Fusion of Antigen to a Dendritic Cell Targeting Chemokine Combined with Adjuvant Yields a Malaria DNA Vaccine with Enhanced Protective Capabilities

<div><p>Although sterilizing immunity to malaria can be elicited by irradiated sporozoite vaccination, no clinically practical subunit vaccine has been shown to be capable of preventing the approximately 600,000 annual deaths attributed to this infection. DNA vaccines offer several potential advantages for a disease that primarily affects the developing world, but new approaches are needed to improve the immunogenicity of these vaccines. By using a novel, lipid-based adjuvant, Vaxfectin, to attract immune cells to the immunization site, in combination with an antigen-chemokine DNA construct designed to target antigen to immature dendritic cells, we elicited a humoral immune response that provided sterilizing immunity to malaria challenge in a mouse model system. The chemokine, MIP3αCCL20, did not significantly enhance the cellular infiltrate or levels of cytokine or chemokine expression at the immunization site but acted with Vaxfectin to reduce liver stage malaria infection by orders of magnitude compared to vaccine constructs lacking the chemokine component. The levels of protection achieved were equivalent to those observed with irradiated sporozoites, a candidate vaccine undergoing development for further large scale clinical trial. Only vaccination with the combined regimen of adjuvant and chemokine provided 80–100% protection against the development of bloodstream infection. Treating the immunization process as requiring the independent steps of 1) attracting antigen-presenting cells to the site of immunization and 2) specifically directing vaccine antigen to the immature dendritic cells that initiate the adaptive immune response may provide a rational strategy for the development of a clinically applicable malaria DNA vaccine.</p></div

Impact of Different Immunization Regimens on Cellular Infiltrate at Site of Immunization.

<p>Impact of Different Immunization Regimens on Cellular Infiltrate at Site of Immunization.</p

Antibody titers in C57Bl/6 mice immunized with <i>P. yoelii</i> CSP DNA constructs Specific antibody concentrations in sera obtained two weeks after the final immunization.

<p>Values shown represent the reciprocal of the endpoint ELISA titer for each mouse (closed circles) and the mean of the reciprocal titers from the 5 mice in each group (horizontal broken lines). The endpoint titer is reported as the highest dilution of serum at which the absorbance was twice the value obtained using pre-immunization serum. The responses to irradiated sporozoite and MIP3α-CSP/Vaxfectin immunization regimens did not differ (p>0.9) and were significantly greater than those to the other immunization regimens (p<0.001), as indicated by ** in the figure.</p

Diagrammatic representation of the DNA constructs, including control plasmids, used for immunization.

<p>The construct for <i>P. yoelii</i> used mouse MIP3α, while the <i>P. falciparum</i> construct employed the sequence for human MIP3α. M refers to MIP3α. CSP refers to a segment of the <i>Plasmodium yoelii</i> circumsporozoite protein; and MCSP refers to the fusion protein of MIP-3α and a segment of the <i>P</i>. <i>yoelii</i> circumsporozoite protein. Also shown are the positions of the leader sequence, spacer, and the myc tag. The leader sequence for the <i>P. yoelii</i> construct is the interferon–γ induced protein 10 secretion leader sequence; the leader sequence for the <i>P. falciparum</i> construct is the human tissue plasminogen activator leader sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090413#pone.0090413-Pennica1" target="_blank">[46]</a>. The MCSP and hMfCSP nucleotide sequences are provided in the supplementary materials.</p

Western blot analysis of expression of constructs of malaria DNA vaccines and controls.

<p><i>P. yoelii</i> (A) and <i>P. falciparum</i> (B) DNA vaccine candidates and control constructs were transfected into and expressed from 293T cells. Protein expression into culture supernatant was detected with anti-myc antibody 48 hours after transfection. In each lane the vaccine constructs are the uppermost bands with lower bands of fCSP representing proteolytic fragments of the expressed proteins. CSP = a fragment of the <i>P. yoelii</i> circumsporozoite protein, fCSP = a fragment of the <i>P. falciparum</i> circumsporozoite protein, M = murine MIP3α, hM = human MIP3α.</p

Ability of different vaccine constructs to protect against the development of blood stage malaria infection.

<p>Ability of different vaccine constructs to protect against the development of blood stage malaria infection.</p

Antibody titers in mice immunized with the <i>P. falciparum</i> construct.

<p>C57BL/6 mice were immunized with 5 µg of the <i>P. falciparum</i> constructs of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090413#pone-0090413-g001" target="_blank">Figure 1</a> with or without Vaxfectin. Specific antibody concentrations. (Closed circles), which are reported as the highest dilution of serum at which the absorbance was twice the value obtained using pre-immunization serum, were determined two weeks after the final immunization. Also shown is the mean of the titers from the 5 mice in each group (lines). The response to the MIP3α-CSP/Vaxfectin immunization regimen was significantly greater than those to the other immunization regimens (p<0.01), as indicated by **.</p

Effect of immunization with the combination of Vaxfectin and MCSP DNA on protection achieved against <i>in vivo</i> challenge with <i>P. yoelii</i> sporozoites.

<p>C57BL/6 mice were immunized with the indicated DNA vaccine or irradiated sporozoites as described in the Methods section. Two weeks after the final immunization, mice were challenged with 5×10³<i>P. yoelii</i> sporozoites, and parasite-specific rRNA levels in the liver were determined by quantitative RT-PCR on samples obtained 48 hours post challenge. All results were normalized against the expression of actin. Results indicate the copy numbers from individual mice (closed squares) and the mean from the 5 mice in each group (horizontal lines). The MCSP/Vaxfectin or irradiated sporozoite groups differed significantly from any other group (p<0.002), but not from each other (p>1.000).</p

CSP-specific Antibody Concentration in Pooled Sera from Different Groups of Immunized Mice.

<p>CSP-specific Antibody Concentration in Pooled Sera from Different Groups of Immunized Mice.</p

Expression of vaccine-induced cytokines and chemokines at immunization site 48 hours after immunization with and without MIP3α.

<p>Scatterplot showing results of an experiment in which 2 µg of Vaxfectin-formulated MCSP or Vaxfectin-formulated CSP were inoculated into the tibialis anterior muscle of C57BL/6 mice. The entire muscle was resected and processed as described in the Methods. Cytokine and chemokine levels of injected muscle samples were analyzed by real-time PCR 24 h (not shown) and 48 h after injection. There were no significant differences between the two time points. In all cases, the cytokine or chemokine levels did not differ between recipients of the CSP vs. the MCSP construct (p>0.5). All cytokine or chemokine levels for either DNA construct were significantly above control levels (p<0.002) except for levels of IL-2, and TGF-β, which did not differ significantly from control levels at either 24 or 48 hours (p>0.2).</p