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Harnessing bioinformatic epitope foresight in combination with peptide microarray for

testing exposure to Simulium damnosum as a useful tool in onchocerciasis control effort.

Onchocerciasis is a parasitic infection caused by the filarial nematode Onchocerca volvulus

which is transmitted through the bites of infected blackflies of the genus Simulium that breed
along fast-flowing waters. It is the second leading cause of blindness after trachoma a (Shey
et al., 2021). Currently, about 218 million people live in areas known to be endemic for
onchocerciasis. Substantial efforts and increasing international funding to eliminate
onchocerciasis through chemotherapy using community-directed treatment with Ivermectin
(CDTI) and vector control measures have contributed to reducing disease prevalence in the
last decades, but the disease remains a public health concern and exerts a serious socio-
economic burden on the affected communities. As of 2017, an estimated 21 million persons
are infected with the disease where, about 1.2 million are suffering from visual impairment
(Koudou et al., 2018).
Following several rounds of public consultation in 2020, the WHO published the draft road
map for neglected tropical disease 2021–2030, which was endorsed by the Seventy-third
WHA. The road map identified as a critical action the mapping of suspected onchocerciasis-
endemic areas, with the launch of MDA wherever indicated. The roadmap further identifies
as critical the development of improved diagnostics to facilitate mapping and decision-
making (Souza et al., 2021). In this regard, new target profiles for diagnostics have been
defined for mapping endemic areas and for stopping treatment.
Presently the definitive proof of active infection due to O. volvulus is by microscopic
demonstration of worms in skin snips or nodules surgically excised from suspects and PCR-
based detection techniques (Vlaminck et al., 2015). Unfortunately, the commonly used skin
snip method has a painful procedure which involves high risk of blood-born infection such as
HIV and may result in refusal of the test by populations understudies. It is also insensitive in
low transmission areas and in areas where long-term use of the microfilaricidal ivermectin
has resulted in a significant reduction of both individual and community skin microfilariae
loads (Osue et al., 2018). The alternative antibody detection assays, although sufficiently
sensitive, are unable to distinguish between active and past infections (Unnasch et al., 2018).
Presently, there is no standardized tool in the onchocerciasis control toolbox certified to
assess the success of vector control efforts and to detect the emergence of recrudescence.
However, the current NTD Roadmap 2030 insists on the need for such novel tools. Though
previous studies targeting onchocerciasis transmission have focused on the use of blackfly
DNA pools to assess the prevalence of infected and infective in the communities (Lloyd et
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al., 2015), there is a dire need to develop tools that could help in assessing the propensity for
recrudescence for the prompt deployment of appropriate diseases and vector control
measures.
Blackflies of the S. damnosum sensu lato (s.l.) complex are the prime vectors of Onchocerca
volvulus. Entomological, parasitological, and clinical assessments are routinely used to
evaluate their exposure to human populations and the risk of onchocerciasis transmission
(Koala et al., n.d.). However, these methods are labor-intensive and difficult to sustain on
large scales, especially when transmission and exposure levels are low (dry season, high
altitude, urban settings, or after vector control) (Hotterbeekx et al., 2020). The density of
human-biting black fly which measures black fly–human transmission intensity of
Onchocerca volvulus (Koala et al., n.d.) is estimated by using trapping methods such as
human-landing catches (HLC) of adult blackflies (Shintouo et al., 2020). Apart from the
ethical concerns raised by this method, estimates thus obtained do not reflect true exposure to
vector bites. Standardized tools to assess vector control efforts and hence possible
recrudescence are needed. Immunological studies have shown that the Simulium damnosum
acts not only as a syringe injecting parasite during a blood meal. It also injects into the human
skin a cocktail of bioactive molecules with anti-hemostatic, anti-inflammatory, and
immunomodulatory activities which assist the vector in the blood-feeding process by
inhibiting several defense mechanisms of the human host. Many of these molecules are
immunogenic and elicit specific acquired cellular or/and humoral responses in human
individuals when exposed to bites of black flies. The intensity of the antibody response
specific to salivary proteins could be a biomarker of the exposure level of humans to the
vector (Marzouki et al., 2015). Previous research on human immune response against salivary
antigens of Simulium damnosum s.l. identified major antigenic proteins of molecular weight
of about 15 and 40 kDa which were mainly apyrase/nucleotidase, and hyaluronidase (Willen
et al., 2021). With the evolvement of technology which can be used to explore this known
blackfly sialome, the rational selection of antigens will be made more feasible. The
successful development of biomarkers for vector control and risk of infection using vector
antigens has been reported for both malaria (gSG6-P1) (Drame et al., 2013) and leishmaniasis
(Teixeira et al., 2010) (PpSP32) and promises to contribute to the development of novel tools
that can be harnessed in the context of the current global elimination goal for onchocerciasis.
Also, IgG responses to An. gambiae whole saliva has been used to predict clinical malaria
cases (Drame et al., 2013). These and many other findings have so far made use of synthetic
peptides for these serological diagnoses, where large amounts of such peptides can be
produced with a high level of purity within a relatively short period. This can then be
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harnessed by arranging the peptides in microarrays to simultaneously analyze up to several

thousands of peptides with a single serum sample (Maksimov et al., 2012). For this study, the
amino acid sequences of some selected proteins from the sialome of the Simulium damnosum
antigenic proteins previously published by Willen et al., 2021 will be obtained from the
simulidae database. These will be fragmented, printed on chips and the peptide microarray
validated by analyzing many well-characterized human serum samples. From microarray
analysis, selected antigenic peptides will be synthesised and used to evaluate seasonal
fluctuation of antibody response to these Simulium damnosum salivary proteins, using serum
from onchocerciasis endemic communities of the Massangam and Mouanko health districts
in the west region and of littoral regions respectively of Cameroon where onchocerciasis
transmission is still ongoing. Although the community-directed treatment with Ivermectin
was launched since in 1996 (Kamga et al., n.d.) in these endemic communities such as the
Masangam health district in the west of Cameroon , transmission is still on going after over
20 years of biannual administration of ivermectin through the CDTI program (Bakajika et al.,
2018). There is therefore a dire need for a change in strategy of community-directed
treatment with Ivermectin. Considering the different seasons can influence the rate of
transmission of onchocerciasis, this seasonal fluctuation will be evaluated through antibody
responses at different period of the year. Studies have shown that secreted peptides or
proteins from the salivary glands of the mosquitoes can be ubiquitous or specific to
arthropods classes, others, families, and genus. In other to test the specificity of the antigenic
peptides of the black fly salivary proteins, an evaluation of any potential cross-reactions with
other bloodsucking arthropod will be done. The results obtained will provide the basis for
easy diagnosis of blackfly exposure which could serve as a tool in assessing the propensity
for recrudescence as well as inform policy on appropriate time of mass drug administration.

Overall Objectives

This project will focus on contributing towards the current global health goal for
onchocerciasis elimination through the development of novel tools to monitor vector
control efforts as well as assess the potential for recrudescence and risk of infection.

Specific objectives

This goal will be achieved through the following specific objectives:

1. Design peptide microarrays using blackfly salivary gland antigenic peptides

and analyze against serum samples from areas endemic for onchocerciasis.
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2. Obtain serum samples from onchocerciasis-infected persons and various

controls during the four seasons of the year which are the starts and peaks of
the dry and rainy seasons.

3. Evaluate the rate of seasonal exposure to simulium blackfly using the selected
peptides form microarray analysis

4. Evaluate the cross reactivity of the lead peptides from the microarray analysis
with antibodies against other arthropods such as anopheles mosquitoes.

Methodology

Ethical Considerations - Ethical approval will be obtained from the Institutional

Review Board of the University of Buea Faculty of Health Sciences. Written informed
consent will be obtained from all participants, or the parent/guardian of minors.

Study Population and blood sample collection –

This study will involve a longitudinal studies of over 10months involving adult male and
females. Blood samples will be collected from consented participants in the Massangam
Health District (where transmission is ongoing) the Bandjoun Health District (reported to
be in near-elimination) and in Buea regional hospital where malaria is endemic but its
onchocerciasis free, all in Cameroon. To exclude and compare unspecific reactions, 6
naïve European sera will be used as controls.

Protein selection- The proteins selected for the study will be sequences easily accessible
to the immune system (salivary proteins). The proteins reported by Willen et al.,2021 will
be selected from the Simuliidae database and used. The BLAST tool on UniprotkB will be
used to assess the level of conservation of the proteins. Selected proteins will be virtually
fragmented into 15-mer peptides with an overlap of 14 amino acids, which is the length
that results in optimum yields in the peptide synthesis. The Peptides with the lowest
similarity to the human proteome will be selected for peptide array production.

Preparation of peptide microarray slides. The peptide will be produced by the company
PEPperPRINT GmbH, Heidelberg, Germany and the procedure for testing, image
acquisition, data transformation, and analysis will follow established protocols.

Examination of sera by peptide microarray.

Well-characterized serum samples will then be examined on the array following the protocol
described by (Maksimov et al., 2012), and the scanning and evaluation of microarray slides
as well as data extraction will then be performed essentially following the appropriate
procedure.
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Analysis of selected peptides for cross reactivity.

Serum samples will be collected from malaria endemic areas such as Buea which are not
endemic for onchocerciasis properly characterized and used analyze the peptides obtained
from microarray analysis.

Expected outcome

At the end of the study we expect to determine the following parameters:

1. Diagnostic specificity and peptide-specific reactivity of sera.
2. Individual diagnostic sensitivities of peptides taken from the literature.
3. Reaction intensity of peptides with diagnostic potential
References

Dadzie, Y., Amazigo, U. V., Boatin, B. A., & Sékétéli, A. (2019). The need for evidence-
based strategies and tools for onchocerciasis elimination in Africa. Infectious Diseases
of Poverty, 8(1), 4–8. https://doi.org/10.1186/s40249-019-0574-0

Drame, P. M., Poinsignon, A., Marie, A., Noukpo, H., Doucoure, S., Cornelie, S., & Remoue,
F. (2013). New Salivary Biomarkers of Human Exposure to Malaria Vector Bites.
Anopheles Mosquitoes - New Insights into Malaria Vectors.
https://doi.org/10.5772/55613

Elimination of human onchocerciasis: progress report, 2019–2020. (n.d.). Retrieved August

15, 2021, from https://www.who.int/publications/i/item/who-wer9545-545-554

Hotterbeekx, A., Perneel, J., Mandro, M., Abhafule, G., Fodjo, J. N. S., Dusabimana, A.,
Abrams, S., Kumar-singh, S., & Colebunders, R. (2020). Comparison of diagnostic tests
for Onchocerca volvulus in the democratic republic of congo. Pathogens, 9(6), 1–8.
https://doi.org/10.3390/pathogens9060435

Koala, L., Nikièma, A. S., Paré, A. B., Drabo, F., Toé, L. D., Belem, A. M. G., Boakye, D. A.,
Traoré, S., & Dabiré, R. K. (n.d.). Entomological assessment of the transmission
following recrudescence of onchocerciasis in the Comoé Valley, Burkina Faso.
https://doi.org/10.1186/s13071-019-3290-5

Lloyd, M. M., Gilbert, R., Taha, N. T., Weil, G. J., Meite, A., Kouakou, I. M. M., & Fischer,
P. U. (2015). Conventional parasitology and DNA-based diagnostic methods for
onchocerciasis elimination programmes. Acta Tropica, 146, 114–118.
https://doi.org/10.1016/j.actatropica.2015.03.019
 6

Maksimov, P., Zerweck, J., Maksimov, A., Hotop, A., Groß, U., Pleyer, U., Spekker, K.,
Däubener, W., Werdermann, S., Niederstrasser, O., Petri, E., Mertens, M., Ulrich, R. G.,
Conraths, F. J., & Schares, G. (2012). Peptide microarray analysis of in silico-predicted
epitopes for serological diagnosis of Toxoplasma gondii infection in humans. Clinical
and Vaccine Immunology, 19(6), 865–874. https://doi.org/10.1128/CVI.00119-12

Marzouki, S., Kammoun-Rebai, W., Bettaieb, J., Abdeladhim, M., Hadj Kacem, S.,
Abdelkader, R., Gritli, S., Chemkhi, J., Aslan, H., Kamhawi, S., Ben Salah, A., Louzir,
H., Valenzuela, J. G., & Ben Ahmed, M. (2015). Validation of Recombinant Salivary
Protein PpSP32 as a Suitable Marker of Human Exposure to Phlebotomus papatasi, the
Vector of Leishmania major in Tunisia. PLoS Neglected Tropical Diseases, 9(9).
https://doi.org/10.1371/journal.pntd.0003991

Osue, H. O., Inabo, H. I., Yakubu, S. E., Audu, P. A., & Mamman, M. (2018). Onchocercal
DNA amplification using beta actin gene primers compared with first internal
transcribed spacer sequences for monitoring onchocerciasis eradication strategy. African
Journal of Biomedical Research, 21(1), 23–28.

Shintouo, C. M., Nguve, J. E., Asa, F. B., Shey, R. A., Kamga, J., Souopgui, J., Ghogomu, S.
M., & Njemini, R. (2020). Entomological assessment of onchocerca species
transmission by black flies in selected communities in the west region of Cameroon.
Pathogens, 9(9), 1–11. https://doi.org/10.3390/pathogens9090722

Teixeira, C., Gomes, R., Collin, N., Reynoso, D., Jochim, R., Oliveira, F., Seitz, A., Elnaiem,
D. E., Caldas, A., De Souza, A. P., Brodskyn, C. I., De Oliveira, C. I., Mendonca, I.,
Costa, C. H. N., Volf, P., Barral, A., Kamhawi, S., & Valenzuela, J. G. (2010). Discovery
of markers of exposure specific to bites of Lutzomyia longipalpis, the vector of
Leishmania infantum chagasiin Latin America. PLoS Neglected Tropical Diseases, 4(3),
13–15. https://doi.org/10.1371/journal.pntd.0000638

Unnasch, T. R., Golden, A., Cama, V., & Cantey, P. T. (2018). Diagnostics for onchocerciasis
in the era of elimination. International Health, 10(March), i20–i26.
https://doi.org/10.1093/inthealth/ihx047

Vlaminck, J., Fischer, P. U., & Weil, G. J. (2015). Diagnostic Tools for Onchocerciasis
Elimination Programs. Trends in Parasitology, 31(11), 571–582.
https://doi.org/10.1016/j.pt.2015.06.007

Who, T. (n.d.). Investing to overcome the global impact of neglected tropical diseases.
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Willen, L., Basáñez, M. G., Dvorak, V., Veriegh, F. B. D., Aboagye, F. T., Idun, B., Osman,
M. E., Osei-Atweneboana, M. Y., Courtenay, O., & Volf, P. (2021). Human immune
response against salivary antigens of Simulium damnosum s.L.: A new epidemiological
marker for exposure to blackfly bites in onchocerciasis endemic areas. PLoS Neglected
Tropical Diseases, 15(6), 1–19. https://doi.org/10.1371/journal.pntd.0009512