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Vaccination is an effective and economically viable means of protection against the influenza virus, but due to rapid viral evolution, modern seasonal vaccines are not effective enough. Next-generation vaccines are designed to provide protection against a wide range of influenza virus strains, including pandemic variants. In our work, we made an epitope-based universal vaccine, rMVA-k1-k2, against the influenza virus based on the modified vaccinia Ankara (MVA) vector and using our own algorithms to select epitopes from conserved fragments of the NP, M1 and HA proteins of influenza A and B. We show that double immunization protects mice with a 67% or greater efficiency against viral influenza pneumonia when infected with various strains of the H1N1, H2N2, H3N2 and H5N1 subtypes of influenza A. In animals, the level of protection provided by the rMVA-k1-k2 vaccine was comparable to that provided by the universal M001 and MVA-NP+M1 (Invictus) vaccines, which have shown success in clinical trials, against strains of the H1N1 and H3N2 subtypes.

26 páginas, 2 tablas, 10 figuras., Bluetongue, caused by bluetongue virus (BTV), is a widespread arthropod-borne disease of ruminants that entails a recurrent threat to the primary sector of developed and developing countries. In this work, we report modified vaccinia virus Ankara (MVA) and ChAdOx1-vectored vaccines designed to simultaneously express the immunogenic NS1 protein and/or NS2-Nt, the N-terminal half of protein NS2 (NS21-180). A single dose of MVA or ChAdOx1 expressing NS1-NS2-Nt improved the protection conferred by NS1 alone in IFNAR(-/-) mice. Moreover, mice immunized with ChAdOx1/MVA-NS1, ChAdOx1/MVA-NS2-Nt, or ChAdOx1/MVA-NS1-NS2-Nt developed strong cytotoxic CD81 T-cell responses against NS1, NS2-Nt, or both proteins and were fully protected against a lethal infection with BTV serotypes 1, 4, and 8. Furthermore, although a single immunization with ChAdOx1-NS1-NS2-Nt partially protected sheep against BTV-4, the administration of a booster dose of MVA-NS1- NS2-Nt promoted a faster viral clearance, reduction of the period and level of viremia and also protected from the pathology produced by BTV infection. IMPORTANCE Current BTV vaccines are effective but they do not allow to distinguish between vaccinated and infected animals (DIVA strategy) and are serotype specific. In this work we have develop a DIVA multiserotype vaccination strategy based on adenoviral (ChAdOx1) and MVA vaccine vectors, the most widely used in current phase I and II clinical trials, and the conserved nonstructural BTV proteins NS1 and NS2. This immunization strategy solves the major drawbacks of the current marketed vaccines., This work was supported by grants AGL2017-82570-R and PID2020-112992RR-I00 from the Spanish Ministry of Science and by the EU Horizon 2020 Program (European Commission grant agreement no. 727393-PALE-Blu). S.U.T. was a recipient of a predoctoral fellowship from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Centro de Investigación en Sanidad Animal (program FPI-SGIT2018). We declare no competing interests. J.O. conceived the study. S.U.T. and L.J.C. wrote the manuscript with contributions from J.O. and inputs from all other authors. S.U.T., L.J.C., S.G., and A.M.L. designed and developed all the vaccines used in this work. S.U.T., L.J.C., E.C.P., G.L., and S.M. conducted mice experiments and cytometric analysis. J.B. and P.S.C. conducted sheep immunizations, postmortem studies, and histopathological analysis. S.U.T. and L.J.C. analyzed antibodies, viremias, and hematological parameters. A.N. helped revising the article

Modified vaccinia Ankara (MVA) is a promising vaccine vector due to its highly attenuated phenotype and good immunogenicity. However, obtaining a new recombinant MVA remains a tedious and laborious procedure involving many rounds of plaque purification. Recombinant MVA generation can be greatly improved and facilitated by different selection techniques. Here, we describe a comparison between techniques based on K1L, F13L and D4R genes.

8 Pag.., Zika virus (ZIKV) is a mosquito-borne flavivirus whose infection in pregnant women is associated with a spectrum of birth defects, which are together referred as Congenital Zika Syndrome. In addition, ZIKV can also induce Guillain-Barré syndrome, which is an autoimmune disease with neurological symptoms. The recent description of the first local infections of ZIKV in the European continent together with the expansion of one of its potential vectors, the Asian tiger mosquito (Aedes albopictus), invite us to be prepared for future outbreaks of ZIKV in this geographical region. However, the antigenic similarities of ZIKV with other flaviviruses can lead to an immune cross-reactivity with other circulating flaviviruses inducing, in some cases, flavivirus-disease exacerbation by antibody-dependent enhancement (ADE) of infection, which is a major concern for ZIKV vaccine development. Until now, West Nile virus (WNV) is the main medically relevant flavivirus circulating in the Mediterranean Basin. Therefore, anticipating the potential scenario of emergency vaccination against ZIKV in areas of Europe where WNV is endemic, in this investigation, we have evaluated the cross-reactivity between WNV and our previously developed ZIKV vaccine candidate based on modified vaccinia virus Ankara (MVA) vector expressing ZIKV structural proteins (MVA-ZIKV). To this end, mice were first immunized with MVA-ZIKV, subsequently challenged with WNV, and then, the ZIKV- and WNV-specific immune responses and protection against WNV were evaluated. Our results indicate low cross-reactivity between the MVA-ZIKV vaccine candidate and WNV and absence of ADE, supporting the safety of this ZIKV vaccine candidate in areas where the circulation of WNV is endemic., This research was funded by the Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación,MCIN/AEI/10.13039/501100011033 grant numbers PID2020-11919JRJ-100 (to N.J.d.O.), PID2019-105117RR-C21 (to M.A.-M.-A.), and E-RTA-2017-00003-C03 (to J.-C.S.); by Comunidad de Madrid, grant number P2018/BAA-4370 PLATESA2-CM (to J.-C.S.); and by Ministerio de Economía y Competitividad grants SAF2013-45232-R and SAF2017-88089-R (AEI/MINECO/FEDER-EU) (to M.E.). P.P. was supported by a Formación de Personal Investigador PhD fellowship from the Spanish Ministry of Health and Education of Spain.

Background Recurrent respiratory papillomatosis (RRP) or laryngeal papillomatosis is a disease caused by papillomavirus infection. Methods In this phase I/II clinical trial, we evaluated the efficacy of the modified vaccinia Ankara (MVA) E2 virus in the treatment of RRP. Twenty‐nine patients (18 female and 11 male) underwent injection of MVA E2 directly into the borders of the vocal cords where lesions were seen and were monitored by direct laryngoscopy. The immune response was assessed by the determination of CD3+, CD4+, and CD8+ lymphocytes counts. The presence of papillomavirus was determined by polymerase chain reaction analysis. Results Lesions were completely eliminated in 13 patients (44.8%). In 16 patients (55.2%), lesions recurred between 6 and 18 months after treatment; these patients received a second round of treatment with MVA E2, and they are not seen with new recurrences. Conclusion The MVA E2 vaccine has excellent potential for generating complete regression of RRP lesions.

This article is made available via the PMC Open Access Subset for unrestricted noncommercial re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic., Vaccines against SARS-CoV-2, the causative agent of the COVID-19 pandemic, are urgently needed. We developed two COVID-19 vaccines based on modified vaccinia virus Ankara (MVA) vectors expressing the entire SARS-CoV-2 spike (S) protein (MVA-CoV2-S); their immunogenicity was evaluated in mice using DNA/MVA or MVA/MVA prime/boost immunizations. Both vaccines induced robust, broad and polyfunctional S-specific CD4+ (mainly Th1) and CD8+ T-cell responses, with a T effector memory phenotype. DNA/MVA immunizations elicited higher T-cell responses. All vaccine regimens triggered high titers of IgG antibodies specific for the S, as well as for the receptor-binding domain; the predominance of the IgG2c isotype was indicative of Th1 immunity. Notably, serum samples from vaccinated mice neutralized SARS-CoV-2 in cell cultures, and those from MVA/MVA immunizations showed a higher neutralizing capacity. Remarkably, one or two doses of MVA-CoV2-S protect humanized K18-hACE2 mice from a lethal dose of SARS-CoV-2. In addition, two doses of MVA-CoV2-S confer full inhibition of virus replication in the lungs. These results demonstrate the robust immunogenicity and full efficacy of MVA-based COVID-19 vaccines in animal models and support its translation to the clinic., This research was supported by Spanish Health Ministry, Instituto de Salud Carlos III (ISCIII), Fondo COVID-19 grant COV20/00151 and Fondo Supera COVID-19 (Crue Universidades-Banco Santander) (to J.G.-A.), CSIC grant 2020E84, Ferrovial and Mapfre donations (to M.E.) and Fondo Solidario Juntos, Banco de Santander (to D.S.). This publication was supported by the European Virus Archive GLOBAL (EVA-GLOBAL) project that has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 871029.

The continuous dissemination of the novel emerging SARS-CoV-2 virus, with more than 78 million infected cases worldwide and higher than 1,700,000 deaths as of 23 December 2020, highlights the urgent need for the development of novel vaccines against COVID-19. With this aim, we have developed novel vaccine candidates based on the poxvirus modified vaccinia virus Ankara (MVA) strain expressing the full-length SARS-CoV-2 spike (S) protein, and we have evaluated their immunogenicity in mice using DNA/MVA or MVA/MVA prime/boost immunization protocols., Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, are urgently needed. We developed two COVID-19 vaccines based on modified vaccinia virus Ankara (MVA) vectors expressing the entire SARS-CoV-2 spike (S) protein (MVA-CoV2-S); their immunogenicity was evaluated in mice using DNA/MVA or MVA/MVA prime/boost immunizations. Both vaccines induced robust, broad, and polyfunctional S-specific CD4+ (mainly Th1) and CD8+ T-cell responses, with a T effector memory phenotype. DNA/MVA immunizations elicited higher T-cell responses. All vaccine regimens triggered high titers of IgG antibodies specific for the S, as well as for the receptor-binding domain; the predominance of the IgG2c isotype was indicative of Th1 immunity. Notably, serum samples from vaccinated mice neutralized SARS-CoV-2 in cell cultures, and those from MVA/MVA immunizations showed a higher neutralizing capacity. Remarkably, one or two doses of MVA-CoV2-S protect humanized K18-hACE2 mice from a lethal dose of SARS-CoV-2. In addition, two doses of MVA-CoV2-S confer full inhibition of virus replication in the lungs. These results demonstrate the robust immunogenicity and full efficacy of MVA-based COVID-19 vaccines in animal models and support its translation to the clinic. IMPORTANCE The continuous dissemination of the novel emerging SARS-CoV-2 virus, with more than 78 million infected cases worldwide and higher than 1,700,000 deaths as of 23 December 2020, highlights the urgent need for the development of novel vaccines against COVID-19. With this aim, we have developed novel vaccine candidates based on the poxvirus modified vaccinia virus Ankara (MVA) strain expressing the full-length SARS-CoV-2 spike (S) protein, and we have evaluated their immunogenicity in mice using DNA/MVA or MVA/MVA prime/boost immunization protocols. The results showed the induction of a potent S-specific T-cell response and high titers of neutralizing antibodies. Remarkably, humanized K18-hACE2 mice immunized with one or two doses of the MVA-based vaccine were 100% protected from SARS-CoV-2 lethality. Moreover, two doses of the vaccine prevented virus replication in lungs. Our findings prove the robust immunogenicity and efficacy of MVA-based COVID-19 vaccines in animal models and support its translation to the clinic.

There is a need to develop a highly effective vaccine against the emerging chikungunya virus (CHIKV), a mosquito-borne Alphavirus that causes severe disease in humans consisting of acute febrile illness, followed by chronic debilitating polyarthralgia and polyarthritis. In this review, we provide a brief history of the development of the first poxvirus vaccines that led to smallpox eradication and its implications for further vaccine development. As an example, we summarize the development of vaccine candidates based on the modified vaccinia virus Ankara (MVA) vector expressing different CHIKV structural proteins, paying special attention to MVA-CHIKV expressing all of the CHIKV structural proteins: C, E3, E2, 6K and E1. We review the characterization of innate and adaptive immune responses induced in mice and nonhuman primates by the MVA-CHIKV vaccine candidate and examine its efficacy in animal models, with promising preclinical findings needed prior to the approval of human clinical trials. This research was supported by the ICRES (Integrated Chikungunya Research), a collaborative project supported by the European Union under the Health Cooperation Work Program of the 7th Framework Program (grant agreement 261202), a grant from the Spanish Ministerio de Ciencia e Innovación (SAF2008-02036), and a grant from Acción Estratégica en Salud from the ISCIII, grant MPY 388/18. Sí

Centro de Biotecnología y Genómica de Plantas (CBGP), Zika virus (ZIKV) is a mosquito-borne pathogen with public health importance due to the high risk of its mosquito vector dissemination and the severe neurological and teratogenic sequelae associated with infection. Vaccines with broad immune specificity and control against this re-emerging virus are needed. Here, we described that mice immunized with a priming dose of a DNA plasmid mammalian expression vector encoding ZIKV prM-E antigens (DNA-ZIKV) followed by a booster dose of a modified vaccinia virus Ankara (MVA) vector expressing the same prM-E ZIKV antigens (MVA-ZIKV) induced broad, polyfunctional and long-lasting ZIKV-specific CD4+ and CD8+ T-cell immune responses, with high levels of CD4+ T follicular helper cells, together with the induction of neutralizing antibodies. All those immune parameters were significantly stronger in the heterologous DNA-ZIKV/MVA-ZIKV immunization group compared to the homologous prime/boost immunizations regimens. Collectively, these results provided an optimized immunization protocol able to induce high levels of ZIKV-specific T-cell responses, as well as neutralizing antibodies and reinforce the combined use of DNA-based vectors and MVA-ZIKV as promising prophylactic vaccination schedule against ZIKV., This research was supported by MINECO Spanish grants SAF-2013-45232-R and SAF-2017-88089-R (to M.E.) and Grant PID2019-105117RR-C21/AEI/10.13039/501100011033 from Agencia Estatal de Investigación to M.A.M.-A. P.P was supported by a fellowship from the Ministry of Science and Innovation of Spain and this work is part of her Thesis defence., 16 Pág.

Mayaro virus (MAYV) is an emerging alphavirus causing acute febrile illness associated with chronic polyarthralgia. Although MAYV is currently restricted to tropical regions in South America around the Amazon basin, it has the potential to spread globally by Aedes species mosquitoes. In addition, there are currently no specific therapeutics or licenced vaccines against MAYV infection. We have previously shown that an adenovirus based Mayaro vaccine (ChAdOx1 May) was able to provide full protection against MAYV challenge in vaccinated A129 mice and induced high neutralising antibody titres. In this study, we have constructed a replication deficient simian adenovirus (ChAdOx2) and a Modified Ankara Virus (MVA) based vaccine expressing the MAYV structural cassette (sMAYV) similar to ChAdOx1 May, and characterised recombinant MAYV E2 glycoprotein expressed in a mammalian system for immune monitoring. We demonstrate that ChAdOx2 May was able to induce high antibody titres similar to ChAdOx1 May, and MVA May was shown to be an effective boosting strategy following prime vaccination with ChAdOx1 or ChAdOx2 May. In order to measure MAYV neutralising ability, we have developed a virus replicon particle-based neutralisation assay which effectively detected neutralising antibodies against MAYV. In summary, our study indicates the potential for further clinical development of the viral vectored MAYV vaccines against MAYV infections.

We evaluated antibody responses to the human immunodeficiency virus (HIV) envelope variable regions 1 and 2 (V1V2) in 29 vaccinees who had received three HIV-1 DNA immunizations and two HIV-modified vaccinia virus Ankara (MVA) boosts in the phase I/II HIVIS03 vaccine trial. Twenty vaccinees received a third HIV-MVA boost after three years in the HIVIS06 trial. IgG and IgG antibody subclasses to gp70V1V2 proteins of HIV-1 A244, CN54, Consensus C, and Case A2 were analysed using an enzyme-linked immunosorbent assay (ELISA). Cyclic V2 peptides of A244, Consensus C, and MN were used in a surface plasmon resonance (SPR) assay. Four weeks after the second HIV-MVA, anti-V1V2 IgG antibodies to A244 were detected in 97% of HIVIS03 vaccinees, in 75% three years later, and in 95% after the third HIV-MVA. Anti-CN54 V1V2 IgG was detectable in 48% four weeks after the second HIV-MVA. The SPR data supported the findings. The IgG response was predominantly IgG1. Four weeks after the second HIV-MVA, 85% of vaccinees had IgG1 antibodies to V1V2 A244, which persisted in 25% for three-years. IgG3 and IgG4 antibodies to V1V2 A244 were rare. In conclusion, the HIV-DNA/MVA vaccine regimen induced durable V1V2 IgG antibody responses in a high proportion of vaccinees.

The lung is the vital target organ of coronavirus disease 2019 (COVID-19) caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the majority of patients the most active virus replication seems to be found in the upper respiratory tract, severe cases however suffer from SARS-like disease associated with virus replication in lung tissues. Due to the current lack of suitable anti-viral drugs the induction of protective immunity such as neutralizing antibodies in the lung is the key aim of the only alternative approach-the development and application of SARS-CoV-2 vaccines. However, past experience from experimental animals, livestock, and humans showed that induction of immunity in the lung is limited following application of vaccines at peripheral sides such as skin or muscles. Based on several considerations we therefore propose here to consider the application of a Modified Vaccinia virus Ankara (MVA)-based vaccine to mucosal surfaces of the respiratory tract as a favorable approach to combat COVID-19.

Chronic hepatitis B virus (HBV) infection affects 257 million people globally. Current therapies suppress HBV but viral rebound occurs on cessation of therapy, novel therapeutic strategies are urgently required. To develop a therapeutic HBV vaccine that can induce high magnitude T cells to all major HBV antigens, we have developed a novel HBV vaccine using chimpanzee adenovirus (ChAd) and modified vaccinia Ankara (MVA) viral vectors encoding multiple HBV antigens. ChAd vaccine alone generated very high magnitude HBV specific T cell responses to all HBV major antigens. The inclusion of a shark Invariant (SIi) chain genetic adjuvant significantly enhanced the magnitude of T-cells against HBV antigens. Compared to ChAd alone vaccination, ChAd-prime followed by MVA-boost vaccination further enhanced the magnitude and breadth of the vaccine induced T cell response. Intra-cellular cytokine staining study showed that HBV specific CD8+ and CD4+ T cells were polyfunctional, producing combinations of IFN&gamma, TNF-&alpha, and IL-2. In summary, we have generated genetically adjuvanted ChAd and MVA vectored HBV vaccines with the potential to induce high-magnitude T cell responses through a prime-boost therapeutic vaccination approach. These pre-clinical studies pave the way for new studies of HBV therapeutic vaccination in humans with chronic hepatitis B infection.

In Europe, cowpox virus (CPXV) infection in South American camelids occurs as a so-called spill-over infection. Although infected animals generally have a mild form of the disease and survive, cases of fatal generalised CPXV infection have also been described. Prevention by prophylactic vaccination is the only way to protect animals from disease. In the present study, modified vaccinia virus Ankara (MVA) vaccine, which has been successfully used in many animal species, was used in a prime-boost vaccination regimen in two alpaca herds with a history of CPXV infection. The focus of the study was the prevention of further clinical cases, and to determine the safety and immunogenicity of the MVA vaccine in alpacas. The MVA vaccine was well tolerated and safe in the 94 animals vaccinated. An indirect immunofluorescence assay (IFA) using MVA as an antigen showed that the seroprevalence of antibody after booster vaccination was 81.3% in herd I and 91.7% in herd II. Detectable antibody titres declined to 15.6% in herd I and 45.8% in herd II over a 12-month period after booster vaccination. Animals could be divided into four groups based on individual antibody titres determined over one year: Group 1 consisted of 19.3% of animals that were seropositive until the end of the trial period, Group 2 consisted of 58.0% of animals that were seropositive after booster vaccination, but seronegative one year later, Group 3 consisted of 14.7% of animals that were not seropositive at any time point, and Group 4 consisted of 7.9% of animals that were seropositive after initial immunisation, seronegative six months later, but seropositive or intermediate in IFA one year after immunisation, likely because of natural exposure. In new-born crias born to MVA-vaccinated mares, specific maternal antibodies were detected in 50.0% of animals up to 14 weeks of age. Our results confirm that MVA vaccination is a feasible tool for the prevention of CPXV disease in alpacas. Long-term studies are needed to verify future vaccination regimen in CPXV affected herds.

Background: Vaccinia is known to induce antibody and cellular responses. Plasmablast, circulating follicular helper T (cTFH) cells, cytokine-expressing CD4 T cells, and memory B cells were compared between subcutaneous (SC) and needle-free jet injection (JI) recipients of non-replicating modified vaccinia Ankara (MVA) vaccine. Methods: Vaccinia-na&iuml, ve adults received MVA SC or by JI on Days 1 and 29. Vaccinia-specific antibodies were quantified by plaque reduction neutralization test (PRNT) and enzyme-linked immunosorbent assay. Plasmablast, cTFH, and cytokine-expressing CD4 T cells were assessed on Days 1, 8, 15, 29, 36, 43 (cTFH and CD4+ only) and 57. Memory B cells were measured on Days 1 and 57. Results: Of the 36 enrolled subjects, only 22 received both vaccinations and had evaluable specimens after the second vaccine. Plasmablasts peaked one week after each vaccine. Day 15 plasmablasts correlated with peak PRNT titers. cTFH peaked on Days 8 and 36 and correlated with Day 36 plasmablasts. CD4+ peaked at Day 29 and one-third produced &ge, 2 cytokines. Day 57 memory B cells ranged from 0.1% to 0.17% of IgG-secreting B cells. Conclusions: This study provides insights into the cellular responses to non-replicating MVA, currently used as a vector for a variety of novel vaccines.

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 &Delta, A40R, 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 &Delta, A40R in infected human macrophages, in comparison to parental MVA-B, revealed an increase in the mRNA expression levels of interferon (IFN)-&beta, 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 &Delta, A40R 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 &Delta, A40R genome (virus termed MVA-B &Delta, A40R-rev) promoted in infected cells high mRNA and protein A40 levels, with A40 protein localized in the cell membrane. MVA-B &Delta, A40R-rev significantly reduced mRNA levels of IFN-&beta, and of several other innate immune-related genes in infected human macrophages. In immunized mice, MVA-B &Delta, A40R-rev reduced the magnitude of the HIV-1-specific CD4+ and CD8+ T cell responses compared to MVA-B &Delta, A40R. These results revealed an immunosuppressive role of the A40 protein, findings relevant for the optimization of poxvirus vectors as vaccines.

Highlights • Reverse vaccinology was applied to identify and rank ASFV immunogenic candidates . • Selected ASFV immunogenic candidate proteins were expressed in HEK-293 mammalian cells and MVA constructs . • Immunizations with antigens purified from HEK-293 cells and MVA constructs in swine were safe . • Immunizations with selected antigens induced ASFV-specific antibodies and T-cell responses in swine., A reverse vaccinology system, Vaxign, was used to identify and select a subset of five African Swine Fever (ASF) antigens that were successfully purified from human embryonic kidney 293 (HEK) cells and produced in Modified vaccinia virus Ankara (MVA) viral vectors. Three HEK-purified antigens [B646L (p72), E183L (p54), and O61R (p12)], and three MVA-vectored antigens [B646L, EP153R, and EP402R (CD2v)] were evaluated using a prime-boost immunization regimen swine safety and immunogenicity study. Antibody responses were detected in pigs following prime-boost immunization four weeks apart with the HEK-293-purified p72, p54, and p12 antigens. Notably, sera from the vaccinees were positive by immunofluorescence on ASFV (Georgia 2007/1)-infected primary macrophages. Although MVA-vectored p72, CD2v, and EP153R failed to induce antibody responses, interferon-gamma (IFN-γ+) spot forming cell responses against all three antigens were detected one week post-boost. The highest IFN-γ+ spot forming cell responses were detected against p72 in pigs primed with MVA-p72 and boosted with the recombinant p72. Antigen-specific (p12, p72, CD2v, and EP153R) T-cell proliferative responses were also detected post-boost. Collectively, these results are the first demonstration that ASFV subunit antigens purified from mammalian cells or expressed in MVA vectors are safe and can induce ASFV-specific antibody and T-cell responses following a prime-boost immunization regimen in swine.

Gammaherpesviruses (gamma HV) are important pathogens causing persistent infections which lead to several malignancies in immunocompromised patients. Murine gamma HV 68 (MHV-68), a homolog to human EBV and KSHV, has been employed as a classical pathogen to investigate the molecular pathogenicity of gamma HV infections. gamma HV express distinct antigens during lytic or latent infection and antigen-specific T cells have a significant role in controlling the acute and latent viral infection, although the quality of anti-viral T cell responses required for protective immunity is not well-understood. We have generated recombinant modified vaccinia virus Ankara (recMVA) vaccines via MVA-BAC homologous recombination technology expressing MHV-68 ORF6 and ORF61 antigens encoding both MHC class I and II-restricted epitopes. After vaccination, we examined T cell responses before and after MHV-68 infection to determine their involvement in latent virus control. We show recognition of recMVA- and MHV-68-infected APC by ORF6 and ORF61 epitope-specific T cell lines in vitro. The recMVA vaccines efficiently induced MHV-68-specific CD8+ and CD4+ T cell responses after a single immunization and more pronounced after homologous prime/boost vaccination in mice. Moreover, we exhibit protective capacity of prophylactic recMVA vaccination during early latency at day 17 after intranasal challenge with MHV-68, but failed to protect from latency at day 45. Further T cell analysis indicated that T cell exhaustion was not responsible for the lack of protection by recMVA vaccination in long-termlatency at day 45. The data support further efforts aiming at improved vaccine development against gamma HV infections with special focus on targeting protective CD4+ T cell responses.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that has spread to more than 70 countries worldwide since 2015. Despite active research, there are currently no licensed vaccines or therapeutics. We have previously reported the development of various adenoviral vectored vaccine candidates (ChAdOx1 ZIKV) with the ability to stimulate effective immunity in mice and provide protection upon a ZIKV challenge model, using a non-adjuvanted single vaccination approach. In this study, we constructed various modified vaccinia Ankara (MVA) viruses to express the ZIKV Envelope (E) with modifications on the precursor membrane (prM) or on the C-terminus envelope transmembrane domain (TM), similar to our ChAdOx1 vaccine candidates. MVA-ZIKV vaccine candidates were evaluated as a non-adjuvanted single vaccination regimen against a ZIKV Brazilian isolate, using viraemia as the correlate of protection. Here, we report the induction of a modest level of anti-ZIKV E antibodies by all MVA vectored vaccines and sub-optimal efficacy in a ZIKV challenge model. Our results indicate the requirement of additional strategies when using MVA-ZIKV vaccines to afford sterile protection upon a non-adjuvanted and single vaccination regime.

Recombinant vaccines based on vaccinia virus and particularly attenuated strains such as MVA are in human clinical trials, but due to the complexity of these large vectors much remains to be understood about the design parameters that alter their immunogenicity. Previous work had found that MVA vectors should be designed to express stable protein in order to induce robust immunity by CD8+ (cytotoxic) T cells. Here, we found that the primacy of stable antigen is not generalizable to all designs of MVA and may depend where a foreign antigen is inserted into the MVA genome. This unexpected finding suggests that there is an interaction between genome location and the best form of antigen for optimal T cell priming in MVA and thus possibly other vaccine vectors. It also highlights that our understanding of antigen presentation by even the best studied of vaccine vectors remains incomplete., A variety of strains of vaccinia virus (VACV) have been used as recombinant vaccine vectors with the aim of inducing robust CD8+ T cell immunity. While much of the pioneering work was done with virulent strains, such as Western Reserve (WR), attenuated strains such as modified vaccinia virus Ankara (MVA) are more realistic vectors for clinical use. To unify this literature, side-by-side comparisons of virus strains are required. Here, we compare the form of antigen that supports optimal CD8+ T cell responses for VACV strains WR and MVA using equivalent constructs. We found that for multiple antigens, minimal antigenic constructs (epitope minigenes) that prime CD8+ T cells via the direct presentation pathway elicited optimal responses from both vectors, which was surprising because this finding contradicts the prevailing view in the literature for MVA. We then went on to explore the discrepancy between current and published data for MVA, finding evidence that the expression locus and in some cases the presence of the viral thymidine kinase may influence the ability of this strain to prime optimal responses from antigens that require direct presentation. This extends our knowledge of the design parameters for VACV vectored vaccines, especially those based on MVA. IMPORTANCE Recombinant vaccines based on vaccinia virus and particularly attenuated strains such as MVA are in human clinical trials, but due to the complexity of these large vectors much remains to be understood about the design parameters that alter their immunogenicity. Previous work had found that MVA vectors should be designed to express stable protein in order to induce robust immunity by CD8+ (cytotoxic) T cells. Here, we found that the primacy of stable antigen is not generalizable to all designs of MVA and may depend where a foreign antigen is inserted into the MVA genome. This unexpected finding suggests that there is an interaction between genome location and the best form of antigen for optimal T cell priming in MVA and thus possibly other vaccine vectors. It also highlights that our understanding of antigen presentation by even the best studied of vaccine vectors remains incomplete.

Herpesviruses can cause lifelong infections in almost 100% of the population. During infection, the virus can enter a "dormant" phase, also known as latency, in which it produces viral proteins to a very limited extent. This latent virus can therefore not be properly recognized by our immune system and as a result we are unable to clear these viruses from our body. There are no drugs available that can recognize and eliminate these dormant viruses. In this thesis, we try to eliminate herpesvirus infections with new techniques. We usurped an anti-viral system from bacteria: CRISPR/Cas9 to specifically inactivate genomes of human herpes viruses. This approach has proven to be highly effective at eliminating virus infections from various herpes viruses, including Epstein-Barr virus (EBV), human Herpes Simplex virus (HSV-1) and cytomegalovirus (CMV). When using multiple anti-herpesvirus CRISPR we were capable of fully blocking replication of HSV-1. In follow-up experiments we have compared the power of this new technique with the treatment method as it is used in the clinic for treatment against HSV-1. This showed that CRISPR/Cas9 was better able to combat HSV-1 infections and to reduce the formation of medication-avoiding mutants. These findings are important because it offers the possibility to further develop anti-HSV-1 CRISPR/Cas9 into a future treatment against HSV-1. Since the virus, in addition to cold sores, can cause recurring eye infections resulting in vision loss, a healing treatment could have a major medical and societal impact. In addition to directly inactivating viral genomes through CRISPR/Cas9, the technique can also be used to identify, host factors involved in the infection of a virus. Through large genome-wide screens, we found human genes that viruses use in the infection process. In addition to HSV-1, we performed a similar screen for the "MVA" smallpox virus and identified human genes used by the viruses in the infection process. The identification of such genes provides more insight into the interaction between host and virus, and can also provide tools for the targeted development of new treatment methods to prevent or treat virus infections in the future.

Herpesviruses can cause lifelong infections in almost 100% of the population. During infection, the virus can enter a "dormant" phase, also known as latency, in which it produces viral proteins to a very limited extent. This latent virus can therefore not be properly recognized by our immune system and as a result we are unable to clear these viruses from our body. There are no drugs available that can recognize and eliminate these dormant viruses. In this thesis, we try to eliminate herpesvirus infections with new techniques. We usurped an anti-viral system from bacteria: CRISPR/Cas9 to specifically inactivate genomes of human herpes viruses. This approach has proven to be highly effective at eliminating virus infections from various herpes viruses, including Epstein-Barr virus (EBV), human Herpes Simplex virus (HSV-1) and cytomegalovirus (CMV). When using multiple anti-herpesvirus CRISPR we were capable of fully blocking replication of HSV-1. In follow-up experiments we have compared the power of this new technique with the treatment method as it is used in the clinic for treatment against HSV-1. This showed that CRISPR/Cas9 was better able to combat HSV-1 infections and to reduce the formation of medication-avoiding mutants. These findings are important because it offers the possibility to further develop anti-HSV-1 CRISPR/Cas9 into a future treatment against HSV-1. Since the virus, in addition to cold sores, can cause recurring eye infections resulting in vision loss, a healing treatment could have a major medical and societal impact. In addition to directly inactivating viral genomes through CRISPR/Cas9, the technique can also be used to identify, host factors involved in the infection of a virus. Through large genome-wide screens, we found human genes that viruses use in the infection process. In addition to HSV-1, we performed a similar screen for the "MVA" smallpox virus and identified human genes used by the viruses in the infection process. The identification of such genes provides more insight into the interaction between host and virus, and can also provide tools for the targeted development of new treatment methods to prevent or treat virus infections in the future.

A vaccine is urgently needed to combat HIV-1, particularly in sub-Saharan Africa, which remains disproportionately affected by the AIDS pandemic and accounts for the majority of new infections and AIDS-related deaths. In this study, two different vaccination regimens were compared. Rabbits that received two DNA primes followed by two modified vaccinia virus Ankara (MVA) and two protein inoculations developed better immune responses than those that received two MVA and three protein inoculations. In addition, DNA and MVA vaccines that expressed mosaic Gag VLPs presenting a stabilized Env antigen elicited better responses than Env alone, which supports the inclusion of Gag VLPs in an HIV-1 vaccine., A vaccine regimen that elicits broadly neutralizing antibodies (bNAbs) is a major goal in HIV-1 vaccine research. In this study, we assessed the immunogenicity of the CAP256 superinfecting viral envelope (CAP256 SU) protein delivered by modified vaccinia virus Ankara (MVA) and DNA vaccines in different prime-boost combinations followed by a soluble protein (P) boost. The envelope protein (Env) contained a flexible glycine linker and I559P mutation. Trimer-specific bNAbs PGT145, PG16, and CAP256 VRC26_08 efficiently bound to the membrane-bound CAP256 envelope expressed on the surface of cells transfected or infected with the DNA and MVA vaccines. The vaccines were tested in two different vaccination regimens in rabbits. Both regimens elicited autologous tier 2 neutralizing antibodies (NAbs) and high-titer binding antibodies to the matching CAP256 Env and CAP256 V1V2 loop scaffold. The immunogenicity of DNA and MVA vaccines expressing membrane-bound Env alone was compared to that of Env stabilized in a more native-like conformation on the surface of Gag virus-like particles (VLPs). The inclusion of Gag in the DNA and MVA vaccines resulted in earlier development of tier 2 NAbs for both vaccination regimens. In addition, a higher proportion of the rabbits primed with DNA and MVA vaccines that included Gag developed tier 2 NAbs than did those primed with vaccine expressing Env alone. Previously, these DNA and MVA vaccines expressing subtype C mosaic HIV-1 Gag were shown to elicit strong T cell responses in mice. Here we show that when the CAP256 SU envelope protein is included, these vaccines elicit autologous tier 2 NAbs. IMPORTANCE A vaccine is urgently needed to combat HIV-1, particularly in sub-Saharan Africa, which remains disproportionately affected by the AIDS pandemic and accounts for the majority of new infections and AIDS-related deaths. In this study, two different vaccination regimens were compared. Rabbits that received two DNA primes followed by two modified vaccinia virus Ankara (MVA) and two protein inoculations developed better immune responses than those that received two MVA and three protein inoculations. In addition, DNA and MVA vaccines that expressed mosaic Gag VLPs presenting a stabilized Env antigen elicited better responses than Env alone, which supports the inclusion of Gag VLPs in an HIV-1 vaccine.

Hepatitis C is a liver disease caused by the hepatitis C virus (HCV) affecting 71 million people worldwide with no licensed vaccines that prevent infection. Here, we have generated four novel alphavirus-based DNA-launched self-amplifying RNA replicon (DREP) vaccines expressing either structural core-E1-E2 or nonstructural p7-NS2-NS3 HCV proteins of genotype 1a placed under the control of an alphavirus promoter, with or without an alphaviral translational enhancer (grouped as DREP-HCV or DREP-e-HCV, respectively). DREP vectors are known to induce cross-priming and further stimulation of immune responses through apoptosis, and here we demonstrate that they efficiently trigger apoptosis-related proteins in transfected cells. Immunization of mice with the DREP vaccines as the priming immunization followed by a heterologous boost with a recombinant modified vaccinia virus Ankara (MVA) vector expressing the nearly full-length genome of HCV (MVA-HCV) induced potent and long-lasting HCV-specific CD4+ and CD8+ T cell immune responses that were significantly stronger than those of a homologous MVA-HCV prime/boost immunization, with the DREP-e-HCV/MVA-HCV combination the most immunogenic regimen. HCV-specific CD4+ and CD8+ T cell responses were highly polyfunctional, had an effector memory phenotype, and were mainly directed against E1-E2 and NS2-NS3, respectively. Additionally, DREP/MVA-HCV immunization regimens induced higher antibody levels against HCV E2 protein than homologous MVA-HCV immunization. Collectively, these results provided an immunization protocol against HCV by inducing high levels of HCV-specific T cell responses as well as humoral responses. These findings reinforce the combined use of DREP-based vectors and MVA-HCV as promising prophylactic and therapeutic vaccines against HCV., This research was supported by MINECO Spanish grants SAF-2013-45232-R and SAF-2017-88089-R to M.E. and by Swedish Research Council SRC grant 2017-03086 to P.L. M.Q.M. received a Formación del Profesorado Universitario PhD fellowship from the Spanish Ministry of Education and a short-term EMBO fellowship.

Antigenic drift of seasonal influenza viruses and the occasional introduction of influenza viruses of novel subtypes into the human population complicate the timely production of effective vaccines that antigenically match the virus strains that cause epidemic or pandemic outbreaks. The development of game-changing vaccines that induce broadly protective immunity against a wide variety of influenza viruses is an unmet need, in which recombinant viral vectors may provide. Use of viral vectors allows the delivery of any influenza virus antigen, or derivative thereof, to the immune system, resulting in the optimal induction of virus-specific B- and T-cell responses against this antigen of choice. This systematic review discusses results obtained with vectored influenza virus vaccines and advantages and disadvantages of the currently available viral vectors.

Background The 2014 West African outbreak of Ebola virus disease highlighted the urgent need to develop an effective Ebola vaccine. Methods We undertook 2 phase 1 studies assessing safety and immunogenicity of the viral vector modified vaccinia Ankara virus vectored Ebola Zaire vaccine (MVA-EBO-Z), manufactured rapidly on a new duck cell line either alone or in a heterologous prime-boost regimen with recombinant chimpanzee adenovirus type 3 vectored Ebola Zaire vaccine (ChAd3-EBO-Z) followed by MVA-EBO-Z. Adult volunteers in the United Kingdom (n = 38) and Senegal (n = 40) were vaccinated and an accelerated 1-week prime-boost regimen was assessed in Senegal. Safety was assessed by active and passive collection of local and systemic adverse events. Results The standard and accelerated heterologous prime-boost regimens were well-tolerated and elicited potent cellular and humoral immunogenicity in the United Kingdom and Senegal, but vaccine-induced antibody responses were significantly lower in Senegal. Cellular immune responses measured by flow cytometry were significantly greater in African vaccinees receiving ChAd3 and MVA vaccines in the same rather than the contralateral limb. Conclusions MVA biomanufactured on an immortalized duck cell line shows potential for very large-scale manufacturing with lower cost of goods. This first trial of MVA-EBO-Z in humans encourages further testing in phase 2 studies, with the 1-week prime-boost interval regimen appearing to be particularly suitable for outbreak control. Clinical Trials Registration NCT02451891; NCT02485912., New vaccines are needed for outbreak pathogens and novel technologies to manufacture them. We describe 2 phase 1 clinical trials demonstrating safety and immunogenicity of a novel Ebola vaccine manufactured using an improved method to reduce costs and increase yield.

An effective vaccine against Human Immunodeficiency Virus (HIV) still remains the best solution to provide a sustainable control and/or eradication of the virus. We have previously generated the HIV-1 vaccine modified vaccinia virus Ankara (MVA)-B, which exhibited good immunogenicity profile in phase I prophylactic and therapeutic clinical trials, but was unable to prevent viral rebound after antiretroviral (ART) removal. To potentiate the immunogenicity of MVA-B, here we described the design and immune responses elicited in mice by a new T cell multi-epitopic B (TMEP-B) immunogen, vectored by DNA, when administered in homologous or heterologous prime/boost regimens in combination with MVA-B. The TMEP-B protein contained conserved regions from Gag, Pol, and Nef proteins including multiple CD4 and CD8 T cell epitopes functionally associated with HIV control. Heterologous DNA-TMEP/MVA-B regimen induced higher HIV-1-specific CD8 T cell responses with broader epitope recognition and higher polyfunctional profile than the homologous DNA-TMEP/DNA-TMEP or the heterologous DNA-GPN/MVA-B combinations. Moreover, higher HIV-1-specific CD4 and Tfh immune responses were also detected using this regimen. After MVA-B boost, the magnitude of the anti-VACV CD8 T cell response was significantly compromised in DNA-TMEP-primed animals. Our results revealed the immunological potential of DNA-TMEP prime/MVA-B boost regimen and supported the application of these combined vectors in HIV-1 prevention and/or therapy., This research was funded by Spanish grants, SAF-2017-88089-R, Red de Sida RD16/0025/0014 and, in part, by EU HIVACAR project. We thank Victoria Jiménez for technical assistance in cell culture, virus growth and purification. We thank NIH AIDS Research and Reference Reagent Program (Bethesda, MD, USA) for HIV peptides.

The highly attenuated Modified Vaccinia virus Ankara (MVA) lacks most of the known vaccinia virus (VACV) virulence and immune evasion genes. Today MVA can serve as a safety-tested next-generation smallpox vaccine. Yet, we still need to learn about regulatory gene functions preserved in the MVA genome, such as the apoptosis inhibitor genes F1L and E3L. Here, we tested MVA vaccine preparations on the basis of the deletion mutant viruses MVA-ΔF1L and MVA-ΔE3L for efficacy against ectromelia virus (ECTV) challenge infections in mice. In non-permissive human tissue culture the MVA deletion mutant viruses produced reduced levels of the VACV envelope antigen B5. Upon mousepox challenge at three weeks after vaccination, MVA-ΔF1L and MVA-ΔE3L exhibited reduced protective capacity in comparison to wildtype MVA. Surprisingly, however, all vaccines proved equally protective against a lethal ECTV infection at two days after vaccination. Accordingly, the deletion mutant MVA vaccines induced high levels of virus-specific CD8+ T cells previously shown to be essential for rapidly protective MVA vaccination. These results suggest that inactivation of the anti-apoptotic genes F1L or E3L modulates the protective capacity of MVA vaccination most likely through the induction of distinct orthopoxvirus specific immunity in the absence of these viral regulatory proteins.

Highlights • Baculovirus-expressed AHS-VP2 and MVA-VP2 vaccines were evaluated in mice. • Clinical protection was complete in mice receiving one or two doses of MVA-VP2. • Clinical protection complete after two doses of baculovirus-expressed VP2. • Significant reduction of viraemia in all vaccinated groups. • Significant levels of immunity were achieved with one dose of either vaccine., African horse sickness is a severe, often fatal, arboviral disease of equids. The control of African horse sickness virus (AHSV) in endemic countries is based currently on the use of live attenuated vaccines despite some biosafety concerns derived from its biological properties. Thus, experimental vaccination platforms have been developed over the years in order to avoid the biosafety concerns associated with the use of attenuated vaccines. Various studies showed that baculovirus-expressed AHSV-VP2 or modified Vaccinia Ankara virus expressing AHSV-VP2 (MVA-VP2) induced virus neutralising antibodies and protective immunity in small animals and horses. AHSV is an antigenically diverse pathogen and immunity against AHS is serotype-specific. Therefore, AHS vaccines for use in endemic countries need to induce an immune response capable of protecting against all existing serotypes. For this reason, current live attenuated vaccines are administered as polyvalent preparations comprising combinations of AHSV attenuated strains of different serotypes. Previous studies have shown that it is possible to induce cross-reactive virus neutralising antibodies against different serotypes of AHSV by using polyvalent vaccines comprising combinations of either different serotype-specific VP2 proteins, or MVA-VP2 viruses. However, these strategies could be difficult to implement if induction of protective immunity is highly dependent on using a two-dose vaccination regime for each serotype the vaccine intends to protect against. In our study, we have tested the protective capacity of MVA-VP2 and baculovirus-expressed VP2 vaccines when a single dose was used. Groups of interferon alpha receptor knock-out mice were inoculated with either MVA-VP2 or baculovirus-expressed VP2 vaccines using one dose or the standard two-dose vaccination regime. After vaccination, all four vaccinated groups were challenged with AHSV and clinical responses, lethality and viraemia compared between the groups. Our results show that complete clinical protection was achieved after a single vaccination with either MVA-VP2 or baculovirus sub-unit VP2 vaccines.

African horse sickness (AHS) is a lethal equine disease transmitted by Culicoides biting midges and caused by African horse sickness virus (AHSV). AHS is endemic to sub-Saharan Africa, but devastating outbreaks have been recorded periodically outside this region. The perceived risk of an AHS outbreak occurring in Europe has increased following the frequent epidemics caused in ruminants by bluetongue virus, closely related to AHSV. Attenuated vaccines for AHS are considered unsuitable for use in non-endemic countries due bio-safety concerns. Further, attenuated and inactivated vaccines are not compatible with DIVA (differentiate infected from vaccinated animals) strategies. All these factors stimulated the development of novel AHS vaccines that are safer, more efficacious and DIVA compatible. We showed previously that recombinant modified Vaccinia Ankara virus (MVA) vaccines encoding the outer capsid protein of AHSV (AHSV-VP2) induced virus neutralising antibodies (VNAb) and protection against AHSV in a mouse model and also in the horse. Passive immunisation studies demonstrated that immunity induced by MVA-VP2 was associated with pre-challenge VNAb titres in the vaccinates. Analyses of the inoculum of these MVA-VP2 experimental vaccines showed that they contained pre-formed AHSV-VP2. We continued studying the influence of pre-formed AHSV-VP2, present in the inoculum of MVA-VP2 vaccines, in the immunogenicity of MVA-VP2 vaccines. Thus, we compared correlates of immunity in challenged mice that were previously vaccinated with: a) MVA-VP2 (live); b) MVA-VP2 (live and sucrose gradient purified); c) MVA-VP2 (UV light inactivated); d) MVA-VP2 (UV light inactivated and diluted); e) MVA-VP2 (heat inactivated); f) MVA-VP2 (UV inactivated) + MVA-VP2 (purified); g) MVA-VP2 (heat inactivated) + MVA-VP2 (purified); and h) wild type-MVA (no insert). The results of these experiments showed that protection was maximal using MVA-VP2 (live) vaccine and that the protection conferred by all other vaccines correlated strongly with the levels of pre-formed AHSV-VP2 in the vaccine inoculum., Highlights • MVA-VP2 vaccines (expressing African horse sickness virus VP2) induce protective immunity in mouse model and in horses. • Experimental MVA-VP2 vaccines contain preformed AHSV-VP2 in the inoculum if they are not sucrose-gradient purified. • MVA-VP2 vaccines express AHSV-VP2 in MVA-VP2 infected cells. • Both pre-formed AHSV-VP2 and ‘de novo’ synthesised AHSV-VP2 in MVA-VP2 vaccinates contribute to MVA-VP2 immunogenicity.

Conventional vaccines have controlled or limited BTV expansion in the past, but they cannot address the need for cross-protection among serotypes and do not allow distinguishing between infected and vaccinated animals (DIVA strategy). There is a need to develop universal vaccines that induce effective protection against multiple BTV serotypes. In this work we have shown the importance of the nonstructural protein NS1, conserved among all the BTV serotypes, in CD8 T cell-mediated protection against multiple BTV serotypes when vectorized as a recombinant MVA vaccine., The development of vaccines against bluetongue, a prevalent livestock disease, has been focused on surface antigens that induce strong neutralizing antibody responses. Because of their antigenic variability, these vaccines are usually serotype restricted. We now show that a single highly conserved nonstructural protein, NS1, expressed in a modified vaccinia Ankara virus (MVA) vector can provide multiserotype protection in IFNAR−/− 129 mice against bluetongue virus (BTV) that is largely dependent on CD8 T cell responses. We found that the protective antigenic capacity of NS1 resides within the N terminus of the protein and is provided in the absence of neutralizing antibodies. The protective CD8 T cell response requires the presence of a specific peptide within the N terminus of NS1, since its deletion ablates the efficacy of the vaccine formulation. These data reveal the importance of the nonstructural protein NS1 in CD8 T cell-mediated protection against multiple BTV serotypes when vectorized as a recombinant MVA vaccine. IMPORTANCE Conventional vaccines have controlled or limited BTV expansion in the past, but they cannot address the need for cross-protection among serotypes and do not allow distinguishing between infected and vaccinated animals (DIVA strategy). There is a need to develop universal vaccines that induce effective protection against multiple BTV serotypes. In this work we have shown the importance of the nonstructural protein NS1, conserved among all the BTV serotypes, in CD8 T cell-mediated protection against multiple BTV serotypes when vectorized as a recombinant MVA vaccine.

Introduction Since the first case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) infection was reported in 2012, the virus has infected more than 1300 individuals in 26 countries, and caused more than 480 deaths. Human-to-human transmission requires close contact, and has typically occurred in the healthcare setting. Improved global awareness, together with improved hygiene practices in healthcare facilities, has been highlighted as key strategies in controlling the spread of MERS-CoV. This study tested the in vitro efficacy of three formulations of povidone iodine (PVP-I: 4% PVP-I skin cleanser, 7.5% PVP-I surgical scrub, and 1% PVP-I gargle/mouthwash) against a reference virus (Modified vaccinia virus Ankara, MVA) and MERS-CoV. Methods According to EN14476, a standard suspension test was used to assess virucidal activity against MVA and large volume plating was used for MERS-CoV. All products were tested under clean (0.3 g/L bovine serum albumin, BSA) and dirty conditions (3.0 g/L BSA + 3.0 mL/L erythrocytes), with application times of 15, 30, and 60 s for MVA, and 15 s for MERS-CoV. The products were tested undiluted, 1:10 and 1:100 diluted against MVA, and undiluted against MERS-CoV. Results A reduction in virus titer of ≥4 log10 (corresponding to an inactivation of ≥99.99%) was regarded as evidence of virucidal activity. This was achieved versus MVA and MERS-CoV, under both clean and dirty conditions, within 15 s of application of each undiluted PVP-I product. Conclusion These data indicate that PVP-I-based hand wash products for potentially contaminated skin, and PVP-I gargle/mouthwash for reduction of viral load in the oral cavity and the oropharynx, may help to support hygiene measures to prevent transmission of MERS-CoV. Funding Mundipharma Research GmbH & Co. Electronic supplementary material The online version of this article (doi:10.1007/s40121-015-0091-9) contains supplementary material, which is available to authorized users.

Bluetongue virus (BTV) is the causative agent of bluetongue disease (BT), which affects domestic and wild ruminants. At the present, 27 different serotypes have been documented. Vaccination has been demonstrated as one of the most effective methods to avoid viral dissemination. To overcome the drawbacks associated with the use of inactivated and attenuated vaccines we engineered a new recombinant BTV vaccine candidate based on proteins VP2, VP7, and NS1 of BTV-4 that were incorporated into avian reovirus muNS-Mi microspheres (MS-VP2/VP7/NS1) and recombinant modified vaccinia virus Ankara (rMVA). The combination of these two antigen delivery systems in a heterologous prime-boost vaccination strategy generated significant levels of neutralizing antibodies in IFNAR(−/−) mice. Furthermore, this immunization strategy increased the ratio of IgG2a/IgG1 in sera, indicating an induction of a Th1 response, and elicited a CD8 T cell response. Immunized mice were protected against lethal challenges with the homologous serotype 4 and the heterologous serotype 1 of BTV. All these results support the strategy based on microspheres in combination with rMVAs as a promising multiserotype vaccine candidate against BTV This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (AGL2011-23506, AGL-2014-57430-R and BFU2013-43513-R). Financial support from the Consellería de Cultura, Educación e Ordenación Universitaria (Centro singular de investigación de Galicia accreditation 2016–2019, ED431G/09) and the European Regional Development Fund (ERDF), is also gratefully acknowledged SI

Modified Vaccinia virus Ankara (MVA) is a promising vaccine vector with an excellent safety profile. However, despite extensive pre-clinical and clinical testing, surprisingly little is known about the cellular tropism of MVA, especially in relevant animal species. Here, we performed in vitro, ex vivo and in vivo experiments with recombinant MVA expressing green fluorescent protein (rMVA-GFP). In both human peripheral blood mononuclear cells and mouse lung explants, rMVA-GFP predominantly infected antigen presenting cells. Subsequent in vivo experiments performed in mice, ferrets and non-human primates indicated that preferential targeting of dendritic cells and alveolar macrophages was observed after respiratory administration, although subtle differences were observed between the respective animal species. Following intramuscular injection, rMVA-GFP was detected in interdigitating cells between myocytes, but also in myocytes themselves. These data are important in advancing our understanding of the basis for the immunogenicity of MVA-based vaccines and aid rational vaccine design and delivery strategies.

The LC16m8 strain of vaccinia virus, the active ingredient in the Japanese smallpox vaccine, was derived from the Lister/Elstree strain. LC16m8 is replication-competent and has been administered to over 100,000 infants and 3,000 adults with no serious adverse reactions. Despite this outstanding safety profile, the occurrence of spontaneously-generated large plaque-forming virulent LC16m8 revertants following passage in cell culture is a major drawback. We identified the gene responsible for the reversion and deleted the gene (B5R) from LC16m8 to derive LC16m8Δ. LC16m8∆ is non-pathogenic in immunodeficient severe combined immunodeficiency (SCID) mice, genetically-stable and does not reverse to a large-plaque phenotype upon passage in cell culture, even under conditions in which most LC16m8 populations are replaced by revertants. Moreover, LC16m8∆ is >500-fold more effective than the non-replicating vaccinia virus (VV), Modified Vaccinia Ankara (MVA), at inducing murine immune responses against pathogenic VV. LC16m8∆, which expresses the SIV gag gene, also induced anti-Gag CD8+ T-cells more efficiently than MVA and another non-replicating VV, Dairen I minute-pock variants (DIs). Moreover, LC16m8∆ expressing HIV-1 Env in combination with a Sendai virus vector induced the production of anti-Env antibodies and CD8+ T-cells. Thus, the safety and efficacy of LC16m8∆ mean that it represents an outstanding platform for the development of human vaccine vectors.

Dengue is a global public health concern and this is aggravated by a lack of vaccines or antiviral therapies. Despite the well-known role of CD8(+) T cells in the immunopathogenesis of Dengue virus (DENV), only recent studies have highlighted the importance of this arm of the immune response in protection against the disease. Thus, the majority of DENV vaccine candidates are designed to achieve protective titers of neutralizing antibodies, with less regard for cellular responses. Here, we used a mouse model to investigate CD8(+) T cell and humoral responses to a set of potential DENV vaccines based on recombinant modified vaccinia virus Ankara (rMVA). To enable this study, we identified two CD8(+) T cell epitopes in the DENV-3 E protein in C57BL/6 mice. Using these we found that all the rMVA vaccines elicited DENV-specific CD8(+) T cells that were cytotoxic in vivo and polyfunctional in vitro. Moreover, vaccines expressing the E protein with an intact signal peptide sequence elicited more DENV-specific CD8(+) T cells than those expressing E proteins in the cytoplasm. Significantly, it was these same ER-targeted E protein vaccines that elicited antibody responses. Our results support the further development of rMVA vaccines expressing DENV E proteins and add to the tools available for dengue vaccine development.

Highly attenuated poxviral vectors, such as modified vaccinia virus ankara (MVA), are promising vaccine candidates against several infectious diseases. One of the approaches developed to enhance the immunogenicity of poxvirus vectors is increasing the promoter strength and accelerating during infection production levels of heterologous antigens. Here, we have generated and characterized the biology and immunogenicity of an optimized MVA-based vaccine candidate against HIV/AIDS expressing HIV-1 clade B gp120 protein under the control of a novel synthetic late/early optimized (LEO) promoter (LEO160 promoter, with a spacer length of 160 nucleotides), termed MVA-LEO160-gp120. In infected cells, MVA-LEO160-gp120 significantly increased the expression levels of HIV-1 gp120 mRNA and protein, compared to the clinical vaccine MVA-B vector expressing HIV-1 gp120 under the control of the commonly used synthetic early/late promoter. When mice were immunized with a heterologous DNA-prime/MVA-boost protocol, the immunization group DNA-gp120/MVA-LEO160-gp120 induced an enhancement in the magnitude of gp120-specific CD4+ and CD8+ T-cell responses, compared to DNA-gp120/MVA-B, with most of the responses being mediated by the CD8+ T-cell compartment, with a T effector memory phenotype. DNA-gp120/MVA-LEO160-gp120 also elicited a trend to a higher magnitude of gp120-specific CD4+ T follicular helper cells, and modest enhanced levels of antibodies against HIV-1 gp120. These findings revealed that this new optimized vaccinia virus promoter could be considered a promising strategy in HIV/AIDS vaccine design, confirming the importance of early expression of heterologous antigen and its impact on the antigen-specific immunogenicity elicited by poxvirus-based vectors.

In 2013, a major chikungunya virus (CHIKV) epidemic reached the Americas. In the past 2 years, >1.7 million people have been infected. In light of the current epidemic, with millions of people in North and South America at risk, efforts to rapidly develop effective vaccines have increased. Here, we focus on CHIKV vaccines that use viral-vector technologies. This group of vaccine candidates shares an ability to potently induce humoral and cellular immune responses by use of highly attenuated and safe vaccine backbones. So far, well-described vectors such as modified vaccinia virus Ankara, complex adenovirus, vesicular stomatitis virus, alphavirus-based chimeras, and measles vaccine Schwarz strain (MV/Schw) have been described as potential vaccines. We summarize here the recent data on these experimental vaccines, with a focus on the preclinical and clinical activities on the MV/Schw-based candidate, which is the first CHIKV-vectored vaccine that has completed a clinical trial.

Highlights ► Current influenza vaccines do not generate heterologous protection. ► Targeting internal influenza antigens may confer cross protection. ► We tested Adenovirus and MVA vectored NP and M1 in chickens. ► Heterologous prime-boost resulted in earlier cessation of viral shedding., Current vaccines targeting surface proteins can drive antigenic variation resulting either in the emergence of more highly pathogenic viruses or of antigenically distinct viruses that escape control by vaccination and thereby persist in the host population. Influenza vaccines typically target the highly mutable surface proteins and do not provide protection against heterologous challenge. Vaccines which induce immune responses against conserved influenza epitopes may confer protection against heterologous challenge. We report here the results of vaccination with recombinant modified Vaccinia virus Ankara (MVA) and Adenovirus (Ad) expressing a fusion construct of nucleoprotein and matrix protein (NP + M1). Prime and boost vaccination regimes were trialled in different ages of chicken and were found to be safe and immunogenic. Interferon-γ (IFN-γ) ELISpot was used to assess the cellular immune response post secondary vaccination. In ovo Ad prime followed by a 4 week post hatch MVA boost was identified as the most immunogenic regime in one outbred and two inbred lines of chicken. Following vaccination, one inbred line (C15I) was challenged with low pathogenic avian influenza (LPAI) H7N7 (A/Turkey/England/1977). Birds receiving a primary vaccination with Ad-NP + M1 and a secondary vaccination with MVA-NP + M1 exhibited reduced cloacal shedding as measured by plaque assay at 7 days post infection compared with birds vaccinated with recombinant viruses containing irrelevant antigen. This preliminary indication of efficacy demonstrates proof of concept in birds; induction of T cell responses in chickens by viral vectors containing internal influenza antigens may be a productive strategy for the development of vaccines to induce heterologous protection against influenza in poultry.

Background: Circumsporozoite protein (CS) is the antigenic target for RTS,S, the most advanced malaria vaccine to date. Heterologous prime-boost with the viral vectors simian adenovirus 63 (ChAd63)-modified vaccinia virus Ankara (MVA) is the most potent inducer of T-cells in humans, demonstrating significant efficacy when expressing the preerythrocytic antigen insert multiple epitope–thrombospondin-related adhesion protein (ME-TRAP). We hypothesized that ChAd63-MVA containing CS may result in a significant clinical protective efficacy. Methods: We conducted an open-label, 2-site, partially randomized Plasmodium falciparum sporozoite controlled human malaria infection (CHMI) study to compare the clinical efficacy of ChAd63-MVA CS with ChAd63-MVA ME-TRAP. Results: One of 15 vaccinees (7%) receiving ChAd63-MVA CS and 2 of 15 (13%) receiving ChAd63-MVA ME-TRAP achieved sterile protection after CHMI. Three of 15 vaccinees (20%) receiving ChAd63-MVA CS and 5 of 15 (33%) receiving ChAd63-MVA ME-TRAP demonstrated a delay in time to treatment, compared with unvaccinated controls. In quantitative polymerase chain reaction analyses, ChAd63-MVA CS was estimated to reduce the liver parasite burden by 69%–79%, compared with 79%–84% for ChAd63-MVA ME-TRAP. Conclusions: ChAd63-MVA CS does reduce the liver parasite burden, but ChAd63-MVA ME-TRAP remains the most promising antigenic insert for a vectored liver-stage vaccine. Detailed analyses of parasite kinetics may allow detection of smaller but biologically important differences in vaccine efficacy that can influence future vaccine development. Clinical Trials Registration: NCT01623557.

The zoonotic transmissions of highly pathogenic avian influenza viruses of the H5N1 subtype hat occur since 1997 have sparked the development of novel influenza vaccines. The advent of reverse genetics technology, cell culture production techniques and novel adjuvants has improved the vaccine strain preparation, the production process and the immunogenicity of the vaccines respectively and would accelerated the availability of pandemic influenza vaccines. However, there is still room for improvement and alternative vaccine preparations are explored such as recombinant antigens (e.g. baculovirus expression) and viral vectors. Modified Vaccinia virus Ankara (MVA), originally developed as a safe smallpox vaccine can be exploited as a viral vector. It has favourable properties, which makes it an attractive candidate as a pandemic influenza vaccine (for review see reference [1]). Recently we have evaluated a MVA-based vaccine for highly pathogenic influenza virus of the H5N1 subtype in mice and macaques. To this end, recombinant MVA was constructed expressing the gene encoding the hemagglutinin of H5N1 influenza virus A/Vietnam/1194/04 (clade 1) (MVA-HA-VN/04) and used to immunize C57BL/6 mice and cynomolgus macaques (macaca fascicularis). Two immunizations induced strong virus specific antibody responses in both species and protected the animals from the development of severe disease observed in control animals inoculated with empty MVA vector or PBS after challenge infection with the homologous or the antigenically distinct influenza virus A/Indonesia/5/05 (clade 2.1). In vaccinated animals virus replication in the respiratory tract was not detectable and the development of histopathological changes in the lungs was prevented. Furthermore, a MVA-based 2009 pandemic H1N1 vaccine protected against severe disease in a pH1N1 ferret model. The preclinical evaluation of MVA-based candidate vaccines indicated that they have potential as vaccines against highly pathogenic H5N1 and pH1N1 influenza viruses. The MVA-based vaccines proved to be immunogenic and induced broad-protective immune responses. MVA has favourable properties for the production, storage and use as a pandemic influenza vaccine and further clinical development seems warranted.

Modified vaccinia virus Ankara (MVA) was generated by serial passaging in chicken embryo fibroblasts. During this attenuation, MVA lost the capacity to productively grow in human and most other mammalian cell lines, as well as acquiring a multitude of deletions and mutations in the MVA genome. This means that the precise molecular basis for the MVA host-range restriction is still unknown. The vaccinia virus (VACV) genes F11L and K1L are mutated or truncated in MVA. F11L was previously implicated in VACV-induced cell motility and virion maturation. Here, we demonstrate that the restoration of F11L gene expression in MVA rescued virus-induced cell motility, but had no impact on MVA virion maturation and host-range restriction. Additional insertion of the K1L gene, which restores MVA replication in RK-13 cells, was not sufficient to extend MVA growth capacity to other mammalian cells.

Bats are reservoir hosts for a spectrum of infectious diseases. Some pathogens (such as Hendra, Nipah and Marburg viruses) appear to use mainly fruit bats as reservoir. We describe designed immortalization of primary fetal cells from the Egyptian fruit bat (Rousettus aegyptiacus) to facilitate isolation and characterization of pathogens associated with these mammals. Three cell lines with different properties were recovered and successful immortalization was confirmed by continuous cultivation for over 18 months. Surprisingly, the cell lines are fully permissive for a highly attenuated poxvirus, modified vaccinia Ankara (MVA). MVA is a safe and well characterized vaccine vector that cannot replicate in most mammalian cells. High permissivity of Rousettus cell lines could justify testing bats for susceptibility to MVA as a replication competent vector with low zoonotic potential to induce herd immunity in bat colonies against viruses causing rabies or haemorrhagic fevers.

Modified vaccinia virus Ankara (MVA), which is a promising replication-defective vaccine vector, is unusual among the orthopoxviruses in activating NF-kappaB transcription factors in cells of several types. In human embryonic kidney (HEK 293T) cells, the MVA-induced depletion of IkappaBalpha required to activate NF-kappaB is inhibited by UV-inactivation of the virus, and begins before viral DNA replication. In HEK 293T, CHO, or RK13 cells, expression of the cowpox virus CP77 early gene, or the vaccinia virus K1L early gene suppresses MVA-induced IkappaBalpha depletion. In mouse embryonic fibroblasts (MEFs), MVA induction of IkappaBalpha depletion is dependent on the expression of mouse or human double-stranded RNA-activated protein kinase (PKR). These results demonstrate that events during the early phase of MVA replication can induce PKR-mediated processes contributing both to the activation of NF-kappaB signaling, and to processes that may restrict viral replication. This property may contribute to the efficacy of this vaccine virus.

A major problem of current vaccines is storage stability, often requiring strict maintenance of cold chains. In the course of the eradication of smallpox, a freeze-dried vaccinia virus (Dryvax), which proved to be very stable, was used to overcome this limitation. However, Dryvax needs to be reconstituted before usage and is administered using a bifurcated needle, procedures that pose a number of additional health risks. We report in this study that a stable, lyophilized, modified vaccinia virus Ankara (MVA) vaccine can be directly applied to the nostrils of mice without previous reconstitution. This direct mucosal application induced systemic Ab and T cell responses comparable to those achieved by i.m. administration. Importantly, mucosal application of lyophilized MVA induced long-lasting protective immunity against lethal bacterial and viral challenges. These data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized MVA.

The porcine reproductive and respiratory syndrome virus (PRRSV) has three major structural proteins which designated as GP5, M, and N. Protein GP5 and M have been considered very important to arouse the humoral and cellular immune responses against PRRSV infection and proposed to be the excellent candidate proteins in the design of PRRS bioengineering vaccine. There were some attempts on expressing GP5 or M in DNA vaccine and adenovirus to arouse humoral and cellular immune responses, but few papers have been reported on that the immune response can be difference because of the expression patterns of GP5 and M proteins in the recombinant virus. In this article, four recombinant viruses that expressed GP5 and M proteins of PRRSV in the modified vaccinia virus ankara (MVA) with different expression patterns were made. In these recombinant virus (rMVAs), GP5 and M proteins were expressed in MVA in the same virus but under the control of two promoters (rMVA-GP5/M), or as a fusion protein under one promoter (rMVA-GP5-M), or separately (rMVA-GP5 and rMVA-M). The humoral and cellular immune responses for the four recombinant viruses were evaluated with mouse model. Every mouse was inoculated with 5 x 10(5) TCID50 of the different rMVAs and boosted 3 weeks later. Neutralizing antibody titers for each group were detected with virus neutralization test assay weekly after the primary inoculation for 13 weeks to evaluate the humoral immune response. The production of gamma interferon (IFN-gamma), interleukin-2 (IL-2), and interleukin-4 (IL-4) was detected in splenocytes of rMVA-inoculated mice at 30, 60, and 90 days post inoculation to evaluate the cellular immune response. Results showed that rMVA-GP5 and rMVA-M cannot induce obvious humoral and cellular immune responses; rMVA-GP5-M inoculated group developed better immune responses than rMVA-GP5 and rMVA-M inoculated groups; however, mice inoculated with rMVA-GP5/M maintained the strongest cellular response against PRRS and consistently enhanced the anti-PRRSV humoral responses. The strategy of co-expressing PRRSV GP5 and M protein in MVA under the control of different promoters might be an attractive method for future PRRSV vaccine design.

El virus vaccinia Ankara modificado (MVA) constituye un buen candidato para el desarrollo de vectores virales de expresión no replicativos porque no replica en la mayoría de las células de mamíferos. Para la producción de MVA recombinantes es fundamental disponer de vectores de transferencia que, por recombinación homóloga con el genoma viral, permitan introducir los genes de interés en regiones no esenciales para la replicación in vitro. En este trabajo se diseñaron y obtuvieron los vectores de transferencia denominados VT-MHA y VT-MTK que portan las regiones correspondientes a las posiciones 1-303 y 608-948 del gen MVA165R y 1-244 y 325-534 del gen MVA086R, respectivamente, las que flanquean un sitio de clonado múltiple para la inserción de los genes foráneos. En dichos vectores se clonaron los casetes para la expresión de los genes lac Z o uid A, y la actividad de las enzimas marcadoras b-galactosidasa y b-glucuronidasa se confirmó in situ. Además, utilizando el vector denominado VT-MTK-GUS, se obtuvieron y aislaron MVA recombinantes puros que portan y expresan el gen uid A. Los resultados obtenidos constituyen las herramientas básicas para establecer la metodología de obtención de MVA recombinantes, con el propósito de desarrollar localmente vectores virales no replicativos candidatos a vacunas.Modified Vaccinia virus Ankara (MVA) constitutes a good candidate for the development of non-replicative expression viral vectors because it does not replicate in most of mammalian cells. It is essential, for the production of recombinant MVA, the availability of transfer vectors which allow the introduction of desired genes into non-essential regions for in vitro viral replication, by homologous recombination with the viral genome. In the present work, the transfer vectors named VT-MHA and VT-MTK were designed and obtained. They carried genomic regions corresponding to 1- 303 and 608-948 positions of the MVA165R gene and 1-244 and 325-534 of the MVA086R gene, respectively, which flank a multiple cloning site for the insertion of foreign genes. In these vectors, the cassettes for the expression of lac Z or uid A genes were cloned, and the activity of the marker enzymes b-galactosidase and b-glucuronidase was confirmed in situ. Furthermore, the vector named VT-MTK-GUS was used to obtain and isolate pure recombinant MVA, which carried and expressed the uid A gene. The results herein constitute the basic tools for establishing the methodology to obtain recombinant MVA with the purpose of locally developing non-replicative viral vectors as candidate vaccines.