Comparative protein profiling of B16 mouse melanoma cells susceptible and non-susceptible to alphavirus infection: Effect of the tumor microenvironment

Alphavirus vectors are promising tools for cancer treatment. However, relevant entry mechanisms and interactions with host cells are still not clearly understood. The first step toward a more effective therapy is the identification of novel intracellular alterations that could be associated with cancer aggressiveness and could affect the therapeutic potential of these vectors. In this study, we observed that alphaviruses efficiently infected B16 mouse melanoma tumors/tumor cells in vivo, whereas their transduction efficiency in B16 cells under in vitro conditions was blocked. Therefore, we further aimed to understand the mechanisms pertaining to the differential transduction efficacy of alp…

Recombinant Semliki Forest virus vectors encoding hepatitis B virus small surface and pre-S1 antigens induce broadly reactive neutralizing antibodies

Most hepatitis B virus (HBV) vaccines consist of viral small surface (S) protein subtype adw2 expressed in yeast cells. In spite of good efficacy, HBV-genotype and subtype differences, escape mutants and insufficient Th1 activation remain potential problems. To address these problems, we generated recombinant Semliki Forest virus (rSFV) vectors encoding S protein, subtype adw2 or ayw2, or a fragment of the large surface protein, amino acids 1-48 of the pre-S1 domain, fused to S (pre-S1.1-48/S). The antigen loop in S protein and the selected pre-S1 sequences are known targets of neutralizing antibodies. BALB/c mice were immunized intravenously with 10(7) rSFV particles and 10(8) rSFV particl…

Reference virus as an internal standard for Semliki forest virus real-time PCR quantification

Eiropas Savienības struktūrfondu projektu administrēšana un grāmatvedības uzskaite Latvijas Biomedicīnas pētījumu un studiju centrā

Pēdējā laikā ES struktūrfondi sniedz nozīmīgu atbalstu Latvijas pētniecības un inovāciju attīstībai. Tomēr fondu apgūšanas process, kas saistīts ar projektu plānošanu, iesniegšanu un realizāciju, ir sarežģīts un prasa plašas zināšanas likumdošanā, kas regulē projektu administrēšanas un grāmatvedības uzskaites principus. Latvijas Biomedicīnas pētījumu un studiju centrs (LBMC) ir vadošā pētniecības iestāde Latvijā, kura īsteno ES struktūrfondu projektus. Šī darba mērķis ir izpētīt ES struktūrfondu projektu administrēšanas un grāmatvedības uzskaites pamatprincipus un sniegt rekomendācijas grāmatvedības uzskaites organizācijas uzlabošanai Latvijas Biomedicīnas pētījumu un studiju centrā. Pētīju…

Enzymatic activity of circular sortase A under denaturing conditions: An advanced tool for protein ligation

Abstract Staphylococcus aureus sortase A is a transpeptidase that is extensively used in various protein research applications. Sortase A is highly selective and does not require any cofactors for the catalysis of protein ligation and, importantly, can be produced in high yields. However, the primary disadvantage of this transpeptidase is its inability to access the recognition site within the highly structured regions of folded substrates. To overcome this problem, we developed an Escherichia coli expression system that produces milligram quantities of circularly closed sortase A; efficient enzyme cyclization was achieved by Synechocystis sp. PCC6803 intein-mediated post-translational spli…

N‐Terminal Modification of Gly‐His‐Tagged Proteins with Azidogluconolactone

Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective in vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pH 7.5 in 1 h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed diffe…

Translation of hepatitis B virus (HBV) surface proteins from the HBV pregenome and precore RNAs in Semliki Forest virus-driven expression.

Hepatitis B virus (HBV) pregenome RNA (pgRNA) serves as a translation template for the HBV core (HBc) protein and viral polymerase (Pol). HBV precore RNA (pcRNA) directs the synthesis of the precore (preC) protein, a precursor of the hepatitis B e antigen (HBeAg). pgRNA and pcRNA were expressed in the Semliki Forest virus (SFV) expression system. Besides the HBc and preC proteins, there was revealed the synthesis of all three forms of HBV surface (HBs) proteins: long (LHBs), middle (MHBs) and short (SHBs), the start codons of which are located more than 1000 nt downstream of the HBc and preC start codons. Moreover, other HBV templates, such as 3′-truncated pgRNA lacking 3′ direct repeat and…