**Maria Malm, Timo Vesikari and Vesna Blazevic \***

Vaccine Research Center, Faculty of Medicine and Health Technology, Tampere University, Biokatu 10, FI-33520 Tampere, Finland; maria.malm@tuni.fi (M.M.); timo.vesikari@tuni.fi (T.V.)

**\*** Correspondence: vesna.blazevic@tuni.fi; Tel.: +358-50-4211-054

Received: 23 September 2019; Accepted: 31 October 2019; Published: 2 November 2019

**Abstract:** Human noroviruses (NoVs) are a genetically diverse, constantly evolving group of viruses. Here, we studied the effect of NoV pre-existing immunity on the success of NoV vaccinations with genetically close and distant genotypes. A sequential immunization as an alternative approach to multivalent NoV virus-like particles (VLPs) vaccine was investigated. Mice were immunized with NoV GI.3, GII.4-1999, GII.17, and GII.4 Sydney as monovalent VLPs or as a single tetravalent mixture combined with rotavirus VP6-protein. Sequentially immunized mice were primed with a trivalent vaccine candidate (GI.3 + GII.4-1999 + VP6) and boosted, first with GII.17 and then with GII.4 Sydney VLPs. NoV serum antibodies were analyzed. Similar NoV genotype-specific immune responses were induced with the monovalent and multivalent mixture immunizations, and no immunological interference was observed. Multivalent immunization with simultaneous mix was found to be superior to sequential immunization, as sequential boost induced strong blocking antibody response against the distant genotype (GII.17), but not against GII.4 Sydney, closely related to GII.4-1999, contained in the priming vaccine. Genetically close antigens may interfere with the immune response generation and thereby immune responses may be differently formed depending on the degree of NoV VLP genotype identity.

**Keywords:** norovirus; VLP; vaccine; genotype; pre-existing immunity; cross-reactivity; blocking antibodies; original antigenic sin (OAS)
