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

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).