Hepatitis E Virus Capsid as a Carrier of Exogenous Antigens for the Development of Chimeric Virus-Like Particles.

INTRODUCTION: Virus-like particles (VLPs), self-assembled multiprotein structures, can stimulate robust immune responses due to their structural similarity to native virions that allow the presentation of multiple copies of the target epitopes. Utilizing VLPs as vaccine platforms to present exogenous antigens is a promising and challenging approach in the vaccine development field. This study investigates the potential of the truncated hepatitis E virus (HEV) capsid as a VLP platform to present foreign antigens. METHODS: The S and M domains of the HEV capsid protein were selected as the optimal carrier (CaSM). The exogenous antigen Seq8 containing 3 neutralizing epitopes from 3 different foot-and-mouth disease virus (FMDV) strains was linked to the C-terminal of CaSM to construct a chimeric VLP (CaSM-Seq8). The chimeric particles were produced in Escherichia coli, and their morphology, physicochemical properties, antigenicity, and immunogenicity were analyzed. RESULTS: Morphological analysis showed that CaSM-Seq8 self-assembled into VLPs similar to CaSM VLPs (∼26 nm in diameter) but smaller than native HEV virions. Further, the thermal stability and the resistance to enzymatic proteolysis of Seq8 were enhanced when it was attached to the CaSM carrier. The antigenicity analysis revealed a more robust reactivity against anti-FMDV antibodies when Seq8 was presented on CaSM particles. Upon injection into mice, FMDV-specific IgGs induced by CaSM-Seq8 appeared earlier, increased faster, and maintained higher levels for a longer time than those induced by Seq8 alone or the inactivated FMDV vaccine. CONCLUSION: This study demonstrated the potential of utilizing the truncated HEV capsid as an antigen-presenting platform for the development of chimeric VLP immunogens.

Design and development of a chimeric vaccine candidate against zoonotic hepatitis E and foot-and-mouth disease.

BACKGROUND: Zoonotic hepatitis E virus (HEV) infection emerged as a serious threat in the industrialized countries. The aim of this study is exploring a new approach for the control of zoonotic HEV in its main host (swine) through the design and development of an economically interesting chimeric vaccine against HEV and against a devastating swine infection: the foot-and-mouth disease virus (FMDV) infection. RESULTS: First, we adopted a computational approach for rational and effective screening of the different HEV-FMDV chimeric proteins. Next, we further expressed and purified the selected chimeric immunogens in Escherichia coli (E. coli) using molecular cloning techniques. Finally, we assessed the antigenicity and immunogenicity profiles of the chimeric vaccine candidates. Following this methodology, we designed and successfully produced an HEV-FMDV chimeric vaccine candidate (Seq 8-P222) that was highly over-expressed in E. coli as a soluble protein and could self-assemble into virus-like particles. Moreover, the vaccine candidate was thermo-stable and exhibited optimal antigenicity and immunogenicity properties. CONCLUSION: This study provides new insights into the vaccine development technology by using bioinformatics for the selection of the best candidates from larger sets prior to experimentation. It also presents the first HEV-FMDV chimeric protein produced in E. coli as a promising chimeric vaccine candidate that could participate in reducing the transmission of zoonotic HEV to humans while preventing the highly contagious foot-and-mouth disease in swine.

Comprehensive analysis of genetic and evolutionary features of the hepatitis E virus.

BACKGROUND: The hepatitis E virus (HEV) is the causative pathogen of hepatitis E, a global public health concern. HEV comprises 8 genotypes with a wide host range and geographic distribution. This study aims to determine the genetic factors influencing the molecular adaptive changes of HEV open reading frames (ORFs) and estimate the HEV origin and evolutionary history. RESULTS: Sequences of HEV strains isolated between 1982 and 2017 were retrieved and multiple analyses were performed to determine overall codon usage patterns, effects of natural selection and/or mutation pressure and host influence on the evolution of HEV ORFs. Besides, Bayesian Coalescent Markov Chain Monte Carlo (MCMC) Analysis was performed to estimate the spatial-temporal evolution of HEV. The results indicated an A/C nucleotide bias and ORF-dependent codon usage bias affected mainly by natural selection. The adaptation of HEV ORFs to their hosts was also ORF-dependent, with ORF1 and ORF2 sharing an almost similar adaptation profile to the different hosts. The discriminant analysis based on the adaptation index suggested that ORF1 and ORF3 could play a pivotal role in viral host tropism. CONCLUSION: In this study, we estimate that the common ancestor of the modern HEV strains emerged ~ 6000 years ago, in the period following the domestication of pigs. Then, natural selection played the major role in the evolution of the codon usage of HEV ORFs. The significant adaptation of ORF1 of genotype 1 to humans, makes ORF1 an evolutionary indicator of HEV host speciation, and could explain the epidemic character of genotype 1 strains in humans.

Effects of mRNA secondary structure on the expression of HEV ORF2 proteins in Escherichia coli.

BACKGROUND: Viral protein expression in Escherichia coli (E. coli) is a powerful tool for structural/functional studies as well as for vaccine and diagnostics development. However, numerous factors such as codon bias, mRNA secondary structure and nucleotides distribution, have been indentified to hamper this heterologous expression. RESULTS: In this study, we combined computational and biochemical methods to analyze the influence of these factors on the expression of different segments of hepatitis E virus (HEV) ORF 2 protein and hepatitis B virus surface antigen (HBsAg). Three out of five HEV antigens were expressed while all three HBsAg fragments were not. The computational analysis revealed a significant difference in nucleotide distribution between expressed and non-expressed genes; and all these non-expressing constructs shared similar stable 5'-end mRNA secondary structures that affected the accessibility of both Shine-Dalgarno (SD) sequence and start codon AUG. By modifying the 5'-end of HEV and HBV non-expressed genes, there was a significant increase in the total free energy of the mRNA secondary structures that permitted the exposure of the SD sequence and the start codon, which in turn, led to the successful expression of these genes in E. coli. CONCLUSIONS: This study demonstrates that the mRNA secondary structure near the start codon is the key limiting factor for an efficient expression of HEV ORF2 proteins in E. coli. It describes also a simple and effective strategy for the production of viral proteins of different lengths for immunogenicity/antigenicity comparative studies during vaccine and diagnostics development.

HPV16 E6/E7 -based mRNA vaccine is therapeutic in mice bearing aggressive HPV-positive lesions.

HPV (Human papillomavirus) affects 600,000 people worldwide each year. Almost all cervical cancers are associated with a past HPV infection. In particular, the positivity to the high-risk type HPV16 is detected in most of the invasive cervical cancers. FDA has approved prophylactic vaccines that protect against new HPV16 infections, but do not induce immunity in those patients with established infections or neoplasms. To date, no therapeutic vaccine targeting HPV16-associated lesions has been authorized. We have developed an mRNA-based vaccine against the HPV16 late oncoproteins E6 and E7, which are abundantly and exclusively expressed in high-grade squamous intraepithelial lesions (HSILs), a stage of the cervical disease that precedes the progression to carcinoma. Our in vitro and in vivo studies demonstrated that the translated mRNA is functional and elicits an antigen-specific adaptive immune response. Upon immunization with the vaccine, mice with HPV16+ lesions exhibited tumor growth inhibition, extension of lifespan, and development of a protective immune memory. In light of these results and the remarkable clinical success of mRNA vaccines against SARS-CoV2, we believe that our mRNA-based therapeutic vaccine has the potential to offer a non-invasive treatment alternative to the current standard of care for HPV16+ HSILs.

Efficient production and characterization of immunogenic HEV-PCV2 chimeric virus-like particles.

Zoonotic hepatitis E virus (HEV) infection is an emerging global public health concern. It is usually transmitted to humans from domestic pigs (main host). Since virus-like particles (VLPs) exhibit unique structural and immunological characteristics that make them of momentous applications in vaccine development, the purpose of the present study was the production of immunogenic chimeric VLPs as vaccine candidates for the control of zoonotic HEV in its main host and the prevention of porcine circovirus associated disease, a multi-factorial disease with major economic repercussions on global pig industry. An immuno-informatics approach was applied for the design and screening of new chimeric antigens presenting the dominant immunogenic domains of both HEV and porcine circovirus 2 (PCV2). Then, using molecular cloning techniques, the chimeric proteins were expressed in Escherichia coli. After purification, full characterization of the physicochemical, morphological, and immunological properties of the target proteins has been conducted. The chimeric immunogens were successfully overexpressed and after the optimization of the expression conditions, 5 chimeric proteins were efficiently purified under native conditions. The purified HEV-PCV2 chimeric proteins were found thermo-stable and able to self-assemble into spherical virus-like particles. Four HEV-PCV2 chimeric proteins have displayed optimal antigenicity and immunogenicity properties, with the nPCV2cp-p166 chimeric immunogen slightly outranking the other designed proteins. In conclusion, this study reports the production of stable HEV-PCV2 chimeric VLPs that exhibited optimal antigenicity and immunogenicity and thus with potential applications in diagnostics and vaccine development. Besides, this study provides a reproducible approach for the design, assessment, and production of chimeric antigens.

Chitosan Nanoparticles Loaded with Truncated ORF2 Protein as an Oral Vaccine Candidate against Hepatitis E.

In a continuous effort to develop effective vaccines against hepatitis E (HE), oral vaccine nanoparticles using the truncated capsid protein p146 (aa460-605) are formulated and characterized. To improve the immunogenicity of p146, chitosan nanoparticles (CSNPs) are used as a mucosal delivery system. Next, the physical-chemical properties, cytotoxic effects in vitro, and immunogenicity in mice of the produced NPs are analyzed. The results show that the produced CS/p146 NPs are stable and well dispersive and display a near-spherical shape with a mean size of 200-300 nm. The findings also demonstrate high encapsulation efficiency (65-73.9%) and loading capacity (27.7-67.5%) of the formulated nanoparticles. Further, the CS/p146 NPs exhibit low cytotoxicity and an obvious sustained-release effect in vitro. Immunogenicity experiments in mice indicate that CS/p146 NPs can induce antigen-specific systemic and mucosal immune responses higher than the purified p146 do. Besides, the expression levels and mRNA transcription of Interleukin (IL)-4 in spleen cells of CS/p146 NPs-immunized mice are higher than those of p146, indicating that a Th2-mediated cellular immune response is activated by the CS/p146 NPs. Overall, the synthesized CS/p146 NPs display promising properties as a potential HE oral vaccine candidate.

In silico identification of strong binders of the SARS-CoV-2 receptor-binding domain.

The world is currently witnessing the spread of the deadly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease 2019 (COVID-19). In less than three months since the first cases were reported, the World Health Organization declared it a pandemic disease. Although several treatment and prevention strategies are currently under investigation, a continuous effort to investigate and develop effective cures is urgently needed. Thus, we performed molecular docking and structure-based virtual screening of libraries of approved drugs, antivirals, inhibitors of protein-protein interactions, and one million other small molecules to identify strong binders of the SARS-CoV-2 receptor-binding domain (RBD) that might interfere with the receptor recognition process, so as to inhibit the viral cellular entry. According to our screening and selection criteria, three approved antivirals (elbasvir, grazoprevir, and sovaprevir) and 4 other drugs (hesperidin, pamaqueside, diosmin, and sitogluside) were identified as potent binders of the RBD. The binding of these molecules involved several RBD residues required for the interaction of the virus with its cellular receptor. Furthermore, this study also discussed the pharmacological action of the 4 non-antiviral drugs on hematological and neurological disorders that, in addition to inhibiting the viral entry, could be beneficial against the neurological disorders identified in COVID-19 patients. Besides, six other small-molecules were identified, with no pharmacological description so far, exhibiting strong binding affinities to the RBD that we believe worth being investigated as inhibitors of the SARS-CoV-2-receptor interaction.

Role of the GTNGTKR motif in the N-terminal receptor-binding domain of the SARS-CoV-2 spike protein.

The 2019 novel coronavirus disease (COVID-19) that emerged in China has been declared as public health emergency of international concern by the World Health Organization and the causative pathogen was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this report, we analyzed the structural characteristics of the N-terminal domain of the S1 subunit (S1-NTD) of the SARS-CoV-2 spike protein in comparison to the SARS-CoV in particular, and to other viruses presenting similar characteristic in general. Given the severity and the wide and rapid spread of the SARS-CoV-2 infection, it is very likely that the virus recognizes other receptors/co-receptors besides the ACE2. The NTD of the SARS-CoV-2 contains a receptor-binding motif different from that of SARS-CoV, with some insertions that could confer to the new coronavirus new receptor binding abilities. In particular, motifs similar to the insertion 72GTNGTKR78 have been found in structural proteins of other viruses; and these motifs were located in putative regions involved in recognizing protein and sugar receptors, suggesting therefore that similar binding abilities could be displayed by the SARS-CoV-2 S1-NTD. Moreover, concerning the origin of these NTD insertions, our findings point towards an evolutionary acquisition rather than the hypothesis of an engineered virus.

Role of the C-terminal cysteines in virus-like particle formation and oligomerization of the hepatitis E virus ORF2 truncated proteins.

The hepatitis E virus (HEV) ORF2 truncated recombinant proteins can self-assemble into virus-like particles (VLPs) and were used as models to investigate the HEV capsid assembly. However, the structural function of the ORF2 C-terminal domain (C52aa from aa 608 to aa 660) remains unclear. Herein, by analyzing a set of ORF2 truncated proteins expressed in Escherichia coli, we found that the highly conserved C-terminal cysteines play a crucial role in the oligomerization of the truncated ORF2 proteins and in their assembly into VLPs, through the formation of dimer-dimer disulfide bonds; and the treatment of native HEV particles with dithiothreitol (DTT) induced the disassembly of the viral capsid, suggesting that the disulfide bonding is required for stabilizing the native HEV capsid. The present study sheds light on the structural role of the C-terminal region of the HEV capsid protein and contributes to the full understating of the viral capsid assembly process.

Design and immunogenicity analysis of the combined vaccine against zoonotic hepatitis E and foot-and-mouth disease.

AIM: Design and immunogenicity assessment of the combined vaccine candidate against zoonotic hepatitis E virus (HEV) and foot-and-mouth disease virus (FMDV). METHODS: Using the molecular cloning technology, we produced and purified 9 HEV ORF2-truncated proteins (HEV genotype 4). Then, we compared their thermal stability, antigenicity, and immunogenicity to select the best HEV immunogen. Next, we used the adjuvant Montanide ISA-206 to prepare different formulations of HEV vaccine alone, FMDV vaccine alone and HEV-FMDV combined vaccine. The formulations were injected into mice and the induced humoral immune responses were monitored up 12 weeks post-immunization. RESULTS: The HEV p222 protein could self-assemble into VLPs (∼34 nm) and showed higher stability and better antigenicity/immunogenicity than the other HEV antigens, thus it was selected as the best HEV immunogen. Mice immunization with the FMDV vaccine alone induced high FMDV-specific antibody titers in a dose-dependent manner; the HEV p222 protein also induced high levels of anti-HEV antibodies but in a dose-independent manner. The HEV-FMDV combination induced anti-FMDV antibody titers 7-16-fold higher than the titers induced by the FMDV vaccine alone, and HEV-specific antibody titers 2.4-fold higher than those induced by the HEV p222 antigen alone. CONCLUSION: Herein, we proposed a new approach for the control of zoonotic HEV infection through its control in its main host (pig). We also designed the first HEV-FMDV combined vaccine and the preliminary analyses revealed a synergistic effect on the immunogenicity of both HEV and FMDV antigens.

Author Correction: Dimerization: a structural feature for the protection of hepatitis E virus capsid protein against trypsinization.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Dimerization: a structural feature for the protection of hepatitis E virus capsid protein against trypsinization.

Orally-transmitted viruses have evolved in a way to resist the extreme conditions of the host's gastrointestinal environment, especially the proteolysis of their structural proteins. However, the mechanisms allowing these viruses to survive these harsh conditions remain unclear. Hepatitis E virus (HEV) is an orally-transmitted human pathogen. Its capsid protein contains three domains S, P1 and P2. The latter forms a homodimer protruding from the virus shell, making it the most exposed part. By combining biochemical and computational methods, we found the trypsin digestion sites to be highly conserved among the HEV strains. Furthermore, the constructs of the HEV capsid protein that contain an extended P2 domain were digested within the extensions leaving the P2 domain intact. The trypsinization seems to occur in three possible double cleavages at R451-R619, R460-R619 or R460-R631.The dimerization disrupts the trypsin action at three main sites in the P2 domain R542, K544 and K554. These sites are very exposed in the monomeric P2 domain constructs which makes the monomeric forms very susceptible to trypsin action. Therefore, we believe that dimerization is a structural feature that has been selected by the evolutionary forces to render the HEV capsid protein resistant to the host's proteases; an evolutionary feature that could be common to some other (if not all) orally-transmitted viruses.

Binding Preference of Anti-HEV Antibodies in Sera Collected in Algeria for Antigens Derived From HEV Genotype 1 .

BACKGROUND: Two hepatitis E virus (HEV) outbreaks occurred in Algeria (1979 - 1980 and 1987 - 1988). However, to date, no study on the prevalence of anti-HEV antibodies has been conducted in Algeria, and the genotype of the circulating strains remains unclear. OBJECTIVES: This study was conducted to investigate the presence of anti- HEV antibodies among outpatients and blood donors in three different hospitals in Northern Algeria and to determine the genotype of the circulating strains through the characterization of the immunoreactivity of anti-HEV antibodies. METHODS: A total of 590 blood samples (379 from blood donors and 211 from outpatients) were collected in three health facilities in Northern Algeria and assessed for anti-HEV antibodies using an in-house double-antigen sandwich immunoassay. HEV open reading frame 2 recombinant proteins p166 (aa 452 - 617) generated from the four HEV genotypes were used as antigens. The genotype of the strains circulating in Algeria was predicted by an indirect ELISA by assessing the anti-HEV antibodies in serially diluted positive sera using the different p166 proteins. RESULTS: Anti-HEV antibodies were detected in 20.17% of the samples. A significant correlation was found between the age of the subjects and the presence of anti-HEV antibodies (P < 0.001). Among blood donors, 83 (21.9%) were diagnosed positive for anti-HEV antibodies with two cases weakly positive for anti-HEV IgM antibodies. Moreover, 9.9% of the subjects aged less than 25 years old (born after the last HEV outbreak) were positive for anti-HEV antibodies. The indirect ELISA revealed that the anti-HEV antibodies within the positive sera reacted more strongly against the p166 antigens generated from genotype 1. CONCLUSIONS: The present findings reveal a relatively high presence of anti-HEV IgGs and clearly indicate that HEV infection is still present in Northern Algeria. Further, the prediction of HEV genotype using different antigens generated from the different HEV genotypes shows that the causative strains are more likely to be of genotype 1.

Role of asparagine at position 562 in dimerization and immunogenicity of the hepatitis E virus capsid protein.

The hepatitis E virus (HEV) capsid protein, pORF2, contains 2 potential N-glycosylation sites, N137 and N310, located in the S domain, and one site, N562, in the P domain. The last domain located at positions 454-606 aa forms a protruding spike from the shell, with N562 being located in the apical center of the spike, which is also a cell-attachment region and neutralizing antigenic site. Here, we expressed in Pichia pastoris a recombinant polypeptide p179 comprising the region of 439-617 aa of the HEV pORF2 as well as a set of 4 mutant proteins containing substitutions of Q, D, P and Y instead of N at position 562. All proteins were shown to be secreted from yeast. Using SDS-PAGE, Western blot analysis and tunicamycin treatment assay, we showed that the wild-type (wt) protein, p179N562, and 2 mutant variants, p179N562Q and p179N562D, formed homodimers but only the wt protein was shown to be glycosylated. As homodimers, all 3 proteins were immunoreactive with a neutralizing monoclonal antibody (5G5); however, they did not immunoreact with 5G5 after denaturation into monomers. Two other mutant variants, p179N562P and p179N562Y, did not form homodimers but were immunoreactive with the 5G5 antibody. The wt protein was shown to be less immunoreactive with 5G5 than the mutant variants in a double-antibody sandwich ELISA, suggesting a role of glycosylation at N562 in reducing antibody binding. In vitro neutralization experiments showed a more efficient neutralization with mouse antibody against p179N562P and p179N562Y than against the other 3 proteins. These findings indicate that specific substitutions at position 562 have a more measurable effect on the activity of the HEV neutralizing epitope than dimerization or glycosylation of the structural protein. Furthermore, the secretion of monomers fully immunoreactive may call into question the importance of dimerization for an effective presentation of HEV neutralization epitopes.

Immunogenicity difference between two hepatitis E vaccines derived from genotype 1 and 4.

We investigated the immunogenicity difference between hepatitis E vaccine p239 derived from hepatitis E virus (HEV) genotype 1 and vaccine p179 derived from HEV genotype 4; and the presence of genotype-specific neutralizing epitopes. HEV ORF2 recombinant proteins (p166W01, p166Mex, p166US and p166Chn) derived from the four HEV genotypes were used to detect anti-HEV IgGs in sera of mice and humans vaccinated with p179 or p239 and in sera of rhesus monkey challenged with HEV genotype 1 or 4 strains. Then monoclonal antibodies (mAbs) against genotype 1 or 4 ORF2 recombinant proteins were prepared and their immunoreactivity was assessed using ELISA and Western blotting; their neutralizing activity was evaluated by an in vitro PCR-based neutralization assay. The results revealed significant immunogenicity difference between the two vaccines: p239-induced IgGs reacted more strongly against p166W01 and p166Mex than against p166US and p166Chn in mice and humans. By contrast, p179-induced IgGs showed a stronger reactivity against p166US and p166Chn than against p166W01 and p166Mex. This difference has also been observed in the sera of rhesus monkeys challenged with HEV genotype 1 or 4 strains. Moreover, besides the two common neutralizing mAbs 3G1 and 5G5, two genotype-specific neutralizing mAbs, 2B1 and 4C5, were obtained. 2B1 could specifically bind to recombinant proteins derived from genotypes 1 and 2 and neutralized only genotypes 1 and 2 strains, while 4C5 immunoreacted specifically against recombinant proteins derived from genotypes 3 and 4 and neutralized only genotypes 3 and 4 strains. These findings revealed the existence of immunogenicity difference between the p179 and p239 vaccines and demonstrated that this difference could be due to the presence of HEV genotype-specific neutralization epitopes.

Antigenic composition and immunoreactivity differences between HEV recombinant capsid proteins generated from different genotypes.

Appreciable variability has been observed in hepatitis E virus (HEV) serological diagnostics. Four recombinant proteins (p166s) were generated from position 452 to 617 aa of ORF2 of different HEV genotypes and used in an indirect ELISA to detect anti-HEV IgMs and IgGs in serially diluted sera of patients infected with different HEV genotypes (genotype 1, n=15; genotype 3, n=12; genotype 4, n=17). To evaluate the differences at a conformational level, 3D-structure models of p166s were predicted, and different bioinformatics tools were used to analyze the antigenic composition. With both anti-HEV IgMs and IgGs antibodies, there was a considerable variability between the four antigens immunoreactivities. In silico results revealed the region 483-533 aa with the highest antigenic potential and contains six key aa at positions 488, 489, 512, 533, 483 and 530. This immunoreactivity variation could affect diagnosis results and seroprevalence estimations and the identification in silico of a region highly antigenic would guide the development of efficient serological assays and epitope-based vaccines.

Genistein: a promising therapeutic agent for obesity and diabetes treatment.

Obesity and type 2 diabetes are serious public health problems worldwide. Considerable efforts have highlighted the link between these two diseases. The high levels of pro-inflammatory cytokines and leptin, secreted by the adipose tissue, contribute actively to the insulin resistance induction; and the high levels of free fatty acids leads to an overproduction of reactive oxygen species that participate in pancreatic ß cells failure and apoptosis. These two induced dysfunctions are the fundamental defects that precede type 2 diabetes. Genistein, an isoflavone present in a number of edible plants, has been reported as a potential therapeutic agent with anti-cancer, anti-oxidant, anti-inflammatory and anti-osteoporosis effects and proposed as a promising compound for the treatment of metabolic disorders. The pleiotropic effects of genistein are due to its multiple mechanisms of action and the multitude of cell signaling pathways involved. Here, we review the effects of genistein on obesity and type 2 diabetes and emphasize on its action on adipocyte life-cycle, obesity-related low-grade inflammation, oxidative stress and the protective effects on pancreatic ß cells.