Monoclonal antibody, expression vector, use, pharmaceutical composition, production process, method for preventing and / or treating flavivirus infections and kit for detecting zika and dengue viruses

Abstract

The present invention pertains to the field of biotechnology, medicine and pharmaceutics. More particularly, the invention discloses an antibody with a surprising neutralising effect against ZIKV and different serotypes of DENV. The present invention further discloses use thereof, a pharmaceutical composition comprising same, a kit, and a method for preventing and / or treating infections caused by the dengue and Zika viruses.

Description

Descriptive Report of Invention Patent MONOCLONAL ANTIBODY, EXPRESSION VECTOR, USE, PHARMACEUTICAL COMPOSITION, PROCESS FOR PRODUCTION, METHOD FOR PREVENTION AND / OR TREATMENT OF FLAVIVIRUS INFECTIONS AND KIT FOR DETECTING ZIKA AND DENGUE VIRUSES Field of the Invention

[0001] The present invention is in the field of Biotechnology, Medicine, and Pharmacy. More specifically, the invention features an antibody with surprising neutralizing effect against ZIKV and different DENV serotypes. Additionally, its use, a pharmaceutical composition comprising the same, a kit, and a method for preventing and / or treating infections caused by dengue and Zika viruses are disclosed. Background of the Invention

[0002] Dengue (DENV) and Zika (ZIKV) viruses, members of the Flaviviridae family, are transmitted primarily by female mosquitoes of the Aedes genus. Infections caused by these agents are extremely important for public health, being endemic in 141 countries. Although most signs and symptoms of the disease are mild and self-limiting, DENV and ZIKV infections are known to lead to serious complications. The Pan American Health Organization reports that the four DENV serotypes cause approximately 500,000 hospitalizations per year due to hemorrhagic syndrome.

[0003] In Brazil, the Dengvaxia® vaccine, which contains a live attenuated virus, was long used to prevent dengue fever. However, this vaccine has disadvantages, such as being contraindicated in seronegative individuals (i.e., those who have never had dengue fever) and immunocompromised individuals. Recently, as an alternative to Dengvaxia®, ANVISA approved the Qdenga® vaccine, from Takeda. This vaccine differs from Dengvaxia® in that can be administered to people who have never come into contact with the dengue virus, but it still has limitations, such as contraindication to immunocompromised patients, since it also contains attenuated live viruses.

[0004] For individuals who develop the viral disease caused by dengue and Zika, there is currently no specific treatment. Existing treatments are primarily aimed at relieving symptoms and carefully monitoring the patient's condition. In mild cases, analgesics, hydration, and adequate rest are recommended. In more severe cases of dengue, such as severe dengue or dengue shock syndrome, intensive care unit support may be necessary, including intravenous fluids, blood transfusions, and continuous monitoring of vital signs. For complications caused by Zika, such as Guillain-Barré syndrome and Congenital Zika syndrome, individualized treatment tailored to these conditions is required. However, these approaches have significant limitations, as the focus is on symptom relief and patient care.

[0005] Given the limitations outlined, there is a need to develop specific prophylactic and therapeutic measures against the dengue and Zika viruses and, thus, prevent the worsening of infections caused by these agents.

[0006] In the search for the state of the art in scientific and patent literature, the following documents were found that deal with the topic:

[0007] Document BR 1120200219282 discloses non-human primate monoclonal antibodies that bind to the DI-DIII binding domain of the Zika virus, as well as a purified inactivated ZIKV vaccine for the immunization of human individuals at risk of Zika infection and who have previously been infected with DENV. This document does not disclose the antibody of the present invention.

[0008] Document BR 11 2019 024643 6 discloses an antibody and its applications in the treatment of dengue virus (DENV) infection. The antibody of BR'643 recognizes non-structural proteins of DENV. Thus, the present patent application differs from the aforementioned document by providing a different antibody targeting a different epitope, in addition to enabling protection against dengue and Zika viruses.

[0009] The present invention is a prophylactic and therapeutic alternative against the dengue and zika viruses.

[0010] Thus, from what can be inferred from the literature researched, no documents were found anticipating or suggesting the teachings of the present invention, so that the solution proposed here has novelty and inventive step compared to the state of the art. Summary of the Invention

[0011] The present invention addresses the problems of the prior art by using a monoclonal antibody comprising the heavy chain sequence defined by SEQ ID NO: 1 and the light chain sequence defined by SEQ ID NO: 2, and compositions comprising the same. This antibody exhibits a high neutralization rate against ZIKV and cross-inhibition against several DENV serotypes. Furthermore, in experiments with immunocompromised mice infected with ZIKV and DENV, administration of the monoclonal antibody according to the present invention demonstrated a protective effect, both as a prophylactic and therapeutic measure. These technical effects are significant, unexpected, and also of surprising magnitude.

[0012] Advantageously, the antibody of the present invention can be used in cases where there is a need for rapid treatment, where individuals are unable to synthesize antibodies because they are immunosuppressed or compromised, and in cases where they are not immune or where there is a likelihood of more serious complications.

[0013] In a first object, the present invention presents a monoclonal antibody comprising complementarity-determining regions that recognize the EDI / II domain of the flavivirus envelope (E). The flavivirus is dengue virus or zika virus, preferably ZIKV, DENV1, DENV2 and / or DENV3.

[0014] In a second object, the present invention presents an expression vector for production of the monoclonal antibody, as defined herein, comprising a nucleotide sequence selected from SEQ ID NO: 8 and SEQ ID NO: 9 and one or more expression promoters functionally linked to said nucleotide sequence.

[0015] In a third object, the present invention presents an expression vector for production of the monoclonal antibody, as defined herein, comprising a nucleotide sequence selected from SEQ ID NO: 10 and SEQ ID NO: 11 and one or more expression promoters functionally linked to said nucleotide sequence, in which: R is A or G; Y is C or T; H is A, C or T; N is A, C, G or T; T is thymine; and Optionally one or more AGY codons are replaced by TCN, one or more TTR are replaced by CTN and one or more CGN are replaced by AGR, reciprocally.

[0016] In a fourth object, the present invention presents the use of the monoclonal antibody, as defined above, to prepare a medicament for the prevention and / or treatment of flavivirus infections, in which, preferably, the flavivirus is selected from the group consisting of ZIKV, DENV1, DENV2 and / or DENV3.

[0017] In a fifth object, the present invention presents a pharmaceutical composition comprising a monoclonal antibody, as defined above, and at least one pharmaceutically acceptable excipient.

[0018] In a sixth object, the present invention presents a process for producing a composition comprising a step of mixing the monoclonal antibody, as defined herein, with at least one excipient pharmaceutically acceptable.

[0019] In a seventh object, the present invention presents a method of preventing and / or treating flavivirus infections comprising the administration of a monoclonal antibody or a pharmaceutical composition, as defined herein.

[0020] In an eighth object, the present invention presents a kit for detecting Zika and dengue viruses comprising the antibody, as defined above, reaction medium and instructions for use.

[0021] In a ninth object, the present invention presents the use of the monoclonal antibody, as defined herein, to prepare a kit for the detection of zika and dengue viruses.

[0022] These and other objects of the invention will be readily appreciated by those skilled in the art and will be described in detail below. Brief Description of the Figures

[0023] The following figures are presented:

[0024] Figures 1A and 1B refer to a schematic demonstration of the plasmids used for the in vitro production of the monoclonal antibody of the present invention. More specifically, they are drawings of the expression vectors (IgG1) containing: 1) the sequence of the constant region of the immunoglobulins and 2) fragments of the variable region inserted into the vectors between the restriction enzyme sites AgeI and SalI for heavy chain (Figure 1A) and AgeI and BsiWI for light chain (Figure 1B).

[0025] Figures 2A, 2B, 2C, and 2D show the binding capacity of the antibody comprising SEQ ID NO: 1 and SEQ ID NO: 2 to ZIKV proteins. More specifically, it shows the analysis of antibody binding to different portions of the ZIKV E protein in native (Figure 2A) or denatured (Figure 2B) form by ELISA. Furthermore, western blotting analysis involves a gel electrophoresis step in the reduced form (Figure 2C) or native gel (without reducing agent or heating, Figure 2D).

[0026] Figure 3AB shows the neutralizing capacity of the antibody comprising SEQ ID NO: 1 and SEQ ID NO: 2 against ZIKV and the four DENV serotypes. (item A) Graphs showing the neutralization percentage curves for the different viruses. (item B) Images of PRNT plates illustrating the foci formed by the ZIKV, DENV1, DENV2, DENV3, and DENV4 viruses.

[0027] Figure 4 shows the protocol of infection with ZIKV and treatment with monoclonal antibody comprising SEQ ID NO: 1 and SEQ ID NO: 2.

[0028] Figures 5AB, 5C, and 5D refer to the administration of the monoclonal antibody comprising SEQ ID NO: 1 and SEQ ID NO: 2 as a prophylactic and therapeutic measure against ZIKV. (Figure 5AB item A) weight change; (Figure 5AB item B) survival rate; (Figure 5C) viral load; (Figure 5D) degree of morbidity according to the levels of signs and symptoms of the disease: ruffled fur, curved spine, paralysis, and moribund. Survival data were analyzed by the Log-Rank (Mantel-Cox) test. The data refer to two independent experiments with 5-6 animals per group, *** p < 0.0003 and **** p < 0.0001. Statistical method: Two-way ANOVA.

[0029] Figure 6 shows the protocol for infection with DENV2 and treatment with monoclonal antibody comprising SEQ ID NO: 1 and SEQ ID NO: 2.

[0030] Figures 7AB, 7C and 7D refer to the efficacy of administering the monoclonal antibody comprising SEQ ID NO: 1 and SEQ ID NO: 2 as a prophylactic and therapeutic measure against DENV2 NGC. (Figure 7AB item A) weight variation; (Figure 7AB item B) survival rate; (Figure 7C) viral load; (Figure 7D) degree of morbidity according to the levels of signs and symptoms of the disease: ruffled fur, curved spine, paralysis and moribund. Survival data were analyzed by the Log-Rank (Mantel-Cox) test. The data refer to two independent experiments with 5-6 animals per group, *** p < 0.0003 and **** p < 0.0001. Statistical method: Two-way ANOVA. Detailed Description of the Invention

[0031] The present invention is based on a genetically engineered monoclonal antibody against ZIKV or DENV. First, a serum sample was investigated for the presence of antibodies specific to the viral envelope (E) protein and their in vitro neutralizing activity. Subsequently, mononuclear cells were obtained from a peripheral blood sample and used for selection and isolation of unique ZIKV-specific memory B lymphocytes. The isolated B lymphocytes were used for RNA extraction, cDNA generation, and amplification of the immunoglobulin variable regions. The fragments obtained from the PCR reaction were sequenced and analyzed for the amino acid composition of the variable portion, identification of the CDR3 region, and V(D)J segments to verify the specific type of each chain.IGHV3-23*01-like heavy chain fragments (SEQ ID NO: 1) comprising the complementarity-determining regions (CDR1; CDR2; CDR3) consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and IGKV1D-39*01-like light chain fragments (SEQ ID NO: 2) comprising the complementarity-determining regions (CDR1; CDR2; CDR3) consisting of SEQ ID NO: 6, AAS sequence and SEQ ID NO: 7 were identified. Subsequently, these fragments were cloned into expression vectors (IgG1) containing the constant region sequences corresponding to each chain (Figures 1A and 1B). These two plasmids (heavy and light chain) were used to transfect Expi293 cells for production of the antibody of the present invention in vitro. Finally, purification was performed using a column containing protein G beads.

[0032] This monoclonal antibody (also called mAb A9Z) exhibits binding activity to conformational epitopes found in the EDI / II domain region of the ZIKV E protein. Furthermore, this antibody surprisingly neutralized ZIKV and DENV serotypes 1, 2, and 3 in viral inhibition assays and protected immunocompromised mice against ZIKV or DENV challenge. More specifically, the antibody The present invention was able to generate high protection in treatment protocols 1 day before infection and 1 day after infection, in addition to increasing the survival of mice treated with it during the peak of viremia when compared to animals that did not receive treatment.

[0033] In a first object, the present invention presents a monoclonal antibody comprising complementarity determining regions that recognize the EDI / II domain of the envelope (E) of flaviviruses selected from ZIKV, DENV1, DENV2 and / or DENV3.

[0034] In one embodiment, the monoclonal antibody against ZIKV, DENV1, DENV2, or DENV3 comprises complementarity determining regions (CDR1; CDR2; CDR3) consisting of SEQ ID NO: 6, AAS sequence, and SEQ ID NO: 7 in the light chain and SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 in the heavy chain.

[0035] In one embodiment, said monoclonal antibody comprises a light chain comprising SEQ ID NO: 2 and a heavy chain comprising SEQ ID NO: 1.

[0036] In one embodiment, said monoclonal antibody comprises human γ chain constant regions.

[0037] In one embodiment, said monoclonal antibody is an IgG antibody.

[0038] In a second object, the present invention presents an expression vector for production of the monoclonal antibody, as previously defined, comprising a nucleotide sequence selected between SEQ ID NO: 8 and SEQ ID NO: 9 and one or more expression promoters functionally linked to said nucleotide sequence.

[0039] In a third object, the present invention presents an expression vector for production of the monoclonal antibody, as previously defined, comprising a nucleotide sequence selected between SEQ ID NO: 10 and SEQ ID NO: 11 and one or more expression promoters functionally linked to said nucleotide sequence, in which: R is A or G; Y is C or T; H is A, C, or T; N is A, C, G, or T; T is thymine; and Optionally, one or more AGY codons are replaced by TCN, one or more TTRs are replaced by CTN, and one or more CGNs are replaced by AGR, reciprocally.

[0040] The genetic code is degenerate, so more than one type of codon can specify the same amino acid in the polypeptide chain. Therefore, a serine can be specified by AGY (which includes AGC and AGT) or TCN (which includes TCA, TCC, TCG, and TCT); a leucine can be specified by TTR (which includes TTA and TTG) or CTN (which includes CTA, CTC, CTG, and CTT); an arginine can be specified by CGN (which includes CGA, CGC, CGG, and CGT) or AGR (which includes AGA and AGG).

[0041] In a fourth object, the present invention provides the use of the monoclonal antibody, as defined above, to prepare a medicament for the prevention and / or treatment of flavivirus infections. More preferably, the flavivirus is Zika virus and / or dengue virus. Even more preferably, the flavivirus is ZIKV, DENV1, DENV2, and / or DENV3.

[0042] In a fifth object, the present invention presents a pharmaceutical composition comprising a monoclonal antibody, as defined above, and at least one pharmaceutically acceptable excipient.

[0043] Pharmaceutically acceptable excipients are any compounds in a composition other than the active ingredient or compound. They aid in the adequate delivery of the ingredient and in enhancing its effect to achieve the desired purpose. Non-limiting examples that may be used in the context of this patent application include: vehicles, such as water, PBS; stabilizers, such as Sodium Citrate Dihydrate, Sodium Chloride, Mannitol (E421), and Polysorbate 80; pH adjusters, such as Sodium Hydroxide and Hydrochloric Acid, among others.

[0044] The medicine may be presented in a form for intramuscular, intravenous and subcutaneous administration.

[0045] In a sixth object, the present invention presents a process for producing a composition comprising a step of mixing the monoclonal antibody, as defined herein, with at least one pharmaceutically acceptable excipient.

[0046] In a seventh object, the present invention provides a method for preventing and / or treating flavivirus infections comprising administering a monoclonal antibody or a pharmaceutical composition, as defined herein. More preferably, the flavivirus refers to Zika virus and / or dengue virus. Even more preferably, the flavivirus is ZIKV, DENV1, DENV2, and / or DENV3.

[0047] Furthermore, the antibody of the present invention can be administered with other drugs to prevent the occurrence of possible side effects of monoclonal antibodies, such as allergic reactions.

[0048] In an eighth object, the present invention provides a kit for detecting Zika and dengue viruses comprising the antibody, as defined above, reaction medium and instructions for use. Preferably, the Zika and dengue viruses detected are ZIKV, DENV1, DENV2 and / or DENV3.

[0049] This kit can be used to detect Zika virus (ZIKV) and dengue virus (DENV) markers present in various matrices, including serum, plasma, cell culture supernatant, or lysate. In one embodiment, the assay uses a monoclonal antibody (primary) against epitopes present on the viral surface and a second antibody (secondary mAb) as a detection ligand conjugated to a fluorophore. The primary mAb, A9Z, of the present invention, is bound to the 96-well plate and serves as a capture antibody, i.e., its function is to bind and remove the virus from the aforementioned matrices. The secondary mAb is used to complete the sandwich effect and provide a signal indicating the presence of the target.

[0050] Additionally, in one embodiment, the kit comprises components such as: test sample diluent solution; assay buffer; wash buffer, chromogen and reaction stop solution.

[0051] In a ninth object, the present invention presents the use of the monoclonal antibody, as previously defined, to prepare a kit for the detection of zika and dengue viruses.

[0052] Surprisingly, administration of the monoclonal antibody described here 1 day before or 1 day after ZIKV or DENV infection resulted in 100% protection in infected immunocompromised animals. Thus, mAb A9Z could be used in the preparation of a drug for prophylaxis or therapy against ZIKV and / or DENV infection, among other applications. Example

[0053] The examples shown here are intended only to illustrate one of the numerous ways of carrying out the invention, without limiting its scope. Example 1 – Determination of the recognized epitope

[0054] To determine the identity of the epitope recognized by the monoclonal antibody of the present invention, binding analyses were performed by ELISA and western blot.

[0055] The viral envelope (E) protein consists of three main sections: domain I, domain II, and domain III. The E protein has highly conserved regions, particularly in the fusion loop (LF) region, a short amino acid sequence located in domain I / II of the protein. This region is clinically relevant among flaviviruses, including ZIKV and DENV. The LF is targeted by cross-reactive antibodies, which may be involved in the process of antibody-dependent enhancement of infection (ADE), especially in areas where viruses co-circulate. This poses a challenge for the development of vaccines and antivirals.

[0056] As a result, it was decided to verify whether the mAb A9Z of the present invention recognizes this highly conserved sequence using a sample of E-mutated protein containing 4 point mutations located in the LF. The protein sequence, as well as point mutations, can be found in the scientific work published by researcher Alexandra Rockstroh's group (Berneck, 2020; Rockstroh, 2015; Rockstroh, 2017; Rockstroh, 2019).

[0057] First, plates were sensitized with 1 µg / mL of wild-type and mutated ZIKV E protein and equimolar amounts of the EDI / II domain (0.8 µg / mL) and the EDIII domain (0.3 µg / mL), in native or denatured form, at 96 °C for 10 minutes and incubated for 16 to 18 hours at room temperature. After washing the plates, different concentrations of the antibody of the present invention comprising SEQ ID NO: 1 and SEQ ID NO: 2 (dilution factor 3x) were added with an initial concentration of 5 µg / mL. Then, the secondary antibody human α-IgG (1:15,000) conjugated with HRP (horseradish peroxidase) was added.

[0058] The results in Figures 2A and 2B show the OD490nm values ​​obtained for ZIKV E proteins in native form (Figure 2A) and for ZIKV E proteins in denatured form (Figure 2B). In Figure 2A, it can be observed that the tested antibody was able to bind to all native protein samples, except for the EDIII domain.

[0059] To confirm the data obtained by ELISA, Western blot analysis was performed. Thus, approximately 1 µg of each of the four proteins was analyzed by gel electrophoresis in the reduced form (Figure 2C) or native gel without reducing agent or heating (Figure 2D), followed by staining with Comassie Blue. The same amount of the four ZIKV proteins was used for Western blotting analysis. Subsequently, the proteins were transferred to nitrocellulose membranes that were incubated with the antibody of the present invention comprising SEQ ID NO: 1 and SEQ ID NO: 2 (10 µg / mL), followed by labeling with human α-IgG-HRP (1:15,000).

[0060] Figure 2D confirms the result obtained previously, since there is a recognition signal in the bands corresponding to the wild-type E, mutated E, and EDI-II proteins. Therefore, the monoclonal antibody comprising SEQ ID NO: 1 and SEQ ID NO: 2 specifically recognize the ZIKV envelope protein (E) domain I / II region. Furthermore, despite recognizing the EDI / II region, it does not bind to LF, which reduces the risk of causing ADE. Example 2 - Cross-reactivity analysis with other viruses of the Flaviviridae family

[0061] To evaluate cross-recognition by the same monoclonal antibody as in example 1, different dilutions of the aforementioned monoclonal antibody (initial concentration of 100µg / mL with a dilution factor of 3x) were incubated for 30 minutes with 100 plaque-forming units (PFU) of the following viruses: (i) ZIKV, (ii) DENV1 (dengue virus serotype 1), (iii) DENV2 (dengue virus serotype 2), (iv) DENV3 (dengue virus serotype 3) and (v) DENV4 (dengue virus serotype 4). In addition, serum from infected mice and mAb 4G2 were used as a positive control. mAb 4G2 is a murine monoclonal antibody that binds to conserved epitopes located in the Fusion Loop of the E protein of flaviviruses, such as Dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV) and Zika virus (ZIKV) (Aubry et al. 2016). The hybridoma used for the production of mAb 4G2 was kindly provided by the research group of the Vaccine Development Laboratory, ICB-USP.The mAb 4G2 hybridoma is commercially supplied and information about the antibody can be found in different suppliers and published scientific works (Aubry et al. 2016; Summers, 1989; Sithiprasasna 1994).

[0062] The mixture was then added to VERO cells in the presence of MEM medium with 2% fetal bovine serum (FBS) and 1% carboxymethyl cellulose (CMC) for 24 hours at 37°C in 5% CO2. The cells were fixed with 4% formalin and stained with crystal violet to verify the formation of viral foci.

[0063] In Figure 3AB item A, it can be seen that the same monoclonal antibody from example 1 (identified as mAb A9Z) is capable of completely neutralizing the ZIKV virus, in addition to DENV serotypes 1, 2 and 3. This result is also observed in Figure 3AB item B, in which PRNT plates that received antibody doses between 1.2 and 100 µg / mL present no or few foci of ZIKV, DENV1, DENV2 and DENV3 when compared with plates incubated with DENV4. Thus, it can be concluded from the results shown so far that the monoclonal antibody comprising SEQ ID NO: 1 and SEQ ID NO: 2 cross-recognizes the envelope (E) proteins of DENV serotypes 1, 2 and 3. Example 3 - Evaluation of the ZIKV infection protocol and treatment with the monoclonal antibody

[0064] To evaluate the infection protocol and treatment with the same monoclonal antibody as in example 1, in vivo experiments were performed with immunocompromised mice of the AG129 lineage, as shown in Figure 4.

[0065] First, these mice were infected with 10 2PFU / mL of ZIKV in the dorsum of the hind paw (50 µL). Furthermore, the animals were treated with 125µg (200µL) of the antibody of the present invention comprising SEQ ID NO: 1 and SEQ ID NO: 2 or the unrelated control at three different times: 1 day before infection (1 dai), 1 day post-infection (1 dpi) or 3 days post-infection (3 dpi). Then, the evaluation of the efficacy of the treatment was performed by monitoring the animals for 30 days, verifying the signs of morbidity through weight variation and survival rate.

[0066] Survival was significantly higher in mice treated with antibody comprising SEQ ID NO: 1 and SEQ ID NO: 2 at times: 1 dai (p < 0.0001); 1 dpi (p < 0.0003) and 3 dpi (p < 0.0022) compared with animals infected only with ZIKV, as shown in Figure 5AB item B and Figure 5D.

[0067] In addition to weight, survival rate, and morbidity levels, viral load was also evaluated throughout treatment with the monoclonal antibody of the present invention. Thus, sera from infected animals were collected at three time points: 3, 7, and 15 days after infection with 10 2 UFP of ZIKV-Br and evaluated for the presence of active viruses by the PRNT technique. To For this, different dilutions of sera (1:10; 1:100 and 1:1,000) were incubated on the VERO cell monolayer for 30 minutes and then replaced with MEM medium containing 2% FBS and 1% CMC. The plates were incubated for 5 days at 37 °C with 5% CO2. Then, the plates were fixed with 4% formalin, developed with crystal violet dye and viremia was defined by the number of foci formed in each well.

[0068] Figure 5C shows that all treatment modalities with the monoclonal antibody of the present invention were able to significantly reduce the presence of active virus. It is therefore concluded that the administration of the monoclonal antibody comprising SEQ ID NO: 1 and SEQ ID NO: 2 as a therapeutic and prophylactic measure leads to the protection of immunocompromised mice infected with ZIKV. Example 4 – Evaluation of the DENV infection protocol and treatment with the monoclonal antibody

[0069] To evaluate the DENV infection protocol and treatment with the monoclonal antibody of the present invention, in vivo experiments were performed with immunocompromised mice of the AG129 lineage, as shown in Figure 6.

[0070] mice were infected with 10 4PFU / mL of DENV2 NGC administered intraperitoneally (200 µL). Additionally, these animals were treated with 125 µg (200 µL) of the same monoclonal antibody as in example 1 or the unrelated control at three different times: 1 day before infection (1 dai), 1 day post-infection (1 dpi), or 3 days post-infection (3 dpi). Treatment efficacy was evaluated by monitoring the animals for 30 days, verifying weight variation, survival rate, and signs and symptoms of the disease, such as ruffled fur, curved spine, paralysis, and moribundity.

[0071] Additionally, sera from infected animals were collected at three times: 3, 7 and 15 days after infection with 10 4 UFP of DENV2 NGC and evaluated for the presence of active viruses by the PRNT technique. For this, different dilutions of sera (1:10; 1:100 and 1:1,000) were incubated in the monolayer of VERO cells for 30 minutes and then replaced with MEM medium containing 2% FBS and 1% CMC. The plates were incubated for 5 days at 37 °C with 5% CO2. The plates were fixed with 4% formalin, developed with crystal violet dye, and the rate of active virus was defined by the number of foci formed in each well.

[0072] From the results in Figures 7AB item A and Figure 7C, it is observed that the mice treated with the monoclonal antibody comprising SEQ ID NO: 1 and SEQ ID NO: 2 maintained the weight of the uninfected control mice and showed viremia control. Furthermore, survival was significantly higher in the mice treated with the monoclonal antibody of the present invention at the following times: 1 dai (p <0.0001); 1 dpi (p <0.0003) and 3 dpi (p <0.0022) compared to animals infected only with DENV2 NGC. Taken together, these results demonstrate that the monoclonal antibody comprising SEQ ID NO: 1 and SEQ ID NO: 2 is also capable of preventing infection and treating immunocompromised mice infected with DENV.

[0073] References

[0074] Berneck, Beatrice at al. 2020. “A Recombinant Zika Virus Envelope Protein with Mutations in the Conserved Fusion Loop Leads to Reduced Antibody Cross-Reactivity upon Vaccination”. Vaccines. .mdpi.com / 2076- 393X / 8 / 4 / 603#B24-vaccines-08-00603;

[0075] Rockstroh, Alexandra et al.2015. “Recombinant Envelope-Proteins with Mutations in the Conserved Fusion Loop Allow Specific Serological Diagnosis of Dengue-Infections.” PLoS Neglected Tropical Diseases 9(11): 1–12. http: / / dx.doi.org / 10.1371 / journal.pntd.0004218

[0076] Rockstroh, Alexandra et al.2017. “Specific Detection of Dengue and Zika Virus Antibodies Using Envelope Proteins with Mutations in the Conserved Fusion Loop.” Emerging microbes & infections 6(11): e99.

[0077] Rockstroh, Alexandra et al., 2019. “Dengue Virus IgM Serotyping by ELISA with Recombinant Mutant Envelope Proteins.” Emerging Infectious Diseases 25(1): 112–15;

[0078] Sithiprasasna, R., D. Strickman, B. L. Innis, and K. J. Linthicum. 1994. “ELISA for Detecting Dengue and Japanese Encephalitis Viral Antigen in Mosquitoes.” Annals of Tropical Medicine and Parasitology 88(4): 397–404.

[0079] Those skilled in the art will appreciate the knowledge presented herein and will be able to reproduce the invention in the embodiments presented and in other variants and alternatives, covered by the scope of the following claims.

Claims

1 / 2 Claims 1. Monoclonal antibody characterized by comprising complementarity determining regions that recognize the EDI / II domain of the envelope (E) of flaviviruses selected from ZIKV, DENV1, DENV2 and / or DENV3.

2. Monoclonal antibody against ZIKV, DENV1, DENV2 or DENV3, according to claim 1, characterized by comprising the complementarity determining regions (CDR1; CDR2; CDR3) consisting of SEQ ID NO: 6, AAS sequence and SEQ ID NO: 7 in the light chain and SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 in the heavy chain.

3. Monoclonal antibody according to claims 1 and 2, characterized by comprising a light chain comprising SEQ ID NO: 2 and a heavy chain comprising SEQ ID NO:

1.

4. Monoclonal antibody according to claims 1 to 3, characterized by comprising constant regions of the human y chain.

5. Monoclonal antibody, according to claims 1 to 4, characterized in that it is an IgG antibody. 6.Expression vector for production of the monoclonal antibody, as defined in any one of claims 1 to 5, characterized in that it comprises a nucleotide sequence selected from SEQ ID NO: 8 and SEQ ID NO: 9 and one or more expression promoters functionally linked to said nucleotide sequence.

7. Expression vector for production of the monoclonal antibody, as defined in any one of claims 1 to 5, characterized by comprising a nucleotide sequence selected between SEQ ID NO: 10 and SEQ ID NO: 11 and one or more expression promoters functionally linked to said nucleotide sequence, where: R is A or G; Y is C or T;. 2 / 2 H is A, C or T; N is A, C, G or T; T is thymine; and Optionally one or more AGY codons are replaced by TCN, one or more TTR are replaced by CTN and one or more CGN are replaced by AGR, reciprocally.

8. Use of the monoclonal antibody as defined in any one of claims 1 to 5, characterized in that it is to prepare a medicament for the prevention and / or treatment of flavivirus infections, in which, preferably, the flavivirus is selected from the group consisting of ZIKV, DENV1, DENV2 and / or DENV3.

9. Pharmaceutical composition characterized by comprising a monoclonal antibody, as defined in any one of claims 1 to 5, and at least one pharmaceutically acceptable excipient.

10. Process for producing a composition characterized by comprising a step of mixing the monoclonal antibody, as defined in any one of claims 1 to 5, with at least one pharmaceutically acceptable excipient. 11.Method of preventing and / or treating flavivirus infections characterized by comprising the administration of a monoclonal antibody, as defined in any one of claims 1 to 5, or of a pharmaceutical composition, as defined in claim 9.

12. Kit for detecting zika and dengue viruses characterized by comprising the antibody, as defined in any one of claims 1 to 5, reaction medium and instructions for use.

13. Use of the monoclonal antibody, as defined in any one of claims 1 to 5, characterized by being to prepare a kit for detecting zika and dengue viruses.

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