Adjuvanted Fusion Protein Vaccine Induces Durable Immunity to Onchocerca volvulus in Mice and Non-Human Primates

Abstract

Onchocerciasis remains a debilitating neglected tropical disease. Due to the many challenges of current control methods, an effective vaccine against the causative agent Onchocerca volvulus is urgently needed. Mice and cynomolgus macaque non-human primates (NHPs) were immunized with a vaccine consisting of a fusion of two O. volvulus protein antigens, Ov-103 and Ov-RAL-2 (Ov-FUS-1), and three different adjuvants: Advax-CpG, alum, and AlT4. All vaccine formulations induced high antigen-specific IgG titers in both mice and NHPs. Challenging mice with O. volvulus L3 contained within subcutaneous diffusion chambers demonstrated that Ov-FUS-1/Advax-CpG-immunized animals developed protective immunity, durable for at least 11 weeks. Passive transfer of sera, collected at several time points, from both mice and NHPs immunized with Ov-FUS-1/Advax-CpG transferred protection to naïve mice. These results demonstrate that Ov-FUS-1 with the adjuvant Advax-CpG induces durable protective immunity against O. volvulus in mice and NHPs that is mediated by vaccine-induced humoral factors.

Keywords: Advax; Onchocerca volvulus; adjuvant; durability; fusion protein; mice; non-human primates; passive immunization; river blindness; vaccine.

Figure 1

Immunization with combination and fusion O. volvulus vaccine antigens formulated with Advax-CpG induced protective immunity in mice. (a) BALB/cByJ mice were immunized with either Ov-103 and Ov-RAL-2 co-administered (n = 22–23), either in combination (n = 12), or as a fusion protein (Ov-FUS-1) (n = 12) formulated with the adjuvant Advax-CpG. Control mice (n = 22–23) received Advax-CpG-only injections. Injections were administered in a prime–boost–boost sequence two weeks apart. Two weeks post-final booster, diffusion chambers containing 25 O. volvulus infective third-stage larvae were implanted, followed by recovery one, two, or three weeks later. (b) Survival of O. volvulus L3 in diffusion chambers. Data are shown as mean percent larval survival, with individual mice represented as points. Error bars indicate standard deviations. Percent reduction in larval survival, when compared to control mice for each time point, is listed below the horizontal axis. (c) Antigen-specific IgG titers measured in the serum of control and immunized mice at the time of recovery. Data shown are mean titers, with individual mice represented as points. Error bars indicate standard deviations. IgG subclass is indicated on the vertical axis, and antigen specificity is indicated above the two associated plots. (d) Ratios of IgG1 to IgG2ab from the serum of individual mice at the time of recovery. Data shown are mean titer ratios, with individual mice represented as points. Error bars indicate standard deviations. IgG subclass is indicated on the vertical axis, and antigen specificity is indicated above the associated plots. Robust regression and outlier removal method with a false discovery rate of 1% was conducted using GraphPad Prism to eliminate 26 outliers. (b–d) * = p ≤ 0.05, indicating significant differences when comparing means to control animals within the time point. ** = p ≤ 0.05, indicating significant differences when comparing means to control animals and co-administered recipients within the time point.

Figure 2

Immunization with Ov-FUS-1 formulated with the adjuvant Advax-CpG induced durable protective immunity in mice. (a) BALB/cByJ mice were immunized with Ov-FUS-1 formulated with either Advax-CpG (n = 12), alum (n = 12), or AlT4 (n = 12) as the adjuvant. Injections were administered in a prime–boost–boost fashion two weeks apart. Two weeks post-final booster, mice were challenged with diffusion chambers containing 25 O. volvulus L3, followed by recovery one week later. A separate group of mice was similarly primed and boosted but challenged 10 weeks post-final booster, followed by recovery one week later. Control mice (n = 24) received adjuvant-only injections that were combined from the early and late time points. (b) Survival of O. volvulus L3 in diffusion chambers. Data are shown as mean percent of surviving larvae, with individual mice represented as points. Error bars indicate standard deviations. Percent reduction in larval survival, when compared to control mice for each adjuvant group, is listed below the horizontal axis. (c) Total and differential counts of immune effector cells from diffusion chambers recovered from control and immunized mice measured by flow cytometry. Data are shown as mean cell counts or percent of total live cells, with individual mice represented as points. Error bars represent standard errors of the mean. (d) Heat map displaying cytokine responses produced by spleen cells, restimulated with antigen (Ov-103 and Ov-RAL-2), from immunized and control mice. Antigen and cytokines are displayed on the vertical axis corresponding to each row, and experimental groups are listed on the horizontal axis corresponding to each column. Data shown are p values generated via linear regression modeling comparing rank-transformed cytokine concentrations between control and immune mice within adjuvant groups. The p values are FDR-adjusted and progress from white (p = 1.00) to blue (p = 0.50) to red (p = 0.05). (e) Antigen-specific IgG titers measured in the serum of control and immunized mice at the time of recovery. Data shown are mean titers, with individual mice represented as points. Error bars indicate standard errors of the mean. IgG subclass is indicated on the vertical axis, and antigen specificity is indicated above the two associated plots. (f) Ratios of IgG1 to IgG2ab from the serum of individual mice at the time of recovery. Data shown are mean titer ratios, with individual mice represented as points. Error bars indicate standard deviations. IgG subclass is indicated on the vertical axis, and antigen specificity is indicated above the associated plots. (b–e) * = p ≤ 0.05, indicating significant differences when comparing means to control animals within the same adjuvant group. (f) * = p ≤ 0.05, indicating significant differences when comparing means to Advax-CpG early and late groups.

Figure 3

Durable protection can be transferred with serum from mice immunized with Ov-FUS-1 and Advax-CpG, regardless of exposure to challenge infection. (a) Larval survival following passive transfer of serum from mice immunized with Ov-FUS-1 and either Advax-CpG (early n = 23, late n = 6), alum (early n = 10, late n = 10), or AlT4 (n = 11) as the adjuvant. Serum was collected at the time of recovery following the early and late challenge period. Control mice received transfer of naïve mouse serum (n = 21), which were combined for early and late time points. Diffusion chambers were recovered one week after implantation and remaining live larvae were counted to determine larval survival. Data are shown as mean percent larval survival, with individual mice represented as points. Error bars indicate standard deviations. Percent reduction in larval survival, when compared to control mice, is listed above the horizontal axis. (b) Total and differential counts of immune effector cells from diffusion chambers recovered from control and immunized mice were measured by microscopy. Data are shown as mean cell counts or percent of total live cells, with individual mice represented as points. Error bars represent standard errors of the mean. (a,b) * = p ≤ 0.05, indicating significant differences when comparing means to control animals within the same adjuvant group.

Figure 4

Ov-FUS-1 is immunogenic in NHPs, regardless of adjuvant formulation. (a) NHPs were immunized with Ov-FUS-1 and either Advax-CpG (n = 3), alum (n = 3), or AlT4 (n = 3) as the adjuvant. Control NHPs received PBS-only injections (n = 3). Injections were administered in a prime–boost–boost fashion 30 days apart. NHPs were challenged with diffusion chambers containing 25 O. volvulus L3, 32 and 33 days post-final booster, followed by recovery 7 days later. Serum was collected from NHPs every two weeks up to Day 99 and again at Day 210. (b) Larval survival measured in diffusion chambers recovered from NHPs. Data are shown as mean larval survival within individual NHPs. Larval survival within individual diffusion chambers (Three per NHP) is represented by points. Shaded columns indicate one NHP from each group with the greatest reduction in larval survival. (c) Total and differential counts of immune effector cells from diffusion chambers recovered from control and immunized NHPs measured by microscopy. Data are shown as mean cell counts or percent of total live cells, with samples from individual NHPs represented as points. Error bars represent standard errors of the mean. (d) Antigen-specific IgG titers measured in the serum of control and immunized NHPs throughout the trial. Data shown are mean titers with error bars indicating standard errors of the mean. IgG subclass and antigen specificity are indicated on the vertical axis. Time course indicating prime, boosters, challenge, and recovery is shown below the plots.

Figure 5

Durable protection can be transferred to mice with serum from NHPs immunized with Ov-FUS-1 and Advax-CpG. (a) Larval survival following passive transfer of pooled serum from NHPs immunized with Ov-FUS-1 and either Advax-CpG (n = 10), alum (n = 10), or AlT4 (n = 10) as the adjuvant into naïve mice. Control mice received transfer of PBS-treated NHP serum (n = 22). Serum was collected from NHPs 6 or 22 weeks post-final booster. Mice received serum at the time of challenge with O. volvulus L3 within diffusion chambers, which were recovered one week after implantation, and remaining larvae were counted to determine larval survival. Data are shown as mean percent larval survival, with individual mice represented as points. Error bars indicate standard deviations. Percent reduction in larval survival, when compared to control mice, is listed above the horizontal axis. (b) Total and differential counts of immune effector cells from diffusion chambers recovered from control and immunized mice measured by microscopy. Data are shown as mean cell counts or percent of total live cells, with individual mice represented as points. Error bars represent standard errors of the mean. * = p ≤ 0.05, indicating significant differences when comparing means to control animals.