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Zika vs Dengue: Minimizing Cross-reactivity in Immunoassay Development

Zika vs Dengue: Minimizing Cross-reactivity in Immunoassay Development content piece image
Spot the difference: Cryo-EM structures of the Zika and Dengue viruses, respectively. Left: Dengue Cryo-EM (Zhang et al. (2012). Cryo-EM structure of the mature dengue virus at 3.5-A resolution. Nat.Struct.Mol.Biol. PDB ID: 3J27). Right: Zika Cryo-EM (Klose et al. (2016). The cryo-EM structure of Zika Virus. Science. PDB ID: 5IRE)
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The Zika virus poses a significant global risk, and there is currently no vaccine or specific treatment available for those infected.

Towards the end of 2019, a series of immunoassays were launched by The Native Antigen Company, providing researchers the capability to work with unprecedented levels of specificity and sensitivity. We spoke to Nick Roesen, Founder and COO, to learn more about Zika-Dengue cross-reactivity, and Zika immunoassay development.

Michele Wilson (MW): Can you please explain the WHO R&D Blueprint for priority emerging diseases, and tell us how it is used to direct research at The Native Antigen Company? 

Nick Roesen (NR): 
In 2015, the World Health Organization (WHO) brought together a coalition of experts from around the world to develop the first "R&D Blueprint" – a guide for prioritising global research on the infectious diseases that pose the most significant risk to global health, and for which no preventative or curative solutions currently exist. 2018’s list may include some examples you’ve heard of:

  • Crimean–Congo haemorrhagic fever (CCHF)
  • Ebola virus disease and Marburg virus disease
  • Lassa fever
  • Middle East respiratory syndrome coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome (SARS)
  • Nipah and henipaviral diseases
  • Rift Valley fever (RVF)
  • Zika virus disease
  • Disease X (indicating an unknown disease that will cause an international epidemic in the near future) 

At The Native Antigen Company, we develop cutting-edge reagents for a range of emerging and endemic diseases. The WHO R&D Blueprint is paramount in guiding our R&D efforts and underpins much of the research that we do at the company.

MW: Can you explain what Zika-Dengue cross-reactivity is and how it threatens disease control? 

The Zika and Dengue viruses are both members of the Flaviviridae family and show a high degree of structural similarity. As they share such similar structural patterns on their surface (epitopes), antibodies produced against one of these viruses will often bind to the other – a phenomenon known as cross-reactivity.

Serological tests, such as ELISAs, are mainstay diagnostics for many infectious diseases. But as these tests use antibodies to detect the presence of viruses in a patient’s bloodstream, cross-reactivity has been a major hurdle – not only threatening effective diagnosis and treatment of the disease, but also the integrity of ongoing academic and epidemiological research. 

MW: Recently, you launched a series of immunoassays for Zika virus. How do these assays improve on existing ELISAs? 

NR: Taking lessons from what we knew about Zika virus’s cross-reactivity, we sought to develop a series of novel ELISAs that specifically overcame this problem by improving the selection and synthesis of antigen targets to achieve better specificity. To select the assay targets, we synthesized a panel of recombinant antigens from Zika, Dengue and other flaviviruses and used them to screen patient sera to identify those antigens preferred in terms of serological specificity and sensitivity. Only antigens with the lowest cross-reactivity were then selected for the new assays. 

The recombinant antigens used were developed in a mammalian cell expression system and show high similarity to the native viral proteins (being fully glycosylated, naturally folded and assembled, and with the same binding activity), ensuring high assay reliability and performance.

The second reason why these immunoassays minimize cross-reactivity is their design. By using an improved assay design, two new and highly specific antibody assays for Zika have been developed: an IgM assay and a total antibody assay (measuring IgM/IgG/IgA). In the IgM assay, excellent specificity is ensured by combining a direct antigen approach with blocking by carefully selected additional reagents. In the total antibody assay, the use of a DABA (double antigen bridging assay) format enables the detection of only higher affinity antibodies, which tend to be more specific and show less cross-reactivity. 

MW: Lateral flow immunoassays are expected to play a huge role in the diagnosis of infectious disease and their relative simplicity and low-cost is certainly desirable in low-resource areas. Can you comment on the expertise required to develop a lateral flow device, and the specific challenges involved? 

The development of lateral flow assays is a highly specialist area, requiring expertise not only in assay design, but also in the physical components of the assay device. The way in which the device is constructed has a major impact on how different sample fluids move through the assay, and can also have a major influence on sensitivity and levels of non-specific binding. This is reflected by the fact that it is not always possible to simply transfer reagents from ELISA assays to lateral flow assays.

That said, the quality of the reagents used to develop lateral flow assays is very important. Where protein antigens are part of the assay design, the use of mammalian-expressed recombinant proteins provides reagents that are matched as closely as possible to the natural antigen, and therefore provides the best possible results. The expertise developed by NAC in preparing such antigens is therefore key in supporting the development of the highest quality lateral flow devices.

MW: It's great to see a company taking initiative and considering the environment in their work. Can you tell us about your sustainability efforts? 

Bringing environmental sustainability into work and personal life is now an important priority for everyone on the planet, and we are committed to showing that it can be done without negatively affecting business. True sustainability for companies in the biotech and pharma areas is difficult, especially when it comes to the use of single-use sterile plasticware which has transformed tissue culture in recent times.

However, we do buy 100% renewable electricity to power our lighting and equipment and use 12% biogas for heating. We also use electronic document signing and storage to reduce paper waste and archiving costs, we use cardboard packaging over plastic and foam wherever practical, and we encourage commuting to and from work by walking, bicycle, bus or electric car. We can always do more and are constantly trying to improve in this area, but I am pleased to say that it really hasn’t had noticeable impact on costs. I am seeing more and more companies going green and am rightly proud of that fact! 

Nick has a PhD in oncolytic adenoviruses from the CRC Institute for Cancer Studies and previously worked as a senior scientist at Hybrid BioSystems (now PsiOxus Therapeutics) helping to develop their lead oncolytic virus candidate. With over a decade of experience in molecular virology and antigen production, Nick co-founded The Native Antigen Company with former CEO Andrew Maxwell in 2010. 

Nick Roesen was speaking to Michele Wilson, Science Writer for Technology Networks.