The dengue virus 4 component of NIAID's tetravalent TV003 vaccine drives its innate immune signature

Abstract

Annually, roughly 2.5 billion people are at risk for dengue virus (DENV) infection, and the incidence of infection has increased 30-fold since its discovery in the 1900s. At present, there are no globally licensed antiviral treatments or vaccines that protect against all four of the DENV serotypes. The NIAID Live Attenuated Tetravalent Vaccine (LATV) dengue vaccine candidate is composed of variants of three DENV serotypes attenuated by a 30 nucleotide (Δ30) deletion in the 3' untranslated region and a fourth component that is a chimeric virus in which the prM and E genes of DENV-2 replace those of DENV-4 on the rDEN4Δ30 backbone. The vaccine candidate encodes the non-structural proteins of DENV-1, DENV-3, and DENV-4, which could be of critical importance in the presentation of DENV-specific epitopes in a manner that facilitates antigen presentation and confers higher protection. Our findings demonstrate that the attenuation mechanism (Δ30) resulted in decreased viral infectivity and replication for each vaccine virus in monocyte-derived dendritic cells but were able to generate a robust innate immune response. When tested as monovalent viruses, DEN-4Δ30 displayed the most immunogenic profile. In addition, we found that the tetravalent DENV formulation induced a significantly greater innate immune response than the trivalent formulation. We demonstrate that the presence of two components with a DENV-4Δ30 backbone is necessary for the induction of RANTES, CD40, IP-10, and Type I IFN by the tetravalent formulation. Finally, we found that the DEN-4Δ30 backbone in the DENV-2 component of the vaccine enhanced its antigenic properties, as evidenced by enhanced ability to induce IP-10 and IFNα2 in monocyte-derived dendritic cells. In sum, our study shows that the Δ30 and Δ30/Δ31 mutations attenuate the DENV vaccine strains in terms of replication and infectivity while still allowing the induction of a robust innate immune response.

Keywords: Dengue; innate immunity; vaccine; vaccine efficacy.

Figure 1.

Viral infectivity and replication of the NAID LATV TV003 vaccine viruses and their wild-type counterparts. (A) The DENV-1 vaccine component was generated via the introduction of a 30-nucleotide (Δ30) deletion into the 3' untranslated region (UTR) of a DENV-1 Western Pacific (WP) strain. The DENV-2 vaccine strain was generated via a chimerization strategy in which the prM and E proteins of a DENV-2 New Guinea C strain were introduced into the backbone of the DENV-4 vaccine virus. The DENV-3 vaccine strain was generated similarly to the DENV-1 vaccine strain, with the introduction of the Δ30 deletion in the 3' UTR of the DENV-3 Sleman/78 strain. To sufficiently attenuate the DENV-3 virus an additional 31 nucleotide deletion was introduced into the 3' UTR of a Sleman/78 isolate. The DENV-4 vaccine virus was generated via the introduction of the Δ30 deletion in the 3' UTR of a DENV-4 Dominica isolate. (B) Viral replication was quantified via RT-qPCR using a primer designed to capture a conserved region of the DENV 3' UTR that is not affected by the presence of the Δ30 deletion. Monocyte-derived dendritic cells (MDDC)s were infected with either DENV-1 WT, rDEN-1Δ30, rDEN-2/4Δ30, DENV-3 WT, rDEN-3Δ30/31, DENV-4 WT or rDEN-4Δ30 at an MOI of 1.0. Cells were lysed and collected at 6, 12, 24, 48, and 72 hpi. Replication levels were standardized to the RPS11 housekeeping gene and relative levels were determined using the 2^-ΔΔCt method. (C) Flow cytometry was performed on MDDCs infected with either mock, DENV-1 WT, rDEN-1Δ30, rDEN-2/4Δ30, DENV-3 WT, rDEN-3Δ30/31, DENV-4 WT, or rDEN-4Δ30 viruses at an MOI of 1.0 for 6, 24, and 48 hpi. Infectivity was measured via the binding and fluorescence of the E protein binding, pan-flaviviral 4G2 antibody. (D) Detailed analysis of viral infectivity at 24 hpi indicating significant differences in infectivity between the DENV-1 WT and vaccine strains, the DENV-3 WT and vaccine strains, the DENV-4 WT and vaccine strains. Hashed colored lines represent significant differences between the DENV-1, rDEN-2/4Δ30, and rDEN-3Δ30/31vaccine strains compared to the rDEN-4Δ30 vaccine strain. Statistical significance between the four vaccine viruses was calculated using two-way analysis of variance (ANOVA) with multiple comparisons and Bonferroni correction (*P ⩽ 0.05, **P ⩽ 0.01, **P ⩽ 0.001). (A color version of this figure is available in the online journal.)

Figure 2.

Cytokine induction of the NAID LATV TV003 vaccine viruses. (A, B) Monocyte-derived dendritic cells (MDDCs) were infected with either mock, rDEN-1Δ30, rDEN-2/4Δ30, rDEN-3Δ30/31, or c vaccine viruses at a multiplicity of infection (MOI) of 1.0 for 6, 24, and 48 hpi. Multiplex ELISAs were performed to evaluate extracellular cytokine secretion. Data shown represent the levels of secreted IFNα2, IP-10, and IL-6 in cell supernatant. Black asterisks represent statistical significance by the two-way analysis of variance (ANOVA), with adjustment for multiple comparisons and the Bonferroni correction (*P ⩽ 0.05, **P ⩽ 0.01). (A color version of this figure is available in the online journal.)

Figure 3.

DENV-2 vaccine and DENV-4 vaccine viruses induce robust levels of IP-10 and IFNα2 but the DENV-2 challenge strain does not. (A) Viral replication was quantified via real-time quantitative PCR (RT-qPCR) using the previously described 3' untranslated region (UTR) primer. Monocyte-derived dendritic cells (MDDCs) were infected at 0 (1 h after infection), 6, 24, and 48 hpi. Viral RNA was quantified as previously described. (B) MDDCs were infected with either Mock, rDEN-2/4Δ30, rDEN-4Δ30 vaccine, or rDEN-2Δ30 challenge strains and cells were stained by flow cytometry (Data shown from one representative donor). The percentages of infected cells were quantified by measuring E protein levels at 6, 24, and 48 hpi. (C) Heat map comparing specific cytokine production levels by MDDCs after mock, trivalent, tetravalent, or DEN-4Δ30 × 2 infection. Calculations were made using data acquired from 10-plex ELISA. The cytokine levels were calculated as the log₂ values obtained in pg/mL (nine DC donors are represented. Red indicates greater expression levels. (D to F) Data are reflective of values represented in panel B. Black asterisks represent statistical significance by the two-way analysis of variance (ANOVA), with adjustment for multiple comparisons and the Bonferroni correction (*P ⩽ 0.05, **P ⩽ 0.01, **P ⩽ 0.001). (A color version of this figure is available in the online journal.)

Figure 4.

The addition of a second DENV-4 component to the trivalent dengue vaccine results in an enhanced viral replication and IP-10 in dendritic cells (DCs). (A) The tetravalent admixture is composed of one component of the rDEN-1Δ30, rDEN-2/4Δ30, rDEN-3Δ30/31, and rDEN-4Δ30 vaccine viruses and was used to infect human monocyte-derived dendritic cells (MDDCs) at a multiplicity of infection (MOI) of 0.25 for each vaccine virus present. (B) The DEN-4Δ30 × 2 admixture is composed of one component of the DENV-1 and DENV-3 vaccines and two components of the DENV-4 vaccine virus and was used to infect human MDDCs at an MOI of 0.25 for each vaccine virus present. (C) The trivalent admixture is composed of one component of the rDEN-1Δ and rDEN-3Δ30/31 vaccines and one component of the rDEN-4Δ30 vaccine virus at an MOI of 0.25 for each vaccine virus present. (D) Real-time quantitative PCR (RT-qPCR) quantifying levels of viral replication in MDDCs infected with the indicated dengue vaccine admixtures. (E) Multiplex ELISA was performed as in previous assays to quantify the presence of IP-10 in the supernatant of MDDC cultures treated with the indicated DENV vaccine admixtures. (F) RT-qPCR capturing the induction of RIG-I. Expression levels were quantified in Raji DC-Sign cells infected with mock, trivalent vaccine, tetravalent vaccine or DEN-4Δ30 × 2 formulation. Statistical significance was calculated using Two-way analysis of variance (ANOVA) with multiple comparisons and Bonferroni correction (*P ⩽ 0.05, **P ⩽ 0.01). (A color version of this figure is available in the online journal.)

Figure 5.

The presence of a second DENV-4 component confers induction of markers that are key in dendritic cell (DC) migration. (A, B) Monocyte-derived DCs (MDDCs) were infected with mock, trivalent vaccine, tetravalent vaccine or DEN-4Δ30 × 2 formulation. Multiplex ELISA was performed as in previous experiments to quantify the levels of MCP-1 and RANTES secretion, respectively, to the culture supernatant at 6, 24, and 48 hpi. (C) Using mass cytometry by time-of-flight (CyTOF) analysis for each mock, trivalent vaccine, tetravalent vaccine, or DEN-4Δ30 × 2 formulation sample. The percent of cells expressing CCR7 was quantified. (D) CyTOF depicting the median expression of CD40 of total cell populations is shown. Black asterisks represent statistical significance by the two-way analysis of variance (ANOVA), with adjustment for multiple comparisons and the Bonferroni correction (*P ⩽ 0.05, **P ⩽ 0.01, **P ⩽ 0.001). (A color version of this figure is available in the online journal.)