BackgroundRecombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs) represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY), the main virulence factor of Gardnerella vaginalis.ResultsThe scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs) that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody.ConclusionsRecombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the scFv-Fc molecules on the surface of pseudotype VLPs was successful and allowed generation of multivalent scFv-Fc proteins with high VLY-neutralizing potency. Our study demonstrated for the first time that large recombinant antibody molecule fused with hamster polyomavirus VP2 protein and co-expressed with VP1 protein in the form of pseudotype VLPs was properly folded and exhibited strong antigen-binding activity. The current study broadens the potential of recombinant VLPs as a highly efficient carrier for functionally active complex proteins.

The B-cell response against measles nucleoprotein (MeN) plays an important role in the control of measles infection. However, the data on B cell epitopes of MeN are still limited. The objective of this study was to identify B cell epitopes in MeN using monoclonal and polyclonal antibodies raised against recombinant yeast-expressed MeN (rMeN) as well as human sera from measles-positive individuals. After immunization of mice, 15 monoclonal antibodies (mAbs) against rMeN were generated. The B cell epitopes were localized using recombinant overlapping MeN fragments, PepScan analysis, and competitive ELISA. The epitopes of 14 mAbs were mapped within the C-terminus of MeN between amino acids (aa) 419 and 525. Four mAbs recognized a linear epitope located within a sequence of aa 440-448. Competitive ELISA revealed a cluster of conformational mAb epitopes. Cross-inhibition studies with human sera demonstrated similar localization of B cell epitopes recognized by serum antibodies from naturally infected individuals. Thus, the majority of B cell epitopes are located at the C-terminal domain of MeN. These findings provide new data on the antigenic structure of MeN and are in agreement with recent experimental evidence indicating that the C-terminal domain of MeN is well accessible on the surface of nucleocapsid-like structures.

Cohort studies are of great importance in defining the mechanism responsible for the development of allergy-associated diseases, such as atopic dermatitis, allergic asthma, and allergic rhinoconjunctivitis. Although these disorders share genetic and environmental risk factors, it is still under debate whether they are linked or develop sequentially along an atopic pathway. The current study was aimed to determine the pattern of allergy sensitization in the Lithuanian birth cohort “Alergemol” (n = 1558) established as a part of the multicenter European birth cohort “EuroPrevall”. Early sensitization to food allergens in the “Alergemol” birth cohort was analysed. The analysis revealed 1.3% and 2.8% of symptomatic-sensitized subjects at 6 and 12 months of age, respectively. The sensitization pattern in response to different allergens in the group of infants with food allergy symptoms was studied using allergological methods in vivo and in vitro. The impact of maternal and environmental risk factors on the early development of food allergy in at 6 and 12 months of age was evaluated. Our data showed that maternal diet, diseases, the use of antibiotics, and tobacco smoke during pregnancy had no significant impact on the early sensitization to food allergens. However, infants of atopic mothers were significantly more often sensitized to egg as compared to the infants of nonatopic mothers.

Human parainfluenza virus type 3 (hPIV3) is a respiratory tract pathogen. The current study aimed to investigate immunodominant regions of hPIV3 nucleocapsid (N) protein by using monoclonal antibodies (mAbs) raised against recombinant N protein and human serum specimens from hPIV3-infected individuals. A panel of murine mAbs was generated following immunization with yeast-expressed hPIV3 N protein self-assembled to nucleocapsid-like particles. All mAbs recognized native viral nucleocapsids in hPIV3-infected cells as confirmed by an indirect immunofluorescence analysis. Antigenic sites recognized by the mAbs were mapped using recombinant overlapping N protein fragments. One major immunodominant site was identified in the carboxy-terminal region (amino acids [aa] 397-486) of hPIV3 N protein. Further analysis with smaller N protein fragments and a synthetic peptide revealed one linear epitope representing aa 437-446 of the N protein located within this antigenic site. This epitope was reactive with 46% of hPIV3 IgG-positive sera. These results suggest that the above antigenic site on the N protein is important in eliciting a humoral immune response against hPIV3.

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