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.     

FL-CTL assay: fluorolysometric determination of cell-mediated cytotoxicity using green fluorescent protein and red fluorescent protein expressing target cells

Cytotoxic T lymphocytes (CTLs) are crucial effectors against intracellular pathogens and cancer. Accurate and efficient assessment of CTL activity is important for basic and clinical studies. Widely used CTL assays, including the chromium release, JAM test and ELISPOT, involve either radioisotopes or lengthy procedures. Here, we developed a new fluorolysometric CTL assay based on cell-mediated cytolysis of fluorescent protein (GFP or DsRed) expressing cells quantified by one of the fluoro-based methods: flow cytometry, fluorescence microplate reader, or fluorescence microscopy. With flexible detection methods and lentiviral vector transduced stable lines of either GFP⁺ or DsRed⁺ cells as targets for antigen presentation and equal number of the other as internal reference for consistency and accuracy, this assay is easy to perform and to scale-up for simultaneous multi-sample analyses. Using two different antigen systems, we demonstrated that this assay is very sensitive to determine primary CTL activity of both in vitro and in vivo primed antigen-specific T cells. Thus, this FL-CTL assay is highly sensitive, reliable, reproducible, economical, convenience and supports broad applications compared to conventional CTL assays.     

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.     

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.     

Studies using a recombinant vaccinia virus expressing the circumsporozoite protein of Plasmodium berghei.

A recombinant vaccinia virus was constructed which expressed the circumsporozoite protein of Plasmodium berghei. Four different strains of mice belonging to different haplotypes were immunized with the recombinant virus. The antibody response to the circumsporozoite protein as well as to vaccinia virus varied among the strains, independently of each other. The anti-circumsporozoite protein titers were comparable to that obtained on immunization with irradiated sporozoites. Spleen cells from H2d mice immunized with P. berghei sporozoites showed a significant proliferative response when cultured in vitro with a low multiplicity of the recombinant vaccinia virus. A weak cytotoxic T lymphocyte response specifically targeting the circumsporozoite protein could be identified in spleens of BALB/c (H2d) mice immunized with vaccinia virus when BALB 3T3 cells transformed with a plasmid expressing the circumsporozoite protein under control of the simian virus 40 promoter were used as target cells in the cytotoxic T lymphocyte assay. However, none of the recombinant virus-immunized animals could be protected from a challenge of sporozoites even at the lowest dose of parasite used.     

Dissecting the humoral immune response to simian immunodeficiency virus

The ultimate goal of an AIDS vaccine is to elicit potent cellular and humoral immune responses that will result in broadly enduring protective immunity. During the past several years, we have focused on characterizing the quantitative and qualitative properties of the antibody response, principally working to define the mechanism(s) of antibody-mediated neutralization in vitro. We have utilized a panel of monoclonal antibodies generated from monkeys infected with attenuated SIV for more than 8 mo to dissect the early events of virus infection involved in antibody-mediated neutralization. Presented herein are highlights from our studies that have identified potential mechanisms by which antibodies neutralize SIV in vitro.     

Heterologous priming-boosting with DNA and modified vaccinia virus Ankara expressing tryparedoxin peroxidase promotes long-term memory against Leishmania major in susceptible BALB/c Mice

Leishmaniasis affects 12 million people, but there are no vaccines in routine clinical use. Th1 polarizing vaccines that elicit long-term protection are required to prevent disease in susceptible populations. We recently showed that heterologous priming-boosting with tryparedoxin peroxidase (TRYP) DNA followed by TRYP-modified vaccinia virus Ankara (TRYP MVA) protected susceptible BALB/c mice from Leishmania major. Here we compared treatment with TRYP DNA with treatment with TRYP DNA/TRYP MVA. We found that equivalent levels of protection during the postvaccination effector phase correlated with equivalent levels of serum immunoglobulin G2a and gamma interferon (IFN-gamma) in draining lymph nodes. In contrast, challenge infection during the memory phase revealed that there was enhanced clinical efficacy with TRYP DNA/TRYP MVA. This correlated with higher levels of effector phase splenic IFN-gamma, sustained prechallenge levels of memory phase IFN-gamma, and a more polarized post-L. major challenge Th1 response compared to the Th2/T(reg) response. Thus, TRYP DNA/TRYP MVA, but not TRYP DNA alone, provides long-term protection against murine leishmaniasis.     

Clinical significance of the humoral immune response to modified LDL

Human low density lipoprotein (LDL) undergoes oxidation and glycation in vivo. By themselves, oxidized LDL (oxLDL) and AGE-LDL have proinflammatory properties and are considered atherogenic. But the atherogenicity of these lipoproteins are significantly increased as a consequence of the formation of immune complexes (IC) involving specific autoantibodies. OxLDL and AGE antibodies have been shown to be predominantly of the IgG1 and IgG3 isotypes. OxLDL antibodies are able to activate the complement system by the classical pathway and to induce FcR-mediated phagocytosis. In vitro and ex vivo studies performed with modified LDL-IC have proven their pro-inflammatory and atherogenic properties. Clinical studies have demonstrated that the levels of circulating modified LDL-IC correlate with parameters indicative of cardiovascular and renal disease in diabetic patients and other patient populations. The possibility that spontaneously formed or induced modified LDL antibodies (particularly IgM oxLDL antibodies) may have a protective effect has been suggested, but the data is unclear and needs to be further investigated.     

Interleukin 17 modulates the immune response to vaccinia virus infection

Interleukin 17 (IL-17) is a newly identified cytokine that has a homolog in herpesvirus saimiri. We inserted murine IL-17 into vaccinia virus to study the role of IL-17 in viral infection. Vaccinia virus expressing IL-17 (vv-IL17) and its parental control virus (vv-pRB) grew to similar titers in vitro; however, vv-IL17 was more virulent in mice with a threefold lower LD(50) than for vv-pRB, and mean time to death of 2.8 days versus 4.5 days. Mice infected with vv-IL17 had higher titers of virus in the ovaries (P < 0.02) and showed a decrease in NK cell cytotoxicity (P < 0.02) on day 3 after infection. No significant difference was found in CTL activity. In addition, a significant decrease in IgG2a (P < 0.01) and increases in IgG1, IgG3, and IgA (P < 0.03) vaccinia virus-specific antibody titers were observed in mice infected with vv-IL17 versus vv-pRB, suggesting a Th-2-like response to infection. These data indicate that IL-17 modulates the immune response during virus infection.     

A neutralization test for specific detection of Nipah virus antibodies using pseudotyped vesicular stomatitis virus expressing green fluorescent protein

Nipah virus (NiV) is a new zoonotic paramyxovirus that emerged in 1998 and is now classified in the genus Henipavirus along with the closely related Hendra virus (HeV). NiV is highly pathogenic in several vertebrate species including humans, and the lack of available vaccines or specific treatment restricts it to biosafety level 4 (BSL4) containment. A serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing green fluorescent protein (GFP) and bearing the F and G proteins of NiV (VSV–NiV–GFP). The neutralization titers were obtained by counting GFP-expressing cells or by measuring fluorescence. The performance of this new assay was compared against the conventional test using live NiV with panels of sera from several mammalian species, including sera from NiV outbreaks, experimental infections, as well as HeV-specific sera. The results obtained with the VSV–NiV–GFP based test correlated with those obtained using live NiV. Using a 50% reduction in VSV–NiV–GFP infected cells as the cut-off for neutralization, this new assay demonstrated its potential as an effective tool for detecting NiV neutralizing antibodies under BSL2 containment with greater speed, sensitivity and safety as compared to the conventional NiV serum neutralization test.     

Subcutaneous administration of modified vaccinia virus Ankara expressing an Ag85B-ESAT6 fusion protein, but not an adenovirus-based vaccine, protects mice against intravenous challenge with Mycobacterium tuberculosis

Recombinant virus-based tuberculosis (TB) vaccines that are strongly immunogenic and elicit robust cellular immunity are considered ideal vaccine candidates. Here, we engineered a poxvirus-based vaccine, MVA85B-E6, and an adenovirus-based vaccine, AD85B-E6, both of which express the fusion protein Ag85B-ESAT6. Subcutaneous vaccination of AD85B-E6 generated strong interferon (IFN)-γ production by both CD4 and CD8 T cells and CD8 cytotoxic T lymphocyte activity; these results indicate that strong T-helper type 1 immune responses were elicited in mice, which is in contrast to the moderate responses induced by vaccination with MVA85B-E6. However, MVA85B-E6 given subcutaneously led to levels of protection comparable with that induced by the bacillus Calmette-Guérin vaccine in the lungs and spleens, whereas AD85B-E6 given subcutaneously did not show any protective efficacy after intravenous challenge of BALB/c mice with Mycobacterium tuberculosis H37Rv. Our study emphasizes that more efficient biomarkers for vaccine efficacy and more appropriate routes of vaccine administration are necessary for the development of a successful TB vaccine.     

Tracing functionally identified neurones in a multisynaptic pathway in the hamster and rat using herpes simplex virus expressing green fluorescent protein

Using a genetically modified herpes simplex virus encoding green fluorescent protein we sought to establish if this viral modification could be used in transneuronal tracing studies of the sympathetic nervous system. The herpes simplex virus encoding green fluorescent protein was injected into the adrenal medulla of three hamsters and six rats. After a suitable survival period, neurones in the sympathetic intermediolateral cell column of the thoracolumbar spinal cord, rostral ventral medulla and paraventricular nucleus of the hypothalamus were clearly identified by the presence of a green fluorescence in the cytoplasm of the neurones of both species. Thus, herpes simplex virus encoding green fluorescent protein labelled chains of sympathetic neurones in the hamster and rat and therefore has the potential to be used in transneuronal tracing studies of autonomic pathways in these species.     

Mucosal immunization with PLGA-microencapsulated DNA primes a SIV-specific CTL response revealed by boosting with cognate recombinant modified vaccinia virus Ankara

Systemically administered DNA encoding a recombinant human immunodeficiency virus (HIV) derived immunogen effectively primes a cytotoxic T lymphocyte (CTL) response in macaques. In this further pilot study we have evaluated mucosal delivery of DNA as an alternative priming strategy. Plasmid DNA, pTH.HW, encoding a multi-CTL epitope gene, was incorporated into poly(D,L-lactic-co-glycolic acid) microparticles of less than 10 microm in diameter. Five intrarectal immunizations failed to stimulate a circulating vaccine-specific CTL response in 2 Mamu-A*01(+) rhesus macaques. However, 1 week after intradermal immunization with a cognate modified vaccinia virus Ankara vaccine MVA.HW, CTL responses were detected in both animals that persisted until analysis postmortem, 12 weeks after the final boost. In contrast, a weaker and less durable response was seen in an animal vaccinated with the MVA construct alone. Analysis of lymphoid tissues revealed a disseminated CTL response in peripheral and regional lymph nodes but not the spleen of both mucosally primed animals.     

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.