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Plasmodium falciparum (Pf) malaria continues to cause considerable morbidity and mortality worldwide. The circumsporozoite protein (CSP) is a particularly attractive candidate for designing vaccines that target sporozoites—the first vertebrate stage in a malaria infection. Current PfCSP‐based vaccines, however, do not include epitopes that have recently been shown to be the target of potent neutralizing antibodies. We report the design of a SpyCatcher‐mi3‐nanoparticle‐based vaccine presenting multiple copies of a chimeric PfCSP (cPfCSP) antigen that incorporates these important "T1/junctional" epitopes as well as a reduced number of (NANP)n repeats. cPfCSP‐SpyCatcher‐mi3 was immunogenic in mice eliciting high and durable IgG antibody levels as well as a balanced antibody response against the T1/junctional region and the (NANP)n repeats. Notably, the antibody concentration elicited by immunization was significantly greater than the reported protective threshold defined in a murine challenge model. Refocusing the immune response toward functionally relevant subdominant epitopes to induce a more balanced and durable immune response may enable the design of a more effective second generation PfCSP‐based vaccine. [ABSTRACT FROM AUTHOR]

Abstract Background Plasmodium spp. sporozoite rates in mosquitoes are used to better understand malaria transmission intensity, the relative importance of vector species and the impact of interventions. These rates are typically estimated using an enzyme-linked immunosorbent assay (ELISA) utilizing antibodies against the circumsporozoite protein of Plasmodium falciparum, Plasmodium vivax VK210 (P. vivax210) or P. vivax VK247 (P. vivax247), employing assays that were developed over three decades ago. The ELISA method requires a separate assay plate for each analyte tested and can be time consuming as well as requiring sample volumes not always available. The bead-based multiplex platform allows simultaneous measurement of multiple analytes and may improve the lower limit of detection for sporozoites. Methods Recombinant positive controls for P. falciparum, P. vivax210 and P. vivax247 and previously developed circumsporozoite (cs) ELISA antibodies were used to optimize conditions for the circumsporozoite multiplex bead assay (csMBA) and to determine the detection range of the csMBA. After optimizing assay conditions, known amounts of sporozoites were used to determine the lower limit of detection for the csELISA and csMBA and alternate cut-off measures were applied to demonstrate how cut-off criteria can impact lower limits of detection. Sporozoite rates from 1275 mosquitoes collected in Madagascar and 255 mosquitoes collected in Guinea were estimated and compared using the established csELISA and newly optimized csMBA. All mosquitoes were tested (initial test), and those that were positive were retested (retest). When sufficient sample volume remained, an aliquot of homogenate was boiled and retested (boiled retest), to denature any heat-unstable cross-reactive proteins. Results Following optimization of the csMBA, the lower limit of detection was 25 sporozoites per mosquito equivalent for P. falciparum, P. vivax210 and P. vivax247 whereas the lower limits of detection for csELISA were found to be 1400 sporozoites for P. falciparum, 425 for P. vivax210 and 1650 for P. vivax247. Combined sporozoite rates after re-testing of samples that initially tested positive for Madagascar mosquitoes by csELISA and csMBA were 1.4 and 10.3%, respectively, and for Guinea mosquitoes 2% by both assays. Boiling of samples followed by csMBA resulted in a decrease in the Madagascar sporozoite rate to 2.8–4.4% while the Guinea csMBA sporozoite rate remained at 2.0%. Using an alternative csMBA cut-off value of median fluorescence intensity (MFI) of 100 yielded a sporozoite rate after confirmational testing of 3.7% for Madagascar samples and 2.0% for Guinea samples. Whether using csMBA or csELISA, the following steps may help minimize false positives: specimens are appropriately stored and bisected anterior to the thorax-abdomen junction, aliquots of homogenate are boiled and retested following initial testing, and an appropriate cut-off value is determined. Conclusions The csMBA is a cost-comparable and time saving alternative to the csELISA and may help eliminate false negatives due to a lower limit of detection, thus increasing sensitivity over the csELISA. The csMBA expands the potential analyses that can be done with a small volume of sample by allowing multiplex testing where analytes in addition to P. falciparum, P. vivax210 and P. vivax247 can be added following optimization.

Navneet Arora, Lokhesh C Anbalagan, Ashok K Pannu Postgraduate Institute of Medical Education and Research, Chandigarh, IndiaCorrespondence: Ashok K PannuDepartment of Internal Medicine, Postgraduate Institute of Medical Education and Research, 4th Floor, F Block, Chandigarh, 160012, IndiaTel +919914291115Email gawaribacchi@gmail.comBackground: Recent advances in mosquito eradication and antimalarial treatments have reduced the malaria burden only modestly. An effective malaria vaccine remains a high priority, but its development has several challenges. Among many potential candidates, the RTS,S/AS01 vaccine (MosquirixTM) remains the leading candidate.Objective and Method: This review aims to understand the advances in the RTS,S/AS01 vaccine, and future comments regarding the vaccine’s effectiveness in malaria eradication. Literature review for the past five decades was performed searching PubMed, EMBASE Ovid, and Cochrane Library, with using the following search items: (“malaria” OR “WHO’s malaria” OR “Plasmodium falciparum” OR “RTS,S” OR “RTS,S/AS01” OR “RTS,S/AS02” OR “pre-erythrocytic malaria” OR “circumsporozoite” OR “Mosquirix”) AND (“vaccine” OR “vaccination”).Results: RTS,S/AS01, a recombinant pre-erythrocytic vaccine containing Plasmodium falciparum surface-protein (circumsporozoite) antigen, is safe, well-tolerated, and immunogenic in children. Three doses, along with a booster, have a modest efficacy of about 36% in children (age 5– 17 months) and about 26% in infants (age 6– 12 weeks) against clinical malaria during a 48-month follow-up. However, the efficacy varies among population subgroups and with the parasite strain, it reduces without a booster and offers protection for a limited duration. Because of its potential cost-effectiveness and positive public health effect, the vaccine is being investigated in a pilot program for mortality benefits and broader deployment.Conclusion: The RTS,S/AS01 vaccine prevents malaria; however, it should be considered another addition to the malaria-control program and not as an eradication tool because of its relatively low to modest efficacy.Keywords: malaria, vaccine, pre-erythrocytic, RTS‚S, RTS‚S/AS01, WHO, circumsporozoite

Background: Plasmodium spp. sporozoite rates in mosquitoes are used to better understand malaria transmission intensity, the relative importance of vector species and the impact of interventions. These rates are typically estimated using an enzyme-linked immunosorbent assay (ELISA) utilizing antibodies against the circumsporozoite protein of Plasmodium falciparum, Plasmodium vivax VK210 (P. vivax210) or P. vivax VK247 (P. vivax247), employing assays that were developed over three decades ago. The ELISA method requires a separate assay plate for each analyte tested and can be time consuming as well as requiring sample volumes not always available. The bead-based multiplex platform allows simultaneous measurement of multiple analytes and may improve the lower limit of detection for sporozoites. Methods: Recombinant positive controls for P. falciparum, P. vivax210 and P. vivax247 and previously developed circumsporozoite (cs) ELISA antibodies were used to optimize conditions for the circumsporozoite multiplex bead assay (csMBA) and to determine the detection range of the csMBA. After optimizing assay conditions, known amounts of sporozoites were used to determine the lower limit of detection for the csELISA and csMBA and alternate cut-off measures were applied to demonstrate how cut-off criteria can impact lower limits of detection. Sporozoite rates from 1275 mosquitoes collected in Madagascar and 255 mosquitoes collected in Guinea were estimated and compared using the established csELISA and newly optimized csMBA. All mosquitoes were tested (initial test), and those that were positive were retested (retest). When sufficient sample volume remained, an aliquot of homogenate was boiled and retested (boiled retest), to denature any heat-unstable cross-reactive proteins. Results: Following optimization of the csMBA, the lower limit of detection was 25 sporozoites per mosquito equivalent for P. falciparum, P. vivax210 and P. vivax247 whereas the lower limits of detection for csELISA were found to be 1400 sporozoites for P. falciparum, 425 for P. vivax210 and 1650 for P. vivax247. Combined sporozoite rates after re-testing of samples that initially tested positive for Madagascar mosquitoes by csELISA and csMBA were 1.4 and 10.3%, respectively, and for Guinea mosquitoes 2% by both assays. Boiling of samples followed by csMBA resulted in a decrease in the Madagascar sporozoite rate to 2.8–4.4% while the Guinea csMBA sporozoite rate remained at 2.0%. Using an alternative csMBA cut-off value of median fluorescence intensity (MFI) of 100 yielded a sporozoite rate after confirmational testing of 3.7% for Madagascar samples and 2.0% for Guinea samples. Whether using csMBA or csELISA, the following steps may help minimize false positives: specimens are appropriately stored and bisected anterior to the thorax-abdomen junction, aliquots of homogenate are boiled and retested following initial testing, and an appropriate cut-off value is determined. Conclusions: The csMBA is a cost-comparable and time saving alternative to the csELISA and may help eliminate false negatives due to a lower limit of detection, thus increasing sensitivity over the csELISA. The csMBA expands the potential analyses that can be done with a small volume of sample by allowing multiplex testing where analytes in addition to P. falciparum, P. vivax210 and P. vivax247 can be added following optimization. [ABSTRACT FROM AUTHOR]

Abstract: Falciparum malaria vaccine candidates have been developed using recombinant, replication-deficient serotype 5 and 35 adenoviruses (Ad5, Ad35) encoding the Plasmodium falciparum circumsporozoite surface protein (CSP) (Ad5.CS, Ad35.CS) (Crucell Holland BV, Leiden, The Netherlands). To evaluate the immunogenicity of these constructs, BALB/cJ mice were immunized twice with either Ad5.CS, Ad35.CS, empty Ad5-vector (eAd5), empty Ad35 vector (eAd35), or saline. Another group received the CSP-based RTS,S malaria vaccine formulated in the proprietary Adjuvant System AS01B (GlaxoSmithKline Biologicals, Rixensart, Belgium). Here we report that Ad5.CS, Ad35.CS, and RTS,S/AS01B, elicited both cellular and serologic CSP antigen-specific responses in mice. These adenoviral vectors induce strong malaria-specific immunity and warrant further evaluation. [Copyright &y& Elsevier]

To date, there is no information on the genetic diversity of the circumsporozoite protein (CSP), a leading vaccine candidate, in Plasmodium vivax populations circulating in Iran. The gene for this protein, Pvcsp, was amplified from 374 P. vivax isolates collected in the temperate northern, and in the tropical southern endemic areas. PCR–RFLP analysis of the repeated central region revealed that the parasites collected in the northern area were almost exclusively of the VK210 type. Parasites collected in the south-eastern areas were of both VK210 and VK247 types. We detected VK210 parasite in 70.5% of the samples, VK247 parasites in 17.5% and mixed type infections in 12% of the isolates. Sequence analysis of 137 isolates obtained from both areas identified a total of 25 distinct genotypes. The degree of genetic diversity was generally higher for the tropical (21 genotypes) than the temperate (7 genotypes) P. vivax populations, a difference possibly reflecting the high cross-border exchanges between Afghanistan and Pakistan and southern Iran. Interestingly, all but two VK210 type isolates sequenced harboured a 36-bp post-repeat insert previously only observed in North Korea and China. This large-scale survey of parasite diversity in the Eastern Mediterranean Region provides a set of baseline data suitable for future molecular epidemiological studies of P. vivax. [ABSTRACT FROM AUTHOR]

Vaccination of malaria-naive humans with recombinant RTS,S/AS02, which includes the C-terminus of the circumsporozoite protein (CS), has been shown to induce strong T cell responses to both the whole protein antigen and to peptides from CS. Here we show that strong T cell responses were also observed in a semi-immune population in The Gambia, West Africa. In a Phase I study, 20 adult male volunteers, lifelong residents in a malaria-endemic region, were given three doses of RTS,S/AS02 at 0, 1 and 6 months. Responses to RTS,S, hepatitis B surface antigen and peptides from CS were tested using lymphocyte proliferation, interferon (IFN)- γ production in microcultures, and IFN- γ ex vivo and cultured ELISPOT, before and after vaccination. Cytotoxic responses were tested only after vaccination and none were detected. Before vaccination, the majority of the volunteers (15/20) had detectable responses in at least one of the tests. After vaccination, responses increased in all assays except cytotoxicity. The increase was most marked for proliferation; all donors responded to RTS,S after the third dose and all except one donor responded to at least one peptide after the second or third dose. There was a lack of close association of peptide responses detected by the different assays, although in microcultures IFN- γ responses were found only when proliferative responses were high, and responses by cultured ELISPOT and proliferation were found together more frequently after vaccination. We have therefore identified several peptide-specific T cell responses induced by RTS,S/AS02 which provides a mechanism to investigate potentially protective immune responses in the field. [ABSTRACT FROM AUTHOR]

We have investigated the genetic diversity of the gene encoding the CS protein. A total of 75 complete and 96 partial sequences are studied. We find high levels of genetic polymorphisms as evidenced by 50 and 24 alleles at the Th2R and Th3R epitopes, respectively. Overall, we find that African isolates are more polymorphic as compared with parasites from other geographic regions. We conclude that the uneven geographic polymorphism may have an adverse impact on the effectiveness of vaccines based on this antigen alone. We find extensive polymorphism in the repeat allotypes, or RATs. In order to explore how the protein structure may impose restrictions in the number of repeats, we have simulated the stability of the structure of the tandem repeat region. Our analysis suggests that the protein structure may play an important role in the observed polymorphism in the number of CS repeats in Plasmodium falciparum. We explored the linkage and recombination events among the polymorphic sites. We found that putative recombination events overlap with linked sites. We discuss how this pattern is explained by the action of positive natural selection, where the recombination events detected are convergent mutations. We conclude that it is inappropriate to use linkage-recombination patterns on genes under positive selection for assessing the structure of parasite populations. [Copyright &y& Elsevier]

Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies. [Display omitted] • Generated a knockin mouse with B cells expressing the inferred germline of CIS43 • Vaccination with junctional-epitope peptide is more effective than full-length PfCSP • Engineered a best-in-class malaria-protective antibody, iGL-CIS43.D3 • Structure and informatics analyses revealed alterations improving protective efficacy CIS43 is a malaria-protective antibody targeting a junctional epitope of the Plasmodium falciparum circumsporozoite protein. Kratochvil et al. engineer a mouse model expressing inferred germline CIS43 antibodies and use an immunofocusing strategy, in tandem with bioinformatic sieving, to generate improved CIS43 antibodies, including iGL-CIS43.D3, a best-in-class malaria-protective antibody. [ABSTRACT FROM AUTHOR]

We have designed a pre-erythrocytic vaccine candidate based on the Plasmodium vivax circumsporozoite (CSV) protein, which includes its N- and C-terminal parts and a truncated region containing repeat sequences from both the VK210 and the VK247 P. vivax subtypes. Two versions of this vaccine candidate were made: a soluble recombinant protein expressed in Escherichia coli, designated VMP001 and a particulate antigen expressed in Saccharomyces cerevisiae, designated CSV-S,S. The latter is composed of CSV-S, a fusion protein between VMP001 and hepatitis B surface antigen (HBsAg), and free HBsAg co-expressed in yeast and self-assembling into mixed particles. Both antigen versions, adjuvanted with AS01, were shown to be immunogenic in rhesus monkeys. CSV-S,S/AS01 induced higher levels of VMP001-specific antibodies than did VMP001/AS01. Antibody responses against the N- and C-terminal regions of CSV and the VK210 repeat motif were of a similar magnitude following immunization with either the soluble or the particulate antigen. However, antibodies against the AGDR region, a potentially protective B cell epitope, were only detected after immunization with CSV-S,S. Analysis of the induced CD4(+) T cells highlighted different cytokine profiles depending on the antigen form. These results warrant further clinical evaluation of these two vaccine candidates to assess the added value of a particulate versus soluble form of CSV, in terms of both immunogenicity and protective efficacy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Malaria results in over 650,000 deaths each year; thus, there is an urgent need for an effective vaccine. Pre-clinical studies and recently reported human trials suggest that pre-erythrocytic stage vaccines can provide protection against infection. A Phase 1, randomized, placebo-controlled, dose-escalation study was conducted with a vaccine composed of a replication-deficient adenovirus-35 backbone with P. falciparum circumsporozoite (CS) surface antigen (Ad35.CS.01). Healthy adult subjects received three doses of 10 (8), 10 (9), 10 (10), or 10 (11) vp/mL Ad35.CS.01 vaccine or saline placebo intramuscularly at 0, 1, and 6-mo intervals. Adverse events were assessed and anti-CS antibody responses were determined by ELISA. Seventy-two individuals were enrolled, with age, gender, and ethnicity similar across each study arm. While the vaccine was generally well tolerated, adverse events were more frequent in the highest dose groups (10 (10) and 10 (11) vp/mL). More robust humoral responses were also noted at the highest doses, with 73% developing a positive ELISA response after the three dose series of 10 (11) vp/mL. The Ad35.CS.01 vaccine was most immunogenic at the highest dosages (10 (10) and 10 (11) vp/mL). Reactogenicity findings were more common after the 10 (11) vp/mL dose, although most were mild or moderate in nature and resolved without therapy.