Juliana Falivene

Improving the MVA Vaccine Potential by Deleting the Viral Gene Coding for the IL-18 Binding Protein

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 MVAs 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.

Early Gag Immunodominance of the HIV-Specific T-Cell Response during Acute/Early Infection Is Associated with Higher CD8+ T-Cell Antiviral Activity and Correlates with Preservation of the CD4+ T-Cell Compartment

ABSTRACT The important role of the CD8+ T-cell response on HIV control is well established. Moreover, the acute phase of infection represents a proper scenario to delineate the antiviral cellular functions that best correlate with control. Here, multiple functional aspects (specificity, ex vivo viral inhibitory activity [VIA] and polyfunctionality) of the HIV-specific CD8+ T-cell subset arising early after infection, and their association with disease progression markers, were examined. Blood samples from 44 subjects recruited within 6 months from infection (primary HIV infection [PHI] group), 16 chronically infected subjects, 11 elite controllers (EC), and 10 healthy donors were obtained. Results indicated that, although Nef dominated the anti-HIV response during acute/early infection, a higher proportion of early anti-Gag T cells correlated with delayed progression. Polyfunctional HIV-specific CD8+ T cells were detected at early time points but did not associate with virus control. Conversely, higher CD4+ T-cell set points were observed in PHI subjects with higher HIV-specific CD8+ T-cell VIA at baseline. Importantly, VIA levels correlated with the magnitude of the anti-Gag cellular response. The advantage of Gag-specific cells may result from their enhanced ability to mediate lysis of infected cells (evidenced by a higher capacity to degranulate and to mediate VIA) and to simultaneously produce IFN-γ. Finally, Gag immunodominance was associated with elevated plasma levels of interleukin 2 (IL-2) and macrophage inflammatory protein 1β (MIP-1β). All together, this study underscores the importance of CD8+ T-cell specificity in the improved control of disease progression, which was related to the capacity of Gag-specific cells to mediate both lytic and nonlytic antiviral mechanisms at early time points postinfection.

Interplay between modified vaccinia virus Ankara and dendritic cells: phenotypic and functional maturation of bystander dendritic cells.

ABSTRACT Modified vaccinia virus Ankara (MVA) is an attenuated poxvirus strain, currently under evaluation as a vaccine vector in various clinical settings. It has been reported that human dendritic cells (DCs) mature after infection with MVA, but reports on the functionality of DCs have so far been controversial. In this work, we studied the phenotype and functionality of MVA-infected DCs. As previously reported, we found that human monocyte-derived DCs upregulated CD86 and HLA-DR in response to MVA infection. Moreover, infected DCs produced a broad array of chemokines and cytokines and were able to activate and induce gamma interferon (IFN-γ) production both in CD4+ and in CD8+ allogeneic T cells and in specific autologous peripheral blood lymphocytes (PBLs). Analysis of DC maturation following infection with a recombinant green fluorescent protein (GFP)-expressing MVA revealed that upregulation of CD86 expression was mainly observed in GFPneg (bystander) cells. While GFPpos (infected) DCs produced tumor necrosis factor alpha (TNF-α), they were unable to produce CXCL10 and were less efficient at inducing IFN-γ production in CEF-specific autologous PBLs. Maturation of bystander DCs could be achieved by incubation with supernatant from infected cultures or with apoptotic infected cells. Type I IFNs were partially responsible for the induction of CXCL10 on bystander DCs. Our findings demonstrate for the first time that, in MVA-infected DC cultures, the leading role with respect to functionality and maturation characteristics is achieved by the bystander DCs.

Th17 and Th17/Treg ratio at early HIV infection associate with protective HIV-specific CD8 + T-cell responses and disease progression

The aim of this study was to analyze Th17 and Treg subsets and their correlation with anti-HIV T-cell responses and clinical parameters during (acute/early) primary HIV infection (PHI) and up to one year post-infection (p.i). Samples from 14 healthy donors (HDs), 40 PHI patients, 17 Chronics, and 13 Elite controllers (ECs) were studied. The percentages of Th17 and Treg subsets were severely altered in Chronics, whereas all HIV-infected individuals (including ECs) showed Th17/Treg imbalance compared to HDs, in concordance with higher frequencies of activated CD8+ T-cells (HLA-DR+/CD38+). Better clinical status (higher CD4 counts, lower viral loads and activation) was associated with higher Th17 and lower Treg levels. We found positive correlations between Th17 at baseline and anti-HIV CD8+ T-cell functionality: viral inhibitory activity (VIA) and key polyfunctions (IFN-γ+/CD107A/B+) at both early and later times p.i, highlighting the prognostic value of Th17 cells to preserve an effective HIV T-cell immunity. Th17/Treg ratio and the IL-17 relative mean fluorescence intensity (rMFI of IL-17) were also positively correlated with VIA. Taken together, our results suggested a potential link between Th17 and Th17/Treg ratio with key HIV-specific CD8+ T-cell responses against the infection.

IL-12 and GM-CSF in DNA/MVA immunizations against HIV-1 CRF12_BF Nef induced T-cell responses with an enhanced magnitude, breadth and quality.

In Argentina, the HIV epidemic is characterized by the co-circulation of subtype B and BF recombinant viral variants. Nef is an HIV protein highly variable among subtypes, making it a good tool to study the impact of HIV variability in the vaccine design setting. We have previously reported a specific cellular response against NefBF with low cross-reactivity to NefB in mice. The aim of this work was to analyze whether the co-administration of IL-12 and GM-CSF, using DNA and MVA vaccine vectors, could improve the final cellular response induced. Mice received three DNA priming doses of a plasmid that express NefBF plus DNAs expressing IL-12 and/or GM-CSF. Afterwards, all the groups were boosted with a MVAnefBF dose. The highest increase in the magnitude of the NefBF response, compared to that induced in the control was found in the IL-12 group. Importantly, a response with higher breadth was detected in groups which received IL-12 or GM-CSF, evidenced as an increased frequency of recognition of homologous (BF) and heterologous (B) Nef peptides, as well as a higher number of other Nef peptide pools representing different viral subtypes. However, these improvements were lost when both DNA cytokines were simultaneously administered, as the response was focused against the immunodominant peptide with a detrimental response towards subdominant epitopes. The pattern of cytokines secreted and the specific-T-cell proliferative capacity were improved in IL-12 and IL-12+GM-CSF groups. Importantly IL-12 generated a significant higher T-cell avidity against a B heterologous peptide. This study indicates that the incorporation of DNA expressing IL-12 in DNA/MVA schemes produced the best results in terms of improvements of T-cell-response key properties such as breadth, cross-reactivity and quality (avidity and pattern of cytokines secreted). These relevant results contribute to the design of strategies aimed to induce T-cell responses against HIV antigens with higher quality.

Early Skewed Distribution of Total and HIV-Specific CD8+ T-Cell Memory Phenotypes during Primary HIV Infection Is Related to Reduced Antiviral Activity and Faster Disease Progression

The important role of the CD8+ T-cells on HIV control is well established. However, correlates of immune protection remain elusive. Although the importance of CD8+ T-cell specificity and functionality in virus control has been underscored, further unraveling the link between CD8+ T-cell differentiation and viral control is needed. Here, an immunophenotypic analysis (in terms of memory markers and Programmed cell death 1 (PD-1) expression) of the CD8+ T-cell subset found in primary HIV infection (PHI) was performed. The aim was to seek for associations with functional properties of the CD8+ T-cell subsets, viral control and subsequent disease progression. Also, results were compared with samples from Chronics and Elite Controllers. It was found that normal maturation of total and HIV-specific CD8+ T-cells into memory subsets is skewed in PHI, but not at the dramatic level observed in Chronics. Within the HIV-specific compartment, this alteration was evidenced by an accumulation of effector memory CD8+ T (TEM) cells over fully differentiated terminal effector CD8+ T (TTE) cells. Furthermore, higher proportions of total and HIV-specific CD8+ TEM cells and higher HIV-specific TEM/(TEM+TTE) ratio correlated with markers of faster progression. Analysis of PD-1 expression on total and HIV-specific CD8+ T-cells from PHI subjects revealed not only an association with disease progression but also with skewed memory CD8+ T-cell differentiation. Most notably, significant direct correlations were obtained between the functional capacity of CD8+ T-cells to inhibit viral replication in vitro with higher proportions of fully-differentiated HIV-specific CD8+ TTE cells, both at baseline and at 12 months post-infection. Thus, a relationship between preservation of CD8+ T-cell differentiation pathway and cell functionality was established. This report presents evidence concerning the link among CD8+ T-cell function, phenotype and virus control, hence supporting the instauration of early interventions to prevent irreversible immune damage.

Env-Specific IgA from Viremic HIV-Infected Subjects Compromises Antibody-Dependent Cellular Cytotoxicity

ABSTRACT Elucidating the factors that modulate HIV-specific antibody-dependent cellular cytotoxicity (ADCC) will help in understanding its role in HIV immunity. The aim of this study was to determine whether IgA could modify the magnitude of ADCC in HIV infection, abrogating its protective role. Plasma samples from 20 HIV-positive (HIV+) subjects enrolled during primary HIV infection (PHI), 10 chronically infected subjects (chronic), and 7 elite controllers (EC) were used. ADCC was determined by using a fluorometric ADCC assay, before and after removal of plasma IgA. Data were analyzed by using nonparametric statistics. ADCC was documented in 80% of PHI enrollment samples and in 100% of PHI 12-month, chronic, and EC samples; it peaked after acute infection, reached a plateau in chronic infection, and decreased after initiation of antiretroviral treatment (ART). Significant associations between ADCC and disease progression were found only after removal of plasma IgA from 12-month PHI samples: the magnitude of ADCC not only increased after IgA removal but also correlated with CD4+ T-cell preservation. This work provides evidence that gp120-specific IgA was capable of modifying ADCC responses during natural HIV infection for the first time and adds to similar evidence provided in other settings. Furthermore, it underscores the complexity of the ADCC phenomenon and will help in an understanding of its underlying mechanisms. IMPORTANCE Although the induction of ADCC-mediating antibodies in HIV-infected subjects has been extensively documented, the association of these antibodies with protection from disease progression is poorly understood. Here, we demonstrate that plasma IgA is a factor capable of modifying the magnitude of IgG-mediated ADCC in HIV infection, mitigating its beneficial effect. These results help in understanding why previous studies failed to demonstrate correlations between ADCC and disease progression, and they also contribute to the notion that an HIV vaccine should stimulate the production of ADCC-mediating IgG antibodies but not IgA.

Novel mucosal DNA-MVA HIV vaccination in which DNA-IL-12 plus cholera toxin B subunit (CTB) cooperates to enhance cellular systemic and mucosal genital tract immunity.

Induction of local antiviral immune responses at the mucosal portal surfaces where HIV-1 and other viral pathogens are usually first encountered remains a primary goal for most vaccines against mucosally acquired viral infections. Exploring mucosal immunization regimes in order to find optimal vector combinations and also appropriate mucosal adjuvants in the HIV vaccine development is decisive. In this study we analyzed the interaction of DNA-IL-12 and cholera toxin B subunit (CTB) after their mucosal administration in DNA prime/MVA boost intranasal regimes, defining the cooperation of both adjuvants to enhance immune responses against the HIV-1 Env antigen. Our results demonstrated that nasal mucosal DNA/MVA immunization schemes can be effectively improved by the co-delivery of DNA-IL-12 plus CTB inducing elevated HIV-specific CD8 responses in spleen and more importantly in genital tract and genito-rectal draining lymph nodes. Remarkably, these CTL responses were of superior quality showing higher avidity, polyfunctionality and a broader cytokine profile. After IL-12+CTB co-delivery, the cellular responses induced showed an enhanced breadth recognizing with higher efficiency Env peptides from different subtypes. Even more, an in vivo CTL cytolytic assay demonstrated the higher specific CD8 T-cell performance after the IL-12+CTB immunization showing in an indirect manner its potential protective capacity. Improvements observed were maintained during the memory phase where we found higher proportions of specific central memory and T memory stem-like cells T-cell subpopulations. Together, our data show that DNA-IL-12 plus CTB can be effectively employed acting as mucosal adjuvants during DNA prime/MVA boost intranasal vaccinations, enhancing magnitude and quality of HIV-specific systemic and mucosal immune responses.

Deletion of A44L, A46R and C12L Vaccinia Virus Genes from the MVA Genome Improved the Vector Immunogenicity by Modifying the Innate Immune Response Generating Enhanced and Optimized Specific T-Cell Responses

MVA is an attenuated vector that still retains immunomodulatory genes. We have previously reported its optimization after deleting the C12L gene, coding for the IL-18 binding-protein. Here, we analyzed the immunogenicity of MVA vectors harboring the simultaneous deletion of A44L, related to steroid synthesis and A46R, a TLR-signaling inhibitor (MVAΔA44L-A46R); or also including a deletion of C12L (MVAΔC12L/ΔA44L-A46R). The absence of biological activities of the deleted genes in the MVA vectors was demonstrated. Adaptive T-cell responses against VACV epitopes, evaluated in spleen and draining lymph-nodes of C57Bl/6 mice at acute/memory phases, were of higher magnitude in those animals that received deleted MVAs compared to MVAwt. MVAΔC12L/ΔA44L-A46R generated cellular specific memory responses of higher quality characterized by bifunctionality (CD107a/b+/IFN-γ+) and proliferation capacity. Deletion of selected genes from MVA generated innate immune responses with higher levels of determining cytokines related to T-cell response generation, such as IL-12, IFN-γ, as well as IL-1β and IFN-β. This study describes for the first time that simultaneous deletion of the A44L, A46R and C12L genes from MVA improved its immunogenicity by enhancing the host adaptive and innate immune responses, suggesting that this approach comprises an appropriate strategy to increase the MVA vaccine potential.

T-cell immune responses against Env from CRF12_BF and subtype B HIV-1 show high clade-specificity that can be overridden by multiclade immunizations.

Background The extreme genetic diversity of the human immunodeficiency virus type 1 (HIV-1) poses a daunting challenge to the generation of an effective AIDS vaccine. In Argentina, the epidemic is characterized by the high prevalence of infections caused by subtype B and BF variants. The aim of this study was to characterize in mice the immunogenic and antigenic properties of the Env protein from CRF12_BF in comparison with clade B, employing prime-boost schemes with the combination of recombinant DNA and vaccinia virus (VV) vectors. Methodology/Principal Findings As determined by ELISPOT from splenocytes of animals immunized with either EnvBF or EnvB antigens, the majority of the cellular responses to Env were found to be clade-specific. A detailed peptide mapping of the responses reveal that when there is cross-reactivity, there are no amino acid changes in the peptide sequence or were minimal and located at the peptide ends. In those cases, analysis of T cell polifunctionality and affinity indicated no differences with respect to the cellular responses found against the original homologous sequence. Significantly, application of a mixed immunization combining both clades (B and BF) induced a broader cellular response, in which the majority of the peptides targeted after the single clade vaccinations generated a positive response. In this group we could also find significant cellular and humoral responses against the whole gp120 protein from subtype B. Conclusions/Significance This work has characterized for the first time the immunogenic peptides of certain EnvBF regions, involved in T cell responses. It provides evidence that to improve immune responses to HIV there is a need to combine Env antigens from different clades, highlighting the convenience of the inclusion of BF antigens in future vaccines for geographic regions where these HIV variants circulate.