Attenuating bacterial virulence by attenuating bacterial folate transport

Abstract

The invention relates to bacterial infections, vaccines directed against those infections and bacterial vaccines. More particularly, the invention relates to vaccines directed against Streptococcus infections in pigs. The invention provides a ΔFolT mutant of a bacterium having reduced capacity to transport folate, wherein said capacity has been reduced by functionally deleting folate transporter (FolT) function. The invention also provides a method to reduce virulence of a bacterium comprising reducing the capacity of said bacterium to transport folate.

Claims

1. A recombinant ΔFolT mutant of a Streptococcus suis (S. suis) bacterium having reduced capacity to transport folate compared to wild type, wherein said capacity has been reduced by deletion or inactivation of a gene of the S. suis encoding folate transporter (FolT) function.

2. The recombinant ΔFolT mutant of claim 1 having the capacity to synthesize folate.

3. The recombinant ΔFolT mutant of claim 1 having reduced expression of FolT.

4. The recombinant ΔFolT mutant of claim 1 having a mutation or deletion of or in the peptide domain FYRKP or an insertion in the peptide domain FYRKP.

5. The recombinant ΔFolT mutant of claim 1 deposited as “CBS 140425 Streptococcus suis ΔFolT mutant” at the Centraalbureau voor Schimmelcultures at Aug. 19, 2015.

6. The ΔFolT mutant of claim 1 deposited as “CBS 143192 Streptococcus suis ΔFolT2 mutant” at the Westerdijk Fungal Biodiversity Institute at Aug. 25, 2017.

7. A composition comprising the bacterium of claim 1.

8. An immunogenic composition comprising the bacterium of claim 1.

9. A vaccine comprising the bacterium of claim 1.

10. A kit for vaccinating a pig, against a disease associated with a Streptococcus suis infection comprising: a dispenser for administering a vaccine to the pig; the recombinant ΔFolT mutant strain according to of claim 1; and optionally an instruction leaflet.

11. The ΔFolT mutant of claim 1, wherein amino acid R in a peptide domain FYRKP has been mutated.

Description

FIGURE LEGENDS

(1) FIG. 1.

(2) [10] depicts the clone that was identified using a complementation strategy containing two incomplete ORFs (greyish blue arrows) and a putative operon (purple) containing orf2[10] and folC[10] preceded by the putative promoter of the operon (for clarification only the sequence of the −35 region (TGGACA) of the putative promoter is depicted in the diagram). Constructs were made that contained either orf2[10] orfo/C[10] (purple) preceded by the putative −35 region of the putative promoter region of the operon (purple). A construct containing orf2 from strain S735 (green) with the −35 region of the putative S735 promoter (TGGTCA) (green) was made. The same construct was mutagenized to contain the −35 region of the putative promoter sequence of strain 10 (TGGACA) (purple) yielding orf2[S735] [t488a].

(3) FIG. 2. In vitro transcription/translation of FolC and ORF2/FolT. In vitro transcription translation of the control construct pCOM1 (lane 1), and constructs pCOM1-V[10] (lane 2), and pCOM1-folc[10] (lane 3). The molecular weight marker is indicated in kDa on the right. FolC expression was detected at the expected weight of 46.8 kDa (closed arrowhead), whereas expression of OR2/FolT was detected at a lower molecular weight, 14 kDa than expected (20.5 kDa) (open arrowhead).

(4) FIG. 3. Predicted riboswitch for tetrahydrofolate using Rfam. Three-dimensional structuring of RNA suggested a riboswitch in which two putative ribosomal binding sites (blue arrows) are inaccessible for ribosomes due to folding.

(5) FIG. 4. Clustal W alignment of different FolT sequences. * indicates identical amino acids; : indicates conservation between groups of strongly similar properties; . indicates conservation between groups of weakly similar properties. CB=Clostridium bolteae; CP=Clostridium phytofermentans; AM=Alkaliphilus metalliredigens; TT=Thermoanaerobacter tengcongensis; EFM=Enterococcus faecium; EFS=Enterococcus faecalis; LB=Lactobacillus brevis; SM=Streptococcus mutans; SG=Streptococcus gallolyticus; SUB=Streptococcus uberis; SSU=Streptococcus suis P1/7.

(6) Red indicates the small and hydrophobic amino acids (including aromatic −Tyr); blue indicates acidic amino acids; Magenta indicates basic amino acids and green indicates hydroxyl, sulphydryl, amine and Gly.

(7) FIG. 5. Folate metabolism in Streptococcus suis.

(8) Schematic presentation of the putative folate metabolism of S. suis.

(9) FIG. 6. Expression levels of orf2 and folC in S. suis wild-type isolates and mutants.

(10) Expression level of orf2 and folC in S. suis wild type isolates strain 10 (black bars) and S735 (white bars) grown exponentially in Todd Hewitt (panel A); and in strain S735 complemented with empty control plasmid pCOM1 (black bars), with orf2[10] (white bars) or with orf2[S735] (hatched bars) grown exponentially in Todd Hewitt (panel B). Expression level of orf2 in S735 complemented with orf2[10], orf2[S735] and orf2[S735] [t488a] after growing in Todd Hewitt until early exponential phase (EEP) (white bars), exponential phase (EP) (small hatched bars), late exponential phase (LEP) (large hatched bars) and stationary phase (SP) (black bars) (panel C). Expression levels were determined using qPCR and expressed as relative expression to housekeeping gene recA. The experiments were performed in triplicate; error bars indicate standard error of the mean. Significance was determined by paired t-tests. * p<0.05; ** p<0.01.

(11) FIG. 7. Predicted 3-dimensional structure for FolT protein of S. suis.

(12) FIG. 8. Body temperature of piglets after S. suis infection, experiment 1. Averaged body temperatures of piglets (n=5) either infected with wild type strain 10 (pink) or with strain 10ΔfolT (CBS 140425) are depicted. Error bars indicate standard error of the mean.

(13) FIG. 9. Bacteraemia of piglets after S. suis infection, experiment 1. Averaged bacteraemia of piglets (n=5) either infected with wild type strain 10 (pink) or with strain 10ΔfolT (CBS 140425) (blue) are depicted. Error bars indicate standard error of the mean.

(14) FIG. 10. Survival curves of pigs infected with S. suis, experiment 1. Pigs were infected either wild type strain 10 or with strain 10ΔfolT (CBS 140425). Pigs were euthanized when they reached predetermined humane end points for ethical reasons. Statistical analysis was done using Log-rank (Mantel-Cox) test.

(15) FIG. 11. Bacteriological examination of piglets infected with S. suis, experiment 1. Pigs were infected either wild type strain 10 or with strain 10ΔfolT (CBS 140425) Bacteria were enumerated by serial dilution and plating. Bacterial counts were calculated as CFU/ml. Different colours indicated different individual piglets.

(16) FIG. 12. Survival curves of piglets infected with S. suis, experiment 2. Piglets (n=10) were infected either with wild type strain 10 or with strain 10ΔfolT (CBS 140425) Pigs were euthanized when they reached predetermined humane end points for ethical reasons.

(17) FIG. 13. Body temperature of piglets after S. suis infection, experiment 2. Averaged body temperatures of piglets (n=10) either infected with wild type strain 10 (blue) or with strain 10ΔfolT (CBS 140425) (green) are depicted.

(18) FIG. 14. Locomotion of piglets after S. suis infection, experiment 2. The percentage of positive observations in piglets either infected with wild type strain 10 (blue) or with strain 10ΔfolT (CBS 140425) are shown. Severity locomotion 1: mild lameness; 2: moderately lameness or reluctance to stand; 3: severe lameness (serving as a human endpoint)

(19) FIG. 15. Consciousness of piglets after S. suis infection, experiment 2. The percentage of positive observations in piglets either infected with wild type strain 10 (blue) or with strain 10ΔfolT (CBS 140425). Severity Consciousness: 1: depression; 2: apathy; 3: coma

(20) FIG. 16. Vaccination of pigs with the ΔFolT 2 strain (CBS 143192) and protection after challenge with S. suis type 2. Pigs were vaccinated at day 1 and 21 with ΔFolT2 strain (CBS 143192). On day 35, the animals were challenged intraperitoneally (ip) with approximately 2×10.sup.9 CFU of a virulent S. suis type 2 isolate. For seven days following challenge, the animals were observed for signs of disease associated with S. suis. Animals found dead or that had to be euthanized prior to off-test for animal welfare reasons were necropsied. The figure shows the percentage of animals that died or were euthanized following challenge (mortality).

(21) FIG. 17. Vaccination of pigs with the ΔFolT strain (CSB140425) and protection after challenge with S. suis type 2.

(22) Pigs were vaccinated at day 1 and 21 with the ΔFolT strain (CBS 140425). On day 36, the animals were challenged intraperitoneally with approximately 2×10.sup.9 CFU of a virulent S. suis type 2 isolate. Following challenge, the animals were observed for signs of disease associated with S. suis for seven days. Animals found dead or that had to be euthanized prior to off-test for animal welfare reasons were necropsied. The figure shows the percentage of animals that died or were euthanized following challenge (mortality).

DETAILED DESCRIPTION

Introduction

(23) Previously, we used a complementation strategy to identify novel virulence factors, which might serve as vaccine candidates. Using this strategy, a hypervirulent S. suis isolate (S735-pCOM1-V[10]) was generated that causes severe toxic shock-like syndrome in piglets after infection resulting in death within 24 h post-infection[14]. S735-pCOM1-V[10] was selected from a library of clones generated in a weakly virulent serotype 2 isolate (S735), after transformation with plasmid DNA isolated from around 30,000 pooled clones carrying randomly cloned genomic DNA fragments from a virulent serotype 2 isolate (strain 10). Isolates with increased virulence were selected by infecting piglets with strain S735 containing the plasmid library of genomic fragments from strain 10. One prevalent clone isolated from the infected piglets contained a 3 kb genomic fragment from strain 10 designated V[10] and was demonstrated to be hypervirulent in subsequent animal experiments. V[10] contained an incomplete open reading frame (ORF), followed by two genes (orf2 and folC) in an operon structure as well as a second incomplete ORF. Assuming that only the full-length ORFs could contribute to the hypervirulence of this isolate, we further characterized the orf2-folC-operon. The first ORF in the operon could not be annotated and was designated orf2, the second ORF in the operon showed homology to the gene encoding polyfolylpolyglutamate synthase (FolC). This operon was present in all S. suis serotypes, including the parent strain S735. Strain S735 with low virulence, contained several single nucleotide polymorphisms (SNP) in orf2-folC and the non-coding regions compared to strain 10. Both genes of the operon that increased the virulence may be putative virulence factors and, if so, could be putative vaccine candidates. Here we investigated 1) whether the hypervirulence of the orf2-folC-operon is caused by orf2 or by folC or both and 2) the effect of a single nucleotide polymorfism in the promotor region of the orf2-folC-operon on virulence.

Materials and Methods

Bacterial Strains and Plasmids

(24) S. suis isolates were grown in Todd-Hewitt broth (Oxoid, London, United Kingdom) and plated on Columbia blood base agar plates (Oxoid) containing 6% (vol/vol) horse blood. Escherichia coli was grown in Luria Broth and plated on Luria Broth containing 1.5% (wt/vol) agar. If required, erythomycin was added at 1 μg ml.sup.−1 for S. suis and at 200 μg ml.sup.−1 for E. coli. S. suis strain S735 complemented with a plasmid containing a 3 kb genomic fragment derived from strain 10 (S735-pCOM1-V[10]) and the other S. suis strains used in this study have been previously described [14] (FIG. 1).

Example 1 Complementation of S. Suis Strain S735

(25) S735 was complemented with plasmid pCOM1 containing one of the two ORFs in the V[10] operon (i.e. orf2[10], or folC[10]) preceded by the putative promoter region of the operon from strain 10 or with plasmid pCOM1 containing orf2 and the cognate upstream promoter from strain S735 (orf2[S735]) (FIG. 1). To construct these plasmids, primers with restriction sites were designed to amplify orf2[10] or orf2[S735] (comE1-comE2), folC[10] (comE4-comE6) or the promoter region of the operon (comE1-comE3) (Table 1). The resulting PCR products orf2[10] and orf2[S735] were digested using restriction enzymes SacI and BamHI, cloned into pKUN19 [15], digested with the same restriction enzymes and subsequently cloned into pCOM1, yielding pCOM1-orf2[10] and pCOM1-orf2[S735], respectively. The PCR amplicon of folC[10] was digested using restriction enzymes SmaI and BamHI and cloned into pKUN19 cleaved with the same restriction enzymes. The PCR product comprising the promoter region of V[10] was cloned in front of folC[10] using restriction enzymes SacI and SmaI. Subsequently, the complete fragment of promoter V[10]-folC[10] was digested from pKUN19 using SacI and BamHI and cloned into pCOM1 digested with the same restriction enzymes, yielding pCOM1-folC[10]. To confirm that the fusion product of promoter—folC[10] was transcribed, in vitro transcription/translation was performed using .sup.35S-methionine. A clear band of the molecular weight of FolC (46.8 kDa) was detected demonstrating that the fusion product could be expressed and translated (FIG. 2). All plasmids were introduced into S. suis strain S735 by electroporation. In addition, pCOM1-V[10] was introduced into the avirulent serotype 2 strain T15 by electroporation to yield T15-pCOM1-V[10].

Example 2 Experimental Infection with Complemented Isolates

(26) Experimental infection of caesarean derived germ-free piglets was performed as previously described [14]. Prior to infection, germ-free status of piglets was confirmed by plating tonsil swabs on Columbia agar plates containing 6% horse blood. Briefly, 4 or 5 one-week-old germ-free pigs were infected intravenously with 10.sup.6 colony-forming units (CFU) of S. suis and then immediately orally administered 40 mg kg.sup.−1 body weight of erythomycin (erythomycin-stearate, Abbott B. V., Amstelveen, The Netherlands) twice a day to keep selective pressure on S. suis isolates harbouring the pCOM plasmids. Infected pigs were monitored twice daily for clinical signs and tonsil swabs collected for bacteriological analysis. Pigs were euthanized when clinical signs of arthritis, meningitis, or sepsis were observed after infection with S. suis. Tissue specimens of CNS, serosae and joints were collected during necropsy, homogenized and bacterial cell counts were determined by plating serial dilutions on Columbia agar plates containing 6% horse blood and 1 μg of erythomycin. To be able to compare results from different animal experiments included herein, a uniform scoring of non-specific and specific symptoms was applied to all animal experiments. Non-specific symptoms included inappetite and depression that were scored 0 (none), 0.5 (mild inappetite/depression) or 1 (severe inappetite/depression). Specific symptoms included lameness, central nervous system (CNS) symptoms (locomotive disorders like cycling, or walking in circles; opistotonus; nystagmus), as well as raised hairs, arched back (kyphosis), and shivering, since these are all symptoms of sepsis or serositis. Based on these observation clinical indices were calculated by dividing the number of observations where either specific or non-specific symptoms were observed by the total number of observations for this parameter. This represents a percentage of observations where either specific or non-specific symptoms were observed. A similar approach was taken for the ‘Fever Index’. Fever was defined as a body temperature>40° C. ‘Mean number of days till death’ was used as a survival parameter. Although animals were euthanized after reaching humane end points (HEP), the time between inoculation and reaching HEPs is still indicative of severity of infection. It is calculated by averaging the survival in days from inoculation until death.

(27) Animal experiments with strain CBS 140425 were performed at the premises of Central Veterinary Institute of Wageningen UR, Lelystad, The Netherlands (now named Wageningen Bioveterinary Research (WBVR)) and were approved by the ethical committee of the Central Veterinary Institute of Wageningen UR, Lelystad, The Netherlands, in accordance with the Dutch law on animal experiments (#809.47126.04/00/01 & #870.47126.04/01/01). Animal experiments with strains CBS 140425 and 143192 were also performed in accordance with the US law on animal experiments.

(28) Statistical analyses were performed on clinical indices of the groups (fever index, specific symptoms and non-specific symptoms) using a non-parametric Kruskal-Wallis test, as there was no homogeneity of variance among groups. In subsequent analyses, all groups were compared pairwise to the control group (S735-pCOM1) on all three parameters, using Mann-Whitney U tests. Differences were considered statistically significant at p<0.05. Calculations were performed using SPSS 19 (IBM, New York, USA).

Example 3 Experimental Infection with Strain 10ΔFolT (CBS 140425), Experiment 1

(29) Ten 4-week-old piglets were housed at CVI animal facility in two groups of five animals. Piglets had ad lib access to feed and fresh water. A light provided animals with warmth and play material was available throughout the experiment. Prior to the start of the experiment, tonsil swabs of piglets were screened by PCR on colonization of S. suis serotype 2. Only PCR-negative piglets were included in the experiment. After ten days, animals were infected intravenously with either 1.1.Math.10.sup.6 CFU of wild type strain 10 or with 9.2.Math.10.sup.5 CFU mutant strain 10ΔfolT in the vena jugularis. Prior to infection basal temperatures of piglets were monitored daily for a period of three days. EDTA blood was collected prior to infection to obtain pre-infection plasma samples, as well as basal levels of white blood cell (WBC) numbers. Infected pigs were monitored three times a day at 8 pm, 3 am and 9 am for clinical signs. Non-specific symptoms included lack of appetite and depression, whereas, specific symptoms included lameness, central nervous system (CNS) symptoms (locomotive disorders like cycling, or walking in circles; opistotonus; nystagmus), as well as raised hairs, arched back (kyphosis), and shivering, all of which are symptoms of sepsis or serositis. Tonsil and faecal swabs were collected daily for bacteriological analysis. Blood was collected daily for bacteriological analysis, WBC counting and plasma collection. Pigs were euthanized when clinical signs of arthritis, meningitis, or sepsis were observed after infection with S. suis. At necropsy, internal organs (kidney, liver, spleen, peritoneum and pericardium) were bacteriologically screened for S. suis by plating on Columbia agar plates containing 6% horse blood. Organs that were macroscopically affected by S. suis, like purulent arthritis joints, pericarditis or peritonitis were plated in serial dilution to determine the bacterial load. Tissue specimens of these organs were fixated in formalin for histological examination. The animal experiment was approved by the ethical committee of the Central Veterinary Institute of Wageningen UR, Lelystad, The Netherlands, in accordance with the Dutch law on animal experiments (#2014011).

Example 4 Experimental Infection with Strain 10ΔFolT (CBS 140425), Experiment 2

(30) In a second experiment, approximately 3-week old piglets (Commercial Cross) were used. The piglets had not been vaccinated against S. suis, had been obtained from a PRRSV negative herd, had never received medicated feed and were tonsil swab negative for S. suis serotype 2 by PCR upon enrolment. Treatment groups (10 piglets each) were housed separately. Animals were inoculated intravenously with either 3.48E+07 CFU of wild type strain 10 or with 1.45E+07 of mutant strain 10ΔfolT. The animals were observed once a day for clinical signs of S. suis associated disease (e.g. increase in body temperature, lameness, and changes in behaviour) for 7 days. Any animals displaying clinical signs that reached humane end-points (e.g. CNS signs, debilitating lameness) were euthanized to minimize suffering. Euthanized animals were necropsied to identify lesions typically associated with S. suis disease. Animals surviving to the end of the observation period were likewise euthanized and necropsied.

Example 5a Vaccination of Pigs with ΔFolT2 Strain (CBS 143192) and Protection after Challenge with S. suis Type 2

(31) The study was conducted in commercial cross pigs; on the day of first vaccination, the pigs were 21±7 days of age. The animals had not been vaccinated against S. suis, were tonsil swab negative for S. suis type 2 by PCR, PRRSV negative by serology and originated from sows that were tonsil swab negative for S. suis type 2 by PCR. The study groups, the vaccination route and dose, the days of vaccination, and the day and route of challenge are listed in Table 6. The media used are described in Table 7.

(32) On day 34, blood and tonsil swabs were collected from all animals, and then the strict control animals were moved to a separate airspace while all other groups were commingled. On day 35, the animals were challenged intraperitoneally (ip) with approximately 2×10.sup.9 CFU of a virulent S. suis type 2 isolate.

(33) For seven days following challenge, the animals were observed for signs of disease associated with S. suis. Animals found dead or that had to be euthanized prior to off-test for animal welfare reasons were necropsied. During necropsy, the animals were assessed for macroscopic signs typically associated with S. suis disease and a CNS (i.e. brain) and joint swab were collected. At off-test, all remaining animals were euthanized, necropsied and samples collected.

(34) The preparation of the vaccines and placebo are listed in Table 7.

(35) The preparation of the challenge material is listed in Table 8.

(36) Vaccination with the S. suis ΔFolT mutant reduced the number of animals that died or had to be euthanized for animal welfare reasons during the post-challenge observation period (see Table 9 and FIG. 16). In addition, vaccination with the ΔFolT reduced findings of severe lameness (ie. the number of animals being unable to stand or being reluctant to stand), as well as findings of apathy in which the animals showed very limited to no interest in the environment (see Tables 10 and 11).

(37) During necropsy, signs of inflammation in the brain, indicated by the presence of fibrin and/or fluid, were less frequently observed in ΔFolT vaccinated animals compared to the negative controls (see Table 12).

(38) The S. suis challenge isolate was less frequently recovered from the brain and the joint swabs collected at necropsy from animals vaccinated with the ΔFolT strain compared to the negative controls (see Tables 13 and 14).

Example 5b Vaccination of Pigs with Strain 10ΔFolT (CBS 140425) and Protection after Challenge with S. Suis Type 2

(39) The study was conducted in commercial cross pigs, 21+/−5 days at the day of the first vaccination. The animals had not been vaccinated against S. suis, were tonsil swab negative for S. suis type 2 by PCR, PRRSV negative by serology and originated from sows that were tonsil swab negative for S. suis type 2 by PCR. The study groups, the number of animals/group at the time of study initiation, the vaccination dose, the days of vaccination, the vaccination route, the day of challenge and the challenge route are listed in Table 15.

(40) On day 35, blood and tonsil swabs were collected from all animals and the strict control animals were euthanized. On day 36, the animals were challenged intraperitoneally with approximately 2×10.sup.9 CFU of a virulent S. suis type 2 isolate.

(41) Following challenge, the animals were observed for signs of disease associated with S. suis for seven days. Animals found dead or that had to be euthanized prior to off-test for animal welfare reasons were necropsied. During necropsy, the animals were assessed for macroscopic signs typically associated with S. suis disease and CNS swabs were collected. At off-test, all remaining animals were euthanized, necropsied and samples collected.

(42) The preparation of the vaccine and placebo is listed in Table 16. The preparation of the challenge material is listed in Table 17.

(43) The S. suis FolT mutant reduced the number of animals showing lameness following challenge, the number of animals showing abnormal behavior (i.e. depression, coma) following challenge as well as the number of animals that died or had to be euthanized for animal welfare reasons during the post-challenge observation period (see Table 18, 19 and 20 and FIG. 17).

(44) At off-test (i.e. at day 7 following challenge or upon removal from the study due to death or euthansia) the animals were observed for abnormal findings in the brain (i.e. fibrin, fluid) as well as in the thoracic cavity (i.e. fibrin, fluid, lung congestion, pneumonia). In addition, samples were collected from the brain for the recovery of S. suis. The results are listed in Table 21, 22 and 23.

Example 6 cDNA Synthesis and Quantitative PCR

RT-PCR

(45) Two hundred ng of RNA was used to synthesize cDNA in a reaction containing 25 ng μl.sup.−1 random primers (Promega, Madison, Wis., USA), 10 mM dNTPs (Promega), 10 mM DTT (Invitrogen), 40 U RNAsin (Promega) and SuperScriptII Reverse Transcriptase (Invitrogen) according to manufacturer's instructions.

qPCR

(46) cDNA was diluted 20 times for qPCR analysis. Primers were designed using PrimerExpress software (Applied Biosystems, Foster City, Calif., USA) (Table 1). Each reaction contained 12.5 pmol forward primer, 12.5 pmol reverse primer and POWR SYBR Green PCR Master Mix (Applied Biosystems) according to manufacturer's instructions. qPCR was performed using an AB17500 (Applied Biosystems). GeNorm software (GeNorm) was used to determine the most stably expressed reference genes. For S. suis recA was the least variable in expression of the 6 potential reference genes (phosphogelycerate dehydrogenase (pgd), acetyl-coA acetyltransferase (aca), mutS, glutamate dehydrogenase (gdh) tested. Genorm combines expression data into a number representing stability of expression, where 1 represents the most stabile gene. Stability numbers for S. suis ranged from 1.667 for gdh to 1.217 for recA. The level of expression of these reference genes was measured to control for variation in RNA-yield and RT-reaction conditions. In each qPCR run a standard curve was incorporated consisting of a vector containing a cloned PCR product of the target gene of that reaction. The standard curve consisted of seven 10-fold dilutions of the control vector. In this way both the expression level of the target gene and the expression levels of external reference genes could be calculated from a standard curve. For each reaction water was included in place of cDNA or template as a negative control. Analysis was performed using the AB17500 Software (Applied Biosystems).

Sequence Analysis

(47) Sequence reactions and analysis were performed by Baseclear (Leiden, The Netherlands).

Example 7 Site-Directed Mutagenesis

(48) Site directed mutagenesis was achieved using the Quick-change II site-directed mutagenesis kit (Agilent Technologies, La Jolla, Calif., USA) according to manufacturer's instructions. PCR primers were designed with the accompanying software (Agilent Technologies) (Table 1). Using primers t448a and t488a_antisense the plasmid pCOM-orf2[S735] was amplified, introducing the desired mutation that changed the −35 region of the putative promotor region of the orf2-folC-operon of S735 from 5′-TGGTCA-3′ to 5′-TGGACA-3′ (FIG. 1). The reaction mixture was digested using DpnI to inactivate the original template vector and subsequently transformed to XL-1-blue competent cells (Invitrogen). To exclude the possibility of introducing PCR errors into the vector backbone, the insert of the plasmid (orf2[S735]) was isolated from the template vector after digestion with restriction enzymes BamHI and SacI and cloned into pCOM1 digested with the same restriction enzymes. The resulting plasmid was introduced into S. suis isolate S735 by electroporation and transformants were selected on Columbia agar containing 1 μg ml.sup.−1 erythomycin, yielding S735-pCOM1-orf2[S735][t488a]. Sequencing was used to exclude presence of PCR errors in the final construct.

Example 8 Construction Knock-Out Mutant of S. Suis Folate Substrate Binding Protein (FolT)

(49) An isogenic folT knock out mutant was constructed in strain 10 by disrupting folT with a Spectinomycin resistance cassette. pCOM1-V[10] [14] was digested with BamHI and ligated into BamHI digested pKUN plasmid, yielding pKUN-V[10]. To remove 3′ part of V[10], pKUN-V[10] was digested with SphI after which the vector fragment was purified and ligated, yielding pKUN-V[10]*. pKUN-V[10]* was partially digested with XmnI, the linear vector fragment was purified and ligated with the blunt end Spectinomycin resistance cassette, yielding pKUN-V[10]*-Spec.sup.R. For construction of the mutant V[10]*-Spec.sup.R was amplified by PCR using V735-fw and M13-rev. The PCR product was purified using the PCR Purification Kit (Qiagen). The purified PCR-product was used transform S. suis strain 10 using ComS as competence inducer as described by Zaccaria et al. [16] to induce homologous recombination. Transformants were selected on Columbia agar plates containing 6% (vol/vol) horse blood and 100 μg ml.sup.−1 spectinomcyin. Double crossovers were checked by PCR and confirmed using Southern blotting. To exclude the possibility of introduction of point mutations, chromosomal DNA of the isogenic knockout mutant was isolated and transformed to strain 10. Again mutants were selected on Columbia agar plates containing 6% (vol/vol) horse blood and 100 μg ml.sup.−1 spectinomcyin, and screened by PCR, yielding strain 10ΔfolT. This prototype recombinant ΔFolT mutant strain has been deposited as “CBS 140425 Streptococcus suis ΔFolT mutant” at the Centraalbureau voor Schimmelcultures for the purpose of patent procedure under the Regulations of the Budapest Treaty at Aug. 19, 2015.

Example 9 ΔFolT Deletion Mutants not Containing the Spectinomycin Resistance Gene

(50) A ΔfolT deletion mutant not containing the Spectinomycin resistance gene was constructed as well. For this the thermosensitive shuttle vector pSET5s (Takamatsu, D., Osaki, M. and Sekizaki, T. 2001. Plasmids 46: 140-148) was used. Plasmid pSET5s contains a temperature sensitive origin of replication and can be propagated at 37° C. in E. coli, but replication of the plasmid is blocked above 37° C. in S. suis (Takamatsu et al). pSET5s contains a cloramphenicol resistance gene (Cm) that can be used for selection of transformants in E. coli as well as in S. suis. A prototype recombinant ΔFolT mutant strain not containing the Spectinomycin resistance gene has been deposited as “CBS 143192 Streptococcus suis ΔFolT2 mutant” at the Westerdijk Fungal Biodiversity Institute for the purpose of patent procedure under the Regulations of the Budapest Treaty at Aug. 25, 2017.

(51) To construct a ΔfolT mutant isolate, a PCR product containing the 5′- and 3′-flanking sequences of the folT gene was generated. This fragment is cloned into pSET5s and Cm resistant transformants are selected at 37° C. in E. coli. The plasmid was then isolated from E. coli and introduced into S. suis strain 10. Transformants were selected on Columbia agar plates at 30° C. containing Cm. A transformed colony was used to inoculate 1 ml of Todd Hewitt Broth (THB) containing Cm and the culture was grown overnight at 30° C. The overnight culture was diluted 100-fold in the same medium and was incubated as above until an optical density at 600 nm of 0.2-0.3 is reached, at which the culture is transferred to 38° C. At this temperature, the plasmid is unable to replicate. This step selects for strains in which the plasmid has integrated into the chromosome via a single recombination event. Serial dilutions of this culture were plated at Columbia horse blood plates containing Cm. Plates were incubated overnight at 38° C. A colony containing the recombinant plasmid integrated into the chromosome was picked and inoculated into 1 ml of Todd Hewitt Broth (THB) with Cm for incubation overnight at 38° C. The culture was diluted 100-fold with Cm-free THB and grown at 28° C. for five subsequent passages. At this temperature, the plasmid is able to replicate and is excised from the chromosome via a second recombination event over the duplicated target gene sequence. The excision of the plasmid can yield the wild type genotype or can result in a folT deletion mutant. Serial dilutions of the culture were plated onto Columbia horse blood plates (without Cm) and incubated overnight at 38° C. Single colonies were then replica plated onto Columbia horse blood plates with and without Cm. Cm sensitive colonies were screened by PCR to identify the ΔfolT mutant isolates not containing the Spectinomycin resistance gene.

Hybridization Studies

(52) Chromosomal DNA was isolated from stationary growing S. suis cultures. Two hundred nanogram of purified DNA was spotted onto Genescreen-Plus (Perkin Elmer, USA). Labelling of probes with .sup.32P, hybridization and washing was done as described before [17]. PCR products of folT and folC were used as a probe, whereas a 16S rRNA probe was used as positive control.

Overexpression of FolT Suffices to Induce Hypervirulence in Strain S735

(53) Introduction of a 3 kb genomic fragment from virulent serotype 2 strain 10, V[10], increased the virulence of the weakly virulent serotype 2 strain S735 [14], creating a hypervirulent isolate (S735-pCOM1-V[10]). All pigs infected with S735-pCOM1-V[10] died within 1 day post infection (p.i.) and a high percentage of the pigs showed severe clinical signs of disease (Table 2), whereas nearly all pigs infected with the control strain S735-pCOM1 survived throughout the experiment. Clinical indices differed significantly (p≤0.01) between pigs infected with S735-pCOM1-V[10] and S735-pCOM1 (Table 2). As a control, we also tested the virulence of S735 transformed with a plasmid containing the homologous 3 kb fragment from strain S735 (S735-pCOM1-V[S735]). A high percentage of the pigs infected with S735-pCOM1-V[S735] survived throughout the experiment. In contrast pigs infected with S735-pCOM1-V[S735] showed significantly more specific clinical signs (p≤0.01) than pigs infected with S735-pCOM1 (Table 2), although differences in clinical indices for fever and non-specific symptoms were not significantly different between the groups (p=0.06). Thus, the increased copy number of V[S735] in S735, due to introduction of plasmid pCOM1-V[S735] increased specific clinical signs of S. suis. Nevertheless, the specific and non-specific clinical signs due to porcine infection with S735-pCOM1-V[10] (p≤0.01) were significantly increased compared to pigs infected with S735-pCOM1-V[S735], demonstrating that the introduction of V[10] in strain S735 increased the virulence more than introduction of V[S735]. This result indicated that hypervirulence of strain S735 pCOM-1-V[10] might be due to the different nucleotide polymorphisms in V[10] compared to V[S735].

(54) To determine if the both the orf2 and the folC-genes are required for the observed increase in virulence, both genes of the operon obtained from strain 10 preceded by its cognate promoter sequence were introduced separately into strain S735 to generate strains S735-pCOM1-orf2[10] and S735-pCOM1-folC[10]. Virulence of these isolates was determined in an experimental infection in piglets, using S735-pCOM1-V[10] and S735-pCOM1 as controls. Table 2 shows that pigs infected with S735-pCOM1-V[10] or with S735-pCOM1-orf2[10] died within one day p.i. with severe clinical signs. Infected pigs developed toxic shock-like syndrome that was not observed using wild-type strain 10 in experimental infections, implying fragment V[10] and orf2[10] increased virulence of S735 yielding more virulent isolates than strain 10 [3]. Both specific and non-specific symptoms were significantly increased (p<0.01) in pigs infected with S735-pCOM1-V[10] or with S735-pCOM1-orf2[10] compared to S735-pCOM1 (Table 2).

(55) Bacteriological examination showed that CNS, serosae and joints were colonized by high CFU of S. suis. In contrast pigs infected with S735-pCOM1-folC[10] or S735-pCOM1 lived throughout the experiment (11 days p.i.) showing mild symptoms of infection, like fever. No significant differences in clinical outcome were observed between pigs infected with S735-pCOM1-folC[10] and with S735-pCOM1. This clearly demonstrates that introduction of folC[10] does not increase the virulence of strain S735, whereas introduction of V[10] and orf2[10] increased the virulence of strain S735. Therefore, we concluded that the observed increased virulence of S735-pCOM1-V[10] compared to S735-pCOM1 was attributed to introduction of orf2[10].

(56) In conclusion, both copy number of V[10] and genetic background of the orf2-folC operon seem to be determinative in the virulence of a given isolate.

Datamining in Silico Data Demonstrates ORF2 is a Substrate Binding Protein Facilitating Folate Transport

(57) Now that the increased virulence was attributed to introduction of multiple copies of orf2[10], the putative function of orf2 was sought. In silico analysis of the 5′ intergenic region preceding the orf2-folC operon revealed the presence of a predicted secondary structure, which showed strong homology to a tetrahydrofolate riboswitch (FIG. 3). Tetrahydrofolate riboswitches are a class of homologous RNAs in certain bacteria that bind tetrahydrofolate (THF) [18]. It is almost exclusively located in the probable 5′ untranslated regions of protein-coding genes, and most of these genes are known to encode either folate transporters or enzymes involved in folate metabolism. For these reasons, it was inferred that the RNAs function as riboswitches. THF riboswitches are found in a variety of Firmicutes, specifically the orders Clostridiales and Lactobacillales, and more rarely in other lineages of bacteria. The THF riboswitch was one of many conserved RNA structures found in a project based on comparative genomics [19]. The relation with folate metabolism was confirmed by Eudes et al, demonstrating that in S. suis the gene upstream of folC encoded a folate transporter, FolT [20]. This annotation was also applied to the new genome sequence of S. suis, SC070731, where the homologous gene of ssu0135 was annotated to encode for FolT (GenBank AGG63648.1). A 3-dimensional structure for the folate energy-coupling factor transport of Lactobacillus brevis was determined [21], leading to the proposal of a multi protein model of the folate transporter. In this model ORF2/FolT functions as the transmembrane substrate-specific binding protein. Together with a more generic transmembrane protein and two nucleotide-binding proteins forming the energy-coupling module this complex facilitates transmembrane transport of folate. Only the substrate-binding protein (FolT) is specific for folate, the other components are also used for transport of other substrates like thiamine or riboflavin. When the protein sequence of FolT of S. suis was compared to putative FolT sequences of other organisms, it was clear that conserved amino acids were also conserved in S. suis [21] (FIG. 4). Interestingly, FIG. 4 also shows that the arginine that was extremely conserved in the human folate transporter as well as in Escherichia coli tetracycline transporters was also conserved in S. suis (Arg99) folT suggesting this residue to be important for transporters ranging from men to bacteria [22]. Taken together, these data strongly suggest that the conserved protein of unknown function, ORF2, identified using a complementation strategy encodes a substrate binding protein facilitating folate transport.

Folate Transport in Streptococcus suis

(58) Sequence analysis of P1/7 (that is highly homologous to the genome of strain 10) indicates that S. suis encodes all enzymes required to synthesize tetrahydrofolate (THF) via the classical folate biosynthesis pathway (FIG. 5). Based on data available in the KEGG database (www.kegg.jp) folate metabolism in S. suis makes use of the classical folate pathways using folE, folQ, folB, folK, folC, folA and substrates GTP, p-aminobenzoate (PABA) and glutamyl (GLU) as depicted in the scheme. However, with the additional genes present on the V[10] operon, S. suis seems also capable of inducing expression of folT to directly import folate and using the simultaneous induced expression of the additional copy of folC, folate can immediately be processed to the endproduct tetrahydrofolate (THF). In this way the combination of folT and folC forms an additional ‘shortcut’ that allows production of THF. The presence of the THF riboswitch upstream of the folT-folC operon suggests tight regulation of this two-gene operon that might imply that the folT-folC operon is only expressed under specific conditions, e.g. folate poor conditions. Based on the results of the animal experiment described above, it is suggested that the folT-folC operon is at least expressed under in vivo conditions.

Presence and Expression of FolT in Streptococcus suis

(59) Presence of the folT gene was demonstrated in all S. suis serotypes tested with exception of serotypes 32, and 34. However, serotypes 32 and 34 were re-assigned to belong to the genus of Streptococcus orisratti, instead of S. suis [1]. So, in conclusion, all S. suis serotypes are deemed to have the genes encoding FolT and FolC.

(60) Sequence analysis of the putative promoter of orf2 revealed a difference at one nucleotide position in the −35 region of the putative promoter in strain 10 (TGGACA) compared to strain S735 (TGGTCA) [14]. The effect of this SNP on expression levels of orf2 and folC in strains 10 and S735 was determined using qPCR analysis. Significantly higher levels of expression of orf2 as well as folC were observed in strain 10 compared to strain S735 (FIG. 6A). This clearly indicates that the SNP in the −35 region of the putative promoter affects the transcription of orf2 and folC. Thereby, it demonstrates that the identified SNP was indeed located in the promoter region co-transcribing orf2 and folC in an operon. Moreover, introduction of pCOM1-orf2[10] into S735 increased expression of orf2 31-fold compared to introduction of pCOM1, whereas introduction of pCOM1-orf2[S735] increased expression of orf2 only 5-fold (FIG. 6B). As expected expression levels of folC were similar for both recombinant strains (FIG. 6B). To confirm that the identified SNP in the −35 region of the promoter is responsible for the differences in transcription of orf2 in strains S735 and 10 the TGGTCA of orf2[S735] was mutated to TGGACA as found in the promoter of orf2[10] (yielding strain S735-pCOM1-orf2[S735][t488a]. Transcript levels of orf2 in S735-pCOM1-orf2[S735][t488a] were shown to be similar to that of strain S735-pCOM1-orf2[10] and four-fold higher than that of strain S735-pCOM1-orf2[S735] in different growth phases (FIG. 6C). Both promoters are most active early in the growth phase of S. suis when grown in Todd Hewitt broth (FIG. 6C). Together, these results clearly demonstrate that in strain 10, the promoter upstream of orf2-folC-operon is stronger than the promoter upstream of this operon in strain S735, due to an SNP in the −35 region.

(61) To determine whether the SNP linked to increased expression of orf2-folC operon was associated with particular clonal types or serotypes of S. suis the promoter regions of a large collection of isolates were sequenced (Table 3). All isolates used were recently characterized and typed by CGH and MLST [23]. Based on the sequence data obtained, isolates could be divided in two main groups (Table 3). The strong −35 promoter region was exclusively found in serotype 1 and 2 isolates that belonged to CGH cluster A and MLST clonal complex 1 and that expressed the EF-protein. The SNP associated with lower promoter activity was found in serotype 7 and 9 isolates belonging to CGH group B (except for two), which are all negative for the expression of EF, as well as in weakly virulent isolates of serotype 2 belonging to CGH group A/Clonal Complex 1 (CC1) that were positive for the expression of the larger form of EF protein (EF*). There were two exceptions; serotype 7 isolate (C126), that belongs to CC1 but does not express the EF-protein contained the SNP linked to a stronger promoter and serotype 7 isolate (7711) which had a different −35 promoter sequence (TTGTCA) for which the promoter strength is undetermined. In conclusion, only CC1 isolates expressing EF protein (and 1 serotype 7 isolate) contain the SNP linked to strong promoter activity. As isolates of this combination of phenotype and genotype are strongly correlated with virulence [23,24], we can conclude that a strong promoter upstream of orf2-folC-operon is associated with virulent isolates of S. suis. This observation, together with the increased virulence observed after introduction of additional copies of folT[10] suggests that increased expression of folT either due to increased copy number or due to a stronger promoter leads to increased virulence in S. suis.

Growth of Streptococcus suis with Additional Copies of FolT or without FolT in Culture

(62) No significant differences were observed in growth in culture of Streptococcus suis with additional copies of folT or without a functional folT in comparison to the parent strain in vitro.

Protein Expression of FolT

(63) Based on the protein sequence of FolT it was predicted that FolT is a very hydrophobic protein with at least 5 transmembrane domains. Homology modeling (Expacy server) using 6 known FolT structures among which the published 3D structure of FolT from Lactobacillus brevis a 3D structure for FolT of S. suis was predicted (FIG. 7).

FolT is Important for Survival In Vivo: Virulence of a FolT Knock-Out Strain 10ΔfolT

(64) Since overexpression of folT in a weakly virulent S. suis strain led to a strong increase of virulence, we hypothesized that FolT plays an important role in vivo. To test whether this hypothesis is true, an isogenic knock-out was constructed in virulent S. suis strain 10 by inserting an spectinomycin-resistance cassette in the folT gene. Since folT and folC are in an operon structure, this knock-out will probably also be knocked out for the additional copy of folC. To determine whether folate transport is essential for virulence in vivo, in experiment 1, ten pigs were intravenously infected with either wild type strain 10 or knock out strain 10ΔfolT. All pigs responded to the inoculation with an increase of body temperature (FIG. 8). However, pigs infected with the wild type strain 10 showed higher temperatures for a longer period of time, compared to pigs infected with the knockout strain 10ΔfolT. This is also reflected by a difference in fever index (percentage of observations where pigs displayed fever) between both groups (p=0.06). This suggests that strain 10ΔfolT is less pyogenic, compared to the wild type strain. This might be a consequence of the fact that significantly fewer bacteria were isolated from the blood of piglets infected with strain 10ΔfolT. Only two pigs infected with strain 10ΔfolT showed a short bacteraemic period, compared to 5 pigs infected with strain 10; pigs infected with strain 10 also had significantly higher numbers of bacteria in their blood for a longer period of time (FIG. 9). This suggests, strain 10ΔfolT is either cleared more efficiently from the blood, or is unable to survive in blood. White blood cell counts revealed that pigs infected with wild types strain 10 showed a stronger increase of WBCs for a longer period of time. All pigs infected with strain 10 displayed increased WBCs, whereas only one of the pigs infected with strain 10ΔfolT showed increased WBCs. The calculated WBC index differs significantly between the groups (Table 5). Survival rates between the two groups differed significantly: pigs infected with strain 10 had an average survival of 2,6 days post infection, whereas pigs infected with strain 10ΔfolT survived 6.2 days p.i. (FIG. 10). Although pigs were euthanized when predetermined humane end points were reached, survival reflects the severity of infection. As is shown in FIG. 10, the survival curves differ significantly between the groups. Gross pathology revealed that ⅘ pigs infected with strain 10 showed clinical signs specific for a S. suis infection like arthritis, pleuritis, pericarditis or peritonitis, whereas ⅗ pigs infected with strain 10ΔfolT showed specific clinical signs. Bacteriological examination of all infected organs revealed that more organs were colonized by higher bacterial loads for the wild type strain 10 compared to strain 10ΔfolT (FIG. 11).

(65) The second animal experiment (experiment 2) generally confirmed the data generated in experiment 1. As in the first experiment, the survival curves of wild type strain 10 and the strain 10ΔfolT isolate differed significantly. In experiment 2, all animals inoculated with strain 10ΔfolT survived until the end of the experiment, whereas 60% of the animals inoculated with strain 10 had to be euthanized in the course of the experiment (FIG. 12). Moreover, the frequency and severity of clinical signs (e.g. temperature, locomotion and consciousness; see FIGS. 13, 14, 15) differed considerably between animals inoculated with wild type strain 10 and strain 10ΔfolT. The frequency of gross pathological lesions in joints and peritoneum obtained at necropsy also differed considerably between the wild type and the 10ΔfolT mutant isolate.

(66) Based on the results of the infection experiments in piglets, it was concluded that the isogenic knock out mutant strain 10ΔfolT was strongly attenuated compared to the wild-type strain. This shows that the folate transporter is required for bacterial survival under in vivo conditions. Taking the result from both studies together, these experiments clearly show that the ΔfolT isolate produced almost no mortality, minimal clinical signs, and a reduced frequency of joint inflammation and peritonitis compared to the parent strain. It can therefore be concluded that a ΔfolT strain is highly attenuated and safe.

Summary Results. A Vaccine Comprising a Bacterium Provided with a Modification Such as a Mutation, Deletion or Insertion in the DNA Region Encoding for the Folate Substrate Binding Protein (a ΔfolT Isolate) of Said Bacterium) of a Bacterium Protects Hosts Against Challenge with a Virulent Isolate of Said Bacterium not having Said Modification

(67) The invention provides a method to produce a bacterium, preferably for use in a vaccine, preferably for use in a vaccine to generate protection against a bacterial infection, comprising selecting a parent bacterial strain generally capable of folate transport and folate synthesis and selecting a bacterium from that parent strain for having a modification such as a mutation, deletion or insertion in the DNA region encoding for the folate substrate binding protein (in Streptococcus suis known as the folT gene) of said bacterium and selecting said bacterium for the capacity to grow to similar rates as said parent strain in vitro but to grow to reduced rates compared with said parent strain in vivo. The invention also provides a method to produce a bacterium, preferably for use in a vaccine, preferably a vaccine for use to generate protection against a bacterial infection, comprising selecting a parent bacterial strain generally capable of folate transport and folate synthesis and transforming, preferably by recombinant means, a bacterium from that parent strain by providing it with a modification such as a mutation, deletion or insertion in the DNA region encoding for the folate substrate binding protein (in Streptococcus suis known as the folT gene) of said bacterium and selecting said bacterium for the capacity to grow to similar rates as said parent strain in vitro but to grow to reduced rates compared with said parent strain in vivo. Such a bacterium, as provided herein, still has the capacity to produce folate for its own use by applying its de novo folate synthesis pathways. Having these synthesis pathways intact leaves its capacity to in vitro growth (in culture) unaffected, surprisingly it was however shown herein that its growth and virulence in the host (in vivo) was strongly reduced.

(68) Such a bacterial strain that grows well in vitro but in vivo grows less than its parent strain and has associated strongly reduced virulence in vivo is very useful as a vaccine strain. Such a strain or mutant as provided by the invention is, on the one hand, essentially unaffected in folate synthesis and thus able to be grown to high titres and thereby relatively easy and inexpensive to produce, while on the other hand it is, due to its reduced growth and reduced virulence in its host as compared to its parent strain, relatively innocuous after in vivo application, making it extremely useful as a vaccine directed against a bacterial infection.

(69) In a first series of experiments herein, approximately three-week old piglets (Commercial Cross) that had not been vaccinated against S. suis and had never received medicated feed were used for the efficacy study. The animals were tonsil swab negative for S. suis serotype 2 by PCR upon enrolment and originated from a PRRSV negative herd. The two treatment groups were housed separately at the study site.

(70) Upon arrival at the study site, blood and tonsil swabs were collected from all animals. On study day 0, following an appropriate acclimation period, one group of the animals were vaccinated with strain 10ΔFolT. Another group of animals was left unvaccinated. The vaccinated animals were revaccinated on day 21 into the right side of the neck with same dose of the mutant isolate, respectively. After each vaccination, the animals were observed for local and systemic reactions. On study day 35, blood and tonsil swabs were collected from all animals before the animals in both groups were challenged. with the challenge strain ATCC700794. The animals were observed for signs of S. suis associated disease (e.g. increase in body temperature, lameness, abnormal behaviour, CNS signs) for 7 days following the challenge. Animals found dead or that had to be euthanized prior to off-test for animal welfare reasons were necropsied. During necropsy, the animals were assessed for macroscopic signs typically associated with S. suis disease (e.g. inflammation of CNS, joints, thoracic cavity). In addition, a CNS swab was collected for recovery of the challenge isolate. On day 42, all remaining animals were euthanized, necropsied and sampled as described above. Vaccinated animals showed considerably less signs of S. suis disease after challenge.

(71) A second series of experiments was conducted in commercial cross pigs; on the day of first vaccination, the pigs were 21±7 days of age. The animals had not been vaccinated against S. suis, were tonsil swab negative for S. suis type 2 PRRSV negative by serology and originated from sows that were tonsil swab negative for S. suis type 2. Upon arrival at the study site, blood and tonsil swabs were collected from all animals. On study day 0, following an appropriate acclimation period, one group of the animals were vaccinated into the left side of the neck with strain ΔFolT2. Another group of animals was left unvaccinated. The vaccinated animals were revaccinated on day 21 into the right side of the neck with the same dose the mutant isolate, respectively. After each vaccination, the animals were observed for local and systemic reactions. On day 34, blood and tonsil swabs were collected from all animals, and then the strict control animals were moved to a separate airspace while all other groups were commingled. On day 35, the animals were challenged intraperitoneally (ip) with approximately a virulent S. suis type 2 isolate.

(72) For seven days following challenge, the animals were observed for signs of disease associated with S. suis. Animals found dead or that had to be euthanized prior to off-test for animal welfare reasons were necropsied. During necropsy, the animals were assessed for macroscopic signs typically associated with S. suis disease and a CNS (i.e. brain) and joint swab were collected. At off-test, all remaining animals were euthanized, necropsied and samples collected. Vaccination with the ΔfolT2 mutant reduced the number of animals that died or had to be euthanized for animal welfare reasons during the post-challenge observation period. During necropsy, signs of inflammation in the brain, indicated by the presence of fibrin and/or fluid, were less frequently observed in ΔfolT2 vaccinated animals compared to the negative controls. The S. suis challenge isolate was less frequently recovered from the brain and the joint swabs collected at necropsy from animals vaccinated with the ΔfolT2 strain compared to the negative controls.

REFERENCES

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TABLES

(74) TABLE-US-00001 TABLE 1 Primer sequences. Primer name Sequence 5′-3′ Target comE1 cgagctcggaagaa orf2[10]-forward-SacI ttggttattgcgcg tg comE2 cgggatcccggggg orf2[10]-reverse-BamHI atgacctgttgctt g comE3 tcccccgggggagt P-orf2-folC[10]- cgtgtgtattcgac reverse-SmaI agcgg comE4 tcccccgggggaca folC[10]-forward-SmaI agcaacaggtcatc ccc comE6 cgggatcccggttg folC[10]-reverse-BamHI aatgcccggcaagc c Orf2-fw ctacggctggttct S. suis orf2 tctatcgaa Orf2-rev gcaatcggtgtcat S. suis orf2 gataaagg folC-fw gtttgtccgtccat S. suis cggttt polyfolylpolyglutamate synthase Folc-rev ctggtcggtcgcat S. suis agatga polyfolylpolyglutamate synthase RecA-fw ggtttgcaggctcg S. suis recombinase A tatgatg RecA-rev accaaacatgacac S. suis recombinase A cgacttttt t488a gaaaggtatagttt Promoter orf2 ttagcaagtggaca aaatatatagtgtg tgatacaat t488a_ attgtatcacacac Promoter orf2 antisense tatatattttgtcc acttgctaaaaact atacctttc V735-fw tatgcgcaatgacg pKUN-V[10]*-Spec.sup.R tagtagaagg M13-rev aacagctatgacca pKUN-V[10]*-Spec.sup.R tg

(75) TABLE-US-00002 TABLE 2 Virulence of complemented S. suis strains in germfree piglets; all strains contained a plasmid (pCOM1) with or without insert. V[10]/V[S735]: original 3 kb fragment from strain 10 or strain S735 that was selected from library; orf2[10]: orf2 from V[10]; folC[10]: orf3 from V[10]encoding dihydrofolate synthase. Mean Clinical index No. of pigs in no. of of the group which S. suis No. days Non- was isolated of Dose Mortality.sup.a till Morbidity.sup.b Specific.sup.c specific.sup.d Fever from Strain pigs (CFU) (%) death (%) symptoms symptoms index.sup.e CNS Serosae.sup.g Joints S735-pCOM1- 4 10.sup.6 100 1 100 100**  100**  38* 4 4 4 V[10] S735-pCOM1- 4 10.sup.6 100 1 100 100**   66** 29 4 4 4 orf2[10] S735-pCOM1- 4 10.sup.6 0 11 0 4 21  1 0 0 0 folC[10] S735-pCOM1 4 10.sup.6 0 11 0 0 21  5 0 0 0 S735-pCOM1- 5 10.sup.6 100 1 100 100**  100**  60* 5 5 5 V[10].sup.f S735-pCOM1- 5 10.sup.6 20 15 100  43** 38 25 1 1 1 V[S735].sup.f S735-pCOM1.sup.f 5 10.sup.6 20 16 60 14  11 12 1 0 0 T15-pCOM1- 5 10.sup.6 0 14 16 4 16 13 1 1 1 V[10] .sup.aPercentage of pigs that died due to infection or had to be killed for animal welfare reasons .sup.bPercentage of pigs with specific symptoms .sup.cPercentage of observations for the experimental group in which specific symptoms (ataxia, lameness of a least one joint and/or stillness) were observed .sup.dPercentage of observations for the experimental group in which non-specific symptoms (inappetite and/or depression) were observed .sup.ePercentage of observations for the experimental group of a body temperature of >40° C. .sup.fPrevious experiments (Smith et al., 2001) were re-analyzed to allow for statistical comparison between experiments, this re-analysis required new stringent definitions of specific and aspecific symptoms as indicated in materials and methods. *p ≤ 0.05 compared to S735-pCOM1 **p ≤ 0.01 compared to S735-pCOM1 .sup.gSerosae are defined as peritoneum, pericardium or pleura

(76) TABLE-US-00003 TABLE 3 Sequence analysis of the −35 region of the orf2/folC promoter among various S. suis isolates and serotypes.sup.1 Pheno- CGH Sero- type clus- Clonal −35 promoter sequence (5′-3′) type MRP.sup.2 EF.sup.3 ter.sup.4 complex TGGACA TGGTCA TTGTCA 1 − − B 13 1/1 1 S + A 1 4/4 2 − − B 16/29/147 6/6 2 + − B 28 1/1 2 + * A 1 7/7 2 − * A 1 1/1 2 + + A 1 9/9 7 − − B 29/1 .sup. 1/8.sup.5 6/8 1/8 9 − − B 16 2/2 9 * − B 16 6/6 9 + − B 16 1/1 .sup.1S. suis isolates were described in de Greeff et al.[23] .sup.2* indicates an higher molecular weight form of MRP; s indicates a lower molecular weight form of MRP .sup.3* indicates an higher molecular weight form of EF .sup.4All isolates were genotyped using Comparative Genome Hybridization (CGH) [23] .sup.5This isolate belongs to clonal complex 1 .sup.6Number of isolates analysed/number of isolates with the respective −35 promoter sequence

(77) TABLE-US-00004 TABLE 4 Clinical parameters of pigs infected with S. suis., experiment 1 Mean no. of No. days of Mortality until Fever WBC Gross Pathology Strain pigs Dose (%) death Index Index Arthritis Pleuritis Pericarditis Peritonitis 10 5 1.1 × 100 2.6   47.sup.  50  11/20 2/5 2/5 1/5 10.sup.6 10ΔfolT 5 9.6 × 20 6.2** 23.sup.# 19*  2/20 1/5 1/5 0/5 10.sup.5 *p ≤ 0.05 compared to 10 **p ≤ 0.01 compared to 10 .sup.#p ≤ 0.1 compared to 10

(78) TABLE-US-00005 TABLE 5 Gross-lesions indicating arthritis and peritonitis: % of positive observations in wild type strain 10 and in 10ΔFolT mutant isolate challenged animals; experiment 2 10 10ΔFolT Joints 100 20 Peritoneum 80 20

(79) TABLE-US-00006 TABLE 6 Study design (for study using CBS 143192) ΔfolT2 CFU Vaccination Challenge Group Treatment per dose (D0, D21) (D35) Off-test 1 Strain 10ΔfolT2 5.5 × 10.sup.7 CFU 0.2 mL id 2 mL ip D42 grown in APS media 2 Strain 10ΔfolT2 1.4 × 10.sup.8 CFU 0.2 mL id grown in THB media 3 Strain 10ΔfolT2 1.4 × 10.sup.8 CFU 2.0 mL im grown in THB media 4 Placebo vaccine N/A 2.0 mL im [Negative Control] 5 No treatment N/A N/A N/A [Strict Control]

(80) TABLE-US-00007 TABLE 7 Vaccine and placebo preparation (for study using CBS 143192) Group Treatment Description 1 Strain On the vaccination day, ACES-buffered Becton Dickinson APS- 10ΔfolT2 TSB media (APS; w/o serum) was inoculated with ΔfolT2 glycerol grown in APS stock and grown with agitation until 0.6 ± 0.1 OD A600 nm. The media culture was centrifuged at 9,000 × g for 5 minutes at 4° C. The supernatant was decanted and then the cells were washed twice in an equal volume of sterile 1X PBS, pH 7.2. The washed cells were suspended in PBS to an OD A600 nm equal to approximately 9 log per mL. Approximately 10 mL of the 9 log washed culture was bottled in sterile vials. Aliquots of the treatment were tested for CFU count prior to vaccination and immediately following vaccination. The vaccine preparations were held on wet ice until administration, no longer than 60 minutes. 2, 3 Strain On the vaccination day, Todd Hewitt broth (THB; w/o serum) 10ΔfolT7 was inoculated with ΔFolT2 glycerol stock and grown with grown in THB agitation until 0.6 ± 0.1 OD A600 nm. The culture was centrifuged media at 9,000 × g for 5 minutes at 4° C. The supernatant was decanted and then the cells were washed twice in an equal volume of sterile 1X PBS, pH 7.2. The washed cells were suspended in PBS to an OD A600 nm equal to approximately 9 log per mL. Approximately 10 mL of the 9 log washed culture was bottled in a sterile vial for the group 2 treatment. An aliquot of the 9 log washed culture was further diluted to the target cell concentration in PBS and bottled in a sterile vial for the group 3 treatment. Aliquots of each treatment were tested for CFU count prior to vaccination and immediately following vaccination. The vaccine preparations were held on wet ice until administration, no longer than 60 minutes. 4 Placebo Approximately 40 mL of sterile phosphate buffered saline (PBS), vaccine pH 7.2 was bottled in a sterile vial and stored at 4° C. until use.

(81) TABLE-US-00008 TABLE 8 Challenge preparation (for study using CBS 143192) Strain S. suis type 2 BIAH #08-06 (ATCC 700794 derivative) Preparation A single colony was inoculated into 10 mL pre-warmed THB + 5% FBS and grown statically to 0.5 ± 0.1 OD A600 nm. The culture was scaled up to 900 mL in THB + 5% FBS, and grown with agitation to 0.7 ± 0.1 OD A600 nm. Sterile glycerol was added to the culture (10% v/v). Aliquots were retained for pre-freeze and post-thaw CFU and for purity. The challenge was dispensed into vaccine bottles and stored at −70° C. until use. Prior to use, the culture was thawed in a 37° C. waterbath, then diluted with sterile THB + 5% FBS to meet the target concentration of 1 × 10.sup.9 cfu/mL. Aliquots of the treatment were tested for CFU count prior to challenge and immediately following challenge.

(82) TABLE-US-00009 TABLE 9 Percentage of animals that died or were euthanized following challenge (mortality) (for study using CBS 143192) # Pigs Group challenged Vaccine Mortality 1 14 Strain 10ΔfolT2 - 35.7% 7 log - APS - id 2 11 Strain 10ΔfolT2 - 45.5% 8 log - THB - id 3 11 Strain 10ΔfolT2 - 27.3% 8 log - THB - im 4 15 Placebo vaccine - 93.3% Negative Control

(83) TABLE-US-00010 TABLE 10 Percentage of animals showing severe lameness following challenge (for study using CBS 143192) Percentage of pigs showing # Pigs severe lameness during Group challenged Vaccine observation period 1 14 Strain 10ΔfolT2 - 4.2% 7 log - APS - id 2 11 Strain 10ΔfolT2 - .sup. 0% 8 log - THB - id 3 11 Strain 10ΔfolT2 - 3.3% 8 log - THB - im 4 15 Placebo vaccine - 41.7%  Negative Control

(84) TABLE-US-00011 TABLE 11 Percentage of animals showing apathy following challenge (for study using CBS 143192) Percentage of pigs showing # Pigs apathy during Group challenged Vaccine observation period 1 14 Strain 10ΔfolT2 - 21.1% 7 log - APS - id 2 11 Strain 10ΔfolT2 - 4.3% 8 log - THB - id 3 11 Strain 10ΔfolT2 - 11.7% 8 log - THB - im 4 15 Placebo vaccine - 50.0% Negative Control

(85) TABLE-US-00012 TABLE 12 Percentage of animals showing signs of inflammation in brain during necropsy (for study using CBS 143192) # Pigs Percentage of pigs showing Group challenged Vaccine inflammation in brain 1 14 Strain 10ΔfolT2 - 21% 7 log - APS - id 2 11 Strain 10ΔfolT2 - 45% 8 log - THB - id 3 11 Strain 10ΔfolT2 - 27% 8 log - THB - im 4 15 Placebo vaccine - 87% Negative Control

(86) TABLE-US-00013 TABLE 13 Percentage of animals from which S. suis was recovered from brain swabs collected at necropsy (for study using CBS 143192) Percentage of pigs from # Pigs which S. suis was Group challenged Vaccine recovered from brain 1 14 Strain 10ΔfolT2 - 35.7% 7 log - APS - id 2 11 Strain 10ΔfolT2 - 27.3% 8 log - THB - id 3 11 Strain 10ΔfolT2 - 27.3% 8 log - THB - im 4 15 Placebo vaccine - 93.3% Negative Control

(87) TABLE-US-00014 TABLE 14 Percentage of animals from which S. suis was recovered from the joints swabs collected at necropsy (for study using CBS 143192) Percentage of pigs from # Pigs which S. suis was Group challenged Vaccine recovered from the joints 1 14 Strain 10ΔfolT2 - 35.7% 7 log - APS - id 2 11 Strain 10ΔfolT2 - 36.4% 8 log - THB - id 3 11 Strain 10ΔfolT2 - 18.2% 8 log - THB - im 4 15 Placebo vaccine - 73.3% Negative Control

(88) TABLE-US-00015 TABLE 15 Vaccination challenge study outline (for study using CBS 140425) Inclusion # Level/2 ml Days of Vacc. Challenge Group Pigs Treatment dose Treatment Route Day Chall. Route 1 15 Strain 10ΔfolT 1.0 × 10.sup.10 0, 21 i.m. 36 i.p. CFU (first vac) 9.8 × 10.sup.9 CFU (second vac) 2 15 Strain 10ΔfolT 9.5 × 10.sup.9 0 i.m. 36 i.p. CFU 3 15 Placebo N/A 0, 21 i.m. 36 i.p. [Negative Control] 4 5 Strict control N/A N/A N/A N/A N/A

(89) TABLE-US-00016 TABLE 16 Vaccine preparation (for study using CBS 140425) Group Treatment Description 1-2 Strain 10ΔfolT A strain 10ΔfolT glycerol stock was transferred into Todd- Hewitt Broth (THB) + 5% Fetal Bovine Serum (FBS) and grown statically to 0.5 ± 0.1 OD A600 nm. The culture was scaled up to 1800 mL in THB + 5% FBS, and grown with agitation to 0.7 ± 0.1 OD A600 nm. The culture was concentrated 6X by centrifugation and removal of supernatant to achieve a 10-log dose. Sterile glycerol was added to the concentrated culture (10% v/v). Aliquots were retained for pre-freeze and post- thaw CFU, identity, and purity. The vaccine was dispensed into vaccine bottles and stored at −70° C. until use. The vaccine was thawed in a 37° C. water bath and diluted to the intended target concentration using storage media, then held on wet ice until administration. 6 Placebo Sterile THB + 5% FBS media, stored at 4° C. until use.

(90) TABLE-US-00017 TABLE 17 Challenge preparation (for study using CBS 140425) Challenge Strain S. suis type 2 BIAH #08-06 (ATCC 700794 derivative) Challenge Preparation A single colony was inoculated into 20 mL pre-warmed THB + 5% FBS and grown statically to 0.5 ± 0.1 OD A600 nm. The culture was scaled up to 900 mL in THB + 5% FBS, and grown with agitation to 0.7 ± 0.1 OD A600 nm. Sterile glycerol was added to the culture (10% v/v). Aliquots were retained for pre-freeze and post-thaw CFU and for purity. The challenge was dispensed into vaccine bottles and stored at −70° C. until use.

(91) TABLE-US-00018 TABLE 18 Percentage of animals showing lameness following challenge (CBS 140425) Percentage of pigs Group # Pigs Vaccine showing lameness 1 13 Strain 10ΔfolT - 7.7% 10 logs - 2 dose 2 15 Strain 10ΔfolT - 40.0% 10 logs - 1 dose 3 15 Placebo 93.3% Negative Control

(92) TABLE-US-00019 TABLE 19 Percentage of animals showing abnormal behavior following challenge (CBS 140425) Percentage of pigs Group # Pigs Vaccine showing abnormal behavior 1 13 Strain 10ΔfolT -  0% 10 logs - 2 dose 2 15 Strain 10ΔfolT - 46.7%  10 logs - 1 dose 3 15 Placebo 100% Negative Control

(93) TABLE-US-00020 TABLE 20 Percentage of animals expired or euthanized following challenge (mortality) (CBS 140425) Group # Pigs Vaccine Mortality (%) 1 13 Strain 10ΔfolT -  0% 10 logs - 2 dose 2 15 Strain 10ΔfolT - 26.7%  10 logs - 1 dose 3 15 Placebo - 100% Negative Control

(94) TABLE-US-00021 TABLE 21 Percentage of animals with abnormal findings in brain upon necropsy (CBS 140425) Percentage of pigs with Group # Pigs Vaccine abnormal findings in CNS (%) 1 13 Strain 10ΔfolT -   0% 10 logs - 2 dose 2 15 Strain 10ΔfolT - 26.7% 10 logs - 1 dose 3 15 Placebo - 93.3% Negative Control

(95) TABLE-US-00022 TABLE 22 Percentage of animals with abnormal findings in thoracic cavity upon necropsy (CBS 140425) Percentage of pig with lesions Group # Pigs Vaccine in thoracic cavity (%) 1 13 Strain 10ΔfolT - 23.1% 10 logs - 2 dose 2 15 Strain 10ΔfolT - 33.3% 10 logs - 1 dose 3 15 Placebo - 93.3% Negative Control

(96) TABLE-US-00023 TABLE 23 Percentage of animals from which S. suis was recovered from brain swab (CBS 140425) S. suis recovered Group # Pigs Vaccine from CNS swab (%) 1 13 Strain 10ΔfolT -   0% 10 logs - 2 dose 2 15 Strain 10ΔfolT -  6.7% 10 logs - 1 dose 3 15 Placebo - 73.3% Negative Control