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Quantitative analysis of striped coat-color patterns in Large White-->Duroc chimeric pigs with special reference to the genetic control mechanisms of the dominant black-eyed white phenotype.

Coat colors of four chimeric pigs produced by the microinjection of dissociated blastomeres of (Landrace x Large White) blastocysts to the blastocyst cavity of Duroc x Duroc) blastocysts (Kashiwazaki et al., 1992) exhibited characteristic horizontal stripe-patterns. We carried out quantitative analysis of those patterns in order to derive information concerning the genetic regulatory mechanisms of the dominant black-eyed white phenotypes in the pig. In the four chimeras, the theoretical mean widths of the single-clone stripe calculated from the estimated widths of minimal recognizable stripe (MRS) (Tachi, 1988) were 2.1 +/- 0.1, 2.23 +/- 0.15, 1.89 +/- 0.06, and 1.93 +/- 0.28 cm respectively. The estimated number of single-clone stripes in the thoracico-lumbar region of those animals were 42.3, 40.7, 46.3, 44.2, and about twice the mean number of vertebrae in the same region (Duroc, 20 or 21; Large White 21 or 22). Furthermore, the mean length of thoracico-lumbar vertebrae in two of the chimeric pigs, as measured on X-ray radiographs, was approximately twice the mean single-clone stripe width. It was concluded that the stripe-patterns of the chimeric pigs probably represented the dermatome patterns of epidermis; and in the pig, a single somite was likely to be derived from the clones of two primordial cells, as originally proposed by Gearhart & Mintz (1972) in the mouse. It was suggested, furthermore, that in the Large White-->Duroc chimeric pigs, melanocytes that migrated into the region of skin formed by a Large White dermatome could not survive, thus creating a clearly demarcated white stripe. Possible involvement of KL or c-kit in the dominant black-eyed white phenotype of the pig is discussed.

Animals↗

Efficacy of a recombinant chimeric anti-hCG antibody to prevent human cytotrophoblasts fusion and block progesterone synthesis.

PROBLEM: A recombinant chimeric antibody against hCG (cPIPP) has been engineered and expressed at high yield in plants. The purpose of this work was to enquire whether this antibody is competent to neutralize the bioactivity of hCG on human trophoblasts. METHODS: Cytotrophoblast cells, isolated from term placentae were maintained in culture for 3 days in presence or absence of humanized chimeric anti-hCG antibodies. Progesterone secreted was quantitated by ELISA. Fusion and cyto-architecture of the cells was studied by light and electron microscopy. Modulation of E-cadherin was investigated using RT-PCR and immunocytochemistry. RESULTS: Recombinant chimeric anti-hCG antibody blocked the synthesis of progesterone by trophoblasts. No fusion of cytotrophoblasts to form syncytium took place. E-cadherin, a vital cell adhesion molecule involved in cell-to-cell interaction did not show differentiation related decline in its expression in presence of the antibody. CONCLUSION: Recombinant chimeric anti-hCG antibody (cPIPP) was effective to neutralize hCG induced bioactivities in the human derived trophoblast cells.

Antibodies, Monoclonal↗

CD154 blockade for induction of mixed chimerism and prolonged renal allograft survival in nonhuman primates.

Costimulatory blockade with anti-CD154 monoclonal antibody (aCD154) prolongs allograft survival in nonhuman primates, but has not reliably induced tolerance when used alone. In the current studies, we evaluated the effect of adding CD154 blockade to a chimerism inducing nonmyeloablative regimen in primates. We observed a significant improvement of donor bone marrow (DBM) engraftment, which has been associated with a lower incidence of acute rejection and long-term survival of renal allografts without the need for previously required splenectomy. Among the long-term survivors, four never showed evidence of rejection, with the longest survival exceeding 1700 days following discontinuation of immunosuppression. Nevertheless, late chronic rejection was observed in three of eight recipients, indicating the necessity of further modifications of the regimen. Control recipients receiving no DBM or donor splenocytes in place of DBM rejected their allografts. Thus, DBM engraftment with, at least, transient mixed chimerism appears essential for induction of allograft tolerance using this conditioning regimen. Modification of the original mixed chimerism approach, by the addition of costimulatory blockade, has been shown to enhance mixed chimerism and induce renal allograft tolerance with less morbidity in nonhuman primates.

Animals↗

Comparative structure-function analysis of VEGFR-1 and VEGFR-2: What have we learned from chimeric systems?

Activation of vascular endothelial growth factor receptor-1 and -2 (VEGFR-1 and VEGFR-2) plays a critical role in vasculogenesis and angiogenesis. However, the mechanism by which activation of VEGFRs elicits these cellular events is not fully understood. We recently constructed chimeric receptors containing the extracellular domain of human CSF-1R/c-fms fused with the entire transmembrane and cytoplasmic domains of murine VEGFR-1 and VEGFR-2. Selective activation of chimeric VEGFR-2, but not chimeric VEGFR-1, stimulated endothelial cell growth, migration, and differentiation. Stimulation of cells coexpressing chimeric VEGFR-1 and VEGFR-2 suppressed VEGFR-2-mediated endothelial cell growth. Site-directed mutagenesis demonstrated that tyrosines 799 and 1173 are required for VEGFR-2-mediated endothelial cell growth and activation of PI3 kinase. Further site-directed mutagenesis demonstrated that tyrosine 1212, located in the carboxyl tail of VEGFR-2, is required for the ligand-dependent autophosphorylation of the receptor and its ability to activate signaling proteins. Collectively, our results suggest that activation of VEGFR-1 and VEGFR-2 differentially regulates endothelial cell function and angiogenesis. Second, activation of VEGFR-2 is associated with many endothelial cell functions, including cell proliferation, migration, and differentiation. Third, activation of PI3 kinase by VEGFR-2 regulates endothelial cell proliferation.

Animals↗

Relaxin-3/insulin-like peptide 5 chimeric peptide, a selective ligand for G protein-coupled receptor (GPCR)135 and GPCR142 over leucine-rich repeat-containing G protein-coupled receptor 7.

Relaxin-3, the most recently identified member of relaxin/insulin family, is an agonist for leucine-rich repeat-containing G protein-coupled receptor (LGR)7, GPCR135, and GPCR142. LGR7 can be pharmacologically differentiated from GPCR135 and GPCR142 by its high affinity for relaxin. Selective ligands that specifically activate GPCR135 or GPCR142 are highly desirable for studying their functional roles. We have created chimeric peptides that consist of the B-chain of human relaxin-3 in combination with various A-chains from other members of the relaxin/insulin family. Pharmacological characterization of these chimeric peptides indicates the A-chain from relaxin-1, relaxin-2, insulin-like peptide (INSL)3, and INSL6 does not change the pharmacological properties of relaxin-3 significantly. In contrast, substitution of the relaxin-3 A-chain with the A-chain from INSL5 results in a chimeric peptide that selectively activates GPCR135 and GPCR142 over LGR7. This study demonstrates that the A-chains among some of the insulin/relaxin family members are pharmacologically exchangeable. The relaxin-3/INSL5 chimeric peptide is a potential tool to study in vivo function of GPCR135. In addition, because of the substitution of a very hydrophobic peptide (the A-chain of relaxin-3) with a very hydrophilic peptide (the A-chain from INSL5), the radiolabeled (125)I-relaxin-3/INSL5 chimera is a suitable ligand (high-affinity, low-nonspecific binding) for receptor autoradiographic studies on tissue sections.

Amino Acid Sequence↗

Selective activation of a chimeric Gi1/Gs G protein alpha subunit by the human IP prostanoid receptor: analysis using agonist stimulation of high affinity GTPase activity and [35S]guanosine-5'-O-(3-thio)triphosphate binding.

A FLAG-tagged form of the human IP prostanoid receptor was expressed stably in HEK 293 cells. This bound [3H]iloprost with high affinity and stimulated cAMP production when exposed to agonist. Iloprost produced weak stimulation of GTPase activity and [35S]guanosine-5'-O-(3-thio)triphosphate binding in membranes of these cells. Pretreatment of cells with pertussis toxin did not modify iloprost-mediated stimulation, but this was blocked by cholera toxin. The effects of iloprost were not increased by coexpression of either Gsalpha or Gi1alpha. In contrast, coexpression of a chimeric G protein alpha subunit in which the carboxyl-terminal six amino acids of Gi1alpha were altered to those of Gsalpha resulted in robust stimulation by iloprost. Because the chimeric G protein alpha subunit (Gi1/Gs6alpha) is not a substrate for either pertussis or cholera toxin, pretreatment of cells coexpressing the IP prostanoid receptor and Gi1/Gs6alpha with a mixture of these toxins resulted in resolution of the signal derived from activation of the chimeric G protein. Agonist-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding and GTPase activity assays are the most commonly used strategies to examine interactions between G protein-coupled receptors and G proteins. These usually are not appropriate for receptors such as the IP prostanoid receptor that interact with G proteins with low rates of guanine nucleotide exchange and hydrolysis. Chimeric G proteins such as Gi1/Gs6alpha that allow appropriate receptor contacts to be converted to the higher nucleotide turnover rates typical of the Gi family G proteins can overcome this and offer a novel means to examine agonist function at such receptors.

Cell Line↗

Chimeric NGF-EGF receptors define domains responsible for neuronal differentiation.

To determine the domains of the low-affinity nerve growth factor (NGF) receptor required for appropriate signal transduction, a series of hybrid receptors were constructed that consisted of the extracellular ligand-binding domain of the human epidermal growth factor (EGF) receptor (EGFR) fused to the transmembrane and cytoplasmic domains of the human low-affinity NGF receptor (NGFR). Transfection of these chimeric receptors into rat pheochromocytoma PC12 cells resulted in appropriate cell surface expression. Biological activity mediated by the EGF-NGF chimeric receptor was assayed by the induction of neurite outgrowth in response to EGF in stably transfected cells. Furthermore, the chimeric receptor mediated nuclear signaling, as evidenced by the specific induction of transin messenger RNA, an NGF-responsive gene. Neurite outgrowth was not observed with chimeric receptors that contained the transmembrane domain from the EGFR, suggesting that the membrane-spanning region and cytoplasmic domain of the low-affinity NGFR are necessary for signal transduction.

Adrenal Gland Neoplasms↗

Development of polyclonal antibodies for detection of aflatoxigenic molds involving culture filtrate and chimeric proteins expressed in Escherichia coli.

Polyclonal antibodies (PAb) were raised against an aflatoxigenic strain of Aspergillus parasiticus by using two different sources for antibody elicitation: (i) filtrate of a culture on which the fungus had been grown (ii) and two chimeric proteins, expressed in Escherichia coli as separate products, of the genes ver-1 and apa-2, which are involved in aflatoxin biosynthesis. The gene products were amplified by PCR, and each was cloned into the E. coli expression vector pGEX2T. Upon induction, the bacteria overexpressed 38- and 33-kDa chimeric proteins corresponding to the N-terminal domains of the genes ver-1 and apa-2, respectively. The chimeric proteins were isolated and affinity purified for use as antigens. The specificity of the raised antibodies was examined by enzyme-linked immunosorbent assay (ELISA). The PAbs raised against the culture filtrate reacted with all the species of Aspergillus and Penicillium tested but not with Fusarium species or corn gain. However, the PAbs elicited against the chimeric proteins were highly specific, showing significantly higher ELISA absorbance values (A405) against A. parasiticus and A. flavus than against the other fungi tested and the corn grain. The approach of utilizing gene products associated with aflatoxin biosynthesis for antibody production therefore appears to be feasible. Such a multiantibody system combined with the PCR technique, could provide a useful tool for the rapid, sensitive, and accurate detection of aflatoxin producers present in grains and foods.

Aflatoxin B1↗

Design and production in Aspergillus niger of a chimeric protein associating a fungal feruloyl esterase and a clostridial dockerin domain.

A chimeric enzyme associating feruloyl esterase A (FAEA) from Aspergillus niger and dockerin from Clostridium thermocellum was produced in A. niger. A completely truncated form was produced when the dockerin domain was located downstream of the FAEA (FAEA-Doc), whereas no chimeric protein was produced when the bacterial dockerin domain was located upstream of the FAEA (Doc-FAEA). Northern blot analysis showed similar transcript levels for the two constructs, indicating a posttranscriptional bottleneck for Doc-FAEA production. The sequence encoding the first 514 amino acids from A. niger glucoamylase and a dibasic proteolytic processing site (kex-2) were fused upstream of the Doc-FAEA sequence. By using this fusion strategy, the esterase activity found in the extracellular medium was 20-fold-higher than that of the wild-type reference strain, and the production yield was estimated to be about 100 mg of chimeric protein/liter. Intracellular and extracellular production was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, dockerin-cohesin interaction assays, and Western blotting. Labeled cohesins detected an intact extracellular Doc-FAEA of about 43 kDa and a cleaved-off dockerin domain of about 8 kDa. In addition, an intracellular 120-kDa protein was recognized by using labeled cohesins and antibodies raised against FAEA. This protein corresponded to the unprocessed Doc-FAEA form fused to glucoamylase. In conclusion, these results indicated that translational fusion to glucoamylase improved the secretion efficiency of a chimeric Doc-FAEA protein and allowed production of the first functional fungal enzyme joined to a bacterial dockerin.

Aspergillus niger↗

Development of recombinant chimeric antigen expressing immunodominant B epitopes of Leishmania infantum for serodiagnosis of visceral leishmaniasis.

Wild canids and domestic dogs are the main reservoir of zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum (syn.: Leishmania chagasi). Serological diagnosis of VL is therefore important in both human and dog leishmaniasis from a clinical and epidemiological point of view. Routine diagnosis of VL is traditionally carried out by immunofluorescent antibody test (IFAT), which is laborious and difficult to standardize and to interpret. In the last decade, however, several specific antigens of Leishmania infantum have been characterized, allowing the development of a recombinant-based immunoassay. Among them, the whole open reading frame encoding K9 antigen, the gene fragment encoding the repetitive sequence of K26, and the 3'-terminal gene fragment of the kinesin-related protein (K39sub) were previously evaluated as diagnostic markers for canine leishmaniasis and proved to be independent in their antibody reactivity. Since sensitivity of serological test is usually higher in multiple-epitope format, in this study the relevant epitopes of K9, K26, and K39 antigens were joined by PCR strategy to produce the chimeric recombinant protein. The resulting mosaic antigen was found highly expressed in Escherichia coli and efficiently purified by affinity chromatography. Antigenic properties of this recombinant antigen were evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using a panel of human and dog sera previously characterized by parasitological and/or serological techniques. Chimeric ELISA showed 99% specificity in both human (n = 180) and canine (n = 343) control groups, while sensitivity was higher in canine VL (96%, n = 213) than in human VL (82%, n = 185). Accordingly, concordance between IFAT and canine chimeric ELISA (k = 0.95, 95% confidence interval = 0.93 to 0.98) was higher than between IFAT and human chimeric ELISA (k = 0.81, 95% confidence interval = 0.76 to 0.87). Results suggest the potential use of this new antigen for routine serodiagnosis of VL in both human and canine hosts.

Adult↗

Mucosal and systemic immune responses to chimeric fimbriae expressed by Salmonella enterica serovar typhimurium vaccine strains.

Recombinant live oral vaccines expressing pathogen-derived antigens offer a unique set of attractive properties. Among these are the simplicity of administration, the capacity to induce mucosal and systemic immunity, and the advantage of permitting genetic manipulation for optimal antigen presentation. In this study, the benefit of having a heterologous antigen expressed on the surface of a live vector rather than intracellularly was evaluated. Accordingly, the immune response of mice immunized with a Salmonella enterica serovar Typhimurium vaccine strain expressing the Escherichia coli 987P fimbrial antigen on its surface (Fas(+)) was compared with the expression in the periplasmic compartment (Fas(-)). Orally immunized BALB/c mice showed that 987P fimbriated Salmonella serovar Typhimurium CS3263 (aroA asd) with pCS151 (fas(+) asd(+)) elicited a significantly higher level of 987P-specific systemic immunoglobulin G (IgG) and mucosal IgA than serovar Typhimurium CS3263 with pCS152 (fasD mutant, asd(+)) expressing 987P periplasmic antigen. Further studies were aimed at determining whether the 987P fimbriae expressed by serovar Typhimurium chi4550 (cya crp asd) could be used as carriers of foreign epitopes. For this, the vaccine strain was genetically engineered to express chimeric fimbriae carrying the transmissible gastroenteritis virus (TGEV) C (379-388) and A (521-531) epitopes of the spike protein inserted into the 987P major fimbrial subunit FasA. BALB/c mice administered orally serovar Typhimurium chi4550 expressing the chimeric fimbriae from the tet promoter in pCS154 (fas(+) asd(+)) produced systemic antibodies against both fimbria and the TGEV C epitope but not against the TGEV A epitope. To improve the immunogenicity of the chimeric fimbriae, the in vivo inducible nirB promoter was inserted into pCS154, upstream of the fas genes, to create pCS155. In comparison with the previously used vaccine, BALB/c mice immunized orally with serovar Typhimurium chi4550/pCS155 demonstrated significantly higher levels of serum IgG and mucosal IgA against 987P fimbria. Moreover, mucosal IgA against the TGEV C epitope was only detected with serovar Typhimurium chi4550/pCS155. The induced antibodies also recognized the epitopes in the context of the full-length TGEV spike protein. Hence, immune responses to heterologous chimeric fimbriae on Salmonella vaccine vectors can be optimized by using promoters known to be activated in vivo.

Adhesins, Escherichia coli↗

Enhanced immunogenicity of a genetic chimeric protein consisting of two virulence antigens of Streptococcus mutans and protection against infection.

The saliva-binding region (SBR) of the cell surface antigen I/II (AgI/II) and the glucan-binding region (GLU) of the glucosyltransferase enzyme of Streptococcus mutans have been implicated in the initial adherence of S. mutans to saliva-coated tooth surfaces and the subsequent sucrose-dependent accumulation of S. mutans, respectively. Here, we describe the construction and characterization of a genetic chimeric protein consisting of the two virulence determinants SBR and GLU (SBR-GLU). The effectiveness of this construct in inducing mucosal and systemic immune responses to each virulence determinant following intranasal immunization was compared to that of each antigen alone or an equal mixture of SBR and GLU (SBR+GLU) in a mouse model. Furthermore, the ability of antibodies induced to SBR-GLU to protect against S. mutans infection was also investigated. Immunization of mice with the chimeric protein SBR-GLU resulted in significantly enhanced (P < 0.001) levels of serum immunoglobulin G (IgG) anti-SBR antibody activity compared to those in the SBR and SBR+GLU groups. The SBR-GLU-immunized mice also demonstrated a significant (P < 0.05) increase in salivary and vaginal IgA antibody responses to SBR and GLU. Analysis of the serum IgG subclass responses to SBR in mice immunized with SBR alone indicated a mixed IgG1 and IgG2a response. A preferential IgG1 response compared to an IgG2a anti-GLU response was induced in mice immunized with GLU alone. Similarly, a preferential IgG1 response was also induced to SBR when GLU was present in either a mixed or conjugated form. Finally, a significant reduction (P < 0.05) in S. mutans colonization was observed only in mice immunized with the SBR-GLU chimeric protein. Taken together, our results indicate that the chimeric protein SBR-GLU significantly enhanced mucosal immune responses to SBR and GLU and systemic immune responses to SBR. The ability of SBR-GLU to induce responses effective in protection against colonization of S. mutans suggests its potential as a vaccine antigen for dental caries.

Administration, Intranasal↗

Effects of chimeric mutants of human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex on nucleolar targeting signals.

Two chimeric mutant genes derived from rev of human immunodeficiency virus type 1 and rex of human T-cell leukemia virus type I were constructed to investigate the functions of the nucleolar-targeting signals (NOS) in Rev and Rex proteins. A chimeric Rex protein whose NOS region was substituted with the NOS of Rev was located predominantly in the cell nucleolus and functioned like the wild-type protein in the Rex assay system. However, a chimeric Rev with the NOS of Rex abolished Rev function despite its nucleolar localization. This nonfunctional nucleolar-targeting chimeric protein inhibited the function of both Rex and Rev. In the same experimental conditions, this mutant interfered with the localization of the functional Rex in the nucleolus.

Amino Acid Sequence↗

Human immunodeficiency virus type 1 clones chimeric for the envelope V3 domain differ in syncytium formation and replication capacity.

Chimeric human immunodeficiency virus type 1 (HIV-1) molecular clones differing only in the envelope V3 region were constructed. The V3 regions were derived from two HIV-1 isolates with a non-syncytium-inducing, non-T-cell-tropic phenotype and from four HIV-1 isolates with a syncytium-inducing, T-cell-tropic phenotype. When assayed in SupT1 cells, the two chimeric viruses with a V3 region derived from the non-syncytium-inducing isolates did not induce syncytia and showed a low level of replication. The four chimeric viruses with a V3 region derived from the syncytium-inducing isolates did induce syncytia and replicated efficiently in SupT1 cells. In A3.01 cells, which do not support syncytium formation, the V3 loop affected replication similarly. Upon prolonged culture in SupT1 cells, the phenotype of a non-syncytium-inducing, low-replicating chimeric HIV-1 converted into a syncytium-inducing, high-replicating phenotype. Mutations within the usually conserved GPGR tip of the loop, which were shown to be responsible for the conversion into the syncytium-inducing, high-replicating phenotype, had occurred. In vitro mutagenesis showed that coupled changes of amino acids at both sides of the tip of the V3 loop were able to convert the viral phenotype from non-syncytium-inducing, low replicating into syncytium inducing, high replicating. Our data show that the V3 loop is involved in both syncytium forming and replicative capacity of HIV-1.

Amino Acid Sequence↗

Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type 1.

Expression vectors based on DNA or plus-stranded RNA viruses are being developed as vaccine carriers directed against various pathogens. Less is known about the use of negative-stranded RNA viruses, whose genomes have been refractory to direct genetic manipulation. Using a recently described reverse genetics method, we investigated whether influenza virus is able to present antigenic structures from other infectious agents. We engineered a chimeric influenza virus which expresses a 12-amino-acid peptide derived from the V3 loop of gp120 of human immunodeficiency virus type 1 (HIV-1) MN. This peptide was inserted into the loop of antigenic site B of the influenza A/WSN/33 virus hemagglutinin (HA). The resulting chimeric virus was recognized by specific anti-V3 peptide antibodies and a human anti-gp120 monoclonal antibody in both hemagglutination inhibition and neutralization assays. Mice immunized with the chimeric influenza virus produced anti-HIV antibodies which were able to bind to synthetic V3 peptide, to precipitate gp120, and to neutralize MN virus in human T-cell culture system. In addition, the chimeric virus was also capable of inducing cytotoxic T cells which specifically recognize the HIV sequence. These results suggest that influenza virus can be used as an expression vector for inducing both B- and T-cell-mediated immunity against other infectious agents.

Amino Acid Sequence↗

Chimeric tick-borne encephalitis and dengue type 4 viruses: effects of mutations on neurovirulence in mice.

Two new chimeric flaviviruses were constructed from full-length cDNAs that contained tick-borne encephalitis virus (TBEV) CME or ME structural protein genes and the remaining genes derived from dengue type 4 virus (DEN4). Studies involving mice inoculated intracerebrally with the ME chimeric virus indicated that it retained the neurovirulence of its TBEV parent from which its pre-M and E genes were derived. However, unlike parental TBEV, the chimeric virus did not produce encephalitis when mice were inoculated peripherally, indicating a loss of neuroinvasiveness. In the present study, the ME chimeric virus (vME) was subjected to mutational analysis in an attempt to reduce or ablate neurovirulence measured by direct inoculation of virus into the brain. We identified three distinct mutations that were each associated independently with a significant reduction of mouse neurovirulence of vME. These mutations ablated (i) the TBEV pre-M cleavage site, (ii) the TBEV E glycosylation site, or (iii) the first DEN4 NS1 glycosylation site. In contrast, ablation of the second DEN4 NS1 glycosylation site or the TBE pre-M glycosylation site or amino acid substitution at two positions in the TBEV E protein increased neurovirulence. The only conserved feature of the three attenuated mutants was restriction of virus yield in both simian and mosquito cells. Following parenteral inoculation, these attenuated mutants induced complete resistance in mice to fatal encephalitis caused by the highly neurovirulent vME.

Amino Acid Sequence↗

Human immunodeficiency virus type 1 Vpu protein induces degradation of chimeric envelope glycoproteins bearing the cytoplasmic and anchor domains of CD4: role of the cytoplasmic domain in Vpu-induced degradation in the endoplasmic reticulum.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is a transmembrane phosphoprotein which induces rapid degradation of CD4 in the endoplasmic reticulum (ER). To identify sequences in CD4 for Vpu-induced degradation, we generated four chimeric envelope glycoproteins having the ectodomain of HIV-1 gp160, the anchor domain of CD4, and 38, 25, 24, and 18 amino acids (aa) of the CD4 cytoplasmic domain. Using the vaccinia virus-T7 RNA polymerase expression system, we analyzed the expression of chimeric proteins in the presence and absence of Vpu. In singly transfected cells, the chimeric envelope glycoproteins having 38, 24, and 18 aa of the CD4 cytoplasmic domain were endoproteolytically cleaved and biologically active in the fusion of HeLa CD4+ cells. However, one of the chimeras having 25 aa of the CD4 cytoplasmic tail was retained in the ER using the transmembrane ER retention signal and was defective in membrane fusion. Furthermore, biochemical analyses of the coexpressing cells revealed that the Vpu protein induced degradation of the envelope glycoproteins having 38, 25, and 24 aa of the CD4 cytoplasmic tail and degradation occurred in the ER. Consequently, the fusion-competent glycoproteins did not induce the formation of syncytia in HeLa CD4+ cells expressing Vpu. However, the HIV-1 gp160 and chimeric envelope glycoprotein having the membrane-proximal 18 aa of the CD4 cytoplasmic tail were stable and fusion competent in cells expressing Vpu. In addition, we examined the stability of CD4 molecules in the presence of Vpu. Coexpression analyses revealed that the Vpu protein induced degradation of CD4 whereas mutant CD4 having the membrane-proximal 18 aa of the cytoplasmic domain was relatively stable in the presence of Vpu. Taken together, these studies have elucidated that the Vpu protein requires sequences or sequence determinants in the cytoplasmic domain of CD4 to induce degradation of the glycoproteins in the cell.

Amino Acid Sequence↗

Human T-lymphotropic virus type 1 peptides in chimeric and multivalent constructs with promiscuous T-cell epitopes enhance immunogenicity and overcome genetic restriction.

Conventional strategies of viral peptide immunizations often elicit low-affinity antibody responses and have limited ability to elicit immune responses in outbred animals of diverse major histocompatibility (MHC) haplotypes. This genetically restricted T-cell-stimulatory activity of peptides is a serious obstacle to vaccine design. However, the use of promiscuous T-cell epitopes may circumvent this problem. Promiscuous T-cell epitopes from tetanus toxin (amino acids [aa] 580 to 599) and the measles virus F protein (aa 288 to 302) that bind to several isotypic and allotypic forms of human MHC class II molecules have been identified and have been used in highly immunogenic constructs to overcome haplotype-restricted immune responses. Chimeric and beta-template peptide constructs incorporating known human T-lymphotropic virus type 1 (HTLV-1) B- and T-cell epitopes from the surface envelope protein gp46 (SP2 [aa 86 to 107] and SP4a [aa 190 to 209]) and promiscuous T-cell peptides were synthesized, and their immunogenicities were evaluated in both rabbits and mouse strains of divergent haplotypes (C3H/HeJ [H-2k], C57BL/6 [H-2b], and BALB/c [H-2d]). In addition, peptide preparations were structurally characterized by analytical high-performance liquid chromatography, mass spectrometry, and circular dichroism. In contrast to their linear forms, the chimeric constructs of both the SP2 and SP4a epitopes displayed alpha-helical secondary structures. Immunogenicity of the peptide constructs was evaluated by direct and competitive enzyme-linked immunosorbant assay (ELISA), as well as by radioimmunoprecipitation, syncytium inhibition, and antigen-induced lymphocyte proliferation assays. Immunization with the SP4a peptide without conjugation to a carrier protein produced antibodies specific for SP4a in two mouse strains (C3H/HeJ and C57BL/6). However, BALB/c mice failed to respond to the peptide, indicating that the T-cell epitope of the SP4a sequence is MHC restricted. In contrast, the chimeric constructs MVF-SP2 and SP4a-measles virus F protein were highly immunogenic, producing elevated ELISA titers after only two immunizations. Elicited antibodies recognized native forms of gp46 in ELISAs and radioimmunoprecipitation assays, as well as inhibited HTLV-1-mediated syncytium formation. In addition, chimeric constructs were effective at induction of lymphocyte proliferation to the T-cell epitope, SP4a, in each strain of immunized mice. Our data demonstrate that the antibody response to retroviral peptides is enhanced by promiscuous peptide constructs, in part because of the ability of such constructs to promote appropriate secondary structural forms of viral epitopes. In addition, these constructs promote virus-specific helper T-cell responses, thereby overcoming genetically restricted immune responses to the synthetic peptides.

Amino Acid Sequence↗