Introduction

Listeria monocytogenes, a Gram-positive, rod-shaped, facultatively anaerobic, intracellular bacterium, is the causative agent of listeriosis, a disease in human acquired from consumption of contaminated foods [16]. Listerial infection, most frequently found in immunocompromised patients, elderly, infants, and pregnant women, is characterized by meningitis, meningoencephalitis, septicemia, gastroenteritis, and abortion or serious infection of the newborn [20]. An understanding of the humoral immune response mounted against L. monocytogenes is of great interest, because its definitive role in protective immunity is unknown. The humoral immune response is normally detected during listerial infection even in the absence of clinical symptoms [1, 13].

Earlier studies by Mackaness and others demonstrated that serum taken from mice infected with a sublethal dose of L. monocytogenes provided no resistance against infection when passively transferred to naive mice [14]. Focus has then been placed on cellular immunity, with almost no attention paid to the role of antibodies in the immune defense against Listeria infection. This has led to the establishment of the crucial role of cell-mediated immunity in resistance to Listeria and intracellular pathogens [7]. These cellular defense mechanisms include cells such as neutrophils, macrophages, NK cells, and targeted T cells [7]. Antibodies are generally considered ineffective in protection against listerial infection.

Listeria-specific antibodies have been demonstrated in patients with listeriosis [8, 10, 19] and in naturally or experimentally infected animals such as sheep [9, 13], goats [18], and cattle [5, 21]. The protein antigens eliciting antibody response include the 58-kDa extracelluar listeriolysin O (LLO), the internalin-related protein IrpA, the internalins InlA and InlB, the ActA protein involved in actin-based intracellular motility of L. monocytogenes, and the autolysin p60 [2, 3, 4, 8, 10]. Other antigens, presently still unidentified and uncharacterized, are those with apparent molecular weights of 160, 106, and 93 kDa recognized by IgG or IgM antibodies in sera of humans or rabbits infected with L. monocytogenes [6, 19] and those with molecular weights of 96, 60, 40, and 14 kDa detected by IgA antibodies from the culture of small intestine fragments of mice orally infected with an actA– mutant of L. monocytogenes [15]. To fully elucidate the role of antibodies in protection against listerial infection, studies aiming to identify and characterize new antibody targets are necessary.

Recently we have identified, through differential immunoscreening of a L. monocytogenes genomic expression library, genes coding for several proteins that react with antibodies in serum (RαL) from rabbits infected with live L. monocytogenes serotype 4b (strain LI0521) but not with antibodies in serum (RαK) from animals immunized with heat-killed bacteria [22, 23]. These proteins, i.e., three internalins (InlA, InlD, InlC2) and five novel proteins of unknown function (designated IspA, IspB, IspC, IspD, and IspE, respectively), were interpreted as in vivo induced or upregulated antigens during infection that may thus be important in Listeria pathogenesis. In fact, InlA is a known virulence factor involved in mediating the entry of bacteria into host cells [20]. Among these identified immunogenic proteins, InlA and InlC2 were found to react much more strongly with RαL during screening of the library. This study was undertaken to further demonstrate that InlA and InlC2 were the important antigens for humoral immune response against listerial infection in several animal species.

Materials and Methods

Bacterial Culture

L. monocytogenes LI0521 (serotype 4b) was cultured in LB broth containing 50 mM MOPS (pH 7.0) at 37°C and enumerated by measuring the OD620 of culture and plating as described [23].

L. monocytogenes Whole-cell Antigens and ELISA

The whole-cell antigens used in enzyme-linked immunosorbent assay (ELISA) to assess the humoral immune response to L. monocytogenes were prepared from live L. monocytogenes serotype 4b by sonication [22]. The ELISA was carried out essentially as described previously [23].

Antisera from Rabbits Infected with L. monocytogenes

Two kinds of rabbit antisera were produced through an intravenous (iv) injection route in a previous study (23): one (RαL) from rabbits infected with live L. monocytogenes and the other (RαK) from rabbits immunized with heat-killed bacterial cells. Also, two New Zealand rabbits, after collection of preimmune sera at day 0, were inoculated orally at day 1 with 1 × 109 viable bacteria in 1 ml saline and at day 30 with the same dose to produce another kind of antisera. The animals were bled weekly at days 7, 14, 21, 28, 35, and 44 and sacrificed at day 45 to collect a large volume of blood (sera).

Antisera from Guinea Pigs Infected with L. monocytogenes

Four guinea pigs were orally administered 1 × 109 viable cells in 1 ml saline after collection of preimmune sera. Another group of guinea pigs was subcutaneously immunized with the same dose of heat-killed bacteria. Animals were bled or scarified to collect antisera at various days as described above for rabbits.

Antisera from Sheep Infected with L. monocytogenes

Four 1-year-old sheep of the Arcott and Oxford breeds (males; approximately 82 kg) were divided into two groups and bled to collect preimmune sera at day 0. In Group 1, two sheep received an oral dosage of viable L. monocytogenes (6 × 1010 cells in 1 ml saline) at day 1 and challenged orally with the same number of live cells at day 30. In Group 2, two sheep were immunized subcutaneously with 6 × 1010 heat-killed L. monocytogenes in 1 ml saline at days 1 and 30, respectively. The animals were bled weekly to monitor the antibody (IgG + IgM) titer using ELISA. At day 44, antibody titers were low for both groups of sheep. These animals were further inoculated with the same doses of bacteria at day 58. Antibody responses were found to have increased considerably in two animals in Group 2, which were sacrificed at day 70 to collect antisera. To obtain a high titer of antibody response to infection with live L. monocytogenes, the two animals in Group 1 were infected at day 135 by iv injection of 6 × 1010 viable cells in 1 ml saline and bled weekly to monitor IgG antibody responses. Following the iv injection, these two sheep developed symptoms of of listeriosis and were treated with tetracycline accordingly. At day 163 a lower dose of live L. monocytogenes (6 × 109 cells) was given intravenously to the two sheep, resulting in the death of one animal at day 184. The other animal was sacrificed to collect antisera at day 216.

Antisera from Bovine Infected with L. monocytogenes

Antisera from two cows experimentally infected with L. monocytogenes [21] were donated by Dr. Irene Wesley, National Animal Disease Center, Ames, Iowa.

Western Blots

Western blots were performed essentially as described [23]. Protein samples for SDS-PAGE (12%) analysis were E. coli Novablue (DE3; Novagen, Madison, WI) cell lysates containing recombinant InlA (amino acids [aa] 397 to 800) with an N-terminal fusion encoded by pSCN34 or recombinant InlC2 (aa 178 to 490) with fusions at both N- and C-terminal ends specified by pSCN92 [23]. Proteins, after electrotransfer onto nitrocellulose membranes, were probed with preimmune sera or antisera at predetermined dilutions from rabbits, guinea pigs, cattle, or sheep and then immunostained with alkaline phosphatase-conjugated goat or donkey anti-species IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in the presence of nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.

Results and Discussion

Antibody (IgG and IgM) responses of rabbits to L. monocytogenes following infection with viable bacteria or immunization with heat-killed cells are shown in Fig. 1. Administration of both bacterial preparations yielded a significant level of anti-L. monocytogenes antibody responses in the rabbits, as revealed by ELISA. The antisera RαL and RαK were used earlier to probe a L. monocytogenes genomic expression library, leading to the discovery of several antibody-reactive bacterial proteins that are believed to be specifically induced or upregulated during infection [23]. Based on the observation that the clones coding for InlA and InlC2 reacted much more strongly with RαL than any other identified proteins during screening of the library, they were hypothesized as two of the major antigens for humoral immune responses during infection. This study aimed to investigate the reactivity of InlA and InlC2 with antisera from several animal species infected with L. monocytogenes. Both truncated InlA (aa 397 to 800) expressed from pSCN34 and truncated InlC2 (aa 178 to 490) expressed from pSCN92 reacted strongly with RαL but not with RαK on Western blots (Figs. 2a and b). Preimmune rabbit sera showed no reaction with the proteins (data not shown). Antisera from two rabbits that were orally administered with 1 × 109 live L. monocytogenes reacted similarly with InlA and InlC2 on Western blots as shown in Fig. 2a.

Fig. 1
figure 1

ELISA of antibody responses of rabbits infected with live L. monocytogenes or immunized with heat-killed bacteria. The microtiter plates were coated with the whole-cell antigen preparation (100 μl, 30 μg/ml) diluted in PBS, and rabbit sera at a dilution of 1:800 were used. Rabbits 1 (○) and 2 (●) were intravenously injected with viable bacteria (1 × 106); rabbits 3 (Δ) and rabbit 4 (▾) were intravenously injected with heat-killed bacteria (1 × 109). The data points represent the mean of two determinations for each serum sample collected at various days postinfection or postimmunization

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Fig. 2
figure 2

Western blot analysis of recombinant InlA and InlC2 with sera from several animal species experimentally infected with L. monocytogenes. Dilutions of 1:400 for rabbit antisera RαL (a) or RαK (b), 1:400 for bovine antisera (c), 1:1200 for guinea pig antisera (d), and 1:800 for sheep antisera (e) were used to probe the blots. Each lane contains total proteins from E. coli cells equivalent to 1 ml of culture with an OD590 of 0.02. Lane 1, E. coli harboring the cloning vector pSCREEN1b+ EcoRV; Lane 2, the pSCRN34 clone expressing truncated InlA; Lane 3, the pSCRN92 clone expressing truncated InlC2. Protein standards with their molecular masses in kilodaltons are indicated on the left

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To further demonstrate whether InlA and InlC2 are targeted by humoral immune responses of other hosts against L. monocytogenes, antisera from cattle (Fig. 2c), guinea pigs (Fig. 2d), and sheep (Fig. 2e) experimentally infected with L. monocytogenes were used to detect InlA and InlC2 on Western blots. Both proteins reacted strongly with the antisera from all infected animals (Fig. 2), while all preimmune sera did not react with either protein (data not shown). Consistent with the findings with RαK, no reaction of InlA and InlC2 with the antisera from guinea pigs or sheep immunized with heat-killed bacteria was observed.

InlA, a known virulence factor, mediates the entry of L. monocytogenes into those normally nonphagocytic epithelial cells through interaction with the E-cadherin receptor [20]. Monoclonal antibodies to the leucine-rich repeat region of InlA can block entry of the bacterium into cells expressing E-cadherin [17]. Demonstration of antibodies to InlA in infected hosts suggests a possible role of these specific antibodies in immune defence against listerial infection. Boerlin et al. [4] described the use of recombinant InlA (aa 2–710) and LLO (aa 26–529) as antigens in ELISAs for the specific detection of anti-L. monocytogenes antibodies in cattle and showed that the InlA ELISA outperformed the LLO ELISA in terms of specificity and sensitivity. The data reported here are consistent with the ELISA results of Boerlin et al. [4] and indicate that antibody response to InlA upon infection is common in various infected hosts.

Although InlC2 belongs to the internalin family, its biological function has yet to be defined. This study demonstrated for the first time the presence of antibodies to InlC2 in various hosts infected with L. monocytogenes. It should be noted that other RαL-reactive L. monocytogenes proteins (i.e., InlD, IspA, IspB, IspC, IspD, and IspE) either reacted weakly or showed no reaction with the antisera from infected bovine or guinea pigs [22, 23]. Collectively the results indicate that InlA and InlC2 are the in vivo induced or upregulated antigens for humoral immune response against L. monocytogenes infection. Similar approaches based on differential antibody responses have been reported in literature for identification of in vivo induced microbial protein antigens during infection for several bacterial pathogens (see references in Ref. 23). Given that rabbits and guinea pigs used in the present study are recognized as good animal models of human infection with L. monocytogenes [11, 12], it may be expected that antibody responses to InlA and InlC2 observed in these animal models would be similarly induced in listeriosis patients. Identification of the two antigens will allow further studies of specific antibodies in protection immunity in the context of listerial infection. In addition, these proteins may be instrumental in the development of diagnostic tests and new antimicrobial agents or vaccines.