• Parasites, Hosts and Diseases
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• 제52권1호
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• pp.93-97
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• 2014

Subolesin (4D8), the ortholog of insect akirins, is a highly conserved protective antigen and thus has the potential for development of a broad-spectrum vaccine against ticks and mosquitoes. To date, no protective antigens have been characterized nor tested as candidate vaccines against Dermacentor silvarum bites and transmission of associated pathogens. In this study, we cloned the open reading frame (ORF) of D. silvarum 4D8 cDNA (Ds4D8), which consisted of 498 bp encoding 165 amino acid residues. The results of sequence alignments and phylogenetic analysis demonstrated that D. silvarum 4D8 (Ds4D8) is highly conserved showing more than 81% identity of amino acid sequences with those of other hard ticks. Additionally, Ds4D8 containing restriction sites was ligated into the pET-32(a+) expression vector and the recombinant plasmid was transformed into Escherichia coli rosetta. The recombinant Ds4D8 (rDs4D8) was induced by isopropyl ${\beta}$-D-thiogalactopyranoside (IPTG) and purified using Ni affinity chromatography. The SDS-PAGE results showed that the molecular weight of rDs4D8 was 40 kDa, which was consistent with the expected molecular mass considering 22 kDa histidine-tagged thioredoxin (TRX) protein from the expression vector. Western blot results showed that rabbit anti-D. silvarum serum recognized the expressed rDs4D8, suggesting an immune response against rDs4D8. These results provided the basis for developing a candidate vaccine against D. silvarum ticks and transmission of associated pathogens.

• 대한수의학회지
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• 제25권1호
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• pp.7-17
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• 1985

Many investigators have been pursuing various attempts so far to produce hepatitis B surface antigen(HBsAg) vaccines using the techniques such as isolation from plasma of chronic HBsAg carrier, recombinant DNA technique or preparation of synthetic peptides specific for immunogenic determinants. Hepatitis B virus can not grow on any cell lines by the tissue culture technique at the present time. The plasma of chronic HBsAg carrier is expensive and its source is limited. The HBsAg from the recombinant DNA technique gave still very low yield. Another approach, therefore, has been initiated to develop a synthetic hepatitis B virus vaccine. The possible use of several distinct synthetic vaccines in prophylaxis can be facilitated by availability of full synthetic immunogens. Peptides synthesized for potential application as antiviral vaccines have been mostly tested in the form of conjugates with carrier proteins, although the free synthetic peptide can be immunogenic. To understand basic knowledges on the antigenicity and immunogenicity of a synthetic peptide specific for major immunogenic determinant of HBsAg, a nonapeptide, $H_2N^{139}Cys-Thr-Lys-Pro-Thr-Asp-Gly-^{146}Asn-Aba$ COOH, which corresponds to HBsAg amino acid residues 139 to 147, was synthesized by the Merrifield's solid-phase method with a slight modification. The antigenicity and immunogenicity of this specific synthetic peptide were examined comparing with purified plasma-derived natural HBsAg. The results obtained are as follows; 1. The peptide synthesized showed the identical amino acid composition to the theoretical value. The degree of purification and molecular weight were acertained by methods of high performance liquid chromatography and mass spectrometry. 2. Using m-maleimidobenzoyl-N-hydroxysuccinimide ester as a conjugating agent, the synthetic peptide was conjugated to rabbit albumin and ${\gamma}$-globulin, tetanus and diphtheria toxoids, and keyhole limpet hemocyanin. Their conjugation yields were 8.3, 9.5, 15.8, 13.5, and 11.2%, respectively. 3. The natural HBsAg was purified from plasma of chronic HBsAg carrier. By the electron microscopic observation of the purified natural HBsAg preparation, no Dane particles were observed and the preparation showed negative DNA polymerase activity. 4. Antigenicity of the synthetic peptide and the plasma-derived natural HBsAg was determined by competition radioimmunoassay using $^{125}I$-natural HBsAg. Their 50% inhibitions appeared as $90{\mu}g/ml$ and $0.12{\mu}g/ml$ for the synthetic peptide and the natural HBsAg, respectively. This indicates that the former was about 750-fold less antigenic than the latter. 5. Immunogenicity of the synthetic peptide was determined by administering the peptide-carrier conjugates into rabbits with and without Freund's complete adjuvant. Regardless the carrier proteins and adjuvant, positive immune responses to the synthetic peptide were observed. The higher antibody titers, however, were shown in the groups administered with Freund's complete adjuvant. 6. Immunizing dose 50% in mice of the various peptide-carrier conjugates was 5.47, 6.00, 65.16, 31.25 and $13.03{\mu}g/dose$ for rabbit albumin and ${\gamma}$-globulin, tetanus and diphtheria toxoids, and keyhole limpet hemocyanin, respectively, while the natural HBsAg showed $0.65{\mu}g/dose$. 7. It was postulated that homologous proteins prefer to heterologous ones as the carriers.

• Clinical and Experimental Pediatrics
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• 제50권5호
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• pp.449-456
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• 2007

목 적 : 우리나라에서 Hib 백신을 기본 예방접종에 포함시키는 것을 고려할 때 우리나라의 Hib 질환의 역학, 우리나라 소아의 Hib 백신에 대한 면역 반응에 대한 연구가 있어야 한다. 본 연구는 우리나라 소아에서 면역원성에 근거한 추가접종의 필요성 여부를 확인하고자 하였다. 방 법 : 2006년 6월부터 2006년 12월까지 이화여자대학교 동대문 병원, 강남 차병원, 관동대 명지병원에 내원하여 검사를 위해 혈액 채취의 기회가 있었던 12-23개월 사이의 소아 144명을 대상으로 하였다. 분리된 혈청으로 항 PRP 항체가를 효소면역법으로 측정하였고, 혈청 살균 능력 측정을 시행하였다. 결 과 : 추가접종까지 완료한 군의 항 PRP 항체의 기하평균은 기초접종을 완료한 군이나 접종하지 않은 군의 기하평균보다 높았다(P<0.05). 항 PRP 항체가가 $1.0{\mu}g/mL$ 이상인 비율은 추가접종까지 완료한 군은 96.5%로 다른 세 군에 비해, 기초접종을 완료한 군은 68.6%로 접종하지 않은 군의 30.0%에 비해 양성률이 높았다(P<0.05). 평균 살균 역가는 추가접종까지 완료한 군이 11,205로 기초접종을 완료한 군의 3,946보다 높았다(P<0.05). 항 PRP 항체가와 살균 역가는 양의 상관관계에 있었다(R=0.60). 결 론 : 우리나라 소아는 Hib 백신의 기초 접종 후 12-23개월의 나이에 많은 경우에서 방어 농도 이상의 항체가를 여전히 가지고 있었다. 추가접종에 대한 면역 기억 반응도 좋아 추가 접종 받은 소아의 항체가는 받지 않은 소아에 비해 의미 있게 높았다. 우리나라에 Hib 백신의 도입과 접종 횟수를 결정하기 위해서 지속적인 연구가 필요하다.

• Pediatric Infection and Vaccine
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• 제8권2호
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• pp.150-159
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• 2001

목 적 : 현재 사용하고 있는 마우스 뇌조직 유래 Nakayama주 사백신 접종 방법의 의학적 타당성을 알아보고자 접종 실태와 부작용 발생 양상과 빈도를 조사하고 추가 접종 방법의 타당성을 확인하기 위하여 중화항체 지속률을 조사하였다. 방 법 : 15,487명의 접종아를 대상으로 이상반응 발생 여부를 조사하였고 초등학교 학생 2,277명을 대상으로 건강기록부와 설문지조사를 통하여 접종실태와 이상반응 발생 빈도를 확인하였으며 접종기록이 일치하는 311명의 학생을 대상으로 중화항체 검사를 시행하였다. 검사는 United States Armed Forces Research Institute of Medical Science/Department of Virology, Bangkok(USAMC-AFRIMS)에서 시행되었고 50% 플라크 감소를 기준으로 1 : 10 이상을 양성으로 하였다. 결 과 : 건강기록부에 의한 초등학생 2,277명의 접종률은 47.5%였으나 설문지 조사에서는 접종률이 93.5%로 큰 차이가 있었다. 건강기록부에 나타난 취학 전 접종률은 남자 48.5%, 여자 46.4%로 차이가 없었다. 연령이 증가함에 따라 일본뇌염의 접종 횟수도 증가하는 양상을 보였다. 예방접종 여부가 건강기록부와 설문지 조사 결과가 일치하는 경우는 95년과 96년에 모두 접종 받았다고 응답한 경우만 75.6%로 높았고 그 외에는 일치도가 낮았다. 일본뇌염 예방접종 장소는 학교가 60.1%로 가장 많았고 그 외 보건소가 25.1%, 병 의원 14.1% 순이었다. 1995년 5~6월 사이 서울 소재 보건소에서 15,487명을 대상으로 일본뇌염 사백신을 접종한 후 0.13%에서 이상반응이 발생하여 의료 기관을 방문하였으며 이 중 57%는 고열 때문이었고 1례에서 접종 후 3분만에 실신 발작이 있었다. 전체 접종자의 0.02%에서 심한 두드러기 반응으로 의료기관을 방문하였으며 0.03%에서 두통 및 구토 등으로 의료 기관을 다시 찾았다. 1996년 봄에 초등학생을 대상으로 시행한 이상반응 설문지 조사에 의하면 주사 부위 발적이 17.4%, 주사 부위 동통이 14.8%, 발열 4.3%였고 그 외에 두통, 구토, 반점 등이 관찰되었다. 초등학생 311명 중 일본뇌염 접종력 조사에서 마지막 접종 후 6개월이 경과한 경우(1군)가 155명, 접종 후 18개월이 경과한 경우(2군) 104명, 30개월이 경과한 경우(3군)이 45명, 42개월이 경과한 경우(4군)가 7명이었다. 이들의 항체 양성률은 1군이 98.1%(152/155), 2군이 99.0%(103/104), 3군이 95.6%(43/45), 4군이 71.4%(5/7)로 양성률에 있어서 각 군간의 차이는 없는 것으로 나타났다. 결 론 : 현재 격년으로 시행하고있는 일본뇌염 사백신 추가 접종은 시기를 늘려 시행하여도 항체 지속률을 유지할 수 있을 것으로 추정된다.

• IMMUNE NETWORK
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• 제4권1호
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• pp.31-37
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• 2004

Background: 1-8D gene is a member of human 1-8 interferon inducible gene family and is shown to be overexpressed in fresh colon cancer tissues. Three peptides 1-6, 3-5 and 3-7 derived from 1-8D gene were shown to have immunogenicity against colon cancer. Methods: To study tumor immunotherapy of these peptides we established an adoptive transfer model. $D^{b-/-}{\times}{\beta}2$ microglobulin (${\beta}2m$) null mice transgenic for a chimeric HLA-A2.1/$D^b-{\beta}2m$ single chain (HHD mice) were immunized with irradiated peptide-loaded RMA-S/HHD/B7.1 transfectants. Spleens were removed after last immunization, and splenocytes were re-stimulated in vitro. Lymphocytes from vaccinated HHD mice were transferred together with IL-2 to the tumor bearing nude mice that were challenged S.C. with the HCT/HHD/B7 colon carcinoma cell line that was found to grow in these mice. Results: Peptide 3-5 was found to be highly effective in CTL activity. Adoptively transferred anti-peptide 3-5 cytolytic T lymphocytes caused significant retardation in tumor growth. Conclusion: This study shows that peptide 3-5 can be the most effective candidate for the vaccine of adoptive immunotherapy against colon cancer.

• 한국동물위생학회지
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• 제21권2호
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• pp.117-126
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• 1998

This study was performed to investigate the immunogenicity of autogenous E coli vaccines and their preventive effects on diarrhea in suckling piglets. Autogenous E coli live and killed vaccines were made from the E coli strains isolated from piglets showing diarrhea in field. In group I, pregnant sows were administered with live and killed vaccines at 4 and 2 weeks before parturition, respectively, Killed vaccines were administered twice to pregnant sows at 4 and 2 weeks before parturition in group II, and saline instead of autogenous E coli vaccines was administered to pregnant sows in group III for the control. After parturition, antibody titers in colostrum and milk from sows, incidence of diarrhea in suckling piglets, and immunoreactivity in the ileum of piglets from each treatment group were examined. The results were as follows ; 1. Sixty-two strains of E coli were isolated from suckling piglets with diarrhea. Of the strains, K88 pilus and K99 pilus antigens were identified in 6(9.8%) and 4(6.5%), respectively. Molecular weights of K88 and K99 pilus were 27,500 and 18,500 daltons, respectively. 2. Antibody titers in colostrum from sows after parturition were 1 : 512 to 1 : 1,024 in group I, 1.256 to 1.512 in group II, and 1 : 4 to 1 : 16 in group III. 3. The incidences of diarrhea In suckling piglets of group I, II and III were 3.3%, 9.4% and 21.4%, respectively. 4. When the immunoreactivity in the ileum of piglets from each group was examined, the proportion of IgG-immunoreactivity cells in group I or II was higher than that in group III. In conclusion, administration of autogenous E coli vaccines to pregnant sows before parturition can be an effective way to prevent diarrhea in suckling piglets.

• Parasites, Hosts and Diseases
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• 제53권6호
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• pp.789-793
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• 2015

In order to explore tick proteins as potential targets for further developing vaccine against ticks, the total proteins of unfed female Dermacentor silvarum were screened with anti-D. silvarum serum produced from rabbits. The results of western blot showed that 3 antigenic proteins of about 100, 68, and 52 kDa were detected by polyclonal antibodies, which means that they probably have immunogenicity. Then, unfed female tick proteins were separated by 12% SDS-PAGE, and target proteins (100, 68, and 52 kDa) were cut and analyzed by LC-MS/MS, respectively. The comparative results of peptide sequences showed that they might be vitellogenin (Vg), heat shock protein 60 (Hsp60), and fructose-1, 6-bisphosphate aldolase (FBA), respectively. These data will lay the foundation for the further validation of antigenic proteins to prevent infestation and diseases transmitted by D. silvarum.

• Parasites, Hosts and Diseases
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• 제51권3호
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• pp.327-334
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• 2013

Vaccination is considered a promising alternative for controlling tick infestations. Haemaphysalis longicornis midgut proteins separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane were screened for protective value against bites. The western blot demonstrated the immunogenicity of 92 kDa protein (P92). The analysis of the P92 amino acid sequence by LC-MS/MS indicated that it was a H. longicornis paramyosin (Hl-Pmy). The full lenghth cDNA of Hl-Pmy was obtained by rapid amplification of cDNA ends (RACE) which consisted of 2,783 bp with a 161 bp 3' untranslated region. Sequence alignment of tick paramyosin (Pmy) showed that Hl-Pmy shared a high level of conservation among ticks. Comparison with the protective epitope sequence of other invertebrate Pmy, it was calculated that the protective epitope of Hl-Pmy was a peptide (LEEAEGSSETVVEMNKKRDTE) named LEE, which was close to the N-terminal of Hl-Pmy protein. The secondary structure analysis suggested that LEE had non-helical segments within an ${\alpha}$-helical structure. These results provide the basis for developing a vaccine against biting H. longicornis ticks.

• Journal of Microbiology and Biotechnology
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• 제22권5호
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• pp.721-728
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• 2012

The human papillomavirus (HPV) is the main cause of cervical cancer in developing countries. Rapid diagnosis and initiation of treatment of the HPV infection are critical. Various methods have been employed to reduce the immunogenicity of antibodies targeting HPV serotypes. Nanobodies are the smallest fragments of naturally occurring single-domain antibodies with their antigen-binding site compromised into a single domain. Nanobodies have remarkable properties such as high stability, solubility, and high homology to the human VH3 domain. In this study, a phagemid library was employed to enrich for nanobodies against the L1 protein of the human papilloma virus. Binding reactivity of the selected clones was evaluated using phage enzyme-linked immunosorbent assay (phage-ELISA). Finally, two nanobodies (sm5 and sm8) with the best reactivity against the Gardasil vaccine and the purified HPV-16 L1 protein were expressed and purified using a $Ni^+$-NTA column. The accuracy of expression and purification of the nanobodies was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting assays. In vitro studies demonstrated that neutralization was achieved by the selected nanobodies. The ease of generation and unique features of these molecules make nanobodies promising molecules for the new generation of HPV diagnosis and therapy.

• IMMUNE NETWORK
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• 제21권6호
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• pp.44.1-44.15
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• 2021

Tumor peptides associated with MHC class I molecules or their synthetic variants have attracted great attention for their potential use as vaccines to induce tumor-specific CTLs. However, the outcome of clinical trials of peptide-based tumor vaccines has been disappointing. There are various reasons for this lack of success, such as difficulties in delivering the peptides specifically to professional Ag-presenting cells, short peptide half-life in vivo, and limited peptide immunogenicity. We report here a novel peptide vaccination strategy that efficiently induces peptide-specific CTLs. Nanoparticles (NPs) were fabricated from a biodegradable polymer, poly(D,L-lactic-co-glycolic acid), attached to H-2K^b molecules, and then the natural peptide epitopes associated with the H-2K^b molecules were exchanged with a model tumor peptide, SIINFEKL (OVA_257-268). These NPs were efficiently phagocytosed by immature dendritic cells (DCs), inducing DC maturation and activation. In addition, the DCs that phagocytosed SIINFEKL-pulsed NPs potently activated SIINFEKL-H2K^b complex-specific CD8⁺ T cells via cross-presentation of SIINFEKL. In vivo studies showed that intravenous administration of SIINFEKL-pulsed NPs effectively generated SIINFEKL-specific CD8⁺ T cells in both normal and tumor-bearing mice. Furthermore, intravenous administration of SIINFEKL-pulsed NPs into EG7.OVA tumor-bearing mice almost completely inhibited the tumor growth. These results demonstrate that vaccination with polymeric NPs coated with tumor peptide-MHC-I complexes is a novel strategy for efficient induction of tumor-specific CTLs.