Evaluation of the Safety, Tolerability, and Immunogenicity of an Oral,Inactivated Whole-Cell Shigella flexneri 2a Vaccine in Healthy AdultSubjects

Subhra Chakraborty,a Clayton Harro,a† Barbara DeNearing,a Jay Bream,b Nicole Bauers,c Len Dally,d Jorge Flores,c

Lillian Van de Verg,c David A. Sack,a Richard Walkerc

Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USAa; Department of Molecular Microbiology andImmunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USAb; PATH, Washington, DC, USAc; The EMMES Corporation, Rockville, Maryland,USAd

Shigella causes high morbidity and mortality worldwide, but there is no licensed vaccine for shigellosis yet. We evaluated thesafety and immunogenicity of a formalin-inactivated whole-cell Shigella flexneri 2a vaccine, Sf2aWC, given orally to adult volun-teers. In a double-blind, placebo-controlled trial, 82 subjects were randomized to receive three doses of vaccine in dose escala-tion (2.6 � 0.8 � 108, � 109, � 1010, and � 1011 vaccine particles/ml). Vaccine safety was actively monitored, and antigen-spe-cific systemic and mucosal immune responses were determined in serum, antibody in lymphocyte supernatant (ALS), and fecalsamples. Cytokines were measured in the serum. Sf2aWC was well tolerated and generally safe at all four dose levels. The vaccineresulted in a dose-dependent immune response. At the highest dose, the vaccine induced robust responses to lipopolysaccharide(LPS) in both serum and ALS samples. The highest magnitude and frequency of responses occurred after the first dose in almostall samples but was delayed for IgG in serum. Fifty percent of the vaccinees had a >4-fold increase in anti-LPS fecal antibodytiters. Responses to invasion plasmid antigens (Ipa) were low. The levels of interleukin-17 (IL-17), IL-2, gamma interferon (IFN-�), tumor necrosis factor alpha (TNF-�), and IL-10 were increased, and IL-8 was decreased immediately after first dose, but thesechanges were very transient. This phase I trial demonstrated that the Sf2aWC vaccine, a relatively simple vaccine concept, wassafe and immunogenic. The vaccine elicited immune responses which were comparable to those induced by a live, attenuatedShigella vaccine that was protective in prior human challenge studies.

Shigella causes bacillary dysentery, a severe, intensely inflamma-tory gastrointestinal disease affecting the distal regions of the

colon and the rectum. Shigellosis is a low-inoculum (10 to 100organisms) infection that is readily transmitted by direct fecal-oral contact. Most of the infections are associated with poor san-itation and contaminated water. Naturally acquired Shigella infec-tion elicits antibody-mediated serotype-specific protective immunity(1, 2). According to the Global Enteric Multicenter Study (GEMS),Shigella was one of the most common pathogens responsible forthe diarrheal disease burden and death in children ages 12 to 59months in areas of Shigella endemicity (3, 4). Mortality from shig-ellosis has diminished significantly in the past 2 to 3 decades (5, 6),largely because of the virtual disappearance of the highly virulentShiga toxin-producing Shigella dysenteriae 1 serotype worldwide.However, the pediatric morbidity due to other species of Shigellaremains substantial (7, 8). In the United States, 10,382 and 10,898cumulative cases of shigellosis were reported in 2014 and 2015(just through August of 2015), respectively (9). World Health Or-ganization guidelines recommend antibiotic treatment for clinicaldysentery (diarrhea with gross blood). However, Shigella fre-quently acquires resistance to antibiotics that were previously ef-fective in reducing disease severity and duration along with patho-gen excretion (8, 10). This background of the enormous globalburden of Shigella, augmented by the growing rate of antimicro-bial resistance, makes a compelling case that development of aShigella vaccine is essential to reduce pediatric shigellosis. The realchallenge in making such a vaccine is that all four species of Shi-gella (Shigella flexneri, Shigella sonnei, Shigella boydii, and Shigelladysenteriae) can cause dysentery, and there are more than 50 se-

rotypes and subserotypes of Shigellae. However, different epide-miological studies have shown that the distribution of species/serotypes that most affect children �5 years of impoverishedpopulations in areas where Shigella is endemic are S. sonnei and S.flexneri 2a (4, 7). Vaccines against these two species/serotypes andagainst S. flexneri 3a and 6 should cover 88% of Shigella infectionsin developing countries through homologous protection andcross protection among the S. flexneri serotypes based on sharedtype and group O antigen determinants (7, 11, 12). In GEMS andin the study conducted by von Seidlein et al., Shigella flexneri wasisolated most frequently among the species, and Shigella flexneri2a was the most frequent serotype (4, 7). Since protective immu-nity is serotype specific, a vaccine against S. flexneri serotype 2a, ora multivalent vaccine which includes S. flexneri 2a, will be requiredto control morbidity and mortality related to shigellosis.

Received 23 October 2015 Returned for modification 17 November 2015Accepted 2 February 2016

Accepted manuscript posted online 10 February 2016

Citation Chakraborty S, Harro C, DeNearing B, Bream J, Bauers N, Dally L, Flores J,Van de Verg L, Sack DA, Walker R. 2016. Evaluation of the safety, tolerability, andimmunogenicity of an oral, inactivated whole-cell Shigella flexneri 2a vaccine inhealthy adult subjects. Clin Vaccine Immunol 23:315–325.doi:10.1128/CVI.00608-15.

Editor: H. F. Staats

Address correspondence to Subhra Chakraborty, schakr11@jhu.edu.

† Deceased.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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A critical question in the evaluation of vaccine candidates iswhich antigen-specific responses are associated with protection.Data from previous studies indicate that O-specific antibodiesplay an important role in protection (13–15). Antibodies againstinvasion plasmid antigens (Ipa) are also produced after naturaland experimental infection and may also contribute to protection(16, 17).

To be acceptable to countries where Shigella is endemic, a vac-cine should be safe, inexpensive, stable, and easy to deliver. Atpresent, there is no licensed vaccine against Shigella, although avariety of vaccines have been shown to confer protection in exper-imental models of shigellosis (18–21). Inactivated whole-cell vac-cines (WCV) are a potentially important complement to thebroad range of approaches that are currently under development(22–25). By this strategy, multiple, often unidentified, antigens aredelivered safely to the gut mucosa to stimulate mucosal and sys-temic immunity. Preparation is relatively easy and inexpensive,and administration does not require needles. Further, this type ofvaccine may not require a buffer for delivery and can be stored inliquid formulations. Although only the inactivated whole-cell oralcholera vaccine Shanchol has been licensed and is in use (26),other WCV have the potential for global use. For example, vacci-nation with inactivated whole cells has shown promise againstenteric diseases caused by enterotoxigenic Escherichia coli (27).

Several early studies reported that an inactivated WCV wasprotective against shigellosis (28, 29), but little information isavailable about the composition of these vaccines, and large oraldoses were required for efficacy. An inactivated WCV vaccine forS. sonnei formulated using a complicated fermentation processwith 2.0 � 1010 vaccine particles (vp)/ml in a three-dose schedulewas found to be effective in a phase I trial (30).

Preclinical studies were undertaken to manufacture forma-lin-inactivated whole-cell vaccine against Shigella flexneri 2a(Sfl2aWC). An immunogenicity and protection study using a mu-rine pulmonary model showed that animals vaccinated with threedoses of 107 or 109 vp/ml of Sfl2aWC were significantly protected(�93%) from an intranasal challenge with a lethal dose (1.5 � 107

CFU) of S. flexneri 2a strain 2457T. Notably, Sfl2aWC formula-tions containing double mutant heat-labile toxin (dmLT) showedminimal improvement or, in some cases, a decrease of the magni-tude of the immune response but were still highly protective inboth the mouse and guinea pig models (31).

The current study was undertaken to evaluate the feasibility ofusing an orally delivered formalin-inactivated whole-cell vaccineSf2aWC against Shigella flexneri 2a. We determined the safety andcharacterized the mucosal and systemic immune responses in adose-escalation study in adult volunteers from the United States.

MATERIALS AND METHODSFormulation and vaccine preparation. Sf2aWC vaccine was prepared atthe pilot production facility at the Walter Reed Army Institute of Re-search, Washington, DC, from S. flexneri 2a strain 2457T, lot 1617S, fol-lowing standard current good manufacturing practice guidelines. S. flex-neri 2a strain 2457T is known to be virulent in North American volunteersand to cause diarrhea, fever, and dysentery. The vaccine was prepared asdescribed by Kaminski et al. in 2014 (31). Viability, stability, and antigenexpression of Ipa proteins (IpaB, IpaC, IpaD) and lipopolysaccharide(LPS) were monitored at 3, 6, 9, 12, and 18 months, and they remained atacceptable levels throughout the course of the phase I trial. Randomlyselected vials were tested for appearance, particle count, sterility, and pHby slide agglutination and Western blotting with specific antiserum for 2a

LPS identity and by Western blotting with specific monoclonal antibodyfor detection of IpaB, IpaC, and IpaD. Vaccine immunogenicity, potency,and efficacy (after challenge with S. flexneri 2a) were assessed in guineapigs. Sf2aWC vaccine lot 1656 was formulated as a sterile liquid suspen-sion of formalin-killed bacterial cells in phosphate-buffered saline (PBS)(pH 6.5 to 7.8). After removal from refrigerated storage, the vaccine waskept on ice until diluted in sodium bicarbonate (1.34%) for administra-tion to volunteers. A 30-ml aliquot of this buffer was added to a separatecontainer, and 1 ml of the Sf2aWC vaccine was added to the container foringestion by individual subjects.

Regulatory approval. The clinical protocol was performed under IND14630 at the Center for Immunization Research, Johns HopkinsBloomberg School of Public Health (JHBSPH). The protocol was ap-proved by the Western Institutional Review Board (Olympia, WA) forJHBSPH and PATH and by the institutional biosafety committee of theJohns Hopkins institutions. The study was registered in clinicaltrials.govunder NCT01509846.

Study design. This single-site, phase I, double-blind, randomized,placebo-controlled trial was designed to evaluate the safety and immuno-genicity of increasing dosages of orally administered Sf2aWC. Healthyadults between ages 18 and 45 years with body mass indices (BMIs) be-tween 19.0 and 34.0 kg/m2 were eligible. Subjects were screened for studyeligibility via medical history review, physical examination, human leu-kocyte antigens (HLA-B27), routine metabolic and hematologic indices,urine toxicity screen, urinalysis by dipstick, and serologic tests for hepa-titis B, hepatitis C, and human immunodeficiency virus. Additional ex-clusion criteria included a history of diarrhea in the 7 days prior to vacci-nation; regular use of laxatives or antacids; history of vaccination againstor ingestion of Shigella within 3 years; symptoms consistent with travelers’diarrhea concurrent with travel to countries where Shigella is endemicwithin 2 years; and use of proton pump inhibitors, H2 blockers, or antac-ids within 48 h prior to dosing. Each participant signed an informedconsent form and was required to score �70% on a study comprehensionassessment prior to undergoing study-specific procedures. Eighty-twosubjects were enrolled into four separate cohorts and were randomized toreceive oral Sf2aWC or placebo. The study was conducted between March2011 and November 2012. The placebo preparation was bicarbonate buf-fer. The number and allocation of subjects in each cohort are shown inTable 1.

Subjects in cohort 1 received a single oral Sf2aWC dose of 2.6 � 0.8 �108 vaccine particles (vp)/ml or placebo. Subjects in cohorts 2, 3, and 4received Sf2aWC in increasing respective dosages of 2.6 � 0.8 � 109,2.6 � 0.8 � 1010, and 2.6 � 0.8 � 1011 vp/ml or placebo at 0, 1, and 2months (Table 1). With each dose, each volunteer drank 120 ml of bicar-bonate buffer solution (1.33%) to neutralize stomach acid; approximately1 min later, they ingested the Sf2aWC vaccine (or placebo) diluted in 30ml of the same buffer. Subjects were required to fast for 90 min before andafter ingestion of the investigational product. The first dose, for each doselevel, was given to volunteers when admitted to the inpatient unit of theCenter for Immunization Research, where they could be carefully ob-served for 72 h following receipt of the vaccine.

Subsequent doses were administered on an outpatient basis. Beforeenrolling subjects in subsequent cohorts, safety data from the previouscohorts through study day 7 were evaluated and reviewed by the safetyreview committee.

TABLE 1 Vaccine dose and subject allocation by cohort

CohortSf2aWC (vaccineparticles/ml) Schedule

No. ofvaccinerecipients

No. ofplaceborecipients

1 2.6 � 0.8 � 108 Day 0 5 22 2.6 � 0.8 � 109 Day 0, 28, 56 20 53 2.6 � 0.8 � 1010 Day 0, 28, 56 20 54 2.6 � 0.8 � 1011 Day 0, 28, 56 20 5

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Safety evaluation. The primary safety endpoints were all adverseevents (AEs) following dosing with Sf2aWC (through day 28 for cohort 1and through day 63 after the last vaccination for cohorts 2, 3, and 4), andthey were evaluated via focused medical interviews, standardized vaccina-tion report cards (VRCs), laboratory tests, vital signs, and physical exam-inations. For all cohorts, systemic reactogenicity data associated with vac-cination 1 were collected by study staff during the inpatient phase onstudy days 0 to 3 and then by study subjects during the outpatient phasevia diary card on study days 4 to 7. For subjects who received two addi-tional vaccinations in cohorts 2 to 4, systemic reactogenicity data wererecorded on diary cards for 7 days after each vaccination. Solicited AEsincluded oral body temperature, loose stools, diarrhea, abdominal pain orcramping, bloating, defecation urgency, nausea, and vomiting. Data onother systemic AEs, including headache, malaise, chills, myalgia, reactivearthritis, excess gas, constipation, and dysentery were also collected. Ap-proximately 6 months following receipt of the last dose of vaccine (orplacebo), subjects were contacted by study staff to inquire about any newchronic health conditions or serious health events.

Immunogenicity evaluation: collection of samples. Blood and fecalsamples were collected from volunteers for immunological evaluations oftotal IgA (fecal IgA only) and antigen-specific IgA or IgG to LPS and Ipaproteins.

Blood specimens. Venous blood was collected from volunteers fromcohorts 2, 3, and 4 on study days �1 (before immunization), 7, 28, 35, 56,63, and 112, and serum samples were separated to measure systemic im-mune responses. Serum IgG and IgA responses were measured using en-zyme-linked immunosorbent assay (ELISA) and were analyzed by thelevel of response (geometric mean titers) as well as the frequency withwhich subjects seroconverted, as measured by the maximum dilution ofsera which showed reactivity to LPS or Ipa protein antigens by more thana 2-fold increase over the preimmunization levels.

Antibody responses from the mucosal immune system were deter-mined by ELISA using antibody from lymphocyte supernatant (ALS)specimens. This assay measures antibodies secreted by peripheral bloodmononuclear cells (PBMCs) circulating in the peripheral blood followingimmunization. Venous blood was collected in a BD vacutainer CPT withheparin (cell preparation tubes) (Becton Dickinson, Franklin Lakes, NJ,USA) on day 0 (before vaccination) and on days 7, 28, 35, 56, and 63 aftervaccination. The PBMCs, isolated by gradient centrifugation, were resus-pended at 1 � 107 viable lymphocytes per milliliter and then incubated at37°C, 5% CO2 for 72 h with no antigenic stimulation. The supernatantfluid was cryopreserved and used later in an IgA ELISA. An ALS responsewas defined as a �4-fold increase over preimmunization levels.

Fecal samples. Stool specimens were collected on the day before vac-cination and on days 7, 35, and 63 days after vaccination. Specimens werestored at �70°C after they were received. Since some specimens werebrought to the clinic during outpatient follow-up visits, the duration be-tween collection and freezing varied up to 18 h. Antibodies were extractedby the following procedure. Four grams of thawed stool was mixed with 16ml of a solution containing soybean trypsin inhibitor (STI) (100 �g/ml)(Sigma, St. Louis, MO), EDTA (0.05 M) (Merck, NJ), and Pefablock (finalconcentration, 0.35 mg/ml) (Roche, NJ) dissolved in PBS (pH 7.2) sup-plemented with 0.05% Tween 20 (PBS-Tween). The mixture was left tostand on the bench at room temperature for 15 min with intermittentshaking. Next, the mixture was centrifuged at 12,000 � g for 30 min.Bovine serum albumin (BSA) (final concentration, 0.1% [wt/vol]) wasadded to the supernatant after the pellet was discarded. Aliquots of thesupernatant were stored at �70°C until they were assayed by ELISA fortotal and specific IgA antibody contents. A fecal response was defined as a�4-fold increase over baseline.

ELISAs with serum, ALS, or fecal samples were performed accordingto standard protocols. Ninety-six well microtiter plates were first coatedwith S. flexneri 2a LPS or Ipa (Ipa B and Ipa D) in PBS. The plates werethen washed twice with PBS, blocked with 0.1% BSA-PBS for 30 min andwashed three times with PBS-0.05% Tween 20. Serum, ALS, and fecal

samples were diluted 3-fold in the plates using 0.1% BSA-PBS-Tween as adiluent. After overnight incubation at 4°C, the plates were washed threetimes with PBS-Tween. Then 100 �l of anti-human IgG or anti-humanIgA conjugated with horseradish peroxidase (KPL, Baltimore, MD) di-luted in 0.1% BSA-PBS-Tween was added to each well, and the plates wereincubated for 90 min. Finally, after washing three times with PBS-Tween,o-phenylenediamine dihydrochloride (OPD) (Sigma, St. Louis, MO) wasadded at a concentration of 100 �l per well. After 20 min, the plates wereread at 450 nm in an automated ELISA reader. Antibody titers for fecalspecimens were expressed as units per milligram of IgA, and these titerswere calculated by using the specific titer (in units per milliliter) dividedby the total IgA contents (in micrograms per milliliter) and then multi-plying by 1,000. The total IgA contents for given samples were determinedby ELISA using a standard IgA preparation (Sigma, St. Louis, MO) with aknown IgA concentration (1 mg/ml).

Titers were calculated to interpolate the dilution of serum, whichyielded an optical density (OD) above baseline of 0.2 for serum samplesand of 0.4 for ALS and fecal samples. Prevaccine and postvaccine sera weretested simultaneously in the same plate. LPS and Ipa were supplied by E.Oaks of the Walter Reed Army Institute for Research.

Serum cytokine measures. We determined serum cytokines as an ex-trapolatory assay in this phase I study. We aimed to understand if therewere any significant levels of cytokine changes after vaccination with aninactivated vaccine Sfl2aWC. We used the ultrasensitive human proin-flammatory 9-plex and the ultrasensitive human IL-17A single-plex assaysfrom Meso-Scale Discovery (MSD), Gaithersburg, MD. Serum sampleswere collected from vaccinees and from those who received placebo incohorts 3 and 4 on the day before vaccination and at 8, 24, 48, and 72 h ondays 56, 58, and 63 postvaccinations. The MSD multispot array was runaccording to the manufacturer’s protocol, with minor modifications. Allsamples were run in duplicate. Briefly, plates were preincubated with 25 �lof supplied human serum diluents for 30 min, with shaking, at roomtemperature. Calibration curves were prepared in the diluents and rangedfrom 2,500 pg/ml to 0.15 pg/ml. Following the 30-min incubation period,25 �l of serum sample or calibrator was added to the wells. Plates werethen incubated at room temperature for 2 h with shaking. Plates were thenwashed with PBS and 0.05% Tween and then incubated with 25 �l ofdetection antibody for 2 h at room temperature with shaking. After wash-ing plates with PBS and 0.05% Tween, 150 �l of detection antibody wasadded. Plates were read using the MS2400 imager (MSD). The lowest limitof quantification (LLOQ) was defined as the lowest calibrator value atwhich the coefficient of variance of concentration was less than 25% andrecovery of calibrator was within 25% of the expected value. All cytokinevalues that were below the LLOQ were considered undetectable and as-signed a value equal to the plate-specific LLOQ for statistical analyses.

Statistical analyses. Clinical and safety data were captured using elec-tronic case report forms (CRFs). For analysis of immune responses, all ofthe placebos were combined and compared with different doses ofSf2aWC. The threshold for definition of a positive serological or ALSresponse was derived by investigating the apparent level of responses inthe placebo recipients. For between-group comparisons of titer magni-tude at each visit, the Kruskal-Wallis test was used; if that test was statis-tically significant, the Wilcoxon rank sum test (2-sided t test approxima-tion) was used to make pairwise comparisons. Within-group change frombaseline at postvaccination follow-up visits was assessed using the signed-rank test. In cytokine analyses, comparisons of dichotomous outcomes,such as fold increase or fold decrease were performed with the chi-squaretest or Fisher’s exact test if cell counts were below 5. For comparisons ofcytokines at baseline versus cytokines at postvaccination follow-up,Student’s t test was performed using the pooled method or the Welch-Satterthwaite method, depending on whether the variance was equalor not. GraphPad Prism (GraphPad, CA) was used to analyze the data.Results of statistical analyses were considered significant only if the Pvalue was less than 0.05.

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RESULTSStudy enrollment, retention, and demographics. A total of 132subjects were screened for this vaccine study, of which 82 subjectswere enrolled. No significant differences between participants(n � 82) and nonparticipants (n � 50) were observed for age,gender, or race/ethnicity (data not shown). Seven subjects werevaccinated in the first cohort. In cohorts 2 to 4, all 75 (100%)subjects completed the first dose, and 72 (96.0%) and 70 (93.3%)subjects completed the second and third doses, respectively (Fig.1). Vaccine administration was discontinued after two doses forone subject in the 1011-vp/ml dose group due to pregnancy. Thereasons for the few dropouts were assessed to be unrelated to thestudy product administration. Of the 82 subjects enrolled, 23 werefemale and 59 were male. The majority (n � 72) race was black orAfrican American. The mean age was 33 years (range, 18 to 45years). The disposition of study subjects is shown in Table 2.

Safety assessment. Overall, the vaccine was well tolerated at allfour dose levels, and no safety signals were identified. At the lowest

dose, in the five subjects who received only one vaccination, noreactogenicity events were reported. At the highest dose, mild/moderate nausea and mild/moderate flatulence were the mostcommon vaccine-related events (Table 3). All laboratory abnor-malities were mild in severity and transient in duration. Therewere no vaccine-related severe adverse events and no other seriousadverse events reported. Of the 77 subjects randomized to receivethree vaccinations or placebo in cohorts 2 to 4, two (3.3%) sub-jects had moderate nausea in the vaccine group compared to one(5.9%) in the placebo group. Three (5.0%) vaccinated and two(11.8%) placebo subjects had moderate abdominal pain. Moder-ate excessive flatulence was found in five (8.3%) of the vaccinatedsubjects and in two (11.8%) of the placebo subjects. One subject ineach group—vaccinated (1.7%) or placebo (5.9%)— had moder-ate anorexia; one (1.7%) vaccinated subject had moderate urgencyof defecation (Table 3). There were no discernible trends in safetylaboratory values, which included basic hematologic and meta-bolic parameters.

FIG 1 Subject enrollment and retention throughout the study. The flow diagram outlines the status (screened, excluded, enrolled, lost to follow-up, withdrawn)of all study participants.

TABLE 2 Subject demographics

Demographic variable

Value by cohort and Sf2aWC dose (vp/ml)

Value for placebo group,all cohorts (n � 17) Total (n � 82)

Cohort 1,2.6 � 0.8 �108

(n � 5)

Cohort 2,2.6 � 0.8 �109

(n � 20)

Cohort 3,2.6 � 0.8 �1010

(n � 20)

Cohort 4,2.6 � 0.8 �1011

(n � 20)

No. (%) by sexMale 3 (60.0) 14 (70.0) 18 (90.0) 12 (60.0) 12 (70.6) 59 (72.0)Female 2 (40.0) 6 (30.0) 2 (10.0) 8 (40.0) 5 (29.4) 23 (28.0)

No. (%) by raceBlack or African American 4 (80.0) 19 (95.0) 14 (70.0) 19 (95.0) 16 (94.1) 72 (87.8)White 1 (20.0) 0 (0.0) 6 (30.0) 1 (5.0) 0 (0.0) 8 (9.8)Other 0 (0.0) 1 (5.0) 0 (0.0) 0 (0.0) 1 (5.9) 2 (2.4)

Mean (range) age, yrs 29.0 (25–34) 31.7 (18–44) 36.5 (20–45) 32.3 (18–44) 31.3 (20–45) 32.7 (18–45)

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Immunology: dose escalation and immune responses. Weinvestigated the magnitude and kinetics of the immune re-sponses to the Sf2aWC vaccine in this dose-escalation study.Analyzing the immune responses, we found that the IgA andIgG responses to LPS in serum and ALS samples were minimalin cohort 3. Therefore, we decided not to perform any immu-nology analysis on the samples from the vaccinees who receivedlower doses, i.e., cohorts 1 (immunization with 108 Sf2aWC)and 2 (immunization with 109 Sf2aWC). Cohorts 3 (immuni-zation with 1010 Sf2aWC) and 4 (immunization with 1011

Sf2aWC) showed dose-dependent immune responses in se-rum, ALS, and fecal samples (Table 4, Fig. 2, 3, and 4).

Serum. Blood samples collected and processed for serum wereassayed by ELISA to determine IgA and IgG endpoint titers. Incohort 4, most vaccinees seroconverted with anti-LPS IgG and IgA(Table 4). Seventy percent (14 of 20) of the vaccinees showed a�2-fold increase for both IgA and IgG to LPS compared to nonefor IgA and three subjects for IgG in the placebo group. The foldincrease of the IgG responses in the three placebo subjects werebelow 2.3-fold and occurred on day 35 (7 days after the seconddose). All three of these placebo subjects were in cohort 3. For LPSIgA, 93% (13 of 14) of the vaccinees who responded had a �2-foldincrease on day 7 after the first dose. In contrast, for LPS IgG, only50% (7 of 14) of the vaccinees who responded had �2-fold in-crease after the first dose. We also analyzed the data by considering�2.5-fold and �4-fold increases as the cut points for response.For LPS IgA in serum samples, 12 of 20 (60%) and 9 of 20 (45%)of the vaccines responded when using �2.5-fold and �4-fold in-creases, respectively, as the cut points. For LPS IgG, 8 of 20 (40%)and 2 of 20 (20%) of the vaccinees responded when using �2.5-

fold and �4-fold increases, respectively, as the cut points. Thegeometric mean titer (GMT) for LPS IgA was highest at 7 daysafter the first dose (range, 176 to 3,242) and was 3.2-fold (P �0.0001) higher than the baseline (highest fold increase of 30) (Fig.2A). Subsequent titers for LPS IgA also significantly increasedfrom prevaccination to 7 days after the second and third doses,although the increase was lower after each one: the fold increasewas 2.3 (P � 0.0006) (highest fold increase of 10) and was 2.0 (P �0.0072) (highest fold increase of 7) at 7 days after vaccinations 2and 3, respectively (Fig. 2B). The GMTs of vaccinees at all the timepoints postvaccination were significantly higher than those forplacebos. For LPS IgG, the GMT was highest 7 days after the sec-ond dose compared to baseline and increased by 1.96-fold (P �0.005) (Fig. 2B). However, the GMT of LPS IgG was not signifi-cantly different from placebo. The highest fold increase was 17compared to the baseline. The titer range on day 35 was 845 to11,361 (Fig. 2A). In placebos, the GMTs for IgA and for IgG didnot change much throughout the study period.

In the cohort 3 immunization with 1010 vp/ml of Sf2aWC, 15%(3 of 20) of the vaccinees seroconverted for both LPS IgA and IgG(Table 4). All of the responders seroconverted on day 7 after thefirst dose for IgA and at 28 days after the first dose for IgG (Fig. 2Aand B).

Although Sf2aWC induced strong serum antibody responsesto LPS, antibody responses directed against IpaB and IpaD werelow. A subset of samples randomly selected from cohort 4 wastested for IgA and IgG of IpaB and IpaD in serum. Twenty-ninepercent of the vaccinees mounted responses, with both IgA andIgG to IpaB. For IpaD, 14% and 29% of the vaccinees respondedfor IgA and IgG, respectively (data not shown).

ALS. With respect to IgA responses directed to LPS in ALSsamples, immunization with 1011 vp/ml of Sf2aWC (cohort 4)resulted in significantly higher endpoint titers than immunizationwith 1010 vp/ml of Sf2aWC (cohort 3). In both cohorts 3 and 4, thetiters for LPS IgA and IgG significantly increased on day 7 after thefirst dose but declined to the baseline level by day 28. The titersagain increased 7 days after the second and third doses, but themagnitudes were much lower than the first peak (Fig. 3A and B).For the IgA to LPS in cohort 4, the GMT increased 24-fold on day

TABLE 4 IgA and IgG responsea rates to S. flexneri 2a LPS or Ipaelicited by vaccine doses

AssaySampletype

No. (%) with response byvaccine dose (vp/ml)

No. with response/total no. tested(%) with placebo1010 (n � 20) 1011 (n � 20)

LPSIgA Serum 3 (15) 14 (70) 0/17 (0)IgG Serum 3 (15) 14 (70) 3/17 (18)IgA ALS 9 (45) 19 (95) 1/17 (6)IgG ALS NDb 15 (75) 0/12 (0)IgA Fecal 8 (40) 10 (50) 2/10 (20)

Ipa proteinIpaB IgA ALS ND 4 (20) 0/12 (0)IpaB IgG ALS ND 2 (10) 0/5 (0)IpaD IgA ALS ND 2 (10) 0/12 (0)

a Greater than 2-fold (in serum) or greater than 4-fold (in ALS and fecal) increase intiters from baseline on any day was considered a response.b ND, not done.

TABLE 3 Proportional maximum gastrointestinal reactogenicity aftervaccination with different doses of Sf2aWC or placebo

Adverse event

No. of reactogenicity events bySf2aWC dose:

No. of reactogenicityevents with placebo(n � 17)

109 vp/ml(n � 20)

1010 vp/ml(n � 20)

1011 vp/ml(n � 20)

NauseaMild 1 1 3Moderate 1 1 1

Vomiting, mild 2 1

Abdominal painMild 1 2 2Moderate 2 1 2

Excess flatulenceMild 2 6 6 2Moderate 2 3 2

UrgencyMild 1 1Moderate 1

Loose stool, mild 1 1 2 1

AnorexiaMild 1 1 1Moderate 1 1

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7 (7 days after the first dose), with the highest fold increases of 290,6-fold on day 35 (7 days after the second dose) and 2.9-fold onday 63 (7 days after the third dose); for LPS IgG, the GMTincreased 7.8-fold on day 7 (with the highest fold increase of76), 2-fold on day 35, and 1.5-fold on day 63 compared to thepreimmunization level. In cohort 4, for LPS IgA and IgG, 95%(19 of 20) and 75% (15 of 20), respectively, of the vaccineesshowed 4-fold or higher increases in their ALS titers. In theplacebo group, one subject responded to LPS IgA, and noneresponded to IgG (Table. 4).

In cohort 3, only 45% (9 of 20) of the vaccinees responded forIgA to LPS in ALS, with the highest peak on day 7 of a GMTincrease of 2.5-fold over the baseline (Table 4, Fig. 3). Due to lower

responses for IgA to LPS in ALS samples, immune responses toIgG in ALS were not done for cohort 3 samples.

The antigen-specific responses for IgA and IgG to IpaB andIpaD in ALS were low. Twenty percent (4 of 20) of the vaccinees incohort 4 had a �4-fold increase and 30% (6 of 20) had a �2-foldincrease in titers from baseline for IgA to IpaB. For IgG to IpaB,10% (2 of 20) of the vaccinees had a �4-fold increase and 55% (11of 20) had a �2-fold increase in titers compared to baseline (Table4). For IgA to IpaD, 10% of vaccinees (2 of 20) had a �4-foldincrease and 20% (4 of 20) had a �2-fold increase in cohort 4.None in the placebo group responded for IgA and IgG to IpaB orfor IgA to IpaD.

Fecal IgA. The fecal extracts were evaluated using ELISA for

FIG 2 IgA (A)- and IgG (B)-to-LPS geometric mean (95% confidence interval) titer increase in serum after immunization with either 2.6 � 0.8 �1010 or 2.6 �0.8 �1011 vp/ml of Sf2aWC or placebo. The x axis indicates days of immune responses measured: day 0 (day before first dose), day 7 (7 days after first dose), day28 (day of the second dose), day 35 (7 days after second dose), day 56 (day of the third dose), day 63 (7 days after third dose), and day 84 and day 112.

FIG 3 IgA- and IgG-to-LPS geometric mean (95% confidence interval) fold increase of titers compared to baseline in ALS after immunization with either 2.6 �0.8 �1010 or 2.6 � 0.8 �1011 vp/ml of Sf2aWC or placebo. The x axis indicates days of immune responses measured: day 7 (7 days after first dose), day 28 (dayof the second dose), day 35 (7 days after second dose), day 56 (day of the third dose), or day 63 (7 days after third dose). Data are presented in logarithmic scale(log10). The dotted line shows 4-fold increase. **, P � 0.001 (responses compared to baseline).

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IgA to LPS in cohorts 3 and 4. In cohorts 3 and 4, 40% (8 of 20) and50% (10 of 20), respectively, of the vaccinees showed 4-fold orhigher responses for IgA to LPS; two subjects responded in theplacebo group (Table 4). In cohorts 3 and 4, the GMTs at 7 daysafter the first and second doses were similar (Fig. 4).

Cytokine responses in serum. We investigated the inflamma-tory response to vaccination by measuring the levels of proinflam-matory cytokines and chemokines in serum. The 9-plex proinflam-matory cytokines included gamma interferon (IFN-), tumornecrosis factor alpha (TNF-), interleukin 1� (IL-1�), IL-2, IL-6,IL-8, IL-10, IL-12p70, and the chemokine granulocyte macro-phage colony-stimulating factor (GM-CSF). We also tested theserum for IL-17A in a singleplex assay. For analysis, we comparedvaccinees in cohorts 3 (n � 20) and 4 (n � 20) with all of theplacebos from both cohorts combined (n � 10).

Among subjects in cohort 4, 16 of 20 (80%) had a �2-foldincrease in levels of IL-17 at 8 h after the first dose, compared toonly one subject in the placebo group (P � 0.0004). Forty percentof the subjects in the vaccine group had a �4-fold increase com-pared to none in the placebo group (P � 0.0288) (Fig. 5A). Themagnitude of the response was also much higher in the vaccinegroup than in the placebo group (P � 0.0001). The increasedlevels of IL-17A were transient and decreased at 24 h after the firstvaccination; IL-17A levels were not substantially increased againfollowing subsequent vaccine dosing.

Similarly, the levels of Th1-type cytokine IL-2 were signifi-cantly increased (P � 0.003) compared to the baseline (Fig. 5B) at8 h after vaccination of the first dose. Forty percent (8 of 20)vaccinees had a �2-fold increase compared to one subject in theplacebo group. IFN- secretion was also significantly higher at 8 h(P � 0.0337) and at 72 h (P � 0.014) after the first vaccinationcompared to baseline (Fig. 5C). Compared to baseline, mean lev-els of TNF- in serum samples were higher at 8, 24, and 72 h afterfirst dose (P � 0.0043, � 0.0482, and � 0.0479, respectively) and

were higher than the levels among placebo recipients (data notshown).

In contrast, the level of IL-8 decreased significantly 8 h after thefirst dose and remained low at 72 h. On day 56 (28 days after thesecond dose), the level was significantly increased and returned tobaseline. The level was again decreased 2 days after the third dose(Fig. 5D). In the placebo group, the changes in the level of IL-8were random.

IL-10 was significantly increased at 8 h after the first dose,remained elevated at 72 h, and then decreased to the baseline levelat day 56 (Fig. 5D). Another cytokine that was upregulated (�2-fold in vaccinees) after the first vaccination was IL-6 (25%). Thetrend of responses to all of the cytokines measured was similar butof lower magnitude in cohort 3. Cytokine responses in serum forGM-CSF, IL-12p70, and IL-1� in cohort 4 and in addition forIFN- in cohort 3, were below detectable limit and were excludedfrom analysis.

We also examined associations between cytokine responses inserum samples and LPS-specific immune response in serum, ALS,and fecal samples after vaccination. Among subjects in cohort 4,subjects with elevated IFN- levels also had �2-fold increases intiters against IgA and IgG to LPS in serum samples and �4-foldincreases in titers in fecal extracts for IgA.

DISCUSSION

The present study demonstrated that an orally delivered, forma-lin-inactivated, whole-cell Shigella flexneri 2a vaccine (Sf2aWC)with a dose of 2.6 � 0.8 � 1011 vaccine particles/ml was safe andwas able to induce both systemic and mucosal immune responses.

Sf2aWC was well tolerated at all four dose levels, ranging from108 to 1011 inactivated cells. There were no significant differencesin the frequency or severity of symptoms in vaccinees compared toplacebo recipients. No fever or serious gastrointestinal symptomswere noted by any of the vaccinated subjects. Thus, our data sug-gest that Sf2aWC, which represents a major Shigella species ofclinical importance, can be used safely at a high dose in humans.

An obvious concern regarding live attenuated candidates isidentifying the appropriate balance between immunogenicity andadverse effects, while the primary concern for an inactivated WCVis immunogenicity. Shigellae are invasive enteric bacteria, and cel-lular invasion is said to be the key to efficient antigen delivery.However, in our study, three doses of Sf2aWC were able to induceconsiderable mucosal and systemic immunity. All vaccinated sub-jects had serum or mucosal IgA or IgG responses to LPS. In serum,most of the vaccinated subjects had IgA responses to LPS, and thehighest GMT was after the first dose. Interestingly, IgG responsesto LPS were delayed, and the highest GMT was after the seconddose. The increase observed in GMTs of IgG to LPS was lower thanthat detected in the IgA isotype. Although IgA titers to LPS in-creased after every dose, the largest increase over baseline oc-curred following the first dose. Since there was little or no boostingof immune responses after the third dose, two doses of 1011 vp/mlof Sf2aWC might be sufficient. The oral inactivated WCV forcholera with a 1011 dose in a two-dose schedule was found to beeffective (26).

Notably, the serum LPS antibody responses elicited by Sf2aWCwere stronger than the response elicited by the live attenuatedvaccine SC602, an S. flexneri strain attenuated by deletions in icsAand iuc, which encode aerobactin. Administration of 104 CFU ofSC602 led to a 3-fold increase in the serum IgA GMT to LPS titers

FIG 4 IgA-to-LPS geometric mean (95% confidence interval) fold increase oftiters compared to baseline in fecal samples after immunization with either2.6 � 0.8 �1010 or 2.6 � 0.8 �1011 vp/ml of Sf2aWC or placebo. The x axisindicates days of immune responses measured: day 7 (7 days after first dose),day 35 (7 days after second dose), or day 63 (7 days after third dose). The dottedline shows 4-fold increase. Data are presented in logarithmic scale (log10). *,P � 0.05 (responses compared to baseline).

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in half of the subjects and to a �2-fold increase in anti-LPS-spe-cific IgG (21, 32). Despite these modest immune responses, SC602was protective in a challenge study in the United States. The im-mune response to LPS with Sf2aWC in our study was also compa-rable to the oral inactivated whole-cell vaccine for Shigella sonnei(SsWC), where four of seven vaccinees (57%) had IgG and IgAresponses to LPS (30).

Based on the human challenge-rechallenge studies (19, 33, 34),primate studies, epidemiological field studies (13), and seroepide-miological surveys (14, 15), LPS is believed to be of critical impor-tance in protection against Shigella infections. In field settings,preexisting IgA and IgG anti-LPS serum antibodies have been as-sociated with protection against shigellosis following natural ex-posure (14). Using active surveillance at the community levels,Ferrecio et al. followed a cohort of children less than 4 years of agein Santiago, Chile, for 30 months and observed that an initial boutof shigellosis afforded 72% protection against subsequent clinicalillness caused by the homologous serotype (P � 0.05). However,protection against a second clinical illness due to a heterologous

serotype was low (�30%; P value not significant) (13). The im-portance of humoral immunity to LPS is also supported by theprotection provided in the SC602 trials (21, 32). It has been pos-tulated that local secretory IgA against LPS may help mediate thisprotection by blocking the interactions that occur between Shi-gella and mucosal epithelial cells which ultimately lead to inva-sion. Another mechanism might be the antibody-dependent cel-lular cytotoxicity against Shigella observed in vitro (35).

A compelling rationale for the oral delivery of live attenuatedvaccines is the induction of mucosal immunity, which has beenshown to correlate well with the transient appearance of LPS-specific IgA antibody-secreting cells (ASC) in the peripheral blood(36). In the SC602 Shigella vaccine trial, a rise in IgA ASCs to LPSwas correlated with protection (21). In our phase I trial, oral ad-ministration of Sf2aWC resulted in high levels of IgA and IgG toLPS in ALS. All but one vaccinee responded to IgA, and 75%responded to IgG in the highest dose. The GMT was increasedafter each dose, but the highest pick was after the first dose.

The immune responses to LPS in fecal samples were modest,

FIG 5 Shigella-specific cytokine production in serum after immunization with three doses of 2.6 � 0.8 �1011 vp/ml. Cytokines IL-17 (A), IL-2 (B), IFN- (C),and IL-10 and IL-8 (D) were measured before vaccination (0 h), at 8, 24, and 72 h after the first dose, at day 56 (28 days after the second dose), and at day 63 (7days after the third dose). *, P � 0.05; **, P � 0.001 (responses compared to baseline).

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with 50% of the vaccinees exhibiting a �4-fold increase after thefirst dose. The other WCV vaccine, SsWC, yielded a relativelysimilar 60% (3 of 5 volunteers) IgA antibody response to LPS infecal samples after immunization. IgA responses measured usingfecal samples might have a role in assessing protection; however,an association has not yet been established (8, 34).

Despite many studies, the potential for other antigens, such asthe Ipa proteins, to elicit protective immunity remains unclear. Inour study, Sf2aWC vaccine elicited low IgA and IgG immune re-sponses to Ipa in serum and ALS samples. The preclinical studieswith Sf2aWC also observed relatively meager humoral responsesfor IpaB, IpaC, or IpaD after immunization with inactivatedwhole Shigella cells in mice regardless of the different conditionsused to inactivate the vaccine (31).

Oral immunization with inactivated Sf2aWC resulted in therapid production of several cytokines, including the signature Th1and Th17 cytokines IFN- and IL-17A, respectively. The serumcytokine responses occurred very quickly (within 8 h) after thefirst vaccine dose and would likely have been missed without this8-h specimen. To our knowledge, this is the first study to measurethe cytokine responses after vaccination with Shigella in humanswhere a time point before 24 h was analyzed. Interestingly, severalcytokines exhibited peak expression at 8 h after the first vaccina-tion and then declined by 24 h postvaccination. Thus, the studieswhere cytokines were measured starting at 24 h or later might havemissed the earlier responses.

Our vaccine induced IL-17A after the first dose. In a pulmo-nary mouse model, Sellge et al. in 2010 showed that IL-17A mightplay an important modulatory role in anti-Shigella protective im-munity to Shigella infection (37). TNF- contributes to epithelialdestruction in experimental shigellosis. Interestingly, in our study,the Sf2aWC vaccine induced only a transient significant increasein TNF- levels after the first dose. IFN- as an indicator of Th1immunity was significantly induced by Sf2aWC. Similar resultswere also observed after experimental challenge of naive U.S. vol-unteers with Shigella sonnei, where IFN-, IL-2, and TNF- weresignificantly increased after infection (38).

We also observed a relationship between IFN- levels and serumand fecal antibody responses. Earlier studies showed that Ifngr1-defi-cient mice have impaired mucosal immune responses (39).

Several studies have revealed the central role played by IL-8 inneutrophil recruitment and inflammation in experimental shigel-losis. In a previous rabbit study using ligated ileal loops, IL-8 wasshown to be expressed in infected intestinal cells exposed to anoninvasive Shigella mutant. This implies that a significant pro-portion of IL-8 was produced in response to extracellular activa-tion either by LPS or, indeed, by inflammatory cytokines (40).Interestingly, in our study, IL-8 expression level was never in-creased over baseline but rather decreased at 8 h after the first doseand came back to baseline at day 56 (28 days after the seconddose). The decrease of IL-8 levels in our study might be due torecruitment of the IL-8-expressing and/or -responding cells fromthe periphery to the tissues, taking free IL-8 with them. A decreaseof IL-8 was also shown to be associated with the trivalent inacti-vated influenza vaccine (41). Notably, the anti-inflammatory cy-tokine IL-10 showed a similar pattern but in the opposite direc-tion. IL-10 levels were significantly increased at 8 h after the firstdose and were decreased at day 56 when the proinflammatorycytokine IL-8 was increased. IL-10 is known to inhibit productionof a wide range of cytokines, including IL-8 (42). Cytokine re-

sponses for GM-CSF, IL-12p70, and IL-1� could not be detectedin this study. This might be because we missed related time points.

Overall, these data suggest that this vaccine rapidly induced aprofile of serum cytokines which are known to play an importantrole in generating protective immunity to infection. For most ofthese cytokines, responses were transient. These short-lived re-sponses were consistent with the excellent safety profile observedfor the vaccine, since persistent increases would have likely beenassociated with a higher degree of systemic and/or local gastroin-testinal side effects. A very similar pattern was observed in volun-teers immunized with injectable influenza vaccine, where fre-quent blood samples postimmunization demonstrated an earlycytokine increase within 3 h of dosing that were back to baselinewithin 24 h (41). In a follow-up to these initial observations, fur-ther studies of this inactivated vaccine will be needed to betterappreciate the significance of these cytokine changes and the im-pact on safety and immunogenicity.

In conclusion, this phase I trial demonstrated that Sf2aWCvaccine, a relatively simple economical and safe approach, effec-tively presented LPS and other accessible antigens of Shigella to themajority of immunized subjects. When given orally, it was ex-tremely well tolerated and induced both systemic and mucosalimmune responses to LPS. These immune responses were compa-rable to those induced by a live attenuated Shigella vaccine whichwas protective in prior human challenge studies performed byother investigators. Thus, the Sf2aWC vaccine deserves furtherevaluation as a Shigella vaccine candidate alone or in combinationwith other clinically important species and serotypes to prevent S.flexneri epidemics.

ACKNOWLEDGMENTS

We thank all of the volunteers who participated in this study. The devotedefforts of Fatuma Mawanda, George Gomes, Alicia Cage, and Denise Ad-ams were invaluable. We thank A. Louis Bourgeois and David Taylor forreviewing the manuscript and providing guidance.

The study was supported by PATH and the United Kingdom Depart-ment for International Development.

FUNDING INFORMATIONPATH provided funding to Subhra Chakraborty, Clayton D. Harro, Bar-bara DeNearing, Jay H. Bream, Nicole Bauers, Len Dally, Jorge Flores,Lillian Van De Verg, David A. Sack, and Richard I. Walker under grantnumber GAT.1371-08887-CTA.

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