Live attenuated Francisella novicida vaccine protects against Francisella tularensis pulmonary challenge in rats and non-human primates

Abstract

Francisella tularensis causes the disease tularemia. Human pulmonary exposure to the most virulent form, F. tularensis subsp. tularensis (Ftt), leads to high morbidity and mortality, resulting in this bacterium being classified as a potential biothreat agent. However, a closely-related species, F. novicida, is avirulent in healthy humans. No tularemia vaccine is currently approved for human use. We demonstrate that a single dose vaccine of a live attenuated F. novicida strain (Fn iglD) protects against subsequent pulmonary challenge with Ftt using two different animal models, Fischer 344 rats and cynomolgus macaques (NHP). The Fn iglD vaccine showed protective efficacy in rats, as did a Ftt iglD vaccine, suggesting no disadvantage to utilizing the low human virulent Francisella species to induce protective immunity. Comparison of specific antibody profiles in vaccinated rat and NHP sera by proteome array identified a core set of immunodominant antigens in vaccinated animals. This is the first report of a defined live attenuated vaccine that demonstrates efficacy against pulmonary tularemia in a NHP, and indicates that the low human virulence F. novicida functions as an effective tularemia vaccine platform.

Figure 1. Fn iglD vaccination is protective against pulmonary Ftt challenge in Fischer 344 rats.

A. Groups of Fischer 344 rats (6 rats/group) were inoculated orally with 10⁷ CFU Fn iglD (open circles) or Ftt iglD (open squares), or mock vaccinated (filled triangles). Rats were challenged 30 days post vaccination with 10⁴ CFU Ftt delivered intratracheally, and monitored for survival. Difference in survival was significant for Fn iglD-vaccinated rats compared to mock vaccinated (P = 0.0439; Kaplan-Meier). Difference in survival of Ftt iglD-vaccinated rats compared to mock vaccinated was not significant. B. Sera from vaccinated rats were analyzed 30 days post-vaccination for Fn- or Ftt-specific antibodies (total Ab, IgG1, and IgG2a).

Figure 2. Fn iglD pulmonary vaccination protects Fischer 344 rats against pulmonary Ftt challenge.

A. Groups of Fischer 344 rats were inoculated intratracheally with 10⁵ (filled circles; n = 4) or 10⁷ CFU (open circles; n = 6) Fn iglD or mock-vaccinated (filled triangles; n = 4). 30 days post vaccination rats were challenged with 10⁴ CFU Ftt delivered intratracheally, and monitored for survival. Difference in survival of Fn iglD-vaccinated rats compared to mock vaccinated was significant (P = 0.0455 for 10⁵ CFU and p = 0.0330 for 10⁷ CFU; Kaplan-Meier). Due to shortage of rats, two group sizes were smaller than optimal n = 6 . B. Sera from vaccinated rats were analyzed 30 days post-vaccination for Fn-specific antibodies (total Ab, IgG1, and IgG2a).

Figure 3. Fn iglD vaccination protects NHP against subsequent pulmonary challenge with Ftt.

A. Groups of cynomolgus macaques were vaccinated with 10⁸ CFU Fn iglD delivered via bronchoscopy (n = 6; open circles), or 10⁸ CFU LVS delivered subcutaneously (n = 4; filled squares), or mock vaccinated (n = 4; filled triangles). 35 days post vaccination NHP were challenged with ∼1000 CFU Ftt via head-only aerosol inhalation; actual presented doses were determined (Table S1). NHP were monitored at intervals shown for 30 days post-challenge for (A) survival, (B) respiration rates, and (C) serum CRP levels. Difference in survival was significant for Fn iglD- and LVS-vaccinated NHPs compared to mock vaccinated (P = 0.0048 and P = 0.0062 respectively; Kaplan-Meier). D. Bacterial burdens (Ftt) were determined in spleen, lung, mesenteric lymph nodes (MesLN), liver, and trachea-bronchial lymph nodes (TBLN) at the time of euthanasia (days 6–10 for mock vaccinees and the single Fn iglD vaccinated NHP that succumbed to pulmonary Ftt challenge [AO8070], day 30 for LVS- and surviving Fn iglD-vaccinated NHPs). The limit of detection (“l.o.d.”; depicted by line) was 70 CFU/g. No bacteria were recovered in the spleens or livers of any surviving Fn iglD vaccinated NHP; organ burdens of the individual vaccinated NHPs are shown in Fig. S5.

Figure 4. Cellular and humoral responses to Francisella spp. in vaccinated NHP.

PBMCs were prepared from (A) Fn iglD- and (B) LVS-vaccinated NHPs either prior to vaccination (naïve), on day 14 post-vaccination, or 30 days post Ftt pulmonary challenge. 200,000 cells/well were stimulated ex vivo with UV-inactivated Fn-iglD (2×10⁶ CFU/ml equivalent) or formalin-fixed LVS (1×10⁵ CFU/ml equivalent) or left unstimulated (medium). IFNγ production was measured by ELISPOT. Assays were performed in triplicate. * indicate significantly (p<0.05; Student t test) more cells produced IFN-γ at the time point indicated as compared to levels measured from naive NHP (Day 0) using the same stimuli. Sera from Fn iglD- (C) and LVS-vaccinated (D) NHPs were analyzed pre-vaccination (naïve), on days 14 and 30 post-vaccination, and 30 days post-Ftt pulmonary challenge by ELISA for total Ab against whole cell killed Fn iglD, LVS, and Ftt antigen. Responses of individual Fn iglD-vaccinated NHPs are shown in Fig. S7 * indicate significantly (p<0.05; Student t test) higher Ab at the time point indicated as compared to levels measured from naive NHP (Day 0) using the same stimuli. (E) Sera from a Fn iglD- (left) and LVS-vaccinated NHP (right) were analyzed on day 30 post-vaccination for reactivity to Fn LPS and LVS LPS by Western immunoblot. Sera were from AO8245 (Fn iglD-vaccinated) and AO8090 (LVS-vaccinated), and equivalent amounts of LPS were loaded in each well.