Efficacy and Immune Correlates of OMP-1B and VirB2-4 Vaccines for Protection of Dogs from Tick Transmission of Ehrlichia chaffeensis

Abstract

Ehrlichia chaffeensis, an obligatory intracellular bacterium, causes human monocytic ehrlichiosis, an emerging disease transmitted by the Lone Star tick, Amblyomma americanum. Here, we investigated the vaccine potential of OMP-1B and VirB2-4. Among the highly expressed and immunodominant E. chaffeensis porin P28s/OMP-1s, OMP-1B is predominantly expressed by E. chaffeensis in A. americanum ticks, whereas VirB2-4 is a pilus protein of the type IV secretion system essential for E. chaffeensis infection of host cells. Immunization with recombinant OMP-1B (rOMP-1B) or recombinant VirB2-4 (rVirB2-4) protected mice from E. chaffeensis infection as effectively as Entry-triggering protein of Ehrlichia immunization. Dogs vaccinated with a nanoparticle vaccine composed of rOMP-1B or rVirB2-4 and an immunostimulating complex developed high antibody titers against the respective antigen. Upon challenge with E. chaffeensis-infected A. americanum ticks, E. chaffeensis was undetectable in the blood of rOMP-1B or rVirB2-4 immunized dogs on day 3 or 6 post-tick attachment and for the duration of the experiment, whereas dogs sham-vaccinated with the complex alone were persistently infected for the duration of the experiment. E. chaffeensis exponentially replicates in blood-feeding ticks to facilitate transmission. Previously infected ticks removed from OMP-1B-immunized dogs showed significantly lower bacterial load relative to ticks removed from sham-immunized dogs, suggesting in-tick neutralization. Peripheral blood leukocytes from rVirB2-4-vaccinated dogs secreted significantly elevated amounts of interferon-γ soon after tick attachment by ELISpot assay and reverse transcription-quantitative PCR, suggesting interferon-γ-mediated Ehrlichia inhibition. Thus, Ehrlichia surface-exposed proteins OMP-1B and VirB2-4 represent new potential vaccine candidates for blocking tick-borne ehrlichial transmission. IMPORTANCE Ehrlichia are tick-borne pathogens that cause a potentially fatal illness-ehrlichiosis-in animals and humans worldwide. Currently, no vaccine is available for ehrlichiosis, and treatment options are limited. Ticks are biological vectors of Ehrlichia, i.e., Ehrlichia exponentially replicates in blood-sucking ticks before infecting animals. Ticks also inoculate immunomodulatory substances into animals. Thus, it is important to study effects of candidate vaccines on Ehrlichia infection in both animals and ticks and the immune responses of animals shortly after infected tick challenge. Here, we investigated the efficacy of vaccination with functionality-defined two surface-exposed outer membrane proteins of Ehrlichia chaffeensis, OMP-1B and VirB2-4, in a mouse infection model and then in a dog-tick transmission model. Our results begin to fill gaps in our understanding of Ehrlichia-derived protective antigens against tick-transmission and immune correlates and mechanisms that could help future development of vaccines for immunization of humans and animals to counter tick-transmitted ehrlichiosis.

Keywords: Ehrlichia; IFN-γ; ISCOM; OMP-1B; VirB2-4; dog; in-tick neutralization; tick transmission; vaccine.

FIG 1

Mice vaccinated with recombinant EtpE-C (rEtpE-C), recombinant OMP-1B (rOMP-1B), or recombinant VirB2-4 (rVirB2-4) develop high antibody titers against the respective antigen. (A) Recombinant proteins (40 ng each of rEtpE-C, rOMP-1B, and rVirB2-4) were subjected to SDS-PAGE and GelCode Blue staining. Molecular size: rEtpE-C, 31 kDa; rOMP-1B, 28 kDa; and rVirB2-4, 15 kDa. The molecular weight (MW) markers are shown in kilodaltons. (B) Enzyme-linked immunosorbent assay (ELISA) titers using the three recombinant proteins as the antigen (underlined). Filled circles, mice vaccinated with rEtpE-C (black), rOMP-1B (red), and rVirB2-4 (green); Sham, sham-vaccinated mice (open circles). Results are shown as means ± standard deviation (SD) from three vaccinated and three sham-vaccinated mice. Black arrows indicate days on which mice were vaccinated, red arrow denotes the day on which mice were challenged with E. chaffeensis.

FIG 2

rOMP-1B or rVirB2-4 immunization protects mice from Ehrlichia chaffeensis infection as effectively as rEtpE-C immunization with low cytokine responses. (A) Relative E. chaffeensis (Ech) 16S rRNA gene/mouse GADPH levels in peripheral blood from three recombinant protein-vaccinated and sham-vaccinated mice 5 days after intraperitoneal challenge with E. chaffeensis (quantitative PCR [qPCR]). The difference between the vaccinated and sham-vaccinated mice was significant (P < 0.05, n = 4 or 5) by one-way analysis of variance (ANOVA), whereas there were no significant differences among the three vaccines. (B) Mouse interferon (IFN)-γ, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-17A gene expression was estimated in spleen samples from sham-vaccinated and recombinant protein-vaccinated mice at 5 days after intraperitoneal challenge with E. chaffeensis using gene-specific primers; the data were normalized using mouse GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (RT-qPCR). Horizontal bars indicate mean values. Cytokine expression was not significantly different between vaccinated and sham-vaccinated mice based on a one-way ANOVA (n = 3).

FIG 3

OMP-1B and VirB2-4 are expressed by E. chaffeensis in adult Amblyomma americanum ticks infected as nymphs. (A and B) Expression of OMP-1B mRNA (A) and VirB2-4 mRNA (B) by E. chaffeensis (Ech) in ISE6 tick cells and in molted, unfed male and female A. americanum ticks normalized by E. chaffeensis 16S rRNA (RT-qPCR). *, P < 0.05; one-way ANOVA. Horizontal bars indicate mean values.

FIG 4

Dogs vaccinated with rOMP-1B or rVirB2-4 develop high antibody titers to their respective immunogens. (A and B) Western blotting for rOMP-1B (A) and rVirB2-4 (B) using plasma from a vaccinated dog (Vac) and a sham-vaccinated dog (Sham). Representative data are shown from one of three vaccinated or one of four sham-vaccinated dogs. dpv, days post-vaccination. (C and D) ELISA titers against rOMP-1B (C), rVirB2-4 (D), or bovine serum antigen (BSA; negative control, panels C and D) as the antigen (underlined). Data are shown for rOMP-1B-vaccinated (red, panel C), rVirB2-4-vaccinated (green, panel D), and sham-vaccinated (blue, panels C and D) dogs. Black arrows indicate days on which dogs were vaccinated, and red arrow denotes day of tick challenge. Data are shown as the means ± SD from three vaccinated dogs and four sham-vaccinated dogs for each condition.

FIG 5

Vaccination of dogs with rOMP-1B or rVirB2-4 prevents tick transmission of E. chaffeensis. E. chaffeensis (Ech) load in peripheral blood from rOMP-1B- and VirB2-4-vaccinated and sham-vaccinated dogs after attachment of E. chaffeensis-infected ticks (RT-qPCR). −ΔC_T (comparative cycle threshold): − (C_T value of E. chaffeensis 16S rRNA −C_T value of dog HPRT mRNA). C_T values of >40 (undetectable) for E. chaffeensis 16S rRNA were capped as 45. Horizontal bars indicate mean values. Asterisks indicate significant differences between the rOMP-1B- or VirB2-4-vaccinated and sham-vaccinated dogs across all days postinfection (P = 0.0039 for rOMP-1B and P = 0.0047 for VirB2-4), as assessed by a mixed-effects model.

FIG 6

Vaccination of dogs with rVirB2-4, but not with rOMP-1B, induces a IFN-γ response to E. chaffeensis challenge. Peripheral blood mononuclear leukocytes (PBMCs) were isolated from three dogs vaccinated with rOMP-1B (A) and three dogs vaccinated with rVirB2-4 (B) and a total of four sham-vaccinated dogs at 1 (A) and 12 (B) days before and 7 days (A and B) after attachment of E. chaffeensis-infected ticks. PBMCs were incubated with medium (negative control), rOMP-1B (A), or rVirB2-4 (B) in triplicates or duplicates. SFU, spot-forming units. Differences in SFU between rOMP-1B- or rVirB2-4-vaccinated and sham-vaccinated dogs were assessed using a negative binomial mixed model. *, P < 0.05.

FIG 7

Expression of IL-12 and IFN-γ is upregulated in rVirB2-4-vaccinated and then challenged dogs. Expression of selected cytokine mRNAs normalized by dog GAPDH mRNA in blood samples from three rOMP-1B- and three rVirB2-4-vaccinated dogs and four sham-vaccinated dogs on day 7 after the attachment of E. chaffeensis-infected ticks (RT-qPCR). *, P < 0.05 based on an unpaired two-tailed Student’s t test. Horizontal bars indicate mean values.

FIG 8

OMP-1B vaccination of dogs reduces E. chaffeensis infection in transmission-fed ticks. (A and B) Eight to ten female ticks each were randomly pulled off from four sham-vaccinated, three rOMP-1B-vaccinated (A), and three rVirB2-4-vaccinated (B) dogs at 10 days after tick infestation. DNA was isolated from each tick for assessment of E. chaffeensis (Ech) 16S rRNA and A. americanum (Aa) actin DNA by a qPCR assay. Actin levels were used to normalize the 16S rRNA levels. *, P < 0.05 based on an unpaired two-tailed Student’s t test; ns, not significant. Horizontal bars indicate mean values.