Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide.

We describe a method to facilitate radioimaging with technetium-99m (99mTc) by genetic incorporation of a 99mTc chelation site in recombinant single-chain Fv (sFv) antibody proteins. This method relies on fusion of the sFv C terminus with a Gly4Cys peptide that specifically coordinates 99mTc. By using analogues of the 26-10 anti-digoxin sFv as our primary model, we find that addition of the chelate peptide, to form 26-10-1 sFv', does not alter the antigen-binding affinity of sFv. We have demonstrated nearly quantitative chelation of 0.5-50 mCi of 99mTc per mg of 26-10-1 sFv' (1 Ci = 37 GBq). These 99mTc-labeled sFv' complexes are highly stable to challenge with saline buffers, plasma, or diethylenetriaminepentaacetic acid. We find that the 99mTc-labeled 741F8-1 sFv', specific for the c-erbB-2 tumor-associated antigen, is effective in imaging human ovarian carcinoma in a scid mouse tumor xenograft model. This fusion chelate methodology should be applicable to diagnostic imaging with 99mTc and radioimmunotherapy with 186Re or 188Re, and its use could extend beyond the sFv' to other engineered antibodies, recombinant proteins, and synthetic peptides.

Upregulation of class I major histocompatibility complex antigens by interferon gamma is necessary for T-cell-mediated elimination of recombinant adenovirus-infected hepatocytes in vivo.

Recombinant adenoviruses are attractive vehicles for liver-directed gene therapy because of the high efficiency with which they transfer genes to hepatocytes in vivo. First generation recombinant adenoviruses deleted of E1 sequences also express recombinant and early and late viral genes, which lead to development of destructive cellular immune responses. Previous studies indicated that class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTLs) play a major role in eliminating virus-infected cells. The present studies utilize mouse models to evaluate the role of T-helper cells in the primary response to adenovirus-mediated gene transfer to the liver. In vivo ablation of CD4+ cells or interferon gamma (IFN-gamma) was sufficient to prevent the elimination of adenovirus-transduced hepatocytes, despite the induction of a measurable CTL response. Mobilization of an effective TH1 response as measured by in vitro proliferation assays was associated with substantial upregulation of MHC class I expression, an effect that was prevented in IFN-gamma-deficient animals. These results suggest that elimination of virus-infected hepatocytes in a primary exposure to recombinant adenovirus requires both induction of antigen-specific CTLs as well as sensitization of the target cell by TH1-mediated activation of MHC class I expression.

Plaque-associated expression of human herpesvirus 6 in multiple sclerosis.

Representational difference analysis was used to search for pathogens in multiple sclerosis brains. We detected a 341-nucleotide fragment that was 99.4% identical to the major DNA binding protein gene of human herpesvirus 6 (HHV-6). Examination of 86 brain specimens by PCR demonstrated that HHV-6 was present in > 70% of MS cases and controls and is thus a commensal virus of the human brain. By DNA sequencing, 36/37 viruses from MS cases and controls were typed as HHV-6 variant B group 2. Other herpesviruses, retroviruses, and measles virus were detected infrequently or not at all. HHV-6 expression was examined by immunocytochemistry with monoclonal antibodies against HHV-6 virion protein 101K and DNA binding protein p41. Nuclear staining of oligodendrocytes was observed in MS cases but not in controls, and in MS cases it was observed around plaques more frequently than in uninvolved white matter. MS cases showed prominent cytoplasmic staining of neurons in gray matter adjacent to plaques, although neurons expressing HHV-6 were also found in certain controls. Since destruction of oligodendrocytes is a hallmark of MS, these studies suggest an association of HHV-6 with the etiology or pathogenesis of MS.

Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells.

We developed a stringently regulated expression system for mammalian cells that uses (i) the RNA polymerase, phi 10 promoter, and T phi transcriptional terminator of bacteriophage T7; (ii) the lac repressor, lac operator, rho-independent transcriptional terminators and the gpt gene of Escherichia coli; (iii) the RNA translational enhancer of encephalomyocarditis virus; and (iv) the genetic background of vaccinia virus. In cells infected with the recombinant vaccinia virus, reporter beta-galactosidase synthesis was not detected in the absence of inducer. An induction of at least 10,000- to 20,000-fold occurred upon addition of isopropyl beta-D-thiogalactopyranoside or by temperature elevation from 30 to 37 degrees C using a temperature-sensitive lac repressor. Regulated synthesis of the secreted and highly glycosylated human immunodeficiency virus 1 envelope protein gp120 was also demonstrated. Yields of both proteins were approximately 2 mg per 10(8) cells in 24 hr. Plasmid transfer vectors for cloning and expression of complete or incomplete open reading frames in recombinant vaccinia viruses are described.

Mucosal and systemic immune responses to a recombinant protein expressed on the surface of the oral commensal bacterium Streptococcus gordonii after oral colonization.

To circumvent the need to engineer pathogenic microorganisms as live vaccine-delivery vehicles, a system was developed which allowed for the stable expression of a wide range of protein antigens on the surface of Gram-positive commensal bacteria. The human oral commensal Streptococcus gordonii was engineered to surface express a 204-amino acid allergen from hornet venom (Ag5.2) as a fusion with the anchor region of the M6 protein of Streptococcus pyogenes. The immunogenicity of the M6-Ag5.2 fusion protein was assessed in mice inoculated orally and intranasally with a single dose of recombinant bacteria, resulting in the colonization of the oral/pharyngeal mucosa for 10-11 weeks. A significant increase of Ag5.2-specific IgA with relation to the total IgA was detected in saliva and lung lavages when compared with mice colonized with wild-type S. gordonii. A systemic IgG response to Ag5.2 was also induced after oral colonization. Thus, recombinant Gram-positive commensal bacteria may be a safe and effective way of inducing a local and systemic immune response.

Directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries.

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

Minimal epitopes expressed in a recombinant polyepitope protein are processed and presented to CD8+ cytotoxic T cells: implications for vaccine design.

The epitopes recognized by CD8+ cytotoxic T lymphocytes (CTL) are generated from cytosolic proteins by proteolytic processing. The nature of the influences exerted by the sequences flanking CTL epitopes on these processing events remains controversial. Here we show that each epitope within an artificial polyepitope protein containing nine minimal CD8+ CTL epitopes in sequence was processed and presented to appropriate CTL clones. Natural flanking sequences were thus not required to direct class I proteolytic processing. In addition, unnatural flanking sequences containing other CTL epitopes did not interfere with processing. The ability of every CTL epitope to be effectively processed from a protein containing only CTL epitopes is likely to find application in the construction of recombinant polyepitope CTL vaccines.

Inflammation-induced recombinant protein expression in vivo using promoters from acute-phase protein genes.

We report that promoters for two murine acute-phase protein (APP) genes, complement factor 3 (C3) and serum amyloid A3 (SAA3), can increase recombinant protein expression in response to inflammatory stimuli in vivo. To deliver APP promoter-luciferase reporter gene constructs to the liver, where most endogenous APP synthesis occurs, we introduced them into a nonreplicating adenovirus vector and injected the purified viruses intravenously into mice. When compared with the low levels of basal luciferase expression observed prior to inflammatory challenge, markedly increased expression from the C3 promoter was detected in liver in response to both lipopolysaccharide (LPS) and turpentine, and lower-level inducible expression was also found in lung. In contrast, expression from the SAA3 promoter was found only in liver and was much more responsive to LPS than to turpentine. After LPS challenge, hepatic luciferase expression increased rapidly and in proportion to the LPS dose. Use of cytokine-inducible promoters in gene transfer vectors may make it possible to produce antiinflammatory proteins in vivo in direct relationship to the intensity and duration of an individual's inflammatory response. By providing endogenously controlled production of recombinant antiinflammatory proteins, this approach might limit the severity of the inflammatory response without interfering with the beneficial components of host defense and immunity.

Rapid RNA polymerase genetics: one-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase.

We present a simple, rapid procedure for reconstitution of Escherichia coli RNA polymerase holoenzyme (RNAP) from individual recombinant alpha, beta, beta', and sigma 70 subunits. Hexahistidine-tagged recombinant alpha subunit purified by batch-mode metal-ion-affinity chromatography is incubated with crude recombinant beta, beta', and sigma 70 subunits from inclusion bodies, and the resulting reconstituted recombinant RNAP is purified by batch-mode metal-ion-affinity chromatography. RNAP prepared by this procedure is indistinguishable from RNAP prepared by conventional methods with respect to subunit stoichiometry, alpha-DNA interaction, catabolite gene activator protein (CAP)-independent transcription, and CAP-dependent transcription. Experiments with alpha (1-235), an alpha subunit C-terminal deletion mutant, establish that the procedure is suitable for biochemical screening of subunit lethal mutants.

Recombinant vesicular stomatitis viruses from DNA.

We assembled a DNA clone containing the 11,161-nt sequence of the prototype rhabdovirus, vesicular stomatitis virus (VSV), such that it could be transcribed by the bacteriophage T7 RNA polymerase to yield a full-length positive-strand RNA complementary to the VSV genome. Expression of this RNA in cells also expressing the VSV nucleocapsid protein and the two VSV polymerase subunits resulted in production of VSV with the growth characteristics of wild-type VSV. Recovery of virus from DNA was verified by (i) the presence of two genetic tags generating restriction sites in DNA derived from the genome, (ii) direct sequencing of the genomic RNA of the recovered virus, and (iii) production of a VSV recombinant in which the glycoprotein was derived from a second serotype. The ability to generate VSV from DNA opens numerous possibilities for the genetic analysis of VSV replication. In addition, because VSV can be grown to very high titers and in large quantities with relative ease, it may be possible to genetically engineer recombinant VSVs displaying foreign antigens. Such modified viruses could be useful as vaccines conferring protection against other viruses.

Potent interleukin 3 receptor agonist with selectively enhanced hematopoietic activity relative to recombinant human interleukin 3.

A systematic evaluation of structure-activity information led to the construction of genetically engineered interleukin 3 (IL-3) receptor agonists (synthokines) with enhanced hematopoietic potency. SC-55494, the most extensively characterized member of this series, exhibits 10- to 20-fold greater biological activity than recombinant human IL-3 (rhIL-3) in human hematopoietic cell proliferation and marrow colony-forming-unit assays. In contrast, SC-55494 is only twice as active as rhIL-3 in priming the synthesis of inflammatory mediators such as leukotriene C4 and triggering the release of histamine from peripheral blood leukocytes. The enhanced hematopoietic activity of SC-55494 correlates with a 60-fold increase in IL-3 alpha-subunit binding affinity and a 20-fold greater affinity for binding to alpha/beta receptor complexes on intact cells relative to rhIL-3. SC-55494 demonstrates a 5- to 10-fold enhanced hematopoietic response relative to its ability to activate the priming and release of inflammatory mediators. Therefore, SC-55494 may ameliorate the myeloablation of cancer therapeutic regimens while minimizing dose-limiting inflammatory side effects.

Recombinant Listeria monocytogenes as a live vaccine vehicle for the induction of protective anti-viral cell-mediated immunity.

Listeria monocytogenes (LM) is a Gram-positive bacterium that is able to enter host cells, escape from the endocytic vesicle, multiply within the cytoplasm, and spread directly from cell to cell without encountering the extracellular milieu. The ability of LM to gain access to the host cell cytosol allows proteins secreted by the bacterium to efficiently enter the pathway for major histocompatibility complex class I antigen processing and presentation. We have established a genetic system for expression and secretion of foreign antigens by recombinant strains, based on stable site-specific integration of expression cassettes into the LM genome. The ability of LM recombinants to induce protective immunity against a heterologous pathogen was demonstrated with lymphocytic choriomeningitis virus (LCMV). LM strains expressing the entire LCMV nucleoprotein or an H-2Ld-restricted nucleoprotein epitope (aa 118-126) were constructed. Immunization of mice with LM vaccine strains conferred protection against challenge with virulent strains of LCMV that otherwise establish chronic infection in naive adult mice. In vivo depletion of CD8+ T cells from vaccinated mice abrogated their ability to clear viral infection, showing that protective anti-viral immunity was due to CD8+ T cells.

Endometrial response toward bovine herpesvirus 4 infection

Metriti ed endometriti sono le patologie maggiormente responsabili delle perdite economiche negli allevamenti bovini da latte, specialmente nel periodo successivo al parto. Mentre le metriti coinvolgono e si sviluppano in tutto l’utero e sono caratterizzate dalla presenza di sintomi sistemici, le endometriti consistono in una infiammazione che riguarda il solo endometrio, con la presenza di perdite purulente, distruzione della superficie epiteliale, congestione vascolare, edema stromale ed accumulo di linfociti e plasmacellule. Queste patologie, inoltre, possono causare, disfunzione ovarica, con conseguente infertilità e riduzione sia dell’efficienza riproduttiva della vacca sia della produzione stessa di latte. Nonostante queste malattie siano, nella maggior parte dei casi, correlate all’instaurarsi di infezioni batteriche, che possono subentrare nell’utero direttamente durante il parto, il ruolo di alcuni virus nello sviluppo di queste patologie è stato recentemente approfondito e la correlazione tra l’ Herpesvirus Bovino 4 e l’insorgere di metriti ed endometriti è stata dimostrata. L’ Herpesvirus Bovino 4 (BoHV-4) è un gamma-herpesvirus ed il suo genoma è costituito da una molecola lineare di DNA a doppio filamento con una struttura genomica di tipo B, caratterizzata dalla presenza di un’unica lunga sequenza centrale (LUR) fiancheggiata da multiple sequenze poli-ripetute (prDNA). BoHV-4 è stato isolato sia da animali sani sia da animali con differenti patologie, tra cui malattie oculari e respiratorie, ma soprattutto da casi di metriti, endometriti, vaginiti o aborti. Generalmente, il ruolo svolto dal virus in questo tipo di patologie è associato alla compresenza di altri tipi di patogeni, che possono essere virus, come nel caso del Virus Della Diarrea Virale Bovina (BVDV), o più frequentemente batteri. Usualmente, l’iniziale difesa dell’endometrio bovino nei confronti dei microbi si fonda sul sistema immunitario innato e l’attivazione di specifici recettori cellulari determina la sintesi e la produzione di citochine e chemochine pro infiammatorie, che possono essere in grado di modulare la replicazione di BoHV-4. Il genoma di BoHV-4 possiede due principali trascritti per i geni Immediate Early (IE), trai quali ORF50/IE2 è il più importante ed il suo prodotto, Rta/ORF50, è fortemente conservato tra tutti gli Herpesvirus. Esso è responsabile della diretta trans-attivazione di numerosi geni virali e cellulari e può essere modulato da differenti stimoli extracellulari. Precedentemente è stato dimostrato come il TNF-, prodotto dalle cellule stromali e dai macrofagi all’interno dell’endometrio, in conseguenza ad infezione batterica, sia in grado di aumentare la replicazione di BoHV-4 attraverso l’attivazione del pathway di NFkB e direttamente agendo sul promotore di IE2. Per queste ragioni, è risultato di forte interesse investigare quali potessero essere, invece, i fattori limitanti la replicazione di BoHV-4. In questo lavoro è stata studiata la relazione tra cellule endometriali stromali bovine infettate con l’Herpesvirus Bovino 4 e l’interferon gamma (IFN-) ed è stata dimostrata la capacità di questa molecola di restringere la replicazione di BoHV-4 in maniera IDO1 indipendente ed IE2 dipendente. Inoltre, la presenza di alcuni elementi in grado di interagire con l’ IFN-γ, all’interno del promotore di IE2 di BoHV-4, ha confermato questa ipotesi. Basandoci su questi dati, abbiamo potuto supporre l’esistenza di uno stretto vincolo tra l’attivazione dell’asse dell’interferon gamma e la ridotta replicazione di BoHV-4, andando a porre le basi per una nuova efficiente cura e prevenzione per le patologie uterine. Poiché il meccanismo corretto attraverso il quale BoHV-4 infetta l’endometrio bovino non è ancora ben compreso, è stato interessante approfondire in maniera più accurata l’interazione presente tra il virus ed il substrato endometriale, analizzando le differenze esistenti tra cellule infettate e non, in termini di espressione genica. Basandoci su dati preliminari ottenuti attraverso analisi con RNA sequencing (RNAseq), abbiamo visto come numerosi geni risultino over-espressi in seguito ad infezione con BoHV-4 e come, tra questi, la Metalloproteasi 1 sia uno dei più interessanti, a causa delle sue possibili implicazioni nello sviluppo delle patologie dell’endometrio uterino bovino. Successive analisi, effettuate tramite westernblotting e real time PCR, sono state in grado di confermare tale dato, sottolineando l’efficacia di un nuovo approccio sperimentale, basato sul RNAseq, per lo studio dell’insorgenza delle patologie.

Detecção dos herpesvirus humanos na mucosa oral de pacientes irradiados para tratamento de carcinoma epidermoide em região de cabeça e pescoço

A radioterapia para tratamento das neoplasias malignas em região de cabeça e pescoço é acompanhada de diversas complicações, decorrentes do comprometimento dos tecidos radiossensíveis localizados próximos ao tumor. Entre essas complicações a mucosite é a que merece maior destaque. A mucosite é uma reação tóxica inflamatória da mucosa oral causada pelo tratamento citorredutivo induzido pela radioterapia (RT) ou pela quimioterapia (QT). Ela manifesta-se com sinais de edema, eritema, úlcera e formação pseudomembrana, resultando em sintomas de ardência, que pode progredir para dor intensa e consequente prejuízo na alimentação e comunicação verbal. Infecções bacterianas, fúngicas ou virais podem acometer a mucosa bucal irradiada e exacerbar a manifestação da mucosite oral por meio da ativação de fatores de transcrição da resposta inflamatória. Existem poucos dados na literatura sobre a participação dos herpesvirus humanos na mucosite oral induzida pela radioterapia. A proposta desse trabalho foi avaliar a excreção oral dos herpesvirus humanos (HSV-1, HSV-2, EBV, CMV, VZV, HHV6, HHV7 e HHV8) e sua possível associação com o desenvolvimento e agravamento da mucosite oral, em pacientes diagnosticados com carcinoma epidermoide (CEC) de boca e orofaringe, submetidos à radioterapia associado à quimioterapia. Nesse estudo foram analisadas 158 amostras de lavado bucal, de 20 pacientes, submetidos à radioterapia para CEC em região de cabeça e pescoço, coletadas semanalmente, durante todo o tratamento. Foi realizada a extração do DNA dessas amostras e em seguida sua amplificação através da PCR utilizando dois conjuntos de primers: HSVP1/P2 para os subtipos HSV-1, HSV-2, EBV, CMV e HHV-8 e o VZVP1/P2 para os subtipos VZV, HHV-6 e HHV-7. As amostras positivas foram submetidas à digestão enzimática com enzimas de restrição BamHI e BstUI para determinação específica de cada um dos oito herpesvirus. Foi também avaliada clinicamente, a mucosite oral, em cada uma das coletas, seguindo os critérios da OMS e NCIC. As análises da amostra mostraram a excreção do EBV, HHV-6 e HHV-7, em todas as semanas de tratamento radioterápico, enquanto que a excreção do HSV1 não pode ser observada no momento da triagem. Considerando-se todos os períodos em conjunto (Triagem, semanas de radioterapia e Controle), a maior frequência foi de pacientes que excretaram EBV (55,0%), seguida daqueles que excretaram HHV-7 (20,5%). A frequência de excreção de EBV foi significativamente maior do que a dos demais vírus (Teste ?2, p<0.001 para todos os cruzamentos). A frequência de excreção de HHV-7 foi significativamente maior do que a de HSV-1 (5,9%) e HHV-6 (5,5%) (Teste ?2, p=0.001 para ambos os cruzamentos). Não houve diferenças estatísticas significantes entre as frequências de HSV-1 e HHV-6. Como conclusão, verificou-se uma correlação positiva entre a excreção oral do EBV e a presença de mucosite induzida pela associação de radioterapia e quimioterapia com graus >=2, sobretudo se considerarmos as três últimas semanas de radioterapia, período este em que a severidade da mucosite foi estatisticamente maior. Esses achados nos possibilitam inferir que o ambiente inflamatório local de mucosites com grau >=2 seja mais favorável para excreção oral do EBV.