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.
Many pathogens evolve extensive genetic variation in virulence proteins as a strategy to evade host immunity. This poses a significant challenge for the host to develop broadly neutralizing ...antibodies. In
, we show that a mechanism to circumvent this challenge is to elicit antibodies to cryptic epitopes that are not under immune pressure. We previously discovered that antibodies to the
invasion protein, PvDBP, cross-react with
VAR2CSA, a distantly related virulence factor that mediates placental malaria. Here, we describe the molecular mechanism underlying this cross-species immunity. We identified an epitope in subdomain 1 (SD1) within the Duffy binding-like (DBL) domain of PvDBP that gives rise to cross-reactive antibodies to VAR2CSA and show that human antibodies affinity purified against a synthetic SD1 peptide block parasite adhesion to chondroitin sulfate A (CSA)
The epitope in SD1 is subdominant and highly conserved in PvDBP, and in turn, SD1 antibodies target cryptic epitopes in
VAR2CSA. The epitopes in VAR2CSA recognized by vivax-derived SD1 antibodies (of human and mouse origin) are distinct from those recognized by VAR2CSA immune serum. We mapped two peptides in the DBL5ε domain of VAR2CSA that are recognized by SD1 antibodies. Both peptides map to regions outside the immunodominant sites, and antibodies to these peptides are not elicited following immunization with VAR2CSA or natural infection with
in pregnancy, consistent with the cryptic nature of these target epitopes.
In this work, we describe a molecular mechanism of heterologous immunity between two distant species of
Our results suggest a mechanism that subverts the classic parasite strategy of presenting highly polymorphic epitopes in surface antigens to evade immunity to that parasite. This alternative immune pathway can be exploited to protect pregnant women from falciparum placental malaria by designing vaccines to cryptic epitopes that elicit broadly inhibitory antibodies against variant parasite strains.
Objective To investigate risk factors associated with the acquisition of antibodies against Plasmodium vivax Duffy binding protein (PvDBP) – a leading malaria vaccine candidate – in a ...well‐consolidated agricultural settlement of the Brazilian Amazon Region and to determine the sequence diversity of the PvDBP ligand domain (DBPII) within the local malaria parasite population.
Methods Demographic, epidemiological and clinical data were collected from 541 volunteers using a structured questionnaire. Malaria parasites were detected by conventional microscopy and PCR, and blood collection was used for antibody assays and molecular characterisation of DBPII.
Results The frequency of malaria infection was 7% (6% for P. vivax and 1% for P. falciparum), with malaria cases clustered near mosquito breeding sites. Nearly 50% of settlers had anti‐PvDBP IgG antibodies, as detected by enzyme‐linked immunosorbent assay (ELISA) with subject’s age being the only strong predictor of seropositivity to PvDBP. Unexpectedly, low levels of DBPII diversity were found within the local malaria parasites, suggesting the existence of low gene flow between P. vivax populations, probably due to the relative isolation of the studied settlement.
Conclusion The recognition of PvDBP by a significant proportion of the community, associated with low levels of DBPII diversity among local P. vivax, reinforces the variety of malaria transmission patterns in communities from frontier settlements. Such studies should provide baseline information for antimalarial vaccines now in development.
Objectif: Investiguer les facteurs de risque associés à l’acquisition d’anticorps contre la protéine de liaison Duffy de Plasmodium vivax (PvDBP) – un candidat vaccin de première ligne contre le paludisme – dans une zone d’habitation agricole bien consolidée de la région amazonienne du Brésil, et déterminer la diversité des séquences du domaine ligand PvDBP (DBPII) au sein de la population locale du parasite du paludisme.
Méthodes: Les données démographiques, épidémiologiques et cliniques ont été recueillies à partir de 541 volontaires à l’aide d’un questionnaire structuré. Les parasites du paludisme ont été détectés par la microscopie conventionnelle et la PCR, et la collecte de sang a été utilisée pour les essais d’anticorps et la caractérisation moléculaire du DBPII.
Résultats: La fréquence de l’infection palustre était de 7% (6% pour P. vivax et 1% pour P. falciparum), avec des cas de paludisme regroupés à proximité des sites de reproduction des moustiques. Près de 50% des résidents avaient des anticorps IgG anti‐PvDBP, tels que détectés par le test ELISA, avec l’âge du sujet étant le seul facteur prédictif fort de la séropositivitéà PvDBP. De façon inattendue, de faibles niveaux de diversité DBPII ont été trouvés parmi les parasites locaux du paludisme, ce qui suggère l’existence de peu de flux de gènes entre les populations de P. vivax, probablement en raison de l’isolement relatif de la zone étudiée.
Conclusion: La reconnaissance de PvDBP par une proportion significative de la communauté, associée à de faibles niveaux de diversité DBPII parmi les moustiques P. vivax locaux, renforce la variété des modes de transmission du paludisme dans les communautés établies dans des zones frontalières. Ces études devraient fournir des informations de base pour les vaccins antipaludiques en cours de développement.
Objetivo: Investigar los factores de riesgo asociados con la adquisición de anticuerpos frente a la proteína de unión al Duffy de Plasmodium vivax (PvDBP) – uno de los principales candidatos a vacuna de malaria – en un asentamiento agrícola bien consolidado de la región Amazónica del Brasil; y determinar la diversidad en la secuencia del dominio del ligando de PvDBP (DBPII) dentro de la población local de parásitos de malaria.
Métodos: Se recolectaron datos demográficos, epidemiológicos y clínicos de 541 voluntarios utilizando un cuestionario estructurado. Los parásitos de malaria se detectaron mediante microscopía convencional y PCR, y se utilizaron muestras de sangre para realizar pruebas de anticuerpos y la caracterización molecular de DBPII.
Resultados: La frecuencia de infección de malaria era del 7% (6% para P. vivax y 1% para P. falciparum), con los casos de malaria agrupados cerca de lugares de cría de mosquitos. Casi un 50% de los pobladores tenían anticuerpos IgG anti‐PvDBP, detectados mediante un ensayo por inmunoabsorción ligado a enzimas (ELISA), siendo la edad del sujeto el único vaticinador importante de seropositividad frente a PvDBP. Sorprendentemente, se halló un bajo nivel de diversidad de DBPII dentro de la población local de parásitos de malaria, sugiriendo la existencia de un flujo genético bajo entre las poblaciones de P. vivax, probablemente debido al aislamiento relativo del emplazamiento estudiado.
Conclusión: El reconocimiento de PvDBP en una proporción significativa de la comunidad, asociada a unos bajos niveles de diversidad del DBPII entre la población local de P. vivax, refuerza la variedad de patrones de trasmisión de malaria en comunidades viviendo en emplazamientos fronterizos. Algunos estudios deberían aportar la información de base para vacunas antimaláricas ahora en desarrollo.
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. Author summary Vaccines might be a crucial component of the current efforts to malaria control and elimination, and much of the vaccine-related research on P. vivax has been focused on region II of the Duffy binding protein (DBPII), a ligand for human blood-stage infection. Recently, the newly described Erythrocyte binding protein 2 (EBP2), a P.vivax DBP paralog that it is antigenically distinct from DBP, was identified as potential vaccine targets. To date, scarce data are available about the naturally acquired immunity to EBP2. In a small outbreak of P.vivax malaria, located in a non-malarious area, we investigated whether a first P.vivax exposure induces antibodies against EBP2 that could be boosted by P.vivax recurrent infections. In parallel, we included an engineered DBPII vaccine (named DEKnull-2) whose antibody response were previously associated with broadly neutralizing P.vivax antibodies. This study shows EBP2, compared with DEKnull-2, was poorly immunogenic among individuals who experienced their first blood-stage P. vivax malaria infection. However, EBP2 was highly immunogenic in long-term malaria exposed individuals, reinforcing its potential as a P. vivax blood-stage vaccine candidate. Finally, our results reinforce that multiple blood-stage antigens should be targeted for the development of efficient vaccines against P. vivax.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Understanding the pathogenesis of Plasmodium vivax malaria is challenging. We hypothesized that susceptibility to P. vivax-induced thrombocytopenia could be associated with polymorphisms on relevant ...platelet membrane integrins: integrin alpha 2 (C807T), and integrin beta 3 (T1565C). Although beta 3 polymorphism was not related with P. vivax malaria, alpha 2 807T carriers, which show high levels of integrin alpha 2 beta 1, had a higher probability for severe thrombocytopenia than wild-type carriers. This evidence of the association of integrin polymorphism and P. vivax morbidity was further demonstrated by a moderate but significant correlation between clinical disease and surface levels of the integrin alpha 2 beta 1.
Understanding the pathogenesis of Plasmodium vivax malaria is challenging. We hypothesized that susceptibility to P. vivax-induced thrombocytopenia could be associated with polymorphisms on relevant ...platelet membrane integrins: integrin α2 (C807T), and integrin β3 (T1565C). Although β3 polymorphism was not related with P. vivax malaria, α2 807T carriers, which show high levels of integrin α2β1, had a higher probability for severe thrombocytopenia than wild-type carriers. This evidence of the association of integrin polymorphism and P. vivax morbidity was further demonstrated by a moderate but significant correlation between clinical disease and surface levels of the integrin α2β1.
Abstract The outer membrane proteins of Anaplasma marginale have been the focus of research to obtain an improved vaccine against bovine anaplasmosis. We evaluated the capacity of the recombinant ...plasmids pcDNA- msp1α, pcDNA -msp1β, and pcDNA-mp5 to express MSP1a, MSP1b, and MSP5 proteins, and to determine the immunogenicity of BALB/c mice immunized with these plasmids individually or in association. Expression of proteins was confirmed in Vero cells by IFA. The combination of recombinant plasmids showed high antibodies response, produced better induction of Th1 response than individual plasmids, and induced significant proliferation of splenocytes. The mice sera immunized with A. marginale showed seroconversion and reacted with all native MSPs, but demonstrated predominance of the humoral IgG1 isotype and did not induce significant proliferation of splenocytes. The use of association of recombinant plasmid can be an effective strategy for the immunoprophylaxis of anaplasmosis.
Anaplasmosis, caused by
Anaplasma marginale, results in significant economic losses of cattle in tropical and subtropical regions worldwide. Six major surface proteins (MSPs) were well characterized ...and designated as MSP1, MSP2, MSP3, MSP4, and MSP5. The objective of this study was to evaluate the humoral immune response of BALB/c mice against the recombinant MSPs, incorporated into immunostimulating complex (ISCOM). The recombinant proteins purified by Ni-NTA columns were incorporated into ISCOM and ISCOMATRIX by the lipid film hydration method. BALB/c mice immunized with ISCOM/rMSPs and ISCOMATRIX/rMSPs vaccines produced whole IgG, IgG1, and IgG2a, in contrast to the negative groups (PBS and ISCOMATRIX adjuvant). All groups that received antigen responded specifically against the rMSPs by Western blotting, showing the rMSP1a (60–105
kDa), rMSP1b (100
kDa), rMSP4 (47
kDa), and rMSP5 (29
kDa). Additional studies will have to be performed in cattle to evaluate the humoral and cellular mechanisms of this subunit vaccine and their possible use as protective vaccines against homologous and heterologous strains of
A. marginale.
The
Anaplasma marginale is a bacterium that has obligate intraerythrocytic multiplication in cattle causing important economic loss. The
A. marginale major surface protein 1 (MSP1) complex, ...heterodimer composed of MSP1a and MSP1b, has been identified as adhesins for bovine erythrocytes. The objectives of this study were to sequences the
msp1β gene and produce and characterize recombinant MSP1a and MSP1b from a Brazilian strain of
A. marginale, PR1. The
msp1α and
msp1β genes from the PR1 strain were cloned and expressed in
E. coli BL21 Star using the vectors pET102 and pET101/D-TOPO. Antibodies were produced against the recombinant proteins and were shown to react with rMSP1a and rMSP1b demonstrating a molecular mass of 70
kDa to 105
kDa and 100
kDa, respectively for these proteins. Bovine erythrocytes were agglutinated by BL21/rMSP1a and BL21/rMSP1b and, this agglutination was inhibited by the presence of the IgY anti-rMSP1a, confirming the adhesion function of these proteins. Additionally, using the IgY anti-rMSP1a and rMSP1b in a IFI, the presence of rMSP1a and rMSP1b was confirmed on the outer membrane of the recombinant
E. coli BL21. Our results show that the
msp1β gene from the PR1 strain has both the conserved region and contain the defined polymorphism regions previously described for other strains of
A. marginale. The results from this study confirm adhesive functions for rMSP1a and rMSP1b from PR1 strain in bovine erythrocytes invasion.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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