Modified Vaccinia Virus Ankara Can Induce Optimal CD8+ T Cell Responses to Directly Primed Antigens Depending on Vaccine Design.

A variety of strains of vaccinia virus (VACV) have been used as recombinant vaccine vectors with the aim of inducing robust CD8+ T cell immunity. While much of the pioneering work was done with virulent strains, such as Western Reserve (WR), attenuated strains such as modified vaccinia virus Ankara (MVA) are more realistic vectors for clinical use. To unify this literature, side-by-side comparisons of virus strains are required. Here, we compare the form of antigen that supports optimal CD8+ T cell responses for VACV strains WR and MVA using equivalent constructs. We found that for multiple antigens, minimal antigenic constructs (epitope minigenes) that prime CD8+ T cells via the direct presentation pathway elicited optimal responses from both vectors, which was surprising because this finding contradicts the prevailing view in the literature for MVA. We then went on to explore the discrepancy between current and published data for MVA, finding evidence that the expression locus and in some cases the presence of the viral thymidine kinase may influence the ability of this strain to prime optimal responses from antigens that require direct presentation. This extends our knowledge of the design parameters for VACV vectored vaccines, especially those based on MVA.IMPORTANCE Recombinant vaccines based on vaccinia virus and particularly attenuated strains such as MVA are in human clinical trials, but due to the complexity of these large vectors much remains to be understood about the design parameters that alter their immunogenicity. Previous work had found that MVA vectors should be designed to express stable protein in order to induce robust immunity by CD8+ (cytotoxic) T cells. Here, we found that the primacy of stable antigen is not generalizable to all designs of MVA and may depend where a foreign antigen is inserted into the MVA genome. This unexpected finding suggests that there is an interaction between genome location and the best form of antigen for optimal T cell priming in MVA and thus possibly other vaccine vectors. It also highlights that our understanding of antigen presentation by even the best studied of vaccine vectors remains incomplete.

Outbreak of Vaccinia Virus Infection from Occupational Exposure, China, 2017.

Human infections with vaccinia virus (VACV), mostly from laboratory accidents or contact with infected animals, have occurred since smallpox was eradicated in 1980. No recent cases have been reported in China. We report on an outbreak of VACV from occupational exposure to rabbit skins inoculated with VACV.

Attenuated and replication-competent vaccinia virus strains M65 and M101 with distinct biology and immunogenicity as potential vaccine candidates against pathogens.

Replication-competent poxvirus vectors with an attenuation phenotype and with a high immunogenic capacity of the foreign expressed antigen are being pursued as novel vaccine vectors against different pathogens. In this investigation, we have examined the replication and immunogenic characteristics of two vaccinia virus (VACV) mutants, M65 and M101. These mutants were generated after 65 and 101 serial passages of persistently infected Friend erythroleukemia (FEL) cells. In cultured cells of different origins, the mutants are replication competent and have growth kinetics similar to or slightly reduced in comparison with those of the parental Western Reserve (WR) virus strain. In normal and immune-suppressed infected mice, the mutants showed different levels of attenuation and pathogenicity in comparison with WR and modified vaccinia Ankara (MVA) strains. Wide genome analysis after deep sequencing revealed selected genomic deletions and mutations in a number of viral open reading frames (ORFs). Mice immunized in a DNA prime/mutant boost regimen with viral vectors expressing the LACK (Leishmania homologue for receptors of activated C kinase) antigen of Leishmania infantum showed protection or a delay in the onset of cutaneous leishmaniasis. Protection was similar to that triggered by MVA-LACK. In immunized mice, both polyfunctional CD4(+) and CD8(+) T cells with an effector memory phenotype were activated by the two mutants, but the DNA-LACK/M65-LACK protocol preferentially induced CD4(+) whereas DNA-LACK/M101-LACK preferentially induced CD8(+) T cell responses. Altogether, our findings showed the adaptive changes of the WR genome during long-term virus-host cell interaction and how the replication competency of M65 and M101 mutants confers distinct biological properties and immunogenicity in mice compared to those of the MVA strain. These mutants could have applicability for understanding VACV biology and as potential vaccine vectors against pathogens and tumors.

Clinical signs, diagnosis, and case reports of Vaccinia virus infections

Vaccinia virus is responsible for a zoonosis that usually affects cattle and human beings in Brazil. The initial clinical signs of the infection are focal red skin areas, fever, and general symptoms similar to those of a cold. Then, pustules and ulcerated lesions surrounded by edema and erythema follow, as well as local lymphadenopathy that can last for weeks. Cure and healing of the lesions occur over several weeks, leaving a typical scar in the skin of people and animals affected. The infection definitive diagnosis is made through morphological characterization of the virus by use of electron microscopy, followed by PCR for specific viral genes. Since 1963, circulating orthopoxviruses in infectious outbreaks in several regions of Brazil have been reported. Later, the etiological agent of those infections was characterized as samples of Vaccinia virus. In addition, the widespread use of those viruses in research laboratories and mass vaccination of militaries have contributed to increase the cases of those infections worldwide. Thus, several epidemiological and clinical studies are required, as well as studies of viral immunology, public health, and economic impact, because little is known about those Vaccinia virus outbreaks in Brazil.

Prime-boost immunization with adenoviral and modified vaccinia virus Ankara vectors enhances the durability and polyfunctionality of protective malaria CD8+ T-cell responses.

Protection against liver-stage malaria relies on the induction of high frequencies of antigen-specific CD8+ T cells. We have previously reported high protective levels against mouse malaria, albeit short-lived, by a single vaccination with adenoviral vectors coding for a liver-stage antigen (ME.TRAP). Here, we report that prime-boost regimens using modified vaccinia virus Ankara (MVA) and adenoviral vectors encoding ME.TRAP can enhance both short- and long-term sterile protection against malaria. Protection persisted for at least 6 months when simian adenoviruses AdCh63 and AdC9 were used as priming vectors. Kinetic analysis showed that the MVA boost made the adenoviral-primed T cells markedly more polyfunctional, with the number of gamma interferon (INF-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-2 (IL-2) triple-positive and INF-gamma and TNF-alpha double-positive cells increasing over time, while INF-gamma single-positive cells declined with time. However, IFN-gamma production prevailed as the main immune correlate of protection, while neither an increase of polyfunctionality nor a high integrated mean fluorescence intensity (iMFI) correlated with protection. These data highlight the ability of optimized viral vector prime-boost regimens to generate more protective and sustained CD8+ T-cell responses, and our results encourage a more nuanced assessment of the importance of inducing polyfunctional CD8(+) T cells by vaccination.

Design and testing of novel oncolytic vaccinia strains.

Oncolytic or replication-selective viruses have been used as powerful tools for the delivery of therapeutic genes to tumors. Because these vectors are capable of replicating within the tumor, the therapeutic gene is amplified within the target tissue itself, resulting in the spread of the virus both within the tumor, and sometimes also between tumors. Vaccinia virus holds many advantages when serving as the backbone for oncolytic viral strains, including a large cloning capacity (at least 25 kbp) (1); a short life-cycle (2, 3); extensive previous use in humans, with contraindications and adverse reactions well described and antivirals available (4); the potential for systemic (intravenous) delivery to distant tumors; and vaccinia strains have previously demonstrated antitumor benefits in clinical trials (5). Because vaccinia has no known receptor and is capable of infecting almost any cell type, tumor selectivity has to be engineered into vaccinia at steps after infection. We will therefore discuss potential viral virulence genes and metabolic targets that result in tumor-selective vaccinia strains. Because the virus has limited natural requirements for host cell proteins, and, instead, contains a large genome and multiple genes involved in virulence, a large number of possible attenuating gene deletions can result in the production of viral strains reliant on inherent properties of the host cell for replication. The protocols for producing viral gene deletions and constructing viral gene expression vectors have been well established for vaccinia and are summarized briefly in this chapter. Basic assays for testing the tumor selectivity and therapeutic index of new oncolytic constructs in vitro will be covered. In addition, we describe how bioluminescence imaging can be incorporated into preclinical testing of vaccinia gene expression strains to examine the timing, biodistribution, and kinetics of viral gene expression noninvasively after delivery of the viral agents to tumor-bearing mice via different routes.

[Construction and biological characteristic for the recombinant modified vaccinia virus ankara co-expressing modified GP5 and M protein of porcine reproductive and respiratory syndrome virus].

Modified ORF5 (MORF5) and ORF6 gene of PRRSV were cloned into two multiple cloning sites of MVA transfer vector pLR-gpt to construct the recombinant plasmid pLR-MORF5/ORF6. Homologous recombination between pLR-MORF5/ORF6 and the wtMVA on BHK-21 cell line was mediated with liposome by infecting the cell with 0.01 MOI wtMVA two hours before transfecting the recombinant plasmid into the cell. When the cytopathic effect (CPE) was obvious, virus was collected from the cell plate and the recombinant virus was selected with drug selecting medium (2% MXHAT). After 12 cycles of selection, rMVA with a selection marker Eco gpt was obtained and named as rMVAgpt-MGP5/M. By infecting BHK-Cre expressing Cre recombinant enzyme, the Eco gpt marker in rMVAgpt-MGP5/M was deleted and this rMVA was named as rMVA-MGP5/M. The insertion of MORF5 and ORF6 into the MVA genome was confirmed with PCR analysis and the expression of MGP5 and M protein was identified with Western blot and IFA. Through biological study on the recombinant MVA, no obvious difference was observed between rMVA-MGP5/M and the wtMVA regarding to the CPE and growth curve. The recombinant MVA constructed in this study could coexpress the modified GP5 and M protein and the expressed product had good immunocompetence. Furthermore, the insertion of the MORF5 and ORF6 into MVA genome had no obvious effect on the replication and biological characteristics of this virus.

Real-time PCR assay to identify variants of Vaccinia virus: implications for the diagnosis of bovine vaccinia in Brazil.

Naturally occurring infections of Vaccinia virus (VACV) have been recognized in Brazil during the past 10 years. Human Brazilian Vaccinia virus (BVV) infections typically occur as a zoonosis transferred from affected dairy cows to their handlers. Outbreaks have caused notable economic losses to the rural community in the region. The origins of BVV are unclear but previous analyses have shown that at least two distinct clades of BVV exist. The aim of this study was to develop a rapid and inexpensive process for identification and differentiation of BVV that should facilitate epidemiological and ecological investigations including the improved diagnosis of Brazilian Orthopoxvirus infections. A SYBR green quantitative real-time polymerase chain reaction (PCR) targeting the hemagglutinin gene was developed to identify different populations of BVV, VACV vaccine strains used in Brazil during the smallpox eradication campaign (Vaccinia Lister (VACV-LIS) and New York City Board of Health (VACV-NYCBH)), and currently available vaccines (VACV-NYCBH DRYVAX and VACV-NYCBH Acambis 2000). Three primer combinations (one to amplify many orthopoxviruses including all vaccinia viruses described so far; one to differentiate BVV from vaccine strains (VACV-LIS, VACV-NYCBH DRYVAX and VACV-NYCBH Acambis 2000); and one to differentiate BVV clades) were designed to work at the same annealing temperature and reaction conditions. In addition, these methods were able to detect orthopoxvirus viral DNA in lesion biopsy material without the need for DNA extraction.

B5r gene based sequence analysis of Indian buffalopox virus isolates in relation to other orthopoxviruses.

We determined complete nucleotide sequence of B5R gene homologue of Vaccinia virus (VACV) in five Buffalopox virus (BPXV) isolates of Indian origin. The obtained sequences were compared with themselves and with corresponding sequences of the other orthopoxviruses. Sequence analysis revealed 99.799.8% and 99.499.7% identities among the BPXV isolates for B5R gene at the nucleotide and amino acid levels, respectively. Sequence identities of B5R gene between BPXV and VACV isolates (98.199.7%) or other orthopoxviruses (95.699.2%) showed highly conserved nature of this protein and a closer relationship of BPXV isolates to VACV than to other orthopoxviruses.

The highly attenuated vaccinia virus strain modified virus Ankara induces apoptosis in melanoma cells and allows bystander dendritic cells to generate a potent anti-tumoral immunity.

Vaccinia virus (VV) has been tested as oncolytic virus against malignant melanoma in clinical trials for more than 40 years. Until now, mainly strains comparable to viral strains used for smallpox vaccination have been probed for anti-tumoral therapy. We have shown recently that the wild-type strain Western Reserve (WR) can interfere with crucial functions of monocyte-derived dendritic cells (DCs). Our aim was to examine whether viral immune evasion mechanisms might be responsible for the ineffectiveness of WR-based vaccination strategies and whether the highly attenuated strain modified virus Ankara (MVA) differs from WR with respect to its possible immunostimulatory capacity after intratumoral injection. Using in vitro experiments, we compared the effect of both strains on melanoma cells and on local bystander DCs. We found that both VV-strains infected melanoma cells efficiently and caused disintegration of the actin cytoskeleton, as shown by fluorescence microscopy. In addition, both VV-strains caused apoptotic cell death in melanoma cells after infection. In contrast to MVA, WR underwent a complete viral replication cycle in melanoma cells. Bystander DCs were consecutively infected by newly generated WR virions and lost their capacity to induce allogeneic T cell proliferation. DCs in contact with MVA-infected melanoma cells retained their capacity to induce T cell proliferation. Immature DCs were capable of phagocytosing MVA-infected melanoma cells. Priming of autologous CD8(+) T cells by DCs that had phagocytosed MVA-infected, MelanA positive melanoma cells resulted in the induction of T cell clones specifically reactive against the model antigen MelanA as shown by enzyme-linked immunospot (ELISPOT) analysis. We conclude that the clinical trials with oncolytic wild-type VV failed probably because of suppression of bystander DCs and consecutive suppression of T cell-mediated anti-melanoma immunity. The attenuated VV-strain MVA facilitates the generation of tumour associated antigen (TAA)-specific T cell response as it is oncolytic for melanoma cells, but non-toxic for DC, and should be a promising candidate for intralesional metastatic melanoma therapy.

Vaccinia virus G9 protein is an essential component of the poxvirus entry-fusion complex.

The vaccinia virus G9R gene (VACWR087) encodes a protein of 340 amino acids with the following structural features that are conserved in all poxviruses: a site for N-terminal myristoylation, 14 cysteines, and a C-terminal transmembrane domain. Previous studies showed that G9 is one of eight proteins associated in a putative entry-fusion complex. Our attempt to isolate a mutant without the G9R gene was unsuccessful, suggesting that it is essential for virus replication. To further investigate its role, we constructed a recombinant vaccinia virus in which G9R is regulated by addition of an inducer. Induced G9 protein was associated with mature infectious virions and could be labeled with a membrane-impermeant biotinylation reagent, indicating surface exposure. Omission of inducer reduced the infectious-virus yield by about 1.5 logs; nevertheless, all stages of virus morphogenesis appeared normal and extracellular virions were present on the cell surface. Purified virions assembled without inducer had a specific infectivity of less than 5% of the normal level and a comparably small amount of G9, whereas their overall polypeptide composition, including other components of the entry-fusion complex, was similar to that of virions made in the presence of inducer or of wild-type virions. G9-deficient virions bound to cells, but penetration of cores into the cytoplasm and early viral RNA synthesis were barely detected, and cell-cell fusion was not triggered by low pH. Of the identified components of the multiprotein complex, G9 is the sixth that has been shown to be required for entry and membrane fusion.

HLA-A2-restricted human CD8(+) cytotoxic T lymphocyte responses to a novel epitope in vaccinia virus that is conserved among orthopox viruses.

Classical major histocompatibility complex class I-restricted CD8(+) cytotoxic T lymphocytes (CTLs) play an important role in protective immunity against infection with smallpox virus. The identification of target antigens is crucial for defining the role played by CD8(+) CTL responses in host resistance to smallpox and for the design of vaccines that produce effective cell-based responses. We report the identification of a novel human leukocyte antigen-A*0201-restricted epitope (J8R(11-19)) within A55R of vaccinia virus (VV) that is conserved in J8R of smallpox virus variola major. The J8R(11-19)-specific CTL line and CTL clone exerted physiologically relevant functions as they recognized VV-infected lymphoblastoid JY cells or autologous B lymphoblastoid cell lines, and the cytolytic activity was accompanied by the production of interferon- gamma , tumor necrosis factor- alpha , and interleukin-2. The CTL response was detected in individuals who had been vaccinated >30 years ago or had recently received a booster of current smallpox vaccine (Dryvax) but not in VV-naive donors.

Comparative sequence analysis of envelope protein genes of Indian buffalopox virus isolates.

Buffalopox virus (BPXV) is considered to be a close variant of vaccinia virus (VACV), the prototype member of the genus Orthopoxvirus. In the present study, we have analyzed the sequences of H3L, A27L, and D8L gene-homologues of VACV in BPXV to elucidate its genetic relationship to VACV and other orthopoxviruses (OPVs). Products of these genes have been shown to be important in attachment of VACV to host cell surface receptors during viral entry. Additionally, the A27L gene is also responsible for cell fusion during infection, while the H3L gene is required for synthesis of the highly immunogenic major envelope protein p35. Full-length nucleotide sequences of H3L, A27L, and D8L genes of three BPXV isolates were determined by PCR amplification, cloning, and sequencing. The nucleotide (nt) sequence and the deduced amino acid (aa) sequences were compared with published sequences from other members of the genus Orthopoxvirus. Comparative sequence analysis of all the three genes revealed high sequence identity of BPXV isolates with VACV (close to 99% sequence identity) at both the nt and aa level. Phylogenetic analysis based on the deduced aa sequences of the H3L, A27L, and D8L genes also showed that BPXVs are more closely related to VACV than to any of the other OPVs.

Vaccinia virus-induced microtubule-dependent cellular rearrangements.

Although infection with vaccinia virus (VV) is known to affect the cytoskeleton, it is not known how this affects the cellular architecture or whether the attenuated modified VV ankara (MVA) behaves similar to wild-type VV (wtVV). In the present study, we therefore compared effects of wtVV and MVA infection on the cellular architecture. WtVV-infection induces cell rounding early in infection, which coincides with the retraction of microtubules (MTs) and intermediate filaments from the cellular periphery, whereas mitochondria and late endosomes cluster around the nucleus. Nocodazole treatment demonstrates that cell rounding and organelle clustering require intact MTs. At the onset of virus assembly late in infection, cells reflatten, a process that coincides with the regrowth of MTs into the cellular periphery. We find that the actin network undergoes several rearrangements that occur sequentially in time and that closely follow the cell-shape changes. Unexpectedly, these actin changes are blocked or reversed upon nocodazole treatment, indicating that intact MTs are also responsible for the wtVV-induced actin rearrangements. Finally, MVA infection does not induce any of these cellular changes. Because this virus lacks a substantial number of VV genes, MVA opens up a system to search for the molecules involved in wtVV-induced cellular changes; in particular, those that may regulate actin/MT interactions.

Host range, growth property, and virulence of the smallpox vaccine: vaccinia virus Tian Tan strain.

Vaccinia Tian Tan (VTT) was used as a vaccine against smallpox in China for millions of people before 1980, yet the biological characteristics of the virus remain unclear. We have characterized VTT with respect to its host cell range, growth properties in vitro, and virulence in vivo. We found that 11 of the 12 mammalian cell lines studied are permissive to VTT infection whereas one, CHO-K1, is non-permissive. Using electron microscopy and sequence analysis, we found that the restriction of VTT replication in CHO-K1 is at a step before viral maturation probably due to the loss of the V025 gene. Moreover, VTT is significantly less virulent than vaccinia WR but remains neurovirulent in mice and causes significant body weight loss after intranasal inoculation. Our data demonstrate the need for further attenuation of VTT to serve either as a safer smallpox vaccine or as a live vaccine vector for other pathogens.

Characterization of a vaccinia virus strain used to produce smallpox vaccine in Argentina between 1937 and 1970.

Due to recent political developments, smallpox has re-emerged as a serious threat. We recovered and characterized an old batch of smallpox vaccine, Malbrán strain, produced between 1945 and 1949. The virus was re-isolated and characterized by sequence analysis and biological activity in animals. Phylogenetic analysis using the hemagglutinin and A45R genes showed that the Malbrán strain was closely related to the Lister strain of vaccinia virus. In animals, the Malbrán strain exhibited low pathogenicity, confirming historical records. Mice immunized with the Malbrán strain survived a lethal challenge with cowpox virus. Thus, this strain of vaccinia virus remains a viable candidate as a smallpox vaccine.

The vaccinia virus K1L gene product inhibits host NF-kappaB activation by preventing IkappaBalpha degradation.

Vaccinia virus wild-type strains such as Ankara and WR synthesize proteins capable of inhibiting the activation of host NF-kappaB, a family of transcription factors that regulate the expression of inflammatory genes. In contrast, an infection by the attenuated MVA strain, whose genome lacks many immunoregulatory genes present in the DNA of its Ankara parent, induces NF-kappaB activation. Insertion of NF-kappaB inhibitory genes into the MVA DNA, then, would alter the MVA phenotype. By this method, a 5.2-kb region of Ankara DNA containing the K1L gene and two other genes that are absent in the MVA genome that was identified as NF-kappaB was inhibited in cells infected with the MVA/5.2kb virus. To determine if K1L was responsible, the relevant biological properties of both a recombinant MVA containing a copy of the WR strain's K1L (MVA/K1L) and a WR deletion mutant lacking the K1L gene (DeltaK1L) were examined. Indeed, unlike its progenitor, the altered MVA halted degradation of the host regulatory protein IkappaBalpha-a key event in the pathway of transcriptional activation by NF-kappaB factors. Moreover, MVA/K1L gained the ability to repress artificially contrived and natural NF-kappaB-regulated expression of a transfected luciferase and the cellular tumor necrosis factor gene, respectively. In contrast, although these functions could also be performed by WR, the DeltaK1L virus lost these abilities. Thus, one apparent molecular function of K1L is to prevent IkappaBalpha degradation. This impediment to NF-kappaB-induced host proinflammatory gene expression, in turn, might enhance virus survival.

Differences in virus-induced cell morphology and in virus maturation between MVA and other strains (WR, Ankara, and NYCBH) of vaccinia virus in infected human cells.

Live recombinants based on attenuated modified vaccinia virus Ankara (MVA) are potential vaccine candidates against a broad spectrum of diseases and tumors. To better understand the efficacy of MVA as a human vaccine, we analyzed by confocal and electron microscopy approaches MVA-induced morphological changes and morphogenetic stages during infection of human HeLa cells in comparison to other strains of vaccinia virus (VV): the wild-type Western Reserve (WR), Ankara, and the New York City Board of Health (NYCBH) strains. Confocal microscopy studies revealed that MVA infection alters the cytoskeleton producing elongated cells (bipolar), which do not form the characteristic actin tails. Few virions are detected in the projections connecting neighboring cells. In contrast, cells infected with the WR, Ankara, and NYCBH strains exhibit a stellated (multipolar) or rounded morphology with actin tails. A detailed transmission electron microscopy analysis of HeLa cells infected with MVA showed important differences in fine ultrastructure and amounts of the viral intermediates compared to cells infected with the other VV strains. In HeLa cells infected with MVA, the most abundant viral forms are intracellular immature virus, with few intermediates reaching the intracellular mature virus (IMV) form, at various stages of maturation, which exhibit a more rounded shape than IMVs from cells infected with the other VV strains. The "IMVs" from MVA-infected cells have an abnormal internal structure ("atypical" viruses) with potential alterations in the core-envelope interactions and are unable to significantly acquire the additional double envelope to render intracellular envelope virus. The presence of potential cell-associated envelope virus is very scarce. Our findings revealed that MVA in human cells promotes characteristic morphological changes to the cells and is able to reach the IMV stage, but these virions were not structurally normal and the subsequent steps in the morphogenetic pathway are blocked.

Tissue distribution of the Ankara strain of vaccinia virus (MVA) after mucosal or systemic administration.

MVA is a candidate vector for vaccination against pathogens and tumors. Little is known about its behaviour in mucosal tissues. We have investigated the fate and biosafety of MVA, when inoculated by different routes in C57BL/6 mice. Intranasal inoculation targeted the virus to the nasal associated lymphoid tissue and the lungs, whereas systemic inoculation led to distribution of MVA in almost all lymphoid organs, lungs and ovaries. Intravaginal, intrarectal and intragastric inoculations failed to induce efficient infection. After 48 h no virus was detectable any more in the organs analyzed. Upon intranasal inoculation, no inflammatory reactions were detected in the central nervous system as well as the upper and lower airways. These results show the tropism of MVA and indicate that high doses of recombinant MVA are safe when nasally administered, a vaccination route known to elicit strong cellular and humoral immune responses in the female genital tract.

Prevention of poxvirus infection by tetrapyrroles.

BACKGROUND: Prevention of poxvirus infection is a topic of great current interest. We report inhibition of vaccinia virus in cell culture by porphyrins and phthalocyanines. Most previous work on the inhibition of viruses with tetrapyrroles has involved photodynamic mechanisms. The current study, however, investigates light-independent inhibition activity. METHODS: The Western Reserve (WR) and International Health Department-J (IHD-J) strains of vaccinia virus were used. Virucidal and antiviral activities as well as the cytotoxicity of test compounds were determined. RESULTS: Examples of active compounds include zinc protoporphyrin, copper hematoporphyrin, meso(2,6-dihydroxyphenyl)porphyrin, the sulfonated tetra-1-naphthyl and tetra-1-anthracenylporphyrins, selected sulfonated derivatives of halogenated tetraphenyl porphyrins and the copper chelate of tetrasulfonated phthalocyanine. EC50 values for the most active compounds are as low as 0.05 microg/mL (40 nM). One of the most active compounds was the neutral meso(2,6-dihydroxyphenyl)porphyrin, indicating that the compounds do not have to be negatively charged to be active. CONCLUSIONS: Porphyrins and phthalocyanines have been found to be potent inhibitors of infection by vaccinia virus in cell culture. These tetrapyrroles were found to be active against two different virus strains, and against both enveloped and non-enveloped forms of the virus, indicating that these compounds may be broadly effective in their ability to inhibit poxvirus infection.