Here we describe the construction and characterization of a recombinant YF17D virus expressing CSP (amino acids 57-344) from Plasmodium yoelii
. A novel insertion site within the YF17D genome was explored, allowing creation of a stable recombinant virus. Previous studies have shown that manipulation of the YF17D capsid is well tolerated as long as the 5′ cyclization sequence, which forms an essential interaction with a region at the 3′ end of viral genome, is unperturbed [22
]. We maintained the cyclization sequence upstream of the inserted P. yoelii
gene, and recoded the duplicated N-terminal portion of the capsid to ablate the second cyclization site. Processing of PyCS from the YF17D polyprotein was achieved by insertion of the FMDV 2A peptide followed by ubiquitin, as has previously been described for hepatitis C virus (HCV) reporter genomes [37
]. We determined that the YF17D/PyCS genome exhibited slightly delayed replication but was genetically stable throughout 6 passages in cell culture. Since were we able to harvest YF17D/PyCS virus with titers comparable to that of YF17D, we hypothesize that the altered kinetics may result from the increased length of the recombinant genome, or from unpredicted factors such aberrant RNA secondary structures. Although, these recombinant viruses were genetically stable for only a limited number of passages in vitro, and in vivo
stability can be unpredictable, virus stocks suitable for vaccination can be generated directly from electroporated cells without passaging to minimize loss of the CSP sequences.
We investigated the ability of the recombinant virus to elicit anti-CSP immune responses and protect against sporozoite challenge in adult mice. Inoculation of mice with 2×105
PFU of YF17D/PyCS, followed by boosting with 5000 irradiated sporozoites conferred protection against infection with live P. yoelii
, while neither of the regimen components alone were completely protective. Previously, YF17D expressing a single CTL H2Kd
restricted epitope derived from P. yoelii
CSP was shown to induce long-lived IFN-γ-producing CD8+
T cells, and achieved sterile immunity in 9 out of 10 mice immunized with 5×105
PFU of virus followed by 10000 irradiated sporozoites [20
]. In the present study, half the dose of recombinant YFV and half the number of irradiated sporozoites resulted in increased efficacy against challenge with three times the number of viable sporozoites. The higher protective efficacy of the YF17D/PyCS genome observed in our study may be a result of the broader repertoire of epitopes represented by the larger CSP insert. In addition to CD8+
T cell epitopes, such as immunodominant aa 280-288 [31
] and subdominant aa 58-67, we included defined CD4+
T cell epitopes (aa 57-70 and aa 59-79), all of which have been associated with reduced parasitemia [39
]. Given that adult mice are generally resistant to YF17D replication [42
], the low level of immunity induced by the recombinant alone is not surprising. We speculate that experimental immunization of rhesus macaques, a broadly accepted model for yellow fever studies, would yield an even more pronounced anti-CSP response.
We observed induction of IFN-γ–producing CD4+
and CD8T cells after YF17D/PyCS immunization, which was further enhanced by a low dose of irradiated sporozoites. Extensive proliferation of CSP epitope-specific T cells from TCR-transgenic mice suggested the expansion included cell subsets targeting CS sequences. Notably, the IFN-γ response to the YF17D/PyCS-based regimen was remarkably superior to that induced by two doses of irradiated sporozoites in our study. High doses of irradiated or attenuated sporozoites, which elicit sterile immunity in experimentally infected rodents, primates, and human volunteers, mediate protection primarily through CD8+
T cell activation and the production of IFN-γ [43
]. The presence of CD8+
T cells is similarly essential for immunity against liver-stage parasites in murine models of malaria [43
]. A critical role for IFN-γ producing CD4+
T cells has also been highlighted in murine and in human malaria. For instance, adoptive transfer of a CD4+
T-cell clone that recognizes an epitope of P. yoelii
CSP protects mice against sporozoite challenge [40
]. Likewise, elevated levels of IFN-γ producing CD4+
T cells have been associated with protection from placental malaria [48
]. IFN-γ is thought to inhibit parasite development thereby reducing re-infection, and high levels of IFN-γ have been associated with protection from parasitemia, clinical malaria, and anemia [49
]. Finally, in contrast to the irradiated sporozoite immunizations, we observed a multifunctional response to YF17D/PyCS vaccination, in which IL-2 and IFN-γ-producing CD4+
T-cells were detected. IL-2 has also been associated with reduced parasitemia in infected individuals [52
]. The strong role of the T cell response in anti-Plasmodium
immunity was emphasized by the observation that the YF17D/PyCS-based regimen conferred protection despite significantly low-levels anti-CSP antibodies as compared to inoculation with irradiated sporozoites.
The efficient priming ability of the recombinant YF vaccine may reflect the activity of DCs. Although YF17D is known to productively infect human DCs [12
], productive infection of mouse DCs or macrophages has not been reported. Mouse BmDCs, however, appeared to become activated upon exposure to the recombinant YFV, as measured by the upregulation of CD86. Previous studies have shown that exposure to YFV induces upregulation of CD86 in infected human DCs and in DCs that are not productively infected [12
]. Activation of exposed but uninfected mouse BmDCs might be the result of abortive viral entry or binding to an unknown viral receptor.
In conclusion, we have developed a recombinant YF17D capable of inducing a strong T-cell mediated immune response targeting P. yoelii in adult mice. Effective priming by YF17D/PyCS substantially reduces the number of irradiated sporozoites required to confer immunity to mice. Fewer doses of a vaccine could result in a more cost-effective schedule, could reduce the number of vaccines in already crowded childhood vaccine schedules and may be especially beneficial for developing countries that have difficulty delivering a complete multiple-dose schedule. This reduction and potential elimination of the highly immunogenic sporozoite boost may hasten development of a much-needed malaria vaccine.