The P. falciparum parasite uses clonal antigenic variation to evade host antibody immune responses and permit the establishment of the fairly long-lasting infections that individual clones require for their transmission back to the mosquito vector. It is difficult to know what the average clonal transit time through the human host is, but it is unlikely to be shorter than three weeks and could be significantly longer. While the important role of PfEMP1 in antigenic variation during the intra-erythrocytic multiplication stages is well established, antigenic variation during merozoite, gametocyte and gamete stages has not been formally demonstrated in P. falciparum, although a role for RIFIN and STEVOR proteins remains plausible.
A-type Rifins are found associated with the surface of infected erythrocytes and gametocytes and thus appear to follow the expression pattern of PfEMP1
, whereas surface exposure of STEVOR so far has only been confirmed on merozoites
. The data presented here show that a specific type B RIFIN variant (PF13_0006) is expressed by free, live merozoites and gametes (activated gametocytes). Transcript analysis presented here and previously
[21, 22] unambiguously show that this RIFIN is upregulated in early ring, schizonts and late stage gametocytes. To investigate PF13_0006 protein expression, rabbit antibodies to the variable region of this protein were generated. These antibodies stained the surface of 3D7 merozoites and gametes, but not the surface of intact 3D7 gametocytes or FCR3 merozoites. Also the antibodies only detected correctly sized protein in western blots of 3D7 schizonts and gametocytes extracts and not of 3D7 rings or FCR3 schizonts. Thus, although the antibodies appear to un-specifically target a number of other proteins in the western blots, collectively the data support the expression of PF13_0006 protein on the surface of merozoites and gametes. The expression of PF13_0006 inside the parasite during schizont and gametocyte stages is in agreement with previous reports using antibodies with specificity for B-type RIFINs
[5, 18]. There is as yet no evidence that expression of RIFIN variants is mutually exclusive
[5, 36]. Transcript analyses published earlier
[21, 22] indicate that transcripts of other B1 RIFINs, in particular PFI0025c, encoding the unique sequence motif identified in this study are present in merozoites and gametes at the same time as PF13_0006. High transcript levels of PF13_0006 were also detected in early rings
 but no protein was detected which could reflect a spill-over of late stages in the synchronization. It should also be noted that the method for detecting transcripts is much more sensitive than the methods used to detect protein.
A proportion of malaria exposed individuals from both East and West Africa had acquired plasma antibodies recognizing recombinant PF13_0006. The seropositive rate was slightly higher among the Gambian children, but this probably reflected that these children participated in a treatment trail and therefore had a recent exposure. The data suggest that RIFIN variants antigenically similar to the variable domain of PF13_0006 occur in the African parasite populations possibly in particular the unique sequence motif of the B1 RIFINs. These antibody responses were more common among older children than in adults. A similar decline in anti-PfEMP1 seropositivity was reported previously
 and probably reflect that the antibody levels to these polymorphic proteins are short lived and that adults who have acquired partial immunity have less parasite exposure than the children. The prevalence of antibodies did not vary consistently with the level of transmission and was higher in the Tanzanian samples collected in 2008 than in the previous years, despite a general decline in transmission in the area
. This could reflect temporal shifts in the relative abundance of parasites carrying RIFIN variants closely related to PF13_0006, as similar fluctuations related to time and geographical location have been observed for antibody recognition of different PfEMP1 variants
Confocal imaging of live, non-fixed, non-permeabilized free merozoites is challenging due to the small size and motility of these stages. However, present data does indicate that PF13_0006 is co-localized with MSP1 on the merozoite surface. This would place RIFIN along with the other variant surface antigens such as SURFINs
 and STEVOR
 which recently have been shown to be expressed on the surface of free merozoites. These studies of diverse multigene families, taken together, suggest that the antigens they encode play a role in evading the immune response against merozoites. However, as group B RIFIN only comprise ~25% (40 genes in the 3D7 genome) of the family, and the B1 sub-group around half of this proportion
, indicating that there is scope for other members of the family to play different roles in the parasite life cycle. It is thus tempting to speculate that the B or B1 type RIFINs have become functionally specialized for a particular role in relation to merozoite egress or erythrocyte invasion.
Similarly, the PF13_0006 B1 RIFIN may serve a particular role either within the developing gametocyte, or in emergent gametes. Whether PF13_0006 serves a role in fertilization, e.g. locating and/or binding to a “partner” cell of opposite mating type, remains to be investigated. However, it is important to note that 3D7 sporozoites have also been found to specifically transcribe the PF13_0006 rif gene
. Extrapolating observations from single parasite isolates is unsound and there is a need to investigate rif expression on other genetic backgrounds, but the finding that PF13_0006 appear to be expressed in all free-living stages of 3D7 highlights the need for functional studies of this RIFIN in extracellular forms of the parasite.
The rif gene family is part of the pir (Plasmodium interspersed repeats) gene super-family of six variant multigene families found in Plasmodium vivax (vir), Plasmodium ovale (oir), Plasmodium knowlesi (kir) and in three rodent malarias (Plasmodium chabaudi, cir; Plasmodium berghei, bir; Plasmodium yoelii, yir). Sequence analysis of these gene families, as for rif genes, show compartmentalization into sub-groups/types indicative of specialized functions rather than sequence variation for antigenic variation alone
. This function may be common to all Plasmodium species which share life cycle, the common challenges of host immunity, and the need for invasion and rupture of host cells. The variable domain of PF13_0006, the domain to which the antibody was raised, seems to be semi-conserved in the C-terminal part for the B1 subtype RIFINs. This part of the protein may serve a common function for the subtype and it may be possible to generate antibodies targeting B1 RIFINs expressed by different parasite clones.