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1.
Vaccine ; 41(42): 6261-6271, 2023 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-37666695

RESUMO

Shigella species cause severe disease among travelers to, and children living in, endemic countries. Although significant efforts have been made to improve sanitation, increased antibiotic resistance and other factors suggest an effective vaccine is a critical need. Artificial Invaplex (InvaplexAR) is a subunit vaccine approach complexing Shigella LPS with invasion plasmid antigens. In pre-clinical studies, the InvaplexAR vaccine demonstrated increased immunogenicity as compared to the first generation product and was subsequently manufactured under cGMP for clinical testing in a first-in-human Phase 1 study. The primary objective of this study was the safety of S. flexneri 2a InvaplexAR given by intranasal (IN) immunization (without adjuvant) in a single-center, open-label, dose-escalating Phase 1 trial and secondarily to assess immunogenicity to identify a dose of InvaplexAR for subsequent clinical evaluations. Subjects received three IN immunizations of InvaplexAR, two weeks apart, in increasing dose cohorts (10 µg, 50 µg, 250 µg, and 500 µg). Adverse events were monitored using symptom surveillance, memory aids, and targeted physical exams. Samples were collected throughout the study to investigate vaccine-induced systemic and mucosal immune responses. There were no adverse events that met vaccination-stopping criteria. The majority (96%) of vaccine-related adverse events were mild in severity (most commonly nasal congestion, rhinorrhea, and post-nasal drip). Vaccination with InvaplexAR induced anti-LPS serum IgG responses and anti-Invaplex IgA and IgG antibody secreting cell (ASC) responses at vaccine doses ≥250 µg. Additionally, mucosal immune responses and functional antibody responses were seen from the serum bactericidal assay measurements. Notably, the responder rates and the kinetics of ASCs and antibody lymphocyte secretion (ALS) were similar, suggesting that either assay may be employed to identify IgG and IgA secreting cells. Further studies with InvaplexAR will evaluate alternative immunization routes, vaccination schedules and formulations to further optimize immunogenicity. (Clinical Trial Registry Number NCT02445963).

2.
mSphere ; 7(3): e0102021, 2022 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-35611657

RESUMO

Diarrheal diseases are a leading cause of global morbidity and mortality, disproportionately affecting children in resource-limited settings. Although improvements in hygiene and access to clean water are helpful, vaccines are considered essential due to the low infectious dose of Shigella species and increasing antibiotic resistance. Building on achievements with conjugate vaccines, a safe and immunogenic novel bioconjugate vaccine linking Shigella O-antigen to Pseudomonas aeruginosa exoprotein A has been developed to induce immunity against Shigella flexneri 2a, 3a, and 6 and S. sonnei. This study evaluated the breadth of reactivity and functionality of pooled serum from rabbits immunized with monovalent and quadrivalent Shigella bioconjugates formulated with or without an adjuvant against Shigella serotypes isolated in Kenya. Rabbit sera were assayed by colony blot for reactivity with 67 isolates of Shigella serotypes targeted by the vaccine, S. flexneri (2a, 3a, and 6) and S. sonnei, and 42 isolates of Shigella serotypes not targeted by the vaccine, S. flexneri (1b, 2b, 4a, and 4b), S. boydii, and S. dysenteriae. Shigella isolates testing positive in the colony blot assay were then used to assess functional activity using a bactericidal assay. Of the 41 Shigella isolates targeted by the vaccine, 22 were reactive with the adjuvanted quadrivalent and the respective monovalent rabbit sera. The S. flexneri 2a and 3a monovalent rabbit serum cross-reacted with S. flexneri 3a, 2b, and 2a, respectively. Immunization with the adjuvanted quadrivalent vaccine also induced cross-reactivity with isolates of S. flexneri 2b, 4a, and 4b. Collectively, these results suggest that the Shigella quadrivalent vaccine may be more broadly protective than designed, offering a promising solution to Shigella infections. IMPORTANCE Diarrheal diseases are the third leading cause of death globally, disproportionally affecting low- to middle-income countries like Kenya, with Shigella species being the leading cause of bacterial diarrhea, especially in children. The low infectious dose and high antibiotic resistance levels complicate treatment, leading to long-term sequelae that necessitate control measures such as vaccines to reduce morbidity and mortality rates, especially among children under 5 years of age. A quadrivalent bioconjugate Shigella vaccine was recently developed to safely and effectively induce immunity against four important Shigella spp. This study demonstrates the breadth of reactivity and functionality of the parenterally administered bioconjugate vaccine by evaluating the ability of rabbit sera to bind and kill Shigella isolates recently collected in Kenya. These results suggest that the Shigella quadrivalent vaccine may be more broadly protective than designed and may offer a promising solution to the morbidity and mortality associated with Shigella infections.


Assuntos
Disenteria Bacilar , Vacinas contra Shigella , Shigella , Animais , Anticorpos Antibacterianos , Antígenos de Bactérias , Diarreia , Quênia , Coelhos , Shigella sonnei , Vacinação , Vacinas Combinadas
3.
EBioMedicine ; 66: 103308, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33813141

RESUMO

BACKGROUND: Diarrheal diseases are a leading cause of global morbidity and mortality affecting all ages, but especially children under the age of five in resource-limited settings. Shigella is a leading contributor to diarrheal diseases caused by bacterial pathogens and is considered a significant antimicrobial resistance threat. While improvements in hygiene, and access to clean water help as control measures, vaccination remains one of the most viable options for significantly reducing morbidity and mortality. METHODS: Flexyn2a is a bioconjugate vaccine manufactured using novel conjugation methodologies enzymatically linking the O-polysaccharide of S. flexneri 2a to exotoxin A of Pseudomonas aeruginosa. The protective capacity of Flexyn2a was assessed in a controlled human infection model after two intramuscular immunizations. Immune responses pre- and post-immunization and/or infection were investigated and are described here. FINDINGS: Flexyn2a induced lipopolysaccharide (LPS)-specific serum IgG responses post-immunization which were associated with protection against shigellosis. Additionally, several other immune parameters, including memory B cell responses, bactericidal antibodies and serum IgA, were also elevated in vaccinees protected against shigellosis. Immunization with Flexyn2a also induced gut-homing, LPS-specific IgG and IgA secreting B cells, indicating the vaccine induced immune effectors functioning at the site of intestinal infection. INTERPRETATION: Collectively, the results of these immunological investigations provide insights into protective immune mechanisms post-immunization with Flexyn2a which can be used to further guide vaccine development and may have applicability to the larger Shigella vaccine field. FUNDING: Funding for this study was provided through a Wellcome Trust grant.


Assuntos
Disenteria Bacilar/imunologia , Disenteria Bacilar/prevenção & controle , Imunidade , Vacinas contra Shigella/imunologia , Shigella flexneri/imunologia , Anticorpos Antibacterianos/imunologia , Especificidade de Anticorpos/imunologia , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Memória Imunológica , Lipopolissacarídeos/imunologia , Linfócitos/imunologia , Linfócitos/metabolismo , Vacinas contra Shigella/administração & dosagem , Vacinação
4.
mSphere ; 5(5)2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968012

RESUMO

Shigella is a major cause of moderate to severe diarrhea largely affecting children (<5 years old) living in low- and middle-income countries. Several vaccine candidates are in development, and controlled human infection models (CHIMs) can be useful tools to provide an early assessment of vaccine efficacy and potentially support licensure. A lyophilized strain of S. sonnei 53G was manufactured and evaluated to establish a dose that safely and reproducibly induced a ≥60% attack rate. Samples were collected pre- and postchallenge to assess intestinal inflammatory responses, antigen-specific serum and mucosal antibody responses, functional antibody responses, and memory B cell responses. Infection with S. sonnei 53G induced a robust intestinal inflammatory response as well as antigen-specific antibodies in serum and mucosal secretions and antigen-specific IgA- and IgG-secreting B cells positive for the α4ß7 gut-homing marker. There was no association between clinical disease outcomes and systemic or functional antibody responses postchallenge; however, higher lipopolysaccharide (LPS)-specific serum IgA- and IgA-secreting memory B cell responses were associated with a reduced risk of disease postchallenge. This study provides unique insights into the immune responses pre- and postinfection with S. sonnei 53G in a CHIM, which could help guide the rational design of future vaccines to induce protective immune responses more analogous to those triggered by infection.IMPORTANCE Correlate(s) of immunity have yet to be defined for shigellosis. As previous disease protects against subsequent infection in a serotype-specific manner, investigating immune response profiles pre- and postinfection provides an opportunity to identify immune markers potentially associated with the development of protective immunity and/or with a reduced risk of developing shigellosis postchallenge. This study is the first to report such an extensive characterization of the immune response after challenge with S. sonnei 53G. Results demonstrate an association of progression to shigellosis with robust intestinal inflammatory and mucosal gut-homing responses. An important finding in this study was the association of elevated Shigella LPS-specific serum IgA and memory B cell IgA responses at baseline with reduced risk of disease. The increased baseline IgA responses may contribute to the lack of dose response observed in the study and suggests that IgA responses should be further investigated as potential correlates of immunity.


Assuntos
Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/imunologia , Disenteria Bacilar/imunologia , Memória Imunológica , Adolescente , Adulto , Linfócitos B/imunologia , Vacinas Bacterianas/administração & dosagem , Fezes/química , Feminino , Liofilização , Humanos , Imunoglobulina A/análise , Imunoglobulina A/sangue , Imunoglobulina G/análise , Imunoglobulina G/sangue , Lipopolissacarídeos/imunologia , Masculino , Pessoa de Meia-Idade , Mucosa/imunologia , Shigella sonnei/imunologia , Adulto Jovem
5.
J Vis Exp ; (144)2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30882773

RESUMO

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.


Assuntos
Anticorpos Antibacterianos/imunologia , Especificidade de Anticorpos , Atividade Bactericida do Sangue , Shigella/efeitos dos fármacos , Antígenos de Bactérias/imunologia , Contagem de Colônia Microbiana , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imunoensaio/métodos , Soro
6.
mSphere ; 3(3)2018 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-29898979

RESUMO

Shigella is an important cause of diarrhea worldwide, with serotypes Shigella flexneri 2a, S. flexneri 3a, and Shigella sonnei demonstrating epidemiological prevalence. Many development efforts are focused on Shigella lipopolysaccharide (LPS)-based vaccines, as O antigen-specific conjugate vaccines are immunogenic and efficacious. Immunization with Shigella vaccines containing LPS can elicit antibodies capable of killing Shigella in a serotype-specific manner. Thus, to facilitate Shigella vaccine development, we have developed a serum bactericidal assay (SBA) specific for three Shigella serotypes 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 against S. flexneri 2a, S. flexneri 3a, and S. 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. The Shigella SBA, combined with a reference serum, should facilitate the development of Shigella vaccines across the field.IMPORTANCEShigella is an important cause of diarrhea worldwide, and efforts are ongoing to produce a safe and effective Shigella vaccine. 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 measuring Shigella-specific functional antibodies in 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 facilitate Shigella vaccine development.


Assuntos
Anticorpos Antibacterianos/sangue , Atividade Bactericida do Sangue , Disenteria Bacilar/imunologia , Ensaios de Triagem em Larga Escala/métodos , Imunoensaio/métodos , Shigella flexneri/imunologia , Shigella sonnei/imunologia , Antígenos de Bactérias/imunologia , Humanos , Lipopolissacarídeos/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Soro/imunologia , Shigella flexneri/fisiologia , Shigella sonnei/fisiologia
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