Elsevier

Vaccine

Volume 30, Issue 39, 24 August 2012, Pages 5761-5769
Vaccine

Development of VAX128, a recombinant hemagglutinin (HA) influenza-flagellin fusion vaccine with improved safety and immune response,☆☆

https://doi.org/10.1016/j.vaccine.2012.06.086Get rights and content

Abstract

Background

We evaluated the safety and immunogenicity profiles of 3 novel influenza vaccine constructs consisting of the globular head of the HA1 domain of the Novel H1N1 genetically fused to the TLR5 ligand, flagellin. HA1 was fused to the C-terminus of flagellin in VAX128A, replaced the D3 domain of flagellin in VAX128B and was fused in both positions in VAX128C.

Methods

In a dose escalation trial, 112 healthy subjects 18–49 and 100 adults ≥65 years old were enrolled in a double blind, placebo controlled clinical trial at two centers. Vaccines were administered IM at doses ranging from 0.5 to 20 μg. VAX128C was selected for second study performed in 100 subjects 18–64 years old comparing 1.25 and 2.5 μg doses. All subjects were followed for safety and sera collected pre- and post-vaccination were tested by hemagglutination-inhibition (HAI). Serum C-reactive protein and cytokine levels were also measured.

Conclusions

In the first study high HAI titers and high seroconversion and seroprotection rates were observed at doses ≥2.5 μg in adults 18–49. In adults ≥65 years, the vaccines doses of ≥4 μg were required to induce a ≥4-fold rise in HAI titer, 50% seroconversion and 70% seroprotection. Based on safety, VAX128A was tested up to 8 μg, VAX128B to 16 μg and VAX128C to 20 μg. Dose escalation for VAX128A was stopped at 8 μg because one subject had temperature 101.6 °F associated with a high CRP response, VAX128B was stopped at 16 μg because of a severe AE associated with a high CRP and IL-6 response. VAX128C was not stopped before reaching the 20 μg dose. In the second study VAX128C was well tolerated among 100 subjects who received 1.25 or 2.5 μg. The peak GMT was 385 (95%CI 272–546), 79% (71–87%) seroconversion and 92% (84–96%) seroprotection.

Discussion

Flagellin adjuvanted vaccines can be designed to minimize reactogenicity and retain immunogenicity, thereby representing a promising next generation vaccine technology.

Highlights

► Recombinant influenza vaccines differ in safety profiles according to configuration. ► VAX128C was well tolerated up to a dose of 20 μg. ► Dose of 1.25 or 2.5 μg of VAX128C gave peak GMT was 385, 79% SC and 92% SP.

Introduction

The worldwide need for seasonal and pandemic influenza vaccines has increased interest in the development of innovative technologies for influenza vaccine production [1]. In addition to improvements in vaccine production efficiency, enhancement of the immunopotency of influenza vaccines is required to meet seasonal and pandemic needs on a global scale. A vigorous immune response to the hemagglutinin (HA) outer surface glycoprotein is widely considered to represent an important surrogate of protection against influenza infection and associated disease.

We developed a novel H1N1 pandemic influenza vaccine designated, VAX128, based on A/California/7/2009. VAX128 is a recombinantly produced vaccine that activates the immune response by coupling a potent immune stimulator, the bacterial protein flagellin (STF2), to the globular head domain of the influenza HA antigen [2]. The globular head domain of HA stably refolds to form the conformationally sensitive antigenic sites that span the majority of the neutralizing epitopes in HA. This vaccine elicits a potent virus-specific neutralizing antibody response and the prokaryotic expression system allows for very efficient manufacture. VAX128 is highly protective in mice [3].

Previously, we tested VAX125, a monovalent seasonal prototype based on the hemagglutinin of H1N1 Solomon Islands, in Phase I studies in healthy young adult and elderly subjects [4], [5]. VAX125 was highly immunogenic at a dose of 1 μg. Although the vaccine was generally well tolerated, there were a few instances of flu-like symptoms (malaise and fever) in the first 24 h following vaccination at doses of 3–8 μg. These symptoms were associated with elevated C-reactive protein (CRP) responses suggesting that they were mediated by cytokine production. Although VAX125 was safe and immunogenic in the 1–2 μg dose level, it was important to ensure that the amount of flagellin in a multivalent vaccine would be well tolerated. In a previous study in mice, we observed that the A/Vietnam/1203/2004 H5 HA, a poor immunogen, was highly immunogenic when substituted for the D3 domain of flagellin [6]. For VAX128 we produced similar modifications in the attachment site(s) of the HA to the flagellin moiety and found that modification of the attachment site could influence reactogenicity in mice without impacting immunogenicity [3]. In the current study we performed a head-to-head comparison of the safety and immunogenicity of three VAX128 constructs first in the rabbit model and then in humans where we evaluated the safety profiles, CRP and cytokine response to doses up to 20 μg.

Section snippets

Vaccine constructs

VAX128A, VAX128B, and VAX128C differ in the site of attachment of the H1 A/CA07/2009 HA subunit to flagellin (Fig. 1). In VAX128A the HA globular head is fused to the C-terminal end of flagellin; in VAX128B the HA globular head is moved from the C-terminus to replace domain three (D3) of flagellin, and in VAX128C the HA globular head is fused to the C-terminal end of flagellin and a second copy replaces D3. The basic cloning, production and analytical assays for the different construct formats

Vaccine immunogenicity and safety in rabbits

All three of the VAX128 vaccine constructs were immunogenic in the rabbit model with ≥4-fold increases in HAI antibody titer observed 7 days post the booster immunization at all dose levels (Fig. 2). At the highest dose (15 μg) VAX128C was significantly more immunogenic than VAX128A and at the lowest dose (0.5 μg) VAX128C was significantly more immunogenic than either VAX128A or VAX128B.

For VAX125 and VAX128A body temperature and serum CRP were affected in a dose-dependent fashion (Fig. 3).

Discussion

In both the rabbit model and in humans we found that replacing the HA antigen in the D3 position of flagellin in VAX128B and VAX128C produced a vaccine that was at least as immunogenic and better tolerated at higher doses than the C terminal fusion construct (VAX128A). These data demonstrate that the HA globular head can be stably expressed in a prokaryotic (E. coli) system positioned in either the C terminal or D3 position of flagellin or both. Most significantly we have demonstrated that the

Acknowledgements

We would like to thank Helena Petridis and Sonya Littlejohn at VaxInnate for clinical trial coordination, our project coordinators Meredith Argulles at MRA and Monica Atwood at JCCT. We are indebted to Arthi Krishnappa for performing the serological studies at VaxInnate and Theresa Fitzgerald for performing the HAI tests and Xia Jin for supervising the CBA assays at University of Rochester.

Contributors: DT, AS and TH designed the clinical trials. ES and CJ enrolled the study subjects and

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This study was presented in part at the 14th Annual Conference on Vaccine Research, May 16, 2011, Baltimore, MD (abstract S-6).

☆☆

The study was approved by the Institutional Review boards at Miami Research Associates, Miami, FL and Johnson County Clin Trials, Lenexa, KS. All subjects gave written informed consent prior to participation. The trial was registered on ClinicalTrials.gov number NCT01172054.

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