Structure based stabilized nipah virus (NIV) structural protein designing and method thereof

Abstract

The present invention is in the field of Nipah virus (NiV) structural protein. Particularly, the invention provides structure based stabilized Nipah virus (NiV) structural protein designing and uses thereof.

Description

[0001] B5105-00330

[0002] STRUCTURE BASED STABILIZED NIP AH VIRUS (NiV) STRUCTURAL PROTEIN

[0003] DESIGNING AND METHOD THEREOF

[0004] FIELD OF THE INVENTION:

[0005] The present invention is in the field of Nipah virus (NiV) structural protein. Particularly, the invention provides structure based stabilized Nipah virus (NiV) structural protein designing and uses thereof.

[0006] BACKGROUND OF THE INVENTION:

[0007] The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0008] The Nipah virus, also known as Henipavirus nipahense or NiV, is a zoonotic pathogen classified as a Biosafety Level-4 agent due to its high pathogenicity in humans and the absence of vaccines or treatments. This enveloped ribonucleic acid (RNA) virus has caused multiple outbreaks of encephalitis with high fatality rates. Fruit bats, specifically those in the Pteropus genus, are the primary reservoirs of NiV. Transmission occurs through biological bat matrices (such as urine and feces) or Ni V-contaminated palm fruits or sap. NiV was first identified in 1998 in Kampung Sungai Nipah (Nipah River Village) in Malaysia. In 1999, it spread to Singapore via exported pigs, affecting abattoir workers. Case fatality rates (CFRs) have risen from smaller, isolated outbreaks in Malaysia to over 300 confirmed cases between 2001 and 2015, with an average CFR of 75%.

[0009] In May 2018, the Nipah virus emerged in India, primarily in the state of Kerala. This outbreak garnered significant attention due to the high death rate and rapid spread of the virus. Fruit bats, the natural carriers, were linked to the initial cases. The outbreak caused severe encephalitis and respiratory symptoms, leading to many fatalities. In September 2023, another outbreak occurred in Kerala, resulting in numerous confirmed cases and deaths, underscoring the virus's high fatality rate and public health threat. The state government quickly implemented containment measures, including mass testing, contact tracing, and isolating infected individuals, particularly in affected districts. This outbreak highlighted the persistent threat of the Nipah virus and the urgent need for B5105-00330 ongoing surveillance, research, and the development of effective vaccines and treatments to prevent future outbreaks.

[0010] Clinically, Nipah virus infection typically presents with fever, encephalitis, and / or respiratory symptoms. According to laboratory-confirmed cases in Malaysia, only 8% of patients are asymptomatic. Currently, there are no specific therapies or vaccines for Nipah virus disease in humans or animals.

[0011] The virus NiV ils a member of the Orthoparamyxovirinae subfamily and Paramyxoviridae family of the Henipavirus genus. Six structural proteins — nucleoprotein (N), phosphoprotein(P), matrix protein (M), fusion protein (F), attachment glycoprotein (G), and the large protein, or RNA polymerase protein (L) — are encoded by the approximately 18.2 kb negative-sense single-stranded RNA that makes up the NiV genome. Using glycoprotein G, which preys onephrin-B2 or, to a lesser degree, ephrin-B3 receptors, is how NiV works. The brain, smooth muscle, lungs, placenta, and prostate are the organs where Ephrin-B2 receptors are most expressed. When the receptor binds, the G protein experiences conformational changes and separates from the F protein, which then goes through more conformational changes that lead to the fusion of the host and viral membranes during the fusion phase. In a variety of preclinical challenge models, such as hamsters, it has been shown that it is feasible to use one or both of the NiV outer-membrane proteins, the glycoprotein (G) and fusion (F) protein, as the antigen(s) to elicit a protective immune response. Infected patients in Malaysia were treated with ribavirin, which is effective against other Paramyxoviruses, including Respiratory Syncytial Virus. Since then, tests on animal models have shown that ribavirin is ineffective. Unlike most other well -characterized paramyxoviruses, the NiV’s G glycoprotein is a type II membrane protein with 602-aa residues that does not bind to carbohydrate moieties and does not have hemagglutinating or neuraminidase activities. The tetrameric protein interlaced beta-sandwich (neck domain) and the N- terminal four-helix bundle (stalk domain) make up the glycoprotein's core. Similar to HRA and HRB or HRN and HRC, respectively, trimeric alpha-helical heptad repeats regions 1 and 2 (HR1 and HR2) are characteristic of class I fusion protein F. Two subunits, the N-terminal F2 and the C-terminal Fl subunits, connected by a disulfide bond, make up the resulting fusion-primed protein. Numerous potential vaccines have been assessed using animal models. The development of strong cellular responses, particularly memory B and T cells capable of effectively combating acute infections and B5105-00330 re-infections throughout an individual's lifetime, is often just as important to a vaccine's efficacy as the induction of high levels of neutralising antibodies. Other vaccination approaches, like the use of live viral vector based vaccines and subunit vaccines, are being researched. Although live-attenuated virus vaccines have the potential to enhance immunity, their application is challenging due to the high virulence and mortality associated with NiV.

[0012] Despite the significant public health threat posed by Nipah virus (NiV) there are notable research gaps in the development of vaccines for these pathogens. Addressing these gaps is crucial for effective prevention and control of outbreaks. In India and its surrounding countries, the reported Nipah outbreaks are pandemic and hence a major health concern to be addressed. Unavailability or limited access to key reagents such as proteins, antibodies, or peptides. Currently there are no approved vaccines, drugs, or biotherapeutics against these viruses. NiV exhibits substantial genetic diversity, complicating the development of a universal vaccine.

[0013] Hence, there is a need to identify conserved viral antigens that can elicit a broad immune response.

[0014] Particularly, there is a need of an effective viral antigens which closely resembles the natural virus, and is capable of eliciting a more potent and specific immune response along with enhanced immunogenicity.

[0015] There is further a need of a technology that can induce the production of broadly neutralizing antibodies, enhancing protection across different strains of the Nipah virus.

[0016] OBJECTIVE OF THE INVENTION:

[0017] The primary object of the present invention is to overcome the drawbacks associated with prior art.

[0018] Another object of the present invention is to provide an Engineered Recombinant Protein Antigen of structural proteins of Nipah virus (NiV) envelope proteins and uses thereof.

[0019] Another object of the present invention is to provide soluble forms of NiV G and F glycoproteins, specifically aimed at generating soluble forms of these glycoproteins.

[0020] Another object of the present invention is to provide NiV vaccine immunogens, the soluble proteins or antigens for the identification of small molecules for antiviral use, and as immunogens that bind specific NiV broad neutralizing antibodies. B5105-00330

[0021] Another object of the present invention is to provide a diagnostic kit and method based on glycoproteins which can be utilized for identifying small molecules that target NiV envelope glycoprotein monomers and / or trimers, as well as serving as antigens for crystallization and electron microscopy (EM) structural analysis.

[0022] Another object of the present invention is to provide a diagnostic kit and method based on glycoproteins for the identification of broad neutralizing antibodies from NiV-infected individuals, vaccinated subjects, or antibody or ligand libraries.

[0023] SUMMARY OF THE INVENTION:

[0024] The Invention provides a chimeric immunogenic construct based on recombinant protein antigen of structural proteins of Nipah virus (NiV) envelope proteins comprising: a) atleast a recombinant NiV G glycoprotein of SEQ ID No: 1; b) atleast a NiV G tetramer of SEQ ID No: 2; c) atleast a fusion (F) ectodomain trimer comprising NiV F trimeric form codon of SEQ ID No: 3.

[0025] In an embodiment, the recombinant NiV G glycoprotein comprises a monomeric NiV G recombinant, His-tagged protein consisting of head domain which is expressed from a polynucleotide comprising a coding region encoding Nipah virus.

[0026] In an embodiment, the recombinant NiV G glycoprotein comprises a tetrameric NiV G recombinant, His-tagged protein consisting of stalk and head domain which is expressed from a polynucleotide comprising a coding region encoding Nipah virus.

[0027] In an embodiment, the fusion (F) ectodomain trimer comprises Nipah F trimeric soluble antigen consisting of ectodomain and folds on trimerization domain which is expressed from a polynucleotide comprising a coding region encoding Nipah virus.

[0028] In an embodiment, the invention also provides a soluble recombinant protein construct comprising a CD5 leader sequence at N-terminal end of the polypeptides for NiV G monomer and tetramer and for NiV F protein a fold on derived from C-terminus of the fibritin domain of T4 bacteriophage and His tag at C terminal end of the polypeptides.

[0029] In an embodiment, the invention also provides a vaccine composition comprising: a) 30 pg of NiV monomer; b) 30 pg of NiV tetramer; B5105-00330 c) Adjuvant comprising AddaVax; wherein the combination of NiV monomer and Ni V tetramer is present along with the adjuvant in the ratio of 1 : 1.

[0030] DETAILED DESCRIPTION OF DRAWINGS:

[0031] To further clarify the advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which are illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting in their scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings in which:

[0032] Fig. 1 : depicts the design of NiV soluble glycoproteins. (A) NiV G monomer soluble protein has been designed by taking the globular soluble head domain of the NiV attachment protein (G) attached to a C-terminal His purification tag B) NiV G tetramer soluble protein has been designed by taking the stalk domain (and globular soluble head domain of the NiV attachment protein (G) attached to a C-terminal His purification tag C.) NiV F soluble trimer like protein has been designed by taking the F ectodomain 2-488 residues and linking C terminally with foldon trimerizing domain and His purification tag. The designed polypeptides are codon optimized and was cloned in commercially available pCDNA 3.4 expression vector. As per the sequence for NiV G monomer, Number of amino acids: 461; Molecular weight: 51795.20; Theoretical pl: 6.85; for NiV G tetramer, Number of amino acids: 568; Molecular weight: 63518.61; Theoretical pl: 7.15 and for NiV F trimer, Number of amino acids: 551; Molecular weight: 60593.94; Theoretical pl: 6.02

[0033] Fig. 2: depicts A.) depicts the schematic of downstream process development of expression, purification of subunit protein based soluble immunogen B) The codon optimized plasmids were expressed in, Expi293F suspension cell culture. Expi293F cells were maintained in suspension cultures in Expi293 Expression Medium (Thermo Fisher Scientific) at 37°C, 8% CO2, and 80% humidity on a shaker incubator set at 110 rpm. Cells were transfected using the ExpiFectamine 293 transfection kit according to the manufacturer's instructions. Proteins were harvested 5-7 days after transfection or upon reaching a cell death of 60% or more. The eluted protein was then dialyzed against PBS at 4°C and then concentrated with a 50 ml amicon filter with a 30 KDa molecular weight cut-off to obtain the final protein. Its purity was analysed by SDS-PAGE on a 10% separating gel and additional staining with Coomassie blue staining. The B5105-00330 final eluted fraction of recombinant NiV G monomer was run under reducing condition and visualised on 10% SDS-PAGE to confirm purity and molecular weight which was around ~ 51 KDa (left panel), NiV G tetramer molecular weight at ~ 63kDa (middle panel), NiV F trimer molecular weight at ~ 60 KDa (right panel).

[0034] Fig. 3: depicts Biochemical characterization of NiV G monomer and tetramer soluble recombinant proteins A. Immunoreactivity as seen by Western blots generated by using commercial monoclonal antibody (Absolute antibody ,Cat no- Ab02865-l .1., dilution - 1 : 1000 against NiV. B.The ability of the NiV G soluble proteins to bind to in-house developed polyclonal mouse sera as were measured by ELISA using NiV G monomer protein coated at a concentration of 2pg per well in the maxsorb 96 well plate in coating buffer incubated over night at 4°C. Antibody titres were calculated as the serum dilution giving OD450 nm readings after subtracting the background levels using prebled control serum at the same dilutions.

[0035] Figure 4: depicts Oligomeric state confirmed by blue Native-PAGE. Sample proteins were prepared by boiling for 10 min with SDS and P-mercaptoethanol, which comprised the 2x loading dye, and separated by reducing 10% SDS-PAGE followed by visualization with Coomassie brilliant blue stain. For Native-PAGE analysis, proteins were separated on 4-15% Native-PAGE gels (Mini-PROTEAN TGXTM, Bio-Rad, Hercules, CA, USA) using Native- PAGE sample preparation buffer (Invitrogen, Waltham), MA, USA) and Bis-Tris working buffer followed by the same protocols as above. Both NiV G momoner and tetramer were found to form higher order oligomers of -molecular weight of 1142kDa and 1200 kDa respectively.

[0036] Figure 5: depicts Negative stain micrographs for diluted NiV G momoner and tetramer samples. Left panel represents selected 2D class averages of NiV monomer and right panel of NiV G tetramers.

[0037] Figure 6: depicts A. Depicts immunogenicity assessment of NiV G monomer and tetramer immunogens in presence of AddaVax™ adjuvant. To determine the immunogenicity of the NiV antigens, the proteins were intramuscularly administered in 6-8 weeks old BALB / c mice. They were divided into four groups of seven mice each. Group one and two was administered with 30 pg of NiV monomer and NiV tetramer with 1 : 1 ratio of adjuvant Addavax™, Group three was administered with only AddaVax™ adjuvant and fourth group was PBS control group serving as negative control. Prime-boost strategy was used for immunization within a gap of 28 days and 14-days post prime and post boost sera were collected for immune response studies. At specified intervals, blood samples were drawn from the orbital venous sinus. The B5105-00330 serum was separated by centrifugation at 3000 rpm for 20 min, and it was stored at -80 °C until needed. B. Binding antibody titers (IgG) were evaluated using ELISA plates coated with lOOpl / well of 2 pg / ml concentration of protein immunogens in carbonate buffer and probed with serial diluted sera collected from animals before and after immunization with prime and boost. Left panel shows the endpoint titers of diluted sera to NiV G monomer coated at a concentration of 2 pg / ml and right panel shows the endpoint titers of diluted sera to NiV G tetramer coated at a concentration of 2 pg / ml.

[0038] DETAILED DESCRIPTION:

[0039] For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

[0040] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

[0041] The present application provides an Engineered Recombinant Protein Antigen of structural proteins of Nipah virus (NiV) envelope proteins and uses thereof.

[0042] The Invention in an embodiment provides various forms of immunogens which are further expressed in the form of recombinant soluble protein derived from a NiV envelope structural proteins, that may be utilized as antigen alone or with other immunogens that will elicit NiV specific as well as cross neutralizing antibodies against other subtypes, or alternately for crystallization and electron microscopy (EM) structural analysis and for the identification of neutralizing antibodies from NiV infected individuals or vaccinated subjects from population, antibody or ligand libraries for identification of small molecules, or as research reagent.

[0043] In an embodiment, the immunogens comprises chimeric sequences containing this recombinant NiV G monomer glycoprotein, NiV G tetramer and fusion (F) ectodomain trimer. The application further covers the nucleic acids responsible for encoding these immunogens and the methods involved in their production. B5105-00330

[0044] Native-like G and F glycoproteins present the viral antigens in a conformation that closely resembles the natural virus, which elicits a more potent and specific immune response. This approach of the present invention induces the production of broadly neutralizing antibodies, enhancing protection across different strains of the Nipah virus. Native-like glycoproteins can induce both humoral (antibody-mediated) and cellular (T-cell-mediated) immune responses, providing comprehensive protection. This is further customized by the present invention to enhance the presentation of conserved epitopes, promoting the generation of broadly neutralizing antibodies.

[0045] Additionally, the present invention uses sequences from Indian clinical isolate to enhance the breadth of protection of vaccines designed from these sequences.

[0046] In the present invention, applicants constructed and designed from the Bangladesh strain GenBank: AY988601.1: i) a monomeric NiV G recombinant, His-tagged protein consisting of head domain which is expressed from a polynucleotide comprising a coding region encoding Nipah virus as described later in the embodiments, ii) a tetrameric NiV G recombinant, His-tagged protein consisting of stalk and head domain which is expressed from a polynucleotide comprising a coding region encoding Nipah virus from Bangladesh strain as described later in the embodiments, iii) Nipah F trimeric soluble antigen consisting of ectodomain and fold on trimerization domain which is expressed from a polynucleotide comprising a coding region encoding Nipah virus from Bangladesh strain. The recombinant protein antigen sequence includes modification that allows for suitable expression system.

[0047] The present invention relates to designing and expression of a monomeric G, stable tetrameric G and stable trimeric F native like viral structural antigen sequence that will express as soluble recombinant proteins a potential as vaccine candidates and possible other utility of being used as antigen for testing anti -NiV antibodies at population in large, but shares the properties of the modified designing and expression of protein of the present invention.

[0048] In another embodiment, the invention provides a plasmid and constructed a recombinant, His- tagged protein which is expressed from a polynucleotide comprising a coding region encoding NiV the embodiments. The recombinant protein may include modification that allows for suitable expression. B5105-00330

[0049] The present invention relates to creation of a stable native like viral NiV structural glycoprotein that has a potential as vaccine candidate and possible other utility of being used as antigen for testing anti-influenza antibodies at population in large, but shares the properties of the modified designing and expression of protein of the present invention.

[0050] In an embodiment, the present invention provides a soluble protein of the present invention which is expressed in mammalian expression system. The soluble recombinant protein is designed with a CD5 leader sequence at N-terminal end of the polypeptides for NiV G monomer and tetramer and for NiV F protein a foldon derived from C-terminus of the fibritin domain of T4 bacteriophage and His tag at C terminal end of the polypeptides. The polypeptides are codon optimized and cloned in pCDNA3.4 vector.

[0051] The soluble monomer and tetramer G and trimer F proteins are expressed, characterized. Additionally, the tetrameric G and F proteins have been found to form oligomeric proteins a property that qualifies this protein antigen to an excellent antigen for immunogenicity studies.

[0052] Therefore, it is an objective of the present invention to provide for a NiV vaccine immunogen. It is another objective of the present invention to provide for a recombinant NiV Env proteins derived from a circulating virus strain in Asia pacific region for use as an immunogen that could induce broad neutralizing antibodies. It is another objective of the present invention to provide for a native G tetramer and F trimer mimic. It is another objective of the present invention to provide for recombinant structural proteins for identification of small molecules, such as small molecules for use as anti-viral compounds that bind specific G or F envelope glycoproteins. It is another objective of the present invention to provide for recombinant structural proteins for crystallization and electron microscopy (EM) structural analysis. It is another objective of the present invention to provide for a recombinant G and F envelope for the identification of broad neutralizing antibodies from NiV infected individuals or vaccinated subjects or antibody or ligand libraries. It is another objective of the present invention to provide for mutations required for recombinant NiV structural proteins to remain in its tetrameric or trimeric conformational state.

[0053] In one aspect, the present invention provides for an engineered or non-naturally occurring NiV structural proteins comprising at least 100% identity with the amino acid sequence encoded by SEQ ID No. 1, 2, and 3.

[0054] In another aspect, the present invention provides methods to develop nanoparticle based or vector or mRNA based immunogen design comprising a nucleotide sequence encoding the NiV B5105-00330

[0055] G and F protein of the present invention. The nucleotide sequence may be codon optimized for expression in a mammalian cell. The vector may be a plasmid. The vector may be a virus. The virus may be a lentivirus, adenovirus, adeno associated virus (AAV), or poxvirus.

[0056] In another aspect, the present invention provides for a method of producing an immune response or eliciting an immune response comprising administering to a mammal the NiV recombinant proteins of the present invention. The method may comprise administering to a mammal an immunogenic composition of the present invention. The NiV G and F protein may be administered with an adjuvant, alum, squalene, alhydrogel or novel adjuvants alone or in different combination.

[0057] The polypeptide can be produced synthetically or by recombinant means. In embodiments in which the polypeptide is produced recombinantly, the present invention provides in another aspect a nucleic acid construct that comprises a coding sequence for a polypeptide as broadly described above and elsewhere herein, operably linked to a regulatory element that is operable in the host cell.

[0058] In another aspect, the present invention provides a method of expression mammalian expression system. For suspension expression, Expi293F cells were cultured in Expi™ Expression Medium (Thermo Fisher Scientific, #A1435101) and transfected with the ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific, #A14524) following the manufacturer’s protocol. Supernatants were collected 5-7 days’ post-transfection or when cell viability fell below 60%. Cultures were clarified by centrifugation and filtration, and proteins were purified by IMAC using Ni-NTA resin (QIAGEN, India, #30210) on an AKTA pure 25 system (Cytiva). Eluates were dialyzed against PBS at 4 °C and concentrated using a 50 mL Amicon centrifugal filter (30 kDa MWCO) to obtain the final preparation. Purity of the expressed protein was assessed by SDS-PAGE (12% resolving gel) followed by Coomassie Brilliant Blue staining. Figure 1 shows the design of the NiV soluble glycoproteins which includes three recombinant constructs optimized for expression in mammalian systems using the pCDNA 3.4 expression vector. First, the NiV G monomer was engineered by isolating the globular head domain of the NiV attachment (G) protein and fusing it to a C-terminal His-tag for purification, resulting in a protein with 461 amino acids, a molecular weight of 51,795.20 Da, and a theoretical pl of 6.85. Second, a NiV G tetramer-like soluble protein was designed by including both the stalk domain and globular head of the G protein, again with a C-terminal His-tag, yielding a construct of 568 amino acids, with a molecular weight of 63,518.61 Da and a theoretical pl of 7.15. Finally, the NiV F trimer-like protein was created by taking the B5105-00330 ectodomain (residues 2-488) of the F protein and fusing it to a foldon trimerization domain and a His-tag to facilitate proper trimer formation and purification. This construct comprises 551 amino acids, has a molecular weight of 60,593.94 Da, and a theoretical pl of 6.02. All constructs were codon-optimized to enhance expression efficiency in mammalian host cells. These soluble glycoprotein designs are well-suited for applications in structural studies, immunogenicity assays, vaccine research, and diagnostic development.

[0059] Figure 2 illustrates the schematic of the downstream process development for the expression and purification of subunit protein-based soluble immunogens derived from the Nipah virus glycoproteins. Codon-optimized plasmids encoding the NiV G monomer, NiV G tetramer, and NiV F trimer constructs were expressed in Expi293F suspension cells. The Expi293F cells were cultured in Expi293 Expression Medium (Thermo Fisher Scientific) under controlled conditions at 37°C, 8% CO2, and 80% humidity, in a shaker incubator set at 110 rpm. Transient transfection was performed using the ExpiFectamine 293 transfection kit following the manufacturer's protocol. Recombinant proteins were harvested between 5 to 7 days posttransfection or when cell viability dropped below 40% (i.e., cell death of 60% or more). The supernatant containing the secreted proteins was collected, and proteins were purified using affinity chromatography via the C-terminal His-tag. The eluted proteins were then dialyzed against PBS at 4°C and concentrated using a 50 mL Amicon ultrafiltration device with a 30 kDa molecular weight cut-off. Protein purity and molecular weight were assessed by SDS- PAGE using a 10% separating gel, followed by Coomassie Brilliant Blue staining. The final purified proteins were analyzed under reducing conditions, with the NiV G monomer appearing at an expected molecular weight of ~51 kDa (left panel), the NiV G tetramer at ~63 kDa (middle panel), and the NiV F trimer at ~60 kDa (right panel), confirming the successful expression and purification of each construct.

[0060] Figure 3 shows the biochemical characterization of the soluble recombinant NiV G monomer and tetramer proteins. In panel A, the immunoreactivity of the purified proteins was assessed via Western blotting using a commercially available monoclonal antibody specific to Nipah virus glycoprotein (Absolute Antibody, Cat. No. Ab02865-l.l), used at a dilution of 1 : 1000. Both the NiV G monomer and tetramer showed specific binding, confirming the retention of antigenic epitopes recognized by the antibody. Panel B illustrates the ability of these soluble proteins to bind to polyclonal antibodies raised in-house in mice. An indirect ELISA was performed using the NiV G monomer coated at a concentration of 2 pg per well on Maxisorp 96-well plates. Plates were coated with antigen in coating buffer and incubated overnight at B5105-00330

[0061] 4°C to allow proper adsorption. Following blocking and incubation with serially diluted mouse sera, the bound antibodies were detected using an appropriate secondary antibody conjugated to an enzyme, and the absorbance was read at 450 nm. Antibody titers were determined by calculating the serum dilution that produced an optical density (OD450) above the background level, which was defined using pre-bleed control serum tested in parallel. The results demonstrate that the recombinant NiV G proteins retain immunogenic conformations capable of eliciting and being recognized by virus-specific polyclonal antibodies, highlighting their potential utility in vaccine development and serological assays.

[0062] Figure 4 illustrates the assessment of the oligomeric state of the NiV G monomer and tetramer proteins using both denaturing SDS-PAGE and native gel electrophoresis. For SDS-PAGE, sample proteins were denatured by boiling for 10 minutes in the presence of SDS and P- mercaptoethanol, as part of the 2X reducing loading dye. These denatured samples were then separated on a 10% reducing SDS-PAGE gel and visualized using Coomassie Brilliant Blue staining, which allowed confirmation of the monomeric molecular weights of the NiV G monomer (~51 kDa) and tetramer (~63 kDa). To determine their native oligomeric states, the proteins were further analyzed by Blue Native-PAGE. Native samples were prepared using Native-PAGE sample preparation buffer (Invitrogen) and run on 4-15% gradient Native- PAGE gels (Mini-PROTEAN TGX™, Bio-Rad) using the Bis-Tris buffer system. The electrophoresis and staining protocols were consistent with those used in the SDS-PAGE. Native-PAGE analysis revealed that both NiV G monomer and tetramer proteins existed predominantly in higher-order oligomeric forms, with estimated molecular weights of approximately 1142 kDa and 1200 kDa, respectively. These findings suggest that the soluble recombinant G proteins retain the capacity to self-associate into oligomeric structures, which may more closely resemble their functional conformation on the viral envelope.

[0063] Figure 6 presents the immunogenicity evaluation of the NiV G monomer and tetramer-based subunit immunogens formulated with AddaVax™ adjuvant in BALB / c mice. In panel A, six- to eight-week-old BALB / c mice were divided into four groups, each comprising seven animals. Groups one and two received 30 pg of NiV G monomer and NiV G tetramer, respectively, formulated in a 1 : 1 ratio with AddaVax™ adjuvant and administered via intramuscular injection. Group three received only AddaVax™ as an adjuvant control, while group four received phosphate-buffered saline (PBS) as a negative control. A prime-boost immunization strategy was followed, with a booster dose administered 28 days after the initial priming. Blood samples were collected at defined intervals i.e. 14 days post-prime and 14 days post-boost via B5105-00330 the orbital venous sinus. Sera were separated by centrifugation at 3000 rpm for 20 minutes and stored at -80 °C until further use. In panel B, IgG binding antibody titers were measured by indirect ELISA using 96-well plates coated with 100 pl per well of 2 pg / ml of either NiV G monomer (left panel) or NiV G tetramer (right panel) in carbonate coating buffer. Serial dilutions of the collected sera were incubated with the coated antigens to evaluate the antibody response pre- and post-immunization. The endpoint titers were calculated based on the last serum dilution giving an OD450 reading above background (defined using pre-immune sera). The results demonstrated a robust antigen-specific IgG response in animals immunized with both the monomeric and tetrameric NiV G constructs, indicating that both immunogens are capable of eliciting a strong humoral immune response when delivered with AddaVax™.

Claims

B5105-00330WE CLAIM:

1. A chimeric immunogenic construct based on recombinant protein antigen of structural proteins of Nipah virus (NiV) envelope proteins comprising: a) atleast a recombinant NiV G glycoprotein of SEQ ID No: 1; b) atleast a NiV G tetramer of SEQ ID No: 2; c) atleast a fusion (F) ectodomain trimer comprising NiV F trimeric form codon of SEQ ID No: 3.

2. The construct as claimed in claim 1, wherein recombinant NiV G glycoprotein comprises a monomeric NiV G recombinant, His-tagged protein consisting of head domain which is expressed from a polynucleotide comprising a coding region encoding Nipah virus.

3. The construct as claimed in claim 1, wherein recombinant NiV G glycoprotein comprises a tetrameric NiV G recombinant, His-tagged protein consisting of stalk and head domain which is expressed from a polynucleotide comprising a coding region encoding Nipah virus.

4. The construct as claimed in claim 1, wherein fusion (F) ectodomain trimer comprises Nipah F trimeric soluble antigen consisting of ectodomain and folds on trimerization domain which is expressed from a polynucleotide comprising a coding region encoding Nipah virus.

5. A soluble recombinant protein construct comprising a CD5 leader sequence at N- terminal end of the polypeptides for NiV G monomer and tetramer and for NiV F protein a fold on derived from C-terminus of the fibritin domain of T4 bacteriophage and His tag at C terminal end of the polypeptides.

6. The soluble recombinant protein construct as claimed in claim 5, wherein polypeptides are codon optimized and cloned in pCDNA vector.

7. The soluble recombinant protein construct as claimed in claim 5, wherein the tetrameric G and F proteins forms oligomeric proteins.

8. A vaccine composition comprising: a) 30 pg of NiV monomer; b) 30 pg of NiV tetramer; c) Adjuvant comprising alum, squalene, alhydrogel; wherein the combination of NiV monomer and NiV tetramer is present along with the adjuvant in the ratio of 1 : 1 volume / volume with respect to antigen;B5105-003309. A method for the identification of small molecules for use as anti-viral compounds comprising the steps of reacting the compound with G or F proteins and determining the binding with the specific G or F envelope glycoproteins; wherein the method identifies small molecules that bind specifically to a target viral envelope glycoprotein (G or F) and inhibit virus entry / fusion.

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