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. 2014 Nov 12;32(48):6537-43.
doi: 10.1016/j.vaccine.2014.08.087. Epub 2014 Sep 17.

Investigating the efficacy of monovalent and tetravalent dengue vaccine formulations against DENV-4 challenge in AG129 mice

Jeremy Fuchs  1 , Haiyan Chu  1 , Peter O'Day  1 , Richard Pyles  2 , Nigel Bourne  2 , Subash C Das  1 , Gregg N Milligan  2 , Alan D T Barrett  2 , Charalambos D Partidos  3 , Jorge E Osorio  1
Affiliations

Investigating the efficacy of monovalent and tetravalent dengue vaccine formulations against DENV-4 challenge in AG129 mice

Jeremy Fuchs et al. Vaccine. .

Abstract

Dengue (DEN) is the most important mosquito-borne viral disease, with a major impact on global health and economics, caused by four serologically and distinct viruses termed DENV-1 to DENV-4. Currently, there is no licensed vaccine to prevent DEN. We have developed a live attenuated tetravalent DENV vaccine candidate (TDV) (formally known as DENVax) that has shown promise in preclinical and clinical studies and elicits neutralizing antibody responses to all four DENVs. As these responses are lowest to DENV-4 we have used the AG129 mouse model to investigate the immunogenicity of monovalent TDV-4 or tetravalent TDV vaccines, and their efficacy against lethal DENV-4 challenge. Since the common backbone of TDV is based on an attenuated DENV-2 strain (TDV-2) we also tested the efficacy of TDV-2 against DENV-4 challenge. Single doses of the tetravalent or monovalent vaccines elicited neutralizing antibodies, anti-NS1 antibodies, and cellular responses to both envelope and nonstructural proteins. All vaccinated animals were protected against challenge at 60 days post-immunization, whereas all control animals died. Investigation of DENV-4 viremias post-challenge showed that only the control animals had high viremias on day 3 post-challenge, whereas vaccinated mice had no detectable viremia. Overall, these data highlight the excellent immunogenicity and efficacy profile of our candidate dengue vaccine in AG129 mice.

Keywords: AG129 mice; Dengue; Dengue 4; Immune protection; Vaccines.

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Figures

Fig. 1
Fig. 1
Neutralizing and anti-NS1 antibodies. (a–c) Neutralizing antibody responses elicited after a single immunization with TDV-4 (a) or TDV-2 (b) vaccines or tetravalent TDV (c). Individual serum samples collected from each group (day 56 post-primary immunization) were tested for neutralizing activity. Sera from group A were tested for neutralizing activity against DENV-4, sera from group B were tested for neutralizing activity against DENV-2 and DENV-4, and those from group C for tetravalent neutralizing antibody responses. Data present mean of neutralizing antibody titers against each virus ± SE. (d) Titration of immune serum collected from animals vaccinated with TDV on day 56 post-immunization or day 28 post-DENV-4 challenges against DENV-2 and DENV-4 NS1 antigens coated on an ELISA plate. Data represent the mean ± SD of titrations of individual serum for each time point tested against the DENV-2 and DENV-4 NS1 antigens ± SD. Normal mouse serum was tested as negative control (green line).
Fig. 2
Fig. 2
T cell responses to DENV E and NS proteins. (a and b) CD4+ and CD8+ T cell responses to E proteins of each DENV serotype elicited after a single subcutaneous immunization with TDV. Immune splenocytes collected 6 weeks post-primary immunization were restimulated in vitro with pools of peptides encompassing the entire sequence of DENV-1–4 E (each pool consisted of the entire peptide array for a given E protein). Cells were stained for surface markers CD4, CD8 and intracellular cytokines (IFNγ, IL-2 or TNF-α). Samples were analyzed by flow cytometry (FACS Calibur). The percentage of cytokine producing cells in medium treated groups was subtracted from each sample. Data are presented as mean ± SD (n = 3). T cells derived from control FTA group did not produce any significant cytokine response above the medium background (data not shown). Peptide arrays representing the entire sequence of E protein for each serotype and NS3 protein of DENV-4 were obtained through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH (http://www.beiresources.org/). For E peptide arrays: Dengue virus type 1, Singapore/S275/1990, NR-4551; Dengue virus type 2, New Guinea C (NGC), NR5-7; Dengue virus type 3, Sleman/1978, NR-511; Dengue type 4, Dominica/814669/1981, NR-512. (c and d) CD4+ and CD8+ T cell responses to the NS3 protein of DENV-4. Immune splenocytes collected seven weeks post-primary immunization with TDV-4 were restimulated in vitro with pools of peptides (10 peptides/pool) encompassing the entire sequence of DENV-4 NS3 protein. Cells were stained for surface markers CD4, CD8 and intracellular cytokines (IFNγ). Samples were analyzed by flow cytometry (FACS Calibur). The percentage of cytokine producing cells in medium treated groups was subtracted from each sample. Data are presented as mean of two individual mice.
Fig. 3
Fig. 3
Impact of immunization on survival following DENV-4 virus challenge. Groups of 15 AG129 mice were immunized with a single dose of TDV-4, TDV-2 or TDV vaccines. Control animals were immunized with FTA (virus diluents). On day 60 post-primary immunization animals were challenged with mouse adapted DENV-4 and morbidity and mortality was monitored over a period of four weeks.
Fig. 4
Fig. 4
Anti-DENV-4 neutralizing antibody responses at day 56 post-primary vaccination or day 28 post-DENV-4 virus challenge. Groups of 15 AG129 mice were immunized with a single dose of TDV-4, TDV-2 or TDV vaccines. Control animals were immunized with FTA (virus diluents). On day 60 post-primary immunization animals were challenged with mouse adapted DENV-4. Individual sera collected post-vaccination or post-challenge (from all surviving animals) were tested for neutralizing activity against DENV-4. Data represent the mean neutralizing titer from each animal in each group ± SE.
Fig. 5
Fig. 5
Viremia data following DENV-4 challenge of animals immunized with TDV-2, TDV-4 or TDV vaccines. Levels of DENV-4 RNA in the serum post-challenge were quantified by qRT-PCR using a Fam labeled 5 prime nuclease assay designed to target the 3 prime non-coding region of dengue virus. Limit of detection is 2.5 log10 genomes/ml.
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