Immunogenicity of the Lyme disease antigen OspA, particleized by cobalt porphyrin-phospholipid liposomes

Abstract

Outer surface protein A (OspA) is a Borrelia lipoprotein and an established Lyme disease vaccine target. Admixing non-lipidated, recombinant B. burgdorferi OspA with liposomes containing cobalt porphyrin-phospholipid (CoPoP) resulted in rapid, particulate surface display of the conformationally intact antigen. Particleization was serum-stable and led to enhanced antigen uptake in murine macrophages in vitro. Mouse immunization using CoPoP liposomes that also contained a synthetic monophosphoryl lipid A (PHAD) elicited a Th1-biased OspA antibody response with higher IgG production compared to other vaccine adjuvants. Antibodies were reactive with intact B. burgdorferi spirochetes and Borrelia lysates, and induced complement-mediated borreliacidal activity in vitro. One year after initial immunization, mice maintained high levels of circulating borreliacidal antibodies capable of blocking B. burgdorferi transmission from infected ticks to human blood in a feeding chamber.

Keywords: Adjuvant; Liposomes; Lyme disease; OspA; Particle vaccine.

Figure 1.. Spontaneous binding of his-tagged OspA to CoPoP/PHAD liposomes.

A) Effect of varying mass ratios of OspA:CoPoP/PHAD liposomes evaluated by native PAGE. The visible bands that migrated in gel represent unbound protein. B) Kinetics of OspA binding to CoPoP/PHAD liposomes incubated at 1:4 mass ratio at room temperature. C) Liposome binding of his-tagged OspA or non-his-tagged lysozyme measured by microBCA assay of the supernatant following high-speed centrifugation. D) Hydrodynamic diameter and polydispersity index of liposomes with or without OspA incubation measured by dynamic light scattering. Error bars represent standard deviations for n=3 measurements. F) Negative-stained electron micrographs of CoPoP/PHAD liposomes with or without bound OspA.

Figure 2.. Serum stability, epitope availability and cellular uptake of particleized OspA.

A) Stability of particleized antigen association with liposomes in 20% (v/v) human serum based on fluorescence quenching of dye-labeled OspA. The arrow shows restoration of OspA fluorescence with detergent and protease treatment. B) Immunoprecipitation of OspA-bound liposomes by OspA-specific monoclonal antibody LA-2. An irrelevant antibody specific for a malaria antigen served as a negative control. CoPoP liposomes included additional PoP for analysis, since CoPoP has weak fluorescence. Uptake of fluorescently labeled OspA (C) or liposomes themselves (D) in RAW 264.7 murine macrophage cells following 2 hr incubation with indicated samples at 37 °C. Cytochalasin B, a phagocytosis inhibitor, was added to medium 1 hr prior to incubation. Error bars represent standard deviations for n=3 experiments.

Figure 3.. Immunogenicity of OspA adjuvanted with CoPoP liposomes.

100 ng OspA, admixed with indicated adjuvants, was injected intramuscularly on day 0 and day 21 and serum was collection on day 42. A) Anti-OspA IgG titers induced by CoPoP/PHAD liposomes compared to other commercial adjuvants. Horizontal lines represent geometric mean. Asterisk shows that the anti-OspA IgG titer was significantly higher in the CoPoP/PHAD adjuvant compared to all others (one-way ANOVA followed by post-hoc Tukey’s test; P<0.05). B) An indirect immunofluorescence assay of B. burgdorferi B31 using goat anti-Mouse IgG DyLight-488 secondary antibody conjugate. C) Immunoblot assay using whole cell lysates of different Borrelia species. CoPoP/PHAD post-immune mouse serum was used. The molecular weight size, in kDa is indicated.

Figure 4.. Th1-biased immune response induced by OspA with CoPoP/PHAD liposomes.

A) IgG isotype profiling for post-immune sera (day 42) using ELISA. Horizontal lines show geometric mean. B) Splenocyte stimulation study to detect interferon-gamma and interleukin-4 secretion after 72-hr stimulation with OspA. Splenocytes were isolated from murine spleen collected on day 42 post-immunization. Error bars represent standard deviations from n=3 triplicate stimulation experiments.

Figure 5.. Borreliacidal antibodies induced by murine immunization with OspA admixed with CoPoP/PHAD liposomes.

(A) Serum bactericidal antibody assay performed using guinea pig complement incubated with varying concentrations of mouse IgG collected on day 42 after priming on day 0 and boosting on day 21 with 100 ng OspA. Survival percentage was derived from normalization of the number of spirochetes after overnight serum treatment to that immediately after incubation. Surviving B. burgdorferi B31-A3 were counted using dark-field microscopy. (B) Average 50% borreliacidal activity (serum dilution rate that effectively eliminated 50% of the bacteria) from three different mice sera. Error bars represent standard error of the mean. “NI”; no inhibition. Statistical significance (P < 0.05, indicated by asterisk) of differences between bactericidal titers is assessed by Kruskal-Wallis test with Dunn’s post-hoc analysis.

Figure 6.. Longevity of anti-OspA response following immunization.

CD-1 mice were immunized on day 0 and 21 with 100 ng OspA with CoPoP/PHAD liposomes A) Anti-OspA IgG titer following immunization. Data points and error bars represent geometric mean and 95% confidence interval. Arrows show days of immunization. B) Serum bactericidal antibody assay on week 52 sera, performed using guinea pig complement incubated with varying post-immune serum dilutions. C) Average 50% borreliacidal activity from three different mice sera. Error bars represent standard error of the mean. No statistical difference of the 50% borreliacidal titer in the serum collected at 6 week or one year post initial immunization. “NI”; no inhibition.

Figure 7.. Assessment of 52 week post-immune mouse sera using infected ticks in a human blood feeding chamber.

A) I. scapularis nymphal ticks carrying B. burgdorferi (Bb) strain B31-A3 were placed in feeding chambers with human blood containing diluted mouse serum (1:424 dilution) from mice immunized 52 weeks earlier with OspA and CoPoP/PHAD liposomes or normal mouse sera. After 5 days, nymphs were pulled from the membrane and spirochete burden was determined in the ticks (B) or in the human blood (C) using qPCR. Geometric mean ± geometric S.D. is shown. For ticks, Bb burden was determined in individual ticks (11 and 14 ticks for dilute normal or post-immune mouse sera, respectively). For human blood, Bb burden was determined on day 5 of feeding, in three feeding chambers. Asterisk indicates a statistically significant difference in spirochete burden (P<0.05, unpaired t-test).