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Post-treatment HIV controllers (PTCs) offer evidence that HIV remission can be achieved in some people after antiretroviral treatment (ART) discontinuation. However, the circumstances and mechanisms leading to PTC remain unclear. We evaluated the impact of early (week 4) vs late (week 24 post infection) ART initiation in SIVmac251-infected cynomolgus macaques that received 2 years of ART before analytical interruption (ATI). Early ART strongly promoted PTC. The divergent outcome of early and late-treated macaques was not related to differences in the frequency of infected cells at ATI. Rather, early treatment favored the development of long-term memory CD8+ T cells with enhanced proliferative and SIV suppressive capacity that were able to mediate a robust secondary-like response upon viral rebound. Our model allowed to formally demonstrate a link between ART initiation during primary infection and the promotion of post-treatment control and provided results that may guide the development of new immunotherapies seeking-HIV remission.

Acquired demyelinating syndrome associated with myelin oligodendrocyte glycoprotein antibodies, named recently myelin oligodendrocyte glycoprotein-associated disease, represents >27% of this paediatric syndrome. Relapses occur in 40% of them, which may be associated with severe outcomes. Aiming to identify biomarker allowing to predict relapse, we measured both myelin oligodendrocyte glycoprotein antibodies and neurofilament light chain levels in blood samples of patients that are known to reflect axonal injuries in neurological diseases including demyelinating autoimmune disorders. Three groups of patients were selected: relapsing myelin oligodendrocyte glycoprotein-associated disease (n = 8), non-relapsing myelin oligodendrocyte glycoprotein-associated disease (n = 7) and control patients with non-inflammatory neurological diseases (n = 12). Neurofilament light chain concentrations were measured in plasma of these three groups of patients using the high-sensitivity single-molecule array method at onset of the disease and 6 months later. At onset of the disease, we found that levels of neurofilament light chain in blood of non-relapsing patients were significantly higher than in control patients (means: 98.36 ± 22.66 versus 12.47 ± 2.47 pg/mL, **P < 0.01, Kruskal–Wallis test). The mean neurofilament light chain value in relapsing patients (82.16 ± 38.41 pg/mL) was not significantly different from that in non-relapsing and in control patients. Plasma myelin oligodendrocyte glycoprotein antibody levels were 2.5-fold higher in relapsing than in non-relapsing patients without reaching significance (means: 15.26 ± 4.87 versus 5.96 ± 1.13; two-tailed Mann–Whitney U-test P = 0.119). Plasma neurofilament light chain correlated significantly with myelin oligodendrocyte glycoprotein antibody levels in relapsing (two-tailed Spearman r = 0.8, P = 0.0218) but not in non-relapsing (two-tailed Spearman r = 0.17, P = 0.71). Interestingly, the ratio of neurofilament light chain-to-myelin oligodendrocyte glycoprotein antibodies was significantly lower in relapsing than in non-relapsing patients (means: 5.19 ± 1.61 versus 21.87 ± 6.13; two-tailed Mann–Whitney U-test P = 0.014). These findings suggest that measuring both neurofilament light chain and myelin oligodendrocyte glycoprotein antibody levels in patients at onset of demyelinating disease could predict relapse of myelin oligodendrocyte glycoprotein-associated disease.

Depot injections comprising aqueous drug suspensions are widely marketed for long-acting administration of contraceptive progestins and antipsychotics. In recent years, there has been considerable interest in developing similar long-acting injectable products containing antiretroviral drugs for treatment or prevention of HIV infection, culminating in 2020/2021 with the approval of Cabenuva® (which combines the integrase strand transfer inhibitor cabotegravir with the non-nucleoside reverse transcriptase inhibitor rilpivirine for treatment of HIV infection) and Apretude (a cabotegravir extended-release injectable suspension for HIV prevention). Here, we describe formulation and preclinical development of a new multipurpose prevention technology product based on a long-acting injectable aqueous suspension formulation combining micronized medroxyprogesterone acetate (the progestin used in the long-acting injectable contraceptive product Depo-Provera®) and a rilpivirine nanosuspension for HIV prevention. Through the application of an extensive range of analytical methods, we have demonstrated that the resulting combination suspension formulation had a bimodal particle distribution, was stable, was sterilised by gamma irradiation, and provided sustained systemic and vaginal exposure of both drugs over at least one month following intramuscular injection in macaques. Further formulation development will be needed to sustain drug concentrations in the systemic/vaginal compartments over longer time periods, and to consider options for use of alternate antiretroviral drugs.

Background Although some integrase strand transfer inhibitors (INSTIs) promote peripheral and central adipose tissue/weight gain in people with human immunodeficiency virus (PHIV), the underlying mechanism has not been identified. Here, we used human and simian models to assess the impact of INSTIs on adipose tissue phenotype and function. Methods Adipocyte size and fibrosis were determined in biopsies of subcutaneous and visceral adipose tissue (SCAT and VAT, respectively) from 14 noninfected macaques and 19 PHIV treated or not treated with an INSTI. Fibrosis, adipogenesis, oxidative stress, mitochondrial function, and insulin sensitivity were assessed in human proliferating or adipocyte-differentiated adipose stem cells after long-term exposure to dolutegravir or raltegravir. Results We observed elevated fibrosis, adipocyte size, and adipogenic marker expression in SCAT and VAT from INSTI-treated noninfected macaques. Adiponectin expression was low in SCAT. Accordingly, SCAT and VAT samples from INSTI-exposed patients displayed higher levels of fibrosis than those from nonexposed patients. In vitro, dolutegravir and, to a lesser extent, raltegravir were associated with greater extracellular matrix production and lipid accumulation in adipose stem cells and/or adipocytes as observed in vivo. Despite the INSTIs’ proadipogenic and prolipogenic effects, these drugs promoted oxidative stress, mitochondrial dysfunction, and insulin resistance. Conclusions Dolutegravir and raltegravir can directly impact adipocytes and adipose tissue. These INSTIs induced adipogenesis, lipogenesis, oxidative stress, fibrosis, and insulin resistance. The present study is the first to shed light on the fat modifications observed in INSTI-treated PHIV.

Clinical trials have demonstrated partial protection against HIV-1 infection by vaginal microbicide formulations based on antiretroviral (ARV) drugs. Improved formulations that will maintain sustained drug concentrations at viral target sites in the cervicovaginal mucosa are needed. We have previously demonstrated that treatment of cervicovaginal cell lines with ARV drugs can alter gene expression of drug transporters, suggesting that the mucosal disposition of ARV drugs delivered vaginally can be modulated by drug transporters. This study aimed to investigate in vivo modulation of drug transporter expression in a nonhuman primate model by tenofovir and darunavir released from film formulations. Cervicovaginal tissues were collected from drug-naive macaques and from macaques vaginally treated with film formulations of tenofovir or darunavir. Drug release in vaginal fluid as well as drug absorption in cervicovaginal tissues and lymph nodes were verified by mass spectrometry. The effects of exposure to drugs on the expression of transporters relevant to ARV drugs were evaluated by quantitative PCR. We showed expression in cervicovaginal tissue of drug-naive macaques of transporters important for distribution of ARV drugs, albeit at lower levels compared to human tissue for key transporters including P-glycoprotein. Concentrations of tenofovir and darunavir well above the EC50 values determined in vitro were detected in vaginal fluid and vaginal tissues of macaques treated with drug-dissolving films over 24 h and were also comparable to those shown previously to modulate drug transporter expression. Accordingly, Multidrug Resistance associated Protein 2 (MRP2) in cervicovaginal tissue was upregulated by both tenofovir and darunavir. The two drugs also differentially induced and/or inhibited expression of key uptake transporters for reverse transcriptase inhibitors and protease inhibitors. The lower expression of key transporters in macaques may result in increased retention of ARV drugs at the simian cervicovaginal mucosa compared to the human mucosa and has implications for translation of preclinical data. Modulation of drug transporter expression by tenofovir and darunavir points to the potential benefit of MRP2 inhibition to increase ARV drug penetration through the cervicovaginal epithelium.

The difficulty to unambiguously identify the various subsets of mononuclear phagocytes (MNPs) of the intestinal

Safety and immunogenicity of BNT162b2 mRNA vaccine are unknown in hematological patients; both were evaluated prospectively in 42 patients with multiple myeloma (MM) and 50 with myeloproliferative malignancies (MPM) (20 chronic myeloid leukemias and 30 myeloproliferative neoplasms), all of them on active anti-cancer treatment, in comparison with 36 elderly controls not suffering from cancer. Subjects serologically and/or molecularly (by nasal/throat swab) positives at basal for SARS-CoV-2 were excluded. Primary endpoint was to compare titers of neutralizing anti-SARS-CoV-2 IgG and seroprotection rates among the cohorts at 3 and 5 weeks from first dose.Titration was done using LIAISON® SARS-CoV-2 S1/S2 IgG test, a quantitative chemiluminescent immunoassay approved by FDA on the basis of robust evidences of concordance (94.4%) between the test at cutoff of 15 AU/mL and the Plaque Reduction Neutralization Test 90% at 1:40 ratio. Cutoff of 15 AU/mL was assumed to discriminate responders to vaccination with a protective titer. Cohorts were compared using Fisher' exact test and the Mann-Whitney test as appropriated. Geometric mean concentrations (GMCs), geometric mean ratios and response rates after 1st and 2nd dose were compared in each cohort by Wilcoxon and McNemar tests, respectively.At 5 weeks, GMC of IgG in elderly controls was 353.3 AU/mL versus 106.7 in MM (p = 0.003) and 172.9 in MPM patients (p = 0.049). Seroprotection rate at cutoff of 15 AU/mL was 100% in controls compared to 78.6% in MM (p = 0.003) and 88% in MPM patients (p = 0.038). In terms of logarithm of IgG titer, in a generalized multivariate linear model, no gender effect was observed (p = 0.913), while there was a significant trend toward lower titers by increasing age (p 0.001) and in disease cohorts with respect to controls (MM: p 0.001 and MPM: p 0.001). An ongoing treatment without daratumumab was associated with higher likelihood of response in MM patients (p = 0.003). No swabs resulted positive on each time point. No safety concerns were observed.BNT162b2 has demonstrated to be immunogenic at different extent among the cohorts. Response was 88% and robust in MPM patients. MM patients responded significantly less, particularly those on anti-CD38-based treatment. These latter patients should be advised to maintain masks and social distancing regardless of vaccination status, and their cohabiting family members need to be vaccinated in order to reduce the risk of contagion from the family. Additional boosters and titer monitoring could be considered. Trial registration Study was formally approved by the IRCCS Central Ethical Committee of Regione Lazio in January 2021 (Prot. N-1463/21).

The association between gene polymorphisms and plasma virus load at the set point (SP-PVL) was investigated in Mauritian macaques inoculated with SIV. Among 44 macaques inoculated with 50 AID50, six individuals were selected: three with SP-PVL among the highest and three with SP-PVL among the lowest. The exons of 390 candidate genes of these six animals were sequenced. Twelve non-synonymous single nucleotide polymorphisms (NS-SNPs) lying in nine genes potentially associated with PVL were genotyped in 23 animals. Three NS-SNPs with probabilities of association with PVL less than 0.05 were genotyped in a total of 44 animals. One NS-SNP lying in exon 1 of the IL37 gene displayed a significant association (p = 3.33 × 10−4) and a strong odds ratio (19.52). Multiple linear regression modeling revealed three significant predictors of SP-PVL, including the IL37 exon 1 NS-SNP (p = 0.0004) and the MHC Class IB haplotypes M2 (p = 0.0007) and M6 (p = 0.0013). These three factors in conjunction explained 48% of the PVL variance (p = 4.8 × 10−6). The potential role of IL37 in the control of SIV infection is discussed.

Summary Some viruses have established an equilibrium with their host. African green monkeys (AGM) display persistent high viral replication in the blood and intestine during Simian immunodeficiency virus (SIV) infection but resolve systemic inflammation after acute infection and lack intestinal immune or tissue damage during chronic infection. We show that NKG2a/c+CD8+ T cells increase in the blood and intestine of AGM in response to SIVagm infection in contrast to SIVmac infection in macaques, the latter modeling HIV infection. NKG2a/c+CD8+ T cells were not expanded in lymph nodes, and CXCR5+NKG2a/c+CD8+ T cell frequencies further decreased after SIV infection. Genome-wide transcriptome analysis of NKG2a/c+CD8+ T cells from AGM revealed the expression of NK cell receptors, and of molecules with cytotoxic effector, gut homing, and immunoregulatory and gut barrier function, including CD73. NKG2a/c+CD8+ T cells correlated negatively with IL-23 in the intestine during SIVmac infection. The data suggest a potential regulatory role of NKG2a/c+CD8+ T cells in intestinal inflammation during SIV/HIV infections., Graphical abstract, Highlights • Molecular determination of NKG2a/c+CD8+ T cells in two species of nonhuman primates • Tissue distribution of NKG2a/c+CD8+ T cell is profoundly sculpted by SIV infections • Intestinal NKG2a/c+CD8+ T cells correlated negatively with IL-23 in SIV infection • NKG2a/c+CD8+ T cells might play a protective gut barrier function in HIV/SIV infection, Immunology ; Viral Microbiology ; Transcriptomics

Highly efficient CD8

Introduction La pandémie mondiale de COVID-19 commence à être contrôlée par une vaccination massive. Les patients atteints de maladies auto-immunes (MAI) traités par rituximab ont un risque de décès 4 fois plus élevé (2,32–7,03) et semblent ne pas avoir la même réponse humorale que les témoins sains au vaccins à ARNm. L’objectif est de comparer les réponses cellulaires et humorales au vaccin ARNm BNT 162b2 contre le COVID-19 de patients atteints de MAI traités par rituximab (RTX) ou d’autres immunosuppresseurs ou immunomodulateurs (autres IS) à des sujets contrôles. Patients et méthodes Les patients et les témoins sains (HC) ont été vaccinés avec le BNT162b2 aux jours 0 et 28 et prélevés aux jours 28 et 56. Les patients présentant une infection antérieure au SARS-CoV 2 ont été exclus. Les patients ont été divisés en 2 groupes : « groupe RTX » s’ils avaient reçu du RTX il y a moins d’1 an, ou « autre IS » s’ils étaient traités avec d’autres traitements. Une évaluation sérologique de la réponse vaccinale (ECLIA Cobas, Roche) et de la neutralisation (iFlash-2019-nCoV Nab, Ylho) a été réalisée. Nous avons défini un seuil ≥ 50 d’IgG anti-Spike comme la réponse au vaccin car c’est la valeur seuil au-delà de laquelle les HC avaient tous des anticorps neutralisants détectables. La réponse cellulaire T contre les peptides de la protéine Spike a été réalisée en détectant le marquage intracellulaire du TNF, de l’IFNg, de l’IL2, sur les lymphocytes T CD4 et CD8 activés. Résultats Vingt-huit contrôles et 57 patients atteints de MAI ont été inclus ; 24 patients dans le groupe rituximab et 33 dans l’autre groupe autre IS. À j56, Le groupe RTX avait significativement moins d’IgG anti-Spike (68,6 ± 110) par rapport au groupe HC (235 ± 58) (p

Objective HIV-infected patients receiving antiretroviral treatment (ART) often present adipose tissue accumulation and/or redistribution. adipose tissue has been shown to be an HIV/SIV reservoir and viral proteins as Tat or Nef can be released by infected immune cells and exert a bystander effect on adipocytes or precursors. Our aim was to demonstrate that SIV/HIV infection per se could alter adipose tissue structure and/or function. Design Morphological and functional alterations of subcutaneous (SCAT) and visceral adipose tissue (VAT) were studied in SIV-infected macaques and HIV-infected ART-controlled patients. To analyze the effect of Tat or Nef, we used human adipose stem cells (ASCs) issued from healthy donors, and analyzed adipogenesis and extracellular matrix component production using two dimensional (2D) and three-dimensional (3D) culture models. Methods Adipocyte size and index of fibrosis were determined on Sirius red-stained adipose tissue samples. Proliferating and adipocyte 2D-differentiating or 3D-differentiating ASCs were treated chronically with Tat or Nef. mRNA, protein expression and secretion were examined by RT-PCR, western-blot and ELISA. Results SCAT and VAT from SIV-infected macaques displayed small adipocytes, decreased adipogenesis and severe fibrosis with collagen deposition. SCAT and VAT from HIV-infected ART-controlled patients presented similar alterations. In vitro, Tat and/or Nef induced a profibrotic phenotype in undifferentiated ASCs and altered adipogenesis and collagen production in adipocyte-differentiating ASCs. Conclusion We demonstrate here a specific role for HIV/SIV infection per se on adipose tissue fibrosis and adipogenesis, probably through the release of viral proteins, which could be involved in adipose tissue dysfunction contributing to cardiometabolic alterations of HIV-infected individuals.

Summary: Highly efficient CD8+ T cells are associated with natural HIV control, but it has remained unclear how these cells are generated and maintained. We have used a macaque model of spontaneous SIVmac251 control to monitor the development of efficient CD8+ T cell responses. Our results show that SIV-specific CD8+ T cells emerge during primary infection in all animals. The ability of CD8+ T cells to suppress SIV is suboptimal in the acute phase but increases progressively in controller macaques before the establishment of sustained low-level viremia. Controller macaques develop optimal memory-like SIV-specific CD8+ T cells early after infection. In contrast, a persistently skewed differentiation phenotype characterizes memory SIV-specific CD8+ T cells in non-controller macaques. Accordingly, the phenotype of SIV-specific CD8+ T cells defined early after infection appears to favor the development of protective immunity in controllers, whereas SIV-specific CD8+ T cells in non-controllers fail to gain antiviral potency, feasibly as a consequence of early defects imprinted in the memory pool.

The infected host fails to eradicate HIV-1, despite significant control of viral replication by combinational antiretroviral therapy. Here, we assessed the impact of HIV infection on immune-cell compartments in a SIVmac251 nonhuman primate infection model, which allowed the choice of contamination route, time of infection, and treatment follow-up. We performed high-throughput multiparameter single-cell phenotyping by mass cytometry to obtain a global vision of the immune system in blood and bone marrow. Circulating polymorphonuclear neutrophils (PMNs) with impaired phagocytosis had altered surface expression of CD62L and CD11b during early chronic infection. The initiation of combinational antiretroviral treatment during primary infection did not restore PMN function. The maturation state of PMNs was highly altered during late chronic SIV infection, showing a primarily immature phenotype. Our results provide new insights into PMN involvement in the pathogenesis of HIV infection and may play a role in the establishment and maintenance of chronic immune activation.

The CD4 and the cryptic coreceptor binding sites of the HIV-1 envelope glycoprotein are key to viral attachment and entry. We developed new molecules comprising a CD4 mimetic peptide linked to anionic compounds (mCD4.1-HS12 and mCD4.1-PS1), that block the CD4-gp120 interaction and simultaneously induce the exposure of the cryptic coreceptor binding site, rendering it accessible to HS12- or PS1- mediated inhibition. Using a cynomolgus macaque model of vaginal challenge with SHIV162P3, we report that mCD4.1-PS1, formulated into a hydroxyethyl-cellulose gel provides 83% protection (5/6 animals). We next engineered the mCD4 moiety of the compound, giving rise to mCD4.2 and mCD4.3 that, when conjugated to PS1, inhibited cell-free and cell-associated HIV-1 with particularly low IC50, in the nM to pM range, including some viral strains that were resistant to the parent molecule mCD4.1. These chemically defined molecules, which target major sites of vulnerability of gp120, are stable for at least 48 hours in conditions replicating the vaginal milieu (37 °C, pH 4.5). They efficiently mimic several large gp120 ligands, including CD4, coreceptor or neutralizing antibodies, to which their efficacy compares very favorably, despite a molecular mass reduced to 5500 Da. Together, these results support the development of such molecules as potential microbicides.

The diversity of the human immune repertoire and how it relates to a functional immune response has not yet been studied in detail in humanized NOD.SCID.gammac(-/-) immunodeficient mice. Here, we used a multiplex PCR on genomic DNA to quantify the combinatorial diversity of all possible V-J rearrangements at the TCR-beta chain and heavy chain Ig locus. We first show that the combinatorial diversity of the TCR-beta chain generated in the thymus was well preserved in the periphery, suggesting that human T cells were not vastly activated in mice, in agreement with phenotypic studies. We then show that the combinatorial diversity in NOD.SCID.gammac(-/-) mice reached 100% of human reference samples for both the TCR and the heavy chain of Ig. To document the functionality of this repertoire, we show that a detectable but weak HLA-restricted cellular immune response could be elicited in reconstituted mice after immunization with an adenoviral vector expressing HCV envelope glycoproteins. Altogether, our results suggest that humanized mice express a diversified repertoire and are able to mount antigen-specific immune responses.

OBJECTIVES: Combination microbicide vaginal rings may be more effective than single microbicide rings at reducing/preventing sexual transmission of HIV. Here we report the pre-clinical development and macaque pharmacokinetics of matrix-type silicone elastomer vaginal rings containing dapivirine and darunavir. METHODS: Macaque rings containing 25 mg dapivirine 100 mg dapivirine 300 mg darunavir or 100 mg dapivirine+300 mg darunavir were manufactured and characterized by differential scanning calorimetry. In vitro release was assessed into isopropanol/water and simulated vaginal fluid. Macaque vaginal fluid and blood serum concentrations for both antiretrovirals were measured during 28 day ring use. Tissue levels were measured on day 28. Ex vivo challenge studies were performed on vaginal fluid samples and IC50 values were calculated. RESULTS: Darunavir caused a concentration-dependent reduction in the dapivirine melting temperature in both solid drug mixes and in the combination ring. In vitro release from rings was dependent on drug loading the number of drugs present and the release medium. In macaques serum concentrations of both microbicides were maintained between 10(1) and 10(2) pg/mL. Vaginal fluid levels ranged between 10(3) and 10(4) ng/g and between 10(4) and 10(5) ng/g for dapivirine and darunavir respectively. Both dapivirine and darunavir showed very similar concentrations in each tissue type; the range of drug tissue concentrations followed the general rank order: vagina (1.8 x 10(3)-3.8 x 10(3) ng/g) > cervix (9.4 x 10(1)-3.9 x 10(2) ng/g) > uterus (0-108 ng/g) > rectum (0-40 ng/g). Measured IC50 values were >2 ng/mL for both compounds. CONCLUSIONS: Based on these results and in light of recent clinical progress of the 25 mg dapivirine ring a combination vaginal ring containing dapivirine and darunavir is a viable second-generation HIV microbicide candidate. (c) The Author 2014. 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.

While the immunological correlates of hepatitis C virus (HCV)-specific immunity are not well understood, it is now admitted that an effective vaccine against HCV will need to induce both cellular and humoral immune responses and address viral heterogeneity to prevent immune escape. We developed a vaccine platform specifically aimed at inducing such responses against HCV antigens displayed by recombinant retrovirus-based virus-like particles (VLPs) made of Gag of murine leukemia virus. Both ex vivo produced VLPs and plasmid DNA encoding VLPs can be used as vaccines. Here, we report that immunizations with plasmid DNA forming VLPs pseudotyped with HCV E1 and E2 envelope glycoproteins (HCV-specific plasmo-retroVLPs) induce strong T-cell-mediated immune responses that can be optimized by using proper DNA delivery methods and/or genetic adjuvants. Additionally, multigenotype or multi-specific T-cell responses were observed after immunization with plasmids that encode VLPs pseudotyped with E1E2 derived from numerous viral genotypes and/or displaying NS3 antigen in capsid proteins. While homologous prime-boost immunizations with HCV-specific plasmo-retroVLPs or ex vivo produced VLPs induce a low level of specific antibody responses, optimal combination of plasmo-retroVLPs and VLPs was identified for inducing HCV-specific T-cell and B-cell responses as well as neutralizing antibodies. Altogether, these results have important meanings for the development of anti-HCV preventive vaccines and exemplify the flexibility and potential of our retrovirus-based platform in inducing broad cellular and humoral immune responses.

International audience; We aimed at improving DNA vaccination efficiency for inducing neutralizing antibodies. We used plasmids encoding Gag of MLV and envelope proteins of VSV or WNV. Upon in vivo injection, they generate retrovirus-derived VLPs pseudotyped with these envelopes expressed in their wild-type conformation. We show that these plasmo-retroVLPs induce potent humoral responses, the efficacy of which could be improved by co-administration of DNA encoding adjuvant cytokines. Antibodies against VSV or WNV were detected earlier than with plasmids not generating VLPs, and had higher neutralizing activities. These results highlight the potential of this approach for vaccination strategies aiming at neutralizing antibody induction.

International audience; BACKGROUND: The expression of Moloney murine leukemia virus (Mo-MLV) gag proteins is sufficient to generate retrovirus-like particles (retroVLPs) that can be used as antigen-display platforms by pseudotyping with heterologous envelope proteins or by insertion of epitopes in structural constituents. To circumvent the in vitro production of such retroVLPs, we used DNA plasmids generating recombinant retroVLPs (plasmo-retroVLPs) as immunogens. We previously demonstrated that plasmo-retroVLPs induce significantly better antigen-specific T cell responses and antiviral immune protection than plasmids bearing a single mutation preventing retroVLPs assembly. In the present study, we investigated the possibility of using such plasmo-retroVLPs in prime-boost immunization strategies for hepatitis C virus (HCV) vaccine development. METHODS: To define the best immunization regimen with plasmo-retroVLPs and serotype 5 recombinant adenovirus vectors (rAd5), we used standardized methodologies measuring immune responses to the GP(33-41) 'gold standard' antigen. The protective efficacy of these immunization schedules was also evaluated in mice after tumor challenge. We then applied the optimal prime-boost immunization strategy using vectors expressing HCV-E1/E2 envelope glycoproteins. RESULTS: Using vectors expressing the model antigen, we demonstrated that rAd5(GP33-41)/plasmo-retroVLP(GP33-41) regimen induced significantly higher cellular immune responses than plasmo-retroVLP(GP33-41)/rAd5(GP33-41). Consequently, HCV-specific plasmo-retroVLPs (plasmo-retroVLP(E1E2)) were used as boost in mice primed with rAd5(E1E2) and we observed that plasmo-retroVLP(E1E2) significantly increased E1/E2-specific interferon-gamma cellular responses and E2-specific antibody generation. By contrast, plasmids unable to form E1/E2-pseudotyped retroVLPs had no boosting effect, revealing the importance of presenting E1/E2 in a particulate form. CONCLUSIONS: Altogether, combining plasmo-retroVLPs that represent a new class of genetic vaccines in a heterologous prime-boost vaccination strategy appears to be a promising strategy for HCV vaccine development.

BackgroundProduct adherence is a pivotal issue in the development of effective vaginal microbicides to reduce sexual transmission of HIV. To date, the six Phase III studies of vaginal gel products have relied primarily on self-reporting of adherence. Accurate and reliable methods for monitoring user adherence to microbicide-releasing vaginal rings have yet to be established.MethodsA silicone elastomer vaginal ring prototype containing an embedded, miniature temperature logger has been developed and tested in vitro and in cynomolgus macaques for its potential to continuously monitor environmental temperature and accurately determine episodes of ring insertion and removal.ResultsIn vitro studies demonstrated that DST nano-T temperature loggers encapsulated in medical grade silicone elastomer were able to accurately and continuously measure environmental temperature. The devices responded quickly to temperature changes despite being embedded in different thickness of silicone elastomer. Prototype vaginal rings measured higher temperatures compared with a subcutaneously implanted device, showed high sensitivity to diurnal fluctuations in vaginal temperature, and accurately detected periods of ring removal when tested in macaques.ConclusionsVaginal rings containing embedded temperature loggers may be useful in the assessment of product adherence in late-stage clinical trials.

Virus-like particle (VLP)-based vaccines have provided highly encouraging results in clinical trials while, in contrast, DNA vaccines expressing non-particulate proteins have proven less successful. Seeking to combine the immunogenicity of VLPs and the ease of production of plasmid DNA, we designed DNA vaccines expressing VLPs consisting of the MLV Gag and modified MLV Env proteins displaying T cell epitopes. We show here that such DNA vaccines are remarkably efficient immunogens for inducing cellular immune responses. In contrast to similar plasmids harboring a point mutation preventing VLP formation, they induce protection against a lethal viral challenge in mice. Thus, these "plasmo-retroVLPs" represent a promising second-generation DNA vaccine.

Abstract 4009 The ex vivo culture of Hematopoietic Stem Cells (HSC) with various combinations of cytokines can increase the number of mature hematopoietic cells that are theoretically capable of rapidly release neutrophils and platelet and reduce recovery duration post transplantation. In patients, the infusion of such cells has been reported, but the short-term effect was not clear. In a randomized, double blind study, we used expanded cells from 4×106/kg peripheral blood hematopoietic selected CD34+cells in comparison to a non manipulated graft containing the same number of CD34+ cells; we designed an ex vivo expansion protocol based on a cocktail of early or late acting cytokines with different culture duration in order to obtain progenitors at various stages of differentiation 1) primitive progenitors obtained from selected CD34+cells cultured for 8 days in presence of fetal liver tyrosine kinase 3 ligand (FLT3-ligand), stem cell factor (SCF), interleukin-3 (IL-3) and thrombopoietin (TPO), 50 ng/ml, each 2) committed megakaryocyte progenitors (Mks) obtained after culture for 10 days in presence of TPO et SCF (50 ng/ml, each) or 3) committed granulocytes and megakaryocyte (GMks) progenitors obtained after culture for 10 days in presence of TPO, SCF and G-CSF (100 ng/ml). Eighteen Non Hodgkin Lymphoma patients submitted to autologous transplantation after a myeloablative regimen consisting of AraC: Day (D)-6 to D-3: 200mg/m2/12h - VP16: 100mg/m2/12h: D-6 to D-3 - BCNU: 300mg/M2: D-6- Melphalan: 140mg/M2: D-2 could be evaluated. Patients in the Expansion Group received graft with 1×106/kg non manipulated cells combined with primitive progenitors issues from 2×106/kg CD34+ cells and Mks progenitors (Mks Expansion Group) or GMks progenitors (GMks Expansion Group) coming from 1×106/kg CD34+cells; unmanipulated cells were used as a source of immunocompetent cells. In the mean, patients of control group (n=10) received 1.3±0.9×108Total Nucleated Cells (TNC)/Kg, 2.7±1.2×106 CD34+/kg, 49±17×104/kg CFU-GM and 17.7±.7104CFU-Mks/kg; in the Mks (n=4) and GMks (n=4) Expansion group, they received respectively in the mean: 61.5±18.5 and 90.1±21.3×108TNC /Kg, 26.5±10.7 et 31± 11.2 x106CD34+/kg, 451± 188 et 557±216×104/kg CFU-GM, 358±212 et 39±18.1 x104/kg CFU-Mk. No cytokines were administered after transplantation. No toxicity was observed after cell infusion. The mean times to reach white blood cell (WBC) recovery (WBC >1x 109/l) was significantly shorter after administration of expanded cells, 14 (10–16), 12 (11–14) and 9 (9–10) days respectively in control, Mks and GMks Expansion Group (p=0.01). Median profound neutropenia (neutrophils25 or50×109/L) despite numerous mature megakaryocytes and CFU-Mks in the Mks Expansion Group; that could be related to the colonies size, very small from expanded cells. No secondary hypoplasia was observed during the 12 months follow-up. This study shows that in comparison with unmanipulated cells, peripheral blood haematopoietic cells expanded from similar doses of CD34+ cells accelerate neutrophil recovery without impairing long-term haematopoiesis and open interesting perspectives in the field of allogeneic cord blood cell transplantation. Disclosures: Leblond: Roche, Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Background: Vaginal ring devices are being actively developed for controlled delivery of HIV microbicides and as multi-purpose prevention technology (MPT) products combining hormonal contraception with prevention of HIV and other sexually transmitted diseases. Presently, there is no reliable method for monitoring user adherence in HIV vaginal ring trials; previous acceptability studies have included some type of participant self-reporting mechanism, which have often been unreliable. More objective, quantitative and accurate methods for assessing adherence are needed.Methods: A silicone elastomer vaginal ring containing an encapsulated miniature temperature recording device has been developed that can capture and store real-time temperature data during the period of designated use. Devices were tested in both simulated vaginal environments and following vaginal placement in cynomolgus macaques. Various use protocols and data sampling rates were tested to simulate typical patient usage scenarios. Results: The temperature logging devices accurately recorded vaginal temperature in macaques, clearly showing the regular diurnal temperature cycle. When environmental temperature and vaginal temperature was significantly different, the device was able to accurately pinpoint the insertion and removal times. Based on the data collected it was possible to infer removal periods as short as 5 min when the external environmental temperature was 25 °C. Accuracy increased with data sampling rate. Conclusions: This work provides proof-of-concept for monitoring adherence using a vaginal ring device containing an encapsulated temperature logger. The addition of one or more active agents into the ring body is not anticipated to affect the temperature monitoring function. A clinical study to compare self- reported user adherence data with that obtained by the device would be highly informative.