Construction and characterization of human adenovirus serotype 3 packaged by serotype 7 hexon

Abstract

Human adenovirus serotype 3 (Ad3) and serotype 7 (Ad7) are important pathogens causing respiratory tract diseases such as acute respiratory disease in pediatric and adult patients, but the immunodominant targets of Ad3- and Ad7-specific neutralizing antibodies (NAbs) remain unclear. A chimeric Ad vector, Ad3/H7, was constructed by replacing the Ad3 hexon gene (H3) with the hexon gene (H7) of Ad7. The chimeric viruses were successfully rescued in HEp-2 cells, and the Ad7 hexon was able to encapsidate the Ad3 genome, and functioned as efficiently as the Ad3 hexon. Furthermore, we tested the host neutralization responses against the viruses using BALB/C mice. Up to 97% of the NAbs produced by mice that were infected with these viruses were specific for the hexon protein in vitro. Preimmunization of mice with one of Ad7 and Ad3/H7 significantly prevented subsequent intranasal infection of the other type in vivo. In contrast, preimmunization of mice with one of Ad3 and Ad3/H7 did not remarkably prevent subsequent infection of the other type. We next evaluated the functional significance of hexon and other structural proteins specific NAbs to suppress the immunogenicity of Ad3/H3 and Ad3/H7 vectors expressing EGFP in mice preimmunized with wild type Ad. Preimmunization of mice with Ad7 evidently suppressed EGFP-specific humoral immune responses elicited by Ad3/H7, and did not exert suppressive effects on Ad3/H3. But contrary to the in vitro neutralization results, EGFP-specific humoral immune responses elicited by Ad3/H7 was remarkably inhibited in Ad3-preimmunization mice. The whole genome of the Ad7 strain was sequenced and aligned with Ad3. The major differences between Ad3 and Ad7 were only observed in the fiber and hexon among all structural proteins, and the variation between the hexons only located in four hypervariable regions (HVRs), HVR-1, -2, -5, and -7. These results thus suggest that Ad3- and Ad7-specific NAbs are directed primarily against the hexon proteins both in vitro and in vivo. But high titer Ad3 fiber-specific NAbs may also play an important role in blunting Ad3 immunogenicity in vivo. These studies contribute to a more profound understanding of Ad immunogenicity and have relevance for the design of novel Ad vaccine.

Introduction

Human adenoviruses (HAdVs) in the alimentary canal and respiratory tract were discovered in 1953, and have been found to cause a broad spectrum of diseases in pediatric and adult patients (Aoki and Tagawa, 2002, Arnold et al., 2010, Kunz and Ottolini, 2010, Louie et al., 2008, Rowe et al., 1953). HAdV can be classified into six species (A–F) consisting of 53 serotypes, based on serum neutralization antibodies and nucleotide sequence. Recently, a new serotype, HAdV52, was reported and defined as a seventh species, species G (Davison et al., 2003, Green et al., 1979, Ishiko et al., 2008, Jones et al., 2007, Walsh et al., 2009). Recently, a broad recombination phenomenon has been discovered between different serotypes of virus strains leading to lethal strains, some of which may be new serotypes (Kajon et al., 2010, Lukashev et al., 2008, Rebelo-de-Andrade et al., 2010). Members of species B, HAdV-3 and -7, have caused severe respiratory disease, such as acute respiratory disease (ARD) pediatric pneumonia epidemics and outbreaks in Asia, Europe and America (Erdman et al., 2002, Frantzidou et al., 2005, Hierholzer, 1992, Li et al., 2005, Tang et al., 2011). As yet, there is no effective medicine or vaccine except for live, oral vaccines used by the US military from 1971 to 1996 (Gooch and Mogabgab, 1972, Lyons et al., 2008). For all these reasons, development of an effective vaccine directing HAdV-3 and -7 is required.

An adequate knowledge of the immunodominant regions of adenovirus NAbs will be helpful for the development of novel adenovirus vaccine. The Ad capsid consists of three major structural proteins: hexon, fiber, and penton base. Research carried out on adenovirus serotype 2 (Ad2) and Ad5 have identified the major coat protein of the HAdV hexon as the main target for neutralizing antibodies (NAbs) (Pichla-Gollon et al., 2007, Sumida et al., 2005, Wu et al., 2002, Youil et al., 2002); however, it is not clear whether this is the case for other HAdV serotypes. Some studies have found that other structural proteins, such as fiber and penton base, also could induce neutralizing antibodies (Fender et al., 1995, Hong et al., 2003, Liebermann et al., 1998). Little is currently known about the neutralizing epitopes of the more than 50 HAdV serotypes (Pichla-Gollon et al., 2007, Rux and Burnett, 2000), although it is possible to locate HVRs using the multiple sequence alignment method (Crawford-Miksza and Schnurr, 1996, Ebner et al., 2005).

Replacement of the immunogenic capsid proteins with those of other serotypes is one of the approaches used to circumvent the problem of pre-existing immunity to an adenovirus delivery vector (Gall et al., 1998, Roberts et al., 2006, Rux et al., 2003, Wu et al., 2002, Youil et al., 2002). Some studies based on the HAdV5 vector have suggested that replacement of the hexon may or may not affect the packaging and gene transfer efficiency (Gall et al., 1998, Wu et al., 2002, Youil et al., 2002). An Ad3 vector has been developed previously as a candidate for vaccine design and gene transfer (Zhang et al., 2009). In the present study, we attempted to construct a chimeric adenovirus, Ad3/H7, by replacing the Ad3 hexon gene with the hexon gene of Ad7 (H7) in the hope that this study could help understand the functional significance of NAbs against major structure proteins, and the strategy used to modify the hexon from Ad3 could be applied to adenovirus vaccine development and gene therapy.