Influenza is a major public health threat worldwide, and current vaccines and antivirals are not always effective or available. We report that intranasal delivery of a novel, computationally engineered small protein (HB36.6) designed to bind the hemagglutinin stalk region neutralizes a broad range of Group 1 influenza strains in vitro and affords protection in mice lethally challenged with H1N1 and H5N1 influenza viruses. HB36.6 afforded 100% protection from lethality in mice when given as a single prophylactic dose up to 72 hrs in advance of viral challenge, and significantly reduced weight loss and accelerated recovery from disease when administered as daily therapeutic doses after challenge. A single dose of HB36.6 administered post-challenge also afforded superior protection when compared to 10 doses (2x daily for 5 days) of a lead marketed antiviral, Oseltamivir. Furthermore, when low doses of HB36.6 were combined with Oseltamivir, we observed synergistic protection against lethal challenge in mice that was superior to either antiviral alone. HB36.6 also suppressed the cytokine storm associated with influenza disease and decreased viral burden. In contrast to monoclonal antibodies and antiviral peptides, protection was independent of engagement with the FcγR or stimulation of a host antiviral cytokines indicating the protein protects via direct interaction with the virus but not the host, a result that suggests potential for use in immune-compromised or elderly who comprise the majority of influenza cases each year. Preliminary studies in ferrets also show that HB36.6 reduces viral replication and disease in ferrets challenged via aerosol with a highly virulent pandemic H1N1 strain. These results support computational design as a new approach for the discovery and development of novel antivirals for influenza and other infectious diseases.
Aim
Recent data suggested that failure to achieve SVR with DAA therapy was usually due to relapse and is mostly associated with the emergence of resistance associated variants (RAVs). The aim of this study was to investigate the prevalence and characterization of NS5A RAVs in 1,500 patients infected with HCV genotype 1, 3 and 4 treated by DAA therapy including anti-NS5A in 4 French referent liver centers.
Materials and methods
From January 2014 to September 2015, 1,500 patients infected with HCV genotype 1, 3 and 4 were exposed to NS5A inhibitors (LDV/SOF, DCV/SOF, DCV/ASU). In the 4 French referent liver centers: Marseille, Lyon, Grenoble, St Laurent du Var, 22 (1.5%) of them relapsed. Prevalence of NS5A RAVs/polymorphisms was analyzed using Sanger sequencing. HCV subtypes and drug-resistance interpretation were identified using DeepChek-HCV software.
Results
Numbers and characteristics of NS5A class RAVs (variants at any position associated with resistance to any NS5A inhibitors) from different genotypes are shown in Table O2.1. Main G1a RAVs detected were M28A, Q30H/K/R, L31M, H58D, Y93F/H/N; main G1b RAVs detected were L31F/M/V and Y93H; main G3a RAVs detected was Y93H; and main G4d RAVs detected was L28V. For only one G3a patient, no mutation was detectable at viral failure. Y93H was the most prevalent RAVs (55%) followed by L31M (23%). Ten out of 22 (45%) patients presented more than one RAVs which confers a higher level of resistance. No specific RAVs profiles based on the different NS5A inhibitors was found.
Conclusions
NS5A RAVs with high level of cross-resistance among NS5A inhibitors were found at the time of viral failure in more than 95% of patients whatever the genotype. These results strongly suggest that for patients who previously failed a regimen containing an NS5A inhibitor, NS5A RAVs screening is recommended when considering retreatment with a regimen containing an NS5A inhibitor.
Table O2.1. Resistance associated variants
|
The cost of direct-acting antivirals (DAA) against chronic hepatitis C viral infection has led the French government to condition their reimbursement to the approval of treatment by a multidisciplinary committee (MC) serving in one of the 34 national hepatitis reference centers. We describe DAA attribution in this setting of care and first data of efficacy on a large cohort of HCV-infected patients in real life.
Physicians were to fill and send an electronic application form respecting patient's anonymity, in order to get approval to start DAA-based treatment. Clinical/biological data, therapeutic strategy, drug regimen/duration were collected and analyzed from March 2014 to July 2015.
113 physicians filled out a total of 1155 forms for 809 men (70%) and 346 women (30%), aging in average 57±10 and 64±12 years, respectively. 257 patients (23%) were HIV-co-infected (HCV/HIV), and 104 (9%) had received (7%) or were waiting for liver transplantation (2%). 508 patients (44%) were HCV treatment-naive and 647 (56%) were pre-treated. MC decided to initiate DAA therapy in 95% of treatment requests.
In HCV/HIV patients, 59% had genotype (G) 1 (36% 1a, 8% 1b) and 23% had G4. In non-HCV/HIV, 68% had G1 (27% 1a, 26% 1b) and 9% had G4. G3 repartition was equal in both populations (17%). 95% of G1/G4 patients received a 12-week (w) treatment (SOF/LDP: 63%; SOF/SIM: 12%; OBT/PTP/r/DBV: 10%; SOF/LDP/RBV: 8%). 75% of G3-patients had 12-w treatment (SOF/DCV: 56%; SOF/LDP/RBV: 26%), and 25% had 24-w (SOF/DCV+/-RBV: 74%). HCV/HIV patients were treated at earlier fibrosis stages compared to non-HCV/HIV (F0–F2:23% vs 9%; F3: 23% vs 35%; F4: 54% vs 56%, respectively). Overall sustained virologic response (SVR) rates were 92% for both 4 and 12 w after treatment completion.
This setting of care allowed harmonization of DAA-regimens with experts’ recommendations and financial issues, an epidemiologic description of HCV-infected people treated in France and the response rate to DAAs in real life practice.
Introduction
Early diagnosis and prompt ART initiation reduce disease progression, prevent secondary transmission and represent the only undisputed strategy to curtail HIV reservoir size. Primary HIV infection (PHI) provides a window of opportunity for early intervention and here we determine the clinical and socio-demographic factors associated with treatment initiation.
Methods
A total of 336 adult HIV-1-infected individuals participating in the Montreal Primary HIV Infection Study (1996–2014) with an estimated date of HIV acquisition of less than 180 days were studied. Clinical and socio-demographic characteristics of PHI study participants initiating (n=219) or not (n=117) ART within the first six months were compared. Patients were recruited in three community clinics (165) and in five hospital centers (171) throughout Montreal.
Results
Majority of the PHI participants were male (95.7%), MSM (78.7%), Caucasian (81.1%), single (45.8%) with an average age of 36±10 years. They presented with a median CD4 T-cell count of 510 cells/uL, CD8 T-cell count of 810 cells/uL and a viral load of 4.6 log-copies/mL. Early ART initiation was observed in 65.4% of PHI participants. The proportion of patients who initiate ART during the first six months was about 63%, 50%, 71% and 91% for every five year interval. Gender, ethnicity, usage of IV drugs and number of sexual partners in the last three months, being followed at community medical clinics or hospitals was not associated with early ART initiation. In contrast, lower income and being unemployed was associated with delayed ART initiation (p=0.021) and was consistent over time.
Conclusions
Lower socio-economic status independently delays ART initiation in recently diagnosed individuals. Advancement in ART and results from clinical trials supporting early ART initiation are key drivers for the very early treatment initiation. Such information will be relevant to scale up strategies aiming at a functional cure.
Aim
Interferon (IFN)-stimulated genes are produced following HIV infection and limit viral replication, but viral proteins or complex mechanisms counteract their effect. The IFN-induced protein kinase RNA-activated (PKR) represses the expression of several viruses and acts as a potent HIV-1 inhibitor by the phosphorylation of the translation initiation factor eIF2α. PKR is activated at the beginning of HIV-1 infection and deactivated when the virus replicates due to the activity of the viral Tat protein, the TAR RNA Binding protein, the adenosine deaminase (ADAR1) and the PKR activator (PACT), which are all RNA binding proteins (Burugu et al., Virus Res. 2014, 193:65). Our aim is to determine the status of PKR activation and ADAR1 expression in patient cells and to elucidate if ADAR1, PACT and Tat act as a multiprotein complex to prevent PKR activation in HIV-replicating cells.
Methods
We compared the activation of PKR and the expression of ADAR1 and PACT in uninfected subjects, HIV-infected naïve patients and HIV-infected treated patients by western blots. We performed immunoprecipitations (IPs) to determine the complex formation around PACT and PKR. We performed in vitro kinase and transient transfection assays to evaluate the role of each protein on PKR activation.
Results
We observed an inverse correlation between PKR activation and ADAR1 expression in HIV-infected patients. IPs from cotransfected cells showed a multiprotein complex between PKR, ADAR1, PACT and Tat. By in vitro kinase assays and transient transfection assays, we observed that ADAR1, PACT, Tat and TAR have a combined inhibition of PKR activation correlated with increased translation.
Conclusions
The strong PKR inhibition during HIV-1 replication can be explained by the formation of a multiprotein complex with ADAR1, PACT and Tat proteins. ADAR1 induction and the formation of this complex in patients may contribute to viral persistence by decreasing the natural innate immunity against HIV.
Introduction
HIV-1 reservoir impedes virus clearance in patients under combination antiretroviral therapy (cART). Activation of HIV expression from latent reservoirs is a part of proposed strategies that may potentially lead to a decrease in the size of the reservoirs. We grouped and analyzed patient data from our two most recent reactivation studies (Darcis G, Kula A, Bouchat S et al. An In-depth comparison of latency-reversing agent combinations in various in vitro and ex vivo HIV-1 latency models identified bryostatin-1+JQ1 and ingenol-B+JQ1 to potently reactivate viral gene expression. PLoS Pathog 2015; 11: e1005063; Bouchat S, Delacourt N, Kula A et al. Sequential treatment with 5-aza-2’-deoxycytidine and deacetylase inhibitors reactivates HIV-1. EMBO Mol Med 2015.) to investigate the role of the size of the HIV-1 reservoir in the reactivation capacity by latency reversing agents (LRAs) ex vivo.
Materials and methods
Reactivation tests were carried out in cultures of CD8+-depleted PBMCs or in resting CD4+ T cell cultures isolated from blood of 43 and 30 cART-treated aviremic HIV+ individuals, respectively. HIV-1 reservoir size was assessed by quantification of total HIV-1 DNA.
Results
We strongly established a statistically significant positive correlation between the size of the HIV-1 reservoir and the frequency of positive HIV-1 recovery measurements in response to various LRAs in ex vivo cultures of CD8+-depleted PBMCs. We next demonstrated a statistically relevant positive correlation between the size of the HIV-1 reservoir and the median level of extracellular HIV RNA measured in the supernatants of tested conditions for each patient. We confirmed these two correlations in ex vivo cultures of resting CD4+ T cells. However, we identified patients characterized by a very low or extremely high reactivation capacity relative to their reservoir size. The understanding of the determinants involved in this patient-specific reactivation variation is a key challenge in the race for HIV durable remission.
Conclusions
The reactivation capacity by LARs ex vivo correlates with the size of the HIV-1 reservoir.
Introduction
Sustained transmission over 1 year and reseeding of Ebola virus during the recent West African outbreak highlight the need for vaccines conferring long-term protection. This study evaluated the safety and immunogenicity of heterologous prime-boost immunization regimens using Ad26.ZEBOV and multivalent modified vaccinia Ankara-vectored (MVA-BN®-Filo) Ebola vaccines aimed at inducing sustained immune responses.
Materials and methods
Phase I, randomized, placebo-controlled, observer-blind, single-center US-based study (NCT02325050). Healthy adults (n=92) were randomized in six groups to receive MVA-BN-Filo prime (1x10ˆ8 50% tissue culture infective dose [TCID50]) / Ad26.ZEBOV boost (5x10ˆ10 viral particles [vp]) at 14-, 28- or 56-day intervals, Ad26.ZEBOV/MVA-BN-Filo (28-day interval), Ad26.ZEBOV/Ad26.ZEBOV or MVA-BN-Filo/MVA-BN-Filo (14-day intervals). Participants were further randomized within groups to vaccine or placebo. We report follow-up of the subjects through 180 days post prime.
Results
No serious adverse events (AEs), AEs leading to discontinuation or deaths occurred. For all schedules, the most frequent local AE was low-grade injection-site pain and systemic AEs were myalgia, fatigue, and headache. Humoral and cellular immune responses were evident after Ad26.ZEBOV prime and increased significantly after MVA-BN-Filo boost. The MVA/Ad26 schedule produced comparable immune responses post-boost. All heterologous regimens induced complete binding antibody responses 21 days post-boost (Table O7.1), with antibody and T-cell responses sustained to 180 days.
Conclusions
Heterologous prime boost regimens with Ad26.ZEBOV and MVA-BN-Filo Ebola vaccines were well tolerated and highly immunogenic in healthy adults, regardless of administration sequence or interval. This approach represents a promising new immunization strategy to induce long-term immune responses against Ebola.
Table O7.1. Binding antibody responses by Ebola vaccine regimen on boost day 15, 29 and 57
| Study vaccine regimen | Boost Day 15 | Boost Day 29 | Boost Day 57 | Combined Placebo Boost Days 15, 29 and 57 | |||
|---|---|---|---|---|---|---|---|
| MVA/Ad26 | MVA/MVA | Ad26/Ad26 | MVA/Ad26 | Ad26/MVA | MVA/Ad26 | ||
| (N=15) | (N=9) | (N=9) | (N=15) | (N=15) | (N=15) | (N=14) | |
| Anti-EBOV glycoprotein IgG ELISA | |||||||
| Day of boost (pre-boost) | |||||||
| GMC (95% CI) ELISA units/ml | 22 | 44 | 142 | 55 | 478 | 54 | N/A |
| (17–28) | (6–340) | (50–409) | (25–122) | (294–776) | (16–175) | ||
| Day 21 post boost | |||||||
| GMC (95% CI) ELISA units/ml | 4418 | 59 | 848 | 6987 | 2977 | 14048 | 18 |
| (3135–6225) | (9–402) | (492–1462) | (4916–9931) | (1951–4541) | (7982–24725) | (18–18) | |
| Responders, % | 100 | 33 | 100 | 100 | 100 | 100 | 0 |
| IFNγ+ T-cell response (ELISPOT) | |||||||
| Day of boost (pre-boost) | |||||||
| Median (IQR) SFU/106 PBMC | 52 | 25 | 95 | 25 | 95 | 25 | N/A |
| (25–117) | (25–25) | (57–307) | (25–57) | (25–510) | (25–25) | ||
| Day 21 post boost | |||||||
| Median (IQR) SFU/106 PBMC | 577 | 25 | 265 | 375 | 600 | 153 | 25 |
| (237–1362) | (25–107) | (107–580) | (152–607) | (225–1298) | (25–358) | (25–25) | |
| Responders, % | 93 | 22 | 67 | 67 | 87 | 47 | 0 |
CI: confidence interval; ELISA: enzyme-linked immunosorbent assay; ELISpot; enzyme-linked immunospot; IQR: interquartile range; BMC: peripheral blood mononuclear cell; SFU: spot-forming units
Aim
In France, the epidemiology of STI has evolved considerably as a result of a rise in gonorrhoea since the end of the 1990s, a resurgence of syphilis since 2000 and the emergence of rectal lymphogranuloma venereum (LGV) since 2002. The objective was to monitor epidemiological trends and characteristics of these 3 STIs in men having sex with men (MSM) from 2012 to 2014.
Materials and methods
National surveillance of gonorrhoea and syphilis (primary, secondary and early latent) is based on a voluntary clinicians network (mainly in STI clinics and hospital outpatient consultations) while LGV are reported by both laboratories and clinicians. Data collected after patient's informed consent included: age, gender, sexual orientation, symptoms, HIV status, dark field and serologic test for syphilis, culture and PCR for gonorrhoea, specific PCR for LGV. Trends were monitored using constantly participating sites.
Results
Between 2012 and 2014, 896 LGV, 3,164 syphilis and 3,648 gonorrhoea were reported. MSM account for 98% (excluded cases with unknown sexual orientation), 85% and 55% of the cases respectively. Number of STI diagnoses among MSM increased from 2012 to 2014 by 95% for LGV, 47% for syphilis and 98% for gonorrhoea. MSM diagnosed with syphilis or gonorrhoea were mostly born in France (Table O8.1). The profile of MSM diagnosed for LGV was specific with a higher median age and a high proportion of HIV-positive people.
Conclusions
Surveillance data indicate high increases in the number of syphilis, gonorrhoea and LGV among MSM. Improvement of testing policies (testing extra-genital strains, nucleic acid testing) might partially explain the rising number of STI detection. Nevertheless, these increases result from the rising of highly risky behavior, particularly among HIV-infected MSM. Within the context of combination HIV prevention (screening, condom, PreP and Tasp), early testing, prompt treatment and condom use still remain the corner stones of STI prevention.
Table O8.1. Characteristics of documented cases of LGV, syphilis and gonorrhea in MSM, France, 2012–2014
| LGV | Syphilis | Gonorrhea | |
|---|---|---|---|
| Number of diagnoses | 438 | 2691 | 2022 |
| Median age (year) | 40 | 36 | 28 |
| Birth in France (%) | – | 79 | 80 |
| Symptoms (%) | 94 | 64 | 64 |
| HIV+ (%) | 79 | 38 | 15 |
Aim
In order to reduce the missed opportunities for HIV diagnosis campaigns are proposed for screening using rapid diagnostic test (RDT), aimed at people most exposed to HIV risks and furthest removed from health concerns. There is one population that is not targeted even though they have quite an appetite for sex, being more multi-partner than the general population: people who frequent shows organised by the erotic industry. Two associations, with the support of healthcare professionals, set up booths inside the shows, providing information on HIV/STI prevention and screening and offering HIV RDT.
Methods
Cross-sectional study through anonymous self-reported questionnaire of people participating in erotic shows in 4 large cities in 2015, all willing to have the HIV RDT. Socio-demographic profile, risk practices and attitude towards HIV/STIs screening were collected as well as RTD results.
Results
In the 4 erotic shows, 943 people took part in the study, 2/3 men, mean age 26 years. >1/3 had never been tested for HIV and >2/3 for STI. 65.8% had had sexual relations without a condom over the past 12 months, 10.7% had used cocaine or snorted over the past 5 years and 73% had experienced abusive consumption of alcohol. Those who had never had an HIV test were statistical younger, more often male, report more frequently no sexual relations over the past 12 months and more often heterosexual relationships and sexual relationships in exchange for money/services. Out of the 943 RDT, 3 were positive. Apart from the demand for testing, people were very keen to get information on HIV/STIs and to discuss their sexual practices.
Conclusion
This study demonstrates that a fun and commercial backdrop as erotic show can be useful as a potential new site for HIV/STI information, screening and to tackle issues of sexuality. People who might not have done the test or sought information elsewhere were encouraged to have themselves screened and to reflect on their risk behavior.
In France, 2 people living with HIV (PLHIV) out of 5 are more than 50 years old. This study was performed to know more about healthcare experience of PLHIV aged over 40 years and their worries about ageing. From July to October 2015, a self-administrated questionnaire was available on Sida Info Service, the French HIV/AIDS helpline, web site. 194 questionnaires constitute the sample. Both quantitative and qualitative data were analyzed with Modalisa v6. This abstract presents only results about comorbidities and healthcare.
75.8% are male. The mean age is 52 years and mean time since HIV diagnosis is 18 years. Almost all participants are on antiretroviral therapy treatment (ART, 97.4%), for 12.5 years on average. 62.9% report one or more comorbidities among dyslipidemia, cardiovascular diseases, osteoporosis, cognitive impairment, renal failure and diabetes. Age, time since HIV diagnosis and time since initiating ART are factors significantly associated with comorbidities development. Depending on the comorbidity, percentage of participants who do not know if they are affected varies between 4.1% and 20.6% and percentage of those do not know if they are regularly screened for varies between 8.4% and 20.9%. Apart from age, screening of comorbidities do not seem to follow the guidelines: 43.3% of participants are not screened every year for renal failure; when on ART 27.3% are not screened once a year for dyslipidemia; female participants are not more screened for osteoporosis than male.
Results show importance of improving PLHIV aged over 40 comordibities screening. In one hand, boosting health professionals’ knowledge about comorbidities screening guidelines appears to be a priority. In another hand, increasing communication between health professionals and patients, in both directions, is important. In that way, PLHIV could develop their knowledge about their own healthcare. It could help raise their empowerment in making informed decisions about their health.
Background
Integrated HIV DNA persists for decades within the genome of HIV-positive individuals, even when they are successfully treated with antiretroviral therapy (ART) with undetectable HIV RNA levels. The persistence of integrated HIV DNA within reservoir cells contributes to our inability to achieve viral eradication in infected individuals who are thus obligated to take life-long treatments.
Although rare, treatment failure in treatment-experienced, integrase inhibitor-naïve individuals treated with dolutegravir (DTG) is commonly associated with the emergence of the R263K substitution in integrase and plasma viral loads that are lower than those observed when treatment failure occurs with ART regimens that do not contain DTG. This is likely due to the fact that R263K confers low-level resistance against DTG and also decreases viral replication capacity and viral integrase activity in short-term infectivity assays.
We sought to determine the effect of the DTG-specific R263K resistance substitution on integration during long-term infections.
Methods
We measured HIV integration by Alu-mediated QPCR over 5 weeks of infection of Jurkat cells with WT, R263K and H51Y/R263K viruses. Levels of integration were measured every week and expressed relative to integration of the WT virus after week 1. Means ± standard deviations were calculated and Student's t-test was used to evaluate significance of differences.
Results
The R263K substitution impaired HIV integration over time and was associated with a progressive decline in levels of integrated HIV DNA. Even further impairments were noted if both the R263K and H51Y substitutions were simultaneously present. Differences were statistically significant.
Conclusions
Our study raises the possibility that emergence of the R263K substitution in individuals who experience treatment failure with DTG might result in a progressive decline in the size of the viral reservoir. Further studies are needed to study this hypothesis.
Introduction
For better understanding of HIV pathogenesis and persistence, it is important to identify viral biomarkers predictive for disease progression and immunological response to ART.
Methods
In 56 patients that participated in a randomized controlled trial of temporary ART vs. no treatment during primary infection (PHI), we have assessed the predictive power of plasma viremia, total HIV-1 DNA, unspliced-gag and multiply spliced (MS)-tat/rev cell-associated HIV-1 RNA, and CD4+ T-cell count, measured at the virological set-point (36 weeks after the PHI diagnosis or discontinuation of early ART), for the time to reach the CD4+ count of 350 cells/mm3. Subsequently, in 27 of these patients who were randomized to receive early ART, we measured the same virological markers, CD4+ count, and CD4:CD8 ratio at PHI, and assessed their predictive power for normalization of the CD4:CD8 ratio by 48 weeks of early ART.
Results
Among the virological markers, MS RNA was the strongest predictor of disease progression: median times to 350 cells/mm3 in patients with MS RNA levels below and above the median were 1180 and 283 days, respectively (P=0.0002, log-rank test). In multivariate Cox regression analysis adjusted for temporary ART vs. no treatment during PHI, CD4+ count (P=0.0004) and MS RNA level (P=0.011) were the only two significant predictors of disease progression. MS RNA level at PHI was also the only marker significantly associated with the CD4:CD8 ratio at 48 weeks ART: the median [IQR] MS RNA levels were 34 [23–186] and 1047 [150–4677] copies/μg total RNA in patients with CD4:CD8 ratios >1 and <1, respectively, at 48 weeks ART (P=0.0017, Mann–Whitney test). MS RNA was the only significant predictor of CD4:CD8 ratio normalization in multivariate logistic regression adjusted for the above parameters (P=0.015).
Conclusions
We identified MS RNA level as an independent predictor of both disease progression without ART and CD4:CD8 ratio normalization on ART.
Human immunodeficiency virus type 1 (HIV-1) can efficiently spread in a cell-free form or by direct cell-to-cell contact, a mechanism termed cell-associated (CA) HIV transmission. To date, most therapeutic and preventive vaccine candidates towards HIV-1 have been evaluated pre-clinically for efficacy against cell-free viral challenges. However, it has been suggested that CA virus is more efficient at transmitting HIV-1 and more difficult to neutralize than cell-free virus.
Prevention from cervico-vaginal transmission in macaques exposed to cell free viral particles of SHIV has been reported after local application or after passive transfer of diverse combinations of broad neutralizing antibodies (bNAbs). However, to date, there is no evidence that strategies that can efficiently prevent macaques exposed to cell-free SHIV, like the use of bNAbs, can also protect from CA-SHIV exposure.
Here we aimed to determine in vitro the extent to which bNAbs inhibit cell-free and CA SHIV162p3 transmission. We developed a cell-to-cell transmission assay and evaluated antibody-mediated virus neutralization using TZM-bl as target cells and in vivo SHIV162pP3-infected spleen cells as donor cells. We used the gp120-directed antibody 2G12, the two gp41-directed antibodies, 2F5 and 4E10, and the anti CD4-binding site IgG1b12 against the two transmission route. The bNAbs were used either alone or in combinations of 3 (2G12+2F5+4E10) and 2 (2G12+4E10, 2G12+2F5, 4E10+2F5). Our results demonstrated that CA SHIV162P3 transmission was less sensitive to neutralizing antibodies compared to cell-free SHIV162p3. Moreover, inhibition of CA transmission was achieved only when 2G12 was present in the NAbs combination. Interestingly the IgG1b12 was very efficient to inhibit cell-cell transmission. Therefore, our findings support the use of antibody combinations against cell-associated virus in future candidate therapeutic regimens.
Little information is available on the generation of HIV-specific T cell responses in newborns early after infection. 12 newborn macaques were intravenously iv infected (48 hrs post-delivery) with pathogenic SIV (n=6) or attenuated SIVΔV1-V2 (n=6). Animals were bled every 2–3 days and plasma viremia monitor at day 10, 14 or 28 post-SIV and until euthanasia where blood, BAL were collected with 25–30 lymphoid and non-lymphoid tissue samples. Analysis of CD8+ T cell responses in SIV-infected animals revealed significant levels of Gag and Tat tetramer-specific binding cells that peaked 2 to 3 wks post SIV (mean values 1% and 0.45 %, respectively) compared to ΔV1-V2 infected animals with levels of Gag tetramer up to 2.5% in PBMC and 1.3% in spleen at day 14. Peak plasma viremia was between days 7–10 in SIV-infected macaques (8.5 log copies/mL) but significantly lower (3–4 log decrease) in ΔV1-V2 infected animals. IFNg ELISPOT revealed early generation of SIV-specific T-cell response to both Gag and Tat that peaked at day 14 (up to 735 SFCs/106 cells) in SIV infected newborns. Stronger T-cell responses to Gag, Env, Tat (up to 1900 SFCs/106 cells) were detected in PBMC from ΔV1-V2 infected animals. Significant increase in the breadth and intensity of SIV-specific T-cell responses was detected at day 14 in spleen, LN, gut of ΔV1-V2 newborns coincident with initial peak viremia (1.2x106copies/ml) at day 14 and rapid decline (1.1x104 copies/ml) at day 28. An expansion of effector SIV-specific CCR7-CD127-IL2- but CD45RA+Perforin+ T-cells was detected in blood of ΔV1-V2-animals, which were mostly CCR7-CD45RA-Perforin-Granzyme B+ in BAL suggesting that newborns can generate strong and broadly directed virus-specific T-cell responses early after infection. Taken together these results suggest, within the limitation of the small animals number studied, that SIV-infected newborns are able to generate functional and virus-specific T cell immune responses early after infection.
Aims
Hepatitis D virus (HDV), in association with hepatitis B, is responsible of faster progression to liver disease and higher mortality rates. HDV exhibits a high level of genetic diversity with eight known genotypes worldwide. Circulating HDV RNA detection is required to confirm viral replication and to evaluate efficiency of treatment. Recent studies have shown that all of available in-house and commercial diagnostic RT-PCR tests failed in detecting or quantifying the 8 clades of HDV. French reference center for HDV (CNR) has developed the first consensus RT-PCR test able to quantify all genotypes. However, this in-house test has several technical limits incompatible with large scale utilization, especially in low-resource countries, including several manual steps, duration and costs.
The aim of this study is to develop and validate a generic one-step RT-PCR kit, based on the CNR test protocol, easier to use and with higher performances than available products.
Materials and methods
Viral RNA was extracted from 0.2 mL of plasma and eluted in 60 μL RNase-free buffer using QIAamp MinElute Kit (Qiagen). A short denaturation step is realized before RT-PCR. Then, 10 μl of the HDV RNA were added to the 15 μL of the reaction mix. Clinical validation was done on 25 selected blinded samples comparatively with the CNR method.
Results
This assay was specific and showed linearity over a wide range from 10 to 107 copies/mL. The 95% detection limit was 100 copies/mL. Intra-assay variability ranged from 0.41 to 1.28%, whereas the inter-assay variability ranged from 3.38 to 4.77%. The kit is able to detect and quantify all genotypes provided and kit showed similar viral load [Figure O15.1].
Conclusions
Collaboration between the CNR and Omunis lead to one of the first validated, standardized and easy-to-use HDV RT-PCR kit compatible with pan-genotypes detection and allowing large distribution worldwide.
Figure O15.1. HDV RNA quantification using Omunis kit in comparison with CNR reference test values
Introduction
HIV-1 integrase (IN) inserts viral DNA in the host cells in two steps: the 3’-processing (3’-P) and the strand transfer (ST). The efficiency of the first integrase strand transfer inhibitors (INSTIs) raltegravir (RAL) and elvitegravir (EVG) is limited by the rapid occurrence of integrase mutations. However, the recently approved dolutegravir (DTG) has a high barrier to resistance. This study aims to understand the increased efficiency of DTG by focusing mainly on its interaction properties with viral DNA. We also suggest DTG derivatives that would interact less with IN and more with the viral DNA.
Materials and methods
Thermodynamic parameters of drug-DNA complexes were determined using fluorescence anisotropy. The intermolecular interaction energies ΔE were computed with the G09 software. NCI-plot program enabled a qualitative visualization of the intermolecular interactions.
Results
In fluorescence anisotropy titrations, DTG shows better interaction with viral DNA than EVG. In addition, NCI-Plot analysis and ΔE values illustrate a higher affinity of DTG's halobenzene moiety for guanine 4 (G4) and cytosine 16 (C16) located at the viral DNA end [Figure O16.1]. Moreover, DTG's derivatives scored more favorable interaction energies for G4/C16 than the parent DTG. The ranking is as follows: b > d > a > c > DTG, with absolute ΔE values in kcal/mol of: 44 > 43.3 > 42.8 > 41.8 > 37.8, respectively; with the derivatives differing by: (a) Substitution of DTG's F in para position by NH2 group, (b) Substitution of DTG's F in para position by CH3NH group, (c) Shift of DTG's F in ortho site to meta position; substitution of DTG's F in para position with NH2 group, (d) Shift of DTG's F in ortho site to meta position; substitution of DTG's F in para position with CH3NH group.
Conclusion
This study would contribute to the design of more potent anti-IN drugs.
Figure O16.1. Representation of the IN-DNA-dolutegravir complex focusing on the recognition site of the DTG's halobenzene ring in interaction with G4 and C16 bases at the viral DNA ends. Dolutegravir is shown in pink, the two cations Mg2+ are represented in two yellow spheres. The green plots are the intermolecular interaction surfaces between G4, C16 and the DTG's halobenzene bearing 2 fluorine atoms shown in green (one in ortho position and the other in meta site). These interactions were generated by the NCI-plot analysis
Introduction
People living with HIV are living longer. However, the proportion of deaths related to chronic comorbid diseases has increased. The aim of this practice survey was to evaluate a systematic screening approach developed in a routine HIV clinic setting in South of France.
Methods
From January to December 2015, HIV patients receiving ART were screened for co-morbidities and risks factors (1) in a one-day procedure at hospital. Socioeconomic status was evaluated using the EPICES score.
Results
During the study period, 163 patients out of cohort of 500 patients were screened. There were 146 male and 17 female, with a mean age of 48.5 years, a mean EPICES score of 35.8/100 and with 125 (85%) MSM. Identified risk factors were cardiovascular in 100 (61%) including 55 (34%) long-term smokers, IST in 65 (40%), hepatitis co-infection in 34 (21%), IVDU in 17 (10%). Overall, we notified 60 (37%) respiratory diseases, 53 (32.5%) high vitamin D deficiency with a need of supplementation, 25 (15%) chronic hepatitis C leading to fibrosis assessment, 23 (14%) cardiovascular diseases, 14 (8.5%) anal lesions identified by anoscopy and 10 (6%) urogenital disorders, 8 (5%) diabetes, 5 (3%) neurological diseases, and one ophthalmologic disease. Among the long term-smokers, 18 (33%) had a clinical event of COPD or asthma (P=0.02). Similarly, cardiovascular risk factor was associated with COPD or asthma (P=0.003), with event of pneumonia (P=0.01), with rheumatologic involvement (P=0.03), and nutritional deficiency (P=0.004). EPICES scores were higher in non-MSM (P=0.02) and tends to be higher in IVDU (P=0.08), but did not show any association with smoking, IST or cardiovascular risk factors.
Conclusion
This survey confirms that, even in a population of HIV patients with an intermediate precarity, such a recommended annual systematic screening approach (1) allow the detection of risk factors and various important comorbidities.
Introduction
The detection and monitoring of hepatic fibrosis allow improved care of patients with chronic liver disease. Biological and radiological non-invasive methods (NIM) have been developed to assess fibrosis. It is recommended to perform at least 2 NIM (including 1 biological) to provide consistent results. The aim of this study was to evaluate in real life the performance of the combination of NIM.
Methods
All consecutive patients undergoing fibrosis assessment in chronic liver disease in our center were included prospectively. Each patient was evaluated concomitantly by bioassay (FibroTest, except if HIV treatment: FibroMax or FibroMeter), Fibroscan (Echosens, Paris, France) and ultrasound (US) (Acuson S2000, Siemens). Measurements were performed by experienced investigators, blind to the results of other NIM. The results were compared for the presence of significant fibrosis (≥F2), severe fibrosis (≥F3) or cirrhosis (F4).
Results
94 patients were enrolled: 77% male, aged 50±7 years, with BMI of 24±6 and alcohol consumption in 12%. The etiology of liver disease was mainly (n=82) viral hepatitis B and/or C, mostly with HIV co-infection (n=73). Overall, we observed good agreement between the biological tests, US and Fibroscan. The direct concordance between US and biological tests to identify ≥F2, ≥F3 or F4 was 63%, 74% and 83% respectively. The concordance between Fibroscan and biological tests was higher: 72%, 82% and 86% respectively. The concordance between Fibroscan and US was excellent: 76%, 90% and 93% respectively. We evaluated a sequential algorithm using first biological tests and Fibroscan and secondly, US, in cases of discrepancy. For discordant cases in the identification of ≥F2, ≥F3 and F4 respectively, US indicates a concordance rate of 73%, 94% and 85% with Fibroscan and 27 %, 6% and 15% with the bioassay.
Conclusion
In real life, because of a better concordance with biological NIM, the choice of Fibroscan may avoid the need for a 3rd test in 3/4 of cases. In case of discrepancy, US mostly confirm the evaluation Fibroscan except in patients with high BMI.
Background and Aims
Recent clinical trials of direct-acting-antiviral agents (DAAs) against hepatitis C virus (HCV) achieved >90% sustained-virological response (SVR) rates, suggesting that cure often took place before the end of treatment (EOT). We sought to evaluate retrospectively whether early response kinetics can provide the basis to individualize therapy to achieve optimal results while reducing duration and cost.
Methods
58 chronic-HCV patients were treated with 12-week sofosbuvir+simeprevir (n=19), sofosbuvir+daclatasvir (n=19), or sofosbuvir+ledipasvir in three French referral centers. HCV was measured at baseline, day 2, every other week, EOT and 12 weeks post EOT. Mathematical modeling was used to predict the time to cure, i.e, <1 virus copy in the entire extracellular-body fluid.
Results
All but one patient who relapsed achieved SVR. Mean age was 60±11 years, 53% were male, 86% HCV genotype-1, 9% HIV co-infected, 43% advanced fibrosis (F3), and 57% had cirrhosis. At weeks 2, 4 and 6, 48%, 88% and 100% of patients had HCV<15 IU/ml, with 27%, 74% and 91% of observations having target-not-detected, respectively. Modeling results predicted that 23(43%), 16(30%), 7(13%), 5(9%) and 3(5%) subjects were predicted to reach cure within 6, 8, 10, 12 and 13 weeks of therapy, respectively. The modeling suggested that the patient who relapsed would have benefitted from an additional week of sofosbuvir+ledipasvir. Adjusting duration of treatment according to the modeling predicts reduced medication costs of 45% and 30% in subjects who had HCV<15 IU/ml at weeks 2 and 4, respectively.
Conclusions
The use of early viral-kinetic analysis has the potential to individualize duration of DAA therapy with a projected average cost-saving of 20% per 100-treated persons.
CCR5 is the major HIV co-receptor, and individuals homozygous for a 32-bp deletion in Ccr5 gene are resistant to infection by CCR5-tropic HIV-1. CCR5 co-receptor provides a unique opportunity to exploit gene knockout technologies for anti-HIV therapy. We are pursuing the use of engineered Zinc Finger Nucleases (ZFNs) to permanently disrupt the CCR5 open-reading frame. We are targeting the second extracellular loop of CCR5 to obtain cells with mutations which mimic a CCR5delta32 mutation. We used a reporter plasmid to check the activity of the ZFNS. CCR5-targeted ZFNs are currently being evaluated in vitro in our laboratory, targeting mature CD4+ T cells and hematopoietic stem cells isolated from naïve-uninfected macaque blood, bone marrow and umbilical cord samples. We engineered SIV-resistant macaque CD4+ T cells using CCR5-ZFNs. After nucleofection of mRNAs encoding for ZFNs into CD4+ cells isolated from macaques, we show that these cells were resistant to in vitro SIVmac239, SIVmac251, and SIVagm infections as shown by the absence of p27 expression. We then focused on the modification of HSC isolated from macaque femoral bone marrow and umbilical cords. We established conditions required to purify and grow macaque CD34+ HSCs in vitro to maximize the efficiency of CCR5 gene disruption while minimizing any adverse effects on cell viability or hematopoietic potential. We successfully engineered CCR5-modified macaque hematopoietic stem cells that were resistant to SIVmac239 infection after in vitro differentiation and expansion on thymocytes. We demonstrated the feasibility of using ZFN technology to establish CD4+ T cells and hematopoietic stem cells resistant to SIV infection in macaque. Our data suggests that genome editing with CCR5-ZFNs may provide therapeutic benefit to HIV-infected individuals.