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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.

Significance Vaccines are required to control COVID-19 during the current pandemic and possibly afterward. Recombinant nucleic acids, proteins, and virus vectors that stimulate immune responses to the SARS-CoV-2 S protein have provided protection in experimental animal and human clinical trials, although questions remain regarding their ability to prevent spread and the duration of immunity. The present study focuses on replication-restricted modified vaccinia virus Ankara (MVA), which has been shown to be a safe, immunogenic, and stable smallpox vaccine and a promising vaccine vector for other infectious diseases and cancer. In a transgenic mouse model, one or two injections of recombinant MVAs that express modified forms of S inhibited SARS-CoV-2 replication in the upper and lower respiratory tracts and prevented severe disease., 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.

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 frameshift mutations in consecutive T’s 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 independent 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 11 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.

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.SignificanceVaccines are required to control COVID-19 during the pandemic and possibly afterwards. Recombinant nucleic acids, proteins and virus vectors that stimulate immune responses to the CoV-2 S protein have provided protection in experimental animal or human clinical trials, though questions remain regarding their ability to prevent spread and the duration of immunity. The present study focuses on replication-restricted modified vaccinia virus Ankara (MVA), which has been shown to be a safe, immunogenic and stable smallpox vaccine and a promising vaccine vector for other infectious diseases and cancer. In a transgenic mouse model, one or two injections of recombinant MVAs that express modified forms of S inhibited CoV-2 replication in the upper and lower respiratory tracts and prevented severe disease.

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.

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.

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.

Classical inbred mice are extensively used for virus research. However, we recently found that some wild-derived inbred mouse strains are more susceptible than classical strains to monkeypox virus. Experiments described here indicated that the 50% lethal dose of vaccinia virus (VACV) and cowpox virus (CPXV) were two logs lower in wild-derived inbred CAST/Ei mice than classical inbred BALB/c mice, whereas there was little difference in the susceptibility of the mouse strains to herpes simplex virus. Live bioluminescence imaging was used to follow spread of pathogenic and attenuated VACV strains and CPXV virus from nasal passages to organs in the chest and abdomen of CAST/Ei mice. Luminescence increased first in the head and then simultaneously in the chest and abdomen in a dose-dependent manner. The spreading kinetics was more rapid with VACV than CPXV although the peak photon flux was similar. These data suggest advantages of CAST/Ei mice for orthopoxvirus studies.

Infection with monkeypox virus (MPXV) causes disease manifestations in humans that are similar, although usually less severe, than those of smallpox. Since routine vaccination for smallpox ceased more than 30 years ago, there is concern that MPXV could be used for bioterrorism. Thus, there is a need to develop animal models to study MPXV infection. Accordingly, we screened 38 inbred mouse strains for susceptibility to MPXV. Three highly susceptible wild-derived inbred strains were identified, of which CAST/EiJ was further developed as a model. Using an intranasal route of infection with an isolate of the Congo Basin clade of MPXV, CAST/EiJ mice exhibited weight loss, morbidity, and death in a dose-dependent manner with a calculated 50% lethal dose (LD 50 ) of 680 PFU, whereas there were no deaths of BALB/c mice at a 10,000-fold higher dose. CAST/EiJ mice exhibited greater MPXV sensitivity when infected via the intraperitoneal route, with an LD 50 of 14 PFU. Both routes resulted in MPXV replication in the lung, spleen, and liver. Intranasal infection with an isolate of the less-pathogenic West African clade yielded an LD 50 of 7,600 PFU. The immune competence of CAST/EiJ mice was established by immunization with vaccinia virus, which induced antigen-specific T- and B-lymphocyte responses and fully protected mice from lethal doses of MPXV. The new mouse model has the following advantages for studying pathogenesis of MPXV, as well as for evaluation of potential vaccines and therapeutics: relative sensitivity to MPXV through multiple routes, genetic homogeneity, available immunological reagents, and commercial production.

The success of the World Health Organization smallpox eradication program three decades ago resulted in termination of routine vaccination and consequent decline in population immunity. Despite concerns regarding the reintroduction of smallpox, there is little enthusiasm for large-scale redeployment of licensed live vaccinia virus vaccines because of medical contraindications and anticipated serious side effects. Therefore, highly attenuated strains such as modified vaccinia virus Ankara (MVA) are under evaluation in humans and animal models. Previous studies showed that priming and boosting with MVA provided protection for >2 years in a monkeypox virus challenge model. If variola virus were used as a biological weapon, however, the ability of a vaccine to quickly induce immunity would be essential. Here, we demonstrate more rapid immune responses after a single vaccination with MVA compared to the licensed Dryvax vaccine. To determine the kinetics of protection of the two vaccines, macaques were challenged intravenously with monkeypox virus at 4, 6, 10, and 30 days after immunization. At 6 or more days after vaccination with MVA or Dryvax, the monkeys were clinically protected (except for 1 of 16 animals vaccinated with MVA), although viral loads and number of skin lesions were generally higher in the MVA vaccinated group. With only 4 days between immunization and intravenous challenge, however, MVA still protected whereas Dryvax failed. Protection correlated with the more rapid immune response to MVA compared to Dryvax, which may be related to the higher dose of MVA that can be tolerated safely.

Three distinct chimpanzee Fabs against the A33 envelope glycoprotein of vaccinia virus were isolated and converted into complete monoclonal antibodies (MAbs) with human γ1 heavy-chain constant regions. The three MAbs (6C, 12C, and 12F) displayed high binding affinities to A33 ( K d of 0.14 nM to 20 nM) and may recognize the same epitope, which was determined to be conformational and located within amino acid residues 99 to 185 at the C terminus of A33. One or more of the MAbs were shown to reduce the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro and to more effectively protect mice when administered before or 2 days after intranasal challenge with virulent vaccinia virus than a previously isolated mouse anti-A33 MAb (1G10) or vaccinia virus immunoglobulin. The protective efficacy afforded by anti-A33 MAb was comparable to that of a previously isolated chimpanzee/human anti-B5 MAb. The combination of anti-A33 MAb and anti-B5 MAb did not synergize the protective efficacy. These chimpanzee/human anti-A33 MAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox and other orthopoxvirus diseases.

Chimpanzee Fabs against the B5 envelope glycoprotein of vaccinia virus were isolated and converted into complete mAbs with human γ1 heavy chain constant regions. The two mAbs (8AH8AL and 8AH7AL) displayed high binding affinities to B5 ( K d of 0.2 and 0.7 nM). The mAb 8AH8AL inhibited the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro , protected mice from subsequent intranasal challenge with virulent vaccinia virus, protected mice when administered 2 days after challenge, and provided significantly greater protection than that afforded by a previously isolated rat anti-B5 mAb (19C2) or by vaccinia immune globulin. The mAb bound to a conformational epitope between amino acids 20 and 130 of B5. These chimpanzee/human anti-B5 mAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox.

While characterizing modified vaccinia virus recombinants (rMVAs) containing human immunodeficiency virus env and gag - pol genes, we detected nonexpressing mutants by immunostaining individual plaques. In many cases, the numbers of mutants increased during successive passages, indicating strong selection pressure. This phenomenon provided an opportunity to investigate the formation of spontaneous mutations in vaccinia virus, which encodes its own cytoplasmic replication system, and a challenge to reduce the occurrence of mutations for vaccine production. Analysis of virus from individual plaques indicated that loss of expression was due to frameshift mutations, mostly by addition or deletion of a single nucleotide in runs of four to six Gs or Cs, and large deletions that included MVA DNA flanking the recombinant gene. Interruption of the runs of Gs and Cs by silent codon alterations and moving the recombinant gene to a site between essential, highly conserved MVA genes eliminated or reduced frameshifts and viable deletion mutants, respectively. The rapidity at which nonexpressing mutants accumulated depended on the individual env and gag - pol genes and their suppressive effects on virus replication. Both the extracellular and transmembrane domains contributed to the selection of nonexpressing Env mutants. Stability of an unstable Env was improved by swapping external or transmembrane domains with a more stable Env. Most dramatically, removal of the transmembrane and cytoplasmic domains stabilized even the most highly unstable Env. Understanding the causes of instability and taking preemptive actions will facilitate the development of rMVA and other poxviruses as human and veterinary recombinant vaccines.

Immunization with recombinant proteins may provide a safer alternative to live vaccinia virus for prophylaxis of poxvirus infections. Although antibody protects against vaccinia virus infection, the mechanism is not understood and the selection of immunogens is daunting as there are dozens of surface proteins and two infectious forms known as the mature virion (MV) and the enveloped virion (EV). Our previous studies showed that mice immunized with soluble forms of EV membrane proteins A33 and B5 and MV membrane protein L1 or passively immunized with antibodies to these proteins survived an intranasal challenge with vaccinia virus. The present study compared MV protein A27, which has a role in virus attachment to glycosaminoglycans on the cell surface, to L1 with respect to immunogenicity and protection. Although mice developed similar levels of neutralizing antibody after immunizations with A27 or L1, A27-immunized mice exhibited more severe disease upon an intranasal challenge with vaccinia virus. In addition, mice immunized with A27 and A33 were not as well protected as mice receiving L1 and A33. Polyclonal rabbit anti-A27 and anti-L1 IgG had equivalent MV-neutralizing activities when measured by the prevention of infection of human or mouse cells or cells deficient in glycosaminoglycans or by adding antibody prior to or after virus adsorption. Nevertheless, the passive administration of antibody to A27 was poorly protective compared to the antibody to L1. These studies raise questions regarding the basis for antibody protection against poxvirus disease and highlight the importance of animal models for the early evaluation of vaccine candidates.

Recombinant proteins are being evaluated as smallpox and monkeypox vaccines because of their perceived safety compared to live vaccinia virus. Previously, we demonstrated that three or more injections of a Ribi-type adjuvant with a combination of three proteins from the outer membranes of intracellular (L1 protein) and extracellular (A33 and B5 proteins) forms of vaccinia virus protected mice against a lethal intranasal challenge with vaccinia virus. Here, we compared several adjuvants and found that QS-21 and to a lesser extent alum+CpG oligodeoxynucleotides accelerated and enhanced neutralizing antibody responses to a mixture of L1 and A33 proteins, provided the highest ratio of IgG2a to IgG1 isotype response, and protected mice against disease and death after only two immunizations 3 weeks apart. In addition, monkeys immunized with recombinant vaccinia virus proteins and QS-21 developed neutralizing antibody to monkeypox virus and had reduced virus load, skin lesions, and morbidity compared to the non-immunized group following monkeypox virus challenge.

The potential use of smallpox as a biological weapon has led to the production and stockpiling of smallpox vaccine and the immunization of some healthcare workers. Another public health goal is the licensing of a safer vaccine that could benefit the millions of people advised not to take the current one because they or their contacts have increased susceptibility to severe vaccine side effects. As vaccines can no longer be tested for their ability to prevent smallpox, licensing will necessarily include comparative immunogenicity and protection studies in non-human primates. Here we compare the highly attenuated modified vaccinia virus Ankara (MVA) with the licensed Dryvax vaccine in a monkey model. After two doses of MVA or one dose of MVA followed by Dryvax, antibody binding and neutralizing titres and T-cell responses were equivalent or higher than those induced by Dryvax alone. After challenge with monkeypox virus, unimmunized animals developed more than 500 pustular skin lesions and became gravely ill or died, whereas vaccinated animals were healthy and asymptomatic, except for a small number of transient skin lesions in animals immunized only with MVA.

A rapid and sensitive neutralization assay is required to evaluate alternative smallpox vaccines. Here we describe the development and use of a 96-well plate, semi-automated, flow cytometric assay that uses a recombinant vaccinia virus expressing enhanced green fluorescent protein and which would be applicable to other viruses.

Modified vaccinia Ankara (MVA) is being tested in humans as an alternative to the current smallpox vaccine Dryvax. Here, we compare the magnitude and longevity of protective immune responses elicited by a DNA/MVA HIV-1 vaccine with those elicited by Dryvax using a monkeypox virus/macaque model. The DNA/MVA vaccine elicited similar levels of vaccinia virus (VV)-specific antibody and 5–10-fold lower levels of VV-specific cellular responses than Dryvax. This MVA-elicited cellular and humoral immunity was long-lived. A subset of the DNA/MVA- and Dryvax-vaccinated macaques were subjected to a lethal monkeypox virus challenge at 3 years after vaccination. All of the vaccinated monkeys survived, whereas the unvaccinated controls succumbed to monkeypox. The viral control correlated with early postchallenge levels of monkeypox-specific neutralizing antibody but not with VV-specific cellular immune response. Thus, our results demonstrate the elicitation of long lasting protective immunity for a lethal monkeypox challenge by a DNA/MVA HIV-1 vaccine.