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We previously reported that mice immunized twice with a lipid nanoparticle vaccine comprising four monkeypox viral mRNAs raised neutralizing antibodies and antigen-specific T cells and were protected against a lethal intranasal challenge with vaccinia virus (VACV). Here we demonstrated that the mRNA vaccine also protects mice against intranasal and intraperitoneal infections with monkeypox virus and bioluminescence imaging showed that vaccination greatly reduces or prevents VACV replication and spread from intranasal, rectal, and dermal inoculation sites. A single vaccination provided considerable protection that was enhanced by boosting for at least 4 months. Protection was related to the amount of mRNA inoculated, which correlated with neutralizing antibody levels. Furthermore, immunocompetent and immunodeficient mice lacking mature B and T cells that received serum from mRNA-immunized macaques before or after VACV challenge were protected. These findings provide insights into the mechanism and extent of mRNA vaccine-induced protection of orthopoxviruses and support clinical testing. [ABSTRACT FROM AUTHOR]

This study assesses mpox-specific immune responses for 12 months after MVA-BN vaccination. [ABSTRACT FROM AUTHOR]

Zoonotic poxviruses such as mpox virus (MPXV) continue to threaten public health safety since the eradication of smallpox. Vaccinia virus (VACV), the prototypic poxvirus used as the vaccine strain for smallpox eradication, is the best-characterized member of the poxvirus family. VACV encodes a serine protease inhibitor 1 (SPI-1) conserved in all orthopoxviruses, which has been recognized as a host range factor for modified VACV Ankara (MVA), an approved smallpox vaccine and a promising vaccine vector. FAM111A (family with sequence similarity 111 member A), a nuclear protein that regulates host DNA replication, was shown to restrict the replication of a VACV SPI-1 deletion mutant (VACV-ΔSPI-1) in human cells. Nevertheless, the detailed antiviral mechanisms of FAM111A were unresolved. Here, we show that FAM111A is a potent restriction factor for VACV-ΔSPI-1 and MVA. Deletion of FAM111A rescued the replication of MVA and VACV-ΔSPI-1 and overexpression of FAM111A significantly reduced viral DNA replication and virus titers but did not affect viral early gene expression. The antiviral effect of FAM111A necessitated its trypsin-like protease domain and DNA-binding domain but not the PCNA-interacting motif. We further identified that FAM111A translocated into the cytoplasm upon VACV infection by degrading the nuclear pore complex via its protease activity, interacted with VACV DNA-binding protein I3, and promoted I3 degradation through autophagy. Moreover, SPI-1 from VACV, MPXV, or lumpy skin disease virus was able to antagonize FAM111A by prohibiting its nuclear export. Our findings reveal the detailed mechanism by which FAM111A inhibits VACV and provide explanations for the immune evasive function of VACV SPI-1. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 vaccines prevent severe disease but are less efficient in averting infection and transmission of variant strains, making it imperative to explore ways of enhancing protection. Use of inbred mice expressing the human SARS-CoV-2 receptor facilitates such investigations. We employed recombinant MVAs (rMVAs) expressing modified S of several SARS-CoV-2 strains and compared their ability to neutralize variants, bind S proteins and protect K18-hACE2 mice against SARS-CoV-2 challenge when administered intramuscularly or intranasally. The rMVAs expressing Wuhan, Beta and Delta S induced substantial cross neutralizing activities to each other but very low neutralization of Omicron; while rMVA expressing Omicon S induced neutralizing antibody predominanly to Omicron. In mice primed and boosted with rMVA expressing the Wuhan S, neutralizing antibodies to Wuhan increased after one immunization with rMVA expressing Omicron S due to original antigenic sin, but substantial neutralizing antibody to Omicron required a second immunization. Nevertheless, monovalent vaccines with S mismatched to the challenge virus still protected against severe disease and reduced the amounts of virus and subgenomic RNAs in the lungs and nasal turbinates, though not as well as vaccines with matched S. Passive transfer of Wuhan immune serum with Omicron S binding but undetectable neutralizing activity reduced infection of the l–ungs by Omicron suggesting additional effector functions. Notably, there was less infectious virus and viral subgenomic RNAs in the nasal turbinates and lungs when the rMVAs were administered intranasally rather than intramuscularly and this held true for vaccines that were matched or mismatched to the challenge strain of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Human mpox (monkeypox), a disease with similarities to smallpox, is endemic in Africa where it has persisted as a zoonosis with limited human-to-human spread. Unexpectedly, the disease expanded globally in 2022 driven by human-to-human transmission outside of Africa. It is not yet known whether the latter is due solely to behavioral and environmental factors or whether the mpox virus is adapting to a new host. Genome sequencing has revealed differences between the current outbreak strains, classified as clade IIb, and the prior clade IIa and clade I viruses, but whether these differences contribute to virulence or transmission has not been determined. We demonstrate that the wild-derived inbred castaneous mouse provides an exceptional animal model for investigating clade differences in mpox virus virulence and show that the order is clade I > clade IIa > clade IIb.1. The greatly reduced replication of the clade IIb.1 major outbreak strain in mice and absence of lethality at 100 times the lethal dose of a closely related clade IIa virus, despite similar multiplication in cell culture, suggest that clade IIb is evolving diminished virulence or adapting to other species. [ABSTRACT FROM AUTHOR]

We have recently shown that the replication of rhinovirus, poliovirus and foot-and-mouth disease virus requires the co-translational N-myristoylation of viral proteins by human host cell N-myristoyltransferases (NMTs), and is inhibited by treatment with IMP-1088, an ultrapotent small molecule NMT inhibitor. Here, we examine the importance of N-myristoylation during vaccinia virus (VACV) infection in primate cells and demonstrate the anti-poxviral effects of IMP-1088. N-myristoylated proteins from VACV and the host were metabolically labelled with myristic acid alkyne during infection using quantitative chemical proteomics. We identified VACV proteins A16, G9 and L1 to be N-myristoylated. Treatment with NMT inhibitor IMP-1088 potently abrogated VACV infection, while VACV gene expression, DNA replication, morphogenesis and EV formation remained unaffected. Importantly, we observed that loss of N-myristoylation resulted in greatly reduced infectivity of assembled mature virus particles, characterized by significantly reduced host cell entry and a decline in membrane fusion activity of progeny virus. While the N-myristoylation of VACV entry proteins L1, A16 and G9 was inhibited by IMP-1088, mutational and genetic studies demonstrated that the N-myristoylation of L1 was the most critical for VACV entry. Given the significant genetic identity between VACV, monkeypox virus and variola virus L1 homologs, our data provides a basis for further investigating the role of N-myristoylation in poxviral infections as well as the potential of selective NMT inhibitors like IMP-1088 as broad-spectrum poxvirus inhibitors. Author summary: N-myristoylation is a lipidic co- or post-translational modification of proteins that is catalyzed by the enzyme N-myristoyltransferase (NMT). In this study, we use IMP-1088, an ultrapotent inhibitor of human NMTs, to query whether targeting the N-myristolation of viral proteins can serve as an effective antiviral strategy against VACV. Through quantitative chemical proteomics, we demonstrated that IMP-1088 blocks the N-myristoylation of L1, a VACV protein essential for viral entry. Progeny virus particles generated in the presence of IMP-1088 lacked N-myrisoylated L1, which considerably reduced their infectivity. Other steps in the viral life cycle, including replication, morphogenesis and viral egress were not directly impacted by IMP-1088-mediated NMT inhibition. While the N-myristoylation of two other EFC proteins, namely A16 and G9, was also abrogated by IMP-1088 treatment, mutational studies revealed they were dispensable for viral entry in the presence of a functional L1 protein. Taken together, these data illustrate the mechanism of action of IMP-1088 as a potential poxviral inhibitor. [ABSTRACT FROM AUTHOR]

Modified vaccinia virus Ankara (MVA) is a replication-restricted smallpox vaccine, and numerous clinical studies of recombinant MVAs (rMVAs) as vectors for prevention of other infectious diseases, including COVID-19, are in progress. Here, we characterize rMVAs expressing the S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Modifications of full-length S individually or in combination included two proline substitutions, mutations of the furin recognition site, and deletion of the endoplasmic retrieval signal. Another rMVA in which the receptor binding domain (RBD) is flanked by the signal peptide and transmembrane domains of S was also constructed. Each modified S protein was displayed on the surface of rMVA-infected cells and was recognized by anti-RBD antibody and soluble hACE2 receptor. Intramuscular injection of mice with the rMVAs induced antibodies, which neutralized a pseudovirus in vitro and, upon passive transfer, protected hACE2 transgenicmice from lethal infection with SARS-CoV-2, as well as S-specific CD3+CD8+IFNγ+ T cells. Antibody boosting occurred following a second rMVA or adjuvanted purified RBD protein. Immunity conferred by a single vaccination of hACE2 mice prevented morbidity and weight loss upon intranasal infection with SARS-CoV-2 3 wk or 7 wk later. One or two rMVA vaccinations also prevented detection of infectious SARS-CoV-2 and subgenomic viral mRNAs in the lungs and greatly reduced induction of cytokine and chemokine mRNAs. A low amount of virus was found in the nasal turbinates of only one of eight rMVA-vaccinated mice on day 2 and none later. Detection of low levels of subgenomic mRNAs in turbinates indicated that replication was aborted in immunized animals. [ABSTRACT FROM AUTHOR]

Modified vaccinia virus Ankara (MVA) is an approved smallpox vaccine and a promising vaccine vector for other pathogens as well as for cancer therapeutics with more than 200 current or completed clinical trials. MVA was derived by passaging the parental Ankara vaccine virus hundreds of times in chick embryo fibroblasts during which it lost the ability to replicate in human and most other mammalian cells. Although this replication deficiency is an important safety feature, the genetic basis of the host restriction is not understood. Here, an unbiased human genome-wide RNAi screen in human A549 cells revealed that the zinc-finger antiviral protein (ZAP), previously shown to inhibit certain RNA viruses, is a host restriction factor for MVA, a DNA virus. Additional studies demonstrated enhanced MVA replication in several human cell lines following knockdown of ZAP. Furthermore, CRISPR-Cas9 knockout of ZAP in human A549 cells increased MVA replication and spread by more than one log but had no effect on a non-attenuated strain of vaccinia virus. The intact viral C16 protein, which had been disrupted in MVA, antagonized ZAP by binding and sequestering the protein in cytoplasmic punctate structures. Studies aimed at exploring the mechanism by which ZAP restricts MVA replication in the absence of C16 showed that knockout of ZAP had no discernible effect on viral DNA or individual mRNA or protein species as determined by droplet digital polymerase chain reaction, deep RNA sequencing and mass spectrometry, respectively. Instead, inactivation of ZAP reduced the number of aberrant, dense, spherical particles that typically form in MVA-infected human cells, suggesting that ZAP has a novel role in interfering with a late step in the assembly of infectious MVA virions in the absence of the C16 protein. Author summary: The attenuated vaccine vector known as modified vaccinia virus Ankara (MVA) was derived by extensively passaging the parental strain of vaccinia virus Ankara in chick embryo fibroblasts and is unable to replicate in most mammalian cells. The MVA host range restriction is exceptional in that synthesis of the abundant viral proteins appears unaffected but morphogenesis of virus particles is abortive. Despite the importance of the host range restriction for vaccine safety, the basis for this antiviral effect has remained an enigma. Here we demonstrate that the zinc finger antiviral protein (ZAP), previously shown to be an inhibitor of RNA viruses, is a specific host restriction factor for replication of MVA in human cells. Moreover, the intact vaccinia virus C16 protein, which was disrupted during the attenuation of MVA, sequesters ZAP in cytoplasmic punctae and effectively counteracts the inhibitory effects of ZAP. [ABSTRACT FROM AUTHOR]

The wild-derived inbred CAST/EiJ mouse, one of eight founder strains in the Collaborative Cross panel, is an exceptional model for studying monkeypox virus (MPXV), an emerging human pathogen, and other orthopoxviruses including vaccinia virus (VACV). Previous studies suggested that the extreme susceptibility of the CAST mouse to orthopoxviruses is due to an insufficient innate immune response. Here, we focused on the low number of natural killer (NK) cells in the naïve CAST mouse as a contributing factor to this condition. Administration of IL-15 to CAST mice transiently increased NK and CD8+ T cells that could express IFN-γ, indicating that the progenitor cells were capable of responding to cytokines. However, the number of NK cells rapidly declined indicating a defect in their homeostasis. Furthermore, IL-15-treated mice were protected from an otherwise lethal challenge with VACV or MPXV. IL-15 decreased virus spread and delayed death even when CD4+/CD8+ T cells were depleted with antibody, supporting an early protective role of the expanded NK cells. Purified splenic NK cells from CAST mice proliferated in vitro in response to IL-15 and could be activated with IL-12/IL-18 to secrete interferon-γ. Passive transfer of non-activated or activated CAST NK cells reduced VACV spread but only the latter completely prevented death at the virus dose used. Moreover, antibodies to interferon-γ abrogated the protection by activated NK cells. Thus, the inherent susceptibility of CAST mice to orthopoxviruses can be explained by a low level of NK cells and this vulnerability can be overcome either by expanding their NK cells in vivo with IL-15 or by passive transfer of purified NK cells that were expanded and activated in vitro. Author summary: With the eradication of smallpox, monkeypox virus (MPXV) remains the only poxvirus causing significant mortality in humans. Although endemic in parts of Africa, human infections have occurred in the United States, the United Kingdom and Israel due to travelers or imported animals. Contrary to its name, MPXV primarily infects rodents and secondarily infects humans and other primates. The wild-derived CAST mouse is an excellent small animal model for studying the pathogenicity of MPXV and related orthopoxviruses including vaccinia virus (VACV) and for evaluating therapeutics. We previously found that the susceptibility of CAST mice is correlated with low numbers of natural killer (NK) cells and a delayed interferon-γ response. Here we showed that in vivo administration of the cytokine IL-15 transiently raised NK cell numbers and protected CAST mice from systemic infections with VACV and MPXV. CAST mouse NK cells that were purified and expanded in vitro with IL-15 also provided protection, further demonstrating the important role of NK cells. The rapid decline in NK cell numbers following cessation of IL-15 administration or NK cell transfer suggests that a low level of NK cell homeostasis contributes to the susceptibility of CAST mice to virus infection. [ABSTRACT FROM AUTHOR]

Modified vaccinia virus Ankara (MVA), a widely used vaccine vector for expression of genes of unrelated pathogens, is safe, immunogenic, and can incorporate large amounts of added DNA. MVA was derived by extensively passaging the chorioallantois vaccinia virus Ankara (CVA) vaccine strain in chicken embryo fibroblasts during which numerous mutations and deletions occurred with loss of replicative ability in most mammalian cells. Restoration of the deleted C12L gene, encoding serine protease inhibitor 1, enhances replication of MVA in human MRC-5 cells but only slightly in other human cells. Here we show that repair of the inactivated C16L/B22R gene of MVA enhances replication in numerous human cell lines. This previously uncharacterized gene is present at both ends of the genome of many orthopoxviruses and is highly conserved in most, including smallpox and monkeypox viruses. The C16L/B22R gene is expressed early in infection from the second methionine of the previously annotated Copenhagen strain open reading frame (ORF) as a 17.4-kDa protein. The C16/B22 and C12 proteins together promote MVA replication in human cells to levels that are comparable to titers in chicken embryo fibroblasts. Both proteins enhance virion assembly, but C16/B22 increases proteolytic processing of core proteins in A549 cells consistent with higher infectious virus titers. Furthermore, human A549 cells expressing codon-optimized C16L/B22R and C12L genes support higher levels of MVA replication than cell lines expressing neither or either alone. Identification of the genes responsible for the host-range defect of MVA may allow more rational engineering of vaccines for efficacy, safety, and propagation. [ABSTRACT FROM AUTHOR]

Modified vaccinia virus Ankara (MVA) is the leading poxvirus vector for development of vaccines against diverse infectious diseases. This distinction is based on high expression of proteins and good immunogenicity despite an inability to assemble infectious progeny in human cells, which together promote efficacy and safety. Nevertheless, the basis for the host-range restriction is unknown despite past systematic attempts to identify the relevant missing viral gene(s). The search for host-range factors is exacerbated by the large number of deletions, truncations and mutations that occurred during the long passage history of MVA in chicken embryo fibroblasts. By whole genome sequencing of a panel of recombinant host-range extended (HRE) MVAs generated by marker rescue with 40 kbp segments of vaccinia virus DNA, we identified serine protease inhibitor 1 (SPI-1) as one of several candidate host-range factors present in those viruses that gained the ability to replicate in human cells. Electron microscopy revealed that the interruption of morphogenesis in human cells infected with MVA occurred at a similar stage as that of a vaccinia virus strain WR SPI-1 deletion mutant. Moreover, the introduction of the SPI-1 gene into the MVA genome led to more than a 2-log enhancement of virus spread in human diploid MRC-5 cells, whereas deletion of the gene diminished the spread of HRE viruses by similar extents. Furthermore, MRC-5 cells stably expressing SPI-1 also enhanced replication of MVA. A role for additional host range genes was suggested by the restoration of MVA replication to a lower level relative to HRE viruses, particularly in other human cell lines. Although multiple sequence alignments revealed genetic changes in addition to SPI-1 common to the HRE MVAs, no evidence for their host-range function was found by analysis thus far. Our finding that SPI-1 is host range factor for MVA should simplify use of high throughput RNAi or CRISPR/Cas single gene methods to identify additional viral and human restriction elements. [ABSTRACT FROM AUTHOR]

The article explores the convoluted path of the formation and structure of the poxvirus envelope through studies of vaccinia virus, the prototype proxvirus. Topics mentioned include the replication of proxviruses within the cytoplasm in which their brick-shaped infectious mature virion facilitates exocytosis, intracellular transport, and dissemination, the hypothesis for the formation of poxviral envelope, and the role of the endoplasmic reticulum in the formation of the viral envelope.

Background: We report the first-in-human safety and immunogenicity evaluation of PENNVAX-G DNA/modified vaccinia Ankara-Chiang Mai double recombinant (MVA-CMDR) prime-boost human immuonodeficiency virus (HIV) vaccine, with intramuscular DNA delivery by either Biojector 2000 needle-free injection system (Biojector) or CELLECTRA electroporation device.Methods: Healthy, HIV-uninfected adults were randomized to receive 4 mg of PENNVAX-G DNA delivered intramuscularly by Biojector or electroporation at baseline and week 4 followed by intramuscular injection of 108 plaque forming units of MVA-CMDR at weeks 12 and 24. The open-label part A was conducted in the United States, followed by a double-blind, placebo-controlled part B in East Africa. Solicited and unsolicited adverse events were recorded, and immune responses were measured.Results: Eighty-eight of 100 enrolled participants completed all study injections, which were generally safe and well tolerated, with more immediate, but transient, pain in the electroporation group. Cellular responses were observed in 57% of vaccine recipients tested and were CD4 predominant. High rates of binding antibody responses to CRF01_AE antigens, including gp70 V1V2 scaffold, were observed. Neutralizing antibodies were detected in a peripheral blood mononuclear cell assay, and moderate antibody-dependent, cell-mediated cytotoxicity activity was demonstrated.Discussion: The PVG/MVA-CMDR HIV-1 vaccine regimen is safe and immunogenic. Substantial differences in safety or immunogenicity between modes of DNA delivery were not observed.Clinical Trials Registration: NCT01260727. [ABSTRACT FROM AUTHOR]

Heterologous prime boost with DNA and recombinant modified vaccinia virus Ankara (rMVA) vaccines is considered as a promising vaccination approach against human immunodeficiency virus (HIV-1). To further enhance the efficacy of DNA-rMVA vaccination, we investigated humoral and cellular immune responses in mice after three sequential immunizations with DNA, a combination of DNA and virus-like particles (VLP), and rMVA expressing HIV-1 89.6 gp120 envelope proteins (Env). DNA prime and boost with a combination of VLP and DNA vaccines followed by an rMVA boost induced over a 100-fold increase in Env-specific IgG antibody titers compared to three sequential immunizations with DNA and rMVA. Cellular immune responses were induced by VLP-DNA and rMVA vaccinations at high levels in CD8 T cells, CD4 T cells, and peripheral blood mononuclear cells secreting interferon (IFN)-, and spleen cells producing interleukin (IL)-2, 4, 5 cytokines. This study suggests that a DNA and VLP combination vaccine with MVA is a promising strategy in enhancing the efficacy of DNA-rMVA vaccination against HIV-1. [ABSTRACT FROM AUTHOR]

Viruses and their hosts can reach balanced states of evolution ensuring mutual survival, which makes it difficult to appreciate the underlying dynamics. To uncover hidden interactions, virus mutants that have lost defense genes may be used. Deletion of the gene that encodes serine protease inhibitor 1 (SPI-1) of rabbit-pox virus and vaccinia virus, two closely related orthopoxviruses, prevents their efficient replication in human cells, whereas certain other mammalian cells remain fully permissive. Our high-throughput genome-wide siRNA screen identified host factors that prevent reproduction and spread of the mutant viruses in human cells. More than 20,000 genes were interrogated with individual siRNAs and those that prominently increased replication of the SPI- 1 deletion mutant were subjected to a secondary screen. The top hits based on the combined data--replication factor C3 (RFC3), FAM111A, and interferon regulatory factor 2 (IRF2)--were confirmed by custom assays. The siRNAs to RFC1, RFC2, RFC4, and RFC5 mRNAs also enhanced spread of the mutant virus, strengthening the biological significance of the RFC complex as a host restriction factor for poxviruses. Whereas association with proliferating cell nuclear antigen and participation in processive genome replication are common features of FAM111A and RFC, IRF2 is a transcriptional regulator. Microarray analysis, quantitative RT-PCR, and immunoblotting revealed that IRF2 regulated the basal level expression of FAM111A, suggesting that the enhancing effect of depleting IRF2 on replication of the SPI-1 mutant was indirect. Thus, the viral SPI-1 protein and the host IRF2, FAM111A, and RFC complex likely form an interaction network that influences the ability of poxviruses to replicate in human cells. [ABSTRACT FROM AUTHOR]

As a primary intervention, raising the topics of faith and religion with individuals traumatised by terrorism and/or natural disasters can be daunting for social workers, because victims often enter the helping relationship with feelings of helplessness, loss of personal control and of doubt about their relationships, environment, and their cultural and belief systems. Just as clients benefit from knowledge and awareness in the aftermath of a traumatic event, insights gleaned from traumatic experiences and from research can be useful for social workers grappling with the challenges associated with designing and deploying appropriate helping strategies with victims of disaster and terrorism. This article draws on extant literature and survey research, to explore how social workers might ethically assess clients' spiritual perspectives and incorporate helping activities that support clients' recovery, in the context of a spiritually sensitive helping relationship with victims of disaster and terrorism. [ABSTRACT FROM AUTHOR]

Summary Poxviruses are enveloped DNA viruses that replicate within the cytoplasm. The first viral structures are crescents and spherical particles, with a lipoprotein membrane bilayer, that are thought to be derived from the ER. We determined that A17, a conserved viral transmembrane protein essential for crescent formation, forms homo-oligomers and shares topological features with cellular reticulon-like proteins. The latter cell proteins promote membrane curvature and contribute to the tubular structure of the ER. When the purified A17 protein was incorporated into liposomes, 25 nm diameter vesicles and tubules formed at low and high A17 concentrations, respectively. In addition, intracellular expression of A17 in the absence of other viral structural proteins transformed the ER into aggregated three-dimensional (3D) tubular networks. We suggest that A17 is a viral reticulon-like protein that contributes to curvature during biogenesis of the poxvirus membrane. [ABSTRACT FROM AUTHOR]

Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNAprimed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome. [ABSTRACT FROM AUTHOR]

The split green fluorescent protein ( GFP) system was adapted for investigation of the topology of ER-associated proteins. A 215-amino acid fragment of GFP (S1-10) was expressed in the cytoplasm as a free protein or fused to the N-terminus of calnexin and in the ER as an intraluminal protein or fused to the C-terminus of calnexin. A 16-amino acid fragment of GFP ( S11) was fused to the N- or C-terminus of the target protein. Fluorescence occurred when both GFP fragments were in the same intracellular compartment. After validation with the cellular proteins PDI and tapasin, we investigated two vaccinia virus proteins ( L2 and A30.5) of unknown topology that localize to the ER and are required for assembly of the viral membrane. Our results indicated that the N- and C-termini of L2 faced the cytoplasmic and luminal sides of the ER, respectively. In contrast both the N- and C-termini of A30.5 faced the cytoplasm. The system offers advantages for quickly determining the topology of intracellular proteins: the S11 tag is similar in length to commonly used epitope tags; multiple options are available for detecting fluorescence in live or fixed cells; transfection protocols are adaptable to numerous expression systems and can enable high throughput applications. [ABSTRACT FROM AUTHOR]

Vaccine-induced HIV antibodies were evaluated in serum samples collected from healthy Tanzanian volunteers participating in a phase I/II placebo-controlled double blind trial using multi-clade, multigene HIV-DNA priming and recombinant modified vaccinia Ankara (HIV-MVA) virus boosting (HIVIS03). The HIV-DNA vaccine contained plasmids expressing HIV-1 gp160 subtypes A, B, C, Rev B, Gag A, B and RTmut B, and the recombinant HIV-MVA boost expressed CRF01_AE HIV-1 Env subtype E and Gag-Pol subtype A. While no neutralizing antibodies were detected using pseudoviruses in the TZM-bl cell assay, this prime-boost vaccination induced neutralizing antibodies in 83% of HIVIS03 vaccinees when a peripheral blood mononuclear cell (PBMC) assay using luciferase reporter-infectious molecular clones (LucR-IMC) was employed. The serum neutralizing activity was significantly (but not completely) reduced upon depletion of natural killer (NK) cells from PBMC (p=0.006), indicating a role for antibody-mediated Fcγ-receptor function. High levels of antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies against CRF01_AE and/or subtype B were subsequently demonstrated in 97% of the sera of vaccinees. The magnitude of ADCC-mediating antibodies against CM235 CRF01_AE IMC-infected cells correlated with neutralizing antibodies against CM235 in the IMC/PBMC assay. In conclusion, HIV-DNA priming, followed by two HIV-MVA boosts elicited potent ADCC responses in a high proportion of Tanzanian vaccinees. Our findings highlight the potential of HIV-DNA prime HIV-MVA boost vaccines for induction of functional antibody responses and suggest this vaccine regimen and ADCC studies as potentially important new avenues in HIV vaccine development. Trial Registration: Controlled-Trials The Pan African Clinical Trials Registry [ABSTRACT FROM AUTHOR]

Background: Intradermal priming with HIV-1 DNA plasmids followed by HIV-1MVA boosting induces strong and broad cellular and humoral immune responses. In our previous HIVIS-03 trial, we used 5 injections with 2 pools of HIV-DNA at separate sites for each priming immunization. The present study explores whether HIV-DNA priming can be simplified by reducing the number of DNA injections and administration of combined versus separated plasmid pools. Methods: In this phase IIa, randomized trial, priming was performed using 5 injections of HIV-DNA, 1000 μg total dose, (3 Env and 2 Gag encoding plasmids) compared to two “simplified” regimens of 2 injections of HIV-DNA, 600 μg total dose, of Env- and Gag-encoding plasmid pools with each pool either administered separately or combined. HIV-DNA immunizations were given intradermally at weeks 0, 4, and 12. Boosting was performed intramuscularly with 108 pfu HIV-MVA at weeks 30 and 46. Results: 129 healthy Tanzanian participants were enrolled. There were no differences in adverse events between the groups. The proportion of IFN-γ ELISpot responders to Gag and/or Env peptides after the second HIV-MVA boost did not differ significantly between the groups primed with 2 injections of combined HIV-DNA pools, 2 injections with separated pools, and 5 injections with separated pools (90%, 97% and 97%). There were no significant differences in the magnitude of Gag and/or Env IFN-γ ELISpot responses, in CD4+ and CD8+ T cell responses measured as IFN-γ/IL-2 production by intracellular cytokine staining (ICS) or in response rates and median titers for binding antibodies to Env gp160 between study groups. Conclusions: A simplified intradermal vaccination regimen with 2 injections of a total of 600 μg with combined HIV-DNA plasmids primed cellular responses as efficiently as the standard regimen of 5 injections of a total of 1000 μg with separated plasmid pools after boosting twice with HIV-MVA. Trial Registration: World Health Organization International Clinical Trials Registry Platform [ABSTRACT FROM AUTHOR]

In this issue of PLOS Pathogens, Hutson and coworkers at the Centers for Disease Control and Prevention (CDC) in Atlanta, USA describe a study with infectious variola virus that was approved by the World Health Organization (WHO) Advisory Committee on Variola Virus Research (ACVVR). In these humanized mice, variola virus can replicate and spread, and although the disease induced is not exactly the same as human smallpox, the model may be useful for testing new therapeutic drugs for smallpox. [Extracted from the article]

Poxviruses are considered less dependent on host functions than other DNA viruses because of their cytoplasmic site of replication and large genomes, which encode enzymes for DNA and mRNA synthesis. Nevertheless, RNAi screens with two independent human genome-scale libraries have identified more than 500 candidate genes that significantly inhibited and a similar number that enhanced replication and spread of infectious vaccinia virus (VACV). Translational, ubiquitin-proteosome, and endoplasmic reticulum-to-Golgi transport functions, known to be important for VACV, were enriched in the siRNA-inhibiting group, and RNA polymerase II and associated functions were enriched in the siRNA-enhancing group. Additional findings, notably the inhibition of VACV spread by siRNAs to several nuclear pore genes, were unanticipated. Knockdown of nucleoporin 62 strongly inhibited viral morphogenesis, with only a modest effect on viral gene expression, recapitulating and providing insight into previous studies with enucleated cells. [ABSTRACT FROM AUTHOR]

The contemporary discussion about spirituality has significant implications for social work education and practice. Recent contributions to the social work literature have deepened and widened our understanding of this domain, although as a profession social workers still remain ambivalent about its value and usefulness. This article reviews some recent contributions to the discussion, and describes how an educational ‘co-creative’ approach can enable spirituality to be located and explored holistically. [ABSTRACT FROM PUBLISHER]

We characterized prime-boost vaccine regimens using heterologous and homologous vector and gene inserts. Heterologous regimens offer a promising approach that focuses the cell-mediated immune response on the insert and away from vector-dominated responses. Ad35-GRIN/ENV (Ad35-GE) vaccine is comprised of two vectors containing sequences from HIV-1 subtype A gag, rt, int, nef (Ad35-GRIN) and env (Ad35-ENV). MVA-CMDR (MVA-C), MVA-KEA (MVA-K) and MVA-TZC (MVA-T) vaccines contain gag, env and pol genes from HIV-1 subtypes CRF01_AE, A and C, respectively. Balb/c mice were immunized with different heterologous and homologous vector and insert prime-boost combinations. HIV and vector-specific immune responses were quantified post-boost vaccination. Gag-specific IFN-c ELISPOT, intracellular cytokine staining (ICS) (CD107a, IFN-c, TNF-a and IL-2), pentamer staining and T-cell phenotyping were used to differentiate responses to inserts and vectors. Ad35-GE prime followed by boost with any of the recombinant MVA constructs (rMVA) induced CD8+ Gag-specific responses superior to Ad35-GE-Ad35-GE or rMVA-rMVA prime-boost combinations. Notably, there was a shift toward insert-focus responses using heterologous vector prime-boost regimens. Gag-specific central and effector memory T cells were generated more rapidly and in greater numbers in the heterologous compared to the homologous prime-boost regimens. These results suggest that heterologous prime-boost vaccination regimens enhance immunity by increasing the magnitude, onset and multifunctionality of the insert-specific cell-mediated immune response compared to homologous vaccination regimens. This study supports the rationale for testing heterologous prime-boost regimens in humans. [ABSTRACT FROM AUTHOR]

For many viruses, one or two proteins enable cell binding, membrane fusion and entry. The large number of proteins employed by poxviruses is unprecedented and may be related to their ability to infect a wide range of cells. There are two main infectious forms of vaccinia virus, the prototype poxvirus: the mature virion (MV), which has a single membrane, and the extracellular enveloped virion (EV), which has an additional outer membrane that is disrupted prior to fusion. Four viral proteins associated with the MV membrane facilitate attachment by binding to glycosaminoglycans or laminin on the cell surface, whereas EV attachment proteins have not yet been identified. Entry can occur at the plasma membrane or in acidified endosomes following macropinocytosis and involves actin dynamics and cell signaling. Regardless of the pathway or whether the MV or EV mediates infection, fusion is dependent on 11 to 12 non-glycosylated, transmembrane proteins ranging in size from 4- to 43-kDa that are associated in a complex. These proteins are conserved in poxviruses making it likely that a common entry mechanism exists. Biochemical studies support a two-step process in which lipid mixing of viral and cellular membranes is followed by pore expansion and core penetration. [ABSTRACT FROM AUTHOR]

Erythromelagia is a condition characterized by attacks of burning pain and inflammation in the extremeties. An epidemic form of this syndrome occurs in secondary students in rural China and a virus referred to as erythromelalgia-associated poxvirus (ERPV) was reported to have been recovered from throat swabs in 1987. Studies performed at the time suggested that ERPV belongs to the orthopoxvirus genus and has similarities with ectromelia virus, the causative agent of mousepox. We have determined the complete genome sequence of ERPV and demonstrated that it has 99.8% identity to the Naval strain of ectromelia virus and a slighly lower identity to the Moscow strain. Small DNA deletions in the Naval genome that are absent from ERPV may suggest that the sequenced strain of Naval was not the immediate progenitor of ERPV. [ABSTRACT FROM AUTHOR]

For many viruses, one or two proteins allow cell attachment and entry, which occurs through the plasma membrane or following endocytosis at low pH. In contrast, vaccinia virus (VACV) enters cells by both neutral and low pH routes; four proteins mediate cell attachment and twelve that are associated in a membrane complex and conserved in all poxviruses are dedicated to entry. The aim of the present study was to determine the roles of cellular and viral proteins in initial stages of entry, specifically fusion of the membranes of the mature virion and cell. For analysis of the role of cellular components, we used well characterized inhibitors and measured binding of a recombinant VACV virion containing Gaussia luciferase fused to a core protein; viral and cellular membrane lipid mixing with a self-quenching fluorescent probe in the virion membrane; and core entry with a recombinant VACV expressing firefly luciferase and electron microscopy. We determined that inhibitors of tyrosine protein kinases, dynamin GTPase and actin dynamics had little effect on binding of virions to cells but impaired membrane fusion, whereas partial cholesterol depletion and inhibitors of endosomal acidification and membrane blebbing had a severe effect at the later stage of core entry. To determine the role of viral proteins, virions lacking individual membrane components were purified from cells infected with members of a panel of ten conditionallethal inducible mutants. Each of the entry protein-deficient virions had severely reduced infectivity and except for A28, L1 and L5 greatly impaired membrane fusion. In addition, a potent neutralizing L1 monoclonal antibody blocked entry at a post-membrane lipid-mixing step. Taken together, these results suggested a 2-step entry model and implicated an unprecedented number of viral proteins and cellular components involved in signaling and actin rearrangement for initiation of virus-cell membrane fusion during poxvirus entry. [ABSTRACT FROM AUTHOR]

A simian immunodeficiency virus (SIV) vaccine coexpressing granulocyte-macrophage colony stimulating factor (GM-CSF) prevented infection in 71% of macaques that received 12 rectal challenges. The SIVsmE660 challenge had the tropism of incident human immunodeficiency virus (HIV) infections and a similar genetic distance from the SIV239 vaccine as intraclade HIV isolates. The heterologous prime-boost vaccine regimen used recombinant DNA for priming and recombinant modified vaccinia Ankara for boosting. Co-expression of GM-CSF in the DNA prime enhanced the avidity of elicited immunoglobulin G for SIV envelope glycoproteins, the titers of neutralizing antibody for easy-to-neutralize SIV isolates, and antibody-dependent cellular cytotoxicity. Impressively, the co-expressed GM-CSF increased vaccine-induced prevention of infection from 25% in the non–GM-CSF co-expressing vaccine group to 71% in the GM-CSF co-expressing vaccine group. The prevention of infection showed a strong correlation with the avidity of the elicited Env-specific antibody for the Env of the SIVsmE660 challenge virus (r = 0.9; P < .0001). [ABSTRACT FROM PUBLISHER]

Background. Recombinant DNA and modified vaccinia virus Ankara (rMVA) vaccines represent a promising approach to an HIV/AIDS vaccine. This Phase 1 clinical trial compared the safety and immunogenicity of a rMVA vaccine administered with and without DNA vaccine primingMethods. GeoVax pGA2/JS7 DNA (D) and MVA/HIV62 (M) vaccines encode noninfectious virus-like particles. Intramuscular needle injections were used to deliver placebo, 2 doses of DNA followed by 2 doses of rMVA (DDMM), one dose of DNA followed by 2 doses of rMVA (DMM), or 3 doses of rMVA (MMM) to HIV-seronegative participants.Results. Local and systemic symptoms were mild or moderate. Immune response rates for CD4 + and CD8 + T cells were highest in the DDMM group and lowest in the MMM group (77% vs 43% CD4 + and 42% vs 17% CD8 +). In contrast, response rates for Env binding and neutralizing Ab were highest in the MMM group. The DMM group had intermediate response rates. A 1/10th-dose DDMM regimen induced similar T cell but reduced Ab response rates compared with the full-dose DDMM.Conclusions. MVA62 was well tolerated and elicited different patterns of T cell and Ab responses when administered alone or in combination with the JS7 DNA vaccine. [ABSTRACT FROM PUBLISHER]

Vaccinia virus (VACV), a member of the chordopox subfamily of the Poxviridae, abortively infects insect cells. We have investigated VACV infection of Drosophila S2 cells, which are useful for protein expression and genome-wide RNAi screening. Biochemical and electron microscopic analyses indicated that VACV entry into Drosophila S2 cells depended on the VACV multiprotein entry-fusion complex but appeared to occur exclusively by a low pH-dependent endocytic mechanism, in contrast to both neutral and low pH entry pathways used in mammalian cells. Deep RNA sequencing revealed that the entire VACV early transcriptome, comprising 118 open reading frames, was robustly expressed but neither intermediate nor late mRNAs were made. Nor was viral late protein synthesis or inhibition of host protein synthesis detected by pulse-labeling with radioactive amino acids. Some reduction in viral early proteins was noted by Western blotting. Nevertheless, synthesis of the multitude of early proteins needed for intermediate gene expression was demonstrated by transfection of a plasmid containing a reporter gene regulated by an intermediate promoter. In addition, expression of a reporter gene with a late promoter was achieved by cotransfection of intermediate genes encoding the late transcription factors. The requirement for transfection of DNA templates for intermediate and late gene expression indicated a defect in viral genome replication in VACV-infected S2 cells, which was confirmed by direct analysis. Furthermore, VACV-infected S2 cells did not support the replication of a transfected plasmid, which occurs in mammalian cells and is dependent on all known viral replication proteins, indicating a primary restriction of DNA synthesis. [ABSTRACT FROM AUTHOR]

Background. Recombinant DNA and modified vaccinia virus Ankara (rMVA) vaccines represent a promising approach to an HIV/AIDS vaccine. This Phase 1 clinical trial compared the safety and immunogenicity of a rMVA vaccine administered with and without DNA vaccine primingMethods. GeoVax pGA2/JS7 DNA (D) and MVA/HIV62 (M) vaccines encode noninfectious virus-like particles. Intramuscular needle injections were used to deliver placebo, 2 doses of DNA followed by 2 doses of rMVA (DDMM), one dose of DNA followed by 2 doses of rMVA (DMM), or 3 doses of rMVA (MMM) to HIV-seronegative participants.Results. Local and systemic symptoms were mild or moderate. Immune response rates for CD4 + and CD8 + T cells were highest in the DDMM group and lowest in the MMM group (77% vs 43% CD4 + and 42% vs 17% CD8 +). In contrast, response rates for Env binding and neutralizing Ab were highest in the MMM group. The DMM group had intermediate response rates. A 1/10th-dose DDMM regimen induced similar T cell but reduced Ab response rates compared with the full-dose DDMM.Conclusions. MVA62 was well tolerated and elicited different patterns of T cell and Ab responses when administered alone or in combination with the JS7 DNA vaccine. [ABSTRACT FROM PUBLISHER]

The eradication of smallpox, one of the great triumphs of medicine, was accomplished through the prophylactic administration of live vaccinia virus, a comparatively benign relative of variola virus, the causative agent of smallpox. Nevertheless, recent fears that variola virus may be used as a biological weapon together with the present susceptibility of unimmunized populations have spurred the development of new-generation vaccines that are safer than the original and can be produced by modern methods. Predicting the efficacy of such vaccines in the absence of human smallpox, however, depends on understanding the correlates of protection. This review outlines the biology of poxviruses with particular relevance to vaccine development, describes protein targets of humoral and cellular immunity, compares animal models of orthopoxvirus disease with human smallpox, and considers the status of second- and third-generation smallpox vaccines. [ABSTRACT FROM AUTHOR]