Elsevier

Vaccine

Volume 20, Issue 16, 15 May 2002, Pages 2045-2052
Vaccine

DNA vaccination of ferrets with chimeric influenza A virus hemagglutinin (H3) genes

https://doi.org/10.1016/S0264-410X(02)00049-XGet rights and content

Abstract

Recently a technology was established based on homologous recombination that allowed the rapid generation of chimeric HA genes of influenza viruses, containing the antigenic determinants obtained from various influenza virus A (H3N2) viruses. In the present report plasmids were generated using a H3 HA vector handle and the hypervariable regions of two genetically distinct influenza A H3N2 viruses, A/Stockholm/7/97 and A/Netherlands/18/94. In a ferret model it was shown that immunisation with plasmid DNA encoding chimeric HA indeed elicited antibody responses specific for the virus from which the hypervariable region with the antigenic determinants were obtained. After DNA-immunisation of the ferrets, protective immunity against infection with influenza virus A/Netherlands/18/94 was evaluated.

Introduction

Infections with influenza viruses belong to the most important causes of respiratory disease and worldwide morbidity and mortality every year. The use of inactivated (split virion or sub-unit) vaccines results in a significant reduction of influenza related morbidity and mortality. However, one of the major problems for the preparation of effective influenza vaccines is the amino acid variation in the antigenic sites predominantly in the hemagglutinin, allowing influenza viruses to escape from virus neutralising antibodies that have been induced by previous infections [1], [2]. This antigenic drift necessitates the adaptation of the influenza vaccine composition when newly emerged drift variants are not sufficiently recognised by antibodies induced by the vaccine.

After it was shown that injection of plasmid DNA encoding the influenza virus nucleoprotein could confer protection against infection in mice [3], numerous studies have demonstrated the induction of influenza virus specific humoral and cell mediated immune responses in animal models [4], [5], [6], [7], [8]. Recently, we have described a technology based on homologous recombination, which allows for rapid alteration of the antigenic determinants of HA in a DNA vaccine [9]. It was shown in a mouse model that a plasmid expressing a chimeric HA molecule was immunogenic. In the present study we performed immunisations of ferrets with different plasmids containing chimeric HA genes. It was shown that immunisation with plasmid DNA encoding chimeric HA indeed elicited antibody responses specific for the virus from which the hypervariable region with the antigenic determinants were obtained. After DNA-immunisation of these ferrets partial protection was observed against challenge infection with influenza virus A/Netherlands/18/94 (H3N2).

Section snippets

DNA constructs

Hemagglutinin plasmid DNA constructs from two genetically and serologically distant influenza A strains (pKCMV HA A/Stockholm/7/97 and pKCMV HA A/NL/18/94) were produced as has been described previously [9]. Briefly, the variable regions of the HA gene of influenza viruses A/Stockholm/7/97 (H3N2) and A/NL/18/94 (H3N2) were amplified by RT-PCR using random 14-mer primers for first strand synthesis and primers 5′-CTATCATTGCCTTTGAGCTAC and 5′-TCCCAACCATTTTCTATG for PCR, yielding a 1048 bp amplicon.

Expression of HA genes

Before immunisation, all plasmids were tested for expression by immunofluorescence in HEK293 cells. Fig. 1 shows the immunostaining of transiently transfected cells at 48 h post-transfection using HA directed monoclonal antibodies and FITC-conjugated secondary antibodies, or FITC-conjugated monoclonal antibodies specific for the nucleoprotein. It was shown that the chimeric HA genes were expressed from pKCMV HA A/Stockholm/7/97 and pKCMV HA A/Netherlands/18/94 (Fig. 1b and c, respectively).

Discussion

Using homologous recombination plasmids were constructed from which chimeric influenza virus HA genes were expressed. Upon immunisation of ferrets with these plasmids antibody responses were induced with specificity for the virus strains from which the regions encoding the hypervariable antigenic sites were obtained. The induction of these strain specific antibodies provides the proof of concept that the vector handle approach can be used for the rapid construction of highly specific HA gene

Acknowledgements

The authors wish to thank Gunnel Engström and Gunilla Jonsson for outstanding technical assistance, Dr. W.E.P. Beyer for statistical analysis and Ger van der Water for continuous support. This work was supported by the Foundation for Strategic Research, the Infection and Vaccinology programme, the Swedish Medical Research Council and the Foundation for Respiratory Virus Infections notably Influenza (SRVI).

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