AGPs were evaluated as adjuvants in vaccine preparations containing F1 capsule protein and/or V-antigen from Y. pestis
to protect against pneumonic plague. Vaccination regimens that varied the amount, the route, the timing, and the number of administrations were tested in a mouse model of pneumonic plague. The most effective vaccine regimen was tested in rats and the mechanism of protection was analyzed using TLR4 mutant mice. When determining vaccine efficacy, the Food and Drug Administration’s Center for Biologics and Evaluation recommends the bacterial challenge doses be 100LD50
]. The LD50
for Y. pestis
CO92 administered intranasally to BALB/c mice was determined to be 2 × 104
CFU (data not shown). This value was consistent with previous determinations by others [18
] and was used in all mouse experiments.
A successful vaccine formulation requires efficacy be established in more than one animal model. For this purpose, we tested the most protective murine vaccine regimen in rats. Although rats have been used extensively as a model for bubonic plague an intranasal LD50
for Y. pestis
CO92 has not been established. Groups of immunologically naïve rats (n = 4 per group) were intranasally challenged with Y. pestis
CO92 at concentrations ranging from 1.5 × 101
to 5 × 107
CFU. All rats challenged with doses of ≥ 5 × 104
CFU succumbed within 4 days, similar to the time-to-death of mice receiving a lethal dose. At a dose of 1.5 × 102
CFU, 1 of 4 rats died, while 3 of 4 rats succumbed to the next higher dilution of 1.5 × 103
CFU. Using the formula of Reed and Muench [34
] we determined the rat LD50
of intranasally administered Y. pestis
CO92 to be 4.7 × 102
3.1. A single i.n. vaccination protects against pneumonic plague
A series of vaccinations were conducted using various concentrations of purified Y. pestis
F1 and V-antigen combined with varying concentrations of AGP adjuvants CRX-524 and CRX-527 (data not shown). A combination of these two lipid A mimetics was used based on our previous results which showed high level induction in murine lung tissue of the pro-inflammatory cytokines TNF-α, IL-12p70 and IFN-γ [32
]. These initial experiments showed that an i.n. vaccine consisting of 2 µg F1 and 6 µg of V-antigen combined with 10 µg of each AGP provided protection. This vaccine formulation was further evaluated. Groups of mice (n = 8) were given a single i.n. vaccine dose, challenged with 100 LD50
of Y. pestis
CO92 at timed intervals extending over 6 months and monitored for survival (). Mice that received either the AGPs alone, or AGPs with antigens, showed 12.5% and 38% survival, respectively, 24 hrs after vaccination. This early protection was likely due to AGP stimulation of innate immunity [32
]. By 2 weeks protection had returned; 38% of the animals receiving a single i.n. vaccine survived challenge. This level of protection increased to 90% 45 days post-immunization and continued to provide ≥ 75% protection through 180 days post vaccine (). The effect of the AGPs as adjuvant was evident because most mice receiving only F1 and V-antigen succumbed to infection; only 2 of 96 mice survived pathogen challenge during the course of this experiment. Mice receiving PBS did not survive bacterial challenge at any time. Statistical analysis comparing all treatment control groups to vaccinated animals showed that vaccine protection was significant (P < 0.0001).
A single i.n. vaccination protects against pneumonic plague
Pooled sera were obtained from each group of animals prior to Y. pestis challenge and IgG titers against F1 or V-antigen were determined (). The data suggested that IgG titers ≥ 10,000 were required for protection against the pneumonic plague challenge. This specific antibody level was achieved 45 days post-vaccination and was maintained for at least six months, the duration of the trial. Animals that received F1 and V-antigen without AGPs had fluctuating IgG titers that never exceeded a titer of 3,000 ().
3.2. Vaccine protection against pneumonic plague is enhanced by a homologous 1°/2° application
The efficacy of a vaccine regimen with 1°/2° doses was tested. A variety of F1 and V-antigen concentrations were tested (data not shown) to determine the effective dose of F1 and V-antigen. Based on this initial data, vaccine formulations containing 1 µg F1 + 3 µg V-antigen mixed with either 10 µg or 20 µg of AGPs, and i.n. doses administered twice (ten days apart) were selected for further study. Protection provided by vaccination with AGPs and V-antigen alone (without F1) was also examined. All animals were challenged 45 days after the 1° dose with 100 LD50 of Y. pestis CO92. Survival of mice receiving 1°/2° doses was compared with mice receiving a 1° vaccine alone, AGPs alone, F1 + V antigens alone, or PBS (). A 1°/2° i.n. vaccine containing 3 µg V-antigen + 10–20 µg AGPs, with or without F1, protected all mice (, P < 0.0001). Also, the two groups of mice which received only a 1° vaccination of 1 µg F1 + 3 µg V-antigen + 10 or 20 µg AGPs had fewer survivors (≤ 42%) compared to mice receiving a 1° vaccine with higher amounts of F1 + V-antigen (2 µg F1+ 6µg V-antigen; 90% survival at day 45; ).
Vaccine protection against pneumonic plague is enhanced by a 2° application
Pooled sera were obtained from each group of animals prior to Y. pestis challenge and total IgG titers against F1 or V-antigen were determined (). The 1° vaccination with F1 + V-antigen + AGPs generated IgG titers to V-antigen >10,000 and to F1 ≤ 1,000. In comparison, animals given a 1°/2° i.n. vaccine (with or without F1) had equivalent or lower IgG titers to V-antigen but had higher survival rates (100% vs. = 42%). A 1°/2° vaccine regimen significantly increased IgG titers to F1 compared to a single i.n exposure. These results indicated that a lower dose i.n. vaccine administered in a 1°/2° schedule provided better protection than a single 1° dose even with equivalent total antigen exposure. Furthermore, IgG titers did not absolutely correlate with the level of protection. In part, this may be artifactual since we measured pooled sera titers from each group vs. titers from individual animals.
3.3. A 1°/2° i.n. vaccination provides early complete protection against pneumonic plague
Given the success observed in the previous set of experiments, we repeated the 1°/2° dose regimen with bacterial challenges at earlier time points to determine how soon protection is achieved. The time interval between 1° and 2° vaccination was also shortened in a series of trials to determine the level of protection obtainable within one week post-1° dose.
Mice were given 20 µg AGPs + 2 µg F1 + 6 µg V-antigen on days 0 and 10. Groups of mice (n = 8) were challenged with 100 LD50 Y. pestis CO92 on days 21, 28, 60, or 90 following the 1° vaccine dose. As seen in , this vaccine regimen provided 100% protection as early as 21 days post-1° vaccination and complete protection was maintained for the 90 day duration of the trial (P <0.0001). Animals receiving AGPs alone or PBS did not survive bacterial challenge (data not shown), and those receiving F1 + V-antigen alone had survival rates that never exceeded 25% ().
A 1° and 2° i.n. vaccination provides early complete plague protection
Pooled sera were obtained from each group of animals prior to Y. pestis challenge and IgG titers were determined (). Titers to F1 and V-antigen were higher among mice receiving the vaccine containing AGP (50,000) compared to mice receiving F1 and V-antigen without AGPs (6,000). We concluded that this i.n. 1°/2° AGP-based plague vaccine provided complete protection against a 100 LD50 pneumonic plague challenge within three weeks.
The timing between 1°/2° vaccinations were further compressed to 24 h or 72 h and protection from pneumonic plague challenge tested at 7 days. shows the results of representative regimens tested. Sixty-three % of the mice survived the challenge administered just one week after the 1° vaccine dose. This high rate of survival was dependent on AGPs in the vaccine preparation as only 12.5% of the animals that did not receive AGPs survived bacterial challenge. In addition, the time-to-death among these mice was 6–10 days. There were no survivors or extended time to death among the control animals receiving PBS or AGPs alone.
Survival of vaccinated mice challenged at seven days with Y. pestis CO92a
The efficacy of administering AGP-based vaccines subcutaneously was also tested. Mice received 2 µg F1 + 6 µg V-antigen + 20 µg AGPs, subcutaneously as a single injection followed by a 100 LD50 Y. pestis i.n. challenge one week later. Among eight mice, 1 expired (87% survival) and the animal that succumbed died 12 days after challenge (data not shown). Control groups included animals that received antigens alone (12% survival), AGPs alone (0 survivors), and a mock PBS vaccine (0 survivors). We concluded that an AGP-based vaccine administered intranasally or subcutaneously provided significant protection (P < 0.0001) within 1 week post-vaccination.
3.4. Vaccination with AGP as adjuvant promotes a TH1 response
To determine if the high IgG titers in immunized animals were indicative of a TH1 or TH2 response, we compared sera IgG1 and IgG2a titers. Animals were given an i.n. vaccination (2 µg F1 + 6 µg V-antigen + 20 µg AGPs) on days 0 and 10 and sera titers analyzed on days 21 or 83 post 1° vaccination. As seen in , animals that received the AGP-based vaccine had high IgG1 and IgG2a titers to both V and F1 (≥80,000). Mice immunized with F1 and V without the AGP adjuvant had at least a 20-fold lower IgG2a response to both V and F1 (≤ 4000). PBS or AGP alone vaccinated control animals had no measurable IgG1 or IgG2a response to V or F1 (data not shown). This data showed that AGPs promoted a cellular immune response, reflected by increased IgG2a titers.
AGP-based vaccine promotes a TH1 response
3.5. AGP-based vaccine protection requires TLR4
To determine if the AGP adjuvant activity was dependant on TLR4 activation, groups of BALB/c TLR4 wild-type and TLR4 mutant mice (n = 8) were vaccinated with 2 µg F1 + 6 µg V-antigen + 20 µg AGPs or mock-vaccinated with PBS in a 1°/2° regimen, administered on days 0 and 10. The mice were challenged with 15 LD50 Y. pestis CO92 on day 21. As seen in , the vaccinated TLR4 mutant animals did not survive challenge and did not exhibit delayed time-to-death. In contrast, 100% of the vaccinated TLR4 competent animals survived challenge. Control animals for both BALB/c TLR4 wild-type and mutant groups receiving mock vaccinations and then challenged with 15 LD50 Y. pestis CO92 all succumbed to infection by day 4. These experiments showed that AGPs employed as adjuvant were dependent upon TLR4.
AGP-based vaccine protection requires Toll-like receptor 4
3.6. A 1°/2° i.n. vaccination protects rats against pneumonic plague
To determine if the vaccine formulation used in the mouse model was efficacious in another mammal, we immunized Sprague-Dawley rats (n = 10) with a 1°/2° i.n. vaccination on day 0 and day 10. To partially compensate for increased body weight, the antigen levels were increased to 4 µg F1 + 12 µg V-antigen + 40 µg AGPs (20 µg each of CRX-524 and CRX-527). Forty-five days after 1° immunization, all rats were intranasally challenged with 1 × 105 CFU Y. pestis CO92, equivalent to just over 1000 LD50. As shown in , this vaccine formulation provided 90% protection compared to 0% protection in a control group receiving PBS. Furthermore, the one vaccinated animal succumbed to infection 12 days post-pathogen challenge compared to control animals all of which died between days 3 and 4. In summary, an effective i.n. vaccine for pneumonic plague developed in a mouse model was effective in rats against a 1000 LD50 intranasal challenge.
A 1° and 2° i.n. vaccination protects rats against pneumonic plague