Peptide Sequence of Pili Subunit Protein 49,8 kDa Shigella flexneri as Antigenic Epitope for Shigellosis Vaccine Development

ABSTRACTShigellais an important cause of diarrhea worldwide, with serotypesShigella flexneri2a,S. flexneri3a, andShigella sonneidemonstrating epidemiological prevalence. Many development efforts are focused onShigellalipopolysaccharide (LPS)-based vaccines, as O antigen-specific conjugate vaccines are immunogenic and efficacious. Immunization withShigellavaccines containing LPS can elicit antibodies capable of killingShigellain a serotype-specific manner. Thus, to facilitateShigellavaccine development, we have developed a serum bactericidal assay (SBA) specific for threeShigellaserotypes that measures killing of target bacteria at multiple serum dilutions and in the presence of exogenous complement. The SBA has a high analytical throughput and uses simple technologies and readily available reagents. The SBA was characterized with human sera with bactericidal antibodies againstS. flexneri2a,S. flexneri3a, andS. sonnei. Purified LPS of a homologous serotype, but not a heterologous serotype, inhibited bacterial killing. Assessment of precision found median intra-assay precision to be 13.3% and median interassay precision to be 19 to 30% for the three serotypes. The SBA is linear, with slight deviations for samples with low (~40) killing indices. The SBA was sensitive enough to allow about 100-fold predilution of serum samples. Repeat assays yielded results with less than 2-fold deviations, indicating the robustness of the assay. Assay results from four different laboratories were highly comparable when normalized with a reference serum. TheShigellaSBA, combined with a reference serum, should facilitate the development ofShigellavaccines across the field.IMPORTANCEShigellais an important cause of diarrhea worldwide, and efforts are ongoing to produce a safe and effectiveShigellavaccine. Although a clear immune correlate of protection has not been established, antibodies with bactericidal capacity may provide one means of protecting against shigellosis. Thus, it is important to measure the functional capacity of antibodies, as opposed to only binding activity. This article describes a simple, robust, and high-throughput serum bactericidal assay capable of measuringShigella-specific functional antibodiesin vitro. We show for the first time that this assay was successfully performed by multiple laboratories and generated highly comparable results, particularly when SBA titers were normalized using a reference standard. The serum bactericidal assay, along with a reference serum, should greatly facilitateShigellavaccine development.

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Shigella Vaccine Development: Finding the Path of Least Resistance

Clinical and Vaccine Immunology ◽

10.1128/cvi.00444-16 ◽

2016 ◽

Vol 23 (12) ◽

pp. 904-907 ◽

Cited By ~ 8

Author(s):

Wilbur H. Chen ◽

Karen L. Kotloff

Keyword(s):

Public Health ◽

Developing Countries ◽

Phase I ◽

Phase I Study ◽

Vaccine Development ◽

Shigella Flexneri ◽

Health Concern ◽

Childhood Diarrhea ◽

Public Health Concern ◽

Content Type

ABSTRACTShigellaspp. represent the second most common etiologic pathogen causing childhood diarrhea in developing countries. There are no licensedShigellavaccines, and progress for such vaccines has been limited. In this issue ofClinical and Vaccine Immunology, Riddle and colleagues (M. S. Riddle, R. W. Kaminski, C. Di Paolo, C. K. Porter, R. L. Gutierrez, et al., Clin Vaccine Immunol 23:908–917, 2016,http://dx.doi.org/10.1128/CVI.00224-16) report results from a phase I study of a parenterally administered monovalent O-polysaccharide “bioconjugate” directed againstShigella flexneri2a. Ultimately, the goal is to develop a broad-spectrumShigellavaccine to address this public health concern. A parenteralShigellavaccine capable of eliciting protection in children of developing countries would be an important tool to reach this goal.

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Monoclonal Antibodies to Shigella Lipopolysaccharide Are Useful for Vaccine Production

Clinical and Vaccine Immunology ◽

10.1128/cvi.00148-16 ◽

2016 ◽

Vol 23 (8) ◽

pp. 681-688 ◽

Cited By ~ 8

Author(s):

Jisheng Lin ◽

Mark A. Smith ◽

William H. Benjamin ◽

Robert W. Kaminski ◽

Heather Wenzel ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Vaccine Development ◽

Shigella Flexneri ◽

High Sensitivity ◽

Epidemiological Data ◽

Enzyme Linked Immunosorbent Assay ◽

Content Type ◽

Bacterial Surface ◽

Bactericidal Assay

ABSTRACTThere is a significant need for an effective multivalentShigellavaccine that targets the most prevalent serotypes. MostShigellavaccines under development utilize serotype-specific lipopolysaccharides (LPSs) as a major component based on protection and epidemiological data. As vaccine formulations advance from monovalent to multivalent, assays and reagents need to be developed to accurately and reproducibly quantitate the amount of LPSs from multiple serotypes in the final product. To facilitate this effort, we produced 36 hybridomas that secrete monoclonal antibodies (MAbs) against the O antigen on the LPS fromShigella flexneri2a,Shigella flexneri3a, andShigella sonnei. We used six of these monoclonal antibodies for an inhibition enzyme-linked immunosorbent assay (iELISA), measuring LPSs with high sensitivity and specificity. It was also demonstrated that theShigellaserotype-specific MAbs were useful for bacterial surface staining detected by flow cytometry. These MAbs are also useful for standardizing the serum bactericidal assay (SBA) forShigella. Functional assays, such as thein vitrobactericidal assay, are necessary for vaccine evaluation and may serve as immunological correlates of immunity. AnS. flexneri2a-specific monoclonal antibody killedS. flexneri2b isolates, suggesting thatS. flexneri2a LPS may induce cross-protection againstS. flexneri2b. Overall, theShigellaLPS-specific MAbs described have potential utility to the vaccine development community for assessing multivalent vaccine composition and as a reliable control for multiple immunoassays used to assess vaccine potency.

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Development of an Aotus nancymaae Model for Shigella Vaccine Immunogenicity and Efficacy Studies

Infection and Immunity ◽

10.1128/iai.01665-13 ◽

2014 ◽

Vol 82 (5) ◽

pp. 2027-2036 ◽

Cited By ~ 6

Author(s):

Michael Gregory ◽

Robert W. Kaminski ◽

Luis A. Lugo-Roman ◽

Hugo Galvez Carrillo ◽

Drake Hamilton Tilley ◽

...

Keyword(s):

Vaccine Strain ◽

Attack Rate ◽

Vaccine Development ◽

Protective Efficacy ◽

Shigella Flexneri ◽

Content Type ◽

Immune Correlates ◽

Study Results ◽

Combination Vaccines ◽

Series Of Experiments

ABSTRACTSeveral animal models exist to evaluate the immunogenicity and protective efficacy of candidateShigellavaccines. The two most widely used nonprimate models for vaccine development include a murine pulmonary challenge model and a guinea pig keratoconjunctivitis model. Nonhuman primate models exhibit clinical features and gross and microscopic colonic lesions that mimic those induced in human shigellosis. Challenge models for enterotoxigenicEscherichia coli(ETEC) andCampylobacterspp. have been successfully developed withAotus nancymaae, and the addition of aShigella-Aotuschallenge model would facilitate the testing of combination vaccines. A series of experiments were designed to identify the dose ofShigella flexneri2a strain 2457T that induces an attack rate of 75% in theAotusmonkey. After primary challenge, the dose required to induce an attack rate of 75% was calculated to be 1 × 1011CFU.Shigella-specific immune responses were low after primary challenge and subsequently boosted upon rechallenge. However, preexisting immunity derived from the primary challenge was insufficient to protect against the homologousShigellaserotype. A successive study inA. nancymaaeevaluated the ability of multiple oral immunizations with live-attenuatedShigellavaccine strain SC602 to protect against challenge. After three oral immunizations, animals were challenged withS. flexneri2a 2457T. A 70% attack rate was demonstrated in control animals, whereas animals immunized with vaccine strain SC602 were protected from challenge (efficacy of 80%;P= 0.05). The overall study results indicate that theShigella-Aotus nancymaaechallenge model may be a valuable tool for evaluating vaccine efficacy and investigating immune correlates of protection.

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The evolutionary history of Shigella flexneri serotype 6 in Asia

Microbial Genomics ◽

10.1099/mgen.0.000736 ◽

2021 ◽

Vol 7 (12) ◽

Author(s):

Si-Nguyen T. Mai ◽

Ladaporn Bodhidatta ◽

Paul Turner ◽

Sonam Wangchuk ◽

Tuyen Ha Thanh ◽

...

Keyword(s):

Type Species ◽

Evolutionary History ◽

Vaccine Development ◽

Phylogenetic Analyses ◽

Shigella Flexneri ◽

Epidemiological Studies ◽

Genomic Research ◽

Phylogenetic Groups ◽

Content Type ◽

Link Type

Shigella flexneri serotype 6 is an understudied cause of diarrhoeal diseases in developing countries, and has been proposed as one of the major targets for vaccine development against shigellosis. Despite being named as S. flexneri , Shigella flexneri serotype 6 is phylogenetically distinct from other S. flexneri serotypes and more closely related to S. boydii . This unique phylogenetic relationship and its low sampling frequency have hampered genomic research on this pathogen. Herein, by utilizing whole genome sequencing (WGS) and analyses of Shigella flexneri serotype 6 collected from epidemiological studies (1987–2013) in four Asian countries, we revealed its population structure and evolutionary history in the region. Phylogenetic analyses supported the delineation of Asian Shigella flexneri serotype 6 into two phylogenetic groups (PG-1 and −2). Notably, temporal phylogenetic approaches showed that extant Asian S. flexneri serotype 6 could be traced back to an inferred common ancestor arising in the 18th century. The dominant lineage PG-1 likely emerged in the 1970s, which coincided with the times to most recent common ancestors (tMRCAs) inferred from other major Southeast Asian S. flexneri serotypes. Similar to other S. flexneri serotypes in the same period in Asia, genomic analyses showed that resistance to first-generation antimicrobials was widespread, while resistance to more recent first-line antimicrobials was rare. These data also showed a number of gene inactivation and gene loss events, particularly on genes related to metabolism and synthesis of cellular appendages, emphasizing the continuing role of reductive evolution in the adaptation of the pathogen to an intracellular lifestyle. Together, our findings reveal insights into the genomic evolution of the understudied Shigella flexneri serotype 6, providing a new piece in the puzzle of Shigella epidemiology and evolution.

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Use of mchI Encoding Immunity to the Antimicrobial Peptide Microcin H47 as a Plasmid Selection Marker in Attenuated Bacterial Live Vectors

Infection and Immunity ◽

10.1128/iai.00487-08 ◽

2008 ◽

Vol 76 (10) ◽

pp. 4422-4430 ◽

Cited By ~ 6

Author(s):

Chee-Mun Fang ◽

Jin Yuan Wang ◽

Magaly Chinchilla ◽

Myron M. Levine ◽

William C. Blackwelder ◽

...

Keyword(s):

Antimicrobial Peptide ◽

Fluorescent Protein ◽

Vaccine Development ◽

Shigella Flexneri ◽

Antibody Responses ◽

Selection Marker ◽

Human Pathogens ◽

Bacterial Strains ◽

Igg Antibody ◽

Expression Plasmids

ABSTRACTLive attenuated bacterial strains expressing heterologous antigens represent an attractive vaccine development strategy. However, the use of drug resistance genes for the selection of expression plasmids introduced into live vectors poses theoretical health risks. Therefore, we developed a novel approach for plasmid selection based on immunity to the antimicrobial peptide microcin H47 (MccH47). Two expression plasmids encoding the reporter green fluorescent protein (GFPuv) were constructed; selection markers comprised eithermchI, conferring immunity to MccH47 (pGEN222I), orbla(encoding β-lactamase), conferring conventional resistance to ampicillin (pGEN222). GFPuv-specific serum immunoglobulin G (IgG) antibody responses were analyzed in mice immunized intranasally either withSalmonella entericaserovar Typhi CVD 908-htrAorShigella flexneri2a CVD 1208S live vector and were boosted parenterally with purified GFPuv. Similar IgG antibody responses were observed for both pGEN222 and pGEN222I when either CVD 1208S or CVD 908-htrA(pGEN222I) was used as the carrier. Interestingly, CVD 908-htrA(pGEN222I) elicited a significantly higher IgG response than CVD 908-htrA(pGEN222). We also compared the priming potential of homologous priming either with CVD 908-htrA(pGEN222I) or CVD 1208S(pGEN222I) to heterologous priming first with CVD 908-htrA(pGEN222I) and then with CVD 1208S(pGEN222I) and vice versa. Immunization with two unrelated live vectors significantly enhanced the IgG responses compared to responses engendered by homologous CVD 908-htrA(pGEN222I) but not to those of CVD 1208S(pGEN222I). MccH47 offers an alternate system for plasmid selection in bacterial live vectors that greatly improves their clinical acceptability. Furthermore, the success of the heterologous priming strategy supports the feasibility of the future development of multivalent live vector-based immunization strategies against multiple human pathogens.

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Safety and Immunogenicity of a Candidate Bioconjugate Vaccine against Shigella flexneri 2a Administered to Healthy Adults: a Single-Blind, Randomized Phase I Study

Clinical and Vaccine Immunology ◽

10.1128/cvi.00224-16 ◽

2016 ◽

Vol 23 (12) ◽

pp. 908-917 ◽

Cited By ~ 55

Author(s):

Mark S. Riddle ◽

Robert W. Kaminski ◽

Claudio Di Paolo ◽

Chad K. Porter ◽

Ramiro L. Gutierrez ◽

...

Keyword(s):

Phase I ◽

Phase I Study ◽

Vaccine Development ◽

Shigella Flexneri ◽

Protein A ◽

Age Groups ◽

Healthy Adults ◽

Candidate Vaccine ◽

Content Type ◽

Single Blind

ABSTRACTSeveral candidate vaccines againstShigellaspp. are in development, but the lack of a clear correlate of protection from challenge with the induction of adequate immune responses among the youngest age groups in the developing world has hamperedShigellavaccine development over the past several decades. Bioconjugation technology, exploited here for anShigella flexneri2a candidate vaccine, offers a novel and potentially cost-effective way to develop and produce vaccines against a major pathogen of global health importance. Flexyn2a, a novelS. flexneri2a bioconjugate vaccine made of the polysaccharide component of theS. flexneri2a O-antigen, conjugated to the exotoxin protein A ofPseudomonas aeruginosa(EPA), was evaluated for safety and immunogenicity among healthy adults in a single-blind, phase I study with a staggered randomization approach. Thirty subjects (12 receiving 10 μg Flexyn2a, 12 receiving Flexyn2a with aluminum adjuvant, and 6 receiving placebo) were administered two injections 4 weeks apart and were followed for 168 days. Flexyn2a was well-tolerated, independently of the adjuvant and number of injections. The Flexyn2a vaccine elicited statistically significantS. flexneri2a lipopolysaccharide (LPS)-specific humoral responses at all time points postimmunization in all groups that received the vaccine. Elicited serum antibodies were functional, as evidenced by bactericidal activity againstS. flexneri2a. The bioconjugate candidate vaccine Flexyn2a has a satisfactory safety profile and elicited a robust humoral response toS. flexneri2a LPS with or without inclusion of an adjuvant. Moreover, the bioconjugate also induced functional antibodies, showing the technology's features in producing a promising candidate vaccine. (This study has been registered at ClinicalTrials.gov under registration no. NCT02388009.)

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Potentiation of the growth-promoting activity of porcine growth hormone (pGH) with an antibody generated in rabbits to the peptide sequence pGH110–118

Journal of Endocrinology ◽

10.1677/joe.0.1270481 ◽

1990 ◽

Vol 127 (3) ◽

pp. 481-485 ◽

Cited By ~ 14

Author(s):

B. S. Wang ◽

E. Szewczyk ◽

H.-M. Shieh ◽

I. C. Hart

Keyword(s):

Amino Acids ◽

Growth Hormone ◽

Growth Rate ◽

The Other ◽

Peptide Sequence ◽

Antigenic Epitope ◽

Peptide Antigens ◽

Epitope Specificity ◽

Hypophysectomized Rats ◽

Growth Promoting

ABSTRACT An effort was made to identify the antigenic epitope(s) of porcine GH (pGH) capable of inducing antibodies which would enhance the growth-promoting activity of the hormone. Several peptide sequences of the pGH molecule were synthesized and the antibodies to these peptides were generated in rabbits. The majority of these antibodies were found to be immunoreactive with both intact pGH and their respective peptide antigens. The biological activity of the antibodies was tested in growth-retarded hypophysectomized rats. Daily injections with pGH alone significantly increased their growth rate and treatment with a combination of pGH and antibody specific to the peptide sequence 110–118 further heightened the somatogenic effect. Administration of this antibody alone did not enhance the growth rate of hypophysectomized rats, neither did antibodies raised to intact pGH or the other peptide sequences. The present findings suggest that antibody with a particular pGH epitope specificity is able to increase the somatogenic activity of pGH and that the peptide corresponding to pGH amino acids 110–118 may prove useful in the development of growth promoting vaccines. Journal of Endocrinology (1990) 127, 481–485

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Shigella flexneri Adherence Factor Expression in In Vivo-Like Conditions

mSphere ◽

10.1128/msphere.00751-19 ◽

2019 ◽

Vol 4 (6) ◽

Cited By ~ 1

Author(s):

Rachael B. Chanin ◽

Kourtney P. Nickerson ◽

Alejandro Llanos-Chea ◽

Jeticia R. Sistrunk ◽

David A. Rasko ◽

...

Keyword(s):

Epithelial Cells ◽

Biofilm Formation ◽

Bile Salts ◽

Vaccine Development ◽

Shigella Flexneri ◽

Colonic Epithelium ◽

Structural Genes ◽

Content Type ◽

Factor Expression

ABSTRACT The Shigella species are Gram-negative, facultative intracellular pathogens that invade the colonic epithelium and cause significant diarrheal disease. Despite extensive research on the pathogen, a comprehensive understanding of how Shigella initiates contact with epithelial cells remains unknown. Shigella maintains many of the same Escherichia coli adherence gene operons; however, at least one critical gene component in each operon is currently annotated as a pseudogene in reference genomes. These annotations, coupled with a lack of structures upon microscopic analysis following growth in laboratory media, have led the field to hypothesize that Shigella is unable to produce fimbriae or other traditional adherence factors. Nevertheless, our previous analyses have demonstrated that a combination of bile salts and glucose induces both biofilm formation and adherence to colonic epithelial cells. The goal of this study was to perform transcriptomic and genetic analyses to demonstrate that adherence gene operons in Shigella flexneri strain 2457T are functional, despite the gene annotations. Our results demonstrate that at least three structural genes facilitate S. flexneri 2457T adherence for epithelial cell contact and biofilm formation. Furthermore, our results demonstrate that host factors, namely, glucose and bile salts at their physiological concentrations in the small intestine, offer key environmental stimuli required for adherence factor expression in S. flexneri. This research may have a significant impact on Shigella vaccine development and further highlights the importance of utilizing in vivo-like conditions to study bacterial pathogenesis. IMPORTANCE Bacterial pathogens have evolved to regulate virulence gene expression at critical points in the colonization and infection processes to successfully cause disease. The Shigella species infect the epithelial cells lining the colon to result in millions of cases of diarrhea and a significant global health burden. As antibiotic resistance rates increase, understanding the mechanisms of infection is vital to ensure successful vaccine development. Despite significant gains in our understanding of Shigella infection, it remains unknown how the bacteria initiate contact with the colonic epithelium. Most pathogens harbor multiple adherence factors to facilitate this process, but Shigella was thought to have lost the ability to produce these factors. Interestingly, we have identified conditions that mimic some features of gastrointestinal transit and that enable Shigella to express adherence structural genes. This work highlights aspects of genetic regulation for Shigella adherence factors and may have a significant impact on future vaccine development.

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Fine mapping of sequential neutralization epitopes on the subunit protein VP8 of human rotavirus

Biochemical Journal ◽

10.1042/bj20021969 ◽

2003 ◽

Vol 376 (1) ◽

pp. 269-275 ◽

Cited By ~ 23

Author(s):

Jennifer KOVACS-NOLAN ◽

Dongwan YOO ◽

Yoshinori MINE

Keyword(s):

Amino Acids ◽

Vaccine Development ◽

Polyclonal Antibodies ◽

Egg Yolk ◽

Virus Neutralization ◽

Amino Acid Residues ◽

Peptide Fragments ◽

Subunit Protein ◽

Human Rotavirus

The epitopes of the HRV (human rotavirus), especially those involved in virus neutralization, have not been determined in their entirety, and would have significant implications for HRV vaccine development. In the present study, we report on the epitope mapping and identification of sequential neutralization epitopes, on the Wa strain HRV subunit protein VP8, using synthetic overlapping peptides. Polyclonal antibodies against recombinant Wa VP8 were produced previously in chicken, and purified from egg yolk, which showed neutralizing activity against HRV in vitro. Overlapping VP8 peptide fragments were synthesized and probed with the anti-VP8 antibodies, revealing five sequential epitopes on VP8. Further analysis suggested that three of the five epitopes detected, M1-L10, I55-D66 and L223-P234, were involved in virus neutralization, indicating that sequential epitopes may also be important for the HRV neutralization. The interactions of the antibodies with the five epitopes were characterized by an examination of the critical amino acids involved in antibody binding. Epitopes comprised primarily of hydrophobic amino acid residues, followed by polar and charged residues. The more critical amino acids appeared to be located near the centre of the epitopes, with proline, isoleucine, serine, glutamine and arginine playing an important role in the binding of antibody to the VP8 epitopes.

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