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Respiratory viruses infections caused by influenza viruses, human parainfluenza virus (hPIV), respiratory syncytial virus (RSV) and coronaviruses are an eminent threat for public health. Currently, there are no licensed vaccines available for hPIV, RSV and coronaviruses, and the available seasonal influenza vaccines have considerable limitations. With regard to pandemic preparedness, it is important that procedures are in place to respond rapidly and produce tailor made vaccines against these respiratory viruses on short notice. Moreover, especially for influenza there is great need for the development of a universal vaccine that induces broad protective immunity against influenza viruses of various subtypes. Modified Vaccinia Virus Ankara (MVA) is a replication-deficient viral vector that holds great promise as a vaccine platform. MVA can encode one or more foreign antigens and thus functions as a multivalent vaccine. The vector can be used at biosafety level 1, has intrinsic adjuvant capacities and induces humoral and cellular immune responses. However, there are some practical and regulatory issues that need to be addressed in order to develop MVA-based vaccines on short notice at the verge of a pandemic. In this review, we discuss promising novel influenza virus vaccine targets and the use of MVA for vaccine development against various respiratory viruses.

T he replication cycle of influenza A virus (IAV) is complex. The virus attaches to susceptible host cells via its hemagglutinin (HA), a homotrimeric type I membrane glycoprotein that recognizes sialoconjugates (1-3). The virus then enters the endocytic pathway, and upon arrival in acidified late endosomes, the HA trimer undergoes a conformational transition that renders it fusogenic. The M2 ion channel is responsible for acidification of the virus lumen, which results in dissociation of the eight viral ribonucleoproteins (vRNPs) (comprised of PB1, PB2, PA, NP, and genomic RNA) from the M1 protein and release of the vRNPs into the host cytosol (4-6). These vRNPs translocate into the nucleus via one of at least two nuclear localization sequences, NLS1 and NLS2, in NP (7-11). mRNA generated from vRNP-dependent synthesis of viral genomic RNA (vRNA) is exported from the nucleus and translated in the cytoplasm. Newly synthesized PB1, PB2, PA, and NP translocate into the nucleus as monomers (NP and PB2) or dimers (PB1-PA), where they assemble with newly synthesized vRNA to yield the vRNP complex (12, 13). These vRNP complexes are exported from the nucleus for incorporation into budding virus particles (14).In the course of a single replication cycle, influenza virus NP interacts with viral RNA and with viral proteins, including PB1, PB2, and M1 (15, 16). Several host proteins also interact with NP, including importin-␣, BAT1,. Mapping such interactions and assessing their relevance for virus replication remains a challenge because of their often-essential role in the replication cycle. With rare exceptions, the influenza virus genome has resisted genetic manipulation, because many such changes cause a complete loss of a particular function (21-23) and compromise viral fitness.The variable domains of heavy-chain-only antibodies (VHHs) isolated from camelids are small, ϳ15 kDa, and their ability to bind their cognate ligand is largely independent of modifications such as disulfide bonds and glycosylation (24,25). These properties allow the VHHs to be expressed in the cytosol of eukaryotic cells with retention of the antigen binding capabilities. This in turn permits the specific targeting of host or viral proteins recognized by VHHs, thus enabling possible perturbation of target protein function (26-32; for a review, see reference 33). VHHs are

Summary Several enveloped viruses exploit host pathways, such as the cellular endosomal sorting complex required for transport (ESCRT) machinery, for their assembly and release. The influenza A virus (IAV) matrix protein binds to the ESCRT-I complex, although the involvement of early ESCRT proteins such as Tsg101 in IAV trafficking remains to be established. We find that Tsg101 can facilitate IAV trafficking but this is effectively restricted by the interferon (IFN) stimulated protein ISG15. Cytosol from type I IFN-treated cells abolished IAV haemagglutinin (HA) transport to the cell surface in infected semi-intact cells. This inhibition required Tsg101 and could be relieved with deISGylases. Tsg101 is itself ISGylated in IFN-treated cells. Upon infection, intact Tsg101-deficient cells obtained by CRISPR/Cas9 genome editing were defective in surface display of HA and for infectious virion release. These data support the IFN-induced generation of a Tsg101/ISG15-dependent checkpoint in the secretory pathway that compromises influenza virus release.

Influenza viruses continuously circulate in the human population and escape recognition by virus neutralizing antibodies induced by prior infection or vaccination through accumulation of mutations in the surface proteins hemagglutinin (HA) and neuraminidase (NA). Various strategies to develop a vaccine that provides broad protection against different influenza A viruses are under investigation, including use of recombinant (r) viral vectors and adjuvants. The replication-deficient modified vaccinia virus Ankara (MVA) is a promising vaccine vector that efficiently induces B and T cell responses specific for the antigen of interest. It is assumed that live vaccine vectors do not require an adjuvant to be immunogenic as the vector already mediates recruitment and activation of immune cells. To address this topic, BALB/c mice were vaccinated with either protein- or rMVA-based HA influenza vaccines, formulated with or without the saponin-based Matrix-M™ adjuvant. Co-formulation with Matrix-M significantly increased HA vaccine immunogenicity, resulting in antigen-specific humoral and cellular immune responses comparable to those induced by unadjuvanted rMVA-HA. Of special interest, rMVA-HA immunogenicity was also enhanced by addition of Matrix-M, demonstrated by enhanced HA inhibition antibody titres and cellular immune responses. Matrix-M added to either protein- or rMVA-based HA vaccines mediated recruitment and activation of antigen-presenting cells and lymphocytes to the draining lymph node 24 and 48 h post-vaccination. Taken together, these results suggest that adjuvants can be used not only with protein-based vaccines but also in combination with rMVA to increase vaccine immunogenicity, which may be a step forward to generate new and more effective influenza vaccines.Electronic supplementary materialThe online version of this article (10.1007/s12026-018-8991-x) contains supplementary material, which is available to authorized users.