USES OF PARASITE MACROPHAGE MIGRATION INHIBITORY FACTORS

Abstract

This invention relates to compositions (e.g. vaccine compositions) which can be used to provide a subject with protective immunity against a parasite infection. The compositions comprise: (i) an immunologically effective amount of a nucleic acid (e.g. a nucleic acid-based vaccine) comprising a sequence which encodes a parasite macrophage migration inhibitory factor (MIF) antigen; (ii) a parasite MIF antigen; or (iii) an antibody which specifically binds to a parasite MIF antigen. The compositions may be used to treat infections and diseases caused by parasitic protozoans, such as a Plasmodium parasite, or parasitic helminths.

Claims

1. A composition for use in a method of providing protective immunity against a parasite infection in a subject in need thereof, which comprises an immunologically effective amount of one or more of: (i) a nucleic acid comprising a sequence which encodes a parasite macrophage migration inhibitory factor (MIF) antigen; (ii) a parasite MIF antigen; or (iii) an antibody which specifically binds to a parasite MIF antigen.

2. The composition for use according to claim 1 (i) wherein the composition comprises an RNA-based vaccine.

3. The composition for use according to claim 1 or claim 2 wherein the protective immunity is characterized by protective immunological memory against the parasite and/or an effective parasite-responsive memory T cell population.

4. The composition for use according to claim 1, 2 or 3 wherein the composition comprises a nucleic acid-based vaccine comprising the nucleic acid sequence which encodes a parasite MIF antigen; for example, wherein the nucleic acid-based vaccine is a RNA-based vaccine, which may comprises a self-replicating RNA molecule, such as an alphavirus-derived RNA replicon.

5. The composition for use according to any preceding claim wherein the composition comprises a cationic nano-emulsion (CNE) delivery system or a lipid nanoparticle (LNP) delivery system.

6. The composition for use according to any preceding claim wherein the composition comprises one or more adjuvants.

7. The composition for use according to claim 1 wherein the antibody which specifically binds to a parasite MIF antigen comprises monoclonal antibody or polyclonal antibody.

8. The composition for use according to claim 1 wherein the antibody which specifically binds to a parasite MIF antigen is a human, humanized or chimeric monoclonal antibody.

9. The composition for use according to any preceding claim wherein the parasite is a parasitic protozoan, for example wherein (i) the protozoan is an apicomplexan parasite; and/or (ii) the protozoan belongs to a genus selected from the group consisting of: Plasmodium, Toxoplasma, Babesia, Eimeria, Theileria, Neospora, Sarcocystis, Leishmania, and Trypanosoma.

10. The composition for use according to any one of claims 1 to 8 wherein the parasite is a parasitic helminth, such as a nematode.

11. The composition for use according to claim 10 wherein the parasitic helminth belongs to a genus selected from the group consisting of: Ancyclostoma, Necator, Brugia, Wuchereria, Loa, Mansonella, Trichinella, Trichuris, Ascaris, Anisakis, Dracunculus, Strongyloides, Haemonchus, Schistosoma and Fasciola.

12. The composition for use according to any preceding claim wherein the subject is a vertebrate, such as a mammal e.g. a human or a veterinary mammal.

13. The composition for use according to any preceding claim wherein: (a) the composition further comprises a nucleic acid sequence which encodes an additional parasite antigen, and/or (b) the composition further comprises an additional parasite antigen, and/or (c) the composition is administered to the subject in combination with a further composition which comprises a nucleic acid comprising a sequence which encodes an additional parasite antigen; and/or (d) the composition is administered to the subject in combination with a further composition which comprises an additional parasite antigen.

14. A composition comprising an immunologically effective amount of: (i) a nucleic acid comprising a sequence which encodes a parasite MIF antigen; or (ii) a parasite MIF antigen; wherein the MIF antigen is from: (a) a parasitic protozoan; or (b) a parasitic helminth which belongs to a genus selected from the group consisting of: Ancyclostoma, Necator, Brugia, Wuchereria, Loa, Mansonella, Trichinella, Trichuris, Ascaris, Anisakis, Dracunculus, Strongyloides, Haemonchus, Schistosoma and Fasciola.

15. The composition of claim 14 wherein the parasitic protozoan is an apicomplexan parasite and/or belongs to a genus selected from the group consisting of: Plasmodium, Toxoplasma, Babesia, Eimeria, Theileria, Neospora, Sarcocystis, Leishmania, and Trypanosoma.

16. The composition of any one of claims 14 to 15 wherein the parasite MIF antigen is a full-length MIF polypeptide or an immunogenic fragment thereof.

17. The composition of any one of claims 14 to 16 which comprises a nucleic acid-based vaccine comprising the nucleic acid sequence which encodes a parasite MIF antigen; for example, wherein the nucleic acid-based vaccine is a RNA-based vaccine, such as a self-replicating RNA molecule, which may be an alphavirus-derived RNA replicon.

18. The composition of any one of claims 14 to 17 which comprises a cationic nano-emulsion (CNE) delivery system or a lipid nanoparticle (LNP) delivery system.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0166] FIGS. 1A-1D show malaria-specific T cell responses in mice immunized with: RNA encoding PbMIF in a CNE delivery vehicle (PbMIF-CNE); RNA encoding GFP in a CNE delivery vehicle (GFP-CNE); or mice immunized with PbMIF protein with Freund's adjuvant (PbMIF-FA); at 7 days after Plasmodium infection on day 40 post-immunization. *P<0.05. FIG. 1A shows % CD4.sup.+ Ki67.sup.+. FIG. 1B shows B-bet MFI. FIG. 1C shows % PbA responding CD4 T cells. FIG. 1D shows IGN- MFI.

[0167] FIGS. 2A-B showneutralization activity of PbMIF immunization on (FIG. 2A) Plasmodium parasitemia and (FIG. 2B) spleen parasite content at 7 days post-infection. *P<0.05

[0168] FIG. 3 shows mean serum anti-PbMIF titers (OD.sub.450 nm) at day 14 (2 weeks after first immunization) for mice immunized with RNA/PMIF CNE or RNA/GFP CNE. Mean titers are expressed as endpoint titers; the reciprocal dilution that yields background OD.sub.450 nm. ****P<0.0001 Mann-Whitney U, n=15 per group.

[0169] FIG. 4 shows mean serum anti-PbMIF titers (OD.sub.450 nm) at day 35 (2 weeks after second immunization) for mice immunized with RNA/PMIF CNE or RNA/GFP CNE. Mean titers are expressed as in FIG. 3. ****P<0.0001 Mann-Whitney U, n=15 per group.

[0170] FIGS. 5A-C show: (FIG. 5A) a scheme for RNA/PbMIF CNE and RNA/GFP CNE vaccination, first parasite challenge, cure (CQ), and second parasite challenge; (FIG. 5B) Plasmodium parasitemia over 7 days following first parasite challenge (days 35 to 42); and (FIG. 5C) serum IFN levels on day 5 post-infection. *P<0.05, **P<0.01 Mann-Whitney U, n=15 per group.

[0171] FIG. 6A shows mean serum anti-PbMIF titers (OD.sub.450 nm) at day 59 (2.5 weeks after first infection) for mice immunized with RNA/PMIF CNE or RNA/GFP CNE (****P<0.0001 Mann-Whitney U, n=15 per group). FIG. 6B shows mean anti-Plasmodium IgG responses at day 59 (***P<0.01 Mann-Whitney U, n=15 per group). Mean titers are expressed as in FIG. 3.

[0172] FIG. 7 shows Plasmodium parasitemia in mice immunized with RNA/PMIF CNE or RNA/GFP CNE over 14 days following second challenge (days 59-73). *P<0.05, **P<0.01 Mann-Whitney U, n=9 total (4RNA/GFP CNE control, 5RNA/PMIF CNE).

[0173] FIGS. 8A-C show the effects of RNA/PMIF CNE versus or RNA/GFP CNE immunization on T cell phenotype. T cells responding to P. berghei (CD4+Ki67.sup.hi) were divided into subsets: Tmem (memory) CD62L.sup.+ IL-7R.sup.+; Tem (effector memory) CD62L.sup. IL-7R.sup.+; and Teff (effector) CD62L.sup. IL-7R.sup.. FIG. 8A shows percentages of individual T cell phenotypes at 7 days after first infection. FIG. 8B shows percentages of individual T cell phenotypes at 7 days after second infection. FIG. 8C shows percentages of Tmem cells which are PD-I.sup.+ (indicating T cell exhaustion). *P<0.05, **P<0.01 Mann-Whitney U.

[0174] FIG. 9 shows Plasmodium parasitemia in mice receiving passive transfer of a polyclonal anti-PbMIF antibody (IgG) compared to mice receiving control IgG from a non-immunized rabbit (Ctrl IgG) at day 7 post-infection. Each data point is a single mouse. P=0.0005 by t-test.

[0175] FIGS. 10A-E show that passive transfer of IgG from PbMIF immunized and blood-stage infected mice provides partial protection in both BALB/c and cerebral malaria-sensitive C57BL/6 Mice. (FIG. 10A) IgG isolated from GFP (control) or PbMIF immunized and PbA-infected mice was administered i.p. to nave BALB/c or C57BL/6 mice and followed by PbA infection. (FIG. 10B) Parasitemia and (FIG. 10C) Kaplan-Meyer survival analysis of BALB/c mice administered immune IgG. p values were generated using a Long-rank test and data are from two independent experiments with 5 mice per group (*p<0.05, **p<0.01). (FIG. 10D) Kaplan-Meyer survival plots and (FIG. 10E) ECM (Experimental Cerebral Malaria) score of C57BL/6 mice administered immune IgG and infected with PbA. Statistical p values were generated using a Long-rank test (**p<0.01) and data are representative of two independent experiments n=10 mice per group.

[0176] FIGS. 11A-G shows that adoptively transferred CD4 T cells from PbMIF immunized mice confer protection to homologous challenge. (FIG. 11A) Immunized CD45.2 BALB/c mice were infected with 10.sup.6 PbA-iRBCs and treated with chloroquine on days 7-12. Four weeks later, the mice were re-infected with PbA and splenocytes isolated 7 days after infection, incubated with chloroquine to eliminate residual Plasmodium, and labeled with CFSE. Purified CD4 T cells (210.sup.7) then were transferred into nave CD45.1 BALB/c mice and infected 3 days later with 10.sup.6 PbA-iRBCs. (FIG. 11B) Parasitemia in mice adoptively transferred with CD4 T cells from GFP () or PbMIF (.circle-solid.) immunized mice (*p<0.05, #p<0.001, by two-way ANOVA). The graph shows % parasitemia against days post-infection. (FIG. 11C) Representative CFSE dilution histogram of adoptively transferred (CD45.2) CD4 T cells from GFP or PbMIF immunized donors, (FIG. 11D) enumeration of recovered CD45.2 CD4 T cells, and (FIG. 11E) proliferation response of transferred CD4 T cells in CD45.1 recipients 7 days after infection. (FIG. 1F) Percent of proliferating CD45.2 CD4 T cells (CFSE.sup.lo) producing IFN after stimulation ex vivo with PbA-iRBC lysates. (FIG. 11G) Mean fluorescence intensity of PD-1 in PbA-responsive CD45.2 CD4 T cells (CFSE.sup.lo) from GFP (control) or PbMIF immunized donors. Results are from two separate experiments with 4 mice per group (*p<0.05, **p<0.01 by Mann-Whitney test.).

MODES FOR CARRYING OUT THE INVENTION

Example 1: Immunization Using P. berghei MIF, Followed by Parasite Challenge

[0177] Groups of 5 female BALB/c mice aged 8-10 weeks were immunized with: (1) RNA encoding P. berghei MIF (PbMIF) in an LNP delivery vehicle (RV01, see references 34 and 36); (2) RNA encoding PbMIF in a CNE delivery vehicle (comprising squalene and DOTAP, as described in reference 35); (3) RNA encoding GFP in a CNE delivery vehicle; or (4) intraperitoneal (i.p.) injection of PbMIF protein with Freund's adjuvant, as set out in Table 1. Immunizations were carried out on day 0 and day 21.

TABLE-US-00002 TABLE 1 Delivery # animals/ Group Antigen system Adjuvant Dose group 1 RNA/PbMIF LNP 1 g 5 2 RNA/PbMIF CNE 15 g 5 3 RNA/GFP CNE 15 g 5 4 PbMIF i.p. FCA/FIA 10/5 g 5 Protein

[0178] Blood samples were taken from immunized mice on day 14 and day 35 (14 days after boosting) and total serum anti-PbMIF IgG titers were measured by anti-PbMIF ELISA assay. Immunized mice were challenged on day 38-40 by i.p. injection of 10.sup.6 P. berghei ANKA (PbA)-infected red blood cells (RBCs). Mouse weights over time (from day 0 to day 40) and clinical appearance were also assessed for the mice in each experimental group.

Serum Anti-PbMIF IgG Titers from Immunized Mice and Tolerability to the Vaccine

[0179] IgG titers were measured 14 days after the first immunization and 14 days after the second boosting immunization. Anti-PbMIF ELISA assays were performed as follows: 96 well plates were coated overnight with 100 ng/ml of recombinant PbMIF. After blocking for 1 hr at room temperature, serial dilutions of sera were incubated for 2 hrs and total bound IgG was detected with a rabbit anti-mouse IgG coupled to horseradish peroxidase (HRP). 3,3,5,5-Tetramethylbenzidine (TMB) was used as substrate. The reaction was stopped with acid and the OD reading performed at 450 nm.

[0180] Immunization with PbMIF self-replicating RNA vaccine or PbMIF protein (Groups 1, 2 and 4) elicited primary and secondary humoral antibody responses to PbMIF. No significant responses were observed in control mice treated with RNA/GFP CNE (Group 3). Secondary responses were observed in 80% of mice treated with RNA/PMIF CNE (Group 2) and 60% of mice treated with RNA/PMIF LNP (Group 1), with comparable titers. Primary responses in Groups 1 and 2 were 100-fold less and secondary responses in Groups 1 and 2 were 1000-fold less than mice treated with PMIF protein FCA/FIA (Group 4).

[0181] Mice tolerated immunization well, with no changes in clinical appearance (clinical observation q3d) and no reductions in weight among the different experimental groups from day 0 to day 40.

Impact of PbMIF Immunization on Malaria-Specific T Cell Responses

[0182] Groups 2, 3 and 4 were selected for study. Immunized mice were challenged on day 40 by i.p. injection of 10.sup.6 PbA-infected RBCs. On day 7 post-infection, splenocytes were isolated and stimulated ex vivo by culturing with infected red blood cell lysates in the presence of anti-CD3/CD28 beads and brefeldin A for 6 hrs. Intracellular cytokine staining was then performed. The following antibodies were used to study CD4 T cell IFN- production, T cell activation (CD11a) and T cell differentiation (T-bet): Ki67 FITC, CD45.2 PerCP-Cy5.5, IFN PE-Cy7, CD4 Alexa 700, CD11a eFluor 405, T-bet Alexa 647 (Life Technologies). PbA-responsive CD4 T cells are defined as CD45.2+, Ki67+, CD4+. Stained cells were analysed by flow cytometry.

[0183] On day 7 post-infection, PbMIF immunized mice (Groups 2 and 4) showed a stronger T cell proliferative response to P. berghei parasites (higher CD4+Ki67+ cells) a stronger memory CD4 T cell response to parasites, as indicated by lower CD11a and lower T-bet (FIGS. 1A and 1B) and fewer inflammatory, terminal effector IFN- producing T cells (FIGS. 1C and 1D) than control mice (Group 3).

[0184] For example, flow cytometry showed that 5.26% of splenic CD4 T cells from RNA/PMIF CNE (Group 2) immunized mice were inflammatory, terminal effector IFN--producing CD4 T cells (labelled with antiCD4 and stained for IFN- with an anti-IFN- antibody), compared to 11.1% of splenic CD4 T cells from control RNA/GFP CNE (Group 3) immunized mice (using pooled cells from 5 mice per group).

[0185] Further studies have shown a >65% increase in the number of Plasmodium-responsive memory CD4 T cells (CD62L+IL7R+) and effector memory CD4 T cell precursors (CD62L-IL7R+), as well as a 20% reduction in the expression of the exhaustion marker PD-1 in PbMIF-CNE immunized mice compared to GFP-CNE control mice at day 7 post-infection (n=5 per group, based on two separate experiments), suggesting a relative preservation of the memory response in the PbMIF immunized mice versus the control group. It has also been shown that, at day 7 post-infection, the number of Plasmodium-responsive follicular helper CD4 T cells (T.sub.FH cellsCD49d.sup.hiCD11a.sup.hi CXCR5.sup.hi CD4) was 50% greater in PbMIF-CNE immunized mice compared to GFP-CNE control mice. Consistent with this observed increase in the population of T.sub.FH cells, there was a corresponding enhancement in splenic B cell numbers, with a 30% increase in (CD19.sup.+B220.sup.+) B cells and a greater than doubling of the B cell plasmablast population (CD19.sup.lo B220.sup.loCD138.sup.hiIgD.sup.). Thus, PbMIF immunization is associated with an improvement in the host T.sub.FH and B cell responses.

Assessment of the Neutralization Activity of PbMIF RNA Vaccinations on P. berghei Parasitemia and Spleen Parasite Content

[0186] On day 7 post-infection, parasitemia was also studied in immunized, challenged mice from Groups 2 and 3. Parasite burden was measured by quantitative PCR detection of P. berghei 18S rRNA copies/L of peripheral blood, and splenic parasite burden was measured by expression of 18S rRNA relative to host GAPDH.

[0187] As shown in FIG. 2, PbMIF-CNE immunization was associated with a significant reduction in parasitemia (30%) and spleen parasite content upon challenge with lethal P. berghei infection.

Parasitemia and Survival During First Infection

[0188] In a separate experiment, BALB/c mice previously immunized with the RNA/PbMIF-CNE vaccine or GFP-CNE were infected with PbA and the parasitemia followed by FACS. Parasitemia was significantly reduced in the RNA/PbMIF-CNE-immunized mice compared to GFP-CNE-immunized control mice. While there was no initial difference in parasitemia between the two groups, there was a more rapid increase in parasitemia after day 5 in the control (GFP-CNE) group, which became moribund on day 21. By contrast, the PbMIF immunized mice showed better control of parasitemia during the first 15 days of infection. Survival was also monitored for up to 30 days post-infection and RNA/PbMIF-CNE-immunized mice showed a 37% prolongation in mean survival time compared to GFP-CNE-immunized control mice.

Conclusions (1)

[0189] PbMIF protein and self-replicating RNA vaccines are well-tolerated and produce a primary and secondary humoral antibody response in BALB/c mice.

[0190] RNA/PbMIF immunization (with CNE) neutralized Plasmodium PbMIF activity, and enhanced CD4 T cell memory differentiation. In addition, the neutralization of PbMIF activity significantly reduced parasitemia and parasite content of spleens and significantly improved mean survival times in infected mice. Thus, PbMIF immunization confers at least partial protection to first challenge infection.

Example 2: Immunization Using P. berghei MIF in CNE RNA Delivery Vehicle, Followed by Parasite Challenge, Cure and Re-Challenge

[0191] Example 2 differs from Example 1 in the addition of a first parasite challenge, followed by cure to expand the Plasmodium-specific memory T cell population.

[0192] Groups of 15 female BALB/c mice aged 8-10 weeks were immunized with: (1) RNA encoding PbMIF in a CNE delivery vehicle; and (2) RNA encoding GFP in a CNE delivery vehicle, as set out in Table 2. Immunizations were carried out on day 0 and day 21.

TABLE-US-00003 TABLE 2 Delivery # animals/ Group Antigen system Adjuvant Dose group 1 RNA/PbMIF CNE 15 g 15 2 RNA/GFP CNE 15 g 15

[0193] Blood samples were taken from immunized mice on days 14 and 35 and total serum anti-PbMIF IgG titers were measured by anti-PbMIF ELISA assay. Immunized mice were challenged on day 35 by i.p. injection of 10.sup.6 PbA-infected RBCs. This was followed by cure with chloroquine (CQ) (50 mg/kg/day) on days 7-10 post-challenge (days 42-45).

[0194] Readouts following first challenge: [0195] Parasitemia; days 3, 5 and 7 post-challenge (days 38, 40, 42). [0196] Total serum anti-PbMIF Ig and total anti-Plasmodium Ig after CQ cure (before 2.sup.na challenge, day 59).

[0197] Immunized mice were re-challenged with P. berghei on day 59 and infection was followed for 7 or 14 days post-re-challenge. 5 mice/group were euthanized at day 4 or 7 and the remaining mice were monitored for parasitemia.

[0198] Readouts: [0199] Parasitemia; days 5, 8, 11 and 14 post-challenge (days 64, 67, 70, 73). [0200] T cell phenotypes (day 66).
Serum Anti-PbMIF IgG Titers on Day 14 (2 Weeks after First Immunization) and Day 35 (2 Weeks after Second Immunization)

[0201] Serum IgG titers were measured by anti-PbMIF ELISA on day 14, following the first immunization and day 35, following the second immunization. Anti-PbMIF ELISA assays were performed as in Example 1.

[0202] Serum anti-PbMIF IgG titers were measured for each mouse at day 14. An anti-PbMIF IgG response (1/2500) was observed in 50% of PbMIF-immunized mice in Group 1 after the first immunization. Mean titers at day 14 are shown in FIG. 3.

[0203] Also, serum anti-PbMIF IgG titers were measured for each mouse at day 35. An increase of the anti-PbMIF IgG titers was observed in 95% of PbMIF-immunized mice in Group 1 after the second immunization in the PbMIF-CNE group and the titers are 5-fold higher compared to those at day 14 (1/14,500).

Challenge Infection on Days 35-42 to Expand Plasmodium-Specific Memory T Cells

[0204] Parasitemia was assessed on days 3, 5, and 7 following first challenge (days 35-42) as described in Example 1. Serum IFN was also assessed by specific ELISA on day 5 post-infection.

[0205] As shown in FIG. 5B, a detectable (0.5 log) difference in parasitemia was observed at 7 days following first Plasmodium infection. This effect is associated with reduced circulating IFN that is consistent with reduced levels of a highly inflammatory, IFN+ T cell response (FIG. 5C).

Serum Anti-PbMIF and Anti-Plasmodium IgG Titers on Day 59, 2.5 Weeks after the First Infection

[0206] Serum anti-PbMIF IgG titers were measured for each mouse at day 59. Plasmodium infection increased the titers of anti-PbMIF Ig by 10-fold (FIG. 6A) in RNA/PbMIF versus RNA/GFP mice. Also, serum anti-Plasmodium IgG titers were measured at day 59 by anti-mouse IgG ELISA on parasitized red cell lysates. Plasmodium infection increased anti-Plasmodium Ig titers by 5-fold (FIG. 6B) in RNA/PbMIF versus RNA/GFP mice.

Parasitemia Following Re-Challenge with Plasmodium on Day 59

[0207] Parasitemia was assessed on days 5, 8, 11 and 14 following second challenge (days 59-73) as described in Example 1.

[0208] As shown in FIG. 7, RNA/PbMIF immunization markedly reduced parasitemia (5 log at day 14 post-infection) after Plasmodium re-challenge on day 59.

Assessment of RNA/PbMIF Effects on Plasmodium-Specific T Cell Phenotype

[0209] Mice were euthanized at day 7 after second infection and the T cell response and phenotype analysed along with the cytokine production. Splenic cells were isolated from 4-5 mice per experimental group and analysed for CD62L and IL-7R staining. CD62L and IL-7R identify different T cell subsets responding to P. berghei (CD4.sup.+Ki67.sup.hi). The results are summarized in Table 3.

TABLE-US-00004 TABLE 3 Tmem: CD4+ P. berghei-responding T memory Antigen cells phenotyped as CD62L+IL-7R+ PbMIF-CNE 29.5% (Group 1) GFP-CNE 17.9% (Group 2 - control)

[0210] Thus, PbMIF neutralization increases the pool of P. berghei-responding CD4+ memory T cells.

Impact of RNA/PbMIF Vaccine on Plasmodium-Specific T Cells

[0211] T cell phenotypes observed after first Plasmodium infection (day 7 post-infection, data from study in Example 1) (FIG. 8A) were compared with T cell phenotypes observed after second Plasmodium infection (day 7) (FIG. 8B).

[0212] As can be seen in FIGS. 8A and 8B, the RNA/PbMIF-CNE vaccine promotes the development of Plasmodium-specific Tmem cells (CD62L IL-7Rac) during a first infection by P. berghei and these Tmem cells increase further during second infection.

[0213] Furthermore, T cell exhaustion, as indicated by PD-1-expressing Plasmodium-specific Tmem cells, was reduced in RNA/PbMIF-immunized mice versus RNA/GFP immunized mice (FIG. 8C).

T Cell Cytokine Production by Plasmodium-Responsive T Cell Subsets

[0214] IFN production by Tem (short-lived effector memory cells) is reduced in PbMIF immunized animals. This may reflect a less inflammatory (and more effective) Plasmodium-specific T cell response. No evident changes in TNF producing T cells were noted. (Data not shown)

Conclusions (2)

[0215] Self-replicating RNA vaccines are well-tolerated and produce strong humoral responses to Plasmodium MIF (PbMIF). First and second immunizations induced a response in 50% and 95% of immunized mice, respectively, with a much higher titer after the second immunization, and again after PbA infection.

[0216] RNA/PbMIF (CNE) immunization neutralized Plasmodium PbMIF activity, as evidenced by enhancement in CD4 T cell memory differentiation. The effects of this neutralization are: [0217] Higher Tmem cell numbers, and a stronger humoral anti-Plasmodium antibody response. After primary challenge with P. berghei, there is a measurable and significant decrease in parasitemia (0.5 log). [0218] Re-challenge after cure of primary infection results in a further expansion of Tmem numbers and a significant reduction in parasitemia (5 log).

[0219] Immunization with RNA/PbMIF allows for the development of memory T cells and provides significant protection to malaria re-challenge. RNA/PbMIF may be a viable vaccine candidate, either as a stand-alone or in combination with other Plasmodium vaccine candidates, where it would act to promote long-lasting memory T cell responses.

Example 3: Passive Transfer of a Polyclonal Anti-PBMIF Antibody

[0220] IgG was purified from a rabbit immunized with recombinant PbMIF (Anti-PbMIF IgG) or from a non-immunized rabbit (Ctrl IgG) and 200 g injected i.p. into C57BL/6 mice at 6 hrs, 24 hrs, 48 hrs, and 72 hrs after infection with 110.sup.6 P. berghei parasitized red cells. Parasitemia was enumerated by quantitative PCR of blood at day 7 post-infection.

[0221] As shown in FIG. 9, passive transfer of the anti-PbMIF antibody significantly reduces parasitemia in the P. berghei infection mouse model of malaria.

[0222] In a further study, 200 g of IgG purified from serum by protein-G chromatography (Pierce) from GFP (control) or PbMIF immunized and PbA-infected mice was administered i.p. to nave BALB/c or C57BL/6 (cerebral malaria-sensitive) mice and followed by PbA infection (FIG. 10A). I.p. administration was on days 1, 1, 2, and 3 post-infection. In C57BL/6 mice, cerebral malaria was monitored and symptoms classified by the Rapid Murine Coma and Behavior Scale (50), where 16=no signs, 15-11=mild symptomatic, 10-08=prodromal signs of Experimental Cerebral Malaria (ECM), 07-04=ECM. Agonal mice were immediately sacrificed.

[0223] Administration of IgG from PbMIF immunized mice into nave BALB/c mice that were infected with PbA provided partial protection, with a delayed rise in parasitemia, a 30% reduction in peak parasitemia, and a 30% prolongation in survival time (FIGS. 10B and C). When studied in the acutely lethal C57BL/6 model of cerebral malaria, IgG from the GFP (control) immunized mice showed no protection: all mice developed neurological signs and became moribund between days 6-7 post-infection (FIGS. 10C and E). By contrast, in C57BL/6 mice that received IgG from PbMIF immunized mice and then were infected with PbA, there was a marked delay in the time to develop neurologic disease, less severe disease, and a survival rate of 30%, with complete protection from neurologic manifestations. Cerebral malaria is a uniformly fatal complication of PbA infection in C57BL/6 mice and the protective effect of immunoglobulin occurred despite equivalent parasitemia between groups until the time of lethality (day 6), after which surviving mice eliminated their parasites (data not shown).

Example 4: Protective Effect of CD4 T Cells from Vaccinated Mice

[0224] BALB/c mice (CD45.2.sup.+) immunized with the RNA/PbMIF-CNE or RNA/GFP-CNE vaccine were infected by i.p. injection of 10.sup.6 PbA-infected RBCs on day 0 and treated with chloroquine on days 7 to 10. At 4 weeks previously infected mice were reinfected and on day 7 post-infection, the splenocytes were isolated and the CD4 T cells were incubated with 10 mM chloroquine for 2h at 37 C. and CFSE labeled. 510.sup.6 CD4 T cells (CD45.2) then were transferred i.v into nave CD45.1 BALB/c mice. All CD45.1 mice were infected with PbA (110.sup.6 iRBCs) at day 3 post-transfer (FIG. 11A). The course of the infection in mice transferred with CD4 T cells from RNA/GFP-CNE or RNA/PMIF-CNE donors was monitored by determining parasitemia by FACS. At day 7 post-infection, mice were sacrificed and CD4.sup.+CD45.2.sup.+ donor cells recovered, quantified, and proliferation assessed by CFSE dilution.

[0225] Infection was established in recipient mice that received CD4 T cells from the GFP (control) group, as evidenced by increasing parasitemia, but not in mice that received CD4 T cells from the PbMIF immunized donors (FIG. 11B).

[0226] Parasitemia was significantly reduced in the mice transferred with CD4 T cells from RNA/PMIF-CNE-immunized donors compared to control mice transferred with CD4 T cells from GFP-CNE-immunized donors. Thus, adoptive transfer of CD4 T cells from PMIF-vaccinated mice also conferred protection to parasite re-challenge. In fact, CD4 T cells from PbMIF immunized mice confer complete protection against blood-stage P. berghei infection.

[0227] The protection conferred by the adoptive transfer of CD4 T cells from the PbMIF immunized donors was associated with a higher number of proliferating CD4 T cells (CFSE.sup.lo) (FIGS. 11C-E), higher levels of IFN- production (FIG. 11F), and reduced expression of the exhaustion marker PD-1 (FIG. 11G) when compared to CD4 T cells adoptively transferred from the control group.

[0228] These data indicate that the augmented CD4 T cell response that develops after PbMIF immunization in infected mice offers complete protection against infection and is sufficient to prevent the establishment of blood-stage infection.

[0229] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

REFERENCES

[0230] [1] Doolan et al., Clin Microbiol Rev, 2009; 22(1): 13-36 [0231] [2] Yazdanbakhsh & Sacks 2010 Nat Rev Immunol, 10(2): 80-81 [0232] [3] Vermeire et al. 2008 Trends Parasitol.; 24(8):355-63 [0233] [4] Dobson et al. 2009 Protein Sci. 18(12):2578-91 [0234] [5] Sun et al. 2012 PNAS 31; 109(31):E2117-26 [0235] [6] Leng et al. 2003 J Exp Med 197:1467-1476 [0236] [7] Kamir et al. 2008 J Immunol.; 180(12):8250-61 [0237] [8] Cho et al. (2011) Chem Biol.; 18(9): 1089-1101. [0238] [9] Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual. [0239] [10] Shortprotocols in molecular biology (4th ed, 1999) Ausubel et al. eds. ISBN 0-471-32938-X. [0240] [11] U.S. Pat. No. 5,707,829 [0241] [12] Current Protocols in Molecular Biology (F. M. Ausubel et al. eds., 1987) Supplement 30. [0242] [13] Geysen et al. (1984) PNAS USA 81:3998-4002. [0243] [14] Carter (1994) Methods Mol Biol 36:207-23. [0244] [15] Jameson, B A et al. 1988, CABIOS 4(1):181-186. [0245] [16] Raddrizzani & Hammer (2000) Brief Bioinform 1(2):179-89. [0246] [17] De Lalla et al. (1999) J. Immunol. 163:1725-29. [0247] [18] Brusic et al. (1998) Bioinformatics 14(2):121-30 [0248] [19] Meister et al. (1995) Vaccine 13(6):581-91. [0249] [20] Roberts et al. (1996) AIDS Res Hum Retroviruses 12(7):593-610. [0250] [21] Maksyutov & Zagrebelnaya (1993) Comput Appl Biosci 9(3):291-7. [0251] [22] Feller & de la Cruz (1991) Nature 349(6311):720-1. [0252] [23] Hopp (1993) Peptide Research 6:183-190. [0253] [24] Welling et al. (1985) FEBS Lett. 188:215-218. [0254] [25] Davenport et al. (1995) Immunogenetics 42:392-297. [0255] [26] U.S. Pat. No. 5,928,902. [0256] [27] WO 90/01496. [0257] [28] Bodanszky (1993) Principles of Peptide Synthesis (ISBN: 0387564314). [0258] [29] Fields et al. (1997) Meth Enzymol 289: Solid-Phase Peptide Synthesis. ISBN: 0121821900. [0259] [30] Chan & White (2000) Fmoc Solid Phase Peptide Synthesis. ISBN: 0199637245. [0260] [31] Kullmann (1987) Enzymatic Peptide Synthesis. ISBN: 0849368413. [0261] [32] Ibba (1996) Biotechnol Genet Eng Rev 13:197-216. [0262] [33] WO2005/113782. [0263] [34] WO2012/006376 [0264] [35] WO2012/006380 [0265] [36] Geall et al. (2012) PNAS USA. September 4; 109(36):14604-9 [0266] [37] WO2013/006834. [0267] [38] WO2013/006837. [0268] [39] WO2012/030901. [0269] [40] WO2012/031046. [0270] [41] WO2012/031043. [0271] [42] WO2013/033563. [0272] [43] WO2013/006825. [0273] [44] Breedveld (2000) Lancet 355(9205):735-740. [0274] [45] Gorman & Clark (1990) Semin. Immunol. 2:457-466. [0275] [46] WO97/18229 [0276] [47] Tang et al. (2012) J Helminthol.; 86(4):430-9. [0277] [48] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472. [0278] [49] WO2011/027222. [0279] [50] Carroll et al., (2010) PLoS ONE 5, e13124.

[0280] The invention includes at least the following numbered embodiments: [0281] 1. A method for providing protective immunity against a parasite infection in a subject in need thereof, comprising administering an immunologically effective amount of a composition to the subject, wherein the composition comprises: [0282] (i) a nucleic acid comprising a sequence which encodes a parasite macrophage migration inhibitory factor (MIF) antigen; [0283] (ii) a parasite MIF antigen; or [0284] (iii) an antibody which specifically binds to a parasite MIF antigen. [0285] 2. The method of embodiment 1 wherein the composition comprises an RNA-based vaccine. [0286] 3. The method of embodiment 1 or 2 wherein the protective immunity is characterized by protective immunological memory against the parasite and/or an effective parasite-responsive memory T cell population. [0287] 4. The method of any preceding embodiment wherein the parasite MIF antigen comprises a full-length MIF polypeptide or an immunogenic fragment thereof. [0288] 5. The method of any one of embodiments 1 and 3 wherein the composition comprises a nucleic acid-based vaccine comprising the nucleic acid sequence which encodes a parasite MIF antigen. [0289] 6. The method of embodiment 5 wherein the nucleic acid-based vaccine is an RNA-based vaccine. [0290] 7. The method of embodiment 6 wherein the nucleic acid-based vaccine comprises a self-replicating RNA molecule. [0291] 8. The method of embodiment 7 wherein the self-replicating RNA is an alphavirus-derived RNA replicon. [0292] 9. The method of any preceding embodiment wherein the composition comprises a cationic nano-emulsion (CNE) delivery system. [0293] 10. The method of any one of embodiments 1 to 8 wherein the composition comprises a lipid nanoparticle (LNP) delivery system. [0294] 11. The method of any preceding embodiment wherein the composition comprises one or more adjuvants. [0295] 12. The method of embodiment 1, 3 or 4 wherein the antibody which specifically binds to a parasite MIF antigen comprises polyclonal antibody. [0296] 13. The method of embodiment 1, 3 or 4 wherein the antibody which specifically binds to a parasite MIF antigen is a humanized or chimeric antibody. [0297] 14. The method of any preceding embodiment wherein the parasite is a parasitic protozoan. [0298] 15. The method of embodiment 14 wherein the protozoan is an apicomplexan parasite. [0299] 16. The method of embodiment 15 wherein the protozoan belongs to the genus Plasmodium. [0300] 17. The method of embodiment 14 wherein the protozoan belongs to a genus selected from the group consisting of: Plasmodium, Toxoplasma, Babesia, Eimeria, Theileria, Neospora, Sarcocystis, Leishmania, and Trypanosoma. [0301] 18. The method of any one of embodiments 1 to 13 wherein the parasite is a parasitic helminth. [0302] 19. The method of embodiment 18 wherein the parasitic helminth is a nematode. [0303] 20. The method of embodiment 18 or embodiment 19 wherein the parasitic helminth belongs to a genus selected from the group consisting of: Ancyclostoma, Necator, Brugia, Wuchereria, Loa, Mansonella, Trichinella, Trichuris, Ascaris, Anisakis, Dracunculus, Strongyloides, Haemonchus, Schistosoma and Fasciola. [0304] 21. The method of any preceding embodiment wherein two or more doses of the composition are administered to the subject. [0305] 22. The method of embodiment 21 wherein the two or more doses are administered at least 1 week apart, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, or about 16 weeks apart. [0306] 23. The method of any preceding embodiment wherein the subject is a vertebrate. [0307] 24. The method of embodiment 23 wherein the subject is a mammal. [0308] 25. The method of embodiment 24 wherein the subject is a human. [0309] 26. The method of embodiment 24 wherein the subject is a veterinary mammal. [0310] 27. The method of embodiment 26 wherein the veterinary mammal is a cat, dog, horse, cow, sheep, deer, goat, or pig. [0311] 28. The method of any preceding embodiment wherein the composition further comprises a nucleic acid sequence which encodes an additional parasite antigen. [0312] 29. The method of any preceding embodiment wherein the composition further comprises an additional parasite antigen. [0313] 30. The method of any preceding embodiment wherein the composition is administered to the subject in combination with a further composition which comprises a nucleic acid comprising a sequence which encodes an additional parasite antigen. [0314] 31. The method of any preceding embodiment wherein the composition is administered to the subject in combination with a further composition which comprises an additional parasite antigen. [0315] 32. A composition for use in a method of providing protective immunity against a parasite infection in a subject in need thereof, which comprises an immunologically effective amount of: [0316] (i) a nucleic acid comprising a sequence which encodes a parasite MIF antigen; [0317] (ii) a parasite MIF antigen; or [0318] (iii) an antibody which specifically binds to a parasite MIF antigen. [0319] 33. The composition of embodiment 32 for use in a method according to any one of embodiments 1 to 31. [0320] 34. A composition comprising an immunologically effective amount of: [0321] (i) a nucleic acid comprising a sequence which encodes a parasite MIF antigen; or [0322] (ii) a parasite MIF antigen; [0323] wherein the MIF antigen is from a parasitic protozoan. [0324] 35. The composition of embodiment 34 wherein the protozoan is an apicomplexan parasite, such as Plasmodium. [0325] 36. The composition of embodiment 34 or 35 wherein the protozoan belongs to a genus selected from the group consisting of: Plasmodium, Toxoplasma, Babesia, Eimeria, Theileria, Neospora, Sarcocystis, Leishmania, and Trypanosoma. [0326] 37. A composition comprising an immunologically effective amount of: [0327] (i) a nucleic acid comprising a sequence which encodes a parasite MIF antigen; or [0328] (ii) a parasite MIF antigen; [0329] wherein the MIF antigen is from a parasitic helminth which belongs to a genus selected from the group consisting of: Ancyclostoma, Necator, Brugia, Wuchereria, Loa, Mansonella, Trichinella, Trichuris, Ascaris, Anisakis, Dracunculus, Strongyloides, Haemonchus, Schistosoma and Fasciola. [0330] 38. The composition of any one of embodiments 34 to 37 wherein the parasite MIF antigen comprises a full-length MIF polypeptide or an immunogenic fragment thereof. [0331] 39. The composition of any one of embodiments 34 to 38 which comprises a nucleic acid-based vaccine comprising the nucleic acid sequence which encodes a parasite MIF antigen. [0332] 40. The composition of embodiment 39 wherein the nucleic acid-based vaccine is an RNA-based vaccine. [0333] 41. The composition of embodiment 40 wherein the nucleic acid-based vaccine comprises a self-replicating RNA molecule. [0334] 42. The composition of embodiment 41 wherein the self-replicating RNA is an alphavirus-derived RNA replicon. [0335] 43. The composition of any one of embodiments 34 to 42 which comprises a cationic nano-emulsion (CNE) delivery system. [0336] 44. The composition of any one of embodiments 34 to 42 which comprises a lipid nanoparticle (LNP) delivery system. [0337] 45. The composition of any one of embodiments 34 to 44 which comprises one or more adjuvants. [0338] 46. The composition of any one or embodiments 34 to 45 wherein the composition further comprises a nucleic acid sequence which encodes an additional parasite antigen. [0339] 47. The composition of any one or embodiments 34 to 46 wherein the composition further comprises an additional parasite antigen. [0340] 48. A method of enhancing an immune response to a non-MIF parasite antigen in a subject, comprising administering an immunologically effective amount of a composition to the subject, wherein the composition comprises: [0341] (i) a nucleic acid comprising a sequence which encodes a parasite MIF antigen; [0342] (ii) a parasite MIF antigen; or [0343] (iii) an antibody which specifically binds to a parasite MIF antigen. [0344] 49. The method of embodiment 48 which further comprises administering the non-MIF parasite antigen to the subject. [0345] 50. The method of embodiment 48 which is a method according to any one of embodiments 1-31. [0346] 51. A method for providing protective immunity against a parasite infection in a subject in need thereof, comprising administering parasite-responsive CD4 T cells isolated from a compatible host, wherein the host has been immunized with a composition comprising: [0347] (i) a nucleic acid comprising a sequence which encodes a parasite MIF antigen; or [0348] (ii) a parasite MIF antigen. [0349] 52. The method of embodiment 51 wherein the compatible host has been administered a composition according to a method of any one of embodiments 1-31.