RESUMO
Ac-TMP-2, an immunodominant hookworm antigen encoding a tissue inhibitor of metalloproteinase (TIMP) was cloned by immunoscreening an Ancylostoma caninum larval cDNA library with sera pooled from dogs immunized with irradiated A. caninum third stage larvae (ir-L3). The open reading frame of Ac-tmp-2 cDNA encoded a 244 amino acids (predicted molecular weight of 27.7 kDa), which shared a common N-terminus with other vertebrate and invertebrate TIMPs, including Ac-TMP-1, the most abundant adult hookworm secreted protein. However Ac-TMP-2 also contains an unusual multicopy (ten) repeat of the amino acid sequence, KTVEENDE. By immunoblotting, Ac-TMP-2 was detected only in adult hookworms and their excretory secretory products although the corresponding mRNA was also detected in L3. Immunolocalization with specific antiserum showed that native Ac-TMP-2 was located in adult worm's esophagus and cephalic glands. Recombinant Ac-TMP-2 expressed in bacteria was highly immunogenic and recognized by ir-L3 immunized dog immune sera. The recombinant Ac-TMP-2 protein inhibited the human matrix metalloproteinases, MMP-2, MMP-7 and MMP-13. As an immunodominant protein having a possible role in the parasite-host relationship of canine hookworm infection, recombinant Ac-TMP-2 represents a plausible target for vaccine development.
Assuntos
Ancylostoma/metabolismo , Antígenos de Helmintos/química , Antígenos de Helmintos/genética , Proteínas de Helminto/química , Proteínas de Helminto/genética , Inibidores Teciduais de Metaloproteinases/química , Inibidores Teciduais de Metaloproteinases/genética , Sequência de Aminoácidos , Ancylostoma/enzimologia , Animais , Antígenos de Helmintos/análise , Clonagem Molecular , Cães , Proteínas de Helminto/análise , Humanos , Metaloproteinases da Matriz Secretadas/antagonistas & inibidores , Metaloproteinases da Matriz Secretadas/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidores Teciduais de Metaloproteinases/análiseRESUMO
Previously, we identified several attenuating mutations in the L polymerase protein of human parainfluenza virus type 2 (HPIV2) and genetically stabilized those mutations using reverse genetics [Nolan SM, Surman S, Amaro-Carambot E, Collins PL, Murphy BR, Skiadopoulos MH. Live-attenuated intranasal parainfluenza virus type 2 vaccine candidates developed by reverse genetics containing L polymerase protein mutations imported from heterologous paramyxoviruses. Vaccine 2005;39(23):4765-74]. Here we describe the discovery of an attenuating mutation at nucleotide 15 (15(T-->C)) in the 3' genomic promoter that was also present in the previously characterized mutants. We evaluated the properties of this promoter mutation alone and in various combinations with the L polymerase mutations. Amino acid substitutions at L protein positions 460 (460A or 460P) or 948 (948L), or deletion of amino acids 1724 and 1725 (Delta1724), each conferred a temperature sensitivity (ts) phenotype whereas the 15(T-->C) mutation did not. The 460A and 948L mutations each contributed to restricted replication in the lower respiratory tract of African green monkeys, but the Delta1724 mutation increased attenuation only in certain combinations with other mutations. We constructed two highly attenuated viruses, rV94(15C)/460A/948L and rV94(15C)/948L/Delta1724, that were immunogenic and protective against challenge with wild-type HPIV2 in African green monkeys and, therefore, appear to be suitable for evaluation in humans.