The impact of IFTIM3 genetic variation on Influenza virus infection, immune responses and disease outcome

Influenza is a global threat. The current avian influenza H7N9 outbreak in China has a high death rate and can transmit from human-to-human. In this study, we will explore how human genetic factors and immune responses influence the severity of the disease. We recently found that people with a particular genetic variant of a gene called IFITM3 are six times more likely to suffer from severe influenza infection than those without (Zhang et al, Nature Communications 2013). This variant gene is very rare in Northern European populations but is common in Chinese people. We found that in 69% of Chinese patients with severe pandemic influenza had only this variant form of IFITM3. This compares to 25% of healthy Chinese individuals.

The IFITM3 protein inhibits the entry of influenza virus into cells; the variant is thought to be unable to do this. One key aim of our project will be to check whether this is true and if so why it lacks this function. If more viruses enter cells the disease may be more severe, but severe influenza is rare in China even though 25% of the population have only this variant of IFITM3. Therefore we suggest that there may be compensating genetic factors snd/or that strong immune responses in people with the variant of IFITM3 protect most of them from severe influenza.

In this study therefore, we will look for other protective genes in Chinese people who have the susceptible IFITM3 gene but who do not get severe influenza. We will also examine their immune responses to the virus and compare those with immune responses to influenza virus in Chinese and UK people who do not carry this gene variant. We will focus particularly on innate immune responses, which are known to be highly active in severe influenza and may actually contribute to the problem. We will also study T cell immune responses, which are specific for the infecting virus and are normally important for rapid viral clearance. Our preliminary data suggest that these T cell responses are more active in Chinese people with the IFITM3 gene variant, which could help compensate for their increased genetic susceptibility to severe infection.

In this way we will gain a better picture of the risk of severe infection in Chinese people, conferred by this gene variant which is very common in the population. It is possible that increased genetic susceptibility in the Chinese helps the spread of influenza virus in South East Asia. We will examine this in the context of the new avian influenza H7N9 threat. We will ask whether this more aggressive virus can override the protection that the common (in Europeans) variant of IFITM3 offers and whether this version of the gene contributes to mild asymptomatic infection which we have previously shown to occur in seasonal, pandemic and avian influenza. Our special links to the infectious disease hospitals You'An and Ditan in Beijing make this unique study possible.

This study addresses important unsolved problems of influenza virus infection, taking a global rather than local view, which is clearly necessary to tackle this threat.

1. The effect of the C variant on IFITM3: virus entry and intracellular location.
We will employ optical microscopy including different fluorescent labeling approaches such as protein-specific antibodies or live-cell tagging technologies such as GFP or SNAP- and HALO-tags. Optical super-resolution microscopes (or nanoscopes), including the STED/RESOLFT or PALM/STORM technologies will enable us to explore the subtle changes in the spatial organization of IFITM3 between variants.

2. IFITM3 genotype impact on innate sensing of influenza A virus and virus entry.
Primary fibroblasts will be grown from skin biopsies. Subsequently, these cells will be infected with IAV. The induction of chemokines and cytokines will be measured by ELISA, bioassays or RT Q-PCR. The accumulation of RIG-I stimulatory RNA in cells of different genotypes will be assessed by RNA extraction from infected cells and re-transfection into RIG-I reporter cells. JR has experience with this approach. In these experiments primary human cells will be used and complemented by human cell lines overexpressing or depleted of IFITM3, as well as IFITM3-deficient mouse fibroblasts.

3. Immune responses in IFITM3 CC/TT patients who do badly compared to those who do well.
a. Trascriptome profiles will be determined using Illumina Human HT-12 V3 Beadchip arrays, which contain more than 48,000 probes. A gene expression analysis software program, Genespring, version 7.1.3 (Agilent), will be used to perform statistical analysis, and hierarchical clustering of samples. A modular analysis strategy will be used as described, enabling the discrimination of clinical groups without reducing the dimensionality of the data.
b. Detailed analysis of influenza specific T cell responses will be performed using overlapping peptides in the ELISPOT assay. In some cases, T cell line and clones will be grown and antiviral efficacy will be evaluated using target cells infected with different viral strains

The likely beneficiaries of this research project will be patients, hospitals, the Health Protection Agency (UK) and the China Centre for Disease Control (China CDC). Vaccine manufacturers will also benefit. Most pandemics and epidemics have originated in South East Asia where farming practices, bird migration patterns and possibly local genetics (the subject of this proposal) may contribute. As influenza, once spreading in humans, rapidly reaches all parts of the world, these factors will impact on the UK. The findings of the proposed study will be important to China and UK residents of Chinese descent if they show that the human genetic factor IFITM3 influences the immune responses to the epidemic, pandemic and novel avian viruses, and consequently the severity of the disease.

Our primary hypothesis is that the risk of severe disease in people with IFITM3 CC genotype can be offset by other genetic factors and/or by improved immune responses. Despite of this, there might be higher incidences of severe influenza in Chinese patients, possibly up to six-fold increase. Furthermore, we hypothesise that subclinical infection will more common in people with the IFITM3 TT genotype, consequently subclinical cases will occur more frequently in the UK than in China. However the influence of the IFTIM3 gene might be different for a virulent avian virus such as the H7N9 virus and this will be determined in this study. Thus we will better understand the factors contributing to disease severity. IFITM3 genotype screening could be utilized for prognosis and guiding early intensification of treatments. Moreover, If the CC genotype carriers possess stronger immune responses, as we propose, they could be prioritised for vaccination. These studies could provide us with crucial information to improve the current vaccine strategies and therapy in different target populations such as in UK and China. Hence, our proposed research could prominently improve quality of life by controlling and preventing life-threatening influenza illness.

The study may also have relevance to vaccination planning. Currently live attenuated vaccines are being given to children. As a live virus vaccine, the IFITM3 genotype could influence the take rate of this vaccine and possibly the incidence of side effects. This is not being tested in this study but once we know more about the role of IFITM3 in influenza virus infection, this could be an important follow on study that would have an important impact on medical practice.

Lastly this study will be a strong UK - China collaboration with significant scientific contributions from both sides. The lead investigator is a Chinese but resident in the UK. She has an honorary joint appointment at the You'an Hospital, Capital University, Beijing and holds grants from Nature Science Foundation China for major international collaboration and is collaborator/partners on many others such as Chinese Ministry of Science and Technology funding for Major International collaboration. The project is covered by a Memorandum of Understanding between the MRC and Beijing Bureu of Science and Tachnology(NJSTB), signed in 2007 by sir Andrew McMichal on behalf of MRC and Director of NJSTB; and between Beijing You'an hospital(BJYA) and MRC Human Immunology Unit(HIU), in 2012 signed by Director HIU Vincenzo Cerundolo and the Director Li of BJYA. Given the importance of influenza, the threat of new pandemics often arising in South East Asia, these very strong links will give the MRC a very strong role in working with China to combat current and future influenza threats. This partnership could be extended to other infectious disease threats, such as a recurrence of SARS, should they arise.