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Background: Understanding cellular responses to SARS-CoV-2 immunisations is important for informing vaccine recommendations in patients with inflammatory bowel disease (IBD) and other vulnerable patients on immunosuppressive therapies. This study investigated the magnitude and quality of T cell responses after multiple SARS-CoV-2 vaccine doses and COVID-19 breakthrough infection., Methods: This prospective, observational study included patients with IBD and arthritis on tumour necrosis factor inhibitors (TNFi) receiving up to four SARS-CoV-2 vaccine doses. T cell responses to SARS-CoV-2 peptides were measured by flow cytometry before and 2-4 weeks after vaccinations and breakthrough infection to assess the frequency and polyfunctionality of responding cells, along with receptor-binding domain (anti-RBD) antibodies., Findings: Between March 2, 2021, and December 20, 2022, 143 patients (118 IBD, 25 arthritis) and 73 healthy controls were included. In patients with either IBD or arthritis, humoral immunity was attenuated compared to healthy controls (median anti-RBD levels 3391 vs. 6280 BAU/ml, p = 0.008) after three SARS-CoV-2 vaccine doses. Patients with IBD had comparable quantities (median CD4 0.11% vs. 0.11%, p = 0.26, CD8 0.031% vs. 0.047%, p = 0.33) and quality (polyfunctionality score: 0.403 vs. 0.371, p = 0.39; 0.105 vs. 0.101, p = 0.87) of spike-specific T cells to healthy controls. Patients with arthritis had lower frequencies but comparable quality of responding T cells to controls. Breakthrough infection increased spike-specific CD8 T cell quality and T cell responses against non-spike peptides., Interpretation: Patients with IBD on TNFi have T cell responses comparable to healthy controls despite attenuated humoral responses following three vaccine doses. Repeated vaccination and breakthrough infection increased the quality of T cell responses. Our study adds evidence that, in the absence of other risk factors, this group may in future be able to follow the general recommendations for COVID-19 vaccines., Funding: South-Eastern Norway Regional Health Authority, Coalition for Epidemic Preparedness Innovations (CEPI), Norwegian Institute of Public Health, Akershus University Hospital, Diakonhjemmet Hospital., Competing Interests: Declaration of interests KHB reports funding from Akershus University Hospital and speaker bureaus for Janssen-Cilag. TKK reports grants from AbbVie, BMS, Galapagos, Novartis, Pfizer, UCB, speakers' bureaus from Grünenthal, Janssen, Sandoz, consultant fees from AbbVie, Gilead, Janssen, Novartis, Pfizer, Sandoz, UCB, and participation on advisory board for AbbVie. JJ reports grants from Boehringer-Ingelheim, speakers bureaus from AbbVie/Abbott, Bristol-Myers, Squibb, Galapagos, Gilead, Janssen, Pfizer, Roche, Sandoz, Takeda, consultant fees from AbbVie/Abbott, Pfizer, and participation in advisory board for AbbVie/Abbott, Bristol-Myers Squibb, Galapagos, Gilead, Janssen, Pfizer, Roche, Sandoz, Takeda. LAM reports funding from KG Jebsen foundation, support for infrastructure and biobanking from the university of Oslo and Oslo University Hospital, grants from the Coalition of Epidemic Preparedness Innovations (CEPI), speakers' bureaus from Incyte, Janssen, and expert testimony for Norwegian Medicines Agency. EAH reports speakers' bureaus from Pfizer, UCB, Novartis, and consulting fees from Abbvie, Pfizer, Eli Lilly. GG reports speaker bureaus from AbbVie/Abbott, Galapagos, Pfizer, UCB, participation in advisory board from AstraZeneca, Janssen, Moderna, Seqirus. and consulting fees from The Norwegian System of Patient Injury Compensation. JTV reports grant from CEPI. FLJ reports funding from South-East Health Authorities in Norway, CEPI and Oslo University Hospital. GLG reports speakers' bureaus from AbbVie/Abbott, Galapagos, Pfizer, UCB, and participation in advisory board from AbbVie/Abbott, Galapagos, Pfizer, UCB, Novartis. KKJ reports speakers’ bureaus from Bristol-Myers Squibb and Janssen, and participation in advisory board for AstraZeneca and IPSEN. SWS reports participation in advisory board for AstraZeneca. ASW reports travel grant of GBP 250 from the British Society of Immunology to support travelling to the BSI Congress 2023 (4–7th Dec 2023) and presenting a poster with some of this data. HSØ, SB, GS, IFK, IJ, UCN, ABK, IEC, SEJ, KPL, AC, ATT, JS, SAP, HK, and SM report nothing to disclose., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: To assess incidence, severity and predictors of COVID-19, including protective post-vaccination levels of antibodies to the receptor-binding domain of SARS-CoV-2 spike protein (anti-RBD), informing further vaccine strategies for patients with immune-mediated inflammatory diseases (IMIDs) on immunosuppressive medication., Methods: IMIDs on immunosuppressives and healthy controls (HC) receiving SARS-CoV-2 vaccines were included in this prospective observational study. COVID-19 and outcome were registered and anti-RBD antibodies measured 2-5 weeks post-immunisation., Results: Between 15 February 2021 and 15 February 2023, 1729 IMIDs and 350 HC provided blood samples and self-reported COVID-19. The incidence of COVID-19 was 66% in patients and 67% in HC, with re-infection occurring in 12% of patients. Severe COVID-19 was recorded in 22 (2%) patients and no HC. No COVID-19-related deaths occurred. Vaccine-induced immunity gave higher risk of COVID-19 (HR 5.89 (95% CI 4.45 to 7.80)) than hybrid immunity. Post-immunisation anti-RBD levels <6000 binding antibody units/mL were associated with an increased risk of COVID-19 following three (HR 1.37 (95% CI 1.08 to 1.74)) and four doses (HR 1.28 (95% CI 1.02 to 1.62)), and of COVID-19 re-infection (HR 4.47 (95% CI 1.87 to 10.67))., Conclusion: Vaccinated patients with IMID have a low risk of severe COVID-19. Hybrid immunity lowers the risk of infection. High post-immunisation anti-RBD levels protect against COVID-19. These results suggest that knowledge on COVID-19 history, and assessment of antibody levels post-immunisation can help individualise vaccination programme series in high-risk individuals., Trial Registration Number: NCT04798625., Competing Interests: Competing interests: KHB reports funding from Akershus University Hospital and speakers bureau for Janssen-Cilag. TKK reports grants from AbbVie, Amgen, Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, Pfizer, Union Chimique Belge; consulting fees from AbbVie, Biogen, Celltrion, Eli Lilly, Gilead, Mylan, Novartis, Pfizer, Sandoz, Sanofi and speakers bureau from Amgen, Celltrion, Egis, Evapharma, Ewopharma, Hikma, Oktal, Sandoz and Sanofi. GG reports speakers bureau from Pfizer, Bayer, Sanofi, Thermo Fisher, GSK and Vitusapotek, and participation on advisory board for Moderna, AstraZeneca, Janssen and Seqirus. JJ reports grants from Boehringer Ingelheim; speakers bureau from AbbVie, Bristol-Myers Squibb, Galapagos, Gilead, Janssen, Pfizer, Roche, Sandoz and Takeda; and participation on advisory board for AbbVie, Bristol-Myers Squibb, Galapagos, Gilead, Janssen, Lilly, Pfizer, Roche, Sandoz and Takeda. EAH reports consultant fees from AbbVie, Boehringer Ingelheim, Eli Lilly and Gilead; and speakers bureau from Pfizer and UCB. LAM reports funding from KG Jebsen foundation, support for infrastructure and biobanking from the University of Oslo and Oslo University Hospital, grants from the Coalition of Epidemic Preparedness Innovations and speakers bureau from Novartis, and Cellgene. JTV reports grants from the Coalition of Epidemic Preparedness Innovations. GLG reports funding from the South-Eastern Norway Regional Health Authority, The Coalition for Epidemic Preparedness Innovations, RCN Covid (312693), a KG Jebsen Foundation (grant 19), Dr Trygve Gythfeldt og frues forskningsfond, Karen Fossum Foundation, the Research Foundation at Diakonhjemmet Hospital and Oslo University Hospital; speakers bureau from AbbVie, Galapagos, Pfizer and Union Chimique Belge; and participation on advisory board of AbbVie, Galapagos, Pfizer and Union Chimique Belge. KKJ reports speakers bureau from Bristol-Myers Squibb and Roche. SWS reported participation on advisory board AstraZeneca. All other authors declare no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Conserved epitopes shared between virus subtypes are often subdominant, making it difficult to induce broadly reactive antibodies by immunization. Here, we generate a plasmid DNA mix vaccine that encodes protein heterodimers with sixteen different influenza A virus hemagglutinins (HA) representing all HA subtypes except H1 (group 1) and H7 (group 2). Each single heterodimer expresses two different HA subtypes and is targeted to MHC class II on antigen presenting cells (APC). Female mice immunized with the plasmid mix produce antibodies not only against the 16 HA subtypes, but also against non-included H1 and H7. We demonstrate that individual antibody molecules cross-react between different HAs. Furthermore, the mix vaccine induces T cell responses to conserved HA epitopes. Immunized mice are partially protected against H1 viruses. The results show that application of valency-based immuno-selection to diversified antigens can be used to direct antibody responses towards conserved (subdominant) epitopes on viral antigens., (© 2024. The Author(s).)

Background: Kidney transplant recipients (KTR) are at high risk for severe COVID-19 and have demonstrated poor response to vaccination, making it unclear whether successive vaccinations offer immunity and protection., Methods: We conducted a serologically guided interventional study where KTR patients that failed to seroconvert were revaccinated and also monitored seroconversion of KTR following the Norwegian vaccination program. We analysed IgG anti-RBD Spike responses from dose 2 (n = 432) up to after the 6th (n = 37) mRNA vaccine dose. The frequency and phenotype of Spike-specific T and B cell responses were assessed in the interventional cohort after 3-4 vaccine doses (n = 30). Additionally, we evaluated the Specific T and B cell response to breakthrough infection (n = 32), measured inflammatory cytokines and broadly cross-neutralizing antibodies, and defined the incidence of COVID-19-related hospitalizations and deaths. The Norwegian KTR cohort has a male dominance (2323 males, 1297 females), PBMC were collected from 114 male and 78 female donors., Findings: After vaccine dose 3, most KTR developed Spike-specific T cell responses but had significantly reduced Spike-binding B cells and few memory cells. The B cell response included a cross-reactive subset that could bind Omicron VOC, which expanded after breakthrough infection (BTI) and gave rise to a memory IgG +  B cell response. After BTI, KTR had increased Spike-specific T cells, emergent non-Spike T and B cell responses, and a systemic inflammatory signature. Late seroconversion occurred after doses 5-6, but 38% (14/37) of KTR had no detectable immunity even after multiple vaccine doses., Interpretation: Boosting vaccination can induce Spike-specific immunity that may expand in breakthrough infections highlighting the benefit of vaccination to protect this vulnerable population., Funding: CEPI and internal funds., Competing Interests: Declaration of interests GG has received speaker bureaus from Pfizer, Sanofi, GSK, Vitusapotek, Bayer, and ThermoFisher; consulting fee from Norsk Pasientskadeerstatning and advisory board for Moderna, Seqirus, and Janssen. GLG has received speaker bureaus from Novartis, AbbVie, and Pfizer. KH has received speaker bureaus and travel support from Astra Zeneca. AÅ has received the support of free study drug/placebo in investigator-initiated RCT from AstraZeneca and has participated in an advisory board for AstraZeneca. LAM has received speaker bureaus from Incyte Biosciences and Janssen and a fee for expert testimony from the Norwegian Medicines Agency. HK, AA, KPL, LT, TdMK, MK, MH, AVR, JTV, FLJ, and KM declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Viral subunit vaccines contain the specific antigen deemed most important for development of protective immune responses. Typically, the chosen antigen is a surface protein involved in cellular entry of the virus, and neutralizing antibodies may prevent this. For influenza, hemagglutinin (HA) is thus a preferred antigen. However, the natural trimeric form of HA is often not considered during subunit vaccine development. Here, we have designed a vaccine format that maintains the trimeric HA conformation while targeting antigen toward major histocompatibility complex class II (MHCII) molecules or chemokine receptors on antigen-presenting cells (APC) for enhanced immunogenicity. Results demonstrated that a single DNA vaccination induced strong antibody and T-cell responses in mice. Importantly, a single DNA vaccination also protected mice from lethal challenges with influenza viruses H1N1 and H5N1. To further evaluate the versatility of the format, we developed MHCII-targeted HA from influenza A/California/04/2009(H1N1) as a protein vaccine and benchmarked this against Pandemrix and Flublok. These vaccine formats are different, but similar immune responses obtained with lower vaccine doses indicated that the MHCII-targeted subunit vaccine has an immunogenicity and efficacy that warrants progression to larger animals and humans. IMPORTANCE Subunit vaccines present only selected viral proteins to the immune system and allow for safe and easy production. Here, we have developed a novel vaccine where influenza hemagglutinin is presented in the natural trimeric form and then steered toward antigen-presenting cells for increased immunogenicity. We demonstrate efficient induction of antibodies and T-cell responses, and demonstrate that the vaccine format can protect mice against influenza subtypes H1N1, H5N1, and H7N1.

Background: Data on response and safety of repeated vaccinations and hybrid immunity in patients with immune-mediated inflammatory diseases on immunosuppressive therapy is needed to further develop vaccination strategies in this vulnerable population. This study aimed to evaluate hybrid immunity and humoral immune response and safety of four SARS-CoV-2 vaccine doses in patients with immune-mediated inflammatory diseases on immunosuppressive therapy., Methods: This prospective observational Norwegian study of vaccine response to COVID-19 (Nor-vaC) included adult patients aged 18 years and older with immune-mediated inflammatory diseases (rheumatoid arthritis, spondyloarthritis, psoriatic arthritis, Crohn's disease, or ulcerative colitis) on immunosuppressive therapy, who had received four SARS-CoV-2 vaccine doses (vaccine group) or three vaccine doses followed by COVID-19 (hybrid group), and healthy controls receiving three vaccine doses (control group). Patients were recruited from the Division of Rheumatology at Diakonhjemmet Hospital, Oslo, and the Department of Gastroenterology at Akershus University Hospital, Lørenskog. Patients who had COVID-19 before the third vaccine dose, and patients with allergies or intolerances to elements of the vaccine were excluded. Antibodies to the receptor-binding domain of SARS-CoV-2 spike protein (anti-RBD antibodies) were assessed 2-4 weeks following vaccination or COVID-19. This study is registered at Clinialtrials.gov, NCT04798625., Findings: Between Nov 12, 2021, and April 19, 2022, 1458 participants with immune-mediated inflammatory diseases provided post-vaccination samples at 2-4 weeks following a third vaccine dose. After 544 participants were excluded, 715 (78%) of the remaining 914 participants received the fourth dose of the vaccine, and of these, 536 (75%) provided post-vaccination samples 2-4 weeks after their fourth vaccination (vaccine group). 199 (22%) of the 914 had COVID-19 after their third dose of the vaccine and of these, 167 (84%) provided samples (hybrid group). 256 of the eligible 703 patients had rheumatoid arthritis, 107 had spondyloarthritis, 115 had psoriatic arthritis, 130 had Crohn's disease, and 95 had ulcerative colitis). Median age was 56 years [IQR 45-65], 398 (57%) were women, and 305 (43%) were men. Patients in the vaccine group had higher anti-RBD antibody concentrations following the fourth vaccine dose (median 6192 BAU/ml [IQR 2878-11 243]) than after the third dose (median 5087 BAU/ml [1250-9081]; p< 0·0001), but lower antibody concentrations than the control group following the third dose (median 7595 BAU/ml [5916-12 001]; p< 0·0001). Antibody concentrations were higher in the patients in the hybrid group (23 548 BAU/ml [IQR 11 440-35 935]) than in the vaccine group (p<0·0001). No difference was found in antibody concentrations between the fourth dose of BNT162b2 (full-dose) and mRNA-1273 (half-dose). Patients and controls had a comparable safety profile after both three and four vaccine doses., Interpretation: Vaccine boosters improve humoral immune responses and are safe in patients with immune-mediated inflammatory diseases on immunosuppressive therapy, and administration should be considered regularly in this patient group. Hybrid immunity with omicron induces a strong humoral response suggesting longer intervals between booster doses in this patient group., Funding: The South-Eastern Norway Regional Health Authority, The Coalition for Epidemic Preparedness Innovations, Akershus University Hospital., Competing Interests: KHB reports funding from Akershus University Hospital and speaker bureaus for Janssen-Cilag. TKK reports grants from AbbVie, Amgen, Bristol Myers Squibb, Merck Sharp & Dohme, Novartis, Pfizer, Union Chimique Belge; consulting fees from AbbVie, Biogen, Celltrion, Eli Lilly, Gilead, Mylan, Novartis, Pfizer, Sandoz, Sanofi; and speakers bureaus from Amgen, Celltrion, Egis, Evapharma, Ewopharma, Hikma, Oktal, Sandoz, and Sanofi. JJ reports grants from Boehringer-Ingelheim; speakers bureaus from AbbVie, Bristol-Myers Squibb, Galapagos, Gilead, Janssen, Pfizer, Roche, Sandoz, and Takeda; and participation on advisory board for AbbVie, Bristol-Myers Squibb, Galapagos, Gilead, Janssen, Pfizer, Roche, Sandoz, and Takeda. LAM reports funding from KG Jebsen foundation, support for infrastructure and biobanking from the university of Oslo and Oslo University Hospital, grants from the Coalition of Epidemic Preparedness Innovations, and speakers bureaus from Novartis, and Cellgene. EAH reports consultant fees from AbbVie, Boehringer-Ingelheim, Eli Lilly, and Gilead; and speakers bureaus from Pfizer and UCB. GG reports speaker bureaus from Bayer, Sanofi, ThermoFisher, and participation on advisory board for AstraZeneca. JTV reports grants from the Coalition of Epidemic Preparedness Innovations. GLG reports funding from the South-Eastern Norway Regional Health Authority, The Coalition for Epidemic Preparedness Innovations, RCN Covid (312693), a KG Jebsen Foundation (grant 19), Dr Trygve Gythfeldt og frues forskningsfond, Karin Fossum Foundation, the Research Foundation at Diakonhjemmet Hospital, and Oslo University Hospital; speakers bureaus from AbbVie, Galapagos, Pfizer, and Union Chimique Belge; and participation on advisory board of AbbVie, Galapagos, Pfizer, and Union Chimique Belge. KKJ reports speakers bureaus from Bristol-Myers Squibb and Roche. All other authors declare no competing interests., (© 2022 Elsevier Ltd. All rights reserved.)

Diagnostic assays currently used to monitor the efficacy of COVID-19 vaccines measure levels of antibodies to the receptor-binding domain of ancestral SARS-CoV-2 (RBDwt). However, the predictive value for protection against new variants of concern (VOCs) has not been firmly established. Here, we used bead-based arrays and flow cytometry to measure binding of antibodies to spike proteins and receptor-binding domains (RBDs) from VOCs in 12,000 serum samples. Effects of sera on RBD-ACE2 interactions were measured as a proxy for neutralizing antibodies. The samples were obtained from healthy individuals or patients on immunosuppressive therapy who had received two to four doses of COVID-19 vaccines and from COVID-19 convalescents. The results show that anti-RBDwt titers correlate with the levels of binding- and neutralizing antibodies against the Alpha, Beta, Gamma, Delta, Epsilon and Omicron variants. The benefit of multiplexed analysis lies in the ability to measure a wide range of anti-RBD titers using a single dilution of serum for each assay. The reactivity patterns also yield an internal reference for neutralizing activity and binding antibody units per milliliter (BAU/ml). Results obtained with sera from vaccinated healthy individuals and patients confirmed and extended results from previous studies on time-dependent waning of antibody levels and effects of immunosuppressive agents. We conclude that anti-RBDwt titers correlate with levels of neutralizing antibodies against VOCs and propose that our method may be implemented to enhance the precision and throughput of immunomonitoring., (© 2022. The Author(s).)

Objectives: Humoral vaccine responses to SARS-CoV-2 vaccines are impaired and short lasting in patients with immune-mediated inflammatory diseases (IMID) following two vaccine doses. To protect these vulnerable patients against severe COVID-19 disease, a three-dose primary vaccination strategy has been implemented in many countries. The aim of this study was to evaluate humoral response and safety of primary vaccination with three doses in patients with IMID., Methods: Patients with IMID on immunosuppressive therapy and healthy controls receiving three-dose and two-dose primary SARS-CoV-2 vaccination, respectively, were included in this prospective observational cohort study. Anti-Spike antibodies were assessed 2-4 weeks, and 12 weeks following each dose. The main outcome was anti-Spike antibody levels 2-4 weeks following three doses in patients with IMID and two doses in controls. Additional outcomes were the antibody decline rate and adverse events., Results: 1100 patients and 303 controls were included. Following three-dose vaccination, patients achieved median (IQR) antibody levels of 5720 BAU/mL (2138-8732) compared with 4495 (1591-6639) in controls receiving two doses, p=0.27. Anti-Spike antibody levels increased with median 1932 BAU/mL (IQR 150-4978) after the third dose. The interval between the vaccine doses and vaccination with mRNA-1273 or a combination of vaccines were associated with antibody levels following the third dose. Antibody levels had a slower decline-rate following the third than the second vaccine dose, p&lt;0.001. Adverse events were reported by 464 (47%) patients and by 196 (78%) controls. Disease flares were reported by 70 (7%) patients., Conclusions: This study shows that additional vaccine doses to patients with IMID contribute to strong and sustained immune-responses comparable to healthy persons vaccinated twice, and supports repeated vaccination of patients with IMID., Trial Registration Number: NCT04798625., Competing Interests: Competing interests: TKK reports grants from AbbVie, Amgen, BMS MSD, Novartis, Pfizer, UCB, consulting fees from AbbVie, Biogen, Celltrion, Eli Lilly, Gilead, Mylan, Novartis, Pfizer, Sandoz, Sanofi, speakers bureaus Amgen, Celltrion, Egis, Evapharma, Ewopharma, Hikma, Oktal, Sandoz, Sanofi, LM reports funding from KG Jebsen foundation, support for infrastructure and biobanking from the university of Oslo and Oslo University Hospital, grants from the Coalition of Epidemic Preparedness Innovations CEPI, speakers bureaus Novartis, Cellgene, JTV reports grant from the Coalition of Epidemic Preparedness Innovations (CEPI), KKJ reports speakers bureaus from Roche and BMS, advisory board Celltrion and Norgine, GLG reports funding from The Karin Fossum foundation, Diakonhjemmet Hospital, Oslo University Hospital, Akershus University Hospital, Trygve Gydtfeldt og frues Foundation, South-East region Health authority, consulting fees AbbVie and Pfizer, speakers fees AbbVie, Pfizer, Sandoz, Orion Pharma, Novartis and UCB, advisory board Pfizer, AbbVie., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The durability of vaccine-induced humoral immunity against SARS-CoV-2 in patients with immune-mediated inflammatory diseases (IMIDs) on immunosuppressive therapy is not known. The aim of this study was to compare the persistence of anti-Spike antibodies following two-dose SARS-CoV-2 vaccination between IMID patients and healthy controls and to identify factors associated with antibody decline., Methods: IMID patients on immunosuppressive medication enrolled in the prospective observational Nor-vaC study were included. Participants received two-dose SARS-CoV-2 vaccination. Serum collected at two time points following vaccination (first assessment within 6-48 days, second within 49-123 days) were analyzed for antibodies binding the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. Multivariable regression models estimated percent reduction in anti-RBD over 30 days and factors associated with reduction., Results: A total of 1108 patients (403 rheumatoid arthritis, 195 psoriatic arthritis, 195 spondyloarthritis, 124 ulcerative colitis, 191 Crohn's disease) and 134 controls provided blood samples within the defined intervals (median 19 days [IQR 15-24] and 97 days [87-105] after second vaccine dose). Antibody levels were lower in patients compared to controls at both time points, with median anti-RBD 2806 BAU/ml [IQR 1018-6068] in patients and 6187 BAU/ml [4105-7496] in controls (p<0.001) at first assessment, and 608 BAU/ml [IQR 58-1053] in patients and 1520 BAU/ml [979-3766] in controls (p<0.001) at second assessment. At second assessment, low anti-RBD antibody levels (defined as <200 BAU/ml) were found in 449 (41%) patients, and 6 (5%) controls (p<0.001). The change was - 83% in patients and - 66% in controls (p<0.001). Patients had a greater estimated 30 days percent reduction in anti-RBD levels compared to controls - 4.9 (95% CI - 7.4 to - 2.4), (p<0.05). Among therapies, mono- or combination treatment with tumor necrosis factor inhibitors was associated with the greatest decline., Conclusions: Within 4 months after vaccination, antibody levels declined considerably in both IMID patients and controls. Patients had lower initial antibody levels and a more pronounced decline compared to healthy controls and were therefore more likely to decline to low antibody levels. These results support that IMID patients need additional vaccine doses at an earlier stage than healthy individuals., (© 2022. The Author(s).)

Background: Results showing that sera from double vaccinated individuals have minimal neutralizing activity against Omicron have been interpreted as indicating the need for a third vaccine dose for protection. However, there is little information about early immune responses to Omicron infection in double vaccinated individuals., Methods: We measured inflammatory mediators, antibodies to the SARS-CoV-2 spike and nucleocapsid proteins, and spike peptide-induced release of interferon gamma in whole blood in 51 double-vaccinated individuals infected with Omicron, in 14 infected with Delta, and in 18 healthy controls. The median time points for the first and second samples were 7 and 14 days after symptom onset, respectively., Findings: Infection with Omicron or Delta led to a rapid and similar increase in antibodies to the receptor-binding domain (RBD) of Omicron protein and spike peptide-induced interferon gamma in whole blood. Both the Omicron- and the Delta-infected patients had a mild and transient increase in inflammatory parameters., Interpretation: The results suggest that two vaccine doses are sufficient to mount a rapid and potent immune response upon infection in healthy individuals of with the Omicron variant., Funding: The study was funded by the Oslo University Hospital, and by grants from The Coalition for Epidemic Preparedness Innovations, Research Council of Norway (no 312780, 324272), South-Eastern Norway Regional Health Authority (no 2019067, 2021071, 10357, 2021047, 33612, 2021087, 2017092), EU Horizon 2020 grant no 848099, a philantropic donation from Vivaldi Invest A/S, and The European Virus Archive Global., Competing Interests: Author AS reports being an employee and shareholder at Age Labs outside of the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial and financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Søraas, Grødeland, Granerud, Ueland, Lind, Fevang, Murphy, Huse, Nygaard, Steffensen, al-Baldawi, Holberg-Petersen, Andresen, Ågnes, Ranheim, Schanke, Istre, Dahl, Chopra, Dudman, Kaarbø, Andersen, Vaage, Tran, Vaage, Michelsen, Müller, Aukrust, Halvorsen, Dahl, Holter and Lund-Johansen.)

Objective: Immunogenicity and safety following receipt of the standard SARS-CoV-2 vaccination regimen in patients with immune-mediated inflammatory diseases (IMIDs) are poorly characterized, and data after receipt of the third vaccine dose are lacking. The aim of the study was to evaluate serologic responses and adverse events following the standard 2-dose regimen and a third dose of SARS-CoV-2 vaccine in IMID patients receiving immunosuppressive therapy., Methods: Adult patients receiving immunosuppressive therapy for rheumatoid arthritis, spondyloarthritis, psoriatic arthritis, Crohn's disease, or ulcerative colitis, as well as healthy adult controls, who received the standard 2-dose SARS-CoV-2 vaccination regimen were included in this prospective observational study. Analyses of antibodies to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein were performed prior to and 2-4 weeks after vaccination. Patients with a weak serologic response, defined as an IgG antibody titer of ≤100 arbitrary units per milliliter (AU/ml) against the receptor-binding domain of the full-length SARS-Cov-2 spike protein, were allotted a third vaccine dose., Results: A total of 1,505 patients (91%) and 1,096 healthy controls (98%) had a serologic response to the standard regimen (P < 0.001). Anti-RBD antibody levels were lower in patients (median 619 AU/ml interquartile range [IQR] 192-4,191) than in controls (median 3,355 AU/ml [IQR 896-7,849]) (P < 0.001). The proportion of responders was lowest among patients receiving tumor necrosis factor inhibitor combination therapy, JAK inhibitors, or abatacept. Younger age and receipt of messenger RNA-1273 vaccine were predictors of serologic response. Of 153 patients who had a weak response to the standard regimen and received a third dose, 129 (84%) became responders. The vaccine safety profile among patients and controls was comparable., Conclusion: IMID patients had an attenuated response to the standard vaccination regimen as compared to healthy controls. A third vaccine dose was safe and resulted in serologic response in most patients. These data facilitate identification of patient groups at risk of an attenuated vaccine response, and they support administering a third vaccine dose to IMID patients with a weak serologic response to the standard regimen., (© 2022 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Antibodies are important for vaccine efficacy. Targeting antigens to antigen-presenting cells (APCs) increases antibody levels. Here, we explore the role of antigen valency in MHC class II (MHCII)-targeted vaccines delivered as DNA. We design heterodimeric proteins that carry either two identical (bivalent vaccines), or two different antigens (monovalent vaccines). Bivalent vaccines with two identical influenza hemagglutinins (HA) elicit higher amounts of anti-HA antibodies in mice than monovalent versions with two different HAs. Bivalent vaccines increase the levels of germinal center (GC) B cells and long-lived plasma cells. Only HA-bivalent vaccines completely protect mice against challenge with homologous influenza virus. Similar results are obtained with other antigens by targeting CD11c and Xcr1 on dendritic cells (DCs) or when administering the vaccine as protein with adjuvant. Bivalency probably increases B cell responses by cross-linking BCRs in readily observable DC-B cell synapses. These results are important for generating potent APC-targeted vaccines., Competing Interests: Declaration of interests The TTO office of the University of Oslo and Oslo University Hospital, Inven2, filed a provisional patent application No. 62/555,305 “Vaccine molecules” on A/B heterodimeric vaccine molecules. R.B. and B.B. are inventors of this patent. B.B. is head of the scientific panel of Nykode AS (previously Vaccibody AS) and holds shares in the company., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The catalytic enzymes tankyrase 1 and 2 (TNKS1/2) alter protein turnover by poly-ADP-ribosylating target proteins, which earmark them for degradation by the ubiquitin-proteasomal system. Prominent targets of the catalytic activity of TNKS1/2 include AXIN proteins, resulting in TNKS1/2 being attractive biotargets for addressing of oncogenic WNT/β-catenin signaling. Although several potent small molecules have been developed to inhibit TNKS1/2, there are currently no TNKS1/2 inhibitors available in clinical practice. The development of tankyrase inhibitors has mainly been disadvantaged by concerns over biotarget-dependent intestinal toxicity and a deficient therapeutic window. Here we show that the novel, potent, and selective 1,2,4-triazole-based TNKS1/2 inhibitor OM-153 reduces WNT/β-catenin signaling and tumor progression in COLO 320DM colon carcinoma xenografts upon oral administration of 0.33-10 mg/kg twice daily. In addition, OM-153 potentiates anti-programmed cell death protein 1 (anti-PD-1) immune checkpoint inhibition and antitumor effect in a B16-F10 mouse melanoma model. A 28-day repeated dose mouse toxicity study documents body weight loss, intestinal damage, and tubular damage in the kidney after oral-twice daily administration of 100 mg/kg. In contrast, mice treated oral-twice daily with 10 mg/kg show an intact intestinal architecture and no atypical histopathologic changes in other organs. In addition, clinical biochemistry and hematologic analyses do not identify changes indicating substantial toxicity. The results demonstrate OM-153-mediated antitumor effects and a therapeutic window in a colon carcinoma mouse model ranging from 0.33 to at least 10 mg/kg, and provide a framework for using OM-153 for further preclinical evaluations., Significance: This study uncovers the effectiveness and therapeutic window for a novel tankyrase inhibitor in mouse tumor models., Competing Interests: G. Grødeland reports personal fees from AstraZeneca, Sanofi, Bayer, Thermo Fisher, and personal fees from Norwegian System for compensation to patients outside the submitted work. S.T. Sowa reports grants from Jane and Aatos Erkko Foundation during the conduct of the study. L. Lehtiö reports a patent to WO2019/243822 issued and a patent to WO2022/008896 issued. R.G. Leenders reports a patent to WO2019/243822 issued and a patent to WO2022/008896 issued (the drug development project is 100% owned by the Oslo University Hospital and administered by the TTO Inven2; Symeres Inc. has a scientific and BD collaboration agreement with Inven2). A. Wegert reports a patent to WO2019/243822 issued and a patent to WO2022/008896 issued (the drug development project is 100% owned by the Oslo University hospital and administered by the TTO Inven2; Symeres B.V. has a scientific and BD collaboration agreement with Inven2). S. Krauss reports a patent to WO2019/243822 issued and a patent to WO2022/008896 issued (the drug development program is 100% owned by the Oslo University Hospital and administered by the TTO Inven2; Symeres Inc. has a scientific and BD collaboration agreement with Inven2 on the drug development program). J. Waaler reports a patent to WO2019/243822 issued and a patent to WO2022/008896 issued (the drug development project is 100% owned by the Oslo University Hospital and administered by the TTO Inven2). Our project collaborator Symeres Inc. has a scientific and BD collaboration agreement with Inven2. No disclosures were reported by the other authors., (© 2022 The Authors; Published by the American Association for Cancer Research.)

Background: In rituximab-treated patients with rheumatoid arthritis, humoral and cellular immune responses after two or three doses of SARS-CoV-2 vaccines are not well characterised. We aimed to address this knowledge gap., Methods: This prospective, cohort study (Nor-vaC) was done at two hospitals in Norway. For this sub-study, we enrolled patients with rheumatoid arthritis on rituximab treatment and healthy controls who received SARS-CoV-2 vaccines according to the Norwegian national vaccination programme. Patients with insufficient serological responses to two doses (antibody to the receptor-binding domain [RBD] of the SARS-CoV-2 spike protein concentration <100 arbitrary units [AU]/mL) were allotted a third vaccine dose. Antibodies to the RBD of the SARS-CoV-2 spike protein were measured in serum 2-4 weeks after the second and third doses. Vaccine-elicited T-cell responses were assessed in vitro using blood samples taken before and 7-10 days after the second dose and 3 weeks after the third dose from a subset of patients by stimulating cryopreserved peripheral blood mononuclear cells with spike protein peptides. The main outcomes were the proportions of participants with serological responses (anti-RBD antibody concentrations of ≥70 AU/mL) and T-cell responses to spike peptides following two and three doses of SARS-CoV-2 vaccines. The study is registered at ClinicalTrials.gov, NCT04798625, and is ongoing., Findings: Between Feb 9, 2021, and May 27, 2021, 90 patients were enrolled, 87 of whom donated serum and were included in our analyses (69 [79·3%] women and 18 [20·7%] men). 1114 healthy controls were included (854 [76·7%] women and 260 [23·3%] men). 49 patients were allotted a third vaccine dose. 19 (21·8%) of 87 patients, compared with 1096 (98·4%) of 1114 healthy controls, had a serological response after two doses (p<0·0001). Time since last rituximab infusion (median 267 days [IQR 222-324] in responders vs 107 days [80-152] in non-responders) and vaccine type (mRNA-1273 vs BNT162b2) were significantly associated with serological response (adjusting for age and sex). After two doses, 10 (53%) of 19 patients had CD4 + T-cell responses and 14 (74%) had CD8 + T-cell responses. A third vaccine dose induced serological responses in eight (16·3%) of 49 patients, but induced CD4 + and CD8 + T-cell responses in all patients assessed (n=12), including responses to the SARS-CoV-2 delta variant (B.1.617.2). Adverse events were reported in 32 (48%) of 67 patients and in 191 (78%) of 244 healthy controls after two doses, with the frequency not increasing after the third dose. There were no serious adverse events or deaths., Interpretation: This study provides important insight into the divergent humoral and cellular responses to two and three doses of SARS-CoV-2 vaccines in rituximab-treated patients with rheumatoid arthritis. A third vaccine dose given 6-9 months after a rituximab infusion might not induce a serological response, but could be considered to boost the cellular immune response., Funding: The Coalition for Epidemic Preparedness Innovations, Research Council of Norway Covid, the KG Jebsen Foundation, Oslo University Hospital, the University of Oslo, the South-Eastern Norway Regional Health Authority, Dr Trygve Gythfeldt og frues forskningsfond, the Karin Fossum Foundation, and the Research Foundation at Diakonhjemmet Hospital., Competing Interests: KKJ reports speakers bureaus from Roche and BMS and advisory board participation for Celltrion and Norgine. JJ reports grants from Abbvie, Pharmacosmos, and Ferring; consulting fees from Abbvie, Boerhinger Ingelheim, BMS, Celltrion, Ferring, Glihead, Janssen Cilag, MSD, Napp Pharma, Novartis, Orion Pharma, Pfeizer, Pharmacosmos, Takeda, Sandoz, and Unimedic Pharma; and speakers bureaus from Abbvie, Astro Pharma, Boerhinger Ingelheim, BMS, Celltrion, Ferring, Glihead, Hikma, Janssen Cilag, Meda, MSD, Napp Pharma, Novartis, Oriuon Pharma, Pfeizer, Pharmacosmos, Roche, Takeda, and Sandoz. TKK reports grants from AbbVie, Amgen, BMS, MSD, Novartis, Pfizer, and UCB; consulting fees from AbbVie, Amgen, Biogen, Celltrion, Eli Lilly, Gilead, Mylan, Novartis, Pfizer, Sandoz, and Sanofi; speakers bureaus from Amgen, Celltrion, Egis, Evapharma, Ewopharma, Hikma, Oktal, Sandoz, and Sanofi; and participation on a data safety monitoring board for AbbVie. LAM reports funding from the KG Jebsen foundation; support for infrastructure and biobanking from the University of Oslo and Oslo University Hospital; grants from the Coalition of Epidemic Preparedness Innovations (CEPI); and speakers bureaus from Novartis and Cellgene. GG reports consulting fees from the Norwegian System of Compensation to Patients and AstraZeneca, and speakers bureaus from Bayer, Sanofi Pasteur, and Thermo Fisher. JTV reports grant from the CEPI. FL-J reports grants from the CEPI and the South-Eastern Norway Regional Health Authority. GLG reports funding from the Karin Fossum foundation, Diakonhjemmet Hospital, Oslo University Hospital, Akershus University Hospital, the Dr Trygve Gydtfeldt og frues Foundation, and the South-Eastern Norway Regional Health Authority; consulting fees from AbbVie and Pfizer; speakers fees from AbbVie, Pfizer, Sandoz, Orion Pharma, Novartis, and UCB; and advisory board participation for Pfizer and AbbVie. All other authors declare no competing interests., (© 2021 Elsevier Ltd. All rights reserved.)

(1) Influenza viruses constantly change and evade prior immune responses, forcing seasonal re-vaccinations with updated vaccines. Current FDA-approved vaccine manufacturing technologies are too slow and/or expensive to quickly adapt to mid-season changes in the virus or to the emergence of pandemic strains. Therefore, cost-effective vaccine technologies that can quickly adapt to newly emerged strains are desirable. (2) The filamentous fungal host Thermothelomyces heterothallica C1 (C1, formerly Myceliophthora thermophila ) offers a highly efficient and cost-effective alternative to reliably produce immunogens of vaccine quality at large scale. (3) We showed the utility of the C1 system expressing hemagglutinin (HA) and a HA fusion protein from different H1N1 influenza A virus strains. Mice vaccinated with the C1-derived HA proteins elicited anti-HA immune responses similar, or stronger than mice vaccinated with HA products derived from prototypical expression systems. A challenge study demonstrated that vaccinated mice were protected against the aggressive homologous viral challenge. (4) The C1 expression system is proposed as part of a set of protein expression systems for plug-and-play vaccine manufacturing platforms. Upon the emergence of pathogens of concern these platforms could serve as a quick solution for producing enough vaccines for immunizing the world population in a much shorter time and more affordably than is possible with current platforms.

Background: There is an urgent need for new antivirals with powerful therapeutic potential and tolerable side effects., Methods: Here, we tested the antiviral properties of interferons (IFNs), alone and with other drugs in vitro., Results: While IFNs alone were insufficient to completely abolish replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), IFNα, in combination with remdesivir, EIDD-2801, camostat, cycloheximide, or convalescent serum, proved to be more effective. Transcriptome and metabolomic analyses revealed that the IFNα-remdesivir combination suppressed SARS-CoV-2-mediated changes in Calu-3 cells and lung organoids, although it altered the homeostasis of uninfected cells and organoids. We also demonstrated that IFNα combinations with sofosbuvir, telaprevir, NITD008, ribavirin, pimodivir, or lamivudine were effective against HCV, HEV, FLuAV, or HIV at lower concentrations, compared to monotherapies., Conclusions: Altogether, our results indicated that IFNα can be combined with drugs that affect viral RNA transcription, protein synthesis, and processing to make synergistic combinations that can be attractive targets for further pre-clinical and clinical development against emerging and re-emerging viral infections.

The highly pathogenic avian influenza H5N1 viruses constantly evolve and give rise to novel variants that have caused widespread zoonotic outbreaks and sporadic human infections. Therefore, vaccines capable of eliciting broadly protective antibody responses are desired and under development. We here investigated the magnitude, kinetics and protective efficacy of the multi-faceted humoral immunity induced by vaccination in healthy adult volunteers with a Matrix M adjuvanted virosomal H5N1 vaccine. Vaccinees were given escalating doses of adjuvanted vaccine (1.5μg, 7.5μg, or 30μg), or a non-adjuvanted vaccine (30μg). An evaluation of sera from vaccinees against pseudotyped viruses covering all (sub)clades isolated from human H5N1 infections demonstrated that the adjuvanted vaccines (7.5μg and 30μg) could elicit rapid and robust increases of broadly cross-neutralizing antibodies against all clades. In addition, the adjuvanted vaccines also induced multifaceted antibody responses including hemagglutinin stalk domain specific, neuraminidase inhibiting, and antibody-dependent cellular cytotoxicity inducing antibodies. The lower adjuvanted dose (1.5µg) showed delayed kinetics, whilst the non-adjuvanted vaccine induced overall lower levels of antibody responses. Importantly, we demonstrate that human sera post vaccination with the adjuvanted (30μg) vaccine provided full protection against a lethal homologous virus challenge in mice. Of note, when combining our data from mice and humans we identified the neutralizing and neuraminidase inhibiting antibody titers as correlates of in vivo protection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhou, Hansen, Pedersen, Grødeland and Cox.)

The 2009 "swine flu" pandemic outbreak demonstrated the limiting capacity for egg-based vaccines with respect to global vaccine supply within a timely fashion. New vaccine platforms that efficiently can quench pandemic influenza emergences are urgently needed. Since 2009, there has been a profound development of new vaccine platform technologies with respect to prophylactic use in the population, including DNA vaccines. These vaccines are particularly well suited for global pandemic responses as the DNA format is temperature stable and the production process is cheap and rapid. Here, we show that by targeting influenza antigens directly to antigen presenting cells (APC), DNA vaccine efficacy equals that of conventional technologies. A single dose of naked DNA encoding hemagglutinin (HA) from influenza/A/California/2009 (H1N1), linked to a targeting moiety directing the vaccine to major histocompatibility complex class II (MHCII) molecules, raised similar humoral immune responses as the adjuvanted split virion vaccine Pandemrix, widely administered in the 2009 pandemic. Both vaccine formats rapidly induced serum antibodies that could protect mice already 8 days after a single immunization, in contrast to the slower kinetics of a seasonal trivalent inactivated influenza vaccine (TIV). Importantly, the DNA vaccine also elicited cytotoxic T-cell responses that reduced morbidity after vaccination, in contrast to very limited T-cell responses seen after immunization with Pandemrix and TIV. These data demonstrate that DNA vaccines has the potential as a single dose platform vaccine, with rapid protective effects without the need for adjuvant, and confirms the relevance of naked DNA vaccines as candidates for pandemic preparedness., Competing Interests: GG and BB are inventors on a patent applications filed on an HLAII-specific targeting moiety according to institutional rules through the TTO offices of the University of Oslo and Oslo University Hospital. Further, BB is inventor of the core patent of Vaccibody AS, and hold shares in the company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Andersen, Bodin, Oftung, Bogen, Mjaaland and Grødeland.)

SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here, we address these challenges by combining Pegasys (IFNα) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNα and that both Serpin E1 and nafamostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world.

Novel and more broadly protective vaccines against influenza are needed to efficiently meet antigenic drift and shift. Relevant to this end, the stem domain of hemagglutinin (HA) is highly conserved, and antibodies specific for epitopes located to the stem have been demonstrated to be able to confer broad protection against various influenza subtypes. However, a remaining challenge is to induce antibodies against the poorly immunogenic stem by vaccination strategies that can be scaled up for prophylactic vaccination of the general population. Here, we have developed DNA vaccines where the conserved stem domain of HA from influenza A/PR/8/34 (H1N1) and A/Shanghai/2/2013 (H7N9) was targeted toward MHC class II molecules on antigen-presenting cells (APC) for increased immunogenicity. Each of these vaccines induced antibodies that cross-reacted with other subtypes in the corresponding phylogenetic influenza groups. Importantly, when mixing the MHCII-targeted stem domains from H1N1 and H7N9 influenza viruses into one vaccine bolus, we observed broad protection against candidate stains from both phylogenetic groups 1 and 2., (Copyright © 2020 Grødeland, Baranowska-Hustad, Abadejos, Blane, Teijaro, Nemazee and Bogen.)

Enhancing the germinal center (GC) reaction is a prime objective in vaccine development. Targeting of antigen to MHCII on APCs has previously been shown to increase antibody responses, but the underlying mechanism has been unclear. We have here investigated the GC reaction after targeting antigen to MHCII in (i) a defined model with T and B cells of known specificity using adjuvant-free vaccine proteins, and (ii) an infectious disease model using a DNA vaccine. MHCII-targeting enhanced presentation of peptide: MHCII on APCs, and increased the numbers of GC B cells, T FH , and plasma cells. Antibodies appeared earlier and levels were increased. BCR of GC B cells and serum antibodies had increased avidity for antigen. The improved responses required cross-linking of BCR and MHCII in either cis or trans . The enhanced GC reaction induced by MHCII-targeting of antigen has clear implications for design of more efficient subunit vaccines., Competing Interests: B.B. and G.G. are inventors on (1) patents (B.B.) and (2) patent applications (G.G., B.B.) on the described vaccine molecules, filed by the TTO office of the University of Oslo and Oslo University Hospital, according to institutional rules. (1) Patent applicant: Inven2/Vaccibody AS. Inventors: Bjarne Bogen, Agnete B. Fredriksen, Inger Sandlie. Application number: European patent No. 1599504. Status of Application: granted in EU and the US. Specific aspect of manuscript covered in patent Application: Homodimeric modular constructs targeting antigens to antigen presenting cells. (2) Patent applicant: Inven2. Inventors: Bjarne Bogen, Gunnveig Grødeland, Agnete B. Fredriksen. Application number: WO2017/221072. Status of Application: National Phase from Dec 20, 2018. Specific aspect of manuscript covered in patent Application: A human version of the MHCII-specific targeting moiety. BB is head of the Scientific panel of Vaccibody AS, and holds shares in the company.

Conventional influenza vaccines are hampered by slow and limited production capabilities, whereas DNA vaccines can be rapidly produced for global coverage in the event of an emerging pandemic. However, a drawback of DNA vaccines is their generally low immunogenicity in non-human primates and humans. We have previously demonstrated that targeting of influenza hemagglutinin to human HLA class II molecules can increase antibody responses in larger animals such as ferrets and pigs. Here, we extend these observations by immunizing non-human primates (rhesus macaques) with a DNA vaccine encoding a bivalent fusion protein that targets influenza virus hemagglutinin (HA) to Mamu class II molecules. Such immunization induced neutralizing antibodies and antigen-specific T cells. The DNA was delivered by pain- and needle-free jet injections intradermally. No adverse effects were observed. Most importantly, the immunized rhesus macaques were protected against a challenge with influenza virus., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Upon APC-targeted DNA vaccination, transfected cells secrete fusion proteins with targeting units specific for surface molecules on APC. In this study, we have tested several different targeting units for their ability to influence the magnitude and subclass of Ab responses to hemagglutinin from influenza A virus. The experiments employed bivalent homodimeric Ig-based molecules (vaccibodies). The overall efficiency in BALB/c mice depended on the targeting units in the following order: αMHC class II > αCD11c > αCD40 > Xcl-1 = MIP-1α > FliC > GM-CSF > Flt-3L > αDEC205. GM-CSF induced mainly IgG1, whereas Xcl1, MIP-1α, αCD40, and αDEC205 induced predominantly IgG2a. A more balanced mixture of IgG1 and IgG2a was observed with αCD11c, αMHC class II, Flt-3L, and FliC. Similar results of IgG subclass-skewing were obtained in Th1-prone C57BL/6 mice with a more limited panel of vaccines. IgG1 responses in BALB/c occurred early after immunization but declined relatively rapidly over time. IgG2a responses appeared later but lasted longer (>252 d) than IgG1 responses. The most efficient targeting units elicited short- and long-term protection against PR8 influenza (H1N1) virus in BALB/c mice. The results suggest that targeting of Xcr1 + conventional type 1 dendritic cells preferentially induces IgG2a responses, whereas simultaneous targeting of several dendritic cell subtypes also induces IgG1 responses. The induction of distinct subclass profiles by different surface molecules supports the APC-B cell synapse hypothesis. The results may contribute to generation of more potent DNA vaccines that elicit high levels of Abs with desired biologic effector functions., (Copyright © 2018 The Authors.)

Zoonotic influenza H7 viral infections have a case fatality rate of about 40%. Currently, no or limited human to human spread has occurred, but we may be facing a severe pandemic threat if the virus acquires the ability to transmit between humans. Novel vaccines that can be rapidly produced for global distribution are urgently needed, and DNA vaccines may be the only type of vaccine that allows for the speed necessary to quench an emerging pandemic. Here, we constructed DNA vaccines encoding the hemagglutinin (HA) from influenza A/chicken/Italy/13474/99 (H7N1). In order to increase the efficacy of DNA vaccination, HA was targeted to either major histocompatibility complex class II molecules or chemokine receptors 1, 3, and 5 (CCR1/3/5) that are expressed on antigen-presenting cells (APC). A single DNA vaccination with APC-targeted HA significantly increased antibody levels in sera compared to nontargeted control vaccines. The antibodies were confirmed neutralizing in an H7 pseudotype-based neutralization assay. Furthermore, the APC-targeted vaccines increased the levels of antigen-specific cytotoxic T cells, and a single DNA vaccination could confer protection against a lethal challenge with influenza A/turkey/Italy/3889/1999 (H7N1) in mice. In conclusion, we have developed a vaccine that rapidly could contribute protection against a pandemic threat from avian influenza. IMPORTANCE Highly pathogenic avian influenza H7 constitute a pandemic threat that can cause severe illness and death in infected individuals. Vaccination is the main method of prophylaxis against influenza, but current vaccine strategies fall short in a pandemic situation due to a prolonged production time and insufficient production capabilities. In contrast, a DNA vaccine can be rapidly produced and deployed to prevent the potential escalation of a highly pathogenic influenza pandemic. We here demonstrate that a single DNA delivery of hemagglutinin from an H7 influenza could mediate full protection against a lethal challenge with H7N1 influenza in mice. Vaccine efficacy was contingent on targeting of the secreted vaccine protein to antigen-presenting cells., (Copyright © 2017 Andersen et al.)

Vaccination is at present the most efficient way of preventing influenza infections. Currently used inactivated influenza vaccines can induce virus-neutralizing antibodies that are protective against a particular influenza strain, but hamper the induction of cross-protective T-cell responses to later infections. Thus, influenza vaccines need to be updated annually in order to confer protection against circulating influenza strains. This study aims at developing an efficient vaccine that can induce broader protection against influenza. For this purpose, we have used the highly conserved nucleoprotein (NP) from an influenza A virus subtype H7N7 strain, and inserted it into a vaccine format that targets an antigen directly to relevant antigen presenting cells (APCs). The vaccine format consists of bivalent antigenic and targeting units, linked via an Ig-based dimerization unit. In this study, NP was linked to MIP-1α, a chemokine that targets the linked antigen to chemokine receptors 1, 3 and 5 expressed on various APCs. The vaccine protein was indirectly delivered by DNA. Mice were vaccinated intradermally with plasmids, in combination with electroporation to enhance cellular uptake of DNA. We found that a single DNA vaccination was sufficient for induction of both antibody and T cell responses in BALB/c mice. Targeting of nucleoprotein to chemokine receptors enhanced T cell responses but not antibody responses. Moreover, a single dose of MIP1α-NP conferred protection in BALB/c mice against a lethal challenge with an H1N1 influenza virus. The observed cross-protection was mediated by CD8(+) T cells., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Current influenza vaccines mostly aim at the induction of specific neutralizing antibodies. While antibodies are important for protection against a particular virus strain, T cells can recognize epitopes that will offer broader protection against influenza. We have previously developed a DNA vaccine format by which protein antigens can be targeted specifically to receptors on antigen presenting cells (APCs). The DNA-encoded vaccine proteins are homodimers, each chain consisting of a targeting unit, a dimerization unit, and an antigen. The strategy of targeting antigen to APCs greatly enhances immune responses as compared to non-targeted controls. Furthermore, targeting of antigen to different receptors on APCs can polarize the immune response to different arms of immunity. Here, we discuss how targeting of hemagglutinin to MHC class II molecules increases Th2 and IgG1 antibody responses, whereas targeting to chemokine receptors XCR1 or CCR1/3/5 increases Th1 and IgG2a responses, in addition to CD8(+) T cell responses. We also discuss these results in relation to work published by others on APC-targeting. Differential targeting of APC surface molecules may allow the induction of tailor-made phenotypes of adaptive immune responses that are optimal for protection against various infectious agents, including influenza virus.

There are two major limitations to vaccine preparedness in the event of devastating influenza pandemics: the time needed to generate a vaccine and rapid generation of sufficient amounts. DNA vaccination could represent a solution to these problems, but efficacy needs to be enhanced. In a separate line of research, it has been established that targeting of vaccine molecules to antigen-presenting cells enhances immune responses. We have combined the two principles by constructing DNA vaccines that encode bivalent fusion proteins; these target hemagglutinin to MHC class II molecules on antigen-presenting cells. Such DNA vaccines rapidly induce hemagglutinin-specific antibodies and T cell responses in immunized mice. Responses are long-lasting and protect mice against challenge with influenza virus. In a pandemic situation, targeted DNA vaccines could be produced and tested within a month. The novel DNA vaccines could represent a solution to pandemic preparedness in the advent of novel influenza pandemics.

Targeting antigens to cross-presenting dendritic cells (DCs) is a promising method for enhancing CD8(+) T-cell responses. However, expression patterns of surface receptors often vary between species, making it difficult to relate observations in mice to other animals. Recent studies have indicated that the chemokine receptor Xcr1 is selectively expressed on cross-presenting murine CD8α(+) DCs, and that the expression is conserved on homologous DC subsets in humans (CD141(+) DCs), sheep (CD26(+) DCs), and macaques (CADM1(+) DCs). We therefore tested if targeting antigens to Xcr1 on cross-presenting DCs using antigen fused to Xcl1, the only known ligand for Xcr1, could enhance immune responses. Bivalent Xcl1 fused to model antigens specifically bound CD8α(+) DCs and increased proliferation of antigen-specific T cells. DNA vaccines encoding dimeric Xcl1-hemagglutinin (HA) fusion proteins induced cytotoxic CD8(+) T-cell responses, and mediated full protection against a lethal challenge with influenza A virus. In addition to enhanced CD8(+) T-cell responses, targeting of antigen to Xcr1 induced CD4(+) Th1 responses and highly selective production of IgG2a antibodies. In conclusion, targeting of dimeric fusion vaccine molecules to CD8α(+) DCs using Xcl1 represents a novel and promising method for induction of protective CD8(+) T-cell responses., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Different diseases require different immune responses for efficient protection. Thus, prophylactic vaccines should prime the immune system for the particular type of response needed for protection against a given infectious agent. We have here tested fusion DNA vaccines which encode proteins that bivalently target influenza hemagglutinins (HA) to different surface molecules on antigen presenting cells (APC). We demonstrate that targeting to MHC class II molecules predominantly induced an antibody/Th2 response, whereas targeting to CCR1/3/5 predominantly induced a CD8(+)/Th1 T cell response. With respect to antibodies, the polarizing effect was even more pronounced upon intramuscular (i.m) delivery as compared to intradermal (i.d.) vaccination. Despite these differences in induced immune responses, both vaccines protected against a viral challenge with influenza H1N1. Substitution of HA with ovalbumin (OVA) demonstrated that polarization of immune responses, as a consequence of APC targeting specificity, could be extended to other antigens. Taken together, the results demonstrate that vaccination can be tailor-made to induce a particular phenotype of adaptive immune responses by specifically targeting different surface molecules on APCs.

Efficacy of DNA vaccination has been improved in mice by fusion vaccines targeting antigen to antigen-presenting cells (APC) via chemokine receptors. Here, we aimed at extending this strategy to large animals and humans. Fusion proteins equipped with human MIP1α (LD78β isoform) retained functional activity and conformational correctness of targeting and antigenic units, respectively. Fusion proteins improved responses of cloned human CD4+ T cells, and a two amino acid NH(2)-truncated version of LD78β outperformed full length LD78β-vaccine proteins in vitro. LD78β DNA fusion vaccines induced improved T cell (both CD4+ and CD8+) and antibody responses in mice following plasmid injection and skin electroporation. Finally, LD78β-vaccine proteins bound Rhesus macaque CCR5, setting the stage for targeted DNA immunization in non-human primates., (Copyright © 2010 Elsevier Ltd. All rights reserved.)