The rLVS ΔcapB/iglABC vaccine provides potent protection in Fischer rats against inhalational tularemia caused by various virulent Francisella tularensis strains

Figures & data

Figure 1. Survival data of rats challenged with aerosolized F. tularensis strains for determination of LD₅₀. Groups of Fischer rats (n = 8/group) were challenged by whole body aerosolization with strains FRAN244, FRAN254, or FRAN255 as indicated, and survival monitored. The calculated LD₅₀ values from these experiments are shown in Table 1.

Table 1. Rat aerosol LD₅₀ analysis of F. tularensis strains.

Strain	Aerosol LD50 (CFU)	Significant Difference from FRAN244
FRAN244	433	–
FRAN254	23	p = 0.0004
FRAN255	5,672	p = 0.003

Figure 2. Efficacy of rLVS against aerosol challenge with F. tularensis (FRAN244) in Fischer rats. (a). Experimental schedule. Fischer rats (n = 7 or 8/group) were immunized subcutaneously (SQ) once with PBS (n = 7/group), 10⁷ CFU LVS (n = 8/group), or 10⁷ CFU rLVS (n = 7/group); twice with 10⁶ CFU rLVS (n = 8/group), 10⁷ CFU rLVS (n = 7/group), or 10⁸ CFU rLVS (n = 7/group) rLVS; or three times with 10⁷ CFU rLVS (n = 8/group), 3 weeks apart; challenged with aerosolized F. tularensis FRAN244 (72 LD₅₀) at Week 9; and monitored for signs of illness, weight change, and death for 3 weeks post challenge, as indicated. (b). Survival post immunization and respiratory challenge. The survival curves are compared by log-rank test for trend, ****, p < 0.0001. (c). All rats were weighed weekly. Weight change post immunization and post respiratory challenge.

Table 2. Vaccine doses for testing protection against FRAN244 in rats.

Group	Vaccine Strain	Week 0 (CFU)	Week 3 (CFU)	Week 6 (CFU)
1	PBS- naïve	–	–	N/A
2	LVS	–	–	1.24 × 10^7
3	rLVS	–	–	1.21 × 10^7
4	rLVS	–	2.71 × 10^6	1.04 × 10^6
5	rLVS	–	1.15 × 10^7	1.21 × 10^7
6	rLVS	–	1.46 × 10^8	1.42 × 10^8
7	rLVS	1.92 × 10^7	1.15 × 10^7	1.21 × 10^7

Figure 3. Efficacy of rLVS against aerosol challenge with F. tularensis FRAN254 and FRAN255 in Fischer rats. (a). Experimental schedule. Fischer rats were immunized subcutaneously (SQ) once at Week 0 with PBS (naïve, n = 6 and 7/group for Type A and Type B challenge, respectively), 10⁷ CFU LVS (n = 7 and 8/group for Type A and Type B challenge, respectively), 10⁷ CFU rLVS (n = 8/group for both Type A and Type B challenge) or twice at Week 0 and Week 3 with 10⁸ CFU rLVS (n = 8/group for both Type A and Type B challenge); challenged with aerosolized F. tularensis Type A (FRAN254, 233 LD₅₀) or Type B (FRAN255, 73 LD₅₀) at Week 6; and monitored for signs of illness, weight change, and death for 3 weeks, as indicated. (b). Weight change and survival after vaccination and challenge. Weight changes were compared between any two groups by one-way ANOVA with Tukey’s post comparison’s test. No significant differences were found. The survival curves are compared by log-rank test for trend (Prism 9.2.0). ***, p < 0.001; ****, p < 0.0001.

Table 3. Vaccine doses for testing protection against FRAN254 and FRAN255 in rats.

Group	Vaccine Strain	Week 0 (CFU)	Week 3 (CFU)
1	PBS- naïve	-	N/A
2	LVS	-	5.2 × 10^7
3	rLVS	-	5.8 × 10^7
4	rLVS	9.7 × 10^Citation7	8.8 × 10^7

Figure 4. Histopathology of lungs from F. tularensis challenged rats. Rats were unvaccinated (naïve) or vaccinated with rLVS, or LVS, as indicated; challenged with FRAN244 (top), FRAN254 (middle), or FRAN255 (bottom); and monitored for survival for 21 days. All naïve rats were necropsied at death or following euthanasia when moribund. Vaccinated rats survived and were euthanized and necropsied at the end of the experiment; however one rLVS (single dose) rat challenged with FRAN255 did succumb). Lungs were examined for histopathology. A representative animal from each group is shown. For the rLVS group, all images shown are from the single dose 10⁷ CFU vaccinated group.

Figure 5. Total serum IgG antibody in rats pre- and post-challenge (survivors) with Type A F. tularensis FRAN244/Schu S4 strain. Animals were immunized and challenged as described in the legend to Figure 2. Serum IgG antibody and subclasses specific to irradiated FRAN244 (Type A antigen) were assayed. a. Total serum antibody and subclasses in immunized rats prior to challenge with aerosolized F. tularensis Type A FRAN244/Schu S4 strain. For IgG, IgG2a, IgG2b, and IgG2c, all vaccinated groups had significantly greater titers than the PBS group (p<0.01-p<0.001). For IgG1, all vaccinated groups except LVS, rLVS 10⁷ x1 rLVS 10⁷ x2 groups had significantly greater titers than the PBS group (p <0.05 - p <0.001). Values represent median with interquartile range (Q1 and Q3 for the whiskers) of serum antibody for n = 7 or 8 per group. b. Total serum antibody and subclasses in immunized rats surviving challenge with aerosolized F. tularensis Type A strains. Values represent median with interquartile range (Q1 and Q3 for the whiskers) of serum antibody. Notes: in Fischer rats, IgG2b/2c indicates a Th1 type response and IgG1/2a indicates a Th2 type response. Values that are significantly different between two groups are marked with asterisk(s) over an open horizontal line crossing above the two groups. * p <0 .05; ** p < 0.01; and *** p < 0.001. Additional descriptive statistical values are included in Supplemental Tables S2–5.

Figure 6. Total serum IgG antibody in rats immunized with rLVS vaccines prior to and post challenge with Type A F. tularensis FRAN254 or Type B FRAN255 strain. Animals were immunized and challenged as described in the legend to Figure 3. Sera were collected in immunized rats prior to and after challenge with Type A or Type B strains and assayed for total IgG antibody specific to irradiated FRAN244 (Type A antigen) and FRAN255 (Type B antigen). (a). Prior to challenge; All vaccinated groups in Panel a had significantly greater titers than the PBS group (p <0.01-p <0.0001). (b). Post challenge (survivors only). Values represent median with interquartile range (Q1 and Q3 for the whiskers) of serum antibody titers for n = 6–8/group. * p < 0.05; ** p <0 .01; and *** p < 0.001. Additional descriptive statistical values are included in Supplemental Tables S6,7.

Markowitz LE, et al. Tick-borne tularemia. An outbreak of lymphadenopathy in children. JAMA. 1985;254(20):2922–5. doi:10.1001/jama.1985.03360200074030.

 PubMed Web of Science ®Google Scholar