV subgenotype A1 and subgenotype D3 replication in vitro and in vivo. Infection with subgenotype A1 is endemic to parts of sub-Saharan Africa, and it is associated with a high risk of hepatocellular carcinoma. Recombinant AAV serotype 2 (AAV2) and 8 (AAV8) vectors bearing greater-than-genome-length sequences of HBV DNA from subgenotype A1 and D3, were produced. Transduced liver-derived cultured cells produced HBV surface antigen and core antigen. Administration of AAV8 carrying HBV subgenotype A1 genome (AAV8-A1) to mice resulted in the sustained production of HBV replication markers over a six-month period, without elevated inflammatory cytokines, expression of interferon response genes or alanine transaminase activity. Markers of replication were generally higher in animals treated with subgenotype D3 genome-bearing AAVs than in those receiving the subgenotype A1-genome-bearing vectors. To validate the use of the AAV8-A1 murine model for anti-HBV drug development, the efficacy of anti-HBV artificial primary-microRNAs was assessed. Significant silencing of HBV markers was observed over a 6-month period after administering AAVs. These data indicate that AAVs conveniently and safely recapitulate the replication of different HBV subgenotypes, and the vectors may be used to assess antivirals’ potency.

Sustained Antiviral and Liver Protection by a Nasal Therapeutic Vaccine (NASVAC, Containing Both HBsAg and HBcAg) in Patients with Chronic Hepatitis B: 2-Year Follow-Up of Phase III Clinical Trial.

A phase III clinical trial in treatment-naïve patients with chronic hepatitis B (CHB) revealed the safety and considerable therapeutic efficacy of a vaccine containing both hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) (NASVAC) at the end of treatment (EOT) and 24 weeks after EOT. Two years after EOT, we checked HBV DNA, alanine aminotransferase (ALT), and hepatitis B e antigen (HBeAg). The data reveal that 33 of 66 NASVAC-recipient CHB patients became negative for HBV DNA in the blood two years after EOT. The ALT levels were within the upper limit of normal (ULN) in 37 patients, although all 66 CHB patients had elevated ALT (above ULN) before the start of therapy. Out of the total twelve HBeAg-positive patients, eight patients became negative for HBeAg. None of the patients developed cirrhosis of the liver within this period. NASVAC is a finite treatment regimen with sustained antiviral and liver-protecting properties. This study is the first to report follow-up data of immune therapy for CHB. NASVAC, an immune therapy of finite duration, is endowed with sustained antiviral and liver protection properties in CHB patients.

Molecular and Serological Characterization of Hepatitis B Virus (HBV)-Positive Samples with Very Low or Undetectable Levels of HBV Surface Antigen.

Background: Gaps remain in the detection of nucleic acid test (NAT) yield and occult hepatitis B virus (HBV) infection (OBI) by current HBV surface antigen (HBsAg) assays. The lack of detection may be due to HBsAg levels below current assay detection limits, mutations affecting HBsAg assays or HBsAg levels, or the masking of HBsAg by antibody to HBsAg (anti-HBs). In this study, we evaluate the incremental detection of NAT yield and OBI from five diverse geographic areas by an improved sensitivity HBsAg assay and characterize the samples relative to the viral load, anti-HBs status, and PreS1–S2–S mutations. Included is a comparison population with HBV DNA levels comparable to OBI, but with readily detectable HBsAg (High Surface–Low DNA, HSLD). Methods: A total of 347 samples collected from the USA, South Africa, Spain, Cameroon, Vietnam, and Cote D’Ivoire representing NAT yield (HBsAg(−), antibody to HBV core antigen (anti-HBc)(−), HBV DNA(+), N = 131), OBI (HBsAg(−), anti-HBc(+), HBV DNA(+), N = 188), and HSLD (HBsAg(+), anti-HBc(+), HBV DNA(+), N = 28) were tested with ARCHITECT HBsAg NEXT (HBsAgNx) (sensitivity 0.005 IU/mL). The sequencing of the PreS1–S2–S genes from a subset of 177 samples was performed to determine the genotype and assess amino acid variability, particularly in anti-HBs(+) samples. Results: HBsAgNx detected 44/131 (33.6%) NAT yield and 42/188 (22.3%) OBI samples. Mean HBV DNA levels for NAT yield and OBI samples were lower in HBsAgNx(−) (50.3 and 25.9 IU/mL) than in HBsAgNx(+) samples (384.1 and 139.5 IU/mL). Anti-HBs ≥ 10 mIU/mL was present in 28.6% HBsAgNx(+) and 45.2% HBsAgNx(−) OBI, and in 3.6% HSLD samples. The genotypes were A1, A2, B, C, D, E, F, and H. There was no significant difference between HBsAgNx(−) and HBsAgNx(+) in the proportion of samples harboring substitutions or in the mean number of substitutions per sample in PreS1, PreS2, or S for the NAT yield or OBI (p range: 0.1231 to >0.9999). A total of 21/27 (77.8%) of HBsAgNx(+) OBI carried S escape mutations, insertions, or stop codons. HSLD had more PreS1 and fewer S substitutions compared to both HBsAgNx(−) and HBsAgNx(+) OBI. Mutations/deletions associated with impaired HBsAg secretion were observed in the OBI group. Conclusions: HBsAgNx provides the improved detection of NAT yield and OBI samples. Samples that remain undetected by HBsAgNx have exceptionally low HBsAg levels below the assay detection limit, likely due to low viremia or the suppression of HBsAg expression by host and viral factors.

Structural and Functional Characterizations of Cancer Targeting Nanoparticles Based on Hepatitis B Virus Capsid.

Cancer targeting nanoparticles have been extensively studied, but stable and applicable agents have yet to be developed. Here, we report stable nanoparticles based on hepatitis B core antigen (HBcAg) for cancer therapy. HBcAg monomers assemble into spherical capsids of 180 or 240 subunits. HBcAg was engineered to present an affibody for binding to human epidermal growth factor receptor 1 (EGFR) and to present histidine and tyrosine tags for binding to gold ions. The HBcAg engineered to present affibody and tags (HAF) bound specifically to EGFR and exterminated the EGFR-overexpressing adenocarcinomas under alternating magnetic field (AMF) after binding with gold ions. Using cryogenic electron microscopy (cryo-EM), we obtained the molecular structures of recombinant HAF and found that the overall structure of HAF was the same as that of HBcAg, except with the affibody on the spike. Therefore, HAF is viable for cancer therapy with the advantage of maintaining a stable capsid form. If the affibody in HAF is replaced with a specific sequence to bind to another targetable disease protein, the nanoparticles can be used for drug development over a wide spectrum.

Cango Lyec (Healing the Elephant): Chronic Hepatitis B Virus among post-conflict affected populations living in mid-Northern Uganda.

BackgroundThe legacy of war in Northern Uganda continues to impact people’s health and wellbeing in the Acholi region. Despite increasing attention to Hepatitis B Virus (HBV) in Uganda and globally, concerns remain that unique drivers of infection, and barriers to screening, and treatment, persist among those affected by conflict.MethodsCango Lyec (Healing the Elephant) cohort survey involved conflict-affected adults aged 13–49 in three mid-Northern Uganda districts (Gulu, Amuru and Nwoya). Baseline (2011–2012) samples were tested for HBV surface antigen (HBsAg), HBV e-antigen (HBeAg), antibodies to HBV surface antigen (HBsAb), antibodies to HBV e-antigen (HBeAb), and antibodies to HBV core antigen (HBcAb). All HBsAg positive samples were tested for IgM antibodies to HBV B core antigen (HBc-IgM) and where available, >6-month follow-up samples were tested for HBeAg and HBV DNA. Data were analyzed using STATA 15 software. Logistic regression accounted for variance due to complex two-stage sampling that included stratification, unequal selection probabilities and community clustering. Odds ratios measured effect potential risk factors associated with chronic HBV infection.ResultsAmong 2,421 participants, 45.7% were still susceptible to HBV infection. HBsAg seropositivity was 11.9% (10.9–13.0), chronic HBV was 11.6% (10.4–12.8), acquired immunity resulting from vaccination was 10.9%, and prior natural infection was 31.5%. Older age (OR:0.570; 95%CI:0.368–0.883) and higher education (OR:0.598; 95%CI:0.412–0.868) were associated with reduced odds of chronic HBV infection. Being male (OR:1.639; 95%CI:1.007–2.669) and having been abducted (OR:1.461; 95%CI:1.055–2.023) were associated with increased odds of infection. Among women, having 1 or 2 pregnancies (compared to none or >2) was associated with increased odds of infection (OR:1.764; 95%CI:1.009–3.084).ConclusionChronic HBV is endemic in Gulu, Amuru and Nwoya districts. Recommended strategies to reduce post-conflict prevalence include establishment of Northern Uganda Liver Wellness Centres, integration of screening and treatment into antenatal care, and roll out of birth-dose vaccination.

Ultrasmall Copper (I) Sulfide Nanoparticles Prevent Hepatitis B Virus Infection

AbstractHepatitis B virus (HBV) poses a severe threat to public health and social development. Here, we synthesized 4±0.5 nm copper (I) sulfide (Cu2S) nanoparticles (NPs) with 46 mdeg chiroptical property at 530 nm to selectively cleavage HBV core antigen (HBcAg) and effectively blocked HBV assembly and prevented HBV infection both in vitro and in vivo under light at 808 nm. Experimental analysis showed that the chiral Cu2S NPs specific bound with the functional domain from phenylalanine23 (F23) to leucine30 (L30) from HBcAg primary sequence and the cutting site was between amino acid residues F24 and proline25 (P25). Under excitation at 808 nm, the intracellular HBcAg concentration was reduced by 95 %, and in HBV transgenic mice, the levels of HBV surface antigen (HBsAg) and HBV DNA were decreased by 93 % and 86 %, respectively. Together, these results reveal the potential nanomedicine for HBV control and provide fresh tools for viral infection.

Ultrasmall Copper (I) Sulfide Nanoparticles Prevent Hepatitis B Virus Infection

AbstractHepatitis B virus (HBV) poses a severe threat to public health and social development. Here, we synthesized 4±0.5 nm copper (I) sulfide (Cu2S) nanoparticles (NPs) with 46 mdeg chiroptical property at 530 nm to selectively cleavage HBV core antigen (HBcAg) and effectively blocked HBV assembly and prevented HBV infection both in vitro and in vivo under light at 808 nm. Experimental analysis showed that the chiral Cu2S NPs specific bound with the functional domain from phenylalanine23 (F23) to leucine30 (L30) from HBcAg primary sequence and the cutting site was between amino acid residues F24 and proline25 (P25). Under excitation at 808 nm, the intracellular HBcAg concentration was reduced by 95 %, and in HBV transgenic mice, the levels of HBV surface antigen (HBsAg) and HBV DNA were decreased by 93 % and 86 %, respectively. Together, these results reveal the potential nanomedicine for HBV control and provide fresh tools for viral infection.

A novel therapeutic HBV vaccine candidate induces strong polyfunctional cytotoxic T cell responses in mice

Background & AimsCurrent standard-of-care suppresses HBV replication, but does not lead to a functional cure. Treatment aiming to cure chronic hepatitis B (CHB) is believed to require the induction of strong cellular immune responses, such as by therapeutic vaccination.MethodsWe designed a therapeutic HBV vaccine candidate (YF17D/HBc-C) using yellow fever vaccine YF17D as a live-attenuated vector to express HBV core antigen (HBc). Its ability to induce potent cellular immune responses was assessed in a mouse model that supports flavivirus replication.ResultsFollowing a HBc protein prime, a booster of YF17D/HBc-C was found to induce vigorous cytotoxic T cell responses. In a direct head-to-head comparison, these HBc-specific responses exceeded those elicited by adenovirus-vectored HBc. Target-specific T cells were not only more abundant, but also showed a higher degree of polyfunctionality, with HBc-specific CD8+ T cells producing interferon γ and tumour necrosis factor α in addition to granzyme B. This immune phenotype translated into a superior cytotoxic effector activity toward HBc-positive cells in YF17D/HBc-C vaccinated animals in vivo.ConclusionsThe results presented here show the potential of YF17D/HBc-C as a vaccine candidate to treat CHB, and warrant follow-up studies in preclinical animal models of HBV persistence in which other candidate vaccines have been unable to achieve a sustained virologic response.Lay summaryResolution of CHB requires the induction of strong cellular immune responses. We used the yellow fever vaccine as a vector for HBV antigens and show that it is capable of inducing high levels of HBV-specific T cells that produce multiple cytokines simultaneously and are cytotoxic in vivo.

Prevalence study of different methods for hepatitis B virus diagnosis with blood donor people in Baghdad city

Background: Globally, infecting with hepatitis B virus (HBV) is a big issue today. The present study was aimed to assess the prevalence and year-wise trend of the HBV surfaces antigens (HBs-Ag), antibodies vs. HBV core antigens (anti-HBcs) while HBV-DNA in serum sample patients and replacement blood donors in Baghdad, Iraq. Methods: During the period between 2017 and 2019, a total of 212 blood donors of ages 18–70 years were screened for HBs-Ag, anti-HBs by enzyme-linked immunosorbent assay (ELISA) technique, and anti-HBc antigen–antibody (Ag–Ab) and HBV-DNA using real-time PCR. Results: A total of 110 (51.8%) of 212 blood donors were positive to all tests: HBs-Ag, anti-Hbs, anti-HBc and HBV-DNA. Forty-six (41.8%) were positive for HBV-DNA, HBs-Ag and anti-HBc, whereas 14 (12.7%) were positive for HBs-Ag and anti-HBc. Although nine (8.1%) blood donors were positive in real-time PCR (HBV-DNA loaded) with a negative result in ELISA. Forty one (37.2%) markers indicated a high number of inveterate or resolvable contagion. The total diffusion increment significance in 2017–2018, stabilized thereafter and started decreasing from 2018 to 2019 marginally. There is a high significance variance at (P < 0.01) while comparison among examined groups. Conclusions: Significance and impact of study: the prevalence of HBV infection in 3 years is lower in Baghdad blood donors (51.8%) compared to other regions of Iraq. A high number of infections with no signs of HBs-Ag or viral DNA were recorded. Therefore, the inclusion of anti-HBc seromarker and DNA in the routine screening could isolate potential infectious blood units.

Occult and active hepatitis B virus detection in donated blood in São Paulo, Brazil.

The present study determined the HBV antigen, antibody, and DNA status in blood donations deemed to be HBV positive. Individuals with an occult HBV infection (OBI), defined as being positive for HBV DNA but negative for HBV surface antigen (HBsAg), as well as those with active infection (HBsAg-positive), were identified and characterized. From a total pool if 198,363 blood donations, we evaluated in a cross-sectional study, 1106 samples that were positive in screening tests for antibody to HBV core antigen (HBcAb), HBsAg, and/or HBV DNA by nucleic acid testing (NAT-HBV). The presence of genetic variants in the HBV pol/S gene in individuals with an active HBV infection was also determined. OBIs were detected in six of 976 samples (0.6%) that were positive only for HBcAb. The rate of HBV active infection was 0.024% (48/198,363) and there was a predominance of HBV sub-genotype A1 (62.2%, 28/45), followed by D3 (17.8%, 8/45). Mutations in the S gene were found in 57.8% (26/45) and immune escape mutations in 37.8% (17/45) of active HBV-infected donors. Among them, T123N, G145A, and D144G high-impact immune escape mutations were identified. Highly sensitive molecular tests improve the capacity to detect OBIs. When NAT is performed in pooled samples, HBcAb test has value in the detection of donors with OBI and improves transfusion safety. Mutations in the S gene are frequent in HBsAg-positive blood, including those associated with diagnostic failure and vaccine escape mutations.

HEPATITIS B VIRUS AND ITS GENOTYPES CIRCULATING IN INDIA

Hepatitis B virus (HBV), a DNA virus, is responsible for the acute and chronic hepatitis and lever carcinoma. Worldwide, there are 300 to 350 million people are chronically infected with the virus and 6 lac death occur due to chronic hepatitis and HBV associated liver cirrhosis and lever carcinoma. HBV show variable nucleotide diversity in its genome on the basis of which the virus is classied into ten genotypes (A-J). The HBV infection is mainly spread parenterally, and through intimate contacts. HBV infection is manifested as different diseases such as acute hepatitis, chronic hepatitis, acute or spontaneous exacerbation of chronic hepatitis, fulminant hepatitis, liver cirrhosis, hepatocellular carcinoma and hepatic decompensation. The HBV genome is a relaxed circular, partially double stranded DNAof approximately 3,200 base pairs. There are four partially overlapping open reading frames encoding the envelope (pre-S/S), core (pre-core /core), polymerase, and X proteins. The pre-S/S open reading frame encodes the large, middle, and small surface glycoproteins. Pre-core / core open reading frame is translated into pre-core polypeptide which is modied in to a soluble protein, the hepatitis B e antigen (HBeAg), and the nucleocapsid core protein hepatitis B core antigen (HBcAg).

Escherichia Coli-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles.

Tandem-core hepatitis B core antigen (HBcAg) virus-like particles (VLPs), in which two HBcAg monomers are joined together by a peptide linker, can be used to display two different antigens on the VLP surface. We produced universal influenza vaccine candidates that use this scaffold in an Escherichia coli-based cell-free protein synthesis (CFPS) platform. We then used the CFPS system to rapidly test modifications to the arginine-rich region typically found in wild-type HBcAg, the peptide linkers around the influenza antigen inserts, and the plasmid vector backbone to improve titer and quality. Using a minimal plasmid vector backbone designed for CFPS improved titers by at least 1.4-fold over the original constructs. When the linker lengths for the influenza inserts were more consistent in length and a greater variety of codons for glycine and serine were utilized, titers were further increased to over 70 μg/mL (4.0-fold greater than the original construct) and the presence of lower molecular weight product-related impurities was significantly reduced, although improvements in particle assembly were not seen. Furthermore, any constructs with the C-terminal arginine-rich region removed resulted in asymmetric particles of poor quality. This demonstrates the potential for CFPS as a screening platform for VLPs.

Seroprevalence of hepatitis B virus infection markers among children in Ukraine, 2017

BackgroundBefore hepatitis B vaccine (HepB) introduction, level of endemicity of hepatitis B virus (HBV) in Ukraine was estimated as intermediate but the prevalence of HBV infection markers has not been measured in population-based serosurveys. Coverage with 3 doses of HepB, introduced in 2002, was 92%-98% during 2004–2007 but declined to 21%-48% during 2010–2016. To obtain data on HBV prevalence among children born after HepB introduction, we tested specimens from a serosurvey conducted in Ukraine in 2017, following circulating vaccine-derived poliovirus outbreak in 2015, among birth cohorts eligible for polio immunization response. MethodsThe serosurvey was conducted in Zakarpattya, Sumy, and Odessa provinces, and Kyiv City, targeting 2006–2015 birth cohorts. One-stage cluster sampling in the provinces and stratified simple random sampling in Kyiv were used for participant selection. All participants were tested for antibodies against HBV core antigen (anti-HBc). Anti-HBc-positive children were tested for HBV surface antigen (HBsAg). We also obtained information on HepB vaccination status for all children. ResultsOf 4,596 children tested, 81 (1.8%) were anti-HBc-positive and eight (0.2%) were HBsAg-positive. HBsAg prevalence was 0.7% (95% confidence interval, 0.3%-1.4%) in Zakarpattya, 0.1% (0.0%-0.4%) in Sumy, 0% (0.0%-03%) in Odessa, and 0.1% (0.0%-0.8%) in Kyiv. Across survey sites, the proportion of recipients of ≥ 3 HepB doses was 53%-80% in the 2006–2009 cohort and 28%-59% in the 2010–2015 cohort. ConclusionHBV prevalence among children in surveyed regions of Ukraine in 2017 was low, including in Zakarpattya—the only site above the 0.5% European Regional target for HBsAg seroprevalence. However, HepB vaccination was suboptimal, particularly among children born after 2009, resulting in large numbers of unvaccinated or incompletely vaccinated children at risk of future HBV infection. HepB coverage should be increased to further reduce HBV transmission among children in Ukraine and achieve regional and global hepatitis B control/elimination targets.

Coverage with Timely Administered Vaccination against Hepatitis B Virus and Its Influence on the Prevalence of HBV Infection in the Regions of Different Endemicity.

Universal hepatitis B vaccination of newborns was implemented in Russia starting from 1998. From 1998 to 2019, the incidence of acute hepatitis B reduced from 43.8 to 0.57 cases per 100,000 population. Here, we assessed the timely coverage of newborns with the birth dose (HepB-BD), second dose (HepB-2nd), and three vaccine doses (HepB3) in two remote regions of Russia with low (Belgorod Oblast) and high (Yakutia) levels of hepatitis B virus (HBV) endemicity. Vaccination data were obtained from the medical records of 1000 children in Yakutia and 2182 children in Belgorod Oblast. Sera of healthy volunteers from Belgorod Oblast (n = 1754) and Yakutia (n = 1072) across all age groups were tested for serological markers of HBV to assess the infection prevalence and herd immunity. Average HepB-BD coverage was 99.2% in Yakutia and 89.4% in Belgorod Oblast (p < 0.0001) and in both regions varied significantly, from 66% to 100%, between medical centers. The principal reason for the absence of HepB-BD was parent refusal, which accounted for 63.5% of cases of non-vaccination (83/123). While timely HepB-2nd coverage was only 55.4%–64.7%: HepB3 coverage by the age of one year exceeded 90% in both study regions. HBV surface antigen (HBsAg) prevalence in the 1998–2019 birth cohort was 0.2% (95% CI: 0.01–1.3%) in Belgorod Oblast and 3.2% (95% CI: 1.9–5.2%) in Yakutia. The proportion of persons testing negative for both antibodies to HBsAg (anti-HBs) and antibodies to HBV core antigen (anti-HBc) in the 1998–2019 birth cohort was 26.2% (125/481) in Belgorod Oblast and 32.3% (162/501) in Yakutia. We also assessed the knowledge of and attitude towards vaccination among 782 students and teachers of both medical and non-medical specialties from Belgorod State University. Only 60% of medical students knew that hepatitis B is a vaccine-preventable disease. Both medical and nonmedical students, 37.8% and 31.3%, respectively, expressed concerns about safety and actual necessity of vaccination. These data indicate the need to introduce a vaccine delivery audit system, improve medical education with respect to vaccination strategies and policies, and reinforce public knowledge on the benefits of vaccination.

Plant-Produced Antigen Displaying Virus-Like Particles Evokes Potent Antibody Responses against West Nile Virus in Mice.

In this study, we developed a hepatitis B core antigen (HBcAg)-based virus-like particle (VLP) that displays the West Nile virus (WNV) Envelope protein domain III (wDIII) as a vaccine candidate for WNV. The HBcAg-wDIII fusion protein was quickly produced in Nicotiana benthamiana plants and reached a high expression level of approximately 1.2 mg of fusion protein per gram of leaf fresh weight within six days post gene infiltration. Electron microscopy and gradient centrifugation analysis indicated that the introduction of wDIII did not interfere with VLP formation and HBcAg-wDIII successfully assembled into VLPs. HBcAg-wDIII VLPs can be easily purified in large quantities from Nicotiana benthamiana leaves to >95% homogeneity. Further analysis revealed that the wDIII was displayed properly and demonstrated specific binding to an anti-wDIII monoclonal antibody that recognizes a conformational epitope of wDIII. Notably, HBcAg-wDIII VLPs were shown to be highly immunogenic and elicited potent humoral responses in mice with antigen-specific IgG titers equivalent to that of protective wDIII antigens in previous studies. Thus, our wDIII-based VLP vaccine offers an attractive option for developing effective, safe, and low-cost vaccines against WNV.

Specific determination of hepatitis B e antigen by antibodies targeting precore unique epitope facilitates clinical diagnosis and drug evaluation against hepatitis B virus infection

ABSTRACT Hepatitis B e antigen (HBeAg) is a widely used marker both for chronic hepatitis B (CHB) clinical management and HBV-related basic research. However, due to its high amino acid sequence homology to hepatitis B core antigen (HBcAg), most of available anti-HBe antibodies are cross-reactive with HBcAg resulting in high interference against accurate measurement of the status and level of HBeAg. In the study, we generated several monoclonal antibodies (mAbs) targeting various epitopes on HBeAg and HBcAg. Among these mAbs, a novel mAb 16D9, which recognizes the SKLCLG (aa −10 to −5) motif on the N-terminal residues of HBeAg that is absent on HBcAg, exhibited excellent detection sensitivity and specificity in pairing with another 14A7 mAb targeting the HBeAg C-terminus (STLPETTVVRRRGR, aa141 to 154). Based on these two mAbs, we developed a novel chemiluminescent HBeAg immunoassay (NTR-HBeAg) which could detect HBeAg derived from various HBV genotypes. In contrast to widely used commercial assays, the NTR-HBeAg completely eliminated the cross-reactivity with secreted HBcAg from precore mutant (G1896A) virus in either cell culture or patient sera. The improved specificity of the NTR-HBeAg assay enables its applicability in cccDNA-targeting drug screening in cell culture systems and also provides an accurate tool for clinical HBeAg detection.

1055. Assessing the Impact of the Routine Childhood Hepatitis B Immunization and the Need for Hepatitis B Vaccine Birth Dose in Sierra Leone, 2018

BackgroundAll African countries recommend 3 doses of hepatitis B vaccine (HepB3), most at 6, 10, and 14 weeks of age, but few recommend a HepB birth dose (HepB-BD). To evaluate the role of mother to child transmission (MTCT) of hepatitis B virus (HBV) with the 3 dose HepB schedule, we conducted a serosurvey in Sierra Leone among 4–30 month old children and their mothers, and 5–9 year old children.MethodsWe conducted a multi-stage cluster survey in 3 districts. Enumeration areas (EA) were selected by probability proportional to size, followed by random selection of eligible households to identify 1901 children per age group. We tested all participants for HBV surface antigen (HBsAg) by rapid test and collected children’s HepB vaccination history. Serum from all HBsAg+ mothers and 1 HBsAg- mother per EA was tested for total antibodies to HBV core antigen (anti-HBc), HBsAg, HBV e antigen (HBeAg), and HBV DNA. We assessed the association of HBsAg prevalence with HepB vaccination and maternal HBV markers.ResultsAmong 1889 children aged 4–30 months, 20 (1.3%; 95% CI:0.8%–2.0%) were HBsAg+; HepB3 coverage was 85%. Among 2025 children aged 5–9 years, 32 (1.6%; 95% CI:1.1%–2.3%) were HBsAg+; HepB3 coverage was 77%. Of HBsAg+ children, 70% (14/20) of younger and 56% (18/32) of older children received HepB3. Among 1776 mothers of younger children, 169 (9.8%; 95% CI:8.1%–11.7%) were HBsAg+. HBsAg prevalence for children with HBsAg+ mothers was 5.9% (10/169) and 0.7% (6/1605) for those with HBsAg- mothers (adjusted OR=10.6 [95% CI:2.8–40.8]). Of 139 HBsAg+ mothers, 13 (9%) were HBeAg+ and 126 (91%) had detectable HBV DNA. Maternal HBsAg (p=0.026), HBeAg (p< 0.001), and HBV DNA levels ≥ 200,000 IU/mL (p< 0.001) were associated with HBsAg positivity in younger children (Table 1).Table 1: Association of maternal HBV serological and molecular markers with HBsAg positivity in 4–30-month old children — Sierra Leone hepatitis B serosurvey, 2018 ConclusionHBsAg prevalence was much lower among children than among mothers, for whom HepB would not have been available, indicating that routine infant HepB vaccination has substantially lowered HBV burden. Increasing HepB3 coverage could further reduce HBsAg prevalence among children. As HBsAg positivity in young children was strongly associated with having a mother with active HBV infection and > 50% of HBsAg+ children received HepB3, HepB-BD is needed to prevent MTCT of HBV and chronic HBV infections in children.Disclosures All Authors: No reported disclosures

Efficient and reproducible depletion of hepatitis B virus from plasma derived extracellular vesicles.

Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral components, mediating immune responses and likely the spread of the virus. However, the obstacles involved in purifying EVs and removing contaminating viral particles in a reliable and effective manner bottlenecks the full potential for the development of clinical and diagnostic treatment options targeting EV. Because of the similarities in size, density, membrane composition and mode of biogenesis of EVs and virions there are no standardized approaches for virus‐removal from EV preparations yet. Functional EV studies also require EV samples that are devoid of antibody contaminants. Consequently, the study of EVs in virology needs reliable and effective protocols to purify EVs and remove contaminating antibodies and viral particles. Here, we established a protocol for EV purification from hepatitis B virus (HBV)‐containing plasma by a combination of size‐exclusion chromatography and affinity‐based purification. After purification, EV samples were free of virus‐sized particles, HBV surface antigen, HBV core antigen, antibodies or infectious material. Viral genomic contamination was also decreased following purification. By using appropriate antibodies and size parameters, this protocol could potentially be applied to purification of EVs from other viral samples. In summary, we established a fast, reproducible and robust approach for the removal of HBV from EV preparations. Looking forward to the point of purifying EVs from clinical samples, this method should enable studies shedding light on the underlying mechanisms of EVs in viral infections and their diagnostic and prognostic potential.

Genetically engineered fusion of allergen and viral-like particle induces a more effective allergen-specific immune response than a combination of them.

Chimeric virus-like particles (VLPs) were developed as a candidate for allergen-specific immunotherapy. In this study, hepatitis B core antigen (HBcAg) that genetically fused to Chenopodium album polcalcin (Che a 3)-derived peptide was expressed in E. coli BL21, purified, and VLP formation was evaluated using native agarose gel electrophoresis (NAGE) and transmission electron microscopy (TEM). Chimeric HBc VLPs were characterized in terms of their reactivity to IgE, the induction of blocking IgG and allergen-specific IgE, basophil-activating capacity, and Th1-type immune responses. Results from IgE reactivity and basophil activation test showed that chimeric HBc VLPs lack IgE-binding capacity and basophil degranulation activity. Although chimeric HBc VLPs induced the highest level of efficient polcalcin-specific IgG antibody in comparison to those induced by recombinant Che a 3 (rChe a 3) mixed either with HBc VLPs or alum, they triggered the lowest level of polcalcin-specific IgE in mice following immunization. Furthermore, in comparison to the other antigens, chimeric HBc VLPs produced a polcalcin-specific Th1 cell response. Taken together, genetically fusion of allergen derivatives to HBc VLPs, in comparison to a mix of them, may be a more effective way to induce appropriate immune responses in allergen-specific immunotherapy. KEY POINTS: • The insertion of allergen-derived peptide into major insertion region (MIR) of hepatitis B virus core (HBc) antigen resulted in nanoparticles displaying allergen-derived peptide upon its expression in prokaryotic host. • The resultant VLPs (chimeric HBc VLPs) did not exhibit IgE reactivity with allergic patients' sera and were not able to degranulate basophils. • Chimeric HBc VLPs dramatically improved protective IgG antibody response compared with those induced by allergen mixed either with HBc VLPs or alum. • Chimeric HBc VLPs induced Th1 responses that were counterparts of Th2 responses (allergic). • Chimeric HBc VLPs increased IgG2a/ IgG1 ratio and the level of IFN-γ compared to those induced by allergen mixed with either HBc VLPs or alum. Graphical Abstract.