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

Abstract Vaccines based on mRNA technology have revolutionized the field. In fact, lipid nanoparticles (LNP) formulated with mRNA are the preferential vaccine platform used in the fight against SARS-CoV-2 infection, with wider application against other diseases. The high demand and property right protection of the most potent cationic/ionizable lipids used for LNP formulation of COVID-19 mRNA vaccines have promoted the design of alternative nanocarriers for nucleic acid delivery. In this study we have evaluated the immunogenicity and efficacy of different rationally designed lipid and polymeric-based nanoparticle prototypes against SARS-CoV-2 infection. An mRNA coding for a trimeric soluble form of the receptor binding domain (RBD) of the spike (S) protein from SARS-CoV-2 was encapsulated using different components to form nanoemulsions (NE), nanocapsules (NC) and lipid nanoparticles (LNP). The toxicity and biological activity of these prototypes were evaluated in cultured cells after transfection and in mice following homologous prime/boost immunization. Our findings reveal good levels of RBD protein expression with most of the formulations. In C57BL/6 mice immunized intramuscularly with two doses of formulated RBD-mRNA, the modified lipid nanoparticle (mLNP) and the classical lipid nanoparticle (LNP-1) were the most effective delivery nanocarriers at inducing binding and neutralizing antibodies against SARS-CoV-2. Both prototypes fully protected susceptible K18-hACE2 transgenic mice from morbidity and mortality following a SARS-CoV-2 challenge. These results highlight that modulation of mRNAs immunogenicity can be achieved by using alternative nanocarriers and support further assessment of mLNP and LNP-1 prototypes as delivery vehicles for mRNA vaccines.

Background: The COVID-19 pandemic, caused by SARS-CoV-2, has highlighted the need for vaccines targeting both neutralizing antibodies (NAbs) and long-lasting cross-reactive T cells covering multiple viral proteins to provide broad and durable protection against emerging variants. Methods: To address this, here we developed two vaccine candidates, namely (i) DNA-CoV2-TMEP, expressing the multiepitopic CoV2-TMEP protein containing immunodominant and conserved T cell regions from SARS-CoV-2 structural proteins, and (ii) MVA-CoV2-B2AT, encoding a bi-cistronic multiepitopic construct that combines conserved B and T cell overlapping regions from SARS-CoV-2 structural proteins. Results: Both candidates were assessed in vitro and in vivo demonstrating their ability to induce robust immune responses. In C57BL/6 mice, DNA-CoV2-TMEP enhanced the recruitment of innate immune cells and stimulated SARS-CoV-2-specific polyfunctional T cells targeting multiple viral proteins. MVA-CoV2-B2AT elicited NAbs against various SARS-CoV-2 variants of concern (VoCs) and reduced viral replication and viral yields against the Beta variant in susceptible K18-hACE2 mice. The combination of MVA-CoV2-B2AT with a mutated ISG15 form as an adjuvant further increased the magnitude, breadth and polyfunctional profile of the response. Conclusion: These findings underscore the potential of these multiepitopic proteins when expressed from DNA or MVA vectors to provide protection against SARS-CoV-2 and its variants, supporting their further development as next-generation COVID-19 vaccines.

IntroductionThe generation of an HIV-1 vaccine able to induce long-lasting protective immunity remains a main challenge. Here, we aimed to modify next-generation soluble, prefusion-stabilized, close-to-native, glycan-engineered clade C gp140 envelope (Env) trimers (sC23v4 KIKO and ConCv5 KIKO) for optimal display on the cell surface following homologous or heterologous vector delivery.MethodsA combination of the following modifications scored best regarding the preservation of closed, native-like Env trimer conformation and antigenicity when using a panel of selected broadly neutralizing (bnAb) and non-neutralizing (nnAb) monoclonal antibodies for flow cytometry: i) replacing the natural cleavage site with a native flexible linker and introducing a single amino acid substitution to prevent CD4 binding (*), ii) fusing a heterologous VSV-G-derived transmembrane moiety to the gp140 C-terminus, and iii) deleting six residues proximal to the membrane.ResultsWhen delivering membrane-tethered sC23v4 KIKO* and ConCv5 KIKO* via DNA, VSV-GP, and NYVAC vectors, the two native-like Env trimers provide differential antigenicity profiles. Whereas such patterns were largely consistent among the different vectors for either Env trimer, the membrane-tethered ConCv5 KIKO* trimer adopted a more closed and native-like structure than sC23v4 KIKO*. In immunized mice, VSV-GP and NYVAC vectors expressing the membrane-tethered ConCv5 KIKO* administered in prime/boost combination were the most effective regimens for the priming of Env-specific CD4 T cells among all tested combinations. The subsequent booster administration of trimeric ConCv5 KIKO* Env protein preserved the T cell activation levels between groups. The evaluation of the HIV-1-specific humoral responses induced in the different immunization groups after protein boosts showed that the various prime/boost protocols elicited broad and potent antibody responses, preferentially of a Th1-associated IgG2a subclass, and that the obtained antibody levels remained high at the memory phase.DiscussionIn summary, we provide a feasible strategy to display multiple copies of native-like Env trimers on the cell surface, which translates into efficient priming of sustained CD4+ T cell responses after vector delivery as well as broad, potent, and sustained antibody responses following booster immunizations with the homologous, prefusion-stabilized, close-to-native ConCv5 KIKO* gp140 Env trimer.

IntroductionWhile there has been considerable progress in the development of vaccines against SARS-CoV-2, largely based on the S (spike) protein of the virus, less progress has been made with vaccines delivering different viral antigens with cross-reactive potential.MethodsIn an effort to develop an immunogen with the capacity to induce broad antigen presentation, we have designed a multi-patch synthetic candidate containing dominant and persistent B cell epitopes from conserved regions of SARS-CoV-2 structural proteins associated with long-term immunity, termed CoV2-BMEP. Here we describe the characterization, immunogenicity and efficacy of CoV2-BMEP using two delivery platforms: nucleic acid DNA and attenuated modified vaccinia virus Ankara (MVA).ResultsIn cultured cells, both vectors produced a main protein of about 37 kDa as well as heterogeneous proteins with size ranging between 25-37 kDa. In C57BL/6 mice, both homologous and heterologous prime/boost combination of vectors induced the activation of SARS-CoV-2-specific CD4 and CD8 T cell responses, with a more balanced CD8+ T cell response detected in lungs. The homologous MVA/MVA immunization regimen elicited the highest specific CD8+ T cell responses in spleen and detectable binding antibodies (bAbs) to S and N antigens of SARS-CoV-2. In SARS-CoV-2 susceptible k18-hACE2 Tg mice, two doses of MVA-CoV2-BMEP elicited S- and N-specific bAbs as well as cross-neutralizing antibodies against different variants of concern (VoC). After SARS-CoV-2 challenge, all animals in the control unvaccinated group succumbed to the infection while vaccinated animals with high titers of neutralizing antibodies were fully protected against mortality, correlating with a reduction of virus infection in the lungs and inhibition of the cytokine storm.DiscussionThese findings revealed a novel immunogen with the capacity to control SARS-CoV-2 infection, using a broader antigen presentation mechanism than the approved vaccines based solely on the S antigen.

The Interferon Stimulated Gene 15 (ISG15), a unique Ubiquitin-like (Ubl) modifier exclusive to vertebrates, plays a crucial role in the immune system. Primarily induced by interferon (IFN) type I, ISG15 functions through diverse mechanisms: (i) covalent protein modification (ISGylation); (ii) non-covalent intracellular action; and (iii) exerting extracellular cytokine activity. These various roles highlight its versatility in influencing numerous cellular pathways, encompassing DNA damage response, autophagy, antiviral response, and cancer-related processes, among others. The well-established antiviral effects of ISGylation contrast with its intriguing dual role in cancer, exhibiting both suppressive and promoting effects depending on the tumour type. The multifaceted functions of ISG15 extend beyond intracellular processes to extracellular cytokine signalling, influencing immune response, chemotaxis, and anti-tumour effects. Moreover, ISG15 emerges as a promising adjuvant in vaccine development, enhancing immune responses against viral antigens and demonstrating efficacy in cancer models. As a therapeutic target in cancer treatment, ISG15 exhibits a double-edged nature, promoting or suppressing oncogenesis depending on the tumour context. This review aims to contribute to future studies exploring the role of ISG15 in immune modulation and cancer therapy, potentially paving the way for the development of novel therapeutic interventions, vaccine development, and precision medicine.

The COVID-19 pandemic has brought significant changes and advances in the field of vaccination, including the implementation and widespread use of encapsidated mRNA vaccines in general healthcare practice. Here, we present two new mRNAs expressing antigenic parts of the SARS-CoV-2 spike protein and provide data supporting their functionality. The first mRNA, called RBD-mRNA, encodes a trimeric form of the virus spike protein receptor binding domain (RBD). The other mRNA, termed T-mRNA, codes for the relevant HLA I and II spike epitopes. The two mRNAs (COVARNA mRNAs) were designed to be used for delivery to cells in combination, with the RBD-mRNA being the primary source of antigen and the T-mRNA working as an enhancer of immunogenicity by supporting CD4 and CD8 T-cell activation. This innovative approach substantially differs from other available mRNA vaccines, which are largely directed to antibody production by the entire spike protein. In this study, we first show that both mRNAs are functionally transfected into human antigen-presenting cells (APCs). We obtained peripheral blood mononuclear cell (PBMC) samples from three groups of voluntary donors differing in their immunity against SARS-CoV-2: non-infected (naïve), infected-recovered (convalescent), and vaccinated. Using an established method of co-culturing autologous human dendritic cells (hDCs) with T-cells, we detected proliferation and cytokine secretion, thus demonstrating the ability of the COVARNA mRNAs to activate T-cells in an antigen-specific way. Interestingly, important differences in the intensity of the response between the infected-recovered (convalescent) and vaccinated donors were observed, with the levels of T-cell proliferation and cytokine secretion (IFNγ, IL-2R, and IL-13) being higher in the vaccinated group. In summary, our data support the further study of these mRNAs as a combined approach for future use as a vaccine.

The human immunodeficiency virus (HIV), responsible of the Acquired Immune Deficiency Syndrome (AIDS), continues to be a major global public health issue with any cure or vaccine available. The Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that is induced by interferons and plays a critical role in the immune response. ISG15 is a modifier protein that covalently binds to its targets via a reversible bond, a process known as ISGylation, which is the best-characterized activity of this protein to date. However, ISG15 can also interact with intracellular proteins via non-covalent binding or act as a cytokine in the extracellular space after secretion. In previous studies we proved the adjuvant effect of ISG15 when delivered by a DNA-vector in heterologous prime-boost combination with a Modified Vaccinia virus Ankara (MVA)-based recombinant virus expressing HIV-1 antigens Env/Gag-Pol-Nef (MVA-B). Here we extended these results evaluating the adjuvant effect of ISG15 when expressed by an MVA vector. For this, we generated and characterized two novel MVA recombinants expressing different forms of ISG15, the wild-type ISG15GG (able to perform ISGylation) or the mutated ISG15AA (unable to perform ISGylation). In mice immunized with the heterologous DNA prime/MVA boost regimen, the expression of the mutant ISG15AA from MVA-Δ3-ISG15AA vector in combination with MVA-B induced an increase in the magnitude and quality of HIV-1-specific CD8 T cells as well as in the levels of IFN-I released, providing a better immunostimulatory activity than the wild-type ISG15GG. Our results confirm the importance of ISG15 as an immune adjuvant in the vaccine field and highlights its role as a potential relevant component in HIV-1 immunization protocols.

The development of new strategies to achieve a functional cure for HIV remains a priority. We tested a novel HIV therapeutic vaccine using unmodified mRNA (TMEP-B) and mRNA modified by 1-methyl-3′-pseudouridylyl (TMEP-Bmod) expressing both a multiepitopic sequences from Gag, Pol, and Nef proteins, including different CD4 and CD8 T-cell epitopes functionally associated with HIV control in transfected monocyte-derived dendritic cells (MDDCs) obtained from HIV infected patients. In vitro assays were used to test the mRNAs alone and in combination with immunomodulator agents, such as the TLR-7 agonist Vesatolimod and the PD-1 antagonist Nivolumab to try to improve HIV-specific cellular immune responses. Combining the mRNAs with the immunomodulators enhanced HIV-specific T-cell responses, together with the secretion of IFNγ, IP10, MIP-1α, and MIP-1β, which are fundamental mediators of viral control. Our data suggest that the mRNA vaccine prototypes TMEP-B and TMEP-Bmod, when combined with Vesatolimod and/or Nivolumab, could achieve functional cure for patients with HIV.

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.

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.

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants in different continents is causing a major concern in human global health. These variants have in common a higher transmissibility, becoming dominant within populations in a short time, and an accumulation of a high number of mutations in the spike (S) protein, especially within the amino terminal domain (NTD) and the receptor binding domain (RBD). These mutations have direct implications on virus infection rates through higher affinity of S RBD for the cellular angiotensin-converting enzyme-2 (ACE-2) receptor. There are also signs of enhanced virulence, re-infection frequency, and increased resistance to the action of monoclonal and polyclonal antibodies from convalescence sera and in vaccinated individuals in regions where the variants spread dominantly. In this review, we describe the different SARS-CoV-2 variants that have thus far been identified in various parts of the world with mutational changes and biological properties as well as their impact in medical countermeasures and human health.

[This corrects the article DOI: 10.1371/journal.pone.0186602.].

A human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) vaccine able to induce long-lasting immunity remains a major challenge. We previously designed a T cell multiepitopic immunogen including protective conserved epitopes from HIV-1 Gag, Pol and Nef proteins (TMEP-B), that induced potent HIV-1-specific CD8 T cells when vectored by DNA and combined with the vaccine candidate modified vaccinia virus Ankara (MVA)-B. Here, we described the vectorization of TMEP-B in MVA (MVA-TMEP) and evaluated the T cell immunogenicity profile elicited in mice when administered in homologous (MVA/MVA) or heterologous (DNA/MVA) prime/boost vector regimens or using homologous or heterologous inserts. The heterologous vector regimen was superior to the homologous protocol in inducing T cell responses. DNA-TMEP-primed animals boosted with MVA-TMEP or MVA-B exhibited the highest magnitudes of HIV-1-specific CD8, CD4 and T follicular helper (Tfh) cells, with MVA-TMEP significantly expanding Gag-specific CD8 T cell responses. In the homologous vector regimen, all groups exhibited similar HIV-1-specific CD8 and CD4 T cell responses, but both MVA-B/MVA-B and MVA-TMEP/MVA-TMEP combinations elicited higher Gag-Pol-Nef (GPN)-specific CD8 T cell responses compared to MVA-TMEP/MVA-B. Our results revealed an enhanced induction of HIV-1-specific T cell responses by TMEP-B when vectored in both DNA and MVA, and supported their use in combined prime/boost strategies for HIV-1 prevention and/or therapy.

We have previously shown that an HIV vaccine regimen including three doses of HIV-modified vaccinia virus Ankara vector expressing HIV-1 antigens from clade B (MVA-B) was safe and elicited moderate and durable (1 year) T-cell and antibody responses in 75% and 95% of HIV-negative volunteers (n = 24), respectively (RISVAC02 study). Here, we describe the long-term durability of vaccine-induced responses and the safety and immunogenicity of an additional MVA-B boost.13 volunteers from the RISVAC02 trial were recruited to receive a fourth dose of MVA-B 4 years after the last immunization. End-points were safety, cellular and humoral immune responses to HIV-1 and vector antigens assessed by ELISPOT, intracellular cytokine staining (ICS) and ELISA performed before and 2, 4 and 12 weeks after receiving the boost.Volunteers reported 64 adverse events (AEs), although none was a vaccine-related serious AE. After 4 years from the 1st dose of the vaccine, only 2 volunteers maintained low HIV-specific T-cell responses. After the late MVA-B boost, a modest increase in IFN-γ T-cell responses, mainly directed against Env, was detected by ELISPOT in 5/13 (38%) volunteers. ICS confirmed similar results with 45% of volunteers showing that CD4+ T-cell responses were mainly directed against Env, whereas CD8+ T cell-responses were similarly distributed against Env, Gag and GPN. In terms of antibody responses, 23.1% of the vaccinees had detectable Env-specific binding antibodies 4 years after the last MVA-B immunization with a mean titer of 96.5. The late MVA-B boost significantly improved both the response rate (92.3%) and the magnitude of the systemic binding antibodies to gp120 (mean titer of 11460). HIV-1 neutralizing antibodies were also enhanced and detected in 77% of volunteers. Moreover, MVA vector-specific T cell and antibody responses were boosted in 80% and 100% of volunteers respectively.One boost of MVA-B four years after receiving 3 doses of the same vaccine was safe, induced moderate increases in HIV-specific T cell responses in 38% of volunteers but significantly boosted the binding and neutralizing antibody responses to HIV-1 and to the MVA vector.ClinicalTrials.gov NCT01923610.

The development of an effective Human Immunodeficiency Virus (HIV) vaccine that is able to stimulate both the humoral and cellular HIV-1-specific immune responses remains a major priority challenge. In this study, we described the generation and preclinical evaluation of single and double modified vaccinia virus Ankara (MVA)-based candidates expressing the HIV-1 clade C membrane-bound gp145(ZM96) trimeric protein and/or the Gag(ZM96)-Pol-Nef(CN54) (GPN) polyprotein that was processed to form Gag-induced virus-like particles (VLPs). In vitro characterization of MVA recombinants revealed the stable integration of HIV-1 genes without affecting its replication capacity. In cells that were infected with Env-expressing viruses, the gp145 protein was inserted into the plasma membrane exposing critical epitopes that were recognized by broadly neutralizing antibodies (bNAbs), whereas Gag-induced VLPs were released from cells that were infected with GPN-expressing viruses. VLP particles as well as purified MVA virions contain Env and Gag visualized by immunoelectron microscopy and western-blot of fractions that were obtained after detergent treatments of purified virus particles. In BALB/c mice, homologous MVA-gp145-GPN prime/boost regimen induced broad and polyfunctional Env- and Gag-specific CD4 T cells and antigen-specific T follicular helper (Tfh) and Germinal Center (GC) B cells, which correlated with robust HIV-1-specific humoral responses. Overall, these results support the consideration of MVA-gp145-GPN vector as a potential vaccine candidate against HIV-1.

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.

Previous studies suggested that poxvirus-based vaccines might be instrumental in the therapeutic HIV field. A phase I clinical trial was conducted in HIV-1-infected patients on highly active antiretroviral therapy (HAART), with CD4 T cell counts above 450 cells/mm3 and undetectable viremia. Thirty participants were randomized (2:1) to receive either 3 intramuscular injections of MVA-B vaccine (coding for clade B HIV-1 Env, Gag, Pol and Nef antigens) or placebo, followed by interruption of HAART.The magnitude, breadth, quality and phenotype of the HIV-1-specific T cell response were assayed by intracellular cytokine staining (ICS) in 22 volunteers pre- and post-vaccination.MVA-B vaccine induced newly detected HIV-1-specific CD4 T cell responses and expanded pre-existing responses (mostly against Gag, Pol and Nef antigens) that were high in magnitude, broadly directed and showed an enhanced polyfunctionality with a T effector memory (TEM) phenotype, while maintaining the magnitude and quality of the pre-existing HIV-1-specific CD8 T cell responses. In addition, vaccination also triggered preferential CD8+ T cell polyfunctional responses to the MVA vector antigens that increase in magnitude after two and three booster doses.MVA-B vaccination represents a feasible strategy to improve T cell responses in individuals with pre-existing HIV-1-specific immunity.ClinicalTrials.gov NCT01571466.

An HIV-1 vaccine continues to be a major target to halt the AIDS pandemic. The limited efficacy of the RV144 phase III clinical trial with the canarypox virus-based vector ALVAC and a gp120 protein component led to the conclusion that improved immune responses to HIV antigens are needed for a more effective vaccine. In non-human primates, the New York vaccinia virus (NYVAC) poxvirus vector has a broader immunogenicity profile than ALVAC and has been tested in clinical trials. We therefore analysed the HIV immune advantage of NYVAC after removing viral genes that act on several signalling pathways (Toll-like receptors—TLR—interferon, cytokines/chemokines), as well as genes of unknown immune function. We generated a series of NYVAC deletion mutants and studied immune behaviour (T and B cell) to HIV antigens and to the NYVAC vector in mice. Our results showed that combined deletion of selected vaccinia virus (VACV) genes is a valuable strategy for improving the immunogenicity of NYVAC-based vaccine candidates. These immune responses were differentially modulated, positive or negative, depending on the combination of gene deletions. The deletions also led to enhanced antigen- or vector-specific cellular and humoral responses. These findings will facilitate the development of optimal NYVAC-based vaccines for HIV and other diseases.

Viruses have developed strategies to counteract signalling through Toll-like receptors (TLRs) that are involved in the detection of viruses and induction of proinflammatory cytokines and IFNs. Vaccinia virus (VACV) encodes A46 protein which disrupts TLR signalling by interfering with TLR: adaptor interactions. Since the innate immune response to viruses is critical to induce protective immunity, we studied whether deletion of A46R gene in a NYVAC vector expressing HIV-1 Env, Gag, Pol and Nef antigens (NYVAC-C) improves immune responses against HIV-1 antigens. This question was examined in human macrophages and in mice infected with a single A46R deletion mutant of the vaccine candidate NYVAC-C (NYVAC-C-ΔA46R). The viral gene A46R is not required for virus replication in primary chicken embryo fibroblast (CEF) cells and its deletion in NYVAC-C markedly increases TNF, IL-6 and IL-8 secretion by human macrophages. Analysis of the immune responses elicited in BALB/c mice after DNA prime/NYVAC boost immunization shows that deletion of A46R improves the magnitude of the HIV-1-specific CD4 and CD8 T cell immune responses during adaptive and memory phases, maintains the functional profile observed with the parental NYVAC-C and enhances anti-gp120 humoral response during the memory phase. These findings establish the immunological role of VACV A46R on innate immune responses of macrophages in vitro and antigen-specific T and B cell immune responses in vivo and suggest that deletion of viral inhibitors of TLR signalling is a useful approach for the improvement of poxvirus-based vaccine candidates.

Vaccinia virus (VACV) encodes an anti-apoptotic Bcl-2-like protein F1 that acts as an inhibitor of caspase-9 and of the Bak/Bax checkpoint but the role of this gene in immune responses is not known. Because dendritic cells that have phagocytosed apoptotic infected cells cross-present viral antigens to cytotoxic T cells inducing an antigen-specific immunity, we hypothesized that deletion of the viral anti-apoptotic F1L gene might have a profound effect on the capacity of poxvirus vectors to activate specific immune responses to virus-expressed recombinant antigens. This has been tested in a mouse model with an F1L deletion mutant of the HIV/AIDS vaccine candidate MVA-C that expresses Env and Gag-Pol-Nef antigens (MVA-C-ΔF1L). The viral gene F1L is not required for virus replication in cultured cells and its deletion in MVA-C induces extensive apoptosis and expression of immunomodulatory genes in infected cells. Analysis of the immune responses induced in BALB/c mice after DNA prime/MVA boost revealed that, in comparison with parental MVA-C, the mutant MVA-C-ΔF1L improves the magnitude of the HIV-1-specific CD8 T cell adaptive immune responses and impacts on the CD8 T cell memory phase by enhancing the magnitude of the response, reducing the contraction phase and changing the memory differentiation pattern. These findings reveal the immunomodulatory role of F1L and that the loss of this gene is a valid strategy for the optimization of MVA as vaccine vector.

Based on the partial efficacy of the HIV/AIDS Thai trial (RV144) with a canarypox vector prime and protein boost, attenuated poxvirus recombinants expressing HIV-1 antigens are increasingly sought as vaccine candidates against HIV/AIDS. Here we describe using systems analysis the biological and immunological characteristics of the attenuated vaccinia virus Ankara strain expressing the HIV-1 antigens Env/Gag-Pol-Nef of HIV-1 of clade C (referred as MVA-C). MVA-C infection of human monocyte derived dendritic cells (moDCs) induced the expression of HIV-1 antigens at high levels from 2 to 8 hpi and triggered moDCs maturation as revealed by enhanced expression of HLA-DR, CD86, CD40, HLA-A2, and CD80 molecules. Infection ex vivo of purified mDC and pDC with MVA-C induced the expression of immunoregulatory pathways associated with antiviral responses, antigen presentation, T cell and B cell responses. Similarly, human whole blood or primary macrophages infected with MVA-C express high levels of proinflammatory cytokines and chemokines involved with T cell activation. The vector MVA-C has the ability to cross-present antigens to HIV-specific CD8 T cells in vitro and to increase CD8 T cell proliferation in a dose-dependent manner. The immunogenic profiling in mice after DNA-C prime/MVA-C boost combination revealed activation of HIV-1-specific CD4 and CD8 T cell memory responses that are polyfunctional and with effector memory phenotype. Env-specific IgG binding antibodies were also produced in animals receiving DNA-C prime/MVA-C boost. Our systems analysis of profiling immune response to MVA-C infection highlights the potential benefit of MVA-C as vaccine candidate against HIV/AIDS for clade C, the prevalent subtype virus in the most affected areas of the world.

Heterologous vaccination based on priming with a plasmid DNA vector and boosting with an attenuated vaccinia virus MVA recombinant, with both vectors expressing the Leishmania infantum LACK antigen (DNA-LACK and MVA-LACK), has shown efficacy conferring protection in murine and canine models against cutaneus and visceral leishmaniasis, but the immune parameters of protection remain ill defined. Here we performed by flow cytometry an in depth analysis of the T cell populations induced in BALB/c mice during the vaccination protocol DNA-LACK/MVA-LACK, as well as after challenge with L. major parasites. In the adaptive response, there is a polyfunctional CD4(+) and CD8(+) T cell activation against LACK antigen. At the memory phase the heterologous vaccination induces high quality LACK-specific long-term CD4(+) and CD8(+) effector memory cells. After parasite challenge, there is a moderate boosting of LACK-specific CD4(+) and CD8(+) T cells. Anti-vector responses were largely CD8(+)-mediated. The immune parameters induced against LACK and triggered by the combined vaccination DNA/MVA protocol, like polyfunctionality of CD4(+) and CD8(+) T cells with an effector phenotype, could be relevant in protection against leishmaniasis.

Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-α, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IP-10, MIG, and IFN-α). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8(+) T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8(+) T cell response including proliferation, secretion of IFN-γ, IL-2, TNF-α, MIP-1β, MIP-1α, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4(+) T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.