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Oral administration of a live attenuated Salmonella vaccine strain expressing the VapA protein induces protection against infection by Rhodococcus equi
Microbes and Infection 9 (2007) 382e390 www.elsevier.com/locate/micinf
Original article
Oral administration of a live attenuated Salmonella vaccine strain expressing the VapA protein induces protection against infection by Rhodococcus equi Aline F. Oliveira a, Luciana C. Ferraz a, Marcelo Brocchi b, Maria-Cristina Roque-Barreira a,* a
Departamento de Biologia Celular e Molecular e Bioagentes Patogeˆnicos, Faculdade de Medicina de Ribeir~ao Preto, Universidade de S~ao Paulo, Av. Bandeirantes, 3900, 14049-900, Ribeir~ao Preto, S~ao Paulo, Brazil b Departamento de Microbiologia e Imunologia, Instituto de Biologia, Universidade Estadual de Campinas, 1308-3862, Campinas, S~ao Paulo, Brazil Received 27 August 2006; accepted 15 December 2006 Available online 12 January 2007
Abstract Rhodococcus equi remains one of the most important pathogens of foals and vaccination strategies to prevent rhodococcosis are under increasing investigation. Attenuated Salmonella strains carrying heterologous antigens offer an advantageous alternative to conventional vaccines, especially because they induce mucosal and systemic immunity. In this work, we expressed the VapA antigen from R. equi in a Salmonella enterica Typhimurium strain, which was able to colonize and persist in the lymphoid tissue of BALB/c mice. Two days after being challenged, oral immunized mice presented a 3- to 7-fold increase in R. equi clearance. This was progressively enhanced during infection and, on the 10th day, a CFU value 50-fold lower than that recovered from non-immunized mice was attained. The number of hepatic granulomas was 2 times lower, and leukocyte infiltration was transiently detected in immunized mice, contrasting with the severe inflammation and necrosis presented by nonimmunized mice. Infection with 1 107 R. equi CFU caused 100% mortality in the control groups, while all immunized mice survived. This protection was associated with the detection of high levels of anti-VapA IgG in the serum of the vaccinated mice, predominantly the IgG2a isotype. Our results suggest that attenuated Salmonella encoding VapA may be used in foals to prevent rhodococcosis. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Rhodococcus equi; VapA antigen; Salmonella enterica; Vaccine
1. Introduction Rhodococcus equi is one of the most important bacterial pathogens affecting young foals world wide [1]. Rhodococcal infection results in pyogranulomatous pneumonia, which is sometimes accompanied by extrapulmonary manifestations [1]. R. equi has also been associated with severe pneumonia in immunocompromised humans [2]. Virulent R. equi strains contain a large 85- to 90-kb plasmid bearing a 27.5-kb pathogenicity island that encodes, among
* Corresponding author. Tel.: þ55 16 36023062; fax: þ55 16 36331786. E-mail address: [email protected] (M.-C. Roque-Barreira). 1286-4579/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2006.12.019
others, 7 genes of the virulence-associated protein (vap) family [3]. One member of this family is VapA, a 15e17 kDa protein that is highly immunogenic, and abundantly expressed on the bacterial surface [3,4]. Deletion of the vapA gene results in attenuation and rapid clearance of the mutant bacteria in mice, showing that VapA is a virulence factor [5]. Furthermore, VapA is thought to be important in inducing immunity against R. equi [6,7]. Current procedures for R. equi control in foals are inconvenient and not always effective. Antibiotic therapy is expensive, prolonged, and not consistently successful [1]. Although many studies have been developed in order to obtain protective vaccines [8], no immunization procedure is currently available for the effective prevention of R. equi pneumonia in foals.
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The use of attenuated Salmonella strains as a vector for the delivery of heterologous antigens had already been shown to induce protective immune responses in a variety of animal models [9,10]. Such studies indicate that Salmonella is a suitable vaccine vector, especially because it is able to elicit all forms of immunity: mucosal, systemic, and humoral responses [11]. Other aspects place oral vaccination with bacterial carriers in advantage when compared with parenteral vaccines. Oral vaccines are easy to administer, safe, adequate for large-scale immunization, and stable without refrigeration (if lyophilized). In the present work we have determined whether mice immunization with attenuated Salmonella enterica Typhimurium c3987 carrying the VapA antigen could provide protection against subsequent challenge with virulent R. equi. 2. Materials and methods 2.1. Bacterial strains, plasmids, and growth conditions Escherichia coli c6212 Dasd strain [12,13] has been used in cloning experiments involving the pYA3137 plasmid. The S. enterica Typhimurium c3987 Dcya Dcrp Dasd attenuated strain [12] was employed here for expression of the VapA antigen. This was accomplished by cloning the vapA gene in the pYA3137 plasmid. The pYA3137 and pYA3137vapA plasmids were mobilized to the S. enterica strain via transduction using the bacteriophage P22HT [14]. Bacterial strains were grown in Luria Broth (LB) medium, in a rotary shaker at 250 rpm, at 37 C. Ampicillin (50 mg/mL) or tetracycline (12.5 mg/mL) was added if required for the selection of transformants or to maintain plasmids. For the preparation of bacterial suspensions for administration in mice, overnight cultures of the S. enterica Typhimurium c3987 strains were precipitated by centrifugation (3000 g; 15 min), and the pellet was re-suspended in phosphate-buffered saline (PBS) to a final cell density of 1e5 1010 CFU/mL. CFU was determined by plating dilutions of the bacterial suspension onto MacConkey agar plates. The virulent R. equi strain ATCC33701, kindly provided by Dr. Shinji Takai (University of Kitasato, Towada, Japan), was cultured in braineheart infusion broth (BHI, Oxoid, Hampshire, England). Cultures were incubated in a rotary shaker at 100 rpm, 37 C, for 60 h. For mice inoculation, bacterial cultures were washed in PBS; actual numbers of inoculated bacteria were confirmed by plate counts at the injection time. Plasmid DNA extraction was performed as described previously [15]. Plasmids were purified using an Eppendorf plasmid purification kit (Eppendorf, Hamburg, Germany).
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GCT-30 ) were used to create the BamHI and the SalI sites flanking this gene. The PCR product was then cloned in the same sites of the pYA3137 plasmid (provided by Dr. Roy Curtiss III e Washington University, Saint Louis, Missouri, USA) in such a way that it would be transcribed constitutively from the strong trc promoter. The presence of mutations in the vapA gene was ruled out by sequencing the entire gene. 2.3. SDS-PAGE and western blotting analysis Cultures of S. enterica Typhimurium c3987 were prepared as described previously [16]. Bacterial extracts were analyzed by SDS-PAGE using 12% polyacrylamide gels. Gels were stained with Coomassie blue or electroblotted onto nitrocellulose membranes for immunological analysis with anti-VapA rabbit antibodies (kindly provided by Dr. Sa€ıd Taouji-Laboratoire d’$E´tudes et de Recherche en Pathologie Equine, IPC, Goustranville, Dozule, France) at a dilution of 1:2000. Horseradish peroxidase-conjugated anti-rabbit antibody (Sigma, Saint Louis, USA) was used for detection. 2.4. Persistence and colonization of S. enterica Typhimurium c3987 recombinant strains in mice organs Two groups of twelve 7-week-old female BALB/c mice were inoculated intragastrically with 1 109 CFU of either c3987-pYA3137vapA or c3987-pYA3137 re-suspended in 200 mL PBS using a gavage needle. On days 3, 10, and 20, groups of 4 mice were sacrificed by cervical dislocation, and spleens and Peyer’s patches were removed. Tissues were homogenized in PBS, and aliquots of 100 mL were plated onto MacConkey agar. Plates were incubated at 37 C for 24 h, for bacterial counting. 2.5. Mice immunization Groups of BALB/c mice were intragastrically immunized, on days 0 and 14, with 1 109 CFU of either c3987pYA3137vapA or c3987-pYA3137 re-suspended in 200 mL PBS. In a 3rd group, mice received 200 mL PBS only. 2.6. Survival of immunized mice following challenge with R. equi Groups of 12 immunized mice were challenged intravenously with a lethal dose (1 107 or 1 108 CFU) of the virulent R. equi strain, on day 14 after the last immunization. All the mice were monitored for survival throughout the experimental period.
2.2. Construction of pYA3137vapA
2.7. Bacterial clearance from tissues of infected mice
A DNA fragment corresponding to the 562-bp vapA gene was amplified by polymerase chain reaction (PCR) using the R. equi virulence plasmid as template. The primers vapA1 (50 -GCGCCGGATCCATGAAGACTCTTCACAAGACGGTT30 ) and vapA2 (50 -GCGCCGTCGACCTAGGCGTTGTGCCA
Orally immunized mice were intravenously challenged with a sublethal dose (4 106 CFU) of virulent R. equi 14 days after the last immunization. On days 2, 4, 8, and 10 postchallenge, groups of 4 mice were sacrificed and their spleens, livers, and lungs were removed. Tissues were homogenized
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and aliquots of 100 mL were plated onto BHI agar. The plates were incubated at 37 C for 24 h, for bacteria counting. 2.8. Histopathological examination of infected mice Groups of 4 mice were sacrificed on days 2, 4, 8, and 10 post-challenge and the spleen, liver, and lung fragments were fixed in 10% formalin. After tissue processing, sections (5 mm thick) were stained with haematoxylineeosin. The number of granuloma/mm2 of hepatic tissue was obtained using an optical microscope with the aid of an integrator lens (Carl Zeiss Jena, Germany). The granuloma area (mm2) was measured by means of KS-100 program (Carl Zeiss Jenamed 2, USA). 2.9. Detection of specific antibodies Blood samples of 4 mice were collected on days 7, 14, 21, 28, 35, and 42 after the 2nd immunization. Antibody responses to R. equi were analyzed by enzyme-linked immunosorbent assay (ELISA), using microtitre plates coated with 1 mg/well of recombinant VapA protein. For IgG detection, sera were diluted 1:30, 1:60, 1:120, 1:240, and 1:480. For isotype detection, sera were diluted 1:20. Reactions were developed by using goat anti-mouse IgG, IgG1, or IgG2a, conjugated with horseradish-peroxidase (Santa Cruz Biotechnology), diluted 1:5000. 2.10. Statistical analysis Statistical analysis was performed using a general linear model (GLM) for repeated measures. Results are presented as the mean and SEM. Tukey’s test was used for multiple comparisons and Student’s t-test was used to compare results for Salmonella persistence in the spleen and Peyer’s patches. Statistical analyses were performed using SPSS statistical software package, version 14.0 (SPSS, Inc., Chicago, IL). 3. Results 3.1. Cloning of the vapA gene into pYA3137 The vapA gene was amplified from the genomic DNA of R. equi and the amplified sequence was cloned into the pYA3137 plasmid. The presence of the intact vapA gene as well as the correct orientation of the cloned gene (Fig. 1A) was confirmed by sequencing this gene and by analyzing the restriction map of the recombinant plasmid using agarose gel electrophoresis (data not shown). 3.2. VapA expression in the S. enterica Typhimurium c3987 strain Oral vaccination with recombinant attenuated Salmonella carrying a foreign antigen is an attractive option for inducing
Fig. 1. Gene cloning and expression of the VapA protein by the transformed S. enterica Typhimurium c3987. Bacterial extracts were separated by SDSPAGE, transferred onto nitrocellulose, and reacted with rabbit anti-VapA serum. (A) Gene cloning. (B) Coomassie blue stained SDS-PAGE. Lanes: (1) whole extracts of control strain (S. enterica Typhimurium c3987-pYA3137); (2) whole extracts of transformed strain (S. enterica Typhimurium c3987pYA3137vapA). (C) Western blotting. Proteins from whole extracts of control strain (lane 1) and transformed strain (lane 2), separated by SDS-PAGE, were transferred onto nitrocellulose membranes. Immunodetection was performed with rabbit anti-VapA serum.
humoral and cellular immune responses. We therefore cloned the vapA gene into the pYA3137 vector and expressed the protein in an S. enterica Typhimurium Dcya Dcrp Dasd strain. According to the SDS-PAGE and Western blotting analyses, the VapA protein was present in the protein extracts of c3987 transformed with pYA3137vapA (Fig. 1B and C, lane 2), but not in the extracts of c3987 transformed with pYA3137 alone (Fig. 1B and C, lane 1). 3.3. Host tissue colonization by transformed strains of S. enterica Typhimurium c3987 The in vivo stability of c3987-pYA3137vapA was assessed by determining the colonization of the Salmonella strains in murine tissues. Following the intragastric inoculation of both transformed S. enterica Typhimurium c3987, namely c3987-pYA3137 and c3987-pYA3137vapA, there was colonization of the spleen and Peyer’s patches during the 20-day
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observation period (Fig. 2A and B). There was no significant difference between the number of bacteria recovered from the organ of mice inoculated with c3987-pYA3137 or c3987-pYA3137vapA (Fig. 2), which indicates that cloning of the vapA gene into pYA3137 did not affect the bacterial ability to colonize the host tissue. The colonization and long persistence of the recombinant Salmonella strains in the mice tissues enable sustained expression of the foreign antigen and triggers vigorous immune response. 3.4. Survival of immunized mice against challenge with R. equi Animals immunized with c3987-pYA3137vapA and control animals inoculated with either c3987-pYA3137 or PBS were intravenously challenged with 1 107 or 1 108 CFU of the virulent R. equi strain and monitored for survival for 20 days (Fig. 3). All control mice infected with 1 108 CFU died by day 6 following the challenge (Fig. 3A). On the other hand, only 20% of the mice immunized with c3987-pYA3137vapA died after the challenge (Fig. 3A). All control mice succumbed to infection with 1 107 CFU by day 8 and 10 post-challenge (Fig. 3B). In contrast, in the group of mice receiving c3987pYA3137vapA, no death had occurred until day 20 after challenge with 1 107 CFU (Fig. 3B).
Fig. 3. Survival of R. equi challenged mice following oral immunization with S. enterica Typhimurium c3987-pYA3137vapA. BALB/c mice (8 per group) were immunized on days 0 and 14 with either S. enterica Typhimurium c3987-pYA3137 (close squares) or S. enterica Typhimurium c3987-pYA3137vapA (close triangles). In animals of a 3rd group, the immunization procedure was replaced by PBS injection (open circles). Mice were intravenously challenged with 1 108 CFU (A) or 1 107 CFU (B) of R. equi ATCC33701 14 days after the last immunization. Graphs show the percentage of immunized animals surviving to challenge.
3.5. Bacterial clearance from tissues of mice infected with R. equi
Fig. 2. Colonization of spleen (A) and Peyer’s patches (B) of BALB/c mice following oral inoculation with S. enterica Typhimurium c3987 carrying plasmids pYA3137 (grey bars) or pYA3137vapA (black bars). Organs collected from 4 animals 3, 10, or 20 days after inoculation were homogenized in PBS and plated onto MacConkey agar for bacteria counting.
In order to investigate if the low mortality observed in the immunized mice was related with decreased bacterial burden in their organs, immunized and control mice were intravenously challenged with a sublethal dose (4 106 CFU/mL) of virulent R. equi 2 weeks after the last immunization. Groups of 4 mice were sacrificed 2, 4, 8, and 10 days post-challenge, and their lung, liver, and spleen were removed for bacteria counting. Fig. 4 shows the kinetics of CFU recovery from the tissues of mice of all the groups. There was no significant difference between the high number of R. equi CFU recovered
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from the 2 control groups. Animal immunization with c3987pYA3137vapA was associated with a lower CFU counting. Bacteria recovery within the first 48 h post-challenge was approximately 7-fold higher in the lung, 3-fold higher in the spleen, and 4-fold higher in the liver of the control mice challenged with R. equi than in the organs of immunized mice. By days 4 and 8, CFU recovered from the lung, liver, and spleen of the control mice reached levels approximately 16-, 7-, and 5-fold higher, respectively, than those recovered from organs of immunized mice. On day 10 post-infection, the bacterial number declined in the 3 groups of animals, a decrease that was much more pronounced in the immunized mice group. Thus, R. equi clearance was more efficient in mice that received c3987-pYA3137vapA than in the 2 control groups. These data suggest that the vaccination procedure enhanced mice resistance to R. equi infection.
3.6. Histopathological examination
Fig. 4. CFU recovered from mice organs following R. equi infection. BALB/c mice were orally immunized with S. enterica Typhimurium c3987-pYA3137vapA (closed triangles), or inoculated with either S. enterica Typhimurium c3987-pYA3137 (closed squares) or PBS (open circles) on days 0 and 14. Mice were intravenously challenged with 4 106 CFU of R. equi ATCC33701 14 days after the last immunization. Bacterial burdens in the lung (A), liver (B), and spleen (C) were determined on days 2, 4, 8, and 10 after infection. The difference between the number of bacteria recovered from the immunized group and the control groups was statistically significant (*P < 0.001 e Tukey’s test).
Histology of the lung, liver, and spleen sections was performed to evaluate whether the resistance of immunized mice was associated with fewer lesions attributable to R. equi infection. For this, immunized mice and control mice were challenged with 4 106 CFU of virulent R. equi. Animals were sacrificed on days 2, 4, 8, and 10 after infection. In the case of the spleen of the control animals, formation of pyogranulomas with extensive areas of central necrosis was detected on days 2 (Fig. 5A) and 4 (data not shown) postinfection. In contrast, the immunized mice presented no apparent inflammatory reaction at any time of infection (Fig. 5B). There was an intense inflammatory process in the liver of the control mice, characterized by several multifocal granulomas on days 2 and 4 (data not shown) post-infection. On days 8 and 10 (data not shown), the inflammatory reaction decreased, although several granulomas were still present in the tissue. During all the analyzed periods, the inflammatory process in the liver of the control mice was associated with extensive necrosis areas, as shown for 10 days post-infection (Fig. 5C). In contrast with the severe hepatic lesions observed in the control mice, the inflammatory process was mild in the liver of VapA-immunized animals. In all the analyzed days, no hepatocyte necrosis was visualized (Fig. 5D). Morphometric analysis demonstrated that the number of hepatic granulomas was lower in the liver of VapA-immunized mice because, by day 2, the density of granulomas/mm2 of tissue was 9 for the immunized mice and 14 for the control mice. Although the granuloma density decreased in the 3 groups, this decrease was much more pronounced in immunized mice. On day 10, immunized mice provided a counting of 2 granulomas/mm2 versus 6 granulomas in the tissue of the control mice (Fig. 5I). Even though the number of granulomas/ mm2 of tissue was lower in the immunized animals, the total area occupied by the granulomas was significantly higher in the liver of these animals (Fig. 5J). On day 4 after challenge, granulomas occupied maximum hepatic area in mice of all the 3 groups. As time elapsed, the occupied hepatic area decreased
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Fig. 5. Histopathology of the spleen, liver and lung sections and morphometric analysis of the hepatic granulomas of BALB/c mice orally immunized with S. enterica Typhimurium c3987-pYA3137vapA, or inoculated with S. enterica Typhimurium c3987-pYA3137 and challenged intravenously with 4 106 CFU of virulent R. equi 14 days after the last immunization. The organs were collected 2 days (A, B, E, and F), 4 days (G and H) and 10 days (C and D) after infection. The 5-mm sections of the spleen (A and B), liver (C and D) and lung (EeH) of the mice were stained with hematoxylineeosin. The asterisks indicate necrosis areas and the arrows indicate the presence of small inflammatory infiltrates. Panel I: sections of the liver obtained on different days of infection (2, 4, 8, and 10) were analyzed by an optical microscopy with the aid of an integrator lens (Carl Zeiss Jena, Germany), to obtain the number of granulomas/mm2 of tissue; each bar represents the average SD of liver sections from 4 animals. Panel J: the granuloma area (mm2) was determined by means of KS-100 program (Carl Zeiss Jenamed 2, USA). The data represent the average SD of liver sections from 4 animals. At least 10 granulomas were analyzed in each tissue section (***P < 0.001; **P < 0.01; *P < 0.05 e Tukey’s test).
in all groups. However, this area was persistently higher in the liver of immunized mice (Fig. 5J). The lungs of the control mice showed extensive lesions characterized by abundant perivascular and peribronchiolar inflammatory infiltrate, obliterating the alveolar spaces of the lung parenchyma, predominantly on days 2 and 4
post-infection (Fig. 5E and G). In the same period, only a discrete infiltration was observed in the lung of immunized mice (Fig. 5F and H). In conclusion, the microscopic examination revealed clearcut differences in the severity of the lesions and in the time course of infection between immunized and control mice.
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3.7. Detection of VapA-specific antibody The anti-VapA IgG titration showed that a strong response was developed by the mice immunized with the recombinant Salmonella c3987-pYA3137vapA, 2 weeks after the last immunization (Fig. 6 upper). Such a response was maintained for more than 6 weeks (data not shown) and
was at least 2-fold higher in the VapA-vaccinated mice than in the control mice. The time course detection of both IgG1 and IgG2a isotypes specific for VapA in the serum of immunized mice showed that IgG2a levels were consistently higher than IgG1 titers (Fig. 6 lower). The IgG2a levels were 2 orders of magnitude higher than the IgG1 levels in all the analyzed days (Fig. 6 lower). The wide dominance of IgG2a strongly suggests that a specific Th1-type response was triggered by immunization with Salmonella c3987-pYA3137vapA. 4. Discussion
Fig. 6. Humoral response elicited by vaccination with Salmonella expressing the VapA protein. BALB/c mice were orally immunized on days 0 and 14 with S. enterica Typhimurium c3987-pYA3137vapA (VapA), or inoculated with either S. enterica Typhimurium c3987-pYA3137 (plasmid) or PBS (PBS). On different days after the last immunization, the serum samples were collected and assayed by ELISA. The recombinant VapA protein was used as the coating antigen. Each bar and symbol represents the average SD of serum from 4 animals in duplicates. (Upper) Total antigen-specific IgG production in different sera dilutions (1:30, 1:60, 1:120, 1:240, and 1:480), 2 weeks after the last immunization. The production of total IgG by immunized mice was significantly higher than that of the control mice (**P < 0.001; *P < 0.01 e Tukey’s test). (Lower) Isotype response at 7, 14, 21, 28, 35, and 42 days after the last immunization. The levels of IgG2a isotype detected were significantly higher than the IgG1 levels in immunized mice (VapA) and it was significantly higher than the production of IgG2a by the 2 control mice (**P < 0.001 e Tukey’s test).
In the present study we have shown that the oral immunization with a live attenuated S. enterica Typhimurium strain expressing the VapA protein confers protection against R. equi challenge in the mouse model of infection. Several vaccination strategies have already been tested as an attempt to prevent the development of rhodococcosis. Immunization procedures using avirulent or killed R. equi did not induce a protective immune response. In spite of the protection conferred against experimental challenge [8,17], virulent R. equi administration cannot be used as a vaccination approach because of the risk of provoking disease and contaminating the environment. VapA has been identified as an important virulence factor required for R. equi replication in host macrophages [5]. Immunization procedures with synthetic peptides representing epitopes of VapA [18] and DNA expressing the vapA gene [19] resulted in the development of a specific immune response associated with variable degrees of protection. Such reports have supported the selection of this R. equi antigen to be expressed in an attenuated S. enterica strain in order to produce a vaccinal preparation. Therefore, the vapA gene was cloned in the pYA3137 vector. The latter is a high-copy-number plasmid, belonging to the asd ‘balanced/lethal system’, which allows a high and stable expression of the heterologous protein [13,20]. Plasmid instability results in reduced bacterial colonization of the host tissues, which will affect the vaccine efficacy [21]. There is evidence that some recombinant Salmonella strains can lose their colonization capacity or can faintly colonize the host tissues [22]. On the other hand, oral immunization with S. enterica Typhimurium expressing heterologous antigens has been associated with bacterial colonization of the host Peyer’s patches, mesenteric lymph nodes and spleen for a long time [23]. In the present study, S. enterica Typhimurium c3987 transformed with either pYA3137vapA or pYA3137 plasmids were inoculated in mice and monitored for their in vivo persistence. In both cases, bacteria were persistently detected in the lymphoid tissues for at least 20 days, indicating that the VapA antigen expression did not affect the ability of Salmonella to colonize host tissues. The fact that bacteria recovered from mice tissues still maintained the pYA3137vapA plasmid demonstrated the stability of the recombinant bacteria. Bacterial clearance is considered a good way to assess the efficacy of a vaccinal approach against rhodococcosis [19,24].
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We compared the R. equi clearance in VapA-immunized mice and in mice of the control groups. Immunization with c3987pYA3137vapA was associated with significant reduction in R. equi recovery from the organs, in all the analyzed periods. A similar reduction in R. equi CFU has been reported for vapA-DNA-immunized mice challenged with R. equi [19]. Mouse protection provided by immunization with c3987pYA3137vapA was manifested early after R. equi challenge. On the 2nd day of infection, 3 times less CFU was recovered from immunized mice than from animals of the control groups. In spite of being challenged with a large inoculum, mice were efficiently protected by the vaccination, which suggests that the humoral and cellular immune responses required to eliminate R. equi [25] were acting in concert. The observations described here are consistent with previous demonstrations that Salmonella vaccine strains are effective in inducing heterologous antigen-specific responses [26]. Histopathological analysis of the organs from infected mice reinforced the ideas raised by the bacterial clearance assay. After being challenged with R. equi, mice immunized with c3987-pYA3137vapA developed a milder inflammatory response only, probably as a consequence of bacterial elimination. In all the analyzed periods, no changes were detected in the spleen tissue and no necrosis was visualized in the liver. In the lung, small inflammatory infiltrates were present on days 2 and 4 post-challenge. In contrast, the tissues of the control animals presented, especially during the first 4 days of infection, all of the most frequent histopathological changes were reported for the mouse model of rhodococcosis, such as intense inflammatory reaction and extensive areas of hepatic necrosis. In the lung, an abundant cellular infiltrate obliterating the alveolar septum was detected. The occurrence of hepatic granuloma formation, which is the most characteristic pathological change in the organs of R. equi infected animals [24], was about 2-fold higher in control mice than in VapAimmunized animals. The area of individual granulomas was persistently lower in the control mice. This suggests that the formation of new granulomas occurred in non-immunized mice only, in an attempt to restrain bacterial proliferation and dissemination to other tissues. In addition, granulomas in the organs of immunized mice were more compact, which corresponds to an adequate organization that avoids dissemination of the non-eliminated bacteria. The time course decrease in R. equi CFU is consistent with the progressive reduction of the granuloma area, observed in all the mice groups studied here. The survival of animals immunized with c3987-pYA3137vapA was 100% in the group challenged with 107 CFU/mL and 80% with 108 CFU/mL of R. equi, whereas all the control animals died before the 10th day post-infection. A similar protection was provided by mice immunization with S. enterica Typhimurium expressing the Sm14 antigen of Schistosoma mansoni [27]. The development of humoral immune response is important to induce protection against R. equi infection [28]. High titers of anti-VapA IgG were detected in the serum of vaccinated mice, with prominence of IgG2a. The differential production
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of cytokines has important effects on the Ig isotype [29], and the high ratio of IgG2a to IgG1 antibodies specific for VapA is consistent with a heightened Th1 response, since IFN-g is required to stimulate IgG2a secretion and is associated with inhibition of IgG1 production [30]. Indeed, the immunization using attenuated recombinant Salmonella has already been reported as inducing a Th1-type response preferentially [26]. In conclusion, we have demonstrated for the first time that the mice vaccination with attenuated S. enterica Typhimurium expressing the VapA protein is able to confer protection against experimental rhodococcosis. The specific immune response triggered by mice immunization and the protective effect induced by foal vaccination are both under investigation in our laboratories. Acknowledgements We thank Vani M.A. Correa, Sandra M.O. Thomaz, Antonio R.M. e Silva, and Patricia E. Vendrusculo for excellent technical assistance. We also acknowledge Julio A. Siqueira, Cristiane C.P. Ribas, Savio H.F. Miranda, and Ednelson A. Mazzotto for expert animal care. We are thankful to Dr. Luciana P. Ruas, Dr. Sandro G. Soares, Dr. Ebert S. Hanna, Dr. Vaˆnia S. Mariano, and Dr. Leandro L. Oliveira for helpful discussions. This work was partially supported by Fundac¸~ao de Amparo a` Pesquisa do Estado de S~ao Paulo (FAPESP) and Conselho Nacional de Pesquisa Cientı´fica e Tecnolo´gica (CNPq). A.F.O. received a scholarship from CNPq. This work generated patent. PI 0506364-7 (filed December 21, 2005). References [1] S. Giguere, J.F. Prescott, Clinical manifestations, diagnosis, treatment, and prevention of Rhodococcus equi infections in foals, Vet. Microbiol. 56 (1997) 313e334. [2] A. Donisi, M.G. Suardi, S. Casari, M. Longo, G.P. Cadeo, G. Carosi, Rhodococcus equi infection in HIV-infected patients, AIDS 10 (1996) 359e362. [3] S. Takai, S.A. Hines, T. Sekizaki, V.M. Nicholson, D.A. Alperin, M. Osaki, D. Takamatsu, M. Nakamura, K. Suzuki, N. Ogino, T. Kakuda, H. Dan, J.F. Prescott, DNA sequence and comparison of virulence plasmids from Rhodococcus equi ATCC33701 and 103, Infect. Immun. 68 (2000) 6840e6847. [4] S. Takai, M. Iie, Y. Watanabe, S. Tsubaki, T. Sekizaki, Virulence-associated 15- to 17-kilodalton antigens in Rhodococcus equi: temperaturedependent expression and location of the antigens, Infect. Immun. 60 (1992) 2995e2997. [5] S. Jain, B.R. Bloom, M.K. Hondalus, Deletion of vapA encoding virulence associated protein A attenuates the intracellular actinomycete R. equi, Mol. Microbiol. 50 (2003) 115e128. [6] J.F. Prescott, M.C. Patterson, V.M. Nicholson, B. Morein, J.A. Yager, Assessment of the immunogenic potential of Rhodococcus equi virulence associated protein (VapA) in mice, Vet. Microbiol. 56 (1997) 213e225. [7] K.E. Hooper-McGrevy, S. Giguere, B.N. Wilkie, J.F. Prescott, Evaluation of equine immunoglobulin specific for Rhodococcus equi virulence-associated proteins A and C for use in protecting foals against Rhodococcus equi-induced pneumonia, Am. J. Vet. Res. 62 (2001) 1307e1313. [8] S. Takai, C. Kobayashi, K. Murakami, S. Yukako, S. Tsubaki, Live virulent Rhodococcus equi, rather than killed or avirulent, elicits protective immunity to R. equi infection in mice, FEMS Immunol. Med. Microbiol. 24 (1999) 1e9.
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[9] M.F. Pasetti, M.M. Levine, M.B. Sztein, Animal models paving the way for clinical trials of attenuated Salmonella enterica serovar Typhi live oral vaccines and live vectors, Vaccine 21 (2003) 401e418. [10] G. Dougan, C.E. Hormaeche, D.J. Maskell, Live oral Salmonella vaccines: potential use of attenuated strains as carriers of heterologous antigens to the immune system, Parasite Immunol. 9 (1987) 151e160. [11] C.N. Kotton, E.L. Hohmann, Enteric pathogens as vaccine vectors for foreign antigen delivery, Infect. Immun. 72 (2004) 5535e5547. [12] R. Curtiss 3rd, S.M. Kelly, Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic, Infect. Immun. 55 (1987) 3035e3043. [13] J.E. Galan, K. Nakayama, R. Curtiss 3rd, Cloning and characterization of the asd gene of Salmonella typhimurium: use in stable maintenance of recombinant plasmids in Salmonella vaccine strains, Gene 94 (1990) 29e35. [14] S.R. Maloy, V.J. Stewart, R.K. Taylor, Generalized transduction with phage P22, in: Genetic Analysis of Pathogenic Bacteria, Cold Spring Harbor Laboratory Press, New York, 1996, pp. 473e492. [15] S. Takahashi, Y. Nagano, Rapid procedure for isolation of plasmid DNA and application to epidemiological analysis, J. Clin. Microbiol. 20 (1984) 608e613. [16] J. Sambrook, E.P. Fritsch, T. Maniatis, Molecular cloning: a laboratory manual, second ed. Cold Spring Harbor Laboratory, New York, 1989. [17] J.M. Chirino-Trejo, J.F. Prescott, J.A. Yager, Protection of foals against experimental Rhodococcus equi pneumonia by oral immunization, Can. J. Vet. Res. 51 (1987) 444e447. [18] S. Taouji, I. Nomura, S. Giguere, S. Tomomitsu, T. Kakuda, V. Ganne, S. Takai, Immunogenicity of synthetic peptides representing linear B-cell epitopes of VapA of Rhodococcus equi, Vaccine 22 (2004) 1114e1123. [19] H.R. Haghighi, J.F. Prescott, Assessment in mice of vapA-DNA vaccination against Rhodococcus equi infection, Vet. Immunol. Immunopathol. 104 (2005) 215e225. [20] K. Nakayama, S.M. Kelly, R. Curtiss 3rd, Construction of an ASDþ expression-cloning vector: stable maintenance and high level expression of cloned genes in a Salmonella vaccine strain, Biotechnology 6 (1988) 693e697.
[21] N.M. Coulson, M. Fulop, R.W. Titball, Effect of different plasmids on colonization of mouse tissues by the aromatic amino acid dependent Salmonella typhimurium SL 3261, Microb. Pathog. 16 (1994) 305e311. [22] H.S. Garmory, K.F. Griffin, K.A. Brown, R.W. Titball, Oral immunisation with live aroA attenuated Salmonella enterica serovar Typhimurium expressing the Yersinia pestis V antigen protects mice against plague, Vaccine 21 (2003) 3051e3057. [23] S.J. Dunstan, C.P. Simmons, R.A. Strugnell, In vitro and in vivo stability of recombinant plasmids in a vaccine strain of Salmonella enterica var. Typhimurium, FEMS Immunol. Med. Microbiol. 37 (2003) 111e119. [24] S. Takai, H. Madareme, C. Matsumoto, M. Inoue, Y. Sasaki, Y. Hasegawa, S. Tsubaki, A. Nakane, Pathogenesis of Rhodococcus equi infection in mice: roles of virulence plasmids and granulogenic activity of bacteria, FEMS Immunol. Med. Microbiol. 11 (1995) 181e190. [25] A.M. Lopez, M.T. Hines, G.H. Palmer, D.C. Alperin, S.A. Hines, Identification of pulmonary T-lymphocyte and serum antibody isotype responses associated with protection against Rhodococcus equi, Clin. Diagn. Lab. Immunol. 9 (2002) 1270e1276. [26] U.G. Lange, P. Mastroeni, J.M. Blackwell, C.B. Stober, DNA-Salmonella enterica serovar Typhimurium primer-booster vaccination biases towards T helper 1 responses and enhances protection against Leishmania major infection in mice, Infect. Immun. 72 (2004) 4924e4928. [27] L.G. Pacheco, E. Zucconi, V.L. Mati, R.M. Garcia, A. Miyoshi, S.C. Oliveira, A.L. de Melo, V. Azevedo, Oral administration of a live Aro attenuated Salmonella vaccine strain expressing 14-kDa Schistosoma mansoni fatty acid-binding protein induced partial protection against experimental schistosomiasis, Acta Trop. 95 (2005) 132e142. [28] S.A. Fernandez, F.J. Prescott, M.V. Nicholson, Protective effect against Rhodococcus equi infection in mice of IgG purified from horses vaccinated with virulence associated protein (VapA)-enriched antigens, Vet. Microbiol. 56 (1997) 187e192. [29] C.M. Snapper, F.D. Finkelman, in: W.E. Paul (Ed.), Fundamental Immunology, third ed. Raven Press, New York, 1993, pp. 845e846 -. [30] F.D. Finkelman, I.M. Katona, T. Mosmann, R.L. Coffman, IFN-g regulates the isotype of Ig secreted during in vivo humoral immune responses, J. Immunol. 140 (1988) 1022e1027.
Original article
Oral administration of a live attenuated Salmonella vaccine strain expressing the VapA protein induces protection against infection by Rhodococcus equi Aline F. Oliveira a, Luciana C. Ferraz a, Marcelo Brocchi b, Maria-Cristina Roque-Barreira a,* a
Departamento de Biologia Celular e Molecular e Bioagentes Patogeˆnicos, Faculdade de Medicina de Ribeir~ao Preto, Universidade de S~ao Paulo, Av. Bandeirantes, 3900, 14049-900, Ribeir~ao Preto, S~ao Paulo, Brazil b Departamento de Microbiologia e Imunologia, Instituto de Biologia, Universidade Estadual de Campinas, 1308-3862, Campinas, S~ao Paulo, Brazil Received 27 August 2006; accepted 15 December 2006 Available online 12 January 2007
Abstract Rhodococcus equi remains one of the most important pathogens of foals and vaccination strategies to prevent rhodococcosis are under increasing investigation. Attenuated Salmonella strains carrying heterologous antigens offer an advantageous alternative to conventional vaccines, especially because they induce mucosal and systemic immunity. In this work, we expressed the VapA antigen from R. equi in a Salmonella enterica Typhimurium strain, which was able to colonize and persist in the lymphoid tissue of BALB/c mice. Two days after being challenged, oral immunized mice presented a 3- to 7-fold increase in R. equi clearance. This was progressively enhanced during infection and, on the 10th day, a CFU value 50-fold lower than that recovered from non-immunized mice was attained. The number of hepatic granulomas was 2 times lower, and leukocyte infiltration was transiently detected in immunized mice, contrasting with the severe inflammation and necrosis presented by nonimmunized mice. Infection with 1 107 R. equi CFU caused 100% mortality in the control groups, while all immunized mice survived. This protection was associated with the detection of high levels of anti-VapA IgG in the serum of the vaccinated mice, predominantly the IgG2a isotype. Our results suggest that attenuated Salmonella encoding VapA may be used in foals to prevent rhodococcosis. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Rhodococcus equi; VapA antigen; Salmonella enterica; Vaccine
1. Introduction Rhodococcus equi is one of the most important bacterial pathogens affecting young foals world wide [1]. Rhodococcal infection results in pyogranulomatous pneumonia, which is sometimes accompanied by extrapulmonary manifestations [1]. R. equi has also been associated with severe pneumonia in immunocompromised humans [2]. Virulent R. equi strains contain a large 85- to 90-kb plasmid bearing a 27.5-kb pathogenicity island that encodes, among
* Corresponding author. Tel.: þ55 16 36023062; fax: þ55 16 36331786. E-mail address: [email protected] (M.-C. Roque-Barreira). 1286-4579/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2006.12.019
others, 7 genes of the virulence-associated protein (vap) family [3]. One member of this family is VapA, a 15e17 kDa protein that is highly immunogenic, and abundantly expressed on the bacterial surface [3,4]. Deletion of the vapA gene results in attenuation and rapid clearance of the mutant bacteria in mice, showing that VapA is a virulence factor [5]. Furthermore, VapA is thought to be important in inducing immunity against R. equi [6,7]. Current procedures for R. equi control in foals are inconvenient and not always effective. Antibiotic therapy is expensive, prolonged, and not consistently successful [1]. Although many studies have been developed in order to obtain protective vaccines [8], no immunization procedure is currently available for the effective prevention of R. equi pneumonia in foals.
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The use of attenuated Salmonella strains as a vector for the delivery of heterologous antigens had already been shown to induce protective immune responses in a variety of animal models [9,10]. Such studies indicate that Salmonella is a suitable vaccine vector, especially because it is able to elicit all forms of immunity: mucosal, systemic, and humoral responses [11]. Other aspects place oral vaccination with bacterial carriers in advantage when compared with parenteral vaccines. Oral vaccines are easy to administer, safe, adequate for large-scale immunization, and stable without refrigeration (if lyophilized). In the present work we have determined whether mice immunization with attenuated Salmonella enterica Typhimurium c3987 carrying the VapA antigen could provide protection against subsequent challenge with virulent R. equi. 2. Materials and methods 2.1. Bacterial strains, plasmids, and growth conditions Escherichia coli c6212 Dasd strain [12,13] has been used in cloning experiments involving the pYA3137 plasmid. The S. enterica Typhimurium c3987 Dcya Dcrp Dasd attenuated strain [12] was employed here for expression of the VapA antigen. This was accomplished by cloning the vapA gene in the pYA3137 plasmid. The pYA3137 and pYA3137vapA plasmids were mobilized to the S. enterica strain via transduction using the bacteriophage P22HT [14]. Bacterial strains were grown in Luria Broth (LB) medium, in a rotary shaker at 250 rpm, at 37 C. Ampicillin (50 mg/mL) or tetracycline (12.5 mg/mL) was added if required for the selection of transformants or to maintain plasmids. For the preparation of bacterial suspensions for administration in mice, overnight cultures of the S. enterica Typhimurium c3987 strains were precipitated by centrifugation (3000 g; 15 min), and the pellet was re-suspended in phosphate-buffered saline (PBS) to a final cell density of 1e5 1010 CFU/mL. CFU was determined by plating dilutions of the bacterial suspension onto MacConkey agar plates. The virulent R. equi strain ATCC33701, kindly provided by Dr. Shinji Takai (University of Kitasato, Towada, Japan), was cultured in braineheart infusion broth (BHI, Oxoid, Hampshire, England). Cultures were incubated in a rotary shaker at 100 rpm, 37 C, for 60 h. For mice inoculation, bacterial cultures were washed in PBS; actual numbers of inoculated bacteria were confirmed by plate counts at the injection time. Plasmid DNA extraction was performed as described previously [15]. Plasmids were purified using an Eppendorf plasmid purification kit (Eppendorf, Hamburg, Germany).
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GCT-30 ) were used to create the BamHI and the SalI sites flanking this gene. The PCR product was then cloned in the same sites of the pYA3137 plasmid (provided by Dr. Roy Curtiss III e Washington University, Saint Louis, Missouri, USA) in such a way that it would be transcribed constitutively from the strong trc promoter. The presence of mutations in the vapA gene was ruled out by sequencing the entire gene. 2.3. SDS-PAGE and western blotting analysis Cultures of S. enterica Typhimurium c3987 were prepared as described previously [16]. Bacterial extracts were analyzed by SDS-PAGE using 12% polyacrylamide gels. Gels were stained with Coomassie blue or electroblotted onto nitrocellulose membranes for immunological analysis with anti-VapA rabbit antibodies (kindly provided by Dr. Sa€ıd Taouji-Laboratoire d’$E´tudes et de Recherche en Pathologie Equine, IPC, Goustranville, Dozule, France) at a dilution of 1:2000. Horseradish peroxidase-conjugated anti-rabbit antibody (Sigma, Saint Louis, USA) was used for detection. 2.4. Persistence and colonization of S. enterica Typhimurium c3987 recombinant strains in mice organs Two groups of twelve 7-week-old female BALB/c mice were inoculated intragastrically with 1 109 CFU of either c3987-pYA3137vapA or c3987-pYA3137 re-suspended in 200 mL PBS using a gavage needle. On days 3, 10, and 20, groups of 4 mice were sacrificed by cervical dislocation, and spleens and Peyer’s patches were removed. Tissues were homogenized in PBS, and aliquots of 100 mL were plated onto MacConkey agar. Plates were incubated at 37 C for 24 h, for bacterial counting. 2.5. Mice immunization Groups of BALB/c mice were intragastrically immunized, on days 0 and 14, with 1 109 CFU of either c3987pYA3137vapA or c3987-pYA3137 re-suspended in 200 mL PBS. In a 3rd group, mice received 200 mL PBS only. 2.6. Survival of immunized mice following challenge with R. equi Groups of 12 immunized mice were challenged intravenously with a lethal dose (1 107 or 1 108 CFU) of the virulent R. equi strain, on day 14 after the last immunization. All the mice were monitored for survival throughout the experimental period.
2.2. Construction of pYA3137vapA
2.7. Bacterial clearance from tissues of infected mice
A DNA fragment corresponding to the 562-bp vapA gene was amplified by polymerase chain reaction (PCR) using the R. equi virulence plasmid as template. The primers vapA1 (50 -GCGCCGGATCCATGAAGACTCTTCACAAGACGGTT30 ) and vapA2 (50 -GCGCCGTCGACCTAGGCGTTGTGCCA
Orally immunized mice were intravenously challenged with a sublethal dose (4 106 CFU) of virulent R. equi 14 days after the last immunization. On days 2, 4, 8, and 10 postchallenge, groups of 4 mice were sacrificed and their spleens, livers, and lungs were removed. Tissues were homogenized
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and aliquots of 100 mL were plated onto BHI agar. The plates were incubated at 37 C for 24 h, for bacteria counting. 2.8. Histopathological examination of infected mice Groups of 4 mice were sacrificed on days 2, 4, 8, and 10 post-challenge and the spleen, liver, and lung fragments were fixed in 10% formalin. After tissue processing, sections (5 mm thick) were stained with haematoxylineeosin. The number of granuloma/mm2 of hepatic tissue was obtained using an optical microscope with the aid of an integrator lens (Carl Zeiss Jena, Germany). The granuloma area (mm2) was measured by means of KS-100 program (Carl Zeiss Jenamed 2, USA). 2.9. Detection of specific antibodies Blood samples of 4 mice were collected on days 7, 14, 21, 28, 35, and 42 after the 2nd immunization. Antibody responses to R. equi were analyzed by enzyme-linked immunosorbent assay (ELISA), using microtitre plates coated with 1 mg/well of recombinant VapA protein. For IgG detection, sera were diluted 1:30, 1:60, 1:120, 1:240, and 1:480. For isotype detection, sera were diluted 1:20. Reactions were developed by using goat anti-mouse IgG, IgG1, or IgG2a, conjugated with horseradish-peroxidase (Santa Cruz Biotechnology), diluted 1:5000. 2.10. Statistical analysis Statistical analysis was performed using a general linear model (GLM) for repeated measures. Results are presented as the mean and SEM. Tukey’s test was used for multiple comparisons and Student’s t-test was used to compare results for Salmonella persistence in the spleen and Peyer’s patches. Statistical analyses were performed using SPSS statistical software package, version 14.0 (SPSS, Inc., Chicago, IL). 3. Results 3.1. Cloning of the vapA gene into pYA3137 The vapA gene was amplified from the genomic DNA of R. equi and the amplified sequence was cloned into the pYA3137 plasmid. The presence of the intact vapA gene as well as the correct orientation of the cloned gene (Fig. 1A) was confirmed by sequencing this gene and by analyzing the restriction map of the recombinant plasmid using agarose gel electrophoresis (data not shown). 3.2. VapA expression in the S. enterica Typhimurium c3987 strain Oral vaccination with recombinant attenuated Salmonella carrying a foreign antigen is an attractive option for inducing
Fig. 1. Gene cloning and expression of the VapA protein by the transformed S. enterica Typhimurium c3987. Bacterial extracts were separated by SDSPAGE, transferred onto nitrocellulose, and reacted with rabbit anti-VapA serum. (A) Gene cloning. (B) Coomassie blue stained SDS-PAGE. Lanes: (1) whole extracts of control strain (S. enterica Typhimurium c3987-pYA3137); (2) whole extracts of transformed strain (S. enterica Typhimurium c3987pYA3137vapA). (C) Western blotting. Proteins from whole extracts of control strain (lane 1) and transformed strain (lane 2), separated by SDS-PAGE, were transferred onto nitrocellulose membranes. Immunodetection was performed with rabbit anti-VapA serum.
humoral and cellular immune responses. We therefore cloned the vapA gene into the pYA3137 vector and expressed the protein in an S. enterica Typhimurium Dcya Dcrp Dasd strain. According to the SDS-PAGE and Western blotting analyses, the VapA protein was present in the protein extracts of c3987 transformed with pYA3137vapA (Fig. 1B and C, lane 2), but not in the extracts of c3987 transformed with pYA3137 alone (Fig. 1B and C, lane 1). 3.3. Host tissue colonization by transformed strains of S. enterica Typhimurium c3987 The in vivo stability of c3987-pYA3137vapA was assessed by determining the colonization of the Salmonella strains in murine tissues. Following the intragastric inoculation of both transformed S. enterica Typhimurium c3987, namely c3987-pYA3137 and c3987-pYA3137vapA, there was colonization of the spleen and Peyer’s patches during the 20-day
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observation period (Fig. 2A and B). There was no significant difference between the number of bacteria recovered from the organ of mice inoculated with c3987-pYA3137 or c3987-pYA3137vapA (Fig. 2), which indicates that cloning of the vapA gene into pYA3137 did not affect the bacterial ability to colonize the host tissue. The colonization and long persistence of the recombinant Salmonella strains in the mice tissues enable sustained expression of the foreign antigen and triggers vigorous immune response. 3.4. Survival of immunized mice against challenge with R. equi Animals immunized with c3987-pYA3137vapA and control animals inoculated with either c3987-pYA3137 or PBS were intravenously challenged with 1 107 or 1 108 CFU of the virulent R. equi strain and monitored for survival for 20 days (Fig. 3). All control mice infected with 1 108 CFU died by day 6 following the challenge (Fig. 3A). On the other hand, only 20% of the mice immunized with c3987-pYA3137vapA died after the challenge (Fig. 3A). All control mice succumbed to infection with 1 107 CFU by day 8 and 10 post-challenge (Fig. 3B). In contrast, in the group of mice receiving c3987pYA3137vapA, no death had occurred until day 20 after challenge with 1 107 CFU (Fig. 3B).
Fig. 3. Survival of R. equi challenged mice following oral immunization with S. enterica Typhimurium c3987-pYA3137vapA. BALB/c mice (8 per group) were immunized on days 0 and 14 with either S. enterica Typhimurium c3987-pYA3137 (close squares) or S. enterica Typhimurium c3987-pYA3137vapA (close triangles). In animals of a 3rd group, the immunization procedure was replaced by PBS injection (open circles). Mice were intravenously challenged with 1 108 CFU (A) or 1 107 CFU (B) of R. equi ATCC33701 14 days after the last immunization. Graphs show the percentage of immunized animals surviving to challenge.
3.5. Bacterial clearance from tissues of mice infected with R. equi
Fig. 2. Colonization of spleen (A) and Peyer’s patches (B) of BALB/c mice following oral inoculation with S. enterica Typhimurium c3987 carrying plasmids pYA3137 (grey bars) or pYA3137vapA (black bars). Organs collected from 4 animals 3, 10, or 20 days after inoculation were homogenized in PBS and plated onto MacConkey agar for bacteria counting.
In order to investigate if the low mortality observed in the immunized mice was related with decreased bacterial burden in their organs, immunized and control mice were intravenously challenged with a sublethal dose (4 106 CFU/mL) of virulent R. equi 2 weeks after the last immunization. Groups of 4 mice were sacrificed 2, 4, 8, and 10 days post-challenge, and their lung, liver, and spleen were removed for bacteria counting. Fig. 4 shows the kinetics of CFU recovery from the tissues of mice of all the groups. There was no significant difference between the high number of R. equi CFU recovered
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from the 2 control groups. Animal immunization with c3987pYA3137vapA was associated with a lower CFU counting. Bacteria recovery within the first 48 h post-challenge was approximately 7-fold higher in the lung, 3-fold higher in the spleen, and 4-fold higher in the liver of the control mice challenged with R. equi than in the organs of immunized mice. By days 4 and 8, CFU recovered from the lung, liver, and spleen of the control mice reached levels approximately 16-, 7-, and 5-fold higher, respectively, than those recovered from organs of immunized mice. On day 10 post-infection, the bacterial number declined in the 3 groups of animals, a decrease that was much more pronounced in the immunized mice group. Thus, R. equi clearance was more efficient in mice that received c3987-pYA3137vapA than in the 2 control groups. These data suggest that the vaccination procedure enhanced mice resistance to R. equi infection.
3.6. Histopathological examination
Fig. 4. CFU recovered from mice organs following R. equi infection. BALB/c mice were orally immunized with S. enterica Typhimurium c3987-pYA3137vapA (closed triangles), or inoculated with either S. enterica Typhimurium c3987-pYA3137 (closed squares) or PBS (open circles) on days 0 and 14. Mice were intravenously challenged with 4 106 CFU of R. equi ATCC33701 14 days after the last immunization. Bacterial burdens in the lung (A), liver (B), and spleen (C) were determined on days 2, 4, 8, and 10 after infection. The difference between the number of bacteria recovered from the immunized group and the control groups was statistically significant (*P < 0.001 e Tukey’s test).
Histology of the lung, liver, and spleen sections was performed to evaluate whether the resistance of immunized mice was associated with fewer lesions attributable to R. equi infection. For this, immunized mice and control mice were challenged with 4 106 CFU of virulent R. equi. Animals were sacrificed on days 2, 4, 8, and 10 after infection. In the case of the spleen of the control animals, formation of pyogranulomas with extensive areas of central necrosis was detected on days 2 (Fig. 5A) and 4 (data not shown) postinfection. In contrast, the immunized mice presented no apparent inflammatory reaction at any time of infection (Fig. 5B). There was an intense inflammatory process in the liver of the control mice, characterized by several multifocal granulomas on days 2 and 4 (data not shown) post-infection. On days 8 and 10 (data not shown), the inflammatory reaction decreased, although several granulomas were still present in the tissue. During all the analyzed periods, the inflammatory process in the liver of the control mice was associated with extensive necrosis areas, as shown for 10 days post-infection (Fig. 5C). In contrast with the severe hepatic lesions observed in the control mice, the inflammatory process was mild in the liver of VapA-immunized animals. In all the analyzed days, no hepatocyte necrosis was visualized (Fig. 5D). Morphometric analysis demonstrated that the number of hepatic granulomas was lower in the liver of VapA-immunized mice because, by day 2, the density of granulomas/mm2 of tissue was 9 for the immunized mice and 14 for the control mice. Although the granuloma density decreased in the 3 groups, this decrease was much more pronounced in immunized mice. On day 10, immunized mice provided a counting of 2 granulomas/mm2 versus 6 granulomas in the tissue of the control mice (Fig. 5I). Even though the number of granulomas/ mm2 of tissue was lower in the immunized animals, the total area occupied by the granulomas was significantly higher in the liver of these animals (Fig. 5J). On day 4 after challenge, granulomas occupied maximum hepatic area in mice of all the 3 groups. As time elapsed, the occupied hepatic area decreased
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Fig. 5. Histopathology of the spleen, liver and lung sections and morphometric analysis of the hepatic granulomas of BALB/c mice orally immunized with S. enterica Typhimurium c3987-pYA3137vapA, or inoculated with S. enterica Typhimurium c3987-pYA3137 and challenged intravenously with 4 106 CFU of virulent R. equi 14 days after the last immunization. The organs were collected 2 days (A, B, E, and F), 4 days (G and H) and 10 days (C and D) after infection. The 5-mm sections of the spleen (A and B), liver (C and D) and lung (EeH) of the mice were stained with hematoxylineeosin. The asterisks indicate necrosis areas and the arrows indicate the presence of small inflammatory infiltrates. Panel I: sections of the liver obtained on different days of infection (2, 4, 8, and 10) were analyzed by an optical microscopy with the aid of an integrator lens (Carl Zeiss Jena, Germany), to obtain the number of granulomas/mm2 of tissue; each bar represents the average SD of liver sections from 4 animals. Panel J: the granuloma area (mm2) was determined by means of KS-100 program (Carl Zeiss Jenamed 2, USA). The data represent the average SD of liver sections from 4 animals. At least 10 granulomas were analyzed in each tissue section (***P < 0.001; **P < 0.01; *P < 0.05 e Tukey’s test).
in all groups. However, this area was persistently higher in the liver of immunized mice (Fig. 5J). The lungs of the control mice showed extensive lesions characterized by abundant perivascular and peribronchiolar inflammatory infiltrate, obliterating the alveolar spaces of the lung parenchyma, predominantly on days 2 and 4
post-infection (Fig. 5E and G). In the same period, only a discrete infiltration was observed in the lung of immunized mice (Fig. 5F and H). In conclusion, the microscopic examination revealed clearcut differences in the severity of the lesions and in the time course of infection between immunized and control mice.
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3.7. Detection of VapA-specific antibody The anti-VapA IgG titration showed that a strong response was developed by the mice immunized with the recombinant Salmonella c3987-pYA3137vapA, 2 weeks after the last immunization (Fig. 6 upper). Such a response was maintained for more than 6 weeks (data not shown) and
was at least 2-fold higher in the VapA-vaccinated mice than in the control mice. The time course detection of both IgG1 and IgG2a isotypes specific for VapA in the serum of immunized mice showed that IgG2a levels were consistently higher than IgG1 titers (Fig. 6 lower). The IgG2a levels were 2 orders of magnitude higher than the IgG1 levels in all the analyzed days (Fig. 6 lower). The wide dominance of IgG2a strongly suggests that a specific Th1-type response was triggered by immunization with Salmonella c3987-pYA3137vapA. 4. Discussion
Fig. 6. Humoral response elicited by vaccination with Salmonella expressing the VapA protein. BALB/c mice were orally immunized on days 0 and 14 with S. enterica Typhimurium c3987-pYA3137vapA (VapA), or inoculated with either S. enterica Typhimurium c3987-pYA3137 (plasmid) or PBS (PBS). On different days after the last immunization, the serum samples were collected and assayed by ELISA. The recombinant VapA protein was used as the coating antigen. Each bar and symbol represents the average SD of serum from 4 animals in duplicates. (Upper) Total antigen-specific IgG production in different sera dilutions (1:30, 1:60, 1:120, 1:240, and 1:480), 2 weeks after the last immunization. The production of total IgG by immunized mice was significantly higher than that of the control mice (**P < 0.001; *P < 0.01 e Tukey’s test). (Lower) Isotype response at 7, 14, 21, 28, 35, and 42 days after the last immunization. The levels of IgG2a isotype detected were significantly higher than the IgG1 levels in immunized mice (VapA) and it was significantly higher than the production of IgG2a by the 2 control mice (**P < 0.001 e Tukey’s test).
In the present study we have shown that the oral immunization with a live attenuated S. enterica Typhimurium strain expressing the VapA protein confers protection against R. equi challenge in the mouse model of infection. Several vaccination strategies have already been tested as an attempt to prevent the development of rhodococcosis. Immunization procedures using avirulent or killed R. equi did not induce a protective immune response. In spite of the protection conferred against experimental challenge [8,17], virulent R. equi administration cannot be used as a vaccination approach because of the risk of provoking disease and contaminating the environment. VapA has been identified as an important virulence factor required for R. equi replication in host macrophages [5]. Immunization procedures with synthetic peptides representing epitopes of VapA [18] and DNA expressing the vapA gene [19] resulted in the development of a specific immune response associated with variable degrees of protection. Such reports have supported the selection of this R. equi antigen to be expressed in an attenuated S. enterica strain in order to produce a vaccinal preparation. Therefore, the vapA gene was cloned in the pYA3137 vector. The latter is a high-copy-number plasmid, belonging to the asd ‘balanced/lethal system’, which allows a high and stable expression of the heterologous protein [13,20]. Plasmid instability results in reduced bacterial colonization of the host tissues, which will affect the vaccine efficacy [21]. There is evidence that some recombinant Salmonella strains can lose their colonization capacity or can faintly colonize the host tissues [22]. On the other hand, oral immunization with S. enterica Typhimurium expressing heterologous antigens has been associated with bacterial colonization of the host Peyer’s patches, mesenteric lymph nodes and spleen for a long time [23]. In the present study, S. enterica Typhimurium c3987 transformed with either pYA3137vapA or pYA3137 plasmids were inoculated in mice and monitored for their in vivo persistence. In both cases, bacteria were persistently detected in the lymphoid tissues for at least 20 days, indicating that the VapA antigen expression did not affect the ability of Salmonella to colonize host tissues. The fact that bacteria recovered from mice tissues still maintained the pYA3137vapA plasmid demonstrated the stability of the recombinant bacteria. Bacterial clearance is considered a good way to assess the efficacy of a vaccinal approach against rhodococcosis [19,24].
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We compared the R. equi clearance in VapA-immunized mice and in mice of the control groups. Immunization with c3987pYA3137vapA was associated with significant reduction in R. equi recovery from the organs, in all the analyzed periods. A similar reduction in R. equi CFU has been reported for vapA-DNA-immunized mice challenged with R. equi [19]. Mouse protection provided by immunization with c3987pYA3137vapA was manifested early after R. equi challenge. On the 2nd day of infection, 3 times less CFU was recovered from immunized mice than from animals of the control groups. In spite of being challenged with a large inoculum, mice were efficiently protected by the vaccination, which suggests that the humoral and cellular immune responses required to eliminate R. equi [25] were acting in concert. The observations described here are consistent with previous demonstrations that Salmonella vaccine strains are effective in inducing heterologous antigen-specific responses [26]. Histopathological analysis of the organs from infected mice reinforced the ideas raised by the bacterial clearance assay. After being challenged with R. equi, mice immunized with c3987-pYA3137vapA developed a milder inflammatory response only, probably as a consequence of bacterial elimination. In all the analyzed periods, no changes were detected in the spleen tissue and no necrosis was visualized in the liver. In the lung, small inflammatory infiltrates were present on days 2 and 4 post-challenge. In contrast, the tissues of the control animals presented, especially during the first 4 days of infection, all of the most frequent histopathological changes were reported for the mouse model of rhodococcosis, such as intense inflammatory reaction and extensive areas of hepatic necrosis. In the lung, an abundant cellular infiltrate obliterating the alveolar septum was detected. The occurrence of hepatic granuloma formation, which is the most characteristic pathological change in the organs of R. equi infected animals [24], was about 2-fold higher in control mice than in VapAimmunized animals. The area of individual granulomas was persistently lower in the control mice. This suggests that the formation of new granulomas occurred in non-immunized mice only, in an attempt to restrain bacterial proliferation and dissemination to other tissues. In addition, granulomas in the organs of immunized mice were more compact, which corresponds to an adequate organization that avoids dissemination of the non-eliminated bacteria. The time course decrease in R. equi CFU is consistent with the progressive reduction of the granuloma area, observed in all the mice groups studied here. The survival of animals immunized with c3987-pYA3137vapA was 100% in the group challenged with 107 CFU/mL and 80% with 108 CFU/mL of R. equi, whereas all the control animals died before the 10th day post-infection. A similar protection was provided by mice immunization with S. enterica Typhimurium expressing the Sm14 antigen of Schistosoma mansoni [27]. The development of humoral immune response is important to induce protection against R. equi infection [28]. High titers of anti-VapA IgG were detected in the serum of vaccinated mice, with prominence of IgG2a. The differential production
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of cytokines has important effects on the Ig isotype [29], and the high ratio of IgG2a to IgG1 antibodies specific for VapA is consistent with a heightened Th1 response, since IFN-g is required to stimulate IgG2a secretion and is associated with inhibition of IgG1 production [30]. Indeed, the immunization using attenuated recombinant Salmonella has already been reported as inducing a Th1-type response preferentially [26]. In conclusion, we have demonstrated for the first time that the mice vaccination with attenuated S. enterica Typhimurium expressing the VapA protein is able to confer protection against experimental rhodococcosis. The specific immune response triggered by mice immunization and the protective effect induced by foal vaccination are both under investigation in our laboratories. Acknowledgements We thank Vani M.A. Correa, Sandra M.O. Thomaz, Antonio R.M. e Silva, and Patricia E. Vendrusculo for excellent technical assistance. We also acknowledge Julio A. Siqueira, Cristiane C.P. Ribas, Savio H.F. Miranda, and Ednelson A. Mazzotto for expert animal care. We are thankful to Dr. Luciana P. Ruas, Dr. Sandro G. Soares, Dr. Ebert S. Hanna, Dr. Vaˆnia S. Mariano, and Dr. Leandro L. Oliveira for helpful discussions. This work was partially supported by Fundac¸~ao de Amparo a` Pesquisa do Estado de S~ao Paulo (FAPESP) and Conselho Nacional de Pesquisa Cientı´fica e Tecnolo´gica (CNPq). A.F.O. received a scholarship from CNPq. This work generated patent. PI 0506364-7 (filed December 21, 2005). References [1] S. Giguere, J.F. Prescott, Clinical manifestations, diagnosis, treatment, and prevention of Rhodococcus equi infections in foals, Vet. Microbiol. 56 (1997) 313e334. [2] A. Donisi, M.G. Suardi, S. Casari, M. Longo, G.P. Cadeo, G. Carosi, Rhodococcus equi infection in HIV-infected patients, AIDS 10 (1996) 359e362. [3] S. Takai, S.A. Hines, T. Sekizaki, V.M. Nicholson, D.A. Alperin, M. Osaki, D. Takamatsu, M. Nakamura, K. Suzuki, N. Ogino, T. Kakuda, H. Dan, J.F. Prescott, DNA sequence and comparison of virulence plasmids from Rhodococcus equi ATCC33701 and 103, Infect. Immun. 68 (2000) 6840e6847. [4] S. Takai, M. Iie, Y. Watanabe, S. Tsubaki, T. Sekizaki, Virulence-associated 15- to 17-kilodalton antigens in Rhodococcus equi: temperaturedependent expression and location of the antigens, Infect. Immun. 60 (1992) 2995e2997. [5] S. Jain, B.R. Bloom, M.K. Hondalus, Deletion of vapA encoding virulence associated protein A attenuates the intracellular actinomycete R. equi, Mol. Microbiol. 50 (2003) 115e128. [6] J.F. Prescott, M.C. Patterson, V.M. Nicholson, B. Morein, J.A. Yager, Assessment of the immunogenic potential of Rhodococcus equi virulence associated protein (VapA) in mice, Vet. Microbiol. 56 (1997) 213e225. [7] K.E. Hooper-McGrevy, S. Giguere, B.N. Wilkie, J.F. Prescott, Evaluation of equine immunoglobulin specific for Rhodococcus equi virulence-associated proteins A and C for use in protecting foals against Rhodococcus equi-induced pneumonia, Am. J. Vet. Res. 62 (2001) 1307e1313. [8] S. Takai, C. Kobayashi, K. Murakami, S. Yukako, S. Tsubaki, Live virulent Rhodococcus equi, rather than killed or avirulent, elicits protective immunity to R. equi infection in mice, FEMS Immunol. Med. Microbiol. 24 (1999) 1e9.
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