IgG Subclass Switch in Volunteers Repeatedly Immunized with the Full-Length Plasmodium falciparum Merozoite Surface Protein 1 (MSP1).

Vaccines are highly effective tools against infectious diseases and are also considered necessary in the fight against malaria. Vaccine-induced immunity is frequently mediated by antibodies. We have recently conducted a first-in-human clinical trial featuring SumayaVac-1, a malaria vaccine based on the recombinant, full-length merozoite surface protein 1 (MSP1FL) formulated with GLA-SE as an adjuvant. Vaccination with MSP1FL was safe and elicited sustainable IgG antibody titers that exceeded those observed in semi-immune populations from Africa. Moreover, IgG antibodies stimulated various Fc-mediated effector mechanisms associated with protection against malaria. However, these functionalities gradually waned. Here, we show that the initial two doses of SumayaVac-1 primarily induced the cytophilic subclasses IgG1 and IgG3. Unexpectedly, a shift in the IgG subclass composition occurred following the third and fourth vaccinations. Specifically, there was a progressive transition to IgG4 antibodies, which displayed a reduced capacity to engage in Fc-mediated effector functions and also exhibited increased avidity. In summary, our analysis of antibody responses to MSP1FL vaccination unveils a temporal shift towards noninflammatory IgG4 antibodies. These findings underscore the importance of considering the impact of IgG subclass composition on vaccine-induced immunity, particularly concerning Fc-mediated effector functions. This knowledge is pivotal in guiding the design of optimal vaccination strategies against malaria, informing decision making for future endeavors in this critical field.

Plasmodium falciparum merozoite surface protein 1 as asexual blood stage malaria vaccine candidate.

INTRODUCTION: Malaria represents a public health challenge in tropical and subtropical regions, and currently deployed control strategies are likely insufficient to drive elimination of malaria. Development and improvement of malaria vaccines might be key to reduce disease burden. Vaccines targeting asexual blood stages of the parasite have shown limited efficacy when studied in human trials conducted over the past decades. AREAS COVERED: Vaccine candidates based on the merozoite surface protein 1 (MSP1) were initially envisioned as one of the most promising approaches to provide immune protection against asexual blood-stage malaria. Successful immunization studies in monkey involved the use of the full-length MSP1 (MSP1FL) as vaccine construct. Vaccines using MSP1FL for immunization have the potential benefit of including numerous conserved B-cell and T-cell epitopes. This could result in improved parasite strain-transcending, protective immunity in the field. We review outcomes of clinical trials that utilized a variety of MSP1 constructs and formulations, including MSP1FL, either alone or in combination with other antigens, in both animal models and humans. EXPERT OPINION: Novel approaches to analyze breadth and magnitude of effector functions of MSP1-targeting antibodies in volunteers undergoing experimental vaccination and controlled human malaria infection will help to define correlates of protective immunity.

Multifunctional IgG/IgM antibodies and cellular cytotoxicity are elicited by the full-length MSP1 SumayaVac-1 malaria vaccine.

Radical control of malaria likely requires a vaccine that targets both the asymptomatic liver stages and the disease-causing blood stages of the human malaria parasite Plasmodium falciparum. While substantial progress has been made towards liver stage vaccines, the development of a blood stage vaccine is lagging behind. We have recently conducted a first-in-human clinical trial to evaluate the safety and immunogenicity of the recombinant, full-length merozoite surface protein 1 (MSP1FL) formulated with GLA-SE as adjuvant. Here, we show that the vaccine, termed SumayaVac-1, elicited both a humoral and cellular immune response as well as a recall T cell memory. The induced IgG and IgM antibodies were able to stimulate various Fc-mediated effector mechanisms associated with protection against malaria, including phagocytosis, release of reactive oxygen species, production of IFN-γ as well as complement activation and fixation. The multifunctional activity of the humoral immune response remained for at least 6 months after vaccination and was comparable to that of naturally acquired anti-MSP1 antibodies from semi-immune adults from Kenya. We further present evidence of SumayaVac-1 eliciting a recallable cellular cytotoxicity by IFN-γ producing CD8+ T cells. Our study revitalizes MSP1FL as a relevant blood stage vaccine candidate and warrants further evaluation of SumayaVac-1 in a phase II efficacy trial.

Plasmodium falciparum transcription in different clinical presentations of malaria associates with circulation time of infected erythrocytes.

Following Plasmodium falciparum infection, individuals can remain asymptomatic, present with mild fever in uncomplicated malaria cases, or show one or more severe malaria symptoms. Several studies have investigated associations between parasite transcription and clinical severity, but no broad conclusions have yet been drawn. Here, we apply a series of bioinformatic approaches based on P. falciparum's tightly regulated transcriptional pattern during its ~48-hour intraerythrocytic developmental cycle (IDC) to publicly available transcriptomes of parasites obtained from malaria cases of differing clinical severity across multiple studies. Our analysis shows that within each IDC, the circulation time of infected erythrocytes without sequestering to endothelial cells decreases with increasing parasitaemia or disease severity. Accordingly, we find that the size of circulating infected erythrocytes is inversely related to parasite density and disease severity. We propose that enhanced adhesiveness of infected erythrocytes leads to a rapid increase in parasite burden, promoting higher parasitaemia and increased disease severity.

Home Sweet Home: Plasmodium vivax-Infected Reticulocytes-The Younger the Better?

After a century of constant failure to produce an in vitro culture of the most widespread human malaria parasite Plasmodium vivax, recent advances have highlighted the difficulties to provide this parasite with a healthy host cell to invade, develop, and multiply under in vitro conditions. The actual level of understanding of the heterogeneous populations of cells-framed under the name 'reticulocytes'-and, importantly, their adequate in vitro progression from very immature reticulocytes to normocytes (mature erythrocytes) is far from complete. The volatility of its individual stability may suggest the reticulocyte as a delusory cell, particularly to be used for stable culture purposes. Yet, the recent relevance gained by a specific subset of highly immature reticulocytes has brought some hope. Very immature reticulocytes are characterized by a peculiar membrane harboring a plethora of molecules potentially involved in P. vivax invasion and by an intracellular complexity dynamically changing upon its quick maturation into normocytes. We analyze the potentialities offered by this youngest reticulocyte subsets as an ideal in vitro host cell for P. vivax.

Increased circulation time of Plasmodium falciparum underlies persistent asymptomatic infection in the dry season.

The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquito vectors to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the human host or compromising host survival, is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5- to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of individuals with febrile malaria in the transmission season, coinciding with longer circulation within each replicative cycle of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication but rather to increased splenic clearance of longer-circulating infected erythrocytes, which likely maintain parasitemias below clinical and immunological radar. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence.

From marginal to essential: the golden thread between nutrient sensing, medium composition and Plasmodium vivax maturation in in vitro culture.

Historically neglected, due to its biological peculiarities, the absence of a continuous long-term in vitro blood stage culture system and a propensity towards high morbidity rather than mortality, Plasmodium vivax was put back on the agenda during the last decade by the paradigm shift in the fight against malaria from malaria control to malaria eradication. While the incidence of the deadliest form of malaria, Plasmodium falciparum malaria, has declined since this paradigm shift took hold, the prospects of eradication are now threatened by the increase in the incidence of other human malaria parasite species. Plasmodium vivax is geographically the most widely distributed human malaria parasite, characterized by millions of clinical cases every year and responsible for a massive economic burden. The urgent need to tackle the unique biological challenges posed by this parasite led to renewed efforts aimed at establishing a continuous, long-term in vitro P. vivax blood stage culture. Based on recent discoveries on the role of nutrient sensing in Plasmodium's pathophysiology, this review article critically assesses the extensive body of literature concerning Plasmodium culture conditions with a specific focus on culture media used in attempts to culture different Plasmodium spp. Hereby, the effect of specific media components on the parasite's in vitro fitness and the maturation of the parasite's host cell, the reticulocyte, is analysed. Challenging the wide-held belief that it is sufficient to find the right parasite isolate and give it the right type of cells to invade for P. vivax to grow in vitro, this review contends that a healthy side-by-side maturation of both the parasite and its host cell, the reticulocyte, is necessary in the adaptation of P. vivax to in vitro growth and argues that culture conditions and the media in particular play an essential role in this maturation process.

Plasmodium vivax readiness to transmit: implication for malaria eradication.

BACKGROUND: The lack of a continuous long-term in vitro culture system for Plasmodium vivax severely limits our knowledge of pathophysiology of the most widespread malaria parasite. To gain direct understanding of P. vivax human infections, we used Next Generation Sequencing data mining to unravel parasite in vivo expression profiles for P. vivax, and P. falciparum as comparison. RESULTS: We performed cloud and local computing to extract parasite transcriptomes from publicly available raw data of human blood samples. We developed a Poisson Modelling (PM) method to confidently identify parasite derived transcripts in mixed RNAseq signals of infected host tissues. We successfully retrieved and reconstructed parasite transcriptomes from infected patient blood as early as the first blood stage cycle; and the same methodology did not recover any significant signal from controls. Surprisingly, these first generation blood parasites already show strong signature of transmission, which indicates the commitment from asexual-to-sexual stages. Further, we place the results within the context of P. vivax's complex life cycle, by developing mathematical models for P. vivax and P. falciparum and using sensitivity analysis assess the relative epidemiological impact of possible early stage transmission. CONCLUSION: The study uncovers the earliest onset of P. vivax blood pathogenesis and highlights the challenges of P. vivax eradication programs.

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.

Author Correction: A comprehensive model for assessment of liver stage therapies targeting Plasmodium vivax and Plasmodium falciparum.

The original version of this Article contained an error in the spelling of Richard Thomson-Luque, which was incorrectly given as Richard Thomson Luque. This error has now been corrected in both the PDF and HTML versions of the Article.

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.

A comprehensive model for assessment of liver stage therapies targeting Plasmodium vivax and Plasmodium falciparum.

Malaria liver stages represent an ideal therapeutic target with a bottleneck in parasite load and reduced clinical symptoms; however, current in vitro pre-erythrocytic (PE) models for Plasmodium vivax and P. falciparum lack the efficiency necessary for rapid identification and effective evaluation of new vaccines and drugs, especially targeting late liver-stage development and hypnozoites. Herein we report the development of a 384-well plate culture system using commercially available materials, including cryopreserved primary human hepatocytes. Hepatocyte physiology is maintained for at least 30 days and supports development of P. vivax hypnozoites and complete maturation of P. vivax and P. falciparum schizonts. Our multimodal analysis in antimalarial therapeutic research identifies important PE inhibition mechanisms: immune antibodies against sporozoite surface proteins functionally inhibit liver stage development and ion homeostasis is essential for schizont and hypnozoite viability. This model can be implemented in laboratories in disease-endemic areas to accelerate vaccine and drug discovery research.

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 Continuous, Long-Term Plasmodium vivax In Vitro Blood-Stage Culture: What Are We Missing?

The recent research efforts to establish a Plasmodium vivax continuous, long-term blood-stage culture have focused on the ideal host cell type. However, this is only part of the story, as the P. vivax intraerythrocytic life cycle is complex. A successful, long-term, robust culture system will depend on a multifaceted approach combining the ideal cell type and parasite isolates, and the culture conditions.

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.

Insights into an Optimization of Plasmodium vivax Sal-1 In Vitro Culture: The Aotus Primate Model.

Malaria is one of the most significant tropical diseases, and of the Plasmodium species that cause human malaria, P. vivax is the most geographically widespread. However, P. vivax remains a relatively neglected human parasite since research is typically limited to laboratories with direct access to parasite isolates from endemic field settings or from non-human primate models. This restricted research capacity is in large part due to the lack of a continuous P. vivax in vitro culture system, which has hampered the ability for experimental research needed to gain biological knowledge and develop new therapies. Consequently, efforts to establish a long-term P. vivax culture system are confounded by our poor knowledge of the preferred host cell and essential nutrients needed for in vitro propagation. Reliance on very heterogeneous P. vivax field isolates makes it difficult to benchmark parasite characteristics and further complicates development of a robust and reliable culture method. In an effort to eliminate parasite variability as a complication, we used a well-defined Aotus-adapted P. vivax Sal-1 strain to empirically evaluate different short-term in vitro culture conditions and compare them with previous reported attempts at P. vivax in vitro culture Most importantly, we suggest that reticulocyte enrichment methods affect invasion efficiency and we identify stabilized forms of nutrients that appear beneficial for parasite growth, indicating that P. vivax may be extremely sensitive to waste products. Leuko-depletion methods did not significantly affect parasite development. Formatting changes such as shaking and static cultures did not seem to have a major impact while; in contrast, the starting haematocrit affected both parasite invasion and growth. These results support the continued use of Aotus-adapted Sal-1 for development of P. vivax laboratory methods; however, further experiments are needed to optimize culture conditions to support long-term parasite development.

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.

Urban and architectural risk factors for malaria in indigenous Amazonian settlements in Brazil: a typological analysis.

In the Amazon, m alaria is highly endemic in indigenous populations, which are often considered one of the last barriers to malaria elimination due to geographic isolation. Although the improvement of housing conditions is a good strategy towards the control and prevention of vector-borne diseases, such as malaria, this preventive practice has been barely undertaken in Latin America. An analysis of the architectural and urban features of indigenous Amazonian populations is essential to define and adapt these vector control measures. A total of 32 villages of 29 different ethnicities were studied and mapped by reviewing literature and visual information, and using a geographic information system. The most important architectural and urban characteristics influencing malaria were analysed according to the following categories: number of households and dimensions, supporting area, openings, materials, lifespan and location. Housing typologies found were classified within each of these variables. The results of this typological analysis included an easy-to-handle working template and revealing of features that benefit or hamper the presence of malaria vectors in Amerindians communities. Among risk factors, presence of open eaves, permeable walls, open-side constructions, large number of sleepers indoors, temporary-ephemeral houses, linear villages along stream banks, houseboats villages, poor urban drainage and villages surrounded by anthropogenic environments were highlighted. Indigenous settlements very permissive for anophelines were identified in ethnic groups, such as the Yanomami, Palikur, Paumari, Waimiri-Atroari and Wajãpi. Positive features were also recognized, including opaque and closed houses, large radial villages on bare soil, highly elevated stilted houses and the fire indoors, found among the Yawalapiti, Ashaninka, and Gavião-Parkatejê tribes. However, as Amazonian indigenous settlement typologies vary greatly even among villages of the same ethnic group, it is imperative to undertake an individual study for each community. Using the working template in Amazonian settlements it is possible to obtain data that will help researchers not only understand how architectural and urban features affect transmission, but also define vector control measures easily applicable by health authorities and acceptable by these communities.