HLA-A*0201, HLA-A*1101, and HLA-B*0702 transgenic mice recognize numerous poxvirus determinants from a wide variety of viral gene products

In virus models explored in detail in mice, CTL typically focus on a few immunodominant determinants. In this study we use a multipronged approach to understand the diversity of CTL responses to vaccinia virus, a prototypic poxvirus with a genome approximately 20-fold larger than that of the model RNA viruses typically studied in mice. Based on predictive computational algorithms for peptide binding to HLA supertypes, we synthesized a panel of 2889 peptides to begin to create an immunomic map of human CTL responses to poxviruses. Using this panel in conjunction with CTLs from vaccinia virus-infected HLA transgenic mice, we identified 14 HLA-A*0201-, 4 HLA-A*1101-, and 3 HLA-B*0702-restricted CD8(+) T cell determinants distributed over 20 distinct proteins. These peptides were capable of binding one or multiple A2, A3, and B7 supertype molecules with affinities typical of viral determinants. Surprisingly, many of the viral proteins recognized are predicted to be late gene products, in addition to the early intermediate gene products expected. Nearly all of the determinants identified have identical counterparts encoded by modified vaccinia virus Ankara as well as variola virus, the agent of smallpox. These findings have implications for the design of new smallpox vaccines and the understanding of immune responses to large DNA viruses in general.     

Rapid and High-Throughput pan-Orthopoxvirus Detection and Identification using PCR and Mass Spectrometry

The genus Orthopoxvirus contains several species of related viruses, including the causative agent of smallpox (Variola virus). In addition to smallpox, several other members of the genus are capable of causing human infection, including monkeypox, cowpox, and other zoonotic rodent-borne poxviruses. Therefore, a single assay that can accurately identify all orthopoxviruses could provide a valuable tool for rapid broad orthopovirus identification. We have developed a pan-Orthopoxvirus assay for identification of all members of the genus based on four PCR reactions targeting Orthopoxvirus DNA and RNA helicase and polymerase genes. The amplicons are detected using electrospray ionization-mass spectrometry (PCR/ESI-MS) on the Ibis T5000 system. We demonstrate that the assay can detect and identify a diverse collection of orthopoxviruses, provide sub-species information and characterize viruses from the blood of rabbitpox infected rabbits. The assay is sensitive at the stochastic limit of PCR and detected virus in blood containing approximately six plaque-forming units per milliliter from a rabbitpox virus-infected rabbit.     

Correlates of protective immunity in poxvirus infection: where does antibody stand?

Even though smallpox has been eradicated, the threat of accidental or intentional release has highlighted the fact there is little consensus about correlates of protective immunity or immunity against re-infection with the causative poxvirus, variola virus (VARV). As the existing vaccine for smallpox has unacceptable rates of side effects and complications, new vaccines are urgently needed. Surrogate animal models of VARV infection in humans, including vaccinia virus (VACV) and ectromelia virus (ECTV) infection in mice, monkeypox virus (MPXV) infection in macaques have been used as tools to dissect the immune response to poxviruses. Mousepox, caused by ECTV, a natural mouse pathogen, is arguably the best surrogate small-animal model, as it shares many aspects of virus biology, pathology and clinical features with smallpox in humans. The requirements for recovery from a primary ECTV infection have been well characterized and include type I and II interferons, natural killer cells, CD4T cells, CD8T cell effector function and antibody. From a vaccine standpoint, it is imperative that the requirements for recovery from secondary infection are also identified. We have investigated host immune parameters in response to a secondary ECTV infection, and have identified that interferon and CD8T cell effector functions are not essential; however, T- and B-cell interaction and antibody are absolutely critical for recovery from a secondary challenge. The central role of antibody has been also been identified in the secondary response to other poxviruses. These findings have important clinical implications and would greatly assist the design of therapeutic interventions and new vaccines for smallpox.     

Poxvirus tropism

Despite the success of the WHO-led smallpox eradication programme a quarter of a century ago, there remains considerable fear that variola virus, or other related pathogenic poxviruses such as monkeypox, could re-emerge and spread disease in the human population. Even today, we are still mostly ignorant about why most poxvirus infections of vertebrate hosts show strict species specificity, or how zoonotic poxvirus infections occur when poxviruses occasionally leap into novel host species. Poxvirus tropism at the cellular level seems to be regulated by intracellular events downstream of virus binding and entry, rather than at the level of specific host receptors as is the case for many other viruses. This review summarizes our current understanding of poxvirus tropism and host range, and discusses the prospects of exploiting host-restricted poxvirus vectors for vaccines, gene therapy or tissue-targeted oncolytic viral therapies for the treatment of human cancers.     

Preparation of monoclonal antibodies cross-reactive with orthopoxviruses and their application for direct immunofluorescence test

Variola virus (smallpox virus), vaccinia virus (VV), cowpox virus (CPV) and ectromelia virus (EV) belong to the genus Orthopoxvirus of the family Poxviridae. To establish the possible diagnosis for smallpox infection, monoclonal antibodies (MAbs) against VV and CPV were produced. The cross-reactivity of seven MAbs with cells infected with various strains of the orthopoxviruses (CPV, VV and EV) was confirmed by an immunofluorescence (IF) test and other immunological analyses. Four and three MAbs reacted with the common antigen of all poxviruses (probably NP antigen) and the antigen involved in neutralization, respectively. We developed the IF test using these MAbs. The direct IF test required only 45 min to perform. Smallpox infection is now eradicated, but it is important to prepare for the diagnosis of smallpox in an emergency. The direct IF assay using MAbs cross-reactive with orthopoxviruses is rapid, simple, specific, applicable for multiple samples, and will make it possible to screen for and detect orthopoxviruses that include variola virus with tissue impression smears from skin lesions in most laboratories or institutes.     

Comparable polyfunctionality of ectromelia virus- and vaccinia virus-specific murine T cells despite markedly different in vivo replication and pathogenicity

Vaccinia virus (VACV) stimulates long-term immunity against highly pathogenic orthopoxvirus infection of humans (smallpox) and mice (mousepox [ectromelia virus {ECTV}]) despite the lack of a natural host-pathogen relationship with either of these species. Previous research revealed that VACV is able to induce polyfunctional CD8(+) T-cell responses after immunization of humans. However, the degree to which the functional profile of T cells induced by VACV is similar to that generated during natural poxvirus infection remains unknown. In this study, we monitored virus-specific T-cell responses following the dermal infection of C57BL/6 mice with ECTV or VACV. Using polychromatic flow cytometry, we measured levels of degranulation, cytokine expression (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-2 [IL-2]), and the cytolytic mediator granzyme B. We observed that the functional capacities of T cells induced by VACV and ECTV were of a similar quality in spite of the markedly different replication abilities and pathogenic outcomes of these viruses. In general, a significant fraction (≥50%) of all T-cell responses were positive for at least three functions both during acute infection and into the memory phase. In vivo killing assays revealed that CD8(+) T cells specific for both viruses were equally cytolytic (～80% target cell lysis after 4 h), consistent with the similar levels of granzyme B and degranulation detected among these cells. Collectively, these data provide a mechanism to explain the ability of VACV to induce protective T-cell responses against pathogenic poxviruses in their natural hosts and provide further support for the use of VACV as a vaccine platform able to induce polyfunctional T cells.     

Poxvirus CD8+ T-cell determinants and cross-reactivity in BALB/c mice

Mouse models of orthopoxvirus disease provide great promise for probing basic questions regarding host responses to this group of pathogens, which includes the causative agents of monkeypox and smallpox. However, some essential tools for their study that are taken for granted with other mouse models are not available for these viruses. Here we map and characterize the initial CD8+ T-cell determinants for poxviruses in H-2d-haplotype mice. CD8+ T cells recognizing these three determinants make up around 40% of the total responses to vaccinia virus during and after resolution of infection. We then use these determinants to test if predicted conservation across orthopoxvirus species matches experimental observation and find an unexpectedly cross-reactive variant peptide encoded by ectromelia (mousepox) virus.     

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.     

非复制型痘苗病毒的生物学性状研究进展

痘病毒被广泛用于针对多种感染性疾病和癌症的疫苗研究,而非复制型痘苗病毒的发展又进一步提高了其安全性。来源于中国天花疫苗株的复制缺陷型痘苗病毒天坛株(NTV)正是一株非复制型痘苗病毒。为了促进未来NTV作为疫苗候选株在临床中的应用,我们对NTV与另外2株非复制型痘苗病毒,即改良痘苗病毒安卡拉株(MVA)和NYVAC的生物学性状做简要综述,主要讨论了它们的基因组结构、体外生长特性、病毒复制和形态发生、组织中的病毒分布和病毒-宿主细胞间的相互作用。     

Immunogenicity and safety of defective vaccinia virus lister: comparison with modified vaccinia virus Ankara

Potent and safe vaccinia virus vectors inducing cell-mediated immunity are needed for clinical use. Replicating vaccinia viruses generally induce strong cell-mediated immunity; however, they may have severe adverse effects. As a vector for clinical use, we assessed the defective vaccinia virus system, in which deletion of an essential gene blocks viral replication, resulting in an infectious virus that does not multiply in the host. The vaccinia virus Lister/Elstree strain, used during worldwide smallpox eradication, was chosen as the parental virus. The immunogenicity and safety of the defective vaccinia virus Lister were evaluated without and with the inserted human p53 gene as a model and compared to parallel constructs based on modified vaccinia virus Ankara (MVA), the present "gold standard" of recombinant vaccinia viruses in clinical development. The defective viruses induced an efficient Th1-type immune response. Antibody and cytotoxic-T-cell responses were comparable to those induced by MVA. Safety of the defective Lister constructs could be demonstrated in vitro in cell culture as well as in vivo in immunodeficient SCID mice. Similar to MVA, the defective viruses were tolerated at doses four orders of magnitude higher than those of the wild-type Lister strain. While current nonreplicating vectors are produced mainly in primary chicken cells, defective vaccinia virus is produced in a permanent safety-tested cell line. Vaccines based on this system have the additional advantage of enhanced product safety. Therefore, a vector system was made which promises to be a valuable tool not only for immunotherapy for diseases such as cancer, human immunodeficiency virus infection, or malaria but also as a basis for a safer smallpox vaccine.     

Engineering and combining oncolytic measles virus for cancer therapy

Cancer immunotherapy using tumor-selective, oncolytic viruses is an emerging therapeutic option for solid and hematologic malignancies. A considerable variety of viruses ranging from small picornaviruses to large poxviruses are currently being investigated as potential candidates. In the early days of virotherapy, non-engineered wild-type or vaccine-strain viruses were employed. However, these viruses often did not fully satisfy the major criteria of safety and efficacy. Since the advent of reverse genetics systems for manipulating various classes of viruses, the field has shifted to developing genetically engineered viruses with an improved therapeutic index. In this review, we will summarize the concepts and strategies of multi-level genetic engineering of oncolytic measles virus, a prime candidate for cancer immunovirotherapy. Furthermore, we will provide a brief overview of measles virus-based multimodal combination therapies for improved tumor control and clinical efficacy.     

Protective effect of Toll-like receptor 4 in pulmonary vaccinia infection

Innate immune responses are essential for controlling poxvirus infection. The threat of a bioterrorist attack using Variola major, the smallpox virus, or zoonotic transmission of other poxviruses has renewed interest in understanding interactions between these viruses and their hosts. We recently determined that TLR3 regulates a detrimental innate immune response that enhances replication, morbidity, and mortality in mice in response to vaccinia virus, a model pathogen for studies of poxviruses. To further investigate Toll-like receptor signaling in vaccinia infection, we first focused on TRIF, the only known adapter protein for TLR3. Unexpectedly, bioluminescence imaging showed that mice lacking TRIF are more susceptible to vaccinia infection than wild-type mice. We then focused on TLR4, the other Toll-like receptor that signals through TRIF. Following respiratory infection with vaccinia, mice lacking TLR4 signaling had greater viral replication, hypothermia, and mortality than control animals. The mechanism of TLR4-mediated protection was not due to increased release of proinflammatory cytokines or changes in total numbers of immune cells recruited to the lung. Challenge of primary bone marrow macrophages isolated from TLR4 mutant and control mice suggested that TLR4 recognizes a viral ligand rather than an endogenous ligand. These data establish that TLR4 mediates a protective innate immune response against vaccinia virus, which informs development of new vaccines and therapeutic agents targeted against poxviruses.     

Monkeypox virus: a re-emergent threat to humans

Human monkeypox (MPX) is a rare zoonotic infection characterized by smallpox-like signs and symptoms. It is caused by monkeypox virus (MPXV), a double stranded DNA virus belonging to the genus Orthopoxvirus. MPX was first identified in 1970 and mostly prevailed in the rural rainforests of Central and West Africa in the past. Outside Africa, MPX was reported in the United Kingdom, the USA, Israel, and Singapore. In 2022, the resurgence of MPX in Europe and elsewhere posed a potential threat to humans. MPXV was transmitted by the animals-human or human-human pathway, and the symptoms of MPXV infection are similar to that of smallpox, but in a milder form and with lower mortality (1%–10%). Although the smallpox vaccination has been shown to provide 85% protection against MPXV infection, and two anti-smallpox virus drugs have been approved to treat MPXV, there are still no specific vaccines and drugs against MPXV infection. Therefore it is urgent to take active measures including the adoption of novel anti-MPXV strategies to control the spread of MPXV and prevent MPX epidemic. In this review, we summarize the biological features, epidemiology, pathogenicity, laboratory diagnosis, and prevention and treatment strategies on MPXV. This review provides the basic knowledge for prevention and control of future outbreaks of this emerging infection.     

The structure of a putative scaffolding protein of immature poxvirus particles as determined by electron microscopy suggests similarity with capsid proteins of large icosahedral DNA viruses

Orf virus, the prototype parapoxvirus, is responsible for contagious ecthyma in sheep and goats. The central region of the viral genome codes for proteins highly conserved among vertebrate poxviruses and which are frequently essential for viral proliferation. Analysis of the recently published genome sequence of orf virus revealed that among such essential proteins, the protein orfv075 is an orthologue of D13, the rifampin resistance gene product critical for vaccinia virus morphogenesis. Previous studies showed that D13, arranged as "spicules," is necessary for the formation of vaccinia virus immature virions, a mandatory intermediate in viral maturation. We have determined the three-dimensional structure of recombinant orfv075 at approximately 25-A resolution by electron microscopy of two-dimensional crystals. orfv075 organizes as trimers with a tripod-like main body and a propeller-like smaller domain. The molecular envelope of orfv075 shows unexpectedly good agreement to that of a distant homologue, VP54, the major capsid protein of Paramecium bursaria Chlorella virus type 1. Our structural analysis suggests that orfv075 belongs in the double-barreled capsid protein family found in many double-stranded DNA icosahedral viruses and supports the hypothesis that the nonicosahedral poxviruses and the large icosahedral DNA viruses are evolutionarily related.     

Repeat sequences from complex ds DNA viruses can be used as minisatellite probes for DNA fingerprinting

In a search for new fingerprinting probes for use with sheep, repeat sequences derived from five poxviruses, an iridovirus and a baculovirus were screened against DNA from sheep pedigrees. Probes constructed from portions of the parapox viruses, orf virus and papular stomatitis virus and the baculovirus from the alfalfa looper, Autographa californica, nuclear polyhedrosis virus all gave fingerprint patterns. Probes from three other poxviruses and an iridovirus did not give useful banding patterns.     

Comparison of immune responses of brown-headed cowbird and related blackbirds to west Nile and other mosquito-borne encephalitis viruses

The rapid geographic spread of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) across the United States has stimulated interest in comparative host infection studies to delineate competent avian hosts critical for viral amplification. We compared the host competence of four taxonomically related blackbird species (Icteridae) after experimental infection with WNV and with two endemic, mosquito-borne encephalitis viruses, western equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, WEEV), and St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV). We predicted differences in disease resistance among the blackbird species based on differences in life history, because they differ in geographic range and life history traits that include mating and breeding systems. Differences were observed among the response of these hosts to all three viruses. Red-winged Blackbirds were more susceptible to SLEV than Brewer's Blackbirds, whereas Brewer's Blackbirds were more susceptible to WEEV than Red-winged Blackbirds. In response to WNV infection, cowbirds showed the lowest mean viremias, cleared their infections faster, and showed lower antibody levels than concurrently infected species. Brown-headed Cowbirds also exhibited significantly lower viremia responses after infection with SLEV and WEEV as well as coinfection with WEEV and WNV than concurrently infected icterids. We concluded that cowbirds may be more resistant to infection to both native and introduced viruses because they experience heightened exposure to a variety of pathogens of parenting birds during the course of their parasitic life style.     

The time scale in poxvirus evolution

Unlike vertebrates and RNA-containing viruses, the objective estimate of molecular clock for DNA-containing viruses was so far absent. An extended central conservative genomic region of orthopoxviruses (about 102 kbp) and the sequence of DNA polymerase gene (about 3 kbp) of the viruses belonging to various genera from the family Poxviridae were analyzed. During this analysis, the known dating of variola virus (VARV) transfer from West Africa to South America (XVI century) and our own data on close phylogenetic relations between the modem West African and South American VARV isolates were used. As a result of this work, it was calculated for the first time that the rate of mutation accumulation in these DNA-containing viruses amounted to 0.9-1.2 x 10(-6) substitutions per site per year. The poxviruses started separating from the ancestor virus to form the modem genera approximately 500 thousand years ago; the ancestor of the genus Orthopoxvirus separated about 300 thousand years ago; and its division into the modem studied species took place approximately 14 thousand years ago.