Intravenous administration of retroviral replicating vector, Toca 511, demonstrates therapeutic efficacy in orthotopic immune-competent mouse glioma model.

Toca 511 (vocimagene amiretrorepvec), a nonlytic, amphotropic retroviral replicating vector (RRV), encodes and delivers a functionally optimized yeast cytosine deaminase (CD) gene to tumors. In orthotopic glioma models treated with Toca 511 and 5-fluorocytosine (5-FC) the CD enzyme within infected cells converts 5-FC to 5-fluorouracil (5-FU), resulting in tumor killing. Toca 511, delivered locally either by intratumoral injection or by injection into the resection bed, in combination with subsequent oral extended-release 5-FC (Toca FC), is under clinical investigation in patients with recurrent high-grade glioma (HGG). If feasible, intravenous administration of vectors is less invasive, can easily be repeated if desired, and may be applicable to other tumor types. Here, we present preclinical data that support the development of an intravenous administration protocol. First we show that intravenous administration of Toca 511 in a preclinical model did not lead to widespread or uncontrolled replication of the RVV. No, or low, viral DNA was found in the blood and most of the tissues examined 180 days after Toca 511 administration. We also show that RRV administered intravenously leads to efficient infection and spread of the vector carrying the green fluorescent protein (GFP)-encoding gene (Toca GFP) through tumors in both immune-competent and immune-compromised animal models. However, initial vector localization within the tumor appeared to depend on the mode of administration. Long-term survival was observed in immune-competent mice when Toca 511 was administered intravenously or intracranially in combination with 5-FC treatment, and this combination was well tolerated in the preclinical models. Enhanced survival could also be achieved in animals with preexisting immune response to vector, supporting the potential for repeated administration. On the basis of these and other supporting data, a clinical trial investigating intravenous administration of Toca 511 in patients with recurrent HGG is currently open and enrolling.

Induction of long-term protective antiviral endogenous immune response by short neutralizing monoclonal antibody treatment.

Long-term immune control of viral replication still remains a major challenge in retroviral diseases. Several monoclonal antibodies (MAbs) have already shown antiviral activities in vivo, including in the clinic but their effects on the immune system of treated individuals are essentially unknown. Using the lethal neurodegeneration induced in mice upon infection of neonates by the FrCas(E) retrovirus as a model, we report here that transient treatment by a neutralizing MAb shortly after infection can, after an immediate antiviral effect, favor the development of a strong protective host immune response containing viral propagation long after the MAb has disappeared. In vitro virus neutralization- and complement-mediated cell lysis assays, as well as in vivo viral challenges and serum transfer experiments, indicate a clear and essential contribution of the humoral response to antiviral protection. Our observation may have important therapeutic consequences as it suggests that short antibody-based therapies early after infection should be considered, at least in the case of maternally infected infants, as adjunctive treatment strategies against human immunodeficiency virus, not only for a direct effect on the viral load but also for favoring the emergence of an endogenous antiviral immune response.

Human airway epithelial cells present antigen to influenza virus-specific CD8+ CTL inefficiently after incubation with viral protein together with ISCOMATRIX.

In the present paper, an in vitro model was established in which the interaction between influenza virus-specific CD8+ T cells and human airway epithelial cells can be studied. To this end, the human lung epithelial cell line A549 was transduced with the HLA-A*0201 gene. This MHC class I allele is involved in the presentation of the immunodominant M158-66 cytotoxic T lymphocyte (CTL) epitope of the influenza A virus matrix protein. The A549-HLA-A2 cells and a CD8+ T cell clone specific for the M158-66 epitope were used to evaluate ISCOMATRIX (IMX), which is considered a potential mucosal adjuvant for influenza vaccines, for its capacity to activate virus-specific CTL after incubation with epithelial cells. It was found that virus infected epithelial cells activated virus-specific CTL efficiently. However, incubation of epithelial cells with ISCOMATRIX and recombinant M1 protein activated CD8+ T cells inefficiently, unlike the incubation of C1R cells expressing a HLA-A2 trans gene or HLA-A2+ B-lymphoblastoid cells with these reagents. It was concluded that this lack of antigen presentation by epithelial cells indicate that these cells are not subject to killing by virus-specific CTL upon instillation with ISCOMATRIX-based vaccines, which may be a favorable property of mucosal vaccines.

KIR2DL4 is an IL-2-regulated NK cell receptor that exhibits limited expression in humans but triggers strong IFN-gamma production.

Killer cell Ig-like receptor (KIR)2DL4 (2DL4, CD158d) was previously described as the only KIR expressed by every human NK cell. It is also structurally atypical among KIRs because it possesses a basic transmembrane residue, which is characteristic of many activating receptors, but also contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). We expressed epitope-tagged 2DL4 in an NK-like cell line to study receptor function. Three distinct 2DL4 cDNA clones were analyzed: one encoding the "conventional" 2DL4 with the cytoplasmic ITIM (2DL4.1) and two encoding different cytoplasmic truncated forms lacking the ITIM (2DL4.2 and 2DL4(*)). Surprisingly, one truncated receptor (2DL4.2), which is the product of a prevalent human 2DL4 allele, was not expressed on the cell surface, indicating that some individuals may lack functional 2DL4 protein expression. Conversely, both 2DL4.1 and 2DL4(*) were expressed on the cell surface and up-regulated by IL-2. Analysis of primary NK cells with anti-2DL4 mAb confirmed the lack of surface expression in a donor with the 2DL4.2 genotype. Donors with the 2DL4.1 genotype occasionally expressed receptor only on CD56(high) NK cells, although their expression was up-regulated by IL-2. Interestingly, Ab engagement of epitope-tagged 2DL4 triggered rapid and robust IFN-gamma production, but weak redirected cytotoxicity in an NK-like cell line, which was the opposite pattern to that observed upon engagement of another NK cell activating receptor, NKp44. Importantly, both 2DL4.1 and 2DL4(*) exhibited similar activation potential, indicating that the ITIM does not influence 2DL4.1 activating function. The unique activation properties of 2DL4 suggest linkage to a distinct signaling pathway.

Inhibition of retroviral RNA production by ZAP, a CCCH-type zinc finger protein.

Cells have evolved multiple mechanisms to inhibit viral replication. To identify previously unknown antiviral activities, we screened mammalian complementary DNA (cDNA) libraries for genes that prevent infection by a genetically marked retrovirus. Virus-resistant cells were selected from pools of transduced clones, and an active antiviral cDNA was recovered. The gene encodes a CCCH-type zinc finger protein designated ZAP. Expression of the gene caused a profound and specific loss of viral messenger RNAs (mRNAs) from the cytoplasm without affecting the levels of nuclear mRNAs. The finding suggests the existence of a previously unknown machinery for the inhibition of virus replication, targeting a step in viral gene expression.

The role of adjuvants in retroviral vaccines.

The global HIV epidemic continues unchecked. Reports to the World Health Organization's Global Programme on AIDS indicate that more than 14 million persons have become infected with HIV and more than two million have died with AIDS. The spread of AIDS has generated a worldwide mandate for the development of safe and effective vaccines against HIV. Vaccines have been the most effective defense against other viral diseases such as polio and smallpox. However, the development of a vaccine against HIV-1 is a formidable task due to the variation of the virus, inadequate animal models of HIV disease, and the lack of correlates of protective immunity. Several candidate HIV vaccines are composed of synthetic, recombinant, or highly purified subunit antigens. Vaccines composed of subunit antigens generally are considered to be safer than traditional whole-killed or live-attenuated vaccines. However, purified subunit vaccines often are inherently less immunogenic than traditional vaccines. Immunologic adjuvants are agents that act generally to enhance specific immune responses to vaccine antigens. Formulation of experimental HIV vaccines with potent immunologic adjuvants is an attractive approach for amplifying and directing immune responses to highly purified antigens. Alum adjuvants, consisting of aluminum salts, first described in the 1920s, remain the only adjuvants in U.S.-licensed vaccine formulations. Novel adjuvants now undergoing preclinical and clinical testing with experimental subunit vaccine include detoxified lipid A, adjuvant emulsions, liposomes, biodegradable microspheres, muramyl peptides, and saponins. Adjuvants have been shown to elicit cytotoxic T-cell responses as well as antibody to subunit antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection of colostral antibodies against bovine leukemia virus infection in calves on a California dairy.

A three-year prospective study involving 244 calves was undertaken on a California dairy to evaluate the protective role of colostral antibodies against bovine leukemia virus (BLV) infection in calves. Calves were followed from birth to the time they left their individual hutch (TLIH), at about 90 days of age. The probability of being infected at TLIH and the daily risk of infection between birth and TLIH were modelled using the logistic and the Cox models, respectively. Calves with no detectable antibodies during the first week of life were up to 2.00 and 2.75 times more likely to be infected at TLIH compared to calves with low and high concentrations of antibodies during the first week of life, respectively (p = 0.01). When the daily risk was modelled, calves without antibodies at the estimated day of infection were up to 3.4 and 11.6 times more likely to become infected than calves with low and high concentrations of antibodies on that day, respectively (p less than 0.001). Results indicated that calfhood infection may be reduced by about 45% through the feeding of colostrum with BLV antibodies. Further reduction in infection may be possible by feeding calves milk powder, milk replacer, and/or milk from noninfected cows. Results also indicated that quantification of the effect of a time-dependent risk factor, such as colostral antibody concentration, might be affected if treated as a fixed factor.

[Bovine leukemia virus antibody levels in the blood and mammary gland secretion in cows in the perinatal period]. / Ksztaltowanie sie miana przeciwcial dla wirusa enzootycznej bialaczki bydla (ebb) w surowicy krwi i wydzielinie gruczoLu mlekowego krów w okresie okoloporodowym.

The titer of BLV-antibodies was estimated in the mammary gland secretion of 18 cows, naturally infected with BLV. Mammary gland secretion samples were collected every week since the 8th week ante partum and every day during the first week post partum. At the same time, blood samples were collected. The examination showed a marked decrease of antibody titer in the blood serum since the 5th week ante partum to the 2nd day post partum. Negative serological results were noticed temporary in the blood serum. The results indicate that serological examination of mammary gland secretion (dry secretion, colostrum) may be helpful because of high concentration of antibodies in the these secretions.

The effect of colostrum-derived antibody on neo-natal transmission of caprine arthritis-encephalitis virus infection.

Two groups of 6 newborn goat kids were artificially fed colostrum containing antibody to caprine arthritis-encephalitis (CAE) virus, obtained from clinically affected does. Kids in group A were fed the colostrum from birth until 7 days of age, while kids in group B were fed colostrum from 1 to 3 days after birth for 7 days. Kids were fed cow's milk at all other times. Serum antibody resulting from the consumption of colostrum, detected by agar gel immunodiffusion (AGID) tests, lasted for up to 8 weeks in group A, but none was detected in group B. Four kids from each group became infected with CAE virus as demonstrated by the emergence of active immunity and by virus isolation procedures. It appeared that uptake of colostral antibody by group A did not prevent viral transmission, interfere with development of active immunity, or modify the outcome of the CAE virus infection.

Transmission of bovine leukaemia virus in milk.

Nineteen calves born to dams free of bovine leukaemia virus (BLV) did not possess maternally derived precipitating antibody to BLV in their sera after the ingestion of colostrum. Eight of these calves remained serologically negative after being fed milk from BLV-free cows while three (27.3%) of 11 similar calves that had been fed milk from BLV-infected cows developed antibody. Forty-four of 47 calves born to BLV-infected dams acquired maternal antibody to BLV after ingesting colostrum. Two (8.7%) of the 23 calves fed milk from BLV-free cows developed antibody to BLV probably as a result of transplacental or colostrum infection whereas four (16.7%) of the 24 calves fed milk from BLV-infected cows developed antibody. It is concluded that milk transmission of BLV is responsible in part for the high rates of infection encountered in our dairy herds and that calves lacking specific maternal antibody are more susceptible to BLV infection through the ingestion of milk than are calves with maternal antibody.

Duration of colostral antibodies to bovine leukemia virus by two serologic tests.

The duration of detectable colostral antibodies to the glycoprotein antigen of bovine leukemia virus was studied in calves which were born to bovine leukemia virus-infected cows, but showed no serologic evidence of prenatal infection. Colostral antibodies detectable by an agar-gel immunodiffusion test (AGIT) persisted for less than 1 month to 6 months (mean 2.9 months) in the 139 calves examined. Colostral antibodies were detectable 1 to 5 months longer by radioimmunoprecipitation assay than by the AGIT in 22 of the 24 calves studied comparatively. The mean duration of colostral antibodies in those 24 calves was 3.8 months (min-max, 2 to 6 months) for the AGIT and 6.0 months (min-max, 4 to 9 months) for the radioimmunoprecipitation assay.

Decay of colostral antibodies to bovine leukemia virus with application to detection of calfhood infection.

An estimated weighted-regression method was used to describe the decay of colostral bovine leukemia virus (BLV) antibodies in the calf, as measured by agar-gel immunodiffusion with glycoprotein antigen. The prediction equation, based on 473 observations from 130 animals, was log10 inverse titer = 1.29 -0.012 age (days). The half-life of BLV antibodies was estimated to be 25.8 days. Ages at which colostral antibodies were last detected were between 51 and 187 days. Normal limits of antibody decay were estimated and used to identify virus-induced active antibodies in calves during the colostral antibody period. Calves known to be infected were identified between 2 and 180 days of age, using 95% limits. Application of this procedure for the early serologic detection of BLV-infected calves in eradication or control programs is discussed.

Eradication of bovine leukemia virus infection from a high-prevalence herd, using radioimmunoassay for identification of infected animals.

Radioimmunoassay (RIA), using the virion glycoprotein antigen, was applied in an attempt to eradicate bovine leukemia virus (BLV) infection from a herd in which virtually all the adult cattle are infected. Considering that most calves born to BLV-infected cows are negative for BLV at birth and remain negative for the first several months of life, the eradication program was based on the identification and isolation of the BLV-free calves born to infected cows. Twenty-five calves raised on colostrum and milk from their infected dams were classified as BLV-free on the basis of negative results in the RIA at 6 to 8 and 9 to 11 months of age. These animals were maintained in either complete (10 calves) or partial (15 calves) isolation from infected cattle and were examined at regular intervals for BLV and BLV antibodies. With the exception of 1 calf in the group raised in partial isolation, the animals have remained free of BLV up to the time of the last evaluation, when they were 32 to 35 months old. At these ages, more than 90% of the nonisolated cattle in the herd are BLV-positive. The data also show that this eradication trial would have failed if, in the initial procedure used to classify the calves as BLV-free, the agar gel immunodiffusion test instead of the RIA had been used. Inasmuch as the 25 calves in this study were fed colostrum and milk from their dams, the fact that only 1 of the calves became infected during the 26 to 29 months of observation provides further evidence that milk-borne transmission of BLV is infrequent and perhaps inconsequential.

Effect of colostral antibody on bovine leukemia virus infection of neonatal calves.

Pairs of newborn calves were exposed to bovine leukemia virus (BLV) when they were given their 1st colostrum feeding. Calves that were given 10(6) BLV-infected lymphocytes in colostrum free of BLV-specific antibody became infected. Calves that were fed 10(7), 10(8), or 10(9) infected lymphocytes in colostrum that contained BLV-specific antibody did not become infected. One of 2 calves inoculated intradermally with 250,000 infected lymphocytes was protected by colostral antibody, but the other was not. Colostral antibody titers in the unprotected calf decreased normally until the calf was 4 months old and then increased markedly; this pattern indicates that the presence of colostral antibody may have prolonged the latent period of the BLV infection.

Biological characterization of the virus causing leukoencephalitis and arthritis in goats.

This study describes the biological properties of a strain of virus isolated from tissues of a goat with leukoencephalomyelitis-arthritis. The agent is a retrovirus, having a virion-associated reverse transcriptase enzyme and an antigenic determinant(s) which cross-reacts with the p30 of visna-maedi viruses. Morphogenesis of the virus is also similar to visna virus in terms of virus assembly and the multinucleated giant cell formation which accompanies replication of the latter virus. Despite its cytopathogenic property the goat agent was not lytic in goat cell culture, causing instead a productive infection which persisted through multiple subcultures of the cells. The virus replicated incompletely in sheep cell cultures but could be rescued from the latter, weeks after inoculation, by co-cultivation with goat cells. Our data suggest that this strain of goat leukoencephalitis virus is a variant of the ovine retroviruses with a host range limited to the goat.

Results of an epidemiological survey of enzootic bovine leukosis in the northern part of Lower Saxony and a preliminary communication of an examination into relationship between BLV-antibody development and calving.

The Agar-Gel-Immunodiffusion-Test as proposed by EEC has been carried out during the last 2 1/2 years within a defined test area of Lower Saxony. About 500 leukosis-infected herds with a total of about 20.000 head of cattle have been tested five times now within three to six months sequences. The AGIDT has proved to be an easy and very practical test for routine diagnosis in large scale surveys. Our results described again show the superiority of the glykoprotein-antigen over the ether-treated p24-antigen in the AGIDT. Slaughter of AGIDT-reactors within this population led to a fast decrease of leukosis-infection as detected by the test. Epidemiological herd-data show, that spreading of infection within a herd depends largely on direct contact between infected and noninfected animals. Moreover our data give rise to the suspicion that the manipulations during blood-sampling seem to be implicated in the spread of the disease within a herd. Possible reasons for inconsistencies of antibody-titer in infected animals are discussed.