The dynamics of Plasmodium falciparum variation in western Kenya
Description
In an area of intense transmission, a malaria vaccine could reduce infection due to the parasite types represented in the vaccine, but have no detectable effect on the overall frequency of infection if there were multiple parasite types and immunization did not protect against heterologous parasites. These studies were performed to determine whether immunization with SPf66 decreased infection with parasites containing the 11 amino acid peptide from merozoite surface protein-1 (MSP-1) present in SPf66, or increased infection due to heterologous parasites containing alternate (heterologous) MSP-1 sequences. Based on this 11 amino acid peptide (YSLFQ KEKMVL) from MSP-1 in SPf66, 3 forward primers (S,Q,V) were designed to amplify the MSP-1 sequence present in SPf66, and 3 additional forward primers (G,H,I) to amplify the alternative MSP-1 sequence (YGLFHKEKMIL). This strategy was validated by PCR amplification and dideoxy sequencing with 14 cloned laboratory isolates, which demonstrated that each of the 6 forward primers amplified one MSP-1 sequence or the other, but not both. This technique was then used to examine filter paper blots from an SPf66 vaccine study of 85 subjects in Saradidi, KENYA. In that study, the prevalence of infection with YSLFQKEKMVL or YGLFHKEKMIL type parasites was unaffected by immunization with SPf66 (based on amplification with the S and G primers $\rm\lbrack p\ge 0.12$ as analyzed by chi-square), the Q and H primers $\rm\lbrack p\ge 0.13\rbrack ,$ or the V and I primers $\rm\lbrack p\ge 0.18\rbrack ).$ These results suggest that SPf66 does not exert a selective effect in vivo. Because the Block 1 sequence in SPf66 is typical for the MAD20 and RO33 allotypes of MSP-1 (YSLFQKEKMVL), immunization with SPf66 should select for the alternative K1 allotype in Block 1 of MSP-1 (YGLFHKEKMIL), and may by linkage also select for K1 (increase the frequency of K1) in the adjacent Block 2 of MSP-1. To test this hypothesis we typed parasites by PCR using MSP-1 allele-specific primers flanking Block 2 to distinguish K1, MAD20 and RO33 parasites. The results of these studies indicate that there were no significant differences in the frequencies of these Block 2 allotypes among persons who received SPf66 im, SPf66 sc or the placebo (hepatitis B vaccine im) $\rm (p\ge 0.17).$ These findings suggest that immunization with SPf66 also does not exert a selective effect on Block 2 of MSP-1 in vivo. As a control we used PCR to amplify the polymorphic Block 3 region of MSP-2. Because MSP-2 is on a different chromosome from MSP-1, there should be no linkage between selection for Block 2 MSP-1 allotypes after immunization with SPf66 and the frequencies of different Block 3 MSP-2 types. We found no significant differences among 10 MSP-2 genotypes between SPf66 im and placebo, or between SPf66 sc and placebo $\rm (p\ge 0.1$ by Fisher's exact test). We found a significant difference between genotype 548/559 for SPf66 im and placebo or between SPf66 sc and placebo, prior to immunization with SPf66 (p = 0.05 and 0.03 respectively). Because these samples were collected before immunization with SPf66, the effect could not be due to immunization with SPf66. These studies have developed a molecular method to indirectly assess vaccine efficacy