I ' ~ U T T E R W O R T H I ' V E I N E M A N N

Vaccine, Vol. 13, No. 1, pp. 88-99, 1995 Copyright ~, 1994 Elsevier Science Ltd

Printed in Great Britain. All rights reserved 0264-410X/95 $10.00 + 0.00

Safety and immunogenicity study of the auxotrophic Shigella flexneri 2a vaccine SFLI070 with a deleted aroD gene in adult Swedish volunteers Anders Kiirnell, An Li, Chun R. Zhao, Kerstin Karlsson, Nguyen B. Minh and Alf A. Lindberg*

The live auxotrophic Shigella flexneri 2a vaccine strain SFLI070 with a deleted aroD gene was given orally to 37 adult Swedish volunteers who received three doses within 5 days. Each dose comprised 1 x 105 (n = 9), 1 × 107 (n = 10), 1 x 10 s (n = 9) or 1 × 109 (n = 9) c.f.u.S, flexneri SFLI070. One volunteer vaccinated with 1 x 107 and three vaccinated with 1 x 108 c.f.u, reported mild gastrointestinal symptoms after the first dose. Vaccination with 1 x 109 c.f.u, caused abdominal pain and watery diarrhoea in four volunteers who all recovered spontaneously within 72 h. S. flexneri SFLI070 was not recovered from volunteers given 1 x 105 c.f.u., but was shed in faeces by six volunteers vaccinated with 1 x 107, by all nine vaccinated with 1 x 108, and by seven volunteers vaccinated with 1 x 109 cf.u. The mean excretion time was 2.6 (range 0-4) days in the 1 x 108 and the 1 x 109 groups. Serum antibody responses against either S. flexneri 2a and Y lipopolysaccharides (LPSs) or Shigella invasion plasmid antigens (Ipa) were seen in eight volunteers vaccinated with 1 x 109 (p < 0.01 to p < 0.05 for mean relative titres o f lgA and IgG against S. flexneri 2a and Y LPSs) , in four vaccinated with 1 × 108, and in two and one volunteers each vaccinated with 1 × 107 and l x 105 c.f.u, o f S. flexneri SFLI070. Intestinal slgA responses to the same antigens were elicited in all volunteers in the 1 x 109 and the 1 x 108 groups, and in six and one volunteers vaccinated with 1 x 107 and I x 105 c.f.u., respectively. The slgA responses against S. flexneri 2a and Y LPSs were significant in all but the 1 x 105 group (p < 0.01 to p < 0.05). Significant antibody-secreting cell (ASC) responses specific to S. flexneri 2a L P S were seen in peripheral blood from eight volunteers each in the 1 x 109 and l x 1 0 s groups and from five volunteers vaccinated with I x 107 cf.u. (p<O.O1 to p<O.05). The number o f volunteers showing anti-Shigella Ipa A S C responses in these groups were five (p < 0.01 to p < 0.05), three and one, respectively. Interferon-7 ( IFN-7 )-secreting T-cell responses were seen in all volunteers vaccinated with i x 108 and 1 x 109 c.f.u. S. flexneri SFLI070 after stimulation o f peripheral blood mononuclear cells with S. flexneri 2a polysaccharide antigen (p<O.01 to p<O.05), and in five volunteers in each o f these groups after stimulation with Shigella Ipa (p<O.O1 to p<O.05). S. flexneri SFLI070 is at least lO0000-fold attenuated compared with its wild-type parent strain 2457T. Immune responses, vaccine excretion and side-effects are dose-dependent in Swedish volunteers orally vaccinated with live S. flexneri 2a SFLI070. The optimal dose ofS. flexneri SFLI070 is 1 x 10 8 c.f.u., which combines immunogenicity in all volunteers with mild gastrointestinal side-effects in three o f nine volunteers only.

Keywords: Shiyella.flexneri; aro-deletion; auxotrophy

Shigellosis is still a major cause of diarrhoea morbidity and mortality, particularly in infants in developing

Karolinska Institute, Department of Immunology, Micro- biology, Pathology and Infectious Diseases, Division of Clinical Bacteriology, F:82 Huddinge Hospital, S-141 86 Huddinge, Sweden. *To whom correspondence should be addressed. (Received 27 January 1994; revised 13 May 1994; accepted 23 May 1994)

countries a. Several attempts have been made to develop vaccines protecting against shigellosis 2. We have previously constructed oral live S. flexneri Y and 2a vaccines by inactivating or deleting the aroD gene from virulent wild-type S. flexneri strains 3 5, thereby making the vaccine strains auxotropic for aromatic compounds not available in mammalian cells. These vaccines have been shown to have a reduced intracellular multiplication in cultured epithelial cells, to be unable to cause

88 Vaccine 1995 Volume 13 Number 1

keratoconjunctivitis in guinea-pigs, and to protect orally vaccinated monkeys against subsequent oral infection with virulent S. flexneri 3-8. The S. flexneri Y vaccine strain SFL124 with a deleted aroD gene was well tolerated by Swedish and Vietnamese volunteers in oral doses of 2 x 10 9 live bacteria and elicited systemic and intestinal immune responses against S. flexneri lipopoly- saccharide (LPS) and invasion plasmid antigens (Ipa) 9'1°. Memory mucosal immune functions were seen 12 months after primary vaccination 1°.

The parent strain of SFL124, strain S. flexneri SFL1, was only moderately virulent, causing dysentery in two of eight volunteers given 7x 103 live bacteria 11. We therefore also wanted to study the safety and immunogenicity of an auxotrophic vaccine strain derived from the highly virulent S. flexneri 2457T, which causes dysentery in a dose of 102 colony-forming units (c.f.u.) ~ 2.

Here we report on the first study using the aromatic-dependent S.flexneri 2a SFL 1070 with a deleted aroD gene as an oral live vaccine in humans. The dose-dependent side-effects, SFL1070 excretion and immune responses were studied in 37 adult Swedish volunteers vaccinated with S. flexneri SFL1070 in doses varying from 1 x 105 to 1 × l 0 9 live bacteria.

MATERIALS AND M E T H O D S

Vaccine strain

Shigellaflexneri 2a strain SFL1070 is the vaccine strain constructed by deletion of gene aroD from the wild-type parent strain S. flexneri 2457T 5. Strain SFL1070 has a reduced intracellular multiplication, is Ser6ny-negative 13 and shows a protective efficacy in monkeys against oral challenge with the wild-type strain 8. The vaccine was produced by the Vaccine Production Unit of the National Bacteriological Laboratory, Stockholm, Sweden, under GMP conditions as described for S. flexneri Y vaccine SFL124 ~°. The vaccine was lyophilized in 5 ml aliquots at a concentration of 2.9 x 10 9 c.f.u, ml- 1 and was stored at -20°C until used. The viable count 4 days after production was 0.8 x l 0 9 c.f.u, ml- 1 and at the time of the study 8-11 months later 0.5 x 10 9 c.f.u, m1-1. The viability and purity of the vaccine was checked on Congo red agar 14 before and after vaccination. Approximately 80% of the colonies were scored as Congo red-positive, which correlates with the invasive phenotype ~5.

Volunteers

Adult Swedish volunteers working at various labora- tory departments at the Huddinge Hospital, Huddinge, Sweden, were recruited on the basis of informed consent after receiving written and oral information about the study. Volunteers were excluded from participation if they had a history of (i) shigellosis; (ii) on-going or recent gastrointestinal disease; (iii) on-going or recent serious disease, e.g. cancer, coronary incompensation, metabolic disturbances etc.; (iv) hypersensitivity to antibiotics that possibly could be used for medical intervention; or (v) if they were pregnant. No physical examination was done prior to participation. Thirty-seven volunteers entered the study, of whom 29 were women and eight were men. The mean age was 35.6 (range 23-54) years. The study was approved by and conducted in accordance with the guidelines from the Ethical Research Committee at

Shigella flexneri 2a vaccine SFLI070: A. K~rnell et al.

Huddinge Hospital, Huddinge, Sweden and the Medical Products Agency, Uppsala, Sweden.

Vaccination schedule and study design

The volunteers were vaccinated orally with live S. flexneri SFL1070 three times, i.e. on days 0, 2 and 5. The volunteers in group 1 (n=9) received 1 x 105 Congo red-positive 14'~s c.f.u, of S. flexneri SFL1070 at each vaccination, the volunteers in group 2 (n= 10) 1 x 107 c.f.u, and those in group 3 (n=9) 1 x 108 c.f.u. The volunteers in group 4 (n =9) received 1 x 109 c.f.u, at each vaccination. Four of them received one dose only. The study was designed so that possible side-effects could be assessed before a higher vaccine dose was given to the next group of volunteers. The vaccination procedure was identical to that used when vaccinating Swedish and Vietnamese volunteers with S.flexneri Y SFL1249'1°, i.e. the volunteers ingested 2 g of NaHCO 3 tablets followed 10 min later by the vaccine strain suspended in 30 ml phosphate-buffered saline (PBS). The volunteers had no food 90min before and 90min after vaccination. Following vaccination the volunteers were seen daily by a physician. Serum, peripheral blood and faecal samples were collected before and at various intervals after vaccination.

Vaccine excretion detected by faecal cultures

Rectal swabs or quantitative faecal cultures were taken before vaccination and daily for 10 days after the third vaccination as before 1°. The vaccine strain S. flexneri SFL1070 was identified by biochemical and serological tests as described 1°. Growth of SFL1070 in quantitative cultures was expressed as c.f.u, g-1 faeces. Growth of SFL1070 in rectal swab cultures was considered to correspond to 10 2 c.f.u, g-1 faeces.

Antigen preparation

Lipopolysaccharides (LPSs) of S.flexneri Y, S.flexneri 2a, Escherichia coli CVD-172, Salmonella typhimurium and Shioella invasion plasmid antigens (Ipa) were the same as in previous investigations a-l°. Polysaccharides (PS) of S.flexneri 2a and E. coli CVD-172 were obtained by weak acid hydrolysis of phenol-water-extracted LPS and characterized as before 9.

Enzyme immune assay (EIA) to estimate serum immune responses

Enzyme immune assays (EIA) were used as before 1° to estimate serum IgA, IgG and IgM titres against S. flexneri 2a and Y LPS, Shigella invasion plasmid antigen (Ipa) and S. typhimurium LPS. The LPSs were coated at a concentration of 5 #g ml- 1. For anti-Ipa titres, water extracts from E. coli K12 SP10 (which harbours the invasion plasmid pWRll016 and therefore expresses Ipa 17) and from its plasmidless parent strain E. coli K12 J53, were coated at a protein concentration of 20/~g ml- 1 Alkaline phosphatase (ALP) conjugated goat anti-human IgA (diluted 1:1500), IgG (1:5000) or IgM (1:2000) (Mabtech, Stockholm, Sweden) were used as secondary antibodies. The absorbance was measured at 405nm using a Titertek Multiskan photometer (Flow Laboratories, UK). Relative titres were calculated by multiplying the absorbance by the serum dilution factor (1000). The

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anti-Ipa titres were obtained by deducting the J53 titres from the SP10 titres. Individual serum immune response following vaccination was defined as at least doubling of the prevaccination titre.

EIA to estimate intestinal immune responses

Fresh faecal samples were diluted 1:5 in a trypsin inhibition buffer 18, homogenized, centrifuged and filtered as before 9. EIA was used to estimate IgA and IgG titres against S. flexneri 2a and Y LPSs, Shigella Ipa and Salmonella typhimurium LPS. The coating doses were the same as in the serum EIA. The plates were blocked with 1% calf serum albumin in PBS for 1 h before the faecal extracts were added. ALP-conjugated goat anti-human IgA (diluted 1:1500) and IgG (1:5000) (Mabtech) horseradish peroxidase (HRP)-conjugated rabbit anti- human secretory component (diluted 1:3000) (Dakopatts a/s, Copenhagen, Denmark) were used as secondary antibodies. Plates with ALP conjugates were read at 405nm and plates with HRP conjugates at 492nm. Relative titres were calculated by multiplying the absorbance by the dilution factor (5). The anti-Ipa titres were obtained as for the serum EIA. Owing to wide day-to-day variation in estimation of faecal IgA titres, an individual intestinal immune response was defined as at least doubling of the prevaccination titre in at least three of seven faecal samples obtained from day 5 onwards following the first vaccine dose.

Preparation of peripheral blood mononuclear cells (PBMCs)

PBMCs were prepared as described in the previous investigation 1°.

Enzyme-linked immunospot assay (ELISPOT) to determine antibody-secreting cell (ASC) responses

The procedure and reagents of the ELISPOT assay were the same as in previous investigations 9'1°. LPSs of S. flexneri 2a and E. coli CVD-172 were coated at a concentration of 20 #g ml-1. Water extracts of E. coli K12 strains SP10 and J53 were coated at a protein concentration of 40 #g ml- 1 Volunteers who had at least five spots/well in at least one of the immunoglobulins IgA, IgG or IgM to the antigens in their postvaccination samples were considered responders.

ELISPOT to determine interferon-gamma (IFN-?) secreting T-cell responses

The assay was done as described 19 with minor modifications. Briefly, 100/~1 well- 1 of monoclonal

mouse anti-human IFN-7 (Genzyme, Cambridge, MA, USA) was coated to nitrocellulose-bottomed microtitre plates at a concentration of 12/~g protein ml-1 in PBS. After washing, 200 #1 of a suspension of PBMC (2 x 105 cells) was added to each well together with one of the following antigens or mitogen: polysaccharides of S. flexneri 2a and E. coli CVD-172 (30 #g), water-extracted proteins of E. coli K12 strains SP10 (3 #g) and J53 (3 #g), phytohaemagglutinin (10#1 of a 1:1000 diluted stock (WeUcome Diagnostics, Dartford, UK). To the controls neither antigen nor mitogen was added. The plates were incubated at 37°C in 7% CO2 in a humidified atmosphere for 40 h. After washing, 100#1 of rabbit anti-human IFN-7 immunoglobulins (Diagnostika, Falkenberg, Sweden) diluted 1:500 was added to each well. The plates were incubated at 4°C overnight, then washed, thereafter 100#1 of 1:1000 diluted biotinylated swine anti-rabbit immunoglobulins (Dakopatts a/s) was added. After 3 h incubation at room temperature, the plates were washed again and 100/~1 of preformed avidin-biotin horseradish peroxidase complex (Vector Laboratories, Burlingame, CA, USA) was added and incubated for 1 h. Spots representing IFN-7-secreting cells were visualized by adding carbazole-dimethylformamide substrate. The assay was performed on samples taken on days 0, 7, 9 and 21. Volunteers with at least twice as many IFN-7-secreting cells after stimulation by Shigella antigens in their postvaccination blood samples than in their prevaccination samples were considered responders. Only volunteers in groups 3 and 4 were tested.

Statistics

The Wilcoxon signed-rank test was used for intragroup comparisons of immune responses. The Mann-Whitney U test was used for intergroup comparisons of immune responses. A p value < 0.05 was considered significant.

RESULTS

Clinical symptoms after vaccination

S.flexneri SFL1070 was tolerated without side-effects by all volunteers in group 1, and by nine of ten volunteers in group 2 (Table 1). The one volunteer in group 2 with side-effects had three loose defaecations the day after the first vaccination.

In group 3, six of nine volunteers tolerated SFL1070 vaccination without any side-effects, whereas mild adverse reactions were seen in three volunteers after the first vaccination and in one volunteer after the second

Table 1 Clinical symptoms in adult Swedish volunteers orally vaccinated three times with S. flexneri SFL1070. Group 1 received 1 x 105, group 2 1 × 10 ~, group 3 1 x 108 and group 4 1 x 109 c.f.u, at each vaccination. Four volunteers in group 4 received the first dose only

Group 1: Group 2: Group 3: Group 4: Vaccinat ion no. Vaccinat ion no. Vaccinat ion no. Vaccine dose no.

1 2 3 1 2 3 1 2 3 1 2 3

Number of vo lunteers 9 9 9 10 10 10 9 9 9 9 5 5 No symptoms 9 9 9 9 10 10 6 8 8 4 5 5 Mild symptoms (%)a 0 0 0 1 (10) 0 0 3 (33) 1 (11) 1 (11) 1 (11) 0 0 Severe symptoms (%)b 0 0 0 0 0 0 0 0 0 4 (44) 0 0 Visible blood in faeces 0 0 0 0 0 0 0 0 0 0 0 0

"One or more of the following symptoms: mild stomach ache, 'noisy stomach', headache, diarrhoea with ~<5 defaecations/day bone or more of the following symptoms: abdominal pains, abdominal tenderness, diarrhoea with > 5 defaecations/day, fever

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Table 2 Culture-verified faecal excretion of vaccine S. flexneri SFL1070 from adult Swedish volunteers orally vaccinated on days 0, 2 and 5. Group 1 (n=9) received 1 x 105 c.f.u., group 2 (n=10) 1 x 107 c.f.u., group 3 (n=9) 1 x 108 c.f.u, and group 4 (n=9) 1 x 109 c.f.u, at each vaccination. Four volunteers in group 4 were vaccinated once only. The f igures denote number of excretors/number of volunteers cultured. The recovery of SFL1070, c.f.u, g-1 faeces, among excretors is given (range)

Day after the last vaccine dose

Group 4

Group 1 Group 2 Group 3 Vaccinated x 1 Vaccinated x 3 All

1 0/9 3/9 7/9 2/4 3/5 5/9 (102-3 x 105) (5 x 102-1 x 105) (102) (1 x 103-1 x 105) (102-1 x 105)

2 0/9 3/10 7/8 1/3 2/5 3/8 (1 x 103-2.5 x 103) (102-1.3 x 105) (102) (4x 104-5 x 104) (102-5 x 104)

3 0/9 0/10 4/9 0/1 3/5 3/6 (1 x 103-5.2 x 105) (1 x 103-3 x 103) (1 x 103-3 × 103)

4 0/9 0/5 1/9 1/3 0/5 1/8 (102 ) (1 x 103 ) (1 x 103 )

5-10 ND 0 0 0 0 0 Total 0/9 6/10 9/9 3/4 4/5 7/9

ND, not done

Table 3 Serum immune responses against S. flexneri LPSs, Shigella Ipa and S. typhimurium LPS as determined by using EIA in Swedish volunteers vaccinated orally three times with S. flexneri SFL1070 (four volunteers in group 4 were vaccinated once only). The f igures denote the number of volunteers showing at least doubling of the prevaccination titres and the total number of volunteers responding against either of the Shigella antigens and against S. typhimurium LPS

Shigella ant igens Salmonella typhimurium

S. flexneri 2a LPS S. flexneri Y LPS Shigella Ipa LPS Vaccine dose

Group (c.f.u.) n IgA IgG IgM IgA IgG IgM IgA IgG IgM Total IgA IgG IgM Total

1 1 x l 0 5 9 0 0 0 0 0 0 0 1 0 1 0 1 0 1 2 1 x 107 10 1 0 0 1 0 0 0 1 0 2 1 0 0 1 3 1 x l 0 8 9 4 2 1 2 1 0 1 1 1 4 0 0 0 0 4 1 x l 0 9 9 5 6 6 5 2 3 3 2 0 8 0 2 0 2

1 dose 4 2 4 3 2 1 2 1 1 0 4 0 1 0 1 3 doses 5 3 2 3 3 1 1 2 1 0 4 0 1 0 1

and the third vaccinations (Table 1). These volunteers had such varying symptoms as mild stomach ache and stomach discomfort lasting up to a few hours, and/or a single loose defaecation. All symptoms appeared within 24 h of vaccination.

In group 4, four of nine volunteers tolerated the vaccination without any side-effects. One volunteer had five loose, non-watery, defaecations within a 3 h period which started 47h after the first vaccine dose. Four volunteers developed more severe symptoms after the first vaccine dose: abdominal distension, slight stomach ache or 'noisy stomach' were the initial symptoms, starting 6-12h after vaccination. In varying degree followed abdominal cramps, colicky pains, continuous abdominal pain, abdominal tenderness and tenesmus which reached maximal intensity within 24 h after vaccination and thereafter gradually declined. Bouts of watery diarrhoea, varying in number from four to 11, occurred between 7 and 48 h after vaccination with a maximal intensity after 10-24 h. Flu-like muscle pains, shivering and a single bout of vomiting were other symptoms reported. In one volunteer the body temperature was elevated to 38.6°C for 12 h starting 20 h after vaccination. Two other volunteers felt feverish but did not measure their body temperature. One of the four volunteers stayed home from work for 2 days. All four volunteers were seen daily by a physician. Antibiotic intervention was considered but not implemented as the symptoms rapidly declined. A complete spontaneous recovery was seen within 60 h after vaccination in one

volunteer and within 72 h after vaccination in the other three volunteers. These four volunteers received only a single dose of SFL1070.

Excretion of the vaccine strain as detected with faecal cultures

The vaccine strain S. flexneri SFL1070 was excreted by 22 of 28 volunteers in groups 2 to 4, whereas no excretion was seen in group 1 (Table 2). The excretors in group 2 shed the vaccine strain for an average of 1.6 days. The excretion time was 2.6 days in both groups 3 and 4. It is notable that of the four volunteers with severe adverse reactions in group 4 only three excreted, and with an average of 1.8 days. One volunteer in each o f groups 3 and 4 excreted S. flexneri SFL1070 for 4 days (Table 2). The recovery rates of S.flexneri SFL 1070 varied from 10 2 to 1 x 10 6 c.f.u, g-1 faeces and were highest in group 3 (Table 2).

Serum immune responses

Serum immune responses correlated with the vaccine dose and were most common in group 4, followed, in decreasing frequency, by groups 3, 2 and 1 (Table 3). In group 4, significant IgA and IgG titre increases were seen against S. flexneri 2a and Y LPSs whereas the anti-Shigella Ipa titre increases were less pronounced (Figure 1C, D). The mean IgA titres against S. flexneri 2a LPS on days 0 and 14 were 170 (range 100-270) and 830 (110-2160). The mean IgG titres on the same days

Vaccine 1995 Volume 13 Number 1 91

Shige l la f lexner i2a vaccine SFL lO70 :A .K~rne l le ta l .

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Rgute 1 Relative serum IgA and IgG titres (mean +s.d.) against S. flexneri 2a and Y LPSs, Shigella Ipa and S. typhimurium LPS as determined with the use of EIA in Swedish volunteers vaccinated with S. flexneri SFL1070. (A) IgA titres in group 3 (n =9) vaccinated with 1 x 10 a c.f.u. SFL1070; (B) IgG titres in group 30; (C) IgA titres in group 4 (n=9) vaccinated with 1 x 109 c.f.u. SFL1070; (D) IgG titres in group 4. O, Anti-S. flexneri 2a LPS antibodies; O, anti-S, flexneri Y LPS antibodies; /k, anti-Shigella Ipa antibodies; rq, anti-S, typhimurium LPS antibodies. *, p <0.05 and **, p <0.01 compared with the prevaccination titre

were 210 (range 30-480) and 640 (50-1510). There was no difference in titres between volunteers who, because of adverse reactions, were vaccinated with a single dose and volunteers given three doses of 1 × 10 9 c.f.u. (data not shown). The only exception was a short-lasting IgA increase against Shigella Ipa on day 14 in the single-dose group (data not shown). Increased titres against the S. Flexneri LPS, though non-significant, were also seen in group 3 (Figure 1A, B). The mean IgA titre against S. flexneri 2a LPS on day 0 was 280 (range 200-390) and 550 (190-1810) on day 14. The mean IgG titres on the same days were 200 (range 40-680) and 350 (range 100-880), respectively. As in group 4, both the IgA and IgG titre curves for S.flexneri 2a and Y LPSs were almost identical (['igure 1). A slight titre increase against Shigella Ipa was seen whereas the anti-S, typhimurium LPS titre, as in group 4, remained low (Figure 1). The average serum

titres were unchanged in volunteers in groups 1 and 2 (data not shown). In both groups 3 and 4 the serum titres started to increase on day 7, peaked on day 14, and declined thereafter (Figure 1). The IgG titres declined the most slowly and 2 months after vaccination were still significantly elevated in group 4 against the homologous S. flexneri 2a LPS (Figure 1D). Altogether 15 volunteers showed IgA, IgG or IgM responses against one of the Shigella antigens (Table 3). IgA and IgG responses were equally common, with 11 responding volunteers each, whereas IgM responses were less frequent. Antibody responses against the homologous S. flexneri 2a LPS were most common, followed by responses against S. flexneri Y LPS and Shigella Ipa (Table 3). The numbers of responding volunteers were 12, nine and seven, respectively. Of the four volunteers with severe adverse reactions in group 4, all responded against S. flexneri 2a

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Shigel la f lexner i 2a vaccine SFLI070: A. K~rnell et al.

Table 4 Intestinal IgA responses against S. flexneri LPSs, Shigella Ipa and Salmonella typhimurium LPS as determined in faecal samples by using EIAs. The volunteers were vaccinated three times with S. flexneri SFL1070 (four volunteers in group 4 were vaccinated once only). The figures denote the number of responding volunteers seen when using goat anti-human secondary antibodies. Response is defined as at least doubling of the prevaccination titre in at least three faecal samples taken from day 5 onwards after the first vaccine dose

Shigella antigens

S. flexneri Salmonella Vaccine dose Shigella typhimurium

Group (c.f.u.) n 2a LPS Y LPS Ipa Total LPS

1 1 ×105 9 0 O 1 1 0 2 1 x 107 10 4 5 4 6 2 3 1 x l08 9 7 7 5 9 3 4 1 x 109 9 8 9 5 9 4

1 dose 4 4 4 4 4 3 3 doses 5 4 5 1 5 1

18 A

15

I

B . C

/

0 . . . . . . 0 7 14 21 28 7 14 21 28 7 14 21 28

Time (day)

Figure 2 Relative intestinal slgA titres (mean+s.d.) against S. flexneri 2a and Y LPSs, Shigella Ipa and S. typhimurium LPS as determined using EIA with goat anti-human IgA secondary antibodies in Swedish volunteers vaccinated with S. flexneri SFL1070. (A) Group 2 (n= 10) vaccinated with 1 x 107 c.f.u. SFL107O; (B) group 3 (n=9) vaccinated with 1 x 108 c.f.u. SFL1070; (C) group 4 (n=9) vaccinated with 1 x 109 c.f.u. SFL1070. slgA directed against ©, S. flexneri 2a LPS; 0, S. flexneri Y LPS; A, Shige/la Ipa; and [~, S. typhimurium LPS. *, p<0.05 and **, p<0.01 compared with the prevaccination titre

LPS, three against S. flexneri Y LPS and one against Shigella Ipa. Three volunteers (one each in groups 1, 2 and 4) developed IgG responses against Shigella Ipa but no responses at all against S. flexneri 2a and Y LPS. Anti-S. typhimurium LPS IgG responses were seen in four volunteers (Table 3), all of whom also responded against either of the Shi#ella antigens. The anti-S. typhimurium responses were low, with relative titres varying from 30 to 180.

Intestinal immune responses

IgA responses to either of the Shigella antigens were seen in all 18 volunteers in groups 3 and 4, in six of ten in group 2, but only in one of nine in group 1 (Table 4). IgA responses against S. flexneri 2a and Y LPSs were most common (Table 4). Significant (p < 0.01 to p < 0.05) and almost parallel IgA titre increases against these antigens were seen in groups 2, 3 and 4, whereas the IgA titres against Shigella Ipa and S. typhimurium LPS were lower (Figure 2). The mean IgA titres against the homologous S. flexneri 2a LPS increased between days

0 and 9 from 1.3 (range 0.8-2.8) to 1.7 (0.5-7.0) in group 1; 1.2 (0.5-2.0) to 4.4 (0.6-12.8) in group 2; 1.0 (0.6-2.1) to 5.4 (1.0-16.5) in group 3; 1.3 (0.5-2.5) to 10.9 (0.6-18.0) in group 4. In group 4, we observed that a single dose of 1 × 109 c.f.u. SFL1070 (i.e. volunteers with adverse reactions) elicited equal or higher titres than the three-dose schedule (Table 5). However, the small number of volunteers did not permit statistical comparisons.

The IgA titres peaked on days 7-9 and remained significantly elevated for a slightly longer period of time in group 4 than in the other groups (Figure 2). After 2 months the IgA titres had declined to prevaccination levels (Table5). Anti-Ipa responses were less common than anti-S, flexneri LPS responses (Table4) and only rarely were the titres significantly increased (Figure 2). It was noteworthy that anti-Ipa responses were seen in all four volunteers in group 4 who, because of severe adverse reactions, were given only a single dose of 1 x 109 c.f.u., but only in one of the five volunteers receiving three doses (Table 4). The relative anti-Shioella Ipa titre increases in the single-dose group on days 5, 9 and 14 were 6.4, 8.7 and 3.5 times higher, respectively, than in the three-dose group (Table5). Responses against the chemically

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Table 5 Intestinal IgA titres, mean (range), against S. flexneri LPSs, Shigella Ipa and S. typhimurium LPS in faecal samples from volunteers in group 4 vaccinated with 1 x 109 c.f.u.S, flexneri SFL1070. Four volunteers received a single dose and five were vaccinated three times. The titres were estimated by using EIA with goat anti-human secondary antibodies

S. flexneri 2a LPS S. flexneri Y LPS Shigella Ipa S. typhimurium LPS

Day 1 dose 3 doses 1 dose 3 doses 1 dose 3 doses 1 dose 3 doses

0 1.4 1.3 1.7 1.5 0.2 0.4 0.5 0.8 (0.7-2.5) (0.5-1.9) (0.8-2.6) (0.6-2.2) (0.0-0.4) (0.1-0.8) (0.5-0.7) (0.5-1.6)

5 9.7 7.7 11.1 11.9 2.9 0.9 1.5 0.9 (1.2-16.9) (1.7-12.6) (2.9-17.4) (3.2-16.3) (0.0-6.6) (0.0-2.3) (0.6-2.3) (0.6-1.3)

9 16.8 6.2 17.4 8.3 4.8 1.1 2.2 0.8 (15.8-18.0) (0.6-10.2) (15.4-20.2) (0.8-13.1) (1.5-7.7) (0.0-2.9) (0.5-3.7) (0.5-1.0)

14 7.7 11.1 8.2 12.6 1.4 0.8 0.9 1.0 (0.9-17.4) (0.6-17.9) ( 1.4-17.4) (0.7-19.8) (0.0-2.5) (0.0-1.4) (0.6-1.8) (0.5-1.7)

28 8.7 7.7 8.9 7.6 1.1 0.6 1.1 0.9 (0.6-16.7) (0.4-18.6) (0.9-16.7) (0.5-17.6) (0.1-2.6) (0.0-2.3) (0.5-1.8) (0.4-1.7)

56 2.7 2.2 1.8 1.9 0.4 0.5 0.7 0.8 (0.6-7.8) (0.9-4.5) (0.8-3.5) (1.3-3.0) (0.0-1.6) (0.0-1.1) (0.5-1.4) (0.5-1.3)

Table 6 ASC responses to S. flexneri 2a LPS, Shigella Ipa and E. coli CVD-172 LPS in peripheral blood from Swedish volunteers orally vaccinated three times with S. flexneri SFL1070 (four of the volunteers in group 4 were vaccinated once only). The figures denote the number of volunteers responding (>5 spots/well) with IgA-ASCs, IgG-ASCs, and IgM-ASCs to each of the antigens, and the total number of volunteers responding

Shigella antigens

S. flexneri 2a LPS Shigella Ipa E. coli CVD-172 LPS Vaccine dose

Group (c.f.u.) n IgA IgG IgM IgA IgG IgM Total IgA IgG IgM Total

1 1 ×105 9 0 0 0 0 0 0 0 0 0 0 0 2 1 x 107 10 5 2 1 1 1 0 5 0 0 0 0 3 1 x 108 9 8 5 5 3 2 0 8 1 0 0 1 4 1 x l 0 9 9 8 5 7 5 4 1 8 2 0 1 2

1 dose 4 3 3 3 3 3 1 3 1 0 1 1 3 doses 5 5 2 4 2 1 0 5 1 0 0 1

unrelated S. typhimurium LPS were both less frequent and of lesser magnitude than either of the responses against the Shioella antigens (Table4). The mean IgA titres against S. typhimurium LPS on day 9 varied from 0.8 (range 0.5-2.9) in group 1 to 1.4 (0.5-3.7) in group 4. The individuals showing anti-S, typhimuriurn LPS responses also responded to either of the Shigella antigens. No intestinal IgG responses were seen in any of the volunteers.

In the EIA for intestinal immune responses, higher numbers of responding volunteers and higher relative titre increases were seen when using goat anti-human than rabbit anti-human secretory component as secondary antibodies (data not shown).

ASC responses

No or very few ( < 5 spots/well) antibody-secreting cells (ASC) specific for the antigens tested were found in peripheral blood before vaccination. Following vaccin- ation, ASC responses ( > 5 spots/well) to Shigella antigens were seen in five of ten volunteers in group 2, in eight of nine of group 3 and in eight of nine in group 4 c.f.u, of S. flexneri SFL1070 (Table6). S. flexneri 2a LPS-specific ASC responses were more common than Shioella Ipa-specific ASC responses, and IgA responses predominated over IgG and IgM responses (Table 6). The ASCs were detectable on day 5, reached the highest number on day 7 and had declined to prevaccination

numbers on day 21 (Figure 3). The number of S.flexneri 2a LPS-specific IgA-ASCs was significantly (p<0.01 to p < 0.05) increased in groups 2, 3 and 4 (Figure 3A, B and D), whereas the number of Shigella Ipa-specific IgA-ASCs was significantly (p <0.01 to p <0.05) increased in group 4 only (Figure 3E). The number of ASCs correlated with the vaccine dose and was significantly higher in groups 3 and 4 than in group 2 (p <0.005) (Figure3). As seen on day 7, the mean number of S. flexneri 2a LPS-specific IgA-ASCs/10 s PBMCs was six (range 0-17) in group 2, 49 (4-110) in group 3 and 99 (4-183) in group 4. The mean number of Shigella Ipa-specific IgA-ASC/105 PBMCs was 0.5 (range 0-3), 2 (0-10) and 8 (3-27), respectively. Comparison of the responses between groups 3 and 4 indicated that only in the early stage after vaccination did group 4 have significantly stronger ACS responses to S. flexneri 2a LPS (p<0.05 for IgM on day 5) and Shigella Ipa (p < 0.05 for IgA on days 5 and 7; p < 0.05 for IgG on day 5). In group 4, there were no differences in the number of ASCs between the volunteers who, because of severe adverse reactions, received a single dose of 1 × l09 c.f.u, and the volunteers vaccinated three times (data not shown). The only non-responder in group 4 received a single dose which, however, caused abdominal pain and four loose defaecations. There were no or very weak responses (<5 spots/well) to the chemically unrelated E. coli CVD-172 LPS in all volunteers during the study (Table 6).

94 Vaccine 1995 Vo lume 13 Number 1

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Figure 3 Number of antibody-secreting cells (ASC) (mean+s.d.) specific to S. flexneri 2a LPS and Shigella Ipa as determined with use of ELISPOT in peripheral blood from Swedish volunteers vaccinated with S. flexneri SFL1070. The volunteers in group 2 (n= 10) were vaccinated with 1 x 107 c.f.u. SFL1070, group 3 (n=9) with 1 x 10 s c.f.u., and group 4 (n=9) with 1 x 109 c.f.u. SFL1070. ASC specific to (A) S. flexneri 2a LPS in group 2; (B) S. flexneri 2a LPS in group 3; (C) S. flexneri 2a LPS in group 4; (D) Shigella Ipa in group 3; and (E) Shigella Ipa in group 4. ~ , IgA-ASC; I , IgG-ASC; ~ , IgM-ASC. *, p<0.05 and **, p<0.01 compared with the prevaccination number

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IFN-?-secreting T-cell responses

Vaccination with S. flexneri SFL1070 elicited almost identical IFN-), T-cell responses in group 3 and in group 4 vaccinated (Figure4). All volunteers responded to stimulation of PBMCs with S.flexneri 2a polysaccharide

(PS) antigen. Five volunteers in each group responded to stimulation with Shigella Ipa, whereas three volunteers in group 3 and four in group 4 responded to stimulation with E. coli CVD-172 PS. The number of IFN-~-secreting T cells was significantly increased in both groups, in

Vaccine 1995 Vo lume 13 Number 1 95

Shige l la f lexner i 2a vaccine SFLI070: A. K~rnell et al.

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Figure 4 Number of IFN-7-secreting T cells (mean+s.d.) as determined with use of ELISPOT in peripheral blood from Swedish volunteers orally vaccinated with S. flexneri SFL1070. (A) Group 3 vaccinated with I x 10 a c.f.u. SFL1070; (B) group 4 vaccinated with 1 x 109 c.f.u. SFL1070. After stimulation of PBMCs with ~ , S. flexneri 2a PS; //., Shigella Ipa; I , E. coil CVD-172 PS. *, p<0.05 and **, p<0.01 compared with the prevaccination number

Table "/ Total number of S. flexneri SFL1070-vaccinated volunteers showing serum antibody responses, intestinal slgA responses, ASC responses and IFN-7-secreting T-cell responses to either of the Shigella antigens tested for

Vaccine dose Serum Intestinal IFN-3,-secreting Group (c.f.u.) n antibody slgA ASC T-cell Total

1 1 x 105 9 1 1 0 ND 2 2 1 x 107 10 2 6 5 ND 7 3 1 x l 0 8 9 4 9 8 9 9 4 1 x109 9 8 9 8 9 9

1 dose 4 4 4 3 4 4 3 doses 5 4 5 5 5 5

Total 15 25 21 18 27

N D , n o t d o n e

particular after stimulation with S. flexneri 2a PS, and peak values were seen on day 9 (Figure 4). Concerning the four volunteers with severe side-effects in group 4, all responded to S. flexneri 2a PS, three to Shigella Ipa and one to E. coli PS stimulation of PMBCs. One of these four volunteers responded to all three antigens. This was the same volunteer who was the only non-responder in the E L I S P O T assay for antibody-secreting cells. There was no difference in number of IFN-7-secreting T cells between volunteers who, because of severe adverse reactions, received a single dose of 1 x 10 9 c.f.u, and the volunteers vaccinated three times (data not shown).

Total immune responses

The number of volunteers showing immune responses to S.flexneri 2a LPS and PS, S.flexneri Y LPS or Shigella Ipa is summarized in Table 7. Regarding the humoral immune responses, slgA responses were the most common, followed by ASC and serum antibody responses. T-cell responses were seen in all volunteers tested. In total, immune responses were seen in all volunteers vaccinated with 1 x 10 s and 1 x 10 9 c.f.u., in seven of ten vaccinated with 1 × 107 c.f.u., but only in two of nine of the volunteers vaccinated with 1 x 105 c.f.u, of S. flexneri SFL1070.

D I S C U S S I O N

The S.flexneri 2a vaccine strain SFL1070 with a deleted aroD gene 5 has a reduced ability for intracellular replication s and is Ser6ny-negative 5'13. S. flexneri SFL 1070 was safe, immunogenic and showed a significant protective efficacy in Macaca fascicularis monkeys vaccinated with 1 × 1011 live bacteria s. The purpose of this first study in humans was to evaluate the safety and immunogenicity of SFL1070 when given orally in doses varying from 1 x 105 to 1 x 10 9 live bacteria. S. flexneri SFL1070 was tolerated without side-effects by all volunteers vaccinated with 1 x 105 c.f.u., whereas one of ten volunteers given 1 x 10 v and three of nine vaccinated with 1 x 10 s c.f.u, developed mild and harmless gastrointestinal symptoms after the first vaccine dose. The highest dose, 1× 10 9 c.f.u., was tolerated without side-effects by four of nine volunteers. Mild and short-lasting diarrhoea was seen in one volunteer, whereas four volunteers experienced abdominal cramps, diarrhoea and fever as the most prominent symptoms. Noticeable dysentery, defined as loose stools containing visible blood, was not seen in any of these individuals and excretion of SFL1070 was neither enhanced nor prolonged. In fact, S. flexneri SFL1070 could be recovered in stool samples from only three of these

96 Vacc ine 1995 V o l u m e 13 Numbe r 1

volunteers, all of whom recovered spontaneously within 72 h of vaccination. However, because of the adverse effects these volunteers received only a single dose.

The attenuated character of S. flexneri SFL1070 was also seen as a dose-dependent and short excretion time, 4 days at the most, which is similar to the excretion times of S. flexneri SFL1070 in monkeys and of the previously tested aromatic-dependent S. flexneri Y vaccine strains S F L l l 4 and SFL124 in monkeys and human volunteers 4'7'9-11. We conclude that S. flexneri SFL1070 is at least 100000-fold attenuated compared with its virulent parent strain S. flexneri 2457T, which in experimental infection caused illness in doses of 102 bacteria 12. The attenuation is thereby equal to or greater than that of S. flexneri SFL124 with a deleted aroD gene x 1. However, volunteers vaccinated with 1 x l09 c.f.u. of S. flexneri SFL1070 developed adverse effects which, although self-limiting, were too severe. They were more frequent and more pronounced than those seen in Swedish volunteers vaccinated with 2 x 109 c.f.u, of S. )qexneri SFL124, presumably because the parent strain of vaccine SFL1070 is more virulent than that of vaccine SFL1248-12. Dose-dependent adverse reactions were also demonstrated in volunteers vaccinated with a live oral E. coli-S, flexneri 2a hybrid strain with a deleted aroD gene, which caused dysentery in doses of 1.8 × 101° c.f.u. 2°. However, S. flexneri SFL1070 did not cause dysentery in any of the volunteers. Dysentery was seen in none of the volunteers vaccinated with the aromatic- dependent S. flexneri Y vaccine strains SFLII4 (50 Vietnamese and five Swedes vaccinated with 1 x 109 c.f.u, and 24 Vietnamese vaccinated with 1 x 101° c.f.u.), or SFL124 (ten Vietnamese vaccinated with 1 x 109 and 30 with 2 x 109 c.f.u., and 21 Swedes vaccinated with 2 x 109 c.f.u.) 9-11'21.

S. flexneri SFL1070 elicited dose-dependent humoral immune responses against Shigella antigens in all volunteers vaccinated with 1 x 108 and 1 x 109 c.f.u., in seven of ten vaccinated with 1 x 107 c.f.u., but only in two of nine volunteers vaccinated with 1 x l0 s c.f.u. (Table 7). Intestinal slgA responses were most common, followed by ASC and serum antibody responses. The numbers of responding volunteers were 25, 21 and 15, respectively. Significant slgA increases against S.flexneri LPS antigens were elicited in volunteers given ~> 1 x 107 c.f.u.: all volunteers in the 1 × 109 group and all but one in the 1 x 10 a group responded to S.flexneri LPSs. slgA responses against Shigella Ipa were less pronounced, something also observed in volunteers vaccinated with S. flexneri SFL1249'1°. A non-specific slgA increase against S. typhimurium LPS, which reached a significant level in the 1 × 10 a group, was seen. However, the number of responding volunteers was less than half the number of Shigella LPS responders (Table4). The finding of almost identical dose-response curves when using the S. flexneri Y and the homologous 2a LPS as antigens (Figure 2) is not surprising, since the O-antigens of all S. flexneri, except serotype 6, have in common a repeating tetrasaccharide unit 22. S. flexneri Y has only the basic backbone O-antigen structure, whereas the serotype specificity of S. flexneri 2a is attributed to the addition of one a-l,3-1inked D-glucose to L-rhamnose z2. We cannot explain why the goat anti-human IgA antibodies were more sensitive than rabbit anti-human secretory component antibodies for detection of intestinal antibodies. It could be argued that the latter should

Shigella flexneri 2a vaccine SFL I070: A. K~rnell et al.

determine secretory antibodies better. Nevertheless, we presume that the responses seen with the goat anti-human IgA represent slgA originating from the intestine and not serum antibodies leaked to the gut. Such a leakage would also result in increased intestinal titres of IgG antibodies, which were not seen in any of the volunteers. Furthermore, the number of volunteers showing intestinal IgA responses was higher in all groups than the number of volunteers with serum IgA responses.

The ability of S. flexneri SFL1070 to elicit intestinal lymphoid tissue reactivity was also seen as significantly increased numbers of S. flexneri 2a LPS-specific, primarily IgA-secreting cells in volunteers vaccinated with >1 1 x 107 c.f.u. All but one volunteer in each of the 1 x 108 and the 1 × 109 groups developed ASC responses (Table 6) and the number of ASCs in these groups was significantly higher (p <0.005) than in the group given 1 x 107 c.f.u. (Figure3). In the studies of S. flexneri SFL124 we showed that three doses of 2 x 109 c.f.u. elicited significant ASC responses specific for the homologous S. flexneri Y LPS antigen. Here we show that vaccination with three doses of 1 x 107 c.f.u, of S. flexneri SFL1070 were sufficient to elicit anti-S.flexneri 2a specific ASC responses of the same 1° or nearly the same 9 magnitude. When given in comparable doses, l x 109 c.f.u, of S.flexneri SFL1070 elicits a considerably higher number of ASCs than did 2 x 109 c.f.u, of S.flexneri SFL124. The mean(s.d.) number of IgA-ASCs specific for S.flexneri 2a LPS was 99(_+ 69)/105 PBMCs 1 week after the first vaccination with S. flexneri SFL1070. The corresponding number of IgA-ASCs specific for S. flexneri Y LPS was 19(_+ 7)/105 PBMCs in Swedish 9 and. eight(+4)/105 PBMCs in Vietnamese 1° volunteers vaccinated with S. flexneri SFL124. The capacity of S. flexneri SFL1070 to elicit anti-Shigella Ipa ASC responses was, however, not superior to that of S.flexneri SFL124 in Vietnamese volunteers 1°. Anti-Ipa ASC responses were not analysed in SFL124-vaccinated Swedes. Significantly increased anti-Ipa ASC numbers were only seen in volunteers vaccinated with 1 x 109 c.f.u. S. flexneri SFL1070, whereas lower and non-significant increases were seen in the 1 x 108 group (Figure 3). Others have also demonstrated that ASC responses specific for S. flexneri 2a LPS exceed the anti-Shigella Ipa responses 2°. Although gut-derived ASCs, as part of the intestinal immune responses 23, are demonstrated in peripheral blood after natural Shigella infection 24, their role in the protective immunity against shigellosis is unclear. Vaccination with 1.2 x 109 to 2.5 x 109 c.f.u, of the live oral E. coli-S, flexneri 2a hybrid strain with a deleted aroD gene elicited an IgA-ASC response against S.flexneri 3a LPS in 97% of the volunteers and still 30% of the volunteers fell ill after challenge infection 2°. Similarly, S. sonnei O-antigen-specific IgA-ASC responses elicited by a bivalent Salmonella typhi-Shigella sonnei vaccine strain did not correlate with protection of vaccinees against challenge infection with S. sonnei 25. Whether the difference was caused by the lower magnitude of the ASC responses elicited by vaccination than by infection with wild-type bacteria was not clear. Neither is it known which of the anti-LPS and anti-Ipa ASC responses are most important for protective immunity. Our study of S. flexneri SFL1070 did not address protective immunity. However, we observed that anti-Ipa ASC responses were less prominent than anti-LPS ASC responses following vaccination with S.

Vaccine 1995 Volume 13 Number 1 97

Shigel la f lexner i 2a vacc ine SFLI070: A. K~rne l l et al.

flexneri SFL1070. In contrast, anti-Ipa ASC responses were as common as anti-LPS ASC responses in Vietnamese volunteers vaccinated with S. flexneri SFL124, presumably because of the previous exposure(s) of the volunteers to shigellae in this endemic area 1°.

S. flexneri SFL1070 elicited dose-dependent Shigella- specific serum antibody responses. For every tenfold increase in vaccine dose a doubling of the number of responding volunteers was seen, from one in the 1 × 105 group to eight in the 1 x 109 group (Table 3). Significant serum IgA responses against S. flexneri LPS antigens were seen in the I x 10 9 group only. This should be compared with the significant slgA and IgA-ASC responses against the same antigens in volunteers vaccinated with >/1 x 107 c.f.u. Only four volunteers vaccinated with 1 x 10 a c.f.u, developed serum antibody responses, whereas six and five vaccinated with 1 x 10 7

c.f.u, developed slgA and ASC responses, respectively. This shows that at least a tenfold higher dose of S.flexneri SFL1070, i,e. 1 × 10 s c.f.u., was required to elicit serum antibody responses than to elicit slgA and ASC responses in comparable numbers of volunteers (Table 7). Antibody responses specific for the homologous S. flexneri 2a LPS and S.flexneri Y LPS were more common than anti-lpa responses, which also was the case with the slgA and ASC responses (Table3, Figure 1). The finding of excellent intestinal immune responses combined with less pronounced serum antibody responses in this study is in agreement with our observations in M. fascicularis monkeys vaccinated with S. flexneri SFL1070 s. Vaccin- ated monkeys were protected against challenge infection, which strongly suggests that local immune response is a key factor in the acquired host defence against shigellosis s. Other studies have suggested that serum antibodies are not protective per se but rather mirror previous exposure to shigeUae 26-2s.

In addition to the humoral immune responses, significantly increased numbers of IFN-?-secreting T cells were seen following vaccination with 1 × 108 and 1 x 10 9

c.f.u, of S. flexneri SFL1070 (Figure4). All volunteers responded after stimulation of PBMCs with S. flexneri 2a PS and there was no significant difference between the numbers of IFN-7-secreting T cells in the two groups. The finding of significant, though lower, responses after stimulation with Shigella Ipa shows that the T-cell responses were not restricted to stimulation with the polysaccharide antigen only (Figure 4). E. coli CVD-172, an enteroaggregative E. coli (EAggEC), was chosen as a non-Shigella control in this assay because its O-antigen has no structural elements and sugars in common with the O-antigen of shigellae (A. Weintraub, Karolinska Institute, personal communication). We saw significant increases in numbers of IFN-7-secreting T cells after stimulation with E. coli CVD-172 PS, which were lower than after stimulation with the S.flexneri 2a PS (Figure 4).

The cellular immune mechanisms in shigellosis are poorly understood. However, by using a peripheral mononuclear cell proliferation test we have previously demonstrated cell-mediated immune responses in Swedes vaccinated with S. flexneri SFL1249. Studies of rectal biopsies from patients with dysentery caused by S. dysenteriae type 1 or S. flexneri indicated a strong cell-mediated immune response during acute shigellosis 29. The IFN-? production seen following vaccination with S.flexneri SFL1070 is a strong indicator of elicitation of cellular immune mechanisms. Human intraepithelial

lymphocytes (IEL) produce IFN-y TM and interferons have been shown in vitro to suppress the intracellular multiplication of shigellae 31. IFN-y and IFN-~t have also been shown in vitro to inhibit epithelial cell invasion by Shigella spp. 32. Others have shown that IFN-fl is produced by fibroblasts in response to invasion by S. flexneri 33. In this study we focused on determining IFN-7-secreting T cells. However, it is unlikely that the cytokine release elicited by vaccination with S. flexneri SFL1070 is restricted to IFN-y only. Increased levels of interleukin (IL-6) and tumour necrosis factor (TNF) were found in serum and stools from S. dysenteriae type 1-infected children 34. Our studies have demonstrated positive staining for IL-lfl, IL-6 and TNF in rectal biopsies from patients with Shigella dysentery (R. Raqib and D. Islam, Karolinska Institute, personal communication).

LPSs of shigeUae and other Gram-negative bacteria cause a wide variety of biological activities 35, including damage to human vascular endothelial cells in Shigella-infected patients 36. Considering that LPS triggers the expression of IL-I~, IL-lfl~ and TNFc~ in macrophages a7, we surmise that the adverse reactions seen following vaccination with 1 x 10 9 c.f.u.S, flexneri SFL1070 can be attributed to cytokine release and subsequent inflammatory reactions induced by invasion of the shigellae. However, administration of S. flexneri SFL1070 in a dose of I x l08 c.f.u, was well tolerated by all volunteers and elicited the same IFN-? T-cell response as a dose of 1 x 109 c.f.u. (Figure 4).

In this study we have demonstrated a narrow safety- immunogenicity profile of S. flexneri SFL1070 in adult Swedish volunteers. The 1 x 108 c.f.u, dose was safe and immunogenic in all volunteers. The 1 × 10 v c.f.u, dose was safe but less immunogenic. The I x 10 9 c.f.u, dose was immunogenic in all volunteers but caused adverse effects. It was shown that the immune responses in four volunteers who, because of severe adverse reactions, received only a single dose of 1 x 10 9 c.f.u, were similar to those seen in five volunteers given three doses. It remains to be established if 1 x 108 c.f.u, of S. flexneri SFL1070 is an optimal dose in adults, and also if a single dose is as immunogenic as three doses. There is also a need to study the safety-immunogenicity profile of S. flexneri SFL1070 in the target population for Shigella vaccine(s), i.e. in small children in the Third World.

At present there is no established correlation between immune response and protection against shigellosis. With further studies of aromatic-dependent S.flexneri vaccines we hope to address this question.

A C K N O W L E D G E M E N T S

This study was supported by the Swedish Medical Research Council (grant no. 16X-656), the Swedish Agency for Research Cooperation with Developing Countries and Stiftelsen Sigurd and Elsa Goljes minne.

R E F E R E N C E S

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2 Lindberg, A.A. and P&I, T. Strategies for development of potential candidate Shigella vaccines. Vaccine 1993, 11, 168-179

98 Vaccine 1995 Vo lume 13 Number 1

3 Lindberg, A.A., K&rnell, A., P&I, T., Sweiha, H., Hultenby, K. and Stocker, B.A.D. Construction of an auxotrophic Shigella flexneri strain for use as a live vaccine. Microb. Pathog. 1990, 8, 433-440

4 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, 10, 389-394

5 Verma, N.K. and Lindberg, A.A. Construction of aromatic dependent Shigella flexneri 2a live vaccine candidate strains: deletion mutations in the aroA and the aroD genes. Vaccine 1991, 9, 6-9

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11 Lindberg, A.A., K&rnell, A. and Stocker, B.A.D. Aromatic-dependent Shigella strains as oral live vaccines. In: New Generation Vaccines. Vaccines against Shigella Part II (Eds Woodrow, G.C. and Levine, M.M.) Marcel Dekker, New York, 1990, pp, 677687

12 DuPont, H.L, Levine, M.M., Hornick, R.B. and Formal, S.B. Inoculum size in shigellosis and implications for expected mode of transmission. J. Infect. Dis. 1989, 159, 1126-1128

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14 Payne, S.M. and Finkelstein, R.A. Detection and differentiation of iron-responsive avirulent mutants on Congo red agar. Infect. Immun. 1977, 18, 94-98

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16 Sansonnetti, P.J., Kopecko, D.J. and Formal, S.B. Involvement of a plasmid in the invasive ability of Shigella flexneri. Infect. Immun. 1982, 35, 852-860

17 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, 53, 57-63

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