Efficient cellular and humoral immune response and production of virus-neutralizing antibodies by the Hepatitis B Virus S/preS1 16-42 antigen

Despite the availability of improved antiviral therapies, infection with Hepatitis B virus (HBV) remains a3 significant health issue, as a curable treatment is yet to be discovered. Current HBV vaccines relaying on the efficient expression of the small (S) envelope protein in yeast and the implementation of mass vaccination programs have clearly contributed to containment of the disease. However, the lack of an efficient immune response in up to 10% of vaccinated adults, the controversies regarding the seroprotection persistence in vaccine responders and the emergence of vaccine escape virus mutations urge for the development of better HBV immunogens. Due to the critical role played by the preS1 domain of the large (L) envelope protein in HBV infection and its ability to trigger virus neutralizing antibodies, including this protein in novel vaccine formulations has been considered a promising strategy to overcome the limitations of S only-based vaccines. In this work we aimed to combine relevant L and S epitopes in chimeric antigens, by inserting preS1 sequences within the external antigenic loop of S, followed by production in mammalian cells and detailed analysis of their antigenic and immunogenic properties. Of the newly designed antigens, the S/preS116–42 protein assembled in subviral particles (SVP) showed the highest expression and secretion levels, therefore, it was selected for further studies in vivo. Analysis of the immune response induced in mice vaccinated with S/preS116–42- and S-SVPs, respectively, demonstrated enhanced immunogenicity of the former and its ability to activate both humoral and cellular immune responses. This combined activation resulted in production of neutralizing antibodies against both wild-type and vaccine-escape HBV variants. Our results validate the design of chimeric HBV antigens and promote the novel S/preS1 protein as a potential vaccine candidate for administration in poor-responders to current HBV vaccines.publishedVersio

Efficient cellular and humoral immune response and production of virus-neutralizing antibodies by the Hepatitis B Virus S/preS116-42 antigen

Despite the availability of improved antiviral therapies, infection with Hepatitis B virus (HBV) remains a3 significant health issue, as a curable treatment is yet to be discovered. Current HBV vaccines relaying on the efficient expression of the small (S) envelope protein in yeast and the implementation of mass vaccination programs have clearly contributed to containment of the disease. However, the lack of an efficient immune response in up to 10% of vaccinated adults, the controversies regarding the seroprotection persistence in vaccine responders and the emergence of vaccine escape virus mutations urge for the development of better HBV immunogens. Due to the critical role played by the preS1 domain of the large (L) envelope protein in HBV infection and its ability to trigger virus neutralizing antibodies, including this protein in novel vaccine formulations has been considered a promising strategy to overcome the limitations of S only-based vaccines. In this work we aimed to combine relevant L and S epitopes in chimeric antigens, by inserting preS1 sequences within the external antigenic loop of S, followed by production in mammalian cells and detailed analysis of their antigenic and immunogenic properties. Of the newly designed antigens, the S/preS116–42 protein assembled in subviral particles (SVP) showed the highest expression and secretion levels, therefore, it was selected for further studies in vivo. Analysis of the immune response induced in mice vaccinated with S/preS116–42- and S-SVPs, respectively, demonstrated enhanced immunogenicity of the former and its ability to activate both humoral and cellular immune responses. This combined activation resulted in production of neutralizing antibodies against both wild-type and vaccine-escape HBV variants. Our results validate the design of chimeric HBV antigens and promote the novel S/preS1 protein as a potential vaccine candidate for administration in poor-responders to current HBV vaccines

A plea for a unified and compatible national security terminology

Security terminology is a contentious and unclear topic in Romania. By analyzing the public speeches of politicians or national strategy texts, we often notice the presence of confusion regarding the correct use of the terms: security, defence, and safety. This confusion gives rise to both theoretical and practical problems, manifested in misunderstandings at the institutional level regarding their respective roles, society’s misconceptions about the functions of certain organizations, and the failure to harness thecollaborative potential between society and the military sector. The appropriate solution to address these issues is the cultivation of a security culture that fosters a grasp of fundamental concepts in this field. To achieve this objective, it is considered essential to have adequate knowledge and usage of the main concepts related to security terminology. This will bring order and enhance understanding of the processes involved in achieving security, defence, and safety.<br/

Bucureºti) ♦ 60♦ Nr

The paper presents the experimental research to redesign the separation stage of a bacterial immune modulator of pharmaceutical use, to be integrated into the GMP (Good Manufacturing Practice) small scale bio product manufacture aseptic, closed flow. It will be studed the separation characteristics for each technological operation, the membrane regeneration capacity and evaluated the period of working time

Influenza Vaccine Manufacturing: Effect of Inactivation, Splitting and Site of Manufacturing. Comparison of Influenza Vaccine Production Processes

<div><p>The aim of this study was to evaluate the impact of different inactivation and splitting procedures on influenza vaccine product composition, stability and recovery to support transfer of process technology. Four split and two whole inactivated virus (WIV) influenza vaccine bulks were produced and compared with respect to release criteria, stability of the bulk and haemagglutinin recovery. One clarified harvest of influenza H3N2 A/Uruguay virus prepared on 25.000 fertilized eggs was divided equally over six downstream processes. The main unit operation for purification was sucrose gradient zonal ultracentrifugation. The inactivation of the virus was performed with either formaldehyde in phosphate buffer or with beta-propiolactone in citrate buffer. For splitting of the viral products in presence of Tween^®, either Triton^™ X-100 or di-ethyl-ether was used. Removal of ether was established by centrifugation and evaporation, whereas removal of Triton-X100 was performed by hydrophobic interaction chromatography. All products were sterile filtered and subjected to a 5 months real time stability study. In all processes, major product losses were measured after sterile filtration; with larger losses for split virus than for WIV. The beta-propiolactone inactivation on average resulted in higher recoveries compared to processes using formaldehyde inactivation. Especially ether split formaldehyde product showed low recovery and least stability over a period of five months.</p></div

The main characteristics of the products from the six different downstream processes.

<p>If applicable the requirements are listed. All products comply with the requirements, except product 5.1FE that has low antigenic HA content</p

SDS PAGE of reduced and reduced plus de-glycosylated samples as a fingerprint of principle proteins present.

<p>Lanes M were loaded with marker proteins, with the corresponding molecular weight presented to the left. The fraction sample identity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150700#pone.0150700.g001" target="_blank">Fig 1</a>) is noted above the lane. Left gel: 1.2 before ZUC, 2.1F after ZUC in phosphate, 2.1B after ZUC in citrate, followed by the six bulks (5.1F, 5.1FE, 5.1FT, 5.1B, 5.1BE and 5.1BT); the migration distance of heavily glycosylated HA proteins varies, causing diffuse bands. In such a case the HA1 band range (~64–79 kD) may be difficult to discriminate from the Nucleoprotein band (~55–66 kD) and the HA2 band range (~23–25 kD) may cover the location of M1 band (~26 kD) as reported by Harvey [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150700#pone.0150700.ref022" target="_blank">22</a>]. After de-glycosylation the HA bands are more distinct and migration distance has increased (right gel, bulks 5.1F, 5.1FE, 5.1FT, 5.1B, 5.1BE and 5.1BT). NP and M1 protein bands have not changed position due to the applied de-glycosylation. In the lanes to the right of the right gel, for comparison products prepared at Intravacc site were applied: 5.1 is WIV BPL inactivated bulk, 5.1S is BPL inactivated Triton split bulk and 3.1 is BPL inactivated influenza before splitting with Triton</p

Overview of the process flows, starting with inoculation of 25.000 eggs and resulting in 6 vaccine products.

<p>In the boxes the unit operations are presented. Fraction identification number is written below the unit operation box. Fraction 1.2 (clarified allantoic fluid) was equally divided over the six process streams. The processes from left to right, with the end product, given in the bottom boxes below the unit operation ‘Sterile Filtration’: 5.1FE standard Cantacuzino Institute process for H3N2 strain, 5.1F Whole Inactivated Virus (WIV) inactivated by formaldehyde, 5.1FT formaldehyde inactivated, Triton split virus product, 5.1BE beta-propiolactone (BPL) inactivated, ether split virus product, 5.1B WIV inactivated by BPL, 5.1BT standard Intravacc process.</p

Representative electron microscope pictures of influenza virus particles, before and after split.

<p>Enlargement pictures to the left 300.000x, pictures to the right 400.000x.Top row whole virus (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150700#pone.0150700.g001" target="_blank">Fig 1</a>, fraction 3.0), bottom row left panel ether split formaldehyde inactivated virus (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150700#pone.0150700.g001" target="_blank">Fig 1</a>, fraction 5.1FE), bottom right panel BPL inactivated Triton split virus (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150700#pone.0150700.g001" target="_blank">Fig 1</a>, fraction 5.1BT). Pictures at top: HA and NA spikes are clearly visible on the outside of the particles. The pictures at the bottom show disrupted, heterologous structures.</p