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

Abstract

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.

Keywords: adjuvants; immunogenicity; protective efficacy; yellow fever inactivated vaccine.

Figure 1

Immunogenicity of different inactivated YF 17DD virus formulations. Neutralizing antibodies titers in pre-(A) and post-challenge (B) times. Total IgG titers in pre-(C) and post-challenge (D) times. Data considering PRNT₅₀ cut off = 1,43 and ELISA IgG cut off = 0.56. Animals from negative control groups died after the challenge, so their post-challenge antibody titers were not measured. (A): non normal data; p < 0.0001 by Kruskal–Wallis test. (B–D): all groups presented normal data; p < 0.0001 by one-way ANOVA.

Figure 2

Distribution of IgG subtypes induced by the formulations tested at pre- and post-challenge times. (A) Sectoral graphs show the contribution of each IgG subtype to total IgG response induced by each vaccine. (B) Quantitative data of IgG subtypes induced by the different formulations.

Figure 3

Cytokine balance in the local response (draining lymph nodes and brain) at pre- (A) and post-challenge (B) times. Sectoral graphs show the contribution of each cytokine to the response induced by each vaccine.

Figure 4

Analysis of the local response (draining lymph nodes and brain) by dosage of activated plasma cells and memory B cells by IgG ELISpot at pre-challenge (A) and post-challenge (B) times. Data expressed in SFCs/10⁶. Samples of brain dosed in pool per group. * Dosage of activated plasma cells not performed due to lack of sufficient available lymph node cells.