DOPS Adjuvant Confers Enhanced Protection against Malaria for VLP-TRAP Based Vaccines
<p>Assessment of CD4<sup>+</sup> T cells polarization. Mice were immunized once every three weeks. One week after the third vaccination the mice were bled to obtain sera for cytokine analysis. To evaluate cytokine production, TNFα, IFNγ (Th1 and CTL), and IL-4 (Th2) were measured by multiplex assays MHSTCMAG-70K kit. Also, for experimental control, standard curves were made of each cytokine and compared with the expected concentration provided by the kit, as seen in the figure. The results were analyzed using GraphPad Prism software applied to assess the means of the groups by unpaired t-tests. Note: *** <span class="html-italic">p</span>-value < 0.0001; ** <span class="html-italic">p</span>-value < 0.001; * <span class="html-italic">p</span>-value < 0.01. MFI = Median Fluorescence Intensity.</p> "> Figure 2
<p>Kinetics of antibody production in BALB/c mice. The figure shows IgG total of all groups of mice bled three weeks after each vaccination, specifically on Days 21, 42, and 59. As seen in the figure, the mice that best responded were those vaccinated with PfTRAP coupled with VLP. Also, when the VLP-PfTRAP was mixed with DOPS adjuvants, it provided an improvement of the antibody response with highly statistical significant (<span class="html-italic">p</span> = 0.0001) when compared with the control group.</p> "> Figure 3
<p>Assessment of IgG subtype in BALB/c mice. The antibody subclasses were tested on day 59 of the immunized mice. VLP-conjugate vaccines induced the more protective isotypes IgG2a and IgG2b compared to TRAP formulated in alum. In other hand, the group of mice vaccinated with TRAP + alum induced IgG1 subtype, as expected. Note: *** <span class="html-italic">p</span>-value < 0.0001; * <span class="html-italic">p</span>-value < 0.01.</p> "> Figure 4
<p>Survival curve and comparative analysis of parasitemia in BALB/c mice infected with <span class="html-italic">Plasmodium berghei</span> replacement expressing TRAP of P. falciparum (PfTRAP). (<b>A</b>) Kaplan–Meyer survival curve that shows the time when the mice reach 1% or more of the erythrocytes infected. (<b>B</b>) Number of days until mice have more than 1% of erythrocytes infected and the comparison of the groups. The experiment did not induce sterile protection, but immunization with PfTRAP-VLP plus DOPS conferred significant protection (<span class="html-italic">p</span> = 0.0005) when compared to the group control vaccinated with TRAP only. Also, infection in mice vaccinated with PfTRAP-VLP was significantly delayed when compared to the group vaccinated with only TRAP (<span class="html-italic">p</span> = 0.0354). *** <span class="html-italic">p</span>-value < 0.0001; * <span class="html-italic">p</span>-value < 0.01.</p> "> Figure 5
<p>Kinetics of antibody production in CD1 mice. CD1 mice were immunized once every three weeks with TRAP, VLP-TRAP, or VLP-TRAP formulated with DOPS. Blood was taken on Days 21, 42, and 59 and serum antibody levels were determined. Total IgG levels against TRAP are shown in this figure; the group of mice vaccinated with PfTRAP coupled with VLP, as well as when the VLP-PfTRAP was mixed with DOPS adjuvants induced significantly antibody response when compared with the control group. Note: *** <span class="html-italic">p</span>-value < 0.0001; ** <span class="html-italic">p</span>-value < 0.001; * <span class="html-italic">p</span>-value < 0.01.</p> "> Figure 6
<p>Assessment of TRAP-specific CD8<sup>+</sup> T cell responses in CD1 mice. As described in <a href="#diseases-06-00107-f001" class="html-fig">Figure 1</a>, mice were immunized three times every three weeks and bled on day 50 (eight days after the third vaccination) and the PBMC cells were stimulated with immunodominant Pb9 peptide to assess production of IFNγ and TNFα by CD8<sup>+</sup> T cells. As seen in the figure above, the group of mice vaccinated with VLP-TRAP + DOPS was able to produce more IFNγ and TNFα than the groups of mice vaccinated with TRAP protein either VLP-TRAP. Note: *** <span class="html-italic">p</span>-value < 0.0001; ** <span class="html-italic">p</span>-value < 0.001.</p> "> Figure 7
<p>Kaplan–Meyer survival curve showing when the CD1 mice reached 1% or more of parasitemia or did not have any infected erythrocytes, which is considered to be sterile protection. The only group of mice showing some sterile protection was the group of mice vaccinated with PfTRAP-VLP plus DOPS (red line). Parasite load was counted daily in blood smears until 14 days after challenge and kept for another two weeks under observation to check for signals of disease. The group of mice immunized with PfTRAP-VLP plus DOPS conferred significant protection (<span class="html-italic">p</span> = 0.0001) when compared to the group control vaccinated with TRAP only.</p> ">
Abstract
:1. Introduction
2. Material and Methods
2.1. PfTRAP Protein Production
2.2. Vaccines Preparation
2.3. Immunization Protocol
2.4. Kinetics of Antibody Production
2.5. Assessment of TCD4 Cells Polarization
2.6. Evaluation of CD8+ T Cells by Intracellular Cytokine Staining (ICS)
2.7. Animals
2.8. Ethics Statement
2.9. Parasite Production
2.10. Parasites
2.11. Generation of DNA Constructs and Genotyping of the Transgenic Parasites
2.12. Phenotyping of Reporter and Transgenic Parasites.
2.13. Efficacy Studies: Determination of Liver Parasite Liver Load by Real Time Imaging and Determination Prepatent Period (after Challenge of Immunized Mice with Transgenic Sporozoites)
2.14. Statistical Analysis and Parasitemia Prediction
3. Results
3.1. DOPS Promotes a Th1 Response in TRAP-Vaccinated BALB/c Mice
3.2. Antibody Production by Vaccinated BALB/c Mice
3.3. TRAP-CuMVtt+DOPS Induces Best Protection Against Infection with P. Bergei Expressing PfTRAP
3.4. Assessing the Efficacy of Vaccine Formulated with TRAP-VLP + DOPS in CD1 Mice
4. Discussion
Supplementary Materials
Author Contributions
Acknowledgments
Conflicts of Interest
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Cabral-Miranda, G.; M. Salman, A.; O. Mohsen, M.; L. Storni, F.; S. Roesti, E.; A. Skinner, M.; D. Heath, M.; F. Kramer, M.; M. Khan, S.; J. Janse, C.; et al. DOPS Adjuvant Confers Enhanced Protection against Malaria for VLP-TRAP Based Vaccines. Diseases 2018, 6, 107. https://doi.org/10.3390/diseases6040107
Cabral-Miranda G, M. Salman A, O. Mohsen M, L. Storni F, S. Roesti E, A. Skinner M, D. Heath M, F. Kramer M, M. Khan S, J. Janse C, et al. DOPS Adjuvant Confers Enhanced Protection against Malaria for VLP-TRAP Based Vaccines. Diseases. 2018; 6(4):107. https://doi.org/10.3390/diseases6040107
Chicago/Turabian StyleCabral-Miranda, Gustavo, Ahmed M. Salman, Mona O. Mohsen, Federico L. Storni, Elisa S. Roesti, Murray A. Skinner, Matthew D. Heath, Matthias F. Kramer, Shahid M. Khan, Chris J. Janse, and et al. 2018. "DOPS Adjuvant Confers Enhanced Protection against Malaria for VLP-TRAP Based Vaccines" Diseases 6, no. 4: 107. https://doi.org/10.3390/diseases6040107