Ludvik Prevec

Manipulation of Adenovirus Vectors

Adenoviruses have been isolated from a large number of different species (mammalian and fowl) and over 100 different serotypes have been reported, some 43 of them human. The human adenoviruses, particularly types 2, 5, and 12, have been the most extensively characterized, and these viruses have served as valuable tools in the study of the molecular biology of DNA replication, transcription, RNA processing, and protein synthesis in mammalian cells. Seeref. 1 for a general review.

Methods for construction of adenovirus vectors.

Adenoviruses are attracting increasing attention as general purpose mammalian cell expression vectors, as recombinant vaccines, and potentially as vectors for gene therapy. Not only is the adenovirus genome relatively easy to manipulate by recombinant DNA techniques, but adenovirus vectors are relatively stable, grow to high titers, and can transduce a variety of cell types in cell culture and in vivo. Vectors can be designed that are either replication competent or replication defective and, in the latter case, are highly efficient at delivering and expressing genes in mammalian cells without resulting in cell killing. Methods are described for growing, titrating, and purifying adenoviruses, for extracting viral DNA from purified virions and from infected cells, for rescuing inserts of foreign DNA into the viral genome, and for assessing expression of inserted genes in adenovirus vectors.

Use of human adenovirus-based vectors for antigen expression in animals.

An infectious recombinant human adenovirus type 5 (Ad5) vector, AdG12, which carries the glycoprotein gene of vesicular stomatitis virus (VSV) and expresses that gene in cultured HeLa cells was used to examine the host range of insert expression by human Ad vectors. The VSV glycoprotein was expressed in bovine, canine and murine cells when infected with AdG12 in culture. These cell lines are respectively permissive, non-permissive and semi-permissive for human Ad5 replication. Administration of the AdG12 vector to calves, piglets or dogs by either the subcutaneous or oral route resulted in the production of high titres of neutralizing antibodies to VSV. Mice injected intraperitoneally with the vector produced neutralizing antibodies and were protected against subsequent intravenous challenge with normally lethal doses of VSV. This work demonstrates the utility of human adenoviral vectors for antigen expression in a number of non-human cell lines and for the induction of an immune response to the delivered antigen in a number of species.

Monitoring foreign gene expression by a human adenovirus-based vector using the firefly luciferase gene as a reporter

We have constructed a helper-independent adenovirus type 5-luciferase recombinant (Ad5-Luc 3) containing the firefly luciferase gene flanked by simian virus 40 (SV40) regulatory sequences inserted in the early region 3 (E3) of the Ad5 genome. Expression of luciferase in cells infected with Ad5-Luc3 was relatively efficient. In HeLa cells approximately 20 micrograms luciferase per 10(6) cells was made by 36 h post-infection and a 62 kilo-Dalton (kDa) luciferase band was clearly visible in a [35S]methionine-labeled Ad5-Luc 3-infected cell extract analyzed directly by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of experiments in which cultured cells were infected with Ad5-Luc 3 in the presence or absence of 1-beta-D-arabinofuranosyl cytosine (AraC) showed that the majority of luciferase expression was dependent on viral DNA replication. This suggested that the enzyme was probably translated primarily from mRNA derived from transcripts expressed from the major late promoter of Ad5. An anti-luciferase antibody was raised in a rabbit and used to further characterize the luciferase expressed in HeLa cells infected with Ad5-Luc 3 by immunoprecipitations and Western blot analyses. The half-life of luciferase expressed in HeLa cells infected with Ad5-Luc 3 was calculated to be approximately 6-8 h by pulse chase analysis. Luciferase is likely to be a useful marker for monitoring virus dissemination and gene expression in experimental animals because assays for enzymatic activity are extremely sensitive and backgrounds are low in all tissues. In mice inoculated intraperitoneally (i.p.) with Ad5-Luc 3, luciferase activity was detected in the liver, spleen, kidney, and lung. A single i.p. inoculation of mice with Ad5-Luc 3 was sufficient to raise anti-luciferase antibody and Ad5 neutralizing antibody which persisted for at least 8 weeks. Even in the presence of circulating anti-luciferase and Ad5 neutralizing antibodies, luciferase activity could be detected in the livers, spleens, and kidneys of mice inoculated i.p. a second time with Ad5-Luc 3.

[2] Techniques for human adenovirus vector construction and characterization

Publisher Summary This chapter describes the techniques for human adenovirus vector construction and characterization. Human adenoviruses (Ads) have attracted considerable attention lately for their potential use as vectors for gene therapy, for recombinant vaccines, and for high-level protein expression in mammalian cells. Among the reasons for these interests are the following: (1) the 36 kilobase pair (kbp) double-stranded DNA genome of Ad is relatively easy to manipulate by recombinant DNA techniques and (2) Ad can infect and direct high levels of protein expression in both proliferating and quiescent cells, an important feature for vectors requiring in vivo expression. The chapter describes several plasmid-based systems for inserting foreign genes into the Ad genome and the methods used to purify, grow, and titrate recombinant viruses. The vectors are based on the human Ad5 genome, the structure of which is presented in the chapter. In a normal infection, early genes (E1A, EIB, E2, E3, and E4) are expressed prior to DNA replication. Late gene expression, driven predominantly by the major late promoter at 16 map units, occurs after DNA replication. The products of the EIA gene are required for the expression of all the other Ad genes. Thus, E1 deletion viruses are defective for replication in all cell types except the E1-complementing 293 cell line. The E3 region, which may be important for virus persistence in vivo, is dispensable for growth of the virus in vitro. Both the E1 and E3 regions, therefore, provide convenient sites for insertions of foreign sequences to generate helper-independent recombinant viruses.

DEVELOPMENT OF A BOVINE ADENOVIRUS TYPE 3-BASED EXPRESSION VECTOR

We constructed a non-defective bovine adenovirus type 3 recombinant (BAd3-Luc) containing the firefly luciferase gene inserted in the early region 3 (E3) of the BAd3 genome. Deletion of a 696 bp XhoI-NcoI E3 segment and insertion of the luciferase gene in E3 was confirmed by Southern blot analyses. Luciferase was expressed in Madin-Darby bovine kidney cells infected with BAd3-Luc as measured by enzymic assays and Western blotting. Analyses of luciferase expression in the presence or absence of 1-beta-D-arabinofuranosylcytosine indicated that approximately 70-75% of luciferase expression was dependent on viral DNA replication, suggesting that transcription of the gene was at least partially under the control of a late promoter. Although yields of infectious virus for BAd3-Luc were approximately 10-fold lower than for wild-type virus, replication of the vector was still relatively efficient. In a Western blot experiment, anti-luciferase antibody reacted with a 62 kDa protein which is of the same molecular mass as the purified firefly luciferase polypeptide. Luciferase was also expressed in the 293 cell line infected with BAd3-Luc for at least 6 days post-infection as monitored by luciferase assays. Based on these observations we suggest that BAd-based expression vectors should have excellent potential for the development of recombinant vaccines for cattle and may also be suitable as vectors for gene transfer into human cells.

Proteins of vesicular stomatitis virus: III. Intracellular synthesis and extracellular appearance of virus-specific proteins

Abstract An examination of the kinetics of synthesis and release of virus-specific proteins from VSV-infected cells showed that the rate of virus protein synthesis was maximal about 4 hr post infection, a constant proportion of the newly synthesized protein being released from the cell at all times. Five virus-specific proteins VP1, VP2, VP3, VP4, and NS1 were identified in infected cells. The intracellular protein VP2 was shown to be distinct by its mobility on polyacrylamide gels from both the virion glycoprotein VP2 and the soluble antigen glycoprotein VP2a and may be a precursor to these proteins. Exposing cells to actinomycin D for 20 hr prior to infection showed that the relative rate of synthesis of NS1 was greatest in the first 2 hr of infection and decreased at later times. In contrast, the virion envelope proteins VP2 and VP4 formed an increasing proportion of virus protein synthesis with time. The result suggests that the protein NS1 may have an early intracellular function in replication. Pulse-chase experiments showed that the proteins VP1 and VP4 could be released from the cell soon after synthesis while newly synthesized VP2 and VP3 appeared in extracellular virus only after some delay.

Human adenovirus type 5 vectors expressing rabies glycoprotein

The prevalence of wildlife rabies throughout the world and the continued spread of this disease in North America highlights the need for oral vaccines which may be used safely and effectively to vaccinate a number of species that are reservoirs or vectors of rabies. We have previously shown that AdRG1, a replication competent recombinant human adenovirus type 5 (Ad5) expressing a rabies glycoprotein (RG), can induce immunity to rabies in rodent, canine, and skunk model systems. To improve the Ad5 vector system as a potential oral vaccine, we have constructed additional Ad5 recombinant vectors and compared RG expression in cell culture and immunogenicity in animals. Two new replication competent vectors are compared. AdRG1.3, which carries RG with accompanying SV40 poly A addition sequences within an E3 deletion, and AdRG4, which has RG in the E3 deletion but under the control of an exogenous Ad2 major late promoter, both express higher levels of RG in permissive cell culture than did AdRG1 and both elicit high levels of serum anti-rabies antibodies by parenteral or oral routes in animals. AdRG1.3 may be a more effective vaccine vector in species which are non-permissive for the replication of human Ad5.

Vesicular stomatitis virus—A new interfering particle, intracellular structures, and virus-specific RNA

Abstract A strain of the Indiana serotype of vesicular stomatitis virus (VSV) designated HR-LT produces a defective particle which differs from the T particle described by others. This “long T” particle is approximately one-half the length of the infectious B particle and contains a single-stranded RNA having a sedimentation coefficient of 30 S. Infection of L cells with the HR-LT strain of VSV results in the intracellular accumulation of viral nucleoprotein structures similar to nucleoprotein structures derived from mature B and “long T” particles. In the infected cell, virus-specific RNA species with sedimentation coefficients of 30 S and 15 S appear to be associated with polyribosomes.

Induction of Antibodies Protecting against Transmissible Gastroenteritis Coronavirus (TGEV) by Recombinant Adenovirus Expressing TGEV Spike Protein

Abstract Ten recombinant adenoviruses expressing either fragments of 1135, 1587, or 3329 nt or the full-length spike gene of transmissible gastroenteritis coronavirus (TGEV) have been constructed. These recombinants produce S polypeptides with apparent molecular masses of 68, 86, 135, and 200 kDa, respectively. Expression of the recombinant antigen driven by Ad5 promoters was inhibited by the insertion of an exogenous SV-40 promoter. Most of the recombinant antigens remain intracytoplasmic in infected cells. All the recombinant-directed expression products contain functional antigenic sites C and B (Gebaueret al.,1991,Virology183, 225–238). The recombinant antigen of 135 kDa and that of 200 kDa, which represents the whole spike protein, also contain antigenic sites D and A, which have previously been shown to be the major inducers of TGEV-neutralizing antibodies. Interestingly, here we show that recombinant S protein fragments expressing only sites C and B also induced TGEV-neutralizing antibodies. The chimeric Ad5–TGEV recombinants elicited lactogenic immunity in hamsters, including the production of TGEV-neutralizing antibodies. The antisera induced in swine by the Ad5 recombinants expressing the amino-terminal 26% of the spike protein (containing sites C and B) or the full-length spike protein, when mixed with a lethal dose of virus prior to administration to susceptible piglets, delayed or completely prevented the induction of symptoms of disease, respectively.