Oka Vaccine Strain Research Articles

New method of differentiating wild-type varicella-zoster virus (VZV) strains from Oka varicella vaccine strain by VZV ORF 6-based PCR and restriction fragment length polymorphism analysis.

A new method was developed to distinguish accurately wild-type varicella-zoster virus (VZV) strains from the Oka vaccine strain. Several DNA fragments covering open reading frame (ORF) 1-37 were amplified from wild-type VZV strains including the Oka parent strain and from the Oka vaccine strain. Restriction fragment length polymorphisms of these regions were compared, and nucleotide differences between the vaccine virus and other wild-type VZV strains were noted in ORFs 6, 10, and 35. In addition, variations of the R2 and R4 reiterated structures of the vaccine and its parent strains were examined. The Oka vaccine strain used in Japan was shown to be a mixture of viruses with different nucleotide sequences that had variations in at least three nucleotide positions in ORF 1-37 and had variable polymorphisms at R2 and R4 repeat regions (two and three patterns, respectively). The Oka parent strain on the other hand showed a single sequence and had only one reiterated structure at these regions. When VZV ORF 6 was amplified and its product was digested with AluI, the Oka vaccine strain could be precisely differentiated from its parent and from 56 other Japanese clinical isolates.

Use of a rodent model to show that varicella-zoster virus ORF61 is dispensable for establishment of latency.

Varicella-zoster virus (VZV) results in a latent infection in humans after primary infection. Latency has also been established in guinea pigs and rats after inoculation with the virus. It was found that infection of cotton rats with the Oka vaccine strain of VZV results in a latent infection. To begin to identify which genes are required for latency, we infected cotton rats with VZV strain Oka that is deleted for ORF61. ORF61 protein transactivates certain VZV promoters and enhances the infectivity of viral DNA in transient transfections. Deletion of ORF61 results in abnormal syncytia and impairs the growth of VZV in vitro. Inoculation of cotton rats with ORF61-deleted Oka virus resulted in latent VZV infection in the nervous system similar to that seen for animals infected with parental virus. Thus, the cotton rat can be used to study the ability of mutants in the Oka vaccine strain of VZV to establish latent infection.

Varicella-zoster virus isolates, but not the vaccine strain OKA, induce sensitivity to alpha-1 and beta-1 adrenergic stimulation of sensory neurones in culture.

The reactivation of varicella-zoster virus (VZV) from its persistent state in sensory neurones causes shingles and induces severe, long-lasting pain and hyperalgesia that often lead to postherpetic neuralgia. To investigate the VZV-induced neuropathic changes, we established conditions for the active infection of sensory neurones from rat dorsal root ganglia in vitro. After 2 days of culture, up to 50% of the cells expressed viral antigens of the immediate-early and late replication phase. The intracellular calcium ion concentration was monitored in individual cells by microfluorimetry. Whereas the calcium response to capsaicin was preserved, the VZV-infected neurones gained an unusual sensitivity to noradrenaline stimulation in contrast to non-infected cells. The adrenergic agonists phenylephrine and isoproterenol had a similar efficacy demonstrating that both alpha(1)- and beta(1)-adrenoreceptors were involved. The sensitivity to adrenergic stimulation was observed after infection with different wildtype isolates, but not with the attenuated vaccine strain OKA. The lack of noradrenaline sensitivity of vaccine-infected neurones demands a structural comparison of wildtype and vaccine viruses with and without phenotype. A partial sequence evaluation (26 kb) of the European OKA vaccine strain surprisingly revealed a series of nucleotide exchanges in comparison to presumably identical OKA strains from other sources, although VZV is generally considered genetically stable. In summary, we report that the infection with wildtype VZV isolates, but not with the vaccine strain, induces noradrenaline sensitivity in sensory neurones, which correlates with clinical and experimental observations of adrenergic effects involved in VZV-induced neuralgia.

DISSEMINATED RASHES AND VARICELLA VACCINE

IN REPLY: Our report stated that “All of 57 vaccinees with breakthrough varicella, clinically diagnosed on the basis of a generalized maculopapular or vesicular rash had wild-type VZV [varicella-zoster virus] infection based on analysis of viral DNA. 1 The Oka vaccine strain of VZV was not identified in any of these cases.” This statement should be amended to say that, “All of 57 vaccinees with breakthrough varicella, clinically diagnosed on the basis of a generalized maculopapular or vesicular rash, in which there was amplifiable DNA had wild-type VZV infection based on analysis of viral DNA.” In fact there were also 36 samples that were VZV PCR-negative and 19 samples that were inadequate. The VZV-negative samples gave negative results by PCR for VZV but were positive for a 268-bp fragment of the human beta-globin gene, indicating that there was amplifiable DNA in the sample. We believe these to be true VZV negatives. The sensitivity of the assay as determined by serial dilution of extracted VZV wild-type and vaccine strain VZV DNA was ≥100. 1 The inadequate samples gave negative results by PCR for both VZV and beta-globin, indicating that there was not even enough DNA to amplify a human gene present in multiple copies in each human cell. We would not agree that assay sensitivity is the problem in these inadequate samples; rather it is the adequacy of sample collection that determines whether there is enough DNA to amplify. If PCR assay sensitivity was the cause of the failure to find the vaccine strain in samples from disseminated rashes in vaccinees, we would have expected even less success in identifying the vaccine strain in samples from zoster lesions which in general are less likely to be vesicular. We were, however, able to identify the vaccine strain in 22 of 32 VZV PCR-positive samples from zoster lesions. In addition even in 9 leukemic children who were vaccinated while receiving chemotherapy and later developed a varicella-like rash, we have only been able to document the wild-type virus as the cause of the rash. Even in these highly immunocompromised children, we were not able to document the vaccine strain as the cause of these rashes. The point of the data presented was that by the most sensitive technique available, we have never been able to find the vaccine strain in a disseminated varicella-like rash in a vaccinee outside of the period when vaccine-associated rash is seen (<42 days postvaccination). Although it is possible that clinical reactivation of the vaccine strain virus could cause disseminated varicella-like rash, we have not been able to document it to date. Philip LaRussa, , M.D. Anne A. Gershon, M.D.

The postmarketing safety profile of varicella vaccine

The postmarketing safety profile of varicella vaccine was evaluated by analyzing selected adverse experience reports temporally associated with the administration of the vaccine. There were 7963 reports voluntarily submitted to Merck for an overall reporting rate of 5.0 per 10 000 doses of vaccine distributed. A varicella zoster virus (VZV) identification program detected the presence of the Oka vaccine strain in three individuals with an immune deficiency — two with pneumonia and one with hepatitis — and in three instances of secondary transmission from vaccinees with vesicular lesions to susceptible household contacts. The Oka vaccine strain was present in 23 patients and wild-type VZV was present in 15 patients with herpes zoster. Vesicular rashes that occurred within 2 weeks of vaccination were more likely to contain the presence of wild-type VZV, while vesicular rashes that occurred more than 2 weeks post-vaccination were more likely to contain the Oka vaccine strain. Eleven patients were hospitalized with complications of breakthrough varicella infection.

Viral strain identification in varicella vaccinees with disseminated rashes.

Approximately 15% of recipients of live attenuated varicella vaccine may develop mild breakthrough varicella months to years after immunization. Although some vaccinees will develop zoster, it is less common in recipients of vaccine than in those who have had natural varicella. To determine the varicella-zoster virus (VZV) strain responsible for breakthrough varicella and zoster in recipients of varicella vaccine. A PCR assay capable of distinguishing wild-type from vaccine strain VZV was performed on samples from skin lesions from vaccinees with breakthrough varicella and zoster. All of 57 vaccinees with breakthrough varicella, clinically diagnosed on the basis of a generalized maculopapular or vesicular rash, in which there was amplifiable DNA [corrected], had wild-type VZV infection based on analysis of viral DNA. The Oka vaccine strain of VZV was not identified in any of these cases. In contrast, in 32 patients with zosteriform rashes, the vaccine strain was identified in 22 samples, and the wild-type strain was identified in 10 samples. Wild-type virus was identified in all generalized rashes occurring after the immediate 6-week postvaccination period. When reactivation of vaccine strain occurred, it presented as typical zoster. We find no evidence that reactivation of vaccine virus occurs with the clinical picture of generalized rash.

Varicella-zoster vaccine: The bad news may be good

Clinically-evident reactivation of the Oka vaccine strain appears common and may have public health benefits for vaccine-induced immunity (pages 451–454 )

Subclinical reactivation of Oka vaccine strain may boost varicella immunity

Aseroepidemiological study has revealed that the Oka varicellazoster virus (VZV) vaccine can reactivate itself as serum antibody titres decrease after vaccination, boosting the host's immunity to the virus. “Lifelong immunity to wild-type VZV may be maintained by a combination of re-exposure to wild-type VZV and repeated subclinical reactivation of latent virus”, say Philip Krause and Dennis Klinman (Food and Drug Administration, Bethesda, MD, USA).

Nucleotide sequences that distinguish Oka vaccine from parental Oka and other varicella-zoster virus isolates.

The sequences of approximately 34 kb from the 3' end of the varicella-zoster virus (VZV) Oka vaccine strain and the previously sequenced Dumas strain were compared. Sequence differences were noted in the coding sequences of several VZV open reading frames (ORFs), including ORFs 48, 51, 52, 55, 56, 58, 59, 60, 62, 64, and 68. Tests based on differences in the ORF62 gene and in the ORF64 poly-A region successfully distinguished the Oka vaccine strain from its wild-type parent and from other Japanese and US clinical isolates. These changes remained stable after passage of the virus in humans.

Infection of human T lymphocytes with varicella-zoster virus: an analysis with viral mutants and clinical isolates.

Varicella-zoster virus (VZV) disseminates in the body in peripheral blood mononuclear cells during chickenpox. Up to 1 in 10,000 mononuclear cells are infected during the viremic phase of the disease. We developed an in vitro system to infect human mononuclear cells with VZV by using umbilical cord blood. In this system, 3 to 4% of T cells were infected with VZV. VZV mutants unable to express certain genes, such as open reading frame 47 (ORF47) or ORF66, were impaired for growth in T cells, while other mutants showed little difference from parental virus. VZV unable to express ORF47 was even more impaired for spread from umbilical cord blood cells to melanoma cells in vitro. Early-passage clinical isolates of VZV infected T cells at a similar rate to the Oka vaccine strain; however, the clinical isolates were more efficient in spreading from infected T cells to melanoma cells. This in vitro system for infecting human T cells with VZV should be useful for identifying cellular and viral proteins that are important for virus replication in T cells and for the spread of virus from T cells to other cells.

Polymerase chain reaction and restriction fragment length polymorphism analysis of varicella-zoster virus isolates from the United States and other parts of the world.

A polymerase chain reaction (PCR) assay that identifies and differentiates wild-type (wt) and vaccine strains of varicella-zoster virus (VZV) was used to determine if VZV strains with restriction fragment length polymorphisms resembling those of the Japanese Oka vaccine strain were present in the wt pool outside of Japan. Virus samples (n = 114) from patients with chickenpox and zoster from various parts of the United States and Australia were analyzed. The assay correctly identified 113 samples as wt strain. The 1 sample identified as Oka vaccine strain came from a child with leukemia who developed a vaccine-associated rash after receiving the live attenuated varicella vaccine. At this point, there is no evidence that wt strains resembling the vaccine are circulating outside of Japan. This indicates that this PCR assay can be utilized to distinguish rashes due to vaccine and wt VZV.

Identification of the Oka strain of the live attenuated varicella vaccine from other clinical isolates by molecular epidemiologic analysis.

A method was developed to distinguish the Oka vaccine strain of varicella-zoster virus (VZV) from other clinical isolates. The molecular characteristics of 52 clinical isolates from varicella or zoster patients with no history of VZV vaccination and the Oka strain, including vaccine and parental viruses, were analyzed by PstI cleavage of the PstI site-less (PSL) region. This was followed by single-strand conformational polymorphism (SSCP) after polymerase chain reaction amplification of repeating region 2 (R2). Most of the clinical isolates tested, especially recent isolates, had a PstI site in the PSL region, but the Oka strain did not. The SSCP patterns of R2 in Oka strain virus differed from those of other viruses. These results suggest that analysis of the PstI site followed by SSCP of R2 will be useful for identifying the Oka vaccine virus in isolates.

Immunization with recombinant varicella-zoster virus expressing herpes simplex virus type 2 glycoprotein D reduces the severity of genital herpes in guinea pigs.

Varicella-zoster virus (VZV) is an attractive candidate for a live-virus vector for the delivery of foreign antigens. The Oka vaccine strain of VZV is safe and effective in humans, and recombinant Oka VZV (ROka) can be generated by transfecting cells with a set of overlapping cosmid DNAs. By this method, the herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) gene was inserted into an intergenic site in the unique short region of the Oka VZV genome. Expression of gD2 in cells infected with the recombinant Oka strain VZV (ROka-gD2) was confirmed by antibody staining of fixed cells and by immunoblot analysis. Immune electron microscopy demonstrated the presence of gD2 in the envelope of ROka-gD2 virions. The ability of ROka-gD2 to protect guinea pigs against HSV-2 challenge was assessed by inoculating animals with three doses of uninfected human fibroblasts, fibroblasts infected with ROka VZV, or fibroblasts infected with ROka-gD2. Neutralizing antibodies specific for HSV-2 developed in animals immunized with ROka-gD2. Forty days after the third inoculation, animals were challenged intravaginally with HSV-2. Inoculation of guinea pigs with ROka-gD2 significantly reduced the severity of primary HSV-2 infection (P < 0.001). These experiments demonstrate that the Oka strain of VZV can be used as a live virus vector to protect animals from disease with a heterologous virus.

Identification of varicella-zoster virus strains by PCR analysis of three repeat elements and a PstI-site-less region

We established a method of identifying varicella-zoster virus (VZV) strains, especially those of the Oka vaccine, in patients with clinical VZV infections. The DNAs of 30 clinically isolated strains and 4 laboratory strains including the Oka vaccine strain and its parent VZV strain, were analyzed by PCR with four sets of primers for the four variable regions, R2, R4, R5, and a region without a PstI site (PS). R4 was unstable in four laboratory VZV strains and was excluded from the study. The other regions were stable in several passages in cell culture. The number of copies in R2 and R5 were distributed from 2 to 13 and from 1 to 3, respectively, in the strains analyzed. The vaccine strain had seven copies in R2 and two copies in R5, and it was PS negative. Among 30 clinical isolates, 3, 23, and 11 strains had the same characteristics as the vaccine strain in R2, R5, and PS, respectively. Therefore, by this method, 97.2% of the isolates were distinguished from the Oka vaccine strain. This strategy will be useful in diagnosing VZV infections induced by the vaccine strain.

Down-Regulation of Glial Fibrillary Acidic Protein Expression during Acute Lytic Varicella-Zoster Virus Infection of Cultured Human Astrocytes

The effects of the varicella-zoster virus (VZV) OKA vaccine strain in producing morphologic and antigenic changes in dissociated cultures of human fetal brain was investigated. Cultures containing 80% glial acidic fibrillary protein (GFAP), GFAP+ (positive) astrocytes and 20% GFAP- (negative) fibroblastic-like cells were infected with cell-free VZV OKA at a multiplicity of infection of 0.1 plaque-forming units per cell. Cytopathic effects and significant viral antigen labeling with antibodies against VZV gpl and immediate-early (IE) protein 62 were first detected 6 to 7 days postinfection. Several observations indicated that astrocyte GFAP expression was altered and diminished as a result of VZV infection itself, thereby raising doubts about the utility of combining cell markers and viral antigenic labeling in assessing the susceptibility of neural cell types to viral infection. The down-regulation of GFAP expression by VZV appears to be mediated by early rather than late events in the viral replication cycle and may not be the result of virally induced global shut-off of host cell protein synthesis. Similar observations were made using VZV Ellen, a multipassaged, nonvaccine strain. These observations have potential in vivo implications related to histologic analysis of VZV-infected tissues and disease pathogenesis.

Restriction fragment differences between the genomes of the Oka varicella vaccine virus and american wild‐type varicella‐zoster virus

The Oka vaccine strains of varicella-zoster virus (VZV) have a significantly different BgII DNA restriction pattern from that of American wild-type isolates of VZV. This difference consists primarily of an additional BgII site, which lies within the BamHI "D" fragment. In conjunction with a study of the efficacy of an experimental Merck/Oka VZV vaccine, the area of the genome from which the most marked restriction pattern alteration arises was studied more closely to determine if there are other significant differences between the Oka strains and American wild-type strains. BamHI "D" fragments from the DNA of the Oka parent strain (the progenitor of the vaccine strain), the RIT/Oka vaccine strain (a derivative of the Oka parent strain), the Merck/Oka vaccine strain, and the EF strain (an American wild type), were submitted to extensive endonuclease digestion studies to ascertain if additional unique restriction sites are present in the Oka parent or vaccine strains. The extra BgII restriction site characteristic of the Merck/Oka vaccine strain is also present in the DNA of the parent virus as well as its derivatives and was therefore not produced by the "attenuation" process. No other novel sites were found in the Oka parent or Oka-derived strains in this section of the genome. The Merck/Oka vaccine strain of VZV, despite its Japanese origin, is therefore quite similar to circulating American varicella-zoster virus strains. Varicella-zoster virus DNA, at least in the area of the BamHI D fragment, also appears to be remarkably stable from strain to strain.

Recombination in tissue culture between varicella‐zoster virus strains

Several clinical varicella-zoster virus isolates obtained during testing of a live varicella vaccine had DNA restriction fragment patterns resembling neither vaccine nor wild-type virus [Gelb et al., J Infect. Dis. 155, 633-640, 1987]. One explanation for these isolates was recombination in vivo. To determine if such recombination is likely, two strains of varicella-zoster virus, distinguishable by restriction endonuclease fragment size differences (wild-type strain EF and the OKA vaccine strain), were grown together in tissue culture. After three passages, the mixed infection virus was plaque-purified. DNA from about 13% of the plaque-purified isolates had one or more BglI fragments found in neither parental virus. Hybridization studies showed that isolates containing one of the new BglI fragments were recombinants of the two parental strains. The BglI restriction fragment pattern of these recombinants resembled those of the unusual varicella isolates from individuals either vaccinated with the live attenuated OKA varicella vaccine and later exposed to natural varicella, or simultaneously exposed to both a recent recipient of the vaccine and natural varicella.

Molecular analysis of the thymidine kinase gene of thymidine kinase-deficient mutants of varicella-zoster virus.

Five thymidine kinase (TK)-deficient mutants of varicella-zoster virus derived from the Oka vaccine strain were examined to determine the state of transcription of the TK gene, and the DNA sequences of two of the mutants and the parent strain were investigated. Restriction endonuclease analysis revealed no major alteration of whole genome organization, but did reveal a minor change of the cleaved fragment in size. Northern blot analysis showed that the transcripts from the TK gene of all five mutants were of the same size as that of the parent strain (1.6 kb). DNA sequences of the TK gene of the parent strain and those of two mutants were determined. One had a base substitution (C to T) in the coding region of TK, resulting in the termination of amino acid elongation in the 68th amino acid of 341. The other had a two-base deletion (AT) in the coding region, resulting in a frame shift from the 126th amino acid and termination at the 162nd amino acid. Both mutants had premature stop codons which might result in the synthesis of nonfunctional TK polypeptides. Thus, the molecular basis of TK deficiency was determined.

Antiviral sensitivities of the acute retinal necrosis syndrome virus.

Varicella zoster was isolated from the vitreous of a patient with the acute retinal necrosis (ARN) syndrome. We utilized a plaque reduction assay to determine the in vitro susceptibility of the ARN isolate to 6 antiviral drugs. The effective doses for 50% inhibition of plaque numbers were 5.3 microM for for acyclovir, 4.7 microM for DHPG, 8.7 microM for ARA-A, 100.7 microM for phosphonoacetic acid, 0.07 microM for BVdU and 2.4 microM for IUdR. Similar inhibitory values were obtained for the OKA vaccine strain of varicella zoster virus. These data do not support the notion that the ARN strain may represent a mutant of varicella zoster virus with significant alterations in either the viral thymidase kinase or DNA polymerase genes based upon its antiviral sensitivities. The implications of these results regarding the role of antiviral chemotherapy in the ARN syndrome are discussed.

Analysis of Varicella-Zoster Virus DNAs of Clinical Isolates by Endonuclease HpaI

The DNAs of 20 strains of varicella-zoster virus (VZV) isolated from epidemiologically unrelated individuals, and of 15 strains isolated from vesicles of vaccinees with varicella or zoster after vaccination, were compared by restriction enzyme cleavage using HpaI. Differences were found in the sizes of the HpaI-F, -G and -K fragments of the wild strains. The gel migration patterns of the HpaI-F and -G fragments, but not of the HpaI-K fragment, were polymorphic in the different strains isolated from the vaccinees. The effects of serial passages in vitro and in humans on the genome stability of VZV were investigated by HpaI analysis. The DNA profiles of the HpaI-K fragments from six isolates recovered from room-mates infected in a single outbreak were identical, but the mobilities of their HpaI-F and -G fragments varied. The DNA profiles of the Oka vaccine virus after 10 and 85 passages in human embryo cells differed only in the HpaI-F fragment. The profiles of these fragments in DNA derived from two isolates obtained at different times from a vaccinee with varicella followed by zoster were compared with those of the Oka (parental) and Oka (vaccine) strains, and identical results were obtained for the two viruses. In addition, the same DNA profiles of HpaI fragments were obtained from three sequential isolates from one person and also from two isolates from another with varicella and zoster. Thus, it was concluded that: three variable fragments (HpaI-K, -F and -G) were not changed in the DNAs of isolates derived from the same patient; HpaI-K was stable both on passage in vitro and after human transmission in the case of the same outbreak, but was different among all wild-type strains isolated in epidemiologically unrelated outbreaks; HpaI-F was very unstable both on passage in vitro and in human infections by either vaccine or wild-type strains; HpaI-G was not influenced by passage in vitro but varied among wild-type strains. Using physical maps of VZV DNA established by others, three variable regions on the viral genome were identified. One was located near the 0.16 coordinate, which is covered by HpaI-K (variable region I, VRI). Another was represented by HpaI-F (VRII), the most unstable fragment, and mapped at about the 0.35 coordinate. The third was VRIII near the right terminus, covered by HpaI-G.