Intratumoral delivery of vector mediated IL-2 in combination with vaccine results in enhanced T cell avidity and anti-tumor activity.

Systemic IL-2 is currently employed in the therapy of several tumor types, but at the price of often severe toxicities. Local vector mediated delivery of IL-2 at the tumor site may enhance local effector cell activity while reducing toxicity. To examine this, a model using CEA-transgenic mice bearing established CEA expressing tumors was employed. The vaccine regimen was a s.c. prime vaccination with recombinant vaccinia (rV) expressing transgenes for CEA and a triad of costimulatory molecules (TRICOM) followed by i.t. boosting with rF-CEA/TRICOM. The addition of intratumoral (i.t.) delivery of IL-2 via a recombinant fowlpox (rF) IL-2 vector greatly enhanced anti-tumor activity of a recombinant vaccine, resulting in complete tumor regression in 70-80% of mice. The anti-tumor activity was shown to be dependent on CD8(+) cells and NK1.1(+). Cellular immune assays revealed that the addition of rF-IL-2 to the vaccination therapy enhanced CEA-specific tetramer(+) cell numbers, cytokine release and CTL lysis of CEA(+) targets. Moreover, tumor-bearing mice vaccinated with the CEA/TRICOM displayed an antigen cascade, i.e., CD8(+) T cell responses to two other antigens expressed on the tumor and not the vaccine: wild-type p53 and endogenous retroviral antigen gp70. Mice receiving rF-IL-2 during vaccination demonstrated higher avidity CEA-specific, as well as higher avidity gp70-specific, CD8(+) T cells when compared with mice vaccinated without rF-IL-2. These studies demonstrate for the first time that the level and avidity of antigen specific CTL, as well as the therapeutic outcome can be improved with the use of i.t. rF-IL-2 with vaccine regimens.

Isolation of poxvirus from debilitating cutaneous lesions on four immature grackles (Quiscalus sp.).

Poxvirus was isolated from nodules on four immature grackles (Quiscalus sp.) collected in two residential areas of Victoria, Texas. All of the birds were emaciated and had nodules on the eyelids, bill, legs, toes, and areas of the skin on the wings, neck, and ventral abdomen. These pox nodules were extensive and probably interfered with both sight and flight. The preliminary diagnosis was confirmed by virus isolation, histopathology, and electron microscopy. Poxvirus was isolated on the chorioallantoic membrane of embryonated hen's eggs and in Muscovy duck embryo fibroblast cell culture. Phaenicia calliphoridae (blowfly) larvae were found in one of the pox nodules, raising the possibility of mechanical transmission of the virus by contaminated adult blowflies.

Comparative studies on the antigenicity of extra- and intracellular viruses of fowl pox.

The intra- and extracellular virus of three strains of fowl pox virus, when precipitated in succession with different saturation of ammonium sulphate revealed three antigens in gel diffusion test in the precipitates obtained at 25%, 50% and 75% of saturation. Further analysis of each positive antigen by dot ELISA revealed that the extracellular virus of FS-8 and HP 1 strains possessed excess antigenic protein at 50% saturation compared to their intracellular viruses. While no difference between extra- and intracellular viruses of FS-4 strain was observed.

Detection of fowlpox virus DNA by in situ hybridization using a biotinylated probe.

In situ hybridization was applied to detect fowlpox virus (FPV) DNA in formalin-fixed paraffin-embedded sections of the skin from infected chickens by using a biotinylated probe and a streptavidin-alkalinephosphatase conjugate. The immunohistochemical examination was applied to compare the distribution of the FPV DNA to that of related antigenic protein in serial sections. In the infected epithelial cells, FPV DNA was detected in cytoplasmic inclusion bodies and in the rest of cytoplasm. Likewise, immunohistochemical examination revealed the virus antigen in cytoplasm. Ultrastructurally, virions were observed in the cytoplasmic inclusion bodies, and immature virus particles were in the rest of the cytoplasm. The study proved restricted distribution of FPV DNA in the cytoplasm.

Pathologic changes in chickens caused by intravenous inoculation with fowlpox virus.

Twenty chickens were inoculated intravenously with fowlpox (FP) virus, and clinical and pathological examinations were carried out chronologically. Upon gross examination, miliary nodules scattered in the kidneys were observed from 10 to 18 days postinoculation (PI), as were papules on the skin and diphtheritic lesions on the mucous membrane of the upper respiratory tract. Microscopically, characteristic FP lesions, composed of swelling and proliferation of cells with formation of Bollinger bodies, were observed in the epithelial cells of renal tubules from 4 to 14 days PI and in the epithelial reticular cells of the thymic medulla from 4 to 10 days PI, as well as in the skin and mucous membrane. Immunofluorescent and electron microscopic observations confirmed the presence of viral antigen and virus particles in the characteristic lesions of FP.

Isolation of Erysipelothrix rhusiopathiae from a red-tailed hawk (Buteo jamaicensis) with a concurrent pox virus infection.

Erysipelothrix rhusiopathiae was isolated from the spleen, liver, lung, heart, kidney, and skin of a red-tailed hawk (Buteo jamaicensis) which had a concurrent avian pox virus infection. The hawk had been housed on a farm with domestic turkeys, providing a possible source of the E. rhusiopathiae.

Studies on pathogenesis of fowl pox: virological study.

One-month-old WLH chickens were inoculated with a field isolate of fowl pox virus (FPV) by intradermal (i.d.) and intratracheal (i.t.) routes. In intradermally infected chickens, the virus in titrable amounts was first detected in the skin at the inoculation site on day 2 and in lungs on day 4 followed by viraemia on the day 5 post-infection (p.i.). Subsequently the virus was recovered from liver, spleen, kidney and brain, but not from the heart. The chickens infected by i.t. route showed an almost similar outcome with minor differences as the virus was first demonstrated in the lungs on day 2 p.i., viraemia occurred on day 4 p.i. Initiation of pocks at the inoculation site in i.d. infected birds was observed on days 3 to 4 p.i., generalized cutaneous pock lesions appeared from 7 to 8 days p.i.

Morphogenesis of canary poxvirus and its entrance into inclusion bodies.

A virus isolated from a natural outbreak of canarypox was replicated on the chorioallantoic membranes of chicken embryos, and its ultrastructure and development were observed. Electron microscopy of thin sections of pocks produced on the chorioallantoic membranes revealed a variety of developmental forms which appear similar to those demonstrated in studies of vaccinia, ie, viroplasm or viral factories; immature, undifferentiated virions partially enclosed by membranes; completely enclosed nondifferentiated spherical or oval virions; immature virions with discrete nucleoids; and the more compact brick-shaped mature virions. Two types of A-type inclusions were noted: those with virions around the periphery, and those filled with virus particles. The appearance of mature viruses within the inclusion bodies and different stages of viruses outside the inclusion indicate that in a course of development, maturing poxvirus may enter the inclusion bodies as they acquire surface tubules on their envelopes. Mature virions also were seen budding out of the cell membrane, apparently enveloped in a portion of the membrane. Studies showing the entrance of poxvirus into inclusion bodies have not been reported. In this report, electron micrographs are shown of viruses entering inclusion bodies.

Psittacine pox virus: virus isolation and identification, transmission, and cross-challenge studies in parrots and chickens.

An avian pox virus was isolated from Amazon parrots dying with severe diphtheritic oral, esophageal, and crop lesions. The virus was propagated on chorioallantoic membranes (CAM) of 10-day-old chicken embryos, and a homogenate of the infected CAM was rubbed vigorously onto the conjunctiva, oral mucosa, and defeathered follicles of two healthy Amazon parrots and three conures. All experimental birds developed cutaneous and ocular pox lesions, and one parrot developed oral pox lesions. Specific-pathogen-free chicks inoculated with the virus isolate developed skin lesions identical to those of the parrots. Chickens vaccinated with fowl and pigeon pox vaccines and inoculated with the psittacine isolate developed lesions typical of avian pox. Chickens vaccinated with the psittacine virus were susceptible to fowl and pigeon pox virus infection. This pox virus isolate may thus be regarded as a potential pathogen for chickens.

Fowlpox virus in the Sudan.

A virulent fowlpox virus was isolated from a natural outbreak of the disease in the Sudan. The virus was used for experimental transmission of the disease to susceptible chickens. Scarification and intravenous methods were used to infect one-month-old chickens. Scarification induced typical pox lesions at 5-7 days postinoculation, whereas intravenous injection induced only swelling of combs or wattles, at 10-11 days postinoculation, although neutralizing antibodies against fowlpox virus were detected 2, 3, and 4 weeks postinoculation. The histopathological features of the pox lesions obtained in both natural and experimental infection were described.

Pox infection among captive peacocks.

An outbreak of avian pox was detected among captive peacocks (Pavo cristatus) at Baghdad Zoological Park during spring, 1978. A total of 45 of the 60 birds in the aviary developed pox lesions around the beaks and eyes. Morbidity was 75% and mortality was 13%. A virus isolated from the skin lesions produced large plaques on the chorioallantoic membrane of developing chicken embryos and induced cytopathic effect characteristic for pox viruses in chicken embryo cell cultures. The virus neither haemagglutinated nor haemadsorbed to chicken erythrocytes. It was ether resistant and chloroform sensitive. Chickens inoculated with the virus by scarification developed localized pox-like lesions, while turkeys had only transient swelling of feather follicles at the site of inoculation. Virus partially purified with Genetron 113 was precipitated by antisera to fowlpox and pigeon pox viruses.

Avian pox virus. An ultrastructural study on a cherrug falcon. Brief report.

The ultrastructure and maturation of avian pox particles is described in the rare case of a naturally infected hawk (Falco cherrug). As in other cells infected by fowlpox virus two types of inclusion bodies are encountered in the cytoplasm: firstly assemblies of fluffy filamentous material apparently giving rise to immature virions. They are thought to present virus factories (inclusion body B). Secondly mature virus particles budded into extensive groupings (Bollinger bodies, inclusion body A) which display a fine structure identical to other strains of fowlpox virus.

Expornitic of avian pox in a zoo.

During a 6-week period at the San Diego Zoo, avian pox occurred in 9 pheasants representing 5 species. Lesions were limited to facial skin and consisted of epithelial cell hyperplasia, secondary inflammatory changes, and intracytoplasmic eosinophilic inclusion bodies which, by electron microscopy, were shown to contain pox virus. The disease was self-limiting in 7 pheasants, but 2 pheasants died. Free-ranging Indian red junglefowl were implicated as the source of the infection.

Poxvirus infection in a spectacled Amazon parrot (Amazona albifrons).

Avian pox was determined to be the cause of death of a spectacled Amazon parrot (Amazona albifrons). Intracytoplasmic inclusion bodies were visualized by light microscopy in esophageal and bronchial epithelial lesions. Electron microscopy revealed pox-virus virions in the inclusions.

Rapid diagnosis of some avian virus diseases.

Direct electron microscopy was used to identify virions of infectious laryngotracheitis in lysed tracheal cells and of fowlpox in scabs and exudates from natural cases. Rapid identification of avian adenovirus and infectious laryngotracheitis by gel diffusion was possible using antigens prepared by simple distilled-water lysis of infected cell cultures. Precipitin lines were often visible within 5 hours.