Influenza NG-34 T cell conserved epitope adjuvanted with CAF01 as a possible influenza vaccine candidate

Conserved epitopes are targets commonly researched to be part of universal vaccine candidates against influenza viruses (IV). These conserved epitopes need to be cross-protecting against distinct IV subtypes and to have a strong immunogenic potential. Nevertheless, subunit vaccines generally require a strong adjuvant to enhance their immunological effects. Herewith, we compare four different adjuvants differing in their immunological signatures that may enhance efficacy of a conserved hemagglutinin (HA)-epitope from IV, the NG-34, to define the most efficient combination of antigen/adjuvant to combat IV infections. Soluble NG-34 was mixed with adjuvants like aluminium hydroxide (AH) and AddaVax, known to induce Th2 and humoral responses; CAF01 which displays a biased Th1/Th17 profile and Diluvac Forte which augments the humoral response. Combinations were tested in different groups of mice which were subjected to immunological analyses. CAF01 + NG-34 induced a complete immune response with the highest IgG1, IgG2c titers and percentages of activated CD4 T cell promoting IFN-γ, IL-2 and TNF-α producing cells. Furthermore, in NG-34 stimulated mice splenocytes, cytokine levels of IFN-γ, IL-1β, IL-6, IL-10, IL-17 and TNF-α were also the highest in the CAF01 + NG-34 mouse group. This complete induced immune response covering the humoral and the cellular arms of the adaptive immunity promoted by CAF01 + NG-34 group suggests that CAF01 could be a good candidate as an adjuvant to combine with NG-34 for an efficacious vaccine against IV. However, more studies performed in IV hosts as well as studies with a challenge model are further required.info:eu-repo/semantics/publishedVersio

Conserved HA-peptides expressed along with flagellin in Trichoplusia ni larvae protects chicken against intranasal H7N1 HPAIV challenge

The immunization of poultry where H5 and H7 influenza viruses (IVs) are endemic is one of the strategies to prevent unexpected zoonoses. Our group has been focused on conserved HA-epitopes as potential vaccine candidates to obtain multivalent immune responses against distinct IV subtypes. In this study, two conserved epitopes (NG-34 and CS-17) fused to flagellin were produced in a Baculovirus platform based on Trichoplusia ni larvae as living biofactories. Soluble extracts obtained from larvae expressing "flagellin-NG34/CS17 antigen" were used to immunize chickens and the efficacy of the vaccine was evaluated against a heterologous H7N1 HPAIV challenge in chickens. The flagellin-NG34/CS17 vaccine protected the vaccinated chickens and blocked viral shedding orally and cloacally. Furthermore, no apparent clinical signs were monitored in 10/12 vaccinated individuals. The mechanism of protection conferred is under investigation.info:eu-repo/semantics/publishedVersio

DNA vaccine based on conserved HA-peptides induces strong immune response and rapidly clears influenza virus infection from vaccinated pigs

Swine influenza virus (SIVs) infections cause a significant economic impact to the pork industry. Moreover, pigs may act as mixing vessel favoring genome reassortment of diverse influenza viruses. Such an example is the pandemic H1N1 (pH1N1) virus that appeared in 2009, harboring a combination of gene segments from avian, pig and human lineages, which rapidly reached pandemic proportions. In order to confront and prevent these possible emergences as well as antigenic drift phenomena, vaccination remains of vital importance. The present work aimed to evaluate a new DNA influenza vaccine based on distinct conserved HA-peptides fused with flagellin and applied together with Diluvac Forte as adjuvant using a needle-free device (IntraDermal Application of Liquids, IDAL®). Two experimental pig studies were performed to test DNAvaccine efficacy against SIVs in pigs. In the first experiment, SIV-seronegative pigs were vaccinated with VC4-flagellin DNA and intranasally challenged with a pH1N1. In the second study, VC4-flagellin DNA vaccine was employed in SIV-seropositive animals and challenged intranasally with an H3N2 SIV-isolate. Both experiments demonstrated a reduction in the viral shedding after challenge, suggesting vaccine efficacy against both the H1 and H3 influenza virus subtypes. In addition, the results proved that maternally derived antibodies (MDA) did not constitute an obstacle to the vaccine approach used. Moreover, elevated titers in antibodies both against H1 and H3 proteins in serum and in bronchoalveolar lavage fluids (BALFs) was detected in the vaccinated animals along with a markedly increased mucosal IgA response. Additionally, vaccinated animals developed stronger neutralizing antibodies in BALFs and higher inhibiting hemagglutination titers in sera against both the pH1N1 and H3N2 influenza viruses compared to unvaccinated, challenged-pigs. It is proposed that the described DNA-vaccine formulation could potentially be used as a multivalent vaccine against SIV infections.info:eu-repo/semantics/publishedVersio

DNA vaccine based on conserved HA-peptides induces strong immune response and rapidly clears influenza virus infection from vaccinated pigs

This work was funded in part by the Spanish Government, Ministerio de Econom?a y Competitividad de España (MINECO), project: AGL2013-48923-C2-2-R, and by the collaborative infrastructure project funded by the European Comission (EC) under Horizon 2020, project Transvac2-730964-INFRAIA-2016-1. IRTA is supported by CERCA Programme/ Generalitat de Catalunya. M.S.O. is supported by MINECO (scholarship n BES-2014-068506). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Swine influenza virus (SIVs) infections cause a significant economic impact to the pork industry. Moreover, pigs may act as mixing vessel favoring genome reassortment of diverse influenza viruses. Such an example is the pandemic H1N1 (pH1N1) virus that appeared in 2009, harboring a combination of gene segments from avian, pig and human lineages, which rapidly reached pandemic proportions. In order to confront and prevent these possible emergences as well as antigenic drift phenomena, vaccination remains of vital importance. The present work aimed to evaluate a new DNA influenza vaccine based on distinct conserved HA-peptides fused with flagellin and applied together with Diluvac Forte as adjuvant using a needle-free device (IntraDermal Application of Liquids, IDAL®). Two experimental pig studies were performed to test DNA-vaccine efficacy against SIVs in pigs. In the first experiment, SIV-seronegative pigs were vaccinated with VC4-flagellin DNA and intranasally challenged with a pH1N1. In the second study, VC4-flagellin DNA vaccine was employed in SIV-seropositive animals and challenged intranasally with an H3N2 SIV-isolate. Both experiments demonstrated a reduction in the viral shedding after challenge, suggesting vaccine efficacy against both the H1 and H3 influenza virus subtypes. In addition, the results proved that maternally derived antibodies (MDA) did not constitute an obstacle to the vaccine approach used. Moreover, elevated titers in antibodies both against H1 and H3 proteins in serum and in bronchoalveolar lavage fluids (BALFs) was detected in the vaccinated animals along with a markedly increased mucosal IgA response. Additionally, vaccinated animals developed stronger neutralizing antibodies in BALFs and higher inhibiting hemagglutination titers in sera against both the pH1N1 and H3N2 influenza viruses compared to unvaccinated, challenged-pigs. It is proposed that the described DNA-vaccine formulation could potentially be used as a multivalent vaccine against SIV infections

Protective effect of a polyvalent influenza DNA vaccine in pigs

Background: Influenza A virus in swine herds represents a major problem for the swine industry and poses a constant threat for the emergence of novel pandemic viruses and the development of more effective influenza vaccines for pigs is desired. By optimizing the vector backbone and using a needle-free delivery method, we have recently demonstrated a polyvalent influenza DNA vaccine that induces a broad immune response, including both humoral and cellular immunity. Objectives: To investigate the protection of our polyvalent influenza DNA vaccine approach in a pig challenge study. Methods: By intradermal needle-free delivery to the skin, we immunized pigs with two different doses (500 μg and 800 μg) of an influenza DNA vaccine based on six genes of pandemic origin, including internally expressed matrix and nucleoprotein and externally expressed hemagglutinin and neuraminidase as previously demonstrated. Two weeks following immunization, the pigs were challenged with the 2009 pandemic H1N1 virus. Results: When challenged with 2009 pandemic H1N1, 0/5 vaccinated pigs (800 μg DNA) became infected whereas 5/5 unvaccinated control pigs were infected. The pigs vaccinated with the low dose (500 μg DNA) were only partially protected. The DNA vaccine elicited binding-, hemagglutination inhibitory (HI) − as well as crossreactive neutralizing antibody activity and neuraminidase inhibiting antibodies in the immunized pigs, in a dosedependent manner. Conclusion: The present data, together with the previously demonstrated immunogenicity of our influenza DNA vaccine, indicate that naked DNA vaccine technology provides a strong approach for the development of improved pig vaccines, applying realistic low doses of DNA and a convenient delivery method for mass vaccination.info:eu-repo/semantics/publishedVersio

Conserved HA-peptide NG34 formulated in pCMV-CTLA4-Ig reduces viral shedding in pigs after a heterosubtypic influenza virus SwH3N2 challenge

Swine influenza viruses (SIVs), the causal agents of swine influenza, are not only important to control due to the economic losses in the swine industry, but also can be pandemic pathogens. Vaccination is one of the most relevant strategies to control and prevent influenza infection. Current human vaccines against influenza induce strain-specific immunity and annual update is required due to the virus antigenic shift phenomena. Previously, our group has reported the use of conserved hemagglutinin peptides (HA-peptides) derived from H1-influenza virus as a potential multivalent vaccine candidate. Immunization of swine with these HA-peptides elicited antibodies that recognized and neutralized heterologous influenza viruses in vitro and demonstrated strong hemagglutination-inhibiting activity. In the present work, we cloned one HA-peptide (named NG34) into a plasmid fused with cytotoxic T lymphocyte-associated antigen (CTLA4) which is a molecule that modifies T cell activation and with an adjuvant activity interfering with the adaptive immune response. The resulting plasmid, named pCMV-CTLA4-Ig-NG34, was administered twice to animals employing a needle-free delivery approach. Two studies were carried out to test the efficacy of pCMV-CTLA4-Ig-NG34 as a potential swine influenza vaccine, one in seronegative and another in seropositive pigs against SIV. The second one was aimed to evaluate whether pCMV-CTLA4-Ig-NG34 vaccination would overcome maternally derived antibodies (MDA). After immunization, all animals were intranasally challenged with an H3N2 influenza strain. A complete elimination or significant reduction in the viral shedding was observed within the first week after the challenge in the vaccinated animals from both studies. In addition, no challenged heterologous virus load was detected in the airways of vaccinated pigs. Overall, it is suggested that the pCMV-CTLA4-Ig-NG34 vaccine formulation could potentially be used as a multivalent vaccine against influenza viruses.info:eu-repo/semantics/publishedVersio

Preclinical evaluation of PHH-1V vaccine candidate against SARS-CoV-2 in non-human primates

SARS-CoV-2 emerged in December 2019 and quickly spread worldwide, continuously striking with an unpredictable evolution. Despite the success in vaccine production and mass vaccination programs, the situation is not still completely controlled, and therefore accessible second-generation vaccines are required to mitigate the pandemic. We previously developed an adjuvanted vaccine candidate coded PHH-1V, based on a heterodimer fusion protein comprising the RBD domain of two SARS-CoV-2 variants. Here, we report data on the efficacy, safety, and immunogenicity of PHH-1V in cynomolgus macaques. PHH-1V prime-boost vaccination induces high levels of RBD-specific IgG binding and neutralizing antibodies against several SARS-CoV-2 variants, as well as a balanced Th1/Th2 cellular immune response. Remarkably, PHH-1V vaccination prevents SARS-CoV-2 replication in the lower respiratory tract and significantly reduces viral load in the upper respiratory tract after an experimental infection. These results highlight the potential use of the PHH-1V vaccine in humans, currently undergoing Phase III clinical trials.Anna Moya and Mireia Muntada for the ELISA analysis; Clara Panosa and Ester Puigvert for her assistance in the production of the vaccine antigen; Glòria Pujol and Eduard Fossas for their assistance in review of the manuscript; and Adrián Lázaro-Frías from Evidenze Health España S.L. for providing medical writing support during the preparation of this paper funded by Hipra Scientific, S.L.U. This project was partially funded by the Centre for the Development of Industrial Technology (CDTI, IDI20210115), a public organization answering to the Spanish Ministry of Science and Innovation.info:eu-repo/semantics/publishedVersio

Towards a universal influenza vaccine: generation and evaluation of vaccine formulations based on HA-epitopes in different influenza hosts

Els virus de la influença tipus A (VIAs), han estat implicats en cinc pandèmies i ocasionen, any rere any, epidèmies estacionals. Tenen un elevat potencial zoonòtic, afectant un ampli ventall d’hostes. Els seus principals reservoris són les aus salvatges aquàtiques que poden transmetre el virus a aus domèstiques, i aquestes, a porcs i humans. A més, els porcs són considerats hostes intermediaris, susceptibles d’infecció tant per virus humans com aviars. La mesura preventiva més efectiva contra els VIAs són les vacunes. La majoria són vacunes inactivades que contenen soques dels subtipus circulants més comuns de VIAs. No obstant, presenten inconvenients, com una limitada capacitat de cross-protecció. Aquestes limitacions requereixen una reformulació de les vacunes per tal que s’ajustin als canvis genètics del virus i exigeixen continua vigilància davant possibles pandèmies. Per superar aquests obstacles, la investigació actual cerca una vacuna universal, implementant diverses estratègies de vacuna/immunització utilitzant immunomodul·ladors i epítops altament conservats dels VIAs. Aquesta tesi doctoral avalua diverses estratègies enfocades en la millora de les formulacions vacunals, com en la seva aplicació/avaluació en hostes naturals de VIAs i consta de tres parts: La primera part descriu la introducció general (Capítol I) i l’objectiu general i específics (Capítol II). La introducció explica (i) els virus de la grip (ii) els VIAs (iii) la resposta immune de l'hoste contra els VIAs (iv) les vacunes comercials disponibles en humans, aviar i porcí contra la grip (v) estratègies per obtenir una vacuna universal i (vi) l’ISM, una eina in silico utilitzada en la tesi per predir pèptids conservats. El capítol II descriu l’objectiu:. crear un prototip de vacuna universal contra diferents subtipus de VIAs utilitzant tècniques que incorporen epítops conservats de la hemaglutinina (HA-epítops), predits in silico, introduïts en plàsmids o en forma soluble amb adjuvants diferents. La part II (Capítols III-V), inclou els tres estudis on el disseny de prototips vacunals amb possible caràcter multivalent i la seva aplicació en porcs i aus és avaluada. En el primer estudi, un HA-epítop: el NF-34, predit per ISM, va ser modificat i formulat en un plàsmid amb CTLA-4 (pCMV-CTLA4-Ig-NG34), promotor de la resposta adaptativa. El pCMV-CTLA4-Ig-NG34 es va utilitzar en porcs seronegatius i seropositius contra la grip porcina i es van desafiar contra el virus heteròleg H3N2. Els vacunats van secretar menys virus, van eliminar el virus a les vies respiratòries, van presentar resposta humoral als subtipus circulants més rellevants i anticossos neutralitzants. No obstant, no hi va haver diferències en el grau de lesions pulmonars ni en els signes clínics. Els anticossos materns no van interferir amb l’efecte de la vacuna. En el segon estudi, es va utilitzar un còctel d’HA-epítops combinats amb flagel·lina (VC-4-flagellin), promotor de la resposta innata, en porcs seronegatius i seropositius i es van desafiar amb soques homòlogues i heteròlogues de VIAs. Els porcs vacunats van reduir l'excreció del virus, van produir resposta humoral front els subtipus H1 i H3 i, anticossos neutralitzants contra ambdós virus. Els anticossos materns no varen ser un obstacle, no obstant, els signes clínics i la patologia dels pulmons no es van reduir. Finalment, en el tercer estudi, es va utilitzar el sistema vectorial d’expressió de baculovirus (BEVS) per obtenir extractes de proteïnes que contenien HA-epítops units a la flagel·lina. La formulació es va utilitzar per immunitzar pollastres que es van desafiar amb H7N1, un virus altament patogènic. Els vacunats van sobreviure sense mostrar signes clínics i sense secretar o, en menor quantitat, el virus. El mecanisme de protecció està sota investigació. La part III (capítols VI-VII), descriu la discussió general i les conclusions. També, inclou les referències bibliogràfiques (Capítol VIII) i els annexos.Influenza viruses (IAVs) have been implicated in five pandemics and are the cause of seasonal epidemics year after year. They are characterized for their high zoonotic potential, affecting a wide range of hosts. Wild aquatic birds are their main reservoirs that transmit the virus to domesticated birds and may spread further to pigs and humans. Moreover, pigs are considered intermediate hosts, susceptible to infection for both humans and avian IAVs. The most effective countermeasure against IVs are the vaccines. Most of them are inactivated vaccines containing strains of the most common circulating subtypes of IVs. However, they present several inconveniences like limiting cross-protection capacity. These limitations require reformulation of vaccines matched to the genetic changes of the virus and demand continuous vigilance for possible pandemics. To overcome these hurdles, current research focuses on seeking for a universal influenza vaccine, implementing several vaccine strategies by using different immunomodulators and highly-conserved IV epitopes in vaccine formulations. This doctoral thesis evaluates distinct approaches both in improving vaccine formulation and its application/evaluation to different IV natural hosts and has been divided into 3 parts: In the Part I, the general introduction (Chapter I) and the general and specific objectives (Chapter II), are described. The introduction explains (i) influenza viruses (ii) the viruses of the genus A (iii) host's immune response against IVs; (iv) the commercial vaccines available against influenza in human, avian and porcine (v) approaches to obtain a universal vaccine and the (vi) ISM, an in silico tool which has been used in the thesis to predict conserved peptides. Chapter II describes the objective of this thesis. Overall, it is intended to create a universal prototype vaccine against different subtypes of IVs using techniques that incorporate in silico predicted conserved HA-epitopes introduced in plasmid or soluble form along with different adjuvants. In Part II (Chapters III-V), three studies that are either published/submitted to international peer-reviewed scientific journals, are included. The design of vaccine prototypes with possible multivalent character and its subsequent application in pigs and birds is evaluated. In the first study, one conserved HA-epitope NF-34, predicted by ISM, was modified and formulated in a plasmid with CTLA-4 (pCMV-CTLA4-Ig-NG34), promoter of the adaptive response. The vaccine approach was used in swine influenza seronegative and seropositive pigs and challenged against heterologous H3N2. Vaccinated pigs secreted fewer viruses, cleared the virus in the respiratory airways, presented humoral response to the most relevant circulating subtypes and elicited neutralizing antibodies. However, there were no differences in the degree of pulmonary lesions and clinical signs. Maternal antibodies did not interfere with the effect of the vaccine. In the second study, a cocktail of HA-epitopes combined with flagellin (VC-4 flagellin), a promoter of the innate response, was used in SIV seronegative and seropositive pigs and were challenged with homologous and heterologous IAV strains. Vaccinated pigs reduced virus excretion, produced humoral response to subtypes H1 and H3, and neutralizing antibodies against both viruses. Maternal antibodies were not an obstacle but, clinical signs and pathology in the lungs were not reduced. Finally, in the third study, baculovirus expression vector system (BEVS) was used to obtain protein extracts containing HA-peptides linked with flagellin. The formulation was used to immunize chickens that were challenged with H7N1, a highly pathogenic virus. Vaccinated animals survived without showing any clinical sign and fewer or no virus secretion. The protection mechanism is under investigation. The general discussion is described in part III (Chapters VI-VII) with possible significance of the results obtained and the relation of conclusions drawn from each study. The bibliographical references (Chapter VIII) and appendices are also included

Towards a universal influenza vaccine : generation and evaluation of vaccine formulations based on HA-epitopes in different influenza hosts /

Els virus de la influença tipus A (VIAs), han estat implicats en cinc pandèmies i ocasionen, any rere any, epidèmies estacionals. Tenen un elevat potencial zoonòtic, afectant un ampli ventall d'hostes. Els seus principals reservoris són les aus salvatges aquàtiques que poden transmetre el virus a aus domèstiques, i aquestes, a porcs i humans. A més, els porcs són considerats hostes intermediaris, susceptibles d'infecció tant per virus humans com aviars. La mesura preventiva més efectiva contra els VIAs són les vacunes. La majoria són vacunes inactivades que contenen soques dels subtipus circulants més comuns de VIAs. No obstant, presenten inconvenients, com una limitada capacitat de cross-protecció. Aquestes limitacions requereixen una reformulació de les vacunes per tal que s'ajustin als canvis genètics del virus i exigeixen continua vigilància davant possibles pandèmies. Per superar aquests obstacles, la investigació actual cerca una vacuna universal, implementant diverses estratègies de vacuna/immunització utilitzant immunomodul·ladors i epítops altament conservats dels VIAs. Aquesta tesi doctoral avalua diverses estratègies enfocades en la millora de les formulacions vacunals, com en la seva aplicació/avaluació en hostes naturals de VIAs i consta de tres parts: La primera part descriu la introducció general (Capítol I) i l'objectiu general i específics (Capítol II). La introducció explica (i) els virus de la grip (ii) els VIAs (iii) la resposta immune de l'hoste contra els VIAs (iv) les vacunes comercials disponibles en humans, aviar i porcí contra la grip (v) estratègies per obtenir una vacuna universal i (vi) l'ISM, una eina in silico utilitzada en la tesi per predir pèptids conservats. El capítol II descriu l'objectiu:. crear un prototip de vacuna universal contra diferents subtipus de VIAs utilitzant tècniques que incorporen epítops conservats de la hemaglutinina (HA-epítops), predits in silico, introduïts en plàsmids o en forma soluble amb adjuvants diferents. La part II (Capítols III-V), inclou els tres estudis on el disseny de prototips vacunals amb possible caràcter multivalent i la seva aplicació en porcs i aus és avaluada. En el primer estudi, un HA-epítop: el NF-34, predit per ISM, va ser modificat i formulat en un plàsmid amb CTLA-4 (pCMV-CTLA4-Ig-NG34), promotor de la resposta adaptativa. El pCMV-CTLA4-Ig-NG34 es va utilitzar en porcs seronegatius i seropositius contra la grip porcina i es van desafiar contra el virus heteròleg H3N2. Els vacunats van secretar menys virus, van eliminar el virus a les vies respiratòries, van presentar resposta humoral als subtipus circulants més rellevants i anticossos neutralitzants. No obstant, no hi va haver diferències en el grau de lesions pulmonars ni en els signes clínics. Els anticossos materns no van interferir amb l'efecte de la vacuna. En el segon estudi, es va utilitzar un còctel d'HA-epítops combinats amb flagel·lina (VC-4-flagellin), promotor de la resposta innata, en porcs seronegatius i seropositius i es van desafiar amb soques homòlogues i heteròlogues de VIAs. Els porcs vacunats van reduir l'excreció del virus, van produir resposta humoral front els subtipus H1 i H3 i, anticossos neutralitzants contra ambdós virus. Els anticossos materns no varen ser un obstacle, no obstant, els signes clínics i la patologia dels pulmons no es van reduir. Finalment, en el tercer estudi, es va utilitzar el sistema vectorial d'expressió de baculovirus (BEVS) per obtenir extractes de proteïnes que contenien HA-epítops units a la flagel·lina. La formulació es va utilitzar per immunitzar pollastres que es van desafiar amb H7N1, un virus altament patogènic. Els vacunats van sobreviure sense mostrar signes clínics i sense secretar o, en menor quantitat, el virus. El mecanisme de protecció està sota investigació. La part III (capítols VI-VII), descriu la discussió general i les conclusions. També, inclou les referències bibliogràfiques (Capítol VIII) i els annexos.Influenza viruses (IAVs) have been implicated in five pandemics and are the cause of seasonal epidemics year after year. They are characterized for their high zoonotic potential, affecting a wide range of hosts. Wild aquatic birds are their main reservoirs that transmit the virus to domesticated birds and may spread further to pigs and humans. Moreover, pigs are considered intermediate hosts, susceptible to infection for both humans and avian IAVs. The most effective countermeasure against IVs are the vaccines. Most of them are inactivated vaccines containing strains of the most common circulating subtypes of IVs. However, they present several inconveniences like limiting cross-protection capacity. These limitations require reformulation of vaccines matched to the genetic changes of the virus and demand continuous vigilance for possible pandemics. To overcome these hurdles, current research focuses on seeking for a universal influenza vaccine, implementing several vaccine strategies by using different immunomodulators and highly-conserved IV epitopes in vaccine formulations. This doctoral thesis evaluates distinct approaches both in improving vaccine formulation and its application/evaluation to different IV natural hosts and has been divided into 3 parts: In the Part I, the general introduction (Chapter I) and the general and specific objectives (Chapter II), are described. The introduction explains (i) influenza viruses (ii) the viruses of the genus A (iii) host's immune response against IVs; (iv) the commercial vaccines available against influenza in human, avian and porcine (v) approaches to obtain a universal vaccine and the (vi) ISM, an in silico tool which has been used in the thesis to predict conserved peptides. Chapter II describes the objective of this thesis. Overall, it is intended to create a universal prototype vaccine against different subtypes of IVs using techniques that incorporate in silico predicted conserved HA-epitopes introduced in plasmid or soluble form along with different adjuvants. In Part II (Chapters III-V), three studies that are either published/submitted to international peer-reviewed scientific journals, are included. The design of vaccine prototypes with possible multivalent character and its subsequent application in pigs and birds is evaluated. In the first study, one conserved HA-epitope NF-34, predicted by ISM, was modified and formulated in a plasmid with CTLA-4 (pCMV-CTLA4-Ig-NG34), promoter of the adaptive response. The vaccine approach was used in swine influenza seronegative and seropositive pigs and challenged against heterologous H3N2. Vaccinated pigs secreted fewer viruses, cleared the virus in the respiratory airways, presented humoral response to the most relevant circulating subtypes and elicited neutralizing antibodies. However, there were no differences in the degree of pulmonary lesions and clinical signs. Maternal antibodies did not interfere with the effect of the vaccine. In the second study, a cocktail of HA-epitopes combined with flagellin (VC-4 flagellin), a promoter of the innate response, was used in SIV seronegative and seropositive pigs and were challenged with homologous and heterologous IAV strains. Vaccinated pigs reduced virus excretion, produced humoral response to subtypes H1 and H3, and neutralizing antibodies against both viruses. Maternal antibodies were not an obstacle but, clinical signs and pathology in the lungs were not reduced. Finally, in the third study, baculovirus expression vector system (BEVS) was used to obtain protein extracts containing HA-peptides linked with flagellin. The formulation was used to immunize chickens that were challenged with H7N1, a highly pathogenic virus. Vaccinated animals survived without showing any clinical sign and fewer or no virus secretion. The protection mechanism is under investigation. The general discussion is described in part III (Chapters VI-VII) with possible significance of the results obtained and the relation of conclusions drawn from each study. The bibliographical references (Chapter VIII) and appendices are also included

Modulating the immune response to SARS-CoV-2 by different nanocarriers delivering an mRNA expressing trimeric RBD of the spike protein: COVARNA Consortium

Vaccines based on mRNA technology have revolutionized the field. In fact, lipid nanoparticles (LNP) formulated with mRNA are the preferential vaccine platform used in the fight against SARS-CoV-2 infection, with wider application against other diseases. The high demand and property right protection of the most potent cationic/ionizable lipids used for LNP formulation of COVID-19 mRNA vaccines have promoted the design of alternative nanocarriers for nucleic acid delivery. In this study we have evaluated the immunogenicity and efficacy of different rationally designed lipid and polymeric-based nanoparticle prototypes against SARS-CoV-2 infection. An mRNA coding for a trimeric soluble form of the receptor binding domain (RBD) of the spike (S) protein from SARS-CoV-2 was encapsulated using different components to form nanoemulsions (NE), nanocapsules (NC) and lipid nanoparticles (LNP). The toxicity and biological activity of these prototypes were evaluated in cultured cells after transfection and in mice following homologous prime/boost immunization. Our findings reveal good levels of RBD protein expression with most of the formulations. In C57BL/6 mice immunized intramuscularly with two doses of formulated RBD-mRNA, the modified lipid nanoparticle (mLNP) and the classical lipid nanoparticle (LNP-1) were the most effective delivery nanocarriers at inducing binding and neutralizing antibodies against SARS-CoV-2. Both prototypes fully protected susceptible K18-hACE2 transgenic mice from morbidity and mortality following a SARS-CoV-2 challenge. These results highlight that modulation of mRNAs immunogenicity can be achieved by using alternative nanocarriers and support further assessment of mLNP and LNP-1 prototypes as delivery vehicles for mRNA vaccines