Introducing adjuvant-loaded particulate hepatitis B core antigen as an alternative therapeutic hepatitis B vaccine component.

Background & Aims: Particulate hepatitis B core antigen (HBcoreAg) is a potent immunogen used as a vaccine carrier platform. HBcoreAg produced in E. coli encapsidates random bacterial RNA (bRNA). Using the heterologous protein-prime, viral-vector-boost therapeutic hepatitis B vaccine TherVacB, we compared the properties of different HBcoreAg forms. We explored how the content of HBcoreAg modulates antigen stability, immunogenicity, and antiviral efficacy. Methods: bRNA was removed from HBcoreAg by capsid disassembly, followed by reassembly in the absence or presence of specific nucleic acid-based adjuvants poly I:C or CpG. The morphology and structure of empty, bRNA-containing and adjuvant-loaded HBcoreAg were monitored by electron microscopy and nuclear magnetic resonance spectroscopy. Empty, bRNA-containing or adjuvant-loaded HBcoreAg were applied together with HBsAg and with or without nucleic acid-based external adjuvants within the TherVacB regimen in both wild-type and HBV-carrier mice. Results: While HBcoreAg retained its structure upon bRNA removal, its stability and immunogenicity decreased significantly. Loading HBcoreAg with nucleic acid-based adjuvants re-established stability of the capsid-like antigen. Immunization with poly I:C- or CpG-loaded HBcoreAg induced high antibody titers against co-administered HBsAg. When applied within the TherVacB regimen, they activated vigorous HBcoreAg- and HBsAg-specific T-cell responses in wild-type and HBV-carrier mice, requiring a significantly lower dose of adjuvant compared to externally added adjuvant. Finally, immunization with adjuvant-loaded HBcoreAg mixed with HBsAg led to long-term control of persistent HBV replication in the HBV-carrier mice. Conclusion: Adjuvant-loaded HBcoreAg retained capsid integrity and stability, was as immunogenic in vivo as externally adjuvanted HBcoreAg, requiring lower adjuvant levels, and supported immunity against co-administered, non-adjuvanted HBsAg. Thus, adjuvant-loaded HBcoreAg represents a promising novel platform for vaccine development. Impact and implications: Hepatitis B core antigen (HBcoreAg) recapitulates the capsid of the HBV that hosts the viral genome. Produced recombinantly, it is not infectious but emerges as a potent immunogen in vaccine development. In this preclinical study, we show that loading HBcoreAg with defined nucleic-acid-based adjuvants on the one hand stabilizes the HBcoreAg with standardized capsid content and, on the other hand, efficiently promotes the immunity of HBcoreAg and a co-administered antigen, allowing for reduced adjuvant doses. Therefore, adjuvant-loaded HBcoreAg not only serves as an encouraging option for therapeutic hepatitis B vaccines, but could also act as an efficient adjuvant delivery system for other types of vaccine.

Immunogenicity of a silica nanoparticle-based SARS-CoV-2 vaccine in mice.

Safe and effective vaccines have been regarded early on as critical in combating the COVID-19 pandemic. Among the deployed vaccine platforms, subunit vaccines have a particularly good safety profile but may suffer from a lower immunogenicity compared to mRNA based or viral vector vaccines. In fact, this phenomenon has also been observed for SARS-CoV-2 subunit vaccines comprising the receptor-binding domain (RBD) of the spike (S) protein. Therefore, RBD-based vaccines have to rely on additional measures to enhance the immune response. It is well accepted that displaying antigens on nanoparticles can improve the quantity and quality of vaccine-mediated both humoral and cell-mediated immune responses. Based on this, we hypothesized that SARS-CoV-2 RBD as immunogen would benefit from being presented to the immune system via silica nanoparticles (SiNPs). Herein we describe the preparation, in vitro characterization, antigenicity and in vivo immunogenicity of SiNPs decorated with properly oriented RBD in mice. We found our RBD-SiNP conjugates show narrow, homogeneous particle distribution with optimal size of about 100 nm for efficient transport to and into the lymph node. The colloidal stability and binding of the antigen was stable for at least 4 months at storage- and in vivo-temperatures. The antigenicity of the RBD was maintained upon binding to the SiNP surface, and the receptor-binding motif was readily accessible due to the spatial orientation of the RBD. The particles were efficiently taken up in vitro by antigen-presenting cells. In a mouse immunization study using an mRNA vaccine and spike protein as benchmarks, we found that the SiNP formulation was able to elicit a stronger RBD-specific humoral response compared to the soluble protein. For the adjuvanted RBD-SiNP we found strong S-specific multifunctional CD4+ T cell responses, a balanced T helper response, improved auto- and heterologous virus neutralization capacity, and increased serum avidity, suggesting increased affinity maturation. In summary, our results provide further evidence for the possibility of optimizing the cellular and humoral immune response through antigen presentation on SiNP.

Efficient stabilization of therapeutic hepatitis B vaccine components by amino-acid formulation maintains its potential to break immune tolerance.

Background & Aims: Induction of potent, HBV-specific immune responses is crucial to control and finally cure HBV. The therapeutic hepatitis B vaccine TherVacB combines protein priming with a Modified Vaccinia virus Ankara (MVA)-vector boost to break immune tolerance in chronic HBV infection. Particulate protein and vector vaccine components, however, require a constant cooling chain for storage and transport, posing logistic and financial challenges to vaccine applications. We aimed to identify an optimal formulation to maintain stability and immunogenicity of the protein and vector components of the vaccine using a systematic approach. Methods: We used stabilizing amino acid (SAA)-based formulations to stabilize HBsAg and HBV core particles (HBcAg), and the MVA-vector. We then investigated the effect of lyophilization and short- and long-term high-temperature storage on their integrity. Immunogenicity and safety of the formulated vaccine was validated in HBV-naïve and adeno-associated virus (AAV)-HBV-infected mice. Results: In vitro analysis proved the vaccine's stability against thermal stress during lyophilization and the long-term stability of SAA-formulated HBsAg, HBcAg and MVA during thermal stress at 40 °C for 3 months and at 25 °C for 12 months. Vaccination of HBV-naïve and AAV-HBV-infected mice demonstrated that the stabilized vaccine was well tolerated and able to brake immune tolerance established in AAV-HBV mice as efficiently as vaccine components constantly stored at 4 °C/-80 °C. Even after long-term exposure to elevated temperatures, stabilized TherVacB induced high titre HBV-specific antibodies and strong CD8+ T-cell responses, resulting in anti-HBs seroconversion and strong suppression of the virus in HBV-replicating mice. Conclusion: SAA-formulation resulted in highly functional and thermostable HBsAg, HBcAg and MVA vaccine components. This will facilitate global vaccine application without the need for cooling chains and is important for the development of prophylactic as well as therapeutic vaccines supporting vaccination campaigns worldwide. Impact and implications: Therapeutic vaccination is a promising therapeutic option for chronic hepatitis B that may enable its cure. However, its application requires functional cooling chains during transport and storage that can hardly be guaranteed in many countries with high demand. In this study, the authors developed thermostable vaccine components that are well tolerated and that induce immune responses and control the virus in preclinical mouse models, even after long-term exposure to high surrounding temperatures. This will lower costs and ease application of a therapeutic vaccine and thus be beneficial for the many people affected by hepatitis B around the world.

Activation of CD4 T cells during prime immunization determines the success of a therapeutic hepatitis B vaccine in HBV-carrier mouse models.

BACKGROUND & AIMS: We recently developed a heterologous therapeutic vaccination scheme (TherVacB) comprising a particulate protein prime followed by a modified vaccinia-virus Ankara (MVA)-vector boost for the treatment of HBV. However, the key determinants required to overcome HBV-specific immune tolerance remain unclear. Herein, we aimed to study new combination adjuvants and unravel factors that are essential for the antiviral efficacy of TherVacB. METHODS: Recombinant hepatitis B surface and core antigen (HBsAg and HBcAg) particles were formulated with different liposome- or oil-in-water emulsion-based combination adjuvants containing saponin QS21 and monophosphoryl lipid A; these formulations were compared to STING-agonist c-di-AMP and conventional aluminium hydroxide formulations. Immunogenicity and the antiviral effects of protein antigen formulations and the MVA-vector boost within TherVacB were evaluated in adeno-associated virus-HBV-infected and HBV-transgenic mice. RESULTS: Combination adjuvant formulations preserved HBsAg and HBcAg integrity for ≥12 weeks, promoted human and mouse dendritic cell activation and, within TherVacB, elicited robust HBV-specific antibody and T-cell responses in wild-type and HBV-carrier mice. Combination adjuvants that prime a balanced HBV-specific type 1 and 2 T helper response induced high-titer anti-HBs antibodies, cytotoxic T-cell responses and long-term control of HBV. In the absence of an MVA-vector boost or following selective CD8 T-cell depletion, HBsAg still declined (mediated mainly by anti-HBs antibodies) but HBV replication was not controlled. Selective CD4 T-cell depletion during the priming phase of TherVacB resulted in a complete loss of vaccine-induced immune responses and its therapeutic antiviral effect in mice. CONCLUSIONS: Our results identify CD4 T-cell activation during the priming phase of TherVacB as a key determinant of HBV-specific antibody and CD8 T-cell responses. IMPACT AND IMPLICATIONS: Therapeutic vaccination is a potentially curative treatment option for chronic hepatitis B. However, it remains unclear which factors are essential for breaking immune tolerance in HBV carriers and determining successful outcomes. Our study provides the first direct evidence that efficient priming of HBV-specific CD4 T cells determines the success of therapeutic hepatitis B vaccination in two preclinical HBV-carrier mouse models. Applying an optimal formulation of HBV antigens that activates CD4 and CD8 T cells during prime immunization provided the foundation for an antiviral effect of therapeutic vaccination, while depletion of CD4 T cells led to a complete loss of vaccine-induced antiviral efficacy. Boosting CD8 T cells was important to finally control HBV in these mouse models. Our findings provide important insights into the rational design of therapeutic vaccines for the cure of chronic hepatitis B.

Identification and modelling of dynamic parameters for round link chains subject to axial loads.

A round link chain subject to axial dynamic loads composes a nonlinear viscoelastic system. Unlike the classical pounding problems, the round link chain will not only suffer linear elastic deformation, but also nonlinear plastic or impacting deformation. Based on theoretical formulation and experiments, a new approach is presented in this paper to model and identify the nonlinear dynamic parameters, namely the stiffness and damping for the round link chain. With linear deformation, nonlinear deformation and energy dissipation considered, a modified nonlinear viscoelastic model is developed to describe the vibrational behavior of the chain with numbers of round links. The linear elastic model and impacting model are combined to derive the equivalent nonlinear stiffness, while experiments and the least square fitting method are employed to identify the nonlinear damping according to the modified nonlinear viscoelastic model. The influences of the key parameters such as the length of the chain, elastic module and loading frequency on the dynamic stiffness and damping are investigated. Another test is performed to validate the identification model and good agreements are observed.

Intramolecular recombination enables the formation of hepatitis B virus (HBV) cccDNA in mice after HBV genome transfer using recombinant AAV vectors.

The mouse is not a natural host of hepatitis B virus (HBV) infection and - despite engraftment of hepatocytes with the HBV receptor - does not support formation of HBV covalently closed circular (ccc) DNA serving as a template for viral transcription and permitting persistent infection. In a recent study, cccDNA formation in mouse hepatocytes has been described following an HBV genome delivery by a recombinant, adeno-associated virus vector (rAAV) (Lucifora et al., 2017). The integrity of HBV cccDNA, its origin and functionality, however, remained open. In this study, we investigated the identity, origin, and functionality of cccDNA established in mice infected with rAAV carrying 1.3-fold overlength HBV genomes. We show that replication of HBV genotypes A, B, C and D can be initiated in mouse livers, and that cccDNA derived from all genotypes is detected. Restriction enzyme and exonuclease digestion as well as sequencing analysis of cccDNA amplicons revealed authentic HBV cccDNA without any detectable alteration compared to cccDNA established after HBV infection of human liver cells. Mouse livers transduced with a core protein-deficient HBV using rAAV still supported cccDNA formation demonstrating that the genesis of cccDNA was independent of HBV replication. When mice were infected with an rAAV-HBV1.3 carrying premature stop codons in the 5' but not in the 3' core protein open reading frame, the stop codon was partially replaced by the wild-type sequence. This strongly indicated that intramolecular recombination, based on >900 identical base pairs residing at the both ends of the HBV1.3 transgene was the origin of cccDNA formation. Accordingly, we observed a constant loss of cccDNA molecules from mouse livers over time, while HBeAg levels increased over the first two weeks after rAAV-HBV1.3 infection and remained constant thereafter, suggesting a minor contribution of the cccDNA molecules formed to viral transcription and protein expression. In summary, our results provide strong evidence that intramolecular recombination of an overlength, linear HBV genome, but not HBV genome recycling, enables cccDNA formation in rAAV-HBV mouse models.

Compressor fault diagnosis system based on PCA-PSO-LSSVM algorithm.

On the basis of the principal components analysis-particle swarm optimization-least squares support vector machine (PCA-PSO-LSSVM) algorithm, a fault diagnosis system is proposed for the compressor system. The relationship between the working principle of a compressor system, the fault phenomenon, and the root cause is analyzed. A fault diagnosis model is established based on the LSSVM optimized using PSO, the compressor fault diagnosis test experimental platform is used to obtain the fault signal of various fault occurrence states, and the PCA algorithm is employed to extract the characteristic data in the fault signal as input to the fault diagnosis model. The back-propagation neural network, the LSSVM algorithm, and the PSO-LSSVM algorithm are analyzed and compared with the proposed fault diagnosis model. Results show that the PCA-PSO-LSSVM fault diagnosis model has a maximum fault recognition efficiency that is 10.4% higher than the other three models, the test sample classification time is reduced by 0.025 s, the PCA algorithm can effectively reduce the input dimension, and the PSO-LSSVM fault diagnosis model based on the PCA algorithm for extracting features has a high recognition rate and accuracy. Therefore, the proposed fault diagnosis system can effectively identify the compressor fault and improve the efficiency of the compressor.

Knockdown of Virus Antigen Expression Increases Therapeutic Vaccine Efficacy in High-Titer Hepatitis B Virus Carrier Mice.

BACKGROUND & AIMS: Hepatitis B virus (HBV) infection persists because the virus-specific immune response is dysfunctional. Therapeutic vaccines might be used to end immune tolerance to the virus in patients with chronic infection, but these have not been effective in patients so far. In patients with chronic HBV infection, high levels of virus antigens might prevent induction of HBV-specific immune responses. We investigated whether knocking down expression levels of HBV antigens in liver might increase the efficacy of HBV vaccines in mice. METHODS: We performed studies with male C57BL/6 mice that persistently replicate HBV (genotype D, serotype ayw)-either from a transgene or after infection with an adeno-associated virus that transferred an overlength HBV genome-and expressed HB surface antigen at levels relevant to patients. Small hairpin or small interfering (si)RNAs against the common 3'-end of all HBV transcripts were used to knock down antigen expression in mouse hepatocytes. siRNAs were chemically stabilized and conjugated to N-acetylgalactosamine to increase liver uptake. Control mice were given either entecavir or non-HBV-specific siRNAs and vaccine components. Eight to 12 weeks later, mice were immunized twice with a mixture of adjuvanted HBV S and core antigen, followed by a modified Vaccinia virus Ankara vector to induce HBV-specific B- and T-cell responses. Serum and liver samples were collected and analyzed for HBV-specific immune responses, liver damage, and viral parameters. RESULTS: In both models of HBV infection, mice that express hepatocyte-specific small hairpin RNAs or that were given subcutaneous injections of siRNAs had reduced levels of HBV antigens, HBV replication, and viremia (1-3 log10 reduction) compared to mice given control RNAs. Vaccination induced production of HBV-neutralizing antibodies and increased numbers and functionality of HBV-specific, CD8+ T cells in mice with low, but not in mice with high, levels of HBV antigen. Mice with initially high titers of HBV and knockdown of HBV antigen expression, but not mice with reduced viremia after administration of entecavir, developed polyfunctional, HBV-specific CD8+ T cells, and HBV was eliminated. CONCLUSIONS: In mice with high levels of HBV replication, knockdown of HBV antigen expression along with a therapeutic vaccination strategy, but not knockdown alone, increased numbers of effector T cells and eliminated the virus. These findings indicate that high titers of virus antigens reduce the efficacy of therapeutic vaccination. Anti-HBV siRNAs and therapeutic vaccines are each being tested in clinical trials-their combination might cure chronic HBV infection.

Synergy of therapeutic heterologous prime-boost hepatitis B vaccination with CpG-application to improve immune control of persistent HBV infection.

Therapeutic vaccination against chronic hepatitis B must overcome high viral antigen load and local regulatory mechanisms that promote immune-tolerance in the liver and curtail hepatitis B virus (HBV)-specific CD8 T cell immunity. Here, we report that therapeutic heterologous HBcore-protein-prime/Modified-Vaccinia-Virus-Ankara (MVA-HBcore) boost vaccination followed by CpG-application augmented vaccine-induced HBcAg-specific CD8 T cell-function in the liver. In HBV-transgenic as well as AAV-HBV-transduced mice with persistent high-level HBV-replication, the combination of therapeutic vaccination with subsequent CpG-application was synergistic to generate more potent HBV-specific CD8 T cell immunity that improved control of hepatocytes replicating HBV.

Structure and Properties of a Metallocene Polypropylene Resin with Low Melting Temperature for Melt Spinning Fiber Application.

An isotactic polypropylene (iPP-1) resin with low melting temperature (Tm) is synthesized by a metallocene catalyst and investigated for melt-spun fiber applications. The structure, thermal and mechanical properties, and feasibility of producing fibers of a commercial metallocene iPP (iPP-2) and a conventional Ziegler-Natta iPP (iPP-3) are carefully examined for comparison. Tm of iPP-1 is about 10 °C lower than the other two samples, which is well addressed both in the resin and the fiber products. Besides, the newly developed iPP-1 possesses higher isotacticity and crystallinity than the commercial ones, which assures the mechanical properties of the fiber products. Thanks to the addition of calcium stearate, its crystal grain size is smaller than those of the two other commercial iPPs. iPP-1 shows a similar rheological behavior as the commercial ones and good spinnability within a wide range of take-up speeds (1200-2750 m/min). The tensile property of fibers from iPP-1 is better than commercial ones, which can fulfill the application requirement. The formation of the mesomorphic phase in iPP-1 during melt spinning is confirmed by the orientation and crystallization investigation with wide angle X-ray diffraction (WAXD), which is responsible for its excellent processing capability and the mechanical properties of the resultant fibers. The work may provide not only a promising candidate for the high-performance PP fiber but also a deep understanding of the formation mechanism of the mesomorphic phase during fiber spinning.

Human antibody 3E1 targets the HA stem region of H1N1 and H5N6 influenza A viruses.

As influenza A viruses remain a major threat to human health worldwide, the discovery of broadly neutralizing monoclonal antibodies that recognize conserved epitopes would facilitate the development of antibody-based therapeutic strategies. Here we report that a VH4-4-encoded human mAb named 3E1 could neutralize H1 and H5 subtype viruses in vitro and protect mice against the H1N1 and H5N6 viruses by inhibiting the low pH-induced conformational rearrangement of haemagglutinin (HA), hence blocking membrane fusion. The crystal structures of 3E1 Fab in complex with HA of two H1N1 strains reveal that 3E1, with both heavy and light chains, binds to a conserved epitope of the HA stem region, comprising parts of the fusion peptide, the F subdomain and the outermost ß-strand preceding helix A. Altogether, these data suggest the potential of 3E1 as a therapeutic drug against H1 and H5 subtype viruses.

Effects of symmetrical foundation on sound radiation from a submarine hull structure.

The effects of a passive noise control method for suppressing sound radiation from a submarine hull structure are investigated. The control method is realized by symmetrizing the foundation about the horizontal plane. The coupled finite element method and boundary element method are adopted to compute the acoustic characteristics of the submerged hull. From the numerical results, the symmetrical foundation has advantages in sound radiation reduction when the hull is subjected to the axial load, but has little influences in the vertical and transverse load cases. Using the modal decomposition technique, the contributions of each individual mode to the sound radiation are analyzed to reveal the mechanism of the control method.