Vaccine delivery by polymeric vehicles in the mouse reproductive tract induces sustained local and systemic immunity

Abstract

Design of easily administered vaccines to protect the female reproductive tract against STIs such as HIV, HPV and HSV is a major step in improving world health standards. However, the effect of immunization routes and regimens (prime/boost) on immune response is not well-understood. Here, we present a systematic study of vaccine delivery by different routes and prime/boosting regimens to produce a robust humoral immune response in the reproductive tract. A model antigen, ovalbumin (OVA), was delivered orally or intranasally via polymer particles, and intravaginally via polymer disks to female mice. Repeated prime/boost at a single site result in high OVA-specific antibody levels in the serum for mice immunized orally (IgA) and invaginally (IgA and IgG) after 3 months. Vaginal antibody titers were the highest for mice immunized by intravaginal routes. Vaginal boosting following intranasal or oral priming did not appear to offer similar advantages to those primed intravaginally. Systemic immunization with OVA in Freund's adjuvant produced robust serum IgG levels, but little serum IgA or antibodies in the vaginal washings. All immunization schemes produced a significant level of IgG in the intestinal mucosa, with the exception of nasal priming followed by intravaginal boost with slow-releasing disks. In contrast, only immunization by nasal priming and intravaginal boost with fast-releasing disks was able to achieve significantly high intestinal IgA titers.

Figure 1

PLGA particles encapsulating OVA. The size distribution of particles was largely small, with 77 % of the population measured < 5 μm in diameter (A). Particles appeared spherical with smooth surface morphology (inset, scale bar = 10 μm). Release of OVA from PLGA particles measured as fraction of theoretical loading showed a bi-phasic release profile with a rapid burst during the first 3 days and a more gradual release over the period of 4 weeks (B). The dashed line indicates the OVA loading efficiency was 82 ± 6 % of theoretical maximum, which corresponded to 3.7 μg OVA/mg PLGA.

Figure 2

EVAc disks encapsulating OVA at different OVA:codispersant ratios. Two formulations of OVA-releasing disks, with 50 % and 30 % (w/w) OVA:codispersant in EVA_c polymer, released proteins at distinct rates: fast-releasing (V_f) disks release ~2× greater OVA during the initial burst (3 days) than slow-releasing disks (V_s). The cumulative OVA released over the period of 2 weeks for both formulations were similar, at 400 μg OVA/disk.

Figure 3

Antibody production in response to immunization via similar prime/boost sites. OVA-specific IgG (A, C) and IgA (B, D) as measured in serum and vaginal washes in mice with primary and secondary immunizations by identical routes: oral (O/O), nasal (N/N), intravaginal (V/V), oral soluble OVA (LIQ/LIQ), and subcutaneous with Freund’s adjuvant (FCA/FIA). The nomenclature scheme was: primary/secondary treatment, as summarized in Table 1. Arrows indicate the times of immunization at week 0 and 4, and filled circles denote statistically significant data from reference sample (LIQ/LIQ) as analyzed by Tukey’s multiple-comparison procedure, confidence level = 0.05, n=3.

Figure 4

Antibody production in response to immunization via different priming sites followed by vaginal boosting. Similar OVA-specific IgG (A, C) and IgA (B, D) levels from serum and vaginal washes of mice immunized by oral, intranasal, or intravaginal routes but received secondary immunization via fast (Vf) or slow (Vs) OVA-releasing disks. Arrows denote the times of primary and secondary immunization at week 0 and 4, and filled circle indicate statistically significant data from reference sample (LIQ/LIQ) as analyzed by Tukey’s multiple-comparison procedure, confidence level = 0.05, n=3.

Figure 5

OVA-specific intestinal IgG (A) and IgA (B) levels for mice immunized with various treatment regimes at week 12. Data analysis is based on Tukey’s multiple-comparison procedure with confidence level = 0.05. IgG levels for all treatment groups, except for N/V_s, indicated with asterisks, are significantly higher than the reference (LIQ).

Figure 6

Serum IgG titers of mice immunized intravaginally with saline (PBS), EVAc disk and saline (EVAc + PBS), EVAc disk and OVA (EVAc + OVA), soluble OVA (OVA) compared to subcutaneously immunized mice in Freund’s adjuvant (FCA/FIA). While immunization with OVA resulted in elevated serum IgG titers over untreated samples, there was no difference between those with and without intravaginal EVAc disk.