The essential malaria protein PfCyRPA targets glycans to invade erythrocytes.

Plasmodium falciparum is a human-adapted apicomplexan parasite that causes the most dangerous form of malaria. P. falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. The precise role of PfCyRPA in this process has not been resolved. Here, we show that PfCyRPA is a lectin targeting glycans terminating with α2-6-linked N-acetylneuraminic acid (Neu5Ac). PfCyRPA has a >50-fold binding preference for human, α2-6-linked Neu5Ac over non-human, α2-6-linked N-glycolylneuraminic acid. PfCyRPA lectin sites were predicted by molecular modeling and validated by mutagenesis studies. Transgenic parasite lines expressing endogenous PfCyRPA with single amino acid exchange mutants indicated that the lectin activity of PfCyRPA has an important role in parasite invasion. Blocking PfCyRPA lectin activity with small molecules or with lectin-site-specific monoclonal antibodies can inhibit blood-stage parasite multiplication. Therefore, targeting PfCyRPA lectin activity with drugs, immunotherapy, or a vaccine-primed immune response is a promising strategy to prevent and treat malaria.

The malaria blood stage antigen PfCyRPA formulated with the TLR-4 agonist adjuvant GLA-SE elicits parasite growth inhibitory antibodies in experimental animals.

BACKGROUND: Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. In contrast to several previously clinically tested merozoite vaccine candidate antigens, PfCyRPA is not polymorphic, making it a promising candidate antigen for blood stage vaccine development. METHODS: Mice and rabbits were immunized with vaccine formulations of recombinantly expressed PfCyRPA adjuvanted either with the glucopyranosyl lipid A (GLA) containing adjuvants GLA-LSQ, GLA-SE, GLA-Alum or with Nanoalum. ELISA and indirect immunofluorescence assays (IFA) were used to analyse elicited IgG titers and the P. falciparum growth inhibitory activity was determined with a standardized in vitro [3H]-hypoxanthine incorporation assay. RESULTS: In the mouse experiments, the GLA adjuvanted formulations were superior to the Nanoalum formulation with respect to antibody titer development, IFA sero-conversion rates and in vitro parasite growth-inhibitory activity. In rabbits, the highest titers of parasite growth inhibitory antibodies were obtained with the GLA-SE formulation. Comparable mean ELISA IgG endpoint titers were reached in rabbits after three immunizations with GLA-SE adjuvanted PfCyRPA doses of 5, 25 and 100 µg, but with 100 µg of antigen, only two immunizations were required to reach this titer. CONCLUSION: PfCyRPA formulated with the human-compatible adjuvant GLA-SE represents an attractive vaccine candidate for early clinical testing in a controlled P. falciparum blood stage challenge trial.

Shell effects and free-electrons in electromigrated oxidized Cu-nanocontacts.

Electromigration in interconnects continues to be an important field of study in integrated circuits as the interconnects are planned to shrink in size at comparable pace as the semiconductor functional elements. Through shrinking the interconnects approach the regime where quantum size effects become important. The observation of quantum size and shell effects is usually restricted either to low-temperatures or vacuum conditions or to chemically inert materials such as Au. Here, we show that in electromigrated Cu nanocontacts such effects can be observed at room temperature and room pressure even in the presence of oxidation. Our data provide evidence that the nanocontacts are nearly spherical objects with a triangular-cylindrical symmetry of their electronic wave functions with a stronger free-electron-like character compared to previous results. We do not observe a detrimental effect of oxygen. The presence of shell effects has implications for the technological use of Cu nanocontacts as interconnects in integrated circuits and could lead to the use of electronic wave functions of shells in such interconnects.

Scalable Process for High-Yield Production of PfCyRPA Using Insect Cells for Inclusion in a Malaria Virosome-Based Vaccine Candidate.

Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) has been identified as a promising blood-stage candidate antigen to include in a broadly cross-reactive malaria vaccine. In the last couple of decades, substantial effort has been committed to the development of scalable cost-effective, robust, and high-yield PfCyRPA production processes. Despite insect cells being a suitable expression system due to their track record for protein production (including vaccine antigens), these are yet to be explored to produce this antigen. In this study, different insect cell lines, culture conditions (baculovirus infection strategy, supplementation schemes, culture temperature modulation), and purification strategies (affinity tags) were explored aiming to develop a scalable, high-yield, and high-quality PfCyRPA for inclusion in a virosome-based malaria vaccine candidate. Supplements with antioxidants improved PfCyRPA volumetric titers by 50% when added at the time of infection. In addition, from three different affinity tags (6x-His, 4x-His, and C-tag) evaluated, the 4x-His affinity tag was the one leading to the highest PfCyRPA purification recovery yields (61%) and production yield (26 mg/L vs. 21 mg/L and 13 mg/L for 6x-His and C-tag, respectively). Noteworthy, PfCyRPA expressed using High Five cells did not show differences in protein quality or stability when compared to its human HEK293 cell counterpart. When formulated in a lipid-based virosome nanoparticle, immunized rabbits developed functional anti-PfCyRPA antibodies that impeded the multiplication of P. falciparum in vitro. This work demonstrates the potential of using IC-BEVS as a qualified platform to produce functional recombinant PfCyRPA protein with the added benefit of being a non-human expression system with short bioprocessing times and high expression levels.

Vaccination with virosomally formulated recombinant CyRPA elicits protective antibodies against Plasmodium falciparum parasites in preclinical in vitro and in vivo models.

The Plasmodium falciparum (Pf) cysteine-rich protective antigen (PfCyRPA) has emerged as a promising blood-stage candidate antigen for inclusion into a broadly cross-reactive malaria vaccine. This highly conserved protein among various geographical strains plays a key role in the red blood cell invasion process by P. falciparum merozoites, and antibodies against PfCyRPA can efficiently prevent the entry of the malaria parasites into red blood cells. The aim of the present study was to develop a human-compatible formulation of the PfCyRPA vaccine candidate and confirming its activity in preclinical studies. Recombinant PfCyRPA expressed in HEK 293 cells was chemically coupled to phosphoethanolamine and then incorporated into the membrane of unadjuvanted influenza virosomes approved as antigen delivery system for humans. Laboratory animals were immunised with the virosome-based PfCyRPA vaccine to determine its immunogenic properties and in particular, its capacity to elicit parasite binding and growth-inhibitory antibodies. The vaccine elicited in mice and rabbits high titers of PfCyRPA-specific antibodies that bound to the blood-stage parasites. At a concentration of 10 mg/mL, purified total serum IgG from immunised rabbits inhibited parasite growth in vitro by about 80%. Furthermore, in a P. falciparum infection mouse model, passive transfer of 10 mg of purified total IgG from PfCyRPA vaccinated rabbits reduced the in vivo parasite load by 77%. Influenza virosomes thus represent a suitable antigen delivery system for the induction of protective antibodies against the recombinant PfCyRPA, designating it as a highly suitable component for inclusion into a multivalent and multi-stage virosomal malaria vaccine.

Loss of Genomic Diversity in a Neisseria meningitidis Clone Through a Colonization Bottleneck.

Neisseria meningitidis is the leading cause of epidemic meningitis in the "meningitis belt" of Africa, where clonal waves of colonization and disease are observed. Point mutations and horizontal gene exchange lead to constant diversification of meningococcal populations during clonal spread. Maintaining a high genomic diversity may be an evolutionary strategy of meningococci that increases chances of fixing occasionally new highly successful "fit genotypes". We have performed a longitudinal study of meningococcal carriage and disease in northern Ghana by analyzing cerebrospinal fluid samples from all suspected meningitis cases and monitoring carriage of meningococci by twice yearly colonization surveys. In the framework of this study, we observed complete replacement of an A: sequence types (ST)-2859 clone by a W: ST-2881 clone. However, after a gap of 1 year, A: ST-2859 meningococci re-emerged both as colonizer and meningitis causing agent. Our whole genome sequencing analyses compared the A population isolated prior to the W colonization and disease wave with the re-emerging A meningococci. This analysis revealed expansion of one clone differing in only one nonsynonymous SNP from several isolates already present in the original A: ST-2859 population. The colonization bottleneck caused by the competing W meningococci thus resulted in a profound reduction in genomic diversity of the A meningococcal population.

Emergence and genomic diversification of a virulent serogroup W:ST-2881(CC175) Neisseria meningitidis clone in the African meningitis belt.

Countries of the African 'meningitis belt' are susceptible to meningococcal meningitis outbreaks. While in the past major epidemics have been primarily caused by serogroup A meningococci, W strains are currently responsible for most of the cases. After an epidemic in Mecca in 2000, W:ST-11 strains have caused many outbreaks worldwide. An unrelated W:ST-2881 clone was described for the first time in 2002, with the first meningitis cases caused by these bacteria reported in 2003. Here we describe results of a comparative whole-genome analysis of 74 W:ST-2881 strains isolated within the framework of two longitudinal colonization and disease studies conducted in Ghana and Burkina Faso. Genomic data indicate that the W:ST-2881 clone has emerged from Y:ST-175(CC175) bacteria by capsule switching. The circulating W:ST-2881 populations were composed of a variety of closely related but distinct genomic variants with no systematic differences between colonization and disease isolates. Two distinct and geographically clustered phylogenetic clonal variants were identified in Burkina Faso and a third in Ghana. On the basis of the presence or absence of 17 recombination fragments, the Ghanaian variant could be differentiated into five clusters. All 25 Ghanaian disease isolates clustered together with 23 out of 40 Ghanaian isolates associated with carriage within one cluster, indicating that W:ST-2881 clusters differ in virulence. More than half of the genes affected by horizontal gene transfer encoded proteins of the 'cell envelope' and the 'transport/binding protein' categories, which indicates that exchange of non-capsular antigens plays an important role in immune evasion.

A Synthetic Virus-Like Particle Streptococcal Vaccine Candidate Using B-Cell Epitopes from the Proline-Rich Region of Pneumococcal Surface Protein A.

Alternatives to the well-established capsular polysaccharide-based vaccines against Streptococcus pneumoniae that circumvent limitations arising from limited serotype coverage and the emergence of resistance due to capsule switching (serotype replacement) are being widely pursued. Much attention is now focused on the development of recombinant subunit vaccines based on highly conserved pneumococcal surface proteins and virulence factors. A further step might involve focusing the host humoral immune response onto protective protein epitopes using as immunogens structurally optimized epitope mimetics. One approach to deliver such epitope mimetics to the immune system is through the use of synthetic virus-like particles (SVLPs). SVLPs are made from synthetic coiled-coil lipopeptides that are designed to spontaneously self-assemble into 20-30 nm diameter nanoparticles in aqueous buffer. Multivalent display of epitope mimetics on the surface of SVLPs generates highly immunogenic nanoparticles that elicit strong epitope-specific humoral immune responses without the need for external adjuvants. Here, we set out to demonstrate that this approach can yield vaccine candidates able to elicit a protective immune response, using epitopes derived from the proline-rich region of pneumococcal surface protein A (PspA). These streptococcal SVLP-based vaccine candidates are shown to elicit strong humoral immune responses in mice. Following active immunization and challenge with lethal doses of streptococcus, SVLP-based immunogens are able to elicit significant protection in mice. Furthermore, a mimetic-specific monoclonal antibody is shown to mediate partial protection upon passive immunization. The results show that SVLPs combined with synthetic epitope mimetics may have potential for the development of an effective vaccine against Streptococcus pneumoniae.

Emergence of a new epidemic Neisseria meningitidis serogroup A Clone in the African meningitis belt: high-resolution picture of genomic changes that mediate immune evasion.

In the African "meningitis belt," outbreaks of meningococcal meningitis occur in cycles, representing a model for the role of host-pathogen interactions in epidemic processes. The periodicity of the epidemics is not well understood, nor is it currently possible to predict them. In our longitudinal colonization and disease surveys, we have observed waves of clonal replacement with the same serogroup, suggesting that immunity to noncapsular antigens plays a significant role in natural herd immunity. Here, through comparative genomic analysis of 100 meningococcal isolates, we provide a high-resolution view of the evolutionary changes that occurred during clonal replacement of a hypervirulent meningococcal clone (ST-7) by a descendant clone (ST-2859). We show that the majority of genetic changes are due to homologous recombination of laterally acquired DNA, with more than 20% of these events involving acquisition of DNA from other species. Signals of adaptation to evade herd immunity were indicated by genomic hot spots of recombination. Most striking is the high frequency of changes involving the pgl locus, which determines the glycosylation patterns of major protein antigens. High-frequency changes were also observed for genes involved in the regulation of pilus expression and the synthesis of Maf3 adhesins, highlighting the importance of these surface features in host-pathogen interaction and immune evasion. Importance: While established meningococcal capsule polysaccharide vaccines are protective through the induction of anticapsular antibodies, findings of our longitudinal studies in the African meningitis belt have indicated that immunity to noncapsular antigens plays a significant role in natural herd immunity. Our results show that meningococci evade herd immunity through the rapid homologous replacement of just a few key genomic loci that affect noncapsular cell surface components. Identification of recombination hot spots thus represents an eminent approach to gain insight into targets of protective natural immune responses. Moreover, our results highlight the role of the dynamics of the protein glycosylation repertoire in immune evasion by Neisseria meningitidis. These results have major implications for the design of next-generation protein-based subunit vaccines.

A broadly-protective vaccine against meningococcal disease in sub-Saharan Africa based on generalized modules for membrane antigens (GMMA).

INTRODUCTION: Neisseria meningitidis causes epidemics of meningitis in sub-Saharan Africa. These have mainly been caused by capsular group A strains, but W and X strains are increasingly contributing to the burden of disease. Therefore, an affordable vaccine that provides broad protection against meningococcal disease in sub-Saharan Africa is required. METHODS: We prepared Generalized Modules for Membrane Antigens (GMMA) from a recombinant serogroup W strain expressing PorA P1.5,2, which is predominant among African W isolates. The strain was engineered with deleted capsule locus genes, lpxL1 and gna33 genes and over-expressed fHbp variant 1, which is expressed by the majority of serogroup A and X isolates. RESULTS: We screened nine W strains with deleted capsule locus and gna33 for high-level GMMA release. A mutant with five-fold increased GMMA release compared with the wild type was further engineered with a lpxL1 deletion and over-expression of fHbp. GMMA from the production strain had 50-fold lower ability to stimulate IL-6 release from human PBMC and caused 1000-fold lower TLR-4 activation in Human Embryonic Kidney cells than non-detoxified GMMA. In mice, the GMMA vaccine induced bactericidal antibody responses against African W strains expressing homologous PorA and fHbp v.1 or v.2 (geometric mean titres [GMT]=80,000-200,000), and invasive African A and X strains expressing a heterologous PorA and fHbp variant 1 (GMT=20-2500 and 18-5500, respectively). Sera from mice immunised with GMMA without over-expressed fHbp v.1 were unable to kill the A and X strains, indicating that bactericidal antibodies against these strains are directed against fHbp. CONCLUSION: A GMMA vaccine produced from a recombinant African N. meningitidis W strain with deleted capsule locus, lpxL1, gna33 and overexpressed fHbp v.1 has potential as an affordable vaccine with broad coverage against strains from all main serogroups currently causing meningococcal meningitis in sub-Saharan Africa.

Optimized DNA preparation from mycobacteria.

Extraction of genomic DNA from mycobacteria requires special consideration because (i) many mycobacterial species exhibit extremely slow growth, and thus produce only small amounts of starting material, and (ii) a robust and waxy cell wall renders mycobacteria difficult to lyse. Hence, mycobacterial DNA extraction often results in low DNA yields of unsuitable quality. Published protocols for mycobacterial DNA preparations and commercially available extraction kits are mainly designed for the isolation of small amounts of genomic material suitable for polymerase chain reaction (PCR)-based applications like species identification. However, such DNA quantities and qualities are usually not sufficient for contemporary genomic analyses such as whole genome sequence analysis, single nucleotide polymorphism (SNP) detection, or DNA microarrays, or for investigations of bacterial evolution, virulence, or epidemiology on a world-wide population level. Moreover, most protocols that achieve a high standard in DNA recovery typically employ large reaction volumes and thus require milliliter-scale plasticware and centrifugal equipment as well as large amounts of chemicals, all of which are costly both in purchase and disposal. The DNA extraction method described here was established to address the challenges that result from the slow growth and distinct cell wall composition of mycobacteria, and to greatly enhance both yield and purity of mycobacterial DNA preparations in a small extraction volume. Designed to be performed using 1.5-mL reaction tubes and the corresponding equipment, the method is economical and practical, and reliably yields large amounts of pure genomic DNA--increases of at least 10-fold as compared to earlier protocols.

Lack of insertional-deletional polymorphism in a collection of Mycobacterium ulcerans isolates from Ghanaian Buruli ulcer patients.

Mycobacterium ulcerans causes the devastating infectious skin disease Buruli ulcer and has a monomorphic population structure. The resolution of conventional genetic fingerprinting methods is therefore not sufficient for microepidemiological studies aiming to characterize transmission pathways. In a previous comparative genomic hybridization analysis with a microarray covering part of the M. ulcerans genome, we have found extensive insertional-deletional sequence polymorphisms among M. ulcerans isolates of diverse geographic origins that allowed us to distinguish between strains coming from different continents. Since large numbers of insertion sequences are spread over the genome of African M. ulcerans strains, we reasoned that these may drive large sequence polymorphisms in otherwise clonal local mycobacterial populations. In this study, we used a printed DNA microarray covering the whole genome of the Ghanaian M. ulcerans reference strain Agy99 for comparative genomic hybridization. The assay identified multiple regions of difference when DNA of a Japanese M. ulcerans strain was analyzed. In contrast, not a single insertional-deletional genomic variation was found within a panel of disease isolates coming from an area of Ghana where Buruli ulcer is endemic. These results indicate that, despite the expectations deduced from other mycobacterial pathogens, only analyses of single nucleotide polymorphisms will have the potential to differentiate local populations of M. ulcerans.

Single nucleotide polymorphisms on the road to strain differentiation in Mycobacterium ulcerans.

The genomic fine-typing of strains of Mycobacterium ulcerans, the causative agent of the emerging human disease Buruli ulcer, is difficult due to the clonal population structure of geographical lineages. Although large sequence polymorphisms (LSPs) resulted in the clustering of patient isolates originating from across the globe, differentiation of strains within continents using conventional typing methods is very limited. In this study, we analyzed M. ulcerans LSP haplotype-specific insertion sequence elements among 83 M. ulcerans strains and identified single nucleotide polymorphisms (SNPs) that differentiate between regional strains. This is the first genetic discrimination based on SNPs of M. ulcerans strains from African countries where Buruli ulcer is endemic, resulting in the highest geographic resolution of genotyping so far. The findings support the concept of genome-wide SNP analyses as tools to study the epidemiology and evolution of M. ulcerans at a local level.

Large sequence polymorphisms unveil the phylogenetic relationship of environmental and pathogenic mycobacteria related to Mycobacterium ulcerans.

Mycolactone is an immunosuppressive cytotoxin responsible for the clinical manifestation of Buruli ulcer in humans. It was believed to be confined to its etiologic agent, Mycobacterium ulcerans. However, the identification of other mycolactone-producing mycobacteria (MPMs) in other species, including Mycobacterium marinum, indicated a more complex taxonomic relationship. This highlighted the need for research on the biology, evolution, and distribution of such emerging and potentially infectious strains. The reliable genetic fingerprinting analyses presented here aim at both the unraveling of phylogenetic relatedness and of dispersal between environmental and pathogenic mycolactone producers and the identification of genetic prerequisites that enable lateral gene transfer of such plasmids. This will allow for the identification of environmental reservoirs of virulence plasmids that encode enzymes required for the synthesis of mycolactone. Based on dynamic chromosomal loci identified earlier in M. ulcerans, we characterized large sequence polymorphisms for the phylogenetic analysis of MPMs. Here, we identify new insertional-deletional events and single-nucleotide polymorphisms that confirm and redefine earlier strain differentiation markers. These results support other data showing that all MPMs share a common ancestry. In addition, we found unique genetic features specific for M. marinum strain M, the genome sequence strain which is used widely in research.

Optimized method for preparation of DNA from pathogenic and environmental mycobacteria.

Genomic studies on pathogenic and environmental mycobacteria are of growing interest for understanding of their evolution, distribution, adaptation, and host-pathogen interaction. Since most mycobacteria are slow growers, material from in vitro cultures is usually scarce. The robust mycobacterial cell wall hinders both experimental cell lysis and efficient DNA extraction. Here, we compare elements of several DNA preparation protocols and describe a method that is economical and practical and reliably yields large amounts--usually 10-fold increased compared to earlier protocols--of highly pure genomic DNA for sophisticated downstream applications. This method was optimized for cultures of a variety of pathogenic and environmental mycobacterial species and proven to be suitable for direct mycobacterial DNA extraction from infected insect specimens.