Combating Pandemics: A Review of the PREDICT Project and the Gibson Ultra Assembly Project: Nancy Khuu, Raymond Rupert MD. MBA.

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Combating Pandemics: A Review of the PREDICT Project and the Gibson Ultra Assembly Project

Nancy Khuu, Raymond Rupert MD. MBA.

Department of Human Biology, University of Toronto, 80 St. George St. Toronto, Ontario, Canada M5S 3H6

ABSTRACT: The adverse global health and economic impacts of pandemics resulting from influenzas demonstrate the need for prevention and control strategies. As such, the PREDICT project led by Dr. Jonna Mazet was developed as a global viral surveillance to detect viruses early, where intervention can be employed before an outbreak occurs.  In the event of an outbreak, Dr. Craig Venter envisions the digitalization of DNA sequences and the use of a “biological printer” or the Gibson Ultra Assembly machine, for rapid distribution of vaccines to enable quick delivery of treatment and to prevent enhanced virulence pathogenicity.

Introduction

Pandemic influenzas are a prominent global health challenge that causes millions of death making prevention and early control of global priority.6 During the twentieth century, the world saw the most devastating pandemic influenza with deaths totaling 40 to 50 million people. Known as the “Spanish flu”, it was caused by the H1N1 Influenza A subtype.1-4 In the following years, two more pandemic transpired resulting in mortality on the order of 1 million: the  “Asian flu” and the “Hong Kong flu” caused by the H2N2 subtype and the H3N2 subtype respectively.1,2

Pandemic influenzas ensue when a highly pathogenic viral subtype, one which the human population does not have immunological resistance to and is easily transmissible, spreads globally and rapidly.2 It is facilitated by an antigenic shift that often occurs through influenza A virus due to its wide pathogenicity and transmissibility of circulating strands.4 An antigenic shift refers to creation of a novel virus through genetic recombination of cells co-infected with human and animal influenza A.4 The recombination process may occur in an intermediate host such as swine, which is susceptible to both influenzas.2 Humans may  contract the novel virus during contact with infected swine species. The resulting virus becomes easily transmissible among humans, contains glycoprotein that the human system lacks serological immunity to and is the cause of a pandemic influenza if not contained.4

PREDICT PROJECT

The inextricable link between humans and wildlife is the core principle for the PREDICT project led by Dr. Jonna Mazet. The PREDICT project is a transdisciplinary team consisting of epidemiologists, wildlife ecologists and virologists that is active in twenty countries including: Tanzania, Uganda, Bangladesh, Cambodia, China, Laos, Mexico and Peru.6 PREDICT serves as a proactive surveillance and virus discovery component of the EPT (Emerging Pandemic Threats) program. It seeks to improve early detection and pandemic threat responses by isolating the emergence of new infectious viruses from wildlife known as “geographic hotspots (Figure 1).” 5,6 These hotspots are high-risk regions for disease transmission. 6 Some of these regions include Southeast Asia, the Gangetic Plain of South Asia, and the Amazonian region of South America.5,6 The generation of hotspots enable resources and efforts to be directed to those site for viral outbreak prevention.

To generate these “geographic hotspots”, surveillance efforts were focused on wildlife that were likely to be a source for zoonotic viruses including rodents and bats at human-animal interfaces.6 These interfaces were characterized by frequent and direct contact with wildlife such as when they share water reservoirs or are hunted by humans. Since interfaces with intense wildlife contact are most likely to facilitate disease emergence, samples were taken and tested for viruses related to taxonomic families known to cause epidemics and pandemics. Human behavioral surveys and were also conducted at various sites to gain a better understanding of the types of human-animal interaction. PREDICT also assessed how land changes (such as: deforestation) could alter the human-animal interfaces to modify the risk of pathogen spillover.  The data was analyzed in light of scientific literature to generate a map of potential disease outbreak. 5

Figure 1. PREDICT Map of “geographic hotspots.” The “geographic hotspots” are pictured as red circles. The map was generated from PREDICT animal sampling data. 6

                  The PREDICT project also aims to build in-country capacity and develop an infrastructure for countries to effectively detect and respond to epidemics and pandemics. It has provided biosafety and disease outbreak investigation training for over 2000 physicians, laboratory technicians and government personnel. Additionally, it has transferred technology and trained staff in 32 diagnostic laboratories on how to safely process wildlife samples for viral pathogens. 6 This fosters a self-sustaining program to ensure continued viral surveillance for outbreak prevention and control measures.

The PREDICT project is accredited with various global achievements. It has led to the discovery of 815 novel viruses and 169 known viruses- an amount surpassing the number of previously recognized viruses by the Internaional Committee on Taxonomy (ICTV). 6 It has prevented a Yellow Fever outbreak in Bolivia. After the death of 5 primates, the PREDICT team promptly investigated. Within 8 days, it was confirmed that they were infected with flavivirus, caused by two yellow fever viral strains. The team’s fast response allowed authorities to quickly implement treatment and disease prevention strategies such that no human cases of illness were reported during the outbreak.6 Furthermore, it has helped control the Ebola outbreak in the Democratic Republic of Congo  by actively engaging with government and public health officials. 6

GIBSON ULTRA ASSEMBLY PROJECT

When pandemics do occur, the need for a treatment to be developed rapidly becomes a vital global priority to not only prevent the spread of the virus but also to reduce the risk of the virus gaining antiviral properties through mutations. 7 An effective treatment tool in the event of a pandemic is a vaccine. Vaccines represent a broad class of altered or weakened pathogens that are introduced into human body to predispose the immune system to the virus.11 It induces the body to form antibodies or cytotoxic cells and memory cells to not only fight the existing pathogen but also to protect the body from future exposure.11

Currently, there are five-types of vaccines: live-attenuated, inactivated microorganisms, subunit, toxoids, and DNA-based.12 Live-attenuated vaccines refer to weakened pathogens through repeated serial passage that can still infect and multiply to elicit both humoral and cellular immunity. Inactivated microorganism vaccines are deceased pathogens caused by chemicals or x-rays that induces a humoral immune response. Subunit vaccines consists of purified antigenic proteins from the pathogen that are poorly immunogenic and produce a humoral response. Toxoid vaccines are inactivated bacterial toxins that induce a humoral response. DNA- based vaccines consist of a bacterial plasmid encoding genes of the pathogen that is injected to elicit a humoral and cellular response. 12

An existing challenge is the prompt manufacture and distribution of vaccines during a pandemic influenza. This spread of the H1N1 virus in Mexico due to delayed efforts serve as a testament to this challenge. Authorities had prohibited the virus from being sent for sequencing preventing a prompt analysis and vaccine development causing the virus to spread. J. Craig Venter, acknowledged for sequencing the human genome, proposes the digitalization of a vaccine blueprint in face of this challenge.8,9 The digitalization of DNA sequences denotes that it can be effectively distributed worldwide rapidly to avoid an epidemic and pandemic influenza. The virus’s genome would be isolated, sequenced and digitalized into a binary code that could be transferred online. The blueprint would be downloaded by a receiving ‘biological printer’. The receiving printer would function as a 3D printer and generate the vaccine from the blueprint.10

This ‘biological printer’, the Gibson Assembly Ultra machine, has been developed by J. Craig Venter’s team to enable the synthesis of the DNA that can serve as the foundation for the vaccine. It is a proficient and automated two-step method for complex assembly of large DNA constructs of up to one thousand kilo-base pairs using just nanograms of DNA.9 DNA constructs are designed by first creating DNA fragments with overlap regions and combining it with the Ultra Master Mix A. The Ultra Master Mix A contains a 5’ exonuclease, which removes the 5’ end to create overhangs. The Ultra Master Mix A is then inactivated at 75°C and the DNA fragments are annealed at 60°C. The resulting fragments are subjected to Ultra Master Mix B, which contains a DNA ligase and a DNA polymerase. The DNA polymerase extends the 3’ end while the DNA ligase catalyzes the repair of nicks to effectively create double stranded DNA (Figure 2).9 Since DNA ligase is used to assemble the subfragments rather than thermocycling, fragmentation of large DNA molecules is prevented.10 

            This robust in-vitro DNA assembly method makes the resulting DNA product feasible for a DNA-based vaccine generation. The DNA plasmid is then transformed into electrically competent Escherichia coli (E. coli) cells for amplification. Since the generated vector is ligated, it is more efficiently transformed into electrically competent (E. Coli) cells compared to un-ligated constructs.10  The E. coli cells are subsequently inoculated on media containing antibiotics to selectively facilitate growth of cells containing the vector with antibiotic resistance. The cells are harvested and the plasmids are purified and dissolved in a saline solution. This solution is then utilized as a DNA vaccine.11

Figure 2. Overview of DNA assembly using the Gibson Assembly Ultra Machine 10

Since the vaccine contains copies of the some of the disease genes and not the entire microbe itself, it is effectively attenuated. When injected into the human body, cells take up the vector and direct the formation of antigens encoded by the vector. These endogenous foreign antigens are degraded and presented on the cell surface by the Major Histocompatibility Complex I (MHC class I) which activates Cytotoxic T-Cells (CD8) to create memory cells for preparation of future exposure and cytotoxic cells which effectively destroys the viral antigen.                

            Alternatively, the DNA generated from the Gibson Ultra Assembly may also be used for the generation of other vaccines such as inactivated microorganism vaccines. The resulting DNA product could be transformed into yeast to generate a synthetic genome that is transplanted into a recipient cell to generate a synthetic cell.10 This synthetic cell would produce the viral proteins encoded by the generated DNA. The proteins could then be inactivated for use in vaccines.

CONCLUSION

The damaging global impacts of pandemics have encouraged the development of proactive and reactive responses to prevent and control pandemic influenzas. Dr. Jonna Mazet’s PREDICT project represents a successful large-scale global initiative to build in-country capacity to detect, prevent and respond to emerging zoonotic disease threats. It established new networks and enhanced communication among different professional sectors to promote global health. It’s proactive approach of recognizing, isolating and responding to viral threats before they emerge is highly impactful given the general lack of vaccinations for new viruses.6 A reactive response is demonstrated by Dr. Craig Venter’s Gibson Ultra Assembly project. Its vision to digitalize DNA sequences is a revolutionary step forward in the field of synthetic genomics. It would enable the basis for vaccines to not only be transported at the speed of light but also globally. However, there is much progress that needs to be made before the implementation of digital genomes for vaccine generation. For genetic combinations to be built and tested at a reasonable price, the cost for DNA synthesis must be lowered. Scientists would also need to ensure the high fidelity of the generated DNA products as even small errors can dramatically alter the properties of the vaccine. Future research should thus focus on developing automated synthesis of DNA fragments from small oligonucleotides to reduce cost and ensuring high fidelity at the level of digitalization to avoid bioterrorism.10

AUTHOR INFORMATION

ACKNOWLEDGMENT

Special thanks to Dr. Rupert for providing me with this incredible learning opportunity.

ABBREVIATIONS

HIV, human immunodeficiency virus; AIDS, acquired immune deficiency syndrome; SARS, severe acute respiratory syndrome; EPT, Emerging Pandemic Threats; PCR, Polymerase Chain Reaction; DNA, Deoxyribonucleic Acids; ICTV, International Committee on Taxonomy of Viruses; MHC I, Major Histocompatibility Complex I; CD8, Cytotoxic T-Cells

REFERENCES

  1. Sambhara, S., Poland, G.A. Lancet., 2006, 367, 1636-1638
  2. MacKeller, L. 2007, 33, 429-435
  3. Girard, M.P., Tam, J.S., Assossou, O.M., Kieny, M.P. Vaccine, 2010, 28, 4895-4902
  4. Yewdell, J.W., Hickman-Miller, H.D., Future Virol, 2006, 1, 161-164
  5. Schwind, J.S., Wolking, D.J., Brownstein, J.S., PREDICT Consortium, Mazet, J.A.K., Smith, W.A. PLOS ONE, 2014, 9
  6. UC Davis Veterinary Medicine http://www.vetmed.ucdavis.edu/ (accessed May 31, 2015)
  7. Girard, M.P., Tam, J.S., Assossou, O.M., Kieny, M.P. Vaccine, 2010, 28, 4895-4902
  8. Shampo, M.A., Kyle, R.A., Mayo Clin Proc., 2011, 86, 26-27
  9. Synthetic Genomics Inc, Company http://www.syntheticgenomics.com/ (accessed May 31, 2015)
  10. Gibson, D.G. Nat. Methods, 2014, 11, 521-526
  11. Donnelly,J.J., Ulmer, J.B., Shiver, J.W., Liu, M.A. Annu. Rev. Immunol., 1997, 15, 617-648
  12. Inglotti, M., Kawalekar, O., Shedlock, D.J., Muthumani, K., Weiner, D.B. Expert Rev. Vaccine, 2010, 9, 747-763