Kill 2 birds with one stone… or many viruses with one egg

Based on: Tseng, Yung-Chieh, et al. "Egg-Based Influenza Split Virus Vaccine with Monoglycosylation Induces Cross-Strain Protection against Influenza Virus Infections." Proceedings of the National Academy of Sciences, vol. 116, no. 10, Mar. 2019, pp.4200-05.DOI.org(Crossref), doi:10.1073/pnas.1819197116

The seasonal flu, or influenza virus, infects ~5% of adults and 20% of children each year (1). This is a virus that mutates relatively quickly and therefore requires a new vaccine to be produced and administered each year. Between flu seasons, scientists will predict how the flu is going to mutate and will create a new vaccine to try and prepare the immune system to combat that variant (2). Unfortunately, this process works better some years than others. Therefore, in this paper, Tseng et al. is looking to create a more general flu vaccine that will protect against the flu virus regardless of how it mutates from year to year. This new vaccine takes the virus and removes its glycosylation sites. Normally, the flu virus can have different glycosylation sites and the vaccines will specifically prime the immune system against specific glycosylation patterns. The vaccine being created is a monoglycosylated flu vaccine made from chicken eggs.

            First, the authors had to show that they could inhibit the glycosylation of the virus to create the monoglycosylated virus. They tested this by using different egg concentrations and different amounts of kifunensine. Then they did SDS/PAGE to analyze the virus and found that the new virus with kifunensine does remove the glycosylation sites without affecting overall protein composition within the virus. They also tested to see if the binding ability of the vaccine was altered after changing the glycosylated virus, and after doing a hemagglutinin (HA) assay it showed that there is no change in the binding ability of the viral HA.

Figure 1. This is figure 2D from the paper and shows that the monoglycosylated virus (X181mg) has the same amount of HA protein as the other virus. This indicates that when the virus is modified, there are no changes to the overall amount of protein present.

            Now that the virus is created, the authors injected mice with either the monoglycosylated vaccine, or other commercial vaccines. After vaccinated the mice, the scientists collected antisera, blood serum which contains the animal’s antibodies, to study. The mice inoculated with the monoglycosylated vaccine had the greatest amount of hemagglutinin inhibition when compared to the other vaccines given. This shows that this vaccine better neutralized the cross-strain influenza viruses. After looking at the antisera after the vaccine was given, the scientists injected a lethal virus dose and the animals were monitored for another 2 weeks to see how effective the vaccines were. The mice vaccinated with the monoglycosylated vaccine showed more than 70% protection against the cross-strained viruses. This indicated this vaccine causes improved antibody response and subsequent protection. Additionally, the mice who received the monoglycosylated vaccine had fewer viral particles in their lungs and had less weight loss after viral infection when compared to the mice who received other viruses.

Figure 2. This figure shows the serum Titer (A, D, G), neutralization (B, E, H), and percent survival (C, F, I) of both the monoglycosylated virus (X-181mg) when compared to other commercial viruses after the mice were injected with one of three viruses. The monoglycosylated virus is the red line and for all three viruses has the highest neutralization percent and survival percent.

            The scientists then wanted to know more about why this new vaccine was so effective. They did an ELISA to look at hemagglutinin levels. It was found that the monoglycosylated virus had more cross-reactive hemagglutinin specific antibodies and had more splenocytes released. Splenocytes are white blood cells that consist of many different cell populations that all propagate an immune response. Therefore, compared to the other vaccines, the monoglycosylated vaccine caused more stem specific antibodies as well as antibody secreting splenocytes. Then, to look at whether this vaccine increased the total number of antibodies they did an ELISA. It showed that the mouse antisera with the monoglycosylated had more antistem antibodies in the total antibody count than the other vaccines.

            The last thing the authors looked at is Fc receptor-mediated immune response. Normally, the main focus when neutralizing a virus is to look at antibody protection. However, recently there has been a greater push to look at the Fc receptor mediated antiviral activities. Fc receptors bind to antibodies that are attached to cells to stimulate phagocytosis. Their last experiment used an antibody dependent cellular cytotoxicity assay to study this response. The assay showed that the monoglycosylated antisera had higher Fc immune activity when compared to the multi glycosylated vaccine. The antibodies produced by the monoglycosylated virus can therefore be shown to provide broader protection for the host.

            Ultimately, this experiment shows that this novel vaccine created from eggs where the virus has the glycosylation sites removed causes improved protection against influenza virus. This vaccine improves the immune response, increase antibodies, and provides a more generalized immune response that can target a variety of flu viruses, not just a single strain. This has the potential to save money because new vaccines would not need to be created annually. Additionally, it would save time for both scientists and the general population to prevent the need for annual vaccinations.

1) Krammer F, Palese P (2015) Advances in the development of influenza virus vaccines. Nat Rev Drug Discov 14:167–182.

2) Dos Santos G, Neumeier E, Bekkat-Berkani R (2016) Influenza: Can we cope better with the unpredictable? Hum Vaccin Immunother 12:699–708

Use of Crispr/Cas9 to Investigate CAR Isoform Function in AdV Infection

Paper: Readler, J. M., AlKahlout, A. S., Sharma, P., & Excoffon, K. J. (2019). Isoform specific editing of the coxsackievirus and adenovirus receptor. Virology, 536, 20-26.

Coxsackievirus infects a range of hosts, including humans, and is linked to a number of diseases such as myocarditis and pancreatic inflammation [2]. Of a different viral family, adenovirus also infects humans, inducing mild diseases such as gastroenteritis and upper respiratory tract infection, in addition to more severe conditions when paired with host immunosuppression. Coxsackievirus is made up of single-stranded RNA, while adenovirus is comprised of double-stranded DNA. Despite having different genomic structures, both coxsackievirus and adenovirus utilize a cell-adhesion protein encoded by the CXADR gene as their primary receptor [2]. This transmembrane receptor exists in two isoforms that are dependent on alternative splicing, a gene expression regulatory process that differentially includes or excludes particular exons of a transcript. The two isoforms, Car^Ex7 and Car^Ex8, have distinct C-terminal ends that lead to different localization in polarized epithelial cells. These polarized cells, as depicted in the image below, are characterized by apical and basal faces that are distinct from one another in structure and function at sites of cell-cell contact [4]. The Car^Ex7  isoform encodes the first seven exons of CXADR and is localized at the basolateral cell surface, while Car^Ex8 includes an additional eighth exon and is localized at the apical cell surface.

Epithelial Cell Polarity: Apical and basolateral surfaces are associated with distinct structures and locations within the cell.
Image source: Figure 4-1. "The Polarity of Epithelial Cells." Adapted from Pearson Education Inc., 2015, p. 116.

            In this study, Readler et al. aimed to better illuminate the functional differences between these two isoforms of the coxsackievirus and adenovirus receptor (CAR). Specifically, researchers chose to focus on the importance of the Car^Ex8 isoform to adenovirus entry at apical cell surfaces. Two key components of this investigation were the Madin Darby Canine Kidney (MDCK) cell line and Crispr/Cas9 gene editing technology. Readler et al. previously created the MDCK cells to stably express human Car^Ex8 cDNA with a promoter that is inducible with the antibiotic doxycycline. With this model cell line established to manipulate the induction of Car^Ex8 expression, researchers then utilized Crispr/Cas9 to knockdown endogenous expression of Car^Ex8 in the MDCK cells. This novel knockdown cell-line thereby isolated the functionality of the Car^Ex8 receptor to a level never achieved before.

           

            To ensure that they successfully targeted the eighth exon of the MDCK CXADR gene, researchers first performed a screen using fluorescence-activated cell sorting and PCR. From this screen, one clone was identified to be a successful knockout, as PCR primers set to recognize the inside of the eighth exon sequence produced no bands. Furthermore, sequencing this clone, renamed JR1-Car^Ex8-KO, verified that the intended deletions were indeed present when compared to MDCK-Car^Ex8 parental cells. After successfully completing this exonic knockout, researchers then investigated total CAR and Car^Ex8  expression levels within the JR1-Car^Ex8-KO cells via western blot analysis. As shown in Figure 3 below, these cells exhibited significantly less expression of Car^Ex8, while showcasing partially reduced levels of total CAR. In both cases, treatment with the antibiotic (Dox), which induced expression of human Car^Ex8 in this cell line, restored expression levels to that of the parental cells. This demonstrated the role of the Car^Ex8 knockdown on the decrease of CAR expression levels, as well as that the insert of human Car^Ex8 was not impacted by the endogenous MDCK Car^Ex8 deletion.

 

Figure 3. CAR expression in JR1-Car^Ex8-KO cells as compared to parental cells. Western blots were performed using polyclonal antibodies detecting A) total CAR expression and B) Car^Ex8 expression alone. Total CAR expression partially decreased while Car^Ex8 reduced significantly in knockout cells (KO) as compared to parental cells (Par). With treatment of 200 ng/mL doxycycline (Dox), KO showed expression restored to the level of Par.

Another important finding of this research was that the less pronounced impact of the knockdown on total CAR expression is likely due to the endogenous levels of Car^Ex7 still maintained in the cell line. As the specificity of CRISPR editing targeted the intronic regions on either side of the eighth exon alone, the seventh exon of CXADR remained intact to generate the majority of CAR expression levels still seen. Another component of this figure worth highlighting is the slight expression of Car^Ex8, despite a seemingly complete knockdown, that was detected via western blot. As compared to the PCR screen (shown below) that showed no amplification of exon 8, the presence of this protein expression in Figure 3B above raises questions as to the true completeness of this knockout. In addressing this discrepancy, Readler et al. offer the possibility that one allele remains, although with primer recognition sites altered, as an explanation for the lack of this Car^Ex8 presence in their PCR analysis. Additionally, they consider non-specific binding within the western blot as a possible reason the fully complete knockdown of Car^Ex8 was not demonstrated within the protein expression levels seen.

 

Figure 1C. PCR knockout screening using primers inside the exon 8 cut site. JR1-Car^Ex8-KO cells (KO) exhibited no band as compared to untreated parental cells (Par) and two haploid knockouts (Hap 1 and 2).

            Despite this inconsistency in their results, Readler et al. determined their knockdown of Car^Ex8 significant enough to investigate the receptor isoform’s importance to adenovirus infection. Using a virus reporter gene, β-galactosidase, researchers quantified the levels of viral-induced luminescence per milligram of protein for MDCK-Car^Ex8 parental and knockout cells. In knockout cells, viral transduction was subjected to a two-fold decrease, suggesting the importance of Car^Ex8 to adenovirus entry. This is supported by Car^Ex8’s known tendency to localize the CAR receptor at apical cell surfaces that are exposed to cellular lumen   where adenovirus is able infect host cells [3]. Further, to look at this apical entry alone, researchers targeted infection to the apical surface of fully polarized MDCK cells and measured genomic levels of adenovirus via qPCR. As shown in Figure 4C below, at 24 hours post-infection a three-fold reduction in adenovirus genome was seen in Car^Ex8-knockouts as compared to parental cells. This difference was restored with the reestablishment of Car^Ex8 induced through Dox antibiotic treatment. This greater fold-change in adenovirus infection when targeted to the apical cell surface supports the notion that Car^Ex8 is essential for the mediation of viral entry at this polarized cell surface, a function that was less pronounced when Car^Ex7 was still allowing adenovirus entry at the basolateral cell surface in these knockouts.

Figure 4C. Cell associated adenovirus genomes in MDCK-Car^Ex8 parental and JR1-Car^Ex8-KO cells. Detected via qPCR, KO cells exhibited a significant reduction in cell-associated viral genomes, as compared to parental and KO cells treated with 200 ng/mL doxycycline.

            Ultimately, this novel Car^Ex8 knockout cell line revealed the importance of this receptor isoform to adenovirus entry at the apical surface of polarized cells. With attenuated expression of Car^Ex8, MDCK cells showed reduced levels of adenovirus transduction and genome levels, which were restored with antibiotic-induced Car^Ex8 expression from a gene insert. The methods utilized here offer a model system for future studies to elucidate further functionalities of the Car^Ex8 receptor using this knockout cell line. Additionally, this methodology could be applied to other genes, such as CD46, which has 14 different receptor isoforms [1]. The use of Crispr/Cas9 editing to create exonic knockouts of this receptor could showcase the interaction between specific CD46 isoforms and the entry of measles or herpesvirus strains [1]. In conclusion, the most notable result of this research is the clinical relevance this method holds in narrowing down receptor isoform correlations to viral entry, as regulation of particular exonic expression could work to inhibit certain strains of viral infection.

References

[1] Excoffon, K. J., Bowers, J. R., & Sharma, P. (2014). 1. Alternative splicing of viral receptors: A review of the diverse morphologies and physiologies of adenoviral receptors. Recent research developments in virology, 9, 1.

[2] He, Y., Chipman, P. R., Howitt, J., Bator, C. M., Whitt, M. A., Baker, T. S., ... & Rossmann, M. G. (2001). Interaction of coxsackievirus B3 with the full length coxsackievirus-adenovirus receptor. Nature Structural & Molecular Biology, 8(10), 874.

[3] Kotha, P. L., Sharma, P., Kolawole, A. O., Yan, R., Alghamri, M. S., Brockman, T. L., ... & Excoffon, K.J. (2015). Adenovirus entry from the apical surface of polarized epithelia is facilitated by the host innate immune response. PLoS pathogens, 11(3), e1004696.

[4] Rappel, W. J., & Edelstein-Keshet, L. (2017). Mechanisms of cell polarization. Current opinion in systems biology, 3, 43-53.

[5] Readler, J. M., AlKahlout, A. S., Sharma, P., & Excoffon, K. J. (2019). Isoform specific editing of the coxsackievirus and adenovirus receptor. Virology, 536, 20-26.

Cardiotonic Steroids May Provide Avenue for the Treatment of Human Adenovirus

In response to: Saha, B., Varette, O., Stanford, W. L., Diallo, J. S., &Parks, R. J. (2019). Development of a novel screening platform for the identification of small molecule inhibitors of human adenovirus. Virology.

           

            Human adenovirus (HAdV) includes a broad group of DNA viruses that can cause infected patients to develop symptoms ranging from respiratory infections or conjunctivitis¹. Over eighty strains of this extremely prevalent nuclear virus have been identified². While this virus is quite prevalent, current anti-viral medications prescribed to people infected with the disease have only limited effectiveness against the disease. Improved anti-viral drug treatments for HAdV are needed because this virus can be fatal in immunocompromised hosts such as patients with AIDS, the elderly or infants³.

           One avenue for developing an effective treatment for human adenovirus involves the identification of small molecules that may disrupt the organization pathways that HAdV uses to infect a cell. When infecting a human host, HAdV must rely on proteins and molecular pathways native to the host cell to generate more viruses that are capable of infecting more host cells. The HAdV hijacks host cell mechanism to multiply and infect other cells in the human host. Small molecules such as selective estrogen receptor modulators have previously been shown to successfully disrupt the propagation of viruses like Ebola by interfering with the viruses’ ability to manipulate host cell proteins and pathways⁴. Until recently, however, very few studies have investigated the effectiveness of small molecules in disrupting the progression of HAdV, and those studies that have investigated the impact of small molecules on the disruption of HAdV have failed to perform a large enough exploration of small molecules⁵. Thus, the Parks laboratory at the Ottawa Hospital designed a new experimental method for visualizing the interaction between HAdV and small molecules that has broad application for testing purposes.

           Researchers at the Parks laboratory ultimately developed a fluoresce tagging protocol that fused a red fluorescent protein (RFP) with the HAdV’s major late promoter (MLP). The MLP is active during the end of HAdV replication process and, consequently, is expressed in greater quantities when the virus is about to spread to other cells. Greater expression of MLP in a cell indicates that the virus is forming more viral particles as the MLP is involved in the construction of structural proteins used for the new viruses to exit the host cell. By fusing a RFP with the MLP, the researchers could determine whether or not specific small molecules were able to disrupt HAdV by visualizing a reduction in MLP activation. HAdV’s that are unable to express MLP would be unable to reproduce effectively and thus the small molecule will have affectively disrupt the virus. 

           After manipulating the HAdV’s genome to include RFP, the virus was incubated with a human lung-derived cell line that was tagged with a green fluorescent protein and infection was allowed to occur. The two different fluorescent colors enabled the researchers to optimize their study and identification technique. The relative amount of green fluorescence permitted the researches to assess whether or not the cells utilized in the experiment were damaged. If the cells were damaged, a decrease in red fluoresce could be the result of a decrease in viable cells instead of a suppression of HAdV replication. Thus, the multiple fluorescent tags improved the validity of the Parks’ study. Researchers quantified the level of RFP in the virally infected cell line after twenty-four hours to determine the base level of RFP in HAdV infected human lung cell lines. 

Figure 1: Imagining of Human Lung Cells Infected with RFP Tagged HAdV. Live-cell images of human lung cells following 18, 21, or 24 respective hours following infection with RFP tagged HAdV. Images display the effectiveness of the tag to increase fluorescence following longer incubation periods in cells and thus higher presence of the virus. 

           After testing the validity of their viral construct through imaging seen in Figure 1, the researchers then used their newly developed fluorescent method to examine the effectiveness of 1200 FDA approved small molecules to inhibit the spread of HAdV. From their screen assays, the Parks laboratory determined that digoxin, digitoxigenin, and lanatoside C were the drugs that yielded the greatest reduction in RFP. All three drugs are cardiotonic steroids that inhibit the activity of Na+/K+ ATPases which are critical for the maintenance of cell membrane potential⁶. Through plaque assays that measure virally induced cell death, the experimenters also showed that exposure to dioxin, digitoxigenin, and lanatoside C lead to a significant reduction in HAdV pathogenicity after twenty-four hours of exposure. Cardiotonic steroids were previously shown in another study to effectively repress the infectious capabilities of HAdV⁷. The combination of the experimenters’ RFP and viral plaque assay findings and the previous work of Grosso et al. provides substantial support for the experimenters’ conclusion that cardiotonic steroids have the potential to be used as effective anti-viral drugs against HAdV. 

Figure 2: Impact of Cardiotonic Steroids on Expression of MLP in HAdV Infected Cells. As the concentration of the cardiotonic steroids (dioxin, digitoxigenin, and lanatoside C) increases in the cell culture there is a decrease in the intensity of RFP in the culture. The decrease in RFP is far greater in the cardiotonic steroid molecules than in the control small molecule (SAHA).

            Ultimately, the Parks’ study is important to the field of virology and the general public for two key reasons. First, the research group was able to construct and execute a new fluorescent technique that could be used to determine the impact of small molecules like cardiotonic steroids on the infectiousness of HAdV. The development of valid visualization methods to monitor HAdV is critically important for the development of effective anti-virals. The Parks’ fluorescence method could potentially be used to monitor the impact of other anti-HAdV treatments beyond small molecules to inhibit the production of late viral proteins in infect cells and thus inhibit the spread of the disease. Additionally, the research group was able to add to the body of literature which has suggested the cardiotonic steroid molecules could be used as an effective anti-viral therapy against HAdV. More research on the impact of cardiotonic steroid molecules on cell cultures infected with HAdV should be performed to determine whether or not these molecules could be used as an effective treatment for people infected with HAdV. Developing an effective anti-viral to HAdV could significantly improve the survival chances and quality of life of immunocompromised people with HAdV. 

1 Lion T. (2014). Adenovirus infections in immunocompetent and immunocompromised patients. Clinical microbiology reviews, 27(3), 441–462. doi:10.1128/CMR.00116-13

2 Binder, A. M., Biggs, H. M., Haynes, A. K., Chommanard, C., Lu, X., Erdman, D. D., … Gerber, S. I. (2017). Human Adenovirus Surveillance - United States, 2003-2016. MMWR. Morbidity and mortality weekly report, 66(39), 1039–1042. doi:10.15585/mmwr.mm6639a2

3 Bhatti, Z., & Dhamoon, A. (2017). Fatal adenovirus infection in an immunocompetent host. The American journal of emergency medicine, 35(7), 1034-e1.

4 Johansen, L. M., Brannan, J. M., Delos, S. E., Shoemaker, C. J., Stossel, A., Lear, C., ... & Lehár, J. (2013). FDA-approved selective estrogen receptor modulators inhibit Ebola virus infection. Science translational medicine, 5(190), 190ra79-190ra79.

5 Duffy, M. R., Parker, A. L., Kalkman, E. R., White, K., Kovalskyy, D., Kelly, S. M., & Baker, A. H. (2013). Identification of novel small molecule inhibitors of adenovirus gene transfer using a high throughput screening approach. Journal of controlled release, 170(1), 132-140.

6 Prassas, I., & Diamandis, E. P. (2008). Novel therapeutic applications of cardiac glycosides. Nature reviews Drug discovery, 7(11), 926.

7 Grosso, F., Stoilov, P., Lingwood, C., Brown, M., & Cochrane, A. (2017). Suppression of adenovirus replication by cardiotonic steroids. Journal of virology, 91(3), e01623-16.