A rapid global review of strategies to improve influenza vaccination uptake in Australia.

This study aimed to identify effective strategies for improving the uptake of influenza vaccination and to inform recommendations for influenza vaccination programs in Australia. A rapid systematic review was conducted to assimilate and synthesize peer-reviewed articles identified in PubMed. The National Health and Medical Research Council (NHMRC) Hierarchy of Evidence was used to appraise the quality of evidence. A systematic search identified 4373 articles and 52 that met the inclusion criteria were included. The evidence suggests influenza vaccination uptake may be improved by interventions that (1) increase community/patient demand and access to influenza vaccine and overcome practice-related barriers; (2) reinforce the critical role healthcare providers play in driving influenza vaccination uptake. Strategies such as standing orders, reminder and recall efforts were successful in improving influenza vaccination rates. Community pharmacies, particularly in regional/remote areas, are well positioned to improve influenza vaccine coverage. The findings of this rapid review can be utilized to improve the performance of influenza immunization programs in Australia and other countries with comparable programs; and recommend priorities for future evaluation of interventions to improve influenza vaccination uptake.

Analysis of diagnostic data for sexually transmissible infections in South Australian Aboriginal Community Controlled Health Services (2008-16).

Australian Aboriginal communities experience a high burden of sexually transmissible infections (STIs). Since 2009, a comprehensive sexual health program has been implemented at nine Aboriginal Community Controlled Health Services in South Australia. This study assessed trends in STI testing and positivity using deidentified diagnostic data from this period (2008-16). METHODS: Testing data for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV) from one urban, three regional and five remote Aboriginal health services were analysed using logistic regression. RESULTS: From 2008 to 2016, testing increased for CT (twofold), NG (threefold) and TV (sixfold). On average, 30% of testing occurred during an annual 6-week screen. Fewer males were tested (range 27-38% annually). Mean annual STI testing coverage was 28% for 16- to 30-year-old clients attending regional or remote services (2013-16). Positivity at first testing episode for all three infections declined during the study period. From 2013 to 2016, when testing was stable and changes in positivity were more likely to indicate changes in prevalence, there were significant reductions in CT positivity (adjusted odds ratio (aOR) 0.4; 95% confidence interval (CI) 0.2-0.5) and TV positivity (aOR 0.6, 95% CI 0.4-0.9), although declines were statistically significant for females only. There was no significant decrease in NG positivity (aOR 0.9; 95% CI 0.5-1.5). CONCLUSIONS: Since the sexual health program began, STI testing increased and STI positivity declined, but significant reductions observed in CT and TV positivity were confined to females. These findings suggest evidence of benefit from sustained, comprehensive sexual health programs in Aboriginal communities with a high STI prevalence, but highlight the need to increase STI testing among men in these communities.

Declining Malaria Transmission Differentially Impacts the Maintenance of Humoral Immunity to Plasmodium falciparum in Children.

Background: We investigated the poorly understood impact of declining malaria transmission on maintenance of antibodies to Plasmodium falciparum merozoite antigens and infected erythrocytes (IEs), including functional immunity. Methods: In a 3-year longitudinal cohort of 300 Kenyan children, antibodies to different AMA1 and MSP2 alleles of merozoites, IE surface antigens, and antibody functional activities were quantified. Results: Over a period in which malaria transmission declined markedly, AMA1 and MSP2 antibodies decreased substantially; estimated half-lives of antibody duration were 0.8 year and 1-3 years, respectively. However, 69%-74% of children maintained their seropositivity to AMA1 alleles and 42%-52% to MSP2 alleles. Levels and prevalence of antimerozoite antibodies were consistently associated with increasing age and concurrent parasitemia. Antibodies promoting opsonic phagocytosis of merozoites declined rapidly (half-life, 0.15 years). In contrast, complement-fixing antibodies to merozoites did not decline and antibodies to IE surface antigens expressing virulent phenotypes were much better maintained (half-life, 4-10 years). Conclusions: A decline in malaria transmission is associated with reduction in naturally acquired immunity. However, loss of immunity is not universal; some key functional responses and antibodies to IEs were better maintained and these may continue to provide some protection. Findings have implications for malaria surveillance and control measures and informing vaccine development.

A novel approach to identifying patterns of human invasion-inhibitory antibodies guides the design of malaria vaccines incorporating polymorphic antigens.

BACKGROUND: The polymorphic nature of many malaria vaccine candidates presents major challenges to achieving highly efficacious vaccines. Presently, there is very little knowledge on the prevalence and patterns of functional immune responses to polymorphic vaccine candidates in populations to guide vaccine design. A leading polymorphic vaccine candidate against blood-stage Plasmodium falciparum is apical membrane antigen 1 (AMA1), which is essential for erythrocyte invasion. The importance of AMA1 as a target of acquired human inhibitory antibodies, their allele specificity and prevalence in populations is unknown, but crucial for vaccine design. METHODS: P. falciparum lines expressing different AMA1 alleles were genetically engineered and used to quantify functional antibodies from two malaria-exposed populations of adults and children. The acquisition of AMA1 antibodies was also detected using enzyme-linked immunosorbent assay (ELISA) and competition ELISA (using different AMA1 alleles) from the same populations. RESULTS: We found that AMA1 was a major target of naturally acquired invasion-inhibitory antibodies that were highly prevalent in malaria-endemic populations and showed a high degree of allele specificity. Significantly, the prevalence of inhibitory antibodies to different alleles varied substantially within populations and between geographic locations. Inhibitory antibodies to three specific alleles were highly prevalent (FVO and W2mef in Papua New Guinea; FVO and XIE in Kenya), identifying them for potential vaccine inclusion. Measurement of antibodies by standard or competition ELISA was not strongly predictive of allele-specific inhibitory antibodies. The patterns of allele-specific functional antibody responses detected with our novel assays may indicate that acquired immunity is elicited towards serotypes that are prevalent in each geographic location. CONCLUSIONS: These findings provide new insights into the nature and acquisition of functional immunity to a polymorphic vaccine candidate and strategies to quantify functional immunity in populations to guide rational vaccine design.

Limited antigenic diversity of Plasmodium falciparum apical membrane antigen 1 supports the development of effective multi-allele vaccines.

BACKGROUND: Polymorphism in antigens is a common mechanism for immune evasion used by many important pathogens, and presents major challenges in vaccine development. In malaria, many key immune targets and vaccine candidates show substantial polymorphism. However, knowledge on antigenic diversity of key antigens, the impact of polymorphism on potential vaccine escape, and how sequence polymorphism relates to antigenic differences is very limited, yet crucial for vaccine development. Plasmodium falciparum apical membrane antigen 1 (AMA1) is an important target of naturally-acquired antibodies in malaria immunity and a leading vaccine candidate. However, AMA1 has extensive allelic diversity with more than 60 polymorphic amino acid residues and more than 200 haplotypes in a single population. Therefore, AMA1 serves as an excellent model to assess antigenic diversity in malaria vaccine antigens and the feasibility of multi-allele vaccine approaches. While most previous research has focused on sequence diversity and antibody responses in laboratory animals, little has been done on the cross-reactivity of human antibodies. METHODS: We aimed to determine the extent of antigenic diversity of AMA1, defined by reactivity with human antibodies, and to aid the identification of specific alleles for potential inclusion in a multi-allele vaccine. We developed an approach using a multiple-antigen-competition enzyme-linked immunosorbent assay (ELISA) to examine cross-reactivity of naturally-acquired antibodies in Papua New Guinea and Kenya, and related this to differences in AMA1 sequence. RESULTS: We found that adults had greater cross-reactivity of antibodies than children, although the patterns of cross-reactivity to alleles were the same. Patterns of antibody cross-reactivity were very similar between populations (Papua New Guinea and Kenya), and over time. Further, our results show that antigenic diversity of AMA1 alleles is surprisingly restricted, despite extensive sequence polymorphism. Our findings suggest that a combination of three different alleles, if selected appropriately, may be sufficient to cover the majority of antigenic diversity in polymorphic AMA1 antigens. Antigenic properties were not strongly related to existing haplotype groupings based on sequence analysis. CONCLUSIONS: Antigenic diversity of AMA1 is limited and a vaccine including a small number of alleles might be sufficient for coverage against naturally-circulating strains, supporting a multi-allele approach for developing polymorphic antigens as malaria vaccines.

Research priorities for the development and implementation of serological tools for malaria surveillance.

Surveillance is a key component of control and elimination programs. Malaria surveillance has been typically reliant on case reporting by health services, entomological estimates and parasitemia (Plasmodium species) point prevalence. However, these techniques become less sensitive and relatively costly as transmission declines. There is great potential for the development and application of serological biomarkers of malaria exposure as sero-surveillance tools to strengthen malaria control and elimination. Antibodies to malaria antigens are sensitive biomarkers of population-level malaria exposure and can be used to identify hotspots of malaria transmission, estimate transmission levels, monitor changes over time or the impact of interventions on transmission, confirm malaria elimination, and monitor re-emergence of malaria. Sero-surveillance tools could be used in reference laboratories or developed as simple point-of-care tests for community-based surveillance, and different applications and target populations dictate the technical performance required from assays that are determined by properties of antigens and antibody responses. To advance the development of sero-surveillance tools for malaria elimination, major gaps in our knowledge need to be addressed through further research. These include greater knowledge of potential antigens, the sensitivity and specificity of antibody responses, and the longevity of these responses and defining antigens and antibodies that differentiate between exposure to Plasmodium falciparum and P. vivax. Additionally, a better understanding of the influence of host factors, such as age, genetics, and comorbidities on antibody responses in different populations is needed.

Antibodies to polymorphic invasion-inhibitory and non-Inhibitory epitopes of Plasmodium falciparum apical membrane antigen 1 in human malaria.

BACKGROUND: Antibodies to P. falciparum apical membrane protein 1 (AMA1) may contribute to protective immunity against clinical malaria by inhibiting blood stage growth of P. falciparum, and AMA1 is a leading malaria vaccine candidate. Currently, there is limited knowledge of the acquisition of strain-specific and cross-reactive antibodies to AMA1 in humans, or the acquisition of invasion-inhibitory antibodies to AMA1. METHODOLOGY/FINDINGS: We examined the acquisition of human antibodies to specific polymorphic invasion-inhibitory and non-inhibitory AMA1 epitopes, defined by the monoclonal antibodies 1F9 and 2C5, respectively. Naturally acquired antibodies were measured in cohorts of Kenyan children and adults. Antibodies to the invasion-inhibitory 1F9 epitope and non-inhibitory 2C5 epitope were measured indirectly by competition ELISA. Antibodies to the 1F9 and 2C5 epitopes were acquired by children and correlated with exposure, and higher antibody levels and prevalence were observed with increasing age and with active P. falciparum infection. Of note, the prevalence of antibodies to the inhibitory 1F9 epitope was lower than antibodies to AMA1 or the 2C5 epitope. Antibodies to AMA1 ectodomain, the 1F9 or 2C5 epitopes, or a combination of responses, showed some association with protection from P. falciparum malaria in a prospective longitudinal study. Furthermore, antibodies to the invasion-inhibitory 1F9 epitope were positively correlated with parasite growth-inhibitory activity of serum antibodies. CONCLUSIONS/SIGNIFICANCE: Individuals acquire antibodies to functional, polymorphic epitopes of AMA1 that may contribute to protective immunity, and these findings have implications for AMA1 vaccine development. Measuring antibodies to the 1F9 epitope by competition ELISA may be a valuable approach to assessing human antibodies with invasion-inhibitory activity in studies of acquired immunity and vaccine trials of AMA1.

New insights into acquisition, boosting, and longevity of immunity to malaria in pregnant women.

BACKGROUND: How antimalarial antibodies are acquired and maintained during pregnancy and boosted after reinfection with Plasmodium falciparum and Plasmodium vivax is unknown. METHODS: A nested case-control study of 467 pregnant women (136 Plasmodium-infected cases and 331 uninfected control subjects) in northwestern Thailand was conducted. Antibody levels to P. falciparum and P. vivax merozoite antigens and the pregnancy-specific PfVAR2CSA antigen were determined at enrollment (median 10 weeks gestation) and throughout pregnancy until delivery. RESULTS: Antibodies to P. falciparum and P. vivax were highly variable over time, and maintenance of high levels of antimalarial antibodies involved highly dynamic responses resulting from intermittent exposure to infection. There was evidence of boosting with each successive infection for P. falciparum responses, suggesting the presence of immunological memory. However, the half-lives of Plasmodium antibody responses were relatively short, compared with measles (457 years), and much shorter for merozoite responses (0.8-7.6 years), compared with PfVAR2CSA responses (36-157 years). The longer half-life of antibodies to PfVAR2CSA suggests that antibodies acquired in one pregnancy may be maintained to protect subsequent pregnancies. CONCLUSIONS: These findings may have important practical implications for predicting the duration of vaccine-induced responses by candidate antigens and supports the development of malaria vaccines to protect pregnant women.

Quantifying the importance of MSP1-19 as a target of growth-inhibitory and protective antibodies against Plasmodium falciparum in humans.

BACKGROUND: Antibodies targeting blood stage antigens are important in protection against malaria, but the key targets and mechanisms of immunity are not well understood. Merozoite surface protein 1 (MSP1) is an abundant and essential protein. The C-terminal 19 kDa region (MSP1-19) is regarded as a promising vaccine candidate and may also be an important target of immunity. METHODOLOGY/FINDINGS: Growth inhibitory antibodies against asexual-stage parasites and IgG to recombinant MSP1-19 were measured in plasma samples from a longitudinal cohort of 206 children in Papua New Guinea. Differential inhibition by samples of mutant P. falciparum lines that expressed either the P. falciparum or P. chabaudi form of MSP1-19 were used to quantify MSP1-19 specific growth-inhibitory antibodies. The great majority of children had detectable IgG to MSP1-19, and high levels of IgG were significantly associated with a reduced risk of symptomatic P. falciparum malaria during the 6-month follow-up period. However, there was little evidence of PfMSP1-19 specific growth inhibition by plasma samples from children. Similar results were found when testing non-dialysed or dialysed plasma, or purified antibodies, or when measuring growth inhibition in flow cytometry or microscopy-based assays. Rabbit antisera generated by immunization with recombinant MSP1-19 demonstrated strong MSP1-19 specific growth-inhibitory activity, which appeared to be due to much higher antibody levels than human samples; antibody avidity was similar between rabbit antisera and human plasma. CONCLUSIONS/SIGNIFICANCE: These data suggest that MSP1-19 is not a major target of growth inhibitory antibodies and that the protective effects of antibodies to MSP1-19 are not due to growth inhibitory activity, but may instead be mediated by other mechanisms. Alternatively, antibodies to MSP1-19 may act as a marker of protective immunity.

Immunization with VAR2CSA-DBL5 recombinant protein elicits broadly cross-reactive antibodies to placental Plasmodium falciparum-infected erythrocytes.

Pregnancy-associated malaria is a severe clinical syndrome associated with the sequestration of Plasmodium falciparum-infected erythrocytes in the placenta. Placental binding is mediated by VAR2CSA, a member of the large and diverse P. falciparum erythrocyte membrane 1 (PfEMP1) protein family. To better understand if conserved regions in VAR2CSA can be targeted by antibodies, we immunized rabbits with VAR2CSA-DBL1 and -DBL5 recombinant proteins produced in Pichia pastoris and developed a panel of seven chondroitin sulfate A (CSA)-binding parasites from diverse geographic origins. Overall, no two parasites in the panel expressed the same VAR2CSA sequence. The DBL1 domains averaged 80% amino acid identity (range, 72 to 89%), and the DBL5 domains averaged 86% amino acid identity (range, 83 to 99%), similar to a broader sampling of VAR2CSA sequences from around the world. Whereas antibodies generated against the VAR2CSA-DBL1 recombinant protein had only limited breadth and reacted with three or four parasites in the panel, immunization with DBL5 recombinant proteins elicited broadly cross-reactive antibodies against all or most parasites in the panel, as well as to fresh clinical isolates from pregnant women. These findings demonstrate that the major PfEMP1 variant expressed by placental isolates exposes strain-transcendent epitopes that can be targeted by vaccination and may have application for pregnancy malaria vaccine development.

Evaluation of the antigenic diversity of placenta-binding Plasmodium falciparum variants and the antibody repertoire among pregnant women.

Pregnant women are infected by specific variants of Plasmodium falciparum that adhere and accumulate in the placenta. Using serological and molecular approaches, we assessed the global antigenic diversity of surface antigens expressed by placenta-binding isolates to better understand immunity to malaria in pregnancy and evolution of polymorphisms and to inform vaccine development. We found that placenta-binding isolates originating from all major regions where malaria occurs were commonly recognized by antibodies in different populations of pregnant women. There was substantial antigenic overlap and sharing of epitopes between isolates, including isolates from distant geographic locations, suggesting that there are limitations to antigenic diversity; however, differences between populations and isolates were also seen. Many women had cross-reactive antibodies and/or a broad repertoire of antibodies to different isolates. Studying VAR2CSA as the major antigen expressed by placenta-binding isolates, we identified antibody epitopes encoded by variable sequence blocks in the DBL3 domain. Analysis of global var2csa DBL3 sequences demonstrated that there was extensive sharing of variable blocks between Africa, Asia, Papua New Guinea, and Latin America, which likely contributes to the high level of antigenic overlap between different isolates. However, there was also evidence of geographic clustering of sequences and differences in VAR2CSA sequences between populations. The results indicate that there is limited antigenic diversity in placenta-binding isolates and may explain why immunity to malaria in pregnancy can be achieved after exposure during one pregnancy. Inclusion of a limited number of variants in a candidate vaccine may be sufficient for broad population coverage, but geographic considerations may also have to be included in vaccine design.

Serum lipoproteins promote efficient presentation of the malaria virulence protein PfEMP1 at the erythrocyte surface.

The virulence of the malaria parasite Plasmodium falciparum is related to its ability to express a family of adhesive proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) at the infected red blood cell surface. The mechanism for the transport and delivery of these adhesins to the erythrocyte membrane is only poorly understood. In this work, we have used specific immune reagents in a flow cytometric assay to monitor the effects of serum components on the surface presentation of PfEMP1. We show that efficient presentation of the A4 and VAR2CSA variants of PfEMP1 is dependent on the presence of serum in the bathing medium during parasite maturation. Lipid-loaded albumin supports parasite growth but allows much less efficient presentation of PfEMP1 at the red blood cell surface. Analysis of the serum components reveals that lipoproteins, especially those of the low-density lipoprotein fraction, promote PfEMP1 presentation. Cytoadhesion of infected erythrocytes to the host cell receptors CD36 and ICAM-1 is also decreased in infected erythrocytes cultured in the absence of serum. The defect appears to be in the transfer of PfEMP1 from parasite-derived structures known as the Maurer's clefts to the erythrocyte membrane or in surface conformation rather than a down-regulation or switching of particular PfEMP1 variants.

Inhibition of dendritic cell maturation by malaria is dose dependent and does not require Plasmodium falciparum erythrocyte membrane protein 1.

Red blood cells infected with Plasmodium falciparum (iRBCs) have been shown to modulate maturation of human monocyte-derived dendritic cells (DCs), interfering with their ability to activate T cells. Interaction between Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) and CD36 expressed by DCs is the proposed mechanism, but we show here that DC modulation does not require CD36 binding, PfEMP1, or contact between DCs and infected RBCs and depends on the iRBC dose. iRBCs expressing a PfEMP1 variant that binds chondroitin sulfate A (CSA) but not CD36 were phagocytosed, inhibited lipopolysaccharide (LPS)-induced phenotypic maturation and cytokine secretion, and abrogated the ability of DCs to stimulate allogeneic T-cell proliferation. CD36- and CSA-binding iRBCs showed comparable inhibition. P. falciparum lines rendered deficient in PfEMP1 expression by targeted gene knockout or knockdown also inhibited LPS-induced phenotypic maturation, and separation of DCs and iRBCs in transwells showed that inhibition was not contact dependent. Inhibition was observed at an iRBC:DC ratio of 100:1 but not at a ratio of 10:1. High doses of iRBCs were associated with apoptosis of DCs, which was not activation induced. Lower doses of iRBCs stimulated DC maturation sufficient to activate autologous T-cell proliferation. In conclusion, modulation of DC maturation by P. falciparum is dose dependent and does not require interaction between PfEMP1 and CD36. Inhibition and apoptosis of DCs by high-dose iRBCs may or may not be physiological. However, our observation that low-dose iRBCs initiate functional DC maturation warrants reevaluation and further investigation of DC interactions with blood-stage P. falciparum.

Antibody recognition of heterologous variant surface antigens after a single Plasmodium falciparum infection in previously naive adults.

Antibodies to variant surface antigens (VSA) including Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) have been associated with protective antimalarial immunity that appears to be variant-specific. This study of adult returned travelers with a single acute P. falciparum infection investigated whether a primary antibody response includes IgG specific for heterologous VSA. We found that 11 of 14 patients had IgG reactive with up to six P. falciparum lines expressing different heterologous PfEMP1 variants, measured by flow cytometry (reactivity greater than two times the mean of the negative control sera was defined as "positive"), with high reactivity (greater than four times the mean of the negative controls) detected in three patients. The dominant variant(s) recognized differed between seropositive individuals. Despite previous evidence that antibody responses to heterologous VSA are short-lived, seven patients had detectable IgG at > 20 weeks post-infection. Together these data suggest that a single P. falciparum infection can be sufficient to induce antibodies reactive with several PfEMP1 variants, although the repertoire of target epitopes they recognize may still be restricted.

Delivery of the malaria virulence protein PfEMP1 to the erythrocyte surface requires cholesterol-rich domains.

The particular virulence of the human malaria parasite Plasmodium falciparum derives from export of parasite-encoded proteins to the surface of the mature erythrocytes in which it resides. The mechanisms and machinery for the export of proteins to the erythrocyte membrane are largely unknown. In other eukaryotic cells, cholesterol-rich membrane microdomains or "rafts" have been shown to play an important role in the export of proteins to the cell surface. Our data suggest that depletion of cholesterol from the erythrocyte membrane with methyl-beta-cyclodextrin significantly inhibits the delivery of the major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). The trafficking defect appears to lie at the level of transfer of PfEMP1 from parasite-derived membranous structures within the infected erythrocyte cytoplasm, known as the Maurer's clefts, to the erythrocyte membrane. Thus our data suggest that delivery of this key cytoadherence-mediating protein to the host erythrocyte membrane involves insertion of PfEMP1 at cholesterol-rich microdomains. GTP-dependent vesicle budding and fusion events are also involved in many trafficking processes. To determine whether GTP-dependent events are involved in PfEMP1 trafficking, we have incorporated non-membrane-permeating GTP analogs inside resealed erythrocytes. Although these nonhydrolyzable GTP analogs reduced erythrocyte invasion efficiency and partially retarded growth of the intracellular parasite, they appeared to have little direct effect on PfEMP1 trafficking.

VAR2CSA is the principal ligand for chondroitin sulfate A in two allogeneic isolates of Plasmodium falciparum.

Malaria during pregnancy causes serious disease that is associated with sequestration in the placenta of Plasmodium falciparum infected erythrocytes that adhere to several host receptors, including chondroitin sulfate A (CSA). The principal CSA binding ligand associated with placental sequestration is the P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var2csa gene. We disrupted the var2csa gene in two allogeneic parasites and ablated CSA binding. However, in one parasite line we were able to re-select for adhesion to bovine trachea CSA associated with transcription of two var genes, var-CS2 and varP. Parasites transcribing parts of var-CS2 and varP were present in the placentae of some infected women but the mutant parasites that transcribed var-CS2 and varP were recognized by sera from men and pregnant women independent of parity. This work raises the possibility that the PfEMP1 molecules encoded by var-CS2 and varP may be minor contributors to placental malaria but also confirms the importance of the immunodominant, conserved var2csa PfEMP1s in pregnancy associated malaria and strengthens the case for var2csa as a pregnancy-specific malaria vaccine.

Antigenic differences and conservation among placental Plasmodium falciparum-infected erythrocytes and acquisition of variant-specific and cross-reactive antibodies.

Background. Pregnant women are infected by Plasmodium falciparum with novel antigenic phenotypes that adhere to chondroitin sulfate A (CSA) and other receptors in the placenta. The diverse and variant parasite protein P. falciparum erythrocyte membrane protein 1 (PfEMP1), which is encoded by var genes, is a ligand for CSA and a major target of antibodies associated with protective immunity.Methods. Serum samples from pregnant women exposed to malaria were tested for immunoglobulin G, adhesion-inhibitory antibodies, and agglutinating antibodies to different CSA-binding isolates expressing conserved var2csa-type genes and to parasite isolates from infected placentas. Parasite isolates also were examined to assess PfEMP1 expression, the effect of trypsin treatment of infected erythrocytes on parasite adhesion and cleavage of PfEMP1, and inhibition of adhesion by rabbit antiserum raised against a CSA-binding isolate.Results. Findings demonstrated that (1) there are significant antigenic differences between CSA-binding isolates that correspond with polymorphisms in var2csa; (2) there are differences in the properties of PfEMP1 and antibody reactivity between CSA-binding and placental isolates, which express multiple PfEMP1 forms; (3) acquired antibodies target diverse and cross-reactive epitopes expressed by CSA-binding infected erythrocytes, and cross-reactive antibodies are not necessarily cross-inhibitory; and (4) the breadth of antibody reactivity is greater among multigravidae than among primigravidae.Conclusions. Immunity may be mediated by a repertoire of antibodies to diverse and common epitopes. Strategies based on vaccination with a single domain or isolate might be hindered by antigenic diversity.