Structural basis of Plasmodium vivax inhibition by antibodies binding to the circumsporozoite protein repeats

Malaria is a global health burden, with Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) responsible for the majority of infections worldwide. Circumsporozoite protein (CSP) is the most abundant protein on the surface of Plasmodium sporozoites, and antibodies targeting the central repeat region of CSP can prevent parasite infection. Although much has been uncovered about the molecular basis of antibody recognition of the PfCSP repeats, data remains scarce for PvCSP. Here, we performed molecular dynamics simulations for peptides comprising the PvCSP repeats from strains VK210 and VK247 to reveal how the PvCSP central repeats are highly disordered, with minor propensities to adopt turn conformations. Next, we solved eight crystal structures to unveil the interactions of two inhibitory monoclonal antibodies (mAbs), 2F2 and 2E10.E9, with PvCSP repeats. Both antibodies can accommodate subtle sequence variances in the repeat motifs and recognize largely coiled peptide conformations that also contain isolated turns. Our structural studies uncover various degrees of Fab-Fab homotypic interactions upon recognition of the PvCSP central repeats by these two inhibitory mAbs, similar to potent mAbs against PfCSP. These findings augment our understanding of host-Plasmodium interactions, and contribute molecular details of Pv inhibition by mAbs to unlock structure-based engineering of PvCSP-based vaccines.

How to induce protective humoral immunity against Plasmodium falciparum circumsporozoite protein

The induction of protective humoral immune responses against sporozoite surface proteins of the human parasite Plasmodium falciparum (Pf) is a prime goal in the development of a preerythrocytic malaria vaccine. The most promising antibody target is circumsporozoite protein (CSP). Although PfCSP induces strong humoral immune responses upon vaccination, vaccine efficacy is overall limited and not durable. Here, we review recent efforts to gain a better molecular and cellular understanding of anti-PfCSP B cell responses in humans and discuss ways to overcome limitations in the induction of stable titers of high-affinity antibodies that might help to increase vaccine efficacy and promote long-lived protection.

Protein interaction analysis of Plasmodium falciparum circumsporozoite protein variants with human immunoproteins explains RTS,S vaccine efficacy in Ghana

The world's first malaria vaccine RTS,S provides only partial protection against Plasmodium falciparum infections. The explanation for such low efficacy is unclear. This study examined the associations of parasite genetic variations with binding affinity to human immunological proteins including human leukocyte antigen (HLA) and T cell receptors (TCR) involved in RTS,S-induced immune responses. Multiplicity of infections was determined by amplicon deep sequencing of merozoite surface protein 1 (PfMSP1). Genetic variations in the C-terminal of circumsporozoite protein (PfMSP1) gene were examined across 88 samples of P. falciparum collected from high and low transmission settings of Ghana. Binding interactions of PfMSP1 variants and HLA/TCR were analyzed using NetChop} and HADDOCK predictions. Anti-CSP IgG levels were measured by ELISA in a subset of 10 samples. High polyclonality was detected among P. falciparum infections. A total 27 CSP haplotypes were detected among samples. A significant correlation was detected between the CSP and MSP multiplicity of infection (MOI). No clear clustering of haplotypes was observed by geographic regions. The number of genetic differences in PfCSP between 3D7 and non-3D7 variants does not influence binding interactions to HLA/T cells nor anti-CSP IgG levels. Nevertheless, PfCSP peptide length significantly affects its molecular weight and binding affinity to the HLA. The presence of multiple non-3D7 strains among P. falciparum infections in Ghana impact the effectiveness of RTS,S. Longer PfCSP peptides may elicit a stronger immune response and should be considered in future version RTS,S. The molecular mechanisms of RTS,S cell-mediated immune responses related to longer CSP peptides warrants further investigations.

Protective effects of combining monoclonal antibodies and vaccines against the Plasmodium falciparum circumsporozoite protein

Combinations of monoclonal antibodies (mAbs) against different epitopes on the same antigen synergistically neutralize many viruses. However, there are limited studies assessing whether combining human mAbs against distinct regions of the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) enhances in vivo protection against malaria compared to each mAb alone or whether passive transfer of PfCSP mAbs would improve protection following vaccination against PfCSP. Here, we isolated a panel of human mAbs against the subdominant C-terminal domain of PfCSP (C-CSP) from a volunteer immunized with radiation-attenuated Pf sporozoites. These C-CSP-specific mAbs had limited binding to sporozoites in vitro that was increased by combination with neutralizing human “repeat” mAbs against the NPDP/NVDP/NANP tetrapeptides in the central repeat region of PfCSP. Nevertheless, passive transfer of repeat- and C-CSP-specific mAb combinations did not provide enhanced protection against in vivo sporozoite challenge compared to repeat mAbs alone. Furthermore, combining potent repeat-specific mAbs (CIS43, L9, and 317) that respectively target the three tetrapeptides (NPDP/NVDP/NANP) did not provide additional protection against in vivo sporozoite challenge. However, administration of either CIS43, L9, or 317 (but not C-CSP-specific mAbs) to mice that had been immunized with R21, a PfCSP-based virus-like particle vaccine that induces polyclonal antibodies against the repeat region and C-CSP, provided enhanced protection against sporozoite challenge when compared to vaccine or mAbs alone. Collectively, this study shows that while combining mAbs against the repeat and C-terminal regions of PfCSP provide no additional protection in vivo, repeat mAbs do provide increased protection when combined with vaccine-induced polyclonal antibodies. These data should inform the implementation of PfCSP human mAbs alone or following vaccination to prevent malaria infection.

Antibody Targets and Properties for Complement-Fixation Against the Circumsporozoite Protein in Malaria Immunity

The Plasmodium falciparum circumsporozoite protein (CSP) forms the basis of leading subunit malaria vaccine candidates. However, the mechanisms and specific targets of immunity are poorly defined. Recent findings suggest that antibody-mediated complement-fixation and activation play an important role in immunity. Here, we investigated the regions of CSP targeted by functional complement-fixing antibodies and the antibody properties associated with this activity. We quantified IgG, IgM, and functional complement-fixing antibody responses to different regions of CSP among Kenyan adults naturally exposed to malaria (n=102) and using a series of rabbit vaccination studies. Individuals who acquired functional complement-fixing antibodies had higher IgG, IgM and IgG1 and IgG3 to CSP. Acquired complement-fixing antibodies targeted the N-terminal, central-repeat, and C-terminal regions of CSP, and positive responders had greater antibody breadth compared to those who were negative for complement-fixing antibodies (p<0.05). Using rabbit vaccinations as a model, we confirmed that IgG specific to the central-repeat and non-repeat regions of CSP could effectively fix complement. However, vaccination with near full length CSP in rabbits poorly induced antibodies to the N-terminal region compared to naturally-acquired immunity in humans. Poor induction of N-terminal antibodies was also observed in a vaccination study performed in mice. IgG and IgM to all three regions of CSP play a role in mediating complement-fixation, which has important implications for malaria vaccine development.

Serological responses to a soluble recombinant circumsporozoite protein-VK210 of Plasmodium vivax (rPvCSP-VK210) among Iranian malaria patients

Background Circumsporozoite protein (CSP) has a central immune domain that includes short regions of repeating amino acid sequences. This immunodynamic region is an epitope of B cells that can elicit an immune response in human and laboratory animals. The aim of the present study was to express the recombinant PvCSP-VK210 antigen and evaluate it for assaying antibodies obtained during human P. vivax infection by Western blotting and indirect ELISA (enzyme-linked immunosorbent assay). Method Genomic DNA of P. vivax was isolated from a blood sample of an Iranian person with vivax malaria, and by PCR, the fragment of the PvCSP-VK210 gene was amplified. The gene fragment was cut after gel purification by BamHI and HindIII enzymes and then cloned into pET28a expression vector. Finally, the recombinant pET28a was transformed into the E.coli BL21 (DE3) as the expression host. In order to produce His-tagged protein, the expression host was cultured in LB medium. The protein was purified by Ni–NTA columns and immobilized metal affinity chromatography, and after confirmation by Western blotting technique, was used as the antigen in the indirect ELISA test. Results The recombinant protein was expressed and purified as a 32-kDa protein. The sensitivity and specificity of the indirect ELISA test with the recombinant PvCSP-VK210 antigen were 61.42% and 97.14%, respectively, based on OD = 0.313. Between the results of the microscopic test and the indirect ELISA test with the recombinant PvCSP-VK210 antigen there was a Kappa coefficient of 0.586. The positive and negative predictive value and validity of the ELISA test with the recombinant PvCSP-VK210 antigen were 95.55%, 71.57%, 79.28%, respectively. Conclusion The sensitivity of the indirect ELISA method with the recombinant PvCSP-VK210 antigen was 61.42%, which is the first report from Iran.

A vaccine targeting the L9 epitope of the malaria circumsporozoite protein confers protection from blood-stage infection in a mouse challenge model

Pre-erythrocytic malaria vaccines that induce high-titer, durable antibody responses can potentially provide protection from infection. Here, we engineered a virus-like particle (VLP)-based vaccine targeting a recently described vulnerable epitope at the N-terminus of the central repeat region of the Plasmodium falciparum circumsporozoite protein (CSP) that is recognized by the potently inhibitory monoclonal antibody L9 and show that immunization with L9 VLPs induces strong antibody responses that provide protection from blood-stage malaria in a mouse infection model.

Assessment of genetic diversity of Plasmodium falciparum circumsporozoite protein in Sudan: the RTS,S leading malaria vaccine candidate

Background The currently used malaria vaccine, RTS,S, is designed based on the Plasmodium falciparum circumsporozoite protein (PfCSP). The pfcsp gene, besides having different polymorphic patterns, can vary between P. falciparum isolates due to geographical origin and host immune response. Such aspects are essential when considering the deployment of the RTS,S vaccine in a certain region. Therefore, this study assessed the genetic diversity of P. falciparum in Sudan based on the pfcsp gene by investigating the diversity at the N-terminal, central repeat, and the C-terminal regions. Methods A cross-sectional molecular study was conducted; P. falciparum isolates were collected from different health centres in Khartoum State between January and December 2019. During the study period, a total of 261 febrile patients were recruited. Malaria diagnosis was made by expert microscopists using Giemsa-stained thick and thin blood films. DNA samples were examined by the semi-nested polymerase chain reaction (PCR). Single clonal infection of the confirmed P. falciparum cases, were used to amplify the pfcsp gene. The amplified amplicons of pfcsp have been sequenced using the Sanger dideoxy method. The obtained sequences of pfcsp nucleotide diversity parameters including the numbers of haplotypes (Hap), haplotypes diversity (Hapd), the average number of nucleotide differences between two sequences (p), and the numbers of segregating sites (S) were obtained. The haplotype networks were constructed using the online tcsBU software. Natural selection theory was also tested on pfcsp using Fuand Li’s D, Fuand Li’s F statistics, and Tajima’s D test using DnaSP. Results In comparison with the different pfcsp reference strains, the Sudanese isolates showed high similarity with other African isolates. The results of the N-terminal region showed the presence of 2 different haplotypes with a Hapd of 0.425 ± 0.00727. The presence of the unique insertion of NNNGDNGREGKDEDKRDGNN was reported. The KLKQP motif was conserved in all the studied isolates. At the central repeat region, 11 haplotypes were seen with a Hapd of 0.779 ± 0.00097. The analysis of the genetic diversity in the C-terminal region showed the presence of 10 haplotypes with a Hapd of 0.457 ± 0.073. Several non-synonymous amino acids changes were also seen at the Th2R and the Th3R T-cell epitope regions including T317K, E317K, Q318E, K321N, I322K, T322K, R322K, K324Q, I327L, G352N, S354P, R355K, N356D, Q357E, and E361A. Conclusions In this study, the results indicated a high conservation at the pfcsp gene. This may further contribute in understanding the genetic polymorphisms of P. falciparum prior to the deployment of the RTS,S vaccine in Sudan.

The P. falciparum CSP repeat region contains three distinct epitopes required for protection by antibodies in vivo

Rare and potent monoclonal antibodies (mAbs) against the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) on infective sporozoites (SPZ) preferentially bind the PfCSP junctional tetrapeptide NPDP or NVDP minor repeats while cross-reacting with NANP major repeats in vitro. The extent to which each of these epitopes is required for protection in vivo is unknown. Here, we assessed whether junction-, minor repeat- and major repeat-preferring human mAbs (CIS43, L9 and 317 respectively) bound and protected against in vivo challenge with transgenic P. berghei (Pb) SPZ expressing either PfCSP with the junction and minor repeats knocked out (KO), or PbCSP with the junction and minor repeats knocked in (KI). In vivo protection studies showed that the junction and minor repeats are necessary and sufficient for CIS43 and L9 to neutralize KO and KI SPZ, respectively. In contrast, 317 required major repeats for in vivo protection. These data establish that human mAbs can prevent malaria infection by targeting three different protective epitopes (NPDP, NVDP, NANP) in the PfCSP repeat region. This report will inform vaccine development and the use of mAbs to passively prevent malaria.