Production, characterization and application of new monoclonal antibodies against viral antigens

Disertacijoje aprašomi monokloniniai antikūnai, sukurti prieš rekombinantinius mielėse susintetintus antigenus: žmogaus paragripo trečiojo tipo viruso, Menangle viruso, hantavirusų bei pasiutligės viruso nukleokapsidės (N) baltymus. Sukurtieji antikūnai buvo visapusiškai charakterizuoti įvairiais imunocheminės analizės metodais, įvertintas jų specifiškumas, afiniškumas, sugebėjimas atpažinti natyvius virusus infekuotų lastelių kultūrose. Buvo nustatyta, kad antikūnai, sukurti prieš rekombinantinius mielėse susintetintus virusų baltymus, tinka virusų nustatymui infekuotose ląstelėse. Šie tyrimai patvirtino, kad rekombinantiniai mielėse susintetinti virusų N baltymai turi panašias antigenines savybes, kaip natyvūs virusų N baltymai, formuojantys nukleokapsides. Sukurtieji monokloniniai antikūnai taip pat buvo panaudoti išsamiems minėtų virusų N baltymų antigeninės struktūros tyrimams bei imunodominuojančių sekų nustatymui. Disertaciniame darbe gauti duomenys svarbūs, kuriant naujas imunodiagnostikos sistemas, skirtas virusų infekcijoms nustatyti. Disertaciją sudaro įvadas, trys skyriai, naudotos literatūros sąrašas ir autorės publikacijų sąrašas. Įvadiniame skyriuje aptariama tiriamoji problema, darbo aktualumas, formuluojamas darbo tikslas bei uždaviniai, darbo mokslinis naujumas ir praktinė reikšmė, pristatomos paskelbtos publikacijos ir pranešimai konferencijose. Pirmasis disertacijos skyrius skirtas literatūros apžvalgai: jame apibūdinamos paragripo virusų, Menangle viruso, hantavirusų ir pasiutligės virusų šeimos ir gentys, jų genomu ypatybės, struktūriniai baltymai, patogenezė ir epidemiologija. Smulkiau aprašomi minėtų virusų N baltymai. Literatūros apžvalgoje taip pat aprašomi rekombinantinių virusinių baltymų sintezės mielėse privalumai, dažniausiai naudojami virusinių infekcijų diagnostikos metodai bei monokloninių antikūnų taikymas virusinių infekcijų diagnostikai. Antrajame skyriuje aprašomi disertaciniame darbe naudoti metodai: hibridomų technologija, imunofermentinė analizė, imunoblotingas, imunofluorescencinė mikroskopija, molekulinės biologijos metodai. Trečiajame skyriuje pateikiami eksperimentiniai duomenys apie naujų monokloninių antikūnų kūrimą, jų specifiškumo tyrimus, sąveika su įvairių virusų N baltymais, infekuotų ląstelių ir audinių tyrimus, virusų N baltymu antigeninės struktūros tyrimus. Disertacijos tema paskelbti 4 straipsniai: visi straipsniai įtraukti į ISI Web of Science sąrašą. Disertacijoje atliktų tyrimų rezultatai paskelbti 6 mokslinėse konferencijose Lietuvoje ir užsienyje

Characterization of the GBoV1 Capsid and Its Antibody Interactions

Human bocavirus 1 (HBoV1) has gained attention as a gene delivery vector with its ability to infect polarized human airway epithelia and 5.5 kb genome packaging capacity. Gorilla bocavirus 1 (GBoV1) VP3 shares 86% amino acid sequence identity with HBoV1 but has better transduction efficiency in several human cell types. Here, we report the capsid structure of GBoV1 determined to 2.76 Å resolution using cryo-electron microscopy (cryo-EM) and its interaction with mouse monoclonal antibodies (mAbs) and human sera. GBoV1 shares capsid surface morphologies with other parvoviruses, with a channel at the 5-fold symmetry axis, protrusions surrounding the 3-fold axis and a depression at the 2-fold axis. A 2/5-fold wall separates the 2-fold and 5-fold axes. Compared to HBoV1, differences are localized to the 3-fold protrusions. Consistently, native dot immunoblots and cryo-EM showed cross-reactivity and binding, respectively, by a 5-fold targeted HBoV1 mAb, 15C6. Surprisingly, recognition was observed for one out of three 3-fold targeted mAbs, 12C1, indicating some structural similarity at this region. In addition, GBoV1, tested against 40 human sera, showed the similar rates of seropositivity as HBoV1. Immunogenic reactivity against parvoviral vectors is a significant barrier to efficient gene delivery. This study is a step towards optimizing bocaparvovirus vectors with antibody escape properties

Persistence of SARS-CoV-2-Specific Antibodies for 13 Months after Infection

Background: Dynamics of antibody responses were investigated after a SARS-CoV-2 outbreak in a private company during the first wave of the pandemic. Methods: Workers of a sewing company (Lithuania) with known SARS-CoV-2 RT-PCR result during the outbreak (April 2020) were invited to participate in the study. Virus-specific IgG and IgM were monitored 2, 6 and 13 months after the outbreak via rapid IgG/IgM serological test and SARS-CoV-2 S protein-specific IgG ELISA. Results: Six months after the outbreak, 95% (CI 86–99%) of 59 previously infected individuals had virus-specific antibodies irrespective of the severity of infection. One-third of seropositive individuals had virus-specific IgM along with IgG indicating that IgM may persist for 6 months. Serological testing 13 months after the outbreak included 47 recovered individuals that remained non-vaccinated despite a wide accessibility of COVID-19 vaccines. The seropositivity rate was 83% (CI 69–91%) excluding one case of confirmed asymptomatic reinfection in this group. Between months 6 and 13, IgG levels either declined or remained stable in 31 individual and increased in 7 individuals possibly indicating an exposure to SARS-CoV-2 during the second wave of the pandemic. Conclusions: Detectable levels of SARS-CoV-2-specific antibodies persist up to 13 months after infection for the majority of the cases

Mapping of recognition sites of monoclonal antibodies responsible for the inhibition of pneumolysin functional activity

The pathogenicity of many bacteria, including Streptococcus pneumoniae, depends on pore-forming toxins (PFTs) that cause host cell lysis by forming large pores in cholesterol-containing cell membranes. Therefore, PFTs-neutralising antibodies may provide useful tools for reducing S. pneumoniae pathogenic effects. This study aimed at the development and characterisation of monoclonal antibodies (MAbs) with neutralising activity to S. pneumoniae PFT pneumolysin (PLY). Five out of 10 produced MAbs were able to neutralise the cytolytic activity of PLY on a lung epithelial cell line. Epitope mapping with a series of recombinant overlapping PLY fragments revealed that neutralising MAbs are directed against PLY loops L1 and L3 within domain 4. The epitopes of MAbs 3A9, 6E5 and 12F11 located at L1 loop (aa 454-471) were crucial for PLY binding to the immobilised cholesterol. In contrast, the MAb 12D10 recognising L3 (aa 403-423) and the MAb 3F3 against the conformational epitope did not interfere with PLY-cholesterol interaction. Due to conformation-dependent binding, the approach to use overlapping peptides for fine epitope mapping of the neutralising MAbs was unsuccessful. Therefore, the epitopes recognised by the MAbs were analysed using computational methods. This study provides new data on PLY sites involved in functional activity

Immunogenic properties and antigenic similarity of virus-like particles derived from human polyomaviruses /

Polyomaviruses (PyVs) are highly prevalent in humans and animals. PyVs cause mild illness, however, they can also elicit severe diseases. Some PyVs are potentially zoonotic, such as simian virus 40 (SV40). However, data are still lacking about their biology, infectivity, and host interaction with different PyVs. We investigated the immunogenic properties of virus-like particles (VLPs) derived from viral protein 1 (VP1) of human PyVs. We immunised mice with recombinant HPyV VP1 VLPs mimicking the structure of viruses and compared their immunogenicity and cross-reactivity of antisera using a broad spectrum of VP1 VLPs derived from the PyVs of humans and animals. We demonstrated a strong immunogenicity of studied VLPs and a high degree of antigenic similarity between VP1 VLPs of different PyVs. PyV-specific monoclonal antibodies were generated and applied for investigation of VLPs phagocytosis. This study demonstrated that HPyV VLPs are highly immunogenic and interact with phagocytes. Data on the cross-reactivity of VP1 VLP-specific antisera revealed antigenic similarities among VP1 VLPs of particular human and animal PyVs and suggested possible cross-immunity. As the VP1 capsid protein is the major viral antigen involved in virus-host interaction, an approach based on the use of recombinant VLPs is relevant for studying PyV biology regarding PyV interaction with the host immune system

Activation of NLRP3 inflammasome by virus-like particles of human polyomaviruses in macrophages

Viral antigens can activate phagocytes, inducing inflammation, but the mechanisms are barely explored. The aim of this study is to investigate how viral oligomeric proteins of different structures induce inflammatory response in macrophages. Human THP-1 cell line was used to prepare macrophages that were treated with filamentous nucleocapsid-like particles (NLPs) of paramyxoviruses and spherical virus-like particles (VLPs) of human polyomaviruses. The effects of viral proteins on cell viability, pro-inflammatory cytokines' production, and NLRP3 inflammasome activation were investigated. Filamentous NLPs did not induce inflammation while spherical VLPs mediated inflammatory response followed by NLRP3 inflammasome activation. Inhibitors of cathepsins and K+ efflux decreased IL-1β release and cell death, indicating a complex inflammasome activation process. A similar activation pattern was observed in primary human macrophages. Single-cell RNAseq analysis of THP-1 cells revealed several cell activation states different in inflammation-related genes. This study provides new insights into the interaction of viral proteins with immune cells and suggests that structural properties of oligomeric proteins may define cell activation pathways

Evaluation of trichodysplasia spinulosa-associated polyomavirus capsid protein as a new carrier for construction of chimeric virus-like particles harboring foreign epitopes

Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes

Molecular characterization of invasive Neisseria meningitidis isolates collected in Lithuania (2009-2019) and estimation of serogroup B meningococcal vaccine 4CMenB and MenB-Fhbp coverage /

Neisseria meningitidis causes invasive meningococcal disease (IMD), which is associated with significant mortality and long-term consequences, especially among young children. The incidence of IMD in Lithuania was among the highest in European Union/European Economic Area countries during the past two decades; however, the characterization of meningococcal isolates by molecular typing methods has not yet been performed. In this study, we characterized invasive meningococcal isolates (n=294) recovered in Lithuania from 2009 to 2019 by multilocus sequence typing (MLST) and typing of antigens FetA and PorA. The more recent (2017-2019) serogroup B isolates (n=60) were genotyped by analyzing vaccine-related antigens to evaluate their coverage by four-component (4CMenB) and two-component (MenB-Fhbp) vaccines using the genetic Meningococcal Antigen Typing System (gMATS) and Meningococcal Deduced Vaccine Antigen Reactivity (MenDeVAR) Index methods, respectively. The vast majority (90.5%) of isolates belonged to serogroup B. MLST revealed a predominance of clonal complex 32 (74.02%). Serogroup B strain P1.19,15: F4-28: ST-34 (cc32) accounted for 64.1% of IMD isolates. The overall level of strain coverage by the 4MenB vaccine was 94.8% (CI 85.9-98.2%). Most serogroup B isolates (87.9%) were covered by a single vaccine antigen, most commonly Fhbp peptide variant 1 (84.5% of isolates). The Fhbp peptides included in the MenB-Fhbp vaccine were not detected among the analyzed invasive isolates; however, the identified predominant variant 1 was considered cross-reactive. In total, 88.1% (CI 77.5-94.1) of isolates were predicted to be covered by the MenB-Fhbp vaccine. In conclusion, both serogroup B vaccines demonstrate potential to protect against IMD in Lithuania

Characterization of the GBoV1 capsid and its antibody interactions

Human bocavirus 1 (HBoV1) has gained attention as a gene delivery vector with its ability to infect polarized human airway epithelia and 5.5 kb genome packaging capacity. Gorilla bocavirus 1 (GBoV1) shares 86% amino acid sequence identity with HBoV1, but has better transduction efficiency in several human cell types. Here, we report the capsid structure of GBoV1 determined to 2.76 Å resolution using cryo-electron microscopy (cryo-EM), and its interaction with mouse monoclonal antibodies (mAbs) and human sera. GBoV1 shares capsid surface morphologies with parvoviruses, with a channel at the 5-fold symmetry axis, protrusions surrounding the 3-fold axis, and a depression at the 2-fold axis. A 2/5-fold wall separates the 2-fold and 5-fold axes. Compared to HBoV1, differences are localized to the 3-fold protrusions. Consistently, native dot immunoblots and cryo-EM showed cross-reactivity and binding, respectively, by a 5-fold targeted HBoV1 mAb, 15C6. Surprisingly, recognition was observed for one out of three 3-fold targeted mAbs, 12C1, indicating some structural similarity at this region. In addition, GBoV1, tested against 40 human sera, showed the same level of seroprevalence as HBoV1. Immunogenic reactivity against parvoviral vectors is a significant barrier to efficient gene delivery. This study is a step towards optimizing bocaparvovirus vectors with antibody escape properties.Human bocavirus 1 (HBoV1) has gained attention as a gene delivery vector with its ability to infect polarized human airway epithelia and 5.5 kb genome packaging capacity. Gorilla bocavirus 1 (GBoV1) VP3 shares 86% amino acid sequence identity with HBoV1 but has better transduction efficiency in several human cell types. Here, we report the capsid structure of GBoV1 determined to 2.76 angstrom resolution using cryo-electron microscopy (cryo-EM) and its interaction with mouse monoclonal antibodies (mAbs) and human sera. GBoV1 shares capsid surface morphologies with other parvoviruses, with a channel at the 5-fold symmetry axis, protrusions surrounding the 3-fold axis and a depression at the 2-fold axis. A 2/5-fold wall separates the 2-fold and 5-fold axes. Compared to HBoV1, differences are localized to the 3-fold protrusions. Consistently, native dot immunoblots and cryo-EM showed cross-reactivity and binding, respectively, by a 5-fold targeted HBoV1 mAb, 15C6. Surprisingly, recognition was observed for one out of three 3-fold targeted mAbs, 12C1, indicating some structural similarity at this region. In addition, GBoV1, tested against 40 human sera, showed the similar rates of seropositivity as HBoV1. Immunogenic reactivity against parvoviral vectors is a significant barrier to efficient gene delivery. This study is a step towards optimizing bocaparvovirus vectors with antibody escape properties.Peer reviewe

Characterization of a Panel of Cross-Reactive Hantavirus Nucleocapsid Protein-Specific Monoclonal Antibodies

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