Robust Th1 cellular and humoral responses generated by the Yersinia pestis rF1-V subunit vaccine formulated to contain an agonist of the CD137 pathway do not translate into increased protection against pneumonic plague

Abstract

Yersinia pestis is the causative agent of plague and is a re-emerging pathogen that also has the potential as a biological weapon, necessitating the development of a preventive vaccine. Despite intense efforts for the last several decades, there is currently not a vaccine approved by the FDA. The rF1-V vaccine adjuvanted with Alhydrogel is a lead candidate subunit vaccine for plague and generates a strong Th2-mediate humoral response with a modest Th1 cellular response. As immune protection against Y. pestis requires both humoral and Th1 cellular responses, modifying the rF1-V subunit vaccine formulation to include a robust inducer of Th1 responses may improve efficacy. Thus, we reformulated the subunit vaccine to include SA-4-1BBL, an agonist of the CD137 costimulatory pathway and a potent inducer of Th1 response, and assessed its protective efficacy against pneumonic plague. We herein show for the first time a sex bias in the prophylactic efficacy of the Alhydrogel adjuvanted rF1-V vaccine, with female mice showing better protection against pneumonic plague than male. The sex bias for protection was irrespective of the generation of comparable levels of rF1-V-specific antibody titers and Th1 cellular responses in both sexes. The subunit vaccine reformulated with SA-4-1BBL generated robust Th1 cellular and humoral responses. A prime-boost vaccination scheme involving prime with rF1-V + Alhydrogel and boost with the rF1-V + SA-4-1BBL provided protection in male mice against pneumonic plague. In marked contrast, prime and boost with rF1-V reformulated with both adjuvants resulted in the loss of protection against pneumonic plague, despite generating high levels of humoral and Th1 cellular responses. While unexpected, these findings demonstrate the complexity of immune mechanisms required for protection. Elucidating mechanisms responsible for these differences in protection will help to guide the development of better prophylactic subunit vaccines effective against pneumonic plague.

Keywords: Plague; SA-4-1BBL; Subunit vaccine; Th1-mediated immunity; Yersinia pestis.

Figure 1.. Single dose of rF1-V + Alhydrogel vaccine better protects female mice against pneumonic plague.

Male and female mice were vaccinated with rF1-V + Alhydrogel and challenged 35 days later intranasally with 10 LD₅₀ of virulent Y. pestis CO92 Lux_PcysZK. Survival was monitored for 14 days. (A) Survival of C57BL/6 mice (compiled data from 4 independent experiments; n=18 and 14 for male and female mice, respectively). (B) Survival of BALB/c mice (data from one experiment; n=5). (C) Anti- rF1-V IgG titers from subset of mice in A and B 7 d prior to infection. (D) Ratio of IgG_2c to IgG₁ antibody subtypes from C57BL/6 mice. Absolute numbers (E) CD4⁺ T cells and (F) CD8⁺ T cells secreting IFN-γ from subset of C57BL/6 mice 35 d post vaccination. Survival data were analyzed by Kaplan-Meier and log-rank test and antibody and T cell phenotypes were analyzed by one-way ANOVA with Tukey post hoc analysis or student’s T-test.

Figure 2.. Addition of SA-4-1BBL improves the Th1 cellular response of the rF1-V + Alhydrogel vaccine.

C57BL/6 male mice were vaccinated with rF1-V or rF1-V + Alhydrogel with increasing concentrations of SA-4-1BBL. Absolute numbers of (A) CD4⁺ T cells and (B) CD8⁺ T cells secreting IFN-γ were determined 35 d post vaccination. (C) Anti-rF1-V IgG titers of vaccinated mice were determined 28 post-vaccination. Data was analyzed by one-way ANOVA with Dunnett’s post hoc analysis compared to 0 µg of SA-4-1BBL: ****= p<0.0001; *** = p<0.001; ** = p<0.01; * = p<0.05.

Figure 3.. Improved Th1 immune response in SA-4-1BBL+ rF1-V + Alhydrogel does not increase protection against pneumonic plague.

C57BL/6 male mice (data from one representative experiment; n=4) were vaccinated with rF1-V antigen alone or with Alhydrogel (ALHD), SA-4-1BBL, or Alhydrogel and SA-4-1BBL (ALHD + SA-4-1BBL) and challenged 35 days later with 10 LD₅₀ of virulent Y. pestis CO92 Lux_PcysZK. (A) Survival was monitored for 14 d. (B) Anti- rF1-V IgG titers from animals in A were determined 7 d prior to Y. pestis challenge. (C) Bacterial proliferation in the lungs as a function of bacterial luminescence of infected mice was monitored by real time live animal optical imagining every 12 h from 24 to 96 h post-infection. Animals that succumbed to infection are represented by x. Survival data were analyzed by Kaplan-Meier and log-rank test and antibody titers by one-way ANOVA with Tukey post hoc analysis: **** = p<0.0001; * = p<0.05; ns = not significant.

Figure 4:. Boost with vaccine containing Alhydrogel or SA-4-1BBL improves survival of male mice against pneumonic plague.

Male C57BL/6 mice were primed with rF1-V + Alhydrogel alone (AL) or rF1-V + Alhydrogel + SA-4-1BBL (AL+BBL). 21 d later, mice were boosted with rF1-V + Alhydrogel (AL; n=12), rF1-V + SA-4-1BBL (BBL; n=8), or rF1-V + Alhydrogel + SA-4-1-BBL (AL+BBL; n=8). Absolute numbers of (A and B) CD4⁺ T cells and (C and D) CD8⁺ T cells secreting IFN-γ were determined 60 d post prime vaccination from a subset of animals. A and C are representative flow cytometer data from one animal in each group. (E) Mice were challenged intranasally 60 d post prime vaccination with 10 LD₅₀ of virulent Y. pestis CO92 Lux_PcysZK and survival was monitored for 14 d (compiled data from 3 independent experiments). (F) Anti- rF1-V IgG titers 7 d prior to infection. (G) Ratio of IgG_2c to IgG₁ antibody subtypes from subset of mice in D. Survival data were analyzed by Kaplan-Meier and log-rank test and cell numbers and antibody titers by one-way ANOVA with Dunnett’s post hoc analysis compared to the AL/AL prime boost group: *** = p<0.001; * = p<0.05.

Figure 5:. Prime-boost vaccination provides long term protection against pneumonic infection.

Male C57BL/6 mice were primed with rF1-V + Alhydrogel alone (AL) or rF1-V + Alhydrogel + SA-4-1BBL (AL+BBL). 21 d later, mice were boosted with rF1-V + Alhydrogel (ALH; n=5), rF1-V + SA-4-1BBL (BBL; n=5), or rF1-V + Alhydrogel + SA-4-1-BBL (AL+BBL; n=5). Mice were challenged 150 d after prime vaccination with 10 LD₅₀ of virulent Y. pestis CO92 Lux_PcysZK and monitored for survival for 14 d. Data was analyzed using Kaplan-Meier survival curve and log-rank test.