A Randomised, Double-blind, Placebo-controlled Phase IIb Trial to Test FLU-v Vaccine

A Randomised, Double-blind, Placebo-controlled, Single-centre Phase IIb Trial as Part of the EU-funded UNISEC Project to Assess the Immunogenicity and Safety of Different Formulations and Dosing Regimens of FLU-v Vaccine in Healthy Adults

Seventh Framework Programme, University of Groningen, University Medical Center Groningen, Robert Koch Institut, Norwegian Institute of Public Health

FLU-v is a vaccine that aims to protect against a wide range of flu viruses. The purpose of this study is to measure the immune responses induced by FLU-v vaccine. This study will look at how safe FLU-v is when administered and how successful it is in preventing flu or reducing the severity of the flu symptoms. The study requires 222 healthy volunteers 18-60 years old. Participation in the study will take a maximum of 7 months and consists of 5 visits. During visit 1, subjects will be examined by a doctor to make sure they are eligible to enter the study. A 15ml blood sample (a tablespoon) will be taken to check general health followed by a general physical exam. Medical history and some personal information will be collected. Subjects that have received the traditional flu vaccine in the past 6 months, and those females who are pregnant or breastfeeding will not be allowed in the study. Subjects of childbearing age must agree to use effective contraceptive methods. At visit 2, subjects will be randomly allocated to one of the four treatment groups summarised below: Treatment 1: FLU-v (test vaccine) at the start of the study (Day 0) and then again 21 days later Treatment 2: FLU-v (test vaccine) with an additional substance added [known as Montanide ISA 51] which improves the effect of the test vaccine. Injection will be given on Day 0 and then Placebo (no test vaccine) alone 21 days later Treatment 3: Placebo (no test vaccine) injection on Day 0 and then 21 days later Treatment 4: Placebo (no test vaccine) with an additional substance added [known as Montanide ISA 51] on Day 0 and then Placebo (no test vaccine) alone 21 days later Treatment will be injected under the skin in the upper arm on day 0 (visit 2) and 21 days later (visit 3). Blood samples will be taken before treatment (day 0), and on days 42 (visit 4) and 180 (visit 5) to the immune responses induced by the vaccine. Subjects will be asked to complete a diary card to write down any side effects that they may experience after vaccination. Subjects will also be asked to complete another diary card to document any flu-like symptoms experienced between December 2016 and March 2017, this time is officially considered as the flu season. During this period, if the subject experiences flu-like symptoms, a collection of a nose and tonsil swab will be arranged by the study site to confirm whether they have the flu or not.

Rationale: Current seasonal influenza vaccines mainly induce immune responses against viral membrane glycoproteins. These proteins, however, undergo continuous mutations by a process called antigenic drift. To prevent immune escape, annual vaccination with the latest predicted viral strains is adopted. Such vaccination strategy not only poses inconvenience and cost-inefficiency, but also results in poor protective effectiveness when the vaccinated strains are mismatched with the actual circulating strains. The latter point is especially of concern during a pandemic outbreak, when a large geographical area is affected and the general population is naïve to the newly re-assorted viral strain due to antigenic shift. Objective: To evaluate the safety and immunogenicity of the influenza vaccine (FLU-v, as a suspension or adjuvanted as emulsion) targeting conserved immunogenic regions of influenza A and B viruses in healthy adults, in particular to show that the TH1 cytokine response at 42 and 180 days after the first injection is greater in the adjuvanted FLU-v and unadjuvanted FLU-v than in the placebo. Study design: A total of 222 study participants will be recruited. The study follows a factorial design where the two factors are treatment (FLU-v / placebo) and formulation (unadjuvanted / suspension, adjuvanted / emulsion). Subjects will be randomised in two strata (age 18 to 40, age 41 to 60) to one of the following treatment regimens: Group 1 (n=74): FLU-v (unadjuvanted) as a suspension in pH neutral HCl/NaOH (0.5mL) on Day 0 and Day 21 Group 2 (n=74): (0.5mL) ISA-51-adjuvanted FLU-v emulsified in water for injection (WFI) on Day 0, saline (0.5mL) on Day 21 Group 3 (n=37): saline solution (0.5mL ) on Day 0 and Day 21 Group 4 (n=37): WFI and ISA-51 emulsion (0.5mL) on Day 0, saline (0.5mL) on Day 21 Each administration will be given subcutaneously. Solicited and unsolicited adverse events (AEs) will be collected by AE questionnaire/diary card until day 42. Adverse events (AEs) and serious adverse events (SAEs) will be collected for the entire study period. The treatments will be administered starting in third quarter of 2016 in order to provide protection for the subsequent influenza season starting in December 2016. Blood samples will be taken from all subjects on day 0 (before FLU-v vaccination), 42 (21 days after the second dosing) and 180 (159 days after the second dosing) for the evaluation of FLU-v-specific cellular and humoral immune responses. Clinical symptom scores to ascertain severity and the incidence of RT-PCR-confirmed influenza A and/or B infection will be recorded during the subsequent influenza season (December 2016 to March 2017) to decide clinical efficacy of the tested vaccines. Study population: Healthy volunteers aged 18-60 years. Intervention: FLU-v investigational influenza vaccine lyophilised product containing 500 micrograms of total peptides reconstituted in either 0.01M HCl (0.25ml) and 0.01M NaOH (0.25ml) to achieve a volume of 0.5ml, or emulsified in WFI (water for injection, 0.25ml) and ISA-51 (0.25ml) to achieve a volume of 0.5ml. Primary study parameters/endpoints: For immunogenicity: To compare the change from baseline (Day 0) between treatments in cellular immune responses, specifically TH1 cytokines, in all groups 42 and 180 days following FLU-v vaccination. For safety: (1) To evaluate the solicited AEs in all subjects until 21 days after the last dosing of the study vaccine (FLU-v); (2) To evaluate the unsolicited AEs and SAEs in all subjects for the entire study period after the first dosing of FLU-v. Secondary study parameters/endpoints: To evaluate the humoral immune responses specific to FLU-v and TH2 cytokines from baseline in all groups 42 and 180 days following FLU-v vaccination. Exploratory study parameters/endpoints: For immunogenicity: To evaluate the change from baseline in cellular immune responses based on additional CMI assays such as ELISPOT (Enzyme-Linked ImmunoSpot) in all groups at 42 and 180 days following FLU-v vaccination, in a subset of subjects chosen at random. Clinical efficacy: (1) To evaluate the efficacy of FLU-v vaccine in reducing the incidence of RT-PCR confirmed influenza A and/or B infections in all subjects during the influenza season 2016-2017. (2) To evaluate the efficacy of FLU-v vaccine in the reduction of symptom score among RT-PCR confirmed influenza A and/or B infection cases during the influenza season 2016-2017. The relationship between efficacy and cellular and humoral response will be explored if possible. The effect of previous influenza vaccination on the immunogenicity of FLU-v will be assessed in a post-hoc exploratory analysis after stratification of the data based on exposure to the influenza vaccine in the previous 24 months or over. Nature and extent of the burden and risks associated with participation, benefit and group relatedness: The intended application of the IMP is as a prophylactic vaccine to prevent influenza virus infection by inducing an enhanced influenza-specific immune response. The FLU-v vaccine is designed to be delivered either as a naturally particulate suspension (i.e. no adjuvant) or emulsified in adjuvant. Particulate and emulsified protein preparations are preferentially taken up by phagocytic cells (e.g. dendritic cells) responsible for inducing primary immune responses. Treatment with FLU-v of up to four doses of 263 μg/occasion or up to two doses of 553 μg/occasion, with or without adjuvant, was well tolerated in animals with no signs of systemic toxicity. In pre-clinical studies, subcutaneous administration of ISA 51 resulted in no systemic toxicity. At the injection sites and occasionally in adjacent tissue the injected material remained surrounded by a mild chronic inflammatory response. As the IMP is provided in one presentation (500 μg of the combined peptide) in lyophilized form for suspension in a sealed single-use vial, overdose is highly unlikely. In a single centre, randomised, double blind phase I study of the safety, tolerability and immunogenicity of FLU-v no safety or tolerability concerns were identified at the doses administered (250 μg and 500 μg FLU-v) to the subjects in this clinical study and no safety or tolerability concerns were identified following administration of the adjuvant to the subjects (1). FLU-v vaccine candidate was demonstrated to be immunogenic in humans, as measured by ex vivo γ-interferon production (1). Clinical experience with Montanide ISA 51 dates back to the 1990s and most trials were related to cancer and Acquired Immune Deficiency Syndrome (AIDS). Currently, cancer trials in melanoma, colorectal, prostate, cervical, brain cancer and leukemia are ongoing. Frequency of vaccination is often between 2 and 4 weeks, and the number of injections can reach up to 40. Route of immunization is most often subcutaneous and volume of injection can reach up to 3mL. Most common local reactions are local pain, tenderness, erythema and granuloma at the injection site. Less frequently, mild to moderate transient indurations and swelling are described. General reactions are mainly 'flu-like symptoms such as chills, fever and headaches. Lethargy and nausea are also observed. The intensity is usually mild or moderate. No biological changes are generally observed. As the Placebo vaccine is provided in a sealed single-use vial, overdose is highly unlikely (Influenza virus (FLU-v) vaccine Investigator's Brochure, Edition 2.0, 07 September 2015).

Subcutaneous injection in the upper arm with 500 ug of FLU-v as 0.5ml suspension in 0.01M HCl and 0.01M NaOH

Subcutaneous injection in the upper arm with 500ug of FLU-v emulsified in 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection

Subcutaneous injection in the upper arm with an emulsion made with 0.25ml of Montanide ISA-51 adjuvant (Seppic, France) and 0.25ml of water for injection

Comparison of the TH1 response in the treatment arms compared to placebo from baseline to day 42 following vaccination, calculated as the median fold change in the number of CD4+ and CD8+ T cells positive for IFN-gamma, TNF-alpha, IL-2 and CD107a. Fold change is calculated as the number of cells on day 42 divided by number of cells on day 0.

Comparison of the TH1 response in the treatment arms compared to placebo from baseline to day 180 following vaccination, calculated as the median fold change in the number of CD4+ and CD8+ T cells positive for IFN-gamma, TNF-alpha, IL-2 and CD107a. Fold change is calculated as the number of cells on day 180 divided by number of cells on day 0.

Percentage of TH1 Cytokine Responders (Responders Defined as Those With >2 Fold Median Increase From Baseline in CD4+ and CD8+ T-cells Positive for Particular Cytokine)

To compare the number of subjects that showed at least a two-fold increase on day 42 and day 180 following vaccination in the number of CD4+and CD8+ T-cells secreting TH1 cytokines in all groups.

Percentage of CD4+ and CD8+ TH1 Cytokine Responders Determined by Mixture Models for Single-cell Assays (MIMOSA)

To compare the number of subjects identified as responders for cytokine markers as determined by MIMOSA analysis (Responders based on a false discovery rate derived P-value <0.05).

Median fold change in IFN-gamma secretion from PBMCs stimulated in vitro with FLU-v antigens. IFN-gamma secretion was measured by ELISA.Fold change was measured as secretion on day 42 divided by secretion on day 0, and secretion on day 180 divided by secretion on day 0.

Responders were defined as subjects having at least a two-fold increase in the amount of IFNg secreted on day 42 and day 180 compared the amount secreted on day 0. IFNg was measured by ELISA

To evaluate the IgG levels as a measure of antibody responses to FLU-v on day 0 (baseline) and on days 42 and 180 following FLU-v vaccination. IgG antibodies were measured by ELISA. The geometric mean for each treatment group was provided.

To evaluate the level of TH2 cytokines (IL-4) from baseline in all groups 42 and 180 days following FLU-v vaccination.

From the start of the vacciantion until study completion for each subject, approximately no more than 7 months

During the influenza season (Dec 2016 to March 2017), fully vaccinated subjects will contact the trial center immediately if they feel unwell for 24h, with a sudden onset of flu-like symptoms. The medical staff will arrange for a nasopharyngeal swab to be performed if the subject has at least one respiratory (cough, sore throat, shortness of breath, runny nose, stuffy nose, sneezing and earache) and one systemic symptom (fever, malaise, headache and myalgia (muscle and joint pain). Swabs should be taken from the reported subjects within 3 days from the trial center being contacted or within 4 days of the onset of symptoms, whatever time is shorter.

Subjects recorded daily influenza symptoms from December 2016 up to March 2017. Subjects with a sudden onset of at least one respiratory and one systemic symptom were swabbed. If the results were positive for influenza then the symptoms were included in the analysis. The duration of symptoms was recorded as the number of symptomatic days during the influenza episode. Fever (≥38oC), malaise, headache, myalgia (muscle and joint pain), cough, sore throat, shortness of breath, runny nose, stuffy nose, sneezing and earache were scored on a severity scale of 0 to 3 (0: no symptom, 1: mild, 2: moderate, 3: severe). The daily severity score was the sum of the severity score for all symptoms listed above on a single day. The total score was the sum of all daily scores during the influenza episode. The peak score was the highest daily score during the influenza episode. The average score was the total score divided by the number of days the influenza episode lasted.

Subjects recorded daily influenza symptoms from December 2016 up to March 2017. Subjects with a sudden onset of at least one respiratory and one systemic symptom were swabbed. If the results were positive for influenza then the symptoms were included in the analysis. The duration of symptoms was recorded as the number of symptomatic days during the influenza episode. The following symptoms were recorded: fever (≥38oC), malaise, headache, myalgia (muscle and joint pain), cough, sore throat, shortness of breath, runny nose, stuffy nose, sneezing and earache.

Inclusion Criteria: Healthy males or healthy non-pregnant females (as indicated by a negative blood pregnancy test done during the screening visit) between the ages of 18 and 60 years, inclusive; Women of childbearing potential (not surgically sterile or postmenopausal for greater than or equal to one year) and men must agree to practice appropriate contraception (a combination of barrier and hormonal methods for women and a condom for men) from screening and until at least 30 days (up to Study Day 51 for females) and 90 days (up to Study Day 111 for males), after the last vaccination. A subject is in good health, as determined by a comprehensive clinical assessment {vital signs (heart rate, blood pressure, oral temperature)}, blood chemistry test (electrolytes, renal/kidney function, liver function, C-reactive protein, complete blood count), medical history, general physical examination, self-reported illness} and the clinical judgment of the investigator; Able to understand and comply with planned study procedures; Provides signed informed consent form Exclusion Criteria: Has a known allergy to any of the components of the vaccine. Has a history of severe reaction following immunization. Persons with immune deficiency/disorder, whether due to genetic defect, immunodeficiency disease, or immunosuppressive therapy. Women who have a positive pregnancy test during the screening visit or who are breastfeeding. Has a history of any of the following (reported by subjects): Acute disseminated encephalomyelitis (ADEM); Neoplastic disease - current or previous; Asthma or severe allergic disease; Bleeding disorders Chronic Hepatitis B and/or C infection; Chronic liver disease; Diabetes mellitus; Guillain-Barré syndrome; HIV; Rheumatoid arthritis or other autoimmune diseases; Severe renal disease; Transplant recipients; Unstable or progressive neurological disorders. Receipt of medicines/treatments that may affect evaluation of immunogenicity such as: Oral or parenteral steroids, high-dose inhaled steroids (greater than 800 micrograms/day of beclomethasone dipropionate or equivalent) or other immunosuppressive or cytotoxic drugs (azathioprine (Imuran), cyclosporine (Neoral, Sandimmune, SangCya); monoclonal antibodies such as basiliximab (Simulect), daclizumab (Zinbryta), infliximab (Remicade), rituximab (MabThera), alemtuzumab (Campath and Lemtrada), omalizumab (Xolair), abatacept (Orencia), adalimumab (Humira and Exemptia) and etanercept (Enbrel)basiliximab (Simulect), daclizumab (Zenapax), and muromonab (Orthoclone OKT3); corticosteroids such as prednisone (Deltasone, Orasone); tacrolimus (Prograf, Advagraf, Protopic); Glatiramer acetate (Copaxone); Mycopehnolate (Cellcept); Sirolimus (Rapamune); (within 6 months of vaccination in this study) Immunoglobulin or other blood products (plasma, blood cells, coagulation factors, haemoglobin)(within 3 months of vaccination in this study); An experimental agent (vaccine, drug, biologic, device, blood product, or medication) within 1 month of vaccination in this study, or expects to receive an experimental agent (during the study period). Influenza antiviral medication (Amantadine (Symmetrel); Rimantadine (Flumadine); Zanamivir (Relenza), Oseltamivir (Tamiflu) (within 4 weeks of vaccination in this study). Has received any influenza vaccine within 6 months of vaccination in this study. Has influenza-like illness (a sudden onset of symptoms and at least one of the four systemic symptoms-fever or feverishness, malaise, headache, myalgia and at least one of the three respiratory symptoms-cough, sore throat, shortness of breath) or acute respiratory infection (a sudden onset of symptoms and at least one of the four respiratory symptoms-cough, sore throat, shortness of breath, coryza (Rhinitis) and a clinician's judgement that the illness is due to an infection) within 6 months prior to vaccination in this study. These symptoms must have stopped the subject from carrying out their normal daily activities such as attending work or school for a period of at least 3 days. Has an acute illness, including an oral temperature greater than 38 degrees Celsius, within 1 week of vaccination. Has a history of alcohol or drug abuse within the last 2 years deemed unsuitable for inclusion by the investigator. Any abnormal haematology values and/or serum chemistries judged by the Investigator as clinically significant.

Pleguezuelos O, Robinson S, Fernandez A, Stoloff GA, Caparros-Wanderley W. Meta-Analysis and Potential Role of Preexisting Heterosubtypic Cellular Immunity Based on Variations in Disease Severity Outcomes for Influenza Live Viral Challenges in Humans. Clin Vaccine Immunol. 2015 Aug;22(8):949-56. doi: 10.1128/CVI.00101-15. Epub 2015 Jun 17.

Pleguezuelos O, Robinson S, Fernandez A, Stoloff GA, Mann A, Gilbert A, Balaratnam G, Wilkinson T, Lambkin-Williams R, Oxford J, Caparros-Wanderley W. A Synthetic Influenza Virus Vaccine Induces a Cellular Immune Response That Correlates with Reduction in Symptomatology and Virus Shedding in a Randomized Phase Ib Live-Virus Challenge in Humans. Clin Vaccine Immunol. 2015 Jul;22(7):828-35. doi: 10.1128/CVI.00098-15. Epub 2015 May 20.

Pleguezuelos O, Robinson S, Stoloff GA, Caparros-Wanderley W. Synthetic Influenza vaccine (FLU-v) stimulates cell mediated immunity in a double-blind, randomised, placebo-controlled Phase I trial. Vaccine. 2012 Jun 29;30(31):4655-60. doi: 10.1016/j.vaccine.2012.04.089. Epub 2012 May 8.

Stoloff GA, Caparros-Wanderley W. Synthetic multi-epitope peptides identified in silico induce protective immunity against multiple influenza serotypes. Eur J Immunol. 2007 Sep;37(9):2441-9. doi: 10.1002/eji.200737254.

van Doorn E, Pleguezuelos O, Liu H, Fernandez A, Bannister R, Stoloff G, Oftung F, Norley S, Huckriede A, Frijlink HW, Hak E. Evaluation of the immunogenicity and safety of different doses and formulations of a broad spectrum influenza vaccine (FLU-v) developed by SEEK: study protocol for a single-center, randomized, double-blind and placebo-controlled clinical phase IIb trial. BMC Infect Dis. 2017 Apr 4;17(1):241. doi: 10.1186/s12879-017-2341-9.

Pleguezuelos O, Dille J, de Groen S, Oftung F, Niesters HGM, Islam MA, Naess LM, Hungnes O, Aldarij N, Idema DL, Perez AF, James E, Frijlink HW, Stoloff G, Groeneveld P, Hak E. Immunogenicity, Safety, and Efficacy of a Standalone Universal Influenza Vaccine, FLU-v, in Healthy Adults: A Randomized Clinical Trial. Ann Intern Med. 2020 Apr 7;172(7):453-462. doi: 10.7326/M19-0735. Epub 2020 Mar 10.