Antibodies raised against an Escherichia coli-produced recombinant protein encoding a 76-kDa section (region C) of malaria transmission-blocking vaccine candidate, Pfs230, have previously been shown to significantly reduce the ability of Plasmodium falciparum parasites to infect mosquitoes (71.2-89.8%). To further define the region of the Pfs230 required for transmission-blocking activity, four recombinant proteins each encoding a section of region C (Pfs230 amino acids 443-1132) were produced using the same E. coli expression system and tested for immunogenicity in mice: (i) r230/MBP.C5' encodes the first half of region C (amino acids 443-791, six cysteines); (ii) r230/MBP.CM1 encodes only cysteine motif (CM) 1 (amino acids 583-913, eight cysteines); (iii) r230/MBP.C1.6 (amino acids 453-913, eight cysteines) also includes all of CM1; and (iv) r230/MBP.C2 encodes only CM2 (amino acids 914-1268, 11 cysteines). All the recombinant proteins induced antibodies that recognized parasite-produced Pfs230, but the titre of the Pfs230 specific-antibodies generated varied, C = C1.6 = C5' > CM1 > CM2. Two recombinants, r230/MBP.C5' and r230/MBP.C1.6, induced antibody titres that were equivalent to or greater than the titre generated by r230/MBP.C. However, in contrast to r230/MBP.C, none of the recombinants induced antibodies that effectively blocked parasite infectivity to mosquitoes. This suggests that the inclusion of amino acids 914-1132 is important for the production of the transmission-blocking epitope present in region C.
To characterize the occurrence, frequency, and kinetics of retroviral recombination in vivo, we intravaginally inoculated rhesus macaques, either simultaneously or sequentially, with attenuated simian immunodeficiency virus (SIV) strains having complementary deletions in their accessory genes and various degrees of replication impairment. In monkeys inoculated simultaneously with SIVmac239⌬vpx/⌬vpr and SIVmac239⌬nef, recombinant wild-type (wt) virus and wild-type levels of plasma viral RNA (vRNA) were detected in blood by 2 weeks postinoculation. In monkeys inoculated first with SIVmac239⌬vpx/⌬vpr and then with SIVmac239⌬nef, recombination occurred but was associated with lower plasma vRNA levels than plasma vRNA levels seen for monkeys inoculated intravaginally with wt SIVmac239. In one monkey, recombination occurred 6 weeks after the challenge with SIVmac239⌬nef when plasma SIVmac239⌬vpx/⌬vpr RNA levels were undetectable. In monkeys inoculated first with the more highly replicating strain, SIVmac239⌬nef, and then with SIVmac239⌬vpx/ ⌬vpr, wild-type recombinant virus was not detected in blood or tissues. Instead, a virus that had repaired the deletion in the nef gene by a compensatory mutation was found in one animal. Overall, recombinant SIV was eventually found in four of six animals intravaginally inoculated with the two SIVmac239 deletion mutants. These findings show that recombination can occur readily in vivo after mucosal SIV exposure and thus contributes to the generation of viral genetic diversity and enhancement of viral fitness.
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