Serological Assays Estimate Highly Variable SARS-CoV-2 Neutralizing Antibody Activity in Recovered COVID-19 Patients.

The development of neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) following infection or vaccination is likely to be critical for the development of sufficient population immunity to drive cessation of the coronavirus disease of 2019 (COVID-19) pandemic. A large number of serologic tests, platforms, and methodologies are being employed to determine seroprevalence in populations to select convalescent plasma samples for therapeutic trials and to guide policies about reopening. However, the tests have substantial variations in sensitivity and specificity, and their ability to quantitatively predict levels of NAbs is unknown. We collected 370 unique donors enrolled in the New York Blood Center Convalescent Plasma Program between April and May of 2020. We measured levels of antibodies in convalescent plasma samples using commercially available SARS-CoV-2 detection tests and in-house enzyme-linked immunosorbent assays (ELISAs) and correlated serological measurements with NAb activity measured using pseudotyped virus particles, which offer the most informative assessment of antiviral activity of patient sera against viral infection. Our data show that a large proportion of convalescent plasma samples have modest antibody levels and that commercially available tests have various degrees of accuracy in predicting NAb activity. We found that the Ortho anti-SARS-CoV-2 total Ig and IgG high-throughput serological assays (HTSAs) and the Abbott SARS-CoV-2 IgG assay quantify levels of antibodies that strongly correlate with the results of NAb assays and are consistent with gold standard ELISA results. These findings provide immediate clinical relevance to serology results that can be equated to NAb activity and could serve as a valuable roadmap to guide the choice and interpretation of serological tests for SARS-CoV-2.

Repurposed Drugs That Activate Autophagy in Filarial Worms Act as Effective Macrofilaricides.

Onchocerciasis and lymphatic filariasis are two neglected tropical diseases caused by filarial nematodes that utilize insect vectors for transmission to their human hosts. Current control strategies are based on annual or biannual mass drug administration (MDA) of the drugs Ivermectin or Ivermectin plus Albendazole, respectively. These drug regimens kill the first-stage larvae of filarial worms (i.e., microfilariae) and interrupt the transmission of infections. MDA programs for these microfilaricidal drugs must be given over the lifetime of the filarial adult worms, which can reach 15 years in the case of Onchocerca volvulus. This is problematic because of suboptimal responses to ivermectin in various endemic regions and inefficient reduction of transmission even after decades of MDA. There is an urgent need for the development of novel alternative treatments to support the 2030 elimination goals of onchocerciasis and lymphatic filariasis. One successful approach has been to target Wolbachia, obligatory endosymbiotic bacteria on which filarial worms are dependent for their survival and reproduction within the human host. A 4-6-week antibiotic therapy with doxycycline, for example, resulted in the loss of Wolbachia that subsequently led to extensive apoptosis of somatic cells, germline, embryos, and microfilariae, as well as inhibition of fourth-stage larval development. However, this long-course regimen has limited use in MDA programs. As an alternative approach to the use of bacteriostatic antibiotics, in this study, we focused on autophagy-inducing compounds, which we hypothesized could disturb various pathways involved in the interdependency between Wolbachia and filarial worms. We demonstrated that several such compounds, including Niclosamide, an FDA-approved drug, Niclosamide ethanolamine (NEN), and Rottlerin, a natural product derived from Kamala trees, significantly reduced the levels of Wolbachia in vitro. Moreover, when these compounds were used in vivo to treat Brugia pahangi-infected gerbils, Niclosamide and NEN significantly decreased adult worm survival, reduced the release of microfilariae, and decreased embryonic development depending on the regimen and dose used. All three drugs given orally significantly reduced Wolbachia loads and induced an increase in levels of lysosome-associated membrane protein in worms from treated animals, suggesting that Niclosamide, NEN, and Rottlerin were effective in causing drug-induced autophagy in these filarial worms. These repurposed drugs provide a new avenue for the clearance of adult worms in filarial infections.

Combinations of the azaquinazoline anti-Wolbachia agent, AWZ1066S, with benzimidazole anthelmintics synergise to mediate sub-seven-day sterilising and curative efficacies in experimental models of filariasis.

Lymphatic filariasis and onchocerciasis are two major neglected tropical diseases that are responsible for causing severe disability in 50 million people worldwide, whilst veterinary filariasis (heartworm) is a potentially lethal parasitic infection of companion animals. There is an urgent need for safe, short-course curative (macrofilaricidal) drugs to eliminate these debilitating parasite infections. We investigated combination treatments of the novel anti-Wolbachia azaquinazoline small molecule, AWZ1066S, with benzimidazole drugs (albendazole or oxfendazole) in up to four different rodent filariasis infection models: Brugia malayi-CB.17 SCID mice, B. malayi-Mongolian gerbils, B. pahangi-Mongolian gerbils, and Litomosoides sigmodontis-Mongolian gerbils. Combination treatments synergised to elicit threshold (>90%) Wolbachia depletion from female worms in 5 days of treatment, using 2-fold lower dose-exposures of AWZ1066S than monotherapy. Short-course lowered dose AWZ1066S-albendazole combination treatments also delivered partial adulticidal activities and/or long-lasting inhibition of embryogenesis, resulting in complete transmission blockade in B. pahangi and L. sigmodontis gerbil models. We determined that short-course AWZ1066S-albendazole co-treatment significantly augmented the depletion of Wolbachia populations within both germline and hypodermal tissues of B. malayi female worms and in hypodermal tissues in male worms, indicating that anti-Wolbachia synergy is not limited to targeting female embryonic tissues. Our data provides pre-clinical proof-of-concept that sub-seven-day combinations of rapid-acting novel anti-Wolbachia agents with benzimidazole anthelmintics are a promising curative and transmission-blocking drug treatment strategy for filarial diseases of medical and veterinary importance.

Repositioning of an existing drug for the neglected tropical disease Onchocerciasis.

Onchocerciasis, or river blindness, is a neglected tropical disease caused by the filarial nematode Onchocerca volvulus that affects more than 37 million people, mainly in third world countries. Currently, the only approved drug available for mass treatment is ivermectin, however, drug resistance is beginning to emerge, thus, new therapeutic targets and agents are desperately needed to treat and cure this devastating disease. Chitin metabolism plays a central role in invertebrate biology due to the critical structural function of chitin for the organism. Taken together with its absence in mammals, targeting chitin is an appealing therapeutic avenue. Importantly, the chitinase OvCHT1 from O. volvulus was recently discovered, however, its exact role in the worm's metabolism remains unknown. A screening effort against OvCHT1 was conducted using the Johns Hopkins Clinical Compound Library that contains over 1,500 existing drugs. Closantel, a veterinary anthelmintic with known proton ionophore activities, was identified as a potent and specific inhibitor of filarial chitinases, an activity not previously reported for this compound. Notably, closantel was found also to completely inhibit molting of O. volvulus infective L3 stage larvae. Closantel appears to target two important biochemical processes essential to filarial parasites. To begin to unravel closantel's effects, a retro-fragment-based study was used to define structural elements critical for closantel's chitinase inhibitor function. As resources towards the development of new agents that target neglected tropical diseases are scant, the finding of an existing drug with impact against O. volvulus provides promise in the hunt for new therapies against river blindness.

Adjuvanted Fusion Protein Vaccine Induces Durable Immunity to Onchocerca volvulus in Mice and Non-Human Primates.

Onchocerciasis remains a debilitating neglected tropical disease. Due to the many challenges of current control methods, an effective vaccine against the causative agent Onchocerca volvulus is urgently needed. Mice and cynomolgus macaque non-human primates (NHPs) were immunized with a vaccine consisting of a fusion of two O. volvulus protein antigens, Ov-103 and Ov-RAL-2 (Ov-FUS-1), and three different adjuvants: Advax-CpG, alum, and AlT4. All vaccine formulations induced high antigen-specific IgG titers in both mice and NHPs. Challenging mice with O. volvulus L3 contained within subcutaneous diffusion chambers demonstrated that Ov-FUS-1/Advax-CpG-immunized animals developed protective immunity, durable for at least 11 weeks. Passive transfer of sera, collected at several time points, from both mice and NHPs immunized with Ov-FUS-1/Advax-CpG transferred protection to naïve mice. These results demonstrate that Ov-FUS-1 with the adjuvant Advax-CpG induces durable protective immunity against O. volvulus in mice and NHPs that is mediated by vaccine-induced humoral factors.

Discovery of New Broad-Spectrum Anti-Infectives for Eukaryotic Pathogens Using Bioorganometallic Chemistry.

Drug resistance observed with many anti-infectives clearly highlights the need for new broad-spectrum agents to treat especially neglected tropical diseases (NTDs) caused by eukaryotic parasitic pathogens including fungal infections. Since these diseases target the most vulnerable communities who are disadvantaged by health and socio-economic factors, new agents should be, if possible, easy-to-prepare to allow for commercialization based on their low cost. In this study, we show that simple modification of one of the most well-known antifungal drugs, fluconazole, with organometallic moieties not only improves the activity of the parent drug but also broadens the scope of application of the new derivatives. These compounds were highly effective in vivo against pathogenic fungal infections and potent against parasitic worms such as Brugia, which causes lymphatic filariasis and Trichuris, one of the soil-transmitted helminths that infects millions of people globally. Notably, the identified molecular targets indicate a mechanism of action that differs greatly from the parental antifungal drug, including targets involved in biosynthetic pathways that are absent in humans, offering great potential to expand our armamentarium against drug-resistant fungal infections and NTDs targeted for elimination by 2030. Overall, the discovery of these new compounds with broad-spectrum activity opens new avenues for the development of treatments for several current human infections, either caused by fungi or by parasites, including other NTDs, as well as newly emerging diseases. ONE-SENTENCE SUMMARY: Simple derivatives of the well-known antifungal drug fluconazole were found to be highly effective in vivo against fungal infections, and also potent against the parasitic nematode Brugia, which causes lymphatic filariasis and against Trichuris, one of the soil-transmitted helminths that infects millions of people globally.

Discovery of New Broad-Spectrum Anti-Infectives for Eukaryotic Pathogens Using Bioorganometallic Chemistry.

Drug resistance observed with many anti-infectives clearly highlights the need for new broad-spectrum agents to treat especially neglected tropical diseases (NTDs) caused by eukaryotic parasitic pathogens, including fungal infections. Herein, we show that the simple modification of one of the most well-known antifungal drugs, fluconazole, with organometallic moieties not only improves the activity of the parent drug but also broadens the scope of application of the new derivatives. These compounds were highly effective in vivo against pathogenic fungal infections and potent against parasitic worms such as Brugia, which causes lymphatic filariasis and Trichuris, one of the soil-transmitted helminths that infects millions of people globally. Notably, the identified molecular targets indicate a mechanism of action that differs greatly from that of the parental antifungal drug, including targets involved in biosynthetic pathways that are absent in humans, offering great potential to expand our armamentarium against drug-resistant fungal infections and neglected tropical diseases (NTDs) targeted for elimination by 2030.

Anti-Plasmodium falciparum invasion ligand antibodies in a low malaria transmission region, Loreto, Peru.

BACKGROUND: Erythrocyte invasion by Plasmodium falciparum is a complex process that involves two families; Erythrocyte Binding-Like (EBL) and the Reticulocyte Binding-Like (PfRh) proteins. Antibodies that inhibit merozoite attachment and invasion are believed to be important in mediating naturally acquired immunity and immunity generated by parasite blood stage vaccine candidates. The hypotheses tested in this study were 1) that antibody responses against specific P. falciparum invasion ligands (EBL and PfRh) differ between symptomatic and asymptomatic individuals living in the low-transmission region of the Peruvian Amazon and 2), such antibody responses might have an association, either direct or indirect, with clinical immunity observed in asymptomatically parasitaemic individuals. METHODS: ELISA was used to assess antibody responses (IgG, IgG1 and IgG3) against recombinant P. falciparum invasion ligands of the EBL (EBA-175, EBA-181, EBA-140) and PfRh families (PfRh1, PfRh2a, PfRh2b, PfRh4 and PfRh5) in 45 individuals infected with P. falciparum from Peruvian Amazon. Individuals were classified as having symptomatic malaria (N=37) or asymptomatic infection (N=8). RESULTS: Antibody responses against both EBL and PfRh family proteins were significantly higher in asymptomatic compared to symptomatic individuals, demonstrating an association with clinical immunity. Significant differences in the total IgG responses were observed with EBA-175, EBA-181, PfRh2b, and MSP119 (as a control). IgG1 responses against EBA-181, PfRh2a and PfRh2b were significantly higher in the asymptomatic individuals. Total IgG antibody responses against PfRh1, PfRh2a, PfRh2b, PfRh5, EBA-175, EBA-181 and MSP119 proteins were negatively correlated with level of parasitaemia. IgG1 responses against EBA-181, PfRh2a and PfRh2b and IgG3 response for PfRh2a were also negatively correlated with parasitaemia. CONCLUSIONS: These data suggest that falciparum malaria patients who develop clinical immunity (asymptomatic parasitaemia) in a low transmission setting such as the Peruvian Amazon have antibody responses to defined P. falciparum invasion ligand proteins higher than those found in symptomatic (non-immune) patients. While these findings will have to be confirmed by larger studies, these results are consistent with a potential role for one or more of these invasion ligands as a component of an anti-P. falciparum vaccine in low-transmission malaria-endemic regions.

Pyrvinium Pamoate and Structural Analogs Are Early Macrofilaricide Leads.

Onchocerciasis and lymphatic filariasis are neglected tropical diseases caused by infection with filarial worms. Annual or biannual mass drug administration with microfilaricidal drugs that kill the microfilarial stages of the parasites has helped reduce infection rates and thus prevent transmission of both infections. However, success depends on high population coverage that is maintained for the duration of the adult worm's lifespan. Given that these filarial worms can live up to 14 years in their human hosts, a macrofilaricidal drug would vastly accelerate elimination efforts. Here, we have evaluated the repurposed drug pyrvinium pamoate as well as newly synthesized analogs of pyrvinium for their efficacy against filarial worms in vitro and in vivo. We found that pyrvinium pamoate, tetrahydropyrvinium and one of the analogs were highly potent in inhibiting worms in in vitro whole-worm screening assays, and that all three compounds reduced female worm fecundity and inhibited embryogenesis in the Brugia pahangi-gerbil in vivo model of infection.

Drugs that target early stages of Onchocerca volvulus: A revisited means to facilitate the elimination goals for onchocerciasis.

Several issues have been identified with the current programs for the elimination of onchocerciasis that target only transmission by using mass drug administration (MDA) of the drug ivermectin. Alternative and/or complementary treatment regimens as part of a more comprehensive strategy to eliminate onchocerciasis are needed. We posit that the addition of "prophylactic" drugs or therapeutic drugs that can be utilized in a prophylactic strategy to the toolbox of present microfilaricidal drugs and/or future macrofilaricidal treatment regimens will not only improve the chances of meeting the elimination goals but may hasten the time to elimination and also will support achieving a sustained elimination of onchocerciasis. These "prophylactic" drugs will target the infective third- (L3) and fourth-stage (L4) larvae of Onchocerca volvulus and consequently prevent the establishment of new infections not only in uninfected individuals but also in already infected individuals and thus reduce the overall adult worm burden and transmission. Importantly, an effective prophylactic treatment regimen can utilize drugs that are already part of the onchocerciasis elimination program (ivermectin), those being considered for MDA (moxidectin), and/or the potential macrofilaricidal drugs (oxfendazole and emodepside) currently under clinical development. Prophylaxis of onchocerciasis is not a new concept. We present new data showing that these drugs can inhibit L3 molting and/or inhibit motility of L4 at IC50 and IC90 that are covered by the concentration of these drugs in plasma based on the corresponding pharmacological profiles obtained in human clinical trials when these drugs were tested using various doses for the therapeutic treatments of various helminth infections.

An Integrated Approach to Identify New Anti-Filarial Leads to Treat River Blindness, a Neglected Tropical Disease.

Filarial worms cause multiple debilitating diseases in millions of people worldwide, including river blindness. Currently available drugs reduce transmission by killing larvae (microfilariae), but there are no effective cures targeting the adult parasites (macrofilaricides) which survive and reproduce in the host for very long periods. To identify effective macrofilaricides, we carried out phenotypic screening of a library of 2121 approved drugs for clinical use against adult Brugia pahangi and prioritized the hits for further studies by integrating those results with a computational prioritization of drugs and associated targets. This resulted in the identification of 18 hits with anti-macrofilaricidal activity, of which two classes, azoles and aspartic protease inhibitors, were further expanded upon. Follow up screening against Onchocerca spp. (adult Onchocerca ochengi and pre-adult O. volvulus) confirmed activity for 13 drugs (the majority having IC50 < 10 µM), and a counter screen of a subset against L. loa microfilariae showed the potential to identify selective drugs that prevent adverse events when co-infected individuals are treated. Stage specific activity was also observed. Many of these drugs are amenable to structural optimization, and also have known canonical targets, making them promising candidates for further optimization that can lead to identifying and characterizing novel anti-macrofilarial drugs.

Preliminary evaluations of 3-dimensional human skin models for their ability to facilitate in vitro the long-term development of the debilitating obligatory human parasite Onchocerca volvulus.

Onchocerciasis also known as river blindness is a neglected tropical disease and the world's second-leading infectious cause of blindness in humans; it is caused by Onchocerca volvulus. Current treatment with ivermectin targets microfilariae and transmission and does not kill the adult parasites, which reside within subcutaneous nodules. To support the development of macrofilaricidal drugs that target the adult worm to further support the elimination of onchocerciasis, an in-depth understanding of O. volvulus biology especially the factors that support the longevity of these worms in the human host (>10 years) is required. However, research is hampered by a lack of access to adult worms. O. volvulus is an obligatory human parasite and no small animal models that can propagate this parasite were successfully developed. The current optimized 2-dimensional (2-D) in vitro culturing method starting with O. volvulus infective larvae does not yet support the development of mature adult worms. To overcome these limitations, we have developed and applied 3-dimensional (3-D) culture systems with O. volvulus larvae that simulate the human in vivo niche using in vitro engineered skin and adipose tissue. Our proof of concept studies have shown that an optimized indirect co-culture of in vitro skin tissue supported a significant increase in growth of the fourth-stage larvae to the pre-adult stage with a median length of 816-831 µm as compared to 767 µm of 2-D cultured larvae. Notably, when larvae were co-cultured directly with adipose tissue models, a significant improvement for larval motility and thus fitness was observed; 95% compared to 26% in the 2-D system. These promising co-culture concepts are a first step to further optimize the culturing conditions and improve the long-term development of adult worms in vitro. Ultimately, it could provide the filarial research community with a valuable source of O. volvulus worms at various developmental stages, which may accelerate innovative unsolved biomedical inquiries into the parasite's biology.

Serological Assays Estimate Highly Variable SARS-CoV-2 Neutralizing Antibody Activity in Recovered COVID19 Patients.

The development of neutralizing antibodies (nAb) against SARS-CoV-2, following infection or vaccination, is likely to be critical for the development of sufficient population immunity to drive cessation of the COVID19 pandemic. A large number of serologic tests, platforms and methodologies are being employed to determine seroprevalence in populations to select convalescent plasmas for therapeutic trials, and to guide policies about reopening. However, tests have substantial variability in sensitivity and specificity, and their ability to quantitatively predict levels of nAb is unknown. We collected 370 unique donors enrolled in the New York Blood Center Convalescent Plasma Program between April and May of 2020. We measured levels of antibodies in convalescent plasma using commercially available SARS-CoV- 2 detection tests and in-house ELISA assays and correlated serological measurements with nAb activity measured using pseudotyped virus particles, which offer the most informative assessment of antiviral activity of patient sera against viral infection. Our data show that a large proportion of convalescent plasma samples have modest antibody levels and that commercially available tests have varying degrees of accuracy in predicting nAb activity. We found the Ortho Anti-SARS-CoV-2 Total Ig and IgG high throughput serological assays (HTSAs), as well as the Abbott SARS-CoV-2 IgG assay, quantify levels of antibodies that strongly correlate with nAb assays and are consistent with gold-standard ELISA assay results. These findings provide immediate clinical relevance to serology results that can be equated to nAb activity and could serve as a valuable 'roadmap' to guide the choice and interpretation of serological tests for SARS-CoV-2.

Macrofilaricidal Benzimidazole-Benzoxaborole Hybrids as an Approach to the Treatment of River Blindness: Part 2. Ketone Linked Analogs.

The optimization of a series of benzimidazole-benzoxaborole hybrid molecules linked via a ketone that exhibit good activity against Onchocerca volvulus, a filarial nematode responsible for the disease onchocerciasis, also known as river blindness, is described. The lead identified in this series, 21 (AN15470), was found to have acceptable pharmacokinetic properties to enable an evaluation following oral dosing in an animal model of onchocerciasis. Compound 21was effective in killing worms implanted in Mongolian gerbils when dosed orally as a suspension at 100 mg/kg/day for 14 days but not when dosed orally at 100 mg/kg/day for 7 days.

Macrofilaricidal Benzimidazole-Benzoxaborole Hybrids as an Approach to the Treatment of River Blindness: Part 1. Amide Linked Analogs.

A series of benzimidazole-benzoxaborole hybrid molecules linked via an amide linker are described that exhibit good in vitro activity against Onchocerca volvulus, a filarial nematode responsible for the disease onchocerciasis, also known as river blindness. The lead identified in this series, 8a (AN8799), was found to have acceptable pharmacokinetic properties to enable evaluation in animal models of human filariasis. Compound 8a was effective in killing Brugia malayi, B. pahangi, and Litomosoides sigmodontis worms present in Mongolian gerbils when dosed subcutaneously as a suspension at 100 mg/kg/day for 14 days but not when dosed orally at 100 mg/kg/day for 28 days. The measurement of plasma levels of 8a at the end of the dosing period and at the time of sacrifice revealed an interesting dependence of activity on the extended exposure for both 8a and the positive control, flubendazole.

Short-course quinazoline drug treatments are effective in the Litomosoides sigmodontis and Brugia pahangi jird models.

The quinazolines CBR417 and CBR490 were previously shown to be potent anti-wolbachials that deplete Wolbachia endosymbionts of filarial nematodes and present promising pre-clinical candidates for human filarial diseases such as onchocerciasis. In the present study we tested both candidates in two models of chronic filarial infection, namely the Litomosoides sigmodontis and Brugia pahangi jird model and assessed their long-term effect on Wolbachia depletion, microfilariae counts and filarial embryogenesis 16-18 weeks after treatment initiation (wpt). Once per day (QD) oral treatment with CBR417 (50 mg/kg) for 4 days or twice per day (BID) with CBR490 (25 mg/kg) for 7 days during patent L. sigmodontis infection reduced the Wolbachia load by >99% and completely cleared peripheral microfilaremia from 10-14 wpt. Similarly, 7 days of QD treatments (40 mg/kg) with CBR417 or CBR490 cleared >99% of Wolbachia from B. pahangi and reduced peritoneal microfilariae counts by 93% in the case of CBR417 treatment. Transmission electron microscopy analysis indicated intensive damage to the B. pahangi ovaries following CBR417 treatment and in accordance filarial embryogenesis was inhibited in both models after CBR417 or CBR490 treatment. Suboptimal treatment regimens of CBR417 or CBR490 did not lead to a maintained reduction of the microfilariae and Wolbachia load. In conclusion, CBR417 or CBR490 are pre-clinical candidates for filarial diseases, which achieve long-term clearance of Wolbachia endosymbionts of filarial nematodes, inhibit filarial embryogenesis and clear microfilaremia with treatments as short as 7 days.

Evidence for protection against chronic hepatitis C virus infection in chimpanzees by immunization with replicating recombinant vaccinia virus.

Given the failures of nonreplicating vaccines against chronic hepatitis C virus (HCV) infection, we hypothesized that a replicating viral vector may provide protective immunity. Four chimpanzees were immunized transdermally twice with recombinant vaccinia viruses (rVV) expressing HCV genes. After challenge with 24 50% chimpanzee infective doses of homologous HCV, the two control animals that had received only the parental VV developed chronic HCV infection. All four immunized animals resolved HCV infection. The difference in the rate of chronicity between the immunized and the control animals was close to statistical significance (P = 0.067). Immunized animals developed vigorous gamma interferon enzyme-linked immunospot responses and moderate proliferative responses. To investigate cross-genotype protection, the immunized recovered chimpanzees were challenged with a pool of six major HCV genotypes. During the acute phase after the multigenotype challenge, all animals had high-titer viremia in which genotype 4 dominated (87%), followed by genotype 5 (13%). However, after fluctuating low-level viremia, the viremia finally turned negative or persisted at very low levels. This study suggests the potential efficacy of replicating recombinant vaccinia virus-based immunization against chronic HCV infection.

Development of a preliminary in vitro drug screening assay based on a newly established culturing system for pre-adult fifth-stage Onchocerca volvulus worms.

BACKGROUND: The human filarial parasite Onchocerca volvulus is the causative agent of onchocerciasis (river blindness). It causes blindness in 270,000 individuals with an additional 6.5 million suffering from severe skin pathologies. Current international control programs focus on the reduction of microfilaridermia by annually administering ivermectin for more than 20 years with the ultimate goal of blocking of transmission. The adult worms of O. volvulus can live within nodules for over 15 years and actively release microfilariae for the majority of their lifespan. Therefore, protracted treatment courses of ivermectin are required to block transmission and eventually eliminate the disease. To shorten the time to elimination of this disease, drugs that successfully target macrofilariae (adult parasites) are needed. Unfortunately, there is no small animal model for the infection that could be used for discovery and screening of drugs against adult O. volvulus parasites. Here, we present an in vitro culturing system that supports the growth and development of O. volvulus young adult worms from the third-stage (L3) infective stage. METHODOLOGY/PRINCIPAL FINDINGS: In this study we optimized the culturing system by testing several monolayer cell lines to support worm growth and development. We have shown that the optimized culturing system allows for the growth of the L3 worms to L5 and that the L5 mature into young adult worms. Moreover, these young O. volvulus worms were used in preliminary assays to test putative macrofilaricidal drugs and FDA-approved repurposed drugs. CONCLUSION: The culture system we have established for O. volvulus young adult worms offers a promising new platform to advance drug discovery against the human filarial parasite, O. volvulus and thus supports the continuous pursuit for effective macrofilaricidal drugs. However, this in vitro culturing system will have to be further validated for reproducibility before it can be rolled out as a drug screen for decision making in macrofilaricide drug development programs.

Antibody responses against the vaccine antigens Ov-103 and Ov-RAL-2 are associated with protective immunity to Onchocerca volvulus infection in both mice and humans.

BACKGROUND: The current strategy for the elimination of onchocerciasis is based on annual or bi-annual mass drug administration with ivermectin. However, due to several limiting factors there is a growing concern that elimination of onchocerciasis cannot be achieved solely through the current strategy. Additional tools are critically needed including a prophylactic vaccine. Presently Ov-103 and Ov-RAL-2 are the most promising vaccine candidates against an Onchocerca volvulus infection. METHODOLOGY/PRINCIPAL FINDINGS: Protection induced by immunization of mice with the alum-adjuvanted Ov-103 or Ov-RAL-2 vaccines appeared to be antibody dependent since AID-/- mice that could not mount antigen-specific IgG antibody responses were not protected from an Onchocerca volvulus challenge. To determine a possible association between antigen-specific antibody responses and anti-larvae protective immunity in humans, we analyzed the presence of anti-Ov-103 and anti-Ov-RAL-2 cytophilic antibody responses (IgG1 and IgG3) in individuals classified as putatively immune, and in infected individuals who developed concomitant immunity with age. It was determined that 86% of putatively immune individuals and 95% individuals with concomitant immunity had elevated IgG1 and IgG3 responses to Ov-103 and Ov-RAL-2. Based on the elevated chemokine levels associated with protection in the Ov-103 or Ov-RAL-2 immunized mice, the profile of these chemokines was also analyzed in putatively immune and infected individuals; both groups contained significantly higher levels of KC, IP-10, MCP-1 and MIP-1ß in comparison to normal human sera. Moreover, human monospecific anti-Ov-103 antibodies but not anti-Ov-RAL-2 significantly inhibited the molting of third-stage larvae (L3) in vitro by 46% in the presence of naïve human neutrophils, while both anti-Ov-103 and anti-Ov-RAL-2 antibodies significantly inhibited the molting by 70-80% when cultured in the presence of naive human monocytes. Interestingly, inhibition of molting by Ov-103 antibodies and monocytes was only in part dependent on contact with the cells, while inhibition of molting with Ov-RAL-2 antibodies was completely dependent on contact with the monocytes. In comparison, significant levels of parasite killing in Ov-103 and Ov-RAL-2 vaccinated mice only occurred when cells enter the parasite microenvironment. Taken together, antibodies to Ov-103 and Ov-RAL-2 and cells are required for protection in mice as well as for the development of immunity in humans. CONCLUSIONS/SIGNIFICANCE: Alum-adjuvanted Ov-103 and Ov-RAL-2 vaccines have the potential of reducing infection and thus morbidity associated with onchocerciasis in humans. The development of cytophilic antibodies, that function in antibody-dependent cellular cytotoxicity, is essential for a successful prophylactic vaccine against this infection.

Ligand binding properties of two Brugia malayi fatty acid and retinol (FAR) binding proteins and their vaccine efficacies against challenge infection in gerbils.

Parasitic nematodes produce an unusual class of fatty acid and retinol (FAR)-binding proteins that may scavenge host fatty acids and retinoids. Two FARs from Brugia malayi (Bm-FAR-1 and Bm-FAR-2) were expressed as recombinant proteins, and their ligand binding, structural characteristics, and immunogenicities examined. Circular dichroism showed that rBm-FAR-1 and rBm-FAR-2 are similarly rich in α-helix structure. Unexpectedly, however, their lipid binding activities were found to be readily differentiated. Both FARs bound retinol and cis-parinaric acid similarly, but, while rBm-FAR-1 induced a dramatic increase in fluorescence emission and blue shift in peak emission by the fluorophore-tagged fatty acid (dansyl-undecanoic acid), rBm-FAR-2 did not. Recombinant forms of the related proteins from Onchocerca volvulus, rOv-FAR-1 and rOv-FAR-2, were found to be similarly distinguishable. This is the first FAR-2 protein from parasitic nematodes that is being characterized. The relative protein abundance of Bm-FAR-1 was higher than Bm-FAR-2 in the lysates of different developmental stages of B. malayi. Both FAR proteins were targets of strong IgG1, IgG3 and IgE antibody in infected individuals and individuals who were classified as endemic normal or putatively immune. In a B. malayi infection model in gerbils, immunization with rBm-FAR-1 and rBm-FAR-2 formulated in a water-in-oil-emulsion (®Montanide-720) or alum elicited high titers of antigen-specific IgG, but only gerbils immunized with rBm-FAR-1 formulated with the former produced a statistically significant reduction in adult worms (68%) following challenge with B. malayi infective larvae. These results suggest that FAR proteins may play important roles in the survival of filarial nematodes in the host, and represent potential candidates for vaccine development against lymphatic filariasis and related filarial infections.