Conditional knockout tools: Application of site-specific incorporation of unnatural amino acid via genetic code expansion in viral and parasite vaccine development.

Recently, Si et al. reported their attempts on generating live, while replication-incompetent influenza virus vaccine [1] as an alternative to inactivated and attenuated virus vaccine. The goal is to make use of the full infectivity of live whole-viron vaccine to elicit protective immunity while remaining as avirulent as possible. In this report, the authors explored the application of the site-specific incorporation of unnatural amino acid approach [2], [3] as a conditional knockout tool. In this method, a pair of tRNA-tRNA synthetase using one of the stop codons to encode for an unnatural amino acid of interest was used. This tRNA-tRNA synthetase pair is orthogonal to all of the other tRNA-tRNA synthetase pairs in the host cell to ensure the site-specific incorporation of the unnatural amino acid of interest (Fig. 1). One of the concerns is that the pre-matural stop codon used for encoding the unnatural amino acid might also be the natural stop codon for some of the host genes. As a result, introducing such tRNA-tRNA synthetase pair and the supplementation of the corresponding unnatural amino acid might impair the functions of the host cell itself. To select a proper cell line for viral packaging, the author integrated the Methanosarcinabarkeri MS pyrolysyl tRNA synthetase/tRNACUA pair, and an amber suppressor codon containing green fluorescent protein gene into the Human embryonic kidney (HEK) 293T cell genome. After 200 generations of passage by selecting the unnatural amino acid dependent GFP production, the authors obtained a cell line that can mediate influenza A/WSN/33 (H1N1: WSN) viral packaging in an efficiency as good as in the parental cells.

Fig. 1

Making use of site-specific unnatural amino acid incorporation as a conditional knockout tool. In this approach, a pre-mature stop-codon is incorporated into the gene of interest. At the same time, a pair of tRNA-tRNA synthetase orthogonal to all tRNA-tRNA synthetase pairs in the host cell was developed for a particular unnatural amino acid of interest. In the presence of the unnatural amino acid, the translation will go through the pre-mature stop codon (A → B → C) in the target gene to produce the protein of interest. In the absence of the unnatural amino acid, translation will terminate pre-maturally and lead to the production of a truncated protein. This can serve as a reversible conditional knockout tool.

The authors then introduced the premature termination codon (PTC) to various positions of the influenza viral genes, including NP, PB1, HA, NA, NS, PA, PB2, M1, and M2. The majority of these PTC viruses show cytopathic effect in the presence of 1 mM of unnatural amino acid in the Methanosarcinabarkeri MS pyrolysyl tRNA synthetase/tRNACUA pair containing transgenic cell line, not the conventional cell line. In some of the positions, PTC substitution has little effect on virus packaging efficiency and replication kinetics relative to the wild type virus. There is indeed a low level of escape frequency (7 × 10−10 to 5.9 × 10−7), which might be attributed to reversion of the amber codon to the sense codon during PTC virus replication and propagation. Incorporating multiple PTC into the viral genome can indeed decrease the escape frequency to an undetectable level (<10−11), while at the same time, compromise the viral packaging and propagation efficiency. The authors then further evaluated the PTC influenza viral vaccine in three different animal models, including BALB/c mice, ferrets, and guinea pigs. Results from these studies indicated that PTC vaccine is safer than cold-adapted live attenuated influenza vaccine (CAIV). In addition, after the second vaccination, PTC vaccine elicits robust humoral, mucosal, and cell-mediated immunity. The PTC hemagglutination inhibition and neutralization antibody titers are at levels comparable to that of CAIV.

The efforts outlined in this work are reminiscent of many of the challenges faced in malaria vaccine development in the last few decades: antigenic variation, efficacy, safety, and being cost-effective [4]. In this study, the PTC influenza viral vaccine demonstrated better safety features relative to CAIV, while PTC vaccine might be more expensive to produce due to attenuated replication efficacy when multiple stop-codons are introduced into the viral genes. In this report, the transgenic host cell (HEK cell) was selected after 200 generations of passage by selecting GFP production in the presence of unnatural amino acid. Due to the fact that many of the host cell genes also make use of the same stop codon, the long-term stability of the cell line and PTC vaccine production reproducibility remain issues to be evaluated.

The above PTC influenza vaccine may be applicable to malaria vaccine development. Five species of Plasmodium parasites can cause malaria infections in human. P. falciparum and P. vivax are the two associated with most human infection incidences. In the last two centuries, obtaining an effective malaria vaccine [4] has been the goal for many scientists because malaria infects a few hundred million people every year and most of the malaria-associated deaths are in children. The World Health Organization (WHO) strategic criteria for malaria vaccines are: 1) at least 50% protection against severe malaria lasting for a minimum of a year; 2) In the long-term, provide >80% protection for 4 years against P. falciparum and P. vivax infection. Thus far, both sub-unit vaccine and whole cell vaccines have been examined and a cost-effective and efficacious malaria vaccine reaching the WHO criteria remains to be developed. For sub-unit malaria vaccine, the most advanced one is the RTS,S, a vaccine that is based on the C-terminal fragment of the circumsporozoite protein (CSP), which contains both B and C-cell epitopes. CSP is the main sporozoite surface protein. RTS,S vaccine is the most advanced one in malaria vaccine development. However, in a large clinical trial in sub-Saharan African children, the protection conferred by RTS,S/AS01 was not satisfactory and the protection efficacy declined rapidly, particularly in infants [5].

Thus far, for malaria vaccine trials, whole cell attenuated vaccines have shown much better protection efficacy [4]. Three different whole cell malaria vaccines have been examined: irradiation attenuated malaria parasites, parasite infection attenuated by drug coverage, and genetically attenuated parasites. For irradiation attenuated malaria vaccine, volunteers are bitten by either ©-gamma-irradiated Plasmodium parasites infected mosquitoes or by direct injection of cryopreserved irradiated sporozoites (PfSPZ) through i.d., s.c. or i. v. delivery. The PfSPZ vaccine is the most advanced one in this category. However, large doses of cryopreserved PfSPZ sporozoites are needed, which makes this type of vaccine very expensive and laborious to produce and prevents its wide application for malaria eradication. The second approach is the vaccination using wild-type malaria sporozoites under the coverage of chloroquine, which is an anti-malaria drug targeting the late blood stage of the parasite. This immunization strategy is also known as Chemoprophylaxis and Sporozoite (CPS) immunization or Immunization-Treatment-Immunization (ITV). Human clinical trials demonstrated that this vaccination approach is effective against subsequent challenges with sporozoites. Depending on the chloroquine treatment protocol, it may also induce cross-stage immunity against the blood stage parasites. However, the wide-spread chloroquine drug resistance in malaria endemic regions is a real concern. One of the most important discoveries is that a whole cell vaccination that allows the parasite to go through the liver stage, while with blocked blood stage, elicits the most effective protection. The third approach is genetic attenuation by gene deletions. Some of the initial attempts focus on the deletion of early liver stage development genes (e.g., UIS3, UIS4, p36, p52 and sap1 etc). In these studies, because it is arrestment at the early liver stage, the immune system is exposed to a limited range of antigens; the protective effect will be less effective than a later stage arresting knockouts.

In summary, for all current malaria vaccine development efforts, whole-cell based vaccine demonstrates much better protection efficacy. The application of site-specific unnatural amino acid incorporation approach [2], [3] might be very useful in malaria parasite gene function characterization and the development of safe, efficacious, and cost effective malaria vaccines. It may have many advantages relative to those for viral vaccines. First, because the blood-stage malaria parasite host is red-blood cell, which does not have DNA, the introduction of orthogonal tRNA-tRNA synthetase pair into the parasite will not have an effect on the host cells. Second, we can introduce stop-codons into various malaria genes, which will allow us to stop the parasite at a specific stage, including UIS3, UIS4, p36, and P52 for early liver stage arresting and the chloroquine target gene for late-erythrocyte stage arrestment when the unnatural amino acid is withdrawn from the culture medium of the animal food supply. Third, besides these known genes that have been explored in literature, we can intentionally introduce toxic genes (e.g., barnase gene or other toxic genes) into the parasite genome under the control of a malaria-parasite development stage specific promoter. Under such a design, supplementing with the unnatural amino acid will induce the parasite death at a very specific stage and expose the antigens to the immune system to elicit immune responses. Fourth, we can incorporate amber suppressor into the CAS9 to create conditional knockout system for CRISPR-Cas9 system [6], which will allow us to have controlled targeting of malaria essential genes to induce malaria cell death.

All of these approaches are being explored in our laboratories. These efforts will provide an easily accessible conditional knockout tool to study malaria gene functions. At the same time, it may allow us to explore whole cell malaria vaccine which might be cost-effective and efficacious. In the last decade, many chemical biology tools have been developed. For both basic and translational study points of views we have also witnessed the increasing applications of these tools in synthetic and systematic biology.