Zika-specific neutralizing antibodies targeting inter-dimer envelope epitopes.

Zika virus (ZIKV) is an emerging pathogen that causes devastating congenital defects. The overlapping epidemiology and immunologic cross-reactivity between ZIKV and dengue virus (DENV) pose complex challenges to vaccine design, given the potential for antibody-dependent enhancement of disease. Therefore, classification of ZIKV-specific antibody targets is of notable value. From a ZIKV-infected rhesus macaque, we identify ZIKV-reactive B cells and isolate potent neutralizing monoclonal antibodies (mAbs) with no cross-reactivity to DENV. We group these mAbs into four distinct antigenic groups targeting ZIKV-specific cross-protomer epitopes on the envelope glycoprotein. Co-crystal structures of representative mAbs in complex with ZIKV envelope glycoprotein reveal envelope-dimer epitope and unique dimer-dimer epitope targeting. All four specificities are serologically identified in convalescent humans following ZIKV infection, and representative mAbs from all four groups protect against ZIKV replication in mice. These results provide key insights into ZIKV-specific antigenicity and have implications for ZIKV vaccine, diagnostic, and therapeutic development.

Low-dose in vivo protection and neutralization across SARS-CoV-2 variants by monoclonal antibody combinations.

Prevention of viral escape and increased coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern require therapeutic monoclonal antibodies (mAbs) targeting multiple sites of vulnerability on the coronavirus spike glycoprotein. Here we identify several potent neutralizing antibodies directed against either the N-terminal domain (NTD) or the receptor-binding domain (RBD) of the spike protein. Administered in combinations, these mAbs provided low-dose protection against SARS-CoV-2 infection in the K18-human angiotensin-converting enzyme 2 mouse model, using both neutralization and Fc effector antibody functions. The RBD mAb WRAIR-2125, which targets residue F486 through a unique heavy-chain and light-chain pairing, demonstrated potent neutralizing activity against all major SARS-CoV-2 variants of concern. In combination with NTD and other RBD mAbs, WRAIR-2125 also prevented viral escape. These data demonstrate that NTD/RBD mAb combinations confer potent protection, likely leveraging complementary mechanisms of viral inactivation and clearance.

Potent Zika and dengue cross-neutralizing antibodies induced by Zika vaccination in a dengue-experienced donor.

Zika virus (ZIKV) has caused significant disease, with widespread cases of neurological pathology and congenital neurologic defects. Rapid vaccine development has led to a number of candidates capable of eliciting potent ZIKV-neutralizing antibodies (reviewed in refs. 1-3). Despite advances in vaccine development, it remains unclear how ZIKV vaccination affects immune responses in humans with prior flavivirus immunity. Here we show that a single-dose immunization of ZIKV purified inactivated vaccine (ZPIV)4-7 in a dengue virus (DENV)-experienced human elicited potent cross-neutralizing antibodies to both ZIKV and DENV. Using a unique ZIKV virion-based sorting strategy, we isolated and characterized multiple antibodies, including one termed MZ4, which targets a novel site of vulnerability centered on the Envelope (E) domain I/III linker region and protects mice from viremia and viral dissemination following ZIKV or DENV-2 challenge. These data demonstrate that Zika vaccination in a DENV-experienced individual can boost pre-existing flavivirus immunity and elicit protective responses against both ZIKV and DENV. ZPIV vaccination in Puerto Rican individuals with prior flavivirus experience yielded similar cross-neutralizing potency after a single vaccination, highlighting the potential benefit of ZIKV vaccination in flavivirus-endemic areas.

Causative agent of canine heartworm (Dirofilaria immitis) detected in wild lemurs.

The lemurs of Madagascar are threatened by human activities. We present the first molecular detection of canine heartworm (Dirofilaria immitis) in a wild non-human primate, the mouse lemur (Microcebus rufus). Zoonotic D. immitis infection has been associated with clinical pathology that includes serious and often fatal cardiac and pulmonary reactions. With human encroachment and associated increases in free-roaming dog populations in Madagascar, we examined lemurs for zoonotic canid pathogens. D. immitis presents a new potential conservation threat to lemurs. We highlight the need for wide-ranging and effective interventions, particularly near protected areas, to address this growing conservation issue.

Backpack PCR: A point-of-collection diagnostic platform for the rapid detection of Brugia parasites in mosquitoes.

BACKGROUND: Currently, molecular xenomonitoring efforts for lymphatic filariasis rely on PCR or real-time PCR-based detection of Brugia malayi, Brugia timori and Wuchereria bancrofti in mosquito vectors. Most commonly, extraction of DNA from mosquitoes is performed using silica column-based technologies. However, such extractions are both time consuming and costly, and the diagnostic testing which follows typically requires expensive thermal cyclers or real-time PCR instruments. These expenses present significant challenges for laboratories in many endemic areas. Accordingly, in such locations, there exists a need for inexpensive, equipment-minimizing diagnostic options that can be transported to the field and implemented in minimal resource settings. Here we present a novel diagnostic approach for molecular xenomonitoring of filarial parasites in mosquitoes that uses a rapid, NaOH-based DNA extraction methodology coupled with a portable, battery powered PCR platform and a test strip-based DNA detection assay. While the research reported here serves as a proof-of-concept for the backpack PCR methodology for the detection of filarial parasites in mosquitoes, the platform should be easily adaptable to the detection of W. bancrofti and other mosquito-transmitted pathogens. METHODOLOGY/PRINCIPAL FINDINGS: Through comparisons with standard silica column-based DNA extraction techniques, we evaluated the performance of a rapid, NaOH-based methodology for the extraction of total DNA from pools of parasite-spiked vector mosquitoes. We also compared our novel test strip-based detection assay to real-time PCR and conventional PCR coupled with gel electrophoresis, and demonstrated that this method provides sensitive and genus-specific detection of parasite DNA from extracted mosquito pools. Finally, by comparing laboratory-based thermal cycling with a field-friendly miniaturized PCR approach, we have demonstrated the potential for the point-of-collection-based use of this entire diagnostic platform that is compact enough to fit into a small backpack. CONCLUSIONS/SIGNIFICANCE: Because this point-of-collection diagnostic platform eliminates reliance on expensive and bulky instrumentation without compromising sensitivity or specificity of detection, it provides an alternative to cost-prohibitive column-dependent DNA extractions that are typically coupled to detection methodologies requiring advanced laboratory infrastructure. In doing so, this field-ready system should increase the feasibility of molecular xenomonitoring within B. malayi-endemic locations. Of greater importance, this backpack PCR system also provides the proof-of-concept framework for the development of a parallel assay for the detection of W. bancrofti.

Profiling the macrofilaricidal effects of flubendazole on adult female Brugia malayi using RNAseq.

The use of microfilaricidal drugs for the control of onchocerciasis and lymphatic filariasis (LF) necessitates prolonged yearly dosing. Prospects for elimination or eradication of these diseases would be enhanced by the availability of a macrofilaricidal drug. Flubendazole (FLBZ), a benzimidazole anthelmintic, is an appealing candidate. FLBZ has demonstrated potent macrofilaricidal effects in a number of experimental rodent models and in one human trial. Unfortunately, FLBZ was deemed unsatisfactory for use in mass drug administration campaigns due to its limited oral bioavailability. A new formulation that enables sufficient bioavailability following oral administration could render FLBZ an effective treatment for onchocerciasis and LF. Identification of drug-derived effects is important in ascertaining a dosage regimen which is predicted to be lethal to the parasite in situ. In previous histological studies, exposure to FLBZ induced damage to tissues required for reproduction and survival at pharmacologically relevant concentrations. However, more precise and quantitative indices of drug effects are needed. This study assessed drug effects using a transcriptomic approach to confirm effects observed histologically and to identify genes which were differentially expressed in treated adult female Brugia malayi. Comparative analysis across different concentrations (1 µM and 5 µM) and durations (48 and 120 h) provided an overview of the processes which are affected by FLBZ exposure. Genes with dysregulated expression were consistent with the reproductive effects observed via histology in our previous studies. This study revealed transcriptional changes in genes involved in embryo development. Additionally, significant downregulation was observed in genes encoding cuticle components, which may reflect changes in developing embryos, the adult worm cuticle or both. These data support the hypothesis that FLBZ acts predominantly on rapidly dividing cells, and provides a basis for selecting molecular markers of drug-induced damage which may be of use in predicting efficacious FLBZ regimens.

The Effects of Ivermectin on Brugia malayi Females In Vitro: A Transcriptomic Approach.

BACKGROUND: Lymphatic filariasis and onchocerciasis are disabling and disfiguring neglected tropical diseases of major importance in developing countries. Ivermectin is the drug of choice for mass drug administration programs for the control of onchocerciasis and lymphatic filariasis in areas where the diseases are co-endemic. Although ivermectin paralyzes somatic and pharyngeal muscles in many nematodes, these actions are poorly characterized in adult filariae. We hypothesize that paralysis of pharyngeal pumping by ivermectin in filariae could result in deprivation of essential nutrients, especially iron, inducing a wide range of responses evidenced by altered gene expression, changes in metabolic pathways, and altered developmental states in embryos. Previous studies have shown that ivermectin treatment significantly reduces microfilariae release from females within four days of exposure in vivo, while not markedly affecting adult worms. However, the mechanisms responsible for reduced production of microfilariae are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed transcriptomic profiles from Brugia malayi adult females, an important model for other filariae, using RNAseq technology after exposure in culture to ivermectin at various concentrations (100 nM, 300 nM and 1 µM) and time points (24, 48, 72 h, and 5 days). Our analysis revealed drug-related changes in expression of genes involved in meiosis, as well as oxidative phosphorylation, which were significantly down-regulated as early as 24 h post-exposure. RNA interference phenotypes of the orthologs of these down-regulated genes in C. elegans include "maternal sterile", "embryonic lethal", "larval arrest", "larval lethal" and "sick". CONCLUSION/SIGNIFICANCE: These changes provide insight into the mechanisms involved in ivermectin-induced reduction in microfilaria output and impaired fertility, embryogenesis, and larval development.

A Novel Xenomonitoring Technique Using Mosquito Excreta/Feces for the Detection of Filarial Parasites and Malaria.

BACKGROUND: Given the continued successes of the world's lymphatic filariasis (LF) elimination programs and the growing successes of many malaria elimination efforts, the necessity of low cost tools and methodologies applicable to long-term disease surveillance is greater than ever before. As many countries reach the end of their LF mass drug administration programs and a growing number of countries realize unprecedented successes in their malaria intervention efforts, the need for practical molecular xenomonitoring (MX), capable of providing surveillance for disease recrudescence in settings of decreased parasite prevalence is increasingly clear. Current protocols, however, require testing of mosquitoes in pools of 25 or fewer, making high-throughput examination a challenge. The new method we present here screens the excreta/feces from hundreds of mosquitoes per pool and provides proof-of-concept for a practical alternative to traditional methodologies resulting in significant cost and labor savings. METHODOLOGY/PRINCIPAL FINDINGS: Excreta/feces of laboratory reared Aedes aegypti or Anopheles stephensi mosquitoes provided with a Brugia malayi microfilaria-positive or Plasmodium vivax-positive blood meal respectively were tested for the presence of parasite DNA using real-time PCR. A titration of samples containing various volumes of B. malayi-negative mosquito feces mixed with positive excreta/feces was also tested to determine sensitivity of detection. Real-time PCR amplification of B. malayi and P. vivax DNA from the excreta/feces of infected mosquitoes was demonstrated, and B. malayi DNA in excreta/feces from one to two mf-positive blood meal-receiving mosquitoes was detected when pooled with volumes of feces from as many as 500 uninfected mosquitoes. CONCLUSIONS/SIGNIFICANCE: While the operationalizing of excreta/feces testing may require the development of new strategies for sample collection, the high-throughput nature of this new methodology has the potential to greatly reduce MX costs. This will prove particularly useful in post-transmission-interruption settings, where this inexpensive approach to long-term surveillance will help to stretch the budgets of LF and malaria elimination programs. Furthermore, as this methodology is adaptable to the detection of both single celled (P. vivax) and multicellular eukaryotic pathogens (B. malayi), exploration of its use for the detection of various other mosquito-borne diseases including viruses should be considered. Additionally, integration strategies utilizing excreta/feces testing for the simultaneous surveillance of multiple diseases should be explored.

Phenotypic and molecular analysis of the effect of 20-hydroxyecdysone on the human filarial parasite Brugia malayi.

A homologue of the ecdysone receptor has been identified and shown to be responsive to 20-hydroxyecdysone in Brugia malayi. However, the role of this master regulator of insect development has not been delineated in filarial nematodes. Gravid adult female B. malayi cultured in the presence of 20-hydroxyecdysone produced significantly more microfilariae and abortive immature progeny than control worms, implicating the ecdysone receptor in regulation of embryogenesis and microfilarial development. Transcriptome analyses identified 30 genes whose expression was significantly up-regulated in 20-hydroxyecdysone-treated parasites compared with untreated controls. Of these, 18% were identified to be regulating transcription. A comparative proteomic analysis revealed 932 proteins to be present in greater amounts in extracts of 20-hydroxyecdysone-treated adult females than in extracts prepared from worms cultured in the absence of the hormone. Of the proteins exhibiting a greater than two-fold difference in the 20-hydroxyecdysone-treated versus untreated parasite extracts, 16% were involved in transcriptional regulation. RNA interference (RNAi) phenotype analysis of Caenorhabditis elegans orthologs revealed that phenotypes involved in developmental processes associated with embryogenesis were significantly over-represented in the transcripts and proteins that were up-regulated by exposure to 20-hydroxyecdysone. Taken together, the transcriptomic, proteomic and phenotypic data suggest that the filarial ecdysone receptor may play a role analogous to that in insects, where it serves as a regulator of egg development.

The Effect of In Vitro Cultivation on the Transcriptome of Adult Brugia malayi.

BACKGROUND: Filarial nematodes cause serious and debilitating infections in human populations of tropical countries, contributing to an entrenched cycle of poverty. Only one human filarial parasite, Brugia malayi, can be maintained in rodents in the laboratory setting. It has been a widely used model organism in experiments that employ culture systems, the impact of which on the worms is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using Illumina RNA sequencing, we characterized changes in gene expression upon in vitro maintenance of adult B. malayi female worms at four time points: immediately upon removal from the host, immediately after receipt following shipment, and after 48 h and 5 days in liquid culture media. The dramatic environmental change and the 24 h time lapse between removal from the host and establishment in culture caused a globally dysregulated gene expression profile. We found a maximum of 562 differentially expressed genes based on pairwise comparison between time points. After an initial shock upon removal from the host and shipping, a few stress fingerprints remained after 48 h in culture and until the experiment was stopped. This was best illustrated by a strong and persistent up-regulation of several genes encoding cuticle collagens, as well as serpins. CONCLUSIONS/SIGNIFICANCE: These findings suggest that B. malayi can be maintained in culture as a valid system for pharmacological and biological studies, at least for several days after removal from the host and adaptation to the new environment. However, genes encoding several stress indicators remained dysregulated until the experiment was stopped.