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Seasonal influenza affects millions of people around the world, with as many as 500,000 deaths annually resulting from influenza-related illnesses. The flu virus undergoes frequent and unpredictable mutations (antigenic drift and shift) that limit the ability of available strain-specific vaccines to protect the population against strains other than those specifically included in a particular season’s flue vaccine. Annual reformulation of the vaccines is needed for annual immunizations.

BiondVax Pharmaceuticals Ltd., an Israeli biotechnology company, is developing a universal influenza vaccine (Multimeric-001, currently in phase 2 clinical testing) designed to be effective against all strains of influenza and eliminate the need for repeated annual immunizations. A phase 2 trial and two phase 1–2 clinical trials have been completed. In all three trials, the vaccine was shown to be safe and immunogenic, successfully stimulating both humoral and cellular immune responses.

Multiepitope Approach

Multimeric-001 vaccine is composed of a combination of nine conserved, nonmutating, linear, immunogenic peptides (epitopes) common to the vast majority of known influenza virus strains, regardless of their antigenic drift and shifts. The epitopes were selected for the following primary distinctive qualities.


Immunogenicity: The selected peptides are immunogenic, inducing specific immune responses when exposed to the immune system.

Conserved Epitopes: The epitopes are not vulnerable to antigenic changes, and they remain intact despite mutations that may change other regions of the viral proteins. This quality enables the vaccine to induce an immune response to a wide variety of virus strains. The epitopes are derived from the inner nucleoprotein (NP) and matrix (M1) proteins as well as from hidden regions of the outer viral protein — the hemagglutinin (HA).

Combination: Both epitopes that stimulate humoral (B-cell or antibody-mediated) immunity and those that stimulate cell-mediated (T-cell) immunity against influenza were selected. Preclinical testing showed that optimal protection against infection is achieved when both humoral and cell-mediated arms of the immune system are used against potential infection (1).

Epitopes from Types A and B Influenza Virus Strains: Of the three influenza virus types (A, B, and C), the vast majority of influenza virus mutations occur in type A viruses, which are responsible for about 80% of human infections with influenza. Type B viruses are less susceptible to mutation and responsible for the other 20% of infection cases annually. Type C influenza viruses are the most uncommon. Annual seasonal influenza vaccines contain two type A virus strains and one type B virus strain. To be considered truly universal, a vaccine would need to provide protection against both type A and type B virus strains.

Linear Epitopes: The peptides represent linear epitopes that can be expressed from a linear DNA sequence. These epitopes are taken in triplicate and combined into a single recombinant protein that is then expressed in Escherichia coli. To avoid manufacturing complexities involved with three-dimensional structures, the chosen epitopes must be linear, thereby avoiding any issues relating to refolding.


The Multimeric-001 vaccine is manufactured by fermentation in E. coli in a standard, robust, and scalable good manufacturing practice (GMP) manufacturing process. In brief, E. coli bacteria containing plasmid with the coding sequence for the Multimeric-001 protein are grown by fermentation. The protein is then purified and formulated as required. BiondVax is testing both adjuvanted and nonadjuvanted formulations in its clinical trials. The entire manufacturing process — from fermentation to recovery of inclusion bodies, purification, formulation, filling, and product release — can be performed on a commercial scale in six to eight weeks.

Because the Multimeric-001 vaccine is not composed of any particular influenza virus strain or mutation, it would not depend on the World Health Oranization–mandated annual strain selection process, as is the case with current influenza vaccines. It could be produced year-round and not be limited to a single production cycle (per hemisphere). As such, manufacturers could produce this vaccine in response to demand, either increasing or potentially even cutting back on production midseason to better manage their inventories, resulting in potential savings for the industry.

Unlike production in eggs or cell culture, a bacterial fermentation process can be rapidly expanded and scaled up in times of emergency, such as during pandemics. In addition, the availability of year-round supply and the ability to immunize well in advance of the influenza season would enable health authorities and public health systems to conduct organized, systematic influenza vaccination campaigns to immunize large population segments regionally or nationwide. National health authorities could significantly increase the level of prepandemic preparedness by stockpiling a strategic supply of vaccine.

Development Status

The Multimeric-001 universal influenza vaccine is currently in phase 2 clinical testing. Two phase 1–2 trials and one phase 2 trial have been completed. The next phase 2 trial is scheduled to begin in the fourth quarter of 2011.

The Phase 1–2 clinical program consisted of two trials in ~60 subjects each, the first in a younger adult group (aged 18–49) and the second in an older adult group (aged 55–75). Both studies were conducted at clinical trial centers in hospitals in Israel. Their purpose was to assess the safety and immunogenicity of various doses (high-dose 500 mcg and low-dose 250 mcg) and formulations (adjuvanted and nonadjuvanted).

In both phase 1–2 trials, Multimeric-001 vaccine had a strong safety profile with no treatment-related severe adverse events, no significant differences between treatment and control groups. The most observed adverse events were mild and all transient. From an immunogenicity perspective, the vaccine stimulated humoral and cellular immune responses. The second study (older adult group) evaluated both immunogenicity and ability to enhance the effectiveness of the standard, commercially available seasonal influenza vaccine when the Multimeric-001 vaccine is used as a primer for a subsequent boost from the seasonal vaccine.

BiondVax recently concluded the first of what is expected to be a series of at least three phase 2 clinical trials. This first of those was a randomized, double-blind, placebo-controlled safety and immunogenicity study in 200 healthy younger adult volunteers (aged 18–49). In the two preceding phase 1–2 trials, researchers found that the best-performing dose and formulation of the vaccine was the adjuvanted 500 mcg formulation. So this formulation was used in the first phase 2 study.

Results showed that the Multimeric-001 vaccine has a very strong safety profile with no treatment-related severe adverse events and no significant differences between treatment and control groups. The most-observed adverse events were
mild and all transient. The vaccine successfully stimulated both humoral and cell-mediated immune responses.

Clinical Trial Results

Standalone Vaccine: Humoral (Antibody-Mediated or B-Cell) Immune Response: In all three clinical studies performed to date, the Multimeric-001 vaccine stimulated production of antibodies (IgG) to the vaccine. Phase 1–2 studies showed a statistically significant increase in IgG levels in all participants immunized with both 500 mcg and 250 mcg adjuvanted doses. Researchers observed a statistically significantly higher number of responders to the vaccine in all participants immunized with the adjuvanted 500 mcg formulation.

In addition, some elevation in IgG titers was observed to various B-cell epitopes contained in the vaccine. Researchers investigated the ability of those antibodies to lyse infected cells and observed elevated complement mediated neutralization of influenza strains from H1N1, H3N2, and type B subtypes. These results provide evidence of the mechanism of action behind the humoral immunity induced by the Multimeric-001 vaccine. Immunization with the adjuvanted 500 mcg Multimeric-001 vaccine significantly increased IgG antibodies to the vaccine.

Cell-mediated Immune (CMI) Response: Phase 1–2 studies investigaed the effect of the Multimeric-001 vaccine on a number of cellular immunogenicity parameters. A statistically significant increase in lymphocyte proliferation in response to the Multimeric-001 vaccine was observed in all young-adult participants immunized with the adjuvanted 500-mcg formulation. Significant lymphocyte proliferation was observed in response to different influenza viruses and T-helper peptides. Results included statistically significant increases in both IFN-γ and interleukin-2 secretion from cells incubated with Multimeric-001.

The phase 2 study confirmed the stimulation of cell-mediated immunity. Immunization with Multimeric-001 vaccine resulted in a statistically significant increase in secretion of IFN-γ to it, to levels exceeding those found in immunization with conventional subunit vaccines (2). Results showed a significant elevation in CD4+ lymphocytes secreting IFN-γ, providing information about the mechanism of action behind the cell-mediated immunity induced by the vaccine.

Combined with Standard Influenza Vaccines: To assess the efficacy of an influenza vaccine and to approve it for use, regulators currently use a particular measure of antibody response as a surrogate marker of protection against influenza. This measure is the hemagglutination inhibition (HI) assay, which measures the presence of particular antibodies to the hemagglutinin protein found on the exterior, changing, surface of the influenza virus. Because the Multimeric-001 vaccine contains only selected epitopes taken from the hidden regions of the hemagglutinin protein, it does not stimulate production of these particular HI antibodies on its own. Therefore the HI surrogate marker cannot be relied upon to show efficacy.

However, in numerous preclinical studies for which the Multimeric-001 vaccine is delivered in combination (either as a prime-boost or co-administered) with a standard trivalent inactivated vaccine (TIV), the level of HI antibodies has been affected. In a study of transgenic mice, Multimeric-001 priming before boosting with the TIV resulted in a fivefold increase in seroprotection rates. In a study of ICR mice, coadministration of Multimeric-001 vaccine with the TIV increased HI titers against both homologous (strains contained in the TIV) and drifting strains (not contained in the TIV).

BiondVax conducted two additional clinical studies. In the first study (phase 1–2 in elderly volunteers), a double prime of the Multimeric-001 vaccine (21 days apart) followed 21 days later by a boost from a TIV resulted in markedly higher rates of seroconversion to all three strains contained within the TIV and to many drifting strains (whether from H1N1, H3N2, or type B influenza) than did the TIV alone. In many strains, seroconversion rates were increased to above the minimum 30% required by regulators for approval of influenza vaccines in elderly patients.

The subsequent phase 2 study again found that administering the Multimeric-001 vaccine once concomitantly with a 50% dose of a seasonal TIV induced higher rates of HI antibody responses than did the TIV alone — against both TIV and drifting strains. These results confirm findings from a previous phase 1–2 trial in older adults. They also show that combining the Multimeric-001 vaccine with TIV (rather than using the TIV alone) can increase the number of people who reach the HI seroconversion levels required by US and European regulatory authorities for licensure of influenza vaccines. That increase in HI seroconversion rates has been observed not only against strains that were included in the TIV, but also against additional type A (H1N1 and H3N2) and type B influenza strains that were not included in the TIV.

Those results suggest that the Multimeric-001 vaccine — when administered in advance of future seasonal or pandemic influenza outbreaks — can raise the general level of preparedness of a population and significantly improve the protection and broaden the cross-strain coverage offered by the strain-dependent influenza vaccines.

Ongoing Clinical Development

In light of the recent successful conclusion of its first phase 2 clinical trial, BiondVax is preparing to conduct the next phase 2 trial with elderly volunteers. We expect that this trial will be conducted in two centers in Israel with ~120 male and female participants aged 65 and over. This trial is expected to begin in the fourth quarter of 2011 and to conclude in the first quarter of 2012.

Notwithstanding major efforts on the part of national health authorities and global pharmaceutical companies, influenza continues to infect hundreds of millions of people every year and kill hundreds of thousands. Many innovative and dynamic companies are striving to develop a universal influenza vaccine that will effectively immunize the entire population against all strains of influenza. BiondVax Pharmaceuticals has made significant progress toward that goal. If the company continues to succeed in its clinical development of the Muiltimeric-001 universal influenza vaccine, we hope such a vaccine will be available on the market within a few years.

About the Author

Author Details
Dr. Tamar Ben Yedidia is chief scientific officer ([email protected]), and Wayne Rudolph is vice-president of corporate development ([email protected]) at BiondVax Pharmaceuticals Ltd.; 972-54-456 2885.


1.) Levi, R, and R Arnon. 1996. Synthetic Recombinant Influenza Vaccine Induces Efficient Long-Term Immunity and Cross-Strain Protection. Vaccine 14:85-92.

2.) Saurwein-Teissl, M. 1998. Whole Virus Influenza Activates Dendritic Cells (DC) and Stimulates Cytokine Production By Peripheral Blood Mononuclear Cells (PBMC) While Subunit Vaccines Support T-cell Proliferation. Clin. Exp Immunol 114:271-276.

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