B-Cell Responses to Pregnancy-Restricted and -Unrestricted Plasmodium falciparum Erythrocyte Membrane Protein 1 Antigens in Ghanaian Women Naturally Exposed to Malaria Parasites

Abstract

Protective immunity to Plasmodium falciparum malaria acquired after natural exposure is largely antibody mediated. IgG-specific P. falciparum EMP1 (PfEMP1) proteins on the infected erythrocyte surface are particularly important. The transient antibody responses and the slowly acquired protective immunity probably reflect the clonal antigenic variation and allelic polymorphism of PfEMP1. However, it is likely that other immune-evasive mechanisms are also involved, such as interference with formation and maintenance of immunological memory. We measured PfEMP1-specific antibody levels by enzyme-linked immunosorbent assay (ELISA) and memory B-cell frequencies by enzyme-linked immunosorbent spot (ELISPOT) assay in a cohort of P. falciparum-exposed nonpregnant Ghanaian women. The antigens used were a VAR2CSA-type PfEMP1 (IT4VAR04) with expression restricted to parasites infecting the placenta, as well as two commonly recognized PfEMP1 proteins (HB3VAR06 and IT4VAR60) implicated in rosetting and not pregnancy restricted. This enabled, for the first time, a direct comparison in the same individuals of immune responses specific for a clinically important parasite antigen expressed only during well-defined periods (pregnancy) to responses specific for comparable antigens expressed independent of pregnancy. Our data indicate that PfEMP1-specific B-cell memory is adequately acquired even when antigen exposure is infrequent (e.g., VAR2CSA-type PfEMP1). Furthermore, immunological memory specific for VAR2CSA-type PfEMP1 can be maintained for many years without antigen reexposure and after circulating antigen-specific IgG has disappeared. The study provides evidence that natural exposure to P. falciparum leads to formation of durable B-cell immunity to clinically important PfEMP1 antigens. This has encouraging implications for current efforts to develop PfEMP1-based vaccines. P rotective immunity to Plasmodium falciparum malaria acquired after natural exposure is mediated to a large extent by IgG antibodies targeting the asexual blood stages of the parasites (reviewed in reference 1). The low rate of acquisition probably reflects the extensive clonal antigenic variation and allelic polymorphism of key antigens. However, other immune-evasive mechanisms may also be involved, such as interference with formation and maintenance of immunological memory. Indeed, it has often been speculated that such subversion is important for the slow and incomplete acquisition of clinical protection following natural exposure to P. falciparum in areas where these parasites are stably transmitted (reviewed in references 2, 3, and 4). The evidence supporting the hypothesis of a fragile or dysfunctional immunological memory to P. falciparum includes the often transient IgG responses in children with malaria (5-9), apparent interference with antigen presentation (10, 11), "masking" of surface-exposed IgG epitopes (12), and expansion of "atypical" or "exhausted" B cells after prolonged exposure to P. falciparum antigens (13,14). Conversely, the hypothe...

Kinetics of B Cell Responses to Plasmodium falciparum Erythrocyte Membrane Protein 1 in Ghanaian Women Naturally Exposed to Malaria Parasites

“…28). Third, the levels of these latter Abs decline fairly rapidly postdelivery, when pregnancy-restricted P. falciparum Ags are no longer present (23,29,30). This contrasts with the postdelivery levels of TT-specific IgG, which remained high throughout follow-up as expected.…”

“…The women lost to follow-up did not differ significantly from the rest for any of the study parameters. Samples from 74 nonpregnant women from the same study area and from 13 nonpregnant Danish women without exposure to P. falciparum malaria collected as part of a previous study (23) were included as P. falciparum-exposed and nonexposed controls for the B cell phenotyping (see later).…”

“…Recombinant proteins representing the entire ectodomains of three PfEMP1 proteins were produced in Baculovirus-infected insect cells as described previously (23). These full-length proteins represented the VAR2CSA-type PfEMP1 protein IT4VAR04 (FV2) and two PfEMP1 proteins involved in rosetting, HB3VAR06 (FV6) and IT4VAR60 (FV60).…”

“…Plasma levels of IgG with specificity for FV2, FV6, and FV60 were measured by ELISA as described (23). In brief, 96-well, flat-bottom microtiter plates were coated with Ag, blocked with BSA, and incubated with prediluted plasma samples for 1 h. Wells were washed and bound Ab was detected with HRP-conjugated rabbit anti-human IgG and ο-phenylene diamine.…”

“…Frequencies of memory B cells with specificity for FV2, FV6, and FV60 were measured by ELISPOT as described previously (23). In brief, cryopreserved PMBCs were thawed, washed, adjusted to 2.5 3 10 6 /ml, and memory B cells were induced to start Ab secretion by stimulating them with IL-2 and the polyclonal activator R-848 for 72 h. The Ags FV2, FV6, FV60, and TT were added to separate wells of prewetted ELISPOT plates, and the plates were incubated overnight (4˚C).…”