The Bw4 public epitope of HLA-B molecules confers reactivity with natural killer cell clones that express NKB1, a putative HLA receptor.

Although inhibition of natural killer (NK) cell-mediated lysis by the class I HLA molecules of target cells is an established phenomenon, knowledge of the features of class I molecules which induce this effect remains rudimentary. Using class I alleles HLA-B*1502 and B*1513 which differ only at residues 77-83 which define the Bw4 and Bw6 serological epitopes, we tested the hypothesis that the presence of the Bw4 epitope on class I molecules determines recognition by NKB1+ NK cells. HLA-B*1513 possesses the Bw4 epitope, whereas B*1502 has the Bw6 epitope. Lysis by NKB1+ NK cell clones of transfected target cells expressing B*1513 as the only HLA-A, -B, or -C molecule was inhibited, whereas killing of transfectants expressing B*1502 was not. Addition of an an anti-NKB1 monoclonal antibody reconstituted lysis of the targets expressing B*1513, but did not affect killing of targets bearing B*1502. The inhibitory effect of B*1513 could be similarly prevented by the addition of an anti-class I monoclonal antibody. These results show that the presence of the Bw4 epitope influences recognition of HLA-B molecules by NK cells that express NKB1, and suggest that the NKB1 molecule may act as a receptor for Bw4+ HLA-B alleles. Sequences outside of the Bw4 region must also affect recognition by NKB1+ NK cells, because lysis of transfectants expressing HLA-A*2403 or A*2501, which possess the Bw4 epitope but are in other ways substantially different from HLA-B molecules, was not increased by addition of the anti-NKB1 antibody. Asparagine 86, the single site of N-linked glycosylation on class I molecules, is in close proximity to the Bw4/Bw6 region. The glycosylation site of the Bw4-positive molecule B*5801 was mutated, and the mutant molecules tested for inhibition of NKB1+ NK cells. Inhibition that could be reversed by addition of the anti-NKB1 monoclonal antibody was observed, showing the presence of the carbohydrate moiety is not essential for class I recognition by NKB1+ NK cell clones

Bravo/Nr-CAM Is Closely Related to the Cell Adhesion Molecules L1 and Ng-CAM and Has a Similar Heterodimer Structure

Diverse cell-surface molecules of the nervous system play an important role in specifying cell interactions during development. Using a method designed to generate mAbs against neural surface molecules of defined molecular weight, we have previously reported on the surface protein, Bravo, found in the developing avian retinotectal system. Bravo is immunologically detected on developing optic fibers in the retina, but absent from distal regions of the same fibers in the tectum. We have isolated cDNA clones encompassing the entire coding region of Bravo, including clones containing five alternative sequences of cDNA. These putative alternatively spliced sequences encode stretches of polypeptide ranging in length from 10-93 amino acids and are predicted to be both extra- and intracellular. The deduced primary structure of Bravo reveals that, like the cell adhesion molecules (CAMs) chicken Ng- CAM and mouse L1, Bravo is composed of six Ig-like domains, five fibronectin type III repeats, a transmembrane domain, and a short cytoplasmic region. Recently, the cDNA sequence of a related molecule, Nr-CAM, was reported and its possible identity with Bravo discussed (Grumet, M., V. Mauro, M. P. Burgoon, G. E. Edelman, and B. A. Cunningham. 1991. J. Cell Biol. 113:1399-1412). Here we confirm this identity and moreover show that Bravo is found on Muller glial processes and end-feet in the developing retina. In contrast to the single polypeptide chain structure of Nr-CAM reported previously, we show that Bravo has a heterodimer structure composed of an alpha chain of M(r) 140/130 and a beta chain of 60-80 kD. As with L1 and Ng-CAM, the two chains of Bravo are generated from an intact polypeptide by cleavage at identical locations and conserved sites within all three molecules (Ser-Arg/Lys-Arg). The similar domain composition and heterodimer structure, as well as the 40% amino acid sequence identity of these molecules, defines them as an evolutionarily related subgroup of CAMs. The relationship of Bravo to molecules known to be involved in cell adhesion and process outgrowth, combined with its pattern of expression and numerous potential isoforms, suggests a complex role for this molecule in cell interactions during neural development

Paths to a malaria vaccine illuminated by parasite genomics.

More human death and disease is caused by malaria parasites than by all other eukaryotic pathogens combined. As early as the sequencing of the first human genome, malaria parasite genomics was prioritized to fuel the discovery of vaccine candidate antigens. This stimulated increased research on malaria, generating new understanding of the cellular and molecular mechanisms of infection and immunity. This review of recent developments illustrates how new approaches in parasite genomics, and increasingly large amounts of data from population studies, are helping to identify antigens that are promising lead targets. Although these results have been encouraging, effective discovery and characterization need to be coupled with more innovation and funding to translate findings into newly designed vaccine products for clinical trials

Mutations in hepatitis C virus E2 located outside the CD81 binding sites lead to escape from broadly neutralizing antibodies but compromise virus infectivity.

Broadly neutralizing antibodies are commonly present in the sera of patients with chronic hepatitis C virus (HCV) infection. To elucidate possible mechanisms of virus escape from these antibodies, retrovirus particles pseudotyped with HCV glycoproteins (HCVpp) isolated from sequential samples collected over a 26-year period from a chronically infected patient, H, were used to characterize the neutralization potential and binding affinity of a panel of anti-HCV E2 human monoclonal antibodies (HMAbs). Moreover, AP33, a neutralizing murine monoclonal antibody (MAb) to a linear epitope in E2, was also tested against selected variants. The HMAbs used were previously shown to broadly neutralize HCV and to recognize a cluster of highly immunogenic overlapping epitopes, designated domain B, containing residues that are also critical for binding of viral E2 glycoprotein to CD81, a receptor essential for virus entry. Escape variants were observed at different time points with some of the HMAbs. Other HMAbs neutralized all variants except for the isolate 02.E10, obtained in 2002, which was also resistant to MAb AP33. The 02.E10 HCVpp that have reduced binding affinities for all antibodies and for CD81 also showed reduced infectivity. Comparison of the 02.E10 nucleotide sequence with that of the strain H-derived consensus variant, H77c, revealed the former to have two mutations in E2, S501N and V506A, located outside the known CD81 binding sites. Substitution A506V in 02.E10 HCVpp restored binding to CD81, but its antibody neutralization sensitivity was only partially restored. Double substitutions comprising N501S and A506V synergistically restored 02.E10 HCVpp infectivity. Other mutations that are not part of the antibody binding epitope in the context of N501S and A506V were able to completely restore neutralization sensitivity. These findings showed that some nonlinear overlapping epitopes are more essential than others for viral fitness and consequently are more invariant during earlier years of chronic infection. Further, the ability of the 02.E10 consensus variant to escape neutralization by the tested antibodies could be a new mechanism of virus escape from immune containment. Mutations that are outside receptor binding sites resulted in structural changes leading to complete escape from domain B neutralizing antibodies, while simultaneously compromising viral fitness by reducing binding to CD81

Characterization of Envelope-Specific Antibody Response Elicited by HIV-1 Vaccines: A Dissertation

Despite 30 years of intensive research，an effective human immunodeficiency virus (HIV) vaccine still remains elusive. The desirable immune response capable of providing protection against HIV acquisition is still not clear. The accumulating evidence learned from a recent vaccine efficacy correlate study not only confirmed the importance of antibody responses, but also highlighted potential protective functions of antibodies with a broad repertoire of HIV-1 epitope specificities and a wide range of different antiviral mechanisms. This necessitates a deep understanding of the complexity and diversity of antibody responses elicited by HIV-1 vaccines. My dissertation characterizes antibody response profiles of HIV-1 Env antibodies elicited by several novel immunogens or different immunization regimens, in terms of magnitude, persistence, epitope specificity, binding affinity, and biological function. First, to overcome the challenge of studying polyclonal sera without established assays, we expanded a novel platform to isolate Env-specific Rabbit mAbs (RmAb) elicited by DNA prime-protein boost immunization. These RmAbs revealed diverse epitope specificity and cross-reactivity against multiple gp120 antigens from more than one subtype, and several had potent and broad neutralizing activities against sensitive Tier 1 viruses. Further, structural analysis of two V3 mAbs demonstrated that a slight shift of the V3 epitope might have a dramatic impact on their neutralization activity. All of these observations provide a useful tool to study the induction of a desired type of antibody by different immunogens or different immunization regimens. Since heavily glycosylated HIV Env protein is a critical component of an HIV vaccine, we wanted to determine the impact of the HIV Env-associated glycan shield on antibody responses. We were able to produce Env proteins with a selective and homogeneous pattern of N-glycosylation using a glycoengineered yeast cell line. Antigenicity of these novel Env proteins was examined by well-characterized human mAbs. Immunogenicity studies showed that they were immunogenic and elicited gp120- specific antibody responses. More significantly, sera elicited by glycan-modified gp120 protein immunogens revealed better neutralizing activities and increased diversity of epitopes compared to sera elicited by traditional gp120 produced in Chinese Hamster Ovary (CHO) cells. Further, we examined the impact of the delivery order of DNA and protein immunization on antibody responses. We found that DNA prime-protein boost induced a comparable level of Env-specific binding Abs at the peak immunogenicity point to codelivery of DNA. However, antibody responses from DNA prime-protein boost had high avidity and diverse specificities, which improved potency and breadth of neutralizing Abs against Tier 1 viruses. Our data indicate that DNA vaccine priming of the immune system is essential for generation of high-quality antibodies. Additionally, we determined the relative immunogenicity of gp120 and gp160 Env in the context of DNA prime-protein boost vaccination to induce high-quality antibody responses. Immunized sera from gp120 DNA primed animals, but not those primed with gp160 DNA, presented with distinct antibody repertoire specificities, a high magnitude of CD4 binding site-directed binding capabilities as well as neutralizing activities. We confirmed the importance of using the gp120 Env form at the DNA priming phase, which directly determined the quality of antibody response

Probing entry inhibitors' activity on HIV and development of new fusion inhibitors : integrating evolutionary biology with virology

Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2011The general aims of this thesis were: 1) to examine the C2, V3 and C3 envelope regions ofHIV-1 and HIV-2 at the molecular, evolutionary and structural levels; 2) to compare HIV-1and HIV-2 susceptibility to entry inhibitors and assess their potential value in HIV-2therapy; 3) to produce a new fusion inhibitor peptide using evolutionary biology basedstrategies.In the first study (Chapter 2), HIV-1 and HIV-2 were compared at the molecular,evolutionary and structural levels in the C2, V3 and C3 envelope regions. We identifiedsignificant structural and functional constrains to the diversification and evolution of C2,V3 and C3 in the HIV-2 envelope but not in HIV-1. In particular, we found that V3 in HIV-2is less exposed and more conserved than in HIV-1, suggesting fundamental differences inthe biology and infection of these viruses as well as in their susceptibility to entryinhibitors.In the second study (Chapter 3) we measured the baseline susceptibility of HIV-1 and HIV-2primary isolates to different fusion inhibitors and coreceptor antagonists, includingenfuvirtide (T-20) and maraviroc (MVC). MVC inhibited HIV-2 R5 variants at significantlyhigher IC90 concentrations than HIV-1 variants. Moreover, as previously found in HIV-1,susceptibility of HIV-2 R5 variants to MVC was inversely related with CD4+ T cell counts attime of virus isolation. These results suggest that the structure of the envelope complex ofR5 variants changes along the course of infection. More importantly, the results call fornew clinical studies to evaluate the efficacy of MVC in HIV-2 infection and to determine itsbest therapeutic dosage in early and late stage disease. We also provide definitiveevidence demonstrating that T-20 is not useful for HIV-2 therapy.In the final study (Chapter 4), we designed a new HIV fusion inhibitor peptide (P3) basedon the ancestral sequences of the HIV-2 and SIV envelope genes. P3 has an a-helixstructure as demonstrated by circular dichroism. It has broad antiviral activity at thenanomolar range against HIV-1 and HIV-2 primary isolates, including HIV-1 variantsresistant to T-20. Binding ELISA assays and selection of resistant mutants suggest that P3prevents viral fusion by binding to the transmembrane protein in the HR1 region. Thesestudies provide proof of concept that viable antiviral peptides can be constructed usingevolutionary biology strategies. Such strategies should be explored to enhance theproduction of peptide drugs and vaccines.O Vírus da Imunodeficiência Humana do tipo 1 e do tipo 2 (VIH-1 e VIH-2) são os agentes etiológicos do Síndrome de Imunodeficiência Adquirida (SIDA). Embora sejam semelhantes na sua organização estrutural e genómica, estes lentivírus humanos apresentam características antigénicas distintas e partilham uma semelhança genética de apenas 50%. Enquanto o VIH-1 é responsável pela pandemia mundial, a infecção pelo VIH-2 localiza-se sobretudo na África Ocidental, em alguns países europeus como Portugal e França, e na Índia. A infecção pelo VIH-2 tem melhor prognóstico, a progressão para a doença é mais lenta e há melhor controlo imunológico do que na infecção pelo VIH-1. Ao contrário do VIH-1, o arsenal terapêutico actualmente disponível para tratar a infecção por VIH-2 é reduzido. Os fármacos antiretrovirais em uso foram especificamente desenvolvidos para o VIH-1 e, consequentemente, a sua actividade pode ser reduzida ou nula no VIH-2. Este é o caso concreto dos inibidores não nucleosídicos da transcriptase reversa e de alguns inibidores da protease. Neste contexto, os inibidores de entrada poderão ser úteis para tratar a infecção por VIH-2. Contudo, a susceptibilidade dos isolados primários de VIH-2 aos inibidores de entrada é actualmente desconhecida. A susceptibilidade do VIH aos inibidores de entrada é determinada pela qualidade da interacção do vírus com os receptores celulares. O VIH-1 e VIH-2 são substancialmente diferentes a este nível. Por exemplo, o VIH-2 pode ligar-se ao co-receptor CCR5 independentemente do receptor CD4 e da região V3 do invólucro. Por outro lado, as regiões C2, V3 e C3 do VIH-2 são substancialmente diferentes do VIH-1 a nível antigénico. Colectivamente, estes dados indicam que a estrutura e conformação das glicoproteínas de superfície do VIH-1 e VIH-2 são substancialmente diferentes e sugerem que a susceptibilidade e resistência dos dois tipos de vírus aos inibidores de entrada podem também ser diferentes. Os principais objectivos desta tese foram: 1) analisar as características moleculares, estruturais e evolutivas das regiões C2, V3 e C3 no VIH-1 e VIH-2; 2) comparar a susceptibilidade do VIH-1 e VIH-2 aos inibidores de entrada e avaliar o seu potencial terapêutico na infecção por VIH-2; 3) produzir um novo inibidor de fusão para o VIH-2. Para melhor compreender as potenciais diferenças destes dois vírus na resposta aos inibidores de entrada começámos por analisar as características moleculares, estruturais e evolutivas da região V3 e as regiões circundantes C2 e C3, num número significativo de vírus VIH-1 e VIH-2 isolados em Portugal e noutras regiões do globo, com recurso a diferentes metodologias de biologia evolutiva e computacional (Capitulo 2). Apesar da menor variabilidade das 3 regiões no VIH-2, verificámos que a região C3 está sob forte selecção positiva e encontra-se exposta à superfície sugerindo que, tal como no VIH-1, esta região poderá constituir um domínio neutralizante. No entanto, ao contrário do VIH-1, a maioria das mutações adaptativas no VIH-2 são prejudiciais e levam à extinção das linhagens virais pelo que o efeito final é um forte constrangimento à variabilidade das regiões analisadas. Ao contrário do VIH-1, verificámos que a ansa V3 do VIH-2 se encontra oclusa no complexo glicoproteico do invólucro, numa conformação que parece ser estabilizada por interacções que mantém com alguns resíduos da regiões C2 e C3. Estes resultados são consistentes com o facto de a V3 não ser imunodominante no VIH-2, ficando assim mais protegida da resposta imunitária e das eventuais mutações que dela resultam. A forte conservação da V3, da C2 e da C3 também é consistente com a sua potencialmente importante actividade imunosupressora. Em conclusão, este primeiro estudo permitiu caracterizar algumas das características estruturais e funcionais que distinguem as glicoproteínas do invólucro do VIH-1 e do VIH-2 e que estão associadas às diferentes características biológicas e fenotípicas destes dois vírus. Estes dados podem ter impacto na resposta dos dois vírus aos inibidores de entrada (analisado no Capítulo 3) e no desenvolvimento de novas vacinas. No segundo estudo (Capítulo 3) comparámos a actividade antiviral dos antagonistas dos coreceptores (AMD3100, TAK-779 e maraviroc) e dos inibidores de fusão (T-20 e T-1249) entre um grupo de 20 isolados de VIH-2 (19 isolados primários + um isolado laboratorial) e nove isolados de VIH-1 (sete isolados primários + dois isolados laboratoriais). Verificámos que a sensibilidade ao AMD3100 e ao TAK-779 é semelhante no VIH-1 e o VIH-2. No entanto, o perfil da curva dose-resposta do maraviroc (MVC) obtido para os isolados R5 foi diferente nos dois tipos de vírus. No VIH-2 os valores de IC90 foram significativamente mais elevados do que no VIH-1; por outro lado, os declives da curva dose-resposta foram mais baixos no VIH-2 do que no VIH-1. Colectivamente, estes resultados sugerem que poderão ser necessárias concentrações mais elevadas de MVC para tratar os doentes infectados pelo VIH-2. Adicionalmente, encontrámos uma correlação forte e de sentido inverso entre as susceptibilidade do VIH-2 ao MVC e o número de células T CD4+ dos doentes quando os vírus foram isolados. Vírus isolados em doentes em fase de SIDA foram menos susceptíveis ao MVC do que os vírus isolados em doentes com uma contagem de células T CD4+ superior a 200 células/ul. Ao contrário do VIH-1 não encontrámos qualquer correlação entre a carga da V3 e a susceptibilidade dos isolados R5 de VIH-2 ao MVC. De um modo geral, os nossos resultados sugerem que são necessários ensaios clínicos para avaliar a efectividade do MVC na infecção pelo VIH-2, determinar a dose terapêutica mais adequada e esclarecer se é necessário fazer um ajuste de dose de acordo com a fase da doença. Adicionalmente, e uma vez que isolados VIH-2 X4 e populações duplas/mistas são totalmente ou parcialmente resistentes ao MVC, é de extrema importância o desenvolvimento de um ensaio de tropismo (genotípico e/ou fenotípico) para o VIH-2 de modo a determinar o tropismo antes do início da terapia com MVC. Sem o conhecimento prévio do tropismo viral, o tratamento com MVC poderá seleccionar espécies X4 minoritárias que estão associadas a maior resistência à neutralização e uma progressão mais rápida da doença. No que diz respeito aos inibidores de fusão, verificámos que o T-20 tem actividade reduzida no VIH-2, confirmando estudos anteriores realizados com dois isolados laboratoriais. Por outro lado, observámos uma elevada susceptibilidade deste vírus ao T- 1249, indicando que os inibidores de fusão são potencialmente eficazes na infecção pelo VIH-2. Assim, o desenvolvimento de um novo inibidor de fusão do VIH-2 foi o objectivo do último estudo desta tese (Capítulo 4). No Capítulo 4, desenvolvemos novos péptidos inibidores de fusão a partir da reconstrução de sequências ancestrais da glicoproteína gp36 do invólucro de VIH-2 e de Vírus de Imunodeficiência dos Símios (VIS). Com esta abordagem inovadora pretendemos incorporar a história evolutiva dos vírus na sequência dos péptidos e desta forma melhorar a tolerância destas moléculas aos polimorfismos naturais da sua região alvo bem como às mutações de resistência seleccionadas na sua presença. Obteve-se um péptido ancestral (P3) constituído por 34 aminoácidos, cuja sequência corresponde às posições homólogas 628 – 661 da proteína Env do isolado VIH-1 HXB2 (ou 623 – 656 do isolado VIH-2 ROD). A sequência do P3 difere em 21 aminoácidos da sequência consenso de VIH-1, 14 aminoácidos da sequência do T-20 e 6 aminoácidos da sequência consenso de VIH-2. Ao contrário da natureza não-estruturada do T-20, o P3 tem uma conformação típica em hélice-a, o que lhe poderá conferir maior a estabilidade contra a degradação proteolítica, bem como maior afinidade para a região alvo. Por outro lado, o P3 foi facilmente solúvel em soluções aquosas o que é uma vantagem num futuro desenvolvimento de uma fórmula farmacêutica. O P3 demonstrou ter uma forte actividade antiviral contra isolados primários e laboratoriais de VIH-1 e VIH-2 (IC50 médio, 11 nM para o HIV-1 e 63.8 nM para o HIV-2), incluindo variantes resistentes ao T-20 (IC50, 0.15 – 11.8 nM). Através da passagem consecutiva de vírus em cultura na presença do péptido, foi seleccionada uma mutação de resistência na região HR1 da gp41 (VIH-1), a qual é responsável pela redução da susceptibilidade do VIH-1 ao P3 em 120x. Nas mesmas condições, e após 60 dias em cultura, não foi possível seleccionar mutações de resistência ao P3 no VIH-2. Estes resultado, em conjugação com a sua forte ligação à glicoproteína transmembranar de um isolado de VIH-2, indicam que, tal como outros péptidos baseados na região HR2 (T-20, T- 1249), o P3 inibe a entrada do VIH pela interacção com a região HR1 da gp41 e sugerem que a barreira genética para a resistência ao P3 é significativamente superior no VIH-2 do que no VIH-1. Neste estudo demonstrámos ainda que o P3 é significativamente menos antigénico do que o T-20 nos doentes infectados pelo VIH-1 o que poderá traduzir-se numa maior duração da eficácia clínica do P3 em comparação com o T-20. Os resultados obtidos com o P3 demonstram pela primeira vez que é possível desenvolver péptidos com actividade antiviral significativa utilizando metodologias de biologia evolutiva, pelo que esta abordagem poderá ser explorada no futuro para a produção de medicamentos peptídicos e, eventualmente, de vacinas

Mass Spectrometry in the Elucidation of the Glycoproteome of Bacterial Pathogens

Presently some three hundred post-translational modifications are known to occur in bacteria in vivo. Many of these modifications play critical roles in the regulation of proteins and control key biological processes. One of the most predominant modifications, N- and O-glycosylations are now known to be present in bacteria (and archaea) although they were long believed to be limited to eukaryotes. In a number of human pathogens these glycans have been found attached to the surfaces of pilin, flagellin and other surface and secreted proteins where it has been demonstrated that they play a role in the virulence of these bacteria. Mass spectrometry characterization of these glycosylation events has been the enabling key technology for these findings. This review will look at the use of mass spectrometry as a key technology for the detection and mapping of these modifications within microorganisms, with particular reference to the human pathogens, Campylobacter jejuni and Mycobacterium tuberculosis. The overall aim of this review will be to give a basic understanding of the current ‘state-of-the-art’ of the key techniques, principles and technologies, including bioinformatics tools, involved in the analysis of the glycosylation modifications

Glycan shifting on hepatitis C virus (HCV) E2 glycoprotein is a mechanism for escape from broadly neutralizing antibodies

Hepatitis C virus (HCV) infection is a major cause of liver disease and hepatocellular carcinoma. Glycan shielding has been proposed to be a mechanism by which HCV masks broadly neutralizing epitopes on its viral glycoproteins. However, the role of altered glycosylation in HCV resistance to broadly neutralizing antibodies is not fully understood. Here, we have generated potent HCV neutralizing antibodies hu5B3.v3 and MRCT10.v362 that, similar to the previously described AP33 and HCV1, bind to a highly conserved linear epitope on E2. We utilize a combination of in vitro resistance selections using the cell culture infectious HCV and structural analyses to identify mechanisms of HCV resistance to hu5B3.v3 and MRCT10.v362. Ultra deep sequencing from in vitro HCV resistance selection studies identified resistance mutations at asparagine N417 (N417S, N417T and N417G) as early as 5 days post treatment. Comparison of the glycosylation status of soluble versions of the E2 glycoprotein containing the respective resistance mutations revealed a glycosylation shift from N417 to N415 in the N417S and N417T E2 proteins. The N417G E2 variant was glycosylated neither at residue 415 nor at residue 417 and remained sensitive to MRCT10.v362. Structural analyses of the E2 epitope bound to hu5B3.v3 Fab and MRCT10.v362 Fab using X-ray crystallography confirmed that residue N415 is buried within the antibody–peptide interface. Thus, in addition to previously described mutations at N415 that abrogate the β-hairpin structure of this E2 linear epitope, we identify a second escape mechanism, termed glycan shifting, that decreases the efficacy of broadly neutralizing HCV antibodies

Automated Detection of Conformational Epitopes Using Phage Display Peptide Sequences

Background: Precise determination of conformational epitopes of neutralizing antibodies represents a key step in the rational design of novel vaccines. A powerful experimental method to gain insights on the physical chemical nature of conformational epitopes is the selection of linear peptides that bind with high affinities to a monoclonal antibody of interest by phage display technology. However, the structural characterization of conformational epitopes from these mimotopes is not straightforward, and in the past the interpretation of peptide sequences from phage display experiments focused on linear sequence analysis to find a consensus sequence or common sequence motifs. Results: We present a fully automated search method, EpiSearch that predicts the possible location of conformational epitopes on the surface of an antigen. The algorithm uses peptide sequences from phage display experiments as input, and ranks all surface exposed patches according to the frequency distribution of similar residues in the peptides and in the patch. We have tested the performance of the EpiSearch algorithm for six experimental data sets of phage display experiments, the human epidermal growth factor receptor-2 (HER-2/neu), the antibody mAb Bo2C11 targeting the C 2 domain of FVIII, antibodies mAb 17b and mAb b12 of the HIV envelope protein gp120, mAb 13b5 targeting HIV-1 capsid protein and 80R of the SARS coronavirus spike protein. In all these examples th

Cell Cycle Control by Xenopus p28^(Kix1) a Developmentally Regulated Inhibitor of Cyclin-dependent Kinases

We have isolated Xenopus p28^(Kix1), a member of the p21^(CIP1)/p27^(KIP1)/p57^(KIP2) family of cyclin-dependent kinase (Cdk) inhibitors. Members of this family negatively regulate cell cycle progression in mammalian cells by inhibiting the activities of Cdks. p28 shows significant sequence homology with p21, p27, and p57 in its N-terminal region, where the Cdk inhibition domain is known to reside. In contrast, the C-terminal domain of p28 is distinct from that of p21, p27, and p57. In co-immunoprecipitation experiments, p28 was found to be associated with Cdk2, cyclin E, and cyclin A, but not the Cdc2/cyclin B complex in Xenopus egg extracts. Xenopus p28 associates with the proliferating cell nuclear antigen, but with a substantially lower affinity than human p21. In kinase assays with recombinant Cdks, p28 inhibits pre-activated Cdk2/cyclin E and Cdk2/cyclin A, but not Cdc2/cyclin B. However, at high concentrations, p28 does prevent the activation of Cdc2/cyclin B by the Cdk-activating kinase. Consistent with the role of p28 as a Cdk inhibitor, recombinant p28 elicits an inhibition of both DNA replication and mitosis upon addition to egg extracts, indicating that it can regulate multiple cell cycle transitions. The level of p28 protein shows a dramatic developmental profile: it is low in Xenopus oocytes, eggs, and embryos up to stage 11, but increases approximately ~ 100-fold between stages 12 and 13, and remains high thereafter. The induction of p28 expression temporally coincides with late gastrulation. Thus, although p28 may play only a limited role during the early embryonic cleavages, it may function later in development to establish a somatic type of cell cycle. Taken together, our results indicate that Xenopus p28 is a new member of the p21/p27/p57 class of Cdk inhibitors, and that it may play a role in developmental processes