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1.
Vaccine ; 41(34): 4967-4977, 2023 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-37400283

RESUMO

There is a need for vaccines effective against shigella infection in young children in resource-limited areas. Protective immunity against shigella infection targets the O-specific polysaccharide (OSP) component of lipopolysaccharide. Inducing immune responses to polysaccharides in young children can be problematic, but high level and durable responses can be induced by presenting polysaccharides conjugated to carrier proteins. An effective shigella vaccine will need to be multivalent, targeting the most common global species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. Here we report the development of shigella conjugate vaccines (SCV) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using squaric acid chemistry to result in single point sun-burst type display of OSP from carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. We confirmed structure and demonstrated that these conjugates were recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG responses, as well as rTTHc-specific IgG responses. Vaccination induced serotype-specific bactericidal antibody responses against S. flexneri, and vaccinated animals were protected against keratoconjunctivitis (Sereny test) and intraperitoneal challenge with virulent S. flexneri 2a and 3a, respectively. Our results support further development of this platform conjugation technology in the development of shigella conjugate vaccines for use in resource-limited settings.


Assuntos
Disenteria Bacilar , Vacinas contra Shigella , Shigella , Humanos , Criança , Animais , Camundongos , Pré-Escolar , Shigella flexneri , Vacinas Conjugadas , Disenteria Bacilar/prevenção & controle , Lipopolissacarídeos , Antígenos O , Anticorpos Antibacterianos , Imunoglobulina G
2.
mSphere ; 8(4): e0007323, 2023 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-37389412

RESUMO

The Shigella artificial invasin complex (InvaplexAR) vaccine is a subunit approach that effectively induces robust immunogenicity directed to serotype-specific lipopolysaccharide and the broadly conserved IpaB and IpaC proteins. One advantage of the vaccine approach is the ability to adjust the constituents to address suboptimal immunogenicity and to change the Shigella serotype targeted by the vaccine. As the vaccine moves through the product development pipeline, substantial modifications have been made to address manufacturing feasibility, acceptability to regulatory authorities, and developing immunogenic and effective products for an expanded list of Shigella serotypes. Modifications of the recombinant clones used to express affinity tag-free proteins using well-established purification methods, changes to detergents utilized in the assembly process, and in vitro and in vivo evaluation of different Invaplex formulations have led to the establishment of a scalable, reproducible manufacturing process and enhanced immunogenicity of Invaplex products designed to protect against four of the most predominant Shigella serotypes responsible for global morbidity and mortality. These adjustments and improvements provide the pathway for the manufacture and clinical testing of a multivalent Invaplex vaccine. IMPORTANCE Shigella species are a major global health concern that cause severe diarrhea and dysentery in children and travelers to endemic areas of the world. Despite significant advancements in access to clean water, the increases in antimicrobial resistance and the risk of post-infection sequelae, including cognitive and physical stunting in children, highlight the urgent need for an efficacious vaccine. One promising vaccine approach, artificial Invaplex, delivers key antigens recognized by the immune system during infection, which results in increased resistance to re-infection. The work presented here describes novel modifications to a previously described vaccine approach resulting in improved methods for manufacturing and regulatory approvals, expansion of the breadth of coverage to all major Shigella serotypes, and an increase in the potency of artificial Invaplex.


Assuntos
Vacinas contra Shigella , Shigella , Vacinas , Criança , Humanos , Shigella flexneri , Lipopolissacarídeos
3.
mSphere ; 3(2)2018.
Artigo em Inglês | MEDLINE | ID: mdl-29600284

RESUMO

The native Invaplex (InvaplexNAT) vaccine and adjuvant is an ion exchange-purified product derived from the water extract of virulent Shigella species. The key component of InvaplexNAT is a high-molecular-mass complex (HMMC) consisting of the Shigella lipopolysaccharide (LPS) and the invasin proteins IpaB and IpaC. To improve product purity and immunogenicity, artificial Invaplex (InvaplexAR) was developed using recombinant IpaB and IpaC proteins and purified Shigella LPS to assemble an HMMC consisting of all three components. Characterization of InvaplexAR by various methods demonstrated similar characteristics as the previously reported HMMC in InvaplexNAT. The well-defined InvaplexAR vaccine consistently contained greater quantities of IpaB, IpaC, and LPS than InvaplexNAT. InvaplexAR and InvaplexNAT immunogenicities were compared in mouse and guinea pig dose escalation studies. In both models, immunization induced antibody responses specific for InvaplexNAT and LPS while InvaplexAR induced markedly higher anti-IpaB and -IpaC serum IgG and IgA endpoint titers. In the murine model, homologous protection was achieved with 10-fold less InvaplexAR than InvaplexNAT and mice receiving InvaplexAR lost significantly less weight than mice receiving the same amount of InvaplexNAT. Moreover, mice immunized with InvaplexAR were protected from challenge with both homologous and heterologous Shigella serotypes. Guinea pigs receiving approximately 5-fold less InvaplexAR compared to cohorts immunized with InvaplexNAT were protected from ocular challenge. Furthermore, adjuvanticity previously attributed to InvaplexNAT was retained with InvaplexAR. The second-generation Shigella Invaplex vaccine, InvaplexAR, offers significant advantages over InvaplexNAT in reproducibility, flexible yet defined composition, immunogenicity, and protective efficacy. IMPORTANCEShigella species are bacteria that cause severe diarrheal disease worldwide, primarily in young children. Treatment of shigellosis includes oral fluids and antibiotics, but the high burden of disease, increasing prevalence of antibiotic resistance, and long-term health consequences clearly warrant the development of an effective vaccine. One Shigella vaccine under development is termed the invasin complex or Invaplex and is designed to drive an immune response to specific antigens of the bacteria in an effort to protect an individual from infection. The work presented here describes the production and evaluation of a new generation of Invaplex. The improved vaccine stimulates the production of antibodies in immunized mice and guinea pigs and protects these animals from Shigella infection. The next step in the product's development will be to test the safety and immune response induced in humans immunized with Invaplex.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Disenteria Bacilar/prevenção & controle , Imunogenicidade da Vacina , Vacinas contra Shigella/imunologia , Shigella/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Disenteria Bacilar/imunologia , Cobaias , Imunização , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Lipopolissacarídeos/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Reprodutibilidade dos Testes
4.
Chem Commun (Camb) ; 47(35): 9792-4, 2011 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-21818500

RESUMO

A water soluble vitamin B(12)-rhenium conjugate was synthesized and used in concert with intrinsic factor to screen for cubilin receptor-mediated uptake in lung cancer cells. Internalization of the conjugate demonstrated that it could be used to rapidly screen for the cubilin receptor in living cells, subsequently confirmed with Western blotting and RT-PCR.


Assuntos
Neoplasias Pulmonares/patologia , Receptores de Superfície Celular/genética , Rênio/química , Vitamina B 12/química , Vitamina B 12/metabolismo , Água/química , Transporte Biológico , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Solubilidade
5.
Dalton Trans ; 40(23): 6216-25, 2011 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-21369609

RESUMO

Nucleoside-derived drugs play an important role in the treatment of cancer. Here, we present the synthesis and characterization of an intriguing series of N3 conjugated Re(CO)(3) thymidine complexes. The complexes were characterized by NMR spectroscopy and mass spectrometry and their cytotoxicity was assessed against A549 cells. A similar dependence on the spacer length and the toxicity has been found for N3 functionalized complexes as recently reported for their C5' counterparts. A remarkable cytotoxic complex 22, carrying a dodecylene spacer at position N3 with a bis-quinoline metal chelate moiety, with an IC(50) value of 3.4 ± 1.6 µM, has been identified. Addition of a 100-fold excess of thymidine did not statistically reduce the observed cytotoxicity of all complexes. Cellular uptake studies of complex 22 have been performed by fluorescent microscopy, showing that compound 22 was clearly internalized into A549 cells. Temperature dependent uptake studies, blocking experiments with thymidine, and endosomal co-localization suggest that uptake of 22 occurs via passive diffusion and endocytosis.


Assuntos
Complexos de Coordenação/síntese química , Compostos Organometálicos/síntese química , Rênio/química , Timidina/química , Linhagem Celular Tumoral , Complexos de Coordenação/química , Complexos de Coordenação/toxicidade , Difusão , Endocitose , Humanos , Microscopia de Fluorescência , Compostos Organometálicos/química , Compostos Organometálicos/toxicidade , Temperatura
6.
ChemMedChem ; 5(9): 1513-29, 2010 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-20652929

RESUMO

Nucleoside analogues are extensively used in the treatment of cancer and viral diseases. The antiproliferative properties of organorhenium(I) complexes, however, have been scarcely explored to date. Herein we present the syntheses, characterization, and in vitro evaluation of Re(I)(CO)(3) core complexes of thymidine and uridine. For the binding of the Re(I)(CO)(3) core, a tridentate dipicolylamine metal chelate was introduced at positions C5', C2', N3, and C5 with spacers of various lengths. The corresponding organometallic thymidine complexes were fully characterized by IR and NMR spectroscopy and mass spectrometry. Their cytotoxicity was assessed against the A549 lung carcinoma cell line. Toxicity is dependent on the site and mode of conjugation as well as on the nature and the length of the tether. Moderate toxicity was observed for conjugates carrying the rhenium moiety at position C5' or N3 (IC(50)=124-160 microM). No toxicity was observed for complexes modified at C2' or C5. Complex 53, with a dodecylene spacer at C5', exhibits remarkable toxicity and is more potent than cisplatin, with an IC(50) value of 6.0 microM. To the best of our knowledge, this is the first report of the antiproliferative properties of [M(CO)(3)](+1)-nucleoside conjugates. In competitive inhibition experiments with A549 cell lysates and purified recombinant human thymidine kinase 1 (hTK-1), enzyme inhibition was observed for complexes modified at either N3 or C5', but our results suggest that the toxicity cannot be attributed solely to interaction with hTK-1.


Assuntos
Antineoplásicos/síntese química , Complexos de Coordenação/síntese química , Rênio/química , Timidina/química , Antineoplásicos/química , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Complexos de Coordenação/química , Complexos de Coordenação/toxicidade , Humanos , Relação Estrutura-Atividade , Timidina Quinase/antagonistas & inibidores , Timidina Quinase/metabolismo , Uridina/química
7.
Inorg Chem ; 49(15): 6790-2, 2010 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-20429603

RESUMO

Four new monomeric pyrophosphate complexes, namely [Co(phen)(2)(H(2)P(2)O(7))] x 4 H(2)O (1 x 4 H(2)O), [Ni(phen)(2)(H(2)P(2)O(7))] x 8 H(2)O (2 x 8 H(2)O), [Cu(phen)(H(2)O)(H(2)P(2)O(7))] (3) and {[Cu(phen)(H(2)O)(P(2)O(7))][Na(2)(H(2)O)(8)]} x 6 H(2)O (4 x 14 H(2)O) have been isolated and structurally characterized. The impact of pH and stoichiometry in obtaining 1-4 is described. These complexes have been tested against the adriamycin-resistant ovarian cancer cell line A2780/AD, revealing highly significant (nM) IC(50) values, compared to microM IC(50) values for cisplatin controls.


Assuntos
Antineoplásicos/química , Difosfatos/química , Compostos Organometálicos/química , Platina/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Conformação Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/farmacologia
8.
Clin Med Oncol ; 3: 19-26, 2009 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-20689607

RESUMO

Conjugates of methotrexate (MTX) and the nucleoside analogs 3-azidodeoxythymidine (AZT), iododeoxyuridine (IUdR) and dideoxycytidine (ddC) linked using poly(ethyleneglycol) are presented. In vitro cytotoxicity assays of the conjugates against drug resistant ovarian cell line A2780/AD are preformed and comparisons made to such assays performed for unconjugated (cocktail) systems. All systems tested were inactive, or had low activity, at 24 h. After 72 hr incubation however, the cocktails of MTX and AZT, IUdR or ddC showed high cytotoxicity in the low nanomolar range. The conjugates were only very moderately active with IC(50) values in the [0.1 to 1.0 mM] range. Conjugation of the antifolate to the nucleoside analogs has it seems reduced the activity significantly when compared to a cocktail of the components, indicating a conjugate approach is unlikely to translate into success in vivo. The positive note comes from the observation that by combining two of the new conjugates, namely those based on MTX with IUdR or AZT, an IC50 at 24 hours of ~ [180 muM] was produced.

9.
Nucleosides Nucleotides Nucleic Acids ; 27(2): 173-85, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18205071

RESUMO

Conjugates of three components namely folic acid, poly(ethyleneglycol) and 3 '-azido-3 '-deoxythymidine (AZT) are presented. Folate-PEG units were coupled to AZT to facilitate delivery of the nucleoside into the cell. A convenient separation of the polydisperse PEGylated-folic acid regioisomers produced upon conjugation is described. This is to select for the active gamma-regioisomer over the inactive alpha-regioisomer. In vitro cytotoxicity assays were conducted against an ovarian cell line (A2780/AD) that overexpresses the folate receptor (FR) and compared to a FR free control cell line. Compared to AZT a approximately 20-fold greater potency against the resistant ovarian line was observed for the conjugates.


Assuntos
Citotoxinas/síntese química , Citotoxinas/farmacologia , Ácido Fólico/síntese química , Ácido Fólico/farmacologia , Zidovudina/síntese química , Zidovudina/farmacologia , Proteínas de Transporte/agonistas , Linhagem Celular , Citotoxinas/química , Avaliação Pré-Clínica de Medicamentos , Resistência a Medicamentos/efeitos dos fármacos , Receptores de Folato com Âncoras de GPI , Ácido Fólico/química , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Receptores de Superfície Celular/agonistas , Zidovudina/química
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