Background Rapid vaccine platforms development is crucial for responding to epidemics and pandemics of emerging infectious diseases, such as Ebola. This study explores the potential of peptide vaccines that self-organize into amyloid-like fibrils, aiming to enhance immunogenicity while considering safety and cross-reactivity. Methods We synthesized two peptides, G33 and G31, corresponding to a segment of the Ebola virus GP2 protein, with G33 known to form amyloid-like fibrils. Their toxicity was assessed in vitro using an MTT assay on MDCK and A549 cell cultures. For in vivo studies, balb/c mice were immunized with these peptides. Immunogenicity was gauged by ELISA for specific antibodies against the recombinant eGP protein. Hematological parameters were determined, and histopathological changes in organs were documented post-euthanasia. Results Both peptides exhibited no cytotoxicity up to 500 μg/mL. G33-immunized mice developed higher antibody titers than those receiving G31 or control, without significant alterations in hematological profiles. However, histological analysis showed periportal infiltration and lymphoid-macrophage clusters in the liver and kidneys, suggesting immune response activation. The homology between the G33 peptide and mammalian proteins poses risks of cross-reactivity. Conclusion The amyloid-like fibrils formed by the G33 peptide elicited a stronger immune response without significant hematological changes, underlining the feasibility of fibril-based peptide vaccines. However, the potential for autoimmune responses due to molecular mimicry warrants further investigation before clinical applications can be considered.

This paper considers the evaluation of uncertainty of quantitative gel electrophoresis. To date, such uncertainty estimation presented in the literature are based on the multiple measurements performed for assessing the intra- and interlaboratory reproducibility using standard samples. This paper shows how to estimate the uncertainty in cases where we cannot study scattering components of the results. The first point is dedicated to a case where we have standard samples (the direct expressions are shown). The second point considers the situation when standard samples are absent (the algorithm for estimating the lower bound for uncertainty is discussed). The role of the data processing algorithm is demonstrated.

In this work, we have extended our previously proposed approach for determining protein concentrations in human serum (using MALDI-TOF mass spectrometry) to include simultaneous analysis of several proteins associated with acute inflammation (alpha-2-macroglobulin, fetuin-A, serum amyloid A1). This technique can be used to diagnose systemic inflammation and provides results in 4-5 h. The developed approach was verified using standard immunological methods (ELISA). Samples from 87 individuals, in specific groups, were used for testing and validation: control; inflammatory soft tissue disease accompanied by sepsis; influenza A infection; or COVID-19. The feasibility of differentiating patient groups with the aforementioned conditions was analyzed using a combination of the inflammatory markers described. For fetuin-A and serum amyloid A1, diagnostically significant concentration ranges were established. (c) 2022 Elsevier B.V. All rights reserved.

Type III interferons exhibit antiviral activity against influenza viruses, coronaviruses, rotaviruses, and others. In addition, this type of interferon theoretically has therapeutic advantages, in comparison with type I interferons, due to its ability to activate a narrower group of genes in a relatively small group of target cells. Hence, it can elicit more targeted antiviral or immunomodulatory responses. Obtaining biologically-active interferon lambda (hIFN-λ1) is fraught with difficulties at the stage of expression in soluble form or, in the case of expression in the form of inclusion bodies, at the stage of refolding. In this work, hIFN-λ1 was expressed in the form of inclusion bodies, and a simple, effective refolding method was developed. Efficient and scalable methods for chromatographic purification of recombinant hIFN-λ1 were also developed. High-yield, high-purity product was obtained through optimization of several processes including: recombinant protein expression; metal affinity chromatography; cation exchange chromatography; and an intermediate protein refolding stage. The obtained protein was shown to feature expected specific biological activity in line with published effects: induction of MxA gene expression in A549 cells and antiviral activity against influenza A virus.

One of the main functions of alpha-2-macroglobulin (A2M) in human blood serum is the binding of all classes of protease. It is known that trypsin, after such interaction, possesses modified proteolytic activity. Trypsin first hydrolyzes two bonds in A2M's 'bait region', and the peptide 705VGFYESDVMGR715 is released from A2M. In this work, specifics of the A2M-trypsin interaction were used to determine A2M concentration directly in human blood serum using MALDI mass-spectrometry. Following exogenous addition of trypsin to human blood serum in vitro, the concentration of the VGFYESDVMGR peptide was measured, using its isotopically-labeled analogue (18O), and A2M concentration was calculated. The optimized mass spectrometric approach was verified using a standard method for A2M concentration determination (ELISA) and the relevant statistical analysis methods. It was also shown that trypsin's modified proteolytic activity in the presence of serum A2M can be used to analyze other serum proteins, including potential biomarkers of pathological processes. Thus, this work describes a promising approach to serum biomarker analysis that can be technically extended in several useful directions.

The influenza A virus genome consists of eight segments of negative-sense RNA that encode up to 18 proteins. During the process of viral replication, positive-sense (+)RNA (cRNA) or messenger RNA (mRNA) is synthesized. Today, there is only a partial understanding of the function of several secondary structures within vRNA and cRNA promoters, and splice sites in the M and NS genes. The most precise secondary structure of (+)RNA has been determined for the NS segment of influenza A virus. The influenza A virus NS gene features two regions with a conserved mRNA secondary structure located near splice sites. Here, we compared 4 variants of the A/Puerto Rico/8/1934 strain featuring different combinations of secondary structures at the NS segment (+)RNA regions 82-148 and 497-564. We found that RNA structures did not affect viral replication in cell culture. However, one of the viruses demonstrated lower NS1 and NEP expression levels during early stage cell infection as well as reduced pathogenicity in mice compared to other variants. In particular, this virus is characterized by an RNA hairpin in the 82-148 region and a stable hairpin in the 497-564 region.

ABSTRACT Type III interferons exhibit antiviral activity against influenza viruses, coronaviruses, rotaviruses, and others. In addition, this type of interferon theoretically has therapeutic advantages, in comparison with type I interferons, due to its ability to activate a narrower group of genes in a relatively small group of target cells. Hence, it can elicit more targeted antiviral or immunomodulatory responses. Obtaining biologically-active interferon lambda (hIFN-λ 1 ) is fraught with difficulties at the stage of expression in soluble form or, in the case of expression in the form of inclusion bodies, at the stage of refolding. In this work, hIFN-λ 1 was expressed in the form of inclusion bodies, and a simple, effective refolding method was developed. Efficient and scalable methods for chromatographic purification of recombinant hIFN-λ 1 were also developed. High-yield, high-purity product was obtained through optimization of several processes including: recombinant protein expression; metal affinity chromatography; cation exchange chromatography; and an intermediate protein refolding stage. The obtained protein was shown to have expected, specific biological activity in line with published effects: induction of MxA gene expression in A549 cells.

Two influenza A nucleoprotein variants (wt: G102R; and mutant: G102R and E292G) were studied with regard to macro-molecular interactions in oligomeric form (24-mers). The E292G mutation has been previously shown to provide cold adaptation. Molecular dynamics simulations of these complexes and trajectory analysis showed that the most significant difference between the obtained models was distance differences between nucleoprotein complex strands. Influenza virus nucleoprotein complexes were isolated from strains bearing the corresponding NP amino acid substitutions. The isolated complexes were characterized by transmission electron microscopy and differential scanning fluorimetry (DSF). Presence of the E292G substitution was shown by DSF to affect nucleoprotein complex melting temperature. In the filament interface peptide model, it was shown that the peptide corresponding in primary structure to the wild-type NP (SGYDF E REGYS, wild type peptide) is prone to temperature-dependent self-association, unlike the peptide carrying the substitution corresponding to E292G (SGYDF G REGYS, mutant peptide). It was also shown that the SGYDF E REGYS peptide (wt) is capable of interacting with a recombinant full-size monomeric nucleoprotein (with primary structure corresponding to wild type); this interaction’s equilibrium dissociation constant is five orders of magnitude lower than for the SGYDF G REGYS peptide. Using small-angle neutron scattering (SANS), the supramolecular structures of isolated complexes of these proteins was studied at temperatures of 15, 32, and 37°C. SANS data show that the structures of the studied complexes (mutant or normal proteins with RNA) at elevated temperature differ from the rod-like particle model and react differently to temperature changes. The data suggest that the mechanism behind cold adaptation with E292G is associated with a weakening of the interaction between strands of the ribonucleoprotein complex and, as a result, the appearance of inter-chain interface flexibility necessary for complex function at low temperature.

Upper respiratory tract infections are the world's most common infectious disease. The etiologic agents behind upper respiratory tract infections (URTIs) are, in fact, a diverse set of pathogens such as influenza, parainfluenza, adenovirus, rhinovirus, and others. More than 200 pathogens are known to be involved. Differential diagnosis of viral infections is sometimes complicated by their diversity or similarity of clinical presentation. This work is devoted to the development of a method which enables simultaneous detection of six common viral URTI pathogens: IAV; IBV; RSV; hAdV; hPIV2; and hPIV3. Antibody microarray technology is utilized to accomplish the analysis. In preparation for protein microchip creation, we produced, characterized, and selected approximately 50 monoclonal antibodies; for each of the aforementioned pathogens, an optimal monoclonal antibody pair was selected. A protein microchip was created, and its core working conditions were optimized. With a balance between convenience and maximal assay sensitivity in mind, a one-step analysis approach was developed for accomplishing the ELISA-like "sandwich" interaction on the manufactured microchip (antibody microarray). Reference viral strains were used to establish the lower limits of detection (LoD) for the assay. For IAV, the LoD was 0.25 ng/ml total viral protein. For other viruses, the LoD ranged from 1 to 2 ng/ml total protein. These sensitivity limits are slightly better than those of standard ELISA, but inferior to those of PCR. Overall, we believe that the developed microchip is a good alternative to existing methods, allowing relatively quick (overnight), inexpensive, simultaneous screening of several pathogens. The design of the antibody microarray is conducive to further development, and the panel of analyzed pathogens can be expanded to include approximately 50 members.

The integration of a biochip with a PDMS fluidic chamber was accomplished. The advantage of dynamic analyte flows, versus static ones, was experimentally demonstrated. This was further clarified by numerical simulation including the approximate mathematical solutions modelling the convective motion and the biotinylated antibody-streptavidin interaction. The possibility of influenza virus A/California/07/09 (H1N1)pdm09 detection by the proposed method was demonstrated.