Search

Highlights • First study on patient acceptability of patient derived cell culture models. • First study to assess the feasibility of using patient derived kidney cancer 3D tumour models. • Study will inform the design of a clinical trial in personalised tumouroid response to therapy., Introduction ‘Personalised medicine’ aims to tailor interventions to the individual, and has become one of the fastest growing areas of cancer research. One of these approaches is to harvest cancer cells from patients and grow them in the laboratory, which can then be subjected to treatments and the response assessed. We have developed a 3D tumour model with a complex protein matrix that mimics the tumour stroma, cell to cell and cell-matrix interactions seen in vivo, called a tumouroid. In this study, we test the acceptability and feasibility of using this model to establish patient-derived tumouroids. Methods and analysis This is a first in-human study using prospective tissue and data collection of adult participants with confirmed or suspected renal cell carcinoma. The goals of the study are to assess patient acceptability to the use of patient-derived tumour models for future treatment decisions, and to assess the feasibility of generating patient-specific renal cancer tumouroids that can be challenged with drugs. These goals will be realised through the collection of tumour samples (expected n = 10), participant-completed questionnaires (expected n = 10), and in-depth semi-structured interviews with patients (expected n = 5). Collected multiregional tumour samples will be dissociated to isolate primary cells which are then expanded in vitro and incorporated into tumouroids. Drug challenge will ensue and the response will be categorised into “responder”, “weak responder”, and “non-responder”. Statistical analysis will be descriptive. Ethics and dissemination The study has ethical approval (REC reference 17/LO/1744). Findings will be made available to patients, clinicians, funders, and the National Health Service (NHS) through presentations at national and international meetings, peer-reviewed publications, social media and patient support groups. Trial registration Registered on ClinicalTrials.gov (NCT03300102).

International audience; Previous studies have shown targeting different tissues via the transcutaneous (TC) and intramuscular injection (IM) with or without electroporation (EP) has the potential to trigger immune responses to DNA vaccination. The CUTHIVTHER 001 Phase I/II randomized controlled clinical trial was designed to determine whether the mode of DNA vaccination delivery (TC+IM or EP+IM) could influence the quality and function of induced cellular immune responses compared to placebo, in an HIV positive clade B cohort on antiretroviral therapy (ART). The GTU®MultiHIV B DNA vaccine DNA vaccine encoded a MultiHIV B clade fusion protein to target the cellular response. Overall the vaccine and regimens were safe and well-tolerated. There were robust pre-vaccination IFN-γ responses with no measurable change following vaccination compared to placebo. However, modest intracellular cytokine staining (ICS) responses were seen in the TC+IM group. A high proportion of individuals demonstrated potent viral inhibition at baseline that was not improved by vaccination. These results show that HIV positive subjects with nadir CD4+ counts ≥250 on suppressive ART display potent levels of cellular immunity and viral inhibition, and that DNA vaccination alone is insufficient to improve such responses. These data suggest that more potent prime-boost vaccination strategies are likely needed to improve pre-existing responses in similar HIV-1 cohorts (This study has been registered at http://ClinicalTrials.gov under registration no. NCT02457689).

We need greater understanding of the mechanisms underlying protection against influenza virus to develop more effective vaccines. To do this, we need better, more reproducible methods of sampling the nasal mucosa. The aim of the current study was to compare levels of influenza virus A subtype-specific IgA collected using three different methods of nasal sampling. Samples were collected from healthy adult volunteers before and after LAIV immunization by nasal wash, flocked swabs and Synthetic Absorptive Matrix (SAM) strips. Influenza A virus subtype-specific IgA levels were measured by haemagglutinin binding ELISA or haemagglutinin binding microarray and the functional response was assessed by microneutralization. Nasosorption using SAM strips lead to the recovery of a more concentrated sample of material, with a significantly higher level of total and influenza H1-specific IgA. However, an equivalent percentage of specific IgA was observed with all sampling methods when normalized to the total IgA. Responses measured using a recently developed antibody microarray platform, which allows evaluation of binding to multiple influenza strains simultaneously with small sample volumes, were compared to ELISA. There was a good correlation between ELISA and microarray values. Material recovered from SAM strips was weakly neutralizing when used in an in vitro assay, with a modest correlation between the level of IgA measured by ELISA and neutralization, but a greater correlation between microarray-measured IgA and neutralizing activity. In conclusion we have tested three different methods of nasal sampling and show that flocked swabs and novel SAM strips are appropriate alternatives to traditional nasal washes for assessment of mucosal influenza humoral immunity.

International audience; Intradermal (i.d.) and intramuscular (i.m.) injections when administered with or without electroporation (EP) have the potential to tailor the immune response to DNA vaccination. This Phase I randomized controlled clinical trial in human immunodeficiency virus type 1-negative volunteers investigated whether the site and mode of DNA vaccination influences the quality of induced cellular and humoral immune responses following the DNA priming phase and subsequent protein boost with recombinant clade C CN54 gp140. A strategy of concurrent i.d. and i.m. DNA immunizations administered with or without EP was adopted. Subtle differences were observed in the shaping of vaccine-induced virus-specific CD4+ and CD8+ T cell-mediated immune responses between groups receiving: i.d.EP + i.m., i.d. + i.m.EP, and i.d.EP + i.m.EP regimens. The DNA priming phase induced 100% seroconversion in all of the groups. A single, non-adjuvanted protein boost induced a rapid and profound increase in binding antibodies in all groups, with a trend for higher responses in i.d.EP + i.m.EP. The magnitude of antigen-specific binding immunoglobulin G correlated with neutralization of closely matched clade C 93MW965 virus and Fc-dimer receptor binding (FcγRIIa and FcγRIIIa). These results offer new perspectives on the use of combined skin and muscle DNA immunization in priming humoral and cellular responses to recombinant protein.

Targeting of different tissues via transcutaneous (TC), intradermal (ID) and intramuscular (IM) injection has the potential to tailor the immune response to DNA vaccination. In this Phase I randomised controlled clinical trial in HIV-1 negative volunteers we investigate whether the site and mode of DNA vaccination influences the quality of the cellular immune responses. We adopted a strategy of concurrent immunization combining IM injection with either ID or TC administration. As a third arm we assessed the response to IM injection administered with electroporation (EP). The DNA plasmid encoded a MultiHIV B clade fusion protein designed to induce cellular immunity. The vaccine and regimens were well tolerated. We observed differential shaping of vaccine induced virus-specific CD4 + and CD8 + cell-mediated immune responses. DNA given by IM + EP promoted strong IFN-γ responses and potent viral inhibition. ID + IM without EP resulted in a similar pattern of response but of lower magnitude. By contrast TC + IM (without EP) shifted responses towards a more Th-17 dominated phenotype, associated with mucosal and epidermal protection. Whilst preliminary, these results offer new perspectives for differential shaping of desired cellular immunity required to fight the wide range of complex and diverse infectious diseases and cancers.

Optimum strategies to elicit and maintain antibodies at mucosal portals of virus entry are critical for the development of vaccines against human immunodeficiency virus (HIV). Here we show in non-human primates that a novel regimen of repeated intravaginal delivery of a non-adjuvanted, soluble recombinant trimeric HIV-1CN54 clade C envelope glycoprotein (gp140) administered in Carbopol gel can prime for B-cell responses even in the absence of seroconversion. Following 3 cycles of repeated intravaginal administration, throughout each intermenses interval, 3 of 4 macaques produced or boosted systemic and mucosally-detected antibodies upon intramuscular immunisation with gp140 formulated in AS01 adjuvant. Reciprocally, a single intramuscular immunisation primed 3 of 4 macaques for antibody boosting after a single cycle of intravaginal immunisation. Virus neutralising activity was detected against clade C and clade B HIV-1 envelopes but was restricted to highly neutralisation sensitive pseudoviruses.

To evaluate antibody specificities induced by simian immunodeficiency virus (SIV) versus human immunodeficiency virus type 1 (HIV-1) envelope antigens in nonhuman primate (NHP), we profiled binding antibody responses to linear epitopes in NHP studies with HIV-1 or SIV immunogens. We found that, overall, HIV-1 Env IgG responses were dominated by V3, with the notable exception of the responses to the vaccine strain A244 Env that were dominated by V2, whereas the anti-SIVmac239 Env responses were dominated by V2 regardless of the vaccine regimen.

Human cytomegalovirus can cause a diverse range of diseases in different immunocompromised hosts. The pathogenic mechanisms underlying these diseases have not been fully elucidated, though the maximal viral load during infection is strongly correlated with the disease. However, concentrating on single viral load measures during infection ignores valuable information contained during the entire replication history up to the onset of disease. We use a statistical model that allows all viral load data sampled during infection to be analysed, and have applied it to four immunocompromised groups exhibiting five distinct cytomegalovirus-related diseases. The results show that for all diseases, peaks in viral load contribute less to disease progression than phases of low virus load with equal amount of viral turnover. The model accurately predicted the time of disease onset for fever, gastrointestinal disease and pneumonitis but not for hepatitis and retinitis, implying that other factors may be involved in the pathology of these diseases.

To further investigate mechanisms of protective immunity that are induced by live, attenuated simian immunodeficiency virus (SIV), three macaques were infected with SIVmacGX2, a nef-disrupted molecular clone. In two of these animals, which expressed the MamuA*01 major histocompatibility complex class I allele, loss of functional activity against an SIV-Gag-encoded immunodominant cytotoxic T lymphocyte (CTL) epitope was observed following prolonged infection. Nonetheless, all three animals were resistant to challenge with an uncloned pool of wild-type SIVmac, whereas four naïve controls became infected. Tetramer staining revealed the rapid generation of CD8+ T-cell responses against gag- and tat-encoded immunodominant epitopes in MamuA*01+ challenge controls. The dynamics of these T-cell responses to the wild-type virus were similar to those observed following primary infection of the vaccine group with attenuated virus. In contrast, neither tetramer staining nor gamma interferon ELISpot assay revealed an immediate, systemic, anamnestic response in the wild-type-challenged, attenuated SIV-infected animals. Functional CTL capacity had not been lost in this group, as lytic activity was still evident 17 weeks after challenge. Both attenuated and wild-type viruses induced a disseminated CD8+ T-cell response, which was of a higher magnitude in lymphoid tissues than in the periphery. These results suggest that, at least as measured in the periphery, protection against wild-type infection that is induced by live, attenuated SIV is not dependent on a rechallenge-driven expansion of immunodominant epitope-specific CD8+ T cells and, therefore, pre-existing activity may be sufficient to prevent superinfection.

Objective: To investigate the effect of highly active antiretroviral therapy (HAART) on circulating Kaposi sarcoma (KS)-associated herpesvirus (KSHV) load in HIV-infected individuals with and without KS. Design: Twenty-nine HIV-infected subjects (21 with KS and 8 without KS) were recruited for the study; they were prospectively studied before and at regular intervals during HAART. Methods: Patients with KS were clinically assessed using Adult Clinical Trials Group (ACTG) criteria, and sequential blood samples were obtained from all patients for determination of plasma HIV-1 load, CD4 cell count, titer of antibody to KSHV, and KSHV load. Results: Ten of 21 patients with KS had a favorable KS response (complete response, 6; partial response, 4) with HAART alone. Of the 20 subjects with detectable KSHV viremia prior to HAART, over one half (12 [60%]) had an undetectable KSHV load with antiretroviral therapy. There was no significant difference between subjects receiving protease inhibitor- or nonnucleoside reverse transcriptase inhibitor-based treatment combinations. Achieving undetectable KSHV viremia is associated with a better clinical outcome in patients with KS. Conclusions: To our knowledge, we demonstrate for the first time that both protease inhibitor-based and nonnucleoside reverse transcriptase inhibitor-based antiretroviral treatment combinations may lead to an undetectable KSHV load and confirm that an undetectable KSHV load is associated with KS regression. There was no clear association between CD4 cell count response and KS response to HAART, but there was a significant relationship between HIV load response to HAART and clinical improvement of KS.

Following the introduction of highly active antiretroviral therapy (HAART), the incidence of Kaposi's sarcoma (KS) has significantly declined in human immunodeficiency virus type 1 (HIV-1)-positive (HIV-1+) individuals and clinical remission is often observed. We hypothesize that these effects are partly due to anti-KS-associated herpesvirus (KSHV) immune restoration. Here, 15-mer overlapping peptides from proteins K12 and K8.1 were used to identify novel KSHV-specific cytotoxic T-lymphocyte epitopes. Three immunogenic peptides, two lytic and one latent, were subsequently used to monitor the anti-KSHV CD8+T-cell responses in a cohort of 19 HIV-1+KSHV+/−KS+/−individuals during 52 weeks of HAART. KSHV and HIV-1 loads, KSHV antibody titers, and both CD4+and CD8+T-lymphocyte counts were enumerated. Prior to HAART, the total number of spot-forming cells (SFC) for all three peptides correlated with both CD4+and CD8+T-lymphocyte counts (P≤ 0.05) in the KSHV-positive KS-positive cohort (n= 11). Following 52 weeks of HAART, significant decreases in HIV-1 and KSHV loads were associated with significant increases in CD4+T-lymphocyte counts and number of SFC for the three KSHV-specific peptides. Although these increases were modest in comparison to the number of SFC observed with the HIV-1gagpeptide SLYNTVATL, they represented a fourfold increase from the baseline, continuing an upward trend to week 52.

It is accepted that an effective prophylactic HIV-1 vaccine is likely to have the greatest impact on viral transmission rates. As previous reports have implicated DNA-priming, protein boost regimens to be efficient activators of humoral responses, we sought to optimize this regimen to further augment vaccine immunogenicity. Here we evaluated single versus concurrent intradermal (i.d.) and intramuscular (i.m.) vaccinations as a DNA-priming strategy for their abilities to elicit humoral and cellular responses against a model HIV-1 vaccine antigen, CN54-gp140. To further augment vaccine-elicited T and B cell responses, we enhanced cellular transfection with electroporation and then boosted the DNA-primed responses with homologous protein delivered subcutaneously (s.c.), intranasally (i.n.), i.m., or transcutaneously (t.c.). In mice, the concurrent priming regimen resulted in significantly elevated gamma interferon T cell responses and high-avidity antigen-specific IgG B cell responses, a hallmark of B cell maturation. Protein boosting of the concurrent DNA strategy further enhanced IgG concentrations but had little impact on T cell reactivity. Interestingly protein boosting by the subcutaneous route increased antibody avidity to a greater extent than protein boosting by either the i.m., i.n., or t.c. route, suggesting that this route may be preferential for driving B cell maturation. Using an alternative and larger animal model, the rabbit, we found the concurrent DNA-priming strategy followed by s.c. protein boosting to again be capable of eliciting high-avidity humoral responses and to also be able to neutralize HIV-1 pseudoviruses from diverse clades (clades A, B, and C). Taken together, we show that concurrent multiple-route DNA vaccinations induce strong cellular immunity, in addition to potent and high-avidity humoral immune responses. IMPORTANCE The route of vaccination has profound effects on prevailing immune responses. Due to the insufficient immunogenicity and protection of current DNA delivery strategies, we evaluated concurrent DNA delivery via simultaneous administration of plasmid DNA by the i.m. and i.d. routes. The rationale behind this study was to provide clear evidence of the utility of concurrent vaccinations for an upcoming human clinical trial. Furthermore, this work will guide future preclinical studies by evaluating the use of model antigens and plasmids for prime-boost strategies. This paper will be of interest not only to virologists and vaccinologists working in the HIV field but also to researchers working in other viral vaccine settings and, critically, to the wider field of vaccine delivery.

Cytomegalovirus (CMV) continues to be a major problem post-transplantation; early markers for predicting patients at risk of CMV disease are needed. Peak CMV load in the blood correlates with CMV disease but frequently occurs too late to provide prognostic information.359 transplant recipients (162 liver, 87 renal, and 110 bone marrow) were prospectively monitored for CMV DNA in the blood with qualitative and quantitative PCR. 3873 samples were analysed. The CMV load in the first PCR-positive sample and the rate of increase in CMV load in blood during the initial phase of replication were assessed as risk factors for CMV disease using logistic regression.127 of the 359 patients had CMV DNA in the blood and 49 developed CMV disease. Initial viral load correlated significantly with peak CMV load (R2=0.47, p=0.001) and with CMV disease (odds ratio 1.82 [95% CI 1.11-2.98; p=0.02; 1.34 [1.07-1.68], p=0.01, and 1.52 [1.13-2.05], p=0.006, per 0.25 log10 increase in viral load for liver, renal, and bone-marrow patients, respectively). The rate of increase in CMV load between the last PCR-negative and first PCR-positive sample was significantly faster in patients with CMV disease (0.33 log10 versus 0.19 log10 genomes/mL daily, p0.001). In multivariate-regression analyses, both initial CMV load and rate of viral load increase were independent risk factors for CMV disease (1.28 [1.06-1.52], p=0.01, per 0.25 log10 increase in CMV load and 1.52 [1.06-2.17], p=0.02, per 0.1 log10 increase in CMV load/mL daily, respectively).CMV load in the initial phase of active infection and the rate of increase in viral load both correlate with CMV disease in transplant recipients; in combination, they have the potential to identify patients at imminent risk of CMV disease.

Cytomegalovirus (CMV) is generally described as a slowly replicating virus. During studies of immunocompromised patients, we observed rapid changes in the quantity of CMV DNA present in serial blood samples by quantitative-competitive polymerase chain reaction commensurate with a doubling time of

Multiplex RT-PCR analysis of human cytomegalovirus (HCMV) replication in human fibroblasts showed transcription of the natural killer (NK) cell decoy gene, UL18, from 72 h onwards. Transcription of glycoprotein B (gpUL55; a late gene) occurred from early time-points and peaked at 24 h post-infection. UL18 mRNA was also detected in the peripheral blood mononuclear cells of organ transplant recipients with HCMV viraemia, especially those with HCMV DNA virus loads greater than 10(5) genomes/ml whole blood. Thus, UL18 is produced via a low abundance transcript late during the infectious cycle at a time coincidental with the increased risk of NK cell lysis as a consequence of class I HLA down-regulation.

Using a unique vaccine antigen matched and single HIV Clade C approach we have assessed the immunogenicity of a DNA-poxvirus-protein strategy in mice and rabbits, administering MVA and protein immunizations either sequentially or simultaneously and in the presence of a novel TLR4 adjuvant, GLA-AF. Mice were vaccinated with combinations of HIV env/gag-pol-nef plasmid DNA followed by MVA-C (HIV env/gag-pol-nef) with HIV CN54gp140 protein (+/-GLA-AF adjuvant) and either co-administered in different muscles of the same animal with MVA-C or given sequentially at 3-week intervals. The DNA prime established a population of B cells that were able to mount a statistically significant anamnestic response to the boost vaccines. The greatest antigen-specific antibody response was observed in animals that received all vaccine components. Moreover, a high proportion of the total mucosal IgG (20 - 50%) present in the vaginal vault of these vaccinated animals was vaccine antigen-specific. The potent elicitation of antigen-specific immune responses to this vaccine modality was also confirmed in rabbits. Importantly, co-administration of MVA-C with the GLA-AF adjuvanted HIV CN54gp140 protein significantly augmented the antigen-specific T cell responses to the Gag antigen, a transgene product expressed by the MVA-C vector in a separate quadriceps muscle. We have demonstrated that co-administration of MVA and GLA-AF adjuvanted HIV CN54gp140 protein was equally effective in the generation of humoral responses as a sequential vaccination modality thus shortening and simplifying the immunization schedule. In addition, a significant further benefit of the condensed vaccination regime was that T cell responses to proteins expressed by the MVA-C were potently enhanced, an effect that was likely due to enhanced immunostimulation in the presence of systemic GLA-AF.

Background Gp41 of the HIV envelope, especially the MPER, contains highly conserved epitopes recognized by neutralizing monoclonal antibodies such as D5, 2F5 and 4E10. The correct presentation of the antigens is considered to be key for eliciting neutralizing immune response. The lipid bilayer of liposomes can mimic the virus surface and therefore provides the appropriate presentation of this membrane protein. In addition, liposomes can integrate the adjuvant monophosphoryl lipid A (MPLA).

Background Prime boost vaccination studies employing plasmid DNA have been shown to significantly increase vaccine-induced immune responses with the added advantage of increasing vaccine-induced immune breadth. Furthermore, recent studies have shown that prevailing immune responses to DNA based vaccines can be substantially augmented by the immediate application of short pulses of electric current (electroporation) at the DNA injection site. Methods We vaccinated mice with DNA via the intradermal (ID), intramuscular (IM) or combined (ID + IM) routes with and without electroporation (EP), using a Gene Transfer Unit (GTU ® ) plasmid expressing CN54gp140. Some mice were further boosted with recombinant trimeric CN54gp140. Female BALB/c mice (n=8) were vaccinated every three weeks. Serum and vaginal lavage samples were collected 1 week post each vaccination and tested by ELISA for anti-HIV-gp140 specific IgG and IgA. Spleen derived T cells were evaluated for antigen reactivity by IFN-g ELISpot using 2 peptide pools of 15mers overlapping by 11, spanning the full length of the gp140 molecule. Results Antigen-specific T lymphocyte IFN-g cytokine expression and humoral antibody profiles were different after either ID or IM immunization or the combined route vaccinations. Moreover, we found that EP added an additional level of complexity by influencing T and B cell responses both quantitatively and qualitatively. As expected, these vaccinations did not elicit biologically significant levels of mucosal antigen-specific antibody responses. Conclusion Multi-route vaccinations can enhance and subtly alter vaccine-induced B and T lymphocyte immunity. All of the vaccine routes benefited from enhancement to the DNA immunization using in vivo EP and the elicited immunity was again influenced by the enhanced transfection efficiency. These strategies are likely to be useful in the future design of vaccine modalities that seek to generate specific immune responses.

We conducted a phase 1 double-blind randomised controlled trial (RCT) of a HIV-1 envelope protein (CN54 gp140) candidate vaccine delivered vaginally to assess immunogenicity and safety. It was hypothesised that repeated delivery of gp140 may facilitate antigen uptake and presentation at this mucosal surface. Twenty two healthy female volunteers aged 18-45 years were entered into the trial, the first receiving open-label active product. Subsequently, 16 women were randomised to receive 9 doses of 100 µg of gp140 in 3 ml of a Carbopol 974P based gel, 5 were randomised to placebo solution in the same gel, delivered vaginally via an applicator. Participants delivered the vaccine three times a week over three weeks during one menstrual cycle, and were followed up for two further months. There were no serious adverse events, and the vaccine was well tolerated. No sustained systemic or local IgG, IgA, or T cell responses to the gp140 were detected following vaginal immunisations. Repeated vaginal immunisation with a HIV-1 envelope protein alone formulated in Carbopol gel was safe, but did not induce local or systemic immune responses in healthy women. TRIAL REGISTRATION: ClinicalTrials.gov NCT00637962.

Background Vaccine-mediated prevention of primary infection with HIV likely requires the sustained production of antibody at mucosal portals of entry. We determined the dynamics of systemic and mucosal antibody production in macaques following a novel approach of repeat mucosal immunisation by delivery of trimeric recombinant HIV1 clade C gp140 in a gel formulated for vaginal delivery either alone or in combination with systemic immunisation via the intramuscular route using adjuvanted gp 140.

Vaccine-mediated prevention of primary infection with human immunodeficiency virus (HIV) may require the sustained production of antibody at mucosal portals of entry. Here, we describe a novel approach of repeated mucosal immunization by delivering an HIV-1 envelope glycoprotein (gp) in a gel formulated for intravaginal delivery. Rabbits were immunized over one to three 19-day cycles of intravaginal dosing with soluble recombinant trimeric HIV-1 clade C gp140 administered in Carbopol gel. The formulation was well tolerated. A single immunization cycle induced immunoglobulin G (IgG) antibody detected in the serum and female genital tract, and titers were boosted on further immunization. Vaccine-induced serum antibodies neutralized the infectivity of a pseudovirus carrying a heterologous clade C envelope. Our data prove the concept that repeated exposure of the female genital tract to HIV envelope can induce mucosally detectable antibody.

Background The rectum is particularly vulnerable to HIV transmission having only a single protective layer of columnar epithelium overlying tissue rich in activated lymphoid cells; thus, unprotected anal intercourse in both women and men carries a higher risk of infection than other sexual routes. In the absence of effective prophylactic vaccines, increasing attention is being given to the use of microbicides and preventative antiretroviral (ARV) drugs. To prevent mucosal transmission of HIV, a microbicide/ARV should ideally act locally at and near the virus portal of entry. As part of an integrated rectal microbicide development programme, we have evaluated rectal application of the nucleotide reverse transcriptase (RT) inhibitor tenofovir (PMPA, 9-[(R)-2-(phosphonomethoxy) propyl] adenine monohydrate), a drug licensed for therapeutic use, for protective efficacy against rectal challenge with simian immunodeficiency virus (SIV) in a well-established and standardised macaque model. Methods and Findings A total of 20 purpose-bred Indian rhesus macaques were used to evaluate the protective efficacy of topical tenofovir. Nine animals received 1% tenofovir gel per rectum up to 2 h prior to virus challenge, four macaques received placebo gel, and four macaques remained untreated. In addition, three macaques were given tenofovir gel 2 h after virus challenge. Following intrarectal instillation of 20 median rectal infectious doses (MID50) of a noncloned, virulent stock of SIVmac251/32H, all animals were analysed for virus infection, by virus isolation from peripheral blood mononuclear cells (PBMC), quantitative proviral DNA load in PBMC, plasma viral RNA (vRNA) load by sensitive quantitative competitive (qc) RT-PCR, and presence of SIV-specific serum antibodies by ELISA. We report here a significant protective effect (p = 0.003; Fisher exact probability test) wherein eight of nine macaques given tenofovir per rectum up to 2 h prior to virus challenge were protected from infection (n = 6) or had modified virus outcomes (n = 2), while all untreated macaques and three of four macaques given placebo gel were infected, as were two of three animals receiving tenofovir gel after challenge. Moreover, analysis of lymphoid tissues post mortem failed to reveal sequestration of SIV in the protected animals. We found a strong positive association between the concentration of tenofovir in the plasma 15 min after rectal application of gel and the degree of protection in the six animals challenged with virus at this time point. Moreover, colorectal explants from non-SIV challenged tenofovir-treated macaques were resistant to infection ex vivo, whereas no inhibition was seen in explants from the small intestine. Tissue-specific inhibition of infection was associated with the intracellular detection of tenofovir. Intriguingly, in the absence of seroconversion, Gag-specific gamma interferon (IFN-γ)-secreting T cells were detected in the blood of four of seven protected animals tested, with frequencies ranging from 144 spot forming cells (SFC)/106 PBMC to 261 spot forming cells (SFC)/106 PBMC. Conclusions These results indicate that colorectal pretreatment with ARV drugs, such as tenofovir, has potential as a clinically relevant strategy for the prevention of HIV transmission. We conclude that plasma tenofovir concentration measured 15 min after rectal administration may serve as a surrogate indicator of protective efficacy. This may prove to be useful in the design of clinical studies. Furthermore, in vitro intestinal explants served as a model for drug distribution in vivo and susceptibility to virus infection. The finding of T cell priming following exposure to virus in the absence of overt infection is provocative. Further studies would reveal if a combined modality microbicide and vaccination strategy is feasible by determining the full extent of local immune responses induced and their protective potential.

Objectives : Despite life-long maintenance therapy, cytomegalovirus (CMV) retinitis frequently progresses in patients with AIDS. Virological markers that could clarify pathogenesis and identify risk factors for progression are required. Design and methods : We prospectively recruited 45 patients with CMV retinitis. Blood and urine samples were collected before and after induction therapy, and on a monthly basis thereafter during routine medical and ophthalmological assessment, and at any time retinitis progressed. CMV load was measured by quantitative-competitive polymerase chain reaction (PCR). Results : The median time to first progression of retinitis was 78 days and to death was 8.7 months. Eighty-five per cent of patients who were PCR-positive at diagnosis of retinitis became PCR-negative after 21 days of ganciclovir induction therapy. Six patients who remained PCR-positive after 21 days of treatment had a significantly higher CMV load at presentation (P = 0.005), and a shorter time to first progression of retinitis of 40 days. High CMV loads in blood at presentation were associated with a shorter time to progression (P = 0.16 ; relative hazard, 1.57) and a significantly shorter time to death (P = 0.004 ; relative hazard, 1.76). This significant relationship with survival remained after adjustment for potential confounding variables (CD4 count, age, method of drug administration). Conclusions : We conclude that CMV load in the blood of AIDS patients is an important factor in the pathogenesis of retinitis, and quantification of CMV could be used to both select patients for controlled clinical trials and to optimize individual anti-CMV induction therapy.

Background We assessed the antigen-specific CD4 and CD8 T lymphocyte responses elicited by a unique vaccine antigen matched and single Clade C DNA-poxvirus-protein regimen in mice and determined the immunomodulatory effect of the novel micellar formulation of a synthetic TLR4 ligand, GLA-AF (GLA) administered either sequentially or simultaneously with the MVA and protein. Methods Groups of 10 BALB/c mice were primed with plasmid DNA encoding CN54 env/gag-pol-nef and then boosted with MVA-C (env-gag-pol-nef) and CN54gp140 protein with or without GLA-AF. The MVA and protein were either given sequentially at 3 weekly intervals or simultaneously in different legs at 3 and 6 weeks. Cellular responses were assessed at necropsy three weeks after the final immunization. Splenocytes were harvested and analysed for antigen-specific T cell responses using peptide pools spanning the Env and Gag proteins by intracellular cytokine staining and CFSE labelling. Multiparametric flow profiles were analysed using FlowJo and data sets were organized and charted using the SPICE software. Results GLA adjuvanted CN54gp140 substantially influenced the antigen-specific T lymphocyte cytokine expression profiles and proliferative responses in animals primed with DNA and boosted by MVA or those immunized with MVA alone. We observed adjuvant-induced changes in the polyfunctional IFN-g ,T NF-a and IL-2 expression profiles of both the CD4 and CD8 T lymphocyte populations and differential responses to separate peptide pools. Conclusion We have shown that the GLA adjuvant alters both the degree and the nature of the antigen-specific T lymphocyte responses and that these immunomodulatory changes are critically dependent upon the timing of application. These effects are likely due to either the presence of systemic GLA providing a general immunostimulatory environment or enhanced T cell priming in the local draining lymph nodes. This ability to tailor CN54gp140-specific T cell immunity is a valuable tool to be exploited in vaccine design.

Methods We have expressed the soluble form of the HIV envelope of the C/B’ strain 97/CN/54 in CHO cells. The production process consisted of a large-scale fed-batch fermentation, an antibody-based affinity chromatography plus additional purifying steps. CN54gp140 was extensively characterized for purity and identity. All glycosylation sites were characterized by mass spectrometry. Immunogenicity and safety were evaluated in mice, rabbits, minipigs, sheep as well as non-human primates. The antigen was formulated for clinical phase I studies in Tris buffer (MUCOVAC I, HIVIS07) in HEC gel (MUCOVAC I) as well as chemically conjugated to hsp70 (MUVAPRED).