Receptor-binding residues lie in central regions of duffy-binding-like domains involved in red cell invasion and cytoadherence by malaria parasites

Erythrocyte invasion by malaria parasites and cytoadherence of Plasmodium falciparum-infected erythrocytes to host capillaries are 2 key pathogenic mechanisms in malaria. The receptor-binding domains of erythrocyte-binding proteins (EBPs) such as Plasmodium falciparum EBA-175, which mediate invasion, and P falciparum erythrocyte membrane protein 1 (PfEMP-1) family members, which are encoded by var genes and mediate cytoadherence, have been mapped to conserved cysteine-rich domains referred to as Duffy-binding-like (DBL) domains. Here, we have mapped regions within DBL domains from EBPs and PfEMP-1 that contain receptor-binding residues. Using biochemical and molecular methods we demonstrate that the receptor-binding residues of parasite ligands that bind sialic acid on glycophorin A for invasion as well as complement receptor-1 and chondroitin sulfate A for cytoadherence map to central regions of DBL domains. In contrast, binding to intercellular adhesion molecule 1 (ICAM-1) requires both the central and terminal regions of DBLβC2 domains. Determination of functional regions within DBL domains is the first step toward understanding the structure-function bases for their interaction with diverse host receptors

Forensics for tracing microbial signatures: Biodefence perspective and preparedness for the unforeseen

23-31Biological weapons are assigned high priority in homeland security, defence, counterproliferation, nonproliferation, intelligence, and counterterrorist programmes. In order to strengthen active defense against development and use of these weapons, several comprehensive technological and forensic capabilities have been developed world over for investigative, intelligence, prosecutive, diplomatic and policy purposes. Microbial forensics is one of such strategies. It is a new discipline combining microbiology and forensic science. Microbial forensics associate the source of the causative agent with a specific individual or group by measuring molecular variations between related microbial strains. In this context, several advanced molecular techniques and practices including molecular phylogeny, whole genome sequencing, microarray analysis, DNA finger printing, etc., can be extremely valuable and effective analytical tools in a microbial forensic investigation. Results from such analyses may be related to the intentional use of microbial agents for bioterrorism or the accidental release of any offensive microorganisms or toxins of public health significance, specifically for the purpose of determining the origin. The new discipline of microbial forensics is an integration of an array of well established fields, such as microbial genomics, phylogenetics, forensic informatics and classical microbiology

Structural analogs of sialic acid interfere with the binding of erythrocyte binding antigen-175 to glycophorin A, an interaction crucial for erythrocyte invasion by Plasmodium falciparum

Plasmodium falciparum causes the most virulent form of malaria and remains a major worldwide health problem. The erythrocytic development of P. falciparum relies on parasite invasion of host erythrocytes, a process mediated in part by the interaction of erythrocyte binding antigen 175 (EBA-175) with the erythrocyte receptor glycophorin A (GA). The binding domain of EBA-175 that interacts with glycophorin A is a ~330 residues module called F2. Several studies have shown that F2 recognizes both sialic acids and the protein backbone on glycophorin A. Here, we have developed ELISA-based quantitative F2-GA binding assays. We also performed a series of competitive inhibition assays to block the F2-GA interaction using a variety of sialic acid analogs. Our data show that both 2,3-didehydro-2-deoxy-N-acetyl neuraminic acid (DANA) and 3'-N-acetyl neuraminyl-N-acetyl lactosamine are excellent inhibitors of the F2-GA interaction. Moderate levels of inhibition were also observed with monomers or oligomers of N-acetyl neuraminic acid (sialic acid). Furthermore, we show that DANA is able to significantly inhibit the invasion of erythrocytes by P. falciparum. Together, our ELISA-based binding assays and in vitro inhibition of erythrocyte invasion data suggest that small variations in the structures of DANA and related inhibitors can result in even more potent invasion inhibitory activities. Our studies provide a platform for the development of high potency inhibitors of the F2-GA interaction using high throughput drug discovery technologies. Such compounds may form part of inhibitor cocktails, which aim to block invasion of erythrocytes by P. falciparum

Structural and functional dissection of the adhesive domains of Plasmodium falciparum thrombospondin-related anonymous protein (TRAP).

TRAP (thrombospondin-related anonymous protein) is a sporozoite surface protein that plays a central role in hepatocyte invasion. We have developed procedures for recombinant production of the entire ECD (extracellular domain) and A domain of TRAP using bacterial- and baculovirus-expression systems respectively. The ECD and A domain were purified to homogeneity and migrated on gel-filtration columns as non-aggregated, monomeric proteins. These adhesive modules bound to HepG2 cells in a dose-dependent and bivalent cation-independent manner. The binding of ECD and the A domain to HepG2 cells was inhibited poorly by an excess of sulphatide analogues, suggesting the presence of as yet unidentified receptors for the A domain on hepatocytes. Using surface-plasmon-resonance-based sensor technology (Biacore), we demonstrate that TRAP ECD has higher affinity for heparin (K(D)=40 nM) compared with the A domain (K(D)=79 nM). We also present a three-dimensional structure of the A domain based on the crystal structure of the homologous von Willebrand factor A1 domain. The TRAP A domain shows two spatially distinct ligand-binding surfaces. One surface on the A domain contains the MIDAS (metal-ion-dependent adhesion site) motif, where point mutations of Thr131 and Asp162 correlate with impairment of cell infectivity by sporozoites. The other surface contains a putative heparin-binding site and consists of a basic residue cluster. Our studies suggest that TRAP interacts with multiple receptors during the hepatocyte invasion process. Our results also pave the way for inclusion of these high-quality recombinant TRAP domains in subunit-based vaccines against malaria

Fusogenic peptide as diagnostic marker for detection of flaviviruses

Background: Dengue, Japanese encephalitis, West Nile encephalitis, yellow fever are the common flaviviral diseases associated with high morbidity and mortality. The initial symptoms of most of the flaviviral infections are similar to each other as well as to some other viral diseases. Making clinical diagnosis, therefore, becomes a challenging task for the clinician. Several studies have been reported on using detection of serum antibodies against flavivirus for the diagnosis of specific flaviviral disease; no field-based pan-flavi virus detection system is available, which can be used in low-endemicity areas for differentiation of flaviviral disease from other viral diseases. Aim: To identify a conserved amino acid sequence among all flaviviruses and evaluate the antibody formed against the conserved peptide to develop pan-flavivirus detection system. Materials and Methods: In the present study we have compared amino acid sequences of several flaviviruses and identified a conserved amino acid sequence lying in domain II of envelope protein. Results : A peptide having the conserved amino acid sequence was used to generate polyclonal antibodies and these antibodies were used to detect several flaviviruses. Anti-peptide polyclonal antibodies selectively recognized flaviviruses and did not detect non-flaviviruses. Anti-peptide antibodies detected presence of virus in serum spiked with pure virus preparations. Conclusion: The study offers a rationale for development of pan-flavivirus capture assay suitable for low endemic areas

Indian Biosimilars and Vaccines at Crossroads–Replicating the Success of Pharmagenerics

Background: The global pharma sector is fast shifting from generics to biologics and biosimilars with the first approval in Europe in 2006 followed by US approval in 2015. In the form of Hepatitis B vaccine, India saw its first recombinant biologics approval in 2000. Around 20% of generic medications and 62% of vaccines are now supplied by the Indian pharmaceutical industry. It is this good position in biologics and biosimilars production that could potentially improve healthcare via decreased treatment cost. India has witnessed large investments in biosimilars over the years. Numerous India-bred new players, e.g., Enzene Biosciences Ltd., are keen on biosimilars and have joined the race alongside the emerging giants, e.g., Biocon and Dr. Reddy’s. A very positive sign was the remarkable disposition during the COVID-19 pandemic by Bharat Biotech and the Serum Institute of India. India’s biopharmaceutical industry has been instrumental in producing and supplying preventives and therapeutics to fight COVID-19. Despite a weak supply chain and workforce pressure, the production was augmented to provide reasonably priced high-quality medications to more than 133 nations. Biosimilars could cost-effectively treat chronic diseases involving expensive conventional therapies, including diabetes, respiratory ailments, cancer, and connective tissue diseases. Biologics and biosimilars have been and are being tested to treat and manage COVID-19 symptoms characterized by inflammation and respiratory distress. Purpose of review: Although India boasts many universities, research centers, and a relatively skilled workforce, its global University–Industry collaboration ranking is 24, IPR ranking remains 47 and innovation ranking 39. This reveals a wide industry-academia gap to bridge. There are gaps in effective translational research in India that must be promptly and appropriately addressed. Innovation demands strong and effective collaborations among universities, techno-incubators, and industries. Methodology: Many successful research findings in academia do not get translation opportunities supposedly due to low industrial collaboration, low IP knowledge, and publication pressure with stringent timelines. In light of this, a detailed review of literature, including policy papers, government initiatives, and corporate reviews, was carried out, and the compilation and synthesis of the secondary data were meticulously summarized for the easy comprehension of the facts and roadmap ahead. For easy comprehension, charts, figures, and compiled tables are presented. Results: This review assesses India’s situation in the biosimilar space, the gaps and areas to improve for Indian investment strategies, development, and innovation, addressing need for a more skilled workforce, industrial collaboration, and business models. Conclusions: This review also proposes forward an approach to empowering technopreneurs to develop MSMEs for large-scale operations to support India in taking innovative thoughts to the global level to ultimately realize a self-reliant India. The limitations of the compilation are also highlighted towards the end

Sequence polymorphisms in the receptor-binding domain of Plasmodium falciparum EBA-175: implications for malaria vaccine development

This article does not have an abstract

Bacterially expressed and refolded receptor binding domain of Plasmodium falciparum EBA-175 elicits invasion inhibitory antibodies

Malaria parasites make specific receptor-ligand interactions to invade erythrocytes. A 175 kDa Plasmodium falciparum erythrocyte binding antigen (EBA-175) binds sialic acid residues on glycophorin A during invasion of human erythrocytes. The receptor-binding domain of EBA-175 lies in a conserved, amino-terminal, cysteine-rich region, region F2 of EBA-175 (PfF2), that is homologous to the binding domains of other erythrocyte binding proteins such as Plasmodium vivax Duffy binding protein. We have developed methods to produce recombinant PfF2 in its functional form. Recombinant PfF2 was expressed in Escherichia coli, purified from inclusion bodies, renatured by oxidative refolding and purified to homogeneity by ion-exchange and gel filtration chromatography. Refolded PfF2 has been characterized using biochemical and biophysical methods and shown to be pure, homogenous and functional in that it binds human erythrocytes with specificity. Immunization with refolded PfF2 yields high titre antibodies that efficiently inhibit P. falciparum invasion of erythrocytes in vitro. Importantly, antibodies raised against PfF2 block invasion by a P. falciparum field isolate that invades erythrocytes using multiple pathways. These observations support the development of recombinant PfF2 as a vaccine candidate for P. falciparum malaria

Microbial forensics

Burlington, US