Composition and uses thereof

Abstract

The present invention provides a particle comprising a fusion protein, wherein the fusion protein comprises at least one NANP repeat (SEQ ID NO: 7), some or all of the C-terminus of the CS protein from Plasmodium falciparum and a hepatitis B surface antigen, and wherein the particle comprises no, or substantially no, free hepatitis B surface antigen protein, and uses thereof.

Claims

1. A virus-like particle comprising a fusion protein, wherein the fusion protein comprises at least one NANP repeat (SEQ ID NO: 7), and the C-terminus of the CS protein from Plasmodium falciparum comprising the sequence : TABLE-US-00008 (SEQ ID NO: 6) NKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKIEKEYLN KIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICK MEKCSSVFN VVNSSIGI; or the sequence (SEQ ID NO: 4) NKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKHIKEYLN KIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICK MEKCSSV, or a conservative substitution variant of SEQ ID No: 6 or SEQ ID NO: 4 with 95% or more sequence identity therewith; and HBsAg, wherein the virus-like particle comprises no free HBsAg.

2. The virus-like particle of claim 1 comprising at least 10 NANP repeats (SEQ ID NO: 13).

3. The virus-like particle of claim 1, wherein the particle comprises at least about 40% or more by mass of its proteinaceous material, the proteinaceous material being derived from Plasmodium falciparum.

4. The virus-like particle of claim 1 comprising a fusion protein consisting of: the sequence of SEQ ID NO: 1 (R21); the sequence of SEQ ID NO: 2 (RTS); or a sequence with at least 95%, or more sequence identity with the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

5. A fusion protein comprising at least one NANP repeat (SEQ ID NO: 7), and the C terminus of the CS protein from Plasmodium falciparum comprising the sequence : TABLE-US-00009 (SEQ ID NO: 6) NKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKIEKEYLN KIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICK MEKCSSVFN VVNSSIGI; or the sequence (SEQ ID NO: 4) NKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKHIKEYLN KIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICK MEKCSSV; or a conservative substitution variant of SEQ ID No: 6 or SEQ ID NO: 4 sequence with 95% or more sequence identity therewith; and HBsAg, wherein the fusion protein comprises no free HBsAg.

6. The fusion protein of claim 5, wherein the fusion protein consists of, a sequence of SEQ ID NO: 1 (R21) or a sequence with at least 95% or more sequence identity with the sequence of SEQ ID NO: 1.

7. The fusion protein of claim 5, wherein the fusion protein is expressed in a Saccharomyces cerevisiae or Pichia pastoris or a methylotrophic yeast cell.

8. The fusion protein of claim 7, wherein the methylotrophic yeast cell is Hansenula polymoipha.

Description

(1) Preferred embodiments of the present invention will now be described, merely by way of example, with reference to the following figures and examples.

(2) FIG. 1—provides the sequence of R21 (Seq ID No: 1) and RTS (Seq ID No: 2);

(3) FIG. 2—shows the results of the analysis of purified R21 particles by transmission electron microscopy—FIG. 2A. In FIG. 2B particles are shown that lack the 105 amino acid of the CS protein C terminus. The particles are negatively stained with 2% uranyl acetate;

(4) FIG. 3—shows the results of the analysis of R21 purified particles by reducing gel electrophoresis. FIG. 3A shows silver stained R21 purified particles. FIG. 3B shows the results of a western blot using monoclonal anti-NANP antibody (“NANP” disclosed as SEQ ID NO: 7; from MR4, named 2A10).

(5) FIG. 4—shows the results of immunisation studies. BALB/c mice were immunised intramuscularly with 0.5 μg R21 with either Alhydrogel (85 ug) or Abisco-100 (12 ug) or AddaVax. Three shots were given 3 weeks apart and NANP-specific antibody (“NANP” disclosed as SEQ ID NO: 7) responses were assayed by ELISA 3 weeks after each immunisation. Median responses are shown. “NANP.sub.6C” disclosed as SEQ ID NO: 12.

(6) FIG. 5—demonstrates that R21 in Abisco adjuvant induced high titre antibodies to the NANP repeat (SEQ ID NO: 7) of the CS protein but modest antibody titres to HBsAg. BALB/c mice were immunised intramuscularly with 5 μg R21 with Abisco-100. Three shots were given 3 weeks apart and HBsAg-specific antibody responses were assayed by ELISA 3 weeks after the 3rd immunisation. Median responses are shown.

(7) FIG. 6—details the results of IFN-γ Spleen ELISpot analysis for BALB/c mice immunised intramuscularly with 0.5 μg R21 with either Alhydrogel (85 ug) or Abisco-100 (12 ug) or AddaVax. Three shots were given 3 weeks apart and NANP-specific antibody (“NANP” disclosed as SEQ ID NO: 7) responses were assayed by ELISA 3 weeks after each immunisation. Median responses are shown.

(8) FIG. 7—demonstrates that R21 in adjuvant can be combined with viral vectors without impairing humoral immunogenicity. BALB/c mice were immunised with either 2 shots of R21+Abisco-100 or the ChAd63 (Ad) ME TRAP-MVA ME TRAP 8 week prime-boost regime alone or combined together, as detailed in Table 1. Humoral responses were assayed in the NANP (SEQ ID NO: 7) ELISA carried out 3 weeks and 8 weeks after the first immunisation, and 3 weeks after the final immunisation. Median responses are shown. No impairment of the humoral immunogenicity of R21 is observed. “NANP.sub.6C” disclosed as SEQ ID NO: 12.

(9) FIG. 8—demonstrates that R21 in adjuvant can be combined with viral vectors without impairing T cell immunogenicity. BALB/c mice were immunised with either 2 shots of R21+Abisco-100 or the ChAd63 (Ad) ME TRAP-MVA ME TRAP 8 week prime-boost regime alone or combined together. Cellular responses were assayed in an IFN-γ spleen ELISpot to a pool of CS peptides (A) or the Pb9 peptide (B) 3 weeks after the final immunisation. Median responses are shown. No impairment of the T cell immunogenicity of R21 or the viral vectors is observed.

(10) FIG. 9—demonstrates that mixing R21 plus adjuvants with vectors can enhance antibody responses to the vector encoded antigen. BALB/c mice were immunised intramuscularly with the ChAd63 (Ad) ME TRAP-MVA ME TRAP 8 week prime-boost regime alone or combined with either Abisco-100 or AddaVax. TRAP-specific antibody responses were assayed by ELISA 3 weeks and 8 weeks after the first immunisation and 3 weeks after the second Median responses are shown.

(11) FIG. 10—demonstrates that the R21 in a ISCOM adjuvant, such as Abisco or Matrix M (available from Isconova, Uppsala, Sweden) provides high level protective efficacy against malaria sporozoite infection in mice. The mice were infected at two immunisations with the R21 particle in adjuvant by a transgenic Plasmodium berghei parasite, transgenic for the P. falciparum CS protein gene. Such parasites are described in the research literature (Tewari R et al. J Biol Chem. 2002 Dec. 6; 277(49):47613-8; Kaba S et al. PLoS One. 2012; 7(10):e48304). The results demonstrate that the R21 particle not only shows excellent immunogenicity against P. falciparum CS but potent efficacy in a very relevant malaria infection model. IM: intramuscular.

(12) FIG. 11—this table illustrates the study design used to consider the immunogenicity of R21 without an adjuvant.

(13) FIG. 12—demonstrates CS-specific IgG responses to R21 with different adjuvants. BALB/c mice were immunised intramuscularly with 0.5 ug R21 alone or formulated with Alhydrogel or Abisco-100. Three immunisations were given three weeks apart and the NANP-specific antibody (“NANP” disclosed as SEQ ID NO: 7) titres were measured by ELISA 3 weeks after each immunisation. Median responses are shown.

(14) FIG. 13—demonstrates IFN-γ Spleen ELISpot responses to R21 with Matrix M. BALB/c mice were immunised intramuscularly with 0.5 μg R21 with Matrix M (12 ug). Three shots were given 3 weeks apart and CS-specific T cell responses were measured in the spleen by IFN-γ ELISpot 3 weeks after the final immunisation. Median responses are shown.

(15) FIG. 14—illustrates the study design for a vaccine immunisation interval study. The illustrated vaccine regimens were used to assess the immunogenicity of R21+Matrix M in BALB/c mice

(16) FIG. 15—demonstrates CS-Specific IgG responses to R21 and Matrix M. BALB/c mice were immunised intramuscularly with 0.5 μg R21 with Matrix M (12 ug). NANP-specific antibody (“NANP” disclosed as SEQ ID NO: 7) titres were assayed by ELISA 3 weeks after each immunisation. Median responses are shown.

(17) FIG. 16—demonstrates IFN-γ Spleen ELISpot responses to R21 and Matrix M. BALB/c mice were immunised intramuscularly with 0.5 μg R21 with Matrix M (12 ug). Three shots were given 3 weeks apart and CS-specific T cell responses were measured in the spleen by IFNg ELISpot 3 weeks after the final immunisation. Median responses are shown.

(18) FIG. 17—shows the study design used to compare R21 and the CSP recombinant protein

(19) FIG. 18—demonstrates CS-specific IgG responses to R21 or CSP with either Matrix M, MF59 or L-AS01. BALB/c mice were immunised intramuscularly with 0.5 μg R21 or CSP with either Matrix M, MF59 or L-AS01. NANP-specific antibody (“NANP” disclosed as SEQ ID NO: 7) titres were assayed by ELISA 3 weeks after each immunisation. Mean responses with SD are shown (Groups compared by One-way ANOVA with Bonferroni's post-test. ns=non-significant, * p<0.05, **p<0.01, ***p<0.001).

(20) FIG. 19—demonstrates CS-specific T cells to R21 or CSP with either Matrix M, MF59 or L-AS01. CS-specific T cells. BALB/c mice were immunised intramuscularly with 0.5 μg R21 or CSP with either Matrix M, MF59 or L-AS01. CS specific T cell responses were measured in the blood by ICS and flow cytometry, 2 weeks after the boost vaccination. Isolated PBMC's were re-stimulated with a pool of CS peptides and frequencies of cytokine secreting CD4+T cells were measured (IFNg, TNF and IL2). Mean responses shown.

(21) FIG. 20—illustrates an R21 vs CSP challenge experiment. BALB/c mice were immunised intramuscularly with two shots of 0.5 μg R21 or CSP with Matrix M, 8 weeks apart. A) Mice were challenged 3 weeks after boost with 1000 sporozoites injected i.v. and time to 1% parasitemia was determined by thin film blood smear from day 5 post challenge. Mice were sterilely protected if they had no blood stage parasites by day 14. Survival curves compared by Log-rank (Mantel-Cox) Test. B) NANP-specific antibody (“NANP” disclosed as SEQ ID NO: 7) titres were assayed by ELISA 3 weeks after each immunisation. Mean responses with SD are shown (Groups compared by One-way ANOVA with Bonferroni's post-test. **p<0.01, ***p<0.001). C) T cell responses were measured in the blood by ICS and flow cytometry, 2 weeks after the boost vaccination. Isolated PBMC's were re-stimulated with a pool of CS peptides and frequencies of cytokine secreting CD4+T cells were measured secreting (IFNg, TNF and IL2). Mean responses with SEM are shown.

(22) FIG. 21—demonstrates the enhanced efficacy of combined immunisation with R21 and viral vectors. BALB/c mice were immunised intramuscularly with R21+MF59 or the ChAd63 PbTRAP-MVA PbTRAP regimen alone (A or B) or combined together (C and D). Mice were challenged 3 weeks after boost with 1000 sporozoites injected i.v. and time to 1% parasitemia was determined by thin film blood smear from day 5 post challenge. Mice were sterilely protected if they had no blood stage parasites by day 14. Survival curves were compared by Log-rank (Mantel-Cox) Test.

(23) FIG. 22—demonstrates the efficacy of IgG passive transfer. BALB/c mice were immunised intramuscularly with R21+Matrix M twice, 8 weeks apart, mice were bled and total IgG was purified. Naïve mice were immunised with either B) 150 ug C) 750 ug or D) 1.5 mg of total IgG from vaccinated mice i.v. A control group also received IgG from unvaccinated mice A). All mice were challenged 5 hours after IgG transfer with 1000 sporozoites injected i.v. and time to 1% parasitemia was determined by thin film blood smear from day 5 post challenge. Mice were sterilely protected if they had no blood stage parasites by day 14. Survival curves were compared by Log-rank (Mantel-Cox) Test.

(24) FIG. 23—demonstrates that NANP-specific IgG (“NANP” disclosed as SEQ ID NO: 7) correlates with efficacy. Groups of BALB/c mice received increasing doses of total IgG from vaccinated mice (R21+Matrix M) and had moderate—good levels of NANP-specific IgG (“NANP” disclosed as SEQ ID NO: 7) titres at the time of challenge (A). The level of NANP-specific IgG (“NANP” disclosed as SEQ ID NO: 7) correlated with the time taken for mice to develop 1% parasitemia (B) (correlation tested using Pearsons correlation, p=0.0008, r=0.659).

(25) In order to exemplify the invention described herein particles comprising the fusion protein R21 (Seq ID no: 1, FIG. 1) were produced and their immunogenic properties were considered.

(26) Development of R21 Expressing Yeast

(27) Materials and Methods

(28) R21 Expression Plasmid

(29) A sequence which encodes for the fusion protein R21 (Seq ID No: 1 and FIG. 1) of was cloned into the pPink-HC expression plasmid from the PichiaPink™ Expression System (Invitrogen, Cat. no. A11150).

(30) The R21 protein comprises the Hepatitis B surface antigen (HBsAg) adw serotype and a C-terminal portion of the circumsporozoite (CS) protein of the Plasmodium falciparum strain NF54. The sequence comprises 410 amino acids from N to C terminus:

(31) 75 amino acids of the NANP repeat (“NANP” disclosed as SEQ ID NO: 7) from the CS protein—comprising MDP followed by 18 NANP repeats (“18 NANP repeats” disclosed as SEQ ID NO: 9):

(32) TABLE-US-00003 (SEQ ID NO: 3) MDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNA NPNANPNANPNANPNANPNANPNANP 
105 amino acid of the CS protein C terminus:

(33) TABLE-US-00004 (SEQ ID NO: 4) NKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKHIKEYLN KIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICK MEKCSSV 
(The 10 C terminal amino acids (FNVVNSSIGL (SEQ ID NO: 10)) of the CS protein have been removed.)
4 amino acids from the pre-S2 region of the hepatitis B virus:

(34) TABLE-US-00005 (SEQ ID NO: 11) PVTN 
followed by 226 amino acids of the HBsAg adw serotype:

(35) TABLE-US-00006 (SEQ ID NO: 5) MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCL GQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLL DYQGMLPVCPLIPGSTTTNTGPCKTCTTPAQGNSMFPSCCCTKPTDGNC TCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIW MMWYWGPSLYSIVSPFIPLLPIFFCLWVYI 
R21 Expressing Yeast

(36) PichiaPink™ Strain 4 (Invitrogen, Cat. no. A11150), which is a double knock-out for proteinases A and B (i.e. pep4 and prb1), was transformed by electroporation with the pPink-HC expression plasmid encoding R21. Positively transformed colonies were selected by growth on adenine deficient agar plates.

(37) Initial Characterisation of Transformed Yeast

(38) Positive colonies were selected and grown in small 1 mL pilot expression cultures and protein expression induced by the addition of methanol. Levels of protein expression in each clone were then analysed by western blot. Induced R21 expressing yeast samples were disrupted in laemmli lysis buffer and western blot analysis was performed using an antibody to the NANP repeat (SEQ ID NO: 7) in the CSP portion of the fusion protein, (monoclonal anti-NANP antibody (“NANP” disclosed as SEQ ID NO: 7) (MR4, 2A10)) and a monoclonal anti-HBsAg antibody (Serotec, MCA4658). Expression of the fusion protein that was recognised by both antibodies at the correct size was confirmed.

(39) Growth of Pichia Pastoris and Induction of R21 Expression

(40) One R21 expressing clone was selected and grown in a 1 L batch culture with BMGY (Buffered complex medium containing glycerol). R21 expression was induced by the addition of methanol by changing the media to BMMY (Buffered complex medium containing methanol). Expression was induced with 0.5% methanol for 3 days at which point the yeast was pelleted by centrifugation at 1,500×g for 5 minutes, the supernatant removed and the yeast frozen at −80° C.

(41) Extraction and Purification of R21

(42) Cell Disruption

(43) Yeast pellets were thawed on ice and resuspended in a lysis buffer containing 10 mM Tris (pH 7.8), 0.1% Triton X-100, 1 mM EDTA. Acid washed glass beads (0.425-600 um) were added and the sample was disrupted by 10 cycles of vortexing for one minute then placing on ice for one minute.

(44) Purification

(45) Clarification

(46) Before purification the yeast debris was removed by centrifugation for 5 minutes at 1,500×g and this lysate was then clarified by ultracentrifugation at 13,000×g for 20 minutes.

(47) CsCl Discontinuous Gradient Centrifugation

(48) The clarified lysate was then layered onto a discontinuous CsCl gradient containing layers of 1.3 g/ml CsCl and 1.1 g/ml CsCl. After ultracentrifugation for 2 hours at 41,000 rpm in a SW41 Ti rotor, (Beckman Coulter Optima L-100 XP) the particle containing fraction was collected.

(49) Gel Filtration

(50) The particle containing fraction was then applied to a PD10 column containing Sephedex G100. The sample was eluted in 10 mM Tris (pH7.8) and particle containing fractions were collected and pooled.

(51) CsCl Isopycnic Gradient Centrifugation

(52) The pooled sample was then added to an isopycnic CsCl gradient containing 1.2 g/ml CsCl. After ultracentrifugation for 20 hours at 41,000 rpm in a SW41 Ti rotor, (Beckman Coulter Optima L-100 XP) the particle containing fraction was collected.

(53) Size Exclusion Chromatography on Sephacryl 500

(54) The sample was then applied to a Hiprep 16/60 Sephacryl S-500 HR gel filtration column (GE Healthcare) to exchange the buffer and remove any remaining lower molecular weight contaminants. The sample was eluted in 10 mM Tris buffer (pH7.8) and pure particle containing fractions were pooled.

(55) R21 Characterisation

(56) Electron Microscopy

(57) To confirm the presence and size of particles, the purified R21 particle preparation was analysed by negative staining with 2% uranyl acetate on a transmission electron microscope (FIG. 2A). Particles appear to be approximately 22 nm in size.

(58) Particles were also made that lacked the 105 amino acid of the CS protein C terminus, these are depicted in FIG. 2B.

(59) Gel Electrophoresis; Silver Stain and Western Blot Analysis

(60) The purified R21 particle preparation was analysed by reducing gel electrophoresis. The sample was silver stained to assess the purity and analysed by western blotting with a monoclonal anti-NANP antibody (“NANP” disclosed as SEQ ID NO: 7; MR4, 2A10) and an anti-HBsAg monoclonal antibody to assess the immunoreactivity of the product. The same band was recognised by both antibodies and the purified product was greater than 90% pure based on the silver stained gel (FIG. 3).

(61) ELISA to CS Repeat Region

(62) The presence and accessibility of the NANP repeat (SEQ ID NO: 7) region on the surface of the particle was assessed by sandwich ELISA using two antibodies to the NANP repeat (“NANP” disclosed as SEQ ID NO: 7) region. Maxisorb 96 well plates (Nunc) were coated with mouse monoclonal anti-NANP antibody (“NANP” disclosed as SEQ ID NO: 7; MR4, 2A10), incubated with purified R21 particle and a rabbit polyclonal serum (MR4, MRA-24) was used for detection. The purified R21 particle prep gave a strong positive signal.

(63) ELISA to CS Repeat Region and HBsAg

(64) The presence and accessibility of the HBsAg portion of fusion protein in the particle was assessed by sandwich ELISA using a monoclonal antibody to the NANP repeat (SEQ ID NO: 7) region (MR4, 2A10) and a cocktail of antibodies to the HBsAg from Monolisa ULTRA HBsAg ELISA kit (Biorad). The purified R21 particle prep gave a weak positive signal thus confirming the presence and accessibility of the HBsAg epitopes on the same NANP (SEQ ID NO: 7) containing R21 particles.

(65) Quantification by Absorbance at 280 nm

(66) The purified particle prep is quantified by measuring absorbance at 280 nm.

(67) Larger Scale Manufacturing of R21

(68) To assess the feasibility of larger scale manufacturing a new process was developed in a GMP facility approved by the UK Medicines and Healthcare products Regulatory Agency (MHRA), the Clinical Biomanufacturing Facility at the University of Oxford. This large scale process provided satisfactory yields of R21 particles at a larger scale that were immunogenic in small animals, indicating that a GMP compatible process is achievable for clinical grade manufacturing of the R21 particle immunogen.

(69) Immunogenicity of R21 Particles in Mice

(70) Immunogenicity was assessed in mice by measuring the antibodies generated to the NANP repeat (SEQ ID NO: 7) in an ELISA and by measuring T cells specific for a pool of CS peptides contained within the R21 vaccine by IFN-γ spleen ELISpot. The antibody response to the HBsAg was also assessed in an ELISA.

(71) Anti-NANP Antibody ELISA (“NANP” Disclosed as SEQ ID NO: 7)

(72) Anti-CSP antibodies induced by R21 immunisation were assessed in an ELISA using NANP.sub.6C peptide (SEQ ID NO: 12). The NANP.sub.6C peptide (SEQ ID NO: 12) consists of 6 copies of the NANP repeat followed by a C. Maxisorb (SEQ ID NO: 12). 96 well plates (Nunc) were coated with this antigen and incubated with serum samples using a 3 fold serial dilution starting at a dilution of 1:1000. Mouse antibodies were detected with alkaline phosphatase conjugated anti-mouse IgG and pNPP (p-Nitrophenyl Phosphate, Disodium Salt) substrate and absorbance read at 405 nm. The results are expressed as endpoint titre which is defined as the dilution at which the OD of the sample is equal to background.

(73) The R21 particle vaccine was found to be highly immunogenic in mice and there is a notable boost effect after each vaccination (FIG. 4). Furthermore, if R21 is administered with the adjuvant Abisco or AddaVax slightly higher antibody titres were induced as compared to R21 administered with Alhydrogel.

(74) IFN-γ spleen ELISpot

(75) T cells induced by immunisation with R21 were measured using IFN-γ ELISpot assay. Fresh splenocytes were isolated and incubated in duplicate wells for 20 hours. Cells were restimulated with a pool of CS peptides spanning the entire CS protein at a final concentration of 2 ug/ml. Plates were coated and INF-γ was detected using antibodies from Mabtech. Spots were developed using an alkaline phosphatase substrate kit from Biorad and counted using an ELISPOT counter (AID). The results are presented in FIG. 6 and are expressed as number of spot forming cells (SFC) per million splenocytes.

(76) T cell responses were only measured after the third vaccination. R21 administered with Abisco is more effective at inducing T cells to the CS peptides in mice than R21 administered with Ahydrogel.

(77) Anti-HBsAg Antibody ELISA

(78) Anti-HBsAg antibodies induced by R21 immunisation with Abisco adjuvant were assessed in an ELISA using HBsAg particle. The HBsAg particle was coated onto maxisorb 96 well plates (Nunc) and the plates were incubated with serum samples using a 3 fold serial dilution starting at a dilution of 1:100. Mouse IgG antibodies were detected and the results expressed as for the anti-NANP antibody ELISA (“NANP” disclosed as SEQ ID NO: 7) above.

(79) HBsAg antibodies were assessed after three immunisations. Low antibody titres to the HBsAg were detected in all of the immunised mice in contrast to high titre antibodies against NANP (SEQ ID NO: 7;_FIG. 5). This result shows that the R21 particle induces preferentially antibodies to the malaria rather than the hepatitis B component of R21.

(80) Further experimentation supported the finding discussed above and in particular demonstrated that R21, when administered alone, is able to induce moderate CS-specific antibodies and T cells after 3 low dose immunisation. More specifically, groups of BALB/c mice were immunised intramuscularly with R21 alone or formulated with adjuvant as detailed in FIG. 11. Three immunisations were given three weeks apart and the immunogenicity was assessed by measuring serum antibody titres 3 weeks after each immunisation and antigen-specific T cell responses in the spleen 3 weeks after the final immunisation. After the third immunisation the CS-specific IgG titres (FIG. 12) and CS-specific T cells (FIG. 13) were not different between the groups receiving R21 alone or R21+Alhydrogel. These results demonstrate that R21 is immunogenic alone, and in the absence of an adjuvant can stimulate cellular immunity, and can still induce low levels of CS-specific IFN-γ producing T cell on its own.

(81) Immunogenicity of R21 Particles with Viral Vector Vaccines in Mice

(82) The immunogenicity of mixtures of R21 plus adjuvant with adenoviral and MVA viral vectors expressing the ME TRAP antigen (O'Hara et al J Infect Dis 2012) were assessed in BALB/c mice as detailed in Table 1. No impairment of humoral (FIG. 7) or T cell immunogenicity (FIG. 8) was observed with the mixtures. This is an important result as it indicates that despite the potential negative impact of an adjuvant on the immunogenicity of viral vectored vaccines, no negative impact was observed. This is surprising as many other adjuvants other than the saponin and emulsion types adjuvants used in this work have been found to negatively impact on the immunogenicity of viral vectors. This observation has potential utility as it could allow these two types of malaria vaccine, R21 which targets sporozoites, and viral vectors encoding the TRAP antigen, which target the liver-stage of malaria, to be combined successfully.

(83) In particular a combination of R21, or a similar RTS or RTS,S like particle, plus a saponin containing adjuvant such as Abisco, matrix M, QS21, AS01 or AS02, plus MVA encoding TRAP or ME TRAP were identified as a novel and particularly preferred vaccine combination for malaria. FIGS. 7 and 8 show that this combination allows both exceptionally high antibody titres (to CSP) and T cell responses to TRAP to be attained. As these are arguably the best characterised correlates of protective immunity to pre-erythrocytic malaria in humans the use of this combination vaccine for boosting malaria immunity should be particularly effective. It is emphasized that MVA is a vector that allows multiple antigens to be expressed so the MVA expressing TRAP in this mixture may also express another malaria antigens, such as for example CSP, from the same or another MVA locus, or may even express an antigen from another pathogen, such as antigen 85A from Mycobacterium tuberculosis. This combination vaccine with an MVA expressing antigens from Mycobacterium tuberculosis as well as Plasmodium falciparum, along with R21 (or a similar particle such as RTS or RTS,S) plus a saponin adjuvant (such as matrix M or Iscomatrix or AS01 or AS02) or an emulsion adjuvant, such as Addavax or MF59, could therefore boost immunity to both tuberculosis and malaria.

(84) TABLE-US-00007 TABLE 1 Vaccine co-administration study Vaccine regimens used to assess the immunogenicity of R21 + adjuvant or ChAd63 ME TRAP - MVA ME TRAP in BALB/c mice administered alone or in combination. Combination vaccines formulated together and administered in the same syringe. No. No. Gp mice Particle + Adjuvant shots Viral vector Interval 1 6 0.5 μg R21 + Abisco IM 2 — 8 weeks 2 6 — — 1 × 10{circumflex over ( )}8 ChAd63 ME TRAP IM 8 weeks 1 × 10{circumflex over ( )}6 MVA ME TRAP IM 3 6 0.5 μg R21 + Abisco IM 2 1 × 10{circumflex over ( )}8 ChAd63 ME TRAP IM 8 weeks 1 × 10{circumflex over ( )}6 MVA ME TRAP IM
Immunogenicity of Viral Vector Insert with Adjuvants

(85) On mixing R21 plus two adjuvants with the same adenoviral and MVA viral vectors an increased antibody response to the encoded antigen, TRAP, was surprisingly observed (FIG. 9). This was found whether or not the R21 particle was included in the mixture, indicating that both the saponin Abisco and the squalene-based emulsion Addavax can enhance antibody responses to a viral vector encoded insert. A variety of similar adjuvants such as QS21 formulations (like Abisco) and MF59 (like Addavax) are available and so may be used to enhance antibody responses to a viral vector encoded antigen.

(86) Anti-TRAP Antibody ELISA

(87) Anti-TRAP antibodies induced by viral vector immunisation were assessed in an ELISA using TRAP recombinant protein. The TRAP protein was coated onto maxisorb 96 well plates (Nunc) and the plates were incubated with serum samples using a 3 fold serial dilution starting at a dilution of 1:100. Mouse IgG antibodies were detected and the results expressed as for the anti-NANP antibody ELISA (“NANP” disclosed as SEQ ID NO: 7) above.

(88) R21 can be Administered in a Number of Regimens for Optimal Immunogenicity

(89) Groups of BALB/c mice were immunised with 4 different regimens as described in FIG. 14 with R21+Abisco-100, CS-specific antibodies were measured 3 weeks after each immunisation and CS-specific T cells were measured 3 weeks after the final immunisation FIGS. 15 & 16 respectively. After the final immunisations there was no difference between any groups in the level of CS-specific antibodies or T cells. This indicates that high titres antibodies and moderate levels of T cells can be generated with multiple regimens so interval will not affect the level of antibodies induced when R21 is combined with viral vectors.

(90) R21 is More Immunogenic than CSP Recombinant Protein

(91) Immunisation with R21 in a range of adjuvants induces higher antibody titres and CS-specific T cells than CSP recombinant protein. A study recently compared four CSP protein vaccines head to head to determine which elicited the highest immune responses and superior efficacy. The most immunogenic recombinant protein evaluated was produced in E. coli by Gennova (Pune, India) and this was obtained from PATH Malaria Vaccine Initiative (MVI, Washington D.C.) for assessment against R21. This assessment was performed with 3 different adjuvants Matrix M (similar to Abisco-100), MF59 (similar to AddaVax) and a biosimilar of the proprietary GSK adjuvant AS01, obtained from Lausanne here called L-AS01.

(92) Groups of BALB/c mice were immunised twice, 8 weeks apart with 0.5 ug or either R21 or CSP formulated with 3 different adjuvants as detailed in FIG. 17. When comparing the two groups that received the same adjuvant, after prime the anti-NANP IgG (“NANP” disclosed as SEQ ID NO: 7) responses were higher in all groups receiving R21 compared to the groups receiving CSP, between, 2.6-4.7 fold higher (FIG. 18-A). The same trend was seen after boost at week 11, with the responses being between 4.5-22 fold greater in the R21 groups (FIG. 18-B). There was also no significant difference between the responses induced by the 3 adjuvants for either R21 or CSP, at any time point. Therefore if the data are pooled together for the R21 groups or the CSP groups at each time point the responses to the particle vaccine, R21 are significantly higher at all time-points than the recombinant protein in the 8 week prime boost regimen (FIG. 18-C). CS-specific T cell responses were also measured and mice immunised with the adjuvants Matrix M or L-AS01 adjuvants developed higher frequencies of T cells than those immunised with MF59 and those immunised with R21 induced higher frequencies of T cells than CSP (FIG. 19).

(93) R21 is More Efficacious than CSP Recombinant Protein

(94) R21 is not only more immunogenic but also more efficacious than CSP recombinant protein. Mice were immunised with R21 or CSP formulated with Matrix M and challenged with transgenic parasites (P. berghei transgenic for P. falciparum CSP) 3 weeks after boost. R21+Matrix M protected 87.5% of the vaccinated mice and CSP+Matrix M protected only 42.5% (FIG. 20-A). The levels of both CS-specific antibodies and T cells were also higher in the R21+Matrix M groups (FIG. 20-B+C).

(95) Protective Efficacy of R21 Particles Against Malaria Sporozite Infection in a Mouse Model

(96) 34 Balb/c mice were divided into four groups. Group 1 was immunised with 0.5 micrograms of R21 in Abisco adjuvant intramuscularly, twice with an eight-week interval. Group 2 received the same dose of R21 with the very similar matrix M adjuvant (also supplied by Isconova, Uppsala, Sweden) with the same interval; the Matrix M is suitable for clinical use. Group 3 received a single dose of 1×108 ifu of a chimpanzee adenovirus (ChAd63) expressing the CS gene followed 8 weeks later by a single dose of 1×106 pfu of an MVA vector expressing the CS gene, both by the intramuscular route. Group 4 were immunised with adjuvant only twice with an 8 week interval, 5 mice receiving Abisco and 5 receiving Matrix M. Three weeks after the day of the booster immunisation all mice were infected by a 1000 sporozoites intravenously of a P. berghei parasite transgenic for the circumsporozoite gene of P. falciparum. Mice were scored for time to 1% parasitaemia by microscopy. 10/10 control adjuvant only mice were infected; 6/8 from Group 3 were infected showing modest efficacy with the vectored vaccines. In contrast only 1/16 mice in Groups 1 and 2 were infected showing 94% sterile efficacy with R21 with the ISCOM adjuvant formulations.

(97) Vaccine Efficacy is Enhanced by Combining R21 and Viral Vectors

(98) Immunisation with either R21+MF59 or the ChAd63 PbTRAP-MVA PbTRAP regimen was protective against sporozoite challenge in BALB/c mice eliciting a significant delay in the time taken to develop 1% parasitemia in the blood (FIGS. 21-A and B). Combining these two vaccine regimens together resulted in an increase in the efficacy of both vaccine regimens (FIGS. 21-C and D).

(99) Vaccine Efficacy is Enhanced by Combining R21 and Viral Vectors

(100) Groups of mice receiving a passive transfer of increasing doses of total IgG from mice immunised with R21+Matrix M were protected against sporozoite challenge in a dose dependant manner (FIG. 22). NANP-specific IgG (“NANP” disclosed as SEQ ID NO: 7) titres were measured 1 day after challenge (FIG. 23-A) and mice had good to moderate titres that correlated with the efficacy measured as delay to 1% parasitemia (FIG. 23-B).