Elsevier

Vaccine

Volume 29, Issue 34, 5 August 2011, Pages 5731-5739
Vaccine

Intranasal immunization with recombinant PspA fused with a flagellin enhances cross-protective immunity against Streptococcus pneumoniae infection in mice

https://doi.org/10.1016/j.vaccine.2011.05.095Get rights and content

Abstract

Streptococcus pneumoniae is a major respiratory pathogen that causes high levels of mortality and morbidity in infants and the elderly. Despite the use of antibiotics and vaccines, fatal pneumococcal disease remains prevalent. Pneumococcal surface protein A (PspA), a highly immunogenic surface protein produced by all strains of S. pneumoniae, can elicit protective immunity against fatal pneumococcal infection. We have previously demonstrated that the Vibrio vulnificus FlaB, a bacterial flagellin protein and agonist of TLR5, has strong mucosal adjuvant activity and induces protective immunity upon co-administration with tetanus toxoid. In this study, we have tested whether intranasal immunization with recombinant fusion proteins consisted of PspA and FlaB (PspA–FlaB and FlaB–PspA) is able to elicit more efficient protective mucosal immune responses against pneumococcal infection than immunization with PspA alone or with a stoichiometric mixture of PspA and FlaB. When mice were intranasally immunized with fusion proteins, significantly higher levels of anti-PspA IgG and IgA were induced in serum and mucosal secretions. The mice immunized intranasally with the FlaB–PspA fusion protein were the most protected from a lethal challenge with live S. pneumoniae, as compared to the mice immunized with PspA only, a mixture of PspA and FlaB, or the PspA–FlaB fusion protein. FlaB–PspA also induced a cross protection against heterologous capsular types. These results suggest that a FlaB–PspA fusion protein alone could be used as an anti-pneumococcal mucosal vaccine or as an effective partner protein for multivalent capsular polysaccharide conjugate vaccines.

Highlights

► Recombinant PspA and FlaB, a TLR5 agonist, fusion proteins were manufactured. ► Intranasal FlaB-PspA vaccine enhanced mucosal anti-PspA immune responses. ► FlaB-PspA vaccine was protective against heterologous serotype pneumococcal infection.

Introduction

Streptococcus pneumonia is a Gram-positive bacteria that is the causative agent of pneumonia, otitis media, bacteremia, and meningitis [1], [2]. The World Health Organization estimates that about 1.6 million cases of fatal pneumococcal disease occur annually worldwide, mostly in infants and the elderly [3]. Recently, antibiotic resistance in Streptococcus has become a worldwide problem, making treatment more difficult and expensive [4]. Therefore, interest in the prevention of pneumococcal disease has increased. A polyvalent polysaccharide vaccine has been efficacious in adults [5], but this vaccine is poorly immunogenic in children because of the weak immunogenicity of the T cell-independent antigens [5], [6]. A 7-valent conjugate vaccine has recently been licensed for use in children in many countries [7]. More recently, new conjugate vaccines covering 10 and 13 serotypes of Streptococcus pneumoniae have been introduced into the vaccine market [8], [9], [10]. These vaccines show good protective efficacy, even in children and aged populations [11], [12]. However, S. pneumoniae species are highly diverse with respect to their capsular types, and the polysaccharide–protein conjugate vaccines will not protect against strains with capsular types not included in the vaccine [13], [14]. Selective pressure exerted by existing vaccines seems to contribute to the emergence of non-vaccine serotype infections [15]. Moreover, the conjugate vaccine is too expensive for widespread use in the developing world, where 1–5 million children die each year of pneumococcal respiratory infections [16], [17]. S. pneumoniae expresses a number of virulence factors. In various host environments, specific virulence factors play specific roles in the development of disease [18]. These virulence factors could serve as potential vaccine targets [19], [20], [21], [22], [23]. Many research groups have been investigating new vaccines targeting these virulence factors [24]. Some of these proteins, such as pneumococcal surface protein A (PspA), have already shown significant promise for the use in alternative vaccine approaches [25], [26]. PspA appears to be essential for the virulence of S. pneumoniae [25] and is found in all pneumococci [27]. Furthermore, PspA has been shown to be highly immunogenic and to possess protective determinants [25], [27], [28], [29]. Although PspA shows antigenic heterogeneity among clinical strains, it appears to elicit cross-protection against strains with different capsular types [21], [30], [31], [32]. Therefore, PspA could be a good candidate antigen for future pneumococcal vaccine development. Based on immunization studies with full-length and truncated fragments of PspA, the α-helical domain was determined to contain protective epitopes [21], [33], [34], [35]. This α-helical domain also exhibited cross-protective immunity similar to that seen following immunization with full PspA [36].

Mucosal immune responses function as a first line of defense against infection. Protective mucosal immune responses are most effectively induced by mucosal immunization through oral, nasal, rectal or vaginal routes [37]. To generate more successful mucosal vaccines, it will be necessary to identify optimal antigenic determinants that elicit stronger protective immune responses in the mucosal compartment and to find better mucosal adjuvants to potentiate the immunogenicity of weak antigens. Recently, flagellin, a ligand for toll-like receptor 5 (TLR5), has been considered as an adjuvant for vaccines and immunotherapy [38], [39], [40], [41]. We have shown that a bacterial flagellin protein, Vibrio vulnificus FlaB, exerts very strong immune reactivity in the mucosal compartments [39], [40]. FlaB binds directly to human TLR5 expressed by human cells and consequently induces NF-κB activation [39]. Our previous studies strongly suggest that V. vulnificus FlaB could be an excellent adjuvant for mucosal vaccines. We hypothesized that physical fusion of FlaB with antigens from mucosal pathogens in a recombinant protein should enable those antigens as mucosal vaccines and provide better protection. In this study, we fused FlaB to PspA and constructed two fusion proteins, FlaB–PspA and PspA–FlaB. The objective of this study was to test the immunogenicity of these two fusion proteins and to compare their protective activity against S. pneumonia infections with PspA alone and an equimolar mixture of PspA and FlaB.

Section snippets

Bacterial strains, plasmids, and media

Bacterial strains and plasmids used in this study are listed in Table 1. S. pneumoniae D39 capsular serotype 2 [42] and WU2 capsular serotype 3 [43] express PspA from clade 2 family 1. S. pneumoniae were routinely grown in Todd–Hewitt broth (Becton Dickinson and Co, le Pont de Claix, France) with 5% yeast extract (THY) or on sheep blood agar plates (Asan Pharmacy Co., Seoul, Korea) at 37 °C in a 5% CO2 atmosphere. Plasmids were maintained in Escherichia coli cells grown on Luria–Bertani (LB)

Protein purification and analysis

The recombinant proteins were purified by the chitin affinity chromatography. The purity of recombinant proteins was confirmed by SDS-PAGE and the Western blot analysis using specific antibodies against FlaB or PspA (Fig. 3). Despite being estimated to have the same molecular mass (68 kDa), the FlaB–PspA and PspA–FlaB fusion proteins showed different mobility on the SDS-PAGE gels. We confirmed that the fusion proteins were correctly expressed by sequencing the expression vectors, MALDI-TOF

Discussion

Despite that many different anti-pneumococcal vaccines were designed and undergone clinical trials, only limited number of vaccine formulations are circulated in the market. Each commercial vaccine has advantages and disadvantages. The 23-valent capsular vaccine could cover many serotypes. But it cannot be given to high-risk groups such as younger children and elderly population and is less immunogenic since the vaccine cannot effectively stimulate T-cell responses. Though conjugate

Acknowledgements

We thank Dr. Ho Young Kang of Pusan National University for kindly providing the pYA3634 plasmid and S. pneumoniae D39 strain. This study was supported by the Regional Technology Innovation Program of the Ministry of Knowledge Economy of the Republic of Korea (No. RTI 05-01-01).

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