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Messenger RNA (mRNA) vaccines against COVID-19 have demonstrated high efficacy and rapid deployment capability to target emerging infectious diseases. However, the need for ultra-low temperature storage made the distribution of LNP/mRNA vaccines to regions with limited resources impractical. This study explores the use of lyophilization to enhance the stability of self-replicating mRNA (repRNA) vaccines, allowing for their storage at non-freezing temperatures such as 2–8 °C or room temperature (25 °C). We lyophilized repRNA molecules complexed to a novel cationic emulsion delivery system, LION™, with different sugar-based lyoprotectants to identify candidates that provided the best vaccine integrity and effectiveness after being thermally stressed. For screening, we used repRNA encoding the reporter protein secreted embryonic alkaline phosphatase (SEAP) and for proof-of-concept, we used repRNA vaccines encoding SARS-CoV-2 full-length spike (WA-1 isolate) or full-length surface protein circumsporozoite (CS) of Plasmodium yoelii (Py). We found that lyophilization of LION/repRNA with sucrose provided the best colloidal stability, preserved in vitro expression, and induced equivalent antigen-specific antibody responses in mice compared to freshly prepared liquid LION/repRNA. Furthermore, lyophilized vaccines were stable for at least one week at 25 °C and at least one year at 2–8 °C. The cumulative analysis of stability-determining physicochemical data, in vitro potency, and in vivo immunogenicity in mice enabled the selection of a lead lyophilized composition containing 10 % w / v sucrose as the lyoprotectant. The data presented here provide a foundation for the clinical evaluation of next-generation thermostable repRNA vaccines that will enable more equitable vaccine access globally. [Display omitted] [ABSTRACT FROM AUTHOR]

Though the circumsporozoite protein (CSP) is the major surface protein of the Plasmodium sporozoite, and the basis for the currently used malaria vaccines, many questions regarding its function and structure remain open. CSP has a conserved domain structure among all Plasmodium species and is important for sporozoite formation, migration, and invasion, with no clear binding partners known. Proteolytic processing of CSP is essential for its function as are dynamic changes to its conformation, but only one domain has so far been crystallized and the enzymes processing CSP are unknown. The circumsporozoite protein (CSP) is one of the most studied proteins of the malaria parasite. It is the target of the only licensed malaria vaccines and is essential for sporozoite formation and infectivity. Yet, the mechanisms by which CSP functions and its interactions with other proteins are only beginning to be understood. Here we review the current state of knowledge of CSP structure and function, as sporozoites develop in the mosquito and establish infection in the mammalian host, and outline outstanding questions that need to be addressed. [ABSTRACT FROM AUTHOR]

Glycosylphosphatidylinositol (GPI) anchor protein modification in Plasmodium species is well known and represents the principal form of glycosylation in these organisms. The structure and biosynthesis of GPI anchors of Plasmodium spp. has been primarily studied in the asexual blood stage of Plasmodium falciparum and is known to contain the typical conserved GPI structure of EtN-P-Man3GlcN-PI. Here, we have investigated the circumsporozoite protein (CSP) for the presence of a GPI anchor. CSP is the major surface protein of Plasmodium sporozoites, the infective stage of the malaria parasite. While it is widely assumed that CSP is a GPI-anchored cell surface protein, compelling biochemical evidence for this supposition is absent. Here, we employed metabolic labeling and mass-spectrometry-based approaches to confirm the presence of a GPI anchor in CSP. Biosynthetic radiolabeling of CSP with [ ³H]-palmitic acid and [³H]-ethanolamine, with the former being base-labile and therefore ester-linked, provided strong evidence for the presence of a GPI anchor on CSP, but these data alone were not definitive. To provide further evidence, immunoprecipitated CSP was analyzed for the presence of myo-inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for highly sensitive and quantitative detection. The single ion monitoring (SIM) method for GC-MS analysis confirmed the presence of the myo-inositol component in CSP. Taken together, these data provide confidence that the long-assumed presence of a GPI anchor on this important parasite protein is correct. [ABSTRACT FROM AUTHOR]

Malaria caused by Plasmodium vivax is a pressing public health problem in tropical and subtropical areas. However, little progress has been made toward developing a P. vivax vaccine, with only three candidates being tested in clinical studies. We previously reported that one chimeric recombinant protein (PvCSP-All epitopes) containing the conserved C-terminus of the P. vivax Circumsporozoite Protein (PvCSP), the three variant repeat domains, and a Toll-like receptor-3 agonist, Poly(I:C), as an adjuvant (polyinosinic-polycytidylic acid, a dsRNA analog mimicking viral RNA), elicits strong antibody-mediated immune responses in mice to each of the three allelic forms of PvCSP. In the present study, a pre-clinical safety evaluation was performed to identify potential local and systemic toxic effects of the PvCSP-All epitopes combined with the Poly-ICLC (Poly I:C plus poly-L-lysine, Hiltonol®) or Poly-ICLC when subcutaneously injected into C57BL/6 mice and New Zealand White Rabbits followed by a 21-day recovery period. Overall, all observations were considered non-adverse and were consistent with the expected inflammatory response and immune stimulation following vaccine administration. High levels of vaccine-induced specific antibodies were detected both in mice and rabbits. Furthermore, mice that received the vaccine formulation were protected after the challenge with Plasmodium berghei sporozoites expressing CSP repeats from P. vivax sporozoites (Pb/Pv-VK210). In conclusion, in these non-clinical models, repeated dose administrations of the PvCSP-All epitopes vaccine adjuvanted with a Poly-ICLC were immunogenic, safe, and well tolerated. [ABSTRACT FROM AUTHOR]

Malaria affects ∼ ¼ billion people globally and requires the development of additional tools to aid in elimination efforts. The recently approved RTS,S/AS01 vaccine represents a positive step, however, the moderate efficacy necessitates the development of more efficacious vaccines. PfCSP is a key target antigen for pre-erythrocytic vaccines aimed at preventing Plasmodium falciparum malaria infections. Epitopes within the central repeat region and at the junction of the repeat and N-terminal domain are well documented as major protective B cell epitopes. On the other hand, a majority of antibodies against the epitopes in the C-terminal domain, have been shown to be non-protective against sporozoite challenge. The C-terminal domain, however, contains CD4+ and CD8+ T cell epitopes previously shown to be important for regulating immune responses. The present study was designed to further explore the immunomodulatory potential of the C-terminal domain using DNA vaccines encoding PfCSP with sequential C-terminal truncations following known T cell epitopes. Five DNA vaccines encoding different truncations of PfCSP within the C-terminal domain were administered via intramuscular route and in vivo electroporation for effective immunogenicity. Protection in mice was evaluated by challenge with transgenic P. berghei expressing PfCSP. In Balb/c mice, antibody responses and protective efficacy were both affected progressively with sequential deletion of C-terminal amino acid residues. Similar studies in C57Bl/6 mice revealed that immunizations with plasmids encoding truncated PfCSP showed partial protection from sporozoite challenge with no significant differences in antibody titers observed compared to full-length PfCSP DNA immunized mice. Further analysis revealed murine strain-specific differences in the recognition of specific epitopes. [ABSTRACT FROM AUTHOR]

Circumsporozoite protein-specific active and passive immunization can protect significantly against Plasmodium falciparum malaria and are being considered as tools to prevent placental malaria. Despite recent encouraging findings, a closer view of the underlying biology indicates significant challenges to preventing placental malaria. [ABSTRACT FROM AUTHOR]

• Prime-and-trap is a two-step heterologous vaccine strategy developed for malaria. • Prime-and-trap aims to induce CD8+ T cells against liver stage Plasmodium parasites. • Prior data show prime-and-trap is immunogenic and protective in mouse models. • Here we report immunogenicity of a prime-and-trap vaccine in non-human primates. Development of next-generation vaccines against Plasmodium falciparum (Pf) is a priority. Many malaria vaccines target the pre-erythrocytic sporozoite (SPZ) and liver stages. These include subunit vaccines based on the Pf circumsporozoite protein (CSP) and attenuated PfSPZ vaccines. However, these strategies require 3–4 doses and have not achieved optimal efficacy against field-transmitted malaria. Prime-and-trap is a recently developed two-step heterologous vaccine strategy that combines priming with DNA encoding CSP followed by a single dose of attenuated SPZ. This strategy aims to induce CD8+ T cells that can eliminate parasites in the liver. Prior data has demonstrated that prime-and-trap with P. yoelii CSP and PySPZ was immunogenic and protective in mice. Here we report preliminary data on the immunogenicity of PfCSP prime and PfSPZ trap vaccine in rhesus macaques. This vaccine induced PfCSP-specific antibodies and T cell responses in all animals. However, response magnitude differed between individuals, suggesting further study is required. [ABSTRACT FROM AUTHOR]

• 135 T cell epitopes identified in four P. falciparum antigen, with 75 % being novel. • Thirty-two percent (32%) of the 135 epitopes show promiscuity in HLA binding. • Fifty-two percent (52%) are conserved across 16 highly diverse parasite variants. • CSP and AMA1 had greater numbers of T cell epitopes compared to TRAP and CelTOS. A malaria vaccine with high efficacy and capable of inducing sterile immunity against malaria within genetically diverse populations is urgently needed to complement ongoing disease control and elimination efforts. Parasite-specific IFN-γ and granzyme B-secreting CD8 + T cells have been identified as key mediators of protection and the rapid identification of malaria antigen targets that elicit these responses will fast-track the development of simpler, cost-effective interventions. This study extends our previous work which used peripheral blood mononuclear cells (PBMCs) from adults with life-long exposure to malaria parasites to identify immunodominant antigen-specific peptide pools composed of overlapping 15mer sequences spanning full length proteins of four malarial antigens. Our current study aimed to identify CD8 + T cell epitopes within these previously identified positive peptide pools. Cryopreserved PBMCs from 109 HLA-typed subjects were stimulated with predicted 9-11mer CD8 + T cell epitopes from P. falciparum circumsporozoite protein (CSP), apical membrane antigen 1 (AMA1), thrombospondin related anonymous protein (TRAP) and cell traversal for ookinetes and sporozoites (CelTOS) in FluoroSpot assays. A total of 135 epitopes out of 297 tested peptides from the four antigens were experimentally identified as positive for IFN-γ and/or granzyme B production in 65 of the 109 subjects. Forty-three of 135 epitopes (32 %) were promiscuous for HLA binding, with 31 of these promiscuous epitopes (72 %) being presented by HLA alleles that fall within at least two different HLA supertypes. Furthermore, about 52 % of identified epitopes were conserved when the respective sequences were aligned with those from 16 highly diverse P. falciparum parasite strains. In summary, we have identified a number of conserved epitopes, immune responses to which could be effective against multiple P. falciparum parasite strains in genetically diverse populations. [ABSTRACT FROM AUTHOR]

[Display omitted] • Genetic diversity and evolution of P. falciparum circumsporozoite (Pfcsp) in Cameroon. • The central region of the Pfcsp gene was most genetically diverse. • Three and eight novel SNPs were found in N- and C-terminal regions, respectively. • The codon site 321 in the C-terminal region was under episodic positive selection. • The SNPs E357 L , K322 I , G349 D , and D359 Y) impact on function/structure. Current understanding of genetic polymorphisms and natural selection in Plasmodium falciparum circumsporozoite (Pf CSP), the leading malaria vaccine, is crucial for the development of next-generation vaccines, and such data is lacking in Africa. Blood samples were collected among Plasmodium -infected individuals living in four Cameroonian areas (Douala, Maroua, Mayo-Oulo, Pette). DNA samples were amplified using nested PCR protocols, sequenced, and BLASTed. Single nucleotide polymorphisms (SNPs) were analysed in each Pf CSP region, and their impact on Pf CSP function/structure was predicted in silico. The N-terminal region showed a limited polymorphism with four haplotypes, and three novel SNPs (N68 Y , R87 W , K93 E) were found. Thirty-five haplotypes were identified in the central region, with several variants (e.g., NV N P and K ANP). The C-terminal region was also highly diverse, with 25 haplotypes and eight novel SNPs (N290 D , N308 I , S312 G , K317 A , V344 I , D356 E , E357 L , D359 Y). Most polymorphic codon sites were mainly observed in the Th2R subregion in isolates from Douala and Pette. The codon site 321 was under episodic positive selection. One novel (E357 L) and three known (K322 I , G349 D , D359 Y) SNPs show an impact on function/structure. This study showed extensive genetic diversity with geographical patterns and evidence of the selection of Cameroonian Pf CSP central and C-terminal regions. [ABSTRACT FROM AUTHOR]

• This is the first-in-human trial of both FMP013 and the ALFQ adjuvant. • The vaccine evinced significant humoral and cell mediated immunogenicity. • The vaccine demonstrated an acceptable safety profile. • The results of this trial have led to an efficacy trial using CHMI. The global burden of malaria remains substantial. Circumsporozoite protein (CSP) has been demonstrated to be an effective target antigen, however, improvements that offer more efficacious and more durable protection are still needed. In support of research and development of next-generation malaria vaccines, Walter Reed Army Institute of Research (WRAIR) has developed a CSP-based antigen (FMP013) and a novel adjuvant ALFQ (Army Liposome Formulation containing QS-21). We present a single center, open-label, dose-escalation Phase 1 clinical trial to evaluate the safety and immunogenicity of the FMP013/ALFQ malaria vaccine candidate. In this first-in-human evaluation of both the antigen and adjuvant, we enrolled ten subjects; five received 20 μg FMP013 / 0.5 mL ALFQ (Low dose group), and five received 40 μg FMP013 / 1.0 mL ALFQ (High dose group) on study days 1, 29, and 57. Adverse events and immune responses were assessed during the study period. The clinical safety profile was acceptable and there were no serious adverse events. Both groups exhibited robust humoral and cellular immunological responses, and compared favorably with historical responses reported for RTS,S/AS01. Based on a lower reactogenicity profile, the 20 μg FMP013 / 0.5 mL ALFQ (Low dose) was selected for follow-on efficacy testing by controlled human malaria infection (CHMI) with a separate cohort. Trial Registration: Clinicaltrials.gov Identifier NCT04268420 (Registered February 13, 2020) [ABSTRACT FROM AUTHOR]

• Plasmodium CSP and AMA1 exhibited more T cell epitopes compared to TRAP and CelTOS. • The most frequent T cell responses to the four antigens were to the CSP C-terminal. • Frequency of IFN-γ responses were 6-fold higher than that of granzyme B responses. Sterile protection against clinical malaria has been achieved in animal models and experimental human challenge studies involving immunization with radiation attenuated Plasmodium falciparum sporozoite vaccines as well as by live sporozoites under chloroquine prophylaxis. Parasite-specific IFN-γ and granzyme B-secreting CD8 + T cells have been identified as key mediators of protection. Although the exact parasite targets of protective CD8 + T cell responses are not fully defined, responses against a handful of vaccine candidate antigens have been associated with protection. Identifying the T cell targets in these antigens will facilitate the development of simpler, cost-effective, and efficacious next generation multi-epitope vaccines. The aim of this study was to identify immunodominant portions of four malaria vaccine candidate antigens using peripheral blood mononuclear cells (PBMCs) from adults with life-long exposure to malaria parasites. Cryopreserved PBMCs from 291 HLA-typed subjects were stimulated with pools of overlapping 15mer peptides spanning the entire sequences of P. falciparum circumsporozoite protein (CSP, 9 pools), apical membrane antigen 1 (AMA1, 12 pools), thrombospondin related anonymous protein (TRAP, 6 pools) and cell traversal for ookinetes and sporozoites (CelTOS, 4 pools) in FluoroSpot assays. 125 of 291 subjects made IFN-γ responses to 30 of the 31 peptide pools tested and 22 of 291 made granzyme B responses, with 20 making dual responses. The most frequent responses were to the CSP C-terminal region and the least frequent responses were to TRAP and CelTOS. There was no association between FluoroSpot responses and active malaria infection, detected by either microscopy, RDT, or PCR. In conclusion, CSP and AMA1 have relatively higher numbers of epitopes that trigger IFN-γ and granzyme B-secreting T cells in adults with life-long malaria parasite exposure compared to the other two antigens tested, and highlights the continued relevance of these two antigens as vaccine candidates. [ABSTRACT FROM AUTHOR]

• rCSP/GLA-LSQ malaria vaccine was associated with only mild/moderate adverse events. • Geometric mean (GM) anti-CSP IgG rose > 4-fold 28 days after 1st dose in all groups. • rCSP/GLA-LSQ resulted in ~90-fold rise GM anti-CSP IgG 28 days after 3rd dose. • GLA-LSQ, a novel adjuvant, showed favorable safety and immunostimulatory results. Plasmodium falciparum circumsporozoite protein (CSP) is a major sporozoite surface protein and a key target of pre-erythrocytic malaria subunit vaccines. A full-length recombinant CSP (rCSP) based strategy could be advantageous, as this antigen includes a region critical to sporozoite cell attachment and hepatocyte invasion. The adjuvant Glucopyranosyl Lipid A-liposome Quillaja saponaria 21 (GLA-LSQ) functions as a TLR4 agonist, promotes antigen-specific T H 1 responses and stimulates cytotoxic T cell production. To date, one study has reported the clinical acceptability of GLA-LSQ. We present interim results of a phase 1 first-in-human dose-escalation clinical trial of full-length rCSP vaccine given with or without GLA-LSQ adjuvant. Participants experienced only mild to moderate related solicited adverse events. The lowest adjuvanted vaccine dose achieved >90-fold rise in geometric mean anti-CSP IgG antibody titer. These favorable safety and immunogenicity results confirm the immunostimulatory capacity of this relatively new adjuvant and support next steps in clinical product development. Trial registration: ClinicalTrials.gov Identifier NCT03589794 (registered 18 July 2018) [ABSTRACT FROM AUTHOR]

• Development of novel multiplex assay to test antigen variants without antigenic competition. • Malaria circumsporozoite protein variants identified in field studies for vaccine evaluation. • Breadth of antibody reactivity to vaccine variant antigens predicts protection from infection. • Data point to critical role of C-terminal Circumsporozoite antibodies in protection. • Reactivity pattern to antigenic variants reflects sequence divergence. • Structural modeling and epitope prediction identified putative non-protective epitopes. The circumsporozoite protein (CSP) is the main surface antigen of malaria sporozoites, a prime vaccine target, and is known to have polymorphisms in the C-terminal region. Vaccines using a single allele may have lower efficacy against genotypic variants. Recent studies have found evidence suggesting the efficacy of the CSP-based RTS,S malaria vaccine may be limited against P. falciparum CSP alleles that diverge from the 3D7 vaccine allele, particularly in this polymorphic C-terminal region. In order to assess the breadth of the RTS,S-induced antibody responses against CSP C-terminal antigenic variants, we used a novel multiplex assay to measure reactivity of serum samples from a recent RTS,S study against C-terminal peptides from 3D7 and seven additional CSP alleles that broadly represent the genetic diversity found in circulating P. falciparum field isolates. We found that responses to the variants showed, on average, a ~ 30-fold reduction in reactivity relative to the vaccine-matched 3D7 allele. The extent of this reduction, ranging from 21 to 69-fold, correlated with the number of polymorphisms between the variants and 3D7. We calculated antibody breadth of each sample as the median relative reactivity to the seven CSP variants compared to 3D7. Surprisingly, protection from 3D7 challenge in the RTS,S study was associated with higher C-terminal antibody breadth. These findings suggest CSP C-terminal-specific avidity or fine-specificity may play a role in RTS,S-mediated protection and that breadth of C-terminal CSP-specific antibody responses may be a marker of protection. [ABSTRACT FROM AUTHOR]

The Plasmodium falciparum circumsporozoite protein (PfCSP) is a sporozoite surface protein whose role in sporozoite motility and cell invasion has made it the leading candidate for a preerythrocytic malaria vaccine. However, production of high yields of soluble recombinant PfCSP, including its extensive NANP and NVDP repeats, has proven problematic. Here, we report on the development and characterization of a secreted, soluble, and stable full-length PfCSP (containing 4 NVDP and 38NANPrepeats) produced in the Lactococcus lactis expression system. The recombinant full-length PfCSP, denoted PfCSP4/38, was produced initially with a histidine tag and purified by a simple two-step procedure. Importantly, the recombinant PfCSP4/38 retained a conformational epitope for antibodies as confirmed by both in vivo and in vitro characterizations. We characterized this complex protein by HPLC, light scattering, MS analysis, differential scanning fluorimetry, CD, SDS-PAGE, and immunoblotting with conformation-dependent and -independent mAbs, which confirmed it to be both pure and soluble. Moreover, we found that the recombinant protein is stable at both frozen and elevated-temperature storage conditions. When we used L. lactis-derived PfCSP4/38 to immunize mice, it elicited high levels of functional antibodies that had the capacity to modify sporozoite motility in vitro. We concluded that the reported yield, purity, results of biophysical analyses, and stability of PfCSP4/38 warrant further consideration of using the L. lactis system for the production of circumsporozoite proteins for preclinical and clinical applications in malaria vaccine development. [ABSTRACT FROM AUTHOR]

• Plasmodium yoelii can repair CRISPR/Cas9-generated DNA breaks by microhomology-mediated end joining. • Size mutants of the central repeat in circumsporozoite surface protein (CSP) were successfully generated. • A minimum size of the CSP central repeat region is required for mosquito stage development. CRISPR/Cas9 is a powerful genome editing method that has greatly facilitated functional studies in many eukaryotic organisms including malaria parasites. Due to the lack of genes encoding enzymes necessary for the non-homologous end joining DNA repair pathway, genetic manipulation of malaria parasite genomes is generally accomplished through homologous recombination requiring the presence of DNA templates. Recently, an alternative double-strand break repair pathway, microhomology-mediated end joining, was found in the Plasmodium falciparum parasite. Taking advantage of the MMEJ pathway, we developed a MMEJ-based CRISPR/Cas9 (mCRISPR) strategy to efficiently generate multiple mutant parasites simultaneously in genes with repetitive sequences. As a proof of principle, we successfully produced various size mutants in the central repeat region of the Plasmodium yoelii circumsporozoite surface protein without the use of template DNA. Monitoring mixed parasite populations and individual parasites with different sizes of CSP-CRR showed that the CSP-CRR plays a role in the development of mosquito stages, with severe developmental defects in parasites with large deletions in the repeat region. However, the majority of the csp mutant parasite clones grew similarly to the wild type P. yoelii 17XL parasite in mice. This study develops a useful technique to efficiently generate mutant parasites with deletions or insertions, and shows that the CSP-CRR plays a role in parasite development in mosquito. [ABSTRACT FROM AUTHOR]

• Adult females have greater immunity than males following vaccination. • Vaccinated adult females are better protected than males following challenged. • Sex differences in immunity and protection are not present prior to puberty. • Depletion of sex steroids in female mice does not alter immunity or protection. • Manipulation of testosterone in adult males affects immunity and protection. In both preclinical animal studies and human clinical trials, adult females tend to develop greater adaptive immune responses than males following receipt of either viral or bacterial vaccines. While there is currently no approved malaria vaccine, several anti-sporozoite vaccines, including RTS,S/AS01 and attenuated sporozoite vaccines, are in development, but the impact of sex and age on their efficacy remains undefined. To examine sex differences in the efficacy of anti-sporozoite stage malaria vaccination, adult (10 weeks of age) or juvenile (11 days of age) male and female C3H mice were twice vaccinated with irradiated transgenic Plasmodium berghei sporozoites expressing the P. falciparum circumsporozoite (CSP) protein and 45 days post boost vaccination, mice were challenged with transgenic P. berghei via mosquito bite or intradermal challenge. Immunization with irradiated sporozoites resulted in greater protection against challenge in adult females, which was associated with greater anti-CSP antibody production and avidity, as well as greater hepatic, but not splenic, CD8+ T cell IFNƴ production in adult females than adult males. No sex differences in adaptive immune responses or protection were observed in mice vaccinated prior to puberty, suggesting a role for sex steroid hormones. Depletion of testosterone in males increased, whereas rescue of testosterone decreased, anti-CSP antibody production, the number of antigen-specific CD8+ T cells isolated from the liver, and protection following parasite challenge. Conversely, depletion of sex steroids in female mice did not alter vaccine-induced responses or protection following challenge. These data suggest that elevated testosterone concentrations in males reduce adaptive immunity and contribute to sex differences in malaria vaccine efficacy. [ABSTRACT FROM AUTHOR]

Antibodies to Circumsporozoite protein (CSP) confer protection against controlled human malaria infection (CHMI) caused by the parasite Plasmodium falciparum. Although CSP is highly immunogenic, it does not induce long lasting protection and efforts to improve CSP-specific immunological memory and duration of protection are underway. We have previously reported that the clinical grade CSP vaccine FMP013 was immunogenic and protective against malaria challenge in mice when combined with the Army Liposomal Formulation adjuvant containing immune modulators 3D-PHAD™ and QS21 (ALFQ). To move forward with clinical evaluation, we now report the safety, toxicity and immunogenicity of clinical grade FMP013 and ALFQ in Rhesus macaques. Three groups of Rhesus (n = 6) received half or full human dose of FMP013 + ALFQ on a 0-1-2 month schedule, which showed mild local site reactions with no hematologic derangements in red blood cell homeostasis, liver function or kidney function. Immunization induced a transient systemic inflammatory response, including elevated white blood cell counts, mild fever, and a few incidences of elevated creatine kinase, receding to normal range by day 7 post vaccination. Optimal immunogenicity in Rhesus was observed using a 1 mL ALFQ + 20 µg FMP013 dose. Doubling the FMP013 antigen dose to 40 µg had no effect while halving the ALFQ adjuvant dose to 0.5 mL lowered immunogenicity. Similar to data generated in mice, FMP013 + ALFQ induced serum antibodies that reacted to all regions of the CSP molecule and a Th1-biased cytokine response in Rhesus. Rhesus antibody response to FMP013 + ALFQ was found to be non-inferior to historical benchmarks including that of RTS,S + AS01 in humans. A four-dose GLP toxicity study in rabbits confirmed no local site reactions and transient systemic inflammation associated with ALFQ adjuvant administration. These safety and immunogenicity data support the clinical progression and testing of FMP013 + ALFQ in a CHMI trial in the near future. [ABSTRACT FROM AUTHOR]

Abstract Imported malaria due to Plasmodium ovale curtisi and P. ovale wallikeri infections from African countries has increased recently (2011–2014) in Chinese travelers. We report novel genotypes, their prevalence and the predominant haplotypes of P. ovale curtisi and P. ovale wallikeri circumsporozoite protein (CSP) from 20 African countries in Chinese travelers. These genotypes should be considered while designing a CSP-based vaccine against P. ovale malaria. Graphical abstract Unlabelled Image Highlights • This is the first to report the csp genotypes and their heterogeneity for P. ovale curtisi and P. ovale wallikeri parasites from African countries. • Ten csp genotypes of P. ovale curtisi and 13 csp genotypes of P. ovale wallikeri based on the arrangement of central repeat motifs were found. • Fifteen csp haplotypes of P. ovale curtisi and 10 csp haplotypes of P. ovale wallikeri on central repeat motifs were identified. • These genotypes should be considered while designing a CSP-based vaccine against P. ovale malaria. [ABSTRACT FROM AUTHOR]

Abstract The repetitive structure of compact virus-like particles (VLPs) provides high density displays of antigenic sequences, which trigger key parts of the immune system. The hepatitis B virus (HBV) and human papilloma virus (HPV) vaccines exploit the assembly competence of structural proteins, which are the effective immunogenic components of the prophylactic HBV and HPV vaccines, respectively. To optimize vaccine designs and to promote immune responses against protective epitopes, the "Asp-Ala-Asp-Pro" (NANP)-repeat from the Plasmodium falciparum circumsporozoite protein (CSP) was expressed within the exposed, main antigenic site of the small HBV envelope protein (HBsAgS); this differs from the RTS,S vaccine, in which CSP epitopes are fused to the N-terminus of HBsAgS. The chimeric HBsAgS proteins are assembly competent, produce VLPs, and provide a high antigenic density of the NANP repeat sequence. Chimeric VLPs with four or nine NANP-repeats (NANP4 and NANP9, respectively) were expressed in mammalian cells, the HBsAgS- and CSP-specific antigenicity of the VLPs was determined, and the immunogenicity of the VLPs assessed in relation to the induction of anti-HBsAgS and anti-CSP antibody responses. The chimeric VLPs induced high anti-CSP titres in BALB/c mice independent of the number of the NANP repeats. However, the number of NANP repeats influenced the activity of vaccine-induced antibodies measured by complement fixation to CSP, one of the proposed effector mechanisms for Plasmodium neutralization in vivo. Sera from mice immunized with VLPs containing nine NANP repeats performed better in the complement fixation assay than the group with four NANP repeats. The effect of the epitope-specific density on the antibody quality may instruct VLP platform designs to optimize immunological outcomes and vaccine efficacy. [ABSTRACT FROM AUTHOR]

IGHV3-33 -encoded antibodies are prevalent in the human humoral response against the Plasmodium falciparum circumsporozoite protein (PfCSP). Among VH3-33 antibodies, cross-reactivity between PfCSP major repeat (NANP), minor (NVDP), and junctional (NPDP) motifs is associated with high affinity and potent parasite inhibition. However, the molecular basis of antibody cross-reactivity and the relationship with efficacy remain unresolved. Here, we perform an extensive structure-function characterization of 12 VH3-33 anti-PfCSP monoclonal antibodies (mAbs) with varying degrees of cross-reactivity induced by immunization of mice expressing a human immunoglobulin gene repertoire. We identify residues in the antibody paratope that mediate cross-reactive binding and delineate four distinct epitope conformations induced by antibody binding, with one consistently associated with high protective efficacy and another that confers comparably potent inhibition of parasite liver invasion. Our data show a link between molecular features of cross-reactive VH3-33 mAb binding to PfCSP and mAb potency, relevant for the development of antibody-based interventions against malaria. [Display omitted] • X-ray crystal structures of 12 VH3-33 mAbs bound to PfCSP peptides reveal four binding modes • Specific HCDR features influence the induced epitope conformation and mAb cross-reactivity • The 1210-like binding mode mediates strong cross-reactivity and inhibitory efficacy • A unique binding conformation confers potent inhibition despite low cross-reactivity Thai et al. provide a comprehensive structure-function assessment of 12 VH3-33 mAbs with varying degrees of cross-reactivity for the PfCSP repeat motifs. High-affinity cross-reactive binding and potent inhibitory function are consistently associated with one PfCSP recognition mode, for which specific HCDR features forming the underlying molecular basis are identified. [ABSTRACT FROM AUTHOR]

Highlights • Cryptosporidium spp. CP15 and CSL peptides stimulate antibody production. • Antibodies induced by CP15-1 and CP15-3 peptides neutralise parasite entry in vitro. • CP15 plays an important role in sporozoite entry to enterocytes. • A multiple-epitope vaccine is required for complete entry blocking. Abstract Cryptosporidium spp. is a zoonotic intracellular protozoan and a significant cause of diarrhoea in humans and animals worldwide. This parasite can cause high morbidity in immunocompromised people and children in developing countries, livestock being the main reservoir. This study was aimed at performing preliminary tests on Swiss albino weaned mice (ICR) to evaluate the humoral immune response induced against peptides derived from Cryptosporidium parvum CP15 (15 kDa sporozoite surface antigen) and CSL (circumsporozoite-like antigen) proteins. Peptides were identified and characterised using bioinformatics tools and were chemically synthesised. The antibody response was determined and the neutralising effect of antibodies was measured in cell culture. Despite all peptides studied here were capable of stimulating antibody production, neutralising antibodies were detected for just two of the CP15-derived ones. Additional studies aimed at evaluating further the potential of such peptides as vaccine candidates are thus recommended. [ABSTRACT FROM AUTHOR]

Highlights • Characterization of protective natural human anti-PfCSP NANP antibodies. • Potent human germline-encoded anti-PfCSP NANP antibodies. • Identification of a non-protective B cell epitope in the PfCSP C-terminus. • Protective B cell epitopes in the N-terminal junction. Malaria is a life-threatening vector-borne disease caused by Plasmodium parasites that infect millions of people in endemic areas every year. The most advanced malaria vaccine candidate RTS,S targets the immune response against circumsporozoite protein of Plasmodium falciparum (PfCSP), the most deadly Plasmodium species in humans. PfCSP plays a fundamental role in parasite development as well as the establishment of the infection and is a molecular target of protective antibodies. However, RTS,S shows overall low efficacy and insufficient long-term protection. Therefore, a major goal in the development of an improved PfCSP-based vaccine remains the reliable and stable induction of protective and ideally sterilizing antibody titers. The molecular and functional characterization of human anti-PfCSP antibody responses paves the way for the rational design of novel immunogens for the development of an improved next-generation PfCSP malaria vaccine. [ABSTRACT FROM AUTHOR]

We have developed FMP014, a vaccine candidate against Plasmodium falciparum malaria, which is comprised of 60 identical monomer protein chains that form an icosahedral shaped self-assembling protein nanoparticle (SAPN). Each monomer contains selected P. falciparum Circumsporozoite Protein (P f CSP) CD4+ and CD8+ epitopes, universal T H epitopes, portions of the α-TSR domain, and 6 repeats of the NANP motifs of the P f CSP. Here we describe the conditions that are required for successful scale-up and cGMP manufacturing of FMP014 with a yield of ≈1.5 g of drug substance per 100 g of wet bacterial paste. When adjuvanted with an Army Liposomal Formulation (ALF) based adjuvant, the nanoparticle vaccine is highly immunogenic and prevents infection of mice by an otherwise lethal dose of transgenic P. berghei sporozoites expressing the full-length P f CSP. [ABSTRACT FROM AUTHOR]

The circumsporozoite protein (CSP) of the malaria parasite Plasmodium vivax is a major pre-erythrocyte vaccine candidate. The protein has a central repeat region that belongs to one of repeat families (VK210, VK247, and the P. vivax -like). In the present study, computer modelling was employed to select chimeric proteins, comprising the conserved regions and different arrangements of the repeat elements (VK210 and VK247), whose structure is similar to that of the native counterparts. DNA encoding the selected chimeras (named CS127 and CS712) were synthetically constructed based on E. coli codons, then cloned and expressed. Mouse monoclonal antibodies (mAbs; anti-Pv-210-CDC and −Pv-247-CDC), recognized the chimeric antigens in ELISA, indicating correct conformation and accessibility of the B-cell epitopes. ELISA using IgG from plasma samples collected from 221 Iranian patients with acute P. vivax showed that only 49.32% of the samples reacted to both CS127 and CS712 proteins. The dominant subclass for the two chimeras was IgG1 (48% of the positive responders, OD 492 = 0.777 ± 0.420 for CS127; 48.41% of the positive responders, OD 492 = 0.862 ± 0.423 for CS712, with no statistically significant difference P > 0.05; Wilcoxon signed ranks test). Binding assays showed that both chimeric proteins bound to immobilized heparan sulphate and HepG2 hepatocyte cells in a concentration-dependent manner, saturable at 80 μg/mL. Additionally, anti-CS127 and −CS712 antibodies raised in mice recognized the native protein on the surface of P. vivax sporozoite with high intensity, confirming the presence of common epitopes between the recombinant forms and the native proteins. In summary, despite structural differences at the molecular level, the expression levels of both chimeras were satisfactory, and their conformational structure retained biological function, thus supporting their potential for use in the development of vivax-based vaccine. [ABSTRACT FROM AUTHOR]

Malaria caused by Plasmodium falciparum continues to threaten millions of people living in the tropical parts of the world. A vaccine that confers sterile and life-long protection remains elusive despite more than 30 years of effort and resources invested in solving this problem. Antibodies to a malaria vaccine candidate circumsporozoite protein (CSP) can block invasion and can protect humans against malaria. We have manufactured the Falciparum Malaria Protein-013 (FMP013) vaccine based on the nearly full-length P. falciparum CSP 3D7 strain sequence. We report here immunogenicity and challenge data on FMP013 antigen in C57BL/6 mice formulated with two novel adjuvants of the Army Liposome Formulation (ALF) series and a commercially available adjuvant Montanide ISA 720 (Montanide) as a control. ALF is a liposomal adjuvant containing a synthetic monophosphoryl lipid A (3D-PHAD®). In our study, FMP013 was adjuvanted with ALF alone, ALF containing aluminum hydroxide (ALFA) or ALF containing QS-21 (ALFQ). Adjuvants ALF and ALFA induced similar antibody titers and protection against transgenic parasite challenge that were comparable to Montanide. ALFQ was superior to the other three adjuvants as it induced higher antibody titers with improved boosting after the third immunization, higher serum IgG2c titers, and enhanced protection. FMP013 + ALFQ also augmented the numbers of splenic germinal center-derived activated B-cells and antibody secreting cells compared to Montanide. Further, FMP013 + ALFQ induced antigen-specific IFN-γ ELISPOT activity, CD4 + T-cells and a T H 1-biased cytokine profile. These results demonstrate that soluble CSP can induce a potent and sterile protective immune response when formulated with the QS-21 containing adjuvant ALFQ. Comparative mouse immunogenicity data presented here were used as the progression criteria for an ongoing non-human primate study and a regulatory toxicology study in preparation for a controlled human malaria infection (CHMI) trial. [ABSTRACT FROM AUTHOR]

Mouse studies evaluating candidate malaria vaccines have typically examined protective efficacy over the relatively short time frames of several weeks after the final of multiple immunizations. The current study examines the protective ability in a mouse model system of a novel protein vaccine construct in which the adjuvant polyinosinic polycytidilic acid (poly(I:C)) is used in combination with a vaccine in which the immature dendritic cell targeting chemokine, macrophage inflammatory protein 3 alpha (MIP3α), is fused to the circumsporozoite protein (CSP) of Plasmodium falciparum ( P. falciparum ). Two vaccinations, three weeks apart, elicited extraordinarily high, MIP3α-dependent antibody responses. MIP3α was able to target the vaccine to the CCR6 receptor found predominantly on immature dendritic cells and significantly enhanced the cellular influx at the vaccination site. At three and 23 weeks after the final of two immunizations, mice were challenged by intravenous injection of 5 × 10 3 transgenic Plasmodium berghei sporozoites expressing P. falciparum CSP, a challenge dose approximately one order of magnitude greater than that which is encountered after mosquito bite in the clinical setting. A ninety-seven percent reduction in liver sporozoite load was observed at both time points, 23 weeks being the last time point tested. [ABSTRACT FROM AUTHOR]

The most effective vaccine candidate of malaria is based on the Plasmodium falciparum circumsporozoite protein (CSP), a major surface protein implicated in the structural strength, motility, and immune evasion properties of the infective sporozoites. It is suspected that reversible conformational changes of CSP are required for infection of the mammalian host, but the detailed structure and dynamic properties of CSP remain incompletely understood, limiting our understanding of its function in the infection. Here, we report the structural and mechanical properties of the CSP studied using single-molecule force spectroscopy on several constructs, one including the central region of CSP, which is rich in NANP amino acid repeats (CSPrep), and a second consisting of a near full-length sequence without the signal and anchor hydrophobic domains (CSPΔHP). Our results show that the CSPrep is heterogeneous, with 40% of molecules requiring virtually no mechanical force to unfold (<10 piconewtons (pN)), suggesting that these molecules are mechanically compliant and perhaps act as entropic springs, whereas the remaining 60% are partially structured with low mechanical resistance (~70 pN). CSPΔHP having multiple force peaks suggests specifically folded domains, with two major populations possibly indicating the open and collapsed forms. Our findings suggest that the overall low mechanical resistance of the repeat region, exposed on the outer surface of the sporozoites, combined with the flexible full-length conformations of CSP, may provide the sporozoites not only with immune evasion properties, but also with lubricating capacity required during its navigation through the mosquito and vertebrate host tissues. We anticipate that these findings would further assist in the design and development of future malarial vaccines. [ABSTRACT FROM AUTHOR]

In October 2021, the world's first malaria vaccine RTS,S was endorsed by WHO for broad use in children, despite its low efficacy. This study examined polyclonal infections and the associations of parasite genetic variations with binding affinity to human leukocyte antigen (HLA). Multiplicity of infection was determined by amplicon deep sequencing of PfMSP1. Genetic variations in PfCSP were examined across 88 samples from Ghana and analyzed together with 1655 PfCSP sequences from other African and non-African isolates. Binding interactions of PfCSP peptide variants and HLA were predicted using NetChop and HADDOCK. High polyclonality was detected among infections, with each infection harboring multiple non-3D7 PfCSP variants. Twenty-seven PfCSP haplotypes were detected in the Ghanaian samples, and they broadly represented PfCSP diversity across Africa. The number of genetic differences between 3D7 and non-3D7 PfCSP variants does not influence binding to HLA. However, CSP peptide length after proteolytic degradation significantly affects its molecular weight and binding affinity to HLA. Despite the high diversity of HLA, the majority of the HLAI and II alleles interacted/bound with all Ghana CSP peptides. Multiple non-3D7 strains among P. falciparum infections could impact the effectiveness of RTS,S. Longer peptides of the Th2R/Th3R CSP regions should be considered in future versions of RTS,S. • The world's first malaria vaccine RTS,S has low efficacy against P. falciparum. • Infections were highly polyclonal each with multiple non-3D7 PfCSP variants. • Genetic differences between 3D7 and non-3D7 PfCSP do not affect binding to HLA. • CSP peptide length significantly affects its binding affinity to the HLA. [ABSTRACT FROM AUTHOR]

Monoclonal antibody L9 recognizes the Plasmodium falciparum circumsporozoite protein (PfCSP) and is highly protective following controlled human malaria challenge. To gain insight into its function, we determined cryoelectron microscopy (cryo-EM) structures of L9 in complex with full-length PfCSP and assessed how this recognition influenced protection by wild-type and mutant L9s. Cryo-EM reconstructions at 3.6- and 3.7-Å resolution revealed L9 to recognize PfCSP as an atypical trimer. Each of the three L9s in the trimer directly recognized an Asn-Pro-Asn-Val (NPNV) tetrapeptide on PfCSP and interacted homotypically to facilitate L9-trimer assembly. We analyzed peptides containing different repeat tetrapeptides for binding to wild-type and mutant L9s to delineate epitope and homotypic components of L9 recognition; we found both components necessary for potent malaria protection. Last, we found the 27-residue stretch recognized by L9 to be highly conserved in P. falciparum isolates, suggesting the newly revealed complete L9 epitope to be an attractive vaccine target. [Display omitted] • Cryo-EM structures of L9 with PfCSP reveal atypical trimer recognition • Each L9 Fab in the atypical trimer mediates direct interactions with an NPNV repeat • Both epitope and homotypic interactions are critical to L9 recognition of PfCSP • The 27 residue L9 epitope is conserved in most P. falciparum isolates Antibody L9 targets the P. falciparum circumsporozoite protein (PfCSP) with high clinical efficacy. Tripathi et al. report cryo-EM structures of L9 in complex with PfCSP and perform structure-function studies. These reveal an atypical trimeric association, implicate homotypic interactions, and delineate a highly conserved 27-residue epitope as a promising vaccine target. [ABSTRACT FROM AUTHOR]

Regulatory T (Treg) cells have been shown in some cases to limit vaccine-specific immune responses and impact efficacy. Very little is known about the regulatory responses to the leading malaria vaccine candidate, RTS,S. The goal of this study was to begin to characterize the regulatory responses to the RTS,S vaccine. Using multi-parameter flow cytometry, we examined responses in 13 malaria naïve adult volunteers who received 2 doses of RTS,S given eight weeks apart. Five of these volunteers had previously received 3 doses of a candidate DNA-CSP vaccine, with the final dose given approximately one year prior to the first dose of the RTS,S vaccine. We found that the frequency of CD25 hi Foxp3 + Treg cells decreased following administration of RTS,S ( p = 0.0195), with no differences based on vaccine regimen. There was a concomitant decrease in CTLA-4 expression on CD25 hi Foxp3 + Treg cells ( p = 0.0093) and PD-1 levels on CD8 + T cells ( p = 0.0002). Additionally, the frequency of anergic CTLA-4 + CCR7 + T cells decreased following vaccination. An inverse correlation was observed between the frequency of Plasmodium falciparum circumsporozoite protein (PfCSP)-specific IFN-γ and PfCSP-specific IL-10, as well as an inverse correlation between IL-10 induced by Hepatitis B surface antigen, the carrier of RTS,S, and PfCSP-specific IFN-γ, suggesting that immunity against the vaccine backbone could impact vaccine immunogenicity. These results have implications for future malaria vaccine design. [ABSTRACT FROM AUTHOR]

In Iran, the prevalence of Plasmodium falciparum and Plasmodium vivax has dropped after a national malaria elimination program was launched. To estimate the likelihood of success and to measure the outcome of malaria intervention tools during elimination programs (2008–2012), the population genetic surveys of Iranian P. vivax isolates (n = 60) were carried out using the CSP genetic marker. The results were compared with a similar work that was carried out during a control phase (2000–2003) in the same study areas. Based on PCR-RFLP analysis, 49 (81.67%) of 60 studied samples were VK210 and 11 (18.33%) were VK247 with no mixed genotypes. However, 10.97% of P. vivax isolates of control phase harbored the mixed genotypes. Sequencing analysis of 50 pvcsp gene showed 14 distinct haplotypes, of which 11 and 3 were VK210 and VK247 types, respectively. However, during the control phase, 19 distinct subtypes (11 VK210 and 8 VK247) were reported. Also, 7 of 11 VK210 and the VK247F subtypes were new, and 3 out of 7 new VK210 and VK247F were isolated from the patients with Pakistani nationality. The lower nucleotide diversity per site (π = 0.02017 ± 0.00436 and π = 0.04525 ± 0.00255) and haplotype diversity (Hd = 0.513 ± 0.093 and Hd = 0.691 ± 0.128) as well as lower In/Del haplotype [Hd(i) = 0.243 and 0] and nucleotide diversity [π(i) = 0.00078 and 0] were recorded for VK210 and VK247of the elimination samples, respectively. In conclusion, the comparison of PRMs and RATs in CRR along with the polymorphism analysis of the sequence lengths, SNPs, and In/Del polymorphisms in all analyzed samples showed lower genetic diversity for PvCSP in the elimination samples. Also, although there is a turnover of P. vivax parasite genotypes in the study areas, reduction in genetic diversity and transmission was detected due to scaling-up of the intervention tools during an elimination program in Iran. This notable challenge of the elimination program must be taken into account and controlled by active surveillance for limiting both reintroductions of new allelic forms as well as the spread of drug-resistant parasite to prevent any disease outbreaks. [ABSTRACT FROM AUTHOR]

Despite reductions in malaria episodes and deaths over the past decade, there is still significant need for more effective tools to combat this serious global disease. The positive results with the Phase III trial of RTS,S directed to the circumsporozoite protein of Plasmodium falciparum have established that a vaccine against malaria can provide partial protection to children in endemic areas, but its limited efficacy and relatively short window of protection mandate that new generations of more efficacious vaccines must be sought. Evidence shows that anti-parasite immune responses can control infection against other stages as well, but translating these experimental findings into vaccines for blood stages has been disappointing and clinical efforts to test a transmission blocking vaccine are just beginning. Difficulties include the biological complexity of the organism with a large array of stage-specific genes many of which in the erythrocytic stages are antigenically diverse. In addition, it appears necessary to elicit high and long-lasting antibody titers, address the redundant pathways of merozoite invasion, and still seek surrogate markers of protective immunity. Most vaccine studies have focused on a single or a few antigens with an apparent functional role, but this is likely to be too restrictive, and broad, multi-antigen, multi-stage vaccines need further investigation. Finally, novel tools and biological insights involving parasite sexual stages and the mosquito vector will provide new avenues for reducing or blocking malaria transmission. [ABSTRACT FROM AUTHOR]

Whole sporozoite vaccine (WSV) approach has been shown to induce efficient CD8 + T cell response, critical for developing of long-lasting sterile protection against Plasmodium . Although WSV was initiated over four decades ago, we still do not fully understand about the absolute requirements for the generation of liver-stage specific CD8 + T memory cells. For more than a decade intravenous (IV) route of immunization has been shown to be protective in pre-clinical studies. However, the intradermal (ID) route is preferred over IV route by many researchers as it is perceived to mimic the natural route of parasite delivery through mosquito bite. Various clinical studies have shown that ID route provokes poor protective responses compared to those seen with IV route of administration. The present study highlights the importance of circumsporozoite (CS) protein in preventing sporozoite entry to the hepatocytes, which however, it is not necessarily sufficient to ensure sterile protection. Instead, this article favors the idea that liver-stage development is a prime requirement for generation of antigen specific CD8 + T cells and suggests the conditions favored by IV inoculation of sporozoite. [ABSTRACT FROM AUTHOR]

The Malaria Vaccine Technology Roadmap calls for a 2015 landmark goal of a first-generation malaria vaccine that has protective efficacy against severe disease and death, lasting longer than one year. This review focuses on product development efforts over the last five years of RTS,S, a pre-erythrocytic, recombinant subunit, adjuvanted, candidate malaria vaccine designed with this goal of a first-generation malaria vaccine in mind. RTS,S recently completed a successful pivotal Phase III safety, efficacy and immunogenicity study. Although vaccine efficacy was found to be modest, a substantial number of cases of clinical malaria were averted over a 3–4 years period, particularly in settings of significant disease burden. European regulators have subsequently adopted a positive opinion under the Article 58 procedure for an indication of active immunization of children aged 6 weeks up to 17 months against malaria caused by Plasmodium falciparum and against hepatitis B. Further evaluations of the benefit, risk, feasibility and cost-effectiveness of RTS,S are now anticipated through policy and financing reviews at the global and national levels. [ABSTRACT FROM AUTHOR]

Prospects for malaria eradication will be greatly enhanced by an effective vaccine, but parasite genetic diversity poses a major impediment to malaria vaccine efficacy. In recent pre-clinical and field trials, vaccines based on polymorphic Plasmodium falciparum antigens have shown efficacy only against homologous strains, raising the specter of allele-specific immunity such as that which plagues vaccines against influenza and HIV. The most advanced malaria vaccine, RTS,S, targets relatively conserved epitopes on the P. falciparum circumsporozoite protein. After more than 40 years of development and testing, RTS,S, has shown significant but modest efficacy against clinical malaria in phase 2 and 3 trials. Ongoing phase 2 studies of an irradiated sporozoite vaccine will ascertain whether the full protection against homologous experimental malaria challenge conferred by high doses of a whole organism vaccine can provide protection against diverse strains in the field. Here we review and evaluate approaches being taken to design broadly cross-protective malaria vaccines. [ABSTRACT FROM AUTHOR]

Background: The RTS,S/AS01 malaria vaccine targets the circumsporozoite protein, inducing antibodies associated with the prevention of Plasmodium falciparum infection. We assessed the association between anti-circumsporozoite antibody titres and the magnitude and duration of vaccine efficacy using data from a phase 3 trial done between 2009 and 2014.Methods: Using data from 8922 African children aged 5-17 months and 6537 African infants aged 6-12 weeks at first vaccination, we analysed the determinants of immunogenicity after RTS,S/AS01 vaccination with or without a booster dose. We assessed the association between the incidence of clinical malaria and anti-circumsporozoite antibody titres using a model of anti-circumsporozoite antibody dynamics and the natural acquisition of protective immunity over time.Findings: RTS,S/AS01-induced anti-circumsporozoite antibody titres were greater in children aged 5-17 months than in those aged 6-12 weeks. Pre-vaccination anti-circumsporozoite titres were associated with lower immunogenicity in children aged 6-12 weeks and higher immunogenicity in those aged 5-17 months. The immunogenicity of the booster dose was strongly associated with immunogenicity after primary vaccination. Anti-circumsporozoite titres wane according to a biphasic exponential distribution. In participants aged 5-17 months, the half-life of the short-lived component of the antibody response was 45 days (95% credible interval 42-48) and that of the long-lived component was 591 days (557-632). After primary vaccination 12% (11-13) of the response was estimated to be long-lived, rising to 30% (28-32%) after a booster dose. An anti-circumsporozoite antibody titre of 121 EU/mL (98-153) was estimated to prevent 50% of infections. Waning anti-circumsporozoite antibody titres predict the duration of efficacy against clinical malaria across different age categories and transmission intensities, and efficacy wanes more rapidly at higher transmission intensity.Interpretation: Anti-circumsporozoite antibody titres are a surrogate of protection for the magnitude and duration of RTS,S/AS01 efficacy, with or without a booster dose, providing a valuable surrogate of effectiveness for new RTS,S formulations in the age groups considered.Funding: UK Medical Research Council. [ABSTRACT FROM AUTHOR]

In this study, we developed human immune system (HIS) mice that possess functional human CD4 + T cells and B cells, named HIS-CD4/B mice. HIS-CD4/B mice were generated by first introducing HLA class II genes, including DR1 and DR4, along with genes encoding various human cytokines and human B cell activation factor (BAFF) to NSG mice by adeno-associated virus serotype 9 (AAV9) vectors, followed by engrafting human hematopoietic stem cells (HSCs). HIS-CD4/B mice, in which the reconstitution of human CD4 + T and B cells resembles to that of humans, produced a significant level of human IgG against Plasmodium falciparum circumsporozoite (PfCS) protein upon immunization. CD4 + T cells in HIS-CD4/B mice, which possess central and effector memory phenotypes like those in humans, are functional, since PfCS protein-specific human CD4 + T cells secreting IFN-γ and IL-2 were detected in immunized HIS-CD4/B mice. Lastly, PfCS protein-immunized HIS-CD4/B mice were protected from in vivo challenge with transgenic P. berghei sporozoites expressing the PfCS protein. The immune sera collected from protected HIS-CD4/B mice reacted against transgenic P. berghei sporozoites expressing the PfCS protein and also inhibited the parasite invasion into hepatocytes in vitro. Taken together, these studies show that our HIS-CD4/B mice could mount protective human anti-malaria immunity, consisting of human IgG and human CD4 + T cell responses both specific for a human malaria antigen. [ABSTRACT FROM AUTHOR]

Efforts to develop a successful malaria vaccine are hampered due to lack of assays that are predictive of protective immunity without conducting large clinical studies. The effect of experimental vaccines and drugs on malaria transmission is yet more difficult to measure. Knowledge on the Plasmodium infection rate in mosquito populations will aid the measurement of effects from intervention measures for malaria control. Here, we report the development of a chemiluminescent sandwich ELISA (ECL-ELISA) that can detect Plasmodium falciparum circumsporozoite protein ( Pf CSP) produced in recombinant form at concentrations of 4.4 pg and in P. falciparum sporozoites ( Pf SPZ) derived from mosquito salivary glands at levels corresponding to 5 Pf SPZ. Most importantly, we demonstrate reliable Pf CSP-based detection of 0.056 day 8 P. falciparum oocysts developing inside mosquito midguts in whole mosquito lysates. Cumulatively, the ECL-ELISA is 47 × more sensitive for the detection of Pf CSP than a colorimetric ELISA while greatly simplifying sample preparation, obviating the need for cumbersome midgut dissections and allowing high throughput screening of Plasmodium infection in mosquito populations. The ECL-ELISA may also have broader application in diagnosis of infectious diseases and the prognostic value in cancer and other diseases such as auto-immunity and genetic disorders based on antigen detection, or quality validation of biological vaccine components. [ABSTRACT FROM AUTHOR]

Highlights: [•] We developed a construct containing the N-terminal and the two repeat variants of the PvCS protein. [•] The polypeptide was recognized by sera from endemic areas of Colombia and PNG. [•] The polypeptide formulated in Montanide adjuvant elicited strong antibody responses in mice. [•] Antibodies from immunized mice and affinity-purified human IgG reacted with the native protein in sporozoites. [•] Specific IgG induced strong in vitro inhibition of sporozoite invasion of hepatoma cells. [Copyright &y& Elsevier]

Highlights: [•] Plasmodium vivax chimeric new vaccines based on the circumsporozoite protein (CSP). [•] CSP chimeras (a VLP and a multi-epitope string) for optimized immune presentation. [•] Vaccines induce potent and long lasting humoral immune responses. [•] One of the highest avidity antibody index obtained with a P. vivax vaccine. [•] Induce long lasting CSP-specific memory T-cells able to secrete IFN-γ and IL-2. [ABSTRACT FROM AUTHOR]

Intranasal (IN) immunization with a Plasmodium circumsporozoite (CS) protein conjugated to flagellin, a Toll-like receptor 5 agonist, was found to elicit antibody-mediated protective immunity in our previous murine studies. To better understand IN-elicited immune responses, we examined the nasopharynx-associated lymphoid tissue (NALT) in immunized mice and the interaction of flagellin-modified CS with murine dendritic cells (DCs) in vitro. NALT of immunized mice contained a predominance of germinal center (GC) B cells and increased numbers of CD11c+ DCs localized beneath the epithelium and within the GC T-cell area. We detected microfold cells distributed throughout the NALT epithelial cell layer and DC dendrites extending into the nasal cavity, which could potentially function in luminal CS antigen uptake. Flagellin-modified CS taken up by DCs in vitro was initially localized within intracellular vesicles followed by a cytosolic distribution. Vaccine modifications to enhance delivery to the NALT and specifically target NALT antigen-presenting cell populations will advance development of an efficacious needle-free vaccine for the 40% of the world's population at risk of malaria. [ABSTRACT FROM AUTHOR]

Highlights: [•] Immunogenicity of Plasmodium vivax circumsporozoite protein-based vaccine candidates was evaluated in rhesus macaques. [•] The study evaluated a soluble versus a particulate form of the vaccine antigen candidate. [•] The particulate form tended to afford enhanced humoral immunogenicity over the soluble antigen. [•] The particulate form induced antibodies to the AGDR epitope. [•] T-cell responses were induced by both formulations, with some quantitative and qualitative differences. [ABSTRACT FROM AUTHOR]

Highlights: [•] We developed a chimeric long synthetic peptide derived from the P. vivax CS protein. [•] PvCS-NRC was recognized at high prevalence by naturally induced human antibodies. [•] Significant immunogenicity was observed in mice by different vaccine formulations. [•] Antibodies to PvCS-NRC showed inhibition of sporozoite invasion into hepatocytes. [ABSTRACT FROM AUTHOR]

Abstract: Highly sensitive and reliable assays based on the quantitation of immunologically relevant component(s) in recombinant or whole parasite-based vaccines would facilitate pre-clinical and clinical phases and the monitoring of malaria vaccine deployment. Here we report a laboratory-grade Western Blot assay for quantitative detection of Plasmodium falciparum circumsporozoite protein (PfCSP) in P. falciparum sporozoite (PfSPZ) and in recombinant (rPfCSP) product. This assay is based on the immuno-reactivity of an anti-P. falciparum CSP monoclonal antibody (mAb 2A10) with the NANP-repeat units on PfCSP. The antigen–antibody complex is detected by reaction with a commercially obtained chemiluminescence-linked Immunodetection system. The linear range for detecting the recombinant P. falciparum CSP (rPfCSP) in this assay is 3–12pg (R2 =0.9399). The range for detecting the day 15 salivary-gland PfSPZ is between 0.0625 and 1 parasite (R2 =0.9448) and approximately 10.0pg of PfCSP was detected on each sporozoite. The assay was highly reproducible in measuring the PfCSP on PfSPZ. The inter-assay Coefficient of Variation (CV%) was 10.31% while the intra-assay CV% on three different days was 6.05%, 2.03% and 1.42% respectively. These results suggest that this ECL-WB assay is highly sensitive and robust with a low degree of inter-assay and intra-assay variations. To our knowledge, this is the most sensitive immunoassay for the detection of a recombinant or native malarial protein and may have a wider range of applications including the quantification of immunological component(s) in a vaccine formulation, determination of the antigenic integrity in adjuvanted-vaccine and in stability studies. In addition, this assay can be applied to measure the mosquito infectivity in malaria transmission areas and to determine the effects of intervention measures on malaria transmission. [Copyright &y& Elsevier]

Abstract: Molecular methods elucidate evolutionary and ecological processes in parasites, where interaction between hosts and parasites enlighten the evolution of parasite lifestyles and host defenses. Population genetics of Plasmodium vivax parasites accurately describe transmission dynamics of the parasites and evaluation of malaria control measures. As a first generation vaccine candidate against malaria, the Circumsporozoite Protein (CSP) has demonstrated significant potential in P. falciparum. Extensive polymorphism hinders the development of a potent malaria vaccine. Hence, the genetic diversity of Pvcsp was investigated for the first time in 60 Sri Lankan clinical isolates by obtaining the nucleotide sequence of the central repeat (CR) domain and examining the polymorphism of the peptide repeat motifs (PRMs), the genetic diversity indices and phylogenetic relationships. PCR amplicons determined size polymorphism of 610, 700 and 710bp in Pvcsp of Sri Lanka where all amino acid sequences obtained were of the VK210 variant, consisting variable repeats of 4 different PRMs. The two most abundant PRMs of the CR domain, GDRADGQPA and GDRAAGQPA consisted ~2-4 repeats, while GNRAAGQPA was unique to the island. Though, different nucleotide sequences termed repeat allotypes (RATs) were observed for each PRM, these were synonymous contributing to a less polymorphic CR domain. The genetic diversity of Pvcsp in Sri Lanka was due to the number of repetitive peptide repeat motifs, point mutations, and intragenic recombination. The 19 amino acid haplotypes defined were exclusive to Sri Lanka, whereas the 194 Pvcsp sequences of global isolates generated 57 more distinct a.a. haplotypes of the VK210 variant. Strikingly, the CR domain of both VK210 and VK247 variants was under purifying selection interpreting the scarcity of CSP non-synonymous polymorphisms. Insights to the distribution of RATs in the CR region with geographic clustering of the P. vivax VK210 variant were revealed. The cladogram reiterated this unique geographic clustering of local (VK210) and global isolates (VK210 and VK247), which was further validated by the elevated fixation index values of the VK210 variant. [Copyright &y& Elsevier]

Abstract: Epitopes of the circumsporozoite (CS) protein of Plasmodium falciparum, the most pathogenic species of the malaria parasite, have been shown to elicit protective immunity in experimental animals and human volunteers. The mechanisms of immunity include parasite-neutralizing antibodies that can inhibit parasite motility in the skin at the site of infection and in the bloodstream during transit to the hepatocyte host cell and also block interaction with host cell receptors on hepatocytes. In addition, specific CD4+ and CD8+ cellular mechanisms target the intracellular hepatic forms, thus preventing release of erythrocytic stage parasites from the infected hepatocyte and the ensuing blood stage cycle responsible for clinical disease. An innovative method for producing particle vaccines, layer-by-layer (LbL) fabrication of polypeptide films on solid CaCO3 cores, was used to produce synthetic malaria vaccines containing a tri-epitope CS peptide T1BT* comprising the antibody epitope of the CS repeat region (B) and two T-cell epitopes, the highly conserved T1 epitope and the universal epitope T*. Mice immunized with microparticles loaded with T1BT* peptide developed parasite-neutralizing antibodies and malaria-specific T-cell responses including cytotoxic effector T-cells. Protection from liver stage infection following challenge with live sporozoites from infected mosquitoes correlated with neutralizing antibody levels. Although some immunized mice with low or undetectable neutralizing antibodies were also protected, depletion of T-cells prior to challenge resulted in the majority of mice remaining resistant to challenge. In addition, mice immunized with microparticles bearing only T-cell epitopes were not protected, demonstrating that cellular immunity alone was not sufficient for protective immunity. Although the microparticles without adjuvant were immunogenic and protective, a simple modification with the lipopeptide TLR2 agonist Pam3Cys increased the potency and efficacy of the LbL vaccine candidate. This study demonstrates the potential of LbL particles as promising malaria vaccine candidates using the T1BT* epitopes from the P. falciparum CS protein. [Copyright &y& Elsevier]

Abstract: Circumsporozoite protein enzyme-linked immunosorbent assays (CSP–ELISAs) are widely used for malaria vector identification throughout the world. However, several studies have reported false-positive results when using this method. The present study was conducted to estimate the frequency of false positives among anopheline species in malaria endemic areas of Bangladesh. In total, 4724 Anopheles females belonging to 25 species were collected and tested for Plasmodium falciparum, Plasmodium vivax-210, and P. vivax-247 CSP. Initially, 144 samples tested positive using routine CSP–ELISA, but the number of positive results declined to 85 (59%) when the samples were tested after heating at 100°C for 10min to remove false-positive specimens. Ten species, Anopheles annularis, Anopheles baimaii, Anopheles barbirostris, Anopheles jeyporiensis, Anopheles karwari, Anopheles kochi, Anopheles minimus s.l., Anopheles peditaeniatus, Anopheles philippinensis, and Anopheles vagus were CSP-positive. The highest and lowest infection rates were found in An. baimaii (4/25, 16.0%) and An. jeyporiensis (1/139, 0.67%), respectively. A significant correlation was found (regression analysis, R 2 =0.49, F =8.25, P <0.05) between human blood index results and the true CSP-positive ratios in 15 Anopheles species. We confirmed that false-positive reactions occurred more frequently in zoophilic species. The relatively high proportion of false positives (40%) that was found in this study should warn malaria epidemiologists working in the field to be cautious when interpreting ELISA results. [Copyright &y& Elsevier]

Abstract: Plasmodium, the causative agent of malaria, is an obligate, intracellular, eukaryotic cell that invades, replicates, and differentiates within hepatocytes and erythrocytes. Inside a host cell, a second membrane delineates the developing pathogen in addition to the parasite plasma membrane, resulting in a distinct cellular compartment, termed parasitophorous vacuole (PV). The PV membrane (PVM) constitutes the parasite–host cell interface and is likely central to nutrient acquisition, host cell remodeling, waste disposal, environmental sensing, and protection from innate defense. Over the past two decades, a number of parasite-encoded PVM proteins have been identified. They include multigene families and protein complexes, such as early-transcribed membrane proteins (ETRAMPs) and the Plasmodium translocon for exported proteins (PTEX). Nearly all Plasmodium PVM proteins are restricted to this genus and display transient and stage-specific expression. Here, we provide an overview of the PVM proteins of Plasmodium blood and liver stages. Biochemical and experimental genetics data suggest that some PVM proteins are ideal targets for novel anti-malarial intervention strategies. [Copyright &y& Elsevier]