Host susceptibility to rotavirus infection and development of antibody-based immunotherapy

Rotavirus infects mature enterocytes of the small intestine of young children and cause gastroenteritis, leading to approximately 500 000 deaths annually worldwide, 85 % of which occur in the developing world. The main objectives of the thesis were to investigate host genetic factors leading to differential susceptibility to rotavirus infections and to develop an antibody-based oral therapy against the infections.

Reduced TLR3 expression was previously suggested to be associated with susceptibility to rotavirus infections. In Paper I, we thus investigated rotavirusspecific IgG antibody responses from individuals (IgA competent or deficient) using two TLR3 SNPs (rs3775291 and rs5743305). We concluded that these two polymorphisms were associated with elevated IgG titers in IgA deficient, but not in IgA competent individuals. In addition, recent in vitro studies have suggested that HBGAs (H type 1 and Lewis antigens) serve as putative receptors for rotavirus VP8*, and play a role in susceptibility to infections in vivo. In Paper II, we therefore studied the effect of SNPs in the FUT2 (rs601338) and FUT3 genes (rs28362459, rs3894326, rs812936 and rs778986) on the serum IgG antibody titers and neutralizing antibody levels to rotavirus P[6] and P[8]. The rotavirus specific serum IgG levels and neutralizing antibody titers to the Wa strain (P[8]) of rotavirus were significantly higher in secretors (individuals with an intact FUT2), suggesting that secretor individuals, expressing the Lewis b antigen, are more prone to rotavirus (P[8]) infections than non-secretors.

We have recently developed an antibody-based therapy against rotavirus, which may confer safe, immediate and efficient viral neutralization and protection. Probiotic bacteria represent an attractive delivery system for antibody fragments and other proteins in the gastrointestinal tract. In Paper III, the combination therapy including engineered L. rhamnosus GG expressing IgG binding domains of protein G and HBC was shown to be more effective in reducing the prevalence, severity, and duration of diarrhea in a mouse pup model of RRV infection in comparison to HBC alone or a combination of wild-type L. rhamnosus GG and HBC. In Paper IV, we developed vectors for co-production of two VHHs: ARP1 and ARP3, by engineered L. paracasei BL23. Both fragments (secreted or anchored) were shown to bind to a broad range of human rotavirus serotypes in vitro. In Paper V, we have shown that the fusion of the mouse IgG1 Fc to ARP1 (Fc-ARP1) confers a markedly increased protection against rotavirus in a neonatal mouse model of rotavirus-induced diarrhea, suggesting a role for Fc-mediated neutralization of rotavirus. These antibodybased treatments could be further developed and used as an alternative or complement to current vaccines.

List of scientific papers

I. Mutations in toll-like receptor 3 are associated with elevated levels of rotavirus-specific IgG antibodies in IgA-deficient but not IgA-sufficient individuals. Günaydin G, Nordgren J, Svensson L, Hammarström L. Clinical and vaccine immunology : CVI 2014; 21:298-301.
https://doi.org/10.1128/CVI.00666-13

II. The FUT2 gene determines susceptibility to rotavirus infections in a Swedish population. Günaydin G, Nordgren J, Sharma S, Svensson L, Hammarström L. [Submitted]

III. Engineered Lactobacillus rhamnosus GG expressing IgG-binding domains of protein G: Capture of hyperimmune bovine colostrum antibodies and protection against diarrhea in a mouse pup rotavirus infection model. Günaydin G, Zhang R, Hammarström L, Marcotte H. Vaccine 2014; 32:470-7.
https://doi.org/10.1016/j.vaccine.2013.11.057

IV. Co-expression of anti-rotavirus proteins (llama VHH antibody fragments) in Lactobacillus: Development and functionality of vectors containing two expression cassettes in tandem. Günaydin G, Álvarez B, Lin Y, Hammarström L, Marcotte H. PloS One 2014; 9(4): e96409.
https://doi.org/10.1371/journal.pone.0096409

V. Fusion of mouse IgG1 Fc to llama single domain VHH (ARP1) confers increased protection against rotavirus in a neonatal mouse model of rotavirus induced diarrhea. Günaydin G, Marcotte H, Hammarström L. [Manuscript]