Vaccinia Virus Nicking-Joining Enzyme Is Encoded by K4L (VACWR035)

Vaccinia virus encodes an enzyme with DNA modifying activity that cleaves and inefficiently cross-links cruciformic DNA. This enzyme is contained within the virion, expressed at late times postinfection, and processes DNA in an energy-independent, Mg(2+) ion-independent manner. Viral nuclease activity was measured in extracts from cells infected with well-defined viral mutants. Since some viral extracts lacked nuclease activity, the gene encoding the activity was postulated to be one of the open reading frames absent in the viruses lacking activity. Inducible expression of each candidate open reading frame revealed that only the gene VACWR035, or K4L, was required for nuclease activity. A recombinant virus missing only the open reading frame for K4L lacked nuclease activity. Extracts from a recombinant virus expressing K4L linked to a FLAG polypeptide were able to cleave and cross-link cruciformic DNA. There were no significant differences between the virus lacking K4L and wild-type vaccinia virus WR with respect to infectivity, growth characteristics, or processing of viral replicative intermediate DNA, including both telomeric and cross-linked forms. Purification of the K4L FLAG polypeptide expressed in bacteria yielded protein containing nicking-joining activity, implying that K4L is the only vaccinia virus protein required for the nicking-joining enzymatic activity

Characterization and Use of Mammalian-Expressed Vaccinia Virus Extracellular Membrane Proteins for Quantification of the Humoral Immune Response to Smallpox Vaccines▿

The licensed smallpox vaccine Dryvax is used as the standard in comparative immunogenicity and protection studies of new smallpox vaccine candidates. Although the correlates of protection against smallpox are unknown, recent studies have shown that a humoral response against the intracellular mature virion and extracellular enveloped virion (EV) forms of vaccinia virus is crucial for protection. Using a recombinant Semliki Forest virus (rSFV) vector system, we expressed a set of full-length EV proteins for the development of EV antigen-specific enzyme-linked immunosorbent assays (ELISAs) and the production of monospecific antisera. The EV-specific ELISAs were used to evaluate the EV humoral response elicited by Dryvax and the nonreplicating modified vaccinia virus Ankara (MVA) in mouse vaccination experiments comparing doses and routes of vaccination. Quantitatively similar titers of antibodies against EV antigens A33R, A56R, and B5R were measured in mice vaccinated with Dryvax and MVA when MVA was administered at a dose of 108 plaque-forming units. Further, a substantial increase in the EV-specific antibody response was induced in mice inoculated with MVA by using a prime-boost schedule. Finally, we investigated the abilities of the EV-expressing rSFV vectors to elicit the production of polyclonal monospecific antisera against the corresponding EV proteins in mice. The monospecific serum antibody levels against A33R, A56R, and B5R were measurably higher than the antibody levels induced by Dryvax. The resulting polyclonal antisera were used in Western blot analysis and immunofluorescence assays, indicating that rSFV particles are useful vectors for generating monospecific antisera

Percutaneous Vaccination as an Effective Method of Delivery of MVA and MVA-Vectored Vaccines

<div><p>The robustness of immune responses to an antigen could be dictated by the route of vaccine inoculation. Traditional smallpox vaccines, essentially vaccinia virus strains, that were used in the eradication of smallpox were administered by percutaneous inoculation (skin scarification). The modified vaccinia virus Ankara is licensed as a smallpox vaccine in Europe and Canada and currently undergoing clinical development in the United States. MVA is also being investigated as a vector for the delivery of heterologous genes for prophylactic or therapeutic immunization. Since MVA is replication-deficient, MVA and MVA-vectored vaccines are often inoculated through the intramuscular, intradermal or subcutaneous routes. Vaccine inoculation via the intramuscular, intradermal or subcutaneous routes requires the use of injection needles, and an estimated 10 to 20% of the population of the United States has needle phobia. Following an observation in our laboratory that a replication-deficient recombinant vaccinia virus derived from the New York City Board of Health strain elicited protective immune responses in a mouse model upon inoculation by tail scarification, we investigated whether MVA and MVA recombinants can elicit protective responses following percutaneous administration in mouse models. Our data suggest that MVA administered by percutaneous inoculation, elicited vaccinia-specific antibody responses, and protected mice from lethal vaccinia virus challenge, at levels comparable to or better than subcutaneous or intramuscular inoculation. High titers of specific neutralizing antibodies were elicited in mice inoculated with a recombinant MVA expressing the herpes simplex type 2 glycoprotein D after scarification. Similarly, a recombinant MVA expressing the hemagglutinin of attenuated influenza virus rgA/Viet Nam/1203/2004 (H5N1) elicited protective immune responses when administered at low doses by scarification. Taken together, our data suggest that MVA and MVA-vectored vaccines inoculated by scarification can elicit protective immune responses that are comparable to subcutaneous vaccination, and may allow for antigen sparing when vaccine supply is limited.</p></div

Protection of mice following single dose percutaneous or subcutaneous vaccination with MVA-HA.

<p>Protection of mice following single dose percutaneous or subcutaneous vaccination with MVA-HA.</p

Pathogenesis of influenza rgA/Viet Nam/1203/04 virus after single low dose vaccination via the SC and PC routes.

<p>Groups of mice were vaccinated with a single dose of MVA-HA at doses of 10², 10³, or 10⁴ pfu, via the subcutaneous and percutaneous routes. A control group was vaccinated subcutaneously with 10⁴ pfu of MVA vector. All mice were challenged with influenza rgA/Viet Nam/1203/2004 virus three weeks after vaccination and weighed daily for 2 weeks. Mean weight changes after challenge are shown. Each data point is the average of two independent experiments. Error bars represent standard deviation.</p

Antibody response and protection after MVA and ACAM2000 inoculation by tail scarification.

<p>Antibody response and protection after MVA and ACAM2000 inoculation by tail scarification.</p

Evaluation of vaccination routes by low dose treatment with MVA-HA.

<p>Mice in groups of 5 were vaccinated with MVA or MVA-HA at the doses indicated in the legend. Booster vaccinations were administered after 3 weeks, and mice were challenged with 10⁶ pfu of influenza rgA/Viet Nam/1203/2004 (H5N1), three weeks after the second vaccination. Mice were weighed for 14 days post challenge. Weight loss as a measure of disease severity is shown in <b>A</b>, and the proportion of surviving animals in the different treatment groups are represented in <b>B</b>.</p

Antibody responses and protection conferred by MVA-HA inoculated via the subcutaneous or percutaneous route.

<p>Mice in groups of five were vaccinated with 10⁴, 10⁵, or 10⁶ pfu of MVA-HA via the SC and PC routes. A control group (5 mice) was vaccinated with 10⁶ pfu of MVA. Mice were vaccinated twice at an interval of three weeks between vaccinations. Post-vaccination antisera obtained 3 weeks after the boost were tested for H5-specific IgG by ELISA. The mean IgG titers are presented (<b>A</b>), with error bars representing standard deviation. Mice were challenged with 10⁶ pfu of rgA/Viet Nam/1203/2004. Weight changes (%) post-challenge are shown in (<b>B)</b>. A “<b>+</b>” sign represents a mouse that succumbed to infection.</p