PROTEÍNAS RECOMBINANTES E VACINA CONTRA OS HERPESVÍRUS BOVINOS 1 E 5

Abstract

This technology comprises vaccine immunogens against bovine herpesviruses 1 and 5, consisting of recombinant multiepitope proteins of the aforementioned viruses covalently associated with carbon nanotubes (CNTs), preferably multi-walled, their production process, as well as their use in immunization protocols, associated with pharmaceutically and pharmacologically acceptable excipients.

Description

1 / 26 “RECOMBINANT PROTEINS AND VACCINE AGAINST BOVINE HERPESVIRUSES 1 AND 5”

[001] This application is a certificate of addition to the patent application PI1101186-6, entitled “Recombinant proteins, polynucleotides and vaccines against bovine herpesvirus”. The technology consists of multiepitope chimeric proteins of bovine herpesviruses 1 and 5 covalently associated with carbon nanotubes (CNTs), immunogenic compositions against bovine herpesviruses 1 and 5, a process for producing these compositions, as well as their use in immunization protocols.

[002] Among the members of the Herpesviridae family, we can highlight two species of great veterinary importance: Bovine Herpesviruses 1 and 5 (Bovine herpesvirus 1 and Bovine herpesvirus 5 - BoHV-1 and BoHV-5, respectively), both members of the subfamily Alphaherpesvirinae, genus Varicellovirus (ROIZMAN et al., Archives of Virology, v.123, n.34, p.425-449, 1992).

[003] The Alphaherpesvirus genome encodes ten glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, gL, and gM), mainly related to the mechanisms of virion entry into cells and its release from them. The glycoproteins gB and gD are essential for the multiplication of bovine herpesviruses; others, although not essential, play important roles in the viral phenotype (KAASHOEK et al., Veterinary Microbiology, vol. 48 (1 / 2), p. 143-153, 1996). Because they are located in the viral envelope and on the surface of infected cells, these proteins play important roles in the interactions between viruses and their hosts (adsorption, penetration, and transmission between cells) as well as in viral pathogenicity. These proteins are also important in interactions with the host's immune system, binding to Petition 870260056791, dated 11 / 06 / 2026, page 9 / 35 2 / 26 components of the complement system or immunoglobulins (JONES et al., Clinical Microbiology Reviews, vol.16 (1), p.79-95, 2003).

[004] The first interaction between the virus and the cell involves the binding of gB and / or gC to cell surface structures, followed by the binding of gD to specific cellular receptors, such as nectin-1, a member of the immunoglobulin superfamily (GERAGHTY, Science, 280:1618-1620, 1998). After this high-affinity interaction between gD and cellular receptors, viral penetration occurs through the fusion of the virion envelope with the plasma membrane, a process that directly depends on the involvement of gB, gD, and the heterodimer formed by gH and gL. Once inside the cell, the viral particle is transported using a microtubule-associated dynein motor complex towards the nuclear pore, where the viral DNA is released and replicated in the nucleus.

[005] BoHV-1, subfamily Alphaherpesvirinae, genus Varicellovirus, is the etiological agent of some syndromes that affect the herd, such as Infectious Bovine Rhinotracheitis (IBR), Infectious Pustular Vulvovaginitis (IPV) and Infectious Pustular Balanoposthitis (IPB), in addition to cases of infertility, conjunctivitis, keratoconjunctivitis and reproductive disorders, characterized by early and / or late embryonic mortality, repeated estrus cycles, fetal mortality with abortion; stillbirths; neonatal mortality and infertility (KAHRS, J. Am. Vet. Med. Assoc. 171:1055-1064, 1977).

[006] BoHV-1 typically presents with high morbidity and low mortality. In the respiratory tract, infection leads to increased body temperature, mucous membrane hyperemia, rhinitis, dyspnea, serous nasal discharge, erosive lesions in the nasal mucosa, and occasionally pneumonia (TAKIUCHI et al., Seminar: Agricultural Sciences, Londrina, v. 22, n.2, p. 203-209, 2001). Complications caused by secondary bacterial infections or other superimposed viral infections may Petition 870260056791, dated 11 / 06 / 2026, page 10 / 35 3 / 26 leading to the animal's death, however the observed mortality rate is quite low (GIBBS & RWEYEMAMU, Vet. Bulletin 47:(5)317-343, 1977). Infectious pustular vulvovaginitis is characterized by the appearance of small vesicles of 1 to 2 mm in diameter that evolve into pustules and erosions in the vulva and vagina of females, also occurring edema and hyperemia of the vulvar epithelium, with secretion that may become mucopurulent due to secondary bacterial contamination (TAKIUCHI et al., Seminar: Agricultural Sciences, Londrina, v. 22, n.2, p. 203-209, 2001). In bulls, similar lesions are found on the prepuce and penis, characterizing balanoposthitis (GIBBS & RWEYEMANN, Vet. Bulletin 47:(5)317-343, 1977). Sporadically, BoHV-1 has been associated with cases of encephalitis, more commonly than previously believed, especially in the Central-South region of Brazil (SILVA et al., Pesquisa Veterinária Brasileira, 27(10):403-408, 2007; RISSI et al., J Vet Diag Invest, 20:346-349, 2008). BoHV-1 is the most commonly found viral pathogen in bovine semen, and its transmission has been greatly favored by the development of semen cryopreservation processes, which create ideal conditions for the preservation of BoHV-1 (GOMES et al., Vet Res Commun 27(6):495-504, 2003).

[007] BoHV-5 is the causative pathogen of non-suppurative meningoencephalitis, a pathology affecting the central nervous system and presenting a high mortality rate, mainly among young animals, with a case fatality rate close to 100% when dealing with animals under one year of age (SALVADOR et al., Pesquisa Veterinária Brasileira, v.18, p.75-82, 1998). In adult animals, BoHV-5 leads to either a subclinical infection or a moderately severe disease. Occasionally, BoHV-5 has been recovered from genital and respiratory infections (BRATANICH et al., J. Vet. Med. B 38:41-48, 1991). The neurological picture resulting from BoHV-5 infection includes several signs. Petition 870260056791, dated 11 / 06 / 2026, page 11 / 35 4 / 26 clinical symptoms, the most frequent being the presence of nasal and ocular discharge, muscle tremors, circling, incoordination, opisthotonus, nystagmus, bruxism, seizures and sometimes anorexia (CLAUS et al., Seminar: Agricultural Sciences, Londrina, v. 23, n. 1, p. 131-141, 2002).

[008] As highlighted by DEL MEDICO ZAJAC et al. (The Veterinary Journal 184. 138-145, 2010), there is a strong association between BoHV-1 and bovine respiratory disease complex (BRDC), causing losses exceeding one billion dollars annually worldwide, placing it at the forefront of research and resource allocation. Economic losses due to BoHV-5, however, are not well defined, and there is a possibility that this virus has been underdiagnosed in the past. The adoption of more specific diagnostic methods will provide a better understanding of the true distribution of BoHV-5 and allow for a real characterization of this virus. BoHV-5 outbreaks are sporadic and apparently restricted in their geographic distribution, being detected mainly in the Southern Hemisphere, a fact that remains unexplained.BoHV-5 infection has been described in a few herds abroad, in Europe, Oceania and North America, and has a high prevalence in Argentina and Brazil, being reported in several states (MG, ES, MT, PR, RS and SP).

[009] BoHV-1 and BoHV-5 infections can be transmitted through direct and indirect contact between animals, via respiratory, ocular, and genital secretions. Initially, infection of epithelial cells occurs at the entry point – nasal or vaginal mucosa, depending on the route of infection. Viral multiplication in the respiratory system leads to a high production of viral progeny that is released in nasal secretions and constitutes a very efficient source of transmission during acute infection. Herpesviruses are excreted in large quantities during the Petition 870260056791, dated 11 / 06 / 2026, page 12 / 35 5 / 26 acute infection, and in lower titers during reactivation episodes. The viruses may also be present in the semen of infected bulls, and can be spread by both natural mating and artificial insemination (GOMES et al., Vet Res Commun 27(6):495-504, 2003; MUYLKENS et al., Veterinary Research 38: 181-209, 2007).

[0010] Currently, cattle and semen contaminated with BoHV-1 and / or BoHV-5 are facing increasing limitations in international trade. The recommended approach for controlling bovine herpesviruses in cattle herds is highly dependent on the prevalence of viral infection. In herds with a low number of infected animals, euthanasia and serological monitoring of the herd are recommended. In areas with a high prevalence of infection by these viruses, such as Brazil, mass vaccination of herds is recommended (ACKERMANN & ENGELS, Veterinary Microbiology 113:293-302, 2006).

[0011] Controlling or even eradicating this disease is of great commercial importance, considering that Brazil has a large beef market. In 2012, more than 31 million cattle were slaughtered in Brazil, an 8% increase compared to the previous year. The total weight of slaughtered carcasses followed this number, reaching a new record of 7.351 million tons. According to data from the Secretariat of Foreign Trade (Secex), in 2012, Brazilian exports of fresh beef totaled 945,482 tons, with revenues of US$4.495 billion. In 2012, Brazil exported fresh beef to 92 countries, of which 16 accounted for 92.4% of imports, with China standing out, having experienced significant increases in imports of Brazilian fresh beef for four consecutive years.Milk production in the same period exceeded 22 million liters, and annual exports of raw milk registered a sharp drop in the quantity exported in 2012 compared to 2011, which... Petition 870260056791, dated 11 / 06 / 2026, page 13 / 35 6 / 26 was also accompanied by a drop in revenue (IBGE Indicators, Livestock Production Statistics, 2013).

[0012] Brazil's participation in international trade has been growing annually, with Brazil being the world's largest exporter of beef, and expectations from the Ministry of Agriculture indicate that by 2020 national meat production will be responsible for supplying approximately 44.5% of the world market (Brazilian Agribusiness Trade Balance, 2011).

[0013] Brazil's tropical climate and vast territory, which allow for the raising of a large portion of livestock on pasture, as well as investment in technology, professional training, and public policies for animal health control and food safety, have contributed to the country meeting the demands of rigorous markets and gaining a foothold on the world stage (Ministry of Agriculture, Livestock and Supply, 2013). However, raising animals in high-density confinement systems, rapid herd turnover, and genetic selection aimed at increasing productivity lead to an increase in health problems and hinder the control of infectious diseases, contributing to reduced production (BARBOSA-STANCIOLI, EF Revista Cultivar Bovinos, n. 1, 2003).

[0014] Due to the already highlighted importance of cattle farming to the Brazilian economy, any disease affecting cattle can interfere with production, leading to a decrease in the sector's economic gains, making it necessary to adopt control, prevention, and eradication programs. Special attention should be given to the causative agents of neuropathies and diseases that induce reproductive losses and abortions, among which we can highlight bovine herpesviruses. Petition 870260056791, dated 11 / 06 / 2026, page 14 / 35 7 / 26

[0015] Vaccines save thousands of human and animal lives annually, contributing to the control of various infectious diseases, both regionally and globally. The explosion of knowledge in diverse areas, such as microbiology, immunology, and biochemistry, among others, has made vaccinology one of the most dynamic areas in biomedical research (LEVINE et al., Lancet 378(9789):439-448, 2011). The development of new vaccines using modern technologies can aid in the prevention and treatment of various diseases, infectious or not, that currently lack available vaccines or whose vaccines have not yet achieved the desired protection.

[0016] The vaccines currently available on the market have aimed at reducing clinical symptoms, with a consequent decrease in economic losses caused by the disease; however, they are not capable of preventing reinfection by field samples (BARBOSASTANCIOLI, Cultivar Bovinos, n. 1, 2003). Furthermore, a recent study using six different commercial vaccines containing inactivated BoHV-1 antigens showed that only one of them was able to induce a high antibody titer against BoHV-1, while none of them were able to induce satisfactory antibody titers against BoHV-5 (SILVA et al., Ciência Rural, v.37, n.5, p.1471-1474, 2007). These results indicate the need for improvements in the immunogenicity of current vaccines, a review of the evaluation criteria, licensing and importation of vaccines, as well as the need to include BoHV-5 samples in existing vaccines (SILVA et al., Ciência Rural, v.37, n.5, p.1471-1474, 2007), which is already being done with some vaccines containing both viruses, already licensed by MAPA.

[0017] The vaccines available today use modified, attenuated, or even inactivated live viruses. Depending on their ability to induce immunogenicity, vaccines can effectively reduce the Petition 870260056791, dated 11 / 06 / 2026, page 15 / 35 8 / 26 clinical manifestations and, consequently, economic losses, however, are not able to completely protect against infection (PIDONE et al., Analecta Veterinaria 19,1 / 2: 40-50, 1999).

[0018] Understanding the factors involved in the immune response during BoHV-1 and BoHV-5 infection shows that viral glycoproteins are of great importance not only in inducing antibody production, but also in promoting the humoral response, and therefore become the main focus in the development of vaccines against these viruses. (BABIUK et al., Veterinary Microbiology 53, 31-42, 1996).

[0019] The technique of producing heterologous proteins, that is, proteins produced in in vitro systems or in organisms that do not naturally express the molecule, was developed in the 1970s and has since been widely used. This technology has simplified, reduced costs and increased the availability, safety and efficiency of the production of several molecules of medical and commercial interest, such as human insulin, interferons, human growth hormone, among others, and has enabled great progress in understanding the pathogenesis of infectious diseases of great importance, in addition to being used in industries as a way of obtaining more efficient recombinant enzymes and vaccines based on recombinant proteins, creating a multi-billion dollar market (RAI & PADH, Current Science, vol.80 (9), 2001; MAHMOUD, Res J Cell & Mol Biol, vol.1 (1) p.9-22, 2007).In academia, it has helped in understanding the structure and function of proteins, as it is a relatively simple and easily reproducible method, generally possessing a high yield of the desired product (MAHMOUD, J Cell & Mol Biol, vol.1 (1) p.9-22, 2007; YIN et al., Journal of Biotechnology, vol.127 p.335-347, 2007), and can be easily mastered and manipulated by the researcher, even for the expression of small fragments of the desired protein. Petition 870260056791, dated 11 / 06 / 2026, page 16 / 35 9 / 26

[0020] Carbon nanotubes (CNTs) exhibit notable structural properties: high stability, strength, and stiffness, combined with low density and elastic deformability (DRESSELHAUS, Carbon nanotubes: Synthesis, Properties and Applications (Spring Velag, Berlin), 2001). Their use as a potential material in biomedicine is due to their flexible structure, propensity for chemical functionalization, biocompatibility with organic systems, and ability to internalize into cells. Nanobiotechnology, the science that studies the interactions between nanocomposites and biological systems, has made significant advances in the use of CNTs as biosensors, biomarkers, vehicles for intracellular transport, and biological delivery of substances such as drugs, proteins, and nucleic acids (FOLDVARI & BAGONLURI, Nanomedicine v.4, n.3, p.183-200, 2008).

[0021] CNTs are cylindrical molecules exhibiting a hexagonal arrangement of carbon atoms with sp2 hybridization; they consist of rolled-up graphite sheets, forming small cylinders with a diameter of 1 to 30 nm. There are 2 forms of perfect CNTs: single-walled (SWNT), which have only a single carbon cylinder; and multi-walled (MWNT), which have from two to 50 concentric cylinders.

[0022] Solubilization is the first step in the use of carbon nanotubes, since, due to the fact that they are formed only by carbon atoms, they are insoluble in water. For this purpose, CNTs are oxidized by a mixture of nitric and sulfuric acids to covalently bind carboxyl and hydroxyl radicals to their walls, through the processes of carboxylation and hydroxylation (CHEN et al., Chem Phys Letters v. 402, p. 312-317, 2005).

[0023] Functionalization itself consists of the covalent bonding between the carboxylic radicals present on the surface of Petition 870260056791, dated 11 / 06 / 2026, p. 17 / 35 10 / 26 MWNT and the amino radicals of proteins are coupled through a diimide-activated amidation reaction (JIANG et al., J Mater Chem v.14, p.37-39, 2004). N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) is used as a coupling agent between the carboxyl radicals present on the surface of the NTC and the proteins. This process is stabilized by the stabilizing agent N-hydroxysuccinimide (NHS), which converts the carboxylic acids into active esters that then react with the amine groups present in the proteins, preventing direct coupling that could lead to undesirable intermolecular bonds between the proteins. At the end of this process, a unique and stable structure is generated, consisting of NTCs covalently linked to the proteins.

[0024] Regarding vaccine development, CNTs are considered excellent vectors due to their large surface area to volume ratio and the possibility of functionalization with peptides and proteins (BIANCO et al., Chem Commun (Camb) v.4, p.571-7, 2005). Studies using CNT technology functionalized with antigens have demonstrated a great potential for generating a protective immune response through the production of neutralizing antibodies (PANTAROTTO et al., Chem Biol v.10, n.10, p.961-6, 2003; YANDAR et al., Vaccine, v.26, n.46, p.5864-73, 2008). These studies have shown that CNTs have great potential for use as antigen-carrying vehicles for presentation to the immune system and subsequent immunization through the production of neutralizing antibodies.

[0025] Other studies have shown that functionalized CNTs are capable of being internalized by B lymphocytes, T lymphocytes, and macrophages without, however, affecting the viability, proliferation capacity in response to an antigenic stimulus, or the immunoregulatory characteristics of these cells. Even so, they were able to activate macrophages in vitro, Petition 870260056791, dated 11 / 06 / 2026, page 18 / 35 11 / 26 leading to the production of pro-inflammatory cytokines such as TNF-α and IL-6 (DUMORTIER et al., Nano Lett v.6, n.7, p.1522-8, 2006).

[0026] Some documents describe the use of nanoparticles associated with viral proteins as well as the vaccine use of recombinant proteins against bovine herpesvirus, however all differ from the present technology and are cited below.

[0027] Document US2004 / 0219655 refers to a method for producing and using nanoparticles optionally enclosed in a capsule comprising one or more viral proteins. However, this application does not refer to the use of carbon nanotubes for generating a vaccine.

[0028] Document WO2007 / 001355 deals with the conjugation of proteins to nanomaterials, such as carbon nanotubes, resulting in improvements in the stability and activity of these proteins, with profound impacts on their application in biosensors, diagnostics, vaccines, drug delivery, and biochips. However, this application does not refer to the use of carbon nanotubes coupled to recombinant BoHV-1 and BoHV-5 proteins for the generation of a vaccine against these viruses.

[0029] Document WO2009 / 117616 refers to compositions based on the ability of carbon nanotubes to activate the cellular immune response, with potential vaccine use. However, it does not characterize the specific use of recombinant BoHV-1 and BoHV-5 proteins for generating a vaccine against these viruses in formulations such as those described in the present technology.

[0030] Protocol document No. PI 1101186-6 called Recombinant proteins, polynucleotides, and vaccines against bovine herpesviruses refers to the production of recombinant proteins containing four antigenic protein epitopes from each of the two viruses (BoHV-1 and BoHV-5), produced in a prokaryotic system, with potential use in... Petition 870260056791, dated 11 / 06 / 2026, page 19 / 35 12 / 26 vaccine production. However, this request does not include the use of carbon nanotubes coupled to them for the generation of vaccines against these viruses.

[0031] Due to the already highlighted importance of cattle farming for the Brazilian economy and considering that bovine herpesvirus infections lead to a decrease in the sector's economic gains, it becomes necessary to adopt control and eradication programs for these infections. Thus, in the present technology, vaccine compositions were developed consisting of two vaccine immunogens against bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5), constituted by two recombinant proteins, each containing immunogenic epitopes of three proteins from these viruses, covalently associated with carbon nanotubes, as well as their use in immunization protocols. Brief Description of the Figures

[0032] Figure 1- SDS-PAGE of recombinant BoHV1 (a) and BoHV-5 (b) proteins purified on a nickel chelate column.

[0033] Figure 2- Functionalized recombinant proteins and their respective washes- (a) MWNT-RecBoHV-1; (b) MWNT-RecBoHV-5.

[0034] Figure 3 - Characterization of the nanotubes used, both by confocal microscopy (a) and by Raman spectroscopy (b).

[0035] Figure 4 - Raman spectra: of the multi-walled carbon nanotube (MWNT), of recombinant proteins, and of the carbon nanotube functionalized with them [MWNT-ReBoHV-1 (a) and MWNTRecBoHV-5 (b)].

[0036] Figure 5- Analysis of the cytotoxicity of recombinant proteins, functionalized or not to carbon nanotubes, through the evaluation of the viability of bovine PBMCs after 24 hours of stimulation with them, in an MTT assay. b vs a, c, d, ep<0.0001. Petition 870260056791, dated 11 / 06 / 2026, p. 20 / 35 13 / 26

[0037] Figure 6 - Indirect ELISA assay developed “in-house” shows the recognition of recombinant proteins, functionalized or not to carbon nanotubes, by IgG and IgM antibodies from cattle naturally infected with BoHV-1 and BoHV-5. (a) Indirect ELISA anti-bovine IgM; (b) Indirect ELISA anti-bovine IgG.

[0038] Figure 7- Lymphocyte activation profile (a) CD4 / CD25+ and (b) CD8 / CD25+ counts, evaluated by flow cytometry. Detailed Description of the Invention

[0039] The present technology consists of a vaccine immunogen against bovine herpesviruses 1 and 5, consisting of recombinant multiepitope proteins (RecBoHV-1 and RecBoHV-5) of the aforementioned viruses covalently associated with carbon nanotubes (CNTs), preferably multi-walled, its production process, as well as its use in immunization protocols, associated with pharmaceutically and pharmacologically acceptable excipients.

[0040] Excipients may be selected from the group comprising water, saline solution, aluminum hydroxide solutions, phosphate-buffered solutions, Ringer's solution, dextrose solution, Hank's solution, biocompatible saline solutions containing or not polyethylene glycol, non-aqueous vehicles such as fixed oils, sesame oil, ethyl oleate or triglycerides, isolated or in mixtures, including pharmaceutical nanoformulations. In addition, excipients may contain additives such as buffers, preservatives, binders, disintegrants, diluents, lubricants and / or surfactants.

[0041] The vaccine immunogen may be presented in solid, semi-solid or liquid forms. In addition, the vaccine immunogen may be administered orally, intramuscularly, intravenously, intraperitoneally, subcutaneously, transdermally or as devices that can be implanted or injected. Petition 870260056791, dated 11 / 06 / 2026, page 21 / 35 14 / 26

[0042] The recombinant proteins RecBoHV-1 and RecBoHV-5 were constructed from an evaluation of the most immunogenic proteins of these viruses and their possible regions recognized by B and T cells. After bibliographic research and in silico analyses, the gene cassettes were designed and their synthesis was commissioned (Entelechon, Germany). The synthetic genes were then cloned into the pQE 30 vector (Qiagen, Germany).

[0043] Subsequently, a transformation was carried out in Chemocompetent Escherichia coli M15 strain and selected clones were incubated to induce protein expression. The multiepitope recombinant proteins of BoHV-1 and BoHV-5 can also be produced in other bacterial vectors, viral vectors, and eukaryotes.

[0044] The purification of the protein extract was carried out on a nickel chelate column, under denaturing conditions, by affinity chromatography.

[0045] The purified proteins were then used for covalent functionalization to multi-walled carbon nanotubes (MWNTs) via diimide-activated amidation reaction. The functionalization product and its respective washes were analyzed on polyacrylamide denaturant gel (SDS-PAGE). The multi-walled carbon nanotubes (MWNTs) were produced in the Crystal Growth Laboratory (Department of Physics - ICEx / UFMG) and made available for this work by the Department of Microbiology ICB / UFMG. The MWNTs were produced by the Electric Arc Discharge (EAD) technique and carboxylated by the nitric acid reflux method (FOLDVARI & BAGONLURI, Nanomedicine v.4, n.3, p.173-82, 2008).

[0046] To characterize the functionalization process of nanotubes with recombinant proteins, the Raman spectroscopy technique was used. The Raman spectrum shows the shift of Petition 870260056791, dated 11 / 06 / 2026, page 22 / 35 15 / 26 peaks after the functionalization process, evidencing the presence of measurable interactions between the compounds, such as covalent bonds, which generate a highly stable product.

[0047] The cytotoxicity of carbon nanotubes functionalized with recombinant proteins (MWNT-RecBoHV-1 and MWNT-RecBoHV5) was evaluated in an MTT assay. Bovine peripheral blood polymorphonuclear cells (PBMCs) were collected and separated from the remaining blood components by a Ficoll gradient and stimulated with 1.0 pg or 10 pg of recombinant proteins, functionalized or not to carbon nanotubes, or RPMI medium. The viability of PBMCs after 24 hours of incubation was analyzed using the MTT colorimetric reagent, which allows the distinction of viable cells, and subsequent reading in a spectrophotometer.

[0048] Bovine sera from a herd naturally infected with BoHV-1 and BoHV-5 were analyzed for recognition of recombinant proteins, functionalized or not to carbon nanotubes, in an indirect ELISA assay developed “in house”, evaluating IgG and IgM immunoglobulins.

[0049] To evaluate the immunogenicity of nanotubes functionalized with recombinant proteins (MWNT-RecBoHV-1 and MWNT-RecBoHV-5), female C57Bl-6 mice were divided into 9 groups, containing 7 animals each, following a 3-dose immunization protocol (dose - booster 1 - booster 2). Each animal in group 1 received 40 pL of PBS; group 2 received 40 pL of a commercial inactivated vaccine for BoHV-1 and BoHV-5, containing alum as an adjuvant; group 3 received 40 pL of a solution containing 1.0 pg of non-functionalized MWNT; group 4 received 1.0 pg of each of the recombinant proteins (RecBoHV-1 and RecBoHV-5); group 5 received 1.0 pg of each of the recombinant proteins (RecBoHV-1 and Petition 870260056791, dated 11 / 06 / 2026, page 23 / 35 16 / 26 Group 5 received MWNT-RecBoHV-1 and MWNT-RecBoHV-5 in a 1:1 ratio, with 1.0 pg of functionalized protein and 1.0 pg of MWNT for each recombinant protein; Group 6 received MWNT-RecBoHV-1 and MWNT-RecBoHV-5 in a 1:1 ratio, with 1.0 pg of functionalized protein and 1.0 pg of MWNT for each recombinant protein; Group 7 received MWNT-RecBoHV-1 and MWNT-RecBoHV-5 in a 1:1 ratio, with 1.0 pg of functionalized protein and 1.0 pg of MWNT for each recombinant protein, associated with alum for each recombinant protein, at a final concentration of 30%; Group 8 received 1.0 pg of each recombinant protein and 1.0 pg of non-functionalized MWNT. Group 9 received 1.0 pg of each recombinant protein and 1.0 pg of MWNT, associated with alum adjuvant at a final concentration of 30%. Doses were administered at 21-day intervals, and 14 days after the last booster, the animals were sacrificed for blood and spleen collection. Splenocytes were collected, and lymphocytes were analyzed by flow cytometry for the presence of CD4+, CD8+, and CD25+ activation markers.

[0050] Only recombinant proteins functionalized to carbon nanotubes, applied at a concentration of 10 pg, promoted a reduction in the viability of PBMCs; however, even this reduction is less than the 20% acceptable to the pharmaceutical industry.

[0051] Recombinant proteins, whether or not associated with carbon nanotubes, were recognized by IgG and IgM antibodies from sera of cattle naturally infected with BoHV-1 and BoHV-5, showing that functionalization does not interfere with the recognition of recombinant proteins.

[0052] In vivo immunogenicity analyses of these tools showed that the frequency of activated CD4+ T lymphocytes was enhanced in animals that received recombinant proteins functionalized to carbon nanotubes, supplemented with alum. The same was observed for CD8+ T lymphocytes, where this group showed Petition 870260056791, dated 11 / 06 / 2026, page 24 / 35 17 / 26 statistically significant difference between the groups that received PBS and the inactivated commercial vaccine.

[0053] In view of this, it can be concluded that nanotubes functionalized with recombinant multiepitope proteins of BoHV-1 and BoHV-5 exhibit high stability, high immunogenic potential and high biocompatibility.

[0054] The present technology, which comprises carbon nanotubes covalently linked to recombinant multiepitope proteins of BoHV-1 and BoHV-5, its production process and its potential use as a vaccine against these viruses, can be better understood through the following examples, which are not limiting to the technology. Example 1 - Production of Recombinant BoHV-1 and BoHV-5 Proteins in a Prokaryotic Vector

[0055] The recombinant proteins RecBoHV-1 and RecBoHV-5 were produced using Escherichia coli M15 clones, each expressing the gene for one of the proteins, which were cloned into the pQE30 expression vector.

[0056] Initially, a 1 mL aliquot of each clone was placed for growth in 45 mL of 2x YT medium (1.6% w / v Bactotryptone, 1% w / v yeast extract, 0.5% 171 mM NaCl), supplemented with antibiotics in 500 mL Erlenmeyer flasks, for 16 h at 37°C and 300 RPM in an orbital shaker (Thermo Scientific, USA). The growth was centrifuged (Jouan, fixed rotor) for 20 minutes at 4000 RPM and 4°C. The supernatant was discarded and the precipitate resuspended in 400 mL of 2x YT medium, supplemented with antibiotics, and incubated at 37°C, 300 RPM, until the optical density OD 600 nm reached a value between 0.4-0.6. A 1.0 mL aliquot was withdrawn and stored as a non-induced control (NIC). To the remainder, 600 pL of 1M IPTG (isopropyl-beta-D-thiogalactopyranoside), resulting in a final concentration of 1.5 mM, were added and incubated for 5 h at 37°C and 300 RPM. Petition 870260056791, dated 11 / 06 / 2026, p. 25 / 35 18 / 26

[0057] After incubation, a 1 mL aliquot was taken as an induced control (IC) and the remainder centrifuged for 20 minutes at 8000 RPM and 4°C. The supernatant was again discarded and the precipitate proceeded to protein purification. Example 2 - Purification of Recombinant BoHV-1 and BoHV-5 Proteins

[0058] On ice, the precipitate was resuspended in 10 mL of lysis buffer (Guanidine 6M, Tris 0.01M, NaH2PO4 0.1M, pH 8) for 20 minutes using an automatic pipette. Then, lysis buffer was added until the volume reached 40 mL, and the solution was incubated in an orbital shaker (Thermo Scientific, USA) for 1 hour at 200 RPM and room temperature to complete lysis. The lysate was then centrifuged for 1 hour at 7150 g and 15°C, and the precipitate was discarded.

[0059] In a new tube, 1 mL of nickel chelate resin (Qiagen, USA) loaded with Ni2+, previously treated with lysis buffer for acclimation, was added, and the tube was incubated for 16 h at 10 RPM and 4°C. The proteins bind to the resin via the histidine tail present in the amino-terminal portion of the proteins. The solution was centrifuged for 3 minutes at 800 RPM and room temperature, and the supernatant was carefully discarded so as not to disturb the resin.

[0060] 10 mL of urea buffer (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris, pH 8) was added slowly to the precipitate so that the resin would not detach, and the tube was centrifuged again for 3 minutes at 800 RPM and room temperature, and the supernatant discarded. On ice, the resin was transferred to the chromatography column and washed with 10 mL of pH 8 urea buffer. Then, the column was washed 10 times with 1 mL of pH 4.5 urea buffer each time, and the eluates were collected. Petition 870260056791, dated 11 / 06 / 2026, page 26 / 35 19 / 26

[0061] To analyze the efficiency of purification under denaturing conditions, ^L of each eluate were applied to 12% SDS-PAGE gel and fractionated by electrophoresis, to determine the best eluate (Figure 1).

[0062] The quantification of the purified proteins was performed using Bradford's reagent, which contains the dye BG-250 that interacts with the proteins present in the sample, absorbing light at a wavelength of 595nm.

[0063] In 96-well plates, 40μL of Bradford reagent (BioRad, USA), 1 μL of sample or standard, and 158 μL of ddH2O were added per well. Light absorbance was measured using a microplate spectrophotometer (Asys Hitech GmbH) at 595 nm, and quantification was performed using a simple rule of three between the results obtained from the samples and the standard (BSA protein with a concentration of 1 mg / mL). Example 3 - Covalent Functionalization of Multi-Walled Carbon Nanotubes (MWNTs) to Recombinant BoHV-1 and BoHV-5 Proteins

[0064] Due to the presence of non-carboxylated portions on the surface of MWNTs, their solubility in water may be reduced. To minimize this effect, 10 mg of MWNTs were added to a glass tube containing 15 mL of deionized water and placed in an ultrasonic bath generator (Branson 1210) for 2 hours, stirring every 30 minutes, and for a further 20 minutes in a sonicator (Ultrasonic Converter, Intercomp). After solubilization, the MWNTs were autoclaved.

[0065] Functionalization was performed following the diimide-activated amidation reaction protocol (JIANG et al., J Mater Chem v.14, p.3739, 2004). 750 μL of carboxylated carbon nanotubes were dissolved in 1875 μL of Milli-Q water, final concentration 0.4 mg / mL, and sonicated for 30 minutes in a bath sonicator and for a further 5 minutes under Petition 870260056791, dated 11 / 06 / 2026, page 27 / 35 20 / 26 direct sonication. Then, 375 pL of MES buffer (2-morpholinoethanesulfonic acid monohydrate, 500 mM pH 6.1) and 863 pL of NHS solution (N-hydroxysuccinimide, 50 mg / mL) were added and vortexed. 450 pL of EDAC solution (N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride, 10 mg / mL) were added, and the mixture was maintained under rapid stirring coupled to a vortex mixer (Phoenix AP56) for 30 minutes at room temperature.

[0066] The solution was centrifuged at 13000 RPM (5415C centrifuge, fixed rotor, Eppendorf) for 10 min at room temperature, and the supernatant was carefully discarded using a pipette to remove EDAC and urea residues from the purification. The Carbon Nanotubes (MWNT) were then resuspended in 3000 µL of MES buffer (50 mM, pH 6.1), thus obtaining a 0.25 mg / mL solution.

[0067] The proteins were functionalized to MWNT following the proportions 1 / 1, 1 / 3, 1 / 5, 1 / 10 and 1 / 20 (weight / weight), keeping the amount of protein constant (200 pg) and varying the amount of MWNT, with the final volume adjusted to 1.0 mL with MES buffer (50 mM, pH 6.1). The solution was kept under agitation for 2 h at 150 RPM and 5°C. Then, the samples were briefly vortexed and centrifuged for 10 minutes at 10000 RPM and 5°C (Eppendorf 5415C centrifuge, fixed rotor). A 0.5 mL aliquot was collected from each wash and stored, and another 0.5 mL of MES buffer (50 mM, pH 6.1) was added, and centrifugation was repeated. The process was performed three times to remove proteins that did not bind to MWNTs, and the samples were then stored at -20°C.10 pL of each aliquot collected from the washes was applied to SDS-PAGE to evaluate the amount of unbound protein in each sample (Figure 2), for the same purpose a dosage was performed using the Bradford method on the samples. Petition 870260056791, dated 11 / 06 / 2026, page 28 / 35 21 / 26 Example 4 - Characterization by Raman Spectrometry of Functionalization of MWNTs with Recombinant BoHV-1 and BoHV-5 Proteins

[0068] To verify the efficiency of MWNT functionalization with recombinant proteins, Raman Spectroscopy was used, in collaboration with Prof. Dr. Ado Jório (Department of Physics ICEx / UFMG).

[0069] On the coverslips, 20 pL of each sample were applied, dried, and then analyzed after excitation by a He-Ne laser (532 nm). The optical signal emitted by the sample passes through the objective lens and is detected by the Avalanche Photodiode Detector (APD) or passes to the spectrograph, being detected by the Charged Integrated Device (CCD).

[0070] This method is one of the most sensitive tools for characterizing nanostructures and is used for the characterization of carbon nanotubes (Figure 3) and to confirm the presence of functionalized groups on MWNT (Figure 4), with the effectiveness of the functionalization being given by the overlap of the spectra (nanotubes and proteins) found in the sample (FOLDVARI, Nanomedicine v.4, n.3, p.173-82, 2008). Example 5 - Evaluation of the Cytotoxicity of MWNTRecBoHV-1 / 5 Tools in an MTT Assay

[0071] Blood was collected from four cattle from a herd seropositive for BoHV-1 and BoHV-5. PBMCs (peripheral blood mononuclear cells) were separated using a density gradient – ​​Ficoll-Paque®, GE, according to the manufacturer's instructions, briefly: collection tubes containing approximately 7 mL of blood were centrifuged at 1200 RPM for 7 minutes at 20°C in a centrifuge (Jouan 4i - S40 High Throughput Swing). Petition 870260056791, dated 11 / 06 / 2026, page 29 / 35 22 / 26 (Out Rotor). Next, plasma was collected using a pipette, transferred to microtubes, and stored at -20°C. The remaining leukocyte sediment in the collection tube was diluted in unsupplemented RPMI medium in a 1:2 ratio. The dilution was carefully transferred to 50 mL tubes containing Ficoll in a 2:1 ratio, and these were centrifuged at 2500 RPM for 40 minutes at 20°C. The obtained PBMCs were then transferred to new 50 mL tubes, to which 40 mL of unsupplemented RPMI was added. The solution was then homogenized, centrifuged at 2500 rpm for 7 minutes at 4°C, and the supernatant discarded. This washing process was repeated three times, and the obtained cells were then diluted in 1 mL of unsupplemented RPMI medium.

[0072] Cells were stained with trypan blue (1:10), transferred to a Neubauer chamber, and counted. PBMCs were distributed in 96-well plates at a concentration of 7.0 x 10⁴ cells / well in a final volume of 200 pL of RPMI medium supplemented with antibiotics. Cells were incubated with recombinant proteins RecBoHV-1 and RecBoHV-5, functionalized or not to carbon nanotubes, at concentrations of 1.0 pg and 10 pg per well, as well as in the absence of stimulus (control with RPMI medium only), for 24 h at 37°C and 5% CO₂.

[0073] After the incubation period, 20 μL of MTT solution (5 mg / mL) were added per well, protected from light, and the plates were incubated for 4 hours at 37°C in a CO2 incubator. At the end of the incubation, 150 μL of the contents of each well were removed and 150 μL of isopropanol (0.04 M HCl) was added. After intense homogenization with the aid of a pipette, the plates were read in a microplate spectrophotometer (Asys Hitech GmbH) at 595 nm (Figure 5). Petition 870260056791, dated 11 / 06 / 2026, pages 30 / 35 23 / 26 Example 6 - In vitro immunogenicity assessment of MWNT-RecBoHV-1 / 5 tools using an indirect ELISA assay.

[0074] The sera from the animals used in the previous experiment were subjected to an indirect ELISA test, using purified viral samples of BoHV-1 (Colorado, ATCC 864) and BoHV-5 (EVI-88) as antigens, recombinant proteins RecBoHV-1, RecBoHV-5 and these functionalized to carbon nanotubes.

[0075] Each plate was sensitized with 250ng of the respective antigen per well, diluted in 100μL of coating buffer solution (0.106g of NaCO3, 0.42g of NaHCO3 dissolved in distilled water - qsp 100mL, pH 9.6), followed by incubation at 4oC “overnight”.

[0076] The plates were washed once with 150μL / well of washing solution (0.1% skimmed milk powder, 0.05% Tween 20 in 1X PBS), then 200μL of blocking solution was added to each well (5% skimmed milk powder in 1X PBS) and the plates were incubated again at 4oC overnight.

[0077] A second wash was performed, and then bovine sera containing primary antibodies were added (1:100 dilution in washing solution; 100μL / well). After incubation for 2 hours at 37°C in a humid chamber, the plates were washed ten times, and secondary antibodies (bovine anti-IgG, 1:5000 dilution or bovine anti-IgM, 1:20,000 dilution) were added. The plates were again incubated at 37°C in a humid chamber for 1 hour, washed 10 times, and developed with OPD solution (100μL / well), incubated for 30 minutes in the dark. The reaction was stopped with 4N H2SO4 (30μL / well), and the reading was performed on a microplate spectrophotometer (Asys Hitech GmbH) at 492nm (Figure 6). Example 7 - In vivo Immunogenicity Assessment of MWNT-RecBoHV-1 / 5 Tools in a Murine Model by Flow Cytometry Petition 870260056791, dated 11 / 06 / 2026, pages 31 / 35 24 / 26

[0078] Female C57BL / 6 mice aged between 4 and 6 weeks were obtained from the Center for Animal Experimentation (CEBIO) of the Federal University of Minas Gerais and were kept in the Animal Experimentation Bioterium of the Microbiology Department of ICB UFMG. Upon arrival, the animals underwent a 14-day deworming period with albendazole sulfoxide (0.6 µg / mL) before the start of immunizations. The animals were housed in plastic cages with micro-isolators, kept in special racks with their own ventilation.

[0079] The protocol for the use of experimental animals was sent to the Animal Experimentation Ethics Committee of UFMG CETEA (CETEA 239 / 11) and all procedures using animals were carried out in accordance with the committee's standards.

[0080] For immunogenicity assessment, animals were immunized subcutaneously with the protein solutions described below, following a booster-dose protocol consisting of three immunizations, with a 21-day interval between each dose. Animals were euthanized 14 days after the last immunization.

[0081] Unpublished data from our group have demonstrated that there is no immunodominance between the recombinant proteins RecBoHV-1 and RecBoHV-5, and, as the vaccine to be developed must contain both, it was decided to always inoculate them together.

[0082] The animals were divided into the following groups, each containing 6 animals, with the final volume adjusted to 40μL with sterile 1x PBS: - Group 1: 40μL of PBS (saline solution); - Group 2: 40μL of inactivated commercial vaccine for BoHV-1 and BoHV-5; - Group 3: 1g of non-functionalized MWNT; Petition 870260056791, dated 11 / 06 / 2026, pages 32 / 35 25 / 26 - Group 4: 1^g of RecBoHV-1 protein + 1^g of Receptor BoHV-5 purified; - Group 5: same solution as group 4, plus ^L of alum (30% of the final volume); - Group 6: MWNT-RecBoHV-1 and MWNT-RecBoHV-5 in a 1:1 ratio, with 1.0pg of functionalized protein to 1.0pg of MWNT, for each recombinant protein; - Group 7: same solution as group 6, plus ^L of alum (30% of the final volume); - Group 8: 1.0 pg of each recombinant protein and 1.0 pg of non-functionalized MWNT; - Group 9: same solution as group 4, plus ^L of alum (30% of the final volume).

[0083] Blood was collected from the animals via the brachial plexus at the time of euthanasia, after anesthesia. The animals were anesthetized with an intraperitoneal injection of ketamine / xylazine solution and euthanized by cervical dislocation, followed by spleen removal and splenocyte collection for T lymphocyte activation profile analysis.

[0084] The cellular immune response was evaluated by analyzing the splenic lymphocyte populations of the immunized animals. CD4+, CD8+ lymphocytes and the activation marker CD25 were quantified using flow cytometry (FACScalibur, BD BioSciences, USA), in which cells are labeled with the fluorescent antibodies CD4-FITC, CD8-PerCP and CD25-PE, and also differentiated by size and granularity.

[0085] The spleen of the immunized animals was collected after euthanasia and stored in 3 mL of RPMI 1640 medium (Cultilab, Brazil) supplemented with 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 U / mL penicillin. The spleens were macerated using... Petition 870260056791, dated 11 / 06 / 2026, pages 33 / 35 26 / 26 frosted-edge slides were centrifuged at 1200 rpm for 10 minutes at 4°C. The red blood cells present were lysed by adding 3 mL of ACK solution (0.15 M NH4Cl, 1 M KHCO3, 0.1 M EDTA) and incubating for 5 minutes on ice, followed by the addition of 5 mL of 5% RPMI to stop the process. After another centrifugation at 1200 rpm for 10 minutes at 4°C, the cells were washed in 5 mL of 5% RPMI, centrifuged again under the same conditions, and resuspended in 1 mL of 10% RPMI. The cells were then stained with Trypan blue and counted using a Neubauer chamber, adding 5x10⁵ cells per well in 96-well plates already containing 20μL of antibody dilutions (CD4-FITC 1 / 50, CD8-PerCP 1 / 100 and CD25-PE 1 / 25) in PBS-Wash (0.5% bovine albumin, 1.0% azide in 1X PBS).

[0086] The plates were incubated at 4°C for 30 min in the dark. The wells were made up to 200 μL with PBS-Wash, and the plates were centrifuged at 1200 rpm for 7 min at 4°C, with the supernatant discarded. The plates were washed again with 200 μL of PBS-Wash per well, and then each well was resuspended in 200 μL of MFF (Max Facs Fix - 2.5 mL of 20x Paraformaldehyde and 2.5 mL of 20x Sodium Cacodylate in distilled water q.s.p. 50 mL). The cell suspension was then transferred to 500 μL tubes and analyzed by flow cytometry (FACScalibur, BD BioSciences, USA). Petition 870260056791, dated 11 / 06 / 2026, pp. 34 / 35

Claims

1 / 2 CLAIMS 1. Recombinant proteins RecBoHV-1 and RecBoHV-5, represented respectively by the sequences SEQ ID No1 and 2, characterized by being associated with carbon nanotubes.

2. Vaccine against bovine herpesvirus 1 and 5 comprising recombinant multiepitope proteins of BoHV-1 and / or BoHV-5, characterized by being covalently associated with carbon nanotubes, and pharmaceutically and pharmacologically acceptable excipients.

3. Vaccine against bovine herpesvirus 1 and 5, according to claim 2, characterized by the carbon nanotubes being single-walled and / or multi-walled, preferably multi-walled (MWNT).

4. Vaccine against bovine herpesvirus 1 and 5, according to any one of claims 2 and 3, characterized by being administered orally, intramuscularly, intravenously, intraperitoneally, subcutaneously, transdermally or as devices that can be implanted or injected.

5. Vaccine against bovine herpesviruses 1 and 5, according to any one of claims 2 to 4, characterized by being presented in solid, semi-solid or liquid forms.

6. Vaccine against bovine herpesviruses 1 and 5, according to claim 2, characterized in that the excipients are selected from the group comprising water, saline solution, aluminum hydroxide solutions, phosphate-buffered solutions, Ringer's solution, dextrose solution, Hank's solution, biocompatible saline solutions containing or not polyethylene glycol, non-aqueous vehicles such as fixed oils, sesame oil, ethyl oleate or triglycerides, isolated or in mixture, including pharmaceutical nanoformulations. Petition 870260056791, dated 11 / 06 / 2026, page 7 / 35 2 / 2 7. Vaccine against bovine herpesvirus 1 and 5, according to claim 6, characterized by the excipients possibly containing additives such as buffers, preservatives, binders, disintegrants, diluents, lubricants and / or surfactants. Petition 870260056791, dated 11 / 06 / 2026, p. 8 / 35

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