An Easy and Efficient Method for Native and Immunoreactive Echinococcus granulosus Antigen 5 Enrichment from Hydatid Cyst Fluid

Background: Currently, the serodiagnosis of cystic echinococcosis relies mostly on crude Echinococcus granulosus hydatid cyst fluid as the antigen. Consequently, available immunodiagnostic tests lack standardization of the target antigen and, in turn, this is reflected on poor sensitivity and specificity of the serological diagnosis. Methodology/Principal Findings: Here, a chromatographic method enabling the generation of highly enriched Antigen 5 (Ag5) is described. The procedure is very easy, efficient and reproducible, since different hydatid cyst fluid (HCF) sources produced very similar chromatograms, notwithstanding the clearly evident and extreme heterogeneity of the starting material. In addition, the performance of the antigen preparation in immunological assays was preliminarily assessed by western immunoblotting and ELISA on a limited panel of cystic echinococcosis patients and healthy controls. Following western immunoblotting and ELISA experiments, a high reactivity of patient sera was seen, with unambiguous and highly specific results. Conclusions/Significance: The methods and results reported open interesting perspectives for the development of sensitive diagnostic tools to enable the timely and unambiguous detection of cystic echinococcosis antibodies in patient sera.This work was supported by Regione Autonoma della Sardegna (http://www.regione.sardegna.it/)Pubblicat

Immunogenicity of Self-Associated Aggregates and Chemically Cross-Linked Conjugates of the 42 kDa Plasmodium falciparum Merozoite Surface Protein-1

Self-associated protein aggregates or cross-linked protein conjugates are, in general, more immunogenic than oligomeric or monomeric forms. In particular, the immunogenicity in mice of a recombinant malaria transmission blocking vaccine candidate, the ookinete specific Plasmodium falciparum 25 kDa protein (Pfs25), was increased more than 1000-fold when evaluated as a chemical cross-linked protein-protein conjugate as compared to a formulated monomer. Whether alternative approaches using protein complexes improve the immunogenicity of other recombinant malaria vaccine candidates is worth assessing. In this work, the immunogenicity of the recombinant 42 kDa processed form of the P. falciparum merozoite surface protein 1 (MSP142) was evaluated as a self-associated, non-covalent aggregate and as a chemical cross-linked protein-protein conjugate to ExoProtein A, which is a recombinant detoxified form of Pseudomonas aeruginosa exotoxin A. MSP142 conjugates were prepared and characterized biochemically and biophysically to determine their molar mass in solution and stoichiometry, when relevant. The immunogenicity of the MSP142 self-associated aggregates, cross-linked chemical conjugates and monomers were compared in BALB/c mice after adsorption to aluminum hydroxide adjuvant, and in one instance in association with the TLR9 agonist CPG7909 with an aluminum hydroxide formulation. Antibody titers were assessed by ELISA. Unlike observations made for Pfs25, no significant enhancement in MSP142 specific antibody titers was observed for any conjugate as compared to the formulated monomer or dimer, except for the addition of the TLR9 agonist CPG7909. Clearly, enhancing the immunogenicity of a recombinant protein vaccine candidate by the formation of protein complexes must be established on an empirical basis

Detection of a Fourth Orbivirus Non-Structural Protein

The genus Orbivirus includes both insect and tick-borne viruses. The orbivirus genome, composed of 10 segments of dsRNA, encodes 7 structural proteins (VP1–VP7) and 3 non-structural proteins (NS1–NS3). An open reading frame (ORF) that spans almost the entire length of genome segment-9 (Seg-9) encodes VP6 (the viral helicase). However, bioinformatic analysis recently identified an overlapping ORF (ORFX) in Seg-9. We show that ORFX encodes a new non-structural protein, identified here as NS4. Western blotting and confocal fluorescence microscopy, using antibodies raised against recombinant NS4 from Bluetongue virus (BTV, which is insect-borne), or Great Island virus (GIV, which is tick-borne), demonstrate that these proteins are synthesised in BTV or GIV infected mammalian cells, respectively. BTV NS4 is also expressed in Culicoides insect cells. NS4 forms aggregates throughout the cytoplasm as well as in the nucleus, consistent with identification of nuclear localisation signals within the NS4 sequence. Bioinformatic analyses indicate that NS4 contains coiled-coils, is related to proteins that bind nucleic acids, or are associated with membranes and shows similarities to nucleolar protein UTP20 (a processome subunit). Recombinant NS4 of GIV protects dsRNA from degradation by endoribonucleases of the RNAse III family, indicating that it interacts with dsRNA. However, BTV NS4, which is only half the putative size of the GIV NS4, did not protect dsRNA from RNAse III cleavage. NS4 of both GIV and BTV protect DNA from degradation by DNAse. NS4 was found to associate with lipid droplets in cells infected with BTV or GIV or transfected with a plasmid expressing NS4

A polyvalent hybrid protein elicits antibodies against the diverse allelic types of block 2 in Plasmodium falciparum merozoite surface protein 1.

Merozoite surface protein 1 (MSP1) of Plasmodium falciparum has been implicated as an important target of acquired immunity, and candidate components for a vaccine include polymorphic epitopes in the N-terminal polymorphic block 2 region. We designed a polyvalent hybrid recombinant protein incorporating sequences of the three major allelic types of block 2 together with a composite repeat sequence of one of the types and N-terminal flanking T cell epitopes, and compared this with a series of recombinant proteins containing modular sub-components and similarly expressed in Escherichia coli. Immunogenicity of the full polyvalent hybrid protein was tested in both mice and rabbits, and comparative immunogenicity studies of the sub-component modules were performed in mice. The full hybrid protein induced high titre antibodies against each of the major block 2 allelic types expressed as separate recombinant proteins and against a wide range of allelic types naturally expressed by a panel of diverse P. falciparum isolates, while the sub-component modules had partial antigenic coverage as expected. This encourages further development and evaluation of the full MSP1 block 2 polyvalent hybrid protein as a candidate blood-stage component of a malaria vaccine

Antibody Responses to Antigenic Targets of Recent Exposure Are Associated With Low-Density Parasitemia in Controlled Human Plasmodium falciparum Infections

Contains fulltext : 200896.pdf (publisher's version ) (Open Access

The acquisition of humoral immune responses targeting Plasmodium falciparum sexual stages in controlled human malaria infections

Individuals infected with P. falciparum develop antibody responses to intra-erythrocytic gametocyte proteins and exported gametocyte proteins present on the surface of infected erythrocytes. However, there is currently limited knowledge on the immunogenicity of gametocyte antigens and the specificity of gametocyte-induced antibody responses. In this study, we assessed antibody responses in participants of two controlled human malaria infection (CHMI) studies by ELISA, multiplexed bead-based antibody assays and protein microarray. By comparing antibody responses in participants with and without gametocyte exposure, we aimed to disentangle the antibody response induced by asexual and sexual stage parasites. We showed that after a single malaria infection, a significant anti-sexual stage humoral response is induced in malaria-naïve individuals, even after exposure to relatively low gametocyte densities (up to ~1,600 gametocytes/mL). In contrast to antibody responses to well-characterised asexual blood stage antigens that were detectable by day 21 after infection, responses to sexual stage antigens (including transmission blocking vaccine candidates Pfs48/45 and Pfs230) were only apparent at 51 days after infection. We found antigens previously associated with early gametocyte or anti-gamete immunity were highly represented among responses linked with gametocyte exposure. Our data provide detailed insights on the induction and kinetics of antibody responses to gametocytes and identify novel antigens that elicit antibody responses exclusively in individuals with gametocyte exposure. Our findings provide target identification for serological assays for surveillance of the malaria infectious reservoir, and support vaccine development by describing the antibody response to leading vaccine antigens after primary infection