Serum antibody assay for determining protection from malaria, and pre-erythrocytic subunit vaccines

Abstract

Disclosed herein are diagnostic methods and compositions for identifying individuals that are protected against Plasmodium falciparum caused malaria. Such methods are particularly useful for determining not only the protective efficacy of Pf whole parasite vaccines for individual subjects, but also within populations of vaccinated subjects. Also disclosed herein are subunit vaccines comprising at least one Pf immunologic determinant for protection against Plasmodium -caused malaria.

1 . A method of detecting antibodies that indicate a state of protective immunity against  P. falciparum -induced malaria in a human subject, comprising: (a) contacting a human serum sample from said subject with a first recombinant polypeptide comprising a sequence that is at least 85%, identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, or an antigenic fragment thereof; and (b) detecting antibodies that are specifically immunoreactive to said first recombinant polypeptide, wherein said immunoreactivity is above a cutoff value in Table 1 for said first recombinant polypeptide. 
     
     
         2 . A method of detecting antibodies to assess the response to a malaria vaccine in a human subject comprising: (a) inoculating the human subject with a  P. falciparum -specific vaccine; (b) contacting a serum sample from said subject with a first recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, or an antigenic fragment thereof, wherein said serum sample is taken from a subject after at least one dose of a  P. falciparum  vaccine is administered to the subject; and (c) detecting antibodies that are immunoreactive to the first recombinant polypeptide is above a cutoff value in Table 1 for said recombinant polypeptide. 
     
     
         3 . A method for immunizing a subject in need of protection against  P. falciparum -induced malaria comprising: (a) determining if a serum sample from a subject comprises an antibody that specifically immunoreacts with at least a first recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, or an antigenic fragment thereof; and (b) administering a  P. falciparum  vaccine to the subject if the sample lacks the antibody detected above a cut off value in Table 1 for the first recombinant polypeptide. 
     
     
         4 - 7 . (canceled) 
     
     
         8 . The method of  claim 1 , further comprising contacting the human serum sample with a second recombinant polypeptide comprising an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO:19 or SEQ ID NO: 20, or an antigenic fragment thereof; and detecting antibodies that are specifically immunoreactive to said second recombinant polypeptide, wherein said immunoreactivity is above a cutoff value in Table 1 for said second recombinant polypeptide. 
     
     
         9 - 15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein the first recombinant polypeptide comprises a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:11, and the method further comprises contacting the human serum sample with a second recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO: 20. 
     
     
         17 . The method of  claim 1 , wherein the first recombinant polypeptide comprises a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:12, and the method further comprises contacting the human serum sample with a second recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO: 20. 
     
     
         18 . The method of  claim 1 , wherein the first recombinant polypeptide comprises a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:13, and the method further comprises contacting the human serum sample with a second recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO: 20. 
     
     
         19 . The method of  claim 1 , wherein the first recombinant polypeptide comprises a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:14, and the method further comprises contacting the human serum sample with a second recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:19, or SEQ ID NO: 20. 
     
     
         20 - 23 . (canceled) 
     
     
         24 . The method of  claim 1 , wherein said antibody immunoreactivity is determined by enzyme-linked immunosorbent assay (ELISA). 
     
     
         25 . The method of  claim 1 , wherein the first recombinant polypeptide is affixed to a solid phase. 
     
     
         26 - 30 . (canceled) 
     
     
         31 . The method of  claim 1 , wherein the first recombinant polypeptide provides a sensitivity of at least about 92% and a specificity of at least about 95%. 
     
     
         32 . The method of  claim 1 , wherein the first recombinant polypeptide provides a sensitivity of about 100% and a specificity of at least about 89%. 
     
     
         33 . (canceled) 
     
     
         34 . The method of  claim 1 , wherein the cutoff is a predictive value in Table 2. 
     
     
         35 . (canceled) 
     
     
         36 . A composition comprising (a) an immunogen comprising a first Pf-specific polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14, or an antigenic fragment thereof; and (b) a carrier. 
     
     
         37 - 39 . (canceled) 
     
     
         40 . The composition of  claim 36  wherein the first Pf-specific polypeptide comprises a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:11, and the composition further comprises a second Pf-specific polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12, SEQ IN NO: 13, SEQ IN NO: 14, SEQ ID NO: 19, or SEQ ID NO: 20. 
     
     
         41 . The composition of  claim 36  wherein the first Pf-specific polypeptide comprises a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:12, and the composition further comprises a second Pf-specific polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 11, SEQ IN NO: 13, SEQ IN NO: 14, SEQ ID NO: 19, or SEQ ID NO: 20. 
     
     
         42 . The composition of  claim 36  wherein the first Pf-specific polypeptide comprises a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:13, and the composition further comprises a second Pf-specific polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 11, SEQ IN NO: 12, SEQ IN NO: 14, SEQ ID NO: 19, or SEQ ID NO: 20. 
     
     
         43 . The composition of  claim 36  wherein the first Pf-specific polypeptide comprises a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:14, and the composition further comprises a second Pf-specific polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 11, SEQ IN NO: 12, SEQ IN NO: 13, SEQ ID NO: 19, or SEQ ID NO: 20. 
     
     
         44 - 63 . (canceled) 
     
     
         64 . The method of  claim 1 , wherein the first recombinant polypeptide comprises a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 13, and wherein the method further comprises contacting the human serum sample with one or more of the following sequences:
 (i) a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 20 or an antigenic fragment thereof, and a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 14 or an antigenic fragment thereof;   (ii) a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 20 or an antigenic fragment thereof, and a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12 or an antigenic fragment thereof;   (iii) a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 11 or an antigenic fragment thereof, and a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12 or an antigenic fragment thereof;   (iv) a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 14 or an antigenic fragment thereof and a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12 or an antigenic fragment thereof;   (v) a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 20 or an antigenic fragment thereof, a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 14 or an antigenic fragment thereof, and a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12 or an antigenic fragment thereof;   (vi) a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 19 or an antigenic fragment thereof, and a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 20 or an antigenic fragment thereof;   (vii) a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 19 or an antigenic fragment thereof, and a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 14 or an antigenic fragment thereof;   (viii) a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 19 or an antigenic fragment thereof, and a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12 or an antigenic fragment thereof;   (ix) a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 19 an antigenic fragment thereof, a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 20 or an antigenic fragment thereof, and a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 14 or an antigenic fragment thereof; or   (x) a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 19 or an antigenic fragment thereof, a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 20 or an antigenic fragment thereof, and a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12 or an antigenic fragment thereof.   
     
     
         65 . The method of  claim 64 , wherein the cutoff value for the first recombinant polypeptide is selected from the cutoff values in Table 1. 
     
     
         66 . The method of  claim 65 , wherein the cutoff is a predictive value in Table 2. 
     
     
         67 - 69 . (canceled) 
     
     
         70 . A composition comprising (a) a first recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 13 or an antigenic fragment thereof, and (b) at least one of the following combinations of recombinant polypeptides:
 (i) a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 20 or an antigenic fragment thereof, and a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 14 GSK3 or an antigenic fragment thereof;   (ii) a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 20 or an antigenic fragment thereof, and a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12 or an antigenic fragment thereof;   (iii) a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 11 or an antigenic fragment thereof, and a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12 or an antigenic fragment thereof;   (iv) a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 14 or an antigenic fragment thereof, and a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12 or an antigenic fragment thereof;   (v) a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 20 or an antigenic fragment thereof, a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 14 or an antigenic fragment thereof, and a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12 or an antigenic fragment thereof;   (vi) a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 19 or an antigenic fragment thereof, and a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 20 or an antigenic fragment thereof;   (vii) a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 19 or an antigenic fragment thereof, and a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 14 or an antigenic fragment thereof;   (viii) a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 19 or an antigenic fragment thereof, and a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12 or an antigenic fragment thereof;   (ix) a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 19 an antigenic fragment thereof, a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 20 or an antigenic fragment thereof, and a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 14 or an antigenic fragment thereof; or   (x) a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 19 or an antigenic fragment thereof, a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 20 or an antigenic fragment thereof, and a recombinant polypeptide comprising a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12 or an antigenic fragment thereof.   
     
     
         71 . (canceled)