Biological Response Modifiers for the Treatment of Individuals with Weakened Immune Systems and Their Compositions

Description

"Biological Response Modifiers for the Treatment of Individuals with Weakened Immune Systems and Their Compositions" Field of invention

[001] The present invention relates to the field of vaccinology, specifically to the area of ​​biological response modifiers capable of improving the immune response of individuals who are poorly or not at all responsive to vaccine antigens or to infections by pathogens. The present invention comprises formulations of cyclic dinucleotides, which can be administered alone, in combination with other biological response modifiers, or in vaccine formulations with specific antigens. The proposed invention also includes a method of preventing or treating acute respiratory infections, preventing disease severity and blocking transmission by inducing an immune response at the entry site (nasopharyngeal mucosa), preventing or reducing person-to-person transmission of pathogens / viruses in preventive vaccine formulations, as well as in formulations used for pre / post-exposure prophylaxis of acute and febrile respiratory infections. Previous Technique

[002] Antibody avidity denotes the strength of binding to its target epitope and reflects the best fit between IgG and epitope. The emergence of the 2019 SARS-CoV-2 coronavirus pandemic (COVID-19) resulted in rapid development of vaccines and therapeutics. The interaction of the SARS-CoV-2 S protein with host cells is a crucial component for the ongoing development of vaccines.

[003] The efficient disruption of the strong interaction established between the The interaction between the SARS-CoV-2 RBD and the human ACE2 protein has been proposed as requiring high-affinity antibodies to bind to multiple RBD sites (at least two molecular epitopes), highlighting the importance of this relationship. Petition 870250087282, dated 09 / 26 / 2025, p. 11 / 80 2 / 50 degree of functional affinity in blocking SARS-CoV2 infection (Khatri, 2020). Other viral infections, such as cytomegalovirus (CMV), Dengue, and Vesicular Stomatitis Virus, require antibodies with high avidity, rather than high titers, to confer effective protection. A similar result was also reported in a study that evaluated the immune response to the malaria vaccine, Dobano C, et al., Nature Communications 2019; 10(1),1-13.

[004] The interaction between the receptor-binding domain of the SARS-CoV-2 spike protein and ACE2 on target cells is the initial step of infection. The binding between the SARS-CoV-2 RBD and ACE2 is characterized by high affinity, Khatri I, et al., Front. Immunol. 2020; 11:570018. Therefore, Khatri and colleagues proposed that efficient neutralization of SARS-CoV-2 would require high-avidity antibodies, adding to the classic concept of neutralizing antibodies the additional requirement of high affinity / avidity.

[005] The association between antibody neutralization titers and antibody avidity contributes to the understanding of humoral responses to SARS-CoV-2 (Benner SE, et al., J Infect Dis. 2020 Nov 13; 222(12):1974-1984). As described for other pathogens, antibody avidity increases throughout the duration of infection; in SARS-CoV-2, the same is observed, since low antibody avidity is demonstrated during the initial infection and avidity increases after 3 weeks from the onset of symptoms in patients recovered from COVID-19. However, the antibody response to SARS-CoV-2 infection and seasonal coronaviruses is characterized by incomplete avidity maturation, as has already been well characterized by several groups.

[006] The analysis of outpatients with Covid-19 and SARS-CoV-2 infection confirmed by positive polymerase chain reaction (PCR) led, however, to a rather unexpected result, Bauer G, et al., J Med Virol. 2021;93:3092-3104. In contrast to the. Petition 870250087282, dated 09 / 26 / 2025, page 12 / 80 3 / 50 regular interaction between the humoral immune system and viruses, IgG avidity towards SARS-CoV-2 antigens appeared to remain low in most patients, even several months after the onset of clinical symptoms.

[007] Decreasing IgG titers after SARS-CoV-2 infection, including neutralizing antibodies, have also been demonstrated by other groups. Compared to Epstein-Barr virus (EBV) (a classic system for applying avidity determination), SARS-CoV-2 infection is similar in low avidity during the first weeks of infection, but completely different in the avidity achieved in previous infections. Less than 15% of SARS-CoV-2 infected patients achieved avidity indices > 0.6 several months after infection, in contrast to more than 90% in the case of EBV. Similar high avidity indices have been reported for previous infections with hepatitis A virus (HAV), hepatitis C virus (HCV), West Nile virus, and other viruses.

[008] Incomplete avidity maturation appears to be an essential strategy of coronaviruses in general. Incomplete avidity maturation has been found for the serological response to SARS-CoV-2 and immune responses to four seasonal coronaviruses (Bauer, 2021, see above; Friedhelm Struck, et al., 2021 J Med Virol; 93(12):6765-6777. doi: 10.1002 / jmv.27270. Epub 2021 Aug 20). It is also known that immune responses to seasonal coronaviruses are characterized by a relatively rapid decline after an initial burst. It appears that these two characteristics of the immune response to seasonal coronaviruses, namely decreasing antibody concentrations and frequently low avidity, are related to the high probability of repeated waves of reinfections with these viruses. Recent data confirm that coronaviruses interfere with avidity maturation to ensure incomplete protection in Petition 870250087282, dated 09 / 26 / 2025, page 13 / 80 4 / 50 in relation to reinfection, allowing replication through repeated waves of infection in the host population. Thus, a valid approach would be to develop vaccines to induce a strong avidity antibody response.

[009] The concept of a coronavirus immune evasion strategy that renders immune responses non-protective, combined with biophysical data on the high affinity between the SARS-CoV-2 RBD and its cellular receptor, leads us to propose a vaccination strategy that exploits certain qualities of IgG to establish protective immunity. Vaccination programs are considered to aim for (i) the stimulation of sufficiently high and long-lasting IgG concentrations that specifically target viral structures relevant for binding to cellular receptors such as the RBD; (ii) complete maturation of neutralizing IgG avidity towards SARS-CoV-2; and (iii) that these antibodies prevent viral binding to cells, ideally in mucosal compartments to prevent both local infection and dissemination / transmission to susceptible hosts.Therefore, the suggested goal is for immunization to achieve a response that surpasses the quality of the immune response achieved after natural infection.

[010] The development of new compounds with the ability to induce high-avidity antibodies represents a challenge in vaccinology. Different adjuvants enhance the immune response by producing a strong antibody response or a Th1 cellular response. However, products with capabilities as biological response modifiers (BRMs) capable of inducing a strong, high-avidity antibody response are limited. New formulations have been developed to address this unmet need of current vaccines. For example, Poly(I:C) and CpG compounds adsorbed onto aluminum hydroxide induce a strong immune response with a significant increase in Petition 870250087282, dated 09 / 26 / 2025, page 14 / 80 5 / 50 effective in antibody avidity. The formulation exhibits different properties compared to the use of the compounds separately (Lu F, et. al., Vaccine. 2019;37(14):1945-1953).

[011] Single intradermal or intramuscular inoculations of GM-CSF DNA with the DNA primer for a simian human immunodeficiency virus (SHIV)-89.6 vaccine, consisting of the DNA primer followed by a booster with modified Ankara vaccinia (MVA), increases blood and intestinal protection against the acute phase of an intrarectal challenge against SHIV-89.6P. GM-CSF appeared to contribute to protection by enhancing two antibody responses: avidity maturation of anti-Env IgG in the blood and the presence of long-lasting antiviral IgA in rectal secretions. Anti-Env IgG avidity showed strong correlations with protection both before and after challenge. Animals with the highest anti-Env Ab avidity showed 1000-fold reductions in peak viremia compared to those with the lowest anti-Env Ab avidity (Lai L, et al., Virology. 2007;369(1):153-167).

[012] TLR agonists and cytokines are involved in affinity maturation; however, these compounds are, as a rule, provided in combination with depot adjuvants to further potentiate this effect. However, in specific scenarios, TLR agonists are unable to increase avidity, creating a confusing scenario that requires case-by-case product optimization. Primary vaccination with TLR adjuvants or oil-in-water TLRs induces high-avidity antibodies – as demonstrated for these Adjuvant Systems (AS) combined with various antigens (e.g., Khurana, 2018), and the induced memory B cells undergo greater maturation and differentiation after antigen retrieval. Affinity maturation parameters appeared to differ compared to Alum. Avidity maturation appeared to be stronger. Petition 870250087282, dated 09 / 26 / 2025, page 15 / 80 6 / 50 mind promoted by AS formulations.

[013] A recent article published by Budroni et al., see above, and the Glaxo-Smith-Kline team of experts specializing in adjuvant development, acknowledged that the effects of adjuvant composition on the durability and functionality of immunological memory, which can be driven by antigen recall, remain poorly investigated and that differences in innate immune imprinting between vaccine adjuvants may mediate different effects on the quantity / quality of persistent adaptive responses. In their studies, avidity maturation appeared to be more strongly promoted by their AS formulations when compared to Alum. After antigen recall, the frequencies of individuals with high-avidity antibodies increased only sharply in the AS groups. Among the AS groups, total antibody responses were lowest for AS04.However, the proportions of high-avidity antibodies were similar between the groups, suggesting that monophosphoryl lipid A (MPL) in AS04 contributes to avidity maturation. Specific combinations of immunostimulants in AS, regardless of their individual nature, increase antibody persistence and avidity maturation. However, antibody avidity remains largely unexplored in current vaccinology and may be considered associated with some adjuvants and not others, regardless of the intensity of the humoral immune response. Furthermore, as is being explored by Budroni et al., the formulation of adjuvants may induce specific effects that may be related to some specific compounds and not others or to the alum.

[014] Regarding protection, recent studies have confirmed that the level of antigen-antibody binding avidity can also correlate with protection. This was demonstrated for the RTS vaccine, con Petition 870250087282, dated 09 / 26 / 2025, p. 16 / 80 7 / 50 for malaria S, among others, and for various treatments with monoclonal antibodies (McAb) [Dobano, 2019, see above]. On the other hand, low-avidity antibodies have been associated with increased antibody-mediated disease after vaccinations against respiratory syncytial virus, dengue, or pandemic influenza [Smatti MK et al., Front. Microbiol. 2018;9:2991].

[015] Cyclic dinucleotides (CDNs), including cyclic diadenosine monophosphate (cyclic di-AMP; CDA), cyclic digunosine monophosphate (cyclic di-GMP; CDG), and cyclic GMP-AMP (cGAMP), are a class of bacterial and mammalian second messengers with potent immunological effector functions [Ebensen T, et al.; Vaccine 2011;29(32),5210-5220]. CDNs can be used as adjuvants to protein subunit vaccines to trigger mucosal immunity and protect mice from respiratory bacterial infections in experimental animal models, such as Mycobacterium tuberculosis, anthrax, Klebsiella pneumoniae and Streptococcus pneumoniae, Acinetobacter baumannii, and methicillin-resistant Staphylococcus aureus. CDNs also induce long-lasting CD8+ T cell immunity and have a therapeutic effect against cancer in mice.Thus, CDNs generate long-lasting humoral and cellular memory responses, both in the systemic and mucosal compartments [Ebensen, 2011, see above].

[016] Superior to other vaccine adjuvants that induce biased immune responses, CDNs produce balanced Th1 / Th2 / Th17 memory cells and cytotoxic CD8+ T cells. Ebensen et al. first reported in 2007 that subcutaneous immunization with CDG / ε-galactosidase (β-Gal) not only triggered a significant humoral response compared with β-Gal alone, but also a cellular response in the spleen of immunized mice [Ebensen T, et al., Clinical and Vaccine Immunology 2007;14(8),952 Petition 870250087282, dated 09 / 26 / 2025, page 17 / 80 8 / 50

[958] . Enzyme-linked immunospot (ELISPOT) analysis of splenic cells showed that CDG immunization increased β-Gal-specific memory Th1 and Th2 cells, thus validating CDG's ability to modulate both humoral and balanced Th responses in a murine model. In 2010 and 2011, CDG and cyclic diinosine monophosphate (CDI) were shown to be potent inducers of interleukin (IL)17a-secreting CD4+ Th cells, along with Th1 and Th2 responses, when immunized intranasally.

[017] CDNs exhibit potent antitumor activity. Demaria et al. demonstrated that intratumoral injection of cGAMP increased the antitumor response of CD8+ T cells, inhibiting the growth of injected tumors in murine models of melanoma and colon cancer. Intratumoral injection of cGAMP into B16F10 lung metastases induced a systemic CD8+ T cell response to restrict the growth of distant tumors. A study by Francica et al. showed that CDN-mediated tumor necrosis factor (TNF)-α production by innate immune cells is responsible for acute tumor clearance, and TNF-α blockade inhibits tumor necrosis and clearance against cold tumors [Francica BJ, et al., Cancer Immunol. Res. 2018, 6, 422-433].

[018] Finally, CDNs effectively induce cross-presentation of dendritic cells (DCs) to MHC class I molecules, producing a CTL response. A potent antigen-specific CD8+ memory T cell response, through in vivo targeting of the DEC-205 receptor on DCs, has provided an effective approach to combat viral infections [Volckmar J, et al., Vaccine 2019;37,4963-4974]. Combinatorial methods, such as intramuscular immunization of CDA with alum, have demonstrated significant enhancement in antigen-specific CD4+ and CD8+ T cell responses, man Petition 870250087282, dated 09 / 26 / 2025, page 18 / 80 9 / 50 having an excellent security profile.

[019] In 2007, Ebensen et al. first demonstrated CDNs as a mucosal immunostimulator. They showed that CDG / e-Gal in immunized mice not only elicited a systemic IgG response, but also generated an enhanced IgA response specific to β-Gal after 42 days in bronchial alveolar lavage fluids (BALF) and vaginal lavage. Intranasal immunization with CDG / e-Gal also enhanced serum IgG2a and IgG1 production compared to mice immunized with β-Gal alone. The systemic cellular response was also enhanced by CDG / e-Gal immunization, as observed with the production of interferon (IFN)-γ, IL-5, and IL-2 in the ex vivo recall assay compared to mice immunized with e-Gal alone [Ebensen, 2007, see above].

[020] CDNs also induce Th17 in the mucosa. The mucosal adjuvant potential of CDNs was further strengthened when Madhun et al. demonstrated that intramuscular administration of a plant-based H5N1 influenza antigen induced high frequencies of multifunctional Th1 cells. Two doses of the CDG adjuvant vaccine were able to trigger a very high mucosal IgA response and protected against antigenically shunted H5N1 viruses. In comparison, the same vaccine, when immunized intramuscularly (IM), did not generate a Th1 response, which is critical for viral infections [Madhun AS, et al., Vaccine 2011;29:4973-4982].

[021] CDNs have been described as useful adjuvants in the patent literature, for example, CDA variants, including thiophosphonate derivatives. Not only c-di-AMP are described, but also c-diGMP or combinations of c-di-AGMP.

[022] Although CDNs have demonstrated promising and safe activity in mucosal vaccines in animal models, currently Petition 870250087282, dated 09 / 26 / 2025, page 19 / 80 10 / 50 there is no evidence regarding the ability of these products to increase the avidity of antibody responses in vaccine studies associated with abrogation to the non-responsive state. In fact, some results are distinct from the CDN-STING-IFNs type I immunity paradigm. Andrew Mellor's group described a CDN-STING-IFNs type I-IDO1-tolerance signaling that mediates potent T-reg responses and suppresses inflammation. They first showed that DNA nanoparticles (DNPs, containing PEI and plasmid DNA without TLR9 ligands) activate STING-dependent IDO-1 production that induces tolerance [Huang L, et al., J. Immunol. 2013;191,3509-3513]. Subsequently, they showed that treatment with CDG delayed the onset of experimental autoimmune encephalitis (EAE) and reduced disease severity [Lemos, 2014]. They identified a novel CDGSTING / IFNs type I / IDO1 / Tregs pathway that suppresses specific autoimmunity of the central nervous system (CNS).Recently, the same group demonstrated that CDN-STING-IDO-1 activity in the tumor microenvironment (TME) promoted the growth of low-antigenicity Lewis lung carcinoma (LLL). Finally, they demonstrated that treatment of pre-diabetic NOD mice with cGAMP delayed the onset of type I diabetes (T1DM), which is dependent on type I IFNs. Thus, CDNs can be used as immunomodulatory drugs to abolish autoimmune responses, as well as to induce strong Th1 immune responses due to their balanced immunomodulation profile. This evidence suggests that the effect and mechanisms of action of CDNs change significantly according to specific conditions, such as the vaccine antigen, vaccine formulation, and vaccination schedule, making it extremely difficult to predict for a person skilled in the art.

[023] In document WO 2021 / 038022 A1, a new use of cyclic dinucleotides is described, namely, the use of CDN in a method Petition 870250087282, dated 09 / 26 / 2025, p. 20 / 80 11 / 50 to induce or promote an immune response in an individual, said individual being a newborn or infant.

[024] Germano MJ et al., J Med Virol. 2023; 95:e28584, describe a SARS-CoV-2 vaccine based on a receptor-binding domain, adjuvanted with cyclic diadenosine monophosphate, which resulted in enhancement of humoral and cellular immunity in mice. It is discussed that mice that received RBD and c-di-AMP showed better neutralizing antibody responses, as determined by the pseudovirus neutralization assay, when administered intramuscularly (IM). A stronger immune response compared to mice vaccinated with aluminum hydroxide-adjuvanted RBD or without adjuvant was demonstrated after IM administration. Detailed description of the invention

[025] The present invention relates to unmet needs in the field of vaccinology, specifically in the area of ​​biological response modifiers (BRMs) capable of abrogating the state of unresponsiveness, administered alone or in combination with other enhancers of innate immunity, aiming at the control of infectious diseases through the stimulation of the adaptive and stimulatory pathways of innate immunity. The object of the present invention is the use of CDN as a biological response modifier to abrogate low or absent responsiveness to specific pathogens / antigens, inducing a high-avidity immune response capable of preventing disease caused by infections. In particular, the present invention targets viruses with the ability to escape or evade immune surveillance mechanisms and, consequently, suppress the induction of a high-avidity and effective adaptive response.This also applies to pathogens with greater genetic plasticity, which are capable of evolving into mutated variants. Petition 870250087282, dated 09 / 26 / 2025, page 21 / 80 12 / 50 tions in critical regions of their antigens, preventing immune elimination.

[026] In a first aspect, the invention relates to CDNs for use as a BRM to enhance the immune response of individuals who need it, with low-performing immune systems, characterized by low / absent response to vaccine antigens or low-performing immune response to pathogens.

[027] When used in this document, the term “biological response modifier” refers to a compound with capacity as an immunomodulatory agent; defined as the class of drugs that target the causative mechanism of disease, which will directly impact the mechanism related to a disease, for example, the absence or reduced maturation of affinity during natural coronavirus infection, or the absence of an immune response to antigens, as in the case of chronic diseases due to defects in innate immunity mechanisms related to antigen presentation, co-stimulation, and cytokine profile of immune responses. In these cases, the effect of the CDN will neutralize the effect of the virus in the context of prevention, post-exposure prophylaxis, or preventive therapy.

[028] The term “underperforming immune system” refers to the immune system of individuals with age-related immunodeficiency (immunosenescence), a chronic condition such as chronic diseases that cause immunosuppression, and also includes the immune response of individuals with other chronic conditions such as obesity, hypertension, and diabetes, which may represent a risk factor for severe illness or death caused by pathogens such as (but not limited to) coronaviruses or hepatitis A, B, C, D, E, or F viruses. It also encompasses individuals with an underperforming immune system as a result of therapeutic interventions such as pharmacological therapies (by Petition 870250087282, dated 09 / 26 / 2025, page 22 / 80 13 / 50 example, treatment with corticosteroids or immunosuppressive drugs) or medical interventions. In the present set of examples, it is presented as a Balb / c mouse with specific deficiencies in its innate immunity receptors.

[029] When used in this document, the term “low / non-responsiveness to vaccine antigens” refers to individuals with an immune response considered suboptimal for protection against specific pathogens, while the term “unsatisfactory immune response to pathogens” identifies individuals with an immune response considered suboptimal for preventing severity or death caused by a natural infection.

[030] The composition according to the present invention is, for example, a compound according to formula (I). R2 in what A, A' is, independently of each other, S or O; X is, independently of each other, S, N, O, Chh; Y, Y' is, independently of each other, NH, Chh, O; Z, Z' is, independently of each other, NH, CH2, O; R1 is, independently of each other, H, NH2, S, or O; R2 is, independently of each other, NH2, O, H, or S; R3 is, independently of each other, absent if there is a covalent bond between Z or Z' and the C atom, or if it is hydrogen, OH, halogen, a straight or branched C1-C1 alkyl group, or Petition 870250087282, dated 09 / 26 / 2025, p. 23 / 80 14 / 50 a straight or branched C1-C6 alkoxy group that may be optionally substituted; R4 is, independently of each other, hydrogen, halogen, or a straight or branched C1-C6 alkyl group, which may be optionally substituted; ... is a single or double bond; or conjugates thereof, and salts or solvates thereof.

[031] In one embodiment, the biological response modifiers (BRMs) present in the immunogenic composition according to the present invention are a compound according to formula (I), wherein any of the purine residues is an adenine, xanthine or hypoxanthine residue or combinations thereof. In another embodiment, both purine residues are adenine. That is, it is preferable that the cyclic dinucleotide be a CDA. For example, c-di-AMP is a 2,3'-c-di-AMP or 3,3'-c-di-AMP. Furthermore, the BRMs may be a bisphosphatethioate analog of c-di-AMP.

[032] In one embodiment, the BRMs of general formula (I) are a compound in which R3 is an OH group, X is an oxygen and Y, Y', Z and Z' are oxygen. Furthermore, the compound according to formula (I) that represents a BRM present in the immunogenic composition is any of the claims, characterized in that the compound according to formula I is a cyclic bis(3'-5') diadenylic acid (CDA or c-di-AMP), c-di-GMP, c-di-GAMP, c-di-IMP, c-IAMP or a thiophosphate of CDA, thiophosphate of c-diMP and thiophosphate of cAMP-IMP.

[033] According to one embodiment of the present invention, a CDN, such as c-di-AMP, is used to increase the avidity of the antibody response in a formulation comprising subunit antigens with physical and chemical natures that result in non-responsiveness after multiple intranasal (IN) immunizations or that result in low immunogenicity after administration to an individual with Petition 870250087282, dated 09 / 26 / 2025, page 24 / 80 15 / 50 deficiency in antigen detection. Thus, CDN, as well as cdi-AMP, is used particularly to overcome the absence of response or a low / weak response, as well as to ensure the quality of the immune response, inducing a strong, rapid, and high-avidity antibody response in an individual who would otherwise show a low / no response. Such antibody responses are characterized by their enhanced functionality, suppressing the binding of an infectious agent to its receptors, particularly a virus, and by strong neutralizing or specific destructive activity against the infectious agent / virus.

[034] The object of the present invention also includes the resulting c-di-AMP formulations and said antigens, as well as the potential combination of c-di-AMP with other immunostimulants to potentiate the effect of c-di-AMP, allowing dose splitting or adding complementary properties to the resulting formulation, in particular in terms of effector functions responsible for eliminating infections, as well as increasing the activation of innate immunity with pattern recognition receptor agonists, such as TLRs.The proposed invention also includes a method for preventing coronavirus infections, disease severity, and transmission by administering the formulations of the present invention to a healthy person, those at risk of infection or severe illness, or a recently infected subject, preferably via the nasal route, in a dosage range capable of eliciting an effective antibody response, blocking the infection process, particularly at the entry site (e.g., nasopharyngeal mucosa), as well as reducing or preventing horizontal person-to-person transmission of the infectious pathogen, such virus, of interest.

[035] The present application relates to the use of CDN, as c-di-AMP, to induce a high-avidity antibody response and, Petition 870250087282, dated 09 / 26 / 2025, p. 25 / 80 16 / 50 Consequently, increase the anti-infective effect, in particular the antiviral effect, of the resulting antibody response. In one embodiment, the formulations of the present invention are administered via the mucosal route, such as intranasal (IN), sublingual, intratracheal or pulmonary, to induce a strong antibody response at the systemic and / or mucosal level, such as in the blood and / or secretions or mucous tissues. The present inventors recognized that the antibody response was superior in strength compared to the administration of the same dose of antigen in aluminum hydroxide as a control vaccine. Formulations that include CDN, as well as the specific use of CDN according to the present invention, induce a mucosal IgA response with inhibitory capacity for ACE2 binding and high levels of viral neutralization titers.The antibody response to SARS-CoV-2, the inhibitory effect of ACE2-RBD binding, and the viral neutralization response depend on the use of c-di-AMP via intravascular administration.

[036] A specific embodiment of the present invention comprises formulations of c-di-AMP and mannosylated subunit antigens produced, and more specifically the RBD proteins and other proteins of SARS-CoV-2 or other infectious agents, in particular coronaviruses, as well as the method of inducing an immune response in mucosal and systemic compartments using these formulations by IN administration. These formulations are capable of overcoming the low or non-responsive status in the specific host with deficiencies in the expression of receptors, those necessary to process the antigens, resembling the process of reduction of innate immunity receptors that occurs in the elderly due to immunological senescence or the affected immune receptors in immunocompromised individuals. In the specific case of the examples, Balb / c mice exhibit specific deficiencies in the expression of Petition 870250087282, dated 09 / 26 / 2025, page 26 / 80 17 / 50 receptors of innate immunity by their professional antigen-presenting cells (APCs), which implies an absence of response to the antigen via the IN route and a weak response after parenteral administration. This effect may, in turn, be translated to the structure of human individuals who exhibit a low response to vaccination in the clinic (as described below).

[037] As demonstrated in the examples, the subject of the present invention is capable of inducing a mucosal IgA response with inhibitory capacity for ACE2 binding, as well as a superior inhibitory effect on ACE2 binding in sera, compared to the response elicited by the same dose of antigen administered intramuscularly (IM) in aluminum hydroxide. The formulations of the present invention induce a stronger viral neutralization response compared to the same antigen administered intramuscularly (IM) without c-diAMP or formulated with TLR agonists in the context of non-responsiveness due to lower expression of receptors in professional antigen-presenting cells. Co-administration, in the same formulation, of a TLR agonist, preferably the hepatitis B virus core antigen (HBcAg), constitutes another modality, provided that the individual requires additional stimulation of the innate immune response, as is the case with immunosuppression caused by disease or immune senescence.

[038] According to the state of the art, it was expected that a compound with proven multiagonist effect for TLR3, TLR7 and TLR8, such as HBcAg and previously used as an adjuvant, could also enhance the immune response to RBD produced in Pichia pastoris; however, mice with low levels of mannose receptors and, consequently, unresponsive to this glycosylated antigen (PICHIA-derived RBD antigen, Pichia being a widely used cellular factory for the production of RBDs) Petition 870250087282, dated 09 / 26 / 2025, p. 27 / 80 18 / 50 vaccine antigen formulations) were refractory to the induction of an immune response in the absence of c-di-AMP. The absence of an immune response from formulations containing HBcAg and RBD, and the high immunogenicity of the CDA comprising the formulation, demonstrated the ability of cdi-AMP to overcome the non-responsiveness status, a completely unexpected and surprising potency and functional activity, given the absence of the recognized HBcAg adjuvant effect. The non-responsiveness of the Balb / c mouse strain, resulting from the low level or absence of mannose and other innate immunity receptors in its APCs, resembles the low level of expression of innate immunity receptors in elderly and other immunocompromised individuals, who naturally have a low response to vaccines compared to the younger population.

[039] Older adults and people with underlying health conditions are at higher risk of developing severe clinical manifestations of COVID-19. This may be associated with less responsive innate immune ligands, altered immune responses—both in quality and quantity—, changes in the cytokine environment, and lower expression of costimulatory signals. Thus, intrinsic host factors, such as reduced ability of the innate immune system to detect PAMPs, may contribute to the severity and mortality of the disease.

[040] No antibody response was detected in Balb / c mice by nasal immunization in groups immunized with HBcAg RBD at both RBD doses studied. Surprisingly, c-di-AMP reversed the low / non-responsive state against the mannosylated antigen via the IN route. Furthermore, it also induced a superior avidity response compared to the alum formulation administered via the IM route; both activities are unprecedented in the scientific literature related to the field of CDN and po Petition 870250087282, dated 09 / 26 / 2025, page 28 / 80 19 / 50 Enhanced avidity is a rare characteristic / property of unique adjuvant formulations. Thus, there are two relevant aspects regarding the use of c-di-AMP. First, the ability of this compound to override the non-responsive state in the experimental environment using the well-established preclinical murine model, with low levels of mannose expression and other innate immunity receptors. Second, the ability of the resulting formulation to induce an antibody response with remarkable avidity, strength, and functionality, alone or in combination with HBcAg.

[041] A relevant feature of the formulation, subject of the present invention, refers to the use of CDN, such as c-di-AMP, to generate an immune response with a rapid titer increase dynamic, which represents a desirable capacity of the immune response triggered by a vaccine, particularly in the context of a pandemic / epidemic outbreak and travelers, the ability to protect most vaccinated individuals in a short period of time and the fewest number of doses required.

[042] In one embodiment, the pathogen or infectious agents are organisms capable of producing infection or infectious disease. They include bacteria, fungi, viruses, and parasites. In one embodiment, the pathogen is a virus and the vaccine is against said virus. The term virus includes the embodiments of coronavirus, influenza virus, respiratory syncytial virus, rhinovirus, including DNA viruses and RNA viruses. In one embodiment, the virus is a virus from the four serotypes of the dengue virus.

[043] In a preferred embodiment of the present application, SARS-CoV-2 RBD or S antigens of different variants or of another coronavirus or infectious agent, one or more, are included in the formulation with c-di-AMP or CDN in general, under appropriate concentration conditions. Petition 870250087282, dated 09 / 26 / 2025, page 29 / 80 20 / 50

[044] In another form of the present request, the formulation of RBD and c-di-AMP or CDN are generally combined with other antigens from the same virus (SARS-CoV-2) or the same virus family (betacoronaviruses) in order to increase the valence of the formulation and extend its properties to other antigens.

[045] In another embodiment of the present invention, the use of CDN vaccination against the hepatitis B virus (HBV) is described, wherein the HBV antigen is described. In one embodiment, the HBV antigen is HBsAg. As demonstrated in the examples, CDNs are particularly useful as biological response modifiers to enhance the immune response of individuals in need thereof with low-performing immune systems due to age-related immune deficiencies, a chronic condition, or due to medical or therapeutic intervention in the case of prophylactic or therapeutic vaccination. As mentioned, particularly in the chronic condition of HBV infection, the present invention demonstrates superior results to other prophylactic or therapeutic vaccination strategies.

[046] In another embodiment of the proposed application, CDNs, as well as c-di-AMPs, are formulated with antigens of coronavirus, influenza virus, respiratory syncytial virus, rhinovirus and any other relevant respiratory pathogen with immune escape / evasion capability or when high-affinity antibody stimulation is critical for clearance, combined in a multivalent vaccine formulation intended for the prevention of acute respiratory infections in general.

[047] In another embodiment of the proposed application, CDN, as well as c-di-AMP, is co-administered with adjuvants that favor the depot effect and slow release of BRMs and antigens, with the aim of reducing or splitting the antigen dose in the vaccine composition. The co-administered adjuvants are (but are not limited to) the following: alum, PLGA, chitosan, ISCOMs, ISCOM matrix and mi Petition 870250087282, dated 09 / 26 / 2025, page 30 / 80 21 / 50 cro or biodegradable nanoparticles.

[048] Furthermore, CDN is useful for enhancing an immune response by accelerating the germinal center reaction in order to further promote antibody hypersomatic mutation and affinity maturation. In this context, the term acceleration of the germinal center reaction refers to the situation in which the antibody repertoire of B cells matures through mutation known as hypersomatic mutation and affinity maturation. This allows the production of antibody-producing B cells, which will eventually become antibody-producing plasma cells (effector B cells), as well as memory B cells with higher affinity antibodies.

[049] In another embodiment of the present application, the formulation is administered via mucosal and / or parenteral routes, with IN and IM routes being preferred. The formulation is intended for mucosal administration, particularly IN; however, it can be administered via mucosal and parenteral routes simultaneously, as well as sequentially in a prime-boost or prime-pull scheme. Similarly, it can be used as a preparation or booster of mRNA, DNA, non-replicative or replicative live vectors, or recombinant protein-based vaccines for SARS-CoV-2, as well as other coronaviruses, in order to promote in immunized patients a partial increase in the properties described for the present formulation.

[050] As used in this document, the term booster refers to the treatment / vaccination of individuals previously treated with another vaccine composition. Prime-boost refers to the rational design of an immunization protocol to induce active immunity based on the administration of two different vaccines, one of which is one of the vaccines described in the present invention. Furthermore, prime-pull protocols aim to attract cells to the mucosal territory of vaccination, thus triggering local / mucosal immune responses capable of Petition 870250087282, dated 09 / 26 / 2025, page 31 / 80 22 / 50 provides protection not only against diseases but also against infections, reducing the risk of horizontal transmission to a susceptible host.

[051] In a preferred embodiment, the formulations will comprise CDN, such as c-di-AMP, in a dose range that allows for a stronger immune response, according to current pharmacological studies. The range comprises doses of 0.01 mg to 0.4 mg of CDN in phosphate-buffered saline (PBS) per immunized individual; a similar range of HBcAg doses may be co-administered, further potentiating the BRM effect of CDN in the formulation of both compounds, as well as in their combination with various antigens in mono- and multivalent formulations.

[052] In a preferred embodiment, the formulations of the present invention can be administered parenterally as part of a formulation with alum or other depot adjuvants, combined with adjuvant-free vaccines, formulated with different adjuvants, or simply mixed at the time of product administration with a different vaccine for SARS-CoV-2, coronavirus, or against one or more infectious agents. Furthermore, the BRMs or mixture of BRMs of the present invention can be provided in isolation, without the administration of antigens.

[053] In a preferred embodiment of the present invention, the formulation will be administered in accelerated immunization schedules, considering weekly application, every 15 days or appropriately adjusted to the need, as well as in long-term schedules adapted to synchronize the booster with the contraction phase of germinal center formation, in order to further promote hypersomatic mutation and affinity maturation. The formulation can be applied to people already exposed to SARS-CoV-2 or other coronaviruses or respiratory infections. Petition 870250087282, dated 09 / 26 / 2025, page 32 / 80 23 / 50

[054] The CDN for use in accordance with the present invention may be in the form of vaccine formulations comprising one or multiple antigens, such as Spikes or RBD, as well as other vaccine-relevant proteins, purified from the pathogen itself or produced as a recombinant protein in a cell factory, such as Pichia pastoris, Escherichia coli or a eukaryotic cell-based protein expression system, in particular from the recombinant SARS group, IAV, RSV, dengue virus, HBV (HBsAg), HIV, HCV, Zika and other pathogens.

[055] In another aspect, the present invention relates to a method for improving the immune response of an individual in need thereof, with low-performing immune systems due to immunodeficiency caused by age, a chronic condition, or due to medical or therapeutic interventions, in which the individual exhibits low / weak or non-responsiveness to vaccine antigens or exhibits a low-performing immune response to pathogens, comprising the step of administering a cyclic dinucleotide (CDN) as a biological response modifier via mucosal route to said individual in need thereof for prophylactic or therapeutic vaccination.

[056] As demonstrated, the method according to the present invention is particularly useful in individuals in need thereof, where the immune response presents deficiencies in their innate immune response. Surprisingly, it has been recognized that the use of CDN according to the present invention in the present method and its administration allows overcoming the limitations in individuals with low-performing immune systems, as described. In one embodiment, the method is a method in which CDN is CDN as defined in this document, in particular, CDN is c-di-AMP.

[057] In another modality, the method is a method for improving Petition 870250087282, dated 09 / 26 / 2025, page 33 / 80 24 / 50 to enhance the immune response against SARS-CoV-2 infection or other coronavirus infections. In another aspect, the present invention relates to a method for enhancing the immune response in people affected by chronic HBV infection. Specifically, the method according to the present invention, comprising administering CDN as a biological response modifier with an HBV antigen via mucosal route, resulted in superior immunostimulatory properties compared to administration with adjuvants known in the art.

[058] In another embodiment of the method according to the present invention, HBcAg is administered together with CDN and antigen. The administration of components can be implemented simultaneously, separately, or sequentially.

[059] The method according to the present invention can be applied as i) a method for establishing epidemic or pandemic immunostimulation, where the biological response modifier is administered alone or in combination with another biological response modifier in a pre / post-exposure prophylaxis setting in the absence of any vaccine antigen, or ii) in formulation with the antigens of an infectious agent from the group of chronic infectious diseases, or iii) in formulation with the antigens of pathogens from the group of acute and / or febrile respiratory infections.

[060] The method according to the present invention is, for example, a prime-booster or prime-pull type vaccination scheme.

[061] In one embodiment, the method according to the present invention is a method in which the administration of the CDN dose corresponds to the range of 0.01 to 0.4 mg per dose, and the vaccine may comprise additional HBcAg in the range of 0.01 to 0.4 mg per dose and may also contain a selected antigen in a dose range of 0.01 to 0.2 mg in a suitable excipient. Petition 870250087282, dated 09 / 26 / 2025, page 34 / 80 25 / 50 Brief Description of the Figures

[062] Fig. 1. Anti-RBD response induced after three immunizations, following the immunization schedule described in example 1. Titers were evaluated using an RBD protein obtained from mammalian cells to prevent the response against homologous glycosylation.

[063] Fig. 2. Avidity of the anti-RBD response induced after three administrations, following the immunization schedule described in example 1. The combined formulations of CDA (G6) or CDA-HBcAg (G10 and G11) significantly stimulated the avidity of the antibody response against RBD. The avidity index is defined by the percentage of antibody bound to RBD relative to the untreated control, according to the molar concentration of Guanidium chloride (GuHCl, the chaotropic agent). The concentration of GuHCl reduces the optical density by 50% by ELISA.

[064] Fig. 3. Inhibitory activity of the antibody response induced by the groups with the strongest anti-RBD response after three administrations, following the immunization schedule described in example 1. A) G2; B) G4; C) G6; D) G11; E) G12 and F) Monoclonal antibody (McAb) CBSSRBD-S.8 used as a standard curve to plot the results on the five ELISA plates.

[065] Fig. 4. SARS-CoV-2 neutralization activity of the antibody response induced by the groups with the strongest anti-RBD response after three administrations, following the immunization schedule described in the example.

[066] Fig. 5. Anti-RBD IgA response induced in the neutralizing activity of the antibody response induced by the groups with the strongest anti-RBD response after three administrations, following the immunization schedule described in the example.

[067] Fig. 6. Anti-RBD response induced after three immunizations, following the immunization schedule described in example 13. The titers Petition 870250087282, dated 09 / 26 / 2025, page 35 / 80 26 / 50 were evaluated using an RBD protein obtained from mammalian cells to prevent the response against homologous glycosylation.

[068] Fig. 7. Kinetics of the anti-RBD IgG response up to day 150.

[069] Fig. 8. Inhibitory activity of the antibody response induced by the groups with the strongest anti-RBD response after three administrations, following the immunization schedule described in example 13.

[070] Fig. 9. Secretion of IFN gamma (A) and IL5 (B), detected in splenocyte supernatants cultured for 96 h from selected groups of mice immunized in Example 1. The cells were stimulated with the homolog (RBD produced by Pichia pastoris), heterolog (RBD produced by CHO), Concanavalin A (ConA) and PBS for 96 h, and the supernatants were studied using the Luminex assay.

[071] Fig. 10. Viral neutralization studies using a specific neutralization assay for the original Wuhan variant, the Delta variant, and the Omicron (subvariant BA4 / BA5). Serum samples from the Example 1 immunization schedule were tested individually.

[072] Fig. 11. Figure 11 shows the dynamics of the anti-RBD IgG response in serum samples from mice primed with the formulations described in Example 13, corresponding to groups 1 to 13, as described in the figure, with the formulation described in Table 5.

[073] Fig. 12. Figure 12 Pseudovirus neutralization assay using the BA.4 / 5 variant of the pseudovirus. Mice boosted with the RBD derived from the Wuhan variant induced a neutralization titer similar to that of mice immunized with the RBD derived from the Omicron isolate. Examples Example 1: The use of CDA negates the unresponsive nature of Petition 870250087282, dated 09 / 26 / 2025, page 36 / 80 27 / 50 Mannosylated RBD in Balb / ce mice induces a rapid and strong immune response.

[074] Several vaccine formulations were studied to select the composition with the most attractive immune response for the prevention of SARS-CoV-2. The receptor-binding domain (RBD) protein was used in combination with other compounds with immunostimulatory activity. The RBD protein was obtained as a recombinant glycoprotein in Pichia pastoris, with the characteristic mannosylated glycosylation pattern.

[075] To explore the effect of RBD on nasal immunogenicity, the CDA, produced by enzymatic synthesis, was used alone or in combination with hepatitis B core antigen (HBcAg) as an innate immunostimulatory formulation, through a simple combination with RBD.

[076] The formulations tested in the present study are described in Table 1. Balb / c mice deficient in the expression of innate immunity receptors were used, specifically females, 8 per group, 12 groups, totaling 96 mice, which received 3 administrations on days 0, 14, and 28 via IN (G1 - G11) and IM (G12) routes. The RBD doses used in the study were 12.5 pg and 50 pg per mouse. The CDA doses were 5 pg and 10 pg per mouse, and the HBcAg dose used was 10 pg / mouse. Serum samples were collected on days -3, 25, and 42. Table 1. Description of immunization formulations and schedules, Example 1. Group Formulations (concentration of components in PBS) Dose (pg / mouse) Total Vol. (Vol. / nostril) Route N 1 PBS (Placebo) - 50pL (25pL) IN 8 2 CDA: 0.1 mg / mL CDA: 5pg 50pL (25pL) IN 8 Petition 870250087282, dated 09 / 26 / 2025, p. 37 / 80 28 / 50 Grupo Formulações (concentração dos componentes em PBS) Dose (pg / camun dongos) Vol. Total (Vol. / narina) Via N 3 RBD: 0,25 mg / mL RBD: 12,5pg 50pL (25pL) IN 8 4 RBD: 1,0 mg / mL RBD: 50pg 50pL (25pL) IN 8 5 RBD: 0,25 mg / mL + CDA: 0,1 mg / mL RBD: 12,5pg CDA: 5pg 50pL (25pL) IN 8 6 RBD: 1,0 mg / mL + CDA: 0,1 mg / mL RBD: 50pg CDA: 5pg 50pL (25pL) IN 8 7 RBD: 1,0 mg / mL + CDA: 0,2 mg / mL RBD: 50pg CDA: 10pg 50pL (25pL) IN 8 8 RBD: 0,25 mg / mL + HBcAg: 0,2 mg / mL RBD: 12,5pg HBcAg: 10pg 50pL (25pL) IN 8 9 RBD: 1,0 mg / mL + HBcAg: 0,2 mg / mL RBD: 50pg HBcAg: 10pg 50pL (25pL) IN 8 10 RBD: 0,25 mg / mL + CDA 0,1 mg / mL+ HBcAg: 0,2 mg / mL RBD: 12,5pg CDA: 5pg HBcAg: 10pg 50pL (25pL) IN 8 11 RBD: 1,0 mg / mL + CDA 0,1 mg / mL+ HBcAg: 0,2 mg / mL RBD: 50pg CDA: 5pg HBcAg: 10pg 50pL (25pL) IN 8 12 RBD 0,5 mg / mL + 0,5mg / mL de Alúmen RBD: 50pg Alúmen: 50pg 100pL IM 8 CDA: c-di-AMP

[077] On day 25, only mice immunized with c-diAMP induced a strong immune response (see Table 2). The responses Petition 870250087282, dated 09 / 26 / 2025, page 38 / 80 The 29 / 50 immune responses induced with the formulations tested in groups 5, 6, 10, and 11 were superior to the immune response induced in the groups with the antigen administered alone and even in the groups where RBD was administered only with HBcAg (groups 8 and 9). The intensity of the response via the intramuscular route was remarkable, considering that the antibody response was tested as early as day 25, 11 days after the second dose. The intensity was even stronger compared to the group immunized via the intramuscular route (G12). Interestingly, the highest (double) dose of CDA tested in group 7 resulted in a weaker immune response after the second dose, demonstrating that the effect of CDA may be limited to a certain dose range and that higher doses do not necessarily represent the most effective scenario for suppressing the immune response. Table 2. Individual levels of anti-RBD IgG in serum samples from individual mice in the 12 study groups. G2 489 Neg Neg Neg Neg 3537 9199 211 Neg Neg 3173 368 454 Neg Neg Neg Neg 895 11996 302 Neg Neg 5345 5544 Neg Neg Neg Neg Neg 87 11802 218 Neg Neg 609 5064 238 neg neg neg neg 80 9589 516 neg neg 6135 4010 218 neg neg neg neg 185 5776 neg neg neg 3894 352 252

[078] The RBD protein, obtained as a recombinant glycoprotein in Pichia pastoris, with the characteristic mannosylated glycosylation pattern, resulted in an undetectable immune response when administered to Balb / c mice. This effect is a result of the characteristic absence of mannose receptors in Balb / c dendritic cells [Autenrieth SE, Autenrieth IB. Immunology 2009;127(4):523-529].

[079] Surprisingly, Balb / c deficient mice Petition 870250087282, dated 09 / 26 / 2025, p. 39 / 80 30 / 50 cias in their innate immunity receptors, immunized with mannosylated RBD, resulted in a valuable model to demonstrate that the lack of response due to lower expression or absence of innate immunity receptors can be abolished with the use of CDA. Only the five groups that used CDA were able to induce an immune response. Even a previously used adjuvant for nasal formulations (HBcAg) was unable to induce an immune response when HBcAg was administered with RBD in the absence of CDA.

[080] In the case of people with immunodeficiencies or immune senescence, the expression level of innate immunity receptors is reduced. The surprising effect of overcoming the unresponsive state and inducing a strong immune response detected in the present study opens a new opportunity for the creation of vaccines for people with reduced levels of immune responsiveness due to lower expression of innate immunity receptors, as is the case for immunocompromised or immunosenescent individuals [Metcalf TU, ​​et al., Aging Cell. 2015 Jun;14(3):421-32. doi: 10.1111 / acel.12320]. Example 2: Combining CDA with HBcAg allows for the splitting of the immunizing dose.

[081] Upon further study of the effect of the different formulations after three immunizations, the absence of a response was reconfirmed in the groups immunized without CDA via the IN route (G3 and G4). Even in the groups immunized with HBcAg, a generalized absence of response was detected (G8 and G9), with the exception of two mice that developed a mild antibody response (Fig. 1). Considering the absence of effect of HBcAg found in G8 and G9 when this immunostimulant compound is administered with RBD alone, it was also surprising to find that the group with the lowest dose of RBD and both compounds (CDA and HBcAg, G10) induced, after three doses, an immune response of similar intensity compared to Petition 870250087282, dated 09 / 26 / 2025, pp. 40 / 80 31 / 50 tion to the groups with the highest dose of RBD (G6 and G11, p>0.05), demonstrating that the RBD dose can be divided into four doses without compromising the effect detected in the groups with the highest doses. This effect was not detected between the group with RBD and CDA alone (G5), which continues to induce significantly lower Ab titers against RBD compared to G6 (p<0.001). These results demonstrate that only after the elimination of non-responsiveness was the HBcAg effect detectable (see Fig. 1). Example 3. The anti-RBD antibody response induced by CDA-containing compositions is characterized by strong avidity.

[082] When studying the avidity of the antibody response induced by the different formulations described in example 1, after three immunizations, a significantly higher avidity was detected in the serum samples of the groups immunized with CDA or with the combined formulation of CDA and HBcAg, compared with the group immunized with the same protein, at the same dose, using the IM route of immunization.

[083] The development of new compounds capable of inducing high-avidity antibodies represents a challenge in vaccinology. Different adjuvants potentiate the immune response, producing a strong antibody response, or Th1 cellular response; however, substances capable of inducing a high-avidity antibody response are limited. New formulations have been developed to address this unmet need of current vaccines. The formulation may present different properties compared to the isolated use of TLR agonists and indicates that the combined composition is a promising formulation to address this unmet need of current vaccines (Lu, 2019). This is the first time that CDA, alone or in combination with HBcAg, has demonstrated an immunostimulatory effect on the avidity of the response. Petition 870250087282, dated 09 / 26 / 2025, page 41 / 80 32 / 50 immune. This characteristic of the resulting immune response is essential, in particular, against viruses with the ability to escape or evade immune surveillance mechanisms and, consequently, suppress the induction of high avidity and an effective adaptive response, demonstrating the industrial applicability of the new object of the present invention (see Figure 2). Example 4. Functionality of the resulting immune response: inhibitory effect of antibodies induced in the antigen-receptor interaction.

[084] A cyclic dinucleotide (CDN) such as c-di-AMP has demonstrated a surprising ability to overcome unresponsiveness and increase the avidity of the antibody response in a formulation comprising subunit antigens with physical and chemical nature that result in unresponsiveness after multiple intranasal (IN) immunizations. Thus, c-di-AMP was able to overcome unresponsiveness in the studied scenario, also inducing a strong, rapid, and highly avid antibody response in the individual who would otherwise exhibit low or no responsiveness. Thus, we studied the effect of the antibody response on the inhibitory activity of ACE2 binding.

[085] The resulting antibody response is capable of inhibiting the binding of the antigen to its cellular receptor ACE2, a process that resembles the interaction of the virus with its cellular receptor. This inhibitory effect is essential to ensure the strong antiviral effect of the immune response against an infectious agent / virus through specific neutralization activity. A strong inhibitory effect induced by CDA or CDA combined with HBcAg (Fig. 3C and D) was demonstrated when compared to the group immunized only with RBD (Fig. 3B). This functional aspect of the antibody response is linked to the higher quality of the same response in terms of avidity. The antibody response induced in G12, the group immunized via the IM route, was notably infectious. Petition 870250087282, dated 09 / 26 / 2025, page 42 / 80 33 / 50 higher in terms of inhibitory effect on RBD-ACE2 recognition. Although the G12 results were demonstrated in four serum sample pools due to limited sample availability, the significantly reduced inhibitory effect on ACE2 binding was clearly demonstrated compared to the groups with CDA or combined CDA / HBcAg formulations (Fig. 3E). The quality of the experiment was ensured by using a McAb on the standard curve of the five plates used in the study and the reproducibility of the inhibitory effect (Fig. 3F), as well as by evaluating the inhibitory ACE2 binding in a group that received CDA placebo but no antigen (Fig. 3A). Example 5. Functionality of the resulting immune response: SARS-CoV-2 neutralization activity of induced antibodies.

[086] In example 1, c-di-AMP (CDA) efficiently averted the lack of response against RBD and was subsequently shown to increase the avidity of the antibody response induced with a formulation containing antigens of a physical and chemical nature, resulting in lack of response after multiple intranasal (IN) immunizations when used in the absence of this BRM.

[087] c-di-AMP has demonstrated a surprising ability to overcome the absence of response, as well as to ensure the quality of the immune response, inducing a strong, rapid, and highly avid antibody response via the nasal immunization route, capable of inhibiting the ACE2 binding capacity of the recombinant RBD protein. In the present example, we study the effect of the formulations described in Example 1 on the resulting SARS-CoV-2 virus neutralization activity.

[088] The viral neutralization assay was conducted in groups 1, 2, 4, 6, and 11. The samples were analyzed individually. Viral neutralization responses (viral neutralization vs. Wuhan isolate) Petition 870250087282, dated 09 / 26 / 2025, page 43 / 80 34 / 50 were detected only in groups: G6 (RBD + CDA) and G11 (RBD + CDA + HBcAg). In both groups, almost all samples showed saturated levels at a dilution of 1:160 (see Fig. 4).

[089] An undetectable response was obtained at a 1:5 dilution in the following groups: G1 (PBS); G2 (CDA Placebo) and G4 (RBD 50 ug IN without CDA) (all mice were negative at a 1:5 dilution). The SARS-CoV-2 neutralization response was consistent with the ACE2 inhibitory activity demonstrated in Example 4.

[090] CDA BRM was able to induce a strong anti-RBD response when mixed with the protein produced in Pichia pastoris and this response demonstrated the functionality of the antibody response by inducing strong neutralization of the virus against the homologous strain isolated from Wuhan. Group 4 was not able to generate an antibody response, even at the RBD dose of 50 ug / mouse, and showed similar behavior to Placebo (G2) and the negative control (G1).

[091] These results further reinforce the idea that Balb / c mice do not respond to Pichia RBD via IN in the absence of CDA and the novelty of the surprising findings that demonstrate that, with the use of CDA or its formulation with HBcAg, the antibody response a) is induced; b) is strong, c) has stronger avidity compared to the response traditionally induced by the IM route in alum, with remarkable functional activity in terms of ACE2 inhibitory activity, as well as virus neutralization capacity. Example 6. Functionality of the resulting immune response: SARS-CoV-2 neutralization activity in various SARS-CoV-2 variants.

[092] c-di-AMP demonstrated a surprising ability to overcome the absence of response, as well as to ensure the quality of the immune response, inducing a strong, rapid, and highly avid antibody response via the nasal immunization route, capable of inhibiting the capacity Petition 870250087282, dated 09 / 26 / 2025, page 44 / 80 35 / 50 ACE2 binding capacity of recombinant RBD protein and neutralization of SARS-CoV-2 virus in vitro. In the present example, we studied the effect of the formulations described in Example 1 on the neutralization of different viral strains of SARS-CoV-2 virus. To this end, different viral variants were tested. The original Wuhan strain and the alpha, delta, and omicron variants were studied. The in vitro study showed a neutralizing effect against the different variants in serum samples from G6 and G11. No neutralizing activity was detected in serum samples from immunized mice without CDA. Example 7. Regarding the immune response induced on the mucosal surfaces of the respiratory tract.

[093] To better characterize the immune response induced by the formulations described in Example 1, nasal and pulmonary lavage samples were collected from mice treated with three doses of RBD or placebo, following the scheme and formulations described for G1, G2, G4, G6 and G11.

[094] Following the same response pattern, only the groups immunized with the RBD formulated with CDA (G6) or with the combined formulation with CDA / HBcAg / RBD were able to induce a positive anti-RBD immune response, both in nasal and pulmonary lavage (Fig. 5A). The intensity of the IgA response was higher in G6, although the inhibitory effect on ACE2 binding was greater in G11 (Fig. 5B). However, the apparent difference may be explained by the detectable levels of IgG in the pulmonary lavage (data not shown).

[095] The current results are consistent with other results, as well as with the state of the art, which considers that, to achieve control of a pathogen that infects the mucosa, the most appropriate scenario is to develop an immune response capable of protecting Petition 870250087282, dated 09 / 26 / 2025, page 45 / 80 36 / 50 both the mucosa and the systemic compartment, since injectable products are not suitable for preventing person-to-person transmission. The mechanism that explains the surprising effect of overcoming non-responsiveness, a novel effect induced by CDA, as well as the resulting impact of CDA or the CDA-HBcAg formulation on the intensity, quality, and functionality of the immune response, deserves further study and understanding. Example 8. Formulations for nasal administration.

[096] Several dose escalation studies were designed and conducted in order to find the ideal CDA dose conditions using RBD with sequence isolates from Wuhan and Omicron. The results reaffirmed the findings of a specific concentration range for the CDA component. c-di-AMP acts within an ideal concentration range, allowing for a stronger immune response from 0.1 mg / mL to 0.2 mg / mL of c-di-AMP, in both single and combined formulations (CDA / HBcAg) for both variants of the same antigen.

[097] Subsequent studies have shown that the formulations in this study could be administered nasally and parenterally. In the case of parenteral administration, preferably intramuscularly, as part of a formulation with alum and other formulations, as well as mixed at the time of product administration. Similar results have been confirmed using the S protein of different coronaviruses, including using the combination of RBD and S antigens and others from the same virus, and in combined vaccines as part of a multivalent vaccine for various respiratory infections.

[098] The formulations of the present invention were administered in accelerated immunization schedules: weekly or every 2 weeks, demonstrating a strong immune response induced in serum and mucosal secretions. The use of CDA or CDA / HBcAg was tested. Petition 870250087282, dated 09 / 26 / 2025, page 46 / 80 37 / 50 satisfactorily as a booster to existing vaccines. The formulations were applied satisfactorily in people already exposed to SARS-CoV-2 or during the early stages of infection, as part of a post-exposure prophylaxis treatment. Example 9. Study of different immunization routes.

[099] To characterize the immune response induced by formulations 6 and 11 described in Example 1, groups of 8 mice were administered via intranasal, sublingual, intratracheal, pulmonary, intramuscular, subcutaneous, intradermal, transcutaneous, and perifollicular routes. As control groups, mice received the excipient(s), RBD, and HBcAg at the dose and concentrations used in groups 6 or 11, in the absence of CDN. Only the groups that received CDN and RBD developed a specific immune response to RBD. The order of immunogenicity according to mucosal routes was: 1) IN, 2) pulmonary, 3) intratracheal. In the case of parenteral routes, 1) IM, 2) subcutaneous, 3) intradermal, 4) transcutaneous, and 5) perifollicular. Example 10. Effect of different CDNs on the resulting biological response.

[0100] To characterize the effect of the nature of CDNs on the resulting biological response, formulations 6 and 11 described in Example 1 were administered intramuscularly (IN) to groups of 8 mice, with or without the RBD antigen, each group consisting of a different CDN.

[0101] Analysis of the immune response revealed that all CDNs can behave as biological response modifiers, capable of subverting non-responsiveness against RBD and promoting antibody responses with strong avidity, inhibitory activity, and viral neutralization effect. However, some differences emerged, demonstrating that cyclic bis(3'-5')diodenillic acid (CDA) or c-di-AMP induced superior responses in terms of antibody response and Petition 870250087282, dated 09 / 26 / 2025, page 47 / 80 38 / 50 avidity / inhibition and neutralization against SARS-CoV-2. The remaining CDNs under study induced a lower response in the following order of intensity: 2) c-di-IMP, 3) c-IAMP or 4) CDA thiophosphate, 5) c-di-IMP thiophosphate and 6) c-IAMP thiophosphate. Example 11. The use of BRM in pre / post-exposure prophylaxis of infectious diseases.

[0102] SARS, influenza A virus (IAV), respiratory syncytial virus, rhinovirus, and dengue virus are pathogens that cause acute and / or febrile respiratory infections and have been adapted to produce infections in animal models. The use of formulations containing exclusively the biological response modifier (CDA) or its combination with HBcAg produced a quantitative reduction in viral titers of SARS and IAV in models of individuals with acute infection and infected and treated cells in vitro. In vivo treatment with BRMs (CDA or CDA / HBcAg) induced a high-avidity antibody response against viral antigens and the induction of antibody titers capable of neutralizing these viruses. The effect was detected when CDA or the CDA / HBcAg combination was administered before or after infection, demonstrating the pre- and post-exposure prophylactic effect.Similar results have been found in the context of chronic infectious diseases associated with non-responsiveness, where viruses and major HBV and HIV antigens are reduced in in vitro studies, expanding the scope of the present invention to the field of acute or febrile respiratory infections, as well as the field of chronic infectious diseases. In vivo concentrations of CDA or its CDA / HBcAg combination are similar to the optimal concentrations obtained with the RBD model antigen, both in the context of vaccine formulations and in the context of post-exposure / early therapy, where CDA or the CDA / HBcAg combination can be used without the presence of the antigen. Petition 870250087282, dated 09 / 26 / 2025, pp. 48 / 80 39 / 50 Example 12. Self-administration of formulations comprising CDN

[0103] Formulations comprising CDN and RBD were administered by a trained person or self-administered in a prospective, controlled, randomized clinical trial. The effect on the resulting immune response was studied in serum samples drawn after a 1-dose booster in individuals immunized via IM and also in a three-dose regimen of the IN formulation at two- or four-week intervals. All groups were studied by partially immunizing individuals with trained nurses or, alternatively, using the same procedure as a self-administered product. No differences were found between immunized and self-immunized individuals, suggesting that the use of compositions containing BRM can be self-administered. Self-administration is an attractive feature of these compositions when administered via IN. Example 13. CDA dose escalation in formulations with HBcAg / RBD or RBD.

[0104] Thirteen groups of female Balb / c mice (6 per group in groups G1 to G3 and 8 per group in groups G4 to G13) were immunized on days 0, 14, and 28 via IN (G1 to G11) and IM (G12 and G13) routes. The dose and concentration of RBD, Alum, CDA, and HBcAg are presented in Table 3, including the formulations studied. The immune response was evaluated after bleeding the mice on day -3 (pre-immune) and after the second (day 25) and third doses (days 39, 67, 95, 131, and 151). In the absence of CDA (G3, Figures 6 to 9), anti-RBD antibodies were not induced, thus reproducing the effect found in previous studies. The application of CDA generates detectable immune responses in CDA-RBD or CDA-RBDHBcAg formulations, reversing the non-responsive state in the recipient organism. Petition 870250087282, dated 09 / 26 / 2025, p. 49 / 80 40 / 50 intramuscularly. The intensity of the anti-RBD IgG antibody response depends on the CDA dose and increases significantly with co-administration of HBcAg (G8 to G11, respectively). RBD formulations containing CDA (G4 to G7), CDA + HBcAg (G8 to G11) and Al(OH)3 (G12), and AL(OH)3 + CDA (G13) induced specific anti-RBD antibodies, which remained in circulation for several months in a follow-up study after the third immunization (Fig. 7).

[0105] Co-administration of CDA + Al(OH)3 and RBD as a combined formulation, via IM, increased the intensity of the anti-RBD response compared to the RBD + Al(OH)3 formulation (without CDA). The discovery of immune responses via the IM route confirmed that the absence of response (non-responsiveness state) depends on the immunization route, suggesting that antigen uptake via the IN route depends on different mechanisms and non-responsiveness depends on the immunization route, although responses are increased with the addition of CDA and, consequently, the functionality of the antibody response has a drastic increase, as demonstrated by the increased inhibitory function of the induced antibodies (G12 vs. G13).It was surprising to discover that similar or higher levels of anti-RBD IgG induced via the IN route induced a stronger ACE2 inhibitory response compared to group 12, immunized with alum hydroxide via the IM route, demonstrating the superiority of the formulation in terms of immune response functionality. This may be due to the increased avidity of the resulting antibody response, as demonstrated in this application, since a similar antibody response is unable to induce a significant ACE2 inhibitory effect compared to the nasally induced groups (G7-G11 vs. G12). The proportion of mice with an antibody inhibitory response above 30% in the groups administered nasally with CDA was significantly higher compared to G12 (Figure 8). Petition 870250087282, dated 09 / 26 / 2025, pages 50 / 80 41 / 50 Table 3. Description of immunization formulations and schedules, Example 13. Grupo Formulações (Concentração dos componentes dissolvidos em PBS) Dose (pg / camundon gos) Vol. / camun dongos (pL) Via N 1 CDA: 0,1mg / mL CDA: 5 50 IN 6 2 HBcAg: 0,2 mg / mL HBcAg: 10 50 IN 6 3 RBD: 0,25 mg / mL RBD: 12,5 50 IN 6 4 RBD: 0,25 mg / mL + CDA: 0,05 mg / mL RBD: 12,5 CDA: 2,5 50 IN 8 5 RBD: 0,25 mg / mL + CDA: 0,1 mg / mL RBD: 12,5 CDA: 5 50 IN 8 6 RBD: 0,25 mg / mL + CDA: 0,15 mg / mL RBD: 12,5 CDA: 7,5 50 IN 8 7 RBD: 0,25 mg / mL + CDA: 0,2 mg / mL CDA: 10 50 IN 8 8 RBD: 0,25 mg / mL + CDA: 0,05 mg / mL + HBcAg: 0,2 mg / mL RBD: 12,5 CDA: 2,5 HBcAg: 10 50 IN 8 9 RBD: 0,25 mg / mL + CDA: 0,1 mg / mL + HBcAg: 0,2 mg / mL RBD: 12,5 CDA: 5 HBcAg: 10 50 IN 8 10 RBD: 0,25 mg / mL + CDA: 0,15 mg / mL + HBcAg: 0,2 mg / mL RBD: 12,5 CDA: 7,5 HBcAg: 10 50 IN 8 11 RBD: 0,25 mg / mL + CDA: 0,2 mg / mL + HBcAg: 0,2 mg / mL RBD: 12,5 CDA: 10 HBcAg: 10 50 IN 8 12 RBD: 0,125 mg / mL + Al(OH)s: 0,5 mg / mL RBD: 12,5 Al(OH)3: 50 100 IM 8 13 RBD: 0,125 mg / mL + Al(OH)3: 0,5 mg / mL + CDA: 0,1 mg / mL RBD: 12,5 Al(OH)3: 50 CDA: 10 100 IM 8 Petition 870250087282, dated 09 / 26 / 2025, pp. 51 / 80 42 / 50

[0106] Only with the inclusion of CDA in RBD formulations via the IN route are anti-RBD antibodies detected, and only with the effect of CDA does the antibody response have sufficient avidity to inhibit the interaction of RBD with its ACE2 receptor (Fig. 8) and neutralize the virus in vitro. Analysis of the ideal dose showed that the use of 10 pg of CDA per mouse was ideal when the antigen was administered in similar proportions (10 pg of CDA and 12.5 pg of RBD), while the dose of 5 pg of CDA induced the strongest immune response when the RBD antigen was administered at a dose of 50 pg (1 mg / mL), a surprising characteristic of the new composition, as demonstrated previously. In this regard, we could consider the preferred concentration ranges for RBD / CDA formulations, as described in this application: from RBD: 0.25 mg / mL / CDA: 0.2 mg / mL up to RBD: 1.0 mg / mL / CDA: 0.1 mg / mL and, in the case of CDA / RBD / HBcAg formulations, adding HBcAg up to 0.2 mg / mL.

[0107] In summary, the results presented further confirm that CDA is necessary to (A) subvert the non-responsiveness to Pichia-derived RBD antigen via IN, as well as to (B) induce a high-intensity / avidity antibody response and also to induce a (C) functional antibody response, considering the effect on the inhibition of ACE2-RBD interaction and viral neutralization. To achieve a robust or even detectable anti-RBD antibody response after intranasal administration, the presence of CDA was necessary. The antibody response induced with the formulations of the present invention is functional in terms of the inhibitory effect on the RBD-ACE2 interaction and in terms of suppressing viral replication in in vitro models of viral neutralization. The biological response-modifying effect of CDA produces an effect on the antigen receptor, resulting in increased avidity of the induced response, as demonstrated previously. Petition 870250087282, dated 09 / 26 / 2025, pp. 52 / 80 43 / 50

[0108] In summary, the interaction of adding CDA, optionally in combination with HBcAg, and the IN route allows for the superior response demonstrated in this example. It is assumed that the combination of these factors contributed to a qualitative change in the antibody response, resulting in a high avidity response, which, in turn, is critical for vaccine efficacy. This aspect of antibody avidity is highly relevant in patients in terms of clinical progression, as already discussed and referenced in the introduction above. Furthermore, in contrast to the previous technique with the intramuscular route, the combined administration of HBcAg IN with CDN has been shown to improve the antibody response. Example 14. CDA and CDA / HBcAg BRM formulations as anti-infective agents.

[0109] In vitro stimulation of cells using CDA or its combinations with HBcAg demonstrated an increase in different channels of stimulatory effects and a synergistic in vivo effect in terms of induction of type I antiviral and type 2 immunomodulatory IFN, resulting in the in vitro elimination of multiple viruses, confirming that combined formulations of both BRMs can be used in post-exposure prophylaxis or early therapy of elderly and immunosuppressed individuals, considering the direct antiviral effect due to their greater interferon-inducing capacity.

[0110] The formulations of CDA and HBcAg BRMs were studied according to the description in Table 4. Antigen X represents antigens of viruses causing acute (SARS, IAV, RSV, RV, Dengue) and chronic (HBV, HIV, HCV, HPV) infections in a dose range of 10 to 200 micrograms, in all cases formulated in phosphate-buffered saline excipients. The antigens studied encompassed the range of inactivated pathogens (such as, for example, the inactivated viruses described above), as well as subunit antigens. Petition 870250087282, dated 09 / 26 / 2025, pp. 53 / 80 44 / 50 The avidity of some of these viruses, for example, the hepatitis B surface antigen, preferably those produced in host cells of Pichia pastoris or E. coli. In general, the avidity of the antibody response, as well as its functional capacity to neutralize or inhibit interaction with its target cellular receptors, proved to be superior in the case of the described formulations from G3 to G8. The use of multiple agonists of innate immune system receptors, synergistic enhancers of IFN responses, has potential in post-exposure prophylaxis of multiple respiratory pathogens, with particular interest in pandemic scenarios, where multiple respiratory pathogens affect humans or other animal species. Table 4. Description of the formulations studied in vivo and their administration schedules. Group Formulations (Concentration of components dissolved in PBS) Treatment Regimen 1 (route) Treatment Regimen 2 (route) 1 CDA: 0.1 mg / mL Days 1 to 7 (IN, SL or combined) 10 doses every 1 / 2 weeks (IN) 2 CDA: 0.2 mg / mL Days 1 to 7 (IN, SL or combined) 10 doses every 1 / 2 weeks (IN) 3 CDA: 0.1 mg / mL + HBcAg: 0.1 mg / mL Days 1 to 7 (IN, SL or combined) 10 doses every 1 / 2 weeks (IN) 4 CDA: 0.2 mg / mL + HBcAg: 0.2 mg / mL Days 1 to 7 (IN, SL or combined) 10 doses every 1 / 2 weeks (IN) 5 CDA: 0.1 mg / mL + HBcAg: 0.1 mg / mL + Ag X Days 1 to 7 (IN, SL or combined) 10 doses every 1 / 2 weeks (IN) 6 CDA: 0.2 mg / mL + HBcAg: 0.2 mg / mL + Ag X Days 1 to 7 (IN, SL or combined) 10 doses every 1 / 2 weeks (IN) Petition 870250087282, dated 09 / 26 / 2025, pp. 54 / 80 45 / 50 Group Formulations (Concentration of components dissolved in PBS) Treatment Regimen 1 (route) Treatment Regimen 2 (route) 7 CDA: 0.1 mg / mL + HBcAg: 0.1 mg / mL + Ag X Days 1 to 7 (IN, SL or combined) 10 doses every 1 / 2 weeks (IN) 8 CDA: 0.2 mg / mL + HBcAg: 0.2 mg / mL + Ag X Days 1 to 7 (IN, SL or combined) 10 doses every 1 / 2 weeks (IN) 9 HBcAg: 0.1 mg / mL Days 1 to 7 (IN, SL or combined) 10 doses every 1 / 2 weeks (IN) 10 HBcAg: 0.2 mg / mL Days 1 to 7 (IN, SL or combined) 10 doses every 1 / 2 weeks (IN) 11 Ag X Days 1 to 7 (IN, SL or combined) 10 doses every 1 / 2 weeks (IN) 12 Ag X Days 1 to 7 (IN, SL or combined) 10 doses every 1 / 2 weeks (IN) Example 15: The use of CDA eliminates the non-responsiveness of the cells- las T in mice immunized with RBD.

[0111] In example 1, several vaccine formulations were studied to select the composition with the most attractive immune response for SARS-CoV-2 prevention. The receptor-binding domain (RBD) protein was used in combination with another MRB (HBcAg). The RBD protein was obtained as a recombinant glycoprotein in Pichia pastoris, with the characteristic mannosylated glycosylation pattern, and used as an immunogen in example 1.

[0112] To explore the effect of CDA and its combination with HBcAg on the T-cell response after nasal administration, CDA and hepatitis B core antigen (HBcAg) were also used in RBD formulations. After the third dose, splenocytes from mice immunized with placebo, CDA, RBD, RBD-CDA, and RBD-CDA-HBcAg were studied for their ability to secrete IFN and other cytokines. Petition 870250087282, dated 09 / 26 / 2025, pp. 55 / 80 46 / 50 nas in response to stimulation with the homologous RBD (produced in Pichia pastoris) and the heterologous antigen (produced in CHO). ConA was used as a positive control and a similar quantity of cells was used as a negative control and received no treatment.

[0113] As a result, nasal administration of RBD in PBS resulted in an undetectable response of secreted cytokines when cells were treated with homologous (Pichia) or heterologous (CHO) RBD antigens. On the other hand, mice immunized with RBD antigen in combination with CDA or CDA-HBcAg induced a strong response in terms of multiple cytokine secretion, at the level of the positive ConA control. These results are described in Figures 9A and 9B, as representative examples of the different cytokines studied, evidencing a mixed Th1 / Th2 response pattern.

[0114] Consistent with the strong Ab response evidenced in examples 1 and 13, and with the T cell response that sustained the high avidity and long duration of the induced titers, the ability of the induced Ab responses to neutralize SARS-CoV-2 of different viral variants was detected, suggesting that increased avidity may sustain the ability of induced antibodies to cross-neutralize against different viral isolates (Fig. 10). Example 16: The use of CDA overrides the non-responsiveness of T cells from mice immunized with dengue virus core proteins.

[0115] Mice with an immune defect in the innate immune receptors described in Example 1 are unable to generate a detectable T-cell immune response specific to dengue virus core antigens of all 4 serotypes when given the antigens administered IN or IM in PBS or alum, respectively. However, co-administration of CDA in the formulation with ca Petition 870250087282, dated 09 / 26 / 2025, pp. 56 / 80 47 / 50 of one of the four core antigens of the 4 distinct serotypes of Dengue virus induced the secretion of high levels of IFN-gamma in the supernatant of splenic cells from immunized mice. Similar responses were detected in the multivalent vaccine with the four different antigens administered together and mixed with CDA and alum or via IN in PBS. The response in formulations comprising CDA as an adjuvant to the monovalent and multivalent composition of dengue virus core antigens was also detected by lymphoproliferative assays for CD8+ and CD4+ T cells. Example 17

[0116] The effect of combining the two compounds HBcAg and CDA on the immune response against Hepatitis B Surface Antigen (HBsAg) was studied in a group of 15 non-human primates (NHP) immunized intranasally with two formulations comprising HBsAg, the surface antigen of the Hepatitis B Virus, obtained from the yeast Pichia pastoris using recombinant DNA technology, and a placebo control group. The first group received HBsAg in combination with two BRMs (HBcAg and CDA), and in the second group, the NHP received a formulation containing only HBcAg as the BRM. As a placebo control group, an unrelated antigen was formulated with the two BRMs. The three groups consisted of 5 animals. The primates (Macaca fascicularis) were inoculated with a dose of 50 pg of HBsAg, as well as 50 pg of HBcAg and CDA, in addition to the phosphate-buffered saline used as an excipient in the three formulations under study.

[0117] After two doses of the formulations, administered two weeks apart, blood samples were collected on day 26. The study of antibody levels against HBsAg showed a strong and widespread response in the group immunized with HBsAg in the formulation containing CDA and HBcAg (100% seroconversion and seroprotection). Petition 870250087282, dated 09 / 26 / 2025, pp. 57 / 80 48 / 50 with an average level well above 10 mIU / mL, while the group without CDA did not induce a detectable response, as was the case with the placebo group. It was concluded that the inclusion of CDA in combination with HBcAg generated a strong maturation of the antibody response at a very early date, while in the absence of CDA the response was undetectable. These results highlighted the effect of CDA, in addition to the effect of HBcAg, which will be adequate to overcome the state of non-responsiveness, as in the case of patients with chronic hepatitis B, as well as in the context of healthy individuals with a subideal antibody response (non-responders to the commercial hepatitis B vaccine).

[0118] The anti-HBsAg response induced in a group of 15 non-human primates (NHP) immunized intranasally with two formulations comprising HBsAg was designed to evaluate the effect of the combination of the two compounds HBcAg and CDA on the immune response against Hepatitis B Surface Antigen (HBsAg). The antigen dose was 50 μg, as was the CDA dose. The inclusion of CDA in combination with HBcAg generated a strong maturation of the response to Ab soon after immunization (day 26), while in the absence of CDA the response was undetectable, as in the placebo control group. Table 5: Summary of experiments Groups No. of NHP Response to Anti-HBsAg IgG (Day 26, 2nd dose) 1: HBsAg + (HBcAg / CDA) 5 100% of NHP responded with anti-HBsAg titers above 10 mIU / mL (seroconversion and seroprotection) 2: HBsAg + HBcAg 5 0% of NHP responded with anti-HBsAg titers above 10 mIU / mL 3: HBcAg / CDA + unrelated antigen (RBD) 5 0% of NHP responded with anti-HBsAg titers above 10 mIU / mL Petition 870250087282, dated 09 / 26 / 2025, pages 58 / 80 49 / 50 Example 18

[0119] The immune response induced after a booster dose after A 220-day study, comprising the RBD in formulation with the BRMs CDA or CDA / HBcAg, was conducted in previously immunized mice, as described in Example 13, Table 3. The formulations used in the booster study are described in Table 5 below. The first four mice in each group were immunized with the recombinant RBD (Wuhan variant) and the second half received the RBD (Omicron BA.1 variant). Table 6: Dose of each antigen or BRM used for booster immunization. All booster doses were administered intranasally. Groups Dose of each antigen or BRM used for boosting (IN route) 1 10 pg CDA, (Placebo) 2 10 pg HBcAg, (Placebo) 3 RBD (Wuhan / Omicron) 12.5 pg (control group, RBD only) 4-7 RBD (Wuhan / Omicron) 12.5 pg + 10 pg CDA 8-11 RBD(Wuhan / Omicron) 12.5 pg + 10 pg CDA+ 10 pg HBcAg 12 and 13 RBD(Wuhan / Omicron) 12.5 pg + 10 pg CDA

[0120] In general, animals vaccinated intranasally (G1-11) and parenterally (G12, 13) showed a booster effect after administration of IN formulations comprising RBD / CDA or RBD / CDA / HBcAg. After the booster dose, the RBD IN group still did not respond (G3), while the booster effect was detected in all groups that received CDA or CDA / HBcAg via IN in the priming immunization schedule, as well as in those that received the IM formulations (Fig. 11), demonstrating the effectiveness of both RBD / CDA and RBD / CDA / HBcAg formulations in reinforcing the response induced by IN or IM routes. However, a stronger booster effect was detected. Petition 870250087282, dated 09 / 26 / 2025, pp. 59 / 80 50 / 50 due to the use of formulations combining CDA and HBcAg in the same formulation. Similar results were also detected at the pseudovirus neutralization assay level, where G11 showed the strongest induction of neutralizing antibodies. Interestingly, the antibody response induced by IN immunization with the Wuhan variant of the RBD boosted with the same variant induced a statistically similar response compared to the groups that received the Omicron variant RBD at the booster dose, see Fig. 12. This suggests that the titers generated with the original variant, under the studied conditions, remain neutralizing against variants that emerged more than two years later, which may be of interest to extend the duration of vaccine efficacy against variants that emerge later and increase the industrial applicability of the product for a longer period.

Claims

1. Cyclic dinucleotide (CDN) for use as a biological response modifier to enhance the immune response of individuals in need thereof, with low-performing immune systems due to immunodeficiency caused by age, a chronic condition, or due to medical or therapeutic interventions, characterized by low / weak or non-responsiveness to vaccine antigens or low-performing immune response to pathogens, wherein the CDN is administered via the mucosal route.

2. CDN for use according to claim 1, characterized in that it is a CDN according to formula I: R2 R2 (I) wherein A, A' are independently of each other, S or O; X is independently of each other, S, N, O, Chh; Y, Y' are independently of each other, NH, Chh, O; Z, Z' are independently of each other, NH, CH2, O; R1 is independently of each other, hydrogen, NH2, S or O; R2 is independently of each other, NH2, O, H or S; R3 is absent, independently of each other, if there is a covalent bond between atom Z or Z' and atom C, or if it is Petition 870250087282, dated 09 / 26 / 2025, p. 61 / 80 2 / 7 hydrogen, OH, halogen, a straight or branched C1-C6 alkyl group, or a straight or branched C1-C6 alkoxy group which may be optionally substituted; R4 is, independently of each other, hydrogen, halogen or a straight or branched C1-C6 alkyl group which may be optionally substituted; ...It is a single or double bond; or conjugates thereof, and salts or solvates thereof.

3. CDN for use according to claim 1 or 2, characterized in that it acts as a biological response modifier in combination with at least one other biological response modifier.

4. CDN for use according to claim 3, characterized in that at least one different biological response modifier is the hepatitis B nucleocapsid antigen (HBcAg).

5. CDN for use according to any one of claims 2 to 4, characterized in that, in the compound according to formula I, any of the purine residues is an adenine, xanthine or hypoxanthine residue or combinations thereof, in particular, both purine residues being adenine.

6. CDN for use according to any one of claims 2 to 5, characterized in that, in formula I, R3 is an OH group, X is an oxygen atom and Y, Y', Z and Z' are oxygen.

7. CDN for use according to any of claims 2 to 6, characterized in that the compound according to formula I is a dimeric bis-(3'-5')cyclic adenosine monophosphate (CDA or c-di-AMP), c-di-GMP, c-di-IMP, c-di-GAMP, c-IAMP or a cyclic di-AMP thiophosphate, c-di-IMP thiophosphate and c-IAMP thiophosphate. Petition 870250087282, dated 09 / 26 / 2025, p. 62 / 80 3 / 7 8. CDN for use in accordance with any of the preceding claims, characterized in that it is administered via the mucosal route, the mucosal route being selected from intranasal, sublingual, intratracheal or pulmonary.

9. CDN for use in accordance with any of the preceding claims, characterized in that it enhances the immune response by boosting the germinal center reaction at the time of contraction to further promote hypersomatic antibody mutation and affinity maturation.

10. CDN for use in accordance with any of the preceding claims, characterized in that it is for generating a rapid immune response as part of vaccine compositions administered in accelerated treatment schedules, specifically weekly, bi-weekly or monthly administration schedules.

11. CDN for use in accordance with any of the preceding claims, characterized in that it is administered as a biological response modifier as part of a prime-booster or prime-pull vaccination schedule.

12. CDN, characterized in that it is for use, according to any one of claims 1 to 11, (i) in establishing epidemic or pandemic immunostimulation, where the biological response modifier is administered alone or in combination with another biological response modifier in a pre / post-exposure prophylaxis context in the absence of any vaccine antigen, or (ii) in formulation with the antigens of an infectious agent from the group of chronic infectious diseases, or (iii) in formulation with the antigens of pathogens from the group of acute and / or febrile respiratory infections.

13. CDN for use in accordance with any of claims 1 to 12, in vaccine formulations, characterized by the fact that Petition 870250087282, dated 09 / 26 / 2025, page 63 / 80 4 / 7 that it comprises one or more viral antigens, such as coronavirus, in particular SARS-CoV-2 antigens.

14. CDN for use in accordance with any of claims 1 to 13, in vaccine formulations, characterized in that it comprises the S (Spike) antigen of the coronavirus or the RBD subunit antigen.

15. CDN for use in accordance with any of claims 1 to 13 for vaccination against HBV, characterized in that the antigen is HBV.

16. CDN for use according to claim 15, characterized in that the HBV antigen is HBsAg.

17. CDN for use according to any of the preceding claims, characterized in that it is for immunizing an individual with reduced responsiveness to RNA viruses and their variants, such as SARS-CoV-2 or other beta-coronaviruses, due to immune senescence or immunosuppression, particularly in combinations with other biological response modifiers capable of further enhancing the effect of the CDN, allowing dose splitting or adding complementary properties to the resulting formulation, such as where the other biological response modifier corresponds to the hepatitis B nucleocapsid antigen or core, with a complementary multi-TLR agonist effect to split the effective dose and further stimulate the innate immune system and nonspecific immunity, of value in immunocompromised or vulnerable patients, or in the elderly or immunosenescent population.

18. Formulations of a biological response modifier according to any of the preceding claims, characterized in that the dose of CDN corresponds to the range between 0.01 and 0.4 mg per dose, and the formula may additionally comprise HBcAg in a similar preferred range and may also contain a selected antigen in a dose range of 0.01 to 0.2 mg, in an excipient such as, among others, phosphate-buffered saline solution.

19. Method for improving the immune response of an individual in need thereof with low-performing immune systems due to immunodeficiency caused by age, a chronic condition, or due to medical or therapeutic interventions, characterized by the fact that the individual presents low / weak or non-responsiveness to vaccine antigens or presents a low-performing immune response to pathogens, comprising the step of administering a cyclic dinucleotide (CDN) as a biological response modifier via mucosal route to said individual in need thereof for prophylactic or therapeutic vaccination.

20. A method for improving the immune response of an individual in need thereof, according to claim 19, characterized in that the CDN is a CDN as defined in any one of claims 2 to 7.

21. A method for improving an individual's immune response, as required by claim 19 or 20, characterized in that the vaccine composition is administered in accelerated treatment schedules, specifically weekly, bi-weekly, or monthly administration schedules.

22. A method for improving the immune response of an individual in need thereof, according to any one of claims 19 to 21, characterized in that the administration is conducted by boosting the reaction time of the germinal center contraction to further promote hypersomatic antibody mutation and affinity maturation.

23. Method for improving the immune response of an individual in need thereof, according to any of claims 19 to 22, characterized in that CDN is administered as a biological response modifier as part of a prime-booster or prime-pull vaccination schedule.

24. Method for enhancing the immune response of an individual in need thereof, according to any one of claims 19 to 23, characterized in that i) the method is a method for establishing epidermal or pandemic immunostimulation, wherein the biological response modifier is administered alone or in combination with another biological response modifier in a pre / post-exposure prophylaxis setting in the absence of any vaccine antigen, or ii) in a formulation with the antigens of an infectious agent from the group of chronic infectious diseases, or iii) in a formulation with the antigens of pathogens from the group of acute and / or febrile respiratory infections.

25. A method for enhancing the immune response of an individual in need thereof, according to any one of claims 19 to 24, characterized in that the CDN is administered as part of vaccine formulations comprising one or more viral antigens.

26. A method for enhancing the immune response of an individual in need of such response against SARS-CoV-2 infection, characterized in that it comprises the S antigen of the coronavirus or the RBD subunit antigen.

27. Method for improving the immune response of an individual in need thereof, according to any one of claims 19 to 25, characterized in that it improves the immune response against HBV in said individual, comprising the administration of vaccine formulations containing one or more HBV antigens, in particular, HBsAg. Petition 870250087282, dated 09 / 26 / 2025, pp. 66 / 80 7 / 7 28. A method for improving the immune response of an individual in need thereof, according to any one of claims 19 to 27, characterized in that it comprises the additional administration of HBcAg as a further modifier of the biological response.

29. A method for improving the immune response of an individual in need thereof, according to any one of claims 19 to 28, characterized in that the administration of the CDN dose corresponds to the range of 0.01 to 0.4 mg per dose, and the vaccine may contain additional HBcAg in the range of 0.01 to 0.4 mg per dose and may also contain a selected antigen in a dose range of 0.01 to 0.2 mg in a suitable excipient.

30. A method for improving the immune response of an individual in need thereof, according to any one of claims 19 to 29, characterized in that the components of the vaccine formulation are administered simultaneously, separately or sequentially.

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