Neutralization Assay Using a Modified Vaccinia Virus Ankara Vector Expressing the Green Fluorescent Protein Is a High-Throughput Method To Monitor the Humoral Immune Response against Vaccinia Virus

Vaccination against smallpox is again considered in order to face a possible bioterrorist threat, but the nature and the level of the immune response needed to protect a person from smallpox after vaccination are not totally understood. Therefore, simple, rapid, and accurate assays to evaluate the immune response to vaccinia virus need to be developed. Neutralization assays are usually considered good predictors of vaccine efficacy and more informative with regard to protection than binding assays. Currently, the presence of neutralizing antibodies to vaccinia virus is measured using a plaque reduction neutralization test, but this method is time-consuming and labor-intensive and has a subjective readout. Here, we describe an innovative neutralization assay based on a modified vaccinia virus Ankara (MVA) vector expressing the green fluorescent protein (MVA-gfp). This MVA-gfp neutralization assay is rapid and sensitive and has a high-throughput potential. Thus, it is suitable to monitor the immune response and eventually the efficacy of a large campaign of vaccination against smallpox and to study the vector-specific immune response in clinical trials that use genetically engineered vaccinia viruses. Most importantly, application of the highly attenuated MVA eliminates the safety concern in using the replication-competent vaccinia virus in the standard clinical laboratory.     

Enhanced immunogenicity and protective effect conferred by vaccination with combinations of modified vaccinia virus Ankara and licensed smallpox vaccine Dryvax in a mouse model

Significant adverse events are associated with vaccination with the currently licensed smallpox vaccine. Candidate new-generation smallpox vaccines such as the replication-defective modified vaccinia virus Ankara (MVA) produce very few adverse events in experimental animals and in limited human clinical trials conducted near the end of the smallpox eradication campaign. Efficacy evaluation of such new-generation vaccines will be extraordinarily complex, however, since the eradication of smallpox precludes a clinical efficacy trial and the correlates of protection against smallpox are unknown. A combination of relevant animal efficacy studies along with thorough comparative immunogenicity studies between traditional and new-generation smallpox vaccines will be necessary for vaccine licensure. In the present study, a variety of immune responses elicited by MVA and the licensed smallpox vaccine Dryvax in a murine model were compared, with a focus on mimicking conditions and strategies likely to be employed in human vaccine trials. Immunization of mice with MVA, using several relevant vaccination routes including needle-free delivery, elicited humoral and cellular immune responses qualitatively similar to those elicited by vaccination with Dryvax. Similar levels of vaccinia-specific IgG and neutralizing antibody were elicited by Dryvax and MVA when higher doses (approximately 1 log) of MVA were used for immunization. Antibody levels peaked at about 6 weeks post-immunization and remained stable for at least 15 weeks. A booster immunization of either MVA or Dryvax following an initial priming immunization with MVA resulted in an enhanced IgG titer and neutralizing antibody response. In addition, both Dryvax and various MVA vaccination protocols elicited antibody responses to the extracellular enveloped form of the virus and afforded protection against a lethal intranasal challenge with vaccinia virus WR.     

Identification and preliminary characterization of vaccinia virus (Dryvax) antigens recognized by vaccinia immune globulin

Using vaccinia immune globulin (VIG), a high-titer antibody preparation from immunized subjects, we demonstrate that the humoral immune response in humans is directed against numerous antigens in the Dryvax vaccine strain. Western blot and immunoprecipitation analyses revealed highly antigenic proteins associated with both the extracellular enveloped virus and intracellular mature virus forms. The modified vaccinia virus Ankara (MVA), a new generation smallpox vaccine that is attenuated for replication in humans, expresses most, but not all, of the major vaccinia antigens recognized by antibodies in VIG, lacking the highly antigenic protein corresponding to the A-type inclusion body protein. Since new-generation smallpox vaccines such as MVA will require extensive comparison to traditional smallpox vaccines in animal models of immunogenicity and protection, we compared the vaccinia virus antigens recognized by VIG to those recognized by sera from Dryvax and MVA immunized mice. The humoral immune response in immunized mice is qualitatively similar to that in humans.     

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 10(8) 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.     

A rapid, high-throughput vaccinia virus neutralization assay for testing smallpox vaccine efficacy based on detection of green fluorescent protein

Virus neutralization remains a vital tool in assessment of vaccine efficacy for smallpox in the absence of animal smallpox models. In this regard, development of a rapid, sensitive, and high-throughput vaccinia neutralization assay has been sought for evaluating alternative smallpox vaccines, use in bridging studies, as well as understanding the effects of anti-viral immunotherapeutic regimes. The most frequently used method of measuring vaccinia virus neutralization by plaque reduction is time, labor, and material intensive, and therefore limiting in its utility for large scale, high-throughput analysis. Recent advances provide alternative methods that are less labor intensive and higher throughput but with limitations in reagents needed and ease of use. An innovative neutralization assay is described based on a modified Western Reserve vaccinia vector expressing green fluorescent protein (WR-GFP) and an adherent cell monolayer in multi-well plate format. The assay is quick, accurate, provides a large dynamic range and is well suited for large-scale vaccination studies using standard adherent cell lines.     

Microarray assay for evaluation of the genetic stability of modified vaccinia virus Ankara B5R gene

Adverse events associated with the use of live smallpox vaccines have led to the development of a new generation of attenuated smallpox vaccines that are prepared in cultured cells as alternatives. The inability to conduct direct clinical evaluation of their efficacy in humans demands that licensure be based on animal studies and exhaustive evaluation of their in vitro properties. One of the most important characteristics of live viral vaccines is their genetic stability, including reversion of the vaccine strain to more virulent forms, recombination with other viral sequences to produce potentially pathogenic viruses, and genetic drift that can result in decrease of immunogenicity and efficacy. To study genetic stability of an immunoessential vaccinia virus gene in a new generation smallpox vaccine, an advanced oligonucleotide microchip was developed and used to assay for mutations that could emerge in B5R gene, a vaccinia virus gene encoding for a protein that contains very important neutralizing epitopes. This microarray contained overlapping oligonucleotides covering the B5R gene of modified vaccinia virus Ankara (MVA), a well-studied candidate smallpox vaccine. The microarray assay was shown to be able to detect even a single point mutation, and to differentiate between vaccinia strains. At the same time, it could detect newly emerged mutations in clones of vaccinia strains. In the work described here, it was shown that MVA B5R gene was stable after 34 passages in Vero and MRC-5 cells that were proposed for use as cell substrates for vaccine manufacture. Potentially, the proposed method could be used as an identity test and could be extended for the entire viral genome and used to monitor consistency of vaccine production.     

DNA/MVA HIV-1/AIDS vaccine elicits long-lived vaccinia virus-specific immunity and confers protection against a lethal monkeypox challenge

Modified vaccinia Ankara (MVA) is being tested in humans as an alternative to the current smallpox vaccine Dryvax. Here, we compare the magnitude and longevity of protective immune responses elicited by a DNA/MVA HIV-1 vaccine with those elicited by Dryvax using a monkeypox virus/macaque model. The DNA/MVA vaccine elicited similar levels of vaccinia virus (VV)-specific antibody and 5-10-fold lower levels of VV-specific cellular responses than Dryvax. This MVA-elicited cellular and humoral immunity was long-lived. A subset of the DNA/MVA- and Dryvax-vaccinated macaques were subjected to a lethal monkeypox virus challenge at 3 years after vaccination. All of the vaccinated monkeys survived, whereas the unvaccinated controls succumbed to monkeypox. The viral control correlated with early postchallenge levels of monkeypox-specific neutralizing antibody but not with VV-specific cellular immune response. Thus, our results demonstrate the elicitation of long lasting protective immunity for a lethal monkeypox challenge by a DNA/MVA HIV-1 vaccine.     

Absence of oropharyngeal vaccinia virus after vaccinia (smallpox) vaccination.

BACKGROUND: With the resumption of the vaccinia (smallpox) vaccination, questions regarding transmission risk prompted this study to determine whether vaccinia virus could be detected in the oropharynx of adults recently vaccinated with vaccinia (smallpox) vaccine. German, Russian, and American studies on the oropharyngeal presence of vaccinia virus revealed conflicting results in different age groups. OBJECTIVE: To measure vaccinia viral particle or antigen presence in the oropharynx of adult health care workers after vaccination with vaccinia (smallpox) vaccine using viral culture and high-sensitivity assays (polymerase chain reaction [PCR] and electrochemiluminescence) and to determine whether there is an association between the presence of vaccinia virus and adverse reactions. METHODS: A total of 155 adults (primary vaccinees and revaccinees) were enrolled for 1 baseline and 5 subsequent throat swabs. The swabs were evaluated using viral culture, PCR, and electrochemiluminescence. RESULTS: Of the 155 participants, 144 had more than 2 throat swabs in the 2 weeks after vaccination. Of the 801 specimens evaluated, there were no positive results by culture, PCR, or electrochemiluminescence except in the control samples (n = 6), which were positive by all 3 methods. CONCLUSIONS: Based on the absence of detectable vaccinia virus in this study population, one can be 95% certain that the true rate of vaccinia virus in the oropharynx of adults during the 2 weeks after vaccination with vaccinia (smallpox) vaccine is 0% to 3.3%. These data should be reassuring to the medical community and support the Advisory Committee on Immunization Practice guidelines that respiratory precautions are not necessary after vaccinia (smallpox) vaccination in healthy adults.     

T cells from a high proportion of apparently naive cattle can be activated by modified vaccinia virus Ankara (MVA)

Modified vaccinia virus Ankara (MVA) was used as a vector to express genes from bovine respiratory syncytial virus (BRSV). Using these recombinant viruses as recall antigens for cells from BRSV-immuned cattle proved to be problematic because non-recombinant MVA itself frequently stimulated high levels of T lymphocyte activation. This phenomenon was observed in a high percentage of cattle from multiple herds. Gamma delta TCR(+) T cells were more sensitive to activation by MVA than other classes of T cells. A serological assay for MVA neutralization detected low, fluctuating titers of serum virus neutralizing (SVN) activity toward MVA in some cattle, but these were lower titers than those observed in cattle that underwent MVA vaccination. T cell reactivity in non-vaccinated cattle did not correlate significantly (p > 0.05) with SVN activity, undermining the notion that any adaptive immune response was responsible for the observed T cell sensitivity. More probable explanations are that MVA has mitogenic or superantigenic properties, or that the virus induces gammadelta TCR(+) T cell activation through interactions with innate pattern recognition receptors.     

B5-deficient vaccinia virus as a vaccine vector for the expression of a foreign antigen in vaccinia immune animals

Recombinant vaccinia viruses have shown promise as vaccine vectors. However, their effectiveness is markedly reduced by pre-existing anti-vaccinia immunity. The possibility of new vaccinia immunizations in the event of a bioterror-related smallpox release poses an additional negative impact on the utility of vaccinia-based vectors. Thus, we aimed to design a vaccinia vector that would enhance the immune response to an expressed foreign protein in a pre-immune animal model. To do this, we made use of the finding that most neutralizing antibodies against the extracellular form of vaccinia virus are directed against the B5 protein. We found that mice immunized with vaccinia, primed with Gag plasmid DNA, and boosted with a recombinant vaccinia virus lacking the majority of the B5 ectodomain expressing a test antigen, HIV Gag, had stronger anti-Gag immune responses than mice that were boosted with a wild-type virus-expressing Gag. These findings are particularly striking given the more attenuated phenotype of this virus, as compared to its wild-type counterpart. Importantly, we found that vaccination with a B5R deletion virus, followed by boosting with the Gag-expressing virus lacking the majority of the B5 ectodomain, resulted in poorer anti-Gag immune responses. Thus, recombinant vaccinia viruses lacking the B5 ectodomain may serve as vaccine vectors in DNA prime-vaccinia boost vaccinations of individuals with pre-existing immunity against vaccinia. These data open the possibility of extending the potential benefit of replication competent recombinant vaccinia virus vectors to a larger population.     

Long-term persistence of the vaccinia virus in a child with a congenital skin disease

Persistence of vaccinia virus which was isolated many times from the blood and skin lesions was discovered in a child vaccinated 6 years before against smallpox and suffering from bullous epidermolysis. The level of immunoglobulins A, M, and G was normal, whereas the titre of virus-neutralizing antibodies against smallpox was low (1:10) in the child, and reached 1:320 in his mother not vaccinated against smallpox. Humoral immunity for other antigens was found to be unimpaired, the skin tests being positive. It was concluded that the child had a selective defect of the immune system. It is emphasized that the virus excretor is epidemically hazardous for subjects with skin diseases, immunodeficient conditions and others, in whom contraction of the infection may result in a disease the identification of the genesis of which would be extremely difficult against the background of discontinued vaccination against smallpox.     

Analysis of Variola and Vaccinia Virus Neutralization Assays for Smallpox Vaccines

Possible smallpox reemergence drives research for third-generation vaccines that effectively neutralize variola virus. A comparison of neutralization assays using different substrates, variola and vaccinia (Dryvax and modified vaccinia Ankara [MVA]), showed significantly different 90% neutralization titers; Dryvax underestimated while MVA overestimated variola neutralization. Third-generation vaccines may rely upon neutralization as a correlate of protection.     

The nonreplicating smallpox candidate vaccines defective vaccinia Lister (dVV-L) and modified vaccinia Ankara (MVA) elicit robust long-term protection

Current smallpox vaccines are live vaccinia viruses that replicate in the vaccinee inducing immunity against the deadly disease smallpox. Replication resulting in virus spread within the host, however, is the major cause of severe postvaccinal adverse events. Therefore, attenuated strains such as modified vaccinia Ankara (MVA) or LC16m8 are candidates as next generation vaccines. These strains are usually grown in primary cells in which mass production is difficult and have an unknown protective potential in humans. Proven vaccine strains of defined origin and modern production techniques are therefore desirable. In this study, defective vaccinia virus (dVV) lacking a gene essential for replication (derived from the Lister vaccine in a complementing cell line) was compared with the Wyeth smallpox vaccine strain and with MVA in mouse animal models using cowpox and ectromelia virus challenge. Similar to MVA, prime-boost immunizations with defective vaccinia induced robust long-term immunity, suggesting it as a promising next generation smallpox vaccine.     

Recombinant modified vaccinia virus Ankara provides durable protection against disease caused by an immunodeficiency virus as well as long-term immunity to an orthopoxvirus in a non-human primate

Recombinant and non-recombinant modified vaccinia virus Ankara (MVA) strains are currently in clinical trials as human immunodeficiency virus-1 (HIV) and attenuated smallpox vaccines, respectively. Here we tested the ability of a recombinant MVA delivered by alternative needle-free routes (intramuscular, intradermal, or into the palatine tonsil) to protect against immunodeficiency and orthopoxvirus diseases in a non-human primate model. Rhesus macaques were immunized twice 1 month apart with MVA expressing 5 genes from a pathogenic simian human immunodeficiency virus (SHIV)/89.6P and challenged intrarectally 9 months later with the pathogenic SHIV/89.6P and intravenously 2.7 years later with monkeypox virus. Irrespective of the route of vaccine delivery, binding and neutralizing antibodies and CD8 responses to SHIV and orthopoxvirus proteins were induced and the monkeys were successively protected against the diseases caused by the challenge viruses in unimmunized controls as determined by viral loads and clinical signs. These non-human primate studies support the clinical testing of recombinant MVA as an HIV vaccine and further demonstrate that MVA can provide long-term poxvirus immunity, essential for use as an alternative smallpox vaccine.     

Enveloped virus is the major virus form produced during productive infection with the modified vaccinia virus Ankara strain

Modified vaccinia virus Ankara (MVA) is a highly attenuated virus strain that may be useful as a vaccine vector. Ultrastructural examination of purified MVA showed that most of the viral particles are enveloped in contrast to the Copenhagen strain (COP). In CsCl gradients, the majority of the MVA particles displayed a light buoyant density characteristic of the enveloped form. Consistent with these results, MVA particles were poorly labeled with antibodies against the surface of intracellular mature virus but strongly labeled with antibodies against an envelope antigen. Furthermore, MVA was more resistant than the COP strain to neutralization by mouse anti-COP antibodies. These results suggest that the MVA strain may be particularly suitable for the engineering of envelope proteins and that MVA may be able to resist the humoral immunity displayed by previously vaccinated individuals.     

Detection of vaccinia virus DNA on the LightCycler by fluorescence melting curve analysis

After eradication of variola virus the worldwide vaccination program was stopped to avoid the severe complications observed in a small fraction of vaccinees. Hence, there is at least one non-vaccinated generation in the human population that is immunologically naive with respect to variola virus infections. The possibility of a deliberate release of variola virus by bioterrorist attacks has led to the resumption of vaccination of hospital employees and military personnel with vaccinia virus in certain parts of the world. However, the appearance of a single confirmed smallpox case worldwide would result in vaccination of possible contact persons with vaccinia virus. Therefore, reliable confirmation of vaccinia virus in patients presenting with smallpox-like syndromes is required. A vaccinia virus-specific single nucleotide polymorphism was identified in the gene B8R coding for a vaccinia virus IFNgamma receptor. Based on this polymorphism, the LightCycler real-time PCR assay detects vaccinia virus DNA in a linear range from 10(6) to 10 genome equivalents and discriminates vaccinia virus from other orthopoxviruses by fluorescence melting curve analysis (DeltaT = 9 degrees C). While the assay amplifies generically DNA of all orthopoxviruses tested, amplification curves are only displayed for vaccinia virus strains including strains formerly used for vaccination. In addition, an internal amplification control is described that allows reliable interpretation of results.     

The N-terminal domain of the vaccinia virus E3L-protein is required for neurovirulence, but not induction of a protective immune response

Encephalitis is a rare, but serious complication from vaccination against smallpox using replication competent strains of vaccinia virus. In this report we describe mutants of vaccinia virus, containing N-terminal deletions of the vaccinia virus interferon resistance gene, E3L, that are attenuated for neuropathogenesis in a mouse model system. These recombinant viruses replicated to high titers in the nasal mucosa after intra-nasal infection of C57BL/6 mice but failed to spread to the lungs or brain. These viruses demonstrated reduced pathogenicity after intra-cranial infection as well, indicating a decrease in neurovirulence. Intra-nasal inoculation or inoculation by scarification with a low dose of recombinant virus containing a deletion of the entire N-terminal domain of E3L protected against challenge with a high dose of wild-type vaccinia virus, suggesting that this replication competent, but attenuated strain of vaccinia virus may have promise as an improved vaccine for protecting against smallpox, and as a vector for inducing mucosal immunity to heterologous pathogenic organisms.     

Development of a tissue-culture-based enzyme-immunoassay method for the quantitation of anti-vaccinia-neutralizing antibodies in human sera

Vaccination with vaccinia virus is carried out in order to induce protection against variola virus, the causative agent of smallpox. Serum titer of vaccinia virus-neutralizing antibodies is considered to be well-correlated with in vivo protection. Plaque reduction neutralization test (PRNT) is the gold standard for detecting and quantifying vaccinia virus-neutralizing antibodies in sera of vaccinees. However, PRNT is time and labor consuming, which does not allow large-scale screening needed for a population survey. A simplified, sensitive, standardized, reproducible and rapid method, neutralization tissue-culture enzyme immunoassay (NTC–EIA) was developed for quantitation of neutralizing antibodies against vaccinia virus. The assay consists of the following steps: neutralization of the virus with serially diluted sera, infection of cells in culture and measurement of residual virus replication using an enzyme immunoassay. The assay can be used for animal (rabbit) or human sera. Titer averages obtained using NTC–EIA were highly correlated (R² = 0.9994) to those obtained using PRNT. The assay is carried out in 96-well plates and takes only 2 days to complete. With the appropriate setup, it can be automated fully to allow screening of a large number of sera.     

Evaluation of a needle-free delivery platform for prime-boost immunization with DNA and modified vaccinia virus ankara vectors expressing herpes simplex virus 2 glycoprotein D

A previous report described a prime-boost immunization strategy using plasmid and modified vaccinia virus Ankara (MVA) vectors expressing herpes simplex virus 2 glycoprotein D (gD). Enhanced humoral and cellular immune responses were elicited by the prime-boost combination compared to plasmid DNA immunization alone. Surprisingly, a more diverse antibody isotype response, and a greater antibody and cellular immune response, was obtained if the gD MVA vector was used as the priming immunization rather than the gD plasmid vector. The present report evaluates the use of a needle-free delivery platform (Biojector) for delivery of plasmid and MVA gD-expressing vectors in a prime-boost immunization strategy. Needle-free delivery of both plasmid and MVA gD expression vectors was efficient, reproducible, and elicited a strong immune response in immunized mice. Biojector delivery of plasmid DNA was able to evoke a broader isotype response and cellular immune response than that obtained by gene gun delivered plasmid DNA. Further, DNA priming by Biojector delivery as part of a prime-boost procedure with MVA-gD2 resulted in a diverse antibody isotype distribution and enhanced cellular immune responses, similar to the responses obtained when MVA-gD2 was used as the priming immunization. Thus, needle-free delivery of plasmid DNA may provide additional flexibility and options for effective prime-boost vaccination.     

A vaccinia virus renaissance: New vaccine and immunotherapeutic uses after smallpox eradication

In 1796, Edward Jenner introduced the concept of vaccination with cowpox virus, an Orthopoxvirus within the family Poxviridae that elicits cross protective immunity against related orthopoxviruses, including smallpox virus (variola virus). Over time, vaccinia virus (VACV) replaced cowpox virus as the smallpox vaccine, and vaccination efforts eventually led to the successful global eradication of smallpox in 1979. VACV has many characteristics that make it an excellent vaccine and that were crucial for the successful eradication of smallpox, including (1) its exceptional thermal stability (a very important but uncommon characteristic in live vaccines), (2) its ability to elicit strong humoral and cell-mediated immune responses, (3) the fact that it is easy to propagate, and (4) that it is not oncogenic, given that VACV replication occurs exclusively within the host cell cytoplasm and there is no evidence that the viral genome integrates into the host genome. Since the eradication of smallpox, VACV has experienced a renaissance of interest as a viral vector for the development of recombinant vaccines, immunotherapies, and oncolytic therapies, as well as the development of next-generation smallpox vaccines. This revival is mainly due to the successful use and extensive characterization of VACV as a vaccine during the smallpox eradication campaign, along with the ability to genetically manipulate its large dsDNA genome while retaining infectivity and immunogenicity, its wide mammalian host range, and its natural tropism for tumor cells that allows its use as an oncolytic vector. This review provides an overview of new uses of VACV that are currently being explored for the development of vaccines, immunotherapeutics, and oncolytic virotherapies.