Norice-Tra CT et al. (2017) J Infect Dis "Insights Into Onchocerca volvulus Population Biology Through Multilocus Immunophenotyping."

Mitreva M, Nutman TB, Norice-Tra CT, Bennuru S, Tyagi R, Ribeiro J, Fay MP

[J Infect Dis, 2017]

We have developed a serologically based immunophenotyping approach to study Onchocerca volvulus (Ov) population diversity. Using genomic sequence data and polymerase chain reaction-based genotyping, we identified nonsynonymous single-nucleotide polymorphisms (SNPs) in the genes of 16 major immunogenic Ov proteins: Ov-CHI-1/Ov-CHI-2, Ov16, Ov-FAR-1, Ov-CPI-1, Ov-B20, Ov-ASP-1, Ov-TMY-1, OvSOD1, OvGST1, Ov-CAL-1, M3/M4, Ov-RAL-1, Ov-RAL-2, Ov-ALT-1, Ov-FBA-1, and Ov-B8. We assessed the immunoreactivity of onchocerciasis patient sera (n = 152) from the Americas, West Africa, Central Africa, and East Africa against peptides derived from 10 of these proteins containing SNPs. Statistically significant variation in immunoreactivity among the regions was seen in SNP-containing peptides derived from 8 of 10 proteins tested: OVOC1192(1-15), OVOC9988(28-42), OVOC9225(320-334), OVOC7453(22-36), OVOC11517(14-28), OVOC3177(283-297), OVOC7911(594-608), and OVOC12628(174-188). Our data show that differences in immunoreactivity to variant antigenic peptides may be used to characterize Ov populations, thereby elucidating features of Ov population biology previously inaccessible because of the limited availability of parasite material.

P JG et al. (2019) PLoS Negl Trop Dis "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."

Zhan T, Tricoche N, Abraham D, Zhan B, Bottazzi ME, Lustigman S, Jain S, Hess JA, P JG, Hotez PJ, Patton JB

[PLoS Negl Trop Dis, 2019]

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-1 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 naive 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 also in humans. The development of cytophilic antibodies, that function in antibody-dependent cellular cytotoxicity, is essential for a successful prophylactic vaccine against this infection.

Liebau E et al. (2000) Mol Biochem Parasitol "Identification of a stress-responsive Onchocerca volvulus glutathione S-transferase (Ov-GST-3) by RT-PCR differential display."

Sommer A, Liebau E, Fischer P, Henkle-Duhrsen K, Tawe W, Eschbach ML, Walter RD

[Mol Biochem Parasitol, 2000]

The effects of oxidative insult on gene transcript levels in the filarial nematode Onchocerca volvulus were investigated using differential display RT-PCR. Oxidative stress was applied with the reagents paraquat, plumbagin and xanthine-xanthine oxidase. In all three cases, a cDNA fragment encoding a novel glutathione S-transferase (GST) resembling members of the theta-class was identified as upregulated (PQ29, PG112, XOD26). The subsequently isolated full-length cDNA harbors a 753-bp open reading frame encoding a GST with 268 amino acid residues and a predicted molecular mass of 31 kDa. This stress-responsive GST (Ov-GST-3) possesses only 14 and 21% sequence identity with the other O. volvulus GSTs (Ov-GST-1 and Ov-GST-2, respectively). Interestingly, Ov-GST-3 shares higher sequence identity with GSTs that are upregulated due to environmental stress. In order to confirm the specific upregulation of the Ov-GST-3 transcripts identified by differential display and to analyze the mRNA levels of the other Ov-GSTs (Ov-GST-1 and Ov-GST-2) under elevated stress conditions, a semi-quantitative polymerase chain reaction-enzyme-linked immunosorbent assay was performed. The Ov-GST-3 gene transcript level increased dramatically in response to xanthine-xanthine oxidase and to a lesser extent with paraquat and plumbagin. In contrast, Ov-GST-1 and Ov-GST-2 did not show any significant alterations in their steady-state mRNA levels in response to oxidative stress when examining the same mRNA samples. The present study clearly demonstrates that Ov-GST-3 is a critical enzyme in the defense against oxidative stress.

Feeser KR et al. (2017) Am J Trop Med Hyg "Characterizing Reactivity to Onchocerca volvulus Antigens in Multiplex Bead Assays."

Cama V, Feeser KR, Lakwo T, Feleke SM, Priest JW, Wiegand RE, Thiele EA, Cantey PT

[Am J Trop Med Hyg, 2017]

Multiplex bead assays (MBAs) may provide a powerful integrated tool for monitoring, evaluation, and post-elimination surveillance of onchocerciasis and co-endemic diseases; however, the specificity and sensitivity of Onchocerca volvulus antigens have not been characterized within this context. An MBA was developed to evaluate three antigens (OV-16, OV-17, and OV-33) for onchocerciasis. Receiver operating characteristics (ROC) analyses were used to characterize antigen performance using a panel of 610 specimens: 109 O. volvulus-positive specimens, 426 non-onchocerciasis controls with filarial and other confirmed parasitic infection, and 75 sera from patients with no other parasitic infection. The IgG and IgG4 assays for OV-16 demonstrated sensitivities of 95.4% and 96.3%, and specificities of 99.4% and 99.8%, respectively. The OV-17 IgG and IgG4 assays had sensitivities of 86.2% and 76.1% and specificities of 79.2% and 82.8%. For OV-33, the IgG and IgG4 assays had sensitivities of 90.8% and 96.3%, and specificities of 96.8% and 98.6%. The OV-16 IgG4-based MBA had the best assay characteristics, followed by OV-33 IgG4. The OV-16 IgG4 assay would be useful for monitoring and evaluation using the MBA platform. Further evaluations are needed to review the potential use of OV-33 as a confirmatory test in the context of program evaluations.

Kampkotter A et al. (2003) J Mol Biol "Functional analysis of the glutathione S-transferase 3 from Onchocerca volvulus (Ov-GST-3): a parasite GST confers increased resistance to oxidative stress in Caenorhabditis elegans."

Klotz LO, Johnson TE, Hegi de Castro S, Kampkotter A, Leiers B, Link CD, Volkmann TE, Henkle-Duhrsen K

[J Mol Biol, 2003]

This study examined the genomic organisation of the coding region of the glutathione S-transferase 3 (Ov-GST-3) from the human parasitic nematode Onchocerca volvulus; alternative splicing leads to three different transcripts (Ov-GST-3/1; Ov-GST-3/2 and Ov-GST-3/3). Since the expression of Ov-GST-3 is inducible by oxidative stress, it is assumed that it is involved in the defense against reactive oxygen species (ROS) resulting from cellular metabolism. Furthermore, we suggest that Ov-GST-3 plays an important role in the protection of the parasite against ROS derived from the host's immune system. To experimentally investigate these speculations, we generated Caenorhabditis elegans lines transgenic for Ov-GST-3 (AK1) and examined their resistance to artificially generated ROS. The AK1 worms extrachromosomal and integrated lines) were found to be much more resistant to internal (juglone) and external (hypoxanthine/xanthine oxidase) oxidative stress than wild-type C.elegans worms. RNA interference experiments targeted to the Ov-GST-3 transcripts resulted in decreased resistance, confirming that this effect is due to the transgenic expression of Ov-GST-3. These results clearly demonstrate that the Ov-GST-3 gene confers an increased resistance to oxidative stress. This study also shows the applicability of C.elegans as a model organism for the functional characterization of genes from (parasitic) nematode species which are not accessible to genetic manipulations.

Lagatie O et al. (2018) Parasite Immunol "Linear epitopes in Onchocerca volvulus vaccine candidate proteins and excretory-secretory proteins."

Stuyver LJ, Van Dorst B, Batsa Debrah L, Verheyen A, Debrah A, Lagatie O

[Parasite Immunol, 2018]

In our previous study, a proteome-wide screen was conducted to identify linear epitopes in this parasite's proteome, resulting in the discovery of three immunodominant motifs. Here we investigated whether such antigenic peptides were found in proteins that were already known as vaccine candidates and excretome/secretome proteins for O. volvulus. This approach led to the identification of 71 immunoreactive stretches in 46 proteins. A deep-dive into the immunoreactivity profiles of eight vaccine candidates that were chosen as most promising candidates for further development (Ov-CPI-2,Ov-ALT-1,Ov-RAL-2,Ov-ASP-1,Ov-103,Ov-RBP-1,Ov-CHI-1, andOv-B20), resulted in the identification of a poly-glutamine stretch in Ov-RAL-2 that has properties for use as a serodiagnostic marker for O. volvulus infection. A peptide ELISA was developed, and the performance of this assay was evaluated. Based on this assessment, it was found that this assay has a sensitivity of 75.0% [95% CI: 64.9%-83.5%] and a specificity of 98.5% [95% CI: 94.6%-99.8%]. Furthermore, 8.7% reactivity in Asian parasite-infected individuals (8 out of 92) was observed. Besides this identification of a linear epitope marker, the information on the presence of linear epitopes in vaccine candidate proteins might be useful in the study of vaccines for river blindness. This article is protected by copyright. All rights reserved.

Steisslinger V et al. (2015) Vaccine "DNA vaccine encoding the moonlighting protein Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) leads to partial protection in a mouse model of human filariasis."

Steisslinger V, Erttmann KD, Brattig NW, Korten S

[Vaccine, 2015]

River blindness, caused by the filarial parasite Onchocerca volvulus, is a major socio-economic and public health problem in Sub-Saharan Africa. In January 2015, The Onchocerciasis Vaccine for Africa (TOVA) Initiative has been launched with the aim of providing new tools to complement mass drug administration (MDA) of ivermectin, thereby promoting elimination of onchocerciasis in Africa. In this context we here present Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) as a possible DNA vaccine candidate. We report that in a laboratory model for filariasis, immunization with Ov-GAPDH led to a significant reduction of adult worm load and microfilaraemia in BALB/c mice after challenge infection with the filarial parasite Litomosoides sigmodontis. Mice were either vaccinated with Ov-GAPDH.DNA plasmid (Ov-pGAPDH.DNA) alone or in combination with recombinantly expressed Ov-GAPDH protein (Ov-rGAPDH). During the following challenge infection of immunized and control mice with L. sigmodontis, those formulations which included the DNA plasmid, led to a significant reduction of adult worm loads (up to 57% median reduction) and microfilaraemia (up to 94% reduction) in immunized animals. In a further experiment, immunization with a mixture of four overlapping, synthetic Ov-GAPDH peptides (Ov-GAPDHpept), with alum as adjuvant, did not significantly reduce worm loads. Our results indicate that DNA vaccination with Ov-GAPDH has protective potential against filarial challenge infection in the mouse model. This suggests a transfer of the approach into the cattle Onchocerca ochengi model, where it is possible to investigate the effects of this vaccination in the context of a natural host-parasite relationship.

Sommer A et al. (2003) Infect Immun "A dominant role for extracellular glutathione S-transferase from Onchocerca volvulus is the production of prostaglandin D2."

Steinhart H, Fischer P, Sommer A, Rickert R, Liebau E, Walter RD

[Infect Immun, 2003]

The extracellular glutathione S-transferase from the filarial parasite Onchocerca volvulus (Ov-GST1) is a glutathione-dependent prostaglandin D synthase. Ov-GST1, located in the outer hypodermal lamellae and in parts of the cuticle, produces prostaglandin D(2) directly at the parasite-host interface. Ov-GST1 therefore has the potential to participate in the modulation of the host immune response by contributing to the production of prostanoids; this supports the predominant hypothesis that parasite-derived eicosanoids influence host inflammatory and immune cells.

Ford L et al. (2005) J Biol Chem "Characterization of a novel filarial serine protease inhibitor, Ov-SPI-1, from Onchocerca volvulus, with potential multifunctional roles during development of the parasite."

Oksov Y, Blaxter ML, Lustigman S, Debnath AK, Liu J, Williams SA, Ford L, Guiliano DB

[J Biol Chem, 2005]

A novel filarial serine protease inhibitor (SPI) from the human parasitic nematode Onchocerca volvulus, Ov-SPI-1, was identified through the analysis of a molting third-stage larvae expressed sequence tag dataset. Subsequent analysis of the expressed sequence tag datasets of O. volvulus and other filariae identified four other members of this family. These proteins are related to the low molecular weight SPIs originally isolated from Ascaris suum where they are believed to protect the parasite from host intestinal proteases. The two Ov-spi transcripts are up-regulated in the molting larvae and adult stages of the development of the parasite. Recombinant Ov-SPI-1 is an active inhibitor of serine proteases, specifically elastase, chymotrypsin, and cathepsin G. Immunolocalization of the Ov-SPI proteins demonstrates that the endogenous proteins are localized to the basal layer of the cuticle of third-stage, molting third-stage, and fourth-stage larvae, the body channels and multivesicular bodies of third-stage larvae and the processed material found between the two cuticles during molting. In O. volvulus adult worms the Ov-SPI proteins are localized to the sperm and to eggshells surrounding the developing embryos. RNA interference targeting the Ov-spi genes resulted in the specific knockdown of the transcript levels of both Ov-spi-1 and Ov-spi-2, a loss of native proteins, and a significant reduction in both molting and viability of third-stage larvae. We suggest the Ov-SPI proteins play a vital role in nematode molting by controlling the activity of an endogenous serine protease(s). The localization data in adults also indicate that these inhibitors may be involved in other processes such as embryogenesis and spermatogenesis.

Wu Y et al. (2003) Filaria J "Human immune responses to infective stage larval-specific chitinase of filarial parasite, Onchocerca volvulus, Ov-CHI-1."

Nutman TB, Bianco AE, McCarthy JS, Wu Y, Egerton G

[Filaria J, 2003]

BACKGROUND: Ov-CHI-1 is a chitinase specifically expressed in the infective stage larvae of the human filarial parasite Onchocerca volvulus. Evidence has show that it could be a vaccine candidate, however, there is no data available regarding the immunological status of people naturally exposed to infective stage larvae and thus provoked by this antigen. METHOD: We analysed the Ov-CHI-1-specific immune response present in four endemic foci of human onchocerciasis (Ecuador, Nigeria, Togo and Cameroon) by enzyme-linked immunosorbent assays and T-cell proliferation assays. RESULTS: In these foci of infection, antibodies to Ov-CHI-1 were found to be present in only 22% of individuals from Ecuador, but were detected in 42-62% of infected individuals in the three foci from West Africa (Nigeria, Togo and Cameroon). There was found to be no relationship between antibody level and age, gender, or infection intensity as indicated by microfilarial density and numbers of skin nodules. The isotype response to Ov-CHI-1 was dominated by the presence of IgG3, IgG1 was present to a lesser extent. Our results show a positive correlation between N- and C-termini of Ov-CHI-1 in their ability to provoke humoral and cellular immune responses in the human. Peripheral blood mononuclear cell (PBMC) proliferative responses to Ov-CHI-1 when assayed, were found to be significantly higher in the individuals from endemic areas and there was a statistically elevated response to Ov-CHI-1 in the infected individuals when compared to putative immune individuals. CONCLUSION: Ov-CHI-1 is an antigen that we have found strongly induces both humoral and cellular immune responses in humans.