Characterization of a candidate tetravalent vaccine based on 2'-O-methyltransferase mutants

<div><p>Dengue virus (DENV) is one of the most widespread arboviruses. The four DENV serotypes infect about 400 million people every year, causing 96 million clinical dengue cases, of which approximately 500’000 are severe and potentially life-threatening. The only licensed vaccine has a limited efficacy and is only recommended in regions with high endemicity. We previously reported that 2’-<i>O</i>-methyltransferase mutations in DENV-1 and DENV-2 block their capacity to inhibit type I IFNs and render the viruses attenuated <i>in vivo</i>, making them amenable as vaccine strains; here we apply this strategy to all four DENV serotypes to generate a tetravalent, non-chimeric live-attenuated dengue vaccine. 2’-<i>O</i>-methyltransferase mutants of all four serotypes are highly sensitive to type I IFN inhibition in human cells. The tetravalent formulation is attenuated and immunogenic in mice and cynomolgus macaques and elicits a response that protects from virus challenge. These results show the potential of 2’<i>-O</i>-methyltransferase mutant viruses as a safe, tetravalent, non-chimeric dengue vaccine.</p></div

Dengue MTase mutants are attenuated and immunogenic in mice.

<p>A) Kinetics of MTase mutants and WT viremia <i>in vivo</i>. Mice were infected i.p. with 1×10⁴ PFU of WT virus or a mix of 1×10⁴ PFU of each MTase mutant (total of 4×10⁴ PFU). Viral titers in the plasma were measured at indicated time points by serotype-specific real-time RT-PCR. No virus was detected for WT and MTase mutant for DENV-3 and DENV-4, respectively. B) IgG titers of immunized mice. Blood was taken 30 days post immunization and total IgG antibody titers against DENV-1-4 were measured by UV-inactivated DENV particle ELISA. C) Neutralizing antibody (nAB) titers of imunized mice. Blood was taken 30 days post immunization and nAB against all four DENV serotypes were measured by flow cytometry-based neutralization assay. D) 30 days post immunization mice were challenged with DENV-1 (1x10⁶ PFU of strain 08K3126), DENV-2 (1x10⁷ PFU of strain D2Y98P), DENV-3 (1.5x10⁶ PFU of strain VN32/96) or DENV-4 (3x10⁶ PFU of strain TVP360). Viremia after challenge was measured by real-time PCR on days 1, 3, 5, and 7 post challenge. Naïve mice challenged with DENV-2 succumbed at day 4 post infection. Data are representative of three experiments with a total of 9–13 mice (A, B) or two experiments with a total of 6–9 mice per group (C, D). Shown are means with SD for all panels. Statistical analysis was performed using student's t-test (B, C, D), **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05.</p

Dengue MTase mutants induce an E-protein targeted T cell response in CM.

<p>IFN-<b>γ</b> ELISPOT after overnight stimulation with peptide pools of E, NS3 and NS5 proteins of DENV-1 and DENV-2. PBMCs from three different time points after vaccination were tested. CD3 beads were used as positive control and values shown are after deduction of spots observed in the negative control. Mean and range of two wells tested per peptide pool is shown for each CM.</p

2’-<i>O</i>-MTase mutants are more sensitive to IFN.

<p>A) DENV WT or MTase mutant infection of HEK-DC-SIGN cells pretreated with increasing amount of IFN-β. HEK-DC-SIGN cells were seeded in a 24-well plate and after incubation overnight, pre-treated for 24 hours with the indicated amount of IFN- β. 24 hours after addition of IFN- β, cells were infected at an MOI of 1. B) DENV WT or MTase mutant infection of monocyte-derived dendritic cells (moDCs) pretreated with increasing amount of IFN-β. Means and SD are shown. Statistical analysis was performed using Student's t-test (****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05). Results shown are from three experiments with n = 4–11 measurements per condition (A) or from two experiments (from two different donors) with n = 4, except for DENV-3 where one experiment is shown from one donor with n = 2 (B).</p

Neutralizing antibody titers one day before challenge (day 97) and six days after challenge (day 104).

<p>Neutralizing antibody titers one day before challenge (day 97) and six days after challenge (day 104).</p

Characterization of NS5-Mtase mutants.

<p>A) Effects of MTase mutations on N7-MTase activities. B) Effects of MTase mutations on 2’-O-MTase activities. Recombinant wild-type (abbreviated as WT) and mutant (abbreviated as Mut). The mutant MTase of each DENV serotype contained double mutations within the <u>K</u>-D-K-<u>E</u> active site (with the underlined residues mutated to Alanine). MTases of all four DENV serotypes were assayed for GpppA-RNA→m7GpppA-RNA and m7GpppA-RNA→m7GpppAm-RNA conversions to indicate N7- and 2′-O-methylation activities, respectively. Relative methylation activities were indicated below each panel with WT activity set as 100%. C) Immunofluorescence analysis (IFA). BHK-21 cells were electroporated with equal amounts of in vitro transcribed WT and Mut genome-length RNAs of DENV-1 to -4. The mutant genome-length RNAs contained double mutations as described above. At indicated days post-transfection, intracellular E proteins were examined by IFA using mouse antibody 4G2 against DENV E protein and goat anti-mouse IgG conjugated with FITC as primary and secondary antibodies, respectively. D) Plaque morphology. WT and Mut viruses recovered from genome-length RNA-transfected cells were analyzed by standard CPE-based plaque assays using BHK cells. Plaques were developed on day 4 (DENV-2) or day 5 (DENV-1, -3 and -4) post-infection. E) Immunostaining. BHK-21 cells were infected with DENV-3 WT or Mut viruses harvested from genome-length RNA-transfected cells. On day 4 post-infection, cells were assayed by immunostaining using mouse antibody 4G2 against DENV E protein and goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) as primary and secondary antibodies, respectively. F) Growth kinetics. BHK-21 and C3/36 cells were infected with WT and mutant viruses at an MOI of 0.01. Viral titers were measured at indicated time points using standard plaque assays (DENV-1, -2 and -4) or immunostaining (DENV-3). Average results and SD of three experiments are presented. Dash line indicates the limitation of detection (10 PFU/ml).</p

Neutralizing antibody titers in CM before and after immunization with TV-MT.

<p>Neutralizing antibody titers in CM before and after immunization with TV-MT.</p

Dengue MTase mutants are immunogenic in monkeys and induce a protective immune response.

<p>A) Kinetics of DENV E protein-specific IgG titer of monkeys immunized with tetravalent MTase mutants formulation or RPMI placebo, respectively. Monkeys were immunized i.d. with a mix of 1×10⁴ PFU of each MTase mutant (total of 4×10⁴ PFU). B) Neutralizing antibody (nAB) titers of monkeys immunized as described above. nAB against all four DENV serotypes were measured by flow cytometry-based neutralization assay. C) 97 days post immunization monkeys were challenged with DENV-2 (1x10⁵ PFU of strain D2Y98P) i.d.. Viremia after challenge was measured by real-time PCR as indicated. Data are representative of one experiment with a total of 3 monkeys per group. Shown are means with SD for all panels. Statistical analysis was performed using Student's t-test (B). *, p < 0.05).</p