新生儿巨细胞病毒感染的检测

建立了用ELISA检测巨细胞病毒(HCMV)IgA抗体的方法,並用于检测北京地区100对母婴的HCMV抗体,母血、脐带血、母乳中HCMV-IgG抗体的阳性率分别为83％,75％和38％,HCMV-IgA抗体的阳性率分别为19％,15％和58％,对其中的16名婴儿半年后追踪观察,5名出生时母、脐血全为阴性的,有2名抗体阳转。8名出生时母、脐血均阳性的,有1名IgA仍阳性並检查发现肝大肋下二指。另1名IgG持续阳性,其他6名婴儿抗体转阴。3名出生时母血HCMV-IgG阳性者中,1名婴儿IgA和‘gG转阳,此时母亲IgA也阳转。随访的16名婴儿中有3名可能是生后半年内受HCMV感染。     

Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

ABSTRACT: BACKGROUND: Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies. METHODS: Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20--57 years (median = 33 years) were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. RESULTS: We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL), suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. CONCLUSIONS: Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, in context of V3 region.     

Detection of Human Cytomegalovirus DNA in Breast Milk by Means of Polymerase Chain Reaction

Three hundred and twenty-five breast milk samples were examined for the occurrence of human cytomegalovirus (HCMV) by cell culture method. Virus was isolated from the milk in 1 of 177 samples collected within 6 days after delivery, 2 of 115 samples collected during the period of 7 days to 1 month after delivery, 10 of 33 samples collected over 1 month after delivery. Next, we tried to amplify HCMV DNA from the breast milk samples from HCMV seropositive mothers and seronegative mothers at 1 month after delivery by polymerase chain reaction. HCMV DNA was detected in 12 of 13 samples from seropositive mothers and in none of 7 samples from seronegative mothers. It was thought that all women seropositive for HCMV principally shed the virus into their breast milk at 1 month after delivery.     

Expression of human cytomegalovirus pp150 gene in transgenic Vicia faba L. and immunogenicity of pp150 protein in mice

The pp150 gene of human cytomegalovirus (HCMV) was transferred into Vicia faba plants by Agrobacterium tumefaciens-mediated transformation. Three of five hygromycin resistant V. faba plants were identified as positive by PCR and dot-blot hybridization. The ELISA results indicated that pp150 protein from three plants of transformed V. faba leaves and seeds made up 0.005–0.015% of the total soluble protein. The results of detection by immunoblot and inhibition of immunofluorescent assay (IFA) showed that pp150 soluble protein had immunity activity. HCMV pp150-specific antibody (IgG, IgA) and IFN-γ producing T cells were detected in 100% of the mice immunized with pp150 transgenic V. faba seeds by ELISA and intracellular staining and flow cytometry analysis, respectively. The transgenic V. faba plants will provide new material for the development of edible vaccination against HCMV infection.     

乳房链球菌GapC蛋白的表达及其B细胞抗原表位的预测与鉴定

乳房链球菌Streptococcus uberis的GapC蛋白是一种位于该菌表面的具有甘油醛-3-磷酸脱氢酶活性的蛋白,其参与细胞活动,表现出多种生物学活性,此外还具有良好的抗原性。文中旨在对乳房链球菌GapC蛋白可能的B细胞抗原表位进行预测,分析和验证候选表位肽的免疫原性。利用S. uberis分离株RF5-1克隆gapC基因,构建重组表达质粒pET-28a-GapC,诱导表达GapC重组蛋白,并以纯化蛋白免疫家兔,获得抗GapC多抗。利用生物信息学软件预测并分析GapCB细胞抗原表位的三维结构和空间位置及对GapC蛋白及表位的同源性比较。结果表明,表达纯化了44kDa的GapC蛋白具有良好的反应性。利用表位预测软件筛选并合成针对S.uberisGapC蛋白的6个线性和3个构象优势B细胞表位多肽,三维结构的分析显示,筛选的多肽具有良好的抗原表位形成条件。以纯化的S.uberis GapC蛋白免疫家兔制备多抗,通过间接ELISA对抗原表位进行鉴定。ELISA检测结果显示,9条抗原表位肽均可不同程度地与抗GapC多抗反应,其中表位266AANDSYGYTEDPIVSSD282与多抗反应...     

Epitope mapping and identification of amino acids critical for mouse IgG-binding to linear epitopes on Gly m Bd 28K

Gly m Bd 28K is one of the major allergens in soybeans, but there is limited information on its IgG-binding epitopes. Thirty-four overlapping peptides that covered the entire sequence of Gly m Bd 28K were synthesized, and 3 monoclonal antibodies against Gly m Bd 28K were utilized to identify the IgG-binding regions of Gly m Bd 28K. Three dominant peptides corresponding to ²⁸GDKKSPKSLFLMSNS⁴²(G28-S42), ⁵⁶LKSHGGRIFYRHMHI⁷⁰(L56-I70), and ¹⁵⁴ETFQSFYIGGGANSH¹⁶⁸(E154-H168) were recognized. L56-I70 is the most important epitope, and a competitive ELISA indicated that it could inhibit the binding of monoclonal antibody to Gly m Bd 28K protein. Alanine scanning of L56-I70 documented that F64, Y65, and R66 were the critical amino acids of this epitope. Two bioinformatics tools, ABCpred and BepiPred, were used to predict the epitopes of Gly m Bd 28K, and the predictions were compared with the epitopes that we had located by monoclonal antibodies.     

The V3-directed immune response in natural human immunodeficiency virus type 1 infection is predominantly directed against a variable, discontinuous epitope presented by the gp120 V3 domain.

The specific binding of antibodies to the V3 loop in sera from human immunodeficiency type 1 (HIV-1)-infected individuals was investigated. Different V3 structures were analyzed as full-length loops or by pepscan. Our data show that on full-length V3 loops, both variable regions on either side of the tip of the loop (GPGRAF) contribute to a common epitope for type-specific antibodies. Type-specific antibodies bound strongly and at high titers to native V3 loops but negligibly once the loop was denatured. In contrast to the type-specific, discontinuous epitope, the linear, conserved epitopes presented by the full-length V3 loop, the tip, the amino-terminal base, and the carboxy-terminal base were not accessible to serum antibody. When the V3 sequences were analyzed with linear peptides, antibodies bound preferentially to peptides containing the conserved GPGRAF sequence. Thus, two different specificities of V3-directed antibodies were detected in patient sera. Unlike group-specific antibodies directed against GPGRAF peptides, lack of type-specific antibodies directed against the discontinuous epitope was correlated with viral escape from autologous neutralization. Our data suggest that the full-length conformation of the V3 loop is accessible predominantly to highly type-specific antibodies present in sera from HIV-1-infected individuals. These antibodies are directed against discontinuous V3 epitopes, not against conserved linear V3 targets. The implications of these findings for viral escape and blockade of infection with V3-based vaccines are discussed.     

Epitope specificity of murine and human bactericidal antibodies against PorA P1.7,16 induced with experimental meningococcal group B vaccines

Synthetic peptides derived from the predicted loops 1 and 4 of meningococcal PorA, sero-subtype P1.7,16, were used to study the epitope specificity of murine and human PorA P1.7,16 bactericidal antibodies. The predicted loops 1 and 4 are surface exposed and carry in their apices the sero-subtype epitopes P1.7 (loop 1) or P1.16 (loop 4), respectively. Peptides were synthesized as mono- and multimeric peptides. Murine monoclonal and polyclonal antibodies were induced with meningococcal whole cell preparations. Polyclonal antibodies were evoked in volunteers after one immunization with 50 μg or 100 μg protein of a hexavalent meningococcal PorA vesicle vaccine. The induction of PorA antibodies was determined in ELISA using purified PorA P1.7,16. The epitope specificity of anti-PorA antibodies for both murine and human antibodies could be demonstrated by direct peptide ELISA using overlapping multimeric peptides almost spanning the entire loops 1 or 4 of the protein. The capacity of peptides to inhibit the bactericidal activity of murine and human antibodies was investigated using meningococcal strain H44/76 (B:15:P1.7,16) as a target strain. Bactericidal activities could be inhibited with both monomeric and multimeric peptides derived from epitopes P1.7 and P1.16.     

Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies

Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA), five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues ⁵AIDITRK¹¹, ⁷²RDELNVL⁷⁸, ²⁵¹KSKHNRREGY²⁶⁰, ²⁶⁹DENGIVLD²⁷⁶, and ³⁴¹DETTLVRS³⁴⁸. Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.     

多聚抗原肽微阵列分析平台的建立与初步应用

基于多肽设计合成技术建立了一个快速、高通量、自动化的多聚抗原肽微阵列分析平台.选取人巨细胞病毒(HCMV)的包膜糖蛋白B和被膜碱性磷酸蛋白PP150,幽门螺杆菌(Helicobacterpylori,Hp)尿素酶(Ure)β亚基为靶蛋白,分析筛选出优势线性表位序列,Fmoc法固相合成上述线形表位的多聚抗原肽(MAPs),高效液相色谱仪(HPLC)纯化后,用机器人点样仪按一定的矩阵排列形式点印至硝酸纤维素膜上,2%的小牛血清封闭,塑料壳体封装制备成MAPs微阵列成品.随机抽样经质控血清鉴定后用于随机人群血清试验并与ELISA检测结果进行比较.筛选、合成并鉴定出4条MAPs.用该MAPs微阵列检测的Hp和HCMV阳性及阴性质控血清结果均与质控血清情况相符,120份随机血清检测结果与用重组抗原和病原微生物裂解物抗原包被的ELISA法检测结果相比具有较好的一致性,Ure-1、Ure-2和PP150三种MAPs的灵敏度和特异性均大于90%.MAPs微阵列片间质控试验结果变异系数小于7%,示重复性良好.MAPs微阵列是一种快速、高通量、自动化的分析平台,该平台在预防性疫苗的开发和蛋白质组学的研究中具有较大的前景.     

应用噬菌体展示技术筛选人巨细胞病毒糖蛋白M新的中和抗原表位

人巨细胞病毒(HCMV)糖蛋白复合物Ⅱ包括两种蛋白,即糖蛋白M(gM)和糖蛋白N(gN)．尽管来自于HCMV阳性病人血清中的糖蛋白复合物Ⅱ的IgG抗体能够中和HCMV粒子,但迄今为止,还没有gM中和性抗原表位的相关研究．应用消减杂交技术,通过噬菌体肽库筛选获得gM抗原的一个表位,即MAD．MAD氨基酸序列与gM第32～38位序列高度同源．将MAD与钥孔血蓝蛋白偶联免疫小鼠可产生抗MAD多抗,该多抗不仅结合天然HCMV病毒粒子,而且特异结合重组表达的gM30～78多肽．ELISA结果表明MAD能够特异结合HCMV阳性的病人血清．病毒中和实验结果进一步证明抗MAD多抗能够抑制HCMV AD169株病毒感染人胚肺细胞．总之,MAD表位有可能成为HCMV病毒疫苗潜在的保护性抗原.     

Modulation of HLA-A*0201-restricted T cell responses by natural polymorphism in the IE1(315-324) epitope of human cytomegalovirus

Cytotoxic T lymphocytes play a central role in the control of persistent human CMV (HCMV) infection and reactivation. In healthy virus carriers, the specific CD8(+) CTL response is almost entirely directed against the virion tegument protein pp65 and/or the 72-kDa major immediate early protein, IE1. Studies that included a large panel of HCMV(+) donors suggested that immunorelevance of pp65 and IE1 was directly related with individual HLA haplotype difference. Nevertheless, there are no data on the incidence of HCMV natural polymorphism on virus-specific CTL responses. To assess the impact of IE1 polymorphism on CTL response, we have sequenced in 103 clinical isolates the DNA region corresponding to IE1(315-324), an immunodominant epitope presented by HLA-A*0201 molecules. Seven peptidic variants were found with extensive difference in their frequencies. The response of four HLA-A*0201-restricted anti-IE1 T lymphocyte clones, which were previously generated from one donor against autologous B lymphoblastoid cells expressing a recombinant clinical variant of IE1, was then evaluated using target cells loaded with mutant synthetic peptides or expressing rIE1 variants. One of four clones, which have been sorted 19 times among 22 clones targeted against IE1(315-324), recognized six of the seven tested variant epitopes. All three other clones showed distinct reactivity patterns to target cells loaded with the different mutant peptides or expressing IE1 variants. Therefore, in the HLA-A2 context, clonal expansions of anti-IE1 memory CTLs may confer a protection against HCMV successive infections and reactivations by killing cells presenting most of the naturally occurring IE1(315-324) epitope variants.     

MAPPING OF HIV-1 GAG EPITOPES RECOGNIZED BY POLYCLONAL ANTIBODIES USING GENE-FRAGMENT PHAGE DISPLAY SYSTEM

Phage display has emerged as a powerful technique for mapping epitopes recognised by monoclonal and polyclonal antibodies. We have recently developed a simple gene-fragment phage display system and have shown its utility in mapping epitope recognised by a monoclonal antibody. In the present study, we have employed this system in mapping epitopes recognised by polyclonal antibodies raised against HIV-1 capsid protein, p24 which is derived from proteolytic cleavage of Gag polyprotein. HIV-1 gag DNA was fragmented by DNase I and the fragments (50–250 bp) were cloned into gene-fragment phage display vector to construct a library of phages displaying peptides. This phage library was used for affinity selection of phages displaying epitopes recognised by rabbit anti-p24 polyclonal antibodies. Selected phages contained sequences from two discrete regions of p24, demonstrating the presence of two antigenic regions.

The DNA sequences encoding these regions were also cloned and expressed as GST fusion proteins. The immunoreactivity of these epitopes as GST fusion proteins, or as phage-displayed peptides, was comparable in ELISA system using same anti-p24 polyclonal antibodies. The results indicate that the gene-fragment based phage display system can be used efficiently to identify epitopes recognised by polyclonal antibodies, and phage displayed epitopes can be directly employed in ELISA to detect antibodies.     

Mapping of hiv-1 Gag epitopes recognized by polyclonal antibodies using gene-fragment phage display system.

Phage display has emerged as a powerful technique for mapping epitopes recognised by monoclonal and polyclonal antibodies. We have recently developed a simple gene-fragment phage display system and have shown its utility in mapping epitope recognised by a monoclonal antibody. In the present study, we have employed this system in mapping epitopes recognised by polyclonal antibodies raised against HIV-1 capsid protein, p24 which is derived from proteolytic cleavage of Gag polyprotein. HIV-1 gag DNA was fragmented by DNase I and the fragments (50-250 bp) were cloned into gene-fragment phage display vector to construct a library of phages displaying peptides. This phage library was used for affinity selection of phages displaying epitopes recognised by rabbit anti-p24 polyclonal antibodies. Selected phages contained sequences from two discrete regions of p24, demonstrating the presence of two antigenic regions. The DNA sequences encoding these regions were also cloned and expressed as GST fusion proteins. The immunoreactivity of these epitopes as GST fusion proteins, or as phage-displayed peptides, was comparable in ELISA system using same anti-p24 polyclonal antibodies. The results indicate that the gene-fragment based phage display system can be used efficiently to identify epitopes recognised by polyclonal antibodies, and phage displayed epitopes can be directly employed in ELISA to detect antibodies.     

基于B细胞表位制备抗肝细胞生成素的抗体

目的：基于B细胞表位制备抗肝细胞生成素（HPO）的抗体。方法：根据HPO的空间结构选择了2个候选B细胞表位,展示在T7噬菌体的表面,将提取的重组噬菌体免疫动物,采用ELISA法检测抗血清的效价,通过杂交瘤技术制备针对HPOC端表位的单克隆抗体。结果：2个候选B细胞表位KDGSCD和DGWKDGSC均能诱导抗相应表位多肽的多克隆抗体的产生,免疫6周后血清中抗体效价均达到1∶103,产生的抗体还能够特异识别HPO全蛋白;针对HPOC端表位KDGSCD的单克隆抗体也能识别HPO全蛋白,且具有良好的特异性。结论：基于T7噬菌体展示的B细胞表位可作为免疫原用于制备识别该B细胞表位来源的全蛋白质的抗体。     

Presentation of native epitopes in the V1/V2 and V3 regions of human immunodeficiency virus type 1 gp120 by fusion glycoproteins containing isolated gp120 domains.

The immune response to viral glycoproteins is often directed against conformation- and/or glycosylation-dependent structures; synthetic peptides and bacterially expressed proteins are inadequate probes for the mapping of such epitopes. This report describes a retroviral vector system that presents such native epitopes on chimeric glycoproteins in which protein fragments of interest are fused to the C terminus of the N-terminal domain of the murine leukemia virus surface protein, gp70. The system was used to express two disulfide-bonded domains from gp120, the surface protein of human immunodeficiency virus type 1 (HIV-1), that include potent neutralization epitopes. The resulting fusion glycoproteins were synthesized at high levels and were efficiently transported and secreted. A fusion protein containing the HXB2 V1/V2 domain was recognized by an HIVIIIB-infected patient serum as well as by 17 of 36 HIV-1 seropositive hemophiliac, homosexual male and intravenous drug user patient sera. Many of these HIV+ human sera reacted with V1/V2 domains from several HIV-1 clones expressed in fusion glycoproteins, indicating the presence of cross-reactive antibodies against epitopes in the V1/V2 domain. Recognition of gp(1-263):V1/V2HXB2 by the HIVIIIB-infected human patient serum was largely blocked by synthetic peptides matching V1 but not V2 sequences, while recognition of this construct by a broadly cross-reactive hemophiliac patient serum was not blocked by individual V1 or V2 peptides or by mixtures of these peptides. A construct containing the V3 domain of the IIIB strain of HIV-1, gp(1-263):V3HXB2, was recognized by sera from a human and a chimpanzee that had been infected by HIVIIIB but not by sera from hemophiliac patients who had been infected with HIV-1 of MN-like V3 serotype. The reactive sera had significantly higher titers when assayed against gp(1-263):V3HXB2 than when assayed against matching V3 peptides. Immunoprecipitation of this fusion glycoprotein by the human serum was only partially blocked by V3 peptide, indicating that this infected individual produced antibodies against epitopes in V3 that were expressed on the fusion glycoprotein but not by synthetic peptides. These data demonstrated that the chimeric glycoproteins described here effectively present native epitopes present in the V1/V2 and V3 domains of gp120 and provide efficient methods for detection of antibodies directed against native epitopes in these regions and for characterization of such epitopes.     

Exploring antibody recognition of sequence space through random-sequence peptide microarrays

A universal platform for efficiently mapping antibody epitopes would be of great use for many applications, ranging from antibody therapeutic development to vaccine design. Here we tested the feasibility of using a random peptide microarray to map antibody epitopes. Although peptide microarrays are physically constrained to ～10(4) peptides per array, compared with 10(8) permitted in library panning approaches such as phage display, they enable a much more high though put and direct measure of binding. Long (20 mer) random sequence peptides were chosen for this study to look at an unbiased sampling of sequence space. This sampling of sequence space is sparse, as an exact epitope sequence is unlikely to appear. Commercial monoclonal antibodies with known linear epitopes or polyclonal antibodies raised against engineered 20-mer peptides were used to evaluate this array as an epitope mapping platform. Remarkably, peptides with the most sequence similarity to known epitopes were only slightly more likely to be recognized by the antibody than other random peptides. We explored the ability of two methods singly and in combination to predict the actual epitope from the random sequence peptides bound. Though the epitopes were not directly evident, subtle motifs were found among the top binding peptides for each antibody. These motifs did have some predictive ability in searching for the known epitopes among a set of decoy sequences. The second approach using a windowing alignment strategy, was able to score known epitopes of monoclonal antibodies well within the test dataset, but did not perform as well on polyclonals. Random peptide microarrays of even limited diversity may serve as a useful tool to prioritize candidates for epitope mapping or antigen identification.     

Study of the V3 Loop as a Target Epitope for Antibodies Involved in the Neutralization of Primary Isolates versus T-Cell-Line-Adapted Strains of Human Immunodeficiency Virus Type 1

Previous studies characterized the third variable (V3) loop of the envelope gp120 as the principal neutralizing determinant for laboratory T-cell-line-adapted (TCLA) strains of human immunodeficiency virus type 1 (HIV-1). However, primary viruses isolated from infected individuals are more refractory to neutralization than TCLA strains, suggesting that qualitatively different neutralizing antibodies may be involved. In this study, we investigated whether the V3 loop constitutes a linear target epitope for antibodies neutralizing primary isolates. By using peptides representative of the V3 regions of various primary isolates, an early, relatively specific and persistent antibody response was detected in sera from HIV-infected patients. To assess the relationship between these antibodies and neutralization, the same peptides were used in competition and depletion experiments. Addition of homologous V3 peptides led to a competitive inhibition in the neutralization of the TCLA strain HIV_MN/MT-4 but had no effect on the neutralization of the autologous primary isolate. Similarly, the removal of antibodies that bind to linear V3 epitopes resulted in a loss of HIV_MN/MT-4 neutralization, whereas no decrease in the autologous neutralization was measured. The different roles of V3-specific antibodies according to the virus considered were thereby brought to light. This confirmed the involvement of V3 antibodies in the neutralization of a TCLA strain but emphasized a more pronounced contribution of either conformational epitopes or epitopes outside the V3 loop as targets for antibodies neutralizing primary HIV-1 isolates. This result underlines the need to focus on new vaccinal immunogens with epitopes able to induce broadly reactive and efficient antibodies that neutralize a wide range of primary HIV-1 isolates.     

Characterization of neutralization epitopes in the V2 region of human immunodeficiency virus type 1 gp120: role of glycosylation in the correct folding of the V1/V2 domain.

A number of monoclonal antibodies (MAbs) with various levels of neutralizing activity that recognize epitopes in the V1/V2 domain of LAI-related gp120s have been described. These include rodent antibodies directed against linear and conformational epitopes and a chimpanzee MAb, C108G, with extremely potent neutralizing activity directed against a glycan-dependent epitope. A fusion glycoprotein expression system that expressed the isolated V1/V2 domain of gp120 in native form was used to analyze the structural characteristics of these epitopes. A number of MAbs (C108G, G3-4, 684-238, SC258, 11/68b, 38/66a, 38/66c, 38/62c, and CRA3) that did not bind with high affinity to peptides immunoprecipitated a fusion glycoprotein expressing the V1/V2 domain of HXB2 gp120 in the absence of other human immunodeficiency virus sequences, establishing that their epitopes were fully specified within this region. Biochemical analyses indicated that in the majority of V1/V2 fusion molecules only five of the six glycosylation signals in the V1/V2 domain were utilized, and the glycoforms were found to be differentially recognized by particular MAbs. Both C108G and MAbs directed against conformational epitopes reacted with large fractions of the fully glycosylated molecules but with only small fractions of the incompletely glycosylated molecules. Mutational analysis of the V1 and V2 glycosylation signals indicated that in most cases the unutilized site was located either at position 156 or at position 160, suggesting the occurrence of competition for glycan addition at these neighboring positions. Mutation of glycosylation site 160 destroyed the C108G epitope but increased the fraction of the molecules that presented the conformational epitopes, while mutation of the highly conserved glycosylation site at position 156 greatly diminished the expression of the conformational epitopes and increased expression of the C108G epitope. Similar heterogeneity in glycosylation was also observed when the HXB2 V1/V2 fusion glycoprotein was expressed without most of the gp70 carrier protein, and thus, this appeared to be an intrinsic property of the V1/V2 domain. Heterogeneity in expression of conformational and glycan-dependent epitopes was also observed for the natural viral env precursor, gPr160, but not for gp120. These results suggested that the closely spaced glycosylation sites 156 and 160 are often alternatively utilized and that the pattern of glycosylation at these positions affects the formation of the conformational structures needed for both expression of native epitopes in this region and processing of gPr160 to mature env products.     

The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL.

Cytotoxic T lymphocytes (CTL) appear to play an important role in the control of human cytomegalovirus (HCMV) in the normal virus carrier: previous studies have identified peripheral blood CD8+ CTL specific for the HCMV major immediate-early gene product (IE1) and more recently, by bulk culture and cloning techniques, have identified CTL specific for a structural gene product, the lower matrix protein pp65. In order to determine the relative contributions of CTL which recognize the HCMV proteins IE1, pp65, and glycoprotein B (gB) to the total HCMV-specific CTL response, we have used a limiting-dilution analysis system to quantify HCMV-specific CTL precursors with different specificities, allowing the antigenic specificity of multiple short-term CTL clones to be assessed, in a group of six healthy seropositive donors. All donors showed high frequencies of HCMV-specific major histocompatibility complex-restricted CTL precursors. There was a very high frequency of CTL specific for pp65 (lower matrix protein); IE1-specific CTL were also detectable at lower frequencies in three of five donors, while CTL directed to gB were undetectable. A pp65 gene deletion mutant of HCMV was then used to estimate the contribution of pp65-specific CTL to the total HCMV-specific CTL response; this showed that between 70 and 90% of all CTL recognizing HCMV-infected cells were pp65 specific. Analysis of the peptide specificity of pp65-specific CTL showed that some donors have a highly focused response recognizing a single peptide; the T-cell receptor Vbeta gene usage in these two donors was shown to be remarkably restricted, with over half of the responding CD8+ T cells utilizing a single Vbeta gene rearrangement. Other subjects recognized multiple pp65 peptides: nine new pp65 CTL peptide epitopes were defined, and for five of these the HLA-presenting allele has been identified. All four of the HLA A2 donors tested in this study recognized the same peptide. This apparent domination of the CTL response to HCMV during persistent infection by a single structural protein, irrespective of major histocompatibility complex haplotype, is not clearly described for other persistent virus infections, and the mechanism requires further investigation.