Vaccine 29 (2011) 5731–5739 Contents lists available at ScienceDirect Vaccine jou rn al h om epa ge: www.elsevier.com/locate/vaccine Intranasal immunization with recombinant PspA fused with a flagellin enhances cross-protective immunity against Streptococcus pneumoniae infection in mice Chung Truong Nguyen a,d , Soo Young Kim a , Myoung Suk Kim a , Shee Eun Lee a,c , Joon Haeng Rhee a,b,∗ a Clinical Vaccine R&D Center, Chonnam National University Hwasun Hospital, 160 Ilsimri, Hwasun-gun, Jeonnam 519-809, South Korea b Department of Microbiology and Research Institute of Vibrio Infections, Chonnam National University Medical School, Gwangju 501-746, South Korea c Dental Science Research Institute, School of Dentistry, Chonnam National University, Gwangju 500-757, South Korea d The Brain Korea 21 Project, Center for Biomedical Human Resources, Chonnam National University Medical School, Gwangju 501-746, South Korea a r t i c l e i n f o Article history: Received 30 December 2010 Received in revised form 25 May 2011 Accepted 28 May 2011 Available online 13 June 2011 Keywords: PspA Flagellin Pneumococcal vaccine a b s t r a c t Streptococcus pneumoniae is a major respiratory pathogen that causes high levels of mortality and mor- bidity 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 pro- duced 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. © 2011 Elsevier Ltd. All rights reserved. 1. 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 annu- ally 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]. There- fore, 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 ∗ Corresponding author at: Clinical Vaccine R&D Center, Chonnam National Uni- versity Hwasun Hospital, 160 Ilsimri, Hwasun-gun, Jeonnam 519-809, South Korea. Tel.: +82 61 379 8479; fax: +82 61 379 8455. E-mail address: jhrhee@chonnam.ac.kr (J.H. Rhee). licensed for use in children in many countries [7]. More recently, new conjugate vaccines covering 10 and 13 serotypes of Strepto- coccus pneumoniae have been introduced into the vaccine market [8–10]. These vaccines show good protective efficacy, even in chil- dren 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]. More- over, 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. pneumo- niae 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 [19–23]. 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 alter- 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.05.095