General Public

After the talk, we had a spike of interest in this issue and our project, with people reaching out for further information and discussion on the topic. There were a few key questions asked:

How can we protect ourselves from bioterrorist attacks? What safety measures are there in different institutions and laboratories preventing the accidental release of these technologies? What are regulatory policies enforced by our government to limit unsupervised access to genetic engineering technologies? How can we keep science open and accessible yet protect information that could be used to develop its hazardous manifestations?

To answer them, we needed to reach out to different communities and hear their perspectives on the issue.

Overview

Our supervisors were working on a research project funded by the NATO Science for Peace and Security Multi-Year Program that focused on the Early Detection and Diagnosis of Emerging Biological Threats. With their help, we determined that among many, poxviruses (specifically orthopoxviruses, OPXV) pose a high risk of being used as a biological weapon due to increased morbidity, the potential for significant diffusion, and relatively-easy development as bioweapons for terrorist attacks. As we discussed that engineering biological organisms would make it harder to detect and treat, we discovered that OPXV could accept up to 25kB of foreign genes in their sequence, making it a high-risk agent for bioterrorism use. Considering the safety measures and restrictions we discussed during the conference and the potential for informational hazards, we declined the idea of working with OPXV agents or even its sequence directly for this project. Instead, we could develop a detection system for poxviruses that would remain efficient even if bioengineered poxviruses are deployed. To do that, our supervisors advised us to look into the vaccinia virus (VACV), which was a source of smallpox vaccine in the 50s, whose proteins are cross-reactive with OPXV. However, we could not and would not want to work with VACV directly. Therefore, we decided to use a non-pathogenic strain of E.coli to produce recombinant proteins of the vaccinia virus that monoclonal antibodies can further detect on functionalized optic fiber. Moreover, we planned to utilize these proteins to develop recombinant vaccine formulations to protect against poxviruses.

Epidemiologists

To receive feedback on the proposed model we met with several representatives of our target audience: epidemiologists, researchers, and biosecurity-focused companies.

As an experienced epidemiologist, Dr. Dinara found our project promising and of urgent need in response to the rapidly emerging problems in biosecurity and biodefense. In particular, Dr. Dinara shared that such a tool would be necessary for rapid results and processing for field epidemiologists for early identification of an outbreak and localization of its source. She also shared her concern about our compliance with regulatory frameworks when considering the installation of such devices on HCBL drainage systems and their efficiency.

Researchers

We presented our biosensor project idea and inquired for help determining the vaccine potential of our recombinant proteins. In this iteration, we envisioned our project to be two-fold - a detection system of threats using biosensors and a prevention system using novel recombinant vaccines. Although we had our hopes high, experts assured us that 1) there are already effective vaccines against different poxviruses, 2) if we cannot proceed with in vivo testing on animals, we cannot prove the efficiency of our model, 3) we have way too tight time restraints to achieve noticeable results supporting our hypothesis. They were supportive and assured us that if we wished to proceed, their center would be glad to help with in-vivo experiments and mentor our team. Experts also shared their experience with informational attacks from anti-vax communities regarding their COVID vaccine development, as well as informed us that Central Reference Laboratory in Almaty (HCBL) and other biosecurity institutions are negatively perceived by the Kazakhstani population (especially if they are affiliated with international organizations, for our project it was NATO). We quickly realized that we needed to halt our project's growth in the direction of vaccine development and focus entirely on our biosensing system. 

Industry

We reached out to Dr. James Diggans both because of his extensive expertise in working with DARPA on Biosecurity and his specific specialization on Biothreat Detection using Biosensors. First, he shared his concerns about the detection capabilities of our system in relation to modified organisms, specifically epitope modifications, since we utilize an antibody-based biosensing system. Second, considering the effort and costs associated with developing our system, it would not be feasible for our customers to use it if it could detect one protein of one specific pathogen of interest at a time. And third, considering the resources needed to train staff to use our system, the lack of automation in our result processing aspect is also a significant limitation. However, he shared that if we come around these limitations effectively, military forces would be interested in installing such a strategic defense system to protect deployed military members in high-risk areas.

Portable Device

As we learned from epidemiologists (Dr. Dinara Alimkhanovna and Dr. Zayurbek Sagiev) they had an unmet need for portable and rapid detection devices to work in the field; however, our initial setup for processing the results aimed at stationary positioning, thus using readily available laboratory equipment (e.g. LUNA), which was not only heavy but also far from being affordable. So we decided to scale down the optical frequency domain reflectometer (OFDR) that analyzes signals from optic fibers before sending them to computing machinery to create a compact mechanism for use in field conditions.

Multiplex and Software

We shortlisted target proteins for recombinant engineering so that only those highly conservative across the OPXV genus are used in our system; regardless of modifications made (insertion of transgenes, epitope modification, etc.), detection efficiency remains high enough. Secondly, we decided to focus on the multiplex detection system, meaning that our functionalized optic fibers are focused on detecting multiple targets simultaneously. And thirdly, we developed software for the automatic and rapid processing of reading results obtained from OFDR that is easy to install and use with no special training needed. 

Aptamers

Also, as advised by our mentors, we sought more sustainable alternatives to using monoclonal antibodies for optic fiber functionalization. Therefore throughout the project, we tried designing a way to utilize aptamers instead. However, there was no record of aptamer-based sensors targeting these specific proteins, considering that even the 3D structure of these proteins was not made before our project. As a result, we built many software tools from scratch or upon existing software made by preceding iGEM teams. For months we made, optimized, and tested in-silico models for optimal aptamer sequence in collaboration with IISER-Tirupati_India and Linköping teams in an iterative process as documented in our Collaboration and Software sections.