Abstract
Advances in synbio allow us to easily manipulate genes for shared benefit, yet it also makes it easier to engineer pathogens for malicious purposes. How can we utilize synbio to protect ourselves from its dangerous manifestations? With that in mind, we are focused on advancing the field of biosecurity, by developing fiber-optic-based biosensors for the rapid detection of engineered pathogens in the environment by virus-specific antibodies bound to the device. We will use a non-pathogenic strain of E. coli BL21(DE3) for the production of recombinant, highly conserved proteins of vaccinia virus, which will further be detected by monoclonal antibodies on functionalized optical fibers. These proteins of the vaccinia virus are cross-reacting with orthopox, monkeypox, and variola viruses, providing our tool with a wide range of detectable pathogens. As a result of the project, a novel, specific, and sensitive device for the fast detection of poxviruses in various fluids will be constructed.
What is the problem?
Advances in synthetic biology offer a great multitude of benefits for humanity across many fields and areas; however, it also raises a safety concern for accidental or deliberate misuse of newly emerging technologies; naturally-spread, leaked, or released infectious agents represent a major issue for humanity. Unlike other types of warfare agents, such as chemical and nuclear, biological agents (BWAs) possess several aggravating characteristics: they are less expensive and space-demanding to store and produce; they are infectious with longer incubation periods; they are transmittable and thus can affect larger masses with smaller doses (1). Viruses are one of the most ominous biological threats - hard to culture and identify, latent during first stages, contagious, inexpensive to engineer. The effect of the biothreat can only be sufficiently mitigated when taken care of at the eraly stages. But how to detect the presence of a biological agent before it infects thousands of people? How to ensure the right level of sensitivity and specificity of an instrument?
Relevance
“The threat of Bioweapons is not only persistent in modern days, it is as relevant as it has never been
before.
Such an attack could kill millions of people and should not be underestimated”
© Ken Alibek
The history of BWAs and other infectious agents is extensive and full of precedents - they can be aerosolized in the air (2), dissolved in drinking and sewage waters (3), and even sent by post (4). Salmonella being used by Germans as a bioweapon during the World War I and by the Rajneen cult in 1986 (5); botulinum toxin, aflatoxin, and anthrax agents being developed in Iraq during the Persian Gulf War in the late nineties (6). Not to mention the outbreaks such as the smallpox pandemic, which killed more than 300 million people since 1900 alone, or more recent coronavirus and monkeypox outbreak, whose casualties are yet to be counted. The issue is no less relevant to the local Kazakhstani community than it is to the world since there is a High Containment Biosafety Laboratory (HCBL) located in the most densely populated city of the country. Considering different political incidents emerging in the Central Asian region, there was an influx of worries among not only Kazakhstani population but also across the entire region about using pathogenic organisms from said laboratories as bioweapons by terrorist organizations (7-9).
Existing detection methods
Methods, such as double agar, PCR, HPLC and enzyme linked immunosorbent assays, that are still known to be conventionally used for detection of infectious agents have some drawbacks - mainly related to the low sensitivity, hight Type 1 and Type 2 error rate, and laborious procedures (1). Latest developed instruments utilize combinations of the wave sensors, immunosensors, biosensors, and microfluidics, surface plasmon resonance, nanoparticles, etc. to avoid these drawbacks. Many of them are capable of real time detection, and exhibit high sensitivity and specificity; yet, the cost efficiency in production and utilization of these methods does not always meet the expectations. Ideal detection mechanisms must be rapid, reliable, portable, suitable for multiple agents and environments, sensitive for low concentrations, and user-friendly.
Goal
Our goal is the early detection of viral proteins related to potential bioterrorism agents to allow for quick medical countermeasures and block consequent damage.
Proposed solution
“Viraless” - a novel method for the rapid detection of viral pathogens in environmental and bodily fluids.
In this project we aim to create an immunosensor based multiplexed system of functionalized optic fibers for
detection of viral particles. To achieve this, we engineer a non-pathogenic E.coli strain to produce
recombinant
proteins of Vaccinia virus and use it to configure and calibrate the optic-fiber-based ball resonator
biosensor
for
detection of the wild type proteins on the free-floating OPXV particles.
We decided to focus on four membrane bound proteins of the Vaccinia virus: A27, A33, L1, and B5R (Figure 2).
The
first three peptides were successfully expressed in E. coli BL21(DE3), so in this project we attempted to
synthesize
B5R protein using the similar technique.
truncated A27 protein
(PDB 3VOP) (10)
truncated A33 variant
PDB 3K7B) (11)
L1 ectodomain
(PDB 1YPY) (12)
t Sushi domains of the B5R
Alpha Fold
How does it work?
The fiber optic technology is based on transmission of the light signal inside of a hollow silicon hollow tube. The photon beam is reflected from the walls, and any interference in this reflection pattern can be recognized and interpreted by an observer. Interaction of molecules with the surface of fiber may introduce such interference - a phenomenon known as the surface plasmon resonance. We make this interference meaningful and easily interpretable by narrowing it down to two states. Antigen-specific antibodies attached to the surface of the fiber -- now named functionalized optic fiber -- represent one state. The second state appears when freely floating proteins of interest bind to fixated antibodies and cause the alternation in reflection pattern of the light within the fiber. As a result, some of the light is being lost on its way, and the returned (decreased) intensity is recorded as an interaction, helping us to draw a conclusion about the presence of certain proteins in a sample.
Why optic fiber?
- Remarkable sensitivity provided by optic fibers allows us to detect viral proteins in nanomolar or even picomolar concentrations, meaning that functionalized optic fibers can effectively detect the presence of virus, if released;
- Real life detection and high detection speed (close to the speed of light)
- Due to low production costs for optic fibers, proposed systems will be accessible and convenient for installation at a large scale;
- Due to low production costs for optic fibers, proposed systems will be accessible and convenient for installation at a large scale;
- The technology is open for multi-channel detection, implying that with time we can increase the list of detectable pathogens by our system.
Practically, the technology can be used to detect important molecules - there is already work being done on cancer detection, etc. One way to use this technology is to detect pathogens - for example in rivers, wastewaters, laboratories, military areas - to prevent epidemics and use of bioweapons - and that is what we are trying to do.
Why recombinant protein?
Orthopoxviruses (OPXVs), including the notorious smallpox and recently naturally-spread monkeypox, are an easily weaponised genus of viruses that thus presents a major threat to humankind. The A27, A33, L1, and B5R are the proteins of vaccinia virus that are highly conserved among many OPXV species, allowing us to expand the detection range by utilizing cross-reacting antibodies. Moreover, B5R membrane protein is considered to be one of the most immunogenic peptides in this genus. Constructs of the complete membrane protein are, however, not very suitable for the expression in prokaryotic cells. Bacteria lack the apparatus for post-translational modifications and protein folding and thus cannot guarantee correct conformation of the final peptide. Synthesis of the Vaccinia virus proteins in prokaryotes is complicated by the multiple cysteine residues that favor formation of the non-specific disulfide bridges. Expressed proteins must be purified, re-solubilized and re-folded in order to produce functional and competitive antigens. To overcome this, the certain modifications were necessary:
- Since B5R contains a leader peptide sequence which is cleaved
- Only ectodomains (Sushi domains) of the proteins were cloned
- Mutation of one extra cysteine to avoid non-specific disulphide bridges
Why our project is good for the world
In response to the danger of newly emerging biological threats, we have developed a highly sensitive and specific instrument for real time detection of natural and engineered pathogens in various fluidic environments. It will help to protect the healthcare system from the drastic impact of the bioterroristic attacks and outbreaks by allowing timely detection of a pathogen as well as ex ante risk assessment. Our solution has successfully addressed all concerns and suggestions proposed by the top experts of this field.
References
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- Takahashi H, Keim P, Kaufmann AF, Keys C, Smith KL, Taniguchi K, et al. Bacillus anthracis Bioterrorism Incident, Kameido, Tokyo, 1993 - Volume 10, Number 1—January 2004 - Emerging Infectious Diseases journal - CDC. [cited 2022 Oct 11]; Available from: https://wwwnc.cdc.gov/eid/article/10/1/03-0238_article
- Drell SD, Sofaer AD, Wilson GD, Hoover Institution on War, Revolution, and Peace, editors. The new terror: facing the threat of biological and chemical weapons. Stanford, Calif: Hoover Institution Press; 1999. 512 p. (Hoover national security forum series).
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- Zilinskas RA. Iraq's Biological Weapons: The Past as Future? JAMA. 1997 Aug 6;278(5):418-24.
- Могут ли опасные штаммы из алматинской “американской” лаборатории стать биологическим оружием в руках террористов? - Аналитика | Караван [Internet]. [cited 2022 Oct 11]. Available from: https://www.caravan.kz/articles/mogut-li-opasnye-shtammy-iz-almatinskojj-amerikanskojj-laboratorii-stat-biologicheskim-oruzhiem-v-rukakh-terroristov-808940/
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- Chang TH, Chang SJ, Hsieh FL, Ko TP, Lin CT, Ho MR, et al. Crystal structure of vaccinia viral A27 protein reveals a novel structure critical for its function and complex formation with A26 protein. PLoS Pathog. 2013;9(8):e1003563.
- Su HP, Singh K, Gittis AG, Garboczi DN. The Structure of the Poxvirus A33 Protein Reveals a Dimer of Unique C-Type Lectin-Like Domains. J Virol. 2010 Mar;84(5):2502-10.
- Su HP, Garman SC, Allison TJ, Fogg C, Moss B, Garboczi DN. The 1.51-Å structure of the poxvirus L1 protein, a target of potent neutralizing antibodies. Proc Natl Acad Sci. 2005 Mar 22;102(12):4240-5.