Nanolipoprotein particle (NLP) vaccine confers protection against Yersinia pestis aerosol challenge in a BALB/c mouse model

Abstract

Introduction: Yersinia pestis is the etiological agent of plague, a disease that remains a concern as demonstrated by recent outbreaks in Madagascar. Infection with Y. pestis results in a rapidly progressing illness that can only be successfully treated with antibiotics given shortly after symptom onset. Live attenuated or whole cell inactivated vaccines confer protection against bubonic plague, but pneumonic plague has been more difficult to prevent. Novel effective subunit vaccine formulations may circumvent some of these shortfalls. Here, we compare the immunogenicity generated by an advanced subunit vaccine (F1V fusion protein) and a nanolipoprotein particle (NLP)-based vaccine.

Methods: The NLP, a high-density lipoprotein mimetic, provides a nanoscale delivery platform for recombinant Y. pestis antigens LcrV (V) and F1. BALB/c mice were immunized via subcutaneous injection twice, three or four weeks apart. Four weeks later, splenocytes and sera were collected for immune profiling, and mice were challenged with aerosolized Y. pestis CO92.

Results: Both formulations induced a strong IgG response against the F1 and V proteins, along with a robust memory B cell response and a balanced cell-mediated immune response as evidenced by both Th1- and Th2-related cytokines. The NLP-based vaccine induced a stronger cytokine response against F1, V, and F1V proteins relative to the F1V vaccine. As with F1V, the inclusion of Alhydrogel (Alu) in NLP vaccine formulations was critical for enhanced immunogenicity and protective efficacy. Mice that received two doses of F1:V:NLP + Alu and CpG were completely protected from a challenge with approximately eight median lethal doses of aerosolized Y. pestis CO92 and this protection confirmed the well-documented synergy between the F1 and V antigens in context of pneumonic plague. The NLPs have defined regions of polarity that facilitates the incorporation of a wide range of adjuvants and antigens with distinct physicochemical properties and are an excellent candidate platform for the development of multi-antigen vaccines.

Keywords: F1; LcrV; Yersinia pestis; mice; nanolipoprotein particle; plague; pneumonic; vaccine.

Figure 1

Overview of the immunization and challenge strategy for direct comparison of benchmark F1V vaccine and various NLP vaccine candidates that were challenged with Y. pestis CO92. The numbers with the degree sign (°) denote vaccine prime and boost.

Figure 2

Development and characterization of NLP-based vaccine. (A) Schematic of NLP assembly and conjugation of His-tagged Y. pestis antigens. (B, C) Representative SEC chromatograms of F1:NLP (B) and V:NLP (C) conjugates, correlating NLP alone (black traces), antigen alone (green traces), and antigen:NLP conjugates (blue traces). Relative retention time regions corresponding to void volume (void), NLP and antigen:NLP (NLP), and unconjugated antigen (F1 or V) are indicated. (D) DLS histograms of NLP alone, F1:NLP and V:NLP formulations (number mean, triplicate measurements). The red, green, and blue histograms represent the replicate measurements for each sample.

Figure 3

Total IgG response against (A) F1V, (B) F1, and (C) V, antigens at 27 days post-vaccination (pre-challenge). The F1V fusion benchmark vaccine is green. The experimental vaccine formulations without Alu are blue and those that include Alu are red. n = 4 animal sera per group. For ELISA, pairwise treatment groups were compared by negative binomial generalized linear mixed model. Complete results from statisitical analyses are described in Supplementary Table 3 . * < 0.05, ** < 0.01, and *** < 0.001.

Figure 4

Serum samples from 28 days post-vaccination (pre-challenge) or 3 days post-infection (dpi) were measured for total serum IgG against F1, V, or TS CO92 antigens using an endpoint ELISA assay on a series of 2-fold dilutions of serum, performed with an additional incubation step with either wash buffer, or 6 M urea to disrupt weak antibody-antigen binding. The avidity index was analyzed by comparing (OD₄₅₀ – OD₅₇₀) values at the lowest dilution that had an (OD₄₅₀ – OD₅₇₀) below 1.5 in the no urea condition. Avidity index was calculated as (OD with urea/OD with wash buffer) x 100%.

Figure 5

Antibody IgG1 and IgG2a subclass response against F1 (A, C) and V (B, D), antigens at 27 days post-vaccination (pre-challenge). The F1V fusion benchmark vaccine is green. The experimental vaccine formulations without Alu are blue and those that include Alu are red. n = 4 animal sera per group. For ELISA, pairwise treatment groups were compared by negative binomial generalized linear mixed model. Complete results from statisitical analyses are described in Supplementary Table 5 . * < 0.05, ** < 0.01, and *** < 0.001.

Figure 6

IgG1-secreting B cells detected 27 days post-vaccination after stimulations with (A) F1V, (B) F1 and (C) V antigens. The F1V fusion benchmark vaccine is green. The experimental vaccine formulations without Alu are blue and those that include Alu are red. For ELISpot results, data were log10 transformed prior to analysis and pairwise treatment groups were compared by linear mixed effects model. n = 4 mice/group. * < 0.05, ** < 0.01, and *** < 0.001.

Figure 7

T cell populations in spleens of mice 27 days after the second vaccine dose. Splenocytes were stained for flow cytometry, fixed in 2% formaldehyde, and run on a FACSCanto II. (A) T cells were defined as viable, CD19-, CD3+ lymphocytes, then gated into CD8+ or CD4+ T cell populations, and defined as either positive or negative for CD62L. (B, C) The CD62L+ percentage of CD8 (B) and CD4 (C) T cells were compared across vaccine groups. The F1V fusion benchmark vaccine is green. The experimental vaccine formulations without Alu are blue and those that include Alu are red. Individual points represent one animal (n = 4 mice/group), and the columns and error bars represent mean ± SEM. Negative binomial regression was used for the flow cytometry data analysis. * < 0.05, ** < 0.01, and *** < 0.001.

Figure 8

Dendritic cell populations in spleens of mice 27 days after the second vaccine dose. Splenocytes were stained for flow cytometry, fixed in 2% formaldehyde, and run on a FACSCanto II. (A) Dendritic cells were defined as viable, CD19-, CD3-, CD11c+ cells, and then defined as CD11b+ (direct presenting) or CD8+ (cross-presenting). CD11b+ DCs were analyzed to measure the number that upregulated costimulatory molecules CD40 and CD80. (B) The CD40/CD80+ double-positive percentage of CD11b+ DCs was compared across vaccine groups. The F1V fusion benchmark vaccine is green. The experimental vaccine formulations without Alu are blue and those that include Alu are red. Individual points represent one animal (n = 4 mice/group), and the columns and error bars represent mean and SEM. Negative binomial regression was used for the flow cytometry data analysis. * < 0.05.

Figure 9

Survival curves of vaccinated and control BALB/c mice challenged with Y. pestis CO92. Mice (n = 9 mice/group) were exposed to 5.25 x 10⁵ CFU (~8 LD₅₀) of Y. pestis CO92 via aerosol exposures. The F1V fusion benchmark vaccine is green. The experimental vaccine formulations without Alu are blue and those that include Alu are red. The mouse survival rates at selected time points were compared by Fisher exact test and the time to death or euthanasia (TTD) were analyzed by Log-rank test for the comparison against PBS group.

Figure 10

The recovery of bacteria as determined by CFU counts from (A) lung, (B) spleen and (C) blood of BALB/c mice three days post-challenge with Y. pestis CO92. The left axis represents CFU/g (Lung and Spleen) or CFU/mL (Blood). The F1V fusion benchmark vaccine is green. The experimental vaccine formulations without Alu are blue and those that include Alu are red. The individual points represent one animal (n = 3 mice/group), and the baseline points indicate the remaining survivors with no detectable CFU. The horizontal lines are the geometric mean. The limits of detection were approximately 100 CFU/mL (blood) and 5 CFU/g (organs).