Extended protection capabilities of an immature dendritic-cell targeting malaria sporozoite vaccine
Introduction
Major problems encountered during the effort to develop an effective pre-erythrocytic malaria vaccine include (1) the need for three or more immunizations to obtain even short term protection in settings in which health care delivery may be problematic and (2) the inability of the vaccine to provide sustained protection over an extended period. These problems were evident in results from the large-scale clinical trial of the RTS,S/AS01 (RTS,S) malaria vaccine. In the first year following completion of the vaccination protocols, vaccine efficacy was at best 50% and this level fell to 16% over the ensuing three years [1], [2], [3].
There is clear evidence that antibody specific for the circumsporozoite protein of the pre-erythrocytic malaria sporozoite stage, at sufficient concentrations, is capable of providing sterilizing immunity against malaria infection. Protection has been demonstrated with passive administration of both monoclonal and polyclonal antibodies, as well as with immunization with adjuvanted peptide vaccines [4], [5], [6], [7], [8]. Studies in humanized mice of passively administered monoclonal antibodies developed from an individual immunized with the RTS,S vaccine concluded that the poor performance of the RTS,S vaccine in clinical trials was attributable to the 95% decline in antibody concentrations over the course of one year [8]. Of note, that rapid decline was presaged by earlier studies in mice using similar vaccine formulations [9].
One line of vaccine development efforts has focused on the use chemokines or cytokines to enhance the inflammatory infiltrate, promote binding of antigen to specific receptors or stimulate differentiation of specific subsets of immune cells [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. In many instances these chemokines or cytokines were used with the goal of eliminating the need for more traditional adjuvants.
We have previously reported on a DNA vaccine in which DNA encoding the chemokine macrophage inflammatory protein 3α, (MIP3α), also known as Chemokine (C-C motif) ligand 20 (CCL20), was fused to DNA encoding a portion of the circumsporozoite protein (CSP) of Plasmodium falciparum (P. falciparum) [21]. This vaccine construct was combined with use of the adjuvant Vaxfectin® (Vical, Inc., San Diego, CA). In a mouse challenge model, this combination yielded complete, antibody-mediated protection against bloodstream infection with transgenic sporozoites three weeks after the last of three immunizations. The enhanced protection was dependent on the presence of the MIP3α component in the vaccine construct. However, unreported follow up studies showed loss of protection three months after the last immunization.
In the current studies we examined the immunogenicity and extended protection capabilities of the protein formulation of this vaccine used with the adjuvant polyinosinic:polycytidylic acid (poly (I:C)). This vaccine formulation yielded significantly higher antibody responses after just two immunizations and protection against bloodstream challenge 23 weeks after the final immunization, the last time point evaluated.
Section snippets
Animals
Six- to eight-week-old female C57BL/6 (H-2b) mice were purchased from Charles River Laboratory (Charles River Laboratories, Inc, Wilmington, MA) and maintained in a pathogen-free micro-isolation facility in accordance with the National Institutes of Health guidelines for the humane use of laboratory animals. All experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University (Protocol MO13H187).
Protein production and purification
The codon-optimized P.
E. coli-derived MCSP binds to CCR6 in vitro
Expression and purification of the cloned and purified human MIP-3α-CSP fusion protein and the CSP protein were confirmed by SDS-PAGE and Western blot analysis using anti-CSP mAb (2A10) (Fig. S1). Both human and murine MIP-3α bind to murine CCR6 [25]. The functional integrity of MCSP was tested by flow cytometric analysis of binding of the MIP3α bearing CSP construct vs. the standard CSP vaccine construct lacking the fused chemokine component. Fig. 1 shows that only the MIP3α-fused CSP vaccine
Discussion
The current studies demonstrate that fusion of the iDC-targeting chemokine MIP3α to a malaria CSP protein vaccine in combination with the adjuvant poly(I:C) results in three fold enhancement of the antibody response compared to a vaccine construct lacking the chemokine fusion component. Further, murine challenge studies 23 weeks after the last of two immunizations indicate that protection is long lasting, consistent with the high antibody concentrations achieved with this immunization regimen.
Conclusion
A malaria vaccine construct which employs adjuvant plus the vaccine antigen fused to the chemokine MIP3α produces significantly greater antibody responses than vaccines lacking the chemokine component. Two immunizations with this construct yields extended protection in a murine challenge model of malaria sporozoite infection.
Potential Conflict of interest statement
Dr. Markham reports an equity interest in Cyvax, Inc., a vaccine development company, during the conduct of the study; in addition, Dr. Markham has a patent 8,557,248 issued, and a patent 20150359869 pending.
Acknowledgements
This work was supported by grants from the National Institutes of Health (Grant #AI073619) and the Johns Hopkins University Malaria Research Institute (http://malaria.jhsph.edu). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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