Flexneri 2a Lipopolysaccharide Research Articles

A Pentavalent Shigella flexneri LPS-Based Vaccine Candidate Is Safe and Immunogenic in Animal Models.

A multivalent vaccine is much needed to achieve protection against predominant Shigella serotypes. Recently, we demonstrated the clinical applicability and immunogenic potential of tri-acylated S. flexneri 2a lipopolysaccharide (Ac3-S-LPS). Using a similar approach, we designed a pentavalent LPS candidate vaccine against S. flexneri 1b, 2a, 3a, 6, and Y (PLVF). In this study, we performed molecular and antigenic characterization of the vaccine candidate and its preclinical evaluation. There were no signs of acute toxicity after subcutaneous administration of PLVF in rabbits at a proposed human dose of 125 μg. No pyrogenic reactions and adverse effects associated with chronic toxicity after repeated administration of PLVF were revealed either. The immunization of mice with PLVF led to ≥16-fold increase in S. flexneri 1b-, 2a-, 3a-, 6-, and Y-specific antibodies. In a serum bactericidal antibody (SBA) assay, we registered 54%, 66%, 35%, 60%, and 60% killing of S. flexneri 1b, 2a, 3a, 6, and Y, respectively. In the guinea pig keratoconjunctivitis model, the efficacy was 50% to 75% against challenge with all five S. flexneri serotypes. These studies demonstrate that PLVF is safe, immunogenic over a wide range of doses, and provides protection against challenge with homologous S. flexneri strains, thus confirming the validity of pentavalent design of the combined vaccine.

Detoxified O-Specific Polysaccharide (O-SP)-Protein Conjugates: Emerging Approach in the Shigella Vaccine Development Scene.

Shigella is the second most common cause of moderate to severe diarrhea among children worldwide and of diarrheal disease-associated mortality in young children in low-and middle-income countries. In spite of many years of attempts to develop Shigella vaccines, no licensed vaccines are yet available. Injectable conjugate vaccines made of the detoxified lipopolysaccharide (LPS) of S. flexneri 2a, S. sonnei, and S. dysenteriae type 1 covalently bound to protein carriers were developed in the early 1990s by John B. Robbins and Rachel Schneerson at the US National Institutes of Health. This approach was novel for a disease of the gut mucosa, at a time when live, rationally attenuated oral vaccine strains that intended to mimic Shigella infection and induce a protective local immune response were extensively investigated. Of keystone support to Shigella glycoconjugates development were the findings of a strong association between pre-existent serum IgG antibodies to S. sonnei or S. flexneri 2a LPS and a lower risk of infection with the homologous Shigella serotypes among Israeli soldiers serving in field units. In view of these findings and of the successful development of the pioneering Haemophilus influenzae type b conjugate vaccines, it was hypothesized that protective immunity may be conferred by serum IgG antibodies to the O-Specific Polysaccharide (O-SP) following parenteral delivery of the conjugates. S. sonnei and S. flexneri 2a glycoconjugates induced high levels of serum IgG against the homologous LPS in phase I and II studies in healthy volunteers. The protective efficacy of a S. sonnei detoxified LPS-conjugate was further demonstrated in field trials in young adults (74%) and in children older than three years of age (71%), but not in younger ones. The evaluation of the Shigella conjugates confirmed that IgG antibodies to Shigella LPS are correlates of protection and provided solid basis for the development of a new generation of glycoconjugates and other injectable LPS-based vaccines that are currently in advanced stages of clinical evaluation.

Repertoire of Naturally Acquired Maternal Antibodies Transferred to Infants for Protection Against Shigellosis.

Shigella is the second leading cause of diarrheal diseases, accounting for >200,000 infections and >50,000 deaths in children under 5 years of age annually worldwide. The incidence of Shigella-induced diarrhea is relatively low during the first year of life and increases substantially, reaching its peak between 11 to 24 months of age. This epidemiological trend hints at an early protective immunity of maternal origin and an increase in disease incidence when maternally acquired immunity wanes. The magnitude, type, antigenic diversity, and antimicrobial activity of maternal antibodies transferred via placenta that can prevent shigellosis during early infancy are not known. To address this knowledge gap, Shigella-specific antibodies directed against the lipopolysaccharide (LPS) and virulence factors (IpaB, IpaC, IpaD, IpaH, and VirG), and antibody-mediated serum bactericidal (SBA) and opsonophagocytic killing antibody (OPKA) activity were measured in maternal and cord blood sera from a longitudinal cohort of mother-infant pairs living in rural Malawi. Protein-specific (very high levels) and Shigella LPS IgG were detected in maternal and cord blood sera; efficiency of placental transfer was 100% and 60%, respectively, and had preferential IgG subclass distribution (protein-specific IgG1 > LPS-specific IgG2). In contrast, SBA and OPKA activity in cord blood was substantially lower as compared to maternal serum and varied among Shigella serotypes. LPS was identified as the primary target of SBA and OPKA activity. Maternal sera had remarkably elevated Shigella flexneri 2a LPS IgM, indicative of recent exposure. Our study revealed a broad repertoire of maternally acquired antibodies in infants living in a Shigella-endemic region and highlights the abundance of protein-specific antibodies and their likely contribution to disease prevention during the first months of life. These results contribute new knowledge on maternal infant immunity and target antigens that can inform the development of vaccines or therapeutics that can extend protection after maternally transferred immunity wanes.

Shigella-specific antibodies in the first year of life among Zambian infants: A longitudinal cohort study.

IntroductionShigellosis, is a leading cause of moderate-to-severe diarrhoea and related mortality in young children in low and middle income countries (LMICs). Knowledge on naturally acquired immunity can support the development of Shigella candidate vaccines mostly needed in LMICs. We aimed to quantify Shigella-specific antibodies of maternal origin and those naturally acquired in Zambian infants.MethodsPlasma samples collected from infants at age 6, 14 and 52-weeks were tested for Shigella (S. sonnei and S. flexneri 2a) lipopolysaccharide (LPS) antigen specific immunoglobulin G (IgG) and A (IgA) by enzyme-linked immunosorbent assay.ResultsAt 6 weeks infant age, the IgG geometric mean titres (GMT) against S. sonnei (N = 159) and S. flexneri 2a (N = 135) LPS were 311 (95% CI 259–372) and 446 (95% CI 343–580) respectively. By 14 weeks, a decline in IgG GMT was observed for both S. sonnei to 104 (95% CI 88–124), and S. flexneri 2a to 183 (95% CI 147–230). Both S. sonnei and S. flexneri 2a specific IgG GMT continued to decrease by 52 weeks infant age when compared to 6 weeks. In 27% and 8% of infants a significant rise in titre (4 fold and greater) against S. flexneri 2a and S. sonnei LPS, respectively, was detected between the ages of 14 and 52 weeks.IgA levels against both species LPS were very low at 6 and 14 weeks and raised significantly against S. flexneri 2a and S. sonnei LPS in 29% and 10% of the infants, respectively.ConclusionIn our setting, transplacental IgG anti-Shigella LPS is present at high levels in early infancy, and begins to decrease by age 14 weeks. Our results are consistent with early exposure to Shigella and indicate naturally acquired IgG and IgA antibodies to S. flexneri 2a and S. sonnei LPS in part of infants between 14 and 52 weeks of age. These results suggest that a potential timing of vaccination would be after 14 and before 52 weeks of age to ensure early infant protection against shigellosis.

Human challenge study with a Shigella bioconjugate vaccine: Analyses of clinical efficacy and correlate of protection

BackgroundShigellosis is a major cause of moderate to severe diarrhoea and dysentery in children under 5 years of age in low and middle-income countries. The Flexyn2a vaccine conjugates the O-polysaccharide of Shigella flexneri 2a to Pseudomonas aeruginosa exotoxin A. We describe a Phase 2b proof-of-concept challenge study that evaluated safety, immunogenicity, and efficacy of the Flexyn2a vaccine to protect against shigellosis. MethodsIn this randomized, double blind, placebo-controlled trial, healthy adults were randomized 1:1 to receive Flexyn2a (10 µg) or placebo intramuscularly, twice, 4 weeks apart, followed by challenge 4 weeks later with 1500 colony forming units (CFUs) of S. flexneri 2a strain 2457T. The primary outcome was vaccine-induced protection. S. flexneri 2a lipopolysaccharide (LPS)-specific immune responses were assessed. FindingsSixty-seven subjects were enrolled, 34 received vaccine and 33 placebo. The vaccine was well tolerated; the majority of adverse events were mild in nature. Thirty vaccinees and 29 placebo recipients received the S. flexneri 2a challenge. Vaccination resulted in a 30.2% reduction in shigellosis compared with placebo (13/30 vs. 18/29; p = 0.11; 95% CI -15 to 62.6). Vaccine efficacy was more robust against severe disease, reaching 51.7% (p = 0.015, 95% CI 5.3 to 77.9) against moderate/severe diarrhoea or dysentery concurrent with fever or severe enteric symptoms and 72.4% (p = 0.07) against more severe diarrhoea (≥10 lose stools or ≥1000 g loose stools/24 h). Vaccinated subjects were less likely to need early antibiotic intervention following challenge (protective efficacy 51.7%, p = 0.01; 95% CI 9 to 76.8). In those who developed shigellosis, vaccinated subjects had a lower disease severity score (p = 0.002) than placebo-recipients. Additionally, LPS-specific serum IgG responses in Flexyn2a recipients were associated with protection against disease (p = 0.0016) and with a decreased shigellosis disease score (p = 0.002). InterpretationThe Flexyn2a bioconjugate vaccine was immunogenic, well tolerated and protected against severe illness after Shigella challenge and is a promising Shigella vaccine construct. We identified a strong association between anti-S. flexneri 2a serum IgG and a reduction in disease outcomes. (Clinicaltrials.gov, NCT02646371.) FundingFunding for this study was through a grant from the Wellcome Trust.

Specific blood group antibodies inhibit Shigella flexneri interaction with human cells in the absence of spinoculation

The classical models of investigating Shigella flexneri adherence and invasion of tissue culture cells involve either bacterial centrifugation (spinoculation) or the use of AfaE adhesin to overcome the low infection rate observed in vitro. However clinically, S. flexneri clearly adheres and invades the human colon in the absence of ‘spinoculation’. Additionally, certain S. flexneri tissue cell based assays (e.g. plaque assays and infection of T84 epithelial cells on Transwells®), do not require spinoculation. In the absence of spinoculation, we recently showed that glycan-glycan interactions play an important role in S. flexneri interaction with host cells, and that in particular the S. flexneri 2a lipopolysaccharide O antigen glycan has a high affinity for the blood group A glycan. During the investigation of the effect of blood group A antibodies on S. flexneri interaction with cells, we discovered that Panc-1 cells exhibited a high rate of infection in the absence of spinoculation. Select blood group A antibodies inhibited invasion of Panc-1 cells, and adherence to T84 cells. The use of Panc-1 cells represents a simplified model to study S. flexneri pathogenesis and does not require either spinoculation or exogenous adhesins.

Highly homogenous tri-acylated S-LPS acts as a novel clinically applicable vaccine against Shigella flexneri 2a infection

Shigellosis, a major cause of diarrhea worldwide, exhibits high morbidity and mortality in children. Specificity of Shigella immunity is determined by the structure of the main protective O-antigen polysaccharide component incorporated into the lipopolysaccharide (LPS) molecule. Endotoxicity, however, precludes LPS clinical use. Thus, there is still no vaccine against the most prevalent shigellosis species (serotype S. flexneri 2a), despite ongoing efforts focused on inducing serotype-specific immunity. As LPS is highly heterogenous, we hypothesized that more homogenous pools of LPS might be less toxic. We developed a method to generate a homogenous S. flexneri 2a LPS subfraction, Ac3-S-LPS, containing long chain O-specific polysaccharide (S-LPS) and mainly tri-acylated lipid A, with no penta- and hexa-acylated, and rare tetra-acylated lipid A. Ac3-S-LPS had dramatically reduced pyrogenicity and protected guinea pigs from shigellosis. In volunteers, 50 µg of injected Ac3-S-LPS vaccine was safe, with low pyrogenicity, no severe and few minor adverse events, and did not induce pro-inflammatory cytokines. In spite of the profound lipid A modification, the vaccine induced a prevalence of IgG and IgA antibodies. Thus, we have developed the first safe immunogenic LPS-based vaccine candidate for human administration. Homogenous underacetylated LPSs may also be useful for treating other LPS-driven human diseases.Clinical trial registry: http://grls.rosminzdrav.ru/

Stable Chromosomal Expression of Shigella flexneri 2a and 3a O-Antigens in the Live Salmonella Oral Vaccine Vector Ty21a.

We have been exploring the use of the live attenuated Salmonella enterica serovar Typhi Ty21a vaccine strain as a versatile oral vaccine vector for the expression and delivery of multiple foreign antigens, including Shigella O-antigens. In this study, we separately cloned genes necessary for the biosynthesis of the Shigella flexneri serotype 2a and 3a O-antigens, which have been shown to provide broad cross-protection to multiple disease-predominant S. flexneri serotypes. The cloned S. flexneri 2a rfb operon, along with bgt and gtrII, contained on the SfII bacteriophage, was sufficient in Ty21a to express the heterologous S. flexneri 2a O-antigen containing the 3,4 antigenic determinants. Further, this rfb operon, along with gtrA, gtrB, and gtrX contained on the Sfx bacteriophage and oac contained on the Sf6 bacteriophage, was sufficient to express S. flexneri 3a O-antigen containing the 6, 7, and 8 antigenic determinants. Ty21a, with these plasmid-carried or chromosomally inserted genes, demonstrated simultaneous and stable expression of homologous S Typhi O-antigen plus the heterologous S. flexneri O-antigen. Candidate Ty21a vaccine strains expressing heterologous S. flexneri 2a or 3a lipopolysaccharide (LPS) elicited significant serum antibody responses against both homologous S Typhi and heterologous Shigella LPS and protected mice against virulent S. flexneri 2a or 3a challenges. These new S. flexneri 2a and 3a O-antigen-expressing Ty21a vaccine strains, together with our previously constructed Ty21a strains expressing Shigella sonnei or Shigella dysenteriae 1 O-antigens, have the potential to be used together for simultaneous protection against the predominant causes of shigellosis worldwide as well as against typhoid fever.

Nasal delivery of Protollin-adjuvanted H5N1 vaccine induces enhanced systemic as well as mucosal immunity in mice

Sporadic, yet frequent human infections with avian H5N1 influenza A viruses continue to pose a potential pandemic threat. Poor immunogenicity of unadjuvanted H5N1 vaccines warrants developing novel adjuvants and formulations as well as alternate delivery systems to improve their immunogenicity and efficacy. Here, we show that Protollin, a nasal adjuvant composed of Neisseria meningitides outer membrane proteins non-covalently linked to Shigella flexneri 2a lipopolysaccharide, is a potent nasal adjuvant for an inactivated split virion H5N1 clade 1 A/Viet Nam1203/2004 (A/VN/1203/04) vaccine in a mouse model. Protollin-adjuvanted vaccines elicited enhanced serum protective hemagglutination inhibition titers, mucosal IgA responses, and H5N1-specific cell-mediated immunity that resulted in complete protection against a lethal challenge with a homologous virus as well as a heterologous clade 2 virus A/Indonesia/05/2005 (A/IN/05/05). Detailed analysis of adaptive immunity revealed that Protollin increased the frequency of lymphoid- as well as local tissue-resident antibody-secreting cells, local germinal center reaction of B cells, broad-spectrum of CD4 T cell response. Our findings suggest that nasal delivery of H5N1 vaccine with Protollin adjuvant can overcome the poor immunogenicity of H5N1 vaccines, induce both cellular and humoral immune responses, enhance protection against challenge with clade 1 and clade 2 H5N1 viruses and achieve significant antigen dose-sparing.

Age-Dependent Association among Helicobacter pylori Infection, Serum Pepsinogen Levels and Immune Response of Children to Live Oral Cholera Vaccine CVD 103-HgR

BackgroundThrough its effects on gastric secretion, we hypothesized that Helicobacter pylori infection may influence oral immunization. Accordingly, we examined the association between H. pylori infection, serum pepsinogen (PG) (measures for H. pylori gastritis) and vibriocidal antibody (a correlate of protection) seroconversion following oral immunization with CVD 103-HgR live cholera vaccine among children of different ages.MethodsSera from 422 Chilean children who were vaccinated with a single dose of CVD 103-HgR were tested by ELISA for serum IgG antibodies to H. pylori, PG I and PG II levels and antibodies to Shigella flexneri 2a lipopolysaccharide and hepatitis A virus (as markers of low socioeconomic status and exposure to enteric pathogens).ResultsThe likelihood of vibriocidal antibody seroconversion following vaccination with CVD 103-HgR was significantly decreased in H. pylori-seropositive children age 6 months to 4 years with PG II>8 µg/L (adjusted OR 0.14 (95% CI 0.03–0.61; P = 0.009), and also in H. pylori seropositives with lower PG II level (adjusted OR 0.34, 95% CI 0.14–0.83; P = 0.017), compared to H. pylori-seronegatives. H. pylori-seropositive children aged 5–9 years with serum PG I>30 µg/L (indicating more severe gastritis) had higher odds of vibriocidal seroconversion than those with lower PG I levels (adjusted OR 4.41, 95%CI 1.26–15.38; P = 0.02). There was no significant association between exposures to S. flexneri 2a or hepatitis A virus and vibriocidal seroconversion.ConclusionsAs H. pylori gastritis progresses with increasing pediatric age in developing country venues, changes in gastric secretion ensue that we believe explain the observed differences in age-related immune responses to immunization with live oral cholera vaccine. The effect of H. pylori and changes of gastric acid secretion on the immunogenicity of various oral vaccines should be studied in different developing, transitional and industrialized country settings.

Safety and immunogenicity of an intranasal Shigella flexneri 2a Invaplex 50 vaccine

Background Shigella flexneri 2a lipopolysaccharide 50 is a nasally delivered subunit vaccine consisting of a macromolecular complex composed of LPS, IpaB, IpaC and IpaD. The current study examined vaccine safety and immunogenicity across a dose range and the clinical performance of a new intranasal delivery device. Methods Volunteers ( N = 36) were randomized to receive vaccine via the Dolphin™ (Valois of America, Congers, New York) intranasal spray device at one of three doses (240, 480, and 690 μg) on days 0, 14, and 28. Another group ( N = 8) received the 240 μg dose via pipette. Vaccine safety was actively monitored and antigen-specific humoral and mucosal immune responses were determined. Results There were no serious adverse events and the majority of adverse events (98%) were mild. Antibody secreting cells (ASC), plasma, and mucosal immune responses to Shigella antigens were detected at all three dose levels with the 690 μg dose inducing the highest magnitude and frequency of responses. Vaccination with comparable doses of Invaplex 50 via the Dolphin™ resulted in higher plasma and ASC immune responses as compared to pipette delivery. Conclusion In this trial the S. flexneri 2a Invaplex 50 vaccine was safe, well-tolerated and induced robust levels of antigen-specific intestinal IgA and ASC responses. The spray device performed well and offered an advantage over pipette intranasal delivery.

Rational design and immunogenicity of liposome-based diepitope constructs: Application to synthetic oligosaccharides mimicking the Shigella flexneri 2a O-antigen

We have designed chemically defined diepitope constructs consisting of liposomes displaying at their surface synthetic oligosaccharides mimicking the O-antigen of the Shigella flexneri 2a lipopolysaccharide (B-cell epitope) and influenza hemagglutinin peptide HA 307–319 (Th epitope). Using well controlled and high-yielding covalent bioconjugation reactions, the two structurally independent epitopes were coupled to the lipopeptide Pam 3CAG, i.e. a TLR2 ligand known for its adjuvant properties, anchored in preformed vesicles. The synthetic construct containing a pentadecasaccharide corresponding to three O-antigen repeating units triggered T-dependent anti-oligosaccharide and anti- S. flexneri 2a LPS antibody responses when administered i.m. to BALB/c mice. Moreover, the long-lasting anti-LPS antibody response afforded protection against a S. flexneri 2a challenge. These results show that liposome diepitope constructs could be attractive alternatives in the development of synthetic carbohydrate-based vaccines.

A novel intranasal Protollin ™-based measles vaccine induces mucosal and systemic neutralizing antibody responses and cell-mediated immunity in mice

Protollin-MV is a vaccine produced by mixing split measles virus (MV) antigen with the novel adjuvant Protollin ™ ( Neisseria meningitidis outer membrane proteins non-covalently complexed with Shigella flexneri 2a lipopolysaccharide). Intranasal immunization of mice with two or three doses of Protollin-MV induces both serum IgG and mucosal IgA with strong neutralizing activity. There is a dose-dependent shift towards lower IgG 1:IgG 2a ratios and MV-specific IFNγ production in splenocytes. Intranasal Protollin-MV can therefore induce systemic and mucosal neutralizing antibody responses as well as elicit a balanced T H1/T H2-type response.

Genetic modulation of Shigella flexneri 2a lipopolysaccharide O antigen modal chain length reveals that it has been optimized for virulence.

The lipopolysaccharide (LPS) molecules of Shigella flexneri 2a have O antigen (Oag) polysaccharides with two modal chain length distributions. The chromosomal wzz(SF) gene results in short (S) type Oag chains [11-17 Oag repeat units (RUs)], and the pHS-2 plasmid-located wzz(pHS2) gene results in very long (VL) type Oag chains (>90 Oag RUs). S. flexneri wzz(SF) mutants are unable to form plaques on HeLa cell monolayers and F-actin comet tails, indicating that IcsA/VirG function in actin-based motility (ABM) is defective. An S. flexneri wzz(SF) wzz(pHS2) double mutant had LPS with relatively short, random length Oag chains and, paradoxically, was able to form plaques and F-actin comet tails. The influence of Oag modal chain length distribution on virulence and related properties was investigated using complementation with different wzz genes. Wzz(O139) from Vibrio cholerae O139 and Wzz(ST) from Salmonella enterica serovar Typhimurium were fully functional in Shigella flexneri, resulting in LPS with either very short (VS) type Oag chains (2-7 Oag RUs) or long (L) type Oag chains (19-35 RUs), respectively. In the absence of VL-type Oag chains, the VS-, S- and L-type Oag chains were permissive for plaque and F-actin comet tail formation. However, in the presence of LPS with VL-type Oag chains, the VS- and S-type Oag chains but not the L-type Oag chains were permissive for plaque and F-actin comet tail formation. These data, and the results of a previous investigation, show that IcsA function in ABM requires LPS Oag chains with at least two but less than 18 RUs when VL-type Oag chains are co-expressed on the cell surface. However, in the absence of the VL-type Oag chains, LPS Oag chains with at least two but less than 90 RUs are able to support IcsA function in ABM. Indirect immunofluorescence staining of IcsA on the cell surface of the S. flexneri strains did not correlate with the observed effect of Oag chain length on plaque and F-actin comet tail formation. However, when intracellular bacteria lacking VL-type Oag chains were examined, an inverse correlation between Oag modal chain length and detection of IcsA was observed, i.e. staining decreased with increased modal length. It is hypothesized that Oag chains can mask IcsA and interfere with its function in ABM, and a model is presented to explain how LPS Oag and IcsA may interact. It is suggested that S. flexneri 2a has evolved to synthesize LPS with two Oag modal chain lengths, as S-type Oag chains allow IcsA to function in ABM in the presence of VL-type Oag chains that confer resistance to serum.

Δ guaBA attenuated Shigella flexneri 2a strain CVD 1204 as a Shigella vaccine and as a live mucosal delivery system for fragment C of tetanus toxin

The Δ guaBA Shigella flexneri 2a vaccine candidate, CVD 1204, was evaluated as a delivery system for the non-toxic C-terminal of tetanus toxin (fragment C), either as a polypeptide expressed in the bacteria or as a DNA vaccine. CVD 1204 was transformed with plasmid pTET nir15 which encodes the fragment C gene ( tetC) under the control of the inducible prokaryotic nir15 promoter or a DNA vaccine plasmid pcDNA3 tetC which encodes tetC under the eukaryotic hCMV promoter. Guinea pigs immunised intranasally (i.n.) with either recombinant strain mounted a secretory immune response against S. flexneri 2a Lipopolysaccharide (LPS) and were protected against ocular challenge with wild-type S. flexneri 2a. Both strains were effective in eliciting a serum IgG response against fragment C in guinea pigs following i.n. immunisation. Furthermore, serum from guinea pigs immunised with CVD 1204(pTET nir15) contained tetanus toxin neutralising antibodies. These results demonstrate that this S. flexneri 2a vaccine candidate can serve as a vehicle for the delivery of foreign antigens to the systemic immune system while retaining its capacity to serve as a mucosal Shigella vaccine.

Vaccination against shigellosis with attenuated Shigella flexneri 2a strain SC602.

The Shigella flexneri 2a SC602 vaccine candidate carries deletions of the plasmid-borne virulence gene icsA (mediating intra- and intercellular spread) and the chromosomal locus iuc (encoding aerobactin) (S. Barzu, A. Fontaine, P. J. Sansonetti, and A. Phalipon, Infect. Immun. 64:1190-1196, 1996). Dose selection studies showed that SC602 causes shigellosis in a majority of volunteers when 3 x 10(8) or 2 x 10(6) CFU are ingested. In contrast, a dose of 10(4) CFU was associated with transient fever or mild diarrhea in 2 of 15 volunteers. All volunteers receiving single doses of >/=10(4) CFU excreted S. flexneri 2a, and this colonization induced significant antibody-secreting cell and enzyme-linked immunosorbent assay responses against S. flexneri 2a lipopolysaccharide in two-thirds of the vaccinees. Seven volunteers who had been vaccinated 8 weeks earlier with a single dose of 10(4) CFU and 7 control subjects were challenged with 2 x 10(3) CFU of virulent S. flexneri 2a organisms. Six of the control volunteers developed shigellosis with fever and severe diarrhea or dysentery, while none of the vaccinees had fever, dysentery, or severe symptoms (P = 0. 005). Three vaccinees experienced mild diarrhea, and these subjects had lower antibody titers than did the fully protected volunteers. Although the apparent window of safety is narrow, SC602 is the first example of an attenuated S. flexneri 2a candidate vaccine that provides protection against shigellosis in a stringent, human challenge model.

Characterization and quantitative analysis of serum IgG class and subclass response to Shigella sonnei and Shigella flexneri 2a lipopolysaccharide following natural Shigella infection.

The IgG subclass response to Shigella sonnei and Shigella flexneri 2a lipopolysaccharide (LPS) was examined in subjects naturally exposed to these organisms. Affinity-purified LPS antibodies obtained using a column of Shigella LPS bound to epoxy-activated Sepharose 6B were used as standards to calibrate the serum antibody response to natural Shigella infection. The geometric mean concentrations of specific IgG in sera from those not exposed to Shigella organisms were 7.9 microg/mL against S. sonnei LPS and 18.6 microg/mL against S. flexneri 2a LPS. After natural exposure to S. sonnei or S. flexneri 2a, the concentrations rose to 30.3 and 127.9 microg/mL, respectively. IgG2 was the major component in the anti-S. flexneri subclass response, while the anti-S. sonnei response was dominated by IgG1. High levels of IgG1 antibodies before exposure to organisms from either Shigella serogroup correlated with a lower risk of developing symptomatic infection.

Specificity of monoclonal antibodies elicited by mucosal infection of BALB/c mice with virulent Shigella flexneri 2a.

Protective immunity against shigellosis is thought to be determined by the O-antigen side chains of the lipopolysaccharide (LPS) molecule. To study possible common protective epitopes, monoclonal antibodies reacting with Shigella flexneri 2a LPS were generated from BALB/c mice infected ocularly with the virulent serotype 2a strain S. flexneri 2457T and tested against a panel of S. flexneri LPSs by enzyme-linked immunosorbent and immunoblot assays. Four monoclonal antibodies were identified, all of which showed restricted specificity patterns. Three different patterns of reactivity to LPS possessing the 3,4 group antigen were seen: (i) 2a only, (ii) 2a and 5a, and (iii) 2a, 4a, 5a, and Y. These results have implications for designing a Shigella vaccine that will be protective against related serotypes. Electron microscopy studies showed that the monoclonal antibodies bind to the bacterial surface in a patchy pattern, suggesting their potential use for examining the LPS distribution on the surface of the bacteria.

Intransal or intragastric immunization with proteosome-Shigella lipopolysaccharide vaccines protects against lethal pneumonia in a murine model of Shigella infection.

Mice immunized intransally or intragastrically with proteosome vaccines containing either Shigella sonnei or S. flexneri 2a lipopolysaccharide were protected against lethal pneumonia caused by homologous organisms in an experimental murine intranasal challenge model of Shigella infection. Histopathological analysis demonstrated that immunization also protected against the progressive lesions resulting from invasion of the pulmonary mucosa by S. sonnei. These data show that mucosal proteosome-lipopolysaccharide vaccines can protect against lethal bacterial pneumonia and indicate that such vaccines are promising candidates for protection against intestinal shigellosis.

Enhancement of anti-Shigella lipopolysaccharide (LPS) response by addition of the cholera toxin B subunit to oral and intranasal proteosome-Shigella flexneri 2a LPS vaccines

Addition of the cholera toxin B subunit to oral and intranasal proteosome-Shigella flexneri 2a lipopolysaccharide vaccines improved their immunogenicities. Enhancement of anti-O-Shigella immunoglobulin A levels was most evident in lung lavages following oral immunization and in lung and intestinal fluids when suboptimal doses were used with either immunization route.