MALARIA VACCINE

Abstract

The invention relates to a composition comprising a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 1 (R21), wherein said polypeptide is in the form of a virus-like particle (VLP), wherein said particle comprises less than 10% free hepatitis B surface antigen protein, for use in the immunisation of a human subject susceptible to Plasmodium falciparum infection, characterised in that said composition is administered in a dosage regimen of at least one dose of 1 g to 20 g R21 per administration for a subject at least 18 years old, or at least one dose of 0.5 g to 10 g R21 per administration for a subject less than 18 years old. The invention also relates to kits, methods and uses.

Claims

1-30. (canceled)

31. A method of immunization of a human subject susceptible to Plasmodium falciparum infection comprising administering a composition to said subject, said composition comprising a pharmaceutically acceptable carrier, diluent or excipient and a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 80% sequence identity to SEQ ID NO: 1 (R21 polypeptide), wherein said R21 polypeptide is in the form of a virus-like particle (VLP), wherein said VLP comprises less than 10% free hepatitis B surface antigen protein, wherein said composition is administered in a dosage regimen of at least one dose of 1 g to 20 g R21 polypeptide per administration when said subject is at least 18 years old, or at least one dose of 0.5 g to 10 g R21 polypeptide per administration when said subject is less than 18 years old.

32. The method of claim 31, wherein said VLP comprises a circumsporozoite protein (CSP) sequence and a Hepatitis B surface antigen (HBsAg) sequence in a 1:1 ratio.

33. The method of claim 31, wherein said dosage regimen is at least one dose of 5 g to 20 g R21 polypeptide per administration when said subject is at least 18 years old, or at least one dose of 2.5 g to 10 g R21 polypeptide per administration when said subject is less than 18 years old.

34. The method of claim 31, wherein said dosage regimen is at least one dose of 10 g R21 polypeptide per administration when said subject is at least 18 years old, or at least one dose of 5 g R21 polypeptide per administration when said subject is less than 18 years old.

35. The method of claim 31, wherein said dosage regimen comprises a first dose, one or more optional additional doses, and a final dose.

36. The method of claim 35, wherein said final dose contains 10%-50% of the amount of R21 polypeptide of the first dose.

37. The method of claim 36, wherein said final dose contains 20% of the amount of R21 polypeptide of said first dose.

38. The method of claim 31, wherein said composition further comprises an adjuvant, wherein said adjuvant is Matrix-M and said adjuvant is present in a ratio in the range 1:1 to 1:50 of R21 polypeptide: Matrix-M.

39. The method of claim 38, wherein said ratio is in the range 1:2 to 1:25 of R21 polypeptide: Matrix-M.

40. The method of claim 39, wherein said ratio is in the range 1:5 to 1:10 of R21 polypeptide: Matrix-M.

41. The method of claim 34, wherein said at least one dose further comprises 10 to 500 g of an adjuvant when said subject is at least 18 years old, or 5 to 250 g of said adjuvant when said subject is less than 18 years old, wherein said adjuvant is Matrix-M.

42. The method of claim 34, wherein said at least one dose further comprises 20 to 200 g of an adjuvant when said subject is at least 18 years old, or 10 to 100 g of said adjuvant when said subject is less than 18 years old, wherein said adjuvant is Matrix-M.

43. The method of claim 34, wherein said at least one dose further comprises 25 to 50 g of an adjuvant when said subject is at least 18 years old, or 5 to 50 g of said adjuvant when said subject is less than 18 years old, wherein said adjuvant is Matrix-M.

44. The method of claim 34, wherein said at least one dose comprises about 10 g R21 polypeptide and about 50 g adjuvant when said subject is at least 18 years old, or comprises about 5 g R21 polypeptide and about 25 g adjuvant when said subject is less than 18 years old, wherein said adjuvant is Matrix-M.

45. The method of claim 35, wherein said doses are administered to said subject at interval(s) of 1 week to 12 weeks.

46. The method of claim 35, wherein said doses are administered to said subject at an interval of 4 weeks.

47. The method of claim 31, wherein the composition further comprises at least one of: a. a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 80% sequence identity to SEQ ID NO: 3); and b. a viral vector, said viral vector comprising a nucleic acid encoding at least one epitope from a malarial antigen, preferably from a P. falciparum or P. vivax antigen.

48. The method of claim 31, wherein said composition is a pharmaceutical composition or a vaccine composition.

49. The method of claim 31, wherein said composition is capable of inducing a protective immune response against P. falciparum in said subject.

50. The method of claim 31, wherein said dosage regimen is at least one dose of R21 polypeptide in the range 0.0000125 to 0.0003333 mg/Kg when said subject is at least 18 years old, or 0.00000625 to 0.001667 mg/Kg when said subject is less than 18 years old.

51. The method of claim 31, wherein said administration is intramuscular.

52. A composition comprising: a. 0.5 g to 20 g of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 80% sequence identity to SEQ ID NO: 1 (R21 polypeptide), wherein said R21 polypeptide is in the form of a virus-like particle (VLP), wherein said VLP comprises less than 10% free hepatitis B surface antigen protein; and b. a pharmaceutically acceptable carrier, diluent or excipient.

53. The composition of claim 52, wherein the VLP comprises a circumsporozoite protein (CSP) sequence and a Hepatitis B surface antigen (HBsAg) sequence in a 1:1 ratio.

54. The composition of claim 52, wherein the composition further comprises an adjuvant, wherein said adjuvant is Matrix-M, and wherein said adjuvant is present in a ratio in the range 1:1 to 1:50 of R21 polypeptide:Matrix-M.

55. The composition of claim 52, wherein the composition further comprises at least one of: a. a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 80% sequence identity to SEQ ID NO: 3; and b. a viral vector, said viral vector comprising a nucleic acid encoding at least one epitope from a malarial antigen, preferably from a P. falciparum or P. vivax antigen.

56. A kit comprising a first composition and a second composition for administration to a human subject: a. said first composition comprising 1 g to 20 g R21 polypeptide per administration when said subject is at least 18 years old, or 0.5 g to 10 g R21 polypeptide per administration when said subject is less than 18 years old, said composition further comprising adjuvant, wherein said adjuvant is Matrix-M and said adjuvant is present in a ratio in the range 1:1 to 1:50 of R21 polypeptide:Matrix-M; b. said final composition comprising 10%-50% of the amount of R21 polypeptide of the first composition per administration, said final composition further comprising adjuvant, wherein said adjuvant is Matrix-M, wherein said adjuvant is present in a ratio in the range 1:1 to 1:50 of R21 polypeptide:Matrix-M; and c. instructions for administration to said subject.

57. The kit according to claim 56 further comprising a second composition, said second composition being identical to said first composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0238] Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which:

[0239] FIG. 1 shows diagrams. Comparison of some characteristics of R21 and RTS,S vaccine virus-like particles. A greater density of CSP sequences on the surface of the R21 VLP compared to the RTS,S particle may relate to favourable characteristics of the R21 vaccine such as the lack of induction of significant levels of antibodies to the HBsAg sequence.

[0240] FIG. 2 shows a flow chart. Vac053 phase I clinical trial of R21 vaccine conducted in the UK with Oxford as the main clinical centre (Venkatraman et al). 75 subjects were screened for eligibility and 31 enrolled. Note that subjects in group 2 did not receive an adjuvant with R21. Doses of R21 as shown in the figure. In groups 1, 3 and 4 the dose of matrix-M was 50 micrograms in all subjects.

[0241] FIG. 3 shows a bar chart. Better Safety Profile for R21/Matrix-M 50 g RTS,S/AS01 vs 10 g R21/Matrix-M. Safety profile of R21 (10 mcgs) in matrix-M adjuvant (50 mcgs) compared to RTS,S/AS01 (50 mcgs of RTS,S. Local and systemic adverse events were graded on a standard severity scale of 1 (least) to 3 (most severe). The reactogenicity profile of R21/matrix-M was statistically significantly better than RTS,S/AS01 after the second dose (data shown) and also after the first dose (data not shown). After the third dose again the safety profile of R21/matrix-M was better but not significantly so.

[0242] FIGS. 4A and 4B show bar charts. Excellent Safety Profile of Low Dose 2 g R21/Matrix-M is shown. The safety profile of R21/matrix-M was improved further by decreasing the dose of R21 (but not matrix-M) further to just 2 micrograms (FIG. 4B).

[0243] FIG. 5 shows graphs. R21 Clinical Immunogenicity Data are shown; FIG. 5A shows Immunogenicity dependent on Matrix-M; FIG. 5B shows 10 g R21/MM comparable with 500 g RTS,S/AS01B.

[0244] FIG. 5A: Median time courses of IgG against the NANP repeat region of CSP in VAC53. Time courses shown are medians for Group 1 (=GI) (10 ug R21 in matrix-M (here abbreviated to MM)), G2 (50 ug (=50 micrograms) R21 with no adjuvant) and G3 (50 ug R21 in MM) and are calculated using data for all volunteers that completed follow-up: G1 n=10, G2 n=3, G3 n=8. Median NANP-specific IgG levels were significantly higher after the second and third vaccinations in the adjuvanted groups compared to the groups that received unadjuvanted R21. NANP-specific IgG was boosted again after the third vaccination, but only in groups that received R21 in MM. The highest median antibody response to NANP was observed in the 10 ug R21/MM group 2 weeks after the third vaccination.

[0245] FIG. 5B: Mean time courses of NANP-specific IgG are shown for G1 and G3 in VAC53 and compared to responses seen in the VAC55 trial (NCT:01883609) in volunteers that received 50 ug RTS,S/AS01B at week 0, 4 and 8. Mean NANP-specific IgG levels are comparable between the 10 ug R21/MM and 50 ug RTS,S/AS01B group after each vaccination.

[0246] Antibodies responses against NANP are analysed using an enzyme-linked immunosorbent assay (ELISA) and were conducted as published by Rampling et al (J Infect Dis (2016) 214 (5): 772-781. DOI: https://doi.org/10.1093/infdis/jiw244). A pool of serum positive for NANP-specific IgG was used to form a standard curve on each plate. Arbitrary ELISA Units (EUs) were calculated for each sample based on the optical density (OD405) of the sample and the parameters of the standard curve.

[0247] FIG. 6 shows plots. Durability of Antibody Response; 10 g R21 generated significantly higher antibody titres than 50 g R21 at 6 months.

[0248] Durability of the NANP-specific IgG responses measured using the standardised ELISA method used in FIG. 5. NANP-specific IgG was measured for VAC53 G1 and G3 volunteers at day 238 (D238). At this late time point, antibody levels were significantly higher in the 10 ug R21/MM group compared to the 50 ug R21/MM group (Mann-Whitney analysis, P=0.02, lines show medians) indicating surprisingly better durability with the lower dose of vaccine.

[0249] FIG. 7 shows plots. Lower dose regimen induces a qualitatively different Tfh response and increased B cells.

[0250] Phenotyping of total circulating T follicular helper cells (cTfh) and B cells in peripheral blood mononucleocytes (PBMC) from VAC65 volunteers at the time point of one day before the malaria challenge, denoted day C-1 (D76). Comparison of 10 ug R21/MM and 50 ug R21/MM. Phenotyping of cTfh is achieved by staining cells with fluorescently labelled antibodies against specific markers and analysed by flow cytometry. Circulating Tfh are defined as single, live, lymphocytes that are PD1+CXCR5+CD45RA-CD4+ T cells and are further divided into subsets by expression of chemokine receptors CXCR3 and CCR6 as: Tfh17 (CXCR3-CCR6+), double positive/dp (CXCR3+CCR6+), Tfh1 (CXCR3+CCR6-) and Tfh2 (CXCR3-CCR6-) as previously published (Schmitt, Bentebibel and Ueno, Trends in Immunology, 2014. DOI: http://dx.doi.org/10.1016/j.it.2014.06.002). B cells were phenotyped in the same manner using markers for CD19, CD20, CD21, CD27, IgD, IgG and IgM. CD19+CD20+ B cells were classified by expression of IgD and CD27 as switched memory (IgD-CD27+), non-switched memory (IgD+CD27+), double negative (IgD-CD27) or nave (IgD+CD27+) as previously published (Sanz et al Semin Immunol 2008 DOI: 100.1016/j.smim.2007.12.006).

[0251] total B cells; switched memory B cells (IgD-CD27+); IgG+ MBC; IgG:IgM.

[0252] FIG. 7A: Percentage of subsets within total cTfh. G1 (10,10,10 ug R21/MM) has a significantly higher proportion of Tfh2 cTfh than G2 (50,50,10 ug R21/MM, Mann-Whitney analysis, P=0.0018). This higher Tfh2 response with the lower dose of R21/MM may account for the better performance of the lower dose of vaccine.

[0253] Boxed section (with arrow) indicates CXCR3-CCR6-cTfh are better at providing help to B cells (Locci et al Immunity 2013)

[0254] FIG. 7B: Percentage of CD19+CD20+ B cells within lymphocytes. Significantly higher percentage of CD19+CD20+ B cells within lymphocytes in the 10,10,10 ug R21/MM group than 50,50,10 ug R21/MM group (Mann-Whitney analysis, P=0.0104).

[0255] FIG. 7C: Percentage of switched memory B cells (CD19+CD20+IgD-CD27+) within lymphocytes. There is a significantly higher percentage of switched memory B cells within lymphocytes in 10,10,10 ug R21/MM group (Mann-Whitney analysis, P=0.0004), which again may relate to the better performance of the lower dose of vaccine.

[0256] FIG. 8 shows plots. Very Low 2 g Dose R21/Matrix-M; Still high immunogenicity with this very low dose; Reactogenicity was found to be minimal at this very low dose. NANP-specific IgG responses measured by ELISA as in FIGS. 5 and 6. Comparison of NANP-specific antibody responses at day 28 (D28), D56 and D84 in VAC53 in volunteers vaccinated with 2, 10 or 50 ug R21 in MM. Assay was completed for all samples that were available at the time of testingsome volunteers had not yet passed these time points. Kruskal-Wallis analysis with Dunn's test for multiple comparisons at each time point shows a significantly lower titre in the 2 ug group at D28 and D56 but not at day 84 (Kruskal-Wallis P=0.022, P=0.050 and P=0.212 at D28, D56 and D84 respectively). Lines indicated medians in each group at each time point. The similar titres at day 84 across the dose groups suggests that even this very low dose of R21 may well be protective.

[0257] FIG. 9 shows a table. VACo65Phase I/IIa Sporozoite Challenge Study; 31 vaccines (11, 11, 9 in Groups 1, 2 and 3) & 6 controls underwent CHMI on 30 and 31 Jan. 2017.

[0258] Summary of the design and groups within Vac065, the phase IIa controlled human malaria infection (CHMI) trial conducted in Oxford in 2016-2017.

[0259] FIG. 10 shows a graph. 82% Efficacy with Low Dose R21/Matrix-M.

TABLE-US-00006 Proportion protected (%) P value Group 1 9/11 (82%) P = 0.0009 10, 10, 10 Group 2 7/11 (64%) P = 0.004 50, 50, 10 Group 3 10, 10, 10 + 6/9 (67%) P = 0.006 vectors

[0260] Summary of the efficacy of the three vaccine regimens in Groups 1-3 in the Vac065 CHMI trial. The highest efficacy was observed, surprisingly, with the simple low dose regimen of group 1, in which three 4 weekly doses of 10 micrograms of R21 in 50 micrograms of matrix-M was used with a vaccine to adjuvant ratio of 1:5. Lower efficacy was observed in the other groups. The Kaplan-Meier curve of time to malaria diagnosis shows that in Group 1 the two (of eleven) vaccines who did develop malaria did so several days later then in the control subjects. This indicates that the vaccine has reduced the number of parasites leading to a blood-stage infection so that parasites are detected later. Hence, in these two subjects there was clear evidence of partial vaccine efficacy.

[0261] FIG. 11 shows a graph. Efficacy of Three Doses of 10 g of R21/Matrix-M vs 500 g of RTS,S/AS01.

[0262] Comparison of the efficacy of the R21 in matrix-M vaccine to the efficacy of the RTS,S/AS01 vaccine, used in a standard 0, 4, 8 week regime with 50 mcg of RTS,S in AS01, in previous CHMI trials in the UK and the USA, compared to non-vaccinated controls in the same trials. The efficacy of the R21 vaccine is higher at 82% than that of the RTS,S vaccine which is 58% and this difference approaches statistical significance (two-tailed P value=0.16; one-tailed P value=0.08).

[0263] FIG. 12 shows a graph. Immunogenicity after Two Doses of R21/Matrix-M May Be Higher than after Three (Day, 0, 28, 56 regime).

[0264] Geometric mean NANP IgG timecourses (measured using the same ELISA method as previous figures). Time courses are shown for 10 ug R21/MM (VAC53 G1) and 50 ug RTS,S/AS01B in VAC55 G2 and VAC59 G1. Antibody responses after the second vaccination are significantly higher in the 10 ug R21/MM group compared to either of the 50 ug RTS,S/AS01B groups. Although these titres are re-boosted by a third vaccination, they are boost to levels comparable to the peak post-second dose and drop more rapidly than after the second vaccination. This high level immunogenicity after just two doses, comparable in titre to the levels observed after three doses, suggests strongly that, unexpectedly, a two dose regime of R21 may provide significant efficacy (as does the three dose regimen).

[0265] FIG. 13 shows graphs.

[0266] FIG. 14 shows a plot.

[0267] FIG. 15 shows a plot.

[0268] FIG. 16 shows a plot.

EXAMPLES

Example 1: A Safety and Efficacy Study of R21+/ChAd63/MVA ME-TRAP

[0269] Sponsor:

[0270] University of Oxford

[0271] Information Provided by (Responsible Party):

[0272] University of Oxford

[0273] ClinicalTrials.gov Identifier:

[0274] NCT02905019

[0275] Purpose

[0276] The purpose of this study is to assess the safety and efficacy of adjuvanted R21 alone and in combination with a viral-vectored vaccine regimen (constituting adjuvanted R21+ChAd63 and MVA encoding ME-TRAP) against malaria sporozoite challenge in healthy malaria-naive volunteers.

[0277] Healthy adult volunteers will be recruited in London, Oxford and Southampton.

[0278] All vaccinations will be administered intramuscularly. The study involves having either two, three or five vaccinations and then undergoing challenge infection with malaria, or receiving no vaccinations then undergoing challenge infection with malaria.

TABLE-US-00007 Condition Intervention Phase Malaria Biological: R21 with Matrix-M1 Phase 1 Biological: ChAd63 ME-TRAP Phase 2 Biological: MVA ME-TRAP [0279] Study Type: Interventional [0280] Study Design: Allocation: Randomized [0281] Intervention Model: Parallel Assignment [0282] Masking: Outcomes Assessor [0283] Primary Purpose: Prevention [0284] Official Title: A Phase I/IIa Sporozoite Challenge Study to Assess the Safety and Protective Efficacy of Adjuvanted R21 at Two Different Doses and the Combination Malaria Vaccine Candidate Regimen of Adjuvanted R21+ChAd63 and MVA Encoding ME-TRAP.

[0285] Resource Links Provided by NLM:

[0286] MedlinePlus related topics: Malaria

[0287] Genetic and Rare Diseases Information Center resources: Malaria

[0288] U.S. FDA Resources

[0289] Further Study Details as Provided by University of Oxford:

[0290] Primary Outcome Measures: [0291] Efficacy of adjuvanted R21 at two different doses and adjuvanted R21+ChAd63 and MVA encoding ME-TRAP in healthy malaria-nave volunteers as assessed by number of completely protected individuals. [Time Frame: 6 months]

[0292] Use statistical analysis to compare number of completely protected individuals (those who do not, by Day 21 following sporozoite challenge, develop blood stage infection measured by occurrence of P. falciparum parasitemia, assessed by blood slide) in the vaccine groups compared to the controls. [0293] Safety of adjuvanted R21 at two different doses and adjuvanted R21+ChAd63 and MVA encoding ME-TRAP in healthy malaria-nave volunteers as assessed by frequency of adverse events. [Time Frame: 6 months]

[0294] Solicited and unsolicited adverse event data will be collected at each clinic visit from diary cards, clinical review, clinical examination (including observations) and laboratory results. This AE data will be tabulated and frequency, duration and severity of AEs compared between groups.

[0295] Secondary Outcome Measures: [0296] Humoral immunogenicity generated in malaria nave individuals with adjuvanted R21 at two different doses [Time Frame: 6 months]

[0297] Antibody response to the circumsporozoite protein generated by vaccination with adjuvanted R21. [0298] Cell-mediated immunogenicity generated in malaria nave individuals with ChAd63 and MVA encoding ME-TRAP [Time Frame: 6 months]

[0299] T-cell responses to the TRAP antigen of the malaria parasite generated by vaccination with ChAd63 and MVA encoding ME-TRAP. [0300] Efficacy measured as time to P. falciparum parasitemia assessed by PCR against malaria sporozoite challenge, in healthy malaria-nave volunteers. [Time Frame: 6 months]

[0301] Statistical analyses using blood stage infection as defined by 500 or more parasites/ml in peripheral blood by quantitative PCR. [0302] Efficacy measured as time to P. falciparum parasitemia assessed by blood slide against malaria sporozoite challenge, in healthy malaria-nave volunteers. [Time Frame: 6 months]

[0303] Statistical analyses using blood stage infection defined by a composite of symptoms, blood film result and parasitaemia. [0304] Efficacy measured as time to P. falciparum parasitemia assessed by parasite density dynamics assessed by PCR against malaria sporozoite challenge, in healthy malaria-nave volunteers. [Time Frame: 6 months]

[0305] Statistical analyses using blood stage malaria infection as defined by 20 or more P. falciparum parasites/ml in peripheral blood by quantitative PCR.

[0306] Other Outcome Measures: [0307] Long term protective efficacy of adjuvanted R21 at two different doses and adjuvanted R21+ChAd63 and MVA encoding ME-TRAP [Time Frame: 12 months]

[0308] Long term efficacy of the vaccination regimens will be assessed by re-challenging any sterilely protected individuals at 5-7 months after the first sporozoite challenge (12 months after the start of the study) and comparing the number of re-challenges who develop blood stage infection with unvaccinated controls.

[0309] Estimated Enrolment: 70

TABLE-US-00008 Assigned Arms Interventions Active Comparator: Group 1 Biological: R21 R21 with Matrix-M1. Three vaccinations with 10 with Matrix-M1 g R21/50 g Matrix-M1 on days 0, 28 and 56. Vaccine Active Comparator: Group 2 Biological: R21 R21 with Matrix-M1. Two vaccinations with 50 g with Matrix-M1 R21/50 g Matrix-M1 on days 0 and 28 and one Vaccine vaccination with 10 g R21/50 g Matrix M1 on day 56. Active Comparator: Group 3 Biological: R21 R21 with Matrix-M1, ChAd63 ME-TRAP and with Matrix-M1 MVA ME-TRAP. Three vaccinations with 10 g Vaccine R21/50 g Matrix-M1 on days 0, 28 and 56. Plus Biological: ChAd63 one vaccination with ChAd63 ME-TRAP on day 7 ME-TRAP and one vaccination with MVA ME-TRAP Vaccine on day 63. Biological: MVA ME-TRAP Vaccine No Intervention: Group 4a These volunteers will not be vaccinated and will serve as infectivity controls when groups 1-3 undergo challenge. No Intervention: Group 4b These volunteers will not be vaccinated and will serve as infectivity controls when group 5-7 and sterilely protected volunteers from groups 1-3 undergo challenge. No Intervention: Group 4c These volunteers will not be vaccinated and will serve as infectivity controls if any volunteers from groups 5 and 7 are rechallenged. Active Comparator: Group 5 Biological: R21 R21 with Matrix-M1. Two vaccinations with with Matrix-M1 10 g R21/50 g Matrix-M1 on days 0 and Vaccine 28 and one vaccination with 2 g R21/50 g Matrix-M1 on day 56. No Intervention: Group 6 Volunteers in this group have received vaccinations in a different malaria vaccine trial. These volunteers will not receive any vaccinations in this trial, but will undergo controlled human malaria infection as part of this study. Active Comparator: Group 7 Biological: R21 R21 with Matrix-M1. Two vaccinations with with Matrix-M1 10 g R21/50 g Matrix-M1 on days 0 and 28. Vaccine

DETAILED DESCRIPTION

[0310] Vaccination phases and challenge procedures have been staggered over the trial period into 2 parts, challenge A and B.

[0311] Challenge A: [0312] Groups 1-3 consist of volunteers receiving either R21 alone or R21+ChAd63-MVA ME-TRAP followed by CHMI by sporozoite challenge (mosquito bite) at week 12. Twelve volunteers will be recruited to each group. [0313] Group 4a will serve as infectivity controls, these volunteers will not be vaccinated.

[0314] Challenge B: [0315] Sterilely protected volunteers in groups 1-3 may be rechallenged to assess durability of efficacy, 5-12 months after the initial challenge. [0316] Groups 5-7 will also be enrolled to participate in challenge B. [0317] Group 5 (8 volunteers) will test the efficacy of standard dose R21 with a fractional third dose followed by CHMI at week 12. [0318] Group 6 will test the long-term efficacy of the standard dose R21 vaccination regimen (volunteers in this group will have already received their vaccinations whilst enrolled in the VACo53 phase I malaria trial which started in 2015 and will therefore not receive any additional vaccinations before undergoing challenge approximately two years after their immunisations). [0319] Group 7 (8 volunteers) will test the efficacy of a two dose R21 vaccination regimen followed by CHMI at week 8. [0320] Group 4b will serve as infectivity controls for groups 5-7 and sterilely protected group 1-3 volunteers. Group 4c volunteers will be used as infectivity controls if any volunteers from groups 5 and 7 are rechallenged.

[0321] Eligibility:

[0322] Ages Eligible for Study: 18 Years to 45 Years (Adult)

[0323] Sexes Eligible for Study: All

[0324] Accepts Healthy Volunteers: Yes

[0325] Criteria

[0326] Inclusion Criteria: [0327] Healthy adults aged 18 to 45 years. [0328] Able and willing (in the Investigator's opinion) to comply with all study requirements. [0329] Willing to allow the investigators to discuss the volunteer's medical history with their General Practitioner. [0330] Women only: Must practice continuous effective contraception* for the duration of the study. [0331] Agreement to refrain from blood donation during the course of the study and for at least 3 years after the end of their involvement in the study. [0332] Written informed consent to participate in the trial. [0333] Reachable (24/7) by mobile phone during the period between CHMI and completion of antimalarial treatment. [0334] Willingness to take a curative anti-malaria regimen following CHMI. [0335] For volunteers not living close to their designated malaria challenge follow-up site (Oxford or Southampton): agreement to stay in a hotel room close to the trial centre during a part of the study (from at least day 6.5 post mosquito bite until anti-malarial treatment is completed). [0336] Answer all questions on the informed consent quiz correctly.

[0337] Exclusion Criteria: [0338] History of clinical malaria (any species). [0339] Travel to a clearly malaria endemic locality during the study period or within the preceding six months [0340] Use of systemic antibiotics with known antimalarial activity within 30 days of CHMI (e.g. trimethoprim-sulfamethoxazole, doxycycline, tetracycline, clindamycin, erythromycin, fluoroquinolones and azithromycin) [0341] Receipt of an investigational product in the 30 days preceding enrolment, or planned receipt during the study period. [0342] Prior receipt of an investigational vaccine likely to impact on interpretation of the trial data as assessed by the investigator. If any volunteers in Group 1-3 undergo rechallenge, this exclusion criterion does not extend to the vaccines previously received in the VACo65 trial [0343] For Group 3 volunteers only: prior receipt of a non-malaria MVA or non-malaria adenovirus vectored experimental vaccine [0344] Any confirmed or suspected immunosuppressive or immunodeficient state, including HIV infection; asplenia; recurrent, severe infections and chronic (more than 14 days) immunosuppressant medication within the past 6 months (inhaled and topical steroids are allowed). [0345] Use of immunoglobulins or blood products within 3 months prior to enrolment. [0346] History of allergic disease or reactions likely to be exacerbated by any component of the vaccine (e.g. egg products, Kathon) or malaria infection. [0347] Any history of anaphylaxis post vaccination. [0348] History of clinically significant contact dermatitis. [0349] History of sickle cell anaemia, sickle cell trait, thalassaemia or thalassaemia trait or any haematological condition that could affect susceptibility to malaria infection. [0350] Pregnancy, lactation or intention to become pregnant during the study. [0351] Use of medications known to cause prolongation of the QT interval and existing contraindication to the use of Malarone [0352] Use of medications known to have a potentially clinically significant interaction with Riamet and Malarone [0353] Any clinical condition known to prolong the QT interval [0354] History of cardiac arrhythmia, including clinically relevant bradycardia [0355] Disturbances of electrolyte balance, eg, hypokalaemia or hypomagnesaemia [0356] Family history of congenital QT prolongation or sudden death [0357] Contraindications to the use of all three proposed anti-malarial medications; Riamet, Malarone and Chloroquine. [0358] History of cancer (except basal cell carcinoma of the skin and cervical carcinoma in situ). [0359] History of serious psychiatric condition that may affect participation in the study. [0360] Any other serious chronic illness requiring hospital specialist supervision. [0361] Suspected or known current alcohol abuse as defined by an alcohol intake of greater than 42 standard UK units every week. [0362] Suspected or known injecting drug abuse in the 5 years preceding enrolment. [0363] Hepatitis B surface antigen (HBsAg) detected in serum. [0364] Seropositive for hepatitis C virus (antibodies to HCV) at screening (unless has taken part in a prior hepatitis C vaccine study with confirmed negative HCV antibodies prior to participation in that study, and negative HCV RNA PCR at screening for this study). [0365] An estimated, ten year risk of fatal cardiovascular disease of 5%, as estimated by the Systematic Coronary Risk Evaluation (SCORE) system. 60 [0366] Positive family history in 1st and 2nd degree relatives <50 years old for cardiac disease. [0367] Volunteers unable to be closely followed for social, geographic or psychological reasons. [0368] Any clinically significant abnormal finding on biochemistry or haematology blood tests, urinalysis or clinical examination. [0369] Any other significant disease, disorder, or finding which may significantly increase the risk to the volunteer because of participation in the study, affect the ability of the volunteer to participate in the study or impair interpretation of the study data.

[0370] ClinicalTrials.gov identifier: NCT02905019

[0371] LocationsUnited Kingdom [0372] NIHR Wellcome Trust Clinical Research Facility, Hammersmith Hospital [0373] London, United Kingdom [0374] Contact: Reshma Sultan+44 (0)20 331 31086 [0375] CCV, University of Oxford, [0376] Oxford, United Kingdom, OX3 7LE [0377] Contact: Volunteer Coordinator vaccinetrials@ndm.ox.ac.uk [0378] Southampton National Institute for Health Research [0379] Southampton, United Kingdom [0380] Contact 02381 204989 UHS.RecruitmentCRF@nhs.net [0381] Sponsors and Collaborators: University of Oxford [0382] Responsible Party: University of Oxford [0383] ClinicalTrials.gov Identifier: NCT02905019 [0384] Other Study ID Numbers: VACo65 [0385] Individual Participant Data (IPD) Sharing Statement: [0386] Plan to Share IPD: Undecided

Example 2

[0387] With reference to the clinical trial outline, there was good reason for believing that either groups 2 or 3 would have been better and surprisingly they were not. It is impressive that the Group 1 result (82% efficacy) with low dose R21 was better than the other two regimens tested (Groups 2 and 3). Group 2 might have been better because in it a larger amount of R21 was administered: 50 mcg rather than to mcg for doses 1 and 2, but the same dose for dose 3: there is evidence in the literature from RTS,S that such a fractional (i.e. reduced) third dose regime might be better than a standard regime (as in group 1) (reference Regules et al. J Infect Dis. 2016; 214:762-71.) Group 3 might have been the best group because of the additional administration of partially effective vectored vaccines, as reported by Rampling et al. (J Infect Dis. 2016 Sep. 1; 214(5):772-81), but strikingly the efficacy in Group 3 was not as high as in Group 1.

Example 3

[0388] Overview:

[0389] R21 is a novel malaria vaccine candidate, which is a biosimilar of the most advanced malaria vaccine candidate, RTS,S/AS01 and is composed of a fusion protein of the malaria circumsporozoite protein and Hepatitis B surface antigen. We assessed the efficacy of R21 administered with Matrix-M (R21/MM) given alone at two different dose schedules and in combination with viral-vectored vaccines using controlled human malaria infection (CHMI) in healthy UK volunteers.

[0390] We undertook this Phase IIa study in healthy UK volunteers to assess the efficacy against malaria sporozoite challenge of R21/MM in different dose schedules and in combination with ChAd63-MVA ME-TRAP.

[0391] Methods

[0392] Volunteers were recruited into this Phase IIa study at three trial centres in the UK and CHMI was undertaken at Imperial College, London. Thirty-one healthy volunteers were vaccinated with either 3 doses of 10/10/10 g of R21/MM (Group 1; n=11), or 3 doses of 50/50/10 g of R21/MM (Group 2; n=11), or 3 doses of 10/10/10 g of R21/MM (Group 1; n=11) given with ChAd63-MVA expressing ME-TRAP.

[0393] Referring to FIG. 2, we described a Phase Ia study; Open-label; Non-randomised; Healthy adults aged 18 to 50 years; Oxford; London (Imperial); 31 volunteers in total.

[0394] As observed in the phase I trial (Vac053, see FIG. 2 for the trial profile) the safety profile of R21 (10 micrograms) in matrix-M (50 micrograms), see FIG. 3, was clearly better than for the RTS,S/AS01 vaccine. As in the phase I trial, vac053see FIG. 5, good immunogenicity was observed for antibodies to CSP with all regimens.

[0395] CHMI was delivered by mosquito bite at week 12 after first vaccination, including 6 unvaccinated controls. The trial is registered with ClinicalTrials.gov (NCT02905019)

[0396] Findings

[0397] This trial was done between 7 Nov. 2016 and 15 May 2017. Of over 70 volunteers screened, 37 volunteers (FIG. 9) underwent malaria sporozoite challenge on the 30 and 31 Jan. 2017. Vaccinations were generally well tolerated, with the majority of local and systemic adverse events being mild in nature and an improved safety profile compared to published RTS,S/As01 datasee FIG. 3. Sterile protection was observed (see FIG. 10) in 9/11 (81.8%) subjects in Group 1, 7/11 (63.6%) subjects in Group 2 and 6/9 (66.7%) subjects in Group 3. All vaccinated volunteers showed a significant delay in patency in comparison to control volunteers. 5/6 control subjects were diagnosed with blood stage malaria. Antibody responses to the NANP repeat region of the circumsporozoite protein were significantly boosted at 14 days after the 2.sup.nd vaccination in all volunteers and comparable to RTS,S/AS01.

[0398] High level efficacy observed in the lower dose group is demonstrated as a benefit of the invention.

[0399] Experimental Details

[0400] Study Design and Participants

[0401] We did a Phase IIa study in healthy malaria-nave adult males and non-pregnant females between the ages of 18 and 45 years. Recruitment and vaccination were conducted at the Centre for Clinical Vaccinology and Tropical Medicine at the University of Oxford and the Wellcome Trust Clinical Research Facility in Southampton and Imperial College in the United Kingdom. This Phase IIa, open-label malaria sporozoite challenge trial consisted of 4 cohorts. The sample sizes reflect practical limitations on volunteer recruitment, ethical considerations limiting the number of volunteers that should receive a vaccine regimen without prior evidence of efficacy, and the desire to describe the efficacy of the immunisation regimes. Allocation to study group was undertaken by the investigators prior to enrolment based on subject preference. Group 1 (n=11) received 3 vaccinations (R21/MM 10 g at 0, 4 and 8 weeks); Group 2 (n=11) received 3 vaccinations (R21/MM 50 g at 0 and 4 weeks and R21/MM 10 g at 8 weeks); Group 3 (n=9) received 5 vaccinations (R21/MM 10 g at 0, 4 and 8 weeks and ChAd63 ME-TRAP 510.sup.10 virus particles (vp) at 1 week, and MVA ME-TRAP 210.sup.8 plaque forming units (pfu) at 9 weeks) and Group 4 (n=6) received no vaccinations. All subjects underwent initial CHMI by mosquito bite at the same time (week 12 after first vaccination for vaccinated subjects).

[0402] The volunteers were infected using five infectious bites from P. falciparum 3D7-strain infected Anopheles stephensi mosquitoes at Imperial College, London. All subjects were infected with a single batch of mosquitoes supplied by the Department of Entomology, Walter Reed Army Institute of Research, Washington D.C., USA. The inclusion and exclusion criteria are listed in the supplementary appendix. All participants gave written informed consent prior to participation, and the study was conducted according to the principles of the Declaration of Helsinki and in accordance with Good Clinical Practice (GCP).

[0403] The study was approved by the UK National Research Ethics Service, Committee South Central-Berkshire (Ref: 16/SC/0261), the Medicines and Healthcare Products Regulatory Agency (Ref: 21584/0360/001-0001), and the Oxford University Clinical Trials and Research Governance team, who independently and externally monitored compliance with Good Clinical Practice guidelines. Viral-vectored vaccine use was authorised by the Genetically Modified Organisms Safety Committee (GMSC) of the Oxford University Hospitals NHS Trust (Reference number GM 462.16.88). The trial was registered with ClinicalTrials.gov (Ref: NCT02905019) and an independent local safety monitor provided safety oversight.

[0404] Procedures

[0405] R21 (Batch no: 01015-01) was manufactured and vialed under Good Manufacturing Practice conditions at the Clinical Biomanufacturing Facility, University of Oxford: the production, manufacture and storage of this product have been previously described in in WO2014/111733; see also Venkatraman et al. Matrix-M (Batch no: M1-103) is a patented adjuvant technology developed by Novavax: the production, manufacture and storage of this product have been previously described [21]. Generation, manufacture and storage of the ChAd63 ME-TRAP (Batch no: 01S11-01) and MVA ME-TRAP (Batch no: 0091013) vaccines has been previously described [12, 22]. On the day of vaccination, R21 was thawed to room temperature and was administered intramuscularly into the deltoid of the non-dominant arm within 1 hour of removal from the freezer, mixed with Matrix-M. The viral vectored vaccines were administered intramuscularly within 1 hour of thawing into the deltoid of the dominant arm (the contralateral arm to R21 administration). All volunteers were observed in the unit for 1 hour after vaccination. Volunteers were provided with an electronic diary card to record their temperature and any solicited local and systemic adverse events for 7 days post-vaccination and unsolicited adverse events for 28 days post-vaccination. Severity grading of adverse events and the assignment of a causal relationship for adverse events were conducted according to predefined guidelines stated in the protocol. An independent Safety Monitoring Committee provided safety oversight during the course of the trial. Safety bloods including full blood count, renal function and liver function tests were done on visits at day 0, 7, 28, 35, 56, 63 and 83 (day before CHMI) in Group 1 and 2 volunteers. Additional safety bloods were done on day 14 and 70 for volunteers in Group 3. Antibody responses measured by anti-NANP IgG ELISA were performed on samples from days 0, 7, 14, 28, 35, 42, 56, 63, 70 and 83. Ex-vivo IFN-ELISpot responses to CSP were assessed on samples from day 0, 42 and 83. In addition, IFN-ELISpot responses to TRAP were assessed on samples from day 0, 28, 70 and 83 in Group 3 volunteers.

[0406] The full methods used for these immunological assays have been previously described [23]. The CHMI procedure was as described by Rampling et al. J Infect Dis. 2016 Sep. 1; 214(5):772-81. Following CHMI, a diagnosis of blood stage malaria infection was made in subjects with symptoms suggestive of malaria and positive thick film microscopy, or qPCR result >500 parasites/ml if either thick film was negative, or symptoms were absent [12]. Vaccinated subjects who had not developed blood stage malaria by day 21 after CHMI were deemed to exhibit sterile protection.

[0407] Outcomes

[0408] The primary outcome measures were to assess the efficacy (occurrence of P. falciparum parasitemia, assessed by blood slide) of the different vaccine regimens against malaria sporozoite challenge, and to assess the safety of the vaccines, in healthy malaria-nave volunteers. The secondary outcome measures were to assess immunogenicity and to assess the efficacy (measured as time to P. falciparum parasitemia assessed by blood slide, by PCR, and parasite density dynamics assessed by PCR) in healthy malaria-nave volunteers.

[0409] Statistical Analysis

[0410] Data were analyzed using GraphPad Prism version 5.03 for Windows (GraphPad Software Inc., California, USA) and Stata 10.0 (Statacorp LP, Texas, USA). Geometric means or medians with interquartile ranges for each group are described. Kruskal-Wallis analysis and the Friedman test were used to compare peak immune responses with the baseline. Significance testing of differences between two groups used Mann-Whitney analysis. A Wilcoxon matched-pairs analysis was used to compare between time points within groups. A chi-squared test for trend was used to compare the safety data between different groups. A statistically significant difference in efficacy between a vaccination regimen and controls was assessed by log rank analysis of Kaplan Meier curves, using a one-tailed log rank test at the different endpoints. A value of p<0.05 was considered significant.

[0411] Results

[0412] Study Population

[0413] From 7 Nov. 2016 to 31 Jan. 2017, a total of 43 volunteers of the 75 who were screened for eligibility were enrolled into this study. One volunteer in Group 1 withdrew after their first vaccination due to a change in personal circumstances and was replaced. Another Group 1 volunteer withdrew after their second vaccination due to a change in personal circumstances. One Group 2 volunteer withdrew after their first vaccination as they were no longer able to commit to the schedule of attendances. Two volunteers in Group 3 withdrew after their third vaccination due to a change in personal circumstances. Another Group 3 volunteer withdrew after their fifth vaccination due to apprehensions about undergoing CHMI. None of the withdrawals were related to the vaccination and there were no ongoing AEs and safety bloods were normal. 37 volunteers (11 Group 1 volunteers, 11 Group 2 volunteers, 9 Group 3 volunteers and 6 unvaccinated controls) underwent CHMI on the 30 and 31 Jan. 2017. All of these 37 volunteers completed follow-up until 90 days post-CHMI. Participant flow and study design is summarised in the table of FIG. 9.

[0414] Efficacy

[0415] There were 9/11 Group 1 volunteers, 7/11 Group 2 volunteers and 6/9 Group 3 volunteers who hadn't developed malaria at 21 days post-CHMI resulting in a sterile efficacy of 81.8% (p=0.0009), 63.6% (p=0.004) and 66.7% (p=0.006), respectively.

[0416] Five out of six control volunteers developed malaria with a mean time to diagnosis of 11.3 days (Median 11, range 11-12, SD 0.45). A significant delay in patency was observed in all three groups with a mean time to diagnosis of 15.3 days (Median 15.3, range 14.5-16, SD 1.1), 16.4 days (Median 16.5, range 14.5-16, SD 1.5) and 15.3 days (Median 16, range 14-16, SD 1.2) in Groups 1, 2 and 3 respectively.

[0417] Adverse Events

[0418] No serious adverse reactions (SARs) or suspected unexpected serious adverse reactions (SUSARs) occurred. There were no withdrawals due to safety concerns and no pre-defined study stopping or holding rules were activated. The majority of adverse events (AEs) reported were self-limiting and mild in severity and reactogenicity profiles observed were similar to previous studies for both the R21/MM (Ref: VAC053, FIG. 2; Venkatraman et al) and the ChAd63_MVA ME-TRAP [23]. Solicited local and systemic adverse events in the first 7 days after each vaccination were mild to moderate and overall all vaccination regimes were well tolerated. Vaccine site pain was the most common local adverse event and was predominantly mild in severity. The reactogenicity profile of the 10/10/10 g R21/MM dose group, which has also been tested in a previous Phase I trial (Venkatraman et al) was significantly improved in comparison to data from two previous trials in our centre using RTS,S/AS01 (Vaccination 1p=0.003; Vaccination 2p=0; Vaccination 3p=0.125). Vaccine site pain was significantly reduced after the first (p=0.02) and second (p=0.02) vaccinations in comparison to RTS,S/AS01. Unsolicited adverse events collected for 28 days after each vaccination deemed definitely, possibly or probably related to vaccination were predominantly mild in nature. Laboratory adverse events were predominantly Grade 1. Immunogenicity profiling showed potent antibody induction to the central NANP repeat region of the circumsporozoite protein as observed in the Vac053 phase I trial (FIG. 5).

[0419] Discussion

[0420] We report here for the first time, high level efficacy of a novel malaria vaccine candidate, R21 adjuvanted with Matrix-M at one-fifth of the dose of the most advanced malaria vaccine candidate, RTS,S/AS01. This will have significant dose-sparing and cost-saving advantages for large scale production of the vaccine if it was proven to be efficacious in subjects living in malaria endemic regions. Our data confirms that this vaccine approach is safe and well tolerated in healthy UK volunteers with adverse events being predominantly mild in nature. The use of a 50 g dose with a fractional (.sup.th) third dose did not improve efficacy, in contrast to the recently published study by Regules et al., where it was shown that a delayed fractional third dose of RTS,S/AS01 improved efficacy in a malaria sporozoite challenge study [10]. The humoral response to the vaccine did not vary between Groups 1-3 and the addition of the viral-vectored vaccines to the regime in Group 3 did not result in reduced CS antibody immunogenicity. The magnitude of the response did not predict efficacy, which suggests that the quality or other aspects of the antibody response may also be relevant for efficacy. There were minimal IFN- ELISpot responses to CSP induced by R21/MM, which is similar to previous experience with RTS,S/AS01 that only induces low level CD4.sup.+ and no CD8.sup.+ T-cell responses [24].

[0421] One of the control volunteers did not develop clinical malaria, which is very unusual and has never occurred previously in CHMI trials at the Oxford centre. However, the failure of one of the controls to develop malaria has been reported in a number of other CHMI trials undertaken by Sanaria Inc. to test the intravenous whole sporozoite vaccine [25, 26]. The mean time to patency in the control group of 11.3 days indicates that this was not an unusually weak challenge, and the vaccination and CHMI methodology used in this trial are largely comparable to other CHMI studies [23].

[0422] In conclusion, this Phase IIa malaria sporozoite challenge study demonstrated high level efficacy with a novel low-cost malaria vaccine, R21/MM. This high efficacy is at least as high or higher than regimes using a higher (50 microgram) dose of the RTS,S/AS01 vaccine used in a similar 0, 4, 8 week schedule (FIG. 11).

[0423] It could also be that a two-dose schedule would be sufficient to protect this primed populationbased on the high immunogenicity observed for anti-CSP antibodies after just two immunisations (FIG. 12). In view of this, are using a two dose-schedule using 10 g of R21/MM in a CHMI study in healthy UK volunteers. If this is proven to also have high level efficacy, this would have significant cost-saving benefits and would be potentially easier to deploy in co-ordination with the EPI program.

Example 4Challenge Study

[0424] Here we show a further challenge study of R21/matrix-M vaccines. We provide data from our completed challenge study with low-dose R21 in matrix-M. In summary we show efficacy in the following groups: [0425] Re-challenged volunteers at 8.5 months post-immunisation: 3/5 protected=60% [0426] A two dose group (10, 10 mcgs): 4/7 protected at 3 weeks=57% [0427] A three dose group (10, 10, 2 mcgs): 5/7 protected at three weeks=71%

[0428] The 57% with a two dose regimen is unprecedented in the whole malaria vaccine field, demonstrating the remarkable technical effects of the invention. The rechallenge efficacy of 60% at 8.5 months is excellent too.

[0429] Study Details

[0430] A further challenge study of R21/matrix-M vaccines was undertaken. Two key objectives are described below.

[0431] The first objective was to evaluate the durability of protection of the to microgram (mcg) R21 dose in 50 mcg of matrix-M administered three times at four weekly intervals. In the original challenge study at the end of January 2017 nine of eleven vaccines administered this regimen were steriley protected. Of the nine protected subjects all were invited for a re-challenge in mid-September 2017 and 5 agreed to participate. Of the five re-challengees three were again steriley protected and two were not, corresponding to 60% sterile efficacy. No booster vaccine dose was administered before the re-challenge so that the re-challenge of these subjects occurred about 8.5 months after their last vaccine dose at the start of January.

[0432] This evidence of durability of sterile protection in most protected subjects out to 8.5 months after last immunisation is very positive and compares favourably with the efficacy of RTS,S/AS01 protection which is generally measured at about 6 months post last dose. Efficacy of R21 at 8.5 month was 49% compared to the reported efficacy of RTS,S/AS vaccine at 5-8 months of 26% (see table).

[0433] The second objective was to assess two new R21 immunisation regimes using the preferred low dosages of R21 in matrix-M. In one group seven subjects were immunised with just two doses of 10 mcg of R21 in 50 mcg of matrix-M adjuvant. Challenge was at 3 to 4 weeks after the last dose. Of these seven individuals 4 were steriley protected amounting to 57% vaccine efficacy, apparently the highest efficacy ever reported with a two dose malaria vaccine (from assessment by detailed literature review).

[0434] A further vaccination group used two doses of 10 mcg of R21/matrix-M at a four week interval followed by a 2 mcg R21 dose in 50 mcg matrix-M after a further four weeks. Seven subjects thus immunised were challenged 3-4 weeks later and 5 were steriley protected, an efficacy rate of 71%. This is little different from the 9/11 protected with three doses of 10 mcg of R21 in 50 mcg of matrix-M. These data with three low doses of R21/matrix-M provide further evidence of its high level efficacy.

[0435] Exemplary data are provided below:

TABLE-US-00009 R21 (Invention) 3 10 mcg doses at 0, 1, 2 months challenge at 4 weeks 9/11 = 82% 5 (of 9) re-challenged at month 8.5 post last dose 3/5 = 60% Overall R21 efficacy at 8.5 months post last dose = 49% RTS,S Regules et al. J Infect Dis 2016 3 week post last dose challenge 10/16 = 62.5% 5 of 10 re-challenged at 8 months post last dose 1/5 = 20% Overall efficacy at 8.5 months post last dose = 12.5% Kester et al. J Infect Dis 2009 0, 1, 2 months: challenge at 2-3 weeks: 18/36 protected = 50% Rechallenge at 5.5 months post last dose: 4/9 protected = 44% Overall efficacy of RTS,S/AS01 at 5.5 months post last dose = 22% (for RTS,S/AS02 0, 1, 2: 14/44 protected = 32% and 4/9 on re- challenge (44%). Overall = 14%) Rampling et al. J Infect Dis 2016 0, 1, 2 months: 12/16 protected at 4 weeks = 75% Re-challenge at 6 months post first CHMI: 5/6 protected = 83% Overall efficacy at 7 months post last dose = 62% To weight/average: 12.5 5, 22 9, 14 9, 62 6: summed = 758.5/29 Averaged Overall RTS,S 5-8 month efficacy: = 26% Table Legend: Durability of protection after immunisation with R21 and RTS,S malaria vaccines. Overall efficacy at 5-8 months post last dose is calculated by multiplying the proportion protected in the initial challenge by the proportion protected in the late challenge, expressed as a percentage. Only those protected in the initial challenge are re-challenged. R21 durable efficacy appears as about double the rate reported for RTS,S.

[0436] In conclusion, this example shows additional useful clinical data on the durability of protection with R21 and also shows that a two dose regimen works in humans.

Example 5R21 Low Dose Further Challenge Study

[0437] In this example we demonstrate good efficacy in a further challenge study. More specifically, we report a further challenge study of R21/matrix-M vaccines. Key findings are good durability of efficacy at 8.5 months after vaccination that appears better than known RTS,S, and also good efficacy with a two dose low-dose regime.

[0438] Experimental:

[0439] A further challenge study of R21/matrix-M vaccines was undertaken in September 2017 with two objectives.

[0440] The first objective was to evaluate the durability of protection of the 10 microgram (mcg) R21 dose in 50 mcg of matrix-M administered three times at four weekly intervals. In the original challenge study (see examples aboveJanuary 2017) nine of eleven vaccines administered this regimen were sterilely protected. Of the nine protected subjects all were invited for a re-challenge in mid-September 2017 and 5 agreed to participate. Of the five re-challengees three were again sterilely protected and two were not, corresponding to 60% sterile efficacy (see Group 1 in FIG. 13).

[0441] FIG. 13 shows outcome of controlled human malaria infection (CHMI) trial in September 2017. In the control group all of almost all the non-vaccinated subjects were infected, in this case 5 of 6. In Group 6 which were two vaccines immunised 20 months before CHMI with lost dose (10 micrograms) R21 in matrix-M, neither was protected. However, in Group 5 three of five individuals undergoing CHMI initially in late January 2017 (after immunisation in November 2016early January 2017) were still sterilely protected at 8.5 months after their last immunisation indicating very useful durability of vaccine efficacy. Moreover, four of seven vaccines receiving just two low 10 microgram doses of R21 one and two months before CHMI in September 2017 were sterilely protected indicating good vaccine efficacy with just two doses of R21 in matrix-M. Also, Group 5 showed that three doses of vaccine with 10, 10 and 2 micrograms generated 72% sterile efficacy (5/6 subjects protected) again showing the efficacy of low doses of R21 in matrix-M. In all vaccines the dose of matrix-M used was 50 micrograms.

[0442] No booster vaccine dose was administered before the re-challenge so that the re-challenge of these subjects occurred about 8.5 months after their last vaccine dose at the start of January.

[0443] This evidence of durability of sterile protection in most protected subjects out to 8.5 months after last immunisation is very positive and compares favourably with the efficacy of RTS,S/AS01 protection which is generally measured at about 6 months post last dose. Efficacy of R21 at 8.5 month was 49% compared to the reported efficacy of RTS,S/AS vaccine at 5-8 months of 26% (see table below).

[0444] The second objective was to assess two new R21 immunisation regimes using low dosages of R21 in matrix-M. In one group (Group 7 in the figure) seven subjects were immunised with just two doses of 10 mcg of R21 in 50 mcg of matrix-M adjuvant. Challenge was at 3 to 4 weeks after the last dose. Of these seven individuals 4 were sterilely protected amounting to 57% vaccine efficacy, apparently the highest efficacy ever reported with a two dose malaria vaccine (from assessment by detailed literature review), showing the technical benefits of the invention.

[0445] A further vaccination group received two doses of 10 mcg of R21/matrix-M at a four week interval followed by a 2 mcg R21 dose in 50 mcg matrix-M after a further four weeks. Seven subjects thus immunised were challenged 3-4 weeks later and 5 were sterilely protected, an efficacy rate of 71% (Group 5 in the figure). This is little different from the 9/11 protected with three doses of 10 mcg of R21 in 50 mcg of matrix-M. These data with three low doses of R21/matrix-M provide further evidence of its high level efficacy.

[0446] A final group (Group 6 in the figure) were just two vaccines receiving R21 20 months earlier: neither was sterilely protected. The sample size in this group was very low.

TABLE-US-00010 TABLE of DATA: Durability of protection after immunisation with R21 and RTS,S malaria vaccines is shown. Overall efficacy at 5-8 months post last dose is calculated by multiplying the proportion protected in the initial challenge by the proportion protected in the late challenge, expressed as a percentage. Only those protected in the initial challenge are re-challenged. R21 durable efficacy (49%) appears as about double the rate reported for RTS,S (26%). Data: R21 (this study) 3 10 mcg doses at 0, 1, 2 months challenge at 4 weeks 9/11 = 82% 5 (of 9) re-challenged at month 8.5 post last dose 3/5 = 60% Overall R21 efficacy at 8.5 months post last dose = 49% RTS,S (known/prior published studies) Regules et al. J Infect Dis 2016 3 week post last dose challenge 10/16 = 62.5% 5 of 10 re-challenged at 8 months post last dose 1/5 = 20% Overall efficacy at 8.5 months post last dose = 12.5% Kester et al. J Infect Dis 2009 0, 1, 2 months: challenge at 2-3 weeks: 18/36 protected = 50% Rechallenge at 5.5 months post last dose: 4/9 protected = 44% Overall efficacy of RTS,S/AS01 at 5.5 months post last dose = 22% (for RTS,S/AS02 0, 1, 2: 14/44 protected = 32% and 4/9 on re-challenge (44%). Overall = 14%) Rampling et al. J Infect Dis 2016 0, 1, 2 months: 12/16 protected at 4 weeks = 75% Re-challenge at 6 months post first CHMI: 5/6 protected = 83% Overall efficacy at 7 months post last dose = 62% To weight/average: 12.5 5, 22 9, 14 9, 62 6: summed = 758.5/29 Averaged Overall RTS,S 5-8 month efficacy: = 26%

Example 6: R21 Low Dose Vaccination is Immunogenic in West African as Well as UK Subjects

[0447] In this example we show good immunogenicity of the low dose regime in West Africa for the first time.

[0448] We refer to FIG. 14.

[0449] This data demonstrates that R21 low dose vaccination is immunogenic in West African as well as UK subjects. R21 at a dose of 10 micrograms in 50 micrograms matrix-M was administered to both UK subjects (VAC53 trial) and to West African subjects from Burkina Faso (Banfora) (in the Vac060 trial). In each trial vaccination was administered as a three dose regime at months 0, 1 and 2. Immunogenicity after two doses was similar in both populations and only slightly lower in Burkina Faso adults after three doses. These data show good immunogenicity of the low dose R21 vaccine even in an area with high level endemic malaria transmission.

Example 7: Low Dose Immunogenicity with 2 Micrograms as Well as 10 Micrograms R21

[0450] In this example we show that 2 micrograms R21 is also an immunogenic dose.

[0451] This demonstrates the utility of low dosesdata in earlier examples was on to micrograms.

[0452] We refer to FIG. 15.

[0453] UK healthy adults subjects were immunised with different doses of R21 in a three dose 0, 1, 2 month immunisation regime. The antibody titres induced after three doses of R21 at day 84 were very similar using 2 micrograms, to micrograms and 50 micrograms of R21 (in each case in 50 micrograms of matrix-M adjuvant). The local and systemic reactogenicity of the 2 microgram dose regime was better (i.e. reduced) compared to that observed with 10 micrograms or 50 micrograms dosages. Thus we demonstrate a benefit of the low doses taught in the present inventionreduced side effects as illustrated by reduced reactogenicityyet still achieving antibody titres similar to far higher doses such as 50 microgram doses.

Example 8: Low Dose Vaccination with R21 Produces Better Durability of Vaccine Responses

[0454] Here we surprisingly show that better immune responses are found at day 238 with 10 compared to 50 micrograms R21: i.e. we show that the lower dose is better.

[0455] We refer to FIG. 16.

[0456] The data demonstrate that low dose vaccination with R21 produces better durability of vaccine responses. UK subjects were immunised at 0, 1, 2 months with R21 with dosages of 10 micrograms or 50 micrograms (always with 50 micrograms matrix-M adjuvant). At day 238 after the first dose antibody levels to the central repeat region of the circumsporozoite protein, a correlate of vaccine efficacy, were higher with the low dose to microgram regime than that higher dose 50 microgram regimen i.e. we show that the lower dose is better.

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[0484] Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiment and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.