Stimulation of long-lasting protection against Streptococcus pyogenes after intranasal vaccination with non adjuvanted fibronectin-binding domain of the SfbI protein

Abstract

Protective immunity against Streptococcus pyogenes can be induced by intranasal vaccination with the fibronectin-binding domain (H12 fragment) of the fibronectin-binding protein I (SfbI) co-administered with the B subunit of the cholera toxin (CTB) as mucosal adjuvant. However, intranasal administration of A–B moiety bacterial toxins or their derivatives has been associated with potentially severe side effects. Since the SfbI protein exhibits adjuvant properties, we investigated whether vaccination with the H12 fragment alone is sufficient to promote long-lasting protection. The obtained results demonstrated that the humoral and cellular immune responses stimulated at both systemic and mucosal levels were almost identical when mice were vaccinated with the H12 fragment in the presence or absence of CTB. Immunized mice were protected against challenge with a lethal dose of S. pyogenes given 36 or 110 days after primary vaccination to the same extent (80% survival), regardless of CTB incorporation. These results demonstrate that vaccination with the H12 fragment stimulates long-lasting protective immunity. The adjuvant properties exhibited by the fibronectin-binding domain of the SfbI protein strength the potential of this antigen for inclusion in multi-component vaccines against S. pyogenes.

Introduction

Streptococcus pyogenes has a mucosal portal of entry, thus, bacterial colonization plays a key role in the pathogenesis of infection [1], [2]. S. pyogenes can cause localized infections, such as pharyngitis, tonsillitis or scarlet fever, as well as invasive and life-threatening diseases, such as sepsis, necrotizing fasciitis and toxic shock-like syndrome [1]. S. pyogenes infections can also lead to severe post streptococcal autoimmune diseases, such as rheumatic fever, rheumatic heart disease and acute post-streptococcal glomerulonephritis [1], [3]. The human suffering and rising costs associated with S. pyogenes infections, as well as the global emergence of antibiotic resistant strains, render necessary the development of a vaccine able to confer protection, without leading to cross reactions with host tissues [4], [5], [6], [7].

The fibronectin-binding protein I (SfbI) of S. pyogenes is a multifunctional protein which (i) mediates bacterial attachment to host cells, thereby promoting the subsequent colonization of the upper respiratory tract, (ii) acts as invasin, mediating bacterial internalization into non phagocytic cells, and (iii) binds to the Fc fragment of human IgG, thereby interfering with Fc-receptor-mediated phagocytosis and antibody-dependent cell cytotoxicity [8], [9], [10], [11], [12], [13], [14]. Since the fibronectin-binding domain of the SfbI protein is (i) highly conserved, (ii) surface exposed, (iii) expressed by a large number of clinical isolates from different serotypes, and (iv) does not cross-react with human tissues [12], [13], [14], [15], it represents a promising candidate antigen for inclusion in vaccine formulations against S. pyogenes.

We have recently shown that intranasal immunization with either purified recombinant SfbI or a truncated derivative spanning the fibronectin-binding domains (H12 fragment) confers protection against lethal challenge with homologous or heterologous S. pyogenes strains [16], [17]. The obtained results suggested that under our experimental conditions the vaccine preparation need to be administered by mucosal route to promote protective immunity. Although similar antibody titers were stimulated in animals vaccinated with SfbI by intraperitoneal route, they were marginally protected, if any at all [16]. This is in agreement with the well-established concept that the elicitation of local immune responses following mucosal vaccination prevents infection and subsequent disease development [18].

Antigens administered by mucosal route are usually poor immunogenic, due to accelerated clearance, degradation by local enzymes or poor penetration. Therefore, they need to be co-administered with mucosal adjuvants. Unfortunately, only a few molecules have been characterized so far, which possess activity as mucosal adjuvants [19], [20], [21], [22], [23]. In our original vaccination protocol, we used as mucosal adjuvant the B subunit of cholera toxin (CTB), since it was successfully employed in different experimental systems [24], [25], [26], [27], [28], [29], [30], [31]. However, recent studies have shown that intranasal administration of bacterial toxins, toxoids or even B subunits alone promote a GM1-dependent retrograde transport of antigen and adjuvant into neuronal tissues [32], [33]. This raises serious concerns regarding the safety of vaccine candidates containing derivatives of bacterial toxins as adjuvants.

Previous studies have shown that the SfbI protein acts per se as a mucosal adjuvant when coupled to an antigen, resulting in improved cellular and humoral responses at both systemic and mucosal level [34]. Recent work has demonstrated that the adjuvanticity of the SfbI protein does not depend on antigen coupling, since immune responses against co-administered antigens are also stimulated [35]. Interestingly, different sub-domains of the SfbI protein (e.g. the H12 fragment) can also act as mucosal adjuvants [35]. In this work, we have investigated if intranasal vaccination with the fibronectin-binding domain alone (i.e. the H12 fragment) is sufficient to stimulate protective immunity against S. pyogenes. The obtained results demonstrated that intranasal vaccination with the H12 fragment alone confers long-lasting protection against bacterial challenge.