Natural genetic diversity of the DBL domain of a novel member of the Plasmodium vivax erythrocyte binding-like proteins (EBP2) in the Amazon rainforest.

In malaria parasites, the erythrocyte binding-like proteins (EBL) are a family of invasion proteins that are attractive vaccine targets. In the case of Plasmodium vivax, the widespread malaria parasite, blood-stage vaccines have been largely focused on a single EBL candidate, the Duffy binding-like domain (DBL) of the Duffy binding protein (DBPII), due to its well-characterized role in the reticulocyte invasion. A novel P. vivax EBL family member, the Erythrocyte binding protein (EBP2, also named EBP or DBP2), binds preferentially to reticulocytes and may mediate an alternative P. vivax invasion pathway. To gain insight into the natural genetic diversity of the DBL domain of EBP2 (region II; EBP2-II), we analyzed ebp2-II gene sequences of 71 P. vivax isolates collected in different endemic settings of the Brazilian Amazon rainforest, where P. vivax is the predominant malaria-associated species. Although most of the substitutions in the ebp2-II gene were non-synonymous and suggested positive selection, the results showed that the DBL domain of the EBP2 was much less polymorphic than that of DBPII. The predominant EBP2 haplotype in the Amazon region corresponded to the C127 reference sequence first described in Cambodia (25% C127-like haplotype). An overview of ebp2-II gene sequences available at GenBank (n = 352) from seven countries (Cambodia, Madagascar, Myanmar, PNG, South Korea, Thailand, Vietnam) confirmed the C127-like haplotype as highly prevalent worldwide. Two out of 43 haplotypes (5 to 20 inferred per country) showed a global frequency of 60%. The results presented here open new avenues of research pursuit while suggesting that a vaccine based on the DBL domain of EBP2 should target a few haplotypes for broad coverage.

Genetic diversity and natural selection of Plasmodium vivax reticulocyte invasion genes in Ecuador.

BACKGROUND: Knowledge of the diversity of invasion ligands in malaria parasites in endemic regions is essential to understand how natural selection influences genetic diversity of these ligands and their feasibility as possible targets for future vaccine development. In this study the diversity of four genes for merozoite invasion ligands was studied in Ecuadorian isolates of Plasmodium vivax. METHODS: Eighty-eight samples from P. vivax infected individuals from the Coast and Amazon region of Ecuador were obtained between 2012 and 2015. The merozoite invasion genes pvmsp-1-19, pvdbpII, pvrbp1a-2 and pvama1 were amplified, sequenced, and compared to the Sal-1 strain. Polymorphisms were mapped and genetic relationships between haplotypes were determined. RESULTS: Only one nonsynonymous polymorphism was detected in pvmsp-1-19, while 44 nonsynonymous polymorphisms were detected in pvdbpII, 56 in pvrbp1a-2 and 33 in pvama1. While haplotypes appeared to be more related within each area of study and there was less relationship between parasites of the coastal and Amazon regions of the country, diversification processes were observed in the two Amazon regions. The highest haplotypic diversity for most genes occurred in the East Amazon of the country. The high diversity observed in Ecuadorian samples is closer to Brazilian and Venezuelan isolates, but lower than reported in other endemic regions. In addition, departure from neutrality was observed in Ecuadorian pvama1. Polymorphisms for pvdbpII and pvama1 were associated to B-cell epitopes. CONCLUSIONS: pvdbpII and pvama1 genetic diversity found in Ecuadorian P. vivax was very similar to that encountered in other malaria endemic countries with varying transmission levels and segregated by geographic region. The highest diversity of P. vivax invasion genes in Ecuador was found in the Amazonian region. Although selection appeared to have small effect on pvdbpII and pvrbp1a-2, pvama1 was influenced by significant balancing selection.

Characterization of Duffy Binding Protein II-specific CD4+T cell responses in Plasmodium vivax patients.

Plasmodium vivax Duffy Binding Protein region II (PvDBPII) is a leading vaccine candidate against blood-stage vivax malaria. Anti-PvDBPII antibodies potentially block parasite invasion by inhibition of erythrocyte binding. However, knowledge of PvDBPII-specific T cell responses is limited. Here, to assess the responses of PvDBPII-specific CD4+T cells in natural P. vivax infection, three cross-sectional studies were conducted in recovered subjects. In silico analysis was used for potential T cell epitope prediction and selection. PBMCs from P. vivax subjects were stimulated with selected peptides and examined for cytokine production by ELISPOT or intracellular cytokine staining. Six dominant T cell epitopes were identified. Peptide-driven T cell responses showed effector memory CD4+T cell phenotype, secreting both IFN-γ and TNF-α cytokines. Single amino acid substitutions in three T cell epitopes altered levels of IFN-γ memory T cell responses. Seropositivity of anti-PvDBPII antibodies were detected during acute malaria (62%) and persisted up to 12 months (11%) following P. vivax infection. Further correlation analysis showed four out of eighteen subjects had positive antibody and CD4+T cell responses to PvDBPII. Altogether, PvDBPII-specific CD4+T cells were developed in natural P. vivax infections. Data on their antigenicity could facilitate development of an efficacious vivax malaria vaccine.

Cross-reactive inhibitory antibody and memory B cell responses to variant strains of Duffy binding protein II at post-Plasmodium vivax infection.

Duffy binding protein region II (DBPII) is considered a strong potential vaccine candidate of blood-stage P. vivax. However, the highly polymorphic nature of this protein often misdirects immune responses, leading them to be strain-specific. Details of cross-reactive humoral immunity to DBPII variants have therefore become an important focus for the development of broadly protective vaccines. Here, cross-reactive humoral immunity against a panel of Thai DBPII variants (DBL-THs) was demonstrated in immunized BALB/c mice and P. vivax patients, by in vitro erythrocyte-binding inhibition assay. Sera from immunized animals showed both strain-transcending (anti-DBL-TH2 and -TH4) and strain-specific (anti-DBL-TH5, -TH6 and -TH9) binding to DBL-TH variants. Using anti-DBL-TH sera at 50% inhibitory concentration (IC50) of the homologous strain, anti-DBL-TH2 sera showed cross inhibition to heterologous DBL-TH strains, whereas anti-DBL-TH5 sera exhibited only strain-specific inhibition. In P. vivax patients, 6 of 15 subjects produced and maintained cross-reactive anti-DBL-TH inhibitory antibodies through the 1-year post-infection timepoint. Cross-reactive memory B cell (MBC) responses to DBL-TH variants were analyzed in subjects recovered from P. vivax infection (RC). The plasma samples from 5 RC subjects showed broad inhibition. However, MBC-derived antibodies of these patients did not reveal cross-inhibition. Altogether, broadly anti-DBP variant inhibitory antibodies developed and persisted in P. vivax infections. However, the presence of cross-reactive anti-DBL-TH inhibitory function post-infection was not related with MBC responses to these variants. More detailed investigation of long-lasting, broadly protective antibodies to DBPII will guide the design of vivax malaria vaccines.

Impact of Epstein-Barr virus co-infection on natural acquired Plasmodium vivax antibody response.

BACKGROUND: The simultaneous infection of Plasmodium falciparum and Epstein-Barr virus (EBV) could promote the development of the aggressive endemic Burkitt's Lymphoma (eBL) in children living in P. falciparum holoendemic areas. While it is well-established that eBL is not related to other human malaria parasites, the impact of EBV infection on the generation of human malaria immunity remains largely unexplored. Considering that this highly prevalent herpesvirus establishes a lifelong persistent infection on B-cells with possible influence on malaria immunity, we hypothesized that EBV co-infection could have impact on the naturally acquired antibody responses to P. vivax, the most widespread human malaria parasite. METHODOLOGY/PRINCIPAL FINDINGS: The study design involved three cross-sectional surveys at six-month intervals (baseline, 6 and 12 months) among long-term P. vivax exposed individuals living in the Amazon rainforest. The approach focused on a group of malaria-exposed individuals whose EBV-DNA (amplification of balf-5 gene) was persistently detected in the peripheral blood (PersVDNA, n = 27), and an age-matched malaria-exposed group whose EBV-DNA could never be detected during the follow-up (NegVDNA, n = 29). During the follow-up period, the serological detection of EBV antibodies to lytic/ latent viral antigens showed that IgG antibodies to viral capsid antigen (VCA-p18) were significantly different between groups (PersVDNA > NegVDNA). A panel of blood-stage P. vivax antigens covering a wide range of immunogenicity confirmed that in general PersVDNA group showed low levels of antibodies as compared with NegVDNA. Interestingly, more significant differences were observed to a novel DBPII immunogen, named DEKnull-2, which has been associated with long-term neutralizing antibody response. Differences between groups were less pronounced with blood-stage antigens (such as MSP1-19) whose levels can fluctuate according to malaria transmission. CONCLUSIONS/SIGNIFICANCE: In a proof-of-concept study we provide evidence that a persistent detection of EBV-DNA in peripheral blood of adults in a P. vivax semi-immune population may impact the long-term immune response to major malaria vaccine candidates.

Antibody response to a new member of the DBL family (EBP2) after a brief Plasmodium vivax exposure.

Plasmodium vivax blood-stage invasion into reticulocyte is critical for parasite development. Thus, validation of novel parasite invasion ligands is essential for malaria vaccine development. Recently, we demonstrated that EBP2, a Duffy binding protein (DBP) paralog, is antigenically distinct from DBP and could not be functionally inhibited by anti-DBP antibodies. Here, we took advantage of a small outbreak of P.vivax malaria, located in a non-malarious area of Brazil, to investigate for the first time IgM/IgG antibodies against EBP2 and DEKnull-2 (an engineering DBPII vaccine) among individuals who had their first and brief exposure to P.vivax (16 cases and 22 non-cases). Our experimental approach included 4 cross sectional surveys at 3-month interval (12-month follow-up). The results demonstrated that while a brief initial P.vivax infection was not efficient to induce IgM/ IgG antibodies to either EBP2 or DEKnull-2, IgG antibodies against DEKnull-2 (but not EBP2) were boosted by recurrent blood-stage infections following treatment. Of interest, in most recurrent P. vivax infections (4 out of 6 patients) DEKnull-2 IgG antibodies were sustained for 6 to 12 months. Polymorphisms in the ebp2 gene does not seem to explain EBP2 low immunogenicity as the ebp2 allele associated with the P.vivax outbreak presented high identity to the original EBP2 isolate used as recombinant protein. Although EBP2 antibodies were barely detectable after a primary episode of P.vivax infection, EBP2 was highly recognized by serum IgG from long-term malaria-exposed Amazonians (range from 35 to 92% according to previous malaria episodes). Taken together, the results showed that individuals with a single and brief exposure to P.vivax infection develop very low anti-EBP2 antibodies, which tend to increase after long-term malaria exposure. Finally, the findings highlighted the potential of DEKnull-2 as a vaccine candidate, as in non-immune individuals anti-DEKnull-2 IgG antibodies were boosted even after a brief exposure to P.vivax blood stages.

The presence of circulating antibody secreting cells and long-lived memory B cell responses to reticulocyte binding protein 1a in Plasmodium vivax patients.

BACKGROUND: Development of an effective vaccine against blood-stage malaria requires the induction of long-term immune responses. Plasmodium vivax Reticulocyte Binding Protein 1a (PvRBP1a) is a blood-stage parasite antigen which is associated with invasion of red blood cells and induces antibody responses. Thus, PvRBP1a is considered as a target for design of a blood-stage vaccine against vivax malaria. METHODS: Both cross-sectional and cohort studies were used to explore the development and persistence of long-lived antibody and memory B cell responses to PvRBP1a in individuals who lived in an area of low malaria endemicity. Antibody titers and frequency of memory B cells specific to PvRBP1a were measured during infection and following recovery for up to 12 months. RESULTS: IgG antibody responses against PvRBP1a were prevalent during acute vivax malaria, predominantly IgG1 subclass responses. High responders to PvRBP1a had persistent antibody responses for at least 12-month post-infection. Further analysis of high responder found a direct relation between antibody titers and frequency of activated and atypical memory B cells. Furthermore, circulating antibody secreting cells and memory B cells specific to PvRBP1a were generated during infection. The PvRBP1a-specific memory B cells were maintained for up to 3-year post-infection, indicating the ability of PvRBP1a to induce long-term humoral immunity. CONCLUSION: The study revealed an ability of PvRBP1a protein to induce the generation and maintenance of antibody and memory B cell responses. Therefore, PvRBP1a could be considered as a vaccine candidate against the blood-stage of P. vivax.

Multiplexed Microsphere-Based Flow Cytometric Assay to Assess Strain Transcending Antibodies to Plasmodium vivax Duffy Binding Protein II Reveals an Efficient Tool to Identify Binding-Inhibitory Antibody Responders.

Malaria remains a major public health problem worldwide, and Plasmodium vivax is the most widely distributed malaria parasite. Naturally acquired binding inhibitory antibodies (BIAbs) to region II of the Duffy binding protein (DBPII), a P. vivax ligand that is critical for reticulocyte invasion, are associated with a reduced risk of clinical malaria. Owing to methodological issues in evaluating antibodies that inhibit the DBPII-DARC interaction, a limited number of studies have investigated DBPII BIAbs in P. vivax-exposed populations. Based on the assumption that individuals with a consistent BIAb response are characterized by strain-transcending immune responses, we hypothesized that detecting broadly reactive DBPII antibodies would indicate the presence of BIAb response. By taking advantage of an engineered DBPII immunogen targeting conserved DBPII neutralizing epitopes (DEKnull-2), we standardized a multiplex flow cytometry-based serological assay to detect broadly neutralizing IgG antibodies. For this study, a standard in vitro cytoadherence assay with COS-7 cells expressing DBPII was used to test for DBPII BIAb response in long-term P. vivax-exposed Amazonian individuals. Taken together, the results demonstrate that this DBPII-based multiplex assay facilitates identifying DBPII BIAb carriers. Of relevance, the ability of the multiplex assay to identify BIAb responders was highly accurate when the positivity for all antigens was considered. In conclusion, the standardized DBPII-based flow cytometric assay confirmed that DBPII-BIAb activity was associated with the breadth rather than the magnitude of anti-DBPII antibodies. Altogether, our results suggest that multiplex detection of broadly DBPII-reactive antibodies facilitates preliminary screening of BIAb responders.

Variable immunogenicity of a vivax malaria blood-stage vaccine candidate.

Relapsing malaria caused by Plasmodium vivax is a neglected tropical disease and an important cause of malaria worldwide. Vaccines to prevent clinical disease and mosquito transmission of vivax malaria are needed to overcome the distinct challenges of this important public health problem. In this vaccine immunogenicity study in mice, we examined key variables of responses to a P. vivax Duffy binding protein vaccine, a leading candidate to prevent the disease-causing blood-stages. Significant sex-dependent differences were observed in B cell (CD80+) and T cell (CD8+) central memory subsets, resulting in significant differences in functional immunogenicity and durability of anti-DBP protective efficacy. These significant sex-dependent differences in inbred mice were in the CD73+CD80+ memory B cell, H2KhiCD38hi/lo, and effector memory subsets. This study highlights sex and immune genes as critical variables that can impact host responses to P. vivax antigens and must be taken into consideration when designing clinical vaccine studies.

Progress towards the development of a P. vivax vaccine.

INTRODUCTION: Plasmodium vivax causes significant public health problems in endemic regions. A vaccine to prevent disease is critical, considering the rapid spread of drug-resistant parasite strains, and the development of hypnozoites in the liver with potential for relapse. A minimally effective vaccine should prevent disease and transmission while an ideal vaccine provides sterile immunity. AREAS COVERED: Despite decades of research, the complex life cycle, technical challenges and a lack of funding have hampered progress of P. vivax vaccine development. Here, we review the progress of potential P. vivax vaccine candidates from different stages of the parasite life cycle. We also highlight the challenges and important strategies for rational vaccine design. These factors can significantly increase immune effector mechanisms and improve the protective efficacy of these candidates in clinical trials to generate sustained protection over longer periods of time. EXPERT OPINION: A vaccine that presents functionally-conserved epitopes from multiple antigens from various stages of the parasite life cycle is key to induce broadly neutralizing strain-transcending protective immunity to effectively disrupt parasite development and transmission.

Dynamics of IgM and IgG responses to the next generation of engineered Duffy binding protein II immunogen: Strain-specific and strain-transcending immune responses over a nine-year period.

BACKGROUND: A low proportion of P. vivax-exposed individuals acquire protective strain-transcending neutralizing IgG antibodies that are able to block the interaction between the Duffy binding protein II (DBPII) and its erythrocyte-specific invasion receptor. In a recent study, a novel surface-engineered DBPII-based vaccine termed DEKnull-2, whose antibody response target conserved DBPII epitopes, was able to induce broadly binding-inhibitory IgG antibodies (BIAbs) that inhibit P. vivax reticulocyte invasion. Toward the development of DEKnull-2 as an effective P. vivax blood-stage vaccine, we investigate the relationship between naturally acquired DBPII-specific IgM response and the profile of IgG antibodies/BIAbs activity over time. METHODOLOGY/PRINCIPAL FINDINGS: A nine-year follow-up study was carried-out among long-term P. vivax-exposed Amazonian individuals and included six cross-sectional surveys at periods of high and low malaria transmission. DBPII immune responses associated with either strain-specific (Sal1, natural DBPII variant circulating in the study area) or conserved epitopes (DEKnull-2) were monitored by conventional serology (ELISA-detected IgM and IgG antibodies), with IgG BIAbs activity evaluated by functional assays (in vitro inhibition of DBPII-erythrocyte binding). The results showed a tendency of IgM antibodies toward Sal1-specific response; the profile of Sal1 over DEKnull-2 was not associated with acute malaria and sustained throughout the observation period. The low malaria incidence in two consecutive years allowed us to demonstrate that variant-specific IgG (but not IgM) antibodies waned over time, which resulted in IgG skewed to the DEKnull-2 response. A persistent DBPII-specific IgM response was not associated with the presence (or absence) of broadly neutralizing IgG antibody response. CONCLUSIONS/SIGNIFICANCE: The current study demonstrates that long-term exposure to low and unstable levels of P. vivax transmission led to a sustained DBPII-specific IgM response against variant-specific epitopes, while sustained IgG responses are skewed to conserved epitopes. Further studies should investigate on the role of a stable and persistent IgM antibody response in the immune response mediated by DBPII.

Identification of an Immunogenic Broadly Inhibitory Surface Epitope of the Plasmodium vivax Duffy Binding Protein Ligand Domain.

The Plasmodium vivax Duffy binding protein region II (DBPII) is a vital ligand for the parasite's invasion of reticulocytes, thereby making this molecule an attractive vaccine candidate against vivax malaria. However, strain-specific immunity due to DBPII allelic variation in Bc epitopes may complicate vaccine efficacy, suggesting that an effective DBPII vaccine needs to target conserved epitopes that are potential targets of strain-transcending neutralizing immunity. The minimal epitopes reactive with functionally inhibitory anti-DBPII monoclonal antibody (MAb) 3C9 and noninhibitory anti-DBPII MAb 3D10 were mapped using phage display expression libraries, since previous attempts to deduce the 3C9 epitope by cocrystallographic methods failed. Inhibitory MAb 3C9 binds to a conserved conformation-dependent epitope in subdomain 3, while noninhibitory MAb 3D10 binds to a linear epitope in subdomain 1 of DBPII, consistent with previous studies. Immunogenicity studies using synthetic linear peptides of the minimal epitopes determined that the 3C9 epitope, but not the 3D10 epitope, could induce functionally inhibitory anti-DBPII antibodies. Therefore, the highly conserved binding-inhibitory 3C9 epitope offers the potential as a component in a broadly inhibitory, strain-transcending DBP subunit vaccine.IMPORTANCE Vivax malaria is the second leading cause of malaria worldwide and the major cause of non-African malaria. Unfortunately, efforts to develop antimalarial vaccines specifically targeting Plasmodium vivax have been largely neglected, and few candidates have progressed into clinical trials. The Duffy binding protein is considered a leading blood-stage vaccine candidate because this ligand's recognition of the Duffy blood group reticulocyte surface receptor is considered essential for infection. This study identifies a new target epitope on the ligand's surface that may serve as the target of vaccine-induced binding-inhibitory antibody (BIAb). Understanding the potential targets of vaccine protection will be important for development of an effective vaccine.

Blood-stage Plasmodium vivax antibody dynamics in a low transmission setting: A nine year follow-up study in the Amazon region.

Plasmodium vivax remains a global health problem and its ability to cause relapses and subpatent infections challenge control and elimination strategies. Even in low malaria transmission settings, such as the Amazon basin, where progress in malaria control has caused a remarkable reduction in case incidence, a recent increase in P. vivax transmission demonstrates the continued vulnerability of P.vivax-exposed populations. As part of a search for complementary approaches to P.vivax surveillance in areas in which adults are the majority of the exposed-population, here we evaluated the potential of serological markers covering a wide range of immunogenicity to estimate malaria transmission trends. For this, antibodies against leading P. vivax blood-stage vaccine candidates were assessed during a 9 year follow-up study among adults exposed to unstable malaria transmission in the Amazon rainforest. Circulating antibody levels against immunogenic P. vivax proteins, such as the Apical Membrane Antigen-1, were a sensitive measure of recent P. vivax exposure, while antibodies against less immunogenic proteins were indicative of naturally-acquired immunity, including the novel engineered Duffy binding protein II immunogen (DEKnull-2). Our results suggest that the robustness of serology to estimate trends in P.vivax malaria transmission will depend on the immunological background of the study population, and that for adult populations exposed to unstable P.vivax malaria transmission, the local heterogeneity of antibody responses should be considered when considering use of serological surveillance.

In-depth phenotypic characterization of reticulocyte maturation using mass cytometry.

Progress towards an in-depth understanding of the final steps of the erythroid lineage development is paramount for many hematological diseases. We have characterized the final stages of reticulocyte maturation from bone marrow to peripheral blood using for the first time single-cell Mass Cytometry (CyTOF). We were able to measure the expression of 31 surface markers within a single red blood cell (RBC). We demonstrate the validity of CyTOF for RBC phenotyping by confirming the progressive reduction of transferrin receptor 1 (CD71) during reticulocyte maturation to mature RBC. We highlight the high-dimensional nature of mass cytometry data by correlating the expression of multiple proteins on individual RBCs. We further describe a more drastic reduction pattern for a component of the alpha4/beta1 integrin CD49d at the very early steps of reticulocyte maturation in bone marrow and directly linked with the mitochondria remnants clearance pattern. The enhanced and accurate RBC phenotyping potential of CyTOF described herein could be beneficial to decipher RBC preferences, as well as still not well understood receptor-ligand interaction of some hemotropic parasites such as the malaria causing agent Plasmodium vivax.

Identification and Immunological Characterization of the Ligand Domain of Plasmodium vivax Reticulocyte Binding Protein 1a.

Background: Erythrocyte invasion by malaria parasites is essential for blood-stage development. Consequently, parasite proteins critically involved in erythrocyte invasion, such as the Plasmodium vivax reticulocyte binding proteins (RBPs) that mediate preferential invasion of reticulocytes, are considered potential vaccine targets. Thus, targeting the RBPs could prevent blood-stage infection and disease. The RBPs are large, and little is known about their functional domains and whether individuals naturally exposed to P. vivax acquire binding-inhibitory antibodies to these critical binding regions. This study aims to functionally and immunologically characterize Plasmodium vivax RBP1a. Methods: Recombinant proteins of overlapping fragments of RBP1a were used to determine binding specificity to erythrocytes and immunogenicity in laboratory animals. The naturally acquired antibody response to these proteins was evaluated using serum samples from individuals in regions of endemicity. Results: The N-terminal extracellular region, RBP1157-650 (RBP1:F8), was determined to bind both reticulocytes and normocytes, with a preference for immature reticulocytes. Antibodies elicited against rRBP1:F8 blocked binding between RBP1:F8 and erythrocytes. Naturally acquired anti-RBP1 binding-inhibitory antibodies were detected in serum specimens from P. vivax-exposed individuals from Papua New Guinea and Brazil. Conclusion: Recombinant RBP1:F8 binds human erythrocytes, elicits artificially induced functional blocking antibodies, and is a target of naturally acquired binding-inhibitory antibodies.

An engineered vaccine of the Plasmodium vivax Duffy binding protein enhances induction of broadly neutralizing antibodies.

Plasmodium vivax invasion into human reticulocytes is a complex process. The Duffy binding protein (DBP) dimerization with its cognate receptor is vital for junction formation in the invasion process. Due to its functional importance, DBP is considered a prime vaccine candidate, but variation in B-cell epitopes at the dimer interface of DBP leads to induction of strain-limited immunity. We believe that the polymorphic residues tend to divert immune responses away from functionally conserved epitopes important for receptor binding or DBP dimerization. As a proof of concept, we engineered the vaccine DEKnull to ablate the dominant Bc epitope to partially overcome strain-specific immune antibody responses. Additional surface engineering on the next generation immunogen, DEKnull-2, provides an immunogenicity breakthrough to conserved protective epitopes. DEKnull-2 elicits a stronger broadly neutralizing response and reactivity with long-term persistent antibody responses of acquired natural immunity. By using novel engineered DBP immunogens, we validate that the prime targets of protective immunity are conformational epitopes at the dimer interface. These successful results indicate a potential approach that can be used generally to improve efficacy of other malaria vaccine candidates.

A Novel Erythrocyte Binding Protein of Plasmodium vivax Suggests an Alternate Invasion Pathway into Duffy-Positive Reticulocytes.

UNLABELLED: Erythrocyte invasion by malaria parasites is essential for blood-stage development and an important determinant of host range. In Plasmodium vivax, the interaction between the Duffy binding protein (DBP) and its cognate receptor, the Duffy antigen receptor for chemokines (DARC), on human erythrocytes is central to blood-stage infection. Contrary to this established pathway of invasion, there is growing evidence of P. vivax infections occurring in Duffy blood group-negative individuals, suggesting that the parasite might have gained an alternative pathway to infect this group of individuals. Supporting this concept, a second distinct erythrocyte binding protein (EBP2), representing a new member of the DBP family, was discovered in P. vivax and may be the ligand in an alternate invasion pathway. Our study characterizes this novel ligand and determines its potential role in reticulocyte invasion by P. vivax merozoites. EBP2 binds preferentially to young (CD71(high)) Duffy-positive (Fy(+)) reticulocytes and has minimal binding capacity for Duffy-negative reticulocytes. Importantly, EBP2 is antigenically distinct from DBP and cannot be functionally inhibited by anti-DBP antibodies. Consequently, our results do not support EBP2 as a ligand for invasion of Duffy-negative blood cells, but instead, EBP2 may represent a novel ligand for an alternate invasion pathway of Duffy-positive reticulocytes. IMPORTANCE: For decades, P. vivax infections in humans have been defined by a unique requirement for the interaction between the Duffy binding protein ligand of the parasite and the Duffy blood group antigen receptor (DARC). Recent reports of P. vivax infections in Duffy-negative individuals challenge this paradigm and suggest an alternate pathway of infection, potentially using the recently discovered EBP2. However, we demonstrate that EBP2 host cell specificity is more restricted than DBP binding and that EBP2 binds preferentially to Duffy-positive, young reticulocytes. This finding indicates that this DBP paralog does mediate a Duffy-independent pathway of infection.

The role of the human Duffy antigen receptor for chemokines in malaria susceptibility: current opinions and future treatment prospects.

The Duffy antigen receptor for chemokine (DARC) is a nonspecific receptor for several proinflammatory cytokines. It is homologous to the G-protein chemokine receptor superfamily, which is suggested to function as a scavenger in many inflammatory-and proinflammatory-related diseases. G-protein chemokine receptors are also known to play a critical role in infectious diseases; they are commonly used as entry vehicles by infectious agents. A typical example is the chemokine receptor CCR5 or CXCR4 used by HIV for infecting target cells. In malaria, DARC is considered an essential receptor that mediates the entry of the human and zoonotic malaria parasites Plasmodium vivax and Plasmodium knowlesi into human reticulocytes and erythrocytes, respectively. This process is mediated through interaction with the parasite ligand known as the Duffy binding protein (DBP). Most therapeutic strategies have been focused on blocking the interaction between DBP and DARC by targeting the parasite ligand, while strategies targeting the receptor, DARC, have not been intensively investigated. The rapid increase in drug resistance and the lack of new effective drugs or a vaccine for malaria constitute a major threat and a need for novel therapeutics to combat disease. This review explores strategies that can be used to target the receptor. Inhibitors of DARC, which block DBP-DARC interaction, can potentially provide an effective strategy for preventing malaria caused by P. vivax.

Structural analysis of the synthetic Duffy Binding Protein (DBP) antigen DEKnull relevant for Plasmodium vivax malaria vaccine design.

The Plasmodium vivax vaccine candidate Duffy Binding Protein (DBP) is a protein necessary for P. vivax invasion of reticulocytes. The polymorphic nature of DBP induces strain-specific immune responses that pose unique challenges for vaccine development. DEKnull is a synthetic DBP based antigen that has been engineered through mutation to enhance induction of blocking inhibitory antibodies. We determined the x-ray crystal structure of DEKnull to identify if any conformational changes had occurred upon mutation. Computational and experimental analyses assessed immunogenicity differences between DBP and DEKnull epitopes. Functional binding assays with monoclonal antibodies were used to interrogate the available epitopes in DEKnull. We demonstrate that DEKnull is structurally similar to the parental Sal1 DBP. The DEKnull mutations do not cause peptide backbone shifts within the polymorphic loop, or at either the DBP dimerization interface or DARC receptor binding pockets, two important structurally conserved protective epitope motifs. All B-cell epitopes, except for the mutated DEK motif, are conserved between DEKnull and DBP. The DEKnull protein retains binding to conformationally dependent inhibitory antibodies. DEKnull is an iterative improvement of DBP as a vaccine candidate. DEKnull has reduced immunogenicity to polymorphic regions responsible for strain-specific immunity while retaining conserved protein folds necessary for induction of strain-transcending blocking inhibitory antibodies.

The association of Duffy binding protein region II polymorphisms and its antigenicity in Plasmodium vivax isolates from Thailand.

Plasmodium vivax Duffy binding protein II (DBPII) plays an important role in reticulocyte invasion and is a potential vaccine candidate against vivax malaria. However, polymorphisms in DBPII are a challenge for the successful design of a broadly protective vaccine. In this study, the genetic diversity of DBPII among Thai isolates was analyzed from Plasmodium vivax-infected blood samples and polymorphism characters were defined with the MEGA4 program. Sequence analysis identified 12 variant residues that are common among Thai DBPII haplotypes with variant residues L333F, L424I, W437R and I503K having the highest frequency. Variant residue D384K occurs in combination with either E385K or K386N/Q. Additionally, variant residue L424I occurs in conjunction with W437R in most Thai DBPII alleles and these variants frequently occur in combination with the I503K variant. The polymorphic patterns of Thai isolates were defined into 9 haplotypes (Thai DBL-1, -2, -3, etc.…). Thai DBL-2, -5, -6 haplotypes are the most common DBPII variants in Thai residents. To study the association of these Thai DBPII polymorphisms with antigenic character, the functional inhibition of anti-DBPII monoclonal antibodies against a panel of Thai DBL variants was characterized by an in vitro erythrocyte binding inhibition assay. The functional inhibition of anti-DBPII monoclonal antibodies 3C9, 2D10 and 2C6 against Thai variants was significantly different, suggesting that polymorphisms of Thai DBPII variants alter the antigenic character of the target epitopes. In contrast, anti-DBPII monoclonal antibody 2H2 inhibited all Thai DBPII variants equally well. Our results suggest that the immune efficacy of a DBPII vaccine will depend on the specificity of the anti-DBPII antibodies induced and that it is preferable to optimize responses to conserved epitopes for broadly neutralizing protection against P. vivax.