Acidic derivatives of homocysteine thiolactone: utility as anionic linkers

Leanza, W. J.; Chupak, Louis S.; Tolman, Richard L.; Marburg, Stephen

doi:10.1021/bc00018a009

Cited by 23 publications

(15 citation statements)

References 10 publications

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“…Maleimide equivalents in an aliquot of the activated Pfs25H were measured with an N-acetylcysteine consumption assay using Ellman's reagent, 5,5Ј-dithionitrobenzoic acid (Pierce) to measure residual thiol (19). N-acetylhomocysteine thiolactone-activated OMPC was aseptically prepared with a 2-h aging time as described (20), except that potassium phosphate buffer was replaced with sterile water. Before use, Nacetylhomocysteinyl-OMPC was made to be 25 mM 2-morpholinoethane sulfonic acid buffer, pH 6.1.…”

Section: Methodsmentioning

confidence: 99%

Sustained high-titer antibody responses induced by conjugating a malarial vaccine candidate to outer-membrane protein complex

Przysiecki²,

Flanagan³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

117

113

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The development of protein subunit vaccines to combat some of the world's deadliest pathogens such as a malaria parasite, Plasmodium falciparum, is stalled, due in part to the inability to induce and sustain high-titer antibody responses. Here, we show the induction of persistent, high-titer antibody responses to recombinant Pfs25H, a human malarial transmission-blocking protein vaccine candidate, after chemical conjugation to the outer-membrane protein complex (OMPC) of Neisseria meningitidis serogroup B and adsorption to aluminum hydroxyphosphate. In mice, the Pfs25H-OMPC conjugate vaccine was >1,000 times more potent in generating anti-Pfs25H ELISA reactivity than a similar 0.5-g dose of Pfs25H alone in Montanide ISA720, a water-in-oil adjuvant. The immune enhancement requires covalent conjugation between Pfs25H and the OMPC, given that physically mixed Pfs25H and OMPC on aluminum hydroxyphosphate failed to induce greater activity than the nonconjugated Pfs25H on aluminum hydroxyphosphate. The conjugate vaccine Pfs25H-OMPC also was highly immunogenic in rabbits and rhesus monkeys. In rhesus monkeys, the antibody responses were sustained over 18 months, at which time another vaccination with nonconjugated Pfs25H induced strong anamnestic responses. The vaccine-induced anti-Pfs25-specific antibodies in all animal species blocked the transmission of parasites to mosquitoes. Protein antigen conjugation to OMPC or other protein carrier may have general application to a spectrum of protein subunit vaccines to increase immunogenicity without the need for potentially reactogenic adjuvants. malaria ͉ Pfs25 ͉ transmission-blocking vaccine A s reported by the World Health Organization in 2004, the worldwide incidence of malaria is Ϸ300 million clinical cases and 1.3 million deaths annually (1). Of the four species of malaria parasites that infect humans, Plasmodium falciparum is responsible for the majority of these deaths, and Plasmodium vivax accounts for Ͼ50% of all malarial infections outside Africa and 10% of those in Africa. Mounting drug resistance by the malaria parasite makes chemotherapy increasingly difficult. Three types of malarial vaccines are under research and development: (i) vaccines targeting the liver-stage parasite development for sterile immunity; (ii) vaccines targeting the blood-stage parasite to reduce disease burden; and (iii) vaccines targeting the parasite development in the mosquito stage to block transmission, called transmission-blocking vaccines (TBVs). For all three types of vaccines, antibody is important. For TBVs, antibody is the only mechanism for providing immune protection. TBVelicited functional antibodies, ingested with the sexual stages of the parasite in a blood meal by a mosquito, will inhibit or block parasite development in the mosquito.Pxs25 proteins encoded by orthologous genes and expressed on the surface of zygotes and ookinetes during the development of the malaria parasite P. falciparum (Pfs25) and P. vivax (Pvs25) are leading candidates for mosquito-stage transmission...

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Section: Methodsmentioning

confidence: 99%

Sustained high-titer antibody responses induced by conjugating a malarial vaccine candidate to outer-membrane protein complex

Przysiecki²,

Flanagan³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

117

113

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“…In 1956 it was found that N-acetyl-Hcy-thiolactone can react with amines and amino acids in alkaline solutions (pH > 9) with the formation of a peptide bond [203]. Nevertheless, N-acetyl-Hcy-thiolactone has been used to introduce a thiol functionality into small molecules and macromolecules, including aminoglycosides, amino lipids, muramyl dipeptide, cell-surface carbohydrates, human serum albumin, lactoglobulin, enzymes, and oligonucleotides for a variety of applications [207]. However, because N-acetyl-Hcy-thiolactone is much less reactive than Hcy-thiolactone, these studies required long reaction times, relatively high pH [205], or the presence of silver ions as a catalyst [205,206].…”

Section: Reactivity Toward Amino Groupsmentioning

confidence: 99%

Homocysteine in Protein Structure/Function and Human Disease

Jakubowski¹

2013

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“…a carrier protein that has been shown to be effective (27) in humans as a conjugate with H. influenzae capsular polysaccharide (PedvaxHIB @). Conjugation of thiolated OMPC with bromoacetyl-Arg-Arg-Arg produced an insoluble product (28), whereas neutral peptide-OMPC conjugates were soluble entities. Assay of the unique thioether in conjugate hydolysates provides a convenient means of determining the degree of covalent attachment of the two macromolecules.…”

Section: Vaccine Conjugationmentioning

confidence: 99%

Cyclic V3‐loop‐related HIV‐1 conjugate vaccines Synthesis, conformation and immunological properties

Tolman

Bednarek

Johnson

et al. 1993

International Journal of Peptide and Protein Research

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Branched undecapeptides with sequences related to the virus glycoprotein V3 domain sequences of the MN and IIIB variants of HIV-1 were synthesized and cyclized with a peptide (amide) closure to cyclic decapeptides. Two-dimensional NMR studies allowed protons for the MN variant-related cycle (L-697,250) to be assigned. Molecular modelling with distance geometry methods permitted a conformation to be identified which showed good agreement with ROESY and 2D NMR study data. A molecular dynamics simulation showed that the highly conserved loop tip sequence (Gly-Pro-Gly-Arg) was in a conventional 8-turn less than 50% of the time. For evaluation of immunogenicity and antibody characterization studies, covalent carrier conjugates were prepared. 3-Maleimidopropionylation of the Nle amino group of the cyclic peptides gave an electrophilic tether which captured a thiol group from a thiolated carrier protein, OMPC (outer membrane protein complex of Neisseritr meningitidis). Through the use of a novel co-conjugation procedure, soluble immunogen-carrier molecules were prepared which had suitable physical properties for use as a vaccine. These V3-loop-based vaccines could elicit neutralizing antibody, but not consistently in all animals. Characterization of sera showed that responses were broadly virus neutralizing.

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Acidic derivatives of homocysteine thiolactone: utility as anionic linkers

Cited by 23 publications

References 10 publications

Sustained high-titer antibody responses induced by conjugating a malarial vaccine candidate to outer-membrane protein complex

Sustained high-titer antibody responses induced by conjugating a malarial vaccine candidate to outer-membrane protein complex

Homocysteine in Protein Structure/Function and Human Disease

Cyclic V3‐loop‐related HIV‐1 conjugate vaccines Synthesis, conformation and immunological properties

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