Expression of rabies VLPs in adherence and suspension conditions: a flexible platform for rabies vaccine production

Rabies is a zoonotic viral disease with a mortality by close to 100%. As there is not an efficacious treatment available, post-exposure vaccination is recommended for individuals in contact with the virus. On the other hand, the most common source of virus transmission is saliva of infected animals, mostly dogs, whereby mass vaccination of pets is the most cost‑effective way to reduce human infections. In this context, availability of both human and veterinary vaccines is critical. In previous works1-2, our group developed immunogenic rabies VLPs, expressing the virus glycoprotein in HEK293 cells. We obtained a producer clone capable of growing in adherence (adhP2E5) and then adapted to suspension conditions (sP2E5). In this work, we analyzed the production of VLPs in both conditions, using two different platforms. On the one hand, adhP2E5 was cultured in 850 cm2 roller bottles (GBO) using medium with 5% FCS, that was exchanged every 48 h during the first 10 days and every 24 h during the last 5 days. RV-VLPs were continuously produced and the harvest obtained (2.5 L per bottle) was analyzed by sandwich ELISA, using the 6th International Standard for rabies vaccine that quantify the glycoprotein content (NIBSC), presenting a value of 19 IU.ml‑1 in average. On the other hand, we cultured sP2E5 in a 5 L bioreactor during 15 days, using EX‑CELL293 SFM (SAFC). The culture reached densities of 2x107 cel.ml-1 and VLPs were continuously secreted to the supernatant. The obtained harvest (28.5 L) presented a glycoprotein content of 28 IU.ml-1, a results that is comparable with the previous one taking into account the number of cells presents in both conditions. These results showed that our clone could be cultured in both platforms depending on the objectives and characteristics of the desired product. For the production of the rabies veterinary vaccine, RV-VLPs can be produced in adherent conditions using medium supplemented with FCS and, for human vaccine production, RV-VLPs can be produced in bioreactors using SFM. 1 Fontana et al. Rabies virus-like particles expressed in HEK293 cells. Vaccine 32 (2014) 2799-2804. 2 Fontana et al. Immunogenic virus-like particles continuously expressed in mammalian cells as a veterinary rabies vaccine candidate. Vaccine 33 (2015) 4238-4246

Laparoscopic repair for perforated peptic ulcers with U-CLIP®

<p>Abstract</p> <p>Background</p> <p>The literature established that, in patients without Boey's risk factors, laparoscopic repair of perforated peptic ulcers, compared to open repair, is associated to lower wound infection rate, less analgesic use, reduction in post operative pain, shorter hospital stay. Some of the main drawbacks are length of operative time and laparoscopic surgeon's experience in intracorporeal knotting.</p> <p>We, for first, report our preliminary experience of perforated peptic ulcers' laparoscopic repair using Medtronic U-Clip^®.</p> <p>Methods</p> <p>From January 2008 to June 2008 we performed laparoscopic repair of perforated peptic ulcers using Medtronic U-Clip^®in 10 consecutive patients (6 men and 4 women, from 20 to 65 years-old of age). All the patients presented with iuxtapyloric perforated peptic ulcer, not greater than 10 mm, without signs of sepsis, free from major illnesses. The mini-invasive procedure was performed both by skilled and non-skilled laparoscopic surgeons under experts' surveillance. After it was recognized, perforation was sutured using U-Clip^®in a full-thickness manner.</p> <p>Results and Discussion</p> <p>We reported no surgical complications in the peri-operative period. The clinical outcome and time needed to perform the intervention didn't change between skilled and non-skilled surgeons. The follow-up at 30 days was good.</p> <p>Conclusion</p> <p>In our experience, the anastomotic device U-Clip^®simplifies laparoscopic repair of perforated peptic ulcer, avoiding the need to perform knots and making the procedure safe and easier.</p

Production and characterization of hepatitis B Virus-like Particles expressed in HEK293 and CHO-K1 recombinant cell lines. Analysis of the humoral immune response

Hepatitis B is a life-threatening liver infection caused by the hepatitis B virus (HBV). Even today, HBV infection constitutes a global health problem, affecting more than 250 million people, and vaccination represents the most cost-effective strategy to control HBV spreading. The main antigen of the HBV its the surface antigen (HBsAg), that consists of three co-carboxyterminal envelope glycoproteins, called Large (L), Middle (M), and Small (S). Current available HBV vaccines are based on the non-glycosylated form of the S protein, expressed in yeast cells. Although efficacious and safe, about 10% of people vaccinated with this vaccine fail to mount an adequate antibody response1. One approach to improve HBV vaccination response is increasing immunogenicity via the inclusion of glycosylated S, M and L proteins in hepatitis B virus-like particles (HB-VLPs) expressed in mammalian cells. This new generation vaccine has proven to be able to elicited higher antibody titers breaking the lack of response to the current vaccine. On this regard, domains present in L and M proteins have shown to be more immunostimulatory than the S protein2. Also, the L protein contains the hepatocyte receptor-binding site and is able to induce the production of infection-neutralizing antibodies3. Please click Download on the upper right corner to see the full abstract

Rational design, expression and characterization of chimeric rabies VLPs displaying the major antigenic site of Foot and Mouth Disease Virus

Virus-like Particles (VLPs) are supramolecular arrangements of one or more viral proteins that resemble the structure of the native virus but are not infective, due the fact that they lack the viral genome. They have become very important in the field of novel recombinant vaccines, because of their biosafety and improved immunogenicity over subunit vaccines due to their particulate nature and highly repetitive epitope display. Moreover, they can elicit immune responses against heterologous antigens by the incorporation of epitopes by genic fusion to the viral proteins, constituting chimeric VLPs (cVLPs). In a previous work, by recombinant expression of the rabies G glycoprotein (RVG) in HEK293 cells, we stablished a platform to produce rabies VLPs as a new generation vaccine candidate 1–3. The main goal of this work was to generate a platform for heterologous antigen presentation based on RVG cVLPs. The heterologous epitope chosen for this study was the immunodominant site of Foot-and-Mouth Disease Virus (FMDV), named G-H loop (that is part of the capsid protein VP1), which is responsible of virus entry into the cell 4. Please click Download on the upper right corner to see the full abstract

RHABDO-LIKE RECOMBINANTE (VLPs), a novel veterinary rabies vaccine: Safety and efficacy trials in pets and cattle

Rabies is a viral fatal disease transmitted to humans from bites of infected animals. Due to nearly 95 % of human infections are related with dog bites, the WHO, OIE and FAO agreed in the implementation of a worldwide plan for the elimination of dog-mediated human rabies death by 2030. On the other hand, in some Latin-American countries paralytic bovine rabies transmitted by vampire bats causes economic losses to the farming industry. In this context, it is important to develop an economic vaccine that could be produced in a short period of time. Our research group have been working in the development and validation of a recombinant rabies vaccine based on virus‑like particles (RV-VLPs), expressing the rabies glycoprotein in HEK293 cells1. Performing a rational design, we carried out the expression vectors construction, stable cell line development and the full biochemical and morphological characterization of the expressed RV-VLPs. On the other hand, the immune response triggered by these VLPs in mice was analysed, demonstrating that this new antigen is able to induce a potent humoral immune response with high titers of neutralizing antibodies. Furthermore, protection against rabies virus challenge was confirmed in NIH potency test assays, demonstrating that RV-VLPs are an excellent new generation and virus-free vaccine candidate. Please click Download on the upper right corner to see the full abstract

Study of rabies VLPs expression in BHK-21 cell line for vaccine applications

In the last decades, virus-like particles (VLPs) have played an essential role in the development of novel vaccines due the fact that they trigger robust and balanced immune responses and, as they lack viral genome, are biosafe. Nowadays, several VLPs are commercially available for human use and one veterinary product was licensed. Besides, other VLP‑based vaccine candidates are in the stages of clinical trials or preclinical evaluation. Our group had previously developed a rabies glycoprotein based-VLP (RV-VLPs) expressed in HEK293 cells. These RV-VLPs were fully characterized and their capacity to induce a protective response and neutralizing antibodies production was confirmed. As inactivated veterinary vaccines for rabies are usually produced using BHK‑21, the goal of the present work was to develop a RV-VLPs expressing BHK‑21 cell line to analyze the characteristics of the VLPs produced using this cell substrate. Therefore, by lentivirus vector-mediated transduction, we generated a rabies virus glycoprotein expressing a stable cell line. The cellular expression of the recombinant protein was analyzed by flow cytometry and the membrane localization was confirmed by fluorescent microscopy. Later, RV-VLPs budding to the supernatant was analyzed by sandwich ELISA. After that, VLPs were purified by density gradient ultracentrifugation and the hydrodynamic diameter of the particles was analyzed by DLS. In a western blot assay, the particles were recognized by specific antibodies present in a rabies polyclonal serum. Finally, the recombinant cell line was cultured in 850 cm2 roller bottles producing RV-VLPs continuously during 25 days of culture. Thus, these results encourage further studies to confirm if BHK-21 is a good cell substrate for the production of RV-VLPs as a veterinary rabies vaccine candidate

Exotic atoms at extremely high magnetic fields: the case of neutron star atmosphere

The presence of exotic states of matter in neutron stars (NSs) is currently an open issue in physics. The appearance of muons, kaons, hyperons, and other exotic particles in the inner regions of the NS, favored by energetic considerations, is considered to be an effective mechanism to soften the equation of state (EoS). In the so-called two-families scenario, the softening of the EoS allows for NSs characterized by very small radii, which become unstable and convert into a quark stars (QSs). In the process of conversion of a NS into a QS material can be ablated by neutrinos from the surface of the star. Not only neutron-rich nuclei, but also more exotic material, such as hypernuclei or deconfined quarks, could be ejected into the atmosphere. In the NS atmosphere, atoms like H, He, and C should exist, and attempts to model the NS thermal emission taking into account their presence, with spectra modified by the extreme magnetic fields, have been done. However, exotic atoms, like muonic hydrogen $(p\,\mu^-)$ or the so-called Sigmium $(\Sigma^+\,e^-)$, could also be present during the conversion process or in its immediate aftermath. At present, analytical expressions of the wave functions and eigenvalues for these atoms have been calculated only for H. In this work, we extend the existing solutions and parametrizations to the exotic atoms $(p\,\mu^-)$ and $(\Sigma^+\,e^-)$, making some predictions on possible transitions. Their detection in the spectra of NS would provide experimental evidence for the existence of hyperons in the interior of these stars.Comment: 10 pages, 6 figures, proceedings of the "International Conference on Exotic Atoms and Related Topics - EXA2017", Austrian Academy of Sciences, Austria, September 11-15, 201

Rational design of novel fusion rabies glycoproteins displaying a major antigenic site of foot-and-mouth disease virus for vaccine applications

Chimeric virus-like particles are self-assembling structures composed of viral proteins that had been modified to incorporate sequences from different organisms, being able to trigger immune responses against the heterologous sequence. However, the identification of suitable sites for that purpose in the carrier protein is not an easy task. In this work, we describe the generation of rabies chimeric VLPs that expose a major antigenic site of foot-and-mouth disease virus (FMDV) by identifying suitable regions in rabies glycoprotein (RVG), as a proof of concept of a novel heterologous display platform for vaccine applications. To identify adequate sites for insertion of heterologous sequences without altering the correct folding of RVG, we identified regions that were evolutionally non-conserved in Lyssavirus glycoproteins and performed a structural analysis of those regions using a 3D model of RVG trimer that we generated. The heterologous sequence was inserted in three different sites within RVG sequence. In every case, it did not affect the correct folding of the protein and was surface exposed, being recognized by anti-FMDV antibodies in expressing cells as well as in the surface of VLPs. This work sets the base for the development of a heterologous antigen display platform based on rabies VLPs.Fil: Garay, Ernesto Sergio. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Fontana, Diego Sebastian. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Leschiutta, Lautaro. Universidad Nacional del Litoral; ArgentinaFil: Kratje, Ricardo Bertoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Prieto, Claudio. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentin