Poxvirus Vectors Research Articles

Preclinical assessment of an anti-HTLV-1 heterologous DNA/MVA vaccine protocol expressing a multiepitope HBZ protein

BackgroundHuman T-lymphotropic virus 1 (HTLV-1) is associated with the development of several pathologies and chronic infection in humans. The inefficiency of the available treatments and the challenge in developing a protective vaccine highlight the need to produce effective immunotherapeutic tools. The HTLV-1 basic leucine zipper (bZIP) factor (HBZ) plays an important role in the HTLV-1 persistence, conferring a survival advantage to infected cells by reducing the HTLV-1 proteins expression, allowing infected cells to evade immune surveillance, and enhancing cell proliferation leading to increased proviral load.MethodsWe have generated a recombinant Modified Virus Vaccinia Ankara (MVA-HBZ) and a plasmid DNA (pcDNA3.1(+)-HBZ) expressing a multiepitope protein based on peptides of HBZ to study the immunogenic potential of this viral-derived protein in BALB/c mice model. Mice were immunized in a prime-boost heterologous protocol and their splenocytes (T CD4+ and T CD8+) were immunophenotyped by flow cytometry and the humoral response was evaluated by ELISA using HBZ protein produced in prokaryotic vector as antigen.ResultsT CD4+ and T CD8+ lymphocytes cells stimulated by HBZ-peptides (HBZ42–50 and HBZ157–176) showed polyfunctional double positive responses for TNF-α/IFN-γ, and TNF-α/IL-2. Moreover, T CD8+ cells presented a tendency in the activation of effector memory cells producing granzyme B (CD44+High/CD62L−Low), and the activation of Cytotoxic T Lymphocytes (CTLs) and cytotoxic responses in immunized mice were inferred through the production of granzyme B by effector memory T cells and the expression of CD107a by CD8+ T cells. The overall data is consistent with a directive and effector recall response, which may be able to operate actively in the elimination of HTLV-1-infected cells and, consequently, in the reduction of the proviral load. Sera from immunized mice, differently from those of control animals, showed IgG-anti-HBZ production by ELISA.ConclusionsOur results highlight the potential of the HBZ multiepitope protein expressed from plasmid DNA and a poxviral vector as candidates for therapeutic vaccine.

The transcriptome of the parapoxvirus Orf virus reveals novel promoters for heterologous gene expression by poxvirus vectors

Orf virus (ORFV) has been used as a vaccine delivery vector for multiple animal species. Several strategies are being used to improve the immunogenicity and efficacy of ORFV vectors, including the use of poxviral promoter(s) with strong early and late activity capable of driving the expression of the heterologous genes for a prolonged time and eliciting a potent immune response. Here, we used RNA-sequencing (RNA-Seq) approach to analyze the transcriptome of ORFV during infection in primary ovine cells. Based on the transcriptional profile of individual ORFV genes, we identified ORFV promoters with strong early and late activity and have shown that they can be used to express heterologous genes in ORFV vectors. Our results show that the intergenic regulatory sequence containing core promoter sequences present upstream of ORF112 (p112) and ORF116 (p116) lead to markedly higher transgene expression than conventional poxviral promoters. Thus, these promoters are valuable alternatives to express transgenes in poxviral vectors.

HB-300, a novel arenavirus-based cancer immunotherapy in patients with metastatic castration-resistant prostate cancer.

TPS5108 Background: Prostate cancer is the most frequent type of malignant neoplasia in men. Immunotherapy targeting self-antigens in prostate cancer is evolving and requires overcoming multiple mechanisms that mediate immune tolerance. Sipuleucel-T, a cell-based therapy recognizing prostatic acid phosphatase (PAP) protein, is approved for treatment of patients with metastatic castration-resistant prostate cancer (mCRPC) and induced PAP-specific T cell responses correlating with overall survival (OS). Poxvirus vectors encoding prostate-specific antigen (PSA) increased PSA-specific T cell levels and were associated with OS benefit, although not confirmed in a Phase 3 study. Arenaviral vectors used in preclinical tumor models could overcome self-tolerance by inducing strong self-antigen specific CD8 T cell responses. In humans, arenaviral vectors encoding HPV16 E7E6 fusion protein (HB-200) induced unprecedented E6E7-specific CD8 T cell levels in patients with recurrent/metastatic HPV16+ cancers. Preliminary clinical activity and favorable safety and tolerability were observed in these heavily pre-treated, checkpoint inhibitor-resistant patients (ref: Fu et al. ASCO 2022; NCT04180215). It is therefore hypothesized that HB-300, a live-attenuated arenaviral vector therapy encoding the self-antigens PAP and PSA, will induce substantially stronger T cell responses in prostate cancer patients than previously tested active immunization approaches. Methods: This is a Phase 1/2 open-label clinical study of HB-300 in ~ 70 patients with advanced mCRPC. A 3+3 Dose Escalation will be followed by a dose-expansion to confirm the recommended Phase 2 dose (RP2D). HB-300 consists of HB-302 (Pichinde virus based) alternating with HB-301 (LCMV based) vectors, administered IV every 3 weeks (Q3W) for 5 doses then Q6W. The program’s entry into the clinic with a mini dose-escalation was supported by the submission of a data master file along with the HB-300 Investigational New Drug application, based on arenavirus vector platform data collected in the HB-200 program. Trial inclusion criteria include male participants age ≥ 18; documented mCRPC (adenocarcinoma without neuroendocrine or small cell features); receipt of at least 1 androgen deprivation therapy and no prior chemotherapy regimens (docetaxel in the castration-sensitive setting is allowed); ≥1 measurable lesion; ECOG PS 0-1. Exclusions are uncontrolled pain or symptoms; active auto-immune disease; immunosuppression; active central nervous system or liver metastasis; and active infection. Phase 1 primary objectives include safety/tolerability and determination of RP2D, secondary objectives include preliminary efficacy, and exploratory assessments include antigen-specific T cell response and circulating tumor cell analysis. Clinical trial# NCT05553639. Clinical trial information: NCT05553639 .

Abstract 697: Extracellular vesicles (EV) - mediators of therapeutic vaccination? In vivo and in vitro characterization of EVs generated after infection of human and murine cells with therapeutic poxviruses

Abstract Virus-based therapies show promise in inducing systemic anti-tumor immunity. Over the past years, modified poxviruses were developed to induce a sustained antitumoral immune response. Several studies confirm this hypothesis and report induction of T cell response against the antigen targeted by these vaccines. We previously reported MVA induced T-cell response against HPV oncoviral antigens E6 and E7 in HPV anogenital cancers (NCT03260023) and against private tumor neoantigen in ovarian and head and neck cancer. Despite these promising results the molecular and cellular mechanisms involved in vaccine response remains to be elucidated. In this work, we inquired whether EVs, well known for mediating intercellular communication, play a role in these therapeutic vaccination process. We first asked whether poxviral infection of human primary cells modifies their EV secretion properties. To this end, peripheral blood mononuclear cells (PBMCs) from healthy donors were infected with poxviral vectors, cell culture supernatants were harvested, and different isolation methods were compared for their ability to separate EVs from viral particles: ultracentrifugation, immunocapture, density gradient, velocity gradient, size exclusion chromatography and filtration. EVs size distribution and concentration were determined by nanoparticle tracking analysis (NTA) and the presence of infectious viral particles was titrated by plaque assays. Our data showed that 0.1µm filtration allowed the complete separation of EVs from infectious viral particles. All other methods showed co-isolation of EVs and infectious viral particles. Using this method, we demonstrate that infection of PBMCs with the poxviruses VACV, MVA and PCPV significantly increased the amount of secreted EVs. Next, we investigated the molecular content of EVs isolated from MVA-infected PBMCs by mass spectrometry. We observed differences in the abundance of at least 57 human proteins between EVs from infected and non-infected cells. In addition, several viral proteins, including virus-encoded payloads were detected in EVs from infected cells. We are now confirming those data with flow cytometry to detect virus-encoded payloads like tumor antigens, antigen-presenting molecules, and EV markers such as tetraspanins. The RNA content of EVs from infected and non-infected cells will be compared. Finally, EVs were isolated from murine dendritic cells infected with therapeutic pox vaccines. We are now evaluating the effects of local and systemic application of EVs in murine tumor models. Citation Format: Lucas Walther, Caroline Tosch, Jules Deforges, Christine Carapito, Magali Rompais, Marie-Christine Claudepierre, Nathalie Silvestre, Kaïdre Bendjama, Eric Quemeneur, Jacky Goetz, Vincent Hyenne, Karola Rittner. Extracellular vesicles (EV) - mediators of therapeutic vaccination? In vivo and in vitro characterization of EVs generated after infection of human and murine cells with therapeutic poxviruses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 697.

CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX.

Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assisted-recombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies.

Efficient production of protein complexes in mammalian cells using a poxvirus vector.

The production of full length, biologically active proteins in mammalian cells is critical for a wide variety of purposes ranging from structural studies to preparation of subunit vaccines. Prior research has shown that Modified vaccinia virus Ankara encoding the bacteriophage T7 RNA polymerase (MVA-T7) is particularly suitable for high level expression of proteins upon infection of mammalian cells. The expression system is safe for users and 10-50 mg of full length, biologically active proteins may be obtained in their native state, from a few litres of infected cell cultures. Here we report further improvements which allow an increase in the ease and speed of recombinant virus isolation, the scale-up of protein production and the simultaneous synthesis of several polypeptides belonging to a protein complex using a single virus vector. Isolation of MVA-T7 viruses encoding foreign proteins was simplified by combining positive selection for virus recombinants and negative selection against parental virus, a process which eliminated the need for tedious plaque purification. Scale-up of protein production was achieved by infecting a BHK 21 suspension cell line and inducing protein expression with previously infected cells instead of virus, thus saving time and effort in handling virus stocks. Protein complexes were produced from infected cells by concatenating the Tobacco Etch Virus (TEV) N1A protease sequence with each of the genes of the complex into a single ORF, each gene being separated from the other by twin TEV protease cleavage sites. We report the application of these methods to the production of a complex formed on the one hand between the HIV-1 integrase and its cell partner LEDGF and on the other between the HIV-1 VIF protein and its cell partners APOBEC3G, CBFβ, Elo B and Elo C. The strategies developed in this study should be valuable for the overexpression and subsequent purification of numerous protein complexes.

From Interferon to Checkpoint Inhibition Therapy-A Systematic Review of New Immune-Modulating Agents in Bacillus Calmette-Guérin (BCG) Refractory Non-Muscle-Invasive Bladder Cancer (NMIBC).

Simple SummaryIn Bacillus Calmette–Guérin (BCG) refractory non-muscle-invasive bladder cancer (NMIBC), radical cystectomy is the gold standard. The advent of immune checkpoint inhibitors (CPI) has permanently changed the therapy landscape of bladder cancer (BC). Immune-modulating (IM) therapies can offer a chance for bladder preservation in the treatment of NMIBC. This systematic review includes four full-text articles and four congress abstracts with IM agents in this setting. Durvalumab plus Oportuzumab monatox, Pembrolizumab, and Nadofaragene firadenovec (NF) show complete response (CR) rates of 41.6%, 40.6%, and 59.6% after 3 months. Instillations with oncolytic viruses such as NF and CG0070 show good efficacy without triggering significant immune-mediated systemic adverse events. Recombinant BCG VPM1002BC could prove to be valid as an alternative to BCG in the future. The recombinant pox-viral vector vaccine PANVAC™ is not convincing in combination with BCG. Interleukin mediating therapies, such as ALT-803, are currently being studied.Background: In Bacillus Calmette–Guérin (BCG) refractory non-muscle-invasive bladder cancer (NMIBC), radical cystectomy is the gold standard. The advent of immune checkpoint inhibitors (CPIs) has permanently changed the therapy landscape of bladder cancer (BC). This article presents a systematic review of immune-modulating (IM) therapies (CPIs and others) in BCG-refractory NMIBC. Methods: In total, 406 articles were identified through data bank research in PubMed/Medline, with data cutoff in October 2021. Four full-text articles and four additional congress abstracts were included in the review. Results: Durvalumab plus Oportuzumab monatox, Pembrolizumab, and Nadofaragene firadenovec (NF) show complete response (CR) rates of 41.6%, 40.6%, and 59.6% after 3 months, with a long-lasting effect, especially for NF (12-month CR rate of 30.5%). Instillations with oncolytic viruses such as NF and CG0070 show good efficacy without triggering significant immune-mediated systemic adverse events. Recombinant BCG VPM1002BC could prove to be valid as an alternative to BCG in the future. The recombinant pox-viral vector vaccine PANVAC™ is not convincing in combination with BCG. Interleukin mediating therapies, such as ALT-803, are currently being studied. Conclusion: CPIs and other IM agents now offer an increasing opportunity for bladder-preserving strategies. Studies on different substances are ongoing and will yield new findings.

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

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

Poxvirus-driven human diseases and emerging therapeutics.

Poxviridae have been successful pathogens throughout recorded history, infecting humans among a variety of other hosts. Although eradication of the notorious smallpox has been a globally successful healthcare phenomenon, the recent emergence of Monkeypox virus, also belonging to the Orthopoxvirus genus and causing human disease, albeit milder than smallpox, is a cause of significant public health concern. The ongoing outbreak of monkeypox, demonstrating human-human transmission, in previously nonendemic countries, calls for critical need into further research in the areas of viral biology, ecology, and epidemiology to better understand, prevent and treat human infections. In the wake of these recent events, it becomes important to revisit poxviral infections, their pathogenesis and ability to cause infection across multiple nonhuman hosts and leap to a human host. The poxviruses that cause human diseases include Monkeypox virus, Molluscum contagiosum virus, and Orf virus. In this review, we summarize the current understanding of various poxviruses causing human diseases, provide insights into their replication and pathogenicity, disease progression and symptoms, preventive and treatment options, and their importance in shaping modern medicine through application in gene therapy, oncolytic viral therapies for human cancers, or as poxvirus vectors for vaccines.

Sequence and vector shapes vaccine induced antibody effector functions in HIV vaccine trials

Despite the advent of long-acting anti-retroviral therapy able to control and prevent infection, a preventative vaccine remains a global priority for the elimination of HIV. The moderately protective RV144 vaccine trial suggested functional IgG1 and IgG3 antibodies were a potential correlate of protection, but the RV144-inspired HVTN702 validation trial failed to demonstrate efficacy despite inducing targeted levels of IgG1/IgG3. Alterations in inserts, and antigens, adjuvant, and regimen also resulted in vaccine induced target quantitative levels of the immune correlates, but drove qualitative changes to the humoral immune response, pointing to the urgent need to define the influence of vaccine strategies on shaping antibody quality, not just quantity. Thus, defining how distinct prime/boost approaches tune long-lived functional antibodies represents an important goal in vaccine development. Here, we compared vaccine responses in Phase I and II studies in humans utilizing various combinations of DNA/vector, vector/vector and DNA/protein HIV vaccines. We found that adenoviral vector immunization, compared to pox-viral vectors, resulted in the most potent IgG1 and IgG3 responses, linked to highly functional antibody activity, including assisting NK cell related functions. Minimal differences were observed in the durability of the functional humoral immune response across vaccine regimens, except for antibody dependent phagocytic function, which persisted for longer periods in the DNA/rAd5 and rAd35/rAd5 regimen, likely driven by higher IgG1 levels. Collectively, these findings suggest adenoviral vectors drive superior antibody quality and durability that could inform future clinical vaccine studies.Trial registration: ClinicalTrials.gov NCT00801697, NCT00961883, NCT02207920, NCT00125970, NCT02852005).

Assessment of an LSDV-Vectored Vaccine for Heterologous Prime-Boost Immunizations against HIV.

The modest protective effects of the RV144 HIV-1 vaccine trial have prompted the further exploration of improved poxvirus vector systems that can yield better immune responses and protection. In this study, a recombinant lumpy skin disease virus (LSDV) expressing HIV-1 CAP256.SU gp150 (Env) and a subtype C mosaic Gag was constructed (LSDVGC5) and compared to the equivalent recombinant modified vaccinia Ankara (MVAGC5). In vitro characterization confirmed that cells infected with recombinant LSDV produced Gag virus-like particles containing Env, and that Env expressed on the surface of the cells infected with LSDV was in a native-like conformation. This candidate HIV-1 vaccine (L) was tested in a rabbit model using different heterologous vaccination regimens, in combination with DNA (D) and MVA (M) vectors expressing the equivalent HIV-1 antigens. The four different vaccination regimens (DDMMLL, DDMLML, DDLMLM, and DDLLMM) all elicited high titers of binding and Tier 1A neutralizing antibodies (NAbs), and some regimens induced Tier 1B NAbs. Furthermore, two rabbits in the DDLMLM group developed low levels of autologous Tier 2 NAbs. The humoral immune responses elicited against HIV-1 Env by the recombinant LSDVGC5 were comparable to those induced by MVAGC5.

Distinct patterns of whole blood transcriptional responses are induced in mice following immunisation with adenoviral and poxviral vector vaccines encoding the same antigen

BackgroundViral vectors, including adenovirus (Ad) and modified vaccinia Ankara (MVA), have gained increasing attention as vaccine platforms in recent years due to their capacity to express antigens from a wide array of pathogens, their rapid induction of humoral and cellular protective immune responses, and their relatively low production costs. In particular, the chimpanzee Ad vector, ChAdOx1, has taken centre stage as a leading COVID-19 vaccine candidate. However, despite mounting data, both clinical and pre-clinical, demonstrating effective induction of adaptive immune responses, the innate immune signals that precede the protective responses that make these vectors attractive vaccine platforms remain poorly understood.ResultsIn this study, a mouse immunisation model was used to evaluate whole blood gene expression changes 24 h after either a single dose or heterologous prime-boost regimen of an Ad and/or MVA vaccine. We demonstrate through comparative analysis of Ad vectors encoding different antigens that a transgene product-specific gene signature can be discerned from the vector-induced transcriptional response. Expression of genes involved in TLR2 stimulation and γδ T cell and natural killer cell activation were induced after a single dose of Ad, while MVA led to greater expression of type I interferon genes. The order of prime-boost combinations was found to influence the magnitude of the gene expression changes, with MVA/Ad eliciting greater transcriptional perturbation than Ad/MVA. Contrasting the two regimens revealed significant enrichment of epigenetic regulation pathways and augmented expression of MHC class I and II molecules associated with MVA/Ad.ConclusionThese data demonstrate that the order in which vaccines from heterologous prime-boost regimens are administered leads to distinct transcriptional responses and may shape the immune response induced by such combinations. The characterisation of early vaccine-induce responses strengthens our understanding of viral vector vaccine mechanisms of action ahead of their characterisation in human clinical trials and are a valuable resource to inform the pre-clinical design of appropriate vaccine constructs for emerging infectious diseases.

Enhancement of the HIV-1-Specific Immune Response Induced by an mRNA Vaccine through Boosting with a Poxvirus MVA Vector Expressing the Same Antigen.

Development of a vaccine against HIV remains a major target goal in the field. The recent success of mRNA vaccines against the coronavirus SARS-CoV-2 is pointing out a new era of vaccine designs against pathogens. Here, we have generated two types of mRNA vaccine candidates against HIV-1; one based on unmodified vectors and the other on 1-methyl-3′-pseudouridylyl modified vectors expressing a T cell multiepitopic construct including protective conserved epitopes from HIV-1 Gag, Pol and Nef proteins (referred to as RNA-TMEP and RNA-TMEPmod, respectively) and defined their biological and immunological properties in cultured cells and in mice. In cultured cells, both mRNA vectors expressed the corresponding protein, with higher levels observed in the unmodified mRNA, leading to activated macrophages with differential induction of innate immune molecules. In mice, intranodal administration of the mRNAs induced the activation of specific T cell (CD4 and CD8) responses, and the levels were markedly enhanced after a booster immunization with the poxvirus vector MVA-TMEP expressing the same antigen. This immune activation was maintained even three months later. These findings revealed a potent combined immunization regimen able to enhance the HIV-1-specific immune responses induced by an mRNA vaccine that might be applicable to human vaccination programs with mRNA and MVA vectors.

Development of a multi-antigenic SARS-CoV-2 vaccine candidate using a synthetic poxvirus platform

Modified Vaccinia Ankara (MVA) is a highly attenuated poxvirus vector that is widely used to develop vaccines for infectious diseases and cancer. We demonstrate the construction of a vaccine platform based on a unique three-plasmid system to efficiently generate recombinant MVA vectors from chemically synthesized DNA. In response to the ongoing global pandemic caused by SARS coronavirus-2 (SARS-CoV-2), we use this vaccine platform to rapidly produce fully synthetic MVA (sMVA) vectors co-expressing SARS-CoV-2 spike and nucleocapsid antigens, two immunodominant antigens implicated in protective immunity. We show that mice immunized with these sMVA vectors develop robust SARS-CoV-2 antigen-specific humoral and cellular immune responses, including potent neutralizing antibodies. These results demonstrate the potential of a vaccine platform based on synthetic DNA to efficiently generate recombinant MVA vectors and to rapidly develop a multi-antigenic poxvirus-based SARS-CoV-2 vaccine candidate.

NF-κB as an Important Factor in Optimizing Poxvirus-Based Vaccines against Viral Infections.

Poxviruses are large dsDNA viruses that are regarded as good candidates for vaccine vectors. Because the members of the Poxviridae family encode numerous immunomodulatory proteins in their genomes, it is necessary to carry out certain modifications in poxviral candidates for vaccine vectors to improve the vaccine. Currently, several poxvirus-based vaccines targeted at viral infections are under development. One of the important aspects of the influence of poxviruses on the immune system is that they encode a large array of inhibitors of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which is the key element of both innate and adaptive immunity. Importantly, the NF-κB transcription factor induces the mechanisms associated with adaptive immunological memory involving the activation of effector and memory T cells upon vaccination. Since poxviruses encode various NF-κB inhibitor proteins, before the use of poxviral vaccine vectors, modifications that influence NF-κB activation and consequently affect the immunogenicity of the vaccine should be carried out. This review focuses on NF-κB as an essential factor in the optimization of poxviral vaccines against viral infections.

Deletion of Vaccinia Virus A40R Gene Improves the Immunogenicity of the HIV-1 Vaccine Candidate MVA-B.

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

MVA-B COMO CANDIDATO VACUNAL FRENTE AL VIH/SIDA: DE LA INVESTIGACIÓN BÁSICA A LOS ENSAYOS CLÍNICOS PROFILÁCTICO Y TERAPÉUTICO

The highly attenuated poxvirus strain modified vaccinia virus Ankara (MVA) has reached maturity as antigen delivery system and as a vaccine candidate against a broad spectrum of infectious diseases. This has been largely recognized from research on virus–host cell interactions, gene expression profiling, virus distribution and immunological studies in preclinical and clinical trials. This review includes our main contributions from the basic knowledge of the biology of the MVA vector, both in vitro and in vivo, in comparison with the attenuated NYVAC strain, to its evaluation as a vaccine candidate against HIV/AIDS in clinical trials. We will detail the generation and characterization of the recombinant poxvirus vector MVA expressing the HIV-1 Env, Gag, Pol and Nef antigens from clade B (referred as MVA-B) and review the preclinical data that supported the evaluation of MVA-B as the first in human HIV-1 prophylactic and therapeutic vaccine in Spain. In addition, we will assess the results of clinical trials and discuss the research projects we are currently working on considering the latest scientific advances in the HIV vaccine field.

Heterologous Combination of VSV-GP and NYVAC Vectors Expressing HIV-1 Trimeric gp145 Env as Vaccination Strategy to Induce Balanced B and T Cell Immune Responses.

The generation of a vaccine against HIV-1 able to induce durable protective immunity continues a major challenge. The modest efficacy (31.2%) of the phase III RV144 clinical trial provided the first demonstration that a prophylactic HIV/AIDS vaccine is achievable but emphasized the need for further refinements of vaccine candidates, formulations, and immunization regimens. Here, we analyzed in mice the immunogenicity profile elicited by different homologous and heterologous prime/boost combinations using the modified rhabdovirus VSV-GP combined with DNA or poxviral NYVAC vectors, all expressing trimeric membrane-bound Env (gp145) of HIV-1 96ZM651 clade C, with or without purified gp140 protein component. In cultured cells infected with recombinant VSV-GP or NYVAC viruses, gp145 epitopes at the plasma membrane were recognized by human HIV-1 broadly neutralizing antibodies (bNAbs). In immunized mice, the heterologous combination of VSV-GP and NYVAC recombinant vectors improved the induction of HIV-1 Env-specific humoral and cellular immune responses compared to homologous prime/boost protocols. Specifically, the combination of VSV-GP in the prime and NYVAC in the boost induced higher HIV-1 Env-specific T cell (CD4/CD8 T cells and T follicular helper -Tfh- cells) immune responses compared to the use of DNA or NYVAC vectors in the prime and VSV-GP in the boost. Such enhanced T cell responses correlated with an enhancement of the Env-specific germinal center (GC) B cell population and with a heavily biased Env-specific response toward the Th1-associated IgG2a and IgG3 subclasses, while the other groups showed a Th2-associated IgG1 bias. In summary, our T and B cell population data demonstrated that VSV-GP-based vectors could be taken into consideration as an optimized immunogenic HIV-1 vaccine candidate component against HIV-1 when used for priming in heterologous combinations with the poxvirus vector NYVAC as a boost.

An MVA Vector Expressing HIV-1 Envelope under the Control of a Potent Vaccinia Virus Promoter as a Promising Strategy in HIV/AIDS Vaccine Design.

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.

The Envelope-Based Fusion Antigen GP120C14K Forming Hexamer-Like Structures Triggers T Cell and Neutralizing Antibody Responses Against HIV-1.

There is an urgent need for the development of potent vaccination regimens that are able to induce specific T and B cell responses against human immunodeficiency virus type 1 (HIV-1). Here, we describe the generation and characterization of a fusion antigen comprised of the HIV-1 envelope GP120 glycoprotein from clade C (GP120C) fused at its C-terminus, with the modified vaccinia virus (VACV) 14K protein (A27L gene) (termed GP120C14K). The design is directed toward improving the immunogenicity of the GP120C protein through its oligomerization facilitated by the fused VACV 14K protein that results in hexamer-like structures. Two different immunogens were generated: a recombinant GP120C14K fusion protein (purified from a stable CHO-K1 cell line) and a recombinant modified vaccinia virus Ankara (MVA) poxvirus vector expressing the GP120C14K fusion protein (termed MVA-GP120C14K). The GP120C14K fusion protein is recognized by broadly neutralizing antibodies (bNAbs) against HIV-1. In a murine model, a heterologous prime/boost immunization regimen with MVA-GP120C14K prime followed by adjuvanted GP120C14K protein boost generated stronger and polyfunctional HIV-1 Env-specific CD8 T cell responses when compared with the delivery of the monomeric GP120C form. Furthermore, the immunization protocol MVA-GP120C14K/GP120C14K elicited higher HIV-1 Env-specific T follicular helper cells, germinal center B cells and antibody responses than monomeric GP120. In addition, a similar MVA-GP120C14K prime/GP120C14K protein boost regimen performed in rabbits triggered high HIV-1-Env-specific IgG binding antibody titers that were capable of neutralizing HIV-1 pseudoviruses. The extent of HIV-1 neutralization was comparable to that elicited by the current standard GP140 SOSIP trimers from clades B and C when immunized as MVA-SOSIP prime/SOSIP protein boost regimen. Overall, the novel fusion antigen and the corresponding immunization scheme provided in this report can therefore be considered as potential vaccine strategies against HIV-1.