A Comparison of Intramuscular and Subcutaneous Administration of LigA Subunit Vaccine Adjuvanted with Neutral Liposomal Formulation Containing Monophosphoryl Lipid A and QS21

Abstract

Leptospirosis vaccines with higher potency and reduced adverse effects are needed for human use. The carboxyl terminal domain of leptospiral immunoglobulin like protein A (LigAc) is currently the most promising candidate antigen for leptospirosis subunit vaccine. However, LigAc-based vaccines were unable to confer sterilizing immunity against Leptospira infection in animal models. Several factors including antigen properties, adjuvant, delivery system, and administration route need optimization to maximize vaccine efficacy. Our previous report demonstrated protective effects of the recombinant LigAc (rLigAc) formulated with liposome-based adjuvant, called LMQ (neutral liposome combined with monophosphoryl lipid A and Quillaja saponaria fraction 21) in hamsters. This study aimed to evaluate the impact of two commonly used administration routes, intramuscular (IM) and subcutaneous (SC), on immunogenicity and protective efficacy of rLigAc-LMQ administrated three times at 2-week interval. Two IM vaccinations triggered significantly higher levels of total anti-rLigAc IgG than two SC injections. However, comparable IgG titers and IgG2/IgG1 ratio was observed for both routes after the third immunization. The route of vaccine administration did not influence the survival rate (60%) and renal colonization against lethal Leptospira challenge. Importantly, the kidneys of IM group showed no pathological lesions while the SC group showed mild damage. In conclusion, IM vaccination with rLigAc-LMQ not only elicited faster antibody production but also protected from kidney damage following leptospiral infection better than SC immunization. However, both tested routes did not influence protective efficacy in terms of survival rate and the level of renal colonization.

Keywords: LMQ adjuvant; Leptospira; LigA subunit vaccine; intramuscular; leptospirosis; subcutaneous; vaccination route.

Figure 1

Analysis of the purified rLigAc by (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE) and Western blotting (WB). Lane M = PageRuler™ Unstained Protein Ladder (Thermo Scientific); lane 1 = the rLigAc stained with Coomassie Brilliant Blue R-250; lane 2 = the rLigAc detected by WB with anti-6× His tag antibody. (B) Circular dichroism (CD) analysis. The CD spectra were measured by a JASCO J-815-150S spectropolarimeter and analyzed with CDPro software. The CD spectrum is represented as an average of more than five spectra from 190 to 260 nm. (C) Binding of rLigAc to purified human FH. The results are shown as mean ± SD absorbance at 450 nm. Student t-test was used to compare the absorbance between coated proteins; *** represents p < 0.001. Heat-killed Leptospira was used as a positive control, and rLipL32 and bovine serum albumin (BSA) were used as negative control.

Figure 2

Antibody levels in the vaccinated hamsters. (A) Total LigAc-specific IgG titers measured 1 week after the second and the third immunizations. (B) Specific IgG1 and IgG2 titers measured at 1 week after the third immunization. The results are shown as mean ± SD. Mann–Whitney U test was used to compare antibody titers or IgG subclasses between groups; ** represents p < 0.01.

Figure 3

Kaplan–Meier plot of survival rates in vaccinated hamsters (n = 5 per group) following lethal challenge by virulent Leptospira. The hamsters were immunized with various vaccine formulations shown in Table 1. Each vaccinated hamster was challenged by 20× LD50 of low passage leptospires. The percent survival was calculated as the number of survivors/total challenged hamsters ×100. Statistical analysis of survival rates between control group and other vaccinated groups was performed by log-rank test.

Figure 4

Leptospiral burden in the kidneys of surviving hamsters after challenge. The leptospiral genome was detected by qPCR. The cycle threshold of each sample was compared with leptospiral DNA standard curve to calculate bacterial load, which is expressed as bacterial DNA per milligram of tissue. Mann–Whitney U test was used to compare bacterial number among vaccination groups; * represents p < 0.05.