Immunization system and use thereof, formulation and use thereof, intranasal vaccine, method to induce systemic and upper airway immunity against respiratory pathogens

Abstract

The present invention refers to a novel immunization platform / system and formulation comprising the targets of neutralizing antibodies against respiratory pathogens in the presence or absence of synthetic peptides containing multiple T-cell epitopes from respiratory pathogens, particularly the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein and T cell epitopes of SARS- CoV-2, wherein the immunization platform / system consists of carrier nanoparticles coated with a mucoadhesive polymer (MaP), selected as a delivery vehicle.

Description

IMMUNIZATION SYSTEM AND USE THEREOF, FORMULATION AND USE THEREOF, INTRANASAL VACCINE , METHOD TO INDUCE SYSTEMIC AND UPPER AIRWAY IMMUNITY AGAINST RESPIRATORY PATHOGENS FIELD OF THE INVENTION

[0001] The present invention refers to a novel immuni zation platf orm / system and formulation comprising the targets of neutralizing antibodies against respiratory pathogens in the presence or absence of synthetic peptides containing multiple T-cell epitopes from respiratory pathogens , particularly the receptor binding domain (RED) of the SARS-CoV-2 Spike protein and T cell epitopes of SARS-CoV-2 , wherein the immunization platf orm / system consists of carrier nanoparticles coated with a mucoadhesive polymer (MaP) , selected as a delivery vehicle .

[0002] Furthermore, the present invention refers to an intranasal nanovaccine comprising the disclosed novel immunization platf orm / system and formulation .

[0003] In addition, the present invention also refers to a method to induce systemic and upper airway immunity against respiratory pathogens and the use of the immunization platf orm / system and formulation herewith disclosed to prepare an intranasal vaccine .BACKGROUND OF THE INVENTION

[0004] Diseases caused by zoonotic respiratory pathogens remain a prominent threat to global public health . The COVID- 19 pandemic serves as the most recent and striking example of the profound impact respiratory infections can have on global populations worldwide .

[0005] In general , said type of virus attacks human cells by binding to the angiotensin-converting enzyme 2 (ACE2 ) cell entry receptor through its receptor binding domain (RED) of the Spike protein . ACE2 receptors are predominantly expressed in respiratory tract cells , making various respiratory pathogens , as SARS-CoV-2 , amucosal pathogen and respiratory diseases like for instance COVID- 19, a mucosal-borne disease .

[0006] When evaluating the response to the COVID-19 pandemic in the last years , it is impossible to overlook how successful the intramuscular vaccines based on traditional or modern platform technologies were in preventing severe COVID-19 and deaths , saving lives , and altering the pandemic course .

[0007] On the other hand, at this point, when the world is no longer dealing with the COVID- 19 pandemic but remains susceptible to the SARS-CoV-2 virus , it is important to reali ze that current intramuscular vaccines offer only short-term protection against respiratory viral replication and transmission, i f any . Moreover, the world population is still faced with the continuous emergence of variants of said respiratory pathogen .

[0008] Notably, a person who experiences breakthrough respiratory infections after intramuscular vaccination has peak nasopharyngeal viral loads comparable to those in unvaccinated individuals , indicating no mucosal protection is achieved with this kind of immunization .

[0009] It is known that a local mucosal response such as nasal secretory IgA is critical for respiratory virus neutralization in the upper respiratory mucosa and is inversely correlated with viral load and systemic disease symptoms in natural infection . However, intramuscular vaccines fail to induce or boost mucosal IgA at the site of virus entry which is the upper airway . This is the reason why intramuscular vaccines are unable to durably prevent breakthrough infection and interrupt onward transmission .

[0010] Therefore, it is critical to develop next-generation vaccines to induce an immune response to respiratory pathogens that can be rapidly deployed and capable of establishing robust IgA mucosal immune responses in the upper respiratory tract, designed tohalt pathogen growth at the first site of infection, to prevent pathogen replication, infection, and transmission, as well as to induce systemic neutralizing antibody and cellular immunity, thus preventing severe disease .

[0011] In this sense, developing mucosal vaccines against respiratory pathogens is an important option since it allows intranasal administration which is a convenient way to potentially stimulate both mucosal and systemic immune protection .

[0012] The intranasal route is of great advantage since trained healthcare personnel is not required and the population, in general , is more willingly bind to be vaccinated than when compared with the intramuscular route, which requires the use of needles . These factors can be of great help in boosting vaccination rates and controlling the outbreak of respiratory infections .

[0013] Regarding specifically the control of SARS-CoV-2 infections , attempts at intranasal delivery of unmodified mRNA, live-attenuated viruses , and viral vectors have not uni formly induced anti-SARS-CoV-2 IgA antibodies in the upper airways leading to a reduction in viral loads or providing protection from infection . The effectiveness of intranasal vaccines relies on carriers or vehicles that can overcome the anatomical and physicochemical barriers of the mucosa .

[0014] Research has focused on nanoscale materials for vaccination, targeting antigen delivery to penetrate the mucosal barriers and reach the nasal-associated lymphoid tissue (NALT ) with nanoparticles , aiming to stimulate the mucosal immune response .

[0015] Moreover, despite nanoparticles being safe, easy to produce, and cost-ef fective, only a limited number of studies have advanced to clinical investigation thus far . Most nanovaccines studied to date, including those approved for emergency use, incorporate nanoparticles that exhibit exclusively mucoadhesiveproperties . This enables them to adhere to the mucosal layer, thereby increasing the vaccine ' s retention time and bioavailability at the site of absorption / action .

[0016] There are , however, notable limitations to this approach : their transit time is dictated by the physiological renewal rate of the mucus layer, and the vaccine will move through the mucus based on its adhesion and diffusion properties .

[0017] In the state of the art, for example, in document EP1872794A1 , it is disclosed a nasal vaccine which would be capable of intranasally inducing an increase in antigen specific immunoglobulin observed in virus infection . Although said document suggests a nasal vaccine, it does not focus on nanoscale materials .

[0018] Document CN111840536A refers to nasal preparations ( sprays and drops ) mainly including microparticles , liposomes and nanoemulsion preparations . However, said document fails to suggest a technical solution to the problem of the transit time dictated by the physiological renewal rate of the mucus layer .

[0019] Therefore, the key requirement for an improved intranasal vaccine is to design a nanoplatform with an extended residence time on mucosal surfaces that can also ef ficiently cross the mucus barrier .

[0020] In this context, carrier nanoparticles with mucuspenetrating properties can overcome the mucus barrier, reach the mucosal epithelium, and di ffuse through the epithelial barrier .

[0021] Thus , combining both mucoadhesive and mucus-penetrating nanoparticulate systems could help achieve optimal intranasal transmucosal delivery .

[0022] The present invention discloses a technical solution which is a novel immuni zation platf orm / system and a formulation combining nanomaterials using muco-penetrating carrier nanoparticles coated with a mucoadhesive polymer, which facilitates the penetration ofsaid carrier nanoparticles and of the novel formulation in the nasal mucosa .

[0023] Moreover, the present invention also reveals an intranasal nanovaccine comprising said novel platform and formulation, a method to induce systemic and upper airway immunity against respiratory pathogens and the use of the formulation herewith disclosed to prepare an intranasal vaccine .BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0024] The present invention will be more clearly understood upon reading the following non-restrictive detailed description and the Sequence Listing as presented herein .SUMMARY OF THE INVENTION

[0025] The present invention refers to an immunization platf orm / system and formulation comprising the targets of neutralizing antibodies to respiratory pathogens - in the case of SARS-CoV-2 , the receptor binding domain (RED) of the Spike protein and multiple T-cell epitopes of the same respiratory pathogen, particularly SARS-CoV-2 antigens , combined with mucus-penetrating carrier nanoparticles coated with a mucoadhesive polymer to enhance mucosal immune responses .

[0026] In one embodiment of the present invention an intranasal nanovaccine comprising the immunization platf orm / system and formulation of the present invention is disclosed .

[0027] Particularly, the formulation of the present invention comprises targets of neutralizing antibodies to respiratory pathogens - receptor binding domain (RED) of the Spike protein- and multiple T-cell epitopes of respiratory pathogens , particularly SARS-CoV-2 antigens or a recombinant protein or other proteins , the most critical region of the Spike or other proteins inducing neutralizing antibodies , combined with synthetic peptides encoding multipleconserved, immunodominant SARS -CoV- 2 T cell epitopes from the enti re viral proteome . The formul ation al so contains an immunostimulatory adj uvant .

[0028] Moreover , the pre sent invention al so embodies a method to induce sys temic and upper airway immunity against respiratory pathogens compri sing admini stering the intranasal nanovaccine to a sub ect .

[0029] Further obj ect o f the present invention i s the use of the immuni zation platf orm / system and the formulation herewi th di sclosed to prepare an intranasal vaccine .BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure s 1 ( a ) to ( e ) - show the characteri zation o f SARS-CoV-2 nanoparti culate formulations . Figures 1 ( a , b ) re fer to the transmi s sion electron microscope analys i s of mucus -penetrating cNP . Regarding Fi gures 1 c to e : the repre sentative ( c ) particle si ze , ( d) pol ydi spersi ty index , and ( e ) zeta potential , as determined by dynami c li ght scattering ( DLS ) , o f empty mucus - penetrating cNP, antigens loaded into NP - NP-Ag , cNP-Ag coated with mucoadhesive polymer - cNP-Ag-MaP , and adj uvant incorporated into cNP-Ag-MaP -cNP-Ag-MaP-Ad j v .

[0031] Figure s 2 ( a ) to ( c ) show the intranasal nanovaccine residence time in the nasal cavi ty and anti gen biodis tribution, in which : ( a ) in vivo fluorescence image s of RED biodi stribution, comparing mice ( n = 3 / group ) nasal ly insti lled wi th a dos e of 1 ) Ag alone , 2 ) MaP-coated Ag, 3 ) cNP- carried Ag, and 4 ) Ag loaded onto cNP and coated by MaP , starting at time zero ( TO ) with reading every 30 minutes for 3 hours ( T 180 ) . The color gradient repre sents the range from the average f luore scence captured in minimum ( 1x10 ^ ) and maximum ( 6x10 ^ ) fluorescence photons / second (p / s ) acquired ( b ) The total flux of f luore scence acquired in the animal s with elapsing time . Figure s ( a , c ) show Ex vivo images of main organs , such as kidney( K) , liver ( LV) , lung ( LG ) and brain ( B ) , at 3 hours post nasal instil lation and corresponding mean fluorescence analys i s in the tested groups . The dotted line in fi gure C indicate s the lower limit of detection ( 4x 10 ^ p / s ) . Data in Fi gures (b , c ) were represented as mean value s ± SD, and are repres entative results from a s ingle experiment . Di f f erences between tested groups per time point or per organ were as ses sed us ing ANOVA followed by Tukey' s multiple compari sons tes t ( three or more groups ) or S tudent ' s t-tes t ( two groups ) . P < 0 . 05 was cons idered stati sticall y signi ficant : * P < 0 . 05 . Wherein TO - time zero ; T30a . 30 minutes ; T 60 - 60 minutes ; T 90 - 90 minutes ; T 120 - 120 minute s ; T 150 - 150 minutes ; T 180 - 180 minutes ; Ag - RBD antigen ; cNP mucus -penetrating inorgani c nanoparti cle ; MaP - mucoadhesive polymer .

[0032] Figure s 3 ( a ) to ( e ) show fluorescence o f nanoparti cle formul ations te sted with labeled antigen and control animal s . Fluore scence images of Figure 3 ( a ) tested formulations containing Cy . 5-l abeled anti gen and Figure 3 ( c ) shows animal s us ed as control ( naive ) . The color gradient represents the range from the average lumine scence captured in minimum ( 1 x 10 ^ ) and maximum ( 6 . 8 x 10 ^ ) fluore scence acquired . And corresponding total flux (photons / s econd [p / s ] ) anal ysi s in Fi gures 3 (b ) , the tested formul ations , ( d) animal s control and ( e ) thei r organs . Ag - RBD anti gen ; cNP - mucuspenetrating inorganic nanoparti cle ; MaP - mucoadhes ive polymer .

[0033] Figure s 4 ( a ) - ( b ) . A 14 -day interval between intranas al doses leads to higher serum I gG titers and enhanced neutral i zing antibody level s . After immuni zing C57BL / 6 mice ( n = 8 ) wi th the intranasal nanovaccine at interval s of 7 , 14 , or 28 days between doses , s era were collected one day be fore each dose , and 7 , 14 , or 28 days after the third dose . Analys i s of the serum : Figure 4 ( a ) shows I gG response speci fi c to the RBD by ELI SA, and Figure 4 (b ) showsneutrali zing capacity against Wuhan ( WT ) pseudovirus . Di f ferences between the animal groups were as ses s ed us ing Tukey' s mul tiple compari sons test ( for three or more groups ) or Student ' s t-test ( for two groups ) . Values of p < 0 . 05 were cons idered s tati s tical ly signi f icant : * P < 0 . 05 , * * P < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 .

[0034] Figure s 5 ( a ) to ( e ) show the RED antigen at 20 pg i s the optimal dose to be incorporated into the intranasal nano vaccine . C57BL / 6 mice ( n = 8 ) were immuni zed with the intranasal nano vaccine containing es calating doses of RED via the intranas al route ( 10 to 100 pg, 10 pL / animal ) three time s at 14 -day interval s . The following parameters were analyzed : Figure 5 ( a ) shows RBD- speci fi c I gG response in sera after each dose , Figures 5 (b- e ) show speci fic I gA responses in, respectively, (b ) serum, ( c ) s aliva , ( d) nasal wash (NAS ) , and ( e ) bronchoalveol ar lavage fluid ( BALF ) . Di f f erences between the animal groups were as ses s ed us ing Tukey' s mul tiple compari sons test ( three or more groups ) or Student ' s t- test ( two groups ) . Values of p < 0 . 05 were considered s tati s ti cal ly signi f icant : * P < 0 . 05 , * * P < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

[0035] Figure s 6 ( a ) to ( h ) show that the addition of synthetic peptides enclosing immunodominant CD4+ and C8+ T cel l epitopes from the SARS-CoV-2 proteome to the nanovaccine l ed to an increase in neutrali zing antibodies and cellular immune response s , but not in systemic and mucos al I gG and I gA antibodi es . C57BL / 6 mice ( n = 10 / group ) were immuni zed with the intranas al nanovaccine containing or not the conj ugated CD4+ and CD8 + peptides ( cNP-MaP and cNP-MaP-conj ugated CD4+ and CD8 + peptides , re spectively ) intranasal ly ( 5 pg, 10 pL / animal ) three times at 14 -day interval s ( DO , D14 and D28 ) . The following parameters were anal yzed from the second dose ( D28 and D42 ) : Figure 6 ( a ) neutral i zing capacity against Wuhan (WT ) ps eudovirus , Figure s (b , c ) I FN-y-producing splenocytes ( per 3 x 10 ^ cell s ) induced by stimulation with 10 pg / mL ( b ) protein RBD and ( c ) conj ugated CD4+ and CD8+ peptides pool was detected by ELI SPOT , wi th a lower limitof detection at 22 . 5 SFU / 10 ^ cell s ( dotted line ) , as well as Figure 6 ( d) RED- speci f ic I gG response in sera , and Fi gures 6 ( e-h ) speci f ic I gA responses in, respectively, ( e ) serum, ( f ) saliva , ( g ) nasal wash (NAS ) , and ( h ) bronchoalveol ar lavage fluid ( BALF ) . Di f f erences between the animal groups were as s es sed us ing Tukey' s multiple compari sons test ( three or more groups ) or Student ' s t-tes t ( two groups ) . Value s of p < 0 . 05 were considered stati stically signi f icant : * P < 0 . 05 and * * * * p < 0 . 0001 .

[0036] Figure s 7 ( a ) to ( j ) show the serum humoral immune responses a fter intranasal vaccination . Fi gure 7 ( a ) Wild-type (WT ) mice ( n = 6 to 10 / group ) were intranasally immuni zed wi th three doses ( DO , D14 and D28 ) of RBD protein and conj ugated CD4 + and CD8+ peptides plus poly ( I : C ) adj uvant without a nanoparticulate delivery system (Ag-Adj v) , or coated with mucoadhe sive pol ymer (MaP ) , or carried on mucus-penetrating inorgani c-bas ed nanoparticl es ( cNP ) , or even the combination of the se two delivery strategies ( cNP+MaP ) . Blood was collected and subj ected to antibody re spons e analysi s - Fi gures 7 ( b , d, e , g , i ) one day be fore and 14 days post the 3rd immuni zation ( n = 10 / group ) , as well as Figures 7 ( c , f , h, j ) 6 months and 1 year after the 3rd dose with the cNP+MaP vaccine ( n = 8 / group ) . Figures 7 ( b , c ) RBD- speci fi c I gG ti ter , Figure s 7 ( d- f ) RBD-speci f ic I gG i sotypes , including I gGl , I gG2b and I gG2 c , and Figures 7 ( g, h ) RBD-speci f ic I gA titer were determined by ELI SA . Al so , Fi gures 7 ( i , j ) show that the neutral i zation of 50 % inhibitory concentration ( IC50 ) values were determined against wi ld-type SARS-CoV-2 pseudovirus infection o f 293T-hACE2 cell s . Al l data are depicted as mean ± SD on a log 10 s cale and are representative re sults of two independent experiments . Di f f erences in antibody level s between animal groups per dose were as se s sed using one-way ANOVA followed by Tukey' s multiple compari sons test ( three or more groups ) or Student ' s t-te st ( two groups ) . P < 0 . 05 was considered s tati s tical ly signi ficant : * P < 0 . 05 , * * P < 0 . 01 , * * * p < 0 . 001 , and * * * * P < 0 . 0001 . DO - day zero ;wherein D14 - day 14 ; D28 - day 28 ; D42 - day 42 ; D200 - day 200 ; D400 - day 400 . And wherein WT - wild type ; Ag - antigens ; Adj v -adj uvant ; cNP - mucus - penetrating inorganic nanoparticle ; MaP -mucoadhesive pol ymer .

[0037] Figure s 8 ( a ) to ( g ) show mucosal humoral immune re spons es after intranasal immuni zation . C57BL / 6 femal e mice were immuni zed with Figure 8 ( a ) two doses ( D14 , n = 10 ) or Figure (b ) three doses ( D28 , n = 10 ) o f RBD protein and conj ugated CD4 + and CD8 + peptides plus poly ( I : C ) adj uvant without a nanoparticulate s ystem (Ag-Adj v) , or coated with a mucoadhe sive pol ymer (MaP ) , or carri ed on mucus penetrating nanoparticl es ( cNP ) , or even the combination of two delivery s trategies ( cNP+MaP ) . Animal s were anestheti zed, and mucosal samples were collected on days 28 and 42 . RBD- speci f ic Figure s 8 ( c , e , g ) I gA ti ters and Figure s 8 ( d, f , h ) I gG titers were determined by ELI SA in Figures 8 ( c , d) s aliva , Fi gures 8 ( e , f ) nasal- as sociated secretion (NAS ) , and Figure 8 ( g ) bronchoalveol ar lavage fluid ( BALF ) . All data are depicted as mean ± SD on a logl O scale and are repres entative resul ts of two independent experiments . Di f ferences in antibody l evel s between animal groups per dose were as ses s ed using one-way ANOVA followed by Tukey' s multiple compari sons test ( three or more groups ) or S tudent ' s t-test ( two groups ) . P < 0 . 05 was considered stati stically signi ficant : * P < 0 . 05 , * * P < 0 . 01 and * * * * p < 0 . 0001 . Wherein DO - day zero ; D14 - day 14 ; D28 - day 28 ; D42 - day 42 ; WT - wild type ; Ag - anti gens ; Adj v - adj uvant ; cNP - mucus-penetrating inorganic nanoparticle ; MaP - mucoadhesive polymer .

[0038] Figure s 9 ( a ) to ( d) show cellul ar immune response s after intranasal vaccination . Animal s were immuni zed as depi cted in Figure 8 ( a ) ( n = 10 in al l groups ) with the same formulations previous ly tested . Animal spleens were coll ected and splenocytes were i solated on days 28 and 42 . I FN-y-producing splenocytes (per 3 x 10 6 cell s ) induced by stimulation with 10 pg / mL Figure s 9 ( a , b ) protein RBDand Figures 9 ( c , d) conj ugated CD4+ and CD8+ peptides pool was detected by ELI SPOT . Fi gures 9 ( a , c ) show images di splaying repres entative results near the average value are pre sented in the le ft panel s . The dotted line indicates the lower limit of detection ( 2 6 . 4 SFU / 106 cell s ) . Data are depicted as the mean ± SD spot- forming cell s ( S FC ) per mill ion spl enocytes and were analyz ed by one-way ANOVA followed by Tukey' s multipl e compari sons test ( three or more groups ) or Student ' s t-tes t ( two groups ) , where * *p < 0 . 01 , * * * *p < 0 . 0001 . And wherein Ag - antigens ; Ad j v - adj uvant ; cNP - mucuspenetrating inorganic nanoparti cle ; MaP - mucoadhes ive polymer .

[0039] Figure s 10 ( a ) to ( i ) show protective e f ficacy of SARS-CoV-2 intranasal nano vaccine against wild-type SARS-CoV-2 infection in hACE-2 -transduced mice . Figure 10 ( a ) The intranasal nano vaccine was admini stered intranasal ly in a 3-dose regimen ( D- 42 , D-28 and D-14 ) to K18 -hACE2 transgenic mice . For compari son , a naive group ( nonimmuni zed and non-inf ected) and a placebo group ( non- immuni z ed and infected) were included . Animal s were intranasally chal lenged wi th 1 x 105 TCID50 ( lethal dose ) live wild-type SARS-CoV-2 on day 0 , and health monitoring was obs erved for 7 days post- inf ection, at whi ch point the surviving animal s were euthani zed and the lungs collected . Oropharyngeal swabs ( days 3 , 5 , and 7 post-infection ) were collected . Figure 10 ( b ) Changes in body weight , Figure 10 ( c ) clinical score , and Fi gure 10 ( d ) survival were observed from day 0 to 7 . Viral load in oropharyngeal swabs on Figure 10 ( e ) 3 dpi and 5 dpi , and Figure 10 ( f ) lungs was analyzed . Figure 10 ( g ) shows pathological change s in the lungs of animal s after chall enge by hi stological analysi s , and Figures 10 ( h, i ) frequency of hi s topathological findings at 4- 7 dpi comparing Fi gure 10 ( h ) al l tested groups and Figure 10 ( i ) viral load pos itive and negative cNP-MaP animal s . Di f ferences between the animal groups were as s es sed us ing Tukey' s multiple compari sons test ( three or more groups ) or S tudent ' s t-test ( two groups ) . Values of p < 0 . 05 were cons idered stati stically signi ficant : *P< 0 . 05 ,* * P < 0 . 01 , * * * p < 0 . 001 . Wherein D- 42 - day minus 42 ; D-28 - day minus 28 ; D- 14 - day minus 14 ; D- l - day minus 1 ; DO - day 0 ; D3 -day 3 ; D5 - day 5 ; D7 - day 7 ; WT - wi ld type ; cNP - mucus -penetrating inorganic nanoparticle ; MaP - mucoadhesive polymer ; DPI - days postinfection .

[0040] Figure 11 shows one embodiment o f the present invention : the formulation characteri zed by the ionic binding of the receptor binding domain (RED ) and multiple T-cell epitopes of respiratory pathogens , an immunostimulatory adj uvant , with a mucoadhesive polymer to an inorganic carri er nanoparticle .

[0041] Figure 12 shows a pre ferred embodiment of the present invention : the formulation characteri z ed by the ionic binding of the receptor binding domain ( RED ) and mul tiple T-cell epitopes of SARS-CoV-2 , Poly ( I : C ) , with 2 -hydroxypropyl- p-cyclodextrin to SiCy nanoparticle .

[0042] Figure 13 ( a ) to ( i ) show the serum humoral immune responses after intranasal booster vaccination . Figure 13 ( a ) Wild-type (WT ) mice ( n = 80 total ) were primed intramus cularly with the Comi rnaty vaccine ( BNT 162b2 , Pf i z er-BioNTech) . After 90 days , the animal s received one or two intranasal boosters ( D91 and D120 ) of the nanoparticulate system wi thout antigens ( placebo group ) , the Wuhan RBD protein or Omicron BA . 2 or BA . 5 and conj ugated CD4+ and CD8 + peptides plus poly ( I : C ) adj uvant , carried on mucus-penetrating inorgani c-bas ed nanoparticles ( cNP ) and coated with mucoadhesive polymer (MaP ) ( n = 10 / group ) . Blood was collected and subj ected to antibody response analysi s - Figure 13 (b ) over 90 days a fter the intramuscul ar mRNA prime ( D30 , D60 and D90 ) ( n = 80 total ) , as well as Figures 13 ( c , d, e , f , g, h, i ) three days be fore and / or 28 days post the 2ndintranasal booster with the intranasal vaccine containing di stinct RBD vari ants ( n = 10 / group ) . Figure s 13 ( b , c ) RBDw - speci f i c I gG titer , Figures 13 ( d, e ) RBD- speci fic I gG titer , including RBDW , RBDBAZ , and RBDBAS , and Figure 13 ( f ) RBDw - speci f icI gA titer were determined by ELI SA. Al so , Fi gures 13 ( g-i ) shows that the neutrali z ation of 50 % inhibitory concentration ( IC50 ) values were determined against infection by wild- type SARS-CoV- 2 pseudovi rus and Omicron BA . 2 and BA . 5 in 293T-hACE2 cel l s . All data are depicted as mean ± SD on a log 10 s cale and are repre sentative results of two independent experiments . Di f ferences in antibody level s between animal groups per dose were as se s sed us ing one-way ANOVA followed by Tukey' s multipl e compari sons te st ( three or more groups ) or Student ' s t-test ( two groups ) . P < 0 . 05 was considered s tati s tical ly signi f icant : * P < 0 . 05 , * * P < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 . Wherein DO - day 0 ; D30 - day 30 ; D60 - day 60 ; D90 - day 90 ; D120 - day 120 ; D150 - day 150 ; I . M . - intramuscular ; I . N . - intranasal . And wherein mucopenetrant , mucoadhesive vaccine - cNP+MaP ; placebo-nanoparticulate system without antigens ; WT - wi ld- type ; BA . 2 -Omicron BA . 2 ; BA . 5 - Omicron BA . 5 ; PSV - pseudovirus .

[0043] Figure 14 ( a ) to ( c ) show mucosal humoral immune re spons es after intranasal booster . Animal s were immuni zed as depi cted in Figure 13 ( a ) ( n = 10 in all groups ) wi th the same formulations previously tested . Animal ' s s aliva was collected on days 118 and 149 . Al so , animal s were anestheti zed, and mucos al sample s were coll ected on days 120 and 150 . Fi gures 14 ( a-c ) RBDw - speci f ic I gA titers were determined by ELI SA in Figure 14 ( a ) saliva , Figure 14 (b ) nasal-as soci ated secretion (NAS ) , and Figure 14 ( c ) bronchoalveolar lavage fluid ( BALF ) . Al l data are depicted as mean ± SD on a log 10 scale and are representative re sults of two independent experiments . Di f ferences in antibody l evel s between animal groups per dose were as ses s ed using one-way ANOVA followed by Tukey' s multiple compari sons test ( three or more groups ) or S tudent ' s t-test ( two groups ) . P < 0 . 05 was considered stati stically signi ficant : * P < 0 . 05 , * * P < 0 . 01 and * * * p < 0 . 001 . Wherein I . N . - intranasal ; mucopenetrant , mucoadhesive vaccine - cNP+MaP ; placebo- nanoparticulate system without antigens ; WT - wild- type ; BA . 2 - Omicron BA . 2 ; BA . 5 - OmicronBA . 5 .

[0044] Figure 15 ( a ) to ( d) show cellul ar immune responses after intranasal booster . Animal s were immuni zed as depicted in Figure 13 ( a ) ( n = 10 in all groups ) with the same formulations previous ly tested . Animal spleens were coll ected and splenocytes were i solated on days 120 and 150 . I FN-y-producing splenocytes (per 3 x 105 cell s ) induced by s timulation wi th 10 pg / mL Fi gures 15 ( a- c ) RED proteins from Figure 15 ( a ) wild-type , Figure 15 (b ) Omicron BA . 2 , and Figure 15 ( c ) Omicron BA . 5 variants . Al so , Figure 15 ( d) conj ugated CD4 + and CD8 + peptides pool were detected by ELI SPOT . The dotted line indicates the lower limit of detection ( 35 . 6 SFU / 105 cell s ) . Data are depicted as the mean ± SD spot- forming cell s ( S FC ) per million splenocytes and were anal yzed by one-way ANOVA fol lowed by Tukey' s multiple compari sons test ( three or more groups ) or Student ' s t-te st ( two groups ) . P < 0 . 05 was considered stati stically s igni f icant : * P < 0 . 05 , * * P < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 . Wherein I . N . - intranas al ; mucopenetrant , mucoadhe sive vaccine - cNP+MaP ; WT -wild-type ; BA . 2 - Omi cron BA . 2 ; BA . 5 - Omicron BA . 5 .DETAILED DESCRIPTION OF THE INVENTIONDe fini tions and abbreviations

[0045] The abbrevi ation " iNP" stands for mucus -penetrating inorganic carrier nanoparticle .

[0046] The abbrevi ation " cNP" stands for mucus -penetrating cerami c carrier nanoparticle .

[0047] The abbreviation "MaP" stands for mucoadhesive semi synthetic polymer .

[0048] The abbreviation "Ag" stands for antigens .

[0049] The abbreviation "Adj v" s tands for adj uvant .

[0050] The abbreviation "WT" stands for wi ld type .

[0051] The abbreviation "BALE" stands for bronchoalveolar lavage fluid .

[0052] The abbreviation "NAS" stands for nasal wash .

[0053] The abbreviation " Poly ( I : C ) " stands for poly ( inosine : cytidine ) .

[0054] The abbreviation " Poly ( I : C / LC ) " stands for polyinosini c-pol ycytidylic acid (poly I : C ) mixed wi th the stabili zers carboxymethylcel lulos e and polylysine .

[0055] The abbreviation "TLR3" stands for Toll-l i ke receptor 3 .

[0056] The abbrevi ation "MPLA" stands for monophosphoryl -lipid A.

[0057] The abbreviation "APCs" stands for antigen-presenting cells .

[0058] The abbreviation "HLA" stands for Human Leukocyte Antigen .

[0059] The expres sions " immuni zation pl atform" and " immuni zation system" are interchangeable in order to cover the physical and biological components of an embodiment of the present invention .Description

[0060] The pre sent invention di sclos es a novel immuni zation platf orm / system and formulation for intranasal del ivery of respiratory pathogens ' antigens , as recombinant proteins compri sing a combination o f carrier nanoparticles ( iNP ) coated with a mucoadhesive polymer (MaP ) , which facilitates the penetration of said carrier nanoparticles and o f the novel formulation in the nas al mucosa , and an immunostimulatory adj uvant whi ch i s an agoni st of innate immune receptors ( Figure 11 ) .

[0061] The recombinant proteins can be recombinant protein subuni ts and other s ynthetic peptide s as it wil l be further described .

[0062] The immunostimulatory adj uvant is for example, poly ( inosine : cytidine ) - Poly ( I : C) or Poly ( I : C / LC) , which is a stabilized dsRNA viral mimic and immunomodulator consisting of two long duplexed strands of Poly-inosinic and Polycytidilic acid stabilized with poly-L-Lysine and Carboxymethylcellulose, an agonist of innate immune receptors Toll-like receptor 3 (TLR3 ) and MDA5 , expressed naturally by the mucosal epithelium and known to be well tolerated at low doses .

[0063] In an embodiment of the present invention, the formulation comprises a recombinant protein - SARS-CoV-2 Spike receptor binding domain (RED) , the most critical region of the Spike protein for inducing neutrali zing antibodies , combined with synthetic peptides encoding multiple conserved, immunodominant SARS-CoV-2 T cell epitopes from the entire viral proteome .

[0064] In order to determine said T cell epitopes , the inventors identi fy multiple CD4+ and CD8+ T cell epitopes , highly recognized by convalescents from mild forms of SARS-CoV-2 infection . A bioinformatics scanning approach was used to select CD4+ T cell epitopes , aiming to find epitopes with predicted binding to multiple HLA-DR molecules ("promiscuous" epitopes ) and with broad population coverage (determined by the frequency of HLA-DR binding to peptides in the world population) .

[0065] For CD8+ epitopes , epitopes capable of binding to class I HLAs (A, B, C) whose frequency in the world population was very high, providing good vaccine coverage were selected .

[0066] In both cases , epitopes highly conserved among the SARS-CoV-2 variants of concern, as well as among various sarbecoviruses , in order to induce a pan-sarbecovirus cellular response, resistant to Spike / RBD variants were selected .

[0067] These epitopes are thus known to be broadly recogni zed by CD4+ and CD8+ T cells among the convalescent population, with highHLA coverage .

[0068] In one embodiment of the pres ent invention the epi topes are selected from Table 1 ( CD4+ ) and Tabl e 2 ( CD8+ ) :Table 1 : CD4+Table 2 : CD8+

[0069] Moreover , in order to obtain the synthetic peptide of the formul ation of the present invention , one CD4+ epitope and one CD8+ epitope were pl aced with a spacer sequence , in each peptide , resulting in long peptide s of 31 to 36 amino acids , whi ch are more immunogenic than short peptides .

[0070] The immunostimulatory adj uvant i s at least one of the groups consi sting of poly I : C ; pol y I : C LC ; MPLA, s aponins , cytokines , bacterial toxins , fl agell in and CpG oligonucl eotides .

[0071] In a particular embodiment o f the invention, the formul ation contains an immunos timulatory adj uvant , particularly poly ( inosine : cytidine ) - Poly ( I : C ) .

[0072] Poly ( I : C ) i s used as a mucosal adj uvant for intranasal subuni t vaccines against respiratory pathogens , el iciting secretoryIgA in the respiratory mucosa as well as systemic antibody responses .

[0073] The mucus-penetrating inorganic nanoparticle ( iNP) can be constituted by silver, gold, copper oxide, aluminum oxides , iron oxides , silicon oxides , zinc oxides , titanium oxides , calcium phosphates , hydroxyapatite , or a combination thereof .

[0074] Another embodiment of the present invention refers to an immuni zation platf orm / system and a formulation which alternatively contains Poly ( I : C / LC) , which is a stabili zed dsRNA viral mimic and immunomodulator consisting of two long duplexed strands of Poly-inosinic and Polycytidilic acid stabilized with poly-L-Lysine and Carboxymethylcellulose, an agonist of innate immune receptors Tolllike receptor 3 (TLR3 ) and MDA5 expressed by the mucosal epithelium and well tolerated at low doses .

[0075] Mucus-penetrating carrier nanoparticles carry the formulation active components to prevent premature degradation, enhance stability, and ensure targeted delivery of the immunogen to antigen-presenting cells (APCs ) . These carrier nanoparticles are coated with a mucoadhesive semi-synthetic polymer, constituted, for example, by cyclodextrins , chitosan or glucans , which allows greater adhesion to the mucosal layer, prolonging residence time, improving absorption and bioavailability of iNPs , and generating localized delivery . In a preferred embodiment, the mucus-penetrating carrier nanoparticles are mucus-penetrating ceramic nanoparticles ( cNP ) . Preferably, the mucus-penetrating carrier nanoparticles are silica ( SiCy) nanoparticles , in the size of less than 200 nm, preferably in the si ze between 10 to lOOnm.

[0076] Thus , after the mucoadhesive semi-synthetic polymer (MaP ) -coated system interacts with the mucosal surface, the MaP dissociates , allowing the iNP to diffuse through the epithelial barrier in the upper respiratory tract, eliciting a mucosal and systemic immune response .

[0077] This technical solution results in an extended residence time on mucosal surfaces that can also efficiently cross the mucus barrier . The reason why the immunization platf orm / system and the formulation of the present invention, including the combination of iNP and MaP as an innovative and versatile delivery system, is the platform for nanovaccines targeting respiratory infectious diseases .

[0078] The formulation of the present invention successfully crossed mucosal barriers , boosting the immunogenicity of antigens in a murine model . It induces upper airway mucosa IgA responses , durable and robust systemic RBD-binding and neutralizing antibody responses , as well as systemic cellular immunity against SARS-CoV- 2 .

[0079] The mucosal immune response leads to the containment of virus load at the site of virus entry and in the lungs , providing protection against challenge with respiratory pathogens , specifically with the ancestral Wuhan variant of SARS-CoV-2 .

[0080] Mucosal subunit vaccines require not only potent adj uvants but also suitable carriers / vehicles to achieve efficacy . The intranasal immunization with the active components in their free form ( i . e . , without a nanoparticle system) failed to trigger a detectable immune response, even after three doses . This occurs because the active vaccine components in their free form dissociate upon entering the body and are rapidly degraded, leading to weak immune stimulation .

[0081] To solve this problem, the inventors developed the immuni zation platf orm / system and formulation of the present invention comprising a combination of nanomaterials capable of crossing cellular barriers and enhancing intracellular delivery .

[0082] Another embodiment of the present invention is an intranasal nanovaccine comprising the immunization platf orm / system and the formulation herein described .

[0083] According to the present invention, the particle si ze is a key factor in eliciting an effective immune response . Thus , the immuni zation platf orm / system and the formulation of the present invention, used to prepare the intranasal nanovaccine, has an average diameter varying between 5 nm and 200 nm, preferably between 10 nm and 40 nm, an appropriate size for intranasal administration .

[0084] The surface charge of nanomaterials also influences nanoparticle-immunogen interactions . Positively charged nanoparticles are typically internali zed by antigen-presenting cells (APCs ) more efficiently than negatively charged ones . The shift in surface charge from negative to positive in the carrier nanoparticles after the addition of the active compounds indicates that they successfully bound to the iNP surface .

[0085] This resulted in a positively charged, mucus-penetrating nanovaccine with enhanced interaction with the negatively charged mucosal epithelium. The abrupt reversal in charge when coating the iNP with MaP as described herein indicates that a polymeric layer has been formed on the surface of the iNP . With these parameters fully controlled, the intranasal nanovaccine of the present invention using a muco-penetrating nanoparticle strategy with a mucoadhesive coating, creates a viable and enhanced option for intranasal instillation .

[0086] The results discussed in the Examples section below show that intranasal instillation of the intranasal nanovaccine with mucoadhesive polymers is associated with increased time of residence in the nasal cavity . Intranasal instillation with mucoadhesive polymers has proven to be effective, but their immunogenicity is associated with the conj ugation of antigens to the polymer .

[0087] In fact, the results show that while the iNP-MaP platform and formulation were highly immunogenic, simply adding the MaP coating to the active components of the formulation failed to generate a detectable immune response along the experimentalprotocol .

[0088] This is because purely mucoadhesive systems adhere strongly to the mucus and cannot penetrate the mucociliary layer to reach the underlying epithelium. Even though iNPs are ideal for adsorbing small molecules and of fer high stability, the iNP formulation for an intranasal nanovaccine triggered a reduced systemic immune response after the third immunization and was unable to activate mucosal immunity .

[0089] Taken together, this suggests that both mucoadhesive and mucopenetrant features are essential for the immunogenicity of the intranasal nanovaccine . The immuni zation plat form / system comprising the combination iNP-MaP for the intranasal nanovaccine establishes close contact with the mucus and penetrates to mucosal tissue or surfaces , warranting increased residence time at the administration site, and improving the delivery efficacy of iNP to the nasal mucosa, as demonstrated in the Examples .

[0090] The induction of upper airway mucosal anti-RBD IgA after nasal instillation, associated with protection and containment of mucosal viral load, is a crucial feature of the intranasal nanovaccine of the present invention .

[0091] It has been reported that in natural SARS-CoV-2 infection, nasal anti-RBD IgA inversely correlates with nasal viral load and systemic disease symptoms . The secreted IgA isotype is more ef fective at preventing infections on the surfaces of the upper respiratory tract than circulating IgG, which is more valuable for preventing severe infections and hospitali zations .

[0092] The finding that it is possible to obtain secretory IgA in upper airway fluids but not in BALE is probably linked to the small volume used for immunizations ( 5 pL per nostril ) , providing ef fective delivery to the upper respiratory tract with minimal exposure to the lower respiratory tract .

[0093] This is also related to the mucoadhesive and muco-penetrating nature of the intranasal nanovaccine, which directs the vaccine to immune system cells rather than epithelial cells . This ensures that it remains at the site of administration, where it is directly captured by APCs .

[0094] The intranasal nanovaccine of the present invention elicited a robust systemic humoral immune response, with high levels of circulating IgG ( IgGl , IgG2b, IgG2c, and neutralizing antibodies ) sustained for at least one year after the 3rc^ intranasal immuni zation . These results were comparable to those achieved with the mRNA vaccine BNT162b2 ( Pf i zer-BioNTech ) , administered systemically in a murine model .

[0095] This indicates that the intranasal nanovaccine of the present invention effectively induces immunity in the systemic compartment, which potently increases anti-RBD T-cell responses , along with responses against the peptides themselves , in addition to boosting neutralizing antibody responses .

[0096] Disclosed herein is a method to induce systemic and upper airway immunity against respiratory pathogens , the method comprising administering the intranasal nanovaccine of the present invention to a subj ect, wherein the intranasal nanovaccine comprises the immunization platf orm / system and formulation of the present invention wherein the immunization platform comprises a combination of mucus-penetrating carrier nanoparticles ( iNP ) coated with a mucoadhesive semi-synthetic polymer (MaP ) , an immunostimulatory adj uvant, for example poly ( inosine : cytidine ) - Poly ( I : C) , an agonist of Toll-like receptor 3 ( TLR3 ) and targets of neutralizing antibodies against respiratory pathogens in the presence or absence of multiple T-cell epitopes from respiratory pathogens , particularly the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein and T cell epitopes of SARS-CoV-2 .

[0097] The subj ect, or patient, being given the vaccine can be a mammal , such as a human . The sub ect may have previously been vaccinated against SARS-CoV-2 , for example by intramuscular inj ection . Such vaccination does not preclude the patient from also receiving the vaccine disclosed herein .

[0098] In a further embodiment of the present invention, it is disclosed the use of the formulation to prepare an intranasal vaccine, wherein the immunization platf orm / system and formulation comprise a combination of mucus-penetrating carrier nanoparticles ( cNP) coated with a mucoadhesive semi-synthetic polymer (MaP) , an immunostimulatory adj uvant, for example poly ( inosine : cytidine ) Poly ( I : C) , an agonist of Toll-like receptor 3 ( TLR3 ) and targets of neutralizing antibodies against respiratory pathogens in the presence or absence of synthetic peptides containing multiple T-cell epitopes from respiratory pathogens , particularly the receptor binding domain (RED) of the SARS-CoV-2 Spike protein and T cell epitopes of SARS-CoV-2 .

[0099] In conclusion, the innovative intranasal nanovaccine of the present invention of fers the advantages of boosting upper airway and systemic immune responses while reducing viral infection in the upper airway and lungs .

[0100] Preliminary evidence indicates that mice receiving two intranasal boosters following priming with mRNA vaccine BNT162b2 ( Pf i zer-BioNTech ) attain the same levels of mucosal and systemic immunity as the three intranasal dose protocols used here as shown in the Examples .

[0101] Finally, the immunization platf orm / system, that is , the iNP-MaP delivery system can be used for other respiratory mucosal pathogens . This platform serves as a universal nanovaccine delivery system to fight emerging or pandemic respiratory pathogens , replacing needles with nasal sprays .

[0102] The following examples are illustrative and provide a clearer and more consistent understanding of the invention but are not intended to limit its scope .EXAMPLESNano vaccine construction

[0103] The monomeric recombinant RED protein from the ancestral sequence (residues 319-537, pCAGGS vector, GenBank: MN908947 ) , or the omicron BA. 2 (residues 319-537, pCAGGS vector, GenBank: UJP23605 ) , or the omicron BA. 5 (residues 319-537, pCAGGS vector, GenBank: UOZ45804 ) variants with a C-terminal 6x-histidine tag (SEQ ID NO: 47 , 48 and 49, respectively) , and a signal sequence of human IgE to ensure protein would be secreted in the medium (YAN et al . , 2007 ) were transfected into Expi293FTM cells (#A14527, Thermo Fisher) . Protein purification was carried out using gravity-flow chromatography with Ni SepharoseTM 6 Fast Flow (#GE17-5318-06, Sigma-Aldrich) .SEQ ID NO: 47 MDWTWILFLVAAATRVHSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVAD YSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDD FTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPL QSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTNLVKNKHHHHHH SEQ ID NO: 48 MDWTWILFLVAAATRVHSRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVL YNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAW NSNKLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYGFRPTYGVG HQPYRVWLSFELLHAPATVCGPKKSTNLVKNKHHHHHH SEQ ID NO: 49 MDWTWILFLVAAATRVHSRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVL YNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAW NSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVG HQPYRVWLSFELLHAPATVCGPKKSTNLVKNKHHHHHH

[0104] In addition to the recombinant RED protein, the intranas al nanovaccine of the present invention al so include s 8 synthetic peptides , each encoding one CD4+ and one CD8+ T-cel l epitope from SARS-CoV-2 , in order to broaden T cel l re spons es . These peptides were selected from the SARS-CoV-2 proteome regions cons erved among sarbecoviruses and among SARS-CoV- 2 variants of concern, acros s 10 di f ferent SARS -CoV- 2 proteins . Selected CD4 + T- cel l epitopes were the most promi s cuous and had ample HLA clas s I I coverage , whi le CD8+ T-cell epitopes were selected that collectively bound to the 10 most frequent HLA clas s I molecules worldwide . The combination of the 8 selected CD4+ and 8 selected CD8 + T cell epitopes was recogni z ed by T cell s from >95 % of COVI D- 19 convalescent subj ects , as previous ly described ( Fernandes E . R . et al . 2022 ) . These peptides are thus call ed conj ugated CD4+ and CD8+ peptides as described in Table 3 below .

[0105] The conj ugated CD4+ and CD8+ peptides of the invention compri se some sequences with cysteines substituted by serine or a-aminobutyri c acid .Table 3 - Amino acid sequence of Conj ugated CD4 + and CD8 + peptides - Sequences o f conj ugated CD4 + and CD8 + peptides encoding immunodominant conserved CD4+ and CD8+ T cell epitopes>>>>Conj ugated CD4+ and CD8+ peptide s equences : N-terminal Lysine ( in bold) for interaction with nanoparticles ; CD4+T cel l epitope ; GPGPG or GGKKK spacer ( in bold) ; CD8+T cell epitope .

[0106] The conj ugated CD4+ and CD8+ peptide s ( 31 to 36-mer ) - SEQ ID NO : 50 , 54 , 56 , 57 , 60 , 62 , 63 , 64 , which increas e HLA coverage , contained an N- terminal lys ine to increase interaction with thenegatively charged carrier nanoparticle , pre ferably sil ica oxide ( SiCy ) , followed by one CD4 + T- cell epitope , a linker sequence ( GPGPG or GGKKK ) and one CD8+ T-cell epitope , and were synthesi zed by Watsonbio ( Houston, USA) with greater than 90 % puri ty .

[0107] These antigens were combined with Poly ( I : C ) ( #P1530 , Sigma-Aldrich ) to obtain a TLR- 3 agoni s t-adj uvanted vaccine . The immuni zation plat form / system and formul ation of the intranasal nanovaccine of the present invention i s characteri z ed by the ionic binding of components ( Figure 12 ) .

[0108] These components were coated with a mucoadhes ive polymer (MaP ) or carried in a nanoparticulate delivery system characteri zed by a mucus -penetrating carrier nanoparticle ( silica oxide nanoparticl es ) with unique changes , or even entrapped in cNP and thi s system coated with MaP ( cNP-MaP ) . The simple mixture o f RED, conj ugated CD4+ and CD8+ peptide s ( SEQ ID NO : 50 , 54 , 56 , 57 , 60 , 62 , 63 , 64 ) and Poly ( I : C ) (Ag-Adj v) without nanoparticulate carriers was admini s tered intranasally for compari son .

[0109] All experiments were conducted wi th placebo groups for each formul ation ( empty cNP and MaP , without anti gens ) s erving as negative control s , but the se were excluded f rom the results due to the absence of detectable immune responses , ensuring greater cl ari ty in the data .

[0110] Detail s of the intranasal nanovaccine o f the present invention content are provided in Table 4 . The phys icochemical characteri s tics of the nanovaccine were determined using appropriate methods .Table 4 - Components of the intranas al nanovaccine formulations FormulationAg-Adjv MaP cNP cNP-MaPVaccine vaccine vaccine vaccine purpOse componentntope d CD4+ antigen and CD8+peptidestcoating Ag - antigens ; Ad v - adj uvant ; cNP - carri er nanoparticle ; MaP -mucoadhesive polymer ; RED - receptor binding domain ; Poly ( I : C ) poly ( inosine : ci tydine ) ; TLR3 - Toll- li ke receptor 3 .Transmi s sion electron microscopy

[0111] The morphology of the cNP nanoparticles was determined by transmi s sion electron mi croscopy . Sampl es were prepared by adding 2 pL of a 20 nm si zed si lica oxide nanoparticl e suspension ( 0 . 01 mg / mL ) onto a 400-me sh copper grid coated with an ultra-thin carbon film ( #01822 -F, TED PELLA, USA) . Exces s li quid was removed from the grid after 10 minute s us ing fil ter paper . Image s were taken using the Jeol JEM-2100 equipment operating at an acceleration vol tage of 200 kV .Particle size , diameter and zeta potential determination

[0112] The particle size distribution, average diameter and zeta potential of formulations were assessed in aqueous dispersion, at 25 °C, with proper dilution using the dynamic light scattering (DLS ) technique (DLS Zetasizer Nano; Malvern Panalytical , UK) in disposable polystyrene cuvettes . All readings were performed in triplicate at different time intervals . The average of three readings was reported as the actual particle size .Virus , biosafety and bioethics statements

[0113] The Sars-CoV-2 / SP02 / human / 2020 / BRA ancestral wild-type virus strain was cultured in vi tro . All experiments involving SARS-CoV-2 were conducted under protocols approved by the Institutional Animal Care and Use Committee (protocol number 8733111121 ) and followed standard rules approved by the laboratory biosafety guidelines required for the novel coronavirus ( 2019-nCoV) by the World Health Organization (WHO) .Mouse strains

[0114] SPF ( specific pathogen- free ) female C57BL / 6 mice and K18-hACE2 transgenic mice (The Jackson Laboratory) , aged six to eight weeks , were used for vaccine immunogenicity and ef ficacy assays , respectively . The animals had ad libi tum access to water and food provided with a 12-hour light / dark cycle, at a temperature of 20-26 °C .Residence time of nano vaccine formulations in the nasal cavity and antigen biodistribution

[0115] C57BL / 6 mice were lightly anesthetized at each reading with ketamine-xylazine intraperitoneally and immunized with a single dose of the different vaccine formulations (RBD alone, RBD entrapped in cNP, RBD coated with MaP, and RBD carried in cNP and coated with MaP ) by nasal instillation ( 5 pL / nostril ) , where the RBD protein waslabeled with Alexa Fluor 647 dye ( Cy . 5 ) ( #A20006 , Invitrogen ) .

[0116] After cleaning the animal s ' nos tril s wi th a paper towel , they were immediately scanned with a real- time , in vi vo imaging s ystem, IVI S Spectrum from PerkinElmer . Scans were performed regularly every 30 minutes over a 3-hour period using the Cy . 5 filter to collect the fluorescence from the labeled protein . Between scans , the mice were returned to their cages to recover from anesthesia . Regions of interes t in the re spiratory tract over time and in organs after the final time point were detected and quanti fied .Animal immuni zation and SARS -CoV- 2 challenge

[0117] Mice were immuni zed intranasally with monomeric wi ld-type RBD as set forth in SEQ ID NO : 47 ( 20 pg / animal / dos e ) , 8 conj ugated CD4+ and CD8+ peptides as s et forth in SEQ ID NO : 50 , 53 , 55 , 57 , 59 , 60 , 62 and 63 ( 5 pg / animal / dos e ) and Poly ( I : C ) ( 20 pg / animal / dos e ) carried in cNP with polymeric coating, in a volume of 5 pl / nostril thrice at 14 -day interval s ( n= 6 to 8 ) . At 14 days pos t- second dose and post-third dose immuni zation, as well as 6 months and 1 year after the third dose , blood samples were collected from the submandibul ar vein to determine antigen- speci fic I gA, I gG antibodies , I gG antibody i sotypes , and pseudovirus neutrali zing antibody ti ters against a wild-type SARS-CoV-2 (Wuhan ) . Mucos al samples were col lected 14 days after the s econd dos e and after the thi rd dose to determine antigenspeci f ic I gA and I gG antibodies .

[0118] Mice were al so primed intramuscul arly with the mRNA vaccine BNT 162b2 , Comirnaty ( Pf i zer-BioNTech, 0 . 5 pg / dose / animal ) in a volume of 25 pL / tibi al mus cle . Ninety days after s ystemic prime , animal s were immuni zed intranasall y wi th wild-type monomeri c RBD (Wuhan ) , Omicron BA . 2 or Omicron BA . 5 , as described in SEQ I D NO : 47 , 48 and 49 , respectivel y ( 20 pg / animal / dose ) , 8 conj ugated CD4+ and CD8+ peptides , as set forth in SEQ I D NO : 50 , 54 , 56 , 57 , 60 , 62 , 63 , 64 ( 5 pg / animal / dose ) and Poly ( I : C ) ( 20 pg / animal / dose ) del ivered inpolymer-coated cNP ( intranasal vaccine ) , in a volume of 5 pL / nostril , twice at 30-day intervals (n = 10 ) . At twenty-seven days after the first and second intranasal boosters , blood samples were collected from the submandibular vein to determine antigen-specific IgA and IgG antibodies , and pseudovirus neutralizing antibody titers against a wild-type SARS-CoV-2 (Wuhan) , Omicron BA. 2 , and BA. 5 . Mucosal samples were collected 28 ( saliva) and 29 (NAS and BALE) days after the first and second intranasal boosters to determine antigenspecific IgA antibodies .

[0119] To collect saliva samples , mice were administered with pilocarpine hydrochloride . To collect nasal-associated secretion (NAS ) and bronchoalveolar lavage fluid (BALE) samples the mice were anesthetized with ketamine-xylazine intraperitoneally. Finally, the spleen was collected under aseptic conditions to as sess the cellular immune responses .

[0120] After three intranasal doses of the intranasal nanovaccine of the present invention, K18-hACE2 transgenic mice (n = 8 ) were lightly sedated with isoflurane administration and intranasally challenged with 105 PFU (plaque-forming units ) of the ancestral Wuhan live SARS-CoV-2 strain in a volume of 10 pL / nostril and observed for 7 days post-infection with daily measurements of body weight and clinical scores .

[0121] Oropharyngeal swabs were collected on days 3 , 5 and 7 . On day 7 post-infection ( 7 dpi ) , surviving animals were euthanized, and lungs were collected . The clinical score was assessed for behavioral changes ( lethargy, dif ficulty breathing, hunched posture, piloerection, eye closure, and death) and recorded based on symptom severity, with scores ranging from 0 to 4 , where 0 indicates the absence of clinical signs and 4 indicates severe signs . Pain signs were monitored according to the Grimace Scale for mice . A humane endpoint was established using criteria such as weight loss exceeding 20% , inactivity, or unresponsiveness to external stimuli .Measurement o f SARS-CoV-2 RBD- speci fic antibodies

[0122] Ninety- six-well high-binding plate s ( #44 -2404 -2 1 , Thermo Scienti fic ) were coated with 200 ng / 50 pL / well ( for serum I gG , I gG i sotypes and I gA) or 1 pg / 50 pL / well ( for saliva , nasal , and bronchoalveolar lavages I gA and I gG ) recombinant SARS-CoV-2 RBD antigen diluted in 0 . 05 M carbonate-bicarbonate buf fer (pH 9 . 6 ) per well and incubated at 4 ° C overnight .

[0123] Plates were blocked for 1 hour at 37 ° C with PBS containing 0 . 25 % (w / v) gel atin and 0 . 05 % Tween-20 ( v / v) ( PBS -T-G ) . Sampl es were serial ly diluted from 1 : 100 ( s erum I gG and I gG i sotypes ) , 1 : 5 ( serum I gA) , or 1 : 2 ( saliva , nasal , and bronchoalveolar lavages I gA and I gG ) starting the dilution in a 2 - fold series and applied to each well for 2 hours at 37 ° C . I gG, I gG i sotypes , and I gA antibody l evel s were detected using the appropriate horseradi sh peroxidase ( HRP ) -conj ugated anti-mouse I gG, I gGl , I gG2b and I gG2 c ( 1 : 8000 , # 1030- 05 , # 1070- 05 , # 1090- 05 and # 1079- 05 , Southern Biotech, respectivel y) or anti-mouse I gA ( 1 : 4000 , # 1040 - 05 , Southern Biotech ) for 1 hour at 37 ° C .

[0124] Between the aforementioned steps , the plates were washed four times with PBS containing 0 . 05 % Tween-20 ( v / v) ( PBS-T ) . Plates were developed for 10 minutes us ing o-phenyl enedi amine ( OPD, #P8787 , Sigma Aldrich ) , followed by the addition of 2 M H2 SO4 to stop the reaction, and opti cal density ( O . D . ) was read at 490 nm us ing an ELI SA plate reader for f inal data acqui sition . Endpoint titers were de fined as the maximum dilution that gave an absorbance reading above the mean of the placebo sample ( untreated mice ) .I FN-y ELI SPOT as say

[0125] ELI SPOT as says were performed using the Mous e Inter feron-y ( I FN-y) ELI SPOT Pair Kit ( # 55188 1 , BD Bios ciences ) . Under aseptic conditions , pre- coated 96-wel l ELI SPOT plates with capture antibody were blocked with R- 10 [ RPMI 1640 ( #31800- 022 , Gibco ) suppl ementedwith 10 % f etal bovine serum ( # 12 657 - 02 9 , Gibco ) , 2 mM L-glutamine ( #25030- 081 , Gibco ) , ImM sodium pyruvate ( # 11360 - 070 , Gibco ) , 1 % vol / vol es sential amino acid solution ( #M7 145 , Sigma- Aldrich ) , 1 % vol / vol vi tamin solution ( # 11120- 052 , Gibco ) , and 5 x 10-5 jy 2 -merca toethanol ( #21985 - 023 , Gibco ) ] for 2 hours at room temperature .[ 0012 6 ] A total of 3 x 10 ^ cell s per wel l from C57BL / 6 mous e spleen were pl ated in each wel l and stimulated for 14 hours at 37 ° C with either recombinant SARS-CoV-2 Spi ke RBD domain protein ( 10 pg / mL ) or 8 pooled conj ugated CD4+ and CD8+ peptides containing SARS-CoV-2 CD4+ and CD8+ T- cell epitopes as set forth in SEQ ID NO : 50 , 53 , 55 , 57 , 59 , 60 , 62 and 63 ( 10 pg / mL ) f rom Table 3 above . R10 was used as a negative control , and DMA ( 50 ng / mL ; #P8139 , Sigma ) and ionomycin ( 1 pg / mL ; #13909 , S igma ) were used as positive control s . The spots were developed according to the manuf acturer ' s instructions . The spots were scanned and quanti fied using an AID ELI SPOT reader (Autoimmun Diagnosti ka GmbH, Germany) . Spot- forming units ( SFUs ) / 105 cel l s werecalculated by subtracting the negative control well s .Neutrali zation as s ay of pseudovirus infection

[0127] The wild-type pSARS-CoV-2 S and pNL4 - 3 AEnv-NanoLuc , carrying the optimi zed SARS-CoV-2 S gene and a backbone of the human immunode ficiency virus type 1 were us ed . At 48 hours posttrans f ection, viral supernatant was coll ected and f roz en at - 80 ° C . Seri ally di luted serum samples ( 5 - fold dilution factor , starting at 1 : 50 ) were incubated with the SARS-CoV-2 pseudovirus at 37 ° C for 1 hour .

[0128] The s erum-virus mixture was then added to a pre- s eeded monolayer of HEK293T-hACE2 cell s . After 48 hours , the infected cell s were lysed wi th 5x Luci feras e Cell Culture Lysi s reagent ( #E 1500 , Promega ) , and luci feras e activity was measured using the Nano-Gio system ( #N1110 , Promega ) in a luminometer ( GloMax® Navigator Microplate Luminometer ) . The 50 % inhibitory concentration ( I C50 ) ofeach sample was calcul ated us ing nonlinear regres si on to re fl ect the anti-SARS-CoV-2 potency ( GraphPad Pri sm 8 . 0 ) .SARS-CoV-2 RNA quanti fi cation in mi ce oropharyngeal swab samples and lung biopsi es

[0129] Total RNA from oropharyngeal swabs and lung biopsie s was extracted using the RNeasy Mini kit ( #7410 6 and #74 136 , Qi agen , respectivel y) and followed by direct ampli fi cation of the SARS- CoV-2 nucl eocapsid gene and Mus musculus Gapdh using the Direct One-Step RT-qPCR kit ( #PCR- 518 L , Jena BioSciences ) for swabs .

[0130] For lung samples , reverse trans cription of total RNA was performed using random primers ( #48190011 , Invitrogen) and Supers cript IV reverse transcriptase ( # 18090010 , ThermoFi sher ) according to the manufacturer ' s instructions . Both direct duplex RT-qPCR and duplex qPCR were performed in tripli cate with total RNA ( oropharyngeal s amples ) or cDNA ( lung biopsies ) and master mix containing TaqMan probes for Gapdh VIC-MGB ( #Mm99999915_gl , ThermoFi sher ) and SARS -CoV- 2_N_gene FAM-MGB ( #Vi O 7918 637 , ThermoFi sher ) .

[0131] For oropharyngeal sampl es , the mix included direct enzyme mix and di rect reaction buf fer , while for lung sample s , the univers al TaqMan master mix ( #4440040 , Applied Biosystems ) was used . The cycle threshold ( CT ) values of the SARS-CoV-2 N gene were normali zed using the CT o f Gapdh with the following formula : 2 x 2ACT ; where ACT = average CT of Gapdh minus average CT of SARS- CoV-2_N_gene

[0058] .Hi stopathological analysi s

[0132] Lungs collected at necropsy were fixed in 10 % neutral buf fered formalin . Sections were evaluated blindly for hi stopathological proces sing . They were dehydrated in a graded alcohol bath, embedded in para f fin, sectioned ( 5 pm) , and stained with hematoxylin and eosin ( H&E ) for examination under light micros copy . Edema , alveolar damage , bronchioliti s , vasculiti s andpneumonia were the hi stopathological findings analyzed .Stati s tical analysi s

[0133] Tests were performed at least in dupli cate and analyzed using GraphPad Pri sm 8 . 0 software . Experimental values were trans formed into logl O for a normal di stribution . One-way or two- way ANOVA followed by Tukey ' s post- tes t was us ed for multipl e group compari sons and Student ' s t-test was appli ed for compari son between two groups .

[0134] The survival curve was analyz ed us ing log-rank s tatis tics . Data are expres s ed as mean ± SD . Values of p < 0 . 05 were considered stati s tical ly signi ficant . ( * ) p < 0 . 05 , ( * * ) p < 0 . 01 , ( * * * ) p < 0 . 001 and ( * * * * ) p < 0 . 0001 .ResultsFormul ation o f the present invention

[0135] Transmi s sion electron microscopy ( TEM) o f sil ica oxide carrier nanoparticles clearly indi cates spherical nanoparti cles , regular in shape , and monodi sperse ( Fi gure 1 ( a ) ) with an average diameter of 20 . 5 nm and minimal si ze vari ation ( Figure 1 (b ) ) .

[0136] In fact , hydrodynamic si ze o f si lica oxide carri er nanoparticl es measured by DLS showed a sl ight increase in the average diameter of the particle s ( 24 . 8 nm) , maintaining i ts low polydi spers ivity index ( 0 . 2 ) ( Figure 1 ( c ) ) .

[0137] After the incorporation o f the active components , the si ze of cNP sli ghtly increased by a factor of 1 . 29 , indi cating hi gh adsorption of anti gens- adj uvant (Ag-Adj v) to cNP , and by a factor of 1 . 36 when the cNP system was coated with polymer Poly ( I : C ) ( cNP-MaP ) , reaching a final average diameter of 33 . 8 nm ( Figure 1 ( c ) ) . The polydi spersi ty index of cNP-MaP i s 0 . 12 , indicating a narrow and favorable particle s i ze di stribution ( PDI < 0 . 3 ) , in addition to the formation of a single particl e population of predominant si zerepres entative o f the formulation for intranas al nanovaccine ( Figure 1 ( d) ) .

[0138] The formulation carried in the cNP wi th a negative zeta potential o f - 19 . 4 mV undergoe s a charge invers ion to pos itive polari ty of 5 . 18 mV ( Fi gure 1 ( e ) ) . Upon coating the cNP system wi th MaP , the zeta potential reverses again to -2 9 . 3 mV ( Figure 1 ( e ) ) . These data indicate that the formulation for the intranasal nanovaccine i s monodi spersed and of adequate s i ze for nasal di stribution .

[0139] The formulation for the intranasal nanovaccine o f the present invention increase s the residence time of the antigen in the nasal cavity and biodi stribution .

[0140] Given the importance of increased nas al residence time for enhancing nas al immune responses , the res idence time of the RED protein, both alone and within di stinct nanoparticle formul ations , was evaluated using an IVI S spectrum in vi vo imaging system ( Figure 2 ( a ) ) .

[0141] I t was confirmed that the antigen labeling in the formul ations ( Figure 3 ( a ) ) and recorded maximum fluorescence in the control (water - 2 . 21 x 10 ^ ) and te st formulations ( 4 . 9 to 6 . 8 x 10 ^ ) ( Figure 3 (b ) ) . To del ineate the cutof f o f emitted fluorescence , naive animal s and their respective organs were used and recorded maximum fluorescence of 1 . 4 x 10 ^ and 4 . 7 x 10 ^ , respectively ( Figures 1 ( c ) to ( e ) ) .

[0142] The formulations were instil led into the nostril s of animal s ( 5 pL / nostril , n = 3 / group ) and analyz ed immediately ( TO ) and every 30 minutes for 3 hours ( T 180 ) . The fluore scence o f the formul ation for the intranasal nano vaccine combining mucoadhesive polymer , poly ( I : C ) , and si lica oxide carrier nanoparticl es ( cNP- MaP ) was higher than that of the s ilica oxide carri er nanoparticle alone ( cNP ) ( Figure 2 (b ) ) , indi cating prolonged nas al re sidence time .

[0143] The e f f icacy of nanovaccines i s well- known to depend critically on their in vi vo di s tribution . At the end of the 3-hour period, the main organs were exci s ed and evaluated the biodi s tribution of the formulated and non- formulated vaccine antigen ( Figure 2 ( a ) , ( c ) ) . The in vi vo imaging data ( Figure 2 ( a ) ) and fluore scence quanti fi cation ( Figure 2 ( c ) ) reveal ed that cNP-MaP was present in al l evaluated organs , particularl y in the lungs . Thes e findings indicate that a mucoadhe sive polymer coating i s es senti al for prolonged re sidence time .

[0144] Intranasal immuni zation with the formulation for the intranasal nanovaccine induce s robust and durable systemic antibody responses .

[0145] The optimal intranasal immuni zation regimens wi th RED and Poly ( I : C ) adj uvant carried in sil ica oxide carrier nanoparticle coated with mucoadhesive pol ymer , Poly ( I : C ) ( cNP-MaP vaccine ) were evaluated ( Figures 3 ) , where an intermediate interval between doses ( 14 days ) was capable o f robustl y inducing high- speci f icity serum antibody response s ( Figure 4 ( a ) ) and a high capaci ty to generate neutrali zing antibodies starting from the 2nd immuni zation ( Figure 4 (b ) ) .[ 0014 6 ] Additionall y, the range of concentrations of the recombinant RED antigen to be carried by the intranasal nanovaccine of the present invention was as ses sed, for whi ch systemic humoral I gG and I gA responses ( Figures 5 ( a ) , (b ) ) , and mucosal I gA ( Suppl ementary Figures 5 ( c ) to ( e ) ) were optimal , establi shing 20 pg RED antigen / animal as the optimal dose .

[0147] Then, the e f fect of the addition of synthetic peptides encoding CD4+ and CD8 + T-cell epitopes al so re ferred to as conj ugated CD4+ and CD8+ peptides as set for in SEQ ID NO : 50 , 53 , 55 , 57 , 59 , 60 , 62 and 63 , to the intranasal nanovaccine on the immunogenici ty readouts was evaluated .

[0148] The addi tion o f synthetic conj ugated CD4+ and CD8+ peptides to the cNP-MaP formulation for intranasal vaccine s igni f icantly increased the anti -RBD and anti-con ugated peptide cellular immune responses starting at the 2nd immuni zation and boos ted at the 3 rd immuni zation ( Fi gures 6 ( a ) , (b ) ) , with no enhancing e f fect on serum neutrali zing antibody and binding I gG and I gA anti-RBD antibodies ( Figures 6 ( c ) to ( e ) ) or mucosal I gA anti-RBD responses ( Figure s 6 ( f ) to ( h ) ) .

[0149] From thi s point on, a combination of at l east 8 of the conj ugated CD4+ and CD8+ peptide s o f Tabl e 3 , for exampl e , SEQ ID NO : 50 , 53 , 55 , 57 , 59 , 60 , 62 and 63 , or all of them, were added to all the formulations for intranasal vaccines tested . To evaluate the systemic humoral immunity eli cited by nanoparticul ate formulations , C57BL / 6 mice ( n = 6 to 8 / group ) were immuni zed two weeks apart with a 3-dose regimen of the vaccine , with antigen and adj uvant ei ther 1 ) carried in mucus -penetrating carrier nanoparti cles ( cNP vaccine ) , 2 ) coated with a mucoadhesive polymer (MaP vaccine ) , or 3 ) carried in cNPs system coated with a mucoadhes ive polymer ( cNP-MaP vaccine ) by intranasal instill ation ( DO , D14 and D28 ; Figure 7 ( a ) ) .

[0150] For compari son purpos es , a group of mice was immuni zed by intranasal instill ation with the f ree active vaccine components , antigens and adj uvant (without nanoparticles , Ag- Adj v) . Immunogenicity was as s es sed one day be fore the fi rst and second immuni zation ( D13 and D27 ) and two weeks ( D42 ) , as well as six months and one year ( D200 and D400 ) after the thi rd immuni zation ( Figure 7 ( a ) ) . The I gGl , I gG2b , I gG2 c and I gA antibody response s were measured by ELI SA, and the titers of serum neutrali zing antibodies were measured us ing pseudovirus as say .

[0151] The intranasal nanovaccine of the present invention elicited anti-RBD I gG response s starting after the prime immuni zation, with an increase s tarting from the 2nd immuni zation, which was robustly increased a fter the 3rd immuni zation ( Figure 7(b ) ) . These anti-RBD I gG l evel s were maintained for at least one year after the 3 rd intranasal dose ( Figure 7 ( c ) ) .

[0152] On the other hand, the cNP vaccine - that i s , the formul ation for an intranasal vaccine without the mucoadhes ive component - was able to induce detectabl e level s of anti-RBD serum I gG only af ter the 3rd immuni z ation , whi le the Ag-Adj v and MaP vaccine groups failed to eli cit serum anti -RBD I gG throughout the protocol ( Figure 7 ( b ) ) .

[0153] Since serum I gG2 c synthesi s i s directed by Thl cytokines , while I gGl production correlates with high l evel s of Th2 cytokine s , the profil e of I gG i sotypes in the sera of mice af ter the thi rd immuni zation were anal yzed . As depicted in Figure s 7 ( d ) to 7 ( f ) , mice immuni z ed wi th the intranasal nanovaccine of the present invention el icited a speci fi c I gGl ( Figure 7 ( d) ) , I gG2b ( Figure 7 ( e ) ) and I gG2 c ( Figure 7 ( f ) ) respons e to RED, signi ficantly higher than the other groups anal yzed .

[0154] Thus , i t implie s that intranas al instil lation of the vaccine carried in cNP and coated with MaP directs both Thl and Th2 responses . The intranas al nanovaccine of the present invention induced RBD- speci fi c serum I gA antibodies starting from the 2nd intranasal immuni zation in some animal s , wi th the magni tude of the response found to be superior to the other groups analyzed ( Figure 7 ( g ) ) ; thi s response l asted les s than 6 months af ter the 3 rd immuni zation ( Figure 7 ( h ) ) . On the other hand, the cNP vaccine induced low titers o f serum I gA, where few animal s seroconverted a fter the 3 rd immuni zation, and the Ag-Adj v and MaP vaccine groups fai led to induce detectable level s o f anti-RBD I gA at the evaluated times ( Fi gure 7 ( g ) ) . In addition to speci fic RED binding ti ters , serum neutral i zing antibody responses were al so evaluated ( Figures 7 ( i ) , 7 ( j ) ) .

[0155] The intranasal nanovaccine o f the present invention induced neutrali zing antibody responses against the Wuhan ances tral strainpseudovirus s tarting from the 2nd intranasal dos e , signi f icantly superior to the other formulations tes ted, and such responses were further increas ed after the 3rd immuni zation ( Figure 7 ( i ) ) , with neutrali zing antibody detection lasting for at l east 1 year after the 3rd immuni zation ( Figure 7 ( j ) ) . Ag-Adj v and MaP vaccine groups f ailed to induce detectable neutrali zing antibody level s ( Figure 7 ( i ) ) .

[0156] The data des cribed above indicate that intranasal sensiti zation wi th a nanovaccine combining muco-penetrating and mucoadhesive strategies i s as sociated with short-l ived serum I gA responses and the induction of superior and long-lasting serum I gG and neutrali zing antibody responses compared to the other vaccines .

[0157] The combined mucoadhesive and mucopenetrant intranasal nanovaccine induce s I gA mucosal antibody responses in the upper respiratory tract .

[0158] To as ses s the RBD- speci fic I gA and I gG humoral response s in the respiratory mucosa , oral ( s al iva ) , nasal ( nasal lavage - NAS ) , and pulmonary (bronchoalveol ar l avage - BALF ) fluids from C57BL / 6 mice ( n = 8 / group ) intranasall y immuni zed with a 3 -dose regimen wi th 14 -day interval s between each dose were coll ected two wee ks after both the 2nd and 3rd intranasal immuni zations and analyz ed by ELI SA ( Figure 8 ( a ) ) .

[0159] The intranasal nanovaccine of the present invention elicited anti-RBD binding I gA antibody ti ters in saliva ( Figure 8 (b ) ) and nasal l avage fluid ( Figure 8 ( d) ) starting from the second intranasal immuni zation which were increas ed after the 3 rd immuni zation ; such mucosal fluid I gA antibodies were undetectable in the Ag-Adj v, MaP vaccine and cNP vaccine groups .

[0160] However , I gA anti-RBD binding antibodies in BALF were poorly induced af ter the 3rd immuni zation with the intranasal nanovaccine of the pre sent invention . Such antibodi es were undetectabl e in the other groups analyzed ( Figure 8 ( f ) ) .

[0161] Intere stingly, whi le RBD- speci fic I gG antibodie s after the 3rd intranasal immuni zation were not detectable in the s aliva o f any of the evaluated groups ( Fi gure 8 ( c ) ) , immuni zation with intranas al nanoparticulate formul ations cNP or cNP-MaP , but not MaP , induced I gG ti ters in the NAS ( Figure 4 e ) and BALF ( Figure 8 ( g ) ) , where the combined cNP-MaP s trategy was s igni f icantly superior to the other groups tested . Thes e data indicate that intranasal sensiti zation with a nanovaccine combining muco- penetrating and mucoadhesive strategies induced an upper airway mucosal I gA re spons e , as well as a lower airway mucosal I gG response .

[0162] The intranasal nanovaccine o f the present invention induces a systemic cell-mediated immune response .

[0163] To as s es s whether nasal mucosa vaccination could el icit a s ystemic cellular immune response af ter two and three rounds of intranasal immuni zation, spl eens were col lected f rom animal s ( n = 8 / group ) immuni z ed with the formulations of the pre sent invention on days 27 and 42 ( Figure 8 ( a ) ) . Splenocyte s were i solated and stimul ated with RBD protein ( Figure 9 ( a ) , Figure 9 (b ) ) and a pool of 8 conj ugated CD4+ and CD8+ peptide s encoding SARS-CoV- 2 CD4 + and CD8+ T-cell epitope s as set forth in SEQ ID NO : 50 , 53 , 55 , 57 , 59 , 60 , 62 and 63 ( Figure 9 ( c ) , Figure 9 ( d ) ) to as ses s whether local instil lation at the mucosal si te could activate a cel l-mediated immune response .

[0164] Antigen- speci fic I FN-y- secreting cell s were quanti fied using the ELI SPOT as s ay, wi th representative ELI SPOT well s shown in Figure 9 ( a ) , Fi gure 9 ( c ) and quanti fi ed I FN-y ELI S POT results in Figure 9 (b ) , Fi gure 9 ( d) .

[0165] All animal s immuni zed intranas ally with the intranasal nanovaccine developed detectabl e spl enic antigen- speci fic I FN-y-secreting cell s af ter stimulation with RBD protein ( Figure 9 ( a ) , Figure 9 ( b ) ) or the conj ugated CD4+ and CD8+ peptides pool ( Figure 9( c) , and Figure 9 ( d) ) since the 2nd immunization; the magnitude of response was greatly expanded after the 3rd immunization .

[0166] This response was superior to the other groups analyzed; cNP vaccine-immuni zed mice showed a low response to RED stimulus after the 3rd immuni zation, while Ad -Adv and MaP vaccines failed to induce IFN-y-secreting cells to any stimulus .

[0167] Importantly, the presence of the conj ugated CD4+ and CD8+ peptides of Table 3 ( SEQ ID NO : 50 to SEQ ID NO : 65 ) encoding T cell epitopes of SARS-CoV-2 in the formulation of the present invention achieved significant ampli fication of cellular responses to RED protein ( Figure 6 (b) ) . In contrast, animals immunized with the cNP vaccine developed a low number of splenic IFN-y-secreting cells , detected in only a few animals after stimulation with RED after the 3rd intranasal immunization, but superior to Ag-Adj v and MaP vaccine groups ( Figure 9 ( a) ) .

[0168] The data above indicate that the cNP-MaP strategy based on the combination of mucoadhesive and mucus-penetrating nanosystems is able to present T-cell epitopes from both RED protein and synthetic peptides , activating a strong cell-mediated immune response mediated by IFN-y-secreting cells starting from the 2nd intranasal immuni zation .

[0169] The intranasal nanovaccine of the present invention induces upper airway and pulmonary protection against SARS-CoV-2 challenge in K18-hACE2 humanized mice while substantially reducing viral loads .

[0170] Since intranasal immunization with intranasal nanovaccine of the present invention against COVID-19 elicited robust local and systemic immune responses , it was also assessed whether immunization with the intranasal nanovaccine of the present invention provided protection against infectious challenge with the ancestral Wuhan strain of SARS-CoV-2 in a highly susceptible K18-hACE2 mouse model ( Figures 10 ) .

[0171] The animal s ( n = 8 / group ) were immuni zed with the intranasal nanovaccine of the pres ent invention intranasally in a 3-dose regimen ( every two weeks ) , and as control s , naive ( nonimmuni zed and non-inf ected, n = 8 ) and a placebo ( non- immuni zed and infected, n = 8 ) groups ( Figure 10 ( a ) ) . K18 -hACE2 mice were intranasall y infected 2 weeks after the 3rd immuni zation and monitored for 7 days post-infection ( dpi ) . Oropharyngeal swabs were collected at 3 , 5 and 7 dpi , and lungs were collected at the time of euthanasia ( Figure 10 ( a ) ) . Placebo-treated animal s succumbed rapidl y and started losing wei ght 1 day a fter viral infection , reaching 19% weight los s on the 6th dpi ( Figure 10 (b ) ) .

[0172] Furthermore , clini cal symptoms s tarted by the 3 rd dpi , reaching maximum cl inical score s on the 6th dpi ( Fi gure 10 ( c ) ) , at which all animal s in the placebo group died ( Figure 10 ( d) ) . In contrast , mice immuni zed wi th the intranas al nanovaccine of the present invention did not show s igni ficant weight los s ( Figure 10 (b ) ) , developed mi ld symptoms from the 4 th to the 6 th dpi , with full recovery on the 7 th dpi ( Fi gure 10 ( c ) ) , and were protected from mortal ity ( Figure 10 ( d) ) .

[0173] [Viral load in oropharyngeal swabs of K18 -hACE2 mice intranasall y immuni zed with the intranasal nanovaccine o f the present invention was 20 - fold lower than that in the placebo group at both 3 and 5 dpi . Signi ficantly, oropharyngeal viral loads were below detection level s in 3 and 4 immuni zed mice at the analyzed time points , respectively ( Figure 10 ( e ) ) .

[0174] Viral loads in the lungs of cNP-MaP immuni z ed mice were approximately 100- fold lower than in the placebo group ; signi f icantly, the viral load was undetectabl e in 5 animal s immuni zed with the intranasal nanovaccine o f the present invention ( Figure 10 ( f ) ) . Pulmonary hi stopathology was analyz ed at 4 to 7 dpi and clas si fied according to the frequency ( % ) of hi stopathological change s ( Fi gure 10 ( g ) , Fi gure 10 ( h ) ) .

[0175] As expected, pulmonary le sions in placebo mi ce were severe and determined the cause of death . Immuni z ation with the intranasal nanovaccine of the present invention induced protection against pneumonia , vasculi ti s and bronchiol iti s caused by infection with the ancestral Wuhan strain of SARS-CoV-2 , with minimal focal hi stopathological changes ( Figure 10 ( g ) ) .[ 0017 6 ] Edema and alveolar damage were the most frequent findings in the placebo group ( 100 % ) , followed by vascul iti s and pneumonia ( 60 % ) and bronchioliti s ( 50 % ) ( Figure 10 ( h ) ) .

[0177] Corroborating with the findings regarding the viral load detected in the lungs of some animal s immuni zed with the intranas al nanovaccine o f the present invention ( Figure 10 ( f ) ) , edema and alveol ar damage were pre sent in 20 % o f these lung biops ies ( Figure 10 ( h ) ) . Thi s could be attributed to ti s sue damage generated at the time of col lection ; however , it occurred at a lower frequency than in the placebo group .

[0178] I t was obs erved that within the intranasal nanovaccine group , lung edema and alveolar damage were onl y noticed in animal s who had detectable lung SARS-CoV-2 RNA ( Figure 10 ( i ) ) .

[0179] The above data indi cate that intranas al immuni z ation wi th the intranasal nanovaccine o f the present invention drasti cally lowered - and in some animal s aboli shed - SARS -CoV- 2 RNA at the oropharyngeal and pulmonary sites , while providing protection against pneumonia , bronchioli ti s and vasculi ti s .

[0180] Intranasal boosting with a nanovaccine formul ation containing RED antigen from di stinct variants after mRNA primer induce s robust s ystemic antibody response s .

[0181] To evaluate s ystemic humoral immuni ty by nanoparticulate formul ations , C57BL / 6 mi ce ( n = 10 / group ) were primed intramuscularly with mRNA vaccine ( Comirnaty, Pf i zer-BioNTech ) and subsequently immuni zed intranasall y at thirty-day interval s with a 2 -boosterregimen o f the nanovaccine ( D91 and D120 ) containing 1 ) RED o f the ancestral s train ( intranasal WT vaccine ) , 2 ) RED of the Omicron BA . 2 strain ( intranasal BA . 2 vaccine ) or 3 ) RBD of the Omicron BA . 5 s train ( intranasal BA . 5 vaccine ) ( Figure 13 ( a ) ) .

[0182] For compari son purpos es , a group of mice was intranasally boosted wi th the nanovaccine without the antigens ( placebo ) . Immunogenicity was as ses s ed over 90 days post-mRNA prime ( D30 , D 60 and D90 ) , three days be fore and 28 days af ter the s econd intranas al boost ( D117 and D148 ) ( Figure 13 ( a ) ) . The I gG and I gA antibody responses were measured by ELI SA, and the titers of serum neutrali zing antibodi es were measured using ps eudovirus as say .

[0183] In order to initiate the booster protocol after a minimum drop of 3 titers of I gG antibodies after priming with Comirnaty, serum samples were collected over 90 days ( D90 ) , at which time the titers of RBD WT- speci fic I gG had fal len 16x ( approximatel y 4 titers ) compared to 30 days a fter priming ( Figure 13 (b ) ) .

[0184] [ The three versions of the intranasal nanovaccine o f the present invention induced equivalent level s of anti -RBDw I gG antibodies , regardles s of the vaccine antigen , and signi ficantly higher than those induced by the placebo group after the firs t and second intranasal booster doses ( Figure 13 ( c ) ) .

[0185] When comparing the magnitude of the serum humoral response induced by the di f ferent formulations against the dis tinct speci f iciti es proposed ( Figures 13 ( d ) , 13 ( e ) ) , I gG antibody level s induced by animal s immuni zed with the intranasal WT vaccine were lower in recogni zing antigens from the Omicron BA . 2 and BA . 5 strains compared to WT after each nasal booster ( Figure 13 ( d) , 13 ( e ) ) . On the other hand, serum I gG antibodie s induced in the groups that received the Omi cron versions of intranasal vaccine , either BA . 2 or BA . 5 , were equivalent in recogni z ing their respective anti gen in addition to the anti gen of the ancestral WT s train af ter the first( Figure 13 ( d) ) and second intranasal boos t ( Fi gure 13 ( e ) ) . [ 0018 6 ] The intranasal WT and BA . 2 vaccine versions , but not BA . 5 , tested as a booster were superior to the placebo group in inducing RBDw - speci f i c s erum I gA antibodies from the f irst intranas al boost ( Figure 13 ( f ) ) . After the s econd boos t , the intranasal vaccine groups , whether WT , BA . 2 or BA . 5 , induced equivalent I gA level s among themselves and were superior to animal s immuni zed with placebo ( Figure 13 ( f ) ) . In addition to RBD- speci fic binding titers , s erumneutrali zing antibody re spons es were al so evaluated ( Fi gures 13 ( g ) , 13 ( h ) , 13 ( i ) ) .

[0187] The intranasal nanovaccine versions of the present invention induced neutrali zing antibody re spons es against the Wuhan ancestral strain pseudovirus starting from the 1stintranasal booster , wi th normali zed NT50 values higher among animal s immuni zed with intranas al WT and BA . 2 vaccine compared to intranas al BA . 5 vaccine , whi ch become equivalent to each other and superior to the placebo group af ter the 2ndintranasal boos ter dose ( Figure 13 ( g ) ) .

[0188] In order to veri fy i f there would be a di f f erence in the potency of thi s response between intranasal vaccine vers ions , the neutrali zing capacity of the sera of animal s immuni zed with the nanoformulations against the di f ferent pseudovirus es af ter each booster dose was compared ( Figures 13 ( h ) , 13 ( i ) ) . The sera o f animal s immuni zed wi th intranasal WT vaccine were superior in neutrali zing the Spi ke protein on the surface of Wuhan ps eudoviral particl es when compared to the two Omicron variants analyzed both af ter the first ( Figure 13 ( h ) ) and af ter the s econd booster ( Figure 13 ( i ) ) . Sera from animal s immuni z ed with the BA . 2 version of intranas al vaccine induced higher l evel s of neutrali z ing antibodi es against WT pseudovirus after the 1stbooster ( Figure 13 ( h ) ) and against WT and BA . 2 compared to BA . 5 after the 2ndbooster ( Fi gure 13 ( i ) ) . On the other hand, serum sample s from animal s immuni zed with intranasal BA . 5 vaccine were abl e to neutrali ze the three di f ferent pseudovi ral parti cles tested in arobust and equivalent manner a fter each boos ter ( Fi gures 13 ( h ) 13 ( i ) ) .

[0189] These resul ts regarding the potency of the intranas al vaccine as a nasal booster dose may indicate that RED epitopes recogni zed by antibodies f rom animal s immuni z ed wi th WT may be suppre s sed or have a di f ferent conformation, and thi s means that the ability of the se antibodi es to e f fectively neutrali ze pseudoviral particles o f other variants may be reduced .

[0190] The combined mucoadhesive and mucopenetrant intranasal nanovaccine containing RED antigen from di stinct strains as an intranasal booster induce s I gA antibody responses in the upper and lower respi ratory tract .

[0191] The intranasal nanovaccine of the present invention elicited anti -RBDw binding I gA antibody titers in sal iva ( Figure 14 ( a ) ) and nasal lavage fluid ( Figure 14 (b ) ) from the first intranasal boos ter ; such I gA antibodies were detected in bronchoalveolar l avage fluids after a second intranasal booster in the intranasal WT and BA . 5 vaccine groups ( Figure 14 ( c ) ) .

[0192] These data indicate that one or two intranasal boos ter doses with a nanovaccine combining muco-penetrating and mucoadhesive strategies induced an upper and lower airway mucosal I gA response .

[0193] The intranasal nanovaccine versions of the present invention as booster regimen induce s a systemic cel l-mediated immune response .

[0194] Splenocytes were i solated and stimulated with RBDW protein ( Figure 15 ( a ) ) , RBDBA.2 protein ( Figure 15 ( b ) ) , RBDBA. 5 protein ( Figure 15 ( c ) ) and a pool of 8 conj ugated CD4 + and CD8+ peptides encoding SARS-CoV-2 CD4 + and CD8+ T-cell epitope s as set forth in SEQ I D NO : 50 , 53 , 55 , 57 , 60 , 61 , 63 and 64 ( Figure 15 ( d) ) to as se s s whether local boost instill ation at the mucos al si te could activate a cel l-mediated immune response . Anti gen- speci fic I FN-y- secreting cell swere quanti fied using the ELI S POT as say ( Figures 15 ( a ) , 15 (b ) , 15 ( c ) 15 ( d) ) .

[0195] Animal s immuni zed wi th the intranas al WT vaccine as a booster induced higher level s of cellular response when splenocytes were stimulated with RBD ( Figure 15 ( a ) ) compared to s timul ation with RBDBA.5 after the firs t boost ( Figure 15 ( c ) ) , and compared to Omicrons RBDs ( Figure s 15 (b ) , 15 ( c ) ) after the second boos t . Intranasal BA . 2 vaccine produced equivalent I FN-y regardl es s o f the s timulus at both time points analyzed ( Figures 15 ( a ) , 15 (b ) , 15 ( c ) ) . Intranasal BA . 5 vaccine produced equivalent I FN-y after the 1stboost and was superior with BA . 5 stimulation ( Figure 15 ( c ) ) compared to stimul ation with RBDBA.2 after the 2ndboost ( Figure 15 (b ) ) . The placebo group was superior when s timulated with RBDW ( Figure 15 ( a ) ) compared to s timulation with RBDBA.5 af ter the fi rst boos t ( Figure 15 ( c ) ) , and equivalent after the s econd boost ( Fi gures 15 ( a ) , 15 (b ) , 15 ( c ) ) .

[0196] Groups that received the intranasal vaccine versions and were stimulated wi th the dipeptide pool induced I FN-y- secreting cell level s above the cut-of f , and below the cut-of f in the placebo group at the evaluated time points ( Figure 15 ( d) ) . After the 1stboos t, animal s immuni zed with the intranasal BA . 5 and WT vaccines were equivalent to each other and superior to BA . 2 , and all intranas al vaccine groups were superior to pl acebo in developing I FN-y- secreting spleni c cel l s af ter s timul ation with the dipeptides ( Figure 15 ( d) ) . After the 2ndboos t , the intranasal vaccine products became equivalent to each other and continued to be superior to the placebo group ( Figure 15 ( d) ) .

[0197] The data above indicate that the iNP+MaP strategy, bas ed on the combination of mucoadhe sive and mucus-penetrating nanosys tems , i s capable of carrying di f f erent vaccine antigens , and demonstrates intranasal vaccine as a potent intranas al booster that complements systemicall y induced immunity .

[0198] It is understood that when a range of parameters is given, all whole numbers and ranges within that range, and tenths and hundredths , are also given by the modalities . Likewise , when a list is given, unless otherwise indicated, it should be understood that each individual element of that list and each combination of components of that list is a separate embodiment .

Claims

CLAIMS1 . Immuni zation system consisting of mucus-penetrating carrier nanoparticles coated with a mucoadhesive polymer .2 . Immuni zation system of claim 1 wherein the mucus- penetrating carrier nanoparticles are in the size of less than 200 nm.3 . Immuni zation system of any one of claims 1 or 2 wherein the mucus-penetrating carrier nanoparticles are ceramic nanoparticles .4 . Immuni zation system of any one of claims 1 - 3 wherein the mucus-penetrating carrier nanoparticles are constituted by silver, gold, copper oxide, aluminum oxides , iron oxides , silicon oxides , zinc oxides , titanium oxides , calcium phosphates , hydroxyapatite, preferably, the mucus-penetrating carriers are silica oxide ( SiCy) nanoparticles .5 . Immuni zation system of claim 1 wherein the mucoadhesive polymer is a semi-synthetic polymer .

6. Immuni zation system of claim 5 wherein the mucoadhesive semisynthetic polymer is constituted by cyclodextrins , chitosan or glucans .7 . Formulation comprising the targets of neutralizing antibodies against respiratory pathogens in the presence or absence of synthetic peptides containing multiple T cell epitopes from respiratory pathogens .8 . Formulation of claim 7 wherein the targets of neutralizing antibodies against respiratory pathogens are recombinant proteins .

9. Formulation of any one of claim 7 wherein the target of neutralizing antibody is the receptor binding domain (RED) of the SARS-CoV-2 Spike protein and T cell epitopes of SARS-CoV-2 .10 . Formulation of claims 7 and 8 wherein the respiratorypathogen i s SARS -CoV-2 and the recombinant protein i s the RED region of Spi ke protein .11 . Formulation of cl aim 7 wherein the T- cell epitope s are CD4+ and CD8+ epitope s from the same pathogen .12 . Formulation of claim 7 wherein the T cel l epitope s are compri sed in a conj ugated CD4+ and CD8+ peptide compri sing 31 to 36 amino acids .13 . Formulation of claim 12 wherein the conj ugated CD4+ and CD8+ peptide encodes the CD4+ and CD8+ epitope s of claim 11 .14 . Formulation of claim 13 wherein the conj ugated CD4+ and CD8+ peptides combine one CD4+ epitope , a spacer sequence and a CD8+ epitope .15 . Formulation of cl aim 14 wherein the spacer sequence i s GPGPG or GGKKK .16 . Formulation of cl aims 11 to 15 wherein the CD4 + and CD8 + epitopes are at l east one of the l i st consi s ting of SEQ ID NO : 1 to SEQ ID NO : 46 .17 . Formulation of claims 11 to 16 wherein it compri s es at least one conj ugated CD4+ and CD8+ peptide .18 . Formulation o f claims 11 to 17 wherein the conj ugated CD4+ and CD8 + peptide s are selected f rom the li st consi sting of SEQ ID NO : 50 to SEQ ID NO : 65 .19 . Formulation of any preceding cl aim wherein it further compri ses an adj uvant .20 . Formulation according to claim 17 wherein the adj uvant i s an immunostimulatory adj uvant , s elected from the group cons i sting of CpG Oligodeoxynucleotide s ( CpG-ODNs ) , R848 ( Res iquimod ) , Imiquimod, Ampligen ( Poly I : Pol y C12U) , Z ymosan, particularly pol yinos inic-polycytidyl ic acid ( Pol y I : C ) and pol yinos inic- polycytidyli c acidcomplex L-l ysine and carboxymethylcellulose (poly ( I : C LC ) ) .21 . Intranasal vaccine compri s ing the immuni zation system as de fined in any o f claims 1 to 6 and the formul ation as de fined in any of claims 7 to 21 .22 . Intranasal vaccine o f claim 21 wherein it has an average total diameter of less than 200 nm, preferably between 5 nm and 90 nm, more pre ferably, between 25 nm and 60 nm .23 . Method to induce systemic and upper airway immunity against respiratory pathogens , the method compri sing admini stering to a subj ect an intranasal vaccine as de f ined in any of claims 21 to 22 .24 . Method of claim 23 wherein the subj ect i s a mammal .25 . Method of claim 24 wherein the mammal i s a human .2 6 . Method of any cl aims 23 to 25 wherein the subj ect has been previously vaccinated against SARS-CoV-2 by intramuscular inj ection .27 . Us e of the immuni z ation system as de f ined in any of claims 1 to 6 and the formulation as de fined in any o f claims 7 to 20 for preparing an intranas al vaccine .

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