Elsevier

Vaccine

Volume 23, Issue 3, 2 December 2004, Pages 366-371
Vaccine

High epitope density in a single recombinant protein molecule of the extracellular domain of influenza A virus M2 protein significantly enhances protective immunity

https://doi.org/10.1016/j.vaccine.2004.05.028Get rights and content

Abstract

The degree of epitope density has been shown to be a critical factor influencing the magnitude of epitope-specific responses. However, whether high epitope density in just a single protein molecule can still enhance the humoral response or, more importantly, the protective immunity, has not been determined. To test this, five glutathione-S-transferase fusion proteins bearing various numbers of copies of the M2e epitope on M2 protein of influenza virus (1, 2, 4, 8 and 16 copies) were prepared, and used to immunize mice and rabbits. Our data show clearly that M2e-specific humoral response was enhanced with increasing epitope density. By lethal challenge assay in mice, it was observed that recombinant proteins with higher M2e epitope densities resulted in higher survival rates and slower weight losses. The survival rate was directly related to the degree of epitope density in the single recombinant protein: 100% in the case of 16 M2e copies; 50% with 4 M2e epitopes; and 0% with one.

Introduction

Traditional vaccines based on intact disease-inducing agents in an inactivated or live-attenuated form have proved effective against a number of diseases, including smallpox, typhoid and hepatitis A [1]. However, there are still no broad spectrum and effective vaccines against viruses with high mutation rates, such as influenza virus and human immunodeficiency virus [2], [3], [4]. Another goal in the development of vaccines is the induction of long-lasting protection by as few vaccine doses as possible, for cost saving considerations [5]. It is obvious that broad-spectrum and long-lasting humoral and cellular responses should be induced for effective vaccination against viruses with high mutation rates [6], [7], [8]. It is known that B-cell epitopes, T-helper cell epitopes and cytotoxic T lymphocyte epitopes all play important roles in these two immune responses [9], [10], [11]. Therefore, it is important to determine the relationship between epitope density and protective immunity, since this might provide a novel strategy for vaccine development that could induce efficient protective immunity.

Close relationships between antigen dose and the efficiency of the specific B cell response, as well as between the in vivo humoral response and the plasma B-cell longevity, have been identified in earlier studies [12], [13], [14]. Some recent studies have focused on the role of epitope density to humoral responses [15], [16], [17]. Using a chemically coupled carrier protein and epitope peptide system, consisting of the same amount of carrier protein coupled with varying amounts of epitope peptide [17], Bachmann and co-workers precisely determined the relationship between single epitope repetitiveness and humoral immune response. They observed clearly that the epitope density dramatically affected Th cell-dependent IgG responses [17]. However, whether high epitope density in just one single protein molecule can also enhance the specific humoral response in mammals has not been determined. More importantly, it is not clear whether high epitope density will enhance the protective immunity [18]. To investigate this, we prepared a series of GST fusion proteins with various epitope density based on the M2e epitope of influenza virus, which has been well proved to induce protective activities against influenza virus replication in vitro and in vivo [19], [20], [21], [22]. Our results demonstrate that high epitope density in one recombinant protein molecule can significantly enhance the protective immunity in mammals.

Section snippets

Cells, virus, peptide and chemical reagents

MDCK (Madin-Darby Canine Kidney) cells and influenza A virus (A/PR/8/34, A/wuhan/359/95) were maintained as described [22]. The peptide of the M2e epitope, N-KSLLTEVETPIRNEWGCRCNDSSD (aa2–24), was synthesized at Genemed Synthesis Inc. (San Francisco, USA). Freund’s adjuvants were obtained from Pierce (Rockford, USA). PGEM-T easy vector plasmid and pGEX-4T plasmids were respectively obtained from Promega (Madison, USA) and Pharmacia (Amersham Biosciences, Sweden).

Preparation of fusion proteins with various epitope densities

Glutathione-S-transferase (GST)

High M2e epitope density significantly enhanced the M2e specific antibody response

Using the epitope cumulation recombinant technique [23], five GST fusion proteins bearing various epitope densities of M2e: GST-(M2e)1, GST-(M2e)2, GST-(M2e)4, GST-(M2e)8 and GST-(M2e)16, were constructed. Fig. 1A shows the BamH I and Xho I double digest results of recombinant pGEX4T-2 plasmids containing these antigen genes in 1% agarose gels, and Fig. 1B the purified fusion proteins in 12.5% SDS-PAGE gels, both of which correspond well with theoretical expectations. Additionally, the

Discussion

Extensive earlier studies have been done to evaluate the influences of epitope density on B-cell responses, either in an organic coupled hapten polymer experimental system [27], [28], [29] or in a natural viral surface protein experimental system [12], [14], [15], [16]. These studies determined that epitope density played an important role in the humoral response. In another ingenious experimental system, which considered the role of Th cell epitopes in B cell activation, focusing the

Acknowledgments

We thank Dr. Xiao Yi for helpful discussion and professor Fang Liang for providing us the influenza virus strain A/PR/8/34. This work was supported by the 863-Program (2003AA219141), the Natural Science Foundation for Outstanding Young Scientist of China (No. 30025038) and the Tsinghua University Foundation for Basic Research.

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