Structure-Based Vaccines Provide Protection in a Mouse Model of Ehrlichiosis

infection. were protected against the pathogen.Our results demonstrate the power of structural vaccines and could be a new strategy in the development of vaccines to provide protection against pathogenic microorganisms

Protection induced by <i>Ehrlichia</i> Hsp60 _43–63 and P28-19 _55–75 peptides was associated with induction of <i>Ehrlichia</i>- specific IgG antibody.

<p>(A) <i>Ehrlichia</i> Hsp60 _43–63 vaccinated mice induced higher IgG antibody levels after challenge with <i>E.</i> muris compared to unvaccinated <i>E. muris</i>-infected mice (***<i>p</i><0.001 as determined by t test). (B) P28-19 _55–75 peptide vaccinated mice induced higher IgG antibody levels after <i>E. muris</i> challenge compared to unvaccinated <i>E. muris-</i>infected mice (***<i>p</i><0.001 as determined by Student <i>t</i> test).</p

P28-19 _55-75 peptide reacted with <i>E. muris</i> antibody.

<p>The peptide corresponding to the predicted hydrophilic sequence of amino acids 55–75 of P28-19 reacted with <i>Ehrlichia</i> antibody. The peptide was found to be more sensitive in reacting with the <i>Ehrlichia</i> antibody than the recombinant P28-19 protein (***<i>p</i><0.001 as determined by Student <i>t</i> test).</p

Antibody isotypes in mice immunized with <i>Ehrlichia</i> Hsp60 _43–63 and P28-19 _55–75 peptides.

<p>(A) Mice vaccinated with <i>Ehrlichia</i> Hsp60 _43–63 peptide had higher levels of IgG1, IgG2c, IgG2b, and IgG3 compared to unvaccinated mice after bacterial challenge. (B) Mice vaccinated with P28-19 _55–75 peptide had higher levels of IgG1, IgG2b, IgG2c, and IgG3 compared to unvaccinated mice after bacterial challenge. The data were expressed as mean plus standard deviation and three mice per group were included for analysis.</p

Detection of <i>E. muris</i> by fluorescence microscopy.

<p>(A) Antibodies produced against peptide P28-19 _55–75 detected <i>E. muris</i> in infected DH82 cells. Left: Infected DH82 cell probed with P28-19 antibody, followed by FITC conjugated goat anti-mouse antibody. Middle: DAPI staining. Right: Merge. Arrows indicate <i>E. muris.</i> (B) Naïve serum did not detect <i>E. muris</i> in DH82 cells. Left: Infected DH82 cell probed with naive serum, followed by FITC-conjugated goat anti-mouse antibody. Middle: DAPI staining. Right: Merge. Arrows indicate <i>E. muris.</i></p

<i>Ehrlichia</i> Hsp60 _43–63 and P28-19 _55–75 peptides reacted with <i>Ehrlichia</i>-specific antibody from dogs infected with <i>E. canis</i> and <i>E. chaffeensis</i>.

<p>(A) <i>Ehrlichia</i> Hsp60 _43–63 peptide reacted with antibodies from five dogs infected with <i>E. chaffeensis</i> and five dogs infected with <i>E. canis.</i> (B) P28-19 _55–75 peptide reacted with antibodies from five dogs infected with <i>E. chaffeensis</i> and five dogs infected with <i>E. canis</i>. Each bar represents the mean of three replicates. The horizontal line in the graphs represents Mean + 3 SD of negative samples. The positive samples are significantly different from negative samples. (C) P28-19 _55–75 peptide did not react with antibodies from mice infected with <i>Rickettsia</i> or <i>Orientia</i>. (D) <i>Ehrlichia</i> Hsp60 _43–63 peptide did not react with antibodies from mice infected with <i>Rickettsia</i> or <i>Orientia</i>.</p

Amino acid sequence of <i>Ehrlichia</i> Hsp60.

<p>(A) Hsp60 peptides corresponding to the underlined predicted hydrophilic sequence were synthesized. The peptide corresponding to the bold underlined (43–63) sequence was found to react with antibodies to <i>Ehrlichia</i> as well as to induce antibody production. (B) Hydrophobicity plot of <i>Ehrlichia</i> Hsp60. The sequences underlined (in red and blue) were used for synthesizing peptides; however, the best peptide sequence selected is underlined in red. (C) (Left) Predicted 3D structure of <i>Ehrlichia</i> Hsp60, (Right) predicted 3D structure of <i>Ehrlichia</i> Hsp60 with the Van der Waals radii of the heavy atoms highlighting the region of interest (Hsp60 _43–63).</p

Amino acid sequence of P28-19.

<p>(A) P28-19 peptides corresponding to the underlined predicted hydrophilic sequence were synthesized. The peptide corresponding to the bold underlined (55–75) sequence was found to react with antibodies to <i>Ehrlichia</i> as well as to induce antibody production. (B) Hydrophobicity plot of P28-19. The sequences underlined (in red and blue) were used for synthesizing peptides; however the best peptide sequence selected is underlined in red. (C) (Left) Predicted 3D structure of P28-19 (side view), (Middle) predicted 3D structure of P28-19 (basal view), (Right) predicted 3D structure of P28-19 with the Van der Waals radii of the heavy atoms highlighting the region of interest (P28-19 ₅₅₋₇₅).</p

<i>Ehrlichia</i> Hsp60 _43–63 and P28-19 _55–75 -specific memory CD4+ T cells develop during <i>E. muris</i> infection.

<p>We determined by flow cytometry the frequencies and absolute numbers of <i>Ehrlichia</i> Hsp60 _43-63- and P28-19–specific IFN-γ-producing CD4+ T cells in the spleen of mice infected with <i>E. muris</i>. (A) Mice infected with <i>E. muris</i> had higher frequency of <i>Ehrlichia</i> Hsp60 _43–63- and P28-19 _55–75 -specific IFN-γ-producing CD4+ T cells in the spleen on day 45 after infection compared to naïve uninfected mice. Representative dot plots were gated on live cells followed by CD3+ T cells (B) Absolute numbers of <i>E. muris</i>-specific IFN-γ-producing CD4+ T cells in the spleen of the same mice detected following <i>in vitro</i> stimulation with the <i>Ehrlichia</i> Hsp60 _43–63, P28-19 _55–75 peptides; rP28-19 and <i>E. muris</i> whole cell lysate are shown for comparison. Horizontal bars represent the mean; data are representative of two independent experiments (n  = 3 animals per group).</p

Immunization with <i>Ehrlichia</i> Hsp60 _43–63 and P28-19 _55–75 peptides protected mice from <i>Ehrlichia</i> infection.

<p>(A) Mice immunized with <i>Ehrlichia</i> Hsp60 _43–63 were protected against <i>E. muris</i> challenge as determined by the bacterial load measured by quantitative real time-PCR on day 14 after <i>E. muris</i> challenge (*<i>p</i><0.05 as determined by t test). (B) Mice immunized with P28-19 _55–75 peptide was protected against <i>E. muris</i> challenge as determined by the bacterial load measured by quantitative real time-PCR on days 7 and 14 after <i>E. muris</i> challenge (**<i>p</i><0.01 as determined by <i>t</i> test).</p