Evaluation of Two Adjuvant Formulations for an Inactivated Yellow Fever 17DD Vaccine Candidate in Mice.

The attenuated yellow fever (YF) vaccine is one of the most successful vaccines ever developed. After a single dose administration YF vaccine can induce balanced Th1/Th2 immune responses and long-lasting neutralizing antibodies. These attributes endorsed it as a model of how to properly stimulate the innate response to target protective immune responses. Despite their longstanding success, attenuated YF vaccines can cause rare fatal adverse events and are contraindicated for persons with immunosuppression, egg allergy and age < 6 months and >60 years. These drawbacks have encouraged the development of a non-live vaccine. The aim of the present study is to characterize and compare the immunological profile of two adjuvant formulations of an inactivated YF 17DD vaccine candidate. Inactivated YF vaccine formulations based on alum (Al(OH)3) or squalene (AddaVax®) were investigated by immunization of C57BL/6 mice in 3-dose or 2-dose schedules, respectively, and compared with a single dose of attenuated YF virus 17DD. Sera were analyzed by ELISA and Plaque Reduction Neutralization Test (PRNT) for detection of total IgG and neutralizing antibodies against YF virus. In addition, splenocytes were collected to evaluate cellular responses by ELISpot. Both inactivated formulations were able to induce high titers of IgG against YF, although neutralizing antibodies levels were borderline on pre-challenge samples. Analysis of IgG subtypes revealed a predominance of IgG2a associated with improved neutralizing capacity in animals immunized with the attenuated YF vaccine, and a predominance of IgG1 in groups immunized with experimental non-live formulations (alum and AddaVax®). After intracerebral (IC) challenge, attenuated and inactivated vaccine formulations showed an increase in neutralizing antibodies. The AddaVax®-based inactivated vaccine and the attenuated vaccine achieved 100% protection, and alum-based equivalent formulation achieved 70% protection.

Safety and immunogenicity of 17DD attenuated yellow fever vaccine in howler monkeys (Alouatta spp.).

BACKGROUND: Alouatta spp. are highly susceptible to yellow fever (YF) infection and develop an often fatal disease. The threat posed by an outbreak started in 2016 leads us to investigate vaccination as a potential tool in preventing YF in non-human primates (NHP). METHODS: Susceptible howler monkeys were immunized with three different concentrations of the human Brazilian commercial YF17DD vaccine. Post-vaccination viremia/RNAemia, immunogenicity, and safety were characterized. RESULTS: The vaccine did not produce YF clinical manifestations in any of the NHPs. After immunization, all animals seroconverted demonstrating the ability of the YF vaccine to induce humoral response in Alouatta species. CONCLUSIONS: The present work has demonstrated the safe and immunogenic profile of the existing YF 17DD vaccine in howler monkeys. This knowledge may support further studies with other susceptible monkey species and provide a possible solution for controlling epizootics and preventing the devastation of endangered species.

Vacinas experimentais para febre amarela como modelo de estudo de novos adjuvantes / Experimental yellow fever vaccines as a study model for new adjuvants

A vacina de febre amarela atenuada é uma das mais bem-sucedidas já desenvolvidas. Entretanto, restrições de administração para pacientes imunodeprimidos e raros eventos adversos associados são desvantagens que motivam o desenvolvimento de vacinas mais seguras. À medida que aumenta a segurança, a imunogenicidade diminui na ausência de replicação viral. Nesse contexto, adjuvantes são elementos chave na ativação da imunidade inata para modulação das respostas adaptativas e proteção. Adjuvantes de diferentes naturezas e mecanismos de ação têm sido estudados: imunoestimuladores como agonistas de TLR, carreadores de antígenos e agentes de efeito depósito. Nesse estudo pretendemos identificar adjuvantes promissores para o desenvolvimento de novos candidatos vacinais para febre amarela. Para isso, camundongos C57BL/6 foram imunizados com diferentes formulações de antígenos modelo (vírus inativado e proteínas de envelope recombinantes produzidas em diferentes sistemas de expressão) com os adjuvantes: Al(OH)3; Addavax (emulsão baseada em esqualeno); combinações de Al(OH)3 e Flagelina FliC (agonista de TLR5); e CAF01 (nanopartícula) em esquema de 2 doses (D0 e D28) ou 3 doses (D0, D14 e D28). Após a imunização, os camundongos foram desafiados com inóculo letal do vírus de febre amarela por via intracerebral para determinar as taxas de sobrevivência. Os soros foram analisados por ELISA e PRNT50 para detecção dos títulos de IgG total e anticorpos neutralizantes

O vírus FA17DD inativado apresentou o melhor desempenho como antígeno modelo, sendo capaz de induzir 100% de proteção ao desafio após imunização com 2 doses na formulação com o adjuvante Addavax e 70% de proteção na formulação com hidróxido de alumínio. Os demais adjuvantes avaliados (Al(OH)3/ Flagelina FliC e CAF01) não foram capazes de gerar incremento de proteção com os antígenos avaliados. As formulações experimentais com melhor desempenho (FA17DD inativado/Addavax e FA17DD inativado/Al(OH3) foram avaliadas em um segundo ensaio para melhor caracterização das respostas imunológicas envolvidas na proteção. Ambas foram capazes de induzir apenas níveis basais de anticorpos neutralizantes; porém altos títulos de IgG para o vírus da febre amarela com predomínio do subtipo IgG1. A caracterização das respostas celulares locais (ELISpot citocinas e células B) no sítio de inoculação nos tempos pré e pós-desafio revelou níveis superiores de IFNγ nos animais sobreviventes. Após o desafio, todos os animais sobreviventes apresentaram altos títulos de anticorpos neutralizante e IgG total, com incremento do subtipo IgG2a. O uso de Addavax como adjuvante para vacinas não vivas para febre amarela surge como uma alternativa promissora de induzir proteção com menor número de doses. A aplicação do modelo de desafio murino para febre amarela na avaliação de novos adjuvantes se mostrou uma abordagem promissora para a avaliação de novos adjuvantes para uso neste modelo, bem como na geração de conhecimentos extrapoláveis para outros candidatos vacinais em desenvolvimento. (AU)

Production of Monoclonal Antibody That Recognizes Zika Virus and Other Flaviviruses in Serum-Free Conditions.

With the recent outbreaks of Zika and Dengue virus infections in various countries worldwide, production of vaccines or diagnostic kits is an urgent public health demand. Production of a monoclonal antibody (mAb) that specifically binds to a common antigen shared by the Flavivirus genus will be necessary for new diagnostic kits or characterization and viral identity tests during vaccine development. This study aimed to cultivate, in serum-free conditions, the 4G2 hybridoma that produces an mAb, which recognizes a shared epitope from the Flavivirus genus. We compared 4G2 hybridoma growth and biochemical profiles between cells cultivated in batch mode over 10 days in roller bottles containing Dulbecco's modified Eagle's medium high glucose containing 10% fetal bovine serum medium or hybridomas directly adapted to Ex-Cell serum-free medium. Cellular parameters such as specific growth rate (µ), maximum cell concentration, specific l-lactate, and glucose and IgG rates were evaluated. Thereafter, we also compared total mAb volumetric productivity, purification yield, and mAb staining of Vero cells infected with Zika and Dengue-2 virus. Direct adaptation to serum-free conditions did not change hybridoma growth rate and mAb production under the conditions tested. Instead, serum-free mAb purification showed a higher yield with no alterations on mAb structure or mAb staining of Zika and Dengue Vero-infected cells.