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Various species of the SARS-CoV-2 host cell receptor, the angiotensin-converting enzyme 2 (ACE2), are present in serum, which may result from virus entry and subsequent proteolytic processing of the membrane receptor. We have recently demonstrated changes of particular ACE2 species in virus infected humans, either cleaved fragments or circulating full-length species. Here, we further explore the potential of serum ACE2 as a biomarker to test SARS-CoV-2 infection and vaccine efficacy in virus susceptible transgenic K18-hACE2 mice expressing human ACE2. First, in serum samples derived from K18-hACE2 mice challenged with a lethal dose of SARS-CoV-2, we observed an increase in the levels of cleaved ACE2 fragment at day 2 post-challenge, which may represent the subsequent proteolytic processing through virus entry. These elevated levels were maintained until the death of the animals at day 6 post-challenge. The circulating full-length ACE2 form displayed a sizable peak at day 4, which declined at day 6 post-challenge. Noticeably, immunization with two doses of the MVA-CoV2-S vaccine candidate prevented ACE2 cleaved changes in serum of animals challenged with a lethal dose of SARS-CoV-2. The efficacy of the MVA-CoV2-S was extended to vaccinated mice after virus re-challenge. These findings highlight that ACE2 could be a potential serum biomarker for disease progression and vaccination against SARS-CoV-2.

Poxvirus vector Modified Vaccinia Virus Ankara (MVA) expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (termed MVA-B) is a promising HIV/AIDS vaccine candidate, as confirmed from results obtained in a prophylactic phase I clinical trial in humans. To improve the immunogenicity elicited by MVA-B, we have generated and characterized the innate immune sensing and the in vivo immunogenicity profile of a vector with a double deletion in two vaccinia virus (VACV) genes (C6L and K7R) coding for inhibitors of interferon (IFN) signaling pathways. The innate immune signals elicited by MVA-B deletion mutants (MVA-B ΔC6L and MVA-B ΔC6L/K7R) in human macrophages and monocyte-derived dendritic cells (moDCs) showed an up-regulation of the expression of IFN-β, IFN-α/β-inducible genes, TNF-α, and other cytokines and chemokines. A DNA prime/MVA boost immunization protocol in mice revealed that these MVA-B deletion mutants were able to improve the magnitude and quality of HIV-1-specific CD4(+) and CD8(+) T cell adaptive and memory immune responses, which were mostly mediated by CD8(+) T cells of an effector phenotype, with MVA-B ΔC6L/K7R being the most immunogenic virus recombinant. CD4(+) T cell responses were mainly directed against Env, while GPN-specific CD8(+) T cell responses were induced preferentially by the MVA-B deletion mutants. Furthermore, antibody levels to Env in the memory phase were slightly enhanced by the MVA-B deletion mutants compared to the parental MVA-B. These findings revealed that double deletion of VACV genes that act blocking intracellularly the IFN signaling pathway confers an immunological benefit, inducing innate immune responses and increases in the magnitude, quality and durability of the HIV-1-specific T cell immune responses. Our observations highlighted the immunomodulatory role of the VACV genes C6L and K7R, and that targeting common pathways, like IRF3/IFN-β signaling, could be a general strategy to improve the immunogenicity of poxvirus-based vaccine candidates.

The mechanisms of viral RNA genome segmentation are unknown. On extensive passage of foot-and-mouth disease virus in baby hamster kidney-21 cells, the virus accumulated multiple point mutations and underwent a transition akin to genome segmentation. The standard single RNA genome molecule was replaced by genomes harboring internal in-frame deletions affecting the L- or capsid-coding region. These genomes were infectious and killed cells by complementation. Here we show that the point mutations in the nonstructural protein-coding region (P2, P3) that accumulated in the standard genome before segmentation increased the relative fitness of the segmented version relative to the standard genome. Fitness increase was documented by intracellular expression of virus-coded proteins and infectious progeny production by RNAs with the internal deletions placed in the sequence context of the parental and evolved genome. The complementation activity involved several viral proteins, one of them being the leader proteinase L. Thus, a history of genetic drift with accumulation of point mutations was needed to allow a major variation in the structure of a viral genome. Thus, exploration of sequence space by a viral genome (in this case an unsegmented RNA) can reach a point of the space in which a totally different genome structure (in this case, a segmented RNA) is favored over the form that performed the exploration.

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.

The immune parameters of HIV/AIDS vaccine candidates that might be relevant in protection against HIV-1 infection are still undefined. The highly attenuated poxvirus strain MVA is one of the most promising vectors to be use as HIV-1 vaccine. We have previously described a recombinant MVA expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (referred as MVA-B), that induced HIV-1-specific immune responses in different animal models and gene signatures in human dendritic cells (DCs) with immunoregulatory function. In an effort to characterize in more detail the immunogenic profile of MVA-B and to improve its immunogenicity we have generated a new vector lacking two genes (A41L and B16R), known to counteract host immune responses by blocking the action of CC-chemokines and of interleukin 1beta, respectively (referred as MVA-B DeltaA41L/DeltaB16R). A DNA prime/MVA boost immunization protocol was used to compare the adaptive and memory HIV-1 specific immune responses induced in mice by the parental MVA-B and by the double deletion mutant MVA-B DeltaA41L/DeltaB16R. Flow cytometry analysis revealed that both vectors triggered HIV-1-specific CD4(+) and CD8(+) T cells, with the CD8(+) T-cell compartment responsible for >91.9% of the total HIV-1 responses in both immunization groups. However, MVA-B DeltaA41L/DeltaB16R enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4(+) and CD8(+) T-cell immune responses. HIV-1-specific CD4(+) T-cell responses were polyfunctional and preferentially Env-specific in both immunization groups. Significantly, while MVA-B induced preferentially Env-specific CD8(+) T-cell responses, MVA-B DeltaA41L/DeltaB16R induced more GPN-specific CD8(+) T-cell responses, with an enhanced polyfunctional pattern. Both vectors were capable of producing similar levels of antibodies against Env. These findings revealed that MVA-B and MVA-B DeltaA41L/DeltaB16R induced in mice robust, polyfunctional and durable T-cell responses to HIV-1 antigens, but the double deletion mutant showed enhanced magnitude and quality of HIV-1 adaptive and memory responses. Our observations are relevant in the immune evaluation of MVA-B and on improvements of MVA vectors as HIV-1 vaccines.

Efficient adaptive antiviral cellular and humoral immune responses require previous recognition of viral antigenic peptides bound to human leukocyte antigen (HLA) class I and II molecules, which are exposed on the surface of infected and antigen presenting cells, respectively. The HLA-restricted immune response to Chikungunya virus (CHIKV), a mosquito-borne Alphavirus of the Togaviridae family responsible for severe chronic polyarthralgia and polyarthritis, is largely unknown. In this study, a high-throughput mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of human cells infected with a vaccinia virus (VACV) recombinant expressing CHIKV structural proteins was carried out. Twelve viral ligands from the CHIKV polyprotein naturally presented by different HLA-A, -B, and -C class I, and HLA-DR and -DP class II molecules were identified. The immunoprevalence of the HLA class II but not the HLA class I-restricted cellular immune response against the CHIKV structural polyprotein was greater than that against the VACV vector itself. In addition, most of the CHIKV HLA class I and II ligands detected by mass spectrometry are not conserved compared to its closely related O'nyong-nyong virus. These findings have clear implications for analysis of both cytotoxic and helper immune responses against CHIKV as well as for the future studies focused in the exacerbated T helper response linked to chronic musculoskeletal disorders in CHIKV patients.

The highly attenuated vaccinia virus strain NYVAC expressing HIV-1 components has been evaluated as a vaccine candidate in preclinical and clinical trials with encouraging results. We have previously described that the presence of C7L in the NYVAC genome prevents the induction of apoptosis and renders the vector capable of replication in human and murine cell lines while maintaining an attenuated phenotype in mice. In an effort to improve the immunogenicity of NYVAC, we have developed a novel poxvirus vector by inserting the VACV host-range C7L gene into the genome of NYVAC-B, a recombinant virus that expresses four HIV-1 antigens from clade B (Env, Gag, Pol and Nef) (referred as NYVAC-B-C7L). In the present study, we have compared the in vitro and in vivo behavior of NYVAC-B and NYVAC-B-C7L. In cultured cells, NYVAC-B-C7L expresses higher levels of heterologous antigen than NYVAC-B as determined by Western blot and fluorescent-activated cell sorting to score Gag expressing cells. In a DNA prime/poxvirus boost approach with BALB/c mice, both recombinants elicited robust, broad and multifunctional antigen-specific T-cell responses to the HIV-1 immunogens expressed from the vectors. However, the use of NYVAC-B-C7L as booster significantly enhanced the magnitude of the T cell responses, and induced a more balanced cellular immune response to the HIV-1 antigens in comparison to that elicited in animals boosted with NYVAC-B. These findings demonstrate the possibility to enhance the immunogenicity of the highly attenuated NYVAC vector by the insertion of the host-range gene C7L and suggest the use of this modified vector as an improved vaccine candidate against HIV/AIDS.

The adaptive cytotoxic T lymphocyte (CTL)-mediated immune response is critical for clearance of many viral infections. These CTL recognize naturally processed short viral antigenic peptides bound to human leukocyte antigen (HLA) class I molecules on the surface of infected cells. This specific recognition allows the killing of virus-infected cells. The T cell immune T cell response to Chikungunya virus (CHIKV), a mosquito-borne Alphavirus of the Togaviridae family responsible for severe musculoskeletal disorders, has not been fully defined; nonetheless, the importance of HLA class I-restricted immune response in this virus has been hypothesized. By infection of HLA-A*0201-transgenic mice with a recombinant vaccinia virus that encodes the CHIKV structural polyprotein (rVACV-CHIKV), we identified the first human T cell epitopes from CHIKV. These three novel 6K transmembrane protein-derived epitopes are presented by the common HLA class I molecule, HLA-A*0201. One of these epitopes is processed and presented via a complex pathway that involves proteases from different subcellular locations. Specific chemical inhibitors blocked these events in rVACV-CHIKV-infected cells. Our data have implications not only for the identification of novel Alphavirus and Togaviridae antiviral CTL responses, but also for analyzing presentation of antigen from viruses of different families and orders that use host proteinases to generate their mature envelope proteins.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive memory loss and cognitive decline that has been associated with an accumulation in the brain of intracellular neurofibrillary tangles (NFTs) formed by hyperphosphorylated tau protein, and extracellular senile plaques formed by β-amyloid peptides. Currently, there is no cure for AD and after the failure of anti β-amyloid therapies, active and passive tau immunotherapeutic approaches have been developed in order to prevent, reduce or ideally reverse the disease. Vaccination is one of the most effective approaches to prevent diseases and poxviruses, particularly modified vaccinia virus Ankara (MVA), are one of the most promising viral vectors used as vaccines against several human diseases. Thus, we present here the generation and characterization of the first MVA vectors expressing human tau genes; the full-length 4R2N tau protein or a 3RC tau fragment containing 3 tubulin-binding motifs and the C-terminal region (termed MVA-Tau4R2N and MVA-Tau3RC, respectively). Both MVA-Tau recombinant viruses efficiently expressed the human tau 4R2N or 3RC proteins in cultured cells, being detected in the cytoplasm of infected cells and co-localized with tubulin. These MVA-Tau vaccines impacted the innate immune responses with a differential recruitment of innate immune cells to the peritoneal cavity of infected mice. However, no tau-specific T cell or humoral immune responses were detected in vaccinated mice. Immunization of transgenic P301S mice, a mouse model for tauopathies, with a DNA-Tau prime/MVA-Tau boost approach showed no significant differences in the hyperphosphorylation of tau, motor capacity and survival rate, when compared to non-vaccinated mice. These findings showed that a well-established and potent protocol of T and B cell activation based on DNA/MVA prime/boost regimens using DNA and MVA vectors expressing tau full-length 4R2N or 3RC proteins is not sufficient to trigger tau-specific T and B cell immune responses and to induce a protective effect against tauopathy in this P301S murine model. In the pursuit of AD vaccines, our results highlight the need for novel optimized tau immunogens and additional modes of presentation of tau protein to the immune system.

The highly attenuated poxvirus strain modified vaccinia virus Ankara (MVA) has reached maturity as a vector delivery system and as a vaccine candidate against a broad spectrum of diseases. This has been largely recognized from research on virus-host cell interactions and immunological studies in pre-clinical and clinical trials. This review addresses the studies of MVA vectors used in phase I/II clinical trials, with the aim to provide the main findings obtained on their behavior when tested against relevant human diseases and cancer and also highlights the strategies currently implemented to improve the MVA immunogenicity. The authors assess that MVA vectors are progressing as strong vaccine candidates either alone or when administered in combination with other vectors.