Purification and Immunogenicity of Nipah Virus-Like Particles from Insect Cells

11 Nipah virus (NiV) is an emerging high-fatality zoonotic threat lacking approved vaccines. Current virus-12 like particle (VLP) production methods rely on costly mammalian cell systems and non -scalable 13 ultracentrifugation purification. We developed an efficient production system for enveloped NiVLPs 14 by co-expressing structural proteins F, G, and M using a baculovirus expression vector system in Sf9 15 cells. A novel multi -step chromatographic purification process was established using monolith 16 convective media, integrating steric exclusion chromatography with sequential cation and anion 17 exchange steps. Purified NiVLPs were characterized by nanoparticle tracking analysis and transmission 18 electron microscopy, then evaluated for immunogenicity in Syrian golden hamsters. The optimized 19 process yielded enveloped particles of approximately 100 -120 nm that morphologically resemble 20 native NiV virions. A single 25 μg NiVLP dose induced robust systemic anti-NiV G IgG responses within 21 14 days, demonstrating rapid immunogenicity suitable for outbreak response . However, neutralizing 22 antibody titers against NiV remained limited compared to total IgG responses. This study establishes 23 the first chromatography -based manufacturing platform for morphologically correct NiVLPs from 24 insect cells. A deeper understanding of the immunity generated is needed to support their potential 25 as a rapidly deployable vaccine platform against NiV. 26 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 2

virus-like particles, Nipah virus, baculovirus expression, monolith chromatography, insect 27 cell culture, vaccine development, pandemic preparedness 28 29

30 Nipah virus (NiV) is a zoonotic paramyxovirus, which was first identified in 1998 during an outbreak in 31 Malaysia and Singapore. Since its emergence, NiV has reappeared almost annually in outbreaks in 32 Bangladesh and India, where it continues to cause severe disease (Khan et al., 2024). NiV is a risk group 33 4 pathogen and is associated with severe respiratory disease and encephalitis, resulting in fatality rates 34 ranging from 40 -100% (Amaya and Broder, 2020) . The natural reservoir of NiV is fruit bats of the 35 Pteropodidae family. Transmission to humans can occur directly, or via intermediate hosts such as pigs 36 and horses (Eaton et al., 2006). Human-to-human transmission has also been documented, notably by 37 the respiratory route. 38 The lack of an approved Nipah vaccine makes NiV a high-priority pathogen with pandemic potential 39 (Moore et al. , 2024) . The development of diverse vaccine platforms offering broad and lasting 40 protection is therefore urgently needed . Various immunization strategies have been explored, 41 including mRNA (Lo et al., 2020; Loomis et al., 2021; Pedrera et al., 2024), DNA (Lu et al., 2023), viral 42 vector (Yoneda et al., 2013; de Wit et al., 2022; Foster et al., 2022; Ithinji et al., 2022; van Doremalen 43 et al., 2022), protein subunit (Mungall et al., 2006; Pallister et al., 2013; Loomis et al., 2020; Geisbert 44 et al., 2021; Gao et al., 2022), and virus-like particles (VLPs) (Walpita et al., 2011, 2017; Welch et al., 45 2023). 46 The surface NiV receptor-binding glycoprotein (G) is the major immunogen that generates neutralizing 47 antibodies and is included in all vaccine candidates that progressed to clinical trials (Kim et al., 2025). 48 NiV G binds to conserved ephrin-B2/B3 receptors (Xu et al., 2008; Wong et al., 2021; Larsen et al., 49 2025), which leads to membrane fusion mediated by the NiV fusion glycoprotein (F). Likewise, NiV F 50 can also generate a neutralizing immune response (Avanzato et al., 2019; Dang et al., 2021), albeit 51 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 3 lower than NiV G (Wit et al., 2023). The matrix (M) and nucleocapsid (N) are the remaining structural 52 proteins that are involved with virion budding an d RNA genome encapsidation, respec tively 53 (Watkinson and Lee, 2016; Ker et al., 2021). 54 VLPs are a proven and safe subunit vaccine platform (Noad and Roy, 2003; Donaldson et al., 2018; 55 Nooraei et al., 2021) that are non-infectious as they do not contain any genetic material. VLPs are self-56 assembled nanoparticles composed of viral structural proteins that closely resemble native pathogens 57 and have the potential to present all key NiV immunogens . Nipah VLPs (NiVLPs) composed of NiV M, 58 F, and G proteins have been previously explored using mammalian cell expression systems (Walpita et 59 al., 2011). Co-expression of these proteins was shown to orchestrate the formation and budding of 60 NiV VLPs that appeared structurally similar to authentic NiV virions and provide protection against viral 61 challenge (Walpita et al. , 2017) . However, so far NiVLP purification strategy relied on 62 ultracentrifugation. This approach is not suitable for vaccine production scale-up (Effio and Hubbuch, 63 2015) and does not resolve the enveloped VLPs from host cell contaminants (Margine et al., 2012; 64 Minh and Kamen, 2021). Instead, a chromatography-based downstream process for NiVLPs needs to 65 be established to support advanced nanoparticle vaccine development (Morenweiser, 2005; Gagnon, 66 2009; Nestola et al., 2015). 67 In this work, we aimed to address purification challenges and develop an alternative production 68 system. We describe the generation of enveloped NiVLPs incorporating key structural proteins (G, F, 69 M) in an insect cell expression system. We developed and optimized a scalable, multi-step purification 70 strategy employing convective interaction media (monoliths). This process integrates steric exclusion 71 chromatography (SXC) with subsequent cation (CEX) and anion (AEX) exchange steps on monolith 72 columns. The purified NiVLPs were characterized by nanoparticle tracking analysis (NTA) and 73 transmission electron microscopy (TEM) . Furthermore, these particles elicited humoral immune 74 responses in Syrian golden hamster model. This outlined downstream process, leveraging the benefits 75 of convective media, provides a robust and scalable platform adaptable for purifying similar enveloped 76 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 4 nanoparticles for diverse applications, including VLP vaccines against emerging viruses with pandemic 77 potential. 78

79 NiVLPs can self -assemble from co -expressed structural proteins in mammalian cells (Walpita et al., 80 2011). However, this system presents challenges for cost -effective, large-scale vaccine production. 81 Insect cells offer a n alternative scalable expression system suitable for VLP production, as 82 demonstrated for influenza (Smith et al., 2013) and coronaviruses (Mortola and Roy, 2004; Bezeljak et 83 al., 2025) . We therefore hypothesized that a baculovirus system could drive NiVLP assembly and 84 secretion in insect cells. To test this, we constructed a single recombinant baculovirus (rBV) to co -85 express NiV M, F, and G proteins in Sf9 cells (Fig. 1a). Following 4 days of expression, culture 86 supernatant was harvested via centrifugation. To capture nanoparticles, including potential NiVLPs, 87 we used SXC principles using polyethylene glycol (PEG) for initial capture of nanoparticles from clarified 88 supernatant (Lee et al., 2012). Unfiltered supernatant containing 6 % PEG6000 was applied to an 8 mL 89 CIM OH column, facilitating binding of larger particles while removing smaller contaminants in flow-90 through. Bound particles were subsequently eluted with a decreasing PEG6000 gradient (Fig. 1b). 91 Analysis of NiV G protein content served as an indicator of NiVLP presence in the collected fractions 92 (Fig. 1c). The detection of NiV G across elution fractions suggests successful NiVLP assembly and 93 secretion in insect cells. Notably, high PEG concentrations in E1 altered the migration of the NiV G on 94 PAGE and western blot, resulting in a lower apparent molecular weight. 95 We proceeded with purification of NiV G -containing fractions from the capture step using CEX 96 chromatography. Diluted SXC elution fractions were loaded onto an 8 mL CIM SO3 column and eluted 97 via a step gradient of ascending NaCl concentration (Fig. 2a). While NiV G was present in all fractions, 98 the majority eluted in fraction E2 (15 to 33 mS/cm conductivity) (Fig. 2b). Finally, NiVLPs were further 99 purified by AEX chromatography. The collected CIM SO3 E2 fraction was applied to a 4 mL CIM QA 100 monolith, and elution was performed using a linear ascending salt gradient (Fig. 2c). Western blot 101 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 5 analysis revealed NiV G eluted in a peak centered around the E2 fraction (Fig. 2d). Finally, buffer was 102 exchanged into PBS with 15 % sucrose and 0.05 % polysorbate-80 using gel filtration group separation. 103 The purified NiVLPs were sterile filtered and analyzed with NTA. The produced vaccine particles display 104 120 nm mean diameter, with 83 nm modal value as determined by NTA (Fig. 3a). This is in agreement 105 with pleomorphic native NiV appearance (Hyatt et al. , 2001; Ang, Lim and Wang, 2018) . NTA 106 measurements were also confirmed by TEM, which revealed characteristic enveloped particles with 107 diameters around 100 nm and evidence of a protein corona, likely corresponding to NiV G and F 108 glycoproteins (Fig. 3b). Thus, NiVLPs expressed in insect cells and purified by this multi-step monolith 109 process closely mimic the morphology of the native pathogen . We used the sterile filtered NiVLPs to 110 determine their immunogenicity in a Syrian hamster model. 111 We immunized Syrian golden hamsters (n = 6) with 25 µg NiVLP intramuscularly (i.m.) with and without 112 AddaVax (ADX) oil-in-water squalene adjuvant (Calabro et al., 2013) (Fig. 4a). Hamsters were boosted 113 21 day s post -prime and we collected serum samples on days 0, 14, 28 and 42, when the study 114 concluded. We quantified IgG titers against recombinant NiV G ectodomain (G ecd) with ELISA and 115 present the data as area under the curve (AUC) (Fig. 4b). NiVLPs, both with and without adjuvant, 116 induced a robust humoral response after a single dose. Interestingly, the ADX adjuvant did not 117 significantly enhance IgG titers, even after the booster dose or at the study's conclusion . We also 118 determined the NiV-B neutralization potential of the final plasma samples in an in vitro neutralization 119 assay (Fig. 5) . Surprisingly, unadjuvanted NiVLPs generated slightly higher, though not statistically 120 significant, neutralizing titers compared to the NiVLP + ADX formulation. However, serum from NiVLP-121 immunized animals (with or without adjuvant) exhibited low neutralizing activity against NiV -B, with 122 only a few samples exceeding the limit of quantification (LOQ) . Interestingly, NiVLPs expressed in 123 mammalian cells induced higher levels of neutralizing antibodies after two doses and conferred 124 complete protection against NiV challenge in hamsters after 1- and 3-dose regimen (Walpita et al., 125 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 6 2017). Altogether, while NiVLPs produced in insect cells are clearly immunogenic in terms of total IgG 126 response, they generate low neutralizing antibody titers in the hamster model against NiV-B strain. 127

128 NiV poses a significant public health threat, yet no approved vaccine exists. VLPs offer a safe and 129 effective vaccine strategy . While mammalian cell -derived NiVLPs are immunogenic (Walpita et al. , 130 2011, 2017; Rajan et al., 2024), their scalable production can be challenging. This study reports, to our 131 knowledge, the first development of NiVLPs expressing F, G, and M proteins using a baculovirus system 132 in Sf9 insect cells, coupled with an efficient, multi-step monolith chromatography purification. 133 Our scalable, chromatography-based purification strategy yielded highly pure NiVLPs of approximately 134 80-120 nm. The particle morphology was consistent with that of native NiV (Hyatt et al., 2001) and 135 previously described NiVLPs (Walpita et al. , 2011) , suggesting correct assembly and surface 136 glycoprotein display. The insect cell-derived NiVLPs induced robust immunogenicity in Syrian golden 137 hamsters. A single 25 µg dose elicited strong systemic anti -NiV G ecd IgG responses, even without 138 adjuvant (Fig. 4b). This rapid induction of high antibody titers is a desirable characteristic for a potential 139 outbreak vaccine. The oil -in-water adjuvant AddaVax (ADX) did not further enhance IgG levels, 140 suggesting the particulate nature of the VLPs themselves is sufficiently immunostimulatory to drive a 141 strong humoral response (Bachmann and Jennings, 2010). 142 Despite high total IgG titers, the neutralizing antibody response against the vaccine homolog Nipah 143 virus Bangladesh strain (NiV-B) was low (Fig. 5). This finding contrasts with studies of mammalian cell-144 derived NiVLPs, which induced higher neutralization titers and protection in hamsters (Walpita et al., 145 2017). This discrepancy is likely attributable to differences in N-glycosylation patterns between insect 146 (typically high -mannose) and mammalian cells (complex N -glycans) (Geisler and Jarvis, 2009) . 147 Glycosylation profoundly impacts viral glycoprotein folding and the presentation of antigens, including 148 neutralizing epitopes. Altered G and F protein conformation on insect cell -derived NiVLPs could lead 149 to limited neutralizing activity. 150 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 7 Interestingly, a structurally similar NiV replicon particle vaccine also failed to generate significant 151 neutralizing titers in hamsters, yet was fully protective against NiV challenge (Welch et al., 2023). This 152 challenges the ke y role of neut ralizing antibod y generation in Paramyxovirus vaccine efficacy 153 determination (He et al., 2016; Pickering et al., 2016; Kingstad-Bakke et al., 2022). Therefore, a more 154 detailed study of NiVLP cellular immune responses and Fc-dependent effector functions in relation to 155 protection against Nipah infection is needed both in humans and in animal models (Bowman, Kaplonek 156 and McNamara, 2023; Zhang et al., 2023). 157 In conclusion, t his study establishes a scalable production and purification platform for 158 morphologically correct NiVLPs that elicit potent systemic humoral immunity after a single dose. 159 Future efforts could focus on evaluation of induced cellular immunity and protection against challenge, 160 head-to-head comparison of insect cell and mammalian -derived antigens, utilizing mammalian-like 161 glycoengineered insect cell lines for NiVLP production (Aumiller et al., 2012) and exploring alternative 162 adjuvants to enhance neutralizing immunogenicity. Furthermore, a direct comparison of the antibody 163 epitope specificity elicited by insect - versus mammalian -derived immunogens would be highly 164 informative for multivalent NiVLP vaccine development. 165

166 Molecular Cloning and Recombinant Baculovirus Preparation 167 NiV-B G sequence was based on GenBank AEZ01397 sequence from 2010 Faridpur outbreak (Lo et al., 168 2012). NiV-M M sequence was based on GenBank AAF73379 sequence (Chua et al., 2000). NiV-M F 169 sequence was based on GenBank AAK29087 with NiVop08 prefusion stabilization mutations (Loomis 170 et al., 2020). The codon-optimized synthetic DNA constructs for insect cell expression were cloned into 171 pLIB shuttle vector (Addgene 80610) in TOP10 E. coli cells (Invitrogen). The NiV M, F and G gene 172 cassettes from pLIB plasmids were then combined into pBIG1a (Addgene 80611) vector using Gibson 173 isothermal assembly according to the biGBac method (Weissmann et al. , 2016) . The expression 174 cassettes were transferred into bacmid backbones in DH10Bac E. coli cells (Invitrogen). We isolated 175 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 8 bacmid DNA with PureLink HiPure Plasmid Miniprep Kit (Invitrogen) and transfected Sf9 insect cells 176 (Oxford Expression Technologies) using ExpiFectamine Sf reagent (Gibco) in a 6 -well culture plate . 177 Recombinant baculovirus (rBV) supernatants were harvested after 5–7 day incubation at 27 °C . We 178 generated V1 baculovirus stocks by infecting Sf9 cells with 1:10 rBV supernatant dilution at 1·106/mL. 179 The viral stocks were harvested after incubation at 27 ° C, 127 rpm for 3-4 days and stored at 4 °C. If 180 necessary, V2 stocks were generated by infecting 2·10 6/mL Sf9 cells with 1:100 V1 dilution. The rBV 181 titers were determined with baculoQUANT qPCR quantification kit (Oxford Expression Technologies). 182 Insect Cell Culture Expression and Harvest 183 Sf9 cells (Oxford Expression Technologies) were grown in a protein -free ESF 921 insect cell culture 184 medium (Expression Systems) at 27 °C and 127 rpm. NiVLPs were expressed in 3 L polycarbonate 185 vented shaker flasks (Corning) by infecting 1 .2 L 1.5·106/mL Sf9 cells at multiplicity of infection (MOI) 186 3. After 4 days of production the cell viability fell below 70 % , at which point we harvested culture 187 supernatants by centrifugation at 4000 rcf for 5 min and 4 °C. The cell culture supernatant was stored 188 at 4 °C prior NiVLP chromatography purification. 189 NiVLP Chromatography Purification 190 NiVLP particles were purified from unfiltered insect cell culture supernatants. A 8 mL CIMmultus® OH 191 column (6 µm; Sartorius BIA Separations) was equilibrated in 50 % buffer OH A (20 mM MES-NaOH pH 192 6.4, 500 mM NaCl, 7 % sucrose) and buffer OH B (20 mM MES-NaOH pH 6.4, 120 mM NaCl, 12 % w/v 193 PEG-6000, 7 % sucrose), resulting in 6 % PEG -6000 concentration. For particle capture with SXC, we 194 loaded the harvested samples on the CIMmultus® OH column with 1:1 in-line dilution with buffer OH 195 B at 40 mL/min. We performed elution at 16 mL/min in a descending linear gradient from 50 % to 0 196 %B over 50 CV . The collected elution fractions were diluted 1:2 in 20 mM MES -NaOH pH 6 .4, 7 % 197 sucrose for nuclease digestion with DENARASE (c-LEcta) at 50 U/mL in presence of 2 mM MgCl2 at room 198 temperature for 2 h to remove contaminating host cell nucleic acids. Digested samples were loaded at 199 40 mL/min on 8 mL CIMmultus® SO3 CEX column (6 µm; Sartorius BIA Separations), which was 200 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 9 equilibrated with buffer SO3 A (20 mM MES-NaOH pH 6.4, 100 mM NaCl, 7 % sucrose). We performed 201 elution in steps of 3-16-40-100 %B at 16 mL/min to buffer SO3 B (20 mM MES-NaOH pH 6.4, 2 M NaCl, 202 7 % sucrose). Selected fractions were diluted 1:2 with 20 mM HEPES-NaOH pH 7.4, 7 % sucrose and 203 loaded at 20 mL/min on 4 mL CIMmultus® QA IEX column (6 µm; Sartorius BIA Separations), which was 204 equilibrated with buffer QA A (20 mM HEPES-NaOH pH 7.4, 100 mM NaCl, 7 % sucrose). NiVLPs were 205 isolated in linear elution gradient to 35 % buffer QA B ( 20 mM HEPES -NaOH pH 7.4, 2 M NaCl, 7 % 206 sucrose) over 25 CVs. Finally, we exchanged buffer to PBS + 15 % sucrose with SEC group separation 207 with Sepharose 6 Fast Flow resin on XK16/20 column (Cytiva) at 2 mL/min. NiVLPs were sterile filtered 208 through 0.22 µm filters (TPP) and stored at -75 °C. The protein content was determined with Bio-Rad 209 Protein Assay (Bio-Rad). 210 SDS-PAGE and Western Blot 211 Collected samples were denatured at 70 °C for 10 min. We performed SDS -PAGE under reducing 212 conditions on 4 -20 % mPAGE™ Bis-Tris precast protein gels ( Merck Millipore) in 1x MOPS running 213 buffer at 200 V. We transferred the samples to activated Immobilion -P PVDF membrane (Merck 214 Millipore) in a Mini -PROTEAN Trans -Blot Module (Bio -Rad) at 350 mA. Immunodetection was 215 performed using SNAP i.d. system (Merck Millipore). The PVDF membrane was blocked with 0.5 % skim 216 milk in TBS T buffer. We used polyclonal anti-Nipah virus/HeV G protein primary antibodies 217 (AntibodySystem, PVV07901). Secondary goat anti-rabbit HRP conjugates (Merck, 12-348) were used 218 for colorimetric detection with 1 -Step Ultra TMB-Blotting Solution (Thermo Scientific). Recombinant 219 NiV G (Proteogenix, PX-P6271-100) protein was used as a positive control. 220 Nanoparticle Tracking Analysis (NTA) 221 Particle size distribution and concentration were determined by Nanoparticle Tracking Analysis (NTA) 222 using a NanoSight N300 (Malvern Instruments, United Kingdom) equipped with a blue laser module 223 (488 nm). NTA software version 3.2 was used for capture and analysis of the data. NiVLP samples were 224 diluted to a working concentration range of 10 7–109 particles/mL (20–100 particles per video frame) 225 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 10 in particle-free water or storage buffer and injected into the device chamber using a syringe pump. 226 Each sample was recorded five times for 45 s at constant capture settings (manually adjusted camera 227 level and focus) and analysis parameters (detection threshold 4 or 5). All measurements were 228 performed at room temperature. 229 Transmission Electron Microscopy (TEM) 230 Formvar-coated grids were placed on drops of NiVLP samples for 5 min and negative staining was 231 performed using 2% phosphotungstic acid (PTA). The grids were examined using a transmission 232 electron microscope JEOL JEM-1400 Plus (Tokyo, Japan) at 120 kV. 233 Hamster Immunogenicity Study 234 The hamster immunogenicity study was performed at the National Biosafety Laboratory, National 235 Center for Public Health and Pharmacy, Budapest. The objective was to evaluate the immunogenicity 236 of insect cell NiVLPs in golden Syrian hamsters. All animal experiments were performed according to 237 the guidelines of the European Communities Council Directive (86/609 EEC) and were approved by the 238 Hungarian National Authority under the license numbers of National Animal Ethical Commitee 239 (PE/EA/00370-6/2024; PE/EA/00958-6/2024). The Syrian hamsters (Janvier) were 6 weeks old and kept 240 under BSL -4 conditions in individually ventilated cages (IsoRat ISO48NFEEU, Techniplast, Italy), the 241 body weight were measured weekly. Groups of 6 animals were assigned to either non-treated control, 242 PBS-treated control, 25 µG NiVLP-only or 25 µg NiVLP + AddaVax™ (Invivogen) experimental groups. 243 The blood samples were taken from orbital vein under inhalational anesthesia with isoflurane 244 (isoflurane: ISOFLUTEK 1000 mg/g, Laboratorios Karizoo S.A.; anesthesia station: MiniHUB -V3, TEM 245 SEGA, France) on dpp 0 (dpp: days post-prime), dpp 14 and dpp 28. At the end of the experiment on 246 dpp 42, blood samples were taken via cardiac puncture after euthanasia. The non -treated control 247 group was euthanized at d pp 0. For immunization 50 µL of NiVLPs were mixed 1:1 with AddaVax ™ 248 adjuvant or PBS (Gibco DPBS) and incubated for 15 minutes at room temperature and then used 249 immediately. For PBS treated control group 100 µL PBS (Gibco DPBS) were used. All immunized 250 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 11 hamsters received an intramuscular (i.m.) injection of 100 µL on dpp 0 and 21. The study was finished 251 on day 42, all hamsters were euthanized and a final blood sample was collected via cardiac puncture. 252 Serum was isolated from the blood and stored at -80° C. 253 ELISA 254 IgG titers against NiV G were determined using an indirect ELISA protocol. Nunc-Immuno™ MicroWell™ 255 96 well plates (Sigma Aldrich) were coated overnight at 4 °C in 1xPBS with 0.5 µg/mL of recombinant 256 NiV Gecd (residues 172-602), which was produced in Sf9 insect cells and purified with Strep-tag affinity 257 chromatography. Next, the plate was washed with wash buffer (0.01 M PBS pH 7.2, Tween-20 0.1% v/v) 258 and blocked with 5% skim milk suspension for 1 h at room temperature. After washing, hamster serum 259 samples were added to the plates at 1: 50 initial dilution. Next, 5-fold serial dilutions were performed 260 and plates were incubated at room temperature for 2 h. After washing, HRP-conjugated secondary goat 261 anti-Syrian Hamster IgG H&L antibodies (Abcam, ab6892) at 1: 10,000 dilution were added and 262 incubated for 1 h at room temperature. The microtiter plates were incubated with TMB substrate 263 (Sigma Aldrich, T4444) for 10 min. After, 2 M HCl stop solution was added and absorbance at 450 nm 264 and 650 nm was determined. For area under the curve (AUC) titer calculation, we determined the 265 baseline from mean blank values plus 3 standard deviations (SD). The negative values were set to 0. 266 The AUC values were determined by integrating the serial dilution data with trapezoidal rule. 267 In vitro Neutralization Assay 268 A seroneutralization assay was performed on hamster serum samples under BSL -4 conditions at the 269 National Biosafety Laboratory, Budapest. Briefly, sera were inactivated at 56 °C for 30 min and were 270 twofold serially diluted starting from 1:20 dilution using serum -free Dulbecco’s Modified Eagle’s 271 Medium (DMEM, VWR) in triplicate in 96-well microtiter plates. Virus-only controls (diluted in DMEM) 272 and cell-only controls (DMEM only) were run in parallel. An equal volume of Nipah virus Bangladesh 273 strain (NiV-B), equivalent to 50 TCID₅₀, was added to each well containing diluted sera and incubated 274 for 1 hour at 37 °C. Subsequently, the mixtures were transferred onto 96-well cell culture plates (TPP , 275 Switzerland) containing Vero E6 cells (Nuvonis) at approximately 2 · 10⁵ cells/mL in serum-free DMEM. 276 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 12 After 5 days of incubation at 37 °C and 5 % CO₂, the plates were inactivated with 10 % formaldehyde 277 solution and stained with crystal violet. The neutralizing antibody titer was defined as the reciprocal of 278 the highest serum dilution that completely inhibited cytopathic effect. The limit of detection for this 279 assay was 1:20 dilution. For calculation purposes, sera that demonstrated partial CPE inhibition but did 280 not achieve complete neutralization were assigned a nominal titer of 6.6 for geometric mean 281 determination. 282 Statistical Analysis 283 The non-parametric Kruskal-Wallis statistical test with Dunn’s multiple comparisons test (Holm 284 mehod) was performed with Python 3.7.11 using pandas, SciPy and scikit-posthocs packages. Asterisks 285 denote correlations that had statistically significant p-value (* p < 0.05; ** p < 0.01). 286 287 Availability of data and materials 288 All data generated or analyzed during this study are available from the corresponding author on 289 reasonable request. 290 291 Funding 292 This work benefited from access to the BSL4 laboratory at National Biosafety Laboratory, National 293 Center for Public Health and Pharmacy, Budapest. The animal study and BSL-4 work are supported by 294 the European Union's Horizon Europe research and innovation program ISIDORE under grant 295 agreement number 101046133 and received funding by the Slovenian Research and Innovation 296 Agency (program No. P3-0083). 297 298 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 13 Declaration of Competing Interest 299 UB and AJ are employees of Sferogen d.o.o., which is a vaccine development company. UB and MP are 300 co-founders of Sferogen d.o.o. Other authors declare no competing interests. 301 302 Authors' contributions 303 UB conceptualized and wrote the manuscript, cloned the genetic constructs, performed 304 chromatography purifications, ELISA and statistical analysis. AJ designed the hamster immunogenicity 305 study and wrote the manuscript. TK cloned the genetic constructs, performed chromatography 306 purifications and ELISA. MLK performed qPCR and western blot analytics. EB maintained insect cell 307 culture, performed qPCR and western blot analytics. MK performed TEM. BP, KZ designed and 308 performed the hamster immunogenicity study and seroneutralization assay. DD performed the 309 hamster immunogenicity study and seroneutralization assay. MP conceptualized the study. All authors 310 read and approved the final manuscript. 311 312

313 We thank M. Leskovec and M. Šnajder of Sartorius BIA Separations for NTA analysis. The pLIB and 314 pBIG1a plasmids were gifts from Jan-Michael Peters. 315 316

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Available at: 481 https://doi.org/10.1038/s41577-022-00813-1. 482 483 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 18 484 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 19 485 Fig. 1. NiVLPs are captured with SXC. (a) Baculovirus expression cassette for the co-expression of NiV 486 M, F and G structural proteins in Sf9 insect cells. HBM : honeybee melittin signal sequence ; polh: 487 polyhedrin promoter; term: SV40-terminator. (b) NiVLP capture step on CIM OH column. Expressed 488 NiVLPs are bound to 8 mL CIM OH column in presence of 6 % PEG6000 from insect cell culture 489 supernatant. Particles were released in descending [PEG6000] gradient. Shaded boxes denote elution 490 fractions E1-5. Fractions E2-4, which were selected for further purification, are light blue. (c) Western 491 blot analysis of capture chromatography step. NiV G protein is detected in E2-E5 elution fractions. SN: 492 cell culture supernatant; FT: chromatography flow-through; E1-5: elution fractions. 493 494 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 20 495 Fig. 2. NiVLP purification with CEX and AEX chromatography. (a) Collected fractions from SXC were 496 loaded on 8 mL CIM SO3 CEX column and purified fractionated using step gradient with increasing NaCl 497 concentration. Shaded area denotes collected elution fractions E1-E4. E2, which was selected for 498 further purification, is light blue. (b) NiVLP detection after CEX with western blot analysis against NiV 499 G. The majority of NiV G is detected in E2 fraction, which was further purified on AEX. (c) The E2 fraction 500 from CEX step was diluted and loaded on 4 mL CIM QA AEX column and separated in ascending salt 501 gradient. (d) NiV G detection with western blot after AEX. The majority of NiVLPs are in E2 fraction. 502 Fractions E1 and E2 were pooled and sterile filtered for immunogenicity study. FT: flow-through; E1-4: 503 elution fractions; rG: recombinant NiV G. 504 505 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 21 506 Fig. 3. Purified NiVLP particles mimic native viral structure. (a) NTA size distribution analysis of sterile 507 filtered NiVLPs . Red shaded area indicates standard error of the mean from 5 runs. SD: standard 508 deviation. (b) TEM images of purified NiVLPs. Scale bars are 100 nm. 509 510 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 22 511 Fig. 4. NiVLPs are immunogenic in hamsters after prime immunization. (a) NiVLP immunogenicity 512 study plan. Syrian golden hamsters (n = 6) were immunized with 25 µg NiVLP with and without AddaVax 513 adjuvant. Hamsters received booster dose 21 days post-prime. Sera samples were collected on days 0, 514 14, 28 and 42. dpp: days post-prime. (b) NiVLP immunogenicity was determined with anti-NiV Gecd IgG 515 ELISA. AUC: area under the curve; PBS: phosphate-buffered saline; ADX: AddaVax adjuvant. * p < 0.05, 516 ** p < 0.01. For statistical comparison, log-transformed AUC titers were evaluated using a Kruskal -517 Wallis test corrected with Dunn’s multiple comparisons test. 518 519 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint 23 520 Fig. 5. Limited NiVLPs stimulation of neutralizing immunity against NiV-B. Hamster serum samples 42 521 days post-prime immunization were used to determine neutralization titers against NiV-B in vitro. 50 522 TCID50 NiV-B was used for neutralization. PBS: phosphate-buffered saline; ADX: AddaVax adjuvant ; 523 LOD: limit of detection. * p < 0.05; ns: not significant. Statistical analysis was performed using Wilcoxon 524 signed-rank test. 525 526 .CC-BY-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted August 24, 2025. ; https://doi.org/10.1101/2025.08.21.671198doi: bioRxiv preprint