Recombinant YopE and LcrV vaccine candidates protect mice against plague and yersiniosis

Abstract

No licensed vaccine exists for the lethal plague and yersiniosis. Therefore, a combination of recombinant YopE and LcrV antigens of Yersinia pestis was evaluated for its vaccine potential in a mouse model. YopE and LcrV in formulation with alum imparted a robust humoral immune response, with isotyping profiles leaning towards the IgG1 and IgG2b subclasses. It was also observed that a significantly enhanced expression of IFN-γ, TNF-α, IL-6, IL-2, and IL-1β from the splenic cells of vaccinated mice, as well as YopE and LcrV-explicit IFN-γ eliciting T-cells. The cocktail of YopE + LcrV formulation conferred complete protection against 100 LD₅₀Y. pestis infection, while individually, LcrV and YopE provided 80 % and 60 % protection, respectively. Similarly, the YopE + LcrV vaccinated animal group had significantly lower colony forming unit (CFU) counts in the spleen and blood compared to the groups administered with YopE or LcrV alone when challenged with Yersinia pseudotuberculosis and Yersinia enterocolitica. Histopathologic evidence reinforces these results, indicating the YopE + LcrV formulation provided superior protection against acute lung injury as early as day 3 post-challenge. In conclusion, the alum-adjuvanted YopE + LcrV is a promising vaccine formulation, eliciting a robust antibody response including a milieu of pro-inflammatory cytokines and T-cell effector functions that contribute to the protective immunity against Yersinia infections. YopE and LcrV, conserved across all three human-pathogenic Yersinia species, provide cross-protection. Therefore, our current vaccine (YopE + LcrV) targets all three pathogens: Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. However, the efficacy should be tested in other higher mammalian models.

Keywords: LcrV; Vaccine; Yersinia enterocolitica; Yersinia pestis; Yersinia pseudotuberculosis; YopE.

Fig. 1

Antibody responses after YopE, LcrV, and YopE + LcrV immunization (A–J). SDS-PAGE profile of purified recombinant YopE and LcrV proteins (A), Uncropped version of gel image for figure [1A] is available in supplementary material. Schematic description of vaccine administration and bleeding schedule for BALB/c mice (B). Serum samples of days 14 and 21 administered boosters were collected after 7 days following each booster, and anti-LcrV and anti-YopE IgG were assessed for mice immunized with individually formulated vaccine candidates (C) along with mice administered with the combination group (D). Mann-Whitney U test was conducted for the comparison of two groups. Serum IgG subclasses following second booster were analyzed including IgG1; IgG2a; IgG2b; and IgG3. The group comparisons for all four IgG isotypes (upper panel) and group comparisons for each isotype induced by different combinations (lower panel) (E). Two-way ANOVA test was conducted to interpret the data, and for multiple comparisons, Tukey's test was conducted. n = 10/group. Mean ± SEM is shown. P < 0.05 was considered statistically significant.

Fig. 2

Cell-mediated immune responses (A–K). Schematic description of the schedule for vaccine administration, bleeding, and spleen harvesting from BALB/c mice (A). Splenocytes were collected one week after the final immunization and stimulated with the corresponding vaccine antigens. After 48 h, supernatants were removed, and cytokine quantities were quantified using ELISA. Concentrations of IFN-γ (B); TNF-α (C); IL-2 (D); IL-1β (E); and IL-6 (F) cytokines were reported as pg/ml. Simultaneously, the proliferative response of the splenocyte was assessed by re-incubating it with Alamar blue dye for 16 h, and optical density was observed at 570 nm and a reference wavelength of 600 nm (G). Bar graph representing the % incidence of CD4⁺ IFN-γ⁺ T-cells in control, YopE, LcrV, and the combination of YopE + LcrV group (H). Bar graph representing the % frequency of CD8⁺ IFN-γ⁺ T cells in control, YopE, LcrV, and the combination of YopE + LcrV group (I). Brown-Forsythe and Welch ANOVA tests were conducted on log-transformed data, followed by Dunnett T3 tests for multiple comparisons. n = 6/group. Mean ± SEM is shown. P < 0.05 was considered statistically significant.

Fig. 3

Protection studies against Y. pestis challenge (A–C). Schematic description of the naive female BALB/c mice vaccine administration, bleeding, and challenge study schedule (A). Kaplan-Meier analysis and the magnitude of protection imparted by alum-adjuvanted YopE, LcrV, and the combination of YopE + LcrV. The percentage of BALB/c mice that survived was plotted against the number of days animals survived post-Y. pestis (LD₅₀) challenge. To ascertain survivability, animals were observed for one-month post-bacterial exposure. P values (<0.0001) represent comparisons to the control group; log-rank (Mantel-Cox) test. P < 0.05 was considered statistically significant (B). Y. pestis was isolated from the carcass following the challenge and was grown on a blood agar plate (C).

Fig. 4

Protection studies against Y. enterocolitica and Y. pseudotuberculosis (A–I). Schematic description of vaccine administration, bleeding, and challenge schedule (A). Graphical representation of CFU count at days 1–3 post-Y. enterocolitica challenge from the blood of BALB/c mice (B). Graphical representation of the unit of protection imparted by vaccine candidates on day 2 in the blood (C). Graphical representation of CFU count at days 1–5 post-Y. enterocolitica challenge from the spleen of BALB/c mice (D). Graphical representation of the unit of protection imparted by vaccine candidates on day 3 in the spleen (E). Graphical representation of CFU count at days 1–3 post-Y. pseudotuberculosis challenge from the blood of BALB/c mice (F). Graphical representation of the unit of protection imparted by vaccine candidates on day 2 in the blood (G). Graphical representation of CFU count at days 1–5 post-Y. pseudotuberculosis challenge from the spleen of BALB/c mice (H). Graphical representation of the unit of protection imparted by vaccine candidates on day 3 in the spleen (I). n = 10/group. Mean ± SEM. One-way ANOVA test was conducted on log-transformed data, for multiple comparisons, Tukey's test was conducted. P < 0.05 was considered statistically significant.

Fig. 5

Histopathological analysis of lungs of Y. pestis infected mice (A–G). Schematic description of schedule for vaccine administration, Y. pestis, and lung harvesting from BALB/c mice (A). Photomicrographs of naive and Y. pestis pestis-challenged lung injuries, day 3 post-challenge after immunization with YopE, LcrV, and YopE + LcrV vaccine candidates. Tissue section of all inoculated groups at day 3 p.i. Naive mice (B), Infected mice (C), YopE-treated (D), LcrV-treated (E), YopE + LcrV-treated mice (F). Blinded histopathological evaluation of lung damage using the American Thoracic Society (ATS) acute lung injury (ALI) scoring systems was done. n = 3 per group. Mean ± SEM. Data were analyzed and interpreted using a one-way ANOVA, for multiple comparisons, Tukey's test was conducted (G). Light microscopy was used to analyze tissue sections from three mice in each group stained with hematoxylin and eosin. 20× magnification; 50 μm scale bar. In the images (a) represents neutrophil's count (alveolar space); (b) represents neutrophil's count (interstitial space); (c) represents formation of hyaline membrane; (d) represents air spaces consist of proteinaceous debris and (e) represents the thickness of the alveolar septae. P < 0.05 was considered statistically significant.

Fig. 6

YopE+LcrV Vaccine Induced protection against plague and Yersiniosis