Subunit Vaccines Using TLR Triagonist Combination Adjuvants Provide Protection Against Coxiella burnetii While Minimizing Reactogenic Responses

Abstract

Q fever is caused by the obligate intracellular bacterium, Coxiella burnetii, a designated potential agent of bioterrorism because of its route of transmission, resistance to disinfectants, and low infectious dose. The only vaccine licensed for human use is Q-VAX^® (Seqirus, licensed in Australia), a formalin-inactivated whole-cell vaccine, which produces severe local and systemic reactogenic responses in previously sensitized individuals. Accordingly, the U.S. Food and Drug Administration and other regulatory bodies around the world, have been reluctant to approve Q-VAX for widespread use. To obviate these adverse reactions, we prepared recombinant protein subunit vaccine candidates containing purified CBU1910, CBU0307, CBU0545, CBU0612, CBU0891, and CBU1398 proteins and TLR triagonist adjuvants. TLR triagonist adjuvants combine different TLR agonists to enhance immune responses to vaccine antigens. We tested both the protective efficacy and reactogenicity of our vaccine candidates in Hartley guinea pigs using intratracheal infection with live C. burnetii. While all of our candidates showed varying degrees of protection during challenge, local reactogenic responses were significantly reduced for one of our vaccine candidates when compared with a formalin-inactivated whole-cell vaccine. Our findings show that subunit vaccines combined with novel TLR triagonist adjuvants can generate protective immunity to C. burnetii infection while reducing reactogenic responses.

Keywords: Coxiella burnetii; TLR agonist; guinea pig model; hypersensitivity; triagonist; vaccine.

Figure 1

Antigen-specific serum IgG responses to vaccination. C. burnetti protein microarrays containing the six antigens from the vaccine candidates and NMI LPS. (A) Guinea pig sera collected from days 7, 21, and 45 post-prime vaccination were probed for total IgG. Values displayed as colorized scale are the means of the intensity from each group (n = 4-5 per group). AU, arbitrary units. (B) Day 45 samples were probed on the protein microarray for antigen-specific IgG1 (blue bar) and IgG2 (pink bar) subtype responses.

Figure 2

TLR agonist vaccines protect against weight loss and fever during aerosol infection. (A) Timeline of challenge experiment in days post-prime vaccination. Guinea pigs were challenged 7 weeks after prime vaccination. (B) Temperatures are presented as change from average individual temperature recorded prior to infection. (C) Change in starting body weight for 14 days post-infection. Body temperature and weight were monitored daily for 14 days post-infection. Graphs show the means for each group (n=5 per group) with error bars indicating the standard error of the mean. Data were analyzed using two-way ANOVA with Dunnett’s correction for multiple comparisons. Asterisks indicate significant differences between candidate- and WCV-vaccinated group (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3

TLR agonist vaccination protects against splenomegaly and reduces pulmonary bacterial burden. (A) Mean spleen weight presented as a percentage of body weight. Vaccines B-F and WCV show significantly less splenomegaly compared to unvaccinated. (B) Mean lung weight presented as a percentage of body weight as an indicator of consolidation. Unvaccinated guinea pigs have significantly heavier lungs than vaccinated groups. (C) Mean genomic equivalents recovered from infected lungs show reduced bacterial burden in all vaccine groups compared to unvaccinated controls. Graphs show the means of each group (n = 4-5 per group) with error bars that represent the standard error of the mean. Data were analyzed using one-way ANOVA with Dunnett’s correction for multiple comparisons. Asterisks indicate significant differences compared to sham-vaccinated group (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 4

TLR agonist vaccines protect against pathologic lesions in the lungs. Histopathologic evaluation of the lungs 14 days after challenge. (A) Histopathologic scoring criteria for lung lesions. (B) Four representative sections of lung were examined and scored for severity of lesions. (C) Representative images of lungs from each experimental group showing foci of consolidation (black arrows) and BALT hyperplasia (arrowheads). Vaccine B and Sham lungs show consolidation of >80% of lung section. 4x magnification, HE stain, scale bar=200 µm. Graphs show the means of each group (n = 4-5 per group) with error bars that represent the standard error of the mean. Data were analyzed using one-way ANOVA with Dunnett’s correction for multiple comparisons. Asterisks indicate significant differences compared to sham-vaccinated group (*p < 0.05, **p < 0.01).

Figure 5

Guinea pigs were sensitized by aerosol infection prior to evaluation of the reactogenicity of TLR agonist vaccines. (A) Timeline of hypersensitivity experiment. Guinea pigs (Groups 2 and 3) were challenged by aerosol infection then rested for 6 weeks before SC injection with vaccine candidates. Uninfected guinea pigs (Group 1) were used to provide sham and unsensitized WCV skin sites as controls. (B) Location of injections for each experimental group. (C) Post-infection changes in body temperature and weight. Infected groups (2 and 3) show transient fever and weight loss compared to uninfected group (1). (D) Post-vaccination changes in weight and temperature. Graphs show the means of each group (n=4-5 per group) with error bars showing the standard error of the mean. Data were analyzed using two-way ANOVA with Dunnett’s correction for multiple comparisons. Asterisks indicate significant differences compared to uninfected group (Group 1) (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 6

TLR agonist vaccines modulate local reactogenic responses to vaccination in pre-sensitized guinea pigs. (A) Histopathologic scoring criteria for vaccination site lesions. (B) Mean histopathologic scores for each experimental group. (C) Representative images of vaccine site lesions from each experimental group. WCV+ produces severe granulomatous inflammation (arrows) with multifocal micro-abscesses (arrowheads). Vaccines B, C, E, and F produce moderate to severe granulomatous inflammation (arrows) with areas of degenerate collagen (asterisks) and occasional foci of hemorrhage (open arrowheads). Vaccine D shows mild lymphohistiocytic inflammation (open arrow). Vaccine A shows minimal perivascular lymphohistiocytic inflammation (open arrow) 4x magnification, HE stain, scale bar = 200 µm. WCV+: WCV vaccination in sensitized guinea pigs. WCV-: WCV vaccination in unsensitized guinea pigs. Graphs show the means of each group (n = 8 for WCV+, n = 4-5 for all other groups) with error bars that represent the standard error of the mean. Data were analyzed using one-way ANOVA with Dunnett’s correction for multiple comparisons. Asterisks indicate significant differences compared to WCV+ vaccinated site in the same group (*p < 0.05, ***p < 0.001, ****p < 0.0001).