Just hanging about

Presumably the question you are asking yourself is what determines persistence in acute RNA viruses? If not, why not?

Viruses have been shown to persist – stay present in the body, potentially after the symptoms of infection have passed. Most of the evidence and mechanism for viral persistence has been collected for DNA viruses and retroviruses (that is RNA viruses that convert their RNA genome into DNA and insert it into the host). However, there is clear evidence that non-retroviral RNA viruses can persist (see the review here). We normally think of these acute RNA viral infections as being short lived, cleared by the host and only succeeding if they can transmit to a new individual. However, this strategy has limitations, particularly if there are no new individuals who haven’t been infected by the virus. Therefore, viruses need to have evolved a way to maintain a reservoir, this is particularly important when we consider that viruses are obligate parasites – they have to use host cells to replicate and survive. It is particularly interesting to think that the viruses can persist in spite of selective pressure from the host immune response which is trying to clear the virus.

The question we set out to answer in a recently published study, led by Prof Rick Randall and Prof Steve Goodbourn, was how RNA viruses can switch between an acute and persistent state. The work focused on parainfluenza virus (PIV), which is a member of the paramyxovirus family. Viruses require specific proteins to make copies of their genetic information, which is described as the polymerase complex. The imaginatively named P protein of parainfluenza virus is a core part of the viral polymerase. If the P protein was phosphorylated (a mechanism by which cells can control protein activity), then the virus no longer replicated in the cells, but and this is important, the viral RNA was maintained within the cell. We then demonstrated that the phosphorylation status of the P protein and was determined by a single amino acid within the protein, if this changed then the protein could be activated or de-activated. Since amino acids are determined by the genetic code of the virus, specifically by 3 nucleotides, a single nucleotide change can alter the amino acid sequence, in turn affecting the phosphorylation of the protein and whether it is active or not. So in essence there is a switch that can control whether the virus makes copies of itself within the cell, given that RNA viruses have leaky polymerases (they make inaccurate copies of their own genes), this flip between active and inactive states can occur readily during the infection/ replication cycle. The switch may be driven by immune pressures, we demonstrated that lytic viral variants replicated to higher levels in a mouse model but were cleared much faster, whereas the persistent variant led to a prolonged infection. We proposed that the virus may start in an active state producing lots of copies of itself, before switching to a persistent state to develop a reservoir.

This was in essence a piece of basic research addressing a fundamental question in virology, but it does have broader impact, understanding why and how RNA viruses persist has implications for infection epidemiology as well as potential for developing novel vaccine platforms.

Breathe it in

Influenza is a serious cause of death and disease, contributing to the winter healthcare burden. One approach to reduce this is vaccination. In addition to the injectable influenza vaccine, which is given as an intramuscular injection there is an intranasal vaccine. This vaccine is also referred to by its initials LAIV – live attenuated influenza vaccine. The vaccine is a live vaccine, that has been adapted to reduce its pathogenicity. Specifically, the vaccine virus was adapted so that it can only replicate at lower temperatures. This is important because there is a temperature differential across the airways: the nose, because it is drawing in cold air is cooler than the lungs. The nose is at approximately 30°C, compared to the lungs which are at 37°C. This means that viruses that can replicate at 30°C are restricted to the upper airways and therefore cannot cause severe disease. The vaccine virus is then administered by a nasal spray syringe, once it gets into the nose, it replicates and this replication is important in the induction of an immune response.

However, one of the problems with influenza is that the virus changes season on season, sometimes in small steps (antigen drift) but sometimes in much bigger jumps (antigen shift). This changing of the viral strain necessitates new vaccines each influenza season. Most of the variation comes through the surface antigens, haemagglutinin and neuraminidase, which are the H and N of influenza virus nomenclature. Luckily the same temperature sensitive attenuated vaccine virus strain can be used as a backbone into which different H and N genes can be substituted. However, to achieve greater coverage three or four (depending on manufacturer) different virus strains in the vaccine, normally two A strains (H1N1 and H3N2) and two B strains.

In the UK, LAIV has been recommend for all primary school age children (up to 11), and some other high risk groups. This decision is based in part on the herd protection that this vaccine could potentially have, protecting the elder generation by reducing the infectious reservoir. However, in recent years there have been some concerns with the efficacy of the LAIV – particularly in the USA where efficacy dropped from 85% before 2009 to 17% in 2013-14 which led to a reversal of the American  Advisory Committee on Immunization Practices to recommend suspension of LAIV between 2016 and 2018. We wanted to understand factors that affected the immunogenicity of this vaccine.

In a previous study, we had described how nasal antibodies, specifically of the IgA type were associated with reduced viral shedding after influenza infection (https://www.frontiersin.org/articles/10.3389/fmicb.2017.00900/full). So  now we wanted to look into the effect of immunisation of children with LAIV on IgA. In our recently published study (https://onlinelibrary.wiley.com/doi/full/10.1111/cei.13395) we saw that three out of the four strains in the vaccine were able to induce a significant increase in IgA. Interestingly the only strain not to induce an increase in IgA – H1N1 – was the one for which concerns have been raised for protective efficacy. Though it was not clear in this study why the H1 strain might behave differently.

In a separate study (https://www.thelancet.com/journals/lanres/article/PIIS2213-2600(19)30086-4/fulltext) in collaboration with Dr Thushan da Silva in Sheffield, we looked into potential reasons for the differences. By happy coincidence, Thushan ran clinical trials with LAIV over 2 seasons and the H1 vaccine strain was changed between the years from A/17/California/2009/38 (Cal09) to A/17/New York/15/5364 (NY15). This gave us an opportunity to understand a bit more about how vaccine strain changes can affect immunogenicity. Strikingly the change in vaccine led to a significant increase in vaccine response and this was linked to how good the vaccine was at replicating – the newer strain (NY15) replicated better both in vivo and in vitro and this was associated with stronger immune responses.

Based on these studies, we want to look at how vaccine replication is associated with immunogenicity and what viral factors enable enough replication to work as a vaccine without causing infection themselves.

Postscript
Interestingly the uptake rate for the free LAIV vaccination in schools in the UK is only 30% (as at 16/12/19). This vaccine has been opt-in rather than opt out. One question is if it was made the default and then people had to opt-out would uptake be higher?

Protective protozoa

Flu, caused by the influenza virus is unpleasant. Even in non-pandemic years, it causes 290,000 to 650,000 deaths. In the absence of a ‘universal’ vaccine that could provide protection against all possible variants of the virus, new vaccines need to be selected and manufactured each year. The majority of these vaccines are manufactured using eggs. Influenza virus is grown in chicken embryos inside the eggs which are then cracked open prior to purify and inactivate the virus for vaccine use.

There are a number of limitations to this approach. Firstly, it is complex to scale up, for example during a pandemic. It can also induce a selective pressure upon the vaccine virus – chicken cell and human cells have slightly different receptors for influenza on their cell surface and co-factors within the cell. This means that in order to replicate efficiently in egg cells the virus may undergo some slight changes. If these changes are in regions of viral proteins recognised by the immune system,  for example haemagglutinin, then the vaccine virus might induce a memory immune response which does not recognise the virus that is actually circulating in the wild. The final problem is that some viruses, in particular the highly pathogenic ones (H5N1 and H7N9) are deadly to birds and kill the chicken embryos before enough virus is made for the vaccine.

Therefore alternative manufacturing approaches are required. One tool that has been widely applied across all fields of biological drug manufacture is the use of recombinant cell culture – where genes from one organism are expressed in cells of another. There is a licensed influenza vaccine (Flucelvax) which is manufactured using the MDCK cell line. These cells were originally isolated in the 1950s from a dog kidney, specifically a cocker spaniel, by S.H. Madin and N.B. Darby – hence Madin-Darby Canine Kidney (MDCK) cells. Growing cells from mammals has advantages compared to embryonated chickens, but there is value in developing alternative methods.

We investigated an alternative manufacturing approach in our recently published study Recombinant Haemagglutinin Derived From the Ciliated Protozoan Tetrahymena thermophila Is Protective Against Influenza Infection in Frontiers in Immunology. Working with a biotech company based in Germany (Cilian, AG) who use a protozon ciliate called Tetrahymena thermophila for the manufacture of biologics. This system has a number of potential advantages, it uses conventional manufacturing equipment, the same as that used for both bacterial and yeast based manufacturing systems.

However, it was possible that viral proteins manufactured using a protozoan might not induce a good vaccine response. We therefore set out to test the immunogenicity of the ciliate derived material. We demonstrated that immunisation with recombinant haemagglutinin could protect against an infection with a matched influenza virus. We saw this with haemagglutinin derived from either influenza A or influenza B viruses.

This proof of principle study therefore opens that path for further development of the Tetrahymena thermophila platform for vaccines. The major next step will be to work the platform up to a good manufacturing practice (GMP) grade material so it can be tested in clinical trials.