Cross-reactivity of rPvs48/45, a recombinant Plasmodium vivax protein, with sera from Plasmodium falciparum endemic areas of Africa.

Background: Ps48/45, a Plasmodium gametocyte surface protein, is a promising candidate for malaria transmission-blocking (TB) vaccine. Due to its relevance for a multispecies vaccine, we explored the cross-reactivity and TB activity of a recombinant P. vivax Ps48/45 protein (rPvs48/45) with sera from P. falciparum-exposed African donors. Methods: rPvs48/45 was produced in Chinese hamster ovary cell lines and tested by ELISA for its cross-reactivity with sera from Burkina Faso, Tanzania, Mali, and Nigeria - In addition, BALB/c mice were immunized with the rPvs48/45 protein formulated in Montanide ISA-51 and inoculated with a crude extract of P. falciparum NF-54 gametocytes to evaluate the parasite-boosting effect on rPvs48/45 antibody titers. Specific anti-rPvs48/45 IgG purified from African sera was used to evaluate the ex vivo TB activity on P. falciparum, using standard mosquito membrane feeding assays (SMFA). Results: rPvs48/45 protein showed cross-reactivity with sera of individuals from all four African countries, in proportions ranging from 94% (Tanzania) to 40% (Nigeria). Also, the level of cross-reactive antibodies varied significantly between countries (p<0.0001), with a higher antibody level in Mali and the lowest in Nigeria. In addition, antibody levels were higher in adults (≥ 17 years) than young children (≤ 5 years) in both Mali and Tanzania, with a higher proportion of responders in adults (90%) than in children (61%) (p<0.0001) in Mali, where male (75%) and female (80%) displayed similar antibody responses. Furthermore, immunization of mice with P. falciparum gametocytes boosted anti-Pvs48/45 antibody responses, recognizing P. falciparum gametocytes in indirect immunofluorescence antibody test. Notably, rPvs48/45 affinity-purified African IgG exhibited a TB activity of 61% against P. falciparum in SMFA. Conclusion: African sera (exposed only to P. falciparum) cross-recognized the rPvs48/45 protein. This, together with the functional activity of IgG, warrants further studies for the potential development of a P. vivax and P. falciparum cross-protective TB vaccine.

A SARS-CoV-2 Spike Receptor Binding Motif Peptide Induces Anti-Spike Antibodies in Mice andIs Recognized by COVID-19 Patients.

The currently devastating pandemic of severe acute respiratory syndrome known as coronavirus disease 2019 or COVID-19 is caused by the coronavirus SARS-CoV-2. Both the virus and the disease have been extensively studied worldwide. A trimeric spike (S) protein expressed on the virus outer bilayer leaflet has been identified as a ligand that allows the virus to penetrate human host cells and cause infection. Its receptor-binding domain (RBD) interacts with the angiotensin-converting enzyme 2 (ACE2), the host-cell viral receptor, and is, therefore, the subject of intense research for the development of virus control means, particularly vaccines. In this work, we search for smaller fragments of the S protein able to elicit virus-neutralizing antibodies, suitable for production by peptide synthesis technology. Based on the analysis of available data, we selected a 72 aa long receptor binding motif (RBM436-507) of RBD. We used ELISA to study the antibody response to each of the three antigens (S protein, its RBD domain and the RBM436-507 synthetic peptide) in humans exposed to the infection and in immunized mice. The seroreactivity analysis showed that anti-RBM antibodies are produced in COVID-19 patients and immunized mice and may exert neutralizing function, although with a frequency lower than anti-S and -RBD. These results provide a basis for further studies towards the development of vaccines or treatments focused on specific regions of the S virus protein, which can benefit from the absence of folding problems, conformational constraints and other advantages of the peptide synthesis production.

Seroreactivity of the Severe Acute Respiratory Syndrome Coronavirus 2 Recombinant S Protein, Receptor-Binding Domain, and Its Receptor-Binding Motif in COVID-19 Patients and Their Cross-Reactivity With Pre-COVID-19 Samples From Malaria-Endemic Areas.

Despite the global interest and the unprecedented number of scientific studies triggered by the COVID-19 pandemic, few data are available from developing and low-income countries. In these regions, communities live under the threat of various transmissible diseases aside from COVID-19, including malaria. This study aims to determine the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroreactivity of antibodies from COVID-19 and pre-COVID-19 samples of individuals in Mali (West Africa). Blood samples from COVID-19 patients (n = 266) at Bamako Dermatology Hospital (HDB) and pre-COVID-19 donors (n = 283) from a previous malaria survey conducted in Dangassa village were tested by ELISA to assess IgG antibodies specific to the full-length spike (S) protein, the receptor-binding domain (RBD), and the receptor-binding motif (RBM436-507). Study participants were categorized by age, gender, treatment duration for COVID-19, and comorbidities. In addition, the cross-seroreactivity of samples from pre-COVID-19, malaria-positive patients against the three antigens was assessed. Recognition of the SARS-CoV-2 proteins by sera from COVID-19 patients was 80.5% for S, 71.1% for RBD, and 31.9% for RBM (p < 0.001). While antibody responses to S and RBD tended to be age-dependent, responses to RBM were not. Responses were not gender-dependent for any of the antigens. Higher antibody levels to S, RBD, and RBM at hospital entry were associated with shorter treatment durations, particularly for RBD (p < 0.01). In contrast, higher body weights negatively influenced the anti-S antibody response, and asthma and diabetes weakened the anti-RBM antibody responses. Although lower, a significant cross-reactive antibody response to S (21.9%), RBD (6.7%), and RBM (8.8%) was detected in the pre-COVID-19 and malaria samples. Cross-reactive antibody responses to RBM were mostly associated (p < 0.01) with the absence of current Plasmodium falciparum infection, warranting further study.

Randomized clinical trial to assess the protective efficacy of a Plasmodium vivax CS synthetic vaccine.

A randomized, double-blind, controlled vaccine clinical trial was conducted to assess, as the primary outcome, the safety and protective efficacy of the Plasmodium vivax circumsporozoite (CS) protein in healthy malaria-naïve (phase IIa) and semi-immune (phase IIb) volunteers. Participants (n = 35) were randomly selected from a larger group (n = 121) and further divided into naïve (n = 17) and semi-immune (n = 18) groups and were immunized at months 0, 2, and 6 with PvCS formulated in Montanide ISA-51 adjuvant or placebo (adjuvant alone). Specific antibodies and IFN-γ responses to PvCS were determined as secondary outcome; all experimental volunteers developed specific IgG and IFN-γ. Three months after the last immunization, all participants were subjected to controlled human malaria infection. All naive controls became infected and drastic parasitemia reduction, including sterile protection, developed in several experimental volunteers in phase IIa (6/11) (54%, 95% CI 0.25-0.84) and phase IIb (7/11) (64%, 95% CI 0.35-0.92). However, no difference in parasitemia was observed between the phase IIb experimental and control subgroups. In conclusion, this study demonstrates significant protection in both naïve and semi-immune volunteers, encouraging further PvCS vaccine clinical development. Trial registration number NCT02083068. This trial was funded by Colciencias (grant 529-2009), NHLBI (grant RHL086488 A), and MVDC/CIV Foundation (grant 2014-1206).

Immunogenicity of full-length P. vivax rPvs48/45 protein formulations in BALB/c mice.

BACKGROUND: Pvs48/45 is a Plasmodium vivax gametocyte surface protein involved in the parasite fertilization process. Previous studies showed that Pvs48/45 proteins expressed in Escherichia coli (E. coli) and Chinese hamster ovary (CHO) cells were highly immunoreactive with sera from malaria-endemic areas and highly immunogenic in animal models. Here the immunogenicity in mice of three different vaccine formulations was compared. METHODS: Recombinant (r) Pvs48/45 proteins were expressed in E. coli and CHO, purified, formulated in Alhydrogel, GLA-SE and Montanide ISA-51 adjuvants and used to immunize BALB/c mice. Animals were immunized on days 0, 20 and 40, and serum samples were collected for serological analyses of specific antibody responses using ELISA and immunofluorescence (IFAT). Additionally, ex-vivo transmission-reducing activity (TRA) of sera on P. vivax gametocyte-infected human blood fed to Anopheles albimanus in direct membrane feeding assays (DMFA) was evaluated. RESULTS: Most immunized animals seroconverted after the first immunization, and some developed antibody peaks of 106 with all adjuvants. However, the three adjuvant formulations induced different antibody responses and TRA efficacy. While GLA-SE formulations of both proteins induced similar antibody profiles, Montanide ISA-51 formulations resulted in higher and longer-lasting antibody titers with CHO-rPvs48/45 than with the E. coli formulation. Although the CHO protein formulated in Alhydrogel generated a high initial antibody peak, antibody responses to both proteins rapidly waned. Likewise, anti-Pvs48/45 antibodies displayed differential recognition of the parasite proteins in IFAT and ex vivo blockade of parasite transmission to mosquitoes. The CHO-rPvs48/45 formulated in Montanide ISA-51 induced the most effective ex vivo parasite blockage. CONCLUSIONS: Three out of six vaccine formulations elicited antibodies with ex vivo TRA. The CHO-rPvs48/45 Montanide ISA-51 formulation induced the most stable antibody response, recognizing the native protein and the most robust ex vivo TRA. These results encourage further testing of the vaccine potential of this protein.

Immunoreactivity of Sera From Low to Moderate Malaria-Endemic Areas Against Plasmodium vivax rPvs48/45 Proteins Produced in Escherichia coli and Chinese Hamster Ovary Systems.

P48/45 is a conserved gametocyte antigen involved in Plasmodium parasite fertilization. A recombinant Plasmodium vivax P48/45 (Pvs48/45) protein expressed in Escherichia coli (E. coli) was highly antigenic and immunogenic in experimental animals and elicited specific transmission-blocking (TB) antibodies in a previous pilot study. Here, a similar Pvs48/45 gene was expressed in Chinese Hamster Ovary (CHO) cells and we compared its immunoreactivity with the E. coli product. Specific antibody titers were determined using plasma from Colombian individuals (n=227) living in endemic areas where both P. vivax and P. falciparum are prevalent and from Guatemala (n=54) where P. vivax is highly prevalent. In Colombia, plasma seroprevalence to CHO-rPvs48/45 protein was 46.3%, while for E. coli-rPvs48/45 protein was 36.1% (p<0.001). In Guatemala, the sero prevalence was 24.1% and 14.8% (p<0.001), respectively. Reactivity index (RI) against both proteins showed an age-dependent increase. IgG2 was the predominant subclass and the antibody avidity index evaluated by ELISA ranged between 4-6 mol/L. Ex vivo P. vivax mosquito direct membrane feeding assays (DMFA) performed in presence of study plasmas, displayed significant parasite transmission-blocking (TB), however, there was no direct correlation between antibody titers and oocysts transmission reduction activity (%TRA). Nevertheless, DMFA with CHO rPvs48/45 affinity purified IgG showed a dose response; 90.2% TRA at 100 µg/mL and 71.8% inhibition at 10 µg/mL. In conclusion, the CHO-rPvs48/45 protein was more immunoreactive in most of the malaria endemic places studied, and CHO-rPvs48/45 specific IgG showed functional activity, supporting further testing of the protein vaccine potential.

Nanotechnological immunoassay for rapid label-free analysis of candidate malaria vaccines.

Malaria is a life-threatening epidemic disease with half of the world's population at risk. Although its incidence rate has fallen since 2010, this ratio dramatically stalled between 2014 and 2018. New fast and optimized tools in vaccine analysis and seroconversion testing are critically needed. We developed a clinical diagnostic device based on piezo-actuated nanoresonators that perform as quantitative in situ calibrated nano-bio sensors for specific detection of multiple target molecules in serum samples. The immunoassay successfully diagnoses humoral immune responses induced by malaria vaccine candidates and reveals the timeline and stage of the infection. We applied the newly developed strategy to a variety of different samples, from pure antibody/vaccine solutions, to blood samples from clinical trials on both naïve and pre-exposed malaria volunteers from sub-Saharan countries. Our nanomechanical assay provides a direct one-step label-free quantitative immunoassay that is on par with the gold-standard, multi-step enzyme-linked immunosorbent assay (ELISA). We achieve a limit of detection of few pg ml-1, or sub-pM concentrations. The 6 µl sample volume allows more than 50 experiments from one finger prick. Furthermore, we simultaneously detected multiple analytes by differential functionalization of multiple sensors in parallel. The inherent differential read-out with in situ controls reduces false positive results. Due to the faster turnaround time, the minimal volume required and the automatized handling system, this technique has great potential for miniaturization and routine diagnostics in pandemic emergencies.

P. falciparum and P. vivax Orthologous Coiled-Coil Candidates for a Potential Cross-Protective Vaccine.

Over the last four decades, significant efforts have been invested to develop vaccines against malaria. Although most efforts are focused on the development of P. falciparum vaccines, the current availability of the parasite genomes, bioinformatics tools, and high throughput systems for both recombinant and synthetic antigen production have helped to accelerate vaccine development against the P. vivax parasite. We have previously in silico identified several P. falciparum and P. vivax proteins containing α-helical coiled-coil motifs that represent novel putative antigens for vaccine development since they are highly immunogenic and have been associated with protection in many in vitro functional assays. Here, we selected five pairs of P. falciparum and P. vivax orthologous peptides to assess their sero-reactivity using plasma samples collected in P. falciparum- endemic African countries. Pf-Pv cross-reactivity was also investigated. The pairs Pf27/Pv27, Pf43/Pv43, and Pf45/Pv45 resulted to be the most promising candidates for a cross-protective vaccine because they showed a high degree of recognition in direct and competition ELISA assays and cross-reactivity with their respective ortholog. The recognition of P. vivax peptides by plasma of P. falciparum infected individuals indicates the existence of a high degree of cross-reactivity between these two Plasmodium species. The design of longer polypeptides combining these epitopes will allow the assessment of their immunogenicity and protective efficacy in animal models.

Designs and Characterization of Subunit Ebola GP Vaccine Candidates: Implications for Immunogenicity.

The humoral responses of Ebola virus (EBOV) survivors mainly target the surface glycoprotein GP, and anti-GP neutralizing antibodies have been associated with protection against EBOV infection. In order to elicit protective neutralizing antibodies through vaccination a native-like conformation of the antigen is required. We therefore engineered and expressed in CHO cells several GP variants from EBOV (species Zaire ebolavirus, Mayinga variant), including a soluble GP ΔTM, a mucin-like domain-deleted GP ΔTM-ΔMUC, as well as two GP ΔTM-ΔMUC variants with C-terminal trimerization motifs in order to favor their native trimeric conformation. Inclusion of the trimerization motifs resulted in proteins mimicking GP metastable trimer and showing increased stability. The mucin-like domain appeared not to be critical for the retention of the native conformation of the GP protein, and its removal unmasked several neutralizing epitopes, especially in the trimers. The soluble GP variants inhibited mAbs neutralizing activity in a pseudotype transduction assay, further confirming the proteins' structural integrity. Interestingly, the trimeric GPs, a native-like GP complex, showed stronger affinity for antibodies raised by natural infection in EBOV disease survivors rather than for antibodies raised in volunteers that received the ChAd3-EBOZ vaccine. These results support our hypothesis that neutralizing antibodies are preferentially induced when using a native-like conformation of the GP antigen. The soluble trimeric recombinant GP proteins we developed represent a novel and promising strategy to develop prophylactic vaccines against EBOV and other filoviruses.

Epitope Mapping and Fine Specificity of Human T and B Cell Responses for Novel Candidate Blood-Stage Malaria Vaccine P27A.

P27A is a novel synthetic malaria vaccine candidate derived from the blood stage Plasmodium falciparum protein Trophozoite Exported Protein 1 (TEX1/PFF0165c). In phase 1a/1b clinical trials in malaria unexposed adults in Switzerland and in malaria pre-exposed adults in Tanzania, P27A formulated with Alhydrogel and GLA-SE adjuvants induced antigen-specific antibodies and T-cell activity. The GLA-SE adjuvant induced significantly stronger humoral responses than the Alhydrogel adjuvant. Groups of pre-exposed and unexposed subjects received identical vaccine formulations, which supported the comparison of the cellular and humoral response to P27A in terms of fine specificity and affinity for populations and adjuvants. Globally, fine specificity of the T and B cell responses exhibited preferred recognized sequences and did not highlight major differences between adjuvants or populations. Affinity of anti-P27A antibodies was around 10-8 M in all groups. Pre-exposed volunteers presented anti-P27A with higher affinity than unexposed volunteers. Increasing the dose of GLA-SE from 2.5 to 5 µg in pre-exposed volunteers improved anti-P27A affinity and decreased the number of recognized epitopes. These results indicate a higher maturation of the humoral response in pre-exposed volunteers, particularly when immunized with P27A formulated with 5 µg GLA-SE.

Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR.

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). In HIV-1, the antisense protein ASP gene is located on the negative strand of env, in the reading frame -2, spanning the junction gp120/gp41. In the sense orientation, the 3' end of the ASP open reading frame overlaps with gp120 hypervariable regions V4 and V5. The study of ASP RNA has been thwarted by a phenomenon known as RT-self-priming, whereby RNA secondary structures have the ability to prime RT in absence of the specific primer, generating non-specific cDNAs. The combined use of high RNA denaturation with biotinylated reverse primers in the RT reaction, together with affinity purification of the cDNA onto streptavidin-coated magnetic beads, has allowed us to selectively amplify ASP RNA in CD4+ T cells derived from individuals infected with HIV-1. Our method is relatively low-cost, simple to perform, highly reliable, and easily reproducible. In this respect, it represents a powerful tool for the study of antisense transcription not only in HIV-1 but also in other biological systems.

Immunogenicity of a recombinant fusion construct composed of intrinsically unstructured, low polymorphic segments derived from merozoite surface protein 2 and trophozoite exported protein 1.

To overcome the extensive polymorphism found in human Plasmodium antigens and to avoid the lengthy characterization of their 3 dimensional structure and subsequent production of the native proteins we have been concentrated in large unstructured, non-or low-polymorphic fragments present in the blood stage of P. falciparum. Three fragments derived from the 2 family-specific and constant regions of merozoite surface protein (MSP2) and PFF0165c protein were previously selected for evaluation as potential single vaccine candidates. In order to increase and optimize their potential efficacy against P. falciparum infection the 3 antigens were combined in a single DNA recombinant product (FusN) and compared its antigenicity with that of single antigens in sera of volunteers living in endemic countries. Immunogenicity of the FusN was then compared with that of the mixture of 3 antigens in 3 strains of mice. Antigen specific, affinity purified human antibodies were then tested in antibody dependent cellular inhibition and merozoite opsonization assays. In addition, the antigen specific antibody response and its association with protection from malaria infection were determined. The data collected indicate that the recombinant product is an equal or better antigen /immunogen than fragments used either alone or as a mixture for vaccination in combination with adjuvant. In addition, antibody response to FusN shows a stronger association with protection than single fragments. The use of a single construct as vaccine would drastically reduce the cost of manufacturing and development of the GMP product.

Detection of antisense protein (ASP) RNA transcripts in individuals infected with human immunodeficiency virus type 1 (HIV-1).

The detection of antisense RNA is hampered by reverse transcription (RT) non-specific priming, due to the ability of RNA secondary structures to prime RT in the absence of specific primers. The detection of antisense RNA by conventional RT-PCR does not allow assessment of the polarity of the initial RNA template, causing the amplification of non-specific cDNAs. In this study we have developed a modified protocol for the detection of human immunodeficiency virus type 1 (HIV-1) antisense protein (ASP) RNA. Using this approach, we have identified ASP transcripts in CD4+ T cells isolated from five HIV-infected individuals, either untreated or under suppressive therapy. We show that ASP RNA can be detected in stimulated CD4+ T cells from both groups of patients, but not in unstimulated cells. We also show that in untreated patients, the patterns of expression of ASP and env are very similar, with the levels of ASP RNA being markedly lower than those of env. Treatment of cells from one viraemic patient with α-amanitin greatly reduces the rate of ASP RNA synthesis, suggesting that it is associated with RNA polymerase II, the central enzyme in the transcription of protein-coding genes. Our data represent the first nucleotide sequences obtained in patients for ASP, demonstrating that its transcription indeed occurs in those HIV-1 lineages in which the ASP open reading frame is present.

The Candidate Blood-stage Malaria Vaccine P27A Induces a Robust Humoral Response in a Fast Track to the Field Phase 1 Trial in Exposed and Nonexposed Volunteers.

Background: P27A is an unstructured 104mer synthetic peptide from Plasmodium falciparum trophozoite exported protein 1 (TEX1), the target of human antibodies inhibiting parasite growth. The present project aimed at evaluating the safety and immunogenicity of P27A peptide vaccine in malaria-nonexposed European and malaria-exposed African adults. Methods: This study was designed as a staggered, fast-track, randomized, antigen and adjuvant dose-finding, multicenter phase 1a/1b trial, conducted in Switzerland and Tanzania. P27A antigen (10 or 50 µg), adjuvanted with Alhydrogel or glucopyranosil lipid adjuvant stable emulsion (GLA-SE; 2.5 or 5 µg), or control rabies vaccine (Verorab) were administered intramuscularly to 16 malaria-nonexposed and 40 malaria-exposed subjects on days 0, 28, and 56. Local and systemic adverse events (AEs) as well as humoral and cellular immune responses were assessed after each injection and during the 34-week follow-up. Results: Most AEs were mild to moderate and resolved completely within 48 hours. Systemic AEs were more frequent in the formulation with alum as compared to GLA-SE, whereas local AEs were more frequent after GLA-SE. No serious AEs occurred. Supported by a mixed Th1/Th2 cell-mediated immunity, P27A induced a marked specific antibody response able to recognize TEX1 in infected erythrocytes and to inhibit parasite growth through an antibody-dependent cellular inhibition mechanism. Incidence of AEs and antibody responses were significantly lower in malaria-exposed Tanzanian subjects than in nonexposed European subjects. Conclusions: The candidate vaccine P27A was safe and induced a particularly robust immunogenic response in combination with GLA-SE. This formulation should be considered for future efficacy trials. Clinical Trials Registration: NCT01949909, PACTR201310000683408.

A public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein.

Immunization with attenuated Plasmodium falciparum sporozoites (PfSPZs) has been shown to be protective against malaria, but the features of the antibody response induced by this treatment remain unclear. To investigate this response in detail, we isolated IgM and IgG monoclonal antibodies from Tanzanian volunteers who were immunized with repeated injection of Sanaria PfSPZ Vaccine and who were found to be protected from controlled human malaria infection with infectious homologous PfSPZs. All isolated IgG monoclonal antibodies bound to P. falciparum circumsporozoite protein (PfCSP) and recognized distinct epitopes in its N terminus, NANP-repeat region, and C terminus. Strikingly, the most effective antibodies, as determined in a humanized mouse model, bound not only to the repeat region, but also to a minimal peptide at the PfCSP N-terminal junction that is not in the RTS,S vaccine. These dual-specific antibodies were isolated from different donors and were encoded by VH3-30 or VH3-33 alleles that encode tryptophan or arginine at position 52. Using structural and mutational data, we describe the elements required for germline recognition and affinity maturation. Our study provides potent neutralizing antibodies and relevant information for lineage-targeted vaccine design and immunization strategies.

The use of a P. falciparum specific coiled-coil domain to construct a self-assembling protein nanoparticle vaccine to prevent malaria.

BACKGROUND: The parasitic disease malaria remains a major global public health concern and no truly effective vaccine exists. One approach to the development of a malaria vaccine is to target the asexual blood stage that results in clinical symptoms. Most attempts have failed. New antigens such as P27A and P27 have emerged as potential new vaccine candidates. Multiple studies have demonstrated that antigens are more immunogenic and are better correlated with protection when presented on particulate delivery systems. One such particulate delivery system is the self-assembling protein nanoparticle (SAPN) that relies on coiled-coil domains of proteins to form stable nanoparticles. In the past we have used de novo designed amino acid domains to drive the formation of the coiled-coil scaffolds which present the antigenic epitopes on the particle surface. RESULTS: Here we use naturally occurring domains found in the tex1 protein to form the coiled-coil scaffolding of the nanoparticle. Thus, by engineering P27A and a new extended form of the coiled-coil domain P27 onto the N and C terminus of the SAPN protein monomer we have developed a particulate delivery system that effectively displays both antigens on a single particle that uses malaria tex1 sequences to form the nanoparticle scaffold. These particles are immunogenic in a murine model and induce immune responses similar to the ones observed in seropositive individuals in malaria endemic regions. CONCLUSIONS: We demonstrate that our P27/P27A-SAPNs induce an immune response akin to the one in seropositive individuals in Burkina Faso. Since P27 is highly conserved among different Plasmodium species, these novel SAPNs may even provide cross-protection between Plasmodium falciparum and Plasmodium vivax the two major human malaria pathogens. As the SAPNs are also easy to manufacture and store they can be delivered to the population in need without complication thus providing a low cost malaria vaccine.

Natural immune response to Plasmodium vivax alpha-helical coiled coil protein motifs and its association with the risk of P. vivax malaria.

Protein α-helical coiled coil structures are known to induce antibodies able to block critical functions in different pathogens. In a previous study, a total of 50 proteins of Plasmodium vivax erythrocytic asexual stages containing α-helical coiled coil structural motifs were identified in silico, and the corresponding peptides were chemically synthesized. A total of 43 peptides were recognized by naturally acquired antibodies in plasma samples from both Papua New Guinea (PNG) and Colombian adult donors. In this study, the association between IgG antibodies to these peptides and clinical immunity was further explored by measuring total IgG antibody levels to 24 peptides in baseline samples from a longitudinal study of children aged 1-3 years (n = 164) followed for 16 months. Samples were reactive to all peptides tested. Eight peptides were recognized by >50% of individuals, whereas only one peptide had < 20% reactivity. Children infected at baseline were seropositive to 23/24 peptides. No significant association was observed between antibody titers and age or molecular force of infection, suggesting that antibody levels had already reached an equilibrium. There was a strong association between antibody levels to all peptides and protection against P. vivax clinical episodes during the 16 months follow-up. These results suggest that the selected coiled coil antigens might be good markers of both exposure and acquired immunity to P. vivax malaria, and further preclinical investigation should be performed to determine their potential as P. vivax vaccine antigens.

Antibody levels against GLURP R2, MSP1 block 2 hybrid and AS202.11 and the risk of malaria in children living in hyperendemic (Burkina Faso) and hypo-endemic (Ghana) areas.

BACKGROUND: Differences in parasite transmission intensity influence the process of acquisition of host immunity to Plasmodium falciparum malaria and ultimately, the rate of malaria related morbidity and mortality. Potential vaccines being designed to complement current intervention efforts therefore need to be evaluated against different malaria endemicity backgrounds. METHODS: The associations between antibody responses to the chimeric merozoite surface protein 1 block 2 hybrid (MSP1 hybrid), glutamate-rich protein region 2 (GLURP R2) and the peptide AS202.11, and the risk of malaria were assessed in children living in malaria hyperendemic (Burkina Faso, n = 354) and hypo-endemic (Ghana, n = 209) areas. Using the same reagent lots and standardized protocols for both study sites, immunoglobulin (Ig) M, IgG and IgG sub-class levels to each antigen were measured by ELISA in plasma from the children (aged 6-72 months). Associations between antibody levels and risk of malaria were assessed using Cox regression models adjusting for covariates. RESULTS: There was a significant association between GLURP R2 IgG3 and reduced risk of malaria after adjusting age of children in both the Burkinabe (hazard ratio 0.82; 95 % CI 0.74-0.91, p < 0.0001) and the Ghanaian (HR 0.48; 95 % CI 0.25-0.91, p = 0.02) cohorts. MSP1 hybrid IgM was associated (HR 0.85; 95 % CI 0.73-0.98, p = 0.02) with reduced risk of malaria in Burkina Faso cohort while IgG against AS202.11 in the Ghanaian children was associated with increased risk of malaria (HR 1.29; 95 % CI 1.01-1.65, p = 0.04). CONCLUSION: These findings support further development of GLURP R2 and MSP1 block 2 hybrid, perhaps as a fusion vaccine antigen targeting malaria blood stage that can be deployed in areas of varying transmission intensity.

Immunogenicity of dimorphic and C-terminal fragments of Plasmodium falciparum MSP2 formulated with different adjuvants in mice.

BACKGROUND: Plasmodium falciparum MSP2 is a blood stage protein that is associated with protection against malaria. It was shown that the MSP2 dimorphic (D) and constant (C) regions were well recognized by immune human antibodies, and were characterized by major conserved epitopes in different endemic areas and age groups. These Abs recognized merozoite-derived proteins in WB and IFA. Here, the goal was to determine in mice the immunogenicity of the two allelic MSP2 D and C domains formulated with different adjuvants, for their possible use in future clinical studies. METHOD: Female A/J, C3H, and ICR mice were immunized subcutaneously 3 times at 3-week interval with a mixture of allelic and conserved MSP2 long synthetic peptides formulated with different adjuvants. One week after the third injection, sera from each group were obtained and stored at -20°C for subsequent testing. RESULTS: Both domains of the two MSP2 families are immunogenic and the fine specificity and intensity of the Ab responses are dependent on mouse strains and adjuvants. The major epitopes were restricted to the 20-mer peptide sequences comprising the last 8aa of D and first 12aa of C of the two allelic families and the first 20aa of the C region, this for most strains and adjuvants. Strong immune responses were associated with GLA-SE adjuvant and its combination with other TLR agonists (CpG or GDQ) compared to alhydrogel and Montanide. Further, the elicited Abs were also capable of recognizing Plasmodium-derived MSP2 and inhibiting parasite growth in ADCI. CONCLUSION: The data provide a valuable opportunity to evaluate in mice different adjuvant and antigen formulations of a candidate vaccine containing both MSP2 D and C fragments. The formulations with GLA-SE seem to be a promising option to be compared with the alhydrogel one in human clinical trials.

Plasmodium falciparum merozoite surface protein 2: epitope mapping and fine specificity of human antibody response against non-polymorphic domains.

BACKGROUND: Two long synthetic peptides representing the dimorphic and constant C-terminal domains of the two allelic families of Plasmodium falciparum merozoite surface proteins 2 are considered promising malaria vaccine candidates. The aim of the current study is to characterize the immune response (epitope mapping) in naturally exposed individuals and relate immune responses to the risk of clinical malaria. METHODS: To optimize their construction, the fine specificity of human serum antibodies from donors of different age, sex and living in four distinct endemic regions was determined in ELISA by using overlapping 20 mer peptides covering the two domains. Immune purified antibodies were used in Western blot and immunofluorescence assay to recognize native parasite derivate proteins. RESULTS: Immunodominant epitopes were characterized, and their distribution was similar irrespective of geographic origin, age group and gender. Acquisition of a 3D7 family and constant region-specific immune response and antibody avidity maturation occur early in life while a longer period is needed for the corresponding FC27 family response. In addition, the antibody response to individual epitopes within the 3D7 family-specific region contributes to protection from malaria infection with different statistical weight. It is also illustrated that affinity-purified antibodies against the dimorphic or constant regions recognized homologous and heterologous parasites in immunofluorescence and homologous and heterologous MSP2 and other polypeptides in Western blot. CONCLUSION: Data from this current study may contribute to a development of MSP2 vaccine candidates based on conserved and dimorphic regions thus bypassing the complexity of vaccine development related to the polymorphism of full-length MSP2.