Figure 1.
Nonintegrative lentiviral vector encoding Plasmodium yoelii CSP used in the study.
Lentiviral vector particles were produced by transient transfection of 293 T cells. The three plasmids used to generate particles are represented here (schematic representation not to scale). The vector expression plasmid pTRIP encodes the vaccine antigen, Plasmodium yoelii CSP. The encapsidation plasmid, p8.74 or pD64V for ILV or NILV respectively, codes for HIV-1 proteins required for particle formation and transduction. The envelope expression plasmid encodes non-crossreacting glycoproteins from Vesiculoviruses used in a specific order to circumvent anti-vector particles antibodies generated after each immunization (Vesicular Stomatitis Virus glycoprotein (VSV-G) Indiana (IND) serotype followed by VSV-G New Jersey (NJ) serotype followed by Cocal virus glycoprotein). Genes coding for structural/enzymatic and regulatory HIV-1 proteins are in dark and light blue respectively, while HIV-1 cis-acting sequences are in yellow and promoter sequences are in grey. The transferred gene, Py CSP, with a human codon-optimized sequence is in green. LTR, long terminal repeat; Ψ, encapsidation signal; cPPT/CTS, central polypurine tract/central termination sequence responsible for the formation of the DNA Flap structure during reverse-transcription which is a determinant of HIV-1 nuclear import; WPRE, Woodchuck hepatitis virus post-transcriptional response element to enhance mRNA nuclear export on a Rev/RRE independent fashion.
Figure 2.
NILV are as immunogenic as ILV when 10-times more particles are injected.
BALB/c mice (n = 5/group) were immunized by IM injection with various doses (expressed as TU/mouse) of lentiviral vector particles encoding Py CSP, either NILV (□) or ILV (▪). Ten days later, specific cellular immune responses were assessed (Figure 2A). The frequency of S9I-specific blood CD8+ cells was assessed by S9I/Kd tetramer staining, and the frequency of IFNg secreting splenocytes in response to overnight restimulation with S9I or I10L peptides was measured by IFNg elispot assay (Figure 2B). Means + SD are shown. BALB/c mice (n = 3/group) were IM immunized with NILV (□) or ILV (▪) at the dose of 5E+08 or 5E+07 TU/mouse respectively (Figure 2C). The frequency of S9I-specific blood CD8+ cells was followed over time by tetramer staining (Figure 2D). At day 24 post-immunization, spleen cellular response was analyzed by S9I/Kd tetramer staining (Figure 2E), by IFNg elispot in response to S9I and I10L peptides (Figure 2F), and by intracellular staining of 3 cytokines, IFNg, IL2 and TNFa, in response to S9I (Figure 2G). Cells secreting individual (green), 2 (blue) or 3 (red) cytokines are shown. Anti-(QGPGAP)2-specific IgG at day 21 post-immunization were quantified by ELISA and expressed as titers (Figure 2H). Medians + range are shown.
Figure 3.
NILV elicit as frequent blood CSP-specific T cells as RAS after 3 injections.
BALB/c mice (n = 6/group) were immunized 3 times by IP injections of NILV. They were primed by administration of NILV particles encoding Py CSP and pseudotyped with VSV-G IND at the dose of 100 or 1500 ng p24/mouse. They were boosted 2 months later with 1500 ng p24 of NILV particles pseudotyped with VSV-G NJ, and boosted again 5 months later with 1500 ng p24 of NILV particles pseudotyped with the glycoprotein from Cocal virus. Additionally, mice (n = 6) from the same batch were immunized 3 times by IV injection with RAS at monthly intervals (Figure 3A). The frequency of S9I-specific blood CD8+ cells was followed over time by S9I/Kd tetramer staining after NILV (Figure 3B) and RAS immunizations (Figure 3C). Data from individual mice and means are shown. The Y-axis uses a logarithmic scale.
Figure 4.
Three immunizations with NILV and RAS induce comparable pre-challenge CSP-specific immune responses.
Groups of BALB/c mice (n = 6/group) were immunized 3 times with NILV by IP injections (in red) or with RAS by IV injections (in black). Immune responses, both cellular and humoral, were compared 28 days after the last immunization. The frequency of IFNg secreting splenocytes in response to restimulation with S9I, I10L or S16I peptides was measured by IFNg elispot assay (Figure 4A). The quality of the S9I specific response was further studied by intracellular staining of 3 cytokines, IFNg, IL2 and TNFa, in response to S9I (Figure 4B). The frequency of IFNg secreting liver cells after restimulation with S9I was analyzed by IFNg elispot (Figure 4C). Additional mice (n = 6/group) were immunized to compare the vaccine-induced in vivo killing capacity of S9I-pulsed target cells (Figure 4D). The presence of IgG directed against (QGPGAP)2 was assessed by ELISA (Figure 4E). Individual responses, means and SD are shown.
Figure 5.
NILV immunizations provide protection against parasitemia after sporozoites challenge, but not as strong as RAS.
Groups of BALB/c mice (n = 8/group) were immunized 3 times with NILV by intraperitoneal injections (in red) or with RAS by intravenous injections (in green), or not (in black) (Figure 5A). They were challenged with 500 spz injected IV 28 days after the last immunization. The protective efficacies of both vaccines against malaria were compared. Parasitemia were followed every other day from day 5 to day 23 post-challenge by Giemsa-stained blood smears. The longitudinal follow-up of individual parasitemia is shown (Figure 5B) as well as means + SD (Figure 5C) and parasitemia at day 9 post-challenge (which corresponds to the peak of viremia in the group of naive animals) (Figure 5D). Among the NILV-immunized mice (in red), fully (•) versus partially (○) protected animals were further distinguished (Figure 5E). The Kruskal-Wallis test was used to compare 3 or 4 groups (Figure 5D and Figure 5E respectively), followed by a Dunn’s multiple comparison post-test. Asterisks denote significance for the post-test (*p<0.05, **p<0.01 and or ***p<0.001). When comparing NILV and naive mice with a Mann-Whitney test (Figure 5D), **p = 0.0072.
Figure 6.
Protection is associated with CSP-specific CD8+ T cells responses.
Immune correlates of protection against malaria were studied by plotting day 28 post-challenge immunity and day 9 post-challenge parasitemia as X and Y variables and using the Spearman test (the rs and p values are shown) and linear regression (r2 is shown) (Figure 6A). Immune responses in challenged mice were compared 28 days post-challenged between the vaccine candidates and their level of protection (fully (•) or partially (○) protected NILV immunized animals in red) by S9I/Kd tetramer staining and IFNg elispot assay with splenocytes and liver cells and elisa (Figure 6B). Means and SD are shown. The Kruskal-Wallis test was used to compare 3 or 4 groups, followed by a Dunn’s post-test.
Figure 7.
The protective efficacy of NILV was confirmed in a second independent study.
Groups of BALB/c mice (n = 8/group) were immunized 3 times with NILV by intraperitoneal injections (in red) or not (in black). They were challenged with 500 spz injected IV one month after the last immunization. The % of parasitized red blood cells was followed every other day from day 5 to day 16 post-challenge by Giemsa-stained blood smears. Individual parasitemia are shown (Figure 7A) as well as means + SD (Figure 7B) and parasitemia at day 10 post-challenge (Figure 7C). Among the NILV-immunized mice (red circles), fully (•) versus partially (○) protected animals were further distinguished (Figure 7D). The Mann-Whitney test was used to compare NILV and naive and the Kruskal-Wallis test followed by a Dunn’s post-test were used to compare fully, partially and naive. The gross morphology of spleens and livers from NILV-immunized and naive mice at necropsy were compared 3 weeks post-challenge (Figure 7E).
Figure 8.
NILV immunizations elicit enduring protective memory responses against malaria.
Groups of BALB/c mice were immunized 3 times with NILV by intraperitoneal injections (n = 7) (in red) or not (n = 9) (in black). They were challenged with 500 spz injected IV six months after the last immunization (Figure 8A). The % of parasitized red blood cells was followed by Giemsa-stained blood smears. Individual parasitemia are shown (Figure 8B) as well as means + SD (Figure 8C) and parasitemia at day 9 post-challenge (Figure 8D). Among the NILV-immunized mice (in red), fully (•) versus partially (○) protected animals were further distinguished (Figure 8E).