Laria naive unvaccinated subjects (blue dots), and malaria naive ChAd63-MVA AKT inhibitor 2 web ME-TRAP vaccinated subjects (red dots). These data were obtained using the linear model; results were similar with the sine model (data not shown). doi:10.1371/journal.pone.0065960.gOptimising CHMI Using Needle SyringeFigure 6. IFN-c ELISPOT responses to individual malaria antigens post CHMI. Median ex-vivo IFN-c ELISPOT responses from the day before CHMI (C-1) and days 7, 11, 14, 35 and 90 post CHMI with cells stimulated against peptide pools to numerous malaria antigens. Each volunteer is represented as a single point. Lines represent the median response at each time point. (A) Individual responses to numerous malaria antigens over time (B) Sum responses to all peptides over time. doi:10.1371/journal.pone.0065960.gmalaria endemic areas and in some cases to individuals who received multiple doses of irradiated purchase Anlotinib sporozoites [15,21,27], although the proportion of individuals responding to some antigens like PfCelTOS (Ag2) was significantly lower than after immunization by the bite of irradiated PfSPZ-infected mosquitoes. [26] The decrease in T-cell immunogenicity by dC+90 supports the notion that repeated low-level antigen exposure would be required to induce and maintain IFN-c responses of a significant protective magnitude. This is the second study to assess the infectivity of 2,500 sporozoites of PfSPZ Challenge administered ID. Our result for this dosing schedule (5/6 participants successfully infected, prepatent period 13.2 days) is near identical to that seen by Roestenberg et al. (5/6 participants successfully infected, pre-patent period 13.days). [12] Given that different batches of PfSPZ Challenge, manufactured two years apart, were used for each of these trials, this result demonstrates the reproducibility of PfSPZ Challenge as a challenge agent. For the first time we have identified an administration regimen for PfSPZ Challenge (25,000 sporozoites IM) that successfully infects 100 of participants. However, given there was no difference in pre-patent period or LBI between 25,000 sporozoites IM and 2,500 sporozoites ID it is likely that the increased proportion of individuals successfully infected with 25,000 sporozoites IM is consistent with chance. In contrast to murine data, administration of 2,500 sporozoites ID was associated with higher infectivity (in terms of proportion of participants infected, duration of pre-patent period and LBI) thanOptimising CHMI Using Needle Syringe2,500 sporozoites administered IM, suggesting ID administration is a more effective route for delivering sporozoites to the liver in humans. The reason for the discrepancy between murine and human models is not clear; however, this finding might be intuitive given that ID injection by needle and syringe most closely resembles injection via a mosquito’s proboscis. Alternatively, other factors such as administration volume could impact on infectivity. In our study, a dose response was seen when PfSPZ Challenge was administered IM, with 25,000 sporozoites appearing more infectious than 2,500. This is in contrast to the study by Roestenberg et al. where increasing the dose of sporozoites ID failed to translate into an increase in the proportion of participants successfully infected. [12] The reason for this difference is unclear but supports the evaluation of higher doses of PfSPZ Challenge IM, which may, in contrast to ID administration, lead to identification of a dose that r.Laria naive unvaccinated subjects (blue dots), and malaria naive ChAd63-MVA ME-TRAP vaccinated subjects (red dots). These data were obtained using the linear model; results were similar with the sine model (data not shown). doi:10.1371/journal.pone.0065960.gOptimising CHMI Using Needle SyringeFigure 6. IFN-c ELISPOT responses to individual malaria antigens post CHMI. Median ex-vivo IFN-c ELISPOT responses from the day before CHMI (C-1) and days 7, 11, 14, 35 and 90 post CHMI with cells stimulated against peptide pools to numerous malaria antigens. Each volunteer is represented as a single point. Lines represent the median response at each time point. (A) Individual responses to numerous malaria antigens over time (B) Sum responses to all peptides over time. doi:10.1371/journal.pone.0065960.gmalaria endemic areas and in some cases to individuals who received multiple doses of irradiated sporozoites [15,21,27], although the proportion of individuals responding to some antigens like PfCelTOS (Ag2) was significantly lower than after immunization by the bite of irradiated PfSPZ-infected mosquitoes. [26] The decrease in T-cell immunogenicity by dC+90 supports the notion that repeated low-level antigen exposure would be required to induce and maintain IFN-c responses of a significant protective magnitude. This is the second study to assess the infectivity of 2,500 sporozoites of PfSPZ Challenge administered ID. Our result for this dosing schedule (5/6 participants successfully infected, prepatent period 13.2 days) is near identical to that seen by Roestenberg et al. (5/6 participants successfully infected, pre-patent period 13.days). [12] Given that different batches of PfSPZ Challenge, manufactured two years apart, were used for each of these trials, this result demonstrates the reproducibility of PfSPZ Challenge as a challenge agent. For the first time we have identified an administration regimen for PfSPZ Challenge (25,000 sporozoites IM) that successfully infects 100 of participants. However, given there was no difference in pre-patent period or LBI between 25,000 sporozoites IM and 2,500 sporozoites ID it is likely that the increased proportion of individuals successfully infected with 25,000 sporozoites IM is consistent with chance. In contrast to murine data, administration of 2,500 sporozoites ID was associated with higher infectivity (in terms of proportion of participants infected, duration of pre-patent period and LBI) thanOptimising CHMI Using Needle Syringe2,500 sporozoites administered IM, suggesting ID administration is a more effective route for delivering sporozoites to the liver in humans. The reason for the discrepancy between murine and human models is not clear; however, this finding might be intuitive given that ID injection by needle and syringe most closely resembles injection via a mosquito’s proboscis. Alternatively, other factors such as administration volume could impact on infectivity. In our study, a dose response was seen when PfSPZ Challenge was administered IM, with 25,000 sporozoites appearing more infectious than 2,500. This is in contrast to the study by Roestenberg et al. where increasing the dose of sporozoites ID failed to translate into an increase in the proportion of participants successfully infected. [12] The reason for this difference is unclear but supports the evaluation of higher doses of PfSPZ Challenge IM, which may, in contrast to ID administration, lead to identification of a dose that r.