Elsevier

Vaccine

Volume 30, Issue 31, 29 June 2012, Pages 4606-4616
Vaccine

Antigenic and immunogenic properties of recombinant hemagglutinin proteins from H1N1 A/Brisbane/59/07 and B/Florida/04/06 when produced in various protein expression systems

https://doi.org/10.1016/j.vaccine.2012.05.005Get rights and content

Abstract

Antibodies directed against the influenza hemagglutinin (HA) protein largely mediate virus neutralization and confer protection against infection. Consequently, many studies and assays of influenza vaccines are focused on HA-specific immune responses. Recombinant HA (rHA) proteins can be produced in a number of protein expression and cell culture systems. These range from baculovirus infection of insect cell cultures, to transient transfection of plants, to stably transfected human cell lines. Furthermore, the rHA proteins may contain genetic modifications, such as histidine tags or trimerization domains, intended to ease purification or enhance protein stability. However, no systematic study of these different forms of the HA protein have been conducted. It is not clear which, if any, of these different protein expression systems or structural modifications improve or diminish the biological behavior of the proteins as immunogens or antigens in immune assays. Therefore we set out to perform systematic evaluation of rHA produced in different proteins expression systems and with varied modifications. Five rHA proteins based on recent strains of seasonal influenza A and five based on influenza B HA were kindly provided by the Biodefense and Emerging Infections Reagent Repository (BEIR). These proteins were evaluated in a combination of biochemical and structural assays, in vitro humoral and cellular immune assays, and in an animal vaccination model. Marked differences in the behavior of the individual proteins was evident suggesting that they are not equal when being used to detect an immune response. They were, nevertheless, similar at eliciting neutralizing antibody responses.

Highlights

► rHA produced and purified by various means are tested as reagents in immune assays. ► rHA produced and purified by various means are tested as immunogens in mice. ► rHA production process impacts the protein's antigenicity in immune assays. ► rHA proteins elicit relevant HAI responses independent of the production process. ► rHA oligomerization correlate with its antigenic and immunogenic properties.

Introduction

Influenza hemagglutinin (HA) glycoproteins are responsible for two major events in the influenza life cycle: (a) binding/attachment to sialic acid containing receptors in the target cells and (b) the fusion of the virus membrane with the endosomal membrane resulting in the release of the viral genome into the cytosol of the target cell [1], [2]. Due to the role of the HA in key aspects of the virus life cycle, and its relative abundance in the surface of the viral membrane, this protein is a relevant target for the humoral response. Indeed, a substantial portion of the antibody response following influenza infection in mice and humans is directed against the HA, with a population of these antibodies capable of neutralizing HA binding and/or fusion [3], [4], [5], [6], [7], [8]. More importantly, these antibodies are sufficient for protection against live virus infection [3], [9], [10], [11], [12] and their concentration shows a negative correlation with disease incidence in humans [12], [13], [14].

In the United States, current FDA licensed influenza vaccines have been optimized to maximize the generation of B cell and antibody responses against the HA of the strains included in the vaccine. In addition, a number of experimental vaccines, are also focused on eliciting antibody responses to the HA component of the vaccine (DNA, recombinant HA, and virus-like particles) [15], [16], [17], [18], [19], [20], [21]. Recombinant HA (rHA) proteins have been widely used to study B cell responses following vaccination and/or infection in humans and animals alike [21], [22], [23]. These proteins have been produce in an assortment of protein expression systems including yeast, bacteria, insect cells, plant cells, and human cell lines [24], [25], [26], [27], [28], [29], [30]. However, to appropriately measure responses to the HA protein of interest it is imperative that the rHA that most resembles the viral HA is chosen. In addition, there are recent efforts to license baculovirus-derived rHA vaccines for seasonal and pandemic strains of influenza [25], [31], [32]. However, while these vaccines have been shown to elicit comparable responses to conventional influenza vaccines, no systematic study of these different forms of the HA protein have been conducted. It is not clear which, if any, of these different protein expression systems improve or diminish the biological behavior of the proteins as viewed by the immune system. Therefore we set out to perform systematic evaluation of rHA produced in different protein expression systems. Five rHA proteins based on recent strains of seasonal influenza A and five based on influenza B HA were obtained through the Biodefense and Emerging Infections Research Resources Repository (BEIR). These proteins were evaluated in a combination of biochemical and structural assays, in vitro humoral and cellular immune assays, and in an animal vaccination model. Marked differences in the behavior of the individual proteins were evident suggesting that they are not equal in being used to detect or elicit an immune response.

Section snippets

Recombinant HA proteins

Purified rHAs from A/Brisbane/59/07 and B/Florida/04/06 listed below (and Table 1) were obtained through the NIH BEIR Repository (NIH/NIAID). For influenza A/Brisbane/59/07: truncated H1 HA protein with a C-terminal trimerization (T4 fibritin foldon) domain, thrombin cut site, and histidine tag produced in Trichoplusia ni High Five insect cells (Tni), NR-15476; truncated H1 HA protein with C-terminal histidine tag produced in human 293 cells (HEK), NR-15477; full-length H1 HA protein produced

Physical properties of recombinant influenza HA proteins

HA purified from the surface of influenza virions aggregates into structures containing 6–8 HA trimers [38]. However, expressed rHA proteins can be observed as oligomers, trimers, and/or monomers [16], [39]. In order to determine the predominant form of the rHA in these preparations we performed size exclusion chromatography. Protein preparations derived from Sf9 cells were primarily composed of high molecular weight oligomers (HMWO) (Fig. 1C, E, H, and J). These oligomers display molecular

Discussion

Long lasting immunity to influenza, whether it is induced by infection or vaccination, depends on the ability of the humoral arm of the immune system to respond to the virus HA protein. Indeed, a substantial portion of the antibody response following influenza infection in mice and humans is directed against the HA. Because of this, understanding the responses against the HA is one of the main focuses of influenza research. Recombinant versions of influenza HAs are widely available as reagents

Acknowledgements

This work was supported by the New York Influenza Center of Excellence, part of the NIH/NIAID network of Centers of Excellence in Influenza Research and Surveillance (CEIRS), contract #HHS-N266200700008C. The rHA reagents used to conduct this research were kindly provided by the Biodefense and Emerging Infections Research Resources Repository.

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