Safety and immunogenicity of the oral E. coil K12-S. flexneri 2a vaccine (EcSf2a-2) among Israeli soldiers

Dani Cohen*;, Shai Ashkenazi*, Manfred S. Green*, Miri Yavzori*, Nadav Orr*, Raphael Slepon*, Yehuda Lerman*, Guy Robin*, Ruhama Ambar*, Colin Block*, David N. Taylor t, Thomas L. Hale ~, Jerald C. Sadoff* and Michael Wiener*

A double-blindplacebo-controlled study was carried out on the sa/'e O, and immunogeniciO~ ()/" the oral Shigella flexneri (EcSj2a-2) vaccine among Israeli soldiers. SixO~ volunteers received the vaccine and 59 received placebo. Fifty-three were given the full vaccine regimen (/bur doses). Doses ranged between 4.1 × 10 ~' and 1.1 × 109 c:#.u. Visits to the unit clinic

jor mild gastrointestinal o'mptoms were common after the first dose in vaecinees (13%) as compared with placebo recipients (5%), but the difference was not significant, p =- 0.12. SimilarO,, there was no d(fference between the groups ./or either gastrointestinal or non-gastrointestinal complaints reported by questionnaire. The vaccine strain was excreted by 69% and 67% (~/the vaccinees one day alter receiving the second and the Jourth doses, respective@. As judged by antibiotic susceptibilio', phage typing and restriction./ragment length po@morphism (RFLP), the vaccine strain emerged as genetically stable aJier replication in human gut and shedding. There was neither bacteriological nor serological evidence o/" transmission o# the vaccine from vaccinees to placebo recipients. Eighteen q# 26 (69.2%) and 11 0.['30 (36.7%) vaccinees had significant lgA secreting cell responses 7 and 21 days qfter the .first dose, respectively. Significant IgA or IgG serum antibody response to S. flexneri 2a LPS was detected in 30% ~['the vaccinees. These results support ./urther evaluation (if EcSJ2a-2 vaccine protective ~f~'ca~ T in field studies.

Keywords: Oral immunization: Shigelhtflexneri: EcSf2a-2 hybrid vaccinc

Israel is highly endemic for shigellosis, having a reported incidence of disease about 20-30 times higher than that in the USA 1. Young children of nursery school age and soldiers serving under field conditions appear to be particularly vulnerable to the disease 2 4. The very low infectious dose of Shiyella, about 102 103 organisms 5, facilitates transmission of the pathogen, and explains the partial failure of routine sanitary and hygiene measures to prevent the sporadic and epidemic occurrence of shigellosis among these populations 3'4. The use of antibiotic treatment in shigellosis is becoming more and more problematic in Israel as clinical isolates are becoming increasingly resistant to antimicrobial agents 6"7. Vaccination of high-risk groups with an effective Shigella vaccine may offer a means of significantly reducing the attack rate of the disease.

*Medical Corps, Israel Defence Force, Israel. +Walter Reed Army Institute of Research, Washington, DC, USA. ++To whom correspondence should be addressed at: Military Post 02149, Medical Corps, Israel Defence Force, Israel. (Received 3 December 1993; revised 18 March 1994; accepted 7 April 1994)

0264-410x/94/15/1436-07 1994 Butterworth-Heinemann Ltd

1436 Vaccine 1994 Volume 12 Number 15

Advances in the understanding of the molecular basis of Shigella pathogenesis have shifted the strategies of Shigella vaccine development from killed whole- cell vaccines 8"9 or spontaneously attenuated Shigella strains 1°'11 towards live, attenuated hybrid Shigella vaccines~2 14. The EcSf2a-2 strain represents a second generation of Escherichia coli carrier vaccine strains containing the invasiveness plasmid of Shi~tella flexneri as well as the chromosomal genes encoding the S.flexneri 2a LPS ~4'15. The aim of the present study was to assess the acceptability of this vaccine in terms of safety and immunogenicity among young soldiers in a region where shigellosis is endemic. We also evaluated the extent and duration of excretion of the vaccine strain, its infectivity (potential transmission from vaccinees to placebo recipients) and its genetic stability.

M A T E R I A L S A N D M E T H O D S

Preparation of the vaccine and placebo

The vaccine was constructed at the Walter Reed Army Institute of Research (WRAIR) by conjugal transfer of the 140 MDa invasiveness plasmid of S.flexneri 5 (strain M90T) into an E. coli K-12 recipient (strain 395-1,

Safety and immunogenicity of EcSf2a-2 oral vaccine: D. Cohen et al.

lactose-negative, streptomycin-resistant) as previously described 14. Subsequent conjugal transfer from the S. .flexneri 2a Hfr donor (strain 256) of the his, pro and arg chromosomal markers in separate matings allowed expression of the S. flexneri 2a LPS surface antigen and stabilized expression of the plasmid-encoded invasiveness phenotype. A further attenuation of the vaccine strain was obtained by PI transduction of a tetracycline- resistance transposon (Tnl0)-inactivated aroD gene followed by selection of a tetracycline-sensitive strain with a growth requirement for p-aminobenzoic acid, a precursor of folic acid not available in mammalian cells 15. A single lot (dated 5-1-90) of fermentor-grown lyophilized vaccine mixed with a dextran sucrose glycerol stabilizer was prepared by the Salk Institute in Swiftwater, PA, USA. The vaccine was stored at -70°C in sealed vials containing 4.0 x 10 l° viable organisms until used in the present study. The placebo was also formulated at the Saik Institute as a lyophilized preparation of phosphate- buffered saline (PBS) with 1% tryptone and containing 7.5% dextran 10, 2% sucrose, 0.75% glycerol, and Dow A (an antifoaming agent, 2 ml per 140 l).

Prevaccination testing of vaccine or placebo

Three vials of EcSf2a-2 vaccine were reconstituted 2 days prior to immunization and colony counts, binding of Congo red and slide agglutination with appropriate antisera were carried out. Concomitantly, the placebo was reconstituted and tested for sterility.

Administration of vaccine or placebo

Volunteers were asked not to eat or drink for 90 min before and after ingestion of vaccine. They were each instructed to drink 120ml of a 1.3% (w/v) solution of NaHCO 3. After one minute, the vaccine was administered orally, suspended in 30ml of the same solution. The desired vaccination dose was 5 x 108 c.f.u. In order to reach this dosage, while controlling for variability between ampoules, the averaged result of previous plate counts was used to adjust the dilution of reconstituted vaccine.

Design of clinical study

The study group comprised male (96%) and female (4%) soldiers who agreed voluntarily to receive four separate doses of the oral, attenuated Shigella vaccine (EcSf2a-2) or placebo. The age of 82% of the volunteers was 18-19 years while the age of the remaining 18% ranged between 19 and 45 years. Informed, witnessed, written consent was obtained after all aspects of the protocol had been explained in depth to the volunteers. The study was approved by the Surgeon General's Human Subjects Research Review Board of the Department of the US Army and by the Human Subjects Research Committee of the Israel Defence Force Medical Corps. The military units in which the volunteers served for the 28-day follow-up were not field units so the risk of natural exposure to Shigella organisms was low. Volunteers underwent a general physical examination and 119 soldiers fulfilling the enrolment criteria were randomly divided into vaccine and placebo groups. Before receiving subsequent vaccine or placebo doses, on days 3, 14 and 17, volunteers were interviewed to verify that they met the criteria for continuing participation in

the study. The subjects kept a symptom record and measured their oral temperature every day for a period of 28 days following the first vaccination. They were required to visit the study medical orderly or physician during this time to report any unusual symptoms.

Bacteriology

In order to attempt recovery of the vaccine strain from the stools of the volunteers after doses 2 and 4, postdefaecation faecal swabs were collected in Car~Blair transport medium. The samples were plated onto MacConkey agar containing streptomycin and incubated for 24 h at 37°C. Lactose non-fermenting colonies were agglutinated with the appropriate S. flexneri antisera (group B antisera). In addition to the special emphasis on the isolation of Shigella, the bacteriological work-up of the stool samples obtained from volunteers complaining of gastrointestinal symptoms during the 28-day follow-up period included identification of Salmonella, Campylobacter, enterotoxigenic E. coli, Aeromonas and Plesiomonas.

Genetic stability

Restriction fragment length polymorphism (RFLP). RFLP analysis of the vaccine strains was carried out as previously reported 16. Briefly, about 2pg of purified chromosomal DNA was digested with restriction endonucleases (EcoRI, Sinai, KpnI, PVulI) (International Biotechnologies Inc., New Haven, USA). The restriction fragments were separated by electrophoresis in 0.8% agarose gel, transferred to Genescreen Plus membranes (DuPont, New England Nuclear, USA) and hybridized. The 32p-labelled 2.5kb EcoRI-HindlII restriction fragment of the pKK3535 plasmid, encoding the 16S rRNA and part of the 23S rRNA, was used as a probe. Lambda DNA digested with HindlII was used as a molecular mass marker on each gel, The hybridization banding patterns of EcSf2a-2 strains before and after shedding were analysed.

Antimicrobial susceptibility (AS). The EcSf2a-2 vaccine strains (before and after shedding) were tested for susceptibility to ampicillin, cotrimoxazole (trimethoprim- sulfamethoxazole), tetracycline, nalidixic acid, norfloxacin, chloramphenicol and cefixime by the Kirby Bauer disc diffusion method 17 (antibiotic discs from Difco, Detroit, MI, USA).

Phage-typing. Vaccine strains plated from the original vials or isolated from stool specimens obtained from volunteers after vaccine doses 2 and 4 were examined for their susceptibility to bacteriophages active on Shigella and E. coli as described previously 18.

Immunology

Detection of serum antibodies. ELISA was performed as previously described 19. Briefly, 100/A of 5 pg ml- 1 S. flexneri 2a LPS in coating buffer (0.05 M carbonate buffer, pH 9.6) were added to each of 96 wells of microtitration plates (Costar, Cambridge, MA, USA) and incubated for 1 h at 37°C. After removal of the coating solution, the plates were incubated for 1 h at 37°C with 0.05M PBS-test buffer supplemented with casein and bovine serum albumin (both 5 g 1-1) and then washed twice in

Vaccine 1994 Volume 12 Number 15 1437

Safety and immunogenicity of EcSf2a-2 oral vaccine: D. Cohen et al.

PBS-Tween 20 washing solution. Sera were added to the wells in twofold dilutions in PBS-test buffer, starting with 1: 50, and incubated overnight at room temperature. After four further washings, goat anti-human IgG, or anti-IgA, conjugated to alkaline phosphatase (Kirkegaard and Perry Laboratories, MD, USA) and diluted 1:500, was added to the wells and incubated overnight at room temperature. The ELISA was completed sequentially by addition of the enzyme substrate solution, paranitro- phenylphosphate (1 mg ml -~) in diethanolamine buffer at pH 9.8, and 3 M NaOH. Absorbance (A) was read at 405nm using an automatic ELISA reader (EL-340, Bio-Tek, USA). The results were expressed as adjusted A calculated from a linear regression analysis of eight doubling dilutions. The antibody response was determined from the ratio (peak A on postvaccination days 7, 14, 21)/(A on day 0), with peak A>~0.15 (IgA) or 0.25 (lgG) at a dilution of 1:100. A significant antibody response was defined as a rise in A higher than the mean rise in A + 2 s.d. found in placebo recipients. The cut-off values for a significant rise were > 2.43 for IgA and > 1.78 for IgG. Control sera were included in every microtitration plate for each of the assays.

Detection of antibody-secreting cells (ASCs)

Mononuclear cells containing mainly lymphocytes were obtained by centrifugation on Ficoll-Hypaque of venous blood taken in EDTA-coated vacuum tubes. Isolated cells were washed three times and then resuspended in culture medium and adjusted to a concentration of 2 x 10 6 ceilsm1-1. The solid-phase enzyme linked immunospot (ELISpot) assay was employed for enumeration of ASCs z° specific to LPS extracted by the phenol-water method 21 from an epidemic strain of S..flexneri.

Statistical analysis

Statistical analysis was performed using the SAS software for PC version 6.04 and included contingency table Z 2 tests, two-tailed Fisher's exact tests and Z 2 tests for trend to detect differences in side-effect rates between the two treatment groups. The immune response in terms of rate of rise in serum antibodies and in number of antibody-secreting cells was analysed by means of )t2 or

Fisher's exact tests. Student's t test was used for evaluation of differences in mean A.

R E S U L T S

Safety of the EeSf2a-2 vaccine

Evaluation of the reactogenicity of the vaccine was based on gastrointestinal (GI) or other symptoms reported by volunteers on the 28-day symptom card, and on the findings of the physician's examination recorded at clinic visits.

Between 0 and 2 days after the first dose, eight of 60 vaccinees (13%) and three of 59 placebo recipients (5%) visited the unit clinic because of mild GI symptoms (p=0.12). One additional placebo recipient visited the unit clinic with similar mild GI complaints one day after the third dose. In none of these volunteers did the (31 complaints interfere with their daily activities or otherwise incapacitate them.

No significant difference in the occurrence of any reported GI or non-GI symptom, following the four doses of vaccine or placebo, was detected between vaccinees and placebo recipients (Table 1).

Abdominal cramps or gurgling, headache, malaise and loss of appetite were the most common symptoms reported by the volunteers in questionnaires. The rates of these symptoms were the highest after dose 1 and decreased at doses 2, 3 and 4. This trend reached statistical significance for all four complaints in vaccinees while only the rate of abdominal cramps or gurgling dropped significantly in placebo recipients (Table 1).

Excretion of the vaccine strain

We examined the extent of vaccine excretion in order to assess the vaccine capacity to replicate in t:ico and to confirm the presumed lack of transmission of the vaccine strain between vaccine and placebo recipients. This was done on stool samples obtained 1~5 days after doses 2 and 4. Peak excretion was found 1 day after administration of the second or fourth doses, when 69% and 67% respectively of the vaccinees examined excreted the vaccine strain (Table 2). Fewer vaccinees excreted the vaccine for 2 days after vaccination and none of them for more than 3 days. The vaccine strain was not detected

Table 1 Rate (%) of symptoms reported by 60 vaccinees and 59 placebo recipients within 3 days of each vaccine dose

Dose 1 Dose 2 Dose 3 Dose 4

Vaccine Placebo Vaccine Placebo Vaccine Placebo Vaccine Placebo

Loose stools 7 5 Diarrhoea 5 2

Abdominal cramps or gurgl ing" 39 25 Vomit ing 3 2 Malaise ~ 37 25 Loss of appetite c 25 24 Headache d 37 22 Fever ( temperature >~37.6' C) 5 0

Visits to unit clinic 13 5

7 5 9 7 9 11 5 2 2 4 4 2

17 17 11 15 11 6 0 3 0 0 0 2

25 24 17 17 11 17 14 19 7 19 9 11 17 17 15 21 15 9 2 0 2 2 2 0

0 0 0 2 0 0

Statistical tests for differences between vaccine and placebo yielded p > 0.05 for all comparisons aManteI-Haenszel Z 2 for trend: p=0.010 (placebo); p <0.001 (vaccine) t'ManteI-Haenszel Z 2 for trend: p=0.205 (placebo); p--0.001 (vaccine) CManteI-Haenszel Z 2 for trend: p=0.109 (placebo); p=0.007 (vaccine) dManteI-Haenszel Z 2 for trend: p=0.131 (placebo); p -0 .006 (vaccine)

1438 V a c c i n e 1994 V o l u m e 12 N u m b e r 15

Table 2 Pattern of vaccine strain excretion a

Days after vaccinat ion

0 1 2 3 4 5 Total

Dose 2 No. of spec imens 7 13 15 11 8 3 57

examined No. (%) posi t ive 2 9 2 0 0 0 13

(29) (69) (13) (23)

Dose 4 No. of spec imens 6 15 15 14 11 0 61

examined No. (%) posi t ive 0 10 3 1 0 0 14

(67) (20) (7) (23)

aNone of the stool specimens obtained from the placebo recipients after dose 2 (n =63) and dose 4 (n=59) were positive for the vaccine strain

Safety and immunogenicity of EcSf2a-2 oral vaccine. D. Cohen et al.

in any of the 63 stool samples obtained from placebo recipients after dose 2 or the 59 samples obtained after dose 4.

Stability of the vaccine strain

The genetic stability of the vaccine strain after shedding was examined according to three parameters: anti- microbial susceptibility, bacteriophage susceptibility and RFLP patterns. All the vaccine strains isolated after direct plating from vials or isolated from stool samples of vaccinees had the same antimicrobial susceptibility. All of them were sensitive to tetracycline, nalidixic acid, cotrimoxazole, chloramphenicol, ampicillin, norfloxacin and cefixime. The phage susceptibility pattern of these strains was likewise identical. All were susceptible to phages active on S. flexneri serotyes 1 5 but were resistant to phages active on S. flexneri serotype 6, S. boydii, S. sonnei or S. dysenteriae. All the EcSf2a-2 strains were also sensitive to E. coli phage O143 which was not active on S..flexneri 2a and were resistant to phage 028 which revealed lytic activiy on S. flexneri 2a. RFLP analysis of the vaccine strains after digestion of chromosomal DNA with four restriction enzymes, before vaccination and after shedding, revealed the same unique pattern (Figure I).

Immunogenicity

ASCresponse. One of 16 vaccinees had a significant IgA ASC response 3 days after the first vaccine dose (Table 3). Eighteen of 26 (69.2%) and 11 of 30 (36.7%) vaccinees showed significant IgA ASC rises at days 7 and 21 respectively (Table 3). The arithmetic mean number of ELISpots detected was 70 spots/106 cells at day 7 and 22 spots/lO 6 cells at day 21. Only one of six vaccinees who showed no ASC response at day 7 had a significant ASC response at day 21. Moreover, in vaccinees with significant ASC responses at both days 7 and 21, the mean number of secreting cells detected at day 21 was lower (99 spots/106 cells on day 7 versus 15 spots/106 cells on day 21). In one placebo recipient, a borderline ASC response (9 spots/106 cells) was detected. The IgG ASC response was much weaker than the IgA response (only 4% (1/26) had a significant IgG ASC response on day 7).

Serum antibodies. Table4 depicts the rate of signifi- cant rises in serum IgG or IgA anti-S, flexneri 2a LPS

Figure 1 RFLP patterns of DNA extracted from vaccine strains before vaccination (lanes 1~), and after shedding (lanes 5-7), following digestion with Smal and hybridization with the pDT4 probe. Lane 8 represents the RFLP pattern of DNA obtained from a virulent S. flexneri 2a strain after digestion with the same restriction enzyme

Table 3 IgA antibody-secreting cell response (ASC) 3, 7 and 21 days after vaccination

No. (%) with Ari thmetic No. s igni f icant mean of tested ASC response a posit ive results

Day 3 Vaccine 16 1 (6.3) Placebo 12 1 (8.3)

Day 7 Vaccine 26 18 (69.2) Placebo 10 1 (10.0)

Day 21 Vaccine 30 11 (36.7) Placebo 18 1 (5.6)

70.0

21.7

aA significant ASC response is an ASC result equal to or higher than 9 spots/106 cells; this f igure is based on the mean (1.58 spots/10 e cel ls)+ 2s.d. (2 x3.62 spots/106 cells) found among those receiving placebo

Table 4 Number (%) of vaccinees and placebo recipients with significant antibody response a

No. tested IgA IgG IgA or IgG

Vaccine Day 7 53 10 (18.9) 1 (1.9) 10 (18.9) Day 14 53 4 (7.6) 2 (3.8) 4 (7.6) Day 28 50 1 (2.0) 3 (6.0) 2 (4.0)

Total 53 15 (28.3) 6 (11.3) 16 (30.2)

Placebo Day 7 52 0 (0.0) 0 (0.0) 0 (0.0) Day 14 54 0 (0.0) 0 (0.0) 0 (0.0) Day 28 48 1 (2.1) 1 (2.1) 1 (2.1)

Total 54 1 (1.9) 1 (1.9) 1 (1.9)

aBased on the ratio (peak A on days 7, 14, 28)/(A on day 0), with peak A~0.15 (IgA) or 0.25 (IgG); a ratio >2.43 (IgA) or 1.78 (IgG) was considered significant, based on mean+2s .d , found in placebo recipients. ELISA results are based on adjusted A calculated from a linear regression analysis of eight doubling dilutions

V a c c i n e 1994 V o l u m e 12 N u m b e r 15 1439

Safety and immunogenicity of EcSf2a-2 oral vaccine: D. Cohen et al.

Table 5 Comparison of IgA serum antibody response (ELISA) and IgA ASC response in vaccinees

ASC negative ASC positive Total

ELISA negative 14 14 28 ELISA positive 0 8 8 Total 14 22 36

p-0.01 by two-tailed Fisher's exact test

antibodies at days 7, 14 and 22 as compared with the prevaccination level. Sixteen of the 53 vaccinees showed a significant IgG or IgA serum antibody response against the S.flexneri 2a LPS in at least one of the serum samples obtained over the 28-day follow-up period. It is clear that the IgA antibody response (28.3%) was higher than the IgG response (11.3%)(Table 4). Of the 16 vaccinees who responded immunologically to the vaccine, 14 showed a significant IgA or IgG response to S. flexneri 2a LPS at day 14 and only two additional vaccinees demonstrated a significant serum antibody rise at day 28. Only one of 54 placebo recipients showed a significant rise in anti-S. .ltexneri 2a antibodies by day 28, without any evidence of an increase in titre between day 0 and day 14. The magnitude of the serum anti-LPS response in this subject was much larger (peak IgA A = 1.683) than those observed among the vaccinees who seroconverted (peak IgA A range = 0.154 0.902). A comparison between the ASC and serum immune responses to S. flexneri 2a LPS revealed that of 22 subjects with a significant IgA ASC response, eight also had a significant rise in serum IgA, while none of 14 subjects with no ASC response showed a significant serum IgA response (Table 5).

D I S C U S S I O N

The EcSf2a-2 vaccine was well tolerated by the volunteers. Visits to the unit clinic for mild GI symptoms were common after the first dose in vaccinees (13%) as compared with placebo recipients (5%), but the difference was not significant, p = 0.12. None of the volunteers lost any training time due to complaints occurring after vaccination. Abdominal cramps or gurgling, headache, malaise and loss of appetite were the most common symptoms reported by volunteers, both those receiving vaccine and those receiving placebo. Symptoms were most frequent after dose 1, and decreased at doses 2, 3 and 4. Since no significant differences in the rate of these reported complaints were identified between vaccinees and placebo recipients at any specific dose, it is possible that the sodium bicabonate, which was given with the vaccine as well as with the placebo, could have been partly responsible. Recent data from a controlled study have shown a significant association between the concentration of the sodium bicarbonate solution administered prior to vaccination with the oral, whole-cell/recombinant B subunit cholera vaccine and the extent of adverse reactions among volunteers 22. The decrease in the occurrence of these mild complaints following doses 2, 3 and 4 observed in our study may reflect physical and psychological accommodation of the volunteers to the vaccination regimen.

The proportion of vaccinees who excreted the EcSf2a-2 vaccine strain after doses 2 and 4 in our study was very similar to those reported among American civilians 23 and military volunteers 24. All the vaccinees stopped shedding the vaccine strain within 3 days after ingestion, demonstrating the limited ability of the EcSf auxotrophic strain to survive because of the aroD mutation. The very close contact between the volunteers due to the special living conditions during their training period, despite the fair level of sanitation of their unit, could have facilitated transmission of the vaccine strain from vaccinees to placebo recipients. The large number of stool specimens available from placebo recipients enabled a detailed examination of this possibility. The findings showed that all these stool samples were negative for the vaccine strain, indicating the lack of such transmission. This conclusion was reinforced by the serological data which, with one exception, showed no significant immune response to the LPS of S. flexneri 2a among placebo recipients during the 28-day follow-up. The sensitivity of the ELISA assay for detection of a significant rise in serum anti-Shiqella LPS antibodies has been previously demonstrated for both symptomatic and asymptomatic Shigella infections 25. The single placebo recipient who seroconverted was asymptomatic during the entire follow-up period. The timing and magnitude of the serological response suggest that the rise in anti-LPS antibodies in the consecutive sera obtained from this volunteer was the result of natural exposure to S. flexneri during the last part of the follow-up period.

The antimicrobial and bacteriophage susceptibility patterns or the RFLP profile may reflect only part of the potential genetic changes that theoretically can occur in the vaccine hybrid before vaccination or after excretion. According to these three characteristics, the vaccine strain appeared to be genetically stable after replication in the human gut and shedding. The phage susceptibility patterns revealed that two bacteriophages active on E. coli, O143 and 028, can be employed as markers for differentiation between the vaccine strain and S./texneri 2a.

While the ASC response was very low on day 3 after vaccination, 69% of the vaccinees showed significant numbers of ASCs in their peripheral blood on day 7. The rate and magnitude of the ASC response to the vaccine strain in our study were very similar to those observed among civilian and military volunteers in the USA 13"24, but much lower than those detected among Israeli soldiers in the convalescent stage of shigellosis (91% response and a mean of 1432 spots per l 0 6 cells) 2°. As measured on day 21, doses 3 and 4 of the vaccine induced a significant ASC response in only one of six vaccinees examined in whom no ASC response was observed on day 7. No booster effect of these doses was seen in vaccinees with previous significant ASC response at day 7. Moreover, in vaccinees with significant ASC responses at both days 7 and 21, the number of secreting cells detected at day 21 was lower. It is possible that day 21 is not the most suitable time to assess the ASC response induced by doses 3 and 4. It is also possible that secretory antibodies elicited after doses 1 and 2 among those who showed a response on day 7 could have inhibited adherence of the vaccine strain to the gut when this was delivered in doses 3 and 4 on days 14 and 17 respectively. The lack of IgA ASC response in about 24% of vaccinees

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Safety and immunogen ic i t y of EcSf2a-2 ora l vaccine: D. Cohen et al.

on both days 7 and 21 may have been a result of pre-existing antibodies to S. flexneri 2a or to organisms that cross-react with the vaccine strain itself. It has recently been reported that the immunogenicity of another live vaccine, Salmonella typhi Ty21a, was impaired by pre-existing cross-reacting intestinal anti- bodies 26. Nevertheless, in our study, we did not detect a higher level of pre-existing serum antibodies to S.flexneri 2a LPS or to the vaccine strain LPS among subjects who showed no immune response to the vaccine (data not shown).

The occurrence of IgG and IgA serum antibody responses to S. flexneri LPS among Israeli volunteers was slightly lower than that found in previous volunteer studies with the EcSf vaccine 23'24 and much lower than the serum antibody response following Shigella natural infection 25. A high systemic immune response to LPS was not anticipated since such a reaction had not been commonly observed in previous studies with live, oral attenuated Shigella and non-Shigella vaccines 27-29. The comparison drawn between the ASC and serum anti-LPS responses in some of the vaccinees revealed that less than half of the 22 vaccinees showing a significant IgA ASC response also had a significant rise in IgA serum antibodies during the 28-day follow-up period. This finding supports the assumption that a live, attenuated oral vaccine induces a local immune response which may be only partially reflected in significant rises in serum antibodies.

In 14 of the 16 vaccinees who seroconverted to S. flexneri 2a LPS, the significant rise in antibodies was already evident at day 14. The rise in serum anti-LPS antibodies in the two additional vaccinees became significant only at day 28. This is consistent with the data on the ASC response, indicating that doses 3 and 4 of the vaccine had a relatively limited contribution to the overall immune response.

In summary, the EcSf vaccine was well tolerated by Israeli volunteers. In terms of rate and duration of excretion, the EcSf vaccine proved its invasive and auxotrophic characteristics. The vaccine strain maintained its genetic stability after replication in and shedding from the gut. Although a good ASC response was induced by the vaccine in the present study, additional vaccination schedules should be evaluated to take better advantage of the four doses of the vaccine or, alternatively, to allow a possible reduction in the number of vaccination doses. Both the safety and immunogenocity data of this study serve to stimulate further evaluation of the EcSf2a-2 vaccine for protective efficacy in field studies in Israel.

A C K N O W L E D G E M ENTS

This study was supported by Grant No. DAMD17-88- z-8010 from US Army Medical Research and Develop- ment Command, Fort Detrick, Frederick, MD. The authors are grateful to Dr I. Sechter for phage-typing of the vaccine strains and thank Mrs Rachel Heller- Mendelzis for very helpful technical assistance.

R E F E R E N C E S

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13 Formal, S.B., Baron, L.S, Kopecko, D.J., Washington, O., Powell, C. and Life, C.A. Construction of a potential bivalent vaccine strain: Introduction of Shigella sonnei form 1 antigen genes into the galE Salmonella typhi Ty21a typhoid vaccine strain. Infect. Immun. 1981, 34, 746-750

14 Formal, S.B., Hale, T.L., Kapfer, C., Cogan, J.P., Snoy, P.J., Chung, Y. et al. Oral vaccination of monkeys with an invasive Escherichia coil K-12 hybrid expressing Shigella flexneri 2a somatic antigen. Infect. Immun. 1984, 46, 465-469

15 Newland, J.W., Hale, T.L. and Formal, S.13. Genotypic and phenotypic characterization of an aroD deletion-attenuated Escherichia coli K12-Shigella flexneri hybrid vaccine expressing S. flexneri 2a somatic antigen. Vaccine 1992, 10, 766-776

16 Yavzori, M., Cohen, D. and Bercovier, H. Molecular epidemiology of Shigella infections in Israel. Epidemiol. Infect. 1992,109, 273-282

17 Bauer, A.W., Kirby, W.W.M., Sherris, J.C. and Turck, M. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 1966, 45, 493496

18 Anderson, E.S. Use of phages in epidemiological studies. In: Bacteriophages, 2nd edn, (Ed. Adams, M.H.) Interscience Publishers, New York, 1959, Chapter 21, pp. 395-420

19 Cohen, D., Green, M.S., Block, C., Rouach, T. and Ofek, I. Serum antibodies to lipopolysaccharide and natural immunity to shigellosis in an Israeli military population.J. Infect. Dis. 1988, 157, 1068-1071

20 Orr, N., Robin, G., Lowell, G. and Cohen, D. Presence of specific immunoglobulin A secreting cells in peripheral blood following natural infection with Shigella sonnei. J. Clin. Microbiol. 1992, 30, 2165-2168

21 Westphal, O. and Jann, K. Bacterial lipopolysaccharides: extraction with phenol-water and further application of the procedures. Methods Carbohydr. Chem. 1965, 5, 83-91

22 Sanchez, J.L., Trofa, A.F., Taylor, D.N., Kushner, R.A., DeFraites, R.F., Craig, S.C. et al. Safety and immunogenicity of the oral, whole cell/recombinant B subunit cholera vaccine in North American volunteers. J. Infect. Dis. 1993, 167, 1446-1449

23 Kotloff, L.K., Herrington, D.A., Hale, T.L., Newland, J.W., Van De Verg, L., Cogan, J.P. et al. Safety, immunogenicity, and efficacy in monkeys and humans of invasive Escherichia coli K-12 hybrid vaccine candidates expressing Shigella flexneri 2a somatic antigen. Infect. Immun. 1992, 60, 2218-2224

24 Taylor, D., Phillip, D.F., Zapor, M., Trofa, A., Van de Verg, L., Hartman, A. et al. Outpatient studies of the safety and immunogenicity of the auxotrophic Escherichia coli/Shigella flexneri 2a hybrid vaccine candidate EcSf2a-2. Vaccine 1994, 12, 565-568

25 Cohen, D., Green, M., Block, C., Lowell, G. and Ofek, I.

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Immunoglobulin M, A, and G antibody response to lipopotysac- charide O antigen in symptomatic and asymptomatic Shigella infections. J. Clin. Microbiol. 1989, 27, 162-167 Forrest, B.D. Impairment of immunogenicity of Salmonella typhi Ty21a due to preexisting cross-reacting intestinal antibodies. J. Infect. Dis. 1992, 166, 210-212 Tramont, E.C., Chung, R., Berman, S., Keren, D., Kapfer, C. and Formal, S.B. Safety and antigenicity of typhoid-Shigella sonnei vaccine (Strain 5086-1C). J. Infect. Dis. 1984, 149, 133-136

28 Levine, M.M., DuPont, H.L., Gangarosa, E.J., Hornick, R.B., Snyder, M.J., Libonati, J.P. et al. Shigellosis in custodial institutions. I1. Clinical immunologic and bacteriologic response of institution- alized children to oral attenuated Shigella vaccines. Am. J. Epidemiol. 1972, 966, 40-49

29 Gilman, R.H., Hornick, R.B., Woodward, W.E., DuPont, H.L., Snyder, M.J., Levine, M.M. and Libonati, J.B. Evaluation of a UDP-glucose-4-epimeraseless mutant of Salmonella typhi as a live oral vaccine. J. Infect. Dis. 1977, 136, 717-723

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