Rationally engineered therapeutic proteins with reduced immunogenicity

Chronic administration of protein therapeutics may elicit unacceptable immune responses to the specific protein. Our hypothesis is that the immunogenicity of protein drugs can be ascribed to a few immunodominant helper T lymphocyte (HTL) epitopes, and that reducing the MHC binding affinity of these HTL epitopes contained within these proteins can generate drugs with lower immunogenicity. To test this hypothesis, we studied the protein therapeutic erythropoietin (Epo). Two regions within Epo, designated Epo 91-120 and Epo 126-155, contained HTL epitopes that were recognized by individuals with numerous HLA-DR types, a property common to immunodominant HTL epitopes. We then engineered analog epitopes with reduced HLA binding affinity. These analog epitopes were associated with reduced in vitro immunogenicity. Two modified forms of Epo containing these substitutions were shown to be bioactive and nonimmunogenic in vitro. These findings support our hypothesis and demonstrate that immunogenicity of protein drugs can be reduced in a systematic and predictable manner.     

人巨细胞病毒gB/AD1基因在CHO细胞中的表达和纯化

人巨细胞病毒(human cytomegalovirus,HCMV)在自然界中普遍存在,g B是该病毒表面的一种重要糖蛋白,其AD1序列是主要的中和抗体表位之一。首先以HCMV基因组为模板,通过PCR技术扩增出g B基因的一个片段,该片段删除了跨膜区和胞内区,仅保留AD1中和抗体表位,然后被连接到哺乳动物细胞表达质粒p SGHV0上,与dhfr基因通过电击转染至CHO/dhfr-细胞,继而用60 nmol/L MTX筛选稳定表达细胞株,用ELISA和Western blot检测AD1重组蛋白的表达,最后用Ni2+-NTA亲和层析进行纯化。获得了真核细胞表达的重组融合蛋白,表达量约为10 mg/L,蛋白纯度可达到80%以上,为HCMV表面糖蛋白g B功能的进一步研究以及基因工程疫苗的开发奠定了基础。     

Palmitoylation Strengthens Cholesterol-dependent Multimerization and Fusion Activity of Human Cytomegalovirus Glycoprotein B (gB)

Herpesviruses are a large order of animal enveloped viruses displaying a virion fusion mechanism of unusual complexity. Their multipartite machinery has a conserved core made of the gH/gL ancillary complexes and the homo-trimeric fusion protein glycoprotein B (gB). Despite its essential role in starting the viral infection, gB interaction with membrane lipids is still poorly understood. Here, evidence is provided demonstrating that human cytomegalovirus (HCMV) gB depends on the S-palmitoylation of its endodomain for an efficient interaction with cholesterol-rich membrane patches. We found that, unique among herpesviral gB proteins, the HCMV fusion factor has a Cys residue in the C-terminal region that is palmitoylated and mediates methyl-β-cyclodextrin-sensitive self-association of purified gB. A cholesterol-dependent virus-like particle trap assay, based on co-expression of the HIV Gag protein, confirmed that this post-translational modification is functional in the context of cellular membranes. Mutation of the palmitoylated Cys residue to Ala or inhibition of protein palmitoylation decreased HCMV gB export via Gag particles. Moreover, purified gB_C777A showed an increased kinetic sensitivity in a cholesterol depletion test, demonstrating that palmitoyl-gB limits outward cholesterol diffusion. Finally, gB palmitoylation was required for full fusogenic activity in human epithelial cells. Altogether, these results uncover the palmitoylation of HCMV gB and its role in gB multimerization and activity.     

Enhancing nicotine vaccine immunogenicity with liposomes

A major liability of existing nicotine vaccine candidates is the wide variation in anti-nicotine immune responses among clinical trial participants. In order to address this liability, significant emphasis has been directed at evaluating adjuvants and delivery systems that confer more robust potentiation of the anti-nicotine immune response. Toward that end, we have initiated work that seeks to exploit the adjuvant effect of liposomes, with or without Toll-like receptor agonist(s). The results of the murine immunization study described herein support the hypothesis that a liposomal nicotine vaccine formulation may provide a means for addressing the immunogenicity challenge.     

Steady-State Plasma Membrane Expression of Human Cytomegalovirus gB Is Determined by the Phosphorylation State of Ser900

Human cytomegalovirus (HCMV) infection of an astrocytoma cell line (U373) or human fibroblast (HF) cells results in a differential cell distribution of the major envelope glycoprotein gB (UL55). This 906-amino-acid type I glycoprotein contains an extracellular domain with a signal sequence, a transmembrane domain, and a 135-amino-acid cytoplasmic tail with a consensus casein kinase II (CKII) site located at Ser₉₀₀. Since phosphorylation of proteins in the secretory pathway is an important determinant of intracellular trafficking, the state of gB phosphorylation in U373 and HF cells was examined. Analysis of cells expressing wild-type gB and gB with site-specific mutations indicated that the glycoprotein was equally phosphorylated at a single site, Ser₉₀₀, in both U373 and HF cells. To assess the effect of charge on gB surface expression in U373 cells, Ser₉₀₀ was replaced with an aspartate (Asp) or alanine (Ala) residue to mimic the phosphorylated and nonphosphorylated states, respectively. Expression of the Asp but not the Ala gB mutation resulted in an increase in the steady-state expression of gB at the plasma membrane (PM) in U373 cells. In addition, treatment of U373 cells with the phosphatase inhibitor tautomycin resulted in the accumulation of gB at the PM. Interestingly, the addition of a charge at Ser₉₀₀ trapped gB in a low-level cycling pathway at the PM, preventing trafficking of the protein to the trans-Golgi network or other intracellular compartments. Therefore, these results suggest that a tautomycin-sensitive phosphatase regulates cell-specific PM retrieval of gB to intracellular compartments.     

Rationally designed hybrid molecules with appreciable COX-2 inhibitory and anti-nociceptive activities

Six molecules were obtained by the combination of three biologically and medicinally significant moieties—indole, chrysin and pyrazole. Bio-evaluation of these hybrid molecules showed significant inhibition of COX-2 enzymatic activity over that of COX-1 and appreciable anti-nociceptive activity, checked at swiss albino mice.     

血液病患者的巨细胞病毒感染

用间接酶联免疫吸附实验(ELISA)对新近诊断的179例血液病患者血清巨细胞病毒(HCMV)IgM和IgA抗体进行了检测。阳性率分别为11,17％11,73％,明显高于对照人群(4,76％和3,97％),提示血液病患者由于免疫功能下降,易于发生HCMV活动性感染。     

Growth in Agarose of Human Cells Infected with Cytomegalovirus

After infection by human cytomegalovirus (CMV), human diploid fibroblasts could grow in agarose medium for several generations. Clones of infected cells grew for weeks, although in every case they ultimately underwent lysis owing to the cytopathic effect of the virus. Virus was inoculated at high dilution and after UV irradiation in an effort to derive cells infected with noninfectious defective particles still capable of inducing cell stimulation. Dilute or irradiated virus occasionally yielded large colonies of replicating cells, although permanent transformation was not observed. One clone derived from UV-CMV-infected cells was passaged four times before undergoing lysis. During these passages the cells exhibited alterations in morphology and orientation.     

Nonproductive Infection of Guinea Pig Cells with Human Cytomegalovirus

Human cytomegalovirus was capable of adsorbing to and penetrating guinea pig cells, but was unable to replicate new virus. Cultures infected with virus inoculum of high titer showed a cytopathic effect (CPE) characterized by cell rounding. This CPE depended upon the presence of infectious virus, and its extent was directly related to the multiplicity of infection. Staining by indirect immunofluorescence by using human convalescent sera was positive as early as 4 h postinfection. Maximal fluorescence was observed 24 h postinfection when 50% of the cells contained fluorescent antigens both in nuclei and cytoplasm. No evidence for viral replication was found, and no defective particles were detected by electron microscopy. Treatment with actinomycin D or with cycloheximide strongly inhibited both the fluorescent antigens and the CPE, whereas 5-fluorodeoxyuridine and bromodeoxyuridine were ineffective.     

Rationally designed interfacial peptides are efficient in vitro inhibitors of HIV-1 capsid assembly with antiviral activity

Virus capsid assembly constitutes an attractive target for the development of antiviral therapies; a few experimental inhibitors of this process for HIV-1 and other viruses have been identified by screening compounds or by selection from chemical libraries. As a different, novel approach we have undertaken the rational design of peptides that could act as competitive assembly inhibitors by mimicking capsid structural elements involved in intersubunit interfaces. Several discrete interfaces involved in formation of the mature HIV-1 capsid through polymerization of the capsid protein CA were targeted. We had previously designed a peptide, CAC1, that represents CA helix 9 (a major part of the dimerization interface) and binds the CA C-terminal domain in solution. Here we have mapped the binding site of CAC1, and shown that it substantially overlaps with the CA dimerization interface. We have also rationally modified CAC1 to increase its solubility and CA-binding affinity, and designed four additional peptides that represent CA helical segments involved in other CA interfaces. We found that peptides CAC1, its derivative CAC1M, and H8 (representing CA helix 8) were able to efficiently inhibit the in vitro assembly of the mature HIV-1 capsid. Cocktails of several peptides, including CAC1 or CAC1M plus H8 or CAI (a previously discovered inhibitor of CA polymerization), or CAC1M+H8+CAI, also abolished capsid assembly, even when every peptide was used at lower, sub-inhibitory doses. To provide a preliminary proof that these designed capsid assembly inhibitors could eventually serve as lead compounds for development of anti-HIV-1 agents, they were transported into cultured cells using a cell-penetrating peptide, and tested for antiviral activity. Peptide cocktails that drastically inhibited capsid assembly in vitro were also able to efficiently inhibit HIV-1 infection ex vivo. This study validates a novel, entirely rational approach for the design of capsid assembly interfacial inhibitors that show antiviral activity.     

Aqueous Cytokine Changes Associated with Posner-Schlossman Syndrome with and without Human Cytomegalovirus

Aim

To study the differences in aqueous cytokines in Posner-Schlossman Syndrome (PSS) patients with and without human cytomegalovirus (CMV) DNA in the aqueous humor.

Methods

This is a prospective study. Fifty-three uveitis patients with clinical signs of PSS were enrolled and aqueous humor samples were collected. Fourteen PSS patients were positive of CMV DNA in the aqueous by polymerase chain reaction (PCR) analysis. These eyes were negative of common ocular pathogens such as herpes simplex virus, varicella-zoster virus, rubella virus and toxoplasma. Twenty-five otherwise healthy cataract patients were enrolled as controls. Cytokine concentration was measured by a magnetic color-bead-based multiplex assay and analyzed using statistical and classification approaches.

Results

The average age of 53 PSS patients was 48.74±13.43 years (yrs) (mean ± standard deviation) and 66.3±15.0 yrs for the controls. The median CMV viral DNA copy number was 26000/mL aqueous (range 1400 to 85000 copies/mL) in 14 CMV positive patients as determined by quantitative PCR. PSS aqueous had significantly higher Interleukin (IL)-8 (CXCL8), monocyte chemotactic protein-1 (CCL2), macrophage inhibitory protein 1-β (CCL4), granulocyte colony-stimulating factor (GCSF) and transforming growth factor-β (TGF-β) levels than controls after adjusted by age and gender. IL-2, IL-12, tumor necrosis factor-α (TNF-α) and interferon-α (IFN-α) levels were significantly lower in PSS aqueous than controls. No difference between CMV positive PSS and CMV negative PSS aqueous was observed. Over 97% of PSS samples were distinguished from controls by elevated CXCL10 (>500 ng/mL), CXCL8 (>30 ng/mL) and CCL2 (>60 ng/mL) levels.

Conclusion

PSS eyes were characterized by elevated aqueous chemokine concentration. The presence of CMV viral DNA was not associated with significant change of the type of cytokine expression in PSS patients.     

A viral nanoparticle with dual function as an anthrax antitoxin and vaccine

The recent use of Bacillus anthracis as a bioweapon has stimulated the search for novel antitoxins and vaccines that act rapidly and with minimal adverse effects. B. anthracis produces an AB-type toxin composed of the receptor-binding moiety protective antigen (PA) and the enzymatic moieties edema factor and lethal factor. PA is a key target for both antitoxin and vaccine development. We used the icosahedral insect virus Flock House virus as a platform to display 180 copies of the high affinity, PA-binding von Willebrand A domain of the ANTXR2 cellular receptor. The chimeric virus-like particles (VLPs) correctly displayed the receptor von Willebrand A domain on their surface and inhibited lethal toxin action in in vitro and in vivo models of anthrax intoxication. Moreover, VLPs complexed with PA elicited a potent toxin-neutralizing antibody response that protected rats from anthrax lethal toxin challenge after a single immunization without adjuvant. This recombinant VLP platform represents a novel and highly effective, dually-acting reagent for treatment and protection against anthrax.     

Synthesis of Proteins and Glycoproteins in Cells Infected with Human Cytomegalovirus

In cytomegalovirus-infected cells, the rate of protein synthesis was detected as two peaks. One occurred during the early phase of infection, 0 to 36 h postinfection, and the other occurred during the late phase, after the initiation of viral DNA synthesis. Double-isotopic-label difference analysis demonstrated that host and viral proteins were synthesized simultaneously during both phases. In the early phase, approximately 70 to 90% of the total proteins synthesized were host proteins, whereas approximately 10 to 30% were viral, even at a multiplicity of infection of 20 PFU/cell. Virus-related proteins or glycoproteins were referred to as infected-cell specific (ICS). Two ICS glycoproteins (gp145 and 100) were clearly detectable and were synthesized preferentially in the early phase of infection. Their synthesis was concomitant with stimulation of the protein synthesis rate. In the late phase of infection, approximately 50 to 60% of the total protein synthesis was viral and approximately 40 to 50% was host. The ICS proteins and glycoproteins detected during the late phase of infection were viral structural proteins. Infectious virus was not detectable until 48 to 72 h postinfection. An inhibitor of viral DNA synthesis, phosphonoacetic acid, prevented the appearance of the late-phase ICS proteins and glycoproteins, but there was little or no effect on early ICS glycoprotein synthesis. Radiolabeled ICS proteins and glycoproteins were identified by their relative rates of synthesis, by their different electrophoretic mobilities compared with those of host proteins and host glycoproteins, and by their similar electrophoretic mobilities compared to those of proteins and glycoproteins associated with virions and dense bodies of cytomegalovirus. Structural viral antigens in the infected-cell extracts were removed by immunoprecipitation, using F(ab')(2) fragments of cytomegalovirus-specific antibodies, and identified as described above. The last two criteria were used to identify viral structural ICS proteins and glycoproteins. Although approximately 35 structural proteins were found to be associated with purified virions and dense bodies, the continued synthesis of host cell proteins complicated their identification in infected cells. Nevertheless, seven of the nine structural glycoproteins were identified as ICS glycoproteins.     

基于gp41近膜端外部区域中和表位天然构象的人类免疫缺陷病毒1型疫苗的设计

疫苗一直被视为终结人类免疫缺陷病毒1型和2型(human immunodeficiency virus type 1/2,HIV-1/2)最有力的武器.位于HIV-1 gp41胞外域C末端的近膜端外部区域(membrane-proximal external region,MPER)是一个重要的抗原位点,但糖蛋白41(...     

Characterization of a Human Cytomegalovirus with Phosphorylation Site Mutations in the Immediate-Early 2 Protein

A human cytomegalovirus mutant (TNsubIE2P) was constructed with alanine substitutions of four residues (T27, S144, T233, and S234) previously shown to be phosphorylated in the immediate-early 2 (IE2) protein. This mutant grew as well as the wild type at both low and high multiplicities of infection. The mutant activated the major immediate-early, UL4, and UL44 promoters to similar levels, and with similar kinetics, as wild-type virus. However, the TNsubIE2P mutant virus transactivated an endogenous simian virus 40 early promoter 4 h earlier and to higher levels than the wild-type virus in infected human fibroblasts. The modification of the IE2 protein by SUMO-1 (i.e., its sumoylated state) was also examined.