Curr Microbiol (2012) 65:813818 DOI 10.

1007/s00284-012-0238-9

A Combined Use of Autolysin p60 and Listeriolysin O Antigens Induces High Protective Immune Responses Against Listeria monocytogenes Infection
Xuenong Luo Xuepeng Cai

Received: 8 August 2012 / Accepted: 31 August 2012 / Published online: 23 September 2012 Springer Science+Business Media New York 2012

Abstract Listeria monocytogenes is an intracellular bacterium responsible for listeriosis in both humans and animals. Infected livestock is believed to be one source of this pathogen. Vaccination is an optimal approach to control the occurrence of this disease in livestock. However, inactivated vaccines have been reported to be insufcient to offer immune protection against L. monocytogenes. Here we evaluated the immune protection capacity of a combination of recombinant p60 and LLO. Mice immunized with p60 and LLO generated a high level of anti-L. monocytogenes antibodies. In addition, the elevated levels of IFN-c and the decreased levels of IL-4 were also observed in these treated mice. Consistent with the colonization of L. monocytogenes post infection, all mice in the control group died within 5 days after infection of L. monocytogenes, while 40, 40, 80, and 100 % of animals immunized with inactivated L. monocytogenes vaccine (ILMV), LLO ? ILMV, p60 ? ILMV, and p60 ? LLO ? ILMV, respectively, survived for 2 weeks. Collectively, the results presented in this study demonstrate the capacity of a combination of LLO and p60 to elicit high protective immune responses against L. monocytogenes infection.

Introduction Listeria monocytogenes is a Gram-positive intracellular bacterial pathogen that infects animals as well as humans
X. Luo (&) X. Cai Key Laboratory of Zoonoses of CAAS, Key Laboratory of Veterinary Parasitology of Gansu Province, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, CAAS, Xujiaping 1, Yanchangbu, Lanzhou 730046, Gansu, China e-mail: xuenongluo@yahoo.com.cn

through food chains, being a great threat to human and animal health [7]. Recently, outbreaks of listeriosis in humans frequently occurred due to contaminated food in Canada and United States of America and other countries [2, 3, 9, 12, 13]. One source of this pathogen is infected livestock. In Northern Spain, a survey of 343 herds including sheep, beef and dairy cattle, and pigs showed that the L. monocytogenes positive rate of dairy cattle, beef cattle, and sheep herds was 46.3, 30.6, and 14.2 %, respectively [5]. A potential approach of controlling listeriosis caused by L. monocytogenes is to eliminate the spread of this disease via vaccination in target animals. Previous studies have shown that inactivated or avirulent L. monocytogenes is not able to elicit protective immune responses largely due to incapacity for induction of IFN-csecreting T cells [23]. Listeriolysin O (LLO) is a main virulent factor of L. monocytogenes and a member of the cholesterol-dependent family of related pore-forming cytolysins expressed by diverse species of Gram-positive bacteria [1]. LLO is primarily responsible for blocking phagosomelysosome fusion by generating small pores that uncouple pH and calcium gradients across the phagosome membrane [19]. In addition, this protein has also shown to promote escape of the bacterium from phagosomes [15]. Another virulent factor of L. monocytogenes is *60 kDa secreted autolysin p60 that is known to be involved in cell division and the generation of long laments [14]. This protein is characterized as a murein hydrolase based on homology to a repeat domain of an autolysin in Enterococcus faecium [8]. p60 is highly immunogenic and its overexpression induces autolysis in L. monocytogenes [10, 22]. p60 may act on infected cells to indirectly enhance NK cell activation and increase innate IFN-gamma production, which presumably promotes early

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bacterial expansion through its immunoregulatory effects on bystander cells. Thus, the simultaneous induction of IFN-gamma production and factors that inhibit the IFNgamma signaling may be a common strategy for misdirection of early antibacterial immunity [11]. It has been shown that these two virulent factors are likely to play a role in induction of protective immunity [16, 21]. LLO induces T cells-mediated protective immunity against L. monocytogenes through induction of IFN-gamma production at an early stage. It is postulated that LLO inhibits differentiation of Th2 cells and the Th2 immune response [24]. The co-administration of extracted LLO with killed bacteria in mice partially induced protective immune responses against L. monocytogenes infection [23]. These results inspire us to investigate the immune response properties of combined use of LLO and p60. Here, we co-administrated puried recombinant LLO and p60 with inactivated L. monocytogenes in mice to evaluate the possibility of this combination being used as a vaccine.

LLO, respectively. After transformation into E coli BL21, recombinant strains were proliferated and induced with IPTG at 37 C for 4 h for pET-p60 and for 3.5 h for pETLLO. Cells were harvested by centrifugation and lysed by three rounds of freezing and thawing. Recombinant p60 and LLO proteins were puried using nickel-charged agarose resin (NiNTA; GE, USA) in the presence of 100 mM or 200 mM imidazole, respectively. Preparation of Vaccine and Immunization in Mice 50 ll of L. monocytogenes was added into 5 ml of brain heart infusion broth (BHI) and incubated with shaking (230 rpm) at 37 C overnight. Formaldehyde was added to a nal concentration of 0.5 % w/v and incubated for 12 h at room temperature to inactivate L. monocytogenes. In order to evaluate inactivation efcacy, treated cells were spread on BHI plates and colony forming units (CFU) were counted after incubation for 18 h at 37 C. Cells were recovered by centrifugation and resuspended in PBS at 1010 cells/ml. Inactivated L. monocytogenes vaccine (ILMV) was prepared by emulsifying with an equal volume of Montanide ISA206 adjuvant (Seppic, France). p60- and LLO-supplemented vaccines were prepared by the addition of puried recombinant proteins to the ILMV (1010 cells/ ml) prior to emulsication: (i) p60-supplemented vaccine (p60 ? ILMV) containing 1 mg/ml of puried p60; (ii) LLO-supplemented vaccine (LLO ? ILMV) containing 0.3 mg/ml of puried LLO; and (iii) p60 and LLO-supplemented vaccine (p60 ? LLO ? ILMV) containing 1 mg/ml of puried p60 and 0.3 mg/ml of puried LLO. Mice were randomly divided into ve groups of ten animals each. Mice in different experimental groups were subcutaneously immunized with 0.2 ml of ILMV, p60 ? ILMV, LLO ? ILMV, and p60 ? LLO ? ILMV. PBS emulsied with adjuvant of Montanide ISA206 was also used to immunize mice as a negative control. Boost was given 21 days post immunization. Blood was collected from mouse tail and sera were stored at -20 C for later use. Serological Antibody Determination Sera from immunized animals were analyzed using indirect enzyme-linked immunosorbent assay (ELISA) to determine the levels of antibodies against L. monocytogenes. In order to prepare L. monocytogenes lysate, cells were recovered by centrifugation, resuspended in PBS, and disrupted using ultrasonication combined with 3 rounds of freezing and thawing, followed by centrifugation to collect the supernatant. 100 ll of the supernatant was coated in each well. After incubation for 2 h at 37 C, plates were

Materials and Methods Strain and Mice L. monocytogenes serotype 4b strain was purchased from American Type Culture Collection (ATCC, 19115). BALB/C mice were purchased from Institute of Biological Products, Lanzhou, China. All animals were housed in an animal facility in Lanzhou Veterinary Research Institute, China. The experimental protocol was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China. Preparation of Recombinant Antigens Genomic DNA of L. monocytogenes was extracted using DNA extraction kit (Omega Biologicals, USA), and LLO and p60 fragments were amplied by PCR using primers containing internal restriction sites as follows: forward 50 ACC GAA TTC ATG AAA AAA GCA ACT ATC GCG GCT-30 and reverse 50 -ACC CTC GAG CTA AGC TTT TCC AAG GTG TTT TTG A-30 for p60; forward 50 -CGG GAT CCA AGG ATG CAT CTG CAT-30 and reverse 50 CCG CTC GAG CTA TTC GAT TGG ATT ATC-30 for LLO. PCR products were digested with EcoR I and Xho I (p60) or by Sal I and Xba I (LLO) and then ligated into predigested expression vector pET30a (Novagen, USA). The positive plasmids were designated as pET-p60 and pET-

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815

rinsed three times with PBS containing 0.5 % Tween-20 (PBST). Mouse sera diluted 1:200 in PBST were then added and incubated for 2 h at 37 C. After washing with PBST, 100 ll of diluted sheep anti-mouse IgG horseradish peroxidase conjugate (1:1000, Sigma) was added to each well, incubated for 1 h at room temperature, and then washed ve times with PBST. Color was developed by addition of 100 ll of O-phenylenediamine dihydrochloride (OPD) peroxidase substrate solution (Sigma) to each well and incubation for 0.5 h at room temperature. Optical density was measured at 490 nm using a microplate reader (Bio-Rad, USA). Detection of IL-4 and IFN-c in Sera The concentration of IL-4 and IFN-c in mouse sera was determined using ELISA kits (RB, USA) strictly according to the manufacturers instructions. Challenge Infection All the mice were intravenously injected with 2 9 106 CFU of viable L. monocytogenes 2 weeks after boost and monitored over a period of 2 weeks. Mice were sacriced and spleens were homogenized, washed with 1 % peptone PBS, and resuspended in 1 % peptone PBS (10 ml/g spleen). Aliquots (100 ll) or serial dilutions were spread in modied McBride Listeria Agar (MMA) plates and CFU were counted after incubation for 18 h at 37 C. The mean titers of live bacteria were expressed as CFU/g and relative geometric growth rates were calculated by equation: R = lg (bacterial number in each experimental group)/lg (bacterial number in group PBS) 3 100 %. Statistical Analysis Graphpad Prism 5 software was employed for data analysis. Signicant differences were determined by Dunnetts test. In all comparisons, a P value less than 0.05 was considered statistically signicant.

Results Serum Antibodies in Immunized Mice As shown in Table 1, the increase in antibody levels in the p60 ? ILMV, LLO ? ILMV, and p60 ? LLO ? ILMV groups was signicantly greater (P \ 0.05) than that in the control and ILMV groups 1 week after rst immunization, and the difference was further enhanced after 2 weeks (P \ 0.01). However, similar high antibody titers were observed in all immunization groups except PBS control 14 days post boost (P [ 0.05). IL-4 and IFN-c Levels in Immunized Mice Compared to the PBS control, a signicant reduction of IL-4 levels was observed in the other groups 14 days post immunization (P \ 0.05). The levels of IL-4 were signicantly lower in the p60 ? LLO ? ILMV and p60 ? ILMV groups than the others 7 days post immunization (P \ 0.05, Fig. 1). Two weeks post boost, the IL-4 levels in the p60 ? LLO ? ILMV and p60 ? ILMV groups decreased to 11.56 pg/ml. Decrease of IL-4 levels was signicantly larger in the groups p60 ? ILMV and p60 ? LLO ? ILMV than that of either the ILMV or LLO ? ILMV group 7 days post immunization and later on (P \ 0.05). The IL-4 levels between the p60 ? ILMV and p60 ? LLO ? ILMV groups were not signicant (P [ 0.05). One week post immunization, IFN-c concentration in mice immunized with p60 ? LLO ? ILMV and LLO ? ILMV was signicantly higher (P \ 0.05) than that in the other groups (Fig. 2). The IFN-c levels in the p60 ? LLO ? ILMV group peaked at 44.58 pg/ml 14 days post boost. Notably, there was no signicant difference of IFN-c levels between the ILMV and PBS groups (P [ 0.05). The increase in the IFN-c levels in the p60 ? ILMV group was not signicantly different from that in the ILMV group, whereas the IFN-c level in the LLO ? ILMV group were signicantly higher than that in the p60 ? ILMV group (P \ 0.05).

Table 1 Geometric mean titers (GMT) of anti-L. monocytogenes antibodies in immunized mice Group PBS ILMV p60 ? ILMV LLO ? ILMV p60 ? LLO ? ILMV BI 1:12 1.32 1:13 2.64 1:11 2.54 1:12 3.75 1:10 2.63 7 days PI 1:8 1.64 1:54 3.15a 1:98 2.76* 1:95 2.56* 1:104 2.28* 14 days PI 1:11 1.82 1:201 2.11** 1:401 3.65**,* 1:396 2.81** * 1:411 3.21** *
, ,

7 days PB 1:14 2.28 1:487 1.79** 1:792 3.86**,* 1:785 3.65** * 1:807 2.68** *
, ,

14 days PB 1:13 2.68 1:821 2.34** 1:591 4.28**,* 1:589 3.68**,* 1:619 2.48**,*

The data were expressed as mean SD (n = 10). GMTs were represented as reactive dilutions PI post immunization, PB post boost, BI before immunization
a

0.01 \ P \ 0.05 compared to group PBS; ** P \ 0.01 compared to group PBS; * 0.01 \ P \ 0.05 compared to group ILMV

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Fig. 3 Survival curve of immunized mice (10 mice per group)

Fig. 1 Serological IL-4 levels in immunized mice. The data were expressed as mean SD (n = 5). BI before immunization, PI post immunization, PB post boost

immunized with p60 ? LLO ? ILMV (*73.1 CFU/g). Difference of the titer of L. monocytogenes between p60 ? ILMV group and LLO ? ILMV group was not signicant.

Discussion In this study, we demonstrated that the combined use of recombinant LLO and p60 with killed bacteria offered complete protection against L. monocytogenes infection, which was further evidenced by the fact that the colony number of L. monocytogenes was signicantly decreased in the spleens of mice administrated with p60 ? LLO ? ILMV in contrast with p60 ? ILMV and LLO ? ILMV. This result suggests that the combination of LLO and p60 is capable of induction of protective immune responses that effectively repress the colonization of L. monocytogenes in immunized animals. It was shown that the levels of IL-4 were signicantly decreased in mice immunized with p60 ? ILMV or p60 ? LLO ? ILMV, while the levels of IFN-c were signicantly up-regulated in mice immunized with LLO ? ILMV or p60 ? LLO ? ILMV. In addition, no signicant increase of IFN-c secretion was observed in the ILMV and p60 ? ILMV groups, suggesting that LLO is a major contributor that induced the elevated secretion of IFN-c in immunized mice. These results are in agreement with a pervious study which reported that LLO plus killed bacteria induced IFN-c and IL-12 expression, which resulted in the generation of protective immunity [23]. IFN-c and IL-4 are key indicators of T cell activation, and changes in the serum levels of these two cytokines reect T helper (Th) cell differentiation [6]. Th cells convert to Th1 cells in response to an increase in the IFN-c/IL-4 ratio, thereby enhancing host defense against infection [4, 18]. In our present experiments, 1 week post immunization the IFN-c/IL-4 ratio was signicantly increased in mice treated with p60 ? LLO ? ILMV, suggesting that the recombinant proteins are likely to promote conversion of Th to Th1, thus enhancing cellular immune responses.

Fig. 2 Serological IFN-c levels in immunized mice. The data were expressed as mean SD (n = 5). BI before immunization, PI post immunization, PB post boost

Challenge Infection and Protection 3 days after injection with L. monocytogenes, dead mice were observed in the PBS group but not in other groups (Fig. 3). All mice in the control group died 5 days post inoculation, while the majority of animals treated with LLO ? ILMV, p60 ? ILMV, and p60 ? LLO ? ILMV survived. After 2 weeks, 60 % (6/10) and 80 % of animals in the LLO ? ILMV and p60 ? ILMV groups survived, respectively, whereas 100 % of animals immunized with p60 ? LLO ? ILMV survived. In order to address whether animal survival was paralleled with reduced in vivo colonization of L. monocytogenes, bacterial titer was measured using the spleens of challenged animals 48 h post infection. As shown in Table 2, the titer of L. monocytogenes was signicantly higher in the PBS control (*1.4 3 108 CFU/g) than the other groups (P \ 0.01). There was a marked reduction in the ILMV group (*1.2 3 106 CFU/g) compared to group PBS, but the lowest titer of L. monocytogenes with a geometric mean count rate of 22.86 % were found in mice

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X. Luo, X. Cai: Autolysin p60 and Listeriolysin O Table 2 Colonization of L. monocytogenes in the spleens of the immunized mice Group Titera R
b

817

PBS 1.42 3 108 5314A

ILMV 1.2 3 106 345.5B 74.57 %

p60 ? ILMV 5.4 3 103 96.6C 45.78 %

LLO ? ILMV 8.3 3 103 112.4C 48.07 %

p60 ? LLO ? ILMV 73.1 6.5D 22.86 %

The data were expressed as mean SD (n = 5) , not applicable

a

CFU/g spleen; b R: geometric growth rate calculated by equation: log (bacterial number in each experimental group)/log (bacterial number in group PBS) 3 100 % P \ 0.01 if any upper right capital letters between groups were different

Although the IFN-c levels were not signicantly different, complete immune protection against L. monocytogenes infection was elicited in mice immunized with p60 ? LLO ? ILMV but not LLO ? ILMV. This suggests that the addition of recombinant p60 antigen signicantly enhances protective immune responses. A recent study showed that p60 activated host innate immune response via the nuclear factor kB (NF-kB) pathway and stimulated mouse macrophages to secrete tumor necrosis factor (TNF)-a potentially via Toll-like receptor 4 [16]. p60 has also been shown to act on infected cells to indirectly enhance natural killer (NK) cell activation and induce IFNc secretion [11], which is closely associated functionally with the N-terminal LysM and SH3 domains of p60 [17]. In rats, NK cells are likely to eliminate L. monocytogenes infection through recognition of alterations in the MHC-I molecules on infected cells [20]. Although no signicant difference of IFN-c secretion between p60 ? LLO ? ILMV and LLO ? ILMV was observed, it is feasible that p60 modulates other immune effector cells such as NK cells and macrophages to facilitate L. monocytogenes clearance. Of particular interest is to explore the roles of NK cells and macrophages in mice immunized with p60 ? LLO ? ILMV in future experiments.

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Conclusions The present study has conrmed the capacity of p60 ? LLO ? ILMV combination to elicit highly protective immune responses against L. monocytogenes infection. The combination vaccine developed in this study is likely to rival live attenuated L. monocytogenes vaccines in terms of efcacy while offering signicant advantages in terms of safety.
Acknowledgments This work was supported by National Key Technology R&D Program (2007BAD40B01), the Ministry of Science and Technology, and Earmarked Fund for China Agriculture Research System (CARS-40-10), the Ministry of Agriculture, China. The authors would like to thank Bangxin Zhou, Xueliang Zhu, Songbo Zhao, and Shuai Wang for their help in animal experiments.

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