BR102022015163A2 - RECOMBINANT PLASMID FOR EXPRESSION OF SURFACE PROTEIN A OF STREPTOCOCCUS PNEUMONIAE, GENETICALLY MODIFIED INFLUENZA VIRUS AND USES - Google Patents
RECOMBINANT PLASMID FOR EXPRESSION OF SURFACE PROTEIN A OF STREPTOCOCCUS PNEUMONIAE, GENETICALLY MODIFIED INFLUENZA VIRUS AND USES Download PDFInfo
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Abstract
plasmídeo recombinante para expressão da proteína de superfície a de streptococcus pneumoniae, vírus influenza geneticamente modificado e usos. a presente tecnologia trata de um plasmídeo recombinante carreando o segmento truncado da neuraminidase e, portanto, defectivo para a multiplicação, e carreando também o gene para expressão da proteína de superfície a (pspa) de streptococcus pneumoniae. a presente tecnologia trata também de um vírus influenza geneticamente modificado carreando este plasmídeo e de sua utilização para produzir uma vacina bivalente contendo tal vírus como vetor vacinal contra a gripe e a pneumonia por pneumococos.recombinant plasmid for expression of the surface protein a of streptococcus pneumoniae, genetically modified influenza virus and uses. The present technology deals with a recombinant plasmid carrying the truncated segment of neuraminidase and, therefore, defective for multiplication, and also carrying the gene for expression of the surface protein a (pspa) of streptococcus pneumoniae. The present technology also deals with a genetically modified influenza virus carrying this plasmid and its use to produce a bivalent vaccine containing such virus as a vaccine vector against influenza and pneumococcal pneumonia.
Description
[01] A presentetecnologiatrata de um plasmídeorecombinantecarreando o segmentotruncado da neuraminidase e, portanto, defectivo para a multiplicação, e carreandotambém o gene para expressão da proteína de superfície A (PspA) de Streptococcus pneumoniae. A presentetecnologiatratatambém de um vírus influenza geneticamentemodificadocarreandoesteplasmídeo e de suautilização para produzirumavacinabivalentecontendotalvíruscomovetorvacinal contra a gripe e a pneumonia porpneumococos.[01] The present technology deals with a recombinant plasmid carrying the truncated segment of neuraminidase and, therefore, defective for multiplication, and also carrying the gene for expression of the surface protein A (PspA) of Streptococcus pneumoniae. The present technology also deals with a genetically modified influenza virus carrying this plasmid and its use to produce a bivalent vaccine containing such a virus as a vaccine vector against influenza and pneumococcal pneumonia.
[02] A infecçãopelo Streptococcus pneumoniae (S. pneumoniae) ocasionadoençascomo pneumonia e meningite, além de outrasinfecçõesfrequentes do tratorespiratório superior, sendoumaimportante causa de mortalidade de crianças e idosos no Brasil e no Mundo. O polissacarídeo capsular é o principal fator de virulência dos pneumococos e apresentampoucareatividadecruzada entre si.[02] Infection by Streptococcus pneumoniae (S. pneumoniae) causes diseases such as pneumonia and meningitis, in addition to other common infections of the upper respiratory tract, being an important cause of mortality in children and the elderly in Brazil and around the world. The capsular polysaccharide is the main virulence factor of pneumococci and has little cross-activity between them.
[03] A primeirageração de vacinas contra ospneumococos era composta dos polissacarídeos dos sorotiposmaisprevalentesnapopulação e possuíam a desvantagem de nãoinduzirrespostasimunológicas T-dependentes e serempoucoimunogênicasemcrianças. A segundageração de vacinas é composta de polissacarídeosconjugados a proteínascarregadoras. Essasvacinaspermitiramcontornarosproblemas das vacinas de polissacarídeo, sendoaindacapazes de inibir a colonização e proteger contra a doençapneumocócicainvasiva (pneumonia invasiva, meningite e bacteremia). Contudo, a proteçãoinduzidaporessasvacinas é sorotipoespecífica, o que resultounasubstituição dos sorotipos de pneumococos que circulamnapopulação. Desta forma, o desenvolvimento de vacinasbaseadasem antígenosproteicosconservados, talcomo o antígeno de superfície A (PspA), osquaisestejampresentesemtodososisolados de pneumococos e sejamcapazes de conferirimunidade contra o maiornúmero o possível de sorotipos, é umaestratégiainteressante para o desenvolvimento de novasvacinas contra essepatógeno.[03] The first generation of vaccines against pneumococci was composed of polysaccharides from the most prevalent serotypes in the population and had the disadvantage of not inducing T-dependent immunological responses and being low-immunogenic in children. The second generation of vaccines is composed of polysaccharides conjugated to carrier proteins. These vaccines made it possible to overcome the problems of polysaccharide vaccines, while still being able to inhibit colonization and protect against invasive pneumococcal disease (invasive pneumonia, meningitis and bacteremia). However, the protection induced by these vaccines is serotype specific, which resulted in the replacement of pneumococcal serotypes circulating in the population. Therefore, the development of vaccines based on conserved protein antigens, such as surface antigen A (PspA), which are present in all pneumococcal isolates and are capable of conferring immunity against as many serotypes as possible, is an interesting strategy for the development of new vaccines against this pathogen.
[04] A PspA é um fator de virulência o qual é encontradoemtodososisolados de pneumococo. Do ponto de vista funcional, a proteínaPspA age nainteração entre o patógeno e o hospedeirointerferindonaativação e deposição de fatores do sistemacomplementonasuperfície da bactéria. Alémdisso, foidescrito que a PspA protege o pneumococo de morte pela ação da apolactoferrinapresenteem mucosa. Estudosdemonstraram que a proteínaPspArecombinanteadministrada com um adjuvantepodeinduzirumaresposta de anticorposprotetoraemdesafiosletaisemmodelosanimais (Briles DE, Hollingshead SK, Paton JC, Ades EW, Novak L, van Ginkel FW, et al. Immunizations with pneumococcal surface protein A and pneumolysin are protective against pneumonia in a murine model of pulmonary infection with Streptococcus pneumoniae. The Journal of infectious diseases. 2003 Aug 1;188(3):339-48. PubMed PMID: 12870114. Epub 2003/07/19. eng).[04] PspA is a virulence factor which is found in all pneumococcal isolates. From a functional point of view, the PspA protein acts in the interaction between the pathogen and the host, interfering in the activation and deposition of factors from the complementary system on the surface of the bacteria. Furthermore, it has been described that PspA protects the pneumococcus from death through the action of apolactoferrin present in the mucosa. Studies have demonstrated that recombinant PspA protein administered with an adjuvant can induce a protective antibody response in lethal challenges in animal models (Briles DE, Hollingshead SK, Paton JC, Ades EW, Novak L, van Ginkel FW, et al. Immunizations with pneumococcal surface protein A and pneumolysin are protective against pneumonia in a murine model of pulmonary infection with Streptococcus pneumoniae. The Journal of infectious diseases. 2003 Aug 1;188(3):339-48. PubMed PMID: 12870114. Epub 2003/07/19. eng).
[05] As infecçõespelovírus influenza A sãooutraimportante causa de morbidade e mortalidade, sobretudoemcrianças e idososemtodo o mundo. A vacinação é a principal maneira de reduzir a morbidade e a mortalidadeassociadas à infecçãopelovírus influenza, sendo a respostaimunedirigidaprincipalmente contra as proteínas de superfíciehemaglutina (HA) e neuraminidase (NA).[05] Influenza A virus infections are another important cause of morbidity and mortality, especially in children and the elderly around the world. Vaccination is the main way to reduce morbidity and mortality associated with influenza virus infection, with the immune response being directed mainly against the surface proteins hemagglutin (HA) and neuraminidase (NA).
[06] Alémdisso, infecçõesbacterianassãocomplicaçõescomunsempacientesinfectados com o vírus influenza, principalmenteemidosos, sendocausadassobretudo pela Streptococcus pneumoniae. Aassociação entre as infecçõespelovírus influenza e as infecções invasivaspelopneumococofoiestabelecidadurante pandemias e na gripe sazonal.[06] Furthermore, bacterial infections are common complications in patients infected with the influenza virus, mainly in elderly people, being caused mainly by Streptococcus pneumoniae. The association between influenza virus infections and invasive pneumococcal infections has been established during pandemics and seasonal influenza.
[07] Atualmente as técnicas de biologia molecular conhecidascomogenéticareversapermitem a manipulaçãogenética do vírus influenza permitindo a construção de vírusrecombinantes, dentreosquais, vírus influenza carreandosegmentostruncados da neuraminidase. Esses vírussãodefectivos para a multiplicação e, portanto, segurosquanto à utilizaçãocomovacinasvivasbivalentes. Desta forma, a utilização do vírus influenza recombinantecarreando a sequênciacodificadora da proteínaPspAcomoplataforma para o desenvolvimento de vacinasbivalentes contra a gripe e a pneumonia porpneumococos é umaabordagemmuitointeressante, nãosomentepelofato de que, conformepreviamentedescrito, as infecçõespor esses doispatógenosestãoestreitamenterelacionadas, mas tambémpelofato de que osidosos, osquaissãoparticularmentesusceptíveisàsinfecçõespelopneumococo, são o principal grupoalvo da vacinação contra o vírus influenza, nãosendonecessárioincluiruma nova vacina no calendário de vacinação, o que reduziriaos custos operacionais.[07] Currently, molecular biology techniques known as reverse genetics allow genetic manipulation of the influenza virus, allowing the construction of recombinant viruses, including influenza viruses carrying truncated segments of neuraminidase. These viruses are defective for multiplication and, therefore, safe for use as live, bivalent vaccines. Thus, the use of the recombinant influenza virus carrying the PspA protein coding sequence as a platform for the development of bivalent vaccines against influenza and pneumococcal pneumonia is a very interesting approach, not only because, as previously described, infections caused by these two pathogens are closely related, but also because elderly people, who are particularly susceptible to pneumococcal infections, are the main target group for vaccination against the influenza virus, it is not necessary to include a new vaccine in the vaccination schedule, which would reduce operational costs.
[08] O documento “Uraki R, Piao Z, Akeda Y, Iwatsuki-Horimoto K, Kiso M, Ozawa M, Oishi K, Kawaoka Y. A Bivalent Vaccine Based on a PB2- Knockout Influenza Virus Protects Mice From Secondary Pneumococcal Pneumonia. J Infect Dis. 2015 Dec 15;212(12):1939-48”, descreve a construção de um vírus influenza knockout para a subunidade de polimerase PB2 e expressando a proteínaPspA de Streptococcus pneumoniae. Neste trabalho é utilizado o sorotipo 4 de Streptococcus pneumoniae para a construção do vírusgeneticamentemodificado.[08] The document “Uraki R, Piao Z, Akeda Y, Iwatsuki-Horimoto K, Kiso M, Ozawa M, Oishi K, Kawaoka Y. A Bivalent Vaccine Based on a PB2- Knockout Influenza Virus Protects Mice From Secondary Pneumococcal Pneumonia. J Infect Dis. 2015 Dec 15;212(12):1939-48”, describes the construction of a knockout influenza virus for the PB2 polymerase subunit and expressing the PspA protein from Streptococcus pneumoniae. In this work, serotype 4 of Streptococcus pneumoniae is used to construct the genetically modified virus.
[09] O documentoKramskaya T, Leontieva G, Desheva Y, Grabovskaya K, Gupalova T, Rudenko L, et al. (2019) Combined immunization with attenuated live influenza vaccine and chimeric pneumococcal recombinant protein improves the outcome of virus- bacterial infection in mice. PLoS ONE 14(9): e0222148”, descreveumavacinabivalente contra gripe e infecçãopneumocócicautilizandoumamistura de vacina viva contra influenza e a proteínaquimérica PSPF.[09] The documentKramskaya T, Leontieva G, Desheva Y, Grabovskaya K, Gupalova T, Rudenko L, et al. (2019) Combined immunization with attenuated live influenza vaccine and chimeric pneumococcal recombinant protein improves the outcome of virus-bacterial infection in mice. PLoS ONE 14(9): e0222148”, described a bivalent vaccine against influenza and pneumococcal infection using a mixture of live influenza vaccine and the chimeric protein PSPF.
[10]O documento “Katsura H, Piao Z, Iwatsuki-Horimoto K, et al. A bivalent vaccine based on a replication-incompetent influenza virus protects against Streptococcus pneumoniae and influenza virus infection. J Virol. 2014;88(22):13410-13417”, descreve a construção de um vírus influenza geneticamentemodificado, knockout para hemaglutinina (HA- KO) e possuindo a sequência para a regiãoantigênica da proteína de superfíciepneumocócica A (PspA) comovacinabivalente contra S. pneumoniae e o vírus influenza.[10]The document “Katsura H, Piao Z, Iwatsuki-Horimoto K, et al. A bivalent vaccine based on a replication-incompetent influenza virus protects against Streptococcus pneumoniae and influenza virus infection. J Virol. 2014;88(22):13410-13417”, describes the construction of a genetically modified influenza virus, knockout for hemagglutinin (HA-KO) and possessing the sequence for the antigenic region of the pneumococcal surface protein A (PspA) as a bivalent vaccine against S. pneumoniae and the influenza virus.
[11]No estado da técnicanãoforamencontradosdocumentos que descrevam um plasmídeo e um vírus influenza recombinantecarreando o segmentotruncado da neuraminidase e a proteínaPspA de S. pneumoniae para usoemvacinabivalente, com a mesmasequêncianucleotídica da presentetecnologia.[11] In the state of the art, no documents were found that describe a plasmid and a recombinant influenza virus carrying the truncated segment of the neuraminidase and the PspA protein from S. pneumoniae for use in a bivalent vaccine, with the same nucleotide sequence as the present technology.
[12]A presentetecnologiautiliza o vírus influenza recombinantecompreendendo um plasmídeocodificando um segmentotruncado da neuraminidase e a proteínaPspA de S. pneumoniae. O vírusrecombinantecarreia a proteína de S. pneumoniae nasuperfície da partícula viral e pode ser utilizadonaprodução de vacina para aimunização tanto contra o vírus influenza, quanto contra infecção pela S. pneumoniae. O antígeno de superfície A (PspA) incorporado no plasmídeo da presentetecnologiaestápresenteemtodososisolados de pneumococosdescritossendo, portanto, um candidatointeressante para o desenvolvimento de vacinascapazes de conferirimunidade contra a maioria dos sorotipos de pneumococosdescritos. Alémdisso, a utilização do vírus influenza comoplataforma para o desenvolvimento de vacinas bivalentes contra a gripe e a pneumonia por S. pneumoniae é umaabordagemmuitointeressante, nãosomentepelofato de que as infecçõespor esses doispatógenosestãoestritamenterelacionadas, mas tambémpelofato de que osgruposmaissusceptíveis à infecçãosecundáriapelo S. pneumoniae sãoosprincipaisgrupos-alvo da vacinação contra o vírus influenza. Desta forma, o público que se beneficia da presentetecnologia seria a parcela da população que já é vacinadaanualmente contra a influenza: criançasmenores de 5 anos de idade, idosos, gestantes, portadores de doençascrônicas. É relevante o fato de que as crianças, osidosos e pacientesportadores de doençascrônicas (ex: asmáticos, portadores de doençaspulmonaresobstrutivas, cardiopatas) estãomaispropensosaapresentarquadros de influenza mais graves e maissusceptíveisàsinfecçõesbacterianassecundáriasnas vias respiratórias.[12] The present technology uses the recombinant influenza virus comprising a plasmid encoding a truncated segment of the neuraminidase and the S. pneumoniae PspA protein. The recombinant virus carries the S. pneumoniae protein on the surface of the viral particle and can be used in the production of vaccines for immunization against both the influenza virus and S. pneumoniae infection. The surface antigen A (PspA) incorporated into the plasmid of the present technology is present in all described pneumococcal isolates and is, therefore, an interesting candidate for the development of vaccines capable of conferring immunity against most of the described pneumococcal serotypes. Furthermore, the use of the influenza virus as a platform for the development of bivalent vaccines against influenza and S. pneumoniae pneumonia is a very interesting approach, not only due to the fact that infections caused by these two pathogens are closely related, but also due to the fact that the groups most susceptible to secondary infection by S. pneumoniae are the main target groups for vaccination. against the influenza virus. In this way, the public that benefits from this technology would be the portion of the population that is already vaccinated annually against influenza: children under 5 years of age, the elderly, pregnant women, people with chronic diseases. It is relevant that children, the elderly and patients with chronic diseases (e.g. asthmatics, patients with obstructive pulmonary diseases, heart disease) are more likely to present more severe cases of influenza and are more susceptible to secondary bacterial infections in the respiratory tract.
[13]A figura 1 é a representaçãoesquemática das regiões que compõemaestrutura da proteínaPspAclado 4 do isolado 255/00 (sorotipo 14) de S. pneumoniae, a qual foiutilizada tanto para a construção do vírus influenza recombinante Flu-PspA (através da clonagem da sequência da PspA4pro) quanto para a produção da proteína PspA4 utilizada para as imunizações de reforço. A sequênciacodificadora da proteína PspA4Pro engloba da região N-terminal em α- hélice (αHD) até a regiãoricaemprolinas da PspA (PRD). Já a sequênciacodificadora da proteína PspA4 englobatodas as regiões. O domínio N-terminal (αHD) estárepresentadoemvermelho; a região do domínio N-terminal que define osclados (CDR) estárepresentadaemvermelhoquadriculado; o domínioricoemprolinas (PRD) estárepresentadoemazul; o domínio de ligação à colinaestárepresentado embranco; a cauda C-terminal hidrofóbic (Cauda) estárepresentadaempreto.[13] Figure 1 is the schematic representation of the regions that make up the structure of the PspAclado 4 protein from isolate 255/00 (serotype 14) of S. pneumoniae, which was used both for the construction of the Flu-PspA recombinant influenza virus (through cloning of the sequence of PspA4pro) and for the production of the PspA4 protein used for booster immunizations. The coding sequence for the PspA4Pro protein ranges from the N-terminal α-helix region (αHD) to the prolin-rich region of PspA (PRD). The coding sequence for the PspA4 protein encompasses all regions. The N-terminal domain (αHD) is represented in red; the region of the N-terminal domain that defines the clades (CDR) is represented in red grid; the prolin-rich domain (PRD) is represented in blue; the choline-binding domain is shown in white; the hydrophobic C-terminal tail (Tail) is shown in black.
[14]A figura 2 é a representaçãoesquemática do plasmídeo pPRNA169-PSPA-178, utilizado para a síntese da neuraminidase (NA) truncadacontendo a sequênciaheteróloga da proteínaPspA, e dos plasmídeos de transferênciaambissenso (Phw2.000), utilizados para a síntese dos demaissegmentos do vírus influenza. A sequência da PspA4Pro (doravantechamadasimplesmente de PspA) foiinserida no sítiomúltiplo de clonagem entre osprimeiros 169 e osúltimos 178 nucleotídeos do segmento da NA. O promotor da polimerase I humana (pPolI) permite a transcrição de um segmento de vRNAsintéticosemadição de 7-Metilguanosina naextremidade 5’ (Cap-5’) e cauda poli A. A sequênciaribozima do vírus da hepatite delta (Rib Hδ) realiza a clivagem do segmentorecémgeradoemposiçõesespecíficas, de tal forma que a sequência do RNA sintetizado é idênticaàquela do segmento de RNA viral original. A transcrição do RNAm e tradução da proteína é realizadapelasproteínasviraisgeradaspelosdemaisplasmídeosambissenso (Phw2.000).[14] Figure 2 is the schematic representation of the plasmid pPRNA169-PSPA-178, used for the synthesis of the truncated neuraminidase (NA) containing the heterologous sequence of the PspA protein, and of the ambisense transfer plasmids (Phw2.000), used for the synthesis of the other segments of the influenza virus. The PspA4Pro sequence (hereinafter simply called PspA) was inserted into the multiple cloning site between the first 169 and the last 178 nucleotides of the NA segment. The promoter of human polymerase I (pPolI) allows the transcription of a segment of synthetic vRNAs with the addition of 7-Methylguanosine at the 5' end (Cap-5') and poly A tail. The ribozyme sequence of the hepatitis delta virus (Rib Hδ) cleaves the newly generated segment in specific positions, such that the sequence of the synthesized RNA is identical to that of the original viral RNA segment. Transcription of mRNA and protein translation is carried out by viral proteins generated by other ambisense plasmids (Phw2.000).
[15]A figura 3 representa o esquema da construção viral a partir de seteplasmídeosbidirecionaiscodificandocada um dos segmentosvirais e o plasmídeodefinido pela SEQ ID No 1, compreendendo a NA truncadacontendo a sequênciaheterólogaPspAfusionada à haste da neuraminidase e ancorada no envelope viral.[15] Figure 3 represents the viral construction scheme from seven bidirectional plasmids encoding each of the viral segments and the plasmid defined by SEQ ID No. 1, comprising the truncated NA containing the heterologous sequence Psp Afused to the neuraminidase stem and anchored in the viral envelope.
[16]A figura 4 representa o fenótipo das placas de liseobservadasemcélulas MDCK apóstitulação sob agarose do vírus influenza selvagem A/PR8/34 (PR8) (A) e dos vírusrecombinantes Flu-CT (B) e Flu-PspA (C). Oscírculosembrancorepresentam as placas de lisecaracterísticas da multiplicação viral.[16] Figure 4 represents the phenotype of the lysis plaques observed in MDCK cells after titration under agarose of the wild influenza virus A/PR8/34 (PR8) (A) and the recombinant viruses Flu-CT (B) and Flu-PspA (C). The white circles represent the lysis plaques characteristic of viral multiplication.
[17]A figura 5 mostraaexpressão da proteína PspA4 emcélulas MDCK nãoinfectadas, MOCK, (A); ouinfectadas com osvírusrecombinantes Flu-CT (B); ou Flu-PspA (C); avaliadaspormicroscopia confocal. Osvírusforamamplificadosnapresença de neuraminidase emuma M.O.I. 0,5 e, após 20 horas de amplificação, as célulasforammarcadas com anticorpopoliclonal anti-PspA4pro e anticorposecundário anti-IgG conjugado a FITC. Osnúcleos das célulasforammarcados com DAPI e estãorepresentadosemazul. As imagens foramadquiridasemmicroscópio confocal Nikon (modelo C2 Plus) em um aumento de 100x. A coluna 1 representa a sobreposição dos corantes DAPI e FITC nascélulas, enquanto as coluna 2 e 3 representam as célulasmarcadassomente com oscorantes DAPI e FITC, respectivamente.[17] Figure 5 shows the expression of PspA4 protein in uninfected MDCK cells, MOCK, (A); or infected with Flu-CT recombinant viruses (B); or Flu-PspA (C); evaluated by confocal microscopy. The viruses were amplified in the presence of neuraminidase in an M.O.I. 0.5 and, after 20 hours of amplification, the cells were labeled with anti-PspA4pro polyclonal antibody and anti-IgG secondary antibody conjugated to FITC. Cell nuclei were labeled with DAPI and are represented in blue. Images were acquired using a Nikon confocal microscope (model C2 Plus) at 100x magnification. Column 1 represents the overlap of DAPI and FITC dyes in the cells, while columns 2 and 3 represent cells labeled only with DAPI and FITC dyes, respectively.
[18]A figura 6 mostraaavaliação da sobrevivência (A) e perda de peso (B) emcamundongosinoculados com vírusrecombinante. Mock (íconeazul): animais que receberamuma dose de PBS, pela via intranasal; Flu-CT (íconevermelho): animais que receberam 105 PFU/mL de vírusrecombinantecontrole, pela via intranasal; Flu-PspA (ícone rosa): animais que receberam 105 PFU/mL de vírusrecombinantecodificando a PspA, pela via intranasal; PR8 (íconeverde): animais que receberam 103 PFU/mL de vírusselvagem A/PR8/34 (H1N1), pela via intranasal. As barras representam as médias ± desviopadrão dos dados de um únicoexperimentoindependente com pelomenos 5 camundongosporgrupo. As curvas de sobrevivênciaforamcomparadasutilizandoos testes Log-rank (Mantel-Cox) e Gehan-Breslow-Wilcoxon e as diferençasnaperda de peso dos animais dos diferentesgruposforamdeterminadaspor ANOVA (p<0,05). *, **, *** e **** indicamdiferençasestatisticamentesignificantesnosvalores de p<0,05,<0,01,<0,001 e <0,0001, respectivamente.[18] Figure 6 shows the evaluation of survival (A) and weight loss (B) in mice inoculated with recombinant virus. Mock (blue icon): animals that received a dose of PBS, intranasally; Flu-CT (red icon): animals that received 105 PFU/mL of control recombinant virus, intranasally; Flu-PspA (pink icon): animals that received 105 PFU/mL of recombinant virus encoding PspA, intranasally; PR8 (green icon): animals that received 103 PFU/mL of wild-type virus A/PR8/34 (H1N1), intranasally. The bars represent the means ± standard deviation of data from a single independent experiment with at least 5 mice per group. Survival curves were compared using the Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests and differences in weight loss of animals from different groups were determined by ANOVA (p<0.05). *, **, *** and **** indicate statistically significant differences at p-values <0.05, <0.01, <0.001 and <0.0001, respectively.
[19]A figura 7 representaostítulos de IgG total (A) e subclasses IgG1 e IgG2c (B) específicos para PspA4 presentes no soro, após a 2a dose do protocolo de imunização. Mock: animais que receberam duas doses de PBS, por via intranasal; Flu-CT/Alum: animais que receberam 105 PFU/mL de vírusrecombinantecontrole, por via intranasal, naprimeira dose e reforço com alúmen, tambémpor via intranasal; Flu- PspA/PspA4+Alum: animais que receberam 105 PFU/mL de vírusrecombinantecodificando a PspA, por via intranasal, naprimeira dose e reforço com 5 μg de proteína PspA4 recombinanteadjuvantada com alúmen, tambémpor via intranasal. As amostrasde soroforamcoletadas 14 diasapós a segunda dose de imunização e ostítulos de anticorposforamrepresentadoscomo o log2 da maiordiluiçãoem que a D.O. a 450 nm apresentou valor > 0,1. As barras representam as médias ± desviopadrão dos dados de trêsexperimentosindependentes com pelomenos 5 camundongosporgrupoemcada. Diferençasnaprodução de anticorpos dos animais do grupovacinalemrelaçãoaos dos gruposcontroleforamdeterminadaspor ANOVA (p<0,05). *, **, *** e **** indicamdiferençasestatisticamentesignificantesnosvalores de p<0,05, <0,01, <0,001 e <0,0001, respectivamente.[19] Figure 7 represents the total IgG titers (A) and IgG1 and IgG2c subclasses (B) specific for PspA4 present in the serum, after the 2nd dose of the immunization protocol. Mock: animals that received two doses of PBS, intranasally; Flu-CT/Alum: animals that received 105 PFU/mL of control recombinant virus, intranasally, in the first dose and boosted with alum, also intranasally; Flu- PspA/PspA4+Alum: animals that received 105 PFU/mL of recombinant virus encoding PspA, intranasally, in the first dose and boosted with 5 μg of recombinant PspA4 protein adjuvanted with alum, also intranasally. Serum samples were collected 14 days after the second dose of immunization and antibody titers were represented as the log2 of the highest dilution at which the O.D. at 450 nm it showed a value > 0.1. The bars represent the means ± standard deviation of data from three independent experiments with at least 5 mice per group in each. Differences in antibody production of animals in the vaccine group in relation to those in the control groups were determined by ANOVA (p<0.05). *, **, *** and **** indicate statistically significant differences in p-values <0.05, <0.01, <0.001 and <0.0001, respectively.
[20]A figura 8 mostra a ligação de anticorpos IgG nasuperfície das bactérias das cepas EF3030 (A), D39 (B), M10 (C), 3JYP2670 (D) e ATCC6303 (E) de S. pneumoniae, após a 2a dose do protocolo de imunização, representadaporcélulaspositivas. Mock (colunaazul): animais que receberam duas doses de PBS, por via intranasal; Flu- CT/Alum (colunacinza): animais que receberam 105 PFU/mL de vírusrecombinantecontrole, por via intranasal, naprimeira dose e reforço com alúmen, tambémpor via intranasal; Flu-PspA/PspA4+Alum (coluna rosa): animais que receberam 105 PFU/mL de vírusrecombinantecodificando a PspA, por via intranasal, naprimeira dose e reforço com 5 μg de proteína PspA4 recombinanteadjuvantada com alúmen, tambémpor via intranasal. As amostras de soroforamcoletadas 14 diasapós a segunda dose de imunização. As barras representam as médias ± desviopadrão dos dados de trêsexperimentosindependentes com pelomenos 5 camundongosporgrupoemcada. Diferençasnaporcentagem de célulaspositivasobservadas no soro dos animais do grupovacinalemrelaçãoaos dos gruposcontroleforamdeterminadaspor ANOVA (p<0,05). *, **, *** e **** indicamdiferençasestatisticamentesignificantesnosvalores de p<0,05, <0,01, <0,001 e <0,0001, respectivamente.[20] Figure 8 shows the binding of IgG antibodies to the surface of bacteria from strains EF3030 (A), D39 (B), M10 (C), 3JYP2670 (D) and ATCC6303 (E) of S. pneumoniae, after the 2nd dose of the immunization protocol, represented by positive cells. Mock (blue column): animals that received two doses of PBS, intranasally; Flu-CT/Alum (gray column): animals that received 105 PFU/mL of control recombinant virus, intranasally, in the first dose and boosted with alum, also intranasally; Flu-PspA/PspA4+Alum (pink column): animals that received 105 PFU/mL of recombinant virus encoding PspA, intranasally, in the first dose and boosted with 5 μg of recombinant PspA4 protein adjuvanted with alum, also intranasally. Serum samples were collected 14 days after the second dose of immunization. The bars represent the means ± standard deviation of data from three independent experiments with at least 5 mice per group in each. Differences in the percentage of positive cells observed in the serum of animals in the vaccine group in relation to those in the control groups were determined by ANOVA (p<0.05). *, **, *** and **** indicate statistically significant differences in p-values <0.05, <0.01, <0.001 and <0.0001, respectively.
[21]A figura 9 representa a sobrevivência (A) e perda de peso (B) observadasnosanimaisimunizados pela via intranasal, apósdesafioletal com S. pneumoniae. Mock: animais que receberam duas doses de PBS, por via intranasal; Flu-CT/Alum: animais que receberam 105 PFU/mL de vírusrecombinantecontrole, por via intranasal, naprimeira dose e reforço com alúmen, tambémpor via intranasal; Flu- PspA/PspA4+Alum: animais que receberam 105 PFU/mL de vírusrecombinantecodificando a PspA, por via intranasal, naprimeira dose e reforço com 5 μg de proteína PspA4 recombinanteadjuvantada com alúmen, tambémpor via intranasal. Osanimaisforamdesafiados com 5x104 UFC/mL (7xDL50) da cepa ATCC6303 de S. pneumoniae, 21 diasapós a última dose. As barras representam as médias ± desviopadrão dos dados de trêsexperimentosindependentes com pelomenos 5 camundongosporgrupoemcada. As curvas de sobrevivênciaforamcomparadasutilizandoos testes Log-rank (Mantel-Cox) e Gehan- Breslow-Wilcoxon e as diferençasnaperda de peso dos animais dos diferentesgruposforamdeterminadaspor ANOVA (p<0,05). *, **, *** e **** indicamdiferençasestatisticamentesignificantesnosvalores de p<0,05, <0,01, <0,001 e <0,0001, respectivamente.[21] Figure 9 represents the survival (A) and weight loss (B) observed in animals immunized via the intranasal route, after lethal challenge with S. pneumoniae. Mock: animals that received two doses of PBS, intranasally; Flu-CT/Alum: animals that received 105 PFU/mL of control recombinant virus, intranasally, in the first dose and boosted with alum, also intranasally; Flu- PspA/PspA4+Alum: animals that received 105 PFU/mL of recombinant virus encoding PspA, intranasally, in the first dose and boosted with 5 μg of recombinant PspA4 protein adjuvanted with alum, also intranasally. The animals were challenged with 5x104 CFU/mL (7xDL50) of the ATCC6303 strain of S. pneumoniae, 21 days after the last dose. The bars represent the means ± standard deviation of data from three independent experiments with at least 5 mice per group in each. Survival curves were compared using the Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests and differences in weight loss of animals from different groups were determined by ANOVA (p<0.05). *, **, *** and **** indicate statistically significant differences in p-values <0.05, <0.01, <0.001 and <0.0001, respectively.
[22]A figura 10 representaostítulos de IgG total específicos para o vírus Influenza A (PR8) presentes no soro, apósaimunização. Mock: animais que receberam PBS, por via intranasal; Flu-CT: animais que receberam 105 PFU de vírusrecombinantecontrole, por via intranasal; Flu-PspA: animais que receberam 105 PFU de vírusrecombinantecodificando a PspA, por via intranasal. As amostras de soroforamcoletadas 14 diasapósaimunização e ostítulos de anticorposrepresentam o log2 da maiordiluição que apresentou D.O. em 450 nm de 0,1. As barras representam as médias ± desviopadrão dos dados de trêsexperimentosindependentes com pelomenos 5 camundongosporgrupoemcada. Diferençasnaprodução de anticorpos dos animais do grupovacinalemrelaçãoaos dos gruposcontroleforamdeterminadaspor ANOVA (p<0,05). *, **, *** e **** indicamdiferençasestatisticamentesignificantesnosvalores de P <0,05, <0,01, <0,001 e <0,0001, respectivamente.[22] Figure 10 represents the total IgG titers specific for the Influenza A virus (PR8) present in the serum after immunization. Mock: animals that received PBS intranasally; Flu-CT: animals that received 105 PFU of control recombinant virus, intranasally; Flu-PspA: animals that received 105 PFU of recombinant virus encoding PspA, intranasally. Serum samples were collected 14 days after immunization and antibody titers represent the log2 of the highest dilution that presented O.D. at 450 nm of 0.1. The bars represent the means ± standard deviation of data from three independent experiments with at least 5 mice per group in each. Differences in antibody production of animals in the vaccine group in relation to those in the control groups were determined by ANOVA (p<0.05). *, **, *** and **** indicate statistically significant differences at P values <0.05, <0.01, <0.001 and <0.0001, respectively.
[23]A figura 11 mostra a sobrevivência (A) e perda de peso (B) observadasapósdesafioletal com vírus influenza A/PR8/34 (H1N1), emanimaisimunizados com uma dose dos vírusrecombinantes. Naive (íconeverde): animaisnãoinfectados; Mock (íconeazul): animais que receberamuma dose de PBS, por via intranasal; Flu-CT (íconevermelho): animais que receberam 105 PFU/mL de vírusrecombinantecontrole, por via intranasal; Flu-PspA (ícone rosa): animais que receberam 105 PFU/mL de vírusrecombinantecodificando a PspA, por via intranasal. Osanimaisforamdesafiados com 105 PFU/mL (100xDL50) do vírus A/PR8/34 (H1N1), 21 diasapósaimunização. As barras representam as médias ± desviopadrão dos dados de um únicoexperimentoindependente com pelomenos 5 camundongosporgrupo. As curvas de sobrevivênciaforamcomparadasutilizandoos testes Logrank (Mantel-Cox) e Gehan-Breslow-Wilcoxon e as diferençasnaperda de peso dos animais dos diferentesgruposforamdeterminadaspor ANOVA (p<0,05). *, **, *** e **** indicamdiferençasestatisticamentesignificantesnosvalores de p<0,05,<0,01,<0,001 e <0,0001, respectivamente.[23] Figure 11 shows the survival (A) and weight loss (B) observed after lethal challenge with influenza A/PR8/34 (H1N1) virus, in animals immunized with a dose of the recombinant viruses. Naive (green icon): uninfected animals; Mock (blue icon): animals that received a dose of PBS intranasally; Flu-CT (red icon): animals that received 105 PFU/mL of control recombinant virus, intranasally; Flu-PspA (pink icon): animals that received 105 PFU/mL of recombinant virus encoding PspA, intranasally. The animals were challenged with 105 PFU/mL (100xLD50) of the A/PR8/34 (H1N1) virus, 21 days after immunization. The bars represent the means ± standard deviation of data from a single independent experiment with at least 5 mice per group. Survival curves were compared using the Logrank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests and differences in weight loss of animals from different groups were determined by ANOVA (p<0.05). *, **, *** and **** indicate statistically significant differences at p-values <0.05, <0.01, <0.001 and <0.0001, respectively.
[24]A figura 12 representaostítulosviraisobtidos com a multiplicação do vírus Flu-PspAemovosembrionados, durante 48 horas. Controleambiental: ovosnãoinoculados; PBS+600 μU: ovosinoculados com 100 μL de PBS contendo 600 μU de neuraminidase de Vibrio cholerae; S/N: ovosinoculados com 100 μL de PBS contendo 103 PFU do vírus Flu- PspA e semadição de neuraminidase; 300 μU: ovosinoculados com 100 μL de PBS contendo 103 PFU do vírus Flu-PspAjuntamente com 300 μU de neuraminidase de Vibrio cholerae; 600 μU: ovosinoculados com 100 μL de PBS contendo 103 PFU do vírus Flu-PspAjuntamente com 600 μU de neuraminidase de Vibrio cholerae. O líquidoalantoicofoicoletadoapós 48 horas de multiplicação. O título viral presente no líquidoalantoicocoletadofoideterminadoportitulaçãoporplaca de lise sob agarose emcélulas MDCK e osresultadosforam expressos em log10 dos títulosviraisem PFU/mL. As barras representam as médias ± desviopadrão dos dados de um experimentoindependente com pelomenos 5 ovosporgrupo. Diferenças entre osgruposforamdeterminadaspor ANOVA (p<0,05). *, **, *** e **** indicamdiferençasestatisticamentesignificantesnosvalores de p<0,05,<0,01,<0,001 e <0,0001, respectivamente.[24] Figure 12 represents the viral titers obtained with the multiplication of the Flu-PspA and embryonated Flu-PspA virus for 48 hours. Environmental control: non-inoculated eggs; PBS+600 μU: eggsinoculated with 100 μL of PBS containing 600 μU of Vibrio cholerae neuraminidase; S/N: eggsinoculated with 100 μL of PBS containing 103 PFU of the Flu-PspA virus and no addition of neuraminidase; 300 μU: eggsinoculated with 100 μL of PBS containing 103 PFU of the Flu-Psp virusAlong with 300 μU of Vibrio cholerae neuraminidase; 600 μU: eggsinoculated with 100 μL of PBS containing 103 PFU of the Flu-Psp virusAlong with 600 μU of Vibrio cholerae neuraminidase. Allantoic liquid was collected after 48 hours of multiplication. The viral titer present in the collected allantoic liquid was determined by titration by lysis plate under agarose in MDCK cells and the results were expressed as log10 of the viral titers in PFU/mL. The bars represent the means ± standard deviation of data from an independent experiment with at least 5 eggs per group. Differences between groups were determined by ANOVA (p<0.05). *, **, *** and **** indicate statistically significant differences at p-values <0.05, <0.01, <0.001 and <0.0001, respectively.
[25]A figura 13 representaostítulos de IgG total específicos para PspA4 presentes no soro, após a primeira (A) e segunda doses (B) do protocolo de imunização, pela via intramuscular. Mock: animais que receberam duas doses de PBS, por via intramuscular; Flu-CT/Alum: animais que receberam 105 PFU/mL de vírusrecombinantecontrole, por via intramuscular, naprimeira dose e reforço com alúmen, tambémpor via intramuscular; Flu-PspA/PspA4+Alum: animais que receberam 105 PFU/mL de vírusrecombinantecodificando a PspA, por via intramuscular, naprimeira dose e reforço com 5 μg de proteína PspA4 recombinanteadjuvantada com alúmen, tambémpor via intramuscular. As amostras de soroforamcoletadas 14 diasapós a primeira e segunda doses de imunização e ostítulos de anticorposforamrepresentadoscomo o log2 da maiordiluiçãoem que a D.O. a 450 nm apresentou valor > 0,1. As barras representam as médias ± desviopadrão dos dados de um experimentoindependente com 6 camundongosemcadagrupo. Diferençasnaprodução de anticorpos dos animais do grupovacinalemrelaçãoaos dos gruposcontroleforamdeterminadaspor ANOVA (p<0,05). *, **, *** e **** indicamdiferençasestatisticamentesignificantesnosvalores de p<0,05, <0,01, <0,001 e <0,0001, respectivamente.[25] Figure 13 represents the total IgG titers specific to PspA4 present in the serum, after the first (A) and second doses (B) of the immunization protocol, via the intramuscular route. Mock: animals that received two doses of PBS, intramuscularly; Flu-CT/Alum: animals that received 105 PFU/mL of control recombinant virus, intramuscularly, in the first dose and boosted with alum, also intramuscularly; Flu-PspA/PspA4+Alum: animals that received 105 PFU/mL of recombinant virus encoding PspA, intramuscularly, in the first dose and boosted with 5 μg of recombinant PspA4 protein adjuvanted with alum, also intramuscularly. Serum samples were collected 14 days after the first and second doses of immunization and antibody titers were represented as the log2 of the highest dilution at which the O.D. at 450 nm it showed a value > 0.1. The bars represent the means ± standard deviation of data from an independent experiment with 6 mice in each group. Differences in antibody production of animals in the vaccine group in relation to those in the control groups were determined by ANOVA (p<0.05). *, **, *** and **** indicate statistically significant differences at p-values <0.05, <0.01, <0.001 and <0.0001, respectively.
[26]A figura 14 representa a sobrevivência (A) e perda de peso (B) observadasnosanimaisimunizados pela via intramuscular, apósdesafioletal com S. pneumoniae. Mock: animais que receberam duas doses de PBS, por via intramuscular; Flu-CT/Alum: animais que receberam 105 PFU/mL de vírusrecombinantecontrole, por via intramuscular, naprimeira dose e reforço com alúmen, tambémpor via intramuscular; Flu- PspA/PspA4+Alum: animais que receberam 105 PFU/mL de vírusrecombinantecodificando a PspA, por via intramuscular, naprimeira dose e reforço com 5 μg de proteína PspA4 recombinanteadjuvantada com alúmen, tambémpor via intramuscular. Osanimaisforamdesafiados com 3x104 UFC/mL (7xDL50) da cepa ATCC6303 de S. pneumoniae, 21 diasapós a última dose. As barras representam as médias ± desviopadrão dos dados de um experimentoindependente com 6 camundongosemcadagrupo. As curvas de sobrevivênciaforamcomparadasutilizandoos testes Log-rank (Mantel-Cox) e Gehan- Breslow-Wilcoxon e as diferençasnaperda de peso dos animais dos diferentesgruposforamdeterminadaspor ANOVA (p<0,05). *, **, *** e **** indicamdiferençasestatisticamentesignificantesnosvalores de p<0,05, <0,01, <0,001 e <0,0001, respectivamente.[26] Figure 14 represents the survival (A) and weight loss (B) observed in animals immunized intramuscularly, after lethal challenge with S. pneumoniae. Mock: animals that received two doses of PBS, intramuscularly; Flu-CT/Alum: animals that received 105 PFU/mL of control recombinant virus, intramuscularly, in the first dose and boosted with alum, also intramuscularly; Flu- PspA/PspA4+Alum: animals that received 105 PFU/mL of recombinant virus encoding PspA, intramuscularly, in the first dose and boosted with 5 μg of recombinant PspA4 protein adjuvanted with alum, also intramuscularly. The animals were challenged with 3x104 CFU/mL (7xDL50) of the ATCC6303 strain of S. pneumoniae, 21 days after the last dose. The bars represent the means ± standard deviation of data from an independent experiment with 6 mice in each group. Survival curves were compared using the Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests and differences in weight loss of animals from different groups were determined by ANOVA (p<0.05). *, **, *** and **** indicate statistically significant differences in p-values <0.05, <0.01, <0.001 and <0.0001, respectively.
[27]A presentetecnologiatrata de um plasmídeorecombinantecarreando o segmentotruncado da neuraminidase e, portanto, defectivo para a multiplicação, e carreandotambém o gene para expressão da proteína de superfície A (PspA) de Streptococcus pneumoniae. A presentetecnologiatratatambém de um vírus influenza geneticamentemodificadocarreandoesteplasmídeo e de suautilização para produzirumavacinabivalentecontendotalvíruscomovetorvacinal contra a gripe e a pneumonia porpneumococos.[27] The present technology deals with a recombinant plasmid carrying the truncated segment of neuraminidase and, therefore, defective for multiplication, and also carrying the gene for expression of the surface protein A (PspA) of Streptococcus pneumoniae. The present technology also deals with a genetically modified influenza virus carrying this plasmid and its use to produce a bivalent vaccine containing such a virus as a vaccine vector against influenza and pneumococcal pneumonia.
[28]O plasmídeorecombinante da presentetecnologia é definido pela sequência de nucleotídeos SEQ ID N°1, a qual compreende um segmentotruncado da neuraminidase, correspondenteaosnucleotídeos do segmento 1 a 1526 da SEQ ID N°1, e a sequênciacodificadora da proteína PspA4Pro de Streptococcus pneumoniae, correspondenteaosnucleotídeos do segmento 182 a 1336 da SEQ ID N°1.[28] The recombinant plasmid of the present technology is defined by the nucleotide sequence SEQ ID No. 1, which comprises a truncated segment of the neuraminidase, corresponding to the nucleotides of segment 1 to 1526 of SEQ ID No. 1, and the coding sequence for the PspA4Pro protein of Streptococcus pneumoniae, corresponding to the nucleotides of segment 182 to 1336 of SEQ ID NO. 1.
[29]O vírus influenza geneticamentemodificado é caracterizadoporcompreender o plasmídeodefinido pela sequência de nucleotídeos SEQ ID N°1, a qual compreende um segmentotruncado da neuraminidase, correspondenteaosnucleotídeos do segmento 1 a 1526 a SEQ ID N°1, e o gene da proteína de superfície A (PspA) de Streptococcus pneumoniae, correspondenteaosnucleotídeos do segmento 182 a 1336 da SEQ ID N°1.[29] The genetically modified influenza virus is characterized by comprising the plasmid defined by the nucleotide sequence SEQ ID NO. 1, which comprises a truncated segment of neuraminidase, corresponding to the nucleotides of segment 1 to 1526 to SEQ ID NO. (PspA) from Streptococcus pneumoniae, corresponding to the nucleotides in segment 182 to 1336 of SEQ ID NO. 1.
[30]Uma forma de concretização, nãolimitante, da tecnologiacompreendeformulaçõesvacinaiscontendo o plasmídeorecombinante SEQ ID No 1 e excipientesfarmacológica e farmaceuticamenteaceitáveis.[30] A non-limiting embodiment of the technology comprises vaccine formulations containing the SEQ ID No. 1 recombinant plasmid and pharmaceutically and pharmaceutically acceptable excipients.
[31]Outra forma de concretização, nãolimitante, da tecnologiacompreendeformulaçõesvacinaiscontendo o vírus influenza geneticamentemodificadoexpressando o plasmídeorecombinante (SEQ ID No 1) e excipientesfarmacológica e farmaceuticamenteaceitáveis.[31] Another non-limiting embodiment of the technology comprises vaccine formulations containing the genetically modified influenza virus expressing the recombinant plasmid (SEQ ID No. 1) and pharmaceutically and pharmaceutically acceptable excipients.
[32]As formulaçõesvacinaispodem ser administradas pela via oral, subcutânea, intradérmica, intranasal e intramuscular.[32] Vaccine formulations can be administered orally, subcutaneously, intradermally, intranasally and intramuscularly.
[33]O plasmídeorecombinante e o vírus influenza geneticamentemodificadopodem ser utilizados para prepararvacinasbivalentes com atividadeimunogênica contra gripe e pneumonia porpneumococos.[33] The recombinant plasmid and the genetically modified influenza virus can be used to prepare bivalent vaccines with immunogenic activity against influenza and pneumococcal pneumonia.
[34]A presentetecnologiapode ser maisbemcompreendidaatravés dos exemplos que se seguem, nãolimitantes.[34] The present technology can be better understood through the following, non-limiting examples.
[35]O plasmídeo pPRNA169x178 codifica um segmentotruncado da NA o qual possuirespectivamenteosprimeiros 169 nucleotídeos da região 3'do segmento da NA seguidopor um sítiomúltiplo de clonagem e pelosúltimos 178 nucleotídeos da região 5'do segmento da NA. A sequênciacodificadora da proteína PspA4Pro, a qual engloba a região N- terminal em a-hélice e a regiãoricaemprolinas da PspAclado 4 do isolado 255/00 sorotipo 14 (Figura 1) de S. pneumoniae foiconstruída sob a forma de um gene sintético pela empresa GENSCRIPT (Hong Kong) e clonada no plasmídeo pPR169x178, resultando no plasmídeo pPR169-PspA-178, correspondente à SEQ ID NO 1 (Figura 2). É importantesalientar que, nessaconstrução, a proteínaheterólogaPspA é produzida sob a forma de proteína de fusão com a neuraminidase e, como a maiorparte da sequência da neuraminidase foiremovida, o polipeptídeoresultante é desprovido de atividade sialidase. Osplasmídeoscodificandoosdemaissegmentos do vírus A/PR8/34 (PR8) foramgentilmentefornecidospelo Dr. Ron Fouchier do Instituto Erasmus de Rotterdam (Holanda).[35] Plasmid pPRNA169x178 encodes a truncated NA segment which has respectively the first 169 nucleotides of the 3' region of the NA segment followed by a multiple cloning site and the last 178 nucleotides of the 5' region of the NA segment. The coding sequence for the PspA4Pro protein, which encompasses the N-terminal α-helical region and the prolin-rich region of PspAclado 4 from S. pneumoniae isolate 255/00 serotype 14 (Figure 1), was constructed in the form of a synthetic gene by the company GENSCRIPT (Hong Kong) and cloned into plasmid pPR169x178, resulting in plasmid pPR169-PspA-178, corresponding to SEQ ID NO 1 (Figure 2). It is important to highlight that, in this construction, the heterologous protein PspA is produced in the form of a fusion protein with neuraminidase and, as most of the neuraminidase sequence has been removed, the resulting polypeptide is devoid of sialidase activity. Plasmids encoding the remaining segments of the A/PR8/34 (PR8) virus were kindly provided by Dr. Ron Fouchier from the Erasmus Institute in Rotterdam (Netherlands).
[36]Osvírus influenza recombinantesforamobtidosporgenéticareversa de acordo com a técnicarepresentadanafigura 3 e descritapreviamentepor Barbosa e colaboradores. Resumidamente, coculturas de monocamadassubconfluentes de células HEK 293T e células MDCK, cultivadasemmeio de Dulbecco modificado (DMEM) empresença de 10% de soro fetal bovino e antibióticos, foramcotransfectadas com: 1) osplasmídeoscodificando o segmentoourecombinantes da NA (NA169x178 ou NA169-PspA-178),2) osplasmídeoscodificandoos demais (sete) segmentos do vírus influenza PR8. Desta forma, oitocomplexosribonucleoproteicosforamreconstituídos in vitro, permitindo a transcrição e a replicação de todosossegmentosvirais e a síntese de novaspartículasvirais. Vinte horas após a transfecção, o meio de culturafoisubstituídopor DMEM suplementado com 1 μg/ml de tripsina TPCK (SIGMA) e 300 μU/ml de NA do Vibrio cholerae (a qual supre a falta da NA viral, permitindo a liberação das partículasviraisneoformadas). Após 72 horas de incubação a 37°C, ossobrenadantes das célulastransfectadasforamcoletados. Osvírusrecombinantesobtidoscarreando a sequência PspA4pro (doravantechamado de Flu-PspA) e o respectivovíruscontrole (doravantechamado de Flu-CT) foramamplificados e purificados duas vezespordiluiçãolimiteemcélulas MDCK, antes de seremsubmetidos a umaamplificação final nestamesmalinhagemcelular. Todos os estoques viraisassimobtidostiveram o seutítuloinfecciosodeterminadoportitulaçãoporplaca de lise sob agarose emcélulas MDCK. O genótipo dos vírusrecombinantesfoiavaliadopor PCR e sequenciamento. Conforme mostradonafigura 4, o vírus Flu-PspAproduzplacas de lise sob agarose menores do que aquelasobservadasnessascélulasquandoinfectadaspelovírus Flu-CT oupelovírusselvagem PR8.[36] Recombinant influenza viruses were obtained by reverse genetics according to the technique represented in figure 3 and previously described by Barbosa and collaborators. Briefly, cocultures of subconfluent monolayers of HEK 293T cells and MDCK cells, grown in modified Dulbecco's medium (DMEM) in the presence of 10% fetal bovine serum and antibiotics, were cotransfected with: 1) plasmids encoding the recombinant NA segment (NA169x178 or NA169-PspA-178) ,2) the plasmids encoding the remaining (seven) segments of the PR8 influenza virus. In this way, eight ribonucleoprotein complexes were reconstituted in vitro, allowing the transcription and replication of all viral segments and the synthesis of new viral particles. Twenty hours after transfection, the culture medium was replaced by DMEM supplemented with 1 μg/ml of TPCK trypsin (SIGMA) and 300 μU/ml of NA from Vibrio cholerae (which makes up for the lack of viral NA, allowing the release of newly formed viral particles). After 72 hours of incubation at 37°C, supernatants from transfected cells were collected. The recombinant viruses obtained carrying the PspA4pro sequence (hereinafter called Flu-PspA) and the respective control virus (hereinafter called Flu-CT) were amplified and purified twice by limiting dilution in MDCK cells, before being subjected to a final amplification in this same cell lineage. All viral stocks thus obtained had their infectious titer determined by lysis plate titration under agarose in MDCK cells. The genotype of the recombinant viruses was evaluated by PCR and sequencing. As shown in Figure 4, the Flu-PspA virus produces smaller agarose lysis plaques than those observed in these cells when infected by the Flu-CT virus or the wild-type PR8 virus.
[37]A expressão da proteína PspA4pro emcélulas MDCK infectadas com o vírus Flu-PspAfoiavaliadapormicroscopia confocal. Resumidamente, as célulasforaminfectadas com o vírus Flu-PspAou Flu-CT (M.O.I.0,5) ouincubadasnapresença de meio de cultura (MOCK). Posteriormente, as célulasforamincubadasdurante 20 horas napresença de NA e, apósesse tempo, as célulasforammarcadas com anticorposespecíficos (anticorpopoliclonal anti-PspA4pro e anticorposecundário Anti-IgG conjugado a FITC). A presença de célulaspositivas para PspAfoiavaliadaatravés da análise de célulasfluorescentesemmicroscópio confocal. Aanálise das células no microscópio de fluorescência confocal (figura 5) demonstrou a presença de célulasfluorescentesinfectadas com o vírus Flu-PspA, confirmando a capacidadedessevírusemexpressar a proteína PspA4, enquantonenhumafluorescênciafoiobservadanascélulasinfectadas com vírus Flu-CT outratadas com meio de cultura (MOCK).[37] The expression of the PspA4pro protein in MDCK cells infected with the Flu-PspA virus was evaluated by confocal microscopy. Briefly, cells were infected with Flu-PspA or Flu-CT virus (M.O.I.0.5) or incubated in the presence of culture medium (MOCK). Subsequently, the cells were incubated for 20 hours in the presence of NA and, after this time, the cells were labeled with specific antibodies (anti-PspA4pro polyclonal antibody and FITC-conjugated Anti-IgG secondary antibody). The presence of PspA-positive cells was assessed through the analysis of fluorescent cells under a confocal microscope. Analysis of the cells under the confocal fluorescence microscope (figure 5) demonstrated the presence of fluorescent cells infected with the Flu-PspA virus, confirming the ability of this virus to express the PspA4 protein, while no fluorescence was observed in cells infected with Flu-CT virus other than culture medium (MOCK).
[38]A segurança do vírusrecombinante Flu-PspAfoiavaliadaemmodelomurino. Para essefim, camundongos da linhagem C57BL/6 fêmeas, de cerca de 8 semanas de vida, foramanestesiados com umamistura de cetamina/xilazina e inoculados pela via intranasal com 105 PFU/mL (do inglês, Plaque Forming Units) do vírus Flu-PspAouvíruscontrole Flu-CT, ou com 103 PFU/mL do vírus PR8, o qual foiutilizadocomocontrolepositivo para avaliação de imunopatogenicidade. Aavaliação da virulênciadessesvírusfoiverificadaaolongo de trêssemanasatravés do acompanhamento da sobrevivência e perda de peso dos animais.[38] The safety of the Flu-PspA recombinant virus was evaluated in a murine model. For this purpose, female C57BL/6 mice, approximately 8 weeks old, were anesthetized with a ketamine/xylazine mixture and inoculated intranasally with 105 PFU/mL (Plaque Forming Units) of the Flu-PspA virus or control Flu virus. -CT, or with 103 PFU/mL of the PR8 virus, which was used as a positive control to evaluate immunopathogenicity. The assessment of the virulence of these viruses was verified over three weeks by monitoring the survival and weight loss of the animals.
[39]Oscamundongosinoculados com o vírus PR8 apresentaramumaperda de peso progressiva superior a 20% entre o 6° e o 12° diaapósainfecção e 50% de mortalidadeapós 20 dias (Figura 6). Em contraste, oscamundongosinoculados com o vírus Flu-PspA, assimcomoosinoculados com o vírus Flu-CT, nãoapresentaramperda de peso significativa e sobreviveram à inoculação (p<0,05). Assim, no seu conjunto, estesresultadosevidenciam a segurança da utilização do vírus Flu-PspAcomoestratégiavacinal.[39] Mice inoculated with the PR8 virus showed a progressive weight loss of more than 20% between the 6th and 12th day after infection and 50% mortality after 20 days (Figure 6). In contrast, mice inoculated with the Flu-PspA virus, as well as those inoculated with the Flu-CT virus, did not show significant weight loss and survived the inoculation (p<0.05). Thus, as a whole, these results demonstrate the safety of using the Flu-PspA virus as a vaccination strategy.
[40]A capacidade dos vírus Flu-PspAeminduziranticorpos contra o S. pneumoniae foiavaliadaem um protocoloheterólogo no qual camundongosforam primo imunizados com o vírus Flu-PspA e receberamumainoculação de reforço com proteínarecombinantePspAadjuvantada com alúmen (30% v/v). Resumidamente, oscamundongosforamanestesiados com umamistura de cetamina/xilazina e inoculados com 105 PFU/mL do vírus Flu-PspA, Flu-CT ouinstilados com PBS (Mock). Vinte e um diasapósaimunização, osanimaisreceberamumavacinação de reforço com proteínarecombinante PspA4, diluídaemsuspensão de alúmen (30% v/v em PBS). O soro dos camundongosfoicoletado 14 diasapós a vacinação de reforço e a presença dos anticorpos IgG anti-PspA4 e as subclasses IgG1 e IgG2 presentes no soro dos animaisvacinadosforamavaliados pela técnica de ELISA.[40] The ability of Flu-PspA viruses to induce antibodies against S. pneumoniae was evaluated in a heterologous protocol in which mice were prime immunized with the Flu-PspA virus and received a booster inoculation with recombinant PspA protein adjuvanted with alum (30% v/v). Briefly, mice were anesthetized with a ketamine/xylazine mixture and inoculated with 105 PFU/mL of Flu-PspA virus, Flu-CT, or instilled with PBS (Mock). Twenty-one days after immunization, the animals received a booster vaccination with PspA4 recombinant protein, diluted in alum suspension (30% v/v in PBS). The serum of the mice was collected 14 days after the booster vaccination and the presence of IgG anti-PspA4 antibodies and the IgG1 and IgG2 subclasses present in the serum of the vaccinated animals were evaluated using the ELISA technique.
[41]Conforme mostradonafigura 7A, aimunização com o protocoloheterólogo Flu-PspA/PspA4+alúmen, resultouemtítuloselevados de anticorpos, diferentemente do observado no soro dos animais dos outros grupos. Ademais, conformemostradonafigura 7B, o protocolo de imunizaçãoutilizadoinduziuníveiscomparáveis de anticorpos das subclasses IgG1 e IgG2.[41] As shown in figure 7A, immunization with the heterologous Flu-PspA/PspA4+alum protocol resulted in high antibody titers, unlike what was observed in the serum of animals from the other groups. Furthermore, as shown in figure 7B, the immunization protocol used induces comparable levels of antibodies of the IgG1 and IgG2 subclasses.
[42]A capacidade de ligação dos anticorposinduzidos pela vacinação à superfície de pneumococostambémfoiavaliada para cepasrepresentativas de cincoclados da PspA (clados que compreendemcerca de 90% de todas as cepas de pneumococosisolados) e osresultadosrepresentadosporporcentagem de célulaspositivas (Figura 8).[42] The binding capacity of antibodies induced by vaccination to the surface of pneumococci was also evaluated for strains representing five PspA clades (clades that comprise approximately 90% of all pneumococcal isolates) and the results represented by the percentage of positive cells (Figure 8).
[43]A maiorcapacidade de ligação dos anticorpos anti-PspA4 induzidos pela imunizaçãofoiobservada à superfície de bactérias da cepa 3JYP2670 (Figura 8D), a qual pertenceaomesmoclado da proteínaPspAutilizada para aimunização (clado 4). Para a cepa utilizadanosdesafiosletaispneumocócicos, ATCC6303 (clado 5), a ligação de anticorposfoiestatisticamentemaior no grupovacinal Flu- PspA/PspA4+Alum (p<0,001) quandocomparada com a dos grupos Mock e Flu-CT/Alum (Figura 8E). Com relaçãoàscepas D39 e M10 (representativas dos clados 2 e 3 respectivamente), apesar da ligação de anticorpos do grupovacinal Flu-PspA/PspA4+Alum ser visivelmentemaior do que a dos demaisgrupos, as diferençasnãoforamestatisticamentediferentes (Figura 8B e 8C). Finalmente, para o isolado EF3030 (clado 1), a ligação de anticorposfoimuitobaixaemtodososgruposavaliados (Figura 8A).[43] The greatest binding capacity of anti-PspA4 antibodies induced by immunization was observed on the surface of bacteria of strain 3JYP2670 (Figure 8D), which belongs to the same clade as the PspA protein used for immunization (clade 4). For the strain used in pneumococcal lethal challenges, ATCC6303 (clade 5), antibody binding was statistically higher in the Flu-PspA/PspA4+Alum vaccine group (p<0.001) when compared to that in the Mock and Flu-CT/Alum groups (Figure 8E). Regarding strains D39 and M10 (representative of clades 2 and 3 respectively), although the binding of antibodies from the Flu-PspA/PspA4+Alum vaccine group was visibly greater than that of the other groups, the differences were not statistically different (Figure 8B and 8C). Finally, for isolate EF3030 (clade 1), antibody binding was very low in all groups evaluated (Figure 8A).
[44]Vinte e um diasapósaimunização de reforço, osanimaisforamdesafiados com umasuspensãocontendo 5x104 UFC/ml (UnidadesFormadoras de Colônia) do isolado ATCC6303 (7 vezes a DL50) diluídoem PBS pela via intranasal. Osanimaisforamacompanhados para avaliação da sobrevivência (Figura 9A) e perda de peso (Figura 9B), durante 10 dias. Após o desafioletal com pneumococos, o grupovacinal Flu-PspA/PspA4+Alum apresentou taxa de sobrevivência de 64,7%, diferentemente do observado no grupo Mock onde se observou 5,5% de sobrevivência e no grupo e Flu-CT/Alum, ondetodososanimaismorreramapóso desafio (Figura 9A). As curvas de sobrevivência de todososgruposforamestatisticamentediferentes (p<0,0001) apósanálises com os testes Log-rank (Mantel-Cox) e Gehan-Breslow- Wilcoxon. Ademais, a perda de peso dos animais do grupo Flu- PspA/PspA4+Alum foisignificativamentemenor do que aquela dos demaisgrupos (Figura 9B).[44] Twenty-one days after booster immunization, the animals were challenged with a suspension containing 5x104 CFU/ml (Colony Forming Units) of the isolate ATCC6303 (7 times the LD50) diluted in PBS intranasally. The animals were monitored to assess survival (Figure 9A) and weight loss (Figure 9B), for 10 days. After the lethal challenge with pneumococci, the Flu-PspA/PspA4+Alum vaccine group presented a survival rate of 64.7%, unlike what was observed in the Mock group where 5.5% survival was observed and in the Flu-CT/Alum group, where all animals died after challenge (Figure 9A). The survival curves of all groups were statistically different (p<0.0001) after analyzes with the Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests. Furthermore, the weight loss of animals in the Flu-PspA/PspA4+Alum group was significantly lower than that of the other groups (Figure 9B).
[45]Dessa forma, nossosresultadosdemonstram a capacidadeaimunização Flu-PspA/PspA4+Alum emreduzirsignificativamente a mortalidadenosanimaisinfectados com S. pneumoniae.[45] Thus, our results demonstrate the ability of Flu-PspA/PspA4+Alum immunization to significantly reduce mortality in animals infected with S. pneumoniae.
[46]A produção de anticorposespecíficos contra o vírus influenza PR8, foiavaliadaapósumaúnicaimunização com osvírus Flu-PspAou o víruscontrole Flu-CT. Resumidamente, osanimaisforamanestesiados com umamistura de cetamina e xilazina e inoculados com 105 PFU/ml do vírus Flu-PspAou Flu-CT ouinoculados com PBS. As amostras de soroforamcoletadas 14 diasapósaimunização. Ostítulos de anticorposforamavaliados pela técnica de ELISA e representadoscomo o log2 da maiordiluição que apresentou D.O. igualoumaior do que 0,1, em 450 nm.[46] The production of specific antibodies against the PR8 influenza virus was evaluated after a single immunization with the Flu-PspA virus or the Flu-CT control virus. Briefly, animals were anesthetized with a mixture of ketamine and xylazine and inoculated with 105 PFU/ml of Flu-PspA or Flu-CT virus or inoculated with PBS. Serum samples were collected 14 days after immunization. Antibody titers were evaluated using the ELISA technique and represented as the log2 of the highest dilution that presented O.D. equaled or greater than 0.1, at 450 nm.
[47]Osresultadosmostradosnafigura 10 evidenciam que tanto o vírus Flu-CT quanto o Flu-PspAforamigualmentecapazes de induzirtítulossignificativos de anticorpos anti-influenza apósumaúnicaimunização. Nãohouvediferençaestatísticanosníveis de anticorposdetectadosnossoros dos animaisinoculados com cada um dessesvírus. Desta forma, nossosresultadosdemonstram que ofato do vírus Flu-PspAcarrearuma proteínaheteróloganasuasuperfícienãodiminui a suacapacidade de induzirresposta humoral contra o vírus influenza.[47] The results shown in figure 10 show that both the Flu-CT and Flu-PspA viruses were equally capable of inducing significant titers of anti-influenza antibodies after a single immunization. There was no statistical difference in the levels of antibodies detected in the sera of animals inoculated with each of these viruses. Thus, our results demonstrate that the fact that the Flu-Psp virus carries a heterologous protein on its surface does not reduce its ability to induce a humoral response against the influenza virus.
[48]A proteção dos animaisdiante de um desafio com o vírus PR8 foiavaliadaatravés da imunização com uma dose dos vírusrecombinantesou PBS, seguido de desafio com uma dose letal (100*DL50) do vírus influenza A/PR8/34 (H1N1). A sobrevivência (Figura 11A) e a perda de peso (Figura 11B) foramacompanhadasdurante 10 dias. Osanimaisimunizados com uma dose do vírus Flu-CT ou do vírus Flu-PspAapresentaram 100% de proteção e ausência de perda de peso ouqualquer outro sinalclínicocaracterístico da infecção viral. Por outro lado, osanimais do grupo Mock, inoculados com PBS, apresentaramperdas de peso significativas (p<0,0001) que resultaramnamorte de 100% dos animais. As curvas de sobrevivência de todososgruposforamestatisticamentediferentes (p<0,0001) apósanálises com os testes Log-rank (Mantel-Cox) e Gehan-Breslow-Wilcoxon.[48] The protection of animals from a challenge with the PR8 virus was evaluated through immunization with a dose of recombinant viruses or PBS, followed by challenge with a lethal dose (100*LD50) of the influenza A/PR8/34 (H1N1) virus. Survival (Figure 11A) and weight loss (Figure 11B) were monitored for 10 days. Animals immunized with a dose of Flu-CT virus or Flu-PspA virus showed 100% protection and no weight loss or any other clinical sign characteristic of viral infection. On the other hand, animals in the Mock group, inoculated with PBS, showed significant weight losses (p<0.0001) that resulted in the death of 100% of the animals. The survival curves of all groups were statistically different (p<0.0001) after analyzes with the Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests.
[49]Como forma de verificar a capacidade do vírus influenza recombinantedefectivo Flu-PspA de multiplicaremovosembrionados de galinha (principal substrato para produção das atuaisvacinas contra influenza), 103 PFU/mL do vírusvacinalfoiinoculadonacavidadealantoide de ovosembrionados SPF de 9 dias de idade. As câmaras de ar dos ovosforamidentificadasporovoscopia e, através delas, foraminoculadosnacavidadealantoide 100 μL de umasuspensãocontendo 103 PFU/mL do vírus Flu-PspAjuntamente com 300 μUou600 μUporovoousem NA em PBS 1X, com auxílio de uma seringa de insulina. Osovosforamentãoincubados a 37°C durante 48 horas. Apósesseperíodo, o líquidoalantóicofoicoletado e o título viral foideterminadoportitulaçãoporplaca de lise sob agarose emcélulas MDCK, conforme previamentedescrito. O título viral presente no líquidoalantoicocoletadofoirepresentadopor log10 em PFU/mL.[49] As a way to verify the ability of the Flu-PspA-defective recombinant influenza virus to multiply and remove chicken embryonates (the main substrate for the production of current influenza vaccines), 103 PFU/mL of the vaccine virus was inoculated into the anthoid cavity of 9-day-old SPF embryonated eggs. The air chambers of the eggs were identified by ovoscopy and, through them, 100 μL of a suspension containing 103 PFU/mL of the Flu-Psp virus was inoculated into the antoid cavity, together with 300 μU or 600 μUporovoous without NA in 1X PBS, with the aid of an insulin syringe. The eggs were then incubated at 37°C for 48 hours. After this period, the allantoic fluid was collected and the viral titer was determined by titration by lysis plate under agarose in MDCK cells, as previously described. The viral titer present in the collected allantoic fluid was represented by log10 in PFU/mL.
[50]Após 48 horas de multiplicaçãoemovosembrionados, foramobservadostítulosviraissignificativos (p<0,0001) emtodas as trêscondiçõestestadas (Figura 12). No teste realizadonaausência de NA o títulomédioobtidoem PFU/mL foi de =5 (log10), enquanto, nas amplificaçõesrealizadasnapresença de neuraminidase, ostítulosobtidosforam de = 5,8 (log10) e = 6,3 (log10) utilizando 300 μU e 600 μU de NA, respectivamente. Em contraste, nãoforamdetectadostítulosvirais no grupo PBS + 600 μU e nem no controleambiental.[50] After 48 hours of multiplication and embryonated eggs, significant viral titers (p<0.0001) were observed in all three conditions tested (Figure 12). In the test carried out in the absence of NA, the average titer obtained in PFU/mL was = 5 (log10), while, in amplifications carried out in the presence of neuraminidase, the titers obtained were = 5.8 (log10) and = 6.3 (log10) using 300 μU and 600 μU of NA , respectively. In contrast, no viral titers were detected in the PBS + 600 μU group nor in the environmental control group.
[51]Alémdisso, ostítulosobtidosnos testes com 300 μU e com 600 μU de NA nãoapresentaramdiferençasignificativa entre si, enquanto o títuloobtido no teste de 600 μUfoisignificativamente (p<0,01) maior que oobtidonaamplificaçãosem NA. Além do mais, o título viral médioobtido no teste semadição de neuraminidase nãoapresentoudiferençaestatística com o teste realizadonapresença de 300 μU de NA.[51] Furthermore, the titers obtained in the tests with 300 μU and 600 μU of NA did not show a significant difference between them, while the titer obtained in the 600 μU test was significantly (p<0.01) higher than that obtained in amplification without NA. Furthermore, the average viral titer obtained in the test without the addition of neuraminidase did not show any statistical difference with the test performed in the presence of 300 μU of NA.
[52]Assim, observamos que ostítulosinfecciososobtidos com a amplificaçãorealizadanapresença de NA (nosdois testes) forambemsemelhantesaosrotineiramenteobtidos com a multiplicaçãoemcultura de células MDCK (cerca de 106 PFU/mL) quandorealizadadurante 72 horas e com a adição de 600 μU de neuraminidase, 4 μg/mL de tripsina e 0,3% de BSA, enquanto a multiplicação do vírus Flu-PspAemovosembrionadosnaausência de neuraminidase exógenaresultouemtítulosmenoresemaproximadamente 1 log (cerca de 105 PFU/mL).[52] Thus, we observed that the infectious titers obtained with amplification performed in the presence of NA (in both tests) were very similar to those routinely obtained with multiplication in culture of MDCK cells (about 106 PFU/mL) when carried out for 72 hours and with the addition of 600 μU of neuraminidase, 4 μg/mL of trypsin and 0.3% BSA, while multiplication of Flu-PspA virus and embryonated in the absence of exogenous neuraminidase resulted in lower titers of approximately 1 log (about 105 PFU/mL).
[53]A capacidade do vírus Flu-PspAeminduziranticorpos contra o S. pneumoniae tambémfoiavaliadaatravés da imunização intramuscular com o protocoloheterólogo de indução e reforço (primo imunização com vírus Flu-PspA e reforço com proteína PspA4 adjuvantada com alúmen - 30% v/v). Resumidamente, oscamundongosforaminoculados com 105 PFU/mL do vírus Flu-PspAou Flu-CT ou com PBS (Mock) por via intramuscular (primeira dose) e, vinte e um diasapósaimunização, osanimaisreceberamumavacinação de reforço com proteínarecombinante PspA4 diluídaemumasuspensão de alúmen (30% v/v em PBS), alúmen e PBS, respectivamente. Em seguida, o sangue dos camundongosfoicoletado 14 diasapós a 1a e 2a doses e a presença dos anticorpos IgG no soro dos animaisvacinadosfoiavaliada pela técnica de ELISA.[53] The ability of the Flu-PspA virus to induce antibodies against S. pneumoniae was also evaluated through intramuscular immunization with the heterologous induction and boost protocol (primary immunization with Flu-PspA virus and boost with PspA4 protein adjuvanted with alum - 30% v/v). Briefly, mice were vaccinated with 105 PFU/mL of Flu-PspA or Flu-CT virus or with PBS (Mock) intramuscularly (first dose) and, twenty-one days after immunization, the animals received a booster vaccination with PspA4 recombinant protein diluted in an alum suspension (30% v/ v in PBS), alum and PBS, respectively. Then, the blood of the mice was collected 14 days after the 1st and 2nd doses and the presence of IgG antibodies in the serum of the vaccinated animals was evaluated using the ELISA technique.
[54]Conforme mostradonafigura 13A e 13B, aimunização intramuscular com o protocoloheterólogo Flu-PspA/PspA4+Alum, resultouemelevadostítulos de anticorposespecíficos, diferentemente do observado no soro dos animais dos gruposcontrole Mock e Flu-CT/Alum.[54] As shown in Figure 13A and 13B, intramuscular immunization with the heterologous protocol Flu-PspA/PspA4+Alum resulted in high titers of specific antibodies, different from what was observed in the serum of animals from the Mock and Flu-CT/Alum control groups.
[55] Vinte e um diasapósaimunização de reforço, osanimaisforamdesafiados com umasuspensãocontendo 3x104 UFC/mL do isolado ATCC6303 (7 vezes a DL50), diluídoem PBS, pela via intranasal. Posteriormente, osanimaisforamacompanhados para avaliação da perda de peso e sobrevivênciadurante 10 dias. Após o desafioletal com pneumococos, o grupovacinal Flu-PspA/PspA4+Alum apresentou 100% de proteção, enquantoosgrupos Mock e Flu-CT/Alum apresentaramapenas 16% de sobrevivência (Figura 14A). As curvas de mortalidadeapresentaramsignificativadiferençaestatística (p<0,01) apósanálisesutilizandoos testes Log-rank (Mantel-Cox) e Gehan-Breslow-Wilcoxon. Alémdisso, osanimais do grupo Flu-PspA/PspA4+Alum não apresentaramperda de peso, diferentemente dos animais dos demaisgrupos (p<0,01) (Figura 14B).[55] Twenty-one days after booster immunization, the animals were challenged with a suspension containing 3x104 CFU/mL of the isolate ATCC6303 (7 times the LD50), diluted in PBS, intranasally. Subsequently, the animals were monitored to evaluate weight loss and survival for 10 days. After lethal challenge with pneumococci, the Flu-PspA/PspA4+Alum vaccine group showed 100% protection, while the Mock and Flu-CT/Alum groups showed only 16% survival (Figure 14A). The mortality curves showed a significant statistical difference (p<0.01) after analyzes using the Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests. Furthermore, animals in the Flu-PspA/PspA4+Alum group did not show weight loss, unlike animals in the other groups (p<0.01) (Figure 14B).
[56]Dessa forma, nossosresultadosdemonstramaeficácia do protocolo de imunizaçãoemproteger contra a infecção com S. pneumoniae tambémquandorealizado pela via intramuscular.[56] Thus, our results demonstrate the effectiveness of the immunization protocol in protecting against infection with S. pneumoniae also when carried out intramuscularly.
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