A Novel Synthetic TLR-4 Agonist Adjuvant Increases the Protective Response to a Clinical-Stage West Nile Virus Vaccine Antigen in Multiple Formulations

Abstract

West Nile virus (WNV) is a mosquito-transmitted member of the Flaviviridae family that has emerged in recent years to become a serious public health threat. Given the sporadic nature of WNV epidemics both temporally and geographically, there is an urgent need for a vaccine that can rapidly provide effective immunity. Protection from WNV infection is correlated with antibodies to the viral envelope (E) protein, which encodes receptor binding and fusion functions. Despite many promising E-protein vaccine candidates, there are currently none licensed for use in humans. This study investigates the ability to improve the immunogenicity and protective capacity of a promising clinical-stage WNV recombinant E-protein vaccine (WN-80E) by combining it with a novel synthetic TLR-4 agonist adjuvant. Using the murine model of WNV disease, we find that inclusion of a TLR-4 agonist in either a stable oil-in-water emulsion (SE) or aluminum hydroxide (Alum) formulation provides both dose and dosage sparing functions, whereby protection can be induced after a single immunization containing only 100 ng of WN-80E. Additionally, we find that inclusion of adjuvant with a single immunization reduced viral titers in sera to levels undetectable by viral plaque assay. The enhanced protection provided by adjuvanted immunization correlated with induction of a Th1 T-cell response and the resultant shaping of the IgG response. These findings suggest that inclusion of a next generation adjuvant may greatly enhance the protective capacity of WNV recombinant subunit vaccines, and establish a baseline for future development.

Fig 1. Induction of a Th1 CD4+ T-Cell Response in SLA-SE Immunized Animals.

Seven days following a single immunization, isolated splenocytes (n = 5 mice/group) were phenotyped by ICS. IFNγ+ CD4 T-cells were induced following immunization with WN-80E in combination with SLA-SE. At decreased antigen doses (100 ng/mouse), inclusion of SLA-SE resulted in a significant increase in cytokine positive cells relative to antigen only controls (p<0.005). Additional cytokine profiling shows that many of the IFNγ cells in the SLA-SE group displayed a Th1 phenotype, and were positive for TNFα and/or IL-2 (B).

Fig 2. ELISA Titers Following A Single Immunization with WN-80E.

Serum antibody titers were determined by ELISA 21 days following a single immunization with WN-80E in combination with adjuvants. Titers of Total IgG (A), IgG1 (B) and IgG2c (C) were determined for all mice (n = 5/group). One way ANOVA was used to evaluate significant differences in antibody levels and PRNT titers between groups. Similar levels of Total IgG and IgG1 were observed in all immunized animals. Significantly elevated levels of IgG2c were detected in mice immunized with SLA-SE compared to those immunized with 10 μg of antigen alone (p<0.0001). Neutralizing antibody titers were also determined by PRNT assay (D) to assess antibody function. There is a trend toward increased titer in SLA-SE immunized animals at all anitgen doses, and significant increases in PRNT titer are observed at the 1μg antigen dose.

Fig 3. SLA Formulated with Alum or SE Increases Functional Antibody Titer Following A Single Immunization with WN-80E.

Serum antibody titers were determined by ELISA 21 days following a single dose of WN-80E in combination with Alum or SE formulations with or without SLA. Anti-WN-80E titers of Total IgG (A), IgG1 (B) and IgG2c (C) were determined for all mice (n = 5/group). One way ANOVA was used to identify significant differences in antibody levels. Significantly elevated levels of IgG2c were detected in mice immunized with SLA containg adjvuants, as well as SE alone compared to those immunized with 1 μg of antigen alone. Mice immunized with SLA-SE showed the highest levels of IgG2c (p<0.0001). Mice receiving SLA-SE also had elevated levels of IgG2c relative to animals immunized with SE alone. Neutralizing antibody titers were also determined by PRNT (D) to assess antibody function. Elevated PRNT titers are observed in mice that received either SE alone, or SLA containing adjuvants in combination with WN-80E.

Fig 4. Immunization with SLA Containing Adjuvants in Combination with WN-80E Enhances Survival and Reduces Viral Titer to Undetectable Levels.

Following a single immunization of WN-80E in combination with the indicated adjuvants, mice (n = 10/group) were challenged with 100 LD₅₀ of WNV via the intraperitoneal route. Surivial of mice was monitored over 14 days following challenge (A,B). Survival curves were compared using a Mantel-Cox test, with p-values compared to immunization with WN-80E shown. Three days post-challenge, serum was collected from all animals in order to assess virus titers. Animals immunized with SLA-Alumhad undetectable titers in all animals (P<0.0005). Those imminzed with SE or SLA-SE had minimal titers while those immunized with Alum, SLA-AF or no adjuvant showed only slightly reduced titers compared to unimmunized controls (P<0.05).

Fig 5. Immunization with SLA Containing Adjuvants in Combination with WN-80E Induces Antibody Secreting Cells in Bone Marrow.

In an independent experiment, we have examined the ability of WN-80E combined with SLA containing adjuvants to induce long lived antibody secreting cells (ASC) in bone marrow. Mice were immunized and bone marrow extracted after 21 days (n = 5/group). The number of antibody secreting cells was assessed by ELISPOT assay on plates coated with WN-80E. Differences between groups were compared by one way ANOVA. Adjuvant formulations containing SLA (SLA-Alum, SLA-SE) as well as emulsion alone induced significantly greater numbers of IgG+ ASC compared with antigen alone. Animals immunized with Alum or unformulated SLA also showed modest increases in numbers of ASC, but differences were not significant relative to antigen only.

Fig 6. Vaccine Formulations which reduce WNV titer post-challenge induce germinal center B-cells and plasmablasts following immunization.

Animals were immunized once with WN-80E (1ug) in combination with Alum or SE with or without SLA. Over a timecourse following this immunization, inguinal lymph nodes were removed and stained for B cell markers by ICS. Gating strategies for germinal center B-cells (A) and plasmablasts (C) are shown. Immunization with SLA-Alum, SE, or SLA-SE resulted in a sustained increase in the number of germinal center B-cells (CD95⁺GL7⁺) (B). Increased numbers of cells were observed relative to both WN-80E alone (***P<0.0005, **** p<0.0001) and relative to WN-80E combined with Alum (# p<0.05, ### p<0.0005) at the same timepoint. Numbers of germinal center cells peaked at D14, and declined by D28 post-immunization. Elevated numbers of plasmablast cells (CD138+B220lo) were also observed at day 7 post-immunization, but declined thereafter (D). Increases in germinal center cell numbers resulted in significant increases in PRNT titers as early as 7 days post immunization (E). Differences between groups were determined by one-way ANOVA using Dunnett’s Multiple comparison test.