Horsepox Virus Research Articles

Recombinant chimeric horsepox virus (TNX-801) is attenuated relative to vaccinia virus strains in both in vitro and in vivo models.

Variola and monkeypox viruses are medically important pathogens that can cause fatal human disease. The two FDA-approved vaccines, ACAM-2000 and JYNNEOS, have important advantages and disadvantages. ACAM-2000 offers durable immunity; however, it has high adverse event rates. In contrast, JYNNEOS has a safer profile but requires two doses 4-weeks apart to achieve comparable immunity. Consequently, there is a need for vaccines offering durable immunity via single immunization with minimal adverse events. TNX-801 is a preclinical stage vaccine that can stimulate potent immunity via a single dose and provides protection against lethal mpox disease in the nonhuman primate model. Here, we show that TNX-801 is >10- to 1,000-fold attenuated in in vitro and in vivo models including human primary cells and immunocompromised murine models than vaccine strains utilized in smallpox eradication. The natural attenuation of TNX-801 and its ability to induce protective immunity via a single vaccination are promising and warrants further development.

Immunogenicity and Efficacy of TNX-1800, A Live Virus Recombinant Poxvirus Vaccine Candidate, against SARS-CoV-2 Challenge in Nonhuman Primates.

TNX-1800 is a synthetically derived live recombinant chimeric horsepox virus (rcHPXV) vaccine candidate expressing Wuhan SARS-CoV-2 spike (S) protein. The primary objective of this study was to evaluate the immunogenicity and efficacy of TNX-1800 in two nonhuman primate species challenged with USA-WA1/2020 SARS-CoV-2. TNX-1800 vaccination was well tolerated with no serious adverse events or significant changes in clinical parameters. A single dose of TNX-1800 generated humoral responses in African Green Monkeys and Cynomolgus Macaques, as measured by the total binding of anti-SARS-CoV-2 S IgG and neutralizing antibody titers against the USA-WA1/2020 strain. In addition, a single dose of TNX-1800 induced an interferon-gamma (IFN-γ)-mediated T-cell response in Cynomolgus Macaques. Following challenge with SARS-CoV-2, African Green and Cynomolgus Macaques exhibited rapid clearance of virus in the upper and lower respiratory tract. Future studies will assess the efficacy of TNX-1800 against newly emerging variants and demonstrate its safety in humans.

Immunogenicity and Tolerability of a SARS-CoV-2 TNX-1800, a Live Recombinant Poxvirus Vaccine Candidate, in Syrian Hamsters and New Zealand White Rabbits.

TNX-1800 is a preclinical stage synthetic-derived live attenuated chimeric horsepox virus vaccine engineered to express the SARS-CoV-2 spike (S) gene. The objectives of this study were to assess the safety, tolerability, and immunogenicity of TNX-1800 administration in Syrian golden hamsters and New Zealand white rabbits. Animals were vaccinated at three doses via percutaneous inoculation. The data showed that the single percutaneous administration of three TNX-1800 vaccine dose levels was well tolerated in both hamsters and rabbits. At all dose levels, rabbits were more decerning regarding vaccine site reaction than hamsters. Lastly, no TNX-1800 genomes could be detected at the site of vaccination. Post-vaccination, all animals had anti-SARS-CoV-2 spike protein IgG specific antibody responses. These data demonstrate that TNX-1800 infection was limited, asymptomatic, and cleared by the end of this study, and a single dose was able to generate immune responses.

Single Dose of Recombinant Chimeric Horsepox Virus (TNX-801) Vaccination Protects Macaques from Lethal Monkeypox Challenge.

The ongoing global Monkeypox outbreak that started in the spring of 2022 has reinforced the importance of protecting the population using live virus vaccines based on the vaccinia virus (VACV). Smallpox also remains a biothreat and although some U.S. military personnel are immunized with VACV, safety concerns limit its use in other vulnerable groups. Consequently, there is a need for an effective and safer, single dose, live replicating vaccine against both viruses. One potential approach is to use the horsepox virus (HPXV) as a vaccine. Contemporary VACV shares a common ancestor with HPXV, which from the time of Edward Jenner and through the 19th century, was extensively used to vaccinate against smallpox. However, it is unknown if early HPXV-based vaccines exhibited different safety and efficacy profiles compared to modern VACV. A deeper understanding of HPXV as a vaccine platform may allow the construction of safer and more effective vaccines against the poxvirus family. In a proof-of-concept study, we vaccinated cynomolgus macaques with TNX-801, a recombinant chimeric horsepox virus (rcHPXV), and showed that the vaccine elicited protective immune responses against a lethal challenge with monkeypox virus (MPXV), strain Zaire. The vaccine was well tolerated and protected animals from the development of lesions and severe disease. These encouraging data support the further development of TNX-801.

Monkeypox Vaccines: Current Update

Monkeypox belonging to the genus Orthopoxvirus (OPV) of the family poxviridiae poses a major human threat even after 30 years after eradicating smallpox. OPV family includes other viruses like vaccinia virus (VACV), variola virus and cowpox virus. VACV smallpox vaccine provided cross protective antibody response against other orthopox viruses.1 The ongoing monkeypox outbreak has affected 31,800 people in more than 89 different countries. India has documented 9 confirmed cases with one death reported.2 Stockpile of smallpox vaccines that contain live vaccinia virus is maintained at two locations, one at Centre for Disease Control laboratory at Atlanta, Georgia and the other at VECTOR institute, Koltsovo, Russia. Data shows effectiveness of Smallpox vaccines against monkeypox to be 85%.3 Vaccine used in smallpox eradication were first generation vaccines which produced multiple side effects like eczema, progressive vaccinia and myopericarditis. This fear of adverse effects and safety concerns had led to the production of second and third generation vaccines like imvanex or ACAM2000.4 The interim guidelines for monkeypox vaccination does not urge the need for mass vaccination considering the side effects and the vaccine production. Countries like Canada, United Kingdom and USA are using ring vaccination to prevent monkeypox. The two vaccines currently being used against monkeypox are Jynneos vaccine and ACAM2000.5 JYNNEOS vaccine (Imvamune in Canada /Imvanex in Europe developed by Bavarian Nordiac) for adults aged 18 years and older was approved in Europe and U.S. The vaccine contains a replication deficient live vaccinia virus (Modified vaccinia Ankara-MVA BN) that is given as subcutaneous injection of 0.5 ml, two doses four weeks apart. The vaccine is used for post exposure prophylaxis against monkeypox virus. CDC recommends the intermediate or high risk group as potential group to receive post exposure propylaxis of the vaccine (Unprotected contact with the body fluid of infected person or contact with less 6 meter distance with the infected person). Vaccine is available in countries like Canada, Europe and Britain.6 ACAM2OOO, containing live replicating vaccinia virus that can replicate as compared to jynneos vaccine. Owning to the side effects associated with the vaccine its only used for laboratory workers handling pox viruses or for military personnel.4,7,8 WHO has stated there is no need for mass vaccination and decision regarding vaccination should be based on the individual case accessing the risk and benefits.5 In India, the Serum Institute of India has confirmed its ongoing research to find vaccine against monkeypox. Monkeypox vaccine pipelines include 1. Tonix Pharmaceuticals Holding Corp TNX-8011, a preclinical live virus, which uses horsepox virus 2. EpiVax, Inc’s-Epitome driven vaccine 3. VennVax, DNA prime peptide boost multi-T cell epitome Pox virus vaccine.9

Genetic ancestry and population structure of vaccinia virus

Vaccinia virus (VACV) was used for smallpox eradication, but its ultimate origin remains unknown. The genetic relationships among vaccine stocks are also poorly understood. We analyzed 63 vaccine strains with different origin, as well horsepox virus (HPXV). Results indicated the genetic diversity of VACV is intermediate between variola and cowpox viruses, and that mutation contributed more than recombination to VACV evolution. STRUCTURE identified 9 contributing subpopulations and showed that the lowest drift was experienced by the ancestry components of Tian Tan and HPXV/Mütter/Mulford genomes. Subpopulations that experienced very strong drift include those that contributed the ancestry of MVA and IHD-W, in good agreement with the very long passage history of these vaccines. Another highly drifted population contributed the full ancestry of viruses sampled from human/cattle infections in Brazil and, partially, to IOC clones, strongly suggesting that the recurrent infections in Brazil derive from the spillback of IOC to the feral state.

COVID‐19, varying genetic resistance to viral disease and immune tolerance checkpoints

Coronavirus disease 2019 (COVID‐19) is a zoonosis like most of the great plagues sculpting human history, from smallpox to pandemic influenza and human immunodeficiency virus. When viruses jump into a new species the outcome of infection ranges from asymptomatic to lethal, historically ascribed to “genetic resistance to viral disease.” People have exploited these differences for good and bad, for developing vaccines from cowpox and horsepox virus, controlling rabbit plagues with myxoma virus and introducing smallpox during colonization of America and Australia. Differences in resistance to viral disease are at the core of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) crisis, yet our understanding of the mechanisms in any interspecies leap falls short of the mark. Here I review how the two key parameters of viral disease are countered by fundamentally different genetic mechanisms for resistance: (1) virus transmission, countered primarily by activation of innate and adaptive immune responses; and (2) pathology, countered primarily by tolerance checkpoints to limit innate and adaptive immune responses. I discuss tolerance thresholds and the role of CD8 T cells to limit pathological immune responses, the problems posed by tolerant superspreaders and the signature coronavirus evasion strategy of eliciting only short‐lived neutralizing antibody responses. Pinpointing and targeting the mechanisms responsible for varying pathology and short‐lived antibody were beyond reach in previous zoonoses, but this time we are armed with genomic technologies and more knowledge of immune checkpoint genes. These known unknowns must now be tackled to solve the current COVID‐19 crisis and the inevitable zoonoses to follow.

Molecular and genomic characterization of a novel equine molluscum contagiosum-like virus.

Cases of pox-like lesions in horses and donkeys have been associated with poxviruses belonging to different genera of the family Poxviridae. These include the orthopoxviruses vaccinia virus (VACV), horsepoxvirus (HPXV) and cowpoxvirus (CPXV), as well as a potentially novel parapoxvirus and molluscum contagiosum virus (MOCV). However, with the exception of VACV, HPXV and CPXV, the genomic characterization of the causative agents remains largely elusive with only single short genome fragments available. Here we present the first full-length genome sequence of an equine molluscum contagiosum-like virus (EMCLV) directly determined from skin biopsies of a horse with generalized papular dermatitis. Histopathological analysis of the lesions revealed severe epidermal hyperplasia with numerous eosinophilic inclusion bodies within keratinocytes. Virions were detected in the lesions in embedded tissue by transmission electron microscopy. The genome sequence determined by next- and third-generation sequencing comprises 166 843 nt with inverted terminal repeats (ITRs) of 3473 nt. Overall, 20 of the predicted 159 ORFs have no equivalents in other poxviruses. Intriguingly, two of these ORFs were identified to encode homologues of mammalian proteins involved in immune signalling pathways, namely secreted and transmembrane protein 1 (SECTM1) and insulin growth factor-like family receptor 1 (IGFLR1), that were not described in any virus family so far. Phylogenetic analysis with all relevant representatives of the Poxviridae suggests that EMCLV should be nominated as a new species within the genus Molluscipoxvirus.

Isolation and phylogenomic analysis of buffalopox virus from human and buffaloes in India

Three genome sequences of Buffalopox virus (BPVX) were retrieved from a human and two buffaloes scab samples. Phylogenomic analysis of the BPXV indicates that it shares a most recent common ancestor with Lister and closely related vaccine strains when compared to potential wild-type VACV strains (like Horsepox virus).

Erratum for DiEuliis and Gronvall, "A Holistic Assessment of the Risks and Benefits of the Synthesis of Horsepox Virus".

Volume 3, no. 2, e00074-18, 2018, [https://doi.org/10.1128/mSphere.00074-18][1]. Page 6 of the PDF, third full paragraph, line 4: “smallpox” should be “horsepox.” The corrected sentence should read “Given much of this information already exists in the literature, the publication of the

A Critical Analysis of the Scientific and Commercial Rationales for the De Novo Synthesis of Horsepox Virus.

This article evaluates the scientific and commercial rationales for the synthesis of horsepox virus. I find that the claimed benefits of using horsepox virus as a smallpox vaccine rest on a weak scientific foundation and an even weaker business case that this project will lead to a licensed medical countermeasure. The combination of questionable benefits and known risks of this dual use research raises serious questions about the wisdom of undertaking research that could be used to recreate variola virus. This analysis also raises important questions about the propriety of a private company sponsoring such dual use research without appropriate oversight and highlights an important gap in United States dual use research regulations.

A Holistic Assessment of the Risks and Benefits of the Synthesis of Horsepox Virus.

The re-creation of horsepox virus, an extinct orthopoxvirus with similarity to smallpox virus, has caused concerns in the biosecurity and biodefense communities that the technical capabilities achieved could advance the re-creation of smallpox virus by nefarious actors. The work is now published. While the authors went through due biosecurity diligence at their research institution and with the proper Canadian federal authorities, now that the experiments have been published, there is an opportunity to discuss the dual use risks and benefits of the research itself, as well as those associated with publication of such research-all of which challenge current policies. Here, an analytical framework is used to assess the risks and benefits of such dual use research, and relevant components of biosecurity policy and the biodefense enterprise (including the acquisition of medical countermeasures) in the United States are discussed. The authors emphasize the need to use such risk/benefit assessments at the onset of research and throughout its development, followed by an assessment for its responsible communication.

Resurrection of a poxvirus causes alarm.

A controversial study in which researchers generated an infectious horsepox virus using synthetic DNA and reverse genetics was recently reported.

Construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments.

Edward Jenner and his contemporaries believed that his variolae vaccinae originated in horses and molecular analyses show that modern vaccinia virus (VACV) strains share common ancestry with horsepox virus (HPXV). Given concerns relating to the toxicity of modern VACV vaccines, we asked whether an HPXV-based vaccine might provide a superior alternative. Since HPXV may be extinct and the only specimen of HPXV that has been identified is unavailable for investigation, we explored whether HPXV could be obtained by large-scale gene synthesis. Ten large (10–30 kb) fragments of DNA were synthesized based on the HPXV sequence along with two 157 nt VACV terminal sequences, and were recombined into a live synthetic chimeric HPXV (scHPXV) in cells infected with Shope fibroma virus (SFV). Sequencing of the 212 kbp scHPXV confirmed it encoded a faithful copy of the input DNA. We believe this is the first complete synthesis of a poxvirus using synthetic biology approaches. This scHPXV produced smaller plaques, produced less extracellular virus and exhibited less virulence in mice than VACV, but still provided vaccine protection against a lethal VACV challenge. Collectively, these findings support further development of scHPXV as a novel replication-proficient smallpox vaccine.

Biosecurity Implications for the Synthesis of Horsepox, an Orthopoxvirus.

This article examines the biosecurity and biodefense implications resulting from the recent creation of horsepox virus, a noncirculating (extinct) species of orthopoxvirus. Here we examine the technical aspects of the horsepox virus synthesis and conclude that orthopox synthesis experiments currently remain technically challenging-and will continue to be so, even once this work is published in the scientific literature. This limits potential misuse by some types of nefarious actors. We also examine the implications of one stated purpose for the recreation of horsepox virus: the development of a smallpox vaccine. If the development is successful, it could take advantage of US government incentives for the priority FDA review of medical countermeasures (MCMs) against biosecurity threats. However, if this case leads to the determination that this incentive is counterproductive for security, the newly created priority review voucher program should be more clearly defined or limited based on need. Limiting the program could have costs that require further consideration, however, as general incentives for biodefense medical countermeasure development are required for MCMs to be available. Finally, while the recreation of horsepox virus was not technically trivial, nor was it cell-free, this experiment was a de facto demonstration of already-assumed scientific capabilities. The ability to recreate horsepox, or smallpox, will remain no matter what policy controls are put into place. It will be impossible to close off all avenues for nefarious misuse of gene synthesis, or misuse of biological materials more broadly. As a result, we advocate for the implementation of policy, regulations, and guidance that will make illicit recreation harder, more burdensome, more detectable, and, thus, more preventable without having sweeping negative consequences for the research enterprise. As part of our biosecurity efforts, we must also encourage and enable scientists to participate actively and to do all they can to safeguard their technical fields from irresponsible or illicit actions.

The De Novo Synthesis of Horsepox Virus: Implications for Biosecurity and Recommendations for Preventing the Reemergence of Smallpox.

In March 2017, the American biotech company Tonix announced that a Canadian scientist had synthesized horsepox virus as part of a project to develop a safer vaccine against smallpox. The first de novo synthesis of an orthopoxvirus, a closely related group of viruses that includes horsepox and the variola virus that causes smallpox, crosses an important Rubicon in the field of biosecurity. The synthesis of horsepox virus takes the world one step closer to the reemergence of smallpox as a threat to global health security. That threat has been held at bay for the past 40 years by the extreme difficulty of obtaining variola virus and the availability of effective medical countermeasures. The techniques demonstrated by the synthesis of horsepox have the potential to erase both of these barriers. The primary risk posed by this research is that it will open the door to the routine and widespread synthesis of other orthopoxviruses, such as vaccinia, for use in research, public health, and medicine. The normalization and globalization of orthopoxvirus synthesis for these beneficial applications will create a cadre of laboratories and scientists that will also have the capability and expertise to create infectious variola virus from synthetic DNA. Unless the safeguards against the synthesis of variola virus are strengthened, the capability to reintroduce smallpox into the human population will be globally distributed and either loosely or completely unregulated, providing the foundation for a disgruntled or radicalized scientist, sophisticated terrorist group, unscrupulous company, or rogue state to recreate one of humanity's most feared microbial enemies. The reemergence of smallpox-because of a laboratory accident or an intentional release-would be a global health disaster. International organizations, national governments, the DNA synthesis industry, and the synthetic biology community all have a role to play in devising new approaches to preventing the reemergence of smallpox.

Labmade smallpox is possible, study shows.

Reconstitution of horsepox virus from mail-order DNA reignites synthetic biology debate

Genomic Analysis, Phenotype, and Virulence of the Historical Brazilian Smallpox Vaccine Strain IOC: Implications for the Origins and Evolutionary Relationships of Vaccinia Virus.

Smallpox was declared eradicated in 1980 after an intensive vaccination program using different strains of vaccinia virus (VACV; Poxviridae). VACV strain IOC (VACV-IOC) was the seed strain of the smallpox vaccine manufactured by the major vaccine producer in Brazil during the smallpox eradication program. However, little is known about the biological and immunological features as well as the phylogenetic relationships of this first-generation vaccine. In this work, we present a comprehensive characterization of two clones of VACV-IOC. Both clones had low virulence in infected mice and induced a protective immune response against a lethal infection comparable to the response of the licensed vaccine ACAM2000 and the parental strain VACV-IOC. Full-genome sequencing revealed the presence of several fragmented virulence genes that probably are nonfunctional, e.g., F1L, B13R, C10L, K3L, and C3L. Most notably, phylogenetic inference supported by the structural analysis of the genome ends provides evidence of a novel, independent cluster in VACV phylogeny formed by VACV-IOC, the Brazilian field strains Cantagalo (CTGV) and Serro 2 viruses, and horsepox virus, a VACV-like virus supposedly related to an ancestor of the VACV lineage. Our data strongly support the hypothesis that CTGV-like viruses represent feral VACV that evolved in parallel with VACV-IOC after splitting from a most recent common ancestor, probably an ancient smallpox vaccine strain related to horsepox virus. Our data, together with an interesting historical investigation, revisit the origins of VACV and propose new evolutionary relationships between ancient and extant VACV strains, mainly horsepox virus, VACV-IOC/CTGV-like viruses, and Dryvax strain. First-generation vaccines used to eradicate smallpox had rates of adverse effects that are not acceptable by current health care standards. Moreover, these vaccines are genetically heterogeneous and consist of a pool of quasispecies of VACV. Therefore, the search for new-generation smallpox vaccines that combine low pathogenicity, immune protection, and genetic homogeneity is extremely important. In addition, the phylogenetic relationships and origins of VACV strains are quite nebulous. We show the characterization of two clones of VACV-IOC, a unique smallpox vaccine strain that contributed to smallpox eradication in Brazil. The immunogenicity and reduced virulence make the IOC clones good options for alternative second-generation smallpox vaccines. More importantly, this study reveals the phylogenetic relationship between VACV-IOC, feral VACV established in nature, and the ancestor-like horsepox virus. Our data expand the discussion on the origins and evolutionary connections of VACV lineages.

The evolution of poxvirus vaccines.

After Edward Jenner established human vaccination over 200 years ago, attenuated poxviruses became key players to contain the deadliest virus of its own family: Variola virus (VARV), the causative agent of smallpox. Cowpox virus (CPXV) and horsepox virus (HSPV) were extensively used to this end, passaged in cattle and humans until the appearance of vaccinia virus (VACV), which was used in the final campaigns aimed to eradicate the disease, an endeavor that was accomplished by the World Health Organization (WHO) in 1980. Ever since, naturally evolved strains used for vaccination were introduced into research laboratories where VACV and other poxviruses with improved safety profiles were generated. Recombinant DNA technology along with the DNA genome features of this virus family allowed the generation of vaccines against heterologous diseases, and the specific insertion and deletion of poxvirus genes generated an even broader spectrum of modified viruses with new properties that increase their immunogenicity and safety profile as vaccine vectors. In this review, we highlight the evolution of poxvirus vaccines, from first generation to the current status, pointing out how different vaccines have emerged and approaches that are being followed up in the development of more rational vaccines against a wide range of diseases.

Has horsepox become extinct?

Mystery surrounds the extent to which horsepox virus may have contributed to the vaccinia virus used to eradicate smallpox. With few documented cases of horsepox in recent years it may never be solved, says José Esparza, who seeks to raise awareness of the potential historical and scientific importance of identifying new cases.