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Protection of mice against experimental murine mycoplasmosis by a Mycoplasma pulmonis immunogen in lysogenized Escherichia coli
P(spers Protection of mice against experimental murine mycoplasmosis by a Mycoplasma pulmonis immunogen in lysogenized Escherichia coli Wayne C. Lai*, Michael Bennett, Brian E. Gordon and Steven P. Pakes A construct of the Mycoplasma pulmonis ( M P ) genomic library, using randomly sheared DNA, was cloned in 2gtll and transfected into C600 Escherichia coli organisms. Clones ofE. coli expressing a fusion protein reactive with anti-MP and monospecific serum were transferred orally or intravenously into Balb / c mice. Expression of the fusion protein was induced by adding isopropyl-[l-D-thiogalactopyranoside to the drinking water. This vaccination protocol led to local and systemic antibody formation, to generation of immune lymphocytes and to protection against large numbers of virulent M P organisms. This approach might be generally successful in preventing infectious disease. Keywords:Mycoplasma pulmonis; Escherichia coli; fusion protein; lysogen
Mycoplasmosis (Mycoplasma pulmonis, MP) is a widespread disease within rodent colonies 1. A dependable vaccine would be useful and beneficial in controlling this disease. It has been reported that significant protection against MP infection can be induced in rats 2 and mice 3 by immunization with live virulent or avirulent MP organisms. Unfortunately, a high percentage of vaccinated animals were culturally positive for MP in their uteri, joints, livers, brains and kidneys. It is possible that live wild-type vaccine MP organisms might lead to a chronic carrier state which could be a potential source of infection. More recently, several attenuated mutants of Salmonella typhimurium and Escherichia coli have been developed as delivery systems to stimulate mucosal immunity to cloned heterologous immunogens4. Mucosal, humoral and cell-mediated immunity to antigens expressed by these strains can be induced by oral or intravenous immunization5,6. Such delivery systems seem quite promising for vaccine development. We have previously produced several monoclonal antibodies 7 (mAbs) which inhibit MP growth in vitro, prevent the attachment of MP to fibroblasts or to red blood cells, and protect against MP infection. Moreover, we used the mAb to purify a MP antigen by affinity column chromatography. The purified antigen was used to vaccinate mice or rabbits which produced antibodies in their sera and tracheolung lavages capable of inhibiting MP growth in vitro. Even though the MP antigen could be purified with a Division of Comparative Medicine, Department of Pathology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9037, USA. *To whom correspondence should be addressed. (Received 27 October 1992; revised 7 June 1993; accepted 7 June 1993) 0264410X/94/04/0291-08 © 1994 Butterworth-Heinemann Ltd
protective mAb by using affinity column chromatography, only a limited amount of the antigen was obtained by this method. Therefore, we cloned this gene into the lacZ portion of the universal expression vector 2gtll. The mAb was used to screen the genomic library. Once the positive clone was found, it was lysogenized into E. coli to serve as a delivery system for an MP vaccine. The E. coli should colonize the gastrointestinal tract of mice. Non-toxic isopropyl-/~-D-thiogalactopyranoside (IPTG) was added to the drinking water to stimulate in vivo production of the fusion protein antigen, and thus host antibody production. This novel strategy to vaccinate animals could certainly be a powerful method to prevent infection. In developing a vaccine, a purified antigen represents a major achievement. During attempts to isolate a purified antigen, it was discovered that the nucleotide sequence contained a TGA stop codon. Mycoplasma utilize the codon TGA for the amino acid tryptophan 8'9, rather than the more common use of TGA to signal termination of translation as found in most other bacteria. This feature has complicated efforts to clone and express mycoplasma genes in E. eolix°'l a. Therefore, production of the long-chain polypeptide is improbable; without the protein product, antibody screening of the genomic library is impossible. Hence it is reasonable to cut genomic DNA of mycoplasma randomly into small fragments (200-500 bp) which would avoid the presence of the TGA stop codon at each end of at least some of the fragments. Thus termination is avoided without the need to mutate the TGA codon. The small fragments should be able to generate immunogenic proteins. This paper reports an approach to deal with the problem of termination of translation of the mycoplasma gene product in E. coil This led to successful production
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M. pulmonis immunogen in E. coli: W.C. Lai et al.
of the vaccine antigen and subsequent protection of mice from M P infection after oral or intravenous (i.v.) transfer of E. coli lysogenized with bacteriophages containing MP DNA.
MATERIALS AND METHODS
Organisms and growth conditions Mycoplasma pulmonis CT strain was grown in the media previously described 12 in all experiments and E. coli Y1090 (Promega Biotech Alac U169 proA + Alon araD 139 strA sup F hsd R- hsd M ÷ pMC9) were grown in LB broth with 0.2% maltose at 37°C overnight, washed and resuspended in one-third of the original culture volume of 10mM MgSO4/phosphate-buffered saline (PBS). These were the competent cells. E. coli K-12 C600 (ATCC no. 23724 F-supE44 lacY1 thr-1 leuB6 mcrB thi-1 tonA21 lambd~) were grown in LB broth containing nalidixic acid (NA), 30/~g ml-1, at 37°C overnight and were selected for NA resistance 13. We attempted to differentiate the endogenous and the lysogenic E. coli recovered from the inoculated host by creating a NA-resistant E. coli strain.
Generation of lysogen of E. coli-2gtl 1 NA-resistant E. coli C600 was lysogenized with the positive recombinant phage which has been described previously 14.
Antiserum preparation Hyperimmune rabbit sera directed against whole M. pulmonis ~5 and monospecific rabbit antisera were
prepared by inoculation of rabbits with the purified MP antigen as described previously7. The hyperimmune polyclonal sera, monospecific antisera, and preimmune rabbit sera were absorbed extensively with E. coli Y1090 (intact cells and lysates) before being used in immunological screening of the clone bank. Test sera were incubated at 4°C overnight with intact E. coli Y1090 at a concentration of 1 x 10 ~ E. coli cells/ml of serum. Aggregates were pelleted at 10000g for 20 min at 4°C. Absorbed sera were then incubated at 4°C overnight with a lysate suspension of 1 x 10~ E. coli cells coated on nitrocellulose paper. E. coli Y1090 lysates were prepared by passing cells which were washed and suspended in PBS containing 1 mM phenylmethylsulfonylfluoride through a French pressure cell twice at 150001bin -2 (1.03 x 103 Pa).
DNA extraction DNA of MP was extracted using the method described previously 16.
Construction of the genomic library The 2gtll expression library was constructed as described by Huynh et al. 17 with the following modifications. The mycoplasmal DNA was sonicated for 4min at 30s intervals into 200-500 base pair (bp) fragments to reduce the inclusion of the TGA stop codon. To remove endogenous EcoR1 sites, the fragmented DNA was then methylated with EcoR1 methylase (BRL, Bethesda Research Laboratories Inc., Gaithersburg, MD). The sheared ends were made flush with T4 DNA
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polymerase (BRL), and EcoR1 linkers (BRL) were ligated to the blunt ends. After digestion with EcoR1, excess linkers were removed by spin-column chromatography 18. These DNA fragments were collected and precipitated in 100%o cold ethanol, spin-dried, and reconstituted in 10 ml TE buffer. The DNA was kinased and ligated into EcoRl-digested and dephosphorylated 2gt 11 DNA (Promega Biotech, Madison, WI ) at a molar ratio of 1:3 (1 mM of 2gt 11 phage DNA arms to 3 mM of mycoplasma DNA). The DNA was ligated overnight at 14°C with T4 DNA ligase (BRL). Recombinant DNA was packaged to produce viable phage with a lambda in vitro packaging system (Gigapack, Strategene, Razolla, CA).
Titration of recombinant clones and efficiency of cloning The total number of recombinant clones and the cloning efficiency were calculated by the method described previously 19.
Immunological screening of the clone bank and analysis of the clones The procedure for screening the genomic library and analysis of the positive clones by electrophoresis and blotting has been described previously 16.
DNA analysis of recombinant clones The recombinant phage DNA of a typical positive clone such as L150 was isolated by a rapid, moderatescale procedure 2°. The inserted MP DNA in 2gtl 1 was rapidly amplified directly from bacteriophage plaques using the polymerase chain reaction 21 (PCR) with forward 5'-GGTGGCGACGACTCCTGGAGCCCG-3' and reverse 5'-TI'GACACCAGACCAACTGGTAATG-3' oligonucleotide primers corresponding to vector DNA sequences flanking the EcoR1 cloning site. The size of inserted MP DNA was rapidly analysed and the fragment was isolated using 1.5% agarose or 5% polyacrylamide gels.
DNA sequencing Total recombinant phage DNA was extracted by using plate lysate techniques 22 and Qiazen column purification (Qiazen Inc., Chatsworth, CA). Nucleotides were sequenced by the dideoxy chain termination method2°'23'24 using Sequenase (US Biochemical, Cleveland, OH ) with the primers described in the previous section. To sequence further into the 529 bp fragment, a second set of primers was synthesized using the initial sequencing data. Both strands of the entire insert were sequenced. Sequencing reactions were resolved on 6% polyacrylamide field gradient gels 25 cast with a wedge spacer using a Bio-Rad sequencer. Nucleic acid and protein computer analyses were performed using the Microgenie Program (Beckman Instruments Inc., Palo Alto, CA).
Mice MP-free Balb/c female mice were purchased from Harlan Sprague-Dawley Inc. (Indianapolis, IN). Care of the animals was in accordance with institutional guidelines, and the mice were maintained in a specific pathogen-free environment.
M. pulmonis immunogen in E. coli: W.C. Lai et al.
Immunization with fusion proteins One (L150) of four positive clones, designated L111, Ll12, Ll13 and L150, which was identified by the monospecific antiserum, was used to infect Y1090 E. coli, and IPTG was added to induce fl-galactosidase protein synthesis. The fusion protein was purified in a 7.5% polyacrylamide gel, fixed and stained with Coomassie blue to determine the size of the fl-galactosidase fusion protein. The total mass of the fusion protein was found to be 117 kDa, with the fl-galactosidase portion of the fusion protein accounting for l l4kDa. The fusion protein band was cut and electroeluted 26, and 10-20 #g of the fusion protein was dissolved in 0.1 ml elution buffer and combined with an equal volume of Freund's complete or incomplete adjuvant. The mixtures were injected subcutaneously into 22 mice on three occasions at intervals of 2 weeks. Complete Freund's adjuvant was used in the first injection and incomplete Freund's adjuvant was used in the second and third injections. The fourth and the last vaccinations involved intranasal inoculation of 10-20 #g protein. One week later, three mice were killed by ether inhalation, and the sera and tracheolung lavages were collected for immunofluorescent antibody (IFA) and enzyme-linked immunosorbent (ELISA) assays 27. Spleen cells were harvested for the lymphocyte transformation test to measure T cellmediated immunity 2s'29. Values are expressed as stimulation indices, (test value of 3H-thymidine incorporation (counts min- ~) divided by control (media alone)). The remaining 19 vaccinated mice were divided into three subgroups (seven, six and six), challenged with 1 x 103, 5 x 104 or 1 x 10 6 c.f.u. MPT2 and killed 2 weeks later
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(Table 1 ). Immunization with iysogen of E. c o l i - 2 g t l l Since live vaccines elicit better protection than killed organisms a, we cloned the protective MP antigenencoded DNA into 2gtll, and induced lysogeny in a wild-type E. coli C600 strain. Then we colonized mice with the bacteria. An overnight broth culture of bacteria was diluted to an absorbance (A6o 0 ) of 0.4 with LB broth containing nalidixic acid and grown at 32°C (with shaking) to an A6o0 of 0.9. The E. cord were induced at 42°C and IPTG was added to a 1.0mu final concentration. The culture was centrifuged and the bacteria were suspended in PBS to an appropriate absorbance (A6o 0 = 2 x 108 c.f.u, ml-~). Mice were inoculated orally with 0.2 ml bacterial suspension by using a feeding needle, or were intravenously injected with 0.2 ml bacterial suspension in a lateral tail vein. Mice were immunized as described in Table I. The animals (except groups 9 and 10) were fed a low dose of IPTG (1.0 mr,l) in their drinking water for 2-3 days after each inoculation. IPTG is not toxic and is not metabolized in the host a°. One mouse each from groups 2 and 3 was killed every 10 days after inoculation; samples from the upper intestine, caecum, colon, lung, spleen and liver were collected and plated out on blood agar media. The bacteria colonies were selected individually using the toothpick method on two LB agar media containing nalidixic acid; one was incubated at 32°C and the other at 42°C. IPTG-impregnated nitrocellulose paper was blotted from the 32°C plate and screened using the methods described previously ~6. Lymphocyte transformation assays of spleen cells of
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control mice (group 8 ) and vaccinated but not challenged proteins from a highly reactive clone (L150) which was mice (groups 3 and 4) were conducted as described processed by SDS-polyacrylamide gel electrophoresis previously 2s'29, using purified MP antigen. Two mice (Figure I ), and transferred to nitrocellulose paper. It was each from groups 4, 5 and 6, and five mice each from further screened with absorbed monospecific antiserum, groups 8, 9 and 10 were killed and the sera and and showed a wide distinct reactive fusion protein band tracheolung lavages were collected for IFA and ELISA slightly above the fl-galactosidase control band. In titres. Group 7 and the rest of the vaccinated animals in addition, none of the proteins from IPTG-induced cells groups 4, 5, 6, 9 and 10 were divided into three subgroups • containing the cloning vector without an MP DNA insert (Table I ) and challenged with various doses of MP reacted with the antibody. This indicated that the (1 x 10 3, 5 X 10 4 and 1 x 106 ) at day 38, and killed on antibody specifically reacted with MP antigen in fusion day 52. Evaluation of the vaccine efficacy was based on: protein. None of the proteins from a control 2gtl 1 lysate (1) microbiological evaluation to compare the numbers reacted with the antibody. Thus, distinct mycoplasma of M. pulmonis recovered from vaccinated- challenged proteins were synthesized in E. coli infected with the mice (groups 1, 4, 5, 6, 9 and 10) with numbers recombinant phage. from the non-vaccinated-challenged group (group 7); (2) pathological evaluation 27 to compare histopathoAnalysis of cloned MP DNA inserts logical changes in lungs of the vaccinated-challenged DNA analyses of the recombinants were performed to mice with the non-vaccinated-challenged group (group examine the size and possible relatedness of the cloned 7); (3) serological evaluation to compare serum and inserts. Because the single EcoR1 site was used as the tracheolung lavage antibody titres with vaccinated alone cloning site in 2gtl 1, the recombinant DNA preparations (groups 1, 2, 3, 4, 5, 6, 9 and 10) and control normal were digested with EcoR1 endonuclease. This digestion mice (group 8 ); (4 ) proliferation responses of lymphocytes resulted in cleavage of the 2gtll phage arms (24.1 kbp to MP antigen to compare vaccinated groups 2 and 3, and 19.6 kbp) away from the complete MP DNA insert. and vaccinated non-challenged groups 4, 5, 6, 9 and 10 However, since the size of the MP DNA insert was small with normal control mice (group 8). Thus, spleen cells or because it was lost from the EcoR1 site, it was not (4 x 10 6 ml- 1) were stimulated with 30/~g MP antigen detected on a 1% agarose gel. Therefore, the polymerase or 2.5/tg concanavalin A (non-specific T-cell nitrogen) chain reaction was used with forward and reverse per well of microtitre plates. Proliferation was measured oligonucleotide primers corresponding to the 2gtll by 3H-thymidine incorporation (counts min -1) 72 h vector DNA sequence flanking the EcoR1 clone sites of later 29. the clones. The amplified insert products were run on a Nucleotide sequence accession numbers The GenBank accession number for L150 nucleotide is M76406. RESULTS Construction of a genomic library A genomic library of recombinant E. coli clones was prepared by ligating small, randomly cut fragments of MP DNA to 2gtl 1 DNA which had been digested with EcoR 1 and dephosphorylated to decrease the possibility of reforming the original vector. Packaging of the recombinant 2 g t l l - M P DNA yielded 1.6 x 105 p.f.u./ 2/tg of DNA when plated on the E. coli lytic strain Y1090. On screening the library with pre-absorbed polyclonal anti-MP serum, 0.03% of the clones containing MP DNA inserts produced proteins that were immunologically reactive. Reactivity among clones with inserts ranged from very intense colour reactions to very light spots or no reactions at all. Forty-eight of the positive clones were spotted in duplicate onto fresh E. coli lawns. Plaques were again screened with pre-absorbed anti-MP serum and with the monospecific antiserum to purified antigen. Only four (designated L l l l , Ll12, Ll13 and L150) reacted with the monospecific antiserum. Reactivity among positive duplicate plaques was equally intense, while the negative plaques were non-reactive. Pre-immune rabbit serum absorbed with E. coli was non-reactive with the genomic library. Electrophoretic analysis of immunologically reactive proteins To characterize the MP protein-producing clones further, a liquid culture method was used to prepare
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Vaccine 1994 Volume 12 Number 4
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determined and is shown in Figure 2. The first TGA stop codon is located at 76 bp followed by two in-frame TAA stop codons at 112 and 235 bp downstream. The first 75 bp sequence could encode for a protein of 25 amino acids with a calculated molecular weight of 3090. The fusion protein has a molecular mass of about 117 kDa as seen in SDS-PAGE (Figure 1 ).
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Figure 2 The nucleotide sequence of the inserted MP DNA. The deduced amino acid sequence is shown below the nucleotide (nt) sequence. Only the nt are numbered, with 1 being the first nt of the inserted MP DNA coding sequence. There are two tryptophan residues which correspond to the TGA codons located at 76 and 523 nt and two stop codons TAb. located at 102 and 240 nt The restriction sites are as follows: the nt position in parentheses refers to the first 5'-bese in the
recognition sequence: ACC1(58), AHA2(49), BCL1(122), ECOR1(258), HINC2(375), HPAl(375), MLU1(117), PVU2(519), SNAB1(41) and XCA1(58)
1% agarose gel and stained with ethidium bromide. The MP DNA inserts could be readily seen near the 615 bp position (data not shown).
Nncleotide sequencing of the insert fragments The nucleotide sequence of the fragments was
The specificity of mycoplasma antigens expressed from cloned genomic fragments was examined in vitro by counter immunoelectrophoresis. A clear white precipitation line was formed between the fusion protein well and the absorbed polyclonal rabbit anti-MP well but no line formed between fl-gal and the rabbit anti-MP well (data not shown). Twenty-two mice were immunized with the fusion proteins (10-20/~g protein). The mice produced high levels of IgG and IgM antibodies against MP in their sera and IgG and IgA antibodies in their tracheolung lavages and gastrointestinal tract (data not shown) by IFA and ELISA assay, using a bicarbonate lysate of MP as antigen (Table2). These results demonstrate that mycoplasmal antigens generated from a recombinant phage are able to induce specific antibodies in naive animals.
Recovery, immunogenicity and ability to vaccinate of lysogenic E. coli E. coli C600 were unable to multiply extensively in the host, but did establish colonization in tissues such as lung, caecum or colon. A large number of transformed lysogenic E. coli were recovered from the caecum and colon (1 x 107 and 1 × l0 s c.f.u, m1-1, respectively, in group 2) of the mice, following oral inoculation at 40 and 50 days (even 7 months after inoculation in another experiment) as demonstrated in blood agar by Western blot analysis. However, no bacteria were isolated from spleen, liver or lung in the group of animals that were inoculated orally. In contrast, animals inoculated i.v. yielded large numbers of bacteria from the lung ( 1 x 107 c.f.u, ml- 1) and a smaller number from the spleen and liver ( 1 x 103 and 1 x 104 c.f.u, ml- *, respectively, group 3). No bacteria were found in the caecum or colon of i.v. inoculated animals. The serum antibody titres (IFA) were > 1:10000 in animals injected i.v. versus 1 : 100 in orally inoculated animals. No MP antibody titres were found in group 6 animals that were vaccinated with E. coli lysogenized with 2gtll without an insert. Antibodies against fl-galactosidase were measured with ELISA using fl-gal as an antigen. They were found in all mice in groups 2 and 3, and in the vaccinated-nonchallenged groups 4, 5 and 6. This indicated that the fl-gal protein had been expressed in oivo and could elicit antibody production. However, no antibodies against fl-galactosidase were found in groups 9 and 10. The vaccinated but unchallenged mice (groups 2, 3, 4, 5, 6, 9 and 10) showed no histopathological changes in the lung. This result suggests that the transformed lysogenic E. coli are not pathogenic for mice. The vaccinated (groups 4, 5, 6, 9 and 10) and non-vaccinated (group 7) Balb/c mice were divided into three subgroups and were challenged with various doses of highly virulent MPT2. The MPT2 results are shown in Figures 3, 4 and 5. All vaccinated mice in groups 1, 4
Vaccine 1994 Volume 12 Number 4
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and 5 were protected at the 1 × 103 c.f.u, dose, i.e. no MP organisms were recovered from tracheolung lavages (Figure3) and no histopathological changes were observed in lung sections (Figure 4). These data indicate that all mice were protected at the 'natural infective dose'. Significant protection (p < 0.05) was also observed even with the highest challenge dose of 1 x 10 6 c.f.u, of MP. In contrast, animals from groups 6, 7, 9 and 10 were not protected against any challenge dose of MP. Data from groups 6, 9 and 10 were similar to group 7; group 7 data are presented in Figure 3. The group 5 mice injected i.v. with lysogenized E. coil demonstrated the greatest protection against mycoplasma colonization at all challenge doses (Figures3 and 4). They also developed the highest serum immunoglobulin levels as determined by ELISA (Table The group 4 mice receiving oral vaccination were protected as well as group 5 mice when the challenge dose was 1 × 103 c.f.u. (Figure 3), but there was less protection if the challenge dose was higher (5 × 104 or 106 c.f.u.) (Figure3). They produced more IgA and lgG antibodies in tracheolung lavages than group 5 mice did. Histopathological lesions were calculated by a grading index score 27. Zero is equal to no lesions and 1.0 is
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Figure 5 Cell-mediated immune response in mice vaccinated with fusion protein, intravenous lysogen or oral lysogen and challenged with wild-type MPT2. Stimulation indices were calculated by dividing 3H-thymidine incorporation (counts min-') of antigen- or mitogenstimulated cultures by the value of control cultures. There was a significant difference (p < 0.05) between controls (group 8) and all vaccinated groups
equal to total pneumonic evolvement, with increments of 0.1. The average of five trials is shown in Figure 4. As can be seen from the figure, all vaccinated groups (groups 1, 4 and 5) were protected, with the greatest protection in group 5. All three groups of vaccinated mice (groups 1, 4 and 5 ) developed cell-mediated immunity to MP (Figure 5) as judged by the proliferative response to MP antigen 29. Group 8 was the control group. DISCUSSION Murine respiratory mycoplasmosis is one of the most pervasive diseases of rodent colonies. To prevent infection, it is desirable to develop vaccines which stimulate not only specific serum antibodies but also specific IgA in the respiratory tract. Such secretory antibodies may interfere with the early events of bacterial attachment and colonization. The prevention of infection and disease is important if vaccines are to facilitate eradication of this disease, for which the rodent is the only reservoir. We have established a simple, inexpensive method (1) to clone a genome fragment of MP DNA encoding the protective epitope, (2) to lysogenize rapidly growing E. coli with a phage containing this fragment, (3) to vaccinate mice by oral or i.v. transfer of E. coli organisms for colonization, and (4) to use the inducing agent, IPTG, to control and to expresss this epitope for immunization. A small fragment of MP DNA has been cloned into a universal expression vector (2gtll) to allow the nucleotide to express the epitope protein as a fl-galactosidase fusion protein, as has been demonstrated in the immunoblotting test against anti-MP or monospecific polyclonal antiserum (Figure1) and antibody against fl-galactosidase in the vaccinated host (ELISA test). These results demonstrate that fl-gal fusion protein is a potent immunogen, fl-galactosidase has a large molecular weight (114 kDa) and can act as a carrier to enhance the animals' immune response to this particular epitope. Determination of corresponding DNA sequences should provide direct assessment of protein structural
features pertinent to the function of these components and may also elucidate genomic structures underlying mycoplasmal antigenic variation. The results of the approach described here suggest various applications. The first is the possibility of producing selected mycoplasma antigens in large quantities by using a bacterial host. Analysis and development of vaccines from pathogenic mycoplasma have been hindered by the necessity of using highly enriched media to grow relatively small quantities of these fastidious organisms. Identification and large-scale production of specific antigens for vaccines to control mycoplasma infections would be ideal using recombinant DNA techniques. The second application is the production of a highly purified epitope of interest. The antigen of MP has not been selectively produced and purified from cultures of the organisms. Recombinant DNA technology using E. coli can produce large quantities of highly purified epitopes recognized by mAbs. This approach could be used to generate vaccines against species of mycoplasma which cause disease in other animals and in humans. Many of the earlier mycoplasma vaccines were inadequate. To achieve an adequate titre, a live vaccine is apparently required. The use of temperature-sensitive mutants 27 led to the objection that reversion to the wild type is always possible. This new method overcomes these objections by offering a vaccine that is safe, effective, non-pathogenic and controllable. Moreover, it can be given orally, which is an advantage for herd immunity. This approach allows observation of the population dynamics of a single foreign epitope after infecting the host with non-pathogenic E. coli. After the bacteria become established in the host, administration of IPTG allows presentation of the experimental epitope to the immune system via a live organism without many of the confounding factors now commonly used, such as adjuvant, hapten carrier or denaturing agents and conditions. The coadministration of a naturally infective dose and IPTG could produce a dose response in the host that would be unique to the specific protein of interest. The transformed E. coli and the unique method of controlling protein expression in vivo offer a new method of studying molecular pathology and antigen presentation issues in infectious disease. ACKNOWLEDGEMENTS This work was supported in part by Public Health Service grants RR00890 and RR08552 from the National Institutes of Health. The authors thank David Steutermann, Geoffrey Tadda and Su-In Ho for excellent technical assistance and Bonnie Mendro and J. Harvey Harris for outstanding secretarial help. They also thank Drs Raymond J. MacDonald and Galvin Swift for the suggestion to shear MP DNA into small pieces to deal with TGA stop codons, and Dr Victor K. Lin for his technical advice concerning the cloning procedure. We also thank Dr Ping Chuan Hu (University of North Carolina, Chapel Hill, NC) for sequencing the insert DNA. REFERENCES Cassell, G.H., Russell Lindsey, J., Davis, J.K., Davison, M.K., Brown, M.B. and Mayo, J.G. Detection of natural Mycoplasma pulmonis infection in rats and mice by an enzyme linked immunosorbent assay (ELISA). Lab. Anim. Sci. 1981, 31,676-682
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6
7 8
9 10 11 12
13 14 15
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Cassell, G.H. and Davis, J.K. Protective effect of vaccination against Mycoplasma pulmonis respiratory disease in rats. Infect. Immun. 1978, 21, 69-75 Taylor, G., Howard, C.J. and Gourlay, R.N. Protective effect of vaccines on Mycoplasma pulmonis-induced respiratory disease of mice. Infect. Immun. 1977, 16, 422-431 Hoiseth, S.K. and Stocker, B.A.D. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature (London) 1981, 291,238-239 Fairweather, N.F., Chatfield, S.N., Makoff, A.J., Strugnell, R.A., Bester, J., Maskell, D.J. and Dougan, G. Oral vaccination of mice against tetanus by use of a live attenuated Salmonella carrier. Infect. Immun. 1990, 58, 1323-1326 Guzman, C.A., Brownlie, R.M., Kadurugamuwa, J., Walker, M.J. and Timmie, T.N. Antibody responses in the lung of mice following oral immunization with Salmonella typhimurium aroA and invasive Escherichia coli strain expressing the filamentous hemagglutinin of Bordetella pertussis. Infect. Immun. 1990, 59, 4391-4397 Lai, W.C., Bennett, M., Pakes, S.P. and Murphree, S.S. Potential subunit vaccine against Mycop/asma pulmonis purified by a protective monoclonal antibody. Vaccine 1991, 9, 177-184 Inamine, J.M., Ho, K.C., Loechel, S. and Hu, P.C. Evidence that TGA is read as a tryptophan codon rather than as a stop codon by Mycop/asma pneumoniae, Mycop/asma genitalium and Mycoplasma gallisepticum. J. Bacterio/. 1990, 172, 504-506 Yamao, Y., Muto, A., Kawandi, Y., Iwami, M., Iwagami, S., Azumi, Y. and Oswa, S. UGA is read as tryptophan in Mycoplasma caprio/um. Proc. Nat/Acad. Sci. USA 1985, 82, 2306-2309 Dallo, S.F., Su, C.J., Horton, J.R. and Bassman, J.B. Identification of P1 gene domain containing epitope(s) mediating Mycoplasma pneumoniae cytoadherence. J. Exp. Med. 1988, 167, 718-723 Frydenbert, J., Lind, K. and Hu, P.C. Cloning of Mycoplasma pneumoniae DNA and expression of Pl-epitomes in Escherichia coil. Israel J. Med. Sci. 1987, 23, 759-762 Lai, W.C., Pakes, S.P., Stefann, C. and Lu, Y.S. Comparison of Chalquest and Hayblide media, with and without ammonium reinekate, for isolating Mycoplasma pulmonis from rats. J. C/in. Microbiol. 1986, 23, 817-821 Hane, M.W. and Wood, T.H. Escherichia coli K 12 mutants resistant to nalidixic acid: genetic mapping and dominance studies. J. Bacteriol. 1969, 99, 238-241 Henry, M.F. and Cronan, J.E. Direct and general selection for lysogens of Escherichia coli by phage recombinant clones. J. Bacteriol. 1991, 173, 3724-3731 Jones, T.C. and Hirsch, J.G. The interaction in vitro of Mycoplasma pulmonis with mouse peritoneal macrophages and L-cells. J. Exp.
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Med. 1971, 133, 231-259 16 Trevino, L.B., Haldewag, W.G. and Baseman, J.B. Expression of Mycoplasma pneumoniae antigens in Escherichia. Infect. Immun. 1986, 53, 129-134 17 Huynh, T.V., Young, R.A. and Davis, R.W. Constructing and screening cDNA libraries in ~gt 10 and ~.gt 11. In: DNA Cloning: Vol. 1 - A Practical Approach (Ed. Glover, D.M.) IRL Press, Oxford, 1985, pp. 49-78 18 Sambrook, J., Fritsch, E.F. and Maniatis, T. In: Molecular Cloning. A Laboratory Manual, 2nd Edn, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, p. E.37 19 Clarke, L. and Carbon, J. A colony bank containing synthetic EcoR1 hybrid plasma representative of the entire E. coli genome. Cell 1976, 9, 91-99 20 Chisholm, D. A convenient moderate-scale procedure for obtaining DNA from bacteriophage lambda. Biotechniques 1989, 7, 21-23 21 Dorfman, D.M., Zon, L.l. and Orkin, S.H. Rapid amplification of lambda gt11 bacteriophage library inserts from plaques using the polymerase chain reaction (PCR). Biotechniques 1989, 7, 568-570 22 Sambrook, J., Fritsch, E.F. and Maniatis, T. Bacteriophage ~. growth, purification and DNA extraction. In: Molecular Cloning. A Laboratory Manual, 2nd Edn, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, pp. 2.64-2.80 23 Biggin, M.D., Gibson, J.J. and Hong, G.F. Buffer gradient gels and ~S label as an aid to rapid DNA sequence determination. Prec. Natl Acad. Sci. USA 1983, 80, 3963-3985 24 Sanger, F., Nicklen, S. and Coulson, A.R. DNA sequencing with chain terminating inhibitions. Proc. Natl Acad. Sci. USA 1977, 74, 5463-5467 25 Olsson, A., Moks, T., Uhlen, M. and Gaal, A.B. Uniformly spaced banding pattern in DNA sequencing gels by use of field-strength gradient. J. Biochem. Biophys. Methods 1984, 10, 83-90 26 Hunkapiller, M.W., Lujan, E., Ostrander, F. and Hood, L.E. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983, 91,227-236 27 Lai, W.C., Bennett, M., Lu, Y.S. and Pakes, S.P. Biological evaluation of Mycoplasma pulmonis temperature-sensitive mutants for use as possible rodent vaccines. Infect. Immun. 1990, 58, 2289-2298 28 Naot, Y. In vitro studies on the mitogenicity activity of mycoplasmal species toward lymphocytes. Rev. Infect. Dis. 1982, 4, $205-209 29 Lai, W.C., Pakes, S.P., Owusu, I. and Wang, S. Mycoplsma pulmonis depresses humoral and cell-mediated response in mice. Lab. Anim. Sci. 1989, 39, 11-15 30 Stryer, L Control of gene expression in procaryotes. In: Biochemistry W.H. Freeman, New York, 1988, pp. 800-822
Mycoplasmosis (Mycoplasma pulmonis, MP) is a widespread disease within rodent colonies 1. A dependable vaccine would be useful and beneficial in controlling this disease. It has been reported that significant protection against MP infection can be induced in rats 2 and mice 3 by immunization with live virulent or avirulent MP organisms. Unfortunately, a high percentage of vaccinated animals were culturally positive for MP in their uteri, joints, livers, brains and kidneys. It is possible that live wild-type vaccine MP organisms might lead to a chronic carrier state which could be a potential source of infection. More recently, several attenuated mutants of Salmonella typhimurium and Escherichia coli have been developed as delivery systems to stimulate mucosal immunity to cloned heterologous immunogens4. Mucosal, humoral and cell-mediated immunity to antigens expressed by these strains can be induced by oral or intravenous immunization5,6. Such delivery systems seem quite promising for vaccine development. We have previously produced several monoclonal antibodies 7 (mAbs) which inhibit MP growth in vitro, prevent the attachment of MP to fibroblasts or to red blood cells, and protect against MP infection. Moreover, we used the mAb to purify a MP antigen by affinity column chromatography. The purified antigen was used to vaccinate mice or rabbits which produced antibodies in their sera and tracheolung lavages capable of inhibiting MP growth in vitro. Even though the MP antigen could be purified with a Division of Comparative Medicine, Department of Pathology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9037, USA. *To whom correspondence should be addressed. (Received 27 October 1992; revised 7 June 1993; accepted 7 June 1993) 0264410X/94/04/0291-08 © 1994 Butterworth-Heinemann Ltd
protective mAb by using affinity column chromatography, only a limited amount of the antigen was obtained by this method. Therefore, we cloned this gene into the lacZ portion of the universal expression vector 2gtll. The mAb was used to screen the genomic library. Once the positive clone was found, it was lysogenized into E. coli to serve as a delivery system for an MP vaccine. The E. coli should colonize the gastrointestinal tract of mice. Non-toxic isopropyl-/~-D-thiogalactopyranoside (IPTG) was added to the drinking water to stimulate in vivo production of the fusion protein antigen, and thus host antibody production. This novel strategy to vaccinate animals could certainly be a powerful method to prevent infection. In developing a vaccine, a purified antigen represents a major achievement. During attempts to isolate a purified antigen, it was discovered that the nucleotide sequence contained a TGA stop codon. Mycoplasma utilize the codon TGA for the amino acid tryptophan 8'9, rather than the more common use of TGA to signal termination of translation as found in most other bacteria. This feature has complicated efforts to clone and express mycoplasma genes in E. eolix°'l a. Therefore, production of the long-chain polypeptide is improbable; without the protein product, antibody screening of the genomic library is impossible. Hence it is reasonable to cut genomic DNA of mycoplasma randomly into small fragments (200-500 bp) which would avoid the presence of the TGA stop codon at each end of at least some of the fragments. Thus termination is avoided without the need to mutate the TGA codon. The small fragments should be able to generate immunogenic proteins. This paper reports an approach to deal with the problem of termination of translation of the mycoplasma gene product in E. coil This led to successful production
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M. pulmonis immunogen in E. coli: W.C. Lai et al.
of the vaccine antigen and subsequent protection of mice from M P infection after oral or intravenous (i.v.) transfer of E. coli lysogenized with bacteriophages containing MP DNA.
MATERIALS AND METHODS
Organisms and growth conditions Mycoplasma pulmonis CT strain was grown in the media previously described 12 in all experiments and E. coli Y1090 (Promega Biotech Alac U169 proA + Alon araD 139 strA sup F hsd R- hsd M ÷ pMC9) were grown in LB broth with 0.2% maltose at 37°C overnight, washed and resuspended in one-third of the original culture volume of 10mM MgSO4/phosphate-buffered saline (PBS). These were the competent cells. E. coli K-12 C600 (ATCC no. 23724 F-supE44 lacY1 thr-1 leuB6 mcrB thi-1 tonA21 lambd~) were grown in LB broth containing nalidixic acid (NA), 30/~g ml-1, at 37°C overnight and were selected for NA resistance 13. We attempted to differentiate the endogenous and the lysogenic E. coli recovered from the inoculated host by creating a NA-resistant E. coli strain.
Generation of lysogen of E. coli-2gtl 1 NA-resistant E. coli C600 was lysogenized with the positive recombinant phage which has been described previously 14.
Antiserum preparation Hyperimmune rabbit sera directed against whole M. pulmonis ~5 and monospecific rabbit antisera were
prepared by inoculation of rabbits with the purified MP antigen as described previously7. The hyperimmune polyclonal sera, monospecific antisera, and preimmune rabbit sera were absorbed extensively with E. coli Y1090 (intact cells and lysates) before being used in immunological screening of the clone bank. Test sera were incubated at 4°C overnight with intact E. coli Y1090 at a concentration of 1 x 10 ~ E. coli cells/ml of serum. Aggregates were pelleted at 10000g for 20 min at 4°C. Absorbed sera were then incubated at 4°C overnight with a lysate suspension of 1 x 10~ E. coli cells coated on nitrocellulose paper. E. coli Y1090 lysates were prepared by passing cells which were washed and suspended in PBS containing 1 mM phenylmethylsulfonylfluoride through a French pressure cell twice at 150001bin -2 (1.03 x 103 Pa).
DNA extraction DNA of MP was extracted using the method described previously 16.
Construction of the genomic library The 2gtll expression library was constructed as described by Huynh et al. 17 with the following modifications. The mycoplasmal DNA was sonicated for 4min at 30s intervals into 200-500 base pair (bp) fragments to reduce the inclusion of the TGA stop codon. To remove endogenous EcoR1 sites, the fragmented DNA was then methylated with EcoR1 methylase (BRL, Bethesda Research Laboratories Inc., Gaithersburg, MD). The sheared ends were made flush with T4 DNA
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polymerase (BRL), and EcoR1 linkers (BRL) were ligated to the blunt ends. After digestion with EcoR1, excess linkers were removed by spin-column chromatography 18. These DNA fragments were collected and precipitated in 100%o cold ethanol, spin-dried, and reconstituted in 10 ml TE buffer. The DNA was kinased and ligated into EcoRl-digested and dephosphorylated 2gt 11 DNA (Promega Biotech, Madison, WI ) at a molar ratio of 1:3 (1 mM of 2gt 11 phage DNA arms to 3 mM of mycoplasma DNA). The DNA was ligated overnight at 14°C with T4 DNA ligase (BRL). Recombinant DNA was packaged to produce viable phage with a lambda in vitro packaging system (Gigapack, Strategene, Razolla, CA).
Titration of recombinant clones and efficiency of cloning The total number of recombinant clones and the cloning efficiency were calculated by the method described previously 19.
Immunological screening of the clone bank and analysis of the clones The procedure for screening the genomic library and analysis of the positive clones by electrophoresis and blotting has been described previously 16.
DNA analysis of recombinant clones The recombinant phage DNA of a typical positive clone such as L150 was isolated by a rapid, moderatescale procedure 2°. The inserted MP DNA in 2gtl 1 was rapidly amplified directly from bacteriophage plaques using the polymerase chain reaction 21 (PCR) with forward 5'-GGTGGCGACGACTCCTGGAGCCCG-3' and reverse 5'-TI'GACACCAGACCAACTGGTAATG-3' oligonucleotide primers corresponding to vector DNA sequences flanking the EcoR1 cloning site. The size of inserted MP DNA was rapidly analysed and the fragment was isolated using 1.5% agarose or 5% polyacrylamide gels.
DNA sequencing Total recombinant phage DNA was extracted by using plate lysate techniques 22 and Qiazen column purification (Qiazen Inc., Chatsworth, CA). Nucleotides were sequenced by the dideoxy chain termination method2°'23'24 using Sequenase (US Biochemical, Cleveland, OH ) with the primers described in the previous section. To sequence further into the 529 bp fragment, a second set of primers was synthesized using the initial sequencing data. Both strands of the entire insert were sequenced. Sequencing reactions were resolved on 6% polyacrylamide field gradient gels 25 cast with a wedge spacer using a Bio-Rad sequencer. Nucleic acid and protein computer analyses were performed using the Microgenie Program (Beckman Instruments Inc., Palo Alto, CA).
Mice MP-free Balb/c female mice were purchased from Harlan Sprague-Dawley Inc. (Indianapolis, IN). Care of the animals was in accordance with institutional guidelines, and the mice were maintained in a specific pathogen-free environment.
M. pulmonis immunogen in E. coli: W.C. Lai et al.
Immunization with fusion proteins One (L150) of four positive clones, designated L111, Ll12, Ll13 and L150, which was identified by the monospecific antiserum, was used to infect Y1090 E. coli, and IPTG was added to induce fl-galactosidase protein synthesis. The fusion protein was purified in a 7.5% polyacrylamide gel, fixed and stained with Coomassie blue to determine the size of the fl-galactosidase fusion protein. The total mass of the fusion protein was found to be 117 kDa, with the fl-galactosidase portion of the fusion protein accounting for l l4kDa. The fusion protein band was cut and electroeluted 26, and 10-20 #g of the fusion protein was dissolved in 0.1 ml elution buffer and combined with an equal volume of Freund's complete or incomplete adjuvant. The mixtures were injected subcutaneously into 22 mice on three occasions at intervals of 2 weeks. Complete Freund's adjuvant was used in the first injection and incomplete Freund's adjuvant was used in the second and third injections. The fourth and the last vaccinations involved intranasal inoculation of 10-20 #g protein. One week later, three mice were killed by ether inhalation, and the sera and tracheolung lavages were collected for immunofluorescent antibody (IFA) and enzyme-linked immunosorbent (ELISA) assays 27. Spleen cells were harvested for the lymphocyte transformation test to measure T cellmediated immunity 2s'29. Values are expressed as stimulation indices, (test value of 3H-thymidine incorporation (counts min- ~) divided by control (media alone)). The remaining 19 vaccinated mice were divided into three subgroups (seven, six and six), challenged with 1 x 103, 5 x 104 or 1 x 10 6 c.f.u. MPT2 and killed 2 weeks later
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control mice (group 8 ) and vaccinated but not challenged proteins from a highly reactive clone (L150) which was mice (groups 3 and 4) were conducted as described processed by SDS-polyacrylamide gel electrophoresis previously 2s'29, using purified MP antigen. Two mice (Figure I ), and transferred to nitrocellulose paper. It was each from groups 4, 5 and 6, and five mice each from further screened with absorbed monospecific antiserum, groups 8, 9 and 10 were killed and the sera and and showed a wide distinct reactive fusion protein band tracheolung lavages were collected for IFA and ELISA slightly above the fl-galactosidase control band. In titres. Group 7 and the rest of the vaccinated animals in addition, none of the proteins from IPTG-induced cells groups 4, 5, 6, 9 and 10 were divided into three subgroups • containing the cloning vector without an MP DNA insert (Table I ) and challenged with various doses of MP reacted with the antibody. This indicated that the (1 x 10 3, 5 X 10 4 and 1 x 106 ) at day 38, and killed on antibody specifically reacted with MP antigen in fusion day 52. Evaluation of the vaccine efficacy was based on: protein. None of the proteins from a control 2gtl 1 lysate (1) microbiological evaluation to compare the numbers reacted with the antibody. Thus, distinct mycoplasma of M. pulmonis recovered from vaccinated- challenged proteins were synthesized in E. coli infected with the mice (groups 1, 4, 5, 6, 9 and 10) with numbers recombinant phage. from the non-vaccinated-challenged group (group 7); (2) pathological evaluation 27 to compare histopathoAnalysis of cloned MP DNA inserts logical changes in lungs of the vaccinated-challenged DNA analyses of the recombinants were performed to mice with the non-vaccinated-challenged group (group examine the size and possible relatedness of the cloned 7); (3) serological evaluation to compare serum and inserts. Because the single EcoR1 site was used as the tracheolung lavage antibody titres with vaccinated alone cloning site in 2gtl 1, the recombinant DNA preparations (groups 1, 2, 3, 4, 5, 6, 9 and 10) and control normal were digested with EcoR1 endonuclease. This digestion mice (group 8 ); (4 ) proliferation responses of lymphocytes resulted in cleavage of the 2gtll phage arms (24.1 kbp to MP antigen to compare vaccinated groups 2 and 3, and 19.6 kbp) away from the complete MP DNA insert. and vaccinated non-challenged groups 4, 5, 6, 9 and 10 However, since the size of the MP DNA insert was small with normal control mice (group 8). Thus, spleen cells or because it was lost from the EcoR1 site, it was not (4 x 10 6 ml- 1) were stimulated with 30/~g MP antigen detected on a 1% agarose gel. Therefore, the polymerase or 2.5/tg concanavalin A (non-specific T-cell nitrogen) chain reaction was used with forward and reverse per well of microtitre plates. Proliferation was measured oligonucleotide primers corresponding to the 2gtll by 3H-thymidine incorporation (counts min -1) 72 h vector DNA sequence flanking the EcoR1 clone sites of later 29. the clones. The amplified insert products were run on a Nucleotide sequence accession numbers The GenBank accession number for L150 nucleotide is M76406. RESULTS Construction of a genomic library A genomic library of recombinant E. coli clones was prepared by ligating small, randomly cut fragments of MP DNA to 2gtl 1 DNA which had been digested with EcoR 1 and dephosphorylated to decrease the possibility of reforming the original vector. Packaging of the recombinant 2 g t l l - M P DNA yielded 1.6 x 105 p.f.u./ 2/tg of DNA when plated on the E. coli lytic strain Y1090. On screening the library with pre-absorbed polyclonal anti-MP serum, 0.03% of the clones containing MP DNA inserts produced proteins that were immunologically reactive. Reactivity among clones with inserts ranged from very intense colour reactions to very light spots or no reactions at all. Forty-eight of the positive clones were spotted in duplicate onto fresh E. coli lawns. Plaques were again screened with pre-absorbed anti-MP serum and with the monospecific antiserum to purified antigen. Only four (designated L l l l , Ll12, Ll13 and L150) reacted with the monospecific antiserum. Reactivity among positive duplicate plaques was equally intense, while the negative plaques were non-reactive. Pre-immune rabbit serum absorbed with E. coli was non-reactive with the genomic library. Electrophoretic analysis of immunologically reactive proteins To characterize the MP protein-producing clones further, a liquid culture method was used to prepare
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determined and is shown in Figure 2. The first TGA stop codon is located at 76 bp followed by two in-frame TAA stop codons at 112 and 235 bp downstream. The first 75 bp sequence could encode for a protein of 25 amino acids with a calculated molecular weight of 3090. The fusion protein has a molecular mass of about 117 kDa as seen in SDS-PAGE (Figure 1 ).
Immunogenicity of antigen expressed in recombinant phage clones
A
Figure 2 The nucleotide sequence of the inserted MP DNA. The deduced amino acid sequence is shown below the nucleotide (nt) sequence. Only the nt are numbered, with 1 being the first nt of the inserted MP DNA coding sequence. There are two tryptophan residues which correspond to the TGA codons located at 76 and 523 nt and two stop codons TAb. located at 102 and 240 nt The restriction sites are as follows: the nt position in parentheses refers to the first 5'-bese in the
recognition sequence: ACC1(58), AHA2(49), BCL1(122), ECOR1(258), HINC2(375), HPAl(375), MLU1(117), PVU2(519), SNAB1(41) and XCA1(58)
1% agarose gel and stained with ethidium bromide. The MP DNA inserts could be readily seen near the 615 bp position (data not shown).
Nncleotide sequencing of the insert fragments The nucleotide sequence of the fragments was
The specificity of mycoplasma antigens expressed from cloned genomic fragments was examined in vitro by counter immunoelectrophoresis. A clear white precipitation line was formed between the fusion protein well and the absorbed polyclonal rabbit anti-MP well but no line formed between fl-gal and the rabbit anti-MP well (data not shown). Twenty-two mice were immunized with the fusion proteins (10-20/~g protein). The mice produced high levels of IgG and IgM antibodies against MP in their sera and IgG and IgA antibodies in their tracheolung lavages and gastrointestinal tract (data not shown) by IFA and ELISA assay, using a bicarbonate lysate of MP as antigen (Table2). These results demonstrate that mycoplasmal antigens generated from a recombinant phage are able to induce specific antibodies in naive animals.
Recovery, immunogenicity and ability to vaccinate of lysogenic E. coli E. coli C600 were unable to multiply extensively in the host, but did establish colonization in tissues such as lung, caecum or colon. A large number of transformed lysogenic E. coli were recovered from the caecum and colon (1 x 107 and 1 × l0 s c.f.u, m1-1, respectively, in group 2) of the mice, following oral inoculation at 40 and 50 days (even 7 months after inoculation in another experiment) as demonstrated in blood agar by Western blot analysis. However, no bacteria were isolated from spleen, liver or lung in the group of animals that were inoculated orally. In contrast, animals inoculated i.v. yielded large numbers of bacteria from the lung ( 1 x 107 c.f.u, ml- 1) and a smaller number from the spleen and liver ( 1 x 103 and 1 x 104 c.f.u, ml- *, respectively, group 3). No bacteria were found in the caecum or colon of i.v. inoculated animals. The serum antibody titres (IFA) were > 1:10000 in animals injected i.v. versus 1 : 100 in orally inoculated animals. No MP antibody titres were found in group 6 animals that were vaccinated with E. coli lysogenized with 2gtll without an insert. Antibodies against fl-galactosidase were measured with ELISA using fl-gal as an antigen. They were found in all mice in groups 2 and 3, and in the vaccinated-nonchallenged groups 4, 5 and 6. This indicated that the fl-gal protein had been expressed in oivo and could elicit antibody production. However, no antibodies against fl-galactosidase were found in groups 9 and 10. The vaccinated but unchallenged mice (groups 2, 3, 4, 5, 6, 9 and 10) showed no histopathological changes in the lung. This result suggests that the transformed lysogenic E. coli are not pathogenic for mice. The vaccinated (groups 4, 5, 6, 9 and 10) and non-vaccinated (group 7) Balb/c mice were divided into three subgroups and were challenged with various doses of highly virulent MPT2. The MPT2 results are shown in Figures 3, 4 and 5. All vaccinated mice in groups 1, 4
Vaccine 1994 Volume 12 Number 4
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and 5 were protected at the 1 × 103 c.f.u, dose, i.e. no MP organisms were recovered from tracheolung lavages (Figure3) and no histopathological changes were observed in lung sections (Figure 4). These data indicate that all mice were protected at the 'natural infective dose'. Significant protection (p < 0.05) was also observed even with the highest challenge dose of 1 x 10 6 c.f.u, of MP. In contrast, animals from groups 6, 7, 9 and 10 were not protected against any challenge dose of MP. Data from groups 6, 9 and 10 were similar to group 7; group 7 data are presented in Figure 3. The group 5 mice injected i.v. with lysogenized E. coil demonstrated the greatest protection against mycoplasma colonization at all challenge doses (Figures3 and 4). They also developed the highest serum immunoglobulin levels as determined by ELISA (Table The group 4 mice receiving oral vaccination were protected as well as group 5 mice when the challenge dose was 1 × 103 c.f.u. (Figure 3), but there was less protection if the challenge dose was higher (5 × 104 or 106 c.f.u.) (Figure3). They produced more IgA and lgG antibodies in tracheolung lavages than group 5 mice did. Histopathological lesions were calculated by a grading index score 27. Zero is equal to no lesions and 1.0 is
2).
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Figure 5 Cell-mediated immune response in mice vaccinated with fusion protein, intravenous lysogen or oral lysogen and challenged with wild-type MPT2. Stimulation indices were calculated by dividing 3H-thymidine incorporation (counts min-') of antigen- or mitogenstimulated cultures by the value of control cultures. There was a significant difference (p < 0.05) between controls (group 8) and all vaccinated groups
equal to total pneumonic evolvement, with increments of 0.1. The average of five trials is shown in Figure 4. As can be seen from the figure, all vaccinated groups (groups 1, 4 and 5) were protected, with the greatest protection in group 5. All three groups of vaccinated mice (groups 1, 4 and 5 ) developed cell-mediated immunity to MP (Figure 5) as judged by the proliferative response to MP antigen 29. Group 8 was the control group. DISCUSSION Murine respiratory mycoplasmosis is one of the most pervasive diseases of rodent colonies. To prevent infection, it is desirable to develop vaccines which stimulate not only specific serum antibodies but also specific IgA in the respiratory tract. Such secretory antibodies may interfere with the early events of bacterial attachment and colonization. The prevention of infection and disease is important if vaccines are to facilitate eradication of this disease, for which the rodent is the only reservoir. We have established a simple, inexpensive method (1) to clone a genome fragment of MP DNA encoding the protective epitope, (2) to lysogenize rapidly growing E. coli with a phage containing this fragment, (3) to vaccinate mice by oral or i.v. transfer of E. coli organisms for colonization, and (4) to use the inducing agent, IPTG, to control and to expresss this epitope for immunization. A small fragment of MP DNA has been cloned into a universal expression vector (2gtll) to allow the nucleotide to express the epitope protein as a fl-galactosidase fusion protein, as has been demonstrated in the immunoblotting test against anti-MP or monospecific polyclonal antiserum (Figure1) and antibody against fl-galactosidase in the vaccinated host (ELISA test). These results demonstrate that fl-gal fusion protein is a potent immunogen, fl-galactosidase has a large molecular weight (114 kDa) and can act as a carrier to enhance the animals' immune response to this particular epitope. Determination of corresponding DNA sequences should provide direct assessment of protein structural
features pertinent to the function of these components and may also elucidate genomic structures underlying mycoplasmal antigenic variation. The results of the approach described here suggest various applications. The first is the possibility of producing selected mycoplasma antigens in large quantities by using a bacterial host. Analysis and development of vaccines from pathogenic mycoplasma have been hindered by the necessity of using highly enriched media to grow relatively small quantities of these fastidious organisms. Identification and large-scale production of specific antigens for vaccines to control mycoplasma infections would be ideal using recombinant DNA techniques. The second application is the production of a highly purified epitope of interest. The antigen of MP has not been selectively produced and purified from cultures of the organisms. Recombinant DNA technology using E. coli can produce large quantities of highly purified epitopes recognized by mAbs. This approach could be used to generate vaccines against species of mycoplasma which cause disease in other animals and in humans. Many of the earlier mycoplasma vaccines were inadequate. To achieve an adequate titre, a live vaccine is apparently required. The use of temperature-sensitive mutants 27 led to the objection that reversion to the wild type is always possible. This new method overcomes these objections by offering a vaccine that is safe, effective, non-pathogenic and controllable. Moreover, it can be given orally, which is an advantage for herd immunity. This approach allows observation of the population dynamics of a single foreign epitope after infecting the host with non-pathogenic E. coli. After the bacteria become established in the host, administration of IPTG allows presentation of the experimental epitope to the immune system via a live organism without many of the confounding factors now commonly used, such as adjuvant, hapten carrier or denaturing agents and conditions. The coadministration of a naturally infective dose and IPTG could produce a dose response in the host that would be unique to the specific protein of interest. The transformed E. coli and the unique method of controlling protein expression in vivo offer a new method of studying molecular pathology and antigen presentation issues in infectious disease. ACKNOWLEDGEMENTS This work was supported in part by Public Health Service grants RR00890 and RR08552 from the National Institutes of Health. The authors thank David Steutermann, Geoffrey Tadda and Su-In Ho for excellent technical assistance and Bonnie Mendro and J. Harvey Harris for outstanding secretarial help. They also thank Drs Raymond J. MacDonald and Galvin Swift for the suggestion to shear MP DNA into small pieces to deal with TGA stop codons, and Dr Victor K. Lin for his technical advice concerning the cloning procedure. We also thank Dr Ping Chuan Hu (University of North Carolina, Chapel Hill, NC) for sequencing the insert DNA. REFERENCES Cassell, G.H., Russell Lindsey, J., Davis, J.K., Davison, M.K., Brown, M.B. and Mayo, J.G. Detection of natural Mycoplasma pulmonis infection in rats and mice by an enzyme linked immunosorbent assay (ELISA). Lab. Anim. Sci. 1981, 31,676-682
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6
7 8
9 10 11 12
13 14 15
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Cassell, G.H. and Davis, J.K. Protective effect of vaccination against Mycoplasma pulmonis respiratory disease in rats. Infect. Immun. 1978, 21, 69-75 Taylor, G., Howard, C.J. and Gourlay, R.N. Protective effect of vaccines on Mycoplasma pulmonis-induced respiratory disease of mice. Infect. Immun. 1977, 16, 422-431 Hoiseth, S.K. and Stocker, B.A.D. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature (London) 1981, 291,238-239 Fairweather, N.F., Chatfield, S.N., Makoff, A.J., Strugnell, R.A., Bester, J., Maskell, D.J. and Dougan, G. Oral vaccination of mice against tetanus by use of a live attenuated Salmonella carrier. Infect. Immun. 1990, 58, 1323-1326 Guzman, C.A., Brownlie, R.M., Kadurugamuwa, J., Walker, M.J. and Timmie, T.N. Antibody responses in the lung of mice following oral immunization with Salmonella typhimurium aroA and invasive Escherichia coli strain expressing the filamentous hemagglutinin of Bordetella pertussis. Infect. Immun. 1990, 59, 4391-4397 Lai, W.C., Bennett, M., Pakes, S.P. and Murphree, S.S. Potential subunit vaccine against Mycop/asma pulmonis purified by a protective monoclonal antibody. Vaccine 1991, 9, 177-184 Inamine, J.M., Ho, K.C., Loechel, S. and Hu, P.C. Evidence that TGA is read as a tryptophan codon rather than as a stop codon by Mycop/asma pneumoniae, Mycop/asma genitalium and Mycoplasma gallisepticum. J. Bacterio/. 1990, 172, 504-506 Yamao, Y., Muto, A., Kawandi, Y., Iwami, M., Iwagami, S., Azumi, Y. and Oswa, S. UGA is read as tryptophan in Mycoplasma caprio/um. Proc. Nat/Acad. Sci. USA 1985, 82, 2306-2309 Dallo, S.F., Su, C.J., Horton, J.R. and Bassman, J.B. Identification of P1 gene domain containing epitope(s) mediating Mycoplasma pneumoniae cytoadherence. J. Exp. Med. 1988, 167, 718-723 Frydenbert, J., Lind, K. and Hu, P.C. Cloning of Mycoplasma pneumoniae DNA and expression of Pl-epitomes in Escherichia coil. Israel J. Med. Sci. 1987, 23, 759-762 Lai, W.C., Pakes, S.P., Stefann, C. and Lu, Y.S. Comparison of Chalquest and Hayblide media, with and without ammonium reinekate, for isolating Mycoplasma pulmonis from rats. J. C/in. Microbiol. 1986, 23, 817-821 Hane, M.W. and Wood, T.H. Escherichia coli K 12 mutants resistant to nalidixic acid: genetic mapping and dominance studies. J. Bacteriol. 1969, 99, 238-241 Henry, M.F. and Cronan, J.E. Direct and general selection for lysogens of Escherichia coli by phage recombinant clones. J. Bacteriol. 1991, 173, 3724-3731 Jones, T.C. and Hirsch, J.G. The interaction in vitro of Mycoplasma pulmonis with mouse peritoneal macrophages and L-cells. J. Exp.
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Med. 1971, 133, 231-259 16 Trevino, L.B., Haldewag, W.G. and Baseman, J.B. Expression of Mycoplasma pneumoniae antigens in Escherichia. Infect. Immun. 1986, 53, 129-134 17 Huynh, T.V., Young, R.A. and Davis, R.W. Constructing and screening cDNA libraries in ~gt 10 and ~.gt 11. In: DNA Cloning: Vol. 1 - A Practical Approach (Ed. Glover, D.M.) IRL Press, Oxford, 1985, pp. 49-78 18 Sambrook, J., Fritsch, E.F. and Maniatis, T. In: Molecular Cloning. A Laboratory Manual, 2nd Edn, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, p. E.37 19 Clarke, L. and Carbon, J. A colony bank containing synthetic EcoR1 hybrid plasma representative of the entire E. coli genome. Cell 1976, 9, 91-99 20 Chisholm, D. A convenient moderate-scale procedure for obtaining DNA from bacteriophage lambda. Biotechniques 1989, 7, 21-23 21 Dorfman, D.M., Zon, L.l. and Orkin, S.H. Rapid amplification of lambda gt11 bacteriophage library inserts from plaques using the polymerase chain reaction (PCR). Biotechniques 1989, 7, 568-570 22 Sambrook, J., Fritsch, E.F. and Maniatis, T. Bacteriophage ~. growth, purification and DNA extraction. In: Molecular Cloning. A Laboratory Manual, 2nd Edn, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, pp. 2.64-2.80 23 Biggin, M.D., Gibson, J.J. and Hong, G.F. Buffer gradient gels and ~S label as an aid to rapid DNA sequence determination. Prec. Natl Acad. Sci. USA 1983, 80, 3963-3985 24 Sanger, F., Nicklen, S. and Coulson, A.R. DNA sequencing with chain terminating inhibitions. Proc. Natl Acad. Sci. USA 1977, 74, 5463-5467 25 Olsson, A., Moks, T., Uhlen, M. and Gaal, A.B. Uniformly spaced banding pattern in DNA sequencing gels by use of field-strength gradient. J. Biochem. Biophys. Methods 1984, 10, 83-90 26 Hunkapiller, M.W., Lujan, E., Ostrander, F. and Hood, L.E. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983, 91,227-236 27 Lai, W.C., Bennett, M., Lu, Y.S. and Pakes, S.P. Biological evaluation of Mycoplasma pulmonis temperature-sensitive mutants for use as possible rodent vaccines. Infect. Immun. 1990, 58, 2289-2298 28 Naot, Y. In vitro studies on the mitogenicity activity of mycoplasmal species toward lymphocytes. Rev. Infect. Dis. 1982, 4, $205-209 29 Lai, W.C., Pakes, S.P., Owusu, I. and Wang, S. Mycoplsma pulmonis depresses humoral and cell-mediated response in mice. Lab. Anim. Sci. 1989, 39, 11-15 30 Stryer, L Control of gene expression in procaryotes. In: Biochemistry W.H. Freeman, New York, 1988, pp. 800-822