Significance of the norovirus GII.4 genotype as a cause of acute gastroenteritis in Finnish children, and the production of a candidate virus-like particle vaccine

Abstract

Norovirus on yleisin virusperäisen ripulitaudin aiheuttaja aikuisilla ja merkittävä pienten lasten vakavan ripulitaudin aiheuttaja. Norovirus jaetaan perimänsä mukaan genotyyppeihin, joista GII.4-genotyyppi on yleisin maailmanlaajuisesti. GII.4-genotyypin perimässä tapahtuu paljon muutoksia, joita esiintyy etenkin kuoriproteiinin epitoopeissa. Nämä muutokset saavat aikaan uusien geneettisten kantojen, varianttien, ilmentymisen muutaman vuoden välein. Tämän tutkimustyön tarkoituksena oli tutkia norovirus GII.4-genotyypin esiintyvyyttä, geneettistä vaihtelua ja kliinistä taudinkuvaa suomalaisilla lapsilla vuosina 1993–2013 sekä näiden tutkimustulosten perusteella tuottaa viruksen kaltaisia partikkeleita (VLP) norovirusrokotekandidaatiksi.

Tutkimuksessani havaitsin GII.4-genotyypin aiheuttaneen 55% lasten norovirusinfektioista Suomessa vuosina 1993–2013. GII.4-genotyyppi oli yleisin (79%) vakavan norovirustaudin aiheuttaja lapsilla Tampereen yliopistollisessa sairaalassa. Havaitsimme uusien GII.4-varianttien saavan aikaan toistuvia epidemioita muutaman vuoden välein. Lisäksi totesimme GII.4-genotyypin aiheuttavan vakavamman ripulitaudin kuin muut norovirusgenotyypit. Koska norovirusinfektioita vastaan ei toistaiseksi ole rokotetta, tuotimme GII.4-genotyypistä VLP-rokotekandidaatin hyönteisoluissa ja vertailimme eri puhdistusmenetelmiä. GII.4 VLP:t saivat hiirissä aikaan immuunivasteen norovirusta vastan. Tutkimme GII-4-varianttien kuoriproteiinissa tapahtuvia muutoksia, joiden havaitsimme olevan palautumattomia varianttien välillä. GII.4:n on todettu muuttuvan varianttien välillä niin, ettei aikaisempi immuniteetti pysty estämään uutta muuntautunutta kantaa.

Tulevaisuudessa norovirusrokotteen tulisi saada aikaan mahdollisimman laaja-alainen immuniteetti useampaa norovirusgenotyyppiä ja uusia variantteja vastaan. Tutkimukseni tulokset ovat olleet tärkeänä pohjatietona norovirusrokotekandidaatin kehittämiselle. Kehitetty GII.4 VLP-rokotekandidaatti näyttää antavan laaja-alaisen immuunivasteen, joka kattaa useampia GII.4-variantteja.

Norovirus (NoV) is the leading causative agent of acute gastroenteritis (AGE) outbreaks among all age groups, and it is the second most common viral agent after rotavirus (RV) to cause clinically severe gastroenteritis in young children. After the introduction of a RV vaccine into the National Immunization Program of Finland in 2009, NoV has become the leading cause of AGE in children. Since the 1990s, NoV GII.4 has been the predominant genotype circulating worldwide in outbreaks and in sporadic gastroenteritis in children, with new variants emerging approximately every two to three years. For the prevention of NoV infections, NoV vaccines based on virus-like particles (VLPs) are being developed. A vaccine against NoV GII.4 is urgently needed.

This dissertation investigates the prevalence of NoV genotypes, and the antigenic changes and clinical features of NoV GII.4 in Finnish children for the general purpose of VLP-based candidate vaccine production and evaluation. The clinical study material is composed of two parts. First, fecal specimens were collected from AGE cases in children who participated in RV vaccine efficacy trials in Finland from 1993 to 2007. Second, fecal specimens were collected from children who were treated at Tampere University Hospital because of AGE from 2006 to 2013.

The distribution of NoV genotypes was first studied in sporadic NoV AGE cases among children in the community between the years 1993 and 2007. NoV GII.4 was the most common genotype (46%) and it predominanted for three seasons (1998–1999, 2002–2003, 2006–2007). Secondly, in children treated at Tampere University Hospital, NoV AGE cases were mainly caused by NoV GII.4 (79%). Altogether, NoV GII.4 was found in 55% of non-outbreak NoV cases in children during the entire period of 1998–2013, with a new variant of GII.4 appearing every two to three years. Six different GII.4 variants were detected, and these coincided with NoV epidemic years. In addition, GII.4 was found to cause more severe cases of gastroenteritis than other NoV genotypes.

For the purpose of vaccine development, NoV GII.4 VLPs were produced using the baculovirus expression system in Sf9 insect cells. The VLPs were purified with several purification procedures and the purity, yield, morphology, and functionality of the NoV VLPs were assessed. To obtain high quality NoV VLPs, two discontinuous sucrose gradient centrifugations were used. However, the density gradient-purified VLP preparations contained impurities from the expression vector. It was thus necessary to purify NoV VLPs further. Chromatographically purified NoV VLP preparations were devoid of impurities and maintained the icosahedral capsid structure. The purified VLPs induced efficient NoV GII.4-specific immune responses in mice.

Antigenic variations in the P2 domain of GII.4 major antigenic epitopes, known as blockade epitopes, are linked to NoV binding profile of human histo-blood group antigens. Genetic analyses of predominant NoV GII.4 variants that circulated in Finland between 1998 and 2013 showed that NoV GII.4 undergo significant variation in the major antigenic epitopes on the surface of the capsid P2 domain. These findings were similar to those seen previously in NoV outbreaks in other populations. Future NoV vaccines should induce either type-specific immunity for each variant – in which case the vaccine would need to be frequently reformulated – or produce broadly cross-protective immunity covering multiple variants. Promisingly, the candidate NoV GII.4 VLP vaccine, based on a 1999 NoV isolate produced in our laboratory appears to induce a broad spectrum immune response in mice covering the new strains of GII.4 up to 2012.