Preclinical characterization of a recombinant adeno-associated virus type 1-pseudotyped vector demonstrates dose-dependent injection site inflammation and dissemination of vector genomes to distant sites.

To translate the potential advantages of recombinant adeno-associated virus type 1 (rAAV1) vectors into a clinical application for muscle-directed gene therapy for alpha1 -antitrypsin (AAT) deficiency, we performed safety studies in 170 C57BL/6 mice and 26 New Zealand White rabbits. A mouse toxicology study included 8 cohorts of 10 mice each (5 per sex). Mice were killed either 21 or 90 days after intramuscular injection of doses ranging up to 1x10(13)vector genomes (VG), equivalent to 4 x 10(14)VG/kg. A mouse biodistribution study was performed in 5 cohorts of 10 mice, receiving intramuscular injections at the same doses; as well as in a lower dose cohort (3 x 10(8) VG; equivalent to 1.2 x 10(10)VG/kg); and in 4 other cohorts (excluding the vehicle control) injected with identical doses intravenously. Finally, biodistribution was examined in rabbits, with serial collection of blood and semen, as well as terminal tissue collection. Two significant findings were present, both of which were dose dependent. First, inflammatory cell infiltrates were detected at the site of injection 21, 60, or 90 days after intramuscular injection of 1 x 10(13)VG. This was not associated with loss of transgene expression. Second, vector DNA sequences were detected in most animals, levels being highest with the highest doses and earliest time points. Vector DNA was also present in liver, spleen, kidneys, and a number of other organs, including the gonads of animals receiving the highest dose. Likewise, vector DNA was present in the semen of male rabbits at higher doses. The copy number of vector DNA in the blood and semen declined over time throughout the study. These two dose-dependent findings have served to guide to the design of a phase 1 human trial of rAAV1-AAT.

Preclinical safety and biodistribution of adenovirus-based cancer vaccines after intradermal delivery.

The recombinant adenoviral (Ad) vector is being considered as a cancer vaccine platform because it efficiently induces immune responses to tumor antigens by intradermal immunization. The aims of this study were to evaluate the potential toxicities and biodistribution after a single dose or six weekly intradermal doses of Ad2/gp100v2 and Ad2/MART-1v2, which encode tumor-associated antigens gp100 and MelanA/MART-1, respectively. The only dose-related toxicities associated with intradermal administration of these Ad vectors were inflammatory cell infiltrates in the draining lymph nodes and injection sites that persisted 83 days after administration. The biodistribution of Ad DNA as detected by real-time polymerase chain reaction was largely confined to the injection sites and draining lymph nodes of mice treated with either a single dose or multiple doses of Ad vector and in the spleens of mice treated with multiple doses of Ad vector. Adenoviral DNA was transiently detected in the bone marrow, lung, or blood of only one animal for each site and was below the limit of assay quantification (<10 copies/microg DNA). The vector persisted in the skin and lymph nodes as long as 92 days after the last dose. We conclude that Ad vectors delivered by intradermal administration provide a safe, genetic vaccine delivery platform that induces desirable immune responses at the immunization sites and the lymph nodes that, ultimately, result in immune responses specific to the tumor antigens.

Preclinical safety assessment of a DNA vaccine using particle-mediated epidermal delivery in domestic pig, minipig and mouse.

DNA vaccination involves the direct injection of genes coding for specific antigenic proteins. One technique known as particle-mediated epidermal delivery (PMED) is a practical approach for epidermal delivery and provides a strong immune response. An important aspect of the preclinical safety assessment of DNA vaccines is the selection of a pharmacologically relevant animal model for the assessment of antigen expression, optimization of delivery and formulation of the plasmid. This paper describes a comparative study of domestic pig, minipig and mouse in regard to local tolerance and antigen expression of HIV immunotherapeutic using PMED. Pig/minipig is considered a good model for the safety assessment of DNA vaccines due to the similarity to human skin. Local reactions were evaluated at 10 min, 4, 24 and 48 h. Histology of administration sites revealed epidermal necrosis with associated dermal inflammation at 10 min and 4h, and subsequent regeneration with repair at 24 and 48 h. The degree and extent of these changes varied according to species. Domestic pig and minipig showed superficial epidermal necrosis and complete repair, while the mouse showed full-thickness epidermal necrosis and partial repair. Expression of HIV antigen was confirmed using immunohistochemistry in all three species at 4, 24 and 48 h. The results showed that PMED is an effective system for DNA vaccine delivery as demonstrated by the antigen expression seen as early as 4 h.

Biological activity of residual cell-substrate DNA.

Vaccines and other biological products manufactured in cells contain contaminating residual DNA derived from that production cell substrate, with the amount and form of this DNA depending mainly on the type of vaccine. The potential risk of this cellular DNA has been debated for over 40 years without resolution. Opinions on residual DNA have varied from it being considered an inert contaminant, and thus its presence should not be deemed to be a risk to vaccine recipients, to it being considered an important risk factor, particularly for vaccines manufactured in certain cell substrates, such as cells derived from tumours or cells that are tumorigenic. We are not of the opinion that DNA can be considered biologically inert, but whether or what risk residual cell-substrate DNA poses remains to be determined. In this paper, we discuss our approaches to address this issue and describe some preliminary work.

Human papillomavirus hpv-16 DNA as an epitheliotropic virus that induces hyperproliferation in squamous penile tissue.

The prevalence of human papillomavirus HPV-16DNA sequences in 57 penile carcinoma biopsies was examined using the polymerase chain reaction (PCR) with type specific internal probes, employing HPV consensus primers from the L1 region. The cases comprised 39 typical squamous cell carcinoma and 18 specimens with different subtype. PCR products were analyzed and HPV-16DNA was detected in a high percentage of specimens. Thirty-eight biopsies were HPV-16DNA positive. This determination was correlated with cellular differentiation and growth pattern. Our data corroborates that squamous cell carcinoma was invariably associated with HPV-16DNA.

No evidence for germ-line transmission following prenatal and early postnatal AAV-mediated gene delivery.

BACKGROUND: Recombinant adeno-associated viruses have been used successfully in a number of pre-clinical and clinical gene therapy studies. Since there is a broad consensus that gene therapy must not lead to germ-line transmission, the potential of such vectors for inadvertent gene transfer into germ cells deserves special attention. This applies in particular to pre- or perinatal vector application which has been considered for diseases presenting with morbidity already at birth. METHODS: AAV serotype 2 derived vectors carrying a beta-galactosidase reporter gene or human clotting factor IX cDNA were injected intraperitoneally or via a yolk sac vein into mouse fetuses or administered intravascularly to newborn mice. Tissue samples of the treated animals including the gonads as well as sperm DNA, obtained by differential lysis of one testis of each male animal, and the offspring of all treated mice were investigated for the presence of vector DNA by nested PCR. In positive samples, the copy number of the vector was determined by quantitative real-time PCR. RESULTS: AAV vectors administered intraperitoneally or intravascularly to fetal or newborn mice reached the gonads of these animals and persisted there for time periods greater than one year. Intravascular injection of the vector resulted more frequently in gene transfer to the gonads than intraperitoneal injection. Vector copy numbers in the gonads ranged from 0.3 to 74 per 10(4) cell equivalents. However, neither in isolated sperm DNA from the treated animals nor in their offspring were vector sequences detectable. CONCLUSIONS: These data suggest the risk of inadvertent germ-line transmission following prenatal or early postnatal AAV type 2 mediated gene delivery to be very low.

Systemic infection and limited replication of SHIV vaccine virus in brains of macaques inoculated intracerebrally with infectious viral DNA.

SHIV deleted in two accessory genes, DeltavpuDeltanef SHIV(PPC), functioned well as a vaccine against later challenge with highly pathogenic SHIV(KU), and it was able to reach the brain after oral inoculation of live virus. In this study, the proviral genome cloned into a plasmid was inoculated as DNA intracerebrally and spread systemically. Few regions of the brain had detectable proviral DNA by real-time PCR. Two measures of virus replication, detection of viral mRNA expression and circular proviral DNA, were negative for those brain regions, with the exception of the infection site in the right parietal lobe, whereas lymphoid tissues were positive by both measures. Histopathological analyses of all the sampled brain and spinal cord regions did not reveal any abnormalities. Despite intracerebral inoculation of the viral DNA, the brain was not targeted for high levels of virus replication.

Human papillomavirus and blastocyst apoptosis.

PURPOSE: The effect of human papillomavirus (HPV) DNA from the E6-E7 region on the integrity of DNA in blastocyst stage embryonic cells was studied. The study design paralleled the event whereby HPV DNA from the infecting virus would target host cell DNA. The objectives were (a) to determine if the DNA of blastocysts were disrupted by the presence of HPV DNA and (b) to determine if the intensity of DNA damage was associated with the type of HPV. METHODS: This study involved superovulating female mice, mating, collecting one-cell embryos, and culturing to the expanded blastocyst stage. The blastocysts were infected with PCR-synthesized DNA fragments from either HPV type 16, 18, 31, or 33. The blastocyst DNA were analyzed by comet assay after 24 h of incubation. The fluorescent images were digitized and the pixel intensity of each blastocyst was measured. RESULTS: Only the DNA of HPV type 16 was associated with significant DNA fragmentation in comparison with the other HPV types. There was no relationship between HPV DNA fragment size and the intensity of DNA fragmentation. CONCLUSIONS: The data suggested that one mode of action of HPV type 16 was to initiate apoptosis of embryonic cells through DNA fragmentation. The effect of HPV 16 occurred rapidly within 24 h. The intensity of DNA damage was not linked to the specific type of HPV. However, the results do not rule out the other HPV types affecting embryos under conditions different from this study.

Preclinical safety testing of DISC-hGMCSF to support phase I clinical trials in cancer patients.

BACKGROUND: DISC-hGMCSF is a gH-deleted HSV-2 based vector expressing human GM-CSF that is being developed for cancer immunotherapy. To support first clinical use, a range of preclinical safety studies were performed using DISC-hGMCSF in addition to DISC-murine-GMCSF and the backbone vector, TA-HSV. METHODS: The toxicity of the DISC vectors was assessed by repeated dose, neurovirulence and neuroinvasiveness studies in mice, and by safety studies in rabbits, guinea pigs and athymic nude mice. Studies were also conducted to determine whether the vector could establish latency in local ganglia in mice following intradermal injection, and whether it could reactivate from the latent state. The vector biodistribution following intravenous administration was also investigated in mice, using PCR to detect vector DNA. RESULTS: The DISC vectors were essentially non-toxic in all the systems studied. No adverse reactions were seen in mice receiving four intravenous doses of DISC-mGMCSF and the results from studies of neurovirulence, neuroinvasiveness, local tolerance in rabbit, general safety in mice and guinea pigs and safety in athymic nude mice were consistent with DISC being unable to replicate and cause disease. The vector could establish latency in local ganglia in mice, but at low efficiency, and could not reactivate infectious virions. Following intravenous administration, vector DNA was widely distributed up to Day 28, but by Day 56 had disappeared from gonads and brain and was only found in blood and liver. CONCLUSION: The panel of safety studies provided evidence that DISC-hGMCSF will be unable to replicate and cause disease, and has low toxicity in man. These data were presented to the Medicines Control Agency and the Gene Therapy Advisory Committee as part of the regulatory submissions for a clinical trial in melanoma patients. These submissions have been approved, and DISC-hGMCSF has now entered a phase I clinical trial in the UK by direct intratumoural injection.

Genetic evidence of an essential role for cytomegalovirus small capsid protein in viral growth.

Many steps in the replication cycle of cytomegalovirus (CMV), like cell entry, capsid assembly, and egress of newly synthesized virions, have not been completely analyzed yet. In order to facilitate these studies, we decided to construct a recombinant CMV that incorporates the green fluorescent protein (GFP) into the nucleocapsid. A comparable herpes simplex virus type 1 (HSV-1) mutant has recently been generated by fusion of the GFP open reading frame (ORF) with the HSV-1 ORF encoding small capsid protein (SCP) VP26 (P. Desai and S. Person, J. Virol. 72:7563-7568, 1998). Recombinant CMV genomes expressing a fusion protein consisting of GFP and the SCP were constructed by the recently established bacterial artificial chromosome mutagenesis procedure. In transfected cells, the SCP-GFP fusion protein localized to distinct foci in the nucleus that may represent sites for capsid assembly (assemblons). However, no viable progeny was reconstituted from these mutant CMV genomes. CMV genomes with deletion of the SCP ORF also did not give rise to infectious virus. Rescue of the mutation by insertion of the SCP gene at an ectopic position in an SCP knockout genome indicates that, in contrast to the HSV-1 SCP, the CMV SCP is essential for viral growth. Expression of the SCP-GFP fusion protein together with the authentic SCP blocked the CMV infection cycle, suggesting that the SCP-GFP fusion protein exerts a dominant-negative effect on the assembly of new virions. The results of this study are discussed with regard to recently published data about the structure of the CMV virion and its differences from the HSV-1 virion.

The major immediate-early gene ie3 of mouse cytomegalovirus is essential for viral growth.

The significance of the major immediate-early gene ie3 of mouse cytomegalovirus (MCMV) and that of the corresponding ie2 gene of human cytomegalovirus to viral replication are not known. To investigate the function of the MCMV IE3 regulatory protein, we generated two different MCMV recombinants that contained a large deletion in the IE3 open reading frame (ORF). The mutant genomes were constructed by the bacterial artificial chromosome mutagenesis technique, and MCMV ie3 deletion mutants were reconstituted on a mouse fibroblast cell line that expresses the MCMV major immediate-early genes. The ie3 deletion mutants failed to replicate on normal mouse fibroblasts even when a high multiplicity of infection was used. The replication defect was rescued when the IE3 protein was provided in trans by a complementing cell line. A revertant virus in which the IE3 ORF was restored was able to replicate with wild-type kinetics in normal mouse fibroblasts, providing evidence that the defective growth phenotype of the ie3 mutants was due to disruption of the ie3 gene. To characterize the point of restriction in viral replication that is controlled by ie3, we analyzed the pattern of expression of selective early (beta) and late (gamma) genes. While we could detect transcripts for the immediate-early gene ie1 in cells infected with the ie3 mutants, we failed to detect transcripts for representative beta and gamma genes. These data demonstrate that the MCMV transactivator IE3 plays an indispensable role during viral replication in tissue culture, implicating a similar role for the human CMV ie2 gene product. To our knowledge, the ie3 deletion mutants represent the first MCMV recombinants isolated that contain a disruption of an essential gene.

Evaluation of the biodistribution, persistence, toxicity, and potential of germ-line transmission of a replication-competent human adenovirus following intraprostatic administration in the mouse.

Adenovirus-mediated gene transfer may hold much promise in the treatment of human cancer. However, concerns regarding vector dissemination beyond the target tissue, particularly with replication-competent viruses, require an evaluation of the persistence of viral infection in collateral tissue and vector-associated toxicities. In addition, for indications such as prostate cancer, the proximity of the point of viral administration to organs of the male reproductive system raises concerns regarding inadvertent germ-line transmission of genes carried by the virus. To address these concerns, the biodistribution, persistence, toxicity, and potential of germ-line transmission of a replication-competent adenovirus (Ad5-CD/TKrep) following intraprostatic administration in the mouse was examined. Ad5-CD/TKrep (10(10) vp, 5 x 10(11) vp/kg) was injected intraprostatically on Day 1 of the study and its presence in the major organs of the male urogenital tract (prostate, testes, seminal vesicles, and urinary bladder) and liver was determined on Days 8 and 29. For comparison, a parallel group of animals was injected with the same dose of a related replication-defective Ad5-FGNR virus. To evaluate germ-line transmission, Ad5-CD/TKrep-injected males were mated to females on Days 8 and 29 and resulting embryos were examined for AdS-CD/TKrep viral DNA. Ad5-CD/TKrep viral DNA was detected in all major organs of the adult male urogenital tract and liver 7 and 28 Days postinjection. Interestingly, relative to the replication-defective Ad5-FGNR adenovirus, the replication-competent Ad5-CD/TKrep virus accumulated to a much greater level (approximately 300-fold) and persisted for a longer period of time in prostate, testes, and liver. This difference could not be explained on the basis of differences in viral infectivity, suggesting that the AdS-CD/TKrep virus may be capable of replicating in mouse tissues in vivo. In vitro infection of six mouse cell lines representing prostate, testes, and liver demonstrated that the Ad5-CD/TKrep virus was indeed capable of replicating in these mouse cell types, albeit with reduced efficiencies relative to human cells. Despite the fact that the Ad5-CD/TKrep vector persisted in the adult male gonads and may have replicated in vivo, we observed no evidence of germ-line transmission in 149 offspring examined. To evaluate the toxicity of combining Ad5-CD/TKrep viral therapy with CD/5-FC and HSV-1 TK/GCV suicide gene therapies as a prerequisite for a human trial, an escalating dose (10(8), 10(9), 10(10) vp) of Ad5-CD/TKrep was administered intraprostatically followed by 7 days of 5-FC and GCV double prodrug therapy. Although the virus persisted in the mouse urogenital tract and liver for up to 28 days postinjection, most of the toxicities observed were expected, minimal, and self-limiting. These results lead us to believe that intraprostatic administration of the Ad5-CD/TKrep virus to humans concomitant with double suicide gene therapy will be associated with acceptable toxicities and will not result in vertical transmission of viral-encoded genes through the germ line.

Safety studies of the intraperitoneal injection of E1A--liposome complex in mice.

The HER-2/neu (also called c-erbB-2) proto-oncogene is overexpressed in many human cancer cells, including those of breast cancer and ovarian cancer. We have previously shown that adenovirus 5 E1A inhibits HER-2/neu transcription and functions as a tumor suppressor gene in HER-2/neu-overexpressing cancer cells. Liposome-mediated E1A gene transfer suppresses tumor development and prolongs survival of tumor-bearing mice. In support of a phase I clinical trial of an E1A-liposome complex administered to patients with HER-2/neu-overexpressing breast of ovarian cancer, we conducted a series of studies to evaluate the safety of intraperitoneal injection of E1A in normal mice. The cumulative doses used were from five to 40 times the DNA-lipid starting dose proposed for the phase I clinical trial. In this dosing range, the administration of the E1A-liposome complex had no adverse effects on renal, hepatic and hematological parameters studied. No major organ pathologic changes were observed. We concluded that intraperitoneal administration of E1A-liposome complex at the proposed dose would not be expected to produce significant toxicity. The E1A-liposome clinical trial was recently approved by the Recombinant DNA Advisory Committee and Food and Drug Administration for a phase I trial in patients with HER-2/neu-overexpressing breast and ovarian cancer.

Risk assessment on the carcinogenic potential of hybridoma cell DNA: implications for residual contaminating cellular DNA in biological products.

The aim of this study was to evaluate the possible carcinogenic potential of residual DNA derived from immortalized and possibly tumorigenic cell lines due to activated oncogenic sequences (oncogenes). These cell lines have been used for the production of biologicals, i.e. monoclonal antibodies, lymphokines and vaccines. The authors used hybridoma DNA as a first model. For this reason experiments in two species were performed, namely in 3-4 week-old female Balb/c mice and newborn Riv:TOX rats. Doses of 250 micrograms DNA, derived from Balb/c hybridoma cells, were injected subcutaneously (s.c.) in 200 mice. These mice also received a s.c. injection of the solvent only (TE buffer) at another site of the back skin (negative control for local tumour development). An additional group of 50 mice was treated intraperitoneally (i.p.) with the solvent only to serve as a negative control group for possible systemic tumorigenic effects. Doses of 5 micrograms plasmid pPy1 DNA, containing the entire Polyoma virus genome, served as positive control and were injected s.c. and i.p. in 20 and 50 mice, respectively. Doses of 50 micrograms hybridoma DNA or 5 micrograms pPy1 DNA were injected s.c. in rats too, using nine animals per group. During the experiment, animals were observed weekly, especially for the occurrence of subcutaneous tumours at the injection sites. The mouse study was terminated after more than 2 years, the rat study after 1 year. Gross necropsy was performed on all animals and histopathological examination of grossly suspected neoplastic lesions was performed. In the mouse experiment, tumour development at the s.c. injection site of the DNA was observed in one out of 20 animals in the pPy1-treated positive control group (neurofibrosarcoma) and one out of 200 animals in the hybridoma DNA-treated group (haemangioma-like lesion). Tumour development at or near the s.c. injection site of the solvent only was observed in two out of 200 animals. In the rat study none out of nine hybridoma DNA-treated rats developed tumours at the injection site, while three out of nine rats of the positive control group, injected with the pPy1 DNA, showed local tumour development (benign and malignant soft tissue tumours.) It is concluded that, at the high dose and numbers of animals tested, parenteral administration of hybridoma DNA does not induce local tumour development. Furthermore, no indications were found for systemic carcinogenic potential of the hybridoma DNA used. Based on a worst case approach of our data, the oncogenic risk of 100 pg residual DNA was estimated to be 2 x 10(-9), a value intermediate of the estimations of the WHO (1987) and the Dutch Health Council (1988) 5 x 10(-11) and 2 x 10(-7), respectively. Therefore, it is unlikely that the risk of 100 pg of DNA derived from other immortalized cell lines will exceed the level of generally accepted cancer risk of 10(-6).

Adenoviral preterminal protein stabilizes mini-adenoviral genomes in vitro and in vivo.

In the absence of host immunity, nonintegrating, first-generation adenoviral vectors remain stable in the nucleus of quiescent transduced cells in mice. A mini-adenoviral genome (9 kb) deleted for viral E1, E2, E3, and late genes, but containing the viral inverted terminal repeats (ITRs), transgene expression cassette (human alpha 1-antitrypsin), and the viral E4 genes was equally efficient at transducing cells in vitro or in vivo as first generation, E1-deleted vectors. In contrast to a first generation vector, gene expression as well as vector DNA was short-lived in cells transduced with the deleted adenoviral genome. We demonstrate that coexpression of the adenoviral E2-preterminal protein from the vector or in trans stabilizes the mini-genome in vitro and in vivo without evidence of cellular toxicity.

Adenoviral-mediated thymidine kinase gene transfer into the primate brain followed by systemic ganciclovir: pathologic, radiologic, and molecular studies.

Transduction of experimental gliomas with the herpes simplex virus thymidine kinase gene (HSV-tk) using a replication-defective adenoviral vector (ADV/RSV-tk) confers sensitivity to ganciclovir (GCV) leading to tumor destruction and prolonged host survival in rodents. To determine treatment tolerance prior to clinical trials, we conducted toxicity studies in 6 adult baboons (Papio sp.). The animals received intracerebral injections of either a high dose of ADV/RSV-tk [1.5 x 10(9) plaque-forming units (pfu)] with or without GCV, or a low dose of ADV/RSV-tk (7.5 x 10(7) pfu) with GCV. The low dose corresponded to the anticipated therapeutic dose; the high dose was expected to be toxic. Magnetic resonance imaging (MRI) of the brain was obtained before treatment and at 3 and 6 weeks after treatment. Animals receiving the high-dose vector and GCV either died or became moribund and required euthanasia during the first 8 days of treatment. Necropsies revealed cavities of coagulative necrosis at the injection sites. Animals receiving only the high-dose vector were clinically normal; however, lesions were detected with MRI at the injection sites corresponding to cystic cavities at necropsy. Animals receiving the low-dose vector and GCV were clinically normal, exhibited small MRI abnormalities, and, although no gross lesions were present at necropsy, microscopic foci of necrosis were present. The vector sequence was detected by polymerase chain reaction (PCR) at the injection sites and in non-adjacent central nervous system tissue in all animals. Recombinant DNA sequence was detected outside of the nervous system in some animals, and persisted up to 6 weeks. The viral vector injections stimulated the production of neutralizing antibodies in the animals. No shedding of the vector was found in urine, feces, or serum 7 days after intracerebral injection. This study suggests that further investigations including clinical toxicity trials of this form of brain tumor therapy are warranted.

Adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase in an ascites model of human breast cancer.

In this study, the growth of locally disseminated breast cancer was modeled using a human breast cancer cell line, MDA-MB-435A, adapted to grow as an ascites tumor in athymic mice. Ex vivo infection of MDA-MB-435A cells with adenovirus containing the herpes simplex virus thymidine kinase gene (HSV-tk) were injected into the intraperitoneal cavity of athymic mice. Ganciclovir (GCV) treatment resulted in prolonged median survival (117 vs. 34 days, p < 0.001) compared to untreated or control animals. Adenovirus containing HSV-tk also demonstrated therapeutic activity after in vivo transduction resulting in prolongation of median survival after GCV treatment (32 vs. 25 days, p < 0.001). However, compared to ex vivo treatment, the effect was modest. In an attempt to increase survival, the viral dose was increased three-fold. Instead of prolonging survival, the increased dose resulted in more toxic deaths. Necropsy demonstrated that the most significant histologic abnormality was marked, diffuse, cytomegalic changes in the liver. Polymerase chain reaction (PCR) analysis of hepatic DNA demonstrated the presence of the virus in the affected tissue. Similar host toxicity and hepatic abnormalities were seen in non-tumor-bearing mice treated with ADV/RSV-tk plus GCV. In conclusion, adenoviral vectors can successfully transfer genes in vivo to cancer cells growing as ascites tumors. Transduction with HSV-tk followed by GCV treatment can prolong survival in this model system of disseminated disease, however toxicity can be substantial. Further refinement in targeting expression of HSV-tk will be required to enhance the therapeutic benefit.

Antibody-forming cell response to virus challenge in mice immunized with DNA encoding the influenza virus hemagglutinin.

Immunization of mice with DNA encoding the influenza virus hemagglutinin (HA) affords complete protection against lethal influenza virus infection and the means to investigate the mechanisms of B-cell responsiveness to virus challenge. Using a single-cell enzyme-linked immunospot assay, we sought to determine the localization of HA-specific antibody-forming cells (AFCs) during the development of humoral immunity in mice given HA DNA vaccine by gene gun. At 33 days postvaccination, populations of AFCs were maintained in the spleen and bone marrow. In response to lethal challenge with influenza virus, the AFCs became localized at the site of antigenic challenge, i.e., within the draining lymph nodes of the lung compartment. Immunoglobulin G (IgG)- and IgA-producing AFCs were detected in lymph nodes of the upper and lower respiratory tracts, underscoring their importance in clearing virus from the lungs. Response to challenge required competent CD4+ T cells, without which no AFCs were generated, even those producing IgM. By contrast, in mice vaccinated with an HA-containing subunit vaccine, fewer AFCs were generated in response to challenge, and these animals were less capable of resisting infection. Our findings demonstrate the comparable localization of AFCs in response to challenge in mice vaccinated with either HA DNA or live virus. Moreover, the former strategy generates both IgG- and IgA-producing plasma cells.

Chromosomal alterations of rat cell lines transformed by human adenovirus type-12 virion, whole DNA and left-end DNA fragments.

A normal rat cell line, 3Y1-B clone 1-6 (3Y1) and its adenovirus (Ad) type-12-transformed derivatives, W4 (transformed by Ad12 Virion), WY3 (transformed by Ad12 whole DNA), CY1-1 (transformed by the Ad12 EcoRI-C fragment, left 16.5%), GY1-1 (transformed by the Ad12 HindIII-G fragment, left 6.8%) and HY1 (transformed by the Ad12 Acd-H fragment, left 4.7%) were studied cytogenetically. 3Y1 and some of the transformed cell lines (W4, WY3 and GY1-1) were diploid or pseudodiploid, while others (CY1-1 and HY1) were hypotetraploid. A metacentric marker M1 was detected in GY1-1 cells and another marker M2 in W4 and CY1-1 cells at a high frequency. By the Giemsa banding technique, the metacentric markers M1 and M2 from these fully transformed cell lines were identified as isochromosomes derived from 1q (M1) and 3q (M2), respectively. On the other hand, the markers were detected only at a low frequency in incompletely transformed HY1 cells. However, hypersomy in chromosome No. 1 was observed at a high frequency in this cell line. It can be concluded that hypersomy of chromosomes No. 1 or 3 found in transformants and metacentric markers found in complete transformants are characteristic features in rat cells transformed by Ad12.