Mosquito saliva biomarkers: Implications for disease risk and epidemic prediction

Diseases spread through the bite of mosquitoes are a major health challenge. Dr Berlin Londono-Renteria, at Kansas State University, is exploring ways to predict the likelihood of an individual developing a mosquito-borne disease after they have been bitten. She has found positive correlations between the levels of antibodies against mosquito salivary biomarkers and the risk of disease. Understanding more about biomarkers of disease is important for public health strategies, disease monitoring and predicting future epidemics.

Despite their small size, mosquitos are one of the deadliest animals on the planet. This is because of their ability to transmit pathogens (organisms that cause disease) through their saliva. When a mosquito feeds on blood, it also swallows any pathogens in the blood and can, after an incubation period, transfer these to the next person it bites through its saliva. Diseases spread in this way, called mosquito-borne diseases, are responsible for causing illness in around 700 million people each year. Of these cases, approximately one million are fatal. Some of the most common mosquito-borne diseases are malaria, dengue, Zika, yellow fever and West Nile virus. There is no specific vaccine for most vector-borne diseases, so prevention relies mainly on interventions to control the vector, e.g mosquitos, ticks or fleas.

When a mosquito bites an animal or human, it deposits saliva in the skin to ensure a good flow of blood by preventing clotting and to modulate the host immune response. Mosquito saliva is immunogenic, meaning that it is able to provoke an immune response in the animal or human it has bitten. It also induces antibody production and the number of antibodies produced is correlated to the intensity of exposure – in other words, the more times a person is bitten, the more antibodies they will produce. There are also factors that influence the composition of mosquito saliva, such as viral infection, proteins (collagen-binding protein) and the origin of the mosquito.

Measuring bi-specific antibodies

Arthropod-borne viruses, also called arboviruses, are deposited in skin along with the mosquito saliva. These viruses have developed a mechanism to benefit from the immunomodulatory properties of the mosquito saliva to guarantee their transmission to the vertebrate host. Dr Berlin Londono-Renteria and her research group at Kansas State University are interested in the link between antibodies induced by mosquito saliva and the chance that the person will develop a mosquito-borne disease. The team use the responses against these salivary proteins to quantify the risk of suffering a disease. Understanding more about these interactions would allow the development of alternative approaches for the control and prevention of mosquito-borne diseases.

“Identification of a mosquito-specific biomarker that is associated with infective bites has huge potential for evaluating the risk of disease transmission.”

There are several different families of antibodies in humans, but it is thought that IgG4 antibodies may be a useful tool to measure exposure to infected mosquito bites. Dr Londono-Renteria showed that IgG4 antibodies, which can recognise two different antigens at the same time, are indeed produced during human infection with mosquito-borne diseases. It was also found that the bi-specific IgG4 antibodies were capable of recognising both mosquito salivary proteins and dengue virus particles. She also concluded that these IgG4 antibodies could be used as an approximate measure of immune responses against virus-containing mosquito saliva.

Antibody levels can predict malaria risk

One of the main barriers to combating malaria is the lack of tools to assess the risk of malaria transmission and the variation in behaviour of the mosquitoes that spread the disease. Identification and characterization of a biomarker for the number and duration of contacts between a human and a vector would allow us to better define the risk of epidemics, to assess and monitor vector-control interventions and to inform the choice of control strategies.

Dr Londono-Renteria hypothesises that human antibodies could be the biomarker we have been waiting for. Alongside colleagues, she has used human antibodies that recognise proteins produced by mosquito salivary gland in an adapted version of a laboratory technique called ELISA (enzyme-linked immunosorbent assay). An ELISA plate contains immobilised target proteins, the mosquito salivary gland protein in this case. The plate is then washed with different solutions of antibodies and the number of complexes between protein and antibody are quantified.

The researchers then used this ELISA-based technique to measure the antibody response in people with different presentations of malaria in order to understand more about the links between antibody levels and disease. They found that there was a positive correlation between the salivary antigens and human antibodies, and statistical analysis showed that people with higher levels of antibodies were up to five times more likely to suffer from malaria.

Anti-viral effects of mosquito saliva

In recent decades, the global incidence of dengue fever has increased, with around 40% of the world’s population now at risk. Dr Londono-Renteria was interested in whether a protein called D7 Long (D7L), found in the saliva of mosquitoes, was related to development of dengue. These proteins are some of the most abundant molecules expressed in the salivary glands and have a wide range of functions, including blocking inflammatory responses, clotting mechanisms and pain when the mosquito is obtaining a blood meal.

The study was done at a hospital in Colombia and evaluated the antibody responses of 63 participants to the D7L protein. As with the malaria study, an ELISA-based method was used to evaluate the antibody concentration. Other factors such as age and living conditions were also taken into consideration. The study showed that people who had dengue infection had higher levels of antibodies against the mosquito D7L protein. In addition, there was a positive trend increasing with age, as people over 20 years old tended to have higher levels of antibodies against D7L.

A link between dengue and socioeconomic factors has already been established in some countries, including Thailand, Brazil and Saudi Arabia. While there was no significant difference between living conditions in this study, there did seem to be a trend towards higher antibody levels in people infected with dengue virus who were living in more deprived conditions (characterized as the upper-low stratum). Dr Londono-Renteria highlights the need for further research exploring predictors of dengue infection, as well as the need to establish a database of specific biomarkers that relate dengue virus infection risk to socioeconomic factors. Her research already suggests that identification of a mosquito-specific biomarker that is associated with infective bites has huge potential for evaluating the risk of disease transmission.

“The researchers measured the antibody response in people with different presentations of malaria to understand more about the links between antibody levels and disease.”

Interestingly, an earlier study lead by her collaborator, Dr Michael Conway at the Central Michigan University, found that the D7L protein inhibited dengue virus infection. Dr Conway, who invited Dr Londono-Renteria to expand this study with specific experiments, was able to show that the D7L protein prevented dengue viruses binding to different types of cells grown in a laboratory and, what’s more, that D7L prevented infection and viral dissemination in a mouse model. Given that some individuals may produce higher antibodies against D7L, their studies suggest that this may actually enhance disease transmission and severity by preventing the anti-viral effects of the D7 protein.

Predicting epidemics

Dr Londono-Renteria reports that their ELISA-based method is suitable for evaluating the efficacy of current vector-control methods, such as the use of insecticide in households, to control transmission of disease by mosquitoes. It can also be used to evaluate efficacy of personal devices that decrease mosquito-human contact, such as bed-nets and insecticide-impregnated uniforms known to protect humans against diseases like malaria. This means it can also be used to guide public health decisions around whether a particular intervention is effective in decreasing the risk of mosquito-borne disease.

These antibody levels can also be plotted on a map, which allows the researchers to see where people are being exposed to mosquito bites. This allows a focused approach to intervention decreasing costs.

Leading on from this, the group at Kanas State University are developing a tracking system that could potentially be linked to an alert system. This alert system could then be utilised by public health agencies to predict where future epidemics may occur, allowing early identification and intervention for disease outbreaks linked to mosquito bites.

Dr Londono-Renteria explains that there is still more work to be done to fully understand the complex interplay of host-virus interactions. However, these studies will lead to the development of better strategies to reduce disease transmission and design of novel therapeutics against mosquito-borne diseases.

References

• Marin-Lopez, A. et al. (2021). AgBR1 and NeSt1 antisera protect mice from Aedes aegypti-borne Zika infection. Vaccine, 39(12), 1675-1679. Available at: https://doi.org/10.1016/j.vaccine.2021.01.072
• Londono-Renteria, B., Montiel, J., Calvo, E., Tobón-Castaño, A., Valdivia, H. O., Escobedo-Vargas, K., Romero, L., Bosantes, M., Fisher, M. L., Conway, M. J., Vásquez, G. M., & Lenhart, A. E. (2020). Antibody Responses Against Anopheles darlingi Immunogenic Peptides in Plasmodium Infected Humans. Frontiers in cellular and infection microbiology, 10, 455. Available at: https://doi.org/10.3389/fcimb.2020.00455
• Manning, J. et al. (2020). Safety and immunogenicity of a first-in-human mosquito saliva peptide vaccine: a randomized, placebo-controlled, double-blind Phase 1 trial. The Lancet, 395(10242). Available at:
  https://doi.org/10.1016/S0140-6736(20)31048-5
• Londono-Renteria, B. L., Shakeri, H., Rozo-Lopez, P., Conway, M. J., Duggan, N., Jaberi-Douraki, M., & Colpitts, T. M. (2018). Serosurvey of Human Antibodies Recognizing Aedes aegypti D7 Salivary Proteins in Colombia. Frontiers in public health, 6, 111. Available at: https://doi.org/10.3389/fpubh.2018.00111
• Conway, M. J., Londono-Renteria, B., Troupin, A., Watson, A. M., Klimstra, W. B., Fikrig, E., & Colpitts, T. M. (2016). Aedes aegypti D7 Saliva Protein Inhibits Dengue Virus Infection. PLoS neglected tropical diseases, 10(9), e0004941. Available at: https://doi.org/10.1371/journal.pntd.0004941
• Londono-Renteria, B., Cardenas, J. C., Troupin, A., & Colpitts, T. M. (2016). Natural Mosquito-Pathogen Hybrid IgG4 Antibodies in Vector-Borne Diseases: A Hypothesis. Frontiers in immunology, 7, 380. Available at: https://doi.org/10.3389/fimmu.2016.00380
• Londono-Renteria, B., Patel, J. C., Vaughn, M., Funkhauser, S., Ponnusamy, L., Grippin, C., Jameson, S. B., Apperson, C., Mores, C. N., Wesson, D. M., Colpitts, T. M., & Meshnick, S. R. (2015). Long-Lasting Permethrin-Impregnated Clothing Protects Against Mosquito Bites in Outdoor Workers. The American journal of tropical medicine and hygiene, 93(4), 869–874. Available at: https://doi.org/10.4269/ajtmh.15-0130

Research Objectives

Dr Londono-Renteria and her lab are developing biomarkers to determine vector-human contact in order to measure disease risk of vector-borne diseases and to predict epidemics.

Funding

The USDA National Institute of Food and Agriculture, Hatch-Multistate, project 1021430 and the COBRE – NIH 5P20GM103638-08, BLR.

Collaborators

• Michael Conway (Central Michigan University)
• Researchers at the Erasmo Meoz Hospital (Cucuta), Emiro Quintero Cañizares Hospital (Ocana) and Villa del Rosario Hospital in Norte de Santander-Colombia.
• Gissella Vásquez (Department of Entomology – U.S. Naval Medical Research Unit No. 6 [NAMRU6])
• Audrey Lenhart (Center for Global Health/Division of Parasitic Diseases and Malaria – CDC)
• Hugo Valdivia (Department of Parasitology – U.S. Naval Medical Research Unit No. 6 [NAMRU6])

Bio

Berlin Londono-Renteria is a microbiologist studying arthropod-host interactions leading to vector-borne diseases. Her research is focused on the characterization of mosquito salivary proteins as markers for disease risk and transmission dynamics. Dr Londono-Renteria has published more than 45 manuscript in high-impact journals including PNAS.

Contact

E: [email protected]
T: +1 785 5322120
W: http://www.vectorbioksu.com/
W: https://www.researchgate.net/profile/Berlin-Londono-Renteria
Linkedin: https://www.linkedin.com/in/berlin-londono-5465b454/
Twitter: @bluxlon