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.

Development of a safe and efficacious vaccine against the HIV/AIDS pandemic remains a major scientific goal. We previously described an HIV/AIDS vaccine based on the modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120 and Gag-Pol-Nef (GPN) of clade B (termed MVA-B), which showed moderate immunogenicity in phase I prophylactic and therapeutic clinical trials. Here, to improve the immunogenicity of MVA-B, we generated a novel recombinant virus, MVA-B ΔA40R, by deleting in the MVA-B genome the vaccinia virus (VACV) A40R gene, which encodes a protein with unknown immune function. The innate immune responses triggered by MVA-B ΔA40R in infected human macrophages, in comparison to parental MVA-B, revealed an increase in the mRNA expression levels of interferon (IFN)-β, IFN-induced genes, and chemokines. Compared to priming with DNA-B (a mixture of DNA-gp120 plus DNA-GPN) and boosting with MVA-B, mice immunized with a DNA-B/MVA-B ΔA40R regimen induced higher magnitude of adaptive and memory HIV-1-specific CD4+ and CD8+ T-cell immune responses that were highly polyfunctional, mainly directed against Env. and of an effector memory phenotype, together with enhanced levels of antibodies against HIV-1 gp120. Reintroduction of the A40R gene into the MVA-B ΔA40R genome (virus termed MVA-B ΔA40R-rev) promoted in infected cells high mRNA and protein A40 levels, with A40 protein localized in the cell membrane. MVA-B ΔA40R-rev significantly reduced mRNA levels of IFN-β and of several other innate immune-related genes in infected human macrophages. In immunized mice, MVA-B ΔA40R-rev reduced the magnitude of the HIV-1-specific CD4+ and CD8+ T cell responses compared to MVA-B ΔA40R. These results revealed an immunosuppressive role of the A40 protein, findings relevant for the optimization of poxvirus vectors as vaccines.

Hepatitis C virus (HCV) represents a major global health problem for which a vaccine is not available. Modified vaccinia virus Ankara (MVA)-HCV is a unique HCV vaccine candidate based in the modified vaccinia virus Ankara (MVA) vector expressing the nearly full-length genome of HCV genotype 1a that elicits CD8⁺ T-cell responses in mice. With the aim to improve the immune response of MVA-HCV and because of the importance of interferon (IFN) in HCV infection, we deleted in MVA-HCV the vaccinia virus (VACV) C6L gene, encoding an inhibitor of IFN-β that prevents activation of the interferon regulatory factors 3 and 7 (IRF3 and IRF7). The resulting vaccine candidate (MVA-HCV ΔC6L) expresses all HCV antigens and deletion of C6L had no effect on viral growth in permissive chicken cells. In human monocyte-derived dendritic cells, infection with MVA-HCV ΔC6L triggered severe down-regulation of IFN-β, IFN-β-induced genes, and cytokines in a manner similar to MVA-HCV, as defined by real-time polymerase chain reaction (PCR) and microarray analysis. In infected mice, both vectors had a similar profile of recruited immune cells and induced comparable levels of adaptive and memory HCV-specific CD8⁺ T-cells, mainly against p7 + NS2 and NS3 HCV proteins, with a T cell effector memory (TEM) phenotype. Furthermore, antibodies against E2 were also induced. Overall, our findings showed that while these vectors had a profound inhibitory effect on gene expression of the host, they strongly elicited CD8⁺ T cell and humoral responses against HCV antigens and to the virus vector. These observations add support to the consideration of these vectors as potential vaccine candidates against HCV.

Despite the decrease in mortality and morbidity due to SARS-CoV-2 infection, the incidence of infections due to Omicron subvariants of SARS-CoV-2 remains high. The mutations acquired by these subvariants, mainly concentrated in the receptor-binding domain (RBD), have caused a shift in infectivity and transmissibility, leading to a loss of effectiveness of the first authorized COVID-19 vaccines, among other reasons, by neutralizing antibody evasion. Hence, the generation of new vaccine candidates adapted to Omicron subvariants is of special interest in an effort to overcome this immune evasion. Here, an optimized COVID-19 vaccine candidate, termed MVA-S(3P_BA.1), was developed using a modified vaccinia virus Ankara (MVA) vector expressing a full-length prefusion-stabilized SARS-CoV-2 spike (S) protein from the Omicron BA.1 variant. The immunogenicity and efficacy induced by MVA-S(3P_BA.1) were evaluated in mice in a head-to-head comparison with the previously generated vaccine candidates MVA-S(3P) and MVA-S(3Pbeta), which express prefusion-stabilized S proteins from Wuhan strain and Beta variant, respectively, and with a bivalent vaccine candidate composed of a combination of MVA-S(3P) and MVA-S(3P_BA.1). The results showed that all four vaccine candidates elicited, after a single intramuscular dose, protection of transgenic K18-hACE2 mice challenged with SARS-CoV-2 Omicron BA.1, reducing viral loads, histopathological lesions, and levels of proinflammatory cytokines in the lungs. They also elicited anti-S IgG and neutralizing antibodies against various Omicron subvariants, with MVA-S(3P_BA.1) and the bivalent vaccine candidate inducing higher titers. Additionally, an intranasal immunization in C57BL/6 mice with all four vaccine candidates induced systemic and mucosal S-specific CD4⁺ and CD8⁺ T-cell and humoral immune responses, and the bivalent vaccine candidate induced broader immune responses, eliciting antibodies against the ancestral Wuhan strain and different Omicron subvariants. These results highlight the use of MVA as a potent and adaptable vaccine vector against new emerging SARS-CoV-2 variants, as well as the promising feature of combining multivalent MVA vaccine candidates.

Hepatitis C virus (HCV) represents a major global health challenge and an efficient vaccine is urgently needed. Many HCV vaccination strategies employ recombinant versions of the viral E2 glycoprotein. However, recombinant E2 readily forms disulfide-bonded aggregates that might not be optimally suited for vaccines. Therefore, we have designed an E2 protein in which we strategically changed eight cysteines to alanines (E2.C8A). E2.C8A formed predominantly monomers and virtually no aggregates. Furthermore, E2.C8A also interacted more efficiently with broadly neutralizing antibodies than conventional E2. We used mice to evaluate different prime/boost immunization strategies involving a modified vaccinia virus Ankara (MVA) expressing the nearly full-length genome of HCV (MVA-HCV) in combination with either the E2 aggregates or the E2.C8A monomers. The combined MVA-HCV/E2 aggregates prime/boost strategy markedly enhanced HCV-specific effector memory CD4⁺ T cell responses and antibody levels compared to MVA-HCV/MVA-HCV. Moreover, the aggregated form of E2 induced higher levels of anti-E2 antibodies in vaccinated mice than E2.C8A monomers. These antibodies were cross-reactive and mainly of the IgG1 isotype. Our findings revealed how two E2 viral proteins that differ in their capacity to form aggregates are able to enhance to different extent the HCV-specific cellular and humoral immune responses, either alone or in combination with MVA-HCV. These combined protocols of MVA-HCV/E2 could serve as a basis for the development of a more effective HCV vaccine.

The constant appearance of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoCs) has jeopardized the protective capacity of approved vaccines against coronavirus disease-19 (COVID-19). For this reason, the generation of new vaccine candidates adapted to the emerging VoCs is of special importance. Here, we developed an optimized COVID-19 vaccine candidate using the modified vaccinia virus Ankara (MVA) vector to express a full-length prefusion-stabilized SARS-CoV-2 spike (S) protein, containing 3 proline (3P) substitutions in the S protein derived from the beta (B.1.351) variant, termed MVA-S(3Pbeta). Preclinical evaluation of MVA-S(3Pbeta) in head-to-head comparison to the previously generated MVA-S(3P) vaccine candidate, expressing a full-length prefusion-stabilized Wuhan S protein (with also 3P substitutions), demonstrated that two intramuscular doses of both vaccine candidates fully protected transgenic K18-hACE2 mice from a lethal challenge with SARS-CoV-2 beta variant, reducing mRNA and infectious viral loads in the lungs and in bronchoalveolar lavages, decreasing lung histopathological lesions and levels of proinflammatory cytokines in the lungs. Vaccination also elicited high titers of anti-S Th1-biased IgGs and neutralizing antibodies against ancestral SARS-CoV-2 Wuhan strain and VoCs alpha, beta, gamma, delta, and omicron. In addition, similar systemic and local SARS-CoV-2 S-specific CD4⁺ and CD8⁺ T-cell immune responses were elicited by both vaccine candidates after a single intranasal immunization in C57BL/6 mice. These preclinical data support clinical evaluation of MVA-S(3Pbeta) and MVA-S(3P), to explore whether they can diversify and potentially increase recognition and protection of SARS-CoV-2 VoCs.