Development and evaluation of a new recombinant protein vaccine (YidR) against Klebsiella pneumoniae infection

Abstract

Vaccination is a promising approach to prevent Klebsiella infection; however, the high heterogeneity of strains is a limiting factor. The best antigenic target for an anti-Klebsiella vaccine should be expressed by all or most of strains. We previously found YidR protein to be highly conserved among K. pneumoniae strains independently of antigen serotype. Therefore, in the present study, we developed a recombinant YidR protein vaccine and evaluated its protective efficacy against lethal challenge with K. pneumoniae in a mouse model. The yidR gene was cloned in Escherichia coli for recombinant expression. The lethal dose (LD₁₀₀) of K. pneumoniae was determined and lethal challenge was carried out after immunization with recombinant purified YidR. After immunization, the concentration of total serum IgG was significantly higher in YidR-immunized mice than in non-immunized mice, indicating strong induction of antibodies. Mice were challenged with LD₁₀₀ of K. pneumoniae, and significantly lower murine sepsis and higher body weight were observed in YidR-immunized mice compared to unvaccinated controls. Moreover, ∼90% of YidR-immunized mice survived beyond 10 days of observation, whereas none of the control mice survived past 48 h. The protective effect of YidR recombinant protein vaccine was demonstrated and YidR may be a promising vaccine candidate to prevent klebsiellosis.

Introduction

Klebsiella pneumoniae is a gram-negative, encapsulated, non-motile bacterium [1] that belongs to the Enterobacteriaceae family [2]. This bacterium is extremely resilient, having the ability to evade, survive, and suppress many components of the immune system and grow at different sites in the host [1]. The bacterium has a large accessory genome of chromosomal gene loci and plasmids, which divides the strains into opportunistic, hypervirulent (hypervirulent K. pneumoniae, kvKP), and multidrug resistant variants [3]. The antibiotic resistance of this pathogen has been increasing [3], [4], [5], [6], [7], [8], [9]. The highly antibiotic-resistant strains can also act as opportunists and are extremely difficult to treat [3]. Consequently, the high prevalence of resistant strains has become a public health concern worldwide [4], [10]. Treatment with antibiotics may have limited efficacy owing to the increased prevalence of infections by drug-resistant strains and adverse reactions caused by the use of high or prolonged doses [4]. According to the Center for Disease Control and Prevention (CDC) (US CDC report – Antibiotic Resistance Threats 2013), more than 9000 healthcare-associated infections were reported in the United States due to carbapenem-resistant Enterobacteriaceae (CRE) each year and it is estimated that 7900 cases were caused by carbapenem-resistant Klebsiella spp. This bacterium is also considered the most common cause of hospital-acquired pneumonia in the United States [11]. Even with optimal therapy, lung infection results in 30–50% mortality [11].

As control methods for K. pneumoniae infection are already challenging, the emergence of multidrug-resistant strains highlights the importance of developing preventive measures [12]. Immunization against Klebsiella has been discussed in the context of its pathogenesis and identification of protective antigens as possible vaccine candidates [13]. Previous studies have attempted to develop vaccines against K. pneumoniae [2], [12], [14], [15], [16], [17], [18], [19], [20], [21]. However, with one exception, none of the preparations have been commercialized due to their cost and approach [4]. The exception, K. pneumoniae siderophore receptor protein (SRP) vaccine (Kleb-SRP) [21], is currently used for reduction of Klebsiella mastitis in lactating cattle. Nevertheless, Klebsiella may be difficult to control by vaccines due to strain heterogeneity [4]. The ideal target for an anti-Klebsiella vaccine should be expressed by all strains, unlike the O- and K-antigens [2].

Previously, we described the pan-genomic profiles, virulence profile, genomic structure, and resistome of 308 K. pneumoniae isolates from dairy cows and humans [22]. A unique IncN-type plasmid (pC5) co-harboring the blaCTX-M-1 and mph(A) genes was identified in two dairy farms and the complete annotated sequence of the plasmid was generated [22]. Furthermore, 177 functional protein families were significant across all isolates, and siderophore systems were prevalent in both the bovine and human isolates [22]. That study also revealed a previously undescribed profile of virulence determinants of K. pneumoniae isolates [22].

From the our existing genomic database [22], we noticed the ubiquitous occurrence of the yidR gene (308/308, 100%) in bovine and human isolates. Moreover, and importantly, the gene was found to be highly conserved between the different isolates, with an overall sequence homology of 97.6%. This prevalence and sequence conservation led us to study yidR as a potential target antigen against K. pneumoniae. The yidR gene encodes a putative ATP/GTP-binding protein which mediates the hyperadherence phenotype [23], [24] and contributes to biofilm formation in Salmonella enterica [24]. The YidR protein contains two conserved domains which are associated with Tol-dependent translocation of colicins into Escherichia coli [25], and it is implicated in the pathogenesis of Enterobacteriaceae [26]. Therefore, in the present study, we developed a new recombinant protein vaccine for prevention of K. pneumoniae infection based on the highly conserved YidR protein, and evaluated the vaccine’s protective efficacy against lethal challenge with K. pneumoniae in a mouse model.