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Abstract Background Timothy grass (Phleum pratense) is a significant source of allergens, and recombinant allergens are increasingly used for diagnostic purposes. However, the performance of different recombinant allergen production systems in diagnostic assays needs further investigation to optimize their use in clinical settings. Objective The main objective of this study was to analyze and compare the diagnostic performance of recombinant timothy grass allergens produced in E. coli and N. benthamiana using a custom-made microarray chip. Methods Recombinant timothy grass allergens Phl p 1, Phl p 2, Phl p 5, Phl p 6, Phl p 11, and Phl p 12 were produced in E. coli and/or N. benthamiana. A total of 113 patient serum samples were tested to evaluate the diagnostic sensitivity, specificity, inter-assay variability, and correlation of allergen-specific IgE detection compared to commercial multiplex tests (ALEX and ISAC). Additionally, the prevalence of sIgE to these allergens was assessed. Results Phl p 1, Phl p 2, Phl p 5, Phl p 6 and Phl p 11 showed high or very high positive correlation in immunoreactivity with other commercial multiplex tests. Notably, Phl p 11 fused with maltose-binding protein (MBP) demonstrated high diagnostic specificity and sensitivity, with a 0.3 arbitrary cut-off value. However, a high intra-assay variation was observed. The study also assessed specific IgE prevalence to timothy grass allergens within the tested patient cohort. Conclusions Recombinant allergens from both E. coli and N. benthamiana demonstrated strong diagnostic potential on the microarray platform, with Phl p 11 (MBP-fused) showing particularly high performance. High intra-assay variation highlights the need for further optimization in allergen formulation and microarray storage conditions. These results highlight the potential of recombinant allergens for diagnostic applications, despite challenges with allergen stability in microarray formats. Specific IgE prevalence to timothy allergens revealed a sensitization profile consistent with findings from multiple studies. Graphical Abstract

Seoul orthohantavirus (SEOV) is a rat-associated zoonotic pathogen with an almost worldwide distribution. In 2019, the first autochthonous human case of SEOV-induced hemorrhagic fever with renal syndrome was reported in Germany, and a pet rat was identified as the source of the zoonotic infection. To further investigate the SEOV reservoir, additional rats from the patient and another owner, all of which were purchased from the same vendor, were tested. SEOV RNA and anti-SEOV antibodies were found in both of the patient’s rats and in two of the three rats belonging to the other owner. The complete coding sequences of the small (S), medium (M), and large (L) segments obtained from one rat per owner exhibited a high sequence similarity to SEOV strains of breeder rat or human origin from the Netherlands, France, the USA, and Great Britain. Serological screening of 490 rats from breeding facilities and 563 wild rats from Germany (2007–2020) as well as 594 wild rats from the Netherlands (2013–2021) revealed 1 and 6 seropositive individuals, respectively. However, SEOV RNA was not detected in any of these animals. Increased surveillance of pet, breeder, and wild rats is needed to identify the origin of the SEOV strain in Europe and to develop measures to prevent transmission to the human population.

Meningitis and meningoencephalitis are neurological inflammatory diseases, and although routine diagnostics include testing of a wide range of pathogens, still in many cases, no causative agent is detected. Human parvovirus B19 (B19V), human bocaviruses 1–4 (HBoV1–4), and human parvovirus 4 (hPARV4) are members of the Parvoviridae family and are associated with a wide range of clinical manifestations including neurological disorders. The main aim of this study was to determine whether human parvoviruses infection markers are present among patients with meningitis/meningoencephalitis in Latvia as well as to clarify the role of these viruses on the clinical course of the mentioned diseases. Our study revealed HBoV1–4 and B19V genomic sequences in 52.38% and 16.67% of patients, respectively. Furthermore, symptoms such as the presence of a headache and its severity, fatigue, disorientation, and difficulties to concentrate were significantly frequently present in patients with active parvovirus infection in comparison with parvoviruses negative patients, therefore we suggest that HBoV1–4 and B19V infection should be included in the diagnostics to reduce the number of meningitis/meningoencephalitis with unknown/unexplained etiology.

Monoclonal antibodies (MAbs) against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have used initially a recombinant yeast-expressed full-length Puumala virus (PUUV) Gc protein. However, this approach was unsuccessful. As an alternative recombinant antigen, chimeric virus-like particles (VLPs) harboring a segment of PUUV Gc glycoprotein were generated in yeast Saccharomyces cerevisiae. A 99 amino acid (aa)-long segment of Gc protein was inserted into the major capsid protein VP1 of hamster polyomavirus at previously defined positions: either site #1 (aa 80–89) or site #4 (aa 280–289). The chimeric proteins were found to self-assemble to VLPs as evidenced by electron microscopy. Chimeric VLPs induced an efficient insert-specific antibody response in immunized mice. Monoclonal antibody (clone #10B8) of IgG isotype specific to hantavirus Gc glycoprotein was generated. It recognized recombinant full-length PUUV Gc glycoprotein both in ELISA and Western blot assay and reacted specifically with hantavirus-infected cells in immunofluorescence assay. Epitope mapping studies revealed the N-terminally located epitope highly conserved among different hantavirus strains. In conclusion, our approach to use chimeric VLPs was proven useful for the generation of virus-reactive MAb against hantavirus Gc glycoprotein. The generated broadly-reactive MAb #10B8 might be useful for various diagnostic applications.

Schmallenberg virus (SBV), discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N) protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs). Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.

Non-infectious virus-like nanoparticles mimic native virus structures and can be modified by inserting foreign protein fragments, making them immunogenic tools for antigen presentation. This study investigated, for the first time, the immunogenicity of long and flexible polytubes formed by yeast-expressed tail tube protein gp39 of bacteriophage vB_EcoS_NBD2 and evaluated their ability to elicit an immune response against the inserted protein fragments. Protein gp39-based polytubes induced humoral immune response in mice, even without the use of adjuvant. Bioinformatics analysis guided the selection of protein fragments from Acinetobacter baumannii for insertion into the C-terminus of gp39. Chimeric polytubes, displaying 28-amino acid long OmpA protein fragment, induced IgG response against OmpA protein fragment in immunized mice. These polytubes demonstrated their effectiveness both as antigen carrier and an adjuvant, when the OmpA fragments were either displayed on chimeric polytubes or used alongside with the unmodified polytubes. Our findings expand the potential applications of long and flexible polytubes, contributing to the development of novel antigen carriers with improved immunogenicity and antigen presentation capabilities.

Hantaviruses are emerging pathogens with a worldwide distribution that can cause life-threatening diseases in humans. Monoclonal antibodies (MAbs) against hantavirus nucleocapsid (N) proteins are important tools in virus diagnostics, epidemiological studies and basic research studies on virus replication and pathogenesis. Here, we extend the collection of previously generated MAbs raised against a segment of Puumala orthohantavirus (PUUV) N protein harbored on virus-like particles (VLPs) and MAbs against N proteins of Sin Nombre orthohantavirus/Andes orthohantavirus by generating nine novel MAbs against N proteins of Dobrava-Belgrade orthohantavirus (DOBV), Tula orthohantavirus (TULV), Thottapalayam thottimvirus (TPMV) and PUUV. In order to have a wide collection of well-described hantavirus-specific MAbs, the cross-reactivity of novel and previously generated MAbs was determined against N proteins of 15 rodent- and shrew-borne hantaviruses by different immunological methods. We found that all MAbs, excluding TPMV-specific MAbs, demonstrated different cross-reactivity patterns with N proteins of hantaviruses and recognized native viral antigens in infected mammalian cells. This well-characterized collection of cross-reactive hantavirus-specific MAbs has a potential application in various fields of hantavirus research, diagnostics and therapy.

Current vaccines against infectious diseases have primarily relied on attenuated or inactivated pathogens. However, self-assembled, virus-based nanoparticles (VNPs) are noninfectious multiprotein structures regarded as safe vaccine platforms for an efficient foreign antigen display within a host immune system. Currently, there is a low diversity of self-assembled, rod-shaped VNPs. Additionally, there is no information regarding the generation of tailed-bacteriophage nanotubes in yeast and their immunogenicity in mice. Here, we developed a novel tubular VNP-based vaccine platform utilizing a yeast-synthesized recombinant tail tube gp39 protein from bacteriophage vB_EcoS_NBD2 (NBD2). The diameter of these extremely flexible polytubes was ~12 nm, while the length varied from 0.1 µm to >3.95 µm. In this study, the immunogenicity of polytubes formed by the recombinant gp39 protein and the elicited antibody response were tested. The tubular structures formed by the recombinant gp39 protein were immunogenic in mice, although the addition of Freund’s adjuvant enhanced the anti-gp39 antibody response compared to the use of tubular structures alone. To further examine the applicability of novel polytubes as a carrier for foreign epitopes, the carboxy-terminal region within the gp39 protein was identified, allowing insertion of six histidine residues with no effect on the recombinant protein synthesis or structure self-assembly. This genetic insertion of a foreign epitope within the surface-exposed gp39 domains resulted in a repetitive display of the insert on the surface of NBD2 tail tube-originated polytubes. The combination of a repetitive, highly ordered display of foreign epitopes as well as tubular shape of nanoparticles can greatly enhance the immune response. Although more studies are needed, the flexible and extremely long polytubes formed by the recombinant tail tube gp39 protein represent a new potential platform for presenting target sequences on the exterior surface of the nanotubes.

Nucleotides, peptides and proteins serve as a scaffold material for self-assembling nanostructures. In this study, the production of siphovirus vB_EcoS_NBD2 (NBD2) recombinant tail tube protein gp39 reached approximately 33% and 27% of the total cell protein level in Escherichia coli and Saccharomyces cerevisiae expression systems, respectively. A simple purification protocol allowed us to produce a recombinant gp39 protein with 85%–90% purity. The yield of gp39 was 2.9 ± 0.36 mg/g of wet E. coli cells and 0.85 ± 0.33 mg/g for S. cerevisiae cells. The recombinant gp39 self-assembled into well-ordered tubular structures (polytubes) in vivo in the absence of other phage proteins. The diameter of these structures was the same as the diameter of the tail of phage NBD2 (~12 nm). The length of these structures varied from 0.1 µm to >3.95 µm, which is 23-fold the normal NBD2 tail length. Stability analysis demonstrated that the polytubes could withstand various chemical and physical conditions. These polytubes show the potential to be used as a nanomaterial in various fields of science.

Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance.