Poxvirus Vaccines: Past, Present, and Future.

Smallpox was a significant cause of mortality for over three thousand years, amounting to 10% of deaths yearly. Edward Jenner discovered smallpox vaccination in 1796, which rapidly became a smallpox infection preventive practice throughout the world and eradicated smallpox infection by 1980. After smallpox eradication, monkeypox vaccines have been used primarily in research and in outbreaks in Africa, where the disease is endemic. In the present, the vaccines are being used for people who work with animals or in high-risk areas, as well as for healthcare workers treating patients with monkeypox. Among all orthopoxviruses (OPXV), monkeypox viral (MPXV) infection occurs mainly in cynomolgus monkeys, natural reservoirs, and occasionally causes severe multi-organ infection in humans, who were the incidental hosts. The first case of the present epidemic of MXPV was identified on May 7, 2022, and rapidly increased the number of cases. In this regard, the WHO declared the outbreak, an international public health emergency on July 23, 2022. The first monkeypox vaccine was developed in the 1960s by the US Army and was based on the vaccinia virus, which is also used in smallpox vaccines. In recent years, newer monkeypox vaccines have been developed based on other viruses such as Modified Vaccinia Ankara (MVA). These newer vaccines are safer and can provide longer-lasting immunity with fewer side effects. For the future, there is ongoing research to improve the current vaccines and to develop new ones. One notable advance has been the development of a recombinant vaccine that uses a genetically modified vaccinia virus to express monkeypox antigens. This vaccine has shown promising results in pre-clinical trials and is currently undergoing further testing in clinical trials. Another recent development has been the use of a DNA vaccine, which delivers genetic material encoding monkeypox antigens directly into cells. This type of vaccine has shown effectiveness in animal studies and is also undergoing clinical testing in humans. Overall, these recent advances in monkeypox vaccine development hold promise for protecting individuals against this potentially serious disease.

In-silico formulation of a next-generation polyvalent vaccine against multiple strains of monkeypox virus and other related poxviruses.

Mpox (formerly known as monkeypox) virus and some related poxviruses including smallpox virus pose a significant threat to public health, and effective prevention and treatment strategies are needed. This study utilized a reverse vaccinology approach to retrieve conserved epitopes for monkeypox virus and construct a vaccine that could provide cross-protection against related viruses with similar antigenic properties. The selected virulent proteins of monkeypox virus, MPXVgp165, and Virion core protein P4a, were subjected to epitope mapping for vaccine construction. Two vaccines were constructed using selected T cell epitopes and B cell epitopes with PADRE and human beta-defensins adjuvants conjugated in the vaccine sequence. Both constructs were found to be highly antigenic, non-allergenic, nontoxic, and soluble, suggesting their potential to generate an adequate immune response and be safe for humans. Vaccine construct 1 was selected for molecular dynamic simulation studies. The simulation studies revealed that the TLR8-vaccine complex was more stable than the TLR3-vaccine complex. The lower RMSD and RMSF values of the TLR8 bound vaccine compared to the TLR3 bound vaccine suggested better stability and consistency of hydrogen bonds. The Rg values of the vaccine chain bound to TLR8 indicated overall stability, whereas the vaccine chain bound to TLR3 showed deviations throughout the simulation. These results suggest that the constructed vaccine could be a potential preventive measure against monkeypox and related viruses however, further experimental validation is required to confirm these findings.

Immune interactions and heterogeneity in transmission drives the pathogen-mediated invasion of grey squirrels in the UK.

Mathematical models highlighted the importance of pathogen-mediated invasion, with the replacement of red squirrels by squirrelpox virus (SQPV) carrying grey squirrels in the UK, a well-known example. In this study, we combine new epidemiological models, with a range of infection characteristics, with recent longitudinal field and experimental studies on the SQPV dynamics in red and grey squirrel populations to better infer the mechanistic basis of the disease interaction. A key finding is that a model with either partial immunity or waning immunity and reinfection, where individuals become seropositive on the second exposure to infection, that up to now has been shown in experimental data only, can capture the key aspects of the field study observations. By fitting to SQPV epidemic observations in isolated red squirrel populations, we can infer that SQPV transmission between red squirrels is significantly (4×) higher than the transmission between grey squirrels and as a result our model shows that disease-mediated replacement of red squirrels by greys is considerably more rapid than replacement in the absence of SQPV. Our findings recover the key results of the previous model studies, which highlights the value of simple strategic models that are appropriate when there are limited data, but also emphasise the likely complexity of immune interactions in wildlife disease and how models can help infer disease processes from field data.

Highly Attenuated Poxvirus-Based Vaccines Against Emerging Viral Diseases.

Although one member of the poxvirus family, variola virus, has caused one of the most devastating human infections worldwide, smallpox, the knowledge gained over the last 30 years on the molecular, virological and immunological mechanisms of these viruses has allowed the use of members of this family as vectors for the generation of recombinant vaccines against numerous pathogens. In this review, we cover different aspects of the history and biology of poxviruses with emphasis on their application as vaccines, from first- to fourth-generation, against smallpox, monkeypox, emerging viral diseases highlighted by the World Health Organization (COVID-19, Crimean-Congo haemorrhagic fever, Ebola and Marburg virus diseases, Lassa fever, Middle East respiratory syndrome and severe acute respiratory syndrome, Nipah and other henipaviral diseases, Rift Valley fever and Zika), as well as against one of the most concerning prevalent virus, the Human Immunodeficiency Virus, the causative agent of Acquired Immunodeficiency Syndrome. We discuss the implications in human health of the 2022 monkeypox epidemic affecting many countries, and the rapid prophylactic and therapeutic measures adopted to control virus dissemination within the human population. We also describe the preclinical and clinical evaluation of the Modified Vaccinia virus Ankara and New York vaccinia virus poxviral strains expressing heterologous antigens from the viral diseases listed above. Finally, we report different approaches to improve the immunogenicity and efficacy of poxvirus-based vaccine candidates, such as deletion of immunomodulatory genes, insertion of host-range genes and enhanced transcription of foreign genes through modified viral promoters. Some future prospects are also highlighted.

Progress on Poxvirus E3 Ubiquitin Ligases and Adaptor Proteins.

Poxviruses have evolved a variety of innate immunity evasion mechanisms, some of which involve poxvirus-encoded E3 ubiquitin ligases and adaptor proteins. Based on their functional domains and ubiquitin transfer mechanisms, these poxvirus-encoded E3 ubiquitin ligases and adaptor proteins can be divided into five categories: PRANC, ANK/BC, BBK, P28/RING, and MARCH proteins. Although the substrates of many poxvirus E3 ubiquitin ligases remain to be discovered, most of the identified substrates are components of the innate immune system. In this review, we discuss the current research progress on poxvirus-encoded E3 ubiquitin ligases and adaptor proteins to provide mechanistic insights into the interplay between these viruses and their hosts.

Polymorphisms in STING Affect Human Innate Immune Responses to Poxviruses.

We conducted a large genome-wide association study (GWAS) of the immune responses to primary smallpox vaccination in a combined cohort of 1,653 subjects. We did not observe any polymorphisms associated with standard vaccine response outcomes (e.g., neutralizing antibody, T cell ELISPOT response, or T cell cytokine production); however, we did identify a cluster of SNPs on chromosome 5 (5q31.2) that were significantly associated (p-value: 1.3 x 10-12 - 1.5x10-36) with IFNα response to in vitro poxvirus stimulation. Examination of these SNPs led to the functional testing of rs1131769, a non-synonymous SNP in TMEM173 causing an Arg-to-His change at position 232 in the STING protein-a major regulator of innate immune responses to viral infections. Our findings demonstrate differences in the ability of the two STING variants to phosphorylate the downstream intermediates TBK1 and IRF3 in response to multiple STING ligands. Further downstream in the STING pathway, we observed significantly reduced expression of type I IFNs (including IFNα) and IFN-response genes in cells carrying the H232 variant. Subsequent molecular modeling of both alleles predicted altered ligand binding characteristics between the two variants, providing a potential mechanism underlying differences in inter-individual responses to poxvirus infection. Our data indicate that possession of the H232 variant may impair STING-mediated innate immunity to poxviruses. These results clarify prior studies evaluating functional effects of genetic variants in TMEM173 and provide novel data regarding genetic control of poxvirus immunity.

DNA-Sensing Antiviral Innate Immunity in Poxvirus Infection.

As pattern recognition receptors, cytosolic DNA sensors quickly induce an effective innate immune response. Poxvirus, a large DNA virus, is capable of evading the host antiviral innate immune response. In this review, we summarize the latest studies on how poxvirus is sensed by the host innate immune system and how poxvirus-encoded proteins antagonize DNA sensors. A comprehensive understanding of the interplay between poxvirus and DNA-sensing antiviral immune responses of the host will contribute to the development of new antiviral therapies and vaccines in the future.

TNF deficiency dysregulates inflammatory cytokine production, leading to lung pathology and death during respiratory poxvirus infection.

Excessive tumor necrosis factor (TNF) is known to cause significant pathology. Paradoxically, deficiency in TNF (TNF-/-) also caused substantial pathology during respiratory ectromelia virus (ECTV) infection, a surrogate model for smallpox. TNF-/- mice succumbed to fulminant disease whereas wild-type mice, and those engineered to express only transmembrane TNF (mTNF), fully recovered. TNF deficiency did not affect viral load or leukocyte recruitment but caused severe lung pathology and excessive production of the cytokines interleukin (IL)-6, IL-10, transforming growth factor beta (TGF-ß), and interferon gamma (IFN-γ). Short-term blockade of these cytokines significantly reduced lung pathology in TNF-/- mice concomitant with induction of protein inhibitor of activated STAT3 (PIAS3) and/or suppressor of cytokine signaling 3 (SOCS3), factors that inhibit STAT3 activation. Consequently, inhibition of STAT3 activation with an inhibitor reduced lung pathology. Long-term neutralization of IL-6 or TGF-ß protected TNF-/- mice from an otherwise lethal infection. Thus, mTNF alone is necessary and sufficient to regulate lung inflammation but it has no direct antiviral activity against ECTV. The data indicate that targeting specific cytokines or cytokine-signaling pathways to reduce or ameliorate lung inflammation during respiratory viral infections is possible but that the timing and duration of the interventive measure are critical.

Optimal priming of poxvirus vector (NYVAC)-based HIV vaccine regimens for T cell responses requires three DNA injections. Results of the randomized multicentre EV03/ANRS VAC20 Phase I/II Trial.

DNA vectors have been widely used as a priming of poxvirus vaccine in prime/boost regimens. Whether the number of DNA impacts qualitatively or quantitatively the immune response is not fully explored. With the aim to reinforce T-cell responses by optimizing the prime-boost regimen, the multicentric EV03/ANRS VAC20 phase I/II trial, randomized 147 HIV-negative volunteers to either 3xDNA plus 1xNYVAC (weeks 0, 4, 8 plus 24; n = 74) or to 2xDNA plus 2xNYVAC (weeks 0, 4 plus 20, 24; n = 73) groups. T-cell responses (IFN-γ ELISPOT) to at least one peptide pool were higher in the 3xDNA than the 2xDNA groups (91% and 80% of vaccinees) (P = 0.049). In the 3xDNA arm, 26 (37%) recipients developed a broader T-cell response (Env plus at least to one of the Gag, Pol, Nef pools) than in the 2xDNA (15; 22%) arms (primary endpoint; P = 0.047) with a higher magnitude against Env (at week 26) (P<0.001). In both groups, vaccine regimens induced HIV-specific polyfunctional CD4 and CD8 T cells and the production of Th1, Th2 and Th17/IL-21 cytokines. Antibody responses were also elicited in up to 81% of vaccines. A higher percentage of IgG responders was noted in the 2xDNA arm compared to the 3xDNA arm, while the 3xDNA group tended to elicit a higher magnitude of IgG3 response against specific Env antigens. We show here that the modulation of the prime strategy, without modifying the route or the dose of administration, or the combination of vectors, may influence the quality of the responses.

Lipid Nanoparticle Formulation Increases Efficiency of DNA-Vectored Vaccines/Immunoprophylaxis in Animals Including Transchromosomic Bovines.

The use of nucleic acid as a drug substance for vaccines and other gene-based medicines continues to evolve. Here, we have used a technology originally developed for mRNA in vivo delivery to enhance the immunogenicity of DNA vaccines. We demonstrate that neutralizing antibodies produced in rabbits and nonhuman primates injected with lipid nanoparticle (LNP)-formulated Andes virus or Zika virus DNA vaccines are elevated over unformulated vaccine. Using a plasmid encoding an anti-poxvirus monoclonal antibody (as a reporter of protein expression), we showed that improved immunogenicity is likely due to increased in vivo DNA delivery, resulting in more target protein. Specifically, after four days, up to 30 ng/mL of functional monoclonal antibody were detected in the serum of rabbits injected with the LNP-formulated DNA. We pragmatically applied the technology to the production of human neutralizing antibodies in a transchromosomic (Tc) bovine for use as a passive immunoprophylactic. Production of neutralizing antibody was increased by >10-fold while utilizing 10 times less DNA in the Tc bovine. This work provides a proof-of-concept that LNP formulation of DNA vaccines can be used to produce more potent active vaccines, passive countermeasures (e.g., Tc bovine), and as a means to produce more potent DNA-launched immunotherapies.

Virus-encoded cytokine and chemokine decoy receptors.

Poxviruses and herpesviruses encode secreted versions of cytokine receptors as a unique strategy to evade the host immune response. Recent advances in the field have shown the great impact of some of these proteins in immune modulation and viral pathogenesis, and have uncovered unique properties of these viral proteins not found in the cellular counterparts. These modifications inspired by viruses lead to improved immune modulatory activity of the soluble cytokine receptors, information that has been used to develop more efficient therapeutics to treat inflammatory conditions.

Three different paths to introduce the smallpox vaccine in early 19th century United States.

The ancient technique of variolation (inoculation of the smallpox) which was introduced in the United States in 1721 was replaced by vaccination (inoculation of the cowpox) soon after the procedure was published by Edward Jenner in 1798. Benjamin Waterhouse is recognized as the introducer of smallpox vaccination in the United States having conducted the first vaccination in Boston on 8 July 1800, although other American physicians also played an important role in extending vaccination in the East Coast of the United States. A different route of introduction brought the smallpox vaccine from Mexico to New Mexico (March 1805) and Texas (April 1806) which at that time where part of the Viceroyalty of New Spain. The vaccine was brought to California in 1817 by Russian merchants who obtained it in Peru, where the vaccine had arrived in 1806 with the Spanish Philanthropic Expedition of the Vaccine. It took almost 150 years of vaccination efforts before the last natural outbreak of smallpox occurred in the United States in 1949.

A chimeric poxvirus with J2R (thymidine kinase) deletion shows safety and anti-tumor activity in lung cancer models.

Oncolytic viruses have shown excellent safety profiles in preclinical and clinical studies; however, in most cases therapeutic benefits have been modest. We have previously reported the generation of a chimeric poxvirus (CF33), with significantly improved oncolytic characteristics, through chimerization among different poxviruses. Here we report the sequence analysis of CF33 and oncolytic potential of a GFP-encoding CF33 virus (CF33-GFP) with a J2R deletion in lung cancer models. Replication of CF33-GFP and the resulting cytotoxicity were higher in cancer cell lines compared to a normal cell line, in vitro. After infection with virus, cancer cells expressed markers for immunogenic cell death in vitro. Furthermore, CF33-GFP was safe and exerted potent anti-tumor effects at a dose as low as 1000 plaque forming units in both virus-injected and un-injected distant tumors in A549 tumor xenograft model in mice. Likewise, in a syngeneic model of lung cancer in mice, the virus showed significant anti-tumor effect and was found to increase tumor infiltration by CD8+ T cells. Collectively, these data warrant further investigation of this novel chimeric poxvirus for its potential use as a cancer bio-therapeutic.

Chicken Embryonic-Stem Cells Are Permissive to Poxvirus Recombinant Vaccine Vectors.

The discovery of mammalian pluripotent embryonic stem cells (ESC) has revolutionised cell research and regenerative medicine. More recently discovered chicken ESC (cESC), though less intensively studied, are increasingly popular as vaccine substrates due to a dearth of avian cell lines. Information on the comparative performance of cESC with common vaccine viruses is limited. Using RNA-sequencing, we compared cESC transcriptional programmes elicited by stimulation with chicken type I interferon or infection with vaccine viruses routinely propagated in primary chicken embryo fibroblasts (CEF). We used poxviruses (fowlpox virus (FWPV) FP9, canarypox virus (CNPV), and modified vaccinia virus Ankara (MVA)) and a birnavirus (infectious bursal disease virus (IBDV) PBG98). Interferon-stimulated genes (ISGs) were induced in cESC to levels comparable to those in CEF and immortalised chicken fibroblast DF-1 cells. cESC are permissive (with distinct host transcriptional responses) to MVA, FP9, and CNPV but, surprisingly, not to PBG98. MVA, CNPV, and FP9 suppressed innate immune responses, while PBG98 induced a subset of ISGs. Dysregulation of signalling pathways (i.e., NFκB, TRAF) was observed, which might affect immune responses and viral replication. In conclusion, we show that cESC are an attractive alternative substrate to study and propagate poxvirus recombinant vaccine vectors.

Harnessing poxviral know-how for anti-cytokine therapies.

Poxviruses have evolved efficient proteins that bind mammalian cytokines and chemokines to suppress host immunity. Here Pontejo et al. examine in detail how one such poxviral protein, CrmD, that has activity against both mammalian tumor necrosis factor and chemokines, interacts with its host targets. They apply their findings to refine a human anti-cytokine therapeutic and increase its specificity, providing an elegant example of the benefits of mining viral proteins for therapeutically useful information.

Humoral and cellular immunity against both ZIKV and poxvirus is elicited by a two-dose regimen using DNA and non-replicating vaccinia virus-based vaccine candidates.

The Zika virus (ZIKV) and poxvirus infection are considered as public health emergencies, necessitating the development of effective vaccines. Here, we report novel recombinant DNA-based and non-replicating vaccinia virus (NTV)-based vaccine candidates that express the precursor membrane-envelope (prME) or envelope (E) glycoproteins of ZIKV. After immunization of BABL/c mice with the vaccines using a homologous protocol (DNA/DNA, NTV/NTV) or heterogeneous (DNA/NTV) protocol, a similar level of anti-E IgG and neutralizing antibodies (microneutralization test) were detected in the mice. However, a significantly higher level of E-specific T cell responses was elicited in mice when a heterogeneous prime-boost protocol was used (DNA/NTV) with either the DNA-based or NTV-based vaccines. Furthermore, neutralizing antibodies and a T cell immune response against the vaccinia virus (VV) were detected in mice that were subjected to the prime-boost protocol (DNA/NTV), whereas those subjected to a homologous NTV/NTV protocol had higher levels of anti-VV IgG and neutralizing antibodies. Lastly, a novel H-2d-restricted CD8 T-cell epitope, VRSYCYEASISDMAS, was identified in the ZIKV E protein. These data demonstrate proof of concept of a bivalent vaccine candidate against ZIKV and orthopoxvirus, and support the use of DNA-prME prime and NTV-E boost protocols to protect against ZIKV and orthopoxvirus infections.

Poor Antigen Processing of Poxvirus Particles Limits CD4+ T Cell Recognition and Impacts Immunogenicity of the Inactivated Vaccine.

CD4+ T cells play critical roles in defending against poxviruses, both by potentiating cellular and humoral responses and by directly killing infected cells. Despite this central role, the basis for pox-specific CD4+ T cell activation, specifically the origin of the poxvirus-derived peptides (epitopes) that activate CD4+ T cells, remains poorly understood. In addition, because the current licensed poxvirus vaccines can cause serious adverse events and even death, elucidating the requirements for MHC class II (MHC-II) processing and presentation of poxviral Ags could be of great use. To address these questions, we explored the CD4+ T cell immunogenicity of ectromelia, the causative agent of mousepox. Having identified a large panel of novel epitopes via a screen of algorithm-selected synthetic peptides, we observed that immunization of mice with inactivated poxvirus primes a virtually undetectable CD4+ T cell response, even when adjuvanted, and is unable to provide protection against disease after a secondary challenge. We postulated that an important contributor to this outcome is the poor processability of whole virions for MHC-II-restricted presentation. In line with this hypothesis, we observed that whole poxvirions are very inefficiently converted into MHC-II-binding peptides by the APC as compared with subviral material. Thus, stability of the virion structure is a critical consideration in the rational design of a safe alternative to the existing live smallpox vaccine.

Proteomics Analysis Reveals That Structural Proteins of the Virion Core and Involved in Gene Expression Are the Main Source for HLA Class II Ligands in Vaccinia Virus-Infected Cells.

Protective cellular and humoral immune responses require previous recognition of viral antigenic peptides complexed with human leukocyte antigen (HLA) class II molecules on the surface of the antigen presenting cells. The HLA class II-restricted immune response is important for the control and the clearance of poxvirus infection including vaccinia virus (VACV), the vaccine used in the worldwide eradication of smallpox. In this study, a mass spectrometry analysis was used to identify VACV ligands bound to HLA-DR and -DP class II molecules present on the surface of VACV-infected cells. Twenty-six naturally processed viral ligands among the tens of thousands of cell peptides bound to HLA class II proteins were identified. These viral ligands arose from 19 parental VACV proteins: A4, A5, A18, A35, A38, B5, B13, D1, D5, D7, D12, D13, E3, E8, H5, I2, I3, J2, and K2. The majority of these VACV proteins yielded one HLA ligand and were generated mainly, but not exclusively, by the classical HLA class II antigen processing pathway. Medium-sized and abundant proteins from the virion core and/or involved in the viral gene expression were the major source of VACV ligands bound to HLA-DR and -DP class II molecules. These findings will help to understand the effectiveness of current poxvirus-based vaccines and will be important in the design of new ones.

A virus-encoded type I interferon decoy receptor enables evasion of host immunity through cell-surface binding.

Soluble cytokine decoy receptors are potent immune modulatory reagents with therapeutic applications. Some virus-encoded secreted cytokine receptors interact with glycosaminoglycans expressed at the cell surface, but the biological significance of this activity in vivo is poorly understood. Here, we show the type I interferon binding protein (IFNα/ßBP) encoded by vaccinia and ectromelia viruses requires of this cell binding activity to confer full virulence to these viruses and to retain immunomodulatory activity. Expression of a variant form of the IFNα/ßBP that inhibits IFN activity, but does not interact with cell surface glycosaminoglycans, results in highly attenuated viruses with a virulence similar to that of the IFNα/ßBP deletion mutant viruses. Transcriptomics analysis and infection of IFN receptor-deficient mice confirmed that the control of IFN activity is the main function of the IFNα/ßBP in vivo. We propose that retention of secreted cytokine receptors at the cell surface may largely enhance their immunomodulatory activity.

ZNF598 Plays Distinct Roles in Interferon-Stimulated Gene Expression and Poxvirus Protein Synthesis.

Post-translational modification of ribosomal subunit proteins (RPs) is emerging as an important means of regulating gene expression. Recently, regulatory ubiquitination of small RPs RPS10 and RPS20 by the ubiquitin ligase ZNF598 was found to function in ribosome sensing and stalling on internally polyadenylated mRNAs during ribosome quality control (RQC). Here, we reveal that ZNF598 and RPS10 negatively regulate interferon-stimulated gene (ISG) expression in primary cells, depletion of which induced ISG expression and a broad antiviral state. However, cell lines lacking interferon responses revealed that ZNF598 E3 ligase activity and ubiquitination of RPS20, but not RPS10, were specifically required for poxvirus replication and synthesis of poxvirus proteins whose encoding mRNAs contain unusual 5' poly(A) leaders. Our findings reveal distinct functions for ZNF598 and its downstream RPS targets, one that negatively regulates ISG expression and infection by a range of viruses while the other is positively exploited by poxviruses.