Heterologous mRNA/MVA delivering trimeric-RBD as effective vaccination regimen against SARS-CoV-2: COVARNA Consortium.

Despite the high efficiency of current SARS-CoV-2 mRNA vaccines in reducing COVID-19 morbidity and mortality, waning immunity and the emergence of resistant variants underscore the need for novel vaccination strategies. This study explores a heterologous mRNA/Modified Vaccinia virus Ankara (MVA) prime/boost regimen employing a trimeric form of the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein compared to a homologous MVA/MVA regimen. In C57BL/6 mice, the RBD was delivered during priming via an mRNA vector encapsulated in nanoemulsions (NE) or lipid nanoparticles (LNP), followed by a booster with a replication-deficient MVA-based recombinant virus (MVA-RBD). This heterologous mRNA/MVA regimen elicited strong anti-RBD binding and neutralizing antibodies (BAbs and NAbs) against both the ancestral SARS-CoV-2 strain and different variants of concern (VoCs). Additionally, this protocol induced robust and polyfunctional RBD-specific CD4 and CD8 T cell responses, particularly in animals primed with mLNP-RBD. In K18-hACE2 transgenic mice, the LNP-RBD/MVA combination provided complete protection from morbidity and mortality following a live SARS-CoV-2 challenge compared with the partial protection observed with mNE-RBD/MVA or MVA/MVA regimens. Although the mNE-RBD/MVA regimen only protects half of the animals, it was able to induce antibodies with Fc-mediated effector functions besides NAbs. Moreover, viral replication and viral load in the respiratory tract were markedly reduced and decreased pro-inflammatory cytokine levels were observed. These results support the efficacy of heterologous mRNA/MVA vaccine combinations over homologous MVA/MVA regimen, using alternative nanocarriers that circumvent intellectual property restrictions of current mRNA vaccine formulations.

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.

Optimized vaccine candidate MVA-S(3P) fully protects against SARS-CoV-2 infection in hamsters.

The development of novel optimized vaccines against coronavirus disease 2019 (COVID-19) that are capable of controlling the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and the appearance of different variants of concern (VoC) is needed to fully prevent the transmission of the virus. In the present study, we describe the enhanced immunogenicity and efficacy elicited in hamsters by a modified vaccinia virus Ankara (MVA) vector expressing a full-length prefusion-stabilized SARS-CoV-2 spike (S) protein [termed MVA-S(3P)]. Hamsters vaccinated with one or two doses of MVA-S(3P) developed high titers of S-binding IgG antibodies and neutralizing antibodies against the ancestral Wuhan SARS-CoV-2 virus and VoC beta, gamma, and delta, as well as against omicron, although with a somewhat lower neutralization activity. After SARS-CoV-2 challenge, vaccinated hamsters did not lose body weight as compared to matched placebo (MVA-WT) controls. Consistently, vaccinated hamsters exhibited significantly reduced viral RNA in the lungs and nasal washes, and no infectious virus was detected in the lungs in comparison to controls. Furthermore, almost no lung histopathology was detected in MVA-S(3P)-vaccinated hamsters, which also showed significantly reduced levels of proinflammatory cytokines in the lungs compared to unvaccinated hamsters. These results reinforce the use of MVA-S(3P) as a vaccine candidate against COVID-19 in clinical trials.

Lung transcriptomics of K18-hACE2 mice highlights mechanisms and genes involved in the MVA-S vaccine-mediated immune response and protection against SARS-CoV-2 infection.

Unravelling the molecular mechanism of COVID-19 vaccines through transcriptomic pathways involved in the host response to SARS-CoV-2 infection is key to understand how vaccines work, and for the development of optimized COVID-19 vaccines that can prevent the emergence of SARS-CoV-2 variants of concern (VoCs) and future outbreaks. In this study, we investigated the effects of vaccination with a modified vaccinia virus Ankara (MVA)-based vector expressing the full-length SARS-CoV-2 spike protein (MVA-S) on the lung transcriptome from susceptible K18-hACE2 mice after SARS-CoV-2 infection. One dose of MVA-S regulated genes related to viral infection control, inflammation processes, T-cell response, cytokine production and IFN-γ signalling. Down-regulation of Rhcg and Tnfsf18 genes post-vaccination with one and two doses of MVA-S may represent a mechanism for controlling infection immunity and vaccine-induced protection. One dose of MVA-S provided partial protection with a distinct lung transcriptomic profile to healthy animals, while two doses of MVA-S fully protected against infection with a transcriptomic profile comparable to that of non-vaccinated healthy animals. This suggests that the MVA-S booster generates a robust and rapid antigen-specific immune response preventing virus infection. Notably, down-regulation of Atf3 and Zbtb16 genes in mice vaccinated with two doses of MVA-S may contribute to vaccine control of innate immune system and inflammation processes in the lungs during SARS-CoV-2 infection. This study shows host transcriptomic mechanisms likely involved in the MVA-S vaccine-mediated immune response against SARS-CoV-2 infection, which could help in improving vaccine dose assessment and developing novel, well-optimized SARS-CoV-2 vaccine candidates against prevalent or emerging VoCs.

Preclinical immune efficacy against SARS-CoV-2 beta B.1.351 variant by MVA-based vaccine candidates.

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.

Full protection from SARS-CoV-2 brain infection and damage in susceptible transgenic mice conferred by MVA-CoV2-S vaccine candidate.

Vaccines against SARS-CoV-2 have been shown to be safe and effective but their protective efficacy against infection in the brain is yet unclear. Here, in the susceptible transgenic K18-hACE2 mouse model of severe coronavirus disease 2019 (COVID-19), we report a spatiotemporal description of SARS-CoV-2 infection and replication through the brain. SARS-CoV-2 brain replication occurs primarily in neurons, leading to neuronal loss, signs of glial activation and vascular damage in mice infected with SARS-CoV-2. One or two doses of a modified vaccinia virus Ankara (MVA) vector expressing the SARS-CoV-2 spike (S) protein (MVA-CoV2-S) conferred full protection against SARS-CoV-2 cerebral infection, preventing virus replication in all areas of the brain and its associated damage. This protection was maintained even after SARS-CoV-2 reinfection. These findings further support the use of MVA-CoV2-S as a promising vaccine candidate against SARS-CoV-2/COVID-19.

Dendritic Cell-Mediated Cross-Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS-CoV-2.

Administration of neutralizing antibodies (nAbs) has proved to be effective by providing immediate protection against SARS-CoV-2. However, dual strategies combining virus neutralization and immune response stimulation to enhance specific cytotoxic T cell responses, such as dendritic cell (DC) cross-priming, represent a promising field but have not yet been explored. Here, a broadly nAb, TNT , are first generated by grafting an anti-RBD biparatopic tandem nanobody onto a trimerbody scaffold. Cryo-EM data show that the TNT structure allows simultaneous binding to all six RBD epitopes, demonstrating a high-avidity neutralizing interaction. Then, by C-terminal fusion of an anti-DNGR-1 scFv to TNT , the bispecific trimerbody TNT DNGR-1 is generated to target neutralized virions to type 1 conventional DCs (cDC1s) and promote T cell cross-priming. Therapeutic administration of TNT DNGR-1, but not TNT , protects K18-hACE2 mice from a lethal SARS-CoV-2 infection, boosting virus-specific humoral responses and CD8+ T cell responses. These results further strengthen the central role of interactions with immune cells in the virus-neutralizing antibody activity and demonstrate the therapeutic potential of the Fc-free strategy that can be used advantageously to provide both immediate and long-term protection against SARS-CoV-2 and other viral infections.

Intranasal administration of a single dose of MVA-based vaccine candidates against COVID-19 induced local and systemic immune responses and protects mice from a lethal SARS-CoV-2 infection.

Current coronavirus disease-19 (COVID-19) vaccines are administered by the intramuscular route, but this vaccine administration failed to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infection in the upper respiratory tract, mainly due to the absence of virus-specific mucosal immune responses. It is hypothesized that intranasal (IN) vaccination could induce both mucosal and systemic immune responses that blocked SARS-CoV-2 transmission and COVID-19 progression. Here, we evaluated in mice IN administration of three modified vaccinia virus Ankara (MVA)-based vaccine candidates expressing the SARS-CoV-2 spike (S) protein, either the full-length native S or a prefusion-stabilized [S(3P)] protein; SARS-CoV-2-specific immune responses and efficacy were determined after a single IN vaccine application. Results showed that in C57BL/6 mice, MVA-based vaccine candidates elicited S-specific IgG and IgA antibodies in serum and bronchoalveolar lavages, respectively, and neutralizing antibodies against parental and SARS-CoV-2 variants of concern (VoC), with MVA-S(3P) being the most immunogenic vaccine candidate. IN vaccine administration also induced polyfunctional S-specific Th1-skewed CD4+ and cytotoxic CD8+ T-cell immune responses locally (in lungs and bronchoalveolar lymph nodes) or systemically (in spleen). Remarkably, a single IN vaccine dose protected susceptible K18-hACE2 transgenic mice from morbidity and mortality caused by SARS-CoV-2 infection, with MVA-S(3P) being the most effective candidate. Infectious SARS-CoV-2 viruses were undetectable in lungs and nasal washes, correlating with high titers of S-specific IgGs and neutralizing antibodies against parental SARS-CoV-2 and several VoC. Moreover, low histopathological lung lesions and low levels of pro-inflammatory cytokines in lungs and nasal washes were detected in vaccinated animals. These results demonstrated that a single IN inoculation of our MVA-based vaccine candidates induced potent immune responses, either locally or systemically, and protected animal models from COVID-19. These results also identified an effective vaccine administration route to induce mucosal immunity that should prevent SARS-CoV-2 host-to-host transmission.

MVA-CoV2-S Vaccine Candidate Neutralizes Distinct Variants of Concern and Protects Against SARS-CoV-2 Infection in Hamsters.

To control the coronavirus disease 2019 (COVID-19) pandemic and the emergence of different variants of concern (VoCs), novel vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed. In this study, we report the potent immunogenicity and efficacy induced in hamsters by a vaccine candidate based on a modified vaccinia virus Ankara (MVA) vector expressing a human codon optimized full-length SARS-CoV-2 spike (S) protein (MVA-S). Immunization with one or two doses of MVA-S elicited high titers of S- and receptor-binding domain (RBD)-binding IgG antibodies and neutralizing antibodies against parental SARS-CoV-2 and VoC alpha, beta, gamma, delta, and omicron. After SARS-CoV-2 challenge, MVA-S-vaccinated hamsters showed a significantly strong reduction of viral RNA and infectious virus in the lungs compared to the MVA-WT control group. Moreover, a marked reduction in lung histopathology was also observed in MVA-S-vaccinated hamsters. These results favor the use of MVA-S as a potential vaccine candidate for SARS-CoV-2 in clinical trials.

A Single Dose of an MVA Vaccine Expressing a Prefusion-Stabilized SARS-CoV-2 Spike Protein Neutralizes Variants of Concern and Protects Mice From a Lethal SARS-CoV-2 Infection.

We generated an optimized COVID-19 vaccine candidate based on the modified vaccinia virus Ankara (MVA) vector expressing a full-length prefusion-stabilized SARS-CoV-2 spike (S) protein, termed MVA-CoV2-S(3P). The S(3P) protein was expressed at higher levels (2-fold) than the non-stabilized S in cells infected with the corresponding recombinant MVA viruses. One single dose of MVA-CoV2-S(3P) induced higher IgG and neutralizing antibody titers against parental SARS-CoV-2 and variants of concern than MVA-CoV2-S in wild-type C57BL/6 and in transgenic K18-hACE2 mice. In immunized C57BL/6 mice, two doses of MVA-CoV2-S or MVA-CoV2-S(3P) induced similar levels of SARS-CoV-2-specific B- and T-cell immune responses. Remarkably, a single administration of MVA-CoV2-S(3P) protected all K18-hACE2 mice from morbidity and mortality caused by SARS-CoV-2 infection, reducing SARS-CoV-2 viral loads, histopathological lesions, and levels of pro-inflammatory cytokines in the lungs. These results demonstrated that expression of a novel full-length prefusion-stabilized SARS-CoV-2 S protein by the MVA poxvirus vector enhanced immunogenicity and efficacy against SARS-CoV-2 in animal models, further supporting MVA-CoV2-S(3P) as an optimized vaccine candidate for clinical trials.