Dengue subunit vaccine composition

Abstract

The present invention discloses a dengue subunit vaccine composition comprising a fusion protein, wherein the fusion protein is a non-structural protein 1 (NS1ΔC or truncated NS1ΔC) with the C-terminus removed that is conjugated or linked to at least one polypeptide, and the at least one polypeptide is a non-structural protein 3c (NS3c or truncated NS3c) and / or a homologous envelope protein third block (cEDIII), thereby enhancing protection against dengue virus attack and alleviating related pathological reactions.

Description

Technical Field

[0001] The present invention relates to a subunit vaccine composition, and in particular to a recombinant dengue subunit vaccine composition comprising a fusion protein of a non-structural protein 1 (NS1ΔC or truncated NS1ΔC) with the C-terminus removed and conjugated or linked to at least one polypeptide of the non-structural protein 3c (NS3c or truncated NS3c) and / or the homologous envelope protein third block (cEDIII), wherein the at least one polypeptide is the non-structural protein 3c (NS3c or truncated NS3c) and / or the homologous envelope protein third block (cEDIII). Background Art

[0002] Dengue virus (DENV) is the most important arbovirus disease in global public health (WHO, 2016). DENV is transmitted to humans by Aedes aegypti and Aedes albopictus mosquitoes, causing an estimated 390 million cases of infection each year, of which 96 million cases have obvious symptoms and 500,000 cases have dengue hemorrhagic fever or dengue shock syndrome (DHF / DSS) (Bhatt et al., 2013; Diamond and Pierson, 2015; Katzelnick et al., 2017). To date, more than 128 countries have reported DENV infections, mainly affecting tropical and subtropical regions in Asia and Latin America. Therefore, in order to control dengue disease, the development of dengue vaccines is a top priority (Wan et al., 2013).

[0003] Sanofi Pasteur launched the world's first dengue vaccine in December 2015 (or CYD-TDV); however, the safety of this vaccine for serum-negative vaccine recipients is still in doubt (Wichmann et al., 2017; Normile, 2017; Sridhar et al., 2018; Gubler and Halstead, 2019). The other two dengue vaccines, TV003 / TV005 developed by the National Institute of Allergy and Infectious Diseases (NIAID) and TAK-003 developed by Takeda, are still in Phase III human trials (Kirkpatrick et al., 2015; Biswal et al., 2019). The above three vaccines are all live attenuated vaccines (Screaton et al., 2015; WHO, 2016). Due to the current dengue vaccine There are still concerns about the safety of the DENV vaccination program, and the safety of other live attenuated vaccine candidates still needs to be carefully evaluated (Normile, 2017). The development of improved vaccines and / or antiviral therapies for DENV remains a global public health priority.

[0004] In the development of dengue subunit vaccines, previous studies have suggested that the envelope (E) protein is the main antigen that elicits protective antibody responses. The third region of the envelope protein (EDIII) contains a cell surface receptor recognition region and is considered a potential target for dengue subunit vaccines (Guzman et al., 2010). However, antibodies against the E protein can not only neutralize DENV, but may also increase DENV infection through antibody-dependent enhancement (ADE) (Guzman et al., 2015). Previous studies have compared the sequences of different isolates of four serotypes of DENV to identify the consensus envelope protein third region (cEDIII) as an immunogen. These studies have shown that cEDIII can elicit antibody responses, thereby cross-neutralizing the four serotypes of DENV (Leng et al., 2009). In non-human primate studies, monkeys vaccinated with cEDIII and aluminum phosphate elicited antibody responses to dengue virus type 2 (DENV2) that were significantly strong and long-lasting, and also elicited specific T cell responses and cytokine production, which correlated with antibody responses (Chen et al., 2013).

[0005] NS1 protein is post-translationally modified to dimerize in the lumen of the endoplasmic reticulum (ER) and can be expressed on the surface of infected cells. NS1 protein can also be secreted as a soluble, lipid-associated hexamer, which becomes the main target of humoral immunity (Flamand et al., 1999; Muller and Young, 2013). It is currently known that high expression of NS1 is associated with DHF, and NS1 is present in patients with vascular leakage for a long time (Adikari et al., 2016; Malavige and Ogg, 2017). Secreted NS1 can activate cells to release proinflammatory cytokines, causing a cytokine storm, leading to vascular leakage (Beatty et al., 2015; Modhiran et al., 2015). In addition, secreted NS1 damages the endothelial glycocalyx, leading to vascular hyperpermeability (Puerta-Guardo et al., 2016; Chen et al., 2016; Chen et al., 2018). Recent studies have shown that NS1 can also activate platelets through TLR4, leading to thrombocytopenia and hemorrhage (Chao et al., 2019). Based on the above results, the attention paid to NS1 as a candidate for vaccine development and therapeutic strategies continues to increase (Halstead, 2013; Amorim et al., 2014; Glasner et al., 2018).

[0006] However, antibodies produced against DENV NS1 also recognize common epitopes on coagulation-related proteins, platelets, and endothelial cells. The inventors' previous studies have shown that antibodies against DENV NS1 cross-react with human platelets and endothelial cells, causing platelet and endothelial cell damage and dysfunction (Lin et al., 2001, 2003, 2005, 2011; Chen et al., 2013). The homology region between DENV NS1 and cell surface antigens is mainly located at amino acid residues 311-352 at the C-terminus (Cheng et al., 2009). NS1ΔC obtained by removing the C-terminal region of NS1 can greatly reduce cross-reactivity (Chen et al., 2009). Passive immunization with polyclonal or monoclonal antibodies against NS1ΔC can reduce the prolonged bleeding time and bleeding caused by DENV (Wan et al., 2014; Wan 2017). Therefore, it is necessary to confirm the protective efficacy of cEDIII-NS1ΔC fusion protein as a potential candidate subunit vaccine. Summary of the invention

[0007] Therefore, one aspect of the present invention is to provide a dengue subunit vaccine composition, which includes a fusion protein, wherein the fusion protein is a non-structural protein 1 (NS1ΔC) polypeptide with the C-terminus removed and conjugated or linked to at least one polypeptide, wherein the at least one polypeptide is a non-structural protein 3c (NS3c) polypeptide and / or a homologous envelope protein third block (cEDIII) polypeptide, and optionally contains one or more pharmaceutically acceptable carriers and / or adjuvants.

[0008] Another aspect of the present invention is to provide a dengue subunit vaccine composition comprising an NS3c polypeptide and a fusion protein comprising a cEDIII polypeptide and an NS1ΔC polypeptide (cEDIII-NS1ΔC), and optionally one or more pharmaceutically acceptable carriers and / or adjuvants.

[0009] Another aspect of the present invention is to provide a dengue subunit vaccine composition comprising a fusion protein comprising NS1ΔC polypeptide and NS3c polypeptide, and optionally one or more pharmaceutically acceptable carriers and / or adjuvants.

[0010] According to the above aspect of the present invention, a dengue subunit vaccine composition is provided, which includes a fusion protein, wherein the fusion protein is a nonstructural protein 1 (NS1ΔC) polypeptide with the C-terminus removed and conjugated or linked to at least one polypeptide, and the at least one polypeptide is a nonstructural protein 3c (NS3c) polypeptide and / or a homologous envelope protein third block (cEDIII) polypeptide. In this embodiment, the NS1ΔC polypeptide may be a sequence as shown in Sequence Identification Number (SEQ ID NO): 2 or 5, the cEDIII polypeptide may be a sequence as shown in SEQ ID NO: 1 or 4, and the NS3c polypeptide may be a sequence as shown in SEQ ID NO: 3 or 6. Furthermore, the fusion protein may optionally contain one or more pharmaceutically acceptable carriers and / or adjuvants.

[0011] According to the above aspect of the present invention, a dengue subunit vaccine composition is further provided. The composition comprises a fusion protein, the fusion protein comprising a cEDIII polypeptide and a NS1ΔC polypeptide, wherein the cEDIII polypeptide may be a sequence as shown in SEQ ID NO: 1 or 4, the NS1ΔC polypeptide may be a sequence as shown in SEQ ID NO: 2 or 5, the NS3c polypeptide may be a sequence as shown in SEQ ID NO: 3 or 6, and optionally contains one or more pharmaceutically acceptable carriers and / or adjuvants.

[0012] In one embodiment, the sequence from the N-terminus to the C-terminus of the fusion protein may be cEDIII polypeptide and NS1ΔC polypeptide (cEDIII-NS1ΔC). In one embodiment, the fusion protein comprises a first linker peptide between the cEDIII polypeptide and the NS1ΔC polypeptide.

[0013] In one embodiment, the fusion protein may be NS1ΔC polypeptide and cEDIII polypeptide (NS1ΔC-cEDIII) in order from N-terminus to C-terminus, and conjugated to NS3c polypeptide. In one embodiment, the fusion protein comprises a second linker peptide between NS1ΔC polypeptide and NS3c polypeptide.

[0014] In one embodiment, the sequence from the N-terminus to the C-terminus of the fusion protein may be NS1ΔC polypeptide and cEDIII polypeptide (NS1ΔC-cEDIII). In one specific example, the fusion protein may include a first linker peptide between the cEDIII polypeptide and the NS1ΔC polypeptide.

[0015] In one embodiment, the fusion protein in the order of NS1ΔC-cEDIII from N-terminus to C-terminus can be conjugated to NS3c polypeptide. In one embodiment, the second linker peptide can be conjugated between the cEDIII polypeptide and the NS1ΔC polypeptide.

[0016] In one embodiment, the fusion protein may optionally contain an adjuvant.

[0017] According to the above aspect of the present invention, a dengue subunit vaccine composition is also provided, comprising a fusion protein, which is a truncated NS1ΔC polypeptide with a sequence as shown in SEQ ID NO: 5 and a truncated NS3c polypeptide with a sequence as shown in SEQ ID NO: 6, and optionally contains one or more pharmaceutically acceptable carriers and / or adjuvants.

[0018] In one embodiment, the sequence from N-terminus to C-terminus of the fusion protein is a truncated NS1ΔC polypeptide and a truncated NS3c polypeptide (truncated NS1ΔC-truncated NS3c).

[0019] In one embodiment, the fusion protein comprises a first linker peptide between the truncated NS1ΔC polypeptide and the truncated NS3c polypeptide.

[0020] In one embodiment, the sequence from N-terminus to C-terminus of the fusion protein is a truncated NS3c polypeptide and a truncated NS1ΔC polypeptide (truncated NS3c-truncated NS1ΔC).

[0021] In one embodiment, the fusion protein comprises a first linker peptide between the truncated NS1ΔC polypeptide and the truncated NS3c polypeptide.

[0022] In one embodiment, the composition further comprises a cEDIII polypeptide having a sequence as shown in SEQ ID NOs: 1 or 4. In this embodiment, the truncated NS1ΔC polypeptide of the fusion protein can be selectively conjugated to the cEDIII polypeptide. In some embodiments, the truncated NS3c polypeptide of the fusion protein can be selectively conjugated to the cEDIII polypeptide. In other embodiments, the cEDIII polypeptide can be selectively conjugated between the truncated NS1ΔC polypeptide and the truncated NS3c polypeptide.

[0023] The dengue subunit vaccine composition of the present invention comprises a fusion protein of cEDIII-NS1ΔC and a NS3c polypeptide, or a fusion protein of a truncated NS1ΔC polypeptide and a NS3c polypeptide, thereby enhancing protection against dengue virus attack and alleviating related pathological reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the detailed description of the accompanying drawings is as follows:

[0025] Figures 1A to 8B Schematic diagram showing various subunits of the dengue subunit vaccine composition according to several embodiments of the present invention.

[0026] 9A to 9CThe results of antibody responses induced by immunization with cEDIII-NS1ΔC, cEDIII plus DJ NS1, and DJ NS1 recombinant protein are shown. Fig. 9A ) This diagram shows cEDIII, NS1ΔC, DJ NS1 and the linear fusion cEDIII-NS1ΔC recombinant protein. ( Fig. 9B ) Each mouse was subcutaneously immunized three times with 25 μg of cEDIII-NS1ΔC, 5 μg of cEDIII plus (+) 20 μg of DJ NS1, or 25 μg of DJ NS1 protein mixed with aluminum salt as an adjuvant. ( Fig. 9C ) Three days after the second and third immunizations, mouse sera were collected and the antibody titers were determined (n=3).

[0027] FIG. 10A to FIG. 10D The results are shown in Figure 2. The serum of mice immunized with aluminum salt mixed with cEDIII-NS1ΔC or DJ NS1 protein has neutralizing antibody titers against four serotypes of DENV. The serum of unimmunized mice (mock serum), serum immunized with aluminum salt (alum serum), serum immunized with cEDIII-NS1ΔC mixed with aluminum salt (cEDIII-NS1ΔC serum) and serum immunized with DJ NS1 mixed with aluminum salt (DJ NS1 serum) were serially diluted twofold and incubated with 50-80 PFU of DENV1 ( Fig. 10A )、DENV2( Fig. 10B )、DENV3( Fig. 10C ) or DENV4( Fig. 10D ) was mixed at room temperature for 1 hour and then inoculated with 1×10 5 BHK-21 cells. Plaque reduction neutralization test (PRNT) was used to determine the neutralizing antibodies against DENV. Each group of data is expressed as the average percentage of plaque production with error bars (SD bar) (n=3).

[0028] FIG. 11A to FIG. 11D To show that the serum of mice immunized with aluminum salt mixed with cEDIII-NS1ΔC or DJ NS1 protein can induce complement-mediated cytolysis of cells infected with four different serotypes of DENV. Fig.11A )、DENV2( Fig. 11B )、DENV3( Fig. 11C ) or DENV4( Fig.11D(MOI = 20) were used to infect HMEC-1 cells individually for 48 hours. Then, 1:200 dilutions of serum from non-immunized mice (mock serum), serum immunized with alum serum, serum immunized with cEDIII-NS1ΔC mixed with alum serum (cEDIII-NS1ΔC serum), or serum immunized with DJ NS1 mixed with alum serum (DJ NS1 serum) were incubated with the cells at 37°C for 6 hours in the presence or absence of complement. Cell supernatants were collected and analyzed for released lactate dehydrogenase (LDH). Mean ± SD of triplicate cultures are presented. **: p < 0.01; ***: p < 0.001 (n = 3).

[0029] FIG. 12A to FIG. 12B To illustrate the experimental design of mice immunized with cEDIII-NS1ΔC protein and the antibody titers before infection with four different serotypes of DENV. ( Fig. 12A ) Mice were immunized three times with 25 μg / mouse cEDIII-NS1ΔC protein mixed with aluminum salt by subcutaneous injection (sc). Three days after the second and third immunizations, mouse sera were collected and the antibody titers were measured. (Non-immunized group, n=2; DENV immunized group, n=3; aluminum salt immunized group, DENV plus cEDIII-NS1ΔC mixed with aluminum salt group, n=4). (FIG.12B) Mice were inoculated with 1×10 8 PFU of DENV1, DENV2, DENV3 and DENV4. Two days after infection, the mice were analyzed for prolonged bleeding time, and serum was collected to determine the viral titer and the content of soluble NS1, such as FIG. 13A to FIG. 14D shown.

[0030] FIG. 13A to FIG. 13D To show that active immunization with cEDIII-NS1ΔC can reduce DENV-induced prolonged bleeding time. Fig. 12A To show the immune pattern of mice. On day 24, mice were given 1×10 8 PFU of DENV1( Fig.13A )、DENV2( Fig. 13B )、DENV3( Fig. 13C )、DENV4( Fig.13D ) were inoculated via intravenous injection (iv). The tail bleeding time of mice was measured 2 days after infection. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.

[0031] FIG. 14A to FIG. 14DTo show that active immunization with cEDIII-NS1ΔC can reduce the viral titer in mouse serum. 8 PFU of DENV1( Fig.14A )、DENV2( Fig. 14B )、DENV3( Fig. 14C )、DENV4( Fig.14D ) were inoculated via intravenous injection (iv). Mice were sacrificed 2 days after infection. Serum was collected and the virus titer was measured by fluorescent focus assay (FFA). **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.

[0032] FIG. 15A to FIG. 15D The results of active immunity with DJ NS1 and NS3 (%) are shown in Figure 2. The results of active immunity with DJ NS1 and NS3 (%) show that CD4 + With CD8 + T cell activation. On day 0, C3H / HeN mice were immunized subcutaneously with different doses of immunogen, i.e., 25 μg DJ NS1 plus 12.5 μg NS3 / mouse, 25 μg DJ NS1 / mouse, 12.5 μg NS3 / mouse, aluminum salt alone or PBS, and boosted on day 14. Mice were sacrificed on day 21, lymphnode (LN) cells were collected and restimulated with 5 μg / mL DJ NS1 or NS3 protein for 3 days, incubated with anti-CD3 / CD28 antibodies for the last 4 hours, and then triple-stained with PE-labeled anti-CD25 antibody, FITC-labeled anti-CD4 antibody, and PE / Cy7-labeled anti-CD8 antibody for analysis of CD4 by flow cytometry. + and CD8 + Expression of CD25 on T cells. Fig.15A To illustrate CD4 + After T cells are stimulated by DJ NS1 antigen, the CD25 + percentage. Fig. 15B To illustrate CD4 + After T cells are stimulated by NS3 antigen, the CD25 + percentage. Fig. 15C To illustrate CD8 + After T cells are stimulated by DJ NS1 antigen, the CD25 + percentage. Fig.15D To illustrate CD8 + After T cells are stimulated by NS3 antigen, the CD25 +Percentage. n = 5 for DJ NS1 plus NS3 group, and n = 4 for other groups. Each group is shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, as determined by one-way ANOVA and Tukey posthoc test.

[0033] FIG. 16A to FIG. 16C The graph shows the results of active immunization with DJ NS1 and NS3 to induce cytotoxic T lymphocyte (CTL) response against L929 cells expressing NS3. Fig.16A To illustrate the experimental design of CTL cytotoxicity assay, lymph node cells were collected and restimulated with 5 μg / mL DJ NS1 or NS3 protein for 3 days to serve as effector cells. Fig. 16B L929 cells (T) expressing NS1 were co-cultured with effector cells (E) at an E:T ratio of 20:1. After 4 hours, target cells (T) were collected and cell death was analyzed by PI staining and flow cytometry. NS1 expression was analyzed by Western blot. Fig. 16C To show that NS2B / 3 with S135A can be used in NS3-expressing L929 cells, the absence of self-processing of NS2B3 confirmed that the mutant construct had impaired protease activity by Western blot analysis. NS3-expressing L929 cells (T) were co-cultured with effector cells (E) at an E:T ratio of 20:1. After 4 hours, target cells (T) were harvested and analyzed for cell death by PI staining and flow cytometry. NS1 expression was analyzed by Western blot. n=5 for DJ NS1 plus NS3 group and n=4 for other groups. Mean ± SD for each group. *p<0.05, **p<0.01, as determined by one-way ANOVA and Tukey post hoc test.

[0034] FIG. 17A to FIG. 17B Figure 2 shows the mice immunized with DJ NS1 and NS3. Fig.17A ) or NS3( Fig. 17B ) antigen in vitro stimulation, the expression of CD107a increased, where CD107a is a cytotoxic CD8 +The mice were sacrificed on day 21, lymph node cells were collected and stimulated with 5 μg / mL DJ NS1 or NS3 protein for 3 days, and then cultured with anti-CD3 / CD28 antibody, monensin and Brefeldin A for the last 4 hours, after which CD8 + T cells were surface stained for CD107a and analyzed by flow cytometry. n = 5 for DJ NS1 plus NS3 group and n = 4 for other groups. Each group is shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, as determined by one-way ANOVA and Tukey posthoc test.

[0035] 18A to 18C The graph shows the results of active immunization with DJ NS1 and NS3 to reduce the viral titer in the serum of DENV-infected mice. Fig.18A Experimental design to show the model of immunized mice (n=3 / group). 8 C3H / HeN mice were inoculated with PFU DENV2 strain 454009A via intravenous injection (iv). Fig.18B To show that active immunization with DJ NS1 and NS3 can reduce the bleeding tendency caused by DENV. 8 PFUDENV2 strain 454009A was inoculated into C3H / HeN mice via intravenous injection (iv). Tail bleeding time was measured 2 days after infection. Fig.18C To show that mice were sacrificed on day 19 and serum samples were collected, and the virus titer was detected by FFA. *p<0.05, **p<0.01, ***p<0.001, as determined by one-way ANOVA and Tukey post hoc test.

[0036] Fig.19 Schematic diagram showing immune responses and protective efficacy induced by active immunization with DJ NS1 and NS3. Immunization with DJ NS1 plus NS3 proteins can synergistically induce a more effective immune response, as demonstrated by the generation of NS3-specific CTL responses, increased CD107 expression, promotion of NS1-specific T cell responses, and antibody titers. The protective mechanism includes direct toxicity of DENV-infected cells by CTLs and neutralization of NS1-induced pathogenicity by anti-NS1 antibodies.

[0037] FIG. 20A to FIG. 20DTo illustrate the truncated NS1ΔC-cEDIII-truncated NS3c fusion protein ( Fig. 20A and Fig. 20B , MW = 49 kDa) and truncated NS1ΔC-truncated NS3c fusion protein ( Fig. 20C and Fig.20D , MW = 35 kDa), which was determined by SDS-PAGE ( Fig. 20A and Fig. 20C ) and Western blot ( Fig. 20B and Fig.20D ) detection.

[0038] FIG. 21A to FIG. 21D To illustrate the experimental design ( Fig.21A ) and the results of active immunization with truncated NS1ΔC-cEDIII-truncated NS3c (or NS1fΔC-cEDIII-NS3cf) fusion protein and truncated NS1ΔC-truncated NS3c (or NS1fΔC-NS3cf) fusion protein ( FIG. 21B to FIG. 21D ), the above two fusion proteins were purified from mammalian 293F cells and can reduce the viral titer of DENV-infected mice ( Fig. 21B ), complement-dependent cytotoxicity (CDC; Fig. 21C ) and bleeding time ( Fig.21D ).

[0039] FIG. 22A to FIG. 22D To illustrate the experimental design ( Fig.22A ) and the results of active immunization with truncated NS1ΔC-cEDIII-truncated NS3c (or NS1fΔC-cEDIII-NS3cf) fusion protein and truncated NS1ΔC-truncated NS3c (or NS1fΔC-NS3cf) fusion protein ( FIG. 22B to FIG. 22D ), the above two fusion proteins were purified from Drosophila S2 cells and can reduce the viral titer of DENV-infected mice ( Fig. 22B ), complement-dependent cytotoxicity (CDC; Fig. 22C ) and bleeding time ( Fig.22D ).

[0040] Wherein, the reference numerals are:

[0041] 101, 103, 201, 203, 205, 207, 301, 303, 401, 403, 501, 503: Curve DETAILED DESCRIPTION

[0042] Embodiments of the present invention will become apparent from the following detailed description with reference to the accompanying drawings, in which like numerals refer to like elements.

[0043] All references cited herein are treated as if each individual reference or patent application was specifically and individually incorporated by reference. If a definition or use of a term in a referenced document is inconsistent or contrary to the definition of that term herein, the definition of that term herein applies and the definition of that term in the referenced document does not apply.

[0044] For purposes of interpreting the specification, the following definitions shall apply and, where appropriate, singular terms shall include the plural and vice versa. Additional definitions are set forth throughout the detailed description.

[0045] Unless the context is inappropriate, "a", "an" and "the", "said" as used herein are defined as "one or more" and include the plural.

[0046] The present invention discloses a dengue subunit vaccine composition, which includes a fusion protein, wherein the fusion protein is a nonstructural protein 1 (NS1ΔC) polypeptide with the C-terminus removed and conjugated or linked to at least one polypeptide, wherein the at least one polypeptide is a nonstructural protein 3c (NS3c) polypeptide and / or a homologous envelope protein third block (cEDIII) polypeptide, thereby enhancing protection against dengue virus attack and alleviating related pathological reactions. Secondly, the present invention discloses a dengue subunit vaccine composition, which includes an NS3c polypeptide and a fusion protein, wherein the fusion protein is a cEDIII polypeptide and an NS1ΔC polypeptide (cEDIII-NS1ΔC). In another embodiment, the present invention discloses a dengue subunit vaccine composition, which includes a fusion protein, wherein the fusion protein is a truncated NS1ΔC polypeptide (also known as NS1fΔC) and a truncated NS3c polypeptide (also known as NS3cf). In another embodiment, the aforementioned composition also includes a plurality of polynucleotide fragments that translate the aforementioned polypeptides.

[0047] The "dengue subunit vaccine" described herein comprises a fusion protein, which is a non-structural protein 1 (NS1ΔC) polypeptide with the C-terminus removed and conjugated or linked to at least one polypeptide, which is a non-structural protein 3c (NS3c) polypeptide and / or a homologous envelope protein third block (cEDIII) polypeptide, and may optionally contain one or more pharmaceutically acceptable carriers and / or adjuvants, thereby enhancing protection against dengue virus attack and alleviating related pathological reactions.

[0048] In some embodiments, the "fusion protein" described herein refers to a NS1ΔC polypeptide (or a truncated NS1ΔC) conjugated or linked to at least one polypeptide, the at least one polypeptide being a NS3c polypeptide (or a truncated NS3c) and / or a cEDIII polypeptide, the two being directly linked via a standard peptide bond (i.e., without an additional linker) in any order, such as cEDIII-NS1ΔC, NS1ΔC-cEDIII, NS1ΔC-NS3c, NS3c-NS1ΔC, cEDIII-NS1ΔC-NS3c, NS1ΔC-cEDIII-NS3c, NS3c-cEDIII-NS1ΔC, or being conjugated or linked via at least one additional linker (e.g., linker-1 and linker-2), such as Figures 1A to 8B Variants of the fusion proteins shown.

[0049] In some embodiments, the "cEDIII" polypeptide may be, for example, a sequence as shown in SEQ ID NO: 1 or 4. The "NS1ΔC" polypeptide may be, for example, a sequence as shown in SEQ ID NO: 2 or 5. The "NS3c" polypeptide may be, for example, a sequence as shown in SEQ ID NO: 3 or 6.

[0050] In other embodiments, the truncated NS1ΔC polypeptide of the fusion protein may be, for example, the sequence shown in SEQ ID NO: 5, and the truncated NS3c polypeptide may be, for example, the sequence shown in SEQ ID NO: 6. In these embodiments, the composition may selectively include a cEDIII polypeptide having a sequence shown in SEQ ID NOs: 1 or 4. In some embodiments, the fusion protein may include a cEDIII polypeptide before, between, or after the NS1ΔC polypeptide and the NS3c polypeptide, and the order is not limited.

[0051] In some embodiments, the linker described herein, such as linker-1 or linker-2, can be unambiguously understood as a "linker" or "spacer" commonly used in general molecular biology. The linker can be any oligopeptide or compound commonly used to conjugate or link NS1ΔC (or truncated NS1ΔC, NS1fΔC) to at least one polypeptide, wherein the at least one polypeptide is NS3c polypeptide (or truncated NS3c, NS3cf) and cEDIII polypeptide. In some embodiments, the linker can contain at least one amino acid, modified amino acid or non-human amino acid element. The aforementioned element can, for example, include one or more of the following: amino-3-oxapentanoic acid, PEG1, PEG2, PEG4, β-amino acid, γ-amino acid, aminohexanoic acid or amino-3,6-dioxaoctanoic acid.

[0052] The term "dengue subunit vaccine" may be used as described above for compositions that enhance protection against dengue virus attack and mitigate associated pathological responses.

[0053] "Subjects" include humans and non-human animals. Non-human animals may include all vertebrates such as mammals and non-mammalian animals, such as non-human primates, sheep, dogs, cows, chickens, amphibians and reptiles. Unless otherwise specified, the terms "patient" or "subject" described herein are interchangeable.

[0054] Several exemplary embodiments are used below to illustrate the application of the present invention, but they are not intended to limit the present invention. A person skilled in the art of the present invention may make various modifications and improvements without departing from the spirit and scope of the present invention.

[0055] Example

[0056] Establishing experimental animal models

[0057] The strain C3H / HeN mice were obtained from Charles River Breeding Laboratories. The mice were raised at the Experimental Animal Center of the College of Medicine, National Cheng Kung University with standard laboratory-grade feed and water. Mice aged 6 to 8 weeks or 3-week-old offspring were used for antibody production and infection experiments. The above animal experiment plan has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The following examples have been approved by the Animal Experiment Ethics Committee of National Cheng Kung University.

[0058] Preparation of recombinant proteins and serum

[0059] This example is to construct a cEDIII-NS1ΔC polypeptide, which is a construct of a DNA fragment fused with NS1ΔC and cEDIII (provided by Dr. Xinwei Chen), and inserted into the pET21b plasmid using the restriction enzyme Not I and Xho I sites. The resulting recombinant protein has an additional 6 consecutive histidine tags (His-Tag) at the C-terminus. In order to express the protein, the above construction system was transformed into Escherichia coli BL21 strains to produce Escherichia coli expressing the cEDIII-NS1ΔC fusion protein. The resulting transformed strain was cultured in Luria broth (LB), induced with 0.5 mM isopropyl thiogalactoside (IPTG), and pelleted by centrifugation. After that, the cells were lysed by ultrasonic treatment. The cEDIII-NS1ΔC fusion protein (or recombinant protein) recovered from the inclusion body is an insoluble aggregate and is then denatured in a denaturing reagent (urea buffer containing 8M urea, 500mM NaCl, and 20mM Tris-HCl). 2+ After purification by using a 50 μM column, the recombinant protein was refolded after slowly diluting out the denaturing agent in the presence of L-arginine, EDTA, PMSF, reduced glutathione, and oxidized glutathione. In this example, the recombinant protein was concentrated using an Amicon Ultra-30 centrifugal filter unit (Millipore) equipped with an Ultracel-30 filter membrane. In addition, cDNAs of NS1ΔC (amino acid residues 271-352 deleted) and DJ NS1 (amino acid residues 1-270 of DENV NS1 and amino acid residues 271-352 of JEV NS1) were cloned into a pET28a vector with a His-Tag. The cEDIII plasmid (obtained by Dr. Hsin-Wei Chen) was expressed in E. coli BL21. After purification, the antibody was tested by 10% or 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The purified cEDIII-NS1ΔC was mixed with alum as an adjuvant and subcutaneously immunized C3H / HeN mice three times. Seven days after the first immunization, three days after the second immunization, and three days after the third immunization, the mouse sera were collected and the antibody titers were determined. After the mice were sacrificed, the mouse sera were collected and stored at -20°C.

[0060] The cDNA of DJ NS1 (amino acid residues 1-270 of DENV NS1 fused with amino acid residues 271-352 of JEV NS1) was cloned into the pET28a vector with a histidine tag (Wan et al., 2014). The above plasmid was then introduced into Escherichia coli BL21, and the recombinant protein was induced with isopropyl BD-1-thiogalactoside (IPTG; Calbiochem, San Diego, CA), dissolved in urea buffer (containing 8 M urea, 500 mM NaCl, 20 mM Tris-HCl), and a nickel ion-nitrilotriacetate (NTA) affinity column (Ni 2+ -NTA affinity column, GE Healthcare Life Science, UK). After purification, the above protein was examined by SDS-PAGE and then stained with Coomassie brilliant blue R250. The purified protein was dialyzed in refolding buffer [containing 50mM Tris-HCl, 250mM NaCl, 1mM EDTA, 250mM L-arginine, 10mM GSH (reduced glutathione) and 1mM GSSG (oxidized glutathione), and 5% glycerol] and concentrated using Amicon Ultra ultrafiltration centrifuge tubes (Centrifugal Filters; Millipore, Billerica, MA).

[0061] DENV NS3 cDNA was cloned into the pET21b vector with His-Tag (provided by Dr. Jiayi Yu, National Institutes of Health). The above plasmid was transformed into E. coli BL21. The transformed cells were cultured overnight at 37°C in LB medium [containing 100μg / mL ampicillin]. The overnight culture was diluted 100 times with LB medium [containing 100μg / mL ampicillin] and cultured again at 37°C until the optical density (OD) at 600nm reached 0.5. Then 1mM IPTG was added and induced at 30°C for 6 hours to express DENV2 NS3. To analyze the expression of NS3 protein, all samples were centrifuged at 8,000×g for 30 minutes at 4°C. Cells were collected by centrifugation and the pellets were stored at -80°C for future use. The bacterial pellets were collected and analyzed by electrophoresis. In order to purify the protein, the bacterial precipitate was resuspended in PBS and placed on ice and sonicated to dissolve the bacteria. The cell suspension was centrifuged at 13,000×g for 30 minutes. The precipitate containing the inclusion bodies was resuspended in binding buffer (containing 8M urea, 0.5M NaCl, and 20mM Tris-HCl, pH 6.95), placed on ice and sonicated again, and then centrifuged at 13,000×g for 45 minutes at 4°C to clarify the reaction solution. The supernatant containing the soluble protein was injected into a nickel ion column (Ni 2+ The column was washed with washing buffer (containing 8 M urea, 0.5 M NaCl, 20 mM Tris-HCl, and 120 mM imidazole, pH 6.95). The His-tagged protein was eluted with elution buffer (containing 8 M urea, 0.5 M NaCl, 20 mM Tris-HCl, and 300 mM imidazole, pH 6.95). After purification, the above protein was examined by SDS-PAGE. The purified protein was dialyzed in refolding buffer (containing 50 mM Tris-HCl, 250 mM NaCl, 1 mM EDTA, 250 mM L-arginine, 10% glycerol, 10 mM GSH and 1 mM GSSG, pH 6.95) to remove urea, and then centrifuged and concentrated using Amicon Ultra (Millipore).

[0062] In order to mix the antigen and aluminum salt, this embodiment first added Imject aluminum salt (Pierce Biotechnology, Rockford, IL) dropwise to the antigen solution and mixed it with it, so that the final volume ratio of Imject aluminum salt to antigen solution was 1:1 (100 μL Imject aluminum salt to 100 μL antigen solution). After that, it was mixed at 4°C overnight to allow the Imject aluminum salt to completely adsorb the antigen. Other clones were selected using the above method, and their sequence identities are shown in Table 1.

[0063] Table 1

[0064]

[0065] Cell culture

[0066] Baby hamster kidney cells (BHK-21) were cultured in DMEM (Invitrogen, Carlsbad, CA) containing antibiotics and 5% FBS. C6 / 36 cells and L929 cells were cultured in DMEM containing antibiotics and 10% FBS. Lymphocytes collected from mouse lymph nodes were cultured in RPMI-1640 medium containing antibiotics, 10% FBS, 1% non-essential amino acids, 1% sodium pyruvate, and 50 μM 2-mercaptoethanol (2-ME) at 37°C and 5% CO2. HMEC-1 cells were obtained from the Centers for Disease Control and Prevention (USA) and cultured in endothelial cell growth medium M200 (Invitrogen) supplemented with 1 μg / mL hydrocortisone, 10 ng / mL epidermal growth factor, 3 ng / mL basic fibroblast growth factor, 10 μg / mL heparin, and antibiotics, and subcultured in culture flasks. The cells were detached using a detaching buffer containing 1000 U / mL trypsin and 0.5 mM EDTA.

[0067] Virus culture

[0068] The second type of dengue virus strain 454009A (DENV2 strain 454009A) was originally isolated from a dengue patient and infected the C6 / 36 cell line to maintain this virus strain. Briefly, DENV2 and C6 / 36 monolayer cells were cultured at 28°C and 5% CO2 at a multiplicity of infection (MOI) of 0.01 for 5 days. Centrifugation was performed at 1000×g for 10 minutes to collect the culture medium and remove cell debris. The virus suspension was collected and stored at -70°C for future use. The virus titer was determined by plaque assay using BHK-21 cells. Briefly, BHK-21 cells were seeded into 12-well cell culture plates (12-well plates, 8×10 4 cells / well) and cultured in DMEM under CO2-enriched conditions. After adding serially diluted viruses and absorbing for 1 hour, the inoculated cells were replaced with fresh DMEM [containing 2% FBS and 0.8% methyl cellulose]. Five days after infection, the cell culture medium was removed, and the cells were fixed and stained with crystal violet solution (composed of 1% crystal violet, 0.64% NaCl and 2% formalin).

[0069] Plaque assay

[0070] BHK-21 cells were seeded into 12-well plates (1×10 5 Cells / well) were cultured in DMEM containing 5% FBS and cultured overnight at 37°C with 5% CO2. The culture medium was removed and the viral supernatant (0.4 mL / well) serially diluted in DMEM containing 2% FBS was added to the cells and cultured at 37°C for 1 hour. Then, the inoculated cells were replaced with DMEM containing 2% FBS and 0.8% methylcellulose, and the culture plates were placed at 37°C for 5 days. Five days after infection, the cell culture medium was removed, the cells were fixed and stained with crystal violet solution (containing 1% crystal violet, 0.64% NaCl and 2% formalin) at room temperature for 1.5 hours to visualize the plaques. The virus concentration is expressed as PFU / mL and is used to calculate the infection multiple (MOI) in the infection experiment.

[0071] Plaque reduction neutralizing test (PRNT)

[0072] Plaque reduction neutralization test (PRNT) was performed using the above-mentioned plaque analysis method using different sera inoculated BHK-21 cells with the first type of dengue virus strain 8700828 (DENV1strain8700828), the second type of dengue virus strain 16681 (DENV2 strain 16681), the third type of dengue virus strain 8700829 (DENV3 strain 8700829) and the fourth type of dengue virus strain 8700544 (DENV4strain8700544). Two-fold serial dilutions of serum (treated at 56°C for 1 hour to inactivate complement) were mixed with 50-80 PFU / well of DENV at room temperature for 1 hour. Then, the virus-serum mixture was added to the BHK-21 monolayer cells for 1 hour, and then replaced with DMEM containing 2% FBS and 0.8% methylcellulose to cover. After 5 days, the coverslip was removed, the cells were fixed and stained with crystal violet solution (containing 1% crystal violet, 0.64% NaCl and 2% formalin) for 3 hours at room temperature to visualize plaques. Finally, the serum concentration that reduced plaques by 50% relative to plaques of serum-free virus was called PRNT50, which can be plotted using Prism software.

[0073] Determine antibody titer

[0074] 0.2 μg / well of DJ NS1 or NS3 protein was dissolved in coating buffer (containing 1.59 g Na2CO3, 2.93 g NaHCO3, pH 9.6, and finally ddH2O was added to 1 L) and added to a 96-well cell culture plate and coated overnight at 4°C. The plate was replaced with PBS containing 5% BSA, blocked overnight at 4°C, and then rinsed three times with PBS containing 0.05% Tween 20. Mouse serum was serially diluted and added to the protein-coated wells and incubated overnight at 4°C. After rinsing three times, HRP-conjugated anti-mouse IgG (Cell Signaling, Danvers, MA) or IgM (KPL, Gaithersburg, MD) was added to each well and incubated at room temperature for 2 hours. After rinsing, ABTS was added to each well of the culture plate, and the absorbance at 405 nm was measured using a microplate reader (Emax microplate reader; Molecular Devices, Sunnyvale, CA).

[0075] Analysis of dendritic cells and CD4 + T cells and CD8 + T cell activation

[0076] In ex vivo studies, pooled lymph node cells were double- or triple-stained with PE-conjugated anti-CD11c antibody (eBioscience, San Diego, CA), combined with APC-conjugated anti-CD40 or anti-CD86 antibody (BioLegend, San Diego, CA), PE-Cy7-conjugated anti-CD80 antibody (BioLegend), and FITC-conjugated anti-MHC class I or II antibody (BioLegend) to determine dendritic cell activation. The cells were analyzed using flow cytometry (CytoFLEXs, Beckman Coulter, Brea, CA).

[0077] The pooled lymph node cells (5×10 5 cells / mL) were cultured in 24-well cell culture plates and stimulated with DJ NS1 or NS3 (5 μg / mL) for 3 days. The cells were harvested and triple-stained with PE-conjugated anti-CD25 antibody (eBioscience), FITC-conjugated anti-CD4 antibody, and PE-Cy7-conjugated anti-CD80 antibody (BD Biosciences, San Jose, CA) to determine T cell activation. The cells were analyzed using a flow cytometer (CytoFLEXs).

[0078] Analysis of CD8 + CD107a on T cells

[0079] The pooled lymph node cells (5×10 5 cells / mL) were cultured in 24-well cell culture plates and stimulated with DJ NS1 or NS3 (5 μg / mL) for 3 days. On the third day, the cells were restimulated with anti-CD3 / CD28 antibodies (0.5 μg / mL; eBioscience), and PerCP / cy5.5-conjugated CD107a antibodies (BioLegend) were added to the cells before stimulation. The above cultures were cultured at 37°C and 5% CO2 for 4 hours in the presence of secretory inhibitors, wherein the secretory inhibitors are Brefeldin (BioLegend) and monensin (BD Biosciences) that inhibit cytotoxic granule acidification and receptor-mediated endocytosis, leaving CD107a on the cell surface. Afterwards, the cells were harvested and triple stained with PE-Cy7-conjugated anti-CD80 antibodies (BD Biosciences) to determine the degranulation of T cells. The above cells were analyzed using a flow cytometer (CytoFLEXs).

[0080] L929 cells show intracellular staining of NS3

[0081] NS3-expressing L929 cells (5×10 5 cells / mL) and fixed the cells with PBS containing 4% paraformaldehyde for 10 minutes at room temperature. After rinsing with PBS, the cells were permeabilized with permeabilized buffer [500mL PBS containing 0.5g saponin, 5g BSA and 0.5g NaN3] and stained with anti-NS3 antibody at 4°C overnight. After rinsing with permeabilized buffer, the cells were incubated with Alexa-488-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA) at room temperature for 1 hour. Finally, after rinsing twice with PBS, the cells were analyzed using a flow cytometer (CytoFLEXs).

[0082] Active immune response and DENV infection mouse model

[0083] Three-week-old mice were immunized subcutaneously (sc) on days 0 and 14 with 25 μg / mouse of DJ NS1 protein mixed with aluminum salt, 12.5 μg / mouse of NS3 protein mixed with aluminum salt, 25 μg / mouse of DJ NS1 protein and 12.5 μg / mouse of NS3 protein mixed with aluminum salt, aluminum salt alone, and PBS control group. Serum and lymph node cells were collected on day 21 for analysis. For the DENV infection model, mice were intravenously (iv) injected with DENV2 (1×10 8 The mice were challenged with virus (PFU / mouse), sacrificed on day 19, and plasma and tissues were collected.

[0084] Virus titer analysis

[0085] To determine the virus titer in mouse serum, a fluorescent focus assay (FFA) was performed. Briefly, serum was serially diluted with RPMI medium containing 2% FBS and cultured with BHK-21 cells at 37°C for 2 hours. Then, the culture medium of the monolayer cells was replaced with DMEM containing 2% FBS and 0.8% methylcellulose and cultured at 37°C. After 2-3 days, the cover was removed and virus foci were stained with anti-NS1 antibody 33D2 (obtained from Dr. Ye Caiming). Alexa488-conjugated goat anti-mouse IgG (Invitrogen) was added to the above cells and observed using a fluorescence microscope.

[0086] NS1 quantitative ELISA

[0087] The concentration of NS1 in mouse blood was quantified using a homemade NS1 sandwich ELISA. Briefly, 5 μg / mL of anti-NS1 monoclonal antibody (mAb) 33D2 was coated on a 96-well cell culture plate and cultured overnight. After blocking the plate with PBS containing 1% BSA for 1 hour, mouse serum (1:5 dilution) and 1 μg / mL of biotin-conjugated anti-NS1 monoclonal antibody 31B2 (obtained from Dr. Ye Caiming) were added to each well and co-cultured at 37°C for 1-2 hours. HRP-labeled streptavidin solution (1:40 dilution) (R&D Systems, Minneapolis, MN) was added to each well and incubated at 37°C for 20 minutes. After rinsing three times with PBST (PBS containing 0.01% Tween 20), tetramethylbenzidine (TMB) was used for color development and observation. After adding the stop solution (2N H2SO4), the absorbance of each well at 450 nm was measured using a microplate reader (Emax microplate reader, Molecular Devices).

[0088] Bleeding time

[0089] Two days after DENV infection, the bleeding time was measured before euthanasia. The tip of the mouse tail was cut off by 3 mm to measure the bleeding time. Blood drops were collected with filter paper every 30 seconds. When the blood spot diameter was less than 0.1 mm, the bleeding time was recorded.

[0090] Enzyme-linked immunosorbent assay (ELISA)

[0091] 100 μL of recombinant protein (cEDIII, NS1ΔC, DJ NS1 or cEDIII-NS1ΔC) was diluted to a concentration of 2 μg / mL with coating buffer and then coated on an ELISA plate. The plate was incubated overnight at 4°C. 200 μL of coating buffer containing 5% BSA was added to the plate, incubated at room temperature for 2 hours, and then rinsed three times with 200 μL of PBST (PBS containing 0.01% Tween 20). Two-fold serial dilutions of antiserum (100 μL) were added and incubated at 37°C for 2 hours or at 4°C overnight. The plate was rinsed three times with 200 μL of PBST. HRP-conjugated secondary antibody (100 μL, diluted 1:5000 in PBS) was added and incubated at 37°C for 2 hours. After the plate was rinsed three times with 200 μL of PBST, ABTS (100 μL) was added to observe the reaction results. After sufficient color development, the absorbance of each well was measured at 415 nm using a microplate reader.

[0092] Antibody-dependent complement-mediated cytolysis assay

[0093] HMEC-1 cells were seeded into 96-well cell culture plates (5×10 3 After the cells were incubated at 37°C and 5% CO2 overnight, the cells were incubated with DENV (MOI = 20) in M200 medium for 48 hours. After the cells were rinsed twice with PBS, the serum of non-immunized mice (mock serum), serum immunized with aluminum salt (alum serum), serum immunized with cEDIII-NS1ΔC mixed with aluminum salt (cEDIII-NS1ΔC serum) or serum immunized with DJ NS1 mixed with aluminum salt (DJ NS1 serum) was diluted 1:200 in M200 medium without phenol red, and incubated with the cells in the presence or absence of Low-Tox-M rabbit complement (1:20 dilution) at 37°C and 5% CO2 for 6 hours to promote complement-mediated cell lysis. The released lactate dehydrogenase (LDH), a cytoplasmic enzyme, was detected using a commercially available kit (Cytotoxicity Detection Kit) to measure cell lysis. The absorbance of each well was measured at 490 nm using a microplate reader.

[0094] Immunization and DENV-infection mouse models

[0095] Recombinant cEDIII-NS1ΔC was mixed with aluminum salt and subcutaneously immunized C3H / HeN mice. Seven days after the first immunization and three days after the second and third immunizations, mouse sera were collected and the antibody titer was determined. Four days after the third immunization, DENV was inoculated intradermally into the upper back of C3H / HeN mice. Three days after infection, the tail bleeding time of the mice was measured and the mice were sacrificed. The mouse sera were also collected and stored at -20°C.

[0096] Using the expression system of mammalian 293F cells and drosophila S2 cells, NS1fΔC-cEDIII-NS3cf and NS1fΔC-NS3cf recombinant proteins were expressed

[0097] Two recombinant proteins (NS1fΔC-cEDIII-NS3cf and NS1fΔC-NS3cf, with molecular weights of 49 kDa and 35 kDa, respectively) were cloned into the pTT5 vector, in which the proteins expressed by the pTT5 vector had His-tags at both ends. The above plasmids were transfected into 293F cells, and the recombinant proteins were secreted into the supernatant by 293F cells. The supernatant containing the recombinant proteins was collected 5 days after transfection and purified by nickel ion column (Ni 2+ The supernatant was purified by PCR using a 4% paraformaldehyde column. After purification, the recombinant protein was lyophilized and stored at -20°C. The recombinant protein obtained from 293F cells was designed and provided by Leadgene Biomedical, Inc. In addition, in order to produce recombinant proteins under more stable and better purified conditions, the protein was cloned into the pMT / BiP / V5 vector, in which the protein expressed by the pMT / BiP / V5 vector has a His-tag and a Strep-tag at both ends, and the plasmid was transfected into Drosophila S2 cells. The recombinant protein-expressing stable cell line of Drosophila S2 cells was established by Professor Ye Caiming's laboratory. When the cells were cultured for the first time, they were cultured in an upright 75T culture flask containing 20 mL of Schneider's Drosophila Medium at room temperature with shaking at 65 rpm. When the cells grew to a concentration of 1.5 to 2×10 7 After reaching 1000 cells / mL, the cells in four 75T culture flasks were transferred to a 1000 mL plastic conical flask for cell culture. The cells were cultured at 80 rpm for 3 days until the cells grew to a concentration of 3 to 5 × 10 in the plastic conical flask containing 500 mL of culture medium. 6cells / mL. 500 μL of 500 mM copper sulfate was added to the above culture medium, and the recombinant protein was induced at room temperature for 3 days. In order to analyze the performance of the recombinant protein, the above sample was centrifuged at 1500 rpm at room temperature to collect the supernatant. The resulting 500 mL supernatant was mixed with 50 mL of binding buffer (containing 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and then passed through a cup filter. The supernatant containing the recombinant protein was injected into a column containing Strep-tactin sepharose. The column was washed with a washing buffer (containing 50 mM NaH2PO4, 300 mM NaCl, pH 8.0). The recombinant protein with Strep-tag was washed out with an extraction buffer (containing 50 mM NaH2PO4, 300 mM NaCl, 2.5 mM desthiobiotin, pH 8.0). After purification, the protein was tested by 12% SDS-PAGE and Western blot analysis, and dialyzed into PBS base overnight using a 10K dialysis membrane. The dialyzed protein was quantified by Bradford protein assay and then freeze-dried.

[0098] Focus reduction neutralization test (FRNT)

[0099] BHK-21 cells were seeded into 96-well cell culture plates (1×10 4Cells / well), placed in a 37°C incubator for 12-16 hours until the cell confluence reaches 60-70%. The serum obtained from the immunized mice was diluted 10 times with DMEM containing 2% FBS, and 50 μL of virus culture solution containing 50-100 plaques was added to the diluted serum and incubated at room temperature for 1 hour. Next, the culture medium of the 96-well culture plate was removed, and 100 μL of the serum-virus mixture was added to each well containing BHK-21 cells and placed in a 37°C incubator for 1-1.5 hours. Then, 100 μL of methyl cellulose medium was directly added to each well and incubated for 2-2.5 days. The cells were fixed with 4% triformaldehyde at 37°C for 1 hour, and then the primary antibody (anti-NS1 monoclonal antibody 33D2 plus anti-DENV serotype 1-4 antibody, prepared in 0.5% Triton X-100 solution) was added to each well and stained at 4°C overnight. Virus foci were stained with FITC-conjugated goat anti-mouse IgG and visualized using a fluorescence microscope.

[0100] Statistical analysis

[0101] Values ​​are expressed as mean ± SD. All values ​​were analyzed using Prism software (GraphPad, San Diego, CA). Bleeding time and viral titer were analyzed using one-way ANOVA. Complement-dependent cell lysis was analyzed using two-way ANOVA. Statistical significance was set at p < 0.05.

[0102] result

[0103] Immunization with cEDIII-NS1ΔC recombinant protein can induce antibodies that specifically respond to cEDII and NS1ΔC proteins

[0104] The protein patterns used in this example include cEDIII, NS1ΔC, DJ NS1, and a linear fusion of cEDIII-NS1ΔC recombinant protein (eg Fig. 9A 6- to 8-week-old C3H / HeN mice (as shown) were subcutaneously immunized with cEDIII-NS1ΔC, DJ NS1, or DJ NS1 plus cEDIII, mixed with aluminum salt as an adjuvant. Fig. 9B After sacrificing the mice, the antibody titer was measured. In this example, cEDIII, DJ NS1 and NS1ΔC proteins were coated on 96-well cell culture plates, and mouse serum was added as primary antibody. Then, ELISA analysis was performed to obtain the antibody titer of the second and third immunization doses (as shown in FIG. Fig. 9C shown).

[0105] Serum from mice immunized with cEDIII-NS1ΔC can neutralize four serotypes of DENV

[0106] Mouse serum induced by cEDIII-NS1ΔC contains anti-NS1ΔC and anti-cEDIII antibodies. Because anti-E antibodies play a role in neutralizing DENV, in this example, it can be used to evaluate whether mouse serum immunized with cEDIII-NS1ΔC can neutralize four serotypes of DENV. In order to verify the neutralizing ability of immunized mouse serum, this example performs a plaque reduction neutralization test (PRNT). Unimmunized serum (mock serum), serum immunized with aluminum salt (alum serum), serum immunized with cEDIII-NS1ΔC (cEDIII-NS1ΔC serum) and serum immunized with DJ NS1 (DJ NS1 serum) were serially diluted by two times, mixed with DENV1, DENV2, DENV3 or DENV4, and added to BHK-21 cells seeded in 12-well cell culture plates. Only the sera from mice immunized with cEDIII-NS1ΔC were able to neutralize DENV1, DENV2, DENV3, and DENV4 in a dose-dependent manner, with sera diluted 1:128 showing a 50% reduction in the number of plaques. However, sera from mice immunized with DJNS1 were unable to neutralize any serotype of DENV (e.g. FIG. 10A to FIG. 10D shown).

[0107] Serum from mice immunized with cEDIII-NS1ΔC destroyed cells infected with four DENV serotypes via antibody-complement-mediated cytolysis

[0108] NS1 is post-translationally modified into dimerized form in the lumen of the endoplasmic reticulum and is expressed on the surface of infected cells. Previous studies by the inventors have shown that anti-NS1ΔC antibodies can provide protection in vitro through complement-mediated cytotoxicity. Therefore, this example aims to find out whether cEDIII-NS1ΔC immune serum (cEDIII-NS1ΔC serum) containing anti-NS1ΔC antibodies can cause complement-mediated cell lysis against four serotypes of DENV. To this end, this example performs an antibody-dependent complement-mediated cytotoxicity assay. HMEC-1 cells were seeded into 96-well cell culture plates and infected with DENV1, DENV2, DENV3 or DENV4. Then, serum from unimmunized mice (mock serum), serum immunized with aluminum salts (alum serum), serum immunized with cEDIII-NS1ΔC mixed with aluminum salts (cEDIII-NS1ΔC serum), or serum immunized with DJ NS1 mixed with aluminum salts (DJ NS1 serum) was diluted 1:200 and incubated with cells in the presence or absence of complement. Cytolysis was measured by detecting the release of lactate dehydrogenase (LDH). Compared with the other groups, serum from mice immunized with cEDIII-NS1ΔC and DJ NS1, when mixed with complement, could cause lysis of cells infected with four different serotypes of DENV (such as FIG. 11A to FIG. 11D Because NS1ΔC is produced by NS1 of DENV2, the above results show that DENV2 NS1ΔC has cross-protective effects against all serotypes of DENV.

[0109] Immunization with cEDIII-NS1ΔC protein can reduce the prolonged bleeding time of DENV-induced mice

[0110] C3H / HeN mice were subcutaneously immunized three times with aluminum salt mixed with cEDIII-NS1ΔC recombinant protein ( Fig. 12A Serum antibodies against cEDIII and DJ NS1 were measured (as shown). Fig. 12B Then, mice were inoculated with different DENV serotypes by intravenous injection (iv) to test the protective effect of cEDIII-NS1ΔC protein. The results showed that compared with mice immunized with DENV alone or DENV mixed with aluminum salt, mice immunized with cEDIII-NS1ΔC could reduce the tail bleeding time caused by four different DENV serotypes (as shown in Figure 2). Figures 13A-13D shown).

[0111] Immunization with cEDIII-NS1ΔC protein can reduce the DENV titer in mouse serum

[0112] In order to confirm the effect of cEDIII-NS1ΔC immunization on viral replication, the viral titer of mouse serum was measured. The results showed that compared with mice immunized with DENV alone or DENV mixed with aluminum salt, mice immunized with cEDIII-NS1ΔC protein could reduce the viral titer measured by different serotypes of DENV infection (such as Figures 14A-14D shown).

[0113] Immunization with DJ NS1 and NS3 can react with DJ NS1 or NS3 antigen in vitro, inducing dendritic cells and specific CD4 + With CD8 + T cell activation

[0114] This example analyzes CD11c + Lymph node dendritic cells (CD11c + The expression of CD40, CD80, CD86, MHC I and MHC II in lymph node dendritic cells (LNDCs) was used to evaluate the activation of dendritic cells (DCs) after immunization. The results showed that most CD11c + LNDCs expressed MHC class I. LNDCs induced by mice immunized with 25 μg DJ NS1 and 12.5 μg NS3 expressed significantly higher MHC class I than those induced by mice immunized with aluminum salt alone, DJ NS1 alone, and NS3 alone. Mice immunized with DJ NS1 alone or NS3 alone expressed MHC class II CD11c + LNDCs increased; although not statistically significant, there was a trend toward an increase in mice immunized with the combination of DJ NS1 and NS3. Although not statistically significant, mice actively immunized with DJ NS1 alone, NS3 alone, or DJ NS1 plus NS3 showed an increase in CD11c + Compared with the PBS immunization control group on day 21, the active immunization groups with DJ NS1 alone, NS3 alone, or the combination of DJ NS1 and NS3 showed an increased trend in the expression of CD40 in LNDCs. + Compared with the aluminum salt immunization group and the PBS immunization control group, only the active immunization group combined with DJNS1 and NS3 increased CD11c + The expression of CD86 by LNDCs was statistically significant. Thus, these results indicate that active immunization with DJ NS1 plus NS3 can induce DC activation, as an increase in DCs expressing co-stimulatory receptors was detected (data not shown).

[0115] To evaluate whether DJ NS1 and NS3 can induce antigen-specific T cell responses, lymph node cells were collected from immunized mice and restimulated with 5 μg / mL of DJ NS1 or NS3 protein for 3 days. Anti-CD3 and anti-CD28 antibodies were administered to all groups to partially mimic TCR messages and amplify responses. Mice actively immunized with DJ NS1 alone or with DJNS1 plus NS3 responded to stimulation with DJ NS1 antigen and significantly induced CD4 + T cell activation (eg Fig.15A Compared with the other groups, only the mice actively immunized with DJ NS1 plus NS3 could respond to the stimulation of NS3 antigen and significantly induce CD4 + T cell activation (eg Fig. 15B Compared with the other groups, mice actively immunized with DJ NS1 plus NS3 also showed a stronger response to DJ NS1 antigen stimulation (as shown in Fig. 15C ) and stimulation of NS3 antigen (as shown Fig.15D ) and significantly induced CD8 + Therefore, active immunization with DJ NS1 plus NS3 can respond to stimulation with DJNS1 or NS3 antigens and simultaneously induce CD4 + With CD8 + T cell activation.

[0116] Active immunization with DJ NS1 and NS3 can induce specific CTL responses

[0117] Previous studies have shown that CD8 + T cells can directly contribute to the prevention of DENV. In our previous studies, active immunization with DJNS1 plus NS3 can induce CD8 + T cell activation (eg FIG. 15C to FIG. 15D In order to detect whether CTL response can be induced, lymph node cells were isolated from immunized mice, restimulated with DJ NS1 or NS3 protein, and then co-cultured with L929 cells expressing DJ NS1 or NS3 (as target) for 4 hours (eg Fig.16AIn order to generate L929 cells expressing NS3, this example established L929 cells expressing wild-type NS2B3 or proteolytically inactive mutant NS2B3-S135A (Rodriguez-Madoz et al., 2015). Since the expression of L929 cells expressing NS2B3-S135A was better (data not shown), these cells were used as target cells. The results showed that only CTLs obtained from mice immunized with DJ NS1 plus NS3 could lyse L929 cells expressing NS1, but there was no statistical significance compared to other groups (such as Fig. 16B On the other hand, CTLs obtained from mice immunized with NS3 alone or with DJ NS1 plus NS3 were able to significantly lyse L929 cells expressing NS3 (as shown in Fig. 16C shown).

[0118] In addition, in order to prove the CTL activity against target cells, this example directly detects the cytotoxic activity of effector cells. + The lipid bilayer of cytotoxic granules in T cells contains lysosomal-associated membrane glycoproteins (LAMPs), including CD107a (LAMP-1); therefore, CTL degranulation results in the accumulation and exposure of CD107a on the cell surface. + Based on the fact that T cells rapidly degranulate, lymph node cells isolated from immunized mice were stimulated with DJ NS1 or NS3 proteins for 3 more days and incubated with anti-CD3 and anti-CD28 antibodies to partially mimic TCR messages during the final 4 hours. Active immunization with DJNS1 plus NS3 resulted in a significant reduction in the expression of DJ NS1 ( Fig.17A ) and NS3( Fig. 17B ) stimulated by CD8 + Compared with the aluminum salt immunization group and the PBS control group, mice immunized with NS3 responded to NS3 stimulation and significantly increased their CD8 + The expression of CD107a on the surface of T cells was lower than that of CD8 + The expression of CD107a on the surface of T cells (such as Fig. 17B ). Figures 16A to 17B The results showed that active immunization with DJ NS1 plus NS3 could induce antigen-specific CTL responses.

[0119] Active immunization with DJ NS1 plus NS3 provides protection against DENV infection

[0120] Next, this example established a DENV infection model to evaluate the protective effect provided by active immunization with DJ NS1 plus NS3. The results are as follows: Fig.18A shown.

[0121] Previous studies by the inventors have demonstrated that inoculation of mice with high titer DENV can produce symptoms such as prolonged bleeding time and localized bleeding (Wan et al., 2014). Therefore, this embodiment evaluates the protective effect provided by active immunization with DJ NS1 plus NS3 by determining the prolonged bleeding time caused by DENV. The results showed that whether infected with DENV alone or combined with aluminum salt infection, the bleeding time of mice was prolonged 48 hours after infection. Compared with the group infected with DENV alone, active immunization with DJ NS1, NS3, or DJ NS1 plus NS3 significantly reduced the prolonged bleeding time of mice caused by DENV (such as Fig.18B shown).

[0122] In this example, the viral titer was measured on day 19. DJ NS1 protein can induce antibody response to induce antibody-dependent complement-mediated cytolysis of DENV-infected cells (Wan et al., 2017). Consistent with the inventors' previous findings, active immunization with DJ NS1 alone can significantly reduce the viral titer (e.g., 1.3% vs. 1.5% for DENV2 infection alone or aluminum salt control infection) Fig.18C As shown in Figure 2 ). Compared with active immunization with DJ NS1 alone, active immunization with NS3 alone can significantly reduce the virus titer. The NS3-specific T cell response shown in this example helps to clear the virus. Active immunization with DJ NS1 plus NS3 and active immunization with NS3 alone can reduce the virus titer after immunization.

[0123] These results suggest that active immunization with DJ NS1 or with DJ NS1 plus NS3 may provide better protection against vascular leakage.

[0124] discuss

[0125] Active immunization with DJ NS1 plus NS3 provides protection against DENV infection

[0126] According to the above examples, for antiviral strategies, the cEDIII-NS1ΔC recombinant protein can be used as a candidate for subunit vaccines. For subunit vaccines based on non-replication proteins, this type of vaccine is considered to be a safer choice.

[0127] Considering that the structural changes of recombinant cEDIII-NS1ΔC may affect the efficacy of immunotherapy, cEDIII and DJNS1 proteins were used together to compare with fusion proteins. In the above example, mice were immunized with cEDIII (5 μg) and DJ NS1 (20 μg) together, and the antibody titer induced to cEDIII and DJ NS1 protein specificity was better, and was similar to the antibody titer induced by cEDIII-NS1ΔC (25 μg) immunization (data not shown). cEDIII-NS1ΔC was selected as the antibody strategy.

[0128] EDIII has a low ability to induce cross-reactive antibodies to heterologous DENV serotypes, suggesting that this is related to the pathogenesis of severe disease (Chin et al., 2007). DENV EDIII recombinant proteins were designed based on four virus strains and mice were immunized with DENV EDIII recombinant proteins to produce serotype-specific IgG1 neutralizing antibodies, and the enhanced activity of these antibodies against DENV in cells bearing FcγR was detected. The results of resurfaced EDIIIs (rsDIIIs) proteins based on genetically engineered DENV2 showed that rsDIIIs reduced the binding of antibodies with inappropriate antigenic determinants, but maintained high affinity for DENV universal neutralizing antibodies. cEDIII contains the consensus sequence of EDIII of four DENV serotypes and can also be considered as a potential subunit vaccine candidate. In the past, it was reported that antibodies from mice immunized with cEDIII showed the ability to neutralize all serotypes of DENV. Individual immunization of monkeys with cEDIII also showed that it could increase the level of neutralizing antibodies in the serum (Chen et al., 2013; Leng et al., 2009). In this example, the neutralizing antibodies induced by cEDIII recombinant protein in C3H / HeN mice not only resisted four serotypes of DENV in vitro, but also protected mice against DENV infection in vivo. In addition, it was observed that after infection with DENV, mice immunized with cEDIII-NS1ΔC had lower viral titers and soluble NS1 levels in serum compared to the group infected with DENV alone.

[0129] Recent studies have highlighted the pathogenicity of DENV NS1 through various mechanisms. Therefore, NS1 is considered a suitable target to combat the pathogenicity caused by DENV. Treatment with anti-NS1 mAb reversed NS1-induced endothelial hyperpermeability (Chen et al., 2016). Vaccination with NS1 also prevented endothelial permeability and vascular leakage caused by DENV NS1 (Beatty et al., 2015). Previous studies identified the C-terminal region of DENV NS1 as the major cross-reactive antigenic determinant (Cheng et al., 2009). Therefore, removal of the C-terminal NS1 protein can avoid autoimmunity and is considered a safer subunit vaccine candidate. The inventors' previous studies showed that antibodies specific for NS1ΔC have antibody-mediated complement-dependent cytotoxicity (Wan et al., 2014). Immunization of mice with cEDIII-NS1ΔC not only induced antibodies to destroy cells infected by four DENV serotypes, but also reduced the prolonged bleeding time induced by DENV in mice.

[0130] Although the exact role of T cells in DENV infection remains to be determined, there is much evidence in current studies in humans and mice to support the protective role of T cells in DENV infection (Katzelnick et al., 2017; Kao 2019). Nonstructural proteins have been reported to contain many antigenic determinants for T cell activation. NS1 has also been mentioned to activate CD4 + and CD8 + T cells play a key role in protecting against DENV. Therefore, the specific T cell response of cEDIII-NS1ΔC protein can be further studied in the future. NS1ΔC and NS3 have been tried as a vaccine strategy against DENV infection (Kao et al., 2019). In the future, cEDIII-NS1ΔC plus NS3 protein can be used as a candidate for vaccine development. It contains prM and E genes but does not contain non-structural proteins of DENV, so it will not produce antibodies specific to DENV NS1 protein, nor will it produce effective T cell responses specific to non-structural proteins. In these embodiments, cEDIII contains the common sequence of EDIII of four serotypes of DENV, and NS1ΔC is produced by NS1 of DENV2. These results show that cEDIII-NS1ΔC has cross-reactivity to four serotypes of DENV.

[0131] Based on the above results, cEDIII-NS1ΔC protein can induce high antibody response specific to outer membrane protein and NS1 protein, and protect against DENV and DENV-infected cells with neutralization and complement-dependent cytolysis functions, respectively. In the mouse protection model, pretreatment with cEDIII-NS1ΔC protein can also induce mice to produce antibodies with high titers specific to membrane protein and NS1 protein. Furthermore, the group immunized with cEDIII-NS1ΔC reduced the prolonged bleeding time caused by DENV and the virus titer in mouse serum after infection with four different serotypes of DENV. Therefore, cEDIII-NS1ΔC protein is a potential vaccine candidate to protect against DENV infection. Dengue fever is an important mosquito-borne viral disease in humans in terms of global incidence and economic impact (Bhatt et al., 2013). Despite the global burden of dengue fever, there is no specific treatment or safer and more effective vaccine for dengue fever, and current prevention is limited to vector control measures. There are still some obstacles to the development of dengue vaccines and antiviral drugs. One is that the pathogenesis is complex and has not yet been fully resolved. Another difficulty is the lack of a suitable animal model (Coller et al., 2011; Wan et al., 2013). Recently, several teams have produced quadrivalent active attenuated vaccines, but safety and protective efficacy remain to be verified. In these embodiments, the aforementioned specific examples produced cEDIII-NS1ΔC fusion protein as a subunit virus for antiviral strategies. Antibodies generated by immunization with cEDIII-NS1ΔC recombinant protein have the ability to neutralize DENV and destroy DENV-infected cells. Immunization with cEDIII-NS1ΔC protein can also reduce the tendency of DENV-induced bleeding and reduce the concentration of soluble NS1 in mouse plasma. The possible mode or role of antibodies caused by cEDIII-NS1ΔC may involve neutralization ability and antibody-mediated complement-dependent cell lysis.

[0132] Several neutralizing antibodies specific for type and subtype have been mapped to EDIII (Gromowski et al., 2007; Wahala et al., 2009; Guzman et al., 2010), and anti-EDIII antibodies have been considered the most potent blockers of viral infectivity (Gromowski et al., 2007). Anti-EDIII antibodies are present in sera from humans immunized with primary and secondary DENV. However, anti-EDIII antibodies represent only a small fraction of the total antibodies in sera bound to DENV (Wahala et al., 2009). EDIII is thought to be responsible for receptor binding (Kuhn et al., 2002; Klein et al., 2013). EDIII exposed on the surface of viral particles is easily accessible, and EDIII recombinant protein can inhibit viral infectivity (Chin et al., 2007; Guzman et al., 2010; Coller et al., 2011). Importantly, EDIII has a low potential to induce cross-reactive antibodies to heterologous DENV serotypes, suggesting that this is related to the pathogenesis of severe disease (Chin et al., 2007; Guzman et al., 2010).

[0133] There are a number of recombinant subunit vaccine candidates based on EDIII in preclinical development. The International Centre for Genetic Engineering and Biotechnology in India has developed a tetravalent chimeric EDIII fusion protein expressed in Pichia pastoris. The tetravalent chimeric EDIII fusion protein consists of the EDIII domains of DENV1, DENV2, DENV3, and DENV4, connected by an elastic peptide linker. Immunization of mice with the tetravalent vaccine candidate in montanide adjuvant resulted in neutralizing antibody responses against all DENV serotypes. The production of a tetravalent dengue vaccine must be cost-effective to make the vaccine affordable in resource-poor regions of the world. To create a single multivalent component, cEDIII, which contains the consensus sequence of EDIII from four DENV serotypes, would be a good vaccine candidate (Leng et al., 2009; Chen et al., 2013). Immunization of mice with recombinant cEDIII resulted in neutralizing antibodies against all DENV serotypes.

[0134] Another group of antibodies induced by immunization with cEDIII-NS1ΔC protein is anti-NS1ΔC antibodies. The inventors of this case have confirmed that the major cross-reactive antigenic determinant is at the C-terminus of DENV NS1 (Cheng et al., 2009; Chen et al., 2009, Wan et al., 2008). Therefore, removing the C-terminus of NS1 can avoid autoimmunity and is a safer subunit vaccine candidate. Secondly, the inventors' previous studies have confirmed that anti-NS1ΔC antibodies have the ability of antibody-mediated complement-dependent cytotoxicity (Wan et al., 2014). In this example, the serum of mice immunized with cEDIII-NS1ΔC can cause cell lysis of HMEC-1 cells infected with four different serotypes of DENV. In addition to antibody-mediated complement-dependent cytotoxicity, other mechanisms by which anti-NS1ΔC antibodies help to resist DENV attack need to be further confirmed, such as preventing blood permeability caused by NS1.

[0135] An ideal vaccine or antiviral drug must be able to protect against any of the four DENV serotypes without the risk of ADE (Wan et al., 2013). Non-neutralizing antibodies or sub-neutralizing antibodies against DENV E and DENV PrM will cross-react with the four DENV serotypes, which will enhance viral entry and infection. DENV NS1 is not a structural protein on the surface of the virus, so anti-NS1 antibodies will not cause ADE. In this embodiment, the above specific examples verify that cEDIII-NS1ΔC serum can neutralize all four DENV serotypes and destroy HMEC-1 cells infected with DENV through antibody-mediated complement-dependent cytolysis. In order to further verify the protective efficacy of cEDIII-NS1ΔC protein, immunization with cEDIII-NS1ΔC protein mixed with aluminum salt can reduce the prolonged bleeding time caused by DENV infection and provide protective efficacy.

[0136] It only contains DENV prM and E genes, but no DENV NS protein, so it cannot produce antibodies specific to NS1 protein, nor does it have the effect of T cell response specific to NS protein. On the contrary, cEDIII-NS1ΔC protein not only produces neutralizing antibodies, but also produces antibodies specific to NS1 protein, thus providing a more comprehensive defense against DENV infection. The specific T cell response of cEDIII-NS1ΔC protein needs further study. In addition, it has been reported that Adverse reactions to vaccination. The cEDIII-NS1ΔC protein is a DENV protein subunit vaccine, but it is not a complete virus particle, so the chance of causing serious side effects is much lower than other live attenuated vaccines.

[0137] Dengue vaccines should be priced to be affordable to individuals who need them most in resource-poor regions of the world. In this example, cEDIII protein was fused to NS1ΔC protein to save costs and speed up protein purification. + T cell responses have been shown to protect against reinfection with heterotypic DENV, thus reducing the risk of ADE. Therefore, cEDIII-NS1ΔC plus NS3 protein is worth trying as a candidate for vaccine development. Since the subunit vaccine expressed in E. coli lacks glycosylation, the above-mentioned specific examples have expressed the above recombinant proteins in mammalian cells and Drosophila cells.

[0138] 9A to 18C The results are summarized in Table 2. Regarding the inhibition of viral titer, reduction of sNS1 content and reduction of prolonged bleeding time, the immune protection provided by the cEDIII-NS1ΔC fusion protein was superior to or similar to that of the mixture of recombinant proteins DJ NS1 and NS3.

[0139] Table 2

[0140]

[0141]

[0142] In the above specific examples, the novel vaccine strategy can use modified NS1 and NS3 together to study the mechanism of the observed protective effect against DENV in mice. Subunit vaccines based on non-replicating recombinant proteins are considered safer and easier to re-formulate. In order to solve the common problem of subunit vaccines, that is, the poor immunogenicity of highly purified antigens, the above specific examples combine NS1 protein with immunodominant NS3 protein. The results showed that NS3 protein can synergistically induce more effective immune responses, including not only generating NS3-specific CTL responses, but also promoting NS1-specific T cell responses and antibody titers. Compared with immunization with NS1 alone or NS3 alone, the immune response and protection of active immunization with DJ NS1 plus NS3 are better (such as Fig.19 shown).

[0143] The protective effect of immunization with recombinant NS1fΔC-cEDIII-NS3cf and NS1fΔC-NS3cf proteins against DENV infection was determined by in vitro analysis of FRNT and in vivo analysis of mouse tail bleeding time.

[0144] As mentioned above, DENV NS3 induces CD8 +T cell responses can protect against reinfection with heterotypic DENV, thereby reducing the risk of antibody-dependent enhancement (ADE). Therefore, fusion proteins of cEDIII, NS1ΔC, and NS3 can also be evaluated as candidates for vaccine development. FIG. 20A to FIG. 20D As shown, NS1fΔC-cEDIII-NS3cf and NS1fΔC-NS3cf proteins were expressed using mammalian cells 293F (as shown in Table 3) and Drosophila cells S2 (as shown in Table 4). Fig.21A and Fig.22A As shown, 25 μg of NS1fΔC-cEDIII-NS3cf or NS1fΔC-NS3cf protein and aluminum salt as adjuvant were subcutaneously injected (sc) into C3H / HeN mice for three immunizations. After the second and third immunizations, mouse sera were collected to detect the antibody titers against DJ NS1, NS3 and cEDIII (as shown in Tables 3 and 4). After the third immunization of mice, the antibody titer stimulated by NS1fΔC-NS3cf fusion protein was higher than that of NS1fΔC-cEDIII-NS3cf fusion protein.

[0145] Table 3

[0146]

[0147]

[0148] Table 4

[0149]

[0150] Focus reduction neutralization test (FRNT) was performed to verify the neutralization ability of immunized mouse sera. Mock serum, serum immunized with aluminum salt alone, serum immunized with NS1fΔC-cEDIII-NS3cf, and serum immunized with NS1fΔC-NS3cf diluted 10 times were mixed with DENV and added to BHK-21 cells seeded in 96-well cell culture plates. Serum from mice immunized with NS1fΔC-cEDIII-NS3cf neutralized DENV, but serum immunized with NS1fΔC-NS3cf, serum immunized with aluminum salt, or non-immunized control serum (mock control serum) did not (e.g. Fig. 21B and Fig. 22B shown).

[0151] NS1 is expressed on the surface of infected cells. The inventors wanted to find out whether the sera of mice immunized with NS1fΔC-cEDIII-NS3cf and those containing anti-NS1ΔC antibodies immunized with NS1fΔC-NS3cf could cause complement-mediated cytolysis of DENV-infected cells. Huh-7 cells were seeded into 96-well cell culture plates and infected with DENV2. Next, the cells were infected with 1:20 diluted non-immune serum, serum immunized with aluminum salt alone, serum immunized with NS1fΔC-cEDIII-NS3cf, and serum immunized with NS1fΔC-NS3cf, mixed with or without complement, respectively. Cell lysis was detected by the released lactate dehydrogenase (LDH). Compared with other groups, both the serum of mice immunized with NS1fΔC-cEDIII-NS3cf and the serum immunized with NS1fΔC-NS3cf mixed with complement could cause cytolysis of DENV-infected cells (such as Fig. 21C and Fig. 22C shown).

[0152] After that, the inventors wanted to test the protective efficacy of mice immunized with NS1fΔC-cEDIII-NS3cf and NS1fΔC-NS3cf proteins after intravenous injection (iv) of DENV. The results showed that compared with the groups infected with DENV alone or DENV plus aluminum salt, mice immunized with NS1fΔC-cEDIII-NS3cf and NS1fΔC-NS3cf proteins could reduce the tail bleeding time caused by DENV (such as Fig.21D and Fig.22D shown).

[0153] Previous studies have shown that Th1-biased responses, which are dominated by IFN-γ production, are associated with less severe secondary DENV infections, while TNFα produced by T cells is associated with more severe infections. Other studies have confirmed that DENV2NS3 recombinant protein induces a higher Th1 response of IFN-γ / TNFα. However, previous clinical studies have demonstrated that strong T cell responses are associated with dengue hemorrhagic fever (DHF), particularly those against NS3. This discrepancy requires further investigation. In the inventors' studies, active immunization with DJ NS1 plus NS3 induced CD8 +CD107a was expressed on the surface of T cells, indicating that the combined use of DJ NS1 and NS3 can promote cytotoxicity against DJ NS1. Consistent with the results of CTL analysis, active immunization with DJ NS1 plus NS3 can induce cytotoxicity against L929 cells expressing DJ NS1, but not against L929 cells expressing only NS3. Previous studies have shown that the expression of IFN-γ and CD107a, together with newly synthesized perforin protein, can promote antigen-specific CD8 + T cells have cytotoxicity. However, the detailed mechanism of cytotoxicity remains to be elucidated.

[0154] Since active immunization with NS3 also induces high titers of anti-NS3 IgM and IgG, the above specific example cannot exclude the possible role of anti-NS3 antibodies in this infection mode. Previous studies have shown that direct antibodies against NS3 can produce a protective effect. Although anti-NS3 antibodies are not neutralizing antibodies, they can be detected in the sera of patients with primary and secondary DENV infections, so the role of anti-NS3 antibodies needs further study.

[0155] Clinical manifestations of DENV infection can range from asymptomatic, typical dengue fever to severe, life-threatening hemorrhagic dengue fever (DHF), which is mainly characterized by increased vascular leakage, leading to bleeding, hypovolemia, hypotension and even shock syndrome. In the DENV infection model, mice showed viremia, prolonged bleeding time, vasculopathy and bleeding. Prolonged bleeding time may reflect coagulation abnormalities, including thrombocytopenia and disruption of the coagulation system. In adults, the number of platelets is significantly correlated with the bleeding manifestations of DENV infection. Although some hypotheses have been proposed during DENV infection, the mechanisms involved in thrombocytopenia and bleeding are not fully understood. In the above DENV infection model, active immunization with either DJ NS1 or NS3 can shorten the DENV-related prolonged bleeding time (such as Fig.18B DJ NS1-induced NS1 neutralizing antibodies can directly block the pathogenesis of sNS1 (data not shown), and can also cause complement-mediated cytolysis of DENV-infected cells with anti-NS1 antibodies (Wan et al., 2017). On the other hand, active immunization with NS3 can induce NS3-specific CTLs, reduce the viral titer and sNS1 content in serum, and thus shorten the prolonged bleeding time. In summary, active immunization with DJ NS1 plus NS3 can significantly shorten the prolonged bleeding time caused by DENV through multiple mechanisms.

[0156] The pathogenesis and defense of vascular leakage and bleeding are very complex. sNS1 can guide complement to attack endothelial cells and induce endothelial cell apoptosis (Amorim et al., 2014). Recent studies have shown that sNS1 can bind to TLR4, prompting cells to release proinflammatory cytokines, leading to vascular leakage (Modhiran et al., 2015). sNS1 has also been shown to activate TLR2 and TLR6, resulting in increased production of proinflammatory cytokines (Chen et al., 2015). In addition, macrophage migration inhibitory factor induced by DENV or NS1 infection can enhance DENV replication through autophagy and promote vascular leakage by destroying tight junctions of endothelial cells in humans and mice (Chen et al., 2018; Chuang et al., 2011; Chen et al., 2016). NS1 can also directly damage the endothelial glycocalyx, leading to hyperpermeability (Puerta-Guardo et al., 2016; Chen et al., 2018). Therefore, sNS1 can be a major factor that directly or indirectly causes vascular leakage and bleeding. As shown in the results, active immunization with DJ NS1 alone or with DJ NS1 plus NS3 can significantly prevent DENV-related vascular permeability changes. Conversely, active immunization with NS3 alone cannot avoid vascular changes. These results highlight that NS1 neutralizing antibodies play an important role in preventing vascular leakage and bleeding.

[0157] According to clinical observations, dengue disease is more severe in secondary heterotypic infections. Any successful vaccine needs to induce protective and sustained immune responses to four DENV serotypes simultaneously to avoid ADE (Pang et al., 2017). Vaccine candidates with DJ NS1 plus NS3 will not cause ADE. Secondly, the above specific examples have confirmed that active immunization with DJ NS1 plus NS3 can induce specific CD8 + T cell responses have recently been shown to protect against reinfection with heterotypic DENV. After the Zika virus (ZIKV) pandemic, some studies have shown that DENV-specific antibodies can cause ADE after reinfection with ZIKV following previous DENV infection. On the other hand, another example demonstrated that five DENV-epitope-specific CD8 +T cells can provide cross-protection against subsequent ZIKV infection (Wen et al., 2017). The candidate vaccine NS3 component contains one of five antigenic epitopes that mediate cross-protection against subsequent ZIKV infection and can avoid ADE of ZIKV. However, the ability of vaccine candidates to provide cross-protection against ZIKV infection requires further study.

[0158] In summary, the present invention discloses a novel DENV vaccine strategy that combines cEDIII-NS1ΔC fusion protein and NS3 protein to enhance protection against dengue virus attack and related pathological reactions.

[0159] Although the present invention has been disclosed as above with several specific embodiments, various modifications, changes and substitutions may be made to the aforementioned disclosure, and it should be understood that in some cases, certain features of the embodiments of the present invention will be adopted but other features will not be used accordingly without departing from the spirit and scope of the present invention. Therefore, the spirit and scope of the claims of the present invention should not be limited to the above exemplary embodiments.

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Sequence Listing <110> Lin Yixing <120> Dengue subunit vaccine composition <130> TWLB11855-20P1 <150> US 16 / 906,134 <151> 2020-06-19 <160> 6 <210> 1 <211> 100 <212> PRT <213> Artificial sequence <220> <223> cEDIII peptide of dengue virus <400> 1 Lys Gly Met Ser Tyr Ala Met Cys Thr Gly Lys Phe Lys Leu Glu Lys 5 10 15 Glu Val Ala Glu Thr Gln His Gly Thr Ile Leu Ile Lys Val Lys Tyr 20 25 30 Glu Gly Asp Gly Ala Pro Cys Lys Ile Pro Phe Glu Ile Gln Asp Val 35 40 45 Glu Lys Lys His Val Asn Gly Arg Leu Ile Thr Ala Asn Pro Ile Val 50 55 60 Thr Asp Lys Glu Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly 65 70 75 80 Asp Ser Tyr Ile Val Ile Gly Val Gly Asp Lys Ala Leu Lys Leu Asn 85 90 95 Trp Phe Lys Lys 100 <210> 2 <211> 275 <212> PRT <213> Artificial Sequence <220> <223> Dengue virus NS1 with C - terminus removed (NS1ΔC) <400> 2 Met Val Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys Asn Lys Glu 5 10 15 Leu Lys Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val His Thr Trp 20 25 30 Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys Leu Ala Ser 35 40 45 Ala Ile Gln Lys Ala His Glu Glu Gly Ile Cys Gly Ile Arg Ser Val 50 55 60 Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro Glu Leu Asn 65 70 75 80 His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met Thr Gly Asp 85 90 95 Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Gln Pro Gln Pro 100 105 110 Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala Lys Met Leu 115 120 125 Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly Pro Glu Thr 130 135 140 Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu Glu Val Glu 145 150 155 160 Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu Lys Leu Arg 165 170 175 Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser Ala Ala Ile 180 185 190 Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp Ile Glu Ser 195 200 205 Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe Ile Glu Val 210 215 220 Lys Ser Cys His Trp Pro Lys Ser His Thr Leu Trp Ser Asn Gly Val 225 230 235 240 Leu Glu Ser Glu Met Ile Ile Pro Lys Asn Phe Ala Gly Pro Val Ser 245 250 255 Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln Thr Ala Gly Pro Trp 260 265 270 His Leu Gly 275 <210> 3 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Dengue virus NS3c protein <400> 3 Ser Ile Glu Asp Asn Pro Glu Ile Glu Asp Asp Ile Phe Arg Lys Arg 5 10 15 Lys Leu Thr Ile Met Asp Leu His Pro Gly Ala Gly Lys Thr Lys Arg 20 25 30 Tyr Leu Pro Ala Ile Val Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr 35 40 45 Leu Ile Leu Ala Pro Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala 50 55 60 Leu Arg Gly Leu Pro Ile Arg Tyr Gln Thr Pro Ala Ile Arg Ala Glu 65 70 75 80 His Thr Gly Arg Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr 85 90 95 Met Arg Leu Leu Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile 100 105 110 Met Asp Glu Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly 115 120 125 Tyr Ile Ser Thr Arg Val Glu Met Gly Glu Ala Ala Gly Ile Phe Met 130 135 140 Thr Ala Thr Pro Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser Asn Ala 145 150 155 160 Pro Ile Met Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser Trp Ser Ser 165 170 175 Gly His Glu Trp Val Thr Asp Phe Lys Gly Lys Thr Val Trp Phe Val 180 185 190 Pro Ser Ile Lys Ala Gly Asn Asp Ile Ala Ala Cys Leu Arg Lys Asn 195 200 205 Gly Lys Lys Val Ile Gln Leu Ser Arg Lys Thr Phe Asp Ser Glu Tyr 210 215 220 Val Lys Thr Arg Thr Asn Asp Trp Asp Phe Val Val Thr Thr Asp Ile 225 230 235 240 Ser Glu Met Gly Ala Asn Phe Lys Ala Glu Arg Val Ile Asp Pro Arg 245 250 255 Arg Cys Met Lys Pro Val Ile Leu Thr Asp Gly Glu Glu Arg Val Ile 260 265 270 Leu Ala Gly Pro Met Pro Val Thr His Ser Ser Ala Ala Gln Arg Arg 275 280 285 Gly Arg Ile Gly Arg Asn Pro Lys Asn Glu Asn Asp Gln Tyr Ile Tyr 290 295 300 Met Gly Glu Pro Leu Glu Asn Asp Glu Asp Cys Ala His Trp Lys Glu 305 310 315 320 Ala Lys Met Leu Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro 325 330 335 Ser Met Phe Glu Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu 340 345 350 Tyr Arg Leu Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg 355 360 365 Arg Gly Asp Leu Pro Val Trp Leu Ala Tyr Arg Val Ala Ala Glu Gly 370 375 380 Ile Asn Tyr Ala Asp Arg Arg Trp Cys Phe Asp Gly Ile Lys Asn Asn 385 390 395 400 Gln Ile Leu Glu Glu Asn Val Glu Val Glu Ile Trp Thr Lys Glu Gly 405 410 415 Glu Arg Lys Lys Leu Lys Pro Arg Trp Leu Asp Ala Arg Ile Tyr Ser 420 425 430 Asp Pro Leu Ala Leu Lys Glu Phe Lys Glu Phe Ala Ala Gly Arg Lys 435 440 445 <210> 4 <211> 101 <212> PRT <213> Artificial sequence <220> <223> Simulation of DENV2 cEDIII (strain 16681, 295-395 aa) <400> 4 Lys Gly Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys Val Val Lys 5 10 15 Glu Ile Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg Val Gln Tyr 20 25 30 Glu Gly Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp Leu 35 40 45 Glu Lys Arg His Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile Val 50 55 60 Thr Glu Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly 65 70 75 80 Asp Ser Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu Asn 85 90 95 Trp Phe Lys Lys Gly 100 <210> 5 <211> 154 <212> PRT <213> Artificial sequence <220> <223> Truncated NS1 with C-terminus removed (DENV2 strain 16681, 91-244 aa) <400> 5 Gly Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg Pro 5 10 15 Gln Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala Lys 20 25 30 Met Leu Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly Pro 35 40 45 Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu Glu 50 55 60 Val Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu Lys 65 70 75 80 Leu Lys Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser Ala 85 90 95 Ala Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp Ile 100 105 110 Glu Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe Ile 115 120 125 Glu Val Lys Asn Cys His Trp Pro Lys Ser His Thr Leu Trp Ser Asn 130 135 140 Gly Val Leu Glu Ser Glu Met Ile Ile Pro 145 150 <210> 6 <211> 91 <212> PRT <213> Artificial sequence <220> <223> Truncated NS3c (DENV3 strain D3 / H / IMTSSA-SRI / 2000 / 1266, 367-457 aa) <400> 6 Lys Ala Gly Asn Asp Ile Ala Asn Cys Leu Arg Lys Asn Gly Lys Lys 5 10 15 Val Ile Gln Leu Ser Arg Lys Thr Phe Asp Thr Glu Tyr Gln Lys Thr 20 25 30 Lys Leu Asn Asp Trp Asp Phe Val Val Thr Thr Asp Ile Ser Glu Met 35 40 45 Gly Ala Asn Phe Lys Ala Asp Arg Val Ile Asp Pro Arg Arg Cys Leu 50 55 60 Lys Pro Val Ile Leu Thr Asp Gly Pro Glu Arg Val Ile Leu Ala Gly 65 70 75 80 Pro Met Pro Val Thr Ala Ala Ser Ala Ala Gln 85 90

Claims

1. A dengue subunit vaccine composition, characterized in that: The dengue subunit vaccine composition comprises: A recombinant protein, which is a truncated NS1ΔC polypeptide as shown in SEQ ID NO: 5 and a truncated NS3c polypeptide as shown in SEQ ID NO: 6, and optionally contains one or more pharmaceutically acceptable carriers and / or adjuvants, wherein the sequence from the N-terminus to the C-terminus of the recombinant protein is truncated NS1ΔC-truncated NS3c.

2. The dengue subunit vaccine composition according to claim 1, characterized in that It also comprises a cEDIII polypeptide having a sequence as shown in SEQ ID NO: 1 or 4.

3. The dengue subunit vaccine composition according to claim 2, characterized in that The cEDIII polypeptide of the recombinant protein is recombinantly linked between the truncated NS1ΔC polypeptide and the truncated NS3c polypeptide.

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