RESUMO
The antibody-dependent enhancement (ADE) of feline infectious peritonitis virus (FIPV) infection has been recognized in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of feline infectious peritonitis (FIP). In the present study, we synthesized eighty-one kinds of peptides derived from the spike (S)2 domain of type I FIPV KU-2 strain, the S2 domain of type II FIPV 79-1146 strain, and the nucleocapcid (N) protein of FIPV KU-2 strain. To detect the T helper (Th)1 epitope, peripheral blood mononuclear cells (PBMCs) obtained from FIPV-infected cats were cultured with each peptide, and Th1-type immune responses were measured using feline interferon (fIFN)-γ production as an index. To detect the linear immunodominant antibody-binding epitope, we investigated the reactivity of plasma collected from FIPV-infected cats against each peptide by ELISA. Four and 2 peptides containing Th1 epitopes were identified in the heptad repeat (HR)1 and inter-helical (IH) regions of the S2 domain of type I FIPV, respectively, and these were located on the N-terminal side of the regions. In the S2 domain of type II FIPV, 2, 3, and 2 peptides containing Th1 epitopes were identified in the HR1, IH, and HR2 regions, respectively, and these were mainly located on the C-terminal side of the regions. In the S2 domain of type I FIPV, 3 and 7 peptides containing linear immunodominant antibody-binding epitopes were identified in the IH and HR2 regions, respectively. In the S2 domain of type II FIPV, 4 peptides containing linear immunodominant antibody-binding epitopes were identified in the HR2 region. The Th1 epitopes in the S2 domain of type I and II FIPV were located in different regions, but the linear immunodominant antibody-binding epitopes were mostly located in the HR2 region. Eight peptides containing Th1 epitopes were identified in N protein, and 3 peptides derived from residues 81 to 100 and 137 to 164 showed strong inductivity of fIFN-γ production in PBMCs isolated from type I FIPV- and type II FIPV-infected non-FIP cats. In N protein, 4 peptides containing linear immunodominant antibody-binding epitopes were identified, and 2 peptides derived from residues 345 to 372 showed strong reactivity with plasma of type I FIPV- and type II FIPV-infected cats. The Th1 and linear immunodominant antibody-binding epitopes were located at different positions in both the S2 domain and N protein. Our results may provide important information for the development of peptide-based vaccine against FIPV infection.
Assuntos
Anticorpos Antivirais/imunologia , Sítios de Ligação de Anticorpos , Coronavirus Felino/imunologia , Epitopos de Linfócito T/imunologia , Testes Genéticos/métodos , Epitopos Imunodominantes/imunologia , Proteínas do Nucleocapsídeo/imunologia , Células Th1/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/metabolismo , Gatos , Coronavirus Felino/metabolismo , Epitopos/imunologia , Epitopos/metabolismo , Epitopos de Linfócito T/metabolismo , Peritonite Infecciosa Felina/imunologia , Peritonite Infecciosa Felina/metabolismo , Epitopos Imunodominantes/metabolismo , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/metabolismo , Estrutura Terciária de Proteína , Células Th1/metabolismoRESUMO
Unmethylated CpG-ODN are known to enhance Th1-type immune response. However, optimal sequences of CpG-ODN for activating Th1-type immune cells vary among species. It is necessary to identify the effective CpG-ODN sequences in each species. In the present study, in order to identify the sequences of CpG-ODN that produce fIFN-γ in cats, 14 kinds of ODN were synthesized and examined regarding their ability to induce fIFN-γ in feline PBMC and splenocytes. It was shown that some CpG-ODN significantly induced fIFN-γ production in splenocytes, but not in PBMC. We found that three kinds of CpG-ODN (no. 2, 5'-ggTGCATCGATGCAGggggG-3'; no. 5, 5'-ggTGCGTCGACGCAGggggG-3'; no. 10, 5'-ggTGCTACGTAGCAGggggG-3') specifically and significantly induced fIFN-γ production in feline splenocytes. The reverse sequences, GpC-ODN, do not cause significant fIFN-γ production. The fIFN-γ production inductivity of a mixture of CpG-ODN nos. 2, 5 and 10 was higher than those of individual CpG-ODN. When the CpG-ODN mixture was encapsulated in an MCL and administrated to cats, the number of fIFN-γ(+) cells in PBMC significantly increased. CpG-ODN nos. 2, 5 and 10 should be useful to elicit a Th1-type immune response as a vaccine adjuvant in cats.
Assuntos
Interferon gama/biossíntese , Leucócitos Mononucleares/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Células Th1/efeitos dos fármacos , Animais , Gatos , Meios de Cultivo Condicionados/análise , Relação Dose-Resposta a Droga , Interferon gama/metabolismo , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/genética , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Células Th1/imunologiaRESUMO
Feline immunodeficiency virus (FIV) vaccine, Fel-O-Vax FIV, was released for sale in the US in 2002. The antibodies of vaccinated cats interfere with serological assays by currently available FIV diagnostic kits. In this study, we investigated whether it is possible to distinguish serologically cats vaccinated with Fel-O-Vax FIV from cats experimentally or naturally infected with FIV. A total of 153 sera taken from 97 cats were used as serum samples. Enzyme linked immunosorbent assay (ELISA) was performed using whole FIV antigen and formalin treated whole FIV antigen, recombinant-gag (r-gag) antigen, and transmembrane (TM) peptide. Statistical analysis was performed using ELISA optical density (O.D.) values obtained with each antigen as variables. Except for the ELISA O.D. values obtained with r-gag antigen, a significant difference in ELISA O.D. values was observed between the vaccinated and the infected groups. However, it was not possible to distinguish both groups unequivocally. Using discriminant analysis, it was possible to distinguish the two groups with an accuracy of 97.1% with two discriminating variables (ELISA O.D. values obtained with formalin treated whole FIV antigen, and TM peptide), 97.8% with three discriminating variables (ELISA O.D. values obtained with whole FIV antigen, formalin treated whole FIV antigen, and TM peptide). Therefore, it was considered possible to distinguish cats vaccinated with Fel-O-Vax FIV from FIV-infected cats by ELISA using two types of antigens including formalin treated whole FIV antigen and TM peptide, or three types of antigens including formalin treated whole FIV antigen, TM peptide and whole FIV antigen.
Assuntos
Anticorpos Antivirais/sangue , Doenças do Gato/imunologia , Vírus da Imunodeficiência Felina/imunologia , Infecções por Lentivirus/veterinária , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/classificação , Antígenos Virais/imunologia , Doenças do Gato/sangue , Doenças do Gato/virologia , Gatos , Análise Discriminante , Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Lentivirus/sangue , Infecções por Lentivirus/imunologia , Infecções por Lentivirus/virologiaRESUMO
Fel-O-Vax FIV is a dual-subtype vaccine consisting of inactivated whole viruses of subtype A (Petaluma strain) and subtype D (Shizuoka strain). The efficacy of this vaccine against heterologous subtype A strain challenge was demonstrated, but it is unclear whether the result reflects efficacy in the field. In this study, we evaluated the efficacy of this vaccine against contact challenge by exposing both vaccinated and unvaccinated control animals with cats infected with Aomori-2 strain belonging to subtype B, a subtype prevalent in many regions of the world. Nineteen specific-pathogen-free (SPF) cats were divided into a vaccinated group (six cats), an unvaccinated control group (eight cats), and a challenge group (five cats), and maintained in the same room. Cats were monitored for FIV proviral DNA by nested PCR and for FIV-specific antibody levels by ELISA. After 1 year of commingling, each cat in the vaccinated group was given a booster dose. In addition, the original challenge group was removed and replaced with another challenge group of SPF cats, which were inoculated with the Aomori-2 strain. FIV infection was confirmed in four of the eight animals in the unvaccinated control group by the 29th week in the second year of commingling. In contrast, all of the animals were negative in the vaccinated group. These findings confirmed the efficacy of this vaccine against heterologous stains classified as subtype B, and suggested that the vaccine exhibits broad efficacy against genetically diverse FIV.