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Exploring the potential applications of live viral vaccine encoded small-hairpin-RNAs in improving both vaccine safety and efficacy through RNA-interference and stimulation of the innate immune system

Periodic Reporting for period 1 - Super-Vaccine (Exploring the potential applications of live viral vaccine encoded small-hairpin-RNAs in improving both vaccine safety and efficacy through RNA-interference and stimulation of the innate immune system)

Reporting period: 2018-09-19 to 2020-09-18

Live viral vaccines consist of attenuated (non-pathogenic) forms of harmful viruses and represent one of the most important and effective interventions against the spread of viral disease. Their widespread use has brought about vast improvements in both human and animal health and also facilitated more cost-effective livestock production. As such, they will continue to be instrumental in addressing ongoing societal challenges in areas such as healthcare and global food security. However, their capacity to genetically recombine with wild type (WT) viruses circulating in the field remains a primary safety concern. Also, efforts to enhance the intrinsic potency of vaccine strains would be very beneficial in terms of reducing the manufacturing resources required to meet market needs and reducing cost of vaccination programs, all of which would represent a tremendous benefit to society.

With this in mind, the objectives of this project were to explore a novel strategy to improve both the safety and efficacy of live viral vaccine strains by modifying them to express small-hairpin-RNAs (or shRNAs, a type of non-coding RNA) that have been specifically designed to:

i) Exclusively inhibit WT virus replication/propagation via RNA-interference (RNAi) during co-infections i.e. improving safety

ii) Increase live vaccine strain stimulation of the innate immune system by functioning as RIG-I agonists and thus act as potent inducers of Type-1 Interferon (IFN-I) expression in host cells during vaccination, essentially acting as adjuvants i.e. enhancing efficacy.

Notably, RIG-I stimulated IFN-I expression represents an important step in the initiation of a Th2 polarized immune response, which is vital for establishing long-term adaptive immunity against viral pathogens. This concept was explored using Cyprinid Herpesvirus-3 (CyHV-3) and its host, carp (Cyprinus carpio), as a virus-host model.

In conclusion, the project represented an important starting point in the exploration of this novel vaccine design strategy. Moreover, it allowed us to rationally scrutinize our widely held assumptions related to our chosen virus-host model which we had factored into our initial plans. Through this process, we have gained extremely valuable insights into the biology of our virus-host model that would have otherwise remained elusive. Taken together, this work puts us in a strong position to proceed towards the testing of several exogenously expressed RIG-I agonists (and our own custom molecules) in carp cells, with the ultimate aim of using such expression systems to improve live vaccine potency through enhanced immune stimulation.
In order to achieve inhibition of CyHV-3 WT strains, the CyHV-3 ORF57 gene – which is partially absent in an existing attenuated vaccine strain (resulting in its attenuation), was chosen as a target for siRNAs processed from vaccine expressed shRNA precursors. Typically, U6 promoters such as the human U6 promoter (HU6) are used for this. Thus, we proposed to establish if this promoter could be used in carp cells, in addition to using the HU6 promoter to express shRNAs that act as anti-WT siRNA precursors, it could also be used to express separate structural variants (blunt-ended-shRNAs) designed to function as RIG-I agonists to provide stimulation of the immune system.

Thus, this project was successful in conducting the first initial characterization the HU6 promoter expression system in carp cells, confirming that it can drive the expression of both protein coding genes (Figure 1A and 1B) and non-coding genes (Figure 1C and 1D) but in both cases activity was lower than in human cells. While these expression levels may possibly be insufficient for RNAi, we reasoned that it may still be sufficient to achieve RIG-I mediated stimulation of the innate immune system.

To offset the potential risk that RNA expression levels from the HU6 promoter may be too low for RNAi (or even immune stimulation), we also conducted the first study aimed at identifying endogenous carp U6 expression systems which should naturally be more active in carp derived cells. Using existing genomic data and our own transcriptomic approach, we conducted the preliminary screening of a putative endogenous carp U6 promoter and uncovered strong evidence for the existence of a second much more active putative carp U6 promoter, which we are currently investigating further.

In parallel, in anticipation of the identification of a U6 promoter system that was sufficiently active to drive shRNA expression in carp cells, we proceeded with the development of methodology to routinely establish co-infected cell monolayers that would exhibit significant number of co-infected cells, which could be used to test inhibition of WT virus via vaccine-derived RNAi mechanism. Despite repeated attempts however, we were unable to observe any substantial amounts of co-infected cells in co-infected monolayers. Further investigations revealed that this was due to the fact that CyHV-3 exhibits rapid superinfection inhibition, thus blocking the main mechanism by which co-infected cells arise.

Despite this, our virus-host model could still be utilized to study the enhancement of live attenuated vaccine potency via expression of RNA-based agonists of the innate immune system. Prior to design of HU6 expression constructs we tested the immuno-stimulatory capacity of several dsRNA molecules in carp cells, revealing that heavily structured 5ppp-non-blunt-ended dsRNA (5ppp-NBE-dsRNA) molecules performed best (Figure 1E). We are currently moving towards testing expression constructs for these molecules (and our own custom molecules) in carp cells, with the aim of inserting functional IFN-I-stimulating expression constructs into the existing live attenuated CyHV-3 vaccine strain.
CyHV-3 is an economically important pathogen of carp, which is one of the most widely cultured freshwater fish species for human consumption making it important in the context of global food security. However, in many regions the long-term sustainability of carp aquaculture has become threatened by the emergence of this devastating virus which can cause up 100% mortality during typical outbreaks. Given the degree of superinfection inhibition exhibited by CyHV-3, recombination between different strains in circulation is extremely difficult, hence such events should be very infrequent. As an extension of this, the same should be true in terms of recombination between the CyHV-3 field strains and live attenuated CyHV-3 vaccine strains during vaccination. This observation further reinforces the safety profile of the current CyHV-3 live attenuated vaccine, emphasizing the low risk associated with its use. As such, these observations may be beneficial in gaining regulatory approval for the existing or future "enhanced-potency" live attenuated CyHV-3 vaccines, which will play a huge role in the control of this virus and helping to ensure the sustainability of carp aquaculture in many communities.
Figure 1