Introduction

DCs are an important bridge between innate and adaptive immune responses. They are able to sense infection and inflammation, and efficiently present pathogen-derived epitopes to T cells1. Once activated, T cells produce cytokines and can help activate antibody producing B cells. In addition, DCs are also able to directly activate B cells to mature and produce high affinity antibodies2.

Because of their central role in the induction of immunity, manipulation of DCs is an interesting strategy to induce adaptive immune responses. Among these strategies, the use of mAbs to directly target DCs in situ has been tested with success in different models3,4,5,6,7. This is accomplished by the use of mAbs that target different DC surface receptors fused to antigens derived from pathogens, cancer cells, etc.8. The C-type lectin DEC205 (CD205) has been used with success to induce both cellular and humoral immune responses5,6. Despite its expression by other cell types as B cells and epithelial cells9,10, the DEC205 expression in DCs is responsible for T cell activation when the antigen is targeted in vivo through a hybrid αDEC205 mAb11,12. The use of a DC maturation stimulus together with the hybrid αDEC205 mAb induces long lasting T cell immunity that can even lead to protection in some mouse models of infection13,14. In addition, the induction of specific antibodies against the targeted antigen has also been observed3,5.

In summary, there is extensive data in the literature showing that antigen targeting to DCs through the DEC205 receptor elicits CD4+ and CD8+ T cell activation as well as antibody responses when the hybrid mAb is administered in the presence of a DC maturation stimulus such as αCD40, polyriboinosinic: polyribocytidylic acid (poly (I:C)) or CpG oligodeoxynucleotides3,5,6,13,15. Among the many antigens delivered to the DEC205+ DC subset we can cite the model antigen ovalbumin13,16,17, the tumor antigens survivin18, HER2/neu19, NY-ESO-120 and melanoma TRP221, and different pathogen-derived antigens such as HIV gag6,7,15, Yersinia pestis LcrV22,23, and Plasmodium yoelii CSP5,24. In all cases, strong CD4+ T cell responses were obtained against previously described peptides or against peptides derived from overlapping peptide libraries. CD8+ T cell activation was also detected when αDEC205 mAb was fused to ovalbumin, NY-ESO-1, TRP2, HIV gag, or CSP, especially when the CD8+ T cells were purified and re-stimulated with single peptides5,6,7,13,21. However, in some cases, the activation of these cells was not detected18,23. Taken together, these results indicated that all these antigens possessed antigenic epitopes recognized by the immune system.

Although much has been published with the use of different proteins, the choice of the antigen has not been fully explored. Would any antigen be able to induce strong T cell and antibody responses if targeted to the DEC205+ DC subpopulation? To start addressing this question, we fused the αDEC205 mAb with two fragments of the merozoite surface protein 1 (MSP1) derived from Plasmodium vivax, the most prevalent species that causes human malaria. MSP1 is expressed during the erythrocytic phase of Plasmodium life cycle and participates in parasite invasion25. It is expressed as an ~200 kDa precursor on the surface of the merozoite, and undergoes successive proteolytic cleavages generating a 42-kDa fragment (MSP142) that is further cleaved into two products: a soluble 33-kDa fragment (MSP133) that corresponds to the N-terminal region of MSP142 and is shed from the free merozoite surface26, and a membrane-bound 19-kDa C-terminal fragment (MSP119), which is the only MSP1 fragment carried with the invading merozoite into the new red blood cell27.

Infection with Plasmodium sp. leads to the induction of antibodies that bind mainly to the MSP119 protein28,29,30 while MSP142 is thought to contain T cell epitopes31 that help B cells to produce anti-MSP119 antibodies32,33. Antibodies and CD4+ T cells directed to MSP1 were shown to be associated with protection against malaria in mice33,34,35 and humans36.

To study the differences in terms of antibody induction and T cell activation in the context of DEC205+ DC targeting, we delivered MSP119 or MSP142 proteins to this subset through two hybrid mAbs, αDEC-MSP119 and αDEC-MSP142. Analysis of the immune response induced by immunization with the two hybrid mAbs in the presence of poly (I:C) showed that T cell epitopes are indeed present in the MSP133 portion of the molecule and that induction of high titres of anti-MSP119 antibodies is obtained mainly when MSP142 is targeted to the DEC205+ DC population.

Results

The hybrid αDEC mAbs containing MSP119 or MSP142 fused proteins were successfully produced and capable of binding to the DEC205 receptor

For the production of αDEC-MSP142 and αDEC-MSP119 mAbs, the open reading frames from msp119 or msp142 genes were cloned in frame with the carboxyl-terminal portion of the heavy chain of the αDEC205 mAb exactly as described in methods and in ref. 14. As a control, we also produced the αDEC205 mAb without any fused antigen. Of note, all our attempts to produce an isotype control fused with the MSP142 protein failed, as the hybrid mAb came out very degraded (data not shown). After transient transfection, the αDEC hybrid mAbs were purified and their integrity was evaluated by SDS-PAGE under reducing and non-reducing conditions. The recombinant proteins MSP119 and MSP133 were also produced and evaluated in the same manner (Fig. 1). A reduced gel showed that the heavy chains of the hybrid mAbs had the expected electrophoretic motilities (~92 kDa for αDEC-MSP142, ~69 kDa for αDEC-MSP119 and ~50 kDa for αDEC, Fig. 1A). All light chains migrated at ~25 kDa. Recombinant (rec.) MSP119 and MSP133 proteins migrated at ~19 kDa and ~33 kDa, respectively. The non-reduced gel showed mainly the presence of a single band for each of the mAbs (Fig. 1B).

Figure 1
figure 1

The hybrid αDEC antibodies fused to P. vivax MSP119 and MSP142 were successfully produced and recognized by serum from a P. vivax infected patient, and retained their ability to bind to DCs expressing the DEC205 receptor.

Approximately 1 μg of each hybrid mAb or recombinant protein was separated under reduced (A) and non-reduced (B) conditions. Both gels were stained with Coomassie Blue dye. A western blot under reduced (C) and non-reduced (D) conditions was performed with serum derived from one patient infected with P. vivax. The membranes were revealed after incubation with goat anti-human total IgG conjugated to HRP. The hybrid mAbs or recombinant proteins are presented in the following order: (1) αDEC, (2) αDEC-MSP119, (3) αDEC-MSP142, (4) recombinant MSP119 protein, and (5) recombinant MSP133 protein. Note that the recombinant proteins were not included in the non-reducing gels. The numbers displayed on the left of each gel indicate the molecular weights in kDa that were cropped out of the final figure. (E) Five million splenocytes from C57BL/6 mice were incubated on ice with 10, 1 or 0.1 μg/mL of the hybrid αDEC-MSP142, αDEC-MSP119 or αDEC mAbs. Splenocytes were then incubated with a pool of fluorescent antibodies and gated as singlets and CD19−DX5−. DCs were selected as CD11c+MHCII+, and subsequently divided in CD8α+ (DEC205 expressing subset) and CD8α−. Binding was detected on 1 × 106 cells using an anti-mouse IgG1-PE antibody. One experiment representative of three is shown. Analysis was performed using FlowJo software.

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In an attempt to verify if both hybrid mAbs and recombinant proteins retained antigenicity, a western blot was performed using sera from a P. vivax infected patient. Figure 1 shows that the patient’s serum recognized the heavy chains (Fig. 1C), or the entire hybrid mAbs (Fig. 1D) containing MSP142 or MSP119. Recognition was also observed for the rec. MSP119 and rec. MSP133 proteins (Fig. 1C).

To verify if the addition of either MSP142 or MSP119 to the αDEC205 mAb altered its binding to the DEC205 receptor, different concentrations (10, 1 and 0.1 μg/mL) of the αDEC-MSP142 or αDEC-MSP119 mAbs were incubated with splenic CD11c+CD8α+ (DCs that express the DEC205 receptor17) or with CD11c+CD8α− DCs (Fig. 1E), or with CHO cells expressing either the murine or the human DEC205 receptors (Supplementary Fig. 1). The empty αDEC205 mAb was used as control. All mAbs bound specifically and in a dose dependent manner to the CD11c+CD8α+ DCs in the spleen (Fig. 1E, CD11c+CD8α+ panel) or to CHO cells expressing the murine DEC205 receptor (Supplementary Fig. 1A). No binding was observed to the CD11c+CD8α− DCs that do not express the DEC205 receptor (Fig. 1E, CD11c+CD8α− panel) or to CHO cells expressing the human DEC205 (Supplementary Fig. 1B).

Targeting of the MSP142 protein to the DEC205+ DC population increases the anti-MSP119 antibody response, is dependent on CD4+ T cell help and promotes class switch and affinity maturation

In an attempt to study the anti-MSP119 antibody response elicited when both hybrid mAbs were administered to mice, two different strains were used throughout this study: C57BL/6 (H-2b haplotype) and B10.A (H-2a(k/d) haplotype). Groups of C57BL/6 or B10.A mice received two doses containing 5 μg of each hybrid mAb or the empty αDEC administered in a 30-day interval in the presence of poly (I:C). Figure 2A shows a schematic representation of the immunization protocol. Five days before or 14 days after the administration of the second dose, mice were bled and anti-MSP119 or anti-MSP133 antibody titres were measured by ELISA (Fig. 2B–E). In both mouse strains, the antibody titres against MSP119 increased after the administration of the second dose (Fig. 2B and C) in mice immunized either with αDEC-MSP142 or αDEC-MSP119. No anti-MSP119 titres were detected in the group immunized with αDEC. Of note was the fact that in both mouse strains, the amount of anti-MSP119 antibodies induced in mice that received αDEC-MSP142 mAb was approximately 100x higher than in mice that received αDEC-MSP119 mAb. As expected, anti-MSP133 antibodies were only detected in mice immunized with αDEC-MSP142 (Fig. 2D and E). To compare the anti-MSP119 antibody response in the presence or absence of DEC205+ DC targeting, we immunized groups of C57BL/6 mice with αDEC-MSP142, αDEC-MSP119, αDEC, rec. MSP119, rec. MSP133 or with a combination of rec. MSP133 and MSP119 (Fig. 3). To our surprise, mice immunized with rec. MSP119 were able to produce anti-MSP119 antibodies that, despite slightly lower, were not statistically different from those observed in mice immunized with αDEC-MSP142 (Fig. 3A). No difference was also observed between the αDEC-MSP142 and rec. MSP119+MSP133 groups. To better characterize the anti-MSP119 response, the IgG subtypes were analysed in both mouse strains and the IgG1/IgG2c ratio was calculated (Fig. 3B). Both C57BL/6 and B10.A mice presented detectable levels of all IgGs in the group immunized with αDEC-MSP142 mAb. An analysis of the IgG1/IgG2c ratio showed that the response in both mouse strains was prone to Th1 (IgG1/IgG2c < 1). On the other hand, in the group immunized with αDEC-MSP119 (in both mouse strains) or with rec. MSP119 or rec. MSP119+MSP133 (in C57BL/6 mice), a more Th2 type of response was observed (IgG1/IgG2c > 1).

Figure 2
figure 2

Immunization with hybrid αDEC-MSP142 mAb induces higher anti-MSP119 antibody titres when compared to immunization with αDEC-MSP119 mAb.

(A) Groups of C57BL/6 or B10.A mice (n = 10) were immunized with 5 μg of hybrid αDEC-MSP142, αDEC-MSP119 or αDEC in the presence of 50 μg of poly (I:C). Thirty days after the first dose, the animals received a booster dose in the same conditions as priming. The anti-MSP119 or anti-MSP133 IgG responses were measured by ELISA 5 days before (pre-boost) and 14 days after administration of the booster dose (post-boost). Total anti-MSP119 IgG antibodies were detected in C57BL/6 (B) and B10.A (C) mice. Anti-MSP133 antibody detection was also performed in C57BL/6 (D) and B10.A (E) mice. Graphs show the antibody titres of different groups normalized in log10 scale. Animals are represented individually (n = 10/group). Experiments were analysed by one-way ANOVA followed by the post-test HSD Tukey. P-value indicators * and *** refer to p < 0.05 and p < 0.001, respectively, while ns = not significant.

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Figure 3
figure 3

Immunization with αDEC-MSP142 promotes class switch and affinity maturation.

(A) Groups (n = 5) of C57BL/6 mice were immunized as described in Fig. 2 with αDEC-MSP142, αDEC-MSP119, αDEC, rec. MSP119, rec. MSP133 or with a combination of rec. MSP133+MSP119 in the presence of poly (I:C). The anti-MSP119 antibody titres were measured by ELISA before and after the administration of the booster dose. Graph shows the antibody titres of different groups normalized in log10 scale, and animals are represented individually. (B) The anti-MSP119 antibody titres for each IgG subclass (IgG1, IgG2b, IgG2c and IgG3) were determined 14 days after the administration of the booster dose in groups of C57BL/6 or B10.A mice. Graphs show the antibody titres plotted in log10 scale. Each bar represents the mean values ± SD of the antibody titres obtained for 5 mice. Numbers above the bars indicate the IgG1/IgG2c ratio calculated for αDEC-MSP142 and αDEC-MSP119 immunized groups. Results are representative of 2 independent experiments. (C) ELISA assessed anti-MSP119 antibody avidities using 7 M urea for 5 min. The avidity index was calculated as the ratio between the OD490 obtained after and before urea treatment multiplied by 100. Results are expressed by the mean ± SD of two distinct experiments performed in triplicates. One-way ANOVA followed by the post-test HSD Tukey was performed. *** Refers to p < 0.001 and ns = not significant. Results are representative of 2 independent experiments.

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The detection of all IgG subclasses in the anti-MSP119 antibody response in both mouse strains, and especially in the αDEC-MSP142 immunized group, suggested that B cells were undergoing class switching and probably affinity maturation. To test this hypothesis, we measured the avidity index of the anti-MSP119 antibodies in C57BL/6 and B10.A mice immunized with the hybrid mAbs or with the rec. proteins (Fig. 3C). We observed that anti-MSP119 antibodies induced in both mouse strains immunized with αDEC-MSP142 showed a higher avidity index when compared to the group immunized with αDEC-MSP119 (Fig. 3C). More importantly, the avidity index presented by C57BL/6 mice immunized with αDEC-MSP142 was higher than the observed in groups that received rec. MSP119 or rec. MSP133+MSP119 proteins (Fig. 3C). Taken together, these results suggest that MSP142 targeting to the DEC205+ DC subset is able to alter the quality of the anti-MSP119 humoral immune response.

It was previously shown that antigen targeting to the DEC205+ DCs induces an antibody response that requires T cell help5. To confirm this requirement in our model, we immunized WT, CD4 KO and MHCII KO mice (C57BL/6 background) with our hybrid mAbs (Fig. 4). As expected, the anti-MSP119 response was abolished in the absence of CD4+ T cells or MHCII presentation.

Figure 4
figure 4

The anti-MSP119 antibody response induced by the immunization with hybrid mAbs is dependent on CD4+ T cell help.

Groups (n = 5) of C57BL/6, CD4 KO and MHCII KO mice were immunized as described in Fig. 2. The anti-MSP119 antibody titres were measured by ELISA before and after the administration of the booster dose. Graph shows the antibody titres of different groups normalized in log10 scale, and animals are represented individually. Results are representative of 2 independent experiments.

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To test if the anti-MSP119 antibodies could bind to the MSP119 protein on the surface of cells, we transfected HEK293T cells with a plasmid capable of expressing the MSP119 as a transmembrane protein (Supplementary Fig. 2A). As a negative control, HEK293T cells were also transfected with a plasmid containing the unrelated Duffy binding protein II (DBPII, Supplementary Fig. 2B). Besides MSP119 or DBPII proteins, both plasmids also expressed the green fluorescence protein (GFP). GFP+ cells were gated for the analysis (Supplementary Fig. 2C and D). We observed that anti-MSP119 antibodies induced in mice immunized with either αDEC-MSP142 or αDEC-MSP119 in both mouse strains (Supplementary Fig. 2E for C57BL/6, and 2G for B10.A) bound to the MSP119 expressing cells while no significant ligation was observed in the DBPII transfected cells (Supplementary Fig. 2F for C57BL/6, and 2H for B10.A). It is important to mention that to perform this assay we normalized the amount of anti-MSP119 antibodies present in the sera.

In summary, the results presented above show that immunization with αDEC-MSP142 is able to induce stronger and broader humoral immune response when compared with αDEC-MSP119, in two different mouse strains.

MSP142 targeting to DEC205+ DCs induces activation and proliferation of CD4+ T cells specific for the 33-kDa fragment

The results described above suggested that MSP142 sequence carried epitopes that might provide help for the B cell mediated antibody production when targeted to the DEC205+ DCs. To start mapping the important MSP142 regions, we performed ELISPOT assays to detect IFN-γ producing cells using splenocytes from C57BL/6 mice immunized with αDEC-MSP142, αDEC-MSP119, αDEC, rec. MSP119, rec. MSP133 or with rec. MSP133+MSP119 (Fig. 5A), or from B10.A immunized with αDEC-MSP142, αDEC-MSP119 or αDEC (Fig. 5B). We detected large numbers of IFN-γ producing cells only in splenocytes derived from mice immunized with αDEC-MSP142 mAb and pulsed with recombinant MSP133. This was observed in C57BL/6 (Fig. 5A) and in B10.A (Fig. 5B) mice. Pulse with rec. MSP119 induced fewer IFN-γ producing cells in C57BL/6 and in B10.A, indicating that the immunodominant T cell epitopes are probably present in the 33-kDa portion of MSP142. Of note, we were able to detect a reasonable number of IFN-γ producing cells in C57BL/6 mice immunized with the rec. MSP119 but not in the animals immunized with αDEC-MSP119 when the splenocytes were pulsed with the same protein. To further analyse the response, we took advantage of the fact that T cells from B10.A mice recognize peptide DYDVVYLKPLAGMYK previously described37. We then pulsed splenocytes from αDEC-MSP142, αDEC-MSP119 or αDEC immunized mice with the DYDVVYLKPLAGMYK peptide and observed that only cells derived from αDEC-MSP142 mice were able to produce IFN-γ (Fig. 5C). However, the number detected (~380 per 106 total splenocytes) was smaller than the number obtained when the pulse was performed with the recombinant MSP133 protein (~1,100 per 106 total splenocytes), indicating that there are probably other epitopes that still need to be mapped.

Figure 5
figure 5

Splenocytes from αDEC-MSP142 immunized mice produce IFN-γ in response to MSP133 recombinant protein.

Groups of C57BL/6 (A) and B10.A (B and C) mice (n = 3) were immunized as described in Fig. 2. The cellular immune response was evaluated by ELISPOT 20 days after administration of the booster dose. Total splenocytes were stimulated with 1 μg/ml of recombinant MSP133 or MSP119 proteins (A and B) or with the DYDVVYLKPLAGMYK or a control unrelated peptide (C). Graphs show the number of IFN-γ producing cells per million cells after subtracting the number of IFN-γ producing cells obtained in the absence of any stimulus. The experiment was performed in triplicates using samples from pooled mice. Bars indicate mean ± SD and the experiment was analysed by one-way ANOVA followed by the post-test HSD Tukey. P-value indicators ** and *** refer to p < 0.01 and p < 0.001, respectively, while ns = not significant. Results are representative of 3 independent experiments.

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To further investigate the cellular immune response induced in mice immunized with αDEC-MSP142 mAb, we evaluated the production of three inflammatory cytokines: IFN-γ, IL-2 and TNFα by CD4+ (Supplementary Fig. 3) and CD8+ T cells (data not shown). CD4+ T cells derived from either C57BL/6 or B10.A mice immunized with αDEC-MSP142 were able to produce the three cytokines when restimulated only with MSP133 recombinant protein. Restimulation with recombinant MSP119 was unable to elicit significant percentages of CD4+ T cells producing IFN-γ, IL-2 or TNFα in C57BL/6 (Supplementary Fig. 3A) or B10.A mice (Supplementary Fig. 3B). Interestingly, very low levels of CD4+ T cells producing any cytokine were observed in C57BL/6 immunized with rec. MSP119 or rec. MSP133 (Supplementary Fig. 3A). Although surprising, we did not detect specific responses elicited in CD8+ T cells (data not shown). Using boolean gating analysis, we were able to detect the simultaneous production of the inflammatory cytokines IFN-γ, IL-2 and TNFα by CD4+ T cells (Fig. 6). We observed that CD4+ T cells derived from mice immunized with αDEC-MSP142 mAb were able to produce combinations of the three tested cytokines in both mouse strains (Fig. 6A, C57BL/6 and 6B, B10.A) when pulsed with MSP133 recombinant protein. In fact, in both mouse strains we were able to detect polyfunctional CD4+ T cells producing all combinations of the three cytokines tested (Fig. 6), especially those producing all three at the same time. To access T cell proliferation, splenocytes from mice immunized with the hybrid mAbs or rec. proteins were stained with CFSE and pulsed with either recombinant MSP119 or MSP133 proteins (Fig. 7). CD4+ T cell proliferation was observed in both mouse strains (Fig. 7A, C57BL/6 and 7B, B10.A) mainly in the animals immunized with αDEC-MSP142 pulsed with recombinant MSP133. CD8+ T cell proliferation was not observed in any case (data not shown). In addition, we observed that CD4+ T cells from B10.A mice also proliferated in response to the DYDVVYLKPLAGMYK peptide (Fig. 7C). However, the percentage of CD3+CD4+ T cells that responded to this peptide was smaller than that obtained when recombinant MSP133 was used (Fig. 7B) indicating the presence of other T cell epitopes in the MSP133 protein sequence.

Figure 6
figure 6

Immunization with the αDEC-MSP142 mAb induces the polyfunctional CD4+ T cells after restimulation with MSP133 recombinant protein in C57BL/6 and B10.A mice.

Groups of mice (n = 3) were immunized as described in Fig. 2. IFN-γ, IL-2 and TNFα were detected by intracellular staining 20 days after the administration of the booster dose in C57BL/6 (A) and B10.A (B) mice. Splenocytes were stimulated with 5 μg/ml of MSP133 recombinant protein. Graphs show the percentage of cells producing IFN-γ, IL-2 and/or TNFα in the CD3+CD4+ gate after subtracting the values obtained in the absence of any stimulus. Boolean combinations were created using FlowJo software to determine the frequency of each cytokine production based on all possible combinations of cytokine expression. The experiment was performed in duplicates using samples from pooled mice. Results are representative of 2 independent experiments.

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Figure 7
figure 7

MSP142 targeting to the DEC205+ DC population stimulates CD4+ T cell proliferation in response to MSP133 protein.

Groups of mice (n = 3) were immunized as described in Fig. 2. Twenty days after the administration of the second dose, total splenocytes from C57BL/6 (A) and B10.A (B and C) mice were labelled with CFSE and placed in culture in the presence or absence of 5 μg/ml of MSP133 or MSP119 recombinant proteins. Graphs show the percentage of CD3+CD4+ T cells that lost CFSE (CFSE low) after subtracting the values obtained in the absence of any stimulus. The experiment was performed in triplicates using samples from pooled mice. Bars indicate mean ± SD and the experiment was analysed by one-way ANOVA followed by the post-test HSD Tukey. *** Refers to p < 0.001, ns = not significant. Results are representative of 2 independent experiments.

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The results presented above show that antigen targeting to the DEC205+ DC population in the presence of poly (I:C) is effective in inducing potent antibody and CD4+ T cell responses when epitopes are present in the protein structure. In the absence of such epitopes, the response is weak.

Discussion

Antigen targeting to the CD8α+ DC population through the use of αDEC205 hybrid mAbs has been successfully used in different models, and was shown to induce both CD8+ and CD4+ T cells5,6,7,13,15,17,18,19,20,21,22,23,24. In addition, the induction of antibodies against the fused antigen was also reported previously3,5. All these studies used antigens previously described as immunogenic. However, to our knowledge, no one has yet targeted different fragments of the same antigen to the DEC205+ DCs, and asked what would be the immune response outcome. To start addressing that question, we produced two mAbs containing fragments of the MSP1 protein derived from P. vivax. The αDEC-MSP119 mAb contains the C-terminal 19-kDa fragment, which is normally target of antibodies in infected individuals28,36,38, while the αDEC-MSP142 mAb contains the 19-kDa fragment (MSP119) fused with the 33-kDa (MSP133) fragment that was shown to elicit T cell responses in the field31. Of note, it was not evaluated which epitopes were recognized by CD4+ or CD8+ T cells31. Both hybrid αDEC-derived mAbs were produced successfully, retained antigenicity and were able to target the DEC205 receptor expressed on the surface of CD8α+ DC population. This is not the first time a Plasmodium derived antigen is fused to the αDEC205 mAb. Previously, the circumsporozoite protein (CSP) from P. yoelii and P. falciparum was also used to immunized mice5 and non-human primates24. However, to our knowledge, this is the first time an antigen expressed by the erythrocytic stage (merozoites) is fused to the αDEC205 mAb. We took advantage of the fact that MSP142 contains MSP119, and immunized mice with both αDEC-derived hybrid mAbs in an attempt to study the anti-MSP119 antibody response elicited when MSP119 was targeted alone or fused to MSP133. As mentioned previously, we were unable to produce an isotype control fused to the MSP142 protein, and then used the recombinant proteins as non-targeted controls. The use of poly (I:C) as a DC maturation stimulus is well documented in the literature and it seems to be very potent when administered together with αDEC205 fusion mAbs3,7,14,15,24,39. An increase in either anti-MSP119 or anti-MSP133 antibody titres was observed after the administration of the second dose, which has been consistently observed in other models5,14. Interestingly, the anti-MSP119 antibody titres were increased about 100x when MSP142 was targeted to the CD8α+ DC population in two different mouse strains. This result suggested that the presence of MSP133 fragment was helping to increase the anti-MSP119 antibody response possibly because of the presence of additional T cell epitopes in the region of 33 kDa. However, to our surprise, immunization with rec. MSP119 or with rec. MSP133+MSP119 was able to induce high anti-MSP119 antibody titres in C57BL/6 mice that were not different from those induced by αDEC-MSP142. This result was unexpected but may reflect a longer persistence of rec. MSP119 in the circulation leading to an increase in the antigen uptake by B cells. We also cannot rule out the possibility that MSP119 contains minor CD4+ T cell epitopes responsible for helping B cells to produce antibodies when the protein is not directly targeted to the DEC205+ DCs. The dependency of T cell help for the induction of antibody responses after targeting to DEC205+ DCs was shown after immunization of CD4 and MHCII KO mice, when complete abrogation of the antibody response was observed in the animals. To characterize in more detail the anti-MSP119 antibody response, we carefully analysed the IgG subclasses induced when the hybrid mAbs or the rec. proteins were used. Surprisingly, immunization with the αDEC-MSP142 mAb induced a very different profile of IgG subclasses when compared to either αDEC-MSP119 or rec. proteins immunized mice. The IgG1/IgG2c ratio in animals immunized with αDEC-MSP142 was <1, indicating a more prone Th1-type of response in both mouse strains analysed. On the other hand, the group immunized with αDEC-MSP119 or with the rec. proteins showed a higher IgG1/IgG2c ratio (>1) in both mouse strains. Previous reports have shown that antigen targeting through DEC205 is able to elicit high titres of IgG2c (or IgG2a, depending on the mouse strain)5,14,23. These results also suggested that immunization with αDEC-MSP142 could be inducing a more pronounced class switch that would imply in an increase in affinity maturation. We then measured the avidity index of the polyclonal sera induced in mice immunized with αDEC-MSP142, αDEC-MSP119 or with the rec. proteins. In both mouse strains, the avidity index was higher in the sera of animals immunized with the αDEC-MSP142 mAb, suggesting that the anti-MSP119 specific B cells were undergoing affinity maturation particularly in this group of mice. On the other hand, the ability to bind to MSP119 expressed on the surface of transiently transfected HEK293T cells was similar between the groups immunized with either αDEC-MSP142 or αDEC-MSP119 mAbs, when we considered dilutions where both sera presented similar OD values. Despite the similar binding observed when similar amounts of anti-MSP119 antibodies were used, the fact that immunization with αDEC-MSP142 induces higher antibody titres with increased avidity may be favourable in the field where higher anti-MSP119 titres have been associated with protection against malaria40.

The results involving the anti-MSP119 antibody response described above indicated that CD4+ T cells were being activated during immunization with the αDEC-MSP142 mAb, as the initial T-B cell interaction leads to germinal centre formation, affinity maturation, and isotype switching. The increased production of IgG2c prompted us to investigate if T cells were able to produce IFN-γ, a cytokine associated with the production of this subclass41. We were able to detect high numbers of IFN-γ producing T cells in both mouse strains immunized with αDEC-MSP142 mAb only when splenocytes were pulsed with the recombinant MSP133 protein. This indicated that the immunodominant epitopes were restricted to the 33-kDa fragment of the molecule. Interestingly, we did not observe a noticeable response when the rec. MSP133 was used as immunogen. On the other hand, we were able to detect IFN-γ producing T cells in animals immunized with rec. MSP119 whose splenocytes were pulsed with the same protein. In addition, we found that CD4+ T cells induced by immunization with αDEC-MSP142 mAb were able to produce different combinations of three inflammatory cytokines (IFN-γ, IL-2 and TNFα), and proliferate, when pulsed with the MSP133 recombinant protein. These results showed that the presence of immunodominant epitopes in the 33-kDa fragment of MSP1 activate polyfunctional CD4+ T cells and the anti-MSP119 antibody response. In B10.A mice, we were able to detect a specific response against the previously defined DYDVVYLKPLAGMYK epitope37, but more epitopes are probably present on the MSP133 sequence as this response was weaker than that observed with the recombinant MSP133 pulse. To completely map the immunodominant epitopes, additional experiments using peptide libraries will be necessary. Induction of CD4+ T cell proliferation and cytokine production after immunization with a hybrid αDEC205 mAb in the presence of poly (I:C) has been reported previously by Trumpfheller et al.15. The use of a αDEC205 mAb fused to a HIV protein elicited mainly CD4+ T cells that proliferated and produced the three inflammatory cytokines (IFN-γ, IL-2 and TNFα). In our case, we detected CD4+ T cells that proliferated vigorously after recombinant MSP133 pulse and were capable of producing not only three cytokines simultaneously, but also two or one. On the other hand, immunization with αDEC-MSP119 mAb or with the rec. proteins was extremely inefficient in inducing either proliferation or cytokine production. These results highlight the importance of the antigen choice when targeting the DEC205 receptor on the surface of CD8α+ DCs.

Of note, we have to mention that contrary to other results previously reported on the literature5,6,7,13,21, we did not detect CD8+ T cell proliferation or cytokine production when MSP142 or MSP119 were targeted to the DEC205+ DCs. The simpler explanation may be that P. vivax MSP142 sequence does not contain CD8+ T cell epitopes or those epitopes are not recognized by C57BL/6 and B10.A haplotypes. We were unable to find in the literature any previous reports mapping CD8+ T cell epitopes to both P. vivax MSP142 and MSP119 proteins. However, we found one report that described CD8+ T cell epitopes in the sequence of the murine P. yoelii MSP14233. It is important to mention that the amino acid sequences of MSP1 from P. vivax and P. yoelii are quite distinct, and that the CD8+ T cells were detected in BALB/c mice after immunization with a recombinant adenovirus. Besides, CD8+ T cells were detected after splenocytes were pulsed with peptide pools33. Indeed, almost every time CD8+ T cells were detected after antigen targeting to CD8α+DEC205+ DCs, detection was measured using overlapping peptide libraries or previously described peptides, and frequently previously enriched CD8+ T cells5,6,7,21. In this work, we did not use overlapping peptide libraries or CD8+ T cell enrichment. Instead, bulk splenocytes were pulsed directly with the rec. proteins. Another explanation that may account for the absence of CD8+ T cell detection may have to do with the timing of analysis. Here we analysed cellular immune responses on day 20 after boost while others normally analyse T cell responses at earlier time points16. In an attempt to maximize our window of detection, we analysed the animals on day 5 after the boost, and still could not detect CD8+ T cell proliferation or cytokine production (data not shown). However, we still cannot rule out the possibility of CD8+ T cell activation because we did not enrich the T cells or used peptide libraries. In fact, we plan to explore those possibilities in the future. Finally, it is important to point out that MSP142 is an antigen expressed during the erythrocytic phase of Plasmodium life cycle, and the evidence points to a role of antibodies and CD4+ T cells in protection, while CD8+ T cells would have a more pronounced role during the pre-erythrocytic phase, before the parasites reach the blood.

Taken together, our results show that the choice of the antigen may be important when designing vaccines targeted to the CD8α+DEC205+ DC subset.

Material and Methods

Mice

Six- to 8-week-old female C57BL/6 and C57BL/6 CD4 KO mice were bred at the Isogenic Mouse Facility of the Parasitology Department, University of São Paulo, Brazil. Female B10.A mice were obtained from the Isogenic Mouse Facility of the Immunology Department, University of São Paulo, Brazil. Female C57BL/6 MHCII KO mice were obtained from the Division of Immunology, Federal University of São Paulo (UNIFESP). All protocols were approved by the Institutional Animal Care and Use Committee (CEUA) of the University of São Paulo (protocol number 082) and all the animals were handled according to the Brazilian College of Animal Experimentation guidelines. In addition, all experimental methods were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Brazilian National Law (11.794/2008).

Plasmid generation

The sequence encoding aminoacids 1326 to 1705 from the P. vivax MSP1 protein (Belem strain, accession number AF435594.142) was synthesized by GenScript (Piscataway, NJ, USA) with codon optimization for expression in mammalian cells. This sequence corresponds to the 42 kDa portion of the MSP1 protein (MSP142). The three putative glycosylation sites (NIT, NES and NVT) were substituted by NII, EES and DVT. Amplification was accomplished using the Phusion High Fidelity DNA Polymerase (New England Biolabs) according to the manufacturer’s instructions. A 1,140 bp fragment was amplified, cloned into the pJET 1.2/blunt vector (ThermoScientific) and then digested with the restriction enzymes Xho I and Not I (New England Biolabs). After digestion, the fragment was ligated in frame with the carboxyl terminus of the heavy chain of mouse αDEC205 (NLDC145 clone) or with an isotype control (GL117 clone) mAb (kindly provided by Dr. Michel C. Nussenzweig, The Rockefeller University), as previously described14. In addition, a 267 bp fragment (amino acids 1617 to 1705) corresponding to the 19 kDa portion of the MSP1 protein (MSP119) was also amplified and cloned as described above. The final plasmids were named pDEC-MSP142, Iso-MSP142, pDEC-MSP119 and Iso-MSP119, and sequenced to confirm the presence of either MSP142 or MSP119 sequences in frame.

For the production of the recombinant MSP119 protein, we used the plasmid pET14b-MSP119 previously described by ref. 43, while the sequence corresponding to amino acids 1326 to 1616 was amplified by PCR as described above and cloned into the pET28a vector. Plasmid pET28a-MSP133 was then generated.

Expression of recombinant hybrid mAbs and proteins

Plasmids containing the heavy chain of the mouse αDEC205 mAbs (pDEC-MSP119, pDEC-MSP142, or pDEC without any fused antigen) or the isotype controls and the respective light chains (pDEC kappa, kindly provided by Dr. Michel C. Nussenzweig, The Rockefeller University, New York, USA) were amplified in DH5α bacteria, and subsequently purified in large scale using the QIAGEN Maxi Prep kit (Qiagen), according to the manufacturer’s instructions.

Transient transfection in human embryonic kidney (HEK) 293T (ATCC No CRL-11268) cells was performed exactly as described in ref. 14. The recombinant fusion mAbs were purified with the aid of Protein G beads (GE Healthcare) according to the manufacturer’s instructions. After purification, all the fractions containing antibodies were pooled together, dialysed against 2 L cold PBS, and sterilized filtered through 0.2 μm membranes (TPP). The fusion mAbs αDEC-MSP142, αDEC-MSP119 and αDEC had their concentrations estimated by Bradford assay (Pierce), and an assay for the detection of LPS (QCL -1000, Lonza) was performed after purification of each batch. Samples containing less than 1 EU/mL were considered clean, and aliquots were stored at −20 °C until use. We were unable to produce the fusion mAb Iso-MSP142 as all our attempts resulted in degradation.

The recombinant MSP119 protein was produced according to the protocol described in ref. 43. A different protocol was developed for the production of the recombinant MSP133 protein. Briefly, a 125 ml bacterial pellet was dissolved in 5 ml of lysis buffer (50 mM Tris, 200 mM Nacl, 10% glycerol, pH 8.0). Next, PMSF to a final concentration of 1 mM was added and the solution was centrifuged at 28,000 × g for 45 minutes. The supernatant was incubated with 5 ml of Ni-NTA (Quiagen) previously equilibrated in lysis buffer for 2 hours at 4 °C, under rotation. After 4 washes with 10 ml of wash buffer (50 mM Tris, 500 mM NaCl, 10% glycerol, 30 mM imidazole, pH 8.0), the protein was eluted from the Ni-NTA matrix using 5 ml of elution buffer (50 mM Tris, 1 M Nacl, 10% glycerol, 500 mM imidazole pH 8.0) for 1 hour at 4 °C.

Immunoblots

Approximately 1 μg of each fusion mAb or the recombinant protein were resolved on 7 or 12% SDS-PAGE gels under non-reducing or reducing conditions, respectively. Gels were either stained with Coomassie Blue (Amresco) or transferred to nitrocellulose membranes (GE Healthcare). Coomassie Blue stained gels were then scanned using a Lexmark 3600–4600 series scanner and transformed into grayscale using AdobePhotoshop CC software (Adobe Systems Incorporated 2013). Nitrocellulose membranes were blocked for an 1 hour at room temperature (rt) in 0.05% PBS-Tween 20 (PBS-T), 5% non-fat milk and 1% BSA, and then incubated with serum (1:2,000 dilution) derived from a patient previously infected with P. vivax (kindly provided by Dr. Claudio R.F. Marinho, University of São Paulo, Brazil). After a 2-hour incubation at rt the membranes were washed twice and incubated for an additional hour using an anti-human IgG-HRP (1:5,000, Jackson Laboratories). After two additional washes, the membranes were developed using quimioluminescence (ECL kit, GE Healthcare) and captured on Kodak film. The films were then scanned and submitted to the same processing described above for Coomassie Blue gels.

Binding assay

Spleens from naïve mice were removed and splenocytes were obtained after erythrocyte lysis with ACK buffer (0.1 mM EDTA, 0.15 mM NH4Cl, 1 mM KHCO3). Five million splenocytes were incubated in PBS-FBS (fetal bovine serum) 2% containing Fc Block (anti-CD16/32, BD Biosciences) at a 1:100 dilution. After a 15-minute incubation on ice, αDEC-MSP142, αDEC-MSP119 or αDEC purified mAbs were diluted to 10, 1, 0.1 μg/ml and added to the wells. After 45 minutes of incubation, the cells were centrifuged and washed twice with PBS-FBS 2%. Another 45-minute incubation on ice followed in the presence of anti-mouse IgG1-PE (clone A85-1), anti-CD11c-APC (clone N418), anti-CD49b-biotin (Clone DX5), anti-CD19-biotin (clone1D3), anti-MHCII-FITC (2G9) and anti-CD8-PE-Cy7 (clone 53-6.7). After 2 more washes, cells were incubated on ice with Streptavidin-PerCP for 30 minutes. All mAbs were purchased from BD biosciences. Finally, after two final washes, half a million events were then read in a FACS Canto flow cytometer (BD biosciences), and analysed using FlowJo software (version 9.3, Tree Star, San Carlo, CA).

Immunization schedule

Groups of 5–10 female mice were immunized intraperitoneally with 5 μg of the following hybrid mAbs: αDEC-MSP142, αDEC-MSP119 or αDEC (as a negative control) in the presence of 50 μg of poly (I:C) (Invivogen). Groups of 5 female C57BL/6 also received 1.5 μg of rec. MSP133 or 1 μg of rec. MSP119 in the presence of the same amount of poly (I:C). This amount corresponds to the same number of molecules of either MSP133 or MSP119 present on 5 μg αDEC-MSP142 mAb. Thirty days after the prime, animals received a second dose containing exactly the same amount of fusion mAbs and adjuvant. Animals were bled 5 days before or 14 days after the administration of the second dose and their sera were used for the analysis of the humoral response. Assays to evaluate the cellular immune response were performed on day 20 after the administration of the second dose, when the animals were euthanized.

Analysis of the antibody responses

For the detection of antibodies against either MSP119 or MSP133, sera from immunized mice were used in ELISA assays, exactly as described previously14. Briefly, high binding ELISA plates (Costar) were coated overnight at room temperature (rt) with 100 ng/well of MSP119 or MSP133 recombinant proteins diluted in PBS. After three washes and one-hour incubation in blocking buffer (PBS-Tween 20 0.02%, non-fat milk 5% and BSA 1%), sera were serially diluted in PBS-Tween 20 0.02%, non-fat milk 5% and BSA 0.25% and incubated for 2–3 h at rt. The secondary antibodies goat anti-mouse IgG Fc-specific-HRP (1:10,000; Jackson ImmunoResearch Laboratories) or anti-mouse IgG subclass-HRP specific antibodies (1:3,000; SouthernBiotech) were added after three additional washes. After one-hour incubation at rt, plates were vigorously washed and the enzymatic reaction was developed by the addition of 1 mg/ml of ortho-phenylenediamine dihydrochloride (Sigma) diluted in phosphate–citrate buffer, pH 5.0, containing 0.03% (v/v) hydrogen peroxide. Reactions were stopped using sulfuric acid 4N. OD490 was measured using a microplate reader (Biotek). Titres represent the highest serum dilution showing an OD490 ≥ 0.1 normalized in a log10 scale. The IgG1/IgG2c ratio was calculated by dividing the mean values of the highest serum dilution obtained for IgG1 by the mean value of the highest serum dilution obtained for IgG2c without normalization. The avidity index was calculated using an extra step of incubation with 7M urea for 5 min, exactly as described previously44,45.

Analysis of T cell responses

Splenocyte isolation

After mice were euthanized, spleens were removed aseptically and processed exactly as described by ref. 14. Bulk splenocytes were ressuspended in R10 [RPMI supplemented with 10% of fetal bovine serum (GIBCO), 2 mM L-glutamine (GIBCO), 10 mM Hepes (GIBCO), 1 mM sodium pyruvate (GIBCO), 1% vol/vol non-essential aminoacid solution (GIBCO), 1% vol/vol vitamin solution (GIBCO), 20 μg/mL of ciprobacter (Isofarma, Brazil) and 5 × 10−5 M 2-mercaptoetanol (GIBCO)]. Cell viability was evaluated using 0, 1% Trypan Blue exclusion dye and cell concentration was estimated using a hemocytometer.

IFN-γ ELISPOT

ELISPOT assays for the detection of IFN-γ producing splenocytes were performed using the Ready-SET-Go kit (eBioscience), according to the manufacturer’s instructions. Three hundred thousand splenocytes were incubated in the presence of 1 μg/mL of the recombinant MSP119 or MSP133 proteins. Control cells were left unpulsed. The AEC kit (BD biosciences) was used to develop the spots that were counted with the aid of an automated stereomicroscope (KS ELISPOT, Zeiss, Oberkochem, Germany). The number of IFN-γ producing cells/106 splenocytes was calculated after subtracting the number of cells in the unpulsed wells.

Detection of IFN-γ, IL-2 and TNFα producing cells by intracellular staining

Splenocytes isolated from immunized mice were obtained as described above, and plated in round-bottomed 96-well plates at a concentration of 1 × 106 cells/well in triplicates. The cells were then incubated with the recombinant proteins MSP133 or MSP119 (5 μg/mL) in R10 medium containing 2 μg/mL of the αCD28 agonist antibody. As negative controls, some cells were left unpulsed while others were incubated with 1 μg/mL αCD3 as positive controls. After one-hour incubation at 37 °C and 5% CO2, 0.5 μg of Golgi Plug (Brefeldin A, BD Pharmingen) was added to each well and plates were re-incubated for another 12 hours. After this period, the plates were centrifuged for 5 min at 1,000 × g and the supernatant was discarded by inversion. Cells were then washed with PBS-FBS and transferred to V-bottomed 96-well plates. The cells were then surface stained with αCD4-PerCP-Cy5.5 mAb (clone RM 4–5) for 45 minutes on ice in PBS-FBS. After 3 washes, cells were resuspended in PharmingenStain buffer (BD Pharmingen) for 10 min on ice, centrifuged, and fixed and permeabilized using the Cytofix/Cytoperm kit (BD Pharmingen). After a 15-min incubation on ice, the plates were centrifuged and washed 3 times with PermWash buffer (BD Pharmingen). The intracellular staining was performed using αCD3-APC-Cy7 (clone 145-2C11), αIFN-γ-APC (clone XMG1.2), αIL2-FITC (clone JES6-5H4), αTNFα-PE (clone MP6-XT22) mAbs for 45 minutes on ice. After three more washes with PermWash buffer, cells were resuspended in PBS-FBS, and one million events were acquired in a FACSCanto flow cytometer (BD biosciences), and analysed using FlowJo software (version 9.3, Tree Star, San Carlo, CA). All antibodies used were purchased from BD Pharmingen.

CFSE-based proliferation assay

Three hundred thousand splenocytes from immunized mice were assayed for their ability to proliferate in vitro using the CFSE dilution based proliferation assay after stimulation with 5 μg/mL of the MSP133 and MSP119 recombinant proteins or with peptides DYDVVYLKPLAGMYK and AKFVAAWTLKAAA, exactly as described in ref. 46.

Data Analysis

One-way ANOVA followed by Tukey’s honestly significantly different (HSD) test were used to calculate statistical significance (p-values). Prism 5 software (GraphPad Software Inc, LA Jolla, CA) was used for all tests and differences were considered significant when p ≤ 0.05.

Additional Information

How to cite this article: Amorim, K. N. S. et al. The presence of T cell epitopes is important for induction of antibody responses against antigens directed to DEC205+ dendritic cells. Sci. Rep. 6, 39250; doi: 10.1038/srep39250 (2016).

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