Influenza is a significant global health problem, causing disease and hospitalisations in elderly individuals and infants. While updated vaccines are available every year, their effectiveness is moderate at best. FLUniversal is a European Union funded consortium, aiming to develop a universal influenza vaccine by bringing together partners with expertise in different areas of vaccine development. An intranasal live attenuated vaccine, DeltaFLU, will be produced using an innovative platform; preclinical assessment in animal models and clinical studies using a controlled human infection model (CHIM) will be conducted for assessment of safety, immunogenicity and protective efficacy; and finally, comprehensive immunological analysis of blood and nasal mucosa will elucidate vaccine responses and potential new correlates of protection (CoPs). In addition to a universal influenza vaccine, listed as a top priority by the EU, FLUniversal seeks to deliver an enhanced vaccine manufacturing technology that is superior in terms of efficiency, production costs and production speed - especially critical in the face of a potential new pandemic. Moreover, an influenza CHIM with a focus on harmonisation of clinical procedures and assays will be established to generate translatable and reproducible data. Newly generated knowledge on mechanisms of protection, CoPs and new molecular analysis tools may significantly contribute to our knowledge on influenza infection and influenza vaccines. In conclusion, FLUniversal is an innovative and ambitious public-private partnership, aiming to present a new development pathway for influenza vaccines, and maximising impact by bringing together leading partners from academy and industry with a shared purpose of collaboration and innovation.

Background, aim: It is well accepted today that papillomaviruses can induce mucocutaneous tumours in vertebrates. Given the high metastatic potential of some of these tumours in humans, and their tendency to recur following treatment, there is a need of more effective therapeutics. Methods: Following the generation of live-attenuated influenza viruses co-expressing bovine papillomavirus type 1 (BPV1)-specific E6 and E7 peptides, we assessed the efficacy of this immunotherapeutic vaccine prototype in a natural tumour model, i.e., horses bearing BPV1 or 2-induced skin tumours termed sarcoids. So far, 21 horses with mild and moderate disease repeatedly received intralesional injections with the vaccine. Ten horses with severe disease (multiple/aggressive sarcoids; 64 in total) were treated by combined therapy, i.e., injections with the vaccine and electrosurgical tumour excision. Where possible, a follow-up was carried out by photo-documentation and BPV1/2 PCR-screening of hair roots/dandruff of the tumour sites. Results: Nine of 21 horses with mild or moderate disease showed complete tumour regression (CR) after six months, and all samples collected from seven individuals scored BPV1/2-negative. Further nine horses exhibited partial sarcoid regression (PR) that is still ongoing, and harboured BPV1 or 2. Three horses showed stable disease and BPV1/2 infection. In one of these horses, repeated surgery and additional booster injections led to freedom from sarcoids and BPV1/2. From the 10 severely affected horses, nine showed CR six months after combined therapy. Three recurrent lesions in two horses were eradicated by repeated immunotherapy or combined therapy. This led to 8/9 horses being tumour- and BPV1/2-free meanwhile. Conclusion: Sarcoid recurrence following standard therapy is a common event because current treatments do not target BPV1/2 infection. Flu-BPV1-E6E7-immunotherapy was effective in 86% of mild and moderate-type sarcoids as well as eradicating BPV infection underlying the disease. Similarly, combined therapy led to CR in 9/10 cases and significantly reduced the risk of post-treatment recurrence due to eradication of BPV infection: only 3/64 treated sarcoids recrudesced, corresponding to a unique recurrence rate of only 4.7%.

Influenza viruses pose a threat to public health as evidenced by severe morbidity and mortality in humans on a yearly basis. Given the constant changes in the viral glycoproteins owing to antigenic drift, seasonal influenza vaccines need to be updated periodically and effectiveness often drops due to mismatches between vaccine and circulating strains. In addition, seasonal influenza vaccines are not protective against antigenically shifted influenza viruses with pandemic potential. Here, we have developed a highly immunogenic vaccination regimen based on live-attenuated influenza vaccines (LAIVs) comprised of an attenuated virus backbone lacking non-structural protein 1 (ΔNS1), the primary host interferon antagonist of influenza viruses, with chimeric hemagglutinins (cHA) composed of exotic avian head domains with a highly conserved stalk domain, to redirect the humoral response towards the HA stalk. In this study, we showed that cHA-LAIV vaccines induce robust serum and mucosal responses against group 1 stalk and confer antibody-dependent cell cytotoxicity activity. Mice that intranasally received cH8/1-ΔNS1 followed by a cH11/1-ΔNS1 heterologous booster had robust humoral responses for influenza A virus group 1 HAs and were protected from seasonal H1N1 influenza virus and heterologous highly pathogenic avian H5N1 lethal challenges. When compared with mice immunized with the standard of care or cold-adapted cHA-LAIV, cHA-ΔNS1 immunized mice had robust antigen-specific CD8+ T-cell responses which also correlated with markedly reduced lung pathology post-challenge. These observations support the development of a trivalent universal influenza vaccine for the protection against group 1 and group 2 influenza A viruses and influenza B viruses.

The influenza A non-structural protein 1 (NS1) is known for its ability to hinder the synthesis of type I interferon (IFN) during viral infection. Influenza viruses lacking NS1 (ΔNS1) are under clinical development as live attenuated human influenza virus vaccines and induce potent influenza virus-specific humoral and cellular adaptive immune responses. Attenuation of ΔNS1 influenza viruses is due to their high IFN inducing properties, that limit their replication in vivo. This study demonstrates that pre-treatment with a ΔNS1 virus results in an immediate antiviral state which prevents subsequent replication of homologous and heterologous viruses, preventing disease from virus respiratory pathogens, including SARS-CoV-2. Our studies suggest that ΔNS1 influenza viruses could be used for the prophylaxis of influenza, SARS-CoV-2 and other human respiratory viral infections, and that an influenza virus vaccine based on ΔNS1 live attenuated viruses would confer broad protection against influenza virus infection from the moment of administration, first by non-specific innate immune induction, followed by specific adaptive immunity.

Bovine papillomaviruses types 1 and 2 (BPV1, BPV2) commonly induce skin tumours termed sarcoids in horses and other equids. Sarcoids seriously compromise the health and welfare of affected individuals due to their propensity to resist treatment and reoccur in a more severe form. We have developed influenza (Flu) A and B virus vectors that harbour a truncated NS1 gene (iNS) assuring interferon induction and co-express shuffled BPV1 E6 and E7 antigens for sarcoid immunotherapy. In a safety trial involving 12 healthy horses, intradermal administration of iNSA/E6E7equ and iNSB/E6E7equ was well tolerated, with the only transient side effect being mild fever in four horses. Repeated screening of secretions and faeces by RT-PCR and plaque assay revealed no virus shedding, thus also confirming biological safety. In a patient trial involving 29 horses bearing BPV1-induced single or multiple sarcoids, at least one lesion per horse was intratumourally injected and then boosted with iNSA/E6E7equ and/or iNSB/E6E7equ. The treatment induced a systemic antitumour response as reflected by the synchronous regression of injected and non-injected lesions. Irrespective of vaccination schemes, complete tumour regression was achieved in 10/29 horses. In 10/29 horses, regression is still ongoing (May 2021). Intriguingly, scrapings collected from former tumour sites in two patients tested negative by BPV1 PCR. Nine severely affected individuals with a history of unsuccessful therapeutic attempts did not (6/29) or only transiently (3/29) respond to the treatment. INSA/E6E7equ and iNSB/E6E7equ proved safe and effective in significantly reducing the tumour burden even in severe cases.

The influenza A non-structural protein 1 (NS1) is known for its ability to hinder the synthesis of type I interferon (IFN) during viral infection. Influenza viruses lacking NS1 (Delta NS1) are under clinical development as live attenuated human influenza virus vaccines and induce potent influenza virus-specific humoral and cellular adaptive immune responses. Attenuation of Delta NS1 influenza viruses is due to their high IFN inducing properties, that limit their replication in vivo. This study demonstrates that pre-treatment with a Delta NS1 virus results in an antiviral state which prevents subsequent replication of homologous and heterologous viruses, preventing disease from virus respiratory pathogens, including SARS-CoV-2. Our studies suggest that Delta NS1 influenza viruses could be used for the prophylaxis of influenza, SARS-CoV-2 and other human respiratory viral infections, and that an influenza virus vaccine based on Delta NS1 live attenuated viruses would confer broad protection against influenza virus infection from the moment of administration, first by non-specific innate immune induction, followed by specific adaptive immunity.

Background: Traditional inactivated influenza vaccines are the type of vaccines that were most frequently developed for immunization against the highly pathogenic avian H5N1 influenza virus. However, clinical trials with inactivated influenza vaccines for H5N1 indicated that high doses and at least two immunizations are required for an effective immune response (Nicholson et al., 2001; Treanor, Campbell et al., 2006; Treanor, Schiff et al., 2006; Ehrlich et al., 2008). We investigated the safety and immunogenicity of a live attenuated H5N1 vaccine (delNS1-H5N1) lacking the interferon antagonist nonstructural protein 1 (NS1). Methods: We conducted a double-blind, placebo-controlled, phase 1 study in healthy adult participants who were randomly assigned at a 2:1 ratio to receive two immunizations of delNS1-H5N1 vaccine at 6.8 log10 50% tissue culture infectious doses (TCID50)/subject or 7.5 log10 TCID50/subject, or placebo. Results: Intranasal vaccination with the live attenuated delNS1-H5N1 vaccine was safe and well tolerated. The most common adverse events identified were symptoms associated with mild influenza infections, such as increased body temperature (>37.0 degrees C), pharyngeal erythema, rhinitis and throat irritation, and were reported within 7 days after the first immunization. delNS1-H5N1 was able to induce significant vaccine specific serum antibody titers even at the lower dose level of 6.8 log10 TCID50/subject. Seroconversion occurred in 75% of study participants after only one immunization with 7.5 log10 TCID50/subject. Vaccine-specific local IgA responses were observed in 41.7% of individuals that showed serum antibody responses after 2nd immunization. Conclusions: We show that vaccination with a live attenuated H5N1 influenza vaccine lacking NS1 is safe and induces significant levels of vaccine-specific antibodies even after one immunization. The safety and immunogenicity data indicate that delNS1-H5N1 has the potential to fulfil the unmet need for an effective influenza vaccine in pandemic situations. (C) 2019 Elsevier Ltd. All rights reserved.

Persistent infection with high-risk human papillomavirus (HPV) types, most often HPV16 and HPV18, causes all cervical and most anal cancers, and a subset of vulvar, vaginal, penile and oropharyngeal carcinomas. Two prophylactic virus-like particle (VLPs)-based vaccines, are available that protect against vaccine type-associated persistent infection and associated disease, yet have no therapeutic effect on existing lesions or infections. We have generated recombinant live-attenuated influenza A viruses expressing the HPV16 oncogenes E6 and E7 as experimental immunotherapeutic vaccine candidates. The influenza A virus life cycle lacks DNA intermediates as important safety feature. Different serotypes were generated to ensure efficient prime and boost immunizations. The immune response to vaccination in C57BL/6 mice was characterized by peptide ELISA and IFN-γ ELISpot, demonstrating induction of cell-mediated immunity to HPV16 E6 and E7 oncoproteins. Prophylactic and therapeutic vaccine efficacy was analyzed in the murine HPV16-positive TC-1 tumor challenge model. Subcutaneous (s.c.) prime and boost vaccinations of mice with recombinant influenza A serotypes H1N1 and H3N2, followed by challenge with TC-1 cells resulted in complete protection or significantly reduced tumor growth as compared to control animals. In a therapeutic setting, s.c. vaccination of mice with established TC-1 tumors decelerated tumor growth and significantly prolonged survival. Importantly, intralesional vaccine administration induced complete tumor regression in 25% of animals, and significantly reduced tumor growth in 50% of mice. These results suggest recombinant E6E7 influenza viruses as a promising new approach for the development of a therapeutic vaccine against HPV-induced disease.

The existence of multiple antigenically distinct types and subtypes of influenza viruses allows the construction of a multivalent vector system for the mucosal delivery of foreign sequences. Influenza A viruses have been exploited successfully for the expression of extraneous antigens as well as immunostimulatory molecules. In this study, we describe the development of an influenza B virus vector whose functional part of the interferon antagonist NS1 was replaced by human interleukin 2 (IL2) as a genetic adjuvant. We demonstrate that IL2 expressed by this viral vector displays immune adjuvant activity in immunized mice. Animals vaccinated with the IL2 viral vector showed an increased hemagglutination inhibition antibody response and higher protective efficacy after challenge with a wild-type influenza B virus when compared to mice vaccinated with a control virus. Our results demonstrate that it is feasible to construct influenza B vaccine strains expressing immune-potentiating foreign sequences from the NS genomic segment. Based on these data, it is now hypothetically possible to create a trivalent (or quadrivalent) live attenuated influenza vaccine in which each component expresses a selected genetic adjuvant with tailored expression levels.

UNLABELLED:We investigated the cross-neutralising potential of serum and nasal wash samples from volunteers who were intranasally immunised once with a monovalent replication-deficient delNS1-H1N1 influenza virus vaccine (7.7log10TCID50/volunteer). Eight out of twelve (8/12) vaccinees responded to vaccination with a significant increase of antibody levels in serum IgG ELISA, mucosal IgA ELISA, MNA or HAI. Four responders showed delNS1-specific ELISA IgA increases and revealed excellent homosubtypic neutralising activity in serum and mucosal washings (4/4). However, 0/4 of the sera but 3/4 of the nasal washings neutralised also heterosubtypic H3N2 and H5N1 influenza viruses. Depletion experiments proved that IgA but not IgG is responsible for the cross-neutralising activity of the nasal wash sample. Our findings indicate that the induction of virus-neutralising IgA may represent a valuable correlate of cross-protection of intranasal influenza vaccines and that the delNS1 concept constitutes a promising approach to protect humans from seasonal and pandemic influenza threats. CLINICAL TRIAL REGISTRATION:NCT00724997.

We explored the possibilities for purification of various ΔNS1 live, replication deficient influenza viruses on ion exchange methacrylate monoliths. Influenza A ΔNS1-H1N1, ΔNS1-H3N2, ΔNS1-H5N1 and ΔNS1-influenza B viruses were propagated in Vero cells and concentrated by tangential flow filtration. All four virus strains adsorbed well to CIM QA and CIM DEAE anion exchangers, with CIM QA producing higher recoveries than CIM DEAE. ΔNS1-influenza A viruses adsorbed well also to CIM SO3 cation exchanger at the same pH, while ΔNS1-influenza B virus adsorption to CIM SO3 was not complete. Dynamic binding capacity (DBC) for CIM QA, DEAE and SO3 methacrylate monoliths for influenza A ΔNS1-H1N1 virus were 1.9E+10 TCID50/ml, 1.0E+10 TCID50/ml and 8.9E+08 TCID50/ml, respectively. Purification of ΔNS1 viruses on CIM QA was scaled up and reproducibility was confirmed. Yields of infectious virus on CIM QA were between 70.8±32.3% and 87±30.8%. Total protein removal varied from 93.3±0.4% to 98.6±0.2% and host cell DNA removal efficiency was ranging from 76.4% to 99.9% and strongly depended on pretreatment with deoxyribonuclease.

Background: The non-structural protein NS1 of the influenza virus counteracts the interferon-mediated immune response of the host. We investigated the safety and immunogenicity of a trivalent formulation containing influenza H1N1, H3N2 and B strains lacking NS1 (delNS1-trivalent).Methods: Healthy adult study participants who were seronegative for at least one strain present in the vaccine formulation were randomized to receive a single intranasal dose of delNS1-trivalent vaccine at 7.0 log10 TCID50/subject (n = 39) or placebo (n = 41).Results: Intranasal vaccination with the live replication-deficient delNS1-trivalent vaccine was well tolerated with no treatment-related serious adverse events. The most common adverse events identified, i.e. headache, oropharyngeal pain and rhinitis-like symptoms, were mainly mild and transient and distributed similarly in the treatment and placebo groups. Significant vaccine-specific immune responses were induced. Pre-existing low antibody titers or seronegativity for the corresponding vaccine strain yielded better response rates.Conclusions: We show that vaccination with a replication-deficient trivalent influenza vaccine containing HI NI, H3N2 and B strains lacking NS1 is safe and induces significant levels of antibodies (ClinicalTrials.gov identifier NCT01369862). (C) 2013 Elsevier Ltd. All rights reserved.

Background: The hemagglutination inhibition assay (HAI) is universally regarded as the gold standard in influenza virus serology. Nevertheless, difficulties in titre readouts are common and interlaboratory variations are frequently reported.Objective: We developed and validated the modified HAI to facilitate reliable, accurate and reproducible analysis of sera derived from influenza vaccination studies.Study design: Clinical and preclinical serum samples, NIBSC reference sera and seasonal influenza virus type A (H1N1 and H3N2) and type B antigens were employed to validate the mHAI. Moreover, pandemic virus strains (H5N1 and H1N1pdm09) were used to prove assay robustness.Results: Utilisation of a 0.08% solution of stabilised human erythrocytes, assay buffer containing bovine serum albumin and microscopical plate readout are the major differences between the modified and standard HAI assay protocols.Validation experiments revealed that the mHAI is linear, specific and up to eightfold more sensitive than the standard HAI. In 95.6% of all measurements mHAI titres were precisely measured irrespective of the assay day, run or operator. Moreover, 96.4% (H1N1) or 95.2% (H3N2 and B), respectively, of all serum samples were determined within one dilution step of the nominal values for spiked samples. Finally, the mHAI results remained unaffected by variations in virus antigens, erythrocytes, reagents, laboratory location, sample storage conditions or matrix components.Conclusion: The modified HAI is easy to analyse, requires only a single source of erythrocytes and allows utilisation of numerous influenza virus antigens, also including virus strains which are difficult to handle by the standard HAI (e.g. H3N2, H5N1 and H1N1pdm09). (c) 2012 Elsevier B.V. All rights reserved.

Egg-derived viruses are the only available seed material for influenza vaccine production. Vaccine manufacturing is done in embryonated chicken eggs, MDCK or Vero cells. In order to contribute to efficient production of influenza vaccines, we investigate whether the quality of inactivated vaccines is influenced by the propagation substrate. We demonstrate that H3N2 egg-derived seed viruses (A/Brisbane/10/07, IVR147, and A/Uruguay/716/07) triggered the hemagglutinin (HA) conformational change under less acidic conditions (0.2-0.6 pH units) than antigenically similar primary isolates. This phenotype was associated with HA1 (A138S, L194P) and HA2 (D160N) substitutions, and strongly related to decreased virus stability towards acidic pH and elevated temperature. The subsequent propagation of H3N2 and H1N1 egg-derived seed viruses in MDCK and Vero cells induced HA2 N50K (H1N1) and D160E (H3N2) mutations, improving virus growth in cell culture but further impairing virus stability. The prevention of the loss or recovery of stability was possible by cultivation at acidified conditions. Viruses carrying less stable HAs are more sensitive for HA conformational change during concentration, purification and storage. This results in decreased detectable HA antigen content - the main potency marker for inactivated influenza vaccines. Thus, virus stability can be a useful marker for predicting the manufacturing scope of seed viruses.

The nonstructural protein 1 (NS1) of influenza A virus (IAV) enables the virus to disarm the host cell type 1 IFN defense system. Mutation or deletion of the NS1 gene leads to attenuation of the virus and enhances host antiviral response making such live-attenuated influenza viruses attractive vaccine candidates. Sublingual (SL) immunization with live influenza virus has been found to be safe and effective for inducing protective immune responses in mucosal and systemic compartments. Here we demonstrate that SL immunization with NS1 deleted IAV (DeltaNS1 H1N1 or DeltaNS1 H5N1) induced protection against challenge with homologous as well as heterosubtypic influenza viruses. Protection was comparable with that induced by intranasal (IN) immunization and was associated with high levels of virus-specific antibodies (Abs). SL immunization with DeltaNS1 virus induced broad Ab responses in mucosal and systemic compartments and stimulated immune cells in mucosa-associated and systemic lymphoid organs. Thus, SL immunization with DeltaNS1 offers a novel potential vaccination strategy for the control of influenza outbreaks including pandemics.