Lampreys use variable lymphocyte receptors (VLR) comprising leucine-rich-repeat (LRR) segments for antigen recognition, distinct from immunoglobulin-based receptors of jawed vertebrates. Lamprey VLRs are as diverse and antigen-specific as immunoglobulin-based antibodies, with unique advantages including high avidity, pH stability, and recognition of novel antigen epitopes. Here we describe the generation of VLR monoclonal antibody against histidine rich protein-2 (HRP-2) of Plasmodium falciparum, a causative agent of malaria. HRP-2, expressed by all parasite stages and secreted into plasma, serves as an effective biomarker of infection. Lamprey larvae immunized with purified HRP-2 protein produced specific VLRB antibodies with relatively high serum titers. Using white blood cells from immunized lampreys, we constructed VLR cDNA libraries expressed on yeast surface. Through yeast display screening, we selected recombinant VLRB antibody 5A10 with high affinity and specificity for HRP-2, recognizing both recombinant and native proteins from P. falciparum culture supernatants and infected patient samples. The antibody retains its binding capacity at temperatures up to 70 °C, significantly outperforming a commercial mouse IgG-based anti-HRP-2 antibody. This HRP-2-specific VLR antibody shows promise for improved malaria diagnostics, particularly in tropical regions requiring heat-stable tests.

BackgroundIn low-transmission settings, accurate estimates of malaria transmission are needed to inform elimination targets. Detection of antimalarial antibodies provides exposure history, but previous studies have mainly relied on species-specific antigens. The use of chimeric antigens that include epitopes from multiple species of malaria parasites in population-based serological surveys could provide data for exposure to multiple Plasmodium species circulating in an area. Here, the utility of P. vivax/P. falciparum chimeric antigen for assessing serological responses was evaluated in Ethiopia, an endemic country for all four human malarias, and Costa Rica, where P. falciparum has been eliminated with reports of sporadic P. vivax cases.MethodsA multiplex bead-based assay was used to determine the seroprevalence of IgG antibodies against a chimeric malaria antigen (PvRMC-MSP1) from blood samples collected from household surveys in Ethiopia in 2015 (n = 7,077) and Costa Rica in 2015 (n = 851). Targets specific for P. falciparum (PfMSP1) and P. vivax (PvMSP1) were also included in the serological panel. Seroprevalence in the population and seroconversion rates were compared among the three IgG targets.ResultsSeroprevalence in Costa Rica was 3.6% for PfMSP1, 41.5% for PvMSP1 and 46.7% for PvRMC-MSP1. In Ethiopia, seroprevalence was 27.6% for PfMSP1, 21.4% for PvMSP1, and 32.6% for PvRMC-MSP1. IgG levels in seropositive individuals were consistently higher for PvRMC-MSP1 when compared to PvMSP1 in both studies. Seroconversion rates were 0.023 for PvMSP1 and 0.03 for PvRMC-MSP1 in Costa Rica. In Ethiopia, seroconversion rates were 0.050 for PfMSP1, 0.044 for PvMSP1 and 0.106 for PvRMC-MSP1.ConclusionsOur data indicate that chimeric antigen PvRMC-MSP1 is able to capture antibodies to multiple epitopes from both prior P. falciparum and P. vivax infections, and suitable chimeric antigens can be considered for use in serosurveys with appropriate validation.

Background Despite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs used in the field. Methods This report describes the purification of native HRP2 (nHRP2) from the HB3 Plasmodium falciparum culture strain. As this culture strain lacks pfhrp3 from its genome , it is an excellent source of HRP2 protein only and does not produce the closely-related HRP3. The nHRP2 protein was isolated from culture supernatant, infected red blood cells (iRBCs), and whole parasite lysate using nickel-metal chelate chromatography. Biochemical characterization of nHRP2 from HB3 culture was conducted by SDS-PAGE and western blotting, and nHRP2 was assayed by RDT, ELISA, and bead-based immunoassay. Results Purified nHRP2 was identified by SDS-PAGE and western blot as a − 60 kDa protein that bound anti-HRP-2 monoclonal antibodies. Mouse anti-HRP2 monoclonal antibody was found to produce high optical density readings between dilutions of 1:100 and 1:3,200 by ELISA with assay signal observed up to a 1:200,000 dilution. nHRP2 yield from HB3 culture by bead-based immunoassay revealed that both culture supernatant and iRBC lysate were practical sources of large quantities of this antigen, producing a total yield of 292.4 µg of nHRP2 from two pooled culture preparations. Assessment of nHRP2 recognition by RDTs revealed that Carestart Pf HRP2 and HRP2/pLDH RDTs detected purified nHRP2 when applied at concentrations between 20.6 and 2060 ng/mL, performing within a log-fold dilution of commercially-available recombinant HRP2. The band intensity observed for the nHRP2 dilutions was equivalent to that observed for P. falciparum culture strain dilutions of 3D7 and US06 F Nigeria XII between 12.5 and 1000 parasites/µL. Conclusions Purified nHRP2 could be a valuable reagent for laboratory applications as well as assessment of new and existing RDTs prior to their use in clinical settings. These results establish that it is possible to extract microgram quantities of the native HRP2 antigen from HB3 culture and that this purified protein is well recognized by existing monoclonal antibody lines and RDTs. Graphical Abstract

The most widespread Plasmodium species, Plasmodium vivax , poses a significant public health threat. An effective vaccine is needed to reduce global malaria burden. Of the erythrocytic stage vaccine candidates, the 19 kDa fragment of the P. vivax Merozoite Surface Protein 1 (PvMSP1 19 ) is one of the most promising. Our group has previously defined several promiscuous T helper epitopes within the PvMSP1 protein, with features that allow them to bind multiple MHC class II alleles. We describe here a P. vivax recombinant modular chimera based on MSP1 (PvRMC-MSP1) that includes defined T cell epitopes genetically fused to PvMSP1 19 . This vaccine candidate preserved structural elements of the native PvMSP1 19 and elicited cytophilic antibody responses, and CD4 + and CD8 + T cells capable of recognizing PvMSP1 19 . Although CD8 + T cells that recognize blood stage antigens have been reported to control blood infection, CD8 + T cell responses induced by P. falciparum or P. vivax vaccine candidates based on MSP1 19 have not been reported. To our knowledge, this is the first time a protein based subunit vaccine has been able to induce CD8 + T cell against PvMSP1 19 . The PvRMC-MSP1 protein was also recognized by naturally acquired antibodies from individuals living in malaria endemic areas with an antibody profile associated with protection from infection. These features make PvRMC-MSP1 a promising vaccine candidate. , plot the predicted protein conformation, amino acid side-chain and chemical bonds peptide-bond and planar molecular and constraint features in a desirable statistical range to be considered allowed conformations for both 3D molecular structures. were also detected in BALB/c mice, the IgG2a/IgG1 ratio suggested a Th2 biased response, which is expected since the genetic regulation of the immune response the BALB/c strain favors the production of IL-4 over IFN- γ 44 . Even when viral vectors are used to present PfMSP1, the IgG2a/IgG1 ratios in BALB/c are still indicative of a Th2 response 34 . Our results suggest that the help provided by the promiscuous T cell epitopes enhances the production of cytophilic antibodies. The production of cytophilic antibodies by PvRMC-MSP1 is encouraging since the inhibition of red blood cell invasion is correlated with the ability of cytophilic antibodies directed against MSP1 to interact with complement 11 . In addition to the ability of PvRMC-MSP1 to elicit the production of cytophilic antibodies in mice, sera samples from humans naturally exposed to P. vivax had a high prevalence of cytophilic IgG1 antibodies that recognize PvRMC-MSP1. The quality of the antibody response elicited by immunization was also confirmed by the assessment of the higher avidity of anti-PvRMC-MSP1 antibodies in C57BL/6 mice. Similarly, phase I clinical studies carried on with a long synthetic peptide based on P. falciparum MSP3 have shown that anti-parasite antibody-dependent cellular inhibition is related to the antibodies avidity 45 . These results demonstrate that immunization with PvRMC-MSP1 could achieve a high avidity cytophilic IgG response that is correlated with protection. Consistent with the high homology ( > 70%) between the promiscuous T cell epitopes and the MSP 19 fragment included in PvRMC-MSP1 and the simian malaria species P. coatney i and P. cynomolgi , we observed that antibodies induced by PvRMC-MSP1 were able to recognize P. coatneyi blood stage parasites by IFA. The antibod- ies recognition of other species confirms that the promiscuous P. vivax T cell epitopes included in PvRMC-MSP1 are conserved in the simian Plasmodium species and preserve their biochemical features. This feature also demon- strates the potential of using P. cynomolgi or P. coatneyi in rhesus macaques as a surrogate model to test efficacy as P. vivax appears to be a species derived from the Macaca lineage of simian parasites 46 . Preclinical trials in NHP have been reported for PvMSP1 based vaccines. PvMSP1 19 expressed in S. cerevisiae linked to two tetanus toxoid promiscuous T-helper epitopes 21 induced partial protection in Saimiri boliviensis monkeys. However, the immunogenicity of the protein was highly dependent on the adjuvant used in the for- mulation 21,47 . This effect was also observed when an MSP1 19 protein associated to two pan allelic CD4 + T cell epitopes was used in Callithrix jacchus , with no evident boosting effect induced by the associated T cell epitopes 22 . Studies using MSP1 33 in Aotus monkeys have shown a protective efficacy between 50 to 80% according to the fragment of the protein used. However, these studies used Freund’s adjuvant, a formulation that is not suitable for human use 23,48 . In another study, recombinant PvMSP1 formulated in Montanide ISA 720 was also able to protect Aotus monkeys from an infectious challenge reducing the parasitemia levels in comparison with control monkeys 49 . These studies demonstrate that achieving protection with PvMSP1 is a feasible goal; but further development is required to obtain a safe, highly immunogenic formulation. Studies with recombinant PvMSP1 19 have shown that this protein is not processed by DCs since the antigen is not soluble and remains attached to the parasite during the ring stage following RBC invasion infection can induce short-lived antibodies not related to protection. In contrast, PvRMC-MSP1 recognition was present in patients that did not have active malaria, which could be an effect of our vaccine design strategy as we use well-defined T and B cell epitopes to avoid the inclusion of regions of low immunogenic potential. We have previously demonstrated that our constructs are better recognized by naturally infected individuals when compared to native proteins and can circumvent the HLA restriction associated to these antigens 27,35 . These characteristics make our proteins not only desirable for vaccination but also useful for seroepidemiological studies. We observed that naturally acquired antibodies against PvRMC-MSP1 exhibited a cytophilic profile. In P. falciparum infection, human cytophilic antibodies against MSP1 and MSP2 have been related to inhibition of the red blood cell invasion 11 . In P. vivax , an antibody profile similar to the one obtained in our study was found to be related to low parasitemias 75,76 . Anti-PvRMC-MSP1 antibodies had a higher prevalence of cytophilic antibodies when compared with previous reports from symptomatic patients 77 , with an IgG1 prevalence of 90% and an IgG3 prevalence of 33.7%. Evidence of the possible PvRMC-MSP1 protective effect is also observed with the correlation of the production of antibodies able to recognize this vaccine candidate and the months passed since the previous malaria episode. There were no associations between the PvRMC-MSP1 responses with the HLA alleles present in the population studied. We have shown that our chimeric proteins can circumvent genetic restriction to the recognition of pre-erythrocytic and erythrocytic P. vivax antigens 27,35 . It is important to test this protein in non-human primates from different genetic backgrounds to confirm the induction of non-genetically restricted immune responses, a highly desirable characteristic for a P. vivax malaria vaccine. The individual linkage of promiscuous T cell epitopes present in PvRMC-MSP1 to the previously defined P. B cell epitope (NANP) of of to block P. falciparum invasion of hepatic in we observed our falciparum show + confirms concerning designing such war-rants further investigation in clinical IgG subclass profiles were determined by ELISA. Following incubation with mouse plates were washed and incubated for 90 minutes with biotinylated anti-mouse IgG1 or IgG2a rat mAbs (BD PharMingen, Franklin Lakes, NJ). Plates were washed again, and the bound antibodies were detected using horseradish peroxidase (HRP)-streptavidin (BD PharMingen) and H 2 O 2 /2,2-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrate (KPL). The avidity of anti-PvRMC-MSP1 antibodies was assessed by a thiocyanate elution-based ELISA using sera samples obtained 20 days after the third immunization (day 60). The assay was conducted in the same manner as described above with a slight modification 24 . In brief, 0–10 M ammonium thiocyanate (NH 4 SCN) in PBS was added to each well after incubation with the sera dilutions. The plates were incubated at room temperature for 15 min and then washed before the addition of the secondary anti-mouse IgG antibody. Serial dilutions of the sera were assayed in the presence or absence of 1 M NH 4 SCN to determine avidity index. For these experiments, the washing step before incubation with anti-mouse IgG antibody was duplicated to remove weakly bound IgG antibodies. The logarithms of reciprocal serum dilutions corresponding to the half-maximum absorbance value in curves obtained with and without NH 4 SCN, termed x 1 and x 2 , were interpolated by third-degree polynomial regression. The avidity index is the ratio between the antilog of x 1 and the antilog of x 2 , or simply antilog (x 1 − x 2 ) as described by Ferreira and Katzin 92 . Absorption ELISA. To test if conformational epitopes are preserved in PvRMC-MSP1, polyclonal antibod- ies obtained from mice immunized with PvMSP1 19 were tested for reactivity against reduced or For human data, analyses were done as previously described 35 using Epi Info 2002 (CDC, Atlanta, GA), and GraphPad Prism 5.0 (GraphPad Software, San Diego, CA) according to the required statistical test. Differences in medians for the study population data were tested by non-parametric Mann–Whitney test when appropriate. Student’s t-test was used to compare the means of normally distributed data or normalized transformations were performed on raw data before testing by one-way ANOVA where appropriate. Differences in the proportions of the frequencies between variables were evaluated by chi-square ( χ 2 ) test. Relationships between the RI again

Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA countries have reported pfhrp2 and pfhrp3 (pfhrp2/3) gene deletions. Blood samples (n = 1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp2/3 deletions. Samples were assayed for HRP2, pan-Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs. The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.

Background: Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA countries have reported pfhrp2 and pfhrp3 (pfhrp2/3) gene deletions. Methods: Blood samples (n=1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp2/3 deletions. Samples were assayed for HRP2, pan-Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs.Results: The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Conclusions: Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.

Background As malaria incidence and transmission in a region decreases, it becomes increasingly difficult to identify areas of active transmission. Improved methods for identifying and monitoring foci of active malaria transmission are needed in areas of low parasite prevalence in order to achieve malaria elimination. Serological assays can provide population-level infection history to inform elimination campaigns. Methods A bead-based multiplex antibody detection assay was used to evaluate a chimeric Plasmodium vivax MSP1 protein (PvRMC-MSP1), designed to be broadly immunogenic for use in vaccine studies, to act as a pan-malaria serological tool based on its ability to capture IgG in plasma samples obtained from naturally exposed individuals. Samples from 236 US travellers with PCR confirmed infection status from all four major Plasmodium species infecting humans, Plasmodium falciparum (n = 181) , Plasmodium vivax (n = 38), Plasmodium malariae (n = 4), and Plasmodium ovale (n = 13) were tested for IgG capture using PvRMC-MSP1 as well as the four recombinant MSP1-19 kD isoforms representative of these Plasmodium species. Results Regardless of infecting Plasmodium species, a large proportion of plasma samples from infected US travellers provided a high assay signal to the PvRMC-MSP1 chimeric protein, with 115 high responders out of 236 samples assessed (48.7%). When grouped by active infection, 38.7% P. falciparum- , 92.1% of P. vivax- , 75.0% P. malariae- , and 53.4% of P. ovale- infected individuals displayed high assay signals in response to PvRMC-MSP1. It was also determined that plasma from P. vivax -infected individuals produced increased assay signals in response to the PvRMC-MSP1 chimera as compared to the recombinant PvMSP1 for 89.5% (34 out of 38) of individuals. PvRMC-MSP1 also showed improved ability to capture IgG antibodies from P. falciparum -infected individuals when compared to the capture by recombinant PvMSP1, with high assay signals observed for 38.7% of P. falciparum- infected travellers in response to PvRMC-MSP1 IgG capture compared to just 1.1% who were high responders to capture by the recombinant PvMSP1 protein. Conclusions These results support further study of designed antigens as an approach for increasing sensitivity or broadening binding capacity to improve existing serological tools for determining population-level exposure to Plasmodium species. Including both broad-reacting and Plasmodium species-specific antigen-coated beads in an assay panel could provide a nuanced view of population-level exposure histories, an extensive IgG profile, and detailed seroestimates. A more sensitive serological tool for detection of P. vivax exposure would aid malaria elimination campaigns in co-endemic areas and regions where P. vivax is the dominant parasite.

The vast majority of malaria mortality is attributed to one parasite species: Plasmodium falciparum. Asexual replication of the parasite within the red blood cell is responsible for the pathology of the disease. In Plasmodium, the endoplasmic reticulum (ER) is a central hub for protein folding and trafficking as well as stress response pathways. In this study, we tested the role of an uncharacterised ER protein, PfGRP170, in regulating these key functions by generating conditional mutants. Our data show that PfGRP170 localises to the ER and is essential for asexual growth, specifically required for proper development of schizonts. PfGRP170 is essential for surviving heat shock, suggesting a critical role in cellular stress response. The data demonstrate that PfGRP170 interacts with the Plasmodium orthologue of the ER chaperone, BiP. Finally, we found that loss of PfGRP170 function leads to the activation of the Plasmodium eIF2α kinase, PK4, suggesting a specific role for this protein in this parasite stress response pathway.

Malaria control and interventions including long-lasting insecticide-treated nets, indoor residual spraying, and intermittent preventative treatment in pregnancy have resulted in a significant reduction in the number of Plasmodium falciparumcases. Considerable efforts have been devoted to P. falciparum vaccines development with much less to P. vivax. Transmission-blocking vaccines, which can elicit antibodies targeting Plasmodium antigens expressed during sexual stage development and interrupt transmission, offer an alternative strategy to achieve malaria control. The post-fertilization antigen P25 mediates several functions essential to ookinete survival but is poorly immunogenic in humans. Previous clinical trials targeting this antigen have suggested that conjugation to a carrier protein could improve the immunogenicity of P25. Here we report the production, and characterization of a vaccine candidate composed of a chimeric P. vivax Merozoite Surface Protein 1 (cPvMSP1) genetically fused to P. vivax P25 (Pvs25) designed to enhance CD4(+) T cell responses and its assessment in a murine model. We demonstrate that antibodies elicited by immunization with this chimeric protein recognize both the erythrocytic and sexual stages and are able to block the transmission of P. vivax field isolates in direct membrane-feeding assays. These findings provide support for the continued development of multi-stage transmission blocking vaccines targeting the life-cycle stage responsible for clinical disease and the sexual-stage development accountable for disease transmission simultaneously.

Introduction: Cellular and humoral immune responses are both involved in protection against Plasmodium infections. The only malaria vaccine available, RTS,S, primarily induces short-lived antibodies and targets only a pre-erythrocytic stage antigen. Inclusion of erythrocytic stage targets and enhancing cellular immunogenicity are likely necessary for developing an effective second-generation malaria vaccine. Adenovirus vectors have been used to improve the immunogenicity of protein-based vaccines. However, the clinical assessment of adenoviral-vectored malaria vaccines candidates has shown the induction of robust Plasmodium-specific CD8+ but not CD4+ T cells. Signal peptides (SP) have been used to enhance the immunogenicity of DNA vaccines, but have not been tested in viral vector vaccine platforms. Objectives: The objective of this study was to determine if the addition of the SP derived from the murine IgG kappa light chain within a recombinant adenovirus vector encoding a multistage P. vivax vaccine candidate could improve the CD4+ T cell response. Methods: In this proof-of-concept study, we immunized CB6F1/J mice with either the recombinant simian adenovirus 36 vector containing the SP (SP-SAd36) upstream from a transgene encoding a chimeric P. vivax multistage protein or the same SAd36 vector without the SP. Mice were subsequently boosted twice with the corresponding recombinant proteins emulsified in Montanide ISA 51 VG. Immunogenicity was assessed by measurement of antibody quantity and quality, and cytokine production by T cells after the final immunization. Results: The SP-SAd36 immunization regimen induced significantly higher antibody avidity against the chimeric P. vivax proteins tested and higher frequencies of IFN-gamma and IL-2 CD4+ and CD8+ secreting T cells, when compared to the unmodified SAd36 vector. Conclusions: The addition of the murine IgG kappa signal peptide significantly enhances the immunogenicity of a SAd36 vectored P. vivax multi-stage vaccine candidate in mice. The potential of this approach to improve upon existing viral vector vaccine platforms warrants further investigation. (C) 2018 Elsevier Ltd. All rights reserved.

The delay in parasite-specific B cell development leaves people in malaria endemic areas vulnerable to repeated Plasmodium infections. Here, we investigated the role of transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), a molecule involved in the generation of antigen-specific antibody secreting cells, in host response to non-lethal Plasmodium yoelii infection. We found that TACI deficiency not only resulted in higher peak parasitemia levels in P. yoelii challenged mice, but also led to a delay in parasite clearance and anti-P. yoelii Merozoite Surface Protein 1(C-terminal 19-kDa fragment [rMSP-119]) protein and anti-rMSP-119 and anti-P. yoelii IgG antibody development. There was also a delay in the generation of splenic high affinity antibody secreting cells that recognize rMSP-119 protein as compared to wild-type mice. Interestingly, coinciding with the delay in parasite clearance there was a delay in the resolution of T follicular helper (TFH) cell and germinal center (GC) B cell responses in TACI -/- mice. The persistence of TFH and GC B cells is likely a result of enhanced interaction between TFH and GC B cells because inducible costimulator ligand (ICOSL) expression was significantly higher on TACI -/- GC B cells than wild-type cells. The difference in the kinetics of GC reaction appeared to also impact the emergence of plasma cells (PC) because there was a delay in the generation of TACI -/- mice PC. Nevertheless, following the recovery from P. yoelii infection, TACI -/- and wild-type mice were both protected from a rechallenge infection. Establishment of protective B cell response was responsible for the resolution of parasitemia because B cells purified from recovered TACI -/- or wild-type mice were equally protective when introduced to naïve wild-type mice prior to P. yoelii challenge. Thus, despite the increased susceptibility of TACI -/- mice to P. yoelii infection and a delay in the development of protective antibody levels, TACI -/- mice are able to clear the infection and resist rechallenge infection.

Plasmodium vivax Merozoite Surface Protein-9 (PvMSP-9) is a malaria vaccine candidate naturally immunogenic in humans and able to induce high antibody titers in animals when delivered as a recombinant protein. Recently, we identified the sequence EAAPENAEPVHENA (PvMSP9E795-A808) as the main linear B-cell epitope in naturally exposed individuals. However, the potential of PvMSP9E795-A808 as an immunogen in experimental animal models remained unexplored. Here we assess the immunogenicity of PvMSP9E795-A808 using synthetic peptides. The peptides tested in BALB/c mice include two repeats of the sequence EAAPENAEPVHENA tested alone (peptide RII), or linked to an autologous (PvMSP9 peptide pL; pLRII) or heterologous (p2 tetanus toxin universal T cell epitope; TTRII) T cell epitope. Immune responses were evaluated by ELISA, FLUOROSPOT, and indirect immunofluorescence. We show that all of the peptide constructs tested were immunogenic eliciting specific IgG antibodies at different levels, with a prevalence of IgG1 and IgG2. Animals immunized with synthetic peptides containing T cell epitopes (pLRII or TTRII) had more efficient antibody responses that resulted in higher antibody titers able to recognize the native protein by immunofluorescence. Relevantly, the frequency of IFN-γ secreting SFC elicited by immunization with TTRII synthetic peptide was comparable to that reported to the PvMSP9-Nt recombinant protein. Taken together, our study indicates that PvMSP9E795-A808 is highly immunogenic in mice and further studies to evaluate its value as promising vaccine target are warranted. Moreover, our study supports the critical role of CD4 T cell epitopes to enhance humoral responses induced by subunit based vaccines.

Malaria remains a considerable burden on public health. In 2015, the WHO estimates there were 212 million malaria cases causing nearly 429,000 deaths globally. A highly effective malaria vaccine is needed to reduce the burden of this disease. We have developed an experimental vaccine candidate (PyCMP) based on pre-erythrocytic (CSP) and erythrocytic (MSP1) stage antigens derived from the rodent malaria parasite P. yoelii. Our protein-based vaccine construct induces protective antibodies and CD4+ T cell responses. Based on evidence that viral vectors increase CD8+ T cell-mediated immunity, we also have tested heterologous prime-boost immunization regimens that included human adenovirus serotype 5 vector (Ad5), obtaining protective CD8+ T cell responses. While Ad5 is commonly used for vaccine studies, the high prevalence of pre-existing immunity to Ad5 severely compromises its utility. Here, we report the use of the novel simian adenovirus 36 (SAd36) as a candidate for a vectored malaria vaccine since this virus is not known to infect humans, and it is not neutralized by anti-Ad5 antibodies. Our study shows that the recombinant SAd36PyCMP can enhance specific CD8+ T cell response and elicit similar antibody titers when compared to an immunization regimen including the recombinant Ad5PyCMP. The robust immune responses induced by SAd36PyCMP are translated into a lower parasite load following P. yoelii infectious challenge when compared to mice immunized with Ad5PyCMP.