Inactivated Vaccinia Virus Research Articles

Intratumoral delivery of inactivated modified vaccinia virus Ankara (iMVA) induces systemic antitumor immunity via STING and Batf3-dependent dendritic cells.

Advanced cancers remain a therapeutic challenge despite recent progress in targeted therapy and immunotherapy. Novel approaches are needed to alter the tumor immunosuppressive microenvironment and to facilitate the recognition of tumor antigens that leads to antitumor immunity. Poxviruses, such as modified vaccinia virus Ankara (MVA), have potential as immunotherapeutic agents. We show that infection of conventional dendritic cells (DCs) with heat- or ultraviolet-inactivated MVA leads to higher levels of interferon induction than MVA alone through the cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase)-STING cytosolic DNA-sensing pathway. Intratumoral injection of inactivated MVA (iMVA) was effective and generated adaptive antitumor immunity in murine melanoma and colon cancer models. iMVA-induced antitumor therapy was less effective in STING- or Batf3-deficient mice than in wild-type mice, indicating that both cytosolic DNA sensing and Batf3-dependent CD103+/CD8α+ DCs are essential for iMVA immunotherapy. The combination of intratumoral delivery of iMVA and systemic delivery of immune checkpoint blockade generated synergistic antitumor effects in bilateral tumor implantation models as well as in a unilateral large established tumor model. Our results suggest that inactivated vaccinia virus could be used as an immunotherapeutic agent for human cancers.

Abstract A007: Intratumoral delivery of inactivated vaccinia virus is more efficacious than live oncolytic vaccinia virus in murine bilateral tumor implantation models

Abstract Poxviruses are large cytoplasmic DNA viruses that have potentials as vaccine vectors and oncolytic agents. Although almost all of the oncolytic vaccinia viruses that are being investigated in clinical trials are replication competent, it is still unclear how these viruses exert antitumor immunologic effects, and whether viral replication is even necessary to achieve systemic antitumor efficacy. In this study, we compared therapeutic efficacy of the intratumoral delivery of the live, attenuated, replication competent oncolytic vaccinia virus expressing mGM-CSF (VC-TK−-mGM-CSF)vs. its heat-inactivated counterpart (iVC-TK−-mGM-CSF) in two bilateral tumor implantation models- B16-F10 murine melanoma and MC38 colon adenocarcinoma. We found that intratumoral injection of inactivated virus exerted stronger systemic antitumor effects than the live virus. FACS analyses of tumor infiltrating immune cells revealed that intratumoral injection of iVC-TK−-mGM-CSF lead to increased percentages and numbers of activating effector CD8+ and CD4+ T cells and reduced percentages and numbers of CD4+Foxp3+ regulatory T cells in the non-injected tumors compared with intratumoral delivery of the live virus. Infection of bone marrow-derived dendritic cells (DCs) with iVC-TK−-mGM-CSF induced type I IFN and proinflammatory cytokine and chemokine production and DC maturation, whereas the live virus did not. Taken together, our results demonstrate that intratumoral delivery of inactivated vaccina virus is safer and more efficacious than live oncolytic vaccinia virus expressing mGM-CSF in two murine bilateral tumor implantation models. Our findings have important implications for the design of poxviral-based immunotherapeutics for patients with metastatic cancers. Note: This abstract was not presented at the conference. Citation Format: Weiyi Wang, Peihong Dai, Ning Yang, Stewart Shuman, Taha Merghoub, Jedd D. Wolchok, Liang Deng. Intratumoral delivery of inactivated vaccinia virus is more efficacious than live oncolytic vaccinia virus in murine bilateral tumor implantation models [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A007.

An interaction quantitative trait loci tool implicates epistatic functional variants in an apoptosis pathway in smallpox vaccine eQTL data.

Expression quantitative trait loci (eQTL) studies have functionalized nucleic acid variants through the regulation of gene expression. Although most eQTL studies only examine the effects of single variants on transcription, a more complex process of variant-variant interaction (epistasis) may regulate transcription. Herein, we describe a tool called interaction QTL (iQTL) designed to efficiently detect epistatic interactions that regulate gene expression. To maximize biological relevance and minimize the computational and hypothesis testing burden, iQTL restricts interactions such that one variant is within a user-defined proximity of the transcript (cis-regulatory). We apply iQTL to a data set of 183 smallpox vaccine study participants with genome-wide association study and gene expression data from unstimulated samples and samples stimulated by inactivated vaccinia virus. While computing only 0.15% of possible interactions, we identify 11 probe sets whose expression is regulated through a variant-variant interaction. We highlight the functional epistatic interactions among apoptosis-related genes, DIABLO, TRAPPC4 and FADD, in the context of smallpox vaccination. We also use an integrative network approach to characterize these iQTL interactions in a posterior network of known prior functional interactions. iQTL is an efficient, open-source tool to analyze variant interactions in eQTL studies, providing better understanding of the function of epistasis in immune response and other complex phenotypes.

Development of a new hydrogen peroxide–based vaccine platform

Safe and effective vaccines are crucial for maintaining public health and reducing the global burden of infectious disease. Here we introduce a new vaccine platform that uses hydrogen peroxide (H(2)O(2)) to inactivate viruses for vaccine production. H(2)O(2) rapidly inactivates both RNA and DNA viruses with minimal damage to antigenic structure or immunogenicity and is a highly effective method when compared with conventional vaccine inactivation approaches such as formaldehyde or β-propiolactone. Mice immunized with H(2)O(2)-inactivated lymphocytic choriomeningitis virus (LCMV) generated cytolytic, multifunctional virus-specific CD8(+) T cells that conferred protection against chronic LCMV infection. Likewise, mice vaccinated with H(2)O(2)-inactivated vaccinia virus or H(2)O(2)-inactivated West Nile virus showed high virus-specific neutralizing antibody titers and were fully protected against lethal challenge. Together, these studies demonstrate that H(2)O(2)-based vaccines are highly immunogenic, provide protection against a range of viral pathogens in mice and represent a promising new approach to future vaccine development.

Advances in the development of vaccines against Marburg and Ebola viruses.

Peter L Collins & Alexander Bukreyev† †Author for correspondence Laboratory of Infectious Disease, National Institute of Allergy & Infectious Diseases, National Institutes of Health, 50 South Drive, Room 6505, Bethesda, MA 20892-8007, USA Tel.: +1 301 594 1854; Fax: +1 301 496 8312; ab176v@nih.gov ‘The development of vaccines against MARV and EBOV ... is a high priority because of their high virulence, ability to spread from person to person and the possibility of their use in bioterrorism.’

Another Road to Interferon: Yasuichi Nagano's Journey

Their discovery marks the dawn of the interferon/cy-tokine field. When Isaacs and Lindenmann were looking into theirinterferon produced in chicken chorioallantoic membranes in re-sponse to inactivated influenza virus in England, another scien-tist, Yasuichi Nagano (Fig. 1), was coming very close to andtouching interferon on the other side of the globe. In 1954, Naganoand his lifelong collaborator, Yasuhiko Kojima (Fig. 2), observedantiviral activity in rabbit skin after injection of inactivated vac-cinia virus in their laboratory in Japan.

Prediction of Residual Immunity to Smallpox, by Means of an Intradermal Skin Test with Inactivated Vaccinia Virus

Intradermal skin testing with inactivated vaccinia virus was evaluated for its prediction of residual immunity to smallpox. An intradermal skin test was performed with heat-inactivated Lancy-Vaxina. Two days later, the subjects were vaccinated with Lancy-Vaxina. The skin lesions resulting from this vaccination were used as a surrogate marker of residual immunity to smallpox, and this surrogate marker was compared with the available indicators of susceptibility to smallpox. Of the 83 subjects, 30 (36%) showed the typical primary response after vaccination (i.e., absence of residual immunity), whereas 34 (41%) showed the typical revaccinee's response (i.e., presence of residual immunity); the remaining 19 (23%) had an indeterminate response and were excluded from the final analysis. The sensitivity and specificity of the intradermal skin test (induration size, >or=4 mm) for prediction of residual immunity to smallpox were 85% and 97%, respectively, whereas those of a positive vaccinia-specific interferon- gamma -producing T cell response (>or=9 spot forming cells/10(6) peripheral-blood mononuclear cells) were 32% and 63%, respectively, and those of a positive neutralizing antibody (titer, >or=1 : 8) were 79% and 80%, respectively. The intradermal skin test appears to be a simple and reliable method for prediction of residual immunity to smallpox.

DETECTION, cDNA CLONING AND SEQUENCING OF CANINE INTERLEUKIN 12

In canine peripheral blood mononuclear cells (PBMC) the mRNAs coding for both subunits of canine interleukin 12 (IL-12) were identified using reverse transcription polymerase chain reaction (RT-PCR). Stimulation of canine PBMC withStaphylococcus aureusstrain Cowan plus Concanavalin A for 5 h resulted in significant mRNA synthesis. Likewise, inactivated vaccinia virus induced IL-12 mRNA synthesis, however with different kinetics. The complete nucleotide sequence for both IL-12 subunits was determined using rapid amplification of cDNA ends (RACE)-PCR and cloning of amplified specific cDNAs. Computer-aided amino acid (aa) sequence comparison of both canine IL-12 subunits revealed more than 80% identity with the amino acid sequences of six other mammalian species. Closest relationship was found to human, porcine, bovine and cervine IL-12. However, no reactivity was found with antibodies directed against human IL-12, when supernatants of stimulated canine PBMC were tested. Supernatants of canine PBMC stimulated for IL-12 release also induced interferon γ (IFN-γ) mRNA as detectable by RT-PCR; however, it was not clear whether IFN-γ mRNA synthesis was due to an IL-12 specific effect or other stimuli. As to the stimulating effect of IL-12 on canine IFN-γ mRNA synthesis, recombinant human IL-12 was found to be a good inducer. Since IL-12 is regarded a major regulatory molecule of T-cell-mediated immune response and cell growth our work on the cloning and sequencing of this cytokine from dogs lays the basis for future investigations on the biological and possible therapeutic role of canine IL-12.

The immunodepressive action of vaccinia virus

The hemolytic plaque test in mice was used to demonstrate that live vaccinia virus has marked immunosuppressant action. Inactivated vaccinia virus produces no such an effect and even stimulates the output of the antibody-forming cells. The experimental data were found to be statistically significant.

Modification of membrane permeability in vaccinia virus-infected cells

Infection of mouse L cells with vaccinia virus leads to a modification in the permeability of the cell membrane. This modification permits the entry of the translation inhibitor hygromycin B (MW 550). On the basis that some inhibition of protein synthesis is also observed in the presence of the protein toxin α-sarcin (MW 16,800), it seems likely that macromolecules can also cross the membrane in vaccinia virus-infected cells. The modification of the cell membrane is observed very early during infection, in the first hour of viral adsorption. After this time the cell membrane gradually reseals, restoring its normal permeability characteristics. The multiplicities of infection needed to observe these modifications in permeability are rather low; the presence of 3 PFU/cell (150 physical particles/cell) are enough to promote the entry of the inhibitor hygromycin B. Ultraviolet (uv)-inactivated vaccinia virus (17,280 ergs/mm 2 promotes the increase in membrane permeability. Moreover, the presence of actinomycin D (25 μg/ml) and cordycepin (100 μg/ml) did not inhibit the passage of hygromycin B suggesting that a virion component is responsible for this phenomenon. That an intact virion is required to permeabilize L cells, is supported by the findings that neither purified core particles, nor virion surface proteins can induce the inhibition of translation by hygromycin B. Since interferon treatment appears to induce chemical, physical, morphological, and immunological alterations in the cell surface, the effect of interferon on changes in membrane permeability following vaccinia virus infection was investigated. We find that the permeability of the cell membrane is enhanced in interferon-treated, vaccinia virus-infected cells when compared to untreated, infected cells.

In vivo immune stimulation by interferon during viral infection

Treatment of Rhesus monkeys with human leukocyte interferon prevents the development of skin lesions after intradermal infection with vaccinia virus. The treatment does not prevent the development of immunity to vaccinia. Inactivated vaccinia virus, which is non-immunogenic in untreated monkeys, induced immunity under interferon treatment, indicating that interferon had an immunestimulating effect.

N-alpha-Cocoyl-L-arginine ethyl ester, DL-pyroglutamic acid salt, as an inactivator of hepatitis B surface antigen

N-alpha-Cocoyl-L-arginine ethyl ester, DL-pyroglutamic acid salt (CAE), exhibited a strong inactivating effect on hepatitis B surface antigen. Concentrations of CAE required for 50 and 100% inactivation of the antigen were 0.01 to 0.025% and 0.025 to 0.05% respectively. CAE completely inactivated hepatitis B surface antigen at the lowest concentration compared with various compounds including about 500 amino acid derivatives, sodium hypochlorite, 2,4,4'-trichloro-2'-hydroxydiphenyl ether, and some detergents. Furthermore, CAE inactivated vaccinia virus, herpes simplex virus, and influenza virus, whereas poliovirus was not inactivated at all. The results suggest that the inactivating effects of CAE are related to interaction with lipid-containing viral envelopes.

An antigenic difference between intracellular and extracellular rabbitpox virus.

SUMMARY Extracellular rabbitpox virus released naturally from infected cells differed antigenically from intracellular virus released by artificial disruption of cells. Intracellular virus was neutralized by antiserum prepared against live rabbitpox virus and by antiserum against inactivated vaccinia virus. In contrast, extracellular virus was neutralized only by rabbitpox antiserum. The antibodies responsible for the neutralization of intracellular and extracellular virus could be absorbed separately from rabbitpox antiserum. Morphologically, extracellular virus differed from intracellular virus in possessing an outer envelope. This envelope was probably the site of the virus antigen characteristic of extracellular virus, and fluorescent antibody staining of infected cells suggested that it was derived from the modified host cell membrane. Antibody directed against extracellular virus was responsible for the ability of rabbitpox antiserum to control the spread of rabbitpox virus in tissue culture and probably for its ability to protect rabbits from rabbitpox infection. Extracellular virus should therefore be used as the test virus in titrations of neutralizing antibody if these are to assess the protective activity of an antiserum.

Poxvirus specific cytopathic effect in interferon-treated L cells

L cells pretreated with homologous interferon preparations and infected with vaccinia strain WR or IHD develop a drastic cytopathic effect, which differs morphologically from the normal cytopathic effect observed in poxvirus-infected cells. In addition, the 51Cr bound to interferon-treated cells is released from the cells after 4 hr of infection. No comparable release occurs in L cells without interferon treatment or in interferon-treated infected chick embryo fibroblasts. Release of 51Cr depends on the multiplicity of infection, the amount of interferon used, and the time of exposure to interferon before infection. The release of 51Cr is not observed if cells are pretreated with heterologous or inactivated interferon. Infection of interferon-treated L cells with cowpox virus or inactivated vacciniavirus does not result in the increased destruction of cells or release of 51Cr. The addition of inhibitors of DNA or protein synthesis or actinomycin D after infection prevents both effects. It is suggested that release of 51Cr might be used to study quantitatively the poxvirus-specific destruction of interferon-treated cells. Several possibilities for the explanation of the degeneration of interferon treated L cells are discussed with respect to the documented inhibitory action of interferon on virus-specific processes.

Non-genetic reactivation of differently inactivated vaccinia virus.

The non-genetic reactivation of vaccinia virus was studied in cultures of GMK cells. Different methods for obtaining reactivating and reactivable virus have been tested. Besides the heat-treatment, treatment with urea, flufenamic acid, indomethacin, PCMB, HgCl2 or iodoacetamid were all found to produce reactivable virus. N-mustard treatment was the only method tested which produced reactivating virus. Neither soluble vaccinial antigens nor antigens from mechanically disintegrated virions were able to cause reactivation of reactivable virus. Kinetic studies showed that the reactivation is about 10 times more dependent upon the dosage of the reactivating virus than of the reactivable one. Furthermore, the intracellular life-time of the components for reactivation was studied. It was found that the reactivable component of heat-treated virions remained intracellularly in a functional form for the longer period, about three days, whereas the reactivating component of N-mustard treated virus lost the specific function after only about one day.

Role of virucides in controlling virus dissemination by fabrics.

AbstractVaccinia virus, a lipophilic agent containing deoxyribonucleic acid, and poliovirus, a hydrophilic ribonucleic acid virus, persisted on wool and cotton fabrics for varying periods up to 20 weeks, which was of sufficient duration to be of epidemiological significance. The length of persistence of each virus varied with the type of fabric, humidity and method of exposure to the virus. A group of quaternary ammonium salts and bromosalicylanilides were evaluated quantitatively for virucidal activity against these viruses in a cell culture system. None of the compounds was active against poliovirus, but three of the quaternary ammonium compounds significantly inactivated vaccinia virus. Impregnation of wool and cotton fabrics with one of these compounds resulted in a marked decrease in vaccinia virus persistence. Both polio and vaccinia viruses persisted for less than five days on a cotton fabric finished with a modified triazone resin to impart a wash‐and‐wear property. Cotton fabric contaminated with vaccinia or with poliovirus was laundered with an anionic detergent and a nonionic detergent. This laundering reduced but did not eliminate the virus. Sterile fabric was contaminated with virus when laundered with the virus‐containing fabrics. Drying the fabrics for 20 hr after laundering reduced the virus titers to below detectable limits.

The development of skin resistance and hypersensitivity following inactivated vaccinia virus vaccines in rabbits.

A study was made of the development of skin resistance and delayed hyper-sensitivity to vaccinia virus after immunization with inactivated vaccinia virus in rabbits. These vaccines are shown to increase skin resistance, and it is also shown that the development of this resistance is dependent upon the virus content of the vaccine. Skin resistance is shown to be related to the titre of circulating antibody rather than to the degree of delayed hypersensitivity. Possible reasons for conflicting reports in the literature on this subject are discussed.An unusual skin reaction was seen in some animals when challenged with live virus after immunization with inactivated vaccine. The nature of these reactions is discussed.

The antibody response of rabbits to inactivated vaccinia virus.

Inactivated vaccinia virus vaccines were prepared from purified virus inactivated by either formalin, hydroxylamine or heat. The immunogenicity of these vaccines was assessed in rabbits by measurement of virus neutralizing antibody following each of two inoculations. It was concluded that inactivated vaccinia virus stimulates the production of neutralizing antibody and that the most important single factor in this immunogenicity is the concentration of virus in the vaccine. Vaccines prepared from virus suspensions containing 107 to 108 pfu/ml. before inactivation give variable antibody responses.

INACTIVATION OF VACCINIA VIRUS BY ASCORBIC ACID.

Ascorbic acid undergoing auto-oxidation inactivated vaccinia virus. Copper ion was shown to have a catalytic effect on the inactivation. Neither unoxidized ascorbic acid nor its oxidation product, dehydroascorbic acid, were inhibitory. When ascorbic acid was oxidized at high pH in the absence of copper ion no inactivation took place. Similarly, enzymic oxidation of ascorbic acid in the absence of copper was without effect on the virus. Catalase prevented inactivation but not the oxidation of ascorbic acid. Glutathione prevented both inactivation and the oxidation of ascorbic acid. Inhibition experiments with ascorbic acid under anaerobic conditions were inconclusive. The mechanism of ascorbic acid inactivation is discussed in the light of these data and that of other authors with different viruses.

Inhibition of the lesions of primary vaccinia and of delayed hypersensitivity through immunological tolerance in rabbits.

In order to gain insight into the pathogenesis of the vesicular lesion of local primary vaccinia infection, newborn rabbits were injected with 0.5 mg of purified inactivated vaccinia virus in an attempt to render them immunologically tolerant. Within a few days these, and control normal rabbits of the same age, were infected on the skin with active vaccinia virus. Most of the tolerant-prepared rabbits failed to develop a local lesion of vaccinia but some developed a very atypical lesion. Successful virus isolation from some, and the presence of inclusions in the tissues of others, indicated successful infection with the virus. Skin allergy to the active virus failed to develop in the test animals but did in the controls. Thus, there was a high degree of correlation between inability to produce delayed hypersensitivity to the viral antigens and failure to develop a vaccinial skin lesion, indicating the probable allergic nature of the primary lesion. There was also a high mortality rate in the group of tolerant-prepared, infected animals. It was associated with a spreading of the virus from the site of infection to the organs, suggesting that generalized vaccinial infection was the cause of death. The observations were compatible with the hypothesis that death was due to viral toxicity. The observations also suggest that, in the animal possessing normal immunological function, active immunity develops rapidly, perhaps at the level of the draining lymph node, to prevent appreciable virus from leaving the site of infection. The absence of detectable immunological activity toward vaccinia virus early in the tolerant-prepared animals and even after 1 month in some of the survivors, indicates that a high degree of immunological tolerance was produced against these microbial antigens.