S9.6-based hybrid capture immunoassay for pathogen detection.

The detection of pathogens is critical for clinical diagnosis and public health surveillance. Detection is usually done with nucleic acid-based tests (NATs) and rapid antigen tests (e.g., lateral flow assays [LFAs]). Although NATs are more sensitive and specific, their use is often limited in resource-poor settings due to specialized requirements. To address this limitation, we developed a rapid DNA-RNA Hybrid Capture immunoassay (HC) that specifically detects RNA from pathogens. This assay utilizes a unique monoclonal antibody, S9.6, which binds DNA-RNA hybrids. Biotinylated single-stranded DNA probes are hybridized to target RNAs, followed by hybrid capture on streptavidin and detection with S9.6. The HC-ELISA assay can detect as few as 104 RNA molecules that are 2.2 kb in length. We also adapted this assay into a LFA format, where captured Bacillus anthracis rpoB RNA of 3.5 kb length was detectable from a bacterial load equivalent to 107 CFU per 100 mg of mouse tissue using either HC-ELISA or HC-LFA. Importantly, we also demonstrated the versatility of HC by detecting other pathogens, including SARS-CoV-2 and Toxoplasma gondii, showing its potential for broad pathogen detection. Notably, HC does not require amplification of the target nucleic acid and utilizes economical formats like ELISA and LFA, making it suitable for use in sentinel labs for pathogen detection or as a molecular tool in basic research laboratories. Our study highlights the potential of HC as a sensitive and versatile method for RNA-based pathogen detection.

Correction: Onchocerciasis: Target product profiles of in vitro diagnostics to support onchocerciasis elimination mapping and mass drug administration stopping decisions.

[This corrects the article DOI: 10.1371/journal.pntd.0010682.].

Onchocerciasis: Target product profiles of in vitro diagnostics to support onchocerciasis elimination mapping and mass drug administration stopping decisions.

In June 2021, the World Health Organization (WHO), recognizing the need for new diagnostics to support the control and elimination of onchocerciasis, published the target product profiles (TPPs) of new tests that would support the two most immediate needs: (a) mapping onchocerciasis in areas of low prevalence and (b) deciding when to stop mass drug administration programs. In both instances, the test should ideally detect an antigen specific for live, adult O. volvulus female worms. The preferred format is a field-deployable rapid test. For mapping, the test needs to be ≥ 60% sensitive and ≥ 99.8% specific, while to support stopping decisions, the test must be ≥ 89% sensitive and ≥ 99.8% specific. The requirement for extremely high specificity is dictated by the need to detect with sufficient statistical confidence the low seroprevalence threshold set by WHO. Surveys designed to detect a 1-2% prevalence of a given biomarker, as is the case here, cannot tolerate more than 0.2% of false-positives. Otherwise, the background noise would drown out the signal. It is recognized that reaching and demonstrating such a stringent specificity criterion will be challenging, but test developers can expect to be assisted by national governments and implementing partners for adequately powered field validation.

Diagnostics and the neglected tropical diseases roadmap: setting the agenda for 2030.

Accurate and reliable diagnostic tools are an essential requirement for neglected tropical diseases (NTDs) programmes. However, the NTD community has historically underinvested in the development and improvement of diagnostic tools, potentially undermining the successes achieved over the last 2 decades. Recognizing this, the WHO, in its newly released draft roadmap for NTD 2021-2030, has identified diagnostics as one of four priority areas requiring concerted action to reach the 2030 targets. As a result, WHO established a Diagnostics Technical Advisory Group (DTAG) to serve as the collaborative mechanism to drive progress in this area. Here, the purpose and role of the DTAG are described in the context of the challenges facing NTD programmes.

Proof-of-Concept Rapid Diagnostic Test for Onchocerciasis: Exploring Peptide Biomarkers and the Use of Gold Nanoshells as Reporter Nanoparticles.

Three O. volvulus immunogenic peptide sequences recently discovered by peptide microarray were adapted to a lateral flow assay (LFA). The LFA employs gold nanoshells as novel high-contrast reporter nanoparticles and detects a serological response against the 3 peptides, found in OvOC9384, OvOC198, and OvOC5528, respectively. When tested on 118 sera from O. volvulus infected patients and 208 control sera, the LFA was 90%, 63%, and 98% sensitive for each peptide, respectively, and 99-100% specific vs samples from healthy volunteers. Samples of other filarial infections cross-reacted by 7-24%. The sensitivity, specificity, and cross-reactivity values matched those obtained by ELISA with the same sample set. While the exact choice of peptide(s) will require fine-tuning, this work establishes that O. volvulus peptides identified by peptide microarray can be translated into an antibody-based LFA and that gold nanoshells provide the same sensitivity, specificity, and cross-reactivity as the corresponding ELISA assays.

A novel rapid test for detecting antibody responses to Loa loa infections.

Ivermectin-based mass drug administration (MDA) programs have achieved remarkable success towards the elimination of onchocerciasis and lymphatic filariasis. However, their full implementation has been hindered in Central Africa by the occurrence of ivermectin-related severe adverse events (SAEs) in a subset of individuals with high circulating levels of Loa loa microfilariae. Extending MDA to areas with coincident L. loa infection is problematic, and inexpensive point-of-care tests for L. loa are acutely needed. Herein, we present a lateral flow assay (LFA) to identify subjects with a serological response to Ll-SXP-1, a specific and validated marker of L. loa. The test was evaluated on serum samples from patients infected with L. loa (n = 109) and other helminths (n = 204), as well as on uninfected controls (n = 77). When read with the naked eye, the test was 94% sensitive for L. loa infection and was 100% specific when sera from healthy endemic and non-endemic controls or from those with S. stercoralis infections were used as the comparators. When sera of patients with O. volvulus, W. bancrofti, or M. perstans were used as the comparators, the specificity of the LFA was 82%, 87%, and 88%, respectively. A companion smartphone reader allowed measurement of the test line intensities and establishment of cutoff values. With a cutoff of 600 Units, the assay sensitivity decreased to 71%, but the specificity increased to 96% for O. volvulus, 100% for W. bancrofti, and 100% for M. perstans-infected individuals. The LFA may find applications in refining the current maps of L. loa prevalence, which are needed to eliminate onchocerciasis and lymphatic filariasis from the African continent.

Recent advances in malaria drug discovery.

This digest covers some of the most relevant progress in malaria drug discovery published between 2010 and 2012. There is an urgent need to develop new antimalarial drugs. Such drugs can target the blood stage of the disease to alleviate the symptoms, the liver stage to prevent relapses, and the transmission stage to protect other humans. The pipeline for the blood stage is becoming robust, but this should not be a source of complacency, as the current therapies set a high standard. Drug discovery efforts directed towards the liver and transmission stages are in their infancy but are receiving increasing attention as targeting these stages could be instrumental in eradicating malaria.

EC144 is a potent inhibitor of the heat shock protein 90.

Alkyne 40, 5-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methylpent-4-yn-2-ol (EC144), is a second generation inhibitor of heat shock protein 90 (Hsp90) and is substantially more potent in vitro and in vivo than the first generation inhibitor 14 (BIIB021) that completed phase II clinical trials. Alkyne 40 is more potent than 14 in an Hsp90α binding assay (IC(50) = 1.1 vs 5.1 nM) as well as in its ability to degrade Her-2 in MCF-7 cells (EC(50) = 14 vs 38 nM). In a mouse model of gastric tumors (N87), 40 stops tumor growth at 5 mg/kg and causes partial tumor regressions at 10 mg/kg (po, qd × 5). Under the same conditions, 14 stops tumor growth only at 120 mg/kg, and does not induce partial regressions. Thus, alkyne 40 is approximately 20-fold more efficacious than 14 in mice.

EC144, a synthetic inhibitor of heat shock protein 90, blocks innate and adaptive immune responses in models of inflammation and autoimmunity.

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding and stabilizing multiple intracellular proteins that have roles in cell activation and proliferation. Many Hsp90 client proteins in tumor cells are mutated or overexpressed oncogenic proteins driving cancer cell growth, leading to the acceptance of Hsp90 as a potential therapeutic target for cancer. Because several signal transduction molecules that are dependent on Hsp90 function are also involved in activation of innate and adaptive cells of the immune system, we investigated the mechanism by which inhibiting Hsp90 leads to therapeutic efficacy in rodent models of inflammation and autoimmunity. EC144, a synthetic Hsp90 inhibitor, blocked LPS-induced TLR4 signaling in RAW 264.7 cells by inhibiting activation of ERK1/2, MEK1/2, JNK, and p38 MAPK but not NF-κB. Ex vivo LPS-stimulated CD11b(+) peritoneal exudate cells from EC144-treated mice were blocked from phosphorylating tumor progression locus 2, MEK1/2, and ERK1/2. Consequently, EC144-treated mice were resistant to LPS administration and had suppressed systemic TNF-α release. Inhibiting Hsp90 also blocked in vitro CD4(+) T cell proliferation in mouse and human MLRs. In vivo, semitherapeutic administration of EC144 blocked disease development in rat collagen-induced arthritis by suppressing the inflammatory response. In a mouse collagen-induced arthritis model, EC144 also suppressed disease development, which correlated with a suppressed Ag-specific Ab response and a block in activation of Ag-specific CD4(+) T cells. Our results describe mechanisms by which blocking Hsp90 function may be applicable to treatment of autoimmune diseases involving inflammation and activation of the adaptive immune response.

Rationally designed high-affinity 2-amino-6-halopurine heat shock protein 90 inhibitors that exhibit potent antitumor activity.

Heat shock protein 90 (Hsp90) is a molecular chaperone protein implicated in stabilizing the conformation and maintaining the function of many cell-signaling proteins. Many oncogenic proteins are more dependent on Hsp90 in maintaining their conformation, stability, and maturation than their normal counterparts. Furthermore, recent data show that Hsp90 exists in an activated form in malignant cells but in a latent inactive form in normal tissues, suggesting that inhibitors selective for the activated form could provide a high therapeutic index. Hence, Hsp90 is emerging as an exciting new target for the treatment of cancer. We now report on a novel series of 2-amino-6-halopurine Hsp90 inhibitors exemplified by 2-amino-6-chloro-9-(4-iodo-3,5-dimethylpyridin-2-ylmethyl)purine (30). These highly potent inhibitors (IC50 of 30 = 0.009 microM in a HER-2 degradation assay) also display excellent antiproliferative activity against various tumor cell lines (IC50 of 30 = 0.03 microM in MCF7 cells). Moreover, this class of inhibitors shows higher affinity for the activated form of Hsp90 compared to our earlier 8-sulfanylpurine Hsp90 inhibitor series. When administered orally to mice, these compounds exhibited potent tumor growth inhibition (>80%) in an N87 xenograft model, similar to that observed with 17-allylamino-17-desmethoxygeldanamycin (17-AAG), which is a compound currently in phase I/II clinical trials.

Orally active purine-based inhibitors of the heat shock protein 90.

Orally active Hsp90 inhibitors are of interest as potential chemotherapeutic agents. Recently, fully synthetic 8-benzyladenines and 8-sulfanyladenines such as 4 were disclosed as Hsp90 inhibitors, but these compounds are not water soluble and consequently have unacceptably low oral bioavailabilities. We now report that water-solubility can be achieved by inserting an amino functionality in the N(9) side chain. This results in compounds that are potent, soluble in aqueous media, and orally bioavailable. In an HER-2 degradation assay, the highest potency was achieved with the neopentylamine 42 (HER-2 IC(50) = 90 nM). In a murine tumor xenograft model (using the gastric cancer cell line N87), the H(3)PO(4) salts of the amines 38, 39, and 42 induced tumor growth inhibition when administered orally at 200 mg/kg/day. The amines 38, 39, and 42 are the first Hsp90 inhibitors shown to inhibit tumor growth upon oral dosage.

Preparation of 8-(arylsulfanyl)adenines with diazonium salts under mild, aerobic conditions.

8-(Arylsulfanyl)adenines 11 were prepared in up to 75% yield by reacting the 8-thionoadenine 6 (acetic acid 3-(6-amino-8-thioxo-7,8-dihydropurin-9-yl)propyl ester) with benzenediazonium tetrafluoroborates in DMSO. Benzenediazonium ions carrying an electron-withdrawing substituent gave the highest yields. The reaction proceeded smoothly at room temperature without any base and could be performed under air atmosphere. The extremely mild conditions are compatible with a wide range of functional groups.