A viral vaccine vector that expresses foreign genes in lymph nodes and protects against mucosal challenge.

A candidate live-virus vaccine strain of Venezuelan equine encephalitis virus (VEE) was configured as a replication-competent vector for in vivo expression of heterologous immunogens. Three features of VEE recommend it for use as a vaccine vector. (i) Most human and animal populations are not already immune to VEE, so preexisting immunity to the vector would not limit expression of the heterologous antigen. (ii) VEE replicates first in local lymphoid tissue, a site favoring the induction of an effective immune response. (iii) Parenteral immunization of rodents and humans with live, attenuated VEE vaccines protects against mucosal challenge, suggesting that VEE vaccine vectors might be used successfully to protect against mucosal pathogens. Upon subcutaneous (s.c.) inoculation into the footpad of mice, a VEE vector containing the complete influenza virus hemagglutinin (HA) gene expressed HA in the draining lymph node and induced anti-HA immunoglobulin G (IgG) and IgA serum antibodies, the levels of which could be increased by s.c. booster inoculation. When immunized mice were challenged intranasally with a virulent strain of influenza virus, replication of challenge virus in their lungs was restricted, and they were completely protected from signs of disease. Significant reduction of influenza virus replication in the nasal epithelia of HA vector-immunized mice suggested an effective immunity at the mucosal surface. VEE vaccine vectors represent an alternative vaccination strategy when killed or subunit vaccines are ineffective or when the use of a live attenuated vaccine might be unsafe.     

Validation and use of an indirect enzyme-linked immunosorbent assay for detection of antibodies to West Nile virus in American Alligators (Alligator mississippiensis) in Florida

In October 2002, West Nile virus (WNV) was identified in farmed American alligators (Alligator mississippiensis) in Florida showing clinical signs and having microscopic lesions indicative of central nervous system disease. To perform seroepidemiologic studies, an indirect enzyme-linked immunosorbent assay (ELISA) was developed to determine exposure of captive and wild alligators to WNV. To validate the test, a group of WNV-seropositive and -seronegative alligators were identified at the affected farm using hemagglutination inhibition (HAI) and the plaque reduction neutralization test (PRNT). The indirect ELISA utilized a rabbit anti-alligator immunoglobulins polyclonal antibody as the secondary antibody, and inactivated WNV-infected Vero cells were used as the coating antigen. For all samples (n=58), the results of the ELISA were consistent with the HAI and PRNT findings. Plasma was collected from 669 free-ranging alligators from 21 sites across Florida in April and October 2003. Four samples collected in April and six in October were positive for WNV antibodies using HAI, PRNT, and the indirect ELISA. This indicated that wild alligators in Florida have been exposed to WNV. These findings can be used as a baseline for future surveys.     

Identification of mutated cyclization sequences that permit efficient replication of West Nile virus genomes: use in safer propagation of a novel vaccine candidate

Existing live-attenuated flavivirus vaccines (LAV) could be improved by reducing their potential to recombine with naturally circulating viruses in the field. Since the highly conserved cyclization sequences (CS) found in the termini of flavivirus genomes must be complementary to each other to support genome replication, we set out to identify paired mutant CS that could support the efficient replication of LAV but would be unable to support replication in recombinant viruses harboring one wild-type (WT) CS. By systematic evaluation of paired mutated CS encoded in West Nile virus (WNV) replicons, we identified variants having single and double mutations in the 5'- and 3'-CS components that could support genome replication at WT levels. Replicons containing only the double-mutated CS in the 5' or the 3' ends of the genome were incapable of replication, indicating that mutated CS could be useful for constructing safer LAV. Despite the identity of the central portion of the CS in all mosquito-borne flaviviruses, viruses carrying complementary the double mutations in both the 5'- and the 3'-CS were indistinguishable from WT WNV in their replication in insect and mammalian cell lines. In addition to the utility of our novel CS pair in constructing safer LAV, we demonstrated that introduction of these mutated CS into one component of a recently described two-component genome system (A. V. Shustov, P. W. Mason, and I. Frolov, J. Virol. 81:11737-11748, 2007) enabled us to engineer a safer single-cycle WNV vaccine candidate with reduced potential for recombination during its propagation.     

Cloned mouse DNA fragments can replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro.

Mouse liver DNA was cut out with BamHI and cloned into YIp5, which contained the URA3 gene of Saccharomyces cerevisiae in pBR322. Of the several plasmids isolated, two plasmids, pMU65 and pMU111, could transform S. cerevisiae from the URA- to the URA+ phenotype and could replicate autonomously within the transformant, indicating that mouse DNA fragments present in pMU65 or pMU111 contain autonomously replicating sequences (ARS) for replication in S. cerevisiae. Furthermore, to determine the correlation between ARS function in yeast cells and that in much higher organisms, we tried to challenge these plasmids with the simian virus 40 (SV40) DNA replication system. Of the two plasmids tested, the EcoRI-BglII region of pMU65 could be hybridized with a chemically synthesized 13-nucleotide fragment corresponding to the origin region of SV40 DNA. Both pMU65 (the EcoRI-BglII region cloned in pBR322) and its subclone pMU65EB could replicate semiconservatively, and initiation of DNA replication started from the EcoRI-BglII region when the replicating activity of these plasmids was tested in the in vitro SV40 DNA replication system we have established before. Furthermore, pMU65 and pMU65EB could replicate autonomously within monkey Cos cells which produce SV40 T antigen constitutively. These results show that a 2.5-kilobase fragment of the EcoRI-BglII region in pMU65 contains the ARS needed for replication in the SV40 DNA replication system.     

A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication.

A recombinant plasmid carrying an infectious adeno-associated viral genome was constructed that differs in several key respects from previously described recombinants. First, the vector is pEMBL8(+), which allows isolation of viral plus and minus strands. Second, the inserted viral sequences contain two XbaI cleavage sites that flank the viral coding domain. These inserts do not affect replication of the virus, and they allow nonviral sequences to be easily inserted between the cis-acting terminal repeats of adeno-associated virus. Third, the viral genome is flanked by PvuII cleavage sites that allow the entire, infectious viral chromosome to be excised from plasmid sequences in vitro. Viral DNA was replicated more efficiently within adenovirus-infected 293 cells if it was excised from the vector with PvuII before transfection. Presumably, the increased efficiency reflects bypass of the excision step which must normally precede replication when a recombinant plasmid enters the nucleus. The ability to bypass the excision step was exploited to search for a viral function required specifically for excision of the viral genome from the integrated state. None of the mutants tested identified a gene product required for excision that was not also essential for replication. The ability to produce pure populations of viral plus and minus strands was used to demonstrate that both strands are infectious.     

Critical role for Env as well as Gag-Pol in control of a simian-human immunodeficiency virus 89.6P challenge by a DNA prime/recombinant modified vaccinia virus Ankara vaccine

Cellular immune responses against epitopes in conserved Gag and Pol sequences of human immunodeficiency virus type 1 have become popular targets for candidate AIDS vaccines. Recently, we used a simian-human immunodeficiency virus model (SHIV 89.6P) with macaques to demonstrate the control of a pathogenic mucosal challenge by priming with Gag-Pol-Env-expressing DNA and boosting with Gag-Pol-Env-expressing recombinant modified vaccinia virus Ankara (rMVA). Here we tested Gag-Pol DNA priming and Gag-Pol rMVA boosting to evaluate the contribution of anti-Env immune responses to viral control. The Gag-Pol vaccine raised frequencies of Gag-specific T cells similar to those raised by the Gag-Pol-Env vaccine. Following challenge, these rapidly expanded to counter the challenge infection. Despite this, the control of the SHIV 89.6P challenge was delayed and inconsistent in the Gag-Pol-vaccinated group and all of the animals underwent severe and, in most cases, sustained loss of CD4(+) cells. Interestingly, most of the CD4(+) cells that were lost in the Gag-Pol-vaccinated group were uninfected cells. We suggest that the rapid appearance of binding antibody for Env in Gag-Pol-Env-vaccinated animals helped protect uninfected CD4(+) cells from Env-induced apoptosis. Our results highlight the importance of immune responses to Env, as well as to Gag-Pol, in the control of immunodeficiency virus challenges and the protection of CD4(+) cells.     

Mosquitoes and West Nile virus along a river corridor from prairie to montane habitats in eastern Colorado

We conducted studies on mosquitoes and West Nile virus (WNV) along a riparian corridor following the South Platte River and Big Thompson River in northeastern Colorado and extending from an elevation of 1,215 m in the prairie landscape of the eastern Colorado plains to 1,840 m in low montane areas at the eastern edge of the Rocky Mountains in the central part of the state. Mosquito collection during June‐September 2007 in 20 sites along this riparian corridor yielded a total of 199,833 identifiable mosquitoes of 17 species. The most commonly collected mosquitoes were, in descending order: Aedes vexans, Culex tarsalis, Ae. dorsalis, Ae. trivittatus, Ae. melanimon, Cx. pipiens, and Culiseta inornata. Species richness was higher in the plains than in foothills‐montane areas, and abundances of several individual species, including the WNV vectors Cx. tarsalis and Cx. pipiens and the nuisance‐biter and potential secondary WNV vector Ae. vexans, decreased dramatically from the plains (1,215‐1,487 m) to foothills‐montane areas (1,524‐1,840 m). Ae. vexans and Cx. tarsalis had a striking pattern of uniformly high abundances between 1,200‐1,450 m followed by a gradual decrease in abundance above 1,450 m to reach very low numbers above 1,550 m. Culex species were commonly infected with WNV in the plains portion of the riparian corridor in 2007, with 14 of 16 sites yielding WNV‐infected Cx. tarsalis and infection rates for Cx. tarsalis females exceeding 2.0 per 1,000 individuals in ten of the sites. The Vector Index for abundance of WNV‐infected Cx. tarsalis females during June‐September exceeded 0.5 in six plains sites along the South Platte River but was uniformly low (0–0.1) in plains, foothills and montane sites above 1,500 m along the Big Thompson River. A population genetic analysis of Cx. tarsalis revealed that all collections from the ≈190 km riparian transect in northeastern Colorado were genetically uniform but that these collections were genetically distinct from collections from Delta County on the western slope of the Continental Divide. This suggests that major waterways in the Great Plains serve as important dispersal corridors for Cx. tarsalis but that the Continental Divide is a formidable barrier to this WNV vector.     

Presence of replicating virus in recombinant hepadnavirus stocks results from recombination and can be eliminated by the use of a packaging cell line

Mutant hepatitis B viruses are useful tools to study the viral life cycle and viral pathogenesis. Furthermore, recombinant hepatitis B viruses are candidate vectors for liver-directed gene therapy. Because wild-type viruses present in recombinant or mutant virus stocks may falsify experimental results and are detrimental for a viral vector, we investigated whether and to what extent wild-type virus is present in recombinant virus stocks and where it originates from. We took advantage of the duck model of hepatitis B virus infection which allows very sensitive detection of replication-competent viruses by infection of primary duck hepatocytes or of ducklings in vivo. Recombinant hepatitis B virus stocks contained significant amounts of wild-type viruses, which were most probably generated by homologous recombination between plasmids containing homologous viral sequences. In addition, replication-competent viral genomes were reconstituted from plasmids which contained replication-deficient but redundant viral sequences. Using a stable cell line for packaging of deficient viral genomes, no wild-type virus was detected, neither by infection of primary hepatocytes nor in vivo.     

Oxazole yellow homodimer YOYO-1-labeled DNA: a fluorescent complex that can be used to assess structural changes in DNA following formation and cellular delivery of cationic lipid DNA complexes.

To improve transfection efficiency following delivery of plasmid expression vectors using lipid-based carriers, it is crucial to define structural characteristics of the lipid/DNA complexes that optimize transgene expression. Due to its strong affinity for DNA and high quantum yield, the fluorescent DNA intercalator YOYO-1 was used as a tool to assess changes in DNA that occur following lipid binding and cell delivery. In this study, the stability of the dye/DNA complex following binding of poly-L-lysine or monocationic lipids is characterized. More than 98% of the fluorescence measured for a defined DNA/YOYO-1 complex was lost when DNA was condensed using poly-L-lysine. This loss in fluorescence could be attributed to displacement of bound dye. In contrast, more than 30% of the fluorescence of the dye-labeled DNA was retained after formation of cationic lipid/DNA complexes. Significantly, the results illustrate differences in structural changes cationic lipids and PLL exert on plasmid DNA. The fluorescent lipid/DNA complex was used to assess DNA delivery to murine B16/BL6 cells in vitro. An assay relying on fluorescence resonance energy transfer between bound YOYO-1 and propidium iodide was used to distinguish between DNA attached to the cell surface and internalized DNA.     

A vector that replicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein-Barr virus.

Epstein-Barr virus (EBV) transforms human B-lymphocytes into proliferating blasts which are efficiently established into cell lines. The viral DNA in these cell lines is usually present as complete, unintegrated plasmid molecules. A cis-acting element of EBV, oriP, permits plasmid maintenance in adherent cells that carry EBV DNA. We constructed a vector, pHEBo, that carries oriP and showed that it is also efficiently maintained as a plasmid when introduced into EBV-transformed B-lymphoblasts. The pHEBo vector carries the coding sequences for the hph gene from Escherichia coli such that it can be expressed in mammalian cells and confers resistance to the antibiotic hygromycin B. Hygromycin B kills EBV-transformed lymphoblasts at concentrations of 50 to 300 micrograms/ml. The combination of oriP plus the expressed hph gene makes pHEBo useful for the stable introduction of genes on plasmids into EBV-transformed lymphoblasts. Because pHEBo is derived from the plasmid pBR322 it can be easily isolated from lymphoblasts by reintroduction into E. coli.     

Simian virus 40 DNA replication in vitro: purification and characterization of replication factors from mouse cells.

We have previously developed simian virus 40 (SV40) DNA replication system in vitro (Ariga and Sugano, J. Virol. 48, 481, 1983). This system is composed of human HeLa or mouse FM3A nuclear extract and cytoplasmic extract of SV40 infected CosI cells. Here FM3A nuclear extract was fractionated by DEAE Sephacel and single-stranded DNA cellulose chromatography into three components required for accurate in vitro SV40 DNA replication. One fraction (A fraction) contained DNA polymerase-primase, and the second component (B fraction) contained DNA topoisomerase. Third component was further purified to near homogenuity using DEAE-Sephacel, single-stranded DNA cellulose, and glycerol gradient centrifugation. The purified protein (named factor I) bound to the origin containing fragment of SV40 DNA. The factor I enhanced the initiation of SV40 DNA replication catalyzed by SV40 infected CosI cytoplasm alone. When all four fractions consisting of A, B fractions, factor I, and SV40 infected CosI cytoplasm were mixed together, the system was reconstituted, meaning that initiation and subsequent elongation were completed to generate the full sized daughter molecules.     

Identification of a naturally occurring recombinant Epstein-Barr virus isolate from New Guinea that encodes both type 1 and type 2 nuclear antigen sequences.

In this report we describe an Epstein-Barr virus isolate, derived from the peripheral blood lymphocytes of a healthy adult from Papua New Guinea, that is a recombinant of the two major Epstein-Barr virus types, encoding type 1 Epstein-Barr nuclear antigen 2 (EBNA2) sequences, and type 2 EBNA3, EBNA4, and EBNA6 sequences.     

Analysis of T antigen and viral DNA in mouse cells transformed by K virus, a nononcogenic murine papovavirus

A tumorigenic line of mouse cells transformed by the nononcogenic murine K papovavirus was established and characterized. Tumor-bearing animals produced antibody specific for K virus-transformed cells; the K virus T antiserum did not react with polyomavirus-transformed cells. Immunoprecipitation of the T antigen in the transformed cells revealed large T antigen (molecular weight 84,000). About 10 to 15 copies of K virus DNA were found to be integrated in the cellular DNA.     

Comparison of simian virus 40 large T antigen recombinant protein and DNA immunization in the induction of protective immunity from experimental pulmonary metastasis

In this report we examine the ability of a recombinant tumor antigen preparation to prevent the establishment of experimental pulmonary metastasis. Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (T-Ag) was injected into BALB/c mice followed by challenge with an intravenous injection of syngeneic SV40-transformed tumorigenic cells. The experimental murine pulmonary metastasis model allows for the accurate measurement of metastatic lessions in the lungs at various times after the challenge, using computer-assisted video image analysis. Following challenge, lung metastasis and survival data for the groups of mice were obtained. Animals immunized with recombinant SV40 T-Ag showed no detectable sign of lung metastasis and survived for more than 120 days after challenge. Antibodies specific for SV40 T-Ag were detected in the serum of immunized mice by enzyme-linked immunosorbent assay. Splenocytes obtained from mice immunized with recombinant SV40 T-Ag did not lyse syngeneic tumor cells, indicating that no cytotoxic T lymphocyte response was induced. Control mice developed extensive lung metastasis and succumbed to lethal tumor within 4 weeks after challenge. These data indicate that immunization with the recombinant SV40␣T-Ag induces protective, T-Ag-specific immunity in an experimental pulmonary tumor metastasis model. Received: 20 August 1998 / Accepted: 25 November 1998     

Zeolin is a recombinant storage protein that can be used to produce value-added proteins in alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.)

The specific features of plants make them particularly suitable for the production of recombinant proteins and alfalfa is one of the recommended plant production systems. We have transformed alfalfa with a gene coding for a chimaeric protein made previously by fusing phaseolin to the N-terminal region of γ-zein and have analyzed the accumulation of this fusion protein, named zeolin. Zeolin was expressed both in T0 Regen SY alfalfa plants and in the progeny resulting from the sexual cross between Regen SY transformants and alfalfa cv. Adriana plants. In some alfalfa plants a 95 kDa zeolin glycosylated polypeptide is the most abundant polypeptide detected by Western-blot analysis, whereas in tobacco the most abundant zeolin polypeptide has a molecular mass around 60 kDa, expected for intact zeolin. Zeolin has been stably accumulated in alfalfa leaves because it forms endoplasmic reticulum-located protein bodies in the cell. As regards zeolin quantisation, in the progeny alfalfa plants a value of about 0.22–0.28 mg of zeolin / g of fresh leaf weight has been estimated. Michele Bellucci and Francesca De Marchis contributed equally to this work.     

Proteins encoded in the 81.2- to 85.0-map-unit fragment of Autographa californica nuclear polyhedrosis virus DNA can be translated in vitro and in Spodoptera frugiperda cells.

We have previously demonstrated that five open reading frames exist in the nucleotide sequence of the 81.2- to 85.0-map-unit (m.u.) segment of plaque isolate E of Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. The corresponding polypeptides are 9.8, 12.1, 36.6, 25.0, and 48.2 kDa in size (C. Oellig, B. Happ, T. Müller, and W. Doerfler, J. Virol. 63:1494, 1989), and we have investigated whether these proteins can be translated in infected cells. On subfragments of this viral DNA segment, mRNAs were selected from AcNPV-infected Spodoptera frugiperda insect cells at different times postinfection (p.i.). The in vitro translation of these RNAs in a rabbit reticulocyte-derived cell-free translation system yielded polypeptides of approximately 10 to 11, 12 to 14, 28, 36 to 38, and 48 to 50-kDa which were commensurate in size with the theoretically expected values. mRNAs for the 28- and 48- to 50-kDa proteins were identified by their translation products at 6 h p.i., and mRNAs for the 10- to 11-, 12- to 14-, and 36- to 38-kDa proteins were identified by their translation products at 12 h p.i. We constructed an AcNPV recombinant which carried in its polyhedrin gene the 3.9-kbp EcoRI-HindIII (81.8 to 84.8 m.u.) subfragment of the EcoRI J segment. Nucleotide sequence determinations revealed that the intact polyhedrin promoter lay adjacent to the additional 81.8- to 84.8-m.u. fragment in this recombinant. In S. frugiperda cells, which were infected with the recombinant AcNPV, a protein of 36 to 38 kDa was detected at 44 h p.i. in larger amounts than after infection with the nonrecombinant virus. However, there was no evidence for larger amounts of RNA derived from the 81.8- to 84.8-m.u. fragment in recombinant-infected cells. Recombinant-infected cells lacked the polyhedrin polypeptide. The synthesis of the 36- to 38-kDa polypeptide in recombinant- or AcNPV-E-infected S. frugiperda cells could be demonstrated by immunoprecipitation experiments. Peculiarly, this polypeptide was present in the cytoplasm as a 64-kDa glycoprotein. These data corroborate the notion that at least some of the open reading frames encoded in the 81.2- to 85.0-m.u. segment of AcNPV can be expressed in S. frugiperda cells.