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Safety and immunogenicity of the live oral auxotrophic Shigella flexneri SFL124 in volunteers
Safety and immunogenicity of the live oral auxotrophic Shigella flexneri SFL124 in volunteers A n L i * , T i b o r Pill**, U r b a n
Forsum*
a n d A l f A. L i n d b e r g *$
The live, aromatic dependent Shigella flexr[eri Y vaccine strain SFL124, with a deleted a r o D gene, was tested for safety and immunogenicity in 21 healthy adult volunteers. A single dose o f 2 × 109 live bacteria was given orally to ten volunteers, whereas 11 received three doses every other day. The vaccine was excreted for 4.2 days and was well tolerated by 90.5% o f the vaccinees. Only 2 of 21 (9.5%) after the first dose had a self-limiting diarrhoea lasting 1 day; o f volunteers given one dose only 3 o f 10 showed anti-lipopolysaccharide ( L P S ) and anti-invasion plasmid coded antigen ( Ipa) responses in serum. A faecal antibody response to L P S and Ipa was seen in six and three persons, respectively. Volunteers given three doses reacted with serum anti-LPS (9/11 ) and anti-Ipa (5/11) antibody responses. In stool, anti-LPS and anti-lpa responses were detected in nine and eight volunteers, respectively. A booster dose of 2 × 1 0 9 bacteria given to six volunteers in the three-dose group 9-10 months later elicited high stool slgA responses, indicating a strong mucosal memory, and was accompanied by a short excretion period o f SFL124 (1.8 versus 4.2 days, p < 0.05). The vaccination also elicited antibody-secreting cell ( A S C ) responses against L P S in peripheral blood." the three doses of the vaccine resulted in a stronger response than did the single dose, while the booster dose elicited only a limited A S C response. Volunteers previously exposed to shigellae exhibited stronger anti-Ipa responses in serum and stool suggestive o f an immunological memory to the Ipa. The results indicate that SFL124 is a safe live vaccine strain inducing specific immune responses against L P S and Ipa with a mucosal immune memory lasting for at least 9 months. Keywords:Shigella.flexneri; oral vaccination; humans; safety; immunogenicity
INTRODUCTION Recently, we developed a Shigella flexneri serotype Y vaccine strain (SFL114), w h i c h - - d u e to an aroD :: T n l 0 m u t a t i o n - - w a s auxotrophic for p-aminobenzoic acid 1. Since this compound is not available in the cytosol, although the strain was able to invade epithelial cells at the wild type level, its ability to grow intracellularly was greatly reduced, making it avirulent 2. This strain induced significant protection in monkeys against homologous challenge 1. Although reversion back to virulence has never been observed with SFL114, for safety reasons a deletion mutant (SFL124) was selected from S F L I I 4 devoid of the T n l 0 transposon and a 1400 base pair (bp) long stretch of the aroD gene making it unable to revert 3. The strain was safe in in vitro and in vivo assays and elicited protective immunity in Macaca fascicularis *Karolinska Institute, Department of Clinical Bacteriology, Huddinge Hospital, S-141 86 Huddinge, Sweden. 1Institute of Microbiology, University Medical School, H-7643 P~cs, Hungary. ~To whom correspondence should be addressed. (Received 22 July 1991 ; revised 20 November 1991 ; accepted 28 November 1991) 0264410X/92/060395-10 © 1992Butterworth-HeinemannLtd
monkeys. Here we report the results of a safety/immunogenicity trial of S. flexneri SFL124 given as an oral live vaccine to 21 adult human volunteers. MATERIALS
AND
METHODS
Volunteers Twenty-one healthy persons, six men and 15 women,
25-55 years of age (mean 39.3), at present residing in Sweden, participated in the study. While none of them had had any episodes of enteric infections in the past 2 years, based on their earlier anamnestic history they formed two groups: 16 of them were not aware of any previous dysentery-like infection when the description of the symptoms was provided by the interviewer, who was medically qualified. This group was tentatively considered to be the previously non-exposed group. Members of the second group (five persons) reported either proven infections (one known laboratory accidental infection with enteroinvasive Escherichia coli 6 years previously), or four of them recalled enteric infections 5-13 years previously which, at the level of symptoms, would qualify as dysentery. It should be noted that these last four
Vaccine, Vol. 10, Issue 6, 1992 395
Live oral auxotrophic Shigella flexneri vaccine in volunteers: A. Li et al.
volunteers were originally from countries where shigellosis is endemic. The group was considered to be previously primed. The recruiting procedure and the trial were approved by the Karolinska Institute Ethics Committee.
Vaccine strain The construction and characteristics of S. flexneri Y AaroD strain SFL124 are described in an accompanying paper 3.
The periplasmic content of shigellae known to be rich in invasion plasmid-coded antigens (lpa) B and C 10 was extracted by distilled water according to Oaks et al. ~1. For this purpose E. coli K12 strains SP10 and J53 were used. SP10 is a derivative of E. coli K12 J53, into which the invasion plasmid (pWR 110)~ 2 of a virulent S. flexneri 5 strain (M90T) (kindly provided by Dr S.B. Formal, Walter Reed Army Institute of Research, Washington, D C ) had been mobilized as described 11.
Enzyme immunoassays (EIA) Vaccination schedule and protocol The volunteers were divided into three groups. G r o u p 1 consisted of ten people receiving a single vaccine dose. G r o u p 2a (11 volunteers) received three doses of the vaccine on days 0, 2 and 4. G r o u p 2b comprised six members of group 2a who were boosted with a single dose 9 - 1 0 months later. On the morning of vaccination the volunteers ingested 2 g of N a H C O 3 tablets. Ten minutes after they drank 2 × 109 cells of the vaccine strain suspended in PBS (pH 7.2). They were not requested to fast before vaccination but were asked to avoid any food and water intake for 2 h after the vaccination. For each vaccination event a new lyophil amouple from the same batch of the vaccine strain was opened, reconstituted with distilled water and diluted in PBS to provide the requested concentration. The ratio of invasion plasmid harbouring clones in the vaccine was above 95%, as judged by the number of Congo red positive colonies 4.
Vaccine excretion Vaccine excretion was monitored daily starting on the first day after the last dose of vaccine and continuing until three consecutive negative results were obtained. The number of colony forming units/gram faeces was determined on DC agar plates (Oxoid Ltd, Basingstoke, Hants, U K ) . The vaccine strain recognized by its morphology was identified by routine biochemical tests ~ and by agglutination in mAb Y-5 monoclonal antibody recognizing the S. flexneri Y O-specific side chain 6. Conversion to the wild ( p r o t o t r o p h ) phenotype was checked on minimal agar plates. Simultaneously, the excretion of the normal E. coil flora was also quantitatively monitored. If faecal samples were not available, rectal swab samples were taken and were processed for qualitative evaluation.
Extraction of faecal samples for slgA-EIA Faecal samples kept at 4°C were always processed on the same day they were delivered. After a 1 : 5 dilution with trypsin inhibition buffer 7, samples were homogenized and centrifuged at 20 0009 for 30 min. The supernatants were filtered and stored at - 7 0 ° C until use.
Antigen preparations Lipopolysaccharide (LPS) of S. flexneri Y, Salmonella typhimurium and E. coli CVD-172 were prepared by hot p h e n o l - w a t e r extraction 8. Polysaccharide (PS) from S. flexneri Y and E. coli CVD-172 was extracted by weak acid hydrolysis 9. No trace of lipid A was detected in the extracts by gas liquid chromatography.
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Antibody titres in serum and faecal extracts against S. flexneri Y LPS and Ipa were examined by EIA. The relative antibody titres, defined as the absorbance (A) values at a given sample dilution were determined as described ~3. To measure the serum antibody responses to S. flexneri Y LPS and sIgA in faecal extracts specific to S..flexneri Y and S. typhimurium LPS, the above LPS antigens were used to coat EIA plates at a concentration of 5 #g m l - 1. To determine the immune response to Ipa, water extracts of SP10 and E. coil K12 J53 were used to coat the plates at a protein concentration of 20 #g m l - 1 To increase the specificity of the assay to Ipa, every sample was tested against extracts made from SP10 as well as from its plasmidless counterpart J53. Then corrected A values obtained by deducting A j53 from AsP~o were used to express relative titres. To measure anti-LPS responses, sera were diluted 1:1000 and for anti-lpa responses, the dilution was 1 : 500. Faecal extracts were diluted 1 : 5 for both antigens. For serum samples rabbit anti-human-IgG (1:4000), -IgA (1 : 1000)and -IgM (1:800)conjugated to alkaline phosphatase were used as secondary antibodies while for sIgA determinations rabbit anti-human secretory component conjugated to horseradish peroxidase (1 : 1000) (all from Dakopatts, Copenhagen, Denmark ) were used. In groups 1 and 2a, 'immune response' was defined as a minimum twofold increase in titres as compared to the individuals' prevaccine titres. Since group 2b was formed by the boostered members of group 2a, in their cases prebooster, instead of prevaccine, values were used as reference. Serum ' n o r m a l ' values were defined as the mean + 2 s.d. of titres against the corresponding antigens of 30 healthy Swedish blood donors.
Western blot of serum samples Some serum samples were analysed by Western blot using whole cell extracts of a virulent S. flexneri 5 strain, M90T and its plasmidless derivative M90T5514. The extraction, polyacrylamide gel electrophoresis and immunoblotting procedures were carried out as described14. Serum samples were diluted 1:50. Identification of plasmid-coded antigen bands on nitrocellulose sheets containing extract of M90T were done on the basis of their reported molecular mass 1~ ( 1 2 0 k D a ; IpaA = 78 k D a ; IpaB = 62 k D a ; IpaC = 43 k D a ; I p a D = 38 k D a ) and by their absence from the extracts of M90T55 run simultaneously. Serum samples of a Vietnamese individual containing antibodies against the above proteins served as positive control.
Preparation of peripheral mononuclear cells Blood was taken into heparinized tubes and diluted 1:1 with Hanks Balanced Salt Solution (HBSS, Flow
Live oral auxotrophic Shigella flexneri vaccine in volunteers: A. Li et al.
Laboratories, Irvine, UK). Mononuclear cells were separated on Ficoll Paque (Pharmacia, Uppsala, Sweden). After washing three times in HBSS, the cell number was set to 10 6 m1-1 in RPMI 1640 (Gibco Limited, Paisley, UK) supplemented with 10 mM HEPES (flow), 2 mM L-glutamine, 1 m~ Na-pyruvate, 100 U ml-x penicillin and 100 #g ml-~ streptomycin. For the assay of antibody-secreting cells, 10% of heat-inactivated fetal calf serum (Gibco) was used; for the cell proliferation assay, 10% of blood type AB human serum was added. Cell viability was always higher than 95% as checked by trypan blue exclusion assay.
Assays for immunoglobulin and for specific antibody-secreting cells The enzyme-linked immunospot assay (ELISPOT) was carried out as described~6'~7 with some modifications. Briefly, to enumerate the immunoglobulin secreting cells (ISC), nitrocellulose-bottomed microtitre plates (Millipore Corporation, Bedford, MA, USA ) were coated with 100/~l/well of goat anti-human IgG, IgA and IgM (Sigma Chemical, St Louis, USA) diluted 1 : 500. To detect the specific antibody-secreting cells (ASC), wells were coated with 100 #1 of S. flexneri Y or E. coli CVD 172 LPS (20 #g ml- 1), or 2 #g ml- 1 teichoic acid from Staphylococcus aureus (National Bacteriology Laboratory, Stockholm, Sweden). After overnight incubation at room temperature wells were washed three times with PBS. Cell suspension ( 100/A) was added to the wells and plates were incubated at 37°C in 7% CO2 in a humidified atmosphere for 16 h. After washing, 100/~1 biotinylated goat anti-human IgA, IgG or IgM were added (Sigma, dilution 1 : 500) and incubation was continued at room temperature for an additional 3 h. Spots representing antibody-secreting cells were visualized by adding preformed avidin-biotinylated horseradish peroxidase complex (Vector Laboratories, Burlingame, CA, USA), then carbazol-dimethylformamide substrate. Spots were counted under a dissection microscope.
Peripheral mononuclear cell proliferation assay Peripheral mononuclear cell suspension was set to a concentration of 5 × 105 m1-1. Suspension (200/d) was added to round-bottomed microtitre plates. The following antigens or mitogens were then added : S. flexneri Y PS (50/2g/well) ; E. coli PS (30 #g/well); tuberculin-purified protein derivative (2~tg/well, Statens Seruminstitute, Copenhagen) and phytohaemagglutinin (10/~l/well of a 1 : 1000 diluted stock, Wellcome Diagnostics, Dartford, UK). Tests were carried out in triplicate. To the control wells no antigen or mitogen was added. After 5 days at Table 1
37°C, 7% CO2, cells were pulsed with methyl-3H thymidine (1/~Ci/well; Amersham International, Buckinghamshire, UK) and harvested 16 h later. Results were counted in a fl-counter. Stimulation indexes (SI) were calculated as counts min-1 in stimulated wells/counts min-1 in unstimulated wells. RESULTS
Clinical symptoms The clinical symptoms following vaccination are summarized in Table 1. Of the 21 volunteers, 13 (61.9%) tolerated the first (in the case of members of group 1, the only) dose of the vaccine without any symptoms. Six volunteers (28.6%) reported one or two of the mild symptoms shown in Table I. Two volunteers (9.5%) developed diarrhoea concomitant with a febrile response, 38.2°C and 39°C, respectively, 14-20 h after vaccination. The diarrhoea was accompanied by fatigue and a slight stomach discomfort. During the late night and early morning, they had four and five episodes of loose to watery stools, respectively. Blood or mucus was not detected in their faeces. On the afternoon of the following day (day 1) their temperatures had dropped to normal (< 37°C). Though they still felt some fatigue during the day, no more diarrhoeal episodes were recorded. On day 2, both of them had recovered spontaneously. After the second vaccine dose (group 2a) one out of 11 volunteers had some fatigue, while the third dose ( 11 volunteers in group 2a) was tolerated without any symptoms. The booster dose given 9-10 months after the primary vaccination to six of the 11 volunteers in group 2a (group 2b) induced two single episodes of loose stool and transient, slight headache in two volunteers.
Excretion of the vaccine strain The excretion of the vaccine strain following the last vaccine dose in each group is shown in Figure 1. The average excretion time in groups 1 and 2a was practically the same (mean + s.d. = 4.4. _+ 1.2 and 4.0 + 1.9 days, respectively), while in group 2b it was significantly shorter (1.8 + 1.8 days, p < 0.05 when compared with either group 1 or group 2a). No significant variation in the normal E. coli flora was observed. No reversion of the vaccine strain to the virulent phenotype was observed.
Immunoglobulin (ISC) and specific antibody-secreting cell (ASC) response in peripheral blood Following vaccination, the total number of IgA, IgG and IgM ISCs slightly increased in all three vaccine
Clinical symptoms in volunteers after oral vaccination with Shigella flexneri SFL124
Number of volunteers No symptoms ( % ) Mild symptoms a ( % ) Severe symptoms b ( % ) Mucus a n d / o r blood in stool
lstdose (group 1, 2a and 2b)
2nd dose (group 2a and 2b)
3rd dose (group 2a and 2b)
4th dose (group 2b)
21 13 (61.9) 6 (28.6) 2 (9.5) 0
11 10 (90.9) 1 (9.1) 0 0
11 11 (100) 0 0 0
6 4 (66.7) 2 (33.3) 0 0
aThe volunteers had one or two of the following symptoms after vaccination: slight headache, muscle pain, fatigue, mild stomach-ache, stomach discomfort or dizziness. bThe volunteers had fever of 38.2°C or 39°C and diarrhoea of more than three episodes for 1 day after the vaccination
Vaccine, Vol. 10, Issue 6, 1992 397
Live oral auxotrophic Shigella flexneri vaccine in volunteers: A. Li et al. ~o~r~
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groups with peaks around days 4-7, but the increases were not significant (p > 0.05) in any case (data not shown). S. flexneri Y LPS-specific ASCs could be found in only a few individuals in low numbers before vaccination. After vaccination, in groups 1 and 2a ASCs started to rise in all three Ig classes on day 4 reaching a peak on day 7 (Figure 2). The strongest responses were observed for IgA. The response appeared to be slightly more prolonged in group 2a than in either of groups 1 and 2b. There were no measurable ASC responses in groups 1 and 2b against heterologous antigens like teichoic acid or E. coli LPS. Due to a possible polyclonal stimulation, a few IgA-secreting clones recognizing these antigens appeared in the blood of some members of group 2a (data not shown), but the increases were not significant (p > 0.05).
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Serum antibody responses The serum IgA and IgG antibody responses against S. flexneri Y LPS are summarized in Figure 3. Responses in the lgM class were low, and generally similar in pattern to those seen in the IgG class (data not shown). Prevaccination IgA titres did not exceed the corresponding 'normal' limits defined as the mean titre ___2s.d. of 30 healthy Swedish blood donors. For IgG, one and for IgM, three out of the 21 volunteers had slightly higher titres than the normal values. After a single dose (group 1 ) anti-LPS titres slightly rose in all three Ig classes reaching a peak around day 14. In the three-dose group (group 2a) the increase in titre was more prominent with a maximum level on day 7. No significant decreases in these titres were observed during the follow-up period (up to day 60). In these two groups there were no significant differences between responses of previously exposed and unexposed volunteers (p > 0.05 for exposed versus unexposed volunteers for group 1 and 2a for samples collected on the same day). Following the booster dose in group 2b, the two volunteers in this group with an anamnestic history of shigellosis responded with a rise of specific lgA titres by day 7, while those without known previous exposure did not exhibit any titre increase. Comparing day 0 with day 14 titres in the groups without dividing them into
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than their prevaccination values and the booster dose caused only a slight further increase. However, by the time of the administration of the booster dose, IgA titres had already fallen back close to prevaccination values, and the booster induced a sharp increase in lpa-specific serum IgA which returned to the prevaccination value in 4 weeks. Data concerning the number of responders are summarized in Table 2. In general, the three vaccine-dose
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that, unlike the LPS responses, after one or three doses of vaccination Ipa responses were considerably higher in the volunteers previously infected with shigellae (Figure 4 ). In their cases, titres rose sharply, reaching a maximum around day 14 and, especially for IgG, remaining high during the 60-day study period. Previously non-exposed members of group 2b still did not respond to Ipa after the booster dose. Owing to the previous three vaccine doses, the prebooster IgG titres of the two previously exposed members of this group were still much higher
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Time (days) Figure 4 Serum IgA and IgG relative EIA titres against Shigella invasion plasmid coded antigens (Ipa) in vaccine groups l(a), 2a(b) and 2b(c) (mean -I- s . d . ) . . , IgA and ,&, IgG titres from volunteers without history of previous exposure to shigellae or enteroinvasive E. coil infections; E3, IgA and /k, IgG titres from volunteers with positive exposure anamnestic history of shigellae or enteroinvasive E. coo infections
Vaccine, Vol. 10, I s s u e 6, 1992
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Live oral auxotrophic S h i g e l l a f l e x n e r i vaccine in volunteers: A. Li et al. Table 2
Antibody responses of volunteers to Shigella flexneri Y LPS and Ipa" Serum antibody to S. flexneri Y LPS
slgA to S. flexneri
Ipa
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2/2 0/8 2/10
1/2 0/8 1/10
0/2 0/8 0/10
2/2 0/8 2/10
1/2 5/8 6/10
1/2 2/8 3/10
1/2 5/8 6/10
Group 2a
Exposed Unexposed Total
2/3 6/8 8/11
2/3 6/8 8/11
1/3 2/8 3/11
2/3 7/8 9/11
2/3 2/8 4/11
3/3 2/8 5/11
1/3 0/8 1/11
3/3 2/8 5/11
3/3 6/8 9/11
3/3 5/8 8/11
3/3 6/8 9/11
Group 2b
Exposed Unexposed Total
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"lpa: invasion plasmid-coded antigens. bExposed: the volunteers had previous exposure history to shigellae or enteroinvasive E. coli infections 5 13 years ago. CUnexposed: the volunteers did not have previous exposure history to shigellae infections. aNumber of responders versus number of volunteers for each immunoglobulin class, or responding in at least one of the classes
schedule was more effective than a single dose in stimulating an immune response against the LPS antigen. In the three-dose group most of the volunteers (8/11) responded to the homologous LPS antigen. For lpa, even in the three-dose group (2a) the response among non-pre-exposed individuals was low (2/8 persons). In group 2b the serum responses against both antigens were low. The booster dose caused further increases in titre in a few volunteers only, mainly in the two pre-exposed persons in this group.
Western blot of serum samples
Figures 5A and B show the Western blot of serum samples in groups 1 and 2a, respectively. With the exception of volunteer no.. 7, prevaccination sera of four out of five volunteers considered to be previously exposed (nos 9, 13, 14 and 18) clearly recognized one or more lpa antigens. In volunteers no. 14 and no. 18 vaccination did not induce the appearance of antibodies recognizing bands not having already been labelled by their prevaccination samples. Postvaccination samples of volunteers no. 7 and no. 13 recognized all Ipa types, as well as the 120 kDa protein and qualitatively a similar response was observed in the previously non-exposed person no. 4. A weak response was seen in volunteer no. 1 to IpaB and to the 120kDa protein. The only band recognized by sera of all the volunteers .showing a response with this technique was IpaB.
Figure 5 Serum antibody responses to Shigella invasion plasmidcoded antigens (Ipa) detected by Western blot. (a) Group 1 (one vaccine dose). (b) Group 2a (three vaccine doses), a, prevaccination sample; b, postvaccination sample; c, control (convalesent human serum)
Only very weak anti-Ipa responses were seen in group 1
Secretory lgA (slgA) response
(Figure 7). However, the three vaccine doses stimulated
Most of the vaccinees in all three groups showed an immune response against the S..flexneri Y LPS antigen (Table 2). The difference between the number of responders as detected in serum and faecal extract was most obvious in groups 1 and 2a. The day to day variation in titres and the low number of exposed/ unexposed individuals in each group did not permit a statistical evaluation. As can be seen in Figure 6, although unexposed individuals also exhibited an increase in relative titres, it was sharper and higher in previously infected persons in all three groups reaching a peak around day 11. In group 2b, both exposed and unexposed individuals showed good responses that were similar to those in group 2a.
a specific slgA response in five out of the eight unexposed individuals (Table 2), although the average titre (as shown in Figure 7) was slightly higher in the previously exposed members of this group. The booster dose (group 2b) did not increase further the average titre in the unexposed persons, while it stimulated a high rise of titre in one exposed individual resulting in a sharp increase in the average titre of this group. To test the non-specific immune stimulant effect of the LPS of the vaccine strain, slgA responses against a heterologous antigen (S. typhimurium LPS) were also measured. The dynamics of these responses were similar to that of the homologous LPS antigen, but the titres were considerably lower (data not shown).
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V a c c i n e , Vol. 10, I s s u e 6, 1992
Live oral auxotrophic Shigella flexneri vaccine in volunteers: A. Li et
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Rgure 6 Faecal secretory IgA EIA titres to Shigella flexneri Y LPS (mean + s.d.) in groups l(a), 2a(b) and 2b(c). [-I, Volunteers with history of previous exposure to shigellae or enteroinvasive E. coli i n f e c t i o n s ; . , volunteers without history of previous exposure to shigellae or enteroinvasive E. coil infections
Peripheral blood mononuclear cell proliferation assay(PBMCP) Nine volunteers in group 2a and six in group 2b were tested for PBMCP. A SI value of 3 was chosen as an arbitrary cut-offlimit to identify responders (see Materials and methods). Based on these criteria, seven out of the
Vaccine, Vol. 10, Issue 6, 1992 401
Live oral auxotrophic Shigella flexneri vaccine in volunteers: A. Li et al. a 20
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nine volunteers in group 2a, and four out of six in group 2b, responded to the homologous S. flexneri Y PS (Figure 8). The responses, however, were much stronger in group 2a with a high plateau between day 7 and day 11 than in the boostered group. Three weeks after vaccination the values returned close to the prevaccination levels. Responses to other mitogens or antigens were within the normal ranges. DISCUSSION The S. flexneri SFL124 vaccine was well tolerated, and no dysentery-like symptoms were recorded among the vaccinees. The first dose of the vaccine caused no symptoms in more than 60% of the volunteers. Only two experienced a self-limiting watery diarrhoea with fever starting 14 h after ingestion of the vaccine and lasting for less than 12 h. No reversion to the virulent, wild phenotype was ever found among colonies recovered from the vaccinees, none of the symptoms recorded were characteristic of dysentery, and all the symptoms disappeared in 1 day without medical intervention. The diarrhoea may have been caused by the endotoxin load due to the invasion of the SFL124 strain given in a dose of 2 x 1 0 9 live bacteria. It should be noted that the second and tlfird doses were even better tolerated, which could be an indirect indication of the stimulation of host defence
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system(s) (Table I ). We surmise that strain SFL124 is safe for humans, and the symptoms recorded can be expected with such a high intake of an invasive endotoxin-containing bacteria is rather than that of the tissue destruction which might have been caused by a virulent, invasive micro-organism. The average excretion time was significantly shorter after the fourth (booster) dose in group 2b than in groups 1 and 2a (Fioure 1). This is in accordance with monkey experiments where SFL124-vaccinated monkeys excreted the challenge strain for a shorter time than unvaccinated monkeys 3 as did monkeys vaccinated with a S. flexneri strain with deleted virulence genes 19. In group 2b the short excretion time of SFL124 is thought to be the result of the strong, 'anamnestic' response observed on the mucosal surface (Fi,qures 6 and 7). Results of the humoral immune assays were evaluated separately for volunteers with or without a previous history of dysentery. As far as the anti-LPS specific humoral immune response is concerned, there was no significant difference between volunteers of different anamnestic history concerning previous shigella infections, with the exception of a strong IgA response in group 2b in the previously exposed individuals. Even after a single vaccine dose, serum anti-LPS titres increased. The increases were more pronounced in volunteers receiving three doses of the vaccine (group 2a). The number of responders and the rise in titres were even higher when the faecal extracts were tested against this antigen. Here, previously exposed individuals showed a stronger response than those without a previous anamnestic history (Fi(4ures 3 and 6, Table 2). These results clearly indicate that the vaccine was immunogenic enough to induce a systemic, as well as a local, antibody response against one of the major surface antigens of shigellae. The serum anti-Ipa response was more prevalent among those who reported a previous shigella infection. To a certain extent that was also true for the sIgA anti-Ipa response. It should be noted, however, that the three doses given in group 2a resulted in a local anti-Ipa response in five out of eight previously non-exposed volunteers. While experimental infection of monkeys was reported to be followed by a serum anti-Ipa response exceeding the one directed against the LPS antigen 2°, our data show that by vaccination it could be more difficult to induce antioIpa than anti-LPS responses. There are reports that anti-Ipa responses were detectable only after challenge, but not after vaccination 11. However, others reported a vaccine trial with anti-Ipa responses of the same magnitude as anti-LPS responses xg. In this latter case, we agree with the authors' suggestion that LPS contamination of the antigen preparation used to measure anti-Ipa responses might have been the reason. The fewer and weaker responses against Ipa than against LPS found by us could be because of the special attention paid to avoid the influence of anti-LPS antibodies when evaluating anti-Ipa responses using water extracts from a rough clone pair with and without Ipa. It is not known how the immune responses against the two major antigen complexes contribute to protection against dysentery. There are data indicating that the immunity against shigellosis is species (or serogroup) specific 21. It was reported that following oral vaccination with a streptomycin-dependent vaccine strain the immunity was strictly serotype-specific22. However, later
Live o r a l a u x o f r e p h i c S h i g e l l a flexneri vaccine in v o l u n t e e r s : A. Li et al.
on it was shown that this vaccine strain did not express Ipa antigens 2°. On the other hand, Meitert et al. 23 reported that repeated doses of S. flexneri 2a vaccine strain T32 also induced strong heterologous protection. While originally this strain was described as invasive 23, presumably expressing Ipa, a recent investigation revealed a deletion in the invasion plasmid, making it noninvasive, and not expressing the plasmid-coded protein 24. Until the exact mechanisms of immunity in shigellosis are understood, responses against both kinds of antigen should be monitored with pure antigen preparations. In general, more volunteers responded against both LPS and Ipa with local sIgA production than with serum antibodies ( Table 2 ). Since the vaccine was administered orally, this observation is not surprising, but still bears a special importance for a vaccine designed against a mucosal infection. The systemic immune response could be an indicator of the overall stimulation of the immune system, and is not necessarily associated with protection 2~. It is known that secretory IgA plays an important protective role in several mucosal infectionsz6 and could be active in antibody-dependent cellular cytotoxicity against invasive bacteria 27. The number of specific ASC reflects the activation of lymphocytes in the gut-associated lymphoid tissues (GALT). Once activated by antigens from the intestine, lymphocytes migrate from the Peyer's patches to the local lymph nodes where they proliferate, differentiate and repopulate the GALT via lymphatics and blood 2s'29. In all three groups of vaccinees the strongest ASC response was seen in the IgA type, with the highest peak in group 2a receiving three doses of the vaccine. This observation is in accordance with the strong sIgA responses we detected against both LPS and Ipa, and also with the reported high IgA responses by peripheral lymphocytes in some clinical observations 29'3°. Interestingly, while the booster dose in group 2b was followed by sIgA responses against both LPS and the Ipa, the number of LPS-specific !gA ASC in peripheral blood was relatively low as compared with those seen in groups 1 and 2a. We surmise that the low ASC response may be a consequence of the rapid elimination of SFL124 in the gut (Figure I) caused by the active mucosat immunity. Then low ASC responses could be considered as a measure of an active mucosal immune defence and be used as a predictor of protective immunity. Data available on cell-mediated immunity against shigellae are rare al and mostly indirect, showing an increased risk of dysentery among those with impaired cell-mediated functions (e.g. AIDS) a2. During the present vaccine trial, most of our attention was directed towards antibody responses against various shigella antigens. However, data obtained on the cellular side of the immune system by testing the proliferation rates of peripheral mononuclear cells after stimulation with homologous and heterologous antigens showed a clear positive, specific response in most of the vaccinees tested (Figure 8 ). This limited observation calls for the necessity of introduction of more sophisticated T-cell specific assays in the evaluation of anti-dysentery vaccines. Although the number of vaccinees with an anamnestic history of shigella or enteroinvasive E. coli infection was low, the reactions detected in their case need special attention. In developing countries, where an antidysentery vaccine is most needed, the target population infants and young children--most probably have already
experienced their first encounter(s) with shigellae 33. A similar phase ! trial is presently being conducted with SFL124 in a developing country and is expected to provide further information about the reaction of previously primed individuals to the vaccine. CONCLUSION This study clearly proved that SFL124 given in multiple doses is attenuated enough to be safe, but still stable and exhibit a stimulant effect strong enough for the host to elicit both systemic and local immune responses. Furthermore, an active mucosal immunity appeared to be still present in the gut 9 months after the primary vaccination. Trials with this vaccine candidate are planned to evaluate its protective efficacy in humans. ACKNOWLEDGEMENTS The authors are grateful to Dr Anders Kfirnell for valuable advice during the course of this investigation. This work was supported by the Swedish Medical Research Council (grant no. 16x-656), Stiftelsen Sigurd and Elsa Goljes Minne and the Swedish Agency for Research Cooperation_ with Developing Countries (SAREC). REFERENCES 1
2
3
4
5 6
7
8 9
10
11
12
13
14
Lindberg, A.A., K&rnell, A., Stocker, B.A.D., Katakura, S., Sweiha, H. and Reinholt, F.P. Development of an auxotrophic oral live Shigella flexneri vaccine. Vaccine 1988, 6, 146 Lindberg, A.A., K&rnell, A., Pal, T., Sweiha, H., Hultenby, K. and Stocker, B.A.D Construction of an auxotrophic Shigella flexneri for use as a live vaccine. Microb. Pathog. 1990, 8, 433 K&rnell, A., Stocker, B.A.D., Katakura, S., Reinholt, F.P. and Lindberg, A.A. Live oral auxotrophic Shigella flexneri SFL124 vaccine with a deleted aroD gene: characterization and monkey protection studies. Vaccine 1992, 11), 389 Maurelli, A.T., Blackmon, B. and Curtiss III, R. Loss of pigmentation in Shigella flexneri 2a is correlated with loss of virulence and virulence-associated plasmid. Infect. Immun. 1984, 43, 397 Ewing, W.H. Edwards and Ewing's Identification of Enterobacteriaceae. 4th Edn, Elsevier, New York, 1986, p. 136 Carlin, N.I.A. and Lindberg, A.A. Monoclonal antibodies specific for Shigella flexneri lipopolysaccharides: clones binding to type IV, V, and VI antigens, group 3, 4 antigen, and an epitope common to all Shigella flexneri and Shigella dysenteriae type 1 strains. Infect. Immun. 1987, 55, 1412 Gaspari, M.M., Brennan, P.T., Solomon, S.M. and Elson, C.O.A. A method of obtaining, processing and analyzing human intestinal secretions for antibody content. J. Immunol. Methods 1988,11{), 85 Westphal, O., L~deritz, O. and Bister, F. 0bet die extraktion von bakterien mit phenol/wasser. Z. Naturforsch. 1952, 7, 148 DrSge, W., Lfideritz, O. and Westphal, O. Biochemical studies on lipopolysaccharides of Salmonella mutants. 3. The linkage of the heptose units. Eur. J. Biochem. 1968, 4, 126 Buysse, J.M., Stover, C.K., Oaks, E., Venkatesan, M. and Kopecko, D.J. Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping. J. Bacteriol. 1987, 169, 2561 Oaks, E.V., Hale, T.L. and Formal, S.B. Serum immune response to Shigella protein antigens in Rhesus monkeys and humans infected with Shigella spp. Infect. Immun. 1986, ~3, 57 Sansonetti, P.J., Kopecko, D.J. and Formal, S.B. Involvement of a plasmid in the invasive ability of Shigella flexneri. Infect. Immun. 1982, 35, 852 Lindberg, A.A., Ekwall, E. and Carlin, N.I.A. Shigella flexneri O-antigen specific enzyme immunoassay: lipopolysaccharides and synthetic glycoconjugates as antigens. Serodiag. Immunother. Inf. Dis. 1988, 2, 63 P~I, T., Newland, J.W., Tall, B.D., Formal, S.B. and Hale, T.L. Intracellular spread of Shigella flexneri associated with the kcpA locus and a 140-kilodalton protein. Infect. Immun. 1989, 57, 477
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Live o r a l a u x o t r o p h i c S h i g e l l a flexneri vaccine in volunteers: A. Li et al.. 15 Hale, T.L., Oaks, E.V. and Formal, S.B. Identification and antigenic characterization of virulence-associated, plasmid-coded proteins of Shigella spp. and enteroinvasive Escherichia coil Infect. Immun. 1985, 50, 620 16 Olsson, T., Shahid, B., H+jeberg, B. and Link, H. Antimyelin basic protein and antimyelin antibody-producing cells in multiple sclerosis. Ann. Neurol. 1990, 27, 132 17 Czerkinsky, C.C., Nilsson, L-~., Nygren, H., Ouchterlony, O. and Tarkowski, A. A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J. Immunol. Methods 1983, 65, 109 18 Laskin, A.I. and Lechevalier, H.A. Handbook of Microbiology. Chemical Rubber Co., Cleveland, 1973, p. 223 19 Sansonetti, P.J. and Arondel, J. Construction and evaluation of a double mutant of Shigella flexneri as a candidate for oral vaccination against shigellosis. Vaccine 1989, 7. 443 20 Hale, T.L., Oaks, E.V., Formal, S.B., Dinari, G. and Echeverria, P. Immune response to Shigella infections and to Shigella vaccines. In: Vaccines 86. New Approaches to Immunization, Developing Vaccines against Parasitic, Bacterial, and Viral Diseases (Eds Brown, F., Chanock, R.M. and Lerner, R.A.) Cold Spring Harbor Laboratory, New York, 1986, p. 181 21 Cohen, D., Green, M.S., Block, C., Slepon, R. and Ofek, I. Prospective study of the association between serum antibodies to lipopelysaccharide O antigen and the attack rate of shigellosis. J. Clin. Microbiol. 1991, 29, 386 22 Mel, D.M., Arsic, B.L., Nikolic, B.D. and Radovanic, M.L. Studies on vaccination against bacillary dysentery. Bull. WHO 1968, 39, 375 23 Meitert, T., Pencu, E., Ciudin, L. and Tonciu, M. Vaccine strain Shigella flexneri T~=-ISTRATI. Studies in animals and in volunteers. Antidysentery immunoprophylaxis and immunotherapy by live vaccine VADIZEN (Sh. flexneri T32-1STRATI). Arch. Roum. Pathol.
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24
25
26
27
28
29
30 31 32
33
Exp. Microbiol. 1984, 43, 251 Venkatesan, M., Fernandez-Prada, C., Buysse, J.M., Formal, S.B. and Hale, T.L. Virulence phenotype and genetic characteristics of the T~-ISTRATI Shigella flexneri 2a vaccine strain. Vaccine 1991, 9,358 Formal, S.B., Maenza, R.M., Austin, S. and Labrec, E.H. Failure of parenteral vaccines to protect monkeys against experimental shigellosis. Proc. Soc. Exp. Biol. Med. 1967, 129, 347 Newby, T.J. Protective immune responses in the intestinal tract. In: Local Immune Responses of the Gut (Eds Newby, T.J. and Stokes, C.R.) CRC Press, Boca Raton, Florida, 1984, p. 153 Tagliabue, A., Boraschi, D., Villa, L., Keren, D.F., Lowell, G.H., Rappuoli, R. and Nencioni, L. IgA-dependent cell-mediated activity against enteropathogenic bacteria: distribution, specificity, and characterization of the effector cells. J. Immunol. 1984, 133, 988 Mestecky, J. The common mucosal immune system and current strategies for induction of immune responses in external secretions. J. Clin. Immunol. 1987, 7, 265 Kantele, A.M., Takanen, R. and Arvilommi, H. Immune response to acute diarrhea seen as circulation antibody-secreting cells. J. Infect. Dis. 1988, 158, 1011 Forrest, B.D. Identification of an intestinal immune response using peripheral blood lymphocytes. Lancet 1988, 1, 81 Zwillich, S.H., Duby, A.D. and Lipsky, P. T-lymphocyte clones responsive to Shigella flexneri. J. Clin. Microbiol. 1989, 27, 417 Blaser, M.J., Hale, T.L. and Formal, S.B. Recurrent shigellosis complicating human immunodeficiency virus infection: failure of pre-existing antibodies to confer protection. Am. J. Med. 1989, 86, 105 Institute of Medicine. The prospects for immunizing against Shigella spp. In: New Vaccine Development: Establishing Priorities. Vol. 2, National Academy Press, Washington, DC, 1986, p. 329
Forsum*
a n d A l f A. L i n d b e r g *$
The live, aromatic dependent Shigella flexr[eri Y vaccine strain SFL124, with a deleted a r o D gene, was tested for safety and immunogenicity in 21 healthy adult volunteers. A single dose o f 2 × 109 live bacteria was given orally to ten volunteers, whereas 11 received three doses every other day. The vaccine was excreted for 4.2 days and was well tolerated by 90.5% o f the vaccinees. Only 2 of 21 (9.5%) after the first dose had a self-limiting diarrhoea lasting 1 day; o f volunteers given one dose only 3 o f 10 showed anti-lipopolysaccharide ( L P S ) and anti-invasion plasmid coded antigen ( Ipa) responses in serum. A faecal antibody response to L P S and Ipa was seen in six and three persons, respectively. Volunteers given three doses reacted with serum anti-LPS (9/11 ) and anti-Ipa (5/11) antibody responses. In stool, anti-LPS and anti-lpa responses were detected in nine and eight volunteers, respectively. A booster dose of 2 × 1 0 9 bacteria given to six volunteers in the three-dose group 9-10 months later elicited high stool slgA responses, indicating a strong mucosal memory, and was accompanied by a short excretion period o f SFL124 (1.8 versus 4.2 days, p < 0.05). The vaccination also elicited antibody-secreting cell ( A S C ) responses against L P S in peripheral blood." the three doses of the vaccine resulted in a stronger response than did the single dose, while the booster dose elicited only a limited A S C response. Volunteers previously exposed to shigellae exhibited stronger anti-Ipa responses in serum and stool suggestive o f an immunological memory to the Ipa. The results indicate that SFL124 is a safe live vaccine strain inducing specific immune responses against L P S and Ipa with a mucosal immune memory lasting for at least 9 months. Keywords:Shigella.flexneri; oral vaccination; humans; safety; immunogenicity
INTRODUCTION Recently, we developed a Shigella flexneri serotype Y vaccine strain (SFL114), w h i c h - - d u e to an aroD :: T n l 0 m u t a t i o n - - w a s auxotrophic for p-aminobenzoic acid 1. Since this compound is not available in the cytosol, although the strain was able to invade epithelial cells at the wild type level, its ability to grow intracellularly was greatly reduced, making it avirulent 2. This strain induced significant protection in monkeys against homologous challenge 1. Although reversion back to virulence has never been observed with SFL114, for safety reasons a deletion mutant (SFL124) was selected from S F L I I 4 devoid of the T n l 0 transposon and a 1400 base pair (bp) long stretch of the aroD gene making it unable to revert 3. The strain was safe in in vitro and in vivo assays and elicited protective immunity in Macaca fascicularis *Karolinska Institute, Department of Clinical Bacteriology, Huddinge Hospital, S-141 86 Huddinge, Sweden. 1Institute of Microbiology, University Medical School, H-7643 P~cs, Hungary. ~To whom correspondence should be addressed. (Received 22 July 1991 ; revised 20 November 1991 ; accepted 28 November 1991) 0264410X/92/060395-10 © 1992Butterworth-HeinemannLtd
monkeys. Here we report the results of a safety/immunogenicity trial of S. flexneri SFL124 given as an oral live vaccine to 21 adult human volunteers. MATERIALS
AND
METHODS
Volunteers Twenty-one healthy persons, six men and 15 women,
25-55 years of age (mean 39.3), at present residing in Sweden, participated in the study. While none of them had had any episodes of enteric infections in the past 2 years, based on their earlier anamnestic history they formed two groups: 16 of them were not aware of any previous dysentery-like infection when the description of the symptoms was provided by the interviewer, who was medically qualified. This group was tentatively considered to be the previously non-exposed group. Members of the second group (five persons) reported either proven infections (one known laboratory accidental infection with enteroinvasive Escherichia coli 6 years previously), or four of them recalled enteric infections 5-13 years previously which, at the level of symptoms, would qualify as dysentery. It should be noted that these last four
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volunteers were originally from countries where shigellosis is endemic. The group was considered to be previously primed. The recruiting procedure and the trial were approved by the Karolinska Institute Ethics Committee.
Vaccine strain The construction and characteristics of S. flexneri Y AaroD strain SFL124 are described in an accompanying paper 3.
The periplasmic content of shigellae known to be rich in invasion plasmid-coded antigens (lpa) B and C 10 was extracted by distilled water according to Oaks et al. ~1. For this purpose E. coli K12 strains SP10 and J53 were used. SP10 is a derivative of E. coli K12 J53, into which the invasion plasmid (pWR 110)~ 2 of a virulent S. flexneri 5 strain (M90T) (kindly provided by Dr S.B. Formal, Walter Reed Army Institute of Research, Washington, D C ) had been mobilized as described 11.
Enzyme immunoassays (EIA) Vaccination schedule and protocol The volunteers were divided into three groups. G r o u p 1 consisted of ten people receiving a single vaccine dose. G r o u p 2a (11 volunteers) received three doses of the vaccine on days 0, 2 and 4. G r o u p 2b comprised six members of group 2a who were boosted with a single dose 9 - 1 0 months later. On the morning of vaccination the volunteers ingested 2 g of N a H C O 3 tablets. Ten minutes after they drank 2 × 109 cells of the vaccine strain suspended in PBS (pH 7.2). They were not requested to fast before vaccination but were asked to avoid any food and water intake for 2 h after the vaccination. For each vaccination event a new lyophil amouple from the same batch of the vaccine strain was opened, reconstituted with distilled water and diluted in PBS to provide the requested concentration. The ratio of invasion plasmid harbouring clones in the vaccine was above 95%, as judged by the number of Congo red positive colonies 4.
Vaccine excretion Vaccine excretion was monitored daily starting on the first day after the last dose of vaccine and continuing until three consecutive negative results were obtained. The number of colony forming units/gram faeces was determined on DC agar plates (Oxoid Ltd, Basingstoke, Hants, U K ) . The vaccine strain recognized by its morphology was identified by routine biochemical tests ~ and by agglutination in mAb Y-5 monoclonal antibody recognizing the S. flexneri Y O-specific side chain 6. Conversion to the wild ( p r o t o t r o p h ) phenotype was checked on minimal agar plates. Simultaneously, the excretion of the normal E. coil flora was also quantitatively monitored. If faecal samples were not available, rectal swab samples were taken and were processed for qualitative evaluation.
Extraction of faecal samples for slgA-EIA Faecal samples kept at 4°C were always processed on the same day they were delivered. After a 1 : 5 dilution with trypsin inhibition buffer 7, samples were homogenized and centrifuged at 20 0009 for 30 min. The supernatants were filtered and stored at - 7 0 ° C until use.
Antigen preparations Lipopolysaccharide (LPS) of S. flexneri Y, Salmonella typhimurium and E. coli CVD-172 were prepared by hot p h e n o l - w a t e r extraction 8. Polysaccharide (PS) from S. flexneri Y and E. coli CVD-172 was extracted by weak acid hydrolysis 9. No trace of lipid A was detected in the extracts by gas liquid chromatography.
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Antibody titres in serum and faecal extracts against S. flexneri Y LPS and Ipa were examined by EIA. The relative antibody titres, defined as the absorbance (A) values at a given sample dilution were determined as described ~3. To measure the serum antibody responses to S. flexneri Y LPS and sIgA in faecal extracts specific to S..flexneri Y and S. typhimurium LPS, the above LPS antigens were used to coat EIA plates at a concentration of 5 #g m l - 1. To determine the immune response to Ipa, water extracts of SP10 and E. coil K12 J53 were used to coat the plates at a protein concentration of 20 #g m l - 1 To increase the specificity of the assay to Ipa, every sample was tested against extracts made from SP10 as well as from its plasmidless counterpart J53. Then corrected A values obtained by deducting A j53 from AsP~o were used to express relative titres. To measure anti-LPS responses, sera were diluted 1:1000 and for anti-lpa responses, the dilution was 1 : 500. Faecal extracts were diluted 1 : 5 for both antigens. For serum samples rabbit anti-human-IgG (1:4000), -IgA (1 : 1000)and -IgM (1:800)conjugated to alkaline phosphatase were used as secondary antibodies while for sIgA determinations rabbit anti-human secretory component conjugated to horseradish peroxidase (1 : 1000) (all from Dakopatts, Copenhagen, Denmark ) were used. In groups 1 and 2a, 'immune response' was defined as a minimum twofold increase in titres as compared to the individuals' prevaccine titres. Since group 2b was formed by the boostered members of group 2a, in their cases prebooster, instead of prevaccine, values were used as reference. Serum ' n o r m a l ' values were defined as the mean + 2 s.d. of titres against the corresponding antigens of 30 healthy Swedish blood donors.
Western blot of serum samples Some serum samples were analysed by Western blot using whole cell extracts of a virulent S. flexneri 5 strain, M90T and its plasmidless derivative M90T5514. The extraction, polyacrylamide gel electrophoresis and immunoblotting procedures were carried out as described14. Serum samples were diluted 1:50. Identification of plasmid-coded antigen bands on nitrocellulose sheets containing extract of M90T were done on the basis of their reported molecular mass 1~ ( 1 2 0 k D a ; IpaA = 78 k D a ; IpaB = 62 k D a ; IpaC = 43 k D a ; I p a D = 38 k D a ) and by their absence from the extracts of M90T55 run simultaneously. Serum samples of a Vietnamese individual containing antibodies against the above proteins served as positive control.
Preparation of peripheral mononuclear cells Blood was taken into heparinized tubes and diluted 1:1 with Hanks Balanced Salt Solution (HBSS, Flow
Live oral auxotrophic Shigella flexneri vaccine in volunteers: A. Li et al.
Laboratories, Irvine, UK). Mononuclear cells were separated on Ficoll Paque (Pharmacia, Uppsala, Sweden). After washing three times in HBSS, the cell number was set to 10 6 m1-1 in RPMI 1640 (Gibco Limited, Paisley, UK) supplemented with 10 mM HEPES (flow), 2 mM L-glutamine, 1 m~ Na-pyruvate, 100 U ml-x penicillin and 100 #g ml-~ streptomycin. For the assay of antibody-secreting cells, 10% of heat-inactivated fetal calf serum (Gibco) was used; for the cell proliferation assay, 10% of blood type AB human serum was added. Cell viability was always higher than 95% as checked by trypan blue exclusion assay.
Assays for immunoglobulin and for specific antibody-secreting cells The enzyme-linked immunospot assay (ELISPOT) was carried out as described~6'~7 with some modifications. Briefly, to enumerate the immunoglobulin secreting cells (ISC), nitrocellulose-bottomed microtitre plates (Millipore Corporation, Bedford, MA, USA ) were coated with 100/~l/well of goat anti-human IgG, IgA and IgM (Sigma Chemical, St Louis, USA) diluted 1 : 500. To detect the specific antibody-secreting cells (ASC), wells were coated with 100 #1 of S. flexneri Y or E. coli CVD 172 LPS (20 #g ml- 1), or 2 #g ml- 1 teichoic acid from Staphylococcus aureus (National Bacteriology Laboratory, Stockholm, Sweden). After overnight incubation at room temperature wells were washed three times with PBS. Cell suspension ( 100/A) was added to the wells and plates were incubated at 37°C in 7% CO2 in a humidified atmosphere for 16 h. After washing, 100/~1 biotinylated goat anti-human IgA, IgG or IgM were added (Sigma, dilution 1 : 500) and incubation was continued at room temperature for an additional 3 h. Spots representing antibody-secreting cells were visualized by adding preformed avidin-biotinylated horseradish peroxidase complex (Vector Laboratories, Burlingame, CA, USA), then carbazol-dimethylformamide substrate. Spots were counted under a dissection microscope.
Peripheral mononuclear cell proliferation assay Peripheral mononuclear cell suspension was set to a concentration of 5 × 105 m1-1. Suspension (200/d) was added to round-bottomed microtitre plates. The following antigens or mitogens were then added : S. flexneri Y PS (50/2g/well) ; E. coli PS (30 #g/well); tuberculin-purified protein derivative (2~tg/well, Statens Seruminstitute, Copenhagen) and phytohaemagglutinin (10/~l/well of a 1 : 1000 diluted stock, Wellcome Diagnostics, Dartford, UK). Tests were carried out in triplicate. To the control wells no antigen or mitogen was added. After 5 days at Table 1
37°C, 7% CO2, cells were pulsed with methyl-3H thymidine (1/~Ci/well; Amersham International, Buckinghamshire, UK) and harvested 16 h later. Results were counted in a fl-counter. Stimulation indexes (SI) were calculated as counts min-1 in stimulated wells/counts min-1 in unstimulated wells. RESULTS
Clinical symptoms The clinical symptoms following vaccination are summarized in Table 1. Of the 21 volunteers, 13 (61.9%) tolerated the first (in the case of members of group 1, the only) dose of the vaccine without any symptoms. Six volunteers (28.6%) reported one or two of the mild symptoms shown in Table I. Two volunteers (9.5%) developed diarrhoea concomitant with a febrile response, 38.2°C and 39°C, respectively, 14-20 h after vaccination. The diarrhoea was accompanied by fatigue and a slight stomach discomfort. During the late night and early morning, they had four and five episodes of loose to watery stools, respectively. Blood or mucus was not detected in their faeces. On the afternoon of the following day (day 1) their temperatures had dropped to normal (< 37°C). Though they still felt some fatigue during the day, no more diarrhoeal episodes were recorded. On day 2, both of them had recovered spontaneously. After the second vaccine dose (group 2a) one out of 11 volunteers had some fatigue, while the third dose ( 11 volunteers in group 2a) was tolerated without any symptoms. The booster dose given 9-10 months after the primary vaccination to six of the 11 volunteers in group 2a (group 2b) induced two single episodes of loose stool and transient, slight headache in two volunteers.
Excretion of the vaccine strain The excretion of the vaccine strain following the last vaccine dose in each group is shown in Figure 1. The average excretion time in groups 1 and 2a was practically the same (mean + s.d. = 4.4. _+ 1.2 and 4.0 + 1.9 days, respectively), while in group 2b it was significantly shorter (1.8 + 1.8 days, p < 0.05 when compared with either group 1 or group 2a). No significant variation in the normal E. coli flora was observed. No reversion of the vaccine strain to the virulent phenotype was observed.
Immunoglobulin (ISC) and specific antibody-secreting cell (ASC) response in peripheral blood Following vaccination, the total number of IgA, IgG and IgM ISCs slightly increased in all three vaccine
Clinical symptoms in volunteers after oral vaccination with Shigella flexneri SFL124
Number of volunteers No symptoms ( % ) Mild symptoms a ( % ) Severe symptoms b ( % ) Mucus a n d / o r blood in stool
lstdose (group 1, 2a and 2b)
2nd dose (group 2a and 2b)
3rd dose (group 2a and 2b)
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Vaccine, Vol. 10, Issue 6, 1992 397
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groups with peaks around days 4-7, but the increases were not significant (p > 0.05) in any case (data not shown). S. flexneri Y LPS-specific ASCs could be found in only a few individuals in low numbers before vaccination. After vaccination, in groups 1 and 2a ASCs started to rise in all three Ig classes on day 4 reaching a peak on day 7 (Figure 2). The strongest responses were observed for IgA. The response appeared to be slightly more prolonged in group 2a than in either of groups 1 and 2b. There were no measurable ASC responses in groups 1 and 2b against heterologous antigens like teichoic acid or E. coli LPS. Due to a possible polyclonal stimulation, a few IgA-secreting clones recognizing these antigens appeared in the blood of some members of group 2a (data not shown), but the increases were not significant (p > 0.05).
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398
Vaccine, Vol. 10, Issue 6, 1992
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Serum antibody responses The serum IgA and IgG antibody responses against S. flexneri Y LPS are summarized in Figure 3. Responses in the lgM class were low, and generally similar in pattern to those seen in the IgG class (data not shown). Prevaccination IgA titres did not exceed the corresponding 'normal' limits defined as the mean titre ___2s.d. of 30 healthy Swedish blood donors. For IgG, one and for IgM, three out of the 21 volunteers had slightly higher titres than the normal values. After a single dose (group 1 ) anti-LPS titres slightly rose in all three Ig classes reaching a peak around day 14. In the three-dose group (group 2a) the increase in titre was more prominent with a maximum level on day 7. No significant decreases in these titres were observed during the follow-up period (up to day 60). In these two groups there were no significant differences between responses of previously exposed and unexposed volunteers (p > 0.05 for exposed versus unexposed volunteers for group 1 and 2a for samples collected on the same day). Following the booster dose in group 2b, the two volunteers in this group with an anamnestic history of shigellosis responded with a rise of specific lgA titres by day 7, while those without known previous exposure did not exhibit any titre increase. Comparing day 0 with day 14 titres in the groups without dividing them into
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than their prevaccination values and the booster dose caused only a slight further increase. However, by the time of the administration of the booster dose, IgA titres had already fallen back close to prevaccination values, and the booster induced a sharp increase in lpa-specific serum IgA which returned to the prevaccination value in 4 weeks. Data concerning the number of responders are summarized in Table 2. In general, the three vaccine-dose
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that, unlike the LPS responses, after one or three doses of vaccination Ipa responses were considerably higher in the volunteers previously infected with shigellae (Figure 4 ). In their cases, titres rose sharply, reaching a maximum around day 14 and, especially for IgG, remaining high during the 60-day study period. Previously non-exposed members of group 2b still did not respond to Ipa after the booster dose. Owing to the previous three vaccine doses, the prebooster IgG titres of the two previously exposed members of this group were still much higher
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Vaccine, Vol. 10, I s s u e 6, 1992
399
Live oral auxotrophic S h i g e l l a f l e x n e r i vaccine in volunteers: A. Li et al. Table 2
Antibody responses of volunteers to Shigella flexneri Y LPS and Ipa" Serum antibody to S. flexneri Y LPS
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Group 2a
Exposed Unexposed Total
2/3 6/8 8/11
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2/3 2/8 4/11
3/3 2/8 5/11
1/3 0/8 1/11
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3/3 5/8 8/11
3/3 6/8 9/11
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2/2 0/4 2/6
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2/2 4/4 6/6
1/2 2/4 3/6
2/2 4/4 6/6
"lpa: invasion plasmid-coded antigens. bExposed: the volunteers had previous exposure history to shigellae or enteroinvasive E. coli infections 5 13 years ago. CUnexposed: the volunteers did not have previous exposure history to shigellae infections. aNumber of responders versus number of volunteers for each immunoglobulin class, or responding in at least one of the classes
schedule was more effective than a single dose in stimulating an immune response against the LPS antigen. In the three-dose group most of the volunteers (8/11) responded to the homologous LPS antigen. For lpa, even in the three-dose group (2a) the response among non-pre-exposed individuals was low (2/8 persons). In group 2b the serum responses against both antigens were low. The booster dose caused further increases in titre in a few volunteers only, mainly in the two pre-exposed persons in this group.
Western blot of serum samples
Figures 5A and B show the Western blot of serum samples in groups 1 and 2a, respectively. With the exception of volunteer no.. 7, prevaccination sera of four out of five volunteers considered to be previously exposed (nos 9, 13, 14 and 18) clearly recognized one or more lpa antigens. In volunteers no. 14 and no. 18 vaccination did not induce the appearance of antibodies recognizing bands not having already been labelled by their prevaccination samples. Postvaccination samples of volunteers no. 7 and no. 13 recognized all Ipa types, as well as the 120 kDa protein and qualitatively a similar response was observed in the previously non-exposed person no. 4. A weak response was seen in volunteer no. 1 to IpaB and to the 120kDa protein. The only band recognized by sera of all the volunteers .showing a response with this technique was IpaB.
Figure 5 Serum antibody responses to Shigella invasion plasmidcoded antigens (Ipa) detected by Western blot. (a) Group 1 (one vaccine dose). (b) Group 2a (three vaccine doses), a, prevaccination sample; b, postvaccination sample; c, control (convalesent human serum)
Only very weak anti-Ipa responses were seen in group 1
Secretory lgA (slgA) response
(Figure 7). However, the three vaccine doses stimulated
Most of the vaccinees in all three groups showed an immune response against the S..flexneri Y LPS antigen (Table 2). The difference between the number of responders as detected in serum and faecal extract was most obvious in groups 1 and 2a. The day to day variation in titres and the low number of exposed/ unexposed individuals in each group did not permit a statistical evaluation. As can be seen in Figure 6, although unexposed individuals also exhibited an increase in relative titres, it was sharper and higher in previously infected persons in all three groups reaching a peak around day 11. In group 2b, both exposed and unexposed individuals showed good responses that were similar to those in group 2a.
a specific slgA response in five out of the eight unexposed individuals (Table 2), although the average titre (as shown in Figure 7) was slightly higher in the previously exposed members of this group. The booster dose (group 2b) did not increase further the average titre in the unexposed persons, while it stimulated a high rise of titre in one exposed individual resulting in a sharp increase in the average titre of this group. To test the non-specific immune stimulant effect of the LPS of the vaccine strain, slgA responses against a heterologous antigen (S. typhimurium LPS) were also measured. The dynamics of these responses were similar to that of the homologous LPS antigen, but the titres were considerably lower (data not shown).
400
V a c c i n e , Vol. 10, I s s u e 6, 1992
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Peripheral blood mononuclear cell proliferation assay(PBMCP) Nine volunteers in group 2a and six in group 2b were tested for PBMCP. A SI value of 3 was chosen as an arbitrary cut-offlimit to identify responders (see Materials and methods). Based on these criteria, seven out of the
Vaccine, Vol. 10, Issue 6, 1992 401
Live oral auxotrophic Shigella flexneri vaccine in volunteers: A. Li et al. a 20
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nine volunteers in group 2a, and four out of six in group 2b, responded to the homologous S. flexneri Y PS (Figure 8). The responses, however, were much stronger in group 2a with a high plateau between day 7 and day 11 than in the boostered group. Three weeks after vaccination the values returned close to the prevaccination levels. Responses to other mitogens or antigens were within the normal ranges. DISCUSSION The S. flexneri SFL124 vaccine was well tolerated, and no dysentery-like symptoms were recorded among the vaccinees. The first dose of the vaccine caused no symptoms in more than 60% of the volunteers. Only two experienced a self-limiting watery diarrhoea with fever starting 14 h after ingestion of the vaccine and lasting for less than 12 h. No reversion to the virulent, wild phenotype was ever found among colonies recovered from the vaccinees, none of the symptoms recorded were characteristic of dysentery, and all the symptoms disappeared in 1 day without medical intervention. The diarrhoea may have been caused by the endotoxin load due to the invasion of the SFL124 strain given in a dose of 2 x 1 0 9 live bacteria. It should be noted that the second and tlfird doses were even better tolerated, which could be an indirect indication of the stimulation of host defence
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Vaccine, Vol. 10, Issue 6, 1992
system(s) (Table I ). We surmise that strain SFL124 is safe for humans, and the symptoms recorded can be expected with such a high intake of an invasive endotoxin-containing bacteria is rather than that of the tissue destruction which might have been caused by a virulent, invasive micro-organism. The average excretion time was significantly shorter after the fourth (booster) dose in group 2b than in groups 1 and 2a (Fioure 1). This is in accordance with monkey experiments where SFL124-vaccinated monkeys excreted the challenge strain for a shorter time than unvaccinated monkeys 3 as did monkeys vaccinated with a S. flexneri strain with deleted virulence genes 19. In group 2b the short excretion time of SFL124 is thought to be the result of the strong, 'anamnestic' response observed on the mucosal surface (Fi,qures 6 and 7). Results of the humoral immune assays were evaluated separately for volunteers with or without a previous history of dysentery. As far as the anti-LPS specific humoral immune response is concerned, there was no significant difference between volunteers of different anamnestic history concerning previous shigella infections, with the exception of a strong IgA response in group 2b in the previously exposed individuals. Even after a single vaccine dose, serum anti-LPS titres increased. The increases were more pronounced in volunteers receiving three doses of the vaccine (group 2a). The number of responders and the rise in titres were even higher when the faecal extracts were tested against this antigen. Here, previously exposed individuals showed a stronger response than those without a previous anamnestic history (Fi(4ures 3 and 6, Table 2). These results clearly indicate that the vaccine was immunogenic enough to induce a systemic, as well as a local, antibody response against one of the major surface antigens of shigellae. The serum anti-Ipa response was more prevalent among those who reported a previous shigella infection. To a certain extent that was also true for the sIgA anti-Ipa response. It should be noted, however, that the three doses given in group 2a resulted in a local anti-Ipa response in five out of eight previously non-exposed volunteers. While experimental infection of monkeys was reported to be followed by a serum anti-Ipa response exceeding the one directed against the LPS antigen 2°, our data show that by vaccination it could be more difficult to induce antioIpa than anti-LPS responses. There are reports that anti-Ipa responses were detectable only after challenge, but not after vaccination 11. However, others reported a vaccine trial with anti-Ipa responses of the same magnitude as anti-LPS responses xg. In this latter case, we agree with the authors' suggestion that LPS contamination of the antigen preparation used to measure anti-Ipa responses might have been the reason. The fewer and weaker responses against Ipa than against LPS found by us could be because of the special attention paid to avoid the influence of anti-LPS antibodies when evaluating anti-Ipa responses using water extracts from a rough clone pair with and without Ipa. It is not known how the immune responses against the two major antigen complexes contribute to protection against dysentery. There are data indicating that the immunity against shigellosis is species (or serogroup) specific 21. It was reported that following oral vaccination with a streptomycin-dependent vaccine strain the immunity was strictly serotype-specific22. However, later
Live o r a l a u x o f r e p h i c S h i g e l l a flexneri vaccine in v o l u n t e e r s : A. Li et al.
on it was shown that this vaccine strain did not express Ipa antigens 2°. On the other hand, Meitert et al. 23 reported that repeated doses of S. flexneri 2a vaccine strain T32 also induced strong heterologous protection. While originally this strain was described as invasive 23, presumably expressing Ipa, a recent investigation revealed a deletion in the invasion plasmid, making it noninvasive, and not expressing the plasmid-coded protein 24. Until the exact mechanisms of immunity in shigellosis are understood, responses against both kinds of antigen should be monitored with pure antigen preparations. In general, more volunteers responded against both LPS and Ipa with local sIgA production than with serum antibodies ( Table 2 ). Since the vaccine was administered orally, this observation is not surprising, but still bears a special importance for a vaccine designed against a mucosal infection. The systemic immune response could be an indicator of the overall stimulation of the immune system, and is not necessarily associated with protection 2~. It is known that secretory IgA plays an important protective role in several mucosal infectionsz6 and could be active in antibody-dependent cellular cytotoxicity against invasive bacteria 27. The number of specific ASC reflects the activation of lymphocytes in the gut-associated lymphoid tissues (GALT). Once activated by antigens from the intestine, lymphocytes migrate from the Peyer's patches to the local lymph nodes where they proliferate, differentiate and repopulate the GALT via lymphatics and blood 2s'29. In all three groups of vaccinees the strongest ASC response was seen in the IgA type, with the highest peak in group 2a receiving three doses of the vaccine. This observation is in accordance with the strong sIgA responses we detected against both LPS and Ipa, and also with the reported high IgA responses by peripheral lymphocytes in some clinical observations 29'3°. Interestingly, while the booster dose in group 2b was followed by sIgA responses against both LPS and the Ipa, the number of LPS-specific !gA ASC in peripheral blood was relatively low as compared with those seen in groups 1 and 2a. We surmise that the low ASC response may be a consequence of the rapid elimination of SFL124 in the gut (Figure I) caused by the active mucosat immunity. Then low ASC responses could be considered as a measure of an active mucosal immune defence and be used as a predictor of protective immunity. Data available on cell-mediated immunity against shigellae are rare al and mostly indirect, showing an increased risk of dysentery among those with impaired cell-mediated functions (e.g. AIDS) a2. During the present vaccine trial, most of our attention was directed towards antibody responses against various shigella antigens. However, data obtained on the cellular side of the immune system by testing the proliferation rates of peripheral mononuclear cells after stimulation with homologous and heterologous antigens showed a clear positive, specific response in most of the vaccinees tested (Figure 8 ). This limited observation calls for the necessity of introduction of more sophisticated T-cell specific assays in the evaluation of anti-dysentery vaccines. Although the number of vaccinees with an anamnestic history of shigella or enteroinvasive E. coli infection was low, the reactions detected in their case need special attention. In developing countries, where an antidysentery vaccine is most needed, the target population infants and young children--most probably have already
experienced their first encounter(s) with shigellae 33. A similar phase ! trial is presently being conducted with SFL124 in a developing country and is expected to provide further information about the reaction of previously primed individuals to the vaccine. CONCLUSION This study clearly proved that SFL124 given in multiple doses is attenuated enough to be safe, but still stable and exhibit a stimulant effect strong enough for the host to elicit both systemic and local immune responses. Furthermore, an active mucosal immunity appeared to be still present in the gut 9 months after the primary vaccination. Trials with this vaccine candidate are planned to evaluate its protective efficacy in humans. ACKNOWLEDGEMENTS The authors are grateful to Dr Anders Kfirnell for valuable advice during the course of this investigation. This work was supported by the Swedish Medical Research Council (grant no. 16x-656), Stiftelsen Sigurd and Elsa Goljes Minne and the Swedish Agency for Research Cooperation_ with Developing Countries (SAREC). REFERENCES 1
2
3
4
5 6
7
8 9
10
11
12
13
14
Lindberg, A.A., K&rnell, A., Stocker, B.A.D., Katakura, S., Sweiha, H. and Reinholt, F.P. Development of an auxotrophic oral live Shigella flexneri vaccine. Vaccine 1988, 6, 146 Lindberg, A.A., K&rnell, A., Pal, T., Sweiha, H., Hultenby, K. and Stocker, B.A.D Construction of an auxotrophic Shigella flexneri for use as a live vaccine. Microb. Pathog. 1990, 8, 433 K&rnell, A., Stocker, B.A.D., Katakura, S., Reinholt, F.P. and Lindberg, A.A. Live oral auxotrophic Shigella flexneri SFL124 vaccine with a deleted aroD gene: characterization and monkey protection studies. Vaccine 1992, 11), 389 Maurelli, A.T., Blackmon, B. and Curtiss III, R. Loss of pigmentation in Shigella flexneri 2a is correlated with loss of virulence and virulence-associated plasmid. Infect. Immun. 1984, 43, 397 Ewing, W.H. Edwards and Ewing's Identification of Enterobacteriaceae. 4th Edn, Elsevier, New York, 1986, p. 136 Carlin, N.I.A. and Lindberg, A.A. Monoclonal antibodies specific for Shigella flexneri lipopolysaccharides: clones binding to type IV, V, and VI antigens, group 3, 4 antigen, and an epitope common to all Shigella flexneri and Shigella dysenteriae type 1 strains. Infect. Immun. 1987, 55, 1412 Gaspari, M.M., Brennan, P.T., Solomon, S.M. and Elson, C.O.A. A method of obtaining, processing and analyzing human intestinal secretions for antibody content. J. Immunol. Methods 1988,11{), 85 Westphal, O., L~deritz, O. and Bister, F. 0bet die extraktion von bakterien mit phenol/wasser. Z. Naturforsch. 1952, 7, 148 DrSge, W., Lfideritz, O. and Westphal, O. Biochemical studies on lipopolysaccharides of Salmonella mutants. 3. The linkage of the heptose units. Eur. J. Biochem. 1968, 4, 126 Buysse, J.M., Stover, C.K., Oaks, E., Venkatesan, M. and Kopecko, D.J. Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping. J. Bacteriol. 1987, 169, 2561 Oaks, E.V., Hale, T.L. and Formal, S.B. Serum immune response to Shigella protein antigens in Rhesus monkeys and humans infected with Shigella spp. Infect. Immun. 1986, ~3, 57 Sansonetti, P.J., Kopecko, D.J. and Formal, S.B. Involvement of a plasmid in the invasive ability of Shigella flexneri. Infect. Immun. 1982, 35, 852 Lindberg, A.A., Ekwall, E. and Carlin, N.I.A. Shigella flexneri O-antigen specific enzyme immunoassay: lipopolysaccharides and synthetic glycoconjugates as antigens. Serodiag. Immunother. Inf. Dis. 1988, 2, 63 P~I, T., Newland, J.W., Tall, B.D., Formal, S.B. and Hale, T.L. Intracellular spread of Shigella flexneri associated with the kcpA locus and a 140-kilodalton protein. Infect. Immun. 1989, 57, 477
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24
25
26
27
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