NYVAC: a highly attenuated strain of vaccinia virus.

A highly attenuated vaccinia virus strain, NYVAC (vP866), was derived from a plaque-cloned isolate of the Copenhagen vaccine strain by the precise deletion of 18 open reading frames (ORFs) from the viral genome. Among the ORFs deleted from NYVAC (vP866) are two genes involved in nucleotide metabolism, the thymidine kinase (ORF J2R) and the large subunit of the ribonucleotide reductase (ORF I4L); the gene encoding the viral hemagglutinin (ORF A56R); the remnant (ORF A26L) of a highly expressed gene responsible for the formation of A-type inclusion bodies; the disrupted gene (ORFs B13R/B14R) normally encoding a serine protease inhibitor; and a block of 12 ORFs bounded by two known viral host range regulatory functions (ORFs C7L through K1L). Within this block a secretory protein (ORF N1L) implicated in viral virulence and a functional complement 4b binding protein (ORF C3L) are encoded. The ORFs were deleted in a manner which prevents the synthesis of undesirable novel gene products. The attenuation characteristics of the derived NYVAC strain were compared in in vitro and in vivo studies with those of the Western Reserve (WR) laboratory strain, the New York City Board of Health vaccine strain (Wyeth), the parental plaque-cloned isolate (VC-2) of the Copenhagen vaccine strain used to derive NYVAC, and the avipox virus canarypox (ALVAC), which is naturally restricted for replication to avian species. The NYVAC strain was demonstrated to be highly attenuated by the following criteria: (a) no detectable induration or ulceration at the site of inoculation on rabbit skin; (b) rapid clearance of infectious virus from the intradermal site of inoculation on rabbit skin; (c) absence of testicular inflammation in nude mice; (d) greatly reduced virulence as demonstrated by the results of intracranial challenge of both 3-week-old or newborn mice; (e) greatly reduced pathogenicity and failure to disseminate in immunodeficient (nude or cyclophosphamide treated) mice; and (f) dramatically reduced ability to replicate on a variety of human tissue culture cells. Despite these highly attenuated characteristics, the NYVAC strain, as a vector, retains the ability to induce strong immune responses to extrinsic antigens.     

Mice immunized with a subviral particle containing the Japanese encephalitis virus prM/M and E proteins are protected from lethal JEV infection.

Extracellular subviral particles produced by HeLa cells infected with a recombinant vaccinia virus encoding the prM and E genes of Japanese encephalitis virus (JEV) were purified and characterized. These particles contained the JEV prM/M and E proteins embedded in a lipid bilayer, and RNA was not detected in particles using the polymerase chain reaction and primers recognizing a part of the JEV E gene. The particles were uniformly spherical with a 20-nm diameter and had 5-nm projections on their surface. Mice that received a single inoculation of the purified extracellular particles emulsified with Freund's complete adjuvant were fully protected against 4.9 x 10(5) LD50 of JEV. Comparison of the neutralizing and hemagglutination-inhibiting antibody titers and radioimmunoprecipitation data showed that immunization with the particles induced an immune response similar to that following inoculation with the recombinant vaccinia virus.     

Comparison of protective immunity elicited by recombinant vaccinia viruses that synthesize E or NS1 of Japanese encephalitis virus.

Immunization with recombinant vaccinia viruses that specified the synthesis of Japanese encephalitis virus (JEV) glycoproteins protected mice from a lethal intraperitoneal challenge with JEV. Recombinants which coexpressed the genes for the structural glycoproteins, prM and E, elicited high levels of neutralizing (NEUT) and hemagglutination inhibiting (HAI) antibodies in mice and protected mice from a lethal challenge by JEV. Recombinants expressing only the gene for the nonstructural glycoprotein, NS1, induced antibodies to NS1 but provided low levels of protection from a similar challenge dose of JEV. Antibodies to the NS3 protein in postchallenge sera, representing the degree of infection with challenge virus, were inversely correlated to NEUT and HAI titers and levels of protection. These results indicate that although vaccinia recombinants expressing NS1 can provide some protection from lethal JEV infection, recombinants expressing prM and E elicited higher levels of protective immunity.     

Recombinant vaccinia virus producing the prM and E proteins of yellow fever virus protects mice from lethal yellow fever encephalitis.

Four recombinant vaccinia viruses were constructed for expression of different portions of the 17D yellow fever virus (YFV-17D) open reading frame. A recombinant, vP869, expressing prM and E induced high titers of neutralizing and hemagglutination inhibiting antibodies in mice and was protective against intracranial challenge with the French neurotropic strain of YFV. Levels of protection were equivalent to those achieved by immunization with the YFV-17D vaccine virus. Recombinant vaccinia viruses expressing E and NS1, C prM, E, NS1, or only NS1 failed to protect mice against challenge with YFV despite eliciting antibodies to NS1. The vP869-infected HeLa cells produced a particulate extracellular hemagglutinin (HA) similar to that produced by YFV-infected cells, supporting previous studies with Japanese encephalitis virus (Mason et al., 1991), suggesting that the ability of recombinant vaccinia virus to produce extracellular HA particles is important for effective flavivirus immunity.     

Localizations of NS3 and E proteins in mouse brain infected with mutant strain of Japanese encephalitis virus

Summary.  Infection with a mutant Japanese encephalitis virus (JEV) strain RP-2ms showed reduced neurovirulence than wild type or RP-9 strains after inoculation in BALB/c mice. However, higher intracellular viral titer was detected in Rp-2ms infected cultured cells. Localizations of non-structural 3 (NS3) and envelope (E) proteins were demonstrated by immunocytochemistry. NS3 protein was primarily found in the pyramidal neurons in cerebrum, in the molecular and granular layers of cerebellum. Neither E nor NS3 protein was detected in Purkinje cells of the cerebellum. Immunoelectron microscopic observations showed that E and NS3 proteins were positive in JEV-induced membranous systems, mainly hypertrophic rough endoplasmic reticulum (rER) and membrane vesicle structure (MVS) but not smooth membrane structure. Virus particles were seen in the Golgi apparatus, rER, nuclear envelope, MVS and cytoplasmic vacuoles. Different mechanisms of intracellular trapping in vivo provide a possible basis for attenuation of RP-2ms strains of JEV. Received May 1, 1998 Accepted July 4, 1998     

A live attenuated turkey rhinotracheitis virus vaccine. 1. Stability of the attenuated strain

Compared with the severe clinical disease caused by two virulent strains of turkey rhinotracheitis virus (TRTV), an attenuated strain has been shown to cause only slight respiratory infection in 7-day-old TRT-free turkey poults, yet still to be capable of stimulating TRT antibodies and of protecting the poults against challenge with virulent TRTV 21 days later. The stability of the attenuation during 12 turkey-turkey passages of the strain has been demonstrated; the poult passaged and prepassaged strains causing only mild respiratory signs even when bacteria, known to be associated with severe outbreaks of TRT in the field, were included in the inoculum.     

A specific and sensitive antigen capture assay for NS1 protein quantitation in Japanese encephalitis virus infection

Japanese encephalitis virus (JEV) is a human pathogenic, mosquito-borne flavivirus that is endemic/epidemic in Asia. JEV is rarely detected or isolated from blood or cerebrospinal fluid (CSF), and detection of IgM is generally diagnostic of the infection. The flavivirus nonstructural glycoprotein NS1 is released transiently during flavivirus replication. The aim of this study was to set up a quantitative JEV NS1 antigen capture assay. A soluble hexameric form of JEV NS1 protein was produced in a stable Drosophila S2 cell clone and purified from supernatant fluids. Two IgG1 monoclonal antibodies (MAbs) with high affinity against two different epitopes of JEV NS1 antigen were used to develop an antigen-capture assay with a limit of detection of 0.2 ng ml⁻¹ NS1. Up to 1 μg ml⁻¹ JEV NS1 protein was released in supernatants of mammalian cells infected with JEV but <10 ng ml⁻¹ was released in sera of virus-infected mice before the onset of encephalitis and death. Moreover, NS1 protein was detected at low levels (<10 ng ml⁻¹) in 23.8% of sera and in 10.5% of CSF of patients diagnosed as IgM-positive for JEV. This quantitative test of NS1 protein is proposed for highly specific diagnosis of acute infection with JEV genotypes I to IV.     

Different contribution of co-stimulatory molecules B7.1 and B7.2 to the immune response to recombinant modified vaccinia virus ankara vaccine expressing prM/E proteins of Japanese encephalitis virus and two hepatitis B virus vaccines

This study clarifies the role of co-stimulatory molecules B7.1 and B7.2 in the immune response to 3 types of vaccines: a/ recombinant modified Vaccinia virus Ankara (MVA) (vJH9) expressing prM/E proteins of Japanese encephalitis virus (JEV), b/ recombinant yeast-expressed Hepatitis B virus (YHBV), c/ human plasma-derived Hepatitis B virus (PHBV). We constructed plasmids expressing B7.1 and B7.2 molecules and found that the expression level of B7.2 protein in transfected CHO-k1 cells was higher than that of B7.1 protein. Mice were co-injected with vaccines vJH9, YHBV and PHBV and plasmids expressing B7.1 or B7.2, respectively, and specific antibody titers for each vaccine were monitored at days 7, 14 and 28 post injection (p.i.). In mice injected with vJH9 vaccine and both B7 plasmids, plasmid B7.2 induced a higher anti-JEV immune response than plasmid B7.1. This implies that the stimulation of the B7.2 immune pathway may be a feasible method of boosting protective immunity against a recombinant viral vaccine. Both B7 molecules were able to induce a specific anti-HBV immune response using YHBV vaccine. On the other hand, B7 molecules had little effect to the specific antibody induction in PHBV vaccination. These results suggested that the contribution of B7.1 and B7.2 molecules in an immune response depended on the character and status of the presenting antigen.     

Temperature sensitive mutations in the genes encoding the NS1, NS2A,NS3, and NS5 nonstructural proteins of dengue virus type 4 restrict replication in the brains of mice

Summary.  Using reverse genetics, it is possible to readily add well-defined attenuating mutations to the genome of wild type or incompletely attenuated dengue (DEN) viruses to generate vaccine candidates that exhibit the desired balance between attenuation and immunogenicity. Here, we describe the identification of eight temperature sensitive missense mutations distributed in four non-structural protein genes that specify a 60- to 10,000-fold range of restricted replication in the suckling mouse brain compared to wild type recombinant DEN4 virus. The usefulness of such mutations in flavivirus vaccine design and development is discussed. Received October 22, 2002; accepted January 8, 2003 Published online March 21, 2003     

The role of the PKR-inhibitory genes,E3L and K3L,in determining vaccinia virus host range

Vaccinia virus encodes two regulators of the cellular antiviral response. The E3L gene is thought to act primarily by sequestering double-stranded RNA, whereas the K3L gene is thought to act as a competitive inhibitor of the double-stranded RNA-dependent protein kinase, PKR. The broad host range associated with vaccinia virus replication appears to be related to the presence of these genes. The E3L gene is required for replication in HeLa cells, but is not required for replication in BHK cells. On the contrary, the K3L gene is required for replication in BHK cells, but is dispensable for replication in HeLa cells. Our results suggest that these cell lines varied in the expression of endogenous activatable PKR and that replication of vaccinia virus in different cell lines led to altered levels of double-stranded RNA synthesis from the virus. Vaccinia virus was able to overcome these cellular variations by regulating PKR activity through the synthesis of either E3L or K3L. The results suggest that vaccinia virus has evolved a broad host range by maintaining both the E3L and the K3L genes.     

Mutations in the NS2B and NS3 genes affect mouse neuroinvasiveness of a Western European field strain of tick-borne encephalitis virus

An attenuated strain (263) of the tick-borne encephalitis virus, isolated from field ticks, was either serially subcultured, 5 times in mice, or at 40 degrees C in PS cells, producing 2 independent strains, 263-m5 and 263-TR with identical genomes; both strains exhibited increased plaque size, neuroinvasiveness and temperature-resistance. Sequencing revealed two unique amino acid substitutions, one mapping close to the catalytic site of the viral protease. These observations imply that virus adaptation from ticks to mammals occurs by selection of pre-existing virulent variants from the quasispecies population rather than by the emergence of new random mutations. The significance of these observations is discussed.     

Nucleotide sequence of the virulent SA-14 strain of Japanese encephalitis virus and its attenuated vaccine derivative, SA-14-14-2

The attenuated SA-14-14-2 strain of Japanese encephalitis (JE) virus has been used to immunize people in the People's Republic of China. Oligonucleotide fingerprints of the parent SA-14 and vaccine strain indicate that multiple genetic changes occurred during attenuation of the virus. We have cloned and sequenced the genomes of both the virulent SA-14 and attenuated SA-14-14-2 viruses to define molecular differences in the genomes. Forty-five nucleotide differences, resulting in 15 amino acid substitutions, were found by comparing sequences of the SA-14 and SA-14-14-2 genomes. Transversion of U to A occurred at position 39 in the 5'-noncoding region of SA-14-14-2 and another SA-14 vaccine derivative SA-14-5-3. A single nucleotide change in the capsid gene of SA-14-14-2 altered a single amino acid which changed its predicted secondary structure. A silent nucleotide change was found in the prM gene sequence and the M-protein was unchanged. There are seven nucleotide differences, resulting in five amino acid changes, in the E glycoprotein sequence of the two viruses. Nine amino acid differences were found in the nonstructural proteins of SA-14 and SA-14-14-2: one in NS2A, two in NS2B, three in NS3, one in ns4a, and two in NS5. A single nucleotide change at position 10,428 in the 3'-noncoding region is vaccine virus-specific. The nucleotide and deduced amino acid sequences of the vaccine strain SA-14-14-2, the parent virus SA-14, and virulent strains JaOArS982 and Beijing-1 have been compared and are highly conserved.     

A plasmid encoding parts of the dengue virus E and NS1 proteins induces an immune response in a mouse model

A DENV-2 plasmid named pEII*EIII/NS1*, containing sequences encoding portions of the envelope protein that are potentially involved in the induction of neutralizing antibodies and a portion of the NS1 sequence that is involved in protection, is reported in this work. The synthesized subunit protein was recognized by human sera from infected patients and had the predicted size. The immunogenicity of this construct was evaluated using a mouse model in a prime-boost vaccination approach. The priming was performed using the plasmid pEII*EIII/NS1*, followed by a boost with recombinant full-length GST–E and GST–NS1 fusion proteins. The mice showed specific antibody responses to the E and NS1 proteins, as detected by ELISA, compared to the response of animals vaccinated with the parental plasmid. Interestingly, some animals had neutralizing antibodies. These results show that EII*, EIII and NS1* sequences could be considered for the design of a recombinant subunit vaccine against dengue disease.     

Expression of functional parvoviral NS1 from recombinant vaccinia virus: effects of mutations in the nucleotide-binding motif.

The gene encoding the major replicative protein, NS1, of minute virus of mice (MVM) was transferred into a recombinant vaccinia virus vector in place of the vaccinia thymidine kinase gene. The NS1 gene was placed under control of a bacteriophage T7 promoter and expressed in cells coinfected with another recombinant vaccinia virus, vTF7-3, which encodes the T7 RNA polymerase. Expression of NS1 was further enhanced by the presence of a 5' untranslated region, derived from encephalomyocarditis virus, which allows efficient cap-independent translation. This system was used to produce and analyze wild-type NS1 and two mutant forms of the protein, NS1K405R and NS1K405M, in which the highly conserved lysine codon located in the putative purine triphosphate binding site of NS1 was changed to arginine and methionine, respectively. Full-length NS1 was expressed efficiently in both human and mouse cells infected with each of the three recombinant viruses, and in each case the NS1 was rapidly and efficiently translocated into the nucleus. Wild-type NS1 expressed in this way was biologically active. It was able to trans-activate an MVM P38 promoter located in a host chromosomal site, whereas the two mutant forms of NS1 showed no significant activity in this assay, and it was capable of resolving palindromic junction fragments cloned from multimeric MVM replicative form DNA molecules. These substrates, representing MVM genomic left-end:left-end and right-end:right-end fusions, were resolved in a DNA synthesis-dependent in vitro reaction supplemented with nuclear extracts containing recombinant wild-type NS1. Neither of the two mutant forms of the polypeptide had any detectable activity in this assay.     

Antigenic variation in the influenza A virus nonstructural protein, NS1

The antigenic structure of the nonstructural (NS1) protein encoded by influenza type A virus was examined using monoclonal antibodies prepared against purified NS1 inclusions isolated from the cytoplasm of infected cells. Topographical analysis by competitive radioimmunoassay indicated that three different overlapping antigenic regions were present on the NS1 of A/WSN/33 (H1N1). Immunoprecipitation studies using infected cell lysates showed that antigenic determinants on A/WSN/33 NS1 are common to NS1 proteins encoded by a wide range of viruses of human, swine, equine, and avian origin. Several avian strains, however, were found to encode antigenically variant NS1 proteins which had either extensive changes in one or more antigenic regions or small changes in epitopes within a region suggestive of antigenic drift. There was no correlation between surface antigen subtype and the antigenic profile of the NS1 protein. The antigenic relationships of NS1 proteins shown in this study are in agreement with the available sequence data.     

A single amino acid substitution in the central portion of the West Nile virus NS4B protein confers a highly attenuated phenotype in mice

West Nile virus (WNV) NS4B is a small hydrophobic nonstructural protein that is hypothesized to participate both in viral replication and evasion of host innate immune defenses. The protein has four cysteine residues (residues 102, 120, 227, and 237). Since cysteines are often critical for the function of proteins, each of the four cysteine residues found in WNV NS4B was mutated to serine by site-directed mutagenesis. While three of these substitutions had little effect on replication or mouse virulence phenotypes, the C102S mutation was associated with a temperature-sensitive phenotype at 41 degrees C as well as attenuation of the neuroinvasive and neurovirulence phenotypes in mice.