Single-dose combination nanovaccine induces both rapid and long-lived protection against pneumonic plague

Abstract

Yersinia pestis, the causative agent of pneumonic plague, induces a highly lethal infection if left untreated. Currently, there is no FDA-approved vaccine against this pathogen; however, USAMRIID has developed a recombinant fusion protein, F1-V, that has been shown to induce protection against pneumonic plague. Many F1-V-based vaccine formulations require prime-boost immunization to achieve protective immunity, and there are limited reports of rapid induction of protective immunity (≤ 14 days post-immunization (DPI)). The STimulator of INterferon Genes agonists cyclic dinucleotides (CDNs) have been shown to be promising vaccine adjuvants. Polyanhydride nanoparticle-based vaccines (i.e., nanovaccines) have also shown to enhance immune responses due to their dual functionality as adjuvants and delivery vehicles. In this work, a combination nanovaccine was designed that comprised F1-V-loaded nanoparticles combined with the CDN, dithio-R_P,R_P-cyclic di-guanosine monophosphate, to induce rapid and long-lived protective immunity against pneumonic plague. All mice immunized with a single dose combination nanovaccine were protected from Y. pestis lethal challenge within 14 DPI and demonstrated enhanced protection over F1-V adjuvanted with CDNs alone at challenge doses ≥7000 CFU Y. pestis CO92. In addition, 75% of mice receiving the single dose of the combination nanovaccine were protected from challenge at 182 DPI, while maintaining high levels of antigen-specific serum IgG. ELISPOT analysis of vaccinated animals at 218 DPI revealed F1-V-specific long-lived plasma cells in bone marrow in mice vaccinated with CDN adjuvanted F1-V or the combination nanovaccine. Microarray analysis of serum from these vaccinated mice revealed the presence of serum antibody that bound to a broad range of F1 and V linear epitopes. These results demonstrate that combining the adjuvanticity of CDNs with a nanovaccine delivery system enables induction of both rapid and long-lived protective immunity against Y. pestis. STATEMENT OF SIGNIFICANCE: • Yersinia pestis, the causative agent of pneumonic plague, induces a highly lethal infection if left untreated. Currently, there is no FDA-approved vaccine against this biodefense pathogen. • We designed a combination nanovaccine comprising of F1-V antigen-loaded polyanhydride nanoparticles and a cyclic dinucleotide adjuvant to induce both rapid and long-lived protective immunity against pneumonic plague. • Animals immunized with the combination nanovaccine maintained high levels of antigen-specific serum IgG and long-lived plasma cells in bone marrow and the serum antibody showed a high affinity for a broad range of F1 and V linear epitopes. • The combination nanovaccine is a promising next-generation vaccine platform against weaponized Y. pestis based on its ability to induce both rapid and long-lived protective immunity.

Keywords: Combination; Cyclic dinucleotide; Nanovaccine; Pneumonic plague; Polyanhydride.

Fig. 1.. Characterization of F1-V Nanovaccine.

A) Representative scanning electron photomicrograph of F1-V nanovaccine (scale bar = 3 μm). B) Nanoparticle size distribution of the F1-V nanovaccine (228 ± 78 nm), determined via ImageJ analysis. C) Cumulative in vitro F1-V release profile from the nanovaccine. Nanoparticles were suspended in PBS (pH 7.4) and released protein was quantified via a microBCA assay.

Fig. 2.. Combination Nanovaccine provides rapid protective immunity against lethal Y. pestis challenge.

Groups of C57BL/6NCrl mice (n = 12–16 per group) were immunized subcutaneously with the following groups: F1-V + CDNs (CDN Vaccine), F1-V encapsulated into nanoparticles (Nanovaccine), F1-V encapsulated into nanoparticles + CDNs (Combination Nanovaccine) or saline. (A,C,E) Serum was collected at 13 DPI and evaluated via ELISA for total anti-F1-V IgG antibody titers. The dashed line represents anti-F1-V IgG (H + L) antibody titers from saline-treated mice as a negative control. * p ≤ 0.0 0 01 compared to Nanovaccine and saline. There was no significant difference in anti-F1-V IgG serum responses observed within each vaccination group between experiments. Mice were challenged at 14 DPI with (B) 5700 CFU, (D) 70 0 0 CFU, or (F) 10,700 CFU Y. pestis CO92 and survival was monitored for two weeks post-challenge. * p ≤ 0.0 0 01 compared to saline-treated mice. + p ≤ 0.0 0 01 compared to Nanovaccine-immunized mice. # p ≤ 0.05 compared to CDN Vaccine-immunized mice.

Fig. 3.. Combination Nanovaccine provides long-lived protective immunity against lethal Y. pestis challenge.

Groups of C57BL/6NCrl mice (n = 12–16 per group) were immunized subcutaneously with the following treatments: F1-V + CDNs (CDN Vaccine), F1-V encapsulated into nanoparticles (Nanovaccine), F1-V encapsulated into nanoparticles + CDNs (Combination Nanovaccine) or saline. (A) Serum samples were collected at 14, 36, 79, 121, and 178 DPI and analyzed for total anti-F1-V IgG (H + L) antibodies via ELISA. The dashed line represents the background anti-F1-V IgG (H + L) antibody response from naive mice. # p ≤ 0.05 compared to CDN Vaccine. * p ≤ 0.0 0 01 compared to Nanovaccine-immunized and saline treated mice. (B) Mice were challenged at 182 DPI with 6500 CFU Y. pestis CO92 and survival was monitored for two weeks post-challenge. # p ≤ 0.01 compared to Nanovaccine-immunized mice. * p ≤ 0.05 compared to Saline-treated mice.

Fig. 4.. Combination Nanovaccine and CDN Vaccine provide broad antibody responses to potentially protective linear epitopes.

Serum samples collected from C57BL/6NCrl mice (n = 12–16 per group) immunized with either saline, CDN Nanovaccine, Nanovaccine, or Combination Nanovaccine at 14, 79, and 178 DPI was analyzed for total anti-F1-V IgG antibodies against twenty-seven 14- to 17-mer linear peptides (11 amino acid overlaps) from the F1 antigen and fifty-three 15- to 17-mer linear peptides (11 or 12 amino acid overlaps) from the V antigen. The peptides were covalently bound to microarray slides as described in Materials and Methods. Each row corresponds to a specific peptide, the top row representing peptide F1 and the proceeding downward rows corresponding to each following linear peptide incrementally through peptide V53. Each column represents responses from a single mouse. The mean fluorescence intensity of serum responses to each peptide is represented by a range of color from white (no response) to purple (maximum response). The full-length F1-V fusion protein was used as a positive control, and Bacillus anthracis protective antigen (PA) and chicken egg ovalbumin (OVA) were used as negative controls. Black arrows indicate significance (p ≤ 0.05) of Combination Nanovaccine and CDN Vaccine serum compared to naïve serum at all time points evaluated. Blue arrows indicate significance (p < –0.05) of Combination Nanovaccine and CDN Vaccine serum compared to naïve serum at 179 DPI. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5.. Long-lived antibody responses to F1-V originate from long-lived plasma cells in the bone marrow.

Bone marrow and splenic lymphocytes were harvested at 213–218 DPI from groups (n = 3–4/group) of C57BL/6NCrl mice immunized with CDN Vaccine, Nanovaccine, Combination Nanovaccine regimens, or treated with saline, and were analyzed via ELISPOT for the number of F1-V-specific antibody secreting cells. Data represent the number of F1-V-specific antibody secreting cells per 50 0,0 0 0 cells in either A) bone marrow or B) spleen from individual mice from one experiment. The dashed line represents the number of F1-V-specific ASCs per 50 0,0 0 0 cells determined from saline-treated mice as a negative control. * p ≤ 0.02 compared to saline-treated mice. # p ≤ 0.03 compared to Nanovaccine-immunized mice.