An engineered polypeptide complex for generating protective immune responses against monkey pox(MPOX)

Abstract

The present invention relates to the development of an engineered polypeptide complex, aimed at inducing protective antibody responses against the Monkey pox virus. The polypeptide complex is structured with antigenic domains connected via specific linkers to a nanocage vehicle. When expressed in a suitable bacterial expression system, the complex generates a recombinant soluble protein that, upon administration in vivo, elicits an immune response that provides protection against Mpox virus infection. The invention also covers the method for producing this engineered polypeptide complex, as well as a vaccine formulation comprising the complex along with pharmaceutically acceptable excipients.

Description

[0001] +

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of biotechnology. In particular, the present invention relates to an engineered polypeptide complex, which is expressed as a recombinant soluble immunogen, for generating protective immune responses against Monkey Pox (Mpox).

[0004] BACKGROUND OF THE INVENTION

[0005] Monkey pox is an important zoonotic disease, which has re-emerged in recent times. It is caused by double stranded DNA monkey pox virus that belongs to the genus Orthopoxvirus, family Poxviridae, and sub-family Chordopoxvirinae. Monkeypox shows symptoms similar to smallpox. Since monkeypox virus share similar genotype and phenotype characteristic to smallpox virus, the same vaccine as that of smallpox are being used to prevent monkeypox.

[0006] Currently, there are two vaccines that are approved and used to provide protection against monkey pox. The Jynneos vaccine contains live attenuated Vaccinia virus and could be used for the prevention of monkeypox disease in adults (18 and older) who are at high risk for infection with either virus. The ACAM2000, FDA licensed Smallpox (Vaccinia) Vaccine is for active immunization against smallpox disease for persons who are at high risk for smallpox infection. The smallpox vaccines available in market provide only 85% protection against Mpox but, there are no specific drugs and vaccines available in market against Mpox. The vaccines availability is limited to LMIC population and the re-emergence of monkeypox virus and other zoonotic poxviruses is causing outbreaks in nonendemic countries over the years; which is a serious concern. In 2022-2023 a global outbreak of Mpox was caused by a strain known as clade Hb and in 2024 WHO again declared mpox outbreak a public health emergency of international concern. Thus, there lies an immediate need for the development of a vaccine that can be effective against recently emerged Mpox, which will have affordability and more accessibility. The Mpox virus genome consists of a linear, double-stranded DNA (dsDNA) of ~200 kilobase pairs and contains ~200 genes. It is important to understand the virus biology for designing and development of novel vaccine candidates. Pox viruses produce two infectious particles: mature virions (MVs) which mainly remain intra cellular (IMV) and extracellular virions (EEVs). Both IMVs and EEVs bind to a variety of host cells using different attachment mechanisms. Approximately, 11+ proteins from IMV and EEV are collectively referred to as the entry fusion complex (EFC), which tightly associates with the viral membrane. Hence, there are more than one antigen which need to be consider for the vaccine design. The initial association of IMV with the cell occurs through glycosaminoglycan binding of the A27, E8, and H3 proteins, which are the surface antigens and suitable for vaccine designing. The association of EV with the cell surface may be mediated in part through a lectin-binding site of the A34 protein. Hence, for development of protein subunit-based vaccine it is necessary to include more than one antigen for the development of suitable vaccine.

[0007] Accordingly, a vaccine platform that can incorporate highly immunogenic or conserved epitopes or domains or antigens in multiple copies in one unit and can be expressed as recombinant soluble protein is highly desirable for the development of subunit protein-based vaccines.

[0008] Further, most of the vaccines currently available are only amenable to mammalian expression systems, have a very low protein yield, cost of production is high, not secreted in the medium as soluble proteins and generally present in inclusion 25 bodies, therefore increasing the steps in downstream processing and therefore very difficult to scale up.

[0009] Hence, there is a need for a vaccine technology that can overcome the problems of the prior art which could be easily producible, ensure scale up and affordability.

[0010] OBJECTIVE OF THE INVENTION

[0011] An object of the present invention is to provide an engineered polypeptide complex, a composition of an engineered polypeptide complex along with carrier as vaccine, process of manufacturing said vaccine and its utility thereof.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention is drawn to an engineered polypeptide complex designed in formula [1] as:

[0014] [First antigenic domain-Linkerl- nanocage Non- Structural Protein (NSP10)-Linker2-second antigenic domain] — Formula [1] wherein the first antigenic domain having SEQ ID NO. 2 linked with Linkerl with SEQ ID NO. 4 which are linked with nano vehicle NSP10 having SEQ. ID No. 8 further linked with linker with SEQ ID No. 4 linked with second antigenic domain having SEQ. ID No. 3. The antigenic domains and linkers are arranged as per Formulafl]. The engineered polypeptide complex, represented by SEQ ID NO: 1, Mpx-V3, expressed in bacterial expression system generates recombinant protein termed as Mpx-V3 which when administered in vivo, induces antibodies that protect against the Mpox virus. The present invention also discloses the process for producing the said polypeptide complex, V3, along with a carrier or a vaccine platform, the final antigen termed as Mpx-V3. The present invention also discloses a vaccine comprising the engineered polypeptide along with pharmaceutically acceptable excipients.

[0015] The complete sequence of the immunogen for Monkey pox virus vaccine Mpx-V3 which consists of the antigens from both E8 and H3 domain sequence represented by SEQ ID NO. 2 and SEQ ID NO. 3 respectively.

[0016] The complete sequence of the immunogen for Mpox V3 vaccine which consists of the linker denoted by SEQ ID NO. 4.

[0017] The administration of the engineered polypeptide complex elicits neutralizing antibodies and T cell responses against Monkey Pox virus, thereby protecting the subject from symptomatic infection, reducing disease severity, and accelerating recovery.

[0018] BRIEF DESCRIPTION OF FIGURES

[0019] Figure 1: depicts designing and expression of four antigens with various linker lengths and selection of the promising candidate for further characterization. The antigenic epitopes of Mpox were used such as fusing the mature virions (MV) E8L surface antigens, at the N terminus and H3L surface antigen at the C terminus of nonstructural protein- 10 (NSP10) vehicle by using respectively using glycine-serine linker combinations linkers as described in Figure 1A. The designed constructs were codon optimized and commercially cloned in a pET28b (+) vector, ThermoFisher Scientific, USA. The vectors containing the polypeptide sequences from VLo (linker GSSGGSSG), VL1 (GSSG), VL2 (GGSGGS) and V3 (GGSGGGSG) was transformed into competent BL-21 E. coli cells, and the expression was checked in SDS-PAGE gel as shown in Figure IB, no expression. Another, construct V3 contains the similar sequence with linker size GGSGGGSG.

[0020] Figure 2: depicts Mpx-V3 Design, purification and characterization. Figure 2(A) depicts the designing of Mpx-V3: fusing E8L surface antigen, at N terminus and H3L surface antigen at C terminus of nonstructural protein-construct 10 (NSP10) vehicle by using respectively using glycine-serine linker GGSGGGSG. The designed constructs were codon optimized and commercially cloned in a pET28b (+) vector, ThermoFisher Scientific, USA. The vector was transformed into competent BL-21 E. coli cells. The Mpox-V3 has been expressed in the inclusion bodies, extensively characterized and the whole polypeptide cassette comprises of 705 amino acids and theoretical molecular weight of 79.276 kDa. Figure 2(B) depicts SDS PAGE of purified Mpx-V3 recombinant protein. Lane 1 : Marker, Lane 2 represents purified Mpx-V3 with a molecular weight of ~79 kDa (>95% pure) and Figure 2(C) represents identification of the whole polypeptide Mpx-V3 protein by Western blot, the protein was developed against anti-E8 antibody, Lane 1 : Blot against anti-H3 antibody, Lane 3 : Blot against anit-NSP-10 polyclonal sera, Lane 5, whereas lane 2, 4, 6 represents protein marker.

[0021] Figure 3 depicts Chromatography and Biophysical characterization of Mpx-V3. Figure 3(A) depicts anion exchange chromatogram of Mpx-V3 in flow through (FT) mode. The column was equilibrated with 5 CV of refolding buffer. The protein was loaded onto the column and elution carried out by 20 CV gradient of 0-1M NaCl. Figure 3(B) depicts size exclusion chromatography of Mpx-V3 using Superdex 200 Increase column. The purified Mpx-V3 recombinant protein eluted at 9.0 ml., suggesting formation of a higher order (dodecamer) structure. Figure 3(C) depicts Intrinsic tryptophan fluorescence of Mpx-V3. The protein was excited at wavelength of 280 nm and emission was measured between 300 nm to 450 nm. The maxima were obtained at 335 nm. Figure 3(D) depicts circular dichroism spectra of Mpx-V3 acquired by Jasco Spectrophotometer. Far UV-CD spectrum showed 80 % alpha helical (208 nm and 220 nm) and 19 % beta sheet (218 nm) structure.

[0022] Figure 4 depicts Mpx-V3 immunogen induces augmented humoral and cell-mediated immune response in BALB / c mice. 6 - 8 weeks old BALB / c mice (n=6) were immunized with 25 pg of Mpx-V3 either with AddaVax™ or Alhydrogel adjuvant and with respective adjuvants alone. The blood sample was drawn from mice before and after prime and boost immunization, serum was separated, heat-inactivated, and stored at -20°C. Individual mice values were plotted, a) The test serum samples (i.e, pre-bleed, prime, and boost) obtained from the immunized animals were used to determine the IgG level using ELISA by endpoint titer of anti-Mpx-V3 antibodies using the Mpx-V3 antigen coated plates. Individual mice (n=6) values were plotted and error bar represent SEM. Statistical analysis between 2 groups was performed by unpaired, two-tailed, Student’s t-test. b) The level of IgG subtypes (IgGl, IgG 2a, IgG 2b, IgG 2c and IgG3) in the boost serum from Mpx-V3 with AddaVax™ and Mpx-V3 with Alhydrogel group was estimated in boost serum samples. The individual mice (n=6) endpoint titer was plotted, c) The ratio of endpoint titer of IgGl to IgG2a was calculated for Mpx- V3+AddaVax™ and Mpx-V3+Alhydrogel groups after averaging the individual values and mean values are plotted on to a graph, d) Neutralizing antibody titres of day 35 anti-Mpx-V3 sera against VACV was determined by PRNT50 method. PRNT50 value from individual mice is plotted, the error bar represents the SEM. The GraphPad Prism 9 was used for calculating statistical analysis. One-way ANOVA was used for comparing multiple groups, Dunnett' s multiple comparison tests was applied to calculate the p values, e) Splenic T-cell response of naive, adjuvant control, and Mpx-V3 along with adjuvants immunized mice against the E8L and H3L stimulation. The data shown here is from three mice and was analyzed using the FlowJo software and graphs were plotted using the GraphPad Prism 9. One-way ANOVA was used for comparing multiple groups, Dunnett's multiple comparison tests was applied to calculate the p values.

[0023] Figure 5 depicts the immunization of Mpx-V3 immunogen confers protection to intranasal challenge of Vaccinia virus in BALB / c mice, a) Schematic of intranasal challenge study with VACV western reserve strain (n=8) for all the groups (n=6 for body weight, survival and n=2 for titer). b & c) The prime-boost immunized mice were intranasally challenged with 50 pl of 1 x 107PFU / ml of vaccinia virus. After the challenge changes in B) body weight and c) survival, were recorded every day for two weeks. The data shown here is mean ± SD from one challenge study having n=6 in each group's and for survival individual animal record was plotted. d) Graphical representation of behavior scores of infected mice. Animals were scored based on the clinical symptoms exhibited by them into 1 - 10. Scoring with 0=no alteration in behavior, l=no restriction of movement; blink frequently; no body stiffening; no hind limb paralysis; 2=dull; 3=piloerection; 4=shivering; 5=hunched posture; 6=restriction of movement; 7= blink frequently; no body stiffening; no hind limb paralysis; 8=restriction of movement; body stiffening; no hind limb paralysis; 9=restriction of movement; eyes closed; 10=body stiffening; hind limb paralysis, sometimes tremor. The data shown here is each group's mean ± standard deviation, e) Heatmap of temperature changes observed after infection up to 14 days. All the groups including normal control, adjuvant control, and Mpx-V3 -immunized groups were assessed for change in body temperature up to 14 days post-infection by the VACV. The data shown here is each group's mean ± standard error of mean (SEM). f & g) Vaccinia virus titer was determined in the lung and trachea samples harvested at 5 days’ post-infection, respectively (n=2 mice). Upon harvesting, organ samples were weighed, homogenized, sonicated, and centrifuged. Later, aliquots of the supernatant from the samples was stored for titer calculation in Vero cells. Confluent Vero cells were infected with different dilutions of organ supernatant for 2 hours and later cells were washed and layered with 2% CMC- in 2% FBS containing DMEM for 48 hours. Then, the media was removed, cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Plaques were counted from the plates to calculate virus titer in pfu / gram. The values plotted are from two biological replicate as mean+SEM, statistical analysis between 2 groups was performed by unpaired, two-tailed, Student’s t-test.

[0024] Figure 6 depicts Mpx-V3 immunogen protects BALB / c mice from skin pock lesions, a) Schematic of skin pock lesion challenge study with VACV western reserve strain, b) Thermal profiling of the immunized mice following the tail scarification technique. All the groups including normal control, adjuvant control, and Mpx-V3 -immunized groups were assessed for change in body temperature up to 21 days post-tail scarification by the VACV. The data shown here is each group's mean ± standard error of the mean (SEM). c) In the tail pock lesion challenge experiment, BALB / c mice were immunized with 25 pg of Mpx-V3 with AddaVax™ or Alhydrogel. After prime and boost immunization, 21 days of boost mice were challenged with VACV (10 pl of 1 x 107PFU / ml) vial tail scarification method as mentioned in methods section. The appearance of “clinical signs” was evaluated on 6-, 12-, and 21-days post-tail scarification. The pictures shown in a red box represent the maximum intensity of skin lesions and scabs in adjuvant control groups whereas, the green box indicate the complete recovery in the Mpx-V3 immunized mice from the skin lesions and scabs.

[0025] Figure 7 depicts Mpx-V3 immunization protects BALB / c mice from intranasal challenge of MPOXV. 35 female BALB / c mice of 6 - 8 weeks age were randomly divided into five groups having n=7 mice in each group, a) Following the previous immunization scheme and dose mice were immunized in a prime and boost dose of 25 pg along with AddaVax™ and Alhydrogel as shown in schematic, b) Neutralizing antibody titres (n=6) of day 35 Mpx-V3 sera against VACV (WR), MPOXV (N-27, from Bel resources) and MPOXV (Clade lib, from NIV Pune) was determined by PRNT50 method. PRNT50 value from individual mice is plotted, the error bar represents that SEM. c) The immunized mice (n=7) were intranasally challenged with 50 pl 1 x 106PFU / ml of MPOXV. Post infection changes in the body weight and clinical scores were recorded every day for two weeks. Animals were scored based on the clinical symptoms exhibited by them into 1 - 10. Scoring with 0=no alteration in behavior, l=no restriction of movement; blink frequently; no body stiffening; no hind limb paralysis; 2=dull; 3=piloerection; 4=shivering; 5=hunched posture; 6=restriction of movement; 7= blink frequently; no body stiffening; no hind limb paralysis; 8=restriction of movement; body stiffening; no hind limb paralysis; 9=restriction of movement; eyes closed; 10=body stiffening; hind limb paralysis, sometimes tremor. The data shown here is each group's mean ± standard deviation, d) The virus titer of MPOXV was determined in the lung and trachea samples (n=6) harvested at 6 days’ post-infection, respectively. Upon harvesting, organ samples were weighed, homogenized, sonicated, and centrifuged. Later, the aliquots of the supernatant from the samples were stored for titer calculation in Vero cells. Confluent Vero cells were infected with different dilutions of organ supernatant for 2 hours and later cells were washed and layered with 2% CMC- in 2% FBS containing DMEM for 48 hours. Then, the media was removed, cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Plaques were counted from the plates to calculate virus titer in pfu / gram. The values are plotted as mean+SEM, statistical analysis between 2 groups was performed by unpaired, two-tailed, Student’s t-test. The GraphPad Prism 9 was used for calculating statistical analysis. One-way ANOVA was used for comparing multiple groups, Dunnett' s multiple comparison tests was applied to calculate the p values.

[0026] DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention discloses an engineered polypeptide complex for generating immune responses against Monkey pox (Mpox), comprising multiple antigenic domains joined to a nanocage through linkers, having formula [1],

[0028] First antigenic domain-Linkerl- nanocage Non- Structural Protein (NSP10)-Linker2-second antigenic domain

[0029] The antigenic domains can be selected from the Mpox strain belongs to Lineage B.1, Clade lib to the west African clade of monkeypox viruses. The selected antigens showed more than 95% homology to other clades (Table 1) The complete genome of monkeypox virus, hMPOXV / USA / MAOO 1 / 2022 has been sequenced (GenBank: ON563414.3 and GISAID: EPI ISL 13052289), comprising E8 and H3 antigens. Mpoxv has a large DNA genome that produces two distinct antigenic virion forms: intracellular mature virus (IMV) and extracellular enveloped virus (EEV), like other Orthopoxviruses. IMVs and EEVs have approximately 25 and 6 surface proteins, respectively, some of which are recognized targets for neutralizing antibodies and are commonly utilized in vaccine design. The current E8 and H3 proteins share sequences with other clades as shown in Table 1. Table 1.

[0030] Sequence alignment and percentage identity of different strains of Monkeypox from clade I and clade Ila and b and VACV. The percentage identity matrix was created by Clustal 2.1 Clade Lineage MPXV Strain GenBank no. Percent Percent

[0031] Identity Identity Matrix Matrix to E8L to H3L

[0032] I 24MPX0239V PP601224.1 99.67 99.38

[0033] I DRC 07-0120 JX878418.1 99.67 99.38

[0034] I Congo S I<J6426I 3. I 99.67 99.38

[0035] Ila LSA 2003 039 DQoTl 157.1 99.67 99.38

[0036] Ila l.iberia l 97O I 84 DQOH 156.1 99.67 99.38 lib A MPXV_Nigeria_2017_2915 OP535339.1 100 100 lib A M5312_HM12_Rivers NC_063383.1 100 100 lib A MPXV-M2940 FCT MT903337.1 100 100 lib A. l MPXV-Singapore MT903342.1 100 100 lib A.1.1 MPXV_USA_2021_MD ON676708.1 100 100 lib A.2 MPXV_USA_2022_VA001 ON675438.1 100 100 lib A.2.2 MPXV_USA_2022_PA0016 OP450997.1 100 100 lib A.3 MPXV_Nigeria_2017_2948 OP535323.1 99.67 100

[0037] VACV VACV AY243312 94.41 93.83

[0038] In the present invention, a viral non-structural protein (NSP-10) of the Coronavirus family, which normally self-assembles into a spherical multimeric structure and could display the foreign epitopes or domains stapled to the N and C terminal ends and an inner hollow hydrophobic chamber which stabilizes the nanocage structure. 4 constructs (Mpx-VLO, Mpx-

[0039] VL1, Mpx-VL2, Mpx-V3). were designed and constructed using different combinations of MPXV surface antigens in a novel Multivalent Self assembled Nanocage (MSN) scaffold protein as a nanocarrier. The nanocage adorned with E8L and H3L antigens elicited robust antigen-specific antibody responses and provided protection against both intranasal and skin pock in vivo preclinical challenge models against the Vaccinia virus as well as intranasal challenge against the Mpox virus strain. The present invention discloses an engineered polypeptide complex, expressed as a recombinant soluble protein wherein, the first antigenic domain is E8 having 255-265 amino acids, preferably 259 amino acids represented by SEQ ID NO: 2.

[0040] The linkerl and linker2 are selected from the group consisting of glycine-serine linkers, having 6-10 amino acids preferably 8 amino acids.

[0041] The linkers can be selected from the group comprising GGSG used in Mpx-VLO represented as SEQ. ID NO. 5, GSSG used in Mpx-VLl represented as SEQ. ID No. 6, GGSGGS used in Mpx-VL2 as SEQ. ID NO. 7, GGSGGGS used in Mpx-V3 represented as SEQ. ID No.4, more preferably linker is selected from GGSGGGSG (glycine-serine linker) with SEQ ID No. 4.

[0042] In the present invention the nanocage is a non- structural protein (NSP), more specifically NSP10 represented as SEQ ID No. 8. The antigenic domains can be positioned at N terminal or C terminal of the nanocage. The nanocage comprises 150-155 amino acids, preferably 152 amino acids and is represented by SEQ ID No. 8.

[0043] The four constructs were designed for combating Mpox virus using four Vector Linkers (VL) Vector Linker o(VLo), Vector Linker 1(VL1), Vector Linker 2(VL2) and Vector Linker 3(VL3) among the four constructs designed — Mpx-VLo, Mpx-VLl, Mpx-VL2, and Mpx- V3 — the V3 construct has demonstrated the most promising results in combating Monkeypox. This was confirmed through a series of rigorous experimental procedures. There is no or minimal expression for the rest of the constructions.

[0044] The construct for Mpx-V3 represented by SEQ. ID No. 15 is capable of producing the Mpx- V3 immunogen represented by SEQ ID NO. 1.

[0045] In the present invention second antigenic domain is H3 having 273-278 amino acids, preferably 276 amino acids and represented by SEQ ID No. 3.

[0046] The engineered polypeptide complex consists of following: i. amino acids in range of 700-710; ii. molecular weight of 75- 80kDa; iii. pl in range of 5-6

[0047] In an embodiment a vaccine comprising: i. the engineered polypeptide complex; ii. excipients selected from adjuvants, stabilizers, and preservatives.

[0048] The excipients are selected from the group comprising adjuvants, stabilizers and preservatives. In an embodiment the adjuvants are selected from the group comprising AddaVax (Squalene based adjuvant which is <6%. given in 1 :1 v / v), Alhydrogel (Alum based adjuvant which is 2%, given in 1 : 1 v / v

[0049] In an embodiment the stabilizers are selected from the group comprising sucrose-based stabilizer 3-7% preferably 5% sucrose, trehalose, polysorbates preferably sucrose.

[0050] In an embodiment the preservatives are selected from (2 -Phenoxyethanol (2 -PE), Thimerosal (thiomersal) and Phenol.

[0051] The present invention also discloses a process for producing the vaccine comprising the engineered polypeptide complex having sequence of SEQ ID NO. 1, comprises: i. identifying antigenic domains of Mpox virus; ii. screening the antigenic domains of Mpox virus identified in step (i) and selecting multiple antigenic domains showing high immunogenicity such as E8, H3 preferably E8 and H3; iii. stapling the selected multiple antigenic domains screened in step (ii) by linkers selected from a group comprising glycine-serine linkers, glycineproline linkers, and glycine-proline-serine linkers preferably glycine-serine linkers; iv. stitching the stapled antigenic domain of step (iii) to a nano-cage NSP10 by glycine-serine linkers, form engineered polypeptide complex; v. optimizing codons for expressing the engineered polypeptide complex of step (iv); vi. cloning the engineered polypeptide complex obtained in step (v) to a vector to form recombinant vector; vii. transforming the recombinant vector cloned with the sequence represented by SEQ ID No. 15 capable of encoding the engineered polypeptide complex obtained in step (vi) into competent E. coli BL21, BL21(DE3) preferably BL21 cells and culturing for protein expression; viii. expressing the engineered polypeptide complex of step (vii) in inclusion bodies and recovering said inclusion bodies from the cultured cells; ix. purifying the engineered polypeptide complex of step (viii) to obtain a preparation having greater than 90-97% preferably 95% purity as determined by SDS-PAGE; x. identifying the purified engineered polypeptide complex of step (ix) by Western blotting using antibodies specific to the antigenic domains and to NSP10 nano-cage; xi. subjecting the engineered polypeptide complex of step (x) to chromatographic purification including anion exchange chromatography and size exclusion chromatography to confirm formation of a higher-order multimeric structure; xii. performing biophysical characterization of the engineered polypeptide complex of step (xi) using intrinsic tryptophan fluorescence and circular dichroism spectroscopy to determine secondary structure content; xiii. formulating the engineered polypeptide complex of step (xii) with an adjuvant selected from Squalene-based adjuvant, Aluminum hydroxide- based adjuvant, or Aluminum hydroxide / phosphate-based adjuvant preferably Squalene-based adjuvant or Aluminum hydroxide-based adjuvant and immunizing a pre-clinical animal models C57BL / 6, BALB / c mice, preferably BALB / c mice.

[0052] The present invention also discloses a vector for protein expression can be selected from the group comprising bacterial expression system expression pET -28a (+), pET28b (+), pET -28c (+) preferably pET28b (+) vector.

[0053] The administration of the engineered polypeptide complex elicits neutralizing antibodies and T cell responses against Monkey Pox virus, thereby protecting the subject from symptomatic infection, reducing disease severity, and accelerating recovery.

[0054] ADVANTAGES OF THE INVENTION

[0055] The present invention offers several advantages, including multivalence, which enables broader and more effective targeting; ease in development with high scalability, allowing smooth and cost-effective transition from laboratory to large-scale production; and high adaptability.

[0056] In the present invention, AddaVax and Alhydrogel are interchangeable used as squalene-based adjuvant in <6%. given in 1 : 1 v / v and alum-based adjuvant in 2%, given in 1 : 1 v / v respectively. Also, Mx-VLO, Mpx-VLl, Mpx-VL2, Mpx-V3 are interchangeably used as VLO, VL1, VL2 and V3.

[0057] Additionally, the engineered polypeptide complex for generating immune responses against Monkey pox consists of the following sequences as shown in table A.

[0058] The present invention is illustrated by examples. The examples are mere embodiments of the present invention and cannot be construed as limiting. EXAMPLE 1: DEVELOPMENT AND DESIGNING OF THE ANTIGEN SEQUENCE Antigens of Monkey virus pox derived from the strain which belongs to the west African clade of monkeypox viruses was used for developing and designing the engineered antigen. The complete genome of monkeypox virus, hMPOXV / USA / MAOOl / 2022 has been sequenced (GenBank: ON563414.3 and GISAID: EPI ISL 13052289). Four antigen constructs were designed named Mpx-VLO denoted by SEQ. ID No. 12, Mpx- VL1 denoted by SEQ. ID No. 13, Mpx-VL2 by SEQ. ID No. 14, Mpx-V3 by SEQ. ID No. 15 using different combinations of MPOXV surface antigens in a novel Multivalent Selfassembled Nanocage (MSN) scaffold protein as a nanocarrier (Figure 1). The antigen constructs further produced engineered polypeptide complex selected from SEQ. ID Nos. 9, 10, 11 and 1. Mpox-V3 construct, the synthetic gene coding for E8 and H3 was fused to NSP 10 nanocage backbone at N terminus and C terminus respectively with different linker lengths (Figure 1A). The SEQ ID No. 1 consists of SEQ ID No. 2 (E8 antigenic epitope) fused N- terminally with NSP-10 SEQ ID No. 8 by linker with SEQ ID No. 4. GGSGGGSG and then again linked C terminally with linker SEQ ID No. 4. GGSGGGSG, to SEQ ID No. 3 (H3 antigenic epitope). The designed construct represented by SEQ. ID No. 15 capable of producing the Mpx-V3 immunogen represented by SEQ ID No. 1 was codon optimized and commercially cloned in a pET28b (+) vector. The vector was transformed into competent BL- 21 E. coli cells. A single colony was picked up from antibiotic plate (kanamycin) and E. coli cells were cultured in Super broth (SB) media containing Kanamycin (25 pg / ml) as a selectable marker. The cells were monitored for absorbance at 600 nm, and the culture was induced ImM isopropyl P-D-l -thiogalactopyranoside (1 mM IPTG) at the OD of 0.4-0.6. The culture was further grown at 37°C, 200 rpm for 4 hours. The induced bacterial cells were harvested by centrifugation at 4000 rpm, 4°C using Sorvall RC6+, USA and stored at -80°C until further use. Out of the four plasmids, only Mpx-V3 plasmid showed the expression, whereas other three plasmids did not express as shown in Figure IB.

[0059] EXAMPLE 2: PURIFICATION AND EXPRESSION OF THE Mpox V3 ANTIGEN SEQUENCE

[0060] MpoxV3 expressed abundantly in A. coli cells by ImM isopropyl P-D-l -thiogalactopyranoside (IPTG) induction in the SB media where the yield was found to be ~30-40mg / litre. The same protocol was repeated using the Luria Broth (LB) and the yield has been improved to ~100mg / litre at lab scale. MpoxV3 mostly accumulated intracellularly as inclusion bodies. The inclusion bodies were washed with 2% Triton-XlOO, 500 mM NaCl and 2 % Deoxy cholate containing >90 % pure MpoxV3. MpoxV3 was then solubilized in low amounts of urea and alkaline pH. The protein was finally folded via pulsatile dilution in refolding buffer containing 50 mM Tris and 5 % Sucrose. The purified refolded MpoxV3 (5 pg) was resolved on 12 % SDS-PAGE along with pertained marker to assess the expression of the protein and was found to be resolved in ~79kDa (Figure 2B). Next the Western blot was carried out with the Mpox V3 protein and resolved against commercial antibodies against Mpox virus antigens anti-E8 (1 :1000), anit-H3 (1 :500) antibodies and against the nanocage vehicle anti-NSPIO antibody (1 :500) (Figure 2C). The membrane was again washed 0.1 % PBST twice followed by incubated at 37°C for 45 minutes with anti-mouse Horseradish Peroxidase (HRP) conjugated secondary antibody (1 :2000). Next, the membrane was washed with PBS and developed using Enhanced Chemiluminescence (ECL) reagent (Biorad laboratories, USA).

[0061] EXAMPLE 3: BIOPHYSICAL CHARACTERIZATION THE ANTIGEN

[0062] The MpoxV3 was further characterized.

[0063] A. The Anion exchange chromatogram of the purified Mpox-V3 immunogen. The chromatogram shown was obtained using the anion exchange chromatography performed at flow-through (FT) mode (Figure 3 A).

[0064] B. The elution profile of Mpox-V3 immunogen on a Superdex 200 column shows a single well- defined peak at 9 ml corresponding to oligomer formation (Figure 3B).

[0065] C. The intrinsic fluorescence of MpoxV3 was measured using Varian Fluorescence spectrophotometer. MpoxV3 gave maximum fluorescence intensity at 335 nm. This maximum is characteristic of MpoxV3 (Figure 3C).

[0066] D. Further, Mpx-V3 was assessed by Far-UV spectroscopy for the prediction of secondary structure. We used Jasco Spectra Manager software to deduce the secondary structure of the purified protein. The spectra were analyzed using Spectra Manager software and compared to Yang’s reference spectra. Assessment by Spectra Manager software showed a presence of 17.0% helix, 32.8% beta, 16.9% turn, and 33.2% random coil structure (Figure 3D).

[0067] EXAMPLE 4: IMMUNOGENICITY ASSESSMENT OF THE ANTIGEN IN BALB / c MICE

[0068] The guidelines by the Committee for the Control and Supervision of Experiments on Animals (CCSEA, New Delhi) were followed faithfully throughout the study. The protocol of the present study was approved by the Institutional Animal Ethics Committee (IAEC) of Translational Health Science and Technology Institute, Faridabad, India (IAEC approval no: IAEC7THSTI / 226 for VACV and IAEC / THSTI / 308 for MPXV). All of the animals were housed in climate- and photoperiod-controlled rooms, supplemented with conventional rodent pellets, and had free access to water. The temperature and humidity were kept at 23 ± 2 °C and 60% respectively. The study adhered to the ARRIVE guidelines.

[0069] To assess the immunogenicity of Mpx-V3 antigen, BALB / c mice were immunized at a dose of 25 pg either with AddaVax™ (<6% Squalene-based adjuvant, given in 1 : 1 v / v) or Alhydrogel (2% Aluminium hydroxide-based adjuvant, given in 1 : 1 v / v) in one prime (0-day) and one boost (28-day) regimen via the intramuscular route. Naive mice were immunized with the adjuvant alone as a control. All Mpx-V3+ AddaVax™ vaccinated mice induced Mpx-V3 specific IgG response, 14 days’ post prime immunization with endpoint titers of >1 : 12150 and post-boost immunization the endpoint titers increased to around 4-logs as compared to prebleed <1 :50, indicating an anamnestic response to Mpx-V3 immunization (Fig 4a). Similarly, Mpx-V3+ Alhydrogel immunized mice sera showed a ~4-fold increase in the dilutions and elicited higher antibody responses after the booster dose. The antibody responses to the backbone NSP-10 were insignificant when comparing the prime immunized sera to the boost immunized sera (Figure 5a). The boost serum collected was also subjected to IgG isotyping to determine the representation of Thl and Th2-type responses. Both the AddaVax™-adjuvanted Mpx-V3 and Alhydrogel-adjuvanted Mpx-V3 vaccine elicited an immune response with a Th2 bias, as demonstrated by an IgGl to IgG2a ratio exceeding 2, which was more prominent in the Alhydrogel-adjuvanted immunized mice (Fig 4 b, c). The neutralization titer of the immunized sera against the Vaccinia virus Western Reserve strain was further analyzed as shown in Fig 4 d. Both the adjuvanted immunized mice sera showed elicitation of neutralization titer as measured against the VACV.

[0070] As T cell immune response also plays an important role in the clearance of viruses, T cell immune responses of Mpx-V3-immunized mice were analyzed (as described in Materials and Methods) by in vitro stimulating the splenocytes with recombinant H3L and E8L soluble commercial proteins. The frequency of intracellular IL-2 and IFN-y cells were measured using the flow cytometry analysis as shown in Fig S6. In both the adjuvanted MpxV3 immunized mice group IFN-y and IL-2 positive cells were present in significantly higher frequency (Fig 4 e), although the E8L specific induction is comparatively high to H3L soluble proteins. The frequency of the cytokine-producing CD4+and CD8+T cells among both the protein stimulation was also measured and presented in Fig 4 e. The observations inferred from the splenic T cell responses strongly support the induction of Th-1 biased cellular immunity for both the antigens (E8L and H3L). This elevated response regulates a Thl -driven cascade of pro-inflammatory mediators, which might result in the recruitment of immune cells for rapid elimination of the viral infection.

[0071] The serum concentration (pg / ml) of IFN-y and IL-2 cytokines were quantified by the ELISA method. The administration of Mpx-V3 adjuvanted with AddaVax™ renders an increased level of IFN-y and IL-2 cytokines with concentrations of 694.3 pg / ml and 240 pg / ml, respectively (Supplementary Figure 4 b). In contrast, the observed value for IFN-y and IL-2 cytokines was 430.4 pg / ml and 84.2 pg / ml in the group immunized with Mpx-V3 and Alhydrogel, respectively (Figure 6 b). The present study suggests that although the adjuvanted antigens primarily induced Th2 -biased responses, they also have the ability to shift an established Th2 response toward a more balanced immune profile by promoting Thl -mediated cell -mediated immunity. This is advantageous for a vaccine capable of eliciting both humoral and T-cell- mediated immune responses. It was further determined whether Mpx-V3 immunized serum could detect the VACV infection. The Vero cells were infected with the VACV Western Reserve (WR) strain and after fixing and permeabilizing, the cells were incubated with Mpx- V3 boost serum and anti-VACV (WR) B5R rabbit polyclonal antiserum (Figure 6 c); in other set of experiment, the infected cells were probed with commercial anti-E8L and anti-H3L antibodies and all were probed with anti-mouse Alexa-flour-488 secondary antibody (Figure 5 d). The results suggested the immunized MpX-V3 mice sera were strongly cross-reactive to VACV.

[0072] EXAMPLE 5: PROTECTIVE EFFICACY ASSESSMENT OF THE ANTIGEN AGAINST THE LIVE VIRUS VACCINA VIRUS IN BALB / c / C MICE

[0073] The protective efficacy of the immunized BALB / c mice was evaluated by challenging them intranasally with the VACV Western Reserve strain. The Mpx-V3 vaccinated BALB / c mice initially had a similar trend of weight loss as that of AddaVax™ and Alhydrogel immunized mice up to 7thdpi, but after 8thdpi, the Mpx-V3 vaccinated animals started to recover with overall general status of healthy appearance (no fur erection or weight loss), proper food intake, and fine mobility (Fig 5 b). Out of six animals in Mpx-V3 vaccinated along with AddaVax™ and Alhydrogel adjuvant group, those were monitored up to 14 days post-challenge, one mortality was witnessed on 8thdpi (80% survival rate), as compared to the adjuvanted control groups where all mice succumbed to death by 8thdpi (zero survival) (Fig 5 c). All the adjuvanted control group animals showed >25% weight loss (were considered dead and hence euthanized) with gross diseased appearance showing altered mobility, weight loss, piloerection, reduced food intake, shivering, and huddling (Fig 5 d). The temperature changes post-challenge was monitored and it was witnessed that adjuvant control animals experienced hypothermia post day 3 infection and Mpx-V3 antigen vaccinated animals either showed hyperthermia or normal body temperature. However, the temperature fluctuations were not more than 2°C of 37°C (Fig 5 e). Organs harvested at 5 days’ post-infection (dpi) were analyzed for viral load using the plaque-forming unit (PFU) assay, with samples from two mice tested in triplicates. Control animals receiving AddaVax™ or Alhydrogel alone exhibited significantly higher viral titers in both lungs (Fig 5 f) and trachea (Fig 5 g). In contrast, the Mpx-V3 vaccinated groups showed marked viral suppression. Notably, the Alhydrogel + Mpx- V3 group exhibited complete viral clearance in the lungs and trachea, with no detectable plaques. The AddaVax™ + Mpx-V3 group also showed a substantial reduction in lung viral titers, with only 4-5 plaques compared to 18-20 plaques in the adjuvant-only controls (pO.0001). The presence of a few plaques in the lungs of the AddaVax™ + Mpx-V3 group, compared to the absence of plaques in the other vaccinated groups, was reflected as a slightly elevated bar in the graph. Further histopathological studies of major organs in the adjuvanted control groups showed severe inflammation as seen by the presence of perivascular cuffing, pneumonitis, and infiltration of monocytes and macrophages (black arrow) (Figure 6 a). Immunohistochemistry revealed the presence of VACV in the adjuvanted -only control group, indicated by reactivity to the anti-E8L monoclonal antibody, in contrast to the vaccinated group (Figure 6 a). These data demonstrated that Mpx-V3 expressing multiple epitopes of E8L and H3L is a potent immunogen and able to elicit significant immune responses for virus clearance and protection. Bioinformatics analysis between different clades of MPOXV and VACV showed a high degree of homology (>90%) and 99-100% homology of the E8L and H3L antigens between the different MPOXV clade strains (Table 1), thus indicating the Mpx-V3 immunogen cross protectiveness to the Vaccinia virus.

[0074] To study, whether the Mpx-V3 immunogen could protect against the skin pock lesions, a tail scarification model using BALB / c mouse was developed. Immunized BALB / c were challenged with 10 pl of 1 x 107PFU / ml of the VACV (WR) using the tail scarification technique (Fig 6 a). The temperature variations among the adjuvant controls and Mpx-V3 + adjuvants were not more than ±3°C of 37°C (Fig 6b). Following tail scarification, a bloody wound on the scarification site and a clot appeared in all groups as the disease progressed. After 6 days of post-tail scarification, the clot started fading in the normal control group inoculated with PBS only (Fig 6 c). The skin lesions in the groups AddaVax™ control and Alhydrogel control tend to appear 7-10 days’ post-tail scarification. The skin lesions eventually developed into scabs on day 12 that peeled off between 18-21 days’ post-scarification, leaving a scar at the scarification site. The group treated with Mpx-V3 antigen adjuvanted with Alhydrogel didnot show any significant skin lesions and scabs after the fading of blood clots and recovered completely after 12-14 days of the VACV infection through scarification (Fig 6 c). In the group treated with Mpx-V3 antigen along with AddaVax™, we observed skin lesions and scabs from day 8 onwards lasting up to 17-19 days, the recovery in this group is delayed as compared to the group treated with Mpx-V3 antigen with Alhydrogel. To reduce variations, a “clinical take” assessment was performed on day 12 post-scarification when all scabs reached their maximal size (Fig 6 c). The tail scarification in the control group with PBS resulted in a bloody wound on the site of scarification that was clotted, it did not develop further and disappeared after 5- 6 days. Apart from the local reaction described above, all vaccinated mice survived and showed no signs of morbidity. Thermal profiling of the body temperature was also measured following the post-tail scarification for 21 days. In the case of VACV-infected animals, the development of skin lesions and scabs was accompanied by a rise in body temperature, which was one of the crucial symptoms. Similar observations have also been reported in our study, the adjuvant control groups showed a high fever between 8-12 days. In contrast, the Mpx-V3 antigen-treated groups along with both the adjuvants showed a low rise in body temperature similar to the normal control group. The challenge study of the Mpx-V3 immunized mice with the MPOXV clade lib A.2.1 strain was performed to evaluate the protective potential of the Mpx-V3 immunogen. Upon receiving the MPOXV clade lib A.2.1 virus from the National Institute of Virology (NIV, ICMR), the virus was cultured in Vero cells and the titre was determined. The challenge model was established in BALB / c mice using different doses of viruses (data not shown). Briefly, 6-8 weeks old BALB / c mice infected with 50 pl of 1 x 106PFU / ml of virus intranasally showed up to 18-20% loss of body weight and developed clinical signs of weight loss up to 20%, including piloerection, dullness and shivering however, no mortality was observed in these mice up to 21 days post-infection. The protective efficacy of Mpx-V3 immunogen was checked by immunizing 6 - 8 weeks-old mice with 25 pg of Mpx-V3 along with AddaVax™ or Alhydrogel, in a prime-boost strategy via intra-muscular immunizations (Fig 7 a). The serum neutralization titre was determined in the boost sera collected from immunized mice. Both the adjuvanted immunized sera showed a significant neutralization against MPOXV strains obtained the Clade lib strain (isolated from India by National Institute of Virology, ICMR) and NR-27 (BEI resources, NIAID, NIH) (Fig 7 b). 21 days after the booster dose mice were shifted to THSTI’s ABSL-3 facility and challenged via an intra-nasal route with MPOXV clade lib A.2.1 (NIV, ICMR) and animals were monitored closely for 14 days post-infection. The blood sample was collected two weeks post every immunization and 6thday post-viral challenge, and the lung, and trachea were harvested to determine the viral load. The Mpx-V3 immunized mice did not show any significant signs of disease or weight loss, however, the AddaVax™ and Alhydrogel immunized mice exhibited dullness, piloerection and hunched posture and loss in body weight up to 14 days post-challenge observation (Fig 7 c). Previous study related to BALB / c mice as a challenge model infected intranasally (i.n.) with Zaire-79 also showed the same set of clinical signs like loss of appetite, hunched posture, piloerection, (Fig 7 c) and transient weight loss of approximately < 20% without mortality

[0075] The viral load in the lungs was determined and the trachea tissue collected on day 6 post-viral challenge. The tissue samples from Mpx-V3 immunized mice had developed fewer or negligible plaques as compared to adjuvant controls (Fig. 7 d). There was a significant reduction in lung and trachea virus titre in Mpx-V3 immunized mice as compared to AddaVax™ and Alhydrogel control groups. When Mpx-V3 adjuvanted groups were compared between Mpx-V3+ AddaVax™ and Mpx-V3+ Alhydrogel groups it was found that alum-based adjuvant Alhydrogel was augmenting more significant viral reduction (p<0.0024). In summary, Mpx-V3 was found to be immunogenic and capable of protecting immunized mice from MPOXV challenge. Furthermore, the lung samples collected on day 6 post-infection were assessed for histopathological analysis, it was found that infiltration of monocytes and macrophages along with perivascular cuffing (pointed in black arrows) in the adjuvant control groups, whereas adjuvanted Mpx-V3 group showed comparatively better morphology (Figure 6 b).

[0076] The present invention study shows that a bivalent formulation with just E8L and H3L, displayed on an NSP-10 nanocage and delivered with squalene- or alum-based adjuvants, is sufficient to induce strong humoral and neutralizing antibody responses. Upon determining the Thl :Th2 ratio in the in vivo study, it was observed that both the AddaVax™-and Alhydrogel- adjuvanted Mpx-V3 vaccine exhibited a Th2-type skewness. The elevation of Th2 cells stimulates B cells to produce antibodies, crucial for neutralizing viruses and preventing their entry into host cells. The Th2-dominated immune response observed with the AddaVax™- and Alhydrogel-adjuvanted Mpx-V3 vaccine suggests a stronger focus on antibody production, which may be effective in generating a strong initial antibody response Further analysis of antigen-specific T-cell responses, showed induction of CD4 and CD8-mediated T-cell responses and secretion of cytokines IFN-y and IL-2 which are critical for virus clearance.

[0077] The present invention findings indicate that while the adjuvanted antigens predominantly triggered Th2 -biased responses, they also demonstrated the capacity to modulate an established Th2 response toward a more balanced immune profile by enhancing Thl -mediated cell- mediated immunity. Such a response is highly advantageous for a vaccine, as it supports the induction of both robust humoral and T cell-mediated immunity.

Claims

Claim:

1. An engineered polypeptide complex expressed as a recombinant soluble protein for generating protective immune responses against Mpox virus infection, having formula [1]:[First antigenic domain-Linkerl- nanocage Non-Structural Protein (NSP10)-Linker2- second antigenic domain]-Formula [1],2. The engineered polypeptide complex as claimed in claim 1, wherein the first antigenic domain is E8 domain and said domain is 255-265 amino acids in length.

3. The engineered polypeptide complex as claimed in claim 2, wherein the first antigenic E8 is 259 amino acids in length and said domain is represented by SEQ ID No. 2.

4. The engineered polypeptide complex as claimed in claim 1, wherein linkerl and linker2 is glycine-serine linkers.

5. The Linkerl and Linker2 as claimed in claim 4, wherein the length of linker is 6-10 amino acids.

6. The Linkerl and Linker2 as claimed in claim 5, wherein the length of linker is 8 amino acids long and represented by SEQ ID No. 4.

7. The engineered polypeptide complex as claimed in claim 1, wherein the NSP10 has ISO- 155 amino acid.

8. The engineered polypeptide complex as claimed in claim 7, wherein the NSP10 has 152 amino acids and is represented by SEQ ID No.

89. The engineered polypeptide complex as claimed in claim 1, wherein the second antigenic domain is H3 and said H3 domain is 273-278 amino acids.

10. The engineered polypeptide complex as claimed in claim 9, wherein the second antigenic domain H3 is 276 amino acids in length and said H3 domain is represented by SEQ IDNo. 3.

11. The engineered polypeptide complex as claimed in claim 1, wherein said polypeptide complex is represented by SEQ ID No. 1.

12. The engineered polypeptide complex as claimed in claim 1 consists of following: i. amino acids in range of 700-710; ii. molecular weight of 75- 80kDa; iii. pi in range of 5-6.

13. A vaccine comprising: i. the engineered polypeptide complex as claimed in claim 1; and ii. excipients selected from adjuvants, stabilizers, and preservatives.

14. The vaccine as claimed in claim 13, wherein the adjuvants are selected from Squalene- based adjuvant, Aluminum hydroxide-based adjuvant, or Aluminum hydroxide / phosphate-based adjuvant.

15. The vaccine as claimed in claim 14, wherein the Squalene-based adjuvant is a <6% Squalene-based adjuvant, given in 1 : 1 v / v.

16. The vaccine as claimed in claim 14, wherein the Aluminum hydroxide-based adjuvant is 2% Aluminum hydroxide-based adjuvant, given in 1 : 1 v / v.

17. The vaccine as claimed in claim 13, wherein the stabilizer is sucrose, sucrose derivatives, trehalose or polysorbates preferably sucrose and sucrose derivatives.

18. The vaccine as claimed in claim 13, wherein the preservatives are selected from (2- Phenoxy ethanol (2 -PE), Thimerosal (thiomersal) and Phenol.

19. A process for producing the vaccine comprising the engineered polypeptide complex having sequence of SEQ ID No. 1, comprises: i. identifying antigenic domains of Mpox virus; ii. screening the antigenic domains of Mpox virus identified in step (i) andselecting multiple antigenic domains showing high immunogenicity such as E8, H3 preferably E8 and H3; iii. stapling the selected multiple antigenic domains screened in step (ii) by glycine-serine linkers; iv. stitching the stapled antigenic domain of step (iii) to a nano-cage NSP10 by glycine-serine linkers, to form engineered polypeptide complex; v. optimizing codons for expressing the engineered polypeptide complex of step (iv); vi. cloning the engineered polypeptide complex obtained in step (v) to a vector to form recombinant vector; vii. transforming the recombinant vector cloned with the sequence represented by SEQ ID No. 15 capable of encoding the engineered polypeptide complex obtained in step (vi) into competent E. coli BL21, BL21(DE3) preferably BL21 cells and culturing for protein expression; viii. expressing the engineered polypeptide complex of step (vii) in inclusion bodies and recovering said inclusion bodies from the cultured cells; ix. purifying the engineered polypeptide complex of step (viii) to obtain a preparation having greater than 90-97% preferably 95% purity as determined by SDS-PAGE; x. identifying the purified engineered polypeptide complex of step (ix) by Western blotting using antibodies specific to the antigenic domains and to the NSP10 nano-cage; xi. subjecting the engineered polypeptide complex of step (x) to chromatographic purification including anion exchange chromatography and size exclusion chromatography to confirm formation of a higher-order multimeric structure; xii. performing biophysical characterization of the engineered polypeptide complex of step (xi) using intrinsic tryptophan fluorescence and circular dichroism spectroscopy to determine secondary structure content; xiii. formulating the engineered polypeptide complex of step (xii) with an adjuvant selected from Squalene-based adjuvant, Aluminium hydroxide-based adjuvant, or Aluminium hydroxide / phosphate based adjuvant preferably Squalene-based adjuvant or Aluminium hydroxide-based adjuvant and immunizing a pre-clinical animal model, C57BL / 6, BALB / c mice, preferably BALB / c mice.

20. A process for producing the vaccine as claimed in claim 17, wherein the vector for protein expression pET -28a(+), pET28b (+), pET -28c(+), preferably pET 28b(+).

21. The engineered polypeptide complex as claimed in claim 11, wherein the administration of the engineered polypeptide complex as represented by SEQ ID NO. 1 which elicits neutralizing antibodies and T cell responses against Monkey Pox virus, thereby protecting the subject from symptomatic infection, reducing disease severity, and accelerating recovery.

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