Analysis of complex formation and immune response of CFP-10 and ESAT-6 mutants

ESAT-6 and CFP-10 form a 1:1 heterodimeric complex which contributes to the virulence of Mycobacterium tuberculosis H37Rv. Based on the structure of CFP-10-ESAT-6 complex, we have selected four point mutations each of CFP-10 and ESAT-6 and have analyzed complex formation for the 25 possible combinations between wild-type and mutant CFP-10 and ESAT-6 proteins. We observed that the mutations L25R or F58R of CFP-10 and L29D or L65D of ESAT-6 lead to disruption of complex formation. We have evaluated the immunogenic responses of the wild-type and mutant CFP-10 and ESAT-6 proteins, the wild-type CFP-10-ESAT-6 complex, six complex-forming and two non-complex-forming combinations of wild-type/mutant CFP-10 and ESAT-6 proteins. CFP-10 mutants I21R, L25R, and W43R were found to have better immunogenic potential than wt-CFP-10, while none of the ESAT-6 mutants were better than wt-ESAT-6. Very interestingly, we have discovered that the non-complex-forming mixture of CFP-10-I21R and ESAT-6-L29D gives a strong immunogenic response.     

Non-homologous end-joining genes are not inactivated in human radiation-induced sarcomas with genomic instability

DNA double-strand break (DSB) repair pathways are implicated in the maintenance of genomic stability. However the alterations of these pathways, as may occur in human tumor cells with strong genomic instability, remain poorly characterized. We analyzed the loss of heterozygosity (LOH) and the presence of mutations for a series of genes implicated in DSB repair by non-homologous end-joining in five radiation-induced sarcomas devoid of both active Tp53 and Rb1. LOH was recurrently observed for 8 of the 9 studied genes (KU70, KU80, XRCC4, LIG4, Artemis, MRE11, RAD50, NBS1) but not for DNA-PKcs. No mutation was found in the remaining allele of the genes with LOH and the mRNA expression did not correlate with the allelic status. Our findings suggest that non-homologous end-joining repair pathway alteration is unlikely to be involved in the high genomic instability observed in these tumors.     

Split dose recovery studies using homologous recombination deficient gene knockout chicken B lymphocyte cells

To understand the role of proteins involved in DSB repair modulating SLD recovery, chicken B lymphoma (DT 40) cell lines either proficient or deficient in RAD52, XRCC2, XRCC3, RAD51C and RAD51D were subjected to fractionated irradiation and their survival curves charted. Survival curves of both WT DT40 and RAD52 (-/-) cells had a big shoulder while all the other cells exhibited small shoulders. However, at the higher doses of radiation, RAD51C(-/-) cells displayed hypersensitivity comparable to the data obtained for the homologous recombination deficient RAD54(-/-) cells. Repair of SLD was measured as an increase in survival after a split dose irradiation with an interval of incubation between the radiation doses. All the cell lines (parental DT40 and genetic knockout cell lines viz., RAD52(-/-), XRCC2(-/-), XRCC3(-/-) RAD51C(-/-) and RAD51D(-/-)) used in this study demonstrated a typical split-dose recovery capacity with a specific peak, which varied depending on the cell type. The maximum survival of WT DT40 and RAD52(-/-) was reached at about 1-2 hours after the first dose of radiation and then decreased to a minimum thereafter (5h). The increase in the survival peaked once again by about 8 hours. The survival trends observed in XRCC2 (-/-), XRCC3(-/-), RAD51C (-/-) and RAD51D(-/-) knockout cells were also similar, except for the difference in the initial delay of a peak survival for RAD51D(-/-) and lower survival ratios. The second phase of increase in the survival in these cell lines was much slower in XRCC2(-/-) , XRCC3(-/-), RAD51C(-/-) and RAD51D(-/-) and further delayed when compared with that of RAD52(-/-) and parental DT40 cells suggesting a dependence on their cell cycle kinetics. This study demonstrates that the participation of RAD52, XRCC2, XRCC3, RAD51C and RAD51D in the DSB repair via homologous recombination is of less importance in comparison to RAD54, as RAD54 deficient cells demonstrated complete absence of SLD recovery.     

Generation and Characterization of Recombinant Influenza A(H1N1) Viruses Resistant to Neuraminidase Inhibitors

ObjectivesTo examine the effect of neuraminidase (NA) mutations on the NA inhibitor (NAI) resistance phenotype, the recombinant influenza A/Chungbuk/4448/2008(H1N1) virus isolated in South Korea during the 2008–2009 season was generated by reverse genetics.MethodsSite-directed mutagenesis was introduced on the NA gene of A/Chungbuk/4448/2008(H1N1) virus, and a total of 23 single, double, and triple mutants were generated. Resistance phenotype of these recombinant viruses was determined by NA-inhibition (NAI) assays based on a fluorometric method using two NAIs (oseltamivir and zanamivir).ResultsNA-inhibition assays showed that all the single and double mutants containing the Y275 except the single Y275-E119V mutant conferred important levels of resistance to oseltamivir, whereas all the single, double, and triple mutants containing the E119V mutation were associated with the resistance to zanamivir.ConclusionConsidering the effect of mutations in NA gene on the resistance to NAIs, it is important to monitor the possible emergence and dissemination of multidrug-resistant variants in the human population due to amino acid changes at NA gene as well as to develop novel NAIs.     

Role of wild-type p53 in apoptotic and non-apoptotic cell death induced by X-irradiation and heat treatment in p53-mutated mouse M10 cells

The sensitizing effects of wild-type p53 on X-ray-induced cell death and on heat-induced apoptosis in M10, a radiosensitive and Trp53 (mouse p53 gene)-mutated lymphoma cell line which dies through necrosis by X-irradiation, were investigated using three M10 derived transfectants with wild-type TP53 (human p53 gene). Cell death was determined by colony formation and/or dye exclusion test, and apoptosis was detected as the changes in nuclear morphology by Giemsa staining. Expression of wild-type p53 protein increased radiosensitivity of cell death as determined by both clonogenic and dye exclusion assays. This increase in radiosensitivity was attributable largely to apoptosis induction in addition to a small enhancement of necrosis. Interestingly neither pathway to cell death was accompanied by caspase-3 activation. On the other hand, heat-induced caspase-3 dependent apoptotic cell death without transfection was further increased by the transfection of wild-type p53. In conclusion, the introduction of wild-type p53 enhanced apoptotic cell death by X-rays or heat via different mechanisms that do or do not activate caspase-3, respectively. In addition, p53 also enhanced the X-ray-induced necrosis in M10 cells.     

循环肿瘤DNA与转移性结直肠癌靶向治疗及耐药机制的相关研究

目的 探讨转移性结直肠癌(metastatic colorectal cancer,mCRC)耐药相关的点突变,明确循环肿瘤DNA(circulating tumour DNA,ctDNA)能否作为mCRC检测和耐药相关的突变分子标志物以及mCRC可能的耐药机制。 方法 通过对连续抗表皮生长因子受体(epidermal growth factor receptor, EGFR)单抗药物治疗的mCRC患者多阶段ctDNA进行KRAS、BRAF和PIK3CA基因点突变动态监测,构建药物作用下的肿瘤基因突变谱变化,筛选耐药相关点突变;培养相应点突变细胞系后,通过体外细胞增殖试验(MTS法)分析细胞系的耐药程度,实时荧光定量PCR( real-time quantitative PCR,RT-qPCR)检测药物作用对突变丰度的影响。 结果 通过临床数据分析,筛选出可能与mCRC耐药相关的点突变KRAS(G12D)、PIK3CA(H1047R)和BRAF(V600E),并纳入后续研究。选择NCI-H747、SW948和SW1417细胞系分别作为KRAS(G12D)、PIK3CA(H1047R)和BRAF(V600E)点突变的细胞系,并选用C2BBe1野生型细胞系作为本底细胞。MTS半数抑制浓度即IC₅₀测定发现,野生型细胞系对西妥昔单抗和帕尼单抗敏感性均较突变细胞高;经与本底细胞稀释后,西妥昔单抗组和帕尼单抗组在培养3 d后突变基因相对水平均比不加药组高,不加药组均比基线组高。 结论 ctDNA检测能够发现与耐药相关的点突变;KRAS(G12D)、PIK3CA(H1047R)和BRAF(V600E)点突变可提高转移性结直肠癌细胞生长速度,并与西妥昔单抗和帕尼单抗耐药相关。     

Global variation in SARS-CoV-2 proteome and its implication in pre-lockdown emergence and dissemination of 5 dominant SARS-CoV-2 clades

SARS-CoV-2 is currently causing major havoc worldwide with its efficient transmission and propagation. To track the emergence as well as the persistence of mutations during the early stage of the pandemic, a comparative analysis of SARS-CoV-2 whole proteome sequences has been performed by considering manually curated 31,389 whole genome sequences from 84 countries. Among the 7 highly recurring (percentage frequency≥10%) mutations (Nsp2:T85I, Nsp6:L37F, Nsp12:P323L, Spike:D614G, ORF3a:Q57H, N protein:R203K and N protein:G204R), N protein:R203K and N protein: G204R are co-occurring (dependent) mutations. Nsp12:P323L and Spike:D614G often appear simultaneously. The highly recurring Spike:D614G, Nsp12:P323L and Nsp6:L37F as well as moderately recurring (percentage frequency between ≥1 and <10%) ORF3a:G251V and ORF8:L84S mutations have led to4 major clades in addition to a clade that lacks high recurring mutations. Further, the occurrence of ORF3a:Q57H&Nsp2:T85I, ORF3a:Q57H and N protein:R203K&G204R along with Nsp12:P323L&Spike:D614G has led to 3 additional sub-clades. Similarly, occurrence of Nsp6:L37F and ORF3a:G251V together has led to the emergence of a sub-clade. Nonetheless, ORF8:L84S does not occur along with ORF3a:G251V or Nsp6:L37F. Intriguingly, ORF3a:G251V and ORF8:L84S are found to occur independent of Nsp12:P323L and Spike:D614G mutations. These clades have evolved during the early stage of the pandemic and have disseminated across several countries. Further, Nsp10 is found to be highly resistant to mutations, thus, it can be exploited for drug/vaccine development and the corresponding gene sequence can be used for the diagnosis. Concisely, the study reports the SARS-CoV-2 antigens diversity across the globe during the early stage of the pandemic and facilitates the understanding of viral evolution.     

N255 is a key residue for recognition by a monoclonal antibody which protects against Yersinia pestis infection

Mab7.3 to Yersinia pestis LcrV antigen (LcrV_Ype) protected J774A.1 macrophages in vitro from killing by a Yersinia pseudotuberculosis strain expressing LcrV_Ype. Of 4 site-directed mutations in the coiled-coil region (148–169) and 7 mutations in the 225–255 sequence of LcrV_Ype, only the mutation of N255 to D255, abrogated the binding of Mab7.3 and reduced its protective capacity against plague. Since the Mab7.3 epitope in LcrV_Ype (135–275) encompasses a region (136–180) thought to be exposed on the injectisome, we suggest that Mab7.3 protects by binding to LcrV_Ype and interfering with protein–protein interactions necessary for type three secretion.     

Differences in sensitivity to DNA-damaging Agents between XRCC4- and Artemis-deficient human cells

Non-homologous end-joining (NHEJ) is the predominant pathway for the repair of DNA double-strand breaks (DSBs) in human cells. XRCC4 is indispensable to NHEJ and functions together with DNA ligase IV in the rejoining of broken DNA ends. Artemis is a nuclease required for trimming of some, but not all, types of broken DNA ends prior to rejoining by the DNA ligase IV/XRCC4 complex. To better understand the roles of these factors, we generated XRCC4- and Artemis-deficient cells from the human colon adenocarcinoma cell line HCT116 by gene targeting and examined their cellular responses to several DNA-damaging agents including X-rays. As anticipated, kinetic analyses of γ-H2AX foci and chromosomal aberrations after ionizing radiation (IR) demonstrated a serious incompetence of DSB repair in the XRCC4-deficient cells, and relatively moderate impairment in the Artemis-deficient cells. The XRCC4-deficient cells were highly sensitive to etoposide and 5-fluoro-2'-deoxyuridine as well as IR, and moderately sensitive to camptothecin, methyl methanesulfonate, cisplatin, mitomycin C, aphidicolin and hydroxyurea, compared to the parental HCT116 cells. The Artemis-deficient cells were not as sensitive as the XRCC4-deficient cells, except to cisplatin and mitomycin C. By contrast, the Artemis-deficient cells were significantly more resistant to hydroxyurea than the parental cells. These observations suggest that Artemis also functions in some DNA damage response pathways other than NHEJ in human cells.     

Individual differences in the radiosensitivity of hematopoietic progenitor cells detected in steady-state human peripheral blood

The aim of this study is to evaluate the individual differences in radiosensitivity of lineage-committed myeloid hematopoietic progenitors, colony-forming cells (CFC), detected in steady-state human peripheral blood (PB). Mononuclear cells were prepared from the buffy-coat of 30 individuals PB, and were assayed for CFC by semi-solid culture supplemented with cytokines. X irradiation was performed in the range of 0.5-4 Gy at a dose rate of about 80 cGy/min. The mean number of hematopoietic progenitor cells is 5866 alpha 3408 in 1 ml of buffy-coat, suggesting that the erythroid progenitor cells are the major population. The total CFC radiosensitivity parameter D(0) and n value are 1.18 alpha 0.24 and 1.89 alpha 0.98, respectively. Using a linear regression analysis, a statistically significant correlation is observed between the D(0) value and the surviving fraction at 4 Gy (r = 0.611 p < 0.001). Furthermore, we evaluate the relationship between individual radiosensitivity and the level of antioxidants, plasma uric acid, plasma bilirubin, and intracellular glutathione. No statistically significant correlations are observed, however, between the D(0) parameter and the level of antioxidants, plasma uric acid, plasma bilirubin, and intracellular glutathione. The present study demonstrates that there are large individual differences in the radiosensitivity of hematopoietic progenitor cells as detected in steady-state human PB. These differences demonstrate almost no correlation with plasma or intracellular antioxidants. The prediction of individual differences in radiosensitivity of CFC can only be measured by 4 Gy irradiation.     

利福霉素耐药结核分枝杆菌rpoB突变与利福布丁耐药水平的关系

目的研究利福霉素耐药结核分枝杆菌 rpoB基因突变与利福布丁耐药水平的相关性。方法倍比稀释法测定64株利福霉素耐药及6株敏感菌株对利福布丁的最低抑菌浓度(MIC),并分析其对异烟肼的耐药情况。同时对rpoB全基因扩增后测序,分析rpoB突变位点和突变性质与利福布丁MICs高低及多重耐药的关系。结果6株敏感株rpoB未突变,MICs为 0.25～0.50 mg/L。64株耐药株rpoB突变率为100％。37株利福布丁高度耐药(MICs≥4 mg/L)株中,S531L突变27株,H526R突变和Y389C突变各2株,S531W、H526Y、Q513K、V176F、D516Y联合Q253R突变与D516G联合L511P突变各1株。17株中度耐药 (MICs 2～4 mg/L) 株中,S531L突变16株,D516G联合L511P和S509R突变1株。10株低度耐药（MICs 0.25～1 mg/L）株中,L533P、H526L、H526S、D516V、D516Y单点突变各2株。93.75％(60/64)的利福霉素耐药株对异烟肼耐药。结论检测rpoB突变即可初步筛选多重耐药结核分枝杆菌;中、高水平利福布丁耐药株以S531L突变占绝对优势,rpoB突变位点及突变类型与利福布丁耐药水平有一定相关性。     

黑龙江省2004～2007年HIV-1毒株耐药基因型检测结果分析

目的了解黑龙江省部分AIDS患者在接受抗病毒治疗中HIV-1耐药基因的变异情况,为更好地开展抗病毒治疗提供科学依据。方法从患者血浆中抽提病毒RNA,用RT-PCR方法扩增HIV—1po1区基因片段,进行核苷酸序列测定及耐药基因型分析。结果2005年的2号样本以及2007年的3号和5号样本各存在～处蛋白酶抑制剂次要耐药突变：A71T、L10I、A71T,但它们单独存在不会产生耐药性。2005年的2号样本以及2007年的5号和6号样本均检出逆转录酶抑制剂的耐药突变,其中核苷类逆转录酶抑制剂（NRTIs）耐药突变位点共有5处：D67N、KTOR、M184V、K219Q、T215FI,非核苷类逆转录酶抑制剂（NNRTIs）耐药突变位点共有7处：K103T、Y181C、K103N、P225H、K238T、V106A、G190A,最终导致三份样本对NRTIs和NNRTIs类中的多种药物产生不同程度的耐受性。结论被检测的AIDS患者中多数对现有的抗病毒药物敏感,但有3例对多数逆转录酶抑制剂产生耐受性,应该加强HIV感染者／AIDS患者体内HIV-1耐药基因型监测,了解治疗前后总体耐药水平。     

Effects of expression level of DNA repair-related genes involved in the NHEJ pathway on radiation-induced cognitive impairment

Cranial radiation therapy can induce cognitive decline. Impairments of hippocampal neurogenesis are thought to be a paramountly important mechanism underlying radiation-induced cognitive dysfunction. In the mature nervous system, DNA double-strand breaks (DSBs) are mainly repaired by non-homologous end-joining (NHEJ) pathways. It has been demonstrated that NHEJ deficiencies are associated with impaired neurogenesis. In our study, rats were randomly divided into five groups to be irradiated by single doses of 0 (control), 0 (anesthesia control), 2, 10, and 20 Gy, respectively. The cognitive function of the irradiated rats was measured by open field, Morris water maze and passive avoidance tests. Real-time PCR was also used to detect the expression level of DNA DSB repair-related genes involved in the NHEJ pathway, such as XRCC4, XRCC5and XRCC6, in the hippocampus. The influence of different radiation doses on cognitive function in rats was investigated. From the results of the behavior tests, we found that rats receiving 20 Gy irradiation revealed poorer learning and memory, while no significant loss of learning and memory existed in rats receiving irradiation from 0–10 Gy. The real-time PCR and Western blot results showed no significant difference in the expression level of DNA repair-related genes between the 10 and 20 Gy groups, which may help to explain the behavioral results, i.e. DNA damage caused by 0–10 Gy exposure was appropriately repaired, however, damage induced by 20 Gy exceeded the body''s maximum DSB repair ability. Ionizing radiation-induced cognitive impairments depend on the radiation dose, and more directly on the body''s own ability to repair DNA DSBs via the NHEJ pathway.     

Identification of a new G-to-A transition mutation at nucleotide position 129 of the Xrcc4 gene in ionizing radiation-hypersensitive mutant LX830 cells

The mouse lymphoma cell line LX830 is an X-ray-hypersensitive mutant. Complementation tests between LX830 cells and radiation-sensitive mutants of M10 (Xrcc4 deficient cells) or SX10 (DNA ligase IV deficient cells) cells showed that M10 cells did not complement LX830 cells, but SX10 cells did, suggesting that LX830 cells would belong to the X-ray-cross complementation group (XRCC4). A sequence analysis of Xrcc4 cDNA in LX830 cells disclosed a transition of G to A at nucleotide position 129, which resulted in a change of tryptophan (43) to a termination codon. Transfection of the mouse Xrcc4 cDNA rescued the X-ray sensitivity of the mutant cells. LX830 is an Xrcc4-deficient cell line bearing a termination codon in exon 2 of the Xrcc4 gene and no wild-type Xrcc4 gene.     

2012-2018年青岛市A型(H1N1)pdm09流感病毒奥司他韦耐药株基因特征

目的 了解2012-2018年青岛市人群A型(H1N1)pdm09流感病毒奥司他韦耐药株基因特征。方法 收集2012年4月-2018年3月间青岛市A(H1N1)pdm09毒株397份,逆转录聚合酶链反应(RT-PCR)扩增神经氨酸酶(Neuraminidase,NA)和血凝素(Hemagglutinin,HA)基因全长,序列测定后进行耐药位点和氨基酸变异及进化分析。结果 5株发生了H275Y突变,为奥司他韦耐药株;另有4株S247N突变,可能为奥司他韦耐药株。2012-2018年H275Y突变株检出率依次为2.8 %、2.0 %、0.0 %、1.1 %、0.0 %和0.7 %。NA和HA进化树显示,2012-2013年青岛H275Y突变株与美国耐药株A/Tennessee/03/2013更接近,2013-2014年青岛H275Y突变株与国内株和日本札幌耐药株更接近,这两个年度耐药株的毒株起源可能有所不同。突变株在酶活性位点、抗原决定簇、受体结合位点及其他功能位点(如HA位点D222、Q223和NA位点V241I、N369K和N386K)的转变与野生敏感株一致。结论 青岛市A型(H1N1)pdm09流感病毒奥司他韦耐药株明显增加且流行起源不同,但并未取得比野生株更强的流行能力。奥司他韦仍可作为流感预防和治疗的有效手段。     

湖北省抗病毒治疗和未治疗的HIV-1感染者耐药基因变异研究

目的研究湖北省流行的HIV-1毒株在经过高效抗逆转录病毒治疗（HAART）及未治疗人群中耐药基因变异情况。方法采集HIV-1感染者抗凝外周血，提取前病毒DNA，用巢式PCR方法扩增HIV-1 pol基因约2kb的核酸序列，进行序列测定并通过斯坦福耐药基因数据库进行耐药基因型分析。结果抗病毒治疗组19例，未治疗组25例。在所有的HIV-1感染者样本序列中，发现针对蛋白酶抑制剂（PIs）的耐药突变：D30N（2．27％），D30G（2．27％），M461（4．55％），M46N（2．27％），147V（4．55％），184V（4．55％），184L（2．27％），N88S（2．27％），L90S（2．27％），以及针对PIs的次要耐药基因突变；A71T（29．55％）。在治疗组中出现针对逆转录酶抑制剂（NRTIs及NNRTIs）的主要耐药基因突变的样本5例，突变主要有M41L（5．26％），A62V（5．26％），D67N（5．26％），L210W（5．26％），T215Y（15．79％）；K103E（5．26％），K103N（10．53％），Y181C（5．26％），G190A（5．26％），K238N（5．26％）。在未治疗组中出现针对逆转录酶抑制剂（NRTIs及NNRTIs）的主要耐药基因突变的样本5例，突变主要有M184V（4％），K65N（4％），Y115M（4％），F116L（4％），M1841（4％）；V179D（4％），G190R（4％）。在逆转录酶（RT）基因中耐药意义不明的突变F214L，与药物的使用有统计学上的相关性（P=0．03）。结论湖北省HIV-1感染者RT基因的耐药突变，在治疗和未治疗人群样本中差异有统计学意义，说明药物治疗已经对HIV耐药基因突变的产生有了一定影响。同时，耐药意义尚未明确的突变位点F214L也可能与治疗或是某些药物的使用有一定的相关性。     

核心家系孤独症患儿FOXP1基因的突变及CNV分析

目的 研究转录因子FOXP1在汉族孤独症患儿核心家系中的外显子突变,并进行初步的功能预测。 方法 288例孤独症患儿及生物学父母进行FOXP1外显子区域基因测序和拷贝数变异(copy number variation,CNV)检测;使用POLYPHEN及SIFT等软件进行功能预测。 结果 4例患儿的FOXP1基因外显子区域发现4个错义突变,包括P42S,H53Q,L68R和M590V。在CNV检测中为阴性结果。通过功能预测发现,FOXP1的四个罕见突变可能不会对FOXP1编码蛋白产生重要影响。 结论 汉族人群中,FOXP1基因可能不是孤独症的主要致病基因。     

辽宁省艾滋病病毒1流行株膜蛋白V3环氨基酸变异特点

目的　了解辽宁省艾滋病病毒 1(HIV 1)感染者 艾滋病 (AIDS)患者体内不同亚型HIV 1膜蛋白V3环氨基酸序列特征 ,突变种类特点。方法　提取含有整合HIV 1前病毒的基因组DNA ,巢式聚合酶链反应 (nest PCR)扩增后直接测序 ,并做序列排列比对、翻译和分析。结果　辽宁省HIV 1感染者感染病毒分属A、B’、C、G四种亚型 ,AIDS患者体内病毒V3环发生与T嗜性 SI表型有关的氨基酸突变形式 (11位出现R ,13位出现S、T、N ,19位出现V ,2 0位出现Y ,2 5、2 9位出现N等 )高于无症状感染组 (P <0 .0 5 ) ,此外还发现GQGR、APGQ、RPGA、GLGR、RPGA等少见的V3环顶端四肽组成形式及第 5位H ,34位S、F等罕见突变形式。结论　辽宁省AIDS人群中 ,A、B、C、G亚型HIV 1毒株V3环各位置出现氨基酸突变情况与国外对B亚型毒株的研究结果总体上相似 ,但发现一些罕见突变和少见V3环顶端四肽组成形式。     

Elimination of luteinizing hormone cross-reactive epitopes from human chorionic gonadotropin

The beta-chain of human chorionic gonadotropin (hCG) has been shown to have efficacy in clinical trials when used as a contraceptive vaccine. This hormone is a heterodimer, the alpha-chain being shared with the other members of the glycoprotein hormone family but the beta-chain being unique to hCG. Nevertheless, there is sequence homology between the hCG beta-chain and the beta-chain of human luteinizing hormone (hLH) which results in cross-reactive antibodies being produced following immunization with wild-type hCGbeta. To reduce or eliminate such cross-reactions we generated a number of mutants of the hCGbeta-chain. One mutant (hCGbeta(R68E)), containing an arginine to glutamic acid replacement at position 68, has been expressed as a recombinant protein in High Five insect cells. The recombinant BAChCGbeta(R68E) form of this molecule was used to immunize rabbits and the antibody response compared to the response following immunization with the recombinant wild-type protein BAChCGbeta and with the native hCGalphabeta heterodimer isolated from pregnancy urine. The mutant elicited the production of antibodies which avidly recognize native hCG. Compared to immunization with wild-type hCG, the response showed very little cross reactivity with hLH. This is demonstrated to be due to a radically altered epitope usage in the response to the mutant, which now focuses mainly upon the C-terminal region of the beta-chain.