by If so, the high immunogenicity of mRNA vaccines could nevertheless be outweighed by several advantages of plasmid delivery such as (i) ease of engineering and production, (ii) independence of a cold chain, and (iii) needle-free jet delivery51. A vaccine that reliably induces protection against multiple influenza strains may eliminate the need for annual updates of seasonal influenza vaccines. heterodimeric molecules, and show that it induces broadly reactive antibodies against subdominant HA epitopes and heterologous protection against influenza A viruses in mice. == Introduction == Seasonal influenza epidemics caused by influenza A viruses (IAVs) and influenza B viruses (IBVs) are responsible for approximately ASP 2151 (Amenamevir) 500,000 deaths worldwide every year1,2. Moreover, IAVs have caused four pandemics during the last century alone3. The main target for neutralizing antibodies against IAVs is viral surface glycoprotein hemagglutinin (HA)4. There are 18 different HA subtypes58. Based on similarities in amino acid sequences, these 18 subtypes are divided into group 1 (H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18), and ASP 2151 (Amenamevir) group 2 (H3, H4, H7, H10, H14 and H15). At present, H1 and H3 viruses circulate in the human population and regularly EM9 cause seasonal epidemics. In addition, a ASP 2151 (Amenamevir) number of zoonotic IAV (H5, H6, H7, H9 and H10) have caused isolated but severe disease outbreaks in humans9. The adaptive immune system often responds to antigen in a hierarchical manner; highly immunogenic epitopes are immunodominant, while less immunogenic epitopes are subdominant10. Current influenza vaccines predominantly elicit antibodies towards immunodominant epitopes in the membrane-distal head domain of HA. Unfortunately, frequent ASP 2151 (Amenamevir) mutations in the HA head allow viral escape from pre-existing immune responses1113, necessitating annual reformulations of seasonal influenza vaccines14. It is therefore important ASP 2151 (Amenamevir) to develop vaccine formats that focus B cell responses towards conserved, subdominant HA epitopes, thus enhancing the generation of broadly cross-reactive antibodies. Human monoclonal antibodies with broad reactivity to many HAs have been isolated15, suggesting that it should be possible to induce broadly reactive antibodies to subdominant HA epitopes by means of innovative vaccination strategies. The main specificities of broadly neutralizing antibodies are the receptor binding site in the HA head domain1618, and epitopes in the fusion peptide in the conserved stem domain1921. We have recently developed a new vaccine format based on an ACID/BASE-modified Fos-Jun zipper where bivalency versus monovalency of vaccine antigens can be controlled22. Using this strategy, we have shown that antigen bivalency enhances the ability of APC-targeted vaccines to induce potent antibody responses23. As an explanation, we hypothesized that antigen bivalency increased B cell stimulation by cross-linking B cell receptors (BCRs) in APC-B cell synapses. According to this hypothesis, if two variants of an antigen are expressed together in a heterodimer, B cells with specificity for conserved epitopes shared by the two variants should be preferentially stimulated23. To test this idea, we here developed a vaccine strategy where single vaccine protein heterodimers should always express two different HA subtypes (e.g. HAx,and HAy), each in a monovalent manner. In such molecules, only conserved epitopes shared between HAxand HAyshould be bivalent while strain-specific HA epitopes should be monovalent. This vaccination strategy should preferentially stimulate B cells (antibodies) with specificity for conserved epitopes, while monovalent strain-specific epitopes should be less immunogenic. To test if such a valency-based immuno-selection strategy could enhance induction of broadly reactive antibody responses against ordinarily subdominant HA epitopes, we have here immunized mice with mixtures of plasmids that together encoded up to 18 different HA subtypes, each HA being monovalently expressed in a large number of heterodimeric vaccine molecules. == Results == == APC-targeted heterodimeric proteins with different HAs delivered as DNA plasmids == We selected HA variants from 18 IAV subtypes based on their commercial availability as proteins and their similarity to strains that presently circulate or have circulated in the past (Table1). A phylogenetic tree (Fig.1a) of selected HA subtypes was generated based on their amino acid sequences (Supplementary Fig.1). We inserted genes for the 18 HA subtypes into A and B plasmids that together encode heterodimeric vaccine proteins due to a centrally located modified Fos-Jun zipper heterodimerization motif called A/B (ACID/BASE) (Fig.1b)22,24. The different HA subtypes were expressed C-terminally of the dimerization unit. To bivalently target proteins to APC, A and B chains both expressed an N-terminal MHCII (I-Ed)-specific scFv. In non-targeted controls a scFv specific for the hapten 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP) replaced the MHCII-specific scFv. == Table 1. == Overview of IAV HA subtype virus strains used in the plasmid mix vaccines, ELISA coats and challenge of mice aH1N1 and H7N1 viruses used for infection were mouse adapted. bIn ELISA only the HA1 subunit of H10 was used. == Fig. 1. Expression of 18 different HA subtypes in.
