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Introduction: The 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., Methods: A 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., Results: When 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., Discussion: In 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Perdiguero, Hauser, Gómez, Peterhoff, Sideris, Sorzano, Wilmschen, Schaber, Stengel, Asbach, Ding, Von Laer, Levy, Pantaleo, Kimpel, Esteban and Wagner.)

To control HIV infection there is a need for vaccines to induce broad, potent and long-term B and T cell immune responses. With the objective to accelerate and maintain the induction of substantial levels of HIV-1 Env-specific antibodies and, at the same time, to enhance balanced CD4 and CD8 T cell responses, we evaluated the effect of concurrent administration of MF59-adjuvanted Env protein together with DNA or NYVAC vectors at priming to establish if early administration of Env leads to early induction of antibody responses. The primary goal was to assess the immunogenicity endpoint at week 26. Secondary endpoints were (i) to determine the quality of responses with regard to RV144 correlates of protection and (ii) to explore a potential impact of two late boosts. In this study, five different prime/boost vaccination regimens were tested in rhesus macaques. Animals received priming immunizations with either NYVAC or DNA alone or in combination with Env protein, followed by NYVAC + protein or DNA + protein boosts. All regimens induced broad, polyfunctional and well-balanced CD4 and CD8 T cell responses, with DNA-primed regimens eliciting higher response rates and magnitudes than NYVAC-primed regimens. Very high plasma binding IgG titers including V1/V2 specific antibodies, modest antibody-dependent cellular cytotoxicity (ADCC) and moderate neutralization activity were observed. Of note, early administration of the MF59-adjuvanted Env protein in parallel with DNA priming leads to more rapid elicitation of humoral responses, without negatively affecting the cellular responses, while responses were rapidly boosted after repeated immunizations, indicating the induction of a robust memory response. In conclusion, our findings support the use of the Env protein component during priming in the context of an heterologous immunization regimen with a DNA and/or NYVAC vector as an optimized immunization protocol against HIV infection., Competing Interests: Author SB was employed by company Novartis Vaccines. Author DW and AC were employed by company ABL Inc. MK is an employee of the GSK group of companies and holds shares in GSK. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Perdiguero, Asbach, Gómez, Köstler, Barnett, Koutsoukos, Weiss, Cristillo, Foulds, Roederer, Montefiori, Yates, Ferrari, Shen, Sawant, Tomaras, Sato, Fulp, Gottardo, Ding, Heeney, Pantaleo, Esteban and Wagner.)

Induction of the endogenous innate immune system by interferon (IFN) triggers the expression of many proteins that serve like alarm bells in the body, activating an immune response. After a viral infection, one of the genes activated by IFN induction is the IFN-stimulated gene 15 ( ISG15 ), which encodes a ubiquitin-like protein that undergoes a reversible posttranslational modification (ISGylation). ISG15 protein can also act unconjugated, intracellularly and secreted, acting as a cytokine. Although ISG15 has an essential role in host defense responses to microbial infection, its role as an immunomodulator in the vaccine field remains to be defined. In this investigation, we showed that ISG15 exerts an immunomodulatory role in human immunodeficiency virus (HIV) vaccines. In mice, after priming with a DNA-ISG15 vector mixed with a DNA expressing HIV-1 gp120 (DNA-gp120), followed by a booster with a modified vaccinia virus Ankara (MVA) vector expressing HIV-1 antigens, both wild-type ISG15-conjugated (ISG15-wt) and mutant unconjugated (ISG15-mut) proteins act as immune adjuvants by increasing the magnitude and quality of HIV-1-specific CD8 T cells, with ISG15-wt providing better immunostimulatory activity than ISG15-mut. The HIV-1 Env-specific CD8 T cell responses showed a predominant T effector memory (TEM) phenotype in all groups. Moreover, the amount of DNA-gp120 used to immunize mice could be reduced 5-fold after mixing with DNA-ISG15 without affecting the potency and the quality of the HIV-1 Env-specific immune responses. Our study clearly highlights the potential use of the IFN-induced ISG15 protein as immune adjuvant to enhance immune responses to HIV antigens, suggesting that this molecule might be exploitable for prophylactic and therapeutic vaccine approaches against pathogens. IMPORTANCE Our study described the potential role of ISG15 as an immunomodulatory molecule in the optimization of HIV/AIDS vaccine candidates. Using a DNA prime-MVA boost immunization protocol, our results indicated an increase in the potency and the quality of the HIV-1 Env-specific CD8 T cell response. These results highlight the adjuvant potency of ISG15 to elicit improved viral antigen presentation to the immune system, resulting in an enhanced HIV-1 vaccine immune response. The DNA-ISG15 vector could find applicability in the vaccine field in combination with other nucleic acid-based vector vaccines., (Copyright © 2020 American Society for Microbiology.)

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., (Copyright © 2019 Perdiguero, Gómez, García-Arriaza, Sánchez-Corzo, Sorzano, Wilmschen, von Laer, Asbach, Schmalzl, Peterhoff, Ding, Wagner, Kimpel, Levy, Pantaleo and Esteban.)

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., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors declare no conflict of interest.

Background: 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., Methods: 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., Results: 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., Conclusions: 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., Trial Registration: ClinicalTrials.gov NCT01923610.

Trial Design: 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., Methods: 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., Results: 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., Conclusion: MVA-B vaccination represents a feasible strategy to improve T cell responses in individuals with pre-existing HIV-1-specific immunity., Trial Registration: ClinicalTrials.gov NCT01571466.

Unlabelled: The generation of vaccines against HIV/AIDS able to induce long-lasting protective immunity remains a major goal in the HIV field. The modest efficacy (31.2%) against HIV infection observed in the RV144 phase III clinical trial highlighted the need for further improvement of HIV vaccine candidates, formulation, and vaccine regimen. In this study, we have generated two novel NYVAC vectors, expressing HIV-1 clade C gp140(ZM96) (NYVAC-gp140) or Gag(ZM96)-Pol-Nef(CN54) (NYVAC-Gag-Pol-Nef), and defined their virological and immunological characteristics in cultured cells and in mice. The insertion of HIV genes does not affect the replication capacity of NYVAC recombinants in primary chicken embryo fibroblast cells, HIV sequences remain stable after multiple passages, and HIV antigens are correctly expressed and released from cells, with Env as a trimer (NYVAC-gp140), while in NYVAC-Gag-Pol-Nef-infected cells Gag-induced virus-like particles (VLPs) are abundant. Electron microscopy revealed that VLPs accumulated with time at the cell surface, with no interference with NYVAC morphogenesis. Both vectors trigger specific innate responses in human cells and show an attenuation profile in immunocompromised adult BALB/c and newborn CD1 mice after intracranial inoculation. Analysis of the immune responses elicited in mice after homologous NYVAC prime/NYVAC boost immunization shows that recombinant viruses induced polyfunctional Env-specific CD4 or Gag-specific CD8 T cell responses. Antibody responses against gp140 and p17/p24 were elicited. Our findings showed important insights into virus-host cell interactions of NYVAC vectors expressing HIV antigens, with the activation of specific immune parameters which will help to unravel potential correlates of protection against HIV in human clinical trials with these vectors., Importance: We have generated two novel NYVAC-based HIV vaccine candidates expressing HIV-1 clade C trimeric soluble gp140 (ZM96) and Gag(ZM96)-Pol-Nef(CN54) as VLPs. These vectors are stable and express high levels of both HIV-1 antigens. Gag-induced VLPs do not interfere with NYVAC morphogenesis, are highly attenuated in immunocompromised and newborn mice after intracranial inoculation, trigger specific innate immune responses in human cells, and activate T (Env-specific CD4 and Gag-specific CD8) and B cell immune responses to the HIV antigens, leading to high antibody titers against gp140. For these reasons, these vectors can be considered vaccine candidates against HIV/AIDS and currently are being tested in macaques and humans., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Objectives: The safety, immunogenicity, impact on the latent reservoir and rebound of viral load after therapeutic HIV-1 vaccination with recombinant modified vaccinia Ankara-based (MVA-B) HIV-1 vaccine expressing monomeric gp120 and the fused Gag-Pol-Nef polyprotein of clade B with or without a drug to reactivate latent HIV-1 (disulfiram) were assessed., Methods: HIV-1-infected patients were randomized to receive three injections of MVA-B (n = 20) or placebo (n = 10). Twelve patients (eight who received vaccine and four who were given placebo) received a fourth dose of MVA-B followed by 3 months of disulfiram. Combined ART (cART) was discontinued 8 weeks after the last dose of MVA-B. Clinical Trials.gov identifier: NCT01571466., Results: MVA-B was safe and well tolerated. A minor, but significant, increase in the T cell responses targeting vaccine inserts of Gag was observed [a median of 290, 403 and 435 spot-forming-cells/10(6) PBMCs at baseline, after two vaccinations and after three vaccinations, respectively; P = 0.02 and P = 0.04]. After interruption of cART, a modest delay in the rebound of the plasma viral load in participants receiving vaccine but not disulfiram was observed compared with placebo recipients (P = 0.01). The dynamics of the viral load rebound did not change in patients receiving MVA-B/disulfiram. No changes in the proviral reservoir were observed after disulfiram treatment., Conclusions: MVA-B vaccination was a safe strategy to increase Gag-specific T cell responses in chronically HIV-1-infected individuals, but it did not have a major impact on the latent reservoir or the rebound of plasma viral load after interruption of cART when given alone or in combination with disulfiram., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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.

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 RV144 phase III clinical trial with the combination of the poxvirus vector ALVAC and the HIV gp120 protein has taught us that a vaccine against HIV/AIDS is possible but further improvements are still needed. Although the HIV protective effect of RV144 was modest (31.2%), these encouraging results reinforce the use of poxvirus vectors as HIV/AIDS vaccine candidates. In this review we focus on the prophylactic clinical studies thus far performed with the more widely studied poxvirus vectors, ALVAC, MVA, NYVAC and fowlpox expressing HIV antigens. We describe the characteristics of each vector administered either alone or in combination with other vectors, with emphasis on the immune parameters evaluated in healthy volunteers, percentage of responders and triggering of humoral and T cell responses. Some of these immunogens induced broad, polyfunctional and long-lasting CD4(+) and CD8(+) T cell responses to HIV-1 antigens in most volunteers, with preference for effector memory T cells, and neutralizing antibodies, immune parameters that might be relevant in protection. Finally, we consider improvements in immunogenicity of the poxvirus vectors by the selective deletion of viral immunomodulatory genes and insertion of host range genes in the poxvirus genome. Overall, the poxvirus vectors have proven to be excellent HIV/AIDS vaccine candidates, with distinct behavior among them, and the future implementation will be dictated by their optimized immune profile in clinical trials.

MVA-B is an attenuated poxvirus vector expressing human immunodeficiency virus type 1 Env, Gag, Pol, and Nef antigens from clade B, and is considered a promising HIV/AIDS vaccine candidate. Recently, a phase I clinical trial in human healthy volunteers has shown that MVA-B is safe and highly immunogenic, inducing broad, polyfunctional, and long-lasting CD4(+) and CD8(+) T cell responses to HIV-1 antigens, with preference for effector memory T cells; and it also triggers the induction of specific antibodies to Env in most of the vaccines. While MVA recombinants expressing HIV-1 antigens are being used or plan to use in therapeutic clinical trials, little is known on the effect of HIV-1 highly active antiretroviral therapy in MVA life cycle. To define this role, here we have evaluated in established cell cultures and human dendritic cells to what extent different HIV-1 protease inhibitors affect virus replication and expression of HIV-1 antigens during MVA-B infection. The results obtained revealed that the most commonly used HIV-1 protease inhibitors (atazanavir, ritonavir, and lopinavir) had no effect on MVA-B virus growth kinetics, even at higher concentrations than those normally used on HAART. Furthermore, expression of gp120 and the fused Gag-Pol-Nef polyprotein in permissive and non-permissive cells infected with MVA-B were also not affected. These findings are relevant information for the therapeutic use of MVA-B as an HIV-1/AIDS vaccine., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Poxviruses encode multiple inhibitors of the interferon (IFN) system, acting at different levels and blocking the induction of host defense mechanisms. Two viral gene products, B19 and B8, have been shown to act as decoy receptors of type I and type II IFNs, blocking the binding of IFN to its receptor. Since IFN plays a major role in innate immune responses, in this investigation we asked to what extent the viral inhibitors of the IFN system impact the capacity of poxvirus vectors to activate immune responses. This was tested in a mouse model with single and double deletion mutants of the vaccine candidate NYVAC-C, which expresses the HIV-1 Env, Gag, Pol, and Nef antigens. When deleted individually or in double, the type I (B19) and type II (B8) IFN binding proteins were not required for virus replication in cultured cells. Studies of immune responses in mice after DNA prime/NYVAC boost revealed that deletion of B8R and/or B19R genes improved the magnitude and quality of HIV-1-specific CD8(+) T cell adaptive immune responses and impacted their memory phase, changing the contraction, the memory differentiation, the effect magnitude, and the functionality profile. For B cell responses, deletion of the viral gene B8R and/or B19R had no effect on antibody levels to HIV-1 Env. These findings revealed that single or double deletion of viral factors (B8 and B19) targeting the IFN pathway is a useful approach in the design of improved poxvirus-based vaccines.

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.

Background: Modified Vaccinia Ankara (MVA) is an attenuated strain of Vaccinia virus (VACV) currently employed in many clinical trials against HIV/AIDS and other diseases. MVA still retains genes involved in host immune response evasion, enabling its optimization by removing some of them. The aim of this study was to evaluate cellular immune responses (CIR) induced by an IL-18 binding protein gene (C12L) deleted vector (MVAΔC12L)., Methodology/principal Findings: BALB/c and C57BL/6 mice were immunized with different doses of MVAΔC12L or MVA wild type (MVAwt), then CIR to VACV epitopes in immunogenic proteins were evaluated in spleen and draining lymph nodes at acute and memory phases (7 and 40 days post-immunization respectively). Compared with parental MVAwt, MVAΔC12L immunization induced a significant increase of two to three-fold in CD8(+) and CD4(+) T-cell responses to different VACV epitopes, with increased percentage of anti-VACV cytotoxic CD8(+) T-cells (CD107a/b(+)) during the acute phase of the response. Importantly, the immunogenicity enhancement was also observed after MVAΔC12L inoculation with different viral doses and by distinct routes (systemic and mucosal). Potentiation of MVA's CIR was also observed during the memory phase, in correlation with a higher protection against an intranasal challenge with VACV WR. Of note, we could also show a significant increase in the CIR against HIV antigens such as Env, Gag, Pol and Nef from different subtypes expressed from two recombinants of MVAΔC12L during heterologous DNA prime/MVA boost vaccination regimens., Conclusions/significance: This study demonstrates the relevance of IL-18 bp contribution in the immune response evasion during MVA infection. Our findings clearly show that the deletion of the viral IL-18 bp gene is an effective approach to increase MVA vaccine efficacy, as immunogenicity improvements were observed against vector antigens and more importantly to HIV antigens.

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.

Attenuated poxvirus vectors expressing human immunodeficiency virus type 1 (HIV-1) antigens are considered promising HIV/AIDS vaccine candidates. Here, we describe the nature of T cell immune responses induced in healthy volunteers participating in a phase I clinical trial in Spain after intramuscular administration of three doses of the recombinant MVA-B-expressing monomeric gp120 and the fused Gag-Pol-Nef (GPN) polyprotein of clade B. The majority (92.3%) of the volunteers immunized had a positive specific T cell response at any time postvaccination as detected by gamma interferon (IFN-γ) intracellular cytokine staining (ICS) assay. The CD4(+) T cell responses were predominantly Env directed, whereas the CD8(+) T cell responses were similarly distributed against Env, Gag, and GPN. The proportion of responders after two doses of MVA-B was similar to that obtained after the third dose of MVA-B vaccination, and the responses were sustained (84.6% at week 48). Vaccine-induced CD8(+) T cells to HIV-1 antigens after 1 year were polyfunctional and distributed mainly within the effector memory (TEM) and terminally differentiated effector memory (TEMRA) T cell populations. Antivector T cell responses were mostly induced by CD8(+) T cells, highly polyfunctional, and of TEMRA phenotype. These findings demonstrate that the poxvirus MVA-B vaccine candidate given alone is highly immunogenic, inducing broad, polyfunctional, and long-lasting CD4 and CD8 T cell responses to HIV-1 antigens, with preference for TEM. Thus, on the basis of the immune profile of MVA-B in humans, this immunogen can be considered a promising HIV/AIDS vaccine candidate.

Background: To investigate the safety and immunogenicity of a modified vaccinia virus Ankara vector expressing HIV-1 antigens from clade B (MVA-B), a phase-I, doubled-blind placebo-controlled trial was performed., Methods: 30 HIV-uninfected volunteers at low risk of HIV-1 infection were randomly allocated to receive 3 intramuscular injections (1×10(8)pfu/dose) of MVA-B (n=24) or placebo (n=6) at weeks 0, 4 and 16. All volunteers were followed 48 weeks. Primary end-points were adverse events and immunogenicity., Results: A total of 169 adverse events were reported, 164 of grade 1-2, and 5 of grade 3 (none related to vaccination). Overall 75% of the volunteers showed positive ELISPOT responses at any time point. The magnitude (median) of the total responses induced was 288SFC/10(6)PBMC at week 18. Antibody responses against Env were observed in 95% and 72% of vaccinees at week 18 and 48, respectively. HIV-1 neutralizing antibodies were detected in 33% of volunteers., Conclusions: MVA-B was safe, well tolerated and elicited strong and durable T-cell and antibody responses in 75% and 95% of volunteers, respectively. These data support further exploration of MVA-B as an HIV-1 vaccine candidate. Clinical Trials.gov identifier: NCT00679497., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The vaccinia virus (VACV) C6 protein has sequence similarities with the poxvirus family Pox_A46, involved in regulation of host immune responses, but its role is unknown. Here, we have characterized the C6 protein and its effects in virus replication, innate immune sensing and immunogenicity in vivo. C6 is a 18.2 kDa protein, which is expressed early during virus infection and localizes to the cytoplasm of infected cells. Deletion of the C6L gene from the poxvirus vector MVA-B expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (MVA-B ΔC6L) had no effect on virus growth kinetics; therefore C6 protein is not essential for virus replication. The innate immune signals elicited by MVA-B ΔC6L in human macrophages and monocyte-derived dendritic cells (moDCs) are characterized by the up-regulation of the expression of IFN-β and IFN-α/β-inducible genes. In a DNA prime/MVA boost immunization protocol in mice, flow cytometry analysis revealed that MVA-B ΔC6L enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4+ and CD8+ T-cell memory immune responses, with most of the HIV-1 responses mediated by the CD8+ T-cell compartment with an effector phenotype. Significantly, while MVA-B induced preferentially Env- and Gag-specific CD8+ T-cell responses, MVA-B ΔC6L induced more Gag-Pol-Nef-specific CD8+ T-cell responses. Furthermore, MVA-B ΔC6L enhanced the levels of antibodies against Env in comparison with MVA-B. These findings revealed that C6 can be considered as an immunomodulator and that deleting C6L gene in MVA-B confers an immunological benefit by enhancing IFN-β-dependent responses and increasing the magnitude and quality of the T-cell memory immune responses to HIV-1 antigens. Our observations are relevant for the improvement of MVA vectors as HIV-1 vaccines.

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 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., Methodology/principal Findings: 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., Conclusions/significance: 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.

The aim of this study was to define the effects on antigen-presenting cells of the expression of HIV antigens from an attenuated poxvirus vector. We have analyzed the transcriptional changes in gene expression following infection of human immature monocyte-derived dendritic cells (DC) with recombinant modified vaccinia virus Ankara (MVA) expressing the genes encoding the gp120 and Gag-Pol-Nef antigens of HIV type 1 clade B (referred to as MVA-B) versus parental MVA infection. Using microarray technology and real-time reverse transcription-PCR, we demonstrated that the HIV proteins induced the expression of cytokines, cytokine receptors, chemokines, chemokine receptors, and molecules involved in antigen uptake and processing, including major histocompatibility complex (MHC) genes. Levels of mRNAs for interleukin-1, beta interferon, CCR8, and SCYA20 were higher after HIV antigen production. MVA-B infection also modulated the expression of antigen processing and presentation genes: the gene for MICA was upregulated, whereas those for HLA-DRA and HSPA5 were downregulated. Indeed, the increased expression of the gene for MICA, a glycoprotein related to major histocompatibility complex class I molecules, was shown to enhance the interaction between MVA-B-infected target cells and cytotoxic lymphocytes. The expression profiles of the genes for protein kinases such as JAK1 and IRAK2 were activated after HIV antigen expression. Several genes included in the JAK-STAT and mitogen-activated protein kinase signaling pathways were regulated after HIV antigen expression. Our findings provide the first gene signatures in DC of a candidate MVA-B vaccine expressing four HIV antigens and identified the biological roles of some of the regulatory genes, like that for MICA, which will help in the design of more effective MVA-derived vaccines.

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.

The attenuated poxvirus vectors MVA and NYVAC are now in clinical trials against HIV/AIDS. Due to the vectors restricted replication capacity in human cells, approaches to enhance their immunogenicity are highly desirable. Here, we have analyzed the ability of a soluble form of hexameric CD40L (sCD40L) to stimulate specific immune responses to HIV antigens when inoculated in mice during priming with DNA and in the booster with MVA or NYVAC, expressing the vectors HIV-1 Env, Gag, Pol and Nef antigens from clade B. Our findings revealed that sCD40L in DNA/poxvirus combination enhanced the magnitude about 2-fold (DNA-B/MVA-B) and 4-fold (DNA-B/NYVAC-B), as well as the breath of the HIV antigen specific cellular immune responses. sCD40L was necessary in both prime and boost inoculations triggering a potent polarization of the Th response towards a Th1 type. In DNA-B/NYVAC-B regime the addition of sCD40L significantly enhanced the humoral immune response against HIV gp160, but not in DNA-B/MVA-B combination. These findings provided evidence for the immunostimulatory benefit of sCD40L when DNA and the poxvirus vectors MVA and NYVAC are used as immunogens.

Poxvirus vectors have proven to be highly effective for boosting immune responses in diverse vaccine settings. Recent reports reveal marked differences in the gene expression of human dendritic cells infected with two leading poxvirus-based human immunodeficiency virus (HIV) vaccine candidates, New York vaccinia virus (NYVAC) and modified vaccinia virus Ankara (MVA). To understand how complex genomic changes in these two vaccine vectors translate into antigen-specific systemic immune responses, we undertook a head-to-head vaccine immunogenicity and efficacy study in the pathogenic HIV type 1 (HIV-1) model of AIDS in Indian rhesus macaques. Differences in the immune responses in outbred animals were not distinguished by enzyme-linked immunospot assays, but differences were distinguished by multiparameter fluorescence-activated cell sorter analysis, revealing a difference between the number of animals with both CD4(+) and CD8(+) T-cell responses to vaccine inserts (MVA) and those that elicit a dominant CD4(+) T-cell response (NYVAC). Remarkably, vector-induced differences in CD4(+)/CD8(+) T-cell immune responses persisted for more than a year after challenge and even accompanied antigenic modulation throughout the control of chronic infection. Importantly, strong preexposure HIV-1/simian immunodeficiency virus-specific CD4(+) T-cell responses did not prove deleterious with respect to accelerated disease progression. In contrast, in this setting, animals with strong vaccine-induced polyfunctional CD4(+) T-cell responses showed efficacies similar to those with stronger CD8(+) T-cell responses.

In this investigation we have generated and defined the immunogenicity of two novel HIV/AIDS vaccine candidates based on the highly attenuated vaccinia virus strains, MVA and NYVAC, efficiently expressing in the same locus (TK) and under the same viral promoter the codon optimized HIV-1 genes encoding gp120 and Gag-Pol-Nef antigens of clade B (referred as MVA-B and NYVAC-B). In infected human HeLa cells, gp120 is released from cells and GPN is produced as a polyprotein; NYVAC-B induces severe apoptosis but not MVA-B. The two poxvirus vectors showed genetic stability of the inserts. In BALB/c and in transgenic HHD mice for human HLA-A2 class I, both vectors are efficient immunogens and induced broad cellular immune responses against peptides represented in the four HIV-1 antigens. Some differences were observed in the magnitude and breadth of the immune response in the mouse models. In DNA prime/poxvirus boost protocols, the strongest immune response, as measured by fresh IFN-gamma and IL-2 ELISPOT, was obtained in BALB/c mice boosted with NYVAC-B, while in HHD mice there were no differences between the poxvirus vectors. When the prime/boost was performed with homologous or with combination of poxvirus vectors, the protocols MVA-B/MVA-B and NYVAC-B/NYVAC-B, or the combination NYVAC-B/MVA-B gave the most consistent broader immune response in both mouse models, although the magnitude of the overall response was higher for the DNA-B/poxvirus-B regime. All of the immunization protocols induced some humoral response against the gp160 protein from HIV-1 clone LAV. Our findings indicate that MVA-B and NYVAC-B meet the criteria to be potentially useful vaccine candidates against HIV/AIDS.

Recombinants based on the attenuated vaccinia virus strains MVA and NYVAC are considered candidate vectors against different human diseases. In this study we have generated and characterized in BALB/c and in transgenic HHD mice the immunogenicity of two attenuated poxvirus vectors expressing in a single locus (TK) the codon optimized HIV-1 genes encoding gp120 and Gag-Pol-Nef (GPN) polyprotein of clade C (referred as MVA-C and NYVAC-C). In HHD mice primed with either MVA-C or NYVAC-C, or primed with DNA-C and boosted with the poxvirus vectors, the splenic T cell responses against clade C peptides spanning gp120/GPN was broad and mainly directed against Gag-1, Env-1 and Env-2 peptide pools. In BALB/c mice immunized with the homologous or the heterologous combination of poxvirus vectors or with Semliki forest virus (SFV) vectors expressing gp120/GPN, the immune response was also broad but the most immunogenic peptides were Env-1, GPN-1 and GPN-2. Differences in the magnitude of the cellular immune responses were observed between the poxvirus vectors depending on the protocol used. The specific cellular immune response triggered by the poxvirus vectors was Th1 type. The cellular response against the vectors was higher for NYVAC than for MVA in both HHD and BALB/c mice, but differences in viral antigen recognition between the vectors was observed in sera from the poxvirus-immunized animals. These results demonstrate the immunogenic potential of MVA-C and NYVAC-C as novel vaccine candidates against clade C of HIV-1.

The cytotoxic T-lymphocyte response (CTL) has been shown to be determinant in the clearance of many viral infections and hence, vaccine candidates against AIDS are designed to enhance this arm of the immune system. In this study, we have analyzed the antigen specific immune responses triggered in mice by different combinations of vaccine vehicles expressing the multiepitope polypeptide TAB13. This chimeric protein contains the V3 region of the gp120 from eight different HIV-1 isolates and was efficiently expressed by a DNA vector (DNA-TAB), and also by vaccinia virus recombinants (rVV) based either on the attenuated modified vaccinia virus Ankara (MVA-TAB) or Western Reserve (VV-TAB) strains. Inoculation of a DNA-TAB vector in priming followed by a booster with VV-TAB or MVA-TAB induces a humoral immune response against TAB13 protein and efficiently enhanced the CD8+ T cell response against V3 epitopes from HIV-1 isolates LR150, MN, and IIIB in comparison with animals immunized with two doses of DNA-TAB. A protocol that incorporates a DNA vector expressing IFN-gamma (DNA-IFN-gamma) with DNA-TAB in the priming, followed by a booster with MVA-TAB, triggered the highest values of specific CD8+ T cell response. By examining the cytokine pattern, the immune response induced by these vaccination approaches was predominantly of Th-1 type. These findings establish safe strategies for the enhanced generation of T cell mediated immunity to HIV-1 that can benefit in the design of an effective vaccine against AIDS.

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. [ABSTRACT FROM AUTHOR]

Background: 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. Methods: 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. Results: 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. Conclusions: 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. Trial registration: ClinicalTrials.gov . [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Poxviruses encode multiple inhibitors of the interferon (IFN) system, acting at different levels and blocking the induction of host defense mechanisms. Two viral gene products, B19 and B8, have been shown to act as decoy receptors of type I and type II IFNs, blocking the binding of IFN to its receptor. Since IFN plays a major role in innate immune responses, in this investigation we asked to what extent the viral inhibitors of the IFN system impact the capacity of poxvirus vectors to activate immune responses. This was tested in a mouse model with single and double deletion mutants of the vaccine candidate NYVAC-C, which expresses the HIV-1 Env, Gag, Pol, and Nef antigens. When deleted individually or in double, the type I (B19) and type II (B8) IFN binding proteins were not required for virus replication in cultured cells. Studies of immune responses in mice after DNA prime/NYVAC boost revealed that deletion of B8R and/or B19R genes improved the magnitude and quality of HIV-1-specific CD8+ T cell adaptive immune responses and impacted their memory phase, changing the contraction, the memory differentiation, the effect magnitude, and the functionality profile. For B cell responses, deletion of the viral gene B8R and/or B19R had no effect on antibody levels to HIV-1 Env. These findings revealed that single or double deletion of viral factors (B8 and B19) targeting the IFN pathway is a useful approach in the design of improved poxvirus-based vaccines. [ABSTRACT FROM AUTHOR]