Human herpesvirus-6 in multiple sclerosis : assay development, immune responses and host genetics

MS is a chronic inflammatory demyelinating disease of the CNS that implies impaired motor- and cognitive functions. Life expectancy is not severely affected but the disease has substantial negative effects on the quality of life of patients and their relatives. The etiology still remains unknown, but both genetic as well as environmental factors are considered to contribute to disease susceptibility. The importance of environmental factors is supported by findings such as the MS latitude gradient with increasing incidence closer to the poles and from findings of twin studies where the healthy twin in monozygotic twin pairs discordant for disease have a less than 30% risk for developing MS. A viral etiology was first proposed based on findings of local MS outbreaks. However, in recent years the focus has shifted to more common pathogens. HHV-6 is a ubiquitous human herpesvirus that most people have been exposed to. With the cumulative body of evidences for an association to MS, HHV-6 is a strong etiological candidate and the focus of this thesis has therefore been to investigate its role in MS.

The main aim of my PhD thesis was to investigate a mechanism by which HHV-6 might induce breakage of tolerance and subsequent autoimmune attacks against myelin, which is the primary target in MS, by constituting an adjuvant effect. Firstly, we present a new Q-PCR based TCID50 read-out method for unequivocal determination of the infectivity of HHV-6 viral stocks. The validation revealed that the new approach is more robust compared to established methods (paper I). Secondly, we show that HHV-6A is not a potent adjuvant as a non-productive infection of HHV-6A in DC reduces IL-8 secretion and reduces the capacity of DC to stimulate allogenic T cell proliferation. However, HHV-6A exposure of DC leads to the up-regulation of HLA-ABC, via autocrine IFN-α signaling, as well as the up-regulation of HLA-DR and CD86, suggesting that DC get partially activated (paper II).

To investigate the clinical relevance of HHV-6 in MS, we characterized MS plasma and CSF for viral DNA and MS plasma for antiviral IgG antibodies. We show no significant difference in the frequency of HHV-6 DNA in plasma or CSF (paper III) or in the status or levels of the antiviral IgG response. However, in paper IV we show that several factors previously associated with MS susceptibility are associated with the antiviral IgG response. Carriership of the MS protective allele HLA-A*02 is associated with lower antibody levels, possibly reflecting efficient cellular antiviral immunity in HLA-A*02 carriers. The MS risk factor smoking is associated with lower antibody levels, which may reflect a previously shown general decrease in IgG levels in smokers. Women had higher antibody levels, possibly due to a more active general humoral immunity, as previously shown. Finally, in paper V using GWAS SNP genotyping we show that carriership of the allele HLA-DQA1*05 is associated with higher antibody levels. Furthermore, we provide a list of 31 host genes with suggestive association to anti-HHV-6 IgG antibody status and 29 host genes with suggestive association to antibody levels that contain or lies within 50 kb of the tagging SNP. The most interesting genes are KSR-2 with suggestive associated to antibody status, and TRBV5-1, CMIP, RUNX1 and MAML3 with suggestive associated to antibody levels. Several of these genes have vital impact on T cell biology and potential importance for steering Th cells into Th1 or Th2 polarization.

To conclude, the results in this thesis provide a robust read-out approach for TCID50 assays of HHV-6A. Furthermore, they expand our understanding for interactions between HHV-6 and the cellular and humoral parts of our immune system and reveal novel insights in cellular pathways with potential importance for anti-HHV-6 immunity.

List of scientific papers

I. Rasmus Gustafsson, Elin Engdahl, Anna Fogdell-Hahn. Development and validation of a Q-PCR based TCID50 method for human herpesvirus 6. Virol J. 2012 Dec; 9: 311.
https://doi.org/10.1186/1743-422X-9-311

II. Rasmus Gustafsson, Elin Engdahl, Oscar Hammarfjord, Sanjaya B. Adikari, Magda Lourda, Jonas Klingström, Mattias Svensson, Anna Fogdell-Hahn. Human herpesvirus 6A partially suppresses functional properties of DC without viral replication. PLoS One. 2013 March;8(3):e58122.
https://doi.org/10.1371/journal.pone.0058122

III. Rasmus Gustafsson, Renate Reitsma, Annelie Strålfors, Andreas Lindholm, Rayomand Press, Anna Fogdell-Hahn. Incidence of human herpesvirus 6 in clinical samples from Swedish patients with demyelinating diseases. J Microbiol Immunol Infect. 2013 March.
https://doi.org/10.1016/j.jmii.2013.03.009

IV. Elin Engdahl, Rasmus Gustafsson, Ryan Ramanujam, Emilie Sundqvist, Tomas Olsson, Jan Hillert, Lars Alfredsson, Ingrid Kockum, Anna Fogdell-Hahn. HLA-A*02 carriership, gender and tobacco smoking, but not multiple sclerosis, affects the IgG antibody response against human herpesvirus 6. [Accepted]
https://pubmed.ncbi.nlm.nih.gov/24662416

V. Rasmus Gustafsson, Elin E. Engdahl, Emilie Sundqvist, Tomas Olsson, Jan Hillert, Lars Alfredsson, International Multiple Sclerosis Genetic Consortium, Anna Fogdell-Hahn, Ingrid Kockum. Host genetics influence on anti-human herpesvirus 6 IgG status and levels. [Manuscript]