Mpox mRNA-1769 Vaccine Inhibits Orthopoxvirus Replication at Intranasal, Intrarectal, and Cutaneous Sites of Inoculation.

The increasing incidence of mpox in Africa and the recent global outbreak with evidence of sexual transmission have stimulated interest in new vaccines and therapeutics. Our previous study demonstrated that mice immunized twice with a quadrivalent lipid nanoparticle vaccine comprising four monkeypox virus mRNAs raised neutralizing antibodies and antigen-specific T cells and were protected against a lethal intranasal challenge with vaccinia virus. Here we extended these findings by using live animal imaging to demonstrate that the mRNA vaccine greatly reduced virus replication and spread from an intranasal site of inoculation and prevented detectable replication at intrarectal and cutaneous inoculation sites. Moreover, considerable protection was achieved with a single vaccination and a booster vaccination enhanced protection for at least 4 months. Protection was related to the amount of mRNA inoculated, which correlated with neutralizing antibody levels. The role of antibody in protection was demonstrated by passive transfer of immune serum pre- or post-challenge to immunocompetent and immunodeficient mice lacking mature B and T cells and therefore unable to mount an adaptive response. These findings provide insights into the mechanism and extent of mRNA vaccine induced protection of orthopoxviruses and support clinical testing.

Comparison of protection against mpox following mRNA or modified vaccinia Ankara vaccination in nonhuman primates.

In 2022, mpox virus (MPXV) spread worldwide, causing 99,581 mpox cases in 121 countries. Modified vaccinia Ankara (MVA) vaccine use reduced disease in at-risk populations but failed to deliver complete protection. Lag in manufacturing and distribution of MVA resulted in additional MPXV spread, with 12,000 reported cases in 2023 and an additional outbreak in Central Africa of clade I virus. These outbreaks highlight the threat of zoonotic spillover by Orthopoxviruses. mRNA-1769, an mRNA-lipid nanoparticle (LNP) vaccine expressing MPXV surface proteins, was tested in a lethal MPXV primate model. Similar to MVA, mRNA-1769 conferred protection against challenge and further mitigated symptoms and disease duration. Antibody profiling revealed a collaborative role between neutralizing and Fc-functional extracellular virion (EV)-specific antibodies in viral restriction and ospinophagocytic and cytotoxic antibody functions in protection against lesions. mRNA-1769 enhanced viral control and disease attenuation compared with MVA, highlighting the potential for mRNA vaccines to mitigate future pandemic threats.

An mpox virus mRNA-lipid nanoparticle vaccine confers protection against lethal orthopoxviral challenge.

Mpox virus (MPXV) caused a global outbreak in 2022. Although smallpox vaccines were rapidly deployed to curb spread and disease among those at highest risk, breakthrough disease was noted after complete immunization. Given the threat of additional zoonotic events and the virus's evolving ability to drive human-to-human transmission, there is an urgent need for an MPXV-specific vaccine that confers protection against evolving MPXV strains and related orthopoxviruses. Here, we demonstrate that an mRNA-lipid nanoparticle vaccine encoding a set of four highly conserved MPXV surface proteins involved in virus attachment, entry, and transmission can induce MPXV-specific immunity and heterologous protection against a lethal vaccinia virus (VACV) challenge. Compared with modified vaccinia virus Ankara (MVA), which forms the basis for the current MPXV vaccine, immunization with an mRNA-based MPXV vaccine generated superior neutralizing activity against MPXV and VACV and more efficiently inhibited spread between cells. We also observed greater Fc effector TH1-biased humoral immunity to the four MPXV antigens encoded by the vaccine, as well as to the four VACV homologs. Single MPXV antigen-encoding mRNA vaccines provided partial protection against VACV challenge, whereas multivalent vaccines combining mRNAs encoding two, three, or four MPXV antigens protected against disease-related weight loss and death equal or superior to MVA vaccination. These data demonstrate that an mRNA-based MPXV vaccine confers robust protection against VACV.

Protection from SARS-CoV-2 Variants by MVAs expressing matched or mismatched S administered intranasally to mice.

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.

Virulence differences of mpox (monkeypox) virus clades I, IIa, and IIb.1 in a small animal model.

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.

Intranasal inoculation of an MVA-based vaccine induces IgA and protects the respiratory tract of hACE2 mice from SARS-CoV-2 infection.

Current vaccines have greatly diminished the severity of the COVID-19 pandemic, even though they do not entirely prevent infection and transmission, likely due to insufficient immunity in the upper respiratory tract. Here, we compare intramuscular and intranasal administration of a live, replication-deficient modified vaccinia virus Ankara (MVA)-based Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike (S) vaccine to raise protective immune responses in the K18-hACE2 mouse model. Using a recombinant MVA expressing firefly luciferase for tracking, live imaging revealed luminescence of the respiratory tract of mice within 6 h and persisting for 3 d following intranasal inoculation, whereas luminescence remained at the site of intramuscular vaccination. Intramuscular vaccination induced S-binding-Immunoglobulin G (IgG) and neutralizing antibodies in the lungs, whereas intranasal vaccination also induced Immunoglobulin A (IgA) and higher levels of antigen-specific CD3+CD8+IFN-γ+ T cells. Similarly, IgG and neutralizing antibodies were present in the blood of mice immunized intranasally and intramuscularly, but IgA was detected only after intranasal inoculation. Intranasal boosting increased IgA after intranasal or intramuscular priming. While intramuscular vaccination prevented morbidity and cleared SARS-CoV-2 from the respiratory tract within several days after challenge, intranasal vaccination was more effective as neither infectious virus nor viral messenger (m)RNAs were detected in the nasal turbinates or lungs as early as 2 d after challenge, indicating prevention or rapid elimination of SARS-CoV-2 infection. Additionally, we determined that neutralizing antibody persisted for more than 6 mo and that serum induced to the Wuhan S protein neutralized pseudoviruses expressing the S proteins of variants, although with less potency, particularly for Beta and Omicron.

Interferon α/ß Decoy Receptor Encoded by a Variant in the Dryvax Smallpox Vaccine Contributes to Virulence and Correlates with Severe Vaccine Side Effects.

Although providing long-lasting immunity, smallpox vaccination was associated with local and systemic reactions and rarely with severe complications, including progressive vaccinia and postvaccinia encephalitis. As the Dryvax smallpox vaccine consists of a population of variants, we investigated a particularly pathogenic isolate called clone 3 (CL3). Virus replication was monitored by inserting the gene encoding firefly luciferase (Luc) into the genomes of CL3 and ACAM2000, the second-generation smallpox vaccine derived from a less virulent clone. Greater luminescence occurred following intranasal or intraperitoneal inoculation of mice with CL3-Luc than ACAM2000-Luc. Previous genome sequencing of CL3 and ACAM2000 revealed numerous differences that could affect pathogenicity. We focused on a 4.2-kbp segment, containing several open reading frames, in CL3 that is absent from ACAM2000 and determined that lower virulence of the latter was associated with a truncation of the interferon α/ß (IFN-α/ß) decoy receptor. Truncation of the decoy receptor in CL3-Luc and repair of the truncated version in ACAM2000-Luc decreased and increased virulence, respectively. Blockade of the mouse type 1 IFN receptor increased the virulence of ACAM2000-Luc to that of CL3-Luc, consistent with the role of IFN in attenuating the former. The severities of disease following intracranial inoculation of immunocompetent mice and intraperitoneal inoculation of T cell-depleted mice were also greater in viruses expressing the full-length decoy receptor. Previous evidence for the low affinity of a similarly truncated decoy receptor for IFN and the presence of a full-length decoy receptor in virus isolated from a patient with progressive vaccinia support our findings. IMPORTANCE Attenuated live viruses make effective vaccines, although concerns exist due to infrequent complications, particularly in individuals with immunological defects. Such complications occurred with smallpox vaccines, which were shown to be comprised of populations of variants. Clone 3, isolated from Dryvax, the vaccine most widely used in the United States during the smallpox eradication campaign, was particularly pathogenic in animal models. We demonstrated that the full-length IFN-α/ß decoy receptor in CL3 and a truncation of the receptor in the clone used for the second-generation smallpox vaccine ACAM2000 account for their difference in pathogenicity. Viruses expressing the full-length decoy receptor were more virulent following intranasal, intraperitoneal, or intracranial inoculation of mice than ACAM2000, and disease was exacerbated following T cell depletion. Correspondingly, the full-length decoy receptor is present in smallpox vaccines with high rates of side effects and in a Dryvax clone obtained from a lesion in a patient with progressive vaccinia.

Spontaneous and Targeted Mutations in the Decapping Enzyme Enhance Replication of Modified Vaccinia Virus Ankara (MVA) in Monkey Cells.

Modified vaccinia virus Ankara (MVA) was derived by repeated passaging in chick fibroblasts, during which deletions and mutations rendered the virus unable to replicate in most mammalian cells. Marker rescue experiments demonstrated that the host range defect could be overcome by replacing DNA that had been deleted from near the left end of the genome. One virus isolate, however, recovered the ability to replicate in monkey BS-C-1 cells but not human cells without added DNA, suggesting that it arose from a spontaneous mutation. Here, we showed that variants with enhanced ability to replicate in BS-C-1 cells could be isolated by blind passaging of MVA and that in each there was a point mutation leading to an amino acid substitution in the D10 decapping enzyme. The sufficiency of these single mutations to enhance host range was confirmed by constructing recombinant viruses. The D10 mutations occurred at N- or C-terminal locations distal to the active site, suggesting an indirect effect on decapping or on another previously unknown role of D10. Although increased amounts of viral mRNA and proteins were found in BS-C-1 cells infected with the mutants compared to those with parental MVA, the increases were much less than the 1- to 2-log-higher virus yields. Nevertheless, a contributing role for diminished decapping in overcoming the host range defect was consistent with increased replication and viral protein synthesis in BS-C-1 cells infected with an MVA engineered to have active-site mutations that abrogate decapping activity entirely. Optimal decapping may vary depending on the biological context. IMPORTANCE Modified vaccinia virus Ankara (MVA) is an attenuated virus that is approved as a smallpox vaccine and is in clinical trials as a vector for other pathogens. The safety of MVA is due in large part to its inability to replicate in mammalian cells. Although host range restriction is considered a stable feature of the virus, we describe the occurrence of spontaneous mutations in MVA that increase replication considerably in monkey BS-C-1 cells but only slightly in human cells. The mutants contain single nucleotide changes that lead to amino acid substitutions in one of the two decapping enzymes. Although the spontaneous mutations are distant from the decapping enzyme active site, engineered active-site mutations also increased virus replication in BS-C-1 cells. The effects of these mutations on the immunogenicity of MVA vectors remain to be determined.

One or two injections of MVA-vectored vaccine shields hACE2 transgenic mice from SARS-CoV-2 upper and lower respiratory tract infection.

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 transgenic mice 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.

Loss of the vaccinia virus 35-amino acid hydrophobic O3 protein is partially compensated by mutations in the transmembrane domains of other entry proteins.

Eleven highly conserved proteins comprise the poxvirus entry-fusion complex (EFC). We focused on vaccinia virus (VACV) O3, a 35-amino acid, largely hydrophobic component of unknown specific function. Experimental evolution was carried out by blindly passaging a virus that was severely impaired in entry due to deletion of the gene encoding O3. Large plaque variants that arose spontaneously were discerned by round four and their numbers increased thereafter. Genome sequencing of individual cloned viruses revealed mutations in predicted transmembrane domains of three open reading frames encoding proteins with roles in entry. There were frame-shift mutations in consecutive Ts in open reading frames F9L and D8L and a nonsynonymous base substitution in L5R. F9 and L5 are EFC proteins and D8 is involved in VACV cell attachment. The F9L mutation occurred by round four in each of three independant passages, whereas the L5R and D8L mutations were detected only after nearly all of the genomes already had the F9L mutation. Viruses with deletions of O3L and single or double F9L, L5R and D8L mutations were constructed by homologous recombination. In a single round of infection, viruses with adaptive mutations including F9L alone or in combination exhibited statistically significant higher virus titers than the parental O3L deletion mutant or the L5R or D8L mutants, consistent with the order of selection during the passages. Further analyses indicated that the adaptive F9L mutants also had higher infectivities, entered cells more rapidly and increased EFC assembly, which partially compensated for the loss of O3.IMPORTANCE Entry into cells is an essential first step in virus replication and an important target of vaccine- elicited immunity. For enveloped viruses, this step involves the fusion of viral and host membranes to form a pore allowing entry of the genome and associated proteins. Poxviruses are unique in that this function is mediated by an entry-fusion complex (EFC) of eleven transmembrane proteins rather than by one or a few. The large number of proteins has hindered investigation of their individual roles. We focused on O3, a predominantly hydrophobic 35 amino acid component of the vaccinia virus EFC, and found that spontaneous mutations in the transmembrane domains of certain other entry proteins can partially compensate for the absence of O3. The mutants exhibited increased infectivity, entry and assembly or stability of the EFC.

MVA Vector Vaccines Inhibit SARS CoV-2 Replication in Upper and Lower Respiratory Tracts of Transgenic Mice and Prevent Lethal Disease.

Replication-restricted modified vaccinia virus Ankara (MVA) is a licensed smallpox vaccine and numerous clinical studies investigating recombinant MVAs (rMVAs) as vectors for prevention of other infectious diseases have been completed or are in progress. Two rMVA COVID-19 vaccine trials are at an initial stage, though no animal protection studies have been reported. Here, we characterize rMVAs expressing the S protein of 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) 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 human cells and was recognized by anti-RBD antibody and by soluble hACE2 receptor. Intramuscular injection of mice with the rMVAs induced S-binding and pseudovirus-neutralizing antibodies. Boosting occurred following a second homologous rMVA but was higher with adjuvanted purified RBD protein. Weight loss and lethality following intranasal infection of transgenic hACE2 mice with CoV-2 was prevented by one or two immunizations with rMVAs or by passive transfer of serum from vaccinated mice. One or two rMVA vaccinations also prevented recovery of infectious CoV-2 from the lungs. A low amount of virus was detected in the nasal turbinates of only one of eight rMVA-vaccinated mice on day 2 and none later. Detection of subgenomic mRNA in turbinates on day 2 only indicated that replication was abortive in immunized animals.

Natural killer cells expanded in vivo or ex vivo with IL-15 overcomes the inherent susceptibility of CAST mice to lethal infection with orthopoxviruses.

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.

Growth and Purification of Vaccinia Virus Stocks for MPM Imaging.

This chapter provides methods for the propagation, purification, and titration of vaccinia virus (VACV) and the highly attenuated strain-modified vaccinia Ankara (MVA). Additionally, we provide information on VACV recombinants we have used for intravital imaging with multiphoton excitation.

Lymph node conduits transport virions for rapid T cell activation.

Despite intense interest in antiviral T cell priming, the routes by which virions move in lymph nodes (LNs) are imperfectly understood. Current models fail to explain how virus-infected cells rapidly appear within the LN interior after viral infection. To better understand virion trafficking in the LN, we determined the locations of virions and infected cells after administration to mice of vaccinia virus or Zika virus. Notably, many rapidly infected cells in the LN interior were adjacent to LN conduits. Through the use of confocal and electron microscopy, we clearly visualized virions within conduits. Functionally, CD8+ T cells rapidly and preferentially associated with vaccinia virus-infected cells in the LN paracortex, which led to T cell activation in the LN interior. These results reveal that it is possible for even large virions to flow through LN conduits and infect dendritic cells within the T cell zone to prime CD8+ T cells.

Human Host Range Restriction of the Vaccinia Virus C7/K1 Double Deletion Mutant Is Mediated by an Atypical Mode of Translation Inhibition.

Replication of vaccinia virus in human cells depends on the viral C7 or K1 protein. A previous human genome-wide short interfering RNA (siRNA) screen with a C7/K1 double deletion mutant revealed SAMD9 as a principal host range restriction factor along with additional candidates, including WDR6 and FTSJ1. To compare their abilities to restrict replication, the cellular genes were individually inactivated by CRISPR/Cas9 mutagenesis. The C7/K1 deletion mutant exhibited enhanced replication in each knockout (KO) cell line but reached wild-type levels only in SAMD9 KO cells. SAMD9 was not depleted in either WDR6 or FTSJ1 KO cells, suggesting less efficient alternative rescue mechanisms. Using the SAMD9 KO cells as controls, we verified a specific block in host and viral intermediate and late protein synthesis in HeLa cells and demonstrated that the inhibition could be triggered by events preceding viral DNA replication. Inhibition of cap-dependent and -independent protein synthesis occurred primarily at the translational level, as supported by DNA and mRNA transfection experiments. Concurrent with collapse of polyribosomes, viral mRNA was predominantly in 80S and lighter ribonucleoprotein fractions. We confirmed the accumulation of cytoplasmic granules in HeLa cells infected with the C7/K1 deletion mutant and further showed that viral mRNA was sequestered with SAMD9. RNA granules were still detected in G3BP KO U2OS cells, which remained nonpermissive for the C7/K1 deletion mutant. Inhibition of cap-dependent and internal ribosome entry site-mediated translation, sequestration of viral mRNA, and failure of PKR, RNase L, or G3BP KO cells to restore protein synthesis support an unusual mechanism of host restriction.IMPORTANCE A dynamic relationship exists between viruses and their hosts in which each ostensibly attempts to exploit the other's vulnerabilities. A window is opened into the established condition, which evolved over millennia, if loss-of-function mutations occur in either the virus or host. Thus, the inability of viral host range mutants to replicate in specific cells can be overcome by identifying and inactivating the opposing cellular gene. Here, we investigated a C7/K1 host range mutant of vaccinia virus in which the cellular gene SAMD9 serves as the principal host restriction factor. Host restriction was triggered early in infection and manifested as a block in translation of viral mRNAs. Features of the block include inhibition of cap-dependent and internal ribosome entry site-mediated translation, sequestration of viral RNA, and inability to overcome the inhibition by inactivation of protein kinase R, ribonuclease L, or G3 binding proteins, suggesting a novel mechanism of host restriction.

Droplet digital PCR for rapid enumeration of viral genomes and particles from cells and animals infected with orthopoxviruses.

Droplet digital polymerase chain reaction (ddPCR) was adapted for quantifying the number of orthopoxviral genomes in purified virus samples, infected cell lysates and tissues of infected animals. In contrast to the more commonly used qPCR, the newer ddPCR provides absolute numbers of DNA copies in samples without need for standard curves and has the ability to detect rare mutants in a population. The genome/infectious unit ratio for several sucrose gradient-purified orthopoxviruses varied from 5 to 10, which correlated well with values obtained using the Virocyt, a dedicated fluorescence flow cytometer. By employing a nuclease step to digest unencapsulated DNA, the genome/infectious unit ratios of virus in crude cell lysates approached that of purified virus particles. The speed, accuracy, sensitivity, and dynamic range of less than one to millions of infectious units in a sample make this semi-automated method well suited to a variety of laboratory, animal and clinical studies.

Insufficient Innate Immunity Contributes to the Susceptibility of the Castaneous Mouse to Orthopoxvirus Infection.

The castaneous (CAST) mouse, a wild-derived inbred strain, is highly susceptible to orthopoxvirus infection by intranasal and systemic routes. The 50% lethal intraperitoneal dose of vaccinia virus (VACV) was 3 PFU for CAST mice, whereas BALB/c mice survived 106 PFU. At all times and in all organs analyzed, virus titers were higher in CAST than in BALB/c mice. In individual CAST mice, luciferase-expressing VACV was seen to replicate rapidly leading to death, whereas virus levels increased for a few days and then declined in BALB/c mice. Increases in gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) were delayed and low in CAST mice compared to BALB/c mice following VACV infection or poly(I-C) inoculation, consistent with differences in innate immune responses. In addition, naive CAST mice had considerably lower numbers of NK and T cells than BALB/c mice. The percentage of IFN-γ-producing CD4+ and CD8+ T cells increased following infection of CAST mice only after considerable virus spread, and the absolute cell numbers remained low. Administration of exogenous IFN-γ or -α to CAST mice before or during the first days of infection suppressed virus replication and prolonged survival, allowing the mice to make adaptive CD4+ and CD8+ T cell responses that were necessary to clear the virus after cessation of interferon treatment. Thus, insufficient innate cytokine and cellular immune responses contribute to the unique susceptibility of CAST mice to VACV, whereas the adaptive immune response can be protective only if virus replication is suppressed during the first several days of infection.IMPORTANCE Most inbred mouse strains are relatively resistant to orthopoxviruses. The castaneous (CAST) mouse is a notable exception, exhibiting extreme vulnerability to monkeypox virus, cowpox virus, and vaccinia virus and thus providing a unique model for studying pathogenicity, immunity, vaccines, and antiviral drugs. To fully utilize the CAST mouse for such purposes, it is necessary to understand the basis for virus susceptibility. We showed that naive CAST mice make low IFN-γ and TNF-α responses and have low levels of NK cells and CD4+ and CD8+ T cells compared to a resistant classical inbred mouse strain. Attenuating virus replication with one or more doses of exogenous IFN-α or -γ before or during the first few days of infection enabled the development of adaptive cellular immunity and clearance of virus. Further genetic studies may reveal the basis for the low innate immunity.

Novel Nonreplicating Vaccinia Virus Vector Enhances Expression of Heterologous Genes and Suppresses Synthesis of Endogenous Viral Proteins.

Viruses are used as expression vectors for protein synthesis, immunology research, vaccines, and therapeutics. Advantages of poxvirus vectors include the accommodation of large amounts of heterologous DNA, the presence of a cytoplasmic site of transcription, and high expression levels. On the other hand, competition of approximately 200 viral genes with the target gene for expression and immune recognition may be disadvantageous. We describe a vaccinia virus (VACV) vector that uses an early promoter to express the bacteriophage T7 RNA polymerase; has the A23R intermediate transcription factor gene deleted, thereby restricting virus replication to complementing cells; and has a heterologous gene regulated by a T7 promoter. In noncomplementing cells, viral early gene expression and DNA replication occurred normally but synthesis of intermediate and late proteins was prevented. Nevertheless, the progeny viral DNA provided templates for abundant expression of heterologous genes regulated by a T7 promoter. Selective expression of the Escherichia coli lac repressor gene from an intermediate promoter reduced transcription of the heterologous gene specifically in complementing cells, where large amounts might adversely impact VACV replication. Expression of heterologous proteins mediated by the A23R deletion vector equaled that of a replicating VACV, was higher than that of a nonreplicating modified vaccinia virus Ankara (MVA) vector used for candidate vaccines in vitro and in vivo, and was similarly immunogenic in mice. Unlike the MVA vector, the A23R deletion vector still expresses numerous early genes that can restrict immunogenicity as demonstrated here by the failure of the prototype vector to induce interferon alpha. By deleting immunomodulatory genes, we anticipate further improvements in the system.IMPORTANCE Vaccines provide an efficient and effective way of preventing infectious diseases. Nevertheless, new and better vaccines are needed. Vaccinia virus, which was used successfully as a live vaccine to eradicate smallpox, has been further attenuated and adapted as a recombinant vector for immunization against other pathogens. However, since the initial description of this vector system, only incremental improvements largely related to safety have been implemented. Here we described novel modifications of the platform that increased expression of the heterologous target gene and decreased expression of endogenous vaccinia virus genes while providing safety by preventing replication of the candidate vaccine except in complementing cells used for vector propagation.

Retrograde Transport from Early Endosomes to the trans-Golgi Network Enables Membrane Wrapping and Egress of Vaccinia Virus Virions.

UNLABELLED: The anterograde pathway, from the endoplasmic reticulum through the trans-Golgi network to the cell surface, is utilized by trans-membrane and secretory proteins. The retrograde pathway, which directs traffic in the opposite direction, is used following endocytosis of exogenous molecules and recycling of membrane proteins. Microbes exploit both routes: viruses typically use the anterograde pathway for envelope formation prior to exiting the cell, whereas ricin and Shiga-like toxins and some nonenveloped viruses use the retrograde pathway for cell entry. Mining a human genome-wide RNA interference (RNAi) screen revealed a need for multiple retrograde pathway components for cell-to-cell spread of vaccinia virus. We confirmed and extended these results while discovering that retrograde trafficking was required for virus egress rather than entry. Retro-2, a specific retrograde trafficking inhibitor of protein toxins, potently prevented spread of vaccinia virus as well as monkeypox virus, a human pathogen. Electron and confocal microscopy studies revealed that Retro-2 prevented wrapping of virions with an additional double-membrane envelope that enables microtubular transport, exocytosis, and actin polymerization. The viral B5 and F13 protein components of this membrane, which are required for wrapping, normally colocalize in the trans-Golgi network. However, only B5 traffics through the secretory pathway, suggesting that F13 uses another route to the trans-Golgi network. The retrograde route was demonstrated by finding that F13 was largely confined to early endosomes and failed to colocalize with B5 in the presence of Retro-2. Thus, vaccinia virus makes novel use of the retrograde transport system for formation of the viral wrapping membrane. IMPORTANCE: Efficient cell-to-cell spread of vaccinia virus and other orthopoxviruses depends on the wrapping of infectious particles with a double membrane that enables microtubular transport, exocytosis, and actin polymerization. Interference with wrapping or subsequent steps results in severe attenuation of the virus. Some previous studies had suggested that the wrapping membrane arises from the trans-Golgi network, whereas others suggested an origin from early endosomes. Some nonenveloped viruses use retrograde trafficking for entry into the cell. In contrast, we provided evidence that retrograde transport from early endosomes to the trans-Golgi network is required for the membrane-wrapping step in morphogenesis of vaccinia virus and egress from the cell. The potent in vitro inhibition of this step by the drug Retro-2 suggests that derivatives with enhanced pharmacological properties might serve as useful antipoxviral agents.

Genetic studies of the susceptibility of classical and wild-derived inbred mouse strains to monkeypox virus.

Previously, we screened 38 inbred mouse strains for susceptibility to monkeypox virus (MPXV) and focused on wild-derived CAST mice because of their extreme vulnerability. Here, we provide further analysis of inbred mouse strains. NZW/Lac and C58 mice exhibited more weight loss than other classical inbred strains but all survived intranasal challenges with 10(4) to 10(6)PFU of MPXV. Mice from three wild derived strains, in addition to CAST, exhibited severe weight loss and died or were euthanized. LD50 values for CASA, MOLF and PERA were 100, 6800 and >10(5)PFU, respectively. CASA was inbred independently from the same founders as CAST, whereas MOLF and PERA are genetically and geographically distinct. The MPXV susceptibility of the F1 progeny of CAST and either C57BL/6 or BALB/c indicated that resistance is dominant. Back-crossing the F1 progeny of C57BL/6 and CAST to CAST suggested more than one independent resistant locus.