Live-attenuated Shigella vaccines

Review

10.1586/14760584.5.5.669 © 2006 Future Drugs Ltd ISSN 1476-0584 669www.future-drugs.com

Live-attenuated Shigella vaccines

Malabi M Venkatesan† and Ryan T Ranallo

†Author for correspondenceDivision of Bacterial and Rickettisal Diseases, Walter Reed Army Institute of Research, 503 Robert Forney Drive, Room 3s12, Silver Spring, MD 20910, USATel.: +1 301 319 9764Fax: +1 301 319 [email protected]

KEYWORDS: challenge studies, immunogenicity, live invasive Shigella vaccine, safety

Several live-attenuated Shigella vaccines, with well-defined mutations in specific genes, have shown great promise in eliciting significant immune responses when given orally to volunteers. These responses have been measured by evaluating antibody-secreting cells, serum antibody levels and fecal immunoglobulin A to bacterial lipopolysaccharide and to individual bacterial invasion plasmid antigens. In this review, data collected from volunteer trials with live Shigella vaccines from three different research groups are described. The attenuating features of the bacterial strains, as well as the immune response following the use of different dosing regimens, are also described. The responses obtained with each vaccine strain are compared with data obtained from challenge trials using wild-type Shigella strains. Although the exact correlates of protection have not been found, some consensus may be derived as to what may constitute a protective immune response. Future directions in the field of live Shigella vaccines are also discussed.

Expert Rev. Vaccines 5(5), 669–686 (2006)

Shigella spp. are the causative agent of bacillarydysentery, a severe and sometimes life-threatening disease whose symptoms rangefrom watery diarrhea, abdominal cramping,fever, nausea, headache and tenesmus, to full-blown dysentery in which there is frequent pas-sage of small-volume bloody, mucoid stools.The areas of the gastrointestinal tract (GIT)most affected during shigellosis are the distalend of the colon and rectum, which showintense and acute mucosal inflammation. Avery low inoculum of 10–100 bacteria is suffi-cient to cause the disease and the bacterium isspread easily, either directly by the fecal–oralroute or by contaminated food and water andflies [1]. Approximately 90% of Shigellainfections occur in the developing world. Inhealthy adults, the illness is generally self-limited, with most severe symptoms occurringin the first 2 days and resolving within 1 week.Prolonged illness and chronic carriage of Shig-ella is unusual but has been described in mal-nourished children. Appropriate antibiotics areused to combat infection; however, as withmost bacterial pathogens, an increase in anti-biotic resistance has dramatically emphasized

the need for a safe and effective vaccine [2,3].The market for such a vaccine would probablyinclude travelers and military service personnel,as well as children living in endemic areas.

Epidemiological data estimate that some165 million cases of shigellosis occur world-wide, predominantly in developing countries,where the morbidity and mortality primarilyaffect children under 5 years of age [4,5].Shigella is classified into serogroups and sero-types based on antigenic differences in theO-antigen polysaccharide of the outer mem-brane-associated lipopolysaccharide (LPS).There are four major serogroups of Shigellaand each serogroup includes several types andsubtypes. Shigella dysenteriae (group A,17 serotypes) includes S. dysenteriae 1, whichproduces a potent cytotoxin, (Shiga toxin,encoded by the stxAB genes) and is responsiblefor epidemic outbreaks of the disease. Systemicspread of the Shiga toxin causes renal endo-thelial cell toxicity and is thought to beresponsible for hemolytic uremic syndrome.Shigella flexneri (group B, 14 serotypes) isendemic in developing countries and accountsfor most Shigella infections worldwide,

CONTENTS

Shigella pathogenesis

Immune response to infection with Shigella spp.

Noninvasive & hybrid Shigella vaccines

Immune responses seen in challenge studies

Immune responses induced with live, invasive Shigella vaccines

Clinical trials with live-attenuated Shigella vaccines at the Center for Vaccine Development

Clinical studies at the Walter Reed Army Instituteof Research

Future directions

Summary

Expert commentary

Five-year view

Key issues

References

Affiliations

http://www.future-drugs.com

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Text Box

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Venkatesan & Ranallo

670 Expert Rev. Vaccines 5(5), (2006)

whereas Shigella sonnei (group C, 1 serotype) is foundprincipally in outbreaks in industrialized countries such as theUSA [4,6]. Shigella boydii (group D, 20 serotypes) is restrictedmostly to the Indian subcontinent [7]. Data from previous clin-ical trials suggest that naïve and endemic populations mayrequire vaccines with highly different potency or characteristicsowing to, among other factors, pre-existing antibody titers inendemic areas [BAQUI ET AL., UNPUBLISHED OBSERVATIONS] [8].These data, along with the multiplicity of serogroups and sero-types, highlight the complexity of inducing immunity toenteric pathogens, such as Shigella.

Shigella is a Gram-negative pathogen and sequencing data ofhousekeeping genes, as well as data from recent genomicsequencing, indicate that Shigella should be considered a patho-type of Escherichia coli [9]. Shigella is capable of orchestrating itsown uptake into nonphagocytic epithelial cells, a process termedinvasion, through secretion of proteins that are highly conservedin all virulent strains. These proteins are encoded by a large213-kb plasmid, referred to as the invasion plasmid. A 30-kbregion on the invasion plasmid encodes the ipa–mxi–spa genes,which are critical for the invasive phenotype. Most of theMxi–Spa proteins assemble into a type III secretion system(TTSS) apparatus through which the invasion plasmid antigen(Ipa) proteins (IpaA, IpaB, IpaC and IpaD), as well as severalother proteins, are secreted into the host cell cytoplasm(reviewed in [10–12]). The Ipa proteins and the VirG, or IcsA,protein (see below) are the major protein antigens against whichantibodies are detected in sera from infected individuals [13].Most of the proteins secreted through the TTSS are encodedoutside the 30-kb region on the invasion plasmid. Currently,these secreted proteins constitute the focus of much of theresearch regarding mechanisms of Shigella pathogenesis.

Recent Shigella vaccine candidates include subcellular com-plexes purified from virulent cultures (Invaplex) [14,15], detox-ified LPS conjugated to carrier proteins [16,17] and live-attenuatedvaccine strains [8,18–28]. Whole-cell inactivated organisms given inhigh and multiple doses continue to undergo clinical testing,although, historically, these vaccines have met with littlesuccess [29]. Live vaccines are predicated to be successful on theobservation that immunity can be achieved naturally following about of shigellosis. This situation is often the case for people,including young children, who live in endemic areas [30,31].Other evidence for natural infection-induced immunity hascome from volunteer challenge studies [32,33]. Thus, live vaccinesseek to mimic the natural infection while subverting the clinicaloutcome. In this review, we will focus primarily on data from theclinical trials of genetically defined Shigella-based, live, invasivevaccines. For a more complete review of pathogenesis and vac-cine development, there are several excellent articles writtenrecently on both of these subjects [12,34–36].

Shigella pathogenesisVarious in vitro and in vivo findings have contributed to thecurrent understanding of Shigella pathogenesis. The concept isderived primarily from data collected using sophisticated cell

culture models of infection [37], as well as animal modelsincluding ligated ileal loops in rabbits, conjunctival infectionin guinea pigs, pulmonary infection in mice and intestinalinfection in nonhuman primates.

In humans, a challenge dose of 10–1000 virulent organisms,preceded by a bicarbonate buffer to reduce gastric acidity, issufficient to consistently induce the symptoms ofshigellosis [33]. The gastrointestinal tract (GIT) is the portal ofentry for Shigella and located within the follicle-associated epi-thelium are thought to be the specialized antigen sampling Mcells. The M cell forms an apparent pocket at the basal mem-brane site where T cells, B cells, macrophages and dendriticcells are situated. The bacteria are delivered by the M cell to theunderlying antigen-presenting cells (APCs), such as macro-phages and dendritic cells (FIGURE 1). This is the initial exposureof the immune cells to bacterial antigens (LPS, Ipa etc.), whichinitiates the innate immune response.

Virulent Shigella spp., with the help of IpaB, move out ofthe phagocytic vacuole and exert a cytotoxic effect on macro-phages that coincides with the release of the bacteria, as wellas proinflammatory cytokines, such as interleukin (IL)-1β,tumor necrosis factor (TNF)-α and IL-18 (FIGURE 1) [38]. Thebacteria invade adjacent intestinal epithelial cells at the baso-lateral side through a process that requires the interaction ofseveral bacterial proteins as well as multiple signaling mole-cules within the host cell (FIGURE 1). In particular, the surface-localized bacterial Ipa proteins, particularly IpaB and -C, playa critical role during the initial contact with the intestinal epi-thelial cell. This interaction activates pathways that lead tohost cytoskeletal rearrangements, which result in the engulf-ment of the bacterium within an endocytic vacuole (host-cellinvasion). Subsequent lysis of the vacuole sets the bacteriumfree within the epithelial cell cytoplasm, where it begins toreplicate with a doubling time of 40 min (FIGURE 1) [37]. At thesame time, the bacteria move within the cell and from one cellto the adjacent epithelial cell with the help of the intercellularspread (Ics) A protein, also known as VirG. The VirG (IcsA)protein is normally expressed at the old pole of the bacterialcell and forms the nucleation site for the polymerization ofhost cell actin filaments, which provides the motive force forbacterial movement [10,12]. During the transition from one cellto the next, the bacterium is enveloped in a double membranefrom which it escapes into the adjacent cell cytoplasm aidedby invasion plasmid protein IcsB [39]. Once into the neighbor-ing cell, the bacteria continue to replicate and spread withoutbeing exposed to the hostile environment of a cascadinginflammatory response. The events described above arepresented in FIGURE 1.

The initial response to infection with Shigella spp. is primarilyinflammatory in nature and the principal factor leading thisresponse is bacterial LPS. In particular, the lipid A componentof LPS, through its multifaceted interactions with numeroussoluble and membrane-bound receptors, is the actual transducerof the proinflammatory cytokine production. Extracellular LPS,present throughout the infection cycle, is picked up by LPS

Live-attenuated Shigella vaccine

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binding protein and presented to CD14 on the macrophageswhere it interacts with the cell surface Toll-like receptor(TLR)-4, and MD–2 [40,41]. Activation of TLR4 leads to activa-tion of the transcription factor NF-κB, which stimulates theproduction of IL-1β, TNF-α and IL-6 by phagocytic and endo-cytic cells [42]. Intracellular LPS and peptidoglycan shed duringintracellular bacterial growth also activate proinflammatorycytokine production through mechanisms independent ofCD14 and TLR4. In the case of peptidoglycan, signalingappears to be through Nod1, which also leads to activation ofNF-κB, as described above [43,44].

In vitro studies have indicated that epithelial cells can beinduced, upon infection with Shigella spp., to secrete IL-8,monocyte chemotactic protein-1, granulocyte-macrophagecolony-stimulating factor and TNF-α, as assessed by mRNAlevels and cytokine secretion (FIGURE 1) [45,46]. All of these fac-tors are associated with chemotaxis and activation of inflam-matory cells. In response to cytokine and chemokine secretionat the site of infection and bacterial dissemination, a massive

infiltration of polymorphonuclear leukocytes or neutrophilsoccurs. The migration of neutrophils from the capillaries onthe basal side of the epithelium through the epithelial barrierto the lumen of the GIT, results in the loosening of the epi-thelial tight junctions and loss of barrier functions, furtherenabling the luminal bacteria to access the basolateral side ofthe epithelium. The VirG(IcsA)-assisted intra- and inter-cellu-lar spread of the bacterium within the epithelial tissue contrib-utes significantly to the loss of epithelial cell integrity andaccompanying tissue injury. Thus, virG(icsA) mutants aresignificantly attenuated in all animal models of virulence [47].The influx of polymorphonuclear leukocytes eventually killsthe bacteria and resolves the infection.

Immune response to infection with Shigella spp.Approximately 7–14 days following exposure, the adaptivehumoral and mucosal response to the bacterial antigens, LPSand the Ipa proteins, can be measured as serum antibodies ofall three immunoglobulin classes (IgA, -G and -M), as

Figure 1. Representation of Shigella uptake at the mucosal epithelium. The bacteria are taken up by M cells that line the gastrointestinal tract and are transcytosed to the underlying phagocytic cells. The bacteria kill the macrophages and invade intestinal epithelial cells. Once inside the host cell, the bacteria replicate and move about the cell, as well as from one cell to another. Some of these steps have been targeted for the attenuation of wild-type strains. These steps are numbered and include: Step 1: secretion of the enterotoxins (sen and set). Step 2: invasion through the basolateral side of enterocytes (guaBA). Step 3: intracellular multiplication (aroA, aroD, guaBA). Step 4: intra- and intercellular spread by virG(icsA). DC: Dendritic cell; IL: Interleukin; TNF: Tumor-necrosis factor.

IL-1βIL-18TNF-α

Macrophages,DC, T and B cells

M cell

Enterotoxin secretion

Enterocyte

Replication

Bacterial invasion

Intracellular spread

IL-8

1

2

3

4



antibody-secreting cells (ASCs) in the blood (measured inperipheral blood mononuclear cells [PBMCs]) and as fecalsecretory IgA (sIgA) [48]. However, the initial innate responseis characterized by elevated levels of interferon (IFN)-γ,which can be observed in stool, serum and rectal biopsies ofpatients with acute shigellosis, while levels of the samecytokine and the mRNA for IFN-γ receptor are upregulatedin the biopsies of patients convalescing from shigellosis [49].More recently, measures of cell-mediated immune responsesin PBMCs in the form of IL-10 and IFN-γ production havealso been detected in challenge studies and vaccinetrials [24,25,50]. Such studies indicate that the correlates of pro-tection for shigellosis, although not defined precisely, proba-bly involve a combination of humoral, mucosal andcell-mediated immune responses to the bacterial antigens.

Immune responses to Shigella antigens in endemic versusnaïve populations demonstrated quantitative differences. Meas-urement of antibodies to LPS and the Ipa proteins in a Chileanpopulation aged 1–16 years old indicated that the antibodylevels to both antigens in this endemic population increased inan age-related manner [51]. Antibody levels to S. flexneri 2a LPSranged from a mean optical density (OD) of 0.4 in infants to apeak OD of 1.2 in the 15-year-old group. The prevalence ofantibody levels against Shigella Ipa also increased with age, from28% in 1-year old children to 100% in children aged 10 years orolder. IpaB was recognized most frequently. Recognition of allfour Ipa proteins was also age-regulated. When similar measure-ments were made in a presumably naïve US population livingoutside Baltimore (MD, USA), antibodies to LPS were low ininfants (mean OD of 0.1) and adults (mean OD of 0.4), whilenone of the US infants and only 38% of the adults tested posi-tive for Ipa. Data similar to that obtained in the Chilean popula-tion have also been gathered from other areas of the world. Suchdata suggest that the general levels of anti-shigella immunity arehigher in adults and children living in an endemic region andthat infants are most susceptible to shigellosis in these areas.Adults living in endemic regions are exposed to Shigella antigensrepeatedly as compared with a naïve population, such as in theUSA [52–54]. Therefore, vaccines that elicit a significant immuneresponse at a particular dose in a naïve population may not do soin an endemic population. One remedy may be to try higherdoses of the same vaccines in an endemic population.

Another significant challenge in developing an effective livevaccine against shigellosis is the need to address the occurrenceof over 50 different Shigella serotypes, considering thatprotection appears to be based on LPS and is mostly serotype-specific [55]. Cross-reactivity of S. flexneri 2a antibodies withother S. flexneri antigens and, more importantly, combinationvaccine studies in guinea pigs immunized with differentS. flexneri serotypes have suggested that a mixture of attenuatedstrains of S. flexneri 2a, 3a and 6 may protect broadly against allS. flexneri [56–58]. With the addition of vaccines against S. sonneiand S. dysenteriae 1, a pentavalent cocktail of five Shigellavaccine strains could protect against most of the disease seenworldwide [58].

Noninvasive & hybrid Shigella vaccinesAttempts in the late 1960s and early 1970s to construct live-attenuated vaccine strains were not based on specific knowl-edge of pathogenesis, but were rather nonspecific approachesto induce high levels of anti-LPS serum antibodies. At thetime, it was known that natural infection induced high levelsof serum anti-LPS antibodies. What was not clear was theimportance of host cell invasion, immune responses to theIpa proteins and the role of mucosal, cell-mediated immuneas well as systemic immune responses in protection. Some ofthe live, noninvasive vaccine candidates that emerged fromthese efforts were the streptomycin-dependent strains,S. flexneri 2a strains 2457O and T32-ISTRATI. Streptomy-cin-dependent vaccines were developed for S. flexneri 1, 2a,3a, 4 and S. sonnei. Large studies in both adults and childrenin the former Yugoslavia showed that the vaccines were pro-tective in 82–100% of cases [59–61]. The protective efficacy ofthe T32-ISTRATI strain given as enteric coated capsulesranged from 70 to 100% over 12 separate field trials in East-ern Europe and China and the efficacy was based on histori-cal data of disease [62]. Another candidate, 2457O, was alsoimmunogenic when given at high doses. Some of these vac-cines were not tested in placebo-controlled studies in theUSA. These vaccine candidates were not pursued further andsuffered from issues related to inadequate genetic characteri-zation, wild-type reversion, genetic instability, repeated highdose regimens, reactogenicity and poor or highly variableprotective efficacy [32,63–65].

The next set of live vaccines tested were E. coli–Shigellahybrid vaccines, designed to maintain the invasive phenotypeof Shigella in an E. coli background. It was becoming clearthat an invasive vaccine strain was necessary to induceimmune responses that mimic responses seen during naturalinfection. The vaccine strain EcSf2a-2 was generated by trans-fer of the S. flexneri 5 invasion plasmid and the S. flexneri 2aLPS biosynthesis genes into an E. coli K-12 strain [66].Although EcSf2a-2 was well tolerated in humans whenadministered in multiple doses at 109–1010 colony formingunits (cfu), it eventually proved to be unstable and demon-strated poor protective efficacy [21]. Another hybrid strain5076–1C consisted of a Salmonella typhi–S. sonnei bivalentvaccine that contained the S. sonnei invasion plasmid, encod-ing the Form I O-antigen, in a S. typhi Ty21a oral vaccinebackground [67]. Although initial studies, administering threedoses of 1–8 × 109 cfu demonstrated protection, lot-to-lotvariability was seen and later, the vaccine candidate wasshown to have lost segments of the invasion plasmid [67,68].

A live, oral bivalent S. flexneri 2a and S. sonnei vaccine (FS)that was developed at the Lanzhou Institute of Biological Prod-ucts (Gansu, People’s Republic of China) remains, since 1999,the only licensed Shigella vaccine available; it can be purchasedin China. The O-antigen genes of S. sonnei were cloned into anexpression vector and the plasmid transformed into the nonin-vasive S. flexneri 2a vaccine strain T32-ISTRATI. Large fieldstudies in China have demonstrated 61–65% protection



against S. flexneri 2a, 57–72% protection against S. sonnei and48–52% protective efficacy against heterologous Shigellaspp. [201]. The vaccine is given in a regimen of three doseswithin a 2-week time period. However, the use of multiple highdoses of the vaccine strain (>2 × 1010 cfu), as well as its price(∼US$10 per vaccination) remains problematic. Further fieldstudies of the FS vaccine in toddlers and infants may helpdefine the public health application of this vaccine inChina [201].

The elucidation of the role of various bacterial virulencefactors has led to a clearer understanding of Shigella patho-genesis, therefore, allowing for the construction of rationallydesigned and genetically well characterized vaccine strains.Each of the candidate strains described below have beenattenuated specifically to eliminate one or more of the stepsinvolved in pathogenesis (FIGURE 1). For practical reasons, ourreview of the literature will be limited to Shigella-based live,invasive vaccine strains that have been tested in clinical trials.To determine efficacy of vaccine strains, a human challengemodel has been developed where volunteers ingest virulentShigella spp. and are monitored for symptoms of the diseaseand the immune responses to the infection. These studies arealso discussed below.

Immune responses seen in challenge studiesSeveral volunteer studies, as well as studies in monkeys andguinea pigs, have demonstrated that infection with a particularserotype confers a high level of protection against the same sero-type, demonstrating that bacterial LPS is the main protective

antigen [55,64,69,70]. Early live Shigella vaccine efficacy trials fol-lowed a protocol in which the challenge strain was administeredorally in skimmed milk [32,67,71]. The collective data from thesestudies revealed an inconsistent attack rate in the naïve controls,leading to compromised efficacy data [72]. Subsequent challengestudies were carried out with the goal of establishing a moreconsistent and higher attack rate so that fewer volunteers wouldbe needed to demonstrate efficacy during vaccine trials [33].

Using the virulent S. flexneri 2a strain 2457T, doses of1.4 × 102 and 1.4 × 103 cfu were administered to naïve and pre-viously immunized adult volunteers designated ‘veterans’ [33].The veterans consisted of two different treatment groupsrecruited from a previous clinical trial. The first treatment group(vaccinated veterans) had been vaccinated with EcSf2a-2 andsubsequently challenged with 2457T. The second treatmentgroup (unvaccinated veterans) consisted of control individualswho had only received 2457T. All of the volunteers in the earlierstudy had developed shigellosis, demonstrating the poor efficacyof EcSf2a-2 [21]. In this later study, naïve and veterans fasted for90 min before and after ingestion of the challenge strain, whichwas administered orally in 30 ml of a bicarbonate buffer and waspreceded by ingestion of 120 ml of the same buffer to reducegastric acidity. The attack rate of any illness (fever, diarrhea ordysentery) in naïve controls given 103 cfu was 92% (11 out of12) compared with 17% (one out of six) in the unvaccinatedveterans and 40% (two out of five) in the vaccinated veteransgroup (FIGURE 2). This not only demonstrated a high attack ratewith 2457T but also a significant reduction in the illness rate(protection) among the veterans group. The attack rate in naïve

Figure 2. Reactogenicity profile to wild-type strains and live-attenuated Shigella vaccine strains tested at CVD and WRAIR. The data were derived from the clinical trials summarized in Table 1 and represent the percentage of volunteers (y-axis) with specific reactogenicity (diarrhea, fever and dysentery) at immunogenic doses for each strain (x-axis). In addition, the reactogenicity to wild-type strains 2457T and 53G is also indicated as a means to compare the relative safety profiles for each vaccine strain [18,20,22–25,28,33,75]. CVD: Center for Vaccine Development; WRAIR: Walter Reed Army Institute of Research.

0

20

40

60

80

100

% w

ith

sym

pto

ms

Fever Diarrhea Dysentery

102 103 103 103/4 104 108 109 1010 108 109 108 109 102 103 104 103 104

2457T SC602 CVD 1207CVD 1203 CVD 1204 CVD 1208 53G WRSS1



controls at the 102 dose was significantly lower (43%). Naïvevolunteers challenged with the 103 cfu dose developed aLPS-specific geometric mean of approximately 240 IgA ASCs,approximately 25 IgM ASCs and 58 IgG ASCs per 106 PBMCs,while the Ipa-specific response in the same category was lower,in the range of 175, 16 and 16 ASCs per 106 PBMCs for thesame three Ig classes, respectively (TABLE 1) [21]. In all cases, theveterans had much lower ASC numbers. For example, the geo-metric mean anti-LPS IgA ASC response on day 7 amongunvaccinated veterans and vaccinated veterans was 9 and 57,respectively, compared with 240 in naïve controls. The geomet-ric mean peak excretion was also significantly lower in veteranscompared with naïve controls, even though 100% of the veter-ans and naïve volunteers adminstered with 1.4 × 103 cfu wereculture positive. The lower excretion rates along with the lowerASC numbers in the veterans group was taken to indicate thatan active mucosal immunity was present in this group, prevent-ing adhesion and invasion of the organisms, reducing furtherantigenic stimulation and minimizing mucosal injury [73].Therefore, this study provided several pieces of useful informa-tion, including an effective challenge dose of 103 cfu of virulent2457T and a protocol for its administration, which is generallyfollowed in most Shigella clinical trials today. 2457T was alsogiven as a challenge to naïve volunteers during efficacy trials ofSC602 (described below) and recently during the testing ofrifaximin, an oral nonabsorbed antibiotic [74].

A second study using a virulent S. sonnei strain described acutesymptoms and immune responses in 11 volunteers challengedwith 5 × 102 cfu of strain 53G, which was administered inskimmed milk [75]. The attack rate at this dose was 55% (6 out of11) with a total of nine, including the six symptomatic volun-teers, excreting the organism (FIGURE 2). All six ill volunteers hadmarked increases in plasma levels of markers associated withinfection and inflammation, such as TNF-α, IFN-γ and C-reac-tive protein, and four volunteers also had increased IL-2 receptorlevels. No significant increases in serum IL-1β were noted in thisstudy. Bacterial excretion in stools was the earliest measure ofinfection, occurring within 24 h of dose administration, followedsequentially by increases in serum TNF-α, IFN-γ and C-reactiveprotein. Eight out of nine culture-positive individuals alsoshowed increased LPS-specific IgA ASCs, while six out of ninehad significant increases in serum IgA (63% response rate) orIgG (83% response rate) [75]. Volunteers who were ill had moresustained increases of all cytokines and immune responses thaninfected but asymptomatic individuals. Since the ill volunteerswere treated with antibiotics soon after either excretion of theorganism in the stool or when the symptoms became evident, theimmune response data were somewhat compromised to reflectonly that occurring during the early phase of shigellosis [75]. Evenso, several interesting observations were made regarding markersof infection and immune responses.

In the early 1970s, virulent epidemic strains of S. dysenteriae 1were administered to adult male volunteers in skimmed milk[76]. As few as ten organisms were sufficient to cause dysenteryin some volunteers. In these studies, a nontoxigenic (Shiga

toxin-negative) but invasive derivative also caused clinical dis-ease in half of the volunteers, while a Shiga toxin-containing,non-invasive strain did not cause dysentery. This finding estab-lished that the invasiveness of Shigella is a prerequisite for diseaseand that Shiga toxin was not required for causing dysentery [76].More recently, another challenge study has been reported usingthe S. dysenteriae 1 strain SC595 [50]. SC595 was derived fromS. dysenteriae 1 strain 7/87 by the deletion of the stxA gene, whileretaining the stxB subunit, which was engineered to be expressedfrom the P1 promoter of pBR322 [50]. Four groups of volunteerswere administered 3 × 102, 7 × 103, 5 × 104 or 7 × 105 cfu ofSC595 in a bicarbonate buffer, in a sequential fashion. At thehighest dose, only one out of six volunteers had low-grade fever,while an additional two had diarrhea, indicating that SC595 wasnot virulent enough to cause shigellosis [50]. There were no anti-LPS or anti-Ipa ASC responses at the lowest dose given, whileASC responses in the remaining groups were of similar magni-tude. There was a dose response in the mean postinoculationanti-LPS IgA, IgG and IgM antibody titers that did not reachstatistical significance given the wide distribution of responsesamong the volunteers in each dose group [50]. There was no refer-ence to elicitation of anti-StxB antibody. One interesting obser-vation made in this study was the detection of significant levels ofIL-10 and IFN-γ when PBMCs from challenged volunteers wereexposed in tissue culture to both bacterial homogenate and par-ticulate prep-arations, as well as to purified IpaC and IpaD [50].At the same time, there was minimal or no production of IL-2,-4, -5, -12 or -15, supporting the idea of a predominantlyT helper (Th)1 response in shigellosis. However, since none ofthe volunteers met the clinical definition of shigellosis, this chal-lenge study needs to be repeated with a more virulentS. dysenteriae 1 strain.

Immune responses induced with live, invasive Shigella vaccinesBy the beginning of the 1960s it was becoming clear that theinvasiveness of Shigella spp. was an important facet of itspathogenesis, which was attributed to the presence of the largeplasmid [77,78]. Rabbit ileal loop studies had shown theimportance of Peyer’s patches, which are part of the gut-associated lymphoid tissue, in the local immune response to livebacteria [79]. Later studies showed that an invasive S. flexneri 5strain M9OT, injected into a rabbit ileal loop, invaded bothPeyer’s patches (through M cells) and villous epithelium andcould be recovered within 2 h, with four-fold more organismsbeing recovered from Peyer’s patches. An isogenic, plasmid-cured, noninvasive derivative, BS176, could only be detectedafter 12 h, at which time eight-fold more organisms of the inva-sive strain was seen in the Peyer’s patches and four-fold more inthe villous epithelium compared with BS176 [80]. In monkeys,failure of protection with parenteral vaccines and local protectionachieved with a live, invasive Shigella strain further establishedthat an invasive strain should be more efficient in delivering LPSand other antigens to the gut-associated lymphoid tissue than anoninvasive or a poorly invasive strain [55,63,81].



Tabl

e 1.

Sum

mar

y of

imm

une

resp

onse

s fr

om c

linic

al t

rials

of

live,

inva

sive

vac

cine

str

ains

tes

ted

by C

VD a

nd W

RAIR

.

Stud

ySt

rain

/vac

cine

Sero

type

Dose

(s)*

Geom

etric

mea

n of

ASC

per 1

06 PBM

C

(%

res

pons

e)%

with

thr

ee-

or f

our-

fold

incr

ease

in

seru

m t

iter

(no

. of

resp

onde

rs/t

otal

no.

)%

wit

h th

ree-

or

four

-fo

ld in

crea

se in

ant

i-LP

S fe

cal/u

rine

tite

r

Ref.

LPS

Ipa

LPS

Ipa

LPS

Ipa

IgA

IgG

IgA

IgG

IgA

IgG

IgA

IgG

sIgA

One

2457

TS.

flex

2a

102

19(7

1)N

DN

DN

DN

/AN

/AN

/AN

/AN

/AN

/A[3

3]

103

239(

92)

58†

175†

19†

N/A

N/A

N/A

N/A

N/A

N/A

[33]

Two

2457

TS.

flex

2a

103

172(

85)§

14(5

7)§

ND

ND

71(5

/7)

57(4

/7)

ND

ND

86(6

/7)

[21]

One

SC60

2S.

flex

2a

103/

4¶30

(75)

#11

(58)

#N

DN

D47

(22/

46)

15(7

/46)

ND

ND

ND

[18]

Two

SC60

2S.

flex

2a

104

17(5

8)§ 42

**6(

41)§ 9*

*N

DN

D50

(6/1

2)33

(4/1

2)N

DN

D33

(4/1

2)[2

0]

One

WRS

S1S.

sonn

ei10

399

307

471

8614

017

83[2

5]

104

395

2827

5050

5025

00

[25]

Two

WRS

S1S.

sonn

ei10

330

40N

DN

D47

20N

DN

DN

DN

D[2

8]

104

7546

ND

ND

6027

ND

ND

ND

ND

[28]

One

CVD1

203

S. fl

ex 2

a10

8††

43(9

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D12

(64)

ND

4045

7070

7040

[22]

109†

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D77

(91)

ND

3636

3654

450

[22]

One

CVD1

207

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1035

(100

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11§§

ND

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[23]

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108

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[24]

109

445(

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310(

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67(1

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71, 2

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109

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(71)

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[24]

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Clinical trials with SFL124 & SFL1070During the late 1980s, investigators at the Karolinska Institutein Sweden developed Shigella vaccines that were attenuated bygene deletions in the common aromatic biosynthesis pathway.SFL124, a S. flexneri serotype Y vaccine strain, was derivedfrom SFL114, which was auxotrophic for p-aminobenzoic acid(PABA), a precursor of folic acid due to an aroD:Tn10mutation [82,83]. aroD encodes the enzyme 3-dehydroquinatedehydratase and folic acid is a cofactor in multiple essential bio-chemical reactions, including the de novo biosynthesis pathwayof purines. Since eukaryotic cells do not contain PABA, abacterial aroD mutant is not expected to multiply withineukaryotic cells and is therefore attenuated. The advantage ofdeveloping a serotype Y vaccine strain is that it can beconverted to the other S. flexneri serotypes, using glucosylatingand/or acetylating phages [84–89]. Owing to a low frequency ofreversion of SFL114, SFL124 was derived from SFL114 by lossof the Tn10 and a 1.4-kb deletion in the aroD gene. SFL124was invasive in HeLa cells and formed plaques (plaque forma-tion in HeLa-cell monolayer is seen as an expression of intra-and intercellular replication) whose sizes were much smallerand more irregular than plaques obtained with the parent wild-type strain SFL1. However, SFL124 was negative in the Serenyreaction in guinea pigs (virulent Shigella strains cause purulentkeratoconjunctivitis when administered ocularly to guinea pigs,termed the Sereny reaction) and no diarrhea was seen inmonkeys given 1 × 1011 cfu.

The lyophilized vaccine SFL124 was reconstituted in distilledwater and diluted in phosphate-buffered saline (PBS) and thepercentage of Congo-red positive colonies, seen as an expres-sion of the number of invasive colonies, in the doses adminis-tered was 95% [27]. During the first clinical trial, SFL124 wasadministered orally to a total of three groups of 21 Swedishadult volunteers who received either one or three doses at2 × 109 cfu (FIGURE 3, dosing regime one and two). A boosterdose was given 9–10 months later to a subset of volunteers whoreceived the three doses (FIGURE 3, dosing regimen three). Five ofthese volunteers had indicated previous exposure (within5–13 years) to an enteric infection with symptoms that wouldqualify as bacillary dysentery. Two out of 21 (10%) volunteershad short-lasting mild fever and diarrhea and six out of21 volunteers (29%) had one or two mild constitutional symp-toms. Between days 7 and 14 there were significant rises inserum antibody titers to S. flexneri Y LPS and a much lowerresponse to the antigens in the water extract (source of Ipa anti-gens) in all three groups. In general, the three-vaccine dose wasmore effective than a single dose in stimulating an immuneresponse against the LPS (FIGURE 3). However, unlike the LPSresponses, Ipa responses, especially of the IgG type, were con-siderably higher in the volunteers exposed to Shigella previ-ously, although the number of volunteers was not large enoughto make a statistical evaluation. The booster dose only slightlyelevated antibody responses. The ASCs increased, beginning onday 4 and peaked on day 7. The number of IgA-specific ASCsto LPS/Ipa per 106 PBMCs in volunteers administered

2 × 109 cfu of SFL124 was 150/40 for the one dose regimen,200/70 for the three-dose regimen and 80/10 for the three-doseregimen plus booster. The lower ASC responses in the boostedgroup was attributed to a vaccination-induced active mucosalimmunity, also reflected by the lowered excretion rate in thisgroup (4 days in the one- and three-dose regimen vs 1.8 days inthe boosted group). Most of the vaccinees showed a fecal sIgAresponse to LPS and Ipa (FIGURE 3). The response was higher inpreviously exposed individuals to both antigens with peak titersbetween days 7 and 14, indicating a mucosal memory responsein this group [27]. In general, more volunteers responded againstboth LPS and Ipa with local sIgA production than with serumantibodies (FIGURE 3). The limited but clearly positive T-cell pro-liferation rates in some vaccinees indicated the activation of acell-mediated immune response (FIGURE 3) [27].

Compared with Swedish volunteers, SFL124 was less reac-togenic and less immunogenic in Vietnamese adultvolunteers [26]. No diarrhea or fever was reported and the excre-tion times were shorter after both primary (FIGURE 3, dosing reg-imens four and seven, average number of days excreted: 2.7)and booster (FIGURE 3, dosing regimens five, six, eight, nine,average number of days excreted: 1) vaccination. That most ofthese volunteers had a probable prior exposure to S. flexneri wasevident from the higher prevaccination serum anti-LPS andanti-Ipa titers. Nonetheless, the mean serum antibody IgA titersagainst S. flexneri Y LPS were significantly elevated after one orthree doses of primary vaccination and again after the boosterdoses given 1 year later. The serum anti-Ipa titer increases weremodest after the booster dose and were significant in only a fewinstances. The strong LPS-specific ASC response seen after pri-mary vaccination was comparable with that seen in Swedishvolunteers (for the three dose group, ASCs for LPS/Ipa were 80out of 150) and the counts after primary vaccination werehigher and persisted longer than after the booster doses [26].These responses suggested that it is more common for the hostto mount a response to LPS than to Ipa antigens and may beone explanation for the observed serotype-specific protectionagainst infection [26]. Conversly, the increases for sIgA to LPSand to the Ipa antigens after the booster dose were significantlyhigher and peaked earlier than after primary vaccination, indi-cating mucosal memory functions that may contribute to thelower excretion rates seen after booster doses [55]. The higheranti-Ipa sIgA responses in the Vietnamese volunteers contrastwith the generally weak responses seen in the Swedish adultvolunteers and may be a reflection of the two populations, oneliving in an endemic area and the other considered naïvevolunteers. Finally, as observed with Swedish volunteers, moreof the volunteers responded with LPS-specific ASCs, sIgA- andIFN-γ-secreting cells than with serum antibody titers (FIGURE 3),indicating that, for a mucosal vaccine, these parameters serve amore useful gauge for an effective primary vaccination thanlevels of serum antibody.

A single dose of SFL124 was also tested in three groups of tenVietnamese children, aged 9–14 years (FIGURE 3, dosing regimenten). The doses ranged from 107 to 109 cfu, with a fourth



group serving as a placebo control [8]. The children drank30 ml sodium bicarbonate–orange juice mixed with or withoutthe vaccine, followed by another 10 ml of just the bicarbo-nate–orange juice (∼pH 7.4). None of the children developedany symptoms that indicated a gastrointestinal infection. Onlymild transient constitutional symptoms were observed in a fewchildren. At the lowest dose, SFL124 could not be recovered byculture from any children. However, using PCR and primerpairs for the ial locus on the invasion plasmid, S. flexneri wasidentified in nine out of ten children with a mean excretiontime of 2.6 days. At the 108-cfu dose, the strain was culturedfrom two children and detected by PCR from nine childrenwith an excretion time of 2.7 days. At the highest dose, therewere three culture-positive children and all ten excreted thestrain based on PCR with a mean excretion time of 3.1 days.

One child who received 108 and five children who received109 cfu showed LPS-specific ASCs and one child and threechildren in each group showed Ipa-specific ASCs (FIGURE 3). Adose-dependent sIgA response to LPS was seen in 60–90% ofthe vaccinated children with peak responses on day 11. An Ipa-specific sIgA response, although significant, was seen in fewerchildren. Since the children had high preexisting antibody titersto S. flexneri Y LPS and water extract, modest and insignificantserum antibody responses were noted [8].

SFL1, the parent strain of SFL124, was only moderately viru-lent by itself, causing dysentery in two out of eight volunteersadministered 7 × 103 cfu. To compare the immunogenicity of avaccine derived from a more virulent strain, S. flexneri 2a strain2457T was used as the parent strain to generate the aroD-deletionstrain SFL1070. SFL1070 has a reduced intracellular

Figure 3. Immune responses to Shigella vaccine strains SFL124 and SFL1070. In each case, only the responses at 2 × 109 cfu are shown. The color-coded bars represent the types of immune responses (shown below the graph). The x-axis shows the percentage of volunteers that responded, while the dosing regimens are shown on the y-axis. The dosing regimens 1–3 and 11–12 refer to testing of SFL124 and SFL1070 in Swedish adults, respectively; dosing regimens 4–9 refer to testing of SFL124 in Vietnamese adults and dosing regimen 10 refers to testing of SFL124 in Vietnamese children. The doses were one or three and in some cases were followed by a booster dose. The dosing regimens are as follows: regimen 1: one dose; regimen 2: three doses on day 0, 2, 4; regimen 3: three doses plus booster dose 9–10 months later; regimen 4: one dose; regimen 5: one dose plus booster at 6 months; regimen 6: one dose plus booster at 12 months; regimen 7: three doses; regimen 8: three doses plus booster at 6 months; regimen 9: three doses plus a booster at 12 months; regimen 10: one dose; regimen 11: one dose; and regimen 12: three doses at day 0, 2, 4. (A) shows the percentage of volunteers who responded to the LPS antigen and (B) shows the percentage of responders to the water extract (or the Ipa proteins) from Shigella. The data have been collated, approximated and reformatted from [8,19,26,27]. ASC: Antibody-secreting cell; CMI: Cell-mediated immunity; LPS: Lipopolysaccharide; sIgA: Secretory immunoglobulin A.

120

100

80

60

40

20

01 2 3 4 5 6 7 8 9 10 11 12

120

100

80

60

40

20

01 2 3 4 5 6 7 8 9 10 11 12

Dosing regime Dosing regime

Serum LPS

sIgA-LPS

ASCs-LPS

CMI-LPS

ASCs-LPS

sIgA-LPS

Serum LPS

A B



multiplication rate compared with 2457T, is Sereny-negative andshows protection against oral challenge in Rhesus monkeys [90].SFL1070 was given to Swedish adult volunteers using a three-dosevaccination protocol similar to that used for SFL124 [19]. Volun-teers were divided into four groups, each receiving three doses of105,107, 108 or 109 cfu (FIGURE 3, dosing regimen 11, 108 cfu;dosing regimen 12, 109 cfu) [19]. At the highest dose, four out ofnine volunteers had diarrhea, fever and severe constitutionalsymptoms after the first dose and these volunteers were not dosedfurther. At 108 cfu, three out of nine volunteers had mild consti-tutional symptoms. A vigorous IgA ASC response to both LPSand water extract was observed in 16 out of 18 volunteers giventhe highest two doses and LPS-specific ASC responses were morecommon [19]. The number of IgA-specific ASCs to LPS/waterextracts or Ipa per 106 PBMCs was 990/80. Significant intestinalsIgA titers to LPS were seen in volunteers given the two highestdoses (78 and 90% responders at the two highest doses), whichpeaked on days 7–9 and declined to prevaccination levels after2 months (FIGURE 3). Although 15 out of 18 volunteers given 108

and 109 cfu of SFL1070 demonstrated serum IgA, IgG and IgMresponses against one of the antigens, these responses were mostcommon at the highest dose and the responses to the Ipa proteinswere less pronounced. All volunteers in the two highest dosesresponded specifically to stimulation of PBMCs with S. flexneri 2apolysaccharide antigen yielding IFN-γ, which was taken to be astrong indicator of the elicitation of cell-mediated immunity [19].In all immune responses, there was no difference seen in volun-teers who had been administered a single dose compared withthose who had received three doses. Overall, sIgA responses werethe most common, followed by ASCs and serum antibodyresponses (FIGURE 3).

The data from the clinical trials of SFL124 and SFL1070illustrated that vaccine candidates attenuated by aromatic auxo-trophy are immunogenic. The reactogenicity of SFL1070 com-pared with SFL124 also pointed out that the virulence of theparent strain is an important consideration in vaccine develop-ment. Furthermore, the safety and immunogenicity profile ofSFL124 in Swedish volunteers versus Vietnamese adults andchildren indicate that naïve and endemic populations react dif-ferently to the same vaccine given at similar doses. This is prob-ably owing to pre-existing titers in the endemic populationwhere there is continuous exposure to the antigens. In such apopulation, children with low maternal antibodies would be themain targets for an oral Shigella vaccine. While SFL124 andSFL1070 were not pursued further, perhaps because of the reac-togenicity seen at immunogenic doses, the concept of incorpo-rating aromatic auxotrophy as a desirable attenuating feature inthe design of Shigella vaccines was utilized subsequently byother investigators (see below).

Clinical trials with live-attenuated Shigella vaccines at the Center for Vaccine DevelopmentBeginning in the 1990s, researchers at the Center for VaccineDevelopment (CVD) at the University of Maryland (MD,USA), designed a series of live-attenuated Shigella vaccines

using the parent S. flexneri 2a strain 2457T. The attenuationswere based on alterations of genes associated with the bio-synthesis of either aromatic amino acids (aroA) or guaninenucleotides (guaBA), with additional deletions in specific viru-lence-associated genes virG(icsA), including the then newlyidentified enterotoxin genes sen and set [23,24]. In all cases, thevaccine strains were harvested freshly from agar plates andadministered to volunteers in a bicarbonate buffer preceded byingestion of the same buffer to reduce gastric acidity.

The first of these to be tested in humans was CVD 1203,which contains a 200-bp deletion in the aroA gene and an addi-tional deletion in the virG(icsA) gene [22]. Like SFL124,CVD 1203 was auxotrophic for PABA and had similar defectsin intracellular growth. The design and construction ofCVD 1203 reflected the safety and immunogenicity dataobtained earlier with SFL124 and SFL1070. Three doses ofCVD 1203 were tested in humans: 1 × 106, 1.5 × 108 and1.5 × 109 cfu (TABLE 1) [22]. The second and third groupsreceived an additional booster dose of 108 cfu on day 14.Approximately 80–95% of the volunteers, given the two higherdoses, excreted the vaccine after the primary vaccination series.Overall, CVD 1203 showed adverse reactions at the two high-est doses (FIGURE 2). Similar to SFL124 and SFL1070, a greaternumber of volunteers responded with ASCs and sIgA to LPSrather than a serum antibody response (TABLE 1). A total of90–100% of the volunteers responded with rises in serumTNF-α titers, while a much lower number of volunteers(18–27%) showed an increase in stool TNF-α levels [22].

Since CVD 1203 proved too reactogenic at doses that pro-vided adequate immunogenicity, alterations in other biosyn-thetic pathways were considered. CVD 1204 contained a spe-cific 918-bp deletion in the guaBA operon [91]. The guaBAoperon is part of the purine metabolic pathway leading to thebiosynthesis of guanine nucleotides. guaB encodes Inosinemonophosphate (IMP) dehydrogenase and guaA encodesGMP synthetase. Compared with 2457T, and unlike CVD1203, CVD 1204 was 2.5 logs lower in invasion efficiencywhen tested in HeLa cells. Additional loss of the virG(icsA)gene generated CVD 1205. Further suicide vector-based dele-tions in the chromosomal gene set, encoding Shigella entero-toxin 1 (ShET1, 459-bp deletion in set 1A encoding the subu-nit with putative enzymatic activity), and the plasmid genesen, encoding Shigella enterotoxin 2 (ShET2, 397-bp deletionin sen), resulted in CVD 1207 [23]. Although CVD 1207 wassignificantly less invasive in HeLa cells and fully attenuated inthe Sereny reaction, it conferred 85% protection against chal-lenge with the wild-type strain in guinea pigs. Groups of threeto seven volunteers were given a single oral dose of CVD1207 starting at 1 × 106 cfu and increasing the dose, one logat a time, to 1010 cfu [23]. The vaccine was well tolerated at thelower doses while at the two highest doses, one out of 12 andone out of six volunteers had diarrhea and emesis, respectively(FIGURE 2). No subject experienced fever or dysentery. All vol-unteers receiving 108–1010 cfu excreted the vaccine strain foran average of 1–3 days and 65–100% of these volunteers also



demonstrated a positive ASC response to LPS. The magni-tude of the LPS-specific IgA ASC responses (35 per 106

PBMCs) was modest compared with the response followingvirulent infection (239 per 106 PBMCs) (TABLE 1). As seenbefore, serum antibody responses to LPS and the Ipa proteinswere modest. Th1 responses in PBMCs, as assayed byincreases in IFN-γ, IL-10 and transforming growth factor-β,were detected in volunteers and proliferative responses toIpaC and IpaD were also observed in a few volunteers [23].

The most recent published clinical trial involved testingCVD 1204 (guaBA) and CVD 1208 (guaBA, set and sen) ingroups of 16–18 volunteers who received a single oral dose ofeither of the two vaccine strains or placebo [24]. A total ofthree doses of each vaccine strain (107, 108, 109 cfu) wereevaluated in sequential fashion. While eight out of 23 recipi-ents ingesting CVD 1204 had an adverse clinical reaction,only one out of 21 recipients receiving CVD 1208 had mildfever, and only at the highest dose tested (FIGURE 2). Bothvaccines elicited significant LPS-specific ASC responses,while responses to the Ipa proteins were lower in magnitudeand less frequent (TABLE 1). Significant serum antibodyresponses to LPS and to the IpaB protein were measured withboth vaccine strains and more than fourfold increases in fecalIgA to LPS were seen in 100 and 86% of volunteers given109 cfu of CVD 1204 and CVD 1208, respectively (TABLE 1).The clinical trial comparing CVD 1204 with CVD 1208 wasimportant to establish the association of sen and set entero-toxins with virulence in humans and suggests the contribu-tion of these two enterotoxins to the diarrheal symptomsobserved previously with live vaccines. It is anticipated thatfurther studies with CVD 1208 will confirm its safety andimmunogenicity profile. Investigators at the CVD have alsoconstructed other Shigella serotypes with the same mutationsas CVD 1208.

Clinical studies at the Walter Reed Army Institute of ResearchIn the late 1990s, researchers at the Walter Reed Army Instituteof Research (WRAIR) initiated the testing of live,Shigella-based vaccines with specific deletions only in virulence-associated genes, whose functions in pathogenesis were fairlywell defined. The virG(icsA) gene seemed an obvious choice,since deletion of this gene did not affect invasiveness in HeLacells but reduced intercellular dissemination.

The vaccine strain SC602 was the first of the virG(icsA)-based vaccine to be tested at WRAIR [18,20]. SC602 wasderived from S. flexneri 2a, strain 454, and constructed byinvestigators at the Institut Pasteur (Paris, France) [92–94]. Inaddition to the loss of the virG(icsA) gene, SC602 alsocontained a deletion in the chromosomal iuc gene (encodingaerobactin). Deletion of the iuc gene by itself gives a slightlyattenuated Sereny reaction, as well as a compromised fluidaccumulation reaction in rabbit ileal loops [95]. SC602 wasmanufactured under current good manufacturing proceduresat WRAIR and stored as a lyophilized product at -80°C.During an initial placebo-controlled, dose-selection trial, the

lyophilized vaccine was hydrated in sterile water and diluted tothe desired concentration in phosphate-buffered saline. Theinoculum was mixed in 30 ml of a bicarbonate solution andgiven to volunteers after they had ingested 120 ml of the samebicarbonate solution. Initially, groups of three volunteers (andthree placebo controls) were vaccinated sequentially, starting at102 cfu and increasing the dose up to 108 cfu [18]. There wereno cases of dysentery at any dose. At the highest dose, two outof three volunteers got diarrhea, fever and severe constitutionalsymptoms. Approximately 20% of the volunteers who ingested102–107 cfu showed transient fever and some reported mild-to-severe headache and abdominal cramps. An expanded safetystudy in 15 volunteers at the 106 cfu dose followed this initialstudy. In this case, a significant number of volunteers haddiarrhea (47%), fever (33%), severe intestinal problems (40%)and severe constitutional symptoms (40%). The 106 cfu dosewas therefore deemed too reactogenic. Finally, an additionalcohort of 12 volunteers was vaccinated with 104 cfu [18]. Onlyone volunteer’s symptoms met the criteria of diarrhea while10–20% of the volunteers reported mild-to-moderateintestinal and constitutional symptoms (FIGURE 2). Based onexcretion of the vaccine strain, which occurred within 24 h ofvaccination in most volunteers, there was robust and pro-longed colonization proportional to the dose given. Forexample, 100% of the volunteers fed 106 cfu of SC602excreted the vaccine for 7 days and 57% excreted the organ-isms until antibiotic treatment began 12 days later. At the104 cfu dose, 92% of the volunteers shed the vaccine strainuntil treated on day 8. A total of seven out of 12 volunteersgiven 104 cfu had significant LPS-specific peak IgA ASCs.Four out of the seven volunteers also had fourfold or greaterrises in serum and urine IgA titer to LPS. Fewer volunteersshowed a response to the Ipa proteins (TABLE 1).

A subset of seven volunteers who were vaccinated with104 cfu of SC602 were subsequently challenged 2 months laterwith 2457T along with seven unvaccinated controls [18]. Fourout of the seven challenged vaccinated volunteers showed nosymptoms of the disease, while the other three met the clinicaldefinition of diarrhea that lasted for 24 h. Three out of the fourvolunteers that were protected completely had demonstratedsignificant LPS-specific IgA ASCs and had a greater than four-fold increase in serum and urine LPS-specific IgA titers aftervaccination. Of the three vaccinated volunteers who haddiarrhea after challenge, two lacked a LPS-specific IgA ASCresponse, as well as an adequate increase in serum or urine IgAtiter after vaccination [18]. The third lacked a serum and urineantibody titer to LPS. In contrast to the vaccinated volunteers,six out of seven unvaccinated volunteers had severe shigellosisafter challenge with 2457T, which included diarrhea, fever anddysentery. Thus, vaccination with SC602 protected volunteersagainst dysentery as well as the severe constitutional and gas-trointestinal symptoms of the disease. The immune correlatesof protection appeared to be a combination of LPS-specificASC responses, three- to fourfold increases in LPS-specificserum antibody titers and a mucosal response.



A community-based evaluation of SC602 followed the initialclinic-based trial. SC602 was administered to 12 inpatient and34 outpatient volunteers in the 103–104 cfu dose range [20].Approximately 15% of the volunteers had transient fever anddiarrhea, while headache and abdominal cramps appeared to bethe main constitutional symptoms reported (FIGURE 2). Approx-imately 50% of the volunteers had a threefold serum IgAenzyme-linked immunosorbant assay response to LPS andapproximately 58% had an ASC response to the sameantigen (TABLE 1). These values were similar to the previous trial.

Clinical trials of SC602 were also conducted in adults,school-age children and toddlers aged between 18 and35 months at the Matlab Diarrhea Treatment Center andsurveillance area at ICDDR B, Bangladesh [BAQUI ET AL., UNPUB-

LISHED OBSERVATIONS]. Two inpatient (one adult, one toddler)and three outpatient trials (one adult, two children) wereconducted. Of the 63 adults who received the vaccine, nonedeveloped fever, diarrhea, abdominal pain or other significantside effects. In the 57 children aged 8–10 years and the34 toddlers aged 18–35 months who received SC602 in dosesranging from 103 to 106 cfu, side effects were uncommon andmild and of no greater frequency than reported in the placeboarm. In the outpatient studies (involving 48 of the adults and48 of the children), monitoring of controls and household con-tacts revealed no evidence of transmissibility [BAQUI ET AL.,

UNPUBLISHED OBSERVATIONS]. However, disappointingly, therewas no evidence of seroconversion in any age group volunteers,perhaps as a consequence of the presence of maternal antibod-ies from breastfeeding in toddlers or owing to over-attenuationof the vaccine candidate for this population. Most of the volun-teers in this population had high pre-existing antibody levels toShigella LPS, indicating the predicament of an enteric vaccinestrain that generates a vigorous immune response in a naïvepopulation but is immunologically ineffective when given to anendemic population [BAQUI ET AL., UNPUBLISHED OBSERVATIONS].These results are reminiscent of those seen earlier with SFL124.

Based on the successful testing of SC602 in US volunteers,WRSS1, a virG(icsA)-based S. sonnei vaccine strain wasdeveloped. WRSS1 was derived from a stable S. sonnei strain,Mosely, and contains a 212-bp deletion in the virG(icsA)gene [96]. WRSS1 was manufactured as a lyophilized productand the protocol for administering it to volunteers was similarto that followed with SC602, except that the vaccine itself wasgiven in 30 ml of water instead of bicarbonate. WRSS1 wastested in a placebo-controlled, dose-escalating study in27 volunteers, who received a single oral dose of 103, 104 105 or106 cfu [25]. No volunteer receiving WRSS1 had dysentery or ahigh fever of more than 102°F. Approximately 10–15% of thevolunteers in the two highest doses developed mild diarrheaand low-grade fever (FIGURE 2). At the highest dose tested, thevaccine was excreted for at least 1 week, showing robust coloni-zation. WRSS1 elicited strong LPS-specific IgA ASCs, serumantibodies and fecal IgA antibodies (TABLE 1) [25]. An increase inIFN-γ production was observed in response to purified Ipa pro-teins, IpaB, IpaC and IpaD at the two highest doses in some of

the volunteers. In general, WRSS1 was considered safe andimmunogenic when given as a single oral dose in the range103–104 cfu.

The initial safety and immunogenicity trial was followed upby a community-based safety study of WRSS1 in Israelivolunteers [28]. The clinical outcome in volunteers given 103,104 and 105 cfu was similar to the observations made in USvolunteers (FIGURE 2) [28]. At the 105 cfu dose, approximately30% of the volunteers had mild fever and/or diarrhea, whereasthe 103–104 cfu dose was well tolerated, with no reported feverand only one report of moderate diarrhea for 1 day. The vac-cine was excreted for an average of 5 days by 80% of the volun-teers ingesting the 103–104 doses and induced robust LPS-specific IgA ASCs and increases in serum IgA titers. Moreimportantly, in spite of the strong colonization seen withWRSS1, the vaccine did not spread to contacts who lived withthe vaccinees over the period of the trial [28].

The low but consistent rate of fever and diarrhea that wasobserved with SC602 and WRSS1 at the 104 cfu dose suggeststhat further attenuation might be needed to reduce these symp-toms. The presence of the set gene (encoding ShET1) on thechromosome of SC602, the sen gene (senA encoding ShET2–1)and a second gene with significant homology to senA (senBencoding ShET2–2) on the invasion plasmid of both SC602and WRSS1 (and WRSd1, see below) indicate that deletions ofthese enterotoxin genes may alleviate the fever and diarrhealsymptoms seen with these vaccine candidates.

Finally, two virG(icsA)-based S. dysenteriae 1 vaccine strains,WRSd1 and SC599, have recently undergone safety and immu-nogenicity trials. WRSd1, developed at WRAIR, was tested at theGeneral Clinical Research Center, Johns Hopkins University(JHU) (MD, USA) [MCKENZIE R, VENKATESAN M, BOURGEOIS ET AL.,

UNPUBLISHED DATA]. The parent strain, 1617, was obtained froman epidemic dysentery outbreak in Guatemala in the late 1960s.WRSd1 was constructed by deletion of the virG(icsA) and stxABgenes [97]. The fnr anerobic regulator gene (linked to stx) was alsodeleted during construction of WRSd1. The vaccine was evalu-ated in 40 volunteers that were divided into five cohorts ofeight volunteers. These cohorts received vaccine doses rangingfrom 103 to 107 cfu. At the 106 and 107 cfu doses, 20% ofvaccinees experienced product-related diarrhea [MCKENZIE R, VEN-

KATESAN M, BOURGEOIS ET AL., UNPUBLISHED DATA]. Nonetheless,there were no Hemocult-positive stools and no fevers, suggestinga minimal inflammatory process in the intestine. At any dosefrom 104 through 107 cfu, approximately half of the volunteershad a strong immune response against S. dysenteriae 1 LPS and anadditional 15–25% had a weak but detectable response [MCKEN-

ZIE R, VENKATESAN M, BOURGEOIS ET AL., UNPUBLISHED DATA]. Over-all, the immune response to WRSd1 was disappointing and aminimal reactogenic dose was not reached in the JHU study. Thefnr gene that was deleted in WRSd1 encodes a transcriptionalregulator that affects the expression of hundreds of genes underanerobic conditions. The loss of the fnr gene in WRSd1 couldconceivably affect the colonization of the vaccine candidate involunteers, since the fnr gene product has been implicated as



having a critical role in the gastrointestinal colonization of E. coliin a rat model [98]. The results from the WRSd1 Phase I trialseem to bear out this prediction [BOURGEOIS AL ET AL., UNPUB-

LISHED OBSERVATION]. A S. dysenteriae 1 vaccine strain, whichretains the fnr gene, may substantially improve the colonizationof this vaccine strain. Such a strain is under construction.

SC599, with deletions in virG(icsA), ent, fep and stxA genes,was derived from strain 7/87, developed at the Institut Pasteur(Paris, France), and tested at the St. George’s Vaccine Institute(London, UK) [99]. The ent and fep genes are bacterial iron scav-enging genes. The SC599 was tested in cohorts of four subjectswho received sequential doses from 102 to 108 cfu. The vaccinewas well tolerated with only mild constitutional symptoms andwas excreted minimally in stools, indicating that SC599 ishighly attenuated. Like WRSd1, the maximum tolerable dosewas not established in this study. Mean ASC responses were 16,50 and 21 ASCs per 106 PBMCs for IgG, IgA and IgM, acrossthe dose range of 105–108 cfu. Serum antibody responses weremodest or absent [99]. A Phase II, double-blind, placebo-controlled study, comparing the immunogenicity and safety oftwo doses, 105 and 107 cfu of SC599 in larger numbers ofhealthy human adult volunteers, sponsored by the InstitutPasteur, is expected to be completed in June 2007.

Future directionsVaccine trials with live, invasive strains have collectively pro-vided important insights and knowledge into the effectivenessof this strategy. At WRAIR, the trials with SC602, WRSS1and WRSd1 have indicated that a single, oral dose of anattenuated virG(icsA)-based Shigella vaccine strain can immu-nize volunteers safely, providing protection against dysenteryand the severe constitutional symptoms of shigellosis. How-ever, residual reactogenicity in 20–30% of the volunteers sug-gests that further attenuation of these vaccine candidates isnecessary. Trials with CVD vaccines, such as CVD1208, haveprovided a strategy that can lead to increased safety withoutcompromising immunogenicity. Second-generationvirG(icsA)-based live Shigella vaccine strains are being con-structed currently to eliminate the enterotoxins present on theplasmid and on the chromosome, as well as to reduce theendotoxicity of the LPS molecule. Mutants are also beingevaluated to determine whether the replication of the bacteriaand subsequent excretion can be reduced without compromis-ing the immune response. The construction of these newderivatives has been aided by the technique of λ-red recombi-nation [100], which has been adapted to Shigella spp. to gener-ate rapid and precisely defined gene deletions. FIGURE 1 indi-cates some of the genes that are being targeted and the stepsin the pathway that will be affected. The behavior of thesemutations in tissue culture studies and animal models ofimmunogenicity and efficacy will determine which of theseand how many mutations will be incorporated into vaccinestrains. Once the attenuating features are established in oneserotype, it can be duplicated resulting in a polyvalent mix-ture that can be used for immunization. Several laboratories,

including those at CVD and WRAIR, are pursuing thisstrategy [101]. Clinical trials with vaccines containing a mix-ture of Shigella serotypes are pending and are required todetermine if such polyvalent vaccines can be equally immuno-genic and protective against individual serotypes. Properlyspaced booster doses of live vaccines, as well as combinationsof live and subunit vaccines, are additional regimens thatmerit clinical investigation.

The advantage of live Shigella vaccines is their ability toinduce a complete immune response, since their delivery tohost target tissue closely mimics the natural route of infec-tion. In live vaccines, the antigens occur in their native formsand are presented directly to the lining of the gut, inducingboth a mucosal and a broad-based humoral and cellularresponse, including a memory response. Other advantagesinclude needle-free administration and economical manufac-turing processes that could be replicated in the developingworld. Live vaccines that can be kept viable at close to roomtemperatures, thus breaking free of the cold chain, would addsubstantial value to the system. Furthermore, a safe, live vac-cine can also be a potential delivery vector for genes encodingvarious antigens from other pathogens, as well as genes thatencode immune modulators, such as cytokines. Several of theCVD Shigella vaccine candidates, as well as SC608, an asdderivative of SC602, have been used to express fimbrial anti-gens as well as derivatives of the heat-labile enterotoxin fromenterotoxigenic E. coli (ETEC), which causes travelers’diarrhea [58,102,103]. In guinea pigs, these constructs elicitedsignificant immune responses to both Shigella spp. and ETECantigens, indicating that multivalent enteric vaccines are adistinct possibility in the future.

Several obstacles remain to be overcome before live Shigellavaccines become a reality. Some of these obstacles have beenenumerated above, such as the need for multiple serotypes, aswell as the difficulty in immunizing endemic populations.Higher doses of the vaccines and different dosing regimensmight be required in endemic areas. A more provocativehypothesis may be to use vaccines with different attenuatingfeatures for endemic populations. Gastrointestinal physiologythat might affect colonization of live vaccines in endemicpopulations needs to be understood. Since infants are the tar-get age group for an anti-shigella vaccine, some thoughtshould also be given to how these vaccines could be adminis-tered effectively to this age group. Furthermore, the diseaseoccurs primarily in less affluent countries and requires inno-vative strategies by governmental, international and privateresources to ensure that there is sufficient funding, motivationand support for their development and distribution. The Billand Melinda Gates Foundation has targeted diseases found inthe most impoverished countries and is an example of onesuch private resource. Finally, a recent survey of policymakersin Asian countries pointed out that factors, such as demon-stration of region-specific disease burden, local manufacturingcapability, evidence of efficacy in endemic populations,economic savings achieved as a result of vaccination and



dissemination of research results in a timely manner to policy-makers, are critical for the acceptability of vaccines againstshigellosis, cholera and typhoid in these regions [104].

SummaryResults from the clinical trials of genetically well character-ized, invasive Shigella vaccines are promising. CVD 1208,SC602, WRSS1 and WRSd1 vaccine candidates, adminis-tered orally, are safe and immunogenic in volunteer trials and,in the case of SC602, have been demonstrated to protectagainst dysentery. Clinical trials with CVD1208 demon-strated that the symptoms of mild fever and diarrhea, whichare seen with some of the live Shigella vaccines, can bereduced by elimination of the sen and set genes from the vac-cine strain. Duplication of a successful strategy in one sero-type to other serotypes is an ongoing area of research and willeventually lead to a polyvalent mixture of Shigella strains ofdifferent serotypes that can protect against most of the shigel-losis seen worldwide. Successful strategies will need to beemployed to ensure that vaccines that look promising in anaïve population are also safe and immunogenic in anendemic one.

Expert commentaryCurrently, strategies to obtain a live Shigella vaccine candidatethat will immunize a naïve population safely and protectagainst dysentery is likely to come to fruition in the near future.Commercial partners will be needed for licensure. Develop-ment of optimal storage conditions to maintain the stability ofthe vaccine at different temperatures and packaging of the vac-cines in single-dose format will be issues that require continuedresearch. Such a vaccine will be useful for military personneland travelers to endemic areas. However, vaccines againstenteric diseases are required mostly in developing countrieswhere the target age-group are infants, whose maternal anti-bodies are minimal and who are therefore susceptible to infec-tions. Live, bacterial vaccines have distinct advantages over sub-unit vaccines for use in developing countries, where a single,oral dose that is easy and cheap to manufacture, can be safelystored and distributed without losing viability and easilyadministered, provide the ideal solution. While several liveShigella vaccines are being tested in the USA and in other coun-tries, these vaccine candidates have to demonstrate immuno-genicity in an endemic population where there is pre-existingimmunity. This has been a challenge, as seen with SFL124 andSC602. Once an effective vaccine is developed for use in anendemic area, other factors have to be addressed as outlined in arecent study, including consistent support from nationalgovernments that have to be persuaded that such a vaccine addsvalue to the healthcare of the local population [104].

Five-year viewThe only licensed Shigella vaccine in use is the FS vaccine thatwas developed in China. Other Shigella vaccines in advanceddevelopment currently include both subunit and live vaccines.

In the next 5 years, data from safety, immunogenicity and effi-cacy studies will become available for some of the live vaccinesthat are being developed and have been discussed in this review.Several subunit vaccine approaches are also in clinical trials andthe fate of some of these will be decided soon.

The parenteral conjugate vaccines developed by John Rob-bins and his colleagues at the National Institutes of ChildHealth and Human Development, NIH (MD, USA), consistof Shigella-detoxified LPS that is conjugated to either succi-nylated recombinant Pseudomonas aeruginosa exotoxin A orto native Corynebacterium diphteriae toxin mutant [9]. Suchconjugates are administered parenterally and have shownsafety, immunogenicity and, in one case, efficacy, in adultsand in 1–4 and 4–7 year old children [17,105,106]. It has beenargued that conjugate vaccines may be the answer in endemicpopulations [17]. At the Sheba Medical Center (TelHashomer, Israel), a major efficacy study in young children isnow underway and is due to be completed within the nextyear. Efficacy data, as well as manufacturing issues, cost andproblems associated with needle-based delivery, are factorsthat will play a role in the further commercial developmentof these conjugate vaccines.

Intranasally administered Invaplex, a purified complex fromShigella water extract, is composed of the Ipa proteins and LPSand is in Phase I trials currently at WRAIR [14,15]. Results arepending and a follow-up challenge trial is being planned forlate 2007 to establish the efficacy of this product.

A parenteral nuclear protein/ribosomal vaccine approach,which is being developed by the International Vaccine Insti-tute (Seoul, Korea) and WRAIR, is still at a preclinical stage.This vaccine approach, developed initially for Shigella spp.by Levenson and colleagues has previously shown promise invarious animal models [107].

New approaches for economical manufacture of conjugatevaccines are being developed by Endobiologics, Inc. (MT,USA), and novel protein antigens that are common to thegenus Shigella are being identified by Intercell AG (Vienna,Austria) for use in subunit vaccines.

AcknowledgementsWe would like to gratefully acknowledge the suggestionsgiven by Thomas L (Larry) Hale, Chief, Division of Bacterialand Rickettsial Diseases at WRAIR. We would also like toacknowledge the many contributions of different investiga-tors in the field of Shigella research (only some of whose ref-erences are included here), whose collective efforts have givenus a better understanding of the mechanism of Shigellapathogenesis and led to the design and testing of varioustypes of Shigella vaccines.

The content of this publication does not necessarily reflectthe views or policies of the Department of Health and HumanServices, the US Department of the Army or the US Depart-ment of Defense, nor does the mention of trade names, com-mercial products or organizations imply endorsement by theUS Government.



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Key issues

• Live Shigella vaccine candidates can be given orally and are cheaper and easier to manufacture than subunit vaccines. These vaccines will provide protective immunity to naïve volunteers. However, the immunogenicity of these candidate strains in endemic populations remains questionable. Higher doses of live vaccines, conjugate vaccines and/or combinations of live and conjugate vaccines may be part of the solution in these regions.

• Manufacturing costs, storage, stability and ease of delivery of the vaccines will be decisive factors for determining which type of vaccine will be commercialized.

• Public health policies that will encourage the application of these vaccines in regions where they are needed the most, still to be developed.



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Website

201 World Health Organizationwww.who.int/vaccine_research/diseasess/diarrhoeal/

Affiliations

• Malabi M Venkatesan, PhD

Division of Bacterial and Rickettsial Diseases, Walter Reed Army Institute of Research, 503 Robert Forney Drive, Room 3s12, Silver Spring, MD 20910, USATel.: +1 301 319 9764Fax: +1 301 319 [email protected]

• Ryan T Ranallo, PhD

Division of Bacterial and Rickettsial Diseases, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USATel.: +1 301 319 9517Fax: +1 3012 319 [email protected]

Live-attenuated Shigella vaccines

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