Protective immune responses to a multi-gene DNA vaccine against Staphylococcus aureus

Abstract

To investigate the strategy of using a multivalent polyprotein DNA vaccine against Staphylococcus aureus, a series of plasmids was used to immunize mice followed by infectious challenge. The plasmid vaccines expressed Clumping factor A (Clfa), fibronectin binding protein A (FnBPA) and the enzyme Sortase (Srt) as single proteins or combined as a polyprotein. All animals produced a mixed Th1 and Th2 response including functional antigen-specific, mostly IgG2a antibodies, sustained production of IFN-γ and a predominantly CD8+ T-cell response. Upon challenge with a virulent S. aureus isolate (Sa042), after 21 days, 55% of the multi-gene vaccinated mice survived infection compared to only 15% of the control groups. Vaccinated mice showed no signs of arthritis when challenged with the less virulent “Newman” strain that caused reactive arthritis in the controls. The results suggest that a multi-gene polyprotein-expressing nucleic acid vaccine alone produces a combined Th1 and Th2 response that can contribute to protection against the complex pathogenesis of S. aureus.

Introduction

Staphylococcus aureus is a common Gram-positive bacterium frequently found on the skin and in the tonsils where it is rarely pathogenic [1]. However, under circumstances of stress or weakened immunity, this bacterium can cause diverse diseases ranging from a mild skin condition to endocarditis, septic arthritis and even septicemia causing death. In the past, these infections were successfully treated with antibiotics. However, in the last 15 years, there has been an increase in the number and the virulence of antibiotic resistant strains of S. aureus commonly called the “super-bug”. These methicillin resistant strains, once confined to hospitals, have recently been found in the general community [2], [3]. Even more ominous are the reports of the isolation of vancomycin resistant strains, although at present they are still confined to hospitals [4].

Few persistent attempts were made to develop vaccines against human S. aureus while antibiotherapy was so successful. Yet, because of the economic importance of the S. aureus bovine mastitis, several bacterin-based vaccines were developed for the prevention of this disease. However, none of these early anti-mastitis vaccines has been consistently effective in preventing infection in cattle. More recently, many countries have adopted policies to reduce the use of antibiotics and consequently renewed efforts have been made to develop vaccines against both bovine and human S. aureus infections [5]. Promising results have been obtained using immunogenic derivatives of the bacterial capsular polysaccharide [5], [6] or using antigens associated with virulence and infectivity [7]. In this latter context, the major group of virulence factors, the bacterial adhesins, has been examined as vaccine candidates. Adhesin proteins such as fibrinogen(Clfa)-, fibrinonectin(FnBp)-, elastin(Eap)- and collagen(CNA)-binding proteins are responsible for the adherence of the bacterium to the extra-cellular matrix [8], [9], [10], [11], [12], [13]. Most of these proteins are cleaved by the protease sortase before being inserted into the cell wall. Sortase is specific for the LPXTG motif present on almost all adhesins [14], [15]. Studies using negative mutants for Clfa, Fnbp A and sortase A have shown reduced septic death and arthritis in mice [16], [17]. Subunit vaccines based on recombinant Clfa or FnBp or fused proteins such as Cna and FnBp [18], [19] have been reported to induce partially protective immune responses against S. aureus infection. However, each type of infection by S. aureus has its own characteristics. The principal way for S. aureus to enter the body is via the bloodstream so the bacteria have easy access to all organs to cause septicemia. In the case of septic arthritis the bacteria must penetrate the joint where it can proliferate in the synovial fluid causing inflammation, infiltration by polymorphonuclear cells and irreversible damage to bone and cartilage. To complicate the situation further, recent studies have confirmed that S. aureus is both an intra-cellular and extra-cellular pathogen [20], [21]. It has the ability to invade and replicate within neutrophils, osteoblasts, and mammary epithelial cells [22]. Bacteria ostensibly hidden from the antibody response may be reactivated when the cell dies and, in cattle, may be the cause of increased susceptibility to mastitis in animals that were thought to have recovered from the disease. These intra-cellular bacteria may also be the cause of chronic susceptibility to septic arthritis in patients who have been cured of obvious signs of infection [23], [24] Thus, an efficient vaccine would need to interfere with both the extra- and intra-cellular forms of the bacterium. A combined Th1 and Th2 response would thus be preferable against S. aureus since the vaccinated animal would be protected against both aspects of the pathogenesis. Previous studies that employed recombinant adhesion proteins as vaccines against S. aureus lead to a mostly humoral response and partial protection against infectious challenge [18], [25].

One of the emerging technologies that can potentially promote a mixed response, is DNA vaccination. Molecular adjuvants can be included as part of the plasmid in order to direct the immune response to the desired cytokine profile. This technology also allows incorporation of more than one antigen into the vaccine. DNA vaccination does not involve expensive recombinant technology or growth of large quantities of bacteria [26], [27]. However, DNA immunization is complicated because the immune response to these vaccines depends on several parameters: the structure of the antigen [28], the type of adjuvant included in the vaccine [29], [30], [31], the vaccine vehicle [32], [33] and the route of vaccine injection [28], [34]. Early successes for plasmid vaccination against viral diseases have been followed by reports of potentially protective vaccination against bacteria such as Clostridum botulinum[35], S. aureus MRSA [36], [37] and Mycobacterium tuberculosis[38], [39]. Recently, DNA vaccines against mastitis strains of S. aureus have been tested with some success by us using adhesin genes [40], [41] and by Kerro-Dego et al. [42] who targeted the cell surface proteins.

Only recently has the first DNA vaccine been approved for commercial use against West Nile Virus in horses because DNA vaccines are normally not very effective in large animals. Thus, most of the research in this field has focused on techniques which improve the efficiency of the immunization [27]. One of these optimization procedures has been the inclusion of multiple genes in the plasmid. This has been reported to induce better protection than a mixture of the individual plasmids [33], [38], [43]. The fusion of two antigens Ag85B and MPT64 in a multi-gene plasmid against M. tuberculosis elicited higher antibody and cytotoxic responses than individual genes [38]. The current project was designed to study the effects of multiple antigens expressed as a polyprotein by a multi-gene vaccine. This report describes the construction and characterization of DNA vaccines containing three genes: clfa, fnbp and sortase A. As previously mentioned, the three corresponding proteins have important roles in the development of S. aurens-induced septic arthritis and septicemia [13], [14], [15], [16], [17]. These were cloned in separate plasmids and in a plasmid that expressed a single fusion polyprotein containing all three antigens. Mice were immunized with the plasmids and the immune responses were characterized. The results of this analysis and the protective effects of the vaccine on mouse models of septic arthritis and septicemia were compared in order to identify the effects of the multi-gene DNA vaccine strategy.