Strategies for vaccine development of COVID-19 / 生物工程学报

An epidemic of acute respiratory syndrome in humans, which appeared in Wuhan, China in December 2019, was caused by a novel coronavirus (SARS-CoV-2). This disease was named as "Coronavirus Disease 2019" (COVID-19). SARS-CoV-2 was first identified as an etiological pathogen of COVID-19, belonging to the species of severe acute respiratory syndrome-related coronaviruses (SARSr-CoV). The speed of both the geographical transmission and the sudden increase in numbers of cases is much faster than SARS and Middle East respiratory syndrome (MERS). COVID-19 is the first global pandemic caused by a coronavirus, which outbreaks in 211 countries/territories/areas. The vaccine against COVID-19, regarded as an effective prophylactic strategy for control and prevention, is being developed in about 90 institutions worldwide. The experiences and lessons encountered in the previous SARS and MERS vaccine research can be used for reference in the development of COVID-19 vaccine. The present paper hopes to provide some insights for COVID-19 vaccines researchers.

Intranasal Immunization Using CTA1-DD as a Mucosal Adjuvant for an Inactivated Influenza Vaccine / 生物医学与环境科学（英文）

OBJECTIVE@#To evaluate the effect of intranasal immunization with CTA1-DD as mucosal adjuvant combined with H3N2 split vaccine.@*METHODS@#Mice were immunized intranasally with PBS (negative control), or H3N2 split vaccine (3 μg/mouse) alone, or CTA1-DD (5 μg/mouse) alone, or H3N2 split vaccine (3 μg/mouse) plus CTA1-DD (5 μg/mouse). Positive control mice were immunized intramuscularly with H3N2 split vaccine (3 μg/mouse) and alum adjuvant. All the mice were immunized twice, two weeks apart. Then sera and mucosal lavages were collected. The specific HI titers, IgM, IgG, IgA, and IgG subtypes were examined by ELISA. IFN-γ and IL-4 were test by ELISpot. In addition, two weeks after the last immunization, surivival after H3N2 virus lethal challenge was measured.@*RESULTS@#H3N2 split vaccine formulated with CTA1-DD could elicit higher IgM, IgG and hemagglutination inhibition titers in sera. Furthermore, using CTA1-DD as adjuvant significantly improved mucosal secretory IgA titers in bronchoalveolar lavages and vaginal lavages. Meanwhile this mucosal adjuvant could enhance Th-1-type responses and induce protective hemagglutination inhibition titers. Notably, the addition of CTA1-DD to split vaccine provided 100% protection against lethal infection by the H3N2 virus.@*CONCLUSION@#CTA1-DD could promote mucosal, humoral and cell-mediated immune responses, which supports the further development of CTA1-DD as a mucosal adjuvant for mucosal vaccines.

Polissacarídeo capsular do Streptococcus agalactiae como antígeno vacinal: desenvolvimento de um modelo vacinal para mucosas com Nanopartícula de quitosana / Capsular polysaccharide of Streptococcus agalactiae as vaccine antigen: development of a mucosal vaccine model with chitosan nanoparticle

A bactéria gram-positiva Streptococcus agalactiae do grupo B (GBS) faz parte da microbiota normal do trato geniturinário humano, sendo um organismo comensal do corpo da mulher. No entanto, em mulheres grávidas, quando há alterações na composição microbiana do canal vaginal, pode ocorrer a proliferação e a infecção pelo GBS. Este microrganismo, em sua forma patogênica oportunista, pode infectar o neonato durante o parto natural, assim como contribuir para infecções urinárias e uterinas durante a gestação. O GBS já foi identificado como um dos responsáveis pela alta taxa de mortalidade neonatal, sendo um dos principais agentes de infecção em recém-nascidos no mundo. Ele também pode ser a causa de infecções nas gestantes, levando a várias complicações, como corioamnionite, endometrite e infecções do trato urinário e do sítio cirúrgico. Pode haver comprometimento da gestação e do feto, com abortamento, morte fetal intrauterina e ruptura da membrana coriônica, levando a parto prematuro - que pode resultar em outras consequências graves. Este trabalho foi desenvolver um modelo vacinal para mucosa sublingual, utilizando o polissacarídeo capsular do Streptococcus agalactiae como antígeno, encapsulado em Nanopartículas de quitosana. Para o estudo de otimização dos parâmetros de fermentação, para aumentar a produtividade de cápsula polissacarídica (PS) presente na superfície celular, utilizou-se o Banco de Dados Kegg (Kyoto Encyclopedia of Genes and Genomes). A adição do suplemento L-Prolina foi o que propiciou a principio, maior relação entre crescimento bacteriano e formação de cápsula polissacarídica. A purificação e extração da cápsula polissacarídica foi realizada com etapas sucessivas de ultra filtração tangencial e precipitação alcóolica dos contaminantes. As caracterizações físico-químicas: difração de raios-X (DRX), cromatografia gasosa (CGMS), ressonância magnética (NMR) e determinação de açúcares pelo método fenol-sulfúrico, foram realizadas para identificação da composição e estrutura monossacarídica de açucares. O PS isolado apresenta ramificações de fucose, manose, glicose, galactose e N-acetil-glucosamina, apresentando estrutura amorfa. A liofilização do polissacarídeo foi realizada para fins de concentração e conservação. A encapsulação do polissacarídeo acoplada quimicamente com OVA, em uma Nanopartícula de quitosana, teve como finalidade aumentar a mucoadesividade e possibilitar maior absorção do antígeno entre as células da junção epitelial das mucosas sublinguais. A partir da análise de DLS (Espalhamento dinâmico de luz), as Nanopartículas apresentaram dimensões entre 200 a 400 nm e o Potencial Zeta acima de 20. O índice de polidispersão (PDI) está dentro do esperado (abaixo de 0.3). A capacidade de encapsulamento em relação à OVA foi de 92,8% dos grupos que continham PS. O teste IgG sérica total mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior reatividade no teste de ELISA, pela Análise de Variância (ANOVA) com ferramenta de Bonferrone. O teste sIgA mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior concentração de anticorpo sIgA total. Como resultado e conclusão, o polissacarídeo capsular do Streptococcus agalactiae é um bom candidato a antígeno vacinal

Gram-positive bacteria Streptococcus agalactiae group B (GBS) is part of the normal microbiota of the human genitourinary tract, being a commensal organism of the female body. However, in pregnant women, when there are changes in the microbial composition of the vaginal canal, GBS proliferation and infection may occur. This microorganism, in its opportunistic pathogenic form, can infect the neonate during natural childbirth, as well as contribute to urinary and uterine infections during pregnancy. The GBS has already been identified as one of the responsible for the high neonatal mortality rate, being one of the main agents of infection in newborns in the world. It can also be the cause of infections in pregnant women, leading to various complications such as chorioamnionitis, endometritis, and urinary tract and surgical site infections. There may be pregnancy and fetal impairment, with abortion, fetal intrauterine death, and rupture of the chorionic membrane, leading to premature labor - which can result in other serious consequences. This work was to develop a vaccine model for sublingual mucosa using the capsular polysaccharide of Streptococcus agalactiae as antigen, encapsulated in chitosan nano particles. For the study of optimization of the fermentation parameters, the Kegg (Kyoto Encyclopedia of Genes and Genomes) database was used to increase the productivity of polysaccharide capsule (PS) present on the cell surface. The addition of the L-Proline supplement gave rise to a higher ratio between bacterial growth and polysaccharide capsule formation. The purification and extraction of the polysaccharide capsule was performed with successive stages of tangential ultrafiltration and alcoholic precipitation of the contaminants. The physicochemical characterization of X-ray diffraction (XRD), gas chromatography (CGMS), magnetic resonance (NMR) and determination of sugars by the phenol-sulfuric method were performed to identify the composition and monosaccharide structure of sugars. The isolated PS presents branches of fucose, mannose, glucose, galactose and N-acetyl-glucosamine, presenting amorphous structure. Lyophilization of the polysaccharide was performed for concentration and conservation purposes. The encapsulation of the polysaccharide coupled chemically with OVA in a chitosan nano particle was aimed at increasing mucoadhesiveness and allowing greater absorption of the antigen between the cells of the sublingual mucosal epithelial junction. From the analysis of DLS (dynamic light scattering), the nanoparticles presented dimensions between 200 to 400 nm and the Zeta potential above 20. The polydispersity index (PDI) is within the expected range (below 0.3). The encapsulation capacity for OVA was 92.8% of the groups containing PS. The total serum IgG test showed that the G2 group (Nano particle with Polysaccharide and Protein coupled) was the one that had the highest reactivity in the ELISA test, by Analysis of Variance (ANOVA) with Bonferrone tool. The sIgA test showed that the G2 group (Nanoparticle with Polysaccharide and Protein coupled) had the highest concentration of total sIgA antibody. As a result and conclusion, the capsular polysaccharide of Streptococcus agalactiae is a good candidate for vaccine antigen

Chitosan and carboxymethylated derivative nanoparticles as delivery systems for biological products: preparation, characterization, stability and in vitro/in vivo evaluation / Nanopartículas de quitosana e derivado carboximetilado como sistemas de fornecimento (delivery) de produtos biológicos: preparo, caracterização, estabilidade e avaliação in vitro/in vivo

Chitosan is a biocompatible and biodegradable mucoadhesive polymer with unique advantages, such as the distinct trait of opening the junctions to allow paracellular transport of antigen and good tolerability. However, the poor solubility of chitosan in neutral or alkalinized media has restricted its applications in the pharmaceutical field. Chitosan can be easily carboxymethylated to improve its solubility in aqueous media, while its biodegradability and biocompatibility are preserved. Apart from this, carboxymethyl chitosan (CMCS) can be easily processed into nanoparticles which highlight its suitability and extensive usage for preparing different drug delivery formulations. The present study deals with the development and characterization of a delivery system based on CMCS nanoparticles using ovalbumin as model protein. We demonstrated that ovalbumin loaded nanoparticles were successfully synthetized using calcium chloride as a cross-linker by ionic gelation. The nanoparticles exhibited an average size of approximately 169 nm and presented a pseudo-spherical shape. The nanoparticles size increased according to the addition of CaCl2 due to the strong electrostatic attraction. During storage the nanoparticles size increased was attributed to swelling and aggregation. The loading efficiency of ovalbumin was found to be 17%. Confocal microscopy clearly showed the association between ovalbumin and CMCS chains into nanoparticles. Therefore, we suggest these nanoparticles can be considered as an attractive and promising carrier candidate for proteins and antigens. The major challenge that limits the use of such carriers is their instability in an aqueous medium. Thus, the next step of this work was to determine the robustness of several formulations using distinct freeze-drying protocols. This study demonstrated that mannitol in concentration of 10% (w/v) is well suited to preserve ovalbumin loaded CMCS nanocapsules from aggregation during lyophilization and subsequent reconstitution. Importantly, the results showed that an annealing step has a huge impact on porosity of freeze-dried cake by nearly complete crystallization of mannitol, once the crystalline matrix prevents the partial collapse and the formation of larger pores observed without annealing. Therefore, the usual observation that annealing increases the pore size due to growth of ice crystal size does not always apply, at least when crystallization of solute is involved. Since all characterizations and stability studies had been performed, the main purpose of this study was to develop a stable antigen delivery system for oral immunization using CMCS and inactivated rabies virus (RV) as the antigen. RV loaded nanoparticles was found to enhance both systemic (IgG) and local (IgA) immune responses against RV after oral delivery in mice. The effective doses 50% were 50-times higher than the negative controls, indicating that the immune response started only after the third boosting dose. Furthermore, enough neutralizing antibodies was produced to be protected against the harmful effects of the rabies virus. It is therefore concluded, that the CMCS nanoparticles formulated in this study, are suitable for oral vaccine delivery, and can be suggested as a promising delivery system for a diverse range of antigens as well as a gene/protein delivery system, especially for those positively charged. Since several approaches show that effective intervention in airway allergic inflammation can be achieved with allergen-activated interleukin-10-secreting cells, the final part of this work was dedicated to assessing whether IL-10 loaded chitosan nanoparticles (IL10-CSNPs) could be used as a possible inhalable therapeutic tool for preventing exacerbations in asthmatic patients. As positive controls, we also assess whether interleukin 17A and interleukin 9 have the ability to stimulate human airway smooth muscle (HASM) cell contractility using magnetic twisting cytometry (MTC). Significant decreased baseline cell stiffness was observed in HASM cells pre-treated with IL-10, but not with IL10-CSNPs, whereas treatment with IL-17A significantly enhanced baseline cell stiffening. Our findings reveal a previously unknown mechanism underlying immunotherapy for prevention and treatment of asthma

A quitosana é um polímero mucoadesivo biocompatível e biodegradável, com vantagens únicas, tais como a característica distinta de abrir as junções que permitim o transporte paracelular de antígenos e boa tolerabilidade. No entanto, sua baixa solubilidade em meios neutros ou alcalinizados tem restringido suas aplicações no campo farmacêutico. A quitosana pode ser facilmente carboximetilada para melhorar de sua solubilidade em meios aquosos, enquanto sua biodegradabilidade e biocompatibilidade são preservadas. Além disso, a carboximetilquitosana (CMCS) pode ser facilmente processada na forma de nanopartículas, o que destaca sua adequabilidade para uso extensivo no preparo de sistemas de delivery de medicamentos. O presente estudo trata do desenvolvimento e caracterização de um sistema de delivery baseado em nanopartículas de CMCS utilizando ovalbumina como proteína modelo. Nós demonstramos que as nanopartículas carregadas com ovalbumina foram sintetizadas com sucesso utilizando cloreto de cálcio como agente de reticulação por gelificação iônica. As nanopartículas exibiram um tamanho médio de aproximadamente 169 nm e apresentaram uma forma pseudo-esférica. O tamanho das nanopartículas aumentou de acordo com a adição de CaCl2 devido à forte atração eletrostática. Durante o armazenamento, o tamanho aumentado das nanopartículas foi atribuído a incorporação de água e agregação. A eficiência de encapsulamento da ovalbumina foi de aproximadamente 17%. A microscopia confocal mostrou claramente a associação entre ovalbumina e a cadeias de CMCS nas nanopartículas. Sugerimos, portanto, que tal sistema pode ser considerado como candidato atraente e promissor para o carreamento de proteínas e antígenos. O principal desafio que limita o uso desses carreadores consiste na instabilidade em meio aquoso. Assim, o próximo passo deste trabalho foi determinar a robustez de várias formulações utilizandose diferentes protocolos de liofilização. Este estudo demonstrou que o manitol em uma concentração de 10% (p/v) é adequado para preservar da agregação as nanocápsulas de CMCS carregadas com ovalbumina durante a liofilização e subsequente reconstituição. Mais importante, os resultados mostraram que uma etapa de annealing tem um enorme impacto sobre a porosidade da amostra liofilizada devido a quase completa cristalização do manitol, uma vez que a matriz cristalina evita o colapso parcial e a formação de poros maiores observados na ausência do annealing. Portanto, a observação comum de que o annealing aumenta o tamanho doporos devido ao crescimento dos cristais de gelo nem sempre se aplica, pelo menos quando a cristalização de um soluto está envolvida. Uma vez que todas as caracterizações e estudos de estabilidade foram realizados, o principal objetivo deste estudo foi desenvolver um sistema estável de delivery de antígeno para imunização oral utilizando CMCS e vírus rábico inativado (RV) como antígeno. Verificou-se que as nanopartículas carregadas com RV aumentam as respostas imune sistêmica (IgG) e local (IgA) contra o RV após administração oral em camundongos. As doses efetivas 50% foram 50 vezes maiores que os controles negativos, indicando que a resposta imune foi iniciada apenas após a terceira dose da vacina. Além disso, foram produzidos anticorpos neutralizantes suficientes para proteção contra os efeitos nocivos do vírus rábico. Conclui-se, portanto, que as nanopartículas de CMCS formuladas neste estudo, são adequadas para o delivery oral de vacinas, e podem ser sugeridas como um sistema promissor de delivery para uma gama diversa de antígenos, bem como para o delivery de genes/proteínas, especialmente para aqueles carregados positivamente. Uma vez que diversas abordagens mostram que uma intervenção efetiva em casos de inflamação alérgica de vias aéreas pode ser conseguida por meio de células secretoras de interleucina 10 (IL-10) mediante ativação por alergenos, a parte final deste trabalho esteve dedicada a avaliação de nanopartículas de quitosana carregadas com IL-10 (IL10-CSNPs) como possível ferramenta terapêutica inalável para prevenção de exacerbações em pacientes asmáticos. Como controles positivos, avaliou-se adicionalmente se as interleucinas 17A (IL-17A) e 9 (IL-9) possuem a capacidade de estimular a contratilidade de células humanas de músculo liso de vias aéreas humanas (HASM) por meio de citometria de torção magnética (MTC). Uma diminuição significativa da rigidez celular basal foi observada em células HASM pré-tratadas com IL-10, mas não com IL10-CSNPs, enquanto que o tratamento com IL-17A aumentou significativamente a magnitude rigidez celular basal. Nossos resultados revelam um mecanismo previamente desconhecido subjacente à imunoterapia para prevenção e tratamento da asma

Intranasal immunization with chitosan-DNA nanoparticles expressing pneumococcal polyamine transport protein D(PotD) protects mice against Streptococcus pneumoniae nasopharyngeal colonization / 中华微生物学和免疫学杂志

Objective To prepare the chitosan-potD nanoparticles and to evaluate its protective efficacy against pneumococcal nasopharyngeal colonization. Methods potD gene was amplificated from pneumococcal genome and was inserted into pVAX1 expression vectors to construct pVAX1-potD recombinant plasmid which was then transfected into 293T cell using LipofectAMINE 2000 to analyze transient potD gene expression in vitro by RT-PCR and Western blot. Chitosan-potD nanoparticles were freshly prepared by coacervation methods at each time and the characterizations of the nanoparticles were then evaluated. BALB/c mice were immunized with chitosan-potD, naked potD DNA or pVAX1 for 4 times at two-week intervals. Anti-PotD IgG, IgG1 and IgG2a levels in serum and IgA levels in nasal washes, bronchoalveolar lavage fluids (BALF) and middle ear lavages(MEL) were detected by indirect enzyme-linked immunosorbent assay (ELISA). IL-17A, IL-4 and IFN-γ levels in splenocytes were determined by double sandwich ELISA. Mice were intrannsally challenged with Streptococcus pneumoniae ATCC6303, and Pneumococci were recovered from the nasopharyngeal niche at the fifth day after challenge. Results potD gene was successfully amplificated by PCR and the sequence was confimed to be consistent with that in the Genbank. The pVAX1-potD recombinant plasmid was successfully constructed and was expressed in eukaryocytes in vitro. The mean size and zeta potential of chitosan-potD nanoparticles was 430 nm and + 20.5 mv, respectively. Chitosan-potD nanoparticles were not digested by DNase Ⅰ , while naked potD DNA was completely digested. The levels of antibodies inculding IgG, IgG1, IgG2a, IgA and cytokines including IL-17A, IL-4 and IFN-γ were significantly higher in mice immunized with chitosan-potD nanoparticles than mice with naked potD or pVAX1 ( P <0.05) only. More importantly, much less Pneumococci were recovered from mice immunized with chitosan-potD nanoparticles than the other groups(P <0.05). Conclusion Chitosan-potD nanoparticles significantly enhanced the immunogenicity and protection efficacy of DNA vaccines by intranasal immunization and could be used as a potential mucosal vaccine to prevent pneumococcal infection.

System and intranasal immunization of group B streptococcal C5a peptide / 中华微生物学和免疫学杂志

Objective To study the system of streptococcal C5a peptidase (ScpB) and the specif-ic antibody levels in serum, lung, vagina and recta after subcutaneous and intranasal immunization with dif-ferent doses of C5a peptide. Methods Recombinant protein C5a peptide was expressed in E. coli strain BL21 and purified by affinity chromatography. The expressed product was identified by SDS-PAGE and pep-tide mass fingerprinting (PMF). BALB/c mice were subcutaneously and intranasally injected with different doses of ScpB. Antibody titer was tested by ELISA. Opsonophagocytosis assay was used to test the function of antibody. Results ScpB protein was successfully expressed and purified. The probability based mouse score of ScpB was 175 by PMF analysis. ELISA data showed that both subcutaneous and intranasal immtmi-zation could elicit significantly higher levels of IgG in immunized mice serum than that of control group (P ＜0.01), 30 μg group waa better than 5 μg and 10 μg group. Intranasal immunization could elicit higher lev-els of IgA in lung, vagina and rectum (P ＜0.001) while system immunization could not. Opsenophagocyto-sis tests indicated that anti-serum of ScpB had opsenophagocytic activity than that of control (P ＜ 0. 05).Conclusion The results demonstrated that intranasal immunization with ScpB could induce significantly higher levels of lgG and IgA, and its anti-serum had better opsenic activity.

Adjuvant requirement for successful immunization with recombinant derivatives of Plasmodium vivax merozoite surface protein-1 delivered via the intranasal route

Recently, we generated two bacterial recombinant proteins expressing 89 amino acids of the C-terminal domain of the Plasmodium vivax merozoite surface protein-1 and the hexa-histidine tag (His6MSP1(19)). One of these recombinant proteins contained also the amino acid sequence of the universal pan allelic T-cell epitope (His6MSP1(19)-PADRE). In the present study, we evaluated the immunogenic properties of these antigens when administered via the intra-nasal route in the presence of distinct adjuvant formulations. We found that C57BL/6 mice immunized with either recombinant proteins in the presence of the adjuvants cholera toxin (CT) or the Escherichia coli heat labile toxin (LT) developed high and long lasting titers of specific serum antibodies. The induced immune responses reached maximum levels after three immunizing doses with a prevailing IgG1 subclass response. In contrast, mice immunized by intranasal route with His6MSP1(19)-PADRE in the presence of the synthetic oligonucleotides adjuvant CpG ODN 1826 developed lower antibody titers but when combined to CT, CpG addition resulted in enhanced IgG responses characterized by lower IgG1 levels. Considering the limitations of antigens formulations that can be used in humans, mucosal adjuvants can be a reliable alternative for the development of new strategies of immunization using recombinant proteins of P. vivax.

Induction of immune responses against rotavirus with liposome coated rotavirus DNA vaccine / 中国免疫学杂志

Objective:To search for the efficient immune adjuvant for rotavirus DNA vaccine by evaluating the efficacy in modifying immune responses to rotavirus.Methods:BALB/c mice were immunized by intramuscular and intranasal routes with 3 doses of naked DNA and liposome-coated DNA vaccine at two-week intervals. Titers of serum IgG and IgA were measured by ELISA.Results:Serum titers of rotavirus specific IgG in naked DNA and liposome-coated DNA vaccine-immunized mice were significantly higher than that of the unimmunized controls. Interestingly, only those mice administrated with liposome-coated DNA vaccine by intramuscular route generated serum rotavirus IgA,and neither by naked plasmid nor by intranasal immunization with DNA-liposome complex.Conclusion:These results indicate that liposome can act as both the vector and adjuvant for DNA vaccine against rotavirus by intramuscular injection,which induces rotavirus-specific IgA in the sera,and may provide partial protection against rotavirus infection.

MUCOSAL VACCINIZATION OF PCDNA3.1/SJTS-1 INDUCED IMMUNE PROTECTION IN MICE AGAINST CHALLENGE INFECTION OF SCHISTOSOMA JAPONICUM / 中国血吸虫病防治杂志

0.05). Conclusion pcDNA3.1/SjTs-1 induced the mucosal and systemic immune response and partial protection against the challenge of S.japonicum by the intranasal vaccinations of mice.

Induction of immune responses against rotavirus by intramuscular and intranasal immunization with rotavirus DNA vaccine / 重庆医科大学学报

Objective:To search for the efficient immune routes for rotavirus DNA vaccine by evaluating efficacy in inducing immune responses to rotavirus.Methods:In this study BALB/c mice were immunized by intramuscular and intranasal routes with rotavirus DNA vaccine,pcDNA 1/VP7,then rotavirus specific systemic immunity was measured.Results:Serum rotavirus specific IgG but not IgA titers of mice immunized by both routes were significantly higher than that of the unimmunized controls.Conclusion:Both intramuscular and intranasal routes are candidates for rotavirus DNA vaccine.