Systems analysis of MVA-C induced immune response reveals its significance as a vaccine candidate against HIV/AIDS of clade C.

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.

Deletion of the viral anti-apoptotic gene F1L in the HIV/AIDS vaccine candidate MVA-C enhances immune responses against HIV-1 antigens.

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.

EV01: a phase I trial in healthy HIV negative volunteers to evaluate a clade C HIV vaccine, NYVAC-C undertaken by the EuroVacc Consortium.

NYVAC-C (vP2010), a recombinant vector expressing HIV subtype C gag, pol, env and nef antigens, was tested in a phase I study in healthy, HIV negative volunteers in London and Lausanne. Twenty-four participants were randomised to receive NYVAC-C (20) or matching placebo (4) at weeks 0 and 4, and assessed for safety and immunogenicity over 48 weeks. There were no serious adverse events, and no clinical or laboratory abnormalities or other events that led to withdrawal, interruption or dose reduction of the NYVAC-C/placebo. Half of the 10 assessed responded in the ELISpot assay under stringent criteria, which informed the sample size for a DNA-NYVAC-C comparison to NYVAC-C alone.

Poxvirus vector-based HIV vaccines.

PURPOSE OF REVIEW: In this review, we will provide the scientific rationale for the use of poxvirus vectors in the field of HIV vaccines, the immunological profile of the vaccine-induced immune responses, an update on the current use of poxvirus vector-based vaccines in HIV vaccine clinical trials, and the development of new modified poxvirus vectors with improved immunological profile. RECENT FINDINGS: An Ad5-HIV vaccine was tested in a phase IIb clinical trial (known as the Step trial). Vaccinations in the Step trial were discontinued because the vaccine did not show any effect on acquisition of infection and on viral load. After the disappointing failure of the Step trial, the field of HIV vaccine has regained enthusiasm and vigour due to the promising protective effect observed in the phase III efficacy trial (known as RV-144) performed in Thailand which has tested a poxvirus-gp120 combination. SUMMARY: The RV-144 phase III has provided for the first time evidence that an HIV vaccine can prevent HIV infection. The results from the RV-144 trial are providing the scientific rationale for the future development of the HIV vaccine field and for designing future efficacy trials.

High constant incidence of second primary colorectal cancer.

Patients who had a colorectal cancer have a 1.5- to 2-fold excess risk of a second colorectal cancer as compared to the general population, the excess being higher at younger age at diagnosis. To further investigate the risk and the age-relation of the incidence of second primary colorectal cancer, we considered 9,389 first colon and rectal cancers registered in the Vaud Cancer Registry, Switzerland, between 1974 and 2008, and followed-up to the end of 2008 for a total of 44,113 person-years. There were 136 second colorectal cancers versus 90.5 expected, corresponding to a standardized incidence ratio (SIR) of 1.5 (95% confidence interval, CI, 1.3-1.8). The SIRs were not heterogeneous between men and women, and in strata of calendar year at diagnosis, duration of follow-up, and subsite. However, the SIR was 7.5 (95% CI 4.2-12.4) for subjects diagnosed below age 50 and declined thereafter to reach 1.0 (95% CI 0.6-1.6) at age 80 or over. Consequently, the incidence of second primary colorectal cancer was stable, and exceedingly high, around 300-400/100,000 between age 30-39 and 70 or over. This age pattern is consistent with the existence of a single mutational event in a population of highly susceptible individuals.

Deletion of the Vaccinia Virus Gene A46R, Encoding for an Inhibitor of TLR Signalling, Is an Effective Approach to Enhance the Immunogenicity in Mice of the HIV/AIDS Vaccine Candidate NYVAC-C.

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.

A Candidate HIV/AIDS Vaccine (MVA-B) that Enhances the Magnitude and Polyfunctionality of Memory HIV-1-Specific T-Cell Responses

Background: The poxvirus vector Modified Vaccinia Virus Ankara (MVA) expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (MVA-B) is currently used as a HIV/AIDS vaccine candidate. A general strategy to try to improve the immunogenicity of poxvirus HIV-1 vaccine candidates is the deletion of known or suggested immunomodulatory vaccinia virus (VACV) genes.Methods: We have generated and characterized the innate immune sensing and the immunogenicity profile of a new HIV-1 vaccine candidate, which contains a deletion in a VACV gene.Results: We show that this VACV protein is expressed early during virus infection and localizes to the cytoplasm of infected cells. Deletion of this VACV gene from the MVA-B had no effect on virus growth kinetics; therefore this VACV protein is not essential for virus replication. The innate immune signals elicited by the MVA-B deletion mutant in human macrophages and monocyte-derived dendritic cells were characterized. In a DNA prime/MVA boost immunization protocol in mice, flow cytometry analysis revealed that the MVA-B deletion mutant 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, the MVA-B deletion mutant induced more GPN-specific CD8 + T-cell responses. Furthermore, the MVA-B deletion mutant enhanced the levels of antibodies against Env in comparison with MVA-B.Conclusion: These findings revealed that this new VACV protein can be considered as an immunomodulator and that deleting this gene in MVA-B confers an immunological benefit by inducing innate immune 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.

A novel HIV vaccine adjuvanted by IC31 induces robust and persistent humoral and cellular immunity.

The HIV vaccine strategy that, to date, generated immune protection consisted of a prime-boost regimen using a canarypox vector and an HIV envelope protein with alum, as shown in the RV144 trial. Since the efficacy was weak, and previous HIV vaccine trials designed to generate antibody responses failed, we hypothesized that generation of T cell responses would result in improved protection. Thus, we tested the immunogenicity of a similar envelope-based vaccine using a mouse model, with two modifications: a clade C CN54gp140 HIV envelope protein was adjuvanted by the TLR9 agonist IC31®, and the viral vector was the vaccinia strain NYVAC-CN54 expressing HIV envelope gp120. The use of IC31® facilitated immunoglobulin isotype switching, leading to the production of Env-specific IgG2a, as compared to protein with alum alone. Boosting with NYVAC-CN54 resulted in the generation of more robust Th1 T cell responses. Moreover, gp140 prime with IC31® and alum followed by NYVAC-CN54 boost resulted in the formation and persistence of central and effector memory populations in the spleen and an effector memory population in the gut. Our data suggest that this regimen is promising and could improve the protection rate by eliciting strong and long-lasting humoral and cellular immune responses.

Improved NYVAC-based vaccine vectors.

While as yet there is no vaccine against HIV/AIDS, the results of the phase III Thai trial (RV144) have been encouraging and suggest that further improvements of the prime/boost vaccine combination of a poxvirus and protein are needed. With this aim, in this investigation we have generated derivatives of the candidate vaccinia virus vaccine vector NYVAC with potentially improved functions. This has been achieved by the re-incorporation into the virus genome of two host range genes, K1L and C7L, in conjunction with the removal of the immunomodulatory viral molecule B19, an antagonist of type I interferon action. These novel virus vectors, referred to as NYVAC-C-KC and NYVAC-C-KC-ΔB19R, have acquired relevant biological characteristics, giving higher levels of antigen expression in infected cells, replication-competency in human keratinocytes and dermal fibroblasts, activation of selective host cell signal transduction pathways, and limited virus spread in tissues. Importantly, these replication-competent viruses have been demonstrated to maintain a highly attenuated phenotype.

An overview of L-2-hydroxyglutarate dehydrogenase gene (L2HGDH) variants: a genotype-phenotype study.

L-2-Hydroxyglutaric aciduria (L2HGA) is a rare, neurometabolic disorder with an autosomal recessive mode of inheritance. Affected individuals only have neurological manifestations, including psychomotor retardation, cerebellar ataxia, and more variably macrocephaly, or epilepsy. The diagnosis of L2HGA can be made based on magnetic resonance imaging (MRI), biochemical analysis, and mutational analysis of L2HGDH. About 200 patients with elevated concentrations of 2-hydroxyglutarate (2HG) in the urine were referred for chiral determination of 2HG and L2HGDH mutational analysis. All patients with increased L2HG (n=106; 83 families) were included. Clinical information on 61 patients was obtained via questionnaires. In 82 families the mutations were detected by direct sequence analysis and/or multiplex ligation dependent probe amplification (MLPA), including one case where MLPA was essential to detect the second allele. In another case RT-PCR followed by deep intronic sequencing was needed to detect the mutation. Thirty-five novel mutations as well as 35 reported mutations and 14 nondisease-related variants are reviewed and included in a novel Leiden Open source Variation Database (LOVD) for L2HGDH variants (http://www.LOVD.nl/L2HGDH). Every user can access the database and submit variants/patients. Furthermore, we report on the phenotype, including neurological manifestations and urinary levels of L2HG, and we evaluate the phenotype-genotype relationship.

Robust vaccine-elicited cellular immune responses in breast milk following systemic Simian Immunodeficiency Virus DNA prime and live virus vector boost vaccination of lactating rhesus monkeys.

Breast milk transmission of HIV remains an important mode of infant HIV acquisition. Enhancement of mucosal HIV-specific immune responses in milk of HIV-infected mothers through vaccination may reduce milk virus load or protect against virus transmission in the infant gastrointestinal tract. However, the ability of HIV/SIV strategies to induce virus-specific immune responses in milk has not been studied. In this study, five uninfected, hormone-induced lactating, Mamu A*01(+) female rhesus monkey were systemically primed and boosted with rDNA and the attenuated poxvirus vector, NYVAC, containing the SIVmac239 gag-pol and envelope genes. The monkeys were boosted a second time with a recombinant Adenovirus serotype 5 vector containing matching immunogens. The vaccine-elicited immunodominant epitope-specific CD8(+) T lymphocyte response in milk was of similar or greater magnitude than that in blood and the vaginal tract but higher than that in the colon. Furthermore, the vaccine-elicited SIV Gag-specific CD4(+) and CD8(+) T lymphocyte polyfunctional cytokine responses were more robust in milk than in blood after each virus vector boost. Finally, SIV envelope-specific IgG responses were detected in milk of all monkeys after vaccination, whereas an SIV envelope-specific IgA response was only detected in one vaccinated monkey. Importantly, only limited and transient increases in the proportion of activated or CCR5-expressing CD4(+) T lymphocytes in milk occurred after vaccination. Therefore, systemic DNA prime and virus vector boost of lactating rhesus monkeys elicits potent virus-specific cellular and humoral immune responses in milk and may warrant further investigation as a strategy to impede breast milk transmission of HIV.

L'ABC des recommandations actuelles : prévention cardiovasculaire par l'activité physique

Cet article fait le point sur les recommandations actuelles en matière d'activité physique bénéfique pour la santé. Il décrit plus spécifiquement les bénéfices cardiovasculaires d'une activité physique régulière. Une approche est finalement proposée pour la pratique du conseil en activité physique au cabinet médical.

Preclinical evaluation of the immunogenicity of C-type HIV-1-based DNA and NYVAC vaccines in the Balb/C mouse model.

As part of a European initiative (EuroVacc), we report the design, construction, and immunogenicity of two HIV-1 vaccine candidates based on a clade C virus strain (CN54) representing the current major epidemic in Asia and parts of Africa. Open reading frames encoding an artificial 160-kDa GagPolNef (GPN) polyprotein and the external glycoprotein gp120 were fully RNA and codon optimized. A DNA vaccine (DNA-GPN and DNA-gp120, referred to as DNA-C), and a replication-deficient vaccinia virus encoding both reading frames (NYVAC-C), were assessed regarding immunogenicity in Balb/C mice. The intramuscular administration of both plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial T-cell responses against both antigens as well as Env-specific antibodies. Whereas low doses of NYVAC-C failed to induce specific CTL or antibodies, high doses generated cellular as well as humoral immune responses, but these did not reach the levels seen following DNA vaccination. The most potent immune responses were detectable using prime:boost protocols, regardless of whether DNA-C or NYVAC-C was used as the priming or boosting agent. These preclinical findings revealed the immunogenic response triggered by DNA-C and its enhancement by combining it with NYVAC-C, thus complementing the macaque preclinical and human phase I clinical studies of EuroVacc.